Modified screen printed electrode for development of a highly sensitive label free impedimetric immunosensor to detect amyloid beta peptides

Analytica Chimica Acta xxx (2015) 1e8

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Analytica Chimica Acta
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Modified screen printed electrode for development of a highly
sensitive label-free impedimetric immunosensor to detect amyloid
beta peptides
Truong T.N. Lien a, b, Yuzuru Takamura a, Eiichi Tamiya c, Mun'delanji C. Vestergaard a, d, *
a

School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi, Ishikawa, 923-1292, Japan
School of Engineering Physics, Hanoi University of Science and Technology (HUST), No.1 Dai Co Viet, Hai Ba Trung, Hanoi, Viet Nam
Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan
d
Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Korimoto-1-21-24, Kagoshima City, Kagoshima,
890-0065, Japan
b
c

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 A label-free impedimetric immunoassay
for
amyloid
beta
was

developed.
 Sensitivity enhanced by elaborate
surface chemistry manipulation using SAM of AuNPs.
 Immobilized Protein G enhanced
sensitivity by directing optimal antibody orientation.
 Lack of interference from highabundant high-molecular weight
BSA demonstrated.

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 24 October 2014
Received in revised form
10 August 2015
Accepted 11 August 2015
Available online xxx

Alzheimer's disease (AD) is a fatal neurodegenerative disease affecting approximately 26 million people
world-wide, and the number is increasing as life expectancy increases. Since the only reliable diagnosis
for the pathology is histochemical post-mortem examination, there is a rather urgent need for reliable,
sensitive and quick detection techniques. Amyloid beta, being one of the established and widely accepted
biomarkers of AD is a target biomolecule.
Herein, we present fabrication of a labelless impedimetric amyloid beta immunosensor on carbon DEP
(disposable electrochemical printed) chip. Three types of amyloid b impedimetric immunosensors were
fabricated in a systematic step-wise manner in order to understand the effects that each surface
modification chemistry had on detection sensitivity. We found that compared to a bare electrode, surface
modification through formation of SAM of AuNPs increased sensitivity by approximately three orders of
magnitude (LoD from 2.04 mM to 2.65 nM). A further modification using protein G, which helps orientate

antibodies to an optimum position for interaction with antigen, lowered the LoD further to 0.57 nM. We
have demonstrated that the presence of one of the most abundance proteins in biological fluids, bovine
serum albumin (BSA), did not interfere with the sensitivity of the sensor. Since the DEP chips are
disposable and the detection platform label-free, the developed sensor is relatively fast and cheap. These
methods could easily be applied for detection of other antigens, with selection of the detection platform
based on the desired for sensitivity.
© 2015 Elsevier B.V. All rights reserved.

Keywords:
Amyloid beta (Ab)
Immunosensor
Electrochemical impedance spectroscopy
(EIS)
Screen-printed electrode (SPE)
Disposable electrochemical printed (DEP)
chip

* Corresponding author. School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi, Ishikawa, 923-1292, Japan.
E-mail address: (M.C. Vestergaard).
/>0003-2670/© 2015 Elsevier B.V. All rights reserved.

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1. Introduction
Alzheimer's disease (AD) is a fatal neurodegenerative disease

affecting approximately 26 million people world-wide and the
number is increasing as life expectancy increases [1]. However,
diagnosis remains in the hands of medical doctors who can only at
best propose ‘probable Alzheimer's or dementia of the Alzheimer
type’ since there is no current test or procedure that is diagnostic.
Although not unilaterally agreed upon, the progressive decline of
patients with AD has been correlated with extracellular deposition
of amyloid plagues, of which amyloid beta is the major constituent
[2]. Amyloid beta (Ab) has therefore become an important
biomarker for the pathology. The main detection method is by
enzyme-linked immunosorbent assay (ELISA) techniques, which
are less flexible, costly, and labour-intensive [3,4]. The past decade
or so has seen tremendous effort put into development of sensitive
and selective detection techniques for this and other peptides/
proteins. They include FRET-based assays [5]; surface Raman
enhanced spectroscopy (SERS) [6]; and several electrochemical
platforms [7,8].
Electrochemical impedance spectroscopy (EIS) recently has
attracted much interest because it has some important advantages
over number of electrochemical methods such as amperometry and
potentiometry. With EIS, developed sensing platforms are (i) labelfree with detection based on direct specific binding events, (ii) less
destructive to the activities of biomolecule due to the small voltage
excitation used during detection, (iii) a simple operation and very
sensitive, with comparable detection limits to optical-based sensors [9e11]. EIS biosensors have been successfully employed for
detection of various biomolecules and biological processes
including DNA hybridization, at very low (femtomolar) detection
limits [12]. Previously, we reported on an impedimetric immunosensor development using DEP chips, and demonstrated its selective detection using a model protein, chorionic gonadotropin
hormone (hCG) (limit of detection (LoD) of 33 pg/mL) [13]. Lien and
colleagues also modified DEP chips using a conducting co-polymer,
polypyrrole-pyrolecarboxylic acid for hCG detection. The LoD was

lowered by an order of magnitude, to 2.3 pg/mL [14]. Most recently,
Rushworth and colleagues have reported on specific detection of
oligomeric amyloid beta using biotylated peptide of prion protein
as the recognition element. The authors have reported an impressive detection limit of 0.5 pM [15].
In this work, we fabricated a labelless EIS immunosensor for
amyloid beta peptide, isoforms 40 and 42. We have used disposable
electrochemical printed (DEP) chips, which have been used in
development of various DNA- and immuno-biosensors, giving very
good reproducibility [13,14,16]. We developed this sensor in a
systematic step-wise fashion so that we could also better understand the effects of surface chemistry modification on sensor
sensitivity. The developed sensors were very reproducible (coefficient of variation <8%). Although the immunosensor's sensitivity
(LOD ~ 0.57 nM) is still lower than the recently reported prionbased sensor [15], it is a good proof-of-principle antibody-based
EIS. With further improvement in surface chemistry modification, it
offers much promise. Besides, since the antibodyeantigen chemistry is well-understood and the fabrication relatively simple and
rapid, this sensor can easily be adaptable for application to other
antigens.

2. Experimental
2.1. Reagents
1-pyrenebutanoic acid, succinimidyl ester was supplied from

Eugene, Oregon (USA). Chloroauric acid (HAuCl4), Bovine serum
albumin (BSA) and Dimethyl Sulfoxide Dehydrated (DMSO) were
purchased from Sigma Aldrich. 1-Ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC) hydrochloride was supplied by Dojindo (USA).
N-hydroxysuccinimide (NHS) and 16-mercaptohexadecanoic acid
(MHDA) were provided by Wako. Amyloid beta (Ab) peptides (trifluoroacetate salt) were purchased from Peptide Institute Inc.,
(Osaka, Japan). N-terminal human monoclonal Ab antibody (anti
mAb) was from Calbiochem (CA, USA). All other reagents used were
of the analytical grade or the highest commercially available purity

and used as supplied without further purification. All solutions
were prepared with deionized water of resistivity no less than
18 MUcm.
2.2. Electrodes
Commercial disposable electrochemical printed (DEP) chips
were obtained from BioDevice Technology Ltd., Japan (http://www.
biodevicetech.com). The chips were fabricated by screen-printing
technology and designed as system with three electrodes containing carbon ink working, carbon ink counter and Ag/AgCl ink
reference electrodes. The carbon ink contained 75% (w/w) graphite
powder and 25% (w/w) mineral oil (Sigma). The surface area of the
working electrode is 2.64 mm2.
2.3. Instrumentation
An AutoLab PGSTAT 30 system (EcoChemie B.V., Ultrecht, The
Netherlands) was used to perform electrochemical impedance
spectroscopy measurements. The spectra was recorded in 0.1 M KCl
solution containing 5 mM of K3[Fe(CN6)]/K4[Fe(CN6)] within frequency range from 100 kHz to 50 mHz. An ac probe amplitude of
10 mV was applied to the system around the open circuit potential.
2.4. Gold nanoparticles fabrication
The carbon ink electrode of DEP chip was modified first by
deposition of gold nanoparticles (AuNPs) on working electrode
using both potential step voltammetry (PV) and cyclic voltammetry
(CV). Tetrachloroauric acid (HAuCl4) was diluted in 100 mM phosphate buffer solution (PBS) to a final concentration of 1 mM. Then
35 mL of the 1 mM HAuCl4 solution was dropped onto DEP chip
electrode surface covering all three electrodes (including Ag/AgCl,
counter and working electrodes). With the PV method, À0.4 V was
applied for different time periods: 5 s, 20 s and 90 s. With the CV
method, À600 to þ500 mV vs. Ag/AgCl were cycled for 5, 10 and 20
cycles at scan rate of 50 mV/s. Following deposition, the AuNPscoated electrodes were washed several times in 10 mM PBS, pH
7.4 solution containing 0.05% Tween 20 followed by deionized
water, and drying under nitrogen (N2) stream. This AuNPs-coated

electrode was then ready for immobilization of anti mAb.
2.5. Electrode fabrication and sensor development
Immunosensors detect signals resulting from specific immunoreactions between antibodies immobilized on a transducer and
the target antigens. In order to have good sensitivity for detection,
the concentration of immobilized antibodies as well as their
orientation on the transducer surface should be as optimal as
possible in order to interact with as many target antigens as
possible. Therefore, surface modification for best possible antibody
immobilization is desirable. In our work, three methods were used
to fabricate the immunosensor based on the physio-chemical
moiety of carbon DEP chip (methods A, B and C). The schematic
diagram of these methods is presented in Fig. 1. In method A,

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Fig. 1. Schematic diagram showing the fabrication of impedimetric immunosensors based on disposable electrochemical printed (DEP) chip following A) Method A, B) Method B
and C) Method C. In method A, carbon ink electrode of the DEP chip was modified first by a functional molecule, 1-pyrenebutanoic acid succinimidyl ester and thence anti mAb
immobilization via succinimidyl ester groups. In methods B and C, the carbon ink electrode of the DEP chip was modified first by deposition of gold nanoparticles (AuNPs) using
cyclic voltammetry method. After that, anti mAb was immobilized onto AuNPs-modified electrodes via COOH group of self-assembled monolayer (SAM) of 16mercaptohexadecanoic acid (MHDA), which can serve as a linker for covalent biomolecule immobilization. Furthermore, in the method C, in order to orientate immobilized antibodies optimally for enhanced antigen detection, protein G was immobilized before anti mAb immobilization. In all sensors ethanolamine was used to block the remaining nonspecific binding sites after antibody immobilization.

N-terminal human monoclonal Ab antibody (anti mAb) was
directly immobilized on carbon DEP chip surface via pyrenyl
groups. In methods B and C, carbon DEP chips were first modified
by an in situ AuNPs synthesis using method described by Lien et al.
[13]. Briefly, AuNPs were electrodeposited on the DEP chips using

either PV or CV. Then anti mAb was immobilized on the AuNPmodified carbon DEP chip via COOH group of self-assembled
monolayer (SAM) of 16-mercaptohexadecanoic acid (MHDA),
which can serve as a linker for covalent biomolecule immobilization. Method C is different from B in that the latter had a predeposition of a protein G layer on SAM AuNPs before immobilization of anti mAb. Since protein G binds specifically to the nonantigenic (Fc) regions of an antibody, it helps immobilize the
antibody in a way that favours its antigen binding sites (Fab) to be
oriented away from the solid phase, towards the antigen [18]. A
schematic diagram of these methods is presented in Fig. 1.

2.5.1. Method A: sensor development on carbon DEP chip
First, a volume of 1.5 mL of 1-pyrenebutanoic acid, succinimidyl
ester (100 mM) was placed onto carbon ink working electrode of
DEP chip for 1 h followed by rinsing several times with deionized
water to wash away excess reagent followed by drying over a
stream N2 gas. In this step, the pyrenyl groups interacted strongly
with the basal plane of carbon graphite via p-stacking. This leads to
the functionalization of carbon surface with succinimidyl ester
groups that are highly reactivated to nucleophilic substitution by
primary and secondary amines [17,18] that exist in abundance on
the surface of most proteins. After, 1.5 mL of 100 mg/mL anti mAb was
dropped onto surface of these electrodes and incubated for 1 h at
room temperature (RT), followed by further washing with 10 mM
PBS to remove any loosely bound antibodies. The surface was then
dried over a gentle stream of N2 gas. Last, the anti mAb-modified
electrodes were subjected to 1.5 mL of 100 mM ethanolamine for 1 h
in order to block the remaining nonspecific binding sites, that is,
any sites that were not antibody-immobilized [12,19]. Last, the

electrodes also were rinsed with PBS followed by deionized water
and then dried over a gentle stream N2 gas. The immunosensors
were ready to use at this point.

2.5.2. Method B: sensor development on Au-NP-modified carbon
DEP chip
In this method, the anti mAb was immobilized onto AuNPsmodified electrode surface via COOH group of self-assembled
monolayer (SAM) of 16-mercaptohexadecanoic acid (MHDA),
which can serve as a linker for covalent biomolecular immobilization. MHDA solution was prepared in deionized water at 0.5 mM
and filtered through a 0.20 mm pore size mesh. To form SAMs, 1.5 mL
of 0.5 mM MHDA was dropped onto AuNPs-modified working
electrode surface for 45 min at RT. In this step, the thiol groups of
the mercaptoalkanes interacted with the AuNPs, forming AueS
bond. The carboxyl groups of MHDA layer need to be activated
before they can react with the amino groups of anti mAb. The
activation is carried out using N-(3-dimethylaminopropyl)- N0 ethylcarbodiimide hydrochloride (EDC, 0.2 M), and N-hydroxysuccinimide (NHS, 0.1 M), which were prepared in deionized
water. A volume of 1.5 mL of the solution was placed onto the
AuNPs-modified working electrode surface for 15 min at RT. The
reason for using both EDC hydrochloride and NHS for activation of
MHDA layer has been explained in more detail in our previous work
[14]. After activation, the electrodes were rinsed with 10 mL of
10 mM PBS containing 0.05% Tween 20 followed by deionized
water, and dried under a gentle stream N2 gas. Then, 1.5 mL of
100 mg/mL anti mAb solution was placed onto the activated SAMAuNPs modified electrode for 1 h at RT. In this procedure, anti
mAb was immobilized successfully onto the AuNPs-modified
electrode. Following this, the electrode was washed with 10 mM
PBS containing 0.05% Tween 20 followed by deionized water to
remove the loosely bound antibodies and dried over a stream N2
gas. In order to block non-specific adsorption, 100 mM ethanolamine was used as described in method A. The electrode was also

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rinsed with 10 mM PBS solution containing 0.05% Tween 20 followed by deionized water, then dried over a gentle stream N2 gas
and used immediately.
2.5.3. Method C: immobilization of protein G on AuNPs-modified
carbon DEP chip for enhanced immuno-sensor sensitivity
In this method, in order to help orientate the immobilized antibodies in an optimum position for Ab interaction, a volume of
1.5 mL of 100 mM protein G was applied onto SAM-AuNPs modified
electrode for 1 h at RT before anti mAb immobilization. The
immobilized protein G helps provide a desirable orientation of
immobilized antibodies for increased sensitivity. This has been
demonstrated before [20]. In the preparation procedure, ethanolamine was also used to block the remaining non-specific binding
sites after antibody immobilization.
2.6. Detection of amyloid b peptides
Amyloid b-peptides were prepared in 0.02% (v/v) ammonia
water at 200 mM concentration by brief vortexing and stored in
0.1 mL vials at À80  C. During preparation, the vials and all the
reagents and chemicals were kept on ice. Just before analysis, the
vials were left to equilibrate to RT (~24 ± 1  C) and subsequently
diluted to the required concentration. In this case, a concentration
range between 10 nM and 200 mM was used. A volume of 2 mL
amyloid b-peptides was dropped on sensor surface, and left for
30 min at RT to let the peptide attach to the immobilized anti mAb.
Then, sensors were rinsed with 10 mM PBS followed by deionized
water and dried over a gentle stream N2 gas. Finally, all sensors
were subjected to electrochemical impedance spectroscopy (EIS)
measurement. The impedance spectra was recorded in 0.1 M KCl
solution containing redox probe solution (5 mM of K3[Fe(CN)6]/
K4[Fe(CN)6]) within frequency range from 100 kHz to 50 mHz

around the open circuit potential with an ac probe amplitude of
10 mV.
2.7. Label-free impedimetric immunosensor
In impedimetric sensors, detection is based on the principle that
any substance attached on the electrode will change the measured
impedance. Therefore, any change in the impedance spectra can be
related to the change in interface properties. In this case, the
binding of amyloid b peptides with anti mAb can be considered as a
coating film, which is expected to affect the sensor impedance
signal. In the present work, Nyquist plots were used to investigate
the change in electron transfer resistance at the interface between
the sensor and the redox probe solution after the binding of amyloid b peptides to the immunosensor chip, and also the change in
electron transfer resistance with changing concentration of amyloid b peptides. Fig. S1 in supporting information shows the typical
Nyquist plot of faradaic impedance spectrum. In general, the
complex impedance is displayed in two parts including a semicircle
(at high frequency region corresponding to the electron transfer
limited process) and a linear part (at lower frequencies resulting
from the diffusion limiting step of the electrochemical process). The
impedance result can be clarified by fitting with Randles equivalent
circuit (inset Fig. S1). This equivalent circuit model consists of the
Ohmic resistance of the electrolyte RS, the double layer capacitance
Cdl, and Warburg impedance ZW. The Warburg resistance describes
the normal diffusion of the redox probe from bulk of solution to the
electrode surface through the complex layer. The last parameter is
the electron transfer resistance RCT which, controls the interfacial
electron transfer rate between the redox probe in solution and the
electrode surface. Ideally, Cdl and RCT are both affected by modification occurring on the electrode surface [9,21]. Thus, Cdl and RCT

are parameters that are mainly used as signals in impedance sensors. However, the value of RCT was found to be strongly affected by
modification occurring on the electrode surface [22e28]. In this

work, RCT parameter was therefore chosen to measure the amyloid
beta concentration.
Based on the principle of an EIS immunosensor, the impedance
signal will be changed due to biomolecular interaction events at the
electrode surface. Through simple systematic surface chemistry
modification of the working electrode affect the impedimetric
measurements, a more sensitive and selective immunosensor can
be developed. In this research, we applied carbon DEP chip with
different modification strategies as our transducer for detecting
amyloid beta. Following our previous work [13,14], carbon DEP chip
surface was modified through an in situ electrochemical synthesis
using HAuCl4 (sensors B and C). The obtained RCT value of in situ
AuNPs-modified electrode was much lower than that of bare DEP
chip (in the supporting Fig. S2). The result suggests that the charge
transfer was mainly performed through AuNPs formed by electrolysis of HAuCl4. Concurrently, hydrochloric acid production after
electrolysis of HAuCl4 could activate the oxidation of free AuNPs on
the surface, under an electric potential. As a whole, AuNPs and
oxidized carbon surface acted as parallel resistors to reduce the
total electrode impedance.
3. Results and discussion
3.1. AuNPs-modified electrode surfaces
Typical scanning electron microscope (SEM) images of AuNPs on
carbon ink electrode are shown in Fig. 2. Using the PV method
where the potential was applied for different time periods, AuNPs
have angular shape and the particle morphology is not so well
defined. When the duration of the applied potential was increased
(up to 90 s), the particle morphology completely changed and the
AuNPs formed Au nano-clusters or Au nano-islands. For our purposes, AuNPs were not successfully formed using the PV method.
However, using the CV method, the AuNPs were formed successfully Fig. 2. Therefore, we selected the CV method to prepare our
AuNPs-modified electrode for biosensor fabrication. The size of the

AuNPs averaged around 40e50 nm. The uniform characteristic and
density distribution of AuNPs significantly depended on the number of cycles. The SEM images showed that for both uniform
characteristics and density distribution of AuNPs formation on
carbon electrode, using 10 and 20 cycles was much better than
using 5 cycles. However, between 10 or 20 cycles, it was difficult to
differentiate between the two. Therefore, the investigation of anti
mAb immobilization efficiency was implemented using both cycles.
For good measure, we also investigated using 5 cycles.
3.2. Evaluation of the fabricated immunosensors
Anti mAb was immobilized onto AuNPs-modified electrodes.
Fig. 3 shows the impedance spectra obtained before and after anti
mAb immobilization onto AuNPs-modified electrode via SAM layer,
with and without protein G. A significant difference in the
impedance spectra of anti mAb immobilization electrodes
compared with bare AuNPs-modified electrodes (RCT values
observed after fitting the experimental spectra using Randle
equivalent circuit) was observed. The diameter of the semicircle is
equal to the electron transfer resistance RCT, which denotes the
blocking behaviour of the electrode surface against the redox
probe, K3[Fe(CN)]6/K4[Fe(CN)]6. As seen from the results, the percentage change in the RCT (%DRCT), which is obtained before and
after anti mAb immobilization, is highest for 20 CVs-AuNPsmodified electrode (61%) and is lowest for 5 CVs-AuNPs-modified

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Fig. 2. Scanning electron microcopy (SEM) images of SAM gold nanoparticles on carbon ink electrode formed using (a) a potential step voltammetry (PV) method, where potential

was applied for 5, 20 and 90 s (Top); and (b) a cyclic voltammetry (CV) method for 5, 10 and 20 cycles (5, 10 and 20 CV) (Bottom).

electrode (24%). This result shows that the anti mAb immobilization
efficiency of 20 CVs-AuNPs-modified electrode was the most optimum. Therefore, the impedimetric biosensor used within this work
was fabricated from electrodes that were modified by AuNPs
deposited using CV method with 20 cycles.
3.3. Performance of the impedimetric amyloid beta immunosensor
We evaluated the performance of the immunosensors fabricated using methods A, B and C discussed in previous sections. The
sensors were termed Sensors A, B, and C, respectively.

3.3.1. Detection of amyloid b (1-40) peptide at anti mAb/pyrenyl
groups-modified carbon DEP chip: Sensor A
To evaluate the performance of anti mAb/pyrenyl groupsmodified carbon DEP chip, sensor A was exposed to various concentrations of amyloid b 1-40 peptide (from 1 nM to 200 mM). The
corresponding Nyquist plots of impedance spectra are shown in Fig.
4a, and the fitted impedance parameters are presented in Table S1
in supporting information SI. It was observed that the Nyquist
semicircle diameter (equal to the electron transfer resistance RCT)
negligibly changed when the peptide concentration was increased
from 1 nM to 0.1 mM. However, when the concentration of the

Fig. 3. Electrochemical impedance spectra after immobilization of anti mAb on protein G-immobilized AuNPs-modified electrode.

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amyloid b peptide (1-40) of this sensor was determined to be

2.04 mM with the sensor area of 2.64 mm2. The LoD was obtained
based on the standard deviation (STDEV) of the blank sample and
slope of calibration curve as:

LOD ¼

3xSTDEV
slope

(1)

The coefficient of variation, which is defined as the ratio of the
STDEV to the mean, changed between 1% and 8% for three repetitive
measurements, indicating good reproducibility. As far as we are
aware, this is one of the few studies on fabrication and detection of
amyloid b peptide impedimetric immunosensor, but the detection
limit was higher than the reported sensors [15,24].

Fig. 4. (a) Impedance spectra of anti mAb/pyrenyl groups-modified electrodes exposed
to difference concentration of Amyloid b 1-40 peptide and (b) the calibration curve of
RCT against Log amyloid b 1-40 antigen concentration. The impedance results were
obtained in solution containing 0.1 M KCl and 5 mM K3[Fe(CN)]6/K4[Fe(CN)]6 at OCP
and frequency range was from 100 kHz to 50 mHz with an ac probe amplitude of
10 mV. All data points are mean response values of three independent electrodes. The
error bars (calculated as standard deviation) provide a measure of the reproducibility
of the system.

peptide increased from 1 mM to 200 mM, the diameter of Nyquist
semicircle increased dramatically due to the binding of a significant
amount of peptide molecules to immobilized anti mAb in higher

concentration of peptide. Thus, the interfacial electron transfer was
hindered significantly, resulting in a correspondingly increased
electron transfer resistance [23e29]. However, the double layer
capacitance Cdl parameter which is expected to decrease when
peptide concentration increases, was found to be less sensitive to
the change of peptide concentration than RCT. This is often observed
in protein detection [23e30] and cell detection [31,32] methods.
We imagine that this could be because the sensitivity of capacitance
depends on obtaining the proper thickness of the original sensing
layer, while measurement of RCT only requires the presence of
redox-active species in the electrolyte [21]. A calibration curve was
obtained by plotting the RCT correlated to the logarithm of peptide
concentration (Fig. 4b). As can be seen, RCT did not change with
increase in peptide concentration within the detected concentration from 1 nM to 100 nM, but increased quickly from 1 mM to
200 mM. Thus, the linear range was obtained from 1 mM to 200 mM
with linear equation of RCT (kU) ¼ 4.06 þ 2.17*log C (mM)
(R2 ¼ 0.9914), that is the correlation between RCT and Log if amyloid
beta concentration is 0.9914. The limit of detection (LoD) for

3.3.2. Detection of amyloid b (1-40) peptide at anti mAb/SAM/
AuNPs-modified carbon DEP chip: Sensor B
It has been reported that the immobilization of antibody molecules is a decisive factor for successful fabrication of immunosensor [13,14]. The immobilization method must maintain the
activity and maintain the stability of biomolecules, and must be
controllable over the distribution and orientation of the immobilized species. In the method A, the DEP chip carbon ink electrode
was modified first by a functional molecule, 1-pyrenebutanoic acid
succinimidyl ester and thence anti mAb immobilization via succinimidyl ester groups. The 1-pyrenebutanoic acid succinimidyl ester
was dissolved in solution containing 70% of DMSO and 30% of
deionized water with 100 mM concentration. In our previous work
[13], we showed that a large amount of DMSO might influence the
carbon ink electrode surface due to its ability to dissolve adhesives

in the carbon ink. Furthermore, the excess DMSO on the electrode
surface can interact with amino group of antibody, which decreases
the anti mAb immobilization efficiency. Therefore, the obtained
LoD of this sensor (Sensor A) was relatively high. In order to
overcome such deficiencies, there are some protocols that are
typically utilized. They include physical absorption, chemical crosslinking and entrapment. Among them, biomolecule immobilization
methods through cross-linking can improve the stability of the
biorecognition component [25e29]. In the case of Sensor B, the
DEP chip carbon electrode was modified first by deposition of
AuNPs on working electrode using cyclic voltammetry over 20
cycles. After, anti mAb was immobilized on to AuNPs-modified
electrodes via COOH group of self-assembled monolayer of
MHDA, which served as a linker for covalent biomolecular immobilization. Fig. 5a illustrates the Nyquist plots of impedance spectra
obtained upon a gradual increase of amyloid b 1-40 peptide concentration (from 1 nM to 200 mM) and the fitted impedance parameters are presented in Table S2 in SI. As can be clearly observed,
the RCT increases with increase in amyloid b peptide concentration.
This result indicates that an insulating layer was formed on the
electrode surface due to the binding of peptide molecules to
immobilized anti mAb receptor. This insulating layer acts by
blocking the electron transfer between the redox probe and the
electrode surface. The calibration curve was obtained by plotting
the RCT value against the logarithm of peptide concentration
(Fig. 5b) which shows a linear region from 1 nM to 1 mM. The RCT
increased slowly with increasing peptide concentration from 1 nM
to 1 mM, and a significant increase was observed between 1 mM and
200 mM peptide concentration.
We suggest the reason why the calibration curve exhibits two
regions. The first is a linear region from 1 nM to 1 mM and the
second starts at a concentration of 1 mM and above. We imagine
that this is due to concentration of the immobilized antibody. As
discussed above, the RCT value denotes the blocking behaviour of

electrode surface for redox probe. The phenomenon of blocking an

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7

modification. This dramatically increased the specific area of
electrode surface which permitted a larger a mount of anti mAb
immobilization. Sensor B also has a good reproducibility with a
coefficient of coefficient of variation between 2.5% and 5.3% for
three repetitive measurements.

Fig. 5. (a) Impedance spectra of anti mAb/SAM/AuNPs-modified electrodes exposed to
difference concentrations of amyloid b 1-40 peptide and (b) the calibration curve of RCT
as against Log amyloid b 1-40 concentration. All data points are mean values of three
independent electrodes. The error bars (calculated as standard deviation) provide a
measure of the reproducibility of the system.

electrode surface is due to occupation of the surface by peptide
molecules when they bind with antibody molecules on the electrode surface. Therefore, the concentration of the antibody on the
surface plays important role. If the concentration of antibody is
relatively high when the amount of antigen is relatively low, there
is a small amount of antibody enough to react with antigen. Thus
the competitive reaction between the antigen and antibody leads
to the change of RCT that is not high. Meanwhile, at high concentration of the peptide, because of large amount of antibody to
react with antigen, the competition of peptide to occupy the space
leads to the dramatically increase of RCT. In our study, a high

concentration (100 mg/mL) of immobilized antibody was used.
This phenomenon was not observed when relatively low concentrations of immobilized antibody was used [19,24,27,29,30].
The advantage of our sensor is that it could work at both low and
high concentration of antigen. The obtained linear equation from
1 nM to 1 mM for this sensor is RCT (kU) ¼ 1.56 þ 0.20*log C (mM)
(R2 ¼ 0.9662). Based on equation (1), the LoD of the sensor B was
determined to be 2.65 nM. The LoD of Sensor B is much lower than
Sensor A. In other words, the anti mAb immobilization efficiency
of sensor B was significantly increased due to electrode surface

3.3.3. Detection of amyloid b (1-42) peptide at anti mAb/protein G/
SAM/AuNPs-modified carbon DEP chip: Sensor C
Protein G is an antibody binding protein, which specifically
binds to the Fc region of an antibody. In other words, protein G
only binds antibodies through the non-antigenic regions, leaving
the antigen binding sites of antibodies available to bind to their
target antigen. Thus, it has been widely used to immobilize antibodies in immunoassays offering important advantages such as
controllable immobilization of the antibodies, resulting in high
sensitivity and low detection limit [30,33,34]. In Sensor C, AuNPsthiolated protein G was used to immobilize anti mAb. Fig. 6 illustrates the Nyquist plots of impedance spectra obtained upon increase in amyloid b 1-42 peptide concentration from 10 pM to
200 mM, and fitted impedance parameters (Please see Table S3 in
SI). Similar to Sensor B, it can be seen that the semicircle diameter
in the Nyquist plots increased with increasing concentration of
amyloid b 1-42, and there are also two regions in the calibration
curve. Notably, the value of RCT increased rapidly even at very low
peptide concentration (10 pMe100 nM), indicating that using
AuNPs-thiolated protein G might offer much more large specific
area to immobilize a larger amount of anti mAb than sensor B.
Based on equation of RCT (kU) ¼ 2.82 þ 0.11*log C (mM)
(R2 ¼ 0.9969), the LoD for amyloid b peptide 1-42 of this sensor
was determined to be 0.57 nM (using equation (1)). Sensitivity of

the developed sensor could be further improved by modifying the
carbon electrode with graphene, for example, in order to increase
conductivity of the electrode.
Human serum albumin is one of the most abundant proteins in
biological fluids and can interfere with detection of low-abundance
biomarkers [35]. Using bovine serum albumin (BSA) we evaluated
the effect that different concentrations of BSA, co-incubated with
amyloid b peptide, had on the sensitivity of the immuno-sensor.
The results show that BSA 2.5 mg/mL did not have any discernable effect on the sensor performance.
4. Conclusion
In this work, we present a novel approach for modifying screenprinted carbon ink electrode for development of a highly sensitive
labelless impedimetric immunosensor for amyloid b peptide. Amyloid beta is an important potential biomarker of Alzheimer's disease. It is produced from a transmembrane protein, amyloid
precursor protein. Above a certain concentration, it starts to selfassemble (aggregate) into oligomers and then into either amorphous aggregates or mature fibrils [36]. The degree of amyloid beta
bioactivity is dependent on its aggregation species [37,38]. In
particular, it is now almost widely accepted that the most potent
neurotic species are the oligomers [39] Veloso and colleagues
developed a species-specific EIS immunosensor for monitoring the
effect of fibril inhibitor/dissociator using a species-specific
antibodies [40]. We have developed a relatively sensitive quantitative EIS immunosensor for amyloid beta. Three types of amyloid b
impedimetric immunosensors were fabricated in a step-wise
manner in order to understand the effects that each surface
modification chemistry had on detection sensitivity. We found that
(i) immobilization of AuNPs, to improve stability of the recognition
element and also increase the surface area for immobilization,
lowered the LOD by both ~ three orders of magnitude (from
2.04 mM to e2.65 nM). A further modification using protein G

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8

T.T.N. Lien et al. / Analytica Chimica Acta xxx (2015) 1e8

Appendix A. Supplementary information
Supplementary information related to this article can be found
at />
References

Fig. 6. (a) Impedance spectra of anti mAb/Protein G-SAM/AuNPs-modified electrodes
exposed to difference concentration of amyloid b 1-42 peptide and (b) the calibration
curve of RCT as against Log amyloid b 1-42 concentration. All data points are mean
values of three independent electrodes. The error bars (calculated as standard deviation) provide a measure of the reproducibility of the system.

towards a desired orientation of the antibody lowered the LOD
further to 0.57 nM. The sensor also demonstrated reduced cost
(cost of carbon ink printed electrode but can use as gold ink printed
electrode), detection platform simplicity, and high reproducibility.
Besides, based on current study, it was found that EIS is an
impressive method for monitoring the interaction of antigen with
antibody that occurred on the electrode surface. These methods
could easily be applied for detection of other antigens, with selection of the detection platform based on the desired for sensitivity.
We are now planning on developing a biosensor for quantitative
detection of oligomeric amyloid beta species.
Acknowledgements
Dr. Truong TN Lien gratefully acknowledges receipt of a grant
from the Japan Society for the Promotion of Science (JSPS). This
work was supported by a Grant-in-Aid for Scientific Research C
from JSPS. We would like to thank Dr. Nguyen Xuan Viet for SEM
imagines.

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Please cite this article in press as: T.T.N. Lien, et al., Modified screen printed electrode for development of a highly sensitive label-free
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