Disposable electrochemical immu nosensor based on ...

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r-GO/Thil AuNPs nanocomposites for detection ofNSE. 1. 1 2. ·. 1 2. Y F 1 2 J. L· 1 d X· . C . 1 2 *. Jinping Luo., Yang Wang .. , HUlren Xu.·, an an.·, untao IU an.
Disposable electrochemical immu nosensor based on r-GO/ThilAuNPs nanocomposites for detection ofNSE . . 1 2* 1 · 1 2 1 2 Juntao L·IU 1 and X·mXIa C at· Jinping Luo., Yang Wang ..1 2 , HUlren Xu.· , Yan Fan.·, . 1 2 University of Chinese Academy of Sciences, State Key Laboratory of Transducer Technology, Beijing, China Institute of Electronics, Chinese Academy of Science, *Corresponding author, [email protected] Beijing, China. Abstract-In immunosensor

this

work,

based

on

a

disposable

screen-printed

electrochemical electrode

with

modification of r-GO/Thi/AuNPs nanocomposites was developed onto the chromatography paper for detection of NSE. The electrochemical

immunosensor

consists

of

two

sheets

of

chromatography paper and one double-side tape. The carbon working electrode (WE) was screen-printed onto the hydrophilic channels separated with wax-printed hydrophobic areas on one sheet, and the counter electrode (CE) and the AglAgCI reference electrode (RE) were on the other sheet. The synthesized r­ GO/Thi/AuNPs

nanocomposites

and

the

anti-NSE

were

successively coated on the carbon working electrode so as to provide an electrochemical interface sensitive to NSE. Finally, the above three electrodes were integrated using the double-side tape. Thus, a capillary-driven flow is generated through the porous medium to connect the three electrodes in the same solution cell on account of the hydrophilic fiber surface. And the modification of the r-GO/Thi/AuNPs nanocomposites on the immunosensor, in which Thi functioned as electrically active substances to generate an electric current and r-GO can accelerate the transfer of electrons to amplify the signal, make it possible to detect NSE through

the

lable-free

immunoreactions.

When

the

immunosensor was incubated with the target NSE antigen, the peak currents of DPVs were decreased because the formation of antibody-antigen immunocomplex onto the working electrode resulted in the enhanced steric hindrance blocking the electron transfer

of

Thi.

Additionally,

the

peak

currents

of

DPVs

decreased with the NSE concentrations increasing. There is a good linear relationship between the peak currents and the NSE concentration in the ranges of 0.01-100 ng/mL and the limit of detection was 10 pg/mL (S/N=3). The results indicated that the immunosensors enabled relatively wide linear range and a low detection limit for NSE. Keywords-Label-free

electrochemical

immunosensor;



GOIThilAuNPs nanocomposites; NSE

1.

INTRODUCTION

According to the latest cancer statistics from the World Health Organization, lung cancer has become the highest cause of cancer morbidity and mortality worldwide with 1.8 million new cases and 1.6 million deaths in 2012 [1]. Based on the appearance of the cancer cells under the microscope and the behavior of the disease, lung cancer is categorized into two basic disease types: small cell lung cancer (SCLC) and non­ small cell lung cancer (NSCLC). Compared to NSCLC, the cancer cells in SCLC are small but grow very quickly into

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large tumors and often spread rapidly to other parts of the body, resulting in not very effective therapies for SCLC especially for patients at advanced stages. Detecting lung cancer at an early and curable stage can improve survival substantially. Tumor marker is a substance secreted or shed into body fluids by cancer cells and could be an indicator to reflect the presence of cancer. Compared to the conventional clinical trials such as chest radiograph and computed tomography, the detection of tumor marker is an effective means in the early diagnosis of lung cancer and easily accepted by patients due to the low cost and the noninvasive way. Neuron-specific enolase (NSE), a phosphopyruvate hydratase presents primarily in the cytoplasm of neurons and neuroendocrine cells, becomes a sensitive, specific, and reliable tumor marker for early diagnostic and prognostic values for monitoring the SCLC state. There are several immunoassay methods applied to detect NSE, such as radioimmunoassay [2], enzyme-linked immunosorbent assay [3], chemiluminescence immunoassay [4] and electrochemical immunoassay [5, 6]. In comparison with other immunoassay methods, the electrochemical immunoassay has gained increasing attentions due to its high sensitivity, good selectivity, easy to integrate with small apparatus and high compatibility with advanced micromachining technologies. Furthermore, with the further development of the microfluidic chip technology, there has been an emerging trend of using paper as a platform for point-of-care testing and then establishing electrochemical immunoassay method on paper-based microfluidic analytical device to detect tumor markers [7-9]. However, the current studies mainly focused on sandwich immune technique, inevitably need more steps solution processes and thus may bring inconvenience to clinical application. Therefore, it is a significant to develop a simple, sensitive, and inexpensive method to detect levels of the biomarkers. Up to date, a series of label-free amperometric immunosensors have been put forward to detect other tumor markers by immobilizing mediator molecules and nanomaterials on glassy carbon electrode or ITO electrode [10 12]. These reports have opened new opportunities for constructing novel amperometric immunosensors on the paper, which reduces the cost and simplifies the assay system. Herein, this work depicts a disposable electrochemical immunosensor based on screen-printed electrode on a wax-printed cellulose filter paper with modification of r-GOIThilAuNPs

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nanocomposites for detection of NSE. Due to the synergistic effect of r-GO and Thi, Thi molecules still keep the property of electroactive redox and grephene could accelerate electron transfer for the signal amplification. Meanwhile, the specific surface area of AuNPs offer active sites for the immobilization of anti-NSE. When the target NSE antigen is absorbed to the immunosensor, the formation of antibody-antigen immunocomplex resulted in the decrease of current response due to the blocking of the electron transfer process, which was directly proportional to the concentrations of corresponding antigens. This novel immunosensor would provide a new platform for low cost, sensitive, and point-of-care diagnosis to public health. II. A.

C. Fabrication ojthe electrochemical immunosensor

The electrochemical immunosensor consists of two sheets of chromatography paper and one double-side tape (shown in Fig.lA).

EXPERIMENTAL

CE

Materials and apparatus

Standard NSE antigen and anti-NSE were purchased from Shanghai linc-bio company (Shanghai, China). Amino­ functional reduced graphene (r-GO) was purchased (purity >95 wt %) from Xianfeng nanomaterials company (Nanjing, China). Thionine acetate (Thi) was obtained from Alfa Aesar. Gold chloride, sodium citrate and bovine serum albumin (BSA) were purchased from Beijing Chemical Reagents Company (Beijing, China). The phosphate-buffered saline (PBS, 0.1 M NaRR2RRHPORR4RR-NaHRR2RRP04-KCl, pH 7.4) was prepared by dissovling a PBS tablet (Sigma, Stlouis, MO, USA) into 200 mL of ultrapure water. Carbon ink (ED 581ss) was purchased from Acheson and AglAgCl ink (CNC-Ol) for screen-printing electrodes were obtained from Henkel Acheson (Shenzhen, China). Whatman chromatography paper NO. 1 (200.0 mm x 200.0 mm, pure cellulose paper) was purchased from GE Healthcare Worldwide (Shanghai, China) and used with further adjustment of size. All other chemicals were of analytical reagent grade was used throughout the experiment. The wax-printing process was accomplished using a wax printer (Xerox Phaser 8570N color printer). The scanning electron microscope (SEM) image of the working electrode surfaces were performed on a scanning electron microscopy (S3500, Hitachi, Tokyo, Japan). All electrochemical experiments including cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were carried out on a Gamry Reference 600 electrochemical workstation (Gamry Instruments, Warminster, PA, USA) and three-electrode system. All experiments were carried out at ambient temperature. B.

mg/mL) were mixed with equal volume and stirred vigorously for 24h. The rGO/Thi nanocomposites were obtained after removing the non-integrated Thi away through centrifugation and further dispersed in ImL water. Subsequently, 5 mL of the as-prepared AuNPs solution was added into this dispersion and react for overnight under stirring. After several washing and centrifugation, the collected samples were re-dispersed in 2mL of CHIT solution (O.l%, w/v) and stored at 4°C for further use.

Synthesis ojr-GOIThiiAuNPs nanocomposites

AuNPs solutions were first prepared by reduction of chloroauric acid (HAuCIRR4RR) with sodium citrate. Briefly, 2.5mL of sodium citrate (0.0 I%, w/v) was rapidly added into 50mL of boiled HAuCIRR4RR solution (0.01%, w/v) under continuous stirring with a reflux device, and the mixture was continued to boil for another 15 min. The resulting wine red solution was cooled to room temperature and stored at 4 °C in refrigerator until needed. Then, the r-GO/ThilAuNPs nanocomposites were prepared according to the reference [13] with little modification. The Thi solution (2 mg/mL) and the ultrasonic treated r-GO solution (1

2016 17th International Conference on Electronic Packaging Technology

,

(c) : WE

(A) I

,

-------------

,

, I

....

_

-----------

-

"

(B) Fig. 1. (A) Construction of the electrochemical immunosensor integrated with the three screen-printed electrodes on the chromatography paper (a, c) using the double-side tape (b). (B) Schematic diagram of the working electrode modified with r-GO/Thi/AuNPs nanocomposites and anti-NSE.

After printed on the paper sheet using the wax printer according to the designed patterns and hot at 150 oC for 120 s, the hydrophobic barriers were formed to separate two hydrophilic circular zones (one is 6 mm and the other is 6 mm in diameter) on the two sheets of chromatography paper. Then, the carbon working electrode (WE) was screen-printed onto the hydrophilic channels separated with wax-printed hydrophobic areas on one sheet, and the counter electrode (CE) and the AglAgCI reference electrode (RE) were on the other sheet. Subsequently, 0.01 mL of the synthesized r-GO/Thi/AuNPs nanocomposites and the anti-NSE (0.2 mg/mL) were in successively coated onto the carbon working electrode and dried at room temperature so as to provide an electrochemical interface sensitive to NSE (seen in Fig.1B). After the block step of the carbon working electrode by addition of BSA, the working electrode, counter electrode and the AglAgCl reference electrode were finally integrated to fabricate the electrochemical immunosensor by conglutination of the above two sheets using the double-side tape. Thus, a capillary-driven

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flow is generated through the porous medium to connect the three electrodes in the same solution cell on account of the hydrophilic fiber surface. And the modification of the r­ GO/Thi/AuNPs nanocomposites on the immunosensor, in which Thi functioned as electrically active substances to generate an electric current and r-GO can accelerate the transfer of electrons to amplifY the signal, make it possible to detect NSE through the lable-free immunoreactions. D.

Electrochemical measurements

To implement the immunoreaction and electrochemical measurement, O.OlmL of detected NSE solutions was dropped onto the as-prepared electrochemical immunosensor. After incubation at room temperature for 10 min, the electrochemical immunosensor was linked with the Gamry electrochemical workstation to record the current signal. CV experiments and DPY experiments were all scanned in O.lM PBS solutions (pH 7.4). The CY scan was conducted with the scan rate of 100mY/s. The DPY scan were taken from -0.5 to 0.3 Y with the modulation amplitude of 0.05 V, the modulation time of 0.025 s, the interval time of 0.5 s and the scan rate of lOmY/s. TIT.

A.

RESULTS AND DISCUSSION

Characterization of the WE surface

The morphology of the WE surface during different stages of modification was examined by the SEM characterization (Fig. 2.).

these elongated strip-shaped nanocomposite are successfully modified in the WE surface to completely cover the screen­ printed carbon particles, resulting the increases of the WE surface area. When the anti-NSE are further immobilized on the surface of the r-GO/ThilAuNPs nanocomposites modified WE, the channel characteristics in the work area is still good to provide a good place for specifical antigen-antibody reaction. Since Thi can be used as electrically active substances to generate an electric current and r-GO can accelerate the transfer of electrons to amplify the signal, the modification of r-GO/Thi/AuNPs nanocomposites and the anti-NSE on the WE surface make it possible to detect NSE through the lable-free immunoreactions. B.

Analytical Performance of the lmmunosensor

To investigate the analytical perfonnance of the immunosensor to detect NSE, the immunosensors were incubated with a series of NSE standard solutions for IS min, respectively, and then the response currents of differential pulse voltammetry (DPy) were measured in pH 7.4 phosphate buffer using a Gamry electrochemical workstation. The results are shown in Fig 3. When the immunosensor was incubated with the target NSE antigen, the peak currents of DPYs were decreased because the fonnation of antibody-antigen immunocomplex onto the working electrode resulted in the enhanced steric hindrance blocking the electron transfer of Thi. Additionally, the peak currents of DPYs decreased with the NSE concentrations increasing. There is a good linear relationship between the peak currents and the NSE concentration in the ranges of 0.0 I-I00 ng/mL and the limit of detection was 10 pg/mL (S/N=3), indicating that the immunosensors enabled relatively wide linear range and a low detection limit for NSE. 6

l-b,ank

-10pgmL -20pgmL -50pgmL O.1ngmL -O.2ngmL 1ngmL -20ngmL -100ngmL

o���� -0.5

-04

-0.3

-0.2

-0.1

0.0

0.1

0.2

Potentia' (V)

(A) 5.0

Fig 2. SEM images of the screen-printed carbon WE on the hydrophilic circular zones of the paper (A), r-GOIThi/AuNPs nanocomposites dropped on a glass (B), the screen-printed carbon WE modified with r­ GOIThi/AuNPs nanocomposites (C) and further modified with anti­ NSE (D).

4.5



Y=-O.458X+4.974 R=O.969

'.0

c



:0

3.5

()

3.0

As is shown in Fig. 2A, there are a large number of carbon particles with sizes about one hundred nanometers evenly distributed over the paper surface, without disruption to the hollow structure of the original filter paper. By comparing the two images of the r-GO/ThilAuNPs nanocomposites dropped on a glass (Fig. 2B) and the screen-printed carbon WE modified with r-GO/ThiiAuNPs nanocomposites (Fig. 2C),

2016 17th International Conference on Electronic Packaging Technology

2.5

3

2

log(CNSE, pglmL) (B)

Fig 3. DPY responses of the proposed immunosensor after incubation with different concentrations of NSE (A) and calibration curves of the immunosensor toward NSE (B). Solution: pH 7.4 phosphate buffer.

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IV.

CONCLUSION

The disposable electrochemical immunosensor based on screen-printed electrodes with modification of r­ GOIThilAuNPs nanocomposites was developed onto the chromatography paper for detection of NSE. The carbon WE, the carbon CE and the AglAgCl RE were all screen-printed onto the different hydrophilic channels separated with wax­ printed hydrophobic areas on two sheets of chromatography paper. After the modification of the synthesized r­ GO/Thi/AuNPs nanocomposites and the anti-NSE were in succession coated on the WE so as to provide an electrochemical interface sensitive to NSE, the above three electrodes were integrated using the double-side tape. Thus, a capillary-driven flow is generated through the porous medium to connect the three electrodes in the same solution cell on account of the hydrophilic fiber surface. And the modification of the r-GOIThilAuNPs nanocomposites on the immunosensor, in which Thi functioned as electrically active substances to generate an electric current and r-GO can accelerate the transfer of electrons to amplify the signal, make it possible to detect NSE through the lable-free immunoreactions. Due to the increasing of antibody-antigen immunocomplex, the peak current of DPVs was decreased and the peak current of THT was directly proportional to the concentrations of corresponding antigens. The proposed immunosensor had a wide linear working range for detection of NSE ranging from 0.01 ng/mL to 100 ng/mL with a detection limit of 10 pg/mL (S/N=3). ACKNOWLEDGMENT

This work was sponsored by the NSFC (No. 61471342, No. 61527815, No. 31500800, No. 61501426), National Science and Technology Major Project (2014CB744600), and the Key Programs of the Chinese Academy of Sciences (No. K JZD­ EW-LlI-2). REFERENCES [I]

[2]

Ferrigno D, Buccheri G, Giordano C. Neuron-specific enolase is an eflective tumor marker in non-small cell lung cancer (NSCLC). Lung Cancer, 2003, 41,pp.311-320.

[3]

Fabrice Barlesia, eline Gimeneza,Jean-Philippe Torre, et at. Prognostic value of combination of Cyfra 21-1, CEA and NSE in patients with advanced non-small cell lung cancer. Respiratory Medicine. 2004, 98, pp.357-362

[4]

Fu X, Meng M, Zhang Y, et aI. Chemiluminescence enzyme immunoassay using magnetic nanoparticles for detection of neuron specific enolase in human serum. Analytica Chimica Acta 2012, 722, pp.1l4-118

[5]

Han J, Zhuo Y, Chai Y, et at. Novel electrochemical catalysis as signal amplified strategy for label-freedetection of neuron-specific enolase. Biosensors and Bioelectronics 2012,31,pp. 399-405

[6]

Yu T, Cheng W, Li Q, et at. Electrochemical immunosensor for competitive detection of neuron specific enolase using fimctional carbon nanotubes and gold nanoprobe. Talanta 2012,93,pp. 433-438

[7]

Li L, Li W, Yang H, et al. Sensitive origami dual-analyte electrochemical immunodevice based on polyaniline/Au-paper electrode and multi-labeled 3D graphene sheets. Electrochimica Acta 2014, 120, pp. 102-109

[8]

Ge S, Sun M, Liu W, et at. Disposable electrochemical immunosensor based on peroxidase-likemagnetic silica-graphene oxide composites for detection of cancerantigen 153. Sensors and Actuators B. 2014, 192, pp.317-326

[9]

Wang P, Ge L, Yan M, et al. Paper-based three-dimensional electrochemical immunodevice based on multi-walled carbon nanotubes fimctionalized paper for sensitive point-of-care testing. Biosensors and Bioelectronics. 2012,32 ,pp. 238- 243

[10] Wang R, Chen X, Ma J, et at. Ultrasensitive detection of carcinoembryonic antigen by a simple label-free Immunosensor. Sensors and Actuators B. 2013,176,pp.1044-1050 [II] Jia X,Liu Z,Liu N,et at. A label-free immunosensor based on graphene nanocomposites for simultaneous multiplexed electrochemical determination of tumor markers. Biosensors and Bioelectronics 2014,53, pp. 160-166. [12] Chen X, Ma Z. Multiplexed electrochemical immunoassay of biomarkers using chitosan nanocomposites. Biosensors and Bioelectronics. 2014,55,pp.343-349 [13] Wang Y, Xu H, Luo J, et at. A novel label-free microfluidic paper­ based immunosensor for highly sensitive electrochemical detection of carcinoembryonic antigen. Biosensors and Bioelectronics,2016,83, pp.319-326

World Health Organization (2014) GLOBOCAN2012: Estimated Cancer Incidence,Mortality and Prevalence Worldwide in 2012.

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