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Simultaneous electrochemical immunosensor based on water-soluble polythiophene derivative and functionalized magnetic material Xiaoyue Zhang, Xiang Ren, Wei Cao, Yueyun Li, Bin Du, Qin Wei * Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong (University of Jinan), School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China

H I G H L I G H T S

G R A P H I C A L A B S T R A C T

 A special water-soluble conducting polymer was been first used in the immunosensor.  PDPMT-Cl has good electrical conductivity and solubility.  Functionalized magnetic material makes the simultaneous detection of two analytes possible.

In this study, a novel, sensitive electrochemical immunosensor for simultaneous determination of squamous cell carcinoma associated antigen (SCC-Ag) and carcinoembryonic antigen (CEA) based on poly [3-(1,10 -dimethyl-4-piperidine-methylene) thiophene-2,5-diylchloride] (PDPMT-Cl) and functionalized mesoporous ferroferric oxide nanoparticles (Fe3O4 NPs) for the combined diagnosis of cervical cancer was designed.

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 March 2014 Received in revised form 10 May 2014 Accepted 14 May 2014 Available online xxx

A novel, sensitive electrochemical immunosensor for simultaneous determination of squamous cell carcinoma associated antigen (SCC-Ag) and carcinoembryonic antigen (CEA) for the combined diagnosis of cervical cancer was designed. The amplification strategy for electrochemical immunoassay was based on poly[3-(1,10 -dimethyl-4-piperidine-methylene) thiophene-2,5-diylchloride] (PDPMT-Cl) and functionalized mesoporous ferroferric oxide nanoparticles (Fe3O4 NPs). PDPMT-Cl dispersed in chitosan solution with enhanced electrical conductivity and solubility was used as matrices to immobilize the first antibodies. Different redox probes (thionine (Th) and ferrocenecarboxylic acid (Fca)) functionalized Fe3O4 NPs incubated with two kinds of secondary antibodies to fabricate the labels. Using an electrochemical analysis technique, two well-separated peaks were generated by Th and Fca, making the simultaneous detection of two analytes on the electrode possible. Under optimized conditions, this method showed wide linear ranges of three orders of magnitude with the detection limits of 4 pg mL1 and 5 pg mL1, respectively. The disposable immunosensor possessed excellent clinical value in cervical cancer screening as well as convenient point-of-care diagnostics. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Cervical cancer Simultaneous determination Polythiophene derivative Functionalized Magnetism

1. Introduction The clinical analysis of cancer biomarker in their early development stage plays an important role in screening and determining

* Corresponding author. Tel.: +86 531 82767872; fax: +86 531 82765969. E-mail address: [email protected] (Q. Wei).

cancer [1,2]. However, the measurement of a single cancer biomarker often has limited diagnostic value because no single cancer biomarker is sensitive and specific enough to meet the strict diagnostic criteria in clinical diagnosis [3–7]. Therefore, simultaneous detection of cancer biomarkers becomes more important in clinical application since it can increase assay throughput, reduce overall cost, improve test efficiency and quantitatively measure the concentrations of multiple cancer biomarkers [8–10].

http://dx.doi.org/10.1016/j.aca.2014.05.025 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.

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Up to now, simultaneous determination of different cancer biomarker based on electrochemical immunoassay using screenprinted electrode [11–14] and different electrochemical redox probes [15–17] are attractive and promising. For example, Wilson described an electrochemical immunosensor for the simultaneous detection of carcinoembryonic antigen (CEA) and alpha fetoprotein. The sensors consisted of two iridium oxide electrodes patterned on a glass substrate. Capture antibodies were immobilized on the porous iridium oxide electrodes by covalent attachment using (3-aminopropyl) triethoxysilane and glutaraldehyde. The spatial separation of the electrodes enabled simultaneous electrochemical immunosensor to be conducted without cross-talk between the electrodes [18]. Zhu's group developed a novel multi-analyte electrochemical immunosensor for ultrasensitive detection of human cardiomyopathy biomarkers cardiac troponin I and human heart-type fatty-acid-binding protein by using metal-ions functionalized titanium phosphate nanospheres as labels. The metal-ions could be detected directly through square wave voltammetry without metal preconcentration and the distinct voltammetric peaks had a close relationship with each sandwich-type immunoreaction [19]. Recently, conducting polymers have been appeared as a promising type of material to fabricate electrochemical immunosensors [20,21]. Among these conducting polymers, a special water-soluble conducting polymer denoted as poly[3-(1,10 -dimethyl-4-piperidine-methylene) thiophene-2,5-diylchloride] (PDPMT-Cl) has many unique amazing features, such as the good electrical conductivity, well biocompatibility, and large specific surface area, good chemical stability and thermal stability [22]. Until now, the usage of PDPMT-Cl in electrochemical immunosensors has not been reported. Chitosan (chit), which contains a large amount of amino and good film-forming property [23,24], can be used to disperse PDPMT-Cl to form a uniform and stable film on the surface of the electrode. Using multifunctional nanoparticles as labels is one of the most popular strategies to amplify the electrochemical responses [25– 27]. Up to now, with respect to electron transfer mediators-based electrochemical immunosensors, different kinds of multifunctional nanomaterials entrapped with antibodies have been investigated. For instance, Yuan's group based on thionine (Th)-doped magnetic gold nanospheres as labels and horseradish peroxidase as enhancer to improve the sensitivity and detection limit of the immunosensor for carcinoembryonic antigen (CEA) detection [28]. S. Viswanathan's group used ferrocene carboxylic acid encapsulated liposome multifunctional nanoparticles labeled anti-CEA to increase the sensitivity of the immunosensor [29]. In this study, thionine and ferrocenecarboxylic acid (Fca) functionalized mesoporous ferroferric oxide nanoparticles (Fe3O4 NPs) were introduced in fabrication labels. Th and Fca as the electrochemical redox probes can generated two well-separated peaks, which made the simultaneous determination in an electrode realized. Fe3O4 NPs not only can immobilize more antibodies and electrochemical redox probes, but also can promote the electron transfer; therefore, functionalized mesoporous ferroferric oxide nanoparticles are selected to fabricate the labels. Herein, simultaneous electrochemical immunosensor for cancer biomarkers based on PDPMT-Cl and functionalized Fe3O4 NPs amplification strategy were established. Different capture antibodies were immobilized on the PDPMT-Cl modified glassy carbon electrodes (GCE) to form an immunosensor. The PDPMT-Cl improves the performance of the electrochemical reaction of substrate and increase the sensitivity of the immunosensor to the targets detection. Th and Fca functionalized Fe3O4 NPs was used as labels of secondary antibodies. Due to the electron transport capacity and the large surface area of Fe3O4 NPs [30,31], using an electrochemical analysis technique, two well-separated peaks

were achieved and the distinct voltametric peaks had a close relationship with each sandwich-type immunoreactions. To demonstrate the workability for simultaneous detection of multiple tumor markers, squamous cell carcinoma associated antigen (SCC-Ag) and CEA for the combined diagnosis of cervical cancer were used as targets, the proposed immunosensor exhibited excellent precision and sensitivity. 2. Experimental 2.1. Reagents and apparatus SCC-Ag and CEA corresponding antibodies were purchased from Shanghai Linc-Bio Science Company Limited (China). Bovine serum albumin (BSA, 96–99%) was from Sigma (USA). Chit, glutaraldehyde (GA), Th and Fca were ordered from Sinopharm Chemical Reagent Company Limited (China). Phosphate buffer saline (PBS) with different pH values were prepared by mixing the stock solution of KH2PO4 and Na2HPO4. All other chemicals were of analytical reagents grade and used without further purification. Ultrapure water was used in all run. All electrochemical measurements were performed on a CHI 760D electrochemical workstation (China). Transmission electron microscope (TEM) images were collected with holey carbon TEM grids on a JEM-2100 microscope operated at an accelerating voltage of 200 kV (Japan). Fourier transform infrared spectroscopy (FTIR) spectrum was obtained from VERTEX 70 (Germany). UV/vis measurements were carried out using a Lambda 35 UV/vis Spectrometer (PerkinElmer, United States). A conventional three-electrode system was used for all electrochemical measurements: a glassy carbon electrode (GCE, 4 mm in diameter) as the working electrode, and saturated calomel electrode as the reference electrode, and a platinum wire electrode as the counter electrode. 2.2. Synthesis of poly[3-(1,10 -dimethyl-4-piperidine-methylene) thiophene-2,5-diylchloride] PDPMT-Cl was synthesized according to the previously reported method [32]. Firstly, 3-methylthiophene was used as starting material and then through bromination, Witting–Horner reaction, and methylation. The monomer was obtained. The

Scheme

1. Structure of PDPMT-Cl.

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polymer was synthesized by oxidative polymerization in chloroform using FeCl3 as oxidant. The structure is described as follows (Scheme 1).

3

electrode surface. The surface was washed once more and the prepared immunosensor was stored at 277 K. A schematic diagram of the stepwise procedure of the immunosensor is shown in Scheme 2.

2.3. Preparation of labels 2.5. Measurement procedure Fe3O4 was prepared according to the previously reported method [33]. Then, 10 mg of Fe3O4 NPs and 10 mg of Th was dispersed in 1 mL of 2.5% (v/v) GA solution and then stirred for 6 h, and then it was magnetic separated and washed for three times. The product Th functionalized Fe3O4 NPs (Fe3O4 NPs-Th) was then added into 1 mL of the second SCC-Ag antibody (Ab2) solution (10 mg mL1) and 1 mL 2.5% (v/v) GA and then stirred for 24 h at 277 K. The Fe3O4 NPs-Th labeled Ab2 (Fe3O4 NPs-Th-Ab2) was then obtained after magnetic separated and washed. Fe3O4 NPs-Th-Ab2 was dispersed in 1 mL of PBS. Fca surface has a number of carboxyl groups; therefore, Fca can be directly combined with the Fe3O4 NPs without any cross-linking agent. The obtained Fe3O4 NPs-Fca was then added into 1 mL of the second CEA antibody (Ab20 ) solution (10 mg mL1) and 1 mL 2.5% (v/v) GA, and then stirred for 24 h at 277 K. The Fe3O4 NPs-Fca labeled Ab20 (Fe3O4 NPs-Fca-Ab20 ) was then obtained after magnetic separated and washed. Fe3O4 NPs-Fca-Ab20 was dispersed in 1 mL of PBS. 2.4. Fabrication of the immunosensor Before modification, the GCE was first polished to a mirror-like surface using 1, 0.3, and 0.05 mm alumina powder sequentially followed by ultrasonic washing in ultrapure water. To fabricate the PDPMT-Cl-chit composite film modified GCE, 6 mL of the proposed PDPMT-Cl (2.0 mg mL1) dispersed in chit (0.25%, wt.) was dropped onto the electrode surface. After drying in room temperature, 3 mL of the first SCC-Ag antibody (Ab1) solution and 3 mL of the first CEA antibody (Ab10 ) solution were added onto the electrode surface and incubated at 277 K. The modified electrode was then thoroughly rinsed with PBS to remove unbounded particles. Subsequently, incubation with 1 wt% BSA solution for 1 h was used to eliminate nonspecific binding between the antigen and the electrode surface. After washing, 3 mL of SCC-Ag and 3 mL of CEA with varying concentrations were added onto the electrode surface and incubated for 1 h at 310 K. Finally, 3 mL of prepared Fe3O4 NPsTh-Ab2 and 3 mL of Fe3O4 NPs-Fca-Ab20 were added onto the

Scheme

Differential pulse voltammetry (DPV) scan was from 0.2 to 0.8 V in PBS to record the electrochemical responses for simultaneously quantitative measurement of SCC-Ag and CEA. 3. Results and discussion 3.1. Analysis of materials In this study, PDPMT-Cl was synthesized. To evaluate the conductivity of PDPMT-Cl, PDPMT-Cl modified bare GCE was characterized by cyclic voltammetry (CV). As shown in Fig. 1A, CV obtained at the GCE in 5 mM Fe(CN)63 is shown in curve a. Welldefined oxidation and reduction peaks are observed. In the case of PDPMT-Cl modified GCE (curve b), there is a drastic increase in the peak current was observed, which reveals that the PDPMT-Cl improved the electron transfer. The PDPMT-Cl was characterized using UV–vis spectroscopy (Fig. 2B). According to the UV–vis spectral analysis, for PDPMT-Cl solution, there was one major peak at approximately 520 nm. This phenomenon is consistent with the literature. Thus, the synthesis of PDPMT-Cl is successful. Fe3O4 NPs was characterized by TEM and FTIR spectrum, as shown in Fig. 1C, Fe3O4 NPs displayed sphere-like shape with high porosity and the size was around 50 nm. And as shown in Fig. 1D, a band at 3450 cm1 in the spectrum showed NH stretching vibrations. And at 1661 cm1 confirms the N H scissoring from the primary amine [34]. The results indicated that amino-group has been grafted on the Fe3O4 NPs successfully [35]. 3.2. Optimization of experimental conditions The pH value of substrate solution was a major factor to the current response. To achieve an optimal electrochemical signal, the response signal toward different pH of PBS (from 5.0 to 9.02) was tested systematically by DPV. It was found that for SCC-Ag, the current response increased from pH 5.0 to 7.4, and it reached the maximum at pH 7.4. Then it decreased from pH 7.4 to 9.02. For CEA,

2. Schematic representation of the preparation of immunosensor.

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Fig. 1. (A) Cyclic votammetric response of (a) GCE and (b) PDPMT-Cl modified bare GCE at 5 mM Fe(CN)63 scan rate: 100 mV s1; (B) UV–vis spectra of PDPMT-Cl; (C) TEM image and (D) FTIR of Fe3O4 NPs.

the peak current also reached the maximum at pH 7.4. It is probable that the highly acidic or alkaline surroundings would damage the immobilized protein [36]. To maintain the physiological environment and obtain high sensitivity, pH 7.4 PBS was selected for further. The DPV response was related to the

concentration of PDPMT-Cl (Fig. 2B). Through experiments, the optimal amperometric response of PDPMT-Cl was achieved at 2 mg mL1. The incubation temperature may significantly affect the performance of the immunosensor. We have done this experiment and found the optimal amperometric response of the incubation

Fig. 2. Effect of pH (A), the concentration of PDPMT-Cl (B), the incubation temperature (C) and the concentration of Fe3O4 NPs (D) on the response of the immunosensor. The concentrations of analytes were both 6 ng mL1.

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temperature was achieved at 310 K. The concentration of Fe3O4 NPs was tested systematically by DPV. Fig. 2D showed the effect of different concentrations of Fe3O4 NPs for the detection of 6 ng mL1 SCC-Ag and CEA. With an increasing concentration, the current response increased sharply and reached a top value after 10 mg mL1. Therefore, 10 mg mL1 Fe3O4 NPs was used as the performed concentration. Subsequently, all the experiments were conducted under optimized conditions. 3.3. Characterization of the immunosensor As an effective tool for characterizing the interface properties of electrode [37], electrochemical impedance spectroscopy (EIS) was utilized to monitor the fabrication process. Fig. 3 showed the typical Nyquist plot (Z00 vs. Z0 ) for the electrode at different stages. The diameter of semicircle at higher frequencies corresponds to the electron transfer resistance Ret. It can be seen that the bare GCE exhibited low resistance (curve a), which was characteristic of a diffusion-limiting step in the electrochemical process. The PDPMTCl-chit modified GCE showed a much lower resistance for the redox probe (curve b), implying that the PDPMT-Cl is an excellent electric conducting material and accelerated the electron transfer. Subsequently, for the PDPMT-Cl-chit/GA modified electrode (curve c), access to the interfacial electron transfer was hindered, which suggests that GA was immobilized on the electrode and blocked electron exchange between the redox probe and the electrode. In the case of GCE/PDPMT-Cl-chit/GA/Ab and Ab0 , the Ret further increased (curve d). Followed by BSA (curve e) resulting in successive increase in Ret. Additionally, after the capture of targets and labels, the resistance increased again (curve f and g), which indicated that the electrode was well-modified [37]. DPV was used to evaluate the performance of the fabricated immunosensor under optimized conditions. The calibration curve of the immunosensor was given in Fig. 4. The calibration plots between the peak current and the concentration displayed good linear relationships in the range of 0.01–10 ng mL1 for SCC-Ag with a regression equation of I(mA) = 7.412 + 5.294cSCC-Ag, R = 0.9975; 0.01–10 ng mL1 for CEA with a regression equation of I(mA) = 10.10 + 6.7417cCEA, R = 0.9939, respectively. The detection limit was estimated to be 4 pg mL1 and 5 pg mL1, respectively, in terms of the rule of three times standard deviation over the blank, n = 11. The low detection limit may be attributed to two factors.

Fig. 3. Complex plane impedance plots in 0.1 mol L1 KCl containing 2.5 mmol L1 [Fe(CN)6]3 and 2.5 mmol L1 [Fe(CN)6]4 at GCE (a), GCE/PDPMT-Cl-chit (b), GCE/ PDPMT-Cl-chit/GA (c), GCE/PDPMT-Cl-chit/GA/Ab and Ab0 (d), GCE/PDPMT-Cl-chit/ GA/Ab and Ab0 /BSA (e); GCE/PDPMT-Cl-chit/GA/Ab and Ab0 /BSA/SCC-Ag and CEA (f); GCE/PDPMT-Cl-chit/GA/Ab and Ab0 /BSA/SCC-Ag and CEA/Fe3O4 NPs-Th-Ab2 and Fe3O4 NPs-Th-Ab20 (g).

Fig. 4. Calibration curves of the multiplexed electrochemical immunosensor toward SCC-Ag and CEA.

Firstly, the good conductivity and electron transfer ability of PDPMT-Cl and the sensitization effect of electron mediator functionalized Fe3O4 increased the sensitivity of the immunoassay and lower the detection limit. Secondly, a relatively large amount of Ab2 and Ab20 had been conjugated onto Fe3O4, which could greatly improve the probability of antibody–antigen interactions and thereby leading to higher sensitivity. 3.4. Reproducibility, stability and selectivity of the immunosensor To elevate the coefficient of variation of intraassay, immunosensors belonging to the same batch were used to detect two different concentrations of mixed antigens. The variation coefficient of five times parallel test were 5.4%, 4.3% and 4.9% for 0.1, 1, and 10 ng mL1 SCC-Ag; 5.3%, 5.7% and 4.1% for 0.1,1, and 10 ng mL1 CEA, respectively. When using different batches of immunosensors, the coefficient of variations were investigated, the results were 5.7%, 5.9% and 5.6% for SCC-Ag; 6.3%, 6.5% and 7.1% for CEA. Thus, the immunosensors showed a desirable reproducibility. Long-term storage stability of the proposed immunosensors was investigated. The immunosensor stored at 277 K for 10 days showed that the steady-state peak current for the detection of 6 ng mL1 SCC-Ag and CEA, was 98.9% and 97.6% of the initial steady-state peak current, respectively. And the average decrease value of peak current was less than 3% compare to that of freshly prepared immunosensor. The good long-term stability maybe ascribed to the good stability of the electron mediator functionalized Fe3O4 itself and the adsorption of the Ab and Ab0 onto the surface of the PDPMT-Cl-chit obtained high activity and stability. An excellent immunosensor must exclude cross-reactivity between analytes and non-specific antibodies. The cross-reactivity was evaluated by comparing the amperometric responses of two analytes to those containing only one analyte. Fig. 5A shows the minimal difference of one or two kinds of analytes and labels. A cuspidal peak appeared at 0.2 V when the incubation solution only contained 6 ng mL1 SCC-Ag (curve a). When both 6 ng mL1 SCC-Ag and CEA added, two signal peaks were obtained in curve c. The DPV peaks (curve a and curve c) had the same current value, indicating that the presence of CEA had no influence on SCC-Ag. Similarly, when the incubation solution only contained 6 ng mL1 CEA (curve b), a peak arose at 0.3 V, which matched the peak at 0.3 V in curve c well. The result indicating that there are no interferences between SCC-Ag and CEA. The influence of cross reactivity is more clearly shown in Fig. 5B by using 6 ng mL1 analytes and their corresponding labels. When

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Fig. 5. (A) Typical DPV immunosensor signals for the investigation of cross-reactivity; (a) 0 ng mL1 CEA and 6 ng mL1 SCC-Ag solution, (b) 0 ng mL1 SCC-Ag solution and 6 ng mL1 CEA solution, (c) 6 ng mL1 SCC-Ag solution and 6 ng mL1 CEA in the sandwich immunosensor; (B) selectivity of the multiplexed electrochemical immunosensor toward BSA (2), CA-125 (3), alpha fetal protein (4), lysine (5), hepatitis B surface antigen (6), L-cysteine (7), ascorbic acid (8).

Table 1 Assay results of clinical serum samples using the proposed and reference methods. Sample

1 2 3 4 5

Proposed method

Reference method

Relative deviation

RSD

SCC-Ag (ng/mL)

CEA (ng/mL)

SCC-Ag (ng/mL)

CEA (ng/mL)

SCC-Ag (%)

CEA (%)

SCC-Ag (%)

CEA (%)

0.61 0.45 0.52 0.37 0.43

2.12 2.97 3.55 1.69 4.97

0.64 0.49 0.55 0.41 0.47

2.26 3.07 3.69 1.54 5.21

4.69 8.16 5.45 9.76 8.51

6.19 3.26 3.79 9.74 4.61

4.68 3.76 5.59 6.73 3.25

7.01 6.53 6.36 7.18 4.33

600 ng mL1 or 1200 U mL1 distractors were injected into the detection system, no apparent change in the current was observed compared with that of the blank test. 3.5. Real sample analysis To evaluate the feasibility of the proposed immunosensor for real sample analysis, a human serum sample using the proposed method were compared with the reference values (the results were provided by Jinan Shizhong People's Hospital) obtained by commercial available electrochemiluminescent analyzer (ROCHE 411, Switzerland). The experimental results were summarized in Table 1. The relative deviation were 4.69–9.76% and 3.26–9.74% for SCC-Ag and CEA, respectively. No significant differences were encountered, thereby revealing a good correlation between the two methods. 4. Conclusions This work demonstrated a convenient and effective method for simultaneous electrochemical immunosensor using a PDPMT-Clbased immunosensor with functionalized magnetic materials as labels. Using SCC-Ag and CEA as a model panel of cancer biomarker for cervical cancer, the immunosensor could be simply prepared by covalently immobilizing the capture antibodies onto the surface of a PDPMT-Cl-chit-modified GCE. The Fe3O4 NPs showed a nanohorn structure for efficient labeling of signal antibodies and electron mediators. The proposed simultaneous electrochemical immunosensor showed high sensitivity, negligible cross-talk, good selectivity and reproducibility, and acceptable stability, providing potential application in clinical diagnostics.

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Please cite this article in press as: X. Zhang, et al., Simultaneous electrochemical immunosensor based on water-soluble polythiophene derivative and functionalized magnetic material, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.05.025

Simultaneous electrochemical immunosensor based on water-soluble polythiophene derivative and functionalized magnetic material.

A novel, sensitive electrochemical immunosensor for simultaneous determination of squamous cell carcinoma associated antigen (SCC-Ag) and carcinoembry...
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