Biosensors and Bioelectronics 102 (2018) 610–616

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Biosensors and Bioelectronics

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

Electrochemical detection of carcinoembryonic antigen T

a,b b,⁎ b,1 a b,c,⁎⁎
Xuefang Gu , Zhe She , Tianxiao Ma , Shu Tian , Heinz-Bernhard Kraatz
a
School of Chemistry and Chemical Engineering, Nantong University, Nantong 226007, PR China
b
Department of Physical and Environmental Science, University of Toronto Scarborough, 1265 Military Trail, Toronto, Ontario, Canada M1C 1A4
c
Department of Chemistry, University of Toronto, 80. St. George Street, Toronto, Canada M5S 3H6

A R T I C L E I N F O A B S T R A C T

Keywords: In this work, a sandwich-type electrochemical immunosensor for carcinoembryonic antigen (CEA) detection has
Ferrocene been constructed and tested. Unlike many other sensors using external electrochemical species in the electrolyte
Gold nanoparticles to generate an electrochemical signal, a ferrocene derivative has been integrated into the design of the sensor to
Carcinoembryonic antigen provide an internal reporting system, allowing detection of CEA in buffers and biological samples. Gold nano-
Electrochemical immunosensor
particles, which have been used to increase the conductivity of sensing surfaces, also carry immobilized sec-
ondary anti-CEA and a ferrocene derivative. The shelf life testing of the sensor shows good performance after
storage for 4 weeks. The sensor has been calibrated against different concentration of the target protein using
square wave voltammetry. The calibration curve has been obtained in the range of 0.05–20 ng mL−1, and the
detection limit for CEA is ~ 0.01 ng mL−1. The capability of the immunosensor has been verified by performing
detection of CEA in human serum samples.

1. Introduction immunoassay, based on the specificity of an antibody to its corre-

The development of an effective diagnostic method for cancer de-
tection, progression, and monitoring of treatment efficacy remains an
important bioanalytical target. With the development of proteomics,
serum tumor markers such as carcinoembryonic antigen (CEA), alpha-
fetoprotein, CA125, CA19-9, and cytokeratin 19 fragment, have been
widely used as screening tools for high-risk populations in annual
medical checkups (Han et al., 2008). The average CEA concentration in
a healthy human is below 5 μg L−1, while serum CEA levels above
20 μg L−1 indicates the presence of cancer (Han et al., 2017). This has
been demonstrated for colon cancer (Vogel et al., 2001), breast cancer
(Han and Magliocco, 2016), lung cancer (Han et al., 2008), gastric
cancer (Tachibana et al., 1998), and ovarian carcinoma (Kudoh et al.,
1999). The coincidence rate increases up to 78% when a combination of
CEA and CYFRA21-1 are used for the diagnosis of non-small cell lung
cancer (Wu et al., 2007). In addition, the preoperative and periodic
postoperative CEA determination provides useful information in pre-
dicting stage, tumor progression and recurrence (Liu et al., 2012).
Therefore, a rapid, accurate and sensitive CEA detection method is of
great interests for the community and may provide an approach to
rapid and facile cancer screening.
At present, the benchmark for single-protein measurement is by
sponding antigen (Pei et al., 2013). Formation of an immunocomplex
has been quantified by colorimetry (Kim et al., 2009), fluorescence (He
et al., 2013), chemiluminescence (Wang et al., 2016; Zhao et al., 2016),
electrochemical methods (Chen et al., 2012; Gao et al., 2011; Han et al.,
2016; Lai et al., 2016; Wang et al., 2013; Weng et al., 2013; Yang et al.,
2017) and by surface-enhanced Raman scattering (SERS) (Chon et al.,
2009). However, traditional fluorescence-based methods, such as en-
zyme-linked immunosorbent assay (ELISA), often suffer from low sen-
sitivity due to a large optical background. Radioimmunoassays have the
potential to expose the operator to a potential safety hazard and require
special waste treatment. While the main problem of Raman-based
readout methods is related to reproducibility issues (Gu et al., 2014).
Amongst conventional immunoassay techniques, electrochemical
methods have attracted considerable interest, since they offer high
sensitivity, short diagnostic time, and the possibility of miniaturization.
Electrochemical responses are readily quantifiable (Li et al., 2014b; Xu
et al., 2017). Detection by amperometric methods are most promising
because of their large linear range, high sensitivity, and low detection
limit (Cevik et al., 2016; He et al., 2008; Ou et al., 2007; Ren et al.,
2014; Zhang et al., 2017).
We have exploited ferrocene (Fc) derivatives as part of electro-
chemical sensors before. Examples of this application are the use of Fc

⁎
Corresponding author.
⁎⁎
Corresponding author at: Department of Physical and Environmental Science, University of Toronto Scarborough, 1265 Military Trail, Toronto, Ontario, Canada M1C 1A4.
E-mail addresses: zhe.she@utoronto.ca (Z. She), bernie.kraatz@utoronto.ca (H.-B. Kraatz).
1
Current address: Department of Chemistry, University of British Columbia, Vancouver Campus, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1.

https://doi.org/10.1016/j.bios.2017.12.014
Received 25 September 2017; Received in revised form 28 November 2017; Accepted 7 December 2017
Available online 08 December 2017
0956-5663/ © 2017 Elsevier B.V. All rights reserved.
X. Gu et al.
as a peptide conjugate for the detection of bacteria (Li et al., 2014b) and
exploiting Fc as a redox relay for monitoring phosphorylation of pep-
tides and proteins, as well as enhancing the electrochemical imaging of
protein receptors (She et al., 2017; Wang et al., 2015). For a protein
immunoassay, the most direct approach would be to conjugate Fc di-
rectly to antibodies. For example, Tang's group reported a proof-of-
concept study of the determination of human IgG protein by integrating
an immunoreaction and hybridization chain reaction (HCR), where Fc
molecules were conjugated to well-designed hairpin DNAs for the HCR
that is triggered by initiator strands on the surface of gold nanoparticles
(Zhang et al., 2012). Feng and coworkers fabricated a sandwich-type
CEA immunosensor using Fc as electrochemical probe. The en-
capsulated HRP was to catalyze H2O2, which further promoted the
electron transfer of Fc-COOH and amplified the electrochemical signal,
and a linear range of 0.001–10 ng mL−1 with a detection limit of
0.0002 ng mL−1 was achieved (Feng et al., 2016). However, no matter
what labeling approach is adopted, the labeling process itself is labor-
ious and time-consuming, and the efficiency of binding is usually quite
low due to steric hindrance.
To solve these issues, various nanoprobes have been prepared that
carry a large number of electroactive molecules, such as methylene
bule, RuHex, and HRP, on a range of different nanocarriers, including
gold nanoparticles (AuNPs) (Bai et al., 2017; Giljohann et al., 2010;
Larguinho and Baptista, 2012; Liu et al., 2016; Xu et al., 2017), gra-
phene (Halder et al., 2017; Weng et al., 2013), carbon nanotubes (Gao
et al., 2011; Han et al., 2016), chitosan (He et al., 2008), polymer
dendrimers (Cevik et al., 2016). This approach has been widely em-
ployed for the enhancement of the electrochemical signal, but is also
fraud with problems including the loss of the label during the electro-
chemical experiment. Additionally, it is also difficult to control the
concentration of deposited electroactive materials, thus reducing the
reproducibility of the result (Gu et al., 2015, 2016). In this regard, in-
troducing thiol groups (–SH or –SCH3) into Fc molecules might be a
feasible approach, because Fc–SH can be immobilized on the surface of
gold nanoparticles via formation of covalent bonds between gold and
sulfur atoms, to form a stable and well-organized films (Love et al.,
2005), where nanoparticles act as nanocarriers and Fc serves as the
signal amplifier.
In this study, we have developed a sensitive and reproducible
electrochemical immunosensor for the detection of CEA. A sandwich-
like immunocomplex containing electroactive Fc was the core of this
sensor, gold nanoparticles (AuNPs) that modified by Fc groups and
carrying immobilized anti-CEA, were then connected on the surface of
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Biosensors and Bioelectronics 102 (2018) 610–616
Scheme 1. Schematic illustration of (a) preparation of Fc-labeled
nanogold probes (Ab2-Au-Fc), and (b) the procedures for the
construction of sandwich-type electrochemical biosensor.
gold electrodes through an immunoreaction. Fc-SH molecules forming a
self-assembled monolayer (SAM) on the surface of AuNPs via a covalent
attachment process have greatly improved the stability and repeat-
ability of the signals in comparison with drop-casting method. In ad-
dition, direct electron transfer could also be achieved through the
monolayer of Fc, thus generating a faster electron transport rate than
through the much thicker drop-dry films, which is essential in fabri-
cating an electrochemical sensor with high sensitivity and rapid re-
sponse (Love et al., 2005). The electrochemistry was used to monitor
the step-by-step fabrication of the sensor, followed by CEA detection by
square-wave voltammetry. Results presented here show that the ap-
proach discussed here has a wide dynamic range and can be used to
detect CEA in human serum.
2. Experimental sections
2.1. Materials and reagents
Carcinoembryonic antigen (CEA), monoclonal anti-CEA (designated
as Ab1), and polyclonal anti-CEA (designated as Ab2) were purchased
from R＆D system. Lipoic acid N-hydroxysuccinimide ester (LPA) was
purchased from Synchem UG & Co (See SI for the detail of other re-
agents and buffers used in this work).
2.2. Preparation of Fc-labeled biofunctionalized AuNPs (Ab2-AuNPs-Fc)
Colloidal gold sol was synthesized by aqueous reduction of chlor-
oauric acid with trisodium citrate according to the previous literature
(Grabar et al., 1995) (See SI for the detail of preparation). Scheme 1a
illustrates the formation of the Fc-labeled nanogold probes (Ab2-
AuNPs-Fc). Polyclonal anti-CEA was first immobilized on the surface of
AuNPs through the electrostatic interaction between negative charged
AuNPs and the protonated amino group of antibody. The electroactive
Fc molecules were then chemisorbed to the gap of the antibody via self-
assembly, which was done by adding a few microliters of 0.2 M K2CO3
to the gold sol for the adjustment of acidity to pH 8.5. Then, 150 μL of
50 μg mL−1 Ab2 was added to 1 mL of the AuNPs solution, and the
mixture was incubated at room temperature for 2 h. Next, 10 μL Fc-SH
ethanol solution (1 mM) was added to the mixture to react with Ab2-
AuNPs for 1 h. Finally, PEG8000 was added to a final concentration of
0.5% for stabilizing the AuNPs. The excess antibody and Fc were re-
moved by repeated centrifugation at 9000 rpm for 20 min and rinsed
with storage buffer. The final deposition (Ab2-AuNPs-Fc) was
X. Gu et al.
suspended in 3 mL of storage buffer and stored at 277 K for further use.
The as-prepared Fc modified gold nanoparticles could be stably stored
in solution for more than three months without aggregation.
2.3. Construction of electrochemical biosensor
Prior to the construction, the electrodes were immersed into a pir-
anha solution (Volume ratio H2SO4/H2O2 = 3:1) for 30 s and rinsed
with a large amount of water. The bare gold electrodes were polished
using aqueous slurries of alumina (1 down to 0.05 µm) to obtain a
mirror surface and then followed by electrochemical cleaning.
Scheme 1b shows the different stages during the fabrication of the
proposed CEA immunosensor. The cleaned electrodes were im-
mediately immersed in a 1 mL of 2 mM LPA-NHS ethanol solution at
277 K overnight. The LPA-NHS monolayer here served as the linker
between the anti-CEA and Au electrodes. The modified electrodes were
then thoroughly rinsed with ethanol and blown dry by N2. A 5 μL
droplet of Ab1 (200 μg mL−1) in PBS buffer (pH 7.4) was carefully
placed over the surface of gold electrode and the electrode was then
placed at 277 K for 48 h. The Ab1-modified electrodes were rinsed with
an excess volume of washing buffer and Milli-Q water and blown dry
followed by soaking them in an ethanolamine solution for 2 h at room
temperature to block the unreacted LPA. The electrodes were incubated
in a solution of CEA antigen (different concentrations in 10 mM PBS
buffer) for 1 h at 310 K, rinsed again. Finally, the gold electrodes with
Ab1-CEA antigen immunocomplexes were immersed in the Ab2-AuNPs-
Fc solution for 45 min at 310 K to construct sandwich structures for the
final electrochemical measurement.
2.4. Electrochemical measurements
Cyclic voltammograms (CV), electrochemical impedance spectro-
scopy (EIS), Chronoamperometry (CA), and square wave voltammetry
(SWV) were recorded on a CHI-660C electrochemical station (CH
Instruments, Austin, Texas) with an enclosed Faraday cage. (See SI for
the detailed electrochemical parameters).
3. Results and discussion
3.1. Characterization of the CEA immunosensor
The step-by-step modification was followed up by TEM and the
images illustrate the formation of antibody modified nanoprobes.
Fig. 1a shows the TEM image of the relatively uniform AuNPs. As ob-
served in Fig. 1b, the TEM image of Fc-SH modified AuNPs, the dark
center (AuNPs) was wrapped by a lighter color shell and the thickness
of the lighter parts was approximately 2 nm, suggesting the self-as-
sembled Fc monolayer on the surface of AuNPs (Petrizza et al., 2016).
Fig. 1c shows the morphology of Ab2-AuNPs-Fc. The thickness of the
lighter parts increased to 5–6 nm, which was in good agreement with
the size of anti-CEA (Hammarström, 1999). Fig. 1d represents the SPR
spectra of the non-aggregated AuNPs and the antibody modified na-
noprobes, which occurred at 520 nm and 530 nm, respectively. Gen-
erally, the locations of SPR strongly depend on the shape, size and the
environment of the metal nanoparticles (Kelf et al., 2006). In this case,
the red shift of the SPR band upon the adsorption of the anti-CEA was
most likely resulted from a change in the dielectric property of the
media surrounding the AuNPs.
EIS is an informative technique to monitor the interfacial properties
of modified electrodes by estimating parameters such as solution re-
sistance (Rs), electron-transfer resistance (Ret), and double layer capa-
citance (Cdl). The EIS measurements were performed in this study to
follow the surface electrochemical property of each step of the im-
munosensor construction; the Nyquist plots results are shown in Fig. 2A
(see Table S1 for the values of the equivalent circuit). The EIS of the
bare Au electrode had a small arc (curve a), implying very low
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Biosensors and Bioelectronics 102 (2018) 610–616
resistance of the surface to [Fe(CN)6]3-/4-. After the bare Au electrode
was modified with LPA (curve b), the Ret value increased to 1684 Ω,
which could be attributed to the insulating effect of the SAM film
formed on the electrode surface, confirming successful immobilization
of the linker on the surface of Au electrodes. When the electrode was
further modified with antibody (curve c), the Ret value dramatically
increased to 3012 Ω (curve c), suggesting that the electron transfer was
hindered by the immobilized Ab1 molecules. Compared to the un-
blocked electrode, Ret value showed a slight decrease after the blocking
stage (curve d). This phenomenon was possibly due to the replacement
of nonspecifically absorbed antibody with the small blocking agent
during the process. As expected, the size of the arc on EIS increased
again (curve e) on the recognition of CEA antigen. When the Ab2-
AuNPs-Fc nanoprobes were captured onto the surface of the modified
Au electrode, the Ret value decreased significantly to 1094 Ω (curve f),
which was ascribed to the fact that AuNPs facilitated the electron-
transfer. The change helps confirming formation of the im-
munocomplex sandwiches on the surface of Au electrodes.
In order to further confirm the preparation process of the im-
munosensor, CVs were carried out using Fe(CN)63-/4- as the redox
probe. Fig. 2B shows CVs of each step (same as Fig. 2a) during the
immunosensor construction in 5 mM [Fe(CN)6]3-/4- solution at a scan
rate of 100 mV/s. As could be expected, the sequential modification of
the electrode blocked the electron transfer between the external redox
probes and the modified electrode. For the CV on a bare Au electrode
(curve a), a couple of reversible redox peaks at 0.302 and 0.234 V were
observed. When the Au electrode was modified with LPA (curve b), an
obvious decrease in peak current and an increase in the peak separation
were observed as the potential of peaks shifted outward. This sup-
pression of current indicates the formation of a LPA monolayer,
blocking electron transfer between the Au surface and the redox couple.
Subsequently, the electrode was modified with antibody (curve c); a
further decrease in peak current and an increase in the peak separation
were observed. However, after we blocked the unreacted active sites of
LPA with ethanolamine to minimize non-specific recognitions, an ob-
servable increase in the peak current and a narrower peak separation
compared with those of curve c occurred (curve d), due to the re-
placement of nonspecifically absorbed macromolecule Ab1 with EA. As
for the recognition CEA antigen (curve e) step, the current lowered
again, this was consistent with the enhanced electron-transfer barriers
introduced by successive modifications. Finally, after the im-
munoreaction with Fc-labeled nanoprobes (curve f), the peak current
showed a dramatic increase (compare to curve e), and such an increase
in current could be basically ascribed to the fact that AuNPs facilitate
the electron transfer (Larguinho and Baptista, 2012). The modification
has been monitored using both CV and EIS to make sure formation of
immunocomplex. These CV results are consistent with the above-
mentioned EIS results, and consequently, all electrochemical results
confirm the successful fabrication of the immunocomplex.
3.2. Electrochemical response of the CEA immunosensor
Generally, the kinetic reversibility of a redox couple can be assessed
according to the separation of the anodic and cathodic peak potentials
in CV. A control experiment was carried out by putting the Fc derivative
onto electrodes and investigated the redox behavior of the Fc firstly. We
have immersed a bare Au electrode in 2 mM Fc-SH ethanol solution for
2 h, and then carried out the CV detection in PBS. A pair of well-defined
redox peaks was clearly observed, corresponding to the oxidation of
ferrocene to ferrocenium ion. The cathodic and anodic peaks are lo-
cated at 0.294 V and 0.360 V (scan rate of 0.1 V s−1), respectively. The
peak-to-peak separation ΔEp = Epa −Epc = 66 mV, and the ratio of
the anodic peak current to the cathodic Ipa/Ipc was 1.32 ± 0.03, sug-
gesting a quasi-reversible one-electron electrochemical reaction for the
immobilized Fc molecules. Fig. 3a shows the CVs of the Fc modified
electrode with various scan rates ranging from 25 mV s−1 to 5 V s−1. As
X. Gu et al.
Fig. 2. (A) EIS spectra and (B) cyclic voltammograms of [Fe(CN)6]3-/4- at the surface of
the modified gold electrode after each modification step: (a) bare gold electrode; (b) after
coating with LPA; (c) after immobilization of Ab1; (d) after blocking with EA; (e) after
immobilization of 1 ng mL−1 CEA and (f) after immobilization of Ab2-AuNPs-Fc. The CVs
were measured in a 10 mM HEPES buffer (pH7.4) containing 1 M NaClO4 and 5 mM/
5 mM [Fe(CN)6]3-/4- at a scan rate of 0.1 V s−1.
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Biosensors and Bioelectronics 102 (2018) 610–616
Fig. 1. TEM images of (a) AuNPs, (b) AuNPs-FcSH, (c) Ab2-
AuNPs-Fc, and UV/Vis spectra of AuNPs and Fc-labeled nanogold
probe.
can be seen, the anodic and cathodic peak currents increased simulta-
neously with increasing scan rate. The inset of Fig. 3a illustrates the
linear dependence of the anodic and cathodic peak currents on the scan
rates, indicating a surface-controlled process (Ou et al., 2007). More-
over, only a slight increase in the potential separation of the cathodic
and anodic peaks ranging from 25 mV s−1 to 5 V s−1 was observed,
suggesting a fast electron transfer rate between the self-assembled
monolayer of Fc molecules and the Au electrode.
The Fc-Gold electrode investigation was followed by probing the Fc
as part of the immunosensor. Fig. 3b presents the CV performance of an
integrated CEA immunosensor with 5 ng mL−1 CEA antigen as the
target. The redox peaks that resulted from Fc to Fc+ are well revealed
by CV. The cathodic and anodic peaks are located at 0.215 V and
0.289 V, respectively. It is clearly evident that in the presence of CEA,
the electrode exhibit reversible electrochemical signal from Fc, similar
to those obtained at Fc modified Au electrodes. Compared to Fig. 3a,
the redox potentials of the CEA immunosensor in Fig. 3b show a ne-
gative shift for ~ 80 mV. This could be attributed to the different way of
linking and different chemical environment around the Fc molecules
(Halder et al., 2017). As reported previously, the immunocomplex
formed on the electrode surface often had a poor electrical conductivity
and the long-range electron transfer would slow down the rate of
electron transfer, thus restrict their analytical performance in some
degree (Lai et al., 2016). In this work, though the peak-to-peak se-
paration had a slight increase, the 74 mV separation and a 1.23 Ipa/Ipc
ratio still implied a reversible and fast electron transfer. More im-
portant, the CV response of the immunosensor has not changed after 20
times of successive scans between 0.0 and 0.6 V, suggesting that the as-
prepared CEA biosensor is stable and the signals are repeatable and
reliable. Overall, the results demonstrate that the attached Fc in na-
nogold probes can reversibly communicate fast electronic transporta-
tion with the supporting electrode, and in turn the feasibility of the
immunosensor. Moreover, the surface coverage of the electroactive
X. Gu et al.
Fig. 3. (a) CV evolutions of Au/Fc obtained in 0.1 M PBS (pH 7.0) containing at scan rates
0.025–5 V s−1 (from inner to outer curves: 0.025, 0.05, 0.075, 0.1, 0.15, 0.2, 0.25, 0.3,
0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 V s−1), the
inset of a is the plot of current (Ip) vs. scan rate, (b) CV of the CEA biosensor in 0.1 M PBS
(pH 7.0) PBS at a scan rate 0.1 V s−1, the concentration of CEA is 5 ng mL−1.
substance was calculated according to the Laviron Eq. (1) (Laviron,
1974), and the amount of the immobilized Fc molecules was calculated
as 2.43 × 10−9 mol·cm−2 (see SI for Fig. S1 and the detailed calcula-
tion process)
n2⋅F 2⋅A⋅Γ ⋅ν n⋅F ⋅Q⋅ν
Ip = =
4RT 4RT
3.3. Optimization of experimental conditions
In order to achieve the optimum analytical performance, the effect
of antibody concentration on the SWV current of the immunosensor was
investigated. The relationship between the concentration of Ab1 and
current is shown in Fig. S2a (see SI). The peak current significantly
increases with an increasing antibody concentration until the peak
current reaches a plateau at 200 μg mL−1. Hence, the Ab1 concentra-
tion of 200 μg mL−1 was selected in this work to have maximum
electrochemical response. The concentration of the Ab2 was also tested
to optimize the conditions. The current response as shown in Fig. S2b
increases quickly at the range of low concentration and reaches the
highest value as the final concentration of Ab2 is 7.5 μg mL−1. How-
ever, when the concentration continues to increase, the current of the
immunosensor shows a slight decrease. This might be due to the com-
petitive adsorption between Ab2 and the labeled Fc molecules. Al-
though a concentration of 7.5 μg mL−1 of Ab2 may not have highest
adsorption on gold nanoparticles, but it is a good balanced point be-
tween Ab2 and the labeled Fc molecules. The trend of current changes
in Fig. S2c (the effect of nanogold probe concentration) is similar to that
Biosensors and Bioelectronics 102 (2018) 610–616
Fig. 4. (a) Typical SWVs of the electrochemical immunobiosensor in 0.1 M PBS (pH~ 7.0)
solution containing increasing concentrations of CEA (0.05, 0.1, 0.5, 1.0, 5.0, 10.0,
20.0 ng mL−1), (b) the calibration curve of the electrochemical biosensor for determi-
nation of CEA standards. Each point is an average of five parallel determination results;
each error bar is the sample-to-sample standard deviation of the currents.
exhibited in Fig. S2a. That is, as the concentration of the nanogold
probe increasing, the current increases accordingly and reached a pla-
teau at 0.6 nM. Therefore, 0.6 nM of the nanogold probe was referred as
the optimum concentration in subsequent experiments.
In addition to the investigation on the concentrations, the incuba-
tion time was explored by monitoring the current response of the im-
(1) munosensor. The SWV current of the immunosensor, shown in Fig. S2d,
increases with increasing incubation time used in the sandwich im-
munoreaction and a constant value is reached at 45 min, indicating that
45 min is enough for the antigen-antibody recognition reaction.
3.4. Analytical performance
Current-time (I/T) curves at different CEA concentrations were
obtained to further investigate the electrochemical characteristics of
this sensor. The signal was also analyzed towards quantifying the
concentration of CEA (see SI for Fig. S3 and the detailed description).
As the figure shows, the charging current of the system increases
slightly with increased CEA concentration and the steady state current
can be reached within 8 s, indicating that the sensor has a faster elec-
tron transfer rate and a shorter response time (Han et al., 2016; Li et al.,
2014a; Ye et al., 2013). Furthermore, calibration was attempted by
analyzing the I/T curves. It is found that the linear range of this
quantitative method was narrow (from 0.5 to 10 ng mL−1) and the
detection limit (~ 0.2 ng mL−1) could not meet the detection require-
ment. Therefore, SWV was used for the quantitative determination of
CEA in this work because it exhibited higher current sensitivity and a
better resolution than CV. Fig. 4a shows the voltammetric responses to
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X. Gu et al. Biosensors and Bioelectronics 102 (2018) 610–616

Table 1
Comparison of the proposed method with reference methods for CEA determination.

Immunoassay substrate Readout method Linear range (ng mL−1) LOD ( ng mL−1) Ref.

Plasma treatment of paper chemiluminscene 0.1–80 0.03 (Zhao et al., 2016)

ZnS-CdS nanodots on MoS2 Electrochemiluminescence 0.05–20 0.03 (Wang et al., 2016)
Carbon nanotube decorated gold nanoclusters Electrochemistry, CV 0.5–5.0 0.1 (Gao et al., 2011)
Nanosilver-coated magnetic beads and gold-graphene nanolabels Electrochemistry, DPV 0.02–60 0.02 (Chen et al., 2012)
Polydopamine modified mesoporous silica nanoparticles Electrochemistry, DPV 0.01–40 0.002 (Wang et al., 2013)
Streptavidin-functionalized nitrogen-doped grapheme Electrochemistry, DPV 0.02–12 0.01 (Yang et al., 2017)
Palladium hybrid vanadium pentoxide/multiwalled carbon nanotubes Electrochemistry, Amperometric 0.01–10 0.003 (Han et al., 2016)
Ferrocene functionalized Fe3O4@SiO2 Electrochemistry, DPV 0.001–10 0.0002 (Feng et al., 2016)
Polymer containing both aldehyde and ferrocene functional groups Electrochemistry, DPV 0.1–20 0.07 (Zhang et al., 2017)
Anti-CEA/AuNP@nafion/FC@CHIT/GCE Electrochemistry, SWV 0.03–100 0.01 (Shi and Ma, 2011)
This work Electrochemistry, SWV 0.05–20 0.01 –

CEA with different concentrations. The electrochemical oxidation peak
of Fc is located at ca. 0.28 V and an increase in CEA concentration re-
sults in the oxidation current increasing at the same time. Moreover,
there is a linear relationship between the current and CEA concentra-
tion ranging from 0.05 to 20 ng mL−1 (Fig. 4b), with the linear re-
gression equation expressed as y = 0.4494 x + 2.043 (r = 0.9968).
The limit of detection was calculated to be 0.01 ng mL−1, using IUPAC
definition LOD = 3 sb/q, where sb is the standard deviation of the blank
signal, and q is the slope of the calibration curve. Comparing to the
calibration curves presented in the existing literatures, the im-
munosensor proposed in this study provides a relatively low limit of
detection. The comparison of different fabrication methods, the range
of linearity, and the detection limit of this method with other reports for
CEA detection is presented in Table 1.
To investigate the specificity of the immunosensor, a high con-
centration (500 ng mL−1) of possible interferents (glucose, cysteine,
bovine serum albumin, human IgG, and prostate specific antigen) was
introduced in two experiments. As shown in Fig. S4A, when sensor is
individually tested in CEA and control interfering samples, dominating
signal was only observed in CEA solution. The test was then carried out
when interfering samples were each mixed into 10 ng mL−1 CEA so-
lution, respectively. The detection signal is very reliable that re-
produced at very similar level of 7 µA as shown in Fig. S4b.
The repeatability of the electrochemical immunosensor was eval-
uated by calculating the relative standard deviation (RSD) of intra-
assay at 0.1, 1.0 and 10.0 ng mL−1 CEA concentration for 8 times of
successive measurements. The RSDs of this sensor were 8.7%, 5.3% and
3.7%, respectively. The reproducibility was estimated by calculating
the inter-assay RSD from various batches (n = 5), for each assay a new
freshly prepared nanogold probe was used. The inter-assay RSDs were
9.8%, 6.5%, and 4.2%, respectively. The results were acceptable for a
trace analysis. The stability of the sensor was tested by comparing
stored sensors with fresh ones. The Fc-AuNPs-Ab2 probe The sensors
was suspended in 10 mM phosphate sodium buffer solution, pH 7.4,
0.1% PEG8000 for 1–4 weeks before using in the detection. n PBS at
277 K and used for CEA detection 3 weeks later, and the current re-
sponse maintained about 91.8% of the original value（Fig. S5, see SI.
The good reproducibility and stability of this electrode could be asso-
ciated with a combination of following factors: (i) the good bio-
compatibility of gold nanoparticles; (ii) self-assembly method allows Fc
molecules to functionalize the surface of gold nanoparticles via Au-S
covalent bonds, thus achieving a more stable film than physically fixed
ones; (iii) closely packed Fc molecules occupy most of the AuNPs after
the modification of antibody, making it difficult for the adsorption of
unwanted interference component; (iv) the internal electrochemical
responses with no external redox species and enzyme conjugates
needed.
3.5. Preliminary analysis of real samples
Human serum samples with spiked CEA were prepared and used to
verify the detection ability of the immunosensor. Samples were made
by adding varying volumes (100.0, 300.0, 500.0, 1000.0 μL) of
1.0 μg mL−1 CEA standard solution firstly to 1.0 mL human serum and
then diluted in a 100 mL volumetric flask with pH 7.4 PBS. SWV and
the calibration curve obtained above were applied to determine the
concentration of each solution; the recovery and RSD are shown in
Table S2. The results suggest that the proposed immunoassay method
may have a great potential in the real-to-life sample analysis of CEA
with high accuracy and good reliability.
4. Conclusion
In conclusion, we have demonstrated a method how to incorporate
the Fc molecules and AuNPs into a sensor towards sensitive detection of
CEA. The Fc-labeled AuNPs provide internal electrochemical signals,
therefore no redox species is needed in the electrolyte. Recognition
antibody (anti-CEA, Ab2) loaded on the AuNPs offers the biological
detection/interaction capability. The Fc–AuNP-anti-CEA probes loaded
a high amount of Fc molecules through self-assembly and realized the
purpose of signal amplification, thus the proposed immunosensor ex-
hibited improved sensitivity. The detection limit was observed to be as
low as 0.01 ng mL−1. The sensor was also tested in biological samples
and good recovery was observed. Moreover, the good specificity, re-
producibility and long-term stability of this immunosensor not only
offer an opportunity for the detection of CEA in low concentration, but
also provide a platform to develop various biosensors for many other
similar proteins determination.
Acknowledgments
Financial support from the Nature Science Foundation of China (No.
21505079), the Natural Science Foundation of Jiangsu Province (Grants
No BK20150402), and the Applied Research Program of Nantong City
(GY12015017, MS12016039) are gratefully acknowledged. X.Gu and S.
Tian are also supported by the Jiangsu Overseas Research and Training
Program. H.-B. Kraatz is grateful for the financial support from Natural
Sciences and Engineering Research Council of Canada and from the
University of Toronto Scarborough.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at http://dx.doi.org/10.1016/j.bios.2017.12.014.
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