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A label-free electrochemical immunosensor for beta-amyloid detection

Article  in  Analytical methods · August 2016

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

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
Received 5th July 2016
Accepted 9th July 2016
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
DOI: 10.1039/c6ay01910b
management of AD. The bio-informatics gathered from such a system could be useful to make timely
www.rsc.org/methods therapeutic decisions.

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

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

This journal is © The Royal Society of Chemistry 2016 Anal. Methods, 2016, 8, 6115–6120 | 6115
Analytical Methods 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)
Fig. 1 Rapid detection of bA needed for the assessment of AD solution (10 mM, pH 7.4) was prepared by dissolving one PBS
progression. 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
reduced form factors.14,15 Furthermore, efforts are being made
IDE-Au (chamber volume 5 mL; electrode width and electrode
for developing these sensing systems for POC applications for
gap 10 mm), performed three-electrode based electrochemical
personalized health monitoring. Although a few electro-
measurement, was procured from Micrux Technologies.
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 2.2. Preparation of bA electrochemical sensor
AD at an early stage is not demonstrated well. Thus, there is an The proposed electrochemical bA immunosensor was fabri-
urgent need to develop a miniaturized electrochemical sensing cated onto a DTSAP-SAM modied IDE-Au using specic An-bA-
protocol for detecting bA at a very low level. The need for rapid Abs for the detection of bA1–40 at the pM level. Prior to the start
bA assessment using an electrochemical immunosensing of sensor fabrication, the IDE-Au was cleaned electrochemically
strategy is illustrated in Fig. 1. using cyclic voltammetry (CV) using 5 mL of 0.1 M H2SO4 at
In this work, we have fabricated an electrochemical immu- 50 mV s
1 scan rate as a function of varying cycles (optimized
nosensor via immobilizing specic An-bA-Abs onto a DTSP-SAM CV cycles were estimated to be 7). The CV studies of the IDE-Au
functionalized IDE-Au to detect bA in the physiological range. exhibited a repeatable and reproducible electrochemical
As a proof of the concept, we selected anti-bA1–40 antibody as response within 1–2% and with no morphological damage.22
a model immobilizing antibody to detect bA1–40 protein. Thus The electrochemically cleaned IDE-Au was dispersed in
the developed sensor is miniaturized and can be integrated to a 2 mg mL
1 solution of DTSP for 2 h to prepare a thin layer of
develop a portable sensing system to detect bA for POC appli- DTSP-SAM via thiol binding. The detailed procedure adopted
cations. The bA/An-bA-Abs/DTSP-SAM/IDE-Au immunosensor for electrochemical cleaning and SAM modication of the IDE-
detected bA at pM levels within 40 minutes. In future, this Au has been published in our previous publication.22 10 mL of
well-characterized and optimized sensor fabrication protocol 1 mg mL
1 An-bA-Abs were immobilized via electrostatic
can be modied to develop an efficient electrochemical interactions onto the DTSP-SAM/IDE-Au for 2 h. The non-
immuno-sensor using antibodies specic to b-A1–42 to detect binding sites of An-bA-Abs/DTSP-SAM-SAM/IDE-Au immuno-
toxic bA1–42 protein. The outcome of this bA sensing device will electrodes were blocked using BSA protein (10 mL of 1 mg mL
1)

Fig. 2 Illustration of the fabrication of an IDE-Au based immunosensor to detect bA1–40 peptide.

6116 | Anal. Methods, 2016, 8, 6115–6120 This journal is © The Royal Society of Chemistry 2016
Paper Analytical Methods

for 30 minutes. The immuno-electrodes were washed using PBS DTSP-SAM/IDE-Au (curve b), An-bA-Abs/DTSP-SAM/IDE-Au
(pH 7.4, 10 mM) to remove any unbound molecules. Such (curve c), and BSA/An-bA-Abs/DTSP-SAM/IDE-Au (curve d). The
fabricated immuno-electrodes i.e., BSA/An-bA-Abs/DTSP-SAM/ electrochemically cleaned IDE-Au showed a typical Nyquist plot,
IDE-Au were refrigerated at 4  C, when not in use. consisting of a semicircle corresponding to charge transfer
resistance (Rct) generated at the electrode followed by a linear
2.3. Characterization technique line (45 to the X axis) corresponding to bulk properties of the
Multiple steps including the electrochemical cleaning of IDE- substrate (curve a). The diameter of the semicircle of the TDE-
Au, fabrication of DTP-SAM on the IDE-Au, and immobilization Au was found to be increased revealing an increase in Rct aer
of An-bA-Abs followed by BSA were characterized using differ- the fabrication of the DTSP-SAM on the IDE-Au (curve b). This
ential pulse voltammetry (DPV) and electrochemical impedance conrmed that the successful fabrication of the SAM on the
spectroscopy (EIS) in PBS (pH 7.4, 5 mM containing 5 mM Fe(II)/ IDE-Au results in hindrance in electron transport due to the
Fe(III)), as redox moieties, using an Autolab potentiostat/galva- insulating nature of the DTSP-SAM. Aer immobilizing the An-
nostat (Eco Chemie, Netherlands)]. Since at pH 7.4 biomole- bA-Abs onto the STSP-SAM/IDE-Au (curve c), the diameter of the
cules retain their biological activity and structure, PBS of pH 7.4 semicircle increases again, suggesting binding of the Abs with
was selected as an electrolyte to conduct all electrochemical DTSP resulting in a high Rct value due to hindered electron
measurements. The electrochemical sensing of the immuno- transport from the medium to the IDE-Au. The value of Rct
sensor as a function of bA concentrations was performed using increases further on immobilizing BSA onto the An-bA-Abs/
EIS in PBS (pH 7.4, 5 mM containing 5 mM Fe(II)/Fe(III)). The DTSP-SAM/IDE-Au conrming the binding of BSA with the
schematic illustration of the electrochemical fabrication and immunoelectrode (curve d). BSA is a well-known non-conduct-
sensing strategy is shown in Fig. 2. ing protein used to block the non-binding sites of the
immunoelectrode.
The ndings of EIS studies were found to be correlated with
3. Results and discussion DPV studies as shown in Fig. 3B. The results of the DPV studies
3.1. Electrochemical characterization of the electrode and showed that the IDE-Au (curve a) is electrochemically respon-
immunosensor sive and exhibits a well-dened oxidative response. The
The results of EIS studies to conrm the stepwise fabrication of magnitude of the electrochemical signal of the IDE-Au
the bA immunosensor are shown in Fig. 3A, IDE-Au (curve a), 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.

Fig. 3 Electrochemical characterization of the bA immuno-sensor.

3.2. Electrochemical sensing of bA
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 The sensing performance of the BSA/An-bA-Abs/DTSP-SAM-
An-bA-Abs/DTSP-SAM/IDE-Au immuno-electrode, and (d) change in SAM/IDE-Au immunosensor was studied as a function of
the electrochemical response of the immuno-electrode on adding 10
various bA concentrations varying from 10 pM to 10 nM using
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 EIS in 5 mL PBS (pH 7.4) containing 5 mM [Fe(II)/Fe(III)] as redox
current while EIS data were analysed in terms of electrochemical moieties at 100 kHz. An increment in charge transfer resistance
charge transfer resistance (Rct). (Rct) was obtained on adding bA due to the specic binding of

This journal is © The Royal Society of Chemistry 2016 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

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
Fig. 4 (A) Optimization of the incubation time for bA binding with the reduces rapidly (Fig. 5B). The sensing parameters achieved
sensor surface. The optimized incubation time was estimated to be 30
using our investigated EIS-based electrochemical bA sensor in
minutes. (B) Electrochemical response studies of the immuno-sensor
as a function of bA known concentrations (10 pM to 100 nM). All comparison with some of the best reported sensors are
measurements were performed in triplicate and an average was used summarized in Table 1.
for analysis.

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,
EIS Electrochemical impedance spectroscopy S. Bhansali and M. Nair, Int. J. Nanomed., 2015, 10, 677.
ELISA Enzyme linked immunosorbent assay 16 J. V. Rushworth, A. Ahmed, H. H. Griffiths, N. M. Pollock,
IDE-Au Interdigitated electrode of gold N. M. Hooper and P. A. Millner, Biosens. Bioelectron., 2014,
M-P Miniaturized potentiostat 56, 83–90.
MRI Magnetic resonance imaging 17 M. d. Vestergaard, K. Kerman, M. Saito, N. Nagatani,
PET Positron emission tomography Y. Takamura and E. Tamiya, J. Am. Chem. Soc., 2005, 127,
POC Point-of-care 11892–11893.
RT-PCR Real time-polymerase chain reaction 18 L.-B. Yang, K. Lindholm, R. Yan, M. Citron, W. Xia,
SAM Self-assembled monolayer X.-L. Yang, T. Beach, L. Sue, P. Wong and D. Price, Nat.
SPECT Single-photon emission computed tomography Med., 2003, 9, 3–4.
bA Beta-amyloid 19 D. Schenk, R. Barbour, W. Dunn, G. Gordon, H. Grajeda,
T. Guido, K. Hu, J. Huang, K. Johnson-Wood and K. Khan,
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21 K. Hegnerová, M. Bockova, H. Vaisocherová, Z. Krištoková,
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6120 | Anal. Methods, 2016, 8, 6115–6120 This journal is © The Royal Society of Chemistry 2016

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