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BIOLOGICAL AND MEDICAL PHYSICS,

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Huangxian Ju Xueji Zhang Joseph Wang

NanoBiosensing
Principles, Development and Application
Huangxian Ju Xueji Zhang
Nanjing University World Precision Instruments, Inc.
Nanjing, P.R. China Sarasota, FL, USA
hxju@nju.edu.cn and
University of Science & Technology
Joseph Wang Beijing, P.R. China
University of California xueji@wpiinc.com
San Diego, CA, USA
josephwang@ucsd.edu

ISSN 1618-7210
ISBN 978-1-4419-9621-3 e-ISBN 978-1-4419-9622-0
DOI 10.1007/978-1-4419-9622-0
Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2011932683

Springer Science+Business Media, LLC 2011


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Preface

The first decade of the 21st century has been labeled the sensing decade. Biosensing,
based on nanomaterials, is one of the hottest topics in nanotechnology and nanosci-
ence. The unique properties of nanomaterials offer excellent platforms as electronic
and optical signal transduction to design a new generation of biosensing devices.
Thus, nanobiosensing opens up the novel concepts for basic research and new tools
for ultrasensitive biosensing in clinical, environmental, and industrial applications.
With the achievements of nanotechnology and nanoscience, a wide variety of
nanoscale materials with different sizes (1100 nm), shapes, and compositions
have been introduced to biosensing. The small sizes of nanoparticles break through
the limitation of structure miniaturization, leading to lower detection limits, even
reaching zeptomolar concentrations. Furthermore, the biofunctional nanoparticles
can produce a synergic effect among catalytic activity, conductivity, and biocom-
patibility to accelerate the signal transduction. Most importantly, nanoscale materials
are in direct contact with the environment, which permits them to act as chemical
and biological sensors in the single molecular detection of biomolecules. As of this
writing, nanobiosensing is routinely being applied in biological systems. Future
efforts on nanomaterial-based biosensing will involve invivo detection with less
cytotoxicity, high sensitivity, and long-term stability for early screening of disease
biomarkers and reliable point-of-care diagnostics.
This book introduces novel principles and detection strategies in the area of bio-
sensing based on nanomaterials. Each chapter provides a theoretical overview of a
different topic and the interesting bioanalytical application of nanobiosensing
devices. The most exciting and unique aspect of the book is that the utilization of
nanomaterials not only enhances the biosensing capabilities, but also brings out
newer approaches such as biomimetic, reagent-less biosensing, and single molecular
detection.
The material is presented in 18 chapters, covering the most successful nanoma-
terials used so far in biosensing. The first four chapters of the book, contributed by
Huangxian Ju (Chap. 1), Songqin Liu and Huangxian Ju (Chap. 2), Zhihui Dai and

v
vi Preface

Huangxian Ju (Chap. 3), and Jianping Lei and Huangxian Ju (Chap. 4), describe the
biofunctionalization of nanomaterials, signal amplification strategies for nanobio-
sensing, and nanostructured mimicking enzymes. The next six chapters focus on
ultrasensitive platforms using various nanomaterials, such as carbon nanofiber,
nanoporous silica, carbon nanotubes, quantum dots, molecularly imprinted nanopar-
ticles, and solgel nanoparticles; they are contributed by Xueji Zhang and Lei Su
(Chap. 5), Shuo Wu and Huangxian Ju (Chap. 6), Jianping Lei and Huangxian Ju
(Chap. 7), Guizheng Zou and Huangxian Ju (Chap. 8), Ruizhuo Ouyang and
Huangxian Ju (Chap. 9), and Jiuhong Yu and Huangxian Ju (Chap. 10). The last eight
chapters illustrate the successful applications of nanobiosensing in environmental
screening and clinical diagnosis and have been contributed by Xueji Zhang and
Chunyan Wang (Chap. 11), Sichun Zhang and Huangxian Ju (Chap. 12), Dan Du and
Huangxian Ju (Chap. 13), Zong Dai, Joseph Wang, and Huangxian Ju (Chap. 14),
Zhifeng Fu and Huangxian Ju (Chap. 15), Yongkang Ye, Joseph Wang, and Huangxian
Ju (Chap. 16), Lin Ding and Huangxian Ju (Chap. 17), and Jie Wu, Joseph Wang, and
Huangxian Ju (Chap. 18). We are very grateful to all the authors who contributed
their high-quality work. We also thank Jie Wu for her help in editing the whole book.
This book involves a broad audience, such as those involved in the research,
teaching, learning, and practice of biosensing, based on various nanomaterials, in
biomedical, military, industrial, and clinical applications. We are fortunate to have
had the opportunity to undertake this enormous project. We warmly acknowledge
the gracious support of our families. Finally, we also thank Springers editors for
doing a remarkable job to publish this book.

Nanjing, P.R. China Huangxian Ju


Sarasota, FL Xueji Zhang
San Diego, CA Joseph Wang

Contents

1 Biofunctionalization of Nanomaterials.................................................. 1
1.1 Introduction....................................................................................... 1
1.2 Biofunctionalization Method of Nanomaterials................................ 2
1.2.1 Biofunctionalization by Noncovalent Assembly................... 2
1.2.2 Covalent Route for the Biofunctionalization
of Nanomaterials................................................................... 5
1.3 Biofunctional Nanomaterials............................................................ 10
1.3.1 Carbon-Based Nanomaterials............................................... 10
1.3.2 Metal Nanoparticles.............................................................. 12
1.3.3 Semiconductor Nanoparticles............................................... 14
1.3.4 Magnetic Nanoparticles........................................................ 15
1.3.5 Other Biofunctional Nanomaterials...................................... 17
1.4 Characterization of Biofunctional Nanomaterials............................. 19
1.5 Applications of Biofunctional Nanomaterials................................... 21
1.5.1 Optical Sensing..................................................................... 21
1.5.2 Electrochemical Sensing....................................................... 27
1.6 Conclusions....................................................................................... 32
References.................................................................................................. 32
2 Signal Amplification for Nanobiosensing............................................... 39
2.1 Introduction....................................................................................... 39
2.2 Nanoparticle-Amplified Optical Assay............................................. 40
2.2.1 Colloidal Gold Nanoparticle-Based Amplification............... 40
2.2.2 Semiconductor Nanoparticle-Based Amplification.............. 48
2.2.3 Nanoparticle-Amplified Chemiluminescence
and Electrogenerated Chemiluminescence Assay................. 49
2.3 Nanoparticle-Amplified Electrochemical Detection......................... 54
2.3.1 Enhanced Conductivity with Nanoparticles.......................... 54
2.3.2 Detection of Nanoparticle Label with
Stripping Voltammetry.......................................................... 57
2.3.3 Nanoparticle-Enhanced Impedance Signal........................... 58
2.3.4 Nanoparticle-Enhanced Voltammetric Signal....................... 62
vii
viii Contents

2.4 Nanoparticles as Carrier for Signal Amplification............................ 65


2.4.1 Gold Nanoparticles as Tracer................................................ 65
2.4.2 Carbon Nanotubes as Carrier................................................ 73
2.4.3 Silica Nanoparticles as Carrier.............................................. 75
2.4.4 Other Materials as Carrier..................................................... 77
2.5 Conclusions....................................................................................... 79
References.................................................................................................. 79
3 Nanostructured Mimic Enzymes for Biocatalysis
and Biosensing.......................................................................................... 85
3.1 Introduction....................................................................................... 85
3.1.1 Need for Nanostructured Mimic Enzymes............................ 85
3.1.2 Developments in Nanostructured Mimic Enzymes............... 86
3.2 Nanostructure Used in Artificial Mimic Enzymes............................ 87
3.2.1 K3Fe(CN)6............................................................................. 87
3.2.2 Fe3O4..................................................................................... 89
3.2.3 FeS........................................................................................ 93
3.2.4 Polystyrene............................................................................ 95
3.2.5 Breslows Mimics.................................................................. 96
3.3 Mimic Enzymes for Sensors............................................................. 98
3.3.1 H2O2 Sensors......................................................................... 98
3.3.2 Glutamate Sensors................................................................. 98
3.3.3 Glucose Sensors.................................................................... 99
3.3.4 Nonelectroactive Cation Sensors.......................................... 100
3.3.5 Easily Oxidizable Compounds and Other
Nontraditional Sensors.......................................................... 100
3.3.6 Transition Metal Hexacyanoferrate Sensors......................... 101
3.4 Conclusions....................................................................................... 102
References.................................................................................................. 102
4 Porphyrin-Based Nanocomposites for Biosensing................................ 111
4.1 Introduction....................................................................................... 111
4.1.1 Porphyrin: A Mimic of Enzymes.......................................... 111
4.1.2 Significance of the Porphyrinic Nanocomposite................... 112
4.2 Assembly of Porphyrins on Carbon-Based Nanomaterials............... 112
4.2.1 Carbon Nanotubes................................................................. 113
4.2.2 Optical and Electrochemical Biosensing.............................. 117
4.2.3 Carbon Nanohorns................................................................ 119
4.2.4 Graphene Sheets.................................................................... 120
4.3 Assembly of Porphyrins on Semiconductor Nanoparticles.............. 122
4.3.1 TiO2Porphyrin Nanocomposite........................................... 122
4.3.2 Quantum Dots....................................................................... 129
4.3.3 Fe3O4 Nanoparticles.............................................................. 131
4.4 Assembly of Porphyrins on Metal Nanoparticles............................. 131
4.4.1 Au Nanoparticles................................................................... 131
4.4.2 Ag Nanoparticles................................................................... 134
4.4.3 Pt Nanoparticles.................................................................... 135
Contents ix

4.5 Other Nanomaterials......................................................................... 137


4.5.1 Polymer Nanoparticles.......................................................... 137
4.5.2 Silica Nanomaterials............................................................. 138
4.5.3 Calcium Phosphate Nanoparticles........................................ 140
4.6 Conclusions....................................................................................... 141
References.................................................................................................. 142
5 Carbon Nanofiber-Based Nanocomposites for Biosensing................... 147
5.1 Introduction....................................................................................... 147
5.2 Synthesis of Carbon Nanofiber......................................................... 149
5.3 Why Carbon Nanofiber?................................................................... 151
5.4 Carbon Nanofiber-Based Electrochemical Biosensors
and Bioassays.................................................................................... 151
5.4.1 Glucose Sensors.................................................................... 151
5.4.2 Ethanol Sensors..................................................................... 154
5.4.3 Acetylthiocholine Sensors..................................................... 155
5.4.4 Phenol Sensors...................................................................... 156
5.4.5 Hydrogen Peroxide Sensors.................................................. 157
5.4.6 NADH Sensors...................................................................... 159
5.4.7 Protein Electron Transfer (ET).............................................. 160
5.4.8 Immunosensors..................................................................... 162
5.4.9 Vertically Aligned Carbon Nanofiber Array-Based
Biosensors............................................................................. 162
5.5 Conclusions....................................................................................... 166
References.................................................................................................. 167
6 Biosensors Based on Nanoporous Materials.......................................... 171
6.1 Introduction....................................................................................... 171
6.2 Why Are Proteins Immobilized?....................................................... 172
6.3 Biosensors Based on Mesoporous Materials.................................... 173
6.3.1 Factors Affecting Protein Immobilization............................. 173
6.3.2 Methods for Protein Immobilization
on Mesoporous Material....................................................... 177
6.3.3 Biosensors Based on Mesoporous Silica.............................. 178
6.3.4 Biosensors Based on Mesoporous Carbon............................ 185
6.3.5 Biosensors Based on Mesoporous Metal Oxide................... 191
6.3.6 Biosensors Based on Mesoporous Hybrid
Nanocomposite...................................................................... 192
6.4 Biosensors Based on Nanoporous Gold............................................ 196
6.4.1 Enzyme Biosensors Based on Nanoporous Gold.................. 196
6.4.2 DNA Biosensors Based on Nanoporous Gold...................... 198
6.4.3 Escherichia coli Biosensors Based on Nanoporous
Platinum-Coated Gold Nanoporous Film............................. 199
6.5 Conclusions....................................................................................... 200
References.................................................................................................. 200
x Contents

7 Electrochemical Biosensing Based on Carbon Nanotubes................... 207


7.1 Introduction....................................................................................... 207
7.1.1 Structure of CNTs................................................................. 207
7.1.2 Advantages of CNT-Based Electrochemical Sensors........... 208
7.2 Functionalization Strategy of CNTs.................................................. 208
7.2.1 Noncovalent Interaction........................................................ 209
7.2.2 Covalent Interaction.............................................................. 213
7.3 Fabrication and Characterization of CNT-Based Sensors................. 214
7.3.1 Scanning Electron Microscopy and Transmission
Electron Microscopy............................................................. 215
7.3.2 Fourier Transform Infrared................................................... 215
7.3.3 Atomic Force Microscopy..................................................... 216
7.3.4 Raman Spectrum................................................................... 216
7.4 Amplification of Signal Transduction............................................... 218
7.5 Electrochemical Biosensing Based on Functional CNTs................. 220
7.5.1 Deoxyribonucleic Acid......................................................... 220
7.5.2 AntigenAntibody................................................................. 222
7.5.3 Cells...................................................................................... 224
7.5.4 Other Biomolecules............................................................... 226
7.6 SWCNT-Based Field-Effect Biosensing........................................... 229
7.6.1 Detection of Proteins by SWCNT-Field-Effect
Transistor............................................................................... 229
7.6.2 Detection of Nucleic Acids by SWCNT-Field-Effect
Transistor............................................................................... 231
7.7 SWCNT Forest in Electrochemical Biosensing................................ 231
7.8 Conclusions....................................................................................... 233
References.................................................................................................. 234
8 Biosensing with Nanoparticles as Electrogenerated
Chemiluminsecence Emitters.................................................................. 241
8.1 Introduction....................................................................................... 241
8.2 Principle of ECL from Nanoparticles............................................... 242
8.2.1 ECL Mechanism of NPs....................................................... 242
8.2.2 Generation Type for NP ECL................................................ 244
8.2.3 Coreactant System for NP ECL............................................ 247
8.3 Biosensing Strategy and Corresponding Application....................... 251
8.3.1 Direct Determination of Biochemical Coreactant................. 252
8.3.2 Improved ECL Performance for Higher Sensitivity............. 253
8.3.3 Analyte-Inhibited (or -Enhanced) ECL Emission................. 254
8.3.4 Determination Based on Resonance Energy Transfer........... 255
8.3.5 Determination with Enzyme-Catalyzed Reaction................. 255
8.3.6 Immunoreactions or DNA
Hybridization-Coupled Sensing............................................ 257
8.4 Conclusions....................................................................................... 260
References.................................................................................................. 260
Contents xi

9 Biosensing Applications of Molecularly


Imprinted Nanomaterials........................................................................ 265
9.1 Introduction..................................................................................... 265
9.2 Molecular Imprinting Technology................................................... 268
9.2.1 Noncovalent Approach...................................................... 269
9.2.2 Covalent Approach............................................................ 270
9.2.3 Other Approaches.............................................................. 271
9.3 Types of MIP Materials................................................................... 271
9.3.1 Organic Materials.............................................................. 272
9.3.2 Inorganic Materials............................................................ 272
9.4 Development of MIP Nanomaterials............................................... 273
9.4.1 Limitation of Traditional MIPs.......................................... 273
9.4.2 Exploration of Novel Molecular
Imprinting Strategies......................................................... 274
9.4.3 Attractiveness of MIP Nanomaterials................................ 275
9.5 MIP-Based Biosensors.................................................................... 276
9.5.1 Molecular Recognition of MIP-Based Biosensors............ 276
9.5.2 Interfacing the MIPs with a Transducer............................ 278
9.5.3 Electrochemical Sensors.................................................... 279
9.5.4 Optical Sensors.................................................................. 282
9.5.5 Mass-Sensitive Devices..................................................... 288
9.6 Conclusions..................................................................................... 294
References.................................................................................................. 294
10 Biosensors Based on SolGel NanoparticleMatrices........................... 305
10.1 Introduction..................................................................................... 305
10.2 SolGel Chemistry.......................................................................... 306
10.2.1 What Is SolGel?............................................................... 306
10.2.2 The SolGel Process.......................................................... 306
10.2.3 Nanoparticles from the SolGel Process........................... 309
10.3 Biosensors Based on SolGel Nanoparticle Matrices..................... 309
10.3.1 Silica Nanoparticle for Biosensing.................................... 310
10.3.2 SolGel-Derived Metal Oxide Nanoparticle..................... 316
10.3.3 SolGel Nanocomposite Matrix for Biosensing................ 319
10.4 Conclusions..................................................................................... 326
References.................................................................................................. 326
11 Nanostructure for Nitric Oxide Electrochemical Sensing.................... 333
11.1 Introduction..................................................................................... 333
11.2 Nanostructure for Nitric Oxide Determination............................... 334
11.3 Nanomaterials for Modification of NO Electrochemical
Sensors............................................................................................. 336
11.4 Conclusions..................................................................................... 344
References.................................................................................................. 345
xii Contents

12 Assembly of Nanostructures for Taste Sensing..................................... 349


12.1 Introduction..................................................................................... 349
12.2 Nanoassembled Films for Taste Sensor Application....................... 350
12.2.1 Nanoassembled Conducting Polymers for
Taste Sensor Application................................................... 350
12.2.2 Carbon Nanotube Polymer Composite
for an Impedimetric Electronic Tongue............................. 352
12.3 Sensor Array Based on Gold NanoparticleFluorophore
Complexes....................................................................................... 352
12.3.1 Detection of Proteins......................................................... 353
12.3.2 Detection of Bacteria and Mammalian Cells..................... 355
12.4 Catalytic Nanomaterial-Based Optical Sensor
and Sensor Array............................................................................. 356
12.4.1 Nanomaterial-Based Cataluminescence Sensors
for Vapor Sensing.............................................................. 356
12.4.2 Catalytic Nanomaterial-Based Optical Chemosensor
Array for Recognition and Discrimination Odors............. 358
12.4.3 Recognition of Organic Compounds in
Aqueous Solutions by Chemiluminescence
on Catalytic Nanoparticle Arrays...................................... 361
12.5 Conclusions..................................................................................... 363
References.................................................................................................. 363
13 Nanostructured Biosensing for Detection of Insecticides..................... 365
13.1 Introduction..................................................................................... 365
13.1.1 Conventional Strategies for Detection
of Pesticides....................................................................... 365
13.1.2 Developments in Pesticide Biosensors.............................. 366
13.2 Enzymes Used in Pesticide Biosensors........................................... 367
13.2.1 ChE-ChO Bienzyme-Based Biosensors
for Pesticides..................................................................... 367
13.2.2 AChE-Based Biosensors for Pesticides............................. 369
13.2.3 OPH-Based Biosensors for Pesticides............................... 371
13.3 Nanostructured Biosensor Design for Pesticide Analysis............... 372
13.3.1 Carbon NanotubeBased Nanobiosensor
for Pesticides..................................................................... 372
13.3.2 Gold Nanoparticles as Transducer for Detection
of Pesticide........................................................................ 375
13.3.3 Quantum Dots as Transducers for Detection
of Pesticides....................................................................... 376
13.4 Pesticide Immunosensors................................................................ 377
13.4.1 Detection Methods for Pesticide
Immunosensors.................................................................. 377
13.4.2 Immunosensors for Pesticides........................................... 378
Contents xiii

13.5 Nanotechnology for Biomonitoring of AChE Activity


and Pesticides.................................................................................. 381
13.5.1 Biomarkers of Organophosphate
Pesticide Exposure............................................................. 381
13.5.2 Biomonitoring of ChE Activity......................................... 382
13.6 Conclusions..................................................................................... 384
References.................................................................................................. 385
14 Carbohydrate Detection Using Nanostructured Biosensing................ 393
14.1 Introduction..................................................................................... 393
14.2 Structural Depiction of Glycans...................................................... 394
14.2.1 Basic Structural Unit of Glycans....................................... 394
14.2.2 Glycoconjugate.................................................................. 394
14.2.3 Major Classes of Glycoconjugates
and Oligosaccharides......................................................... 394
14.3 Biological Roles of Glycans............................................................ 396
14.4 Difficulty in Studying Genetic Glycosylation Defects.................... 397
14.5 ProteinGlycan Interactions............................................................ 397
14.6 Techniques Used to Identify a Carbohydrate
and Its Derivates.............................................................................. 398
14.6.1 General Considerations for Analyzing the Primary
Structure of a Carbohydrate............................................... 398
14.6.2 Detection of Carbohydrates............................................... 399
14.6.3 Linkage Analysis............................................................... 400
14.7 Nanotechnology............................................................................... 402
14.7.1 Analysis of Carbohydrates in Biological System
with Nanostructure Device/Components........................... 402
14.7.2 Other Principles of Direct Carbohydrate
Detection............................................................................ 404
14.7.3 Carbohydrate Components in Biosensors......................... 413
14.8 Conclusions..................................................................................... 420
References.................................................................................................. 420
15 Nanomaterials for Immunosensors and Immunoassays...................... 425
15.1 Introduction..................................................................................... 425
15.2 Principle of Immunoassays and Immunosensors............................ 426
15.2.1 Antigen, Antibody, and Their Recognition
Reaction............................................................................. 426
15.2.2 Immunoassays and Immunosensors.................................. 427
15.3 Immunosensors Based on Biocompatible Nanomaterials............... 429
15.3.1 Nanomaterials Used as Immobilization Substrates........... 429
15.3.2 Nanomaterials Used as Signal Tags................................... 436
15.3.3 Nanomaterials Used as Probe Carriers.............................. 444
15.4 Conclusions..................................................................................... 447
References.................................................................................................. 448
xiv Contents

16 Nanostructured Biosensing and Biochips for DNA Analysis............... 453


16.1 Introduction..................................................................................... 453
16.2 Nanostructures in DNA Biosensing................................................ 455
16.2.1 Carbon Nanotubes for DNA Analysis............................... 455
16.2.2 Metal Nanoparticles for DNA Analysis............................ 464
16.2.3 Semiconductor Nanostructures for DNA Analysis........... 467
16.3 Nanostructures for DNA Biochips.................................................. 471
16.3.1 Optical Techniques for DNA Biochips.............................. 471
16.3.2 Electrochemical Methods for DNA Biochips.................... 474
16.4 Conclusions..................................................................................... 475
References.................................................................................................. 479
17 Cytosensing and Cell Surface Carbohydrate Assay
by Assembly of Nanoparticles................................................................. 485
17.1 Introduction..................................................................................... 485
17.2 Why Use Nanomaterials in Cytosensing?....................................... 486
17.3 Cytosensing by Assembly of Nanomaterials................................... 487
17.3.1 Fluorescence Imaging by Assembly
of Nanoparticles................................................................. 488
17.3.2 Magnetic Resonance Imaging by Assembly
of Magnetic Nanoparticles................................................ 499
17.3.3 Cellular Surface-Enhanced Raman Scattering (SERS)
Detection by Assembly of Nanoparticles.......................... 506
17.3.4 Colorimetric Cytosensing by Assembly
of Nanoparticles................................................................. 511
17.3.5 Electrochemical Cytosensing by Assembly
of Nanomaterials............................................................... 511
17.4 Cell Surface Carbohydrate Assay by Assembly
of Nanoparticles.............................................................................. 517
17.4.1 Cell Surface Carbohydrate Assay Based
on Nanomaterial Substrates............................................... 518
17.4.2 Cell Surface Carbohydrate Assay Based
on Nanoprobes................................................................... 520
17.5 Conclusions..................................................................................... 527
References.................................................................................................. 528
18 Nanobiosensing for Clinical Diagnosis................................................... 535
18.1 Introduction..................................................................................... 535
18.2 Nanotechnologies in Biosensing..................................................... 536
18.2.1 Nanomaterials for Biological Detection............................ 536
18.2.2 Nanofabrication................................................................. 544
18.2.3 Nanodevices....................................................................... 548
18.3 Nanobiosensing for Clinical Diagnosis........................................... 555
18.3.1 Glucose Detection............................................................. 555
18.3.2 Disease Protein Biomarker Detection............................... 556
Contents xv

18.3.3 DNA Detection.................................................................. 558


18.3.4 Virus Detection.................................................................. 560
18.3.5 Bacteria Detection............................................................. 561
18.3.6 Cancer Cell Detection........................................................ 561
18.4 Conclusions..................................................................................... 562
References.................................................................................................. 562

Index.................................................................................................................. 569
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Chapter 1
Biofunctionalization of Nanomaterials

1.1Introduction

The unique properties of nanoscale materials (1200nm) offer excellent platforms


for electronic or optical signal transduction and the design of a new generation of
bioelectronic and biosensing devices. However, the drawbacks of nanoparticles
(NPs) in biocompatibility and biological recognition ability limit their application
in analytical chemistry. The biofunctionalization of nanomaterials can endow them
with good biocompatibility for the immobilization of biomolecules, tissue, or cells
and high specificity for biological recognition [16], which led to stable biosensing
systems with good selectivity and reproducibility. Particularly, the biofunctional
NPs can produce a synergic effect among catalytic activity, conductivity, and bio-
compatibility to accelerate signal transduction and achieve a rapid response to target
with a very high sensitivity by signal amplification. The need for ultrasensitive bio-
assays and the trend toward miniaturized assays have made the biofunctionalization
of nanomaterials one of the hottest fields. Therefore, seeking suitable methods for
the functionalization of nanomaterials with biomolecules such as protein, DNA,
small organic molecules, polymer films, and even entire living cells has attracted
considerable attention.
Two approaches for the functionalization of NPs have been involved: noncova-
lent interaction, including physical adsorption and entrapment of biomolecules
around the NPs, and covalent interaction to the functional groups on the NPs surface
[710]. The noncovalent approach via electrostatic interaction, pp stacking, or van
der Waals force, is an efficient immobilization method of biomolecules, which can
avoid destruction of the conjugated skeleton and loss of electronic properties of the
NPs. The covalent functionalizations of NPs are subdivided into three general strat-
egies: direct chemical reaction, linker strategies, and click chemistry. Covalent
binding in general should be preferable to unspecific physisorption in terms of the
stability and reproducibility of the surface functionalization.

H. Ju et al., NanoBiosensing: Principles, Development and Application, 1


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0_1, Springer Science+Business Media, LLC 2011
2 1 Biofunctionalization of Nanomaterials

Functional nanomaterials offer manifold perspectives for the increasing


iniaturization, novel properties and functions, and complexity of technical devel-
m
opments. The conjugation of NPs with biomolecules could provide excellent signal
transduction of biological phenomena in the development of electronic or optical
biosensors [1114]. On the one hand, optical detection is advantageous in biosensor
design because of its high sensitivity, wide dynamic range, and multiplexing capa-
bilities. In comparison with organic dyes and fluorescent proteins, NP probes such
as fluorescence energy-transfer nanobeads and quantum dots (QDs) provide signifi-
cant advantages in signal brightness, photostability, and emission of multicolored
light. On the other hand, electrochemical assays based on molecular nanoprobes are
attractive because of their low cost, high sensitivity, and simplicity. An electro-
chemical DNA assay exhibits a sensitivity of approximately 100aM by detecting
the amplified electrochemical signal from a microbead embedded with electroactive
molecules [15]. Moreover, NPs are in direct contact with the environment, which
permits them to act as chemical and biological sensors in the single-molecule detec-
tion of biomolecules for an electrochemical assay.
This chapter focuses on the fundamental strategies for the biofunctionalization
of nanomaterials with biomolecules. Moreover, we highlight some elegant applica-
tions of biofunctionalized nanomaterials as excellent electronic or optical signal
transducers in bioanalysis.

1.2Biofunctionalization Method of Nanomaterials

On the basis of the tremendous success in supramolecular chemistry, NPs function-


alized with various molecular and biomolecular units have been assembled into
complex hybrid systems through a variety of techniques, including physical adsorp-
tion, electrostatic binding, specific recognition, and covalent coupling.

1.2.1Biofunctionalization by Noncovalent Assembly

1.2.1.1Physical Adsorption

The simple adsorption of biomolecules on NPs has frequently been performed for
the biofunctionalization of NPs with biomolecules, which range from small organic
substances to large protein/enzyme molecules [2]. In the case of NPs that are stabi-
lized by anionic ligands, such as carboxylic acid derivatives, citrate, tartrate, and
lipoic acid, the functional NPs allow effective binding to the positively charged
amino acid side chains of the protein by the negatively charged anionic groups on
their surface (Fig.1.1). For example, gold (Au) NPs produced by citrate reduction
can be functionalized with carcinoembryonic antibody molecules at pH values that
lie slightly above the isoelectric point of the citrate ligand [16]. Another example of
1.2 Biofunctionalization Method of Nanomaterials 3

Fig.1.1 Biofunctionalization method of nanomaterials by noncovalent assembly. Reprinted with


permission from Veiseh etal. [2]. 2010, Elsevier

protein coating through electrostatic interactions includes the direct adsorption of


oxidases at thioglycolic acid (TGA)-capped CdSe QD particles [17].
The electrostatically driven adsorption of negatively charged DNA on positively
charged Cd2+-rich CdS NPs has been used for the preparation of inorganic pro-
teins [18]. Single-stranded DNA (ssDNA) can wrap around single-walled carbon
nanotubes (SWNTs) through an aromatic interaction to form a soluble DNA-SWNT
complex, which has been used to construct a highly sensitive sensor for the target
protein and opens up an avenue to carbon nanotube (CNT)-based applications in
biotechnology [19].
For the carbon-based nanomaterials, aromatic molecules, such as pyrene,
porphyrin, and their derivatives, can interact with the sidewalls of carbon nanoma-
terials by means of pp stacking interactions. A general and attractive approach has
been designed for the noncovalent functionalization of SWNT sidewalls and the
subsequent immobilization of biological molecules onto SWNTs via N-succinimidyl-
1-pyrenebutanoate [20]. The direct pp interaction between porphyrins and SWNTs
plays an important role in achieving an ordered assembly of protonated porphyrin
in the form of J- and H-type aggregates on the SWNTs surface [21].
Another method for immobilizing biomolecules on NPs is to entrap them in a
biocompatible polymer such as poly(ethylene glycol), Nafion, chitosan, and
copolymer. The coating polymers not only prevent the aggregation of NPs but
also provide abundant positions for functionalization with second biomole-
cules. Inparticular, an electroactive polymer, which can generate a large number
4 1 Biofunctionalization of Nanomaterials

Fig.1.2 Schematic structure of PDDA-MWNTs/GOx films on gold electrodes. Reprinted with


permission from Zhao and Ju [24]. 2006, Elsevier

of electrons during electrochemical oxidation, can serve to amplify the electro


chemicalsignal and therefore enhance the detection sensitivity. For example, poly-
tyrosine has been used as an electroactive label for the detection of prostate-specific
antigen (PSA) with the limit of detection of about 1nM [22]. A redox polymer,
poly(vinylimidazole), complexed with Os(4,4-dimethylbpy)2Cl, has been elec-
trodeposited on Ta-supported multiwalled carbon nanotubes (MWNTs) and
enhanced the sensing sensitivity toward the glucose [23].
The electrostatic deposition of biomolecules, particularly proteins or enzymes,
can also be extended to multilayer-level assemblies. This strategy permits the prepa-
ration of functional films on NPs with a high density of enzyme molecules. For
example, a uniform and stable multilayer membrane of MWNTs and glucose oxidase
(GOx) can be prepared through the layer-by-layer (LBL) assembly technique after
modification of the MWNTs with polyelectrolytes poly(dimethyldiallylammonium
chloride) (PDDA) (Fig. 1.2). This membrane shows a porous structure, and the
assembled MWNTs show an electrocatalytic activity to the reduction of dissolved
oxygen [24]. The electrostatic LBL self-assembly onto CNT carriers maximizes the
ratio of enzyme tags per binding event to offer the greatest amplification factor in
connection with alkaline phosphatase tracers. Such amplified bioelectronic assays
allow the detection of DNA and proteins down to 80 copies (5.4aM) and 2,000 pro-
tein molecules (67aM), respectively [15]. The CdSe QDCNT conjugate has been
fabricated by electrostatic adsorption of the QDs on CNTs for preparation of the
electrochemiluminescence (ECL) immunosensor [25]. A simple electrostatic method
for polyelectrolytes coating submicrometer-size latex spheres has been assembled
with Au NPs for the ultrasensitive detection of DNA [26].

1.2.1.2Specific Affinity Interactions

Affinity interaction is very effective for the bioconjugation of targeting ligands to NPs
due to specific and strong complementary recognition interactions such as
antigenantibody, nucleic acidDNA, lectinglycan, streptavidinbiotin, aptamer
protein, aptamersmall biomolecule, and hormonereceptor interactions. Moreover,
1.2 Biofunctionalization Method of Nanomaterials 5

various biomolecules contain several binding sites; for example, antibodies exhibit two
Fab (antigen-binding fragment) sites, whereas streptavidin or concanavalin A each dis-
plays four binding domains. This allows the multidirectional growth of NP structures.
As shown in Fig.1.1, the surfaces of NPs can be modified with streptavidin, which
specifically binds biotinylated molecules. The linkage formed is highly stable and the
strongest of all noncovalent linkages. Unlike hydrophobic and electrostatic interac-
tions, affinity binding is not sensitive to environmental conditions such as changes in
pH, salinity, or hydrophilicity. For example, avidin-functionalized Ce/Tb-doped LaPO4
NPs have been used for the superassembly of biotinylated proteins and oligonucle-
otides [27]. Since biotinylated molecules are easily obtained, the applicability of these
bioconjugates in biosensing and biolabeling has proved to be useful.
Aptamers, the short, single-stranded nucleic acid sequences originated from
in vitro selection (SELEX), have attracted much interest as new recognition ele-
ments of proteins and other small biomolecules. The competitive advantages of
aptamers over antibodies, such as high specificity and affinity, chemical stability,
ready availability, and high flexibility, make them promising tools for protein detec-
tion. Many explorations have been achieved in the development of aptamer-based
technologies for highly sensitive protein detection by combining them with poly-
merase chain reaction (PCR), electrochemistry, capillary electrophoresis, mass
spectrometry, and quartz crystal microbalance. The aptamer-functionalized NPs
will be a promising platform for constructing new biosensing and bioanalytical sys-
tems by the specific recognition of the aptamer toward target biomolecules.
Recently, based on specific recognition between lectins and sugar epitopes, a
concanavalin Afunctionalized Au NP as nanoprobe was developed for the highly
sensitive and selective in situ evaluation of carbohydrates on living cells by electro-
chemical tracing of the enzyme coimmobilized on the NPs [28]. Coupled with the
efficient capture of cells on electrodes modified with arginine-glycine-aspartic acid-
serine tetrapeptidefunctionalized SWNTs by the specific affinity interaction
between the tetrapeptides and the integrins on the cell surface, the dynamic changes
in four kinds of cell surface carbohydrates during drug treatment can be monitored
by four horseradish peroxidase (HRP)-labeled lectins [29].

1.2.2Covalent Route for the Biofunctionalization


of Nanomaterials

1.2.2.1Direct Chemical Reaction

Controlled chemisorption via covalent binding in general should be preferable to


unspecific physisorption in terms of the stability and reproducibility of the surface
functionalization. In direct-reaction strategies, functional groups at the NP surfaces
can be directly bonded to reactive ligands by a linkage reaction facilitated with the
aid of catalysts. NP surfaces functionalized with sulfhydryl, amine aldehyde, and
carboxylic functional groups can be targeted.
6 1 Biofunctionalization of Nanomaterials

Fig. 1.3 Preparation of Ab2-MWNT-HRP bioconjugates via amide bond formation. Reprinted
with permission from Yu etal. [34]. 2006, American Chemical Society

First, the primary binding of thiolated molecules, such as oligopeptides, to Au


NPs can provide a means for the covalent tethering of biomolecules to NPs [30].
Elsewhere, CdTe NPs capped with dimercaptosuccinic acid through the strong
binding of its thiol groups to Cd2+ ionic sites on the surface of the NPs can be func-
tionalized with biomolecules by direct chemical reaction [31]. In some cases, the
tight chemisorption of proteins on Au NPs can originate from the binding of thiol
groups from cysteine residues that exist in the proteins [32] to the Au NPs surface.
When no thiolated residue is available in the native proteins, thiol groups can be
incorporated with 2-iminothiolane by chemical means [3].
Second, NPs decorated with carboxylic acid groups can be covalently bonded to
biomolecules bearing primary amines through 1-ethyl-3-(3-dimethylaminopropyl) car-
bodiimide (EDC)/N-hydroxysuccinimide (NHS) linkers [33]. This approach has been
used in the attachment of DNA, aptamers, and antibodyantigen to NPs. Typically,
MWNTs can be shortened by sonication in 3:1 H2SO4/HNO3 for 46h to introduce
hydrophilic carboxylate groups for functionalization (Fig. 1.3). After the activation
with 1mL of 400mM EDC and 100mM NHS in pH 6.0, the protein molecules such
as secondary anti-PSA antibody (Ab2) can be attached on the surface of MWNTs [34].
Similarly, amino-decorated NPs can conjugate with biomolecules bearing carboxy-
lated groups for the attachment of peptides, proteins, antibodies, and enzymes to NPs.
Third, the main compounds used for modifying metal oxide NPs are silanes, car-
boxylates, and phosphonates. Silanes are the most frequently used modifiers for metal
oxide surfaces, since silanes can bear numerous functionalities, including amino,
cyano, carboxylic acid, epoxy groups, etc., for subsequent functionalization. On the
other hand, it was one useful way to biofunctionalize the surface of metal oxide NPs
with the binding of carboxylate ligands and phosphonate groups of biomolecules via
monodentate coordination, bridging chemisorption, or chelating chemisorption [8].

1.2.2.2Linker Chemical Reaction

Biomolecules, especially for protein, in direct contact with an unprotected solid


substrate are subject to denaturing of varying severity and the ensuing loss of their
1.2 Biofunctionalization Method of Nanomaterials 7

Fig. 1.4 Bioconjugation strategies for chemical modification on a nanoparticle (NP) surface.
Reprinted with permission from Liu etal. [6]. 2010, Hindawi Publishing Corporation

specific biochemical functionality. Low-molecular bifunctional linkers, which have


anchor groups for their attachment to NP surfaces and functional groups for their
further covalent coupling to the target biomolecules, have been extensively used in
the generation of covalent-tethered conjugates of biomolecules with various NPs.
Anchor groups such as thiols, disulfides, or phosphine ligands are often used for the
binding of the bifunctional linkers to Au, Ag, CdS, and CdSe NPs. These anchor
groups may readily substitute weakly adsorbed molecules to stabilize the NPs or
may be incorporated in the synthesis of the NP to yield a NP surface that is function-
alized for further reactions.
A wide variety of terminal functional groups are available in different bifunc-
tional linkers. The most common amine, active ester, isothiocyanate, and maleimide
groups are used to couple biological compounds covalently by means of carbodiim-
ide-mediated esterification and amidation reactions or through reactions with thiol
groups (Fig.1.4) [6]. The main role of the linker molecules is not only to provide a
high density of docking sites for the specific attachment of biomolecules but also to
maintain a sufficiently low density of electronic defects at the NPs surface.
Using phosphorothioate-modified DNA coupled with a short bifunctional
fastener (BF), a novel method to assemble NPs along DNA strands has been devel-
oped to precisely control the position and distance between NPs, since the BF has
an alkane thiol group at one end that can bind to an Au NP and an iodoacetamide
group at the other end that can bind to a phosphorothioate group on a modified DNA
backbone [35].
8 1 Biofunctionalization of Nanomaterials

Fig.1.5 Schematic illustration of modification of alkyne-functionalized NPs with azide-functionalized


biomolecules via click chemistry. Reprinted with permission from Krovi etal. [38]. 2010, Royal
Society of Chemistry

1.2.2.3Biofunctionalization by Click Chemistry

Click chemistry, a Cu-catalyzed azidealkyne cycloaddition, is a relatively new


approach of direct conjugation, developed by Sharpless etal. almost a decade ago
[36]. Click reactions are fast and efficient, require mild reaction conditions (aqueous
environment, relatively neutral pH), and create water-soluble and biocompatible
linkages. Compared to other direct-conjugation strategies, this method of attachment
offers several unique features. First, azide and alkyne reactive groups are highly
specific for one another, and unreactive with most functional groups, ensuring
specific conjugation at the desired location on the reactive moiety. Second, the
formed bonds are highly stable. This is in contrast to amide bonds, which can be
cleaved by hydrolysis reactions, and disulfide linkages, which are susceptible to
cleavage under reducing environments. Third, the formed linkages are extremely
rigid, which helps to maintain conformation of the reacted moieties at the NP surface
and prevents their cross-interactions.
Click chemistry methodologies are currently being used in polymer and mate-
rials science for easy and almost quantitative functionalization [37]. The NP surfaces
can be decorated with either alkyne or azide functionalities for conjugation to the
complementarily functionalized bioactive molecules. Via a one-step click reaction,
the drug-loaded polymer NPs can be functionalized with folate, biotin, and Au NPs
(Fig. 1.5) [38]. A gradient of a glycinearginineglycineaspartateserine linear
peptide has been fabricated on a versatile substrate by using click chemistry tech-
nology as a tool for screening surface-directed cell function [39].
A general approach for the functionalization of low-fouling, nanoengineered
polymer capsules with antibodies has been presented using click chemistry.
Antibody-functionalized capsules specifically bind to colorectal cancer cells even
when the target cells constitute less than 0.1% of the total cell population [40]. The
efficient grafting of oligo(ethylene glycol) chains on nonoxidized silicon via a click
reaction also provides a highly resistant surface to the nonspecific adsorption of
proteins [41].
1.2 Biofunctionalization Method of Nanomaterials 9

1.2.2.4Activation Method for the Biofunctionalization of Nanomaterials

When the NP surfaces are primarily functionalized with some groups, such as the
sulfhydryl, amine, carboxylic, and hydroxyl groups, the further biofunctionalization
of these NPs sometimes needs a step to activate these groups, which enables the
groups to react with ligands of choice. The method selected for the activation of a
functional NP surface must be compatible with both the ligand and the NPs. The
general methods can be summarized as follows.
The amine reactive chemistries are the first methods introduced to activate the NP
surfaces. Here cyanogen bromide (CNBr) can be used for the activation of hydroxyl
groups. It can react with the OH group at a high pH to produce a very reactive
cyanate ester, which then reacts directly with amine groups. This method can be used
almost universally to activate NPs containing hydroxyl groups. CNBr-activated NPs
can be used to couple to small ligands as well as to high-molecular-weight biopolymers
containing primary amine groups. The procedure is relatively simple to carry out and
is very reproducible for coupling sensitive biomolecules such as enzymes and anti-
bodies to NPs. Another method of amine reactive chemistries is the use of N-hydroxy
succinimide (NHS) esters as active groups to form amide bonds with primary amines.
NHS can activate both OH and COOH groups. The produced NHS ester will react
quite efficiently with primary amine-containing ligands to form a stable amide bond.
For the activation of a COOH group, N,N-carbonyl diimidazole (CDI) and EDC are
highly reactive carbonylating reagents. In the presence of a primary amine-containing
compound, the CDI-introduced imidazole is displaced to form a stable amide bond.
Sometimes, CDI can also be used to activate hydroxyl-containing NPs. The EDC-
mediated linkages can be done in two ways: Amine-containing NPs can be used to
couple a carboxyl-containing ligand, or NPs with the carboxyl group available can be
used to couple an amine-containing ligand.
Aldehydes and ketones can react with the primary and secondary amino groups to
form reversible Schiff bases, which can then be reduced and stabilized as covalent
linkages by using a reducing agent such as sodium borohydride. Thus, the primary or
secondary amino group-containing biomolecules can be introduced to NPs by these
reactions. The key step is the generation of aldehyde groups on the NP surface, which
can be created by the mild oxidation of adjacent diols of agarose with NaIO4. Thus,
the first step is to functionalize the NPs with agarose-based gels or glycidol. The
latter can react with OH to introduce adjacent hydroxyls to the NP surface.
Hydrazide reactive chemistry permits the coupling of aldehyde- or ketone-
containing ligands through the formation of stable hydrazone linkages. Thus, glyco-
proteins in particular may be introduced to the NP surface using this procedure after
being oxidized with NaIO4 to produce formyl groups on their carbohydrate chains.
This method is powerful for functionalizing hydrazide-activated NPs with proteins
while leaving critical active sites free.
Sulfhydryl reactive chemistry is another way to introduce biofunctional molecules
to the NP surface. It can be carried out by the iodoacetyl and bromoacetyl activation
method. The iodoacetyl- and bromoacetyl-activated NPs containing the NH2 group
can be coupled to sulfhydryl groupcontaining ligands by extremely stable thioether
10 1 Biofunctionalization of Nanomaterials

bonds. Maleimide is another efficient group for binding sulfhydryl-containing


proteins to amino-containing NPs. One of the most popular cross-linkers is succini
midyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), which has an
amine reactive NHS ester on one end and a maleimide group on the other. The
excellent stability and slow hydrolysis rate of the SMCC maleimide end allow for
the primary modification of one protein or NP through amine groups, and a secondary
couple to the sulfhydryl group of another protein.
The NP surface can also be activated by hydroxyl and active hydrogen reactive
chemistries. The previous methods contain epoxy (bisoxirane) activation,
divinylsulfone activation, and cyanuric chloride activation, and the latter contains
diazonium chemistries and Mannich condensation. Diazonium chemistries have
long been used in organic synthesis for conjugations involving active aromatic
hydrogens or other compounds susceptible to electrophilic attack. However, some
problems, such as an extremely rapid reaction rate, lead to poor reproducibility of
the functionalization of NPs. Mannich condensation can be an optional method
forthe functionalization of NPs with biomolecules containing no easy reaction or
low-reactivity functional groups, but having certain sufficiently active hydrogens
that can be condensed with formaldehyde and an amine in the Mannich reaction.
Particular hydrogens in phenols, esters, ketones, acetylenes, and a host of other
compounds can be aminoalkylated using this reaction.
Photoreactive cross-linkers can also be considered as an efficient way for the bio-
functionalization of NPs, though this process has not been reported yet. The aryl
azide group is an important photoreactive cross-linker that was introduced in 1969 by
Fleet etal. It uses a bright light source in the range of 265275nm to activate a pho-
tolysis conversion for the formation of an extremely reactive nitrene. The generated
nitrene reacts with covalent bonds to nonspecifically insert in the structure of a target
molecule, leading to yields of the desired reaction product. Thus, this reaction can be
used to couple two target molecules. Other photoreactive cross-linking reagents con-
tain hydrazide derivatives, NHS esters, pyridyl disulfides, and formyl derivatives.

1.3Biofunctional Nanomaterials

1.3.1Carbon-Based Nanomaterials

Carbon-based nanomaterials (CNTs, carbon nanohorns, graphene sheets, etc.) pres-


ently attract much attention as prospective technological materials. For biological
applications, the lack of solubility of CNTs in aqueous or organic solvents is a
major technical barrier. Great efforts have been devoted to the search for cost-
effective approaches to functionalize CNTs for the attachment of biomolecules as
recognition elements [9]. Generally, this procedure can be performed by two func-
tionalization routes, noncovalent interaction and covalent binding [42].
The main advantage of the noncovalent functionalization of CNTs is the conser-
vation of their electronic structure by preventing disruption of the intrinsic nanotube
1.3 Biofunctional Nanomaterials 11

Fig.1.6 1-Pyrenebutanoic acid, succinimidyl ester irreversibly adsorbing onto the sidewall of an
SWNT via pp stacking. Reprinted with permission from Chen et al. [20]. 2001, American
Chemical Society

sp2 structure and conjugation. Most notably, small aromatic molecules and conjugated
polymers have been used to decorate the nanotube surface via electrostatic interac-
tion, pp stacking, or van der Waals force, and to improve the solubility and elec-
tronic properties of CNTs. The first demonstration of SWNT functionalization with
proteins via pp stacking was reported by Dais group at Stanford University [20]. A
bifunctional molecule, 1-pyrenebutanoic acid, succinimidyl ester, was first irrevers-
ibly adsorbed onto an SWNT via pp stacking between the pyrenyl group and the
sidewall of the SWNT (Fig.1.6). Proteins were then immobilized through a nucleo-
philic substitution of NHS by the amino group of the proteins to form an amide bond.
Alternatively, a direct assembly of water-insoluble porphyrin on SWNT was designed
based on the pp noncovalent interaction between porphyrin and CNTs, resulting in
a novel biosensor for trichloroacetic acid [43].
12 1 Biofunctionalization of Nanomaterials

Fig.1.7 Schematic illustration of the dehydration and carbonization process of fructose and the
following enzyme-antibody-functionalized CNSs. Reprinted with permission from Du etal. [46].
2010, American Chemical Society

The covalent attachment of bioactive molecules on CNTs is considered to be a


useful tool for biological applications. Typically, CNTs are first treated by oxidation
in strong acid, which introduces a number of carboxyl groups on the CNTs surface.
Then, using EDC as a cross-linker, biomolecules can be covalently conjugated onto
carboxylated CNTs via an amide bond [44, 45]. To generate carboxylic groups on
the surface of carbon nanospheres (CNSs) (Fig.1.7), CNSs can be treated with a
mixture of concentrated H2SO4 and HNO3. The functionalized CNSs can then be
grafted with HRP-Ab2 by mixing with EDC and NHS overnight at room tempera-
ture. The developed immunosensor shows a seven-fold increase in detection signal
compared to the immunosensor without modification and CNS labeling [46].
The covalent functionalization of radionuclide-filled SWNTs is also used as a
radioprobe for invivo radioemitter localization and imaging [47]. The regiospecific
covalent functionalization of SWNTs with oligonucleotides can provide new hybrid
materials with sensing and self-assembly properties for the precise positioning of
biomolecules and NPs for new biodiagnostic sensory devices [48].
Graphene as a single-layered two-dimensional sheet has attracted enthusiastic
interest in many areas of nanoscience and nanotechnology. Since oxidized graphene
sheets in aqueous dispersion are negatively charged, it is expected that the cationic
polyelectrolyte and porphyrin derivatives can be assembled onto their surfaces
through electrostatic and pp stacking interactions, respectively [49]. Based on the
supramolecular assembly of free-base TMPyP on reduced graphene, graphene-
porphyrin hybrid as an optical probe has been constructed for the rapid and selective
sensing of Cd2+ ions in aqueous media [50].

1.3.2Metal Nanoparticles

Metal NPs involving Au, Ag, Pd, and Pt NPs can provide three-dimensional architec-
tures, which have attracted widespread interest since their nanosized physical
1.3 Biofunctional Nanomaterials 13

Fig.1.8 Schematic illustration of the different stability of Au NPs with folded and unfolded ade-
nosine binding DNA aptamer. Reprinted with permission from Zhao etal. [32]. 2008, American
Chemical Society

p roperties are quite different from those of the bulk materials. Metal NPs have mainly
been modified with thiols, disulfides, amines, nitriles, carboxylic acids, and phos-
phines [8]. First, multipoint metalS bond formation is a straight-forward way to fix
functional molecules in the desired geometry. Second, some of the thiolate ligands in
alkanethiolate-stabilized Au NPs can be substituted by reaction with other thiols at
rates depending on the chain length and steric bulk of the leaving thiolate and incom-
ing thiols and on the charge of the Au NPs. Thiolthiol exchange mostly requires a
considerable excess of incoming ligand to render the exchange complete, but a delib-
erate partial exchange may also be useful. Third, carboxylic acidamine coupling
can be used to link any protein bearing a primary amine to Au NPs [7].
Conjugates of Au NPDNA are of great current interest because of the potential
use of the programmability of DNA base pairing to organize nanocrystals (NCs) in
space and the multiple ways of providing a signature for the detection of precise
DNA sequences. Adenosine aptamers have been chemically coupled onto Au NPs
using AuS chemistry. Before the addition of adenosine, the ssDNA aptamer on Au
NPs adopts a loose random coil structure, since there is no strong intramolecular
base pairing. By contrast, the aptamer folds into a well-characterized tertiary struc-
ture in the presence of adenosine (1 mM) in a buffer containing 4 mM MgCl2,
100mM NaCl, and 20mM Tris-HCl (Fig.1.8). On the basis of this unique phenom-
enon, colorimetric biosensors have been developed for the detection of adenosine,
K+, adenosine deaminase, and its inhibitors [32]. Further, coupled with PCR and
rolling-circle amplification, the three-dimensional positioning of Au NPs has been
realized for the development of negative-index materials [51] and unique scaffolds
in nanotechnology and biodiagnostics [52].
Metal NPs are often combined with other materials, such as solgel matrices,
polymers, and other nanomaterials, which can provide a network structure or a basal
matrix that immobilizes metal NPs onto the electrode surface. For example, a novel
Au NPbacteria cellulose nanofiber has been synthesized using bacterial cellulose
nanofibers as robust biotemplates via a one-step method for the detection of H2O2
14 1 Biofunctionalization of Nanomaterials

Fig.1.9 Modification of semiconductor QDs with functional encapsulating layers for water solu-
bilization and preservation of luminescence properties and/or secondary covalent modification of
the surface with biomolecules. Reprinted with permission from Gill etal. [5]. 2008, Wiley

with a detection limit lower than 1mM [53]. Pd NPs have also been electrodeposited
on SWNTs from a PdCl2 solution, leading to 1,000-fold increases in resistance to H2
gas [54].

1.3.3Semiconductor Nanoparticles

Methods to stabilize the fluorescence properties of semiconductor QDs in aqueous


media are very important for bioanalysis. Several methods have been used for the
attachment of biomolecules to QDs (Fig.1.9). The most common methods are to
prepare QD bioconjugates via covalent bond formation between carboxylic acids
and biomolecules. In addition, biomolecules containing basic functional groups,
1.3 Biofunctional Nanomaterials 15

such as amines or thiols, may interact directly with the QDs surface as ligands.
When biomolecules do not contain the groups for direct QD binding, they may be
modified to introduce this functionality. For example, nucleic acids and peptides
have been modified to introduce thiol groups for binding to QDs. Since the QDs are
negatively charged in neutral or basic buffers, the positively charged molecules can
be used for the electrostatic functionalization of QDs, especially for macromole-
cules such as enzymes and antigenantibody. Another method for the surface modi-
fication of semiconductor NPs has also become modular through high-affinity
streptavidinbiotin binding. QDstreptavidin conjugates are convenient for indirect
binding to a broad range of biotinylated biomolecules. The coating of the QDs with
protective silicon oxide films or polymer films has also been an alternative method
to keep the biocompatibility of QDs [5].
In addition, amine-modified QD705 (emission maximum at 705nm) has been
conjugated to a heterobifunctional cross-linker, 4-maleimidobutyric acid-NHS ester,
yielding a maleimide-NC surface for integrin-targeted near-infrared optical imaging,
which will be useful in cancer detection [55].
The main compounds used for modifying metal oxide NPs are phosphonates,
carboxylate, and silanes. Carboxylate ligands, especially fatty acids, are often used
for the functionalization of metal oxide NPs. The binding of carboxylate ligands can
be formed on the surface of titania NPs via physical adsorption, monodentate coor-
dination, bridging chemisorption, and chelating chemisorption [8]. A new proce-
dure based on photodeposition of nano-Ag on a TiO2-coated piezoelectric quartz
crystal (PQC) electrode has been developed to fabricate a highly sensitive PQC/
DNA biosensor [56].

1.3.4Magnetic Nanoparticles

The surface functionalization and modification of magnetic NPs (microspheres,


nanospheres, and ferrofluids) are widely studied and applied in various fields of
biology and medicine [57]. A major challenge is still protection against corrosion,
and therefore suitable protection strategies will be emphasized here. Currently, there
are two strategies to fabricate magnetic NP-based multifunctional nanostructures.
The first one is molecular functionalization, which involves attaching antibodies,
proteins, and dyes to the magnetic NPs. The second method integrates the magnetic
NPs with other functional nanocomponents, such as QDs or metallic NPs. Because
they can exhibit several features synergistically and deliver more than one function
simultaneously, such multifunctional magnetic NPs could have unique advantages
in biomedical applications [58].
Figure1.10 shows the schematic illustration of the typical fabrication process of
functionalized magnetic particles. The Prussian blue (PB)Fe3O4 NPs are prepared
using the Fe3O4 NP-suspended solution as the seeds under stirring. Next,
0.10molL1 K3[Fe(CN)6] containing 10mmolL1 HCl is added and stirred to form
a mixed solution, and then a further 10mL of 0.10molL1 FeCl3 solution is added
16 1 Biofunctionalization of Nanomaterials

Fig.1.10 Schematic illustration of the functionalized magnetic particle fabrication process. (a)
Fe3O4 NPs; (b) PBFe3O4 NPs; (c) the Au NP seeds coating PBFe3O4 NPs; and (d) AuPBFe3O4
NPs. Reprinted with permission from Zhuo etal. [59]. 2009, Elsevier

drop by drop with the help of a slight excess of H2O2. Subsequently, the surface of
PBFe3O4 NPs is chemically modified with bovine serum albumin (BSA) to obtain
the amido and disulfide groupmodified PBFe3O4 NP. Finally, the PBFe3O4 par-
ticles covalently attract 13-nm Au NP seeds, which act in a next step as nucleation
sites for the formation of a continuous gold outer layer by citrate reduction of Au3+
to Au0. The multilabeled AuPBFe3O4 NPs exhibit satisfying redox electrochemi-
cal activity and high enzymatic activity for an ultrasensitive and reproducible elec-
trochemical immunosensor [59]. Based on magnetic beads (MBs) and HRP-labeled
anti-AFP antibody-modified Au NPs, a novel and sensitive chemiluminescence
(CL) immunoassay has been developed by employing a new CL enhancer, bro-
mophenol blue, for the determination of AFP, a tumor marker, with a linear range
from 0.1 to 5.0ngmL1 and a detection limit of 0.01ngmL1 [60]. With a multiple-
enzyme-labeled antibody-MB bioconjugate (7,500 HRP on 1mm MBs), an ultra-
sensitive electrochemical immunosensor has been constructed to detect cancer
biomarkers in serum [61].
The nucleic acid- or antigen-functionalized magnetic particles, together with the
naphthoquinone 4-modified magnetic particles, can yield enhanced electrogenerated
CL in the presence of HRP-bioconjugates and luminol. Using higher rotation speeds,
the sensitivity of the antibody detection can be further improved, with a detection
limit of 50100pgmL1 at 2,000rpm [62]. A signal amplification strategy based on
bio-barcodefunctionalized magnetic NPs as labels holds promise to improve the
sensitivity and detection limit of the detection of DNA hybridization and single-
nucleotide polymorphisms by flow-injection CL assays [63]. A sandwich-type
detection strategy is employed as shown in Fig.1.11. Biotinylated capture probes
are loaded on avidin-modified magnetic microparticles (MMPs), while thiolated
detection probes are assembled on Au NPs via AuS bonds. In the presence of
targetDNA, the capture probe recognizes the target DNA, along with the detection
probe, to the proximity of MMPs, and this complex is then magnetically separated
1.3 Biofunctional Nanomaterials 17

Fig. 1.11 The multicomponent nanoprobe-based sandwich-type DNA detection strategy.


Reprinted with permission from Li etal. [64]. 2008, Wiley

for subsequent optical detection. This novel method can conveniently detect as few
as 100pM target DNA with the naked eye, and this sensitivity can be significantly
improved by instrument-based assays [64].

1.3.5Other Biofunctional Nanomaterials

Among these inorganic-based materials, mesoporous silica nanoparticles (MSNs)


have attracted much research attention for their potential application in the fields of
biotechnology and nanomedicine due to superior biocompatibility and two func-
tional surfaces (exterior particle and interior pore faces). Mesoporous silica shows a
high density of silanol groups, which can be used to obtain functionalized surfaces
by grafting organic silanes [6567].
Using the conventional coupling reagent EDC and NHS, a novel electrochemical
immunosensor has been developed for the detection of tumor necrosis factor-alpha
(TNF-a) based on a poly-(guanine)-functionalized silica NP label. The detection
limit for TNF-a is found to be 5.01011gmL1 (2.0pM), which corresponds to
60aM of TNF-a in 30mL of sample [68]. Hepatitis B virus (HBV) DNA has been
detected using a silica NP-enhanced dynamic microcantilever biosensor via a two-
step process. First, 3-amino-propyltrimethoxysilane is added to the ethanol solution
of silica NPs to generate an NH2-modified surface. It is followed by a further activa-
tion with 2,4,6-trichloro-1,3,5-triazine to generate a triazine-functionalized surface.
The triazine-functionalized NPs are subsequently conjugated with amine-modified
DNA [69] to produce a simple method, which can provide label-free sequence-
specific DNA detection with single-nucleotide polymorphism detection selectivity
via fluorescence resonance energy transfer (FRET) on silica NPs [70].
18 1 Biofunctionalization of Nanomaterials

The ECL of doped silica NPs [71], prepared by a reverse-microemulsion


method that leads to covalent incorporation of Ru(bpy)32+, has been investigated
in acetonitrile and aqueous buffers. Using tripropylamine as coreactant, it is worth
noting that the functionalized NPs provide a more than 1,000-fold increase of the
ECL signal compared to that of a single dye [72]. To construct the immunoassay,
thionine-horseradish peroxidase conjugation (TH-HRP) has initially been doped
into nanosilica particles using the reverse-micelle method, and then the HRP-
labeled anti-CA125 antibodies (HRP-anti-CA125) are bound onto the surface of
the synthesized NPs as recognition elements. Under optimal conditions, the elec-
trochemical immunoassay exhibits a wide working range from 0.1 to 450UmL1.
with a detection limit of 0.1UmL1 CA125 [73]. A simple and sensitive method
for in situ amplified electrochemical immunoassay of human serum IgG has been
developed by using double-codified nanosilica particles as labels based on HRP-
doped nanosilica particles with the conjugation of anti-IgG antibodies [74].
Coupled with SWNT forests, an immunosensor array has been constructed via
water-in-oil (W/O) microemulsions for PSA detection, with a detection limit of
40pgmL1 [75].
The conjugated polymer-amplified silica NP-based immunoassay has been
also constructed for IgG detection. After immobilization of the prime antibody on
the NP surface, the NPs are used to capture antigen and Cy3-labeled secondary
antibody in a sandwich assay format. The presence of target antigen in solution
brings the fluorescent Cy3 molecules to the NP surface. The addition of a cationic
conjugated polymer further amplifies the fluorescence signal of the dye, which
improves the assay sensitivity and selectivity. Within the tested concentration
range, a linear response for IgG detection is observed from 0 to 1mgmL1, with a
detection limit of ~1.1ngmL1 [76]. In the same group, an assay with aptamer-
functionalized silica NPs was used as the sensory platform for thrombin detection
in blood serum [77].
The polymer matrix is biocompatible with most biological species [7880]. A
conducting polymer-based interface can prevent the protein conformation change
and alleviate this problem. Therefore, many polymer NPs have been employed as
amplification support in bioanalysis. For example, an ultrasensitive electrochemical
nucleic acid biosensor has been fabricated using the conducting polyaniline (PANI)
nanotube array as the signal enhancement element. Compared with Au NP or
CNTbased DNA biosensors, the PANI nanotube array-based DNA biosensor can
achieve similar sensitivity without catalytic enhancement, purification, or end-
opening processing, thus allowing the target oligonucleotide to be readily detected
at a concentration as low as 1.0fM [81].
A conducting NP monolayer film has been formed by polymeric dendrimer, as
shown in Fig.1.12. Due to specific WatsonCrick base pairing, Au NPs, functional-
ized with oligonucleotides of complementary base sequences, are immobilized on
substrate surfaces to improve the electrical transport properties [82]. A DNA den-
drimer is introduced to further improve the sensor performance. The limit of detec-
tion of 100200 fg mL1 for protein has been achieved, which is three orders of
magnitude better than that without the DNA dendrimer interface [83].
1.4 Characterization of Biofunctional Nanomaterials 19

Fig.1.12 Schematic of the immobilization of gold nanoparticles (Au NPs) by DNA hybridization
on silicon dioxide surfaces. Reprinted with permission from Koplin et al. [82]. 2006, Royal
Society of Chemistry

1.4Characterization of Biofunctional Nanomaterials

The morphology of biofunctional nanomaterials can usually be characterized by


atomic force microscopy (AFM), scanning electron microscopy (SEM), transmis-
sion electron microscopy (TEM), and Raman spectroscopy.
Figure1.13 shows the AFM images of carboxylated CNTs before and after bio-
conjugation with HRP and Ab2. Size analysis shows that the image before biocon-
jugation has an average height of 24.5nm, whereas the images of nanotubes with
HRP and Ab2 attached show average heights of 34.2nm. The 10-nm increase in
height for the bioconjugated nanotubes is consistent with the average thickness of a
monolayer of the major coating component HRP (4.06.711.7nm, Brookhaven
Protein Database) on the 25-nm nanotubes. Furthermore, after protein binding,
globular shapes suggestive of protein aggregates appear on the sidewalls of the nan-
otubes [34].
Figure1.14 shows the typical TEM and SEM images of the colloidal CNSs and
the Au NP/CNS hybrid material. The obtained colloidal CNSs, with an average
diameter of 250nm, are uniform in size and morphology. The numerous individual
dark nanodots spread along the gray nanospheres and the corresponding outside
particles in Fig.1.14 are Au NPs, which indicates that well-dispersed Au NPs deco-
rate the colloidal carbon surface quite uniformly. Using Au NP/CNS as a label, the
sensor provided a linear response range between 0.01 and 250 ng mL1, with a
detection limit of 5.6pgmL1 for the detection of protein [84].
The antibody-immobilized carbon nanoparticle (CNP)/poly(ethylene imine)
(PEI)-modified screen-printed graphite electrode (CNP-PEI/SPGE) has been char-
acterized through XPS analysis, which exhibits a signal peak at 288.7eV (Fig.1.15a),
indicating the presence of COOH units at the surface, in addition to signals repre-
senting CC (285.4eV) and CN (286.6eV) groups. No such signal for COOH
groups is detected on the bare CNP-PEI/SPGE (Fig.1.15b), confirming the success-
ful deposition of anti-CEA antibody molecules on the surface of the SPGE [85].
20 1 Biofunctionalization of Nanomaterials

Fig. 1.13 Tapping-mode AFM images of surface-carboxylated MWNTs isolated on smooth,


freshly cleaved mica surfaces (a) not derivatized with protein and (b) derivatized as Ab2CNT
HRP bioconjugate. Reprinted with permission from Yu etal. [34]. 2006, American Chemical
Society

Fig.1.14 (a) TEM and (c) SEM images of colloidal CNSs, and (b) TEM and (d) SEM images of
the Au NP/CNS hybrids. Reprinted with permission from Cui etal. [84]. 2008, Wiley
1.5 Applications of Biofunctional Nanomaterials 21

Fig.1.15 X-ray
photoelectron spectra
of the surfaces of the
(a) antibody-deposited
CNP-PEI/SPGE and the
(b) CNP-PEI/SPGE.
Reprinted with permission
from Ho etal. [85].
2009, American Chemical
Society

Raman spectrum has been employed to characterize a goldsilver coreshell


nanodumbbell for the assembly of Cy3-modified oligonucleotides [86].

1.5Applications of Biofunctional Nanomaterials

1.5.1Optical Sensing

Optical detection is advantageous in biosensor design because of its high sensitivity,


wide dynamic range, and multiplexing capabilities. Moreover, NP probes provide
significant advantages in signal brightness, photostability, and multicolored-light
emission. In addition, noble metal NPs, such as gold and silver NPs, have attracted
much interest in optical biosensor fabrication because of their unique sizes, compo-
sition, optical properties, and ease of functionalization [87].

1.5.1.1Colorimetric Detection of Bioanalytes

Metal NPbased homogeneous colorimetric detection of oligonucleotides holds


great promise due to the low cost, low volume, and rapid readout of a target DNA
22 1 Biofunctionalization of Nanomaterials

Fig.1.16 DNA-modified Au NPbased barcode DNA detection and quantification. Au NP aggre-


gates are spotted on a TLC plate. Reprinted with permission from Nam etal. [93]. 2007, Nature
Publishing Group

sequence [88]. A homogeneous colorimetric DNA biosensor has been developed by


a novel nicking endonuclease-assisted NP amplification process that is capable of
recognizing long single-stranded oligonucleotides with single-base mismatch selec-
tivity and a 103-fold improvement in amplification (ca. 10 pM) [89]. The use of
polyvalent oligonucleotide Au NPs provides a novel means of signal amplification,
with the limit of detection comparable to the PCR-based telomeric repeat amplifica-
tion protocol. The assay can detect telomerase activity with as few as ten HeLa
cells, with on-chip positive and negative controls [90]. Since the results can be
clearly seen with the naked eye without any complicated step such as surface modi-
fication of Au NPs or PCR amplification, this method can be easily applied to point-
of-care diagnosis.
Based on the scattering properties of silver-amplified Au NP probes, a simpler
and more sensitive detection system has been developed for DNA detection. The
sensitivity of this scanometric array detection system exceeds that of the analogous
fluorophore system by one order of magnitude. The assay can be measured and
quantified with a UV-visible spectrometer and, in some cases, visually monitored
with the unaided eye [91]. An ultrasensitive glycans array using iron oxidegold core
shell NPs conjugated with antibodies or proteins has been developed and reached
the subattomole level in carbohydrate detection [92].
The bio-barcode-based colorimetric assay is an important advance in that this
method offers both the attomolar sensitivity of the bio-barcode amplification scheme
and the simplicity, portability, and low cost of Au NPbased colorimetric DNA
detection method. As shown in Fig.1.16, barcode DNA-mediated Au NP aggre-
gates on a thin-layer chromatography (TLC) plate are spotted for quantification
using a graphic processing method [93]. Based on porous microparticles, which
enable loading a large number of barcode DNA per particle, a colorimetric
bio-barcode method minimizes the above requirements while detecting 30-aM con-
centrations of cytokines (about three orders of magnitude more sensitive than
conventional nonenzymatic cytokine detection assays) [94].
1.5 Applications of Biofunctional Nanomaterials 23

Fig.1.17 Schematic illustration showing a cascade procedure used to amplify fluorescence signal
depending on the proteolytic activity of MMP-2. Reprinted with permission from Kim and Chung
[95]. 2010, Wiley

1.5.1.2Fluorescence Detection

A new strategy for highly sensitive and rapid protease assay is developed by
mediating the proteolytic formation of oligonucleotide duplexes and using the
duplexes for signal amplification (Fig.1.17). In the presence of matrix metallopro-
tease-2 (MMP-2), fragmentation of the intact DNApeptide on Au NPs by hydro-
lytic cleavage of a peptide bond within the substrate allows diffusion of the DNA
away from the Au NPs and the formation of a DNA/RNA heteroduplex, leading to
digestion of RNA by RNase H. Because of the high quenching efficacy of Au NPs
to the fluorophore in RNA and multiple digestions of the RNA, the fluorescence
signal recovery is amplified. This method permits the assessment of the activity of
MMP-2 at concentrations as low as 10pM within 4h. Compared with the reported
protease nanosensors using QDs, Au NPs, and magnetic NPs with the same peptide
sequence, the assay time of this method is 6 times faster and the limit of detection
is 100 times more sensitive [95]. A near-infrared fluorescence imaging probe based
on Au NPs functionalized with self-assembled heterogeneous monolayers of dye-
labeled peptides and poly(ethylene glycol) has been developed to visualize prote-
olytic activity invivo [96]. And a metal-enhanced fluorescence platform has been
designed to develop a sensing surface by water-soluble polyelectrolytes with under-
lying Ag NP arrays for optically amplified DNA detection [97].
A much simpler and milder strategy to amplify fluorescence signals by using ionic
NCs with no special optical properties has been suggested. The cation-exchange reac-
tion with ionic NCs can release thousands of divalent cations, which can in turn trigger
the fluorescence from thousands of nonfluorescent metal-sensitive dyes to obtain large
fluorescence amplification. The NCdye set of CdSe and fluo-4 used in the present
study led to a 60-fold enhancement of the fluorescence signal and a limit in protein
detection 100 times lower than that of the organic fluorophore Alexa 488 [98]. Lately,
the cation exchangebased fluorescence amplification method has been designed for
24 1 Biofunctionalization of Nanomaterials

Fig. 1.18 Schematic presentation of the small RNA detection assay using CXFluoAmp. MP
magnetic particles; CP capture probe; DP detection probe. Reprinted with permission from Li
etal. [99]. 2009, American Chemical Society

Fig.1.19 Fluorescence emission from Cy5 on illumination on a QD caused by FRET between


Cy5 acceptors and a QD donor in a nanosensor assembly. Reprinted with permission from Zhang
etal. [100]. 2005, Nature Publishing Group

the detection of low abundance and short strand length of small RNA molecules
(Fig.1.18). Nonfluorescent, ionic NCs of CdSe are conjugated to detection probes and
immobilized onto the array surface via ligation with the target small RNA, miR21,
which is bound to the capture probe complementarily. Each binding event induced by
one target miR21 molecule is then amplified by the release of thousands of Cd2+ from
one NC. The free Cd2+ immediately turns on the fluorescence of thousands of fluoro-
genic Rhod-5N molecules. With such a powerful signal-amplification strategy, the
assay achieves a limit of detection of 35fM and the signals are detectible with analyte
concentrations spanning over seven orders of magnitude [99].
FRET-based probes incorporated with single-molecule fluorescence detection
technologies have allowed the detection of DNA with low abundance without unam-
plification. As shown in Fig.1.19, the QD acts as both a FRET energy donor and a
1.5 Applications of Biofunctional Nanomaterials 25

target concentrator. When a target DNA is present in solution, it is sandwiched by


the two probes. Several sandwiched hybrids are then captured by a single QD
through biotinstreptavidin binding, resulting in a local concentration of targets in
a nanoscale domain. The resulting assembly brings the fluorophore acceptors and
the QD donor into close proximity, leading to fluorescence emission from the accep-
tors by means of FRET on illumination of the donor. Unbound nanosensors produce
near-zero background fluorescence, but binding to even a small amount of target
DNA (~50 copies or less) can generate a very distinct FRET signal [100]. Based on
FRET, an ultrasensitive and reliable nanotechnology assay is set up for the detection
and quantification of DNA methylation. This approach detects as little as 15pg of
methylated DNA in the presence of a 10,000-fold excess of unmethylated alleles
and allows for multiplexed analyses [101].
Combining invivo biotinylation of engineered host-specific bacteriophage and
conjugation of the phage to streptavidin-coated QDs, a rapid and simple method has
been constructed for the detection of antibiotic-resistant bacteria. The method pro-
vides the specific detection of as few as ten bacterial cells per milliliter in experi-
mental samples, with an approximately 100-fold amplification of the signal over
background in 1h [102].
Using color-coded NPs to simultaneously recognize two binding sites on a single
target, the individual molecules of genes, proteins, and intact viruses can be detected
and identified in complex mixtures without target amplification or probe/target sep-
aration. With a data acquisition time of 80s and 2-mm flow channels, single mole-
cules can be detected from target concentrations as low as 2030fM [103]. Based
on dual-color imaging and the automated colocalization of bioconjugated NP
probes, routine two-color superresolution imaging and single-molecule detection
have been achieved at nanometer precision with standard fluorescence microscopes
and inexpensive digital color cameras [104].

1.5.1.3Other Spectroscopic Measurements

Surface-enhanced Raman scattering (SERS) is a powerful spectroscopy technique


that can provide nondestructive and ultrasensitive characterization down to the sin-
gle-molecule level, comparable to single-molecule fluorescence spectroscopy.
Colloidal NPs are of interest as SERS substrates because of their strong light scat-
terings and tunable optical properties. The controlled fabrication results in the SERS
signal amplification of ~1010, exceeding what was previously thought possible only
at randomly occurring particle aggregates [105].
A shell-isolated, NP-enhanced Raman spectroscopy is provided for Raman signal
amplification by Au NPs with an ultrathin silica or alumina shell. This approach
could be used for inspecting pesticide residues on food and fruit. Figure1.20 shows
normal Raman spectra recorded on fresh oranges with clean pericarps (curve I) or
contaminated by parathion (curve II). The clean pericarps show only two bands, at
about 1,155 and 1,525cm1, attributed to carotenoid molecules contained in citrus
fruits. By spreading shell-isolated NPs on the same surface, two bands can clearly
be detected, at 1,108 and 1,341cm1 (curve III), which are characteristic bands of
26 1 Biofunctionalization of Nanomaterials

Fig.1.20 In situ inspection


of pesticide residues on food/
fruit. Normal Raman spectra
on fresh citrus fruits. Curve I,
with clean pericarps; curve II,
contaminated by parathion.
Curve III, SHINERS
spectrum of contaminated
orange modified by Au/SiO2
NPs. Curve IV, Raman
spectrum of solid methyl
parathion. The laser power on
the sample was 0.5mW, and
the collected times were 30s.
Reprinted with permission
from Li etal. [106]. 2010,
Nature Publishing Group

parathion residues. This demonstrates that the shell-isolated, NP-enhanced Raman


spectroscopy can have tremendous scope as a simple-to-use, field-portable, and
cost-effective analyzer [106]. Furthermore, by coupling electronic resonant Raman
of the osmium complex with the SERS amplification by Au NPs, an extra enhanced
resonant Raman scattering signal has been used for the sensitive detection of glu-
cose [107].
Two-photon Rayleigh scattering properties of gold nanorods can be used for the
rapid, highly sensitive, and selective detection of Escherichia coli bacteria from
aqueous solution. With covalent immobilization of the antibody onto the amine-
modified gold nanorod surface, the two-photon Rayleigh scattering intensity
increases by 40 times when anti-E. coli antibody-conjugated nanorods are mixed
with various concentrations of E. coli O157:H7 bacterium. The detection limit a
bionanotechnology assay can reach is a 50 colony forming units (CFU) mL1 level,
with excellent discrimination against any other bacteria [108].
Since photoacoustic imaging has a higher spatial resolution in deep tissues (up to
3cm) than other optical modalities, a new platform has been developed for invivo
magnetic enrichment and detection of rare circulating tumor cells (CTCs) from a
large pool of blood using targeted MNPs in combination with two-color photoa-
coustic flow cytometry. This approach has potential for the early diagnosis of cancer
and the prevention of metastasis in humans [109]. With the addition of magnetic
nanotag amplification, an inexpensive, giant magnetoresistive sensor has been con-
structed for multiplexed protein detection of potential cancer markers at subpicomo-
lar concentration levels and with a dynamic range of more than four decades [110].
SWNTs have several advantages when used as optical sensors, such as photo-
stable near-infrared emission for prolonged detection through biological media and
1.5 Applications of Biofunctional Nanomaterials 27

single-molecule sensitivity. Molecular adsorption can be transduced into an optical


signal by perturbing the electronic structure of the nanotubes. For example, d(GT)15
oligonucleotide-bound nanotube (DNASWNT) provides at least four modes that
can be modulated to uniquely fingerprint agents by the degree to which they alter
either the emission band intensity or the wavelength. This identification method can
identify six genotoxic analytes and single-molecule sensitivity in detecting
hydrogen peroxide in real time within live 3T3 cells [12].

1.5.2Electrochemical Sensing

Electrochemical transducers are very attractive for such bioassays, due to their high
sensitivity, inherent simplicity and miniaturization, and low cost and power require-
ments. The biofunctionalized nanomaterials provide an excellent platform to con-
struct ultrasensitive electrochemical sensors for the detection of biomolecules such
as DNA, proteins, cells, and other signal biomolecules.

1.5.2.1DNA

The development of highly sensitive and selective DNA sensors to pico- and femto-
molar levels is a field of ever-increasing interest for various applications, including
the diagnosis and treatment of genetic diseases, drug discovery, and warning against
bio-warfare agents. CNTs make them extremely attractive for electrochemical DNA
sensors due to the unique electronic, chemical, and mechanical properties. Most
CNT-sensing work has focused on the ability of surface-confined CNTs to promote
electron-transfer reactions involved in biocatalytic devices. Wangs group [13] has
demonstrated that CNTs play a dual-amplification role in both the recognition and
transduction events, namely, as carriers for numerous enzyme tags and for accumu-
lating the product of the enzymatic reaction. The 104-fold improvement in the sen-
sitivity is in good agreement with the estimated ALP loading per CNT. The favorable
response of the 5fgmL1 DNA target indicates a remarkably low detection limit of
around 1fgmL1 (54aM), i.e., 820 copies or 1.3zM in the 25-mL sample [13].
Asensitive electrochemical DNA biosensor has been successfully realized on poly-
aniline nanofiber (PANI)-coated MWNTs in chitosan filmmodified carbon paste
electrode (CPE). The immobilization of the probe DNA on the surface of the elec-
trode is largely improved due to the unique synergistic effect of PANI and MWNTs.
Under the optimal conditions, the dynamic detection range of this DNA electro-
chemical biosensor is from 1.01013 to 1.0107molL1, with a detection limit of
2.71014molL1, for the detection of DNA-specific sequences of the phosphino-
thricin acetyltransferase gene [111].
Figure1.21 represents a schematic view of a sandwich-type DNA sensor employ-
ing Pd NPs as electrocatalytic labels. To achieve low levels of nonspecific binding
of DNA-conjugated Pd NPs, ITO electrodes are modified with a silane copolymer
28 1 Biofunctionalization of Nanomaterials

Fig.1.21 Schematic view of DNA detection using the catalytic and electrocatalytic oxidation of
NaBH4 on Pd NPs and the rapid enhancement of the electrocatalytic activity of DNA-conjugated
Pd NPs. Reprinted with permission from Das etal. [112]. 2009, Royal Society of Chemistry

containing poly(ethylene glycol) and carboxylic acid. In this solution, the fast
catalytic hydrolysis of NaBH4 on Pd NPs generates many atomic hydrogens, which
are rapidly sorbed into Pd NPs (Fig.1.21). The fast hydrogen sorption induces the
rapid enhancement of the electrocatalytic activity of Pd NPs. Finally, the electro-
catalytic oxidation current of NaBH4 by Pd NPs is measured. Because NaBH4
undergoes multielectron (maximum 8e) oxidation on Pd NPs, higher current signals
can be obtained than those in the electrochemical reactions involving one- or two-
electron oxidation [112]. A DNA biosensor has been fabricated by immobilizing
capture-probe DNA on the nanoporous gold (NPG) electrode and hybridization
with target DNA, which further hybridizes with the reporter DNA loaded on the Au
NPs. Electrochemical signals of [Ru(NH3)6]3+ bound to the reporter DNA via elec-
trostatic interactions are measured by chronocoulometry. Taking advantage of the
dual-amplification effects of the NPG electrode and multifunctional encoded Au
NPs, this DNA biosensor can detect the DNA target quantitatively, in the range of
8.010171.61012 M, with a limit of detection as low as 28 aM, and exhibits
excellent selectivity even for single-mismatched DNA detection [30].
A new metal sulfide NPbased electrochemical detection method has been pro-
vided with the detection capability down to 100aM of target DNA. The setup is
constructed to give a signal-off response with a built-in control signal. The control
signal eliminates the disadvantages commonly associated with signal-off sensors
[113]. Multianalyte aptamer-based devices, with lower detection limits, are highly
desired for measuring a large panel of disease markers present at ultralow levels
during early stages of the disease progress. Four encoding NPs (cadmium sulfide,
zinc sulfide, copper sulfide, and lead sulfide) have been used to differentiate the
signals of four DNA targets in connection to stripping voltammetric measurements
of the corresponding metals [14].
The remarkable synergistic effects of the ZnO NPs and MWNTs have been devel-
oped for the ssDNA probe immobilization and fabrication of the electrochemical
1.5 Applications of Biofunctional Nanomaterials 29

DNA biosensor. Under optimal conditions, the dynamic detection range of the sensor
to PAT gene complementary target sequence is from 1.01011 to 1.0106molL1,
with a detection limit of 2.81012molL1 [114].
Of the diverse DNA detection techniques, ECL-based biosensing has received
considerable attention due to its versatility, simplified optical setup, and good tem-
poral and spatial control [115]. The quenching of ECL from a CdS:Mn NC film by
proximal Au NPs is observed as a result of Frster energy transfer, while an enhance-
ment of ECL takes place after hybridization with target DNA due to the energy
transfer of ECL-excited surface plasmon resonances in Au NPs to the CdS:Mn NCs
at large separation, based on which an ultrasensitive and specific DNA biosensor
has been constructed. The relationship between the increase in ECL peak height
before and after hybridization and target DNA concentration shows a linear range
from 50aM to 5.0fM. The favorable response of 50-aM target DNA indicates a
remarkably low detection limit (S/N=3), i.e., 2,100 copies in 70 mL of sample
[116]. A novel PCR-free ECL-based bio-barcode assay is also reported for the
quantitative detection of genetically modified organisms from raw materials without
additional purification [117].
A novel and sensitive flow-injection CL assay is reported for sequence-specific
DNA detection. The hybridization events are monitored by the CL intensity of lumi-
nolH2O2Cu2+ after the cupric ions are dissolved from the hybrids. The CL inten-
sity increases with the increase in the concentration of target DNA in the range of
2.010142.01012M, with a detection limit of 4.81015M [118].

1.5.2.2AntibodyAntigen

An ultrasensitive and simple method for detecting and quantifying biomarkers is


essential for the early diagnosis of diseases. A novel tracer, GOx-functionalized nano-
composite, has been designed to label the signal antibodies for ultrasensitive multi-
plexed measurement of tumor markers using a disposable immunosensor array. The
immunosensor array is constructed by coating LBL colloidal Prussian blue, Au NPs,
and capture antibodies on screen-printed carbon electrodes. The simultaneous multi-
plexed immunoassay method using the immunosensor array and the designed tracer
shows linear ranges of three orders of magnitude, with the detection limits down to 1.4
and 2.2pgmL1 for carcinoembryonic antigen and a-fetoprotein, respectively [16].
Much attention has been focused on signal amplification without using enzymes.
For example, DNA-functionalized Au NPs have been used to enhance the sensitivity
of the aptasensor because a DNAAu NP-modified interface can load more
[Ru(NH3)6]3+ cations. Thus, the assembly of two aptamer-contained DNA strands
integrated with the DNAAu NP amplification not only improves the sensitivity of
the electrochemical aptasensor but also presents a simple and general model for bio-
functional aptasensor. The proposed aptasensor has a low detection limit (0.02nM
for adenosine and 0.01mgmL1 for lysozyme) and exhibits several advantages, such
as high sensitivity and biofunctional recognition [119]. A novel electrochemical assay
based on the aptamer and the signal of amplification of NPs has been constructed for
30 1 Biofunctionalization of Nanomaterials

Fig.1.22 Schematic representation of electrochemical immunoassay strategy using phospholipid-coated


MWNTs as electrochemical labels. Reprinted with permission from Nie etal. [121]. 2009, Wiley

the determination of thrombin. Differential pulse voltammetry is employed to detect


the CdS NPs loaded on the surface of the Au NPs through the linker DNA, which are
related to the concentration of the target protein. The assay takes the advantages of
the amplification ability of Au NPs carrying multiplex CdS NPs and the specific
affinity of aptamers. Thrombin can be detected in the linear range of 1.01015
1.01011M, with a detection limit of 5.51016M of the target protein [4].
NPs have received wide attention as electrocatalysts for electrochemical reaction
in protein detection. An ultrasensitive and simple electrochemical method for signal
amplification is achieved by the catalytic reduction of p-nitrophenol to p-aminophenol
using gold-nanocatalyst labels. The electrochemical signal is amplified by the
chemical reduction of p-quinone imine to p-aminophenol by NaBH4. The detection
limit for mouse IgG is 1fgmL1, which corresponds to about 7aM. Importantly, the
concentration of mouse IgG can be detected ranging from 1fgmL1 to 10mgmL1
with a single assay format, which covers a 10-order concentration range [120].
A novel electrochemical immunoassay strategy has been developed using phospho-
lipid-coated CNTs as the electrochemical labels. In the presence of PSA, magnetic
separation collects the immunocomplex formed between MWNT-labeled Ab1 and
magnetic-bead (MB)-modified Ab2 on an isolating SAM-modified electrode. Treatment
with DMF dissociates the phospholipid from the MWNTs, leaving the MWNTs
assembled on the SAM (Fig.1.22). With the MBs removed by a magnet, the assembled
MWNTs mediate electron transfer between electroactive species and the electrode,
triggering the electrochemical signal. In the absence of antigen (PSA), the collection of
the labels by magnetic separation leads to the lack of MWNTs on the isolating SAM;
thus, no current signal is obtained. A quasilinear response is obtained in a logarithmic
concentration scale within a four-order-of-magnitude concentration range from
5pgmL1 to 50ngmL1, with a readily achieved detection limit of 3pgmL1 [121].
Similarly, this strategy is demonstrated for the quantitative analysis of the interaction of
folate with a tumor biomarker of folate receptor, and a detection limit of 3pM of folate
receptor is readily achieved with a desirable specificity and sensitivity [122].
1.5 Applications of Biofunctional Nanomaterials 31

1.5.2.3Cells

A novel electrochemical cytosensing strategy was designed based on the specific


recognition of integrin receptors on a cell surface to arginineglycineaspartic acid
serinefunctionalized SWNTs via covalent interaction. On the basis of the dual-
signal amplification of SWNTs and enzymatic catalysis, the cytosensor can respond
down to 620cellsmL1 of BGC-823 human gastric carcinoma cells, with a linear
calibration range from 1.0103 to 1.0107 cells mL1, showing a very high
sensitivity. The dual-signal amplification can be further used to evaluate the man-
nosyl groups on the cell surface, and the mannosyl groups on a single, living, intact
BGC cell are detected to correspond to 5.3107 molecules of mannose [123].
Further, four nanoscaffolds of nanohorns functionalized with four kinds of lectins
are also prepared on an electrode surface for cell capture and to enhance the electri-
cal connectivity [124]. The constructed three-dimensional recognition interface can
maintain the biological activity of the immobilized proteins, increase the cell-bind-
ing capacity, and enhance the sensitivity for impedimetric analysis of the cell sur-
face glycosignature. This method can be used for monitoring alterations of cell
surface glycans in response to drugs and during biological events. Another method
for cytosensing by ECL of QDs has also been developed [125]. Here, the thiogly-
colic acidcapped CdSe QDs with a diameter of 1.8nm are first covalently bound
to chitosan (CS)-Au NP-composite-modified electrodes and then further functional-
ized with four lectins individually. The resulting biofunctional films with four kinds
of lectins show excellent ECL behavior, which is decreased upon the specific bind-
ing of the lectin-functionalized QDs to cell surface carbohydrates on the electrodes
surface. The detection limit for cell concentration at WGA-, Con A-, and PNA-
modified electrodes are 1.1103, 1.6103, and 2.1103 cells mL1, respectively.
This strategy has been used to monitor the dynamic changes of cell surface carbo-
hydrates in response to drugs.

1.5.2.4Other Biological Compounds

CNTs and a redox mediator toluidine blue O (TBO) have been coimmobilized in a
matrix of the biopolymer chitosan and used for the oxidation of the enzyme cofactor
b-nicotinamide adenine dinucleotide (NADH) [126]. The integration of CNTs and
redox mediators can provide a remarkable synergistic augmentation of the current
because of the oxidation of redox-active species. In particular, it amplifies the NADH
current approximately 60 times while reducing the response time from approximately
50s for CHIT-TBO to approximately 5s for CHIT-TBO/CNT films.
A highly sensitive electrochemical sensor has been developed for the detection
of Hg2+ ions in aqueous solution by using a thymine (T)-rich, mercury-specific oli-
gonucleotide probe and Au NP-based signal amplification via the Hg2+-mediated
coordination of THg2+T base pairs [127]. This Au NP-based sensing strategy
brings about an amplification factor of more than three orders of magnitude, leading
to a limit of detection of 0.5nM [128].
32 1 Biofunctionalization of Nanomaterials

1.6Conclusions

Biological molecules have been immobilized on polymer matrices and inorganic


supports through a variety of techniques, including physical adsorption, electro-
static binding, specific recognition, and covalent coupling. The expectation is that
biofunctional hybrids will be able to perform some specific functions better than
either purely organic or purely inorganic systems. In fact, the biofunctional hybrids,
coupled with biological molecules such as proteins/enzymes, antigens/antibodies,
and DNA/oligonucleotides, have been used as excellent signal transducers in
numerous biotechnological applications. Nanometer-sized particles such as semi-
conductor QDs and energy-transfer NPs have novel optical properties, such as tun-
able light emission, signal brightness, and multicolor excitation, that are not available
from traditional organic dyes and fluorescent proteins. Meanwhile, conductive
nanomaterials such as metallic and carbon-based materials show the excellent elec-
trochemical performances of miniaturization, high sensitivity, and easy in vivo
detection.
However, since simultaneous analysis is required in practice, it is of key interest
for the development of high-throughput techniques for the parallel analysis of
numerous components in samples. The possibility to control and tune these unique
optical and electronic properties of metal or semiconductor NPs through their
dimensions paves the way for the application of NPs as versatile analytical probes.
On the other hand, many proteins or other complex biological systems require a
physiological environment and a minimum degree of biocompatibility on the solid
substrate. Therefore, it is a significant direction to explore a suitable biofunctional
way to increase the biocompatibility in nanomaterials chemistry. The ultimate goals
of this endeavor are the creation of novel biofunctionalized nanomaterials and real-
izing high-throughput, multicomponent analysis on a nanometer scale.

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Chapter 2
Signal Amplification for Nanobiosensing

2.1Introduction

One of the major goals in developing novel biological assay methods for the
detection of biomolecules and DNA hybridization is achieving high sensitivity. The
need for ultrasensitive bioassays is of major importance in view of the growing
trend toward miniaturized assays. Highly sensitive methods, which are urgently
required for measuring disease diagnosis markers present at ultralow levels during
early stages of disease progression, can facilitate the treatment of diseases. For
example, polymerase chain reaction (PCR) amplification has revolutionized genetic
testing. However, it is somewhat restricted because of its complexity, potential con-
tamination, and cost. On the other hand, the ultrasensitive monitoring of proteins is
particularly challenging due to the absence of PCR-like amplification protocols.
Conventional (optical and electronic) sandwich bioaffinity assays have the disad-
vantage of capturing a small number of labels per binding event. Recently, signal
amplification has attracted considerable attention for developing ultrasensitive
detection methods for biothreats and infectious agents. Such kinds of highly sensi-
tive bioagent detection schemes provide an early warning of their release and pre-
vent outbreaks of foodborne illnesses, hence minimizing human casualties.
The achievement of ultrahigh sensitivity requires innovative approaches that
couple with different amplification platforms and amplification processes.
Nanotechnology offers unique opportunities for creating highly sensitive innovative
biosensing devices and ultrasensitive bioassays. The unique optical [14],
photophysical [5], electronic [6], and catalytic [79] properties of metal and semi-
conductor nanoparticles (NPs) turn them into ideal labels for biorecognition and
biosensing processes. For example, the unique plasmon-absorbance features of gold
(Au) NPs and specifically the interparticle-coupled plasmon absorbance of conju-
gated particles have been widely used for DNA [10] and antibodyantigen [1113]
analyses. Similarly, the tunable fluorescence properties of semiconductor NPs have

H. Ju et al., NanoBiosensing: Principles, Development and Application, 39


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0_2, Springer Science+Business Media, LLC 2011
40 2 Signal Amplification for Nanobiosensing

been used for the photonic detection of biorecognition processes [14]. This chapter
focuses on signal amplification based on nanobiotechnologies for highly sensitive
nanobiosensing.

2.2Nanoparticle-Amplified Optical Assay

Among all detection methods used in biosensors, the optical-based technique is the
most popular one because of its high sensitivity and the ability to remotely interro-
gate the information on the biosensor using light or laser. Metal NPs, such as Au and
silver NPs, exhibit plasmon absorbance bands in the visible spectral region that are
controlled by the size of the respective particles. Numerous studies on the labeling of
biomaterials and the staining of biological tissues by metal particles as a means to
image and visualize biological processes have been reported [15, 16]. The spectral
shifts originating from adjacent or aggregated metal NPs, such as Au NPs [17], have
led to increasing interest in the development of optical biosensors based on
biomaterial-NP hybrid systems. Similarly, semiconductor NPs exhibit size-dependent
tunable absorbance and fluorescence. Due to the high-fluorescence quantum yields,
photostability, and tunable fluorescence bands, semiconductor NPs are attracting
substantial research interest as fluorescence labels for biorecognition processes.

2.2.1Colloidal Gold Nanoparticle-Based Amplification

The unique plasmon-absorbance features of Au NPs, and specifically the interparticle-


coupled plasmon absorbance of conjugated particles, have been widely used for DNA
[18] and antibodyantigen [1922] analyses. For example, a cationic Au NP has been
used for signal amplification by ionic interaction with 16S rRNA hybridized on the
peptide nucleic acid probe-immobilized surface plasmon resonance (SPR) sensor chip
[23]. Peptide nucleic acid has a neutral backbone structure; therefore, hybridization
with 16S rRNA results in the ionic condition being changed from neutral to negative.
16S rRNA has been used as a genetic marker for the identification of organisms and
can be analyzed directly without PCR amplification due to the relatively high number
of copies. This method results in an Escherichia coli rRNA detection limit of 58.2
(1.37)pg/mL. With this analytical method, Staphylococcus aureus can be detected
without purification of rRNA. Yao etal. [24] used oligonucleotide (ODN)-capped Au
NPs in a sandwich assay of ODN or polynucleotide by flow-injection SPR. A car-
boxylated dextran film was immobilized onto the SPR sensor surface to eliminate the
nonspecific adsorption of ODN-capped Au NPs. The tandem use of signal amplifica-
tion via the adlayer of the ODN-capped Au NPs and the differential signal detection
by the bicell detector on the SPR resulted in a remarkable detection limit of DNA.
A 39-mer target at a quantity as low as 2.11020mol, corresponding to 1.38fM, can
be measured. The method is shown to be reproducible (relative standard deviation
values<16%) and to possess high sequence specificity. Drastic sensitivity enhancement
2.2 Nanoparticle-Amplified Optical Assay 41

Fig.2.1 Diagram of the three types of fluorescence probes used for the LSPCF biosensor. Reprinted
with permission from Hsieh etal. [27]. 2007, American Chemical Society

was maximized using longitudinal plasmonic resonance of Au nanorods for


ultrasensitiveSPR biosensing with functionalized Au nanorods as amplification labels
due to the electromagnetic interaction between the nanotag and the sensing film
[25,26]. The detection sensitivity of the nanorod-conjugated antibody was estimated
to be ~40pg/mL, which was 25100 times more sensitive than the current reported
values. A novel fiber optic biosensor based on a localized surface plasmon-coupled
fluorescence (LSPCF) system also has been developed [27]. As shown in Fig.2.1, the
biosensor consists of a biomolecular complex in a sandwich format of antibody/
antigen/Cy5antibodyAu NP. It is immobilized on the surface of an optical fiber,
where a Cy5antibodyAu NP complex forms the fluorescence probe. The LSPCF is
excited by localized surface plasmon on the Au NPs surface, where the evanescent
field is applied near the core surface of the optical fiber. At the same time, the fluores-
cence signal is detected, with high collection efficiency, by a photomultiplier tube
located beside the unclad optical fiber. This LSPCF biosensor is able to detect mouse
immunoglobulin G (IgG) as low as 1pg/mL (7fM) during the biomolecular interac-
tion of the IgG with antimouse IgG, indicating a very high sensitivity due to the
amplification of the LSPCF intensity by Au NP coupling.
A highly sensitive and specific colorimetry-based rolling-circle amplification
(RCA) assay method for single-nucleotide polymorphism (SNP) genotyping has
been developed [28]. As shown in Fig.2.2, a circular template is generated by liga-
tion upon the recognition of a point mutation on DNA targets. An RCA amplifica-
tion is then initiated using the circular template in the presence of Phi29 polymerase.
The RCA product can be digested by a restricting endonuclease, and the cleaved
DNA fragments can mediate the aggregation of Au NP-tagged DNA probes. This
causes a colorimetric change of the solution as the indicator of the mutation occur-
rence, which can be detected using UV-vis spectroscopy or viewed by naked eyes.
On the basis of the high amplification efficiency of Phi29 polymerase, a mutated
target of 70fM can be detected in this assay. In addition, the protection of the circle
template using phosphorothioated nucleotides allows the digestion reaction to be
performed simultaneously in RCA. Moreover, DNA ligase offers high fidelity in
distinguishing the mismatched bases at the ligation site, resulting in the positive
42 2 Signal Amplification for Nanobiosensing

Fig. 2.2 Schematic illustration of the RCA reaction and Au NP assembly-based assay: (1)
hybridization between padlock probe and target and ligation; (2) RCA and digestion; (3) colori-
metric detection via the assembly of Au NP-tagged DNA probes. Reprinted with permission from
Li etal. [28]. 2010, American Chemical Society

detection of mutant targets even when the ratio of the wild type to the mutant is
10,000:1. The developed RCA-based colorimetric detection scheme has been dem-
onstrated for SNP typing of the thalassemia gene at position 28 in genomic DNA.
A sandwich-type assay for the optical detection of DNA using multicomponent
cross-linked Au NP aggregates was reported by Fan (Fig.2.3) [29]. In their work, a
DNA-bridged multi-functional Au NP aggregate, which integrated DNA recogni-
tion (detection probe [DP]), signal amplification (enzyme, horseradish peroxidase,
HRP), and nonspecific blocking (bovine serum albumin, BSA) section, was
employed as the DP. In a typical sensing process, the Au aggregates DP was brought
to the proximity of magnetic particles through the DNA hybridization. Once the
magnetic field was added, these sandwich complexes were magnetically separated.
As a result, HRP that was confined at the surface of Au aggregates could catalyze
the enzyme substrate and generate an optical signal. This assay was employed for
the detection of breast cancerassociated BRCA-1 gene. The detection limit was
about 1fM, which was significantly improved compared with the results obtained
from individual Au NP-labeled assays.
Figure2.4 shows a sensitive detection method for adenosine (AD) in human urine
by using enhanced-resonance light scattering (RLS) [30]. It is based on the specific
recognition and signal amplification of AD aptamer (Apt) coupled with Au NPs via
G-quartetinduced NP assembly, which is fabricated by triggering a structureswitch-
ing of the 30 terminus G-rich sequence and Apt duplex. The RLS signal linearly
2.2 Nanoparticle-Amplified Optical Assay 43

Fig.2.3 Schematic for the amplified sandwich-type detection assay of the BRCA-1 gene using
multifunctional cross-linked Au aggregates and magnetic particles. Note: The drawing is not to
scale. Reprinted with permission from Li etal. [29]. 2009, Elsevier

Fig.2.4 Schematic diagram for analytical principle. (a) DNA duplex; (b) procedure of Au NPs
aggregate. (a) G-quartets stacked perpendicularly to the column axis; (b) four guanines fold into a
G-quadruplex structure by Hoogsteen hydrogen bonds. Reprinted with permission from Zhang
etal. [30]. 2010, Elsevier

correlates with the concentration of AD over the range of 6115nM. It has been
applied to detect AD in real human urine, and the obtained results are in good agree-
ment with those obtained by the HPLC method. This study illustrates that the com-
bination of the excellent selectivity of Apt with the high sensitivity of the RLS
technique provides promising potential for Apt-based small-molecule detection, and
may be beneficial in extending the applications of RLS.
Based on Au NP probes, a one-step, washing-free, and amplification-free assay for
protein analysis via dynamic light scattering (DLS) has been developed (Fig.2.5) [31].
The concentration of the target protein is determined by analyzing the level of Au NP
44 2 Signal Amplification for Nanobiosensing

Fig.2.5 Illustration of a one-step homogeneous biomolecular assay using gold nanoparticle (NP)
probes as light-scattering enhancers coupled with dynamic light-scattering detection. Reprinted
with permission from Liu and Huo [31]. 2009, Elsevier

Fig. 2.6 Scanometric immunoassay. Reprinted with permission from Kim et al. [32]. 2009,
American Chemical Society

aggregation caused by antibodyantigen interactions using DLS. The mouse IgG is


directly mixed with Au NPs conjugated to goat antimouse IgG. Due to the multiple
binding sites of primary mouse IgG by the secondary antibody, mouse IgG causes NP
aggregation. Mouse IgG can be detected at a concentration as low as 0.5ng/mL, and
the dynamic range of this assay is between 0.5 and 50ng/mL. On the other hand, with
the use of both mouse IgG and goat antimouse IgG-conjugated Au NPs, this study
designs a competitive assay, in which mouse IgG is detected within a dynamic range of
100ng/mL to 10mg/mL.
The use of gold development results in greater signal enhancement than the typical
silver development, and multiple rounds of metal development have been found to
increase the resulting signal compared to one development (Fig.2.6) [32]. An anti-
body microarray is fabricated by spotting monoclonal capture antibodies to the surface
2.2 Nanoparticle-Amplified Optical Assay 45

of N-hydroxysuccinimide-activated glass slides (CodeLink, SurModics). Six spots, all


with antibodies for PSA, are used in each assay well. The use of six spots allows sta-
tistically significant data to be obtained in each assay. The slides are then passivated
with ethanolamine. Probes are prepared by first modifying 13-nm-diameter Au NPs
with 3-propylthiol and 5-decanoic acidmodified oligonucleotides and then cova-
lently immobilizing antibodies for PSA via carbodiimide coupling. The assay begins
by incubating the test solution with PSA at a designated concentration with capture
antibodies on the chip (assay buffer: Dulbeccos PBS with 0.1% Tween-20, 0.1%
BSA, and 1% poly(acrylic acid)). After that, Au NP probes are incubated with the
microarray-bound targets. To increase the light-scattering signal of the immobilized
Au NP probes, gold or silver is catalytically deposited on the chip using electroless
deposition techniques. Finally, the light scattering is quantified with a Verigene Reader
system, which is a device that captures evanescent wave-induced light scattering from
the amplified Au NPs. Under these conditions, the assay is capable of detecting
300aM of PSA in buffer and 3fM in 10% serum. Additionally, the highly selective
detection of three tumor markers at low picomolar concentrations in buffer and 10%
serum has been demonstrated. The use of gold deposition may have significant utility
in scanometric detection schemes and in broader clinical and research applications.
A homogeneous colorimetric DNA detection by a novel nicking endonuclease-
assisted nanoparticle-amplification (NEANA) process has been reported to recognize
long single-stranded oligonucleotides with single-base mismatch selectivity and a
100-fold improvement in amplification [33]. A three-component sandwich assay for-
mat that included a target DNA (tDNA) and two sets of oligonucleotide-modified NP
probes is typically used in conventional homogeneous NP-based colorimetric DNA
detection. tDNA serves as a linker strand that triggers particle aggregation and a con-
comitant color change. The colorimetric detection limit is directly associated with the
minimum number of the linkers required to initiate particle aggregation that can be
visualized with the naked eye. However, at low linker concentrations (e.g., 10nM),
the aggregation of 14-nm NPs cannot exhibit sharp colorimetric melting transitions.
Normally, there are two ways to improve the sensitivities of the colorimetric assay:
Enlarge the size of particle probes, or reduce the surface coverages of the oligonucle-
otide. The former is dominant in sedimentation. To increase the sensitivity of homo-
geneous NP-based assays, the nicking endonuclease is specifically designed to cleave
only the linker strand [34]. As shown in Fig.2.7, after nicking, the fragments of the
linker strand spontaneously dissociate from the tDNA at an elevated temperature.
Subsequently, another linker strand hybridizes to the target to continue the strand-
scission cycle, which results in the cleavage of a large molar excess of linkers. Upon
completion of the strand-scission cycle, two sets of different oligonucleotide-modified
gold NPs with sequences complementary to that of the linker strand are added to the
solution to detect the presence of a tDNA (Fig.2.8). If the linker DNA is noncomple-
mentary to the tDNA, particle aggregation will occur. This system offers handling
convenience and ultrahigh detection sensitivity and selectivity, and provides addi-
tional detection versatility for long-stranded DNA sequences.
Another approach to increase the signal intensity of dye-based assays is metal-
enhanced fluorescence (MEF) [3538]. MEF is the result of interactions between
46 2 Signal Amplification for Nanobiosensing

Fig. 2.7 Nicking endonuclease-assisted NP amplification for target DNA detection. Reprinted
with permission from Xu etal. [34]. 2009, Wiley

Fig.2.8 Photograph showing colorimetric responses of a NEANA detection system. The labeled
concentrations (20nM, 2nM, 200pM, 20pM, and 10pM) are the calculated final target concen-
trations in solutions. The NEase recognition site of the target is highlighted in red. Reprinted with
permission from Xu etal. [34]. 2009, Wiley

fluorophores and surface plasmons in metallic nanostructures, most typically in Ag


[39, 40] and Au [41, 42]. Enhanced emissions can be obtained as a result of an ampli-
fication of the incident electric field, which effectively increases the fluorophores
absorption cross-section, or by an acceleration of the radiative decay rate [4347].
These desirable effects need to counterbalance possible emission suppression by
energy transfer to the metallic surfaces [4850]. Nevertheless, differences in spatial
and orientational dependencies between amplification and quenching mechanisms
allow one to find appropriate emitter-surface environments and organizationswhere an
improved performance is obtained [37]. The examples include MEF-based assays for
DNA [51], RNA [52], and immunological applications [53].
2.2 Nanoparticle-Amplified Optical Assay 47

Fig. 2.9 (a) Chemical structure of OFP; (b) HR-TEM image of OFP; (c) normalized UVvis
absorption spectra of the arm 4, OFP, and EB (dashed lines), and PL spectra of 4 and OFP (solid
lines) in water. Reprinted with permission from Pu etal. [54]. 2010, Wiley

Using hybrid nanomaterials as the signal amplifiers, Pu etal. provided a new way
to improve the performance of fluorescence technologies for biological imaging
through a fluorescence resonance energy transfer (FRET) approach [54]. From the
materials viewpoint, the emission wavelength, charge nature, and diameter of poly-
hedral oligomeric silsesquioxane (POSS)-based fluorescent NPs can be easily
adjusted through chemical modification of fluorescent arms so as to fulfill the dif-
ferent requirements of specific applications. In terms of materials applications, the
high quantum yields and good signal amplification capability of POSS-based mol-
ecules allow high-quality biological imaging even with a small amount of indicator
dyes, consequently avoiding the side effect of elevated dye concentrations. In view
of their aggregation-inhabited nanostructures and environment-resistant fluores-
cence, POSS-based nanomaterials are also appropriate for signal amplification in
various biological assays, such as DNA and protein microarrays (Fig.2.9).
48 2 Signal Amplification for Nanobiosensing

Fig.2.10 (a) SEM photomicrograph of beads in anisotropically etched silicon chip. (b) Chip (iv)
is fitted between double-sided adhesive layer (ii) and cover slip (i) with laminate layers (iii, v, vi)
included to direct fluid flow through the PMMA base (viii) and inlet and outlet ports (vii). (c)
Sealed LOC assembly. (d) Fluorescent image of beads after immunoassay, including negative
controls as imaged with 1s of CCD camera integration (exposure) time. Reprinted with permission
from Jokerst etal. [55]. 2009, Elsevier

2.2.2Semiconductor Nanoparticle-Based Amplification

Similarly, semiconductor NPs exhibit size-dependent tunable absorbance and


fluorescence. The high-fluorescence quantum yields, photostability, and tunable fluo-
rescence bands of semiconductor NPs have attracted substantial research efforts
directed toward the use of semiconductor NPs as fluorescence labels for biorecogni-
tion processes.
With the integration of semiconductor NP quantum dots (QDs) into a modular,
Jokerst etal., designed a microfluidic biosensor for the multiplexed quantitation of
three important cancer markers: carcinoembryonic antigen (CEA), cancer antigen
125 (CA125), and Her-2/Neu (C-erbB-2) (Fig. 2.10) [55]. Nanobiochips that
employed a fluorescence transduction signal with a QD-labeled detecting antibody
were used in combination with antigen capture by a microporous agarose bead array
supported within a microfluidic ensemble so as to complete the sandwich-type
immunoassay. The utilization of QD probes in this miniaturized biosensor format
2.2 Nanoparticle-Amplified Optical Assay 49

Fig.2.11 Schematic of CL SNP quantitative assay based on Au and CuS NP probe and one-step
DNA hybridization reaction. Reprinted with permission from Ding etal. [56]. 2010, Elsevier

resulted in a 30-fold signal amplification relative to that of standard molecular


fluorophores as well as reduced observed limits of detection by nearly two orders of
magnitude (0.02ng/mL CEA; 0.11pM CEA) relative to enzyme-linked immuno-
sorbent assay (ELISA). Assay validation studies indicated that measurements by the
nanobiochip system correlated to standard methods at R2=0.94 and 0.95 for saliva
and serum, respectively. This integrated nanobiochip assay system, in tandem with
next-generation fluorophores, would be a sensitive, multiplexed tool for important
diagnostic and prognostic applications.

2.2.3Nanoparticle-Amplified Chemiluminescence
and Electrogenerated Chemiluminescence Assay

The functionalized NPs have been used to enhance the chemiluminescence (CL) or
electrogenerated chemiluminescence (ECL) intensity. An ultrasensitive CL method
based on the Au NP amplification for the quantitative detection of SNPs in genomic
DNA has been accomplished by the DNA polymerase I (Klenow fragment)-induced
coupling of the nucleotide-modified NP probe to the mutant sites of duplex DNA
under the WatsonCrick base-pairing rule [56]. As shown in Fig. 2.11, Au NPs
50 2 Signal Amplification for Nanobiosensing

Fig. 2.12 (a) Schematic representation of the CL detection of DNA hybridization based on
bio-barcode-functionalized magnetic nanoparticle labels (bbcMNPs) (upper part: carboxyl-coated
MNPs (carboxyl-MNPs) are functionalized with amino-modified probe DNA (amino-pDNA) and
amino-modified bio-barcode DNA (amino-bbcDNA), fabricating bbc-p-DNA-MNPs). (b)
Schematic diagram of the FI-CL detection system for the determination of Fe3+. Reprinted with
permission from Bi etal. [58]. 2009, Royal Society of Chemistry

are first electrodeposited on the surface of the Au electrode for DNA probe
immobilization, followed by the hybridization between the DNA probe and the mix-
ture of the single-base-mismatched tDNA and complementary tDNA. Au NP probes
modified with CuS NPs and a base (guanine, G) that is complementary to the muta-
tion site (cytosine, C) are coupled to the formed duplex DNA in the presence of DNA
polymerase. CuS NPs and Au NPs are linked by an amidization reaction between
mercaptoacetic acid on the surface of Au NPs and aminoethanethiol on the surface
of CuS NPs. The base G and the Au NP are linked by DNA with a sequence of
5-SH-(CH2)6-ATG TCC CTC AGA CCC TTT-(CH2)6-NH2-3. The amount of the
SNPs is monitored by the CL intensity of luminol-CN-Cu2+ after the cupric ions are
dissolved from the hybrid (Fig.2.11). A preconcentration processing of cupric ions
is performed by anodic stripping voltammetric (ASV) technology to improve the
sensitivity of the method. The mechanism of the luminolCNCu2+ CL system to
produce CL signal is based on coupling the complex-formation reaction of cupric
ions and cyanide with the CL reaction of luminal and Cu(CN)42, which has a high
oxidation potential. As a single Au NP can be loaded with 77 CuS NPs, the incorpo-
ration of Au NPs significantly enhances the sensitivity. Moreover, the preconcentra-
tion processingof cupric ions can further increase the sensitivity about tenfold. As a
result of these two combined effects, this method could detect as low as 19aM SNPs,
and the linear range for SNPs was from 8.01017 to 1.01014M. Based on this
2.2 Nanoparticle-Amplified Optical Assay 51

Fig. 2.13 CL immunoassay of IgG using CdTe QDs as label. Reprinted with permission from
Wang etal. [59]. 2009, Elsevier

strategy, Zhangs group also demonstrated a dual amplification with thrombin-


labeled PbS NPs and thiocyanuric acidgold NP network for DNA detection [57].
On the other hand, Zhangs group developed an FI-CL detection platform of
DNA hybridization based on bio-barcode-functionalized magnetic nanoparticle
(MNPs) labels [58]. As shown in Fig.2.12, thiolated cDNA was assembled on the
Au electrode surface via sulfurgold affinity, and bbcDNA and pDNA were labeled
with carboxyl-modified MNPs on the 5-NH2 end, both of which flanked the tDNA,
resulting in the fabrication of a sandwich-type detection protocol. Compared with
single DNA probe strands, MNPs containing pDNA and bbcDNA could avoid
cross-reaction and improve the detection sensitivity of tDNA. Once the ferric ions
were dissolved from the hybrids, the large amount of released ferric ions could be
sensitively determined by the luminolH2O2Fe3+ CL reaction system and generated
a strong CL signal. The CL intensity was proportional to the amount of tDNA based
on the concentration of dissolved ferric ions. A detection limit of 0.32fM could be
achieved without any preconcentration process.
Li and his coworkers [59] found that mixing CdTe QDs with luminol in the pres-
ence of KMnO4 could induce a greatly sensitized effect on CL emission. The CL
spectra displayed that there was only one peak emission, around 425 nm, for the
luminolCdTe QDKMnO4 system when scanned from 300 to 700nm, which was in
agreement with that of the luminolKMnO4 system. Moreover, the CL intensity of the
luminolKMnO4CdTe QD system was obviously stronger than that of the luminol
KMnO4 system, indicating the sensitized effect of CdTe QDs on the luminolKMnO4
CL reaction due to the accelerated luminol CL induced by the oxidized species of
CdTe QDs. Based on this finding, using thioglycolic acid (TGA)-capped CdTe QDs
as a label and IgG as a model analyte, Lis group designed a CL immunoassay (CLIA)
protocol for IgG content detection (Figs.2.13 and 2.14). Overall, the studies strongly
demonstrated the possibility that QDs induced CL for more practical applications.
52 2 Signal Amplification for Nanobiosensing

Fig.2.14 CL spectra of luminolKMnO4 in the presence of CdTe QDs (CL1) and luminolKMnO4 in
the absence of CdTe QDs (CL2). Conditions: luminol, 1105 M (in 0.01 M NaOH); KMnO4,
1105M; CdTe QDs, 1103M. Reprinted with permission from Wang etal. [59]. 2009, Elsevier

Zhus group [60] indicated that the ECL of CdSe QDs could be greatly enhanced
by combining carbon nanotubes (CNTs) and poly (diallyldimethylammonium chlo-
ride) (PDDA) in CdSe QD film. Based on this phenomenon, they developed a sensi-
tive ECL immunosensor for the detection of human IgG (Ag). The fabrication
procedures for CdSe QDCNT conjugates and the ECL immunosensor were shown
in Fig.2.15. Where PDDA as a binding linker was conjugated to the CdSe QDCNT
composite film on the electrode, the ECL signal was significantly enhanced.
Subsequently, Au NPs assembled onto the CdSe QDCNT/PDDAmodified elec-
trode amplified the ECL signal once again. After antibody (Ab) was immobilized
onto the electrode through Au NPs, the ECL immunosensor was fabricated. The
principle of ECL detection for target Ag was based on the increment of steric hin-
drance after immunoreaction, which resulted in a decrease in the ECL intensity
(Fig. 2.16). The Ag concentration was determined in the linear range of 0.002
500ng/L, with a detection limit of 0.6pg/mL.
When nanoporous gold leaf (NPGL) electrodes are used, the sensitivity of the
ECL assay can be remarkably increased due to ultrathin nanopores. Based on
this phenomenon, Hu et al. [61] developed a sensitive ECL DNA assay
(Fig. 2.17). In this assay, tDNA was hybridized with capture DNA (cDNA)
bound on the NPGL electrode, which was fabricated by conjugating amino-
modified cDNA to TGA modified at the activated NPGL electrode. Following
that, amino-modified probe DNA was hybridized with the tDNA, yielding
2.2 Nanoparticle-Amplified Optical Assay 53

Fig.2.15 The fabrication


procedures for (a) CdSe
QDsCNTs conjugates and
(b) the ECL immunosensor.
Reprinted with permission
from Jie etal. [60]. 2009,
Elsevier

sandwich hybrids on the NPGL electrode. Then, mercaptopropionic acidcapped


CdTe QDs were labeled to the amino group end of the sandwich hybrids. Finally,
in the presence of S2O82 as coreactant, the ECL emission of the QD-labeled
DNA hybrids on the NPGL electrode was measured by scanning the potential
from 0 to 2V to record the curve of ECL intensity vs. potential. The maximum
ECL intensity on the curve was proportional to the tDNA concentration, with a
linear range of 5101511011mol/L (Fig.2.18).
The broad-spectrum expression of telomerase in most malignancies makes it a
promising target as a cancer diagnostic and prognostic tool. Conventional PCR-
based telomerase activity assay is highly sensitive but susceptible to amplification-
related errors. The ECL detection method for telomerase activity is accomplished
by the hybridization of ECL nanoprobes to telomerase reaction products, the subse-
quent capture by magnetic beads (MBs), and in situ measurement of the light signal
from ECL nanoprobes [62]. The ECL intensity directly reflects the quantity of
telomerase reaction products, which corresponds to the telomerase activity. The
high sensitivity afforded by the current MB and NP-based ECL detection platform
allows the measurement of telomerase activity from as little as 500 cultured cancer
cells in crude cell extracts without the PCR amplification of telomerase reaction
products. In addition, a comparative study of the ECL nanoprobe and linear telom-
ere antisense ECL probe has been executed. By using the ECL nanoprobe, one
obtains about a 100-fold elevation in sensitivity. This method is ideal for telomerase
activity analysis due to its reliability and high sensitivity.
54 2 Signal Amplification for Nanobiosensing

Fig. 2.16 ECLpotential curves of (a) CdSe QDsCNTs; (b) (a)+PDDA; (c) (b)+GNPs;
(d) (c)+Ab; (e) (d)+BSA; and (f) (e)+Ag-modified Au electrodes in 0.1 M PBS (pH 7.4)
containing 0.1M KCl and 0.1M K2S2O8. Scan rate: 100mV/s. Reprinted with permission from Jie
etal. [60]. 2009, Elsevier

2.3Nanoparticle-Amplified Electrochemical Detection

2.3.1Enhanced Conductivity with Nanoparticles

The formation of conductive domains, as a result of biomolecular interactions of


proteins and the enlargement of gold NP tags, provides an attractive route for
electrochemical transduction of biorecognition events. For example, based on
NP-induced changes in the conductivity across a microelectrode gap, Velev
and Kaler [63] developed conductivity immunoassays of proteins in connec-
tion to antibody-functionalized latex spheres placed between two microelectrodes.
2.3 Nanoparticle-Amplified Electrochemical Detection 55

Fig.2.17 Schematic representation of the process of DNA determination. Reprinted with permis-
sion from Hu etal. [61]. 2010, Elsevier

Sandwich immunoassay led to the binding of a secondary gold-labeled antibody,


followed by the catalytic deposition of a silver layer to bridge the two electrodes.
Such a formation of conductive paths across interdigitated electrodes led to a mea-
surable conductivity signal and enabled the ultrasensitive detection of human IgG
down to the 21013M level. This method holds promise for creating miniaturized
on-chip protein arrays. Analogous measurements of DNA hybridization were also
reported by Mirkins group [64].
The extensive knowledge in the preparation of metal and semiconductor NPs
functionalized with biomaterials suggests that the unique catalytic or photoelectro-
chemical properties of the NPs can be used to develop electrochemical and photo-
electrochemical biosensors [63]. For example, the catalytic electroless deposition of
metals on NP-hybrid labels can be used to generate conductive domains on func-
tionalized or patterned surfaces, and the conductivity properties of the systems then
transduce the biosensing processes.
Silver-enhanced labeling is frequently employed in immunochromatographic
assays to improve the sensitivity of detecting pathogens. For example, Liu etal. [65]
56 2 Signal Amplification for Nanobiosensing

Fig. 2.18 (a) IECLE curves of CdTe QD-labeled DNA hybrids immobilized on an NPGL
electrode in PB (pH 7.4) containing 0.1mol/L K2S2O8 and 0.1mol/L KNO3 for different t-DNA
concentrations (1015mol/L): (1) 0, (2) 5.0, (3) 10, (4) 30, (5) 100, (6) 500, (7) 1,000, (8) 2,000,
(9) 4,000, (10) 6,000, (11) 8,000, and (12) 10,000. (b) Magnification of the IECLE curves
indicated in (15). Inset: Relationship between IECL and t-DNA concentration. Reprinted with per-
mission from Hu etal. [61]. 2010, Elsevier

applied a silver enhancement technique for biomolecular signal amplification in a


gold NPbased conductimetric biochip. The response of the silver-enhanced bio-
chip comprised two distinct regions: (1) a subthreshold region, where conduction
occurred due to electron hopping between silver islands and the electrolyte; and (2)
an above-threshold region, where the conduction was due to a direct flow of elec-
trons. These two regions were characterized by different conduction slopes. Results
from fabricated prototypes showed a dynamic range of more than 40dB and with a
detection limit of 240pg/mL.
2.3 Nanoparticle-Amplified Electrochemical Detection 57

2.3.2Detection of Nanoparticle Label with Stripping


Voltammetry

Powerful NP-based electrochemical DNA hybridization assays have been developed


using Au and Ag metal tracers [6669]. Such protocols rely on capturing the gold
[66, 67] or silver [68] NPs to the hybridized target and use ASV to measuring the
metal tracer electrochemically. The probe or target immobilization is accomplished
directly on a carbon or indium-tin oxide (ITO) electrode [70, 71]. Picomolar and sub-
nanomolar levels of the DNA target can be detected. For example, an electrochemical
method is employed for the Au NPbased quantitative detection of the 406-base
human cytomegalovirus DNA sequence (HCMV DNA) [67]. The HCMV DNA is
immobilized on a microwell surface and hybridizes with the complementary oligonu-
cleotide-modified Au NP. The resulting surface-immobilized Au NP double-stranded
assembly is treated with HBr/Br2 for oxidative dissolution of the gold particles. The
solubilized Au3+ ions are then electrochemically reduced and accumulated on the elec-
trode and subsequently determined by ASV using a sandwich-type screen-printed
microband electrode (SPMBE). As the nonlinear mass transport of the ions and the
release of a large number of Au3+ ions upon the dissolution of the particle associate
with a single recognition event, this method enables detection of the HCMV DNA as
low as 51012 M. Further sensitivity enhancements can be obtained by catalytic
enlargement of the gold tracer in connection to NP-promoted precipitation of gold
[66] or silver [72, 73]. Combining the metal particle tags with the electrochemical
stripping analysis paved the way to subpicomolar detection limits.
Another example is presented as an ultrasensitive technique for the electrochem-
ical detection of the mutated BRAF gene associated with papillary thyroid carcino-
mas (PTC) [74]. The biotinylated 30-nucleotide probe DNA is immobilized in a
streptavidin-modified 96-well microtiter plate, and the free active sites of the
streptavidin are blocked by biotinylated BSA. The biotinylated tDNA is then added
and allowed to hybridize with the immobilized probe DNA. Subsequently, strepta-
vidin-labeled gold NPs are added, and a NP enlargement process is performed using
gold ion solution and formaldehyde reductant. The gold NPs are then dissolved in
bromide, and the detection process of DNA hybridization is performed using a
square-wave stripping voltammetry (SWSV) technique. The coefficient of determi-
nation (R2) of the semilog plot of the SWSV response current against the tDNA
concentration (0.521,300aM) is 0.9982. The detection limit is 0.35aM (based on
a signal-to-noise ratio of 3:1). This value is approximately three orders of magni-
tude lower than that obtained using a similar method without the gold amplification
process.
Dequaire etal. [75] demonstrated an electrochemical metal immunoassay based
on stripping voltammetric detection of the colloidal gold label. Such a heterogeneous
sandwich immunoassay involved capture of the gold-tagged secondary antibody, fol-
lowed by acid dissolution and anodic-stripping electrochemical measurement of the
solubilized metal tracer. This protocol could detect the target IgG protein down to
the 3-pM level using a 35-mL sample volume. Such a high sensitivity competed
58 2 Signal Amplification for Nanobiosensing

favorably with colorimetric ELISA assays. This study also indicated that labeling the
antibody with gold NPs had no apparent effect on their interaction with their antigen.
Further enhancements to sensitivity could be achieved by the cyclic accumulation of
gold NPs [76] or the catalytic deposition of metals on core gold NP tags [77, 78].
Recent activities have demonstrated that inorganic nanocrystals offer an elec-
trodiverse population of electrical tags for multiplexed bioanalysis. For example,
Wangs group used encoding NPs (cadmium sulfide, zinc sulfide, copper sulfide,
and lead sulfide) to the multiplexed detection of DNA targets [79], SNPs [80], and
antigens [81]. The multitarget electrical detection capability was coupled to the
amplification feature of electrochemical stripping transduction (to yield fM detec-
tion limits) and with an efficient magnetic separation (to minimize nonspecific
adsorption effects). Each biorecognition event yielded a distinct voltammetric peak,
whose position and size reflected the identity and level of the corresponding target.
Recently, Wangs group designed a QD/aptamer-based ultrasensitive electrochemi-
cal biosensor to detect multiple protein targets [82]. As shown in Fig. 2.19, the
protocol was based on a simple single-step displacement assay involving the coim-
mobilization of several thiolated aptamers, along with binding of the corresponding
QD-tagged proteins on a gold surface (a), addition of the protein sample (b), and
monitoring of the displacement through electrochemical detection of the remaining
nanocrystals (c). Such an electronic transduction of aptamerprotein interactions
was extremely attractive for meeting the low-power, size, and cost requirements of
decentralized diagnostic systems. A detection limit of 20ng/L (0.5pM) was obtained
by this biosensor, which was 34 orders of magnitude lower than those (16.4nM)
obtained with other advanced aptamer biosensors.
With the use of the RCA technique, a cascade signal amplification strategy was
proposed for detection of the protein target at an ultralow concentration [83]. In this
assay, the ultrasensitive detection was achieved by combining the RCA technique
with oligonucleotide-functionalized QDs, multiplex binding of the biotin
streptavidin system, and ASV measurement. As shown in Fig.2.20, the RCA prod-
uct containing tandem-repeat sequences could serve as an excellent template for the
periodic assembly of QDs, which present per protein recognition event to numerous
QD tags for electrochemical readout. Both the RCA and the multiplex binding sys-
tem showed remarkable amplification efficiency, with very little nonspecific adsorp-
tion and low background signal (Fig.2.21). With human vascular endothelial growth
factor (VEGF) as a model protein, the designed strategy could quantitatively detect
protein down to 16 molecules in a 100-mL sample with a linear calibration range
from 1aM to 1pM and was amenable to quantification of the protein target in com-
plex biological matrices. The proposed cascade signal-amplification strategy seems
to be a powerful tool for proteomics research and clinical diagnostics.

2.3.3Nanoparticle-Enhanced Impedance Signal

Due to the electrochemical properties of Au NPs, they have been used as signal
amplifiers in many electrochemical DNA biosensors. Wang etal. [84] demonstrated
2.3 Nanoparticle-Amplified Electrochemical Detection 59

Fig.2.19 Operation of the


aptamer/quantum-dot-based
dual-analyte biosensor,
involving displacement of the
tagged proteins by the target
analytes: (a) mixed
monolayer of thiolated
aptamers on the gold
substrate with the bound
proteinQD conjugates;
(b) sample addition and
displacement of the tagged
proteins; (c) dissolution of
the remaining captured
nanocrystals followed by
their electrochemical-
stripping detection at a coated
glassy carbon electrode.
Reprinted with permission
from Hansen etal. [82].
2006, American Chemical
Society

that Au NPs could amplify the electrochemical impedance and capacitance signals
for the model fluorescein/antifluorescein system. Following the immobilization of
fluorescein onto Au through the formation of a self-assembled monolayer, goat anti-
fluorescein conjugated with 10-nm Au NPs was introduced into the system. This
resulted in an increase of 400nF/cm2 in the capacitance, whereas no change could
be observed for goat antifluorescein without the Au NP conjugate. This allowed the
construction of high-sensitivity electrochemical impedance biosensors at a single
low frequency, where the signal was sensitive to the interfacial Rct.
The impedance detection of CEA, a glycoprotein involved in cell adhesion
produced only during fetal development, was recently reported [85]. The CEA
antibodywas first bound through its surface amino groups to glutathione-modified Au
NPsof151.5-nmdiameterbyamide-bondformationusing N-(3-dimethylaminopropyl)-
N-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfylsuccinimide sodium
60 2 Signal Amplification for Nanobiosensing

Fig. 2.20 Schematic representation of the cascade signal-amplification strategy for protein
detection. Reprinted with permission from Cheng etal. [83]. 2010, American Chemical Society

Fig.2.21 Anodic stripping voltammograms of cadmic cation responding to 1fM of VEGF (a)
with and (b) without RCA. Reprinted with permission from Cheng etal. [83]. 2010, American
Chemical Society
2.3 Nanoparticle-Amplified Electrochemical Detection 61

Fig.2.22 Schematic representation of experimental protocol. (a) Representation of the avidin-


modified electrode and its surface; (b) immobilization of double-labeled IS200 amplicon on the
electrode surface through the formation of the biotinavidin complex; (c1) addition of gold-Ab/
anti-DIG complex; (c2) addition of Protein G. Reprinted with permission from Bonanni etal. [86].
2009, Royal Society of Chemistry

salt (NHSS). The sensing interface was formed by copolymerizing a mixture


of o-aminophenol and the Au-NP-conjugated CEA antibodies. The Rct increased
6.3105 W on the sensing interface with Au NPs, while an increase of only
0.59105W on the sensing interface without Au NPs was observed.
Double-tagged DNA coming from the PCR amplification of a Salmonella spp.
sample has been detected by an electrochemical impedimetric genosensor based on
avidin bulk-modified graphite-epoxy biocomposite (Av-GEB) [86]. As shown in
Fig.2.22, the double-tagging PCR strategy provides the amplicon with both biotin
and digoxigenin (DIG) moieties. The immobilization of the double-tagged DNA is
based on its biotin moiety, while the DIG label is used for signal amplification.
Impedance spectra are recorded to detect the change in interfacial charge-transfer
resistance, experimented by the redox marker ferri-/ferro-cyanide after the avidin
biotin fixation of the sample DNA onto the electrode surface. A further step in the
genosensing strategy is the amplification of impedimetric signal by the use of an
enhancing procedure. The latter is based on the reaction of the DIG moiety belonging
to the amplicon with an anti-DIG antibody from mouse. Two different secondary
enhancing steps, based on gold NPlabeled antimouse IgG or on Protein G, are
performed and compared to improve the assay sensitivity.
A renewable, site-selective immobilization platform of microelectrode array
(MEA) for multiplexed immunoassays has been developed using pencil graphite
62 2 Signal Amplification for Nanobiosensing

Fig.2.23 (a) Schematic conformation of four individually addressable MEA platforms (left) with
graphite particles coated with gold layers by electrodeposition (right); (b) a typical SEM image of
the MEA platform after gold modification; (c) CV characterization of the above MEA platform
measured in the presence of 10mM [Fe(CN)6]3/4 with a scan rate of 0.1V/s. Reprinted with per-
mission from Zhang etal. [87]. 2009, Elsevier

particles coated with gold layers as microelectrodes [87]. As shown in Fig.2.23, the
graphite particles available on the common pencil are utilized to direct the
electrodeposition of gold layers with uniform microstructures, which displays a
well-defined sigmoidal voltammetric response. In the concept-of-proof experi-
ments, the resulting MEA platform is modified with a functionalized monolayer, on
which antihuman IgG antibodies can be stably immobilized in a site-selective way
through binding chemistry to selectively capture human IgG antigens from the sample
media. The subsequent introduction of antihuman IgG antibodies (conjugated with
15-nm electroactive gold NPs), which recognize the captured IgG proteins, results
in a significant decrease in the interfacial electron-transfer resistance. As shown in
Fig.2.24, a highly sensitive electrochemical quantification can be obtained through
gold NPamplified impedance responses. Thus, the MEA sensor can detect human
IgG with a wider linear range (0.05100ng/mL) and a sensitivity over 103 larger
than that of the conventional, bulk gold electrode.

2.3.4Nanoparticle-Enhanced Voltammetric Signal

The Au NP-based amplification of voltammetric signals has also been characterized


[8890]. Li and Hu [91] developed an electrochemical determination method for
2.3 Nanoparticle-Amplified Electrochemical Detection 63

Fig.2.24 (a) Schematic representation of successive fabrication, human IgG detection (5ng/mL),
and regeneration of an MEA platform-based immunosensor. (b) The corresponding Nyquist plots
(Zim vs. Zre) for Faradaic impedance spectra in the presence of 10mM [Fe(CN)6]3/4 at the MEA
platform after (a) electrodeposition of the gold layer, (b) assembly of the 11-MUA monolayer, (c)
covalent immobilization of antihuman IgG antibody, (d) binding of human IgG antigen, and (e)
regeneration treatment. Reprinted with permission from Zhang etal. [87]. 2009, Elsevier

analyzing sequence-specific DNA using ferrocene-capped gold NPstreptavidin


conjugates. Thiolated DNA probes were covalently immobilized on a gold electrode,
with hexanethiol forming a mixed self-assembled monolayer. After hybridization
with tDNA, duplex DNA was formed on the gold surface. Then functional gold NPs
were introduced via the strong interaction effect between biotin and streptavidin.
The electrochemical signal of the ferrocene covering on the gold NPs was obviously
enhanced in cyclic voltammetric and differential pulse voltammetric detection.
64 2 Signal Amplification for Nanobiosensing

Fig.2.25 Cyclic
voltammograms obtained at
(a) an ITO electrode modified
with detection probe
(DP)-conjugated Au NPs,
(b) an Au NP-modified ITO
electrode, and (c) an ITO
electrode sequentially
modified with Au NPs and
DPs, in a 0.1M phosphate
buffer solution (pH 8)
containing 2mM hydrazine
(at a scan rate of 50mV/s)
before and after NaBH4
treatment for 15min in Tris
buffer (pH 9) containing
10mM NaBH4. Reprinted
with permission from Das
and Yang [92]. 2009,
American Chemical Society

Hydrazine can be electroxidated on bare Au NPs, while the electrooxidation is


not observed on DNA-conjugated Au NPs (Fig. 2.25) [92]. Thus, when DNA-
conjugated Au NPs are used as electrocatalytic labels in electrochemical DNA
detection, the anodic current of hydrazine cannot be observed within the potential
window because of the high overpotential caused by the slow electron-transfer
kinetics on DNA-conjugated Au NPs as well as the slow electron tunneling between
the Au NP and the ITO electrode. As NaBH4 treatment can significantly enhance the
electrocatalytic activity of DNA-conjugated AuNPs, it substantially decreases the
overpotential caused by the slow electron-transfer kinetics; hence, the anodic cur-
rent of hydrazine can be measured within the potential window if the distance
between the Au NP and the ITO electrode is not too large. The enhancement with
2.4 Nanoparticles as Carrier for Signal Amplification 65

Fig.2.26 Sensing assembly: (a) top three bilayers of PAH/GOD; (b) three bilayers of PAH/PSS;
and (c) 12 bilayers of PAH/CdTe QDs. Reprinted with permission from Li etal. [93]. 2009,
American Chemical Society

NaBH4 treatment produces a high signal current, and the low intrinsic electrocata-
lytic activity of ITO electrodes results in a low background current. This high sig-
nal-to-background ratio enables a detection limit of 1 fM DNA without target
amplification or enzymatic signal amplification.
Li and coworkers [93] developed a blood glucose sensor based on the multilayer
films of CdTe QDs and glucose oxidase (GOD) by using a layer-by-layer assembly
technique (Fig.2.26). When the composite films were contacted with glucose solu-
tion, the photoluminescence of QDs in the films was quickly quenched because the
enzyme-catalyzed reaction product (H2O2) of GOD and glucose gave rise to the for-
mation of surface defects on QDs. The quenching rate was a function of the concen-
tration of glucose. The linear range and sensitivity for glucose determination could be
adjusted by controlling the layers of QDs and GOD (Fig.2.27). The biosensor could
determine the concentration of blood glucose in real serum samples without sample
pretreatment and exhibited satisfactory reproducibility and accuracy.

2.4Nanoparticles as Carrier for Signal Amplification

2.4.1Gold Nanoparticles as Tracer

Au NPs can also be used as carriers of the signaling molecules for amplification
detection of DNA [94] and protein targets [9597]. For example, Au NPs have
been used as carriers of the signaling antibody anti-CA 15-3-HRP in order to
achieve an amplification analysis of CA 15-3 antigen (Fig.2.28) [97]. In the range
Fig.2.27 UV-vis spectra of
growing PAH/CdTe QD
multilayers (112). Inset
shows a plot of l=567nm
vs. the number of bilayers.
Reprinted with permission
from Li etal. [93]. 2009,
American Chemical Society

Fig.2.28 Schematic (not to scale) of (a) the preparation of the Auanti-CA153-HRP complex
and (b) the sandwich-type ELISA procedure without (IIIa) and with (IIIb) the application of
AuNPs as the signal enhancer. Reprinted with permission from Ambrosi et al. [97]. 2010,
American Chemical Society
2.4 Nanoparticles as Carrier for Signal Amplification 67

between 0 and 60 U/mL, the assay adopting Au NPs as an enhancer results in


higher sensitivityand shorter assay time when compared to classical ELISA proce-
dures. Used an enzyme-labeled Au NP probe, Liu etal. developed a highly sensi-
tive protein detection method [98]. As shown in Fig.2.29, the enzyme-labeled Au
NP probe is prepared by coating Au NP with antibody, single-stranded DNA
(ssDNA), and HRP. Magnetic microparticle (MMP) functionalized with another
antibody is used as the capture probe. The target protein is sandwiched by the
enzyme-labeled Au NP probe and the capture probe through immunoreaction, and
the target immunoreaction event can be sensitively transduced via the enzymati-
cally amplified optical signal. The detection limit of CEA for this strategy is
12ng/L, which is approximately 130 times more sensitive than the conventional
ELISA.
Using a nanoporous gold (NPG) electrode and HRP-labeled secondary anti-
bodyAu NP bioconjugates, a highly sensitive protein detection method has
been described (Fig. 2.30) [99]. The electroactive product of ophenylenedi-
amine (OPD) oxidized with H2O2 catalyzed by HRP is reduced in the Britton
Robinson buffer, the peak current of which is used to determine the concentration
of antigen in the sample. The active surface area of the NPG electrode is larger
than that of a bare flat one, and the presence of Au NPs enhances the immobi-
lized amount of HRP-labeled antibody. The sensitivity of the immunoassay for
the determination of the target protein is increased significantly. A detection
range of 0.011.0ng/mL is obtained for hepatitis B surface antigen (HBs-Ag),
and the detection limit is 2.3pg/mL, which is about 100 times more sensitive
than ELISA.
Figure2.31 shows a sensitive CLIA based on MBs and HRP-labeled anti-AFP-
modified Au NPs for the determination of AFP antigen [100]. A new CL enhancer,
bromophenol blue (BPB), is employed in the CLIA. Due to the magnetic separa-
tion and amplification feature of Au NPs as HRP labels, the CLIA offers a linear
detectionrange from 0.1 to 5.0ng/mL (R=0.9997) and a detection limit of 0.01ng/
mL (3s) for AFP. This detection limit is one order of magnitude lower than that
obtained without using Au NPs, and much lower than that typically achieved by
ELISA.
Using Au NPs to immobilize capture aptamers, Fang etal. [101] proposed an
electrochemiluminescence aptasensor for the sensitive and cost-effective detection
of the target thrombin. As shown in Fig.2.32, capture aptamers labeled with Au
NPs were first immobilized onto the thio-silanized ITO electrode surface. After
catching the target thrombin, signal aptamers tagged with ECL labels were attached
to the assembled electrode surface, forming an Au NP-captureaptamer/thrombin/
ECL-tagged signalaptamer sandwich type. Treating the resulting electrode surface
with tri-n-propylamine (TPA) and applying a swept potential to the electrode, an
ECL response was generated that detected the target protein. The signal-to-dose
curve followed a sandwich format equation, and a detection limit of 10-nM throm-
bin could be estimated by this strategy.
68 2 Signal Amplification for Nanobiosensing

Fig.2.29 Schematic diagrams of the preparation of the (a) enzyme-labeled Au NP probes and (b)
MMP probes. (c) Schematic illustration of the enzyme-labeled Au NP probe-based immunoassay
processes. Reprinted with permission from Liu etal. [98]. 2010, Royal Society of Chemistry

Fig.2.30 Schematic illustration of the stepwise process of the modified electrode. Reprinted with
permission from Ding etal. [99]. 2010, Elsevier
2.4 Nanoparticles as Carrier for Signal Amplification 69

Fig.2.31 The schematic illustration of the determination of AFP based on the sandwich-type chemi-
luminescence immunoassay. Reprinted with permission from Bi etal. [100]. 2009, Wiley

Fig.2.32 The schematic diagram of the fabrication of the ECL aptasensor. Reprinted with permis-
sion from Fang etal. [101]. 2008, Elsevier

Using Au NP-based signal amplification, an electrochemical sensor has been


designed for the highly sensitive detection of Hg2+ ions in aqueous solution [102].
A T-rich mercury-specific oligonucleotide probe (MSO) (5-TTCTTTCTTCCCCTT
GTTTGTT-3) is used to selectively bind with Hg2+. In the presence of Hg2+, the
complex of Hg2+ with thymines yields a stable hairpin structure [103]. Normally, the
MSO probe is directly immobilized on Au electrode surfaces to capture Hg2+ in
aqueous solution (Fig.2.33a), and the electrochemical reduction of surface-confined
Hg2+ provides a readout signal for the quantitative detection of Hg2+. However, the
sensitivity of this Hg2+ sensor can be improved by more than three orders of
magnitude with Au NP-based signal amplification, in which Au NPs are comodified
70 2 Signal Amplification for Nanobiosensing

Fig. 2.33 (a) Directly immobilized MSO probe and (b) Au NP-mediated immobilized MSO
probe. Reprinted with permission from Zhu etal. [102]. 2009, American Chemical Society

with the MSO probe and a linking probe that is complementary to a cDNA probe
immobilized on gold electrodes (Fig.2.33b).
Using Au NPs to load many CdS NP-labeled linker DNA, a significant amplifi-
cation for the detection of thrombin has been obtained [104]. As shown in Fig.2.34,
aptamers are immobilized on the Au NP-modified electrode to construct the sand-
wich-type detection strategy. The concentration of thrombin is monitored based
upon the concentration of dissolved Cd2+ formed in the dissolution of CdS by acid
treatment and quantified by differential pulse voltammetry. Thrombin can be
detected in the linear range of 1.010151.01011 M, with a detection limit of
5.51016 M. A similar strategy has been conducted by Zhangs group based on
bio-barcode techniques [105].
2.4 Nanoparticles as Carrier for Signal Amplification 71

Fig. 2.34 The scheme of the electrochemical determination of thrombin based on aptamer.
Reprinted with permission from Ding etal. [105]. 2010, Elsevier

Taking the advantage of the catalytic reaction of DNAzyme upon its binding to
Pb2+ and the use of DNAAu bio-barcodes to achieve signal enhancement, an electro-
chemical DNAzyme sensor for the sensitive and selective detection of Pb2+ has been
developed [106]. As shown in Fig.2.35, a specific DNAzyme for Pb2+ is immobilized
onto the Au electrode surface via a thiolAu interaction. The DNAzyme hybridizes to
a specially designed complementary substrate strand that has an overhang, which in
turn hybridizes to the DNAAu bio-barcode. A redox mediator, Ru(NH3)63+, which
can bind to the anionic phosphate of DNA through electrostatic interactions, serves as
the electrochemical signal transducer. Upon binding of Pb2+ to the DNAzyme, the
DNAzyme catalyzes the hydrolytic cleavage of the substrate, resulting in the removal
of the substrate strand along with the DNAAu bio-barcode and the bound Ru(NH3)63+
from the Au electrodes surface. The release of Ru(NH3)63+ results in a lower electro-
chemical signal of Ru(NH3)63+ confined on the electrodes surface. The differential
pulse voltammetric signals of Ru(NH3)63+ provide quantitative measurements of the
Pb2+ concentrations, with a linear calibration range from 5nM to 0.1mM. Because
each NP carries a large number of DNA strands that bind to the signal transducer
molecule Ru(NH3)63+, the use of DNAAu bio-barcodes enhances the detection sensi-
tivity, enabling the detection of Pb2+ at a very low level (1nM).
A densely packed gold NP platform combined with a multiple-enzyme-labeled
detection antibodyMB bioconjugate has been used as the basis for preparing an
ultrasensitive electrochemical immunosensor to detect cancer biomarkers in serum
[107]. As shown in Fig.2.36, the sensor is fabricated by alternate layer-by-layer
electrostatic adsorption of a dense glutathione-decorated Au NP and an underlying
layer of cationic poly(diallyldimethyl ammonium chloride) on a pyrolytic graphite
72 2 Signal Amplification for Nanobiosensing

Fig.2.35 The principle of the electrochemical DNAzyme sensor for Pb2+. Reprinted with permission
from Shen etal. [106]. 2008, American Chemical Society

Fig.2.36 Au NP immunosensor with Ab1 attached that has captured an antigen from a sample
after treating with Ab2magnetic bead (MB)HRP providing multiple enzyme labels for each
PSA. The detection step involves immersing the immunosensor into buffer containing mediator,
applying voltage, and injecting H2O2. Reprinted with permission from Mani etal. [107]. 2009,
American Chemical Society
2.4 Nanoparticles as Carrier for Signal Amplification 73

Fig.2.37 Schematic representation of the (a) preparation procedure of GODAu NPs/CNTsAb2


tracer and (b) preparation of immunosensors and sandwich-type electrochemical immunoassay.
Reprinted with permission from Lai etal. [109]. 2009, American Chemical Society

electrode. The capture antibody is attached to the Au NP-modified electrode to


detect PSA in serum. Highly amplified detection is achieved by using multilabeled
bioconjugates made by linked multiple HRP and detection antibodies to carboxy-
lated MBs for signal development. This represents an ultralow mass detection limit
of 5fg of PSA.

2.4.2Carbon Nanotubes as Carrier

CNTs are a good carrier for biomolecules and signal molecules [108, 109]. As shown
in Fig.2.37, the immunosensor array is constructed by coating layer by layer colloi-
dal Prussian blue (PB), gold NPs, and capture antibodies on screen-printed carbon
electrodes. The preparation of GOD-functionalized nanocomposites and the labeling
of antibody are performed by the one-pot assembly of GOD and antibody on gold
NP-attached CNTs (Fig.2.37b). The PB immobilized on the immunosensors sur-
face acts as a mediator to catalyze the reduction of H2O2 produced in the enzymatic
cycle. Both the high-content GOD and CNTs in the tracer amplify the detectable
signal for the sandwich-type immunoassay. The simultaneous multiplexed immuno-
assay method can offer linear ranges of three orders of magnitude, with the detection
limits down to 1.4 and 2.2pg/mL for CEA and a-fetoprotein, respectively.
Using CNT-based labels, Lee etal. developed an amplified nucleic acid detection
[110]. As shown in Fig.2.38, the CNT-based labels were synthesized based on diim-
ideactivated amidation. The DPs were prelabeled with HRP enzymes cross-linked by
glutaraldehyde. The carboxylated SWNTs were covalently functionalized with prela-
beled DPs by reacting with the amine group at the 5-end of DPs with carboxylic acid
74 2 Signal Amplification for Nanobiosensing

Fig.2.38 Diimide-activated amidation for conjugation of SWNTHRPDP. Reprinted with permission


from Lee etal. [110]. 2007, IOP Publishing Ltd.

Fig.2.39 Schematic representation of the signal amplification for sandwich hybridization assay
performed on MBs. The signal-to-single hybridization event ratio of (a) the conventional HRP
label is amplified by (b) multiple HRP and DP-conjugated CNT-based labels. Reprinted with per-
mission from Lee etal. [110]. 2007, IOP Publishing Ltd.

groups on SWNTs in the presence of the cross-linker N-(3-dimethylaminopropyl)-


N-ethylcarbodiimide hydrochloride. The resulting CNT labels significantly enhanced
the nucleic acid assay sensitivity by at least 1,000 times compared to that of conven-
tional labels used in enzyme-linked oligosorbent assay (Fig. 2.39), enabling the
detection of a four-order-wide dynamic range of target concentrations, with a detec-
tion limit of 11012M (601018mol in 60mL).
2.4 Nanoparticles as Carrier for Signal Amplification 75

Fig.2.40 Schematic illustration of the CCP-amplified NP-based fluoroimmunoassay. Reprinted


with permission from Wang and Liu [111]. 2009, Elsevier

2.4.3Silica Nanoparticles as Carrier

Due to the small size, high surface-to-volume ratio, and good biocompatibility,
silica NPs have become another normally used carrier for biomolecule
immobilization. For example, based on silica NPs, Wang and Liu [111] developed a
fluoroimmunoassay for antigen detection and quantification. As shown in Fig.2.40,
after immobilization of the prime antibody on the silica NP surface, the NPs were
used to capture antigen and Cy3-labeled secondary antibody in a sandwich assay
format. The presence of target antigen in solution brought the fluorescent Cy3 mol-
ecules to the NPs surface. The addition of a cationic conjugated polymer (CCP)
further amplified the fluorescence signal of the dye and improved the assay sensitiv-
ity. Due to the pink color of the Cy3 molecules, the assay allowed a detection limit
of 50ng/mL of IgG by naked-eye detection.
Mesoporous silica nanoparticles (MSN) have also been used as molecule carrier
to improve detection sensitivity. For example, Yang etal. [112] designed an MSN-
based label by loading MSN with mediator thionine (TH), enzyme HRP, and
secondary antihuman IgG antibody for IgG detection (Fig.2.41). The sensitivity of
the sandwich-type immunosensor could greatly improved, leading to a detection
range of 0.0110ng/mL of human IgG.
The electrochemiluminescence of doped silica nanoparticles (DSNPs), prepared
by a reverse microemulsion method that leads to the covalent incorporation of the
Ru(bpy)32+, has been investigated in acetonitrile and aqueous buffers [113].
76 2 Signal Amplification for Nanobiosensing

Fig. 2.41 Schematic representation of the preparation of the (a) MSNTHHRPAb2 and (b)
immunosensor. Reprinted with permission from Yang etal. [112]. 2010, Elsevier

The ECL intensity obtained by such functionalized substrates in aqueous media,


using tripropylamine (TPrA) as coreactant, is surprisingly increased with respect to
direct electrochemical oxidation because of the ability of oxidized TPrA to diffuse
within the DSNPs structure and reach a higher number of emitting units with
respect to direct electron tunneling. A more than 1,000-fold increase in the ECL
signal can be received by the chemically and electrochemically stable DSNP com-
pared to that of a single dye, suggesting that this nanostructure as luminescent labels
represents a very promising system for ultrasensitive bioanalysis.
Using CdTe QD-functionalized silica nanosphere labels, Chen et al. [114] designed
an electrochemical immunsensor for protein detection. As shown in Fig.2.42, AFP
antibody was covalently binded to CdTe QDs on the surface of silica NPs. Enhanced
sensitivity can be achieved based on an increase in CdTe QDs loading per sandwiched
immunoreaction (Fig.2.43).
2.4 Nanoparticles as Carrier for Signal Amplification 77

Fig. 2.42 Preparation process of Si/QD/Ab2. Inset: TEM image of the resultant Si/QD/Ab2.
Reprinted with permission from Chen etal. [114]. 2009, Royal Society of Chemistry

Fig. 2.43 Sandwiched immunoassay process using Si/QD/Ab2 as labels. Inset: TEM image of
MB/Ab1AgSi/QD/Ab2. Reprinted with permission from Chen etal. [114]. 2009, Royal Society
of Chemistry

2.4.4Other Materials as Carrier

A NP label capable of amplifying the electrochemical signal of DNA hybridization


has been fabricated by functionalizing poly(styrene-co-acrylic acid) microbeads
with CdTe QDs [115]. As shown in Fig.2.44, CdTe-tagged polybeads are prepared
by a layer-by-layer self-assembly of the CdTe QDs and polyelectrolyte on the poly-
beads. The CdTe-tagged polybeads are then attached to DNA probes specific to
breast cancer by streptavidinbiotin binding to construct a DNA biosensor. The
detection of the DNA hybridization process is achieved by the square-wave voltam-
metry of Cd2+ after the dissolution of the CdTe tags with HNO3. The efficient car-
rier-bead amplification platform, coupled with the highly sensitive stripping
voltammetric measurement, gives rise to a detection limit of 0.52 fmol/L and a
dynamic range spanning five orders of magnitude.
Figure2.45 shows a novel NP-based electrochemical immunoassay of CA125
coupling with a microfluidic strategy [116]. To construct the immunoassay,
78 2 Signal Amplification for Nanobiosensing

Fig.2.44 Schematic diagram of the assembly process for the preparation of streptavidin/CdTe-
tagged polybeads. Reprinted with permission from Dong etal. [115]. 2010, Wiley

Fig.2.45 Construction of the immunosensing probe and recognition element and measurement
protocol of the NP-based electrochemical immunoassay with a sandwich-type format. Reprinted
with permission from Tang etal. [116]. 2010, American Chemical Society

t hioninehorseradish peroxidase conjugation (TH-HRP) is initially doped into


nanosilica particles using the reverse-micelle method, and then HRP-labeled anti-
CA125 antibodies (HRP-anti-CA125) are bound onto the surface of the synthe-
sized NPs. The sandwich-type immunoassay format is used for the online formation
of the immunocomplex in an incubation cell and captured in the detection cell with
an external magnet. The electrochemical signal is derived from the carried HRP
toward the reduction of H2O2 using the doped TH as electron mediator. A wide
working range of 0.1450U/mL, with a detection limit of 0.1U/mL CA125, can
be obtained. The assay has been evaluated for clinical serum samples and has
yielded results that are in excellent accordance with the results obtained from the
standard ELISA method.
References 79

2.5Conclusions

Nanotechnology offers unique opportunities for designing ultrasensitive bioassays.


The studies demonstrate the broad potential of bioconjugated NPs for the amplified
transduction of biomolecular recognition events. Given the enormous amplification
afforded by NP tracers, such nanomaterials provide the basis for ultrasensitive
assays of proteins and nucleic acids. The remarkable sensitivity of the new nanoma-
terial-based sensing protocols opens up the possibility of detecting disease markers,
biothreat agents, or infectious agents that cannot be measured by conventional
methods. Due to the diverse properties of different nanomaterials, utilizing two or
more types of nanomaterials can enhance the good qualities as well as offset the
insufficiency of each individual nanomaterial, which can produce better results than
those obtained using only one type of nanomaterial. The use of NP tags to detect
proteins is still in its infancy, but the lessons learned in ultrasensitive DNA detection
should provide useful starting points. The successful realization of the new signal-
amplification strategies requires proper attention to nonspecific adsorption issues
that commonly control the detectability of bioaffinity assays. Proper washing and
surface blocking steps should thus be employed to avoid the amplification of back-
ground signals (associated with nonspecific adsorption of the nanoparticle amplifi-
ers). A wide range of newly introduced nanomaterials is expected to further expand
the realm of nanomaterial-based biosensors.

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Chapter 3
Nanostructured Mimic Enzymes
for Biocatalysis and Biosensing

3.1Introduction

3.1.1Need for Nanostructured Mimic Enzymes

Accurate, rapid, inexpensive, and selective analysis is required today for use in
clinical diagnostics and the food industry. The majority of known electrochemical
biosensors are based on immobilized specific biomolecules, such as proteins,
enzymes, nuclear acids, antibodies, and antigens on the modified electrodes [18].
These resulting biomolecule-based devices usually show high sensitivity and spec-
ificity due to the high loading of enzymes on the nanoparticles [912]. However,
their stability is limited due to easy denaturation [13] and leakage of biomolecules
during their storage and immobilization procedure. Furthermore, the preparation
and purification of biomolecules are usually time-consuming and expensive [14].
Therefore, the syntheses of artificial biomolecules with highly catalytic properties
and a wide range of practical applications are becoming a significant field for dif-
ferent purposes.
Artificial enzyme mimetics have attracted considerable interest because they can
overcome the disadvantages of the natural enzyme [1518]. Up to now, some coor-
dination compounds have been prepared as artificial enzyme mimetics [1923].
Through invitro selection, nonnatural ribozymes, deoxyribozymes (catalytic DNA
molecules) [2427], and many other enzyme mimetics [2836] have been devel-
oped, such as cytochrome P450 mimetics [3739], serine proteases mimetics [40],
dioxygenase mimetics [41, 42], phosphodiesterase mimetics [43], ligase mimetics
[44], nuclease mimetics [45], and methanogenesis mimetics [46]. Among these, a
lot of research has focused on peroxidase mimetics, including hemin [47, 48],
hematin [49], hemoglobin [50], cyclodextrin [51], and porphyrin [52, 53], which
have been used for hydrogen peroxide (H2O2) and ascorbic acid detection [54].
Due to their unique optical, electric, chemical, and mechanical properties,
nanostructured materials have been widely applied in biocatalysis and biosensing

H. Ju et al., NanoBiosensing: Principles, Development and Application, 85


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0_3, Springer Science+Business Media, LLC 2011
86 3 Nanostructured Mimic Enzymes for Biocatalysis and Biosensing

assays [5559]. Nanomaterials mainly used as supports for the immobilization of


biomolecules can amplify the detection signals of biomolecules [6062]. In addi-
tion, nanoparticles, especially metal and semiconductor nanoparticles, can be used
directly as electroactive labels for the electrochemical amplification detection of
DNA and proteins [6368]. Nanostructured materials are generally considered to be
biologically and chemically inert, while the catalytic behaviors of some carbon-
based nanomaterials and metal nanoparticles have been used to accelerate the elec-
tron transfer of amperometric biosensors [6972].

3.1.2Developments in Nanostructured Mimic Enzymes

Peroxidase mimetics, including hematin [49], porphyrin [52], and cyclodextrin [42],
were already applied in different fields. Peroxidases are responsible in nature for the
reduction of H2O2. Some iron-based compounds, such as Prussian blue (PB)
[7380], have been denoted as electrocatalysts in H2O2 reduction due to the expected
conversion of Fe3+/Fe2+, which occurs in the reaction centers of a number of peroxi-
dase enzymes [8183]. PB has good stability and highly catalytic properties. Hence,
it has a wide range of practical applications.
Although Fe3O4 nanoparticles had been used for the cathodic determination of
H2O2 [84], their peroxidase activity was always ignored until the release of a recent
report in which the intrinsic peroxidase-like activity of Fe3O4 nanoparticles was
observed to catalyze the breakdown of H2O2 [85]. Afterward, Fe3O4 nanoparticles
were presented as a mimic peroxidase for the detection of H2O2 and glucose [86].
Because FeS shows typical MichelisMenten kinetics and a good affinity to
both H2O2 and 3,3,5,5-tetramethyl benzidine (TMB), it is also used as a mimic
enzyme for the development of biocatalysts and amperometric biosensors. The
H2O2 sensor based on FeS shows better stability than that based on horseradish
peroxidase when they are exposed to solutions with different pHs and tempera-
tures. These excellent characters make the nanostructured FeS powerful for a
wide range of potential applications as an artificial peroxidase in biosensors and
biotechnology [87].
The new nanoenzyme model with a glutathione peroxidase-like active site has
been constructed on the polystyrene nanoparticle via microemulsion polymeriza-
tion. The polystyrene nanoparticle exhibits an obvious enhancement in catalytic
activity and can be developed as an excellent model for combining most of the
catalytic factors of the enzyme into one scaffold [88].
Natural enzymes are macromolecules, while many enzyme models and mimics
are small molecules. Breslow etal. have done extensive studies on reactions related
to those catalyzed by enzymes that used pyridoxal phosphate and pyridoxamine
phosphate, the central coenzymes for amino acid metabolism [89106]. As a typi-
cal example, they catalyzed the transamination between an amino acid and a keto
acid, which is the most important form of nitrogen transfer in diverse biological
systems.
3.2 Nanostructure Used in Artificial Mimic Enzymes 87

3.2Nanostructure Used in Artificial Mimic Enzymes

3.2.1K3Fe(CN)6

PB can be electrochemically reduced to Prussian white (PW), which is capable of


catalyzing the reduction of H2O2 at low potentials. The mechanism is as follows:

Fe 4 [Fe(CN)6 ]3 (PB) + 4e + 4K + Fe 4 K 4 [Fe(CN)6 ]3 (PW) (3.1)


Fe 4 K 4 [Fe(CN)6 ]3 (PW) + 2H 2 O2 Fe 4 [Fe(CN)6 ]3 (PB) + 4OH + 4K + (3.2)


PB has been intensively investigated in the form of thin polycrystalline


electrodeposited films. The use of nanoparticles can improve the analytical perfor-
mance for H2O2 detection; nanosized particles have unique physical and chemical
properties, often showing very interesting peculiarities unmatched by their bulk
counterpart. The large surface-to-volume ratio and the increased surface activity of
nanoparticles, when compared to those of bulk materials, enable their use in cataly-
sis and sensing [78].
The possibility for the selective detection of H2O2 by its reduction in the presence
of oxygen on PB-modified electrodes was first demonstrated by Karyakin [107].
Thereafter several articles on the application of PB as an H2O2 transducer appeared
[108110]. The approach, however, was the oxidation of H2O2 at high anodic poten-
tials rather than its reduction [110]. The electrocatalyic reduction of H2O2 by PB has
been thoroughly investigated [108, 111, 112]. The comparative activities of the
inorganic polycrystal in oxygen and H2O2 reduction are highly dependent on the PB
structure [108, 113]. Optimizing the deposition procedure for PB, a selective elec-
trocatalyst for H2O2 reduction in the presence of oxygen, which is able to operate in
a wide potential range, has been synthesized [108]. At an optimal potential for sen-
sor and biosensor applications (0.0V, Ag/AgCl), the current of H2O2 reduction was
several 100 times higher than that of oxygen reduction.
The stability of the PB-based H2O2 transducer is a crucial point commonly raised
against its practical applications. Indeed, PB (the redox state of PB at 0.0V) is ther-
modynamically unstable on electrode surfaces. In addition, hydroxyl ions, being the
products of H2O2 reduction in neutral media [111], are able to solubilize the inor-
ganic polycrystal. However, due to continuous efforts to improve the crystalline
structure of the deposited PB, its additional excellent posttreatment operational
stability, which even exceeds the stability of the known H2O2 transducers, has been
achieved [77, 114]. The kinetics of H2O2 reduction catalyzed by PB has been inves-
tigated [111, 115]. In neutral media, the reaction scheme of H2O2 reduction has been
found to be as follows:

H 2 O 2 + 2e 2OH
k
cat
(3.3)

88 3 Nanostructured Mimic Enzymes for Biocatalysis and Biosensing

The electrochemical rate constants for H2O2 reduction have been found to be
dependent on the amount of PB deposited on the electrode, confirming that the H2O2
penetrates the films, and inner layers of the polycrystal take part in catalysis. For
46 nmol/cm2 of PB, the electrochemical rate constant exceeds 0.01 cm/s [115],
which is higher than that for all the other known H2O2 transducers. For comparison,
the electrochemical rate constant for H2O2 oxidation on platinum in neutral media is
less than 7106cm/s [116]. The activity of Pt in H2O2 reduction is even lower.
Due to both high activity and high selectivity, PB was denoted as an artificial
peroxidase [77]. Using PB as a transducer for H2O2, it was possible to achieve a
sensitivity of 0.6/AM/cm2 in flow-injection mode [75, 77], which takes into account
the dispersion coefficient [117] corresponding to the sensitivity of 1/AM/cm2, either
in batch regime or under continuous flow.
This is pertinent to the chemical synthesis of a PB-based H2O2 transducer, which
can be used in both screen-printed and carbon paste electrodes. Both the commer-
cially available and commonly precipitated PB showed a minor electrocatalytic
activity in H2O2 reduction [118]. A successful chemical synthesis of the electrocata-
lyst has recently been carried out by open-circuit deposition onto graphite powder
from a ferricyanide solution [119]. The resulting carbon pastePB transducer for
H2O2 exhibited high operational stability in neutral and weakly basic media up to
pH 9. The sensitivity of the PB-based carbon paste sensors for H2O2 was rather
satisfactory, but 20200 times lower than the sensitivity of the PB-modified glassy
carbon electrodes.
There were several attempts to use other metal hexacyanoferrates as H2O2 trans-
ducers. Cupric hexacyanoferrate was integrated in a biosensor, but the resulting
sensitivity was three orders of magnitude lower than that of a similar biosensor
based on PB [120]. Moreover, cupric hexacyanoferrate has to be poised to high
cathodic overvoltages (>0.7 V vs. its redox potential) to achieve a considerable
rate of H2O2 reduction. These observations indicated that cupric hexacyanoferrate
was a poorer electrocatalyst than PB.
Another transition metal-based transducer for H2O2 was made by cycling a tita-
nium dioxide electrode in ferricyanide solution [121]. Although the authors did not
claim the synthesis of a new transition metal hexacyanoferrate, the cyclic voltam-
mograms were similar to PB. Unfortunately, the titanium dioxide electrode modi-
fied with hexacyanoferrate did not show high activity to H2O2 reduction: The
sensitivity calculated from the data in the paper was 0.8 mA/M/cm2. Two metal
hexacyanoferrates with extremely high sensitivity to H2O2 (>1/AM/cm2) were
recently reported: Co-hexacyanoferrate [112] and Cr-hexacyanoferrate [29].
There was also doubt concerning the high catalytic activity of cohexacyanofer-
rate. Lin and Jan reported two redox potentials for the electroactivity of cobalt
hexacyanoferrate: at 449 and 595mV [112]. However, after the addition of H2O2,
electrocatalysis was observed at a shoulder, with a potential of about 0.2V [112],
which has never been reported for Co-hexacyanoferrate, but was similar to one
attributed to PB with the precision of the reference electrode used. It should be
noted that the deposition of Co-hexacyanoferrate [112] included 6h of cycling in
solution containing ferricyanide, and, thus, the resulting film obviously contained
3.2 Nanostructure Used in Artificial Mimic Enzymes 89

some amount of PB being deposited even from a single ferricyanide solution [122].
There was no experimental evidence showing that the high catalytic activity in H2O2
reduction was peculiar to Co-hexacyanoferrate rather than to PB. This study con-
firmed the unique catalytic properties of PB. As a conclusion from this section, PB
has to be considered the most advantageous H2O2 transducer over all other existing
systems.
Overall, a PB-based electrocatalyst is a truly good choice for the development of
oxidase-based biosensors. PB-modified electrodes are (1) selective electrocatalysts
for electrochemical reduction of H2O2 in the presence of oxygen, which is not a
particular property of platinum; (2) more stable and active than peroxidase-modified
electrodes; and (3) less expensive than both platinum and peroxidase electrodes.

3.2.2Fe3O4

Nanoparticles containing magnetic materials, such as magnetite (Fe3O4), are par-


ticularly useful for imaging and separation techniques. Iron oxides possess distinct
roles in chemical, medical, and industrial fields, and there are approximately six
different forms, such as a-Fe2O3 (hematite), g-Fe2O3 (maghemite), b-Fe2O3, e-Fe2O3,
FeO, and Fe3O4 (magnetite) [123]. Fe3O4, with the valence state of Fe(II) and Fe(III),
is identified as the catalyst for H2O2 detection with limited interference. The reduc-
tion of H2O2 follows a two-electron, two-proton reduction step to give the product
of H2O, and the reductive valence state of Fe(II) on Fe3O4 is oxidized to Fe(III). At
this moment, the oxidative valence state of Fe(III) is regenerated back to the origi-
nal state of Fe(II) at the applied potential of 0.2V, and the response is simultane-
ously recorded. The above electron-transfer process is used for the H2O2 chemical
sensor by an Fe3O4-modified electrode. Based on the mixed-valence characteristic,
Fe3O4 is also called a binary iron oxide. The mixed-valence compound is defined by
a polynuclear cluster with two or more metal centers linked by a bridging ligand
[124], and the electrons are delocalized in the whole cluster. This cluster is utilized
as an artificial peroxidase to catalyze the reduction of H2O2, such as metal hexacy-
anoferrates [125128]. The reductive mode of H2O2 quantitative analysis probably
is one of the most important advantages for these inorganic compounds to be used
in oxidase-based biomedical sensors [129, 130].
A simple method to develop the interference-free electrochemical H2O2 sensor
was described [86]. The applied potential was around 0V, and the Fe3O4/chitosan
modified glassy carbon rotating disk electrode has been used to reach the unique
features of electrocatalytic reduction and interference elimination. The H2O2 sensor
has been demonstrated at cathodic potential, and the chitosan-coated thin film pro-
vided several characteristic enhancements in both biological and environmental
situations. This Fe3O4-based H2O2 sensor has shown the advantages of low applied
potential, low background current, rapid response, limited interference, and long-
term stability. In optimum pH, the linearity of the Fe3O4/chitosanmodified
electrode for H2O2 was up to 4.0mM, with a sensitivity of 16.8mA/mM (r=0.999).
90 3 Nanostructured Mimic Enzymes for Biocatalysis and Biosensing

Fig. 3.1 Fe3O4-catalyzed oxidation of various peroxidase substrates in the presence of H2O2 to
produce different color reactions. Reprinted with permission from Gao etal. [85]. 2007, Nature

A detection limit of 7.6mM was also obtained (S/N=3). The precision value from
RSD was 2.2% by 20 successive measurements of 0.5mM H2O2. A typical response
time between 10 and 90% of the steady-state response was 5.2s at the injection of
0.5mM H2O2.
Fe3O4 possesses an intrinsic enzyme mimetic activity similar to that found in
natural peroxidases. Peroxidase activity has a wide range of practical applications.
For example, the ability to catalyze the oxidation of organic substrates to reduce
their toxicity and/or to produce a color change was frequently used in wastewater
treatment or as a detection tool. Fe3+/Fe2+ ions in solution (Fentons reagent) are
known to catalyze the breakdown of H2O2. A number of peroxidase enzymes
(including the heme-containing enzyme HRP) and enzyme mimetics contain Fe2+ or
Fe3+ in their reaction centers. However, the fact that Fe3O4 nanoparticles have been
conjugated to HRP to introduce peroxidase activity in a number of applications,
including commercially available magnetic enzyme-linked immunosorbent assay
(ELISA) kits, demonstrates that the presence of this activity has so far been ignored.
Taking HRP as a comparison, the peroxidase-like activity of Fe3O4 was character-
ized by Gao etal. [85].
Fe3O4 catalyzed the reaction of TMB in the presence of H2O2 to produce a blue-
colored reaction (Fig.3.1 left), with a maximum absorbance at 652nm. Like enzy-
matic peroxidase activity, such as that observed for the commonly used enzyme
HRP, this color reaction was quenched by adding H2SO4. To further characterize the
peroxidase-like activity of Fe3O4, other peroxidase substrates, including di-azo-
aminobenzene (DAB) and o-phenylenediamine (OPD), were used in place of TMB.
The middle photo in Fig.3.1 shows that the Fe3O4 not only catalyzed the oxidation
of TMB, producing a blue color, but also catalyzed DAB to give a brown color and
OPD to give an orange color. These results indicated that the Fe3O4 had peroxidase-
like activity toward typical peroxidase substrates (Fig.3.1, far right).
To investigate the mechanism of the peroxidase activity of Fe3O4, the apparent
steady-state kinetic parameters for the reaction were determined. Within a suitable
3.2 Nanostructure Used in Artificial Mimic Enzymes 91

Fig. 3.2 Immunoassays based on the peroxidase activity of Fe3O4 magnetic nanoparticles. (a)
immunosensor preparation, and (b) immunoassay process. Reprinted with permission from
Gao etal. [85]. 2007, Nature

range of H2O2 concentrations, typical MichaelisMenten curves were observed for


both Fe3O4 and HRP. The apparent Km value of the Fe3O4 with H2O2 as substrate was
significantly higher than that of HRP, consistent with the observation that a higher
concentration of H2O2 was required to observe maximal activity for the Fe3O4. The
apparent Km value of the Fe3O4 with TMB as substrate was about 4 times lower than
that of HRP, suggesting that the Fe3O4 has a higher affinity for TMB than HRP. At
the same molar concentration, the activity of Fe3O4 was 40 times higher than HRP.
This may be due to the fact that an HRP molecule has only one iron ion, in contrast
to the surface of an Fe3O4. The presence of ferrous and ferric ions in the nanoparti-
cles is likely to be the key to their catalysis.
Because Fe3O4 is an inorganic nanomaterial, it is expected to be more stable than
the enzyme HRP. The Fe3O4 was indeed found to remain stable over a wide range of
pH, from 1 to 12, and temperatures from 4 to 90C. In contrast, the enzyme HRP did
not show any activity after treatment at a pH lower than 5 or at temperatures greater
than 40C. The robustness of Fe3O4 makes it suitable for a broad range of applica-
tions in the biomedicine and environmental chemistry fields.
Two immunoassays using the intrinsic dual functionality of the Fe3O4 as a peroxidase
and magnetic separator were developed [85]. Fe3O4 was modified with different
compounds, including SiO2, 3-aminopropyltriethoxy silane (APTES), polyethylene
glycol (PEG), or dextran, to make them biocompatible. The enzyme activity of the
modified Fe3O4 decreased. A number of factors, such as the size and density of
packing of the modifying groups and the thickness of the coating layer, may influ-
ence the extent to which surface-modifying groups shield the surface from the sub-
strate and hence affect activity. For a given type of modifying group, variation in the
modification protocol can produce coats of different thickness.
In the first immunoassay format (Fig.3.2a), protein A was immobilized on Fe3O4
and used in place of an enzyme-conjugated secondary antibody. After several washes,
92 3 Nanostructured Mimic Enzymes for Biocatalysis and Biosensing

nonspecific binding was removed. The Fe3O4 with immobilized protein A and the
substrate TMB were then added, so that protein A bound to the primary anti-pre
antibody and Fe3O4 catalyzed a color reaction in the presence of H2O2. The reaction
was measured using an ELISA reader at 652nm. The results demonstrated that the
intrinsic peroxidase-like activity of the Fe3O4 can still be detected after surface modi-
fication. On the other hand, the magnetic properties of the Fe3O4 could potentially be
used for recovery or recycling of the Fe3O4. As shown in Fig.3.2b, the two intrinsic
properties of Fe3O4, namely, magnetism and peroxidase activity, were combined in a
novel capturedetection immunoassay format. First, an antibody to cardiac troponin
I (TnI), a well-known biomarker for myocardial infarction, was immobilized on the
Fe3O4. The antibody-labeled Fe3O4 was then mixed with serum, allowing capture of
the target TnI in the sample. The TnI captured by Fe3O4 was easily separated from
the sample using a magnet. After washing off contaminants, the Fe3O4 with target
bound was transferred onto a plate coated with another anti-TnI antibody. After
washing off nonbound Fe3O4, the substrate TMB was added in the presence of H2O2
and the bound Fe3O4 catalyzed a color reaction. These assays demonstrated the versa-
tility and power of Fe3O4 as both a capture agent and a detection tool, due to its intrinsic
dual functionality. This can be compared with traditional magnetic ELISA, in which
Fe3O4 captures targets and an additional step is required to introduce a secondary
antibody carrying, for example, HRP to allow detection. This method was easier,
faster, and more economical and provided greater sensitivity. Furthermore, the intrin-
sic peroxidase-like activity of Fe3O4 should be taken into account when it is used in
standard magnetic ELISA: The conjugation of the secondary antibody to HRP for
detection is likely to lead to high background. In fact, it was this observation that
discovered the peroxidase-like activity of the Fe3O4.
More importantly, a sensitive and selective method for glucose detection was
developed using glucose oxidase (GOx) and Fe3O4 [86]. Such detection platforms
for H2O2 and glucose not only confirmed the intrinsic peroxidase-like activity of
Fe3O4, but also showed great potential applications in varieties of simple, robust,
and easy-to-make analytical approaches in the future. With a combination of the
catalytic reaction of glucose with GOx and the Fe3O4 catalytic reaction ((3.4) and
(3.5)), the developed method exhibited a sensitive and selective response toward
glucose detection.

Fe3 O4
H 2 O 2 + ABTS 2H 2 O + oxidized ABTS (3.4)

O 2 + glucose
GOx
H 2 O 2 + gluconic acid (3.5)

The Fe3O4 was prepared via a coprecipitation method. The as-prepared Fe3O4
was then used to catalyze the oxidation of a peroxidase substrate 2,2-azino-bis
(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) by H2O2. The
oxidized colored product (3.4) provided a colorimetric detection of H2O2. A linear
range from 5106 to 1104mol/L, with a detection limit of 3106mol/L H2O2,
could be detected.
3.2 Nanostructure Used in Artificial Mimic Enzymes 93

Fig.3.3 Typical absorption


profiles for glucose detection
with the colorimetric method
using GOx and the
as-prepared Fe3O4 (black line,
500mM glucose; red line,
buffer; green line, 5mM
maltose; blue line, 5mM
fructose; and cyan line, 5mM
lactose). Reprinted with
permission from Wei and
Wang [86]. 2008,
American Chemical Society

When the catalytic reaction shown in (3.4) is coupled with the glucose catalytic
reaction by GOx (3.5), the observation of a colorimetric glucose detection occurred
as a result. A typical absorption profile for glucose detection using the colorimetric
method is shown in Fig.3.3. Because GOx could be denatured in pH 4.0 buffer solu-
tion, the glucose detection was performed in two separate steps. When the reaction
of (3.5) was finished in a pH 7.0 buffer solution, the H2O2 produced by the glucose
oxidation with GOx was detected using the as-prepared Fe3O4 (3.4). A typical glu-
cose concentration response curve as low as 3105mol/L glucose could be detected,
with a linear range from 5105 to 1103mol/L. In order to test if the detection of
glucose is specific, control experiments were taken using fructose, lactose, and
maltose. As high as 5mM control samples were investigated, and no detectable sig-
nals were obtained, showing high selectivity toward glucose detection.

3.2.3FeS

Because of its high activity and selectivity toward the reduction of H2O2, FeS can
also be considered an artificial enzyme peroxidase and has been used in the con-
struction of electrochemical biosensors [87]. FeS possesses specific electron-
transfer ability [12] and good adsorption and, more importantly, has a lower band gap
than FeO [13], which is in favor of facilitating the electron transfer. A nanostructure
of sheet-like FeS that was prepared by a simple micelles-assisted synthetic method
was designed as a novel mimic peroxidase. This nanostructured FeS could provide
the enzymatic active center of Fe2+/Fe3+ for electron transfer. Such a nanostructure
had a large specific surface area and high peroxidase-like activity, allowing it to be
used as a mimic enzyme for the development of biocatalysts and amperometric bio-
sensors. The synthesized sheet-like FeS nanostructure showed attractive performance
of intrinsic peroxidase-like activity, which was confirmed by evaluating the ability to
catalyze the oxidation of organic substrates to produce a color change and developing
94 3 Nanostructured Mimic Enzymes for Biocatalysis and Biosensing

Fig.3.4 The electrocatalytic activity of sheet-like FeS nanostructure (red line) and HRP (black
line) to H2O2 at pH 7.0 and 40C after exposure to different pHs (a) and temperatures (b) for 2h.
Reprinted with permission from Dai etal. [87]. 2009, Wiley

a fast, sensitive, and low-cost electrochemical sensor for H2O2. It is well known that
the peroxidase can catalyze the oxidation of a peroxidase substrate to produce a color
change and the color reaction can generally be quenched by H2SO4. The sheet-like
FeS nanostructure showed typical MichelisMenten kinetics and a good affinity to
both H2O2 and TMB. The Kmapp for the sheet-like FeS with TMB was 0.13mM, while
the value of Kmapp for HRP with TMB was 0.4mM. The latter coincided with that
reported previously, and the former was slightly larger than that of Fe3O4 nanoparti-
cles. The Kmapp value of the sheet-like FeS nanostructure suggested that it had a higher
affinity to TMB than to HRP. This may be due to the fact that an HRP molecule has
only one iron ion, in contrast to the surface of a sheet-like FeS nanostructure. On the
other hand, the Kmapp value of the FeS with H2O2 was 7.2mM, slightly higher than
that of 3.7mM for HRP in solution. But the Kmapp value was much lower than that of
154mM for Fe3O4 nanoparticles, indicating a better affinity of the sheet-like FeS
nanostructure to H2O2 than Fe3O4 nanoparticles.
At pH 7.0, the constructed amperometric sensor showed a linear range for the
detection of H2O2 from 0.5 to 150 mM, with a correlation coefficient of 0.9998,
without the aid of any electron-transfer mediator. Also, this H2O2 sensor had a more
sensitive response than those based on spherical FeS nanoparticles.
The FeS nanostructure is an inorganic nanomaterial and is expected to be more
stable than natural peroxidases. To examine its stability, both HRP and the sheet-
like FeS nanostructuremodified electrodes were exposed to different temperatures,
ranging from 10 to 70C, and solutions, with pH ranging from 2.0 to 10.0 for 2h,
and their relative activities toward the electrocatalytic reduction of H2O2 were then
measured at pH 7.0 and 40C. The sheet-like FeS nanostructure showed much better
stability than HRP in the measured temperature and pH ranges (Fig. 3.4). After
exposure to pH 2.0 and 10.0 solutions for 2h, the nanostructure could maintain 40
and 56% of the peroxidase activity, respectively, while the HRP denatured
completely at pH lower than 3.0 and maintained only 30% of its activity at pH 10.0.
3.2 Nanostructure Used in Artificial Mimic Enzymes 95

The effect of temperature on the stability showed similar phenomena. In the


temperature range of 2060C, the nanostructure was almost stable, but the HRP
showed a very narrow temperature range for maintaining its activity, and when it
was exposed to temperatures higher than 70C, the complete denaturation was
observed. The good stability, ease of production, and special properties made the
sheet-like nanostructured FeS powerful for a wide range of potential applications as
an artificial peroxidase in biosensors and biotechnology.
This FeS nanostructure can be considered a mimic peroxidase. Its peroxidase
activity has been demonstrated by the following facts: (1) It could catalyze the oxi-
dation reaction of TMB by H2O2; (2) the FeS nanostructure-catalyzed reaction
showed behavior similar to the general peroxidase- and nanoparticles-catalyzed
reactions; (3) it showed electrocatalytic activity toward the reduction of H2O2 to
produce a fast, sensitive, and low-cost H2O2 sensor; (4) the catalytic behaviors
offered typical MichelisMenten kinetics and a good affinity to both H2O2 and
TMB; (5) the H2O2 sensor based on the sheet-like FeS had a more sensitive response
than those based on spherical FeS; (6) the electrocatalytic activity was dependent on
pH and temperature; and (7) the sheet-like FeS nanostructure had better stability
than HRP when exposed to solutions with different pHs and temperatures, and the
resulting sensor for H2O2 had good stability and reproducibility. Overall, the novel
sheet-like FeS nanostructure can be used as an artificial peroxidase for the poten-
tial development of amperometric transducers and biocatalysts.

3.2.4Polystyrene

A new nanoenzyme model with a glutathione peroxidase-like active site was con-
structed on a polystyrene nanoparticle (PN1) via microemulsion polymerization
[88]. In this model system, two functional monomers were designed: One was a
tellurium-containing compound that was introduced on the surface of the nanopar-
ticle and acted as a catalytic center; the other was an arginine-containing compound
designed as a binding site for the complexation of the carboxyl group of substrate
3-carboxy-4-nitrobenzenethiol (ArSH, 1). As a new glutathione peroxidase (GPx)
mimic, it demonstrated excellent catalytic activity and substrate specificity. In the
ArSH assay system, it was at least 316,000-fold more efficient than diphenyl disele-
nide (PhSeSePh) for the reduction of cumene hydroperoxide (CUOOH) by ArSH.
To further promote the catalytic efficiency, a substrate ArSH surface-imprinted
nanoenzyme model (I-PN) was developed. The polymerization process is depicted
in Fig.3.5. By correctly incorporating and positioning the catalytic center tellurium
and functional binding factor guanidinium, a 596,000-fold continuative activity
enhancement for the reduction of CUOOH was observed by catalyst I-PN rather
than PhSeSePh. The results clearly show that a polymeric nanoparticle can be devel-
oped as an excellent model for combining most of the catalytic factors of an enzyme
into one scaffold.
96 3 Nanostructured Mimic Enzymes for Biocatalysis and Biosensing

Fig. 3.5 Polymerization process of the surface-imprinted nanoenzyme model. Reprinted with
permission from Huang etal. [88]. 2008, American Chemical Society

3.2.5Breslows Mimics

Natural enzymes are macromolecules. Their macromolecular structures offer ideal


frames for the construction of versatile and robust catalytic sites. Strong and selec-
tive binding of the substrate is attained through a combination of the hydrophobic
effect and specific substrate-enzyme interactions such as hydrogen bonding. The
macromolecular structure can also create regions in which the catalyzed reactions
occur in a less-than-fully aqueous medium. In comparison with natural enzymes,
most enzyme models are small molecules. Although many features of the real
enzymes have been well mimicked by these models, it remains to mimic the role of
the macromolecular character of enzymes in catalysis. Thus, Liu and Breslow [96]
3.2 Nanostructure Used in Artificial Mimic Enzymes 97

studied some polymeric enzyme models. They reported a great increase in the
transamination rate for the pyridoxamine-keto acid system when they attached pyri-
doxamine to polyethylenimine (PEI) carrying some attached lauryl groups. They
also reported that hydrophobic effects exert profound effects on rates and substrate
selectivities in the PEIpyridoxamine transaminase mimics.
A variety of mono- and unsymmetrical bifunctional b-CD have been developed
as efficient mimics of aldolases, some of which have shown a large rate of accelera-
tion and substrate selectivity [91]. A novel catalyst has been synthesized in which a
manganeseporphyrin unit was linked to four hydrophobic cyclophane-binding
groups [92]. The Cu(II) complex of a cyclodextrin dimer linked by a bipyridyl unit
catalyzed the hydrolysis of an unactivated doubly bound benzyl ester [93]. A cyclo-
dextrin dimer with a linking bipyridyl group was synthesized as a catalyst precursor,
a holoenzyme mimic. It bound both ends of potential substrates into the two differ-
ent cyclodextrin cavities, holding the substrate ester carbonyl group directly above
a metal ion bound to the bipyridyl unit. The result was very effective ester hydroly-
sis with good turnover catalysis. For example, a Cu(II) complex accelerated the rate
of hydrolysis of several nitrophenyl esters, with at least 50 turnovers and no sign of
product inhibition. In the best case, with an added nucleophile that also bound to the
metal ion, a rate acceleration of 1.45107 over the background reaction rate was
observed. Hydrolysis by a catalyst with only one cyclodextrin-binding group was
significantly slower than in the bidentate-binding cases. As expected, the binding of
a transition state analog to these catalysts with the presence of the metal ion was
stronger than those without the metal ion. This and kinetic evidence point to a
mechanism in which the metal ion played a bifunctional acid-base role, enforced by
the binding geometry that held the substrate functionality right on top of the cata-
lytic metal ion [98]. Zn(II) complexes of monomers and dimers derived from
1,4,7-triazacyclododecane and 1,5,9-triazacyclotetradecane were examined as cata-
lysts for the hydrolyses of p-nitrophenyl phosphate and for the cyclizations of
p-nitrophenyl 2-hydroxypropyl phosphate and 3,5-uridyluridine (UpU). The dim-
ers with 1,3-phenyl linkers were more effective than monomers or a longer dimer
with a 4,4-biphenyl linker in the hydrolysis of p-nitrophenyl phosphate, suggesting
that two Zn(II) ions coordinated to the phosphate group, as in the enzyme alkaline
phosphatase. However, for the hydrolysis or cyclization of the phosphate diesters,
the longer biphenyl linker was preferred. In this case, one Zn(II) coordinated to the
phosphate group, while the other delivered a nucleophilic oxide anion. Bell-shaped
pH vs. rate profiles were seen in both cases [101].
Synthetic organic chemistry normally achieves selectivity by manipulating the
intrinsic reactivity of the substrate, but enzyme use is quite a different principle. The
geometry of the enzymesubstrate complex determines enzymatic selectivity, com-
pletely overwhelming any normal selective reactivities. Biomimetic chemistry aims
to imitate the enzymatic style. Some early approaches used attached reagents or
templates to direct photochemical and free radical processes, with a combination of
geometric and reactivity control. Recent work used a mimic of the enzyme class
cytochrome P-450 to achieve the selective hydroxylations of steroids with complete
domination by the geometry of the catalystsubstrate complex [103]. The ability of
98 3 Nanostructured Mimic Enzymes for Biocatalysis and Biosensing

oxidizing enzymes, particularly those of the cytochrome P-450 class, to perform


selective hydroxylations of unactivated carbons in substrates, such as steroids, was
of great practical importance. It also represented a great challenge for biomimetic
chemistry. The relevant enzymatic reactions involve oxidation by metalloporphy-
rins, with reversible enzyme binding of the substrate, in such a geometry that spe-
cific substrate positions were within reach of the oxygen atom on the metal. After
oxidation, the product was released, so catalytic turnover was observed [98].

3.3Mimic Enzymes for Sensors

3.3.1H2O2 Sensors

An alternative way for a low-potential and selective detection of H2O2 was recently
demonstrated by Karyakin [7477, 111, 115, 125]. PB polycrystals deposited in a
defined way onto the electrode surface were shown to be active and selective electro-
catalysts for H2O2 reduction. Due to their catalytic ability, which is reminiscent of
biological catalysts, the specially deposited PB was denoted artificial peroxidase.
Figure3.6 shows the experimental curves obtained for the amperometric detec-
tion of H2O2 performed with electrodes of different amounts of PAH/PB nanoparti-
cle bilayers. A good linear range up to 0.4 mmol/L of H2O2 was obtained on all
electrodes. On the other hand, the analytical response depended strongly on the
number of bilayers. The sensitivities obtained by the electrode containing 5, 10, and
15 bilayers were 34.5, 76.8, and 103.5 mA mmol/L/cm2, respectively, showing a
linear increase in sensitivities with the amount of immobilized PB. These results
clearly indicated electrical connections among nanoparticles in different bilayers in
the sense of obtaining an amplification of the analytical response with the amount
of catalyst controlled at the nanoscale level.

3.3.2Glutamate Sensors

A glutamate biosensor was fabricated based on the specially deposited PB [75].


Glutamate oxidase was immobilized on the surface of the PB-modified electrode in
a Nafion layer using a nonaqueous enzymology approach. PB-based artificial per-
oxidase was an attractive transducer for the selective low-potential detection of
H2O2. The electrocatalyst was stable, highly active, and selective to H2O2 reduction
in the presence of oxygen, which provided attractive performance characteristics of
the corresponding biological sensors. In a flow-injection mode, a detection range of
11071104M glutamate was achieved. The sensitivity of the PB-based biosen-
sor was 0.21/AM/cm2, which was several times higher compared to similar bioana-
lytical devices. The detection limit of 107M was markedly lower than that recently
3.3 Mimic Enzymes for Sensors 99

Fig.3.6 Linear-range
analytical curves obtained for
H2O2 detection for LBL films
of 5, 10, and 15 PAH/PB
nanoparticle bilayers.
Reprinted with permission
from Fiorito etal. [78].
2005, Royal Society of
Chemistry

obtained for platinum- and peroxidase-based biosensors and comparable for


glutamate biosensors using the substrate amplification principle. In addition, the
influence of reductants could be avoided in practice by applying low potential on an
indicator electrode (0.0V Ag/AgCl). The attractive performance characteristics of
the glutamate biosensor illustrated the advantages of PB-based artificial peroxi-
dase as a transducer for H2O2 detection.

3.3.3Glucose Sensors

Recently, an oxalate biosensor based on the immobilization of oxalate oxidase onto


a PB-modified electrode was reported to show high performance and stability [78].
Furthermore, an electrodeposited PB film was used as the substrate for the effective
immobilization of GOx by the layer-by-layer (LBL) technique to prepare a reliable
glucose biosensor. Because of its high activity and selectivity toward the reduction
of H2O2 and oxygen, PB is usually considered an artificial enzyme peroxidase and
has been extensively used in the construction of electrochemical biosensors. The
LBL technique opened enormous possibilities for preparing different molecular
architectures with the use of PB nanoparticles. The construction of amperometric
biosensors via grafting PB nanoparticles on the polymeric matrix of multiwalled
carbon nanotubes (MWCNTs) and poly(4-vinylpyridine) (PVP) was reported [79].
The MWCNT/PVP/PB composite films were synthesized by casting films of
MWCNTs wrapped with PVP on gold electrodes, followed by the electrochemical
deposition of PB on the MWCNT/PVP matrix. The electrode modified with the
MWCNT/PVP/PB composite film showed prominent electrocatalytic activity
toward the reduction of H2O2, indicating the remarkable synergistic effect of the
100 3 Nanostructured Mimic Enzymes for Biocatalysis and Biosensing

MWCNTs and PB. A fast amperometric response to H2O2 was observed, with a
detection sensitivity of 1.3mA/mM/cm2 and a detection limit of 25nM. These results
were much better than those reported for PB-based amperometric sensors. In addi-
tion, a glucose biosensor fabricated by casting an additional GOx-contained Nafion
film above the MWCNT/PVP/PB composite film showed promise for the sensitive
and fast detection of glucose. The high stability, high sensitivity, and high reproduc-
ibility of the MWCNT/PVP/PB composite films are promising for the reliable and
durable detection of H2O2 and glucose.

3.3.4Nonelectroactive Cation Sensors

The participation of cations in redox reactions of metal hexacyanoferrates provides


a unique opportunity for the development of chemical sensors for nonelectroactive
ions. The development of sensors for thallium [131], cesium [132], and potassium
[133, 134] pioneered the analytical applications of metal hexacyanoferrates. Later,
the number of cationic analytes was enlarged, including ammonium [135], rubidium
[136], and other mono- and divalent cations [137]. In most cases the electrochemi-
cal techniques used were potentiometry and amperometry, either under constant
potential or in a cyclic voltammetric regime. However, some monovalent ions simi-
larly promoted the electroactivity of metal hexacyanoferrates, which affected the
selectivity of the corresponding sensors. In particular, it was rather hard to distin-
guish alkali metal and ammonium ions. Indeed, the same metal hexacyanoferrates
were used in potassium, cesium, and rubidium sensors, as well as in ammonium
sensors. Particular cases were potassium-selective potentiometric sensors based on
cobalt [138] and nickel [136, 139] hexacyanoferrates. These hexacyanoferrates pos-
sessed quite satisfactory redox activity, with sodium as the counter cation [140].
According to the two possible mechanisms of such redox activity (either sodium
ions penetrate the lattice or charge compensation occurs due to entrapment of
anions), there was no thermodynamic background for selectivity of these sensors. In
these cases, electroactive films seemed to operate as smart materials, similarly to
conductive polymers in electronic noses. Except for the above applications, the
intercalation of alkali metal ions in metal hexacyanoferrates was used for the adsorp-
tion and separation of cesium ions from different aqueous solutions with PB [141,
142] and cupric hexacyanoferrate [143, 144].

3.3.5Easily Oxidizable Compounds and Other Nontraditional


Sensors

The ability of metal hexacyanoferrates to oxidize some organic and inorganic com-
pounds was used in the 1990s for analytical applications. Despite some of the compounds
being tested in real objects, the cross-selectivity of such sensors must be low.
3.3 Mimic Enzymes for Sensors 101

The PB films grown through interdigitated arrays were used as a humidity sensor
[145] as well as a sensor for vapors of methanol and dichloroethane [146]. A non-
conventional optical pH sensor based on PB was also reported [147, 148].

3.3.6Transition Metal Hexacyanoferrate Sensors

More than 90% of commercially available enzyme-based biosensors and analytical


kits contain oxidases as terminal enzymes responsible for the generation of the ana-
lytical signal. These enzymes catalyze the oxidation of a specific analyte with
molecular oxygen producing H2O2 according to the reaction:

Oxidase
Analyte Oxidized
Analyte

O2 H2O2

Among different kinds of oxidase-based biosensors, the detection of H2O2 produc-


tion was found to be the most progressive, allowing the detection of low levels of
analytes [149]. However, the detection of H2O2 has to be carried out at low poten-
tials in order to reduce the interference of easily oxidizable compounds [150].
The application of metal hexacyanoferrates for the development of biosensors
was first announced in 1994 [107]. The goal was to substitute platinum as the most
commonly used H2O2 transducer for the PB-modified electrode.
Another approach for the development of PB-based biosensors was published in
1995 and involved enzyme immobilization by entrapment into PB films during its
deposition [119]. However, as mentioned, the best media for the deposition of PB is
0.1 M HCl, which is not tolerable of enzymes, in particular, for GOx [151].
Moreover, the entrapment of the enzyme in metal hexacyanoferrates during their
deposition does not provide enough enzyme activity in the resulting film, which
resulted in a rather low sensitivity of the resulting biosensor [130] compared to the
sensitivity of the corresponding H2O2 transducer [126].
Except for a low-potential H2O2 transducer, PB was integrated in biosensors as
an electrocatalyst for H2O2 oxidation [110]. However, metal hexacyanoferrates
are ideal electrocatalysts for the oxidation of easily oxidizable compounds like
ascorbate [152], which would definitely interfere in the biosensor response. The
detection of H2O2 at 0.45V by its oxidation on PB-modified electrodes seems to
be doubtful, because the PB oxidation state has not been found active in either the
oxidation or reduction of H2O2 [76, 113]. Metal hexacyanoferrate-based biosen-
sors were developed for the analysis of glucose [153158], ethanol [115], d-
alanine [108], oxalate [159], glutamate [75], and choline [119]. The sensitivity of
cupric hexacyanoferrate is several orders of magnitude lower than that of PB.
Moreover, it is important to compare the properties of the related biosensors with
bioanalytical systems based on different detection principles. Take glutamate
102 3 Nanostructured Mimic Enzymes for Biocatalysis and Biosensing

biosensors as an example: Due to the low potential of the indicator electrode, the
influence of interferents on the PB-based biosensor [75] was similarly low as that
in the case of a biosensor based on peroxidase wired in osmium hydrogel [160].
However, PB-based electrodes offered a detection limit of 1107M glutamate in
the flow-injection mode, which was one order of magnitude lower than that for
known biosensors.

3.4Conclusions

Some iron-based compounds, such as PB, Fe3O4, FeS, polystyrene, pyridoxal


phosphate, and pyridoxamine phosphate, have been denoted as artificial enzyme
mimetics. They can be used as electrocatalysts in H2O2 reduction and can be applied
in H2O2, glutamate, glucose, nonelectroactive cation sensors, and sensors for easily
oxidizable compounds and other nontraditional sensors transition metal hexacyano-
ferrates. Nanostructure artificial enzyme mimetics have good stabilities, highly cata-
lytic properties, and a wide range of practical applications. The particular importance
of their application is expected in certain areas of clinical diagnostics, where high
sensitivity and selectivity as well as the possibility of miniaturization are required,
such as brain research and noninvasive monitoring of blood chemistry.

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Chapter 4
Porphyrin-Based Nanocomposites
for Biosensing

4.1Introduction

4.1.1Porphyrin: A Mimic of Enzymes

Porphyrins are an important class of conjugated organic molecules, which can be


employed to mimic the active site of many important enzymes, such as hemoglobin,
myoglobin, cytochrome c oxidase (CcO), nitric oxide reductase, vitamin B12, and
chlorophyll [13]. The macrocyclic structure of porphyrin can conjugate many
metal elements to form stable metalloporphyrins, which have remarkable photo-,
catalytic-, electro-, and biochemical properties. Among these complexes, iron por-
phyrins can be used well as electron media based on the reversible redox of Fe3+/Fe2+
and exhibit good electrocatalysis to many small molecules related to life processes
[4, 5], including dissolved oxygen, NO, neurotransmitters, hydrogen peroxide, and
nitrite. On the other hand, high-valent iron(IV)porphyrin as a strong oxidant has
been utilized to catalyze the mono-oxygenation of organic substrates and biomole-
cules in many chemical reactions [6, 7].
In comparison with the simple porphyrin, a picket-fence porphyrin has a macro-
cyclic porphyrin ring with four column-like phenyl substitutes, which simulates the
active center of some proteins and enzymes, thus leading to improved catalysis
toward biomolecules [8]. A great deal of creative effort has been expended in
designing, synthesizing, and studying functional models of heme enzymes by
Collmans group. The typical example is the biomimetic study of CcO (Fig.4.1),
which has identified that the distal Cu is obligatory for O2 reduction activity under
biologically relevant turnover-determining electron flux [3]. The design of biomi-
metic analogs of ever-closer structural similarity to the active sites of enzymes is
continuing progress in the area.

H. Ju et al., NanoBiosensing: Principles, Development and Application, 111


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0_4, Springer Science+Business Media, LLC 2011
112 4 Porphyrin-Based Nanocomposites for Biosensing

Fig.4.1 (a) Crystal structure of the active site of cytochrome c oxidase (CcO) from the bovine
heart; (b) biomimetic analogs of the heme/Cu site of CcO. Reprinted with permission from
Collman etal. [3]. 2007, Science

4.1.2Significance of the Porphyrinic Nanocomposite

Porphyrin molecules also are used in self-assembling processes to prepare soft


nanostructures such as spheres and tubes in natural photosystems. For the artificial
photoactive molecular devices, nanoscaled particles composed of porphyrins are
expected to have chemical activities significantly different from those of the free
porphyrins or of those immobilized onto/into supports. Porphyrin-functionalized
nanoparticles are promising components of advanced materials because of the rich
photochemistry, stability, and proven catalytic activity. Thus, the controlled organi-
zation of functional porphyrins into highly ordered nanomaterials is an area of
research with great potential application in materials science and is expected to
yield new materials for biosensing [9, 10].
Nanometer-scale particles composed of carbon, metals, metal oxides, and other
inorganic materials have been reported as functional platforms to assemble many
organic molecules. Covalent or noncovalent assemblies are the main approach for
the interaction between porphyrin and nanomaterials. The nanocomposites of por-
phyrin nanomaterials should have unique photonic and electrochemical properties
and enhanced stability and catalytic rate due to the aggregated structure and the
greater surface area for biosensing. This chapter discusses the design, characteriza-
tion, and application of porphyrin-functionalized nanomaterials.

4.2Assembly of Porphyrins on Carbon-Based Nanomaterials

Carbon-based nanomaterials (carbon nanotube [CNT], carbon nanohorn [CNH],


graphene sheet, etc.) presently attract much attention as prospective technological
materials. They constitute a major part of the emerging field of nanoscience and
nanotechnology. In particular, functionalized carbon nanomaterials with an electron
4.2 Assembly of Porphyrins on Carbon-Based Nanomaterials 113

donor moiety have been expected to lead the construction of a novel electron
donoracceptor composite system for the development of photoelectrochemical and
electrochemical biosensing.

4.2.1Carbon Nanotubes

4.2.1.1Assembly in Organic Solvent

The unique structural, mechanical, and electronic properties of CNTs have made
these promising materials for device fabrication. To effectively utilize CNTs as
building blocks for nanotechnology, nanotubes have been covalently and noncova-
lently functionalized in a number of ways to render them soluble in aqueous or
organic solutions and to gain precise control over nanotube orientation and location.
CNTs are usually divided into single-walled carbon nanotubes (SWCNTs) and mul-
tiwalled carbon nanotubes (MWCNTs) according to the number of layers of curved
graphene sheets. The functionalization of SWCNTs with porphyrins has been inves-
tigated with increasing frequency because these flat, planar aromatic structures are
ideal for p-stacking interactions with the sidewalls of SWCNTs. Generally, this
procedure can be performed by covalent or noncovalent routes.
Noncovalent methods, including electrostatic interactions, pp interactions, and
axial coordination, are more appealing because they do not significantly perturb the
electronic structure of the nanotubes. In 2003, the first hybrid nanomaterials of
SWCNTsporphyrin were reported in Nakashimas group. As shown in Fig.4.2a, b,
the solid purified SWCNTs solely are insoluble in DMF, and the DMF solution of
zinc protoporphyrin IX (ZnPP) shows a red color. After the sonication of SWCNTs
in ZnPP DMF solution, a reddish-black-colored transparent solution is observed
(Fig. 4.2c), strongly suggesting that ZnPP can disperse/dissolve p-SWCNTs. No
precipitation is noticed in the SWCNTZnPP DMF solution even after a 2-month
storage at 5C [11]. Density functional theory calculation proves that the chemical
reactivity of semiconducting SWCNTs toward metalloporphyrin is stronger than
that of metallic SWCNTs [12]. Therefore, semiconducting SWCNTs can be sepa-
rated from metallic SWCNTs through the adsorption of metalloporphyrin due to the
difference in charge transfer and hybridization between metalloporphyrin molecules
and SWCNTs [13].
Protonated porphyrin provides another convenient way to construct ordered
molecular assemblies. The ordered assembly of SWCNTs-protonated porphyrin
can be obtained by adding 1 mg of purified SWCNTs in tetrahydrofuran (THF)
containing 1.0% H2SO4 (v/v) and 0.2mM porphyrin. The pp interaction between
porphyrins and SWCNTs plays an important role in achieving the ordered assembly
of protonated porphyrin in the form of J- and H-type aggregates on the SWCNTs
surface (Fig. 4.3). This unusual molecular aggregation phenomenon driven by
SWCNTs further assembles in the form of linear bundles [14]. This simple method
of designing supramolecular assembly can pave the way for developing light-
harvesting assemblies and optoelectronic devices.
114 4 Porphyrin-Based Nanocomposites for Biosensing

Fig.4.2 Photos of (a) DMF dispersion of SWCNTs, (b) DMF solution of ZnPP, and (c) a transparent
DMF dispersion of SWCNTsZnPP. Reprinted with permission from Murakami etal. [11]. 2003,
Elsevier

Fig.4.3 Illustration of supramolecular assembly between protonated porphyrins and SWCNTs.


Reprinted with permission from Hasobe etal. [14]. 2005, American Chemical Society

Axial coordination provides a possibility for the functionalization of CNTs. In


general, the functionalization of CNTs with porphyrins by axial coordination is very
difficult due to the absence of a sufficient binding site on CNTs. In order to achieve
the process, pyridyl group [15] and 4-aminopyridine [16] have been covalently bound
and imidazole ligand has been assembled by pp stacking [17] to the surface of
4.2 Assembly of Porphyrins on Carbon-Based Nanomaterials 115

Fig. 4.4 Schematic diagram of CNxMWCNTFeTpivPP-modified GCE. Reprinted with


permissionfrom Tu etal. [18]. 2010, Wiley

CNTs to form electron donor for the self-assembly of porphyrin molecules. Similarly,
nitrogen-doped MWCNTs (CNx-MWCNTs) containing the nitrogen atom in a CNT
structure provide the electron donor to form axial coordination for the preparation of
a functional nanocomposite of picket-fence porphyrin, bromo-[iron(III)-5,10,15,20-
tetrakis(a,a,a,a-2-pivalamidopheny) porphyrin] (FeTpivPP). The functional nano-
composite exhibits a promising tool to assemble the CNTs via Fe-N axial coordination
(Fig.4.4). This approach provides a facile avenue for the direct axial assembly of
porphyrin and the design of novel biofunctional materials [18].
Compared to noncovalent methodologies, the formation of SWCNT conjugates
employing covalent methods bears a number of advantages. First, the spacer that is
used to link SWCNT and the photoactive molecule is stable and well defined.
Second, the number of functional groups is controlled by fine-tuning the function-
alization processes. The covalently connected porphyrins on CNTs can enhance the
efficiency of photoinduced electron transfer and energy transfer [19]. Typically,
SWCNTs are purified using stepwise wet-air oxidation and shortened using a
sulfuric acid/nitric acid (3:1) treatment to introduce carboxylic acid groups on the
surface of CNTs. Then, CNTs treated with thionyl chloride are reacted with excess
5-phydroxyphenyl-10,15,20-tritolylporphyrin (por-OH) in toluene in the presence
of triethylamine at 100C for 24h under a pure nitrogen atmosphere. To remove the
unreacted por-OH, the tubes are washed thoroughly with plenty of methanol, fol-
lowed by a small amount of acetic acid and triethylamine, and finally with THF. The
final products are then dried at 40C for 5h under vacuum (Fig.4.5). Steady-state
fluorescence reveals that covalently connected porphyrins act as energy-absorbing
and electron-transferring antennae, and the CNTs act as electron acceptors [20].
116 4 Porphyrin-Based Nanocomposites for Biosensing

Fig.4.5 Porphyrin-grafted CNTs and the photoinduced electron transfer. Reprinted with permission
from Baskaran etal. [20]. 2005, American Chemical Society

4.2.1.2Assembly in Aqueous Solution

Water-soluble porphyrin is the mimic of enzyme model systems. Thus, it is


necessary to develop the conjugate structure of CNTs and porphyrin in aqueous
solution. Awater-soluble porphyrin, meso-(tetrakis-4-sulfonatophenyl) porphyrin
(H2TPPS4), has solubilized SWCNTs via pp interactions, producing an aqueous
solution that is stable for several weeks. The resulting nanocomposite can be pre-
cisely aligned on hydrophilic poly(dimethylsiloxane) (PDMS) surface by combing
SWCNT solution along a desired direction to form parallel SWCNT patterns for
future device fabrication [21].
Electrostatic interaction is another force to dissolve the SWCNTs in aqueous
solution. For example, SWCNTs are first treated with 1-(trimethylammonium
acetyl)-charged pyrene derivatives in water. The resulting SWCNT/pyrene is found
to form a stable nanohybrid structure through the formation of pp interactions in
aqueous media. Then a series of water-soluble metalloporphyrins (MP8+) have
evolved into functional nanohybrids through a combination of associative van der
4.2 Assembly of Porphyrins on Carbon-Based Nanomaterials 117

Fig.4.6 Fabrication process of self-assembled GNP/MWCNTFeTMAPP monolayer. Reprinted


with permission from Liu etal. [28]. 2007, Elsevier

Waals and electrostatic interactions [22]. A crucial feature of SWCNT/pyrene/


MP8+ is that an efficient exfoliation of the initial bundles brings about isolated nano-
hybrid structures to improve the photoconversion efficiency [23], which is promising
for the construction of photoactive electrodes with monochromatic solar-energy
conversion efficiencies of up to 8.5% internal photoconversion efficiencies [24].

4.2.2Optical and Electrochemical Biosensing

Coupled with the high electrocatalysis of porphyrin, the nanocomposites of porphyrin


with nanomaterials show efficient electrocatalysis toward many biological molecules.

4.2.2.1Detection of O2 with Four-Electron Reduction

The measurement of dissolved oxygen is very important in various areas of chemi-


cal, physical, and environmental monitoring. The MWCNTcobalt porphyrin com-
positions, mixed with Nafion, display excellent catalytic performance for oxygen
reduction in acidic media (pH range, 0.05.0) at room temperature [2527]. With
low catalyst loading, the oxygen reduction rates are more than one order of magni-
tude higher than previously reported values for free cobalt porphyrin catalysts.
However, the acidic conditions limit their applications in biological systems. A kind
of nanocomposite with good dispersion has been prepared through the non-
covalent adsorption of iron picket-fence porphyrin (FeTMAPP), iron-5,10,15,20-
tetrakis[a,a,a,a-2-trismethylammoniomethyl-phenyl] porphyrin, on MWCNTs in
neutral phosphate buffer (PBS). A gold nanoparticle (GNP)/nanocomposite self-
assembled monolayer (SAM) is formed on gold electrodes via the electrostatic
interactions (Fig.4.6) and shows highly synergetic behavior toward the electrocata-
lytic reduction of O2, with a 200-mV decrease in the overpotential. The resulting
biosensor exhibits a good response to oxygen, with a linear range from 0.52 to
180mM and a detection limit of 0.38mM, without the interference of ascorbic acid
and uric acid. This method provides an application potential of the proposed nano-
composite in the detection of dissolved oxygen and oxidase substrates [28].
118 4 Porphyrin-Based Nanocomposites for Biosensing

Furthermore, the combination of MWCNTs, cobalt porphyrin, and peroxidase


(horseradish, cabbage) enzyme in the film (deposited onto glassy carbon electrode
[GCE] substrate) can produce a bio-electrocatalytic system capable of the effective
reduction of oxygen in such neutral media as 0.1 mol/L KCl+0.01 citrate buffer
(pH 6). The multicomponent bio-electrocatalytic film leads to a synergistic effect for
some positive shift of the oxygen reduction potentials and a significant (ca. twice)
increase in voltammetric currents compared to that of the enzyme-free system [29].

4.2.2.2H2O2

H2O2 is a product of the enzymatic reactions between most oxidases and their sub-
strates; thus, its detection is very interesting for the development of biosensors for
oxidase substrates. Iron(III) protoporphyrin IX [Fe(III)PP], adsorbed either on
SWCNTs or on hydroxyl-functionalized SWCNTs (SWCNTs-OH), has been
incorporated within a Nafion matriximmobilized graphite electrode. Both the
SWCNTFe(III)PP- and SWCNTOHFe(III)PP-modified graphite electrodes exhibit
electrocatalytic activity toward H2O2 reduction. The sensitivities of the modified elec-
trodes for H2O2 are found to vary in the following sequences: SWCNTOHFe(III)
PP=2.45 mA/MSWCNTFe(III)PP=2.95 mA/M>free Fe(III)PP=1.34 mA/M
[30]. In addition, an electrochemiluminescent (ECL) biosensor of H2O2 has been con-
structed based on cobalt(II) meso-tetraphenylporphrine/MWCNT (CoTPP/MWCNT)
modified GCE. Under the optimum conditions, the enhanced ECL intensity shows a
linear relationship with the concentration of H2O2 in the range of 1.0107
8.0108mol/L, with a detection limit of 5.0109mol/L [31].

4.2.2.3DNA

Considering the different adsorption properties of single-stranded DNA (ssDNA)


and double-stranded DNA (dsDNA) on the surface of CNTs and the unique intrinsic
properties of porphyrins, a detection method of specific DNA with noncovalent
anionic porphyrin-functionalized MWCNT suspension has been proposed. When a
dye-tagged ssDNA is added to the water-soluble anionic tetra (p-carboxyphenyl)
porphyrin (TCPP)/MWCNTs suspension, the fluorescence from a dye-tagged ssDNA
is quenched. However, the fluorescence can be retained if the dye-tagged ssDNA is
first hybridized with its complementary target DNA to form a dsDNA hybrid and
added into the TCPP/MWCNT suspension. Thus, target DNA in a DNA sample and
single-base mismatches in DNA sequences can easily be detected [32].

4.2.2.4Sudan I

The lipophilic Sudan I (1-phenylazo-2-naphthol) is a synthetic azo-colorant that has


been widely used as an additive in everyday foods. Thus, developing a sensitive,
4.2 Assembly of Porphyrins on Carbon-Based Nanomaterials 119

Fig.4.7 Analytical curve for


the electrooxidation of GSH
in PBS at pH 7.4 in the
concentrations: 5, 20, 85,
160, 280, 400, and
500mmol/L, 1, 2, 3, 4, and
5mmol/L. Inset: the
square-wave voltammograms.
Reprinted with permission
from Luz etal. [34]. 2008,
Elsevier

rapid, and convenient method for the determination of Sudan dyes is of great
importanceand interest. In pH 7.0 TrisHCl buffers, Sudan I shows a sensitive cata-
lytic reduction peak at 0.08V on the iron-porphyrin (5,10,15,20-tetraphenyl-21H,
23H-porphine iron(III) chloride)SWCNTDMFmodified GCE. Using square-
wave voltammetry, the linear relationship of Sudan I is 5.031082.01106mol/L,
with a detection limit of 1108mol/L [33]. This biosensor has been successfully
applied in the determination of Sudan I in hot chili powder, hot chili juice, and
ketchup samples.

4.2.2.5l-Glutathione

l-Glutathione (GSH) is an essential compound in many biological processes such as


catabolism and transportation. A highly sensitive voltammetric sensor for reduced
l-glutathione has been developed using iron(III) tetra-(N-methyl-4-pyridyl)-porphyrin
(FeTMPyP)/MWCNTmodified basal plane pyrolytic graphite electrode. The modi-
fied electrode shows very efficient electrocatalytic activity for l-glutathione oxida-
tion, substantially decreasing the oxidation peak at 0.025V vs. Ag/AgCl (Fig.4.7).
A linear response range from 5mmol/L to 5mmol/L is obtained with a sensitivity of
703.41mAL/mmol. The detection limit for GSH is 0.5mmol/L, and the relative stan-
dard deviation (RSD) for ten determinations of 250mmol/L GSH is 1.4% [34]. The
modified electrode has been applied for GSH determination in erythrocyte samples,
and the results are in agreement with those obtained by a reference method.

4.2.3Carbon Nanohorns

CNHs, as a dahlia-flowerlike spherical superstructure of aggregated nanosized gra-


phitic tubes, represent promising alternatives to CNTs and have started emerging as
120 4 Porphyrin-Based Nanocomposites for Biosensing

interesting nanometer-sized building blocks for the construction of novel materials


with potential applications in nanotechnology. There are three critical points that dif-
ferentiate CNHs from CNTs: (1) high purity, due to the absence of any metal nano-
particles during the laser ablation production; (2) heterogeneous surface structure,
due to highly strained conical ends; and (3) aggregation in spherical superstructures,
typically ranging between 50 and 100nm. Moreover, the rough surface structure of
CNH aggregates with minimum van der Waals interactions between the superstruc-
tures gives rise to a better dispersion of CNH in liquid media, as compared to the
tightly bundled CNTs, which do not show significant dispersions.
Similar to CNTs, covalent functionalization of CNHs has been achieved using
two different synthetic protocols: (1) direct attack of a free amino group on the nano-
horn sidewalls (nucleophilic addition); and (2) amidation reaction of the carboxylic
functions in oxidized nanohorns. On the other hand, supramolecular approaches uti-
lizing noncovalent pp stacking interactions between the sidewalls of CNH with
aromatic organic materials and/or synergistic electrostatic interactions have been
developed [35]. In addition, the combination of pp stacking and electrostatic inter-
actions has been used to integrate an anionic porphyrin with CNHs, mediated by
positively charged pyrene units. The strong fluorescence emission of anionic
porphyrin is significantly quenched by CNHs, suggesting effective energy and
electron transfer between the photoexcited porphyrin and the extended p-electronic
network of nanohorns [36].
More recently, a sandwich nanohybrid of single-walled carbon nanohorn
(SWNH)TiO2porphyrin has been prepared via the dentate binding of TiO2 nano-
particles to carboxylate groups (Fig.4.8). The resulting nanocomposite shows excel-
lent electrocatalytic activity toward the reduction of chloramphenicol (CAP) in
neutral media. The modified GCE can respond very rapidly to the change of CAP
concentration within only 5s. The linear detection range can be up to 135.7mM,
with a detection limit of 0.9nM at a signal-to-noise ratio of 3. The sandwich nano-
structure of SWNHTiO2porphyrin provides a functional electrocatalyst to construct
a sensitive biosensor [37].

4.2.4Graphene Sheets

Graphene as a single-layered two-dimensional (2D) sheet has attracted enthusiastic


interest in many areas of nanoscience and nanotechnology due to its fascinating
physical properties, such as great mechanical strength, fast electron transfer,
universal optical absorption, and high specific surface area. Usually, graphene oxide
(GO) can be easily deposited on different substrates to produce continuous films for
constructing transparent conductors, photovoltaic devices, and biosensors, since
GO sheet decorated with oxygen functional groups can be readily exfoliated to form
a stable aqueous dispersion [38].
However, the numerous oxygen-containing groups of GO render it too electrically
insulating for a conductance-based device. Thus, reduced graphene oxide (RGO), via
chemical reduction using hydrazine hydrate to remove oxygen and recover the
4.2 Assembly of Porphyrins on Carbon-Based Nanomaterials 121

Fig.4.8 Schematic illustration of the structure of SWNHTiO2porphyrin nanohybrids. Reprinted


with permission from Tu etal. [37]. 2009, Royal Society of Chemistry

a romatic double-bonded carbons, is another promising candidate for constructing


electronic and optical devices. Due to the lack of solubility and active sites on the
sheet, it is urgent to functionalize RGO for further applications. Based on the supra-
molecular assembly of free-base TMPyP on RGO through electrostatic and pp
stacking-cooperative interactions, an optical probe has been constructed for the rapid
and selective sensing of Cd2+ ions in aqueous media [39]. More recently, the func-
tionalization of RGO with water-soluble 5,10,15,20-tetrakis [aaaa-2-trismethylam-
moniomethyl-phenyl]porphyrin iron(III) pentachloride (FeTMAPP), a kind of
picket-fence porphyrin with one planar side and another positively charged side, has
been reported via pp noncovalent interactions by Jus group [40]. The obtained
porphyrin/RGO nanocomposite shows good dispersion. The synergistic effect
between RGO and porphyrin leads to highly efficient electrocatalytic activity for the
reduction of chlorite (Fig.4.9). The current-time curve of FeTMAPP/RGO-modified
ITO electrode upon the successive addition of chlorite at an applied potential of
0.36V clearly illustrates the rapid response of the modified electrode to chlorite.
The response reaches steady signal within only 4s, and displays a linear increase
with increasing chlorite concentrations, from 5.0108 to 1.2104 mol/L. The
detection limit is 2.4108mol/L at a signal-to-noise ratio of 3 [40]. Thisamperometric
122 4 Porphyrin-Based Nanocomposites for Biosensing

Fig.4.9 Schematic representation of the noncovalent assembly and electrocatalysis of FeTMAPP


on RGO. Reprinted with permission from Tu etal. [40]. 2010, Wiley

biosensor prepared with FeTMAPP/RGO shows promising application in monitoring


chlorite, with high sensitivity and a wide concentration range.

4.3Assembly of Porphyrins on Semiconductor Nanoparticles

Semiconductors are an attractive material for a broad range of electronic, optical, and
piezoelectric applications due to their direct band gap and excellent thermal, chemical,
and structural properties. There are various ways to anchor porphyrins onto semicon-
ductors host surfaces: (1) covalent attachment by anchoring groups; (2) electrostatic
interactions, via ion exchange, ion pairing, or donoracceptor interactions; (3) hydro-
phobic interactions; (4) hydrogen bonding; (5) van der Waals interactions; and
(6) physical entrapment inside the pores or cavities of hosts. Semiconductors are
usually divided into two parts: metal oxide and quantum dots (QDs).

4.3.1TiO2Porphyrin Nanocomposite

4.3.1.1Porphyrin Bound to TiO2

TiO2 is a most useful semiconductor, but its wide band gap (3.2eV) limits its use as
a visible-light photocatalyst. The main drawback is low quantum yield, and the lack
4.3 Assembly of Porphyrins on Semiconductor Nanoparticles 123

Fig.4.10 Structures of the porphyrins and the anticipated binding geometries of the COOH and
COOEt3NH derivatives on metal oxide surfaces. Reprinted with permission from Rochford etal.
[41]. 2007, American Chemical Society

of visible-light utilization hinders its practical application. To overcome these


problems, the sensitization of TiO2 using visible-light-absorbing organic dyes has
been a topic of interest for the past few years. The covalent bonding is the most stable
form of attachment between TiO2 and porphyrin. Usually, the covalent attachment is
realized by a variety of anchoring groups with different affinities to the metal oxide
surfaces. The best anchoring groups for metal oxides are phosphonic acids, followed
by carboxylic acids and their derivatives. For example, four Zn(II)-TCPP derivatives
(p-COOH, m-COOH, m-COOMe, and m-COOEt3NH) binding to TiO2 nanoparticle
films result in the appearance of strong and broad bands in the 1,3901,410/cm
region, which are characteristic of the symmetric v (CO2) stretch. A broad band
observed at approximately 1,540/cm for the porphyrin acids and salts bound to TiO2
is assigned to the v (CO2) asymmetric stretching mode. Overall, both porphyrin
acids/TiO2 and salts/TiO2 are consistent with the chelating and/or bidentate binding
modes of the carboxylate groups on the TiO2 surface (Fig.4.10) [41]. Lately, in the
same group, a series of Zn(II) tetraphenylporphyrins (ZnTPP), with a phenyl or oli-
gophenyleneethynylene rigid-rod bridge varying in length (930) and terminated
124 4 Porphyrin-Based Nanocomposites for Biosensing

with an isophthalic acid anchoring unit, have been prepared as model dyes for the
study of sensitization processes on metal oxide semiconductor nanoparticle (such as
TiO2, ZnO, and insulating ZrO2) surfaces for photoelectrochemical cells [42].
In an alternative approach, short aromatic amines have been tethered to TiO2
nanoparticles and used to scavenge porphyrin (Ru(CO)OEP, Ru(CO)TPP, and
ZnTPP) from solution and anchor the porphyrin, via axial ligation, in close proxim-
ity to the electrode surface. The attachment of porphyrins via axial coordination
provides a number of advantages, including the ability to control chromophore ori-
entation relative to the surface, and simplifies syntheses using commercially avail-
able materials for the modular assembly of porphyrin sensitization, reduction of
surface aggregation, and a controllable mechanism for stepwise construction of
multiporphyrin arrays. The simple method is highly adaptable for use in dye-
sensitized solar cells [43]. In addition, TiO2 nanoparticles have been found to
enhance the formation of J-aggregates of water-soluble porphyrin [44].
The main disadvantage of these anchoring groups is instability against water,
acids, and bases in their applications. To overcome this limitation, catechol, ethers,
acetylacetonate, and salicylates have been explored. For example, the introduction
of catechol-anchoring groups (3,4-dihydroxybenzo compounds) for grafting por-
phyrins onto metal oxide surfaces is relatively stable and soluble [45]. Using cate-
chol groupterminated Zn(II)-porphyrin to functionalize the single-crystalline TiO2
nanoleaves along the face results in a facet-selective, self-assembled, 2D stacking
structure [46]. Tyrosine methyl ester can be used as a bridge between TiO2 nano-
clusters and tetratolylporphyrin in neutral ethanol solution to enhance the photoin-
duced electron transfer in a heterogeneous system [47].

4.3.1.2Characterization of the Nanocomposite

The morphology of the nanocomposite is usually characterized by transmission elec-


tron microscopy (TEM), scanning electron microscopy (SEM), X-ray photoelectron
spectroscopy (XPS), X-ray diffraction (XRD), and electron paramagnetic resonance
(EPR). Figure4.11a, b show the TEM images of free-base porphyrin (H2P)modified
TiO2 nanotubes and nanoparticles (H2P-COO-TiO2 tubes and H2P-COO-TiO2 NPs),
respectively. These two images show the distinguishable structural features, that is,
the tubular and particle structures of the H2P-modified TiO2 precursors employed
prior to complexation with C60. Any aggregation of H2P-COO-TiO2 NPs and H2P-
COO-TiO2 tubes seen on the TEM grid is likely to arise from the close packing of the
molecules [48]. Figure4.11c, d show the SEM images of OTE/SnO2/(H2P-COOTiO2
tube+C60)n and OTE/SnO2/(H2P-COO-TiO2 NPs+C60)n, respectively. The TiO2 nan-
otubes and nanoparticles are assembled on the electrode surface as packed nano-
structures. A comparison of the SEM images with the TEM images demonstrates
that composite molecular clusters with TiO2 nanotubes or nanoparticles as deposited
onto OTE/SnO2 electrodes retain their morphology without exhibiting a significant
bundling effect or particle growth. The maximum photon-conversion efficiency
obtained with TiO2 nanotube architecture is higher that obtained with nanoparticle
4.3 Assembly of Porphyrins on Semiconductor Nanoparticles 125

Fig. 4.11 TEM image of (a) H2P-COO-TiO2 tube, and (b) H2P-COO-TiO2Pa. (c, d) are SEM
images of OTE/SnO2/(H2P-COOTiO2 tube+C60)n and OTE/SnO2/(H2P-COO-TiO2Pa+C60)n.
Reprinted with permission from Hasobe etal. [48]. 2007, Wiley

architecture. The efficiency of light energy conversion of these solar cells has been
explained on the basis of the geometrical orientation of the porphyrins with respect
to the TiO2 surface and the supramolecular complex formed with C60.
XPS has been used to distinguish the surface change of pure TiO2 and N-doped
TiO2 (N-TiO2) nanoparticles after they adsorbed Zn porphyrin. Figure4.12 shows O
1s XPS spectra of pure TiO2 and N-TiO2 before and after they adsorb ZnTPP and
ZnTCPP, respectively. The O 1s peaks of N-TiO2-ZnTCPP are 0.50.7eV higher
than those of pure TiO2 and N-TiO2, while no shift of O 1s peaks for the N-TiO2-
ZnTPP is observed. This result suggests that the ZnTCPP molecules bind more
slightly on the surface of TiO2 samples than ZnTPP, and their structure may change
after their adsorption on the surface of TiO2. The characterization shows that
the ZnTCPP is chemisorbed on the surface of TiO2 through an O=COTi bond,
while the ZnTPP is physically adsorbed. N-TiO2 sensitized by Zn porphyrin exhib-
its higher absorption in the visible-light region and higher photocatalytic degrada-
tion efficiency of methylene blue under visible-light irradiation than N-TiO2 and
TiO2 sensitized by ZnTCPP [49].
Figure4.13 displays the XRD patterns of the bare TiO2 and the Nb-, Ge-, and
Zr-added TiO2. All the peaks in each sample can be assigned to anatase. No XRD
pattern arising from rutile is observed. It should be noted here that all the samples
126 4 Porphyrin-Based Nanocomposites for Biosensing

Fig.4.12 O 1s high-resolution XPS spectra of TiO2-based samples. Reprinted with permission


from Chen etal. [49]. 2008, Elsevier

Fig.4.13 XRD patterns of (a) TiO2, (b) Nb-added TiO2, (c) Ge-added TiO2, and (d) Zr-added
TiO2. Reprinted with permission from Imahori etal. [50]. 2006, American Chemical Society
4.3 Assembly of Porphyrins on Semiconductor Nanoparticles 127

exhibit a similar XRD pattern. This implies that the TiO2 anatase nanocrystalline
structure is retained after doping a small amount (5mol%) of Nb, Ge, or Zr in the
TiO2 structure [50]. It is the first systematic comparison of the electrode structures
and photovoltaic properties of porphyrin-sensitized solar cells with TiO2 composite
electrodes in which the Ti atom is partially substituted with other metals (i.e., Nb,
Ge, and Zr).

4.3.1.3Photoelectrochemical and Electrochemical Sensing

The electron transfer at the semiconductordye interface has been successfully uti-
lized in the development of solar cells, electronic devices, heterogeneous photoca-
talysis, and biosensing. Manganese oxides, having economic and environmental
advantages, have been used for a long time in air electrodes as electrocatalysts for
the reduction of O2. Combined with porphyrin, a binary catalyst composed of elec-
trodeposited manganese oxide nanoparticles (nano-MnOx) and cobalt porphyrin
has been proposed for the efficient four-electron reduction of molecular oxygen to
water in acidic media [51]. The modification of GCE with cobalt porphyrin alone
results in a significant positive shift of the oxygen reduction reaction (ORR) com-
pared to the unmodified GCE, which maintained a two-electron reduction. A posi-
tive shift of the onset potential of the ORR of ca. 450mV is achieved at the former
electrode. The modification of the GCE with nano-MnOx alone does not affect the
ORR peak potential, but causes a remarkable increase in the reduction peak current
due to the catalytic disproportionation of the electrogenerated hydrogen peroxide
into water and oxygen. The modification of a GCE with both cobalt porphyrin and
nano-MnOx results in the occurrence of the ORR at a significantly positive potential
with almost double the peak current compared to the unmodified GCE (Fig.4.14),
suggesting a promising procedure for developing electrocatalysts to oxygen reduc-
tion in replacement of costly Pt.
An improved photocurrent generator can be prepared easily by spin coating a
Nafion/porphyrin/TiO2 mixture onto an ITO substrate. The generated photocurrent
density is about 10 times higher than that in the absence of TiO2 (Fig.4.15). The
photocurrent density increases linearly concomitantly with high surface concentra-
tions of porphyrin and high membrane thickness. It becomes evident that TiO2 con-
tributes to more efficient photocurrent generation by intramembrane electron
mediation [52].
SiO2/TiO2/phosphate can be obtained by the solgel processing method, and then
H2TMPyP is immobilized on the matrix surface by an ion-exchange reaction and
metallated in situ with Co(II), resulting in an SiO2/TiO2/phosphate/CoTMPyP mate-
rial. The amount of CoTMPyP incorporated into the matrix is 35.0 mmol/g. The
immobilized complex catalyzes O2 reduction to H2O at 0.22 V in 1 mol/L KCl
solution at pH 6.8 (Fig.4.16). The cathodic current intensities plotted against O2
concentrations between 1 and 11ppm show a linear correlation [53]. Hematoporphyrin
IX and protoporphyrin IX are efficiently immobilized on a cellulose/titanium (IV)
oxide composite fiber surface by the reaction of the porphyrin COOH groups with
128 4 Porphyrin-Based Nanocomposites for Biosensing

Fig. 4.14 Cyclic voltammetries obtained at (a) bare GC and modified with (b) nano-MnOx,
(c) cobalt porphyrin, and (d) cobalt porphyrin and nano-MnOx GCE in O2-saturated 0.1M H2SO4.
Scan rate: 100mV/s. Reprinted with permission from EI-Deab etal. [51]. 2008, Springer

Fig. 4.15 Photoelectrochemical responses of the Nafion-TiO2-porphyrin (solid line) and the
Nafion-porphyrin (dashed line) membranes on ITO electrodes. Reprinted with permission from
Ikeda etal. [52]. 2005, American Chemical Society

TiO2, presumably by forming the COO-Ti chemical bond. The resulting sensors
show a linear range, from 0.5 to 13mg/L, to the O2 reduction [54].
The quantification of phenolic derivatives is of great importance since many of
these compounds, even in small proportions, easily penetrate through the skin and
4.3 Assembly of Porphyrins on Semiconductor Nanoparticles 129

Fig.4.16 Cyclic
voltammetric curves obtained
for SiO2/TiO2/phosphate/
CoTMPyP in various
dioxygen concentrations: (a)
desaerated; (b) 3.6, (c) 7.1,
and (d) 10.0ppm. Reprinted
with permission from
Castellani and Gushikem
[53]. 2000, Academic
Press

membranes of animals and plants, causing genotoxic, mutagenic, and hepatotoxic


effects. A carbon paste electrode with SiO2/Nb2O5 oxideadsorbed Ni-porphyrin
can be used to investigate the detection of hydroquinone and 4-aminophenol by
electrochemical methods in KCl-supporting electrolyte solution. The as-prepared
biosensor shows a higher catalytic efficiency than the electrode whose oxidation is
mediated by the semiconducting property of Nb2O5. The reason for this is that the
former presented remarkably less positive potential for phenolic compounds. The
concentration ranges of linear chronoamperometric answers are 11,000 and
1900mmol/L for hydroquinone and 4-aminophenol, respectively [55].

4.3.2Quantum Dots

IIVI semiconductor nanoparticles, due to the wide band gap and controlling emis-
sion, are particularly interesting for the development of novel optoelectronic devices
like light-emitting diodes, lasers, and transistors. Hetero-nanoassemblies in toluene
solution have been formed via anchoring pyridyl substituted free-base porphyrin mol-
ecules (Pyr)nH2P on the colloidal core-shell semiconductor nanocrystals CdSe/ZnS
QDs. Only one molecule is estimated to anchor on one nanocrystal even at high molar
ratios [56]. Then, the above-outlined self-assembly principle is organized where
molecular arrays anchor on semiconductor QD surfaces in a systematic way. The
quenching of the fluorescence is partly related to fluorescence resonance energy trans-
fer from the QD to H2P and can be explained according to the Frster model [57].
A new type of self-assembled film has been prepared by alternating the deposi-
tion of oppositely charged meso-tetra-(4-trimethylaminophenyl) porphyrin
nickel iodide(NiTAPPI) and citrate-stabilized CdSe nanoparticles [58]. The SEM
images show the formation of densely packed 2D arrays and the conversion
from disorder to order of CdSe nanoparticles on the quartz substrate modified by
poly(diallyldimethylammonium) chloride (PDDA) when depositing positively
130 4 Porphyrin-Based Nanocomposites for Biosensing

Fig. 4.17 Mode of interaction between porphyrins and thioglycolic acid-capped CdTe QDs.
Reprinted with permission from Jhonsi and Renganathan [59]. 2010, Academic Press

Fig.4.18 Synthesis of Fe3O4porphyrin nanocomposite. Reprinted with permission from Gu etal.


[60]. 2005, Royal Society of Chemistry

charged NiTAPPI. Placed in ambient air, the self-assembled film exhibits a significant
enhancement in fluorescence intensity.
The photoinduced interaction of thioglycolic acid-capped CdTe QDs with
porphyrins provides a promising way to assemble porphyrin on QDs (Fig.4.17). The
QDs surface is negatively charged since the thiol capping agent contains a carboxylic
group. Positively charged TMPyP interacts with QDs through charge-transfer mech-
anism, negatively charged porphyrins (TCPP and TSPP) interact through an energy-
transfer mechanism, and the neutral TPP does not have any interaction [59].
4.4 Assembly of Porphyrins on Metal Nanoparticles 131

4.3.3Fe3O4 Nanoparticles

Porphyrin derivatives and iron oxides are complementary in both properties and
functions. The conjugation of porphyrin and iron oxide nanoparticles may lead to a
bimodal anticancer agent that can be used in the combinational treatment of photo-
dynamic therapy and hyperthermia therapy. Figure 4.18 illustrates the synthetic
pathway for making the conjugation of Fe3O4porphyrin nanoparticles. After an
N-hydroxysuccinimide-activated derivative of dopamine reacts with the diaminopo-
rphyrin, a simple deprotection is used to remove the benzyl groups and affords com-
pound 4 in good yield (65%). Reacting compound 4 (5 mg, in 2 mL of MeOH/
CHCl3 1:1) with magnetite nanoparticles (30mg, in 5mL of hexane) in an ultrasonic
bath for 60min gives a reddish-brown mixture, which is centrifuged and redissolved
in methanol. After the methanol solution is washed 3 times using chloroform, high-
speed centrifugation yields the final product for bimodal anticancer therapy [60].

4.4Assembly of Porphyrins on Metal Nanoparticles

The SAMs of porphyrins on flat metal substrates or equivalents have been exten-
sively studied, with the aim of developing artificial photosynthetic materials.
However, the light-harvesting efficiency in the 2D systems has so far been limited
due to the porphyrin monolayer, which can absorb little light. Metal nanoparticles
involving Au, Ag, and Pt nanoparticles can provide three-dimensional (3D) archi-
tectures, which have attracted widespread interest, since their nanosized physical
properties are quite different from those of the bulk materials. The potential applica-
tions of metal nanoparticles have been extensively applied in biochemical sensors,
nanostructure fabrication, and optoelectronic devices.

4.4.1Au Nanoparticles

4.4.1.1Functionalization and Characterization

Multipoint AuS bond formation is a straightforward way to fix functional mole-


cules in the desired geometry. One-pot reduction of HAuCl4 in a DMF solution
containing a porphyrin-cored tetradentate passivant produces horizontal porphy-
rin monolayer-coated gold nanoparticles. The porphyrin is expected to build fast
and tight connections with the gold surface via four AuS bonds since all four S
atoms are preoriented to form AuS bonds and do not rotate away due to steric
hindrance between the amide moiety and the porphyrin plane (Fig.4.19). Particle
analysis reveals that the nanocomposite has a mean diameter of 3.50.7 nm,
whose particle size is significantly smaller than that made by using monodentate
passivants under identical conditions [61]. These nanoparticles exhibit signifi-
cantly enhanced anion-binding capability compared with the corresponding free
132 4 Porphyrin-Based Nanocomposites for Biosensing

Fig.4.19 The structure of the composite and TEM images with size distribution. Reprinted with
permission from Ohyama etal. [61]. 2008, Royal Society of Chemistry

porphyrin receptors in solution, and have been shown to recognize anions for
chloride and dihydrogen phosphate [62]. To assess the quantitative significance of
multiple interactions, monolayer-protected gold nanoclusters (MPCs) with a
mixed monolayer containing different loadings of N-methylimidazole as ligand
have been exploited for the recognition of discrete porphyrin arrays, which
increases the binding strength by up to three orders of magnitude with respect to
a monovalent system [63].
In addition, alkanethiolate MPCs are stable in air, and soluble in both nonpolar
and polar organic solvents; therefore, they are capable of facile modification with
other functional thiols through exchange reactions or by couplings and nucleophilic
substitutions. Thus, constructing the 3D architectures of porphyrin MPCs, which
have a large surface area, would improve the light-harvesting efficiency as com-
pared to the 2D porphyrin SAMs [64]. The first successful synthesis and the photo-
physical properties of porphyrin MPC were reported by Fukuzumis group. The
gold nanoparticles, unlike their bulk counterparts, do not quench the fluorescence of
porphyrin MPCs intensively [65]. Furthermore, under IR light irradiation, a mixed
toluene solution of ammonium saltstabilized gold nanoparticles with (3.80.8)-
nm core diameter and a porphyrin thioacetate derivative afford a thin photoactive
film of the clusterporphyrin network [66].
Gold nanoparticles functionalized with imidazolylporphyrinatozinc(II) have
been bridged by successive imidazole-to-zinc coordinations of bidirectional
porphyrinatozinc(II) units. AFM images of samples prepared from two different
concentrations in (CHCl2)2 (9103 and 3104 M) are shown in Fig. 4.20. The
higher-concentration sample formed long and overlain wires in AFM images,
whereas much shorter and nonoverlain wires are mostly observed in the other AFM
image. The average length of the supramolecular wires in the right-hand AFM
image is 7635nm, but wires with lengths greater than 200nm are also observed
[67]. The supermolecular electronics is ready to use in next-generation electronic
devices.
4.4 Assembly of Porphyrins on Metal Nanoparticles 133

Fig.4.20 AFM images of the composite on mica; (left) 9103M and (right) 3104M (CHCl2)2
solutions were deposited. Reprinted with permission from Satake etal. [67]. 2009, Royal Society
of Chemistry

4.4.1.2Electrocatalysis and Electrochemical Biosensing

The first example of the cocatalyst effects of gold clusters in the enhanced activity
of Mn-porphyrin catalyst was reported by Konishi in 2007. The Au cluster led to
appreciable acceleration of the catalytic reaction of Mn(TPP)Cl (TPP) tetraphe-
nylporphinato toward styrene oxidation (Fig.4.21). The major role of the Au cluster
was to regenerate the active catalytic path involving Mn(III) and Mn(V) from the
deactivated Mn(IV) species [68].
Through electrostatic layer-by-layer (LBL) assembly, AuCl4 anions and CoTMPyP
cations have been alternately deposited on ITO substrates and 4-aminobenzoic acid
modified GCE. The electrochemical reduction of AuCl4 anions sandwiched between
CoTMPyP layers leads to the in situ formation of Au nanoparticles in the multilayer
films. The resulting composite films containing Au nanoparticles with high stability
exhibit high electrocatalytic activity with the two-electron reduction of O2 to H2O2 in
O2-saturated 0.1M H2SO4 solution [69]. A simple, efficient, and sensitive sensor for
dissolved oxygen has been proposed by combining a self-assembly monolayer of
mono-(6-deoxy-6-mercapto)-b-cyclodextrin, FeTMPyP, and cyclodextrin-functional-
ized gold nanoparticles. The supramolecular-modified electrode shows excellent cata-
lytic activity for oxygen reduction, with a 200-mV positive shift of the reduction
potential compared with bare gold electrode. The ORR probably involves four elec-
trons with a rate constant of 7104 mol/L/s. Alinear response range from 0.2 to
6.5mg/L, with a sensitivity of 5.5mAL/mg and a detection limit of 0.02mg/L, is
obtained. The repeatability of the sensor, evaluated in terms of RSD, is 3.0% for ten
measurements of a solution of 6.5mg/L of oxygen [70].
134 4 Porphyrin-Based Nanocomposites for Biosensing

Fig.4.21 Schematic illustration of a possible catalytic mechanism in the Au cluster/Mn(TPP)Cl


system. Reprinted with permission from Murakami and Konishi [68]. 2007, American Chemical
Society

An amperometric artemisinin (ARN) sensor has been developed based on the


supramolecular recognition of glycosylated metalloporphyrin, which is included in
the Au NPchitosan film coated on GCE. The proposed glycosylated metallopor
phyrin/Au nanoparticle-modified electrodes show excellent selectivity and sensitivity
toward ARN with respect to a number of interferents and exhibited stable current
response. The calibration range for ARN is from 1.8107 to 1.7109mol/L, with
a detection limit of 1.7109mol/L. Significant advantages of the proposed proce-
dure over the conventional reductive electrochemical methods are the selective detec-
tion and the relatively low applied potential requirement of the ARN sensor [71].

4.4.2Ag Nanoparticles

Ag nanoparticles are the enhancer of surface-enhanced (resonance) Raman scatter-


ing [SER(R)S] spectroscopy, which is widely employed for porphyrin study at low
concentrations [72]. The SERRS of three differently charged free-base porphyrins,
namely, cationic TMPyP, anionic TSPP, and neutral TPP, have been measured on
SERS-active substrates prepared by immobilizing Ag or Au nanoparticles on
silanized glass plates. Au surfaces are suitable for TMPyP and TSPP detection,
while Ag surfaces are suitable for TMPyP and TPP detection. SERRS spectra
exhibit excellent reproducibility and very good stability (RSD lower than 10%) and
do not contain any signal of the porphyrin metalation or perturbation of its native
structure. Even SERRS spectra of water-insoluble TPP can be obtained without any
4.4 Assembly of Porphyrins on Metal Nanoparticles 135

Fig.4.22 SERRS spectra of TPP measured from Ag surfaces; soaking concentrations from bot-
tom to top: 1107M, 4107M, 8107M, 2106M, 3106M, and 6106M. Reprinted with
permission from Hajdukov etal. [73]. 2008, Elsevier

interference of solvent Raman signal. Estimated limits of detection (LOD) are


between 3109 and 8108 M porphyrin concentrations in soaking solution
(Fig. 4.22). SERRS on immobilized Ag and Au nanoparticles is found to be a
suitable analytical method for detecting free-base (including water-insoluble)
porphyrins [73].
The electrostatic LBL adsorption technique can be used to fabricate Ag
nanoparticleporphyrin composite films on ITO electrodes. A remarkable enhance-
ment in the photocurrent action and fluorescence excitation spectra is observed for
porphyrin when considerable amounts of Ag nanoparticles are deposited onto the
ITO electrode [74].

4.4.3Pt Nanoparticles

Pt nanoparticles (Pt NPs) are usually used as catalysts for hydrogen evolution.
A tetraphenylporphyrin bearing four naphthalene donor moieties 5,10,15,20-
tetrakis(4-(naphthalen-1-ylmethoxy)phenyl)porphyrin has been synthesized to form
functionalized platinum nanocomposite. The photoreceptive dye forms a shell con-
taining a nanosized Pt core (~2.8nm). The photocatalytic activity of the Pt nano-
composite toward water reduction to hydrogen is twice stronger than that of a
Ptnaphthalene-free porphyrin system [75].
136 4 Porphyrin-Based Nanocomposites for Biosensing

Fig. 4.23 Amperometric current response vs. concentration for five organohalides using a Pt
NPZn porphyrin nanocomposite in 0.1M TBAP/ACN. Reprinted with permission from Wiyaratn
etal. [78]. 2005, American Chemical Society

The LBL self-assembly method, initially developed for pairs of oppositely


charged polyelectrolytes, is a facile and versatile technique that has recently emerged
as a visible approach for the preparation of a 3D superstructure array of nanoparti-
cles. Through electrostatic LBL assembly, negatively charged citrate-stabilized Pt
NPs and positively charged CoTMPyP are alternately deposited on both a 4-amin-
obenzoic acid-modified GCE and ITO substrates, directly forming the 3D nano-
structured materials. The Pt NPs containing multilayer films exhibit high
electrocatalytic activity for the reduction of dioxygen with high stability. Rotating
disk electrode and rotating ring-disk electrode voltammetric analyses demonstrate
that the Pt NP-containing multilayer films can catalyze an almost four-electron
reduction of O2 to H2O in an air-saturated 0.5M H2SO4 solution. The high electro-
catalytic activity and good stability for dioxygen reduction make the Pt NP-containing
multilayer films potential candidates for the efficient cathode material in fuel cells
[76, 77].
Pt NPZn porphyrin nanocomposites have been synthesized using zinc porphy-
rin and dihydrogen hexachloroplatinate in the presence of light and ascorbic
acid. The Pt NPZn porphyrin nanocomposite, with an average diameter of ~3.5nm,
exhibits catalytic activity for the reduction of five organohalides, involving carbon
tetrachloride, chloroform, pentachlorophenol, chlorobenzene, and hexachloroben-
zene, at 1.0V vs. Ag/AgCl. The above two aliphatic and three aromatic organoha-
lides have detection limits of 0.5mM, with linearity up to 8mM (Fig.4.23). The
modified electrode is good for at least 80 repeated measurements of 4 mM chlo-
robenzene with a storage stability of 1 month at room temperature [78].
4.5 Other Nanomaterials 137

4.5Other Nanomaterials

4.5.1Polymer Nanoparticles

Polymers permit sufficient flexibility to assist the transport of the substrates, which
may be essential for artificial enzymes. For example, a new anionic water-soluble
polythiophene and a cationic porphyrin have been synthesized through electrostatic
interactions. Based on the enhanced energy transfer offered by light-harvesting con-
jugated polymers, the singlet oxygen is produced and effectively kills the bacteria,
with about a 70% reduction of bacterial viability in only 5min of irradiation under
white light (400800nm) [79]. The polymer nanocomposite of the polyelectrolytes
poly(allylamine hydrochloride) and poly(styrene sulfonate) and TPPS are fabricated
as photoactive microcapsules via LBL self-assembly [80]. In addition, a formylpor-
phyrin has been covalently bound to poly(allylamine hydrochloride) via a Schiff base
intermediate for the electrocatalytical oxidation of the sulfite and nitrite [81].
Oxygen is a critical component for many physiological and pathological processes in
living cells. A novel nanoparticle architecture, consisting of p-conjugated polymer mol-
ecules doped with an oxygen-sensitive phosphorescent dye, has been described for oxy-
gen sensing. The conjugated polymers employed as the doping host are the polyfluorene
derivatives poly(9,9-dihexylfluorene) (PDHF) and poly(9,9-dioctylfluorene) (PFO).
Platinum(II) octaethylporphine (PtOEP) serves as the oxygen-sensitive dye (Fig.4.24).
The rapid addition of a solution of polyfluorene and PtOEP in THF to water results in
nanoparticle formation and the simultaneous entrapment of the hydrophobic PtOEP
molecules inside the nanoparticles via the collapse of polymer chains. AFM results for
PDHF-based particles indicate that the resulting particles are approximately spherical in
shape, with particle heights (diameters) of 255nm. Upon the light excitation, the poly-
mer efficiently transfers the energy to phosphorescent dye, resulting in a bright phospho-
rescence that is highly sensitive to the concentration of dissolved oxygen [82]. A
polystyrene-based oxygen nanosensor has been constructed using platinum(II)meso-
tetra(pentafluorophenyl)porphine via spin-coating the polymer on glass plates. The sen-
sor response is assessed in aqueous solution as well as in yeast culture [83].
A porphyrin-containing copolymer having dual sensing in response to metal ions
and temperature has been used to fabricate a novel nanosensor [84]. First, a specific
triblock copolymer is designed and synthesized by sequential reversible-addition-
fragmentation chain-transfer polymerization. Below 32C, the addition of different
metal ions to the solution of copolymer leads to an unprecedented full spectral color
range (Fig. 4.25). Most surprisingly, upon heating, the multicolored nanosensors
display discrete thermochromic characteristics in the temperature range of 3561C,
with the phase-transition point dependent on the metal ion. The thermochromism
speed was typically less than ~30s, and the temperature range of the color transition
is smaller than 0.4C. This sensor displays an isothermal thermochromic point as
an ultrasensitive thermometer.
Of recent interest are the magnetic polymer nanospheres, where the core is com-
posed of numerous Fe3O4 particles and the shell is composed of a copolymer of
138 4 Porphyrin-Based Nanocomposites for Biosensing

Fig. 4.24 (a) Schematic illustration of the formation of conjugated polymer dots for oxygen
sensing. (b) AFM image of PtOEP-doped PDHF dots dispersed on a mica substrate. (c) Histogram
of particle-height data obtained from AFM image in (b). Reprinted with permission from Wu etal.
[82]. 2009, Wiley

styrene and Mn(III) porphyrin. The catalytic efficiencies of these magnetic


nanospheres to hydroxylate cyclohexane with molecular oxygen are much higher
than those of the nonsupported Mn(III) porphyrin analogs. It is also found that these
nanospheres have good magnetic responsiveness and thus can be completely recov-
ered by applying an external magnetic field. The magnetic polymer nanospheres
may be helpful in designing new, highly efficient metalloporphyrin catalysts [85].

4.5.2Silica Nanomaterials

Amorphous silica is one of the popular traditional materials used as a matrix for
incorporating functional molecules. The most attractive features of silica include
nontoxicity, high porosity, and effective transparency. In general, the 3D space in
silica is sufficiently adjustable to hold various functional molecules. Porphyrins
embedded in silica are used as biomimetic catalysts in various sensor devices for
4.5 Other Nanomaterials 139

Fig.4.25 Simplex-stimuli-sensing thermochromic sensor in the absence of metal ions (left), and
dual-sensing optical sensors (full-spectrum colorimeter and ultrasensitive thermometer) upon
introduction of different metal ions (right). Reprinted with permission from Yan et al. [84].
2010, Royal Society of Chemistry

clinical analysis. For example, a transparent hybrid silica material encapsulating


5,10,15,20-tetrakis(3,4-dimethoxyphenyl)-21H,23H-porphyrin has been designated
via acidbase-catalyzed hydrolysis and the condensation of tetraethylorthosilicate,
for advanced optoelectronic devices [86]. The entrapment of protoporphyrin IX in
silica spheres generates singlet oxygen for use in photodynamic therapy by modify-
ing protoporphyrin IX molecules with an organosilane reagent [87].
On the other hand, the encapsulated molecules may be affected by physical and
chemical properties, including reaction kinetics by the nanomatrix. The metalation
of meso-tetra(4-N,N,N-trimethylanilinium) porphyrin tetrachloride (TTMAPP) with
Cu(II) was selected as a model for a kinetic study [88]. The results demonstrated
that the encapsulated molecules retain reactivity, but the reaction-rate constant
greatly declines in the silica nanomatrix in comparison to free TTMAPP molecules
in a bulk solution.
The silica nanocomposites are potentially useful chemosensory materials for
rapidly detecting trace analysts. Two kinds of porphyrin-doped silica films with
mesoporous structures have been fabricated using the evaporation-induced self-
assembly approach and examined for chemosensor applications to detect explosive
compounds such as 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, and nitrobenzene by
fluorescence quenching with a high sensitivity of 10ppb [89].
140 4 Porphyrin-Based Nanocomposites for Biosensing

Fig.4.26 (a) Chemical structure of the zinc(II) porphyrin derivative; (b) schematic drawing of the
SiNW-FET device from silicon-on-insulator. Reprinted with permission from Winkelmann etal.
[90]. 2007, American Chemical Society

Porphyrin derivative-coated silicon nanowire is promising as field-effect


transistors(SiNW-FETs) (Fig.4.26), as it displays a large, stable, and reproduc-
ible conductance increase upon illumination. The decay kinetics from the
high- to the low-conductance state is governed by charge recombination via
tunneling, with the rate depending on the state of the SiNW-FET. The compari-
son to porphyrin-sensitized CNT FETs allows the environment- and molecule-
dependent photoconversion process to be distinguished from the charge-to-current
transducing effect of the semiconducting channel [90]. Porphyrin-sensitized
SiNW-FETs are, furthermore, extremely promising tools for sensing applications
such as artificial eyes.

4.5.3Calcium Phosphate Nanoparticles

Calcium phosphate nanoparticles have gained increasing interest in recent years


from their high biocompatibility, which is due to the fact that calcium phosphate
constitutes the inorganic mineral of mammalian bone and teeth. Calcium phosphate
nanoparticles can act as drug carriers, e.g., for nucleic acids or for antitumor drugs.
For example, calcium phosphate nanoparticles are functionalized with 5,10,15,20-
tetrakis(4-phosphonooxyphenyl)porphine (p-TPPP) as biocompatible fluorescing
agents [91]. No other stabilizing agent is necessary, and all dissolved counterions
and excess porphyrin can be removed by ultracentrifugation. The resultingporphy-
rin-functionalized nanoparticles are spherical with a diameter of about 250 nm
(Fig.4.27). In a cell culture with NIH 3T3 fibroblast cells, the particles show excel-
lent biocompatibility and good fluorescence in the visible region of the spectrum,
which foretells promising applications in biosensing.
4.6 Conclusions 141

Fig.4.27 SEM picture of


p-TPPP-functionalized
calcium phosphate
nanoparticles. Reprinted with
permission from Ganesan
etal. [91]. 2008, Royal
Society of Chemistry

4.6Conclusions

Porphyrins are the mimics of many important enzymes and can be modified with
other functional moiety on the porphyrin ring to enhance the selectivity toward
biomolecular catalysis. Furthermore, depending on the nanomaterials properties,
the hybrid nanocomposite of porphyrin nanomaterials can be designed and achieved
via either noncovalent or covalent interactions. The former can keep the electronic
structure of the nanomaterials, and the latter can more efficiently obtain the defined
3D superstructure of the hybrid nanocomposite. Combined with the unique struc-
ture of nanomaterials, the resulting nanocomposite demonstrated excellent optical
and electrochemical biosensing toward life-relating molecules and provided a prom-
ising application potential in bioanalysis.
In order to improve the performance of the nanocomposite in biosensing, two
problems should be addressed: The first is improving the binding between porphy-
rins and nanomaterials since many anchoring groups for assembly are instable
against water, acids, and bases in their applications. The emerging field of click
chemistry has the potential to provide an elegant protocol to prepare porphyrin-
based functional nanomaterials since the reaction is versatile and clean, and can be
operated in very mild conditions without significantly disturbing the conjugated
p-system [92]. Second, it is highly desirable to seek a novel material with a suffi-
cient binding site for functionalization. The doping heteroatom nanostructure, such
as nitrogen-doping CNTs, may be the best candidate for functionalization. The
ordered assembly of porphyrins on the nanomaterials provides a powerful platform
to construct the biosensor with high selectivity and sensitivity and shows promising
applications in biosensing.
142 4 Porphyrin-Based Nanocomposites for Biosensing

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Chapter 5
Carbon Nanofiber-Based Nanocomposites
for Biosensing

5.1Introduction

The history of carbon nanofiber (CNF) can go back more than a century. It was
reported in a patent published in 1889 that carbon filaments are grown from carbon-
containing gases using a metallic crucible as the probably unintentional
catalyst [1]. In 1950, a Russian group performed the first electron microscopy
observations of CNFs. For the first 80 years of the twentieth century, however, the
occurrence of CNFs then often referred to as carbon filaments or filamentous
carbon was considered a nuisance. For example, in FischerTropsch or steam-
methane reforming reactions, the fibers often occurred in metallic catalysts used for
the conversion of carbon-containing gases. In 1991, carbon nanotubes (CNTs) were
first discovered as a new member of the carbon allotrope family. This discovery and
other nanostructures triggered an outburst of interest in CNTs and nanofibers [2].
Generally, the nanotubes can be divided into two categories: single-walled car-
bon nanotubes (SWNTs) and multiwalled carbon nanotubes (MWNTs). MWNTs
are composed of coaxial, multilayer graphene tubes with an interlayer space of
0.34nm, and the diameter for MWNTs varies from 1.4 to 100nm. CNFs are similar
to a large-diameter MWNT; however, CNFs are not continuous like the tubes, and
their surfaces show steps at the termination of each tube wall, forming cylindrical
nanostructures. CNFs can be divided into platelet CNFs, tubular CNFs, and her-
ringbone CNFs, according to the different arrangement of graphene layers. As
shown in Fig.5.1, the graphene layers of platelet CNFs are vertical in relation to the
fiber axis, and the exposed surfaces are mainly occupied by edge atoms. The gra-
phene layers of tubular CNFs are parallel to the fiber axis, and many basal atoms are
exposed; the graphene layers of herringbone CNF incline toward the fiber axis, and
the ratio of edge atoms to basal atoms can be adjusted by controlling the angle of
graphene layers to the fiber axis [3].
CNFs can be produced by various methods, such as arc-discharge [4], laser
ablation [5], chemical vapor deposition (CVD) methods [6, 7], and others [8, 9].

H. Ju et al., NanoBiosensing: Principles, Development and Application, 147


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0_5, Springer Science+Business Media, LLC 2011
148 5 Carbon Nanofiber-Based Nanocomposites for Biosensing

Fig.5.1 Schematic representation of different types of CNTs and CNFs. Reprinted with permission
from Serp etal. [3]. 2003, Elsevier

The arc-discharge and laser ablation methods lead to mixtures of carbon materials
and thus to a cumbersome purification to obtain nanofibers or nanotubes. From an
application point of view, the catalytic growth of nanofibers should be most promis-
ing [1, 6]. CNFs also can be grown in such a way that the nanofibers are all
vertically aligned to form vertically aligned carbon nanofibers (VACNFs), which
are emerging as a useful material for applications such as chemical/biochemical
sensing [1012]. VACNFs are aligned with each fiber approximately perpendicular
to the underlying growth substrate, providing each nanofiber with a direct electrical
connection to an underlying electrode. Although the more commonly studied
SWNTs and MWNTs expose primarily basal-plane graphite, VACNFs consist of
nested cones of graphene that expose large amounts of edge-plane graphite along
their sidewalls. Electron transfer (ET) rates at edge-plane graphite are ~105 times
faster than those at the basal plane [13]; this implies that VACNFs may have out-
standing properties as supports, for example, for electrocatalytic reactions.
CNFs are very promising materials for the development of biosensor systems
since they possess several striking properties that have proven to be very suitable for
the construction of biosensor systems, such as excellent electrical conductivity,
unique structural and catalytic properties, high loading of biocatalysts, good stabil-
ity, and so on. These striking properties of CNFs have triggered intensive research
efforts to utilize them to develop various kinds of advanced biosensors with higher
sensitivity, faster response, biocompatibility, and cost-effectiveness [1417]. This
chapter surveys the current status of CNF-based nanocomposites for biosensor
5.2 Synthesis of Carbon Nanofiber 149

applications, with a particular emphasis on electrochemical biosensors. The scope


of this chapter is mainly limited to the electrochemical detection of common and
important biological compounds such as glucose, ethanol, acetylthiocholine, phe-
nol, and hydrogen peroxide in addition to the catalytic oxidation reaction of NADH
at CNFs, which is one of the indispensable reactions of dehydrogenase-based elec-
trochemical biosensors, and to the ET process of redox proteins at CNFs.
Additionally, CNF-based electrochemical immune sensors are also discussed,
because CNF-based electrochemical immunosensors have several advantages and
have attracted extensive interest as bioanalytical devices in recent years. Finally,
because the functionalization of VACNFs is essential for the applications of VACNFs
in the development of electrochemical biosensors, the advances in the functional-
ization of VACNFs are discussed. Representative examples that use CNF-based
nanocomposites to improve analytical performances of biosensor systems will be
presented.

5.2Synthesis of Carbon Nanofiber

In CVD methods, the most important metals to catalyze the growth of graphitic
CNFs are (alloys of) iron, cobalt, and nickel; chromium, vanadium, and molybde-
num have also been studied [1, 6, 18]. The metals have been used both as bulk
particles (size typically 100nm) and as supported particles (1050nm). All of these
metals can dissolve carbon and/or form metal carbonides. Typically, methane,
carbon monoxide, synthesis gas (H2/CO), ethyne, and ethene in the temperature
range of 7001,200K are employed to provide carbon atoms [1].
A model for the nucleation and growth of CNFs is shown in Fig.5.2 [1]. Methane
decomposes into carbon and hydrogen atoms at the nickel surface (see step 2 in
Fig.5.2). H2 molecules desorb and carbon dissolves and forms (substoichiometric)
nickel carbide (see step 3 in Fig.5.2). This nickel carbide is metastable with respect
to nickel metal and graphite. After, say, 10min, the carbide phase decomposes into
metallic nickel and graphite, which encapsulates the nickel particle in question (see
step 4 in Fig. 5.2). According to this model, the metal particle is squeezed out
because of pressure buildup due to the formation of graphite layers at the internal
surface of the graphite envelope (see step 5 in Fig.5.2). As soon as the metal is
pushed out, the fresh surface is exposed to the methane and growth continues.
Finally, a steady-state process occurs with either pulsed growth (see step 6a in
Fig.5.2) or smooth growth of a straight fiber (see step 6b in Fig.5.2). This model
also explains why, more often than not, metal particles are found at the tip of the
carbon fiber: The graphite fiber pushes the metal particle from the support and con-
tinues to grow at the back of the particle.
It is known that the diameter of the nanofibers is governed by that of the catalyst
particle responsible for their growth, and the structure of CNFs can vary depending
on the type of catalyst and precursor used during synthesis. It has been reported that
the CNF morphology consists of revealed graphite platelets stacked perpendicularly
150 5 Carbon Nanofiber-Based Nanocomposites for Biosensing

Fig.5.2 Mechanism for the nucleation and growth of a carbon nanofiber (CNF) from methane
catalyzed by a supported metal particle. Reprinted with permission from De Jong and Geus [1].
2000, Marcel Dekker

to the fiber axis when produced from iron catalyst and carbon monoxide/hydrogen
precursor (4:1) at 600C. The graphite platelets were parallel to the fiber axis with
silica-supported iron catalyst, even though the same carbon precursor and synthesis
conditions were used [19, 20]. Currently, most of the catalyst methods for growing
CNTs and CNFs use transition metal particles (Fe, Co, Ni) in the presence of hydro-
carbons at high substrate temperatures ranging between 450 and 1,250C. Boskovic
etal. reported a large-area synthesis of CNFs at room temperature for the first time
[6]. They used hydrocarbon plasmas to provide the energy dynamics necessary for
the dissociation of carbon and the subsequent catalytic growth of CNF on transition
metal particles at room temperature. CNFs were synthesized from radio-frequency
plasma-enhanced CVD at room temperature with Ni powder catalyst placed on
graphite, silicon and plastic substrates at the earthed electrode, and methane or
methane/hydrogen as carbon sources. CNFs synthesized by this method are consi
dered to be potential candidates for many possible applications, such as large-area
flat-panel displays, electrochemical cells, and nanoelectronics.
Very recently, the effects of catalyst support and carbon source on the yield and
structure of carbon were reported [18, 21]. Yamada etal. quantitatively examined
CNF formation on an iron group metal loaded on spherical silica. They found that,
at optimal conditions, the amount of CNF increased in the following order:
FeCo<Ni, irrespective of carbon sources. They also found that on Ni/SiO2, poorly
crystallized CNFs were produced by the tip-growth mechanism. However, Co/SiO2
gave tip- and bottom-growth CNFs, and on Fe/SiO2 small amounts of bottom-growth
5.4 Carbon Nanofiber-Based Electrochemical Biosensors and Bioassays 151

CNFs were produced with cylindrical graphene sheets. Yamada concluded that the
growth mechanism of CNF was strongly affected by the interaction between iron
group metals and SiO2 [18].

5.3Why Carbon Nanofiber?

Of the carbon-based nanomaterials, such as CNTs, CNFs, fullerenes, graphite and


graphene, carbon nanohorns, and nanoporous structures, CNTs are most widely
used in electrochemical biosensor systems. Compared with CNTs, however, CNFs
have a higher ratio of surface-active groups to volume [22], better mechanical
stability [23], easier mass production, and lower production cost; for example,
the CNF materials are 100 times lower in cost than SWNTs [17]. Particularly,
CNFs possess more edge sites on the out wall than CNTs; thus, a range of oxygen-
containing groups can be produced at these sites, e.g., by the oxidation treatment
of CNFs with nitric acid without degradation of the structural integrity of its back-
bone. These unique properties make CNFs extremely attractive for the development
of biosensors [1417, 22, 24]. For example, the high conductivity of CNFs seems to
be ideal for the electrochemical signal transduction and the surface-active groups-
to-volume ratio of CNFs. When combined with the fact that the number and type of
functional groups on the outer surface of CNFs can be well controlled, it is expected
to allow for the selective immobilization and stabilization of functional biomole-
cules such as proteins, enzymes, and DNA. Based on these unique properties, CNFs
have recently emerged as a potential candidate to replace CNTs, and CNF-based
biosensors are expected to have a higher sensitivity as well as improved operational
and storage stabilities over those prepared with CNTs [1517, 22, 24, 25].

5.4Carbon Nanofiber-Based Electrochemical Biosensors


and Bioassays

5.4.1Glucose Sensors

Glucose determination has attracted considerable interest in clinical, biological, and


chemical samples, as well as in food processing and fermentation. Especially in the
diagnosis and management of diabetes mellitus, because of the large number of
diabetic people, no assay is performed more frequently than that of glucose [26].
Basically, there are two kinds of strategies for the determination of glucose: One is
the direct, nonenzymatic determination of glucose, and the other is the enzymatic
electrooxidation of glucose.
Many efforts have been directed toward the amperometric measurements of glucose
without using enzymes. There are several advantages when using electrochemical
methods for the detection of glucose without using enzymes, including stability,
152 5 Carbon Nanofiber-Based Nanocomposites for Biosensing

Fig. 5.3 (a) SEM image, (b) TEM image, and (c) EDX spectra of the NiCF nanocomposite.
Reprinted with permission from Liu etal. [35]. 2009, Elsevier

simplicity, reproducibility, cost reduction, and the fact that it is free of oxygen
limitations [27]. For an amperometric glucose sensor, however, there are two key
issues that should be addressed properly: enhancing the sensitivity to glucose, and
the interference by electroactive species. Generally, the direct oxidation of most
carbohydrates, including glucose, requires a large overpotential at conventional
electrodes. Such an overpotential limits the selectivity of the determination of glu-
cose, causing tough interference problems [27]. Recently, combining the unique
properties of CNF with the catalytic activity of electrocatalysts has been expected to
oxidize glucose effectively. These electrocatalyst candidates consist of precious
metals such as Pt, Au, and alloy [28] and relatively low-cost catalysts such as metal
oxide and complex catalysts, i.e., CuO [29], NiO [30], MnO2 [31], Co(II) phthalo-
cyanine tetrasulfonate (CoPcTS) [32], Cu nanocluster/MWNTs [33, 34], and MWNTs
[34]. Liu et al. demonstrated such expectations by fabricating Ni nanoparticle-
loaded CNF paste electrode for the nonenzymatic oxidation of glucose [35].
They used SEM, TEM, and EDX to investigate the morphology and microstructure
of the as-prepared NiCF nanocomposite, and each element existed in the nano-
composite (see Fig.5.3). They found that Ni nanoparticles with a diameter of about
5.4 Carbon Nanofiber-Based Electrochemical Biosensors and Bioassays 153

Fig.5.4 Schematic picture of the immobilization of the model enzyme GOx on CNFs and single-
walled carbon nanotubes. Reprinted with permission from Vamvakaki etal. [22]. 2006, American
Chemical Society

50nm are embedded in the CNF matrix, and although the concentration of Ni-based
catalyst on the Ni-CNF paste electrode is much less than that on the Ni bulk elec-
trode, the sensitivity of the Ni-CNF paste electrode is about 1.5 times higher than
the bulk electrode. This phenomenon was attributed to the high electrocatalytic
activity of the Ni nanoparticles embedded in the Ni-CNF paste electrode with a
large electroactive surface area.
Though the electrochemical determination of glucose concentration without
using an enzyme is one of the dreams that many researchers have been trying to
realize, at present, the most common strategy for glucose determination is based on
glucose oxidation-catalyzing enzymes [26]. Two families of enzymes, glucose oxi-
dases (GOx) and PQQ-glucose dehydrogenases (PQQ-GDH), are most widely used
in the electrooxidation of glucose. Because the immobilization procedure of
enzymes affects the bioactivity of enzymes, several research groups have examined
the immobilization and stabilization efficiency of these enzymes on the CNF. For
example, in 2006, Vamvakaki etal. used GOx as the model enzyme to compare the
potentiality of CNF, CNT and graphite powder as the matrix for the development of
biosensors. They demonstrated that CNFs have a much larger functionalized surface
area compared to that of single-walled CNTs and are expected to immobilize and
better stabilize the enzyme GOx, as illustrated in Fig.5.4. Their experimental results
led to an interesting conclusion that CNF is the best matrix so far for the develop-
ment of biosensors, far superior to CNT or graphite powder [22].
154 5 Carbon Nanofiber-Based Nanocomposites for Biosensing

Furthermore, in 2007, Wu etal. demonstrated the versatility of the CNF using


GOx as a model, in which CNF served not only as the matrix for the immobilization
of GOx, but also as the excellent electrocatalyst for the reduction of dissolved oxy-
gen involved in the enzymatic cycle of glucose oxidation. Based on the catalytic
activity of CNF toward oxygen reduction in the neutral solution, they demonstrated
a kind of first-generation glucose biosensor [25].

5.4.2Ethanol Sensors

The measurement of trace ethanol plays an important role in different industries and
biotechnological processes such as the production of alcoholic beverages, food-
stuffs, pharmaceutical products, and clinical and forensic analysis [36]. Two
enzymes have been extensively used in the determination of alcohols, namely,
alcohol oxidase (AOX) and alcohol dehydrogenase (ADH). Almost all AOX-based
ethanol sensors developed so far have been based on monitoring O2 consumption or
H2O2 formation. This has been mostly achieved using amperometric electrodes set
at appropriate potentials, namely, 600mV for O2 monitoring or +600mV for H2O2
monitoring. When a potential of 600 mV relative to Ag/AgCl is applied to the
platinum electrode, O2 is reduced according to the equation O2 + 4H + + 4e - 2H 2 O
and a current proportional to O2 concentration is produced [36]. However, the
cathodic current observed at 600mV often expresses the combined reduction of
both dissolved oxygen and other redox species, such as hydrogen peroxide pro-
duced by the enzyme cycle. Therefore, a decreased overpotential for the dissolved
oxygen-reduction reaction is required to eliminate the interference. Recently, Wu
etal. demonstrated a new kind of CNF-based ethanol amperometric biosensor. CNFs
were functionalized noncovalently with the thionine molecule. The noncovalent
functionalization of CNFs with thionine improved the dispersion of CNFs due to the
fact that thionine can be electrochemically polymerized, which is similar to some
kinds of heterocyclic aromatic dyes such as methylene blue (MB) and methylene
green (MG). AOx could be effectively incorporated to form a stable poly(thionine)
CNF/AOx biocomposite film on an electrode surface. Though poly(thionine) did
not show any catalytic activity toward the reduction of oxygen, the CNFs could
effectively catalyze the oxygen reduction reaction at a relatively low overpotential,
such as 0.3V vs. Ag/AgCl in pH 7.0 phosphate buffer solution [37]. Furthermore,
Wus group demonstrated the noncovalent functionalization of CNFs with water-
soluble porphyrin, i.e., iron(III) mesotetrakis(N-methylpyridinum-4-yl) porphyrin
(FeTMPyP), to construct another kind of CNF-based ethanol biosensor. The formed
CNFFeTMPyP nanocomposite (see Fig. 5.5) showed better catalytic activity
toward the reduction of dissolved oxygen than the bare CNFs, so that the constructed
ethanol biosensor could be operated at a lower overpotential (0.2V vs. Ag/AgCl
in the same conditions) [38]. The detection limit was found to be lower than that
obtained at an ADH/CNF-modified electrode by monitoring the electrocatalytic
oxidation current of NADH [39].
5.4 Carbon Nanofiber-Based Electrochemical Biosensors and Bioassays 155

Fig.5.5 Steady-state
absorption spectra of
(a) 5 mM FeTMPyP and
(b) 0.5mgmL/L CNF-
FeTMPyP in aqueous
solution. Inset: Chemical
structure of FeTMPyP.
Reprinted with permission
from Wu etal. [38]. 2008,
Elsevier

Ethanol biosensors based on ADH have been reported to be more stable and
specific than those based on AOX [36]. Recently, the biocompatibility of CNFs
toward ADH has been studied with the FT-IR spectrum, of which the amide I and
amide II infrared absorption bands of ADH can provide detailed information on the
secondary structure of the polypeptide chain. It was found that the absorption bands
of ADH in the CNF film were nearly the same as those at 1650.8 and 1541.4/cm
obtained for the protein itself, indicating that CNF film showed good biocompatibility
and retained the native structure of the ADH [39]. Such biocompatibility of CNF
makes it very attractive in the development of an ADH-based ethanol biosensor.

5.4.3Acetylthiocholine Sensors

One of the products of hydrolysis of acetylthiocholine chloride (ATCl) with acetyl-


cholinesterase (AChE) is thiocholine. The detection of thiocholine can be used to
assess the activity of AChE, a biomarker of the effect of pesticides (organophos-
phates [OPs] and carbamates) that inhibit cholinesterases. The analysis of ATCl is,
therefore, of great importance, particularly in the development of sensors for the
detection of environmental pollutants such as OPs and carbamates [40]. Recently,
Chaniotakiss group utilized the synergistic role of two different nanomaterials,
CNFs and biomimetically synthesized silica, to propose a novel acetylthiocholine
electrochemical biosensor, based on the direct oxidation of the enzymes hydrolysis
product thiocholine on the surface of CNF. The formed silica/CNF superstructure
was demonstrated to be an ideal architecture for the development of electrochemical
biosensor systems that can withstand exposure to extreme operational conditions,
156 5 Carbon Nanofiber-Based Nanocomposites for Biosensing

Fig.5.6 Preparation of Try/PANIILCNFbased biosensor. (a) GCE pretreatment; (b) the for-
mation of PANI-IL-CNF composite film on GCE surface; (c) the addition of tyrosinase solution
onto the composite film; (d) the as-prepared Tyr/PANI-IL-CNF-based biosensor. Reprinted with
permission from Zhang etal. [43]. 2009, Elsevier

such as high temperatures or the presence of proteases. With a superstructure, CNF


was considered to act as a very efficient electron transfer in nanochannels.
Comparatively, the silica/AChE biosensor without CNFs showed a lower sensitivity,
a limited linear range of response, and an increased response time, indicating that
the presence of CNFs in silica/AChE superstructure leads to the development of
biosensors with enhanced analytical characteristics [41, 42].

5.4.4Phenol Sensors

Phenolic compounds, which are highly toxic, are widely used in wood preserva-
tives, textiles, herbicides, and pesticides and are released into the ground and sur-
face water. The determination of these compounds is of great importance in
environment monitoring. Such a determination is commonly based on monitoring
the reduction signal of their enzymatic oxidation products, o-quinones, by molecu-
lar oxygen in the presence of tyrosinase (Tyr). Thus, for the construction of a phenol
biosensor with CNFs, it is essential to examine the biocompatibility of the CNFs
toward Tyr and to immobilize Tyr on CNF effectively. Recently, Zhang etal. dem-
onstrated a novel PANI-nanocomposite, polyanilineionic liquidcarbon nanofiber
(PANIILCNF) composite, by the in situ one-step electropolymerization of aniline
in the presence of IL and CNFs (see Fig.5.6). The newly designed PANIILCNF
composite showed a fibrillar morphology, which was beneficial to the loading of
enzyme, and thus improved the capacity for immobilization of Tyr. The prepared
Tyr/PANIILCNF-modified electrode exhibited highly sensitive amperometric
responses to the analogs of phenolic compounds such as catechol, p-cresol, phenol,
and m-cresol. With 0.3mM catechol as a model, the amperometric response of the
Tyr/PANIILCNF-modified electrode showed good resistance against 3mM ascor-
bic acid, 30mM uric acid, and 30mM caffeine, which was attributed to the use of a
relatively low operating potential (0.05V vs. Ag/AgCl, pH 7.0) [43].
The electrochemical determination of phenol without using enzyme is also
attractive. For example, Jamal et al. prepared a series of conductive polymer-
modified CNF electrodes and examined the ability of these electrodes to detect
para-aminophenol (p-AP). After poly[N-vinylcarbazole-co-vinylbenzene sulfonic
acid], poly[carbazole-co-methylthiophene], and polycarbazole were coated electro-
chemically on CNF microelectrodes, they found that these modified CNF electrodes
were effective systems for the determination of p-AP and thin-film-coated
5.4 Carbon Nanofiber-Based Electrochemical Biosensors and Bioassays 157

Fig.5.7 Cyclic
voltammograms obtained at
(a) p[NVCzVBSA]-coated
carbon fibers and (b)
untreated carbon fibers in
buffer solution containing
1mM p-aminophenol at scan
rates of 20, 40, 60, 100, 120,
140, and 160mV/s.
Reprinted with permission
from Jamal etal. [44].
2004, Elsevier

p[NVCzVBSA] was the most suitable modified electrode for the detection of p-AP.
It can be seen in Fig.5.7 that quasi-reversible behavior was obtained (anodic to a
cathodic peak current ratio of 1.0) and plots of peak current vs. v1/2 were linear,
indicating that the process was controlled under a diffusion step [44].

5.4.5Hydrogen Peroxide Sensors

H2O2 is a product of the enzymatic reactions between most oxidases and their sub-
strates; its detection is very interesting for the development of biosensors for oxi-
dase substrates and monitoring the activity of oxidases. Generally, high overpotentials
are required for the reduction or oxidation of H2O2 on many electrode materials.
Such a high overpotential causes a problem of electrochemical interference due to
the presence of reducing compounds present in real sample matrices (e.g., ascorbic
acid, uric acid, and acetaminophen), which are also oxidized at that potential.
Furthermore, slower responses are observed.
It is well known that the electrocatalytic reactions are very common on carbon
materials, and the negative surface charge resulting from some surface oxides on a
158 5 Carbon Nanofiber-Based Nanocomposites for Biosensing

Fig.5.8 CVs of 5.0mM


H2O2 at (a) bare and (b)
CNF-modified GCE in pH
7.0 PBS, and (c) the
CNF-modified GCE in pH
7.0 PBS. Scan rate: 0.01V/s.
Reprinted with permission
from Wu etal. [45]. 2007,
Royal Society of Chemistry

carbon surface can have significant electrochemical effects on ET rates. Due to the
existence of a large number of edge sites and the oxygen-containing groups at the
CNF surface, CNF is strongly expected to show catalytic ability for the reduction or
oxidation of hydrogen peroxide. Recently, Wu etal. demonstrated the electrocata-
lytic ability of the CNF for the reduction of hydrogen peroxide in phosphate buffer
solution (pH 7.0). They found that upon adding H2O2, the reduction current at the
CNF-modified glassy carbon electrode dramatically increased and the oxidation
peak current of the oxygen-containing groups on the CNF surface decreased, while
the naked glassy carbon electrode did not show any response to H2O2 (see Fig.5.8).
They attributed such changes in both the reduction and oxidation peak currents to
the synergetic effect of the electrocatalytic action of the oxygen-containing groups
to the reduction of H2O2 and the facilitation of ET kinetics of the electroactive H2O2
by the edge sites on the outer wall of CNFs. This is similar to the observation at
other CNF-modified electrodes. The prepared CNF-modified glassy carbon elec-
trode showed a high sensitivity and good selectivity for the determination of H2O2,
with two common physiological compounds such as uric acid and ascorbic acid as
interferences. The demonstrated electrocatalytic activity of CNF for the reduction
of H2O2 gives CNF a promising application for the development of a nonenzymatic
H2O2 sensor [45].
Additionally, Li etal. studied the effect of CNF microstructure on the catalysis
of CNF for H2O2. CNFs with three microstructures, including platelet-carbon nano-
fibers (PCNFs), herringbone-carbon nanofibers (HCNFs), and tube-carbon nanofi-
bers (TCNFs), were synthesized, characterized, and evaluated for electrochemical
sensing of hydrogen peroxide. Sensors based on PCNFs/GC, HCNFs/GC, and
TCNFs/GC were used in the amperometric detection of H2O2 in solution by applying
a potential of +0.65V vs. Ag/AgCl at the working electrode. The highest electrocatalytic
performance was observed for PCNFs/GC among the three types of hydrogen
peroxide sensors, which was attributed to the highest ratio of edge atoms to basal
atoms of PCNFs [46].
5.4 Carbon Nanofiber-Based Electrochemical Biosensors and Bioassays 159

Fig.5.9 CVs of CNF-modified electrode in (a) 0.2M pH 7.0 PBS and (b) (a) +2.0mM NADH.
Inset: CVs of bare (lower) and untreated CNF-modified (upper) electrodes in (a) 0.2M pH 7.0
PBS and (b) (a) +2.0mM NADH. Scan rate, 10mV/s. Reprinted with permission from Wu etal.
[39]. 2007, American Chemical Society

5.4.6NADH Sensors

The electrochemical oxidation of NADH at the electrode surface has received


considerable interest due to its significance as a cofactor for dehydrogenase enzymes
and its role in the ET chain in a biological system, and also due to the need to
develop amperometric biosensors for substrates of NAD+-dependent dehydroge-
nases. However, the oxidation of NADH at a conventional solid electrode surface is
highly irreversible and takes place at considerable overpotentials, which limits the
selectivity of the determination in a real sample. Furthermore, the reaction at a high
overpotential involves radical intermediates that cause electrode fouling and the
loss of analytical sensitivity, reproducibility, and operational lifetime [39].
The oxidation treatment of CNF with nitric acid can produce a range of oxygen-
containing groups without degradation of the structural integrity of its backbone.
These formed oxygen-rich groups can be expected to facilitate the electrocatalytic
behavior of NADH as well as that of CNT. Recently, Jus group demonstrated the
first biosensing application of CNF with excellent catalytic activity for the conve-
nient preparation of highly sensitive sensors for NADH and substrates of dehydro-
genase. From Fig.5.9, it can be seen that in the presence of 2.0mM NADH, the CVs
160 5 Carbon Nanofiber-Based Nanocomposites for Biosensing

showed two oxidation peaks, at +0.062 and +0.361V, which could be ascribed to
the oxidation of NADH at the edge plane sites on the CNF and those of the underly-
ing GCE. The catalytic activity was generally evident from the defined peak at
+0.062V. Compared to peptide nanotube-based and CNT-based NADH sensors, the
oxidation overpotential and applied potential are further decreased by more than
340 and 240 mV, respectively. They attributed the accelerated ET kinetics to the
formation of a high amount of oxygen-rich groups on the CNF, which resulted from
the oxidation treatment of CNF with nitric acid [39].
Furthermore, Perez et al. compared CNTs, CNFs, and carbon microparticles
(CMPs) as an electrocatalysts to modify the electrode substrate for the oxidation of
NADH using CVs technology. They found that though higher currents for the
NADH oxidation peak have been observed for these electrodes, the CNF film pro-
motes better electron transfer (ET) of NADH, minimizing the oxidation potential at
+0.352V. Figure5.10 shows the possible sites of attachment of NADH molecules
that can be introduced onto the surface of the carbon materials [24].

5.4.7Protein Electron Transfer (ET)

Most of the electrochemical biosensors at present can be categorized into the so-
called first- or second-generation biosensors, in which enzymes or proteins utilized
as the biocatalyst do not directly communicate electron transfer with the electrode
substrate. They are based on the consumption of the natural cosubstrate, such as O2,
or the formation of the product of enzymatic catalytic cycles, or the introduction of
artificial redox mediators. The third-generation electrochemical biosensors are
based on the concept of the direct electron transfer between the enzymes and the
electrode substrate, and a few examples of the third-generation electrochemical
biosensors have demonstrated their advantages over the former two kinds of biosensors
[47, 48]. These include horseradish peroxidase [49], cellobiose dehydrogenases
[48, 50], superoxide dismutase [51], laccase [52], and others [47]. However, the
redox centers of most enzymes are located sufficiently far from the outermost sur-
face to be electrically inaccessible, and consequently these enzymes cannot
efficiently communicate electron transfer with conventional electrode materials
[53, 54] and be applied to construct the third-generation electrochemical biosensors.
CNTs have been demonstrated to exhibit the promotion for the direct ET of enzymes
such as hemoglobin [55], cytochrome c [55], horseradish peroxidase [56], laccase
[52], and so on, and such promotion has been attributed both to CNTs serving as the
nanoscaled electrical wire and to the interaction between enzymes and CNTs
strongly hydrophobic sidewall. CNF can also act as nanoscaled electrical wire and
has the hydrophobic sidewall, just like CNTs; thus, CNF is expected to prompt the
direct ET of enzymes. Recently, Wu etal. observed the direct ET of AOx confined
onto CNF, which has never been directly observed, probably due to steric hindrances
and large reaction barriers [37]. Such an observation indicated CNF could provide
5.4 Carbon Nanofiber-Based Electrochemical Biosensors and Bioassays 161

Fig.5.10 Schematic representation (not to scale) of the possible interactions of the NADH mol-
ecules with graphene layers for the (a) CMPs and (b) CNFs. Reprinted with permission from Perez
etal. [24]. 2007, Elsevier

a microenvironment for preserving the natural structure and accelerating the ET of


the immobilized AOx [38].
Hemoglobin (Hb), an important respiratory protein in red cells, consists of four
polypeptide chains, each with one electroactive iron heme group. Hb functions
physiologically in the storage and transport of molecular oxygen in the blood of
vertebrates, and has catalytic activity for H2O2 similar to horseradish peroxidase. It
is an ideal model molecule for the study of the ET reactions of heme proteins and
also for biosensing and biocatalysis. Since the redox-active center of Hb is deeply
embedded in its protein shell, direct electron transfer between the protein and the
electrode surface has been a great challenge for decades. The possibility of direct
electron transfer between protein and electrode surface could pave the way for supe-
rior reagentless and mediator-free biosensing devices, as it obviates the need for
cosubstrates or mediators and allows efficient transduction of the biorecognition
event [57]. CNFs may act as nanoscaled electrical wires, just like CNTs, to facilitate
direct ET between the redox proteins and the underlying electrode. For example,
Lu etal. reported that CNF could be successfully combined with Nafion and HB to
162 5 Carbon Nanofiber-Based Nanocomposites for Biosensing

construct a reagentless mediator-free H2O2 biosensor. The result revealed that


hemoglobin retained its essential secondary structure in the CNF-based composite
film. With the advantages of organicinorganic hybrid materials, dramatically facil-
itated direct electron transfer of hemoglobin and good bioelectrocatalytic activity
toward H2O2 were obtained [58].

5.4.8Immunosensors

The principle of electrochemical immunosensors and immunoassays (EII) is based


on a specific reaction of the antibody and antigen. Electrochemical immunosensors
have several advantages, such as high sensitivity and low cost of the resulting sen-
sors and instrumentations; they have attracted extensive interest as bioanalytical
devices in recent years [59]. For electrochemical immunosensors, the immunologic
materials are immobilized on an electrochemical transducer. After a sandwich or
competitive immunoreaction, electrochemical labels are attached to the transducers
surface. Quantification is generally achieved by measuring the specific activity of an
electrochemical label after a releasing step or adding a substrate, i.e., its redox activ-
ities and enzyme activities. The applications of nanomaterials in electrochemical
sensors can be classified into two categories according to their functions: (1) nano-
material-modified electrochemical transducers to facilitate antibody immobilization
or improve electrochemical properties of transducers, such as low-background cur-
rent, high signal-to-noise ratio, and fast ET; (2) nanomaterial-biomolecular conju-
gates as labels for electrochemical immunosensors [60].
Recently, Jus group used CNF for the first time to construct an immunosensor
for a rapid separation-free immunoassay. The acidic oxidation of the CNF provided
its solubility and wettability for the convenient preparation of a porous CNF mem-
brane and a larger number of active sites for covalent binding of carcinoma antigen-
125 (CA125) and thionine as the ET mediator. This matrix provided a suitable
environment for the immobilized protein. The immobilized HRP-labeled immuno-
conjugate showed good enzymatic activity for the oxidation of thionine by hydro-
gen peroxide. Figure5.11 shows the preparation and detection procedures of the
CA125 immunosensor [61].

5.4.9Vertically Aligned Carbon Nanofiber Array-Based


Biosensors

VACNFs are self-assembling, catalytically synthesized structures that span across


multiple length scales, featuring nanoscale tip radii (2050nm) and lengths up to
tens of microns (see Fig.5.12). The vertical alignment of the fibers may provide a
very high degree of surface area that is easily accessible to molecules in the solution
phase. This accessibility is important for sensing applications; for example, it may
5.4 Carbon Nanofiber-Based Electrochemical Biosensors and Bioassays 163

Fig.5.11 Preparation and detection procedures of the CA125 immunosensor. Reprinted with per-
mission from Wu etal. [61]. 2007, Elsevier

Fig.5.12 Scanning electron


microscope images of
VACNFs at (a) low and (b)
high magnification. Reprinted
with permission from Baker
etal. [64]. 2005, American
Chemical Society
164 5 Carbon Nanofiber-Based Nanocomposites for Biosensing

be important to detect molecules of a wide range of sizes. The ends and sidewalls of
the nanofibers provide a very high surface area and, consequently, a very high
number of biological binding sites. This high density of binding sites may increase
sensitivity in the same manner as in porous materials, such as porous carbon and
silicon [11, 12, 62]. The sidewalls of nanofibers can also be insulated, leaving
exposed electrodes of extremely small size that have been used for highly sensitive
detection of DNA and glucose via measurements of the electrochemically active
molecules in solution. These striking properties make VACNF very promising for
the development of electrochemical biosensors [63].
Most applications of CNFs have been based on bare fibers with little or no
chemical modification of the surface. In many applications, however, it is important
to functionalize the electrodes with molecules of interest in order to provide them
with specific chemical and/or electrical properties [63]. Therefore, we will focus on
the functionalization of CNF for the development of CNF-based biosensors.
Baker etal. studied covalent functionalization of VACNFs for biomolecular rec-
ognition and compared two different strategies for covalently modifying CNFs with
biological molecules, such as DNA. One method begins with a photochemical reac-
tion between the nanofibers and molecules bearing both a terminal olefin group and
a protected amine group, followed by deprotection to yield the free primary amine.
The second method uses a chemical reaction of an aryldiazonium salt with the nano-
fibers, followed by electrochemical reduction to the primary amine. Both methods
then link the primary amines to thio-terminated DNA oligonucleotides. Their mea-
surements show that both methods yield DNA-modified CNFs exhibiting excellent
specificity and reversibility in binding to DNA probe molecules in solution having
complementary vs. noncomplementary sequences [64].
Landis and Hamers used ferrocene as a model system to understand the ET prop-
erties of redox-active molecules covalently linked to the surface of VACNFs.
Ultraviolet-initiated grafting of organic alkenes was used to prepare carboxylic
acid-terminated layers, and ferrocene was then linked to these layers via amide
groups (see Fig.5.13). Their results showed that molecular layers grafted to CNFs
were sparse and disordered compared with those commonly studied on planar sur-
faces [65].
Previous studies have grafted molecular layers to VACNFs using the photo-
chemical grafting of alkenes or via reaction with diazonium compounds. The
photochemical method uses ultraviolet light to link terminal alkenes to the surface,
but is limited to alkenes that are stable under ultraviolet light and may not be effective
on thick nanofiber arrays because of the strong optical absorption of the nanofibers.
The diazonium method frequently forms multilayers due to the radical intermediates
involved. Although several other techniques, including oxidative methods and acyla-
tion in concentrated nitric acid, have been used to functionalize VACNF surfaces,
these methods require harsh reaction conditions and are difficult to control [66].
Landis and Hamers proposed a gentle method for covalently functionalizing
VACNFs through a copper-catalyzed azidealkyne cycloaddition (CuAAC) reac-
tion in which an azide group is bound to the surface and then linked with an alkyne
[66]. The CuAAC has come into wide use since its introduction in 2002 [67].
5.4 Carbon Nanofiber-Based Electrochemical Biosensors and Bioassays 165

Fig.5.13 (a) SEM image of


the as-grown VACNF
surfaces; (b) reaction scheme
of the ferrocene attachment to
the VACNF surface.
Reprinted with permission
from Landis and Hamers
[65]. 2008, American
Chemical Society

The reaction coupled a terminal azide with an alkyne, creating a 1,4-disubstituted


1,2,3-triazole linkage via a [3+2] Huisgen cycloaddition. They demonstrated that
electrochemically active ferrocene groups covalently linked to VACNFs via the
Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC), as shown schematically in
Fig.5.14. Stability tests showed that the covalently grafted ferrocene groups were
stable for more than 1,500 repeated cyclic voltammograms and over a potential
window of greater than 1.5V, limited by the solvent, which suggested that the use
of click chemistry with VACNFs provided a facile route toward the synthesis of
high-surface-area electrodes with high stability and tailored electrochemical
properties.
Most of the functionalization of the bulk of nanofiber has been directed toward
functionalization of the entire nanofiber surface. However, in 2006, McKnight etal.
demonstrated a site-specific biochemical functionalization along the height of
166 5 Carbon Nanofiber-Based Nanocomposites for Biosensing

Fig.5.14 (a) SEM image of


vertically aligned CNF
cross-section; (b) reaction
scheme of ferrocene
attachment to the VACNF
surface. Reprinted with
permission from Landis and
Hamers [66]. 2009,
American Chemical Society

VACNF arrays (Fig.5.15). They demonstrated that flexible and convenient photoresist
techniques provided site-specific physical, chemical, and electrochemical function-
alization of unprotected, exposed regions of the nanofiber both along its length as
well as at discrete locations across an array of nanofibers [68].

5.5Conclusions

This chapter introduced the synthesis of CNF and VACNF and their applications in
the development of electrochemical biosensors. CNF has been demonstrated to be
very promising as a material for the development of biosensor systems since it pos-
sesses several striking properties that have been proven to be very suitable for the
construction of biosensor systems, such as excellent electrical conductivity, unique
structural and catalytic properties, high loading of biocatalysts, good stability, and
so on. These demonstrations propose that CNF should be better than CNTs for the
development of electrochemical biosensors. VACNFs consist of nested cones of
References 167

Fig.5.15 General scheme for photoresist-based blocking of chemical or electrochemical func-


tionalization of arrays of VACNFs. Resist layers may be used to block functionalization sites spe-
cifically along the nanofiber height (two fibers depicted at the left of each drawing) or site
specifically at different regions of an array (single fiber depicted at the right of each drawing).
Reprinted with permission from McKnight etal. [68]. 2006, American Chemical Society

graphene that expose large amounts of edge-plane graphite along their sidewalls.
VACNFs may also have outstanding properties for electrocatalytic reactions.
Therefore, the functionalization of VACNFs will endow VACNFs with more
striking properties suitable for the development of electrochemical biosensors.

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Chapter 6
Biosensors Based on Nanoporous Materials

6.1Introduction

According to the International Union of Pure and Applied Chemistry (IUPAC)


definition, porous materials are divided into three classes: microporous (<2 nm),
mesoporous (250nm), and macroporous (>50nm). When the pore dimension is in
the nanometer range, such materials can be denoted as nanoporous materials [1].
Recently, nanoporous materials have found increasing applications in many areas,
including bioengineering [2], catalysis [3], and biosensing [4], due to their large
surface area, tailored pore size distribution, controllable pore structure, and versatile
composition. Especially for biosensors, the use of nanoporous materials can signifi-
cantly improve the analysis performance of the biosensors since their large surface
area and versatile porous structure are beneficial for the loading of large amounts of
active catalysts and they have a fast diffusion rate. In fact, various kinds of biosensors
have been designated and fabricated on the basis of nanoporous materials [510]
in the past decade and used for the detection of various biocomponents, including
glucose, DNA, antibodies, and bacteria, with improved sensitivity. The composition
of the nanoporous materials is versatile [1116], including silica, carbon, metal,
metal oxide, and hybrid composition. In addition, their applications cover both the
electrochemical [1116] and optical fields [17, 18].
The applications of nanoporous materials in biosensors can be classified into two
categories according to their functions: (1) as a protein-immobilized host; (2) both
as a protein-immobilized host and as a catalyst taking part in the reaction. Indeed,
there are several main advantages for the biosensing application of nanoporous
materials. First, the large surface area and the uniform pores can provide more cata-
lytic sites via its catalytic mesostructure or the loading of a large amount of catalyst,
hence making highly sensitive detection possible [19]; second, a large amount of
hydroxyls left on the nanoporous metal oxides after removing the template make

H. Ju et al., NanoBiosensing: Principles, Development and Application, 171


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0_6, Springer Science+Business Media, LLC 2011
172 6 Biosensors Based on Nanoporous Materials

the introduction of functional groups into the porous walls possible. And the
modified nanoporous materials could be rendered with many new functions
[2023]. Third, the great porosity and uniform structure would facilitate the fast
transport of the target analytes to the active sites in the nanopores [14, 15]. Fourth,
the inorganic matrices of most nanoporous materials are stable due to their highly
cross-linked structure and therefore can resist biodegradation under extreme condi-
tions. In addition, the structure, pore size, hydrophilic/hydrophobic character, water
insolubility, charge distribution, pH environment, conductivity, and catalytic ability
can be tailored to satisfy the practical applications by using different kinds of tem-
plates or by changing the composition of the material.
In this chapter, we intend to review some of the major advances and milestones
in nanoporous material-based electro-biosensors, classify their functions, and sum-
marize the influence of their components, structure, pore size, and particle size on
the final biosensor performance. In addition, the applications of mesoporous mate-
rial in particular are focused on in a separate section. Recent advances in other
nanoporous metals or metal hybridization are also mentioned.

6.2Why Are Proteins Immobilized?

A biosensor is an analytical device composed of a biological sensing element


(enzyme, antibody, DNA) in intimate contact with a physical transducer (optical,
mass, or electrochemical), which together relate the concentration of the analyte
to a measurable electrical signal [2426]. The stability of biomolecules and signal
transfer to transducer surface are crucial factors in the stability and sensitivity of
the biosensor. In aqueous solutions, biomolecules such as enzymes lose their
catalytic activity rather rapidly because enzymes can suffer oxidation reactions or
their tertiary structure can be destroyed at the airwater interface, hence making
the use of enzymes and reagents both expensive and complex [27]. This problem
can be dissolved by the immobilization of biomolecules to some inert matrix.
Byattachment to an inert support material, bioactive molecules may be rendered,
retaining catalytic activity and thereby extending their useful life [28, 29].
Inaddition, the immobilized materials with good biocompatibility and good
mechanical strength have the potential to improve the stability of enzymes under
extreme conditions [30]. Therefore, the immobilization of biomolecules in a suit-
able matrix is essential.
In the earlier stage, scientists used silica solgel [26], polyacrylamide hydrogel
[31], didodecyldimethylammonium bromide [32], Nafion [33], poly(ester sulfonic
acid) [34], and some inorganic materials [3537] for the entrapment of proteins.
Although these matrices can maintain the activity of biomolecules, the surface areas
are small and the problems of aggregation and leakage are unavoidable. Therefore,
it is essential to develop new matrix materials with a nanoscale porous structure for
the immobilization of proteins.
6.3 Biosensors Based on Mesoporous Materials 173

6.3Biosensors Based on Mesoporous Materials

In 1992, scientists at Mobile reported the successful preparation of a new kind of


mesoporous silica material with a large surface area and pore volume [38, 39]. The
pore size of this kind of material can be well controlled in the nanometer range, which
is quite consistent with the dimension of the enzyme, antigen, and DNA [40]. That
means that more biomolecules can be effectively loaded into and separated by the
mesopores, which is advantageous for improving their activities [4]. In addition,
through adjustments to the synthesis conditions, both their mesostructure and
composition can be well controlled. In the last two decades, various compositions
[1116] including metal, metal oxide, silica, and carbon hybrid component and
structures including one-dimensional layer, two-dimensional (2D) hexagonal, and
three interconnected mesostructures have been prepared [38, 39, 41]. It is easy to
understand that the difference in both the structure and the composition can lead to
differences in their interactions with proteins, the substrates diffusion, and the signal
transition, therefore resulting in a different analytical performance. The achievements
in mesoporous material indeed provide us with more chances to look for the most
suitable protein-immobilized matrix and the development of excellent biosensors.
Many efforts have been devoted to protein immobilization using these mesoporous
materials, and factors involving the pore diameter, mesostructure, surface area,
surface-modified conditions, material morphology, isoelectric point, ionic strength,
protein dimension, morphology, and composition have been studied in depth.

6.3.1Factors Affecting Protein Immobilization

6.3.1.1Pore Size

Pore size is the fundamental factor influencing protein immobilization, which is


closely related to the protein stability, loading amount, and protein activity. Above
all, the pore size should be large enough to enable the protein to enter into the
mesopores [4244]; otherwise, the protein can only be adsorbed on the outer sur-
faces or partly entrapped into the pores [8], therefore resulting in a small loading
amount and poor stability. However, too large a pore diameter will result in protein
leaching when the interaction between the protein and the materials is not strong
enough [45, 46]. Enzyme leaching can be minimized by cross-linking the proteins
in the mesopore [45], decreasing the size of the pore openings by silylation [46, 47],
and modifying the pore walls with thiol moieties [48] and carboxylic acid groups
[46, 49]. However, either activity loss or complex operations are unavoidable. Therefore,
a good match between the mesopore size and the protein dimension is essential. For
example, Deere etal. studied the adsorption and activity of cytochrome c on mes-
oporous materials with different pore diameters [42]. They found cytochrome c was
only adsorbed in the mesopores of MCM-41 with a large pore diameter. Diaz and
174 6 Biosensors Based on Nanoporous Materials

Table6.1 Property of normally used proteins and enzymes


Molecular weight Molecular dimensions
Protein in solution (Da) (nm3) pI
Cytochrome c 12,400 2.63.23.3 10.0
Lysozyme 14,388 1.92.54.3 10.8
Myoglobin 17,000 2.13.54.4 7.0
Papain 20,700 3.6 8.8
Trypsin 23,400 3.8 10.5
Pepsin 33,000 n.a. 1.0
b-Lactoglobulin 35,000 2.93.44.0 5.2
Ovalbumin 43,000 4.05.07.0 4.9
Chloroperoxidase n.a. 4.1
Horseradish peroxidase 44,000 4.04.46.8 8.8
Manganese peroxidase 45,000 n.a. 3.6
Bovine serum albumin 66,400 5.07.07.0 4.7
Conalbumin 76,000 5.05.69.5 6.0
Glucose oxidase 160,000 7.05.58.0 4.3
Reprinted with permission from Hartman et al. [52]. 2005, Elsevier

Balkus found that the protein loading amount into mesoporous silica MCM-41 in a
limited contact time decreased with increasing protein molecular weight [50].
Wright and coworkers [46] studied the adsorption and leaching of trypsin on MCM-
41, MCM-48, and SBA-15 and found that about 72% of the trypsin was leached
from MCM-48 due to the smaller pore size dimension (2.4nm) than that of trypsin
(3.8 nm). However, due to the similar dimension of trypsin and MCM-41 pores
(3.5nm), only 46% of the trypsin was leached from MCM-41, which was much
lower than those of MCM-48. All these results proved that the pore size should be
in good match with the biomolecules size. By varying the template with different
lengths or by introducing some swelling agent with strong hydrophobicity, the pore
size of the mesoporous material can be tailored [41, 51]. In general, the pore size of
MCM series mesoporous materials is relatively small due to the use of short-chain
templates. Normally, they are used to immobilize small proteins with dimensions
less than 4nm. While the pore size dimension of the mesoporous materials synthe-
sized by polymers involving SBA-15, SBA-16, and FDU-5 is in the range of
525nm, they could be applied for the entrapment of relatively large proteins. Once
the protein dimension is larger than 10nm, the mesocellular foam with a window
size greater than 10nm is preferred. For convenience, the normally used protein
dimensions are summarized in Table6.1 [52].
Except for the adsorbed amount and stability, there are close relationships
between the pore size and the enzyme activity [53]. The majority of enzymes
undergo inactivation upon isolation from their native biological environment. Recent
theoretical models suggest that the stabilization of proteins against unfolding can be
achieved by physical confinement inside relatively small cages [54], therefore main-
taining the activity of enzymes. The calculation has recently been proven by an
experimental study in which a stabilization effect was indeed observed based on the
6.3 Biosensors Based on Mesoporous Materials 175

foldingunfolding equilibrium of proteins when enzymes were introduced to


nanosized cages [5557]. This stabilization effect is attributed to the fact that in
such confined spaces the unfolded configurations of the chain are not thermody-
namically favored. According to theoretical calculations, the maximum stabiliza-
tion of proteins can be obtained in spherical cages with a diameter two to six times
the diameter of the native protein. Considering that the average diameter of a
hydrated protein is within the range of 10nm, the use of mesoporous materials [58]
will provide the maximum stabilization for the proteins. Therefore, by selecting
mesoporous materials with a suitable pore size, the maximum activity of immobi-
lized proteins can be achieved.

6.3.1.2Surface Characteristics

The surface characteristics, including hydrophilicity/hydrophobicity and surface


charge, are another key factors influencing the stability, activity, and loading amount
of proteins. Four kinds of interactions, involving the electrostatic interaction, hydro-
gen bonding, coordinated interaction, and hydrophobic/hydrophilic interaction, can
affect the protein adsorption, which are also affected by solution pH value [50],
ionic strength [59], and the synthesized conditions [60]. Some scientists considered
that the electrostatic interaction is one of the important factors for the adsorption
and desorption of proteins in mesoporous molecular sieves; based on this principle,
the surface charges of mesoporous materials and the proteins must be complemen-
tary [6164]. For example, SBA-15 synthesized through the hydrogen bonding
interaction using the nonionic surfactant as template has relatively lower charges
than those of FSM-16 and MCM-41 synthesized with ionic surfactants, so when
those materials are applied for the immobilization of horseradish peroxidase (HRP)
and subtilisin, the protein-adsorbed amount on MCM-41 and FSM-16 is higher than
that on SBA-15 [60, 65] due to the relatively strong electrostatic interactions.
However, some other scientists considered that the hydrophobic interaction
rather than the electrostatic interaction dominated the final adsorption amount and
the adsorption rate. Hartmanns group observed the absorption behavior of cyto-
chrome c and lysozyme in MCM-41 and SBA-15 and found when the solution pH
was near the isoelectric point (pI) of the protein, the adsorption amount reached the
maximum. They demonstrated that under these conditions, the net charge of the
protein was low and the Coulombic repulsive force between the protein molecules
was minimal. Thus, closer packing of the protein molecules was possible and the
monolayer capacity increased [52, 66]. Czeslik and colleagues studied the adsorp-
tion of lysozyme at the silicawater interface and concluded that the attractive
Coulombic interactions were overcompensated by repulsive proteinprotein inter-
actions. They found that the adsorption process was endothermic, while the attrac-
tive Coulombic interaction between lysozyme molecules and the silica surface
should lead to an exothermic adsorption process [67].
As is well known, the primary structure of protein is amide acids, which are full
of carboxyl and amino groups. The large number of functional groups can interact
176 6 Biosensors Based on Nanoporous Materials

with transition metal elements [68], such as Ti, V, Pt, Pd, and so on, thus making the
enzyme stably adsorbed on the mesowalls containing transition elements. For
example, Jus group [14] prepared a conductive nanocage comprised of palladium
nanoparticles (PdNPs) homogeneously distributed on the mesowall and used it for
the entrapment and biosensing of glucose oxidase (GOD). They found the introduc-
tion of PdNPs on the mesowalls could provide sufficient catalytic sites for lowering
the detection overpotential of glucose. In addition, the interaction between palla-
dium and COOH or NH2 led to stable immobilization of enzymes even on meso-
cellular foam with relatively large mesopore size.
Besides that, organic groups can be introduced into the mesoporous surfaces via
postgrafting, therefore changing the surface characteristics. Lei etal. [62] reported
that a suitable organically functionalized mesoporous host could provide a higher
affinity for protein molecules and a more favored microenvironment, resulting in
exceptional immobilizing efficiency. Wright and coworkers [48] investigated the
adsorption and desorption behavior of protein on SBA-15 functionalized by thiol,
chloride, amine, and carboxyl groups and found that the interactions of the enzyme-
support depended strongly on the nature of the functional groups attached to the
surface.

6.3.1.3Morphology

The morphology of the mesoporous material affects the final protein adsorption
amount. Usually, mesoporous material with a relatively small particle size can pro-
vide more entrances to entrap enzymes and will result in an exceptionally high
immobilization capacity and very rapid adsorption rate. Zhao and coworkers stud-
ied the adsorption of lysozyme in mesoporous silica with controlled morphologies
(including conventional SBA-15, 20 mm in length; rod like SBA-15, 12 mm in
length; mesoporous monolith; and the macroporous-mesoporous membranes) [69].
They found that the lysozyme entrance amount increased with the decreasing size
of mesoporous nanoparticles. The rod-like SBA-15 showed a faster adsorption rate
and larger immobilization amount than those of conventional SBA-15. Although no
systemic study on the application of nanoscale mesoporous materials in protein
adsorption has been reported [70], from the advantages the rod-like SBA-15 showed,
it could be concluded that the nanoparticle with a shorter nanoscale mesochannel
will be an advantage for a high protein loading amount and fast substrate diffusion.

6.3.1.4Mesostructure

The structure of mesoporous material is versatile. Among those mesostructures,


hexagonal, cubic, and layer structures are the most familiar, typical of MCM-41/
SBA-15, MCM-48, and MCM-50, as shown in Fig.6.1. The layer mesostructure
isunstable and is easily collapsed after the removal of the template; therefore, it is
seldom used in protein immobilization and biosensing. The mesoporous materials
with a 2D hexagonal mesostructure, including MCM-41, hexagonal mesoporous
6.3 Biosensors Based on Mesoporous Materials 177

Fig. 6.1 Structure models of 2D hexagonal p6mm (MCM-41), 3D cubic Ia3d (MCM-48), and
layer mesophase (MCM-50)

silica (HMS), and SBA-15, are mostly used. Their channels are straight and parallel
with each other, which are unfavorable for protein adsorption and substrate diffu-
sion. Since their channel length is always in the micrometer range, when proteins
are loaded onto the mesopores, they block the channel and make further protein
adsorption difficult, resulting in the lowly effective occupation of the mesopores. In
comparison, three-dimensional mesostructures can facilitate protein immobilization
and substrate diffusion due to their interconnected mesopores [4] and therefore are
more suitable for protein immobilization and biosensing applications.

6.3.2Methods for Protein Immobilization


on Mesoporous Material

Four methods are usually used for the preparation of mesoporous-based electro-
chemical biosensors. In the first method, mesoporous molecular sieves are added to
protein solutions, followed by stirring or shaking in a shaking bath and centrifuging
the mixture. Then the sediment is washed with deionized water and vacuum-dried.
Thus, the protein- or enzyme-adsorbed mesoporous molecular sieves are obtained,
which are cast on an electrode surface via an adhesive polymer.
The second method mixes mesoporous molecular sieves in carbon paste (CP) to
prepare mesoporous molecular sievemodified CP electrodes. Mesoporous molecu-
lar sieve suspension is thoroughly mixed with graphite carbon powder. Then paraf-
fin oil is added to the mixture, followed by evaporation of water for 3h in air. The
immobilization of protein on the surface of the mesoporous molecular sieve
modified CP electrode can be achieved by dropping the protein solution onto the
pretreated mesoporous molecular sievemodified CP electrode or dipping this mod-
ified CP electrode into the protein solution [71].
The third method is to prepare mesoporous molecular sieves colloid in polyvi-
nyl alcohol (PVA) for immobilization of protein. First, mesoporous molecular
sieves are dispersed into water to obtain a suspension. The suspension is then
mixed with a PVA solution of ethanol and water to produce mesoporous molecular
sieve solution. Then mesoporous molecular sieve solution and the protein solution
are dropped and mixed on a pretreated glassy carbon electrode surface and allowed to
178 6 Biosensors Based on Nanoporous Materials

Fig. 6.2 (a) The immobilization scheme based on magnetic materials on an Au electrode.
Reprinted with permission from Lee etal. [45]. 2005, Wiley. (b) The immobilization scheme
based on magnetic materials on a magnetic electrode. (a) Water drop with CNT/Fe3O4 composite
dispersed inside; (b) graphite-epoxy composite electrode; (c) water drop; and (d) CNT/Fe3O4 com-
posite loaded on the electrode. Reprinted with permission from Qu etal. [74]. 2007, Elsevier

dry under ambient conditions. Finally, the modified electrode should be rinsed with
doubly distilled water two or three times to get rid of the nonfirmly adsorbed pro-
teins [19, 72, 73].
The fourth method is based on the special mesoporous material with favorable
magnetism, as shown in Fig. 6.2. Usually, a homemade magnetic electrode first
needs to be prepared with the help of a magnet, and then the magnetic mesoporous
material is loaded onto the electrodes surface by the magnetic attraction. Compared
with the methods mentioned above, the immobilization method with the help of an
external field has the advantages of easy regeneration and reposition and can be well
controlled, which holds promise in the near future for the fabrication of magneti-
cally controlled biosystems.

6.3.3Biosensors Based on Mesoporous Silica

6.3.3.1Direct Electrochemistry of Redox Proteins and Their Applications

Due to the good hydrophilicity and biocompatibility, mesoporous silica has exten-
sively been used in protein immobilization and biosensing. The use of mesoporous
silica as an enzyme-immobilized matrix was pioneered by Jus group in 2004 [19].
6.3 Biosensors Based on Mesoporous Materials 179

Fig.6.3 Cyclic voltammetric


(CV) curves of (a) GCE,
(b)HMS-modified GCE,
(c)Hb-modified GCE, and
(d)Hb/HMS-modified GCE
in 0.1M pH 7.0 PBS at
100mVs1. Reprinted with
permission from Dai etal.
[19]. 2004, Elsevier

They immobilized hemoglobin (Hb) in the mesopores of HMS-modified glassy


carbon, and observed the direct electron transfer (ET) of the Hb. As shown in
Fig.6.3, the Hb/HMS electrode exhibited two pairs of stable redox peaks that were
attributed to the redox of immobilized Hb. For the first pair, the anodic and cathodic
peak potentials were at 22 and 96mV, respectively, and for the second pair, they
were at 186 and 278mV, respectively. The two pairs of redox peaks in Fig.6.3d
with the formal potentials of 0.037 and 0.232V resulted from the adsorbed Hb
and the intercalated Hb, respectively. No peak was observed at both bare and the
HMS-modified glassy carbon electrodes, showing that HMS was electroinactive in
the potential window. Although the Hb-modified glassy carbon electrode also
showed the response of Hb, there was only an irreversible reduction peak and the
response was smaller than that of the Hb/HMS-modified electrode. The average
electron-transfer rate ks of hemoglobin was 0.920.18s1, as estimated from the
peak-to-peak separation (Fig.6.4) and the Laviron equation.
As is well known, the study of the direct ET between redox proteins and elec-
trode surfaces is of practical and theoretical importance in biological and energy
sciences as well as analytical chemistry. Determining the redox potentials and elec-
tron-transfer rates of these proteins can help us to understand the effect of the envi-
ronment on their biochemical function [75]. However, the ET of the redox enzyme
is rather difficult to obtain due to the unfavorable factors involving the deep immo-
bilization of the electroactive prosthetic groups, adsorption denaturation of proteins
onto electrodes, asymmetric distribution of surface charges on protein molecule,
low rate of mass-transfer process of proteins [76], and the high activation energy
required for the conformational changes of proteins [7781]. To achieve the ET of
proteins, the property of electrode material, modification matrix, and immobiliza-
tion process of proteins are important. Jus group ascribed the fast electron-transfer
rate of Hb to the strong interaction between Hb and HMS, and indicated HMS could
provide a good microenvironment for Hb to undergo a simple electron-transfer
reaction. In the following work, Ju and coworkers observed the direct electrochem-
istry of HRP [82] and myoglobin [73] by the mesoporous material-modified
180 6 Biosensors Based on Nanoporous Materials

Fig.6.4 CV curves of Hb/HMS-modified GCE in 0.1M pH 7.0 PBS at 20, 30, 50, 80, 100, 120,
150, 180, and 200mVs1. Inset: plot of peak current vs. scan rate. Reprinted with permission from
Dai etal. [19]. 2004, Elsevier

e lectrodes. These proteins were encapsulated into the mesopores through physical
adsorption and hydrophobic/hydrophilic or electrostatic interaction without the con-
ventional cross-linking interaction, which are favorable for enzymes to maintain
their bioactivities.
Additionally, Lis group [83] also reported the ET of Hb in bimodal mesoporous
silica (BMS) and chitosan inorganicorganic hybrid film. The BMS possesses a
three-dimensional disordered pore structure with bimodal pore sizes, i.e., smaller
pores in the 23-nm range and larger pores between 10 and 40nm. They demon-
strated that the larger pore size compared with that of conventional protein-facilitated
BMS to effectively immobilize a large amount of proteins.
Significantly, the direct ET between immobilized redox protein and the electrode
surface without the need for any electron-transfer mediator or promoter offers an
opportunity to build mediator-free sensors for hydrogen peroxide (H2O2), nitrite, and
other biocomponents, which is promising for the construction of the third-generation
electrochemical biosensors. As shown in Fig.6.5, typical cyclic voltammetric (CV)
curves for the electrocatalysis response of Mb/HMS/GCE to H2O2 are observed [73].
Upon the addition of H2O2 to the solution, the shape of the CV for Mb changes
dramatically, with an increase in reduction current and a decrease in oxidation
current, while no electrocatalytic current is observable at a bare GCE or the HMS/
GCE (inset), displaying an obvious electrocatalytic behavior of the Mb intercalated
in the mesopores of HMS to the reduction of H2O2. Under optimal conditions
6.3 Biosensors Based on Mesoporous Materials 181

Fig.6.5 (a) CVs of Mb/HMS/GCE in 0.1M pH 7.0 PBS containing 0, 0.01, 0.02, 0.03, 0.04, 0.05,
0.07, and 0.09mM H2O2 at 20mVs1. Inset: cyclic voltammograms of HMS/GCE in 0.1M 7.0
PBS containing 0, 0.02, 0.04, 0.07, and 0.09mM H2O2 at 20mVs1, and (b) in 0.1M pH 7.0 PBS
containing 0 and 0.1mM NaNO2 at 20mVs1. Reprinted with permission from Dai etal. [73].
2004, Elsevier

(400mV, pH 7.0 PBS), the linear response range of the sensor to H2O2 concentration
is from 4.0 to 124mM, with a correlation coefficient of 0.9999. The enzyme elec-
trodes achieve 95% of the steady-state current in less than 10s, indicating HMS is an
effective material for the construction of sensors. The detection limit is 6.2108M
at a signal-to-noise ratio of 3. When the H2O2 concentration is higher than 124mM,
a response platform is observed, showing a characteristic of the MichaelisMenten
kinetics mechanism. The apparent MichaelisMenten constant for the electrocata-
lytic activity of Mb/HMS/GCE to H2O2 is determined to be 0.0650.005mM. After
a month, the Hb/HMS/GCE can retain 96% of its initial current response to H2O2.
The fabrication reproducibility of six Mb/HMS/GCE electrodes shows a RSD of
4.1% for the current determined at 4.0mM H2O2. Similar results are obtained in the
Hb/HMS/GCE and HRP/HMS/GCE electrodes, indicating proteins entrapped in the
HMS matrix are suitable for constructing H2O2 sensors [73].
Xings group found that the mesoporous silica could also facilitate the ET of
GOD [84]. They immobilized GOD on the SBA-15 and Nafion matrices; the immo-
bilized GOD had undergone a direct and nearly reversible electrochemical reaction,
containing a two-electron and two-proton exchange, with good stability. GOD
immobilized on the SBA-15 and Nafion matrices had a wide linear response to glu-
cose in the positive potential range.

6.3.3.2Coimmobilization of Two Enzymes and Their Application

The large surface areas of mesoporous molecular sieves make the simultaneous
immobilization of multiple kinds of proteins possible. Jus group prepared two
highly sensitive electrochemical sensors based on the coimmobilization of two
enzymes for the detection of phenol and glucose, respectively. Compared with
182 6 Biosensors Based on Nanoporous Materials

the detection based on a single enzyme, the two-enzyme synergy always exhibits
some exciting performance such as an extremely extended linear range and high
sensitivity. For example, a phenol electrochemical biosensor [85] without the addi-
tion of mediator was fabricated by the coimmobilization of tyrosinase and HRP in
the mesoporous silica. In air-saturated PBS solution, the electrochemical response
of phenol on the enzyme electrode was based on the following equations:

phenol + tyrosinase (O2 ) catechol


catechol + tyrosinase(O 2 ) o-quinone + H 2 O


o-quinone + 2H + + 2e - catechol (at electrode)


The immobilization of HRP results in an increase in the sensitivity, since the catechol
formed from the electrochemical reduction of o-quinone could be oxidized by
theH2O2 produced from the reduction of dissolved O2 [86], which was catalyzed
bythe coimmobilized HRP. And the regeneration of o-quinone would result in an
increase in reduction current. As shown in Fig. 6.6, the response of tyrosinase/
MCM-41/GCE to 0.2mM phenol was 5.4 times larger than that of tyrosinase/GCE
(Fig. 6.6a, b), while being about two times smaller than that of tyrosinase-HRP/
MCM-41/GCE (Fig.6.6b, c), indicating that the MCM-41 played an important role
in enhancing the enzymatic activity of tyrosinase and the two-enzyme system
possessed better electrocatalytic efficiency.
Recently, another electrochemical biosensing system based on the coimmobiliza-
tion of GOD and HRP was reported by Jus group for the detection of glucose [87].
The HRP immobilized in the mesopores of SBA-15 showed the direct ET. In pres-
ence of glucose, the enzymatic reaction of the GOD-glucose-dissolved oxygen system
could generate hydrogen peroxide, which was immediately reduced at 0.40V by an
electrocatalytic reaction with the HRP entrapped in the same mesopore, leading to a
highly sensitive and selective glucose biosensor without the addition of any mediator.
The mechanism could be shown by (6.1)(6.5):

glucose + GOD(FAD) gluconolactone + GOD(FADH 2 ) (6.1)


GOD(FADH 2 ) + O 2 GOD(FAD) + H 2 O 2 (6.2)


HRPred + H 2 O 2 HRPox + H 2 O (6.3)


HRPox + 2H + 2e HRPred + H 2 O (6.4)



In total:

glucose + O 2 + 2H + 2e gluconolactone + 2H 2 O (6.5)


6.3 Biosensors Based on Mesoporous Materials 183

Fig. 6.6 Cyclic voltammograms of (a) tyrosinase/GCE, (b) tyrosinase/MCM-41/GCE, and (c)
tyrosinase-HRP/MCM-41/GCE in the absence (solid line) and presence (dotted line) of 0.2mM
phenol in air-saturated 0.1M pH 7.0 PBS at 100mVs1. Scan from negative to positive direction.
Reprinted with permission from Dai etal. [85]. 2005, Wiley

The detection limit of the biosensor was down to 2.7107M. Compared with the
glucose biosensor based on a single GOD, the bienzyme system showed an extremely
wide linear range from 3.0106 to 3.4102M, proving the advantage of SBA-15
as enzyme a host material for the fabrication of electrochemical biosensors.

6.3.3.3Immuno- and DNA Biosensors Based on Mesoporous Silica

Antigenantibody and DNA could also be incorporated into the mesoporous matrix
with a suitable pore size and applied to biosensing. He and coworkers [10] prepared
a mesoporous material-modified CP electrode for the determination of cardiac tro-
ponin I (cTnI) by anodic stripping voltammetry. The cTnI in the serum of patients
has been considered as the gold standard for the diagnosis of myocardial injury
[88]. Detection of cTnI in the serum is helpful for the diagnosis of acute myocardial
infarction (AMI), the identification of myocardial injury, and the stratification of
184 6 Biosensors Based on Nanoporous Materials

Fig.6.7 Protocol format of cTnI analytical procedure. Reprinted with permission from Guo etal.
[10]. 2005, Elsevier

cardiac risk. The influence of different kinds of mesoporous materials with different
pore sizes was studied on the response of the immunosensor, and SBA-15 was found
to be more suitable for the immobilization of IgG1 than MCM-41 and Y-type zeolite
due to the larger mesopores as well as the larger dimension of cTnI (10145nm)
[10]. The pore size was the crucial factor for the current response, since the larger
the pore size, the easier the immobilization of the IgG1 and the higher IgG1 immo-
bilization efficiency. Figure6.7 shows the protocol of the cTnI analytical procedure;
the response of the cTnI is based on the anodic stripping peak of the adsorbed silver
on the cAu-IgG2. The nonspecific adsorption of the multipore materials is controlled
by selecting suitable concentration of cAu-IgG2 and adding gelatin as blocking
reagent. Under optimal conditions, a linear relationship between the anodic strip-
ping peak current of silver and the concentration of cTnI from 0.5 to 5.0ng/mL is
acquired. In addition, the established method is tested by determining cTnI in AMI
samples using enzyme-linked immunoadsorbent assay for comparison analysis,
with good results.
Recently, based on the decrease in conductivity resulting from pore blocking,
mesoporous silica film-modified electrodes have been applied to labeling-free
biomolecular recognition [89]. The working principle is as follows: First, the
electrodes modified with amine functional groups of hybrid mesoporous silica film
(0.1mm thick) are attached with biotin groups or oligonucleotides, and then they
react with streptavidin or complementary DNA molecules, respectively, followed
by measurement in an electrolyte solution containing indicator molecules. Before
specific binding, the indicator molecules can freely access the conductive surface
and produce a typical redox response. After specific binding, the indicator molecules
can no longer freely access the conductive surface, resulting in a reduction in,
6.3 Biosensors Based on Mesoporous Materials 185

ordisappearance of, the redox current. From the decrease or disappearance of the
indicator current response before and after the specific binding, the target biomole-
cules from nanomole to femtomole levels can be recognized. In addition, the
response due to the complementary DNA is much lower than that of the noncomple-
mentary DNA, indicating a good selectivity.

6.3.4Biosensors Based on Mesoporous Carbon

6.3.4.1Biosensors Based on Amorphous Mesoporous Carbon

Ordered mesoporous carbon materials (OMCs) are another important member of


the mesoporous group that are mostly synthesized by using mesoporous silica as
hard template [9093]. The conductivity of OMCs is better than mesoporous silica,
which is favorable for the fast electrical communication between the enzyme and
electrode. In addition, some amorphous OMCs contain edge-plane-like defective
sites, which have good electrocatalytical activity [93]. However, their applications
for enzyme immobilization and electrode modification are obviously delayed com-
pared with mesoporous silica. The major problem comes from the fact that it is
difficult to disperse homogeneously in the aqueous solution, which may result in
both poor reproducibility and slow response rate [94]. To solve this problem, some
scientists try to squeeze macroporous carbon into a tube (e.g., polytetrafluoroethyl-
ene) to form a carbon rod electrode, and the enzyme molecules are immobilized on
the carbon electrode by physically absorbing in the enzyme solutions [95]. However,
their poor and noncontrollable enzyme load and the complicated and time-consum-
ing enzyme immobilization process (typically 16 or 20h) are unfavorable.
The first electrochemical biosensor based on OMC was reported by Chens group
in 2007 [90]. The biosensor was fabricated by the alternative assembly of Hb and a
hexagonal OMC (CMK-3) on the chitosan-modified glassy electrode surface. In
their work, CMK-3 was first treated by 1M H2SO4 for 3h at 80C under refluence.
After the treatment, the surface of CMK-3 was full of COOH groups, which could
improve its hydrophilicity. Also, the negatively charged surface was also favorable
to immobilize Hb, which was positively charged in pH 7.0 PBS (Hb, IEP=7.4).
Figure6.8 shows the electrochemical impedance spectroscopy (EIS) to monitor
the surface changes of the modified electrode during the modification process.
Compared to bare glassy carbon, after the electrodeposition of chitosan, the semi-
circular diameter of the Nyquist plot that represents the electron-transfer resistance
becomes much larger, indicating that chitosan was successfully deposited on the
electrode surface and hinders the ET of the redox probe. In contrast, upon assembly
of CMK-3 on the outer layer of chitosan, the semicircular diameter becomes much
smaller, proving the better conductivity of CMK-3. The good conductivity favors
the accelerated ET of Hb.
Figure 6.9 shows the cyclic voltammograms of the biosensors fabricated with
different layers of assembly. Before the modification of Hb, nearly no observable
186 6 Biosensors Based on Nanoporous Materials

Fig.6.8 EIS for (a) bare,


(b) chitosan, (c) CMK-3/
chitosan, and (d) Hb/CMK-3/
chitosan-modified GCE.
Reprinted with permission
from Feng etal. [90].
2007, Elsevier

Fig.6.9 CVs of the {Hb/


CMK-3}n film electrode in
pH 7.0 PBS with n=16
(af). Reprinted with
permission from Feng etal.
[90]. 2007, Elsevier

peak is present at either the bare or chitosan-modified electrode from 0.3 to 0.9V.
With the introduction of Hb and the CMK-3 layer onto the electrode surface
(Fig.6.9), a pair of well-defined redox peaks is observed at about 377 and 296mV
in pH 7.0 PBS under the optimized modification conditions, which is attributed to
the ET of Hb. In addition, the {Hb/CMK-3}n film electrode shows strong catalytic
reduction toward both H2O2 and O2. The better CV performance and the good cata-
lytic behavior of the electrode suggest that CMK-3 has an obvious effect to improve
the reversibility of the electrode reaction for Hb.
Liu etal. [91, 92] confirmed the advantage of OMC in promoting the ET of pro-
teins. Her group dispersed bicontinuous gyroidal mesoporous carbon (BGMC) or
mesoporous carbon nanocomposite (CMM) in the Nafion solution and used them for
the immobilization of Mb and GOD, respectively. The cyclic voltammograms showed
those OMCs could facilitate the ET of Mb and GOD. The Mb surface coverage on
Mb/BGMC(2.8nm)/GCE was calculated to be 2.01010molcm2. The value was 19
times larger than the theoretical value of a full-packed monolayer (1.081011molcm2).
From the value, they assumed that the long axis of the Mb molecule was parallel to the
electrode surface. In addition, they also observed the influence of pore size on the
electron-transfer behavior of Mb, as shown in Table6.2. They found that the surface
6.3 Biosensors Based on Mesoporous Materials 187

Table6.2 (A) The characteristic results of BGMCs and (B) the loading amounts and electron-transfer
properties of Mb immobilized on BGMCs (at 10mVs1)
Pore size (nm) 2.8 3.4 4.6 7.1
(A) BET surface area (m2g1) 1,472 1,410 1,241 1,116
Mesopore volume (cm3g1) 1.22 2.1 1.59 1.87
Electroactive surface area (cm2)a 2.720.04 3.350.06 1.690.12 1.440.08
(B) GMb (1010molcm2) 2.00.4 1.80.3 1.70.3 1.80.4
0
EMb (mV vs. SCE) 3291 3371 3383 3425
DEpMb (mV) 80 160 331 481
ket0 Mb (s1) 10.40.3 8.70.2 7.00.2 4.70.1
Reprinted with permission from You etal. [91]. 2009, Elsevier
a
Electroactive surface area and apparent geometric surface area of bare GCE are 1.10.01,
0.07cm2, respectively

coverage of Mb immobilized onto BGMC matrices with various pore sizes was
similar, indicating that the immobilization capacity for proteins was mainly influ-
enced by the close surface areas of BGMCs, and not very related to the pore sizes. The
immobilized Mb exhibited the smallest peak potential separation (DEp) and the largest
apparent heterogeneous electron-transfer rate constant ket0 in the BGMC matrix with
a pore size of 2.8nm, suggesting that such pore size made a good match to the size
of the Mb molecule (2.13.54.4 nm); thus, the shortest distance between the
redox center of Mb and the electrode surface could be achieved, giving the fastest
ET. The hypothesis was confirmed by the fact that the larger GOD molecule achieved
the smallest DEp and the largest ket0 in the BGMC matrix of a corresponding pore
size of 4.6nm.
Dongs group [93] recently presented an advanced electrochemical biosensing
platform for the detection of ethanol and glucose based on OMCs with a 2D
hexagonal p6mm mesostructure. They found that except for the advantage of con-
ductivity, OMCs also showed excited electrochemical responses to NADH and H2O2
at low oxidation overpotential, which exhibited more favorable electron-transfer
kinetics compared with carbon nanotube (CNT). In detail, Fig.6.10ac show the
cyclic voltammograms for NADH oxidation at glassy carbon electrode, CNT/GCE,
and OMC/GCE, respectively. The oxidation of NADH occurs at +0.65, +0.45, and
+0.2V, respectively. In addition, the peak current for NADH at the OMC/GCE is
2.35 times larger than that of the CNT/GCE, while the peak current at CNT/GCE
increases 2.44-fold compared with the bare electrode. Figure 6.10df show the
cyclic voltammograms of (d) GCE, (e) CNT/GCE, and (f) OMC/GCE in the pres-
ence of H2O2. With the addition of H2O2 to the solution, the anodic current begins
increasing from +0.30, +0.50, and +0.70V at OMC/GCE, CNT/GCE, and GCE,
respectively. These results confirmed that OMC/GCE had higher electrocatalytic
activity for NADH and H2O2 oxidation compared with CNT/GCE and GCE.
Obviously, the presence of OMCs can accelerate the ET.
Several factors may contribute to the good electrochemical performance of
OMCs. The first factor is the nanostructured channel of OMCs. The large surface
188 6 Biosensors Based on Nanoporous Materials

Fig.6.10 CVs for 2mmolL1 NADH at (a) GE, (b) CNTs/GE, and (c) OMCs/GE. CVs for
4.2mmolL1 H2O2 at (d) GE, (e) CNTs/GE, and (f) OMCs/GE. Dotted lines represent the background
response. Scan rate: 50mVs1. Reprinted with permission from Zhou etal. [93]. 2008, Elsevier

area may increase the apparent electroactive surface area of OMCs/GCE, which
offers a favorable microenvironment for transferring species in solution through the
pores of OMCs, and also is beneficial for accelerating ET between the electrode and
species in solution. The second factor is the uniform-pore-size ordered arrangement
of carbon nanorods and pieces of OMCs homogeneously covered on GCE, which
are very attractive for various electrochemical applications, especially for the devel-
opment of electrochemical sensors and biosensors. Additionally, the good electro-
catalytic ability is also important, which can be increased by increasing the number
of edge-plane-like defective sites, proved by FT-IR, XPS, and Raman spectra. As
shown in Fig.6.11, FT-IR spectra (a) indicated that the bands around 3,435, 1,688,
1,550, 1,515, and 1,106cm1 were attributed to the oxygen-containing functional
groups (OFGs) on CNT and OMC. XPS spectra (b) showed the O/C ratio from XPS
for CNTs and OMCs was 0.098 and 0.032, respectively, which means CNTs contain
more OFGs than OMCs. The Raman spectra of CNTs (e) and OMC exhibited the
presence of two scattering peaks centered at 1,348 and 1,595 cm1, which were
attributed to the sp3-hybridized (D band) and E2g zone center mode of crystalline
graphite (G band) [96, 97], respectively. The relative intensity ratio of the D and G
lines (ID/IG ratio) was proportional to the number of defective sites in carbon materi-
als, which was responsible for the electrochemical activity of the materials. The
ID/IG ratio of OMCs (f) and CNTs (e) was 1.82 and 0.58, respectively. This means
the OMCs contained more edge-plane-like defective sites than CNTs without puri-
fication or end-opening processing, and hence had better catalytic activity. Based on
6.3 Biosensors Based on Mesoporous Materials 189

Fig.6.11 (a) FT-IR spectra


of (a) CNTs and (b) OMCs;
(b) XPS spectra of (c) CNTs
and (d) OMCs; and
(c) Raman spectra of
(e) CNTs and (f) OMCs.
Reprinted with permission
from Zhou etal. [93].
2008, Elsevier

the greatly enhanced electrochemical reactivity of NADH and H2O2, two biosensors
were fabricated by the entrapment of GOD and alcohol dehydrogenase (ADH),
respectively, in the mesopores of OMCs for the detection of glucose and alcohol,
respectively. Both of them showed a good analytical performance, indicating OMCs
are good matrices for protein immobilization and biosensing.

6.3.4.2Biosensors Based on Graphitic Mesoporous Carbon

The fundamental aspects of an electrochemical biosensor involve the enzyme immo-


bilization onto an electrode surface and the formation of efficient electrical com-
munication between the enzyme and the electrode while retaining the enzymatic
stability and bioactivity [98, 99]. Graphitized ordered mesoporous carbon (GMC) is
190 6 Biosensors Based on Nanoporous Materials

Fig.6.12 XRD patterns of GMC-13, GMC-6, and CNTs. Reprinted with permission from Lu etal.
[94]. 2009, Royal Society of Chemistry

a promising alternative for the construction of enzyme-based electrochemical


biosensors as it has the advantage of good electrical conductivity due to the good
graphitization degree. However, very few reports have explored the electrochemical
biosensing applications of these fascinating materials [94, 100].
In 2009, Chens group [94] prepared two kinds of three-dimensional ordered
graphitized mesoporous carbon designated as GMC-6 (pore diameter=6nm) and
GMC-13 (pore diameter=13nm) by a nickel-catalyzed template-assisted method,
and explored them systematically for the immobilization of Hb and the construction
of electrochemical biosensors. Different from the earlier-reported OMCs, the GMC
has a high degree of crystallinity and graphitization, which has been proven by the
XRD pattern (Fig.6.12). Two intensive peaks at 26 and 44.5 indexed to the typi-
cal (002) and (100) diffractions of the graphite are observed in the XRD pattern.
The high graphitization degree of GMC reveals the good electrical conductivity,
which is essential for the electrical transducing of biorecognition events.
Figure6.13 shows the typical cyclic voltammograms of different enzyme elec-
trodes in 50mM pH 7.0 PBS. A pair of stable and well-defined Hb redox peaks is
observed at the HbNafionGMC-6/GC electrode (Fig.6.13a). The peak current is
much larger than those at HbNafionCNT/GC and the HbNafion-GMC-13/GC.
The ket0 of Hb immobilized on HbNafionGMC-6/GC and HbNafionGMC-13/
GC are estimated to be about 1.32 and 1.14s1, respectively, which are comparable
to that of Hb immobilized on HbNafionCNTs/GC (1.18 s1), suggesting a fast
electron-transfer process. The surface concentration of Hb is estimated to be
6.251010 mol cm2 at HbNafionGMC-6/GC, which is more than four times
larger than those obtained at the HbNafionCNTs/GC- (1.731010molcm2) and
Hb-Nafion-BGMC/GC (less than 21010molcm2) modified electrodes, suggesting
6.3 Biosensors Based on Mesoporous Materials 191

Fig.6.13 CVs of
(a) HbNafionGMC6/GC,
(b) HbNafionGMC13/
GC, (c) HbNafionCNT/
GC, and (d) HbNafion/GC
in pH 7.0 PBS with scan rate
of 0.2Vs1. Reprinted with
permission from Lu etal.
[94]. 2009, Royal Society
of Chemistry

the larger effective conductive surface areas of the GMC. GMC-6 offers significant
advantages over GMC-13 and CNTs in facilitating the ET of entrapped Hb and
improving the performance of the fabricated biosensors, indicating that suitable
pore size is important for Hb immobilization. The biosensor based on GMC-6 dis-
plays excellent analytical performance over a wide linear range (1180mM, n=19,
R=0.9894) along with good stability and selectivity for the detection of H2O2, and
suggesting graphitized-ordered mesoporous carbons with good pore size matching
for enzymes will be a promising electrochemical biosensing platform.

6.3.5Biosensors Based on Mesoporous Metal Oxide

Besides the large surface area and narrow pore size distribution, mesoporous metal
oxides may have some specific characteristics. For example, it has been reported that
TiO2 can coordinate with COOH and NH2 groups, thus facilitating the immobiliza-
tion of proteins without leaching from the pores [76]. MnO2 can catalyze the electro-
chemical oxidation of H2O2 at pH values close to neutral [101, 102]. Once those
metal oxides own the mesostructure, those specific characteristics can be amplified
due to the large surface area, therefore indicating that mesoporous metal oxides are
promising enzyme-immobilized hosts for electrochemical biosensing applications.
In 2008, mesoporous MnO2 [103] was synthesized by Zengs group through a sol
gel process using nonionic surfactant polyxyethylene fatty alcohol as template.
Although the obtained MnO2 material presented disordered porous structure, the
pore size was appropriate and suitable for the immobilization of GOD. An ampero-
metric glucose biosensor based on GOD entrapped in MnO2 was fabricated, in which
MnO2 also acted as catalyst for the electrochemical oxidation of H2O2 produced by
enzymatic reactions. The biosensor showed a fast and sensitive current response to
glucose in the linear range of 0.00092.73mM with a sensitivity of 24.2mAcm2mM1.
192 6 Biosensors Based on Nanoporous Materials

The good performance indicated MnO2 has promising application in enzyme


immobilization and biosensor construction.
Zhaos group synthesized a kind of mesoporous Nb2O5 and applied it in the
immobilization of cytochrome c [104]. They demonstrated that at potential higher
than the band gap, semiconductor Nb2O5 exhibited a certain electrical conductivity
and had the possibility to promote electrical transfer. Jias group [16] reported the
use of highly ordered mesoporous titanium oxide (mesoTiO2) materials as an Hb
immobilization matrix. Hb in the mesoTiO2 matrix could retain its native secondary
structure, and achieved direct ET between the electrode and the enzyme. The results
demonstrated that the mesoTiO2 matrix could improve the protein loading with the
retention of bioactivity and greatly promote the direct ET.

6.3.6Biosensors Based on Mesoporous Hybrid Nanocomposite

Recently, hybrid mesoporous materials with two or more compositions have


attracted considerable attentions, since the synergism between the different compo-
nents always leads to surprising phenomena. Nowadays, inorganicorganic hybrid-
ization and inorganicinorganic hybridization nanoporous materials have been
prepared for enzyme immobilization. The inorganicorganic composition synthe-
sized by functional group grafting can be used for protein immobilization and pro-
vides the protein with a suitable microenvironment and good stability. The
introduction of additional inorganic components into the mesowalls always leads to
specific optical, electrical, and catalytic abilities and thus has promising aspects in
electrochemical biosensing areas.
In 2007, Jus group [4] synthesized a kind of mesocellular silicacarbon nano-
composite foam (MSCF) and used it for the immobilization and biosensing of GOD.
Figure6.14 shows the characteristics of the MSCF. From the transmission electron
micrograph (TEM), X-ray spectroscopic (EDX), and nitrogen sorption isotherms, it
can be concluded that the as-prepared MSCF has an ordered mesostructure, large
surface area, and ordered mesopore size distribution. The pore size distributions
calculated using the BarrettJoynerHalenda method are 7 and 20nm, indicating
the MSCF possesses ink-bottlelike pores [41] and that the MSCF is composed of
main cells with a diameter of 20nm interconnected by 7-nm windows.
Comparing with OMC, the MSCF possesses better hydrophilicity: The water con-
tact angle of the MSCF of 32.6 is similar to that of 30.3 for the mesoporous silicate
SBA-15, and much less than that of 79.1 for pure carbon mesoporous material [105].
The presence of silicate greatly improves the hydrophilicity of the nanocomposite,
which is beneficial to the approach of analytes to enzyme-active sites [106] and the
enhancement of the biocompatibility for the material. Furthermore, compared with
OMS composed of insulated SiO2, the conductivity of MSCF is significantly
improved, which can accelerate the ET between the immobilized enzyme and the
electrode. Jus group compared the conductivity of MSCF with single (SWCNT) and
mutiwalled CNTs (MWCNTs), OMC, and MSF, and found that the conductivity of
6.3 Biosensors Based on Mesoporous Materials 193

Fig.6.14 (a) TEM, (b) EDX spectrum, (c) nitrogen adsorption/desorption isotherms, and (d) pore
size distributions of the MSCF. Reprinted with permission from Wu etal. [4]. 2007, Wiley

MSCF was comparable to those carbon materials. Electrochemical impedance


spectra showed the electron-transfer resistances at these electrodes were in the range
of 2030W except for one at MSF-modified GCE, indicating the MSCF possessed
good conductivity similar to OMC, SWCNT, and MWCNT.
Considering the large surface area, suitable pore size, good hydrophilicity, and
good conductivity, the MSCF seems to be a promising candidate for the immobili-
zation and electrochemical biosensing of enzyme. In fact, the immobilized GOD
showed fast direct electrochemistry corresponding to its FAD/FADH2 redox couple.
As shown in Fig.6.15, the cyclic voltammogram of the MSCF/GOD/Nafion shows
a pair of well-defined redox peaks at 468 and 444mV. These peaks result from
the direct ET of the immobilized GOD for the conversion of GOD(FAD) to
GOD(FADH2) [107]. The formal potential of the GOD is 456mV, which is very
close to the standard electrode potential of 460mV (vs. saturated calomel elec-
trode) for FAD/FADH2 at pH 7.0 (25C) [107], indicating that most GOD molecules
preserve their native structure after impregnating the MSCF. In comparison, the
MSCF/FAD/Nafion-modified electrode shows a pair of redox peaks at 519 and
468mV, respectively (Fig.6.15), producing a formal potential of 493mV, which
is more negative than that of 456mV for the immobilized GOD. Considering the
194 6 Biosensors Based on Nanoporous Materials

Fig.6.15 CVs of the MSCF/


GOD/Nafion, MSCF/Nafion,
GOD/Nafion, and MSCF/
FAD/Nafion-modified GCE
in 0.2M pH 7.0 PBS at
100mVs1. Reprinted with
permission from Wu etal.
[4]. 2007, Wiley

fact that the reduction of FAD groups or the oxidation of FADH2 groups inside the
peptides of GOD is easier or more difficult than free FAD or FADH2 due to the pres-
ence of positively charged amino acid residues of these peptides at pH 7.0, the redox
couple of the MSCF/GOD/Nafion-modified electrode is not free FAD. The ket0 is
calculated to be 16.4s1, and it is the largest value reported, confirming the good
microenvironment and conductivity MSCF provided for GOD loading and biosens-
ing. Based on a decrease in the electrocatalytic response of the reduced form of
GOD to dissolved oxygen, the proposed biosensor shows a linear response to glu-
cose concentration ranging from 50mM to 5.0mM, with a detection limit of 34mM
at an applied potential of 0.4V. It has good stability and selectivity, and can exclude
the interferences from ascorbic acid and uric acid that always coexist with glucose
in real samples. All these results confirm that the nanocomposite foam is a good
matrix for protein immobilization and biosensor preparation.
By introducing transition metal elements into the mesostructure, the catalytic abil-
ity of mesoporous hybrid material can be improved. For example, Jus group designed
a conductive nanocage composed of PdNPsdoped MSCF [14] by a designed wetness
chemical process followed by electrochemical reduction. The TEM image (Fig.6.16a)
demonstrated the formation of PdNPs (black dots) with sizes of 13nm on the meso-
walls. The corresponding EDX (Fig. 6.16b) showed the typical peak of palladium
with the content of 6.1% at 2.85keV, which confirmed the existence of palladium. The
introduction of PdNPs significantly improved the electrochemical activity of the
nanocage. At the Pd-MSCF-modified electrode, the anodic oxidation of H2O2 occurred
at +0.25V, while it occurred at potentials more positive than +0.51V at the MSCF-
modified electrode (Fig.6.17a). Obviously, the presence of PdNPs made the oxidation
overpotential decrease for 260mV. In addition, the amperometric responses of the
Pd-MSCF-modified electrodes to 1.0mM H2O2 in pH 7.0 PBS at +0.3V was 24.3mA,
more than 120 times larger than 0.201mA obtained at the MSCF-modified electrode
(Fig.6.17b). After GOD entrapment and under the optimized conditions, the glucose
6.3 Biosensors Based on Mesoporous Materials 195

Fig. 6.16 Morphology and composition of Pd-MSCF. (a) TEM of Pd-MSCF; (b) EDXS of
Pd-MSCF. Reprinted with permission from Wu etal. [14]. 2008, American Chemical Society\

Fig.6.17 (a) CVs of Pd-MSCF- and MSCF- (inset) modified electrodes in 0.2M pH 7.0 PBS in
the absence (solid) and presence (dash) of 3.0 mM H2O2. (b) Amperometric responses of the
MSCF- (dash) and Pd-MSCF- (solid) modified electrodes to 1.0mM H2O2 in 0.2M PBS at +0.3V.
Reprinted with permission from Wu etal. [14]. 2008, American Chemical Society

biosensor showed a surprisingly fast response, which achieved 95% of the steady-state
current in less than 3s, a linear range from 5.0mM to 4.0mM, a limit of detection of
1.0 mM, and a high sensitivity of 2.12 mA mM1. All those results confirmed the
advantages of the hybrid nanocomposites.
Another example of hybrid nanoporous materials with improved catalytic ability
was reported by Dai etal. [108], who prepared Ti-containing MCM-41 and found
the material could significantly catalyze the electrochemical oxidation of NADH
with an overpotential decrease of about 400mV. The author ascribed the decrease
in the potential of NADH oxidation to the higher hydrophilicity induced by
Ti-MCM-41 at the modified electrode, which offered the electrode surface a better
contact with the solution containing NADH and consequently could participate in
increasing the electron-transfer rate.
196 6 Biosensors Based on Nanoporous Materials

6.4Biosensors Based on Nanoporous Gold

Gold nanomaterials involving nanoparticle, nanofiber, nanotube, nanopore, and nan-


orod are the most widely used nanomaterials in the biosensing fields due to their
good biocompatibility, excellent catalytic ability, good conductivity, and strong inter-
action with thiol groups. As an important member of the gold nano-group, nanopo-
rous gold (NPG) not only possesses all the merits of Au nanomaterials, but it also has
a series of unique characteristics [5]. In detail, (1) it possesses a three-dimensional
spongy morphology with tunable nanopore and large surface area [109, 110]; (2) it is
bulk in nature yet nanoscale in microstructure, which means it can be easily applied
and recovered; (3) its pore size is tunable in a wide range, from a few nanometers to
many microns, which facilitates the study on the pore size-dependent effect; (4) it
can be prepared by simply dealloying Ag from Ag/Au alloys in concentrated HNO3;
NPG has very clean surfaces, which can eliminate the possible poisoning or
passivatingeffects from unwanted molecules or ions such as polymer surfactants and
Cl, as often used in Au nanoparticle preparation; (5) NPG can avoid particle aggre-
gation, thus improving the stability of the NPG-based electrode and prolonging its
lifetime. Up to now, the biosensors based on NPG almost spanned all biosensor
aspects, including DNA, antigens, enzymes, and bacteria. The good sensitivity indi-
cates that NPG is a promising platform for biosensor fabrication.

6.4.1Enzyme Biosensors Based on Nanoporous Gold

NPG is a good conductor and has a suitable pore size distribution and large surface.
It can greatly enhance the electrochemical response toward the enzymatic substrates
NADH and H2O2 based on their low-coordinated Au atoms. All these advantages
make it attractive for the construction of dehydrogenase- and oxidase-based biosen-
sors [111, 112], which may have improved sensitivity and anti-interference ability.
The great application of NPG was reported by Huang etal. [5], who developed two
NPG-based electrochemical biosensors. The NPG was prepared simply by dealloy-
ing Ag from Au/Ag alloy. The selective dissolution of silver from Ag/Au alloy
resulted in an open bicontinuous nanoporous microstructure comprised almost
entirely of gold, as shown in the inset of Fig.6.18. The dealloying mechanism was in
accord with an atomistic model proposed by Erlebacher etal. [109]. The nanoporous
structure made NPG more active than gold sheet based on the fact that both the oxida-
tion and reduction peaks of the NPG/GCE were negatively shifted as compared with
the gold sheet electrode. The NPG-modified glassy carbon electrode (NPG/GCE)
exhibited high electrocatalytic activity toward the oxidation of NADH and H2O2. The
anodic peaks of NADH and H2O2 occurred at 0.52 and 0.4 V at the NPG/GCE-
modified electrode, which were more negative than the value of 0.72 and 0.8V at the
gold sheet-modified electrodes. In addition, the peak currents of NADH and H2O2 at
the NPG/GCE were much larger than those at the gold sheet electrode (Fig.6.19).
The high density of edge-plane-like defective sites and large specific surface area of
NPG should be responsible for the electrocatalytic behavior. Such electrocatalytic
6.4 Biosensors Based on Nanoporous Gold 197

Fig.6.18 CVs of (a) NPG/GCE and (b) gold sheet electrode in 0.1M PBS. Scan rate: 50mVs1.
The inset is the SEM image of the NPG with a pore size of 40nm. Reprinted with permission from
Qiu etal. [5]. 2009, Elsevier

Fig.6.19 CVs of (a) NADH (1mM) and (b) H2O2 (4mM) at (a) NPG/GCE and (b) gold sheet
electrode in 0.1M PBS (pH 7.2). Scan rate: 50mVs1. Reprinted with permission from Qiu etal.
[5]. 2009, Elsevier

behavior of the NPG/GCE permitted the effective low-potential amperometric


biosensing of ethanol or glucose via the incorporation of ADH or GOD within the
three-dimensional matrix of NPG. The ADH- and GOD-modified NPG-based bio-
sensors showed good analytical performance for biosensing ethanol and glucose due
to the clean, reproducible, and uniformly distributed microstructure of NPG. The
stabilization effect of NPG on the incorporated enzymes also made the constructed
biosensors very stable. After 1 months storage at 4C, the ADH- and GOD-based
biosensors lost only 5.0 and 4.2% of the original current response. All these results
made NPG another popular material for electrochemical biosensor construction.
198 6 Biosensors Based on Nanoporous Materials

Fig.6.20 Chronocoulometry determination of DNA hybridization through two steps of amplifica-


tion. Reprinted with permission from Hu etal. [115]. 2008, American Chemical Society

6.4.2DNA Biosensors Based on Nanoporous Gold

In recent years, the development of highly sensitive and selective DNA sensors to
bring down the limit of detection to pico- and femtomolar levels has been a field of
ever-increasing interest since the genoassays are suitable for various applications,
including clinical diagnosis, environmental control, and forensic analysis [113
115]. For the DNA biosensor preparation, the immobilization of biomolecular
probes on a desired substrate is a very important process since the sensitivity, detec-
tion resolution, and reproducibility are all significantly affected by these steps. Gold
substrates have attracted special attention as an electrode material for the construc-
tion of DNA electrochemical biosensors due to their strong interaction with thio-
lated DNA via Authiol binding. Thiolated DNA can be monolayered on gold in a
self-assembly manner, which provides stable and structurally well-defined electro-
chemical interfaces [116]. Recently, Zhangs group [115] developed a sensitive
electrochemical DNA sensor based on an NPG electrode prepared by dealloying Ag
from Au/Ag alloy and multifunctional encoded Au nanoparticles (Au NPs)
(Fig.6.20). The active surface area of the NPG electrode was 9.2 times higher than
that of a bare flat electrode as characterized by CVs. A DNA biosensor was fabricated
6.4 Biosensors Based on Nanoporous Gold 199

Fig.6.21 SEM images of (a) Au nanoporous film and (b) PGNF. Reprinted with permission from
Yang etal. [9]. 2009, Elsevier

by immobilizing capture probe DNA on the NPG electrode and hybridization with
target DNA, which further hybridized with the reporter DNA loaded on the Au NPs.
The Au NP contained two kinds of bio-barcode DNA; one was complementary to
the target DNA, while the other was reducing the cross-reaction between the targets
and reporter DNA on the same Au NP. Electrochemical signals of [Ru(NH3)6]3+
bound to the reporter DNA via electrostatic interactions were measured by
chronocoulometry.
Taking advantage of the dual-amplification effects of the NPG electrode and
multifunctional encoded Au NP, this DNA biosensor could detect the DNA target
quantitatively, in the range of 8.010171.61012M, with a limit of detection as
low as 28aM, and exhibited excellent selectivity even for single-mismatched DNA
detection.

6.4.3Escherichia coli Biosensors Based on Nanoporous


Platinum-Coated Gold Nanoporous Film

Bimetallic nanoparticles always feature interesting catalytic behavior with respect


to monometallic systems; therefore, considerable efforts have recently been devoted
to this field. Jin etal. [9] recently prepared a Pt nanoparticlecoated Au nanoporous
film (PGNF) [117] and used it for the detection of Escherichia coli. Figure 6.21
shows the SEMs of Au nanoporous film and the PGNF. An irregular and porous
structure of the Au nanoparticles film is shown in Fig.6.21a. Figure6.21b shows
that the Pt nanoparticles cover the Au nanoporous film uniformly and the Pt/Au film
become denser. The porous structure facilitates the fast ET and increases the cata-
lytic ability of the PGNF; thus, the anodic peak currents of K3Fe(CN)6 at the PGNF
electrode is 1.7 times larger than that of the bare Au electrode. The resulting PGNF
electrode exhibited high electrocatalytic activity toward p-aminophenol (PAP),
200 6 Biosensors Based on Nanoporous Materials

which was the electroactive substance produced when b-galactosidase catalyzed the
hydrolysis of p-aminophenyl-b-d-galactopyranoside (PAPG) in the culture medium
of E. coli, and the quantity of PAP was proportional to the concentration of E. coli.
The linear range for PAP was from 10 nM to 40 mM, with a detection limit of
1108M. Based on a sensitive detection of PAP, this PGNF electrode could suc-
ceed in detecting E. coli rapidly and sensitively. The linear range for E. coli was
from 2101 to 1106CFUmL1, with a detection limit of 10CFUmL1 (S/N=3).
The analytical performances as well as the repeatability of its E. coli response dem-
onstrated that this PGNF electrode would be suitable for the fabrication of portable
amperometric E. coli sensor.

6.5Conclusions

In conclusion, this chapter describes the recent advances in the development of


nanoporous material-based biosensors. Due to the inherent advantages of a large
surface area and little diffusion limitation, nanoporous materials have been widely
used for biomaterial immobilization and biosensor fabrication. Today, nanoporous
materials with various compositions, different pore sizes, and multiple structures
have been used for biomaterial immobilization, and their application range has cov-
ered almost all the biomaterials (enzyme, protein, antigen, DNA, and bacteria).
Additionally, the role of nanoporous materials has not been limited to being just an
immobilized matrix; some noble metal- and carbon-based nanoporous materials
have acted as good electron-transfer media and highly catalytic materials, which
could further improve the performance of the biosensors. Due to their obvious mer-
its, nanoporous materials have been widely established as a kind of robust biosensor
fabrication material, which would have great prospects.

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wwwwwwwwwwwww
Chapter 7
Electrochemical Biosensing Based
on Carbon Nanotubes

7.1Introduction

7.1.1Structure of CNTs

Since their discovery, carbon nanotubes (CNTs) have been extensively investigated
as essential platforms in constructing electrochemical biosensors. CNTs can be
classified into two basic varieties: single-wall carbon nanotubes (SWCNTs), which
are a single layer of graphene sheet rolled into cylindrical tubes, and multiwall car-
bon nanotubes (MWCNTs) comprised of multiple layers of concentric cylinders
with a spacing of about 0.34nm between the adjacent cyclinders (Fig.7.1). The
lengths of the nanotubes can range from several hundred nanometers to several
micrometers, and the diameters from 0.22nm for SWCNTs and from 2 to 100nm
for MWCNTs [1]. CNT synthesis techniques can be classified into three major
categories: laser ablation, catalytic arc discharge, and chemical vapor deposition [2].
Due to the diameters being similar to or smaller than those of individual biomole-
cules, CNTs are expected to serve as high-performance electrical conduits for
interfacing with biological systems. Therefore, there is enormous interest in utiliz-
ing CNTs for biosensor applications [3, 4].
The insolubility of the SWCNTs in most solvents and the difficulties of handling
these highly intractable carbon nanostructures, however, are restricting their real-
life applications. In order to improve the properties of CNTs, two approaches for the
functionalization of CNTs have been involved: noncovalent interaction, including
physical adsorption and entrapment of biomolecules around the nanotubes [5], and
covalent interaction to the functional groups produced via chemical reactions on the
p-conjugated skeleton of a CNTs sidewall [6]. The noncovalent functionalization
of the SWCNTs can preserve the desired electronic properties of the CNTs while
remarkably improving their solubility. Coupled with immobilized biomolecules as
the recognition element, the functionalized CNTs as a sensing platform exhibit high
sensitivity and selectivity in electrochemical biosensing.

H. Ju et al., NanoBiosensing: Principles, Development and Application, 207


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0_7, Springer Science+Business Media, LLC 2011
208 7 Electrochemical Biosensing Based on Carbon Nanotubes

Fig.7.1 The structures of SWCNTs (left) and MWCNTs (right). Reprinted with permission from
Kim etal. [1], 2007, Wiley

7.1.2Advantages of CNT-Based Electrochemical Sensors

Typically, there are three advantages for CNT-based electrochemical sensors: (1)
The high length-to-diameter ratios provide high surface-to-volume ratios for the
immobilization of functional molecules. In fact, the high surface area of SWCNTs
is estimated to be as high as 1,600m2/g; (2) CNTs have an outstanding ability to
mediate fast electron-transfer kinetics for a wide range of electroactive species,
leading to an enhanced electrochemical signal; (3) the small size of CNTs is suitable
to miniaturize the device for in situ detection.
Due to these unique properties, CNTs have attracted much attention on their
potential application in biosensing. In this chapter, two functionalization categories,
noncovalent interaction and covalent binding, are introduced to enhance the CNTs
hydrophobility for bioaffinity and realize efficient electrical communication, leading
to the amplification of signal transduction. Further, CNT-based biosensing method-
ologies are highlighted and discussed for the biosensing of DNA, antigenantibody,
cells, and other biological molecules. Finally, field-effect transistors and CNT
forests are also summarized for ultrasensitive detection. Electrochemical biosensing
based on CNTs provides a powerful way to miniaturize the device and achieve the
real-time detection of biomolecules.

7.2Functionalization Strategy of CNTs

Chemical functionalization offers an efficient route not only to modify the chemical
and physical properties of SWCNTs but also to further expand their potential
application areas as recognition elements [7]. The main approaches for the
functionalization of SWCNTs can be divided into two broad categories, namely,
covalent and noncovalent approaches.
7.2 Functionalization Strategy of CNTs 209

Fig.7.2 Schematic
representation of
N-succinimidyl-1-
pyrenebutanoate-decorated
SWCNTs. Reprinted with
permission from Zhao and
Stoddart [10], 2009,
American Chemical Society

7.2.1Noncovalent Interaction

The main advantage of noncovalent functionalization of CNTs is the conservation of


the CNTs electronic structure by preventing disruption of the intrinsic nanotube sp2
structure and conjugation. Most notably, small aromatic molecules and conjugated
polymers have been used to decorate the nanotube surface via electrostatic interaction,
pp stacking, or van der Waals force, and modify solubility and electronic properties
of CNTs, leading to highly specific electrochemical biosensors [8].

7.2.1.1Aromatic Small-Molecule-Based Noncovalent Functionalization

Aromatic molecules, such as pyrene, porphyrin, and their derivatives, can interact
with the sidewalls of SWCNTs by means of pp stacking interactions, thus opening
up the way for the noncovalent functionalization of SWNTs. Dai and coworkers
reported a general and attractive approach to the noncovalent functionalization of
SWCNTs sidewalls and the subsequent immobilization of biological molecules onto
SWCNTs with a high degree of control and specificity [9]. The biofunctional mole-
cule N-succinimidyl-1-pyrenebutanoate was irreversibly adsorbed (Fig.7.2) onto the
hydrophobic surfaces of SWCNTs in either N,N-dimethylformamide (DMF) or
MeOH, which led to the further functionalization of SWCNTs with succinimidyl
ester groups that were reactive to nucleophilic substitution by primary and secondary
amines of some proteins, such as ferritin, streptavidin, and biotiny l-3,6-dioxaoctane-
diamine [10]. This technique has enabled the immobilization of a wide range of
biomolecules on the SWCNTs sidewalls, with high specificities and efficiencies,
and applications that may be useful for the development of biosensors.
Recently, Guldis group designed several new hybrid materials of CNTs and
electron donors via two steps. They first prepared SWCNT/pyrene+ hybrids via pp
stacking, and then immobilized negatively charged electron donors onto SWCNT/pyrene+
via electrostatic interaction to yield the electron donoracceptor nanohybrids [11].
210 7 Electrochemical Biosensing Based on Carbon Nanotubes

Fig.7.3 Photographs of vials containing 0.5mg/mL of SWCNTs in different solutions: (a) phos-
phate buffer (0.05M, pH 7.4); (b) 98% ethanol; (c) 10% ethanol in phosphate buffer; (d) 0.1%
Nafion in phosphate buffer; (e) 0.5% Nafion in phosphate buffer; and (f) 5% Nafion in ethanol.
Reprinted with permission from Wang etal. [14], 2003, American Chemical Society

The direct pp interaction between porphyrins and SWCNTs played an important


role in achieving the ordered assembly of protonated porphyrin in the form of J- and
H-type aggregates on the SWCNTs surface [12]. A functional composite of
SWCNTs with hematin, a water-insoluble porphyrin, was prepared in 1-butyl-3-
methylimidazolium hexafluorophosphate (BMIMPF6) ionic liquid, which provided
an easy way for accelerating the electron transfer, constructing highly sensitive bio-
sensors, and extending the application of water-insoluble porphyrin [13].

7.2.1.2Entrapment

Entrapment is an alternative method of solubilizing CNTs for biosensing. The first


example of solubilizing SWCNT in Nafion was described by Wangs group in 2003
[14]. Figure7.3 displays photographs of vials containing SWCNT in the presence
of Nafion along with the corresponding control solutions. Increasing the Nafion
content (from 0.15 wt%; Fig.7.3df) results in dramatic enhancement of the solu-
bility of SWCNTs. A homogeneous, well-distributed solution of the NafionCNTs
complex is observed in the 5% polymer solution. Such solubilization is not observed
using the corresponding ethanol or phosphate-buffer control solutions (Fig.7.3ac).
The resulting homogeneous CNTsNafion solution can be doped on electrode to
produce a strong and stable electrocatalytic response toward hydrogen peroxide at a
low potential [15].
SWCNTs can be readily wrapped with single-stranded DNA (ssDNA) through
aromatic interaction for biosensing [16, 17]. Further, long ssDNAs synthesized by
rolling-circle amplification were used to functionalize CNTs [18]. In comparison to
genomic DNAs, the sequence of a rolling-circle amplification product can be easily
controlled because a defined circular DNA is used as the template to generate the
required ssDNA. This long ssDNA, with length comparable with that of SWCNTs,
will potentially be useful for scaffolding SWCNTs to fabricate precisely assembled
larger-scale microelectronics.
7.2 Functionalization Strategy of CNTs 211

Conducting polymers, such as polypyrrole, polyaniline, and polythiophene, are


attractive for sensor applications because their electronic and electrochemical prop-
erties are highly sensitive to molecular interactions. For example, a hybrid nano-
composite has been fabricated on a gold electrode by the in situ electrochemical
polymerization of 3-aminophenylboronic acid monomers in the presence of ssDNA/
SWCNTs [19]. The resulting biosensor can detect nanomolar dopamine with a lin-
ear range of four orders of magnitude. A poly(3-methylthiophene)-modified glassy
carbon electrode (GCE) coated with Nafion/SWCNT film was fabricated and used
for the highly selective and sensitive determination of dopamine with a detection
limit of 5.0nM [20].
The first example of the incorporation of SWCNTs into redox polymer hydrogels-
enzyme films was reported by Schmidtkes group [21, 22]. The composite films
were constructed by first incubating an enzyme in an SWCNT solution and then
cross-linking within a poly[(vinylpyridine)Os(bipyridyl)2Cl2+/3+] polymer film. The
incorporation of SWCNTs, modified with glucose oxidase (GOD), into the redox
polymer films resulted in a two- to tenfold increase in the oxidation and reduction
peak currents during cyclic voltammetry, while the glucose electrooxidation current
was increased three-fold to ~1mA/cm2 for glucose sensors [21]. A redox polymer,
poly(vinylimidazole), complexed with Os(4,4-dimethylbpy)2Cl has been electrode-
posited on Ta-supported MWCNTs for glucose sensing [23]. In addition, a simple
one-step electrodeposition method was developed for constructing an enzyme
chitosan (CHIT) CNT composite by the electrolysis of H+ at a direct-current power
supply (3.0V) to decrease the solubility of CHIT on electrode surface. The nano-
composite exhibited good electrocatalytic ability in the reduction and oxidation of
hydrogen peroxide [24].
In addition, many inorganic materials, such as CHIT, surfactant, peptide
amphiphiles, phospholipids, and solgel, have been involved in the functionaliza-
tion of CNTs. For example, the biocompatible CHIT or polyamino-saccharide chi-
tosan was used to solubilize the nanotubes, followed with covalent immobilization
of glucose dehydrogenase (GDH) [25, 26] and acetylcholinesterase (AChE) [27]
using glutaric dialdehyde (GDI) as a cross-linking reagent. The surfactant of sodium
dodecyl sulfate (SDS) is most frequently used for the noncovalent functionalization
of CNTs [28], which constitutes a simple and versatile protocol for the noncovalent
functionalization of nanotubes for the development of new biosensors [29, 30].
Peptide amphiphiles show more advantages for the dispersion and noncovalent
functionalization of CNTs in water due to their short hydrophobic alkyl tails cou-
pled with a more hydrophilic peptide sequence [31]. A highly nontoxic and conduc-
tive ormosil composite film has been prepared using (3-aminopropyl)triethoxysilane
and 2-(3,4-epoxycyclohexyl)-ethyltrimethoxysilane by doping ferrocenemonocar-
boxylic acidbovine serum albumin conjugate and MWCNTs. After immobilizing
GOD in the film, the resulting biosensor can detect glucose down to 20mM with a
wide linear range of 0.0520.0mM [32]. Similarly, horseradish peroxidase (HRP)
can be immobilized in this film for constructing a H2O2 biosensor [33]. The enzyme
entrapped in solgel was highly stable and showed good affinity toward its substrate
due to the low diffusion barrier.
212 7 Electrochemical Biosensing Based on Carbon Nanotubes

Interestingly, metallic nanoparticles such as platinum, palladium, and gold have


been dispersed on the solubilized CNTs to form metallic nanoparticle/CNT nano-
composite [3436]. Since these nanoparticles can be easily modified with a wide
range of biomolecules, they provide a versatile method for the fabrication of CNT-
based biosensors. For example, a PtMWCNT composite was electrochemically
deposited on the electrode surface for sensor fabrication. The resulting sensor
showed a linear range of 5.01071.5105 mol/L for estradiol, 2.0106
5.0105mol/L for estrone, and 1.01067.5105mol/L for estriol [37]. When
SWCNT surfaces are decorated with Pd metal, individual SWCNTs become approx-
imately twice as sensitive to H2 gas [38].
Carboxylic acid- and amino-functionalized ionic liquids acted as a linker to con-
nect metal nanoparticles with CNTs. The imidazolium ring moiety of ionic liquids
might interact with the p-electronic nanotube surface by virtue of cationp and/or
pp interactions, and the functionalized group moiety of ionic liquids might interact
with the metal NPs surface. It was observed that platinum nanoparticleMWCNT
hybrids could be well dispersed in water, which may find future applications in
fields such as catalysis, nanoscale electronics, as well as sensors [39].

7.2.1.3Layer-by-Layer Fabrication of CNT Electrode

One simple and versatile method to assemble dispersed CNTs into thin films is
using layer-by-layer (LBL) assembly, which consists of the repeated, sequential
immersion of a substrate into aqueous solutions of complementarily functionalized
materials. This technique can produce conformal ultrathin films and highly tunable
surfaces using various nanomaterials on geometric surfaces. For example, positively
charged PtCNTCHIT solution and negatively charged poly(sodium-p-styrenesul-
fonate) salt (PSS) have been employed to fabricate stable ultrathin multilayer films
on gold electrode and quartz glass slides in an LBL fashion. With the immobiliza-
tion of cholesterol oxidase onto the electrode surface using GDI, a biosensor that
responds sensitively to cholesterol has been constructed due to the synergistic action
of Pt nanoparticles and CNTs. In pH 6.98 phosphate buffer, the almost interference-
free determination of cholesterol has been realized at 0.1V versus SCE with a linear
range from 0.01 to 3mM and response time <30s [40].
More recently, negatively and positively charged MWCNTs have been function-
alized on their exterior walls with carboxylic acid groups yielding MWCNTs-
COOH, and with amine groups yielding MWCNTs-NH2. LBL MWCNT films,
which consist of well-dispersed MWCNTs, are produced by LBL using stable dis-
persions of negatively and positively charged MWCNTs (Fig. 7.4). Unlike other
multilayer assemblies that contain CNTs as one of two or more components, these
systems incorporate MWCNTs without the incorporation of additional organic
materials. This difference enables the development of 100% CNT thin films with
properties that can be manipulated using assembly conditions. In particular, the
irregular shape of the CNTs enables the direct formation of porous, all-carbon
nanostructures with a high surface area. Sheet resistance and cyclic voltammetric
7.2 Functionalization Strategy of CNTs 213

Fig. 7.4 Layer-by-layer assembled MWCNT thin film with positively and negatively charged
MWCNTs. Reprinted with permission from Lee etal. [41], 2009, American Chemical Society

measurements show that these MWCNT thin films are promising electrode materials
for high-power and high-energy electrochemical devices [41].
A glucose biosensor was constructed based on an electrostatic LBL technique.
Gold electrodes were initially functionalized with negatively charged 11-mercap-
toundecanoic acid followed by alternate immersion in solutions of a positively
charged redox polymer, poly[(vinylpyridine)Os(bipyridyl)2Cl2+/3+], and a negatively
charged enzyme, GOD, or a GOD solution containing SWCNTs. The incorporation
of SWCNTs led to a dramatic (617-fold) increase in the current response, which
depended on the number of multilayers [42].

7.2.2Covalent Interaction

The covalent attachment of bioactive molecules on CNTs is considered to be a


useful tool for biological application. Two general strategies are involved in the
covalent functionalization of CNTs: amide bond formation at nanotube ends using
a carboxyl group [43], and nonselective attack of nanotube sidewalls by highly reac-
tive species such as nitrenes [44] and aryl diazonium salts [45]. Typically, CNTs are
first treated by oxidation in strong acid, leading to a number of carboxyl groups on
the CNTs surface. Then, using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
(EDC) as cross-linker, biomolecules [46, 47] can be covalently conjugated onto
carboxylated CNTs via amide bond. Experiments with surfactant-free or SDS-
protected SWCNTs can show a great extent of nonspecific adsorption of the biomol-
ecules onto the wall of CNTs in biosensing.
A relatively general route to MWCNT-based electrochemical biosensors
was reported by confining ferricyanide redox mediator onto MWCNTs [48]. The
strategy was essentially based on the creation of positively charged moieties onto
CNTs by first grafting CNTs with polyether via an in situ cationic ring-opening
214 7 Electrochemical Biosensing Based on Carbon Nanotubes

Fig.7.5 Synthetic route of the polyether-grafted and MIM-tethered MWCNTs. Reprinted with
permission from Xiang etal. [48], 2008, American Chemical Society

polymerization of epoxy chloropropane and then introducing positively charged


methylimidazolium (MIM) moieties through a quaternization reaction between
methylimidazole and polyether-grafted CNTs (Fig. 7.5). The positively charged
MIM moieties introduced onto the CNTs could essentially interact with Fe(CN)63
through an electrostatic interaction and consequently confine such kinds of redox
mediators onto CNTs. The prepared nanocomposite with surface-confined Fe(CN)63
redox mediator could be used as efficient electronic transducers for the general
development of redox enzyme-based electrochemical biosensors.
Since the relatively fewer defect sites on the pristine CNTs limit the introduction
of the sidewall substituents, heteroatom-doped carbon nanotubes, especially nitrogen-
doped carbon nanotubes (CNx-MWCNTs), are expected to be excellent candi-
dates for biosensor design [49, 50]. The doped nitrogen atom can act as an electron
donor and introduce some defective sites or active sites on the nanotube surface for
functionalization. For example, the direct noncovalent assembly of porphyrin on
CNx-MWCNTs via the FeN coordination provides a facile way for designing
novel biofunctional materials for highly efficient biosensing [51]. CNx nanotubes
can be more biocompatible when compared to CNTs and might be more advanta-
geous for practical applications.

7.3Fabrication and Characterization of CNT-Based Sensors

The morphology of CNT-based sensors is usually characterized by scanning electron


microscopy (SEM), transmission electron microscopy (TEM), Fourier transform
infrared (FT-IR) spectroscopy, atomic force microscopy (AFM), and resonance
Raman spectroscopy.
7.3 Fabrication and Characterization of CNT-Based Sensors 215

Fig.7.6 (a) SEM and (b) TEM images of aligned carbon nanotubes. Reprinted with permission
from Alonso-Lomillo etal. [52], 2007, American Chemical Society

7.3.1Scanning Electron Microscopy and Transmission


Electron Microscopy

When MWCNTS are grown on gold electrodes by microtechnology, the SEM image
can display their length of approximately 50mm (Fig.7.6a). The TEM study verifies
the presence of MWCNTs with average outer and inner diameters of 1525 and
512nm, respectively (Fig.7.6b). The vertically aligned CNTs with a high density
can be employed as a platform for the oriented and stable immobilization of an
Ni-Fe hydrogenase. A sigmoidal shape of the cyclic voltammogram is observed, in
which the current density above 550mV almost reaches a plateau, which is related
to the H2 concentration in the range of 1.3310.74mM [52].

7.3.2Fourier Transform Infrared

Arginine-glycine-aspartic acid-serine (RGDS)-functionalized SWCNTs have been


prepared for electrochemical cytosensing [53]. The covalent conjugation of the
RGDS tetrapeptide to SWNTs can be proved with FT-IR spectra. The oxidized
SWCNTs display two peaks, at 1,718 and 1,586 cm1 (Fig. 7.7a), which can be
216 7 Electrochemical Biosensing Based on Carbon Nanotubes

Fig.7.7 FT-IR spectra of


(a) oxidized SWCNTs and
(b) RGDS-SWCNTs.
Reprinted with permission
from Cheng etal. [53],
2008, American
Chemical Society

assigned to the carbonyl stretch mode of -COOH and -COO. After covalent conju-
gation of the RGDS tetrapeptide to the SWCNT, the vibration of amide I and amide
II of the RGDS tetrapeptide can be observed at 1,645 and 1,533cm1 (Fig.7.7b),
whereas the peaks assigned to the carbonyl stretch mode decreased greatly, indicat-
ing the effective reaction of the carboxylic acid groups of SWNTs with the amino
groups of the RGDS. The conjugated RGDS shows a predominant ability to capture
cells on the electrode surface by the specific combination of RGD domains with
integrin receptors on the cell surface [53].

7.3.3Atomic Force Microscopy

To achieve direct electron transfer, SWCNTs act as a nanoconnector that electri-


cally contacts the active site of the enzyme and the electrode. Figure7.8 shows the
AFM images of the GODSWCNTs hybrid. SWCNTs with one or two enzyme
units are observed at the edges of the tubes. The height of the enzyme units is about
5nm, which is consistent with the dimensions of GOD. The high-resolution TEM
image is also verified in a CNT modified with two GOD units (negatively stained
with uranyl acetate) at the edges of the tube. These results suggest that the reconsti-
tution of GOD units takes place preferentially at the edges of the FAD-modified
SWCNTs. An interfacial electron-transfer-rate constant of 42s1 is estimated for
50-nm SWCNTs [54], which provides a significant potential for constructing an
electrochemical biosensor.

7.3.4Raman Spectrum

Poly-l-lysine-functionalized SWCNTs are useful in promoting cell adhesion. The


covalent attachment of poly-l-lysine to SWCNTs can be confirmed by Raman spec-
tra. Both spectra of SWCNTs-PLL (Fig. 7.9a) and SWCNTs (Fig. 7.9b) contain
7.3 Fabrication and Characterization of CNT-Based Sensors 217

Fig.7.8 AFM image of


SWCNTs reconstituted at
their ends with GOD units.
Reprinted with permission
from Patolsky etal. [54],
2004, Wiley

Fig.7.9 Raman spectra of (a) SWCNTsPLL assembly and (b) shortened SWCNTs. Reprinted
with permission from Zhang etal. [55], 2004, American Chemical Society

characteristic peaks at 1,558 and 1,581cm1 (tangential modes) and at 1,336cm1


(disorder mode). As the disorder mode is the diagnostic of disruptions in the hex-
agonal framework of the SWCNTs, the increase in relative intensity of this mode
provides direct evidence of the covalent modification of SWCNTs. SWCNTs play
an important role as connectors to assemble these active amino groups of poly-L-
lysine, which provides a relative friendly and soft environment for further deri-
vation, such as attaching bioactive molecules. As a potential application, HRP is
successfully immobilized on the SWCNT-PLL assembly, and the chemically modi-
fied electrode exhibits amplification for H2O2 biosensing [55].
218 7 Electrochemical Biosensing Based on Carbon Nanotubes

7.4Amplification of Signal Transduction

It is very important to achieve ultrasensitive measurements in biological recognition


events. Electrochemical transducers are very attractive for such bioassays due to
their high sensitivity, inherent simplicity, and miniaturization as well as their low
cost and power requirements. Two strategies are involved to enhance the signal
transduction, which are based on the biocatalysis of labeled enzymes and excellent
electronic conductor of CNTs.
Alkaline phosphatase (ALP)-loaded CNTs dual-amplification route has been
demonstrated for ultrasensitive electrical bioassays of proteins and DNA [56, 57].
The ALP-loaded CNT tags are captured to the streptavidin-modified magnetic beads
by a sandwich DNA hybridization. Electrochemical detection of the product of the
enzymatic reaction yields a dramatic enhancement of the sensitivity at the CNT-
modified GCE. The remarkably low detection limit is around 1fg/mL (54aM) for
the sensing of DNA. Similarly, IgG can be determined with a detection limit of
500fg/mL (160zmol in 25-mL samples) [56].
Compared with the conventional immunosensor providing one label per binding
event (Fig.7.10a), a multilabel strategy provides advantages of enhanced sensitivity
and selectivity. Greatly amplified sensitivity was achieved by using bioconjugates
featuring HRP labels and secondary antibodies (Ab2) linked to MWCNTs at a high
HRP/Ab2 ratio to replace single-HRP-labeled secondary antibodies (Fig. 7.10b).
This resulted in a detection limit of 4pg/mL (100amol/mL) for prostate-specific
antigen (PSA) in 10mL of undiluted calf serum. PSA in laser-microdissected cancer
cells from prostate tissue was measured more quantitatively than measured by the
gold standard immunohistochemical method [58]. These easily fabricated SWCNT
immunosensors showed excellent promise for the clinical screening of cancer bio-
markers and point-of-care diagnostics.
Except for ALP and HRP, GOD has been immobilized on CNTs to enhance the
enzymatic signal [59]. Typically, a novel immunosensor array was constructed by
coating LBL colloidal Prussian blue (PB), gold nanoparticles (Au NPs), and capture
antibodies on screen-printed carbon electrodes and coupling with a new tracer nano-
particle probe-labeled antibody (Ab2) that was prepared by one-pot assembly of
GOD and the antibodies on Au NP-attached CNTs. The CNT-based probe could
provide dual signal amplification for the multiplexed immunoassay by a PB-mediated
electron-transfer process to catalyze the reduction of H2O2 produced in a GOD cycle
at an individual working electrode. The immobilized PB could not only eliminate
the electrochemical cross-talk but also avoid the interference of dissolved oxygen.
Using carcinoembryonic antigen and a-fetoprotein as model analytes, the simulta-
neous multiplexed immunoassay method showed linear ranges of three orders of
magnitude, with detection limits down to 1.4 and 2.2pg/mL, respectively [59]. The
great potential application of this assay approach is to detect low-abundant proteins
in clinical sample.
On the other hand, based on the excellent electronic conductor of CNTs, a signal
amplification for the terminal protection assay of small-molecule-linked ssDNA
7.4 Amplification of Signal Transduction 219

Fig.7.10 Illustration of detection principles of SWCNTs immunosensors. (A) treatment with a


conventional HRP-Ab2 (a) to provide one label for per binding event. (B) treatment with HRP-
CNT-Ab2 (b) to provide numerous enzyme labels for per binding event. Reprinted with permission
from Yu etal. [58], 2006, American Chemical Society

Fig.7.11 Terminal protection assay of small-molecule-linked ssDNA. Reprinted with permission


from Wu etal. [60], 2009, American Chemical Society

was developed. As shown in Fig. 7.11, the SWCNT-wrapping ssDNA can be


digested stepwisely from the 3 end by exonuclease I (Exo I), producing SWCNTs
with no surface-tethered ssDNA. These naked SWCNTs can precipitate from the
220 7 Electrochemical Biosensing Based on Carbon Nanotubes

solution and are assembled on the 16-mercaptohexadecanoic acid (MHA)


self-assembly monolayer (SAM). Because the SWCNTs adsorbed on the isolating
MHA SAM can mediate efficient electron transfer between the electrode and an
electroactive species such as ferrocenecarboxylic acid, a strong redox current is
generated due to the signal amplification from single SWCNTs. In the presence of
small-molecule-binding proteins, the ssDNA-wrapped SWCNTs can bind to the
protein target through the small-molecule moiety at the 3 terminus, thus preventing
the degradation of the ssDNA by Exo I. Because of strong electrostatic and hydra-
tion repulsions between the DNA-SWCNTs and the negatively charged SAM, the
SWCNTs wrapped by the protected ssDNA cannot be adsorbed on the MHA SAM,
keeping the MHA SAM isolated with no background signal. According to the
mechanism, a tumor biomarker of folate receptor is detected within the range
10pM5.0nM and a detection limit of 3pM [60].
In the same group, a novel electrochemical immunoassay strategy was proposed
by using phospholipid-coated CNTs as the electrochemical labels. In contrast to
conventional electrochemical labels that were immediately converted into electroactive
species for electrochemical readouts, the CNT-based labels controlled assembly on
an electrode was blocked by an insulating SAM. This CNT assembly then mediated
the electrochemistry of certain redox indicators, creating a very sensitive and spe-
cific tactic for signal transduction [61].
In addition, integrating the hydrophilic ion-conducting matrix of CHIT with a
two-electron mediator Toluidine blue O (TBO) and an electronic conductor CNT,
the produced NADH sensor showed very low oxidation overpotential and good ana-
lytical performance due to the synergy between CNTs and redox mediators.
Significantly, it amplified the NADH current by ~60 times, while reducing the
response time from ~50s for CHIT-TBO to ~5s for CHIT-TBO/CNT films [62].

7.5Electrochemical Biosensing Based on Functional CNTs

7.5.1Deoxyribonucleic Acid

Electrochemical DNA sensors are regarded as a particularly suitable tool for direct
and fast biosensing since they can convert the hybridization event into an electro-
chemical signal. DNA-sensing approaches include the intrinsic electroactivity of
DNA, electrochemistry of DNA-specific redox indicators, electrochemistry of
enzymes, and conducting polymers and ionic liquid (RTIL).
The direct electrochemical oxidation of guanine or adenine residues of ssDNA
leads to an indicator-free DNA biosensor. A composite screen-printed carbon elec-
trode modified with MWCNTs has been used for the detection of calf thymus
ssDNA ranging from 17.0 to 345mg/mL, with a detection limit of 2.0mg/mL at 3s,
and yeast tRNA ranging from 8.2mg/mL4.1mg/mL [63].
Many cationic metal complexes and intercalating organic compounds can be
used as redox indicators for the development of highly sensitive DNA biosensors.
7.5 Electrochemical Biosensing Based on Functional CNTs 221

Fig.7.12 (af) Square-wave voltammograms for 0 (control), 1, 5, 10, 20, and 40fg/mL, DNA
target. Also shown (insets) are the resulting calibration plot and the response for a 0.2-fg/mL target
solution, and the protocol. Reprinted with permission from Munge etal. [66], 2005, American
Chemical Society

For example, a novel approach was used to fabricate DNA biosensors, in which self-
assembled MWCNTs directly grew vertically to the Au substrates, followed by
DNA adsorption. Hybridization between the probe and target DNA oligonucleotides
was confirmed by the changes in the voltammetric peak of the indicator of methyl-
ene blue. The DNA biosensors based on self-assembled MWCNTs achieved a
higher hybridization efficiency than those based on random MWCNTs [64].
Signal amplification using enzyme multilayers on CNT templates has been
shown to yield a remarkably sensitive electrochemical detection of nucleic acids.
Hellers group demonstrated that a highly sensitive amperometric monitoring of
DNA hybridization (down to 5zmol) could be achieved in connection with an HRP-
labeled target [65]. Figure7.12 displays the potential of the ALP-LBL-CNT ampli-
fication route for the ultrasensitive electrochemical detection of DNA hybridization
in connection with sandwich assays, with a second probe conjugated to the CNT-
(PDDA/ALP)4PDDA/streptavidin label, along with enzymatic reaction of the
R-naphthyl phosphate substrate. It shows the voltammetric hybridization response
for extremely low DNA target concentrations, ranging from 1 to 40 fg/mL
(542,160aM). These aM concentration changes result in well-defined square-wave
voltammetric signals for the R-naphthol product. The favorable response for the
0.2-fg/mL target solution indicates a detection limit of approximately three
copies/mL (0.1fg/mL, 5.4aM) based on the signal-to-noise characteristics (S/N=3).
222 7 Electrochemical Biosensing Based on Carbon Nanotubes

Given the enormous amplification afforded by the new CNT-LBL biolabel, such a
route offers great promise for the ultrasensitive detection of infectious agents and
disease markers [66].
A sensitive electrochemical DNA biosensor was successfully realized on poly-
aniline nanofibers (PANI), MWCNTs, and CHIT-modified carbon paste electrode
(CPE) based on the synergistic effect between PANI and MWCNTs in CHIT film.
PANI and MWCNT nanocomposites resulted in highly enhanced electron conduc-
tive and biocompatible nanostructured film. The immobilization of the probe DNA
on the surface of the electrode was largely improved due to the unique synergistic
effect of PANI and MWCNTs. Under optimal conditions, the dynamic detection
range of this DNA electrochemical biosensor for the phosphinothricin acetyltrans-
ferase gene was from 1.010131.0107 mol/L, with a detection limit of
2.71014mol/L [67].
Hydrophobic room-temperature ionic liquid of BMIMPF 6, combined with
nanosized shuttle-shaped cerium oxide (CeO2) and SWCNTs, has been applied
in electrochemical sensing of the immobilization and hybridization of DNA.
The remarkable difference between the Ret value at the probe DNA-immobilized
electrode and that at the hybridized electrode can be used for the label-free
electrochemical impedance spectroscopy detection of the target DNA. Under
optimal conditions, the dynamic range for detecting the sequence-specific
DNA was from 1.010121.0107 mol/L, and the detection limit was
2.31013mol/L [68].

7.5.2AntigenAntibody

Two strategies in CNT-based immunoassays are involved: labeled with enzyme or


electroactive mediators, and label-free immunosensors. As one of the most popular
tracer labels, enzymes, including ALP, HRP, and GOD, have been immobilized on
CNTs to enhance the enzymatic signal. Typically, a sandwich-type immunosensor
is designed for detecting mouse IgG (Fig. 7.13). Biotinylated antimouse IgG is
immobilized on avidin-modified CNTs to allow the specific binding of a mouse
IgG. Next, ALP-conjugated antimouse IgG is bound to the mouse IgG. The bound
ALP catalyzes the conversion of p-aminophenyl phosphate to p-aminophenol,
which is electrooxidized on the MWCNTs. The detection limit for mouse IgG is
10pg/mL [57]. The sensitive electrochemical immunosensor should take advantage
of the low background current of an indium tin oxide electrode, the good electro-
catalytic properties of MWCNTs, and the low biofouling properties of poly(ethylene
glycol)-silane copolymer.
The picomolar electrochemical detection of human immunodeficiency virus
type-1 protease (HIV-1PR) using ferrocene (Fc) -pepstatin-modified surfaces has
been presented by modifying a gold electrode with Au NPs or thiolated SWCNTs/
Au NPs. The sensing electrode modified with thiolated SWCNTs/Au NPs shows
remarkable detection sensitivity, with an estimated detection limit of 0.8pM [69].
7.5 Electrochemical Biosensing Based on Functional CNTs 223

Fig.7.13 Schematic
illustration of an
electrochemical
immunosensor for detecting
mouse IgG. Reprinted with
permission from Aziz etal.
[57], 2007, Royal Society
of Chemistry

In addition, a sensitive method for the detection of cholera toxin (CT) using an
electrochemical immunosensor with liposomic magnification has been proposed
[70]. The sensing interface consisted of monoclonal antibody against the B subunit
of CT that was linked to poly(3,4-ethylenedioxythiophene) coated on Nafion-
supported MWCNT caste film on a GCE. The sandwich assay provided the ampli-
fication route for the detection of CT ranging from 1014107g/mL, with a detection
limit of 1015g/mL [70]. A disposable electrochemical immunosensor for carcino-
embryonic antigen using ferrocene liposome and MWCNT-modified screen-printed
carbon electrode was also developed recently [71].
The label-free immunosensor shows a convenient fabricating and detection pro-
cedure. For example, several label-free peptide-coated CNT-based immunosensors
have been proposed for the direct assay of human serum sample using square-wave
stripping voltammetry [72], quartz crystal microbalance measurements [73], and
differential pulse voltammetry [74]. Based on carbon nanotube field-effect transis-
tor (CNT-FET), a label-free protein biosensor functionalized with a solution con-
taining various linker-to-spacer ratios was prepared for the detection of a prostate
cancer marker [75].
Recently, protein arrays that measure multiple protein cancer biomarkers in clin-
ical samples have been seen to hold great promise for reliable early cancer detec-
tion. A prototype four-unit electrochemical immunoarray was reported based on
SWCNT forests for the simultaneous detection of multiple protein biomarkers for
prostate cancer. After the final washing, the immunoarray was placed into an elec-
trochemical cell containing the mediator hydroquinone in buffer, and hydrogen per-
oxide was injected to develop the amperometric response (Fig.7.14). The steady-state
current increased linearly in clinically relevant ranges for PSA (140 ng/mL),
prostate-specific membrane antigen (PSMA) (10250ng/mL), and platelet factor-4
(PF-4) (140ng/mL). The calibration plot for interleukin-6 (IL-6) (50500pg/mL)
was biphasic, with a better sensitivity below 350pg/mL, but the overall sensitivity
was appropriate for accurate measurements in the clinical range [76].
224 7 Electrochemical Biosensing Based on Carbon Nanotubes

Fig. 7.14 Amperometry at 0.3 V and 2,500 rpm for SWCNTs immunoarrays: (a) PSA;
(b) PSMA; (c) PF-4; and (d) IL-6. Reprinted with permission from Chikkaveeraiah etal. [76],
2009, American Chemical Society

7.5.3Cells

The functionalization of CNTs with biomolecules such as peptide and monosac-


charides is a useful way to achieve biocompatible interactions between CNTs and
living cells. For example, the thin-film networks of SWCNTs were glycosylated
with monosaccharides such as N-acetyl-d-glucosamine, d-glucose, or d-mannose.
These sugar moieties are anchored onto nanotubes by either a pyrene or lipid tail.
Glycosylated SWNTs-net can directly interface with living cells by supporting their
adhesion and growth for monitoring dynamic biomolecular release from these cells
[77]. Using a CNT network, the label-free detection of ATP release from living
astrocytes was developed with high temporal resolution [78].
Based on the functionalization of SWCNTs with RGDS, a cytosensor array for
multiplex evaluation of both the glycan expression profile on an intact cell surface
and the dynamic changes in the glycome during drug treatment has been presented
[79]. The RGDS-functionalized SWCNTs are immobilized on the electrode surface
for capture of cells by the specific recognition of RGDS to integrin on cell surface.
7.5 Electrochemical Biosensing Based on Functional CNTs 225

Fig.7.15 Schematic representation of the electrochemical cytosensor array for cell-surface gly-
can analysis, and close-up illustrations of (a) cells captured on RGDS-SWCNTs/SPCE and (b)
HRP-lectin binding with cell-surface glycans. Reprinted with permission from Cheng etal. [79],
2009, Wiley

Four HRP-labeled lectins (concanavalin A, dolichos biflorus agglutinin, peanut


agglutinin, and wheat germ agglutinin) are used for simultaneous multiple analysis
at four different working electrodes. As shown in Fig.7.15, glycans on cancer cell
surfaces could be selectively conjugated to corresponding HRP lectins. The immo-
bilized HRP, the amount of which depended on the specific carbohydrate sites on
the cell surface, produced characteristic electrochemical signals from enzyme catal-
ysis in the solution containing 8.0mM H2O2 and 10mM o-phenylenediamine (o-PD)
for sensitive readout of cell-surface glycans. The optimal concentration of K562
cells used for the capture of cells on the cytosensor array was 6106 cells/mL. The
designed electrochemical cytosensor array was used for analyzing the dynamic
change of the K562 cell-surface glycome during erythroid differentiation induced
by sodium butyrate [79].
A metal-cluster-decoration CNT-based biosensor was constructed by evaporat-
ing 3 Cr and 5 Au using an e-beam evaporator for real-time cell detection.
Monoclonal antibody for Aureococcus anophagefferens was used to functionalize
the device, which could detect A. anophagefferens at a concentration of 104 cells/
mL with a signal-to-noise ratio of 7. The further functionalization of the metal-
cluster-decoration CNTs with Tween-20 could suppress nonspecific binding and
enable the label-free and selective detection of A. anophagefferens [80]. To improve
the detection sensitivity and specificity, gold-plated CNT conjugated with folic acid
were used as a second contrast agent for rapid photoacoustic detection of circulating
tumor cells in the bloodstream of mice [81].
226 7 Electrochemical Biosensing Based on Carbon Nanotubes

7.5.4Other Biomolecules

7.5.4.1Nitric Oxide

Nitric oxide (NO), a simple gas molecule and a free radical, was biologically
identified as an endothelium-derived relaxing factor in 1987. Some pathological
processes, such as acute hypertension, diabetes, ischemia, and atherosclerosis, are
found to be associated with abnormalities of NO release. However, it is hard to
accurately measure the NO level due to its fast reaction with oxygen and short
lifetime in biological systems. The noncovalent nanoassembly of porphyrin on
SWCNTs manifested the efficiently electrocatalytic reduction of nitric oxide [82].
In that case, NO sensors were made by modifying the surface of GCE. However,
the big size of GCE is not suitable for the accurate quantitative determination of
NO on the single-cell level. A novel NO electrochemical microsensor was fabri-
cated by modifying the surface of a carbon fiber microdisk electrode (diameter:
57mm) with SWCNTs and a Nafion membrane. The modification of SWCNTs
dramatically improved the sensitivity, with a detection limit of 4.3nM for NO,
which was nearly 10 times lower than that from the bare one and lower than most
NO electrochemical sensors previously reported [83]. The Nafion membrane
offered a good barrier to some interferents, such as nitrite and ascorbic acid, with-
out losing response speed to NO. The sensor has been successfully applied to the
measurement of NO release from single isolated human umbilical vein endothe-
lial cells.

7.5.4.2Glucose

The ultimate goal of glucose sensing is to develop the third-generation biosensor,


which is based on the direct electron transfer between the cofactor FAD of GOD
and the electrode [84]. However, the direct electron transfer for the oxidation of
FADH2 [85] or the reduction of FAD [86] is hard to realize at conventional elec-
trodes, because the FAD is deeply seated in a cavity and not easily accessible for
the conduction of electrons from the electrode surface. Thus, many CNT-based
nanohybrids have been explored to immobilize GOD for glucose biosensing [87].
For example, a glucose biosensor based on the deposition of only 32 units of GOD
onto crystalline Au nanoparticle-modified MWCNT electrode was prepared by a
very simple chemical reduction method. The fabricated biosensor exhibited a lin-
ear range from 0.05 to 22mM, with a detection limit of 20mM and sensitivity of
0.4mA/mM [88]. The GC electrode modified by both Pt nanoparticles and CNTs
responded even more sensitively to glucose than the GC/GOx electrode modified
by Pt nanoparticles or CNTs alone. The response time and detection limit of this
biosensor were determined to be 3s and 0.5mM, respectively [89]. Quantum dots
(QDs) are now extremely attractive and important nanomaterials in bioanalytical
7.5 Electrochemical Biosensing Based on Functional CNTs 227

applications. Due to the synergy between the CdTe QDs and CNTs, this novel
biosensing platform based on a QD/CNT electrodes responded even more sensi-
tively to glucose than that based on a GC electrode modified by CdTe QDs or
CNTs alone [90].
A bienzymatic glucose biosensor was proposed for the selective and sensitive
detection of glucose [91, 92]. This mediatorless biosensor was made by the simul-
taneous immobilization of GOD and HRP in an electropolymerized pyrrole film on
SWCNT-coated electrode. The linear response range of the bienzymatic sensor was
from 0.03 to 2.43mM, with a correlation coefficient of 0.998, while the monoenzy-
matic sensor was only from 0.93 to 4.93mM, with a correlation coefficient of 0.996.
The detection limits, based on a signal-to-noise ratio of 3, were estimated to be
around 0.030.009 and 0.90.07 mM for the bienzymatic and monoenzymatic
sensors, respectively [92].

7.5.4.3Hydrogen Peroxide

It is very interesting to detect H2O2 since H2O2 is a product of the enzymatic reac-
tions between most oxidases and their substrates. An H2O2 biosensor has been fab-
ricated based on the direct electrochemistry and electrocatalysis of myoglobin (Mb)
immobilized on silver nanoparticle-doped CNT film with hybrid solgel techniques.
A pair of redox peaks with a peak separation of 160mV and a formal potential of
0.295V were observed at this composite film, corresponding to the direct electro-
chemistry of Mb. The heterogeneous rate constant was estimated to be 0.41 s1.
Under optimum conditions, the amperometric determination of H2O2 was performed
with a linear range of 2.01061.2103 mol/L and a detection limit of
3.6107 mol/L (S/N=3). The MichealisMenten constant was estimated to be
1.62mmol/L [93].
The polyanilinePB/MWCNT hybrid amplified the sensitivity greatly due to the
synergy between the polyanilinePB and MWCNTs [94]. Recently, a new composite
electrode has been fabricated using MWCNTs and the ionic liquid n-octylpyridinum
hexafluorophosphate (OPFP). Compared to other electrodes modified with CNTs
and other ionic liquids, one major advantage of this electrode is its extremely low
capacitance and background currents. The MWCNT/OPFP electrode responded
very rapidly to changes in the level of H2O2, producing steady-state signals within
46s [95].
The utilization of iron-based species (mainly metallic iron, hematite, and
magnetite) encapsulated into MWCNTs as reactants provides a new platform to
monitor H2O2. PB is electrosynthesized in a heterogeneous reaction between
ferricyanide ions in aqueous solution and the iron species encapsulated into
CNTs, resulting in an intimate contact between the PB and the CNTs. The elec-
trode exhibited a low detection limit (1.94108mol/L) and a high sensitivity
(15.3A/cm2M) [96].
228 7 Electrochemical Biosensing Based on Carbon Nanotubes

Fig.7.16 Successive amperometric response of porphyrin/SWCNT-BMIM PF6-modified GCE to


TCA in PBS (0.1M, pH 7.0) at 0.40V. Insets: linear calibration curve and amplified response.
Reprinted with permission from Tu etal. [13], 2009, Wiley

7.5.4.4Organohalide Pollutant

Trichloroacetic acid (TCA), as an organohalide pollutant, is of major environmental


concern due to its extensive use in agriculture and public health programs. Thus, the
World Health Organization has urgently appealed for the development of rapid, reli-
able, and accurate analytical methods for the detection of TCA. A direct assembly
of water-insoluble porphyrin on SWCNTs was designed via the pp noncovalent
interaction between porphyrin and CNTs in the presence of BMIMPF6 ionic liquid
[13]. The resulting sensor showed excellent electrocatalytic activity toward the
reduction of TCA, producing a highly sensitive biosensor for TCA due to the syn-
ergic effect of SWCNTs, ionic liquid, and porphyrin. A linear TCA concentration
range from 9.01071.4104M, with a detection limit of 3.8107M at a signal-
to-noise ratio of 3, was obtained (Fig.7.16).

7.5.4.5Epinephrine

Epinephrine (EP) is an important catecholamine neurotransmitter in the mammalian


central nervous system. Many life phenomena are related to the concentration of
EP in blood. A novel modified CNT paste electrode with 2-(4-oxo-3-phenyl-3,
4-dihydro-quinazolinyl)-N-phenyl-hydrazinecarbothioamide as a new electrocatalyst
was fabricated. The modified electrode displayed strong catalytic function for the
oxidation of EP and norepinephrine (NE) and resolved the overlapping voltammet-
ric response of EP and NE into two well-defined voltammetric peaks. A linear
response was obtained in the range of 51085.5104M, with a detection limit of
9.4nM for EP [97].
7.6 SWCNT-Based Field-Effect Biosensing 229

7.6SWCNT-Based Field-Effect Biosensing

SWCNT-FET is a promising device for the specific recognition of biomolecules.


A CNT-based FET relies on the shift of the threshold voltage and the change in electri-
cal conductance of the CNTs [98101]. Since the charge transport in SWCNTs
directly contacts the environment, it is extremely sensitive to its chemical and physi-
cal environment. Potentially, a single biomolecule, present on the surface or in close
proximity to the SWCNT, can alter its electronic properties, leading to a sensitive
biosensor.

7.6.1Detection of Proteins by SWCNT-Field-Effect Transistor

The first biosensor based on an individual SWCNT-FET was reported by Dekkers


group [102]. GOD was attached to the sidewalls of a semiconductive CNT by a
biofunctional reagent with a pyrene group (Fig. 7.17a). GOD immobilization
appears to have a strong effect on the conductance of a semiconducting SWCNT.
Figure 7.17(b) shows that DMF decreased the conductance of a semiconducting
SWCNT, which may be the result of the electron-donating power of DMF upon
adsorption to the tube. Most importantly, the measurements showed that attachment
of only about 50 molecules of GOD (the tube length is, on average, 600nm) signifi-
cantly decreased the conductance of a semiconducting SWCNT. This demonstrated
that the sensor has potential for measuring GOD proteins. Moreover, a change of
conductance of GOD-coated semiconducting SWCNT upon the addition of glucose
indicated that an enzyme-activity sensor could be constructed at the single-molecule
level of an individual SWCNT [102]. In the presence of redox mediators, such as
K3Fe(CN)6/K4Fe(CN)6 and K2IrCl6/K3IrCl6, the SWCNT-FETs were shown to lin-
early detect the enzyme activity of the blue copper oxidase, laccase, varied over two
orders of magnitude of enzyme concentration in the picomolar range [103].
SWCNT-FET devices have been fabricated for highly sensitive detecting of spe-
cific proteinprotein interactions [104, 105]. Figure 7.18 shows the electrical
responses of CNTFETs after the introduction of PBS solution, nontarget proteins
(bovine serum albumin, fibrinogen, streptavidin, each 10mg/mL), and target protein
(10pg/mL IgG) onto the IgG Fab-modified CNT-FET. When the target protein was
introduced on the CNT channel, the electrical signal rapidly decreased. In contrast,
upon addition of PBS buffer as control or nontarget proteins, the devices showed a
slight increase in electrical conductance. The CNT-FET biosensors had very low
sensitivity (detection limit ~1,000ng/mL), whereas those based on small Fab frag-
ments could detect 1pg/mL (~7-fM level) [106]. Significantly, this strategy can be
applied to general antibody-based detection schemes, and it should enable the pro-
duction of label-free ultrasensitive electronic biosensors to detect clinically impor-
tant biomarkers for disease diagnosis. In addition, based on aptamer-modified
CNT, a protein CNT-FET was fabricated for the detection of immunoglobulin E (IgE).
230 7 Electrochemical Biosensing Based on Carbon Nanotubes

Fig.7.17 (a) Schematic


picture of two electrodes
connecting a semiconducting
SWCNT with GOD enzymes
immobilized on its surface.
(b) Conductance of a
semiconducting SWCNT as a
function of the liquid-gate
voltage in milli-Q water.
Reprinted with permission
from Besteman etal. [102],
2003, American Chemical
Society

Fig.7.18 Electrical conductance of CNTFET after the introduction of PBS buffer solution, non-
target proteins (bovine serum albumin, fibrinogen, streptavidin), and target protein onto the IgG
Fab-modified CNTFET. Reprinted with permission from Kim etal. [106], 2008, Elsevier
7.7 SWCNT Forest in Electrochemical Biosensing 231

The detection limit for IgE was determined as 250 pM [107]. The label-free
SWCNT-FETs with molecular-scale sensitivity are attractive for point-of-care
applications in immunoassay.

7.6.2Detection of Nucleic Acids by SWCNT-Field-Effect


Transistor

The label-free CNT-based filed-effect sensor offers a new approach for a next gen-
eration of DNA biosensing [108, 109]. For example, a simple and generic protocol
for the label-free detection of DNA hybridization was demonstrated with a random
sequence of 15 and 30 mer oligonucleotides by detecting the charge transfer
inherent to the hybridization reaction [110]. A network CNT-based field-effect
transistor (NTNFETs) was also reported by Star et al. as selective detectors of
DNA immobilization and hybridization [111]. NTNFETs with immobilized syn-
thetic oligonucleotides have been shown to specifically recognize target DNA
sequences for label-free DNA detection at picomolar to micromolar concentra-
tions. The sensing mechanism attributed the strong effect of DNA counterions on
the electronic response, thus suggesting a charge-based mechanism of DNA detec-
tion using NTNFET devices [111].
On the other hand, aptamers are artificial oligonucleotides that can bind to a wide
variety of entities with high selectivity, specificity, and affinity, equal to or often
superior to those of antibodies. The first SWCNT-FET-based biosensor comprising
aptamer was proposed by Lees group [112]. Adding thrombin to the thrombin
aptamer-functionalized SWCNT-FET surface caused a sharp decrease in conduc-
tance, thereby demonstrating the selectivity of the immobilized thrombin aptamers
[112]. The aptamer-modified SWCNT-FETs are promising candidates for the devel-
opment of label-free protein biosensors.
Au- and Cr-contacted FET based on SWCNT networks was designed for the
detection of DNA hybridization. The contribution of electrode-SWCNT junctions
to DNA sensing was up to 6 times more pronounced than that of the SWCNT
channel [113].

7.7SWCNT Forest in Electrochemical Biosensing

For electrochemical biosensors, the ordered CNT forest will be necessary to obtain
the best sensitivities and detection limits. The highly ordered CNT array was fabri-
cated by chemical vapor deposition within a hexagonally ordered, anodized, alumi-
num oxide nanopore template. These vertically aligned MWCNT arrays showed
dense packing on the order of 1010/cm2, and a tight, even plane of nanotube tips,
which are highly suitable for interfacing with biomolecules (Fig.7.19). The site-
selective, covalent docking of the enzyme GOD on the CNT tips had a marked
232 7 Electrochemical Biosensing Based on Carbon Nanotubes

Fig.7.19 SEM image of highly ordered CNT array. Reprinted with permission from Withey etal.
[114], 2006, Elsevier

effect on enhancing electron-transfer properties. A molecular electron-transfer rate


of 1,500s1 has been measured for this system, exceeding the rate of oxygen reduc-
tion by GOD [114]. The redox enzyme-CNT array conjugate can be utilized as a
quantitative substrate-specific biosensor.
The patternable, conductive, nanoscale structure of an SWCNT forest provides
new opportunities in the development of nanoimmunosensor arrays. Based on the
coordination of the carboxylic acid groups of acid-oxidized SWCNTs to Fe(OH)3
adsorbed on a Nafion-coated pyrolytic graphite electrode (PGE), the resulting
SWCNT forest was employed to immobilize HRP or Mb, leading to fast electron
transfer from the redox sites of the enzymes to the electrode. The detection limit of
H2O2 was obtained to be 50 and 70nM for HRP or Mb, respectively [115]. Further,
a prototype amperometric immunosensor was evaluated based on the adsorption of
antibodies onto perpendicularly oriented assemblies of SWCNT forests in the same
group [116]. The forests were self-assembled from oxidatively shortened SWCNTs
onto Nafion/iron oxide-coated PGE. In the presence of a soluble mediator, the detec-
tion limit for HRP labeled biotin was 2.5pmol/mL. Unlabeled biotin was detected
in a competitive approach with a detection limit of 16nmol/mL and a relative stan-
dard deviation of 12%. The immunosensor showed low nonspecific adsorption of
biotinHRP (approx. 0.1%) when blocked with bovine serum albumin.
Recently, the aligned SWCNT array electrode has been developed for fabricating
a DNA hybridization biosensor based on the direct current response of guanine.
Figure7.20 shows the AFM images of the surface of GCE after the assembly of
SWCNTs. The AFM height cross-sectional analysis indicates that the diameters of
the majority of the tubes are shorter than 2nm. In most cases, the aligned SWCNTs
are observed to occur vertically as individual needles with a length less than 80nm.
The result indicates that carboxylic acid groups are only formed at the tip of the
SWCNTs, and therefore the SWCNTs can be accomplished to align vertically on
7.8 Conclusions 233

Fig. 7.20 Tapping-mode AFM of the surface of SWCNT array electrode: left, height images;
right, 3D images. Reprinted with permission from Zhang etal. [117], 2009, American Chemical
Society

the GCE surface. Under optimum conditions, the response is proportional to the
concentration of target DNA in the range of 40110nM, with a detection limit of
20nM. Ready renewal is the outstanding merit of this label-free biosensor, which
can be reused more than 3,000 times [117].

7.8Conclusions

Based on the unique properties of CNTs, a number of biosensing methodologies


with fast response, high sensitivity, and easy miniaturization have been developed
for DNA, antigenantibody, cells, and other biomolecules. Meanwhile, CNTs are in
direct contact with the environment, which permits them to act as chemical and
biological sensors in the single-molecule detection of biomolecules, especially for
the SWCNT-based FET. However, for biological applications, the lack of solubility
of CNTs in aqueous or organic solvents is a major technical barrier. Great efforts
have been devoted to searching for cost-effective approaches to functionalize CNTs
with biomolecules. Recently, a 1,3-dipolar cycloaddition, the so-called click reac-
tion, has shown potential to provide an elegant protocol to functionalize CNTs due
to high yields, an absence of byproducts, and perfect regioselectivity. Moreover,
further immobilization of biomolecules can be carried out in a variety of solvents at
a wide range of pH values over a broad temperature range. On the other hand, in
order to eliminate the false results, it is highly likely to simultaneously detect two or
more biomarkers in clinic diagnoses. Ordered SWCNT forests will achieve the
simultaneous measurement of multiple biomarkers with the best sensitivities and
detection limits. Future research should be directed toward developing an elegant
protocol to functionalize CNTs in the mild conditions and multiplexed bioassays of
SWCNT forests in CNT-based electrochemical biosensing.
234 7 Electrochemical Biosensing Based on Carbon Nanotubes

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wwwwwwwwwwwww
Chapter 8
Biosensing with Nanoparticles as
Electrogenerated Chemiluminsecence Emitters

8.1Introduction

Electrogenerated chemiluminescence (ECL), also known as electrochemiluminescence,


is the luminescence generated by relaxation of exited-state molecules that are produced
during an electrochemically initiated reaction [1]. The phenomenon of ECL has been
known for a long time. Reports date back as far as 1927 for the light emission of Grignard
compounds at applied potentials [2] and 1929 for the ECL of luminol [3]. Along with
subsequent publications concentrated on the investigation of the mechanism and nature
of ECL, especially of polyaromatic hydrocarbons (PAHs) and metal complexes [46],
ECL has now become a very powerful analytical technique and been widely used in the
areas of, for example, immunoassay, food and water testing, and biowarfare agent detec-
tion [7, 8]. As a method of producing light at an electrode, ECL represents a marriage
between electrochemical and spectroscopic methods. This gives ECL many distinct
advantages over other spectroscopy-based detection systems. For example, ECL do not
involve a light source as fluorescence methods do; thus, the attendant problems of scat-
tered light and luminescent impurities are absent without the presence of a background
signal. Moreover, the specificity of the ECL reaction associated with the ECL label and
the coreactant species decreases problems with side reactions, such as self-quenching.
Among various events in the development of ECL, as shown in Fig. 8.1, the
evolvement of ECL luminophores is of great importance, because the application of
ECL technology is mainly based on the ECL-emitting species. For example, ECL
from Ru(bpy)32+ was first reported in 1972, and then Ru(bpy)32+ was quickly devel-
oped as an important ECL emitter with outstanding applications. A number of new
ECL-emitting species were synthesized, and their ECL properties were investigated
[1]. The driving forces behind these kinds of studies included (1) finding new lumi-
nophores with higher ECL efficiencies and (2) modifying a moiety of the emitter so
that it can be used for labeling of bimolecular [8]. The numerous ECL-emitting spe-
cies are usually classified into three categories: (1) inorganic systems, whichmainly

H. Ju et al., NanoBiosensing: Principles, Development and Application, 241


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0_8, Springer Science+Business Media, LLC 2011
242 8 Biosensing with Nanoparticles as Electrogenerated Chemiluminsecence Emitters

Fig.8.1 Time line of ECL: 19641965, first experiments; 1965, theory; 1966, transients; 1969,
magnetic field effects; 1972, Ru(bpy)32+; 1977, oxalate; 1981, aqueous; 1982, Ru(bpy)32+ polymer
and persulfate; 1984, Ru(bpy)32+ label; 1987, tri-n-propylamine (TPrA); 1989, bioassay; 1993,
ultramicroelectrodes; 1998, laser action; 2002, semiconductive nanocrystals. Reprinted with per-
mission from Miao [8], 2008, American Chemical Society

contain organometallic complexes; (2) organic systems, which cover polycyclic


aromatic hydrocarbons; and (3) nanomaterial systems, especially semiconductor
nanoparticles (NPs, also known as nanocrystals; quantum dots).
It is well known that quantum dots (QDs) possess size-dependent optical, elec-
tronic, and chemical properties and have been widely used for biosensing applica-
tions [911]. For example, since the electrochemistry and ECL study of NPs were
first reported in 2002 for Si NPs [12], several hundreds of papers, patents, and book
chapters have been published on the ECL of NPs, ranging from the very fundamental
to the very applied. Also, several excellent reviews covering various aspects of ECL
have appeared [8, 1317]. The advances concerning the electrochemistry and ECL
of NPs not only open a promising field for the development of a new generation of
ECL-emitting species, but also complement the optical (spectroscopic, microscopic)
methods that are usually employed for QD applications [13]. This chapter covers
the reports from 2002 to 2009 on the principles, systems, and biosensing applica-
tions of NPs as ECL-emitting species.

8.2Principle of ECL from Nanoparticles

8.2.1ECL Mechanism of NPs

Although the ECL of NPs was first reported for the elemental semiconductor Si NPs
[12], the following research soon proved that many compound semiconductors (e.g.,
CdS [18], CdSe [19, 20], CdTe [21], ZnS [22], PbS [23, 24], CuSe [25], carbon NPs
[26, 27], CdO [28], core/shell CdSe/ZnS [29], nanotube CdSe [30], and thiol-capped
CdSe [31]) as well as the elemental semiconductor NPs (e.g., Si [12] and Ge [32])
can also produce ECL emission. Similarly to other common ECL emitters [7, 8], the
NP ECL is usually generated in two routines as reported by Bards group [12].
8.2 Principle of ECL from Nanoparticles 243

Annihilation routine. This kind of ECL emission involves electrochemically oxidizing


and reducing NPs to electrogenerated anion and cation radicals. For example, the
ECL occurs by charge injection into freely diffusing Si NPs under repetitive elec-
trode potential cycling or pulsing between oxidation and reduction of NPs [12]. As
Si NPs can be oxidized or reduced by injecting holes or electrons under electrochem-
istry conditions, the electron-transfer annihilation of electrogenerated anion and
cation radicals then results in the production of excited states for ECL emission.
Scheme8.1 summarizes the possible mechanism for the annihilation routine. Rand
R refer to negatively and positively charged Si NPs, respectively, electrogenerated at
+

the electrode surface, which then react in solution to give the excited state R*.

Scheme8.1

As semiconductor NPs can undergo redox chemistry by injecting holes or electron


under electrochemistry conditions [12, 3337], ECL from NPs can occur in the anni-
hilation routine if the resulting charged states are sufficiently stable to survive until
colliding with oppositely charged species in an annihilation reaction. From this point,
it is clear that not only are sufficient applied electrode potentials required to generate
both the negatively and positively charged species necessary for the annihilation rou-
tine, but also required is sufficient energy in the electron-transfer reaction to produce
the excited state. Thus, the NPs must be chemically stable and maintain their charged
states long enough to transfer charge upon colliding with oppositely charged NPs. No
ECL is observed through an annihilation-type mechanism 1 for thioglycerol-capped
CdS quantum dots due to the instability of the electrogenerated reactants [38].
Coreactant routine. The coreactant is a species that, upon oxidation or reduction,
produces an intermediate that can react with an ECL luminophore to produce excited
states. Unlike the ion-annihilation routine, in which electrolytic generation of both
the oxidized and reduced NPs is required, the coreactant routine involves electron
transfer only between the electrochemically generated nanocrystal species and a
deliberately added coreactant. Because all commercially available ECL analytical
instruments are based on coreactant ECL technology [8], understanding the mecha-
nisms of the coreactant ECL of NPs is of great importance for their applications.
By the coreactant routine, ECL can be generated with one directional potential
scanning at an electrode in a solution containing NP species (the emitter) in the
presence of coreactants, as coreactants can form strong oxidants or reductants by
bond cleavage under given conditions. For example, when the annihilation reaction
between oxidized and reduced species is not efficient, the use of a coreactant may
produce more intense ECL. In the first research for the ECL feature of NPs, Bards
group demonstrated that the higher-intensity light emission from the Si NP solution
was observed when coreactants were added [12]. If excess C2O42 is added to the NP
244 8 Biosensing with Nanoparticles as Electrogenerated Chemiluminsecence Emitters

solution, ECL will only require hole injection and can be easily obtained by simply
oxidizing the NPs. In this case, the oxidation of oxalate produces a strong reducing
agent, CO2, which can inject an electron into the LUMO of an oxidized Si NP to
produce an excited state that then emits light. The corresponding mechanism is
shown in Scheme8.2.

Scheme8.2 (5)
(6)
(7)

Similar to the coreactant for common ECL emitters [8], the coreactant is useful
especially when either R+ or R is not stable enough for NP ECL reaction or when
the ECL solvent has a narrow potential window so that R+ or R cannot be formed.
However, a number of criteria also need to be met for a good coreactant of NP ECL
[8], which includes solubility, stability, electrochemical properties, kinetics, quenching
effect, ECL background, etc. For example, the coreactant should be easily oxidized
or reduced at or near the electrode and undergo a rapid chemical reaction to form an
intermediate that has sufficient reducing or oxidizing energy to react with the oxi-
dized or reduced NPs to form the excited state. As many NP-based ECL sensors
have been developed with the coreactant routine [8, 1416], the coreactant routine
is of great importance for the biosensing application of NP ECL.

8.2.2Generation Type for NP ECL

The electron and hole injections for NP ECL favorably occur on the NC surface due
to the large surface area of NPs and many possible dangling bonds on the surfaces.
ECL mainly results from electron and hole functions at the particle surface. As the
electrochemical potentials found for reduction and oxidation can provide data for
the band gap of the NPs, the stepwise addition (or removal) of charge from NPs by
the electrochemical method can yield information for the energy required for elec-
tron transfer and ECL emission. With different energies required for the ECL emis-
sions, there are two different generation types for the ECL reaction of NPs. Also,
the different spectra features of NP ECL also provide unambiguous evidence that
NP ECL occurs in the two different models.
One type is the ECL originated from surface states of NPs. The spectra of this type
of NP ECL show red-shifted ECL maxima with respect to their PL spectra. This
surface-state model for ECL is demonstrated nicely in Fig.8.2. Photoluminescence
(PL) mainly occurs through excitation and emission within the NP core, while the
electron and hole wave functions can interact strongly with the NC surface. Different
energies are required to achieve the emitting states of PL and ECL processes
(Fig.8.3). Because less energy is required for the electron (or hole) injections, red-
shifted ECL maxima with respect to their PL spectra are featured for this type of
8.2 Principle of ECL from Nanoparticles 245

Fig.8.2 Schematic
representation of PL and ECL
in semiconductor NPs.
Reprinted with permission
from Miao [8], 2008,
American Chemical Society

Fig.8.3 ECL spectrum of


CdSe/ZnSe core/shell NPs in
a CH2Cl2 solution containing
0.1M TBAP. Reprinted with
permission from Myung etal.
[29], 2003, American
Chemical Society

ECL emission. The red-shifted ECL maxima with respect to their PL obtained from
Si [12], CdSe [19], and Ge [32] NPs have also proved that the emitting states of ECL
from NP surface states are different from those of photoluminescence (PL) from
NPs. As PL occurs mainly from the interior of the NPs, PL spectroscopy can be used
to probe the interior of the particle and provide information about the electronic tran-
sition (band gap) of material. Because charge injection into the NPs generally occurs
via surface states, electrochemistry and ECL studies are mainly used to probe the
particle surface. This also means that ECL that originated from surface states gener-
ally is not sensitive to the NP size and capping agent used, but depends more sensi-
tively on the surface chemistry and the presence of surface states [13].
246 8 Biosensing with Nanoparticles as Electrogenerated Chemiluminsecence Emitters

Fig.8.4 (a) ECL spectrum


of TGA-capped CdSe QDs/
H2O2 system at a constant
potential of 1.8V obtained
with an accumulation of five
fast scans (50nm/s) from
450700nm and (b) PL
spectrum of the film for the
ECL emission. Reprinted
with permission from Jiang
and Ju [31], 2007,
American Chemical Society

This surface-state model for ECL has been further verified via the ECL study of
CdSe/ZnSe core-/shell-type NPs [29]. Unlike the spectra from the ECL of Si or CdSe
NPs, where the emission occurs in a single peak that is significantly red-shifted from
the PL peak, the CdSe NPs are well passivated with a shell of ZnSe, and thus show a
large ECL peak at the wavelength of band-edge PL plus a red shift by ~200nm from
the PL peak (Fig.8.3). The red shift is deduced from the unpassivated CdSe surface
because the same red-shift extent can be found in the ECL spectra from CdSe [19] and
Si [12] NPs. The large ECL peak at the wavelength of band-edge PL suggests that the
surfaces of CdSe/ZnSe core-/shell-type NPs have been largely passivated; ECL emis-
sion cannot be generated from the surface states on the NPs. Thus, ECL emission
from CdSe NPs whose surfaces have been largely passivated is dependent on the bulk
of NPs, which is another ECL generation type, known as band-gap ECL.
As another type of NP ECL, band-gap ECL mainly corresponds to the bulk of NPs:
Its ECL spectrum matches the PL spectrum, and therefore is size-dependent and tun-
able [21, 29, 31, 39, 40]. To achieve band-gap ECL, a common method is to passivate
the surface state of NPs [29], because the charge injection in an NP is generally assumed
to occur via its surface states, which results in a surface-state model as the main genera-
tion model for NP ECL. Bard and coworkers found that surface states of CdTe NPs
could be easily passivated and then result in band-gap ECL [21]. Recently, Ju and
coworkers also observed the band-gap ECL from CdSe and CdTe QDs in aqueous
solution by capping the QDs with thioglycolic acid (TGA) [31] and mercaptopropionic
acid (MPA), respectively [39, 40]. As shown in Fig.8.4, the ECL maximum of TGA-
capped CdSe QDs is located at the peak position of the PL, indicating that the excited
state in the ECL emission was the same as that in the PL, in which the CdSe/TGA*
could be considered the emitter to produce 1Se1Sh transition emission [31].
The two generation types of NP ECL have many different applications. The
surface-state model ECL is sensitive and can be used to probe the surface states of
NPs, while the band-gap model ECL cannot. However, the ECL spectrum of the
surface-state model will be red-shifted and not size-dependent, which may limit its
8.2 Principle of ECL from Nanoparticles 247

applications for multiplexing. The band-gap model ECL has potential in corresponding
applications. Recently, Han demonstrated that the surface-state model ECL is as effi-
cient and sensitive as the band-gap model ECL for general biosensing schemes, such
as H2O2 sensing [41]. As to the great importance of biosensing in modern times, the
applied system for NP ECL will be discussed in the following section.

8.2.3Coreactant System for NP ECL

There are a wide variety of NPs that exhibit ECL, suggesting NP ECL systems can
be classified by the different features of emitters. For example, NP ECL systems can
be classified as the ECL of bare QDs [12, 19], spherical QDs [42], hollow spherical
QDs [43, 44], QD film [28, 31, 4547], shell/core structured QDs [29, 48, 49],
nanotubes [30, 50, 51], nanorods [52], and nanoflakes [53] by the NP structure. NP
ECL systems can also be classified as the ECL of elemental NPs (e.g., Si [12] and
Ge [32]), of compound NPs (e.g., CdSe [19]), and even of aromatic hydrocarbon
NPs [52] and of polymer NPs [54].
A more scientific way to define different NP ECL is based on the ECL mecha-
nism; that is, NP ECL systems are reasonably classified as annihilation NP ECL
systems and coreactant NP ECL systems. Due to the merits discussed above, the
overwhelming majority of publications concern coreactant ECL and its analytical
applications. This section mainly concerns the typical coreactant NP ECL systems
and their mechanisms.
Oxalate (C2O42) system. Oxalate is the first account of a coreactant ECL system
reported by Bards group in 1977 [55]. It is often referred to as an oxidative or
oxidative-reductive coreactant due to its ability to form a strong reducing agent
(CO2) upon electrochemical oxidation. As a classical coreactant ECL system, the
electrode oxidizes both the oxalate and the ECL reactant D; the reducant, CO2, is then
generated upon bond cleavage of oxalate. This strategy has been widely used in ana-
lytical and biotechnological fields, with the reactant D being Ru(bpy)32+ [7, 8]. By
adding excess C2O42 to the NP solution, Bard and his coworkers reported that this
typical coreactant can result in coreactant anodic ECL for Si NPs by the electron trans-
fer between CO2 and oxidized Si NPs [12]. We detailed the general mechanism for an
oxalate system in Scheme8.2. Recent work further proves that oxalate can also work
as an efficient ECL coreactant for 9,10-diphenylanthracene (DPA) nanorods [45]. The
classical coreactant, oxalate, works well as the coreactant for NP ECL emissions.
Tri-n-propylamine (TPrA) system. TPrA is another important popular oxidative
reductive coreactant for ECL systems, especially the Ru(bpy)32+. The majority of
ECL applications reported so far involve Ru(bpy)32+ or its derivatives as an emitter
(or label) and TPrA as a coreactant because the Ru(bpy)32+/TPrA system exhibits the
highest ECL efficiency. This system has become the basis of commercial systems for
ECL immunoassay and DNA analysis [8]. Electrochemical studies of various ali-
phatic amines indicate a possible reaction pathway for oxidizing TPrA to produce a
248 8 Biosensing with Nanoparticles as Electrogenerated Chemiluminsecence Emitters

Fig.8.5 Proposed TPrA


oxidation/reaction sequence
with abbreviations in
parentheses. Reprinted with
permission from Richter [7],
2004, American Chemical
Society

Fig.8.6 ECL emissions of


0.95mM QDs in the presence
of 0.052M TPrA in 0.1M
Tris buffer. The scan rate is
100mV/s. Reprinted with
permission from Mei etal.
[49], 2010, Wiley

strong reducing agent [56]. Upon oxidation, the short-lived TPrA radical cation
(TPrA+) is believed to lose a proton from an a-carbon to form the strongly reducing
intermediate TPrA (Fig. 8.5). ECL emission resulted from the electrooxidized
rubrene NPs (R+), and TPrA suggests that TPrA can also work as a coreactant for
NP ECL emissions [52].
Recently, Wang and coworkers demonstrated the anodic ECL emission of 3-mer-
captopropionic acid (MPA)-capped CdTe/CdS QDs with TPrA as the coreactant in
aqueous solution [49], as shown in Fig.8.6.
The whole procedure for the TPrA-involved NP ECL is deduced as follows:
The cation QDs+ and TPrA+ are anodically produced by the electrochemical oxi-
dation of CdTe/CdS QDs and TPrA, respectively; TPrA+ loses a proton from an
a-carbon to form TPrA, and then reduces QD+ to QDs*; finally, the ECL is gener-
ated from QDs*. The general mechanism for the TPrA system can be depicted as
Scheme8.3.
8.2 Principle of ECL from Nanoparticles 249

Scheme8.3

R+ refers to positively charged NPs electrogenerated at the electrode surface.


Besides the TPrA system, the anodic ECL of QDs toward different kinds of amines
has also been investigated [57], and an anodic ECL for thioglycerol-stabilized CdSe
NPs/tertiary amine is obtained [58].
Peroxydisulfate (Persulfate, S2O82) system. Peroxydisulfate is the first example of
the so-called reductive oxidation coreactant reported previously [59], because the
reduction of peroxydisulfate produces the strong oxidant SO4, which can undergo
an electron-transfer reaction with an ECL luminophore such as Ru(bpy)32+ to generate
light. Thus, peroxydisulfate is used to generate ECL in a different way from those
of oxalate and TPrA. Bard and coworkers first demonstrated that peroxydisulfate
can be used as the coreactant for the NP ECL emission [12]. By adding peroxydis-
ulfate to the Si NP solutions, ECL emission was observed in the potential region for
NC reduction, because the SO4, generated by electrochemically reducing peroxy-
disulfate, can react with the negatively charged NPs by injecting a hole into the
HOMO and producing an excited state of the NPs. The general mechanism for per-
oxydisulfate system can be depicted as Scheme8.4.

Scheme8.4

R refers to negatively charged NPs electrogenerated at the electrode surface.


Recently, many mechanisms and biosensing studies concerning the ECL of NPs with
peroxydisulfate as the coreactant have been carried out [22, 26, 46, 50, 6062].
Hydrogen peroxide (H2O2) system. Hydrogen peroxide is an important coreactant
for the ECL of luminol [1, 7, 8]. Many bioanalytical applications of ECL have been
developed based on this typical coreactant. Zou and Ju first demonstrated that the
electron-transfer reaction between electrochemically reduced nanocrystal species
and oxidant coreactants such as H2O2 and reduced dissolved oxygen can result in
ECL emission from NPs [45] (Fig.8.7).
Because the dissolved oxygen can be electrochemically reduced to H2O2 and
then work as coreactant for ECL emission, the general mechanism for the H2O2
(or dissolved oxygen) system can be drawn as Scheme8.5 according to Ref. [45].

Scheme8.5
250 8 Biosensing with Nanoparticles as Electrogenerated Chemiluminsecence Emitters

Fig.8.7 Cyclic voltammograms and ECL curves of (a) bare and (b) CdSe nanocrystal thin-film-
modified PIGE in air-saturated 0.1M pH 9.3 PBS containing 0.1 M KNO3, (c) solution (b) bubbled
with N2 for 25min, and (d) solution (b) +100mM H2O2. Scan rate: 20mV/s. Reprinted with per-
mission from Zou and Ju [45], 2004, American Chemical Society

R refers to the negatively charged NPs electrogenerated at the electrode surface.


Due to the bioanalytical feature of H2O2, hydrogen peroxide- or dissolved oxygen-
involved coreactant ECL from NPs has been extensively carried out with different NPs
and techniques [41, 45, 6372]. For example, by using a controlled solution-precipita-
tion method, hierarchical CdS nanotube arrays assembled in an anodic aluminum oxide
template can display considerably enhanced solid-state ECL in H2O2 solution com-
pared with those of random nanoparticle aggregates [66]. Many NP ECL biosensors
for different analytes have been developed based on the H2O2 system [1417].
Sulfite (SO32) system. Recently, Liu and Ju demonstrated a new coreactant, SO32,
to enhance the anodic ECL of MPA-capped CdTe QDs [39]. As the sulfite could
accelerate the formation of superoxide anion [39], an 18-fold greater ECL emission
was obtained with great sensing application potentials (Fig. 8.8). The proposed
mechanism for the sulfite system may be depicted as Scheme8.6.

Scheme8.6
8.3 Biosensing Strategy and Corresponding Application 251

Fig.8.8 ECL curves of


0.2mM MPA-modified CdTe
QDs (black), 1.0mM Na2SO3
(blue), 0.2mM QDs+0.4mM
Na2SO3 (red), and 0.2mM
QDs+1.0mM Na2SO3
(green) in air-saturated pH
7.5 PBS at 100mV/s.
Reprinted with permission
from Liu and Ju [39],
2008, American Chemical
Society

Dichloromethane (CH2Cl2) system. A unique coreactant was proposed during the


ECL study of CdTe in CH2Cl2 containing 0.1M (TBA)PF6 [21]. In this case, a large
ECL signal was detected at the first negative potential, which could not be explained
by the annihilation of redox species of NPs, because there were only reduced
particles; there was no oxidized particle to act as an electron acceptor. A much
weaker ECL signal was obtained when the reaction medium was changed to a
mixture of benzene-MeCN at the first negative potential, which was attributed to
some impurities in the cell. Ushida and coworkers [22] revealed that CH2Cl radicals
produced under the irradiation of CH2Cl2 played a role as an electron acceptor to
oxidize aromatic hydrocarbons. Based on this reference, CH2Cl was proposed as an
oxidant. The CH2Cl2, which was reduced from CH2Cl2, decomposes into CH2Cl
and Cl, and then the oxidant, CH2Cl, accepted an electron from the reduced NPs to
form the emitting state (Scheme8.7).

Scheme8.7

Although ECL analytical techniques coupled with QDs have been rapidly developed
and extensively studied in both organic and aqueous media, it is clear that a normal
coreactant for common molecules as ECL emitters still works for the study of NP
ECL. Since the ECL of NPs has only recently been explored, it is hard to overstate
the importance of coreactants to the growth and development of NP ECL.

8.3Biosensing Strategy and Corresponding Application

The ECL emission of NPs involving electron transfer between electrochemically


generated nanocrystal species and coreactants suggests that QDs can act as the
emitters for the preparation of ECL sensors. Thus, the biosensing application of NP
252 8 Biosensing with Nanoparticles as Electrogenerated Chemiluminsecence Emitters

ECL is mainly realized with the coreactant ECL routine. According to the different
ECL mechanisms of NPs [8, 1517], their biosensing is mainly realized by the fol-
lowing strategy.

8.3.1Direct Determination of Biochemical Coreactant

The direct determination strategy mainly concerns hydrogen peroxide determina-


tion, as H2O2 is an important biological molecule as NP ECL coreactant [41, 45, 50,
64, 68, 71].
In the earlier NP ECL sensing studies, NP films on the surface of electrodes
received much attention because NPs could be applied in optoelectronic systems or
as components in future nanoelectronic devices. Additionally, because the solution-
phase NPs often suffer from low solubility, low concentration, and small diffusion
coefficient [8], semiconductor NP thin films may also offer better electrochemical
and ECL signals. The first example of an ECL sensor based on QDs was fabricated
by depositing a CdSe QD film on a graphite electrode [45]. By scanning to negative
potentials, the CdSe nanocrystal thin film exhibited two ECL peaks, at 1.20 (ECL-1)
and 1.50V (ECL-2), in pH 9.3, 0.1M PBS during the cyclic sweep between 0 and
1.8V at 20mV/s (Fig.8.7). ECL-1 showed a higher sensitivity to the concentra-
tion of oxidant coreactants than ECL-2 and was used for ECL detection of the H2O2
coreactant. A linear response of ECL-1 to H2O2 was observed in the concentration
range of 2.51076105M, with a detection limit of 1.0107M. The fabrica-
tion of 10 CdSe nanocrystal thin-film-modified graphite electrodes displayed an
acceptable reproducibility, with an RSD of 1.18% obtained at an H2O2 level of
10mM [45].
Entrapping NPs also presents a way for the study and application of NP ECL
[43]. The ECL emission of CdS nanotubes entrapped in carbon paste electrode has
been used for ECL detection of H2O2 in the presence of S2O82 as coreactant [50].
Interestingly, two ECL peaks are found, corresponding to two different ECL states
excited by different mechanisms. The first peak is attributed to the annihilation
process between reduced and oxidized species of CdS, while the second one is
attributed to the electron-transfer reaction between a reduced species of CdS and the
coreactants (S2O82 or H2O2). The architecture of the NPs may also have an impor-
tant effect on their ECL-sensing applications. Dai etal. reported a sensing applica-
tion of ECL from the CdS hollow spheres to H2O2 [44], which showed much better
responses than those from CdS solid nanoparticles.
Chen etal. developed a sensor for H2O2 based on CdS quantum dots and hemo-
globin multilayer film [68]. Due to the high stability of ECL of {Hb/CdS}n and the
excellent electrocatalytical ability of Hb to H2O2, this multilayer film sensor would
have great merit in the application of biosensors to life science and environmental
science.
8.3 Biosensing Strategy and Corresponding Application 253

With the development of QD synthesis techniques, thiol-capped QDs with high


solubility in aqueous solution have been presented as an alternative for the ECL-
sensing strategy. An MPA-capped CdTe NP is synthesized for the ECL determina-
tion of H2O2 in aqueous solutions, with a detection limit of 0.06mM [41]. Based on
the ECL of blue-emitting ZnSe QDs, a sensor for H2O2 was developed and used to
detect H2O2 in the pig kidney (pk-15) cell, veto cell, and mineral water, respectively.
The good reproducibility showed its potential application in real samples [71].
For more possible applications in biological detection, less toxic ZnS nanocrys-
tals doped with Mn2+ were modified on a glassy carbon electrode and displayed a
specific ECL emission at ca. 1.50V versus SCE for the direct determination of
H2O2 [64].

8.3.2Improved ECL Performance for Higher Sensitivity

In ECL-sensing procedures, an enhanced ECL intensity is preferred for its high


sensitivity and detection performance, especially when the ECL intensity is not
efficient for sensing purposes. Efficient ECL emission from QDs can be achieved
via these common methods:
1. Choice of efficient coreactant. For example, the anodic ECL emission of MPA-
capped CdTe QDs is very weak and cannot be used for sensing purposes in air-
saturated pH 7.5 PBS. However, with a new coreactant, such as sulfite, the greatly
enhanced ECL emission can lead to a sensitive sensing for analytes [39], such as
tyrosine.
2. Modification of ECL system with functional materials [65, 67, 7378]. For
example, QDs composited with carbon nanotubes can dramatically enhance their
ECL intensity [67]. Such a property promotes the application of QD ECL in
fabricating sensors [65]. For example, CNT film can greatly enhance the ECL of
CdTe QDs dispersed in aqueous solution; consequently, a method for the deter-
mination of methimazole is developed with CNT film-modified electrode [73].
Jie at el. found the ECL of CdSe QDs could be greatly enhanced by the combina-
tion of CNTs and poly (diallyldimethylammonium chloride) (PDDA) in the
CdSe QD film, and could successfully be used to develop a sensitive ECL immu-
nosensor for the detection of human IgG(Ag) [75]. By facilitating the CdTe QD
oxidation and triggering O2 generation, graphene oxide can also result in an
enhanced ECL for sensing glutathione [74].
3. Optimum architecture or shape of ECL emitter. For example, hierarchical CdS
nanotube arrays with high porosity and uniform alignment display considerably
enhanced solid-state ECL in H2O2 solution compared with those of random
nanoparticle aggregates, and a linear relationship between the ECL intensity and
H2O2 concentration is obtained from 1.01071.5104 M, with a detection
limit of 6.0108M, which demonstrates that CdS nanotube arrays can be used
to develop sensitive ECL sensors [66].
254 8 Biosensing with Nanoparticles as Electrogenerated Chemiluminsecence Emitters

4. Nanocomposite. Wang et al. successfully prepared CdSAg nanocomposite


arrays by the electrochemical approach using CdS hierarchical nanoarrays as
templates. The enhanced ECL of the CdSAg nanocomposite arrays presented a
sensitive hydrogen peroxide sensor with a low detection limit of 1.5108 M
[77]. Ju and coworkers prepared a nanocomposite of CdSe quantum dots with
nitrogen-doped carbon nanotubes [78]. With hydrogen peroxide as the coreac-
tant, the cathodic ECL emission from the nanocomposite-modified electrode was
five times stronger than that from pure CdSe QDs, and three times stronger than
that from CdSe QDs composited with carbon nanotubes.

8.3.3Analyte-Inhibited (or -Enhanced) ECL Emission

This strategy is mainly based on either the enhancing or inhibiting effect of the
analyte on the NP ECL. According to this strategy, a series of ECL sensors have
been developed with the interaction between analytes and the species in the ECL
process [31, 39, 7984].
For exampleit is believed that the intermediate OH radical in the TGA-capped
CdSe QD film/peroxide ECL system is the key species for producing hole-injected
QDs. ECL sensors for biologically important scavengers of hydroxyl radicals, such
as g-l-glutamyl-l-cysteine-glycine (GSH), are thus developed based on inhibition
effect [31]. As l-cysteine is involved in many biological processes, such as
Parkinsons and Alzheimers diseases, the high sensitivity obtained suggests their
sensing potential for both scavengers and generators of hydroxyl radicals in clinical
samples.
A stable anodic ECL emission can be detected from MPA-capped CdTe QD/
sulfite system. Using tyrosine as a model compound, whose electrooxidized product
can quench the excited QDs and thus the ECL emission, an MPA-capped CdTe
QD-based ECL sensor for tyrosine with a wide concentration range has been devel-
oped [39]. Based on the ECL of CdTe QDs using carbon nanotube-modified glassy
carbon electrode, a highly sensitive method for the determination of methimazole
has also been developed. The ECL intensity decreases linearly in the concentration
range of 1.01094.0107M for methimazole, with a relative coefficient of 0.995,
which shows a finer sensitivity than that at a bare electrode [73].
As room-temperature ionic liquid (RTIL) film on the glassy carbon electrode can
greatly enhance the ECL intensity of CdTe QDs, the highly sensitive sensing of gos-
sypol content using CdTe QD ECL with RTIL-modified glassy carbon electrode has
been achieved. The sensor shows a good linear relationship in the gossypol concen-
tration range of 5.01075.0109M, with a detection limit of 5.0109M. The
sensor has been used to detect gossypol in cottonseed oil with satisfactory results.
The RTIL-modified electrode may extend the analytical applications of QD ECL
systems [82].
An anodic ECL-sensing strategy based on a TGA-capped CdSe QD/sulfite
system in a neutral medium has been developed in a similar way for detecting ECL
8.3 Biosensing Strategy and Corresponding Application 255

quenchers, using dopamine as a model molecule [79]. Based on an electrochemical


oxidation inhibition process, a method for the ECL detection of nitrite with a CdSe
QD/sulfite system has been proposed with high sensitivity and good selectivity [80].

8.3.4Determination Based on Resonance Energy Transfer

As the ECL emitter for NP ECL is the excited NPs, the possible energy transfer
from excited NPs can also provide a strategy for biosensing [40, 47]. This detection
strategy mainly works in two ways:
1. ECL energy is directly transferred from the excited CdTe QDs to analyte, the
quencher. For example, intensive anodic ECL emission from MPA-capped CdTe
QDs can be obtained with a peak value at +1.17V (vs. Ag/AgCl) in pH 9.3 PBS
at an indium tin oxide (ITO) electrode, and the ECL energy transfer from the
excited CdTe QDs to catechol derivatives, such as dopamine or l-adrenalin, can
produce decreased ECL emission for sensing [40]. In the case of dopamine and
l-adrenalin, this ECL method shows wide linear ranges, from 50nM5mM and
80nM30mM, respectively. Both ascorbic acid and uric acid, which are common
interferences, do not interfere with the detection of catechol derivatives in practical
biological samples.
2. ECL energy is transferred to a quencher (not to analyte). The amount of analytes
plays an important role for the energy transfer. For example, ECL energy transfer
from CdS:Mn NP film to proximal Au NPs quenched the ECL emission. Ahybrid-
ization with target DNA (t-DNA) results in an enlarged distance between Au NPs
and CdS:Mn NPs, and hence enhanced the ECL emission of the CdS:Mn NP
film, which can be used for sensing the t-DNA [47] (Fig.8.9).

8.3.5Determination with Enzyme-Catalyzed Reaction

Detection with an enzyme-catalyzed reaction is based on the ECL reactions of NPs


with H2O2 produced from the enzymatic reactions of analytes in the presence of
their corresponding oxidases.
Biosensors based on the ECL of QDs coupled with an enzymatic reaction were
first developed for glucose detection, with glucose oxidase as a model [85]. The
TGA-capped CdSe QDs were coimmobilized with glucose oxidase. Glucose could
be detected by measuring the decrease in the ECL response of glucose oxidase
CdSe QDs after the addition of glucose. This strategy could be applied in more
bioanalytical systems for oxidase substrates.
Because the electrooxidized product of tyrosine could quench the excited QDs,
tyrosine could be detected by ECL emissions of QDs. In order to further increase
the detection sensitivity, tyrosinase was used to catalyze the oxidation of tyrosine by
256 8 Biosensing with Nanoparticles as Electrogenerated Chemiluminsecence Emitters

Fig.8.9 ECL sensing strategy with energy transfer between CdS:Mn NPs and Au NPs. Reprinted
with permission from Shan etal. [47], 2009, Royal Soc Chemistry

Fig.8.10 Procedure of the highly sensitive detection of tyrosine based on the enhanced anodic
ECL and catalytic oxidation of tyrosinase in air-saturated PBS. Reprinted with permission from
Liu and Ju [39], 2008, American Chemical Society

dissolved O2 to produce o-quinone, the quencher (Fig. 8.10). By combining the


enzymatic cycle of trace tyrosinase to produce the oxidized product, an extremely
sensitive method for the ECL detection of tyrosine with a subpicomolar limit of
detection was achieved [39]. This strategy could be easily realized and opened new
avenues for the applications of QDs in ECL biosensing.
Based on the functional feature of multiwalled carbon nanotubes (MWCNTs), an
ECL enzyme biosensor based on the ECL from CdS NPs formed in situ on MWCNTs
was proposed for choline and acetylcholine. The MWCNTCdS could react with
H2O2 to generate strong and stable ECL emission in neutral solution. Compared
with pure CdS NPs, the MWCNTCdS enhanced the ECL intensity by 5.3-fold and
moved the onset ECL potential more positively for about 400mV, which reduced
H2O2 decomposition at the electrode surface and increased the detection sensitivity
8.3 Biosensing Strategy and Corresponding Application 257

of H2O2. Benefiting from these properties, the enzyme-based biosensors were


fabricated by cross-linking choline oxidase and/or acetylcholinesterase with glutar-
aldehyde on MWCNTCdS-modified electrodes. The resulting ECL biosensors
showed wide linear ranges, from 1.7332 and 3.3216mM, with a low detection
limit of 0.8 and 1.7mM for choline and acetylcholine, respectively [65].

8.3.6Immunoreactions or DNA Hybridization-Coupled Sensing

The selective feature of immunoreactions or DNA hybridization has been extensively


exploited for the sensitive determination of analytes in common ECL studies [1, 7, 8].
NP ECL biosensors coupled with immunoreactions or DNA hybridization techniques
provide a strategy for the selective detection of analytes [60, 61, 75, 76, 8689].

8.3.6.1DNA Assay

A common way for ECL DNA sensing is using ECL emitter as label. This technique
can work for the QD-based ECL assays.
For example, Hu etal. [87] developed a DNA assay by using TGA-capped CdTe
QDs as DNA labels. t-DNA was hybridized with capture-DNA (c-DNA) bound on
the nanoporous gold leaf (NPGL) electrode, which was fabricated by conjugating
amino-modified c-DNA to TGA-modified NPGL electrode. Following that, amino-
modified probe DNA was hybridized with t-DNA, yielding sandwich hybrids on the
NPGL electrode. Then, MPA-capped CdTe QDs were labeled to the amino group
end of the sandwich hybrids. ECL emission of the QD-labeled DNA hybrids on the
NPGL electrode was measured for DNA sensing (see Fig. 2.17).
Huang developed a novel biosensor for thrombin by the QD ECL technique. The
thiol-terminated aptamer with 15 nucleotides (probe I) was first immobilized on an
Au electrode, and then thrombin was imported to form the aptamerthrombin bioaf-
finity complexes. Another 5-biotin-modified aptamer (29 nucleotides, probe II) was
next hybridized with the combined thrombin to form a sandwich-type structure.
Streptavidin-modified QDs (avidin-QDs) were bound to probe II via the biotinavidin
system. The QD ECL signal was responsive to the amount of probe II, which was
indirectly proportional to the amount of combined thrombin. In addition, the biosensor
exhibited excellent selectivity responses and good stability toward the target analyte
[88]. A facile strategy for the fabrication of aptamer-based adenosine 5-triphosphate
(ATP) biosensor was developed by a QD ECL technique in a similar way [88].
A QD ECL biosensor for the detection of lysozyme has been developed by forming
the aptamerlysozyme bioaffinity complexes at the Au electrode. The free probes
are hybridized with the 5-biotin-modified cDNA oligonucleotides to form double-
stranded DNA (ds-DNA) oligonucleotides. Avidin QDs are bound to these hybridized
cDNA through the biotinavidin system. The ECL signal of the biosensor is
responsive to the amount of QDs bonded to the cDNA oligonucleotides, which
isindirectly inversely proportional to the combined target protein [89].
258 8 Biosensing with Nanoparticles as Electrogenerated Chemiluminsecence Emitters

8.3.6.2Immunoassay Based on NP ECL

A proposed method for fabricating QD ECL immunosensor is to quantify the inhibition


of ECL emission caused by the immunocomplex [60, 61, 75, 76]. Zhu etal. reported a
label-free NP ECL immunoassay strategy for the sensitive detection of human IgG
(HIgG) based on a combination of CdSe NP/MWNTchitosan/3-aminopropryl-
triethoxysilane (APS) [76]. As shown in Fig.8.11, colloidal solution containing CdSe
NP/CNTCHIT composite was first covered on the Au electrode surface to form a
robust biocompatible film. After APS as a cross-linker was covalently conjugated to
the CdSe NP/CNTCHIT film, the ECL intensity was about 20-fold higher than that of
the CdSe NP/CNTCHIT film. After antibody was bound to the functionalized film via
glutaraldehyde, the specific immunoreactions between HIgG and antibody resulted in
a decreased ECL intensity for the detection of HIgG.
A recent investigation for fabricating label-free immunosensor with NP-ampli
fication techniques has been reported by the use of CdSe NP/Au nanoparticles/anti-
PAB for the detection of human prealbumin (PAB, antigen). In this case, a detec-
tion limit of 1.01011 g/mL was obtained [61]. With self-assembly and gold
NP-amplification techniques, a novel label-free ECL biosensor for low-density
lipoprotein (LDL) was developed. The biosensor was prepared as follows: The
gold nanoparticles were first assembled onto a cysteamine monolayer on the gold
electrode surface. This gold nanoparticle-covered electrode was next treated with
cysteine and then reacted with CdS NPs to afford a CdS NC-electrode. Finally,
apoB-100 (ligand of LDL receptor) was covalently conjugated to the CdS
NC-electrode. The resulting modified electrode was tested as an ECL biosensor for
LDL detection. The ECL peak intensity of the biosensor decreased linearly with
the LDL concentration in the range of 0.02516ng/mL, with a detection limit of
0.006ng/mL [60].

Fig.8.11 Scheme for the label-free NP ECL immunoassay strategy. Reprinted with permission
from Gie etal. [76], 2008, American Chemical Society
8.3 Biosensing Strategy and Corresponding Application 259

The ECL of CdSe QDs could be greatly enhanced by the combination of CNTs
and PDDA in the CdSe QD film. A sensitive ECL immunosensor for the detection
of human IgG (Ag) was proposed with CdSe QDCNT composites [75]. After
PDDA as a binding linker was conjugated to the CdSe QDCNT composite film on
the electrode, the ECL signal was significantly enhanced. Subsequently, gold nano-
particles AuNPs assembled onto the CdSe QDCNT/PDDA-modified electrode
could further amplify the ECL signal. After antibody (Ab) was immobilized onto
the electrode through AuNPs, the ECL immunosensor was successfully fabricated.
The principle of ECL detection for target Ag was based on the increment of steric
hindrance after immunoreaction, which resulted in a decrease in ECL intensity. The
Ag concentration was determined in the linear range of 0.002500 ng/L, with a
detection limit of 0.6pg/mL. This work opened up new avenues for applying QD
ECL in highly sensitive bioassays [75].
More recently, based on the ECL emission of bidentate chelate CdTe QDs at
relatively low cathodic potential [90], a quantum dot-based electrochemiluminescent
immunosensor was developed by coupling enzymatic amplification with self-
produced coreactant from oxygen reduction [91]. The bidentate chelate CdTe QDs
exhibit surface traps to produce a narrower band gap than the core. Using the elec-
trochemically produced H2O2 as coreactant, a strong ECL emission is observed,
which can be quenched by introducing hydroquinone and horseradish peroxidase in
the solution. The quenching effect has been applied for the ECL detection of hydro-
quinone [90]. After the QDs and human IgG (HIgG) are coimmobilized on an elec-
trode surface, upon the immunorecognition of the immobilized HIgG to its antibody
labeled with horseradish peroxidase, the enzyme is introduced to the electrode surface,
as shown in Fig.8.12. In the presence of hydroquinone, the enzymatic cycle thus
consumes the self-produced coreactant H2O2, leading to a wide calibration range of

Fig.8.12 (a) Construction and (b) incubation of the immunosensor, and ECL detection (c) with-
out and (d, e) with the enzymatic amplification by the consumption of H2O2 as the coreactant.
Reprinted with permission from Liu etal. [91], 2010, American Chemical Society
260 8 Biosensing with Nanoparticles as Electrogenerated Chemiluminsecence Emitters

0.05ngmL1~5mgmL1 for competitive immunoassay of HIgG. This immunosensor


shows good stability and fabrication reproducibility. The immunoassays of practical
samples show acceptable results. This simple immunosensing strategy opens new
paths for the detection of proteins and application of QDs in ECL biosensing.

8.4Conclusions

The fundamental and biosensing applications with NPs as ECL emitters have been
reviewed in this chapter. As a new kind of ECL emitter, NPs have provided an
entirely different sensing paradigm. There are still many disadvantages to over-
come; for instance, a high negative potential is required before ECL is produced,
and the intensity is not at the level of Ru(bpy)32+ or luminol ECL. With further
understanding of NP ECL mechanisms, new highly efficient and tunable NP ECL
systems (emitters and coreactants) and combined analytical techniques should be
developed to further improve the analytical performance of NP-based ECL
biosensors.

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Chapter 9
Biosensing Applications of Molecularly
Imprinted Nanomaterials

9.1Introduction

Chemical sensors and biosensors have attracted considerable attention within the
field of modern analytical chemistry, as seen both from the number of publica-
tions and from the diversity of approaches and techniques. This is essentially due
to new demands and opportunities that are appearing particularly in clinical settings,
(e.g., routine blood testing), environmental monitoring, warfare protection, home-
land security, food analysis, illicit drug detection, and genotoxicity. Sensor devel-
opment has been driven, in large part, by the need for new devices that are less
expensive and simpler to construct and operate, that provide adequate detection
limits and selectivity, and that are accurate and reliable.
The central part of a biosensor is the recognition element (e.g., an antibody,
aptamer, cell, DNA oligonucleotide, enzyme, lectin, protein), which is in close contact
with an interrogative transducer and serves to recognize specifically the target analyte
(Fig.9.1a). The binding or conversion (if the analyte is a substrate) event is used to
produce an optical, mass, thermal, or electrochemical signal that is related to the
analyte concentration in the sample [1, 2]. The biosensor concept has been developed
in parallel to the development of elegant biosensor arrays [36] for simultaneous
multianalyte detection. However, despite the progress that has been made on bio-
sensors and microarrays based on biological recognition elements, there are well-
documented limitations associated with many types of biorecognition elements
[79]. Aptamers can address several of these issues [10], but they are not yet available
for a wide range of analytes and can be expensive to produce for most analyses.
Although biologically based recognition elements are clearly important and attractive
for the design of biosensors for continuous process or environmental monitoring,
the poor chemical and physical stability of biomolecules sometimes generally pre-
vents their use in harsh environments, which ultimately leads to a compelling case
for developing inexpensive, robust, and reusable alternatives for these expensive/
labile biorecognition elements. Molecular imprinted polymers (MIPs), as purely

H. Ju et al., NanoBiosensing: Principles, Development and Application, 265


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0_9, Springer Science+Business Media, LLC 2011
266 9 Biosensing Applications of Molecularly Imprinted Nanomaterials

Fig.9.1 (a) Schematic representations of antibody-based chemosensor and (b) MIP-based biomi-
metic sensor. Inset in (b) shows the concept of molecular imprinting. Reprinted with permission
from Guan etal. [2]. 2008, Molecular Diversity Preservation Int

synthetic materials, can, in principle, be designed from scratch to act not only as
simple affinity adsorbents, but also as smart reporters responsive both to targeted
molecules [11] and to different environmental stimuli (Fig.9.1b) [1215]. Table9.1
shows a basic comparison of MIPs with antibodies and aptamers [16].
Molecular imprinting has been widely recognized as the most promising meth-
odology for the preparation of a tailor-made artificial receptor that can selectively
bind predetermined target molecules [9]. Molecular imprinting is a technique
involving the formation of binding sites in a synthetic polymer matrix that are of a
complementary functional and structural character to its substrate molecule. The
principle of molecular imprinting is illustrated in Fig.9.2, which refers to arranging
polymerizable functional monomers around a template, followed by polymerization
and template removal. The arrangement is typically achieved by noncovalent inter-
actions (e.g., H-bonds, ion pairing) or reversible covalent interactions. The binding
sites that are generated during the imprinting process often have affinities and selec-
tivities approaching those of antibodyantigen systems, and the properly designed
MIPs have therefore been dubbed antibody mimics [17], which can then bind the
template or structurally similar analytes. When the combination of MIP materials
with a transducer is in a suitable format, the sensors with MIP recognition can iden-
tify and quantify a target species by converting the analyteMIP binding event into
a physically readable signal, as shown in Fig.9.1b. Except for possessing antibody-
like molecular selectivity, the major advantages of using MIPs over their biological
counterparts include physical robustness, high strength, resistance to elevated
temperatures and pressures, and inertness to acids, bases, metal ions, and organic
solvents, as well as low production cost and easy preparation [18].
Table9.1 Comparison of MIPs with antibodies and aptamers
9.1 Introduction

MIPs Antibodies Aptamers


Structural Synthetic polymers in which binding sites Polypeptides folded into defined Single-stranded DNA and RNA
characteristics are stabilized by cross-linked 3D 3D structures sequences folded into defined 3D
network structures structures
Preparation Synthetic chemistry based on rational Use of an animals immune response In vitro screening and amplification of
design; MIPs can be produced on by means of a complicated combinatorial DNA or RNA
a large scale; production costs are combinatorial process; sequences; rational design can be
low and are often determined by the monoclonal antibodies can be used to introduce new functions;
template molecules produced on a large scale large-scale production is limited
using the hybridoma technique because of the high costs
Stability High stability; can be used in both Low stability; used only under Low stability; used only under aqueous
aqueous and organic solvents aqueous conditions conditions
Binding site Noncovalently imprinted polymers often Monoclonal antibodies have Homogeneous binding sites
characteristics have heterogeneous binding sites homogeneous binding sites
showing broad affinity distributions
Structural Difficult for cross-linked amorphous Possible Possible
characterization materials
Main target Low-molecular-weight (<1,000) Biomacromolecules and small Small and large molecules
molecules compounds and metal ions immunogenic molecules that can
be conjugated to protein carriers
Reprinted with permission from Ye etal. [16]. 2008, American Chemical Society
267
268 9 Biosensing Applications of Molecularly Imprinted Nanomaterials

Fig. 9.2 Schematic representation of the two most common MIP fabrication strategies.
(a)Noncovalent imprinting; (b) covalent imprinting. Reprinted with permission from Holthoff and
Bright [44]. 2007, American Chemical Society

These mimics display some clear advantages over real antibodies for sensor
technology [19]: (1) binding affinities comparable to a biological recognition ele-
ment; (2) robustness and stability under a wide range of chemical and physical
conditions; and (3) easy design of recognition sites for analytes that lack suitable
biorecognition elements [9]. Even though the great potential of this technology has
only been recognized recently, in particular after the introduction of synthetic
organic polymers as imprinting matrices, there is now a strong development toward
the use of MIPs as recognition elements in biosensing techniques [20, 21].

9.2Molecular Imprinting Technology

As illustrated in Fig.9.2, the molecular imprinting of synthetic polymers is a process


in which functional and cross-linking monomers are copolymerized in the presence
of the target analyte (the imprint molecule), which acts as a molecular template. The
functional monomers initially form a complex with the imprint molecule. After
polymerization, their functional groups are held in position by the highly cross-linked
polymeric structure. The subsequent removal of the imprint molecule reveals bind-
ing sites that are complementary in size and shape to the analyte. In that way, a
molecular memory is introduced into the polymer, which is now capable of rebinding
the analyte with a very high specificity. The interaction between the functional groups
of the template molecule and the complementary functionality of polymer-forming
components is of major importance in this technique [22].
9.2 Molecular Imprinting Technology 269

Fig.9.3 (a) Examples of organic polymerizable functional monomers that can be used alone or in
combination in noncovalent molecular imprinting. (b) Examples of cross-linking monomers that
can be used for the synthesis of molecularly imprinted polymers. (c) Examples of organic polym-
erizable functional monomers that can be used in covalent molecular imprinting. Reprinted with
permission from Holthoff and Bright [19]. 2007, Elsevier

Two distinct approaches have been followed for obtaining MIPs, as depicted in
Fig.9.2a, b. The selection depends upon interactions between the template and the
functional monomers involved in the imprinting and rebinding steps. These
approaches include (1) self-assembly or noncovalent approaches a prepolymer-
ization complex between the imprint molecule and functional monomers can be
formed via noncovalent interactions; and (2) covalent approach monomers can be
covalently coupled to the imprint molecule, that is, a polymerizable derivative of
the imprint molecule is synthesized.

9.2.1Noncovalent Approach

The noncovalent approach (Fig.9.2a) was first reported by Mosbach [17]; various
polymerizable functional monomers commonly used for noncovalent molecular
imprinting are illustrated in Fig.9.3a [9]. A cross-linking monomer is generally
used to form a three-dimensional rigid structure around the template molecule and
produce stable binding cavities. Figure9.3b illustrates several of the more common
cross-linker monomers that have been used [9]. Noncovalent forces, such as
hydrogen bonds, van der Waals forces, ionic interactions, and hydrophobic effects,
270 9 Biosensing Applications of Molecularly Imprinted Nanomaterials

are utilized. This process is more similar to natural processes, in which most
biomolecular interactions are noncovalent [17, 23]. Following the polymerization
and template removal, the functional groups within the templated polymeric
matrix can subsequently recognize and bind the target analyte using the same
noncovalent interactions. Arrangement using noncovalent interactions requires
the template and target analyte to form a sufficient number of noncovalent inter-
molecular interactions to produce the binding pocket during polymerization.
Because the relative interactions involved are weak, an excess of functional
monomers is often added to stabilize the templatefunctional monomer complex
during polymerization, which can result in heterogeneous binding sites sometimes
requiring subsequent purification. However, the commercial availability of a large
number of functional monomers and the ease of preparation have attracted the
widespread use of this approach. The combinatorial synthesis [24, 25] and silica
screening methodology [26] developed recently have also accelerated the process
of obtaining optimal noncovalently imprinted polymers. The rational design of
more potent functional monomers, such as via metal coordination interactions to
bind specific amino acid sequences, leads to the binding sites becoming more
homogeneous [27]. New potent functional monomers based on polymerizable
amidines and urea have been developed, which allow stoichiometric amounts of
functional groups to be incorporated into imprinted polymers to reduce nonspe-
cific adsorption [28, 29].

9.2.2Covalent Approach

In the covalent approach, pioneered by Wulff etal. [30], reversible chemical bonds
are maintained between the template and the functional monomers during imprinting
polymerization, which can be used to overcome the limitations of noncovalent
imprinting (Fig.9.2b). The same driving force is used for the MIPs to subsequently
bind the template. This approach creates strong interactions due to the restoration of
the covalent bond between the matrix and the target; however, it is limited by the
rather small number of useful reversible covalent interactions that can be used [31].
Binding site monomers having a boronic acid, diol, aldehyde, or amine functional
group have been successfully used for covalent molecular imprinting (Fig.9.3c) [9].
Cross-linking monomers (Fig.9.3b) are also utilized in this approach to help stabi-
lize and define the molecularly templated sites within the final material. This
approach can lead to homogeneous binding sites, since the template-functional
monomer complex can be kept intact during the polymerization reaction. However,
removal of the chemically bonded template from the highly cross-linked polymer
matrix is difficult, and the rebinding process is normally very slow because of the
necessary formation of covalent bonds between the target compound and the MIP.
Furthermore, prior modification of the template is needed, which can require strin-
gent synthetic conditions.
9.3 Types of MIP Materials 271

Fig.9.4 The sacrificial spacer approach to molecular imprinting. A cholesterol-responsive MIP is


fabricated by using cholesteryl (4-vinyl)phenylcarbonate ester as the template. Reprinted with
permission from Holthoff and Bright [44]. 2007, American Chemical Society

9.2.3Other Approaches

In addition to the noncovalent and covalent approaches mentioned above, attempts


have been made to combine the advantages of the two approaches so as to overcome
the drawbacks associated with both approaches. In one of the approaches, imprinting
is carried out by polymerization of a functional monomer covalently coupled to a
template, together with selective rebinding by carefully designed noncovalent inter-
actions. For example, a tripeptide (Lys-Trp-Asp) has been imprinted using both
covalent and noncovalent interactions and recognized only via noncovalent interac-
tions [32]. Whitcombe et al. [33] also described an alternative approach that
exploited the positive aspects of covalent and noncovalent imprinting. In the sacri-
ficial spacer approach, the authors used cholesteryl (4-vinyl)phenylcarbonate ester
as a functional monomer, which operated as the covalently bound template mono-
mer but was easily cleaved hydrolytically with the loss of CO2 (Fig.9.4). This com-
bination resulted in the formation of a noncovalent recognition site within the final
material bearing a phenolic residue capable of interacting with the template (i.e.,
cholesterol) through hydrogen bonding.

9.3Types of MIP Materials

An important step in preparing molecularly imprinted materials is to choose the


suitable polymerizable functional building blocks for the template. In general, these
components are reactive monomers that are capable of forming stable network polymers
or gels that maintain a memory for the template or template analog [9].
272 9 Biosensing Applications of Molecularly Imprinted Nanomaterials

9.3.1Organic Materials

As illustrated in Fig. 9.3, polystyrenes/polyacrylates and inorganic polysiloxanes


represent two major classes of precursors that have been successfully used for
molecular imprinting, especially for the noncovalent imprinting approach. The
assortment of functional monomers makes the vinyl and acrylic polymers pretty
attractive because these monomers can provide a variety of noncovalent interac-
tions, including electrostatic interactions, hydrogen bonding, hydrophobic interactions,
metal coordinating, etc. [9]. Methacrylic acid is the most widely used functional
monomer because it can form hydrogen bonds with a wide variety of functional
groups on a target analyte. In addition, such MIP materials can be used under mild
conditions [34].
To date, many approaches have been used to prepare MIPs. Usually, MIPs are
produced by solution polymerization and then mechanically ground to give smaller
particles with micrometer diameters. Dispersion polymerization can also be used to
fabricate MIPs. This approach obviates mechanical grinding and yields aggregates
of spherical particles [35] or uniformly sized microspheres [36]. MIPs can also be
prepared as thin cross-linked imprinted polymer films [37, 38] by precipitation from
solutions of linear polymers in the presence of the analyte [39] or by casting an
imprinted polymer in the pores of an inert support membrane [40, 41]. Another
possibility for the formation of ultrathin polymer films on the electrode surface is
using the electropolymerization technique. This method is generally used for in situ
preparation of MIPs and is one of the most interesting methods for developing
piezoelectric sensors [42].

9.3.2Inorganic Materials

The solgel technique offers a wide range of processing approaches that can pro-
duce three-dimensional matrices in different configurations such as thin films,
porous materials, and bulk structures [43], and therefore is attractive for the prepa-
ration of MIPs [4451]. The application of imprinted solgel materials to the pro-
duction of sensors is in its infancy. Most of the accounts describing these materials
end with a statement that suggests the potential application for chemical sensing. So far,
several research groups have dealt with the application of imprinted solgel films as
recognition layers applied on various transduction systems. For example, in 1949,
Dickey [52] first imprinted silica to create materials that were 420 times more effec-
tive than nonimprinted silica controls on binding the target molecules. Much has trans-
pired since Dickeys seminal work, and solgel processing has allowed researchers
to create a wide variety of molecularly imprinted solgel-based sensors. Lulka etal.
[53] described the properties of surface-imprinted silicas using NATA or fluorescein
as templates. Silica gel was mixed with bis(2-hydroxyethyl) aminopropyl triethox-
ysilane as functional monomer and TEOS as the template. The slurry was allowed
to polymerize. After extraction of the template and nonspecifically bound material,
9.4 Development of MIP Nanomaterials 273

the polymers were glued to cards and their properties were examined in a right-angle
fluorescence-detection configuration. Quenching experiments using KI and acryl-
amide suggested that MIP-bound substrates were somewhat protected from their
solvent environments. The results illustrated an alternative method for the determi-
nation of inherently fluorescent molecules.

9.4Development of MIP Nanomaterials

9.4.1Limitation of Traditional MIPs

As mentioned above, molecular imprinting can produce MIPs with a built-in func-
tionality for the recognition of a particular chemical substance (template molecule)
with its complementary cavity. The synthesis of MIPs typically consists of the copo-
lymerization of functional and cross-linking monomers in the presence of a template
molecule [9]. However, the effectiveness of this technique is greatly dependent on
three major factors: the interaction nature between the template and monomer [5456];
the formation of imprinted materials [5760]; and the rigidity of the polymeric matrix
[6167]. Because of the nature of the highly cross-linked polymer network, it is dif-
ficult to thoroughly remove the template taken up in the preparation stage. Therefore,
crushed materials are needed in the application, during which the MIPs suffer from
the gradual elution of the template. This incomplete extraction of the template from
the network can easily result in significant problems in the assessment of the rebind-
ing assay, separation efficiency, etc. [68]. Therefore, the imprinted materials that are
ideally suitable for molecular recognition elements have yet to be explored.
The most common methodology for molecular imprinting is the monolithic
approach, where MIPs are prepared in bulk and subsequently ground and sieved to
the desired size [6972]. Despite being a simple and convenient approach, the wide
applications of MIPs as artificial receptors in analytical chemistry are still limited
by several critical factors. First, because of the highly cross-linked structure with its
irregular shape, most imprinted bulky polymers usually need a grinding process to
make the removal of the template easier and more efficient. However, the extraction
of original templates located at the interior area of bulky MIPs is still quite difficult.
Sometimes the imprinted cavities in the cross-linking network can be damaged by
the grinding process [6163]. The issue of poor accessibility of the imprinted cavities,
which are often created within the polymer bulk, is a major problem of this method.
It could result in slow kinetics of the target analyte binding [64, 65]. This is especially
important in the imprinting of macromolecules, such as oligosaccharides and pro-
teins. Additionally, the rigidity of the polymeric matrix greatly reduces the confor-
mational freedom of molecular recognition by excluding any further chain mobility
[66, 67]. Second, the uncontrollable random polymerization always gives rise to the
heterogeneity of the imprinted sites in the formed polymer matrix [73, 74], which
can be ascribed to the different polymerizing abilities of vinyl functional monomer
and divinyl cross-linking agent, and to the much larger amount of the cross-linking
274 9 Biosensing Applications of Molecularly Imprinted Nanomaterials

agent than that of the functional monomer. The final problem is the lack of signal
output of the analyte binding at MIPs due to the poor assembly ability at the surface
of the transducer, which has a negative effect on their use in chemical detections or
bioassays [19]. Therefore, the design and development of MIP materials at a molecular
level so as to provide a better antibody/enzyme mimic are of necessity and a great
challenge.

9.4.2Exploration of Novel Molecular Imprinting Strategies

Many efforts have been made to prepare MIPs in an optimizing form that controls
templates to be situated at the surface or in the proximity of the materials surface
[7587] so as to solve the problems of limited mass transfer and template removal
often associated with the traditional technique of molecular imprinting. Surface
imprinting has been widely studied as an effective approach. For example, Mosbach
and coworkers [75] first reported the surface molecular imprinting strategy by the
covalent immobilization of template molecules at the surface of a solid substrate.
After the imprinting polymerization and the removal of substrate, all the templates
were located on the surface of imprinted materials, providing a possibility for the
complete removal of templates, excellent accessibility to target species, and confor-
mational flexibility of recognition. This improvement is especially valuable when
imprinting macromolecules, such as proteins [7678], cells [79, 80], and viruses
[81]. As templates, these molecules with large sizes are more difficult to remove
from traditional highly cross-linked MIPs. The common approach of surface
imprinting is the creation of thin MIP films on suitable substrates. An advantage of
this approach is the thin film could be formed directly on different sensor substrates,
such as gold and glassy carbon electrodes [88, 89], quartz crystal microbalance
(QCM) [90, 91], and surface plasmon resonance (SPR) [92]. Various methodologies
have been used to prepare such MIP films, including spin-coating, surface-initiated
atom-transfer radical polymerization (ATRP), and electropolymerization.
In this approach, the control of MIP film thickness is the major issue, as it should
be sufficiently thin for rapid mass transfer. This is especially important in sensor
application, where a high sensitivity and a short response time are desired [87].
Accordingly, the controlled deposition of MIP nanostructures for sensor applications
has also been investigated. For example, Huang etal. constructed an on-chip sensor
device by photoirradiation of a cross-linkable polymer [93]. Such a cross-linkable
polymer in solution can be deposited more easily than a simple monomer mixture
on a flat surface by spin-coating and other printing techniques. By combing solgel
chemistry with spin-coating deposition, Marx et al. [94] coated a thin MIP film
(70nm) on ITO electrodes to fabricate electrochemical sensors. The designed sen-
sors displayed very high sensitivity and selectivity for the model analytes. By using
ATRP, Li etal. [95] synthesized a series of linear copolymers of 2-methacryloyl-
ethyl methacrylate and methacrylic acid. The linear copolymers were subsequently
allowed to form stable complexes with the templates before the formation of a
9.4 Development of MIP Nanomaterials 275

cross-linking structure. The use of ATRP to produce a surface-bound ultrathin


(<10nm) MIP layer was reported recently by Husson and coworkers [96], using
fluorescent amino acid derivatives as templates. The result showed that the use of
ATRP provided a microscopically smooth surface (rms roughness <2 nm) and a
precise control of film thickness, which were the two important matters in developing
optical sensing devices. However, the direct use of ATRP for molecular imprinting
in a noncovalent system may be complicated, because the catalytic complex formed
by the Cu ion and acidic or basic ligands can be easily disrupted by various template
molecules, resulting in the polymerization process not really being living. In this
regard, the other controlled radical polymerization techniques using iniferters [97],
RAFT reagents [84], and nitroxide-mediated polymerization [98] seem to be more
appropriate [11]. In addition, Jus group synthesized an MIP nanofilm on a gold
electrode by the electrochemical copolymerization of o-phenylenediamine and
dopamine. Using glutamic acid (Glu) as the template molecule, it was used
successfully to fabricate a capacitive sensor for the enantioselective recognition of
Glu [89].

9.4.3Attractiveness of MIP Nanomaterials

Recently, nanosized materials have been widely used as a material foundation of


chemosensors and have achieved tremendous success in improving the detection
sensitivity and selectivity [2]. Accordingly, the novel nanotechnologies and surface
chemistry have been introduced into molecular imprinting strategy and have
attracted considerable research interest because of the wider applications of
imprinted nanomaterials in bioassays and chemosensors [5759, 99, 100]. The
molecular imprinting nanotechnologies are expected to greatly enhance the
molecularaffinity of MIP materials, and thus provide a wider range of applications
approaching biological receptors [89]. The benefit of nanosized MIP materials is
not only the size itself, but also their fast equilibration with the analyte. Most of the
template molecules are situated at the surface and in the proximity of the materials
surface because of the small dimension of the nanostructured MIP materials with an
extremely high surface-to-volume ratio.
Figure9.5 illustrates the difference in the distribution of effective binding sites
between the bulky MIP materials and MIP nanoparticles after the extraction of tem-
plates is done [59]. Here, it can be assumed that these templates located within x
nanometers from the surface can be removed in the bulky materials with a scale of
d, and the target species can access the resulting imprinted sites. The effective vol-
ume of imprinted materials that can rebind target species is calculated as
[d3(d2x)3]. For highly cross-linked bulky materials, the x value is generally very
small, although either porogens or solvents are usually used in the imprinting
process. However, if the imprinted materials with the same size are prepared in the
form of a nanostructure with a scale of 2xnm, all of the templates can be removed
from the highly cross-linked matrix, and these resultant sites are all effective for the
276 9 Biosensing Applications of Molecularly Imprinted Nanomaterials

Fig.9.5 Schematic illustration of the distribution of effective binding sites in the imprinted bulky
materials and the nanosized, imprinted particles after the removal of templates. Reprinted with
permission from Gao etal. [59]. 2007, American Chemical Society

binding of target species. In the case of nanosized particles, most of the imprinted
sites are located at the surface or in the proximity of surface. Therefore, the forms
of imprinted materials are expected to greatly improve the binding capacity, kinetics,
and site accessibility of imprinted materials. In comparison with bulky MIP
materials, the imprinted nanomaterials have a higher affinity and sensitivity to target
analyte, and a more homogeneous distribution of recognition sites [2].
For the low-dimensional MIPs with nanostructures, it is easy to obtain regular
shapes and sizes, and the tunable flexibility of shapes and sizes. The nanosized MIP
materials also have better dispersibility in analyte solutions and thus greatly improve
the mass transfer, exhibiting fast binding kinetics [57, 58, 82, 83, 101]. In particular,
novel nanostructure assembly technologies have achieved wide success in building
various nanodevices [102, 103]. The imprinted nanomaterials with well-defined
morphologies can be feasibly installed onto the surface of devices in a required
form for many applications in nanosensors and molecular detection.

9.5MIP-Based Biosensors

9.5.1Molecular Recognition of MIP-Based Biosensors

As promising alternatives to enzymes, antibodies, and other natural receptors, MIP


materials have been increasingly applied in the development of biosensors [44, 104, 105]
due to their advantages of high specificity and stability, low cost, and ease of
9.5 MIP-Based Biosensors 277

Table9.2 Different approaches to the transduction of the binding signal in MIP sensors
Signal is generated
Directly through the
binding event By the analyte By the polymer
What is measured Change in general Specific property Change in the signal
physiochemical of the analyte emitted by reporter
properties of the groups incorporated
system into the polymer
Examples Mass change (QCM), Fluorescence, Fluorescence, scintillation,
capacitance change electrochemical spectral shift, proton
activity, IR release (pH)
spectrum
Reprinted with permission from Ye etal. [106]. 2004, Springer

p reparation. Three issues are critically important for the successful design of MIP
sensors: (1) the development of a highly sensitive transducer, capable of monitoring
the binding process and transforming it into a processable signal; (2) the develop-
ment of MIPs capable of interacting with the template-analyte under the required
conditions with the required affinity and specificity; (3) the integration of the MIP
with the transducer. Therefore, a major concern for the development of MIP-based
sensors is how to measure the analyte binding at MIP materials. Typically, MIP-
based sensors are fabricated by assembling MIP materials onto the surface of trans-
ducer; thus, the analyte binding is converted into a measurable signal (Fig.9.1b).
Table9.2 depicts the three different possibilities for the transduction of the binding
events in MIP sensors [106]. In the simplest case, a change in one or more physico-
chemical parameters of the system upon analyte binding (such as mass accumula-
tion) is used for detection. This principle is widely applicable and more or less
independent of the nature of the analyte. In general, the efficiency of sensors depends
not only on the selectivity and sensitivity of MIPs to target species, but also on the
approaches of signal output. The optimal transduction approach to a readable signal
output can be expected to maximize the selectivity and sensitivity of sensors.
In principle, many physical measurements, such as electrochemical voltammetry,
fluorescence, piezoelectricity, and SPR, can be used for the signal detection in MIP-
based sensors [12, 20, 44, 105]. The choice of the transducer is based on the proper-
ties of target analytes and the forms of MIPs. For example, mass-sensitive acoustic
transducers, such as QCM [107112], have become increasingly popular for the
design of MIP sensors based on mass-accumulation binding. An alternative way of
detecting mass accumulation at a surface is by optical means, such as ellipsometry
or SPR [113, 114]. If the target analyte exhibits a special property such as fluores-
cence or electrochemical activity, this can be exploited for the design of MIP-based
optical or electrochemical sensors. For example, a fluorescence sensor for dansyl-
phenylalanine, a fluorescent analyte, has been constructed [115]. The fluorescence
of the MIP after analyte binding is measured using fiber optics, and the signal is
found to be a function of the analyte concentration. The sensor shows a certain
degree of stereoselectivity for the l-form of the analyte. Electrochemical voltammetry
278 9 Biosensing Applications of Molecularly Imprinted Nanomaterials

is an excellent approach for measuring the binding of electroactive analytes at the


MIP nanofilms [88, 89, 116]. An amperometric morphine sensor has been devel-
oped where the analyte morphine is selectively enriched on an MIP film and subse-
quently quantified by electrooxidation [117]. So far, the studies on the development
of MIP-based sensors, in particularly electrochemical [88, 89, 116126] and optical
[127137] sensors, have been dramatically reported. At the same time, mass-sensi-
tive transducers are increasing in popularity since the approach has the advantage,
theoretically at least, that it can be universally applied to a broad range of targets
[42, 138145].

9.5.2Interfacing the MIPs with a Transducer

An important aspect in the design of a MIP-based sensor is to find an appropriate way


of interfacing the polymer with the transducer. In most cases, the MIP has to be
brought into close contact with the transducers surface. Otherwise, the signal output
of the MIP sensor will be greatly affected, resulting in low sensing efficiency. The
common way to dissolve this problem is that either the polymer can be synthesized in
situ at the transducer surface or the surface can be coated with a preformed polymer.
MIPs can be integrated with a transducer surface by electropolymerization on
conducting surfaces such as gold [118, 146, 147]. Compared to other immobiliza-
tion techniques, the unique advantage of electropolymerization lies in its ability to
deposit a recognition film at a precise spot on the detectors surface. This provides
the possibility of coating an electrode with a complex geometry with homogeneous
film. But this technique requires special electropolymerizable monomers, which, at
least to date, have been less thoroughly studied than the common acrylic and vinyl
polymers with respect to the possibility of being molecularly imprinted. However,
the standard surface-coating techniques such as spin- and spray-coating are more
generally applicable, which have been used to apply a thin film of monomer solution
to acoustic transducer surfaces. With these two techniques, thin and even polymer
layers can be produced, despite the fact that the use of radically polymerized vinyl
or acrylic systems requires oxygen-free conditions for the coating due to the radical
scavenging effect of oxygen, which would inhibit polymerization. A simple way to
synthesize MIP monolayer is the self-assembly technique. Piletsky et al. [148]
prepared a self-assembled monolayer (SAM) of hexadecyl mercaptan on a gold
electrode in the presence of a cholesterol template and used the imprinted SAM to
measure the cholesterol concentration using potassium ferricyanide as the electro-
chemical probe. The use of an organic SAM to create molecular recognition sites on
gold electrodes for developing various chemical sensors was reported by several
laboratories [149154]. Using nanoparticles as scaffolds, Boal and Rotello [155]
assembled multivalent binding sites for flavin on gold colloids, where diaminopyri-
dine- and pyrene-functionalized thiols were chosen as building blocks. The limited
mobility of thiols on gold colloids allowed optimal binding between the recognition
elements and the flavin template.
9.5 MIP-Based Biosensors 279

Preformed MIP particles with nano- or microsized structures can be interfaced


with the transducer in different ways. The entrapping of MIP particles into gels
[117] or behind a membrane [115, 156] has been suggested for electrochemical
transducers. In this case, a suspension of MIP particles in a solution of inert, soluble
polymer (PVC) acting as glue was spin-coated onto an acoustic transducer surface
[157]. But the incorporation of MIP nanoparticles can cause diffusion limitations,
resulting in long response times of the sensor, nonspecific analyte binding, or a
decrease in binding capacity. On the other hand, MIP materials exhibiting electrical
conductivity facilitate their assembly with an electrochemical transducer in an inte-
grated device. So the preparation of composite particles consisting of an electrically
conducting polymer (such as polypyrrole) and an acrylic MIP was examined. The
polypyrrole that was grown into the preformed porous MIP did not change its
recognition properties. Moreover, MIP particles could be mechanically and electri-
cally connected to a gold-covered silica substrate in this way.

9.5.3Electrochemical Sensors

Electrochemical devices usually monitor the current at a fixed voltage (amperometry),


monitor the voltage at an aero current (potentiometry), or measure conductivity or
impedance changes. Amperometric sensors are the most popular due to their simplic-
ity and ease of production, and the low cost of the devices and instruments. MIP-
based electrochemical sensors were first reported in the early 1990s by Mosbachs
group [17, 116]. They described the integration of a phenylalanine anilide-imprinted
polymer into a field-effect capacitance sensor and reported a significant reduction in
the overall capacitance when the sensor was exposed to the target species. The capac-
itance sensors based on MIPs were also fabricated and used to detect many other
analytes, such as amino acid derivatives with a detection limit of 500ppm [118], and
barbituric acid with a detection limit of 3.5ppm [158]. Tabushi etal. were the first to
use molecular imprinting to develop an high-affinity immunosensor-like device
[159]. They performed chemosorption of octadecylchlorosilane in the presence of
inert template hosts (n-hexadecane) to tin dioxide or silicon dioxide. After the hosts
were extracted, vitamins K1, K2, and E as well as cholesterol and adamantine were
detected and yielded strong electrochemical signals. Recently, Pesavento et al.
designed an MIP sensor for the amperometric determination of the nonelectroactive
substances cyanuric acid and atrazine by glassy carbon or graphite electrodes [160].
The current linearly depended on the concentration of the template molecules in
aqueous solution up to 104M. Sode etal. described an amperometric fructosylamine
sensor that used a catalytic fructosylamine dehydrogenase mimicking MIP [161].
Catalytic hydrolysis of the target resulted in the oxidation of an electron acceptor
(1-methoxy-5 methylphenazinium methylsulfate [m-PMS]), which was subsequently
reduced at the electrode to give rise to a signal.
During the past decade, remarkable progress in MIP-based electrochemical
sensors has been achieved by the use of conductometric/potentiometric measurements
280 9 Biosensing Applications of Molecularly Imprinted Nanomaterials

and MIP nanomaterials, greatly extending the range of detected targets and improving
the sensitivity, selectivity, and simplicity of electrochemical sensors [20, 118124,
162165]. For example, a successful potentiometric sensor was reported for the
detection of glucose [166]. The sensor operation was based on measuring the con-
centration of protons released during the interaction of metal-complexing imprinted
polymer with glucose. The proposed sensor was capable of measuring the glucose
concentration in a clinically relevant range (025mM) in plasma. Kitade etal. [162]
prepared an interesting potentiometric artificial immunosensor based on an MIP as
the detecting element in micro-total analysis systems with the intent of providing
easy clinical analysis. In this study, the sensing element of serotonin-imprinted
polymer was absorbed and immobilized in a plasma-polymer layer by a method of
swelling and polymerization. The obtained sensor was highly responsive to sero-
tonin in water but not to tryptamine, acetaminophen, or procainamide, with a detec-
tion limit of 100pmol/L. The electrochemical sensors are most commonly fabricated
by installing MIP nanomaterials, as recognition elements, onto the surface of the
electrode [88, 119]. The changes of current and peak voltage at cyclic voltammetry
upon the analyte binding can sensitively respond to the concentration and kind of
analytes, respectively, due to the oxidation or reduction of analytes at the MIP-
modified electrode. Kan etal. [119] constructed a novel electrochemical sensor to
monitor the neurotransmitter dopamine by modifying the glassy carbon electrode
with a composite of multiwalled carbon nanotubes (MWNTs) and dopamine-imprinted
polymers. The MWNT-MIP-modified electrode not only possessed a rapid dynamic
binding with an equilibrium period of 30min, but also exhibited a high selectivity
and sensitivity toward dopamine, with a linear range of 5.01072.0104 M.
Zhang and coauthors [88] reported a surface molecular self-assembly strategy for
molecular imprinting in electropolymerized polyaminothiophenol (PATP) mem-
branes at the surface of gold nanoparticle (Au NP)modified glassy carbon elec-
trode for the electrochemical detection of the pesticide chlorpyrifos (CPF), as shown
in Fig.9.6a, b. The SEM image (Fig.9.6c) confirmed the formation of PATP/Au NP
membranes on the Au NPGC electrode surface, where the rough surface provided
a large surface area for the adsorption of target species at the modified electrode.
The cyclic voltammetric response of the imprinted PATPAuNP-based sensor to
CPF was about 3.2-fold more than that of the imprinted PAPTAu sensor (Fig.9.6d),
and the detection limit for CPF was about two orders of magnitude lower than by
the imprinted PAPTAu sensor.
However, not all analytes can be electroactive and hence detected using amperom-
etry. A method for the displacement of nonspecific electroactive markers from an
MIP has been developed for the detection and quantification of ligand-polymer bind-
ing events, which can be used for the development of multisensors [167]. Additionally,
the direct detection of an inert template required MIPs to possess the ability to
change conformation or surface potential upon binding with template. Sensors spe-
cific for l-phenylalanine and cholesterol [168] and sugars show high selectivity and
sensitivity at the micromolarnanomolar range. Another approach in MIP sensor
design for inert analyte detection is based on capacitance measurements [169, 170].
9.5 MIP-Based Biosensors 281

Fig.9.6 (a) Preparation procedures of the imprinted PATPAu NPGC electrode; (b) schematic
illustrations for the adsorption of the ATP molecule at the Au NP surface and the further self-
assembly of CPF at ATP-modified Au NP electrode; (c) typical SEM image of the imprinted PATP
membrane; and (d) cyclic voltammograms of (a) imprinted PATPAu NPGC electrode, (b) non-
imprinted PATPAu NPGC electrode, and (c) the imprinted PATPAu electrode in the presence
of CPF. Reprinted with permission from Xie etal. [88]. 2010, American Chemical Society

Mosbach et al. reported the first capacitive sensor by using MIP membranes as a
sensing layer in a field-effect device consisting of silicon wafers with an SiO2 coating
[116]. Upon specific binding of the original print molecule to the MIP membrane, a
reduction in the capacitance was observed. Based on this detection technique, Jus
group also designed an MIP capacitive sensor for enantioselective recognition of Glu
[169]. In this study, o-phenylenediamine and dopamine were chosen as monomers
and electrochemically copolymerized on gold electrode surface in the presence of
nonelectroactive template l- or d-Glu (Fig. 9.7a). The designed MIP capacitance
sensor displayed high enantioselectivity and sensitivity to the stereoselective rebind-
ing of l- or d- to their corresponding receptor-mimicking MIP films due to the exact
definition of the imprint cavity (Fig.9.7b, c).
Although many successful studies on the development of MIP-based electro-
chemical sensors have been reported, the current level of commercial activity related
to their development and marketing still cannot meet the requirements. Several critical
problems associated with MIP development need to be addressed before successful
commercialization can start [20]. They include (1) the development and validation
of a general protocol for MIP design; (2) the need for a substantial increase in poly-
mer affinity and improvement of the ratio between specific and nonspecific binding;
(3) the development of effective immobilization protocols. To realize these goals, a
better understanding needs to be established between polymer chemists working on
MIP development and engineers developing sensors.
282 9 Biosensing Applications of Molecularly Imprinted Nanomaterials

Fig.9.7 (a) Fabrication process of MIP film and the dependences of relative capacitance change
on the concentrations of (b) l-Glu at (a) l-Glu-imprinted and (b) nonimprinted sensors, and (c)
d-Glu at l-Glu-imprinted sensor as well as (c) (a) d- and (b) l-Glu at the d-Glu-imprinted sensors.
Reprinted with permission from Ouyang etal. [169]. 2007, Wiley

9.5.4Optical Sensors

One of the most challenging tasks in developing biomimetic transduction systems


based on MIPs is transforming binding events into measurable signals [21]. Among
various signal transducers, optically addressable sensors based on fluorescent turn-
on or turn-off mechanism have been demonstrated to be highly desirable for a
variety of small-molecule analytes in many challenging environments, due to their
high signal output and feasible measurements [12, 20, 44]. Great efforts have
recently been made to prepare the fluorescent sensors using molecular imprinting
methods [120, 127132, 171]. The reason for this lies in the great flexibility and
breadth of information obtained by applying optical methods to the analysis of
polymer structure, affinity, selectivity, and binding kinetics in the interrogation
of polymer-binding mechanisms.
The earliest MIP sensor based on an optical device was developed for sensing
l-dansyl phenylalanine [115]. In this study, polymer particles imprinted with the
fluorescent template l-dansyl phenylalanine were sealed beneath a quartz window
and reexposed to the template. The fluorescence response of the systems was shown
to be related to the concentration of template, and, importantly, this response was
9.5 MIP-Based Biosensors 283

shown to be stereoselective. The important issue to any sensing platform is


establishing a reliable link between the target-binding event and transduction [123].
As the composition and structure of a molecularly imprinted site are, to some degree,
controlled, a number of studies have aimed to incorporate reporter monomers into
the binding pocket in order to establish a direct link between binding and transduc-
tion. Since the fluorescence characteristics of a particular molecule are influenced
by the local molecular environment, researchers have used fluorescence as a means
to observe the template-binding process.
For example, Liao etal. [172] used a competition approach to develop a sensor
for l-tryptophan. A fluorescent monomer (2-N-dansyl) ethyl 3,3-dimethylacrylate,
was incorporated into an l-tryptophan MIP to develop a competition between the
template and an efficient fluorescent quencher p-nitrobenzaldehyde. Takeuchi and
coworkers [127] designed and synthesized 2-acrylamidoquinoline as a fluorescent
functional monomer with a polymerizable acrylate moiety and a fluorescent hydro-
gen-bonding moiety. The template cyclobarbital was imprinted into a polymer
matrix by using the fluorescent functional monomer, in which the hydrogen bonding
of the target into the imprinted sites remarkably enhanced the fluorescence. The
fluorescent sensor showed the ability to selectively monitor the binding of the ana-
lyte with a detection range of 0.12.0 mM. The exposure of the sensor to other
structurally related interferences caused almost no fluorescence enhancement; the
resulting fluorescence enhancement was very small. Wangs group [128] developed
a system that responded to the binding event with a significant fluorescence inten-
sity change without the use of an external quencher. In this system, the formation of
the boronic ester bond between the boronic ligand and cis diols at the fructose-
imprinted sites could lead to the significant fluorescence enhancement. This was
attributed to the reformation of the boronic ester with the cis-diol of the fructose.
Such an MIP-based fluorescent sensor can provide a sensitive detection for d-fruc-
tose by the fluorescent enhancement of 80% for 100mM d-fructose.
On the other hand, Turkewitsch etal. [173] first used a fluorescent monomer to
prepare a fluorescence-quenching sensor for the detection of cAMP. The incorpora-
tion of the fluorescence probe into the prepolymerization system resulted in MIPs
that could produce a quenching response as rebinding of the target. The MIP-based
fluorescent sensor displayed a 20% fluorescence quenching in the presence of 1mM
cAMP, whereas almost no effect was observed for similar structural molecules.
A main drawback of the imprinted fluorescent polymer was its high background
signal. Another alternative fluorescent system was described by Rathbone and
coworkers [129, 174]. They synthesized a novel fluorescent monomer 3-acrylamido-
5-(2-methoxy-l-napthylidene) rhodanine that was cross-linked into MIPs for the
fluorescence-quenching detection of the template and range of cross-reactants [174].
Dramatic fluorescence quenching approaching background levels was observed
upon the binding of target species. The rebinding of template led to a 24.4-fold
reduction in fluorescence intensity, while similar analytes gave a 1.7-fold quenching.
Additionally, Rathbone and Bains [129] further extended the range of polymeric
fluorescent probes by describing a surface immobilization approach for preparing
fluorescence-responsive MIPs using quinine acrylate. Recently, Li et al. [137]
284 9 Biosensing Applications of Molecularly Imprinted Nanomaterials

Fig.9.8 Schematic illustration for the molecular imprinting (a) process and (b) mechanism of
LC-imprinted silica nanospheres embedded CdSe quantum dots, and (c) TEM images of (a) origi-
nal CdSe QDs, (b) CdSe-SiO2-MIP, inset: TEM image of a single CdSe-SiO2-MIP particle.
(c) SEM image of CdSe-SiO2-MIP, inset: SEM image of single CdSe-SiO2-MIP particle. Reprinted
with permission from Li etal. [137]. 2010, American Chemical Society

reported an MIP-based fluorescence nanosensor by anchoring the MIP layer on the


surface of silica nanosphere-embedded CdSe quantum dots (QDs) via a surface
molecular imprinting process. Figure9.8a illustrates the major steps involved in the
imprinting synthesis on the surface of silica nanospheres. In the first step, CdSe QDs
(1) were chemically modified with APTs according to the reported method. The
resultant APTS-CdSe (2) was further reacted with LC through hydrogen bond inter-
actions, driving template molecules into the formed silica matrix (Fig. 9.8b). The
synthesized CdSe-SiO2-MIP nanoparticles not only exhibited highly spherical mor-
phology and rough surface (Fig.9.8c), but also showed higher photostability. After
removal of the original templates, the CdSe-SiO2-MIP displayed a highly selective
and sensitive determination of LC via the intensity decrease of FL, and it could be
applied to detect trace LC in water without the interference of other pyrethroids and
ions in the concentration range of 0.11,000mM, with a detection limit of 3.6mg/L.
9.5 MIP-Based Biosensors 285

It was found that LC can quench the luminescence of CdSe-SiO2-MIP in a concen-


tration-dependent manner that was best described by a SternVolmer-type equation.
Except for the fluorescence enhancement and quenching mentioned above,
photo-induced electron transfer [175, 176], quencher-analyte competition adsorp-
tion [172], and chemiluminescence (CL) [177179] have been extensively explored
to monitor the analyte-binding events. Photo-induced electron transfer is a very
popular sensing mode for fluorescent molecular recognition [131, 154, 175, 176].
The electron-transfer mechanism is also applicable for the fluorescence detection of
nonfluorescent analyte. Cywinski etal. [176] prepared the fluorescent, molecularly
imprinted polymer thin films with cyclic guanosine 3,5-monophosphate (cGMP)
as a template and 1,2-diphenyl-6-vinyl-1H-pyrazole-[3,4-b]-quinoline as a fluores-
cent receptor according to a method based on commercially available poly (methyl
methacrylate). This method predicted photo-induced cross-linking in the mixture of
polymer chains and involved components. The advantages of this method were the
relatively simple preparation and the use of a common polymer. A strong fluores-
cence-quenching effect was observed when a cGMP-imprinted film was incubated
in aqueous solutions of cGMP. Leung etal. [175] reported a solgel molecularly
imprinted luminescent sensor fabricated by using a tailor-made organosilane as
fluorescent functional monomer and 2,4-D as template molecule. The luminescence
was greatly enhanced by the formation of acidbase ion pairs with 2,4-D because of
the suppression of photo-induced electron-transfer quenching on the anthryl fluoro-
phore emission. A gradually rising trend in luminescent intensity was observed by
increasing the 2,4-D concentration from 10 to 166.6g/mL, while the control materi-
als showed a negligible response in luminescent intensity.
Chemiluminance (CL) is a very attractive detection technique for the study of
MIP materials. During the rebinding events, the intensity of the emitted light is
directly proportional to the concentration of analyte. Once a suitable CL system is
chosen, the MIP-CL sensors show a high sensitivity and selectivity. Lin and cowork-
ers [130, 180] synthesized 1,10-phenanthrolinethe (Fig.9.9a) and dansyl-l-pheny-
lalanine (Fig. 9.9b). The polymer particles with the imprinted sites of
dansyl-l-phenylalanine were used as imprinted MIPs in a CL flowthrough sensor.
The catalytic decomposition of both hydrogen peroxide and peroxomonosulfate
could be linked to radical-induced decomposition of bound template, resulting in a
CL output. When the HSO4/Co2+ solution flowed through the polymer particles, the
target analytes adsorbed on the MIP particles were oxidized to decompose and gave
out CL emission with a low detection of 4107M.
Du etal. [181] used CL for the detection of epinephrine. Once epinephrine was
bound on an epinephrine MIP, the CL reagents, luminol and potassium ferricyanide,
flowed through the polymer packed into a capillary. Epinephrine binding to the
polymer produced a strong concentration-dependent CL signal. The detection limit
achieved using this setup was 0.2nM. More recently, Yang etal. [179] developed a
new MIP-CL method for the detection of proline. The MIP microspheres were syn-
thesized using precipitation polymerization with hydroxyproline, a structural ana-
log, as the template. Polymer microspheres were immobilized in microtiter plates
(96 wells), which selectively adsorbed the analyte (dansyl-proline). After washing,
286 9 Biosensing Applications of Molecularly Imprinted Nanomaterials

Fig. 9.9 The recognition of dansyl-proline with (a) proline MIP and (b) dansyl-proline MIP.
Reprinted with permission from Lin and Yamada [130]. 2000, American Chemical Society

the bound fraction was quantified based on peroxyoxalate CL reaction enhanced by


imidazole. A linear relationship was obtained between the relative CL intensity and
the concentration of proline in the range from 1106 to 4105mol/L, with a limit
of detection 3107mol/L.
Infrared spectroscopy (IR) has also been applied to design MIP sensors, which is
helpful for the identification and confirmation of the interaction mechanisms between
the template and functional entities before and after polymerization. For example,
Yoshida etal. [182, 183] studied the interactions between l-tryptophan ethyl ester
and the functional molecules with IR spectroscopy. Similarly, Egan et al. [184]
designed an MIP-IR sensor to detect calcium oxalate monohydrate or dihydrate, the
most common components of human kidney stones. The obtained MIP layers induced
the nucleation of their morphologically matched crystals. Gong etal. [185] have also
used IR spectroscopy to study acrylonitrile-acrylic acid terpolymer membrane
imprinted with uric acid. In their work, the MIP membrane displayed good thermal
stability and a good molecular recognition behavior toward uric acid. The MIP mate-
rial also showed the potential of application in blood filtration devices.
SPR is well known for its better signal reliability and stability than other transduc-
ers [186, 187] and is highly sensitive even for the detection of subnanomolar concen-
trations due to the change in refractive index on the metallic surface. Since the late
1990s, SPR has been rapidly developed as a nonlabeling detection method in biosen-
sors and has evolved as a good alternative to ELISAs. Several encouraging results of
SPR-MIPs were published. Lai et al. [114] first reported an SPR-MIP system in
1998. They prepared MIP particles imprinted with theophylline, caffeine, and xan-
thine using a monolithic approach and deposited them onto silver-coated SPR
surfaces by solvent evaporation. The prepared SPR-MIP sensor displayed good
selectivity for their template and demonstrated little cross-reactivity in their response
to a range of other analytes. Taniwaki et al. [188] prepared a 9-ethylademine-
imprinted polysulfone backbone and successfully fabricated an SPR-MIP sensor
capable of discriminating adenosine and guanosine. The obtained SPR-MIP sensor
9.5 MIP-Based Biosensors 287

showed good recognition behavior toward the analyte. Similarly, Li et al. [189]
developed an SPR-MIP senor by using the nanosized MIP films for the detection of
l-phenylalanine ethyl ester. The sensor showed good enantioselectivity during
rebinding events. However, in these experiments, the sensitivity of the SPR-MIP
sensors was not enough for the analysis of real samples. So an improved method
was developed by Kugimiya and Takeuchi [190]. They used a covalent approach
to graft a sialic acid MIP directly onto an allyl derivatized gold-coated SPR sur-
face to prepare an SPR-MIP sensor for ganglioside GM1, a sialic acidterminating
glycolipid. The resulting sialic acidimprinted SPR sensor responded linearly to
GM1, while it could not be used to detect concentrations lower than 0.1mg/mL.
Using a similar approach, Nishimura etal. [191] prepared an MIP-SPR sensor for
tetracaine. They grafted a methacrylic acidco-ethyleneglycol dimethacrylate
cinchonidineimprinted film onto an SPR transducer by using a covalent approach.
The sensor showed a linear response to tetracanine in the concentration range of
0.010.04mol/L.
In 2007, Raitman etal. [192] reported a very interesting SPR-MIP sensor for the
measurement of NADH. They combined the NADHimprinted polyacrylamide
polyacrylamidophenylboronic acid copolymer with SPR transducer to measure
NADH unambiguously at the 10-mM level. The catalytic properties of the MIP film
have been studied by detecting the oxidation of lactate to pyruvate by NAD+ in the
presence of lactate dehydrogenase. A number of recent studies have further
demonstrated the efficacy and adaptability of MIP-SPR sensor [193, 194]. For
example, Choi etal. [193] investigated the feasibility of employing the imprinting
technique for the detection of the mycotoxin zearalenone using an SPR transducer.
The molecularly imprinted polypyrrole (MIPPy) film was prepared by electropoly-
merization of pyrrole onto the bare Au chip in the presence of a template zearale-
none molecule (Fig.9.10). The MIPPy SPR sensor exhibited a linear response for
zearalenone in the range of 0.33,000ng/mL, with a detection limit of 0.3ng/mL.
The selectivity efficiencies of zearalenone and other structurally related analogs
were 1.0 and 0.150.27, respectively. These results suggested that a combination
of SPR sensing with MIPPy film was a promising alternative method for the detection
of zearalenone.
Willner etal. [194] first functionalized Au nanoparticles with thioaniline elec-
tropolymerizable groups and (mercaptophenyl)boronic acid, and then elctropoly-
merized the functionalized Au NPs in the presence of antibiotic substrate neomycin
(NE), kanamycin (KA), or streptomycin (ST) to yield bisaniline-cross-linked Au
NP composites. After the removal of the ligated antibiotics, the molecularly
imprinted matrixes were prepared and the antibiotics were sensed by SPR spectros-
copy. The resulting SPR-MIP sensor revealed high sensitivities and a linear response
toward the sensing of the antibiotic analytes, with detection limits of 2.00.21pM
for NE, 1.00.1pM for KA, and 20030fM for ST, respectively. The imprinted
Au NP composites were successfully used to analyze the antibiotics in milk samples.
Despite the inherent limitations in sensitivity arising when targeting low-molecular-
weight molecules, the combination of the two technologies still provided a promising
potential for its wide applications in biosensors.
288 9 Biosensing Applications of Molecularly Imprinted Nanomaterials

Fig.9.10 Schematic diagrams of the setup for electropolymerization and SPR detecting zearale-
none. (a) Preparation of molecularly imprinted polypyrrole on bare Au using a three-electrode
electrochemical system; (b) the shift in resonance angle of the SPR sensor resulting from the
rebinding of zearalenone to the MIPPy film. Reprinted with permission from Choi etal. [193].
2009, American Chemical Society

9.5.5Mass-Sensitive Devices

Mass-sensitive signal transduction has increased in popularity since the measure-


ment of mass is the most general method suitable for the detection of any analyte.
Piezoelectric devices such as QCM can provide an extremely sensitive measurement
to the mass of the analyte binding at the surface of piezoelectric materials through an
accompanying decrease in the oscillation frequency of a piezoelectric crystal. A
general rule of thumb is that for a system resonating at 10MHz, a change in mass of
1ng results in a 1Hz change in resonance frequency. Therefore, when an MIP layer
is attached to the surface of a QCM, the system can be used to measure template-
specific binding with a high degree of sensitivity. The synergetic advantages of the
selectivity provided by MIP and the sensitivity provided by piezoelectric sensing
make the sensors almost universally applicable, with good limits of detection, low
cost, and the possibility of easy miniaturization and automation [138145]. The three
most common application areas for QCM-MIP sensors are clinical diagnosis, envi-
ronmental monitoring, and control of enantiomeric separation. In these applications,
MIP synthesis is usually performed either in situ on the sensor surface or via pre-
pared MIP particles/beads that are immobilized on the sensor surface using a PVC
matrix. The thickness of the MIP layer varies between 18nm and 5mm [195].
In 1999, Haupt et al. [140] reported an enantioselective QCM-MIP sensor.
UsingS-propranolol as a template, a poly(trimethylolpropane trimethacrylate-co-
methacrylic acid) membrane was prepared by sandwiching the prepolymerization
9.5 MIP-Based Biosensors 289

Fig.9.11 (a) Tapping-mode AFM picture showing the ongoing imprinting process on a coated
QCM electrode and (b) solgel layer from titaniumIV ethylate imprinted with S. cetrvisiae.
Reprinted with permission from Dickert and Hayden [196]. 2002, American Chemical Society

solution between a gold-coated QCM chip and the surface of a UV lamp. The
resultantsensor showed good enantioselectivity for the S enantiomer when com-
pared to the R enantiomer. Dickert and Hayden [196] modified a QCM surface
with a surfaced-imprinted solgel using whole yeast cells. In this study, the MIP
layer showed honeycomb-like structures (Fig.9.11). Figure9.11a shows the atomic
force microscopy (AFM) image of the ongoing imprinting process on a coated
QCM electrode. The polymerizing material incorporates half of single S. cerevisiae
cells and forms packed and highly ordered biomimetic receptors. Figure 9.11b
shows the solgel layer from titaniumIV ethylate imprinted with S. cerevisiae. The
coating is extremely robust and scratch-resistant. The reinclusion of cells allows a
selective online monitoring of these microorganism concentrations in water over
five orders of magnitude. The sensitivity to cells held up in growth media up to
21g/L. Even cell fragments could be detected in flowing conditions. Krozer etal.
[138] reported the QCM sensor with dissipation (QCM-D) by coating the sensor
surface with premade molecularly imprinted nanoparticles. The nanoparticles were
physically entrapped into a thin poly(ethylene terephthalate) (PET) layer spin-
coated on the transducer surface. By controlling the deposition conditions, a high
nanoparticle loading can be gained in the stable PET layer, allowing the recognition
sites in nanoparticles to be easily accessed by the test analytes. The highest uptake
of the nanoparticle film to propranolol corresponded to approximately 2nmol/cm2,
or about 11015molecules/cm2. The detection limit of the QCM-MIP sensor was
about 10mM, and chiral recognition and discrimination between R- and S-propranolol
can also be achieved. Similarly, Chan etal. [111] described the use of the thin per-
meable films of MIPs as biomimetic recognition materials on QCM surface. The
sensor can provide a high enantioselectivity and sensitivity for the discrimination of
l- and d-tryptophan enantiomers, with a detection limit of 8.8mM.
290 9 Biosensing Applications of Molecularly Imprinted Nanomaterials

On the other hand, the in situ polymerization of a self-assembled alkanethiol


monolayer on a gold-coated QCM resonator can also produce the thin MIP coat-
ing layer [91, 139, 197]. For example, Ersz etal. [197] combined QCM with
MIP to prepare a sensor using the ability of glucose to chelate the copper (II) ion
of methacrylamidohistidine monomer to create a ligand exchange-assembled
monolayer suitable for glucose determination. Because the ligand exchange on a
copper (II) complex is very fast and reversible, the obtained QCM-MIP sensor
showed highly selective detection of a-d-glucose in the liquid phase, with a
detection limit of 0.07mM. Piacham etal. [91] studied the preparation of thin
MIP nanofilms on the surface of QCM. The thickness of the MIP film was below
50nm. The selective recognition of target analytes can be easily detected by an
underlying quartz crystal resonator. When used in a flow-injection analysis sys-
tem, the assembled QCM sensor generated a large frequency change upon
encountering a small amount of analyte (0.19mM), had a very short response
time (<1min), and displayed certain chiral selectivity toward the original tem-
plate, S-propranolol, at a concentration higher than 0.38mM. Yang etal. [141]
fabricated an MIP-based QCM using the glucose-sensitive TiO2 ultrathin com-
posite films as recognition element for the detection of monosaccharides. The
largest binding capacity was 2.3-fold more than that of the corresponding nonim-
printed films.
The determination of proteins is very important in quick medical diagnostics
and modern drug discovery [198, 199]. The traditional detection approaches, such
as dye labeling and electrophoresis, usually suffer from poor selectivity and sensi-
tivity when the specimen is a protein mixture. The MIP film coupled with QCM
may be a potential analytical method for the detection of proteins. In the case of
protein imprinting, MIP film is a most desirable form of materials since the removal
of the high-molecular protein template becomes much easier than in normal bulky
MIP monoliths [26, 145, 198, 199]. In 2004, Chou and coworkers [145] prepared
albumin MIP film to analyze albumin protein mixture by QCM-MIP sensor. The
albumin MIP film was successfully coated on the QCM gold electrodes with amino,
hydroxyl, and carboxyl ligands by ultraviolet light-inducing polymerization. With
respect to adsorption selectivity, the QCM-MIP sensor exhibited a higher response
to albumin than other serum proteins, in which the adsorption mass ratio of cyto-
chrome c/lysozyme/albumin/myoglobin was 160/1/1,942/30. In addition, the
QCM-MIP results also showed a good match with the data of the clinical assay in
the test of human serum. Tai et al. [110] combined the surface-imprinting and
epitope-mediated modification with QCM for the detection of dengue virus pro-
tein. As shown in Fig.9.12, a 15-mer peptide known as the linear epitope of the
dengue virus NS1 protein was used as the template. By comparing with protein-
engaged imprinting on the surface, the epitope-mediated imprinting process pro-
vided a convenient invitro cellar assay for quantitatively recognizing proteins and
offered several advantages, such as the avoidance of embedding and bleeding the
target protein, minimizing the nonspecific interaction between the MIP and target
protein, and a good arrangement, in the proper orientation, of all the proteins bound
to the MIP film.
9.5 MIP-Based Biosensors 291

Fig.9.12 Comparison of epitope-mediated imprinting with protein-engaged imprinting. Reprinted


with permission from Tai etal. [110]. 2005, American Chemical Society

Based on this approach, this group recently fabricated another QCM-MIP sensor
in the presence of epitope peptides for the detection of the protective antigen of
anthrax [200]. Those five peptides, such as (N-Acr-l-Cys-NHBn)2, are linear or
conformational epitopes of the anthrax-protective antigen PA83, which made the
resulting epitope-cavity show high affinity for the corresponding template, as shown
in Tables9.3 and 9.4. The affinities of the peptide to their corresponding MIPs were
more closely related to the molecular weight of the analyte than to the number of
residues. All epitope-cavities differentiated their epitope region on the protective
antigen PA83 as well as the corresponding furin-cleavage fragments PA63 and PA20.
The differential response to the protective antigen fragment could be observed in
the picomolar range.
292 9 Biosensing Applications of Molecularly Imprinted Nanomaterials

Table9.3 Amino acid sequences for epitope-peptides as a template


QCM chip Sequence no. Epitope sequence Region8 CD structurea
PA7179 chip 7179 36
VKKSDEYTF 1b5 Type I b-turn
PA659672 chip 65967236 RYDMLNISSLRQDG 4b7 Random-coil
PA681694 chip 68169434 YNDKLPLYISNPNY 4b8> <4b9 Random-coil
PA683694 chip 68369431 DKLPLYISNPNY 4b8> <4b9 Random-coil
PA713722 chip 71372236 NGDTSTNGIK 4b9> <4b10 Random-coil
Reprinted with permission from Tai etal. [200]. 2010, American Chemical Society
a
CD circular dichroism

Table9.4 Comparison of the affinities (Kd) of epitope-peptides to their QCM chipa


QCM chip Analyte Kd (nM) No. of residue pI37 MW (Da)
PA7179 chip VKKSDEYTF 10 9-mer 6.01 1115.55
PA659672 chip RYDMLNISSLRQDG 5 14-mer 5.57 1666.81
PA681694 chip YNDKLPLYISNPNY 3 14-mer 5.45 1712.84
PA683694 chip DKLPLYISNPNY 7 12-mer 5.88 1435.73
PA713722 chip NGDTSTNGIK 20 10-mer 5.64 1005.47
Reprinted with permission from Tai etal. [200]. 2010, American Chemical Society
a
AHB/AM/ATA/EBAA=1:1:2:4

In order to prevent the spread of infection in animal and plant populations, the
rapid and inexpensive screening of viral and bacterial infections is urgently needed
[201204]. However, the detection of viruses and bacteria is generally time-
consuming and expensive because the biological detection needs a complex process,
including incubation, separation, dyeing, and microscopy. The bioimprinted QCM
sensors may provide a fast and selective biodetection. Hayden and coworkers
recently developed surface-imprinting techniques on polymer-coated QCM to detect
tobacco mosaic viruses (TMV) [109, 205] and living yeasts [196] in aqueous
media. The imprinting cavities or trenches on the polymer surface mimicking the
shape and surface functionality of the viruses and bacteria served as recognition
sites for their rebinding. The sensors were applicable to TMV detection ranging
from 100ng/mL to 1mg/mL [109], and allowed a selective online monitoring of the
yeast cell concentrations in water over five orders of magnitude [196]. Moreover,
they expended the bioimprinting concept to the recognition of mammalian cells,
which was a greater challenge because of their lower mechanical stability compared
to microorganisms [54, 130, 196, 206]. Erythrocyte-specific interactions with rec-
ognition sites on surface-imprinted polyurethane were applicable for blood-group
typing of the main ABO antigens [54]. The interesting finding in QCM-MIP sensors
was highly relevant for clinical applications in serology.
Recently, Pietrzyk etal. [112] devised a histamine piezoelectric sensor using an
electrodeposited MIP film as the recognition element. The preparation of the
sensing film involved two consecutive electrochemical polymerizations. First, a
poly(bithiophene) barrier film was deposited by electropolymerization on the Pt/
quartz resonator to prevent histamine electrooxidation and avoid possible
9.5 MIP-Based Biosensors 293

Fig.9.13 (a) Schematic of pollen stamp preparation: The pollen grains adhere to PDMS due to
adhesive forces. Excess pollen is blown off with pressurized air, resulting in a monolayer stamp;
(b) contact-mode AFM image of a polyurethane imprinted with pollen. Reprinted with permission
from Jenik etal. [206]. 2009, Springer

c ontamination of the Pt electrodes surface. Next, the histamine-templated MIP


film was deposited by electropolymerization on top of this barrier film. The con-
secutive growth of both overlaid films was monitored with an electrochemical
quartz crystal microbalance (EQCM). The analytical performance of the biosensor
was assessed under flow-injection analysis conditions. The negative peaks of reso-
nant frequency linearly decreased with the increase in the histamine concentration
in the range of 10100 mM with 150 mL/min flow rates, and 100-mL injected
sample. The sensitivity of the MIP sensor (0.33Hz/mM) was more than twice that
of the sensor without the poly(bithiophene) barrier film (0.15Hz/mM). The sen-
sors performance was superior for selective recognition of histamine if the
poly(bithiophene) barrier film thickness exceeded 200nm. The concentration limit
of detection was as low as 5nM histamine if the carrier solution flow rate was as
low as 35mL/min and the injection sample volume as large as 1mL. The sensor
was selective with respect to functionally and structurally related interfering com-
pounds, such as tryptamine, dopamine, and ididaxole. Dickert etal. [207] designed
pollen-imprinted polyurethanes for QCM allergen sensors in the gas phase. In this
study, imprints of birch and nettle pollen (major allergens) could be generated by a
polydimethylsiloxane stamping technique (Fig. 9.13a), as proven by AFM
(Fig.9.13b). Such materials showed mass effects as expected until all interaction
sites were occupied, and they revealed good selectivity toward target molecules.
The cross-selectivity assessment showed birch MIP gave rise to negative effects
for both birch and nettle pollen.
Although many successful examples of the development of MIP-based PZ sen-
sors exist, their current level of development is still low. Several key problems asso-
ciated with MIP development need to be solved before successful commercialization
can start. First, a general protocol for MIP design needs to be developed and vali-
dated; second, further study on the exploration of MIPs capable of effective func-
tioning in aqueous solution should be made; finally, novel imprinting techniques are
urgently needed to meet the requirement of a substantial increase in polymer affinity
and to improve the ratio between specific and nonspecific binding.
294 9 Biosensing Applications of Molecularly Imprinted Nanomaterials

9.6Conclusions

Molecular imprinting is an exciting and promising technique. It is being increasingly


adopted as a platform for creating responsive materials for biosensors. MIPs appear
to offer an inexpensive, robust, and reusable alternative to expensive and labile
biorecognition elements. These materials exhibit binding affinities that are compa-
rable to antibodyantigen and offer the ability to design recognition sites for a pleth-
ora of analytes, including those without their own natural receptors. Moreover, as
described in this chapter, various nanotechnologies are increasingly being adopted in
the preparation of MIP materials and the fabrication of MIP-based sensors. The
imprinting of molecular recognition sites at nanostructures has greatly improved the
removal of templates and the binding capacities and kinetics of molecular recogni-
tion, compared with the traditional imprinted bulky materials. Nanosized MIP mate-
rials with a high surface-to-volume ratio are characterized by their capability to bind
target molecules with similar affinity and selectivity to those of antibody, whereas
they offer a higher physical and chemical stability and better engineering possibility
than natural receptors. Nanosized MIP materials as molecular recognition elements
have particularly exhibited remarkable advantages for applications in biomimetic
sensors. Although the combination of MIP nanomaterials with transducers gives rise
to ultrasensitive detections of some analytes, replacing natural counterparts by MIP
nanomaterials still requires substantial research effort. First of all, the specific molec-
ular affinity and reduction of nonspecific adsorption should be enhanced by the
design of MIP nanostructure. Second, the exploration of a general synthesis protocol
for MIP nanomaterials with uniform shape and size has to be made. Third, further
study on the engineering combination of MIP nanoarrays with transducers in a suit-
able format is needed. Finally, future explorations are necessary for the development
of multisensors with multiplexing capabilities and high integration through the use
of nanofabrication. Fortunately, national and international funding agencies have
recognized the potential of MIP-based sensors for analytical chemistry, and several
large research projects aimed at demonstrating the validity and practical usefulness
of MIP-based analytical methods and devices are currently underway. Thus, future
efforts to solve these problems by multidisciplinary approaches will hopefully extend
the real applications of MIP nanomaterials in biosensors.

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wwwwwwwwwwwww
Chapter 10
Biosensors Based on SolGel
NanoparticleMatrices

10.1Introduction

In recent years, many researchers have been engaged in the development of


biosensors for environmental and biomedical monitoring. A biosensor is an analytical
device composed of a biological sensing element such as a protein, antigen, anti-
body, DNA, or RNA in intimate contact with a physical, optical, mass, or
electrochemical transducers, which can generate a measurable signal relating to the
concentration ofan analyte. Many review articles have discussed the development
of this field [15].
The stability of biomolecules and signal transfer to the transducers surface are
crucial factors in the stability and sensitivity of the biosensor. In aqueous solutions,
biomolecules such as enzymes lose their catalytic activity rather rapidly, because
enzymes can suffer oxidation reactions or their tertiary structure can be destroyed at the
airwater interface. This problem can be avoided by immobilization of enzymes [6].
By attachment to an inert support material, enzymes may be rendered, retaining
catalytic activity and thereby extending their useful life. In view of the above neces-
sity and advantages, different techniques have been developed to immobilize
biomolecules, including adsorption, covalent attachment, and entrapment in various
polymers [79].
Nanoparticles (NPs) with a specific size and morphology have attracted increasing
interest due to the influence of their shape and size on their electronic, optical,
magnetic, and catalytic properties [1012]. NPs play important roles in the devel-
opment of the biosensing field, because they exhibit a higher surface area-to-volume
ratio than their bulk counterparts. They can be conjugated with the biomolecules
such as redox enzymes and also act as nano-connectors (e.g., activate redox
enzymes or electrical labels) for biorecognition, leading to a variety of architec-
tures for biosensing [1316].
The solgel technique is a most widely used method for NP preparation [1720].
The solgel process can be carried out at low temperatures, and it results in chemically

H. Ju et al., NanoBiosensing: Principles, Development and Application, 305


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0_10, Springer Science+Business Media, LLC 2011
306 10 Biosensors Based on SolGel Nanoparticle Matrices

inert, porous, transparent, and mechanically stable nanostructures [19, 21, 22],
which are very suitable for biosensor fabrication [2325]. This chapter highlights
the advantages, recent developments, and future perspectives of biosensing applica-
tions based on solgel nanoparticle matrices.

10.2SolGel Chemistry

10.2.1What Is SolGel?

Sols are dispersions of colloidal particles in a liquid. Colloids are solid particles
with diameters of 1100nm. A gel is an interconnected, rigid network with pores of
submicrometer dimensions and polymeric chains whose average length is greater
than a micrometer. Three approaches are normally used to make solgel monoliths
[26]: (1) gelation of a solution of colloidal powders; (2) hydrolysis and polyconden-
sation of alkoxide or nitrate precursors followed by hypercritical drying of gels; (3)
hydrolysis and polycondensation of alkoxide precursors followed by aging and drying
under ambient atmospheres.

10.2.2The SolGel Process

Most commonly, solgel process refers to the hydrolysis and condensation of


metal or semimetal (e.g., Si, Al, Ti, Zr, etc.) alkoxide-based precursors such as
Si(OEt)4 (tetraethyl orthosilicate, or TEOS), which was just mentioned as method (3).
The earliest examples of such reactions date to the work of Ebelmen in 1846. The
versatility and general usefulness of the modern solgel process is reflected in many
available papers, which provide an excellent overview of the theory concerning
each step of the solgel process [19, 2633].
Disregarding the nature of the precursor, using silicon alkoxide as an example, a
complete solgel process can be characterized by a series of distinct steps [26].
Step 1: Mixing. A liquid alkoxide precursor, such as Si(OR)4, where R is CH3, C2H5,
or C3H7, is hydrolyzed by mixing with water (Eq.10.1).

Eq.10.1 Hydrolysis, from [26]


10.2 SolGel Chemistry 307

The hydrated silica tetrahedra interact in a condensation reaction (Eq. 10.2),


forming SiOSi bonds.

Eq.10.2 Condensation, from [26]

The linkage of additional SiOH tetrahedra occurs as a polycondensation


r eaction (Eq.10.3) and eventually results in an SiO2 network. The H2O and alcohol
expelled from the reaction remain in the pores of the network.

Eq.10.3 Polycondensation, from [26]

The hydrolysis and polycondensation reactions initiate at numerous sites within


the TMOS water solution as mixing occurs. When sufficient interconnected SiOSi
bonds are formed in a region, they respond cooperatively as colloidal particles or a
sol. The size of the sol particles and the cross-linking within the particles depend
upon the pH and R ratio [R=H2O/Si(OR)4].
Step 2: Casting. Since the sol is a low-viscosity liquid, it can be cast into a mold.
The mold must be selected to avoid adhesion of the gel.
Step 3: Gelation. Gelation is a process in which the colloidal particles and condensed
silica species link together to become a 3D network [34]. The physical characteristics
of the gel network depend greatly upon the size of particles and the extent of cross-
linking prior to gelation. During gelation, the viscosity increases sharply, and a solid
object results in the shape of the mold. With appropriate control of the time-dependent
change of viscosity of the sol, fibers can be pulled or spun as gelation occurs.
Step 4: Aging. Aging of a gel, also called syneresis, involves maintaining the
cast object for a period of time, hours to days, completely immersed in liquid.
During aging, polycondensation continues along with localized solution and
reprecipitation of the gel network, which increases the thickness of the
308 10 Biosensors Based on SolGel Nanoparticle Matrices

Fig.10.1 Structural relationship among a gel, a xerogel, and an aerogel (reprinted with permission
from Cushing etal. [33]. 2004, American Chemical Society)

i nterparticle necks and decreases the porosity. The strength of the gel thereby
increases with aging. An aged gel must develop sufficient strength to resist
cracking during drying.
Step 5: Drying. During drying, the liquid is removed from the interconnected pore
network. When a gel is evaporated to dryness, either by thermal evaporation or by
supercritical solvent extraction, the gel structure changes substantially in some
cases. During thermal drying or room-temperature evaporation, capillary forces
induce stresses on the gel that increase the coordination numbers of the particles and
induce collapse of the network. The increase in particle coordination numbers
results in the formation of additional linkages that strengthen the structure against
further collapse and eventually lead to the formation of a rigid pore structure.
Thestructure can therefore be considered as a collapsed and highly distorted form
of the original gel network, which results in xerogel.
The supercritical extraction of solvent from a gel does not induce capillary
stresses due to the lack of solventvapor interfaces. As a result, the compressive
forces exerted on the gel network are significantly diminished relative to those
created during formation of a xerogel, and lead to aerogels [33] (Fig.10.1).
Step 6: Dehydration or chemical stabilization. Through impregnated with optically
active polymers such as fluors, wavelength shifters, dyes, or nonlinear polymer,
surface silanol (SiOH) bonds can be removed from the pore network. This results
in a chemically stable ultraporous solid.
Step 7: Densification. Heating the porous gel at high temperatures causes densifica-
tion. The pores are eliminated, and the density ultimately becomes equivalent to
fused quartz or fused silica. The densification temperature depends considerably on
the dimensions of the pore network, the connectivity of the pores, and the surface
area [35]. The purity and homogeneity of dense gel-silica made by method (3) are
superior to other silica glass-processing methods. The ability to produce optics with
nearly theoretical limits of optical transmission, lower coefficients of thermal expan-
sion, and greater homogeneity, along with net shape casting, represents major
advances from solgel processing of monolith.
10.3 Biosensors Based on SolGel Nanoparticle Matrices 309

Fig.10.2 Schematic diagram showing the solgel process and its various products (reprinted with
permission from Gupta and Kumar [36]. 2008, IOP)

10.2.3Nanoparticles from the SolGel Process

The solgel technique is the most widely used method to prepare various kinds of
nanoparticles (Fig.10.2) [36, 37]. First, sols themselves are dispersions of colloi-
dal particles with diameters of 1100nm in liquids, which can be used directly or
coated on a substrate though spinning, dipping, and spraying to get a nanoparticle
film [3840]. In addition, colloidal particles can be isolated from sols by drying,
centrifugation, and coprecipitation [4143] and so on. Second, by covering a com-
plete solgel process, through hydrolysis, gelation, aging, drying, stabilization,
and densification at high temperature, we can obtain nanoparticles from xerogel or
aerogel [26, 33].

10.3Biosensors Based on SolGel Nanoparticle Matrices

Solgel biosensors, especially based on bioactive molecules entrapped in a gel


network, have seen tremendously research interest [7, 4452]. In this section,
we mainly discuss the biosensors based on nanoparticles related to solgel
techniques.
310 10 Biosensors Based on SolGel Nanoparticle Matrices

10.3.1Silica Nanoparticle for Biosensing

Solgel-based silica nanoparticles used for biosensing are mainly synthesized


through the Stber method [37] and the reverse-micelle method [53, 54]. The Stber
process is the most common method for synthesizing colloidal silica nanoparticles
with a size under 100nm [37]. The process involves the hydrolysis of a Si alkoxide
precursor (such as tetraethoxysilane, TEOS) in a mixture of ethanol and aqueous
ammonium hydroxide. Silicic acid is produced during hydrolysis, and when its
concentration is above its solubility in ethanol, it nucleates homogeneously and
forms silica particles of a submicron size. Primarily, this method is used for the
preparation of pure silica nanoparticles.
Reverse-micelle methods, also known as water-in-oil (W/O) microemulsion
systems, create thermodynamically stable aggregates of amphiphilic surfactants.
Ahydrophilic head region is formed that contains numerous nanometer-sized water
cores, each with a hydrophobic tail that extends into an apolar continuous phase.
The nanodroplets of water in the bulk-oil phase act as nanoreactors for discrete
particle formation. As in the Stber method, silica particles are formed by the hydro-
lysis of silane precursors. The diameter of the prepared particles is controlled by the
size of the water cores and the water-to-surfactant molar ratio (WO), since the reac-
tion is restricted in the water core. In comparison to the Stber method, particle
synthesis needs a much longer time frame, i.e., 12 days vs. a few hours. This
method has been shown to be highly efficient for the fabrication of highly spherical
and monodispersed nanoparticles. In addition to pure silica particles, the reverse-
micelle method can also be used to prepare doped nanoparticles.

10.3.1.1Pure Silica Nanoparticle for Biosensing

Silica nanoparticles prepared by a solgel process have a high specific surface area
as well as good biocompatibility, which is favorable to biomolecule loading for
biosensors [13, 5559]. Biomolecules can attach to silica nanoparticles through an
electrostatic force. He et al. [55] assembled heme protein hemoglobin (Hb) or
myoglobin (Mb) and silica nanoparticles in a variety of charge states layer by layer
into films on solid surfaces to investigate the driving forces for film assembly. They
found that when the proteins and silicas were both negatively charged, stable layer-
by-layer films were successfully fabricated, although the amounts of proteins were
smaller than that in the films assembled by opposite charged nanoparticles and pro-
teins. This demonstrated the importance of localized Coulombic attractions between
the negatively charged nanoparticle surface and the positively charged amino acid
residues on the Mb or Hb surfaces in the assembly and for the stability of films.
Based on this finding, many kinds of biosensing methods have been developed.
Qhobosheane etal. [13] used freshly prepared pure silica nanoparticles to immo-
bilize two different enzymes, glutamate dehydrogenase (GDH) and lactate dehy-
drogenase (LDH), for biosensors, in which enzymes were involved in a similar
enzymatic reaction with their cosubstrates, glutamate and lactate, respectively.
10.3 Biosensors Based on SolGel Nanoparticle Matrices 311

Luckarift etal. [56] reported a simple and rapid method for the deposition of silica
nanoparticles coated with lysozyme onto a gold surface. The method was based
on the ability of lysozyme to mediate the formation of silica nanoparticles.
A monolayer of lysozyme was deposited via nonspecific binding to gold. The
lysozyme then mediated the self-assembled formation of the silica monolayer.
The silica layer significantly increased the surface area compared to the gold sub-
strate and was directly compatible with a detection system. Organophosphate
hydrolase was successfully encapsulated within the silica particles, and a detec-
tion limit for the substrate, paraoxon, using the surface-encapsulated enzyme was
found to be 20mM.
Grant etal. [57] utilized silica nanoparticles as an optical platform for the devel-
opment of a protease biosensor based on the chemical transduction method and
fluorescence resonance energy transfer (FRET). Tang et al. [58] formed glucose
biosensors with glucose oxidase (GOD) immobilized in a composite matrix, which
was composed of hydrophobic silica nanoparticles and polyvinyl butyral (PVB) by
a solgel method. The experiments showed that nanoparticles can significantly
enhance the catalytic activity of the immobilized enzyme. The current response can
be increased from tens of nanoamperometers to thousands of nanoamperometers
with the same glucose concentration, and the electrodes responded very quickly, to
about 1min. Tsagkogeorgas etal. [59] presented the encapsulation of anti-diclofenac
antibodies in silica nanoparticles by a combination of reverse-micelle and solgel
techniques. The antibody source was a purified IgG fraction originating from a
polyclonal rabbit antiserum. Tetramethyl orthosilicate was used as the precursor.
Rather uniform, monodispersed, and spherical silica particles of about 70-nm-diam-
eter size were fabricated. The biological activity of the encapsulated antibodies was
evaluated, which demonstrated an obvious advantage of this approach for a mild
immobilization of different antibody species.

10.3.1.2Doped Silica Nanoparticle for Biosensing

Some small organic molecules, with optical and electrochemical activities, can
be doped into the mesopores of silica nanoparticles during the solgel process
[60, 61]. The doping procedures are shown in Fig.10.3. The dopant can enhance
the silica nanoparticle stability and generate or amplify useful biosensing signals
[13, 54, 62, 63].
Tapec etal. [62] utilized the combination of two silica precursors, tetraethy-
lorthosilicate and phenyltriethoxysilane, to synthesize organic dye-doped silica
nanoparticles. The nanoparticles could be synthesized in the nanometer range
with high photostability and minimal dye leakage. The silica matrix of the nano-
particles allowed different surface biomolecular modifications for biosensor and
bioanalysis applications. Biotin interaction of avidin-coated nanoparticles can
be used for the determination of biotinylated bovine serum albumin, and the
immobilization of GDH on the nanoparticle surfaces enabled the determination
of glutamate.
312 10 Biosensors Based on SolGel Nanoparticle Matrices

Fig.10.3 Procedure for the surface modification of dye-doped silica nanoparticles using a water-
in-oil microemulsion (reprinted with permission from Bagwe et al. [61]. 2006, American
Chemical Society)

Zhang et al. [63] developed an amperometric glucose biosensor based on


f errocene-doped silica (FcDS) nanoparticles conjugated with a biopolymer
chitosan (CHIT). The FcDS nanoparticles were prepared by a water-in-oil (W/O)
microemulsion method. The nanosilica surface exhibited high biocompatibility
and the ferrocene doped inside maintained its high electron-transfer efficiency as
a mediator. He etal. [64] reported a method for cell recognition of system lupus
erythematosus (SLE) patients. Photostable luminescent silica nanoparticles pre-
pared with a water-in-oil microemulsion technique was used as biological labels.
Tris (2,2-bipyridyl) dichlororuthenium(II) hexahydrate Ru(bpy) was doped
inside as a luminescent signaling element. The luminescent silica nanoparticles
were covalently immobilized with goat antihuman immunoglobulin G (IgG),
which can recognize SmIgG(+) B lymphocytes. They have used antibody-labeled
nanoparticles to recognize target SmIgG(+) B lymphocytes isolated from the
circulating blood of SLE patients. This bioassay based on fluorescent nanopar-

ticles can identify target cells selectively and efficiently.


Zhang and Zheng [65] synthesized luminol-doped SiO2 nanoparticles
immobilized on the surface of chitosan film-coated graphite electrode by the self-
assembled technique to develop a novel electrogenerated chemiluminescence
(ECL) sensor for pyrogallol. This ECL sensor showed less than a 5% decrease in
continuums over 100 times ECL measurements. The linear range extended from
3.0109 to 2.0105 mol/L for pyrogallol, and with a detection limit of
1.0109mol/L. Qin etal. [66] developed a method of fluorescent nanoparticle-
based indirect immunofluorescence microscopy (FNP-IIFM) for the rapid detec-
tion of Mycobacterium tuberculosis. An anti-M. tuberculosis antibody was used
as primary antibody to recognize M. tuberculosis, and then an antibody binding
protein (Protein A) labeled with Ru(bpy)-doped silica nanoparticles was used to
10.3 Biosensors Based on SolGel Nanoparticle Matrices 313

Fig.10.4 Coimmobilization of HRP and anti-AFP on monodisperse SiO2 spheres (reprinted with
permission from Wu etal. [41]. 2009, American Chemical Society)

generate a fluorescent signal for microscopic examination. The use of the


fluorescent nanoparticles revealed amplified signal intensity and higher photo-
stability than conventional fluorescent dye.

10.3.1.3Chemically Bonded Silica Nanoparticle for Biosensing

Due to some functional groups, for example, hydroxyl residues on the surface, silica
nanoparticle is easily derived through cross-linking [61, 67, 68]. This makes the
biofunctionalization process more convenient and diversified by using some chemi-
cal linkages.
Qian et al. [69] synthesized and characterized silica nanoparticles doped with
Nile red and further conjugated with biomolecules (such as apo-transferrin and folic
acid). In vitro experiments revealed that these functionalized nanoparticles can
serve as effective optical probes for specific targeting of cancer cells such as HeLa
cells. These doped silica nanoparticles may serve as a robust tool for early diagnosis
and therapy of cancer and other diseases. Lius group [41] reported a novel strategy
for the sensitive detection of biomarkers using horseradish peroxidase (HRP)-
functionalized silica nanoparticles as the label, which were fabricated by the
coimmobilization of HRP and r-fetoprotein antibody onto the surface of SiO2 nano-
particles using g-glycidoxypropyltrimethoxysilane (GPMS) as the linkage. The
electrochemical and chemiluminescence measurements showed 29.5- and 61-fold
increases in detection signals, respectively, in comparison with the traditional sand-
wich immunoassay (Fig.10.4).
Endo etal. [70] developed a localized surface plasmon resonance (LSPR)-based
label-free optical DNA biosensor based on gold-capped silica nanoparticles using a
silane-coupling reagent. The detection of PNA-DNA hybridization with target
oligonucleotides and PCR-amplified real samples were performed with a limit of
detection value of 0.677pM target DNA. Selective discrimination against a single-
base mismatch was also achieved. The LSPR-based biosensor was applicable to
monitor the interaction of biomolecules, such as proteins, whole cells, or receptors,
with a massively parallel detection capability in a highly miniaturized package.
In addition, the surface-functionalized mesoporous silica nanoparticle materials
can be readily internalized by animal and plant cells without posing any cytotoxicity
314 10 Biosensors Based on SolGel Nanoparticle Matrices

Fig. 10.5 Transmission electron microscopy images of human cervical cancer (HeLa) cells in
contact with MSN. (a) an MSN entering through the cell membrane. (b) an MSN trapped in vesicles
inside the cell (reprinted with permission from Slowing etal. [71]. 2007, Wiley)

issue in vitro [71]. The functionalization of the external surface of mesoporous


silica nanoparticles with groups for these cells does express specific receptors, like
folic acid, and notably enhances the uptake efficiency of the material by cells
(Fig.10.5).

10.3.1.4Silica Core-Shell Nanostructure for Biosensing

Here, core-shell nanostructures specifically refer to those nanomaterials with


significant core and shell structures, unlike some examples mentioned previously
where nanoparticles are coated with some organic and inorganic layers. In the pres-
ence of impregnated metal or magnetic nanoparticle during the solgel process,
colloidal silica nanoparticles can form a core-shell nanostructure, as shown in
Fig. 10.6. These novel nanostructures can be used as avenues to create generic
approaches for the fabrication of biofunctional materials with magnetic capabilities
to design highly stable and magnetically separable biosensing systems [72].
Many biosensing systems based on this kind of nanostructure have been
reported to date. Zhou and Zhou [73] developed a new class of silica core-shell
nanoparticles that encapsulated with cyanine dyes and applied the dye-doped
nanoparticles as labeling in the DNA microarray-based bioanalysis. The developed
core-shell nanostructure contained 15-nm Au colloidal cores with 95 dye-alkane-
thiol (dT) (20) oligomers chemisorbed on each Au particle surface and 1015-nm
silica coatings bearing thiol functional groups. To be utilized for microarray
detection, the dye-doped nanoparticles were conjugated with DNA signaling
probes by using a heterobifunctional cross-linker. The use of the fluorophore-
doped nanoparticles in high-throughput microarray detection revealed a higher
sensitivity, with a detection limit of 1pM for target DNA in a sandwich hybridiza-
tion and greater photostable signals than the use of organic fluorophore as label.
A wide dynamic range similar to four orders of magnitude was found when the
dye-doped nanoparticles were applied in microarray-based DNA bioanalysis.
10.3 Biosensors Based on SolGel Nanoparticle Matrices 315

Fig.10.6 TEM images of nanoparticle with 13nm Fe3O4 core, and (a) 5 and (b) 20nm silica shell,
(c) TEM image at high magnification of (b) (reprinted with permission from Won etal. [72].
2010, American Chemical Society)

Inaddition, the use of these dye-doped nanoparticles improved the differentiation


of single-nucleotide polymorphisms.
Borchers etal. [74] reported nanostructured core-shell particles with tailor-made
affinity surfaces to form densely packed amorphous nanoparticle layers for the
specific binding of protein ligands from aqueous solution. Biofunctional core-shell
particles consisted of a silica core with a diameter of 100nm and an organic shell a
few nanometers thick. The nanoparticle core was prepared by solgel chemistry,
and the shell was formed in suspension by organosilane chemistry. The shell
provided amino groups or carbonyl groups at its outer surface for the subsequent
covalent immobilization of streptavidin, rabbit IgG antibodies, or goat IgG antibod-
ies. AlexaFluor 647((R))-conjugated and biotinylated cytochrome c and CyDye-
labeled antirabbit IgG and antigoat IgG were probed as model analytes. The
core-shell nanoparticles were spotted using a pin-ring micro-arrayer onto micro-
scope glass slides that were coated with a polycation monolayer by dip-coating
prior to nanoparticle deposition. Amorphous particle layers of well-defined thick-
nesses in the range of 100 nm2 mm were obtained by printing aqueous particle
suspensions containing 5500mg/mL (0.550wt.%) of silica particles. The specific
affinity of the plotted nanoparticulate capture surface was demonstrated by binding
Cy3-labeled donkey antirabbit IgG- and Cy5-labeled mouse antigoat IgG to immo-
bilized rabbit IgG and goat IgG particles. The signal intensity per spot increased for
any given analyte concentration when the amount of particles per spot was aug-
mented. The nanoparticulate microchip surface resulted in a wide dynamic range of
4fM20nM, which covers six orders of magnitude.
Aslan etal. [75] developed versatile highly fluorescent and photostable core-shell
Ag@SiO2 nanocomposites, three different fluorescent probes, including organic
fluorophore (Rh800), lanthanide probe, and organic fluorophore (Alexa 647), cova-
lently linked to the silica shell. Compared to the control fluorescent nanoparticles
(nanobubbles), fluorescent nanoparticles with silver core-silica shell architecture
yielded up to a 20-fold (with Rh800) enhancement of the fluorescence signal.
In terms of nanoparticle detectability for sensing and cellular imaging applications,
a 20-fold increase in fluorescence intensity coupled with a 10-fold drop in lifetime
316 10 Biosensors Based on SolGel Nanoparticle Matrices

afforded a total increased detectability similar to 200-fold as compared to the control


sample nanobubbles containing the same number of fluorophores.
Zhang etal. [76] developed a biofunctional nanoarchitecture by combining the
magnetic iron oxide and the luminescent Ru(bpy)32+ encapsulated in silica. First, the
iron oxide nanoparticles were synthesized and coated with silica, which was used to
isolate the magnetic nanoparticles from the outer-shell-encapsulated Ru(bpy)32+ to
prevent luminescence quenching. Then, onto this core, an outer shell of silica
containing encapsulated Ru(bpy)32+ was grown through the Stber method. Highly
luminescent Ru(bpy)32+ served as a luminescent marker, while magnetic Fe3O4
nanoparticles allowed external manipulation by a magnetic field. Since Ru(bpy)32+
is a typical ECL reagent and it could maintain such a property when encapsulated in
the biofunctional nanoparticle, Zhang etal. explored the feasibility of applying the
as-prepared nanostructure to fabricate an ECL sensor. The prepared ECL sensor
was applied not only to the typical Ru(bpy)32+ coreactant tripropylamine (TPA), but
also to the practically important polyamines. The ECL sensor showed a wide linear
range, high sensitivity, and good stability.
Wang et al. [77] fabricated a composite of C@SiO2 with gold nanoparticles
(Au NPs) by a layer-by-layer assembly technique and used it to fabricate a novel
hydrogen peroxide biosensor. The composite was composed of a colloidal carbon
sphere core with an average diameter of 200nm and a porous silica shell with a
uniform thickness of about 50nm, and decorated with Au NPs on the surface. The
porous silica shell could promote the composite to show high biocompatibility
and chemical stability. The Au NPC@SiO 2 composite combined with Hb was
used to construct a novel biosensor for the determination of H2O2, displaying a
wide linear range from 5.0 to 80mM, with a detection limit of 0.08mM at 3S/N.
The Kmapp value for the biosensor was determined to be 71.49mM.

10.3.2SolGel-Derived Metal Oxide Nanoparticle

Using some metal alkoxide, such as Ti, Al, and Zr, as precursors, through a solgel
process, metal oxide nanoparticles can be obtained. These oxide nanoparticles can
easily adsorb biomolecules for biosensing constructions. This has been discussed in
some review articles [36, 45, 78].
Topoglidis etal. [79, 80] prepared solgel-derived porous TiO2 nanoparticles for
protein immobilization. As optically transparent, semiconductor TiO2 could be used
to carry out direct spectroelectrochemistry of Hb (Fig. 10.7) and cytochrome c,
showing that the immobilized Hb retained its chemical functions of oxidation/
reduction (achieved using chemical oxidants and reductants) and ligand binding
(O2, CO, NO). Such solgel nanoparticle-immobilized Hb films could be used to
determine quantitatively the concentration of these dissolved gases.
Zhang etal. [81] used solgel-derived TiO2 nanoparticle film to immobilize HRP
on a pyrolytic graphite (PG) electrode. The enzyme incorporated in TiO2 nanopar-
ticle film retained its bioactivity. The direct electron transfer between HRP and PG
electrodes was greatly enhanced, indicating that the TiO2 nanoparticle could provide
10.3 Biosensors Based on SolGel Nanoparticle Matrices 317

Fig.10.7 Absorption spectra are shown for both (a) oxidized met Fe(III) and (b) electrochemi-
cally reduced deoxy Fe(II) -immobilized Hb/TiO films (reprinted with permission from Topoglidis
etal. [79]. 2000, Royal Society of Chemistry)

Fig.10.8 Cyclic voltammetric response of TiO2 nanoparticle/HRP biosensor to (A) H2O2 and
(B) dissolved O2 at different concentrations (reprinted with permission from Zhang etal. [81].
2004, Elsevier)

a favorable microenvironment for HRP to exchange electrons with the electrode and
facilitate the rate of electron transportation. Based on the direct electrochemistry of
HRP, a third-generation biosensor for H2O2 and dissolved O2 was presented
(Fig. 10.8). Obviously, in the above case, the biomolecules were immobilized
through direct physical adsorption.
Yu etal. [82] synthesized facilely mesoporous MnO2 through a solgel process
using nonionic surfactant polyxyethylene fatty alcohol as a template. The mesopo-
rous MnO2 material presented a disordered porous structure and an appropriate pore
size suitable for the immobilization of GOD. An amperometric glucose biosensor
based on GOD entrapped in mesoporous MnO2 was fabricated, in which mesopo-
rous MnO2 also acted as a catalyst for the electrochemical oxidation of H2O2
318 10 Biosensors Based on SolGel Nanoparticle Matrices

Fig.10.9 (A) Amperometric response of (a) gelatin/GCE and (b) mesoMnO2gelatin/GCE to the
addition of 0.98mM H2O2; (B) amperometric response of (c) GODgelatin/GCE, (d) mesoMnO2
gelatin/GCE, and (e) GODmesoMnO2gelatin/GCE (reprinted with permission from Yu et al.
[82]. 2008, Elsevier)

p roduced by enzyme reaction. The biosensor showed a fast and sensitive current
response to glucose in the linear range of 0.00092.73mM. The response time (t95%)
was less than 7 s (Fig. 10.9). The sensitivity and detection limit were 24.2 mA/
cm2mM and 1.8107M (S/N=3), respectively, indicating that mesoporous MnO2
has promising application in enzyme immobilization and biosensor construction.
Liu etal. [83] investigated a biosensor based on the use of ZrO2 solgel matrix
for enzyme immobilization in the mild condition. This bioceramic zirconia alcogel
was prepared by the novel alcohothermal route with a cheap inorganic salt
Zr(NO3)45H2O with several desirable features, including a large surface area (about
460m2/g) as well as pore volume and a well-developed textural mesoporosity, and
HRP was selected as a model enzyme. The results showed that the as-prepared zir-
conia matrix has an advantageous microenvironment and a large surface area avail-
able for high enzyme loading. The resulting biosensor exhibited a high sensitivity
of 111 mA/mM for hydrogen peroxide over a wide range of concentration from
2.5107 to 1.5104mol/L, a quick response of less than 10s, and good stability.
Kim etal. [84] developed an amperometric glucose biosensor by using the nano-
porous composite film of solgel-derived zirconia and perfluorosulfonated ionomer,
Nafion, for the encapsulation of GOD on a platinized glassy carbon electrode.
Zirconium isopropoxide (ZrOPr) was used as a solgel precursor for the preparation
of zirconia/Nafion composite film. The glucose biosensor based on the zirconia/
Nafion composite film could reach 95% of steady-state current in less than 5s. In
addition, the biosensor responded to glucose linearly in the range of 0.0315.08mM,
with a sensitivity of 3.40mA/mM and the detection limit of 0.037mM (S/N=3).
Moreover, the biosensor exhibited good sensor-to-sensor reproducibility (similar to 5%)
and long-term stability (90% of its original activity retained after 4 weeks) when
stored in 50mM phosphate buffer at pH 7 at 4C.
10.3 Biosensors Based on SolGel Nanoparticle Matrices 319

Fig.10.10 GNPs embedded in a 3D solgel network (reprinted with permission from Li etal.
[87]. 2010, World Gold Council)

Ansari etal. [85] dip-coated solgel-derived nanostructured tin oxide (Nano-SnO2)


film onto ITO-coated glass for immobilization of GOD. GOD/NanoSnO2/ITO bio-
electrode exhibited a response time of 5s, detection limit of 0.169mg/L, linearity of
10300mg/L, correlation coefficient of 0.899, and sensitivity of 2.687mAL/mgcm2;
moreover, it retained about 80% of its initial response current after 7 weeks. The value
of the MichaelisMenten constant (Km) has been estimated as 0.16mM.

10.3.3SolGel Nanocomposite Matrix for Biosensing

As solgel matrix has a porous structure, nanoparticles can be incorporated inside the
pores in a solgel network to form nanocomposites or hybrids [8688], e.g., as shown
in Fig.10.10. Using organically modified solgel, termed Ormosil, to incorporate
nanoparticles, one can realize the surface assembly of solgel, which supplies an
alternative method for nanoparticle binding to solgel matrix [89, 90]. Moreover,
solgel matrices are highly biocompatible, which do not harm the bioactivities of
biomolecules. Nanoparticles in a solgel matrix can not only realize a better biomol-
ecule immobilization, but also play their own important roles, such as accelerating
electron transfer. In recent years, most reported solgel nanoparticle biosensors have
been built upon nanoparticle incorporation in solgel matrices [9196].

10.3.3.1Metal Nanoparticle SolGel Composite Matrix for Biosensing

Among the metal nanoparticles used in biosensors, Au NPs have received greatest
interest because they have intriguing properties that can be applied to many fields,
320 10 Biosensors Based on SolGel Nanoparticle Matrices

such as electronics, optoelectronics, catalysis, and biology [25, 9799]. Au NPs,


with the diameter of 1100nm, have a high surface-to-volume ratio and high surface
energy to provide a stable immobilization of a large amount of biomolecules while
retain their bioactivity. Moreover, Au NPs have an ability to permit fast and direct
electron transfer between a wide range of electroactive species and electrode mate-
rials. In addition, the light-scattering properties and extremely large enhancement
ability of the local electromagnetic field enable Au NPs to be used as signal ampli-
fication tags in diverse biosensors [87].
Au NPs have been extensively studied as a biosensor construction element [25,
87, 98, 100104]. The combination of Au NPs in a solgel matrix can be realized
through entrapment or self-assembly. With the addition to a sol solution, Au NPs
can be incorporated into the pores of a gel during the gelation process. If precursor
contains a thiol group, hydrolysis can produce a modified sol, to which Au NPs can
be assembled through thiol residue. Through gelation, an organically modified sol
gel matrix, called Ormosil, with the entrapment of Au NPs can be prepared. Au NPs
incorporated in the solgel matrix do not lose their natural properties [105108].
Hence, the biosensors constructed based on Au NPs naturally can be transplanted
into solgel matrices [91, 109, 110].
Based on the change in the electrochemical behavior of enzymatic activity
induced by pesticide, Du etal. [95, 111, 112] developed an electrochemical method
for investigating pesticides using biosensors based on acetylcholinesterase-assem-
bled Au nanoparticle embedded in solgel-derived silicate network. Other enzymes,
such as HRP [105, 113, 114], superoxide dismutase [115], and GOD [116], and
proteins, such as cytochrome c [106, 117] and Mb [118], were immobilized on Au
NPs incorporated in silica solgel matrices to achieve the direct electrochemistry
and hence construct biosensors.
Deng et al. [119] fabricated an ECL NADH and ethanol biosensor based on
partial sulfonation of a solgel network with Au NPs. Fu etal. [108] fabricated a
novel indicator-free DNA biosensor by self-assembling bilayer 2D 3-mercaptopro-
pyltrimethoxysilane, Au NPs, and oligonucleotide on gold substrate and used the
impedance spectroscopy as a platform for reagentless DNA sensing. In addition, the
experiment results indicated that oligonucleotide immobilized this way exhibited
good sensitivity, selectivity, and stability and a long-term maintenance of bioactiv-
ity. Liang etal. [90] developed a hepatitis B surface antigen (HBsAg) immunosen-
sor by self-assembling Au NPs to a thiol-containing 3D silica gel. Hepatitis B
surface antibody (HBsAb) was adsorbed onto the surface of the Au NPs. An inter-
facial design of bare gold electrode (BGE)/MPS/Au/HBsAb was prepared to detect
HBsAg in human serum based on the specific reaction of HBsAb and HBsAg. This
immunosensor system was evaluated on several clinical samples. The analytical
results obtained by this method were in agreement with those detected by the
enzyme-linked immunosorbent assay (ELISA) method.
In addition, Au NPs without biofunctionalization that are embedded in solgel
matrices can also be used directly for biosensing. For example, Liu et al. [120]
proposed an ECL glucose biosensor based on Au NPs-catalyzed luminol ECL. Au
NPs were self-assembled onto a silica solgel network, and then GOD was adsorbed
10.3 Biosensors Based on SolGel Nanoparticle Matrices 321

on the surface of Au NPs. The assembled Au NPs could efficiently electrocatalyze


luminol ECL. The response of the ECL biosensor was linear over the range of
1mM5mM, with a detection limit of 0.2mM glucose, and showed satisfying repro-
ducibility, stability, and selectivity.
Nonsilica matrix, such as titania solgel film, is found to embed Au NPs for
sensingapplications. Buso etal. [121] used TiO2 solgel films doped with Au NPs
as both optical and conductometric sensors for the detection of CO and H2. Tests
showed that CO and H2 induced reversible variations in the optical absorption of
thin TiO2/Au solgel films. The absorbance changes were strongly dependent on
both the testing wavelength and the film microstructure. Together with the sensing
dynamics, it was possible to obtain a better understanding of the mechanisms
involved in the detection of both gases. The presence of H2 elicited almost ideal,
step-like dynamic responses using conductometric detection.
Other transition metal (Ag, Pd, Pt, etc.) nanoparticles have also been reported in
solgel matrices for biosensor construction. Volkan etal. [122] reported the devel-
opment of a surface-enhanced Raman scattering substrate based on silver nanopar-
ticles in solgel. Guo and Tao [123] coated Ag nanoparticledoped silica
nanocomposites on an optical fiber for ammonia sensing. Xu etal. [124] used nano-
sized silver particles to dope into the solgel film to prepare a biosensor.
The silver nanoparticles in the solgel film can adsorb the enzyme molecules and
improve the conductivity of the solgel film. Zuo etal. [96] developed a disposable
glucose biosensor by immobilizing GOD into silver nanoparticle-doped silica
solgel and polyvinyl alcohol hybrid film on a Prussian blue-modified screen-
printed electrode.
Ag nanoparticles embedded in a solgel matrix can also enhance the direct
electrochemistry of protein. Zhao etal. [94] entrapped both Hb and colloidal silver
nanoparticles in a titania solgel matrix by a vapor deposition method on the surface
of a GCE. Silver nanoparticles could greatly enhance the electron-transfer reactivity
of Hb and its catalytic ability toward nitrite. The direct fast electron transfer between
Hb and the GCE was achieved.
Fu et al. [125] developed a method for the fabrication of DNA biosensors by
means of self-assembling colloidal Ag to a thiol-containing solgel network. They
utilized the impedance spectroscopy as a platform for DNA sensing assay. The linear
range of the biosensor was 8.0 1091.0106 M, with a detection limit of
4.0109M at 3s. The experiment results indicated that oligonucleotide immobi-
lized this way exhibited good sensitivity and selectivity, high stability, and a long-
term maintenance of bioactivity. Cioffi et al. [126] embedded Pd nanoparticles in
SnOx films through the solgel technique to fabricate chemiresistor-type sensing
devices (Fig.10.11).

10.3.3.2Oxide Nanoparticle SolGel Matrix for Biosensing

Similar to metal nanoparticles, oxide nanoparticles can also be entrapped in the


solgel 2D or 3D network. Cheng etal. [127] investigated the promotion effect of
322 10 Biosensors Based on SolGel Nanoparticle Matrices

Fig.10.11 Detection of ethanol vapors by a Pd/SnOx (2%) chemiresistor. Top: typical response
curve; Bottom: sequence of analyte injections in the measuring chamber (reprinted with permis-
sion from Cioffi etal. [126]. 2006, Elsevier)

titania nanoparticles (nano-TiO2) on the direct electron transfer between LDH and
the silica solgel-modified gold electrode by adding nano-TiO2 (50nm) in the modi-
fication process. This nano-TiO2-LDH electrode showed a pair of quasi-reversible
cyclic voltammetry peaks with the formal potential of 70mV (vs. SCE). Compared
to the pure LDH solgel-modified electrode, on this nano-TiO2-LDH solgel elec-
trode, results demonstrated that the direct electrochemistry of LDH was enhanced
by nano-TiO2. This electrode can be used as a biosensor for the determination of
lactic acid. The calibration range of lactic acid was 1.020mmol/L, and the detec-
tion limit was 0.4mmol/L. Meanwhile, the small Kmapp value (2.2mmol/L) suggested
that LDH possessed high enzymatic activity and good affinity to lactic acid due to
the promotion effect of nano-TiO2.

10.3.3.3Carbon Nanotube SolGel Matrix for Biosensing

Carbon nanotubes are widely used for biosensor constructions [104, 128, 129]. The
presence of carbon nanotubes in solgel can facilitate electron transfer [92]. On the
other hand, the carbon/sol composite, called carbon ink, can be easily filled into the
tubes of different shapes or screened onto surfaces [130] (Fig.10.12). As the gela-
tion process is going, carbon nanomaterials, biofunctionalized or not, can be incor-
porated in the solgel matrix, to supply a versatile platform for biosensor construction.
Moreover, the composite surface can be renewed through polish, which provides a
chance to fabricate disposable biosensors, which are convenient for everyday life.
Many researchers use carbon nanotube solgel composite to construct biosen-
sors, especially electrochemical biosensors [131134]. For example, based on
CNTs self-catalytic activity, Abbaspour and Ghaffarinejad [131] used microwave
10.3 Biosensors Based on SolGel Nanoparticle Matrices 323

Fig.10.12 (a) Eight biogel-based strip electrodes produced by a screen-printing process; (b) SEM
image of the strip electrodes in (a) (reprinted with permission from Wang etal. [130]. 1996,
American Chemical Society)

irradiation for the preparation of a solgel-derived multiwall CNT ceramic electrode


(MW-CNCE) to investigate the electrochemical behavior of MW-CNCE in nicotin-
amide adenine dinucleotide (NADH), l-cysteine, adenine, and guanine, which was
successfully used for the oxidation of adenine and guanine in DNA.
Surface-renewable and mechanically rigid CNT ceramic composite electrodes
(NCE), reported by Zhu etal. [132], could be fabricated by dispersion of multiwall
carbon nanotubes in solgel solution. The electrochemical behavior of such elec-
trodes has been compared with graphite ceramic composite electrodes (CCE) and
evaluated with respect to the electrochemistry of some biological molecules, such
as ascorbic acid (AA), uric acid (UA), acetaminophenol (AP), b-nicotinamide ade-
nine dinucleotide (NADH), epinephrine (EP), dopamine (DA), cysteine (CySH),
and hydrogen peroxide with cyclic voltammetry. In all cases, NCE provided better
reversibility, with a substantially decreased overpotential, better-defined peak shape,
and higher sensitivity compared with CCE (Table10.1).
Choi etal. [135] developed a highly sensitive and stable Ru(bpy)32+ ECL sensor
based on CNT dispersed in mesoporous composite films of solgel titania and per-
fluorosulfonated ionomer (Nafion). The hydrophobic CNT in the titaniaNafion
composite films coated on a glassy carbon electrode increased the amount of
Ru(bpy)32+ immobilized in the ECL sensor, the electrocatalytic activity toward the
oxidation of hydrophobic analytes, and the electronic conductivity of the composite
films. Therefore, the present ECL sensor based on the CNTtitaniaNafion showed
improved ECL sensitivity for TPA compared to the ECL sensors based on both
titaniaNafion composite films without CNT and pure Nafion films.
Alternatively, carbon nanomaterials entrapped in the solgel network can be
functionalized. Wang et al. [136] published a third-generation H2O2 biosensor in
which HRP was immobilized by the solgel technology on CNT-modified elec-
trode, which promoted effects on the direct electron transfer between HRP and the
electrode surface. Tan etal. [93] presented an amperometric cholesterol biosensor
by immobilizing cholesterol oxidase (COX) and Prussian blue (PB) on MWNTs in
324 10 Biosensors Based on SolGel Nanoparticle Matrices

Table10.1 Summary of the cyclic voltammetric data for several redox systems at
NCE and CCE
NCE CCE
Ep (mV) Ip (mA) Ep (mV) Ip (mA)
CySH 0.432 9.951 0.632 3.991
AA 0.161 21.22 0.242 11.94
UA 0.335 9.032 0.370 6.807
DA 0.176/0.123 13.99/2.723 0.193/0.130 8.654/0.6477
EP 0.430/0.09 63.94/40.22 0.460/0.070 43.02/22.61
AP 0.385/0.249 32.34/16.22 0.434/0.098 18.78/9.882
Reprinted with permission from Zhu etal. [132]. 2007, Elsevier

Fig.10.13 (A) Typical currenttime response curve and (B) calibration curve for successive addi-
tion of 0.5 mM cholesterol using (A) GC/PB/CS-SiO2-COX and (B) GC/PB/CS-SiO2-COX-
MWCNT electrode (reprinted with permission from Tan etal. [93]. 2005, Elsevier)

solgel chitosan (CS)/silica organicinorganic hybrid composite. The analytical


performances of the biosensor, including response time, sensitivity, linear range,
limit of detection, and apparent MichaelisMenten constant, can be improved
greatly after the introduction of MWNTs (Fig.10.13).
Similar to nanoparticles, carbon nanotubes in solgel matrices can accelerate the
electron transfer between the proteins and electrodes surface, and thus can be used
for biosensor constructions. For example, Chen and Dong [137] fabricated a solgel-
derived ceramic-carbon nanotube (SGCCN) nanocomposite film by doping MWNTs
into a silicate gel matrix and used it to immobilize protein. The SGCCN film can
provide a favorable microenvironment for HRP to perform direct electron transfer at
a glassy carbon electrode. The HRP immobilized in the SGCCN film showed a pair of
well-defined redox waves and retained its bioelectrocatalytic activity to the reduction
of O2 and H2O2, which was superior to that immobilized in silica solgel film.
Lim et al. [138] encapsulated bilirubin oxidase within a silica solgel/carbon
nanotube composite to catalyze the reduction of molecular oxygen into water
10.3 Biosensors Based on SolGel Nanoparticle Matrices 325

through direct electron transfer at the carbon nanotube electrode surface. In this
nanocomposite approach, the silica matrix was designed to be sufficiently porous
for substrate molecules to have access to the enzyme and yet provided a protective
cage for immobilization without affecting biological activity. The incorporation of
carbon nanotubes enhanced the electrical connectivity and increased the active elec-
trode surface area. The carbon nanotube sidewalls are primarily responsible for
direct electron-transfer processes.

10.3.3.4Multinanoparticle SolGel Matrix for Biosensing

In some cases, very complex solgel composites, which contain two or more kinds
of nanoparticles, are used for biosensor fabrication. Liu and Hu [139] fabricated an
H2O2 biosensor based on the direct electrochemistry and electrocatalysis of Mb
immobilized on silver nanoparticle-doped carbon nanotube film with hybrid solgel
techniques. A pair of redox peaks with peak separation of 160mV and formal poten-
tial of 0.295V was observed at this composite film, corresponding to the direct
electrochemistry of Mb. Under optimum conditions, the amperometric determination
of H2O2 was performed, with a linear range of 2.0 1061.2103 mol/L and a
detection limit of 3.6107mol/L (S/N=3). The MichaelisMenten constant was
also estimated to be 1.62mmol/L.
Kang et al. [24] presented a sensitivity-enhanced glucose biosensor based on
multiwalled carbon nanotubes, Pt nanoparticles (Pt NPs), and a solgel of chitosan
(CS)/silica organicinorganic hybrid composite. The CS/silica hybrid solgel was
produced by mixing methyltrimethoxysilane (MTOS) with the CNTPt NPCS
solution. With the immobilization of GOD into the solgel, the glucose biosensor of
GODCNTPt NPCSMTOSGCE was fabricated. The properties of the result-
ing glucose biosensor were measured by electrochemical impedance spectroscopy
and cyclic voltammetry. In phosphate buffer solutions (PBS, pH 6.8), the nearly
interference-free determination of glucose was realized at a low applied potential of
0.1V, with a wide linear range of 1.21066.0103M, a low detection limit of
3.0107M, a high sensitivity of 2.08mA/mM, and a fast response time (within
5s). The biosensor provided a highly synergistic electrocatalytic action, and exhib-
ited good reproducibility and long-term stability.
Guo etal. [140] employed an approach combining sonication and solgel chem-
istry to synthesize silica-coated CNT coaxial nanocables. It was found that a homo-
geneous silica layer can be coated on the surface of the CNTs. Au NPs-supported
coaxial nanocables were facilely obtained using amino-functionalized silica as the
interlinker. Furthermore, to reduce the cost of Pt in fuel cells, designing a Pt shell on
the surface of a noble metal such as gold or silver is necessary. High-density gold/
platinum hybrid nanoparticles were located on the surface of 1D coaxial nanocables
with high surface-to-volume ratios. It was found that this hybrid nanomaterial
exhibited a high electrocatalytic activity for enhancing oxygen reduction (a low
overpotential was associated with the oxygen reduction reaction and an almost four-
electron electroreduction of dioxygen to water).
326 10 Biosensors Based on SolGel Nanoparticle Matrices

Huang et al. [141] used platinum nanoparticles to develop an amperometric


l-lactate biosensor based on the immobilization of l-lactate oxidase through silica
solgel film on multiwalled carbon nanotube/platinum nanoparticle-modified
GCE. Yang etal. [142] prepared platinum nanoparticle-doped solgel solution and
used as a binder for multiwalled CNT for the fabrication of electrochemical sensors.
Solgel solution containing an amine group was selected for stabilizing the nano-
particles in solution. The resulting CNT-silicate material brought new capabilities
for electrochemical devices due to the synergistic action of the electrocatalytic
activity of Pt nanoparticles and CNT. The combined electrocatalytic activity per-
mitted the low-potential detection of hydrogen peroxide with remarkably improved
sensitivity. With the incorporation of GOD within the PtCNTsilicate matrix, a
PtCNT paste-based biosensor was constructed, which is more sensitive to glucose
than a CNT-based biosensor.

10.4Conclusions

The solgel technique plays an important role in biosensor construction based on a


nanoparticle matrix. It is the most widely used technique to prepare various nano-
particles. Sols themselves are dispersions of colloidal particles with diameters of
1100nm in liquids, which can be used directly or coated on a substrate though
spinning, dipping, and spraying to get a nanoparticle film. In addition, colloidal
particles can be isolated from sols by drying, centrifugation, coprecipitation, and so
forth. Additionally, covering a complete solgel process, through hydrolysis, gela-
tion, aging, drying, stabilization, and densification at high temperature, can also
produce nanoparticles from xerogel or aerogel.
On the other hand, the solgel matrix has a porous structure. It can be used as a
support backbone for nanoparticle incorporation in a solgel network to form nano-
composites or hybrids, which supply an alternative method for nanoparticle binding
to a solgel matrix. Moreover, solgel matrices are highly biocompatible, which do
not harm the bioactivities of biomolecules. Compared to pure solgel, nanoparticles
in a solgel matrix cannot only realize a better biomolecule immobilization, but also
play their own important roles to accelerate electron transfer.

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Chapter 11
Nanostructure for Nitric Oxide Electrochemical
Sensing

11.1 Introduction

Since nitric oxide (NO) was biologically identified as an endothelium-derived relaxing


factor in 1987 [1], there has been a great increase in the research of its chemistry,
biology, and therapeutic actions. In 1992, NO was declared molecule of the year
in Science [2]. Since then, in addition to acting as the molecular messenger and
vasodilator, NO has been found to be involved in a wide range of biological pro-
cesses, including penile erection [3], neurotransmission [4], inhibition of platelet
aggregation and immune response [5]. In 1998, in recognition of their contribution
to the field of NO research, three American scientists, Robert F. Furchgott, Louis J.
Ignarro, and Ferid Murad, who first unraveled the complex nature of this simple
molecule, were jointly awarded the Nobel Prize for Physiology or Medicine. Due to
the importance of NO in biology, it is very important to accurately measure the con-
centration of NO in situ and in real time. However, its short half-life (~6s) and high
reactivity with biological compounds such as superoxide, oxygen, thiols and others,
made the detection of NO very difficult. Many different techniques have been devel-
oped for the detection of NO in biological samples, including chemiluminescence,
paramagnetic resonance spectrometry and imaging, and bioassay and electrochemi-
cal sensors [2]. Among them, electrochemical sensors are of significance in NO
detection because of its high sensitivity, good selectivity, fast response time, and
long-term stability. In addition, these sensors are the only available approach for in
situ and real-time detection. In 1992, World Precision Instruments (WPI) made the
first commercial electrochemical NO detection system, ISO-NO. Since then, they
have continued to develop highly specialized and sensitive NO electrodes to detect
the NO in a very small volume or single cell. In 2002, they successfully fabricated
a nanometer-sized sensor with a tip diameter of just 100nm [6]. In addition to mak-
ing extremely small NO electrochemical sensors, extensive efforts have been directed

H. Ju et al., NanoBiosensing: Principles, Development and Application, 333


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0_11, Springer Science+Business Media, LLC 2011
334 11 Nanostructure for Nitric Oxide Electrochemical Sensing

to improve the sensitivity and selectivity through electrode surface modification


using nanomaterials. In this chapter, the nanostructure of the NO sensors will be
addressed in the aseptic manner of nanometer-sized electrode and nanomaterials
used for the modification of electrode.

11.2 Nanostructure for Nitric Oxide Determination

NO electrochemical sensors have been fabricated and utilized to measure NO for-


mation and release from cells [7, 8], in brain slices [9, 10], in renal tubule [11],
from the saphenous vein [12], and in rat hearts [13]. However, the signal transduc-
tion mechanisms of NO have not been understood yet. To clarify these mecha-
nisms, the kinetics and localization of NO release and its action targets within a cell
need to be studied. This has attracted much interest in the manufacturing of an NO
sensor to investigate the NO in single cells or ultrasmall volumes [6, 14, 15]. The
ultra-micro-electrodes need to be developed to monitor NO in extremely small
spaces in biological microenvironments, such as single cells or certain locations in
tissues. The carbon fiber microelectrode with a diameter of several micrometers
has great advantages over other electrodes because of its ultrasmall size and large
electrode area. In addition, the fast response time(msms) and excellent biocom-
patibility of the carbon fiber microelectrode make it a powerful tool for the real-
time monitoring of biological events. The shape of the carbon fiber microelectrode
can be cylindrical, which can offer a high response to NO due to its large electrode
area. The shape of the carbon fiber microelectrode can also be a microdisk, which
can provide accurate information about the NO concentration at the spot where it
is positioned, avoiding the average of the signal as a cylindrical shape [14, 16].
However, the nanometer size of the carbon fiber electrode was only realized by its
cylindrical shape.
In 1996, Zhang etal. reported the carbon fiber cone nanometer-sized electrodes
with total tip sizes as small as 50 nm [17]. The electrode was fabricated by the
carbon fiber etched by an argon ion beam, which has higher mechanical properties
than that of the carbon fibers etched by flames. However, the overall physical tip
dimensions with large insulating and supporting electrodes are in the millimeter
range and cannot be used for measurement at the single-cell level. In 2002, Zhangs
group reported a nanometer-sized electrode for nitric oxide with a tip diameter of
just 100nm [6]. This NO sensor consisted of a single etched carbon fiber working
electrode combined with an Ag/AgCl reference electrode. Figure11.1 demonstrates
the structure of this nanometer-sized electrode.
To prepare this integrated nanometer-sized NO sensor, the carbon fibers are first
etched with an argon beam to obtain sharpened tips with diameters ranging from
100nm to several hundred nanometers. The etched carbon fiber is mounted on the
end of a copper wire and fixed by silver epoxy. The mounted carbon fiber silver wire
is inserted into a glass capillary and sealed with epoxy. A 1.5-cm Ag/AgCl layer is
coated on the tip of the capillary to serve as the reference electrode. Such a design
11.2 Nanostructure for Nitric Oxide Determination 335

Fig.11.1 Schematic drawing of an integrated NO nanoelectrode showing the surface coated with
Nafion (dashed line) and WPI selective membrane (solid line). Reprinted with permission from
Zhang etal. [6], 2002, Elsevier

Fig.11.2 Typical amperograms at +860mV for additions of 50mM (AA), 50mM nitrite, 100mM
l-arginine (l-arg), 10mM DA, 100nM NO, and 200nM and 400nM NO, respectively, in a stirred,
saturated CuCl solution with an NO nanosensor coated with Nafion/WPI membranes. Reprinted
with permission from Zhang etal. [6], 2002, Elsevier

enables a two-electrode configuration to be free in the use of an external reference


electrode. Leaving 2mm of Ag/AgCl exposed at the very tip, the remainder of the
Ag/AgCl is coated with insulation and further coated with carbon ink to form a
shielding layer. This shielding layer can eliminate the electronic noise and therefore
enhance the detection limit to a low-nM range. The sensor tip is dipped into a 3%
Nafion solution three times each for 20s and dried at 90C for 20min to form the
perm-selective coating membrane. The negatively charged Nafion layer can elimi-
nate the interference of anionic molecules, such as ascorbic acid and nitrite.
Furthermore, it can stabilize the NO+ formed upon the oxidation of NO and prevent
the formation of nitrite and nitrate. However, Nafion cannot eliminate the interfer-
ence of cationic molecules such as dopamine, serotonin, and neutral molecules
(e.g., hydrogen peroxide, acetaminophen, etc.).
In 2000, the same group reported the use of the hydrophobic WPI coating mem-
brane to improve the selectivity of the microelectrode sensor [18]. The Nafion-
coated electrode was immersed in the 5% WPI membrane in acetone solution two
times each for 30s to form the hydrophobic coating layer. Figure11.2 shows the
336 11 Nanostructure for Nitric Oxide Electrochemical Sensing

Fig.11.3 Amperometric response of a Nafion/WPI membrane-coated NO microsensor upon the


addition of 400nM NO and 1mM of oxyhemoglobin to a stirred 0.1M phosphate buffer solution
(pH 7.4). Reprinted with permission from Zhang etal. [6], 2002, Elsevier

response of the NO nanosensor to NO and other interference species. It can be seen


that 50mM ascorbic acid, 10mM dopamine and nitrite have no effect on the mea-
surement of NO.
The response time is critical for NO sensors for invivo measurement, since the
NOs half-life is on the order of seconds. The thickness of Nafion and the WPI
selective membrane, which can decide the diffusion rate of NO, is essential for the
response time of the NO nanosensor. Figure11.3 shows the 90% response time of
the sensor to a step from 0 to 400nM NO and injection of 1mM of oxyhemoglobin
with a filter. It indicates that the response time was less than 5s, which was similar
to the microsensor and within the timeframe required to measure NO. The linear
range of the NO nanosensor was excellent, at a 101000 nM concentration range.
The sensitivity of the nanosensor was approximately 0.5pA/nM. The lower limit of
determination (defined as a signal-to-noise ratio of 2) was about 2nM, which was
lower than that of the microsensor.
This nanosized NO electrode, with a wide linear range, high selectivity, fast
response time, low detection limit, and high sensitivity, can be potentially applied
for the accurate, real-time, selective detection of NO, which is required at the
single-cell and micro-capillary levels.

11.3Nanomaterials for Modification of NO


Electrochemical Sensors

Besides the ultrasmall size of the electrodes that need to be developed, the sensitivity
and selectivity should be improved by modifying the electrodes. Malinski and Taha
developed a sensitive NO sensor by modifying the carbon fiber electrode with
Ni-porphyrin and Nafion and found that Ni-porphyrin had a catalytic effect on the
electrooxidation of NO [19]. Since then, many materials have been found to catalyze
11.3 Nanomaterials for Modification of NO Electrochemical Sensors 337

the NO electrooxidation and used to modify the electrode for an improvement in


sensitivity. Due to their small size and large surface area, nanomaterials such as multi-
walled carbon nanotubes (MWNTs), single-walled carbon nanotubes (SWNTs), gold
nanoparticles, nanoTiO2, TiO2Au nanocomposite, nano-alumina, poly-CuTAPc
(copper metal tetraaminophthalocyanine), and nanoSnO2 have been widely used as
mediators to catalyze the reduction or oxidation of NO. The main role of these nano-
materials as the modifiers of the electrode is to improve the sensitivity and selectivity
of the electrochemical detection of NO. Generally, the electrocatalytic properties of
nanoparticles can enhance the peak current of NO and lower the potential peak.
Carbon nanotubes have been widely used as scanning probes, batteries, electron
field emission sources, nanoelectronic devices, and chemical sensors [2024]. Two
forms of carbon nanotubes exist: SWNTs and multi-walled carbon nanotubes.
Carbon nanotubes can promote electron-transfer reaction when used as an electrode
in an electrochemical reaction due to their subtle electronic properties [25].
Furthermore, carbon nanotubes have a high porous structure and small dimension,
which provide a new application in the electrode surface modification to design new
electrochemical sensors and novel electrocatalytic materials. Both MWNTs and
SWNTs have been studied for the electrocatalytic oxidation of NO [2628]. An
obvious anodic peak at +0.72V on the cyclic voltammogram of NO is observed on
the multi-walled carbon nanotubes modified glassy carbon (GC) electrode with
Nafion coating. This is similar to a poly-CuTAPc-modified electrode, which sug-
gests that MWNTs can act as a new electrode material to catalyze the oxidation of
NO. The peak current is observed to increase significantly, which may be due to the
large surface area and good adsorptive property of MWNTs for NO. The mecha-
nism of NO oxidation at the MWNT-modified electrode is assumed: Nitric oxide is
absorbed by carbon nanotubes first, and then it loses an electron to the electrode and
NO is oxidized. The MWNT-modified electrode showed a good linear relationship
in the NO concentration range of 0.2150mL. The detection limit has been esti-
mated to be 80nM [26]. In order to prepare an ultra-microelectrode for the determi-
nation of NO in a biological system, the MWNT-modified carbon fiber
ultra-microelectrode was fabricated by Wang etal. to determine NO in liver mito-
chondria [27]. The oxidation peak potential of MWNT/Nafion-modified carbon
fiber is at +0.78V, with the linearity of 0.286mM. The MWNT has also been stud-
ied with other polymers to enhance electrocatalytic abilities.
Zheng etal. reported that the cofunction of MWNTsACB (azocarmine B) and
PACB (polyazocarmine) made the oxidation peak current of NO increase greatly
[28]. The MWNT was noncovalently functionalized with ACB and cast onto the
glassy carbon electrode. The surface-modified electrode was further covered by
PACB via electropolymerization and followed by Nafion solution casting. The dif-
ferential pulse voltammograms of the obtained Nafion/PACB/MWNTACB/glassy
carbon electrode showed a much higher peak current compared to Nafion/MWNT/
glassy carbon electrode or Nafion/PACB/glassy carbon electrode. The PACB can
decrease MWNTs proton-donor ability and lead to the enhancement of its electro-
catalytic ability. The current response of the Nafion/PACB/MWNTACB/glassy
carbon electrode exhibited an excellent linear relationship with the concentration of
338 11 Nanostructure for Nitric Oxide Electrochemical Sensing

Fig.11.4 Formation of the NO sensor and measurement of NO in rat liver cells. Reprinted with
permission from Zheng etal. [28], 2008, Elsevier

NO in the range of 0.22120mM. The sensitivity and the determination limit were
0.25mA/(mmol/L) and 28nM, respectively. The sensor also showed good selectiv-
ity, stability (95% of its initial response remained after being kept for one week),
and reproducibility (parallel detection 10 times, with a relative standard deviation of
2.1%). This sensor was further used for measuring NO released from rat liver cells,
and results showed that the sensor could have practical applications in an NO moni-
toring system (Fig.11.4).
Not only carbon nanotubes, but also the organic nanotubes, such as Poly-
CuTAPc nanotube, can catalyze the electrooxidation of NO. Phthalocyanines are
weakly semiconducting organic dye materials and can be substituted with different
metals. CuPc is one of the most interesting MPc materials studied in the literature.
The structure of CuTAPc is demonstrated in Fig.11.5. Poly-CuTAPc has been used
for the determination of oxidizing gases due to the good p-electron donating prop-
erties of the Pc rings [29, 30]. Poly-CuTAPc is electropolymerized on the surface
of the electrode for NO sensor application [31]. Poly-MTAPc nanotubes are pre-
pared through Pt-coated anodic aluminum oxide (AAO) template-assisted elec-
tropolymerization. The SEM pictures of the synthesized Poly-MTAPc nanotubes
after the Pt and AAO have been washed out are shown in Fig.11.6. The oxidation
peak of NO on the Nafion/poly-CuTAPc nanotube-modified electrode is at +0.72V,
which is much lower than the values for bare Pt or carbon fiber electrodes
(0.90.95V). The negative shift in oxidation potential implied that poly-CuTAPc
11.3 Nanomaterials for Modification of NO Electrochemical Sensors 339

Fig.11.5 Copper metal


tetraaminophthalocyanine
(CuTAPc). Reprinted with
permission from Gu etal. [33],
2008, IOP

Fig.11.6 FE-SEM images of poly-CuTAPc nanotubes [A, B top views]. Reprinted with permis-
sion from Gu etal. [31], 2009, IOP

acts as a catalyst in the NO oxidation process. The electrooxidation process was


proposed as follows [32]:

[M(II) TAPc] + NO [M(II) TAPc](NO)


[M(II) TAPc](NO) [M(II) TAPc](NO)+ + e

[M(II) TAPc](NO)+ [M(II) TAPc]+N O +

The differential potential voltammograms show two linear concentration ranges of


0.11.0 and 1.010mM on the Nafion/Poly-CuTAPc nanotube-modified electrode.
Compared with the lower concentration range, the oxidation potential at a higher
concentration range is found to be slightly positively shifted to 0.76V and the peak
shapes are less sharp and distinct. A detection limit as low as 20nM is obtained with
the Nafion/poly-CuTAPc-modified electrode by the voltammetric analysis.
340 11 Nanostructure for Nitric Oxide Electrochemical Sensing

Gold nanoparticles have been applied in electrocatalysis for biosensor devices.


They also have been used for electrocatalysis toward the oxidation of NO. The gold
nanoparticles can be self-assembled on the glassy carbon electrode or incorporated to
polyelectrolyte film with the aid of 4-(dimethylamino) pyridine stabilization [34, 35].
At the Au nanoparticle/polyelectrolyte film-modified ITO electrode, the oxidation
peak occurs at +0.82V, the linear dependence of the peak current on NO is in the
range from 0.05 to 0.5mM, with a detection limit of about 0.010mM. Zhu etal. stud-
ied the similar electrocatalytic behavior of NO at a platinum electrode modified by a
monolayer of gold nanoparticles and Nafion [36]. The bilayer self-assembly nanopar-
ticle-modified GC electrode also showed the electrocatalytic behavior of NO. The
linear concentration range of NO was 0.0510mM, and the detection limit was 27nM,
which was much lower than the Au nanoparticle/polyelectrolyte film composite [34].
The nanocrystalline TiO2 can also act as the electrocatalysis for the oxidation of
NO. A nano TiO2/Nafion composite film-modified GC electrode demonstrates a
potential peak at 0.64V, which is due to the good electrocatalytic activity of TiO2 and
the stabilization of NO+ by Nafion film to prevent the formation of NO2 and NO3.
The peak current of the modified electrode is found to be linear in the range of 0.36
54mM [37]. Due to the photovoltaic properties, nano titanium dioxide can also effi-
ciently enhance the enzyme activities of the proteins to some extent, which is useful
for developing more sensitive biosensors. Hemoglobin (Hb) can be used to detect
nitric oxide. Hb solution (6 mg/ml) is mixed with nano titanium dioxide solution
(1mg/mL), and the mixture is cast onto the surface of the pyrolytic graphite electrode
to form a uniform film [38]. Before detecting NO, nano TiO2Hb film has been irradi-
ated by UV for 5h to activate Hb by the photovoltaic effect of nano TiO2, which then
shows a reduction peak at 0.78V. The reduction peak current is linear with the NO
concentration in the range of 5.0400mM. The detection limit is 1mM [38]. However,
when TiO2 is incorporated with an Au nanoparticle, it has been implied that TiO2 acts
only as an inert mesoporous support, while Au nanoparticles electrocatalyze the oxi-
dation of NO [39]. The TiO2Au nanocomposite film electrode is prepared by a layer-
by-layer deposition procedure [39]. The cyclic voltammograms show that the current
observed on the TiO2Au nanocomposite electrode is 670mA, which is much higher
than the current peak observed on the gold disk electrode, which is 14 mA. The
increase in anodic peak current is consistent with the increase in the active gold nano-
particle surface. The qualitative experiments conducted with only gold nanoparticles
in the absence of TiO2 gave similar current peaks with the TiO2Au nanocomposite,
which clearly suggested that TiO2 acted only as an inert mesoporous support. All
these results implied that the Au nanoparticles could electrocatalytically oxidize nitric
oxide, and the mechanism was proposed as the following process:

NO(aq) NO(Au)
NO(Au) NO + (Au) + e -1
NO + (Au) NO + (aq)
NO + (aq) + H 2 O NO 2 - (aq) + 2H + (aq)
4NO + (Au) + 2OH - (Au) 4NO(Au) + O 2 (aq) + 2H + (aq)

11.3 Nanomaterials for Modification of NO Electrochemical Sensors 341

Table11.1 List of nano-materials for the determination of NO by an electrochemical sensor


Modifiers inner/outer Working potential/ Detection Linear range Lifetime
layer reference electrode limit (nM) (mM) (days) Ref.
Nafion/MWNTACB/ +0.72V/SCE (oxidation) 28 0.22120 [28]
PACB/GC
Nafion/MWNT/GC +0.78V/SCE (oxidation) 20 0.286 [27]
Nafion/Nano-TiO2/GC 0.67V/SCE (oxidation) 54 0.3654 10 [37]
Myoglobin/MWNT/GC 0.7V/SCE (reduction) 80 0.240 77 [41]
Nano Au bilayer/GC +0.86V/SCE (oxidation) 27 0.0510 [34]
Nafion/Nano Au/Pt +0.74V/SCE (oxidation) 50 0.140 50 [36]
Nano-Al2O3/GC +0.69V/SCE (oxidation) 7.2 0.04164 14 [40]
Nano-TiO2/ 0.78V/SCE (reduction) 1,000 5.0400 [38]
hemoglobin/PG

Nano-alumina has also attracted much attention in electrochemical sensors and


electroanalysis. The nano-alumina-modified glassy carbon electrode shows a sensi-
tive oxidation peak at +0.69V in the presence of NO [40]. The mechanism of NO
oxidation at the nano-Al2O3 assumes that NO is absorbed by nano-Al2O3 film fist,
which is then oxidized by losing an electron to the electrode. The linear range of the
sensor is 0.04210mM. The sensitivity and detection limit are 0.037mA/mM and
7.2nM, respectively [40].
The linear ranges and detection limits of various nanomaterials modified elec-
trodes is summarized in Table11.1.
The nanomaterial-modified ultrasmall electrodes have also been fabricated to
determine the NO from small-volume or single cells, such as MWNT- or SWNT-
modified carbon fiber microelectrodes. An SWNT-modified carbon fiber microdisk
electrode was reported to monitor NO release from single cells in real time [14].
Figure11.7 demonstrates the process of modifying the carbon fiber microelectrode
and the SEM images of the modified microdisk electrode. To modify the surface of
the carbon fiber microdisk electrode, Du etal. developed a novel method that can
avoid modifying the whole surface of the sensor. The bare carbon fiber microdisk
electrode was hung vertically above the SWNT-modifying solution with Ag/AgCl
as the reference electrode [Fig.11.7a (a)]. The double-pulse test, with a width of
5ms and an amplitude of 10mV, was started and administered to the carbon fiber
microdisk electrode. There was no current flow to Fig. 11.7a (a), as shown in
Fig.11.7a (b), because of the open circuit. When the carbon fiber microdisk elec-
trode moved downward to touch the solution [Fig.11.7a (c)], the electric circuit was
complete, which resulted in the obvious change in the current trace [Fig.11.7a (d)].
When the carbon fiber microdisk electrode was lifted up [Fig.11.7a (e)], no current
was observed, as in Fig.11.7a (f). The dipping time was around 1s, and then the tip
was dried in the air and the same procedure was repeated twice. After the modifica-
tion of SWNTs, the Nafion coating was applied using the same method with 0.5%
Nafion-ethanol solution. From the SEM images in Fig.11.7b (a, b), it could be seen
that an SWNT/Nafion-modified carbon fiber microdisk electrode was more rough
than the insulated bare carbon fiber microdisk electrode, which increased the surface
area of the electrode.
342 11 Nanostructure for Nitric Oxide Electrochemical Sensing

Fig.11.7 (a) (a, c, e) Schematic diagram illustrating the process of modifying the carbon fiber
microdisk electrode (CFMDE). (b, d, f) The signal display provided by an oscilloscope-like win-
dow in PLUSE corresponding with three processes in (a), (c), and (e), respectively. (b) SEM
images of (a) the surface of a CFMDE and (b) the surface of an SWNT/Nafion modified CFMDE.
Reprinted with permission from Du etal. [14], 2008, Elsevier

The cyclic voltammograms of bare or SWNT/Nafion-modified carbon fiber micro-


disk electrode showed that the limiting current of the NO oxidation on the modified
electrode was about five times higher than on the bare one. The detection limit of the
SWNT/Nafion-modified carbon fiber microdisk electrode for NO was 4.3nM, which
was much smaller than the platinum-deposited carbon fiber microdisk electrode
11.3 Nanomaterials for Modification of NO Electrochemical Sensors 343

Fig.11.8 (a) View of the calibration system to calibrate the CFMDE modified by SWNTs and
Nafion with NO solution of different concentrations. The microcapillary and the microelectrode
were both placed in a chamber filled with 1mL of PBS. The PBS was aerated to be in accordance
with physiological conditions in cell experiments. (b) View of the detection system in single-cell
experiments. A CFMDE modified by SWNTs and Nafion was placed at a fixed distance (generally
1 mm) away from the surface of a cell (HUVEC, human umbilical vein endothelial cells). The
outlet of the microcapillary rightly faced the cell to inject a stimulus toward it. Reprinted with
permission from Du etal. [14], 2008, Elsevier

(100nM) [16]. The calibration system with NO solution of different concentrations


is shown in Fig.11.8(a). The linear response for NO was tested in the concentration
range of 0.10.8mM. The response time of the sensor was only 125ms when the
response time was defined as the interval between 10 and 90% of the maximum cur-
rent. The SWNT/Nafion-modified electrode was used to detect the NO release from
single isolated human umbilical vein endothelial cells, as shown in Fig.11.8(b). The
SWNT/Nafion-modified electrode was placed at a fixed distance (generally 1mm)
away from the surface of a cell and observed at real-time monitored NO release.
The multi-walled carbon nanotube-modified carbon fiber ultramicroelectrode
was fabricated and used for the determination of nitric oxide radical in liver mito-
chondria [27]. The single carbon fiber (7.8 mm diameter) was connected to the cop-
per wire and inserted into a glass capillary (0.9 mm inner diameter), leaving 1cm in
length exposed. The copper wire and glass capillary were fixed with epoxy and the
capillary tip was fused on the flame to seal the carbon fiber. The protruded carbon
fiber was etched on the flame to the desired length (about 300500mm). After rest-
ing the electronic contact and potential leaks, the carbon fiber ultra-microelectrode
was rinsed with redistilled water. The MWNT suspension was deposited onto the
surface of the cleaned carbon fiber electrode. Then the suspension was evaporated
under an infrared lamp to obtain the MWNT-modified carbon fiber ultra-microelec-
trode. The modified carbon fiber electrode was then coated with 3% (w/v) Nafion
solution to obtain the MWNT/Nafion-modified carbon fiber electrode. Figure11.9
shows the SEM images of the carbon fiber ultra-microelectrode (CFUE) and
MWNT-modified carbon fiber ultra-microelectrode. Compared with the smooth
surface of the bare carbon fiber ultra-microelectrode, the MWNT-modified carbon
fiber ultra-microelectrode showed a porous interfacial layer. The square wave of the
voltammograms of NO showed a small anodic peak at the CFUE/Nafion electrode
344 11 Nanostructure for Nitric Oxide Electrochemical Sensing

Fig.11.9 SEM images of (a) the general view of carbon fiber ultramicroelectrode (CFUE), (b) the
midsection of bare CFUE, (c) the midsection of CFUE/MWNTs, and (d) the tip and cross-section
of CFUE/MWNTs. Reprinted with permission from Wang etal. [27], 2005, Elsevier

but a significant increased peak at the CFUE/MWNT/Nafion electrode, and the


oxidation peak potential shifted negatively to around 0.78V. The current response
of the MWNT-modified carbon fiber electrode exhibited a good linear relationship
with the NO concentration in the range of 0.2~86mM. The sensitivity and minimum
detection were 2.291nA and 0.02mM, respectively.

11.4 Conclusions

Nitric oxide acts as a very important role in organisms, such as vasodilatory mes-
senger, endothelium-derived relaxing factor, neurotransmitter, among other roles.
In order to elucidate the function of NO in the biology, different electrochemical
References 345

biosensors have been developed to determine NO in the ultrasmall volume or single


cell. On the other hand, the nanosized NO electrochemical sensor, based on a car-
bon fiber electrode, has been manufactured. The NO nanosensor shows an excellent
linear range of NO at a 101,000-nM concentration range. The sensitivity of the
nanosensor is about 0.5pA/nM. The low limit of determination (defined as a signal-
to-noise ratio of 2) is about 2nM. Moreover, in order to improve the sensitivity and
selectivity of the NO sensor, many nanomaterials, such as nano Au, MWNT, nano
TiO2, etc., have been used as the electrocatalyst to modify different electrodes, such
as GC, PG, and Pt. However, these electrodes are too large for the determination of
NO in an ultrasmall volume. The nanomaterials, such as SWNT- or MWNT-
modified ultra-microelectrode carbon fiber, have been manufactured and used for
the determination of NO in the cells.

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wwwwwwwwwwwww
Chapter 12
Assembly of Nanostructures for Taste Sensing

12.1Introduction

Taste and smell are the two human senses that are chemical in nature. However, they
have not been as successfully replicated with sensors, probably because of the
complexity of the human system [1]. Although the science behind taste is still not fully
elucidated, it is known that it relies on a series of taste cells densely packed in taste buds
mainly located on the tongue and palate and in the pharynx. These taste buds are
connected to nerve fibers that carry the signals of the chemical environment to the brain
stem, where central processing of the information occurs [2]. It is commonly accepted
that humans distinguish at least five basic taste qualities: saltiness, sourness, bitterness,
sweetness, and umami, although some consider this model too simplified [3].
The electronic counterpart of tongues is an array of chemical sensors through which
a raw signal output is transferred to a computer system and the data are processed. As
an analogy to nature, it is generally assumed that taste sensors have to rely on sensors
with a rather broad or relatively low chemical selectivity [4]. This philosophy is also
supported by IUPAC in a technical report on electronic tongues [5]. The artificial
tongues are dedicated to the automatic analysis of complicated composition samples,
to the recognition of their characteristic properties, and to fast qualitative analysis.
Several approaches have been discussed for the transduction mechanism and
chemical selectivity. A wide variety of chemical sensors can be employed in the
design of electronic tongues: electrochemical (voltammetric, potentiometric), opti-
cal, or enzymatic sensors (biosensors). However, the majority of taste-sensing
systems for food and beverage analyses rely on arrays of electrochemical sensors.
In 1985, the first system for liquid analysis based on the multisensor array was
presented by Otto and Thomas [6]. Since then, a few devices of electronic tongues
have been presented. Most of these systems are based on potentiometric sensors,
especially ion-selective electrodes. The potential of the ion-selective electrodes is a
function of the activity of ionic species in a sample solution and is formed in the
ion-sensitive membrane, where selective complexation (ion recognition) of the

H. Ju et al., NanoBiosensing: Principles, Development and Application, 349


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0_12, Springer Science+Business Media, LLC 2011
350 12 Assembly of Nanostructures for Taste Sensing

a nalyte molecules occurs. In contrast with potentiometric methods, voltammetric


measurements are performed when equilibrium is not reached, and the signal obtained
is the currentpotential relationship. The surface of the electrodes used for voltam-
metric measurements also can be modified with various chemosensitive materials.
Another modification of voltammetric electrodes involves the application of enzy-
matic layers, providing the possibility of monitoring analytes such as glucose.
Optical electronic tongues involve various optical sensors, usually based on
polymeric microspheres with a chemically modified surface that allows covalent
bonding to various receptors. There is a wide range of indicators used in optical sen-
sors, and thus more analytes can be detected in electrochemical sensor technology
[7, 8]. There are many possible modes of operation of optical sensors: the acquisition
of fluorescence intensity, lifetime signals, absorbance, reflectance, and so forth.
A variety of mass sensors, such as surface acoustic wave (SAW) and quartz
crystal microbalance (QCM), have been proposed for the development of elec-
tronic nose systems [9].
Nanotechnology has recently become one of the most exciting fields in the
forefront of biosensor fabrication. Nanotechnology is defined as the creation of
functional materials, devices, and systems through control of matter at the 1100nm
scale. A wide variety of nanoscale materials of different sizes, shapes, and composi-
tions are now available. The huge interest in nanomaterials is driven by their desir-
able properties. The use of nanomaterials in biosensors allows the use of many new
signal transduction technologies in their manufacture. Because of their size, nano-
sensors, nanoprobes, and other nanosystems are revolutionizing the fields of chemi-
cal and biological analyses. In particular, the ability to tailor the size and structure
and hence the properties of nanomaterials offers excellent prospects for designing
novel sensing systems and enhancing the performance of the bioanalytical assay.
In this chapter, we intend to review some of the major advances in nanomaterial-
based chemical sensor array for taste sensing. The nanoassembled films are the
most common materials for taste sensor application. Both conducting polymers and
carbon nanotube (CNT) polymer composites have been employed to construct the
taste sensor with electrical signal detection. In recent years, optical detection sensor
arrays also have been used to attempt sample identification, to function as an
electronic tongue. Nanomaterials have an important role in the sensor array
construction based on their unique optical properties.

12.2Nanoassembled Films for Taste Sensor Application

12.2.1Nanoassembled Conducting Polymers for Taste Sensor


Application

The field of nanoscience and nanotechnology is a broad and interdisciplinary area


with worldwide research, developing activities that have been growing explosively
in the last few years. Through the use of nanostructured thin films of conducting
12.2 Nanoassembled Films for Taste Sensor Application 351

polymers and a lipid-like material coating gold microelectrodes, we are able to


mimic the human perception of taste. An electronic tongue composed of nanostruc-
tured films of polyaniline and polypyrrole (PPy) and their mixtures with a lipid-like
material as transducers has been reported [10]. Because a single material is unlikely
to be sensitive to all basic tastes, a combination of sensors has been used. The nano-
assembled films are built using the LangmuirBlodgett (LB) technique due to the
feasibility of depositing different conducting polymers with a thickness of about
2nm per layer, which also provides excellent control of the film thickness.
Nanostructured conducting polymer films of poly(o-ethoxyaniline) (POEA) and
sodium sulfonated polystyrene (PSS) produced by the layer-by-layer (LBL) tech-
nique have been employed as part of sensory units in a taste sensor system [11]. The
sensitivity of these films to standard taste solutions has been investigated by means
of electrical capacitance measurements. The film architecture plays an important
role on the film electrical response to different tastes, which also depend on the
frequency of the input signal. The performance of these films in terms of the repro-
ducibility and repeatability of their electrical response to different tastes has been
studied. In general, the electrical response of POEA/PSS films is highly reproduc-
ible and repetitive independently of the film architecture and frequency of interroga-
tion. An array containing five LBL POEA/PSS films with different numbers of
polymeric layers is able to distinguish liquids according to their taste and is also
able to quantify NaCl at concentrations much lower than the human detection capac-
ity. Such results have demonstrated the potentiality of these films on the develop-
ment of taste sensor systems.
An artificial tongue composed of four sensors made from ultrathin films (of the
order of nanometers) deposited onto gold interdigitated electrodes has been able to
distinguish the four basic tastes (salty, sour, sweet, and bitter) easily, in addition to
determination of inorganic contaminants in ultrapure water and identification of
different brands of coconut water [12]. Some tastants have been detected below the
human threshold values, for example, 5 mM of NaCl or sucrose. Suppression of
quinine by sucrose has also been detected. The high sensitivity may be partially
attributed to the ultrathin nature of the films as the sensors are produced with LB
films of the 16-mer polyaniline oligomer, PPy, and a ruthenium complex and with
self-assembled films of an azobenzene-containing polymer. The sensor response is
evaluated with AC measurements taken at various frequencies, with the admittance
being treated theoretically with an equivalent circuit representing the sensor
immersed in a polyelectrolyte solution. Subsidiary measurements have shown that
the sensor sensitivity falls as the number of layers in an LB film of PPy is increased
from 5 to 15. In principle, an even higher sensitivity can be obtained with thinner
films, but stability may become a limitation. Furthermore, full coverage of the elec-
trode must be guaranteed. This may not be achieved if only a few layers are depos-
ited. The capability to detect and in some cases distinguish inorganic contaminants
in water is an added advantage of the electronic tongue. This is particularly impor-
tant in cases where humans cannot be used in tests due to the toxic nature of the
contaminants. The electronic tongue is also a simple and inexpensive way to quan-
tify the amount of a sweet substance that should be added to a bitter medicine to
make it palatable, without the need for human testing.
352 12 Assembly of Nanostructures for Taste Sensing

12.2.2Carbon Nanotube Polymer Composite for an Impedimetric


Electronic Tongue

A study aimed at the characterization of five compounds with different chemical


characteristics and gustative perceptions by measuring the variations of the electrical
impedance of a composite sensor array was presented [13]. The array was com-
posed of five sensors of three different types based on CNTs or carbon black
dispersed in polymeric matrices and doped polythiophenes. Measurements were
carried out by evaluating the electrical impedance of the sensor array at a frequency
of 150Hz, and the data acquisition process was automated; a mechanical arm and a
rotating platform controlled by a data acquisition card and dedicated software
allowed the sequential dipping of sensors in the test solutions. Fifty different solu-
tions eliciting the five basic tastes (sodium chloride, citric acid, glucose, glutamic
acid, and sodium dehydrocholate for salty, sour, sweet, umami, and bitter, respec-
tively) at ten concentration levels comprising the human perceptive range were ana-
lyzed. More than 100 measurements were carried out for each sample during a
4-month period to evaluate the systems repeatability and robustness. The impeden-
tiometric composite sensor array was shown to be sensitive, selective, and stable for
use as an electronic tongue. The experimental setup was designed in order to allow
the automatic selection of a test solution and dipping of the sensor array following
a dedicated measurement protocol. Measurements were carried out on 15 different
solutions eliciting the five different tastes listed above at three concentration levels
comprising the human perceptive range. In order to avoid overfitting, more than 100
repetitions for each sample were carried out over a 4-month period. Principal com-
ponent analysis (PCA) was used to detect and remove outliers. Classification was
performed by linear discriminant analysis (LDA). A fairly good degree of discrimi-
nation was obtained.

12.3Sensor Array Based on Gold NanoparticleFluorophore


Complexes

The detection and quantification of cells, proteins, and other biosystems in complex
matrices are important for disease detection. A wealth of methods is available to
attain this goal, including antibodies used in ELISA-type tests, proteomics and
related approaches coupled with mass spectrometry, as well as widely employed
techniques such as gel electrophoresis to detect serum imbalances in patients with
liver failure or other gross metabolic problems. The chemical nose/tongue
approach provides an alternative for the sensing protocols for analyte detection.
Strategically, the array is able to present chemical diversity to respond differentially
to a variety of analytes.
Bunz and Rotello created a sort of sensor array with noncovalent gold nanopar-
ticle (NP)fluorescent polymer conjugates to detect, identify, and quantify protein
12.3 Sensor Array Based on Gold NanoparticleFluorophore Complexes 353

targets [14]. Monolayer-protected gold NPs were polyvalent by design and can be
combined with any type of fluorophore. They covered positively charged gold NPs
with negatively charged conjugated polymers of the poly(para-phenyleneethy-
nylene) (PPE) type and with the likewise anionic green fluorescent protein (GFP).
In both cases, the combination of polyvalent fluorophore with the polyvalent gold
NP generated attractive self-assembled sensors that discerned proteins, bacteria,
and cells by analyte-induced fluorescence turn-on. This work demonstrated the
construction of novel nanomaterial-based protein detector arrays with potential
applications in medical diagnostics.
The combination of ammonium-functionalized 2-nm-core gold NPs with conju-
gated polymers or with GFP in water gave rise to a diverse set of self-assembled
hydrophobic or electrostatic complexes or hybrids. Depending upon the ratio of
conjugated polymer or GFP to gold NPs, the fluorescence of the construct can be
precisely tuned; the higher the concentration of NPs, the lower the emission inten-
sity. While the quenching mechanism was not fully understood, the overlap of the
absorption spectrum of the gold NP with the emission spectra of the fluorophores
suggested efficient energy-transfer quenching. The formation of these nonfluores-
cent constructs was driven by electrostatic interactions, so that the ammonium
head groups can carry a significant number of different substituents. The chemical
nature of the used ammonium groups somewhat modulated the fluorescence quench-
ing through the binding or association constants (Ka) between particle and fluoro-
phore. However, regardless of the NP used, Ka is always high and averages between
107 and 1011 in water, with ammonium salts sporting aromatic head groups display-
ing the highest binding. The high Ka values allowed for the efficient assembly of the
complexes at relatively low analytically and diagnostically relevant concentrations,
with fluorophores in high-nanomolar (100500nm) concentrations. The interaction
of the self-assembled hybrids with (negatively charged) biological analytes such as
proteins in water or in serum, inorganic ions such as PPi, and bacterial or mamma-
lian cells led to a fluorescence modulation of the constructs; in most cases, fluores-
cence turn-on results.

12.3.1Detection of Proteins

A sensor array containing six noncovalent gold NPfluorescent polymer conjugates


has been created [15, 16]. The polymer fluorescence was quenched by gold NPs; the
presence of proteins disrupted the NPpolymer interaction, producing distinct fluo-
rescence-response patterns. These patterns are highly repeatable, are characteristic
for individual proteins at nanomolar concentrations, and can be quantitatively dif-
ferentiated by LDA. Based on a training matrix generated at protein concentrations
of an identical ultraviolet absorbance at 280nm, LDA, combined with ultraviolet
measurements, has been successfully used to identify 52 unknown protein samples
(seven different proteins) with an accuracy of 94.2%. This strategy exploited the
size and tunability of the NP surface to provide selective interactions with proteins,
354 12 Assembly of Nanostructures for Taste Sensing

and the efficient quenching of fluorophores by the metallic core to impart efficient
transduction of the binding event. Through application of LDA, they were able to
use these fluorescence changes to identify and quantify proteins in a rapid, efficient,
and general fashion. The robust characteristics of the NP and polymer components,
coupled with the diversity of surface functionality that can be readily obtained using
NPs, made this array approach a promising technique for biomedical diagnostics.
The discrimination of proteins by the nonbiological NPPPE constructs is
powerful, perhaps because each NPPPE construct has an analog response toward
each protein. The sensing of single proteins in water, albeit of fundamental interest,
is not sufficient to use these types of constructs to determine analytes in complex
matrices such as serum, sperm, urine, or sweat. The most diagnostically important
biological matrix is human serum, the proteinacious solution that remains when
blood is freed from white and red blood cells.
Medicinal diagnostics of serum samples is generally done by simple electro-
phoresis to determine large-scale protein imbalances in serum that arise for liver
malfunction and other disease states, while for the detection of proteins such as
troponin, which are present in trace amounts, antibody assays are used. LDA
demonstrates that the proteins, with the exception of IgG and antitrypsin, are well
resolved in two dimensions, forming nonoverlapping patterns. Further experiments
indicated that mixtures of different proteins and the addition of one protein in
different concentrations also led to a specific and reproducible change in the LDA-
based patterns. Mixtures of proteins spiked into the serum could be detected and
gave specific responses.
Miranda etal. developed an enzyme-NP sensor array where the sensitivity was
amplified through enzymatic catalysis [17]. In this approach cationic gold NPs were
electrostatically bound to an enzyme (beta-galactosidase, beta-Gal), inhibiting
enzyme activity. Analyte proteins released the beta-Gal, restoring activity and
providing an amplified readout of the binding event. Using this strategy, we have
been able to identify proteins in buffer at a concentration of 1nM, which was lower
than current strategies for array-based protein sensing.
To minimize nonspecific interactions between the fluorophore and the serum
proteins, GFP was selected as the fluorophore in the sensing of serum proteins, as
PPEs can display nonspecific interactions with different proteins. Furthermore, GFP
has a defined size and molecular weight, and its fluorophore core is embedded in a
barrel-shaped protein, thus significantly reducing aggregation-induced quenching
and excimer formation, which can occur with conjugated polymers.
There is a direct correlation between protein levels and disease states in human
serum, which makes it an attractive target for sensors and diagnostics. However, this
is challenging because serum features more than 20,000 proteins, with an overall
protein content greater than 1mM. Rotello etal. reported a sensor based on a hybrid
synthetic biomolecule that used arrays of GFP and NPs to detect proteins at biorel-
evant concentrations in both buffer and human serum [18]. Distinct and reproduc-
ible fluorescence-response patterns were obtained from five serum proteins (human
serum albumin, immunoglobulin G, transferrin, fibrinogen, and alpha-antitrypsin),
both in buffer and when spiked into human serum. Using LDA, they identified these
12.3 Sensor Array Based on Gold NanoparticleFluorophore Complexes 355

proteins with an identification accuracy of 100% in buffer and 97% in human serum.
The arrays were also able to discriminate between different concentrations of the
same protein, as well as a mixture of different proteins in human serum.
These results suggested that simple libraries made from differently functional-
ized gold NPs and either biofluorophores such as GFP or conjugated polymers
recognized and detected proteins and protein imbalances in serum as a complex
matrix. This is a significant achievement for these fairly simple constructs, which
only employ electrostatic and hydrophobic interactions for the facile detection and
quantification of proteins in water, buffer, and serum.

12.3.2Detection of Bacteria and Mammalian Cells

The successful exploitation of protein sensing suggests that the functionality on


bacterial surfaces should respond to nanoconstructs formed from gold NPs and
PPEs [19, 20]. Bacterial cell walls are negatively charged and should furnish a poly-
valent environment that can interact strongly with NPPPE constructs under fluo-
rescence turn-on, similarly to the effect that was observed for the proteins. Gold
nanorods were functionalized by electrostatic interactions or through covalent
attachment with an antibody against Pseudomonas aeruginosa. Incubation with
these bacteria led to hybrids, in which the nanorods covered the bacterial surface.
All 12 microorganisms group distinctly. Importantly, three different Escherichia
coli strains [XL1 Blue, BL21(DE3), DH5a] were easily discerned. The three strains
were not grouped particularly closely together by their fluorescence responses,
which suggested that there must be some differences in their surface chemistries.
Also, Gram-negative (E. coli, Pseudomonas putida) and Gram-positive bacteria
(Aechmea azurea, Bacillus subtilis, Bacillus lichenformis) could easily be discerned
but were not grouped in any particular way in the LDA plot. Random solutions of
bacteria from the training set were identified in 95% of all cases, thus demonstrating
the robustness of this small NPPPE library. It is speculated that hydrophobic
hotspots on the surface of the bacteria were a reason for their binding to hydrophobic
NPs, and while there was significant literature on the function of bacterial surface
proteins and surface structure, the mesoscopic landscape of the bacterial surface,
that is, how the protein complexes, lipid rafts, and glycans self-organize into struc-
tures that are 10100nm in size, is much less known but probably plays a signifi-
cant role for the surprising success of this simple NPPPE-based assay.
The detection and identification of bacteria by the preformed NPfluorescent
polymer complexes presented the possibility that mammalian cells might also be
identified. In other cases, mannose-substituted gold NPs were specifically com-
plexed by the pili, that is, the fine hairs that emanate from the surface of E. coli
bacteria. The pili are rich in lectins (sugar-binding proteins) and therefore interact
preferentially with these parts of the bacterial anatomy. Mammalian cells and their
interactions with gold NPfluorophore constructs were studied to differentiate normal
from cancerous and metastatic cells and also to differentiate cancerous and
356 12 Assembly of Nanostructures for Taste Sensing

metastatic cells. A successful differentiation would generate potential tools for the
development of simple assays for the early diagnosis of neoplastic growth and its
classification into metastatic or nonmetastatic cells. These three NPs in combination
with PPE generated the largest responses toward mammalian cells and were obtained
from the library; other NPs were tested but did not show significant responses when
exposed to mammalian cells. The three NPPPE constructs are able to differentiate
among four different human cancer cell lines but are also capable of discerning
breast tissue that is normal, cancerous, or metastatic.

12.4Catalytic Nanomaterial-Based Optical Sensor


and Sensor Array

Chemiluminescence (CL) is defined as the emission of electromagnetic radiation


(usually in the visible or near-infrared region) produced by a chemical reaction that
generally yields one of the reaction products in an electronic excited state, produc-
ing light on falling to the ground state. CL resulting from the interaction between
gases and solid surfaces has been studied for decades. The phenomenon was
observed during the catalytic oxidation of carbon monoxide on a thoria surface by
Breysse etal. and was defined as cataluminescence (CTL) [21]. Applications of the
luminescence have been developed as a means of revealing intermediate stages in
adsorption and catalysis [22].
The nanosized material-based sensing mode utilizing the phenomenon of CTL
shows a high sensitivity and wide linear response for the determination of volatile
organic vapors (VOCs) and gas samples. Gas sensors based on CTL not only provide
direct, sensitive, selective, and rapid analysis but also show a greener advantage
attributed to the following factors: (1) No CL reagents or other reagents are needed
for CL generation; (2) exhausts after detection are usually of low toxicity; and (3)
the apparatus can be simple and potentially portable for field analytical chemistry,
thus avoiding sampling and potential pollution from the reagents used to preserve
the samples. Furthermore, many kinds of nanosized materials suitable for preparing
this type of gas sensor and a group of CTL gas sensors can construct a sensor array
for sample determination and discrimination.

12.4.1Nanomaterial-Based Cataluminescence Sensors


for Vapor Sensing

The first report to study the CTL emission of organic vapors on nanosized materials
was published by Zhang and coworkers [23]. Seven nanosized materials in their
work were investigated, and CTL was detected on six of them, including MgO
(28nm), TiO2 (20nm), Al2O3 (18nm), Y2O3 (90nm), LaCoO3:Sr2+ (50nm), and
SrCO3 (25nm) [ZnO (68nm) did not emit CTL] while organic vapor passed through.
12.4 Catalytic Nanomaterial-Based Optical Sensor and Sensor Array 357

They applied this phenomenon to the development of several sensitive, stable gas
sensors. To demonstrate their original idea, a nanosized TiO2 was chosen as the
sensing material for preparing the gas sensor. Strong CTL emission was generated
by the catalytic oxidation of organic molecules on the surface of TiO2 NPs. The
analytical characteristics were evaluated by the examination of CTL emission from
trace ethanol and acetone on the sensor. The results indicated that this new sensing
mode could be developed by utilizing CTL on nanosized materials.
They also developed a catalytic chemiluminescent trimethylamine (TMA) sen-
sor [24]. Intensive CTL was detected when TMA was introduced over the surface of
nanosized catalysts and subsequently catalytically oxidized by O2 from the air, and
four catalysts were investigated, with the strongest CTL intensity obtained on nano-
sized Y2O3. This effect was utilized to develop a novel nanosized Y2O3-based cata-
lytic CTL sensor for TMA, which under optimal conditions exhibited a wide linear
range of 6042,000ppm and a detection limit of 10ppm. An attractive advantage of
this novel CTL sensor is its high selectivity to TMA with negligible responses to
many other gases such as NH3 and organic vapors. This CTL sensor had a short
response time of less than 3s, and showed good stability when examined by con-
tinual introduction of TMA into the sensor for 96h. The applicability of this sensor
to actual fish samples was also demonstrated in Zhang and coworkers report.
The released energy during a catalytic reaction can also be transformed into rare
earth metal ions to generate emission instead of excited intermediates. Zhang etal.
reported the observation of an energy-transfer process between excited intermedi-
ates and the nanosized catalysts [25]. The CTL was quenched when Ho3+, Co2+, and
Cu2+ were introduced into the catalyst, while new intensive CTL peaks appeared
when the catalyst was doped with Eu3+ or Tb3+. Further study indicated that the new
CTL peak on Eu3+- or Tb3+-doped catalyst originated from the luminescence of the
doped ions, excited by the energy transferred from excited intermediates produced
during the reaction. An ethanol sensor was developed with Eu3+-doped nanosized
ZrO2 that was linearly response to ethanol concentrations from 45 to 550 ppm.
Theenergy transfer led to 72 times higher sensitivity than the CTL from excited
intermediates in the sensor. High selectivity and stability were also obtained for this
sensor. The results indicated that the main factor limiting the sensitivity of a CTL
sensor on pure catalyst may be the inevitable energy quenching of excited inter
mediates with the catalyst, which was artfully utilized in the work by the intro
duction of Eu3+ that effectively absorbed this part of energy and transferred it into
light energy.
Hu etal. reported a gas sensor by using the CTL emission from the oxidation of
ethyl ether by oxygen in the air on the surface of borate glass [26]. Theoretical
calculations, together with experimental investigations, revealed that ethyl ether
was first oxidized to acetaldehyde and then to acetic acid, during which the main
luminous intermediates such as CH3CO were generated and emitted light with a
peak at 493nm. At a reaction temperature of 245C, the overall maximal emission
was found around 460nm. Interference from foreign substances, including alcohol
(methanol, ethanol and isopropanol), acetone, ethyl acetate, n-hexane, cyclo-
hexane, dichloromethane, or ether (n-butyl ether, tetrahydrofuran, propylene oxide,
358 12 Assembly of Nanostructures for Taste Sensing

isopropyl ether, and methyl tert-butyl ether), was not significant except for a minimal
signal from n-butyl ether (<2%). The proposed ethyl ether gas sensor offered the
following distinct advantages: (1) Compared with GC and GC/MS, it is a simple
and cost-effective sensor; (2) it is a greener sensor, since no toxic reagent/solvent
is needed, and the exhaust is of low toxicity and at low concentrations; and (3) it
shows much better sensitivity and selectivity, and faster response than many com-
mon VOC sensors.
Yang etal. described the development of a novel CTL sensor coupled with ionic
liquids (ILs)based headspace solid-phase microextraction (HS-SPME) technolo-
gies for the quantification of human plasma acetone levels associated with diabetic
disease ex vivo [27]. The unique properties of ILs, such as their nonvolatile and
nonflammable nature, coupled with their high thermal stability allowed ILs to be
conveniently adopted as pseudo-solid carriers for direct loading of acetone into a
CTL sensor without matrix interference. Acetone from diabetic patient plasma and
plasma samples spiked with acetone along with methanol, ethanol, and formalde-
hyde was conveniently and rapidly extracted and enriched in 3mL of IL and then
rapidly quantified by a CTL sensor. The presence of plasma alone or spiked plasma
containing methanol, ethanol, or formaldehyde did not interfere with acetone mea-
surements. HS-SPME-CTL provided higher enrichment efficiency than headspace
single-drop microextraction-based CTL (HS-SDMECTL) methods, possibly due to
the fact that the thin film formed in HS-SPME instead of the single IL drop in
HS-SDME increased the exchange area for extracted acetone. The enrichment effi-
ciency by HS-SPME-CTL was almost 80-fold higher than that with direct injection
using the same volume of aqueous samples and more than sixfold higher than that
using HS-SDME-CTL. Considering that ILs can be easily prepared from inexpen-
sive materials and tuned by the combination of different anions and cations for the
extraction of specific analytes from various solvent media, this proposed technology
raises an exciting possibility by employing HS-SPME-CTL for the fast determina-
tion of specific targets in many fields.

12.4.2Catalytic Nanomaterial-Based Optical Chemosensor


Array for Recognition and Discrimination Odors

The different organic vapors can be discriminated from the different CTL responses
in the presence of the different nanosized materials. These materials are potentially
suitable for processing as a chip-mounted sensor array.
Na et al. found that luminescent efficiencies of the CTL are different for a
compound on different nanomaterials [28]. As shown in Fig.12.1a, ethanol gave the
strongest emission signal on ZrO2:Tb3+ (ZrO2 doped with 5% Tb3+), while giving no
signal on nanosized WO3 and Fe2O3. Hydrogen sulfide generated an obvious emis-
sion on WO3, Fe2O3, and Y2O3, but no emission on others. TMA produced strong
signals on ZrO2:Eu3+ (ZrO2 doped with 5% Eu3+), Al2O3, and ZrO2:Tb3+, but only
weak signals on others. Similarly, the same nanomaterial exhibited different CTL
12.4 Catalytic Nanomaterial-Based Optical Sensor and Sensor Array 359

Fig.12.1 Chemoselective response of CL on nanomaterials. (a) The fingerprint profiles of the


CTL on nine nanomaterials for (a) ethanol, (b) hydrogen sulfide, and (c) TMA. The nanomaterials
are as follows: ZrO2:Eu3+, MgO, Al2O3, WO3, ZrO2:Tb3+, SrCO3, Fe2O3, Y2O3, and ZrO2 from left
to right. (b) The comparison of CL spectra on the sensing materials of Al2O3, ZrO2:Eu3+, Y2O3, and
ZrO2. (a) Ethanol; (b) hydrogen sulfide; and (c) TMA. Reprinted with permission from Na etal.
[28]. 2006, American Chemical Society

properties upon exposure to different analytes. A strong signal can be obtained on


Fe2O3 for the detection of hydrogen sulfide, while there was only a weak response
for TMA and no signal for ethanol. As for ZrO2:Tb3+, ethanol and TMA gave bright
luminescence, but hydrogen sulfide gave nearly no luminescence.
The CTL spectral shapes of a compound are distinct on various nanomaterials. As
shown in Fig.12.1b, the spectral shapes of ethanol were different on Al2O3, ZrO2:Eu3+,
Y2O3, and ZrO2. The maximum emission of ethanol on ZrO2:Eu3+ was at the wave-
length of 620nm, while that on Al2O3 was 425nm. In the same way, the spectral
shapes of different compounds on the same nanomaterial were also different, such as
that of ethanol, hydrogen sulfide, and TMA. The chemoselective responses of CTL
on catalytic nanomaterials and its abundant optical information inherently coincide
with the requirements to fabricate a cross-reactive chemical sensor array.
For demonstration purposes, a 33 array with nine catalytic nanomaterials were
fabricated on a piece ceramic chip with temperature controller (Fig.12.2a). They
examined three kinds of compounds (alcohols, amines, and thiols) to represent a
wide range of chemical functionality. When the array was exposed to air without
sample, a blank image was recorded. The CTL images for ethanol, hydrogen sul-
fide, and TMA on the sensor array are distinct, as shown in Fig.12.2b. Therefore, a
compound is represented by the position of luminous spots and the relative CTL
intensity of each spot.
Zhangs group also showed the results of discriminating the four alcohols, namely
methanol, ethanol, n-propanol, and n-butanol, with the CTL sensor array. A com-
parison of images for the four alcohols with the same concentration is shown in
Fig.12.2c. Methanol and ethanol were easily distinguished from the others by the
images. Though the luminous spots appeared at the same position for n-propanol
and n-butanol, their brightnesses were different.
360 12 Assembly of Nanostructures for Taste Sensing

Fig.12.2 Schematic diagrams of the CTL sensor array and the recorded images upon exposure to
various samples. (a) Schematic diagrams of the CTL sensor array; (b) images obtained by the sen-
sor array after exposure to air for 1min (a) without sample, (b) with ethanol vapor, (c) hydrogen
sulfide, and (d) TMA vapor. (c) Images obtained by the sensor array upon exposure to four alcohol
vapors: (a) methanol; (b) ethanol; (c) n-propanol; and (d) n-butanol. Reprinted with permission
from Na etal. [28]. 2006, American Chemical Society

The images of the sensor array can be adjusted by changing the working
temperature, because the route and rate of catalytic reaction are dependent on
temperature, which leads to different luminescent efficiencies and spectral shapes.
Their results indicated that even if similar images were recorded with the sensor
array for two analytes at one temperature, they may be differentiated according to
the images at another temperature.
The sensor array can also be used to fulfill quantification of a given analyte
by the CTL emission intensity, because the CTL intensities vary linearly with
the change in the analyte concentration. The linear range for the determination
of ethanol was 45550 ppm with a detection limit of 15 ppm on the spot of
ZrO2:Eu3+; for hydrogen sulfide on Fe2O3 it was 8.02,000ppm with a detection
limit of 3.0ppm; while for TMA on Y2O3, it was 6042,000ppm with a detec-
tion limit of 10ppm. It should be pointed out that the linear range and detection
limit for each analyte vary significantly from different catalytic nanomaterials.
12.4 Catalytic Nanomaterial-Based Optical Sensor and Sensor Array 361

The reversible response and relative long-term stability of the sensor array
indicated its perspective in real applications.
This sensor array was applied for the discrimination and identification of flavors
in cigarettes [29]. Twenty-one nanomaterials, including metal oxides, metal oxides
deposited on CNTs, gold NPs deposited on metal oxides, and carbonate, have been
carefully selected as sensing elements of the array. Eleven flavors commonly used
in the cigarette industry were examined, including ethyl acetate, butyl acetate,
benzyl benzoate, benzyl alcohol, phenylethyl alcohol, furfural, eugenol, butane-
dione, anisaldehyde, benzaldehyde, and iso-valeric acid. The unique patterns of the
11 flavors were obtained individually on the sensor array, indicating the different
CTL properties of each flavor on the catalytic nanomaterials. The results were given
numerically, which functioned as fingerprints of each flavor and gave us the
intuitionistic information of the differences among flavors. With these results, facile
discrimination of one flavor from the others can be achieved by the naked eye from
their unique patterns. Hierarchical cluster analysis (HCA) and LDA were used to
analyze the patterns. The obtained CTL patterns were temperature-dependent; thus,
additional discrimination power could be provided by changing the working
temperature of the array. Quantification of the flavors has been performed according
to the emission intensity on the specific sensing element. The linear range of the
sensor array for the flavors was 202,000ppmv, with the limits of detection below
10ppmv, which varied with the kinds of flavors.
Six brands of cigarettes were analyzed in order to demonstrate the potential of
the sensor array for complex compounds. The tobacco was heated at 80C for
15min to evaporate the flavors, and the vapors were injected into the sensor array
by the carrier gas for the detection. Six brands of cigarettes were discerned by their
CTL patterns obtained with the present sensor array. The results demonstrated the
potential analytical applications of a catalytic nanomaterial-based CTL sensor array
for the discrimination and identification of flavors. The robust and reversible
response of this array, combined with its simple instrumentation, indicated the
promise of this array to real-world applications.

12.4.3Recognition of Organic Compounds in Aqueous Solutions


by Chemiluminescence on Catalytic Nanoparticle Arrays

The sensor array based on nanomaterials has been extended to the discrimination of
samples in solution, since amino acids, saccharides, and steroid pharmaceuticals
can produce CL emission on the surface of nanomaterials.
A novel aerosol CTL detector based on porous alumina was developed according
to the generated CTL emission from catalytic oxidation of the analytes [30]. This
aerosol CTL-based detector has three main processes: nebulization of solution; CTL
emission on surface of porous alumina material; and optical detection. To demon-
strate the utility of the aerosol CTL detector, some compounds such as saccharides,
poly(ethylene glycol)s, amino acids, and steroid pharmaceuticals were determined
362 12 Assembly of Nanostructures for Taste Sensing

by the present aerosol CTL-detection method. The CL emission could be generated


due to the catalyzing oxidization of saccharides on the surface of porous alumina.
The saccharides, such as sucrose, a-lactose, maltose, raffinose, galactose, xylose,
and glucose, can be successfully detected. The linear ranges of those saccharides
were from 302,000 to 502,000mg/L; relative standard deviations ranged from 2.1
to 3.7% (200mg/L, n=11). The detector showed the following features: (1) exten-
sive CTL emissions on porous alumina by many compounds tested, which leads to
the potential application for the determination of volatile and nonvolatile chemicals;
(2) a CTL mechanism based on the catalytic oxidation of analytes suggests the pres-
ent detector be free from the interference of light scattering.
An aerosol CTL-based sensor array containing six kinds of catalytic NPs for the
pattern recognition of organic compounds in aqueous solutions has been developed,
functioning as mammalian tongues [31]. The catalytic oxidization of sample aero-
sols produces distinct CTL-response patterns on the array that are highly repeatable
and can be differentiated by LDA. Fourteen organic compounds including three sac-
charides, two organic acids, and nine amino acids, which are common small biomol-
ecules, have been classified successfully with their characteristic patterns. A training
matrix (6 NPs14 analytes5 replicates) has been generated and used to identify
126 unknown solution samples at three different concentrations with an accuracy of
100%. The applicability of this array for real-life samples has been demonstrated by
discriminating eight beverages. The stable and reversible response of this array,
combined with its simple instrumentation and long lifetime, indicates the promise
of this array for real-world application.
The unique CTL responses patterns generated from 14 organic aerosols formed
a fingerprint. To evaluate whether this fingerprint can be used for the identifica-
tion of the solutions, the raw data were subjected to LDA, which is normally used
in statistics to recognize the linear combination of features and differentiate two or
more classes of objects or events. The CTL responses were transformed into canoni-
cal scores that were visualized as a well-clustered, two-dimensional plot. The first
two canonical factors contained 70 and 19% of the variation, respectively. All 70 of
the cases (14 solutions5 replicates) were correctly assigned to their respective
groups using LDA. LDA converted the patterns of the training matrix (6 NPs8
beverages5 replicates) into canonical scores. The first two canonical factors con-
tained 79.9 and 14.7% of the variation, respectively, occupying 94.6% of the total
variation. Obviously, the canonical patterns were clustered into eight different
groups, and the classification accuracy was 100%. Next, 24 30% beverage samples
were prepared for the blind tests. After the data matrix was processed by LDA, the
unambiguous identification of 14 organic compounds, including saccharides, amino
acids, and small organic acids, as well as a series of common beverages, was
achieved by the present array, which demonstrated its potential applications for
solutions discrimination. Moreover, it is attractive that unknown samples at differ-
ent concentrations can be identified utilizing the training matrix generated by sam-
ples at only one concentration. With the very simple sensing elements and
instrumentation, as well as the reversible response and long-term stability, this sen-
sor array has prospects for use in real applications.
References 363

12.5Conclusions

Chemical sensor arrays based on nanomaterials have achieved a rather obvious


improvement in odor and taste sensing. Despite the success in some areas, the efforts
to arrive at a universal device that can make subtle discriminations of odor and taste
eventually replace the human nose or tongue are disappointing. The initial hope was
to approach the ability of human odor or taste sensing by increasing the number of
individual sensors. However, the reason for the nose or tongues unequaled perfor-
mance has turned out to be not only the high number of different human receptor
cells, but also their selectivity and their unsurpassed sensitivity for some analytes.
Nanomaterials open the door to solve this problem. Sensors with new sensitive lay-
ers based on nanomaterials are under development. Moving in this direction, sensor
arrays have the potential to enter our daily life far away from well-equipped chemi-
cal laboratories and skilled specialists.

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Chapter 13
Nanostructured Biosensing for Detection of
Insecticides

13.1Introduction

Organophosphorous pesticides (OPs) are widely used in agriculture due to their


high effectiveness and low toxicity for pest control and protecting crops and seeds
[1, 2]. Their residuals in crops, livestock, and poultry products are clearly dangerous
to human health. The related clinical signs include negative effects on the visual
system, sensory function, cognitive function, and nervous system [3, 4]. Specifically,
exposure to OPs has been shown to cause headache, dizziness, profuse sweating,
blurred vision, nausea, vomiting, reduced heartbeat, diarrhea, loss of coordination,
slow and weak breathing, fever, coma, and even death [5]. The toxicity of OPs
mainly arises from their irreversible inhibition on acetylcholinesterase (AChE),
which is essential for the central nervous system to function, often causing respira-
tory paralysis and death [6]. Because of the high toxicities, rapid detection of these
agents in the environment, public places, or workplaces and the monitoring of
individual exposures to OPs have become increasingly important for homeland
security and health protection [7].

13.1.1Conventional Strategies for Detection of Pesticides

Great efforts have been devoted to develop highly sensitive methods for the deter-
mination of pesticide residues in food and water. The identification and quantifica-
tion of pesticides are generally based on chromatographic methods, such as gas
chromatography (GC) or high-performance liquid chromatography (HPLC) cou-
pled with mass spectroscopy (MS). These analytical techniques have been described
and reviewed extensively in the literature [810].
These methods are highly efficient and allow for multiresidue analysis; how-
ever, they require tedious sample pretreatments, highly qualified technicians, and

H. Ju et al., NanoBiosensing: Principles, Development and Application, 365


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0_13, Springer Science+Business Media, LLC 2011
366 13 Nanostructured Biosensing for Detection of Insecticides

sophisticated instrumentation. Therefore, several rapid, relatively inexpensive,


sensitive analytical screening techniques that need almost no sample pretreatment
have been developed for the identification and quantification of such pesticides
[11, 12]. These techniques can be divided into three main types: (1) inhibition
mechanism of OPs on cholinesterase (ChE); (2) catalyzation of the hydrolysis
mechanism of OPs by organophosphorous hydrolase (OPH); (3) immunoassays of
OPs based on anti-OP antibodies [13]. In the inhibition mode, OPs are determined
by measuring the ChE activity before and after an incubation step with pesticide.
The presence of OPs can block the enzyme activity, thereby leading to a decreased
device response. If ChE activity is blocked, acetylcholine (ACh) accumulates at
cholinergic receptor sites, thereby excessively stimulating the cholinergic recep-
tors. Most sensors are based on this inhibition mode, where AChE acts as the rec-
ognition component [14]. Unlike ChE, OPH catalyzes the hydrolysis of OPs with
a high turnover rate. It exhibits the capability to hydrolyze a large variety of OPs
by producing less toxic products, such as p-nitrophenol and diethyl phosphate [15].
Biosensors based on catalytic reaction are superior to the inhibition mode since
they can potentially be reused and are also suitable for continuous monitoring.
Several immunosensors based on antipesticide antibodies [1619] offer selective,
sensitive, and low-cost tools for pesticide analysis. Obtaining high-affinity mono-
clonal antibodies (mAbs) toward OPs pesticide has dramatically promoted the
development of the immunoassay technique, because it has the advantages of an
unlimited preparation of a single and homogeneous type of antibody.

13.1.2Developments in Pesticide Biosensors

OPs have been detected based on various principles, including spectroscopy, chro-
matography, microgravimetry, and electrical and electrochemical techniques.
Among the devices used for detecting OPs, electrochemical devices such as modi-
fied electrodes are relatively inexpensive, small in size, and easy to operate [2022].
One of the characteristic features of electrochemical devices is that the function and
performance of electrodes can arbitrarily be regulated by modifying the surface
with functional molecules, proteins, DNA, etc. [2325]. Thus, electrochemical
devices can be developed for detecting chemicals in sample solutions and in a gas
phase. The modified electrode with a surface modified with proteins and other bio-
logical molecules is often called a biosensor. For this reason, modified electrodes or
biosensors have been widely used for detecting OP agents [2628].
It is well established that electrochemical techniques are useful for determining
ions and molecules in solution and in a gas phase. Oxidizable and reducible chemical
species dissolved in solution can be detected by measuring oxidation or reduction
current that is produced upon electrolysis of the species on the surface of electrodes.
Usually, the output current of the system depends on the concentration of species in
the solution. Thus, one can quantify the concentration of the analyte. Another merit
of electrochemical techniques is the easy identification of analyte by recording the
13.2 Enzymes Used in Pesticide Biosensors 367

redox potential of the analyte, because the redox species can be characterized by its
own redox potential. Electrochemical biosensors are devices that are fabricated by
combining proteins or other biological molecules and electrodes. In recent years,
electrochemical biosensors with high sensitivity, long-term stability, and low-cost
detection of specific biological binding events have extensively reduced sampling
and testing times in pesticide determinations [29]. Satisfactory results were obtained
when AChE was immobilized on modified electrode matrices. However, the enzyme-
immobilization technique remains rather complicated and often involves very com-
plex matrices [15]. Further, the incorporation of AChE into certain mediator matrices
also lowered the stability of the enzyme [30], and the reproducibility after pesticide
inhibition was also poor in such AChE sensors. In recent years, AChE has been
immobilized onto various nanomaterial surfaces to improve the response and stabil-
ity in trace pesticide detection. These nanomaterial matrices include carbon nano-
tubes (CNTs) [1722], gold nanoparticles (Au NPs), and others [2326]. These
matrices significantly promote the stability, sensitivity, and detection limit in OP
determination in the picomolar (pM) to nanomolar (nM) concentration range.

13.2Enzymes Used in Pesticide Biosensors

The electrochemical biosensors used for detecting OP compounds can be divided into
three types depending on the type of enzymes used for constructing biosensors: (1)
(ChE)-choline oxidase (ChO) or bienzyme-modified biosensors; (2) ChE-modified
biosensors; and (3) OPH-modified biosensors.

13.2.1ChE-ChO Bienzyme-Based Biosensors for Pesticides

The most general approach for the determination of OPs is based on their inhibition
of the activity of ChE. The very interesting method for determination of choline
esterase activity is based on coupling two consecutive reactions catalyzed by AChE
and choline oxidase (ChO) enzymes.
The primary objective of ChE-ChO bienzyme biosensors is to determine
neurotransmitter ACh in biological samples [31]. The redox potential of ACh is too
high to be determined directly using electrochemical reaction. For this purpose,
AChE is employed as ChE. AChE and ChO are immobilized on the surface of the
electrode to perform electrochemical analysis. These sensors are based on the
enzymatic reaction of ChO to detect ACh or the use of AChE with ChO to measure
ACh, shown as follows:

ChO sensor:

Choline + O 2
ChO
Betaine aldehyde + H 2 O 2 (13.1)
368 13 Nanostructured Biosensing for Detection of Insecticides

AChE-ChO bienzyme sensor:

Acetylcholine + H 2 O
AChE
Choline + acetate (13.2)

Choline + O 2
ChO
Betaine aldehyde + H 2 O 2 (13.3)

The enzymatic reactions of AChE-ChO bienzyme biosensors consist of AChE-


catalyzed hydrolysis of ACh into choline and acetic acid (13.1) and oxidation reac-
tion of choline catalyzed by ChO (13.3). The latter reaction produces H2O2, which
can be electrochemically oxidized on the electrode to generate output signal depend-
ing on the concentration of ACh. It is also possible to use an oxygen electrode in
place of a metal or carbon electrode because ChO consumes dissolved O2 during the
oxidation of choline. The reason why AChE-ChO bienzyme biosensors can be used
for detecting OP compounds is that the catalytic activity of AChE is inhibited by OP
compounds. OP compounds are known to irreversibly bind to AChE to disturb its
catalytic activity. It is thus clear that (13.2) is suppressed in part depending on the
concentration of OP and the rate of choline production decreases in the presence of
OP compounds, resulting in reduced response in the output current of the sensor.
Butylcholine esterase (BChE) is sometimes used in place of AChE, in which bufryl-
choline is used as substrate.
According to the above protocol, an amperometric sensor was developed for the
determination of choline and ACh using polyethyleneglycol-modified ChO and
native AChE immobilized on polyvinylacetate membranes obtained by means of a
cyclic freezing-thawing process [27]. The working electrode was a Pt wire (0.5-mm
diameter) polished with alumina powder. Electrochemical measurements were done
amperometrically at a polarization voltage of +650 mV. Another amperometric
biosensor was constructed [28] by means of the enzymes AChE and ChO immobi-
lized on polyhydroxyethylmethacrylic acid membranes for the detection of the pes-
ticide Aldicarb. The immobilization of the enzyme was done either by entrapment
or surface attachment via a hybrid immobilization method. All of these studies used
model toxic compounds and administered preliminary laboratory investigations
aimed at environmental pesticide control.
A key point for constructing high-performance OPs sensors in bienzyme systems
may be to suitably control the ratio of the catalytic activity of AChE and ChO in the
sensor. In the present case, the catalytic activity of ChO should be higher than that
of AChE the overall rate of the reactions is determined by the rate of AChE-
catalyzed reaction because OP compounds disturb this step. Therefore, usually,
excess amounts of ChO are mixed with AChE and the ChO-AChE mixture is immo-
bilized on the surface of the electrode to make the AChE-catalyzed reaction a rate-
limiting step. In this situation, the overall reaction rate can be determined by the rate
of the ChE-catalyzed reaction because the following electrochemical oxidation of
H2O2 is sufficiently fast.
13.2 Enzymes Used in Pesticide Biosensors 369

13.2.2AChE-Based Biosensors for Pesticides

As described in the previous section, the use of a bienzyme system sometimes


induces complexity in the design of biosensors originating from different catalytic
activity of the enzymes. In the ChE-ChO systems, ChO is used to oxidize choline
that is generated through ChE-catalyzed reaction because the oxidation potential
of choline is too high to be electrochemically directly oxidized. It is reason
able to assume that if the reaction products can be oxidized on the electrode
directly,onecan remove the second enzyme ChO from the sensor design, and a
ChE-modified electrode may be used for detecting OPs. This concept uses an
AChE-modified electrode as sensor in the presence of acetylthiocholine (ATCh) in
place of ACh as the substrate of AChE. The enzyme-inhibition mechanism pro-
ceeds through the formation of a stable complex through reversible and irreversible
reaction of OPs with the active site of AChE [32].
When AChE was immobilized on the working electrode surface, its interaction
with the substrate of ATCh obtained the electroactive product of thiocholine, which
produced an irreversible oxidation peak. The inhibition of OPs to AChE was moni-
tored by measuring the oxidation current of thiocholine. The reactions that occurred
on the surface of a biosensor were as follows:
AChE

Acetylthiocholine + H 2 O Thiocholine + HA(acetic acid)


AChE

2Thiocholine(red) Thiocholine(ox)(dimeric) + 2H + + 2e -

The presence of low concentrations of inhibitors strongly and specifically affects


enzyme activity. The activity of the AChE can be determined directly using tradi-
tional spectrophotometric methods or electrochemical techniques. Amperometric
AChE biosensors based on the inhibition of AChE have shown satisfactory results
for pesticides analysis [33, 34] in which the enzymatic activity was employed as an
indicator of the quantitative measurement of insecticides. Recently, Du and coau-
thors have reported a novel AChE biosensor that was constructed by modifying
glassy carbon electrode with CdTe quantum dots (QDs) and excellent conductive
gold nanoparticles (Au NPs) through chitosan microspheres to immobilize AChE
[26]. The combination of CdTe QDs and Au NPs promoted electron transfer and
catalyzed the electrooxidation of thiocholine, thus amplifying the detection sensi-
tivity (Fig. 13.1). This novel biosensing platform based on CdTe QDs Au NPs
composite responded even more sensitively than that on CdTe QDs or Au NPs alone
because of the presence of synergistic effects in CdTe-Au NPs film. The inhibition
of monocrotophos was proportional to its concentration in two ranges, 11,000ng/mL
and 215mg/mL, with a detection limit of 0.3ng/mL.
Similarly, Dus group also presented a novel strategy for a sensitive investigation
of the interaction between AChE and its small-molecule carbamate inhibitors [35].
370 13 Nanostructured Biosensing for Detection of Insecticides

Fig.13.1 Schematic diagram


for biosensor fabrication and
determination of
monocrotophos. Reprinted
with permission from Du
etal. [26]. 2008, Elsevier

With the signal amplification of Au NPs, the specific interactions between the Au
NP-labeled carbamate inhibitors (ALC1 and ALC2) and the immobilized AChE on
the sensor chip surface were readily examined. The association/dissociation con-
stants for the binding interaction between carbamate inhibitors and AChE were
reported for the first time. This Au NP labeling strategy is versatile and may be
applicable for the direct or competitive SPR kinetic assay of the interaction between
small-molecule inhibitors and their target proteins with high sensitivity.
The key aspect in the construction of this kind of biosensor is the immobilization
of AChE on the solid electrode surface with a high electron-transfer rate and bioac-
tivity. In order to settle it, a variety of matrix materials have been employed, such as
cobalt phthalocyanine (CoPc) [36], Au NPs [37, 38], CdTe QDs [39], multiwalled
carbon nanotubes (MWNTs) [21], Al2O3 [40], Prussian blue [41], polyaniline [42],
chitosan [43, 44], and so on.
Despite the significant focus of scientific research dedicated to AChE biosen-
sors, little success has been realized through real practical applications and com-
mercialization of these devices for solving real-world problems. A major limitation
of existing AChE biosensors is related to their inability to correctly differentiate
and identify particular analytes, so the selectivity for measuring AChE inhibitors is
very poor [45, 46]. In general, all organophosphorous and carbamate pesticides
and heavy metals inhibit the AChE activity with a different degree of inhibition,
which makes proper identification difficult. In other cases, the inhibition power
registered with AChE biosensors does not follow the same pattern as obtained with
the enzyme in solution. This has been attributed to the matrix used to immobilize
the enzyme [4749]. Due to this important limitation, AChE biosensors are mainly
attractive for measuring the total toxicity of the sample, rather than measuring a
13.2 Enzymes Used in Pesticide Biosensors 371

specific inhibitor. This will reflect the sum of all AChE inhibiting species present
in the sample. The most important challenge in the development of AChE biosen-
sors for practical applications is the transfer of these devices from pristine research
laboratory conditions to real-life and commercial applications. In this direction,
some critical parameters such as enzyme stability, reliability, and selectivity still
have to be improved.

13.2.3OPH-Based Biosensors for Pesticides

The detection of OPs using biosensors modified with ChE-ChO or ChE enzymes
relies on an inhibition of catalytic activity of ChE enzymes by OPs. Therefore, it is
a drawback of the sensors that the magnitude of the output signal correlates inversely
with the concentration of OPs in the sample. The protocol in measurements is some-
what complicated because the substrate of ChE has to be added in the sample solu-
tion before measurements, resulting in multiple steps needed for OP detection. In
addition, the inhibition of ChE by OPs is usually irreversible, and thus reactivation
of ChE activity is required for repeated use of the sensors, although this problem
can be circumvented by using disposable sensor tips.
In contrast to the inhibition mode of detection in the above biosensors, an
alternative direct biosensing route for the detection of OPs has been proposed by the
use of OPH [50], which can be used for continuous monitoring of OPs in the envi-
ronment. The foundation of these devices is the hydrolysis of OPs, producing two
protons as a result of the cleavage of the PO, PF, PS, or PCN bonds and an
alcohol, which in many cases is chromophoric and/or electroactive, catalyzed in a
highly specific manner by OPH [5153].
Because the organophosphate is the substrate for OPH, this process leads to
a direct determination of analyte, as the rate of signal generation is directly
proportional to the concentration of organophosphate. Among a variety of bio-
logical methods based on the biocatalytic activity of OPH, amperometric, poten-
tiometric, and optical biosensing devices have been developed for detecting OPs
[54]. Electrochemical biosensors in particular have been widely investigated to
monitor various pesticides, including OP compounds such as paraoxon [55],
parathion [56], sarin [57], and soman [58], via an enzyme-catalyzed hydrolysis
reaction by OPH due to their fast speed, high efficiency, low cost, and small
sample size [55].
Recently, Deo etal. [59] described an amperometric biosensor for organophosphate
pesticides based on a CNTmodified transducer and OPH biocatalyst. Chen et al.
developed OPH amperometric biosensors with carbon electrodes for monitoring OPs
[57]. The widespread interest in OPH-based electrochemical biosensors stems from
their simplicity, directness, and speed of determining OPs. The detection limit of OPH-
based biosensors could be further improved by either lowering the enzyme Michaelis
constant (Km) or increasing the bimolecular rate constant.
372 13 Nanostructured Biosensing for Detection of Insecticides

13.3Nanostructured Biosensor Design for Pesticide Analysis

Despite the fact that the above biosensors provide great advantages, their emergence
from the research laboratory to the marketplace has been slow. The obstacles to
exploitation have been fundamentally related to the presence of biomaterial in the
biosensor (immobilization of biomolecules on transducers, stability and selectivity
of enzymes and antibodies), the development of the sensor device (sensitivity and
reproducibility issues), and the integration of biosensors into complete systems.
Major fundamental and technological advances should be made in the near future
toward enhancing the sensitivity, selectivity, and reliability of OP biosensing
devices, and the emergence of nanotechnology may open new horizons and satisfy
the above targets [60].
In recent years, nanomaterials have been widely employed in electrode modifica-
tion and electrochemical sensor development. The nanomaterials and their applica-
tions have been the subject of many reviews [61]. The advantages of application of
nanotechnology in the field of biosensors are multiple. First, their extremely small
sizes offer high surface-to-volume ratios, leading to loading of more enzyme as well
as high interface sensitivity. Second, due to the unique nanosize effect, nanomaterials
such as metal and semiconductor nanoparticles have excellent optical or electrical
properties, which can be used as the highly sensitive transducer signals in biosen-
sors. Finally, enzyme engineering at the nanoscale may be one of the most promis-
ing techniques with respect to the design of more sensitive and selective AChE and
OPH variants. Altogether, nanotechnology can be expected to be an effective
approach to develop more sophisticated multianalyte detection systems with a low
cost.

13.3.1Carbon NanotubeBased Nanobiosensor for Pesticides

Since their discovery in 1991 [62], CNTs have been the target of numerous
investigations, due to their extraordinary conductivity, large surface areas, excellent
biocompatibility, and easy chemical functionalities, which make them ideal for the
development of compound-specific biosensors [63]. Recent studies have demon-
strated that CNTs are promising materials for electrochemical biosensors and show
great potential for use in the next generation of biosensors [18]. CNTs have been
considered potential electrode materials of electrochemical biosensors due to their
highly accessible surface area, electronic conductivity, stability, and capacity to
immobilize enzymes [20].
Most reported CNTs-based biosensors for detecting OPs are enzyme-modified
electrodes. These types of sensors take advantage of enzymatic reaction-generated
electroactive products, which can be sensitively detected on CNTs-modified elec-
trodes. Here, the CNTs on the electrode surface functions as both an electrode for
attaching enzymes and a transducer for enhancing the electrochemical response.
Moreover, the CNTs can promote the electron-transfer reaction between the signaling
molecules and the electrode.
13.3 Nanostructured Biosensor Design for Pesticide Analysis 373

Fig.13.2 Schematic
representation of layer-by-
layer electrostatic self-
assembly of AChE on
MWCNTs: (a) assembling
positively charged PDDA on
negatively charged
MWCNTs; (b) assembling
negatively charged AChE; (c)
assembling the second PDDA
layer. Reprinted with
permission from Liu and Lin
[19]. 2006, American
Chemical Society

Lins group reported a highly sensitive flow-injection amperometric biosensor


for paraoxon detection [19]. The layer-by-layer (LBL) electrostatic self-assembly
of AChE on multiwalled carbon nanotube (MWCNT)modified GCE is shown in
Fig. 13.2. Initially, about 20 mL of the acid-treated MWCNTs was cast on the
374 13 Nanostructured Biosensing for Detection of Insecticides

GCE surface and dried. More negative charges were introduced above this
MWCNT-modified GCE by dipping in 1M NaOH solution for 5min. This nega-
tively charged MWCNT/GCE was dipped into an aqueous solution of 1mg/mL
PDDA containing 0.5M NaCl for 20min, which led to the adsorption of a posi-
tively charged polycation layer (Fig.13.2a). Above this polycation layer, a nega-
tively charged AChE layer was adsorbed by dipping the PDDA/MWCNTs/GCE
in 0.2unit/mL of AChE in TrisHCl buffer solution pH 8.0 (Fig.13.2b). Finally,
in order to prevent the leakage of AChE from the electrodes surface, another
PDDA layer was adsorbed above this AChE layer (Fig.13.2c). This results in a
sandwich-like structure of PDDA/AChE/PDDA on the MWCNTs surface.
Transmission electron microscopy (TEM) results confirm the formation of LBL
nanostructures on MWCNTs. The flow-injection analysis (FIA) results show that
PDDA/AChE/PDDA/MWCNT/GCE exhibits good reproducibility and stability,
with no surface fouling effect. The relative inhibition of AChE activity was linear
with-log [paraoxon] at the concentration range 110120.1109M. The detec-
tion limit was reported as 0.41012M.
Similarly, Joshi etal. reported nM paraoxon detection at AChE-functionalized
MWCNT-modified screen-printed electrode (SPE) [17]. They immobilized AChE
by physical adsorption without any mediators. Further, Du et al. reported that
incorporation of silica solgel (SiSG) into an MWCNT matrix improved triazo-
phos detection [21]. The MWCNT-incorporated solgel matrix provided a bio-
compatible microenvironment around the enzyme, which efficiently prevented
the leakage of the enzyme from the film. Under optimum experimental condi-
tions, the inhibition of triazophos was proportional to its concentration, from 20
to 1,000 nM and 530 mM, respectively. The detection limit was 5 nM. The
determination of triazophos in garlic samples showed an acceptable accuracy.
The fabrication reproducibility of this sensor was good, and the stability was also
acceptable. The same group developed MWCNT as a new sorbent for the solid-
phase extraction (SPE) of OPs [64]. A combination of SPE with square-wave
voltammetric (SWV) analysis resulted in a fast, sensitive, and selective electro-
chemical method for the determination of OPs using methyl parathion (MP) as a
representative. Because of the strong affinity of MWCNT for phosphoric group,
nitroaromatic OP compounds can strongly bind to the MWCNT surface. The
macroporosity and heterogeneity of MWCNT allow a large amount of MP to be
extracted in less than 5min. The stripping response was highly linear over the
MP range of 0.052.0mg/mL, with a detection limit of 5ng/mL. The determina-
tion of MP in garlic samples showed an acceptable accuracy. The fast extraction
ability of MWCNT makes it a promising sorbent for various solid-phase
extractions.
Jus group reported a novel method for the immobilization of AChE by binding
covalently to a cross-linked chitosanMWCNT composite [65]. As shown in
Fig.13.3, the MWNTs were homogeneously distributed in the chitosan membrane,
which showed a homogeneous porous structure. The immobilized AChE could
catalyze the hydrolysis of ATCl with a Km of 177mm to form thiocholine, which
was then oxidized to produce a detectable signal in a linear range of 1.0500mm
13.3 Nanostructured Biosensor Design for Pesticide Analysis 375

Fig.13.3 Schematic representation of binding of AChE to MWCNTcross-linked chitosan com-


posite for flow-injection amperometric detection of organophosphorous insecticide. Reprinted
with permission from Kandimalla and Ju [65]. 2006, Wiley

and a fast response. MWCNT catalyzed the electrooxidation of thiocholine, thus


increasing the detection sensitivity. Based on the inhibition of OPs on the enzy-
matic activity of AChE, using sulfotep as a model compound, the conditions for the
flow-injection detection of the insecticide were optimized. Both the biocompatibil-
ity of chitosan and the inherent conductive properties of MWCNTs favored the
detection of the insecticide from 1.5 to 80mM along with good stability and repro-
ducibility. A 95% reactivation from inhibited AChE could be regenerated by using
2-pyridinealdoxime methiodide within 15min.

13.3.2Gold Nanoparticles as Transducer


for Detection of Pesticide

Au NPs have also been widely used in biosensors to immobilize biomolecules. The
ability of Au NPs to provide a stable immobilization of biomolecules retaining their
bioactivity is a major advantage for the preparation of biosensors. Furthermore, Au
NPs permit the direct electron transfer between redox proteins and bulk electrode
materials, thus allowing electrochemical sensing to be performed with no need for
electron-transfer mediators. Characteristics of gold nanoparticles, such as high
surface-to-volume ratio, high surface energy, ability to decrease the proteinmetal
particle distance, and functioning as electron-conducting pathways between pros-
thetic groups and the electrode surface, have been claimed as reasons to facilitate
electron transfer between redox proteins and electrode surfaces [66, 67]. With the
use of Au NPs, the efficiency and stability of the pesticide sensor have been greatly
amplified. Moreover, the nanoparticle matrix offers a very friendly environment to
the immobilized enzyme.
A simple strategy for the design of an electrochemical AChE sensor based on the
enzyme-induced growth of Au NPs without adding gold nanoseeds was proposed [24].
376 13 Nanostructured Biosensing for Detection of Insecticides

In this method, immobilized AChE mediated hydrolysis of ATCl and yielded a reducing
agent, thiocholine. In the presence of AuCl4, Au NPs were formed without the addi-
tion of any metal nanoparticles. Atomic force microscopy (AFM) images proved that
small spherical nanoparticles could grow on the enzyme-modified surface after incu-
bation in HAuCl4 and ATCl solution. An [Fe(CN)6]3/4 redox system was used as
a probe to indicate the process of electron transfer across the interface and also to
analyze the enzyme inhibitor quantitatively. The inhibition effect of malathion on
AChE was proportional to its concentration in the range 0.1500ng/mL, with detec-
tion limit of 0.03ng/mL.

13.3.3Quantum Dots as Transducers for Detection of Pesticides

QDs, with unique photophysical properties such as narrow and size-tunable


emission spectra, robust signal intensity, broad excitation band, and high
photochemical stability, have been shown to be ideal for use as fluorophores in
multiplexed bioanalysis. The unique electronic and photonic properties of semi-
conductor QDs have been used in a range of optoelectronic applications [68].
Specifically, the photophysical features of semiconductor nanoparticles are
employed to develop sensor [69] and biosensor systems [70], light-emitting
diodes [71], and lasers [72].
Willner and coworkers reported on an AChECdS nanoparticle hybrid system
used for the photoelectrochemical detection of AChE inhibitors [73]. CdS nanopar-
ticles were capped with a protecting monolayer of cysteamine and mercaptoethane
sulfonic acid. The enzymeCdS nanoparticle system may be employed as a versa-
tile photoelectrochemical label for biosensing events. Blocking the enzyme stimu-
lates nerve conduction that leads to the rapid paralysis of vital functions of living
systems. The capped CdS nanoparticles were covalently linked to an Au-electrode
functionalized with an N-hydroxysuccinimide active ester cysteic acid (Fig.13.4).
They demonstrated that enzyme inhibitors decreased the photocurrents, and thus the
nanoparticleAChE system acted as a biosensor for the respective inhibitor. The
CdS nanoparticleAChEATCh system may be a versatile photoelectrochemical
label for different biosensors, and the system may be further developed for the
biosensing of biological warfare.
Similarly, Du et al. reported a novel AChE biosensor that was constructed by
modifying glassy carbon electrode with QDs and gold nanoparticles through chito-
san microspheres to immobilize AChE [26]. The combination of CdTe QDs and
Au NPs promoted electron transfer and catalyzed the electrooxidation of thiocho-
line, thus amplifying the detection sensitivity. This novel biosensing platform based
on CdTe QDs-GNPs composite responded even more sensitively than that of CdTe
QDs or Au NPs alone because of the presence of synergistic effects in CdTe QDs-Au
NPs film. The inhibition of monocrotophos was proportional to its concentration in two
ranges, from 1 to 1,000 ng/mL, and 215 mg/mL, with a detection limit of 0.3ng/mL.
The proposed biosensor showed good precision and reproducibility and acceptable
stability and accuracy in garlic samples analysis.
13.4 Pesticide Immunosensors 377

Fig.13.4 Assembly of the CdS nanoparticle/AChE hybrid system used for the photoelectrochem-
ical detection of the enzyme activity. Reprinted with permission from Pardo-Yissar et al. [73].
2003, American Chemical Society

13.4Pesticide Immunosensors

Immunoassay-based sensors have been developed to exploit the high degree of


specificity and affinity of antibodies for specific antigens. In this particular applica-
tion, a given chemical, metabolite, or modified protein can act as the antigen, and a
pesticide response linked to antibody binding with the antigen. Based upon trans-
duction mechanism, immunosensors can be classified into electrochemical, optical,
piezoelectric, and nanomechanic immunosensors.

13.4.1Detection Methods for Pesticide Immunosensors

Electrochemical transducers are the oldest and most common methods used in
biosensors. The principle is based on the electrical properties of the electrode or

buffer that is affected by AbAg interaction. They can determine the level of pesti-
cides by measuring the change of potential, current, conductance, or impedance
378 13 Nanostructured Biosensing for Detection of Insecticides

caused by the immunoreaction. Electrochemical transducers combine the high


specificity of traditional immunoassay methods with the low detection limits and
low expenses of electrochemical measurement system, thus exhibiting great advan-
tages. They are not affected by sample turbidity, quenching, or interference from
absorbing and fluorescing compounds commonly found in biological samples.
Optical devices are the second most commonly used transducers. A general
optical sensor system consists of a light source and a number of optical compo-
nents to generate a light beam with specific characteristics. To direct this light to
a modulating agent, the system also has a modified sensing head and a photode-
tector. Optical immunosensors have been shown to be able to measure adsorbed
molecular layers, which utilize the evanescent wave to form the sensing device.
Different techniques can be used for creating an optical change, such as reflecto-
metric interference spectroscopy, interferometry, optical waveguide lightmode
spectroscopy, total internal reflection fluorescence, and surface plasmon resonance.
Optical immunosensors offer advantages of compactness, flexibility, resistance to
electrical noise, and small probe size. A label-free approach of detection is
preferred and is of high value. In addition, the use of very small amounts of
reagents makes optical immunosensors advantageous. It is important to wash the
surface before reading the signal.
Potentiometric immunosensors are based on measuring the changes in potential
induced by the label used, which occur after the specific binding of the AbAg.
They measure the potential across an electrochemical cell containing the Ab or Ag,
usually by measuring the activity of either a product or a reactant in the recognition
reaction monitored.

13.4.2Immunosensors for Pesticides

Immunochemical methods have already gained a place in the analytical benchtop as


alternative or complementary methods for routine pesticide analysis. They are fast,
economic, and at least as sensitive as usual chromatographic techniques. The number
of pesticides for which immunoassays have been developed is continually increasing
worldwide [74, 75].
Since pesticide molecules are organic compounds of molecular mass less than
1kD, the formation of an immunocomplex (AbAg) on a transducer surface with
these molecules in a direct immunoassay format does not usually produce signifi-
cant shifts in the physical parameters of transducer devices, so the sensitivity of this
type of assay for low-molecular-mass pesticide is limited. These molecules are,
therefore, coupled with some macromolecules (e.g., proteins or enzyme tracers) and
then used in a competitive approach based on binding-inhibition tests. This assay
format monitors the competition between labeled and unlabeled Ag for the limited
available binding sites of Abs immobolized on the transducers surface. By using a
fixed amount of immobilized Ab and labeled Ag, an inhibition curve is drawn by
13.4 Pesticide Immunosensors 379

introducing different amounts of unlabeled Ag. The concentration of unlabeled Ag


in a test sample can then be determined by comparison with the inhibition curve
plotted. Competitive immunosensor formats rely on the use of an Ag-tracer con-
jugate, which competes with the analyte for a limited number of binding sites of the
Ab immobilized on the transducer surface. Free binding sites of the Ab exposed to
the Ag are then monitored by binding to the immobilized Ab [76]. This can also be
accomplished by immobilizing Ag on the carrier surface.
In another approach, based on a flow-immunodetection system using a fluores-
cent detector, atrazine was screened with a high-throughput-sample capacity [77].
The immunochemical-detection principle was based on chromatographic separation
of the immunocomplex formed (AbAg or AbAg*) and the free Ag (Ag) by a
restricted access (RA) column, utilizing size-exclusion and reversed-phase mecha-
nisms. A fluorescein-labeled analyte (Ag*) was used in the competitive assay for-
mat with fluorescence detection. The speed and simplicity of the assay were its
greatest advantages, allowing measurement of the analyte to be carried out in less
than 1min. The LOD was 1.4nM (300pg/mL) for atrazine.
Recently, several interesting new technologies have emerged that are potentially
suitable for implementation into point-of-care (POC) diagnosis methods of nerve
agent exposure, and they could circumvent the nonavailability of highly specific
antibodies. For instance, a magnetic electrochemical immunoassay has been
reported based on the detection of phosphorylated AChE in plasma [78]. Basically,
this assay is based on the following two-step principle. In the first step, phospho-
serine-containing proteins are captured from the plasma sample by commercially
available antiphosphoserine antibodies that have been immobilized on magnetic
particles. In the second phase, the captured OP-inhibited AChE is detected with
antihuman AChE monoclonal antibodies, labeled with cadmium-source QDs. The
subsequent detection of the cadmium component with square wave voltammetry
(SWV) showed a linear response over a broad concentration range of phosphory-
lated AChE (Fig.13.5).
A related multistep procedure for the detection of phosphorylated AChE was
published by Liu et al. [79], consisting of a nanoparticle-based electrochemical
immunosensor. Instead of antiphosphoserine antibodies, zirconia nanoparticles
coated on SPE were employed for the first capturing step (Fig.13.6). This surpasses
the drawback of the scarce commercial availability of the antiphosphoserine anti-
bodies, as described above. Then cadmium QDlabeled antihuman AChE antibody
was used in the subsequent immunoreaction. The captured QDs were then deter-
mined by SWV detection, after release of the cadmium ions by the addition of acid.
In this way, the immunosensor could successfully detect phosphorylated AChE
down to a concentration of 8pM, and in preliminary experiments it was shown that
it could also deal with the detection of phosphorylated AChE in human plasma,
down to relevant inhibition levels. Immunosensors have great potential to become
cost-effective, sensitive devices for on-site monitoring of pesticide pollutants.
They are becoming an attractive option because Abs can be produced against
low-molecular-mass pesticides.
380 13 Nanostructured Biosensing for Detection of Insecticides

Fig. 13.5 Schematic illustration of magnetic electrochemical immunoassays of OP-AChE:


(a) plasma samples; (b) magnetic capture of OPAChE using amorphous MP-Ab1 conjugates;
(c) selective recognition of bound OPAChE using QD-Ab2 labels; (d) electrochemical SWV
analysis of cadmium released by acid from the captured QDs; and (e) representative SWV signal
output. Reprinted with permission from Wang etal. [78]. 2008, American Chemical Society

Fig.13.6 The principle of electrochemical immunosensing of phosphorylated AChE. (a) ZrO2


nanoparticle-modified SPE; (b) selective capture of phosphorylated AChE adducts; (c) immu-
noreaction between bound phosphorylated AChE adducts and QD-labeled anti-AChE antibody;
(d) dissolution of nanoparticles with acid following an electrochemical stripping analysis.
AChE: purple, ZnO2: green, electrode: yellow. Reprinted with permission from Liu etal. [79].
2008, Wiley
13.5 Nanotechnology for Biomonitoring of AChE Activity and Pesticides 381

13.5Nanotechnology for Biomonitoring of AChE Activity


and Pesticides

Biomarkers are used in many aspects of health surveillance to identify disease


presence, track disease progression, monitor drug delivery or metabolism, or moni-
tor chemical exposure. There is a recognized need to expand and improve biomarker
identification and quantification. The covalent bond between OPs and the active site
serine of intact ChE is stable. OP-AChE adduct becomes irreversibly bound to the
enzyme after one of the alkyl groups on the OPs is lost in a step called aging [80].
The dealkylated OP makes a stable salt bridge with the protonated histidine of the
catalytic triad, so that histidine is no longer available for the dephosphorylation step
that would otherwise have restored the enzyme to an uninhibited state [81]. Hundreds
of scientists have contributed to this understanding of the mechanism of OP inhibi-
tion on AChE and BChE activity. Their studies are the foundation for the use of
AChE and BChE as biomarkers of OP exposure.

13.5.1Biomarkers of Organophosphate Pesticide Exposure

OP-inhibited AChE and BChE have been established as biomarkers of OP expo-


sure. The special features that make them good biomarkers are (1) they react rapidly
with OPs at low OPs concentrations, (2) symptoms of acute toxicity always corre-
late with the inhibition of AChE and BChE, (3) AChE is present in human red blood
cells, while BChE is present in human plasma, making it possible to test for OP
exposure by measuring enzyme activity in a blood sample, (4) enzyme activity
assays for AChE and BChE are simple and inexpensive, (5) the OP adduct for com-
mon pesticides is relatively stable, making it possible to detect exposure days after
the actual event, (6) the mechanism of irreversible inhibition of AChE and BChE
activity by OPs is understood [82].
Human plasma can serve as an ideal source for biomarkers of exposure, due to the
ease of sample collection and the expansive range of proteins held within the plasma
compartment. In terms of monitoring OP exposure, the esterases present in plasma
are ideal targets for assessing their inhibition and modification. In humans, measure-
ment of AChE inhibition is one standard method of detecting OP exposure [83], with
the caveat that the inhibition of AChE usually overestimates the inhibition of AChE
in the central nervous system, depending on the pharmacokinetics of the agent and
the amount of time since exposure. In addition, there are other difficulties with this
method. The first difficulty is due to interindividual variability. While the intraindi-
vidual coefficient of variation (CV) is about 10%, the interindividual CV is between
10 and 40% [84]. Measuring the individuals preexposure activity levels would
improve precision, but this is only practical in certain situations, such as monitoring
agricultural worker exposure during the growing season. Preexposure activity levels
are rarely available for cases of nonagricultural exposure. Second, measuring AChE
382 13 Nanostructured Biosensing for Detection of Insecticides

activity levels will not identify the specific inhibitory OP agent. Many animals,
including pig and rodent species, have abundant carboxylesterase (CE) in plasma. In
humans, CE is not free in plasma, but can be purified from a membrane-bound form
on monocytes [85]. While perhaps not an ideal biomarker, CE can be inhibited by an
array of OPs. Due to its freely soluble presence in plasma, BChE has been a popular
target for biomarker research. Newer methods have been developed that can identify
and quantify OP exposure based on BChE modification. MS detection is a major
advance in diagnosing OP exposure.
A disadvantage of AChE or BChE as biomarkers is that the OP residues are
prone to aging, in which the O-alkyl group is cleaved, thus losing key structural
information; this occurs particularly rapidly with soman. Also, the entire OP
moiety may be partially displaced from nonaged residues if therapeutic oximes
have been administered. Albumin adducts, which are much more stable with
regard to aging or oxime displacement, have been identified in experimental
animals but appear to be valuable biomarkers at higher exposure levels in com-
parison to BChE adducts.

13.5.2Biomonitoring of ChE Activity

Biomonitoring is an efficient approach for the detection and evaluation of exposures


to OPs. It primarily focuses on measuring OP concentrations or their metabolites in
blood and urine or, alternatively, measuring ChE activities in blood [8689].
Measuring OPs or the associated metabolites is very complicated because it requires
sophisticated analytical methods, such as liquid chromatography coupled with mass
spectrometry (LC-MS), GC-MS, or high-pressure liquid chromatography (HPLC)-MS
[87, 8991]. Although these methods are sensitive and accurate, they are very expen-
sive, time-consuming, and laboratory-oriented, and they require a pretreatment of the
sample and highly qualified technicians. For the alternative approach to measure
ChE activities, a blood assay is usually used, but it is invasive and time-consuming,
and the results are not immediately available [86, 92]. In contrast, a saliva ChE
enzyme-activity assay offers a simpler approach for biomonitoring OPs because of
the convenience of sampling. Moreover, it benefits greatly from less interference
because saliva contains fewer proteins than blood.
An assay for salivary ChE activity has recently been reported, and it shows
potential for biomonitoring exposures to OPs [9395]. The ChE activities are con-
ventionally measured with the Ellman assay using Ellman reagent [90, 95, 96].
Some alternative approaches, such as radiometric assay [94], immunoassay [97],
fluorescence [98, 99], chemiluminescence [100, 101] or MS [102], have been
reported for the sensitive detection of enzyme activities. However, these assays are
tedious, time-consuming, or both, and they require expensive and sophisticated
instruments, which makes these methods unsuitable for rapid and on-site applica-
tions. Therefore, it is imperative to develop simple, rapid, and sensitive biomoni-
toring methods for on-site and emergency use.
13.5 Nanotechnology for Biomonitoring of AChE Activity and Pesticides 383

Electrochemical techniques combined with a flow-injection system offer a simple


and inexpensive approach for rapid and on-site biomonitoring of enzyme activities
[103]. They have been widely used in various chemo/biosensors because of their
simplicity, low cost, and high sensitivity and, particularly, because the device can be
miniaturized [103108]. The sensitivity of these types of chemo/biosensors can be
further enhanced by using various nanotechnology-based amplifications [109, 110].
Lins group reported the development of a CNT-based amperometric sensor with a
flow-injection system for the characterization of enzyme activities in rat saliva sam-
ples [111]. The flow-injection system provides great advantages for the on-site, real-
time, and continuous detection of biomolecules and requires only a small volume of
the sample. The sensor takes advantage of the electrocatalytic properties of CNTs,
which makes it feasible for a sensitive electrochemical detection of the products from
enzymatic reactions at low potential. It offers a new biomonitoring approach for a
rapid, inexpensive, and highly sensitive detection of exposures to OP compounds.
These methods have an inherent disadvantage since a control or baseline is
needed because the measured value of enzyme activity has to be compared with the
unexposed normal (baseline) values. To circumvent this problem, a statistically
derived value of enzyme activity measured from a large sample size of population
generally serves as the control. However, these methods are not accurate because of
a large variability in baseline values derived from the variation of enzyme activity
between individuals (e.g., sex, age, ethnicity, etc.) and the deviation of measure-
ment methods from different laboratories [112].
In general, considering inter- and intraindividual variations in the normal levels
of ChE, the exposure that results in inhibition of less than about 20% (especially if
clinical symptoms are absent) may not be easily detectable or provide reliable evi-
dence for current available screening methods unless recent control values of that
particular individual are available [112]. In a worst-case situation, these methods
may provide ambiguous results. Moreover, biomonitoring of individuals with a low
level of OP exposure (<10% inhibition) may be problematic without a preexposure
baseline enzyme determination with these methods.
It was found that some compounds such as nucleophiles [21, 113116] or fluo-
ride ions [117120] can reactivate the phosphorylated complex, yielding a restored
enzyme activity. This process is called reactivation. Based on the reactivation, a
number of assay methods such as measuring released OP from phosphorylated
enzyme (fluoride ion-induced reactivation) using MS have been developed for the
detection and identification of exposures to OPs [117, 118].
In parallel, oxime-induced reactivation has been used to evaluate drug efficacy
and for therapeutic intervention as a treatment for OP poisoning. The most com-
monly used pyridinium oxime is pralidoxime iodide (2-PAM) [114]. Current
biomonitoring methods for OP exposures ignore evaluation of the restored enzyme
activity via reactivation. If the activity of phosphorylated enzyme could be regener-
ated completely to its original state and activity determined, this restored enzyme
could serve as its own control or baseline. Therefore, a double test of enzyme activ-
ity in biological samples before and after reactivation could be exploited to quantify
the extent of enzyme activity/inhibition.
384 13 Nanostructured Biosensing for Detection of Insecticides

Fig. 13.7 Schematic illustration of OP-AChE formation and AChE regeneration process by
reactivator. Reprinted with permission from Du etal. [121]. 2009, American Chemical Society

Motivated by this idea, Du, Lin, and colleagues [121] developed a portable,
rapid, and sensitive assessment of subclinical OP exposure based on the reactivation
of cholinesterase (ChE) from OP-inhibited ChE using rat saliva (invitro) using an
electrochemical sensor coupled with a microflow-injection system (Fig.13.7). The
sensor was based on a CNTmodified screen-printed carbon electrode (SPE), which
was integrated into a flow cell. Due to the extent of interindividual ChE activity
variability, ChE biomonitoring often requires an initial baseline determination
(noninhibited) of enzyme activity, which is then directly compared with activity
after OP exposure. They describe an alternative strategy where the reactivation of
the phosphorylated enzyme was exploited to enable the measurement of both inhib-
ited and baseline ChE activity in the same sample. Paraoxon was selected as a model
OP compound for invitro inhibition studies. A level of 9295% ChE reactivation
can be achieved over a broad range of ChE inhibition (594%) with paraoxon. The
extent of enzyme inhibition using this electrochemical sensor correlates well with
conventional enzyme-activity measurements. Based on the double determinations
of enzyme activity, this flow-injection device has been successfully used to detect
paraoxon inhibition efficiency in saliva samples, which demonstrated its promise as
a sensitive monitor of OP exposure in biological fluids.

13.6Conclusions

This chapter describes recent developments in biosensors for detecting OP pesticides.


Different detection modes using different enzymes are applicable for constructing
OP biosensors using electrochemical principles. A merit of electrochemical biosen-
sors in OP detection is that a portable device can easily be prepared for in-field
References 385

analysis. The easy operation of biosensors is another merit as compared to


chromatographic or spectroscopic measurements. Therefore, biosensors have a high
potential for the fast on-site screening of OP pesticides.
Current efforts in pesticide biosensors are directed toward the development of
more reliable systems with increased selectivity and sensitivity. Most pesticide
biosensors have shown excellent characteristics for synthetic samples, but they are
not sufficiently robust in real samples, and they do not offer adequate selectivity.
Most of the existing limitations could be directly related to the selectivity in multi-
composite mixtures and complex matrices and the inability of identifying a specific
OP analyte. However, their sensitivity is sufficient to detect a minimum level of
pesticides and heavy metals imposed by regulatory agencies, and they remain attrac-
tive as alarms or screening devices.
The identification of new biomarkers of OP exposure may also lead to an under-
standing of why some people are intoxicated by low doses of OP that have no effect
on the majority of the population. The new motif for OP binding to tyrosine may
lead to new antidotes for OP poisoning; for example, peptides containing several
tyrosines and several arginines may be effective OP scavengers.

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wwwwwwwwwwwww
Chapter 14
Carbohydrate Detection Using Nanostructured
Biosensing

14.1Introduction

Carbohydrates, which are defined as polyhydroxyaldehydes or polyhydroxyke-


tones, or larger compounds that can be hydrolyzed into such units, are ubiquitous
in the living world. Carbohydrates act as the sources of energy and carbon in
plants and animals and are the important structural elements in plant cell walls as
well as in the extracellular matrix of animal and human tissues. Like nucleic acids
and proteins, carbohydrates are involved in many biological processes and metab-
olism and seem to play critical roles in determining biological functions [1].
Glycosylation also affects the biological activity, lifetime, cellular uptake, and
specificity of these proteins [2]. Therefore, the study and characterization of car-
bohydrates has become increasingly important and has emerged as the new fron-
tier for elucidating fundamental biochemical processes and for identifying new
pharmaceutical substances.
This chapter describes the basic structure and biological roles of glycans, sum-
marizes the techniques used to identify carbon hydrate and its derivates, and spe-
cifically focuses on addressing the recent accomplishments in the use of nanoscale
materials for the detection and quantitation of carbohydrates with amplified readout
and higher signal throughput. Because glucose is technically a monosaccharide
rather than a carbohydrate, glucose sensing with a nanostructure is not discussed,
although it is an active area of research due to the profound health effects of aber-
rant glucose levels in diabetics. Readers can refer to the previous reviews that
addressed on the construction of glucose biosensors [3, 4].

H. Ju et al., NanoBiosensing: Principles, Development and Application, 393


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0_14, Springer Science+Business Media, LLC 2011
394 14 Carbohydrate Detection Using Nanostructured Biosensing

14.2Structural Depiction of Glycans

14.2.1Basic Structural Unit of Glycans

The basic structural unit of carbohydrate, which cannot be hydrolyzed into a simple
unit, is a monosaccharide. There are two types of monosaccharides: aldoses and
ketoses. A monosaccharide with an aldehyde group at the end of the carbon chain is
called an aldose, whereas that with a ketone group at an inner carbon of the carbon
chain is called a ketose. Free monosaccharides can exist in open chain or ring forms
(Fig. 14.1). Ring forms of the monosaccharides are the rule in oligosaccharides,
which are branched or linear chains of monosaccharides attached to one another via
glycosidic linkages. The ring form of a monosaccharide generates a chiral center at
C-1 for aldo sugars or at C-2 for keto sugars. A glycosidic linkage involves the
attachment of a monosaccharide to another residue, typically via the hydroxyl group
of this chiral center, which can be a or b linkages. These two types of linkages lead
to very different structural properties and biological functions upon sequences [5].

14.2.2Glycoconjugate

A glycoconjugate is a compound in which one or more monosaccharide or oligosac-


charide units (glycone) are covalently linked to a noncarbohydrate moiety (agly-
cone). The portions of the molecule comprising glycans vary greatly, from being
minor in amount to being the dominant component. It is striking that sugar chains
make up a substantial portion of the mass of most glycoconjugates (Fig. 14.2).
Unlike nucleotides and proteins, which are linear polymers having only one basic
type of linkage, each monosaccharide can theoretically generate an a or a b linkage
to any position on another monosaccharide in a chain. As the number of units in the
polymer increases, this difference in complexity becomes even greater. Fortunately,
naturally occurring biological macromolecules contain relatively few of the possible
monosaccharide units in a limited number of combinations [6, 7].

14.2.3Major Classes of Glycoconjugates and Oligosaccharides

Varieties of modifications on oligosaccharides enhance the diversity of oligosac-


charides in nature and frequently serve to mediate specific biological functions. The
hydroxyl groups of different monosaccharides can be subject to phosphorylation,
sulfation, methylation, O-acetylation, or fatty acylation. Amino groups can remain
free or be N-acetylated or N-sulfated. Carboxyl groups are occasionally subject to
lactonization to nearby hydroxyl groups [5, 8].
14.2 Structural Depiction of Glycans 395

Fig.14.1 Open chain and


ring forms of galactose

Fig.14.2 Schematic
representation of the
Thy-1-glycoprotein,
including the three N-glycans
and a glycophospholipid
(GPI-glycan) anchor whose
acyl chains would normally
be embedded in the
membrane bilayer

The common classes of oligosaccharides found on eukaryotic cells are primarily


defined according to the nature of the linkage (core) regions to the aglycone (protein
or lipid). An N-glycan [N-linked oligosaccharide, N-(Asn)-linked oligosaccharide] is
a sugar chain covalently linked to an asparagine residue of a polypeptide chain within
the consensus peptide sequence: Asn-X-Ser/Thr. N-glycans share a common penta-
saccharide core region and can be generally divided into a high-mannose type, a
complex type, and a hybrid type. An O-glycan [O-linked oligosaccharide, O-(Ser/
Thr)-linked oligosaccharide] is typically linked to the polypeptide via N-acetyl-
galactosamine (GalNAc) to a serine or threonine residue and can be extended into a
variety of different structural core classes. However, since the O-glycan linkage is the
best known, it is often describe by the generic term O-glycan. A glycophospholipid
anchor is a glycan bridge between phosphatidylinositol and a phosphoethanolamine
in amide linkage to the carboxyl terminus of a protein. A glycoprotein is a glycocon-
jugate in which a protein carries one or more oligosaccharide chains covalently
attached to a polypeptide backbone, usually via N- or O-linkages. A mucin is a large
glycoprotein that carries many O-glycans that are often closely spaced (clustered).
A glycosphingolipid (glycolipid) is an oligosaccharide usually attached via glucose
396 14 Carbohydrate Detection Using Nanostructured Biosensing

or galactose to the terminal primary hydroxyl group of the lipid moiety ceramide,
which is composed of a long chain base and a fatty acid. These are only the most
common classes of glycans reported in eukaryotic cells. These are several other less
common types found on both sides of the cell membrane in animal cells [912].

14.3Biological Roles of Glycans

Carbohydrates are involved in many biological processes and metabolism. They


appear to have critical roles in determining biological functions and affecting wide-
ranging physiological processes [1]. A variety of specific genetic defects that express
specific defects in glycan biosynthesis were defined in mutant variants of cultured
cells. These spatially and temporally controlled patterns of glycan expression imply
the mechanistic involvement of the glycans in many processes [1217]. The more
specific biological roles of oligosaccharides are often mediated by unusual glycan
structures, unusual presentations of common structures, or further modifications of
the saccharides themselves.
The biological roles of glycans span the spectrum from those that are trivial to
those that are crucial for the development, growth, function, or survival of organism.
Because of the ubiquitous and complex nature of glycans, the biological roles of
glycans are quite varied [8, 1823]. The biological roles of glycans can be broadly
divided into two groups (Fig.14.3). One group relies on the structural and modula-
tory properties of glycans, and the other relies on the specific recognition of glycan
structures by other molecules (generally, receptor proteins or lectins). The latter
category can be further subdivided into two major groups: those involving recogni-
tion by endogenous receptors within the same organism and those resulting from
recognition by endogenous agents. The last group consists mostly of pathogen
receptors and toxins, but it can also include symbiotic agents.

Fig.14.3 General classification of


the biological roles of glycans
14.5 ProteinGlycan Interactions 397

14.4Difficulty in Studying Genetic Glycosylation Defects

Unlike oligonucleotides and proteins, glycan chains are rarely expressed in a linear,
unbranched fashion, and even when they are, such chains are often subject to various
modifications [23, 24]. Changes in expression of glycans are also found in the set-
ting of transformation and progression to malignancy. The evolution of glycosyla-
tion is clearly shared, although there are unique features of glycosylation in different
animals, and an increasing complexity is often seen in higher forms. Intra- and
interspecies variations in gycosylation are also relatively common [1217].
Whenever a new probe (e.g., antibody or lectin), specific for a particular glycan, is
developed for fully understanding the biology of glycoconjugates, it requires
in-depth knowledge of the primary sequence and three-dimensional structure of
carbohydrate chains in relation to cellular activation, embryonic development,
organogenesis, and differentiation. These spatially and temporally controlled
patterns of glycan expression imply the mechanistic involvement of the glycan in
many processes. However, the biological consequences of altering glycosylation in
various systems seem to be highly variable and unpredictable. A given glycan can
also have different roles in different tissues or at different times in development.
Such unusual glycans or modifications are also more likely to be targets for
pathogens and toxins. Finally, since genetic defects in glycosylation are easily
obtained in cultured cells, but often have limited consequences, many major func-
tions of glycans may be imperative only within an intact organism [21, 22, 25].

14.5ProteinGlycan Interactions

The concept of glycans being specifically recognized by proteins dates back to


Emil Fisher, who used the phrase lock and key to refer to enzymes that recog-
nize specific glycan substrates [26]. The first glycanprotein interactions revolved
around enzymes, such as the endoglycosidase lysozyme, and enzymes involved in
intermediary metabolism, such as glycogen and starch synthases and phosphory-
lases. Lysozyme, which was the first carbohydrate-binding protein to be crys-
tallized, was subsequently shown to be a highly specific endoglycosidase, capable
of specifically cleaving b14 linkages in bacterial peptidoglycan. Glycogen syn-
thetase was found to generate a14 glucosyl residues in glycogen, whereas other
branching and debranching enzymes recognized a16-branched glucose residues
[2730]. Other glycan-binding proteins whose three-dimensional structures are
of historical significance are concanavalin A and influenza virus hemagglutinin
[3133]. In addition, critical information to the development of this field was
gathered by Lemieux, Kabat and coworkers in studies on the combining sites of
lectins and antibodies toward specific blood group antigens [34]. In discussing
these interactions, the term lectin is now generally used to denote proteins with
glycan-binding activity.
398 14 Carbohydrate Detection Using Nanostructured Biosensing

14.6Techniques Used to Identify a Carbohydrate


and Its Derivates

To fully understand the biology of glycoconjugates requires an in-depth knowledge


of the primary sequence and three-dimensional structure of carbohydrate chains.
The complete sequencing of oligosaccharides is difficult to accomplish by a single
method and therefore requires iterative combinations of physical and chemical
approaches that eventually yield the details of the structure under study. Likewise,
glycosylation can be used to study enzymes (endoglycosidases and exoglycosi-
dases), lectins (carbohydrate-binding proteins), chemical modification or cleavage,
metabolic radioactive labeling, glycosylation inhibitors and primers, antibodies,
molecular cloning of glycosyltransferases, and the genetic manipulation of glycosy-
lation in intact cells and organisms. The direct invitro synthesis of glycans using
chemical and enzymatic methods has also taken great strides forward in recent
years, providing many new tools for exploring glycobiology. The generation of
complex oligosaccharide libraries by a variety of routes has further enhanced this
interface of chemistry and biology.
Many strategies have been devoted to the detection and quantification of carbo-
hydrates, including electrochemical analysis coupled with capillary electrophoresis
(CE) and/or liquid chromatography (LC) [35], fluorescence spectroscopy [3638],
molecularly imprinted polymer coupled with quartz crystal microbalance (MIP-
QCM) [39], ion-selective field-effect transistor (ISFET) [40], nuclear magnetic
resonance (NMR) [41], circular dichroism (CD) spectroscopy [42], fluorescence
resonance energy transfer (FRET) [43], mass spectrometry (MS) [44, 45], and so
on. The construction and the mechanism of carbohydrate-based biosensors have
been specifically summarized by the previous reviews [1, 4648].

14.6.1General Considerations for Analyzing the Primary


Structure of a Carbohydrate

For most carbohydrate, the secondary and higher-order structures in solution are not
readily defined, due to their inherent flexibility. The structural diversity of naturally
occurring glycans is great, making it difficult to define a universal protocol for their
analysis. The building blocks of complex carbohydrates (i.e., the monosaccharides)
exhibit very similar structures; thus, their differentiation is more involved than that
of amino acids. The structural analysis of carbohydrates requires a flexible approach,
which all but precludes full automation of the process. Only within a particular sub-
class of carbohydrate structures (e.g., for N- and O-glycans commonly found in gly-
coproteins) can some general techniques be applied universally. At any given juncture
in the procedure, the choice of methodology is dictated by the amount and purity of
carbohydrate material available. If pure carbohydrate material is abundantly
available, the goals for structural characterization can be set as high as possible:
14.6 Techniques Used to Identify a Carbohydrate and Its Derivates 399

Investigators should be able to delineate the complete primary structure. In situations


where purity and/or amounts are limiting factors, partial characterization may still be
possible. Characterization of carbohydrate dynamics by experimental and theoretical
methods remains an area of active research.

14.6.2Detection of Carbohydrates

The approaches taken to detect carbohydrates in glycoconjugates include chemical


reactions and radioactive labeling, as well as detection with specific lectins or anti-
bodies. Detection methods based on the susceptibility of sugars to periodate oxida-
tion have traditionally been used to identify glycoproteins in gels with the periodic
acidSchiff (PAS) reaction. Commercially available kits now allow the detection of
510ng of glycoprotein, using the same periodate reaction with subsequent ampli-
fication by means of biltinhydrazide/streptavidinalkaline phosphatase. Lectin
overlays of blots can also be used to detect the presence of carbohydrates, with
comparable sensitivity.
Alternatively, proteins metabolically labeled with radioactive sugar precursors
can be detected directly by autoradiography. Cells included with 3H- or 14C-labeled
mannose, glucosamine, galactose, or fucose will incorporate the label into the gly-
can chains of glycoproteins, glycolipids, and proteoglycans. Such molecules can be
detected as radioactively labeled bands when a mixture of proteins is analyzed by
SDS-PAGE, or the lipid extract is analyzed by TLC. The ability to label proteins
with specific radioactive precursors (e.g., myo-inositol, ethanolamine) may suggest
that they are GPI-anchored. Proteoglycans containing radioactively labeled GAGs
can be detected using polysaccharide lyases. Another approach is to transfer a label
from radioactive sugar nucleotides to endogenous glycans using purified glycosyl-
transferases (e.g., O-linked GlcNAc residues can be detected by using galactosyl-
transferases and UDP-[3H] Gal). Glycosaminoglycan chains on proteoglycans can
be liberated with a nonspecific protease or by alkaline b-elimination. GAGs are then
isolated by anion-exchange chromatography or cetyl pyridinium chloride/ethanol
precipitation. In radioactively labeled samples (e.g., with 35SO4 or [3H] GlcNH2),
GAGs are quantitated by directed scintillation counting, whereas nonradioactive
GAGs are usually quantified by measuring the content of uronic acid.
When chemical radioactive labeling reactions are applied, glycoconjugates will
incorporate radioactively if specific structural features are present (e.g., mild perio-
date oxidation of terminal sialic acid side chains followed by reduction of the
resulting aldehyde with sodium [3H] borohydride). These radioactive labeling
approaches have the advantage of being easy to perform and to monitor. Radioactively
labeled sugar chains can also be isolated following the same methodology used for
nonlabeled sugars with the advantage that radiometric purity is much easier to
achieve and sufficient for further analysis. However, the information obtained from
such analyses is limited, and a complete structural identification requires the
isolation of nonlabeled material.
400 14 Carbohydrate Detection Using Nanostructured Biosensing

14.6.3Linkage Analysis

The structural analysis of complex carbohydrate is a labor-intensive and time-


consuming endeavor, and multiple iterative methods are often needed to obtain a
complete picture of the structure.

14.6.3.1Determination of Linkage Positions

The next question en route to the complex structural characterization of an iso-


lated oligosaccharide concerns the linkages of the individual units to one another.
Methylation analysis, an ingenious approach for determining linkage positions,
was developed in the late 1960s and early 1970s. The principle of this method is
to introduce on each free hydroxyl group of the native oligosaccharide a stable
substituent (methyl group). The glycosidic linkages are then cleaved, producing
individual monosaccharide residues with new free hydroxyl groups that appear at
the positions that were previously involved in a linkage. The partially methylated
monosaccharides were originally analyzed by cellulose column chromatography
and identified by a laborious process. Today, the monosaccharides are derivatized
to produce volatile molecules amenable to GLC-MS analysis. The most common
derivatization strategy involves reduction of the monosaccharides to produce
alcohols at C-1 (eliminating the formation of ring structures), followed by deriva-
tization (e.g., acetylation) of free hydroxyl groups to produce volatile molecules.
Individual components of the mixture of partially methylated (methyl groups
mark the hydroxyl groups that were originally free), partially acetylated (acetyl
groups mark hydroxyl groups originally involved in glycosidic linkages and ring
structures) monosaccharide alditols can be identified by GLC-MS. However,
methylation analysis indicates neither which residues are attached to each other
(i.e., does not provide sequence information) nor if a particular linkage is of the a
or b configuration.

14.6.3.2Determination of Chirality

Some information of linkage configuration of each residue (chirality, a or b) can


be obtained from NMR profiling and sequential exoglycosidase digestions. The
NMR spectrum typically indicates how many residues there are (counting the chi-
ral signals) and how many of them belong to each chiral type. Also, the exact posi-
tions of H-1 signals in NMR spectrum are more or less characteristic of the type of
monosaccharide and its substitution pattern. Cleavages by a or b exoglycosidases
not only indicates the chirality of specific terminal sugar residues, but also gives
added information regarding internal regions of the chain. Some glycosidases will
cleave linkages of residue at specific positions of monosaccharide, allowing
detailed structural conclusions. Now, the number of such available enzymes is
14.6 Techniques Used to Identify a Carbohydrate and Its Derivates 401

limited. If sufficient quantities of carbohydrate are available, the chirality of a


particular monosaccharide residue in a glycan is more reliably determined by 1H
NMR spectroscopy.

14.6.3.3Sequence Analysis

NMR spectroscopy is the only technique that has the potential for full structural
characterization of a glycan with little or no assistance from other methods. A com-
plete structural investigation requires both the 1H and 13C NMR spectra of the oligo-
saccharide by a combination of two-dimensional NMR techniques such as correlated
spectroscopy (COSY) and total correlation spectroscopy (TOCSY) for 1H, and then
heteronuclear single-quantum coherence (HSQC) for 13C. However, in instances of
inadequate sample for these two-dimensional 1H13C NMR experiments, data pro-
vided by other techniques are required. A less rigorous NMR approach for glycan
sequencing relies on two-dimensional 1H NMR spectroscopy, using through-space
effects (nuclear Overhauser effects [NOEs]) as the sole source of evidence for link-
ing position and sequence. The sequence information of intact derivatized oligosac-
charides was provided by various MS approaches, such as GLC-MS, direct-inlet
EI-MS, fast atom bombardment (FAB-MS), and liquid secondary ion-mass spec-
trometry (LSIMS). These techniques yield molecular ions and only some fragmen-
tation. The spectra can be interpreted to obtain some sequence information of
carbohydrates much larger than those amenable to EI- and chemical ionization
(CI)-MS. In some instances, the sequence information of native glycoconjugate
molecules can be obtained because their physical properties make them good pro-
ducers of ions on the surface of the matrix used for the analysis. These MS tech-
niques are capable of analyzing mixtures of oligosaccharides and provide (1) the
molecular weight (MW) of the oligosaccharides, (2) the heterogeneity of the sam-
ple, (3) structural information, for example, on branching, and (4) structural infor-
mation on individual components (using MS/MS). Newer ionization approaches
introduced in the 1990s include electrospray ionization (ESI) and matrix-assisted
laser-desorption ionization (MALDI). Today, ESI-MS has mostly replaced FAB-MS
for peptide- and glycopeptide-mapping purposes. Although MALDI-time of flight
[TOF]-MS still lags behind other MS approaches in terms of resolution and mass
accuracy, its results are far better than those obtained by SDS-PAGE or SEC.

14.6.3.4Mapping of Glycosylation Sites

If each of the glycans isolated from a glycoconjugate is analyzed using the appro-
priate combination of approaches, a complete picture of the glycoconjugate can be
achieved. The ultimate level of glycoprotein primary structure analysis involves
the detailed determination of the particular glycans present at each glycosylation
site. This could be accomplished, for instance, by the production and mapping of
peptides, the identification and preparative isolation of those peptides containing
402 14 Carbohydrate Detection Using Nanostructured Biosensing

sugars, and the study of each peak using the methods described above. Alternatively,
current state-of-the-art direct MS analysis makes it possible to accomplish this
task at the level of the intact glycoconjugate.

14.7Nanotechnology

Nanobiotechnology describes the use of nanotechnology in biological systems [49].


The unique properties of nanoscale materials offer excellent prospects for designing
ultrasensitive and selective bioassays for nucleic acids, proteins, and carbohydrates.
The creation of nanomaterials for specific sensing applications greatly benefits from
the ability to vary the size, composition, and shape of the materials, and hence their
physical properties [50]. Particular recent attention in designing new bioassays has
been given to nanoparticles and nano-carbon materials. Nanoparticles of different
compositions and dimensions have been widely used as versatile and sensitive
tracers for the electronic, optical, and microgravimetric transduction of different
biomolecular recognition events [5155]. Benefitting from the signal amplifying
protocol of nanoparticle labels, the formation of nanoparticlebiomolecule
assemblies provides the basis for ultrasensitive optical and electrical detection
protocols, which can meet the high sensitivity demands of modern bioassays [50].
Carbon-based nanomaterials, for example, carbon nanotubes and carbon nanofibers,
have been applied to modify metal or carbon surfaces in connection with
electrochemical [5658] and field-effect transistor [5961] sensing. With recent
advances in nanotechnology, nanomaterial-based biosensors have become simple
yet efficient tools to measure the concentration of analytes and the response-time
functions of target biomolecules to the drugs or the toxic reagents on microsurfaces
and nanosurfaces using miniaturized, portable devices [6264].

14.7.1Analysis of Carbohydrates in Biological System with


Nanostructure Device/Components

There are a variety of difficulties associated with the detection of carbohydrates in


the established methods, including monosaccharide determination in any possible
matrix, lack of sensitivity and selectivity, difficulty in assessing the different glyco-
forms of a glycoprotein, oligosaccharide mapping, and separation of highly poly-
meric polysaccharides and proteoglycans [46, 65]. Since generally carbohydrates
do not contain intrinsic chromophores (neither fluorescent nor emitting in the
UV-visible range), electrochemical approaches for carbohydrate determination have
become increasingly important in a variety of biological and pharmaceutical
samples [35]. Historically, enzyme-based electrochemical detection strategies for
carbohydrates are developed because direct electrochemical detection of saccharide
14.7 Nanotechnology 403

Fig. 14.4 The structure of hesperidin (top) and below the proposed ECirrevE mechanism to
explain the observed voltammetry in aqueous solution. Reprinted with permission from Sims etal.
[66]. 2009, Elsevier

compounds are hampered by the unfavorable redox properties of many sugars


[1, 35]. However, benefitting from the electrocatalytic and signal-amplified proper-
ties of nanomaterials, the resulting modified electrodes have now been successfully
imported in the direct quantitation of several carbohydrates.
Hesperidin is a flavone glycoside found in the skins and juices of citrus fruits.
The concentration level of hesperidin can be used as an indication of the citrus fruit
juices freshness. Recently, in combination with adsorptive stripping voltammetry
(AdSV) with accumulation at open circuit potential, hesperidin was detected using
multiwalled carbon nanotubemodified basel-plane pyrolytic graphite electrode
(MWCNT-BPPGE) and MWCNT-modified mass producible screen-printed elec-
trodes (MWCNT-SPEs) [66]. The much larger electroactive surface area of these
MWCNT-modified electrodes allows for a greater degree of hesperidin adsorption
compared to bare carbon surfaces, while being much less toxic and much more
practical than mercury drop electrodes. The detection limit of hesperidin by this
protocol is down to 7nM. The oxidation mechanism of the observed voltammetry
corresponds to the redox chemistry of the guaiacol subunit within the hesperidin
molecular structure, as in Fig.14.4 [66].
Carbohydrates can be oxidized in strongly alkaline conditions at the surfaces of
transition metal electrodes, including Cu [67, 68], Ni [69], Co [70], and Ru [71].
Therefore, several transition metals nanoparticles/nanostructures were synthesized
404 14 Carbohydrate Detection Using Nanostructured Biosensing

to construct electrodes with a high electrocatalytic capability toward carbohydrates.


For example, various Ni-based nanostructures have been applied to the detection of
sugars, such as nanostructured Ni(II)-curcumin (curcumin: 1,7-bis[4-hydroxy-3-
methoxyphenyl]-1,6-heptadiene-3,5-dione) (NC) film was prepared and electrode-
posited on a glassy carbon electrode. According to electrochemical characterizations,
the nanostructured NC film could act as an efficient material for the electrocatalytic
oxidation of fructose [72]. Ni nanoparticles were prepared and dispersed in disor-
dered graphite-like carbon (Ni-NDC) film simply, in one step, by a radiofrequency
sputtering method at 200C. The resulting films were developed as a working
electrode for high-performance liquid chromatography (HPLC). A good separation
of four sugars (glucose, fructose, sucrose, lactose) at a relatively low-constant
detection potential (0.40 V vs. Ag/AgCl) and a linearity of over three orders of
magnitude was obtained. The detection limits of sugars on the proposed Ni-NDC
film were at least one order of magnitude lower than those obtained on the bulk
Ni film electrode [73].

[Ni(II) - curcumin] + OH - [Ni(II) - curcumin(OH)]-

[Ni(II) - curcumin(OH)]- + Fructose [Ni(II) - curcumin(OH)Fructose]-


[Ni(II) - curcumin(OH)Fructose]- + 2OH - [Ni(III) - curcumin(O)
(OH)Fructose]2 - + H 2 O + e
[Ni(III) - curcumin(O)(OH)Fructose]2 - [Ni(III)-curcumin(OH)
Fructose]- + OH -

[Ni(III) - curcumin(OH)Fructose] - [Ni(II) - curcumin
(OH)] + intermediate(s)
Intermediate + 2nOH - product + nH 2 O + 2ne

14.7.2Other Principles of Direct Carbohydrate Detection

14.7.2.1Lectin-Based Carbohydrate Biosensors

Lectins are a broad family of proteins involved in diverse biological processes.


Lectins can reversibly bind to mono- and oligosaccharides and to glycans with high
specificity [74]. Furthermore, particular structural profiles of glycans and their rec-
ognition by lectins have been attributed to disease progression, making the analysis
of saccharidelectin binding processes important as a diagnostic tool [1]. Based on
such properties, lectins have been extensively exploited as recognition components
for carbohydrate sensing. Lectins are proteins of nonimmune origin that can recog-
nize and bind to specific carbohydrate structural epitopes. Because of their ability to
recognize saccharide motifs, lectins are invaluable tools to interpret the sugar codes
14.7 Nanotechnology 405

Fig.14.5 (a) Mixed SAM on the gold substrate; (b) covalent immobilization of the lectin; (c)
addition of the tagged and untagged sugars; (d) dissolution of the captured nanocrystals, followed
by their stripping voltammetric detection at a mercury-coated glassy carbon electrode. Reprinted
with permission from Dai etal. [75]. 2006, American Chemical Society

on cell surfaces [35]. Two recent studies have described the use of lectin arrays to
analyze the carbohydrate motifs expressed on glycoproteins [36, 37].
The protocol of electrochemical recognition of sugars was established by com-
petitions of target sugars and the quantum dotlabeled sugars. As shown in Fig.14.5,
the bioassay process involves the immobilization of the lectin onto the gold surface,
competition between a nanocrystal (CdS)-labeled sugar and the target sugar for the
carbohydrate-binding sites on lectins, and monitoring the extent of competition
through highly sensitive electrochemical stripping detection of the captured nano-
crystal. EDAC/NHS coupling was also used for conjugating the CdS tracer to the
4-aminophenyl-a-d-galactopyranoside sugar. The protocol relies on a one-step
competitive assay, which is more suitable for monitoring small sugar molecules and
lectinsugar interactions. The lectinsugar recognition event thus yields a distinct
cadmium-stripping voltammetric current peak, whose size decreases upon increas-
ing the level and affinity of the target glycan [75].
Based on integrating the specific carbohydrate recognition and enzymatic signal
amplification of proteins on Au nanoparticles, a dual-functionalized nanoprobe was
designed for highly sensitive and selective in situ evaluation of carbohydrates on
living cells. As shown in Fig. 14.6, a nano-scaffold of nanohorns functionalized
with arginine-glycine-aspartic acid-serine tetrapeptide was immobilized on the sur-
face of an electrode for capturing cells and enhancing the electrical connectivity.
Combined with the nanoprobe and peptide-nanohorns, a highly sensitive electro-
chemical strategy was developed for cytosensing, which showed a detection limit
down to 15 cells, broad dynamic range, acceptable rapidity, and low cost. The pro-
posed method was further used for monitoring the dynamic variation of carbohy-
drate expression on cancer cells in response to drugs, which obviated the destruction
or labeling of cells and the covalent tagging of lectin and enzyme. The one-pot con-
jugation of three components was very convenient and could be extended for prepa-
ration of other lectin-functionalized nanoprobes. Further development of this
technique would contribute considerably to meeting the challenges in a comprehen-
sive understanding of the glycomic codes [76].
Nanoparticle-based probes are emerging as alternatives to molecular probes due
to their various advantages, such as bright and tunable optical property, enhanced
406 14 Carbohydrate Detection Using Nanostructured Biosensing

Fig. 14.6 Scheme of nanoprobe assembly and electrochemical strategy for in situ detection of
mannose groups on living cells. Reprinted with permission from Ding etal. [76]. 2010, American
Chemical Society

Fig.14.7 DSC-mediated dextran conjugation to amine-functionalized nanoparticles. Represents


phosphonate or PEG functional group. The nanoparticles can be Ag, Fe3O4, or ZnS-CdSe. They are
first coated with silica and then functionalized with dextran

chemical and photochemical stability, and ease of introduction of multifunctionality.


A simple and general approach for functionalizing various nanoparticle systems
for use as glycobiological probes was provided by Ying et al. A silica coating
scheme was developed for various nanoparticles and QDs [77]. The silica coating
provides a cross-linked shell that protects the core nanoparticle from adverse
experimental conditions, and the coated particles can be further functionalized with
various molecules of interest. Silica-coated nanoparticles of Ag, Fe3O4, and ZnS-
CdSe were synthesized and a novel dextran functionalization scheme was proposed
(Fig. 14.7). The resulting 1040-nm-sized particles were robust, water-soluble,
colloidally stable, and biochemically active. The biochemical activity of dextran
functionalized nanoparticles was tested using Con A and glucose. Upon the addi-
tion of Con A to dextran-functionalized nanoparticles, visible aggregates of parti-
cles were observed within minutes. Over time, the suspended aggregates precipitated
14.7 Nanotechnology 407

Fig.14.8 Con A-induced nanoparticle aggregation of (a) dextran1K-Ag, (b) dextran 6K-Fe3O4,
and (c) dextran 40K-QD. In each sample, Con A was added with a final concentration of 10mM,
and the absorption spectrum was obtained after 0min (black line), 30min (blue line), 1h (green
line), and 2h (red line). A magnet is shown to remove the aggregated Fe3O4 from the solution in
(b). Reprinted with permission from Earhart etal. [77]. 2008, American Chemical Society

from the solution. Complete precipitation of all nanoparticle systems was observed
within 4h. This precipitation process was associated with a significant red-shifting
of the plasmon absorption band and reduced peak absorbance for the Ag nanopar-
ticles (Fig. 14.8a). No significant red shift was observed with Con Adextran
ZnSCdSe aggregation, but both the absorbance and fluorescence were reduced
with time as the particle precipitated from the solution (Fig.14.8c). Con Adextran
Fe3O4 aggregates were removed from the solution over time using a magnetic field,
resulting in a decrease in absorbance (Fig. 14.8b). The dextranFe3O4 particles
could not be separated by a magnetic field unless Con A was added. This approach
is simple, and widely applicable to different types of nanoparticles and dextran of
different MWs [77].
A simple and easy-to-perform detection assay for lectins using a two-step-
synthesized, water-soluble, carbohydrate-conjugated core-shell CdSe ZnS QDs was
developed. As shown in Fig.14.9, several neoglycoconjugates with a reactive thiol
group were synthesized for the solubilization and stabilization of CdSe-ZnS QDs in
aqueous solution. Three different sizes of QDs with lactose, melibiose, and maltotriose
on their surface were utilized for the first time for lectin detection through agglutina-
tion assay. The sugarQDs were characterized by TEM, fluorescence, and absorption
spectroscopy. Agglutination of sugarQDs by three different lectins occurred through
specific multivalent carbohydratelectin interactions and was studied extensively by
monitoring the scattered light at 600nm. At first, soluble smaller sugarQD lectin
aggregates are formed through the specific interaction of carbohydrates and multiva-
lent lectins, which leads to an increase in turbidity and consequently an increase in
the scattering. Further association of the smaller aggregates into much larger aggre-
gates induces the precipitation and subsequent decrease in the absorbance at longer
time scales as observed experimentally (Fig.14.10). The detection sensitivity of the
maltotrioseQD was as little as 100nM of the lectin using light scattering. Further
enhancement of the detection sensitivity of the protocol could be considerable from
the fluorescence properties of QDs. This agglutination process seems to occur
through formation of smaller soluble aggregates, which further associate to form
larger aggregates [78].
408 14 Carbohydrate Detection Using Nanostructured Biosensing

Fig.14.9 Scheme of synthesis of neoglycoconjugated CdSeZnS QD constructs. Reprinted with


permission from Babu etal. [78]. 2006, American Chemical Society

Fig.14.10 Graphical representation of agglutination of MT QD-589 by ConA tetramer. Reprinted


with permission from Babu etal. [78]. 2006, American Chemical Society

Glycans have great potential as disease biomarkers and therapeutic targets.


However, the major challenge for glycan biomarker identification from clinical
samples is the low abundance of key glycosylated proteins. To demonstrate the
potential for glycan analysis with nanoliter amounts of glycoprotein, a new technol-
ogy (Lectin NanoProbeArray) based on piezoelectric liquid dispensing for noncon-
tact printing and probing of a lectin array was developed [79]. Instead of flooding
the glycoprotein probe on the lectin array surface, a piezoelectric printer was used
to dispense nanoliters of fluorescently labeled glycoprotein probe over the lectin
spots on the array. The ability of Lectin NanoProbeArrays to precisely identify and
reliably distinguish between the closely related glycoforms of fetuin is illustrated in
Fig.14.11. The glycoproteins ASF and 6SF were labeled with HiLyte Fluor 555
14.7 Nanotechnology 409

Fig.14.11 Comparative analysis of lectin microarray and lectin nanoprobe array. Error bars in
the graph represent standard error of the mean (N=5). Reprinted with permission from Nagaraj
etal. [79]. 2008, Elsevier

dye and used for probing the lectin array either by flooding the array (Lectin
Microarray) or by piezoelectric liquid dispensing (Lectin NanoProbeArray). The
reactivity of lectins (SNA, MAA, and DSA) on the array with a range of concentra-
tions for ASF and 6SF were analyzed. A sample image for one block on the array
was shown with the graph. Each block consisted of five identical rows of six spots
with alternate spots of lectins and PBS buffer (control). Sensitivity levels compa-
rable to lectin arrays that use evanescent-field scanners were achieved along with a
several orders of magnitude reduction in the amount of probe required for glycosy-
lation analysis [79].
A strategy for profiling cell surface carbohydrate expression patterns by
evaluating cell-binding patterns on lectin arrays was present using an inverted
optical microscope. The approach was demonstrated using a small array of six com-
mercially available lectins with well-characterized binding specificities. The lectin
array was fabricated on gold thin-film substrates functionalized with NHS ester
alkyl disulfide (Fig.14.12). Different lectins were spotted onto the substrate to react
for 3h and the chip was then incubated in bovine serum albumin (BSA) to minimize
nonspecific binding. The lectin arrays were used to study cell surface carbohydrate
expression. The cells employed were BHK-21. Lectin arrays were placed in the
wells of a 6-well tissue culture plate, and 2mL of cell suspension was added to each
410 14 Carbohydrate Detection Using Nanostructured Biosensing

Fig. 14.12 Surface fabrication: self-assembled NHS-ester monolayer on a gold substrate.


Reprinted with permission from Zheng etal. [80]. 2005, American Chemical Society

Fig.14.13 BHK-21 cells and Caco-2 cells showed distinct binding patterns to the lectin array. The
lectins were spotted according to the pattern in (c). (a) BHK-21 cells bound to three of the lectins,
while (b) Caco-2 cells bound to every lectin except GSL-II. Reprinted with permission from Zheng
etal. [80]. 2005, American Chemical Society

well to cover the lectin chips. After washes, the chips were observed with an optical
microscope. The two cell lines exhibited distinct patterns of binding to the lectin
arrays; BHK-21 cells bound to only three of the lectins, ConA, WGA, and RCA,
while Caco-2 cells bound to every lectin except GSL-II (Fig.14.13). These results
indicated that BHK-21 cells have mannose, galactose, and GlcNAc expressed on
their surfaces, and no detectable expression [80]. The work provides a proof-of-
principle demonstration of the direct qualitative profiling of cell surface carbohy-
drate expression patterns by simple microscopic observation of cell binding to lectin
arrays, and may readily be expanded to encompass a wider variety of carbohydrate
structural motifs by increasing the number of lectins or other specific ligands pres-
ent in the array.
14.7 Nanotechnology 411

Fig.14.14 LDI-TOF mass spectra of (a) 2a deposited on the surface of the gold-coated MALDI
plate and (b) 2a on the GCNPs. Reprinted with permission from Noriko and Shin-Ichiro [81].
2006, Wiley-VCH

The laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS)


was used to assay the glycosyltransferase activity. Gold colloidal nanoparticles
(GCNPs) modified with multivalent sugars were enzymatically synthesized and
proved to be excellent acceptor substrates for the glycosyltransferase reaction. LDI-
TOF MS analysis of chemisorbed materials on GCNPs occurred by directly monitor-
ing glycosyltransferase reactions using recombinant human b-1,4-GalT, recombinant
human a-1,3-FucTVI, and crude cell extracts of Escherichia coli. When the ioniza-
tion efficiency of the LDI-TOF MS of 2a adsorbed on the GCNPs was compared with
that of 2a adsorbed on a gold-coated plate, drastically amplified spectrometric signa-
tures in the results from the GCNPs was observed (Fig.14.14). LDI analysis by repet-
itive laser irradiation at the same position on the gold-coated MALDI plate did not
show significant ionization (Fig.14.14a). In the case of the LDI of GCNPs, irradia-
tion performed 10 times at the same position still generated the satisfactory ion peak
at m/z 781 signifying the homo-disulfide compound 2a (Fig.14.14b). This indicated
that the LDI-TOF/TOF MS analysis of GCNPs containing a variety of biomolecules
is an extremely versatile and efficient method for directly and rapidly monitoring the
metabolism (metabolomics) of chemisorbed small-molecule compounds on GCNPs
in living cells. This LDI-TOF MS approach using chemisorbed small-molecule com-
pounds on GCNPs displays a high tolerance to the nonspecific ionization of contami-
nants and no matrix interferences because GCNPs allow the selective desorption/
ionization of the substrates/ligands covalently bound onto the surface of the GCNPs
through an Au-S linkage in the absence of a conventional organic matrix [81].
A simple and highly specific protein detection system using glycoconjugated
gold nanoparticles has been investigated. Gold nanoparticles coated with carbohydrate
412 14 Carbohydrate Detection Using Nanostructured Biosensing

Fig.14.15 Gold colloid solutions (a) before and (b) after Mal-C12S modification; (c) MalC12S-
modified gold nanoparticle solution after adding Con A (2.6mM); (d) unmodified gold nanoparti-
cle solution after adding Con A (2.6mM). Models of each step are also shown. Reprinted with
permission from Sato etal. [82]. 2008, Springer

alkanethiols are first prepared. The functioned gold nanoparticles aggregate in the
presence of corresponding proteins (lectins) that have specific recognition for cer-
tain carbohydrates. Therefore, the corresponding lectins in sample solutions can be
estimated. As shown in Fig.14.15, maltoside alkanethiol, which has a disaccharide
group and a flexible long alkyl chain, is selected as an effective sensing modifier.
The surface modification of gold nanoparticles with maltoside alkanethiol results in
a shift and broadening (from 520 to 610nm) of the absorption peak. Monodispersed
maltoside-adsorbed gold nanoparticles aggregate in the presence of Con A, which
can be used to detect the presence of Con A in sample solutions. The precipitate of
the maltosidegold nanoparticleCon A mixture can be further redispersed by the
addition of methyl a-d-mannopyranoside, whose adsorption coefficient is larger
than that of maltoside with Con A [82].

14.7.2.2Estimation of Glycolation of Protein

Cell surface glycoproteins are shown to play important roles as receptors for hor-
mones and as mediators of immunological specificity in intracellular recognition
and adhesion, and in cell development and differentiation [47, 83]. Therefore, in
order to develop a novel high-throughput tool for monitoring carbohydrate-protein
interactions, a series of carbohydrate or glycoprotein microarrays were prepared by
14.7 Nanotechnology 413

Fig.14.16 Schematic representation of one spot on the microarray. From left to right, the binding
of a biotin-modified lectin to the (a) spotted monosaccharide and (b) glycoprotein, then labeled by
avidin-FITC followed by the attachment of peptide stabilized gold nanoparticles, the silver
enhancement step, and reading by detection of resonant light scattering. Reprinted with permission
from Gao etal. [84]. 2008, American Chemical Society

immobilizing amino-modified carbohydrates on aldehyde-derivatized glass slides


or glycoprotein on epoxide-derivatized glass slides and lectin-binding experiments
were carried out using these microarrays. As shown in Fig.14.16, biotinylated lec-
tins were added on the monosaccharide or glycoprotein assay. The binding events
between lectins and glycoproteins were traced by the attachment of gold nanopar-
ticles followed with silver enhancement. The attachment of gold nanoparticles was
achieved by standard avidinbiotin chemistry through the addition of avidin-FITC.
Three monosaccharides (Man-, Glc-, and Gal-) or three glycoproteins (Asf, RNase
A, and RNase B) with two lectins (Con A and RCA120) were chosen to establish
the assay, and the signal was detected by resonance light scattering (RLS), respec-
tively. The method showed a high selectivity of recognition of carbohydrateprotein
on the microarray spots with the detection limit for RCA120, Gal-, and Asf in solu-
tion of 25.6pg/mL, 8mM, and 32ng/mL, respectively [84].

14.7.3Carbohydrate Components in Biosensors

The technical advantages of applying gold nanoparticles in biological systems


have been well recognized. The covalent binding between gold nanoparticles and
biomolecules can be easily achieved by self-assembled thiolated molecules onto the
414 14 Carbohydrate Detection Using Nanostructured Biosensing

Fig.14.17 (a) Schematic illustration of the interactions of carbohydrateAu NPs and Con A on
the biosensor chip used in the competition assays. (b) Five different carbohydrates (sugar-R)
encapsulated on two different sizes of gold nanoparticles are summarized. Specifically, gluco- and
galatopyranoside are encapsulated on nanoparticles of 6 nm and abbreviated as 6-g-AuNP and
6-t-AuNP, respectively. Mannopyranoside clustered on nanoparticles of 6 and 20nm in diameter
are abbreviated as 6-m-AuNP and 20-m-AuNP, respectively. Mannopyranoside with short or long
linker lengths are encapsulated on 20-nm nanoparticles to form s-20-m-AuNP and l-20-m-AuNP,
respectively. Reprinted with permission from Lin etal. [85]. 2003, Royal Society of Chemistry

nanoparticles surface. In addition, gold nanoparticles exhibit an intense color in the


visible region for spectroscopic detection and also a great contrast for electron
microscopic imaging. Moreover, a single nanoparticle with a large surface-to-
volume ratio is ready for the covalent attachment of multiple ligands, which provides
a great possibility for the enhancement of some biomolecule interactions. In par-
ticular, carbohydrateprotein interaction is generally identified with a very low
affinity between them. However, in nature, the low affinity can be compensated for
by the presentation of multiple ligands to individual receptors. The polyvalent inter-
actions between multiligands and their receptors can be collectively much stronger
than corresponding monovalent interactions. Recently, multiple-carbohydrate
ligands have been assembled on linear polymers, two-dimensional gold surface, and
liposomes to enhance carbohydrateprotein interactions.
Recently, a method for targeting specific proteins in living bacteria was presented
by using mannose-encapsulated gold nanoparticles (m-AuNPs) as a probe [43]. The
multivalent interactions between Con A and mannose-, glucose-, and galactose-
encapsulated gold nanoparticles (abbreviated as m-AuNP, g-AuNP, and t-AuNP,
respectively, Fig.14.17) were quantitatively analyzed by SPR technique. The results
indicated that nanoparticles can be excellent multivalent carbohydrate carriers for
lectins. The binding of m-AuNP to Con A exhibited a strong multivalent effect, and
14.7 Nanotechnology 415

Fig.14.18 Schematic representation of the self-assembled multilayer system for the investigation
of biomolecular binding. Reprinted with permission from Guo etal. [86]. 2007, Elsevier

the binding specificity of Con A for the multivalent carbohydrate-encapsulated


gold nanoparticles (carbohydrate-AuNP) was similar to that of the monovalent
counterparts. The affinity of m-AuNP for Con A could be also adjusted by altering
the nanoparticles size or sugar moiety. The carbohydrateAu NP has great
potentialas an effective inhibitor of proteincarbohydrate interactions in biological
systems[85].
Another label-free optical biosensor for the detection of Con A was reported.
1-Dodecanethiol (DDT) was self-assembled onto gold nanoparticles that were
deposited on glass slides. Glycolipid molecules were then inserted into dodecane-
thiol by physical interactions (Fig.14.18). The recognition between Con A and car-
bohydrate was monitored by UVvis spectrophotometry. The absorption spectrum
shifted when Con A was bound to the sugar residues of glycolipids immobilized on
nanogold slides, while almost no spectrum change was observed when another non-
specific protein molecule reached the nanogold slides. The self-assembled bilayer
on nanogold substrates had very high sensitivity for Con A. The detection limit of
Con A was 0.1 nM. The simplicity and sensitivity of this biosensor architecture
once again show the prospects for nanogold application in biosensors [86].
Another approach for the determination of FimH adhesin of bacterial type 1 pili
was investigated by the synthesis and characterization of m-AuNPs and by monitor-
ing the binding between m-AuNP and FimH on bacteria using TEM. As shown in
Fig.14.19, compound 3 was synthesized by the glycosylation of 2 with alcohol and
then hydrolyzed to obtain thiomannosyl dimer 4.12. The m-AuNP 1 was prepared
by treating HAuCl4 with 4 in the presence of NaBH4. A single m-AuNP consists of
approximately 200 attached mannoses. m-AuNP was then tested for its ability to
bind mannose-specific adhesin FimH of type 1 pili in E. coli. Two E. coli strains,
ORN178 and ORN208, were used to confirm the specific binding of m-AuNP to
FimH. Two bacterial strains were incubated with m-AuNP separately in different
buffers (PBS, LB, and H2O) and at varied temperatures (4, 25, and 37C), and the
416 14 Carbohydrate Detection Using Nanostructured Biosensing

Fig.14.19 Synthesis of m-AuNP. (a) Ac2O, pyr. DMAP, 90%; (b) HBr/HOAc, 80%; (c) 4-pentenyl
alcohol, Hg(CN)2, 88%; (d) HSAc, AIBN, dioxane, 80%; (e) NaOMe (cat.), MeOH, 97%;
(f)HAuCl4, NaBH4. Reprinted with permission from Lin etal. [87]. 2002, American Chemical
Society

Fig.14.20 Typical TEM images of sectioned areas of (a) pili of the Escherichia coli ORN178
strain bound with m-AuNP; (b) the E. coli ORN208 strain deficient of the fimH gene without
m-AuNP binding. The experiments were performed in LB at room temperature. Scale bar: 100nm.
Reprinted with permission from Lin etal. [87]. 2002, American Chemical Society

binding of m-AuNP to bacterial pili in each condition was examined by TEM. The
TEM results showed that m-AuNP selectively bound the pili of the ORN178 strain
but not those of the ORN208 strain (Fig.14.20), demonstrating specific binding of
m-AuNP to FimH. This work further demonstrates that carbohydrate-attached gold
nanoparticles can be used as an efficient labeling probe and multiligand carrier in a
biological system [87].
A novel approach to the optical detection of viruses using gold nanoparticles was
proposed. Although inorganic nanostructures prepared with viruses as templates
have often been reported recently, there are no reports on the use of their inherent
molecular recognitions for the spatially ordered arrangement of metal nanoparticles,
which can be applied to virus detection. Since viruses have multiple recognition sites
on their surface for ligands, the ligand-displaying nanoparticles could be specifically
14.7 Nanotechnology 417

Fig.14.21 Schematic representation of the detection of virus particles. (a) The formation of the
sialylAu particle array based on virus-like particles (VLPs) through viral ligand recognition;
(b)structures of the sialic acid-linked and carboxyl lipids used in the preparation of sialylAu and
carboxylAu particles. Reprinted with permission from Niikura et al. [88]. 2009, American
Chemical Society

arranged on the viral surface (Fig.14.21a). Therefore, viruses can be detected from
the plasmon shift derived from the assembled gold nanoparticles on the virus surface
rather than the analyte-induced aggregation of metallic nanoparticles. The surface
plasmon resonance shift strongly depends on both the interparticle distance and the
number of coupled particles. The plasmon shift for multiple (more than two) nano-
particles in a linear arrangement is larger than that for two nanoparticles; thus, the
multiple coupling of nanoparticles on the virus is expected to cause a large shift of
the plasmon band. In addition, the assembly of metallic nanoparticles into a shell-
shaped structure is expected to lead to a large plasmon shift. Metallic clusters formed
on a spherical body, such as silica particles (diameter~100nm) or liposomes (diam-
eter~50 nm), show a large red shift (~200 nm) of the plasmon band. The virus-
templated assembly of metallic nanoparticles in a shell-shaped structure is thought
to contribute to this large plasmon shift [88].
A single-walled carbon nanotube (SWNT) is a pseudo-one-dimensional nano-
structure capable of carrying/displaying a large number of bioactive molecules and
species in aqueous solution. A series of dendritic b-d-galactopyranosides and a-d-
mannopyranosides with a terminal amino group have been synthesized and used for
the functionalization of SWNTs, which targeted the defect-derived carboxylic acid
moieties on the nanotube surface (Fig.14.22a). The higher-order sugar dendrons
were more effective in the solubilization of SWNTs, with the corresponding func-
tionalized nanotube samples of improved aqueous solubility characteristics. Through
the functionalization, the nanotube apparently serves as a unique scaffold for dis-
playing multiple copies of the sugar molecules in pairs or quartets. Results from the
biological evaluation of these sugar-functionalized SWNTs in binding assays with
pathogenic E. coli and with Bacillus subtilis are shown in Fig.14.22b. Obviously,
418 14 Carbohydrate Detection Using Nanostructured Biosensing
14.7 Nanotechnology 419

Fig.14.23 Schematic illustration of the nanobioelectronic detection system for Alzheimers Ab


peptides. The sugar immobilized substrate was prepared through steps iiii. (i) Deposition of gold
nanoparticle on carbon electrode; (ii) formation of acetylenyl-terminated SAM; and (iii) saccha-
ride immobilization for Ab detection. The attachment of the Ab peptide to the sugar layer and the
electrochemical detection were realized on a single electrode. The peak oxidation current response
of the Tyr residue of Ab was utilized as the analytical signal. Reprinted with permission from
Chikae etal. [90]. 2008, Elsevier

significant aggregation of the cells in the presence of Gal2SWNT could be


observed. The amount of aggregates (precipitates at the bottom of the centrifuge
tube) was larger for a higher starting E. coli concentration. Between Gal2SWNT
and GalSWNT under similar experimental conditions, the amount of recovered
aggregates was clearly larger in the former than in the latter [89]. These multivalent
carbohydrate configurations may potentially offer interesting chemical and bio-
chemical properties and functions, despite some of the complications that are yet to
be understood [89].
The electrochemical sensing of saccharideprotein interactions using a couple of
sialic acid derivatives and Alzheimers amyloid-beta (Ab) has been described [90].
The densely packed saccharide area for the recognition of protein was fabricated
onto a carbon electrode by three steps, which were electrochemical deposition of
Au nanoparticles on a screen-printed strip, SAM formation of the acetylenyl group
on Au nanoparticles, and the cycloaddition reaction of an azide-terminated sialic
acid to the acetylenyl group (Fig.14.23). The attachment of Ab peptides to the sialic

Fig.14.22 (a) Carbon nanotubes functionalized with Gal-, Man-, or their dendrons. (b) Fluorescence
microscopy images (10 mm for all scale bars) on the agglutination of E. coli O157:H7 (GFP-
expressing) cells by (a) Gal2SWNT, (b) GalSWNT, and (c) the control (cells only). (d) A visual
comparison on the amount of precipitates (in centrifuge tubes) associated with the agglutination of E.
coli O157:H7 cells (top, 5107cells/mL; bottom, 108cells/mL) by Gal2SWNT (left) and Gal
SWNT (right). Reprinted with permission from Gu etal. [89]. 2008, American Chemical Society
420 14 Carbohydrate Detection Using Nanostructured Biosensing

acid layer was confirmed by electrochemistry and atomic force microscopy imaging.
The intrinsic oxidation signal of the captured Ab(140) and (142) peptides, con-
taining a single tyrosine (Tyr) residue, was monitored at a peak potential of 0.6V
(vs. Ag/AgCl within this sensor) in connection with differential pulse voltammetry.
The peak current intensities were concentration-dependent. The proposed process
provides new routes for the analysis of saccharideprotein interactions and for elec-
trochemical biosensor development [90].

14.8Conclusions

The growing interest in the biological importance and applications of oligosaccha-


ride derivatives has led to the development of new techniques for carbohydrate char-
acterization. The complexity and high variability of carbohydrate structures make
their practical analytical applications very challenging. Many carbohydrate biosen-
sors were designed based on the molecular recognition and specific binding encoun-
tered between polysaccharides and other macromolecules, particularly proteins.
Such molecular interactions, including carbohydrate lectin, carbohydrate toxin, or
saccharide enzyme affinities, play significant roles in diverse biosensor devices and
bioassays, either those aiming to detect and/or analyze oligosaccharides or others
that rely on embedded carbohydrates for the detection of other biomolecules.
Overall, understanding and harnessing the intrinsic complexity of carbohydrate
structures is the underlying factor for the development and utilization of oligosac-
charide biosensor designs and their future applications.
Ideally, the detail of the glycan structure can be assessed by the coming new
techniques that will allow total glycan analysis to be performed on intact glycocon-
jugates. Eliminating the need for the prior isolation of individual glycans will elimi-
nate selective and nonselective losses and the production of artifacts inherent to
degradative procedures. Contributions from a variety of disciplines will be required
for such technologies to materialize and come to fruition. Putting together the pieces
of the puzzle provided by primary structural analysis promises to be as interesting a
challenge for the generations to come as it is for todays carbohydrate chemists.

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Chapter 15
Nanomaterials for Immunosensors and
Immunoassays

15.1Introduction

There is a continuously increasing demand for the specific and sensitive


determination of trace amounts of analytes in complex matrices for various
purposes. In this respect, immunoassays and immunosensors that rely on
antibodyantigen binding provide a promising approach of analysis for their
remarkable specificity and sensitivity. High specificity of immunoassays and
immunosensors is achieved solely by the molecular recognition of target analytes
by antibodies or antigens to form stable immunocomplexes. On the other hand,
sensitivity depends on several factors, including the affinity of antibodies, the
amount of immobilized immunological recognition elements, and the choice of
transducer and signal probe. Therefore, the improvement of immunoassay and
immunosensor performance mainly relies on the development of antibody prepa-
ration techniques, the improvement of immobilization and tagging methods, and
the adoption of a high-performance transduction method.
In recent years, unique optical, electronic, and mechanical properties of
nanomaterials have offered excellent prospects for designing highly sensitive
and selective biosensing methods. Various biocompatible nanomaterials (e.g.,
noble metals, magnetic oxides, and carbon nanoparticles) with unique physical
and chemical properties have been successfully applied in immunosensing
interface fabrication to achieve greatly improved immobilization of antibodies
or antigens, as well as to improve transduction efficiency. The enormous signal
enhancement associated with using nanoparticles as signal tags and forming
nanoparticlebiomolecule assemblies also provides a great probability for ultra-
sensitive immunoassays and immunosensors.
This chapter briefly introduces the main principle of immunoassays and
immunosensors. More of the content focuses on the development and application of
biocompatible nanomaterials in developing immunoassays and immunosensors.

H. Ju et al., NanoBiosensing: Principles, Development and Application, 425


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0_15, Springer Science+Business Media, LLC 2011
426 15 Nanomaterials for Immunosensors and Immunoassays

15.2Principle of Immunoassays and Immunosensors

15.2.1Antigen, Antibody, and Their Recognition Reaction

The science of immunology is based upon an organisms ability to generate the


biological effect known as the immune response. The immune response can be
defined as any mechanism of identifying nonself substances from self sub-
stances in an organism, which usually results in a more rapid destruction of those
substances identified as nonself [1]. Some degree of this ability to identify and
respond to foreign substances has been found in very simple life forms, such as
microbes. In higher forms of life, particularly in mammals, the immune system is a
complex mechanism in which identification and communication take place in the
blood and lymph.
When a foreign substance enters the body of an advanced animal, certain pro-
teins are synthesized to identify the invader and to prohibit its harmful effects.
Antibodies are biologically defined as the proteins that are formed when an animal
is immunized with an antigen (nonself substance). Antibodies show very high
specificity and binding constants toward their corresponding antigens. An antigen
has been defined as any agent that gives rise to antibody formation specific for
that agent when transferred to a living cell system containing cells of the
immunologically competent type [2]. The natural antigens may be such macro-
molecule substances as proteins and nucleic acids. The haptens are defined as some
substances with low molecular weight, typically less than 1,000Da, that contain
specific sites or functionalities against which the antibodies are directed. Contrary
to antigens, haptens cannot directly activate the immune system of animals to
produce antibodies unless they conjugate with such protein carriers as bovine
serum albumin and ovalbumin.
Antibodies are divided into five classes (immunoglobulin [Ig] G, IgA, IgE, IgM,
and IgD) based on their structures and biological functions. IgM and IgG are the
first and second antibodies produced in response to an invading nonself substance.
IgA protects mucous membranes, and IgE protects against parasites. However, the
main function of IgD is still unknown. Of the five classes of antibodies, IgG is the
class used the most frequently for immunoassays because it exists at the highest
level and is readily available. Generally, the structure of IgG is represented by a
Y-shaped figure consisting of four polypeptide units. Two of them are identified
and known as the heavy chains with a molecular weight of 55,00060,000Da. The
other two sequences are light chains with a molecular weight of 20,00024,000Da.
The two double-ended segments of the Y are denoted as Fab fragments and are the
sites at which antibody binds with antigen. The variable and hypervariable regions
of Fab create an active portion that recognizes a specific area of the antigen. The
singular segment at the other end of Y shape is known as the Fc fragment, which
cannot bind with antigen but has the ability to affix to the cell surface and to pass
through the placenta [3].
15.2 Principle of Immunoassays and Immunosensors 427

Many different types of antibodies exist in the serum of animals immunized with
specific antigens. The mixture of these different types of antibodies is a so-called
polyclonal antibody. Because these antibodies arise from the clones of a number of
separate B cells, they are heterogeneous, and different antibodies in this mixture
react with different antigenic determinants. With the ever-increasing sophisticated
genetic techniques, the production and use of monoclonal antibodies have attracted
more interest since this technique was first developed by Khler and Milstein in
1975, who won the Nobel Prize in 1984 [4]. Monoclonal antibodies are produced by
the fusion of myeloma (tumor) cells cultivated in vitro with mouse spleen
B-lymphocytes immunized with a specific antigen. The fusion called a hybridoma
is cultivated and screened. Once we are sure that a certain hybridoma is producing
the right antibody, we can culture that hybridoma indefinitely and harvest monoclo-
nal antibody from it. Because a monoclonal antibody reacts only with one specific
antigenic determinant, it shows a higher sensitivity and better specificity than the
conventional polyclonal antibodies for immunoassays.
The principle of antigenantibody interaction lies in the specific combination of
the antigen determinant with the Fab fragment of the antibody. The specific binding
between antigen and antibody is a collection of noncovalent forces, including
electrostatic forces, hydrophobic attractions, hydrogen bonding, and van der Waals
interactions. The interaction between antigen and antibody is quite strong, as
indicated by the large association constant of 1051012 M1 [5]. Therefore, the
antibodyantigen complex does not dissociate so readily unless some harsh solu-
tions such as buffers at pH higher than 10 or lower than 3, organic solvents, and
saline solutions at high concentration are used to regenerate it [6].

15.2.2Immunoassays and Immunosensors

Immunoassays usually regard the analytes to be detected as antigens, and antibodies


for the analytes as considered specific recognition reagents. Immunoassays based on
highly specific antigenantibody binding have been customized in a variety of formats
adapted to particular applications and experimental procedures. In some formats,
referred to as homogeneous immunoassays, the assay strategies do not require the
separation of the immunocomplexes from unbound immune reagents. This approach
includes agglutination [7], capillary electrophoresis [8], fluorescence polarization [9],
and fluorescence-resonance energy-transferbased immunoassays [10]. The other
formats described as heterogeneous immunoassays impose the initial separation of
the immunocomplexes from the unbound immune reagents. In heterogeneous immu-
noassays, the immunocomplexes are bound to a solid substrate, allowing the retention
of the molecules of interest while the unbound ones are washed out of the system.
Heterogeneous assays, although requiring a longer run time and more complex manip-
ulations, are more versatile, more sensitive, and more specific. Thus, heterogeneous
immunoassays are inevitably more popular than homogeneous ones. Most of the
428 15 Nanomaterials for Immunosensors and Immunoassays

Fig.15.1 (a) Schematic


illustration of competitive
immunoassay and (b)
sandwich immunoassay

current works concerning immunoassays are performed in heterogeneous format.


The solid substrates for heterogeneous immunoassays can be biocompatible rigid
materials such as polymer membrane or bead [11], microtiter plate [12], and magnetic
bead [13]. In general, a solid substrate is prepared with an excess of immunological
recognition elements (either antigen or antibody). Immobilization procedures vary for
each solid substrate and include simple physical adsorption, embedding, and covalent
bonding. The resulting solid substrate is then blocked to reduce the nonspecific bind-
ing of the analytes and labeled antibodies by using some protein-based blocking
agents. The most f requently used blocking agents include bovine serum albumin and
casein at concentrations higher than 1%.
Competitive and sandwich methods are the two most popular heterogeneous
immunoassay strategies. In the both strategies, the label or tag is utilized as the
signal probe for quantifying the antigenantibody reaction. In a typical competitive
immunoassay, shown schematically in Fig.15.1a, the mixture of sample antigens
(Ag) and labeled antigens (Ag*) is added to the surface of the substrate immobilized
with antibodies (Ab). A competitive binding to the immobilized antibodies occurs
between the sample antigens and the labeled antigens. After an antigenantibody
binding equilibrium is reached, the solid substrate surface is rinsed with buffer to
remove unbound antigens, and the bound labels signal is detected. Therefore, the
measured signal is inversely proportional to the antigens concentration in the
sample for competitive assay. In a sandwich immunoassay (Fig.15.1b), the target
analyte (antigens) is exposed to the substrate and captured by the immobilized
primary antibodies (Ab1). Then the captured antigens bind the labeled secondary
antibodies (Ab2*) used as the tracer and are rinsed to remove extra tracer. These
tracer antibodies provide a signal that is directly proportional to the analytes
concentration. The use of two different antibodies for the sandwich immunoassay
often imparts greater selectivity since cross-reacting species rarely bind both the
capture and the tracer antibodies. In general, sandwich immunoassays are used for
macromolecule analytes rather than analytes with a low molecular weight.
15.3 Immunosensors Based on Biocompatible Nanomaterials 429

As a type of biosensor, an immunosensor is defined as a compact analytical


device incorporating immunological recognition elements either intimately
connected to or integrated within the signal transducer [14]. As with other types of
biosensors, an immunosensor contains three basic components: recognizing element,
transducer, and detector. Either antigens or antibodies are immobilized on the
surface of a solid substrate and participate in the specific binding, allowing
recognition of the target analyte. The transducer monitors physicochemical changes
resulting from the immunoreaction between immunological recognition elements
and target molecules and converts them to a detectable physical signal. Then the
signal is collected and amplified by the detector to indicate the identification and
quantity of the target analyte. Therefore, almost all immunosensors reported have
been based on a heterogeneous immunoassay format up to now.
Many types of transduce modes for immunoassays and immunosensors, including
optical (fluorescent, chemiluminescent [CL], electrochemiluminescent [ECL],
refractive index), electrochemical (amperometric, potentiometric, capacitative,
electrochemical impedance spectroscopic), and mass-sensitive (surface plasmon
resonance [SPR], quartz crystal microbalance) transducer have been developed for
immunosensing. These various detection approaches based on immunoreaction
have been widely used for detection of trace amount of analytes in complex matrices
such as biological and environment samples due to their high selectivity and
sensitivity.

15.3Immunosensors Based on Biocompatible Nanomaterials

15.3.1Nanomaterials Used as Immobilization Substrates

In immunosensors and heterogeneous immunoassays, immunological recognition


elements (either antigens or antibodies) are usually presented as immobilized forms
on solid substrates. The immobilization form and the immobilization amount of the
immunological recognition element may play an important role to determine their
sensitivity, reliability, and reproducibility since the recognition of target molecules
is the first step of the detection. The surface characteristics of solid substrates are a
crucial factor for obtaining a good assay performance. An ideal solid immobiliza-
tion substrate for immunosensors and heterogeneous immunoassays should meet
the following key requirements: (1) It must provide a functional surface to enable
the appropriate immobilization of immunological recognition elements; (2) its
immobilization manner should not severely affect the spatial structure of the
immunological recognition elements, to retain their immunoactivity; (3) it should
have a high surface-area-to-volume ratio to immobilize abundant immunological
recognition elements; (4) it should be hydrophilic to avoid nonspecific interaction
with the analytes and the sample matrix; and (5) it should have the proper mechanical
intensity in order to retain its physical shape during the manipulation process. The
continuous progress of nanotechnology in material science has led to the development
430 15 Nanomaterials for Immunosensors and Immunoassays

of nanosized materials fit for acting as biomolecule immobilization substrate.


Besides the conventional substrates based on polystyrene and sepharose, some
particular nanomaterials, such as novel metal and carbon nanomaterials, have been
widely used for the fabrication of immunosensing interfaces due to their good
biocompatibility and surprising immobilization ability. Over the last two decades,
considerable attention has been paid to the development of new biocompatible
nanomaterials with suitable hydrophilicity, high porosity, and large surface area for
immunological recognition element immobilization.
Gold nanoparticles (Au NPs) are a kind of nanomaterial with surprising electro-
static adsorption ability for proteins, and they have been extensively used as an
immobilized matrix for retaining the bioactivity of antigens and antibodies and for
promoting the direct electron transfer of the immobilized proteins. These characters
allow Au NPs to be widely used for the fabrication of various types of electrochemi-
cal immunosensors, especially amperometric immunosensors. For example, Jus
group [15] employed colloidal Au NPdoped chitosan composite to immobilize
immunological recognition elements on screen-printed carbon electrode (SPCE)
and fabricated a disposable electrochemical immunosensor for carcinoembryonic
antigen (CEA). The Au NPs improved the reversibility of the electrochemical
reaction of substrate and increased the detection sensitivity to a detection limit of
0.22ng/mL CEA. In addition, the presence of Au NPs could provide a congenial
microenvironment for adsorbed biomolecules and decrease the electron-transfer
impedance. Thus, Au NPs have widely been used for the fabrication of reagentless
immunosensors, in which the immunoassay can be carried out by measuring the
direct electrochemical signal of labeled enzymes, such as horseradish peroxidase
(HRP). For example, the same group fabricated a reagentless immunosensor for
human chorionic gonadotrophin (hCG) by encapsulating HRP-labeled hCG anti-
body in a designed hydrophilic, nontoxic, and conductive Au NP/titania solgel
composite membrane [16]. The presence of the Au NPs facilitated the electron
transfer between HRP and the electrode, leading to the direct electrochemical
behavior of the immobilized HRP without the need for peroxide or mediator. The
formation of immunoconjugates by a simple one-step immunoreaction between
hCG in sample solution and the immobilized HRP-hCG antibodies introduced a
barrier of direct electrical communication between the immobilized HRP and the
electrodes surface. Thus, the concentration of sample could be detected by the
decreased current. Under optimal conditions, the hCG analyte could be determined
with a detection limit of 0.3mIU/mL.
Further, Jus group [17, 18] developed several disposable reagentless immu-
nosensor arrays for a simple immunoassay of panels of tumor markers by individu-
ally embedding different kinds of HRP-labeled antibody-modified Au NPs in a
designed biopolymer/solgel matrix formed on SPCEs (Fig.15.2). The presence of
Au NPs both accelerated the electron transfer between immobilized HRP and the
electrode and increased the hole size for improving the permeability of the solgel
matrix so that the antigens in solution could easily penetrate into the solgel film for
immunoreaction. Upon formation of the immunocomplex, the direct electrochemi-
cal signal of the HRP decreased due to the increasing spatial blocking, and the
15.3 Immunosensors Based on Biocompatible Nanomaterials 431

Fig.15.2 Schematic diagrams of immunosensor array and multianalyte electrochemical immuno-


assay system. (a) Nylon sheet; (b) silver ink; (c) graphite auxiliary electrode; (d) Ag/AgCl refer-
ence electrode; (e) graphite working electrode; (f) insulating dielectric. Reprinted with permission
from Wu etal. [17]. 2008, American Association of Clinical Chemistry

analytes could be simultaneously determined by monitoring the signal changes.


Such a type of immunosensor arrays provided a simple multi-analyte immunoassay
with no need for a substrate and no between-electrode cross-talk, offering the
capability of point-of-care testing.
Au NPs can be deposited electrochemically on the surface of electrode directly
in a short time, and the size of the nanoparticles can be controlled using different
conditions of electrochemical deposition. Typically, Au NPs are prepared by the
simple chemical reduction of HAuCl4 using NaBH4, citrate, or chitosan. The size of
Au NPs can also be conveniently controlled by adjusting the ratio of HAuCl4 and
reductant. However, tiresome procedures are required, unfortunately, to assemble
Au NPs onto the electrodes surface to fabricate an electrochemical immunosensing
interface. The electrochemical reduction of HAuCl4 is a simple and promising alter-
native method to form a nanostructure gold film on the electrode. The electrochemi-
cally deposited Au NPs provide a stable and porous surface for immunological
recognition elements immobilization. Moreover, the high specific area of this porous
gold shows a higher sensitivity than the original flat surface device. This strategy
has been applied for the preparation of a CEA amperometric immunosensor [19].
However, Liang etal. [20] mentioned that Au NPs prepared by chemical reduction
are hydrophilic. When antibodies were adsorbed on the surface of a polyvinyl
butyral solgel membrane containing Au NPs, they could leak out from the hydro-
phobic solgel film. To avoid the leakage of biomolecules, the gold nanowires were
prepared by the electrochemical reduction of HAuCl4 with the aid of the polycar-
bonate template. The antibody-functionalized gold nanowires can form a crossing
network with polyvinyl butyral solgel, and greatly enhance the stability of the
composite membrane.
Silver nanoparticle (Ag NPs) prepared by the chemical reduction of AgNO3 is
another noble metal nanoparticle with similar physical and chemical properties as
Au NPs. Ag NPs also have been reported to fabricate various electrochemical
432 15 Nanomaterials for Immunosensors and Immunoassays

immunosensors as immobilization substrates. A label-free capacitive immunosensor


was developed for the detection of microcystin-LR (MC-LR) using a gold elec-
trodemodified with self-assembled thiourea monolayer incorporated with Ag NPs.
The interaction of MC-LR and its antibodies can be directly detected by capacitance
measurement. Comparing to the modified electrode without Ag NPs, its signal could
be obtained up to 43 times [21].
Besides noble metal nanoparticles, some carbon nanomaterials, including nano-
tubes, nanofibers, and nanohorns, show good immobilization abilities, as well as
surprising electrocatalytic effect. Therefore, carbon nanomaterials have been
widely used in the development of immunosensors. Wohlstadters group [22] used
carbon nanotubes (CNTs) as both an electrode and an immobilization phase to fab-
ricate an ECL-based sensing device for the immunoassay of a-fetoprotein (AFP).
The nanotube-polymer electrode was fabricated by compounding carbon nanotubes
with poly(ethylene vinylacetate). With a sandwich immunoassay, this nanotube-
based system achieved a sensitivity and dynamic range that spanned the clinically
relevant values for AFP. This work demonstrated that ECL-mediated assays based
on CNTs provided a complete system to quantitatively measure a wide range of
biologically relevant analytes. CNTs were also widely used for the fabrication of
amperometric immunosensors, which are described in Chap. 7.
Carbon nanofiber (CNF) has been recognized as a very promising material
based on its nanostructure and properties. The oxidation of CNF with nitric acid
can produce carboxyl groups without degradation of the structural integrity of its
backbone. Compared to CNTs, CNF has a much larger functional surface area and
higher ratio of surface active groups to volume; therefore, it can be used for cova-
lent binding of proteins and mediators with the help of a cross-linking reagent. Jus
group [23] used soluble CNF to construct an immunosensor for a rapid separation-
free immunoassay for carcinoma antigen 125 (CA 125). As shown in Fig.15.3, the
acidic oxidation of the CNF provided its solubility and wettability for the conve-
nient preparation of a porous CNF membrane and a larger number of active sites
for covalent binding of CA 125 and thionine as the electron-transfer mediator. The
covalent attachment of proteins to the surface overcame the problems of instability
and inactivation. With a competitive mechanism, the CNFbased immunosensor
was able to detect CA 125 concentrations from 2 to 75U/mL.
Single-walled carbon nanohorns (SWNHs), as dahlia flowerlike spherical
aggregates (diameters of about 80120nm), are composed of thousands of gra-
phitic tubule closed ends with cone-shaped horns and have a large surface area,
excellent conductivity, plentiful inner nanospaces, and highly defective horns.
The oxidation treatment of SWNHs can produce extensive oxygen-functionalized
sites exposed on the cone-shaped tips for the immobilization of proteins. Using
SWNHs as an immobilization scaffold of small antigen molecules, Jus group
[24] developed a novel immunosensor for MC-LR. As shown in Fig. 15.4, the
functionalization of nanohorns was performed by covalently binding MC-LR to
the abundant carboxylic groups on the cone-shaped tips of the nanohorns in the
presence of linkage reagents. Using HRP-labeled MC-LR antibody for the com-
petitive immunoassay, the immunosensor exhibited a wide linear response to
MC-LR ranging from 0.05 to 20mg/L, with a detection limit of 0.03mg/L.
15.3 Immunosensors Based on Biocompatible Nanomaterials 433

Fig. 15.3 Preparation and detection procedures of the CNF-based CA 125 immunosensor.
Reprinted with permission from Wu etal. [23]. 2007, Elsevier

Fig.15.4 Schematic representation of the preparation and detection procedure of MC-LR immu-
nosensor. Reprinted with permission from Zhang etal. [24]. 2010, American Chemical Society

Silica nanoparticles (Si NPs) are typically synthesized by using a water-in-oil


microemulsion procedure, where the hydrolysis and polycondensation of tetraethox-
ysilane precursor occur in the ammonium hydroxide/cyclohexane system. Si NPs
have no obvious electrochemical activity as noble metal and carbon nanoparticles.
434 15 Nanomaterials for Immunosensors and Immunoassays

However, they still can be considered for the immobilization of antigens/antibodies


to fabricate immunosensors due to their high surface-to-volume ratio and good
biocompatibility. A highly hydrophilic and nontoxic colloidal Si NPs/titania solgel
composite membrane was assembled on a gold electrode via a chemical vapor-
deposition method [25]. With CEA as a model analyte and the encapsulation of
anti-CEA in the composite architecture, this membrane could be used for potentio-
metric immunoassay. The formation of immunoconjugate by a simple one-step
immunoreaction between CEA in sample solution and the immobilized anti-CEA
led to the change in the potential. The proposed immunosensor exhibited high
sensitivity, rapid response, good reproducibility, and acceptable stability toward
CEA because the presence of Si NPs provided a congenial microenvironment for
adsorbed biomolecules. A direct and highly sensitive piezoelectric immunoassay for
Toxoplasma gondii-specific IgG in infected rabbit serum was proposed on the basis
of a biomolecular immobilization strategy incorporating an Si NP matrix and the
plasma-polymerized film of n-butyl amine [26]. Si NPs were chemically activated
and then used to conjugate T. gondii antigens onto the plasma-polymerized film-
deposited crystal. Compared to the commonly applied methods, in particular, the
glutaraldehyde cross-linking procedure, this strategy allowed for antigens immobilized
with a higher loading amount and better retained immunoactivity. Recently, Jus group
[27] synthesized a biofunctionalized three-dimensional ordered nanoporous silica
film to construct a CL immunosensing device. The nanoporous silica film was pre-
pared with self-assembly of polystyrene spheres as a template and 5-nm Si NPs on a
glass slide followed by a calcination process. As shown in Fig.15.5, the film was
then functionalized with streptavidin by using 3-glycidoxypropyltrimethoxysilane
(GPTMS) as a linker. Based on the high-selectivity recognition of streptavidin to
biotin-labeled antibodies, a novel immunosensor was constructed for highly efficient
CL immunoassay of carbohydrate antigen 125. The three-dimensional ordered nano-
pores had a high capacity for loading of streptavidin and antibodies and for improving
the mass transport of immunoreagents for immunoreaction. Thus, the resulting CL
immunosensor showed a wide dynamic range for fast immunoassay and good
reproducibility and stability. The highly efficient immunosensing system showed a
dynamic range of three orders of magnitude, from 0.5 to 400U/mL.
Paramagnetic nanoparticles (PNPs) are currently widely used in immunoassays
as immobilization substrates for their advantages over conventional solid-phase
substrates: The large surface area allows for the capture of more antibodies to
enhance the sensitivity; the free suspension in the immunoreagents leads to a rapid
reaction speed; the isolation of the particles can be easily and rapidly performed
under a simple magnetic field; the total amount of analyte will be effectively
concentrated when the particles are collected. The current applied PNPs are often
prepared in a core-shell structure with an Fe3O4 core. Li et al. [28] proposed a
piezoelectric immunoassay method for IgG detection using PNPs to immobilize
goat-anti-IgG antibodies. The antibodies were first covalently immobilized to PNPs
via surface-modified amino groups. The magnetic bio-nanoparticles formed were
attached to the surfaces of quartz crystal with the aid of a permanent magnet.
Thedetection of IgG could be performed with the magnetic piezoelectric sensor.
15.3 Immunosensors Based on Biocompatible Nanomaterials 435

Fig.15.5 Schematic diagram for biofunctionalization of 3D nanoporous SiO2 film and one-step
sandwich immunoassay procedure for CA 125. Reprinted with permission from Yang etal. [27].
2008, Wiley

After detection, the PNPs carrying immunocomplex layers could be released by


withdrawing the magnetic field. Therefore, the piezoelectric sensor could be
expediently regenerated for reuse. As known, this regeneration was of practical
significance for immunoassays, especially for those with expensive base transducers.
Tang et al. [29] synthesized magnetic core-shell NiFe2O4/SiO2 nanoparticles and
fabricated a magnetic controlled microfluidic device for the electrochemical detec-
tion of four tumor markers. The immunoassay system consisted of five working
electrodes and an Ag/AgCl reference electrode integrated on a glass substrate. Each
working electrode attached PNPs with different antibodies and was capable of
measuring a specific tumor marker by a noncompetitive mode.
Park etal. [30] proved that by combining viral nanoparticles engineered to have
dual affinity for troponin antibodies and nickel, with three-dimensional nanostruc-
tures including nickel nanohairs, one could detect troponin levels in human serum
samples. The detection limit was six to seven orders of magnitude lower than that of
the conventional enzyme-linked immunosorbent assays. The viral nanoparticles
oriented the antibodies for maximum capture of the troponin markers. High densi-
ties of antibodies on the surfaces of the nanoparticles and nanohairs led to a greater
binding amount of the troponin markers, which remarkably improved the detection
sensitivity. The nickel nanohairs were reusable and can reproducibly distinguish
healthy serum from unhealthy ones.
436 15 Nanomaterials for Immunosensors and Immunoassays

15.3.2Nanomaterials Used as Signal Tags

For immunorecognition events, quantification is generally achieved by measuring


the specific activity of a tag substance, that is, its optical activity and enzyme
activity, after an immunoreaction between antibodies and antigens. Therefore, tags
with a high sensitivity are obviously advantageous to improve the signal intensity
and lower the detection limits of immunosensors and immunoassays. Compared
with the conventional signal tags such as enzyme and organic fluorescent dye,
nanomaterial tags are showing greater promise for developing ultrasensitive immu-
noassay methods. Two main reasons may be responsible for the improved sensitivity
of immunoassays by using nanomaterials as tags. One is the use of special electrical,
optical, catalytic, and magnetic properties of nanosized materials for signal
transduction. For example, the high fluorescence intensity of quantum dots (QDs)
and the enhanced SPR from the use of Au NPs have resulted in improved sensitivity
of fluorescent and surface-enhanced Raman spectroscopy (SERS) detection.
Thesecond reason for the improved sensitivity is attributed to numerous signal
molecules contained in a single nanosized tag. The single nanosized tag containing
a large number of signal molecules thus produces a much higher signal than the
single tag containing only one or several signal molecules (traditional tag). For
example, europium compound nanoparticles show much stronger time-resolved
fluorescence than single-europium compound as the tag.
The applications of colloidal Au NPs and Ag NPs in immunoassays date back to
the early 1970s when 550nm Au NPs were first used as electron-dense tags in
electron microscopy and thus enabled sensitive, high-resolution immunocytochem-
istry [31]. The subsequent development of silver-enhanced techniques then allowed
Au NPs to provide very specific and sensitive immunocytochemistry. Among all
metal-based tags, Au NP is an ideal one in immunological systems for its inherent
advantages: Au NPs can be easily prepared in a wide range of sizes, from 2 to
100nm; the biochemical activity of the immunoreagents, such as antigens or anti-
bodies, could be well retained after coupled to the surfaces of Au NPs; Au NPs
show unique optical and electronic properties when used as the signal probe.
Au NP-based immunochromatographic strip is one of the most frequently used
portable immunoassay devices for the purpose of screening. In this approach, Au
NPs are usually used to tag antibodies while the whole assay process is performed
on a disposable and low-cost strip in several minutes. The formed immunocom-
plexes on the strip lead to an observable red line resulting from the optical absor-
bance of Au NPs. Thus, qualitative and semiquantitative assays can be easily
achieved by the naked eye, while accurate quantitative colorimetric assay can be
performed on an instrument. With this portable and low-cost immunoassay strip,
various analytes, such as zearalenone [32], TNT [33], Alexandrium minutum [34],
deoxynivalenol [35], ochratoxin A [36], clenbuterol, and ractopamine [37], have
been rapidly detected for screening or quantitative purposes.
Besides these immunochromatographic strips, some other immunoassay strategies
were also proposed to detect various analytes based on the absorbance characteristics
of Au NPs. Zhang etal. [38] immobilized antibodies onto a glass slide with the aid of
15.3 Immunosensors Based on Biocompatible Nanomaterials 437

chitosan to fabricate an optical immunosensor for human serum albumin. After


incubating in sample solution, the obtained substrate was immersed in Au NP-labeled
antibodies for signal generation. The two steps were repeated alternatively three times
to form a multilayer of Au NPs via antigenantibody-specific binding. Ultraviolet-
visible absorption spectrum was recorded to obtain quantitative information about the
target analyte. Compared to the previous reported works, the proposed immunosensor
showed an improved sensitivity, as many more Au NPs can be coupled to the func-
tionalized surface by making use of the abundant amino groups of chitosan.
Homogeneous colorimetric immunoassays have also been developed based on
the unique phenomenon that different aggregation states of the Au NPs can result
in distinctive color changes, in which Au NPs functionalized with antigens aggre-
gate in the presence of antibodies. This method was much simpler than those
heterogeneous methods. However, the main disadvantage of this approach is its
low sensitivity [39].
Au NPs can be dissolved to form AuCl4 in some oxidative acid solution, and the
resulting AuCl4 can react with luminol to generate strong CL emission. Each Au NP
contains thousands of gold atoms (e.g., 1.1105 gold atoms are theoretically con-
tained in a 15-nm spherical Au NP), and consequently, low detection limits can be
achieved by using Au NPs as tags for CL immunoassays. Hu etal. [40] described a
CL immunoassay method based on AuCl4-enhanced luminol CL reaction for the
highly sensitive detection of ApxIV antibodies of Actinobacillus pleuropneumoniae.
The optimal condition of gold dissolution was composed of a 5.0102 M HCl,
1.5102M NaCl, and 2.5104M Br2 solution. The proposed method provided a
new tool for the indirect determination of antibodies against ApxIV in pig serum
samples and showed great potential for numerous applications in immunoassays.
With the similar strategy, goat-antihuman IgG can also be CL-detected in the range
of 5ng/mL to 10mg/mL, with a detection limit of 1.5ng/mL [41]. A sensitive CL
immunoassay that combined the inherent high sensitivity of CL analysis with the
dramatic signal amplification of silver precipitation on Au NP tags was developed for
human IgG detection. Au NPs labeled on sandwich complexes were treated by silver
reduction solution, which resulted in the catalytic precipitation of silver on the surface
of colloidal gold. Then a large number of Ag+ ions were oxidatively released in
HNO3 solution from the silver metal anchored on the immunocomplexes. Thus,
human IgG was indirectly determined by a sensitive Ag+K2S2O8Mn2+H3PO4
luminol CL reaction over the concentration range of 0.0250ng/mL [42].
These methods should be further improved since the stripping procedure,
namely, the dissolution of Au NPs or Ag NPs, was performed under extremely
harsh conditions (highly concentrated HNO3-HCl or poisonous HBr-Br2), which
resulted in a high background. A nonstripping CL immunoassay using Au NPs as
tag was also reported based on the phenomenon that the irregular Au NPs could
greatly enhance the CL intensity of the luminolH2O2 system. Although this
protocol avoided the harsh stripping procedure, the synthesis of irregular Au NPs
was difficult to control, requiring stirring for a long time with a temperature control
(40C for 24h), purging of oxygen, and relatively low monodispersity. This fact
may influence the repeatability among different batches, limiting the practical
application of this method [43].
438 15 Nanomaterials for Immunosensors and Immunoassays

Fig. 15.6 Schematic representation of the preparation of immunosensor array and analytical
p rocedure for the simultaneous detection of human IgG and goat IgG. Reprinted with permission
from Leng etal. [45]. 2010, Elsevier

Duan etal. [44] found that Au NPs surrounded by protein could trigger the CL
reaction between AgNO3 and luminol. Based on this finding, this novel CL system,
that is, luminolAgNO3Au NP system, was utilized to develop a novel nonstrip-
ping CL immunoassay protocol. Besides the application in optical immunosensors,
Au NPs have also been utilized as signal probes for immunoassays with electro-
chemical analysis approaches, such as amperometric, potentiometric, stripping
voltammetric, and piezoelectric methods. For example, Jus group [45] proposed a
simple, sensitive, and low-cost, inherently cross-talk-free multiplexed immunoas-
say by combining a disposable chip with Au NP as an electrochemical label.
As shown in Fig.15.6, the immunosensor array was first prepared by immobilizing
capture antibodies on different SPCEs by passive adsorption. Following a sandwich
immunoassay format, the Au NP-labeled antibodies were conjugated on the immu-
nosensors surface. The analytes were detected by electrooxidization of Au NPs in
0.1 M HCl. This method eliminated electrochemical cross-talk between adjacent
immunosensors due to the strong adsorption of the AuCl4 on the printed carbon
surfaces. Since each Au NP contains thousands of atoms, the immunoassay pos-
sesses a relatively high sensitivity. Using human IgG and goat IgG as model targets,
under optimal conditions this method achieved linear ranges from 5.0 to 500 and
5.0400ng/mL with limits of detection of 1.1 and 1.6ng/mL, respectively.
Selvaraju etal. [46] developed a nanocatalyst-based electrochemical immunoas-
say using Au NPs. Au NP tags of the immunocomplexes on the electrode produced
15.3 Immunosensors Based on Biocompatible Nanomaterials 439

p-aminophenol from p-nitrophenol by catalytic reduction in the presence of NaBH4,


and the generated p-aminophenol was then electrooxidized. The oxidized product,
p-quinone imine, was reduced back to p-aminophenol by NaBH4 and then re-elec-
trooxidized at the electrode. This redox cycling greatly amplified the electrochemi-
cal signal. Accordingly, the high signal-to-background ratio allowed an extremely
low detection limit in cyclic voltammetric experiments.
Mao etal. [47] demonstrated a novel electrochemical protocol for quantification
of human IgG based on the precipitation of copper on Au NP tags and a subsequent
electrochemical stripping detection of the dissolved copper. In this work, the copper
enhancer solution was added to deposit copper on the Au NP tags of the sandwich
complexes. After dissolution with HNO3, the released copper ions were then quanti-
fied by anodic stripping voltammetry.
Chumbimuni-Torres etal. [48] reported for the first time on the use of potentiom-
etry for the ultrasensitive nanoparticle-based detection of protein interactions. It was
based on a sandwich immunoassay forming Au NP-tagged immunocomplexes. Then
Ag NPs formed by catalytic silver enlargement onto the gold tags. The precipitated
silver was oxidatively dissolved with hydrogen peroxide to yield silver ions, which
were potentiometrically detected with a polymer membrane silver ion-selective
microelectrode. The detection reported here was made without a preconcentration
step typically used in other electrochemical techniques.
Zhang etal. [49] fabricated a patterned ITO-arrayed impedimetric immunosensor
by the use of Au NP tags. Based on the immunological reactions occurring on the
electrodes, a large amount of silver deposition could be produced specifically on
the Au NP tags, which resulted in the amplification of the impedance signal. The
signal amplification of the silver precipitation was found to provide ultrasensitive
impedimetric detection of multiple antibodies.
A highly sensitive piezoelectric immunosensor was proposed using Au NP tag
and applied to detect aflatoxin B1 in contaminated milk [50]. It was unlikely that the
direct binding of small molecules such as aflatoxin B1 to the piezoelectric sensor
surface could result in a satisfactory sensitivity. Thus, an indirect competitive immu-
noassay strategy was applied for the detection of the target using Au NPs as a
weight tag to the secondary antibodies for amplifying the response. This method
was proven in its ability to detect the analyte down to a level of 0.01ng/mL due to
the fact that the Au NP-labeled antibodies were very heavy compared to small mol-
ecules or biospecies.
Au NPs are known to have a large light absorption and scattering cross-section
in the SER wavelength regions. The magnitude of light scattering from Au NPs
can be orders of magnitude higher than light emission from strongly organic
fluorescent dyes. This unique property has enabled many important and promis-
ing applications of Au NPs in the homogeneous immunoassay field. As known,
homogeneous immunoassays always require simpler manipulation and a shorter
assay time than heterogeneous immunoassays. Thus, the light-scatteringbased
immunoassay methods are really advantageous in their assay speed and labor
cost. A homogeneous noncompetitive immunoassay method was developed using
human IgG as a model analyte. The assay strategy was based on the aggregation
440 15 Nanomaterials for Immunosensors and Immunoassays

Fig. 15.7 A schematic illustration of a homogeneous immunoassay using antibody-conjugated


gold nanoparticles and nanorods coupled with dynamic light-scattering measurement. Reprinted
with permission from Liu etal. [51]. 2008, American Chemical Society

of antibody-functionalized Au NPs directed by the immunoreaction coupled with


light-scattering detection. The light-scattering intensity of Au NPs functional-
ized with goat-antihuman IgG could be greatly enhanced by the addition of the
human IgG. Based on this fact, a wide dynamic range of 0.0510mg/mL for the
immunoassay of human IgG was obtained [39].
Liu etal. [51] reported a highly sensitive, one-step homogeneous immunoassay
for free prostate-specific antigen (PSA) detection using Au NP tags coupled with
dynamic light-scattering analysis. As illustrated in Fig.15.7, two different types of
Au NPs (one is a spherical nanoparticle and the other is a nanorod) were conjugated
with an anti-PSA antibody pair, one with the capture antibodies and one with the
detector antibodies. When these two bioconjugated antibodies were mixed in a
sample solution that contains free PSA, the binding of free PSA caused nanoparticles
to form dimers, oligomers, or aggregates, depending on the concentration of the
antigens. The relative ratio of nanoparticle dimers, oligomers, or aggregates vs. indi-
vidual nanoparticles can be measured to quantitate the concentration of antigens.
In another work concerning homogeneous immunoassays, Au NPs strongly
catalyzed the redox reaction between hydrazine and Cu(II) to form Cu particles,
which exhibited a strong resonance scattering peak at 602 nm. Thus, an Au
NP-catalytic resonance-scattering spectral assay was established for the detection
of microalbumin using 10-nm Au NPs to label antibodies. This method showed very
high sensitivity, with a detection limit of 0.1 pg/mL [52]. The same group also
established a selective and highly sensitive resonance scattering assay for IgG using
Cu(II)ascorbic acidAu NP reaction with a similar principle [53].
SERS-based immunosensors and immunoassays have attracted noticeable interest
in recent years because of their inherently high sensitivity. In SERS, the Raman
signal of a molecule adsorbed on the gold/silver nanoparticles surface can be
enhanced more than a million-fold due to the electromagnetic enhancement mecha-
nism. However, the use of SERS in protein detection requires special labels where
15.3 Immunosensors Based on Biocompatible Nanomaterials 441

Raman-active molecules (as well as the antibodies) need to be attached to gold or


silver nanoparticles since protein molecules usually produce weak SERS signals.
Manimaran and Jana [54] conjugated 25-nm Au NPs with fluorescein isothiocya-
nate and antibodies in order to obtain the Raman tracer antibodies. This Raman tag
acted as an efficient label, and the SERS signal was greatly amplified followed by the
silver enhancement. It is possible to identify the presence of human IgG down to
the level of ng/mL by using this detection scheme. The detection sensitivity of the
described SERS-based technique was similar to Au NP-based immunoassays.
However, each Raman-active molecule has a unique vibrational fingerprint, and thus
many of the SERS tags can be prepared for the simultaneous immunoassay of many
proteins. Grubisha etal. [55] synthesized a reagent that consisted of Au NPs modi-
fied to antibodies with 5,5-dithiobis(succinimidyl-2-nitrobenzoate) for the genera-
tion of intense, biolyte-selective SERS responses in immunoassays. The reagent was
constructed by coating Au NPs (30nm) with a monolayer of the strong Raman scat-
terer. This strategy both minimized the separation between the scatterer and the par-
ticle surface and maximized the number of scatterers on each particle. It also showed
several other advantages: narrow spectral bandwidth, resistance to photobleaching
and quenching, and long-wavelength excitation of multiple tags with a single excita-
tion source. Detection limits of about 1pg/mL in human serum and about 4pg/mL in
bovine serum albumin have been achieved for PSA.
SPR is a flowthrough biosensor technology that measures very small changes in
the refractive index on a noble metal surface when mass binds to that surface and
is used as a transduction approach. This technique has been used to fabricate vari-
ous immunosensors. Nanoparticles have been employed as high-mass tags for
enhancing the binding signals and improving the detection sensitivity of SPR
immunosensors. A specialized linker conjugate of testosterone has been synthe-
sized for covalent immobilization to a dextran SPR sensor surface as the coating
antigen. This surface was then used for the development of an ultrasensitive immu-
nosensor for testosterone by using secondary antibodies labeled with Au NP signal
enhancement. The increased signal can attribute to increased binding mass and a
gold plasmon-coupling effect [56]. The signal enhancement of immunoassays
using nanoparticles is affected by the size and distance of the nanoparticles from
the sensing surface. High signal amplifications are expected with an increased par-
ticle size and decreased distance between the sensor surface and the particle. Based
on this fact, Yuan et al. [57] described an ultrasensitive SPR immunoassay
(4.9ng/L) for small molecules (progesterone) by reducing the distance of 10-nm
Au NP from the sensor surface using a stable mixed self-assembled monolayer/
progesteroneoligoethylene glycolovalbumin configuration.
A highly sensitive immunoassay has been proposed based on time-resolved
inductively coupled plasma mass spectrometry with Au NPs (15nm) as tags to anti-
bodies [58]. In this work, Hu etal. recorded in a time-resolved mode the transient
signals induced by the flash of ions in the plasma torch from the ionization of nano-
particles tagged on antibodies. Since the frequency of transient signals was directly
correlated to the concentration of Au NP tags, the concentration of the sample could
be quantified by the frequency of transient signals. This protocol was evaluated for
442 15 Nanomaterials for Immunosensors and Immunoassays

a competitive immunoassay, and the linear range for AFP was 0.0166.8mg/L [58].
A similar mass spectrometric immunoassay strategy also has been proposed by the
same group to detect human IgG in immunomicroarray mode with a detection limit
of 0.012ng/mL [59].
Xie etal. [60] first presented a highly sensitive immunoassay method based on a
single Au NP counter in solution. This strategy was based on the photon burst
counting in a small detection volume by utilizing strong resonance scattering and
Brownian motion of a single Au NP. The relationship between the photon burst
counts and the Au NP amount showed an excellent linearity. Based on this phenom-
enon, this group developed an ultrasensitive and highly selective detection platform
for homogeneous immunoassay, which was two to five orders of magnitude more
sensitive than the current homogeneous immunoassays. Moreover, the detection
volume was less than 1fL, and the sample requirement can be easily reduced to the
nanoliter level by using a droplets array. Thus, this method had the potential to
evolve a high-throughput detection platform similar to currently used microarray
immunochips.
QDs, also known as nanocrystals, are the most eye-catching fluorophores devel-
oped for fluorescent images and bioconjugates in the past two decades. They exhibit
some important differences compared to traditional fluorophores, such as organic
fluorescent dyes and naturally fluorescent proteins. QDs are nanometer-scale atom
clusters, containing from a few hundred to a few thousand atoms of semiconductor
material such as CaSe or CaTe, which sometimes have been coated with an addi-
tional semiconductor shell such as ZnS to improve their optical properties. Besides
their excellent quantum efficiency, QDs also show several other advantages over
conventional organic dyes. Their emission spectra are symmetric, narrow, and tun-
able according to their size and chemical composition, permitting closer spacing of
different probes without substantial spectral overlap. They exhibit excellent photo-
stability, tolerating long-time excitation. QDs also display broad absorption spectra,
making it possible to minimize sample autofluorescence by choosing an appropriate
excitation wavelength. Thus, QDs have attracted increasing interest as tags for
immunoassays besides their application in bioimage in the past decade.
Vinayaka etal. [61] developed a reliable and rapid fluoroimmunoassay method
for analysis of 2,4-dichlorophenoxyacetic acid by using CdTe QDs to tag 2,4-dichlo-
rophenoxyacetic acid. Antibodies were immobilized in an immunoreactor column
using Sepharose CL-4B as an inert matrix. The detection of 2,4-dichlorophenoxya-
cetic acid was carried out by competitive binding between conjugated 2,4-dichloro-
phenoxyacetic acidCdTe and free 2,4-dichlorophenoxyacetic acid with immobilized
antibodies in an immunoreactor column. 2,4-Dichlorophenoxyacetic acid can be
detected in the range of 250pg/mL to 1,000ng/mL. Another fluorescent sandwich
immunoassay using high-affinity antibodies and QD optical reporters have been
developed for the detection of botulinum neurotoxin serotype A using a renewable
surface column and 96-well plate formats. Using QDs with a maximum emission of
455nm, detection limits of 31 and 5pM were obtained for the two formats, respec-
tively [62]. Chen etal. [63] described a rapid and ultrasensitive detection method
using a microfluidic chip for analyzing 7-aminoclonazepam residues in human urine.
15.3 Immunosensors Based on Biocompatible Nanomaterials 443

The microfluidic chip-based immunoassay with laser-induced fluorescence detection


based on the water-soluble denatured bovine serum albumincoated CdTe QDs
was performed for the ultrasensitive detection of this analyte. Under the optimal
conditions, 7-aminoclonazepam residues could be detected with a linear range of
1.160.1ng/mL. Soman and Giorgio [64] proposed a novel approach to sensitive
and rapid antigen detection based on QD aggregation phenomenon. In the presence
of a specific antigen, QDantibody conjugates can rapidly self-assemble into
agglomerates, which were one order of magnitude larger than their individual
components. The relative concentration of QD conjugates and antigen molecules
affected the size distribution of the agglomerated colloids and their emission
characteristics. With this strategy, angiopoietin-2 and mouse IgG can be detected to
sub-picomolar concentrations. This simple technique also enabled the potential
simultaneous detection of multiple antigenic biomarkers. Goldman etal. [65] used
antibody-conjugated QDs with emission maximums at 510, 555, 590, and 610nm
to demonstrate multiplex assays for four protein toxins present in the same sample.
Such a four-color QD-based multianalyte immunoassay was limited by signal
overlapping and the deconvolution of composite spectra was necessary.
The intrinsic redox properties and the sensitive electrochemical stripping analysis
of the metal components of QDs make the tags in the electrochemical biosensors
very sensitive. This concept was first proposed by Wangs group for the electro-
chemical DNA hybridization assay [66], and then extended to the immunoassay
[67]. In this pioneering paper, an anodic stripping voltammetric immunoassay
protocol for the simultaneous measurements of multiple protein targets was designed
based on the use of different QDs as tags (CdS, ZnS, and PbS). Each biorecognition
event yielded a distinct voltammetric peak whose position and size reflected the
identity and concentration, respectively, for the corresponding antigen. This novel
concept has been demonstrated for a simultaneous immunoassay of a2-microglob-
ulin, IgG, bovine serum albumin, and C-reactive protein. With a similar strategy,
ZnS@CdS QDs were also widely used in anodic stripping voltammetric immunoas-
says and disposable immunosensors for various analytes, including interleukin-1a
[68], PSA [69, 70], and cyanobacterial hepatotoxin Microcystin-LR [71], by
dissolving Cd2+ using acid after immunoreaction.
More recently, by combining the rolling-circle amplification (RCA) technique
with QDs and multiplex binding of the biotinstreptavidin system, Jus group [72]
proposed an ultrasensitive immunosensor for the detection of protein target at an
ultralow concentration. The RCA product containing tandem-repeat sequences can
serve as an excellent template for the periodic assembly of QDs, which present per
protein recognition event to numerous QD tags for electrochemical readout (Fig. 2.21).
With a sandwich immunoassay, the designed immunosensor can quantitatively
detect human vascular endothelial growth factor protein down to 16 molecules in a
100-mL sample with a linear calibration range from 1aM to 1pM.
QDs have also been used as signal tags for potentiometric immunoassays.
Thurer etal. [73] reported a potentiometric immunoassay of IgG in a microtiter
plate format using CdSe as tags. After sandwich immunoreaction on the microtiter plate,
the captured QDs were dissolved in a matter of minutes with hydrogen peroxide.
444 15 Nanomaterials for Immunosensors and Immunoassays

The released Cd2+ ions can be sensitively detected by Cd2+-selective micropipette


electrodes. The potentiometric immunoassay method for IgG exhibited a log-linear
response ranging from 0.15 to 4.0pM, with a detection limit under 10fM in 150-mL
sample wells.
Eu2O3 can be simply and cheaply prepared as inorganic phosphor nanoparticles
for the use of immunoassay tags by a simple microwave-assisted surface chemistry.
A silane layer can be capped over the particles surface to provide amine groups
that can be applied for biological conjugation. The silane shell can also protect the
Eu2O3 nanoparticles during conjugation chemistry, while their desirable optical
properties will be well retained. The use of the Eu2O3 nanoparticle tags in
immunoassay yielded very good sensitivity for atrazine, with a detection limit of
sub-parts-per-billion [74].

15.3.3Nanomaterials Used as Probe Carriers

As mentioned earlier, one of the most efficient strategies to improve the label
amount for antibodies or antigens is to prepare nanosized tags, that is, to prepare
nanoparticles composed of a large amount of signal molecules. Unfortunately, the
preparation of nanosized tags is sometimes challenging and difficult. Up to now,
only several of the usual tags have been prepared in a nano size and used for
labeling biomolecules. An alternative approach for the same purpose is the use of
currently existing nanomaterials as nanovehicles to load a large number of signal
molecules for highly sensitive immunoassays.
Si NPs have shown many advantages in the development of carrier vehicles
for the signal probe, such as ease of fabrication and functionalization, and high
stability in a variety of environments. Different strategies, including covalent
binding, entrapment, and electrostatic interaction, have already been adopted to
prepare functionalized Si NPs for bioconjugation. With the inverse microemul-
sion polymerization protocol, Ru(bpy)3Cl2 was doped into Si NPs by Hun and
Zhang [75]. Then core-shell structured fluorescent Si NP-labeled anti-TNF-a
monoclonal antibodies were prepared and used for the fluoroimmunoassay of
TNF-a in human serum samples, with a detection limit of 0.1 pg/mL. With a
similar fluoroimmunoassay strategy, some other analytes such as staphylococcal
enterotoxin C1 [76], and IL-6 [77] have also been detected by using the nanopar-
ticles with a core of Ru(bpy)3Cl2 and a shell of silica.
Luminescent europium(III) and terbium(III) chelates can be covalently immobi-
lized on the surface of prepared Si NPs to which reporter antibodies or bridging
proteins for antibody binding are conjugated. Xu and Li [78] utilized the resulting
conjugates in time-resolved fluorescent immunoassays for hepatitis B surface
antigen and hepatitis B antigen, both individually and simultaneously. The prepared
nanoparticle conjugates were homogeneous in size at 55nm in diameter, and stable
for long-term storage (>2 years). The proposed method showed good sensitivity and
repeatability. The europium chelate-loaded Si NPs were also successfully adopted
15.3 Immunosensors Based on Biocompatible Nanomaterials 445

to prepare a portable lateral flow immunoassay strip with better sensitivity than the
conventional strip using Au NP tags [79].
Wang et al. [80] prepared an electroactive Si NP in which poly(guanine) was
used to functionalize Si NPs. The Si NPs loading poly(guanine) could serve as a
biological tag for a sensitive electrochemical immunoassay. It was found that there
were ca. 60 strands of poly(guanine)20 per Si NP, which meant that the average
surface coverage of poly(guanine)20 on an Si NP was ca. 8.51012 molecules/cm2.
The detection limit for TNF-a was found to be 5.01011g/mL (2.0pM) for an
immunosensor using the nanoparticles as tags, which corresponds to 60 aM of
TNF-a in 30mL of sample. This immunosensor based on the poly(guanine)-func-
tionalized Si NP tags offered great promise for the rapid, simple, cost-effective
analysis of biological samples.
Au NPs can be used as a probe carrier for their excellent adsorption ability. They
are generally used for loading enzymes or enzyme-labeled antibodies by the adsorp-
tion of these biomolecules on their surface. Due to their excellent biocompatibility,
the activity of the adsorbed enzymes can be well retained to generate a strong
signal. Zhou etal. [81] described a novel immunoassay strategy for the amplified
detection of AFP using DNAzyme-functionalized Au NPs as catalytic tags. Since
the Au NPs carried a large number of DNAzyme units per protein-binding event,
there was substantial amplification. A chromogenic reagent ABTS showed a green
color in the presence of DNAzyme catalysis. This method resulted in a linear
working curve in the concentration range of 0.220 ng/mL. Wang et al. [82]
described a very simple and easily operated colorimetric multiplexed immunoassay
method for the sequential detection of tumor markers. Magnetic microparticles con-
jugated with antibodies were used to capture antigens. Through different enzymatic
reactions of 3,3,5,5-tetramethylbenzidine and o-phenylenediamine catalyzed by
HRP molecules that were loaded on the surfaces of Au NPs, two antigens, CEA and
AFP, can be detected even with the naked eye. The detection limit obtained from the
spectrophotometric measurements is as low as 0.02 ng/mL. Ambrosi et al. [83]
developed an optical ELISA for the analysis of the CA15-3 antigen. Amplification
of the optical signal was achieved by using Au NPs as carriers of the signaling anti-
bodies anti-CA15-3-HRP. In the range of 060U/mL, the method using Au NPs as
enhancers resulted in a higher sensitivity and shorter assay time when compared to
classical ELISA procedures. Tang etal. [84] proposed a new signal amplification
strategy based on thionine-doped magnetic Au NPs as tags and HRP as enhancer for
the immunoassay of CEA. This immunoassay system was performed on a carbon
fiber microelectrode covered with a well-ordered anti-CEAprotein Ananogold
architecture. The reverse-micelle method was applied for the preparation of thionine-
doped magnetic Au NPs, and the nanoparticles were then used to load HRP-bound
anti-CEA. A sandwich-type protocol was successfully introduced to develop a high-
efficiency electrochemical immunoassay with the labeled bionanospheres toward
the reduction of H2O2. CEA can be detected in the range 0.01160ng/mL by using
the Au NP-based tags.
Polystyrene nanoparticles (PNPs) is a polymer mainly used for loading
europium(III) chelate, a time-resolved fluorescent probe. Europium(III) chelate-dyed
446 15 Nanomaterials for Immunosensors and Immunoassays

fluorescent PNPs with a functionalized surface have already been commercially


prepared and provided by Seradyn Inc. for immunoassay tags. Kakko et al. [85]
compared the characteristics of two homogeneous competitive immunoassays using
either europium(III)-chelates loaded in PNPs or soluble europium(III)-chelates as
donors in a fluorescence resonance energy-transferbased method. The use of the
nanoparticle tags significantly increased the obtained fluorescent signal, which was
generated by a single binding event. This phenomenon attributed to the extremely
high specific activity of the nanosized tag and also in some extent the longer Frster
radius between the donor and the acceptor. The amount of the binder protein used in
the method could be decreased by tenfold without impairing the obtainable sensi-
tized emission, which subsequently led to improved sensitivity. Pelkkikangas etal.
[86] described a rapid thyroid-stimulating hormone assay that enabled diagnosis
during the first visit at the doctors office, leading to faster and cost-effective medical
treatment. To accomplish such an assay method, europium(III) chelate nanoparticles
were coated with thyroid-stimulating hormone tracer antibodies, and the captured
antibodies were immobilized onto wells by streptavidinbiotin chemistry. The assay
was performed in dry chemistry mode using 5mL of sample in a 30-mL assay volume
in the commercial immunoassay system. The sensitivity of the proposed time-
resolved fluorescent immunoassay method was 0.0012mIU/L, corresponding to the
fourth-generation thyroid-stimulating hormone assay, and less than 0.02 mIU/L
when a serum-based matrix was used for calibration. With the use of the same PNP-
based fluorescent tags, some other analytes, such as free PSA [87], total PSA [88],
and atrazine [89], have also been detected with high sensitivity.
CNTs are particularly exciting one-dimensional nanomaterials. Recent works
have utilized CNT coated with enzymes as labels for amplified bio-detection.
Wangs group [90] demonstrated the use of CNTs for dramatically amplifying the
enzyme-based bioaffinity electrical sensing of proteins and DNA. A detection limit
of 500fg/mL IgG was provided by this method. A greatly amplified sensitivity was
achieved by using bioconjugates featuring HRP labels and secondary antibodies
(Ab2) linked to CNTs at a high HRP/Ab2 ratio to replace singly HRP-labeled
secondary antibodies [91]. This resulted in a detection limit of 4pg/mL (100aM/
mL) for prostate specific antigen in 10mL of undiluted calf serum. Jus group [92]
functionalized a GOD-loaded CNT probe to enhance the enzymatic signal in immu-
noassays. As shown in Fig. 2.37, the tracer probe was prepared by the one-pot
assembly of GOD and the antibodies on Au NPs attached to CNTs. The CNT-based
probe can provide dual signal amplification for the multiplexed immunoassay by a
PB-mediated electron-transfer process to catalyze the reduction of H2O2 produced
in a GOD cycle at an individual working electrode. The simultaneous multiplexed
immunoassay was used to detect both CEA and AFP with linear ranges of three
orders of magnitude, showing great potential clinical applications for low-abundant
protein detection.
Apoferritin is a native protein composed of 24 polypeptide subunits that interact
to form a hollow cagelike nanostructure with a diameter of 12.5nm. The interior
cavity of apoferritin is about 8 nm in diameter and capable of accommodating
around 4,500 iron atoms. The protein nanocage of apoferritin can be disassociated
15.4 Conclusions 447

Fig.15.8 Preparation of probe-loaded apoferritin nanovehicle. Reprinted with permission from


Liu etal. [93]. 2006, American Chemical Society

into 24 subunits at a low pH of 2.0 and reconstituted at a high pH of 8.5. Due to this
unique property, it is possible to pack different small-molecule probes into
apoferritin cages by adjusting the pH to form nanosized tags for immunoassays.
Hexacyanoferrate (electroactive probe) and fluorescein (fluorescence probe) were
chosen as model probes for loading into the apoferritin cages to develop versatile
nanosized tags by Liu etal. [93]. The process for preparing probe-loaded apoferritin
nanocages is briefly depicted in Fig.15.8. Apoferritin was dissociated into subunits
at a low pH and then reconstituted at a high pH, thereby packing the probes in solu-
tion within its interior. The protein shell of the apoferritin remained a substantially
stable structure and sustained no obvious alteration during the process, which ascer-
tained the formation of shell-core structure nanoparticles containing a large number
of probe molecules. These nanocage-based tags enabled the immunoassay of IgG
with detection limits of 0.39 and 0.52 pM by hexacyanoferrate and fluorescein,
respectively. The same authors also proposed a simple and facile apoferritin-
templated synthesis of metallic phosphate nanoparticle tags for immunoassay. In
this strategy, metal ions diffused into the cavity of apoferritin and precipitated in the
presence of phosphate. The protein shells of the resulting metal phosphate particles
were then surface-biotinylated to form anodic stripping voltammetric immunoassay
tags. The release of metal ions from apoferritin nanocages in an acetate buffer at pH
4.6 avoided the harsh conditions in the traditional metallic nanoparticles dissolu-
tion. This assay method was ultrasensitive, and its detection limit for biomarkers
was as low as 77fM [94].

15.4Conclusions

As promising approaches for selective and sensitive analysis, immunoassays and


immunosensors have already gained increasing attention in different fields, including
environmental monitoring, clinical diagnosis, food safety, pharmaceutical analysis,
and bacterial identification. The rapid development of nanotechnology offers unique
opportunities for designing ultrasensitive immunosensing methods. The published
works described in this chapter demonstrate the huge potential of biocompatible
nanomaterials for the amplified transduction of biomolecular recognition events.
The remarkable sensitivity of the novel nanoparticle-based immunosensing proto-
cols opens up a new possibility for analyzing very low levels of disease markers,
biothreat agents, or environmental pollutants that cannot be detected by other,
448 15 Nanomaterials for Immunosensors and Immunoassays

c onventional methods. Such highly sensitive biodetection schemes provide useful


techniques for the early diagnosis of disease or warning of terrorist attack.
However, two problems arising from the use of nanomaterials are worthy of
serious consideration. First, severely nonspecific adsorption is often observed in
immunoassays and immunosensors using nanoparticles for either antigenantibody
immobilization or signal tags due to the strong adsorption ability of nanomaterials.
Although signals are significantly amplified by nanomaterials, the simultaneous
increase of the background noise may be rather disadvantageous to obtain low
detection limits. Proper washing and surface blocking steps should be employed as
a matter of course to avoid increasing the background noise associated with nonspe-
cific adsorption of the nanoparticles. A very careful study of the optimal dosage of
nanoparticles is also extremely important for controlling the background noise of
immunoassays and immunosensors. Second, most of the current developed
nanomaterials suffer from poor stability under ordinary storage conditions. As we
know, some nanomaterials are even more unstable than biomolecules such as
proteins and nucleic acids. Minor changes in storage and manipulation conditions
often lead to irreversible aggregation and transformation. Therefore, nanoparticle-
based signal tags and the immunosensing interface must be very carefully manipu-
lated and stored to avoid a decrease in analysis performance resulting from the
instability of nanoparticles.

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Chapter 16
Nanostructured Biosensing and Biochips
for DNA Analysis

16.1Introduction

Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA),
are required for the storage and expression of genetic information. DNA is present
not only in chromosomes in the nucleus of eukaryotic organism, but also in mito-
chondria and in the chloroplasts of plants. Prokaryotic cells, which lack nuclei, have
a single chromosome but also contain nonchromosomal DNA in the form of plas-
mids. The DNA contained in a fertilized egg encodes the information that directs
the development of an organism. This development may involve the production of
billions of cells. Each of these cells is specialized, expressing only those functions
that are required for it to perform its role in maintaining the organism. Therefore,
the DNA must be able not only to replicate precisely each time a cell divides, but
also to have the information that it contains be selectively expressed. RNA partici-
pates in the expression of the genetic information stored in the DNA [1].
DNA-based tests, known as genetic tests, allow the genetic diagnosis of
vulnerabilities to inherit diseases and can also be used to determine a childs pater-
nity (genetic father) or a persons ancestry. Normally, every person carries two
copies of every gene, one inherited from the mother, and another inherited from the
father. The human genome is believed to contain around 20,00025,000 genes. In
addition to studying chromosomes to the level of individual genes, genetic testing
in a broader sense includes biochemical tests for the possible presence of genetic
diseases, or mutant forms of genes associated with increased risk of developing
genetic disorders. Genetic testing identifies changes in chromosomes, genes, or
proteins [2]. Most of the time, testing is used to find changes that are associated with
inherited disorders. The results of a genetic test can confirm or rule out a suspected
genetic condition or help determine a persons chance of developing or passing on

H. Ju et al., NanoBiosensing: Principles, Development and Application, 453


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0_16, Springer Science+Business Media, LLC 2011
454 16 Nanostructured Biosensing and Biochips for DNA Analysis

a genetic disorder. Several hundred genetic tests are currently in use, and more are
being developed [3]. Available types of testing include the following [4]:
1. Newborn screening: Newborn screening is used just after birth to identify genetic
disorders that can be treated early in life. Many countries currently test infants
for phenylketonuria (a genetic disorder that causes mental illness if left untreated)
and congenital hypothyroidism (a disorder of the thyroid gland).
2. Diagnostic testing: Diagnostic testing is used to diagnose or rule out a specific
genetic or chromosomal condition. In many cases, genetic testing is used to
confirm a diagnosis when a particular condition is suspected based on physical
mutations and symptoms.
3. Carrier testing: Carrier testing is used to identify people who carry one copy of a
gene mutation that, when present in two copies, causes a genetic disorder. This type
of testing is offered to individuals who have a family history of a genetic disorder
and to people in ethnic groups with an increased risk of specific genetic conditions.
4. Prenatal testing: Prenatal testing is used to detect changes in a fetuss genes or
chromosomes before birth. This type of testing is offered to couples with an
increased risk of having a baby with a genetic or chromosomal disorder.
5. Preimplantation genetic diagnosis: These are genetic testing procedures that are
performed on human embryos prior to the implantation as part of an in vitro
fertilization procedure.
6. Forensic testing: Forensic testing uses DNA sequences to identify an individual
for legal purposes. This type of testing can identify crime or catastrophe victims,
rule out or implicate a crime suspect, or establish biological relationships between
people (e.g., paternity).
7. Research testing: Research testing includes finding unknown genes, learning
how genes work, and advancing our understanding of genetic conditions.
DNA analysis has a particular interest in genetics, molecular diagnosis, pathology,
food safety, biothreat detection, criminology, environmental protection, and so on.
The main principle of DNA biosensors is immobilizing one single-stranded DNA (or
PNA), a probe-target DNA, onto a transducer surface, and these DNA sequences have
the ability to precisely recognize its partner of complementary base sequence, target-
probe DNA. Consequently, a DNA biosensor conveys this recognition event into a
useful readable signal. In the early stage of DNA analysis, scientists used different
tags to label specific DNA or oligonucleotides for specific recognition, including
radioactive-tag [5], molecular beacons [6], fluoresceins [7], dyes, metal complexes,
biomolecules (enzyme, etc.), pharmic molecules [8], and so on. Nowadays, the nano-
technology and hybridization indication techniques are playing important roles in
developing sensitive, selective, and miniaturized DNA biosensors for fast and reliable
DNA sequence analysis in many applications, such as newborn screening, diagnostic
testing, presymptomatic testing, and forensic testing [9]. Due to their huge surface
area, nanoscale, excellent optical/electrochemical/magnetic properties, and flexible
surface chemistry, nanostructures have received considerable attention in the DNA
biosensing field. The large surface area of nanostructures can increase the attached
DNA amount on the nanomaterial substrates surface, and concentrate a large number
of enzyme molecules or different nanoparticles to indicate DNA hybridization.
16.2 Nanostructures in DNA Biosensing 455

In this chapter, we discuss recent advances in DNA analysis and DNA biochips
based on nanomaterials and nanostructures, and summarize the nanostructures for
DNA-labeling or label-free techniques, various detection methods, and DNA-
sensing strategies.

16.2Nanostructures in DNA Biosensing

Over the past two decades, many important nanomaterials and technologies have
provided us with the key tools to design novel DNA-sensing methods and devices,
which have led to enormous improvements in sensitivity, selectivity, multiplexing
capacity, and simplicity. Many studies still focus on various combinations of DNA
with different types of transducers. Electrical/electrochemical, optical, and acoustic
sensing techniques have emerged as some of the most promising DNA-biosensing
technologies.
Moreover, different types of nanostructures can be associated together to achieve
versatile sensing units and/or microarrays, to meet the demands of fast, simple, and
inexpensive methods for specific DNA analysis.

16.2.1Carbon Nanotubes for DNA Analysis

Carbon nanotubes (CNTs) are often considered as quasi-one-dimensional (Q1D)


nanomaterials and have been constructed with a length-to-diameter ratio of up to
28,000,000:1 [10], which is significantly larger than other materials. These cylin
drical carbon molecules have novel properties that make them potentially useful in
many applications in electronics, optics, and other fields of nanotechnology science.
CNTs exhibit extraordinary strength and unique electrical properties and are efficient
thermal conductors. Some aspects of the analytical applications of CNTs have been
briefly discussed by Trojanowicz [11], including gas sensors based on their sorption,
electrical, and thermoelectric properties; their voltammetric/stripping voltammetric
analytical systems for inorganic and organic molecules; CNT-based enzyme biosen-
sors and DNA hybridization biosensors; in solid-phase extractions and chromato-
graphic applications. Rivas etal. [12] summarized the relevant applications in the
development of CNT-based electrochemical biosensors for different substrates such
as catecholamines, homocysteine, carbohydrates, nitroaromatic explosives, amino
acids, proteins,. CNT-basedelectrochemical enzymatic biosensors, and CNT-based
DNA biosensors. Fang etal. [13] reviewed the applications of CNTs in electrochemi-
cal DNA biosensors specifically, placing emphasis on two main aspects: (1) using
CNTs as substrates immobilizing DNA molecules as well as powerful amplifiers to
amplify signal transduction events of DNA hybridizations; (2) CNTs serve as effec-
tive carriers and/or indicators to concentrate enzymes or electroactive substrates for
electrochemical sensing of DNA hybridizations. Rao and coworkers [14] recently
456 16 Nanostructured Biosensing and Biochips for DNA Analysis

Fig. 16.1 Schematics of layer-by-layer electrostatic self-assembly of protein-polyion on carbon


nanotube template. Also shown are the TEM images, single-wall carbon nanotube coated with a
monolayer of PDDA bilayer (left top), and CNT modified with four bilayers of protein-polyion (left
bottom). Reprinted with permission from Munge etal. [15], 2005, American Chemical Society

reviewed the DNA-functionalized/grafted CNTs and their characterization methods,


including UV-Vis-near IR spectrometry, fluorescence imaging, FT-IR, surface-
enhanced IR absorption (SEIRA), Raman spectra, transmission electron microscopy
(TEM), atomic force microscopy (AFM), scanning electron microscopy (SEM),
X-ray photoelectron spectroscopy (XPS), and electrochemical characterizations.
Basically, DNA biosensors can be used for the detection of hybridization events,
DNA damage, and the interaction of DNA with other molecules [12].

16.2.1.1DNA Hybridization Sensors

The preparations of DNA hybridization biosensors consist of three steps: immobili-


zation of DNA (or PNA) probes onto the certain surfaces; hybridization of the probe
and target under optimized solution conditions (such as hybrid temperature, pH, ion
strength, time, and washing conditions, etc.); detection of formed DNA double helix
by optical/electrical (or electrochemical)/magnetic methodologies, which allow one
to obtain significant signals that are directly/indirectly caused by the sequence-spe-
cific recognition events [12, 13].
Layer-by-layer immobilization: Wangs group [15] developed a method of enzyme
multilayers on CNT template carriers (Fig.16.1), which showed ultrasensitive elec-
trochemical detection of proteins and nucleic acids. By employing square-wave
voltammetric measurement of the reaction product, Wangs group could detect the
hybridization response for extremely low DNA target concentrations, ranging from
1 to 40fg/mL, with a detection limit of 0.135zmol (about 80 copies in the 25mL of
solution).
Jiao recently reported [16] layer-by-layer films of poly-l-lysine and Au-CNTs for
preparation of a DNA hybridization biosensor, shown in Fig.16.2, with a detection
16.2 Nanostructures in DNA Biosensing 457

Fig.16.2 Alternate self-assembly procedure of layer-by-layer films of poly-l-lysine and Au-CNT


hybrid. Reprinted with permission from Du etal. [16], 2009, Wiley

limit of 2.451011mol/L target DNA. Compared with other immobilization meth-


ods, the layer-by-layer method shows greatly enhanced amplification and hence an
improved sensitivity for DNA detection compared to single-layer detection
schemes.
CNTs associated with other materials: Wangs group [17] presented a CNT-based
amplified bioelectronic protocol (Fig.16.3), which involved the sandwich hybrid-
ization (a) or antigenantibody (b) binding along with magnetic separation of the
analyte-linked magnetic-bead/CNT assembly (A), followed by enzymatic amplifi-
cation (B), and chronopotentiometric stripping detection of the product at the CNT-
modified electrode (C). The TEM observations (Fig. 16.4) indicated that the
hybridization event led to cross-linking of the ALP-loaded CNTs and the magnetic
beads (with the DNA duplex acting as glue).
By using a chronopotentiometry method, Wangs group further demonstrated a
CNT-based dual amplification route for ultrasensitive electrical bioassays of DNA
(Fig. 16.5). The use of CNT amplifiers (loaded with numerous ALP tags) was
combined with the preconcentration feature of CNT transducers to yield a dramatic
enhancement of the sensitivity. Such a coupling of several CNT-derived amplifica-
tion processes resulted in the highly sensitive detection of proteins and DNA and
hence indicated great promise for PCR-free DNA assays.
Yang etal. [18] found that SWNTs could form a nanoscale, noncovalent adduct
with ferrocene (Fc). This nanoscale adduct greatly enhanced the reduction response of
hydrogen peroxide and displayed high stability in aqueous solution under sonication
458 16 Nanostructured Biosensing and Biochips for DNA Analysis

Fig. 16.3 Schematic representation of the analytical protocol: (a) Capture of the ALP-loaded
CNT tags to the streptavidin-modified magnetic beads by (a) a sandwich DNA hybridization or (b)
AbAgAb interaction. (b) Enzymatic reaction. (c) Electrochemical detection of the product of the
enzymatic reaction at the CNT-modified glassy carbon electrode. MB, magnetic beads; P, DNA
probe 1; T, DNA target; P2, DNA probe 2; Ab1, first antibody; Ag, antigen; Ab2, secondary anti-
body; S and P, substrate and product, respectively, of the enzymatic reaction; GC, glassy carbon
electrode; CNT, carbon nanotube layer. Reprinted with permission from Wang etal. [17], 2004,
American Chemical Society

Fig.16.4 TEM images of the magnetic beads-DNA-CNT assembly produced following a 20-min
hybridization with the (a) 10- and (b) 0- pg/mL target sample. Reprinted with permission from
Wang etal. [17], 2004, American Chemical Society

due to the strong pp stacking interaction between Fc and SWNT (Fig.16.6). In this
case, SWNT played a dual role in both the recognition and transduction events. Using
the electrocatalytic properties of Fc/SWNT toward H2O2, they succeeded in designing
a sandwich-type gene-detection system that recognized target DNA. The different
responses toward c-DNA and n-DNA sequences, combined with the Fc/SWNT stabil-
ity, led to a useful approach for the preparation of CNT-based DNA biosensors.
Jiaos group reported a series of nanoparticleCNT complexes for developing
electrochemical DNA hybridization sensors [19, 20], which yielded excellent sensi-
tivities and considerable wide ranges for target DNA concentration from 1.01011
1.0106 mol/L, with a detection limit of 2.81012 mol/L, and from 1.0107
1.01012mol/L, with a detection limit of 2.81013mol/L, respectively.
16.2 Nanostructures in DNA Biosensing 459

Fig.16.5 Chronopotentiometric signals for increasing levels of the DNA target: (a) 0.01, (b) 0.1,
(c) 1, (d) 50, (e) 100pg/mL. Also shown (inset) are (A) the resulting calibration plot, and (B) the
response for 5 fg/mL-target DNA and (C) 10-ng/mL noncomplementary (NC) oligonucleotide.
Sample volume, 25ml (B) and 50ml (C). Reprinted with permission from Wang etal. [17], 2004,
American Chemical Society.

Fig.16.6 Schematic illustration of the electrochemical gene sensing system based on the forma-
tion of a complementary sandwich-type complex. Reprinted with permission from Yang etal. [18],
2007, Springer
460 16 Nanostructured Biosensing and Biochips for DNA Analysis

Fig.16.7 (a) Optical image of the central region of a single sensor chip with four SWNT devices
(scale bar 200mm). Electrodes extending out of the liquid cell (dashed circle) connect to large
wire-bonding pads. (b) Schematic illustration of a single device during electrical measurement.
Complementary ssDNA oligos hybridize to thiolated ssDNA coimmobilized with MCH on the
gold electrodes. Real-time monitoring of (c) 15-mer and (d) 30-mer DNA hybridization in PBS,
pH 7.4, is shown. Two liquid cells were used in parallel for simultaneous drop, adding 5mL of
complementary and mismatched target oligo solutions to 500mL of buffer. Reprinted with permis-
sion from Tang etal. [22], 2006, American Chemical Society

Label-free assay: Ye and Ju [21] reported a method for the rapid sensitive detection
of DNA or RNA using a screen-printed carbon electrode modified with multiwalled
carbon nanotubes (MWNTs), where the MWNTs displayed catalytic characteristics
for the direct electrochemical oxidation of guanine or adenine residues of ssDNA
and adenine residues of RNA, leading to an indicator-free detection of ssDNA and
RNA concentrations.
Tang etal. fabricated virtually two terminal SWNT-DNA sensor arrays (Fig.16.7)
and the simple and yet generic protocol for direct label-free detection of DNA
hybridization [22]. They also carried out a systematic study of sensing mechanism,
involving XPS, quartz crystal microbalance (QCM) (Fig.16.8), and fluorescence
measurements (Fig.16.9).
Recently, Mao described a method for label-free and sequence-specific DNA
detection with a low detection limit by using CNTs as support to increase the surface
loading of probe DNA. Figure16.10 shows the preparation of the label-free DNA
16.2 Nanostructures in DNA Biosensing 461

Fig.16.8 QCM frequency shift (F3) versus time curves showing that (a) thiolated 15-mer ssDNA
(p15) absorbs onto SWNT sidewall spontaneously and (b) no further binding to mismatched
(MM15) and complementary (CM15) ssDNAs. (c) Phosphilipid-PEG maleimide (PL-PEG-M)
self-assembles onto SWNTs and allow effective linkage to p15. (d) The SWNT-PL-PEG-M linked
ssDNA probes selectively hybridize to complementary strands. The inserts are schematic drawings
of the SWNTssDNA complexes. Reprinted with permission from Tang et al. [22], 2006,
American Chemical Society

Fig. 16.9 Fluorescence images showing DNA hybridization on patterned (circle) SWNT film.
Thiolated ssDNA (probe) was conjugated to phospholipid-maleimide wrapped on an SWNT side-
wall. Fluorescence intensity from the cy3-labeled complementary target (b) is about five times
higher than the mismatched target (a). Insert is the atomic force microscopy image of one circle
region (scale bar 5 mm). Reprinted with permission from Tang et al. [22], 2006, American
Chemical Society
462 16 Nanostructured Biosensing and Biochips for DNA Analysis

Fig.16.10 Schematic of label-free and sequence-specific DNA detection with CNTs as support
for probe DNA. Reprinted with permission from Zhu etal. [23], 2009, Elsevier

sequence biosensor [23]. By using the electrochemical impedance spectroscopy


(EIS) method, they demonstrated a sensitive and label-free electrochemical DNA
hybridization sensor. Moreover, Marrazza and coworkers [24] demonstrated CNT
thin films that were prepared by chemical vapor deposition (CVD) using acetylene
and ammonia as precursor gases and nickel particles as the catalyst (Fig.16.11).
Such a route offered defined guanine oxidation peaks in the presence of different
DNA targets, over the concentration range from 0.5 to 10mM.

16.2.1.2DNA Damage Sensors

DNA damage sensors, namely, a type of sensors containing DNA as biorecognition


substrates, are designed for the detection of physical/chemical/biological damage
induced on DNA. Generally, the preparation of this kind of sensors involves three
steps: immobilization of dsDNA on the transducers surfaces; interaction between
the given damage agents and the DNA layers within the controlled conditions;
collecting the detectable variational signals, from the nucleo-bases, unwinding of
the DNA strands, or damage agents, etc.
Jiao and Wang proposed an approach for in situ detection of the DNA damage
by electro-Fenton reaction based on Fe@Fe2O3 core-shell nanonecklace and
multiwalled CNT composite. Figure16.12 shows the design of the DNA damage
sensor [25].
16.2 Nanostructures in DNA Biosensing 463

Fig.16.11 Aligned carbon nanotube thin films obtained by CVD. (a) SEM image of the CNT film
grown on a SiO2 substrate (CNT/Ni/SiO2); (b) SEM image of the CNT film grown on an Al/SiO2
substrate (CNT/Ni/Al/SiO2); (c) scheme of CNT/Ni/SiO2; (d) scheme of CNT/Ni/Al/SiO2.
Reprinted with permission from Berti etal. [24], 2009, Elsevier

Fig.16.12 Schematic diagram illustrating the DNA damage induced by the E-Fenton reaction and
the detection method. Reprinted with permission from Wang and Jiao [25], 2010, Elsevier

The DNA damage in situ induced by the E-Fenton reaction can be detected by
differential pulse voltammetry (DPV) with 20mM Co(phen)33+. Due to the interca-
lative binding of Co(phen)33+ to dsDNA, Co(phen)33+ binds more strongly to intact
dsDNA than the damaged DNA. After the sensor is treated by E-Fenton reaction for
464 16 Nanostructured Biosensing and Biochips for DNA Analysis

Fig.16.13 Differential pulse voltammograms of DNA/PDDA/Fe@Fe2O3-MCNT/PDDA/GCE in


0.1M pH 5.5 PBS containing (a) 20mM Co(phen)33+ or (b) 50mM Ru(NH3)63+ after the electrode
was treated by E-Fenton reaction for different times. Reprinted with permission from Wang and
Jiao [25], 2010, Elsevier

a given time, the DPV response of Co(phen)33+ on the biosensor is obtained and the
results are shown in Fig.16.13(a).
The DNA damage can also be detected using another electrochemical indicator,
Ru(NH3)63+. After the biosensor is treated by the E-Fenton reaction, an increasing DPV
peak current is obtained with an extended reaction time, as shown in Fig.16.13(b).

16.2.2Metal Nanoparticles for DNA Analysis

16.2.2.1Gold Nanoparticles

The excellent physical and chemical properties of Au NPs (colloidal gold) offer
excellent prospects for a wide range of biosensing applications, especially toward
signal amplications in biorecognition systems. Wittenberg and Haynes [26]
discussed different detection methods of Au NP-enhanced sensing, including
extinction/scattering/reflectivity (localized surface plasmon resonance, LSPR;
surface-enhanced Raman scattering, SERS), electricity/electrochemistry, gravimetry,
and so on. Wang reviewed the recent developments of nanoparticle-based biosensing
methods [2729], where Au nanoparticles were used for nanoparticle-amplified
optical bioaffinity assays [27], tags for electrochemical detection of proteins and
DNAs, ultrasensitive particle-based bioassay-based multiple-amplification schemes
[28], and amplified microgravimetric monitoring of bioaffinity events [27]. The
aspect of electrochemical biosensing also was emphasized [29]. Liu and Lin also
introduced the application of colloidal gold as nanomaterial labels in immunosensors
and immunoassays [30].
Au NPs can play multiple roles in DNA sensing for immobilization, signal
amplification, and labeling. These aspects of Au NPs are discussed in Chap. 2 in
detail. Table 16.1 summarizes some aspects of the applications of Au NP-based
DNA-biosensing amplifying strategies and techniques.
16.2 Nanostructures in DNA Biosensing 465

Table16.1 Applications of Au NP-based DNA biosensing


Sensing Amplification Detection
strategies Methods times Targets limit Ref.
Optical Surface-enhanced 18 DNA [31]
plasmon
resonance (SEPR)
Optical signal DNA: 1fM [32]
BRCA-1
gene
Colorimetric detection DNA 0.5fmol [33]
SERS-plasmonic 40200 DNA [34]
coupling
Scanometric image Comparable to PCR HeLa cell 10 cell [35]
Photoelectrochemical DNA 1nM [36]
method
Surface plasmon 2 7.82aM [37]
resonance (SPR)
Localized surface DNA [38]
plasmon
resonance (LSPR)
Scanometric image, 8 to agarose gel 2fmol [39]
strip assay electrophoresis
Electro Square-wave 1000 DNA: BRAF 0.35aM [40]
chemical stripping gene
voltammetry
(SWSV)
Anodic stripping 200 DNA 0.5fM [41]
voltammetry
(ASV)
Cyclic voltammetry DNA 1fM [42]
(CV)
Acoustic Surface acoustic DNA ~ 10.9ag [43]
wave (SAW)

16.2.2.2Other Metal Nanoparticles

Recent studies have demonstrated that metal nanoparticles such as silver can be
readily assembled on DNA via the chemical or photoinduced reduction of DNA-
complexed metal ions in aqueous media. Dongs team presented the studies on
fabricating hybrid nanobiomaterials containing thin films, where multilayer films
were utilized as nanoreactors to prepare DNA-silver nanohybrids in situ [44].
Zhang etal. proposed a strategy of using copper(II) complex of Luteolin C30H18CuO12
(CuL2) as an electroactive indicator based on silver nanoparticle- and multiwalled
carbon nanotubs (Ag/MWCNT) -modified glassy carbon electrode (GCE). The
proposed method dramatically increased the DNA attachment quantity and
complementary ssDNA detection sensitivity for its large surface area and good
charge-transport characteristics [45].
Table 16.2 summarizes different metal nanoparticles used in DNA-related
biosensing.
466

Table16.2 Applications of sensing strategies and detection methods of metal nanoparticle-based DNA biosensoring.
Sensing
strategies Methods Signal Immobilization method Label Nanostructure Targets DL Ref.
+
Optical UV-Vis, SERS, DNA-Ag ITO-PDDA immerse SERS: Rhodamine Ag NPs [44]
XPS DNA-Ag+ 6G, 4-aminothio
phenol
Fluorescence EB-DNA Ag NPMWCNT Cu-Luteolin Ag NPs DNA 65pM [45]
spectroscopy modified GCE complex
Colorimetric Pt-Aptamer Pt NPs Thrombin [46]
detection
Electro CV, DPV, EIS CuL2 Ag NPMWCNT Cu-Luteolin (CuL2) Ag NPs DNA 0.65pM [45]
chemical modified GCE complex
CV, DPV Ag-oxidation, Passive adsorption Ag Ag NPs DNA [47]
Guanine using amino linked
DNA onto Ag NPs
CV, i-t AgNPs-DNA Electrodeposition H2O2, glucose [48]
CV Ag+ reduction Thiolated DoxorubicinAg Ag NPs DNA of avian 1:00 PM [49]
oligonucleotide NPs flu virus
probe on Au H5N1
electrode
CV, DPV Co(phen)33+ DNA-PDDA-Fe@ Fe@Fe2O3, DNA damage 0.16mg [25]
Ru(NH3)63+ Fe2O3-MWCNT- MWCNTs
PDDA-GCE
CV, DPV Co(phen)33+ DNA-PDDA-Fe@ Fe@Fe2O3 DNA damage 0.4mg [50]
Ru(NH3)63+ Fe2O3-AuNP- nanonecklace,
PDDA-GCE Au NPs
16 Nanostructured Biosensing and Biochips for DNA Analysis
16.2 Nanostructures in DNA Biosensing 467

16.2.3Semiconductor Nanostructures for DNA Analysis

Jiaos team proposed an electrochemical DNA biosensor based on ZnO nanoparticles


and MWNTs for DNA immobilization and enhanced hybridization detection [19].
The hybridization events were monitored by DPV using methylene blue (MB) as an
indicator. The sensor could effectively discriminate different DNA sequences related
to the PAT gene in the transgenic corn, with a detection limit of 2.81012mol/L of
target sequence.
Mathur et al. presented an amplified electrochemical sensing method for the
detection of DNA based on the current readout resulting from chemical oxidation of
guanine on nanoscaled metal oxides (TiO2, SnO2, and Fe3O4) obtained by CVD onto
pencil graphite electrode (PGE) as the electrochemical transducer [51]. The pro-
posed strategy was suitable to produce cost-effective disposable sensor elements
enabling the quantitative detection of nanomolar concentrations of DNA. The detec-
tion limit estimated for signal-to-noise ratios greater than 3 corresponded to 21.3,
53.9, and 45.8nmole/mL of 20-mer-bases DNA oligonucleotide for TiO2, SnO2, and
Fe3O4 films, respectively.
Quantum dots (QDs) are another frequently used type of semiconductor nano-
particles for biological detection of DNA. For example, based on the fluorescence
resonance energy transfer (FRET) between QDs and graphene oxide, Jus group
[52] designed an effective sensing platform for DNA target. The QDs modified on
molecular beacons were used as probes to recognize the target analyte. As shown
in Fig. 16.14, the strong interaction between molecular beacon and graphene
oxide led to the fluorescent quenching of QDs. Upon the recognition of the target,
the distance between the QDs and graphene oxide increased, and the interaction
between target-bound molecular beacon and graphene oxide became weaker,
which significantly hindered the FRET, leading to an increase in the fluorescence
of QDs. The change in fluorescent intensity was used for the detection of the
target, with a detection limit down to 12 nM.
On the other hand, QD tags are also widely used in ultrasensitive electro-
chemical DNA assays due to their intrinsic redox properties and the sensitive
electrochemical stripping analysis of the metal components of QDs. Recently,
Jus group [53] synthesized QD-functionalized poly(styrene-co-acrylic acid)
microbeads to fabricate an ultrasensitive electrochemical biosensor for DNA
detection. The CdTe-tagged polybeads were prepared by a layer-by-layer self-
assembly of the CdTe QDs and polyelectrolyte on the polybeads. The mean QD
coverage was (9.541.2)103 per polybead. As shown in Fig.16.15, under a
sandwich hybridization format, the efficient carrier-bead amplification platform,
coupled with the highly sensitive stripping voltammetric measurement, provided
a detection limit of 0.52 fmol/L and a dynamic range spanning five orders of
magnitude.
The optical/electrochemical/gravity detections of DNA onto metal oxide
nanoparticles are listed in Table16.3.
468 16 Nanostructured Biosensing and Biochips for DNA Analysis

Fig. 16.14 Schematic representation of graphene oxide-induced fluorescence quenching of


molecular beacon-QDs and biosensing mechanism. Reprinted with permission from Dong etal.
[52], 2010, American Chemical Society

Fig. 16.15 Procedure for detection of DNA hybridization using streptavidin/CdTe-tagged


polybeads. Reprinted with permission from Dong etal. [53], 2010, Wiley
Table16.3 Applications of sensing strategies and detection methods of metal oxide nanoparticle-based biosensoring
Sensing strategies Methods Indicator/signal Immobilization method Label Nanostructure Targets DOL Ref.
Electrochemical CV, EIS [Fe(CN)6]3/4 CeO2SWNTs- CNTs, CeO2 DNA 0.23pM [20]
BMIMPF6/GCE nanoshuttle
DPV Methylene blue Chitosan MCNTZnO NP DNA 2.8pM [19]
DPV Guanine Chemical vapor Label-free TiO2, SnO2, Fe2O3 DNA 21.3, 53.9, [51]
deposition on PGE 45.8
nmol/mL
Electrochemical ThiolDNAMTPS Label-free GaN nanowires [54]
impedance GaNNWs
spectroscopy
16.2 Nanostructures in DNA Biosensing

CV, DPV [Fe(CN)6]3/4 ThiolDNACdS CdS CdS cDNA 1pM [55]


DroppedGCE
CV, EIS [Fe(CN)6]3/4 Fe2O3 microsohere Label-free Self-doped DNA 21fM [56]
SPAN polyaniline
(SPAN)
nanofibers
Electrodeposition DNA hybridization AuPdAu [57]
assisted multisege- nanobarcode
mented nanowires
FET ThiolDNAAu Label-free CdTeAuCdTe DNA <1mM [58]
NW FET Nanowires PSA 1pM [59]
DPASV Thrombin Au/aptamerI/thrombin/ PbSNPAu NP Thrombin 6.2fM [60]
DNAAu NPs barcode
SWV CdTe Layer-by-layer self- CdTe/polybeads DNA 0.52fM [53]
assembly
(continued)
469
470

Table16.3 (continued)
Sensing strategies Methods Indicator/signal Immobilization method Label Nanostructure Targets DOL Ref.
Optical UV-Vis, FTIR, ZnO NPs ZnODNA [61]
XPS, AFM interaction
Photoluminescence ThiolDNAMTPS Label-free GaN nanowires [54]
spectroscopy GaNNWs
Time-resolved Europium DNA covalently immobi- Silica NPs Silica NPs DNA [62]
fluorescence complex lized on the common
glass slides surface
SPR, CV, CdS Nps, Pt AptamerNPscocaine CdS Nps, Pt NPs, Cocaine [63]
photocurrent NPs, Au NPs Au NPs
Fluorescence CdTe CdTemolecular beacon CdTe, graphene DNA 12nM [52]
oxide
Gravity QCM Absorption Label-free ZnO nanorod 60-fold, [64]
mercaptohexanol 30-fold
for DNA
16 Nanostructured Biosensing and Biochips for DNA Analysis
16.3 Nanostructures for DNA Biochips 471

16.3Nanostructures for DNA Biochips

The rapid technological advances in biochemistry and semiconductor science in


the 1980 led to the large-scale development of biochips in the 1990. The actual
sensing component (or chip) is just one piece of a complete analysis system.
Transduction must be done to translate the actual sensing event (DNA binding,
oxidation/reduction, etc.) into a format understandable by a computer, which then
enables additional analysis and processing to produce a final, human-readable
output. The multiple technologies needed to make a successful biochip from sensing
chemistry, to microarraying, to signal processing require a true multidisciplinary
approach, making the barrier to entry steep. One of the first commercial biochips
was introduced by Affymetrix. Their GeneChip products contain thousands of
individual DNA sensors for use in sensing defects, or single-nucleotide polymor-
phisms (SNPs), in genes such as p53 and BRCA1 and BRCA2 [65]. Microarrays
provide a powerful analytical tool for the simultaneous analyses of up to thousands
of parameters of nucleic acids in a single experiment. DNA microarrays are now
fairly well established as diagnostic and pharmaceutical tools. Microarrays are
fabricated by immobilizing biologically relevant moieties (from several to many
thousands) into discrete spatial arrangements (spot sizes from 10 to 500mm in size).
Such microdevices have given researchers significant benefits, including higher
throughput, more automation, cost savings, and ideally ease of use [66].
Advances in biochip/microarray areas are giving scientists and researchers new
methods for unraveling the complex biochemical processes occurring inside cells,
with the larger goal of understanding and treating human diseases. At the same
time, the semiconductor industry has been steadily perfecting the science of micro-
miniaturization and even nano-miniaturization. The merging of these two fields in
recent years has enabled researchers to begin packing the traditional bulky sensing
tools into micro-, submicro-, even nano-spaces, onto so-called biochips. Here, we
focus our vision on the nanostructures and their roles in DNA biochips/microarrays
in recent years.

16.3.1Optical Techniques for DNA Biochips

The DNA probe, or oligonucleotide, is immobilized on a solid support either cova-


lently or otherwise in micron-sized patterns typically used in photolithography, ink-
jet printing (spotting), or contact printing. The probe molecule is then used to
capture the target molecule through specific interactions, such as hybridization for
DNA and RNA or ligand-receptor binding for proteins. Many of the commercially
available microarrays rely on fluorescent labels and fluorescent detection, though
alternative labeling strategies as well as label-free detection methods are also being
developed [67].
472 16 Nanostructured Biosensing and Biochips for DNA Analysis

Label-based microarrays: Although much effort is being contributed to the


development of label-free detection methods for monitoring binding processes, the
use of labels still dominates microarray technology. Currently, the most common
labeling technique remains fluorescent tags, due to the availability of a great variety
of dyes with defined emission spectra and their compatibility with established
microscopy or colorimetric detection techniques. The demand for even higher
sensitivity, specificity, and detection reliability for microarray applications has
stimulated the further development of both new labeling strategies as well as new
optical detection methods. The most promising labeling strategies are direct
approaches (in which the entire analytical sample is marked) and indirect approaches
(or sandwich assay, which makes use of secondary labeled detection elements inter-
acting specifically with the target) [67]. These labels are described below:
Fluorescent dye molecules: Fluorescent dye molecules remain the dominant label
used in conjunction with microarrays. The most commonly fluorophores used are
Cy3 and Cy5 [6872] and CyP2D6 [73].
Quantum dot labels: Fluorescent semiconductor QDs are considered to be 20 times
as bright and 100 times as stable as organic dyes and have therefore emerged as a
promising new type of label [67]. QDs exhibit a broad absorption band in the ultra-
violet (UV) range, with a narrow, well-defined emission wavelength that can be
tuned by controlling the size and chemical composition of the QDs. This makes
them particularly well suited for multiplexing [74].
Metal colloid labels: Another labeling approach that has appeared only recently in
connection with microarray technology makes use of metal colloids, especially gold
[7583] and silver-enhanced gold nanoparticles [8489]. Various optical and elec-
trical techniques have been proposed to detect such tags.
Magnetic nanoparticle (MP) labels: Compared to the micron sizes of general mag-
netoresistive labels, MPs are more attractive in terms of size, but result in smaller
sensor signals per label. Accord-ingly, biodetection systems utilizing magnetic
nanoparticles normally require the use of high label concentrations in order to avoid
slow, low-gradient increases in signal due to the gradual movement of the nanopar-
ticles to the sensor. Graham and his coworkers overcame the diffusion barrier
through the use of on-chip generated magnetic field gradients [90]. Martins etal.
used 250-nm MPs as labels of target DNA. The biological detection limit of the
device was significantly improved by three orders of magnitude, from picomolar
down to femtomolar, by the use of an efficient magnetic focusing system associated
with prehybridization labeled samples [91].
Label-free microarrays: Because of the drawbacks of labeling methods, such as
being time-consuming (additional steps are required), labeled reagents are expen-
sive, and one must deal with the photobleaching or quenching effects of fluorescent
dyes. Label-free detection methods may be the alternative. But there are still some
disadvantages with label-free methods, including false-positive results due to non-
specific adsorption and a lower sensitivity. However, the different label strategies
and therefore detection techniques provide different apparent results and solution
16.3 Nanostructures for DNA Biochips 473

Fig.16.16 Nanomechanical detection of DNA hybridization. Schematic of the (a) experiment and
(b) stiffness map. After hybridization, the surface of the array spot is scanned with the AFM to
generate the stiffness map. Scan size, 3mm. Hybridized molecules are measured to be less stiff and
appear as dark brown spots. dsDNA, double-stranded DNA; ssDNA, single-stranded DNA.
(c)stiffness at each pixel in (b) is calculated from forcedistance curves. Reprinted with permis-
sion from Husale etal. [92], 2009, Nature

information about the system. Thus, the choice of the detection technique heavily
depends on what one is interested in and the system under investigation.
Husale and coworkers [92] used a label-free atomic force microscope (AFM) tech-
nique to investigate the stiffness of single- and double-stranded DNA molecules, as
illustrated in Fig.16.16. The resulting stiffness maps show hybridized DNA molecules
as dark brown spots. This identification is verified by a series of experiments with
noncomplementary targets and complementary targets with varying concentrations.
Golovlev etal. [93] developed two computational models to study the ionic inter-
action of colloidal particles and biopolymer molecules on a microarray surface. The
theoretical considerations are in qualitative agreement with the experimental results
for detecting large target DNA and a 50-base-long synthetic oligonucleotide on the
amino-modified microarray substrate. The experimental and theoretical results of
this study allowed further development of the label-free microarray technology
demonstrated previously for gene expression analysis that is free of reverse tran-
scription and dye labeling [93].
Tamiya etal. reported an Au-coated nanostructured biochip with functional-
ized thiolated primers on its surface for the label-free and real-time optical detec-
tion of polymerase chain reaction (PCR) [94]. The immobilization of 5-end
474 16 Nanostructured Biosensing and Biochips for DNA Analysis

thiolated primers and blocking agent (6-mercaptohexanol) on the gold surface


resulted in significant changes in the refractive index of the layer medium and
therefore could be detected by the increment in the RRI of the nanostructured
surface spectra.

16.3.2Electrochemical Methods for DNA Biochips

The advantage of fully electrical chips is the intrinsic high spatial resolution and
direct signal coupling of the biosensing element and the transducer. Numerous stud-
ies have been devoted to the construction of miniaturized biosensing devices
enabling the electrical monitoring of metabolites as well as the evaluation of biomo-
lecular interactions [95].
The use of metal nanoparticles in bioanalytical applications has recently attracted
increased attention. Besides using optical properties of nanoparticles, such as
changes in light scattering due to the induced aggregation of particles by interac-
tions between biomolecules, colorimetric measurements [78, 79, 89], electrochemi-
cal detection, and quantification principles have been successfully adapted for
biochip analysis, including stripping voltammetry [75, 95, 96], cyclic voltammetry
[94, 95, 97], amperometry [98], electrical/FET [77, 79, 81, 83, 88, 91], impedance
spectra [97, 99], and resistance [96].
Label-based detection methods: Signal-enhancement strategies such as silver
deposition on gold particles can further increase the sensitivity and facilitate the
use of nanoparticles in highly sensitive biomolecule detection [75]. Hsing and
coworkers presented a complete DNA-based assay in a single siliconglass micro-
chamber for multiple pathogen (Escherichia coli and Bacillus subtilis) detection.
The assay involves the following steps: (1) sample preparation using thermal cell
lysis and magnetic particle-based target genome isolation; (2) target DNA amplifi-
cation by the PCR; (3) hybridization of the amplicons to their complementary
oligonucleotide capture probes immobilized onto individual detection electrode
surfaces; and (4) electrochemical transduction of the recognition event via gold
nanoparticles and silver enhanced with signal amplification using electrocataytic
silver deposition [75].
Chens team designed and developed an array-based CMOS biochip to replace
the conventional DNA microarray for DNA identification by electrical detection
[77]. They used a self-assembly gold nanoparticle multilayer to conduct and amplify
electrical signals. The electric current passing through the gold nanoparticle multi-
layer, which is formed by complementary target oligonucleotide strands, exceeded
that of the gold nanoparticle monolayer by three orders of magnitude. Chen also
demonstrated an electrical detection method on a DNA biochip that employed an
approach for the ultrasensitive detection of DNA using self-assembled gold nano-
particles and bio-bar-code-based amplification (BCA) DNA [81]. The detective
16.4 Conclusions 475

concentration of target DNA with electrical DNA biosensor was as low as 1fM for
the analysis of current-voltage curves.
Schuler etal. demonstrated that it was possible to utilize screen-printed electrode
structures for a chip-based electrical DNA detection, along with silver nanoparticles
as amplification agents [83]. The screen printing of electrode structures for DNA-
chip with electrical detection offered an interesting and cost-efficient possibility to
produce DNA chips with microstructured electrodes.
Label-free detection methods: Wang and coworkers showed that the nanopore alu-
minum anodized oxide (AAO) membrane could be employed to immobilize specific
ss-probes by aminosilanes and glutaraldehyde linker, and then used for the label-
free electrical detection of complementary target bacteria DNA without additional
modification [97]. This AAO membrane-based biosensor provided a detection limit
of 0.5nM for complementary target DNA.
Shiigi etal. fabricated a highly controlled nanogap electrode, consisting of an Au
nanoparticlealkylchainAu nanoparticle repeated sequence, in which the gap
between particles was precisely controlled with the binder molecule [96]. Combining
this with the conductive DNA enabled a label-free measurement of the very small
electrical property change in DNA with a high S/N ratio (>30). Upon adding a
complementary oligonucleotide on the nanoparticle film chip, an immediate
decrease in the film resistance (ca. 1.4W) due to a hybridization event occurred in
a reproducible manner with this simple setup.
Table 16.4 summarizes the applications of nanostructures in DNA biochips/
microarrays.

16.4Conclusions

A variety of nanostructures have been introduced into DNA biosensing and DNA
microarrays for investigating DNA hybridizations, DNA damage, interactions
between DNA and other molecules, and so on. This chapter overviews a few
strategies using nanostructures for the preparation of DNA biosensors and microar-
rays, which may contribute to improving DNA biosensing, including a simple and
low-cost design, and sensitivity enhancement. These nanostructures are believed to
be very promising in the stages such as fictionalization, specific binding, and biorec-
ognition. Moreover, multifunctional nanostructures can be composited together to
achieve versatile sensing units, to meet the demands of fast, simple, and inexpensive
methods for DNA biosensing.
DNA microarrays clearly demonstrate the value of miniaturization and automa-
tion. More and more researchers and commercial organizations have started apply-
ing techniques to manipulate from micro- to nano- even picoliter sample volumes
into micro-, even nano-space automated biochip systems. Such microdevices will
give various science researchers and even ordinary consumers significant benefits,
including automation, cost savings, high throughput, and ease of use.
Table16.4 Using nanostructures binding DNA biochip technology
476

Biochip purpose Detection methods Nanostructure Labels Arrays Targets Ref.


DNA detection Total internal reflection Nanoarray Fluorescein, DNA molecules [68]
fluorescence microscopy Cy5 (fM to aM range)
DNA detection Fluorescence detection Si nanostructures Cy3 Homemade Oligonucleotide [69]
Gene expression Fluorescence detection Au NPs Dye label ScanArray DNA (2pg) [70]
SERS NPs, NWs Cy3 [71]
DNA detection Fluorescence image Nano-scale controlled Streptavidin-Cy3 Oligonucleotide [72]
surface (~50fM)
SNP Nanofluidic platform Fluorescent dyes TaqMan assay [73]
DNA detection FRET CdSe/ZnS [74]
Pathogen identification Stripping voltammetry Au NPs Ag-enhanced Au ITO electrodes E coli; B. subtilis [75]
NPs
Addressable chip Light microscopy: Au NP Au NPs GOx-HRP Microfluidics Oligonucleotide [76]
HPLC fluorescent detector: chip A24, C19
GOx-HRP
DNA detection Electrical: i-v Au NPs, nanogap chip Au NPs CMOS chip DNA [77]
DNA detection Colorimetric method Au NPs Au NPs stain microRNA M13mp18 DNA [78]
microarray
(Invitrogen,
Carlsbad, CA
USA)
DNA detection Electrical: FET nanosphere Au NPs Si 3N4 100aM (10 zmol) [79]
16 FET+1T sensor DNA
DNA detection Au NPs, nanogap chip Au NPs CMOS chip DNA [80]
DNA detection Electrical detection Au NPs, magnetic NPs, Au NPs, MNPs Homemade Bio-bar-code DNA [81]
nanogap electrodes (1 fM)
DNA detection (Field emission) FE-SEM Au NPs Au NPs DNA (1 pM) [82]
DNA detection Electrical detection Ag NPs Ag NPs SPE Viral DNA [83]
Diagnosis Colorimetric method Au NPs Ag-enhanced Au PixSys5500 0.1 fmol DNA [84]
NPs
16 Nanostructured Biosensing and Biochips for DNA Analysis
Biochip purpose Detection methods Nanostructure Labels Arrays Targets Ref.
Diagnosis Photodiode: photocurrent Au NPs Ag-enhanced Au Homemade 8*6 Human papilloma [85]
NPs photodiode array virus (HPV) DNA
DNA detection Optical detection, Au NPs Ag-enhanced Au Affymetrix GMS DNA (200 fM) [86]
fluorescence detection NPs
DNA binder Light-scattering image Au NPs Ag-enhanced Au Affymetrix GME DNA binders [87]
NPs 418
16.4 Conclusions

SNP Electrical detection Au NPs Ag-enhanced Au SNP [88]


NPs
DNA detection Colorimetric image Au NPs Ag-enhanced Viral DNA [89]
AuNP
streptavidin
DNA detection Magnetic signal detection MNPs Magnetic label Magnetoresistive DNA (2 fmol/cm 2) [90]
biochip
DNA detection Electronic circuitry MNPs Magnetic label Magnetoresistive DNA (1 fM) [91]
biochip
DNA detection AFM Nanoarray Label-free cDNA, microRNA [92]
(aM)
NA detection Computational models Label-free DNA, RNA [93]
DNA detection Optical detection: Au-coated NPs Label-free PCR chamber [94]
CV
DNA detection Electrochemical detection: Polyaniline PANI NT array 1 fM (~300 zmol) [95]
CV, DPV (PANI) NTs Oligonucleotide
DNA detection Resistance, LSV AuNPs, Nanogapped Label-free Oligonucleotide [96]
array
DNA detection CV, EIS Aluminum anodized Label-free Potential E. coli O157:H7 [97]
oxide (AAO)
nanopore
memebranes
DNA detection Amperometric detection AuNPs SPE array Oligonucleotide [98]
AC impedance spectra Au nanoelectrodes Potential for single [99]
Nanofluidic channels DNA molecule
477

(continued)
Table16.4 (continued)
478

Biochip purpose Detection methods Nanostructure Labels Arrays Targets Ref.


DNA expression Fluorescent signal Viral NPs Cowpea mosaic Affymetrix [100]
viral-dye NPs microarray
(threefold
enhancement
over Cy5)
DNA detection FT-IR Si nanoporous DNA [101]
Molecule detection TIRFM Nanoarray Fluorescence Single protein [102]
dyes molecules
Gene expression Fluorescence detection SWCNTs Fluorescence Cell [103]
DNA detection Magnetic signal detection MNPs Giant magnetoresistive HPV DNA [104]
(GMR) biochip (10 pM)
Optical detection Nanoarray [105]
Aptamer, DNA used Fluorescence detection Nanomaterials [106]
as tool assembly
DNA detection Fluorescence detection Nanocystal DNA [107]
SNP mutation
DNA detection SERS Microfluidic chamber DNA [108]
DNA detection Fluorescence detection Si/SiO 2 nanostructures DNA [109]
Nanochannels [110]
Silica nanowire templates
DNA separation Size-exclusion Al nanopores [111]
chromatography
DNA separation Nanogel [112]
Temperature Fluorescent images DNA molecular beacon Fluorescent COMS chip BRCA1 gene [113]
used as nanoscale component
temperature probe
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Chapter 17
Cytosensing and Cell Surface Carbohydrate
Assay by Assembly of Nanoparticles

17.1Introduction

Cells are the basic units of life. All organisms are made up of cells. Thus, developing
sensing platforms for probing the chemistry and physics in or at a living cell is one
of the basic goals in understanding the intricate processes that ultimately contribute
to life and life processes. Moreover, in the field of medicine, each cell type has a
unique molecular signature that distinguishes between healthy and diseased tissues
[1]. Cancerous cells are differentiated from noncancerous ones on the basis of
intracellular or extracellular (cell surface) biomarkers. In most cases, the distinc-
tions between normal vs. tumor and benign vs. metastatic cells are often subtle.
Thus, the identification of cellular signatures for early cancer cell detection is a
major hurdle for cancer therapy. The earlier these signatures can be established, the
more effectively they can be treated [2]. In this regard, technological systems that
provide sensors with high sensitivity, selectivity, and stability are in high demand
to identify the unique cellular characteristics such as proteins and nucleic acids in
and on living cells at early disease states, providing the prospects of better health
and more effective therapy [3].
In the context of cytosensing, researchers require the ability to track molecules
within their native environs. Thus, the questions relating quantity, timing, and loca-
tion become as important as knowing the molecular targets of enzymes or receptors.
The efficiency of sensing systems consists of recognition elements and the trans-
duction process and is critically related to the outcome of the detection process in
terms of the response time, signal-to-noise (S/N) characteristics, sensitivity, and
selectivity of the system.
Current methods for cell interrogation are mainly based on antibody-, nuclear
acid-, and aptamer-based recognition techniques to introduce a unique probe into
the cellular components under investigation. This is due to the fact that few biomol-
ecules are naturally endowed with features that permit their direct detection in
complex milieus. Due to the flexibility in implementation, noninvasiveness, and

H. Ju et al., NanoBiosensing: Principles, Development and Application, 485


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0_17, Springer Science+Business Media, LLC 2011
486 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

high sensitivity, fluorescence microscopes and flow cytometers have been considered
the key tools for probing cells. Although notable progress has been made in this
field, challenges in improving the recognition process as well as designing new
signal-transduction mechanisms still exist.
To achieve ultrasensitive detection and imaging for cytosensing, the combination
of nanotechnology with chemistry, biology, physics, engineering, and medicine has
emerged as a cornerstone solution. Nanoscale materials are typically smaller than
several hundred nanometers and are comparable to the size of large biological
molecules such as enzymes, receptors, and antibodies. With sizes about 10010,000
times smaller than those of human cells, these nanomaterials can offer unprece-
dented interactions with biomolecules both on the surface of and inside cells [4].
The most well-studied nanomaterials include metal nanoparticles (NPs) [5, 6],
quantum dots (QDs) [7, 8], carbon nanomaterials [9, 10], silica nanomaterials [11],
and paramagnetic NPs [12]. In addition to the large surface-to-volume ratio that
favors miniaturization, NPs possess unique optical, electronic, and magnetic prop-
erties depending on their core materials. Furthermore, these properties of the nano-
materials depend on their size and shape and vary with their surrounding chemical
environment. In addition, NPs can be fashioned with a wide range of small organic
ligands and large biomacromolecules by using tools and techniques of surface
modification [3]. These distinctive physical and chemical properties of nanomateri-
als make them a promising scaffold for the pursuit of new recognition and transduc-
tion processes, as well as increasing the sensitivity, selectivity, reliability, and
practicality of detection [13]. The progress may revolutionize research on cell adhe-
sion, glycobiology, cell trafficking, and signal transduction along with structural
biology, transcriptional regulation, and disease diagnosis and treatment.
This chapter describes recent advances in research involving the use of NPs in
the detection of cells and diagnosis of analytes at or within a cell. A significant
number of modern technologies and instrumentations involved in the progression of
cytosensing are reviewed here. Because cell surface carbohydrates, in the form of
glycopeptides, glycolipids, glycosaminoglycans, proteoglycans, or other glycocon-
jugates, have long been known to participate in the plethora of biological processes,
this chapter will also give a brief introduction to the application of nanomaterials in
cell surface carbohydrate assay.

17.2Why Use Nanomaterials in Cytosensing?

The applications of nanotechnology in cytosensing play important roles in the


development of biology and biomedicine. NPs can be engineered as nanoplatforms
for the effective and targeted delivery of reporting tags, imaging labels, and drugs
into cells by overcoming many biological, biophysical, and biomedical barriers [4].
The use of nanoplatforms affords many advantages over conventional approaches.
One major merit of using nanomaterials is that their size and composition are
advantageous over the corresponding bulky structure because a target binding event
17.3 Cytosensing by Assembly of Nanomaterials 487

(i.e., DNA hybridization or immunoreaction) involving NPs can significantly affect


their optical or electrochemical properties. This offers novel options for in situ
fabrication of signal-transduction modes for cytosensing [14].

The surface coating of nanomaterials can be readily adjusted to achieve not only
nontoxic and biocompatible but also specific localization on a living cell. The nature
of surface coatings and their subsequent geometric arrangement on the NPs deter-
mine the overall size of the colloid, which plays a significant role in the biokinetics
and biodistribution of NPs in the body. Nevertheless, the biological or molecular
coating acts as a bioactive and selective interface. Moreover, multiple, potentially
different targeting ligands on the NPs can provide enhanced binding affinity and
specificity. The types of specific coatings or derivatization of these NPs depend on
the end application and should be chosen by keeping a particular application in
mind [15]. This will ultimately lead to further improvements in early cancer diagno-
sis and treatment.
Nanomaterials can be designed to integrate multiple functions in a very predict-
able manner to meet the needs of specific applications. There are two strategies to
fabricate multifunctional nanostructures. The first, molecular functionalization,
involves attaching antibodies, proteins, and dyes to the NPs. The second method
integrates one NP with other functional nanocomponents, such as encapsulating
magnetic NPs in QDs or metallic NPs. Because they can exhibit several features
synergistically and deliver more than one function simultaneously, such multifunc-
tional nanomaterials can have unique advantages in real-time cytosensing [12].
NPs hold immense promise as versatile amplifying labels for cytosensing. There
are two central and related concepts that have been used for the amplified transduc-
tion of biorecognition events. One is the simultaneous immobilization of the recog-
nizing component and reporter tag on nanomaterials; the latter is often species with
catalytic ability, such as enzymes. The other is the multiple loading of the reporter
tags or a combination of tags on the surface of nanomaterials or by entrapping them
within the nanomaterials, which provides the opportunity for dramatic cascade
signal amplification.
The nanomaterial-based techniques, combined with nanofluidics, single-
molecule detection, and multiplexing methodologies, provide exciting new possi-
bilities for ultrasensitive cytosensing. In particular, benefiting significantly from
technological advancements in nanotechnology, emerging biomedical sciences and
biotechnology will inevitably shed new light on acquiring a deep understanding of
biochemical processes of life and improving disease diagnosis and treatment.

17.3Cytosensing by Assembly of Nanomaterials

Cytosensing refers to the characterization and measurement of biological processes


at the cellular and/or molecular level [4]. It can give whole-body readout in an intact
system, dramatically decrease the workload, and reduce the cost of biomedical
research and drug development. It also provides more statistically relevant results
488 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

since longitudinal studies can be performed in the same animals, aids in early lesion
detection in patients and patient stratification, and helps in individualized treatment
monitoring and dose optimization [16]. Cytosensing modalities include optical
fluorescence, molecular magnetic resonance imaging (MRI), surface-enhanced
Raman scattering (SERS), electrochemistry, and colorimetry. Many hybrid systems
that combine two or more of these modalities are already commercially available,
and a few others are under active development [12].

17.3.1Fluorescence Imaging by Assembly of Nanoparticles

Fluorescence imaging is an important approach to extract information on processes


and functions in living cells. In fluorescence imaging, excitation light illuminates the
subject and the emission light is collected at a shifted wavelength [17]. The major
disadvantage of fluorescence imaging is that it is typically not quantitative and the
image information is surface-weighted due to tissue absorption [18]. In most cases,
significant background signal is also observed because of autofluorescence emanating
from superficial tissue layers, which restricts the sensitivity and the depth to which
fluorescence imaging can be used. Only a limited number of fluorescent molecular
imaging agents are available in the near-infrared (NIR) window, with a large spectral
overlap between them, which restricts the ability to interrogate multiple targets simul-
taneously (multiplexing). Moreover, rapidly photobleaching fluorescent molecules
limits their useful lifetime and prevents studies of a prolonged duration [19].
Although state-of-the-art fluorescence dyes provide high sensitivity, current cellular
molecular research indicates the strong need for better optical labels. Fluorophores are
increasingly being replaced with NPs since they have several advantages. NPs tend to
have superior optical properties, substantially greater chemical stability, and stability
against photobleaching. Moreover, the ability to systematically vary their optical prop-
erties via modification of particle size or dimension can lead to an array of new applica-
tions. Several types of NPs have been prepared using semiconductor, metal, magnetic,
or organic material [11]. The general preconditions for the application of NPs in molec-
ular biology and medicine are (1) high biocompatibility, (2) inexpensive materials, (3)
simple detection, and (4) the presence of a highly specific emission that does not over-
lap with the autofluorescence of tissues and cells [20].
The most widely studied NPs for optical imaging applications are QDs. QDs are
inorganic fluorescent semiconductor NPs with many superior properties for biological
imaging than organic fluorophores, such as high quantum yields, high molar extinc-
tion coefficients, strong resistance to photobleaching and chemical degradation,
continuous absorption spectra spanning UV to NIR (700900nm), long fluorescence
lifetimes (>10ns), narrow emission spectra (typically 2030nm full-width at half-
maximum), and large effective Stokes shifts [2123]. Numerous efforts to employ
QDs as replacements for currently utilized organic dyes have been made [8].
To improve the stability and biocompatibility of QDs, a silica layer/shell has
been used to encapsulate QD aggregates or a single QD. For example, Bakalovas
17.3 Cytosensing by Assembly of Nanomaterials 489

Fig. 17.1 (a) Model structure of a silica-shelled single QD micelle; (b) intracellular delivery
(fluorescence confocal microscopic image) of the micelles in viable HeLa cells. Reprinted with
permission from Zhelev etal. [24]. 2006, American Chemical Society

group developed silica-shelled single-QD micelles, which possess a high quantum


yield in aqueous solution, a comparatively small size (up to 18nm in diameter), a
sharp size distribution (~12%), and a comparatively good intracellular delivery [24].
As shown in Fig.17.1, the NPs can be highly concentrated into the cells (cytoplasm
and/or nucleus), probably because of the large positive charge on their surface and
comparatively small size.
In order to make NPs more useful for biomedical applications, NPs need to be
effectively, specifically, and reliably directed to a specific organ or disease site with-
out alteration [4]. Specific targeting can be achieved by attaching targeting mole-
cules to the NP surface, such as peptides, antibodies, aptamer, and so on.

17.3.1.1Peptide-Conjugated Nanoparticles

Although aptamer-conjugated NPs have been ideal candidates for cell surface pro-
tein recognition and therefore cancer cell targeting [25], the targeting of QD conju-
gates in living subjects was first reported using peptide-coated ZnS-capped CdSe
QDs to bind the lung, blood vessels, and the lymph, respectively [26]. In this work,
polyethylene glycol (PEG) was used to eliminate the nonspecific uptake of the con-
jugate by the reticuloendothelial system, which opened up a new field of QD-based
nanoplatforms for cellular specific targeting. A variety of targeting peptides have
been explored to label QDs for improving the stability and target ability during
invitro studies, such as cellular nuclear localizing sequence (NLS) (Pro-Pro-Lys-
Lys-Lys-Arg-Lys-Val), cell-penetrating Tat peptides (Arg-Arg-Arg-Gln-Arg-Arg-
Lys-Lys-Arg-Gly-Tyr), neuropeptide AST1 (Ala-Pro-Ser-Gly-Ala -Gln-Arg-Leu-T
yr-Gly-Phe-Gly-Leu-NH2), and neurotoxins such as chlorotoxin (CTX) and
490 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

Fig. 17.2 In vitro staining of human glioblastoma U87MG cells using 1 nM QD705-RGD.
Staining of U87MG cells with 1nM QD705 (denoted U87MG+QD705) or 1nM QD705-RGD
in the presence of 2mM c(RGDyK) (denoted U87MG+Block) are also shown. Reprinted with
permission from Cai etal. [29]. 2006, American Chemical Society

d endrotoxin-1 (DTX-1) [27]. Neurotoxins are high-affinity peptides that interact


and bind to endogenously expressed cell membrane proteins and ion channels. A
multiplexing labeling method of endogenously expressed cellular proteins within
living cells has been developed by combining CTX and DTX-1-modified QDs,
demonstrating the capability of neurotoxins as a live assessment of markers toward
identifying the presence of cancer cells [28].
Several peptide-associated QDs have been reported for invivo applications. Cai
etal. reported the invivo targeted imaging of tumor vasculature in a U87MG tumor-
bearing mice using arginine-glycine-aspartic acid (RGD, potent integrin avb3 antag-
onist) peptide analog-conjugated QDs [29]. In this study, the RGD-containing
peptides exhibited high affinity to integrin avb3. Because the majority of tumor
vasculature overexpresses integrin avb3 during angiogenesis, the proposed RGD-

QDs showed great potential as nanoprobes for detecting tumor vasculature invivo
for most cancer types (Fig.17.2). More recently, Choi etal. fabricated zwitterionic
QDs with an overall diameter of 5.5nm coupled with RGD [30]. Those particles
17.3 Cytosensing by Assembly of Nanomaterials 491

Fig.17.3 In vivo detection


of prostate cancer in a mouse
model before and after
adding antibody-marked
CdSe@ZnS QDs. Reprinted
with permission from Gao
etal. [31]. 2004, Nature

demonstrated good tumor targeting and, because of their extremely compact size,
showed renal clearance by the kidneys within 4h postinjection. The results of this
study suggested a set of design rules for tumor-targeted NPs that could be elimi-
nated through renal clearance.

17.3.1.2Antibody-Conjugated Nanoparticles

QDs functionalized with antibody have also fulfilled their promise as nano-
probes for molecular imaging. Gao et al. conjugated CdSe@ZnS QDs with
prostate-specific antigen (PSA)specific monoclonal antibodies for prostate
cancer targeting and imaging in mice [31]. The QD probes were accumulated at
tumors both by the enhanced permeability and retention of tumor sites and by
antibody binding to cancer-specific cell surface biomarkers (Fig.17.3). These
results raised new possibilities for the ultrasensitive and multiplexed imaging of
molecular targets invivo.
In a recent study, by incorporating antibody-coated QDs within biodegradable
polymeric nanospheres, a hybrid nanocomposite (QDNC) was developed and a
noninvasive method to image subcellular structures in living cells was designed
[32]. The bioresponsive delivery system underwent endolysosomal to cytosolic
translocation via the pH-dependent reversal of nanocomposite surface charge polar-
ity (Fig. 17.4). Upon entering the cytosol, the polymer nanospheres underwent
hydrolysis, thus releasing the QD bioconjugates. This approach facilitated multi-
plexed labeling of subcellular structures inside live cells without the requirement of
cell fixation or membrane permeabilization. More importantly, this work demon-
strated an important rational strategy for the design of a multifunctional nanosystem
for biological applications.
492 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

Fig.17.4 Mechanism of cytosolic delivery and subcellular targeting of QDNCs. Schematic repre-
sentation depicting QDNC escape from the endolysosomal compartment upon cellular internaliza-
tion with cytosolic release of the encapsulated cargo. Antibody-conjugated QDs can be delivered
in this manner to allow the labeling of subcellular organelles or other molecular targets. Reprinted
with permission from Kim etal. [32]. 2008, American Chemical Society

17.3.1.3Aptamer-Conjugated Nanomaterials

As is well known, the early detection of cancer greatly increases the chances for
successful treatment. Consequently, research on accurate and sensitive recognition
and detection of cancer cells is extremely important for cancer diagnosis and ther-
apy. Cancer cells, especially those in the early stages of disease development, may
have a very low density of the target on the cell surface. Recent research discovered
that aptamers represent an attractive alternative to the routinely used antibodies to
recognize these targets because of their small sizes, chemical robustness, and ease
of synthesis [33]. As the DNA molecules with predicable structures and easy site-
specific chemical modification, aptamers can readily link to advanced signaling
mechanisms [33, 34].
Bagalkot et al. developed a QD-aptamer (Apt)doxorubicin (Dox) conjugate
[QD-Apt(Dox)] for a targeted cancer imaging, therapy, and sensing system [35].
The A10 RNA aptamers used were specific for prostate-specific membrane anti-
gen (PSMA) expressing on the LNCaP cell surface, and the Dox, a fluorescent
17.3 Cytosensing by Assembly of Nanomaterials 493

Fig.17.5 Schematic illustration of (a) QD-Apt(Dox) Bi-FRET system and (b) specific uptake of
QD-Apt(Dox) conjugates into target cancer cell through PSMA-mediated endocytosis. Reprinted
with permission from Bagalkot etal. [35]. 2007, American Chemical Society

anthracycline drug emitting at 520640 nm, was then intercalated into the
double-stranded CG sequences in the PSMA aptamer. As shown in Fig.17.5,
the formation of this nanostructure resulted in quenching of both QDs and Dox
based on a bifluorescence resonance energy-transfer (Bi-FRET) mechanism. After
incubating the aptamerQDDox conjugates with target cells, Dox was released
from the conjugates, resulting in fluorescence recovery of both QDs and Dox in
cells. This strategy was sensitive to detecting cancer at a single-cell level since both
QDs and Dox gave sharp images of cancer cells with low background noise.
To increase the signal and enhance the binding affinity, multivalent binding,
instead of single-aptamer binding, is usually considered to be an effective approach.
Nanomaterials are thus considered to be particularly advantageous as multivalent
ligand scaffolds by virtue of their large surface area and variable sizes. Tans group
synthesized AuAg nanorods (NRs) as nanoplatforms for multivalent binding by
multiple aptamers on the rods to increase both the signal and binding strengths of
these aptamers in cancer cell recognition [36]. Up to 80 fluorophore-labeled aptam-
ers could be attached on one Nile red (NR), leading to a 26-fold higher affinity and
over 300-fold higher fluorescence signal. The molecular assembly of aptamers on
nanomaterials achieved multivalency, and showed the potential application for the
elucidation of cells with low density of binding sites. This also illustrated how
molecular assemblies could integrate the properties of aptamers and nanomaterials
to improve cancer cell targeting.
494 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

17.3.1.4Streptavidin-Conjugated Nanoparticles

As a popularly used recognition pair, biotin-avidin (or streptavidin) constitutes an


alternative option to specifically bind QDs to living cells. The coupling of biotin
and avidin (or streptavidin) yields an interaction with femtomolar affinity and a
long half-life in solution. Orndorff etal. developed a QD-based strategy to label
nicotinic receptors (nAchRs) on the postsynaptic membrane at neuromuscular
junctions (NMJs) in mature ex vivo mouse diaphragm [37]. The nAchRs were first
bound by a high-affinity nAchR antagonist biotinylated r-Bungarotoxin (r-BTX),
which could be subsequently quantified and visualized ex vivo using streptavidin-
conjugated QDs. This approach provided a novel methodology for the direct
assessment of the presence and mobility of neurotransmitter receptors in native
tissue. Recently, Lequeuxs group described a novel QD surface chemistry based
on bidentate sulfobetaine zwitterionic ligands [38]. These dihydrolipoic acid-
sulfobetaine (DHLA-SB) QDs were small and exhibited excellent stability with
time, pH, and salinity, and also could easily be functionalized with biotin or
streptavidin by mixing DHLA-SB with other functional ligands in controlled
ratios, allowing specific staining of cannabinoid receptor 1 (CB1R) and their
tracking during recycling into living HEK cells.

17.3.1.5Carbohydrate-Conjugated Nanomaterials

NP-based multivalent platforms offer valuable tools for evaluating multivalent


oligosaccharide interactions with cell surface lectins based on carbohydrate-
modified nanomaterials. These systems can present multiple oligosaccharide
monomers (>100) on a single particle. Given the high quantum yields in aque-
ous systems and unique photophysical properties, QDcarbohydrate conjugates
have become a powerful tool in studying the cell biology of cell surface lectins.
The number of carbohydrates per QD can be controlled by adjusting the linking
chemistry.
Different sugar-capped PEGylated QDs have been developed for in vitro and
invivo targeting applications [39]. The d-galactose-coated QDs are preferentially
taken up via asialoglycoprotein receptor (ASGP-R)-mediated endocytosis invitro,
and QDs capped with d-mannose and d-galactosamine sequester specifically in the
liver in the mouse model.
Fluorescent water-soluble mannose-protected Au nanodots (Man-Au NDs) have
also been developed for the facile detection of Escherichia coli (E. coli) [40].
Incubation of the nanodots with E. coli reveals that the Man-Au NDs bind to the
bacteria, yielding brightly fluorescent cell clusters. The relationship between the
fluorescence signal and the E. coli concentration is linear, from 1.00106 to
5.00107cells/mL (R2=0.96), with the limit of detection (LOD) of E. coli being
7.20105cells/mL.
17.3 Cytosensing by Assembly of Nanomaterials 495

17.3.1.6Fluorescent Polymer-Conjugated Nanoparticles

The cell membrane surface consists primarily of a thin layer of amphipathic phos-
pholipids, carbohydrates, and many integral membrane proteins. Their amount and
types differ between species according to the function of cells [1]. Current detection
methods for the specific recognition of intracellular/extracellular biomarkers (e.g.,
DNA/RNA/proteins) require previous knowledge of specific mutations in genome
or changes in protein biomarkers. However, there has been no single marker or a
combination of biomarkers that has sufficient sensitivity and specificity to differen-
tiate among normal, cancerous, and metastatic cell types [41].
An NP-conjugated polymer sensor array, which relies on selective interactions
between multiple reporter elements and the target cell, represents a new method
for diagnostic, biophysical, and surface science processes involving cell surfaces
[1, 42]. The multivalent capabilities and sensitivity of conjugated polymers to
minor conformational or environmental changes make them ideal candidates for
biosensing applications. Combining polymer-conjugated NPs with array technol-
ogy, this platform holds particularly promise for cell sensing, such as bacteria
identification [42] and mammalian cancer cell detection [1].
For example, a multivalent fluorescent polymer (PPECO2) conjugated differentially
with functionalized gold NPs has been subjected to incubation with cell sample. The
quenched fluorescent polymer can be displaced by cells with the concomitant restora-
tion of fluorescence. Through detection of the fluorescent pattern, (1) different cell
types, (2) normal, cancerous, and metastatic human breast cells, and (3) isogenic
normal, cancerous, and metastatic murine epithelial cell lines can be rapidly and effec-
tively distinguished [1]. A similar method has also been developed for bacterial sensing
with a conjugated polymer (Sw-CO2) featuring a branched oligo(ethylene glycol) side
chain to suppress nonspecific polymer-microorganism interactions (Fig.17.6) [42].
Unlike small-molecule fluorophores, fluorescent polymers feature a molecular-
wire effect and polyvalent modes of interactions that can enhance signal generation
[43, 44]. Moreover, conjugated polymer chains with multiple recognition elements
can bind to one analyte molecule, thereby increasing both the binding efficiency and
recognition selectivity for specific analytes [44].

17.3.1.7Luminescent Porous Silicon Nanoparticles

Since the discovery of photoluminescence of porous silicon in 1990 [45], lumines-


cent (porous) silicon NPs have been produced by several methods [46, 47], some of
which are amenable to biological applications [47]. For in vivo use, silicon NPs
provide attractive chemical alternatives to heavy-metal-containing QDs, which have
been shown to be toxic in biological environments. In addition, silicon is a common
trace element in humans, and silicic acid administered to humans is efficiently
excreted from the body through the urine [48].
496 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

Fig.17.6 Design of the NPconjugated polymer sensor array. (a) Schematic representation of the
displacement of anionic conjugated polymers from cationic NPs by negatively charged bacterial
surfaces. (b) Schematic illustration of fluorescence pattern generation on a microplate. The fluo-
rescence response is dependent upon the level of displacement determined by the relative NP-PPE
binding strength and bacteriaNP interactions. In the diagram, AG on the microplate represents
bacteria of different types, and codes 14 represent the different PPENP constructs. Reprinted
with permission from Phillips etal. [42]. 2008, Wiley

Park et al. developed dextran-coated luminescent porous silicon NPs (D-LPSi


NPs) that could carry a drug payload and of which the intrinsic NIR photolumines-
cence enabled monitoring of both accumulation and degradation invivo (Fig.17.7)
[48]. This study represented the first example of the imaging of a tumor and other
organs using biodegradable silicon NPs in live animals. It can be expected that this
type of multifunctional nanostructure with a low-toxicity degradation pathway will
provide a promising application of clinical translation.
17.3 Cytosensing by Assembly of Nanomaterials 497

Fig.17.7 In vitro and invivo fluorescence imaging with LPSi NPs. (a) In vitro cellular imaging
with LPSiNPs. Red and blue indicate LPSi NPs and cell nuclei, respectively. The scale bar is
20mm. (b) In vivo fluorescence image of LPSi NPs injected subcutaneously and intramuscularly
on each flank of a mouse. Reprinted with permission from Park etal. [48]. 2009, Nature

17.3.1.8Fluorescent Hydrogel Nanoparticles

Hydrogel NPs (nanogels), colloidally stable NPs made from hydrogels, are appealing
probes for use in chemical and biochemical sensing because of their stability, bio-
compatibility, and softness [49]. Nanogels have been employed to develop detection
technology for protein [50] and carbohydrates [51]. Recently, fluorescent nanogels
were reported to be capable of sensing temperature and pH in the cytoplasm of liv-
ing cells [52]. The nanogel was fabricated by the combination of a thermorespon-
sive polyNIPAM unit with a water-sensitive fluorophore, which could transduce
volume changes into a change in fluorescence intensity (FI) [5].
In a later work by Peng et al., a ratiometric fluorescent nanogel was prepared
rather simply from an inert but biocompatible polyurethane polymer that was
made pH sensitive by loading it with the pH indicator Bromothymol blue (BTB)
[53]. Furthermore, efficient fluorescence resonance energy transfer (FRET)
inside the nanogel was achieved by the addition of two standard fluorophores,
coumarin 6 (C6) and NR, and constituted the sensing mechanism. This nanogel
could be incorporated by living epithelial normal rat kidney (NRK) cells by
vesicular uptake mechanisms and was capable of intracellularly sensing pH val-
ues in the physiological range. By replacing the respective indicator dyes (probes)
by indicators for other ions, various other kinds of sensing nanogels could be
constructed and were likely to have a wide scope in terms of intracellular chemical
sensing.
498 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

17.3.1.9Carbon NanotubeBased Near-Infrared Fluorescent Cytosensing

The NIR (wavelength 0.82mm) fluorescent technique promises to be of great use


in biological systems due to little background signals caused by autofluorescence
from cells, tissues, and other biological molecules in this spectral range, as most
autofluorescence is confined to the visible range. Furthermore, biological tissues
allow for the relatively high transmission and penetration of NIR light near ~1mm
for detection within an organism or under the surface of tissues [54].
The unique optical properties, in particular, the NIR photoluminescence of
semiconducting single-walled carbon nanotubes (SWNTs) [55], have made them
promising as NIR fluorescent contrast agents in biological systems [54, 56, 57].
Individual semiconducting SWNTs have small band gaps of the order of ~1 eV,
depending on the diameter and chirality of a given nanotube [10]. This band gap
allows for emission mostly in the infrared-A range (11.4mm), which is ideal for
biological imaging, due to the high tissue penetration near 8001,000nm and the
inherently low autofluorescence from tissue in the NIR range [58]. SWNTs have a
further advantage due to the large Stokes shift between the excitation (550850nm)
and emission bands (9001,600nm). This spacing allows excitation in the biologi-
cal transparency window near 800nm while further reducing the background effects
of autofluorescence and Raman scattering [57].
However, raw carbon nanotubes (CNTs) have highly hydrophobic surfaces and
are not soluble for biomedical applications. Thus, surface chemistry or functional-
ization is required to solubilize CNTs, and to render biocompatibility and low
toxicity. A noncovalent functionalization of SWNTs by PEGylated phospholipids
(PL-PEG) has been developed to meet the requirements of high water solubility of
nanotubes and versatile functionalities [10]. Phospholipids are the major compo-
nent of cell membranes and are safe to use in biological systems. The two hydrocar-
bon chains of the lipid strongly anchor onto the nanotube surface with the hydrophilic
PEG chain extending into the aqueous phase, imparting water solubility and
biocompatibility. Unlike nanotubes suspended by typical surfactants, PEGylated
SWNTs prepared by this method are highly stable in various biological solutions,
including serum, and even under harsh conditions [10]. In order to use CNTs for
potential cancer imaging and/or treatment, targeting nanotubes to tumors is highly
desirable. Commonly used targeting ligands include folic acid, peptides, and
antibodies.
In recent work, Jin etal. used NIR photoluminescence to track endocytosis and
exocytosis of SWNTs in NIH-3T3 cells in real time [59]. Welsher et al. reported the
use of semiconducting SWNTs as NIR fluorescent tags for selective probing of cell
surface receptors [54]. The SWNTs were functionalized with phospholipid-polyeth-
yleneglycol (PEG)-amine (PL-PEG-NH2) and conjugated to antibodies such as
Rituxan to selectively recognize CD20 cell surface receptor on B-cells and Herceptin
to recognize HER2/neu-positive breast cancer cells.
Although SWNT has been demonstrated successfully in NIR imaging invitro,
there are still challenges for invivo NIR photoluminescence imaging. The biggest
stumbling block is the development of bright SWNTs with high biocompatibility.
17.3 Cytosensing by Assembly of Nanomaterials 499

Fig.17.8 (a) In vitro NIR photoluminescence images of human malignant glioma cells (U87 MG)
treated with exchange-SWNT/RGD conjugates. Arginineglycineaspartic acid (RGD) peptide
ligand binds to avb3-integrin overexpressed on the cells. (b) Human breast cancer cell line
(MDA-MB-468) is used as a negative. Cells treated with exchange-SWNT/RGD show a high posi-
tive signal with very low nonspecific binding to the negative cell line. Reprinted with permission
from Welsher etal. [57]. 2009, Nature

In order to overcome the issue, Welsher et al. has shown that sonicating SWNTs
with sodium cholate, followed by surfactant exchange to form PL-PEG-coated nan-
otubes, produces in vivo imaging agents that are both bright and biocompatible
[57]. Using the obtained nanoprobes, targeted cell imaging has been carried out in
the 1,1001,700-nm range using the intrinsic NIR photoluminescence of the
exchange-SWNTs (Fig.17.8), and the high-resolution intravital microscopy imag-
ing of tumor vessels beneath thick skin is also achieved.

17.3.2Magnetic Resonance Imaging by Assembly


of Magnetic Nanoparticles

Cell labeling with ferro/paramagnetic substances is an increasingly common method


for invivo cell study as the labeled cells can be detected by MRI [15]. MRI is a
noninvasive medical diagnostic technique based on the interaction of certain nuclei
(typically protons) with each other and with surrounding molecules in a tissue of
interest [4]. MRI provides much greater contrast between the different soft tissues
of the body than computed tomography (CT) does, making it especially useful in
neurological (brain), musculoskeletal, cardiovascular, and oncological (cancer)
imaging. Exogenous contrast agents can further enhance the contrast by selectively
shortening either the longitudinal (T1) or transverse (T2) relaxation time [4, 60].
500 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

Traditionally, gadolinium chelates have been used as positive contrasts to shorten


the relaxation time T1 and generate a bright image [4, 60]. Unfortunately, this
powerful imaging modality is still limited by its inability to accurately delineate
tumor boundaries and quantify tumor volumes [61]. Iron oxide particles are mainly
employed to shorten the transverse relaxation time T2, thus leading to signal reduc-
tion, and a dark image can be obtained [60]. In comparison to gadolinium chelates,
NP-based contrast agents offer prolonged delineation of tumor margins because of
enhanced cellular internalization and slower clearance from the tumor site [61]. The
major advantages of MRI over radionuclide-based imaging (such as CT scans and
traditional X-rays) are the absence of ionizing radiation and better contrast resolution
(usually sub-millimeter level). The disadvantage of MRI is its inherent low sensitiv-
ity, which can only be partially compensated for by working at higher magnetic
fields (4.714T), acquiring data for much longer periods during imaging, and using
exogenous contrast agents [4].
The labeling of cell by magnetic NPs can be achieved mainly by two routes: One
is attaching magnetic particles to the cell surface, and the other is internalizing
biocompatible magnetic particles by fluid-phase endocytosis, receptor-mediated
endocytosis, or phagocytosis [15]. Iron oxide NPs are the most widely used magnetic
NPs, and various biological molecules, such as antibodies, proteins, carbohydrate,
targeting ligands, etc., have been used as ligands to functionalize NPs.

17.3.2.1Cell Surface Targeting by Magnetic Nanoparticles

The iron oxide NPs targeted to a cell surface molecule can remain bound to their
target, thus being useful as molecular MRI probes [62]. To target endothelial markers
of inflammation, anti-VCAM-1 antibodies have been conjugated to microparticles of
iron oxide, allowing specific and quantitative binding to activated endothelial cells.
The nanoprobes can be used for invivo detection of vascular cell adhesion mole-
cule-1 (VCAM-1) expression in a mouse model of acute brain inflammation [63].
In order to improve bioavailability for molecular imaging of extravascular targets,
extremely small but potent NPs open up possibilities to develop targeted MRI probes.
In a recent work, a strongly positive charged peptide, protamine, was used to coat
citrate-stabilized iron oxide NPs by electrostatic attraction [64]. The protamine
allowed the chemical decoration of the NPs by Annexin A5, a cell apoptosis-
detection marker. The nanoprobes with a hydrodynamic diameter of 15nm have been
successfully tested on apoptotic human lymphoblastic T-cell (Jurkat) cultures. The sub-
stantially reduced hydrodynamic diameter is interesting for invivo applications since
it facilitates extravasation, thereby enabling MRI of extravascular targets.
Although various types of cancer cells can be detected both invitro and invivo
by using antibody-immobilized magnetic NPs, there is still the need to develop
other recognition element-based nanoprobes, considering the fact that particular
antigenic structures may be absent in some types of cancer cells. An attractive
ligand for receptor-mediated interaction is carbohydrates and, in particular, glyco-
conjugates, which play important roles in cancer development and metastasis [65].
17.3 Cytosensing by Assembly of Nanomaterials 501

Fig.17.9 The concept of targeted magnetomechanical cancer-cell destruction using disc-shaped


magnetic particles possessing a spin-vortex ground state. Reprinted with permission from Kim
etal. [68]. 2010, Nature

The ability to characterize and distinguish carbohydrate-binding profiles of a variety


of cells can expedite both the mechanistic understanding of their roles in disease
development and the expansion of diagnostic and therapeutic tools [66]. El-Boubbou
etal. synthesized different types of carbohydrates modified magnetic NPs, called
magnetic glyconanoparticles (MGNPs), to detect and differentiate cancer cells [66].
The binding ability of the proposed nanoprobes was closely related to the cell type,
which could be quantitatively monitored by MRI, thus offering MRI signature for
each cell type. The MGNPs have also been demonstrated to allow presymptomatic
invivo imaging and acute detection of brain disease [67].
Although the targeting application of magnetic NPs has met great success,
there are also inherent problems in translating magnetic targeting to the clinic.
Recently, a novel clinical application based on interfacing cells with lithographi-
cally defined microdiscs has been reported [68]. Microdiscs biofunctionalized
with antihuman-IL13 2R antibody can specifically target human glioblastoma
cells. As shown in Fig.17.9, when an alternating magnetic field is applied, the
magnetic discs oscillate, compromising membrane integrity and initiating spin-
vortex-mediated programmed cell death. A low-frequency field of a few tens of
hertz applied for only 10min is sufficient to achieve ~90% cancer-cell destruction
invitro. Although this technique holds great promise for the long-awaited transla-
tion of magnetic targeting to a clinical application, more biocompatible and
smaller discs may still need to be developed.
502 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

17.3.2.2Intracellular Trapping of Magnetic Nanoparticles

The targeting of cell surface molecules with adequately modified iron oxide
particles has allowed cellular delivery of the magnetic tag and MR imaging of
specific cell types [62]. For cell surface receptor targeting, transferrin, lactoferrin,
transforming growth factor-a (TGF-a), insulin, nerve growth factor (NGF),
ceruloplasmin, pullulan, elastin, albumin, RGD peptide, and folic acid etc. have
been used to modify magnetic NPs for various biomedical applications. The
receptors for these ligands are frequently overexpressed on the surface of mam-
malian cells [15]. These receptors are not only cellular markers, but also have
been shown to efficiently internalize the NPs targeted to these receptors via receptor-
mediated endocytosis [15].
Integrin avb3 is the one of the most well-studied targets for molecular MRI [4].
The avb3-integrin is the receptor for a variety of extracellular matrix proteins
containing an RGD sequence. The avb3-integrin plays a key role in angiogenesis
and is an attractive target for the treatment of certain tumor types (melanoma,
glioblastoma, ovarian, and breast cancer) [69].
In a recent study, cyclic peptide c(RGDyK), with high selectivity and strongly
enhanced binding activity, was employed to modify Fe3O4 NPs using a new ligand
4-methylcatechol (4-MC) for coating NPs and conjugating the peptide [70]. The
c(RGDyK)-conjugated NPs showed good binding ability to integrin on U87MG
human glioblastoma cell lines. The enhanced binding, compared with monomeric
RGD peptide, was due to the multivalent effect. The Fe3O4 NPs synthesized in this
work were ultrasmall, with a 4.5-nm core size, and the obtained c(RGDyK)-MC-
Fe3O4 NPs had an overall diameter of ~8.4nm. This smaller hydrodynamic size was
desirable for overcoming the nonspecific uptake problem and enhancing the
extravascular ability.
Tumor cells expressing the membrane-bound matrix metalloproteinase MMP-2
can also be labeled with MRI nanoprobes with a high level of specificity both
invitro and invivo using a 9L gliosarcoma cell line and mice bearing xenografts
as models [61]. The nanoprobe is composed of an iron oxide core coated by PEG
and functionalized by a 36-amino-acid peptide, CTX. The CTX is of attractive
interest due to its specific binding capability to glioma, medulloblastoma, prostate
cancer, sarcoma, and intestinal cancer. The nanoprobes show a profound improve-
ment in tumor labeling efficiency compared with nontargeting nanoprobes, which
may be attributed to the ligandreceptor-mediated nanoprobe internalization by
target cells.
Besides decreasing the NP size, an alternative strategy for improving the
MRI sensitivity is controlling the NP shape [63]. The mononuclear phagocytic
system (MPS) is one of the most important pathways for clearance of NPs. The
design of elongated NPs offers a new way to prolong blood half-life due to the
low MPS uptake. Another approach to enhance MRI detection is the develop-
ment of new magnetic core materials [71]. For example, a better sensitivity of
MnFe2O4 than Fe3O4 in MRI applications has recently been demonstrated for
individual NPs [72].
17.3 Cytosensing by Assembly of Nanomaterials 503

Fig.17.10 Schematic illustration for the preparation of bimodal imaging nanoprobes having both
19F-based multispectral magnetic resonance and QD-based multicolor optical imaging capabili-
ties. Reprinted with permission from Lim etal. [73]. 2009, American Chemical Society

17.3.2.3Multifunctional Magnetic Nanoparticles

The possibility of bringing together several different functional materials into a


single nanostructure offers great potential for increased efficiency and versatility in
the numerous applications of nanomaterials in practical science. The combination
of magnetic nanomaterials and other functional nanostructures produces one single
object of nanoscale dimensions that can perform several tasks in parallel. The
obtained core-shell or heterodimer nanostructures with both magnetic and other
properties are a promising candidate for multimodality molecular imaging [12].
The combination of superparamagnetism and fluorescence at a nanometer scale
provides robust fluorescent NPs that can be physically manipulated by an external
magnetic field or can enhance magnetic resonance imaging [12]. Lim etal. devel-
oped a dual-function imaging nanoprobe, perfluorocarbon (PFC)QD nanocompos-
ite emulsions, to integrate the advantages of MR with the optical imaging capabilities
[73]. The strategy exploited the combination of the multispectral MR properties of
four different PFC materials and the multicolor emission properties of three differ-
ent-color CdSe/ZnS QDs (Fig. 17.10). Through the detection of the distinct
504 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

19F-based MRI and the specific fluorescence image of PFCQD nanocomposite,


the uptake of nanocomposite emulsions in different phagocytic cells could be
monitored, demonstrating the feasibility of using multimodality nanoprobes for the
multiplexed detection and imaging of therapeutic cells both invitro and invivo.
Fluorescent dye-doped silica NPs offer a powerful platform for integration
fluorescence detection with magnetic NPs. These silica NPs have a high FI, good
photostability (due to the exclusion of oxygen by the silica encapsulation), and good
potential for surface modification [11]. Because the fluorescence signal is up to
30,000 times higher than that of a single organic fluorophore, a signal enhancement
of up to five magnitudes may be obtained [11], thus negating the need for additional
signal-amplification steps.
Recently, Lee et al. developed a multifunctional nanomedical platform for
multimodal imaging and simultaneous drug delivery [74]. The nanomaterials were
synthesized by decorating the surface of mesoporous dye-doped silica NPs with
multiple magnetite nanocrystals. The magnetite nanocrystals enabled the NPs to be
used as a contrast agent in MRI, and the dye molecule in the silica framework
imparted fluorescence imaging modality. An anticancer drug, Dox, could be loaded
in the pores and induced efficient cell death. In vivo passive targeting and accumula-
tion of the NPs at the tumor sites were confirmed by both T2 MR and fluorescence
imaging (Fig.17.11).
As an alternative way to solve the problem of photobleaching of fluorescent
dyes, gold nanorods decorated by Fe3O4 NPs have been fabricated for MRI and
fluorescence imaging as well as photothermal therapy [75]. The obtained nano-
pearlnecklace-structured multifunctional NPs are functionalized with Herceptin
for the specific targeting of SK-BR-3 cells.
Besides the attachment of magnetic NPs on the fluorescent component, another
way to fabricate dual functional nanoplatforms is the incorporation of the two
components within biomaterials, such as lipoproteins and phospholipid.
Lipoproteins are micelles responsible for the transport of lipids and other hydro-
phobic substances such as vitamins and drugs in the aqueous environment of the
blood. Bruns etal. embedded QDs and superparamagnetic iron oxide nanocrystals
(SPIO) in the core of lipoproteins (Fig.17.12) and showed that it was possible to
image and quantify the kinetics of lipoprotein metabolism invivo using fluores-
cence and dynamic magnetic resonance imaging [76]. Using this strategy, it was
possible to study the clearance of lipoproteins in metabolic disorders and to
improve the contrast in clinical imaging.
A novel type of interaction, barnase and barstar system, has been introduced into
the fabrication of nanostructures with multifunctions consisting of different single-
function particles: labels, carriers, recognition, and targeting agents. The link
between the NPs is based on the very specific and strong noncovalent interaction
between the two proteins [77]. The in-advance programmable properties of the
superstructures have been achieved through a bioengineering method [78]. The
method employs barnase and barstar noncovalent binding system (BBS) to rapidly
join the structural components together directly in water solutions. The properties
of the superstructures can be designed on demand by linking different agents of
17.3 Cytosensing by Assembly of Nanomaterials 505

Fig. 17.11 In vivo accumulation of nanoparticles at tumor site. (a) In vivo T2-weighted MR
images (upper) and color-mapped (lower) images of tumor site before and 3h after intravenous
injection of the NPs (arrows indicate tumor site). (b) Confocal laser scanning microscopic images
of sectioned tumor tissue harvested 24 h after injection. Left: Red fluorescence showing
NP-internalized cells. Right: Merged image with DAPI-stained nuclei (blue) (scale bar 10mm).
Reprinted with permission from Lee etal. [74]. 2010, American Chemical Society

various sizes and chemical nature, designated for specific goals. As a proof of
concept, colloidally stable trifunctional structures have been assembled by binding
together magnetic particles, QDs, and antibodies using barnase and barstar. The
specific interaction of such superstructures with human ovarian carcinoma SKOV-3
cells, which overexpress the HER2/neu receptor on their surface, resulted in fluores-
cent labeling of the cells and their responsiveness to magnetic field.
This new generic method, protein-assisted nanoassembly, can be regarded not as a
substitution of other most general approaches, (strept)avidinbiotin system and
complementary DNA-assembled scaffolds, but as an alternative that broadens the range
of available nanoagents. It can be employed either on its own or in combination with
other techniques allowing the synergistic use of inorganic moieties, organic particles,
and single biomolecules for self-assembly of a wide variety of superstructures with
506 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

Fig.17.12 Schematic of pure and functionalized QD- and SPIO-nanosomes. Nanosomes were
generated from lipids extracted from human triglyceride-rich lipoproteins. QD- and SPIO-
nanosomes provide information on the location and distribution of the lipoproteins within the cells
or tissues. Radiolabeled SPIO-nanosomes allow quantification of cell uptake and organ distribu-
tion. Functionalizing the nanosomes with apolipoprotein E and lipoprotein lipase allows specific
targeting to the liver lipoprotein receptors. Reprinted with permission from Bruns etal. [76].
2009, Nature

programmable properties such as specific functionality, size, and composition


(Fig. 17.13). A remarkable feature of the strategy is the opportunity to attach any
protein in its functional form to the key components, barnase and barstar, by gene engi-
neering methods to create desirable recognition, visualization, or cytotoxic modules.

17.3.3Cellular Surface-Enhanced Raman Scattering (SERS)


Detection by Assembly of Nanoparticles

SERS is a promising analytical technique that allows intracellular sensing and


diagnosis and treatment of cancer [79]. The primary mechanism of SERS is the
increase in Raman scattering signals from molecules in close proximity to a noble
17.3 Cytosensing by Assembly of Nanomaterials 507

Fig.17.13 Possible variants for assembling of three particles into a superstructure by the protein-
assisted nanoassembler method. The correspondence of different combinations of BBS proteins
conjugated with the green and gray particles and the structure number (ac) are shown in the table.
Reprinted with permission from Nikitin etal. [78]. 2010, National Academy of Sciences

metal nanostructure [80]. Since the first reported SERS on silver and gold colloids
in 1979 [81], they have become the most commonly used metal substrates for SERS.
Colloidal gold has been found to amplify the efficiency of Raman scattering by
1415 orders of magnitude [82]. These nanostructures must satisfy certain condi-
tions to provide a strong Raman signal. First of all, the roughness of the metal
surface should be smaller than the wavelength of the excitation field. Second, the
wavelength of the excitation laser should correspond to the plasmon resonance
wavelength of the SERS substrate. The latter can be controlled by changing the size,
shape, and material of the metallic structures [83]. SWNTs also show an intense
Raman peak produced by the strong electronphoton coupling that causes efficient
excitation of tangential vibration in the nanotubes quasi-one-dimensional structure
upon light exposure. In a recent work reported by Keren et al., SWNTs were used to
demonstrate whole-body Raman imaging, NP pharmacokinetics, multiplexing, and
invivo tumor targeting [84].
Besides substrates, SERS labels and probes need reporter molecules to attach
substrate, which provide the Raman signature of the analyte. These reporter mole-
cules are in many cases fluorescent dyes. In practice, it is necessary to distinguish
two types of SERS labels. In the first type, the substrates are enwrapped in mono-
layers of reporter molecules, or further embedded in a protective coating of glass or
508 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

PEG [80]. These SERS labels are physically robust and immune to their biological
and chemical environment. Based on the optical signature of the reporter, biological
structures can be highlighted and imaged. The second type does not use a protective
cover, and the surface-enhanced Raman signal generated by other molecules present
in the local optical fields can also be obtained [80]. The main advantage of these
SERS probes lies in the ability to obtain spectra from target analytes present in
subcellular compartments. In order to deliver the SERS labels into cells and control
their intracellular aggregation and distribution, they can be functionalized with
specific antibodies, peptides, and DNA [85].
The high sensitivity of SERS is due to the electromagnetic field coupling on
roughened metal surfaces and junctions, which can cause the scattering signal of the
molecule to increase by several orders of magnitude compared to fluorescence [86].
Moreover, in SERS, excitation can be out of resonance with electronic transitions of a
reporter molecule. Thus, SERS detection is considerably less sensitive to photobleach-
ing, ensuring the stability of SERS labels. The characteristic Raman bands are up to
two orders of magnitude narrower than fluorescence bands [86]. The fingerprint-like
nature of the sharp and distinguishable vibrational spectrum provides an enormous
multiplexing platform for invivo SERS imaging even under stringent physiological
conditions [87]. SERS probes fulfill the requirements of dynamic invivo systems
the use of very low laser powers and very short data acquisition times. Additionally, it
provides the free choice of excitation wavelength (and therefore, also detection range)
as yet another significant advantage over fluorescence labels [80].

17.3.3.1Cytosensing Based on SERS

Capitalizing on these unique features, recent studies have focused on the investiga-
tion of molecular phenomena inside living cells. These experiments are performed
by first introducing SERS probes into organisms and attaching them to the surface
of cells as targeting agents of specific surface markers, and then detecting the SERS
spectral signature of the reporter molecules [80].
A class of biocompatible and nontoxic pegylated gold NPs (Au NPs coated with
a protective layer of PEG) has been developed for in vivo tumor targeting and
surface-enhanced Raman detection with large optical enhancements [82]. When
conjugated to tumor-targeting ligands such as single-chain variable fragment (ScFv)
antibodies, the conjugated NPs are able to target tumor biomarkers such as epider-
mal growth factor (EGF) receptors on human cancer cells and in xenograft tumor
models (Fig.17.14). The advantage of this NP platform is the facile conjugation of
tumor-targeting ligands to heterofunctional PEG linkers.
The actual mechanism of particle uptake can be investigated by SERS. Using
gold nanoshells as a surface-enhanced Raman platform for intracellular sensing in
NIH-3T3 fibroblast cells, the voluntary, controllable cellular uptake of single gold
nanoshells has been studied by using a NIR Raman system [79]. This work demon-
strates that the uptake is independent of the known endocytotic mechanisms, and
shows that nanoshells can be used as a viable and versatile platform for intracellular
SERS nanosensors.
17.3 Cytosensing by Assembly of Nanomaterials 509

Fig.17.14 Cancer cell targeting by using antibody-conjugated SERS NPs comprising SH-PEG
shell and a heterofunctional PEG. Reprinted with permission from Qian et al. [82]. 2008,
Nature

SERS signals are known to be highly sensitive to exact NP configuration, and


thus the uncontrollable aggregation of NPs in cells poses difficulties. A solution to
this problem is using the SERS substrates with a fixed NP geometry. A SERS-active
nanopipette has been designed by incorporating gold NPs on the outer surface of the
pipette tip for intracellular monitoring of living cell function in real time [83]. The
characteristic SERS spectra from the tip of the nanopipette inserted in the cell
nucleus and cytoplasm can be collected. This nanopipette can be fabricated based
on any standard equipment for cell microinjection.

17.3.3.2Multiplexed Cytosensing Based on SERS

The greatest potential of SERS labels and probes lies in the efficiency provided by mul-
tivariate methods for their fingerprint-based imaging. In several cellular processes, the
simultaneous investigation of different locations is favorable. To achieve this, it is useful
to introduce with each probe type a probe-specific signature that allows identification.
Zavaleta et al. demonstrated the ability of Raman spectroscopy to separate the
spectral fingerprints of up to ten different types of SERS NPs in a living mouse [88].
Based on the results, they simultaneously injected the five most intense and spec-
trally unique SERS NPs to image their natural accumulation in the liver (Fig.17.15).
This work combined the ultrasensitive properties of Raman spectroscopy with the
multiplexing capabilities of SERS NPs, allowing better detection of multiple
biomarkers associated with a specific disease.

17.3.3.3In Vivo SERS Imaging Using Zebrafish as a Model

More recent efforts have been reported on the biocompatibility and localization
dynamics of NPs in zebrafish embryos. Zebrafish is chosen as a model for sensor
510 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

Fig. 17.15 Demonstration of deep-tissue multiplexed imaging 24 h after IV injection of five


unique SERS NP batches simultaneously. (a) Graph depicting five unique Raman spectra, each
associated with its own SERS batch: S420 (red), S421 (green), S440 (blue), S466 (yellow), and
S470 (orange). (b) Raman image of liver overlaid on digital photo of mouse, showing accumula-
tion of all five SERS batches accumulating in the liver after 24h post-IV injection. Panels to the
right of (b) depict separate channels associated with each of the injected SERS NP batches.
Reprinted with permission from Zavaleta etal. [88]. 2009, National Academy of Sciences

platform development because of its unique optical clarity, rapid growth and
development, high fecundity, and availability of large numbers of genetic mutant
zebrafish lines [86]. Through microinjected SERS NPs in vivo in developing
zebrafish embryos, their localization as the embryos develop from the one-cell stage
into desirable stages as organs or tissues form has been detected and imaged [86].
The NPs appear to be spread throughout the cytoplasmic bridges that connect all
zebrafish blastomeres, and do not affect the development from 4h (40% epibobly
stage) to 7 days postfertilization. This is the first report on multiplex SERS imaging
of zebrafish embryos to study its localization during development in conjunction
with toxicity and gene expression studies. This research can be further applied to
track the fate of targeted NPs in dividing cells as they develop into tissues.
17.3 Cytosensing by Assembly of Nanomaterials 511

17.3.4Colorimetric Cytosensing by Assembly of Nanoparticles

Early and accurate detection of cancer often requires time-consuming techniques


and expensive instrumentation. The ability to rapidly identify rare and low-abundance
disease markers on the cell surface with simple procedures is a tremendous
challenge in clinical pathology. Colorimetric methods, in particular, are extremely
attractive for this purpose because they can be easily read with the naked eye, in
some cases at the point of use. The unique physical properties, particularly localized
surface plasmon resonance (LSPR), make gold NPs (Au NPs) attractive building
blocks for nanoscale signal transducers and/or signal amplifiers in a variety of colo-
rimetric platforms. The color change arises from the interparticle plasmon coupling
during Au NP assembly and disassembly [89].
Aptamer-functionalized Au NPs (ACGNP) are mostly used for colorimetric
sensing, where Au NPs act as the color indicators while aptamers act as functional
targeting molecules [34]. A direct colorimetric assay for direct detection of
diseased cells was developed by Medley et al. (Fig.17.16) [90]. Au NPs modi-
fied with aptamers specific for CCRF-CEM cells (CCL-119 T-cell, human acute
lymphoblastic leukemia) were incubated with target cells. The aptamer-directed
assembly of Au NPs on the membrane surfaces acted like a larger micrometer-
scaled gold structure, which interacted efficiently with the light and exhibited
significantly increased scattering and absorption coefficients compared to that of
individual Au NP due to the surface plasmon interaction. Thus, the target cells
were labeled with distinct color change, while nontarget samples did not elicit
any change in color. The assay was quite sensitive, since 1,000 target cells could
be readily detected by the naked eye, and the detection limit based on simple
absorbance measurements was 90 cells. In addition, the assay was able to dif-
ferentiate between different types of target and control cells based on the aptamer
used in the assay. It can be expected that the aptamer-conjugated gold NPs could
become a powerful tool for point-of-care diagnostics and large-scale cancer
screening.

17.3.5Electrochemical Cytosensing by Assembly


of Nanomaterials

Electrochemical biosensing technology, with intimately coupled biological


recognition elements and electrochemical transduction units, has been extensively
used to obtain quantitative or semiquantitative information concerning a large
number of analytes [91]. Electrochemical detection offers several advantages over
conventional fluorescence measurements, such as portability, higher performance
with lower background, less expensive components, and the ability to carry out
measurements in turbid samples [92]. In addition, its importance has been mani-
fested by eliminating or simplifying sample preparation steps. Because living cell
detection has a close relationship with life science research, clinical diagnostics,
512 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

Fig. 17.16 (a) Schematic representation of the ACGNP-based colorimetric assay; (b) plots
depicting the absorption spectra obtained for various samples analyzed using ACGNPs. The
spectra illustrate the differences in spectral characteristics observed after the ACGNPs bind to
the target cells. Reprinted with permission from Medley et al. [90]. 2008, American
Chemical Society

and public health protection, an emerging trend of electrochemical biosensor is


moving away from just sensing molecules towards interactions with, and sensing
of, whole cells [92].
There has been an explosion of interest in the use of various kinds of zero-, one-,
two-, and three-dimensional nanomaterials for electrochemical cytosensing. Examples
of such materials include semiconductor QDs, metallic NPs, CNTs, nanostructured
conductive polymers or nanocomposites thereof, and various other nanomaterials. The
significant roles of nanomaterials in cytosensing lie in the ability to address some of the
key issues, including design of the cell-compatible interface, facilitation of the electron
transfer in electrochemical reactions, achievement of efficient transduction of the
biorecognition event, increases in the sensitivity and selectivity, and improvement in
response times [92].
17.3 Cytosensing by Assembly of Nanomaterials 513

17.3.5.1Electrochemical Cytosensing Based on Cellular


Voltammetric Behaviors

A living cell can be properly described as an electrochemical dynamic system [93].


Electron generation and charge transfer exist in all living cells due to the redox
reactions and the changes of ionic composition and concentration in life processes
[94], which have been used to characterize the viability of cells in solution. The
voltammetric responses of the redox centers in living cells usually show irrevers-
ible electron-transfer processes, which is related to the oxidation of guanine
[95100]. The presence of nanomaterials on the electrode for cell immobilization
can both significantly reinforce the electrochemical response and maintain cell
viability [95100].
Jus group has developed a series of nanomaterials, such as Au NPs, multiwall
carbon nanotubes (MWNTs), and nanocomposites, to modify electrodes for the
electrochemical investigation of exogenous effects [96] and electrochemical antitu-
mor drug sensitivity test [98, 100]. For example, colloidal gold NPs were employed
to construct a nontoxic biomimetic interface for immobilization of AsPC-1 cells
(pancreatic adenocarcinoma cells derived from ascites) on a carbon paste electrode
surface, which provided an environment similar to a native system and allowed
more freedom in orientation of the biomolecules [101], thus efficiently retaining the
activity of living tumor cells and preventing cell leakage from the electrode inter-
face. The living AsPC-1 cells immobilized on colloidal gold NPs exhibited an irre-
versible voltammetric response, which was used to investigate the influence of
exogenous factors on physiological function of living cells, evaluate antitumor drug
effect, and differentiate between normal and disease cells [96].
In a similar study using an MWNT-modified glassy carbon electrode, a simple,
invitro, electrochemical antitumor drug sensitivity test was developed [98]. MWNTs
promoted electron transfer between the electroactive centers of cells and the
electrode. The cytotoxicity curves and results obtained corresponded well with the
results of MTT assays. Compared with conventional methods, the developed drug
sensitivity test exhibits good performance, such as high sensitivity, desirable
accuracy, low cost, and simplified procedures.
In order to further improve the electrochemical signal, some novel types of
nanocomposites have been fabricated by incorporating nanomaterials into biocom-
patible polymer matrices [97, 99, 100]. These hybrid materials, displaying superior
properties to both elements, have attracted increasing interest because of the
improvement of the stability and biocompatibility of nanomaterials and enhance-
ment of the capability for cell immobilization. Ideally, these polymeric materials
should be biocompatible and have reactive functional groups for further attachment
with biomolecules, for example, microporous cellulose and chitosan (CS).
Hao etal. designed a novel architecture by combining the biocompatibility of CS
with the excellent conductivity of carbon nanofiber (CNF) [97]. The controllable
electrodeposition of soluble CNF-doped CS colloidal solution formed a robust
CNF-CS nanocomposite film with good biocompatibility for the immobilization
and cytosensing of K562 cells on an electrode. The presence of CNF facilitated the
electrochemical behavior of K562 cells, which exhibited a well-defined anodic peak
514 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

of guanine. A nanocomposite gel was also prepared by neutralizing a designer


nanocomposite solution of CS-encapsulated Au NPs formed by reducing in situ
tetrachloroauric acid in CS [99]. The bio-inspired gel was designed for the
immobilization and electrochemical study of cells and for monitoring the adhesion,
proliferation, and apoptosis of cells on electrodes.

17.3.5.2Impedance Cytosensing by Assembly of Nanomaterials

During electrochemical impedance spectroscopic (EIS) measurements, a small


sinusoidal AC voltage probe (typically 210mV) is applied, and the current response
is determined. The in-phase current response determines the real (resistive) compo-
nent of the impedance, while the out-of-phase current response determines the
imaginary (capacitive) component [102]. EIS holds important advantages for appli-
cation in biosensing, especially for the sensitive and direct detection of binding
events, detection of cells in buffer solution, cellcell interactions, and cellmatrix
interactions [103]. This technique is inexpensive and relatively easy to perform in
comparison with surface plasmon resonance and quartz crystal microbalance tech-
niques. Theoretically, it can dynamically measure cellular movement at the nano-
scale with better resolution than conventional optical methods [104].
Due to the unique properties of nanomaterials, they have been widely used for
the fabrication of an EIS-based cytosensor to improve cell immobilization, retain
cell viability, increase the electrode surface area, and decrease the background
impedance, thus enhancing the detection sensitivity. Au NP-CS nanocomposite gel
[99], CNFCS film [97], and CNF doped polypyrrole (PPy) film [105] have been
reported by Jus group to be useful in composite substrates for impedance sensing
of cells. In these studies using EIS, the binding of K562 leukemia cells was
monitored as an increase in electron-transfer resistance (Rct). In the study using
Au NP-CS nanocomposite gel to monitor the adhesion of K562 leukemia cells, the
Rct was reported to correlate to the logarithm of the cell concentration over the
range 104108cells/mL, with a LOD of 8.7102cells/mL [99]. The nanocomposite
gel increased the sensitivity to the cell binding, which was attributed to increased
electrode surface area. Because the capture efficiency for cells was a major issue
associated with cellular EIS detection, optimal utilization of the functional surface
area (where attached cells are detected) of an electrode was thus of vital impor-
tance for detection [106]. Magnetic NPantibody conjugates (MNAC) could be
utilized to concentrate separated cells into a small volume, and further into the
active layer of interdigitated array microelectrode (IDAM), at which cells were
then detected [106].
The EIS measurement cannot only be used to detect cells in suspensions but also
changes on the electrode surface upon the proliferation of living tumor cells [99,
105]. As shown in Fig.17.17, a significant change of the diameter of the semicircu-
lar part of spectra, which represents the electron-transfer resistance, can be observed
when cells are proliferating on the modified surface. With the increase of culture
time, the electron-transfer resistance increases. After a culture time of 120 h, a
17.3 Cytosensing by Assembly of Nanomaterials 515

Fig.17.17 EIS
measurements of K562 cells
proliferated on Au NP-CS
gel/GCE after cell incubation
for (a) 24h, (b) 48h, (c)
72h, and (d) 96h. Inset:
relationship between
electron-transfer resistance
and proliferation time of
K562 cells on Au NP-CS
gel/GCE. Reprinted with
permission from Ding etal.
[99]. 2007, American
Chemical Society

d rastically increasing resistance is observed, which is related to the apoptosis of


cells. This electrochemical strategy is an effective and simple way for continuous
online monitoring of cell proliferation and apoptosis when disposable electrodes are
used. In a later report by Jus group, a novel nanoscaffold of CNFdoped PPy was
combined with disposable indium-tin oxide electrodes for the development of an
EIS-based cytosensor [105]. This highly conducting matrix provided a biocompat-
ible and robust substrate for the adhesion and subsequent proliferation of ECA-109
cells and thus led to method for cell immobilization and sensitive monitoring of cell
growth. This strategy offered advantages of simple and low-cost fabrication and
economic and convenient detection, and has potential application in cytological
study [107].
Although these EIS-based strategies have provided wide applications for cellular
assay and biological research, they all depend on conventional electrodes. Recently,
with the advances in nanotechnology, electrochemical impedance measurement
using nanoelectrodes has offered advantages such as reduced double-layer capaci-
tance, fast convergence to a steady-state signal, enhanced current density arising
from increased mass transport at the working electrode interface, low detection
limits, and improved S/N ratios [108]. In particular, ordered arrays of CNTs have
been studied rigorously [103].
Highly aligned multiwalled CNTs are synthesized in the shape of towers and
embedded into fluidic channels as electrodes for the impedance measurement of
LNCaP human prostate cancer cells [103]. As shown in Fig. 17.18, the tower
electrodes up to 8-mm high are grown and easily peel off a silicon substrate. The
nanotube electrodes are then successfully soldered onto patterned printed circuit
boards and cast into epoxy under pressure. Electrodeposition of Au particles on the
plasma-treated tower electrodes is done at a controlled density. Finally, the nanotube
electrodes are embedded into a polydimethylsiloxane (PDMS) channel and electro-
chemical impedance spectroscopy is carried out with different conditions. Preliminary
516 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

Fig.17.18 Schematic representation of a microfabricated flow cell with an adjustable gap elec-
trode. Reprinted with permission from Yan etal. [103]. 2007, IOP

electrochemical impedance spectroscopy results using deionized water, buffer


solution, and LNCaP prostate cancer cells show that nanotube electrodes can distin-
guish the different solutions and be used in future cell-based biosensor development.

17.3.5.3Neural Cytosensing by Assembly of Nanomaterials

In recent years, strategies for the diagnosis, therapy, and treatment of neural disor-
ders have increasingly relied on electrical stimulation techniques [109]. For the
effective transmission of charge from electrodes to cells, the interface for cell adhe-
sion must have a low electrical impedance [110]. Inert metals, including platinum
and iridium oxide, and organic conducting polymers have been widely explored for
electrodecellular interfaces. Nanostructuring such electrodes can improve perfor-
mance by reducing impedance and influencing cell compatibility. Beginning in
2006, nanostructured carbons, such as CNTs and graphene, emerged as novel elec-
trode structures, providing an inherently electrochemically stable organic interface.
CNT-based interfaces produce electrodes with high capacitance and low imped-
ance, which make them one of the most promising materials for applications in
neural biosensing [110]. Interactions between various cell lines and CNTs have
been previously reported and have included the adhesion, growth, and differentia-
tion of neuronal cells on CNT-based substrates [92]. Providing electrical stimula-
tion to neurons via a CNTbased platform has led to the formation of a functional
neural network of neural stem cells (NSCs).
17.4 Cell Surface Carbohydrate Assay by Assembly of Nanoparticles 517

Keefer etal. used CNTs to coat conventional tungsten and stainless steel wire
electrodes, leading to enhanced recording and electrical stimulation of neurons in
culture, rats, and monkeys [111]. Another significant advance is the work by Kam
et al., in which layer-by-layer-assembled composites from SWNTs and laminin
were fabricated and found to be conducive to NSC differentiation and suitable for
their successful excitation [109]. These results indicate that the proteinSWNT
composites can serve as the material foundation of neural electrodes with a chemi-
cal structure better adapted with long-term integration with the neural tissue.

17.4Cell Surface Carbohydrate Assay by Assembly


of Nanoparticles

Carbohydrates, a class of intricate and informative biomacromolecules, exist on all


eukaryotic cell surfaces. Glycosylation is one of the most common posttranscrip-
tional modifications in eukaryotes. Virtually every class of biomolecules, such as
proteins, lipids, tRNA, and many secondary metabolites, can be found in glycosy-
lated forms [112]. Glycans account for a large degree of cell surface structural varia-
tions and form complicated codes for cellular physiology, including protein folding,
trafficking, and stability, organ development, cellular adhesion, cell signaling,
immune response, and pathological processes [113115]. Dynamic changes in the
glycosylation status on carcinoma cell surfaces have been observed to play impor-
tant roles in oncogenic transformation, cell differentiation, and metastasis. Therefore,
sensitive analysis of carbohydrates on living cells in various events is keenly
desirable for basic science advancement and clinical diagnostics [116, 117].
Glycomics, which studies the structure and function of diverse glycans, is quickly
becoming a driving force for deciphering various cellular pathophysiological
processes and discovering potential biomarkers and therapeutic targets. Current
efforts for decoding cell surface glycosylation have focused on mass-spectrometric
methods [118]. Although this technique can provide molecular details, it is time-
consuming and not amenable to living cell interrogation due to the destructive
sample preparation methods. The use of lectins and antibodies with defined glycan
specificities [119] offers an alternative nondestructive tool to profile cell surface
glycans and correlate global changes in their expression with developmental stages
and disease [112]. These methods are mainly high-throughput lectin-array-based
microscopic approaches, which usually involve the fluorescent labeling of cells.
However, the issues of active-site accessibility and lectin denaturation in the surface
immobilization format with high density usually impair the sensitivity and stability of
this method. Moreover, the reproducibility of lectin array data needs to be improved,
and a reliable quality-control methodology is also being developed [120].
Nanomaterial-based approaches open an avenue for the sensitive and selective
analysis of carbohydrates on living cells. The applications of nanotechnology play
an important role in cell surface carbohydrate assay because of the capabilities for
tagging, signal amplification, integration of multiple functions, and effective cell
518 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

capture. Up to now, there have been mainly two types of nanomaterial-based


platforms in the field of cell surface carbohydrate sensing. One is engineered as
nanoscaffolds for the immobilization of lectins and cells under investigation, while
the other is designed as nanoprobes to combine the carbohydrate recognition, signal
transduction, and signal amplification abilities.

17.4.1Cell Surface Carbohydrate Assay Based on Nanomaterial


Substrates

Current development in cell surface carbohydrate-sensing strategies using nano-


material substrate is mostly performed through the combination with electro-
chemical detection. These methods can be categorized into two classes: One uses
nanomaterial-modified electrodes to capture cells and then detecting the signal
from the reporter molecules linked to the recognition element by voltammetry
[121, 122]; the other immobilizes the recognition element on electrodes and then
detects the cell-binding events by EIS [123, 124] or electrochemiluminescence
(ECL) [125].
The application of enzyme-linked lectin and nanomaterials for specific recogni-
tion of carbohydrates on electrode-captured cells provides enhanced sensitivity for
evaluation of the expression extent of carbohydrates, due to the dual signal amplifi-
cation of enzymes to produce abundant electroactive enzymatic catalysis product,
and the nanomaterials to increase the electrodes surface and accelerate electron
transfer. Based on such a scheme, an electrochemical cytosensing strategy was
designed to detect mannose groups on BGC cells based on the specific recognition
of integrin receptors on the cell surface to arginine-glycine-aspartic acid-serine
(RGDS)functionalized SWNTs [122]. The conjugated RGDS showed a predomi-
nant ability to capture cells on the electrode surface. The cell surface mannosyl
groups could specifically bind with horseradish peroxidase (HRP)-labeled conca-
navalin A (Con A), producing an electrochemical cytosensor. On the basis of the
dual signal amplification of SWNTs and enzymatic catalysis, the cytosensor could
respond down to 620cells/mL of BGC cells with a linear calibration range from
1.0103 to 1.0107cells/mL, showing a very high sensitivity. The cytosensor could
be further used to evaluate the mannosyl groups on the cell surface, and the man-
nosyl groups on a single living intact BGC cell were detected to correspond to
5.3107 molecules of mannose. This strategy presents a promising platform for the
highly sensitive cytosensing and convenient evaluation of surface carbohydrates on
living cells. Later, this work was further expanded for the development of electro-
chemical cytosensor array for dynamic analysis of carcinoma cell surface glycans
[121]. Combined with screen-printed carbon electrodes, the developed disposable
electrochemical cytosensor array can simultaneously analyze multiple glycans on
intact human leukemic K562 cell surfaces (see Fig.7.15), showing excellent perfor-
mance in sensitivity, stability, and practicality. The results reflected the expression
extent of different glycans on cell surfaces and suggested high expression of
(GlcNAc)2 and/or sialic acid, moderate expression of mannose, less Galb1-3GalNAc
17.4 Cell Surface Carbohydrate Assay by Assembly of Nanoparticles 519

and little GalNAc residues on the K562 cell surface, which were in good agreement
with flow-cytometric results. The strategy was further used for effective monitoring
of the dynamic variation of glycans on cancer cell surfaces during both drug induce-
ment and erythroid differentiation of K562 cells.
For cellular viability and bodily function, glycosylation are in the context of
glycoproteins, glycolipids, glycosylphosphatidylinositol anchors, glycosaminogly-
cans, and polysaccharides. The overproduction of certain glycoproteins is a common
feature of tumors. For example, an energy-dependent transport protein named
P-glycoprotein (P-gp), which is often overexpressed at the tumor cell, is closely
related to multidrug resistance phenomenon [126]. Accordingly, the ability to
characterize the cell surface glycoprotein expression status is critical to advance
chemotherapy of the malignant tumor.
A strategy to detect P-gp on K562/ADM cell and quantify the cell number has
been developed by Jus group using electrochemical immunoassay combined with
the immobilization of cells on a highly hydrophilic interface [127]. The interface
was constructed by adsorption of Au NPs on a methoxysilyl-terminated butyrylchi-
tosan modified glassy carbon electrode (Au-CS/GCE). The incubation with P-gp
monoclonal antibody and then the secondary alkaline phosphatase (AP)conjugated
antibody introduced AP onto the electrode-immobilized cells. The bound AP led to
an amperometric response of 1-naphthyl phosphate, which was proportional to the
logarithm of cell concentration in the range from 5.0104 to 1.0107 cells/mL,
with a detection limit of 1.0104 cells/mL. The results were comparable to the
flow-cytometric analysis of P-gp expression. Later, some groups expanded this
strategy to investigate the expression extent of P-gp on HeLa cells [126] and BGC
823 cells [128].
An alternative method for cell surface carbohydrate assay has been developed
based on the comparison of cell-binding extents to different lectin-modified
electrodes, which could be monitored conveniently by EIS and ECL. These strate-
gies obviate the cell immobilization step and cell labeling, and enable the in situ
investigation of cell surface glycan without disturbing cellular nature.
For example, label-free strategies for facile and specific electrochemical analysis
of cell surface glycan expression have been developed by combining rapid and
sensitive EIS measurement with single-walled carbon nanohorns (SWNHs) [123]
or MWNTs [124] and using lectins as recognition units. An effective three-
dimensional (3D) recognition interface toward cell surface glycan motifs was
constructed on glassy carbon electrode through stable immobilization of lectins
on uniquely sphere-structured SWNHs [123] and poly(diallyldimethylammonium)
functionalized MWNTs [124]. Using unprocessed mammalian living tumor cells
as a model, the glycan expression on cell surfaces was monitored through evalu-
ation of the cell-capturing ability of the recognition interface. When more of
certain glycans were expressed on the cell surface, a larger increase of Rct could
be obtained during EIS detection, resulting from more cells being captured by
the recognition interface.
In order to adopt the above strategy into ECL-based detection, the SWNHs were
replaced by CdSe QDs, which could act as ECL-emitting species and functionalized
by various kind of lectins [125]. These lectins included Con A, dolichos bifows
520 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

Fig.17.19 Schematic representation of ECL cytosensor for monitoring cell surface carbo-
hydrate expression. Reprinted with permission from Han etal. [125]. 2010, Royal Society
of Chemistry

agglutinin (DBA), peanut agglutinin (PNA), and wheat germ agglutinin (WGA).
As shown in Fig.17.19, the specific capture of cells on the modified electrode led to
a decrease in the ECL intensity, the amplitude of which could be used to investigate
the expression extent of carbohydrates. The above two approaches could both be
used for the sensitive dynamic monitoring of carbohydrate expression on living
cells in response to drugs.

17.4.2Cell Surface Carbohydrate Assay Based on Nanoprobes

NPs have been extensively employed to couple with biomolecules for the
development of biological nanoprobes [5, 129, 130]. The widespread use of these
hybrid bionanostructures in bioassays and diagnostics is due to an elegant combi-
nation of the superior physical and chemical features of nanomaterials with the
highly selective catalytic and recognition properties of biomolecules. Indeed,
these nanoprobes have been used to develop various sensing and imaging platforms
[131133] and have contributed significantly to applications of bioanalytical
technology in various areas, including medical diagnostics, environmental moni-
toring, and antiterrorism [134].
17.4 Cell Surface Carbohydrate Assay by Assembly of Nanoparticles 521

The development of nanoprobes suitable for carbohydrate sensing, especially in


situ cell surface carbohydrate monitoring, has emerged as an important area in
bioassays. Current nanoprobe-based sensing platforms include electrochemical
[135, 136], fluorescent [137139], flow cytometric [117], scanometric [140], ECL
[141], and scanning electrochemical microscopic (SECM) [142] methods. Among
these platforms, electrochemical methods have received increasing and consider-
able attention due to their operational simplicity, flexibility, low cost, and accept-
able sensitivity [143].

17.4.2.1Electrochemical Strategies

An earlier work for the fabrication of a nanostructure to recognize cell surface


glycans was performed by Chen et al. [144]. Glycopolymers decorated with a-N-
acetylgalactosamine (a-GalNAc) residues, which mimic cell surface mucin glyco-
proteins, were used to coat CNTs. The nanocomposite was further bound by Helix
pomatia agglutinin (HPA), a hexavalent lectin that is specific for a-GalNAc resi-
dues, and recognized by cell surface glycan through the available HPA-binding
sites. This work offered a strategy for interfacing biocompatible CNTs with cell
surfaces by virtue of carbohydratereceptor interactions and opened new opportuni-
ties for probing biological processes.
Different from the above-mentioned fabrication of cell surface mimics to
interface with the cell surface, Xue et al. prepared an electrochemical lectin-probe,
ferrocene-Con A, for in situ monitoring of cell surface glycan using the electroac-
tive ferrocenyl group to reflect the probe binding extent by the cells [145]. In order
to increase the detection sensitivity, a mannan carbohydrate monolayer was devel-
oped to compete with cell surface carbohydrate to recognize lectin-functionalized
QD probes [135]. The nanoprobes captured by the mannan monolayer could then be
detected by anodic stripping voltammetric technique (Fig.17.20). This first-reported
one moleculetwo surfaces competition format allowed in situ analysis of cell
surface mannose moieties. This work did not require cell labeling and overcame the
problems of active site inaccessibility and lectin denaturation, which are often
encountered in the surface immobilization of proteins with high density. The anodic
stripping analysis of QDs is a powerful electrochemical detection technique because
of its effective built-in preconcentration (deposition) step [14]. By combining the
signal amplification from stripping analysis with the competition format, this
method could reach the detection limit of 102 cells/mL, and the average Con
A-binding capacity of a single K562 cell could be estimated to correspond to
2.31010 mannose moieties.
Besides QDs, Au NPs are also widely used nanosubstrates for the fabrication of
nanoprobes [6]. A dual-functionalized nanoprobe has been designed for the highly
sensitive and selective in situ evaluation of carbohydrates on living cells by integrat-
ing the specific carbohydrate recognition and enzymatic signal amplification of
proteins on Au NPs [136]. A nanoscaffold of nanohorns functionalized with RGDS
tetrapeptide has also been prepared on an electrode surface for cell capture and
522 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

Fig. 17.20 Schematic representation of the monolayer fabrication and the competitive assay.
Reprinted with permission from Ding etal. [135]. 2008, American Chemical Society

enhancing the electrical connectivity. Through the combination of the nanoprobes


and peptide-nanohorns, a highly sensitive electrochemical strategy has been devel-
oped for cytosensing, which shows a detection limit down to 15 cells, broad dynamic
range, acceptable rapidity, and low cost. The proposed method can be further used
for monitoring dynamic variation of mannose expression on cancer cells in response
to drugs, using swainsonine (SW), a mannosidase II inhibitor, as the model.
Compared with other methods for monitoring dynamic glycan expression on cell
surface, this strategy offers the following advantages: (1) It obviates the need for
cell lysis and cell labeling; (2) the preparation of the nanoprobe does not involve
covalent coupling, so it can thus maintain the biological activity of proteins; (3) the
measurement of voltammetric peak current is more quantitative and facile than
microscopy-based observation; (4) the dual signal amplification coupled with
enhanced electrical connectivity provides high sensitivity; (5) this method also
obviates the need for complicated equipment, such as mass spectrometry and flow
cytometry, thus providing the opportunity for miniaturization.

17.4.2.2Fluorescent Strategies

To develop fluorescence-based nanoprobes, semiconductor QDs, which have broad


excitation and size-dependent photoluminescence with a narrow emission band-
width covering a wide spectral range, have been considered excellent candidates
[146]. On the other hand, magnetic NPs are also extensively studied and widely
17.4 Cell Surface Carbohydrate Assay by Assembly of Nanoparticles 523

used for drug delivery, magnetic treatment, and magnetic separation due to their
small size, single magnetic domain structures, and superparamagnetism [147].
However, in many biomedical and clinical diagnostic fields, there has always been
some need for materials with the functions of both magnetic separation and
fluorescence tracking.
A type of trifunctional nanospheres with excellent fluorescence, magnetism, and
recognition of cancer cells surface-expressed with sialic acid and N-acetylglucosamine
has been developed [138]. The nanoprobes are fabricated by the encapsulation of
QDs and nano-g-Fe2O3 in poly(styrene/acrylamide) copolymer and then biofunc-
tionalized by WGA. After incubating the nanoprobes with cells for 30min, the cells
captured by the nanoprobes can exhibit obvious orange fluorescence. The success in
capturing DU-145 cells by the nanoprobes has opened up a new field of visualizable
and recognizable manipulation and sorting of cells.
In a later work reported by the same group, different types of lectins WGA,
PNA, and DBA were used to functionalize fluorescent-magnetic nanospheres
[137]. The obtained nanoprobes could be used for qualitative and quantitative
analysis of the glycoconjugates on A549 cell surface. These lectin-modified
trifunctional nanoprobes not only could quantify the different glycoconjugates on
the A549 cell surface, but also could recognize and isolate A549 cells. Using 0.5mg
of WGA-modified fluorescent-magnetic trifunctional nanobiosensors allowed the
capture of 7.0104 A549 cells. Therefore, these nanoprobes might be applied in
mapping the glycoconjugates on cell surfaces and for recognition and isolation of
targeted cells.
The major drawbacks of fluorescence microscopic strategies for cell surface
glycan assay lie in the indirect quantification method and interference by autofluo-
rescence. To solve the problems, the mercaptopropionic acid (MPA)capped CdTe
QDs have been linked to Con A for the fabrication of mannose-specific nanoprobes
[139]. The prepared QDlectin nanoprobe can bind to cells in cell suspensions by
the specific recognition of Con A for cell surface mannosyl groups using leukemic
K562 cells as a model. After homogeneous incubation of cells with the nanoprobe
solution and subsequent removal of the nanoprobe-bound cells, the decrease in FI of
the nanoprobe solution is related to the cell amount and the expression extent of the
mannosyl groups on the K562 cells. This method has further been applied for
dynamically monitoring the change in carbohydrate expression on cancer cells in
the presence of a drug.

17.4.2.3Flow-Cytometric Strategies

Flow cytometry is a powerful technique for probing cell surface carbohydrate


expression using fluorescein-conjugated lectins for recognition. The optical proper-
ties of QDs let this fluorescent marker be introduced into ultrasensitive biological
detection using flow cytometry. Zhelev et al. developed water-soluble COOH-
functionalized CdSe QDs with plant-derived lectins for the identification of leuke-
mia cells from normal lymphocytes [117]. The application of QDlectin conjugates
524 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

Fig.17.21 Scheme of the scanometric strategy for in situ detection of mannose groups on living
cells. Reprinted with permission from Ding etal. [140]. 2010, American Chemical Society

for distinguishing cancer cells from normal ones was based on the ability of some
lectins to interact with specific target oligosaccharides on the cancer cell surface and
to recognize cancer cells from normal ones. These lectin characteristics made them
versatile primary detection reagents in the histochemical and flow-cytometric detec-
tion of cancer cells. The results demonstrate that QD-SBA and QD-DBA conjugates
are appropriate fluorescent markers for the identification of several leukemia cell
lines (especially Jurkat, MOLT-4, Raji, and Daudi cells).

17.4.2.4Scanometric Strategies

Au NP-based colorimetric assay is now of particular interest due to the simple


readout of signal from interparticle plasmon coupling. The scanometric technique
combines the advantage of colorimetric detection with the array format; it has
become an FDA-approved detection method and has spurred the development of
many related assays [148]. Here, the scanometric technology implies that the infor-
mation-recording and acquirement procedure is performed with a flat-bed scanner
[140]. This assay utilizes the catalytic properties of the NPs in a subsequent ampli-
fication event to affect the reduction of Ag+ in the presence of hydroquinone, which
simply needs to measure the grayscale value of the resulting silver spots to quantify
the amount of target present [149].
A convenient and label-free scanometric approach for in situ cell surface carbo-
hydrate assay was designed by integrating the bioconjugation and aggregation of
glyconanoparticles, silver signal amplification, and spot test [140]. As shown in
Fig. 17.21, in the presence of lectin, using Con A and mannose as a couple of
models, the glyconanoparticles exhibited fast aggregation. The aggregation process
could be inhibited by the specific recognition of lectin by the carbohydrate on the
cell surface. Combining the Au NP-catalyzed silver enhancement and scanometric
17.4 Cell Surface Carbohydrate Assay by Assembly of Nanoparticles 525

detection, the number of cell surface carbohydrate groups could be conveniently


read out. The average number of mannose units on a single living intact BGC cell
was detected to be (4.50.4)107. This largely noninstrumental method took the
advantages of an NP-based recognition and an aggregation regulated signal ampli-
fication, and avoided cell pretreatment and labeling processes. It possesses potential
applications in clinical diagnosis and in the elucidation of carbohydrate functions
on living cells.

17.4.2.5Electrochemiluminescent Strategies

Due to the low background noise and high sensitivity of the ECL technique, the
combination of nanoprobes with this technique can greatly improve in situ assay of
cell surface carbohydrate expression. A facile ECL strategy for label-free monitor-
ing of carbohydrate expression on living cells has been designed based on carbohy-
drate-functionalized CdS QDs/CNT nanocomposites, which act as ECL-emitting
species [141]. The ECL biosensor was fabricated by combining CNTs and mercap-
topropionic acidcapped CdS QDs via a layer-by-layer method, and mannan was
then coupled to QDs. The carbohydrate-functionalized CdS nanocomposites showed
high ECL sensitivity and good stability, and provided an effective 3D architecture
for Con A recognition, which resulted in a decrease in ECL intensity. When BGC
cells, whose surfaces have abundant mannose moieties, were used for competition
with the mannan-derivatized electrode to bind the Con A, the ECL intensity of the
electrode increased. The increase in magnitude depended on both the cell amount
and the expression level of cell surface carbohydrate. This ECL strategy for
evaluating cell surface carbohydrate expression was demonstrated to possess high
sensitivity, perfect stability, and acceptable reproducibility, and the average amount
of mannosyl groups on the cell surface could be obtained by this protocol. This
method obviated cell and lectin labeling; thus, the biological activity of cell and
protein could be maintained to the largest extent. Thus, this method could become a
powerful tool to decode the complex mechanisms underlying carbohydrate-related
biological processes.

17.4.2.6Scanning Electrochemical Microscopic Strategies

The above-mentioned methods only offer the information of average expression


level of a cell population and, thus, possibly enshroud the heterogeneity of single
cell, which might be indicative of disease in the early stage [150]. Moreover, these
methods require the usage of cell suspension, which are obtained by peeling cells
from the culture dish, leading to unexpected changes in the state of the cell surface
[151]. Thus, noninvasive and sensitive methods at the single-cell level are urgently
required for in situ detection of membrane glycans on adherent cells.
The SECM method is a scanning probe technique by which the localized chemical
reaction under near-physiological conditions can be quantitatively monitored in a
526 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

Fig. 17.22 (a) Scheme of SECM imaging for cell surface glycan expression. (b) Single-cell
SECM images in situ recorded in PBS containing ferrocenylmethanol (FMA) and H2O2 for BGC
cells after incubation with (a) HRP-WGA, (b) HRP-ConA, (c) HRP-PNA, and (d) HRP-DBA.
Reprinted with permission from Xue etal. [142]. 2010, American Chemical Society

noninvasive manner [152]. Therefore, it has been successfully used for real-time
evaluation of cell viability at the single-cell level by using oxygen as an endogenous
indicator [153]. The nanoscale height change of a single cell has been monitored in
a physiological environment with a novel Pt nanodisk electrode and a newly
designed step-approaching strategy. Most recently, the SECM method has been
employed for in situ electrochemical imaging of different surface glycan motifs on
adherent cells at the single-cell level using HRP-tagged lectins coupled with cell
micropatterning [142]. In this work, these adherent cells were first micropatterned
in the microwell of poly(dimethylsiloxane) membrane for precisely controlling the
localized surface interaction, and the membrane glycans were then specifically
17.5 Conclusions 527

r ecognized with corresponding lectins labeled with HRP. The glycan expression
level was obtained by detecting enzymatic catalysis product at the SECM tip. The
cell surface glycans could thus be in situ imaged by SECM at a single-cell level
without peeling the cells from the culture dish. Four types of membrane glycan
motifs showed statistically distinguishable expression levels (Fig.17.22). Thus, this
work afforded a promising protocol for the study of cell biology related to cell
surface carbohydrate expression of single living cells, and could be applied in cell
biology study based on cell surface carbohydrate expression.

17.5Conclusions

In this chapter, we comprehensively reviewed the current research regarding the use
of nanomaterials for applications of cytosensing and cell surface carbohydrate assay.
Various types of functionalized nanomaterials and several related techniques have
been discussed. NPs are on the threshold of widespread use and almost touch upon
every single modality of the cellular sensing arena. For many sensor applications,
NPs exhibit distinctive attributes that can increase the sensitivity and selectivity of
assays relative to conventional diagnostic techniques. Advanced nano-diagnostic
techniques have opened a promising avenue to provide rapid, low-cost, easy, and
multiplexed cytosensing strategies.
The further development of cytosensing will lie in the fabrication of novel
nanoprobes for both recognition and signal transduction. Surface functionalization
is the most essential and fundamental factor as far as cytosensing is concerned. The
newer conjugation strategies, such as a barnase and barstar noncovalent binding
system or a click chemistrybased covalent linker, will greatly accelerate the devel-
opment of this field. The optimization of the surface composition of nanomaterials
will also enable the development of efficient sensing strategies with the ability to
detect analytes in complex biological fluids like blood, urine, serum, and so forth.
Although tremendous success has been made, the concern of long-term toxicity of
nanomaterials is still an important issue to be addressed.
Moreover, there is tremendous incentive for developing technologies that detect
cancer at its earliest stages. In most cases, the detection of stage 1 cancers is associ-
ated with a greater than 90% 5-year survival rate. Conventional anatomic imaging
techniques typically detect cancers when they are a centimeter or greater in
diameter, at which point they already consist of more than 109 cells [154]. Thus,
cytosensing, especially cell surface assay, which can allow the sensitive and spe-
cific monitoring of the development of diseased cells and the expression of key
biomarkers on the cell surface, is expected to play an important role in biological,
medical, and clinical research. Given the remarkable parallel progress in nanotech-
nology, chemical biology, organic chemistry, and molecular biology, multifunc-
tional nanoprobes for both sensitive diagnostics and delivery of drugs without
systemic toxicity will bring unprecedented opportunities for the future of cancer
diagnosis and therapy.
528 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles

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Chapter 18
Nanobiosensing for Clinical Diagnosis

18.1Introduction

Technological platforms that provide the reliable, rapid, quantitative, cheap, and
high-throughput identification of biomolecules play a significant role in the clinical
deployment of personalized treatment [1]. A biosensor is a small device employing
biochemical molecular-recognition properties as the basis for a selective analysis
[2]. Three basic parts are involved in any biosensor system: biosensing, signal trans-
duction, and signal readout. The biosensing element is capable of recognizing the
presence, activity, or concentration of a specific analyte; it could be either a binding
process (affinity ligand-based biosensor with the recognition element of a protein,
peptide, DNA, RNA, whole cell, or tissue) or a biocatalytic reaction (enzyme-based
biosensor). Over the past decades, due to their advantages of specificity, speed,
portability, and low cost, we have witnessed a tremendous amount of activity in the
area of biosensors as well as their clinical applications [3], especially for cancer
diagnosis [410].
The early diagnosis of cancer is crucial for the successful treatment of the disease.
Highly sensitive methods are urgently needed for measuring cancer diagnosis markers
present at ultralow levels during early stages of the disease. Moreover, small, fast,
and high-throughput devices are also highly desired for replacing time-consuming
laboratory analyses to be able to screen large populations for signs of precancerous
developments or the presence of early malignant lesions. Such methods should
facilitate early detection and an adequate selection of treatment options and should
lead to increased patient survival rates. However, existing diagnostic biosensors are
neither sensitive enough to detect biomolecules at levels corresponding to advanced
stages of the disease nor integrated for the fast, automatic, and multiplexed detection
of diverse biomarkers [11].
The emergence of nanotechnology the creation and utilization of materials,
devices, and systems through the control of matter on the nanometer scale is open-
ing new horizons for the development of nanoprobes, nanosensors, and nanosystems

H. Ju et al., NanoBiosensing: Principles, Development and Application, 535


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0_18, Springer Science+Business Media, LLC 2011
536 18 Nanobiosensing for Clinical Diagnosis

with submicron-sized dimensions that are suitable for molecular diagnostics


[1216]. For example, nanomaterials offer improved biocompatibility, additional
binding sites, and higher signal intensity compared with traditional materials.
Hence, nanomaterial-based biosensors can markedly improve the sensitivity and
specificity of biomolecule detection. Nanofabrication allows for miniaturization of
the sensor, which improves the sensitivity and reduces the sample and reagent vol-
umes, making the detection process more efficient. Nanosystems integrating sen-
sors, fluidics, optics, and electronics on a chip are used for automatic biochemical
diagnostics. Nanoarrays and biochips, including miniaturized multi-analyte sensor
devices with high-throughput capabilities, help screening and guide treatment.
Nanodevices enable diagnosis at the single-cell and molecule levels.

18.2Nanotechnologies in Biosensing

18.2.1Nanomaterials for Biological Detection

To date, modern material science has reached a high degree of sophistication. A wide
variety of nanoscale materials with different sizes, shapes, and compositions are
now available [17]. Due to the novel properties of their small size (1100nm) and
correspondingly large surface-to-volume ratio, tailorable chemical and physical
properties, unusual target binding properties, and overall structural robustness,
nanomaterials are being gradually applied to biosensors for markedly enhancing the
sensitivity and specificity of detection [18, 19].

18.2.1.1Nanoparticles

Nanoparticles (NPs) are generally defined as isolable particles between 1 and 50nm
in size and are prevented from agglomerating by protective shells. In recent years,
NPs of different compositions and dimensions have been widely used as versatile
and sensitive tracers in a broad spectrum of highly innovative approaches for assays
of small molecules, proteins, and nucleic acid biomarkers [1926]. The most fre-
quently used NPs for biological detection are gold (Au) NPs, quantum dots (QDs),
and magnetic NPs.
Au NPs are particularly attractive labels for sensors because a variety of analytical
techniques can be used to detect them, including optical absorption, fluorescence,
Raman scattering, atomic and magnetic force, and electrical conductivity. The intro-
duction of Au NPs into some well-studied bioassays can lead to improved sensitivity
and specificity. For example, in a real-time DNA surface plasmon resonance detec-
tion system [27], a 1,000-fold lower detection limit is achieved with the use of an
Au NP-labeled oligonucleotide [28]. Moreover, the enormous signal enhancement
associated with the use of NP-amplifying labels and with the formation of
NPbiomolecule assemblies, such as bio-bar-code assay (Fig.18.1), provides the
18.2 Nanotechnologies in Biosensing 537

Fig.18.1 General bio-bar-code assay scheme. (a) Nanoparticle and magnetic microparticle probe
preparation; (b) nanoparticle-based PCR-less DNA amplification scheme. The target can be DNA,
RNA, or protein. Reprinted with permission from Nam etal. [32]. 2004, American Chemical
Society

basis for ultrasensitive optical and electrical detection with polymerase chain
reaction (PCR) or fluorescent taglike sensitivity [2934].
QDs are inorganic fluorophores. With advantages of high sensitivity, broad exci-
tation spectra, sharp emission spectra, easily tunable emission properties, and no
need for lasers, QDs have been used as possible alternatives to the conventional fluo-
rescent markers for molecular diagnostics and genotyping. A major challenge is that
QDs have an oily surface, whereas the cellular environment is water-based. Attempts
are being made to produce biocompatible water-soluble QDs so that they can act as
538 18 Nanobiosensing for Clinical Diagnosis

molecule-sized light-emitting diodes and can be used as probes to track antibodies,


viruses, proteins, and DNA within the human body [35, 36]. Biomolecules labeled
with luminescent QDs have various applications to fluorescent immunoassays and
biological imaging [3539]. Because of their small size, QDs can be used to visualize,
measure, and track individual molecular events using fluorescence techniques. They
also have a unique ability to visualize and track dynamic molecular processes over
long time scales [40]. QDs can be designed to emit light at any wavelength from
infrared to visible to ultraviolet. This enables the use of a large number of colors, and
thus multiplexed assays can be performed [41]. However, QDs have the problem of
toxic effects, which prevent them from being used in invivo applications.
Magnetic NPs are a powerful and versatile diagnostic tool in biology and medicine.
Upon aggregation, magnetic NPs can act as magnetic relaxation switches by dephasing
the spin of the proton in the surrounding water, resulting in an enhancement of the
T2 relaxation time. This phenomenon has been exploited for the detection of DNA,
proteins, and viruses [42, 43]. Based on hybridization of magnetic NPs to telom-
erase-associated TTAGGG repeats, a magnetic NP-based nanosensor has been
developed for rapid screening of telomerase activity in biological samples [44].
High-throughput adaptation of the technique by magnetic resonance imaging allows
processing of hundreds of samples within tens of minutes at an ultrahigh sensitivity.
These studies establish and validate a novel and powerful tool for invivo and patient
sample diagnostics because the magnetic signal is not affected by the turbidity of
the clinical sample.

18.2.1.2Nanotubes

Carbon nanotubes (CNTs) are particularly exciting one-dimensional nanomaterials.


Due to their unique structure-dependent electronic and mechanical properties, CNTs
have generated considerable interest for biological detections [18, 19]. For example,
the surface-confined CNTs can facilitate the adsorptive accumulation of the guanine
nucleobase and greatly enhance its oxidation signal. Wang et al. exploited this
phenomenon by showing that label-free electrochemical detection of DNA could be
performed by CNTmodified electrodes at nanomolar concentrations [45]. Recent
works have utilized CNTs coated with enzymes as labels for amplified biological
detection [4649]. These schemes can detect target DNA and proteins at concentra-
tions as low as 5.4 aM and 67 aM, respectively [46]. Furthermore, CNT are being
explored as new signal-transduction motifs in electrical biological detections.
Woolley et al. implemented a method for multiplexed detection of polymorphic
sites and direct determination of haplotypes in 10-kb DNA fragments using single-
walled carbon nanotubes (SWCNTs) as atomic force microscopy (AFM) probes
(Fig. 18.2) [50]. Specifically, they marked particular sequences along the DNA
strand with streptavidin-labeled complementary DNA probes, then used AFM to
identify the streptavidin, and the positions of the tagged sequences were detected by
direct SWCNT tip imaging. Thistechnique enabled the detection of specific haplo-
types that code for genetic disorders.
18.2 Nanotechnologies in Biosensing 539

Fig.18.2 Schematic illustration of the method for labeling specific DNA sites and detection with
SWNT AFM probes. Labeled DNA molecules are deposited on freshly cleaved mica and imaged
by AFM using SWNT probes. The presence and locations of the sequence-specific tags ( and )
can be readily observed in the AFM image. Reprinted with permission from Woolley etal. [50].
2000, Nature

CNTs have also been used to develop highly specific electronic biomolecule
detectors for investigating surfaceprotein and proteinprotein binding [51].
Nonspecific binding on nanotubes, a phenomenon found with a wide range of proteins,
is overcome by immobilization of polyethylene oxide chains. A general approach is
then advanced to enable the selective recognition and binding of target proteins by
conjugation of their specific receptors to polyethylene oxide-functionalized nano-
tubes. These arrays are attractive because no labeling is required and the entire
assay can be done in the solution phase. This scheme, combined with the sensitivity
of nanotube electronic devices, enables highly specific electronic sensors for detecting
clinically important biomolecules such as antibodies associated with human auto-
immune diseases.

18.2.1.3Nanowires

Another type of attractive one-dimensional nanostructures for biological detection


is semiconductor or conducting polymer nanowires. Metal and conducting polymer
nanowires can be readily prepared by a template-directed electrochemical synthesis
involving electrodeposition into the cylindrical pores of a membrane template [52].
Because of their high surface-to-volume ratio and novel electron-transport properties,
the electronic conductance of nanowires can be strongly influenced by minor surface
perturbations (such as those associated with the binding of macromolecules). Such
nanowires thus offer the prospect of rapid (real-time) and sensitive label-free
bioelectronic detection and a massive redundancy in nanosensor arrays [5356].
The extreme dimensions of these nanomaterials allow a huge number of sensing
elements to be packed onto a small footprint of an array device. The tailored
electronic conductivity of conducting polymers, coupled with their ease of processing/
modification and rich chemistry, makes them extremely attractive as one-dimensional
540 18 Nanobiosensing for Clinical Diagnosis

Fig.18.3 Self-electrophoresis (bipolar electrochemical) mechanism for the propulsion of catalytic


nanowire motors in the presence of hydrogen peroxide. The mechanism involves an internal
electron flow from one end to the other end of the nanowire, along with migration of protons in the
double layer surrounding the wires. Reprinted with permission from Wang [60]. 2009, American
Chemical Society

sensing nanomaterials [57, 58]. For example, newly introduced CNTs/conducting


polymer nanowires, based on incorporating oxidized CNTs as the charge-balancing
dopants within electro-polymerized wires, further enhance the sensing capabilities
of nanowire materials [59].
Bi-segment nanowires, such as bimetal (e.g., Au/Pt) catalytic nanowires, are also
promising materials for creating synthetic nanomachines due to their autonomous
propulsion in the presence of hydrogen peroxide fuel [60], and their practical realiza-
tion as a bioanalytical tool is in development [61]. Such fuel-driven bimetal catalytic
nanowire motors are prepared by template-directed electro-deposition within the
cylindrical nanopores of a porous membrane followed by the template removal. This
template-assisted electrochemical synthesis allows the convenient preparation of
multisegment nanowires of a variety of sizes or compositions. The sequential deposi-
tion of the platinum and gold segments thus leads to asymmetric nanowires with
spatially defined catalytic zones. Such asymmetry is essential for generating a
directional force. The resulting nanomotors are propelled by electrocatalytic decom-
position of the hydrogen peroxide fuel (on both ends of the wire), with oxidation of
the peroxide fuel occurring at the platinum anode and its reduction to water on the
gold cathode. Various mechanisms have been proposed for the self-propulsion of
bimetallic catalytic nanomotors, the most accepted one being an electrokinetic self-
electrophoresis [62]. This mechanism suggests that in addition to the hydrogen
peroxide reduction, the cathodic reaction on the gold segment also involves the four-
electron reduction of oxygen to water. These cathodic reactions, along with the
oxidation of the peroxide fuel at the platinum segment, result in an electron flux
within the wire (toward the gold cathode) and the generation of an electric field
(Fig.18.3). These lead to electro-migration of protons in the electrical double layer
(surrounding the nanowire) from the platinum end to the gold end, and hence the
self-electrophoresis and propulsion of the nanomotors in a random autonomous non-
Brownian movement at speeds around 1015mm/s toward their platinum end [63, 64].
Recently, Wangs group discovered an unusual increase in the speed of catalytic
nanowire motors in the presence of silver ions [65]. Based on this strategy, they
18.2 Nanotechnologies in Biosensing 541

Fig.18.4 Motion-based nucleic acid detection. (a) Hybridization of the target and capture of the
Ag nanoparticletagged detector probe in a typical sandwich assay on the ternary SH-CP/
DTT+MCH surface, including washing of unbound SHDPAg NPs. (b) Dissolution of silver
nanoparticle tags in the peroxide fuel, leading to Ag+-enriched fuel. (c) Visual detection of the
motion of the catalytic nanowire motors in the resulting Ag+-enriched fuel. C1, C2, and C3 repre-
sent hypothetical and increasing target nucleic acid concentrations. Reprinted with permission
from Wu etal. [61]. 2010, Nature

further presented a fundamentally new nanomotor-based biodetection platform for


specific DNA and bacterial ribosomal RNA detection (Fig.18.4). The motion-based
hybridization sandwich assay relied on the duplex formation of the nucleic acid
target with a thiolated DNA capture probe and a silver nanoparticletagged detector
probe. Subsequent dissolution of the Ag nanoparticle tags in the hydrogen peroxide
fuel released Ag ions, which caused a substantial increase in the nanomotors speed.
The higher the concentration of the nucleic acid target, the more silver nanoparticles
that were captured, and the greater the nanomotor speed. The resulting distance
signals allowed convenient measurements of the DNA target down to the attomole
level. This new nanomotor concept may be readily expanded to the detection of a
broad range of target biomolecules in connection with different biomolecular inter-
actions and motion-transduction principles. Such motion-driven biosensing repre-
sents a new paradigm in bioanalysis, and the use of artificial nanomachines addresses
the limitations of using biological motors, including a limited lifetime invitro and a
narrow functioning range of environmental conditions [66].
542 18 Nanobiosensing for Clinical Diagnosis

18.2.1.4Dendrimers

Dendrimers are a novel class of three-dimensional, nanoscale, and core-shell struc-


tures that can be synthesized for a wide range of applications. Specialized chemistry
techniques allow precise control over the physical and chemical properties of the den-
drimers. The techniques are especially useful in drug delivery but can also be used for
the development of new pharmaceuticals with novel activities. For example, dendrim-
ers can be conjugated to different biofunctional moieties, such as folic acid using
cDNA oligonucleotides, to produce clustered molecules targeting cancer cells that
overexpress the high-affinity folate receptor [67, 68]. This DNA-linked dendrimer nano-
cluster platform is considered to allow the delivery of drugs, genetic materials, and
imaging agents to cancer cells, offering the potential for developing combination ther-
apeutics. Polyamidoamine dendrimers can be used to modify magnetic nanoparticles
(MNP) and to form antisense survivin oligodeoxynucleotidedendrimerMNP com-
posites [69]. The results show that such a dendrimer-modified MNP can enter into
tumor cells within 15min, cause obvious downregulation of the survivin gene and
protein, and inhibit cell growth in dose- and time-dependent means.
Dendrimers have been successfully incorporated into DNA assays in efforts to
improve upon or replace the molecular fluorophore-based assay. Wang et al.
employed dendrimers possessing about 30 single-stranded arms specific to the
waterborne pathogen Cryptosporidium parvum, to modify the surface of mass-
sensitive piezoelectric transducers [70]. The immobilized dendrimers, with the
numerous probes on the outermost layer accessible to the Cryptosporidium DNA
target, yielded three-dimensional surface hybridization and, consequently, increased
the hybridization capacity and detection capability and directly monitored (without
an indicator) the kinetics of hybridization. Specifically, dendrimers have primarily
been used as a means to increase the number of labels associated with each target-
binding event [71]. Using a dendrimer probe that contains approximately 250 fluo-
rophores instead of a conventional molecular fluorophore probe, the fluorescent
assay sensitivity increases by a factor of ~16. However, the dendrimer introduces an
additional level of synthetic complexity to the assay, which might negate the
improvement in sensitivity.

18.2.1.5Fullerenes

The unique feature of fullerene molecules is that they have numerous points of
attachment, allowing for the precise grafting of active chemical groups in three-
dimensional orientations. This attribute, the hallmark of rational drug design, allows
positional control in matching fullerene compounds to biological targets. Together
with other attributes, such as the size of the fullerene molecules, the redox potential,
and the relative inertness in biological systems, it is possible to tailor requisite phar-
macokinetic characteristics to fullerene-based compounds and to optimize their
therapeutic effect [72].
18.2 Nanotechnologies in Biosensing 543

Fullerene antioxidants bind and inactivate multiple, circulating, intracellular free


radicals. This binding gives them unusual power to stop free-radical injury and to halt
the progression of diseases caused by excess free-radical production. C60 fullerene has
30 conjugated carboncarbon double bonds; all of them can react with a radical spe-
cies. In addition, fullerene forms a bond with a radical every time it encounters one.
Numerous studies demonstrate that fullerene antioxidants work much better as thera-
peutics than other natural and synthetic antioxidants do, at least for CNS-degenerative
diseases. In oxidative injury or disease, fullerene antioxidants enter cells and modu-
late free-radical levels, thereby substantially reducing or preventing permanent cell
injury and cell death [73]. The mechanism of action of fullerenes can be described as
follows: (1) Fullerenes can capture multiple electrons derived from oxygen free radi-
cals in unoccupied orbitals; (2) when an attacking radical forms a bond with fullerene,
it creates a stable and relatively nonreactive fullerene radical; (3) a tris-malonic acid
derivative of the fullerene C60 molecule (C3) is capable of removing the biologically
important superoxide radical; (4) C3 localizes to mitochondria, suggesting that C3
functionally replaces manganese superoxide dismutase (SOD), acting as a biologically
effective SOD mimic [73, 74].
Fullerenes have potential applications in the treatment of diseases in which
oxidative stress plays a role in the pathogenesis, such as neurodegenerative diseases.
For example, the malonic acid C-60 derivatives (carboxyfullerenes) are capable of
eliminating both superoxide anion and H2O2 and are effective inhibitors of lipid
peroxidation. Additionally, carboxyfullerenes demonstrate robust neuroprotection
against excitotoxic, apoptotic, and metabolic insults in cortical cell cultures [75].
They are also capable of rescuing mesencephalic dopaminergic neurons from both
MPP+ and 6-hydroxydopamine-induced degeneration. The results from animal
models indicate that systemic administration of the C-3 carboxyfullerene isomer
delays motor deterioration and death in a mouse model of familial amyotrophic
lateral sclerosis.
Another possible application of fullerenes is in nuclear medicine, as an alternative
to chelating compounds that prevent the direct binding of toxic metal ions to serum
components. This application can increase the therapeutic potency of radiation
treatments and decrease their adverse effects, because fullerenes are resistant to
biochemical degradation within the body [74].

18.2.1.6Nanobiological Molecules

Nanobodies are the smallest fragments of naturally occurring heavy-chain antibodies


that have evolved to be fully functional in the absence of a light chain. Heavy-
chain-only antibodies have recently been discovered in the blood of camelids.
These heavy-chain antibodies contain a single variable domain (VHH) and two
constant domains (CH2 and CH3). Importantly, the cloned and isolated VHH
domain is a perfectly stable polypeptide harboring the full antigen-binding capacity
of the original heavy-chain antibody.
544 18 Nanobiosensing for Clinical Diagnosis

These antibody fragments have several remarkable features. For example, they
are small and stable and can bind antigen with nanomolar affinity. They have high
target specificity and low inherent toxicity. They can address therapeutic targets not
easily recognized by conventional antibodies (e.g., active sites of enzyme). They
have an extremely low immunogenic potential. Particularly appealing is their ability
to simultaneously inhibit various crucial growth-factor receptors or their ligands
with a single molecule. In addition, they are easy to clone and express on the tip of
filamentous phage, which opens the possibility to select for nanobodies inducing
particular biological effects. The unique and well-characterized properties enable
nanobodies to excel as conventional therapeutic antibodies, making them ideal can-
didates as next-generation cancer therapeutics and for use in diagnostics, for dis-
eases such as cancer [74, 7678]. It is reported that the successful use of untagged
nanobodies for the in vivo treatment of solid tumors [79]. Based on functional
phage-antibody selection using competitive elution with the ligand epidermal
growth factor (EGF), antagonistic anti-EGFR nanobodies for cancer therapy have
been developed. The selected antibody fragments are found to efficiently inhibit
EGF binding to the EGF receptor without acting as receptor agonists themselves. In
addition, they can also block EGF-mediated signaling and EGF-induced cell prolif-
eration. These nanobodies are effective in delaying the outgrowth of A431-derived
solid tumors in an invivo model for solid tumors.

18.2.2Nanofabrication

The growing interest in the development of new biosensing and high-throughput


screening technologies has been largely driven for biotechnology applications. For
miniaturization and integration, appropriately designed nanometric systems should
be developed and fabricated for high-sensitivity, homogeneous assays.

18.2.2.1Nanoarrays

Currently, the microarray technologies are of great interest for providing a platform
for multiplexed DNA and protein detection in small areas with small amounts of
sample and reagent, hence offering a variety of applications in biomedical diagnostics
and proteomics. Nanoarrays are the next stage in the evolution of the miniaturization
of microarrays because they would allow for orders of magnitude more massively
paralleled multiplexed detection in the same array area as a microarray and poten-
tially improved detection limits resulting from the smaller analyte capture area [19, 80].
Various methods have been developed to fabricate nanoarrays, such as dip-pen
nanolithography (DPN) [81], nanografting [82], scanning probe lithography [83],
and other lithography strategies.
DPN is a direct-write scanning probe-based lithography in which an AFM tip is
used to deliver chemical reagents directly to nanoscopic regions of a target substrate.
18.2 Nanotechnologies in Biosensing 545

Fig.18.5 Direct patterning of multiple-DNA inks by DPN. (a) Combined red-green epifluores-
cence image of two different fluorophore-labeled sequences (Oregon Green 488-X and Texas Red-
X) simultaneously hybridized to a two-sequence array deposited on an SiOx substrate by DPN. (b)
Tapping-mode AFM image of 5- (dark) and 13- (light) nm-diameter gold nanoparticles (Au NPs)
assembled on the same pattern after dehybridization of the fluorophore-labeled DNA. Reprinted
with permission from Demers etal. [84]. 2002, Science

Demers et al. has used direct-write DPN to fabricate oligonucleotide nanoarrays on


both metal and insulating surfaces [84]. Such DNA nanoarrays can recognize both
fluorophore-labeled complementary target DNA as well as DNA-functionalized Au
NPs (Fig.18.5). In the case of fluorophore labeling, the presence of the target can
be detected with a fluorescence microscope; and for nanoparticle labeling, the tar-
gets presence can be monitored using AFM. Nanoparticle labels are particularly
promising tags for nanoscale detection applications. They exhibit a wide variety of
properties, from their high molar extinction coefficients to their physical shapes,
which can be used to encode information. As shown in Fig.18.5b, nanoparticle size
can conceivably be used as a tag in an AFM-based screening procedure, which is
much simpler than using different-color fluorophores in optical screening. DPN has
also enabled the creation of protein nanoarrays, for example, protein arrays of rabbit
IgG and lysozyme [85]. IgG has been arrayed indirectly by patterning 16-mercapto-
hexadecanoic acid on a gold substrate. This strategy has been used in a sandwich
immunoassay for detection of human immunodeficiency virus type 1 (HIV-1)
(Fig. 18.6) [86]. First, DPN is used to generate nanoscale patterns of antibodies
against the HIV-1 p24 antigen on gold surface. HIV-1 p24 antigen in plasma obtained
directly from HIV-1-infected patients is hybridized to the antibody array in situ. To
amplify the signal, the nanoarray is rinsed with anti-p24-modified Au NPs, which
are bound to the spots only when p24 is present and increase the height of the spots.
The nanoarray features in the three-component sandwich assay can be confirmed by
AFM. In this assay, only 1mL of sample is required, which is critical in cases where
sample volumes are small and limited. The detection limit for p24 using this assay
is 0.025pg/mL, which is much better than conventional ELISAs (5pg/mL).
546 18 Nanobiosensing for Clinical Diagnosis

Fig.18.6 Schematic representation of the sandwich immunoassay format used to detect HIV-1 p24
antigen with an anti-p24 antibody nanoarray made by DPN. The HIV-1 p24 antigen was sand-
wiched between anti-p24 antibody bound to the MHA patterned surface and Au NP probes coated
with anti-p24 antibody. The change in height due to the nanoparticle binding event could be detected
by AFM. Reprinted with permission from Lee etal. [86]. 2004, American Chemical Society

18.2.2.2Nanopore Technology

Nanopore sensors, whose ionic conductivity can be diminished by the passage of


target molecules, can transduce the passage of a single macromolecule into a discrete
electrical signal. Remarkable progress has been made in the study of the electrical
ionic conduction signals from voltage-biased nanoscale biopores [87, 88]. For
example, it can distinguish between hybridized or unhybridized unknown RNA and
DNA molecules that differ only by a single nucleotide (Fig.18.7) [88]. Each biosensor
element consists of an individual DNA oligonucleotide covalently attached within
the lumen of the a-hemolysin pore to form a DNA-nanopore. The binding of
single-stranded DNA (ssDNA) molecules to the tethered DNA strand causes changes
in the ionic current flowing through a nanopore. On the basis of DNA duplex life-
times, the DNA nanopores are able to discriminate between individual DNA strands
up to 30 nucleotides in length differing by a single base substitution. This can be
illustrated by the detection of a drug resistance-conferring mutation in the reverse-
transcriptase gene of HIV. In addition, the approach can be used to sequence a
complete codon in an individual DNA strand tethered to a nanopore.
18.2 Nanotechnologies in Biosensing 547

Fig.18.7 A single-base mismatch abolishes the binding of an oligonucleotide to a tethered DNA


strand within a DNA nanopore. (a) Representative single-channel current trace of H6(17C-oligo-1)1
interacting with 267nM oligo-2G, a 7-mer of complementary sequence. Negative current deflec-
tions (b) are individual binding events of oligo-2G (black) to the tethered oligo-1 (blue). The short
downward spike (s) in the trace is a translocation event of oligo-2G that did not bind to the tethered
oligonucleotide. (b) Representative trace of the same channel as in (a) after perfusion of the cis
chamber and the addition of 267nM oligo-2C (black with a red peak). Only short translocation
events (s) were observed. Reprinted with permission from Howorka etal. [88]. 2001, Nature

Nanopore biosensors can enable direct, microsecond-time scale nucleic acid


characterization without the need for amplification, chemical modification, surface
adsorption, or the binding of probes. However, this system has limits for studies of
biological molecules because the pore is of a fixed size, and its stability and noise
characteristics are restricted by chemical, mechanical, electrical, and thermal con-
straints. A nanometer-scale pore in a solid-state membrane provides a new way of
electronically probing the structure of single linear polymers. Golovchenkos group
developed a solid-state nanopore microscope for electronically characterizing
single long-chain polymers such as DNA molecules [89]. The microscope consisted
of a voltage-biased nanopore, fabricated in a silicon nitride membrane by ion-beam
sculpting technology. The membrane separated two chambers of conducting elec-
trolyte solution. The only electrical conduction path from one chamber to the other
passed through the nanopore. To resolve the interesting molecular structure, the
nanopore dimensions must be small enough to avoid averaging over continuous
single-molecule configurations induced by thermal fluctuations and large enough to
pass the smallest dimensions of the molecule to be probed. The count of single
nucleotides is accomplished by registering in time the changes in electrical resis-
tance of the nanopores.
The development of material science also provides a significant number of new
substrates that offer potential platforms for very high-density nanopore arrays. One
of the most promising of these substrates is anodically etched alumina. Martin and
coworkers recently used the alumina nanopore membrane as a mask by overlaying it
on a polymer during a plasma etching process [90]. After removing the alumina
mask, a regular array of nanopores was created. These alumina membranes could
also be etched to produce an array of conical nanopores, which have been used as
548 18 Nanobiosensing for Clinical Diagnosis

synthetic resistive-pulse sensors for the stochastic measurement of biomedical


analytes [91]. As the fabrication of these conical pores has becomes more reproducible,
the use of artificial-nanopore biosensors will become more widespread.

18.2.2.3Nanofluidic Channels

Future molecular diagnostic tools should possess the ability to separate, isolate, and
investigate a small number of molecules in a nanoscale-confined environment. With
recent advances in nanoscience and nanotechnology, the use of nanoscale channels in
place of microfluidic channels for fluid flow have brought this goal closer to reality.
Nanofluidic systems enable the study of physical principles, such as kinetic profiling,
and biomolecular interactions in a controllable fluidic environment at the nanoscale.
In addition, nanofluidic channels are important elements for lab-on-a-chip devices,
which have promise for achieving absolute single-molecule sensitivity combined with
high selectivity along with the advantages of high throughput, low access times, and
extremely small sample volumes (pL to fL range). Nanofluidic devices allow for many
applications, including biomolecule separation, concentration, reaction/hybridization,
sequencing (in the case of DNA), and detection [92]. At the time of this writing, the
main application of nanofluidic devices is biomolecular separation, which can occur
at the nanoscale due to many different phenomena, including steric, hydrodynamic,
entropic, electrical, and ratcheting. Craigheads group designed a nanofluidic channel
device, consisting of many entropic traps, for the separation of long DNA molecules
[93]. The channel comprises narrow constrictions and wider regions that cause size-
dependent trapping of DNA at the onset of a constriction. This process creates elec-
trophoretic mobility differences, thus enabling the efficient separation of long DNA
molecules (5,000 to ~160,000 base pairs) in 15-mm-long channels. This device can be
modified to separate smaller or larger DNA molecules as well as various proteins and
other polymers. Moreover, multiple-channel devices, integrating several separation
channels optimized for different length ranges of DNA in parallel, can realize single-run
sorting and analysis of various DNA samples. Recently, a simple nanofluidic device
was developed to perform free-solution hybridization and separation of single-stranded
and double-stranded oligonucleotides [94]. The device can identify target DNA
sequences and quantitatively measure hybridization kinetics through the electrokinetic
separation of hybridized and unhybridized probe DNA in a nanochannel. In this
device, on-chip fluid control is achieved using only electric fields, no sample labeling
is required, and no sieving matrix is needed. These characteristics make the device
well suited for integration into microfabricated lab-on-a-chip systems.

18.2.3Nanodevices

Next-generation biosensor platforms will be required to meet the needs of a variety


of fields ranging from invitro molecular diagnostics, pharmaceutical discovery, to
18.2 Nanotechnologies in Biosensing 549

pathogen detection. Nanobiosensors are nanosensors used for the selective detection
of biological materials. Nanobiosensors adopt inexpensive low-voltage measurement
schemes for the direct detection of a binding event and can be electronically gated
to respond the single molecule-binding event. There is no need for costly, compli-
cated, and time-consuming labeling chemistries such as fluorescent dyes or the use
of bulky and expensive optical detection systems. Nanobiosensors provide great
possible to develop implantable detection and monitoring devices.

18.2.3.1Electrical Nanobiosensors

Electrical transducers, in which electrical resistance (conductance) or capacitance is


measured in relation to a biological recognition event, have been investigated
because of their compatibility with advanced semiconductor technology and large-
scale reproducible fabrication and miniaturization. Initial studies of electrical bio-
sensors included the use of the microelectrode gap. By biological binding events,
the gold colloids were localized to the gap between the two microelectrodes, and a
subsequent silver enhancement results in a conductive metal layer across the gap,
leading to a measurable conductivity signal [95]. Very recently, a nanogap biosensor,
consisting of two electron-conducting electrodes separated by an insulating gap/
layer of nanometer dimension, is explored for highly sensitive and selective detection
of biological species [9698].
A nano-gapped microelectrode-based biosensor array is fabricated for the ultra-
sensitive electrical detection of microRNAs (a class of small, nonprotein-coding
RNA molecules that play roles in gene expression regulation in a broad range of
plants, viruses, and mammals) [96]. Based on the in situ hybrid-templated and enzy-
matically catalyzed formation of electrically conducting polyaniline nanowires, this
strategy could detect the target miRNA as low as 5fM. Recently, a unique nanoelec-
tronic platform, based on SWCNTs, has been fabricated for measuring electrical
transport in single-molecule DNA [97]. A total of 80 base pairs of single- and double-
stranded DNA (ssDNA and dsDNA, respectively) of complex base sequences was
tested. This work exploited a true dimensional compatibility between the SWCNT
electrode (diameter ~1.2nm) and the DNA double helix (diameter ~2nm), with the
argument that steric hindrance would prevent more than one DNA molecule from
bridging the gap between the SWCNT electrodes, ensuring that the electrical signal
solely originates from a single molecule. Based on the estimated length of the DNA-
capture probe, a nanogap of 27nm was created at the center of a single SWCNT,
electrically addressable by Ti/Au electrodes deposited at its ends (Fig.18.8a). In the
absence of base-pair stacking, one ssDNA carries a feeble current of similar to 1pA
or less (Fig.18.8b). In contrast, when a dsDNA molecule covalently attached to the
SWNT electrode, a current about 2540pA (at 1V) was measured (Fig.18.8c).
A nanogap-based, high-frequency (1.28GHz) impedance biosensor with nanometer
gaps has been prepared for the detection of biomolecular interactions such as protein
antibody and proteinaptamer binding [98]. The measurements rely on changes in
the electrical impedance due to changes in the effective dielectric constant within
550 18 Nanobiosensing for Clinical Diagnosis

Fig.18.8 (a) The upper figure represents an arbitrarily shaped ssDNA molecule, which is stretched
and attached to a pair of functionalized SWNT electrodes in the presence of a DEP field. The lower
figure depicts a covalently attached dsDNA molecule, which has a definite conformation. The
nanoelectrodes are separated by a gap of 272nm (the contour length of the 80-bp DNA molecule
is ~27nm). The bridging DNA molecule suspends over a trench without touching the silicon diox-
ide surface. (b, c) Current flow at room temperature through single ssDNA and dsDNA molecules,
respectively, in ambient and vacuum conditions. Reprinted with permission from Roy etal. [97].
2008, American Chemical Society

the 68-nm gaps between two gold electrodes. As a model system, the specific binding
of the blood clotting factor human thrombin with different concentrations to its
ribonucleic acid (RNA) alpha-thrombin aptamer, as well as the immobilization pro-
cess of the RNA-aptamer, have been detected in real time.

18.2.3.2Nanowire Nanobiosensors

As the surface of nanowires can be easily modified, nanowires have been used to
decorate with a different potential chemical or biological molecular recognition
unit, making the wires themselves analyte-independent. Nanowire sensors operate
on the basis that the change in chemical potential accompanying a target/analyte-
binding event can act as a field-effect gate upon the nanowire, thereby changing its
conductance. Such a detection format is similar to how a field-effect transistor oper-
ates. The ideal nanowire sensor is a lightly doped, high-aspect ratio, single-crystal
nanowire with a diameter between 10 and 20nm. This is because the nanowire sensor
is too noisy with much smaller wires, and unsensitive with much larger wires.
As the nanomaterials transduce the biological binding event in an extremely sensitive,
real-time, and quantitative fashion, nanowire sensors have great potential for both
biological research and clinical applications. For example, Lieber and coworkers
demonstrated that silicon nanowires functionalized with PNA could be used for
real-time, label-free detection of DNA [54]. In their assay, the conductance of a
PNA-functionalized silicon nanowire bridging two electrodes was measured in the
presence of target DNA and mutant DNA with three consecutive base deletions.
The introduction of target DNA into the assay caused a rapid and immediate change
in conductance, while the effect of mutant DNA was negligible. The concentration-
dependent measurements showed the detection of DNA could be carried out to at
least the tens of femtomolar range. Furthermore, Liebers group also functionalized
18.2 Nanotechnologies in Biosensing 551

Fig. 18.9 Nanowire-based detection of single viruses. (Left) Schematic shows two nanowire
devices, 1 and 2, where the nanowires are modified with different antibody receptors. Specific
binding of a single virus to the receptors on nanowire 2 produces a conductance change (right)
characteristic of the surface charge of the virus only in nanowire 2. When the virus unbinds from
the surface, the conductance returns to the baseline value. Reprinted with permission from Patolsky
etal. [55]. 2004, National Academy of Sciences

the interface of nanowires with antibodies specific for influenza A virus particles
and used a microfluidic sampling system to demonstrate that single-virus/nanowire
recognition events could be detected by measuring real-time changes in nanowire
conductivity [55]. As shown in Fig.18.9, when a virus particle binds to the antibody
receptor on a nanowire device, the conductance of the device changes from the
baseline value. In contrast, when the virus drops off the nanowire surface, the con-
ductance returns to the baseline value. The conductance of a second nanowire device
at which binding does not occur during the same time period showed no change and
can serve as an internal control. One compelling advantage of nanowire sensors is
that the number and density of the sensor elements are limited only by the ability to
electronically address individual nanowires. Very dense nanowire sensor circuits,
with modification of different nanowires within the array with receptors specific for
different analytes, may be addressed. Thus, integrated circuits can be constructed
552 18 Nanobiosensing for Clinical Diagnosis

within very small environments, thereby enabling high-throughput measurements


of large numbers of different genes and proteins from very small tissue samples, or
even single cells.

18.2.3.3Fiber Optic Nanobiosensors

Recent advances in nanotechnology have led to the development of fiber optic


nanosensors with nanoscale dimensions suitable for intracellular measurements
[99]. The possibility of monitoring invivo biological processes within single living
cells could greatly improve the understanding of cellular function, thereby revolu-
tionizing cell biology. Fiber optical nanosensors are generally defined as nanome-
ter-scale measurement devices that consist of a biologically or chemically sensitive
layer. This layer can either contain biological recognition elements or be made of
chemical recognition elements covalently attached to an optical transducer.
Interaction between the target analyte and the sensing layer is designed to produce
a physicochemical perturbation that can be converted into a measurable effect.
Vo-Dinh etal. developed fiber optical nanosensors with antibody-based probes to
measure specific fluorescent targets inside a single cell [100]. The nanoprobe
employs antibody-based receptors targeted to a fluorescent analyte, benzopyrenetet-
rol (BPT), a metabolite of the carcinogen benzo[a]pyrene (BaP) and of the BaP
DNA adduct. Detection of BPT is of great biomedical interest, since this species can
serve as a biomarker for monitoring DNA damage due to BaP exposure and for pos-
sible precancer diagnosis. Before making measurements, the cells were treated with
BPT. Nanoprobes were then inserted into individual cells and incubated for 5min.
The fluorescent BPT molecules were bound by interaction with the immobilized
antibody receptor, forming a receptorligand complex. Upon laser excitation of this
complex, a fluorescence signal emitted by BPT molecules provided a basis for the
quantification of BPT concentration in the cell being monitored. This method could
determine a concentration of 9.60.21011 M for BPT in the individual cells
investigated. Here, different antibodies as well as other bioreceptors such as DNA
probes targeted to species of biological and medical interest could be used in nano-
probe arrays to provide highly multiplexed probes for high-throughput drug discov-
ery. In addition, this technique could be extended to nonfluorescent antigen species
by using a second, fluorescently tagged antibody in a sandwich-type immunoassay,
or by performing a competitive binding assay with a fluorescently labeled antigen.
These features bring great potential applications of fiber optical nanobiosensors in
biotechnology, molecular biology research, and medical diagnostics.

18.2.3.4Cantilever Nanobiosensors

Recently, it has been demonstrated that molecular adsorption also results in measur-
able mechanical forces, which can be detected by micro- and nanoelectromechanical
systems. Detecting biomolecular interactions by measuring nanomechanical forces
18.2 Nanotechnologies in Biosensing 553

Fig.18.10 Specific biomolecular interactions between target and probe molecules alter intermolecular
interactions within a self-assembled monolayer on one side of a cantilever beam. This can produce a
sufficiently large force to bend the cantilever beam and generate motion. The origin of this nanome-
chanical motion lies in the interplay between changes in the configurational entropy and intermolecular
energetics. Reprinted with permission from Wu etal. [101]. 2001, National Academy of Sciences

offers an exciting opportunity for the development of highly sensitive, miniature, and
label-free biological sensors. Microcantilevers with nanoscale thickness allow the
detection of biomolecules and microorganisms due to surface stresses created by
molecular adsorption, in which the resonance frequency was used as a function of
target binding (Fig. 18.10) [101]. A microfabricated cantilever with an internal
piezoresistive component has been sensitized with thiolated ssDNA strands and uti-
lized to investigate DNA hybridization. The generation of a differential surface stress
onto the functionalized cantilever surface upon target recognition has allowed the
nanomechanical identification of 12-nucleotide complementary DNA probes with
single-base-mismatch discrimination and a low sensitivity of 0.2 mM [102]. For
amplification, microcantilevers have been used to detect DNA strands modified with
Au NPs [103]. The gold labels provide a site for silver ion reduction, which increases
the mass on the cantilever and results in a detectable frequency shift that signals the
binding events. This strategy can detect target DNA as low as 0.05nM. The detection
of proteins, such as cancer biomarkers, viruses, and bacteria is also possible using
cantilever nanobiosensors. For example, prostate-specific antigen (PSA) has been
successfully detected on cantilevers using immobilized specific antibody [104].
Craighead and coworkers modified the microcantilevers with antibodies specific for
either viruses or bacteria to detect their corresponding target [105, 106].
With the development of lithographic technologies and micro- and nano-fabrication
techniques, multitarget sensor arrays involving tens of cantilevers and analog pro-
cessing on a single chip can be manufactured. Increasing the number of sensing
elements in an array can lower noise, increase selectivity, and enhance robustness.
Due to the advantages of simplicity, low power consumption, low cost, inherent
compatibility with array designs, and label-free detection, cantilever nanobiosensors
are very attractive for future clinical applications.
554 18 Nanobiosensing for Clinical Diagnosis

Fig. 18.11 Schematic of the ion-channel switch biosensor with two antibody Fab fragments
r eacting with the target antigen. The binding of the target antigen (#) to the antibody fragments (*)
causes the conformation of gramicidin A to shift from conductive dimers to nonconductive mono-
mers (ac). This causes a loss of conduction of ions across the membrane and a change in the
electrical output of the reader. Reprinted with permission from Oh etal. [108]. 2008, Elsevier

18.2.3.5Ion-Channel Switch Nanobiosensors

The ion-channel switch (ICS) is a recently developed biosensor technology that


offers a rapid and sensitive immunodiagnostic for viral detection at point of care
[107, 108]. As shown in Fig.18.11, the ICS biosensor is based upon a synthetic
self-assembling membrane, composited with a bilayer lipid membrane (BLM) and
the ion-channel molecule gramicidin A, on a gold surface. The membrane is separated
from the gold surface by ethylene glycol spacer molecules, which provides a reser-
voir for ions that pass through the gramicidin ion channel. The flow of ions through
gramicidin only occurs when two monomers align to form a dimer. Gramicidins
within the tethered inner leaflet of the lipid bilayer are linked to the gold surface of
the electrode. Target-specific antibody Fab fragments are attached to the mobile
gramicidin molecules in the outer monolayer leaflet and to other non-ion-channel
sites tethered to the electrodes surface. The arrival of the target analyte cross-links
the antibodies attached to the mobile outer layer channels with those attached to
other fixed sites on the membrane surface. Due to the low density of tethered chan-
nels within the inner membrane leaflet, the cross-linking of the antibodies by the
target molecules causes the outer layer channels to be anchored away from their
tethered inner layer partners. Gramicidin dimer alignment is thus prevented, dis-
rupting the current across the membrane, hence signaling the presence of specific
molecules by detecting the change in the electrical current. The sensitivity and spec-
ificity for detection of influenza A virus by the ICS biosensor are comparable to the
commercially available Medix test, while the ICS biosensor can perform rapid
point-of-care tests for influenza A virus in specimen inoculation within 10 min,
without the need for chemical or other pretreatments for the clinical specimens. The
incorporation of appropriate antibodies into the ICS biosensor will enable adaptation
of this diagnostic device for rapid detection and surveillance in the monitoring of
potential pandemic and influenza outbreaks.
18.3 Nanobiosensing for Clinical Diagnosis 555

18.3Nanobiosensing for Clinical Diagnosis

Some clinical applications of nanodiagnostics mentioned above have already been


described, along with a description of nanotechnologies. Here, we briefly describe
a few examples of nanobiosensing in the clinical diagnosis of glucose, disease bio-
markers, gene, viruses, microorganisms, and cancer cells.

18.3.1Glucose Detection

Glucose plays a critical role in the bodys metabolism. The dysfunction of glucose
handling from insulin deficiency or resistance can lead to diabetes. Recently, with
the development of nanoscience, it is possible to engineer micro/nanoscale devices
for invivo glucose sensing, which opens the way to monitor the glucose of diabetes
patients in real time. A new type of glucose nanosensor has been fabricated by using
SWCNTs to modulate their emission in response to the adsorption of specific
biomolecules [109]. The signal transduction includes two distinct mechanisms:
fluorescence quenching and charge transfer. Glucose oxidase is coated on CNTs;
then ferricyanide is sprinkled onto the surface of nanotubes. Ferricyanide draws
electrons from the nanotubes, quenching their capacity to glow when excited by
infrared light. When glucose is present, it reacts with the oxidase, producing hydrogen
peroxide, which can react with ferricyanide in a way that reduces that molecules
hunger for electrons. The higher the glucose level, the greater is the infrared fluores-
cence of nanotubes. To test the feasibility of implanting the sensors in the body, the
nanotubes, modified with both glucose oxidase and ferricyanide, are placed inside a
sealed glass tube 1cm long and 200mm thick (Fig.18.12). The tube is riddled with
pores large enough to let glucose enter but small enough to keep the nanotubes
inside. The tube could implant in a sample of human skin and the sensor can be
excited with infrared light to detect fluorescence.
Probes encapsulated by biologically localized embedding (PEBBLEs) nanosensors,
with a size of 45nm in diameter, have been fabricated for real-time glucose imaging
in living cells [110]. The nanosensors incorporate glucose oxidase, an oxygen-
sensitive fluorescent indicator, and an oxygen-insensitive fluorescent dye, as a refer-
ence for ratiometric intensity measurements. The enzymatic oxidation of glucose to
gluconic acid results in the local depletion of oxygen, which is measured by the oxy-
gen-sensitive dye. Due to the PEBBLE matrix, this kind of nanosensor is insensitive
to interference from proteins in cells, enabling reliable invivo chemical analysis.
The matrix also significantly reduces the toxicity of the indicator and reference dyes
to the cells, so that a larger variety of dyes can be used in an optimal fashion.
Furthermore, it also enables a synergistic approach in which there is a steady state
of local oxygen consumption, and this cannot be achieved by separately introducing
free enzyme and dyes into a cell.
556 18 Nanobiosensing for Clinical Diagnosis

Fig.18.12 Glucose detection using a carbon-nanotube optical sensor. (a) Reaction at the enzyme
converts glucose to gluconolactone, with the hydrogen peroxide coproduct detected by interaction
with the Fe(CN)63 functional groups on the exposed nanotube surface between enzyme mono-
mers. (b) A 200-mm1-cm, 13-kDa microdialysis capillary, shown to size on a human finger, is
loaded with the nanotube solution allowing glucose to diffuse through the membrane with the
containment of the sensing medium. On placing the capillary beneath a human epidermal tissue
sample, we can clearly map the nanotube fluorescence from the capillary, as seen in this two-
dimensional profile. The dotted square in the picture of the tissue sample symbolizes the area used
for the fluorescence map. Reprinted with permission from Barone etal. [109]. 2005, Nature

18.3.2Disease Protein Biomarker Detection

As is well known, early detection is particularly important in the case of cancer and
other pathologies, because the early stages of disease are typically treated with the
greatest probability of success. As the levels of the biomarkers, such as tumor-related
antigens, in serum are associated with the stages of tumors, the requirements of
technology platforms that provide the reliable, rapid, quantitative, low-cost, and
multiplexed identification of biomarkers are raising. Nanotechnology is helpful in
this effort, as shown by the following examples.
Nanomaterials are widely used for signal amplifications. For example, Mirkins
group developed a nanoparticle-based bio-barcode amplification strategy for detecting
PSA with a detection limit of 30aM [29]. Rusling etal. used CNTs, modified with
both enzymes and secondary antibodies at a high enzyme/antibody ratio, to fabricate
18.3 Nanobiosensing for Clinical Diagnosis 557

Fig. 18.13 (a) Schematic illustration and (b) photograph of disposable electrochemical immu-
nosensor diagnosis device. Reprinted with permission from Liu etal. [111]. 2007, American
Chemical Society

electrochemical immunosensors for PSA [48]. A greatly amplified detection


sensitivity of 40fg PSA could be achieved within 10mL of undiluted calf serum. By
assembling glucose oxidase and antibody on Au NPattached CNTs, Jus group
designed a disposable immunosensor array for the ultrasensitive multiplexed
measurement of tumor markers [49]. This assay could simultaneously detect carci-
noembryonic antigen and a-fetoprotein as low as 1.4 and 2.2pg/mL, respectively.
Using QDs (CdS@ZnS) as labels, Lins group designed a disposable electro-
chemical immunosensor diagnosis device for in-field and point-of-care quantitative
testing of disease-related protein biomarkers [111]. A sandwich immunoreaction
was performed on the immunochromatographic strip, and the captured QD labels in
the test zone were determined by highly sensitive stripping voltammetric measure-
ment of the dissolved metallic component (cadmium) with a disposable screen-
printed electrode, which is embedded underneath the membrane on the test zone
(Fig.18.13). The device, taking advantage of the speed and low cost of the conven-
tional immunochromatographic strip test and the high sensitivity of the nanoparticle-
based electrochemical immunoassay, could detect PSA antigen in human serum
sample with a detection limit of 20pg/mL.
Besides these applications of nanomaterial-based amplification assays, miniaturized
multiplexed immunoassay nanodevices based on nanowires have also attracted much
attention. Liebers group described an innovative approach for the real-time, label-free,
multiplexed electrical detection of four cancer markers using a silicon-nanowire array
[112]. They developed integrated nanowire arrays (Fig.18.14) in which distinct nano-
wires and surface receptors could be incorporated as individual device elements. Protein
markers were routinely detected at femtomolar concentrations with high selectivity, and
simultaneous control by nanowires discriminated against false positives.
558 18 Nanobiosensing for Clinical Diagnosis

Fig. 18.14 (a) Optical image (top) of a nanowire device array. The schematic (bottom) shows
details of metal electrodes (golden lines) connecting nanowires (blue lines) in this region with
orientation rotated 90 relative to the red rectangle. (b) Schematic illustrating multiplexed protein
detection by three silicon-nanowire devices in an array. Devices 1, 2, and 3 are fabricated from
similar nanowires and then differentiated with distinct mAb receptors (1 red; 2 green; 3 blue)
specific to three different cancer markers. Reprinted with permission from Zheng etal. [112].
2005, Nature

18.3.3DNA Detection

The recent discovery and sequencing of the human genome has provided valuable
insight into understanding how genetic factors contribute to the development of disease.
Specifically, the sensitive detection of DNA sequence variations plays an important role
in the diagnosis of genetic-related disease and conditions, especially for early-stage
treatment and monitoring. Nanoscale materials offer excellent prospects for designing
highly sensitive and selective bioassays of nucleic acid. For example, relying on nano-
particle-based amplification, Mullers group presented a microarray-based method for
multiplex single nucleotide polymorphism (SNP) genotyping in total human genomic
DNA without the need for target amplification or complexity reduction [113]. Specificity
is derived from two sequential oligonucleotide hybridizations to the target DNA SNP
segments by allele-specific surface-immobilized capture probes and gene-specific
oligonucleotide-functionalized Au NPs. The assay format is simple, rapid, and robust
pointing to its suitability for multiplex SNP profiling at the point of care.
Using QDs, Wangs group has developed an ultrasensitive and reliable nanotech-
nology assay for the detection and quantification of DNA methylation, which
contributes to carcinogenesis by silencing key tumor-suppressor genes [114].
Bisulfite-modified DNA is subjected to PCR amplification with primers that would
differentiate between methylated and unmethylated DNA. QDs are then used to cap-
ture PCR amplicons and determine the methylation status via fluorescence resonance
energy transfer (Fig.18.15). This approach detects as little as 15pg of methylated
DNA in the presence of a 10,000-fold excess of unmethylated alleles, enables reduced
use of PCR (as low as eight cycles), and allows for multiplexed analyses. The high
sensitivity of this method enables one-step detection of methylation at PYCARD,
CDKN2B, and CDKN2A genes in patient sputum samples that contain low concen-
trations of methylated DNA, which normally would require a nested PCR approach.
Nanostructured materials also constitute new platforms for biomolecular sensing
that may provide increased sensitivity and amenability to miniaturization. Tans
18.3 Nanobiosensing for Clinical Diagnosis 559

Fig. 18.15 Principle of MS-qFRET for detection of DNA methylation. In step 1, extracted
genomic DNA is subject to sodium bisufite conversion, wherein unmethylated cytosines are con-
verted to uracil while methylated cytosines remain unaffected. In step 2, DNA is amplified using
PCR wherein the forward and reverse primers are labeled with a biotin (black dot) and a fluoro-
phore (red dot), respectively. In step 3, the resulting labeled PCR product is captured by streptavi-
din-functionalized QDs through streptavidinbiotin affinity. In step 4, upon suitably exciting the
QD, the nanoassembly formed allows for FRET to occur between the QD donor and the fluoro-
phore acceptor. Consequently, the labeled PCR products are detected by emissions of fluorophores
accompanied by quenching of QDs to reveal the status of DNA methylation. Reprinted with per-
mission from Bailey etal. [114]. 2009, Cold Spring Harbor Laboratory Press

group developed a novel genomagnetic nanocapturer (GMNC) for the collection,


separation, and detection of trace amounts of DNA/RNA molecules with one
single-base difference [115]. The GMNC, constructed by bioconjugating molecular
beacon DNA probes onto magnetic nanoparticle surfaces (Fig.18.16), could be suc-
cessfully applied in artificial buffer solution samples and in cancer cell samples, both
containing different proteins and random DNA sequences. This method has distinctly
useful features, such as highly efficient collection of trace amounts of DNA/mRNA
samples down to femtomolar concentrations, excellent ability to differentiate single-
base-mismatched DNA/mRNA samples by combining the exceptional specificity of
molecular beacons and the separation power of magnetic nanoparticles, and real-time
560 18 Nanobiosensing for Clinical Diagnosis

Fig.18.16 Structure of the genomagnetic nanocapturer (GMNC). (a) Schematic representation of


a GMNC: 1 magnetic nanoparticles; 2 silica layer; 3 biotinavidin linkage; 4 molecular beacon
DNA probe. (b) TEM image of a silica-coated magnetic nanoparticle. Reprinted with permission
from Zhao etal. [115]. 2003, American Chemical Society

monitoring and confirmation of the collected gene products. Kelleys group fabri-
cated gold nanoelectrode ensembles (NEEs) for ultrasensitive biosensing of DNA
probe, oligonucleotide sequences that correspond to a portion of the 23S rRNA gene
from Helicobacter pylori (a pathogen implicated in gastric ulcers and cancer), with
an attomole-level detection limit [116]. The label-free system reports on the binding
of a target DNA sequence to an probe oligonucleotide, immobilized on Au NEEs,
using a catalytic reaction between two transition-metal ions, Ru(NH3)63+ and
Fe(CN)63. The Ru(III) electron acceptor is reduced at the electrode surface and then
reoxidized by excess Fe(III), making the electrochemical catalytic process. The
increased concentration of anionic phosphates at the electrode surface that accompa-
nies DNA hybridization increases the local concentration of Ru(NH3)63+, and there-
fore produces large changes in the electrocatalytic signal.

18.3.4Virus Detection

Rapid, selective, and sensitive detection of viruses is crucial for implementing an


effective response to viral infection, such as through medication or quarantine. The
established methods for viral analysis, such as plaque assays, immunological assays,
and PCR-based testing of viral nucleic acids, cannot achieve rapid detection at a
single-virus level. With the development of nanoscience, the assays of virus detec-
tion have been significantly improved. For example, by using nanowire field-effect
transistors, Liebers group presented a method for the direct, real-time electrical
detection of single virus particles [55]. Recently, Sandoghdars group developed a
colocalization methodology that combines scattering interferometry and single-
molecule fluorescence microscopy to visualize both position and orientation of
single QDlabeled Simian virus 40 (SV40) particles [117]. By achieving nanometer
18.3 Nanobiosensing for Clinical Diagnosis 561

Fig.18.17 Images of bacterial cells. (a) Scanning electron microscope image of Escherichia coli
O157:H7 cell incubated with antibody-conjugated nanoparticles; (b) scanning electron microscope
image of E. coli DH5a cell (negative control) incubated with nanoparticles conjugated with anti-
body for E. coli O157:H7; (c) fluorescence image of E. coli O157:H7 after incubation with anti-
body-conjugated nanoparticles. The fluorescence intensity is strong, enabling single-bacterium
cell identification in aqueous solution. Reprinted with permission from Zhao etal. [118]. 2004,
National Academy of Sciences

spatial and 8-ms temporal resolution, they observed sliding and tumbling motions
during rapid lateral diffusion on supported lipid bilayers, and repeated back-and-
forth rocking between nanoscopic regions separated by 9nm. These findings suggest
the recurrent swap of receptors and viral pentamers as well as receptor aggregation
in nanodomains.

18.3.5Bacteria Detection

The rapid and sensitive detection of pathogenic bacteria is extremely important in


medical diagnosis and measures against bioterrorism. Recently, Tans group devel-
oped a bioassay for the accurate determination of a single bacterial cell within 20min
by using bioconjugated nanoparticles [118]. The nanoparticle provides an extremely
high fluorescent signal for bioanalysis and can be easily incorporated in a biorecogni-
tion molecule such as an antibody. Through antibodyantigen interaction and recogni-
tion, the antibody-conjugated nanoparticles can readily and specifically identify a
variety of bacterium, such as Escherichia coli O157:H7 (Fig.18.17). This method can
be applied to multiple bacterial samples with high throughput by using a 384-well
microplate format. It has a potential for application in ultrasensitive detection of dis-
ease markers and infectious agents. One limitation of QD technology is that it gives
qualitative information such as in the detection of a small number of microorganisms
but does not provide quantitative information about the number of organisms.

18.3.6Cancer Cell Detection

The early diagnosis of cancer is the critical element in successful treatment and long-
term favorable patient prognosis. Late diagnosis is often associated with the lack of
timely sensitive imaging modalities. The promise of nanotechnology is presently
562 18 Nanobiosensing for Clinical Diagnosis

limited by the inability to simultaneously seek, treat, and image cancerous lesions.
Adairs group synthesized fluorescent calcium phosphosilicate nanocomposite par-
ticles (CPNPs) for systemically targeting breast and pancreatic cancer lesions [119].
The CPNPs with a diameter of 20 nm were composed of an amorphous calcium
phosphate matrix doped with silicate in which a near-infrared imaging agent was
embedded. The conjugation of biotinylated human holotransferrin (diferric transfer-
rin) and biotinylated anti-CD71 antibody (anti-transferrin receptor antibody) to
avidin-conjugated CPNPs (Avidin-CPNPs) permits targeting of transferrin receptors,
which are highly expressed on breast cancer cells. Similarly, the conjugation of bioti-
nylated pentagastrin to Avidin-CPNPs and decagastrin (gastrin-10) to PEG-CPNPs
via PEG-maleimide coupling permits targeting of gastrin receptors, which are over-
expressed in pancreatic cancer lesions. These results showed that the bioconjugated
CPNPs could perform as a theranostic modality, simultaneously enhancing drug
delivery, targeting, and imaging of breast and pancreatic cancer tumors.
QDs have the potential to become a new class of fluorescent probes for many
biological and biomedical applications, especially cellular imaging. Wus group
used QDs linked to immunoglobulin G and streptavidin to label the breast cancer
marker Her2 on the surface of fixed and live cancer cells, to stain actin and microtu-
bule fibers in the cytoplasm, and to detect nuclear antigens inside the nucleus [120].
The results showed all labeling signals from QDs were specific for the intended
targets and were brighter and considerably more photostable than comparable
organic dyes. They also realized simultaneously the detection of two cellular targets
with one excitation wavelength by using QDs with different emission spectra con-
jugated to IgG and streptavidin.

18.4Conclusions

Nanotechnologies promise to extend the limits of current molecular diagnostics and


enable point-of-care diagnosis, the integration of diagnostics with therapeutics, and
the development of personalized medicine. Primary examples of emerging approaches
include the nanomaterial-based amplification assays such as bio-barcode assay,
nanowire-based molecular electronics, and nanoscale biosensors and bioarrays.
Through their individual or combined uses, it is envisioned that progress will be
accomplished in the high-throughput multiplexing of analyses of nucleic acids and
proteins, resulting in commensurate advances in clinical diagnostics.

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wwwwwwwwwwwww
Index

A label-free detection methods, 475


Acute myocardial infarction (AMI), 183, 184 metal nanoparticles, 474
Adsorptive stripping voltammetry (AdSV), 403 large-scale development, 471
Affinity interactions, 45 optical techniques
Antibodies computational models, 472473
classes fluorescent dye molecules, 472
anti-TNF-a, 444 label-free detection methods, 471
IgA and IgE, 426 label-free microarrays, 472473
IgM, IgG and IgD, 426 magnetic nanoparticle (MP) labels, 472
monoclonal and polyclonal, 427 metal colloid labels, 472
HRP-labeled anti-CA125, 18 micron-sized patterns, 471
Antigenantibody interaction, 2930, polymerase chain reaction (PCR), 473474
222224, 427 quantum dot labels, 472
Antigens single-nucleotide polymorphisms (SNPs), 471
antibody interaction (see Antigenantibody Biocompatible nanomaterials
interaction) immobilization substrates
defined, 426 AuNPs, 430
recognition reaction, 426427 biofunctionalization, 435
Arginine-glycine-aspartic acid (RGD), 502 biopolymer/solgel matrix, 430
AuNPs. see Gold nanoparticles CA 125 immunosensor, 433
carbon nanofiber (CNF), 432
carcinoembryonic antigen, 430
B HAuCl4 chemical reduction, 431
Barstar noncovalent binding system (BBS), HRP-hCG antibody, 430
504, 527 immunosensors and heterogeneous
Bicontinuous gyroidal mesoporous carbon requirements, 429
(BGMC), 186187 microcystin-LR detection, 432
Bilayer lipid membrane (BLM), 554 nickel nanohairs, 435
Bimodal mesoporous silica (BMS), 180 PNPs, 435
Bioanalysis, 2, 14, 18, 58, 76, 141 silica nanoparticles (Si NPs), 433
Biochips, nanostructures SWNHs, 432
complex biochemical processes, 471 Toxoplasma gondii antigens, 434
electrochemical methods probe carriers
DNA biochip technology, 476478 apoferritin, 446447
label-based detection methods, CNTs, 446
474475 DNAzyme catalysis, 445

H. Ju et al., NanoBiosensing: Principles, Development and Application, 569


Biological and Medical Physics, Biomedical Engineering,
DOI 10.1007/978-1-4419-9622-0, Springer Science+Business Media, LLC 2011
570 Index

Biocompatible nanomaterials (cont.) characterization


europium(III) and terbium(III) chelates, carboxylated CNTs, tapping-mode
444445 AFM images, 19, 20
Frster radius, 446 colloidal CNSs, TEM and SEM images,
inverse microemulsion polymerization 19, 20
protocol, 444 x-ray photoelectron spectra, 19, 21
polystyrene nanoparticles, 445446 ECL and HRP, 18
signal tags electrochemical sensing
Actinobacillus pleuropneumoniae, 437 antibody-antigen, 2930
advantages, 436 DNA, 2729
amplification, 441 b-nicotinamide adenine dinucleotide
anti-PSA antibody pair, 440 (NADH), 31
colorimetric immunoassay, 437 MSNs, 17
electrooxidization, 438 NPs
harsh stripping procedure, 437 magnetic, 1517
immunochromatographic strip, AuNPs, metal, 1214
436 semiconductor, 1415
intensity and detection limits, 436 optical sensing
light-scatteringbased immunoassay, colorimetric detection, bioanalytes,
439440 2122
microalbumin detection, 440 fluorescence detection, 2325
preconcentration step, 439 spectroscopic measurements, 2527
QDs/nanocrystals, 442 polyaniline (PANI) nanotube array, 18
Raman tracer antibodies, 441 thionine-horseradish peroxidase
SERS detection, 436 conjugation (TH-HRP), 18
signal-to-background ratio, 439 Biological system, carbohydrates
Biofunctionalization AdSV, 403
affinity interactions, 45 alkaline conditions, 403
covalent route detection, 402
activation method, 910 hesperidin structure, 402403
click chemistry, 8 HPLC, 404
direct chemical reaction, 56 Ni-based nanostructures, 403404
linker chemical reaction, 67 Ni-NDC film, 404
NPs functionalization approaches, 1 Biomarkers
physical adsorption clinic diagnoses, 233
entrapping, biocompatible polymer, disease protein detection
34 electrochemical immunosensor
method, nanomaterials, 23 diagnosis device, 557
MWNTs, 4 nanomaterials, 556
PDDA-MWNTs/GOx films, gold nanowire arrays, 557558
electrodes, 4 intracellular/extracellular, 495
p-p stacking interaction, 3 myocardial infarction, 92
SWNT, 3 organophosphate pesticide exposure, 381382
QDs, 2 protein, 223, 495
Biofunctional nanomaterials tumor, 508
Au NPs, DNA hybridization, 18, 19 Biopolymer chitosan (CHIT), 312
carbon-based Biosensing. see also Electrochemical
CNTs, 10 biosensing; Insecticides detection,
fructose dehydration and carbonization nanostructured biosensing;
process, 12 Nanostructured mimic enzymes
graphene, 12 advantages, 171172
p-pstacking, 11 analyte inhibited/enhanced ECL emission
1-pyrenebutanoic acid, succinimidyl CdTe QDs/sulfite system, 254
ester, 11 RTIL, 254
Index 571

aptamer-functionalized NPs, 5 bienzymatic glucose, 227


biochemical coreactant direct biocatalysts and amperometric, 93
determination biomaterial-nanoparticle hybrid systems, 40
hydrogen peroxide, 252 carbohydrate components
NP ECL, 252 acetylenyl group, 419420
bioconjugates, 5 carbon nanotubes, 418, 419
carbon nanofiber (see Carbon nanofiber) competition assays, 414
ECL-based, 29 gold nanoparticles, 413
ECL performance, higher sensitivity, inorganic nanostructures, 416
253254 label-free optical biosensor, 415
electrocatalysis and electrochemical, m-AuNP synthesis, 415416
133134 multiple-carbohydrate ligands, 414
electrochemical, 182, 187, 190193 nanobioelectronic detection system,
enzyme catalyzed reaction, 255257 419
immunoreactions/DNA hybridization self-assembled bilayer and multilayer
coupled sensing system, 414415
DNA assay, 257 surface-to-volume ratio, 414
immunoassay based on NP ECL, SWNT, 417
258260 TEM images, 416
molecularly imprinted nanomaterials virus particles, 417
(see Molecularly imprinted colorimetric, 13
nanomaterials) components, immunosensor, 429
nanobiosensing (see Signal amplification) development, 209
optical and electrochemical, 117119 DNA, 15, 18, 22, 2829, 58, 77, 220222
resonance energy transfer determination, ECL, QDs, 255257
255 electrochemical
Biosensing, nanostructures construction, 99
CNTs impedance, 59
damage sensors, 462464 and photoelectrochemical, 5859
hybridization sensors, 456462 sensors, 279282
layer-by-layer electrostatic electrostatic LBL technique, 213
self-assembly, 456 enzyme-based, 257
Q1D nanomaterials, 455 ethanol
metal nanoparticles alcohol dehydrogenase (ADH), 155
gold nanoparticles, 464465 AOX-based, 154
photoinduced reduction, 465 CNF-FeTMPyP nanocomposite,
sensing strategies and detection 154155
methods, 465466 glucose
semiconductor nanostructures amperometric measurements, 151152
amplified electrochemical sensing GOx immobilization, 153154
method, 467 NiCF nanocomposite, 152153
graphene oxide-induced fluorescence H2O2
quenching, 467468 CNF-modified GCE, 157158
QDs, 467 oxidation, 101
sensing strategies and detection PCNFs, HCNFs and TCNFs, 158
methods, 469470 immobilized specific biomolecules, 85
streptavidin/CdTe-tagged polybeads, immunosensors, 162, 163
467468 LSPCF, 41
Biosensors mass-sensitive devices, 288293
acetylthiocholine, 155156 mesoporous carbon
advantages, nanoporous materials, 171 amorphous, 185189
applications, 207 graphitic, 189191
aptamer, 58 mesoporous hybrid nanocomposite,
artificial peroxidase, 86 192195
572 Index

Biosensors (cont.) VACNF (see Vertically aligned carbon


mesoporous materials nanofiber)
carbon, 185191 Biosensors enzymes
hybrid nanocomposite, 192195 AChE-based, 369371
mesostructure, 176177 ChE-ChO
metal oxide, 191192 butylcholine esterase (BChE), 369
morphology, 176 hybrid immobilization method, 368
pore size, 173175 OP compounds, 368
silica, 178185 OPH-based, 371
surface characteristics, 175176 BLM. see Bilayer lipid membrane
mesoporous metal oxide, 191192 Breslows mimics
mesoporous silica biomimetic chemistry, 97
coimmobilization, two enzymes, 181183 Cu(II) complex, 97
direct electrochemistry, redox proteins, hydrogen bonding, 96
178181 polyethylenimine (PEI), 97
immuno-and DNA biosensors, 183185 steroids, 98
metal hexacyanoferrates, 101
MIPs interfacing, transducer
electropolymerization, 278 C
nano/micro-sized structures, 279 Calcium phosphate nanoparticles, 140141
SAM, 278 Calcium phosphosilicate nanocomposite
surface coating techniques, 278 particles (CPNPs), 562
molecular recognition Carbohydrate detection, nanostructured
approaches, binding signal biosensing
transduction, 277 biological roles
issues, 276277 classification, 396
QCM and SPR, 277278 genetic defects, 396
NADH processes and metabolism., 396
CNTs, CNFs and CMPs, 160, 161 description, 393
CVs, CNF-modified electrode, 159160 genetic glycosylation defects
electrochemical oxidation, 159 intra-and interspecies variations, 397
nanoporous gold mechanistic involvement, 397
DNA, 198199 transformation and progression, 397
enzyme, 196198 identification techniques
Escherichia coli, 199200 detection, 399
optical detection, 2, 21 linkage analysis, 400402
optical sensors, 282288 primary structure analysis, 398399
PANI nanotube array-based DNA, 18 molecular recognition, 416
PB-based electrocatalyst, 89 nanotechnology
phenol carbohydrate analysis, biological
p[NVCzVBSA], 156157 system, 402404
Tyr/PANI-IL-CNF preparation, 156 components, biosensors, 413420
porous materials classes, 171 direct carbohydrate detection,
porphyrins ordered assembly, 404413
nanomaterials, 141 protein-glycan interactions, 397
preparation, 251 structural depiction
protein electron transfer glycans, 394
AOx, 160161 glycoconjugates, 394396
hemoglobin (Hb), 161162 oligosaccharides, 394396
third-generation electrochemical, 160 Carbohydrates
proteins immobilization, 172 carbohydrate-conjugated nanomaterials, 494
QDs, 443 cell surface assay
sol gel (see Solgel) nanomaterial substrate, 518520
SWNHs-TiO2-porphyrin sandwich nanoprobes, 520527
nanostructure, 120, 121 defined, 393
Index 573

Carbon nanofiber (CNF) DNA


vs. CNTs, 151 amperometric response, 223
electrochemical biosensors and bioassays biosensors, 18
acetylthiocholine, 155156 carbon paste electrode (CPE), 222
ethanol, 154155 electrochemical spectroscopy, 222
glucose, 151154 hybridization efficiency, 221
hydrogen peroxide, 157158 intrinsic electroactivity, 220
immunosensors, 162 protein arrays, 223
NADH, 159160 square-wave voltammograms, 221
phenol, 156157 voltammetric hybridization, 221
protein electron transfer, 160162 dual-amplification role, 27
VACNF array-based, 162167 electrochemical DNA sensors, 27
synthesis electrode and immobilization phase, 432
catalyst type and precursor, 149150 electronic tongue, 352
iron group metals and SiO2, 150151 fabrication and characterization
nucleation and growth, 149, 150 AFM, 216
VACNFs, 148 FT-IR, 215216
Carbon nanohorns (CNHs) Raman spectrum, 216217
vs. CNTs, 119120 SEM and TEM, 215
SWNHs-TiO2-porphyrin nanohybrids functionalization
structure, 121 covalent interaction, 213214
synthetic protocols, 120 noncovalent interaction, 209213
Carbon nanotubes (CNTs) GOx immobilization, 153
advantages hybridization sensors
electron-transfer kinetics, 208 analytical protocol, 457458
in-situ detection, 208 chronopotentiometric signals,
real-time detection, biomolecules, 208 457, 459
signal transduction amplification, 208 electrochemical gene sensing system, 459
surface-to-volume ratios, 208 label-free assay, 460462
aqueous solution assembly, 116117 layer-by-layer immobilization, 456
bilirubin oxidase, 324325 self-assembly procedure, 456457
bio-detection amplification, 446 steps, 456
biogel-based strip electrodes, 322, 323 SWNT devices, 460
biomolecules TEM images, 457458
epinephrine (EP), 228 infra-red fuorescent cytosensing, 498499
glucose, 226227 interfaces, 516
hydrogen peroxide, 227 layer-by-layer electrostatic self-assembly,
nitric oxide, 226 456
organohalide pollutant, 228 MW-CNCE, 322323
categories, 147 MWNTs, 323324, 513, 519
cells MWNTs-ACB and PACB, 337338
biocompatible interactions, 224 noncovalent functionalization, 1011
cytosensor array, 224 vs. OMCs, 17
monoclonal antibody, 225 one-dimensional nanomaterials, 538
CFUE/MWNT/Nafion electrode, optical sensor, 555556
343344 organic solvent assembly
cyclic voltammetric data, NCE and CCE, CNTs, 113114
323, 324 CNx-MWCNTs-FeTpivPP modified
damage sensors GCE, 115
biorecognition substrates, 462 covalent methods advantages, 115
CVD, 463 DMF photos, 113, 114
differential pulse voltammograms, porphyrin-grafted CNTs and photoinduced
464 electron transfer, 115, 116
E-Fenton reaction, 463 supramolecular assembly, protonated
in situ detection, 462 porphyrins and SWCNTs, 113, 114
574 Index

Carbon nanotubes (CNTs) (cont.) nanoprobes development, 520


Q1D nanomaterials, 455 nanotechnology applications, 517
Raman spectra, 188 scanning electrochemical microscopic
and redox mediators, 31 strategies, 525527
Ru(bpy)32+ ECL sensor, 323 scanometric strategies, 524525
SGCCN, 324 Chemosensor
structure antibody-based, 266
discovery, 207 CTL
functionalization, 207 cigarettes, 361
laser ablation, 207 CL response, nanomaterials,
SWCNTs and MWCNTs, 207 358359
surface chemistry/functionalization, 498 ethanol, hydrogen sulfide, and TMA,
SWCNT and MWCNT, 192193 359360
SWNTs linear range and detection limit, 360
advantages, 2627 Chlorotoxin (CTX), 489490
dual-signal amplification, 31 Click chemistry, 8, 141, 165, 527
p-p interaction, porphyrins, 3 Clinical diagnosis, nanobiosensing
radionuclide-filled, 12 bacteria, 561
Carcinoembryonic antigen (CEA) cancer cell
amperometric immunosensor preparation, CPNPs, 562
431 QDs, 562
antigen, 434 disease protein biomarkers
composite architecture, 434 electrochemical immunosensor
detection sensitivity, 430 diagnosis device, 557
magnitude ranges, 446 nanomaterials, 556
Cataluminescence (CTL) nanowire arrays, 557558
definition, 356 DNA
nanomaterial-based sensors, vapour genomagnetic nanocapturer, 559
sensing Helicobacter pylori, 560
energy transfer process, 357 MS-qFRET principle, 559
ethyl ether gas, 357358 glucose
HS-SPME-CTL, 358 carbon-nanotube optical sensor,
nanosized materials, 356357 555556
trimethylamine (TMA), 357 dysfunction, 555
organic compounds recognition, 361362 PEBBLEs, 555
CEA. see Carcinoembryonic antigen signal transduction mechanism, 555
Cell surface carbohydrate assay nanotechnologies
electrochemical nanodevices, 548554
advantages, 511 nanofabrication, 553
microscopic scanning, 504, 505 nanomaterials, 535543
monolayer fabrication and the virus, 560561
competitive assay, 521, 522 CNF. see Carbon nanofiber
QDs anodic stripping analysis, 521 CNTs. see Carbon nanotubes
electrochemiluminescent strategies, 525 CPNPs. see Calcium phosphosilicate
flow cytometric strategies, 523524 nanocomposite particles
fluorescent strategies, 522523 CTL. see Cataluminescence
glycosylation and mass spectrometric Cyclic voltammetric (CV) curves, 179, 180,
methods, 517 198199, 212213
nanomaterial substrate Cytosensing
cell-binding comparison, 519 colorimetric
ECL cytosensor, 520 ACGNP, 511, 512
electrochemical detection and BGC AuNPs, 524
cells, 518 electrochemical
and enzyme-linked lectin application, 518 advantages, 511
P-gp, 519 CNF and CS, 513
Index 575

electron generation and charge transfer, fabrication and characterization


living cells, 513 AFM, 216
MWNTs, 513 FT-IR, 215216
fluorescence imaging, 488499 Raman spectrum, 216217
impedance SEM and TEM, 215
AuNPs-chitosan, 514 functional CNTs
EIS measurements, K562 cells, 514515 antigen-antibody, 222223
microfabricated flow cell, adjustable biomolecules, 226228
gap electrode, 515516 cells, 224225
MRI, 499506 DNA, 220222
nanomaterials use, 486487 functionalization strategy
neural, 516517 covalent interaction, 213214
SERS detection, 506510 noncovalent interaction, 209213
regioselectivity, 233
signal transduction amplification
D alkaline phosphatase (ALP), 218
Deoxyribonucleic acid (DNA) analysis analytical performance, 220
description, 453 AuNPs, 218
genetic test types, 453454 biocatalysis, enzymes, 218
hybridization, 454 point-of-care diagnostics, 218
nanostructures, biochips prostate specific antigen (PSA), 218
electrochemical methods, 474478 self-assembly monolayer (SAM),
optical techniques, 471474 219220
nanostructures, biosensing SWCNTs immunosensors, 218
CNTs, 455464 terminal protection assay, 218219
metal nanoparticles, 464466 ultrasensitive measurements, 218
semiconductor nanostructures, 467473 SWCNTs-based field-effect
Dip-pen nanolithography (DPN) nucleic acids detection, 231
fabricate nanoarrays, 544 proteins detection, 229231
multiple-DNA inks, 544545 SWCNTs forest
Direct carbohydrate detection chemical vapor deposition, 231
lectin-based biosensors (see Lectin, based pyrolytic graphite electrode (PGE),
biosensors) 232
protein glycolation relative standard deviation, 232
aldehyde-derivatized glass slides, 413 SEM image, CNTs array, 231232
biotin-modified lectin, 412413 tapping mode AFM, 232233
cell surface glycoproteins, 412 Electrochemical impedance spectroscopy
DNA biosensors (EIS), 185, 186, 514
Au NP/CNT-based, 18 Electrochemical sensing
electrochemical, 2729 antibody-antigen, 2930
hybridization events, 456 biological compounds, 31
mesoporous silica, 183185 biosensors
metal nanoparticle-based, 466 acetylthiocholine, 155156
nanoporous gold, 198199 ethanol, 154155
principles, 454 glucose, 151154
readable signal, 454 hydrogen peroxide, 157158
ZnO nanoparticles, 467 immunosensors, 162
DPN. see Dip-pen nanolithography NADH, 159160
phenol, 156157
protein electron transfer, 160162
E VACNF array-based, 162167
Electrochemical biosensing DNA
CNT CNTs, 27
advantages, 208 detection, 2728
structure, 207 ECL-based biosensing, 29
576 Index

Electrochemical sensing (cont.) F


hybridizations, 455456 Ferrocene-doped silica (FCDs), 312
Pd NPs, 2728 Fluorescence imaging
electron-transfer mediators, 375 advantages and disadvantages, 488
nitric oxide (see Nitric oxide) antibody-conjugated NPs
and photoelectrochemical prostate cancer in vivo detection, mouse
CVs, 127, 128 model, 491
ORR, 127 QDNCs cytosolic delivery and
responses, nafion-TiO2-porphyrin and subcellular targeting mechanism,
nafion-porphyrin membranes, 127, 491, 492
128 aptamer-conjugated nanomaterials
SiO2/TiO2/phosphate/CoTMPyP, CV cancer detection, 492
curves, 127, 1329 nanomaterials, 493
saccharideprotein interactions, 419 QD-Apt(Dox) Bi-FRET system, 493
Electrochemical sensors carbohydrate-conjugated nanomaterials, 494
critical problems, 281 CNTs, infra-red cytosensing
current, 279 in vitro NIR photoluminescence
detection technique, 281 images, human malignant glioma
L-Glu, D-Glu, 281 cells, 499
MIP based, 279280 optical properties, 498
neurotransmitter dopamine, 280 SWNTs, 498
potentiometric, 280 hydrogel NPs, 497
preparatin procedures, PATP-AuNP-gc, luminescent porous silicon NPs, 495496
280, 281 NPs application preconditions, 488
Electrochemiluminescent (ECL) strategies, peptide-conjugated NPs
525 aptamer, 489
Electrogenerated chemiluminsecence (ECL) human glioblastoma U87MG cells
biosensing strategy and application, in vitro staining, 490
251260 polymer-conjugated NPs
coreactant system cell types, 495
dichloromethane system, 251 sensor array design, 495, 496
hydrogen peroxide system, 249250 silica-shelled single QD micelle structure,
oxalate system, 247 489
peroxydisulfate system, 249 streptavidin-conjugated NPs, 494
sulfite system, 250 Functionalized magnetic nanoparticle (MNPs),
TPrA system, 247249 50, 51
description, 241
driving forces, 241
generation type, NP G
applications, 246247 Glassy carbon electrode (GCE), 115, 211
band-gap, 246 Glucose oxidase (GOD), 193194
CdSe/ZnSe core/shell spectrum, 245, Glycans
246 biological processes and metabolism, 396
and PL, 245 protein interactions, 397
spectrum, TGA-capped CdSe QDs/ structural and modulatory properties, 396
H2O2 system, 246 Gold nanoparticle-fluorophore complexes
mechanism, NPs bacteria and mammalian cells, 355356
annihilation routine, 243 chemical nose/tongue approach, 352353
coreactant routine, 243244 conjugated polymer/GFP, 353
time line, 242 protein detection
ELISA. see Enzyme-linked immunosorbent assay cationic, 354
Enzyme-linked immunosorbent assay (ELISA) fluorescence-response patterns,
carcinoembryonic antigen (CEA), 48 353354
colorimetric, 5758 human serum, 354355
nano-bio-chip system, 49 NP-PPE construct, 354
Index 577

Gold nanoparticles (AuNPs) I


AChE mediated hydrolysis, 376 ICS. see Ion channel switch
advantages, 413414 Immune response, 426, 517
anti-p24-modified, 545 Immunoassay, NP ECL
aptamer-functionalized, 511 biosensor preparation, 251
biocompatible and nontoxic pegylated immunosensor construction and
class, 508 incubation, 259
biosensing applications, 464 label-free strategy, 258
carbohydrate-attached, 416 Immunosensors. see also Immunosensors
carbon electrode, 419 and immunoassays, nanomaterials;
characteristics, 375 Pesticides, immunosensors
DNA sensing, 464 array construction, 16, 29
electrocatalysis and electrochemical ECL, 4, 52, 53
biosensing NP, 72
ARN sensor, 134 preparation, 73
catalytic mechanism, cluster/Mn(TPP) Immunosensors and immunoassays,
Cl system, 133, 134 nanomaterials
supramolecular modified electrode, 133 antibodyantigen binding, 425
electrocatalysis, NO oxidation, 340 antigen and antibody reaction
electrochemical AChE sensor, 375376 classification, 426
functionalization and characterization haptens, defined, 426
composite AFM images, mica, 132, 133 hybridoma fusion, 427
composite and TEM images structure, immune response, 426
131, 132 polyclonal antibody, 427
MPCs, 132 biocompatible
glycoconjugated, 411412 immobilization substrates, 429435
immobilize biomolecules, 375 probe carriers, 444447
immunoassay, 440 signal tags, 436444
maltoside alkanethiol, 412 competitive and sandwich method, 428
multivalent fluorescent polymer, 495 components, 429
nanomaterial, 430 imobilization procedure, 428
optical detection, 416 Insecticides detection, nanostructured
SERS-active nanopipette, 509 biosensing
standard avidinbiotin chemistry, 413 AChE activity and pesticides
tracer ChE activity biomonitoring, 382384
AFP determination, 67, 69 organophosphate pesticide exposure,
anti-CA 15-3-HRP, 65, 66 381382
Au NP and MMP probes, 67, 68 carbon nanotube-based
differential pulse voltammetry, 70 compound-specific biosensors, 372
DNA-Au bio-bar code, 71 enzyme-modified electrodes, 372
hepatitis B surface antigen (HBs-Ag), 67 flow-injection amperometric biosensor,
linear calibration range, 71 373
magnetic microparticle, 67 organophosphorous insecticides,
magnetic separation and amplification, 67 374375
MSO probe, 69, 70 solid-phase extraction (SPE), 374
serum biomarkers, 71 conventional strategies
tri-n-propylamine (TPA), 67 chromatographic methods, 365
virus surface, 417 immunosensors, 366
screening techniques, 366
developments
H biological binding events, 367
Helix pomatia agglutinin (HPA), 521 electrical and electrochemical
High-performance liquid chromatography techniques, 366
(HPLC), 404 enzyme-immobilization technique, 367
Hybridoma fusion, 427 picomolar (pM) to nanomolar (nM), 367
578 Index

Insecticides detection, nanostructured gold colloid solutions, 412


biosensing (cont.) LDI-TOF mass spectra, 411
electrochemical sensor development, 372 microarray and nanoprobe array,
enzymes 408409
AChE-based biosensors, 369371 mono and oligosaccharides, 404
ChE-ChO bienzyme-based biosensors, MT QD-589 agglutination, 407408
367368 nanoprobe assembly and
OPH-based biosensors, 371 electrochemical strategy, 405406
gold nanoparticles, 375376 plasmon absorption band, 407
pesticide immunosensors (see Pesticides, protein signal amplification, 405
immunosensors) saccharide motifs, 404405
quantum dots, 376377 sensitivity levels, 409
Ion channel switch (ICS) silica-coated nanoparticles, 406
BLM and gramicidin A, 554 surface fabrication, 409410
Medix test, 554 cell-surface, 494
Iron oxide (Fe3O4) QD, 523
colorimetric method, 93 Linker chemical reaction
dual functionality, 91 bioconjugation strategies, 7
electron-transfer process, 89 biomolecules, 67
glassy carbon disk electrode, 89 Localized surface plasmon coupled
GOx, 92 fluorescence (LSPCF), 41
immunoassays, peroxidase activity, 90, 91 LSPCF. see Localized surface plasmon
intrinsic properties, 92 coupled fluorescence
MichaelisMenten curves, 9091
mixed-valence compound, defined, 89
steady-state kinetic parameters, 90 M
Iron(II) sulfide (FeS) Magnetic nanoparticles
electrocatalytic activity, 94 ELISA, 92
micelles-assisted synthetic method, 93 functionalized fabrication process, 1516
MichelisMenten kinetics, 94, 95 MRI
peroxidase activity, 95 cell surface targeting, 500501
intracellular trapping, 502
multifunctional, 503506
K nucleic acid/antigen-functionalized, 16
K3Fe(CN)6 PB-Fe3O4, 1516
artificial peroxidase, 88 peroxidase activity, Fe3O4, 91
cupric hexacyanoferrate, 88 protection, 15
electrocatalyst reduction, H2O2, 87 sandwich-type DNA detection strategy,
PB-modified electrodes, 89 1617
redox potentials, 88 Magnetic resonance imaging (MRI)
advantages and disadvantages, 500
cell surface targeting
L cancer cells types, 500
Lectin magnetomechanical cancer-cell
based biosensors destruction, 501
BHK-21 and Caco-2 cells, 410 intracellular trapping
CdSe-ZnS QDs, 407408 integrin avb3, 502
cell-binding patterns, 409 RGD and CTX, 502
direct qualitative profiling, 410 multifunctional magnetic NPs
disease biomarkers and therapeutic BBS, 504
targets, 408 bimodal imaging nanoprobes
DSC-mediated dextran conjugation, 406 preparation, 503
electrochemical recognition protocol, 405 fluorescent dye-doped silica, 504
GCNPs, 411 in vivo accumulation, tumor site, 504, 505
Index 579

lipoproteins and phospholipid, 504 metal oxide


possible variants, protein-assisted characteristics, 191
nanoassembler method, 506, 507 MnO2, 191192
pure and functionalized QD and morphology, 176
SPIO-nanosomes, 504, 506 pore size
noninvasive medical diagnostic technique, biomolecules, 174
499 enzyme and stabilization effect,
Mass-sensitive devices 174175
amino acid sequences and affinities vs. MCM, 173174
QCM chip, 291292 protein and enzyme property, 174
development problems, MIP, 293 protein immobilization, 173
epitope-mediated vs. protein-engaged silica
imprinting, 290291 biosensors based, 178185
MIP layer, honeycomb-like structures, 289 protein immobilization methods,
pollen stamp preparation and contact mode 177178
AFM image, 293 surface characteristics
proteins determination, 290 adsorption and desorption behavior, 176
QCM-MIP sensors, 289 hydrophilicity/hydrophobicity and
sensing film preparation, 292293 charge, 175
viral and bacterial infections, 292 interactions types, 175
Mesocellular silica-carbon nanocomposite protein structure, 175176
foam (MSCF), 192, 193 Mesoporous silica
Mesoporous carbon dye-doped, 504
amorphous immuno-and DNA biosensors
BGMC, 186187 AMI, 184
CVs, 185186 cTnI analytical procedure protocol
EIS, 185, 186 format, 184
electrochemical biosensor, 185 working principle, 184185
ethanol and glucose detection, 187 protein immobilization methods, 177178
FT-IR spectra, CNTs, 188, 189 redox proteins direct electrochemistry and
GCE, CVs, 187 applications
OMCs, 185 BMS, 180
graphitic CVs, Hb/HMS-modified GCE, 179,
CVs, 190, 191 180
enzyme immobilization, 189 ET, 179
Hb-Nafion-CNTs/GC, 190191 Hb/HMS electrode, 179
XRD patterns, 190 Mb/HMS/GCE, CVs, 180, 181
Mesoporous materials MichaelisMenten kinetic mechanism,
carbon 181
amorphous, 185189 two-enzyme system co-immobilization and
graphitic, 189191 application
compositions, 173 cyclic voltammograms, 182, 183
hybrid nanocomposite enzyme electrode equations, 182
amperometric responses, MSCF and mechanism, 182
Pd-MSCF, 194 Mesoporous silica nanoparticles (MSNs),
CVs, 193194 17, 75
enzyme and protein immobilization, Metal nanoparticles
192 adenosine aptamers, 13
GOD, 193194 Au NPs-bacteria cellulose nanofiber, 1314
MSCF characteristics, 192, 193 gold, 464465
OMC, SWCNT and MWCNT, 192193 photoinduced reduction, 465
Pd-MSCF and MSCF, CVs, 194195 sensing strategies and detection methods,
Pd-MSCF morphology and 465466
composition, 194, 195 three-dimensional architectures, 1213
580 Index

Metal oxide nanoparticles Multi-walled carbon nanotubes (MWCNTs),


bioceramic zirconia alcogel, 318 113, 192193, 513, 519
GOD/Nano-SnO2/ITO bioelectrode, 319
mesoporous MnO2, 317318
spectroelectrochemistry, Hb, 316, 317 N
TiO2/HRP biosensor, H2O2, 316317 Nanocomposite matrix, sol gel
zirconia/nafion composite film, 318 carbon nanotube (see Carbon nanotubes)
Mimic enzymes GNPs, 3D network, 319
CcO crystal structure, bovine heart, 111112 metal nanoparticle
conjugated organic molecules, 111 AuNPs, 319320
vs. picket-fence porphyrin, 111 ECL glucose biosensor, 320321
Mimic enzyme sensors ethanol vapor detection, chemiresistor,
glucose, 99100 321, 322
glutamate, 9899 HBsAb immunosensor, 320
H2O2, 98 silver, 321
nonelectroactive cations, 100 TiO2/Au, 321
oxidizable and nontraditional compounds, multi-nanoparticle
100101 glucose biosensor, 325
transition metal hexacyanoferrates, 101102 gold/platinum hybrid, 325
Molecularly imprinted nanomaterials H2O2 biosensor, 325
advantages, sensor technology, 268 platinum, 326
antibody-based chemosensor, 265266 oxide nanoparticle, 321322
chemical sensors and biosensors, 265 Nanodevices
MIP cantilever nanobiosensors, 552554
biosensors, 276293 electrical nanobiosensors
comparison, antibodies and aptamers, microelectrode gap, 549
265266 nano-gapped microelectrode-based
development, 273276 array, 549
fabrication strategies, 266, 268 fiber-optic nanobiosensors
types, 271273 BPT detection, 552
molecular recognition, 294 nanometer scale measurement, 552
technology ICS nanobiosensors
covalent approach, 270 BLM and gramicidin A, 554
non-covalent approach, 269270 Medix test, 554
tripeptide (Lys-Trp-Asp), 271 nanowire nanobiosensors, 550552
Molecularly imprinted polymers (MIP) Nanofabrication
attractiveness, nanomaterials nanoarrays
effective binding sites distribution, DPN, 544545
275276 multiple-DNA inks, 544545
nanosized materials, 275 sandwich immunoassay, 545
biosensors (see Biosensors)novel nanofluidic channel
exploration, strategies electrophoretic mobility, 548
approach, surface, 274 physical principles, 548
ATRP, 274275 nanopore technology
limited mass transfer and template DNA-nanopore, 546
removal, 274 single-base mismatch, 546547
sensor application, 274 Nanomaterials
traditional limitation applications, electrochemical sensors, 162
factors, 273274 biofunctional
monolithic approach, 273 applications, 2131
types Au NPs immobilization, 18, 19
inorganic materials, 272273 carbon-based, 1012
organic materials, 272 characterization, 1921
Multiwall CNT ceramic electrode magnetic, 1517
(MW-CNCE), 322323 metal NPs, 1214
Index 581

MSNs, 17 Nanoparticle-amplified electrochemical


polyaniline (PANI) nanotube array, 18 detection
semiconductor, 1415 enhanced conductivity
biofunctionalization method biomolecular interaction, 54
covalent route, 510 on-chip protein array, 55
noncovalent assembly, 25 sandwich immunoassay, 55
calcium phosphate nanoparticles, silver-enhanced labeling method, 5556
1401441 impedance signal
carbon-based anti-human IgG antibodies, 62
CNHs, 119120 carcinoembryonic antigen (CEA), 59, 61
CNTs, 113116 DNA biosensors, 58
graphene sheets, 120122 microelectrode array, 6162
optical and electrochemical biosensing, Salmonella spp., 61
117119 stripping voltammetry
cell surface carbohydrate assay, substrate, Au and Ag metal tracers, 57
535544 biorecognition, 58
cytosensing cascade signal amplification, 58
colorimetric, 511 cyclic accumulation, 58
electrochemical, 511517 multiplexed bioanalysis, 58
fluorescence imaging, NPs, papillary thyroid carcinomas, 57
488499 picomolar and sub-nanomolar levels,
MRI, 499506 DNA, 57
SERS detection, 505510 vascular endothelial growth factor
dendrimers (VEGF), 58
chemistry techniques, 541 voltammetric signal
DNA assays, 542 Au NP-based amplification, 62
fullerenes hydrazine, 64
application and mechanism, 543 layer-by-layer assembly technique, 65
features, 542 thiolated DNA probes, 63
inorganic UV-vis spectra, 66
Fe3O4, 91 voltammograms, 64
FeS, 94 Nanoparticle-amplified optical assay
metal hexacyanoferrates, 100 biorecognition process, 40
nanomaterial-based biosensor, 79 chemiluminescence (CL) and ECL
nanoparticles (see Nanoparticles) amidization reaction, 50
nanotubes Au nanoparticles amplification, 4950
CNTs, 538 bio-bar-code, 50, 51
surface-protein and protein-protein CdSe QDsCNTs conjugation, 5254
binding, 539 dual-amplification, 5051
SWNT AFM probes, 538 ECL DNA assay, 52, 55
nanowires IECLE curve, 53, 56
bi-segment, 540 immunosensor, 53
motion-based nucleic acid detection, luminolKMnO4, 51, 52
540541 NPGL electrode, 53
self-electrophoresis mechanism, tDNA sensitivity, 51
539540 thioglycolic acid (TGA), 51
nitric oxide (see Nitric oxide (NO)) colloidal gold
optical sensor and array antibody microarray, 4445
chemosensor, 358361 DNA and antibody-antigen analyses, 40
CTL, vapour sensing, 356358 dynamic light scattering (DLS), 4344
organic compounds recognition, CTL, enhanced resonance light scattering, 42
361362 fluorescence signal, 41
polymer nanoparticles, 137138 LSPCF, 41
signal amplifiers, 47 metal-enhanced fluorescence, 4546
silica nanomaterials, 138140 NEANA, 4546
582 Index

Nanoparticle-amplifi ed optical assay (cont.) Pt, 135136


polyhedral oligomeric silsesquioxane sensing strategies and detection
(POSS), 47 methods, 465466
radiative decay rate acceleration, 46 nanomaterials, cytosensing
rolling-circle amplification (RCA), assembly, 487517
4142 usages, 525
sandwich type assay, 42, 43 Ni, 152153
scanometric immunoassay, 44 palladium, 176
sensitive detection method, adenosine, polymer, 137138
4243 POSS, 47
SNP genotyping, 41 properties, 486
Verigene reader system, 45 QD, 537538
high sensitivity and ability, 40 semiconductor (see also Semiconductor
semiconductor nanoparticles)
beads, anisotropically etched silicon Fe3O4, 131
chip, 48 quantum dots, 129130
ELISA, 49 TiO2-porphyrin nanocomposite, 122129
quantum dots (QDs), 48 silver, 541
spectral shifts, 40 spectral shifts, 40
Nanoparticles (NPs) stripping voltammetry, 5758
amplified optical assay Nanoporous gold (NPG)
colloidal gold nanoparticle, 4047 characteristics, 196
nanoparticle-amplified DNA
chemiluminescence, 4954 biosensor preparation, 198
semiconductor nanoparticle, 4849 chronocoulometry determination, 198
antibody-conjugated, 561 dual-amplification effects advantage,
bimetallic, 199 199
bio-bar-code assay, 536537 enzyme
calcium phosphate, 140141 advantages, 196
cancer detection, 527 CVs, NADH and H2O2, 196, 197
cancerous vs. noncancerous cells, 485 CVs, NPG/GCE and gold sheet
carrier, signal amplification electrode, 196, 197
carbon nanotubes, 7374 Escherichia coli
gold nanoparticles as tracer, 6573 PGNF, 199200
silica nanoparticles, 7577 SEM images, 199
cell surface carbohydrate assay, 518520 Nanostructured mimic enzymes
coreactant system, artificial
NP ECL, 247251 Breslows mimics, 9698
description, 486 Fe3O4, 8993
ECL mechanism, 242244 FeS, 9395
enhanced conductivity, 5456 K3Fe(CN)6, 8789
enhanced impedance signal, 5862 polystyrene, 9596
enhanced voltammetric signal, 6265 biomolecules immobilization, 86
fluorescence labels, 48 development, 86
generation type, 244247 disadvantages, 85
gold, 536 electrochemical biosensors, 85
magnetic, 1517, 538 sensors
mesoporous, 176 glucose, 99100
metal glutamate, 9899
Ag, 134135 H2O2, 98
Au, 131134 nonelectroactive cation, 100
gold nanoparticles, 464465 oxidizable compounds and
particles, 40 nontraditional, 100101
photoinduced reduction, 465 transition metal hexacyanoferrate, 101102
Index 583

Nanostructures H2O2, 118


biochips L-glutathione, 119
electrochemical methods, 474478 O2 detection, 4-electron reduction
optical techniques, 471474 dissolved oxygen measurement, 117
biosensing GNP/MWCNTs-FeTMAPP monolayer
CNTs, 455464 fabrication process, 117
metal nanoparticles, 464466 Sudan I, 118119
semiconductor nanostructures, 467473 Optical sensing
NEANA. see Nicking endonuclease assisted bioanalytes colorimetric detection
nanoparticle amplification barcode DNA-mediated Au NP, 22
Nicking endonuclease assisted nanoparticle oligonucleotides, 2122
amplification (NEANA), 45, 46 fluorescence detection
Nitric oxide (NO) Au NPs and DNA/RNA, 23
electrochemical sensor, nanomaterials cascade procedure, signal amplification,
carbon nanotubes, 337 23
CFMDE, 341342 emission, Cy5, 2425
CFUE, 352353 FRET, 2425
CFUE/MWNT/Nafion electrode, small RNA detection assay,
343344 CXFluoAmp, 2324
CuTAPc, 338339 spectroscopic measurements
electrooxidation process, 339 normal Raman spectra, fresh citrus
gold nanoparticles, 340 fruits, 2526
linear ranges and detection limits, 341 SERS, 2526
MWNTs-ACB and PACB, 337338 SWNTs advantages, 2627
nano-alumina, 341 two-photon Rayleigh scattering
nanocrystalline TiO2, 340 properties, 26
Ni-porphyrin, 336337 Optical sensors
SWNTs/Nafion modified CFMDE, Au NPs, 287
342343 catalytic nanomaterial-based
endothelium-derived factor, 226 chemosensor (see Chemosensor)
nanostructure CTL (see Cataluminescence)
carbon fiber microelectrode, 334 CL, 285286
nanometer-sized NO sensor, 334335 competition approach, 283
nanosensor response, 335336 dansyl-proline recognition, 285, 286
NO. see Nitric oxide electropolymerization and SPR detecting
Noncovalent interaction zearalenone setup, 287, 288
aromatic molecule epinephrine detection, 285286
N-succinimidyl-1-pyrenebutanoate, 209 fluorescence enhancement and quenching,
nucleophilic substitution, 209 283
entrapment IR, 286
electrocatalytic ability, 211 LC-imprinted silica nanosphere CdSe
imidazolium ring moiety, 212 quantum dots, 283284
rolling circle amplification, 210 NADH measurement, 287
sensor fabrication, 212 SPR-MIPs, 287
vials photographs, 210 target binding event and transduction,
layer-by-layer fabrication 282283
charged MWCNTs, 212213 turn-on/turn-off mechanism, 282
LBL assembly, 212 Ordered mesoporous carbon materials (OMCs)
molecule-based functionalization, 209210 advantage, 187
electrochemical biosensor, 185
mesoporous group, 185
O Organophosphorous pesticides (OPs), 365
Optical and electrochemical biosensing Oxygen reduction reaction (ORR),
DNA, 118 127, 154, 325
584 Index

P nanometer-scale particles, 112


PEBBLEs. see Probes encapsulated by Ni-porphyrin, 336337
biologically localized embedding noncovalent nanoassembly, 226
Periodic acidSchiff (PAS) reaction, 399 peroxidase mimetics, 86
Pesticides photoelectrochemical and electrochemical
AChE-based biosensors, 369371 sensing, 127129
biosensors development, 366367 polymer nanoparticles, 137138
ChE-ChO bienzyme-based biosensors, quantum dots
367368 semiconductor nanoparticles, 129
conventional strategies, detection, 365366 thioglycolic acid capped CdTe QDs,
in situ inspection, 26 interaction mode, 130
immunosensors semiconductors host surfaces, 122
assay format monitors, 378 silica nanomaterials, 138140
detection methods, 377378 TiO2
electrochemical immunosensing, 380 anchoring groups, 123, 124
flow-immunodetection system, 379 and anticipated binding geometries
immunochemical methods, 378 structures, 123
OP-AChE, magnetic electrochemical drawback, 122123
immunoassays, 379380 water-insoluble, 210, 228
point-of-care (POC) diagnosis methods, POSS. see Polyhedral oligomeric
379 silsesquioxane
OPH-based biosensors, 371 Probes encapsulated by biologically localized
Polyhedral oligomeric silsesquioxane (POSS), embedding (PEBBLEs), 555
47
Polymer nanoparticles
magnetic nanospheres, 138 Q
PtOEP, oxygen sensitive dye, 137, 138 QDs. see Quantum dots
thermochromic and dual-sensing optical Quantum dots (QDs)
sensors, 137138 biosensing mechanism, 468
Polystyrene, 9596 fluorescent semiconductor, 472
Porphyrin inorganic fluorophores, 537
amperometric response, 223, 228 intrinsic redox properties, 467
biosensing, problems, 141 molecular beacons, 467
calcium phosphate nanoparticles, 140141 nanomaterials, 486
carbon-based nanomaterials semiconductor nanoparticles, 467
CNHs, 119120 silica layer/shell, 488
CNTs, 113116 toxic effects problem, 538
graphene sheets, 120122 Quasi-one-dimensional (Q1D) nanomaterials,
optical and electrochemical biosensing, 455
117119
crystal structure and biomimetic analogs,
113 S
Fe3O4 nanoparticle, 131 Semiconductor nanoparticles
H2O2 and ascorbic acid detection, 85 amine-modified QD705, 15
macrocyclic structure, 111112 amplification
metal nanoparticles assay validation studies, 49
Ag, 134135 biorecognition, 48
Au, 131134 quantum dots (QDs), 48
Pt, 135136 carboxylate ligands, 15
nanocomposite characterization porphyrins assembly
O 1s high-resolution XPS spectra, Fe3O4, 131
TiO2-based samples, 125, 126 quantum dots, 129130
TEM images, 124, 125 TiO2-porphyrin nanocomposite, 122129
XRD patterns, 125126 preparation, 55
Index 585

QDs, 1415, 467 LSPR-based biosensor, 313


tunable absorbance and fluorescence, 40, core-shell nanostructure
48 amorphous layers, 315
Signal amplification AuNPs-C @ SiO2, 316
Au NP-based, 31 dye-doped, 314
bio-barcode-functionalized magnetic NPs, 16 fluorescent, 315316
carrier magnetic iron oxide and Ru(bpy)32+,
carbon nanotubes, 7374 316
CdTe-tagged polybeads, 77, 78 doped
clinical serum samples, 78 ECL sensor, 312
gold nanoparticles, tracer, 6573 FCDs, CHIT & SLE, 312
silica nanoparticles, 7577 fluorescent, 312313
thionine-horseradish peroxidase surface modification, 311, 312
conjugation (TH-HRP), 7778 mesoporous, 17
cascade procedure, 23 pure
DNA and antibody-antigen analyses, 40 anti-diclofenac antibodies
dual, SWNTs, 31 encapsulation, 311
electrochemical detection, nucleic acids, 221 GDH and LDH, 310
fluorescence, 521 lysozyme, 311
multiplexed immunoassay, 218 Mb and Hb, 310
nanoparticle-amplified electrochemical reverse micelle method, 310
detection Stber method, 310
enhanced conductivity, nanoparticles, Single-walled carbon nanotubes (SWNTs),
5456 113, 192193, 498, 499, 507,
impedance signal, 5862 517, 518
stripping voltammetry, 5758 Solgel
voltammetric signal, 6265 biosensors, nanoparticle matrices
nanoparticle-amplified optical assay metal oxide, 316319
chemiluminescence and nanocomposite, 319326
electrogenerated silica, 310316
chemiluminescence, 4954 description, 306
colloidal gold, 4047 nanoparticle, 309
semiconductor, 4849 process, steps
nanotechnology applications, 504 aging, 307308
revolutionized genetic testing, 39 casting and gelation, 307
ultrasensitive and simple electrochemical dehydration and densification, 308
method, 30 drying, 308
without enzymes, 29 mixing, 306307
Silica nanomaterials SPR. see Surface plasmon resonance
amorphous, 138 Structural analysis, carbohydrates
porphyrins, 138139 chirality, 400401
zinc(II) porphyrin derivative and SiNW- glycosylation sites mapping, 401402
FET device, 140 positions determination, 400
Silica nanoparticles sequence analysis, 401
carrier Surface-enhanced Raman scattering (SERS)
CdTe QD-functionalized silica cytosensing based
nanosphere labels, 76, 77 cancer cell target, 508509
doped silica nanoparticles (DSNPs), multiplex, 509
7576 NP, 509
mesoporous silica nanoparticles in vivo imaging, zebrafish model,
(MSN), 75 509510
chemically bonded labels types, 507
HeLa cells, MSN, 313314 mechanism, 508
HRP and anti-AFP coimmobilization, 313 nanostructures, conditions, 508
586 Index

Surface plasmon resonance (SPR) LB technique, 350351


biosensing, 41 POEA and PSS, LBL, 351
signal amplification, 40 TiO2-porphyrin nanocomposite
SWCNTs-based field-effect biosensing characterization, 124127
charge transport, 229 photoelectrochemical and electrochemical
nucleic acids detection sensing, 127129
DNA sensing, 231 porphyrin bound, 122124
immobilized thrombin aptamers, 231 Tobacco mosaic viruses (TMV), 292
network CNTs-based field-effect Transmission electron microscopy (TEM),
transistor (NTNFETs), 231 124, 125
proteins detection Tri-n-propylamine (TPrA) system
antibody-based detection, 229 emissions, 248
electrical conductance, 229, 230 oxidation/reaction sequence, abbreviations,
GOD enzymes, 229 248
liquid-gate voltage, 229, 230 oxidative-reductive coreactant, 247
System lupus erythematosus (SLE), 312

V
T VACNF. see Vertically aligned carbon
Taste sensing nanofiber
carbon nanotube polymer composite, 352 Vertically aligned carbon nanofiber
catalytic nanomaterial-based optical (VACNF)
sensor covalent functionalization, 164
chemiluminescence (CTL), 356358 CuAAC reaction, 164
chemosensor array, 358361 electron microscope image scanning,
organic compounds recognition, CTL, 162, 163
361362 ferrocene attachment, 165, 166
gold nanoparticle-fluorophore complexes molecules detection, 162, 164
bacteria and mammalian cell detection, photochemical grafting and CuAAC
355356 reaction, 164
chemical nose/tongue approach, 352 photoresist-based blocking, 165167
conjugated polymer/GFP, 353
protein detection, 353355
nanoassembled conducting polymers X
electronic tongue, 351 X-ray diffraction (XRD), 125126

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