A High Performance Electrochemical Sensor for Sulfite Based on MOWS₂ Nanocomposite Modified Electrode

INTRODUCTION
For many years, sulfite (SO₃^2-) has had a global widespread usage as a kind of food additives as sulfite, bisulfite, sulfur dioxide, and metabisulfite. It has been widely applied as an anti-oxidant, food additives, and bacterial growth. Moreover, it modulates enzymatic and non-enzymatic browning responses while protecting and storing food [1,2]. Conventional SO₃^2- with beverages are cider, alcoholic and non-alcoholic beer, wine, bottled fruit juices, and concentrates. However, SO3^2- damages to DNA and chromosomes, and is an uncertain allergen, so that a lot of people may be subjected to allergic reactions or even deadly asthma attacks. As stated by the Food and Drug Administration (FDA), inclusion of caution labels on all foodstuff and beverages containing more than 10 ppm of SO32- with maximal concentrations of 50 mg L-1 in beer and 350 mg L-1 in wine shall be obligatory [3-5]. Hence, one of the prominent aspects of food quality control and safety is to have a sulfite detection technique with more sensitivity, selectivity, and higher speed [6, 7].
Numerous techniques of sulfite determination exist such as ion-pair chromatography [8,9],high-performance liquid chromatography [10], flow injection analysis [11] and spectrophotometery method [12]. Yet, the techniques have usually defects (i.e., laborious, tiresome sample pre-treatment, expensiveness and in some cases lower level of sensitiveness and selectivities).
As a complementary choice, electro-chemical techniques measure the current responses produced by direct sulfite oxidation [13]. It has been confirmed that they are easy, cost-effective, and adjustable, have greater selectivities, and readily automated for routine analyses [14-18]. Nonetheless, sulfite behaves electrochemically poorly at typical solid electrode surfaces (glassy carbon, gold, & platinum), because numerous samples can be poisonous for the surfaces of electrode and decline sensitiveness and precision of the electrodes [19,20]. However, modifying the surface of electrodes significantly mitigates overpotentials and augments the electron transfer rates [21-26].
Appropriateness of determining minute concentrations analytes may additionally be increased rapidly via incorporation of nano-materials that significantly enhances surface area, electrical conductivity of electrodes, and performances. Recently, nano-material based electro-chemical sensors have been greatly attracted [27-36].There are different hopeful materials in nanotechnology; however, MoS2nano-sheets gained a certain attention because of their specific electrical and physical features and simple syntheses. One of the mostly attended methods is Molybdenum disulphide (MoS₂) based nano-materials because of the related manifold useful properties. MoS₂ contains S-Mo-S triple layers of certain semi-conducting features of metal dichalcogenide compounds. Extraordinary electro-chemical and luminescence features emphasized on MoS₂ based nano-materials as new sensing probes in order to carefully detect a series of analytes. The respective multi dimensional structures have been the key reason for attention with their multi-faceted application potential. The ultrathin 2D MoS₂ layered structure exhibits higher surface area and generates a prominent supporting material to generate metallic nano-composites.
The charge transfer features of a composite can be enhanced by the synergic impact of metal nano-particles, including MoS₂ and tungsten nano-sheets [37-40].
Screen printed electrodes (SPEs) have functional applications as sensors for chemical analytes. They are advantageous for field analyses, including higher efficiency, little sample sizes, portable capacity and higher velocity. In addition, such SPEs are inexpensive, allowing them for disposability. In any case, such a characteristic is clearly important while testing biological samples. Therefore, they avoid surface fouling side effects [41-44].
This study showed the benefits of MOWS₂ nanocomposites for modifying a screen-printed electrode for examining the sulfite electro-chemical behaviors.

MATERIALS AND METHODS
Apparatus and chemicals
An Autolab potentiostat/galvanostat (PGSTAT 302N, Eco Chemie, the Netherlands) was applied for measuring electrochemicals. General Purpose Electrochemical System (GPES) software was employed to control conditions of experiments. The screen-printed electrode (DropSens, DRP-110, Spain) includes 3 main sections that contain a silver pseudo-reference electrode, a graphite working electrode, and graphite counter electrode. pH was measured by a Metrohm 710 pH meter.
Sulfite and all the remaining reagents had an analytical grade. They have been prepared via Merck (Darmstadt, Germany). Orthophosphoric acid and the related salts that were above the pH range of 2.0–9.0 were used for preparing the buffer solutions.

Hydrothermal synthesis of MoWS₂ nanocomposite
The following process was applied to produce MoWS₂. 3.85 mmol of Na₂MoO₄.2H₂O and 0.15 mmol of Na₂WO₄·2H₂O were dissolved in 40 mL of de-ionized water. In the next step, while continuously stirring the obtained aqueous solution was added with 15 mmol CH₄N₂S and 3 mmol C₂H₂O₄.2H₂O. As precursor solution was converted into a transparent liquid, it has been transported to a 60 mL Teflon liner, followed by loading in a stainless steel autoclave, heating at 200°C for 24 hours, repeatedly cleaning by distilled water and ethanol for removing impurities, and finally vacuum-drying for 4 hours at 80 °C.

Preparation of the electrode
MoWS₂ nano-particles have been used to coat the bare screen-printed electrode. A stock solution of MoWS₂ nanoparticles in 1 mL of aqueous solution has been prepared by distributing 1 mg of MoWS2 nano-particles via ultra-sonication for 30 minutes, whereas 5 µl of aliquots of the MoWS₂ suspension solution has been cast on carbon working electrodes. Then, we waited until the solvent evaporation at room temperature.

RESULTS AND DISCUSSION
Characterization
The FESEM images (MIRA3 TESCAN with EDX microanalysis) of the hydrothermally synthesized MoWS₂ composite is illustrated in Fig. 1, showing a 2 μm averaged diameter for each individual MoWS₂ flower-like microsphere. Densely stacked 2D curved nanopetals with 5 to 15-nm thickness were predominantly observed on the surface of this uniform spherical morphology. Therefore, these structures are called as ‘‘MoWS₂ microflowers with nanopetals’’. A crosswise and random way was found in such secondary structures, suggesting the ability of these heavily staggered 2D nanopetals to grow perpendicularly towards the surface and eventually to form the spherical shape. In conclusion, the synthesize of nanopetal-structured 3D hierarchical architecture can be observed obviously and definitely in accordance with the FESEM images prepared for the produced MoWS2 composite.It can be said that the hydrothermal environment has completely affected MoWS₂ nanopetals in justifying the mechanism of producing the nanopetal structure. The primary duration of hydrothermal reaction at 200 °C caused a significant alteration in the structure of amorphous MoS₂/WS₂. These amorphous nanoparticles became spheroid with dense surface curls after descending trend from Na₂MoO₄·2H₂O/Na₂WO₄·2H₂O to MoS₂/WS₂, thereby eliminating the swinging links and lowering total energies. The spherical morphology can be formed for these primary structures spontaneously following the layered 2D characteristic of MoS2/WS2. The structure of composite may be in the form of curls in the hydrothermal environment because of completely matched lattice constants of MoS₂ and WS2, thereby driving the confined growth of such hierarchical structure without remarkable alteration.
The EDX spectra prepared for the layers verify the existence of Mo, W and S in MoWS₂ with no additional impurities of the source ingredients, as shown in Fig. 2.
The XRD pattern prepared from the synthesized MoWS₂ composite structure is shown in Fig. 3. The distinct sharp diffraction peaks for the MoWS₂ composite means a high level of crystallinity regarding such hydrothermally fabricated samples. The distinct sharp diffraction peaks for the composite also means a higher level of crystallinity regarding such hydrothermally fabricated samples. The standard XRD peaks at 14.38° and 14.32°, respectively, show (002) reflections of MoS₂ and WS₂. Joint Committee on Powder Diffraction Standards (JCPDS=37-1492) card was implemented to index the X-ray diffraction pattern. Based on the Bragg’s equation, 2d sin θ = nλ, so that d represents inter-planar spacing, θ is diffraction angle, n is diffraction series, and λ indicates X-ray wavelengths. Standard values are slightly higher than those of all peaks of diffraction (002) calculated for such species, and this demonstrates that the interlayer spacing is larger and a strain exists between layers for curve hierarchical structure of the fabricated specimens.

Electrochemical behaviour of sulfite at the surface of different electrodes
The electrochemical behaviour of sulfite depends on the pH value of the aqueous solution. Thus, it is essential to optimize the solution pH in order to gain more useful results for electro-oxidation of sulfite. Therefore, sulfite electrochemical behaviour was examined in 0.1 M PBS at distinct pH numbers (2.0–9.0) at MOWS₂/SPE surface by voltammetry. The results indicated more advantagousness of neutral conditions for sulfite electro-oxidation at MOWS₂/SPE surface in comparison to the basic or acidic medium. Here, pH 7.0 was selected as an optimal pH for sulfite electro-oxidation at MOWS₂/SPE surface.
Fig. 4 shows responses of CV to electro-oxidation of 100.0 μM Sulfite at the unmodified SPE (curve a) and MOWS₂/SPE (curve b). The peak potential occurs at 660 mV due to sulfite oxidation, which is around 160 mV more negative than the unchanged SPE. Furthermore, MOWS₂/SPE exhibits very high anodic peak currents for sulfite oxidation than that of the unchanged SPE. This showed a significant improvement of the electrode performance toward sulfite oxidation by changing the constant SPE with MOWS₂ nanocomposite.
Impact of scan rate
Researchers investigated the impact of the rates of potential scan on sulfite oxidation current (Fig. 5). Findings indicated induction of enhancement in the current of the peak by the increased potential scan rate. Additionally, diffusion in oxidation processes are monitored, as inferred by the linear dependence of the anodic peak current (Ip) on the square root of the potential scan rate (ν^1/2) for sulfite [45-47].

Chronoamperometric measurements
Chronoamperometric measurements of sulfite at MOWS2/SPE were conducted by adjusting the working electrode potential at 0.71 V for different concentrations of sulfite (Fig. 6) in PBS (pH 7.0), respectively. For electroactive materials (sulfite in this case) with a diffusion coefficient of D, the Cottrell equation describes current seen for electrochemical reaction at the mass transport limited condition:

I =nFAD^1/2C_bπ^-1/2t^-1/2                                       (1)

where D and Cb respectively represent diffusion coefficient (cm² s^-1) and bulk concentration (mol cm⁻³). Experimental plots of I versus t^−1/2 were used with the best fits for various concentrations of sulfite (Fig. 6A). Then, the resultant straight lines slopes were drawn against sulfite concentrations (Fig. 6B). According to the resultant slope and the Cottrell equation, mean values of D was 8.4×10-6 cm²/s for sulfite.

Calibration plots and detection limits
The electro-oxidation peak currents of sulfite at MOWS_2/SPE surface can be applied to define sulfite in the solution. Since the increased sensitivity and more suitable properties for analytical utilizations are considered as the benefits of differential pulse voltammetry (DPV), MOWS2/SPE in 0.1 M PBS consisting of different distinct concentrations of sulfite was used to conduct DPV experiments (Fig. 7). It was found that the electrocatalytic peak currents of sulfite oxidation at MOWS₂/SPE surface linearly depended on sulfite concentrations above the range of 0.08 to 700.0 µM (with a correlation coefficient of 0.9997), while determination limit (3σ) was achieved to be 0.02 µM.These values are comparable with values reported by other research groups for the determination of sulfiteat the surface of modified electrodes (see Table 1).

Stability and repeatability of MOWS2/SPE
Testing stability of MOWS₂/SPE has been done by maintaining the suggested sensor at pH=7.0 in PBS for 15 days and then recording cyclic voltammogram of the solution containing 70.0 µM of sulfite for comparison with cyclic voltammogram obtained before immersion. Oxidation peak of sulfite has not modified, and the current showed a less than 2.7 % decline in signals in comparison to the initial response, which suggests that MOWS₂/SPE have good stability.
Examining the anti-fouling characteristic of the modified SPE towards sulfite oxidation and the respective products have been performed by cyclic voltammetry for the modified SPE before and after using in the presence of sulfite. Recording the cyclic voltammograms have been done in the presence of sulfite after cycling the potential fifteen times at a 50 mV s⁻¹. The currents declined by less than 2.2%, and the peak potential has not altered.

Interference study
The effects of different materials that can have interference with the 50.0 µM sulfite detection, including biological fluids and pharmaceuticals, were evaluated at the optimal conditions. The maximum level of the interfering materials with error of <±5% for the sulfite detection was regarded as the tolerance limit. These materials were L -lysine, S²⁻, glucose, Fe⁺³, NH⁴⁺, sucrose,  Mg²⁺, fructose, hydroxylamine, hydrazine, lactose, benzoic acid, Al³⁺, ascorbic acid, F⁻, SO₄²⁻, and sucrose, which exhibited no interference in the sulfite detection.

Analyzing real sample
The method illustrated above was used to evaluate MOWS₂/SPE usability for determining sulfite in real samples in order to determine sulfite in river water and well water samples. Therefore, the standard addition technique was applied. Table 2 reports the results. Acceptable recoveries of sulfite were observed, and reproducible results were shown with regard to the mean relative standard deviation (R.S.D.).

CONCLUSION
The present research examined electro-chemical oxidation behaviors of sulfite at MOWS2/SPE. The research findings indicated facilitation of electro-chemical responses of sulfite via MOWS₂ because of its great surface area, higher absorptive capacity, and very good catalytic ability. Additionally, it is effortless and uncomplicated to be fabricated. The peak current exhibited acceptable linear relationship with sulfite concentrations between 0.08 to 700.0 µM. Determination limit of sulfite has been found to be 0.02 μM. Functional features of the proposed sensor (e.g., acceptable stability and reproducible capacity) are a promising fact, indicating that it can be applied as an influential device to determine the sulfite in real samples.

ACKNOWLEDGEMENTS
The authors acknowledge the financial support provided for this project (Project No. 9800552 and ethics code 1398.365) by the Kerman University of Medical Sciences, Kerman, Iran.

CONFLICT OF INTERESTS
The authors declare that there is no conflict of interests regarding the publication of this paper.