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A label-free electrochemical immunosensor for beta-amyloid detection

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DOI: 10.1039/C6AY01910B

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Analytical
Methods
PAPER

A label-free electrochemical immunosensor for

beta-amyloid detection
Cite this: Anal. Methods, 2016, 8, 6115
Ajeet Kaushik,*a Pratikkumar Shah,b Phani Kiran Vabbina,b Rahul Dev Jayant,a
Sneham Tiwari,a Arti Vashist,a Adriana Yndarta and Madhavan Nair*a

Received 5th July 2016
Accepted 9th July 2016
DOI: 10.1039/c6ay01910b
www.rsc.org/methods
A label-free detection of beta-amyloid (bA) protein using an electrochemical immunosensor fabricated via
immobilizing specific anti-beta-amyloid antibodies (An-bA-Abs) onto an interdigitated electrode of gold
(IDE-Au) modified using a self-assembled monolayer (SAM) of dithiobis(succinimidyl propionate) [DTSP]
is presented here. The bA has been investigated as a potential biomarker for monitoring Alzheimer's
disease (AD), permanent irreversible and progressive brain damage. Thus bA detection at the pM level is
of high significance for AD diagnostics. The IDE-Au modification and covalent immobilization of An-bA-
Abs onto electrodes were characterized by differential pulse voltammetry (DPV) and electrochemical
impedance spectroscopy (EIS) as a function of electrical response variation in each step involved in
sensor fabrication. The EIS studies confirmed that the developed bA immunosensor is selective and
exhibits a detection limit of 10 pM, its detection range varies from 10 pM to 100 nM, and it has a high
sensitivity of 11 kU M
1 with a regression coefficient of 0.99. Thus, the developed sensitive and selective
immunosensor with the features of the IDE-Au can be integrated with a miniaturized potentiostat (M-P)
to develop a sensing system to detect bA for point-of-care (POC) applications for the assessment and
management of AD. The bio-informatics gathered from such a system could be useful to make timely
therapeutic decisions.

monitoring.4 These markers are total tau protein and beta-

1. Introduction
Alzheimer's disease (AD) is an irreversible progressive brain
disorder that causes severe incurable impairments.1 Recent
studies conrmed that the increase in AD incidence, especially
in aging populations, will be three times greater by 2060.2 The
risk factors associated with AD in an aged population are very
serious and unfortunately no cure is available. Therefore,
continuous monitoring of AD progression becomes essential for
therapeutics and disease management strategies.
The conventional method to diagnose AD is the post-mortem
examination of a patient's brain via the identication of senile
plaques and neurobrillary tangles.1 The AD diagnosis is based
on clinical history and in vivo brain imaging using magnetic
resonance imaging (MRI),3 single-photon emission computed
tomography (SPECT) and metabolic positron emission tomog-
raphy (PET) of a patient.4 The neuropsychological, cognitive,
and neurological tests are also used to identify AD. Unfortu-
nately these methods are time consuming and expensive, and
their accuracy is limited to only 85%. Recently, detection of
soluble markers for AD is also being explored for AD detection/
a
Center for Personalized Nanomedicine, Institute of NeuroImmune Pharmacology,
Department of Immunology, Herbert Wertheim College of Medicine, Miami, FL, USA.
E-mail: ajeet.npl@gmail.com; nairm@u.edu
b
Department of Electrical and Computer Engineering, Florida International University,
Miami, FL, USA
amyloid (bA) protein in cerebrospinal uid or plasma.5 As per
the amyloid hypothesis, bA (peptides with 10-40/42 amino acids
in length) accumulation in the AD brain, which cleaves out of
the neuronally expressed amyloid precursor protein (APP), has
been investigated as a major causative agent for AD progres-
sion.6,7 Abnormal bA levels cause neurotoxicity and induce
oxidative stress in the brain resulting in neurodegeneration and
nally dementia. At present, efficient and desired AD moni-
toring tools are not available.8–11 Conventional techniques
including enzyme linked immunosorbent assay (ELISA) and
real time-polymerase chain reaction (RT-PCR) are in practice to
detect bA concentration at the clinical level. However, the
available methods for bA detection to monitor AD need signif-
icant effort to engineer them for rapid and selective detection of
bA at the pM level.
Recently, an electrochemical immunosensing methodology
due to its fast, selective and ultrasensitive detection capabilities
has been adopted for target biomarker detection at the pg mL
1
level.12,13 The introduction of nano/micro electrodes, nano-
structured sensing materials, microelectronics, and miniatur-
ized sensing transducers into electrochemical sensor
fabrication has been shown to improve the sensing perfor-
mance.14,15 The integration of a miniaturized highly sensitive
sensor with a microuidic system and miniaturized potentio-
stat (M-P) has been reported to monitor biomarkers with

This journal is © The Royal Society of Chemistry 2016 Anal. Methods, 2016, 8, 6115–6120 | 6115
Analytical Methods
Fig. 1 Rapid detection of bA needed for the assessment of AD
progression.
reduced form factors.14,15 Furthermore, efforts are being made
for developing these sensing systems for POC applications for
personalized health monitoring. Although a few electro-
chemical,7,16,17 ELISA assay18,19 and surface plasma resonance20,21
based sensors have demonstrated bA detection at micro- and
sub-micro levels, the bA detection at the pM level to diagnose
AD at an early stage is not demonstrated well. Thus, there is an
urgent need to develop a miniaturized electrochemical sensing
protocol for detecting bA at a very low level. The need for rapid
bA assessment using an electrochemical immunosensing
strategy is illustrated in Fig. 1.
In this work, we have fabricated an electrochemical immu-
nosensor via immobilizing specic An-bA-Abs onto a DTSP-SAM
functionalized IDE-Au to detect bA in the physiological range.
As a proof of the concept, we selected anti-bA1–40 antibody as
a model immobilizing antibody to detect bA1–40 protein. Thus
the developed sensor is miniaturized and can be integrated to
develop a portable sensing system to detect bA for POC appli-
cations. The bA/An-bA-Abs/DTSP-SAM/IDE-Au immunosensor
detected bA at pM levels within 40 minutes. In future, this
well-characterized and optimized sensor fabrication protocol
can be modied to develop an efficient electrochemical
immuno-sensor using antibodies specic to b-A1–42 to detect
toxic bA1–42 protein. The outcome of this bA sensing device will
Fig. 2 Illustration of the fabrication of an IDE-Au based immunosensor to detect bA1–40 peptide.
6116 | Anal. Methods, 2016, 8, 6115–6120
Paper
enable screening and monitoring of AD in patients. Thus the
generated informatics can be utilized to decide therapeutics.
2. Experimental methods
2.1. Materials and methods
DTSP, sodium borohydride (NaBH4), bA peptides of 1–40 amino
acid chains (#131438-79-4), and bovine serum albumin (BSA)
were purchased from Sigma-Aldrich and were used without any
further purication. Highly specic polyclonal anti-beta-
amyloid antibodies (An-bA-Abs) were obtained from Thermo
Fisher Scientic (PA3-16760). Phosphate buffer saline (PBS)
solution (10 mM, pH 7.4) was prepared by dissolving one PBS
tablet in 200 mL of de-ionized (DI) water. The PBS (pH 7.4) water
was used to prepare stock solutions of An-bA-Abs (1 mg mL
1)
and bA (1 mg mL
1, incubated for 4 days at 37  C) and both the
solutions were stored at
20  C for future experiments. The
IDE-Au (chamber volume 5 mL; electrode width and electrode
gap 10 mm), performed three-electrode based electrochemical
measurement, was procured from Micrux Technologies.
2.2. Preparation of bA electrochemical sensor
The proposed electrochemical bA immunosensor was fabri-
cated onto a DTSAP-SAM modied IDE-Au using specic An-bA-
Abs for the detection of bA1–40 at the pM level. Prior to the start
of sensor fabrication, the IDE-Au was cleaned electrochemically
using cyclic voltammetry (CV) using 5 mL of 0.1 M H2SO4 at
50 mV s
1 scan rate as a function of varying cycles (optimized
CV cycles were estimated to be 7). The CV studies of the IDE-Au
exhibited a repeatable and reproducible electrochemical
response within 1–2% and with no morphological damage.22
The electrochemically cleaned IDE-Au was dispersed in
a 2 mg mL
1 solution of DTSP for 2 h to prepare a thin layer of
DTSP-SAM via thiol binding. The detailed procedure adopted
for electrochemical cleaning and SAM modication of the IDE-
Au has been published in our previous publication.22 10 mL of
1 mg mL
1 An-bA-Abs were immobilized via electrostatic
interactions onto the DTSP-SAM/IDE-Au for 2 h. The non-
binding sites of An-bA-Abs/DTSP-SAM-SAM/IDE-Au immuno-
electrodes were blocked using BSA protein (10 mL of 1 mg mL
1)
This journal is © The Royal Society of Chemistry 2016
Paper
for 30 minutes. The immuno-electrodes were washed using PBS
(pH 7.4, 10 mM) to remove any unbound molecules. Such
fabricated immuno-electrodes i.e., BSA/An-bA-Abs/DTSP-SAM/
IDE-Au were refrigerated at 4  C, when not in use.
2.3. Characterization technique
Multiple steps including the electrochemical cleaning of IDE-
Au, fabrication of DTP-SAM on the IDE-Au, and immobilization
of An-bA-Abs followed by BSA were characterized using differ-
ential pulse voltammetry (DPV) and electrochemical impedance
spectroscopy (EIS) in PBS (pH 7.4, 5 mM containing 5 mM Fe(II)/
Fe(III)), as redox moieties, using an Autolab potentiostat/galva-
nostat (Eco Chemie, Netherlands)]. Since at pH 7.4 biomole-
cules retain their biological activity and structure, PBS of pH 7.4
was selected as an electrolyte to conduct all electrochemical
measurements. The electrochemical sensing of the immuno-
sensor as a function of bA concentrations was performed using
EIS in PBS (pH 7.4, 5 mM containing 5 mM Fe(II)/Fe(III)). The
schematic illustration of the electrochemical fabrication and
sensing strategy is shown in Fig. 2.
3. Results and discussion
3.1. Electrochemical characterization of the electrode and
immunosensor
The results of EIS studies to conrm the stepwise fabrication of
the bA immunosensor are shown in Fig. 3A, IDE-Au (curve a),
Fig. 3 Electrochemical characterization of the bA immuno-sensor.
EIS (A) and DPV (B) studies of the (a) DTSP-SAM modified IDE-Au, (b)
anti-bA-Abs immobilized DTSP-SAM/IDE-Au, and (c) BSA immobilized
An-bA-Abs/DTSP-SAM/IDE-Au immuno-electrode, and (d) change in
the electrochemical response of the immuno-electrode on adding 10
pM bA (e) in 5 mL PBS (pH 7.4) containing 5 mM [Fe(CV)6]3
/4
at
50 mV s
1. Results of DPV were analysed in terms of the magnitude of
current while EIS data were analysed in terms of electrochemical
charge transfer resistance (Rct).
This journal is © The Royal Society of Chemistry 2016
Analytical Methods
DTSP-SAM/IDE-Au (curve b), An-bA-Abs/DTSP-SAM/IDE-Au
(curve c), and BSA/An-bA-Abs/DTSP-SAM/IDE-Au (curve d). The
electrochemically cleaned IDE-Au showed a typical Nyquist plot,
consisting of a semicircle corresponding to charge transfer
resistance (Rct) generated at the electrode followed by a linear
line (45 to the X axis) corresponding to bulk properties of the
substrate (curve a). The diameter of the semicircle of the TDE-
Au was found to be increased revealing an increase in Rct aer
the fabrication of the DTSP-SAM on the IDE-Au (curve b). This
conrmed that the successful fabrication of the SAM on the
IDE-Au results in hindrance in electron transport due to the
insulating nature of the DTSP-SAM. Aer immobilizing the An-
bA-Abs onto the STSP-SAM/IDE-Au (curve c), the diameter of the
semicircle increases again, suggesting binding of the Abs with
DTSP resulting in a high Rct value due to hindered electron
transport from the medium to the IDE-Au. The value of Rct
increases further on immobilizing BSA onto the An-bA-Abs/
DTSP-SAM/IDE-Au conrming the binding of BSA with the
immunoelectrode (curve d). BSA is a well-known non-conduct-
ing protein used to block the non-binding sites of the
immunoelectrode.
The ndings of EIS studies were found to be correlated with
DPV studies as shown in Fig. 3B. The results of the DPV studies
showed that the IDE-Au (curve a) is electrochemically respon-
sive and exhibits a well-dened oxidative response. The
magnitude of the electrochemical signal of the IDE-Au
decreases on deposition of the DTSP-SAM (curve b) due to the
insulating nature of DTSP which hinders electron transport
from the medium to the IDE-Au. On immobilization of the Abs
onto the DTSP-SAM electrode, the electrochemical response
decreases due to binding of An-bA-Abs with DTSP which affects
the electron transport (curve c). The immobilization of BSA onto
the An-bA-Abs/DTSP-SAM/IDE (curve d) also reduces the
magnitude of the electrochemical response suggesting BSA
immobilization and conrming the blocking of non-binding
sites of the immunosensor. As a proof-of-the-concept, the
electrochemical response of the immuno-electrode was
measured by adding 10 pM bA (10 mL) for 30 minutes. A
reduction in the current magnitude (Fig. 3B curve e) conrmed
that the sensor is responsive to bA only.
The EIS method is one of the best non-invasive analytical
tools utilized for monitoring biological phenomena such as
protein–protein interaction,23–25 bio-sensing,26 nano-toxicity27
and tumour–drug interaction.28 Therefore, EIS was selected for
bA sensing, as described in the next section, due to its above
mentioned salient features and the availability of a M-P capable
of performing EIS and the feature of interfacing with a mobile
needed for data storage and POC applications.
3.2. Electrochemical sensing of bA
The sensing performance of the BSA/An-bA-Abs/DTSP-SAM-
SAM/IDE-Au immunosensor was studied as a function of
various bA concentrations varying from 10 pM to 10 nM using
EIS in 5 mL PBS (pH 7.4) containing 5 mM [Fe(II)/Fe(III)] as redox
moieties at 100 kHz. An increment in charge transfer resistance
(Rct) was obtained on adding bA due to the specic binding of
Anal. Methods, 2016, 8, 6115–6120 | 6117
Analytical Methods Paper

bA with An-bA-Abs. During sensing, based on the immuno-

sensing strategy, the magnitude of the identical electrochemical
response is dependent on binding kinetics, i.e., binding
between the targeted Abs and biomarkers. Thus, this is very
crucial to optimize the incubation time, an appropriate time
needed to establish a binding between bio-recognition and
targeted markers at the saturation level.29 The variation in the
response in terms of Rct with respect to time, used to bind An-
bA-Abs and each bA (100 pM), is shown in Fig. 4A. The magni-
tude of the Rct value increased on increasing the incubation
time from 5 to 25 minutes due to the formation of a non-con-
ducting immune-complex between An-bA-Abs and bA which
affects the electron transport. Aer 30 minutes, the magnitude
of Rct achieved a steady state suggesting that binding between
Abs and bA reached the saturation level. Thus the incubation
time for the immunosensing of each bA concentration was
selected as 30 minutes.
For bA sensing using EIS, 10 mL of each bA concentration
was spread onto the sensor surface for 30 minutes. Then, the
bio-electrodes were washed to remove unbound bA particles.
The EIS measurements were performed under identical elec-
trochemical measurement conditions. At each bA concentra-
tion, the EIS measurements were conducted 3 times and an
average Rct value was calculated for further analysis. A

Fig. 5 Selectivity (A) and shelf-life (B) assessment of the electro-

chemical bA immunosensor using EIS in 5 mL PBS (pH 7.4) containing
5 mM [Fe(II)/Fe(III)] as redox moieties at 100 kHz. All measurements
were performed in triplicate and an average was used for analysis.

calibration curve [y ¼ 916 + 11  log(bA concentration)] was

Fig. 4 (A) Optimization of the incubation time for bA binding with the
sensor surface. The optimized incubation time was estimated to be 30
minutes. (B) Electrochemical response studies of the immuno-sensor
as a function of bA known concentrations (10 pM to 100 nM). All
measurements were performed in triplicate and an average was used
for analysis.
plotted between Rct and bA concentration with an r2 of 0.99
(Fig. 4B). The BSA/An-bA-Abs/DTSP-SAM/IDE-Au immuno-
sensor exhibited a low detection limit of 10 pM and a high
sensitivity of 11 kU M
1.
The results of EIS studies showed that the BSA/An-bA-Abs/
DTSP-SAM/IDE-Au immunosensor is not affected (2–3%) by
interferents such as prostate specic antigen (PSA) (100 pM)
and cortisol (100 pM). The results of the EIS interference study
showed that the sensor exhibited a change in Rct (14) with
respect to bA only (Fig. 5A). This demonstrates that the effect
of interferents on the BSA/An-bA-Abs/DTSP-SAM/IDE immu-
nosensor is minimal and that the selected antibody is selective
to the bA molecule only. An EIS study was also carried out to
study the shelf-life of the BSA/An-bA-Abs/DTSP-SAM/IDE-Au
immuno-sensor at intervals of 1 week. The obtained results
conrm that the sensor is stable for 30 days at 4  C and beyond
that time the magnitude of the electrochemical response
reduces rapidly (Fig. 5B). The sensing parameters achieved
using our investigated EIS-based electrochemical bA sensor in
comparison with some of the best reported sensors are
summarized in Table 1.

6118 | Anal. Methods, 2016, 8, 6115–6120 This journal is © The Royal Society of Chemistry 2016
Paper Analytical Methods

Table 1 Comparison of the obtained sensing parameters with the reported sensorsa

Sensing strategy Sensing parameters Ref.

SM ¼ 3,30 -dithiobis(sulfosuccinimidyl)propionate (DTSSP)/Au Study explored mA (5 mM) aggregation over time (0.5 to 48 7
RM ¼ bA1–40 via specic antibodies (OC and A11) h)
ST ¼ EIS using [Fe(CN)6]3
/4
as a mediator R ¼ this research explored bA aggregation and docking.
T ¼ Rct However, all sensing parameters need optimization
SM ¼ LBL sensor construction from SPE/co-polymer, derived DL ¼ 0.5 pM 16
from a mixture of tyramine and its carboxylic acid analogue, 3- Other sensing parameters need optimization
(4-hydroxyphenyl) propionic acid
RM ¼ bA oligomers via a mediator using the synthetic non- R ¼ the presented multi-component system is for oligomer
antibody PrPC fragment as a bioreceptor only. The study needs to be performed on bA
ST ¼ EIS
T ¼ Rct
SM ¼ glassy carbon electrode (GCE) RSD ¼ 0.3 to 1.4 (bA1–40), 0.4 to 3% (bA1–42) 17
RM ¼ bA1–40/42 via antibody DL ¼ 0.7 mg mL
1
ST ¼ square wave voltammetry (SWV) DT ¼ 300 (bA1–40) 700 (bA1–42)
T ¼ I(a) R ¼ detection limit is high
SM ¼ screen printed electrode modied using Au DR ¼ 0.5 to 500 ng mL
1 30
nanoparticles
RM ¼ bA1–42 protein detection using biotin labeling DL ¼ 0.1 ng mL
1
ST ¼ electrochemical/CV R ¼ very efficient sensor and there is scope to improve
T ¼ I (a) sensing parameters
SM ¼ SAM of Au nanoparticles prepared by screen printing DR ¼ 0.001 to 200 mM, for bA1–40 and bA1–42 31
RM ¼ bA1–40/bA1–42 protein detection using specic
antibodies
ST ¼ electrochemical/EIS DL ¼ 2.04 mM for bA1–40, 0.57 mM for bA1–42
T ¼ Rct R ¼ there is scope to improve the sensitivity of this sensor
SM ¼ SAM on a Au-coated SPR chip DR ¼ 50 to 10 000 ng mL
1 21
RM ¼ 17-beta-hydroxysteroid dehydrogenase type 10 (17b- DL ¼ 0.5 nM
HSD10) enzyme/peptide onto a bA1–40 immobilized surface
ST ¼ SPR R ¼ DL is very high
T ¼ optical
SM ¼ polymer modied Au-coated SPR DL ¼ 50 mM 32
RM ¼ bA1–40/aggregation in the presence of metals R ¼ this sensor needs more optimization
ST ¼ SPR imaging
T ¼ uorescence intensity
SM ¼ silicon/silicon oxide microarray DR ¼ 0.1 to 100 ng mL
1 33
RM ¼ bA1–42 binding with Cov-12F14/Cov-6E10 DL ¼ 73 pg mL
1
ST ¼ interferometric reectance imaging sensor R ¼ DL is very high
T ¼ uorescence intensity
SM ¼ DTSP/IDE-Au DR ¼ 10 pM to 100 nM Present
RM ¼ bA1–40 via specic Abs DL ¼ 10 pM research
ST ¼ EIS using [Fe(CN)6]3
/4
as a mediator DT ¼ 40 minutes
T ¼ Rct S ¼ 11 kU M
1
R ¼ obtained sensing parameters are in the physiological
range. The sensor fabricated on the IDE-Au can be used to
design a miniaturized system for POC applications
a
Sensing material [SM], Recognition Marker [RM], Sensing Technique [ST], Transduction [T], Detection Range [DR], Detection Limit [DL],
Sensitivity [S], Detection Time [DT], Relative standard deviation (RSD), and Remark [R].

4. Conclusion a package sensing component to develop a portable electro-

chemical bA sensing system.
In this work, a label-free selective electrochemical immuno-
sensor was fabricated to detect bA at the 10 pM level. This Conflict of interest
investigated sensor is sensitive and selective and exhibits the
ability to detect bA in the physiological range within 40 minutes. The authors declare no conict of interest.
The miniaturized architecture and ability to integrate this
sensor with a M-P make this sensor suitable for POC applica- Abbreviations
tions. Thus the developed sensor can serve as an analytical tool
for the AD management program to obtain bio-informatics
needed to optimize its therapeutics. For future research, efforts AD Alzheimer's disease
will be made to validate this sensor using ELISA and also An-bA-Abs Anti-beta-amyloid antibodies

This journal is © The Royal Society of Chemistry 2016 Anal. Methods, 2016, 8, 6115–6120 | 6119
Analytical Methods Paper

APP Amyloid precursor protein 14 A. F. D. Cruz, N. Norena, A. Kaushik and S. Bhansali, Biosens.
DPV Differential pulse voltammetry Bioelectron., 2014, 62, 249–254.
DTSP Dithiobis (succinimidyl propionate) 15 A. Kaushik, A. Yndart, R. D. Jayant, V. Sagar, V. Atluri,
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SPECT Single-photon emission computed tomography Med., 2003, 9, 3–4.
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6120 | Anal. Methods, 2016, 8, 6115–6120 This journal is © The Royal Society of Chemistry 2016

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