Simultaneous electrochemical detection of multiple tumor markers using functionalized graphene nanocomposites as non-enzymatic labels

Accepted Manuscript Title: Simultaneous electrochemical detection of multiple tumor markers using functionalized graphene nanocomposites as non-enzymatic labels Author: Dexiang Feng Lihua Li Xiaowei Han Xian Fang Xiangzi Li Yuzhong Zhang PII: DOI: Reference:

S0925-4005(14)00534-6 http://dx.doi.org/doi:10.1016/j.snb.2014.05.015 SNB 16889

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

26-11-2013 9-3-2014 3-5-2014

Please cite this article as: D. Feng, L. Li, X. Han, X. Fang, X.L. ,Yuzhong Zhang, Simultaneous electrochemical detection of multiple tumor markers using functionalized graphene nanocomposites as non-enzymatic labels, Sensors and Actuators B: Chemical (2014), http://dx.doi.org/10.1016/j.snb.2014.05.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Simultaneous electrochemical detection of multiple tumor markers using functionalized graphene nanocomposites as non-enzymatic labels

ip t

Dexiang Fenga,b, Lihua Lia,b, Xiaowei Hana, Xian Fanga, Xiangzi Lib,Yuzhong Zhanga*

cr

a College of Chemistry and Materials Science, Anhui Normal University, Wuhu

us

241000,People’s Republic of China

b Department of Chemistry, Wannan Medical College, Wuhu 241002, People’s Republic of

Ac ce p

te

d

M

an

China

* Corresponding author. Tel: +86 553 3869303; Fax: +86 553 3869303 E-mail address: [email protected] (Y. Zhang).

1   

Page 1 of 35

Abstract: In this work, we designed a novel sandwich-type electrochemical immunosensor for simultaneous detection of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) using

ip t

non-enzymatic multiple-label method. Firstly, gold nanoparticles (AuNPs) decorated reduced

cr

graphene oxide (rGO)-carried prussian blue (PB) and AuNPs decorated rGO-carried thionine

us

(Thi) (denoted as AuNPs-PB@rGO and AuNPs-Thi@rGO, respectively) were employed as distinguishable signal tags, which were utilized to load anti-CEA (Ab2,1) and anti-AFP (Ab2,2),

an

respectively. Subsequently, hierarchically aloe-like gold microstructures (HAG) with large effective area were used to immobilize the capture antibodies anti-CEA (Ab1,1) and anti-AFP

M

(Ab1,2), which effectively enhanced the performance of the immunosensor. Experimental results revealed that the sandwich-type immunosensor can permit the simultaneous detection of CEA

d

and AFP with linear range of 0.6-80 ng mL−1 for two analytes, the detection limit was 0.12 ng

te

mL−1 for CEA and 0.08 ng mL−1 for AFP (at a signal to noise ratio of 3), respectively. The

Ac ce p

immunosensor was applied to real sample analysis, and the recovery was within 95.9%-105.6% for CEA and 95.6%-105.4% for AFP. In addition, the immunosensor exhibited good reproducibility and stability, suggesting the immunosensor offered potential in analysis of biological samples.                                                 

Keywords: Immunosensor,  Reduced graphene oxide-carried thionine,  Reduced graphene oxide-carried prussian blue, Aloe-like gold microstructures, Simultaneous detection

2   

Page 2 of 35

1. Introduction The determination of cancer biomarkers plays an important role in screening and diagnosis of cancer [1, 2]. As is we known, no single tumor marker is sensitive and specific enough to

ip t

meet the strict diagnostic standard, single tumor marker detection is often easily to cause false

cr

positives and negatives phenomenon. Therefore, simultaneous detection of multiplex tumor

us

markers related to a certain cancer in human serum has become an interesting and promising analytical method for reliable diagnosis of cancer [3-7]. Compared with the traditional

an

single-analyte immunoassay, the simultaneous multiplexed immunoassay (SMIAs) exhibited many advantages including short analytical time, small sample volume and high accuracy

M

[8-10]. Thus, different measurement techniques have been developed for SMIAs. Among these techniques, electrochemical immunosensor has been applied to multi-analyte assays due to its

te

d

high sensitivity, low cost, simple instrumentation and good portability.  For multi-analyte simultaneous detection, one of the most major problems is how to

Ac ce p

discriminate each electrochemical signal for one special analyte from the multiple antigen-antibody reactions. The present methods are mainly adopted either multiple labels or spatial resolution assay protocols [11, 12]. However, spatial resolution  in clinical diagnosis is limited due to its high cost and cross-talk between the adjacent electrodes. The detection based on multiple labels improved the ability of discriminating electrochemical signal to some extent. The reason attribute to two aspects:  one of the reasons is different antibodies are immobilized simultaneously on the same electrode, which avoids  the use of multiple electrodes. Another reason is non-enzymatic reaction can eliminate cross-talk due to no substrate or mediator in the detection solution. For those reasons, some researchers have paid attention to redox-probe 3   

Page 3 of 35

tagged electrochemical sensors for simultaneous detection multi-analyte due to attractive electrochemical activity of redox-probe and distinguishable ability from each other. Furthermore, compared with traditional metal nanoparticle or QD based as labels, redox-probe

ip t

tagged SMIAs avoided a complicated and time-consuming acid dissolution step and metal

cr

preconcentration before electrochemical detection [13]. Thus, it is vitally important to search

us

distinguishable redox probes as trace labels for multiple-label electrochemical immunoassay. Recently, nanomaterials loaded with electroactive species including organic dyes and

an

enzymes have been reported as multi-label carriers to amplify detection signals, such as carbon nanotube [14, 15], carbon nanosphere [16] and gold nanoparticles [17, 18]. For example, tang

M

et al. developed horseradish peroxide (HRP)-thionine and HRP-ferrocene functional gold hollow microspheres as distinguishable signal tags for simultaneous determination of two

d

biomarkers [19]. Yuan and coworkers used three redox-probes containing thionine, ferrocene

te

and 2,2’-bipyridine-4,4’-dicarboxylic acid [Co(bpy) ]33+ functionalized graphene sheets as

Ac ce p

signal labels to simultaneously detect three liver cancer biomarkers [20]. Most recently, graphene has been widely used in electrochemical sensors due to its large surface area and good conductivity [21]. Research showed that some redox probes can directly interact with graphene through the π-π stacking [22], and effectively overcome graphene aggregation. Inspired from the advantage of the nanocomposites of graphene with redox probes, we prepared two novel reduced graphene oxide-based nanocomposites AuNPs-thionine (Thi)@rGO and AuNPs-prussian blue (PB)@rGO as signal tags. The nanocomposites not only provided a biocompatible microenvironment for the immobilization of antibody, but also could accelerate the electron transfer.  In contrast to the earlier related methods, our approach showed 4   

Page 4 of 35

some advantages. First, hierarchically aloe-like gold microstructures (HAG) and polymeric aniline (PANI) and rGO as sensing interface provide good biocompatibility and high surface area, which could greatly enhance the immobilization of primary antibody. Second,

us

cr

the secondary antibody, which greatly improved the signal intensity.

ip t

AuNPs-PB@rGO and AuNPs-Thi@rGO nanocomposites could immobilize a large amount of

2. Experimental

an

2.1. Materials

Carcinoembryonic antigen (CEA), CEA antibody (anti-CEA), α-fetoprotein (AFP), human

M

chorionic gonadotropin (HCG) and prostate specific antigen (PSA) were purchased from Biocell Biotech. Co., Ltd (Zhengzhou, China) and stored in refrigerator at 4 °C. Bovine serum

te

d

albumin (BSA) was purchased from the Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Reduced graphene oxide (purity ≥98%) was purchased from Institute of Coal

Ac ce p

Chemistry, Chinese academy of sciences (Taiyuan, China). Poly (diallyldimethylammonium chloride) (PDDA) solution (average Mw100000-200000 low molecular weight, 20 wt %) and thionine (Thi) were provided by Aldrich (St. Louis USA).  Potassium ferricyanid (K3Fe(CN)6), potassium ferrocyanide (K4Fe(CN)6.3H2O), and chloride hexahydrate (FeCl3.6H2O) were obtained from Shanghai Ling Feng Chemical Reagent Co., Ltd. (Shanghai, China). Chloroauric acid (HAuCl4.4H2O) was obtained from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Aniline was obtained from Accela Chem Bio Co., Ltd. (Shanghai, China) and was distilled under reduced pressure before use. Phosphate buffer saline (PBS) with various pH values were prepared by mixing the stock solutions of 0.1 M Na2HPO4, 0.1 M NaH2PO4 and 5   

Page 5 of 35

0.1 M KCl. The washing buffer was pH 7.0 PBS containing 0.05% (W/V) Tween (PBST). Blocking solution was 1% BSA. The clinical serum samples were from the clinical laboratory of the Yiji Shan Hospital (Wuhu, China). Twice-quartz-distilled water was used through the

cr

ip t

study.

us

2.2. Apparatus

All electrochemical measurements were performed on a CHI 650C electrochemical

an

analyzer (CH Instruments Inc., China) with a conventional three-electrode system composed of a platinum auxiliary, an silver-silver chloride (Ag/AgCl) (with 3.0 M KCl) reference, and a bare

M

GCE or modified GCE working electrode. All potentials in this work are referenced to the Ag/AgCl.

d

Electrochemical impedance spectra (EIS) were performed in 0.1 M PBS containing 5.0

te

mM [Fe(CN)63−/4−] and 0.1 M KCl. The frequency ranged from 0.1 to 100 kHz, the amplitude

Ac ce p

of the alternate voltage was 5 mV.

Morphologies of various nanomatrials and nanocomposites were obtained with scanning electron microscopy (SEM) (JEOLJSM-6700F, Hitachi, Japan).

2.3. Preparation of AuNPs-Thi@rGO and AuNPs-PB@rGO Scheme.1 (A) displayed the preparation process of immunosensing probes. In order to prepare immunoprobe, AuNPs was firstly synthesized according to literature [23].  Thi@rGO nanocomposites were synthesized as follows: 4.0 mL of rGO dispersion (0.5 mg mL−1) was mixed with 4.0 mL Thi (1.0 mM) and stirred vigorously for at least 12 h. During this process, 6   

Page 6 of 35

Thi was bound to the rGO sheet via π-π stacking [24], the Thi@rGO nanocomposites were purified by centrifugation and washed with ultrapure water  to remove the non-integrated Thi. Next, the as-prepared Thi@rGO nanocomposites were added dropwise into 20.0 mL of the

ip t

as-prepared AuNPs and stirred for 24 h to give a homogeneous suspension. Thus, the

cr

negatively charged AuNPs were absorbed on the surface of Thi@rGO because of the

us

interaction between the amine groups in THi and AuNPs together with the electrostatic interaction. After centrifugation under 10,000 rpm for 10 min, the complex was washed with

an

water for at least three times and re-dispered in 2.0 mL of 0.1 M pH 7.0 PBS buffer solution. We synthesized PB@rGO nanocomposites according to the literature with slight

M

modification [25]. The PB nanoparticles were attached to the rGO surfaces by reduction reaction. Briefly, 4.0 ml aqueous solution containing 10.0 mM FeCl3 and 10 mM K3Fe(CN)6

te

d

was adjusted to pH 1.5 with HCl, and then it was added into 4.0 mL rGO (0.5 mg mL−1) dispersion. After stirring for 2 h, the color of the mixture gradually changed from yellow brown

Ac ce p

to dark cyan, suggesting the formation of PB@rGO nanocomposites. The size of PB nanoparticles was about 30 nm (Seen in Fig. S2). AuNPs-PB@rGO nanocomposites were prepared as follows: the as-prepared PB@rGO nanocomposites were dispersed in 4.0 mL PDDA solution (1%) and stirred for 30 min to give a homogeneous suspension. Next, the above dispersion were mixed with 20.0 mL AuNPs solution and sonicated for 30 min. After centrifugation, the mixture was washed with water for at least three times and re-dispered in 2.0 mL PBS.

2.4. 

Preparation

of

anti-AFP2,2-AuNPs-Thi@rGO

and

anti-CEA2,1-AuNPs-PB@rGO bioconjugates 7   

Page 7 of 35

100 μL  (1mg mL−1) anti-AFP2,2 was added to the above AuNPs-Thi@rGO composites solution and gently mixed at 4

◦

C for 12 h.  After centrifugation, the obtained

anti-AFP2,2-AuNPs-Thi@rGO bioconjugates were reacted with 1% (w/w) BSA solution for 2 h

ip t

to block any possible remaining active sites to avoid any nonspecific absorption. After

cr

centrifuged and washed for several times, this bioconjugates were re-dispersed in 2.0 mL PBS

us

(0.1 M, pH 7.0) and stored at 4 °C when not in use.

For the anti-CEA2,1-AuNPs-PB@rGO bioconjugates, anti-CEA2,1-AuNPs-PB@rGO

an

bioconjugates were also prepared using the similar method.  This bioconjugates  were

M

re-dispersed in 2.0 mL PBS (0.1 M, pH 7.0) and stored at 4 °C.

2.5. Fabrication of the immunosensor

te

d

Before modification, the bare glassy carbon electrode (GCE) was successively polished with 1.0, 0.3 and 0.05 μm alumina slurry, and it was then rinsed with distilled water, and

Ac ce p

cleaned ultrasonically sequentially in water and 95% ethanol for 5 minutes. Finally, and the electrode was dried in nitrogen atmosphere for further use. 3.0 μL of rGO (0.5 M) composites were dropped onto the surface of pre-treated GCE and left to dry naturally at room temperature to form thin film. Afterwards, the electrode modified with rGO was placed into electrolyte solution (0.5 M H2SO4 and 0.05 M aniline). PANI was in situ electropolymerized on the rGO surface at constant potential of 0.75 V for 60 s. After that, the electrode modified with PANI/rGO was washed with distilled water and dried.  The modified electrode was immersed in 0.1 M KNO3 solution containing 12 mM HAuCl4 and electrochemical deposited 10 min at - 200 mV [26]. Thus, HAG was distributed on the surface 8   

Page 8 of 35

of the PANI/rGO. The obtained electrode was denoted as HAG/PANI/rGO/GCE.  Immobilization of anti-CEA (Ab1,1) and anti-AFP (Ab1,2) was performed by dropping a mixture of anti-CEA (Ab1,1) and anti-AFP (Ab1,2) (10.0 μL, 200 μg mL−1) solution onto the

ip t

surface of the HAG/PANI/rGO/GCE, and kept it for 12 h in a refrigerator at 4 °C. Following

cr

that, the modified electrode was incubated in 1% BSA solution for 1 h at 37 °C to block any

us

possible remaining active sites against non-specific adsorption, and washed several times with

an

PBST. The obtained immunosensor was stored at 4 °C prior to use.

2.6. Electrochemical measurements

M

Scheme 1(B) showed the fabrication process of the electrochemical immunosensor of CEA and AFP determination. Initially, the immunosensor was incubated with the mixture of

was

then

incubated

te

d

containing various concentrations CEA and AFP or serum samples for 50 min at 37 °C,  and it with 

mixture

containing

Ab2,1-AuNPs-PB@rGO

and

Ac ce p

Ab2,2-AuNPs-Thi@rGO solution (1:1) for another 50 min at 37 °C. Finally, it was transferred into pH 6.5 PBS  for electrochemical detection using DPV technique. The experiment parameters are listed: potential range, 600 to -600 mV; pulse amplitude, 50 mV.

Scheme 1.

3. Results and discussion 3.1. Characterization of the Thi@rGO and PB@rGO nanocomposites

9   

Page 9 of 35

rGO, which has large specific surface area, high electrical conductivity and fracture strength [27, 28], has made it as a promising nanomaterial in various fields’ applications, especially in electrochemical immunosensor [29]. It could be observed in Fig. 1A, rGO showed

ip t

the irregularly crumpled and wrinkled sheet-like structures, which could increase the electrode

cr

effective surface. When PB was attached onto the rGO (Seen in Fig. 1B), it could be clearly

us

observed PB cubic nanoparticles covered to the surface of rGO tightly,  indicating  PB@rGO nanocomposites were successfully obtained.  Fig. 1C showed the morphology of Thi@rGO

an

nanocomposites, which is similar to that of rGO. In order to improve the biocompatibility of PB@rGO nanocomposites, PDDA were used to functionalize  PB@rGO nanocomposites via a

M

simple sonication-induced assembly, PDDA functionalized PB@rGO not only increased the solubility of nanocomposites but also allowed its further decoration with AuNPs. Fig. 1D

te

d

showed the morphology of AuNPs-PB@rGO. It also could be clearly observed that AuNPs were distributed on the surface of PB@rGO surface. Fig. 1E showed the morphology of

Ac ce p

AuNPs-Thi@rGO nanocomposites, it was also observed that AuNPs distributed on the surface of Thi@rGO nanocomposites, indicating Thi not only acted as an electrochemical active species but also acted as a “glue” to connect the AuNPs to the rGO. Fig. 1.

UV-vis spectrometry was used to investigate the interactions of rGO and PB or rGO and thionine in the prepared nanocomposites. Fig. 2A, B and C showed the absorption spectra of pure rGO, PB@rGO and Thi@rGO, respectively. As shown in Fig. 2A, rGO appeared a strong absorption peak at about 266 nm, which corresponded to the reduced GO [30]. After rGO were modified with PB nanoparticle, two addition peaks were observed at 300 nm and 750 nm,  10   

Page 10 of 35

which belonged to the typical absorption of the PB [25] (Seen in Fig. 2B),  suggesting PB nanoparticles could be adhered to rGO successfully. It was also observed that the absorption peaks of Thi could be appeared in Thi@rGO nanocomposite (Seen in Fig. 2C),  indicating that

cr

ip t

the Thi@rGO nanocomposites were prepared successfully.

us

Fig. 2.

an

3.2. Characterization of the assemble process of the immunosensor SEM was used to characterize the morphologies of rGO, PANI/rGO and HAG/PANI/rGO,

M

respectively. As could be seen in Fig. 3A that rGO showed the irregularly crumpled and wrinkled sheet-like structures, which could provide more active nucleation sites for PANI [31].

te

d

A  dendritic structure was observed after the electro-polymerization of aniline in Fig. 3B, indicating the formation of PANI on the surface of rGO. When HAG were electrodeposited

Ac ce p

onto the modified electrode, the superstructures of aloe-like HAG with diameter of about 200-300 nm  were homogeneously dispersed on the surface of PANI (Fig. 3C).  This aloe structure could greatly increase the electrode effective surface area, which largely enhanced the probe loading amount. Moreover, the aloe structure also greatly improved the electron transfer ability.

Fig. 3.

3.3. Electrochemical characterization of the assemble process of the 11   

Page 11 of 35

immunosensor EIS was used to monitor the stepwise construction process of the immunosensor. The electron transfer resistances (Ret) were determined from the diameter of the semicircular parts

ip t

in the high frequency regions of the Nyquist plot, and the linear part at lower frequencies

cr

corresponds to the diffusion process (the equivalent electrical circuit is shown in the inset in

us

Fig. 4). It could be observed that the bare electrode displayed a small semicircle with a Ret of about 103Ω (curve a). After the bare electrode was modified with  HAG/PANI/rGO, the

an

semicircle disappeared (curve b), indicating that the HAG/PANI/rGO can accelerate the electron transfer. However, when Ab1 was loaded on the surface of the HAG/PANI/rGO, the Ret

M

increased to 152 Ω (curve c). The reason is that the Ab1 formed an block layer on the surface of modified electrode, which further blocked the electron transfer of [Fe(CN)6]3−/4−. When BSA

te

d

was used to block non-specific sites of electrode surface, the Ret increased up to 190 Ω (curve d). Furthermore,  when the immunosensor was recognized CEA or AFP, the Ret increased to

Ac ce p

about 280 Ω (curve e), the clearly increased in Ret is ascribed to the insulating layers formation of immunocomplex and block the electron transfer of [Fe(CN)6]3−/4−. When it was incubated with the mixture of Ab2,2-AuNPs-Thi@rGO and Ab2,1-AuNPs-PB@rGO solution, the Ret increased up to 318Ω (curve f).

Fig. 4.

3.4. Optimization of experimental conditions In order to obtain good performance of immunosensor, some experiment parameters were investigated (such as pH, incubation time). Fig. 5A and C showed the pH value can affect 12   

Page 12 of 35

electrochemical behavior of the redox mediator Thi and PB. It could be observed clearly that  the peak current of PB or Thi was increased with the pH value increased from 4.0 to 6.5, and then decreased. When the pH is 6.5, the peak current of PB and Thi reached the maximum

ip t

value. Herein, So, PBS of pH 6.5 was chosen as the detection solution for further experiments

cr

in this study.

us

Fig. 5B and D showed the response of the immunosensor changed with the incubation times range from 10-80 min. It could be clearly observed that the  reduction current of PB and

an

Thi increased with increasing incubation time and trended to reach a plateau after 50 min, exhibiting a saturated binding between the antigen and the primary antibody on electrode

M

surface. Therefore, subsequent experiments employed 50 min as the optimum time for all the 

Fig. 5.

Ac ce p

te

d

incubation steps of the assay.

3.5. Evaluation of cross-reactivity An excellent immunosensor for simultaneous detection of two tumor markers must exclude cross-reactivity. The cross-reactivity between the analytes and bioconjugates antibodies was investigated by carrying out the two control tests as follows:  (i) the immunosensor incubated

with

blank

solution,

which

was

analyzed

using

the

mixture

of

Ab2,1-AuNPs-PB@rGO and Ab2,2-AuNPs-Thi@rGO bioconjugates as signal tags and (ii) single analyte, 20.0 ng mL−1 CEA or AFP, which was analyzed using the mixture of Ab2,1-AuNPs-PB@rGO and Ab2,2-AuNPs-Thi@rGO bioconjugates as signal tags. The results 13   

Page 13 of 35

were shown in Fig. 6B and C, the electrochemical performance of immunosensor was obviously larger in the presence of CEA or AFP than that in the absence of CEA and AFP,

ip t

suggesting that the cross-reactivity could be ignored.

us

cr

Fig. 6.

3.6. Analytical performance

an

In this study, DPV was employed to obtain the electrochemical response of the immunosensor. Fig. 7A showed the DPVs response of the immunosensor in different

M

concentrations of CEA and AFP. The DPV signal was obviously enhanced with the concentrations of CEA and AFP increased. Under the optimum conditions, the peak currents of

d

PB and Thi had a good linear relationship with the concentration of CEA and AFP from 0.6-80

te

ng mL−1. For CEA, the linear regression equation were I = 1.0655+0.0188C (unit of C is ng

Ac ce p

mL−1, unit of I is μA) with a regression coefficients of 0.9908 (seen Fig. 7B). For AFP, the linear regression equation were I =1.3292+0.0273C (unit of C is ng mL−1, unit of I is μA) with a regression coefficients of 0.9936 (seen Fig. 7C). The detection limit of CEA and AFP were 0.12 ng mL−1 and 0.08 ng mL−1 (at 3σ), respectively. Compared with other CEA or AFP immunosensors, this immunosensor displayed a better performance than some earlier reported [32-36] (seen in Table 1.). The proposed immunosensor exhibited a good electrochemical performance, ascribing to so large amount of PB/Thi and AuNPs can load on the surface of rGO. The signal tages could effectively amplify the electrochemical signal of immunosensor, so the immunosensor possessed higher sensitivity than previously works. 14   

Page 14 of 35

Fig. 7.

ip t

Table 1.

cr

3.7. Reproducibility, specificity and stability of the immunosensor

us

The reproducibility of the immunosensor was evaluated by using identical immunosensor detecting five times per run in 5 h at four different concentrations of CEA and AFP (0.6, 6, 20,

an

and 60 ng mL−1). The coefficient of variations was 5.8%, 6.2%, 8.2%, and 7.8% at 0.6, 6, 20, and 60 ng mL−1 of CEA and AFP, respectively. Similarly, the inter-assay precision was

M

investigated by measuring four different concentrations of CEA and AFP (0.6, 6, 20 and 60 ng mL−1) using five immunosensors. The coefficient of variations was 7.2%, 9.8%, 7.8% and 8.6%,

te

d

respectively, suggesting the immunosensor possessed acceptable precision and reproducibility. As electrochemical immunosensors, the selectivity is obviously a crucial factor to be

Ac ce p

considered. Some proteins such as human chorionic gonadotropin (HCG), bovine serum albumin (BSA), and prostate specific antigen (PSA) were used as the possible interferences to evaluate the specificity of the proposed immunosensor. The electrochemical signals was obtained by immunosensors to detect the electrochemical response of 10 ng mL-1 CEA and 10 ng mL-1AFP in absence and presence of 100 ng mL-1 interferential substance. The results were shown with histograms in Fig. S3.  The response signal changed a little in contrast to that of CEA or AFP alone, which indicated that BSA, HCG and PSA could not interfere with the detection of CEA and AFP. Indicating that the immunosensor possesses good specificity for CEA and AFP (Seen in in Fig. S3). 15   

Page 15 of 35

The stability of the immunosensor was examined by testing the response value every five day. The response value of the immunosensor at 5, 10, 15, 20, 25 and 30 days could maintain 97.6%, 95.2%, 93.8 %, 90.7%, 89.3% and 88.6% of its initial response. The electrochemical

us

cr

the composites, indicating the immunosensor had good stability.

ip t

performance decrease was related to the gradual deactivation of the incorporated antibody in

3.8. Application of the immunosensor in human serum

an

In order to examine the applicability of the immunosensor for practical analyses, the recovery experiments were performed by standard addition methods. The standard samples of

M

CEA and AFP were dissolved in the healthy human serum and detected the CEA and AFP in serum. As shown in the following steps: the antigen concentrations of CEA and AFP were 2.5

te

d

ng mL-1, and 20.0 μL of serum sample was injected into 2.0 mL PBS (pH 7.4) to dilute the serum. Then, several freeze-drying standard samples of CEA or AFP were dissolved in the

Ac ce p

as-diluted human serum samples (the concentrations range of the CEA and AFP are within 0.6 to 80 ng mL-1). CEA or AFP was detected with the method described in section 3.6. The recovery is within 95.9%-105.6% and 95.6%-105.4%, respectively, indicating the method is suitable for serum analysis. (Seen in Table S4) The blood samples were from the venous blood and without anticoagulant during preparation, then, it was placed in dry test tube to stand for one hour. Next, blood samples were centrifuged (2000 rpm×5 min) and took the upper solution. Finally, the samples were detected and these results are shown in Table 2. It can be seen that the value obtained is in agreement with that of the ELISA, indicating the immunosensor can be applied to serum analysis. 16   

Page 16 of 35

Table 2.

ip t

4. Conclusions In summary,  this study described a facile and highly sensitive electrochemical

cr

immunosensor for the simultaneous detection of CEA and AFP. HAG decorated PANI/rGO

us

was used as the sensing platform and  AuNPs-PB@rGO and AuNPs-Thi@rGO bioconjugates were used as signal tags, respectively. Well-defined HAG provided a large effective surface for

an

assembly of the primary antibody and enormously promoted the electron transfer on the

M

electrode surface.  Moreover, the prepared AuNPs-PB@rGO and AuNPs-Thi@rGO  nanocomposites possessed favorable bioactivity to accelerate the electron transfer and

d

enhanced the immobilization amount of Ab2.  Experimental results indicated that the

te

immunosensor showed good precision, high sensitivity, acceptable stability and reproducibility.

Ac ce p

Thus, the convenient operation and sensitivity of this method provides a promising potential in clinical diagnosis for the accurate quantitative analysis of biological samples.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 20675002), the Young Foundation of Wannan Medical College (No.WK201210) and the Natural Science Foundation of Anhui province of China (No.1208085QE102).

[1] Y. Zhang, W.J. Dai, F. Liu, L. Li, M. Li, S.G. Ge, M. Yan, J.H. Yu, Ultrasensitive immunosensor based on dual signal amplication electrochemiluminescent of gold 17   

Page 17 of 35

nanoparticles-dotted graphene composites and CdTe quantum strategy dots coated

silica

nanoparticles, Analytical and Bioanalytical Chemistry 405 (2013) 4921-4929. [2] T.A. Stamey, N. Yang, A.R. Hay, J.E. McNeal, F.S. Freiha, E. Redwine,

Prostate-antigen

ip t

as a serum marker for adenocarcinoma of the prostate, New England Journal of Medicine 317

cr

(1987) 909-916.

us

[3] J.P. Li, Q. Xu, X.P. Wei, Z.B. Hao, Electrogenerated chemiluminescence immunosensor for bacillus thuringiensis cry1Ac based on Fe3O4@Au nanoparticles, Journal of Agricultural and

an

Food Chemistry 61 (2013) 1435-1440.

[4] T. Kreisig, R. Hoffmann, T. Zuchner, Homogeneous fluorescence-based immunoassay

M

detects antigens within 90 seconds, Analytical Chemistry 83 (2011) 4281-4287. [5] Y. Wang, J. Dostalek, W. Knoll, Magnetic nanoparticle-enhanced biosensor based on

te

d

grating-coupled surface plasmon resonance, Analytical Chemistry 83 (2011) 6202-6207. [6] Y. Yang, Y.C. Zhu, Q. Chen, Y.Y. Liu, Y. Zeng, F.F. Xu, Carbon-nanotube-activated Pt

Ac ce p

quartz-crystal microbalance for the immunoassay of human IgG, Small 5 (2009) 351-355. [7] D.P. Tang, R. Yuan, Y.Q. Chai, Quartz crystal microbalance immunoassay for carcinoma antigen 125 based on gold nanowire-functionalized biomimetic interface, Analyst 133 (2008) 933-938.

[8]  T. Li, B. Shu, B. Jiang, L. Ding, H.Z. Qi, M.H. Yang, F.L. Qu, Ultrasensitive multiplexed protein biomarker detection based on electrochemical tag incorporated polystyrene spheres as label, Sensors and Actuators B: Chemical 186 (2013) 768-773. [9] H. Wu, G.D. Liu, J. Wang, Y.H. Lin, Quantum-dots based on electrochemical immunoassay of interleukin-1α, Electrochemistry Communications 9 (2007) 1573-1577. 18   

Page 18 of 35

[10]  G.S. Lai, F. Yan, J. Wu, C. Leng, H.X. Ju, Ultrasensitive multiplexed immunoassay with electrochemical stripping analysis of silver nanoparticles catalytically deposited gold nanoparticles and enzymatic reaction, Analytical Chemistry 83 (2011) 2762-2732.

ip t

[11] J. Wang, X. Mi, H. Guan, X. Wang, Y. Wu,  Assembly of folate-polyoxometalate hybrid

cr

spheres for colorimetric immunoassay like oxidase, Chemical Communications 47 (2011)

us

2940-2942.

[12] Z.G. Yang, Y. Chevolot, Y. A. Onal, G.C. Kastylevsky, E. Laurenceau,  Cancer biomarkers

an

detection using 3D microstructured protein chip: implementation of customized multiplex immunoassay, Sensors and Actuators B: Chemical 175 (2012) 22-28.

M

[13] L.N. Feng, Z.P. Bian, J. Peng, F. Jiang, G.H. Yang, Y.D. Zhu, D. Yang, L.P. Jiang, J.J. Zhu, Ultrasensitive multianalyte electrochemical immunoassay based on metal ion functionalized

te

d

titanium phosphate nanospheres, Analytical Chemistry 84 (2012) 7810-7815. [14] R. Akter, M.A. Rahman, C.K. Rhee, Amplified electrochemical detection of a cancer

Ac ce p

biomarker by enhanced precipitation using horseradish peroxidase attached on carbon nanotubes, Analytical Chemistry 84 (2012) 6407-6415. [15] X. Yu, B. Munge, V. Patel, J. Jensen, A. Bhirde, J.D. Gong, S.N. Kim, J. Gillespie, J.S. Gutkind, F. Papadimitrakopoulos, J.F. Rusling, Carbon nanotube amplification strategies for highly sensitive immunodetection of cancer biomarkers, Journal of the American Chemical Society 128 (2006) 11199-11205. [16] Q.N. Xu, F. Yan, J.P. Lei, C. Leng, H.X. Ju, Disposable electrochemical immunosensor by using carbon sphere/gold nanoparticle composites as labels for signal amplification, ChemistryA European Journal l18 (2012) 4994-4998. 19   

Page 19 of 35

[17] Y. Liu, Q. Cheng, Detection of membrane-binding proteins by surface plasmon resonance with an all-aqueous amplification scheme, Analytical Chemistry 84 (2012) 3179-3186. [18] A. De La Escosura-Muniz, C. Parolo, F. Maran, A. Mekoci, Size-dependent direct

ip t

electrochemical detection of gold nanoparticles: application in magneto immunoassays,

cr

Nanoscale 3 (2011) 3350-3356.

us

[19]  J. Tang, D.P. Tang, R.H. Niessner, G.N. Chen, D.M. Knopp, Magneto-controlled graphene immunosensing platform for simultaneous multiplexed electrochemical immunoassay using

an

distinguishable signal tags, Analytical Chemistry 83 (2011) 5407-5414.

[20] Q. Zhu, Y.Q. Chai, R. Yuan, Y. Zhuo, J. Han, Y. Li, N. Liao, Amperometric immunosensor

M

for simultaneous detection of three analytes in one interface using dual functionalized graphene sheets integrated with redox-probes as tracer matrixes, Biosensors and Bioelectronics 43 (2013)

te

d

440-445.

[21] S.X. Wu, Q.Y. He, C.L. Tan, Y.D. Wang, H. Zhang, Graphene-based electrochemical

Ac ce p

sensors, Small 9 ( 2013) 1160-1172.

[22] F.Y. Kong, B.Y. Xu, Y. Du, J.J. Xu, H.Y. Chen, A branched electrode based electrochemical platform: towards new label-free and reagentless simultaneous detection of two biomarkers, Chemical Communications 49 (2013) 1052-1054. [23] Enustum B, Turkevich J, Coagulation of colloidal gold, Journal of the American Chemical Society (1963) 85:3317-3328. [24] F.Y. Kong, M.T. Xu, J.J. Xu, H.Y. Chen, A novel lable-free electrochemical immunosensor for carcinoembryonic antigen based on gold nanoparticles-thionine-reduced graphene oxide nanocomposite film modified glassy carbon electrode, Talanta 85 (2011) 2620-2625. 20   

Page 20 of 35

[25] F.Y. Kong, B.Y. Xu, Y. Du, J.J. Xu, H.Y. Chen, A branched electrode based electrochemical platform: towards new label-free and reagentless simultaneous detection of two biomarkers, Chemical Communications 49 (2013) 1052-1054.

ip t

[26]  L. Shi, Z.Y. Chu, Y. Liu, W.Q. Jin, X.J. Chen, Facile synthesis of hierarchically aloe-like

cr

gold micro/nanostructures for ultrasensitive DNA recognition, Biosensors and Bioelectronics,

us

49(2013)184-191.

[27] W. Lv, M. Guo, M.H. Liang, F.M. Jin, L. Cui, L.J. Zhi, Q.H. Yang, Graphene-DNA hybrids:

an

self-assembly and electrochemical detection performance, Journal of Materials Chemistry 20 (2010) 6668-6673.

M

[28] F.N. Xia, D.B. Farmer, Y.M. Lin, P. Avouris, Graphene field-effect transistors with high on/off current ratio and large transport band gap at room temperature, Nano Letters 10 (2010)

te

d

715-718.

[29] A.K. Geim, K.S. Novoselov, The rise of graphene, Nature materials 6 (2007) 183-191.

Ac ce p

[30]  H. Liu, J. Gao, M.Q. Xue, N. Zhu, M.N. Zhang, T.B. Cao, Processing of graphene for electrochemical application: noncovalently functionalize graphene sheets with water-soluble electroactive methylene green, Langmuir 25 (2009)12006-12010. [31] D.W. Wang, F. Li, J. Zhao, W. Ren, Z.G. Chen, J. Tan, Z.S Wu, I. Gentle, G.Q. Lu, H.M. Cheng,

Fabrication

of

graphene/polyaniline

composite

paper

via

in

situ

anodic

electropolymerization for high-performance flexible electrode, ACS Nano 3 (2009) 1745-1752. [32] L. Ge, J.X. Yan, X.R. Song, M. Yan, S.G. Ge, J.H. Yu, Three-dimensional paper-based electrochemiluminescence immunodevice for multiplexed measurement of biomarkers and point-of-care, Biomaterials 33 (2012) 1024-1031. 21   

Page 21 of 35

[33] J.H. Lin, Z.J. Wei, C.M. Mao, A label-free immunosensor based on modified mesoporous silica for determination of simultaneous tumor markers, Biosensors and Bioelectronics 29 (2011) 40-45.

ip t

[34]  X. Chen, X.L. Jia, J.M. Han, J. Ma, Z. F. Ma, Electrochemical immunosensor for

cr

simultaneous detection of multiplex cancer biomarkers based on graphene nanocomposites,

us

Biosensors and Bioelectronics 50 (2013) 356-361.

[35] Z.J. Yang, H. Liu, C. Zong, F. Yan, H.X. Ju, Automated support-resolution strategy for a

an

one-way chemiluminescent multiplex immunoassay, Analytical Chemistry 81 (2009) 5484-5489.

M

[36] Z.J. Song, R. Yuan, Y.Q. Chai, Y. Zhuo, W. Jiang, H.L. Su, X. Che, J.J. Li, Horseradish peroxidase-functionalized Pt hollow nanospheres and multiple redox probes as trace labels for a

Ac ce p

46 (2010) 6750-6752.

te

d

sensitive simultaneous multianalyte electrochemical immunoassay, Chemical Communications

22   

Page 22 of 35

Figure captions: Table 1. The analytical performance of this immunosensor compared with others reported works.

ip t

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

cr

Scheme 1. (A) displayed the preparation process of immunosensing probes. (B) Fabrication

us

process of the electrochemical CEA and AFP immunosensors.

Fig. 1. SEM images of rGO (A), PB@rGO nanocomposites (B), Thi@rGO nanocomposites (C),

an

AuNPs-PB@rGO nanocomposites (D) and AuNPs-Thi@rGO nanocomposites (E).

nanocomposites (C).

M

Fig. 2. UV vis absorption spectra of rGO (A), PB@rGO nanocomposites (B) and Thi@rGO

Fig. 3. The SEM images of rGO (A), PANI/rGO (B) and HAG/PANI/rGO (C). 4.

EIS

of

(a)

(f)

(b)

HAG/PANI/rGO,

(c)

Ag/BSA/Ab1/HAG/PANI/rGO,

Ab1/HAG/PANI/rGO (e)

(d)

Ab2/AuNPs-Thi(PB)

te

BSA/Ab1/HAG/PANI/rGO,

bare,

d

Fig.

Ac ce p

@rGO/Ag/BSA/Ab1/HAG/PANI/rGO modified GCE in 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] containing 0.1 M KCl. CCAE=CAFP = 20 ng mL−1. Inset: equivalent electrical circuit diagrams for the impedance plots.

Fig. 5. Effect of the pH (A) and (C), the incubation time of (B) and (D) on the response of the immunosensor to CCAE=CAFP = 20 ng mL−1. Fig. 6. DPV responses of the CEA and AFP immunosensors for blank control (A), 20 ng mL−1 AFP (B) and 20 ng mL−1 CEA (C) in 0.1 M PBS, pH 6.5. Fig. 7. Quantitative analysis performance of the immunosensor. (A) Determination of solutions containing different concentrations of two antigens. From (a) to (f): 0.6, 2, 38, 50, 60, 80 ng mL−1. Calibration curves for simultaneous determination of CEA (B) and AFP (C) using the 23   

Page 23 of 35

proposed immunosensor. The error bars represent the standard deviations of three parallel tests.

Biographies: Dexiang Feng received her MS degree in  the College of Chemistry and Materials Science of

ip t

Anhui Normal University,  P. R. China in 2005. She started his Ph.D. thesis in the College of

cr

Chemistry and Materials Science of Anhui Normal University from 2011. Her research interests

us

include chemical sensors and nanomaterials.

Lihua Li received her MS degree in Nanjing University of Chinese Medicine, P. R. China in

an

2006. Presently, she started her Ph.D. thesis in the College of Chemistry and Materials Science of Anhui Normal University from 2012. Her research interests include biomedical sensors,

M

optical sensors and biomaterials.

Xiaowei Han is a master student in College of Chemistry and Materials Science from Anhui

electrodes.

te

d

Normal University. Her research interests are electrochemical sensors and chemically modified

Ac ce p

Xian Fang is a master student in College of Chemistry and Materials Science from Anhui Normal University. Her research interests are DNA biosensors. Xiangzi Li received his M.S. in 2007 and obtained his Ph.D. in 2011 from in the College of Chemistry and Materials Science of Anhui Normal University Sciences. He then joined Wannan Medical College, as an associate professor working on the synthesis and performance of advanced functional materials. Yuzhong Zhang is a professor in the College of Chemistry and Materials Science of Anhui Normal University Sciences, P.R. China. He received his Ph.D. degree in 2002 from University of Chinese Academy of Science.  His research interests include  bioelectrochemistry, 24   

Page 24 of 35

Ac ce p

te

d

M

an

us

cr

ip t

electrochemical sensors, nanomaterials and optical sensors.

25   

Page 25 of 35

Immunosensor fabrication

probes

Liner range (ng mL-1)

DL (ng mL-1)

CEA

CEA AFP

AFP

Ref.

CS/GCE

[Ru(bpy)3]2+

1.0-100, 0.5-100

0.5

0.15

APTS-SBA-15/ITO

MPS-Fc/HRP

0.5-45, 1.0-90

0.2

0.5

CGS-PB/CGS-TB

0.5-60, 0.5-60

0.1

0.05 0.89

[35]

0.6

[36]

HRP

1.0-60, 1.0-80

0.6

CSSH

Fc/Thi

1.5-20, 1.8-2.5

0.5,

HAG/PANI/GS

AuNPs-PB/Thi@GS

0. 6-80, 0. 6-80

0. 12

0.08

ip t This work

Ac ce p

te

d

M

an

 

[34]

us

CL

[33]

cr

Chit-AuNPs/GCE

[32]

26   

Page 26 of 35

Sample no.

Multiplexed immunoassaya (ng mL-1) CEA

AFP

CEA

AFP

Relative deviation (%) CEA

AFP

0.8±0.12

0.8±0.21

0.8

0.8

+2.5

-2.8

2

6.2±0.28

6.1±0.32

6.0

6.0

+3.0

+1.7

3

18.8±0.39

19.1±0.45

20.0

20.0

-6.0

-4.7

4

57.3±0.68

59.6±0.53

60.0

60.0

5

78.8±0.76

81.2±0.66

80.0

80.0

cr -4.0

+2.0

-3.6

+1.5

us

Mean value ± SD of five measurements serum sample

ip t

1

an

a

ELISA (ng mL-1)

Ac ce p

te

d

M

Table 2.

27   

Page 27 of 35

Ac

ce

pt

ed

M

an

us

cr

i

Figure(s)

Page 28 of 35

Ac

ce pt

ed

M

an

us

cr

ip t

Figure(s)

Fig. 1.

Page 29 of 35

Ac

ce pt

ed

M

an

us

cr

ip t

Figure(s)

Fig. 2.

Page 30 of 35

Ac

ce pt

ed

M

an

us

cr

ip t

Figure(s)

Fig. 3.

Page 31 of 35

an

us

cr

ip t

Figure(s)

Ac

ce pt

ed

M

Fig. 4.

Page 32 of 35

ed

M

an

us

cr

ip t

Figure(s)

Ac

ce pt

Fig. 5.

Page 33 of 35

M

an

us

cr

ip t

Figure(s)

Ac

ce pt

ed

Fig. 6.

Page 34 of 35

Ac

ce pt

ed

M

an

us

cr

ip t

Figure(s)

Fig. 7.

Page 35 of 35