AuNPs nanocomposite for label-free detection of bisphenol A

Accepted Manuscript Title: A novel electrochemical aptasensor based on f-MWCNTs/AuNPs nanocomposite for label-free detection of bisphenol A Author: Behjat Deiminiat Gholam Hossein Rounaghi Mohammad Hossein Arbab-Zavar Iman Razavipanah PII: DOI: Reference:

S0925-4005(16)31832-9 http://dx.doi.org/doi:10.1016/j.snb.2016.11.041 SNB 21251

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

11-9-2016 4-11-2016 8-11-2016

Please cite this article as: Behjat Deiminiat, Gholam Hossein Rounaghi, Mohammad Hossein Arbab-Zavar, Iman Razavipanah, A novel electrochemical aptasensor based on f-MWCNTs/AuNPs nanocomposite for label-free detection of bisphenol A, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.11.041 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A novel electrochemical aptasensor based on f-MWCNTs/AuNPs nanocomposite for label-free detection of bisphenol A

Behjat Deiminiat, Gholam Hossein Rounaghi*1, Mohammad Hossein Arbab-Zavar, Iman Razavipanah

Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad-Iran

*

Corresponding author: Tel: +98 5137626388

E-mail address: [email protected]; [email protected]

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Highlights 

A new electrochemical aptasensor was constructed for determination of bisphenol A.



F-MWCNTs/AuNPs nanocomposite was introduced to improve performance of the sensor.



The proposed sensor was used successfully for bisphenol A determination in real samples such as mineral water, orange juice and milk.

Abstract A new label-free electrochemical aptasensor was constructed for sensitive and selective determination of bisphenol A (BPA) based on functionalized multiwall carbon nanotubes /gold nanoparticles (f-MWCNTs/AuNPs) nanocomposite film modified gold electrode. The fMWCNTs/AuNPs nanocomposite was synthesized chemically and the structure of the prepared nanocomposite was characterized by UV-Vis spectrophotometry, Fourier transform infrared (FT-IR) spectrometry, X-ray diffraction (XRD) and transmission electron microscopy (TEM). The fabrication process of the electrochemical sensor was also investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in the presence of [Fe(CN)6]3−/[Fe(CN)6]4− as an electrochemical active probe. The molecular dynamic (MD) simulations were used to study the interaction between BPA and its aptamer molecules. The effect of several parameters influencing the performance of the aptasensor was investigated and they were optimized. Under the optimized experimental conditions, the square wave voltammetry (SWV) was applied as a sensitive analytical method for determination of BPA in solutions and a good linear relationship was observed between the BPA concentration and the peak current within the range of 0.1–10 nM with a detection limit of 0.05 nM. The effect of interfering species on the determination of BPA was investigated and it was found that the proposed aptasensor is highly selective to BPA. Also, the reproducibility and stability of the sensor were all found to be satisfactory. Finally, the developed aptasensor was successfully applied for determination of BPA in real samples such as mineral water, orange juice and milk.

Keywords: Label-free electrochemical aptasensor, Bisphenol A, Nanocomposite, Molecular dynamic simulations, Aptamer.

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1. Introduction Bisphenol A [BPA, 2,2-bis (4-hydroxyphenyl)propane] is an important organic compound that is widely used as an intermediate for the manufacture of polycarbonate plastic, epoxy resin, flame retardants and the other special products. The plastic products and epoxy resins are extensively employed for baby bottles, food can linings, beverage containers and thermal papers used in sales receipts, from which the BPA molecules can release into the food, drink and environment, therefore, the humans may daily ingest trace amounts of BPA [1,2]. Recently, BPA has attracted more attention from regulatory agencies and scientists throughout the world, because it has hormone-like properties, estrogenic activity and acts as an environmental endocrine disruptor. Furthermore, many kinds of adverse effects, including the cancers, decrease fertility, reduce immune function and the other diverse pleiotropic actions in the brain and cardiovascular system, have been reported in human and animals due to the long exposure to BPA [3-5]. Therefore, development of a rapid, simple, sensitive, selective and economic analytical method for determination of BPA is of significant importance from the industrial, environmental and health viewpoints. Until now, a variety of analytical methods have been reported for determination of BPA including fluorimetry [6,7], liquid chromatography [8-10], gas chromatography [11-13], enzyme-linked

immunosorbent

assay

(ELISA)

[14-16],

and

flow

injection

chemiluminescence [17]. Although these techniques can offer a good sensitivity and accuracy, but some of the factors such as expensive instrumentation, time consuming and complicated procedures, have restricted their application. Some electrochemical methods also have been developed for BPA detection using chemically modified electrodes [18-21]. However, these electrochemical sensors suffer from a moderate selectivity. Aptamers are bio-recognition elements with a high specificity and affinity for the various targets, ranging from large molecules such as proteins, peptides, amino acids, and complex molecules of drugs to organic small molecules or even metal cations [22-24]. It has been recognized that the aptamer affinity toward the target molecules, is comparable to or even higher than that of antibodies [25,26]. Aptamers are small single-stranded DNA (ssDNA) or RNA sequences (oligonucleotides of ~100 nucleotides or less) that are selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX) [27-29]. They are thermally stable, reusable, resistant to denaturation and degradation, and they can be easily modified for their detection and immobilization by introducing of certain functional groups. Therefore, the aptamer-based sensors, have been widely used for detection of cancer cells,

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drugs, some of the organic molecules, and also a variety of proteins. Among the various aptasensors, the electrochemical aptasensors have received a great deal of attention due to their excellent sensitivity, rapid response, simplicity and low cost [30-32]. In order to improve the sensitivity of the electrochemical aptasensors, the most effective way is the use of conducting nanomaterials to modify the surface of the electrodes. Gold nanoparticles (AuNPs) and carbon nanotubes (CNTs) are the two most common nanosized materials for construction of electrochemical sensors and biosensors. Gold nanoparticles are one of the most stable metal nanoparticles, easy to synthesize in a variety of sizes; there is a protocol to modify these nanoparticles with additional functionality, and they possess unique properties such as strong adsorption ability, large specific surface area, good biocompatibility and conductivity [33]. Since the discovery of the carbon nanotubes in 1991 [34], they have been employed for the development of hybrid nanomaterials because of their excellent mechanical, chemical, electrical, and thermal properties [35]. In order to enhance the features of both of these types of materials, there have been much efforts in recent years to fabricate the nanocomposites where the CNT acts as a support for AuNPs [36-38]. Preparation of carbon nanotubes/gold nanoparticles (CNTs/AuNPs) nanocomposites, requires the design of the methods that lead to robust the structure without significantly compromising the integrity of the underlying CNT framework. Thus, development of a simple and efficient method for preparation of CNTs/AuNPs nanocomposites with uniform dispersion of the AuNPs onto the CNT and tuning the size of AuNPs is a main challenge of CNTs/AuNPs nanocomposites fabrication and their application. In this study, a new sensitive and selective electrochemical aptasensor was developed for the indirect determination of BPA based on f-MWCNTs/AuNPs nanocomposite film. The aptamer was immobilized on the surface of f-MWCNTs/AuNPs nanocomposite film modified gold electrode through the formation of thiol–gold (S–Au) bonds between the gold nanoparticles and the thiol group of the aptamer at 5′ end. The interaction between BPA and its aptamer was investigated theoretically by molecular dynamic (MD) simulations. To our knowledge, the synthesis and use of f-MWCNTs/AuNPs nanocomposite and aptamer in construction of an electrochemical sensor for determination of BPA have not been reported until yet. The function of the f-MWCNTs/AuNPs nanocomposite and the performance of the proposed sensor were investigated by electrochemical methods in detail. Moreover, the developed sensor was applied for determination of BPA in real samples. The incorporation of f-MWCNTs, AuNPs and aptamer, led to enhance the sensitivity and selectivity of the fabricated electrochemical sensor. 4

2. Experimental 2.1. Chemicals The synthetic aptamer against BPA molecules with the sequence: 5'-SH-(CH2)6-CCG GTG GGT GGT CAG GTG GGA TAG CGT TCC GCG TAT GGC CCA GCG CAT CAC GGG TTC GCA CCA-3' which has been designed by Jo and et al. [39], was purchased from Sangon Biotechnology Co. Ltd. China. MWCNTs (>95٪, OD 5-15 nm) were obtained from US research nanomaterials, Houston, USA. Hydrogen tetrachloroaurate trihydrate (HAuCl4.3H2O),

6-mercapto-1-hexanol

(MCH)

and

tris-(2-carboxyethyl)

phosphine

hydrochloride (TCEP) were purchased from Sigma-Aldrich chemical company (USA). Bisphenol A (BPA), bisphenol B (BPB) and tetrakis (hydroxymethyl) phosphonium chloride (THPC) were supplied from Merck chemical company (Darmstadt, Germany). Bisphenol AF (6F-BFA) and 4,4'-biphenol (BP) were obtained from Exir (Germany) and Alfa Aeser (Germany), respectively. All of the solvents and the other reagents used in this study were of analytical grade and were obtained from Merck (Darmstadt, Germany). 2.2. Instrumentation The electrochemical measurements were performed using an Autolab PGSTAT 101 (Metrohm Autolab, Utrecht, The Netherlands, NOVA software) connected to a conventional three-electrode cell. A modified gold electrode, a platinum wire and a saturated Ag/AgCl electrode were used as working, counter and reference electrodes, respectively. Gill AC potentioastate (ACM instruments) was used for the electrochemical impedance spectroscopic measurements. A Metrohm pH meter (Model 827) was used for measurement the pH of the solutions. The transmission electron microscopy (TEM) images were taken with a LEO 912 AB transmission electron microscope. Surface elemental analysis was performed by the energy dispersive X–ray (EDX) technique using Oxford-7353 EDX microanalyzer. X-ray diffraction (XRD) analysis was carried out on an X-ray diffractometer (Philips analytical Xray). The Fourier transform infrared (FT-IR) spectra were obtained by an AVATAR-370 Fourier transform infrared spectrometer. Also, the UV-Vis spectra were recorded by an Agilent 8453 UV/Vis spectrophotometer. 2.3. Synthesis of f-MWCNTs/AuNPs nanocomposite The f-MWCNTs/AuNPs nanocomposite was synthesized chemically by the following procedure: at the first step, the carboxylic functional group (COOH) was grafted on the surface of the MWCNTs to enhance their dispersion and compatibility. For this purpose, 0.3 g of raw MWCNTs and 50 ml of nitric acid were added into a round-bottomed glass flask,

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and the resulting mixture was sonicated for 30 min in an ultrasonic bath. Next, it was refluxed at 120 °C with a vigorous stirring rate for 24 h. After cooling to room temperature, the mixture was filtered on a 0.22 µm polycarbonate membrane and washed thoroughly with distilled water for several times until the pH of the filtrate was neutral. The filtered solid was dried under vacuum for 12 h to obtain the carboxylic acid-functionalized MWCNTs (fMWCNTs). Then, the f-MWCNTs/ AuNPs nanocomposite was prepared by the following three step procedure. At the first step, for formation of the colloidal AuNPs, 0.5 mL of 1 M NaOH and 1 mL of THPC solution were added to 50 mL of HPLC grade water. The mixture was stirred for 5 min under a strong vortex in a reaction flask. Then, 1.0 ml of 1% HAuCl4 aqueous solution was added to the stirred solution, and it was stirred further for 30 min. Mean-while, the color of the solution changed quickly from colorless to dark reddish yellow and the resulting solution was stored at 4 ºC for at least 3 days before further use. For attachment the colloidal AuNPs to f-MWCNTs, 0.3 g of f-MWCNTs was dispersed in 6 mL ethanol under sonication. Then, 45 mL of gold colloid solution was added to the above mixture and it was gently stirred for 3 h and then centrifuged at 10000 rpm. The resulted sediment was redispersed in 15 ml HPLC grade water by sonication and centrifuged for at least 3 times. In order to grow the gold NPs on the f-MWCNTs/AuNPs, 0.0375 g of potassium carbonate, as a strong reducing agent, was dissolved in 150 mL of HPLC grade water by stirring for 3 min. Then, 3 mL of a solution of 1% HAuCl4 in water and 15 mL of the solution containing the f-MWCNTs/AuNPs were added while the solution was stirred vigorously. Finally, 225μl of formaldehyde was injected slowly to the solution and it was stirred for 30 min at 65 0C. After cooling the solution to room temperature, it was centrifuged and redispersed in HPLC grade water to remove the residual reactants. At the last step, the solid products were dried for 24 h under vacuum at 60 ◦C to obtain the f-MWCNTs /AuNPs nanocomposite powder. 2.4. Preparation of f-MWCNTs/AuNPs based aptasensor Prior to modification the gold electrode surface, it was polished thoroughly with 0.3 µm and 0.05 µm alumina slurry and sonicated in double distilled water and ethanol for 5 min, respectively. Then, the electrode surface was cleaned with piranha solution (1:3 mixture of 30% H2O2 and concentrated H2SO4) and rinsed with double distilled water and then allowed to dry. Finally, it was subjected to sweep the potential cyclically between -0.2 and 1.5 V in 1.0 M H2SO4 solution until a stable cyclic voltammogram was obtained. Then, 5.0 µL of 1.0 mg mL-1 suspension of f-MWCNTs/AuNPs nanocomposite in dimethylformamide (DMF) was dropped onto the surface of pretreated gold electrode and the solvent was evaporated at 6

room temperature. Then, the aptamer was immobilized onto the surface of the fMWCNTs/AuNPs nanocomposite modified gold electrode by self-assembly technique. For this purpose, the aptamer solution was reduced in 1 mM tris-(2-carboxyethyl ) phosphine hydrochloride (TCEP) for 1 h to cleave the disulfide bonds. Then the modified gold electrode was incubated in 10 mM Tris-HCl solution (pH 7.6) containing 1 μM aptamer for 18 h at 4 °C in a 100% moisture-saturated environment and subsequently it was immersed in a solution of 2 μM 6-mercapto hexanol (MCH) for 1 h. Finally, the prepared electrode was rinsed with double distilled water and dried under N2 stream. Then, the fabricated electrode was applied as an electrochemical aptasensor for determination of BPA in the subsequent studies. 2.5. Electrochemical measurements The interaction between BPA molecule and its aptamer was performed by incubating the modified electrode into a buffer solution (25 mM Tris-HCl, pH 8.0 with 100 mM NaCl, 10 mM MgCl2 and 25 mM KCl) containing 2.0 nM BPA for 40 min at room temperature. Then, electrochemical measurements were carried out in a probe solution containing 0.1 molL-1 KCl and 5.0 mmolL-1 K3[Fe(CN)6]/K4 [Fe(CN)6] redox pair. The cyclic voltammograms were recorded in the potential range from -0.10 to 0.50 V with a scan rate of 50 mV s-1. Electrochemical impedance spectroscopy (EIS) was used at an applied potential of 0.20 V, amplitude of 10 mV and in a frequency range of 0.1–30000 Hz. The square wave voltammograms (SWVs) were obtained under the step potential of 1 mV with amplitude of 20 mV and frequency of 15 Hz. All electrochemical measurements were performed at room temperature. 2.6. Sample preparation The mineral water (Nestle), orange juice (Minute Maid) and milk (Pegah) samples were purchased from a local supermarket (Mashhad, Iran). The mineral water sample was mixed with Tris–HCl buffer solution and tested without any further pretreatment. Prior to analysis, the orange juice sample was centrifuged for 15 min in order to remove the precipitated materials. Then the sample was diluted with Tris-HCl buffer solution to obtain the required concentration for assay. 5.0 mL of fresh liquid milk was first added to 20 mL of Tris-HCl buffer solution. After 15 min sonication and 10 min shaking, the resulting mixture was centrifuged for 10 min, and then the supernatant of the solution was diluted with Tris-HCl buffer solution. Finally, the prepared samples were spiked with certain amounts of BPA.

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3. Results and discussion 3.1. Characterization of the synthesized f-MWCNTs/AuNPs nanocomposite The spectra of UV-Vis, FT-IR and the XRD pattern of f-MWCNTs/AuNPs nanocomposite are shown in Fig. 1. Figure 1A, shows the UV-Vis spectrum of f-MWCNTs/AuNPs nanocomposite. As is evident in this Figure, two characteristic absorption bands are observed in this spectrum. One of them is located at about 261 nm, which is due to the absorption peak of f-MWCNTs, and the second peak is appeared at about 538 nm, corresponding to the absorption peak of the AuNPs [36]. The synthesized f-MWCNTs/AuNPs nanocomposite demonstrates a slight blue shift in the absorption band in comparison with the previously reported values for the CNTs/Au hybrids [40]. The FT-IR spectra of f-MWCNTs and fMWCNTs/AuNPs nanocomposite are shown in Figure 1B. The peak at 3400 cm-1 can be attributed to the stretching vibration of –OH bond. After coating the f-MWCNTs with the AuNPs, the intensity of –OH peak reduces significantly which confirms the formation of the AuNPs onto the f-MWCNTs surface. The XRD patterns of f-MWCNTs and fMWCNTs/AuNPs nanocomposite are displayed in Figure 1C. As is evident in this Figure, the f-MWCNTs show a typical peak at 26°, and the peaks which are appeared at about 38.2°, 44.4°, 64.7°, 77.7° and 81.8,° can be assigned to the gold face-centered cubic (fcc) phase according to the data of the JCPDS file (04-0784) [36]. The morphology, structure and composition of the f-MWCNTs/AuNPs nanocomposite were investigated using transmission electron microscopy (TEM) and energy dispersive X-ray analysis (EDX). As is shown in Fig. 2A, the AuNPs with the size of about 12 nm, are uniformly dispersed on the f-MWCNTs. Fig. 2B, shows the energy dispersive X-ray (EDX) spectrum of the synthesized f-MWCNT/AuNPs nanocomposite which confirms that the AuNPs have been successfully attached onto the f-MWCNTs surface. The advantages of the method used in this research work for fabrication of the f-MWCNTs/AuNPs nanocomposite, include its simple route, easy preparation of similar size of AuNPs and also the uniform attachment of the AuNPs to the f-MWCNTs compared to the other methods for fabrication of metal NPs on the surface of CNTs or the other carbon substrates [41]. 3.2. Design strategy of the aptasensor Fig.3 shows a schematic presentation of the sensing strategy of BPA molecules using singlestrand DNA (ssDNA) aptamer-embedded on f-MWCNTs/AuNPs nanocomposite. As shown in this Figure, the aptamer molecules that are directly immobilized on the surface of fMWCNTs/AuNPs nanocomposite modified gold electrode, act as a gate of the long tunnels

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from which the redox probe [Fe(CN)6]3−/4− can reach the electrode surface. In the absence of BPA molecules, the aptamer molecules remain unfolded and the gate remains open, resulting in the passage of the electrons to the electrode surface. After exposition of the fabricated electrode to BPA molecules, the conformation of the aptamer is changed which limits the chance for the electron transfer of the [Fe(CN)6]3−/4− redox probe on the electrode surface. In addition, the binding of BPA molecules with the aptamer, exposes the negatively charged back bone of the aptamer to the redox probe, which leads to a further increase in the repulsion toward the electron transfer [42]. The molecular dynamic (MD) simulations were used for investigation the interactions between the aptamer and BPA molecules. Since the tertiary structure of the aptamer is not available, we designed a B-DNA composed of 63 nucleotides, and this structure was used as a model for aptamer (Fig. 4A). In order to obtain the tertiary structure of the aptamer molecules, 200 ns MD simulations in water were carried out on this structure. The folded structure of the aptamer after 200 ns MD simulations is shown in Fig. 4B. In order to study the effect of the BPA on the aptamer, the dynamical behavior of the folded aptamer in the presence and absence of BPA molecules during the 25 ns MD simulations was investigated. Fig. 4C, shows the structure of the aptamer with BPA after 25 ns MD simulations. According to this Figure, the BPA molecules have been absorbed by the aptamer molecule, and the structural analysis indicates that 6 BPA molecules have been trapped by the aptamer. The values of root mean square deviation (RMSD) of the folded aptamer with and without BPA during the 25 ns MD simulations were calculated and the results are depicted in Fig. 4D. As can be seen from this Figure, in the presence of the BPA molecules, the aptamer molecule has a lower fluctuation and the maximum value of the RMSD in this situation is 13.9 Å, which is a small value. Analysis of the radii of gyration (Rg) of the structures of aptamer (Fig. 4E), confirms the RMSD results and it reveals that the BPA molecules, reduces the aptamer fluctuations. The root mean square fluctuation (RMSF) plots (Fig. S1A) indicate that due to the BPA molecules interactions with the aptamer, the fluctuation of its residues decreases. Fig. S1B, shows the calculated electrostatic interaction energy between the BPA molecules and the aptamer, which indicates a remarkable interaction between the aptamer and BPA molecules that confirms the results of RMSD, Rg and RMSF. In order to show the effect of BPA molecules on the behavior of aptamer, the number of hydrogen bonds (H-bonds) of the aptamer with and without presence of BPA was calculated (Fig. S1C). As is evident in this Figure, in the absence of BPA, the average number of H-bonds is 110, while in presence of 9

the BPA molecules, it reduces to 95. Fig. S1D, shows the radial distribution function (RDF) between the BPA molecule and its aptamer. As is evident in this Figure, the first peak is around 1 Å, which demonstrates that the BPA molecules are very close to the aptamer molecule and, therefore, there is an effective interaction between the BPA molecules and the aptamer molecule. 3.3. Electrochemical characterization of the aptasensor Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) are valuable and convenient techniques to monitor the electrochemical properties of the modified electrodes. To investigate the electrochemical behavior of the stepwise fabrication process of the aptasensor, the CV and EIS measurements were carried out in 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] solution containing 0.1 M KCl at the bare and modified gold electrodes, and the results are shown in Figs. 5 and 6, respectively. At the bare Au electrode (curve 5a), a pair of redox peaks are obtained for [Fe(CN)6]3−/4−. As can be seen in curve 5b, after modification of the surface of the Au electrode with f-MWCNTs/AuNPs nanocomposite, the peak current increases sharply, which is due to the fact that the fMWCNTs/AuNPs nanocomposite, can significantly enhance the electrical conductivity of the electrode, which facilitates the electron transfer process. When the aptamer is immobilized on the electrode surface, a significant decrease in the peak current of the [Fe(CN)6]3−/4− with an increase in the peak separation (ΔE) is observed compared to the f-MWCNTs/AuNPs nanocomposite modified Au electrode. This behavior can be attributed to the electrostatic repulsion between the negatively charged deoxyribose-phosphate back bone of aptamer and [Fe(CN)6]3−/4− redox couple. In addition, when the aptamer/f-MWCNT/AuNPs nanocompsite/ Au electrode is incubated in 2.0 nM of BPA solution, the current response of the sensor decreases. It seems that the conformational change of the aptamer after binding to the analyte, blocks the arrival of the [Fe(CN)6]3−/4− probe anions onto the electrode surface. Fig. 6, shows the Nyquist diagrams of the modified electrode at different modification stages. As can be seen from this Figure, the charge-transfer resistance (Ret) is lower for fMWCNTs/AuNPs nanocomposite modified electrode than that for the bare electrode. The bare Au electrode exhibits a semicircle part at high frequencies (Ret=129.09 Ω) and a linear part at low frequencies. After modification of the Au electrode with f-MWCNTs/AuNPs nanocomposite (curve 6b), the Ret decreases to 3.0 Ω, which is an evidence for a faster electron transfer kinetics of [Fe(CN) 6]3−/4− anions on the electrode surface. Subsequently, when the aptamer is immobilized on the modified electrode surface, the EIS shows an increase in diameter (curve 6c), which confirms the successful self-assembly process of the 10

aptamer on the surface of the electrode. The binding of BPA molecules to the surface of aptasensor, results in a small increase in Ret. The increase in the EIS signal is due to a partial change of the aptamer conformation upon binding to BPA molecules, which results in a more retardation of [Fe(CN)6]3−/4− anions to the electrode surface and thus increasing the R et value. 3.4. Effect of different parameters on the sensor performance In order to obtain the best performance for the fabricated aptasensor, the effect of several parameters influencing the response of the sensor, such as: the aptamer and MCH concentration, aptamer and MCH incubation times, BPA detection time and the pH of the solution was investigated. The change in the current response of [Fe(CN)6]3−/4− redox probe (Δip) was calculated by subtracting the current which was obtained in the presence of BPA from the current recorded in the absence of BPA molecules. Effect of the aptamer and MCH concentration In order to achieve a high sensitivity for BPA detection, the concentrations of the aptamer and MCH were optimized. The performance of the aptamer/f-MWCNTs/AuNPs nanocomposite/ Au electrode was examined by varying the aptamer concentration and also MCH concentration in the range of 0.5-10 µM. As is shown in Figs. S2A and S2B, the optimum concentrations were found to be: 1 µM and 2 µM with respect to aptamer and MCH, respectively which they were chosen in the subsequent experiments. Effect of the aptamer and MCH incubation time The incubation time of the aptamer and MCH on the surface of the prepared aptasensor was also investigated. It was observed that the response current (Δi) increases with increasing the incubation time and it reaches a plateau at 18 h for the aptamer and 1 h for MCH, which indicates that the aptamer and MCH are saturated on the modified gold electrode surface beyond these times. Therefore, the optimum incubation times for the aptamer and MCH were chosen18 h and 1 h, respectively. Effect of BPA detection time The relationship between the current response and the BPA binding time was studied. The prepared sensor was incubated in a 2.0 nM BPA solution containing Tris-HCl buffer solution (pH 8.0) for different incubation times (Fig. S2C). As is evident in this Figure, the response current (Δi) increases with incubation time up to 40 min and then it remains nearly constant. The results obtained in this study, show that more BPA molecules are accumulated on the surface of the aptamer/f-MWCNTs/AuNPs nanocomposite/Au electrode with increasing the accumulation time up to 40 min, but it seems that beyond this time, the accumulation of BPA

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molecules tends to be saturated. Therefore, an accumulation time of 40 min was chosen for further studies. Effect of pH It was found that the pH of the solutions is an effective parameter on the BPA binding at the surface of the modified electrode. Therefore, the pH of the solutions needs to be optimized in the incubation step in order to make a suitable molecular conformation of BPA for better interaction with the aptamer. For this purpose, a series of solution of 2.0 nM BPA in the pH range of 5.0 to 10.0 was examined with an incubation time of 40 min (Fig. S2D). The maximum current response was obtained at pH 8.0, which was selected as a suitable pH in the subsequent studies. 3.5. Analytical performance of the electrochemical aptasensor Under optimized experimental conditions, the analytical performance of the present biosensor was evaluated by detecting BPA in standard solutions using the square wave voltammetry (SWV) technique. The aptasensor was incubated in different concentrations of BPA solution and the corresponding redox peak currents were recorded. The inset of Fig. 7, shows the decrease of the redox peak current of [Fe(CN)6]3−/4− with increasing the BPA concentration in solution. When the aptasensor is immersed in the solutions containing BPA, the BPA molecules combine with the aptamer molecule on the surface of the electrode and the redox peak current decreases with the sensing of BPA molecules. As is shown in Fig. 7, a linear range from 0.1 to 10.0 nM for concentration of BPA is observed with the regression equation of Δi = 3.2356C + 20.714 (R2= 0.9928), where C is the BPA concentration (nM) and Δi is the current difference (µA). The limit of detection (LOD) was found to be 0.05 nM based on 3Sb/S, where S and Sb represent the slope of the calibration curve and the standard deviation of the blank (n = 5), respectively. As is evident in Table1, our fabricated sensor has a lower detection limit compared to the other electrochemical sensors constructed by the other research groups for determination of BPA in solutions. The main purpose of the proposed aptasensor is to improve its selectivity so that the BPA molecules can be detected even in the presence of the other closely related compounds in solutions. In order to investigate the selectivity of the aptasensor, the bisphenol B (BPB), 4,4'-biphenol (BP) and 6F bisphenol A (6F-BFA) which have a similar structure to BPA were chosen as interfering species with an identical concentration (2.0 nM) in solutions. As is evident in Fig. 8, the effect of the interfering species on the electrochemical response to 2.0 nM bisphenol A is negligible. The good selectivity of the aptasensor can be attributed to the high affinity of the aptamer against BPA molecules. 12

The reproducibility of the aptasensor was estimated with determining the current response of 2.0 nM BPA solution using five fresh electrodes prepared independently under identical experimental conditions. The relative standard deviation (RSD) which was 3.5%, shows the good sensor-to-sensor reproducibility. The long term stability of the aptasensor was also investigated by measurement of 2.0 nM BPA solution at regular intervals for a period of two weeks. At first, the aptasensor was incubated in 2.0 nM BPA solution and its response was recorded. After regenerating the electrode, it was stored in a refrigerator at 4 °C, and then examined at regular intervals after it was incubated with 2.0 nM BPA. When the constructed sensor was stored at 4 °C, its electrical response retained 94% to its initial value which demonstrates that the developed sensor has an acceptable stability. The proposed sensor was successfully applied for determination of BPA in mineral water, orange juice and milk samples, which were prepared by the procedure described in section 2.6. The prepared samples, were spiked with two different concentrations of BPA solution (0.5 and 1.0 nM) and three replicate measurements were performed at each concentration. The experimental results are summarized in Table 2. As is seen in Table 2, the samples which obtained from the market, are free of BPA. In addition, the values of recovery are between 96% and 106% with RSDs of 2.5–4.2%, which indicate that the aptasensor can be successfully applied for determination of BPA in real samples.

4. Conclusion A novel label-free electrochemical aptasensor was developed for determination of bisphenol A (BPA) by immobilizing its aptamer on the surface of f-MWCNTs/AuNPs nanocomposite film modified gold electrode. Molecular dynamic (MD) simulations on the folded aptamer structures in the presence and absence of BPA reveal that the aptamer can absorb the BPA molecules and the structural analysis indicates that 6 BPA molecules are trapped by the aptamer. The results obtained from molecular dynamic (MD) simulations, show that BPA molecule has a remarkable interaction with its aptamer and it has important effect on the aptamer structural properties. These results were used to develop the label-free aptasensor with excellent analytical performance monitored by square wave voltammetry (SWV). The excellent performance of the proposed aptasensor can be attributed to the f-MWCNTs/AuNPs nanocomposite layer with a high conductivity and large specific surface area and also the high affinity of the aptamer molecule toward the BPA molecules. The proposed aptasensor shows an excellent selectivity toward BPA molecules with no considerable cross-reactivity

13

with its similar congeners such bisphenol B (BPB), 4,4'-biphenol (BP) and 6F bisphenol A (6F-BFA). In addition, it has a good stability, reproducibility and a linear dynamic range from 0.1 to 10 nM with a limit of detection of 0.05 nM. Moreover, the fabricated sensor can be applied successfully for the analysis of real samples and the obtained results, confirm that the matrix of the real samples has no significant interference for the determination of the BPA using this fabricated electrochemical biosensor.

Acknowledgment The authors acknowledge Ferdowsi University of Mashhad, Mashhad, Iran for generous financial support to carry out this research work (Grant No. 3.32159).

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18

Biographies Behjat Deiminiat is a Ph.D. student of Analytical Chemistry at Ferdowsi University of Mashhad, Mashhad, Iran. She received her M.Sc. degree in Analytical Chemistry from the same university in 2010. Her research interests include the development of electrochemical sensors based on molecularly imprinted polymers and aptasensors. Gholam Hossein Rounaghi is a full distinguished professor of Chemistry at Ferdowsi University of Mashhad, Mashhad, Iran. He received his B.Sc. degree in Chemistry from Ferdowsi University of Mashhad, in 1970, his M.Sc. degree in Analytical Chemistry at the same university in 1973, and his PhD degree from Michigan State University (MSU) in Analytical Chemistry in 1980. His research interests focus on the study of complexation of macrocyclic ligands with metal cations in non-aqueous solvents and development of electrochemical sensors for cations and pharmaceutical compounds. Mohammad Hossein Arbab-Zavar is a full distinguished professor of Chemistry at Ferdowsi University of Mashhad, Mashhad, Iran. He was graduated in Chemistry (B.Sc. degree) from Shahid Beheshti University, Tehran, (Iran) in 1968 and then he received M.Sc. and Ph.D. in Analytical Chemistry from University of Southampton (England) in 1978 and 1982, respectively. His research interests include electrochemical hydride generation for measurement of some toxic elements and development of electrochemical sensors and their application in electrochemical analysis. Iman Razavipanah (born Mashhad, Iran, 1982) received his B.Sc., M.Sc. and Ph.D. degrees from Ferdowsi University of Mashhad, in Mashhad (Iran). His research is focused on the development

of

electrochemical

sensors,

synthesis

of

electrocatalysts

and

photoelectrocatalysts for fuel cells and new electrochemically prepared SPME coatings. He has published more than 40 peer-reviewed and conference papers on these topics.

19

Figures Caption Figure 1. A) UV-Vis spectrum of f-MWCNTs/AuNPs nanocomposite, B) FT-IR spectra of (a) f-MWCNTs and (b) f-MWCNTs/AuNPs nanocomposite and C) XRD patterns of (a) fMWCNTs and (b) f-MWCNTs/AuNPs nanocomposite. Figure 2. A) Transmission electron microscopy (TEM) image and B) energy dispersive X-ray analysis (EDX) spectrum of f-MWCNTs/AuNPs nanocomposite. Figure 3. Schematic illustration of the proposed aptasensor for determination of BPA. Figure 4. A) Initial structure of the aptamer, B) folded structure of the aptamer after 200 ns MD simulations in water, C) structure of the aptamer in the presence of BPA after 25 ns MD simulations, D) RMSD and E) Rg plots of the folded aptamer with and without BPA during the 25 ns MD simulations in water. Figure 5. Cyclic voltammograms (CVs) of (a) bare Au electrode, (b) f-MWCNTs/AuNPs nanocomposite modified electrode, (c) aptamer/f-MWCNTs/AuNPs nanocomposite modified electrode and (d) aptamer/f-MWCNTs/AuNPs nanocomposite modified electrode after 40 min incubating in 2.0 nM BPA solution. Conditions: Potential scan range -0.1V to +0.5 V and scan rate 50 mVs−1. Figure 6. Electrochemical impedance spectroscopy (EIS) of (a) bare Au electrode, (b) fMWCNTs/AuNPs nanocomposite modified electrode, (c) aptamer/f-MWCNTs/AuNPs nanocomposite modified electrode (d) aptamer/f-MWCNTs/AuNPs nanocomposite modified electrode after 40 min incubating in 2.0 nM BPA solution. Conditions: Potential 0.20 V, frequency range of 0.1-30000 Hz and at amplitude of 10 mV. Figure 7. Calibration curve of aptasensor in different concentrations of BPA solutions. Inset is the square wave voltammograms of aptamer/f-MWCNTs/AuNPs nanocomposite/Au electrode before (a) and after incubation in 0.1 (b), 1.0 (c), 2.0 (d), 5.0 (e), 7.0 (f) and 10.0 (g) nM of BPA solutions. Conditions: Potential scan range -0.1 V to +0.50 V, Step potential 1 mV, Frequency15 Hz, Amplitude, 20 mV in Tris-HCl buffer pH 8.0 containing 0.1 M KCl and 1.0 mM [Fe(CN)6].3−/4− Figure 8. Selectivity of the aptasensor to BPA molecules and some interferences including BPB, BP and 6F-BPA.

20

0.6

A

Absorbance

0.5 0.4 0.3 0.2 0.1 0 0

200

400 600 800 Wavelength (nm)

1000

1200

Transmittance

B

b

a

4000

3000 2000 1000 -1 Wavenumber(Cm )

0

Intensity(a.u.)

C

b a 0

20

40 60 2 Theta(degree)

Figure 1 21

80

100

A

B

Figure 2

22

Fe(CN)63Fe(CN)63-

Fe(CN)64-

Fe(CN)6 4-

FFe(CN)64Fe(CN)64-

Binding

f- MWCNTs/Au

Aptamer

MCH

Figure 3

23

BPA

D A

B

C

E

Figure 4

24

80

b

60 40

c d

I(µA)

20

a

0 -20 -40 -60 -80 -0.2

0

0.2 E(V)

Figure 5

25

0.4

0.6

60

a 50

Z" (ohm)

40

d

30

c b

20 10 0 0

50

100 Z' (ohm)

Figure 6

26

150

200

60 50

100

I(µA)

40

ΔI(µA)

a

80

30 y = 3.2356x + 20.714 R² = 0.9928

20

60

g

40 20 0

0.1

10

0.3 E (V)

0.5

0 0

2

4

6

8 10 12 Concentration (nM)

Figure 7

27

14

16

18

20

30 25

ΔI(µA)

20 15 10 5 0 BPA

BPB

BP

Figure 8

28

6F-BPA

Table 1.Comparison of the dynamic range and detection limit of the different electrodes for determination of BPA in solutions. Method

Linear range (µmol L-1)

Detection limit (µmol L-1)

Reference

f-SWCNT/PC4/GCE

0.099–5.794

0.032

[43]

CS-Fe3O4/GCE

0.05–30

0.008

[44]

MWCNT/MAM/GCE

0.01–40.8

0.005

[45]

sol-gel MIP /GNPsMWCNTs/ Au

0.113–8.21 ×103

3.6×10-3

[46]

PEDOT/GCE

22-410

22

[47]

f-MWCNTs/AuNPs 1×10-4-0.01 5×10-5 This work nanocomposite/aptasensor f-SWCNT/PC4/GCE: single-walled carbon nanotubes/carboxylic-functionalized poly (3,4-ethylenedioxythiophene) complex modified glassy carbon electrode. CS-Fe3O4/GCE: chitosan–Fe3O4 modified glassy carbon electrode. MWCNT/melamine/GCE: multi-walled carbon nanotubes/melamine complex modified glassy carbon electrode. sol-gel MIP /GNPs-MWCNTs/ Au : sol-gel molecular imprinting/ gold nanoparticles and multi-walled carbon nanotubes modified gold electrode. PEDOT/GCE: poly(3,4-ethylenedioxythiophene) modified glassy carbon electrode.

29

Table 2. Results of BPA determination in real samples (n = 3). Sample

Added (nM)

Detected (nM)

Recovery(٪)

RSD(٪)

Mineral water

0 0.5 1.0 0 0.5 1.0 0 0.5 1.0

Not detected 0.51 0.98 Not detected 0.53 0.96 Not detected 0.48 1.03

102 98 106 96 96 103

2.9 2.5 3.4 3.0 4.2 3.7

Orange juice

Milk

30