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Author’s Accepted Manuscript
Improved screen-printed carbon electrode for
multiplexed
label-free
amperometric
immuniosensor: Addressing its conductivity and
reproducibility challenges
Lihua Zhao, Hongliang Han, Zhanfang Ma
www.elsevier.com/locate/bios
PII:
DOI:
Reference:
S0956-5663(17)30709-1
https://doi.org/10.1016/j.bios.2017.10.041
BIOS10066
To appear in: Biosensors and Bioelectronic
Received date: 9 August 2017
Revised date: 12 October 2017
Accepted date: 17 October 2017
Cite this article as: Lihua Zhao, Hongliang Han and Zhanfang Ma, Improved
screen-printed carbon electrode for multiplexed label-free amperometric
immuniosensor: Addressing its conductivity and reproducibility challenges,
Biosensors and Bioelectronic, https://doi.org/10.1016/j.bios.2017.10.041
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Improved screen-printed carbon electrode for multiplexed label-free
amperometric immuniosensor: addressing its conductivity and
reproducibility challenges
Lihua Zhao, Hongliang Han, Zhanfang Ma*
Department of Chemistry, Capital Normal University, Beijing 100048, China
E-mail: mazhanfang@cnu.edu.cn
1
Abstract
A new screen-printed carbon electrode (SPCE) with multiple working electrodes
and one signal output channel without counter and reference electrodes was designed.
The multiple working electrodes can be individually modified for each target of
interest. The SPCE contained one signal output channel, making the immuniosensor
be realized by common single-channel electrochemical workstation. The counter and
reference electrodes were independent of disposable SPCE, reducing costs and
eliminating precious metal pollution. Platinum network as counter electrode improved
the reproducibility of the SPCE. Moreover, method of generating hydrogel on
working electrode was used to enhance the conductivity of SPCE. Based on this, a
multiplexed single channel label-free amperometric immuniosensor for four tumor
markers, namely, squamous cell carcinoma antigen (SCCA), fragment antigen 21-1
(Cyfra21-1), carbohydrate antigen 125 (CA125), and neuron specific enolase (NSE)
was developed, and the corresponding detection limits were 5.5 pg mL-1, 4.8 pg
mL-1, 0.0054 U mL-1 and 2.3 pg mL-1, respectively. The sensitivity of this
immunosensor was 0.83 µA (lg(ng ml-1))-1 for SCCA, 1.92 µA (lg(ng ml-1))-1 for
Cyfra21-1, 4.75 µA (lg(U ml-1))-1 for CA125 and 2.40 µA (lg(ng ml-1))-1 for NSE.
Among them, the sensitivities of CA125 and NSE were four-fold higher than those
of the previous works.
Keywords: screen-printed carbon electrode, multiplexed label-free amperometric
2
immuniosensor, multifunctional redox hydrogel, tumor marker
1. Introduction
The growing interest in the exploration of complicated biological systems, which
function was completed by biological recognition between the different interacting
molecules (e.g., in immune system investigations) (Bogle and Dunbar 2012), and the
increasing demand for multiplexed sensors (e.g., in clinical diagnostics) (Liu et al.
2016; Scott et al. 2017) have made the simultaneous monitoring of different binding
events in a convenient way a powerful method for promoting biosensing applications.
Multiplexed electrochemical sensor with the advantage of requiring comparatively
inexpensive, miniaturized and easy to use instruments has been widely used (Rong et
al. 2016; Tarasov et al. 2016). Among them, immunosensors based on screen-printed
carbon electrode (SPCE) which are easy to fabricate, portable, and able to mass
production is the core to promote the practical application of electrochemical sensors
(Corgier et al. 2007; Ge et al. 2016).
In SPCE-based multiplexed immuniosensor, multiple working electrodes on SPCE
were individually modified of each target and the corresponding signals were
recorded by multi-channel electrochemical workstation (Du et al. 2011; Wan et al.
2014). Although this type of immunosensors has made considerable advances, they
have some problems: (1) due to the used SPCE with multiple working electrodes and
only one counter electrode, the area of the counter electrode is not much larger than
that of working electrodes and cannot avoid the interference of the current
measurement from the counter electrode (Scholz 2010), leading to a poor repeatability
3
of the immunoassay; (2) reference electrode and counter electrode containing precious
metals like silver and platinum were printed on disposable SPCEs (Agrisuelas et al.
2017), resulting in the cost increase of SPCEs and precious metal pollution; (3) the
presence of the polymeric binder in the carbon ink used for printing SPCEs influenced
conductivity of SPCEs (Mistry et al. 2014), seriously affecting the sensitivity of the
immunoassay; (4) expensive multi-channel electrochemical workstation is required,
resulting in a limitation of the immunoassay wide application. If these problems can
be overcome by designing new structure of SPCE and attaching high conductive
material on working electrodes, this will widely increase the potential applications of
this type of immunosensor.
In order to improve this situation, a new type of SPCE with multiple working
electrodes and one signal output channel but without counter and reference electrodes
was designed. The multiple working electrodes were able to be individually modified
for each target of interest, enabling the developed SPCE to be used in the construction
of multiplexed label-free immuniosensor. One signal output channel could make the
immuniosensor to be realized by a common single-channel electrochemical
workstation. The counter and reference electrodes containing precious metals like
silver and platinum were independent of disposable SPCE, reducing the costs and
eliminating precious metal pollution of SPCE. Platinum network as counter electrode
ensured that the area of counter electrode was larger than that of working electrodes
and thus improved the reproducibility of SPCE. Moreover, the method of generating
three-dimensional (3D) network hydrogel on working electrode was used to enhance
the conductivity of SPCE.
2. Experimental
2.1 Reagents and materials
4
Neuron specific enolase (NSE), cytokeratin 19 fragment antigen 21-1 (Cyfra21-1),
carbohydrate antigen 125 (CA125), squamous cell carcinoma antigen (SCCA) and
corresponding antibodies as well as carcino embryonie antigen (CEA) were purchased
from Shanghai Linc-Bio Science Co. Ltd. (Shanghai) O-phemylenediamine was
obtained from Tianjin Guangfu Fine Chemical Co. Ltd (Tianjin, China). N,
N’-diphenyl-p-phenylediamine (PPD), 3, 3’, 5, 5’-tetramethylbenzidine (TMB) and
methylene blue (MB) were obtained from Aladdin (Tianjin, China). H2O2 (30%),
HAuCl4·xH2O and K2PdCl4, ascorbic acid (AA), dopamine (DA) were obtained from
Alfa Aesar (Tianjin, China). Human immunoglobulin G (IgG) and bovine serum
protein (BSA) was achieved from Beijing Xinjinke Bioechnology Co. Ltd (Beijing,
China). Sodium alginate (SA) (alginic acid sodium salt from brown algea, low
viscosity) was purchased from Sigma-Aldrich Co (Beijing, China). phosphate buffer
solutions (PBS) was prepared by mixing NaH2PO4, Na2HPO4 and KCl. CaCl2,
NaH2PO4, Na2HPO4, KCl, K3Fe(CN)6 and K4Fe(CN)6 were from Beijing Chemical
Reagents Company (Beijing, China). SPCE (containing four graphite working and a
single signal output channel) was obtained from Beijing Union Trust technology Co.
Ltd (Beijing, China). Clinical human serum samples were obtained from Capital
Normal University Hospital (Beijing, China). All chemicals were analytical grade and
used without further purification.
2.2 Apparatus
Scanning electron microscopy (SEM) images and energy dispersive X-ray
microanalysis (EDX mapping) characterizations were determined with a Hitachi
S-4800 scanning electron microscope. The multi-point Brunauer-Emmet-Teller (BET)
was obtained by a volumetric gas analyzer Autosorb IQ (Quantachrome Instruments,
5
American). Thermogravimetry (TGA) was performed on TGA/SDTA851 from
METTLER TOLEDO. X-ray photoelectron spectroscopy (XPS) was obtained on an
Escalab 250 X-ray Photoelectron Spectroscope (Thermofisher, American). All
electrochemical experiments were realized through a CHI-830 electrochemical
workstation (Chenhua, Shanghai, China). A conventional three-electrode system was
used in the experiment with a modified SPCE (with four graphite working electrodes,
Φ = 3 mm) as the working electrode, a platinum network as auxiliary electrode, and
Ag/AgCl electrode (saturated KCl) as the reference electrode.
2.3 Synthesis of poly (o-phemylenediamine)-Au/Pd (PoPD-Au/Pd)
The PoPD-Au/Pd was prepared by the previous method with minor modifications
(Wang et al. 2015b). HCl aqueous solution (1 M) was added to 2 mL PoPD (20mM)
aqueous solution until pH=1. Then a homogeneous solution of 150 µL HAuCl4 (101.4
mM) and 150 µL K2PdCl4 (101.4 mM) was injected in the PoPD aqueous solution
under vigorous stirring. The color of the solution immediately turned black. After
stirring for 2 h at room temperature, the obtained PoPD-Au/Pd were thoroughly
washed by centrifugation at 8000 rpm with plenty of water. The final production was
dispersed in 2 mL ultrapure water.
2.4 Synthesis of poly (methylene blue)-Au/Pd (PMB-Au/Pd)
The PMB-Au/Pd composites were synthesized by the previous method with some
modifications (Shan et al. 2016). First, 0.5 mL MB (10 mM) was added to 3 mL
ultrapure water. Then the mixture of 500 μL HAuCl4 (10mM) and 500 μL K2PdCl4
(10mM) aqueous solution was injected into the MB solution under vigorous stirring
and the solution was kept stirring at room temperature for 2 h. The obtained
PMB-Au/Pd composites were washed with water for 3 times, collected by
centrifugation at 10000 rpm and then diluted with 2 mL water.
6
2.5 Synthesis of poly (N, N’-diphenyl-p-phenylediamine)-Au/Pd (PPPD-Au/Pd)
The synthesis of the PPPD-Au/Pd was according to the previous method with some
modifications (Wang et al. 2016). First, 300 µL PPD ethanol solution (5.5 mg mL-1)
was mixed with 1.7 mL ultrapure water and sonicated for 1 min. Then the mixture of
150 µL HAuCl4 (101.4 mM) and 150 µL K2PdCl4 (101.4 mM) was quickly injected
under vigorous stirring and the solution was kept stirring at room temperature for 2 h.
The composites of PPPD-Au/Pd were thoroughly washed by centrifugation at 8000
rpm with plenty of water. The final production was dispersed in 2 mL ultrapure water.
2.6 Synthesis of poly (3, 3’, 5, 5’-tetramethylbenzidine)-Au/Pd (PTMB-Au/Pd)
The synthesis of PTMB-Au/Pd was according to the previous method with little
modifications (Wang et al. 2015a). First, 1 mL TMB ethanol solution (9.28 mg mL-1)
was mixed with 1 mL ultrapure water and sonicated for 1 min. Following, the mixture
of 500 µL HAuCl4 (10 mM) and 500 µL K2PdCl4 (10 mM) was in injected under
vigorous string. After stirring for 2 h at room temperature, the obtained PTMB-Au/Pd
were thoroughly washed by centrifugation at 8000 rpm with plenty of water. The final
production was dispersed in 2 mL ultrapure water.
2.7 Preparation of sodium alginate-Au nanoparticle (SA-AuNP) composites
In brief, 4 mL 0.2% SA (wt. %) aqueous solution was quickly mixed with 5 μL
HAuCl4 solution (101.4 mM). The obtained homogeneous solution was reacted in
microwave reaction instrument (250 W) at 100 C for 10 min and then cooled down
to the room temperature. And the SA-AuNP composites were obtained and stored at
4C for further use.
2.8 Modification of the electrodes
Prior to modification, the SPCEs were electrochemically cleaned by cyclic
voltammetry (CV) in 0.5 M H2SO4 aqueous solution with the scanning range of
7
-1.0~1.0 V until a stable and remarkable voltammetric peak was observed. Then, the
SPCEs was thoroughly washed with ultrapure water and dried with nitrogen. The
prepared PoPD-Au/Pd, PMB-Au/Pd, PPPD-Au/Pd and PTMB-Au/Pd were
mixed with SA-AuNP at 1:2 (v/v) respectively. Then 10 μL the above mixtures
were dropped into each working electrode of the SPCEs and dried at 37C.
After that, 5μL CaCl2 (20 nmol) was dropped on the each working electrodes of
SPCEs and a thin homogeneous multifunctional hydrogels film was
immediately achieved. Finally, the resulting SPCEs were thoroughly washed by
ultrapure water.
2.9 Fabrication of the immunosensor
The capture antibody solutions (10 μL, 200 µg mL-1) of anti-SCCA,
anti-Cyfra21-1, anti-CA125, and anti-NSE were dropped on the four working
electrodes of the SPCEs modified by PoPD-Au/Pd, PMB-Au/Pd, PPPD-Au/Pd
and PTMB-Au/Pd respectively and incubated 12 h to fix the antibodies on the
surface of SPCEs. After that, each working electrodes of the SPCEs was
incubated with 5 L of 1 % BSA (w/w) for 45 min to block the non-specific
active sites. And after each step, the resulting SPCEs were washed thoroughly
with ultrapure water (Tang et al. 2017; He et al. 2015).
2.10 Measurement procedure
The total detection of SCCA, Cyfra21-1, CA125 and NSE was performed at
SPCEs by one step. In brief, the modified working electrodes of the SPCEs were
covered with 10 µL of their corresponding target solutions (SCCA, Cyfra21-1,
CA125 and NSE) at various concentrations at 37C for 45 min and washed by
ultrapure water. Then detection of tumour markers can be achieved by a
traditional three-electrode system through square wave voltammetry (SWV)
8
measurement from -1.3 to 1.0 V (vs. Ag/AgCl) in 0.2 M PBS buffer (pH 6.5)
with pulse amplitude of 50 mV and a frequency of 15Hz.
3. Results and discussion
3.1 Principle of the proposed electrochemical biosensor
Scheme 1 illustrated the principle of the proposed method. To achieve simultaneous
detection of multiple targets, the SPCE was designed with multiple graphite working
electrodes. In order to reduce the cost, eliminate precious metal pollution and enhance
reproducibility of the SPCE, the counter and reference electrodes were independent of
the disposable SPCEs and a platinum network was used as the counter electrode. To
avoid the requirement of expensive instruments, the SPCE was designed with a signal
output channel.
As a proof-of-concept, the developed SPCE was used to constructe a multiplexed
label-free amperometric immuniosensor for four tumor markers (SCCA, Cyfra21-1,
CA125, and NSE). Four redox species with distinguishable current signals were
synthesised by previous method namely PoPD-Au/Pd, PMB-Au/Pd, PPPD-Au/Pd,
and PTMB-Au/Pd (Shan et al. 2016; Wang et al. 2015b; Wang et al. 2016). Four
different multifunctional hydrogels were obtained by activating SA-AuNP hydrogels
with PoPD-Au/Pd, PMB-Au/Pd, PPPD-Au/Pd and PTMB-Au/Pd, and they were used
as the substrates for SCCA, Cyfra21-1, CA125, and NSE, respectively. The biological
event of the tumor marker (antigen-antibody interaction) generated a difference of its
corresponding redox current signal (ΔI) and the simultaneous detection of the tumor
markers was realized.
The above hydrogels have multiple functions: (1) the 3D continuous network and
large specific surface area effectively enhanced charge collection and electron transfer
of the SPCE (Zhai et al. 2013). (2) distinguishable redox signals at -0.58 V, -0.1 V,
9
0.25 V and 0.8 V endowed each biomerker with an independent current signal. (3)
admirable electrocatalytic ability for the decomposition of H2O2 was obtained because
of the presence of Au/Pd nanoparticle (Gao and Goodman 2012). (4) the hydrogel
synthesis was capable of being realized by low cost patterning technologies (Pan et al.
2012). Therefore, the multifunctional hydrogels were used to enhance the
performance of SPCE.
Scheme 1
3.2 Characterization of the used nanomaterials
The synthesis of multifunctional hydrogels was realized by simply mixing
SA-AuNP, redox species, and Ca2+. After mixing, the mixed solution
immediately gelated through the interaction between SA-AuNP, redox species
and Ca2+ (inserts in Fig. 1). The morphologies and the composites of the
multifunctional hydrogels were characterized by SEM and EDX mapping, as shown
in Fig. S1. The nanostructure of the multifunctional hydrogels showed 3D porous
network nanostructures (Fig. S1A). The EDX mapping images of Pd and Au elements
in hydrogels were shown in Fig. S1B and Fig. S1C, respectively. The red and green
color indicated the existence of Pd element and Au element, respectively. It illustrated
that Au and Pd elements were evenly dispersed on the hydrogels. In order to
investigate the specific surface areas of the multifunctional hydrogels, the
frozen hydrogels was placed in a freeze-dryer that removed all the water from it.
After
that,
the
specific
surface
areas
of
the
freeze-dried
hydrogels
(PoPD-Au/Pd-SA-AuNP, PMB-Au/Pd-SA-AuNP, PPPD-Au/Pd-SA-AuNP and
PTMB-Au/Pd-SA-AuNP) were calculated at 55.9, 60.54, 68.1 and 132.9 m2 g-1
by the multi-point BET method, respectively (Kostoglou et al. 2015). The BET linear
plot (Fig. 1) was fitted by three points between 0.05<P/P0<0.30. The correlation
10
coefficient was close to unity. It is a typical relative pressure range selected in the test
of specific surface areas in macroporous or non-porous materials because in that
specific range a monolayer of adsorbed N2 can be formed. The chemical
compositions of these multifunctional hydrogels were analyzed by XPS (Fig.
S2).
The water contents of the multifunctional hydrogels were measured by
thermogravimetry (Fig. S3). After purification through extensive rinsing with
ultrapure water, the multifunctional hydrogels were swollen and the water contents
were
90%,
91%,
PMB-Au/Pd-SA-AuNP,
83%
and
90%
(w/w)
PPPD-Au/Pd-SA-AuNP,
for
and
PoPD-Au/Pd-SA-AuNP,
PTMB-Au/Pd-SA-AuNP),
respectively.
Fig. 1
3.3 Electorchemical characterization of the designed SPCEs and redox hydrogels
To investigate the stability of the counter and reference electrodes, the cyclic
voltammetry measurements for conventional SPCEs (with the counter and
reference electrodes on it) and the designed SPCEs (without the counter and
reference electrodes on it) were performed for 50 cycles in 5 mM [Fe(CN)6]3-/4containing 0.1 M KCl, respectively (Fig. S4). Clearly, the counter and reference
electrodes on the SPCEs showed a poor stability, which might lead to a great
influence on the current response and a poor reproducibility of the
immunosensor (Fig S4A). In contrast, platinum network as counter electrode
avoided the interference of the current response from the counter electrode (Fig
S4B), which might be explain that the large surface area of platinum network
ensured that the area of the counter electrode is much larger than that of
working electrodes on the SPCEs (Scholz 2010).
11
The electrochemical characterization of the multifunctional hydrogels was
investigated in Fig. 2A. Only one working electrode of SPCE was modified by
one hydrogel (curve a-d) (PoPD-Au/Pd-SA-AuNP, PMB-Au/Pd-SA-AuNP,
PPPD-Au/Pd-SA-AuNP and PTMB-Au/Pd-SA-AuNP). The four working
electrodes of the SPCE were separately modified by four different redox
hydrogels (curve e). Only one obvious current response was detected
corresponding to the redox species in the presence of only one redox hydrogel,
and four current signals at -0.58 V, -0.1 V, 0.25 V and 0.8 V were appeared
simultaneously in the four redox hydrogels modified SPCE, indicating that the
current signals of the immunoassay platforms were distinguishable to each
other.
In this assay, SA-AuNP hydrogel was introduced for electron-transfer
acceleration. Fig. 2B clearly showed that the redox species doped SA-AuNP
hydrogel, the multifunctional hydrogel, endowed a higher response compared
to redox species alone. Additionally, a further signal amplification was realized
by electrocatalytic ability toward H2O2 of the multifunctional hydrogels (Fig.
S5). It was observed that the SWV cure with H2O2 amplification exhibited
higher current response (curve b) than that without H 2O2 amplification (curve
a), indicating that H2O2 effectively amplified the signals of the redox hydrogels.
Fig. 2
3.4 The layer-by-layer assembling of the immunosensor
The layer-by-layer assembling of the immunosensor was monitored by SWV
measurements (Fig. 3). A high current signal (-0.58V, -0.1V, 0.25V and 0.8 V)
was
produced
by
the
PoPD-Au/Pd-SA-AuNP,
PMB-Au/Pd-SA-AuNP,
PPPD-Au/Pd-SA-AuNP and PTMB-Au/Pd-SA-AuNP modified SPCE (curve a).
12
After the assembly of the capture antibodies of SCCA, Cyfra21-1, CA125 and
NSE the corresponding current peaks were significantly reduced (curve b).
Subsequently, when the remaining nonspecific sites were blocked by BSA, a
further decrease of the current peaks was appeared (curve c). Finally, after
SCCA, Cyfra21-1, CA125 and NSE were incubated in their corresponding
immune surface, the current peaks was decreased (curve d), because the
immunocomplexes formed by antibodies and antigens retarded the electron
conduction of the electrode surface.
Fig. 3
3.5 Optimization of the experimental condition
In order to achieve the optimal performance of the immunosensor, the pH of
the detection solution and the incubation time were optimized. To optimize the
pH, the immunosensor platform was incubated with antigens (0.1 ng mL-1
SCCA, 0.1 ng mL-1 Cyfra21-1, 0.1 U mL-1 CA125 and 0.1 ng mL-1 NSE) was
tested in a series of PBS. The SWV current increased from 5.5 to 6.5 and then
decreased at higher pH values (Fig. S6A). Thus, pH 6.5 for PBS was adopted.
To optimize the incubation time, immunosensor platform was incubated with
the target antigens of 0.1 ng mL-1 SCCA, 0.1 ng mL-1 Cyfra21-1, 0.1 U mL-1
CA125 and 0.1 ng mL-1 NSE. The current peaks of immunosensor response to
the incubation time increased from 15 min to 45 min and then tended to remain
constant after 45 min (Fig. S6B). Hence, 45 min was chosen as the optimal
incubation time. To further investigate H2O2 influence, the optimization of the
quantity of H2O2 was carried out in Fig. S7. The current signal increased with
the increase of H2O2 concentration from 2 mM to 5 mM. Then the current
signal remained constant at higher concentration of H2O2. Therefore, 5 mM was
13
adopted as the optimum concentration of H2O2.
3.6 Analytical performance of the immunoassay
Under the optimal conditions, after incubated with different concentrations of
the four target antigens, the sensitivity and dynamic range of the proposed
immunoassay were estimated by detecting the SWV responses (Fig. 4A). With
the increase of target antigens (SCCA, Cyfra21-1, CA125, and NSE)
concentration, their corresponding current peaks at (-0.58 V, -0.1 V, 0.25 V,
and 0.8 V) decreased as shown in Fig. 4A. The calibration plot exhibited a good
linear relationship between the current peaks and the logarithm of the analytes
concentrations in the range from 0.01 to 100 ng mL -1 for SCCA (Fig. 4B) and
Cyfra21-1 (Fig. 4C), from 0.01 to 200 U mL -1 for CA125 (Fig. 4D) and from
0.01 to 200 ng mL-1 for NSE (Fig. 4E). The detection limits were 5.5 pg mL-1
for SCCA, 4.8 pg mL-1 for Cyfra21-1, 0.0054 U mL-1 for CA125 and 2.3 pg
mL-1 for NSE, according to three times of the standard deviation above the
blank (S/N=3) (Jiang et al. 2017).
Fig. 4
3.7 Evaluation of specificity, reproducibility and stability of immunosensor
To assess the specificity, UA, AA, IgG and AFP were chosen as the
interfering species (Fig. S8). The analysis tests were conducted as follows: the
immunosensor was incubated with the four antigens (0.1 ng mL-1 for NSE,
Cyfra21-1 and SCCA, 0.1 U mL-1 for CA125); the control groups were
incubated by 10 ng mL-1 DA, AA, IgG, CEA; the blank group was incubate
with ultrapure water. The signals of the target were far less than that of control
groups. And the signals of the control groups were as same as the signal
obtained from the control groups, demonstrating the good specificity of the
14
immunosensor. To assess the reproducibility, four as-prepared immunosensor
were used to investigate the repeatability and the concentration of the incubated
target antigens was 0.1 ng mL-1 for SCCA, 0.1 ng mL-1 for Cyfra21-1, 0.1 ng
mL-1 for NSE and 0.1 U mL-1 for CA125. The relative standard deviations
(RSDs) were 2.2% for SCCA, 3.5% for Cyfra21-1, 4.2% for CA125, and 5.2%
for NSE, demonstrating the good repeatability of the immunosensor. To assess
the reproducibility, the well-modified immunosensors was stored at 4C for one
month and evaluated once a week. The SWV current retained 98.6% for SCCA,
98.7% for Cyfra21-1, 99.2% for CA125, and 98.5% for NSE after one week,
97.3% for SCCA, 97.8% for Cyfra21-1, 97.5% for CA125, and 97.7% for NSE
after two weeks, 96.7% for SCCA, 97.0% for Cyfra21-1, 97.1% for CA125,
and 97.4% for NSE after three weeks and 95.2% for SCCA, 96.4% for
Cyfra21-1, 96.2% for CA125, and 95.8% for NSE after four weeks, indicating
the good stability of the immunosensor.
Compared with some recent literature of the immunosensors based on SPCEs,
the proposed immunosensor showed a wider linear detection range, a lower
detection limit, and a higher sensitivity (Table S1). The sensitivity of CA125
was 4.75 µA (lg(U ml-1))-1 and NSE was 2.40 µA (lg(ng ml-1))-1 , which was
four-fold increase than that of the previous work. The relative standard
deviations between parallel experiments were less than 5.2%, thereby rivaling
the reproducibility of the previous sensors based on SPCE.
3.8 Analysis of clinical serum samples
In order to evaluate the practicability of the immunosensor, with the
Enzyme-linked immunosorbent assay (Mahou et al.) as reference method, ten
clinical human serum samples were measured by the proposed immunosensor
15
and ELISA in Table 1. The relative errors between the two methods ranged
from -8.16% to 8.33%, demonstrating the detection results of proposed
immunosensor for simultaneous detection of four tumor markers were well
consistent with that of ELISA method.
Table 1
4. Conclusion
In summary, a novel SPCE was designed with multiple working electrodes and a
signal output channel but without counter and reference electrodes. Based on the
developed SPCE, a multiplexed label-free amperometric immunosensor for four
tumor markers (NSE, CA125, Cyfra21-1 and SCCA) reported in this work offered
several advantages over existing methods. First, the counter and reference electrodes
were independent of SPCEs and reusable, which enhanced the reproducibility and
eliminated precious metal pollution of the immunosensor. Second, multifunctional
hydrogels with large specific surface area, admirable electrocatalytic ability as
substrates enhanced sensitivity of the immunosensor. Third, this method was easier to
use and did not require probes for targets labeling and other costly instruments for
signal output. Moreover, this method was able to be extended for almost any
molecules with identification element and recognition reactions, which opened the
door for simple, efficient and highly sensitive in biological systems investigations and
clinical diagnostics.
Acknowledgements
This work was supported by the National Natural Science Foundation of
China (21673143, 21273153), Natural Science Foundation of Beijing
Municipality (2172016, 2132008), and the Project of the Construction of
Scientific Research Base by the Beijing Municipal Education Commission.
16
References
Agrisuelas, J., González-Sánchez, M.-I., Valero, E., 2017. Sens. Actuators B 249,
499-505.
Bogle, G., Dunbar, P.R., 2012. PLoS One 7, 13.
Corgier, B.P., Laurent, A., Perriat, P., Blum, L.J., Marquette, C.A., 2007. Angew.
Chem. Int. Ed. 46, 4108-4110.
Du, D., Wang, J., Lu, D.L., Dohnalkova, A., Lin, Y.H., 2011. Anal. Chem. 83,
6580-6585.
Gao, F., Goodman, D.W., 2012. Chem. Soc. Rev. 41, 8009-8020.
Ge, X.X., Zhang, A.D., Lin, Y.H., Du, D., 2016. Biosens. Bioelectron. 80, 201-207.
He, Y., Xie, S.B., Yang, X., Yuan, R., Chai, Y.Q., 2015. ACS Appl. Mater. Interfaces
7, 13360-13366.
Jiang, B.Y., Li, F.Z., Yang, C.Y., Xie, J.Q., Xiang, Y., Yuan, R., 2017. Sens.
Actuators B 244, 61-66.
Kostoglou, N., Polychronopoulou, K., Rebholz, C., 2015. Vacuum 112, 42-45.
Liu, G.Z., Qi, M., Hutchinson, M.R., Yang, G.F., Goldys, E.M., 2016. Biosens.
Bioelectron. 79, 810-821.
Mahou, R., Borcard, F., Crivelli, V., Montanari, E., Passemard, S., Noverraz, F.,
Gerber-Lemaire, S., Bühler, L., Wandrey, C., 2015. Chem. Mater. 27, 4380-4389.
Mistry, K.K., Layek, K., Mahapatra, A., RoyChaudhuri, C., Saha, H., 2014. Analyst
139, 2289-2311.
Pan, L.J., Yu, G.H., Zhai, D.Y., Lee, H.R., Zhao, W.T., Liu, N., Wang, H.L, Tee,
B.C.K., Shi, Y., Cui, Y., Bao, Z.N., 2012. PNAS 109, 9287-9292.
Rong, Q.F., Feng, F., Ma, Z.F., 2016. Biosens. Bioelectron. 75, 148-154.
Scholz, F., 2010. Electroanalytical methods (Vol. 1). Berlin-Heidelberg: Springer.
17
Scott, A.W., Garimella, V., Calabrese, C.M., Mirkin, C.A., 2017. Bioconjugate Chem.
28, 203-211.
Shan, J., Ma, Z.F., 2016. Microchim. Acta 183, 2889-2897.
Tang, Z.X., Fu, Y.Y., Ma, Z.F., 2017. Biosens. Bioelectron. 94, 394-399.
Tarasov, A., Gray, D.W., Tsai, M.Y., Shields, N., Montrose, A., Creedon, N., Lovera,
P., O'Riordan, A., Mooney, M.H., Vogel, E.M., 2016. Biosens. Bioelectron. 79,
669-678.
Wan, Y., Zhou, Y.-G., Poudineh, M., Safaei, T.S., Mohamadi, R.M., Sargent, E.H.,
Kelley, S.O., 2014. Angew. Chem. Int. Ed. 53(48), 13145-13149.
Wang, L.Y., Feng, F., Ma, Z.F., 2015a. Sci. Rep. 5, 16855.
Wang, L.Y., Liu, N., Ma, Z.F., 2015b. J. Mater. Chem. B 3, 2867-2872.
Wang, L.Y., Shan, J., Feng, F., Ma, Z.F., 2016. Anal. Chim. Acta 911, 108-113.
Zhai, D.Y., Liu, B.R., Shi, Y., Pan, L.J., Wang, Y.Q., Li, W.B., Zhang, R., Yu, G.H.,
2013. ACS Nano 7, 3540-3546.
18
Figure Captions
Scheme 1. Schematic diagram of the proposed label-free amperometric
immunosensor by using improved screen-printed carbon electrode as working
electrodes.
Fig. 1. Nitrogen adsorption-desorption isotherm of multifunctional hydrogels:
PoPD-Au/Pd-SA-AuNP (A), PMB-Au/Pd-SA-AuNP (B), PPPD-Au/Pd-SA-AuNP (C)
and PTMB-Au/Pd-SA-AuNP (D). And the inserts are the photographs of the four
multifunctional hydrogels inside a glass vial.
Fig. 2. The cross interference of the SWV signals of the four multifunctional hydrogels
(A) and the SWV signals of the four redox species (a) and the four multifunctional
hydrogels (b) ( B).
Fig. 3. The SWV characterization of the modified procedure of the electrodes in 2
mM PBS (pH 6.5).
Fig. 4. SWV responses (A) and calibration curves of the immunosensor for different
concentrations of SCCA (B), Cyfra21-1 (C), CA125 (D) and NSE (E) (n=3).
19
Table 1. Assay results of clinical serum samples using the proposed
immunosensor and ELISA (n=3).
Sample No.
This
work
ELAS
A
SCCA
(ng mL-1)
Cyfra211
(ng
-1
mL )
CA125
(U mL-1)
NSE (ng
mL-1)
SCCA
(ng mL-1)
Cyfra211
(ng
-1
mL )
CA125
(U mL-1)
NSE (ng
mL-1)
SCCA
(ng mL-1)
Relativ
e error
Cyfra21(%)
1
(ng
-1
mL )
CA125
(U mL-1)
NSE (ng
mL-1)
1
2
3
4
5
6
7
8
9
10
0.30
0.36
0.38
1.01
0.19
0.69
0.34
0.18
0.13
0.15
1.81
1.01
2.28
1.68
0.96
8
3.24
1.82
1.79
0.45
2.61
2.28
4.83
6.66
1.78
1.79
4.13
5.42
3.36
3.99
14.1
7
0.31
10.8
9
0.37
1.59
5.89
0.18
10.0
1
0.72
15
0.37
11.0
1
1.04
14.8
9
9.26
0.37
0.17
10.8
9
0.12
18.1
2
0.16
1.95
1.06
2.19
1.75
0.9
3.08
1.76
1.87
0.49
2.71
2.37
4.61
7.01
1.87
1.68
3.91
5.72
3.15
3.01
14.9
7
3.
23
-6.70
10.5
5
-2.70
1.68
6.19
2.70
10.5
8
-2.88
5.56
10.3
8
4.17
14.2
3
8.89
-8.11
15.6
0
5.59
10.2
6
8.33
17.5
5
-62.5
-4.71
4.11
-4.00
7.56
5.20
3.41
-4.28
-8.16
-3.69
-3.80
4.77
-4.81
6.55
5.63
4.64
-5.24
6.67
3.26
-5.34
3.22
-4.9
9
-5.3
6
4.06
-4.84
-3.56
4.16
-3.85
6.14
3.25
20
Highlights
1. Screen-printed carbon electrode with multi-working electrodes and single-channel
without counter and reference electrodes was designed.
2. Conductive hydrogel generating on working electrode was used to enhance
the conductivity of screen-printed carbon electrode.
3. Single-channel label-free amprometric immunosensor for simultaneous detection
of tumor markers was developed.
21
fig 1
22
fig 2
23
fig 3
24
fig 4
25
scheme 1
26
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