Week 2 Bioisensors 21sep2009 Bio Catalysis

Biocatalysis Based Biosensors, Bioaffinity Based Biosensors & Microorganisms Based Biosensors, Biologically Active Material and Analytes
H 3C CH 2 H3C H3C CH H3C 2 H3C S S S S S S S
T R A N S D U C E R
AMPLIFIER
DISPLAY
ANALYTE
BIOMOLECULE
MATRIX
Centre for NanoBioengineering & Spintronics, Chungnam National University,Daejeon,Korea

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Biosensor Biocatalysis based Biosensors Biaffinity based Biosensors Micoorganisms Based Biosensors Conclusions Literature
10/5/2009 WCU Project, CNU,bansi.malhotra@gmail.com 2
BIOSENSOR
What is a BIOSENSOR ?
IUPAC Nomenclature: A biosensor is a self-contained integrated device which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is in direct spatial contact with a transducer element.
It consists of 3 parts
The bioreceptor. The transducer or the detector element Associated electronics or signal processors that is primarily responsible for the display of the results in a user-friendly way.
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Classification of Biosensors
Classification based on transducer system
Calorimetric (detect on the basis of heat evolved in biological reaction) Piezoelectric (detect on the basis of electric dipoles generated due to mechanical stress) Optical (detect on the basis of change in light received ) Electrochemical such as Potentiometric, Conductometric and Amperometric
Classification based on bio-recognition element

Enzyme DNA Antigen-antibody Cell
Characteristics of a Biosensor
(i) Selectivity, (ii) Recovery time (iii) Shelf-life (iv)Stability, (v) Response time, (vi) Accuracy, (vii)Reusability
Biocatalysis based sensor

Biocatalysis-based biosensors depend universally on the use of enzymes. The field of biocatalysis is open. This frontier of research is racing ahead, propelled by advances in the database-supported analysis of sequences and structures as well as the designability of genes & proteins.
Biocatalytic processes differ from conventional chemical processes, owing mainly to enzyme kinetics, protein stability under technical conditions and catalyst features that derive from their role in the cells physiology, such as growth, induction of enzyme activity or the use of metabolic pathways for multistep reactions.
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Catalysts are substances that speed up chemical reactions. Organic/bio-catalysts are called enzymes. Reactions with enzymes are up to 10 billion times faster than those without enzymes. Enzymes are specific for one particular reaction or group of related reactions.
An enzyme-substrate complex forms when the enzymes active site binds with the substrate like a key fitting a lock. The shape of the enzyme must match the shape of the substrate. Enzymes are ,therefore, very specific; they will only function correctly if the shape of the substrate matches the active site
The enzyme does not form a chemical bond with the substrate. After the reaction, the products are released and the enzyme returns to its normal shape. The enzyme molecule can be reused. Only a small amount of enzyme is needed because they can be used repeatedly.
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Effect of Temperature Increase in the temperature of a system results from increases in the kinetic energy of the system. This has several effects on the rates of reactions. 1. More energetic collisions
The greater the kinetic energy of the molecules in a system, the greater is the resulting chemical potential energy when two molecules collide .
2 The number of collisions per unit time will increase.

In order to convert substrate into product, enzymes must collide with and bind to the substrate at the active site. Increasing the temperature of a system will increase the number of collisions of enzyme and substrate per unit time. Thus, within limits, the rate of the reaction will increase.
3 The heat of the molecules in the system will increase

As the temperature of the system is increased, internal energy of the molecules in the system will increase. The internal energy of the molecules may include the translational energy, vibrational energy and rotational energy of the molecules. Some of this may be converted into chemical potential energy. If this chemical potential energy increase is great enough , some of the weak bonds that determine the three dimensional shape of the active proteins may be broken. This could lead to a thermal denaturation of the protein and thus inactivate the protein. Thus too much heat can cause the rate of an enzyme catalyzed reaction to decrease because the enzyme or substrate becomes denatured and inactive.
Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the temperature is raised. A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%. This figure shows that the reaction rate increases with temperature to a maximum level, then abruptly declines with further increase of temperature. Because most animal enzymes rapidly become denatured at temperatures above 40C, most enzyme determinations are carried out somewhat below that temperature. Effect of pH pH can affect the ionization of the amino acid side chain, which in turn change the secondary, tertiary and quaternary structures of the protein molecule. This will change the enzyme's active site and consequently its activity.
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Enzyme Classification
There are approximately 3000 known enzymes. These enzymes are classified into six categories based on the type of reaction they catalyze. 1. Oxido- reductase: Oxidizes or reduces by transfer of hydrogen or electrons. (a) Dehydrogenases: SH2 + A S + AH2 (S: Substrate, A: acceptor) Example:Lactate dehydrogenase: L-lactate + NAD Pyruvate + NADH + H+ (b) Oxidases: SH2 + 1/2 O2 S + H2O or SH2 + O2 S + H2O2 Example:Glucose oxidase: -D-glucose + O2 Gluconolactone + H2O2 (c) Peroxidases: 2SH + H2O2 2S + 2H2O or 2S + 2H+ + H2O2 2S+ + 2H2O Example: Horse radish peroxidase: 2[Fe(CN)6]4- + 2H+ 2[Fe(CN)6]3- + 2H2O (d) Oxygenases: SH + DH + O2 S-OH + D + H2O Example: Lactate 2-monooxygenase: L-lactate + O2 acetate + CO2 + H2O
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2. Transferase: Transfers C-, N-, P-, or S-containing functional groups such as aldehydes and ketones, glycosils, acyls, phosphates, and sulfur containing groups. AX + B A + BX Example: Hexokinase: D-hexose + ATP D-hexose-6-phosphate + ADP 3. Hydrolase: Hydrolyses esters, anhydrides, peptide bonds, other C-N bonds, glycosides Example: Cholesterol esterase: Cholesterol ester + H2O cholesterol + fatty acid Glucoamylase: Amylose + n H2O n -D-glucose 4. Lyase: Adds to double bonds: >C=C< >C=O >C=N 5. Isomerase: Isomerizes optical iomers Example Glucose isomerase: D-glucose D-fructose
6. Ligase: Splits C-C, C-O, C-N, C-S and C-halogen bonds without hydrolysis or oxidation, mostly with ATP Example : Pyruvate Carboxylase: Pyruvate + HCO3- + ATP oxaloacetate + ADP + Pi
Coenzymes, Prosthetic group, Effectors

Sometimes the surface cavity does not act as a catalytic site until it is modified by a second incoming molecule. These participants known as the coenzymes are non-peptide molecules capable of completing the binding site for the transition state. Other molecules that do the similar function are prothetic group, and effectors.
Coenzyme: Coenzyme is a non-peptide molecule capable of completing the binding site for
the transition state. Examples include many vitamin derivative such as coenzyme A, thiamine, pyrophosphate, vitamin B12
Prosthetic Group: Prosthetic group is the same as the coenzyme but are tightly bound to
the enzyme. When they are split off, the enzyme is mostly denatured. Examples include flavin nucleotides and heme.
Effectors: Effectors accelerate (activators) or block (inhibitors) enzyme reaction

Examples of activators include Mg++, Ca++, Zn++, K+, and Na+, Examples for the inhibitors include Hg, and substrate analogs. Table 1 lists functions of some of the important coenzymes and prostshetic groups.
Table1:Function of some important coenzymes and prosthetic groups
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ENZYME KINETICS
Let us consider a catalytic reaction, k1 k2
E+S ES E+P
k-1
where E, S, P and ES represent the enzyme, substrate, product and transient complexes of the enzyme and k1 , k-1 k2 are rate constants (formation) and (breakdown),
So, Rate of ES formation d[ES]/dt= k1([E] - [ES])[S] Rate of ES breakdown = k-1[ES] + k2[ES] Now, the initial rate of reaction reflects a steady state in which [ES] is constant, e.g. the rate of formation of ES is equal to the rate of its breakdown. This is called the steady-state assumption.
k1([E] - [ES])[S] = k-1[ES] + k2[ES] Or, k1[E] [S] - k1[ES][S] = (k-1 + k2 )[ES] Or, k1[E] [S] = (k1[S] + k-1 + k2) [ES], Or, [ES] = (k1[S] + k-1 + k2)
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{by adding k1[ES][S]} [E] [S] Or, [ES] =
k1[E] [S]
[S] + (k-1 + k2)/ k1 The term (k2 + k-1) / k1 is defined as the Michaelisconstant, Km
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Assumptions
The first key assumption in this derivation is the quasi-steady-state assumption (or pseudo-steady-state hypothesis), namely that the concentration of the substrate-bound enzyme ([ES]) changes much more slowly than those of the product ([P]) and substrate ([S]). This allows us to set the rate of change of [ES] to zero and also write down the rate of product formation:
The second key assumption is that the total enzyme concentration ([E]0) does not change over time, thus we can write the total concentration of enzyme [E]0 as the sum of the free enzyme in solution [E] and that which is bound to the substrate [ES]:
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The validity of the following derivation rests on the reaction Scheme given below and two key assumptions: that the total enzyme concentration and the concentration of the intermediate complex do not change over time.
The most convenient derivation of the MichaelisMenten equation, described by Briggs and Haldane, is obtained as follows (Note that often the experimental parameter kcat is used but in this simple case it is equal to the kinetic parameter k2):
The enzymatic reaction is assumed to be irreversible, and the product does not bind to the enzyme.
BiocatalysisbasedBiosensoratNPL
GLUCOSE
CHOLESTEROL
UREA
GLUCOSE SENSOR
GLUCOSE + GLUCOSE OXIDASEOXIDIZED PRODUCT +GLUCOSE OXIDASE REDUCED GLUCOSE OXIDASEREDUCED +MEDIATOROXIDIZED MEDIATORREDUCED + GLUCOSE OXIDASEOXIDIZED + MEDIATOR REDUCED MEDIATOROXIDIZED+2e-
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Glucose Oxidase and the biochemical reactions involved during the Glucose sensing
Structure of glucose oxidase
The enzymatic reaction catalysed by glucose oxidase (GOx) Active site structure of GOx enzyme
Au-nanoparticles/ Polypyrrole Composite
Iron Oxide NanoparticlesChitosan Composite
Matrices for Glucose biosensor
Au nanoparticle/ Polyaniline Composite
Polythiophene Gold Nanoparticles Composite
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Au Nanoparticles / Polypyrrole Composite
20 ml of 1mm chloro auric acid solution
Heat up to boiling
Gold nanoparticles-polypyrrole thin film Covalently immobilized glucose oxidase (GOx) on Gold nano particles-polypyrrole thin film
Faint blue to wine red color Tri sodium citrate solution

I(A)
800
600
(iii)
400
(ii) (i)
200
Size ~ 10-20 nm
-2 0 0
-1 0 0 0
-5 0 0
500
1000
1500
UV-vis spectra: peak at 520nm

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E (m V )
(i) PPy/ITO (ii) GOx/AuNPs-PPy/ITO bioelectrode (iii) AuNPs-PPy/ITO electrode in phosphate buffer .05M, pH7.0, at scan rate 20mV/s 20
Cyclic voltammograms of GOx-PPy/ITO electrode as a function of different conc of glucose (25mg/dL-200mg/dL) in phosphate buffer .05M,pH 7.0, scan rate of 20mV/s
Cyclic voltammograms of GOx/AuNPs-PPy/ITO bioelectrode as a function of glucose concentration (25mg/dL300mg/dL).Gold nanoparticles enhance the sensitivity of the bioelectrode. (i-vii) - current
Au-nanoparticles / Polyaniline Composite
Journal of Nanoscience and Nanotechnology, 2008, 8, 3158.
Aniline
(AuNPs) PANI/ITO
HOOC-Enz EDC/NHS
H N +
N H
N H
ITO
N
H
N
H H N + N H
NH-CO-Enz
AuNPs/PANI/ITO
H N + N H N N H N H H +N N N + H N + N H H N + N H N H N2 H
N H
N H
N H N + H N +
N H N H
N N H N H N H N H
N H
2.5x10 2.0x10 1.5x10

Current (A)
-3
a
-3 -3
c a
2927
1615
1.0x10 5.0x10
-3
1247
800
Transmittance (%)
3186 1627 1259 3434 800
b
463
-4
0.0 -5.0x10 -1.0x10 -1.5x10

-4
c
1290 3434 1640 800 463
-3
-3
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
0.2
0.4
0.6
0.8
1.0
3434
4000 3500 3000 2500 2000
-1
Voltage (V)
1500
1000
500
Cyclic voltammogrammes (a) PANI/ITO film (b)AuNPS-PANI/ITO electrode (c) 10/5/2009 GOx/AuNPS-PANI/ITO bioelectrode
W avenum ber (Cm )
FTIR spectra of (a) PANI film (b) AuNPs-PANI (c) GOx/AuNPs-PANI film on ITO 22 WCU Project, CNU,bansi.malhotra@gmail.com
Glucose Oxidase Activity Immobilized on AuNPs-PANI/ITO

GOx/PANI/AuNPs/ITO
Glucose + O2 H2O2
Electrochemical oxidation
Gluconic acid + H2O2 2H+ + O2 + 2e-
.....Eq.1
.........Eq.2
Current response of GOx/AuNPs-PANI/ITO bioelectrode as a function of glucose concentration. 10/5/2009
Hanes plot of GOx/AuNPs-PANI/ITO bioelectrode as a function of glucose concentration

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Shelf Life
2.0x10
-3
1.0x10
Current (A)
-3
0.0
-1.0x10
-3
-2.0x10
-3
Shelf life of GOx/AuNPsPANI/ITO bioelectrode with time.

0 2 4 6 Weeks 8 10 12
Summary
Km value of immobilized enzyme on gold nanoparticles polyaniline composite films 2.2 mM (39.64 mg/dl) Response time of GOx/AuNPsPANI/ITO bioelectrode ~ 10 s clearly indicate that self assembled gold nanoparticles in PANI matrix provide biocompatible environment to enzyme Sensing property to glucose concentration 50300 mg/dl GOx/AuNPsPANI/ITO bioelectrode retains more than 85% of the GOx activity even after 11 weeks.
J. Applied Poly. Sci., 2008, DOI 10.1002/app.
FT-IR spectra of (a) regP3HT-AuNPs/Au film and (b) bifunctionalized gold nanoparticles (regP3HT-DTSPAuNPs-Au) film.
DTSP : dithiobissuccinnimidyl propionate Schematic of Covalent immobilization of glucose oxidase on bifunctionalized gold nanoparticles
Peak at 450 nm ~ P3HT moieties Peak at 557 nm~ P3HT capped AuNPs
UV-Vis absorption of (a) citrate capped gold nanoparticles (b) P3HT in toluene (c) P3HT - AuNPs in toluene. 25 WCU Project, CNU,bansi.malhotra@gmail.com
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Enzyme activity studies using UV-visible spectrophotometer

0.014 0.012
Absorbance (500 nm)
0.010
0.008
0.006
0.004
0.002 0 50 100 150 200 250 300 350 400 450
Conc (mg/dL)
Photometeric response of GOx-regP3HT-AuNPs DTSP/Au) bioelectrode as a function of analyte (glucose) concentration.
Hanes plots of GOx-regP3HT-AuNPsDTSP/Au bioelectrode as a function of analyte (glucose) concentration.
Absorbance response of GOx-regP3HT-AuNPs Effect of temperature on response of GOxDTSP/Au bioelectrode in PBS buffer (50mM, 0.9 regP3HT-AuNPsDTSP/Au bioelectrode 10/5/2009 NaCl) of pH (i) 6.0 (ii) 6.5 (iii) 7.0 (iv) 7.4 (v) 8 WCU Project, CNU,bansi.malhotra@gmail.com
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Iron Oxide NanoparticlesChitosan Composite

Biosens. Bioelectron., DOI. 10.1016/j.bios.2008.06.032
Iron oxide nanoparticles (Fe3O4) has been prepared using co-precipitation method. Nanocomposite of chitosan and Fe3O4 has been prepared using electrostatic interaction between positively charged CH and negatively charged Fe3O4 nanoparticles.
WCU Project, 10/5/2009 27 CNU,bansi.malhotra@gmail.com glucose oxidase on nanocomposite matrix Schematic of formation of CH-Fe3O4 Nanocomposite and immobilization of
SEM, CH/ITO
SEM, CH-Fe3O4/ITO
SEM, GOx/CH-Fe3O4/ITO
Km Value = 0.141 mM
50 nm Transmission Electron Micrograph of Fe3O4 nanoparticles
The activity of the GOx/CH-Fe3O4/ITO bioelectrode stored at 4 o C has been measured at different time interval. Stability curve of GOx/CH-Fe3O4/ITO It has been observed that the activity of glucosel oxidase bioelectrode as a function of absorbance with immobilized onto the ITO surface shows stability upto 8 weeks 10/5/2009 28 WCU Project, CNU,bansi.malhotra@gmail.com respect to time (weeks)
Electrochemical Response Studies of GOx/PANI/ITO Bioelectrodes
The bioelectrode shows linearity within the range of 50 to 400mg/dl of glucose with corelation factor of 0.99 and sensitivity of 0.1 x 10-3 mA/ (mg/dl).
DPV for GOx/NS-PANI/ITO bio-electrode as function of Glucose concentration
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Urea Biosensor
Estimation of urea in serum/blood/urine is important for diagnosis of renal and liver diseases. An increase in urea level normal range is 820 mg/ dl in blood and urine causes renal failure, urinary tract obstruction, dehydration, shock, burns, and gastrointestinal bleeding. Moreover, reduced urea level may cause hepatic failure, nephritic syndrome, and cachexia low-protein and high-carbohydrate diets.
Most urea biosensors are based on urease Urs and n catalytic conversion of urea to hydrogen bicarbonate and ammonium. It has been observed that ammonium ions easily diffuse in solution. Thus, glutamate dehydrogenase, GLDH has been used as an alternate since it catalyzes the reaction between ammonium ions, -ketoglutarate -KG and nicotinamide adenine dinucleotide NADH to produce Lglutamate and NAD+.
Zinc oxide-chitosan nanobiocomposite for P3HTsensor urea - SAM
Matrices for Urea biosensor
Iron oxide-chitosan nanobiocomposite for urea sensor

Zinc oxide-chitosan nanobiocomposite
APPLIED PHYSICS LETTERS 93, 163903 2008
XRD pattern of ZnO-CH nanobiocomposite film. b
Scanning electron micrograph of Urs-GLDH/ZnO-CH/ITO bioelectrode
Linearity =5100 mg/ dl, Detection limit = 3 mg/ dl Km = 4.92 mg/ dl
EIS of i) CH/ITO, ii) ZnO-CH/ITO, iii) UrsGLDH/ZnO-CH/ITO electrode, 10/5/2009
Electrochemical response of Urs-GLDH/ZnOCH/ ITO bioelectrode with respect to urea concentration 5100 mg CNU,bansi.malhotra@gmail.com 32 WCU Project, dl1 at scan rate of 10 mV s1
Iron oxide-chitosan nanobiocomposite
Sensors and Actuators B 138 (2009) 572580
SEM images of CH-Fe3O4 nanobiocomposite/ITO electrode
10/5/2009
X-ray diffraction pattern of Fe3O4 nanoparticles. WCU Project, CNU,bansi.malhotra@gmail.com
Ur-GLDH/CH-Fe3O4 nanobiocomposite/ITO electrode. 33
DPV of (a) CH/ITO, (b) CH-FeO4 nanobiocomposite/ITO and (c) Ur-GLDH/CH-Fe3O4 nanobiocomposite/ITO bioelectrode
Cyclic voltammograms of (a) CH/ITO, (b) CH-Fe3O4 nanobiocomposite/ITO and (c) Ur-GLDH/CH-Fe3O4 nanobiocomposite/ITO bioelectrode
Electrochemical response of Ur-GLDH/CH-Fe3O4 nanobiocomposite/ITO 10/5/2009 bioelectrode as a function of urea concentration (5100 mg/dL).
The effect of interferents on electrctrochemical response of 34 Ur-GLDH/CH-Fe3O4 nanobiocomposite/ITO bioelectrode
Cholesterol & Triglyceride Biosensor
Triglyceride
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Electrophoretically deposited nanostructured P3HT - SAM polyaniline film
Cholesterol
Polyaniline Langmuir Blodgett Films
Matrices for Cholesterol & Triglyceride biosensor
Electrophoretically deposited MWCNTc/polyaniline

Electrophoretically deposited nano-structured polyaniline film
analytica chimica acta 6 0 2 ( 2 0 0 7 ) 244251
Electrophoretic deposition of polyaniline from its colloidal suspension at 80V Formation of polyaniline colloidal suspension
0.35 0.30 Absorbance (Abs) 0.25 0.20 0.15 0.10 0.05 0.00
Colloidal suspension
(i)
(ii)
Film
Polyaniline chain
-NH+ ITO
400
600
800
1000
1200
Wavelengh ()
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Analytica Chimica Acta 6 0 2 ( 2 0 0 7 ) 244251
Conformational analysis of polyaniline
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Covalent immobilization of cholesterol oxidase on electrophoretically deposited polyaniline films

OH Enz O CH3CH2-N=C=N-(CH2)3N(CH3)2
Enz O NH-CH2CH3 O N N-(CH2)3N(CH3)2
Enzyme
EDC
Adduct-I
O
OH
N
O O Enz P NH
Amide bond formation
NHS
NH2
Enz O
Polyaniline
Adduct-II
100 nm
50 nm
SEM micrograph of electrophoretically deposited nanostructured polyaniline film

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SEM micrograph of PANI/ChOx/ITO bio-electrode
Transmission electron microscope image of polyaniline fibre with the associated protonating acid 38
Photometric Response Studies of ChOx/PANI/ITO Bio-electrode

ChOxoxiNS-PANIITO + Cholesterol + O2 ChOxredNS-PANIITO + 4-cholesten-3-one + H2O2
HRP H2O2 + O- dianisidine (reduced) 2H2O + O- dianisidine (oxidized) orange color
Optimum pH 6.5
Photometric response of ChOx/NS-PANI/ITO bioelectrode as a function of cholesterol concentration
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Electrophoretically deposited MWCNT-c/ Polyaniline Composite

Carbon 46 (2008) 1727-1735
(i)
(ii)
10/5/2009 submitted to Carbon Revision
40
Application of electrophoretically deposited MWCNT-c/polyaniline composite to free cholesterol sensing
UV-Vis spectra of electrophoretically deposited ES and ES/MWNT-c films
CV comparing the electrochemical hysteresis of a pure Polyaniline (ES) film to that of ES/MWNT-c composite film
AFM of Electrophoretically deposited Nanostructured polyaniline and ES /MWNT-c/Composite
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Amperometric Response Studies of ChOx/PANI-CNT/ITO Bio-electrodes
Linearity 1.3 to 13mM
Sensitivity: 6700nA/mM
CV for ChOx/PANI-MWCNT/ITO bioelectrode as function of cholesterol concentration
Amperometric response of ChOx/PANI-MWCNT/ITO bioelectrode as a function of cholesterol concentration
Schematic of reaction taking place at ChOx/PANI-MWCNT Bioelectrode 10/5/2009 WCU Project, CNU,bansi.malhotra@gmail.com 42
Polyaniline Langmuir-Blodgett Films
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Schematic for electrode preparation
43
0.12
0.10
sensitivity of 88.9 nA mg-1 dL (vii)

(vi) (v) (iv) (iii)
Current(mA)
0.08
0.06
(ii) (i)
0.04
0.02
0.00
-0.02 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8
Potential(V)
FT-IR spectra of PANI/SA and PANI/Glut/ChOx LB film bioelectrode
55 50 45
Change in Current [A]
40 35 30 25 20 15 10 5 0 50 1 00 1 50 2 00 25 0 30 0 3 50 4 00 4 50
Linearity 25-400mg/dl
C o le ste ro l C o n ce n tra tio n [m g /d l]
SEM of PANI/SA LB film bioelectrode 10/5/2009
SEM of ChOx/Glut/PANI-SA LB film bioelectrode
1) 2)
LSV for ChOx/Glu/PANI-SA LB film as a function of cholesterol concentration 44 Linear regression curve of ChOx /Glu/PANI-SA LB film bioelectrode.
Polyaniline Nanotubes
Triglyceride detection in blood is considered important since its high concentration is associated with increased risk of atherosclerotic events and is useful for diagnosis and treatment of diabetes mellitus, nephrosis, liver obstruction and other diseases involving lipid metabolism of various endocrine disorder
Schematic for electrode preparation

(i)
(ii)
Linearity : 25300 mg dL1,

300 nm
Low Km :0.62 mM
Mag: 1.0 K X 3 m Mag: 1.0 K X 3 m
SEM images of PANI-NT/ITO(i) and LIP/Glu/PANINT/ITO
Impedimetric response of LIP/Glu/PANI-NT/ITO bioelectrode for tributyrin detection; inset shows the calibration plots derived from (B) Effects of different interferents on the response the impedimetric measurements as a function of tributyrin of LIP/Glu/PANI-NT/ITO bioelectrode. concentration 10/5/2009 46 WCU Project, CNU,bansi.malhotra@gmail.com
Bioaffinity Based Sensor

O N
O HO P OH O
NH N NH2
CH3 NH2 N
NH
O
O HO P
NH O
O CH3
OH
NH2 N N
CH3
H 3C O NH NH O
N
O HO P OH
+
+
NH
O O
Deoxyribose Sugar Phosphoric Acid Nitrogenous Bases

Adenine Guanine Cytosine and Thymine 10/5/2009
O HO P
O CH3
OH
O HO P
N NH
O O
O NH
OH
N
CH3 O HO P OH
+
H2N
NH2
O N O N
OH
47
DNA Biosensor
Among various affinity biorecognition elements DNA is known to have interesting chemical and physical properties. DNA is a double stranded helix structure made up to sugar phosphate backbone with specific sequences made up of nitrogen bases. since phosphate group of the backbone is negatively charged, DNA is usually surrounded by positive counter-ion like hydrogen , sodium or potassium in the solid state. In water, these so called counter ions can freely diffuse away leaving behind a negatively charged DNA strand. This property of DNA makes it ideal for electron transfer. Physical properties of DNA is also very important as with the change in temperature and pH, two strands of DNA double helix can be separated. The two complementary strands of DNA anneal when the conditions are slowly brought to normal and this process is called DNA hybridization or annealing. This process of annealing occurs due to formation of hydrogen bonds between the nitrogen bases of the complementary strands
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Polyaniline based DNA biosensor for P3HT - SAM Escherichia coli
Nanostructured Cerium Oxide Film Based Immunosensor for Ochratoxin-A Detection
Matrices for DNA biosensor
Electrochemically deposited Polyaniline film for N. Gonorrhoea

Polyaniline based DNA biosensor for Escherichia coli
Arora et.al., Analytical Chemistry 2007, 79, 6152-6158
Immobilization of avidin onto PANI films coated onto Pt disc electrode using EDC-NHS coupling Immobilization of E. coli specific 5-biotin labelled BdE probe indirectly onto avidin-PANI Characterization of prepared BdE-avidin-PANI bioelectrodes using DPV, SEM, Impedance spectroscopy, FT-IR etc. Hybridization detection of complementary, one base mismatched and non complementary sequences via monitoring guanine and methylene blue oxidation. Detection of complementary sequences in E. coli genomic DNA and lysed E.coli cells. Complementary target DNA
5biotin end labeled BdE probe
Hybridization with complementary DNA
Avidin C= O N C= O N C= O N C= Covalent bond between O COOH of avidin and -NH of N PANI PANI film onto Pt disc electrode
C=O C=O C=O C=O C=O N N N N
E.coli is responsible for three types of infections in humans: urinary tract infections (UTI), Neonatal meningitis, and intestinal diseases(gastroenteritis). These diseases depend on a specific array of pathogenic(virulence) determinants.
Preparation of Electrochemically deposited Polyaniline film
Film PANI
Wavenumber (cm-1) 1602 1482 1305
Assignment C=C double bond of quinoid rings. C=C double bond associated with the benzoid ring. Not as yet completely understood. Perhaps linked with various stretching and bending vibrations associated with C-C single bond. C-N double bond indicative of protonation. N-H amide bond. Assymmetric stretching of P-O-C vibration. Stretching vibration of P=O of the phosphoric acid group. Carbonyl Stretching vibration band of C double bonds in the purine & pyrimidine rings. Peak becomes more intense due to complementry DNA 52 association.
1171 Avidin-PANI BdNGAvidin-PANI 1565 & 1658 1067 1243
1492 & 1606
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dNG-AvidinPANI
Polyaniline (PANI)
BdNG-Avidin-PANI bioelectrode
DPV shows oxidation peak of methylene blue at - 0.25V in Phosphate buffer (0.05M, pH 7.0, 0.9% NaCl). There is increase in the oxidation peak of methylene blue observed with the decrease in complementary DNA concentration. This bioelectrode can detect the DNA upto 2 x 10-15g/l concentration
2 .0 x 1 0 1 .8 x 1 0 1 .6 x 1 0
-5 -5 -5 -5 -5 -5 -6 -6 -6 -6
B d E -a v id in -P A N I H yb rid iz a tio n w ith d E ' H yb rid iz a tio n w ih t d E '1 H yb rid iz a tio n w ith d E 'n c
I (A)
1 .4 x 1 0 1 .2 x 1 0 1 .0 x 1 0 8 .0 x 1 0 6 .0 x 1 0 4 .0 x 1 0 2 .0 x 1 0
0 .8
0 .9
1 .0
1 .1
1 .2
1 .3
1 .4
1 .5
1 .6
V (v o lts )
DPV curves of BdE-avidin-PANI bioelectrodes in 0.05 M phosphate buffer pH 7.0 at at pulse height of 50 mV and pulse width of 70 ms after hybridization with complementary probe (dE), one base mismatch probe (dE1) and non-complementary probe (dEnc); (a) monitoring guanine oxidation, (b) monitoring methylene blue oxidation.
5 0
I (A)
DPV curves of BdE-avidin-PANI electrodes after hybridization with dE probes (0.00050.25 fmol) after 20 M MB pretreatment in 0.05 M phosphate buffer pH 7.0 at pulse height of 50 mV and pulse width of 70 ms. (Inset shows the linear plot of 1/ln(dE fmol) as a function of peak height of MB in A.
-5 -10
-15 -20 -25 -30 -35
16 14 12 10 8 6 4
I-dE' = 0.0007 fmol I-dE' = 0.001 fmol I-dE' = 0.005 fmol I-dE' = 0.007 fmol I-dE' = 0.01 fmol I-dE' = 0.05 fmol I-dE' = 0.125 fmol I-dE' = 0.25 fmol
Detection limit of BdE-avidin-PANI = 0.001 fmol IdE = - 0.49622 [1/ln (dE concentration)] + 0.0901
10/5/2009
I (A)
2 -35 -30 -25 -20 -15 -10 -5
1/ln(II-dE' concentration in fmol)
-600
-500
-400
-300
-200
-100
100
200
V (mV)
54
Surface Plasmon Resonance based Nucleic Acid Biosensor for detection of M. Tuberculosis
BK7 gold film Immobilization of 20 mer thiolated DNA and 24 mer PNA probes specific to M.Tuberculosis for 8500 sec. by SPR technique
Washing with acetone, Ethanol and Piranha solution
Nucleic acid immobilized onto gold electrode Characterization of electrode by contact angle measurement, Impedance, Cyclic Voltametry, Atomic force microscopy techniques.
Study with the complementary, one base mismatch and non complementary targets Hybridization signal due to change in refractive index upon the binding
10/5/2009
55
Total immobilized thiolated DNA is 1200 of refractive angle change is corresponds to 1 nanogram of immobilized DNA ( 2380 = 1.98 ng/mm2 i.e., 16.83ng/ spot or 0 = 1.7 ng/mm2 PNA(204 i.e., 14.45 ng/spot)
Contact angle measurement with sesile drop method: bare gold 760, Thiolated DNA self assambled monolayer (600), Thiolated PNA self assambled monolayer 54.570
-1430
-500
Refractive angle (millidegrees)
One base mismatch non complementary Complementary
-1440 -1450 -1460 -1470 -1480 -1490
complementary
Refractive angle (millidegrees)
-600
one base mismatch
non complementary
-1500 -1510 -1520
-700
-800
100
200
300
400
500
600
700
200
400
600
800
Time (seconds)
Time (Seconds)
SPR sensorgram with complementary, non complementary and one base mismatch of (a) DNA probe, (b) PNA probe
PNA/Au bioelectrode can be used to detect complementary target sequence using genomic DNA of M. tuberculosis (10 ng mL-1)
10/5/2009
57
Immunosensor
biological molecule (protein) that specifically recognizes a foreign substance (antigen) as a means of natural defence
ANTIBODY (immunoglobulin):A
Immunosensors transduce antigen-antibody interactions directly into physical signals. The design and preparation of an optimum interface between the biocomponents and the detector material is the key part of immunosensor development.
Antibodies
Polyclonal
Antibodies that are collected from sera of exposed animal
Monoclonal
Individual B lymphocyte hybridoma is cloned and cultured. Secreted antibodies are collected from culture media
Recognize multiple antigenic sites of injected biochemical.
Recognize ONE antigenic site of injected biochemical
10/5/2009
59
Immunosensor becomes important when

Fast , accurate and sensitive measurement are required, Highest possible detection strength is required , Large numbers of samples are to be expected , Alternate of available expensive analytical methods.
10/5/2009
60
Nanostructured Iron Oxide Film Based Immunosensor for ochratoxin-A Detection

Ochratoxin-A (OTA) is one of the most abundant food contaminating mycotoxins. OTA is found in tissues and organs of animals including human blood and breast milk and is known to produce nephrotoxic, teratogenic, carcinogenic and immune toxic activity in several animal species.
It affects humans mainly through consumption of improperly stored food products and causes carcinogenicity (Group 2B, possibly by induction of oxidative DNA damage). OTA can also cause immunosupression and immuno toxicity. Why Cerium oxide (FeO2) ? Superparamagnetics, Surface charged, High adsorption capability, High electron transfer capability, High affinity with the oxygen atom of enzymes , Biocompatability
FTIR spectra of (a) CH/ITO electrode (b) CH-Fe3O4 nanobiocomposite (c) IgGs/CH-Fe3O4 nanobiocomposite/ITO bioelectrode (d) BSA/IgGs/CH-Fe3O4 nanobiocomposite/ITO bioelectrode
10/5/2009
62
X-ray diffraction pattern and transmission electron microscopic studies of Fe3O4 nanoparticles
10/5/2009
CH-Fe3O4/ITO ; IgGs/ CH-Fe3O4/ITO and BSA/IgGs/ CH-Fe3O4/ITO WCU Project, CNU,bansi.malhotra@gmail.com
63
3.5x10 3.0x10 Current (A) 2.5x10 2.0x10 1.5x10 1.0x10
-4
-4
-4
-4
a) CH/ITO electrode, b) CH-Fe3O4/ITO c) r-IgGs /CH-Fe3O4/ITO immunoelectrode d) BSA/IgGs/CH-Fe3O4/ITO immunoelectrode
-4
-4
a
-4
5.0x10
-5
4.5x10 4.0x10
3.5x10 3.0x10 Current (A)
-4
Current (A)
0.0 -0.2
-4
0.0
0.2 0.4 Potential (V)

-1
1.9x10 1.8x10 1.8x10 Current (A) 1.7x10 1.7x10 1.6x10 1.6x10
-6 -6
0.6
0.8
Linear range: 0.5-6 ng dL -1 Detection limit: 0.5 ng dL -1 -2 Sensitivity: 36 A/ng dL cm Response time: 18 s
-1
3.8x10 3.6x10 3.4x10 3.2x10 3.0x10 2.8x10 2.6x10 2.4x10 2.2x10
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
2.4x10 2.2x10 2.0x10 1.8x10 Current (A) 1.6x10 1.4x10 1.2x10 1.0x10 8.0x10 6.0x10 4.0x10
-6 -6 -6 -6 -6 -6 -6
Linear range: 1-6 ng dL B -1 Detection limit: 1 ng dL -8 -1 -2 Sensitivity: 4.68 x 10 A/ng dL cm Response time: 35 s
2.5x10 2.0x10 1.5x10 1.0x10 5.0x10
-4
a
0 1 2 3 4
-1
2.0x10
-4
-6
-6
-4
Concentration (ng dL )
-6
-6
a
-4
Potential (V)
1.2 1.0 0.8 0.6 0.4 0.2
-6
1.6x10
-6
a
0 1
0.175
1.5x10
-6
-4
Concentration (ng)
b
20 40 60 80 100 Time (Second)
0.170
-6 -7
Potential (V)
-5
0.0
0.165
0.160
0.0 -0.1
0 20 40 60 80
0.155
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
-7 -7 -7
0.150
Time (s)
Potential (V)
2.0x10
-0.2
0.0
0.2
0.4
0.6
0.8
10/5/2009
Potential (A)
64
Micro-organism based sensor
10/5/2009
65
Smallest to largest micro-organisms..

Prions Viruses Bacteria Fungi
10/5/2009
66
Many types of microbial sensors have been developed as analytical tools since the first microbial sensor was studied by Karube et al. in 1977.
The microbial sensor consists of a transducer and microbe as a sensing element. The characteristics of the microbial sensors are a complete contrast to those of enzyme sensors or immunosensors, which are highly specific for the substrates of interest, although the specificity of the microbial sensor has been improved by genetic modification of the microbe used as the sensing element.
Microbial sensors have the advantages of tolerance to measuring conditions, a long lifetime, and cost effective performance, and have the disadvantage of a long response time.
Microbial sensors result from the combination of a microorganism with a transducer capable of detecting the metabolite involved. Microorganisms possess enzymatic systems that effect biological transformations. The immobilization of microorganism on a transducer is first step in the construction of a biosensor.
A self-assembled monolayer based conductometric algal whole cell biosensor for water monitoring
Microchim Acta (2008) 163:179184
This unicellular green algae has been chosen due to its considerable ecological advantages (it is ubiquist in all dulcicol environments and is able to accumulate large quantities of pollutants).
Schematic representation of the immobilization of algal cells on the platinum electrode modified by SAMs.
Bacterial whole-cell biosensors are very useful for toxicity measurements of various samples. Semi-specific biosensors, containing fusions of stress-regulated promoters and reporter genes, have several advantages over the traditional, general biosensors that are based on constitutively expressed reporter genes. Semi-specific biosensors are constantly being refined to lower their sensitivity and, in combination, are able to detect a wide range of toxic agents.
APA res (residual) for 30 min exposure to Cd2+ (10 mmol l1 TrisHCl, 50 mol l1 pNPP, pH 8.5)
The platinum electrodes modified by self-assembled monolayer without (a) and with (b) immobilized algal cells
Biosensor is sensitive to the presence of cadmium with a detection limit of 1 ppb. It has been demonstrated that immobilization on a monolayer improves the repeatability (RSD<5%) and the reproducibility (RSD<10%) of the response. The lifetime of the sensor is 17 days.
Conclusions:
Biocatalysis & Bioaffinity Sensors Glucose,Urea,Cholesterol DNA Immunosensor Whole Cell Immunosensor
10/5/2009
71
Some literature for Studies ( Week 2):

Prospects of conducting polymers in biosensors, B.D Malhotra, A. Chaubey and S. P. Singh, Analytica Chmica Acta , 578 (2006) 5974. Electrophoretically deposited conducting polymers for applications in organic electronics,Chetna Dhand and B.D.Malhotra,Organic Electronics in Sensors & Biotechology, J.Shinar & Ruth Shinar( Editors),McGraw-Hill),2008 Recent developments in urea biosensor, Gunjan Dhawan, G.Sumana and B.D.Malhotra, Biochemical Engineering Journal ,2009 ,44 , pp. 42-52. Electrocatalytic oxidation of hydrazine and hydroxylamine at gold nanoparticlepolypyrrole nanowire modified glassy carbon electrode Jing Li, Xiangqin Lin, Sensors and Actuators B 126 (2007) 527535 Application of Polyaniline as glucose biosensor, K. Ramanathan, S. Annapoorni and B. D. Malhotra, Sensors & Acturators B, 21, 1994, 165 69. Polythiophene gold nanoparticles composite film for application to glucose sensor, Pratibha Pandey, Sunil K. Arya , Zimple Matharu, S. P. Singh, Monika Datta and B. D. Malhotra, Journal of Applied Polymer Science , Vol. 110, 988994 (2008), Cholesterol biosensor based on cholesterol esterase, cholesterol oxidase and peroxidase Immobilized on conducting polyaniline films, Suman Singh, P. R. Solanki, M. K. Pandey and B. D. Malhotra, Sensors & Actuators B, 115,2006,pp534-541. Microchim Acta (2008) 163:179184. Fully integrated biocatalytic electrodes based on bioaffinity interactions, E Katz, V Heleg-Shabtai, A Bardea, I Willner, Biosensors and Bioelectronics, 1998
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10/5/2009
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MichaelisMentonEquation
10/5/2009
WCU Project,Chungnam National University, Daejeon, Korea,bansi.malhotra@gmail.com
75
Now,V0 is determined by the breakdown of ES to form product, which is determined by [ES] V0 = k2[ES]
Or, V0 = Vmax [S] Km +[S]
k2[E] [S] Km+ [S]

Substituting the value of V0, we have
Or, V0 =
This is the Michaelis-Menten equation, the rate equation for a one-substrate enzyme-catalyzed reaction.
Km value determine s the affinity of biomolecule with the analyte . Lower is the value, higher is
the affinity 1
Now,
Km +[S] Vmax [S] Km Vmax [S] Km Vmax [S]

+ +
V0
1
[S] Vmax [S] 1 Vmax
= =
V0
1
This form of the Michaelis-Menten equation is called the Lineweaver-Burk equation

V0
MichaelisMentenKinetics
MichaelisMentenkinetics (alsoreferredtoasMichaelis MentenHenrikinetics)approximatelydescribesthekinetics ofmany enzymes. ItisnamedafterLeonorMichaelis andMaudMenten.This kineticmodelisrelevanttosituationswhereverysimple kineticscanbeassumed,(i.e.thereisnointermediateor productinhibition,andthereisnoallostericity or cooperativity). Morecomplexmodelsexistforthecaseswherethe assumptionsofMichaelisMentenkineticsarenolonger appropriate. TheMichaelisMentenequationrelatestheinitialreaction ratev0 tothesubstrateconcentration[S].Thecorresponding graphisahyperbolicfunction;themaximumrateisdescribed asvmax.
TheMichaelisMentenequationdescribestheratesofirreversible reactions.Asteadystatesolutionforachemicalequilibrium modeledwithMichaelisMentenkineticscanbeobtainedwiththe GoldbeterKoshland equation.
10/5/2009 WCU Project,Chungnam National University, Daejeon, Korea,bansi.malhotra@gmail.com 77

Week 2 Bioisensors 21sep2009 Bio Catalysis

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Week 2 Bioisensors 21sep2009 Bio Catalysis

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Biocatalysis Based Biosensors, Bioaffinity Based Biosensors & Microorganisms Based Biosensors, Biologically Active Material and Analytes

H 3C CH 2 H3C H3C CH H3C 2 H3C S S S S S S S

Centre for NanoBioengineering & Spintronics, Chungnam National University,Daejeon,Korea

WCU Project, CNU,bansi.malhotra@gmail.com

Classification based on bio-recognition element

Biocatalysis based sensor

WCU Project, CNU,bansi.malhotra@gmail.com

WCU Project, CNU,bansi.malhotra@gmail.com

2 The number of collisions per unit time will increase.

3 The heat of the molecules in the system will increase

WCU Project, CNU,bansi.malhotra@gmail.com

Coenzymes, Prosthetic group, Effectors

Effectors: Effectors accelerate (activators) or block (inhibitors) enzyme reaction

Table1:Function of some important coenzymes and prosthetic groups

WCU Project, CNU,bansi.malhotra@gmail.com

{by adding k1[ES][S]} [E] [S] Or, [ES] =

WCU Project, CNU,bansi.malhotra@gmail.com

WCU Project, CNU,bansi.malhotra@gmail.com

Au-nanoparticles/ Polypyrrole Composite

Iron Oxide NanoparticlesChitosan Composite

Matrices for Glucose biosensor

Au nanoparticle/ Polyaniline Composite

Polythiophene Gold Nanoparticles Composite

WCU Project, CNU,bansi.malhotra@gmail.com

Au Nanoparticles / Polypyrrole Composite

20 ml of 1mm chloro auric acid solution

Faint blue to wine red color Tri sodium citrate solution

UV-vis spectra: peak at 520nm

WCU Project, CNU,bansi.malhotra@gmail.com

Au-nanoparticles / Polyaniline Composite

Journal of Nanoscience and Nanotechnology, 2008, 8, 3158.

2.5x10 2.0x10 1.5x10

3186 1627 1259 3434 800

0.0 -5.0x10 -1.0x10 -1.5x10

-1.0 -0.8 -0.6 -0.4 -0.2 0.0

W avenum ber (Cm )

Glucose Oxidase Activity Immobilized on AuNPs-PANI/ITO

Gluconic acid + H2O2 2H+ + O2 + 2e-

Current response of GOx/AuNPs-PANI/ITO bioelectrode as a function of glucose concentration. 10/5/2009

Hanes plot of GOx/AuNPs-PANI/ITO bioelectrode as a function of glucose concentration

WCU Project, CNU,bansi.malhotra@gmail.com

Shelf life of GOx/AuNPsPANI/ITO bioelectrode with time.

Polythiophene Gold Nanoparticles Composite

J. Applied Poly. Sci., 2008, DOI 10.1002/app.

Enzyme activity studies using UV-visible spectrophotometer

Absorbance (500 nm)

0.002 0 50 100 150 200 250 300 350 400 450

Photometeric response of GOx-regP3HT-AuNPs DTSP/Au) bioelectrode as a function of analyte (glucose) concentration.

Hanes plots of GOx-regP3HT-AuNPsDTSP/Au bioelectrode as a function of analyte (glucose) concentration.

Iron Oxide NanoparticlesChitosan Composite

50 nm Transmission Electron Micrograph of Fe3O4 nanoparticles

Electrochemical Response Studies of GOx/PANI/ITO Bioelectrodes

WCU Project, CNU,bansi.malhotra@gmail.com

Zinc oxide-chitosan nanobiocomposite for P3HTsensor urea - SAM

Matrices for Urea biosensor

Iron oxide-chitosan nanobiocomposite for urea sensor

Zinc oxide-chitosan nanobiocomposite

APPLIED PHYSICS LETTERS 93, 163903 2008

XRD pattern of ZnO-CH nanobiocomposite film. b

Scanning electron micrograph of Urs-GLDH/ZnO-CH/ITO bioelectrode

Linearity =5100 mg/ dl, Detection limit = 3 mg/ dl Km = 4.92 mg/ dl

EIS of i) CH/ITO, ii) ZnO-CH/ITO, iii) UrsGLDH/ZnO-CH/ITO electrode, 10/5/2009