Biosensors and Bioelectronics 77 (2016) 1078–1085

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Biosensors and Bioelectronics

journal homepage: www.elsevier.com/locate/bios

A double signal electrochemical human immunoglobulin G

immunosensor based on gold nanoparticles-polydopamine
functionalized reduced graphene oxide as a sensor platform and
AgNPs/carbon nanocomposite as signal probe and catalytic substrate
Si Zhang, Na Huang, Qiujun Lu, Meiling Liu n, Haitao Li, Youyu Zhang, Shouzhuo Yao
Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering,
Hunan Normal University, Changsha 410081, PR China

art ic l e i nf o a b s t r a c t

Article history: In this paper, a double signal electrochemical Human immunoglobulin G (HIgG) immunosensor based on
Received 5 August 2015 AgNPs/carbon nanocomposite (Ag/C NC) as the signal probe and catalytic substrate was developed for
Received in revised form fast and sensitive detection of HIgG. The as-prepared AuNPs-PDA-rGO nanocomposite and Ag/C NC were
29 October 2015
confirmed by UV–vis, Fourier transform infrared spectroscopy, scanning electron microscopy and
Accepted 30 October 2015
transmission electron microscopy. Electrochemical impedance spectroscopy, cyclic voltammetry and
Available online 31 October 2015
differential pulse voltammetry were used to investigate the electrochemical properties of the proposed
Keywords: immunosensor. The AuNPs-PDA-rGO nanocomposite can improve the electron transfer rate and capture
Double signal electrochemical im- more Ab1. In the sandwich-type immunoassay process, the Ag/C NC functionalized bioconjugates were
munosensor
captured on HIgG/Ab1/AuNPs-PDA-rGO surface and the electrochemical double-signal strategy was
AuNPs-PDA-rGO
employed. These double electrochemical detection signals were directly monitored the oxidation current
Ag/C NC
Sandwich-type immunoassay originated from Ag/C NC and indirectly detected the reduction current of benzoquinone which was
produced from the reaction of H2O2 and HQ by catalysis of Ag/C NC in electrochemical detection of HIgG.
Under the optimized conditions, the current responses were changed with the concentrations of HIgG for
the proposed immunosensor with wide linear ranges of 0.1 to 100 ng mL
1 and 0.01–100 ng mL
1 with
the lowest detection concentration of 0.001 ng mL
1 in the absence and presence of H2O2 and HQ. The
double-signal strategy is used for detection of HIgG, and the results came from the two signals were well
consistent with each other. The proposed immunosensor was successfully applied in analysis of human
IgG in real samples and this strategy may provide a relative simple and effective method for construction
of other immunsensors in detection of other biomarkers in clinical medicine.
& 2015 Elsevier B.V. All rights reserved.

1. Introduction clinical diagnose. Immunoassay, which based on the highly spe-

Human immunoglobulin G (HIgG) is a class of important
component mainly existing in human serum, which plays a vital
role in recognizing some diseases (Campanella et al., 2008; Liu
et al., 2015). The HIgG concentration in normal adults ranges from
9.4 to 15.1 g/L (Li et al., 2014). High immunoglobulin levels will
cause rheumatoid arthritis, liver diseases and infectious diseases.
However, low immunoglobulin levels will also cause humoral
immunodeficiencies and metabolic diseases (Li et al., 2014).
Therefore, the development of ultrasensitive and reliable analy-
tical methods for detecting HIgG in human serum is necessary in
n
Corresponding author. Fax: þ86 731 88872531.
E-mail address: liumeilingww@126.com (M. Liu).
cific recognition of antigens by antibodies, has attracted much
attention in recent years owing to its high selectivity and low
detection limit. Enzyme-linked immunoassays (Zhao et al., 2007),
electrochemiluminescent immunoassays (Chen et al., 2014; Xu
et al., 2011) and fluorescent immunoassays (Ahn et al., 2015; Ii-
zumi et al., 2013) as the conventional immunoassays have been
commonly used in clinical diagnosis. Although some significant
researches have been reported, they are often subject to expensive
instruments, complicated operations and long assay time. Thus,
the development of new methods and strategies to enhance the
sensitivity and simplicity of clinical immunoassay is still of great
necessity.
Electrochemical immunosensors based on highly specific re-
cognition between the antigen and antibody, can give

http://dx.doi.org/10.1016/j.bios.2015.10.089
0956-5663/& 2015 Elsevier B.V. All rights reserved.
 S. Zhang et al. / Biosensors and Bioelectronics 77 (2016) 1078–1085
electrochemical signals by the transducer. Electrochemical im-
munoassay has been widely studied because of its intrinsic ad-
vantages, such as low cost, high sensitivity and simple operation
processes compared with other conventional immunoassay tech-
niques. Especially, the electrochemical sandwich-type im-
munosensors have been received striking attention in recent years
due to their improved sensitivity by different signal amplified
strategies (Yang et al., 2015; Du et al., 2010). However, these im-
munosensors must rely on the use of electroactive substance as
the second antibody's marker (Zhu et al., 2013). Therefore, the
preparation of the bioconjugates must be carefully controlled and
the electroactive substance may easily leak out from electrode
surface to the solution, which may result in unstable output sig-
nals and unreliable experimental results. Thus, it is necessary to
develop new materials to construct novel immunosensors for
sensitive and fast detection of antigen.
Nanomaterial-based electrochemical immunosensors have
been widely studied in recent years owing to their high selectivity
and sensitivity. Among the various types of nanomaterials, AgNPs
have gained significant attention because of their catalytic activity
and good electrical conductivity (Mazloum-Ardakani et al., 2015).
However, the relatively poor biocompatibility and stability of
AgNPs limits their use in immunoassay (Sun et al., 2015). There-
fore, the development of novel AgNPs-based nanomaterials with
high catalytic activity, good stability and biocompatibility is ne-
cessary. To the best of our knowledge, carbon dots (C-dots), which
is one of the new members of carbon nanomaterials family, have
attracted tremendous attention in nanotechnology field recently
(Zhao et al., 2013; Liu et al., 2012). C-dots show excellent bio-
compatibility and low toxicity in biomedical applications. Recently,
our group has successfully synthesized C-dots by one-pot elec-
trochemical carbonization of ethylene glycol (Lu et al., 2015). The
as synthesized C-dots do not need any further surface functiona-
lization and possess excellent stability and reducibility. It can act
as the reductant for direct synthesis of AgNPs to obtain silver/
carbon nanocomposite (Ag/C NC). This method is fast and simple
without using any additional reductant and stabilizing agent
compared with other conventional synthesis method of AgNPs.
More importantly, the as synthesized Ag/C NC shows excellent
peroxidase-like activity (Lu et al., 2015) and can give electro-
chemical signal (Gao et al., 2015). Furthermore, it may also afford
abundant active sites for the attachment of biomolecules owing to
the presence of AgNPs. To date, Ag/C NC-based immunoassay that
simultaneously using its peroxidase-like property and employing
it as a signal label has not been reported.
Herein, in this work, a simple and high selectivity im-
munosensor for determination of human IgG was successfully
constructed. Based on the sandwich-type immunoassay, rabbit
anti-human IgG (Ab1) was immobilized onto AuNPs-PDA-rGO
modified GCE, and goat anti-human IgG (Ab2) was integrated with
Ag/C NC as a signal label through AgNPs or carboxyl group of
C-dots. Compared with the previously reported approaches, our
protocol presents several advantages: firstly, more Ab1 can be
immobilized on the electrode surface by using AuNPs-PDA-rGO as
the immobilizing platform which has many unique properties,
including good biocompatibility, large surface area and excellent
conductivity. Secondly, the Ag/C NC/Ab2 biocomposite can result in
double electrochemical signals, one is directly originated from
oxidation of Ag/C NC, and the other is indirectly coming from re-
duction of benzoquinone which was produced from the reaction of
H2O2 and HQ by catalysis of Ag/C NC. And both of the currents
response changed with the concentration of HIgG. Thirdly, the
proposed electrochemical immunosensor based on the double
signal strategy is reliable and the detection results can be sup-
ported by each other. This double-signal electrochemical im-
munosensor exhibited excellent sensitivity, wide detection range
1079
and low detection limit and it may provide a new mode for pro-
posing other sandwich-type immunosensor for detection of other
biomarkers.
2. Experimental section
2.1. Reagents and apparatus
Human IgG, rabbit anti-human IgG and goat anti-human IgG
were purchased from Bioss (Beijing, China). Dopamine hydro-
chloride (DA), chloroauric acid (HAuCl4), bovine serum albumin
(BSA), silver nitrate (AgNO3) and ethylene glycol (C2H6O2) were
purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai,
China). Graphene oxide (GO) was purchased from XFNANO Co.,
Ltd. (Nanjing, China) and used without further purification. All
other chemicals were of analytical grade and used as received.
Tris–HCl buffer solution (10 mM, pH 8.5) was used for PDA pre-
paration and functionalization. Phosphate buffer solution (PBS,
0.1 M) was prepared with Na2HPO4, NaH2PO4 and 0.1 M Na2SO4.
All aqueous solutions were prepared with ultrapure water.
The UV–vis spectra (UV) were obtained on a UV-2450 spec-
trophotometer (Shimadzu, Japan). FT-IR spectra (IR) were collected
on a Nexus-670 Fourier transform-infrared spectrophotometer
(Nicolet Instrument Co, USA) with the KBr pressed pellet trans-
mission mode. The morphology and composition of nanocompo-
site were characterized by scanning electron microscopy (SEM)
and energy dispersive X-ray spectroscopy (EDS) (Zeiss Sigma,
Germany) and the size of the nanoparticles was characterized by
transmission electron microscopy (TEM) (JEOL, Japan). Electro-
chemical experiments, including cyclic voltammetry (CV), elec-
trochemical impedance spectra (EIS), differential pulse voltam-
metry (DPV) were carried out with a research electrochemical
workstation of SP-150 (BioLogic Science Instruments, France)
controlled by EC-Lab software. A three-electrode system was used
which contained a glassy carbon electrode as working electrode, a
saturated calomel reference electrode (SCE) and a carbon rod
electrode as counter electrode. All potentials here were cited
versus SCE. All experiments were performed at room temperature.
2.2. Synthesis of AuNPs-PDA-rGO nanocomposite
PDA-rGO was synthesized by previous method (Hu et al., 2014).
Firstly, 10 mg of GO and 10 mg of dopamine hydrochloride were
added into 20 mL of 10 mM Tris–HCl solution (pH¼8.5) and dis-
persed by sonication for 30 min in ice bath. Then, the reaction
mixture was stirred vigorously at 60 °C for 20 h. After cooling to
room temperature, 600 μL of 2% HAuCl4 was added into the above
mixture under vigorous stirring. 1 mL of 60 mM DA was added
dropwisely to the above solution, and the reaction mixture was
stirred 4 h to allow complete reaction and form AuNPs-PDA-rGO
nanocomposite, followed by centrifugation and washed with wa-
ter. Finally, the obtained AuNPs-PDA-rGO nanocomposite was re-
dispersed in pH 8.5 Tris–HCl buffer solution before further use.
2.3. Synthesis of Ag/C NC/Ab2 composite
C-dots were electrochemically synthesized based on our pre-
vious work (Lu et al., 2015). Briefly, ethylene glycol and sodium
hydroxide solution were used as the precursor and electrolyte,
platinum sheets employed as the positive and negative electrodes.
The C-dots were synthesized by electrolyzing in the electrolyte
solution under 30 V for 40 min. After electrolysis, the electrolyte
solution was adjusted to neutral with hydrochloric acid and then
dialyzed (Mw 1000) against water for two days, followed by
centrifuging at 12000 rpm for 10 min to remove the precipitate
1080 S. Zhang et al. / Biosensors and Bioelectronics 77 (2016) 1078–1085
particles.
The Ag/C NC was synthesized according to our previous work
(Lu et al., 2015). A certain amount of AgNO3 aqueous solution
(0.1 mM) was added into 1 mL of the as-synthesized C-dots (NaOH
treated) aqueous solution at room temperature for 40 min.
Ag/C NC/Ab2 was prepared typically as follows: firstly, 100 μL of
100 μg mL
1 goat anti-HIgG (Ab2) was added into 1 mL of the as-
prepared Ag/C NC solution under vigorous stirring at 4 °C for 5 h,
followed by centrifugation and washing with ultrapure water. Fi-
nally, the obtained Ag/C NC/Ab2 was redispersed in ultrapure
water before further use.
2.4. Fabrication of the immunosensor
Glassy carbon electrode (GCE, 3 mm in diameter) was firstly
polished with emery paper and 50 nm alumina slurries followed
by rinsing thoroughly with ultrapure water. The electrodes were
successively ultrasonicated in ethanol and water and then washed
thoroughly with ultrapure water to obtain fresh and clear surface,
and dried in the air. 10 μL of 0.1 mg mL
1 AuNPs-PDA-rGO solu-
tion was dropped onto the GCE and dried in air. Subsequently,
10 μL of 100 μg mL
1 primary antibody (Ab1) was attached onto
the surface of AuNPs-PDA-rGO/GCE for 12 h at 4 °C. Successively,
the modified immunosensor was blocked with 10 μL of
50 mg mL
1 BSA for 40 min at room temperature. After every step,
the modified electrode was thoroughly cleaned with ultrapure
water to remove the physically adsorbed species. Based on the
sandwich-type immunoassay strategy, the immunosensor was
incubated with target antigens (HIgG) solution for 35 min at room
temperature, and then the as-prepared 10 μL of Ag/C NC/Ab2 was
dropped onto the modified electrode surface for immune reaction
at room temperature. The fabrication procedures of the im-
munosensor are presented in Scheme 1.
Scheme 1. The fabrication processes for the electrochemical immunosensor.
2.5. Electrochemical measurements
Electrochemical experiments were performed in a conventional
three-electrode system. DPV was used to monitor the current re-
sponses after the sandwich reaction to detect HIgG in the absence
or presence of HQ and H2O2. The DPV were carried out in 0.1 M-pH
7.4 PBS at room temperature. The DPV parameters applied were:
pulse amplitude, 2.5 mV; pulse width, 100 ms; pulse period, 0.2 s;
scanning potential,
0.2 to 0.5 V and 0.4 to
0.3 V.
3. Results and discussion
3.1. Characterization of the materials and immunosensor
UV spectra of C dots (a), Ag/C NC (b) and Ag/C NC/Ab2 (c) are
displayed in Fig. 1A. The as-synthesized C-dots (a) shows a strong
absorption peak at 257 nm, which can be attributed to the π–π*
transition (Jaiswal et al., 2012). After reaction with AgNO3, besides
the strong absorption at 257 nm, a broad peak at around 410 nm
(b) is observed, which is the characteristic absorption peak of AgNPs
(Tan and Chen, 2012). The color of the reaction solution changed
from pale yellow to bright yellow during the reaction. After goat anti-
HIgG conjugation with Ag/C NC, the intensity of the absorption at
410 nm decreased. At the same time, the absorption intensity at
257 nm increased with conjugation time. All results illustrated that
Ag/C NC was formed and the goat anti-HIgG was successfully im-
mobilized on Ag/C NC. The successful preparation and modification
were further confirmed by FTIR. The results are shown in Fig. 1B, the
characteristic peaks at 3408 and 1074 cm
1 are corresponding to the
stretching vibration and bending vibration of the hydroxyl groups of
C-dots (Fig. 1B, a) (Lu et al., 2015). Compared with the spectrum of
C-dots, the relative intensity of hydroxyl groups (–OH) slightly in-
creased and the carbonyl group decreased which was attributed to
the reduction of C-dots during the NaOH-treated process. While
 S. Zhang et al. / Biosensors and Bioelectronics 77 (2016) 1078–1085 1081

0.8 a
a

Transmittance(%)
b
0.6

Absorbance 0.4 c

1571
1674
0.2

867
985
b
0.0

1126
200 300 400 500 600 700 800 4000 3500 3000 2500 2000 1500 1000 500
Wavelength(nm) Wavenumber(cm ) -1

a
1.5
b

Transmittance(%)
Absorbance

1.0

3200
b

0.5 c
c

1729
0.0 a

1613
1511
200 300 400 500 600 700 800 4000 3500 3000 2500 2000 1500 1000 500
Wavelength(nm) Wavenumber(cm )
-1

Fig. 1. The UV (A) and FTIR spectra (B) of C dots (a), Ag/C NC (b) and Ag/C NC/Ab2 (c); the UV (C) and FTIR spectra (D) of PDA (a), PDA-AuNPs (b), AuNPs-PDA-rGO (c).

Fig. 2. TEM images of C-dots (A), Ag/C NC (B) and SEM image (C) and EDS (D) of AuNPs-PDA-rGO.
1082 S. Zhang et al. / Biosensors and Bioelectronics 77 (2016) 1078–1085

comparing with the C-dots, the intensity of carbonyl groups (C¼O)
(1721 cm
1) slightly decreased after reaction with AgNO3 and this
was because the C-dots was only partially oxidized in the AgNO3
solution to produce AgNPs. After goat anti-HIgG was conjugated with
Ag/C, the new characteristic peaks at 1674 cm
1 for amide I and
1571 cm
1 for amide II absorption bands were found (Wu et al.,
2007). Therefore, this indicated that goat anti-HIgG was successfully
conjugated with Ag/C NC.
UV spectra have been employed to monitor the preparation of
AuNPs-PDA-rGO nanocomposite. As shown in Fig. 1C, the spec-
trum of PDA shows two strong characteristic absorption peaks at
around 215 nm, 278 nm (Fig. 1C, a) and a small peak at 410 nm
(which can be seen clearly in Fig. S1A, a). After addition of HAuCl4,
there was a new absorption peak around 500 nm (Fig. 1C, b),
which was the characteristic absorption peak of AuNPs (Hu et al.,
2014). When the AuNPs-PDA-rGO nanocomposite was synthe-
sized, the characteristic absorption peaks at 215 nm and 278 nm
shifted to 208 nm and 280 nm (Fig. 1C, c). Meanwhile, a new ab-
sorption peak around 510 nm was observed, which can be seen
clearly in Fig. S1A, (b). Thus, all results illustrated that the AuNPs-
PDA-rGO nanocomposite was synthesized successfully.
The as-prepared AuNPs-PDA-rGO nanocomposite can also be
characterized by FTIR. As shown in Fig. 1D (b), the characteristic
peaks at 1519, 1316 and 3200 cm
1 originating from the scissoring
vibration of N-H, bending vibration of –CH2 and –NH group of PDA
(Hu et al., 2014) increased significantly after DA reaction with
HAuCl4. The results showed that DA was self-polymerized and
HAuCl4 was reduced. No obvious peak for C ¼O around 1729 cm
1
was observed in the spectrum of PDA-rGO (Fig. S1B, b), indicating
that the carboxyl groups on GO were disappeared and the GO was
reduced during the self-polymeriztion of DA (Kang et al., 2011).
Besides, as shown in Fig. 1D (c), the peak intensity at 1729 cm
1
for C ¼O in GO obviously decreased, and the characteristic peak of
PDA was almost remained in the resultant composite. Thus, the
above results indicated that the AuNPs-PDA-rGO nanocomposite
was successfully prepared.
The morphologies and nanostructures of C-dots and Ag/C NC
were investigated by transmission electron microscopy (TEM). As
shown in Fig. 2A, the C-dots were of low multiple dispersity with
the diameters ranging from 2 to 4 nm (Lu et al., 2015). After re-
action with AgNO3, the as-synthesized Ag/C NC diameters ranged
from 10 to 12 nm in diameters (Fig. 2B). The Ag/C NC shows good
dispersion without obvious aggregation. This may result from the
C-dots that acted as the stabilizing agents to attach to the Ag
surface through hydrogen bonding to protect AgNPs from
aggregation.
Fig. 2 shows the SEM (C) and EDS (D) of the AuNPs-PDA-rGO. It
was found that gold nanostructures distributed on the PDA-rGO
nanosheets on SEM image. EDS was used to analyze the chemical
composition of AuNPs-PDA-rGO, and it was found that the peaks
of C, N, O and Au were obviously found as shown in Fig. 2D.
Therefore, the above results showed that AuNPs-PDA-rGO nano-
composite was successfully prepared.
CVs were performed to characterize the surface features of the
electrodes in 2 mM Fe(CN)63
/4
containing 0.1 M K2SO4 solution.
As shown in Fig. 3A, a pair of well-defined redox peaks for
Fe(CN)63
/4
was obtained at bare GCE (Fig. 3A, a). It is obvious
that the peak current was the highest on AuNPs-PDA-rGO mod-
ified electrode (Fig. 3A, b). This may result from the excellent
conductivity and larger specific surface area of AuNPs-PDA-rGO
nanocomposite. After the immobilization of anti-HIgG, BSA and
HIgG layer by layer in Fig. 3A (c, d and e), the peak currents de-
creased gradually. After incubation of Ag/C NC/Ab2 bioconjugates
(Fig. 3A, f), the peak current decreased obviously, this may result

60 5000
a
a
b
40 c 4000 b
c
d
d
e 3000 e
20 f
-Z'' /Ω

f
2000
0 C
1000
R
-20
0
R Z
-40
-0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0 2000 4000 6000
E /V (vs.SCE) Z' /Ω

14 0
a B
12 b
c
10 d -10
e
8 f
-20
6
4 -30 a
d
2
c
0 -40 b

-0.2 0.0 0.2 0.4 0.6 -0.4 -0.2 0.0 0.2 0.4
E /V (vs.SCE) E /V (vs.SCE)

Fig. 3. CV (A) and EIS (B) of bare GCE (a), AuNPs-PDA-rGO/GCE (b), Ab1/AuNPs-PDA-rGO/GCE (c), BSA/Ab1/AuNPs-PDA-rGO/GCE (d), HIgG/BSA/Ab1/AuNPs-PDA-rGO/GCE
(e) and Ag/C NC/Ab2/HIgG/BSA/Ab1/AuNPs-PDA-rGO/GCE (f) in 2 mM Fe(CN)63
/4
. (C) DPVs of bare GCE (a) Ag/C NC/Ab2/HIgG/GCE (b), HIgG/BSA/Ab1/AuNPs-PDA-rGO/GCE
(c) and Ag/C NC/Ab2/HIgG/BSA/Ab1/AuNPs-PDA-rGO/GCE with different concentrations of HIgG (d: 1 ng mL
1, e:10 ng mL
1, f: 100 ng mL
1) and (D) Ag/C NC/Ab2/HIgG/GCE
(a), Ag/C NC/Ab2/HIgG/BSA/Ab1/AuNPs-PDA-rGO/GCE with different concentrations of HIgG (b: 1 ng mL
1, c: 10 ng mL
1, d: 100 ng mL
1) in 0.1 M-pH 7.4 PBS (C: in absence
of HQ and H2O2, D: in presence of HQ and H2O2) at a scan rate of 50 mVs
1 and 100 mVs
1, respectively.
 S. Zhang et al. / Biosensors and Bioelectronics 77 (2016) 1078–1085
from the weak electron transfer speed of Ag/C NC/Ab2. Therefore,
the above results indicated that each layer was modified on the
electrode successfully.
The electron transfer properties of the modified electrodes
after each modification process were further characterized by EIS.
As shown in Fig. 3B, the Nyquist plots were fitted by the Randles
equivalent circuit (Fig. 3B, inset), where Rs is the solution re-
sistance, Cd is the capacitance, Rct is the charge transfer resistance
and ZW is the Warburg impedance. The Rct of the electrode is equal
to the semicircle diameter of the Nyquist diagram. Well-defined
semicircles with Rct of 307 Ω and 199.6 Ω at higher frequencies
were obtained for the bare GCE (Fig. 3B, a) and AuNPs-PDA-rGO/
GCE (Fig. 3B, b), indicating that the bare GCE had relatively larger
interface electron transfer resistance than that of AuNPs-PDA-rGO/
GCE. It suggested that AuNPs-PDA-rGO/GCE could improve the
conductivity and accelerate the electron transfer. With the fol-
lowing immobilization, blocking and incubation steps, the Rct va-
lues increased gradually. Especially, after Ag/C NC/Ab2 was grafted
onto the HIgG/BSA/Ab1/AuNPs-PDA-rGO/GCE, a remarkable in-
crease in the interfacial resistance (Fig. 3B, f) was found. These
results indicated that the immunocomplex layers were modified
on the electrode surface successfully. This maybe result from that
the immunocomplex layers on the electrode can act as the elec-
tron transfer blocking layers to hinder the electron transfer. The
above results were agreed with the CV results.
3.2. Analytical feasibility of the electrochemical immunosensor
In this study, we choose the double-signal strategy to in-
vestigate the feasibility of the electrochemical immunosensor.
Method A is directly using Ag/C NC/Ab2 composite to produce
electrochemical signal to detect HIgG. In this process, the experi-
ment was controlled by monitoring the oxidation currents of Ag/C
NC with different HIgG concentrations without introduction of any
other electroactive substance in PBS. Method B is using Ag/C NC to
catalyze the reaction of H2O2 and HQ for indirectly electrochemical
detection of HIgG in the presence of HQ and H2O2. To prove the
catalysis properties of Ag/C NC, the catalysis of the reduction of
H2O2 to oxidize HQ by Ag/C NC was conducted. The reduction of
benzoquinone (the oxidation product of HQ) in the presence of
H2O2 was studied by DPV on the Ag/C NC modified electrode. As
shown in Fig. S2, the peak currents of benzoquinone increased
with concentrations of HQ at the Ag/C NC modified electrode in pH
7.4 PBS containing 1.25 mM H2O2. The results illustrated that Ag/C
NC can catalyze the reduction of H2O2 to oxidize HQ.
The electrochemical immunosensor was then conducted on the
basis of Ag/C NC and AuNPs-PDA-rGO. As shown in Fig. 3C, the
experiment was performed by comparing their current responses
of different modified electrodes under the same conditions. It was
found that there was not any obvious peak for the oxidation of Ag
was observed on bare GCE (a), Ag/C NC/Ab2/HIgG (b) and
HIgG/BSA/Ab1/AuNPs-PDA-rGO (c) modified electrodes. The rea-
sons may be that the Ag/C NC was not stably immobilized on the
electrodes or there were not form sandwich structures on those
electrodes (Li et al., 2015). On the other hand, the oxidation peak
currents of the Ag (Fig. 3C, d, e, f) increased with the concentra-
tions of HIgG in PBS when Ag/C NC/Ab2/HIgG/BSA/Ab1/AuNPs-
PDA-rGO was used. This may indicated that this method may be
used to quantitatively detect of HIgG.
Secondly, the indirectly detect of HIgG is shown in Fig. 3D, and
the current responses for different electrodes in the presence of
1 mM HQ and 1.25 mM H2O2 were investigated. The reduction
current of benzoquinone (the oxidation product of HQ) on the Ag/
C NC/Ab2/HIgG/BSA/Ab1/AuNPs-PDA-rGO modified electrode was
obviously improved in comparison with the Ag/C NC/Ab2/HIgG
(Fig. 3D, a) modified electrode. And the reduction peak currents of
1083
benzoquinone (Q) increased with concentrations of HIgG in the
same condition (Fig. 3D, b, c, d). This may ascribe to the catalytic
property of Ag/C NC towards the reaction of H2O2 and HQ, the
reaction mechanism may be as follows (Ionescu et al., 2007):
Ag/C
HQ + H2 O2 → Q + H2 O (1)
Q + H+ + e− → HQ (2)
In this process, Ag/C NC catalyzed the reduction of H2O2 which
was transferred into H2O, and meanwhile, HQ was oxidized into
benzoquinone (Q) (1). And then Q got an electron and was reduced
to HQ on the electrode surface (2). With different amount detec-
tion objects, the different amount of Ag/C NC conjugates will be
immobilized on the electrode, which may show different current
responses for Q (Zhao et al., 2014). Therefore, the quantitative
detection of HIgG can also be realized. Thus, this may indicated
that method B may also can be used to quantitatively detect of
HIgG.
3.3. Optimization of experimental conditions
To get the optimal experimental results, the optimization of
experimental condition is necessary. The volume of PDA-rGO-
AuNPs nanocomposite will affect the binding sites for HIgG and
the current of the immunosensor. As shown in Fig. S3A, the peak
current increases with the volume of PDA-rGO-AuNPs from 4 to
10 μL and then decreases when the volume is over 10 μL. In fact,
the PDA-rGO-AuNPs nanocomposite can capture antibodies and
improve the performance of the immunosensor. But excess PDA-
rGO-AuNPs nanocomposite may hinder interface electron transfer
between nanocomposite and electrode. Therefore, 10 μL was the
optimal volume for immunosensor.
The incubation time for the immune-reaction is also studied as
shown in Fig. S3B. Upon the addition of HIgG solution, the peak
current obviously increased within 35 min, and then decreased
over 35 min. So the optimal incubation time was about 35 min for
next investigation.
The properties of biomolecules were affected by pH, so the pH
values of PBS ranging from 6.0 to 8.0 were studied. As shown in
Fig. S3C, the current value increased with pH from 6.0 to 7.4, and
then decreased in the range of 7.4 to 8.0. Therefore, pH 7.4 was the
optimal pH value for the immunosensor, which was also close to
the pH of the human body.
In addition, the ratio of the Ab2 and Ag/C NC in preparation of
the Ag/C NC and the concentration of H2O2 and HQ in the detec-
tion process were also conducted. As shown in Fig. S3D–F, the
optimal ratio of the Ab2 (100 μg mL
1) and Ag/C NC was 1:10
(V1:V2), the optimal concentration of HQ was 1 mM, the optimal
concentration of H2O2 was 1.25 mM.
3.4. Electrochemical detection of HIgG based on the double signal
strategy
Under the optimal experimental conditions, the relationship
between peak current values and HIgG concentrations was studied
with DPV using the double-signal strategy for detecting of HIgG.
The proposed immunosensor based on method A was eval-
uated by measuring the current generated from the oxidation of
AgNPs in Ag/C NC. As shown in Fig. 4A, the oxidation peak currents
of Ag increased with concentrations of HIgG in pH 7.4 PBS. This can
be attributed the increasing amount of Ag/C NC on the platform
after the sandwich reaction. It was indicated that the adsorbed
HIgG is directly related to the amount of the immobilized Ag/C
NC/Ab2 on electrode. Therefore, through the monitor the oxidation
current of Ag/C NC, we can evaluate the amount of the detected
1084 S. Zhang et al. / Biosensors and Bioelectronics 77 (2016) 1078–1085
10
8
6 6.0
4
2 4.5
-0.2 0.0 0.2 0.4
E /V (vs.SCE)
0 -16
-10
-20
-30
-40
-0.4 -0.2 0.0 0.2
E /V (vs.SCE)
Fig. 4. DPVs of immunosensor to different HIgG concentrations (A, C) and the calibration curves (B, D) for HIgG determination in 0.1 M PBS ( pH 7.4) in the absence (A, B) and
in the presence of (C, D) 1.0 mM HQ and 1.25 mM H2O2.
HIgG. The calibration plots showed a good linear relationship be-
tween the currents and the logarithmic values of the HIgG con-
centrations from 0.1 to 100 ng mL
1, as shown in Fig. 4B. The re-
gression equation was I (μA)¼5.439 þ 0.7461 log c (ng mL
1) with
the correlation coefficient of 0.9908 and a detection limit of 0.0075
ng mL
1 (signal/noise ¼3).
The proposed immunosensor based on method B was in-
vestigated by measuring the current generated from the reduction
of benzoquinone. As shown in Fig. 4C, the peak current of ben-
zoquinone increased with concentrations of HIgG in the pH 7.4 PBS
in the presence of 1 mM HQ and 1.25 mM H2O2. It was indicated
that the amount of the immobilized Ag/C NC/Ab2 will indirectly
affected the reduction of the benzoquinone which was an oxida-
tion product of HQ catalyzed by Ag/C NC in presence of H2O2. Si-
milarly, the amount of the HIgG will directly lead to change of Ag/C
NC and then affected the response of benzoquinone. Through the
monitor the reduction current of benzoquinone, the amount of the
detected HIgG can be obtained. It showed a good linear relation-
ship between currents and the logarithm concentrations of HIgG
from 0.01 to 100 ng mL
1 (Fig. 4D). The regression equation was I
(μA) ¼
22.89 to 2.211 log c (ng mL
1) with the correlation coef-
ficient of 0.9921 and the lowest detection concentration of
0.001 ng mL
1.
Comparing of the results of the two methods, we can know that
the detection ranges for HIgG were overlapped from 0.1 to
100 ng mL
1 when detecting of HIgG. Based on the double-signal
strategy, according to the two linear equations, quantitative de-
tection of HIgG could be realized. Meanwhile, the detection results
can be supported by each other simply and conveniently. Fur-
thermore, the linear ranges and detection limits of the proposed
immunosensor were compared with previous reports, and the
results are listed in Table S1. It was found that the immunosensor
exhibited good performance. The results demonstrated that the
proposed double-signal immunosensor could be used to detect
7.5
7.0
6.5
5.5
5.0
4.0
0.6 -2 -1 0 1 2 3
logc/ng mL-1
-18
-20
-22
-24
-26
-28
-30
0.4 -3 -2 -1 0 1 2 3
logc/ng mL-1
HIgG quantitatively in real samples.
3.5. Selectivity and stability of the immunosensor
Possible interfering substances were used to evaluate the se-
lectivity of the proposed immunosensor. As shown in Fig. S4A–B,
the immunosensors were incubated with 1.0 ng mL
1 HIgG so-
lution containing 100 ng mL
1 of the interference of BSA, L-cy-
steine, tyrosinase (TYR) and prostate specific antigen (PSA), re-
spectively. There is no remarkable change in DPV peak current
based on the two methods for the mixture in comparison with
individual HIgG solution. Therefore, it can be concluded that the
selectivity of the proposed immunosensors was acceptable. The
stability of the immunosensor is a key factor in developing of good
immunosensor. As shown in Fig. S4C–D, the immunosensor was
stored at 4 °C when it was not in use, and it retained 94.53%
(method A) and 96.63% (method B) of its initial response when
detecion of 10 ng mL
1 HIgG after 15 days of storage. The fact
indicates the good stability of the proposed immunosensor.
3.6. Application of the immunosensor
In order to further investigate the applicability of the proposed
immunosensor for real samples. The three serum samples were
diluted to proper concentrations with PBS (pH 7.4, 0.1 M), then the
immunosensor was used to detect of the concentration of HIgG.
The standard addition method was employed to validate the ac-
curacy and precision of the proposed immunosensor (Wang et al.,
2015). By adding different standard concentrations of HIgG to the
diluted human serum samples (Zhao et al., 2015; Mazloum-Ar-
dakani et al., 2015) and then dropped on the modified electrode to
form the sandwich-type structure followed by the conjugation
with the Ab2 complex, the samples were detected based on the
double signals strategy. The detection results come from the two
 S. Zhang et al. / Biosensors and Bioelectronics 77 (2016) 1078–1085 1085

Table 1 and Technology Department (14JJ4030, 13JJ2020), the Opening

Comparison of the responses obtained by the proposed immunosensor (containing Fund of state key Laboratory of Chemo/Biosensing and Chemo-
method A and method B) towards HIgG in serum samples.
metrics (Hunan University, 2013017), the Construct Program of the
Sample Method Added Found: mean 7SD Recovery RSD Key Discipline and Aid Program for Science and Technology In-
(ng mL
1) (ng mL
1 n ¼6) (%) (%, n ¼6) novative Research Team in Higher Educational Institutions of Hu-
nan Province.
Human serum A 0.00 9.39 7 0.28 – 2.98
B 0.00 9.42 7 0.21 – 2.23
A 0.10 9.50 7 0.31 110.0 3.26
B 0.10 9.517 0.34 90.0 3.58
Appendix A. Supplementary material
A 50.0 58.34 7 1.31 97.9 2.25
B 50.0 60.81 7 1.59 102.3 2.61
A 100.0 112.687 3.76 103.3 3.34 Supplementary data associated with this article can be found in
B 100.0 107.83 7 2.14 98.41 1.98 the online version at http://dx.doi.org/0.1016/j.bios.2015.10.089.

signals were consistent with each other when detection of the

same sample, as shown in Table 1. The recovery range was from
97.9 to 110.0% and the relative standard deviations range from 2.25
to 3.34% by method A. While for method B, the recovery rate was
90.0–102.3% and the relative standard deviations ranged from 1.98
to 3.58%. Meanwhile, the results based on the proposed method
were compared with that of the method in hospital, which was
shown in Table S2 in the supporting information. It can be con-
cluded that results originated from the proposed strategy is sa-
tisfactory. Thus, we can conclude that the proposed immunosensor
could be used for clinical application.
4. Conclusions
In summary, a relatively simple, sensitive and convenient
electrochemical double-signal immunosensor for the detection of
HIgG based on PDA-rGO-AuNPs and Ag/C nanocomposite was
proposed for the first time. In this study, Ag/C NC acting as a signal
label and antibodies carrier, could not only give electrochemical
signal directly from Ag/C NC but also catalyze the reduction of
H2O2 to oxidize HQ for detection of HIgG. From the experiment
results, we can conclude that the proposed immunosensor possess
excellent selectivity, acceptable stability and good reliability. The
double signals strategy based electrochemical immunosensor can
be used to evaluate the HIgG in real samples and the results were
satisfactory. Therefore, this double signals strategy might provide a
promising and reliable method for construction other im-
munosensor of biomarkers in clinical medicine in the future.
Acknowledgment
This work was supported by the National Natural Science
Foundation of China (21305042, 21375037), Scientific Research
Fund of Hunan Provincial Education Department (14B116), Science
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