Electrochemical determination of T2 toxin by graphite/polyacrylonitrile nanofiber electrode

Abstract Fabricating graphite electrode corrected with nanofiber by electrospinning as a considerable procedure for utilization in the fluid materials, milk, and syrup for detection of T2 mycotoxin is a significant technique. The modern biosensor was fabricated at normal degrees of room and utilized via buffer Britton–Robinson (B‐R) in pH = 5 to refine the chemico‐mechanical specifications. The electrochemical manner of the modified surface was surveyed using the scanning electron microscopy (SEM), cyclic voltammetry (CV), square wave voltammetry (SQWV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV). The corrected electrode displayed a linear reply to T2 toxin in two distinct concentration ranges of 30–100 nM with correlation coefficients of 0.99. The greatest signals in the square wave spectrums for the B‐R buffer created on the uttermost signals of the obtained streams were pH = 5 and 0.5 M of KNO3 for T2 toxin. The modified electrode has a big signal, broad dynamic concentration and high sensitivity and selectivity.

Popular method for quantitative recognition of mycotoxin includes the following: high-performance liquid chromatography coupled with ultraviolet detector (HPLC-UV), gas chromatography (GC) coupled with electron capture (ECD), flame ionization (FID), enzyme-linked immunosorbent assays (ELISA), and thin-layer chromatography (TLC) (Meneely et al., 2011;Pascale, 2009;Rodriguez-Mozaz et al., 2007). These techniques have some disadvantages including complicate specimen procurement, being expensive, requiring lots of the time and solvent and professional operator (Manasa et al., 2019). Hence, based on the influences of T 2 toxin versus human safety, there is a powerful requirement to expand a trusty, simple, susceptible, and efficient and valuable analysis procedure to classified mycotoxin in the food substances (Babakhanian et al., 2015;Rodriguez-Mozaz et al., 2007).
Electrochemical biosensors is a new functional method which was developed as a promising strategy to evaluate the bio-molecules (Meneely et al., 2011;Rahmani et al., 2009). This biosensor work based on the interaction between bio-substances and analytes, which are the most common conventional biosensor (Husain et al., 2016).
Electrochemical biosensors are considerably noteworthy due to the small size, simpleness function, high sensivity and selectivity, cheapness, biodegradability, transportability, capability to continuous respond, and preparing an exact information (D'Souza et al., 2017).
Electrochemical biosensors have a wide application in farming , food technologies (Tahernejad-Javazmi et al., 2019), and medical fields (Khodadadi et al., 2019). Therefore, detection of cost-effective electrochemical biosensors with the aims of increasing electrocatalytic sensitivity and declining potential oxidation is a novel and promising method. Recently, biosensor with modified surface electrode has been done as a new method for improving the limits of detection of materials (Shetti, Malode, Nayak, Aminabhavi, et al., 2019). Chemically modified electrodes such as carbon paste electrodes have gained the great attention because of producing the high surface, decline the fouling impact and facilitated of surface renewal (Shikandar et al., 2018). Thus recognition of biomolecules can be conducted by different chemically modified sensors. It should be note that surface correction can be applied in the shape of thick or thin layer and or mono films. By this process, modifier practices as an intermediary for quick electron movement among analytes and modified surface. Large modified surface increase sensivity and electrocatalytic activities and the analyte can diffuse more rapid on surface of electrode (Yola, 2019;Yola & Atar, 2019a, 2019b. Shetti et al. (2020) designing the skin-patchable electrodes for wearable biosensors which can measure heart beat and blood pressure (Kim et al., 2018) as well as permit convenience examination of essential bio-chemical markers by sweat, saliva, tears etc (Amjadi et al., 2016). Therefore, thus, the noninvasive recognition by using of this process high degree of health can be provided (Shetti, Bukkitgar, Reddy, et al., 2019). Another modified carbon paste electrode for detection of ferulic acid including screen-printed electrodes (Blanco et al., 2015), screen-printed carbon electrodes, graphite pencil electrodes (David et al., 2016), laccase-modified graphite electrode, and electrochemically reduced graphene oxide-based electrochemical sensor (Liu et al., 2014) etc. To get favorable conclusion, complex process for production of electrode, low pH, and precise evaluating of the electrode stability are important factors. Shetti, Malode, Nayak, Reddy, et al. (2019) designed a new biosensor with silica gel as a modifier in order to increase the electrocatalytic efficiency of paracetamol. The silica gel has three dimensional structure and considered as a strong inorganic materials, as well as has high surface area. The silanol agent (Si -OH) in this compounds exert great ion exchange in them in a bit alkaline condition (Zaazaa et al., 2015). As results, diffusion rate increase and more binding sites are created for analytes (Shetti, Bukkitgar, Reddy, et al., 2019).
Monoclonal antibody label-free immunoassay in contrast to immune sensor through enzyme label has industrialized to straight identify toxins via substantially altering the immune compound (Tang et al., 2010;Vidal et al., 2011). The substantial benefits for the biologically sensors remains susceptibility, its transportability, and simple equipment. Electrochemical immune sensors have a great application in portable devices in two aspects: direct and sandwich methods (Catanante et al., 2016;Hosseini et al., 2013;Liu et al., 2012). Sandwich methods have high sensitivity and selectivity in detection of T 2 toxin by hydrodynamic fluid stream (Okuno et al., 1987). Also, electro-chemiluminescence (ECL) can efficiently detect T 2 toxin because of high sensitivity, simple instrumentation, controlling ability of ECL, and low cost (Chu et al., 2012;Lin et al., 2006).
Electrospinning is a modern procedure with important function in corrected electrodes to discover T 2 toxin (Chu et al., 2012;Samadian et al., 2017). Electrospinning is a simple, and cost-effective technique which can made fibers and particles by vast surface, flexibility, better morphologically, and mechanical properties. In the electrospinning procedure, a big voltage is exerted to a polymeric matrix to remove the surface stress. By vaporizing the solvent, the electrospun micrometer-nanometer fiber is formed (Figure 1a; Mohammadi et al., 2018). Electrospinning has attracted significant attention thanks to its easy production procedure and a variety of appropriate substances (Shaulsky et al., 2017). Different electrospun nanomaterial sensors such as resistant, electrochemical, fluorescent, acoustic wave, colorimetric, and photoelectric sensors have been designed (Cai et al., 2018;Zhang et al., 2017). The electrochemical sensor has several benefits such as high precision, simple and easy equipment; accordingly they have good ap-

| Reagents and chemicals
T 2 toxin as tested sample, KI, KNO 3 , NaNO 3 , NaCl, and KCl salt as a sponsor electrode, CH₃COOH, H 3 PO 4 , H 3 BO 3 , and NaOH as the primary material for buffer preparation are required. All chemical substances were bought from Merck and Sigma-Aldrich corporations (Parzhak Shimi laboratory). Nanofibers were applied to modify the electrode area. Pan, C 5 H 8 O 2 , and H 2 SO 4 as an auxiliary factor were applied by nanofibers.

| Equipments
Electrochemistry tests were done by an Auto lab PGSTAT101 and NOVA.1.11 software. A conservative 3 electrode method was used to integrate an occupied modified electrode as an electrospun G/Pan-synthesized constituent electrode, an orientation electrode as a soaked Ag/AgCl electrode, and a counter electrode as a graphite electrode. The pH capacities were presented by a 781 Metrohom pH meter prepared with a joint glass electrode. The surface morphology was calculated by an SEM (Philips XL30, Philips Panalytical, California). In this method, firstly, the samples were covered with gold and then SEM was acted at 5 kV.

| Characterization of nanofibercorrected electrode
To provide the corrected electrode, the electrospinning method was applied. At first, the exterior of un-corrected graphite electrode was varnished fine up to it was without of chemical contamination.
Next, electrospinning procedure with the volume of 0.0008 ml/min was used to produce the nanofibers containing surface modifier (Bukkitgar et al., 2015;Guha et al., 2014). The best situations in electrospinning device were used the 25 kV voltage, spinning period for 20 min, page gap of 25 cm and moisture amount of 56% ( Figure 2).
To prepare T 2 toxin samples, distilled deionized water was applied for all samples overall the trials. B-R buffer matrix was provided via incorporation 0.2 M NaOH (aq) to the matrix comprising 0.04 M H 3 BO 3 (aq) , CH₃COOH (aq) , and H 3 PO 4 (aq) to create pH = 3-12.
Finally, the matrix were polluted by a various amounts of T 2 toxin (0.5-300 nM).
To procurement nanofibers as electrode corrector cover; electrospinning procedure were performed as previously mentioned for the corrected electrode. Pan along with nano-clay substance was applied to produce a layer of nanofibers, for this manner syringe pump operated as the fiber sprayer. The best situations for nanofibers film on a graphite electrode area were the 25 kV voltage, spinning period for 20 min, page gap of 25 cm and moisture amount of 56%. In addition, C 5 H 8 O 2 (7%) and H 2 SO 4 (5%) were used as a fiber couplers. At last, the fabricated nanofibers were were washed with sulfate buffer 5 times for 7 min each time (Pascale, 2009).

| Preparation of T 2 toxin sample
A modified electrode was applied to measure T 2 toxin in the con-

| pH analysis
The manner which is applied for assessing the influence of pH continuously the respond for nanofiber-corrected graphite electrode in each analyze were as bellows: Ten milliliter of B-R buffer (pH = 3-12) and 30 μM of T 2 toxin were incorporated to the 25-ml tube. The matrix were agitated greatly and conveyed to the electrochemical vial of the Auto lab device. Next, the matrix with various pH amounts was occupied from a cyclic voltammogram. It should be noted that the best pH in these analyses were the pH which fabricated the highest flow velocity (Adriano, 2017).

| Salt concentration investigation
In these procedures, the impact of electrolyte salt amount on the sensor reply was evaluated applying 30 mM of T 2 toxin at pH 5. The important factor in this manner was the highest signal which caught up by the sensor (Adriano, 2017).

| Evaluations of nanofiber-corrected electrode
The nanofiber-corrected electrode was applied to determine T 2 toxin in the contaminated products. The corrected electrode was gotten at the existence of 30 μl of T 2 toxin and silver chloride (standard electrode) in the CV. This procedure enhance the peak altitude relevant to the oxidization of T 2 toxin on the corrected electrode area in the matrix eventually, by using of this manner T 2 toxin can be detect along with designed sensor (Figure 2). This outcome is in accordance with the findings of moradi et al. (Moradi et al., 2020;Palmisano et al., 1981).

| Influence of scanning electron microscopy from corrected graphite electrode area
The SEM patterns were caught from the area of un-corrected graphite and nanofiber-corrected graphite (Figure 3). For un-corrected graphite electrode, an unsuitable flat surface was seen (Figure 3a).

| Impact of scan speed
The impact for SEM was evaluated in the existence of the corrected electrode at 10-100 mV/s and optimum chemical situations. Then, 10 milliliter of B-R buffer in pH = 5 and 50 μM for T 2 toxin in the existence of corrected graphite electrode and optimum chemical substances were carried into the vial. The compounds were surveyed in the existence of the proper sensor in the potential extend. According to the results, 100 mV/s was selected as the suitable speediness for any sample (Figure 4; Bukkitgar et al., 2016;.

| Impact of pH
The influence of pH matrix thru the sample on the cathodic peak of the square wave spectrum at the existence of 30 μM T 2 toxin for the corrected electrode was assessed at pH = 3-12 in B-R buffer ( Figure 5a and b). High-level acid/alkaline situation, that is associated to pH = 3-12, intrusion of H-(pH = 3) and OH-ions (pH = 12) and its assault into the electrode area lead the altering of analyte peak and subsequently eliminated the peak since the pH schedule. Figure 5 displays that T 2 toxin cathode peak is associate with the pH variations proton receiver agents on the T 2 toxin structure. The optimized signals pro the square wave spectra in the B-R buffer according to the highest signal of the obtained stream were pH = 5 for T 2 toxin. As well as, 10 milliliter of B-R buffer were determined such as a suitable buffer content. An amount more than 10 ml did not influence the increasing manner of the obtained signal. The pH diagram at pH = 4 into 7 showed a linear manner with the (−19) mV slope for the pH, which proposed the T 2 toxin reaction on the area of the corrected electrode were a proto/electron manners (Figure 5a and b) (Moradi et al., 2020). The pH chart slopes could be applied to define the electron convey rate by the below formulation: pH amounts also powerfully influenced the oxidizing possibility of T 2 toxin, and enhancing of pH, can decrease oxidation rate to negative amounts Shetti et al., 2009). As a result, a great linear connections was established among the pH and oxidizing rate.

| Influence of salt amount
Choosing appropriate electrolyte in voltammetric responses is ex- displayed which 0.5 M of KNO 3 as a backer electrolyte was propered to define T 2 toxin at the existence of the optimal electrode. The reason for this choice was the most signal obtained, as seen in Figure 6a and b.

| Calibration diagram in the matrix
In the appropriate chemico-mechanical situations, various matrix of T 2 toxin were collected at the variety of 0.5-550 nM. By using advanced seeking in the variety of 0.0-0.4 V, the voltammetric spectrum of the square wave was obtained and the calibration chart of T 2 toxin was drawn at the existence of the corrected electrode. The flow-concentration diagram was taken in linear areas, from 30 to 100 nM (Figure 7a and b). Consequently, the correlation coefficient for whole areas was described to be 0.99. As observed in Figure 5, the content of the conveyed flow was increased via raising the analyte volume, which demonstrated enhancing in the electrode area at the existence of T 2 toxin in the bed fluid and also show a flow enhance from the exterior of the corrected electrode within the procedure.
Via applying the information gotten from the standard chart and its chart formulation, the limit of quantitation (LOQ) and limit of detection (LOD) were determined. The outcomes of flow/potential and flow/concentration curves were observed in Figure 5. Also, the To calculate the LOD, LOQ (three repeated test), the flow cycles were occupied from every concentration and by trebling the quantity of SD, and interrupt of calibration curve was analyzed as a middling parameter.

| Interference
Assessing the impact of kinds of coexisting and analyte is the most considerable controlled factors. To survey the influence of interfering, the impacts of various volumes of the matrix of possible organic and inorganic substances in simulant on the reply of the corrected electrode with 30 μM T 2 toxin and modified sensor were investigated (Table 1). In addition, 5% fault in the signals was presented as a border of disorders. Under optimized situations, the findings demonstrated that these substances did not have any specific disorder for the corrected electrode. As a result, it can approve that the corrected electrode has a good capability to recognize T 2 toxin in the polluting materials.

| CON CLUS ION
In this study, square wave voltammetry procedures were applied as a susceptible electrochemistry manner to discover and evaluate

CO N FLI C T O F I NTE R E S T
All authors declare that there is no conflict of interest.

E TH I C A L A PPROVA L
This article does not contain any studies with human participants or animals performed by any of the authors.

TA B L E 1
The effect of interfering organic and inorganic species on the response of the modified electrode to 30 mM T 2 toxin (three repetitions)