Analytica Chimica Acta 806 (2014) 128–135

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Amplified impedimetric aptasensor based on gold nanoparticles covalently bound graphene sheet for the picomolar detection of ochratoxin A Ling Jiang 1 , Jing Qian 1 , Xingwang Yang, Yuting Yan, Qian Liu, Kan Wang, Kun Wang ∗ Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, 212013, PR 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

• AuNPs–rGO was fabricated to provide vast binding sites for DNA strands.

• A sandwich aptasensor was designed with AuNPs–rGO as signal amplification platform. • 7∼ orders of magnitude in Rct was obtained compared with the AuNPs–rGO absence one. • The impedimetric aptasensor could detect OTA with picomolar sensitivity. • The sensitivity is 200∼ fold lower than that of most existed aptasensors using EIS.

a r t i c l e

i n f o

Article history: Received 8 August 2013 Received in revised form 30 October 2013 Accepted 2 November 2013 Available online 14 November 2013 Keywords: Gold nanoparticles covalently bound graphene Signal amplification Impedimetric Aptasensor Ochratoxin A

a b s t r a c t An amplified electrochemical impedimetric aptasensor for ochratoxin A (OTA) was developed with picomolar sensitivity. A facile route to fabricate gold nanoparticles covalently bound reduced graphene oxide (AuNPs–rGO) resulted in a large number of well-dispersed AuNPs on graphene sheets with tremendous binding sites for DNA, since the single rGO sheet and each AuNP can be loaded with hundreds of DNA strands. An aptasensor with sandwich model was fabricated which involved thiolated capture DNA immobilized on a gold electrode to capture the aptamer, then the sensing interface was incubated with OTA at a desired concentration, followed by AuNPs–rGO functionalized reporter DNA hybridized with the residual aptamers. By exploiting the AuNPs–rGO as an excellent signal amplified platform, a single hybridization event between aptamer and reporter DNA was translated into more than 107 redox events, leading to a substantial increase in charge-transfer resistance (Rct ) by 7∼ orders of magnitude compared with that of the free aptamer modified electrode. Such designed aptasensor showed a decreased response of Rct to the increase of OTA concentrations over a wide range of 1 pg mL−1 –50 ng mL−1 and could detect extremely low OTA concentration, namely, 0.3 pg mL−1 or 0.74 pM, which was much lower than that of most other existed impedimetric aptasensors. The signal amplification platform presented here would provide a promising model for the aptamer-based detection with a direct impedimetric method. © 2013 Elsevier B.V. All rights reserved.

1. Introduction ∗ Corresponding author. Tel.: +86 511 88791800; fax: +86 511 88791708. E-mail address: [email protected] (K. Wang). 1 These authors contributed equally to this work. 0003-2670/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2013.11.003

The occurrence of mycotoxins in agricultural staples is of great concern in food assurance due to human and animal health hazards such as nephrotoxicity, teratogenicity, cytotoxicity, and

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genotoxicity [1]. Ochratoxin A (OTA), one of the most toxic foodborne mycotoxins, can contaminate food commodities including cereals, wheat, barley, corn, coffee, and wine [2]. As a consequence, maximum tolerated levels of OTA have been fixed by different countries and organizations. For example, the European Commission has established the limited level of OTA in different food products including dried fruits (10.0 ng g−1 ), raw cereal grains (5.0 ng g−1 ), cereals (3.0 ng g−1 ), and wine and grape juice (2.0 ng g−1 ) [3,4]. Therefore, developing sensitive detection method to ensure food safety issues and prevent the risk of human OTA consumption is of great significance. The conventional analytical methods are based on high performance liquid chromatography [5], liquid chromatography coupled with tandem mass spectrometry [6], and fluorescence [7]. Despite their accuracy and low detection limit, these techniques suffer from high cost, long processing time, and requirement of technical skills and sophisticated equipments [8]. A common alternative for OTA detection is the immunoassay on the basis of antigen–antibody interactions such as enzyme linked immunosorbent assay and immunochromatographic strip [9]. These assays possess obvious advantages such as simplicity and reliability, but the preparation of antibodies is generally very expensive, labour-intensive, and time-consuming, while antibodies are susceptible to problems with their stability or modification [10]. These may be overcome by the use of OTA’s aptamers, which are artificial nucleic acids first selected by Cruz-Aguado and Penner in 2008 [11]. As an alternative to antibodies, aptamer exhibits many merits such as remarkable target diversity, high binding affinity, convenient automated-synthesis, ease-of labeling, and high stability [12]. Over five years, a series of assays using aptamers against to OTA have been developed, combining with a variety of signal transduction techniques such as fluorescent [13,14], electrochemical [15,16], and colorimetric transducers [17,18]. Among them, the analysis based upon electrochemistry is of particular interest due to their high sensitivity, simple instrumentation, low production cost, rapid response, and portability. Since OTA is always present at ultralow levels in samples, there are increasing demands for ultrasensitive electrochemical method, while this goal is difficult to achieve by a basic aptasensor. In this context, several amperometric detection schemes have been demonstrated on the basis of signal amplification such as rolling circle amplification [19], aptamer displacement amplification [20], and enzyme labeling amplification [21]. Although these methods offer advantages in terms of signal amplification, their practical use might be hampered by their high operation cost, complicated operating process or relatively poor stability of enzymes. Besides, DNA-functionalized gold nanoparticles (AuNPs) have been reported for the amplified amperometric detection of OTA, taking the advantage of their high stability, low cost, and labeling convenience [22]. By exploiting the signal amplification feature of AuNPs, the reported sensitivity for OTA was as low as 30 pg mL−1 . As one of the electrochemical technologies, electrochemical impedance spectroscopy (EIS) has been demonstrated to be one of the most powerful analytical tools for interfacial investigation [23,24]. Numerous EIS based bio-affinity sensors have been described for proteins, DNA-DNA, and antibody–antigen interactions [25]. Recently, several papers were also reported based on the use of impedance transduction, following changes in charge-transfer resistance (Rct ) on electrode surface based on the structure switch property of OTA’s aptamer [26–28]. Typically, an impedimetric aptasensor based on the covalently immobilized aptamers onto mixed Langmuir–Blodgett film has been explored to detect OTA. The results of EIS studies revealed the resulted aptasensor exhibited a detection limit of 100 pg mL−1 [26]. Apparently, the impedimetric aptasensor is simple, cost-effective, and requires no external modification of aptamer with enzyme or

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redox species, while its major obstacle lies in its lower sensitivity as compared with other technologies [29]. The reason for this predicament is the lack of efficient signal amplified platform adaptable for EIS method [30]. The sandwich system of aptamerI/thrombin/aptamerII-functionalized AuNPs (Apt-AuNPs) has been successfully demonstrated as the sensing platform for amplified impedimetric aptasensor for thrombin [30,31]. With the sole Apt-AuNPs or enlargement of Apt-AuNPs as a signal enhancer, allowed the detection of thrombin at a concentration down to 0.1 pM [30] and 20 pM [31]. However, the exploration of EIS to identify affinity complexes between aptamers and small molecules (e.g., OTA) is of great difficulty, since the reorganization of the aptamer-target complex on electrode surface produces a minute change in Rct compared to the free aptamer-modified electrode [21]. In this context, an efficient signal amplified platform for sensing impedance properties of the interface should be envisioned for the detection of OTA. With the aim to enhance the sensitivity of impedimetric aptasensor for OTA, we herein described a facile route to fabricate AuNPs covalently bound reduced graphene oxide (AuNPs–rGO), where a large number of AuNPs were homogeneously and densely planted on rGO surface via strong covalent bonding. Since a single rGO sheet and each AuNP could be loaded with hundreds of reporter DNA, AuNPs–rGO was used as an excellent carrier of reporter DNA. In this case, the negative assembled reporter DNA almost covered the whole surface of AuNPs–rGO and effectively repelled the [Fe(CN)6 ]4−/3− anions, and this offered a significant amplification method for the impedimetric detection of OTA by adopting a sandwich manner. Meanwhile, the proposed aptasensor has good regeneration, reproducibility, selectivity, sensitivity, and can detect OTA in real wine samples.

2. Experimental 2.1. Materials and chemicals Graphene oxide (GO) was synthesized from natural graphite powder by a modified Hummers method [32]. 1-Eethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), Nhydroxy succinimide (NHS), 2-aminothiophenol (2-ATP), chloroauric acid (HAuCl4 ·4H2 O), ethylenediaminetetraacetic acid disodium salt (EDTA), and 6-mercapto-1-hexanol (MCH) were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). Capture DNA (DNA1): 5 -HS-TGT CCG ATG CTC, aptamer: 5 -GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-3 , and reporter DNA (DNA2): 5 -CCA CAC CCG ATC-SH-3 were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). OTA, ochratoxin B (OTB), and fumonisin B1 (FB1) were obtained from Sigma–Aldrich. DNA oligonucleotide stock solutions were prepared with 50 mM Tris–HCl buffer (pH 7.4, containing 0.2 M NaCl, 1.0 mM EDTA) and kept frozen in dark. All of the reagents were used as purchased without further purification. Double-distilled water was used throughout the study.

2.2. Apparatus Transmission electron microscopy (TEM) was conducted using JEOL 2100 TEM technique (JEOL, Japan). X-ray photoelectron spectroscopy (XPS) was performed on ESCALAB 250 multitechnique surface analysis system (Thermo Electron Co., USA). A conventional three-electrode system was used with an Au electrode (2 mm in diameter) as working electrode, an Ag–AgCl reference electrode, and a platinum wire counter electrode. All the EIS measurements with a Zennium electrochemical workstation (Zahner, Germany) were performed under an oscillation potential of 5 mV

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Fig. 1. Overview of stepwise systhesis of AuNPs–rGO (top) and the preparation of AuNPs–rGO-DNA2 bioconjugation (bottom).

over the frequency range of 10 kHz–0.01 Hz, in a solution of 5 mM [Fe(CN)6 ]3−/4− containing 0.1 M KCl at room temperature.

2.3. Synthesis of AuNPs covalently bound rGO sheets 25 mg of GO was firstly activated with excess EDC and NHS in ethanol and the mixture was stirred for 8 h at room temperature. In the next step, excess of 2-ATP was added to the above mixture and stirred at room temperature for another 12 h. The 2-ATP functionalized GO (GO-SH) was washed repeatedly with ethanol to remove unbound 2-ATP and dried under vacuum. Subsequently, the freshly prepared sodium citrate aqueous solution (10 mL, 25 mg mL−1 ) was slowly added into the GO-SH suspension (50 mL, 0.2 mg mL−1 ) under refluxing and stirring. The mixture was further refluxed and stirred for 2.5 h, whereupon 150 ␮L HAuCl4 (2 wt.% in water) was quickly added to the above solution and reflux was continued for another 30 min. Finally, the resulting homogeneous black AuNPs–rGO suspension was centrifuged at 12,000 rpm and washed with water; it was re-dispersed into 10 mL water by sonication for further use. The process for the synthesis of AuNPs–rGO is illustrated in Fig. 1 (top).

2.5. Fabrication of the aptasensor A clean Au electrode was firstly immersed into 5 ␮M DNA1 solution in order to assemble the monolayer of DNA1 on the electrode surface. The assembly was kept for 6 h at room temperature, followed by rinsing with Tris–HCl buffer for several times. Then, the electrode was dried in a nitrogen stream, after which the interface was covered with 5 ␮L of 10 ␮M MCH and kept at room temperature for 1 h, followed by rinsing with Tris–HCl buffer. Then the electrode was covered with 5 ␮L of 5 ␮M aptamer solution for 2 h in order to assemble the monolayer of aptamer. After rinsing and drying as above, the sensing interface (aptamer/DNA1-Au) was immersed in different concentrations of OTA and incubated for 2 h, followed by washing with Tris–HCl buffer. Then, 5 ␮L of AuNPs–rGO-DNA2 was placed on the electrode surface for 1 h to obtain the AuNPs–rGO-DNA2/aptamer/DNA1-Au sandwich structure followed by rinsing with Tris–HCl buffer for several times. The whole process for the aptasensor fabrication is illustrated in Fig. 2.

3. Results and discussion 3.1. Preparation and characterization of AuNPs–rGO nanocomposites

2.4. Preparation of AuNPs–rGO functionalized reporter DNA The conjugation of the reporter DNA (DNA2) to the AuNPs–rGO (AuNPs–rGO-DNA2) was performed according to the literature [22]. Briefly, DNA2 solution (45 ␮L, 100 ␮M) was added to 1 mL of the as-prepared AuNPs–rGO suspension and incubated overnight before incubating with NaCl (100 ␮L, 2 M) for 24 h. After centrifugation at 13,000 rpm for 10 min, as much supernatant as possible was adsorbed with a pipet to remove the free DNA2. The precipitate was rinsed and centrifuged twice, and re-dispersed in 10 mL of 50 mM Tris–HCl buffer.

A proposed mechanism model for the formation of AuNPs–rGO was shown as top of Fig. 1. GO obtained with the modified Hummers method was easily suspended in water to form a stable colloidal solution due to the abundant surface oxygen-containing groups such as carboxyl and hydroxyl. Addition of EDC/NHS allowed the effective binding of the NH2 group of 2-ATP with the COOH group on the GO surface. This reaction would modify GO sheets with SH groups. Then, AuNPs–rGO hybrid was synthesized by a one-step reduction of the mixture of GO-SH and HAuCl4 . During this process, AuNPs were in situ attached on graphene surface through the

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Fig. 2. Schematic illustration of the impedimetric aptasensor with AuNPs-rGO as an excellent signal amplified platform.

well-developed Au S chemistry, while GO was also reduced into rGO by sodium citrate at the same time. The direct evidence for the successful immobilization of AuNPs onto the rGO surface was given by the TEM images of GO-SH (Fig. 3A) and AuNPs–rGO (Fig. 3B and C) at different magnifications. As can be seen from Fig. 3B and C, a large number of AuNPs with an average size of 15 nm were homogeneously and densely planted on the rGO surface, which would provide tremendous binding sites for DNA strands. Furthermore, no free AuNPs was observed outside of the rGO surface, indicating that AuNPs were well bound to rGO sheets via strong covalent binding. Important information on the chemical composition of AuNPs–rGO nanocomposites and the reduction of GO can be provided by XPS measurements. As shown in Fig. 4A, the wide scan XPS spectrum of AuNPs–rGO composites clearly indicated that the sample was composed of Au, S, C, N, and O elements. Fig. 4B and C displayed the high-resolution XPS spectra of C rGO and GO samples, respectively. Deconvolution of the C1s peak of AuNPs–rGO and GO showed the presence of three peaks at 284.8, 286.8, and 288.4 eV associated with C C, C O (epoxyl and hydroxyl), and C O (carbonyl), respectively [33]. It was obvious that the peak intensity of C O was strong in GO, in contrast, after the reduction process, the peak intensity of C O in AuNPs–rGO nanocomposites tremendously decreased, suggesting that most of the oxygen-containing functional groups were successfully removed. All the observations confirmed the successful synthesis of AuNPs–rGO nanocomposites. 3.2. Conjugation of reporter DNA to AuNPs–rGO surface AuNPs are well known to bind strongly with thiolated molecules, and the formation of self-assembled monolayers on AuNPs surface has been attributing to the famous Au S linkage.

Besides, graphene could strongly bind with DNA, as a result of hydrophobic and ␲-stacking interactions between the nucleobases and graphene sheets [34]. The combination of the aromatic scaffold as well as AuNPs was beneficial to the bioconjugation of thiolated reporter DNA (DNA2) with AuNPs–rGO via both covalent coupling reactions and noncovalent ␲–␲ stacking. On the basis of the carrier possessed dual functionality, a large number of DNA2 could be stably adsorbed on the graphene sheets, with the nucleobases “lying” nearly flat on the surface due to ␲–␲ stacking attraction, while the others were “standing” on the AuNPs surface through the Au S covalent binding (bottom of Fig. 1). XPS was used to confirm the thiolated DNA2 was successfully conjugated to the surface of AuNPs–rGO and obtain the key information concerning the binding modes of the DNA2. The Au 4f signal shown in Fig. 4D could be fitted with a pair of doublet peaks at 84.0 and 87.7 eV for both AuNPs–rGO (curve a) and AuNPs–rGO-DNA2 (curve b), corresponding to the 4f7/2 and 4f5/2 levels of metallic Au atoms in the both samples [35]. A maximum P 2p signal at 132.6 eV was observed for AuNPs–rGO-DNA2 (curve b, Fig. 4E); yet no clear peak was observed in the same region for AuNPs–rGO (curve a, Fig. 4E). These results indicated that the characterized P 2p line was from the phosphate backbones of DNA2, confirming DNA2 was immobilized onto the surface of AuNPs–rGO successfully [36]. Be different from the sole sulfur S 2p signal of AuNPs–rGO (curve a in Fig. 4F), the S 2p3/2 and S 2p1/2 doublet peaks at 163.7 and 164.6 eV were obviously observed for AuNPs–rGO-DNA2. According to previously published data, we assigned the peak at 163.7 eV (S 2p3/2 ) to Au S bond between DNA2 or 2-ATP and AuNPs [37]. Further, the remaining signal at 164.6 eV (S 2p1/2 ) was assigned to sulfur of free thiol groups (S H), which were not bound to the AuNPs [38]. Therefore, the anchoring of the thiolated DNA2 could be achieved by both models readily due to the presence of AuNPs as well as ␲ conjugated domains.

Fig. 3. TEM images of GO-SH (A) and AuNPs–rGO at low (B) and high (C) magnification.

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Fig. 4. The wide scan XPS spectrum of AuNPs–rGO (A). C 1s XPS spectra of AuNPs–rGO (B) and GO (C). Au 4f (D), P 2p (E), and S 2p (F) XPS spectra of AuNPs–rGO (a) and AuNPs–rGO-DNA2 (b).

3.3. AuNPs–rGO as an excellent signal amplification platform for the impedimetric aptasensor A typical sandwich aprasensor was designed for OTA, as illustrated in Fig. 2. Specifically, the thiolated capture DNA (DNA1) was immobilized on the gold electrode through the formation of Au S bond. After assembled with MCH, the aptamer against OTA was linked to the electrode through partial hybridization with DNA1 to obtain the sensing interface of aptamer/DNA1-Au. The properties of bare electrode (curve a), DNA1-Au (curve b), and aptamer/DNA1-Au (curve c) were investigated by EIS (Fig. 5A), exploiting the solutionbased redox probe [Fe(CN)6 ]3−/4− . In the EIS, the semicircle portion observed at high frequencies corresponded to the electron transfer limiting process. The Rct value can be directly measured as the semicircle diameter. When DNA1 was modified on the electrode surface and then treated with MCH, the Rct value increased obviously as compared to that of bare electrode, due to the presence of negatively charged backbones of DNA strands and the blocking effect of MCH which retarded the interfacial electron-transfer kinetics of the probes [39–41]. When the aptamers were partially hybridized with DNA1, a half-duplex structure was formed and more negatively charged phosphate backbones could repel the negatively charged redox probe, leading to a further enhancement of Rct value, which demonstrated the aptamer had been successfully immobilized on the electrode [42]. To illustrate the signal amplification from AuNPs–rGO-DNA2, the aptamer/DNA1 modified electrode was not treated with OTA for convenience, followed by the conjugation of AuNPs–rGO-DNA2 to form a sandwich manner of AuNPs–rGO-DNA2/aptamer/DNA1Au, and the EIS was thus recorded as curve d in Fig. 5A. In this case, the AuNPs and rGO were no longer well conductors (here for [Fe (CN)6 ]4−/3− probe) but negatively charged complexes capped with tremendous negatively charged phosphate backbones, which caused effective repulsion between the similarly charged redox

couple. That was largely attributed to the fact that the single rGO sheet and each AuNP on rGO sheet could be loaded with hundreds of DNA2. Both the bulky effect of AuNPs–rGO and the charge-effect of DNA2 contributed to the dramatic enhancement of Rct value from 4626  (curve c) to 32687  (curve d), at least 7 orders of magnitude than that of aptamer/DNA1-Au without functionalized AuNPs–rGO. These results indicated that the signal amplification based on AuNPs–rGO was effective for the sandwich aptasensor by EIS. 3.4. The optimization of important factors To generate a sensitive and stable aptasensor with a low detection limit for OTA, it is significant to optimize important parameters in the sensor fabrication process. As shown in Fig. 5B, the DNA1 modification in the first step had a saturation value for the amount of DNA1 on the electrode surface. Before saturation, the Rct of AuNPs–rGO-DNA2/aptamer/DNA1-Au (without the incubation with OTA) increased with the increasing amounts of DNA1, and the Rct value tended to be stable when the concentration of DNA1 was above 5 ␮M. Therefore, 5 ␮M was selected to be the optimal concentration of DNA1. The normal ratio of the hybrid events between two complementary DNA strands is 1, it is reasonable to choose 5 ␮M as the suitable concentration of aptamer for our following experiments. Because DNA hybridization is a time-dependent process, the hybridization time between DNA1 and aptamer was also optimized (without the incubation with OTA for convenience). The hybridization time from 30 to 180 min was studied as shown in Fig. 5C. It was apparent that Rct value of AuNPs–rGO-DNA2/aptamer/DNA1-Au obviously increased with the increasing interaction time from 30 to 90 min and then reached a plateau in 120 min. This suggested that 120 min was enough and thus chosen as the hybridization time in our following research. Since the reaction time of OTA with its aptamer was closely related

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Fig. 5. (A) EIS responses of bare (a), DNA1-Au (b), aptamer/DNA1-Au (c), and AuNPs–rGO-DNA2/aptamer/DNA1-Au (d). (B) EIS responses of AuNPs–rGO-DNA2/aptamer/DNA1Au obtained by different concentrations of DNA1. (C) The different hybridization time of DNA1 with aptamer. (D) The different binding time of OTA with the aptamer.

to the aptasensor fabrication, the effect of the binding time was also optimized. As shown in Fig. 5D, it was apparent that Rct value of AuNPs–rGO-DNA2/aptamer/DNA1-Au (incubated with 1 ng mL−1 of OTA) obviously decreased with the increasing binding time from 30 to 90 min and then reached a plateau in 120 min. This suggested that 120 min was enough and thus chosen as the incubation time in the following research. 3.5. Impedimetric detection of OTA with picomolar sensitivity As shown in Fig. 2, the aptamers were firstly immobilized on Au electrode surface by complementary pairing to DNA1, followed by incubating with the OTA solution with desired concentrations. The more target molecules in the tested solution, the more aptamers combined with the targets and released into solution, followed by a reducing amount of AuNPs–rGO-DNA2 linked to the electrode, leading to a substantial decrease in Rct . This explanation was consistent with the Nyquist plots which showed a decrease in Rct as the OTA concentration increased in the detection system as shown in Fig. 6A. These EIS data were fitted well with the Randles equivalent circuit (inset of Fig. 6A) as evident from the low percentage error values. The fitting parameters involved the resistance of the solution (Rs ), Rct , Warburg impedance (Zw ) attributed to the contribution of diffusion, and the constant phase element (Q). The obtained Rct values after fitting were used to perform the calibration curves. By analyzing the Rct value with the concentrations of OTA (Fig. 6B), we obtained a linear relationship (inset in Fig. 6B) between the logarithm of Rct value and the logarithm of the concentration of OTA over a range of 1 pg mL−1 –50 ng mL−1

with an extremely low detection limit at 3, namely 0.3 pg mL−1 or 0.74 pM. The reproducibility of the aptasensor was also investigated at the OTA concentration of 1 ng mL−1 , and the relative standard deviation (RSD) for five times was 6.3%. Besides, the aptasensor can be regenerated by adding new aptamer solution onto the sensing interface through hybridization, followed by incubated with OTA and AuNPs–rGO-DNA2, accordingly. The investigation of the repetitive use was carried out by the recovery of EIS at the OTA concentration of 1 ng mL−1 , the results demonstrated that the aptasensor could be regenerated at least five times with the RSD of 7.9%. The selectivity of the as-prepared aptasensor was illustrated in Fig. S1. As can be seen, the response signals to the FB1 and OTB were almost neglectable while an obvious decrease in Rct was observed at the same concentration of OTA, indicating that the aptasensor was specific to OTA and possessed high selectivity. Characteristics such as the detection strategy, the linear range of the corresponding calibration graph, and the limit of detection achieved were all summarized in Table 1. As can be seen, the present aptasensor possessed a broader linear range and lower detection limit, its sensitivity was approximately 200∼ fold higher than most other existed impedimetric aptasensors for OTA, the detection limit of which was down to 50 pg mL−1 [25] or 100 pg mL−1 [26]. The binding constant, namely the association constant (Ka ), between OTA and its aptamer can be obtained by Langmuir adsorption isotherm [43]. The Ka value obtained in our work was 1.44 × 107 M−1 , similar with that reported by Malhotra’s group [26] in the absence of Ca2+ (1.21 × 107 M−1 ). It was reported that the presence of Ca2+ can enhance the binding between OTA and its aptamer, so the Ka value was about 6∼ fold lower than that

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Fig. 6. (A) EIS responses for the detection of different concentrations of OTA, from a to h: 0.001, 0.005, 0.01, 0.05, 0.1, 1, 10, and 50 ng mL−1 . Inset: the equivalent circuit. (B) The relationship between Rct and the concentration of OTA. Inset: the calibration curve of the detection. Table 1 Characteristics of the present aptasensor along with others reported in the literatures. Sensor a

FAM ssDNA/aptamer-AuNPs dsDNA/MCH/ssDNA-Au ABEIb -DNA2/DNA1/AuNP-Au Thiolated DNA aptamer-Au Apt-DNA/PANIc –SAd -ITO AuNPs–rGO-DNA2/aptamer/DNA1-Au a b c d e f g

Analytical method e

FL DPVf ECLg EIS EIS EIS

Liner range (ng mL−1 )

Detection limit (pg mL−1 )

References

0.005–5 0.005–10 0.02–3 0.04–40 0.10–10 0.001–50

2 1 7 50 100 0.3

[44] [20] [45] [25] [26] This work

Fluorescein amidite. N-(4-aminobutyl)-N-ethyl-isoluminol. Polyaniline. Stearic acid. Fluorescence. Differential pulse voltammetry. Electrochemiluminescence.

calculated in the presence of 20 mM Ca2+ (8.3 × 107 M−1 ) [11]. However, the obtained Ka value was even much lower in comparison with that determined in the binding solution without Ca2+ , e.g., 1.10 × 108 and 1.2 × 108 M−1 [22,25]. As the Ka value of the present work was not large, we may conclude that the high sensitivity of the present aptasensor benefit from the signal amplification strategy with AuNPs–rGO as an excellent carrier of reporter DNA. 3.6. Analytical application in wine samples The analytical performance of the impedimetric aptasensor has been evaluated by the standard addition method in real red wine samples. Theses samples purchased from the local supermarket were subjected to a simple pre-treatment process according to the reported method [14]. After filtrating, the solution was adjusted to pH 7.4 and diluted 10∼ fold for later use. Subsequently, OTA was added to the treated real wine samples at three concentrations of 0.05, 0.1, and 10 ng mL−1 . The recovery in the range of 90–97% was acceptable according to the analytical results shown in Table S1, while the RSD values for the three concentrations were below 7.1%. This implies that the as-prepared aptasensor has a promising feature for the practical use in red wine samples. 4. Conclusions A novel AuNPs–rGO platform was constructed and used as an excellent signal amplified platform for impedance aptasensor. The decoration of AuNPs on graphene provided tremendous binding sites for reporter DNA, since a single rGO sheet and each AuNP could be loaded with hundreds of reporter DNA. In this sense, a small amount of residual aptamers was expected to efficiently

bind a tremendous amount of negatively charged phosphate backbones, resulting in an amplified Rct signal of the redox probes compared with that of the aptamer simply without functionalized AuNPs–rGO. Through such amplified strategy, a detection limit of 0.3 pg mL−1 , or namely 0.74 pM, was realized, which was approximately 200∼ fold lower than that of most other existed impedimetric aptasensors. As the AuNPs and/or rGO can be functionalized with other reporter DNA and signal probes, the sensor designed here provides a promising signal amplification strategy for aptamer-based detection for other molecules with even other techniques.

Acknowledgements The present work was supported by the National Natural Science Foundation of China (No. 21375050 and 21175061), the Natural Science Foundation of Jiangsu province (No.BK20130481), China Postdoctoral Science Foundation (No.2012M520998.), major Program of Natural Science Foundation of Education Bureau of Jiangsu Province, China (Grant No.340 10KJA470007), Key Laboratory of Modern Agriculture Equipment and Technology 341 (No.NZ201109), Jiangsu Planned Projects for Postdoctoral Research Funds (1301141C), and Research Foundation of Jiangsu University (12JDG087).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2013.11.003.

L. Jiang et al. / Analytica Chimica Acta 806 (2014) 128–135

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