Analytical Biochemistry 482 (2015) 48–54

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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

Simultaneous electrochemical detection of multiple biomarkers using

gold nanoparticles decorated multiwall carbon nanotubes as signal
enhancers
Dexiang Feng a,b, Lihua Li a,b, Junqing Zhao a, Yuzhong Zhang a,⇑
a
College of Chemistry and Materials Science, Anhui Normal University, Wuhu 241000, People’s Republic of China
b
Department of Chemistry, Wannan Medical College, Wuhu 241002, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: In this work, a novel sandwich-type electrochemical immunosensor has been developed for simulta-
Received 17 January 2015 neous detection of carcinoembryonic antigen (CEA) and a-fetoprotein (AFP) based on metal ion labels.
Received in revised form 13 April 2015 Gold nanoparticles decorated multiwall carbon nanotubes (AuNPs@MWCNTs) were used as carriers to
Accepted 16 April 2015
immobilize secondary antibodies and distinguishable electrochemical tags of Pb2+ and Cd2+ to amplify
Available online 22 April 2015
the signals. Due to the intrinsic property of high surface-to-volume ratio, the AuNPs@MWCNTs could
load numerous secondary antibodies and labels. Therefore, the multiplexed immunoassay exhibited
Keywords:
good sensitivity and selectivity. Experimental results revealed that this sandwich-type immunoassay
Gold nanoparticles decorated multiwall
carbon nanotubes
displayed an excellent linear response, with a linear range of 0.01 to 60 ng mL–1 for both analytes
Pb2+ and Cd2+ and detection limits of 3.0 pg mL–1 for CEA and 4.5 pg mL–1 for AFP (at a signal-to-noise ratio of 3).
Multiplex assay The method was successfully applied for the determination of AFP and CEA levels in clinical serum
Carcinoembryonic antigen samples.
a-Fetoprotein Ó 2015 Elsevier Inc. All rights reserved.

Precise and early determination of multiplex tumor markers
could greatly improve the treatment efficiency of many cancers
in clinical diagnosis. Therefore, simultaneous detection of mul-
tiplex tumor markers related to a certain cancer in human
serum has attracted great attention in the biomedical field.
During recent years, various immunoassay methods for simulta-
neous detection of multiplex tumor markers have been devel-
oped [1–5]. Among these methods, electrochemical
immunoassay has become one of the predominant analytical
methods due to high sensitivity, inherent simplicity, and low
cost [6–8].
To realize successfully electrochemical simultaneous multi-
plexed immunoassays, an important issue is to search distinguish-
able signal probe as trace labels [9,10] in multiple labels mode. As
ideal multiple tags for proteins label, it should meet two demands;
one is based on the independent response achieved by each target
on the identical sensing interface, and the other is based on distin-
guishable voltammetric signals to avoid interaction with analytes
and the sample matrix [11]. Nowadays, a large number of signal
⇑ Corresponding author. Fax: +86 553 3869303.
E-mail address: zhyz65@mail.ahnu.edu.cn (Y. Zhang).
tags such as enzymes, quantum dots (QDs),1 oligonucleotide, and
dyes by loading on carriers for the preparation of labels have been
reported in electrochemical immunoassay [12]. Among them, QDs
have been proved to be promising due to their comparable size
and feasibility for surface modification [13]. However, QD-based
labels often not only require a tedious preparation process but also
involve a complicated detection step of acid-dissolving process to
obtain the electrochemical signals [14,15]. As a result, there is still
a great challenge in developing novel probes with a simple prepara-
tion process and easy detection steps. Recently, Feng and coworkers
developed a novel multianalyte electrochemical immunosensor for
ultrasensitive detection of cardiac troponin and fatty-acid-binding
protein using metal ions Zn2+ and Cd2+ functionalized titanium phos-
1
Abbreviations used: QD, quantum dot; AFP, a-fetoprotein; CEA, carcinoembryonic
antigen; PDDA, poly(diallyldimethylammonium chloride); AuNPs@MWCNT, gold
nanoparticles decorated multiwall carbon nanotube; anti-CEA, CEA antibody;
anti-AFP, AFP antibody; BSA, bovine serum albumin; HAc, acetic acid; NaAc, sodium
acetate anhydrous; HAuCl4
4H2O, chloroauric acid; PBS, phosphate-buffered saline;
PBST, PBS containing Tween; SEM, scanning electron microscopy; Ab2,1, anti-CEA2,1;
Ab2,2, anti-AFP2,2; SWV, square wave voltammetry; CV, cyclic voltammetry; EIS,
electrochemical impedance spectrum; IgG, immunoglobulin G; Glu, glucose; AA,
ascorbic acid; ELISA, enzyme-linked immunosorbent assay.

http://dx.doi.org/10.1016/j.ab.2015.04.018
0003-2697/Ó 2015 Elsevier Inc. All rights reserved.
 Electrochemical detection of two tumor markers / D. Feng et al. / Anal. Biochem. 482 (2015) 48–54
phate nanospheres as labels [16]. Due to metal ions’ highly sensitive
electrochemical response and easy signal obtaining process, the
metal ions as labels are beneficial as reported [17], but this method
still involves time-consuming fabrication of titanium phosphate
nanospheres for metal ions exchange. Thus, the development of a
metal ion label with simple carriers in electrochemical immunoassay
is urgently needed.
Here, we have developed a simple electrochemical immunosen-
sor for simultaneous detection of a-fetoprotein (AFP) and carci-
noembryonic antigen (CEA) using Cd2+ and Pb2+ as multiple
labels with a simple carrier. Using poly(diallyldimethylammonium
chloride) (PDDA) as linkage reagents, the gold nanoparticles deco-
rated multiwall carbon nanotubes (AuNPs@MWCNTs) nanocom-
posites with good electric conductivity and biocompatibility
[18,7,19] were easily fabricated not only as an ideal simple carrier
but also as an excellent signal enhancer. As expected, the
immunosensor can achieve simultaneous detection of CEA and
AFP in one detection platform with different voltammetric peaks
through square wave voltammetry. The peak currents and peak
positions were dependent on the concentration and type of the
corresponding analytes, respectively. In addition, the peak poten-
tials were not too far, which may save the detection time. The
obtained immunosensor exhibited sensitive and stable response
for detection of multiple tumor markers and showed great poten-
tial in clinical applications.
Materials and methods
Materials
CEA, CEA antibody (anti-CEA), AFP, and AFP antibody (anti-AFP)
were purchased from Biocell Biotechnology (Zhengzhou, China)
and stored in a refrigerator at 4 °C. Bovine serum albumin (BSA)
and PDDA (20% [w/w] in water, MW 200,000–350,000) were pur-
chased from Sinopharm Chemical Reagent (Shanghai, China).
MWCNTs with carboxylic acid groups (purity >95%, diameter 20–
30 nm, length 10–30 lm) were obtained from Chengdu Institute
of Organic Chemistry (Chengdu, China). Acetic acid (HAc), sodium
acetate anhydrous (NaAc), Cd(NO3)2, Pb(NO3)2, and chloroauric
acid (HAuCl4
4H2O) were obtained from Shanghai Chemical
Reagent (Shanghai, China). Phosphate-buffered saline (PBS) with
various pH values was prepared by mixing the stock solutions of
0.10 mol L–1 Na2HPO4, 0.10 mol L–1 NaH2PO4, and 0.10 mol L–1
KCl and then adjusting the pH with 0.10 mol L–1 NaOH and
H3PO4. The washing buffer was PBS (pH 7.0) containing 0.05%
(w/v) Tween (PBST). The blocking solution was 1% BSA. The clinical
serum samples were obtained from the clinical laboratory of Yiji
Shan Hospital (Wuhu, China). Twice-quartz-distilled water was
used throughout the study.
Apparatus
All electrochemical measurements were performed on a CHI
650C electrochemical analyzer (CH Instruments, China) with a con-
ventional three-electrode system composed of a platinum auxiliary
electrode, a saturated calomel electrode as reference electrode, and
a bare Au or modified Au electrode as working electrode.
Electrochemical impedance spectra were performed in 0.1 M
PBS containing 5.0 mM [Fe(CN)3–/4–
6 ] and 0.1 M KCl at pH 7.0. The
frequency ranged from 0.1 to 100 kHz, and the amplitude of the
alternate voltage was 5 mV.
Morphologies of AuNPs, PDDA functionalized MWCNTs, AuNPs–
MWCNTs, and AuNPs@Au were obtained with scanning electron
microscopy (SEM) (JEOL JSM-6700F, Hitachi, Japan). The morpholo-
gies of the materials were measured on a transmission electron
microscope (Hitachi 800).
49
Preparation of AuNPs@MWCNTs nanocomposites
The colloidal AuNPs of 16 nm diameter were prepared accord-
ing to the previous protocol (see Fig. S1 in online supplementary
material) [20]. The acid-treated MWCNTs were further functional-
ized with PDDA according to the reported method [21]. The
obtained PDDA–MWCNTs (0.5 mg) (see Fig. S2) were dispersed in
5.0 mL of as-prepared colloidal AuNPs and stirred for 20 min, and
the AuNPs were assembled on the surface of PDDA@MWCNTs
nanocomposites via electrostatic interaction. After centrifugation,
the AuNPs@MWCNTs composites were obtained (see Fig. S3), and
these were further washed with water and redispersed in 2 ml of
50 mM Tris–HCl solution (pH 9.0) for further use.
Preparation of immunosensing probes
First, the signal anti-CEA2,1 (Ab2,1) (200 lL, 1 mg mL–1) was
added to the above AuNPs@MWCNTs dispersion and stirred at
room temperature for 2 h. Second, 400 lL of 1% BSA solution was
added to the obtained bioconjugates and allowed to react for 2 h.
After centrifugation, the resulting immunocomplex was further
washed with 0.01 M PBS (pH 7.0) three times. Finally, the prepared
immunocomplex was dispersed in 2 mL of 10 mM Pb(NO3)2 aque-
ous solution and shaken overnight; thus, the Pb2+–Ab2,1–
AuNPs@MWCNTs bioconjugates were synthesized. The Cd2+–
anti-AFP2,2 (Ab2,2)–AuNPs@MWCNTs were synthesized using a
similar method.
Fabrication of immunosensor
Before modification, the gold electrode was treated according to
our previous reported procedure [22]. The modified electrode was
immersed in 0.1 M KNO3 solution containing 5.0 mM HAuCl4 and
electrochemically deposited for 300 s at –200 mV. Thus, AuNPs
were distributed on the surface of the gold electrode. The modified
electrode was denoted as AuNPs/Au.
Immobilization of Ab1,1 and Ab1,2 was performed by dropping a
mixture of Ab1,1 and Ab1,2 (10.0 lL, 200 lg mL–1) solution onto the
surface of the AuNPs/Au and keeping it in a refrigerator for 12 h at
4 °C. The resulting electrode was washed with PBST and PBS to
remove physically absorbed Ab1. Following that, the modified elec-
trode was incubated with 1% BSA solution for 50 min at 37 °C to
block any possible remaining active sites against nonspecific
adsorption and was washed several times with PBST and PBS.
The obtained immunosensor was stored at 4 °C prior to use.
Electrochemical measurements
A schematic preparation of the immunosensing probes is illus-
trated in Scheme 1. SEM was performed to characterize the shape
of the AuNPs/MWCNTs (inset of Scheme 1A). The preparation pro-
cedure of the immunosensors for CEA and AFP determination was
as follows. The immunosensor was incubated with the mixture of
CEA and AFP solution or serum samples with various concentra-
tions for 50 min at 37 °C, followed by washing with PBST and
PBS. Then, it was incubated with a mixture of 1:1 diluted Pb2+–
Ab2,1–AuNPs@MWCNTs and Cd2+–Ab2,2–AuNPs@MWCNTs solution
for another 50 min at 37 °C, followed by washing with PBST and
PBS. Finally, the electrode was transferred into 0.2 M acetate buffer
solution (pH 4.5). Square wave voltammetry (SWV) was used to
obtain the response signal of the immunosensor. SWV scanning from
0 to –800 mV (vs. Ag/AgCl) with a pulse amplitude of 25 mV, a
pulse frequency of 15 Hz, and a quiet time of 2 s was performed
to record the electrochemical responses at –0.54 and –0.71 V for
simultaneous quantitative measurement of CEA and AFP.
50 Electrochemical detection of two tumor markers / D. Feng et al. / Anal. Biochem. 482 (2015) 48–54
Scheme 1. (A) Preparation procedure of immunosensing probes. (B) Fabrication process of immunosensors.
Results and discussion
Investigation of assembled process of immunosensor with SEM and
cyclic voltammetry techniques
Fig. 1A and B showed the surface morphologies of bare Au elec-
trode and AuNPs modified Au electrode, respectively. After the
electrode was electrodeposited at –0.2 V for 300 s in 0.1 M KNO3
solution containing 5.0 mM HAuCl4, the spherical AuNPs were
assembled on the surface of Au electrode, which was beneficial
for improving the immobilized amounts of capture antibody due
to the good affinity of AuNPs for biomolecules on the electrode sur-
face. The SEM showed a more uniform spherical structure with a
regular distribution of AuNPs. As shown in Fig. 1C, the cyclic
voltammograms of bare Au electrode and AuNPs modified Au elec-
trode in 0.5 M H2SO4 solution, the peak current at AuNPs modified
electrode was much higher than that at bare Au electrode. The
results revealed that the AuNPs increased the effective surface area
of electrode significantly, at the same time, the effective surface
areas of electrodes can be obtained from the coulombic integration
of the reductive waves of gold oxide.
Electrochemical characterization of assembling process of
immunosensor
Cyclic voltammetry (CV) characterization of the immunosensor
at different steps is exhibited in Fig. 2A. A pair of distinct redox
peaks was observed due to the oxidation and reduction of the
redox couple Fe(CN)–4/–3
6 on the bare Au electrode (curve a). After
the electrode was modified with AuNPs, the peak current of cyclic
voltammograms increased sharply (curve b) due to the excellent
ability of electron transfer of the AuNPs. When the electrode was
incubated in the mixture of capture Ab1,1 and Ab1,2, the mixture
of CEA and AFP, and the mixture of 1:1 diluted Pb2+–Ab2,1–
AuNPs@MWCNTs and Cd2+–Ab2,2–AuNPs@MWCNTs bioconjugates
successively, the redox peaks decreased step by step (curves c, d,
and e). This can be ascribed to form a sandwich-type immunocom-
plex, and the immunocomplex increases with the increment of the
CEA and AFP concentration in the sample. The insulating layer of
proteins hinders interfacial electron transfer. On the other hand,
the stepwise construction process of the immunosensor was char-
acterized by an electrochemical impedance spectrum (EIS)
(Fig. 2B). The EIS includes a semicircular portion and a linear
portion; the diameter of the semicircle at higher frequencies corre-
sponds to the electron transfer resistance (Ret), and the linear part
at lower frequencies corresponds to the diffusion process. It can be
observed that the bare electrode displayed a small semicircle with
an Ret of approximately 250 X (curve a). After the bare electrode
modified with AuNPs, the Ret decreased to 120 X (curve b), indicat-
ing that the AuNPs were assembled on the electrode surface.
However, as the electrode was incubated in the mixture of capture
Ab1,1 and Ab1,2, the mixture of CEA and AFP, and the mixture of 1:1
diluted Pb2+–Ab2,1–AuNPs@MWCNTs and Cd2+–Ab2,2–
AuNPs@MWCNTs bioconjugates successively, the Ret increased
step by step (curves c, d, and e). The results are in good agreement
with the results obtained from CV. Based on the above results, the
simultaneous detection of CEA and AFP was possible.
Optimization of experimental conditions
The electrochemical performance of the immunosensor is influ-
enced by many factors. Therefore, some experimental parameters
were investigated (e.g., pH, incubation time). Fig. 3A and B show
that the pH value could affect electrochemical behavior of Cd2+
and Pb2+. The pH value of detection solution has great influence
not only on the activity of the antigens and antibodies but also
on the electrochemical behavior of Cd2+ and Pb2+. It can be clearly
observed that the reduction peak current of Cd2+ and Pb2+ was
increased when the pH value increased from 3.0 to 4.5 and then
decreased. When the pH was 4.5, the reduction peak current of
Cd2+ and Pb2+ reached the maximum value. This might be because
metal ions combined with hydroxide ions and became inactive at
pH above 4.5. Here, pH 4.5 was chosen for this study.
Fig. 3C and D show that the response of the immunosensor
changed with changes in the incubation times, which ranged from
10 to 60 min. It can be clearly observed that the reduction current
of Cd2+ and Pb2+ increased with increasing incubation times and
tended to reach a plateau after 50 min, exhibiting saturated bind-
ing between the antigen and the primary antibody on electrode
surface. Therefore, subsequent experiments employed 50 min as
the optimal time for all of the incubation steps of the assay.
Analytical performance
Under optimized assay conditions, the reduction peaks of Pb2+
and Cd2+ were found to be proportional to the concentrations of
 Electrochemical detection of two tumor markers / D. Feng et al. / Anal. Biochem. 482 (2015) 48–54 51

results indicated that the multiplexed electrochemical immunoas-

say enabled wide linear ranges and low detection limits.
Furthermore, the proposed immunosensor exhibited satisfactory
electrochemical performance. Some possible explanations may con-
tribute to these observations. First, the AuNPs@MWCNTs nanocom-
posites as good carriers have a large area to provide a biocompatible
microenvironment for the immobilization of antibody and further
loading of a large amount of labels [28]. Second, the outstanding
electric conductivity of AuNPs@MWCNTs nanocomposites can also
accelerate the electron transfer. Finally, the amplification of the
amperometric signal output was mainly ascribed to the excess
Pb2+ and Cd2+, with favorable electron conductivity and chemical
stability being loaded on the surface of AuNPs@MWCNTs
nanocomposites.

Cross-reactivity, specificity, reproducibility, and stability of

immunosensor

The cross-reactivity of the immunosensor was examined by

comparing the SWV responses of two analytes with those contain-
ing only one analyte. First, antibodies of CEA and AFP were immo-
bilized on the electrodes. Then, two control tests were carried out
as follows: (i) immunosensors were incubated with CEA only or
with AFP only. (ii) AFP and CEA were monitored simultaneously.
Finally, they were bioconjugated with Pb2+–anti-CEA–AuNPs@M
WCNTs or Cd2+–anti-AFP–AuNPs@MWCNTs probes to perform
the sandwich-type immunoreaction. The responses of SWV are
listed in Table 1. The results indicate that the detection of CEA

Fig.1. (A,B) SEM images of bare Au electrode (A) and AuNPs/Au (B). (C) Cyclic
voltammograms of bare Au electrode (a) and AuNPs modified electrode (b) in 0.5 M
H2SO4 solution.

CEA and AFP in the incubation solution, as shown in Fig. 4A. Under
the optimal conditions, the SWV peaks for simultaneous detection
of CEA and AFP increased with the increment of CEA and AFP concen-
trations. The calibration plots display a good linear relationship
between the reduction peaks and the concentration of analytes in
the range of 0.01 to 60 ng mL–1 for both CEA and AFP
(Fig. 4B and C). The regression coefficients were 0.9911 and
0.9962, respectively. The detection limits of CEA and AFP were 3.0
and 4.5 pg mL–1 (at 3r), respectively. These were lower than those
of metal ions tagged immunocolloidal gold (4.6 pg mL–1 for CEA
and 3.1 pg mL–1 for AFP) [8], horseradish peroxidase functionalized
platinum hollow nanospheres (50 pg mL–1 for CEA and 80 pg m for
AFP) [23], CdS/DNA and PbS/DNA nanochains as labels (3.3 pg mL–1
for CEA and 7.8 pg mL–1 for AFP) [24], SiO2@C-dots label–electroche
miluminescence (6.0 pg mL–1 for CEA and 5.0 pg mL–1 for AFP) [25], Fig.2. (A) CV responses and (B) electrochemical impedance spectra of the different
immunochromatographic test strip (2.0 ng mL–1 for CEA and modified electrodes in 0.1 M PBS containing 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] and
0.1 M KCl, respectively: (a) bare Au; (b) electrode modified successively with
3.0 ng mL–1 for AFP) [26], and time-resolved immunofluorometric AuNPs; (c) mixture of capture anti-CEA and anti-AFP; (d) mixture of 10 ng mL–1 CEA
assay (240 pg mL–1) QD barcode-based electrochemical immunoas- and AFP; (e) mixture of Pb2+–Ab2,1–AuNPs@MWCNTs and Cd2+-Ab2,2-
say (3.3 pg mL–1) for CEA [13,27] reported in previous studies. The AuNPs@MWCNTs.
52 Electrochemical detection of two tumor markers / D. Feng et al. / Anal. Biochem. 482 (2015) 48–54
Fig.3. Effect of pH (A,B) and incubation time (C,D) on response of immunosensor to CCEA = CAFP = 20 ng mL–1.
and AFP exhibited low interference and that the cross-reactivity
between two analytes was negligible.
The specificity of the immunosensor played an important role in
analyzing biological samples without separation; some interfer-
ences such as human immunoglobulin G (IgG), BSA, glucose
(Glu), and ascorbic acid (AA) were used as interferences to evaluate
the specificity. To test the specificity of the immunosensor,
1.0 ng mL–1 CEA and AFP was mixed with 50 ng mL–1 IgG, BSA,
Glu, and AA, respectively. Fig. 5 shows the SWV response of pure
CEA and AFP and that obtained from CEA and AFP containing an
interferential substance. When the immunosensor detected the
mixture of 1 ng mL–1 CEA or AFP and 50 ng mL–1 of another inter-
ference, the response signal of the mixture changed a little in con-
trast to CEA or AFP alone, which showed that the proposed
immunosensor had good selectivity for CEA and AFP.
To estimate the reproducibility of the simultaneous multiana-
lyte immunoassay, the intra-assay precision was investigated by
detecting five times every 5 h at four different concentrations of
CEA and AFP (0.05, 2, 20, and 60 ng mL–1) using identical
immunosensors. The coefficients of variation were 5.5, 6.8, 7.9,
and 9.2% at 0.05, 2, 20, and 60 ng mL–1 CEA and AFP, respectively.
Similarly, the inter-assay precision was investigated by measuring
four different concentrations of CEA and AFP (0.05, 2, 20. and
60 ng mL–1) using five immunosensors. The coefficients of varia-
tion were 9.6, 8.8, 6.1, and 8.6%, respectively, suggesting that the
immunosensor possessed acceptable precision and reproducibility.
In addition, when the immunosensor was stored at 4 °C, the stabil-
ity of the immunosensor was examined by testing the response
after 3 weeks; more than 90% of the initial responses remained
after 3 weeks for both CEA and AFP. The slow decrease in response
seemed to be related to the gradual deactivation of the
immobilized antibody on the sensor platform. The immunosensor
showed acceptable stability and was favored as dual labels for
simultaneous detection of dual biomarkers based on SWV
measurements.
Application of immunosensor in human serum
The capability to detect practical samples is a major concern
during the development of a clinical diagnostic platform. The
detailed process of practical samples prepared was as follows.
The blood samples were from venous blood without added antico-
agulant; they were placed in dry test tubes to stand for 1 h. Next,
blood samples were centrifuged (2000 rpm for 5 min) and precip-
itated, and we took the upper solution to detect. To examine the
applicability of the immunosensor for practical analyses, the recov-
ery experiments were performed by standard addition methods.
The standard samples of CEA and AFP were dissolved in the healthy
human serum (the concentration range of the CEA and AFP was
0.01–60 ng mL–1) and detected the CEA and AFP in serum. The
recoveries obtained were 94.65 to 108% and 94.2 to 105.7%, respec-
tively, indicating that the method is suitable for serum sample
analysis (see Table S1 in supplementary material).
Importantly, to investigate the possibility of the newly devel-
oped method being applied for clinical analysis, several real sam-
ples were examined by the developed immunoassay and the
enzyme-linked immunosorbent assay (ELISA) methods for deter-
mination of CEA and AFP. The serum samples came from normal
persons and cancer patients. These results are shown in Table 2.
The value obtained was in agreement with that of the ELISA meth-
ods, indicating that the immunosensor could be applied to serum
analysis.
 Electrochemical detection of two tumor markers / D. Feng et al. / Anal. Biochem. 482 (2015) 48–54 53

Fig.5. Specificity of immunosensor conditions: CCEA = 1 ng mL–1,

Cinterferents = 50 ng mL–1 (BSA, Glu, IgG, and AA).

Table 2
Comparison of CEA and AFP using the proposed immunosensor and reference
methods.

Sample Multiplexed immunoassay ELISA (ng mL–1) Relative

(ng mL–1) deviation (%)
CEA AFP CEA AFP CEA AFP
1 0.63 ± 0.23 0.58 ± 0.45 0.60 0.60 +5.00 1.67
2 0.98 ± 0.36 1.10 ± 0.25 1.00 1.00 +2.00 1.00
3 18.45 ± 0.42 21.21 ± 0.32 20.00 20.00 7.75 +6.05
4 41.12 ± 0.72 38.58 ± 0.24 40.00 40.00 +2.80 +3.55
5 58.36 ± 0.64 61.08 ± 0.56 60.00 60.00 2.77 +1.80

Conclusions

We have developed a simple and reliable electrochemical

Fig.4. (A) SWV of immunosensors for different concentrations of CEA and AFP (0.01,
0.05, 2.0, 8, 20, 30, 45, and 60 ng mL–1). (B,C) Calibration curves of multiplexed
immunoassay toward CEA (B) and AFP (C) in 0.2 M HAc–NaAc (pH 4.5).
immunosensor for simultaneous detection of CEA and AFP based
on metal ion as labels and AuNPs@MWCNTs as simple carrier
and signal enhancers. Highlights of the developed immunoassay
are as follows. First, AuNPs@MWCNTs nanocomposites with their
exceptionally high surface area, good conductivity, and biocompat-
ibility were used as an excellent carrier for immobilizing antibod-
ies and further loading a large amount of labels. Second,
high-content metal ion labels could be detected without acid dis-
solution using the SWV analysis technique, thereby amplifying
the current response effectively. Moreover, the immunosensor
showed good precision, high sensitivity, acceptable stability and
reproducibility.

Acknowledgments
Table 1
Interference degree or cross-talk level.
This work was supported by the the Foundation of Anhui
Sample Concentration Current shift at CEA Current shift at AFP
Normal University Excellent Doctoral – Organic Chemisty
type (ng mL–1) position (lA)a,b position (lA)a,c
(NO. 003061425), the National Natural Science Foundation of
CEA 2 8.72 0.07
China (NO. 20675002), and the Natural Science Foundation of
60 58.98 0.02
Anhui (NO. 1408085QB40).
AFP 2 0.03 6.32
60 0.06 50.31
CE+AFP 2+2 8.43 6.01
60 + 60 59.76 49.97 Appendix A. Supplementary data
a
The average value of three measurements in 0.2 M HAc–NaAc (pH 4.5).
b
The SWV peak current was 23.12 lA for zero CEA analyte. Supplementary data associated with this article can be found, in
c
The SWV peak current was 14.02 lA for zero AFP analyte. the online version, at http://dx.doi.org/10.1016/j.ab.2015.04.018.
54 Electrochemical detection of two tumor markers / D. Feng et al. / Anal. Biochem. 482 (2015) 48–54

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