Talanta 132 (2015) 803–808

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Ultrasensitive electrochemical immunosensor for squamous cell carcinoma antigen detection using lamellar montmorillonite-gold nanostructures as signal amplification Hongying Jia, Picheng Gao, Hongmin Ma, Yueyun Li, Jian Gao, Bin Du, Qin Wei n Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 26 August 2014 Received in revised form 13 October 2014 Accepted 15 October 2014 Available online 23 October 2014

Sodium montmorillonites (Na-Mont), which could be transformed from nano-montmorillonites, have large surface area, chemical stability, nontoxicity, high cation exchange property and superior adsorption ability. In this paper, Na-Mont were used as a support of polyaniline (PANI) and gold nanoparticles (AuNPs) via the interaction of aniline and HAuCl4 solution. A sandwich-type electrochemical immunosensor was developed to detect squamous cell carcinoma antigen (SCC-Ag). It used nitrogen-doped graphene sheets (N-GS) for the immobilization of primary anti-SCC antibodies (Ab1) and the combined Na-Mont-PANIAuNPs nanocomposites as labels. Na-Mont-PANI-AuNPs have excellent catalytic ability towards the reduction of H2O2, thus enhance the sensitivity of the immunosensor. The immunosensor exhibits a wide linear range (1 pg/mL–5 ng/mL), a low detection limit (0.3 pg/mL), good reproducibility, selectivity and stability. This new type of immunosensor with Na-Mont-PANI-AuNPs as labels may provide potential application for the detection of SCC-Ag. & 2014 Elsevier B.V. All rights reserved.

Keywords: Electrochemical immunosensor Lamellar montmorillonite-gold nanostructures Nitrogen-doped graphene sheets Squamous cell carcinoma antigen

1. Introduction Squamous cell carcinoma antigen (SCC-Ag), a glycoprotein with isoforms ranging from 45 to 55 kDa, is a subtype of TA-4 and belongs to the ovalbumin family of serine proteinase inhibitors [1–3]. There is a close relationship between high level of SCC-Ag in human serum and cervical cancer, squamous cell carcinoma of the lungs, esophageal cancer, and so on [4]. The serum level of SCC-Ag would increase in parallel to the growth of the tumor size or the recurrence of the disease [5]. Recently, radioimmunoassay [6], enzyme-linked immunosorbent assay [7] and chemiluminescence enzyme immunoassay [8] have been used to detect SCC-Ag in serum. Although these methods are widely used, there is still a highly demand for developing a sensitive, simple and low-cost method for the early detection of SCC-Ag. Electrochemical immunosensor is a miniaturized analytical device. Due to its many merits such as high sensitivity, precise current measurement and the cost-effectiveness, it has attracted much attention in analytical chemistry. To aim at this demand, herein, we had developed a convenient sandwich-type immunosensor based on Na-montmorillonite-polyaniline-gold nanoparticles (Na-Mont-PANIAuNPs) and nitrogen doped graphene sheets (N-GS) for the sensitive detection of SCC-Ag. There are two basic issues for increasing the

n

Corresponding author. Tel.: þ 86 531 82765730; fax: þ 86 531 82765969. E-mail address: [email protected] (Q. Wei).

http://dx.doi.org/10.1016/j.talanta.2014.10.033 0039-9140/& 2014 Elsevier B.V. All rights reserved.

sensitivity of the immunosensor, one is the capture of primary antibody (Ab1) onto electrode surface, and the other is the signal amplification strategy. In recent years, many kinds of nanomaterials, such as silica nanoparticles, carbon nanotubes, titanium dioxide nanoparticles and nano-montmorillonites have been used as labels for signal amplification in electrochemical immunosensors [9–12]. Nanomontmorillonites belong to aluminosilicate clay minerals that could be converted to sodium montmorillonites (Na-Mont) by reacting with NaCl solution [13]. Na-Mont which consisted of layered structures have proved to be an ideal host due to their inherent advantages, such as large surface area, high cation exchange capacity, superior adsorption ability, nontoxicity and chemical stability and so on [14–18]. Based on the cation exchange ability of Na-Mont, many organic molecules with polar functional groups and ions can be intercalated into the interlayer space of montmorillonites [15]. Polyaniline (PANI) have aroused extensive attention in the application of electrochemical immunosensor due to their interesting conductivity, electronic and catalytic characteristics [19,20]. Gold nanoparticles (AuNPs) also have many advantages including its good catalytic performances, easy preparation, good biocompatibility and strong bonding ability [21–23]. Usually, aniline would exist in the form of cations in acidic solution, so it can insert into Na-Mont and react with the added HAuCl4 solution. Due to the large surface and cation exchange ability of Na-Mont [24,25], AuNPs and PANI could form largely in the interlayer structures based on the

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reaction of HAuCl4 and aniline. The secondary anti-SCC antibodies (Ab2) can be adsorbed onto the Na-Mont surface based on its excellent adsorption ability and the Ab1 could be immobilized by N-GS due to its excellent electronic conductivity, large surface areas and amounts of edge sites [26–28]. Hence, we proposed a new type electrochemical immunosensor for the sensitive detection of SCC-Ag by using Na-Mont-PANIAuNPs nanostructures as labels. To the best of our knowledge, this is the first time that Na-Mont-PANI-AuNPs nanostructures have been prepared through intercalation strategy and used as labels for electrochemical immunosensor construction.

2. Materials and methods 2.1. Apparatus and reagents The montmorillonites were purchased from the Fangzi Bentonite Plant (Weifang, Shandong Province, China). Bovine serum albumin (BSA, 96–99%) was purchased from Sigma. The SCC-Ag, Ab1 and Ab2 were purchased from Beijing Kwinbon Biotechnology Co., Ltd. (Beijing, China). Aniline, HAuCl4  4H2O, chitosan and K3[Fe(CN)6] were obtained from Sinopharm Chemical Reagent Shanghai Co., Ltd., China (Beijing,

China). KH2PO4 and Na2HPO4 were of analytical reagents grade and used without further purification. Phosphate buffered solutions (PBS) were prepared using 0.067 mol/L Na2HPO4 and 0.067 mol/L KH2PO4 stock solution. All other chemicals and solvents were of analytical grade. Ultrapure water was used throughout the experiment. All electrochemical measurements were performed on a CHI760D electrochemical workstation (Chenhua Instrument Shanghai Co., Ltd., China). Scanning electron microscope (SEM) images were recorded using field emission SEM (ZEISS, Germany). Transmission electron microscope (TEM) images were obtained from a Hitachi H-800 microscope (Japan). Conventional three-electrode system was used for all electrochemical measurements: a glassy carbon electrode (GCE, 4 mm in diameter) as working electrode, a platinum wire electrode as the counter electrode and a saturated calomel electrode (SCE) as the reference electrode.

2.2. Synthesis of nitrogen-doped graphene sheets (N-GS) N-GS were synthesized through thermal annealing of graphite oxide in ammonia [29]. Graphite oxides (GO) were prepared from graphite powder by a modified Hummers’ method [30]. In brief, a 9:1 (v:v) mixture of 36 mL H2SO4 and 4 mL H3PO4 solution were added

Fig. 1. Schematic representation of the preparation of the Na-Mont-PANI-AuNPs-Ab2 (a) and the fabrication processes of the immunosensor (b).

H. Jia et al. / Talanta 132 (2015) 803–808

into the mixture of 1.8 g KMnO4 and 0.3 g graphite flakes and the reaction was under 50 1C for 12 h in a three flask. Then 30 μL H2O2 was added into the solution after stirring for 0.5 h and the products were washed by hydrochloric acid (0.2 M) and ethyl alcohol by drying after reaction. The synthesized GO (50 mg) were dispersed in 25 mL ultrapure water with a concentration of 2 mg/mL, then the pH of the solution was raised up to 10 by the addition of 30% NH3  H2O. After stirring for 10 min, 100 mL N,N-dimethyl formamide was added to the mixture and they were reacted at 153 1C for 1 h. Finally, the black sediment was filtered and washed with ultrapure water to neutral. The solids were dried to gain N-GS and the obtained N-GS were then dispersed in chitosan (0.5 wt%) until used. 2.3. Synthesis of sodium montmorillonites (Na-Mont) According to Yan’s report [31], 25.0 g natural montmorillonites with 29.3 g NaCl powder were dispersed in 0.5 L ultrapure water. The solution was shaken for 12 h. After that, the remaining solids were washed with ultrapure water repeatedly until no free of chloride ions were detected by AgNO3 solution. The products were dried at 85 1C for 24 h. They were ground in an agate mortar and then kept in a sealed bottle. 2.4. Synthesis of Na-Mont-PANI-AuNPs About 10.0 μL aniline were added into 50.0 mL ultrapure water to form 0.0022 M aniline solution, then they were added into 100 mL 0.2 M sulphuric acid solution for the protonation of aniline. Next, 1.06 g Na-Mont was dispersed in the acidic solution along with shaking uniformly for 3 h. In this process, the protonated aniline would insert into the interlayer of Na-Mont. 300 μL 1% HAuCl4 solution was added into above mixture with gently

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stirring, it can be observed that the solution color changed from yellow to light green and then to dark green. After discarding the supernatant, the Na-Mont-PANI-AuNPs nanocomposites were obtained. 2.5. Preparation of Na-Mont-PANI-AuNPs-Ab2 labels Fig. 1a showed the schematic illustration for the preparation of Na-Mont-PANI-AuNPs-Ab2 labels. Typically, 0.25 mL of 10 μg/mL Ab2 was added into the Na-Mont-PANI-AuNPs solution and the mixture were placed at 4 1C under shaking for 12 h. Ab2 could be loaded into channels of the Na-Mont-PANI-AuNPs. The obtained Na-Mont-PANI-AuNPs-Ab2 was washed with PBS (pH 7.4). The resulting bioconjugates were redispersed in 1 mL of PBS at 4 1C until used. 2.6. Fabrication of the immunosensor Fig. 1b showed the preparation process of the immunosensor. A GCE with 4 mm diameter was polished with 1.0, 0.3 and 0.05 μm alumina powder and thoroughly cleaned with ultrapure water before use. First, 6.0 μL of N-GS solution (2 mg/mL) which was dispersed in chitosan (0.5 wt%) was modified onto the electrode. After drying, the electrode was thoroughly cleaned with PBS to remove unbounded N-GS. Then 3 μL of glutaraldehyde (2.5%) was dropped onto the electrode surface to immobilize the Ab1 and incubated for 1 h. Next, 6.0 μL of Ab1 (10 μg/mL) solution was added onto electrode surface and incubated for another 1 h. The excess Ab1 was washed by PBS. The modified electrode was then incubated with 1 wt% BSA solution to block nonspecific binding sites. Subsequently, different concentrations of SCC-Ag solution were added to the electrode surface and incubated for another 1 h.

Fig. 2. SEM images of N-GS (a) Na-Mont-PANI-AuNPs (b) and TEM image of Na-Mont (c).

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Finally, the prepared Na-Mont-PANI-AuNPs-Ab2 solution was dropped onto electrode surface and incubated for another 1 h via specific antibody-antigen interaction. The modified electrode was washed three times to remove unbound Na-Mont-PANIAuNPs-Ab2. The well-prepared electrode was stored at 4 1C prior to use.

2.7. Detection of SCC-Ag Electrochemical characteristics of modified electrode were measured by amperometric measurements in PBS (pH 6.4). A detection potential of  0.4 V was selected for amperometric measurement. After the background current was stabilized, 5 mM H2O2 was added into PBS and the changed current was recorded. All electrochemical measurements were conducted in a harmony electrochemical condition at 25 1C.

3. Results and discussion 3.1. Characterization of N-GS and Na-Mont-PANI-AuNPs nanostructures In this study, SEM and TEM were used to characterize N-GS and Na-Mont-PANI-AuNPs nanostructures. N-GS were used to increase Ab1 loading because of its large surface area. Fig. 2a shows the SEM image of a wrinkle paper-like structure of N-GS. Na-Mont-PANIAuNPs were used to incubate Ab2 and Fig. 2b displays the SEM image of Na-Mont-PANI-AuNPs. It can be seen that the layered wrinkle structured Na-Mont and AuNPs were dispersed evenly in the interlamination of Na-Mont. From the TEM image of Na-Mont (Fig. 2c), it could be concluded that the layered structure with large specific surface area can immobilize more Ab2 and improve the sensitivity of immunosensor.

3.2. Characterization of the immunosensor fabrication

Fig. 3. Nyqiust plots of the electrochemical impedance spectroscopy (EIS) for each immobilized step. The bare GCE (a), N-GS/GCE (b), glutaraldehyde/N-GS/GCE (c), Ab1/glutaraldehyde/N-GS/GCE (d), BSA/Ab1/glutaraldehyde/N-GS/GCE (e), SCC-Ag/ BSA/Ab1/glutaraldehyde/N-GS/GCE (f), Na-Mont-PANI-AuNPs-Ab2/SCC-Ag/BSA/Ab1/ glutaraldehyde/N-GS/GCE (g).

Electrochemical impedance spectroscopy (EIS) has been employed to be a powerful tool to characterize the interface properties of surface-modified immunosensor during the process of fabrication and assembly. It is well known that the changes in electrochemical impedance show the chemical obstruction occurring in the electrochemical sensors [32]. The impedance spectra consist of a semicircle portion and a linear portion. The semicircle portion at higher frequencies indicates the electron-transfer limited process and the linear portion at low frequencies represents the diffusion-limited process. The semicircle diameter corresponds to the electrontransfer resistance (Ret). As shown in the Nyquist curves (Fig. 3), both GCE (curve a) and N-GS modified electrode (curve b) appear to be a very small semicircle domain nearly linear, which implies of a diffusion-limiting step in the electron-transfer process. Subsequently, for Ab1 modified electrode (curve c), the larger semicircle indicated that Ab1 was immobilized onto the electrode and the hydrophobic

Fig. 4. Effect of pH (a), the concentration of N-GS (b), and incubation time (c) on the response of the immunosensor to 0.5 ng/mL SCC-Ag.

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layer of protein was insulated the conductive support, thus hindering interfacial electron transfer. The Ret of the electrode further increased after modification of BSA, which indicated the successful immobilization of BSA. Additionally, after successful incubation of SCC-Ag and NaMont-PANI-AuNPs-Ab2, the Ret elevated again (curves e and f). EIS Nyquist plots demonstrated that the immunosensor had been fabricated successfully and could be used for the detection of SCC-Ag. 3.3. Optimization of experimental conditions The immunosensor was fabricated based on the chemical reaction of Na-Mont-PANI-AuNPs labels towards H2O2 reduction. The injected H2O2 solution in the electrolyte would result in saltation of current response, then SCC-Ag could be quantified based on specific recognized immunoreaction between antibody and antigen. In order to obtain the best electrochemical signal responses, the experimental conditions were optimized. For the immunosensor with the same concentration of SCC-Ag (0.5 ng/mL), the pH value of working solution plays an important role in the current response. As shown in Fig. 4a, it could be found that the amperometric responses increased with the increase of pH values from 5.0 to 6.4 and then decreased from 6.4 to 9.0. These results indicated that Na-Mont-PANI-AuNPs displayed best catalytic performance towards H2O2 reduction at pH 6.4, then pH 6.4 PBS was used as the optimal electrolyte solution in the following experiments. In addition, the amount of N-GS has a great effect on the electrochemical behavior of immunosensor. N-GS influenced the electrochemical signals of both the coated electrode and the loaded of Ab1. As seen in Fig. 4b, with the increased concentration of N-GS, the current response increased and then decreased. Therefore, the optimal concentration of N-GS was 1.5 mg/mL. Furthermore, incubation time has a great influence on the sensitivity of the immunosensor. In the fabricating process of the immunosensor, after adding SCC-Ag onto electrode surface, various incubation times were investigated for the detection of 0.5 ng/mL SCC-Ag. As shown in Fig. 4c, the current response increased with the incubation time increasing initially, but further increasing incubation

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time did not significantly change the signal. Hence, 45 min was selected for the optimal antigen-antibody interaction time. Under the optimal conditions, the immunosensor using NaMont-PANI-AuNPs as labels was used to detect different concentrations of SCC-Ag. The relationship between the current responses and different concentrations of SCC-Ag towards 5 mM H2O2 by reduction in pH 6.4 PBS at 0.4 V was shown in Fig. 5. The current responses increased linearly with the increasing of SCC-Ag concentrations in the range from 1 pg/mL to 5 ng/mL. The equation of the calibration curve was I (μA)¼0.8432þ1.686 c SCC-Ag (ng/mL) (r¼0.998) with a detection limit of 0.3 pg/mL. The sensitivity of our immunosensor is favorable in detecting cancer marker compared with previous reports [33–37] in Table 1. The low detection limit may be attributed to three factors: (1) N-GS could greatly increase the loading number of Ab1 and facilitate electron transfer due to its good biocompatibility and high conductivity; (2) as discussed above, Na-Mont-PANI-AuNPs with large surface area could immobilize amounts of Ab2, which greatly increases the sensitivity of the immunosensor; (3) PANI and AuNPs with good electrochemical conductivity could accelerate electron transfer and possess good catalytic performance towards H2O2 reduction. 3.4. Reproducibility, stability and selectivity of the immunosensor In order to evaluate the reproducibility of immunosensor, five immunosensors were prepared to detect 0.5 ng/mL SCC-Ag under the same conditions. The relative standard deviation (RSD) achieved from these five measurements was 3.18%. The result suggested the precision and reproducibility of the proposed immunosensor were both good. The study on the stability of immunosensor was measured by periodically checking current response. When the immunosensor was not in use, it was stored in a refrigerator at 4 1C. The current response of the immunosensor still retained 94% and 85% of the initial value after one week and three weeks, respectively. The slow decrease in the current response may be due to the gradual denature of SCC-Ag and anti-SCC antibody. The selectivity of the immunosensor was tested for its important role in analyzing biological samples. The effect of possible inhibitors that might interfere with the response of immunosensor had been investigated. SCC-Ag (0.5 ng/mL) solution containing 100 ng/mL of interfering substances (BSA, glucose, vitamin C and AFP) was measured by immunosensor. As a result, the current variation due to the interfering substances was less than 5% of that without interferences. These results indicated the selectivity of the immunosensor was acceptable. 3.5. Real sample analysis

Fig. 5. Calibration curve of the immunosensor toward different concentrations of SCC-Ag, errorbar¼ RSD (n ¼5).

In order to evaluate the feasibility of the electrochemical immunosensor for practical application, the detection of SCC-Ag in human serum samples was evaluated using standard addition methods. 1.00 ng/mL, 2.00 ng/mL and 4.00 ng/mL of SCC-Ag solutions were added into serum samples and analyzed by the proposed immunosensor. The results are shown in Table 2. It can be seen that the RSD

Table 1 A comparison of the performance of the described and referenced immunosensors for the detection of SCC-Ag. Electrode materials

Linear range

Detection limit

R-square

References

Magnetic mesoporous nanogold/thionine/NiCo2O4 HPR-nanogold/graphene nanosheets Fluorescein isothiocyanate (FITC) and N-(aminobutyl)-N-(ethylisoluminol) (ABEI) Bimetallic gold-silver nanoclusters (AuAg NCs) Semiconductor carboxylated graphitic carbonnitride (g-C3N4) Carbon-supported Pd-Au (Pd-Au/C) Lamellar montmorillonite-PANI-Gold

2.5 pg/mL–15 ng/mL 0.5–20 ng/mL 0.005–20 ng/mL 0.025–10 ng/mL 0.005–2 ng/mL 0.001–5 ng/mL

1.0 pg/mL 0.02 ng/mL 1.3 pg/mL 8.53 pg/mL 1.7 pg/mL 0.33 pg/mL

0.9867 0.9964 0.9899 0.9917 0.995 0.998

[33] [34] [35] [36] [37] This work

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Table 2 Results for determination of SCC-Ag in human serum samples. Initial SCC-Ag concentration in sample (ng/mL)

0.800

0.500

Added SCC-Ag concentration (ng/mL)

Recovery The detection content (ng/ RSD (%) mL) (%, n¼ 5)

1.00 2.00 4.00 1.00 2.00 4.00

1.77, 1.79, 1.82, 1.83, 1.76 2.83, 2.86, 2.84, 2.78, 2.79 4.62, 4.71, 4.81, 4.89, 4.75 1.48, 1.54, 1.53, 1.44, 1.47 2.54, 2.50, 2.56, 2.66, 2.59 4.47, 4.42, 4.53, 4.59, 4.35

1.70 1.20 2.14 2.82 3.30 2.08

99.7 101 99.1 99.5 102 99.3

was from 1.20% to 3.30% and the recoveries were between 99.0% and 102%. Thus, the presented method could meet the demand for clinical determination of SCC-Ag in serum samples. 4. Conclusions In this work, a novel sandwich-type immunosensor for the det ection of SCC-Ag had been developed. The immunosensor was fabricated by immobilizing Ab1 onto N-GS and using Na-Mont-PANIAuNPs as labels. N-GS with large surface area enhanced the amount of Ab1 immobilized onto electrode surface and the Na-Mont-PANIAuNPs showed excellent catalytic performance towards H2O2 reduction. Then the proposed immunosensor showed low detection limit for the detection of SCC-Ag. The simplicity, good sensitivity and reproducibility of the immunosensing platform may be quite promising in clinical application for the detection of SCC-Ag.

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Ultrasensitive electrochemical immunosensor for squamous cell carcinoma antigen detection using lamellar montmorillonite-gold nanostructures as signal amplification.

Sodium montmorillonites (Na-Mont), which could be transformed from nano-montmorillonites, have large surface area, chemical stability, nontoxicity, hi...
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