Biosensors and Bioelectronics 64 (2015) 51–56

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Ultrasensitive electrochemical immunosensor for carbohydrate antigen 72-4 based on dual signal amplification strategy of nanoporous gold and polyaniline–Au asymmetric multicomponent nanoparticles Haixia Fan, Zhankui Guo, Liang Gao, Yong Zhang, Dawei Fan, Guanglei Ji, 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, China

art ic l e i nf o

a b s t r a c t

Article history: Received 21 May 2014 Received in revised form 26 July 2014 Accepted 5 August 2014 Available online 23 August 2014

A sandwich electrochemical immunosensor is described for carbohydrate antigen 72-4 (CA72-4) based on a dual amplification strategy with nanoporous gold (NPG) film as the sensor platform and polyaniline–Au asymmetric multicomponent nanoparticles (PANi–Au AMNPs) as labels. In this study, the second anti-CA72-4 antibody (Ab2) adsorbed onto the Au of the PANi–Au AMNPs, which could be simply synthesized by interfacial reaction and have many characteristics of polyaniline and Au nanoparticle, such as well-controlled size, high conductivity, biocompatibility and catalysis. NPG film was used as electrode substrate material to fix a large number of antibodies, due to its unique properties: good biocompatibility, high conductivity, large surface area, and stability. The synergetic of NPG film and PANi–Au AMNPs could increase signal response, and significantly improve sensitivity of the immunosensor. The proposed immunosensor exhibited a wide linear range from 2 to 200 U/mL, with a detection limit of 0.10 U/mL CA72-4, good reproducibility, selectivity and stability. This new type of labels for immunosensors may provide many potential applications in the detection of carbohydrate antigen in immunoassays. & 2014 Elsevier B.V. All rights reserved.

Keywords: Immunosensor Nanoporous gold film Asymmetric gold nanocomposite particles Carbohydrate antigen 72-4

1. Introduction Carbohydrate antigen 72-4 (CA72-4), considered to be a kind of high molecular weight tumor-associated glycoprotein (220– 400 kDa), was reported by Colcher in 1981. CA72-4 is known to be overexpressed in various carcinomas including ovarian, gastric, colorectal, and breast cancers, and is rarely expressed in most benign tumors and normal adult tissues in contrast to malignant tumors (Sharifzadeh et al., 2013; Tang et al., 2007; Jin et al., 2008). The preoperative levels of CA72-4 can assist in prediction, diagnosis of tumor and providing prognostic information (Brandwein et al., 1992; Epivatianos et al., 2000; Sheng et al., 2007). The cutoff limit for CA72-4 has been set at 6 U/mL, the CA72-4 concentration is lower than this limit in normal human serum and elevated levels of CA72-4 are correlated with cancer incidents (Guadagni et al., 1991). The traditional methods for CA72-4 determination include enzyme-linked immunosorbent assay, radioimmunoassay (Gero et al., 1989; Ferroni et al., 1990), time-resolved immunofluorometric assay (Sheng et al., 2007), immunohistochemical 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.bios.2014.08.043 0956-5663/& 2014 Elsevier B.V. All rights reserved.

detection (Ouyang et al., 2010), chemiluminescence enzyme immunoassay (Sharifzadeh et al., 2013). The above techniques are low-efficiency, time-consuming, expensive and complex. Therefore, a rapid, specific and inexpensive analytical method of high sensitivity and easy operation is urgently needed to monitor CA724 in human serum, which will have a massive effect on a number of aspects of the diagnostics, treatment, and prognosis monitoring of a cancer, and has great potential applications in medicine domain. Electrochemical immunosensors, which combine traditional immunoassay methods with electrochemical transduction, have drawn considerable attention in the past few years (Wang et al., 2011a, 2011b; Qian et al., 2010a, 2010b; Tothill, 2009). Especially, sandwich-type immunoassay protocol is regarded as a more sensitive platform. The sensitivity of immunosensors is related to the substrate material for electrode modification and is mainly determined by what kind of the label used. Many types of nanomaterials, including metal nanoparticles, quantum dots, carbon nanomaterials, semiconductor nanoparticles, and hybrid nanostructures, have been investigated as labels for these immunosensors (Pei et al., 2013; Wu et al., 2009; Chen et al., 2009; Qian et al., 2010a, 2010b; Yuan et al., 2012; Wu et al., 2012). There is a

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necessity to choose a favorable nanomaterial as label, for the purpose of the enormous signal enhancement. Multicomponent nanoparticles, consisting of two or more different nanoscale components, have aroused strong interests due to their excellent properties of the compositional multifunctionality, novel functions and enhanced properties compared with each individual component (Hao et al., 2010; Zeng and Sun, 2008; Shtansky et al., 2010; Zhang et al., 2010; Chen et al., 2011; He et al., 2012). Polymer-inorganic nanocomposites have a wide range of the potential applications, such as energy (Zhao and Lin, 2012), sensing (Jia et al., 2011; Wang et al., 2011a, 2011b; Hu et al., 2012), supercapacitors (Murugan, 2006) and optoelectronics (Zhang et al., 2012; Thakur et al., 2012; Goodman et al., 2009). In particular, much effort has been focused on polyaniline–Au (PANi–Au) multicomponent nanoparticles. Compared with other labels reported so far for immunosensors, PANi–Au nanocomposites have obvious superiority used as labels for the electrochemical immunosensors, due to the combined properties of polyaniline and Au nano-particles, such as innocuity, excellent catalytic activity, high conductivity (Hung et al., 2010; Stoyanova et al., 2011), large surface area (Xu et al., 2010) and good biocompatibility (Feng et al., 2011), which can improve the sensitivity of the immunosensor. For electrochemical sensors, nanoporous gold (NPG) has attracted considerable attention as an attractive substrate material, which can fix more antibodies and is important for the increase of sensitivity of the immunosensor. NPG has been extensively applied to many fields because of its unique properties: a bicontinuous porous structure, large specific surface area, good biocompatibility, good conductivity and catalysis (Ruffato et al., 2012; Wei et al., 2011; Feng et al., 2013). The good conductivity can enhance the electron transfer on the electrode surface, and also amplify the electrochemical signal. In this work, in order to overcome the limitations of enzyme activity, the polyaniline–Au asymmetric multicomponent nanoparticles (PANi–Au AMNPs) were prepared through an interfacial reaction, which showed strong effect in catalyzing H2O2 reduction. With the secondary antibody (Ab2) adsorbed onto Au, the resulting PANi–Au AMNPs-Ab2 were used as label for the preparation of immunosensor. NPG film was used as electrode substrate material to capture a large number of the primary antibody (Ab1), so as to increase of the electrochemical signal. CA72-4 was used as model, and the sandwich-type structure is prepared by immobilizing the primary anti-CA72-4 antibody (Ab1) onto NPG film surface, the CA72-4 in the sample captured, and PANi–Au AMNPs-Ab2 as label. This immunosensing approach is simple, economic and sensitive, which may find wide potential application for the ultrasensitive detection of different tumor markers in clinical analysis.

2. Materials and methods 2.1. Apparatus and reagents Monoclonal antibody to human CA72-4 (anti-CA72-4 Ab1, Ab2), human cancer antigen CA72-4 were purchased from Shanghai Linc-Bio Science Co., Ltd. (Shanghai, China) Sigma (USA). Bovine serum albumin (BSA, 96–99%) was purchased from Sigma (USA). Gold(III) chloride (99%), n-hexane, aniline, HNO3, K3Fe(CN)6 and K4Fe(CN)6 were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). The alloy silver/gold was purchased from Sepp Leaf Products (New York). All the chemicals were used directly without further purification. All other chemicals and solvents were of analytical grade. Phosphate buffered solutions (PBS) were prepared using 0.067 mol/L Na2HPO4 and 0.067 mol/L KH2PO4 stock solution. Ultrapure water was used throughout the experiments. All electrochemical measurements were performed on a CHI 760D electrochemical workstation (Chenhua Instrument Shanghai Co., Ltd., China). Electrochemical impedance spectroscopy (EIS) was obtained from the IM6e impedance measurement unit (ZAHNER Elektrik, Germany). Transmission electron microscope (TEM) image was obtained from H-800 microscope (Hitachi, Japan). Scanning electron microscope (SEM) image was obtained using field emission SEM (ZEISS, Germany). A 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. Preparation of NPG NPG was made by selective dealloying of silver from silver/gold alloy according to the report (Ding et al., 2004). The alloy was corroded in concentrated HNO3 at the room temperature, and the pore sizes of NPG could be controlled by the reaction time. The NPG was then thoroughly washed to the neutral pH with ultrapure water. 2.3. Preparation of the PANi–Au AMNPs PANi–Au AMNPs were prepared by interfacial reactions (He et al., 2012). 2.75 mg of HAuCl4 was dissolved in 5 mL of ultrapure water. After homogeneous mixing, the solution was placed into a pre-heated water bath at 45 ˚C for 10 min. Then 0.5 mL of aniline in hexane solution (20 mmol/L) was carefully added on the top of the HAuCl4 aqueous solution. The mixture was then incubated overnight at 45 ˚C for the further growth of PANi–Au AMNPs. The hexane in the surface of aqueous phase was abandoned after the solution was cooled down to room temperature. The PANi–Au AMNPs were collected by centrifugation and washed with water for several times, and dried under vacuum. 2.4. Preparation of the PANi–AU AMNPs-Ab2 labels As shown in Fig. 1A, the PANi–Au AMNPs (2 mg) were dispersed in 1 mL of phosphate buffer at pH 7.4. This dispersion was then mixed with 1 mL 10 μg/mL of anti-CA72-4 Ab2. The mixture was allowed to react at room temperature under stirring for 24 h, followed by centrifugation. The resulting PANi–Au AMNPs-Ab2 were washed with buffer solution (pH 7.4) and then re-dispersed in 1 mL of buffer and stored at 4 °C before use. 2.5. Fabrication of the immunosensor

Fig. 1. Schematic representation of the preparation of the PANi–Au AMNPs-Ab2 (A) and immunosensor (B).

Fig. 1B shows the fabrication procedure of the immunosensor. The GCE was polished carefully with 0.3, 0.1 and 0.05 μm alumina

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slurry, and sonicated in water and ethanol, respectively. The NPG film was carefully coated onto the electrode surface to form NPG modified GCE. Then the NPG coated electrode was dried and washed with buffer solution. After drying, 6 μL of Ab1 (10 μg/mL) was added onto electrode surface and incubated for 1 h. Subsequently, the electrode was washed and incubated in BSA solution (w/w, 1%) for 1 h to block nonspecific binding and avoid the nonspecific adsorption. Following that, the modified electrode was incubated with different concentrations of CA72-4 solution for 1 h. Finally, 6 μL of the prepared PANi–Au AMNPs-Ab2 buffer solution was dropped onto the electrode surface. The finished immunosensor was stored at 4 °C until use. 2.6. Detection of CA72-4 The pH 7.4 PBS was used for all the electrochemical measurements. For amperometric measurement of the immunosensor, a detection potential of  0.4 V was selected. After the background current was stabilized, 5.0 mmol/L H2O2 was added into the buffer solution and the current change was recorded. Electrochemical impedance spectroscopy (EIS) was obtained in 2.5 mmol/L Fe(CN)63 /4 solution.

3. Results and discussion 3.1. Characterization of PANi–Au AMNPs-Ab2 and NPG In this study, the PANi–Au AMNPs were used to label antiCA72-4 Ab2 because of its great catalytic activity toward H2O2, and good conductivity. Fig. 2A–C shows the characterizations of PANi– Au AMNPs. From the TEM and SEM images of PANi–Au AMNPs in

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Fig. 2A and B, it was confirmed that the shape of PANi–Au AMNPs was three Au domains on single polyaniline (PANP), the diameter of PANPs was approximately150 nm, while that of AuNPs was about 85 nm. In order to confirm the composition of the AMNPs, EDS spectra were collected during SEM imaging (Fig. 2C). This spectrum confirms the presence of Au in the AMNPs. Fig. 2D–F shows the single-crystal nature of NPG; NPG presents an interconnected network of pores and ligaments, because gold atoms readjustment occurs only by diffusion along the alloy/ electrolyte interface, and does not involve nucleation of new grains. Fig. 2D–F shows a series of TEM images of NPG sample left in nitric acid for different periods of time. TEM images of Au/ Ag foil dealloyed in concentrated nitric acid at room temperature under free corrosion conditions for 15 s, 25 s and 3 min, respectively. Au/Ag alloy sample was dealloyed for 15 s, and a uniform pore size distribution centered around 9 nm can be seen (Fig. 2D). In addition, samples dealloyed for 25 s with about 15 nm sized pore structure (Fig. 2E), and 3 min with about 50 nm pore size (Fig. 2F) were obtained. Furthermore, testing PANi–Au AMNPs as labels for immunosensor, we investigated the performance of PANi–Au AMNPs toward the detection of H2O2. As shown in Fig. 3A, for the Au NPs (the Au NPs were prepared by the sodium citrate reduction method) modified electrode, after the addition of 1.0 mmol/L H2O2 in PBS, only a very small reduction current was observed. For PANi–Au AMNPs modified electrode (Fig. 3B), after the addition of the same concentration of H2O2, there is a much larger reduction current. Since the high sensitivity of PANi–Au AMNPs toward the detection of H2O2 has been demonstrated, immunosensors for detecting CA72-4 using PANi–Au AMNPs-Ab2 as labels were built and characterized.

Fig. 2. TEM image (A), SEM image (B) and EDS spectra (C) of PANi–Au AMNPs; TEM images of NPG samples with 15 s (D), 25 s (E) and 3 min (F) in concentrated nitric acid.

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Fig. 3. Cyclic voltammograms of (A) Au NPs, (B) PANi–Au AMNPs modified electrode in PBS (pH 7.40) without (a) and with (b) 1.0 mmol/L H2O2.

The stability of the PANi–Au AMNPs-Ab2 was investigated. When not in use, the NPs were stored in PBS at 4 °C. Two weeks later, there is almost no change of the current response of the immunosensor prepared using the same PANi–Au AMNPs-Ab2 solution. It presumedly means that the good long-term stability maybe due to the good stability of the PANi–Au AMNPs itself, and the adsorption of Ab2 onto Au obtained high activity and stability. 3.2. Characterization of the immunosensor In order to characterize the fabrication process of the immunosensor, CVs of the different modified electrode conducted in 5.0 mmol/L K3Fe(CN)6 with a scan rate of 100 mV/s are shown in Fig. 4A. A pair of well-defined reduction/oxidation peaks of K3Fe(CN)6 was observed at the bare GCE (curve a). The peak current on NPG film modified electrode (curve b) was observably increased, because of the large specific surface area of NPG. After Ab1 was assembled onto the electrode surface, the peak current clearly decreased (curve c), implying that Ab1 was successfully immobilized onto the electrode surface. The reason for the decrease of amperometric current is that protein will hinder the electron transfer. After BSA blocked nonspecific sites, a decrease of

current was also obtained (curve d). Then the BSA/Ab1/NPG/GCE modified electrode incubated with CA72-4 solution (50 U/mL); the peak current was further decreased (curve e), because the antigen–antibody complex on the immunosensor surface inhibited the electron-transfer. At last, after the capture of Ab2, the peak current increased (curve f). Although the formed sandwich-type immune complex may hinder the electron transfer, PANi–Au AMNPs as labels may improve the electron transfer because of its high conductivity. EIS can give further information about the impedance changes of surface-modified electrodes. Fig. 4B shows the electrochemical impedance of a bare GCE and the modified electrodes in 2.5 mmol/ L Fe(CN)63  /4  at room temperature. It was observed that the bare GCE revealed almost a straight line (curve a). After the NPG film was deposited onto the GCE, the electrode showed a similar straight line as that of the bare GCE electrode (curve b), implying that NPG exhibits an excellent electronic conduction ability and accelerated the electron transfer. Subsequently, the immobilization of Ab1 on the electrode surface generated an insulating protein hydrophobic layer, which hindered electron transfer and increased the resistance (curve c). Followed by blocking nonspecific sites with BSA, the EIS showed a higher resistance (curve d).

Fig. 4. (A) CVs of bare GCE (a), NPG/GCE (b), Ab1/NPG/GCE (c), BSA/Ab1/NPG/GCE (d), CA72-4/BSA/Ab1/NPG/GCE (e), PANi–Au AMNPs-Ab2/CA72-4/BSA/Ab1/NPG/GCE (f) in 5.0 mmol/L K3Fe(CN)6, with a scan rate of 100 mV/s. (B) Nyquist diagrams of electrochemical impedance spectroscopy of the different modified electrodes in 2.5 mmol/L Fe(CN)63  /4  , and the composition of the electrode is the same as (A). All potentials are given vs. SCE.

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Additionally, after the capture of CA72-4, the resistance increased again (curve e), which may be ascribed to the inhibition of electron transfer by the formed immune complex. Finally, when PANi–Au AMNPs labeled Ab2 interacted with CA72-4, the resistance decreased (curve f); it may be that the electronic conductivity of PANi–Au AMNPs was higher than the ability to inhibit electron transfer of the sandwich-type immune complex. The above results could clearly confirme the success of the assembly of the electrode. 3.3. Optimization of experimental conditions To achieve an optimal electrochemical signaling, the pH value of substrate solution was investigated. As shown in Fig. 5A, the response of the immunosensor to 5.0 mmol/L H2O2 for the detection of 150 U/mL CA72-4 at different pH was studied from 5.40 to 9.18. It can be seen that the current response was first increased and then decreased, and reached the maximum at 7.40. Thus, pH value of 7.40 was chosen and selected in our subsequent test. The time of etching Ag/Au was also investigated. Fig. 5B shows the effect of etching time of NPG film. The response of the immunosensor at different etching times was studied from 15 s to 60 s, and the current response of the electrode reached a maximum at 25 s. The reason maybe that the pore size of NPG increased along with the corrosion time growth, and the NPG after 25 s of etching was suitable for the loading of the antibody and sensing. Moreover, in the experiment, we chose Ag/Au alloy different from our previous work (Wei et al., 2011), and the corrosion time reduced greatly and the efficiency improved. 3.4. Performance of the immunosensor Under the optimum conditions, the amperometric response of the prepared immunosensor to different concentrations of CA72-4 is shown in Fig. 6A, which exhibited good linear relationships between the current change and the concentration of CA72-4 in the range from 2 to 200 U/mL. And the equation of the calibration curve: Y ¼0.814X þ120.46, r ¼ 0.9945. Based on S/N ¼3, a detection limit of 0.10 U/mL was obtained. The low detection limit may be attributed to two factors: (1) the large specific area of NPG film could be used to fix a large amount of Ab1, and the good catalytic activity of NPG film towards H2O2 can help electrochemical signal greatly improved; (2) the large amounts of Ab2 could bind on Au of PANi–Au AMNPs, and the high catalytic activity of

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PANi–Au AMNPs-Ab2 towards H2O2 can also increase the sensitivity. 3.5. Selectivity, reproducibility, and stability of the immunosensor The selectivity of the immunosensor was also tested by 25 U/mL of CA72-4 solution containing 1000 ng/mL of interfering substances (alpha fetoprotein, vitamin C, BSA, glucose) was measured by the immunosensor, and the designed immunosensor incubating the blank solution (without CA72-4) was also measured; the results are shown in Fig. 6B. The current variation due to the interfering substances was less than 5% of that without interferences, indicating that the selectivity of the immunosensor was acceptable. To evaluate the reproducibility of the immunosensor, a series of five electrodes were prepared for the detection of 50 U/mL of CA72-4. The relative standard deviation (RSD) of the measurements for the five electrodes was 4.5%, suggesting that the precision and reproducibility of the proposed immunosensor was quite good. To test the stability of the immunosensor, the sensor was stored in pH 7.40 PBS when not in use. After five and twenty days, the current response of the as-prepared immunosensor decreased respectively to about 93% and 89% of its initial value, suggesting that the stability of the proposed immunosensor was also good. The decrease in the current response may be due to the gradual denature of Ab1 and Ab2. 3.6. Determination of CA72-4 in serum samples In order to evaluate the feasibility of the proposed immunosensor for real sample analysis, the immunosensor was used for the determination of CA72-4 by standard addition methods in serum samples. The result showed that the recovery was from 99.8% to 101% and the RSD was from 0.9% to 2.9% (Table S1).

4. Conclusions In conclusion, a simple and effective enzyme-free electrochemical immunosensing platform based on NPG and PANi–Au AMNPs for CA72-4 detection was developed. The large surface area of NPG increased the amount of Ab1 immobilized onto electrode surface, and the combined properties of polyaniline and Au nano-particles present in PANi–Au AMNPs enhanced the reduction ability towards H2O2, which together with excellent catalytic activity of NPG improved the sensitivity and detection

Fig. 5. The effect of (A) pH and (B) the time of etching Ag/Au on the response of the immunosensor to 150 U/mL CA72-4.

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Fig. 6. (A) Calibration curve of the immunosensor toward different concentrations of CA72-4, Error bar¼ RSD (n¼ 5). (B) Amperometric response of the immunosensors to (1) 25 U/mL CA72-4; (2) 25 U/mL CA7-24þ1000 ng/mL alpha fetoprotein; (3) 25 U/mL CA72-4 þ1000 ng/mL vitamin C; (4) 25 U/mL CA72-4 þ1000 ng/mL BSA; (5) 25 U/mL CA72-4þ 1000 ng/mL glucose; (6) 0 U/mL CA72-4.

limit of the immunosensor. The immunosensor displayed a linear response for detection CA72-4 within a wide range (2–200 U/mL). The proposed biosensor shows a low detection limit (0.10 U/mL), good reproducibility, selectivity and acceptable stability. This ultrasensitive immunosensor may be quite promising in many potential applications for the detection of cancer biomarkers in clinical diagnostics.

Acknowledgment This study was supported by the National Natural Science Foundation of China (Nos. 21175057, 21375047, and 21377046), the Natural Science Foundation of Shandong Province (ZR2010ZR063), the Science and Technology Plan Project of Jinan (No. 201307010), and QW thanks the Special Foundation for Taishan Scholar Professorship of Shandong Province and UJN (No. ts20130937). All the authors express their deep thanks.

Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.08.043.

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