Description:FIELD OF INVENTION:
The present invention relates to the determination of the presence of unauthorized food colorant rhodamine B in cotton candy at the nanomolar level. More particularly, the present invention discloses a method to quantify rhodamine B through cyclic and differential pulse voltametric techniques by exploiting the transition metal dichalcogenide, molybdenum disulfide, combined with biocompatible silicic acid.

BACKGROUND OF INVENTION:
Rhodamine B (RhB), chemically [9-(2-carboxyphenyl)-6-(diethylamino) xanthen-3-ylidene]-diethylazanium chloride is a water-soluble, basic xanthene dye widely used in textile, leather and printing industries. It is also used as a fluorescent reagent in analytical chemistry and the green powdered RhB turns fluorescent pink in contact with water. The extensive use of RhB as a colouring agent in the food industry is due to its low cost and high stability. However, there are serious health concerns related to its use. The International Agency for Research on Cancer, has classified RhB as a group III carcinogenic agent, being hepatotoxic, genotoxic, and connected with reproductive toxicity. Rh B has been reported to adversely affect aquatic life as well. The photosynthetic and antioxidant activity of certain aquatic species were reduced on exposure to RhB. As a result, many countries have prohibited its use in food and beverages. However, other industries continue to use this toxic dye. Therefore, there arises a need for stringent monitoring of the presence of this dye in food and water that is used by the common folk.

The detection of RhB is possible with the help of numerous analytical methods like capillary electrophoresis, ultraviolet-visible spectrometry, high-performance liquid chromatography, ion-exchange chromatography, and electrochemical methods. The electrochemical methods offer a fast, simple, and affordable detection platform. However, the other quantification methods require professional expertise and pre-treatment.

Research on the detection of Rhodamine B has been undertaken in the past using different types of composite materials and sensors. Patent application no. CN109535494B titled “Composite material for detecting organic pollutant rhodamine b and preparation and application thereof” explores the synthesis of a composite material for detecting Rhodamine b in which graphite oxide and ferrocene are connected through pi-pi action, and chitosan is loaded on the graphene oxide-ferrocene and then is reduced, so that the reduced graphene oxide-ferrocene-chitosan is synthesized. The electrode modified by this composite material is taken as a working electrode and is placed in [ Fe (CN)6 containing rhodamine b]4-/3-solvent, and identifying by differential pulse voltammetry under the conditions that the scanning speed is 50mV/s and the scanning range is-0.2V-0.6V. However, it is silent on the sensitivity of detection of Rhodamine B in the presence of other interfering chemicals in the sample.

Another application CN-116930300-A titled “Molecular imprinting electrochemical sensor based on nanomaterial modification and application of molecular imprinting electrochemical sensor in rhodamine B detection” discloses a molecular imprinting electrochemical sensor modified based on a nano material for rhodamine B detection. The sensor comprises of a working electrode, a reference electrode and a counter electrode; the working electrode being a glassy carbon electrode modified with a molecularly imprinted polymer and a nano material f-MWCNT, the reference electrode being a saturated calomel electrode and the counter electrode being a platinum wire electrode.

The use of screen-printed electrodes (SPE) as an analytical tool has revolutionized the health sector. The time-to-time monitoring of blood glucose levels in diabetic patients was made possible with the invention of a glucometer. Similar point-of-care diagnostic tools are necessary for the improvement in the quality of life of the people. SPEs offer a platform for the detection of a variety of analytes and are used in many fields like biosensors, sensors in food and drink analysis, and forensic science. Cost-effectiveness combined with portability, electrical and thermal conductivity, reproducibility, and ease of modification leading to improved selectivity and sensitivity make screen-printed carbon electrodes (SPCEs) the centre of interest.

Molybdenum disulfide (MoS2), a transition metal dichalcogenide having appreciable chemical and thermal stability is an indirect bandgap semiconductor with tunable physical and chemical properties. Chalcogens are the Group 16 elements of the modern periodic table consisting of 5 elements oxygen, sulphur, selenium, tellurium and polonium. The elements in this group are also known as chalcogens or ore-forming elements because many elements can be extracted from sulphide or oxide ores.

The stacked monolayers of bulk MoS2 are held together by van der Waals force of attraction between the S layers and a strong covalent bond between Mo-S layers. The electric conductivity across two adjacent S-Mo-S layers can be increased by the development of MoS2-based hybrids using mesoporous materials. Liquid exfoliation through ultrasonication can transform bulk MoS2 into two-dimensional sheets and reduce the energy gap and make it a direct band gap semiconductor. Moreover, the biological safety associated with the use of MoS2 makes it a versatile material from a sustainable point of view. It has been widely used in making hybrids and composites for applications in various fields, including electrochemical and optical sensors.

However, the incorporation of biocompatible materials with MoS2 has been less explored in the field of chemical sensing. The electrocatalytic property and the selective sensing of analytes can be enhanced by the assimilation of biopolymers in MoS2. The porous nature of the polymeric materials improves the charge transfer process at the electrode surface. Moreover, biopolymeric materials exhibit great biocompatibility, biodegradability, adhesiveness, and film-forming ability, making them either drop casted, spin-coated, or electrodeposited on the working electrode surface.

OBJECT OF THE INVENTION:
To obviate the drawbacks of the existing state of the art, the present invention discloses a method of determination of unauthorized food colorant rhodamine B in cotton candy at the nanomolar level.

The main object of the invention is to provide a method for quantification of rhodamine B through cyclic and differential pulse voltametric techniques by exploiting the transition metal dichalcogenide, molybdenum disulfide, combined with biocompatible silicic acid.

Yet another object of the invention is to provide a method for selective detection and quantification of rhodamine B by using modified SPCEs with MoS2 and silicic acid (SA).

Yet another object of the invention is to provide a method for selective detection and quantification of rhodamine B.

Yet another object of the invention is to provide a method of detection of food colourants with the lowest ever reported Limit of Detection (LOD) such that the sensor shows good performance in the nanomolar concentration range of the analyte.

Yet another object of the invention is to provide a method of detection of food colourants wherein the sensors detect even trace amounts of the analyte in food samples, especially in cotton candies.

Yet another object of the invention is to provide the sensor showing appreciable selectivity and stability with remarkable reproducibility and repeatability.

Yet another object of the invention is to provide the screen-printed electrodes as sensors for quantifying the food colourants in a sample wherein said sensors can be deployed in a portable device.
SUMMARY OF THE INVENTION:
The present invention discloses the fabrication of sensor electrodes for selective detection and quantification of food colorants, especially Rhodamine B. The sensor electrodes are fabricated by modification of Screen-Printed Carbon Electrodes (SPCEs) by drop casting of MoS2 and silicic acid (SA) on the electrodes for the selective detection of RhB. The morphological and topological characterization of the sensor was done using scanning electron microscopy and atomic force microscopy followed by performing electrochemical quantification of RhB on the proposed electrode using differential pulse voltammetry.

The fabricated MoS2-SA hybrids were observed to form three-dimensional architectures with an increased rate of diffusion of RhB resulting from enhanced electrical conductance. The limit of detection, linear concentration range, and selectivity toward RhB of the sensors from other potential interferents were evaluated under various experimental conditions. The present invention facilitates the quantification of lowest-ever reported nanomolar detection of the unauthorized food colorant RhB in cotton candy samples.

BRIEF DESCRIPTION OF DRAWINGS:
Fig 1: depicts the Fabrication of MoS2/SA/SPCE sensor and possible mechanism for the electrochemical oxidation of RhB on the fabricated electrode.
Fig. 2: depicts the MoS2/SA/SPCE a) ATR-IR spectrum b) XRD spectrum
Fig. 3: depicts the ATR-IR spectra of different electrodes
Fig. 4: depicts the SEM images of the bare and modified electrodes Fig. 5: depicts the EDS spectra and mapping images of the different electrodes
Fig. 6: depicts the AFM images of the bare and fabricated electrodes
Fig. 7: depicts the XRD plots obtained for the different electrodes
Fig. 8: Depicts the a) CV response of the electrodes in 5 mM K3[Fe (CN)6)] and 0.1M KCl b) Nyquist plots obtained for the electrodes in 5 mM K3[Fe (CN)6)] and 0.1M KCl c) Equivalent circuit obtained using Zsimpwin software
Fig. 9: depicts the a) Effect of concentration of MoS2 and silicic acid on oxidation current and potential (DPV response of 1 µM RhB in 0.1 M PBS of pH 7) b) Effect of varying pH from 5 to 8 on oxidation current and potential (DPV response of 1 µM RhB) c) Calibration plot showing the linear relationship between pH and oxidation potential (DPV response of 1 µM RhB in 0.1 M PBS of pH 5 to 8) d) DPV responses of 1 µM RhB on MoS2/SA/SPCE, MoS2/SPCE and SA/SPCE, and SPCE electrodes in 0.1 M PBS buffer of pH 7
Fig. 10: depicts the a) CV data showing the scan rate study from 5 mV to 250 mV of 1 µM RhB in 0.1 M PBS of pH 7 b) Peak current versus square root of scan rate in CV study of 1 µM RhB in 0.1 M PBS of pH 7 c) log current versus log square root in 0.1 M PBS buffer of pH 7 and d) Peak potential versus log scan rate study in 0.1M PBS buffer of pH 7
Fig. 11: depicts the Plot of scan rate vs peak potential Ep
Fig. 12: depicts the Linear concentration range of 20 - 2000 nM of RhB using DPV on MoS2/SA/SPCE a) DPV response of RhB in 0.1M PBS of pH 7 b) Calibration plot
Fig. 13: depicts the Linear concentration range of 10-70 µM of RhB using DPV on MoS2/SA/SPCE a) DPV response of RhB in 0.1M PBS b) Calibration plot
Fig. 14: depicts the a) Stability of MoS2/SA/SPCE in 0.1 M of PBS buffer of pH 7 through DPV technique and b) selectivity of 1 µM RhB in 0.1 M PBS of pH 7 on MoS2/SA/SPCE through DPV technique
Fig. 15: depicts the UV-Visible absorption spectra of a) 0.06 µM of RhB before electrochemical oxidation at MoS2/SA/SPCE, b) after oxidation.
Fig. 16: depicts the Proposed mechanism of electrochemical oxidation of RhB on MoS2/SA/SPCE

DETAILED DESCRIPTION OF THE INVENTION:
The present invention discloses the design and fabrication of a sensor for the selective detection and quantification of nanomolar quantities of food colourant Rhodamine B (RhB). The fabricated sensors can also be modified for similar analysis of other food colourants and dyes. The sensor is fabricated by modification of screen-printed carbon electrodes (SCEs) with Molybdenum disulfide (MoS2) and Silicic Acid (SA). To assess the efficacy of the fabricated sensor, its limit of detection, linear concentration range, and selectivity for RhB from other potential interferents has been experimentally evaluated under different conditions.

The sensor is fabricated using analytical grade reagents. All the reagents and chemicals were procured from authentic sources to maintain the reproducibility of results:
Molybdenum disulfide (MoS2) and silicic acid (H4SiO4) were procured from Sigma Aldrich, USA.
Sodium hydroxide pellets, potassium ferricyanide, and potassium chloride were procured from Nice Chemicals Pvt Ltd, India.
Dimethyl sulfoxide (DMSO) was purchased from Spectrochem Pvt Ltd, India.
Phosphate buffer solution (1M) was prepared from sodium dihydrogen phosphate and disodium hydrogen phosphate purchased from Spectrochem Pvt Ltd, India. All the solutions were prepared in double distilled water.
For real sample analysis, a pack of cotton candy was purchased from a nearby shop.

The Analytical methods and instruments were selected based on the specificity of the analysis to be undertaken:
The Zensor screen printed electrodes were purchased from CH Instruments, USA.
Electrochemical measurements like cyclic voltammetry (CV), and differential pulse voltammetry (DPV) were performed using a CHI 610E electrochemical analyzer, CH Instruments USA.
A conventional three-electrode system consisting of a carbon counter electrode, Ag/AgCl as the reference electrode, and MoS2/SA/SPCE as the working electrode were used.
Electrochemical Impedance (EIS) measurements were performed using CH instrument CHI6087E, USA.
The infrared spectroscopic analysis was accomplished by attenuated total reflection (ATR-IR) spectroscopy of Perkin Elmer.
Scanning Electron Microscopy-Electron Dispersive X-ray Spectroscopy (SEM-EDS) analysis to understand the surface morphology and the elemental constitution of the electrode was performed using Jeol 6390LV Scanning Electron Microscope with EDS [An accelerating voltage of 0.5 kV to 30 kV and a resolution of 4 nm (30 kV) and magnification of 300,000 EDS].
Atomic Force Microscopy (AFM) analysis to study the topography of the electrode was done using Alpha 300RA (WITec, GmbH, Ulm, Germany) AFM System.
A Rigaku Mini Flex Benchtop Powder X-ray diffraction (XRD) Instrument was used for XRD studies.
The Milli-Q water filtration system was used for double-distilled water.

FABRICATION OF THE SENSOR:
A homogeneous suspension of 2 mM of molybdenum disulfide and 2 mM of silicic acid was prepared in 5 mL DMSO as solvent as the stock solution. For preparing the homogeneous suspension, 0.0016 g MoS2 (molecular weight= 160.07 g/mol) and 0.0008 g silicic acid (molecular weight= 78.10 g/mol) was weighed accurately and sonicated for 20 minutes in an ultra sonicator. From the stock solution various equimolar concentrations of MoS2 and silicic acid ranging from 0.8 to 1.5 mM were prepared through the method of dilution and 5µl was drop casted to the working electrode surface of the Zensor screen printed carbon electrode. The electrode was then kept for drying at room temperature. All the steps involved were conducted at room temperature (~ 28-30°C).

Schematic diagram of the fabrication process has been depicted in Fig. 1. Solution having 1 mM each of Molybdenum Sulphide (MoS2) and Silicic Acid (SA) was prepared using DMSO as the solvent through ultrasonication. 5 µL of the well-dissolved solution thus obtained, was carefully drop-casted on the surface of the Zensor screen-printed electrode resulting in the fabrication of MoS2/SA/SPCE electrode sensor. The fabricated electrode was dried at room temperature for analysis and use.

VOLTAMMETRIC TECHNIQUES:
Electrochemical experiments were conducted to analyze the characteristics of the fabricated electrodes. These experiments were performed in a restricted light environment to prevent the photodegradation of dye solution in an aqueous medium. Cyclic voltametric measurements of the fabricated MoS2/SA/SPCE electrode were performed from an initial potential of -1 V to 1 V at a scan rate of 100 mV/s. Differential Pulse Voltammetry (DPV) used for the quantification of RhB at MoS2/SA/SPCE was done at a pulse width of 0.025 s from a potential of 0 to 1.0 V at an amplitude of 0.05 V/s. Cotton candy was purchased from a nearby shop for to be used as real sample and 2g of cotton candy was weighed and dissolved in 0.1 M PBS solution for the analysis without any pretreatment.

CHARACTERIZATION OF THE ELECTRODE
Attenuated Total Reflectance-Infrared Spectroscopy (ATR-IR):
The chemical modifications on the screen-printed electrodes were identified by infrared spectroscopy. The ATR-IR spectra of MoS2/SA/SPCE electrodes have been depicted in Fig. 2a and the spectra of bulk MoS2, SA, unmodified SCPE, near-infrared spectrum of MoS2/SA/SPCE have depicted in Fig. 3. The bands observed at 211 cm-1 and 264 cm-1, 496 cm-1, 620 cm-1 and 3392 cm-1 in Fig. 3 indicate the characteristic absorptions of MoS2 bending and stretching vibrations. The symmetric stretching vibrations of Si-O bonds of SA were observed at 1264 cm-1 and 847 cm-1 (Fig 2a). The absence of the strong and broad O-H absorption frequency in Fig. 2a confirms the participation of the O-H groups of SA with MoS2 for the hybrid formation. The characteristic S-H stretching frequency of 2660 cm-1 is also present, confirming the presence of hybrid modification of SA and MoS2.

Scanning Electron Microscopy (SEM)-Energy Dispersive X-Ray Spectroscopy (EDS)
The surface morphology of the bare and modified electrodes was studied using Scanning Electron Microscopy (SEM), which has been depicted in Fig. 4. The elemental constitution of the modified electrodes has been revealed through Energy Dispersive X-Ray Spectroscopy (EDS) mapping, which has been depicted in Fig. 5. The graphitic carbon nature of the electrode surface is identified by the flake-like structures covered in a polymeric binder (from the ink during screen printing) giving it a webbed appearance in the SEM image of the SPCE. The slight disruption and non-uniformity in the flake-like porous structure of the SEM image of the modified electrodes indicate the presence of MoS2/SA hybrid formation and was further confirmed by the presence of the respective elements by EDS mapping.

No evidence of crystal or flower-shaped clusters has been reported in the SEM images of the deposited MoS2 of the fabricated electrode when compared with the state-of-the-art. This may be because the drop-casted mixture of MoS2/SA hybrid forms a comparatively thinner mesoporous layer on the SPCE. The EDS data depicted in Fig. 5 confirms the presence of micro-level concentrations of molybdenum (1.86 %), sulphur (0.58 %), silicon (0.52 %) and oxygen (5.29 %) in the modified electrodes with a high percentage of carbon (77.8 %).

Atomic Force Microscopy (AFM) and X-Ray Diffraction (XRD)
The topological characterization of the fabricated electrodes was obtained from Atomic Force Microscopy (AFM). The surface topography of the electrodes, as visualized, supports the modification on the surface of SPCE. The AFM images of SPCE, MoS2/SA/SPCE, MoS2/SPCE and SA/SPCE have been depicted in Fig. 6. The change in the roughness value from 56.73 nm (SPCE) to 68.85 nm (MoS2/SA/SPCE) is an indication of the surface modification with increased roughness of MoS2/SA/SPCE. Notably, surface roughness has a direct relation with the number of exposed edge plane-like sites or defects that mark the origin of electron transfer in graphite electrodes. X-Ray Diffraction (XRD) analysis revealed the crystallinity, chemical composition, and defects in a sample. The sharp and narrow peaks in the XRD pattern observed for the bare and modified SPCEs, as indicated in Fig. 2 b and Fig. 7 indicate the crystalline nature of the electrodes. The sharp peaks at 38.2°, 44.3°, and 64.8° on the bare SPCE, represent the graphitic nature thus confirming the the basic carbon nature of SPCE which was not disturbed by the hybrid formation of SA and MoS2. Additionally, the presence of a peak at 2Ѳ = 7.3o, and 14.4o, are the characteristic MoS2 diffraction peaks.

ELECTROCHEMICAL CHARACTERIZATION OF THE FABRICATED ELECTRODE
Cyclic voltammetry
Cyclic voltammetry was used for elucidating the electrochemical characteristics of bare and modified electrodes, studied in a mixture of 5 mM K3[Fe (CN)6)] and 0.1M KCl solution as the redox probe. Fig. 8a confirms the conducting nature of MoS2/SA/SPCE electrode through the current response of the redox probe. SPCE was observed to be less conductive when compared to MoS2/SA/SPCE. The rate of the redox reaction Fe2+/Fe3+ is enhanced at the MoS2/SA/SPCE, i.e., the quick electron movement of [Fe (CN)6)]3- on the surface was made possible with the composite modification.

The surface area of the electrodes was calculated using the Randles Sevcik equation, which is given as:
Ip = 2.69×105 A(n)3/2(D)1/2(ν)1/2C,

where C denotes the reactant concentration (5 mM K3[Fe(CN)6)]); n, represents the number of electrons involved in the redox system (n = 1); A is the electrode surface area; ν represents the scan rate and D depicts the diffusion coefficient of the ferricyanide system. Using the Randles-Sevcik equation and the slope of the Ip vs υ 1/2 plot, the surface areas of the unmodified and modified SPCE were calculated to be 0.0638 cm2 and 0.0805 cm2 respectively. Thus, it was concluded that there was a tremendous increase in the surface area of the modified electrode, indicating that the fabricated electrode can effectively sense RhB.

Electrochemical impedance spectroscopy (EIS)
The conducting nature of the fabricated electrode was further confirmed by Electrochemical impedance spectroscopy (EIS), the results for which, have been depicted in Fig. 8b. Electrochemical impedance spectroscopy is an important tool that is used for the elucidation of electron transfer kinetics and interfacial properties wherein the corresponding Nyquist plot is utilized for comparing the electrochemical properties of the electrodes. The Nyquist plot consists of two parts: a semicircular part at high frequencies of impedance spectra corresponding to the electron transfer-limited process, and a linear part at lower frequencies representing the diffusion-limiting step of the electrochemical process. The Nyquist plot of the electrodes in 5 mM K3[Fe(CN)6)]/0.1M KCl, as depicted in Fig. 8b, confirms that MoS2/SA/SPCE has lower charge transfer resistance/Warburg resistance than MoS2/SPCE and SA/SPCE.

The charge transfer resistance of the electrodes was calculated by fixing them to an appropriate equivalent circuit, R (CR), using ZSimpwin software. It was found to be 1.549 × 105 Ω for MoS2/SA/SPCE, the lowest among the four electrodes, as depicted in Fig. 8b and Table 1. Despite being a semiconductor, resistance is offered by MoS2, but the thin film of MoS2/SA increased the conductivity and electron transfer rates because of the synergic effect of MoS2 and SA. The dominance of Warburg resistance rather than charge transfer resistance is evident from the straight-line graph, indicating diffusion-controlled kinetics on the electrode surface.

Table 1: EIS quantification through equivalent circuit R (CR)
Electrode RS (Ω) RS (Ω) C (F)
SPCE 200 5.493 E5 4.179 E5
MoS2/ SA/SPCE 209.2 1.549 E5 4.068 E5
MoS2/ SPCE 224.9 2.284 E5 2.328 E5
SA/SPCE 239.2 1.732 E5 3.167 E5
Rs- Solution resistance, Rct- Charge transfer resistance, C –Capacitance

OPTIMIZATION OF REACTION PARAMETERS
Effect of monomer concentration
The concentrations of SA and MoS2 for drop casting on the SPCE were optimized. DMSO was chosen as the solvent for preparing the hybrid solution of SA and MoS2. A 2 mM stock solution of SA and MoS2 in 5 mL DMSO was prepared, and from the stock solution, various dilutions were prepared to optimize the monomer concentration. 5 µL of the various hybrid solutions ranging from 0.8 mM to 1.5 mM of SA and MoS2 were drop casted on the SPCE and dried at ambient temperature. The current-voltage response of each fabricated MoS2/SA/SPCE towards the oxidation of 1 µM RhB was analyzed through DPV. From the plot of concentration versus the oxidative current and potential, as indicated in Fig. 9a, it was evident that the highest response was for the 1 mM solution of SA and MoS2 drop-cast on MoS2/SA/SPCE.

Effect of pH of supporting electrolyte
The pH of the supporting electrolyte greatly influences the redox behaviour that exists between the analyte and the modified electrode. Differential pulse voltammetry (DPV) was used to study the oxidation rate of RhB at MoS2/SA/SPCE in 0.1 M PBS within a pH range of 5 to 8 (Fig. 9b). The current-voltage response of the MoS2/SA/SPCE electrode at various pH levels was studied, and pH 7 was shown to be optimum for the oxidation of RhB. The regression equation for the relation between peak potential and pH of electrolyte for the oxidation of RhB at MoS2/SA/SPCE is given by EPa (V) = -0.55 pH + 0.72; R2 = 0.94. It is evident from the graph in Fig. 9c that there exists a linear inverse relation between pH and the peak potential, i.e., an increase in pH leads to a decrease in the oxidation peak potentials. The involvement of protons during the RhB oxidation process on the modified electrode is evident from this inverse relationship. The magnitude of the slope of EP versus pH agrees with the theoretical value, indicating that the oxidative transfer involves an equal number of electrons and protons.

Effect of scan rate
The electrode process or the electrochemical reaction mechanism involved in the irreversible oxidation of RhB is revealed from the scan rate study. The voltammograms of irreversible oxidation of 1 µM RhB were recorded on the MoS2/SA/SPCE surface at varying scan rates from 5 - 250 mV by CV under optimized electrode conditions. As depicted in Fig. 10a, with an increase in the scan rate, anodic peak current (Ip) at the MoS2/SA/SPCE increases linearly indicating that the electrochemical process is diffusion-controlled (Fig. 10b) with a linear regression equation: Ip (μA) = 2.398 υ1/2 (mV/s) + 0.1954.

The observation was confirmed from the slope of the plot between the log of anodic peak current vs log υ, given by log Ip (μA) = 0.465 log υ + 0.444, as depicted in Fig. 10c. This fact has also been confirmed from the Nyquist plot of Fig. 9b. The straight line extending along the real and imaginary axes confirms a diffusion-controlled process. It was observed from the graphs that, as the scan rate increases, the peak potential Ep is shifted to more positive values, which confirms the kinetic limitation in the electrochemical process. The linear regression equation obtained is:

Ep (V) = 0.229 log υ + 0.446 (Fig. 10d).

The Laviron equation (eqn. 4) for Ep is as follows:
Ep = E0 + [ 2.303RT/ α nF] [ log (RTk0 /αnF)] + [2.303RT/ αnF] log ν eqn (1)

According to Bard and Faulkner, peak potential Ep and α are related as:
47.7/((Ep-Ep/2))=αn eqn (2)
Where α is the transfer coefficient and was found to be 0.12, R is the universal gas constant, T (temp.) is 298 K, k0 is the standard heterogeneous rate constant of the reaction, n stands for the number of electrons transferred, ‘υ’ is the scan rate and E0 stands for the formal redox potential.

From the slope of the Ep vs log ν plot, as depicted in Fig. 10d and equation (2), the value of the number of electrons involved in the irreversible oxidation was found to be 2. The value of E0 (0.713 V) can be obtained from the intercept of peak potential vs scan rate, as shown in Fig. 11 and therefore, the value of the heterogeneous rate constant obtained using the above equation is 0.692 s-1. It is to be noted that the rate of electrochemical oxidation is the rate of transport of each analyte molecule through the supporting electrolyte toward the electrode surface.

EVALUATION OF ELECTRODE SENSOR:
The peak current corresponding to the oxidation of RhB under optimized conditions was observed at + 0.68 V using DPV. The linear concentration range for the irreversible oxidation of RhB on MoS2/SA/SPCE was quantified using the developed sensor. As depicted in Fig. 12, RhB exhibited two linear ranges with concentration from 20 nM - 2000 nM, according to the regression equations, IPa = -0.055 C (μM) + 20.17 (R = 0.986). Using the slope value (m) of the linear regression equation, as depicted in Fig. 12b and the active electrode area (A), the sensitivity (S) of the fabricated electrode toward RhB oxidation was found to be 250.59 µA/µM/cm2.

The standard deviation of 6 blank experiments (σ) of the peak current corresponding to the peak potential of RhB was calculated. The limit of detection (LOD) (3 σ/S) and limit of quantification (LOQ) (10 σ/S) were found to be 0.141 nM and 0.471 nM, respectively. The sensor can be employed for the quantification of RhB in a higher concentration from 10-70 µM, as depicted in Fig. 13. It is clear from the results that the sensor showed good performance in terms of linear range at the nanomolar concentration of the analyte, with appreciable LOD and LOQ values. In short, the sensor offers the best performance towards nanomolar detection and appreciable detection in the micromolar range for RhB electrochemically.

REPEATABILITY AND REPRODUCIBILITY STUDIES:
By using the fabricated electrode in quick succession on the same day for 0.06 µM RhB oxidation, a repeatability of Relative Standard Deviation (RSD) = 3.88 %, n = 3 was achieved. The detection of 0.06 µM Rh B using the fabricated electrode on different days under varied laboratory conditions results in reproducibility of RSD = 1.7 %, n = 3. Thus, it can be concluded that the proposed MoS2/SA/SPCE electrode exhibited high reproducibility and repeatability for the nanomolar quantification of RhB.

STABILITY AND SELECTIVITY OF MOS2/SA/SPCE ELECTRODES:
The stability of the fabricated electrode was appreciably assessed for storage time by keeping the proposed MoS2/SA/SPCE electrodes at room temperature in 0.1 M PBS solution. The current response to different concentrations (1 µM, 11 µM and 21 µM) of RhB oxidation for the electrode was measured at regular intervals of days. Fig. 14 a represents the stability of the fabricated electrode after 45 days of fabrication. It clearly explains the variation in current voltage response of the fabricated electrode from day 1 to day 45. As depicted in Fig. 14a, the analysed signal response showed that the oxidation potential of the electrodes remains almost the same, indicating the stability of the fabricated electrode. Thus, the proposed MoS2/SA/SPCE electrode showed appreciable stability for almost 45 days for RhB quantification.

Fig. 14b represents the behaviour of the fabricated sensor in the presence of species that may particularly interfere with the detection of RhB using the fabricated sensor. These species include structurally similar chemicals with similar functional groups.

Selectivity is the aspect that differentiates a sensor from an electrode therefore, the impact of other molecules with similar structures must be considered when evaluating the practical utility of any sensor. Most often, synthetic colours are employed in combination with many other chemical constituents. The MoS2/SA/SPCE sensor was examined for its selectivity for different colouring agents such as 21 µM indigo carmine (IC), 21 µM ponceau (P4R), 21 µM tartrazine (TZ), 21 µM carmoisine (CM), and 21 µM sunset yellow (SY), as depicted in Fig. 14b. Thus, the fabricated sensor can be suitable for the individual quantification of RhB in the presence of other interferent species in food samples, even with 21-fold the concentration of RhB.

QUANTIFICATION OF RhB SAMPLES BY MOS2/SA/SPCE SENSORS IN REAL SAMPLE ANALYSIS
The developed sensor was employed effectively for the quantification of RhB in cotton candy samples by the standard addition method. For analysing real sample, about 5g of cotton candy was accurately weighed and dissolved in phosphate buffer solution. From this 10 µl was directly added to the electrochemical cell set up with the fabricated MoS2/SA/SPCE for the detection and the DPV analysis was carried out. Upon the addition of 10 µL of the cotton candy solution to the electrolyte, no signal was obtained corresponding to RhB. However, after spiking the samples with known concentrations of (0.05 µM, 0.06 µM and 2 µM) RhB, DPV response was visible and the recovery obtained was in the range of 95 to 102 %. The results have been depicted in Table 2.

Spectrophotometric method was also employed to validate the electrochemical performance of the developed MoS2/SA/SPCE for the quantification of RhB. As has been revealed in Table 2, the recovery obtained for the spiked quantification of RhB from the proposed method is more than the spectrophotometric method. This underscores the practical usefulness of the proposed MoS2/SA/SPCE for the trace quantification of RhB in cotton candy samples.

Table 1: Electrochemical quantification of spiked RhB from cotton candy samples on the proposed MoS2/SA/SPCE
Electrochemical Method Spectrophotometric Method
Sl. no Cotton candy+RhB added (µM) Obtained (µM) *Recovery ± RSD (µM) Cotton candy+RhB added (µM) Obtained (µM) *Recovery ± RSD (µM)
1 0.05 0.05 100 ± 0.0014 0.05 0.044 88 ± 0.02
2 0.06 0.057 96 ±0.0011 0.06 0.049 81 ± 0.01
3 2 2.07 103 ±0.0012 2 1.5 75 ± 0.024
*Mean of 4 replicate analysis, Recovery (%) = [calculated amount/added amount] *100

Table 3 compares the performance of the fabricated electrode in electrochemical quantification of RhB with respect to the sensitivity and Limit of Detection (LOD) with respect to various other electrodes reported in literature. The highlight of the fabricated electrode is that it offers high sensitivity with the ever-reported value of LOD for RhB determination electrochemically. Thus, the sensor employing MoS2/SA/SPCE can be effectively utilized as a medium for nanomolar detection of RhB.

Table 2: Comparison of MoS2/SA/SPCE sensor electrochemical parameters with the previously reported RhB electrochemical sensor
Electrode Technique Linear range (µM) LOD (nM) Sensitivity µA/µM/cm2) Real sample
SPCE DPV 60-4000 10 - Surfaces water
AgNP/GPLs/SPCE SWP 2-100 1940 - Crackers
Ag/ZnO/SPCE DPV 0.06-12.11 0.8 151 Red chili sauce
MIP/SPCE-MSPE DPV 5-100 1440 - Tomato juice, chili powder, tomato sauce
GOQDs/GCE DPV 5-50 800 - Chili powder
Cu@CS/GCE DPV 0.3-30 100 - Tomato & soya sauce
GS/GCE DPV 5-120 1.5 - Soy sauce
MoS2/SA/SPCE DPV 0.02-2
10-70 0.14 250 Cotton candy
SPCE-screen printed carbon electrode, MIP-molecularly imprinted polymer, MSPE-magnetic solid phase extraction, CS-carbon sphere, GS-graphene nanosheets, GCE-glassy carbon electrode, DPV-differential pulse voltammetry, SWV-square wave voltammetry

EVIDENCE FOR THE OXIDATION OF RHB AT MOS2/SA/SPCE SURFACE THROUGH UV VISIBLE SPECTROSCOPY
The oxidation of RhB at MoS2/SA/SPCE has been confirmed from the UV spectrum of the analyte solution before and after oxidation. RhB is characterized by two distinctive peaks in the UV-visible absorption spectrum, one at 256 nm and the other at 555 nm. It has been reported that the absorbance of RhB in a neutral environment of phosphate buffer solution occurred as a sharp peak at 555 nm corresponding to the n → π transition of C=N, C=O groups, which gives the dye its colour in the aqueous medium. As depicted in Figs. 15a and 15b, the absorbance at 555 nm showed a marked increase after electrochemical oxidation of 0.06 µM of RhB on MoS2/SA/SPCE by DPV. This indicates that a possible oxidation of the dye happened at the tertiary amine part of RhB.

The retention of the colour of the RhB solution even after oxidation also confirms that the tertiary amine is oxidized to amine oxide at MoS2/SA/SPCE. A possible mechanism for the electrochemical oxidation of RhB on the fabricated electrode is depicted in Fig. 16. The MoS2/SA hybrid acts as a medium for the charge transfer from the bulk of the electrolyte solution to the SPCE surface to facilitate the oxidation of RhB. The enhanced electrocatalytic property is due to the synergic effect of the transition metal chalcogenide and silicic acid at the modified MoS2/SA/SPCE electrode. The CV and DPV responses of MoS2/SA/SPCE towards RhB accentuate the proposed mechanism of oxidation.

The fabricated MoS2/SA/SPCE electrode of the present invention can be employed as an effective tool for the trace-level quantification of unauthorized RhB in food samples. The limit of detection (LOD) of the fabricated electrode sensor is as low as nanomoles of RhB which is considered to be the lowest till date. This low LOD value emphasizes the importance of the fabricated sensor in detecting even trace amounts of the analyte in food samples, especially in cotton candies. Notably, the fabricated electrode sensor shows appreciable selectivity and stability with remarkable reproducibility and repeatability. The fabricated sensor can further be converted to a portable device thus enhancing its functionality and ease of use.
, Claims:WE CLAIM:
1. Sensor electrodes for the selective detection and quantification of food colorant dyes and fabrication method thereof, said sensor comprising:
- at least one transition metal dichalcogenide (TMDC),
- molybdenum disulfide (MoS2)
- biocompatible silicic acid, and
- screen printed carbon electrodes (SPCEs)
characterized in that the 3D architecture of said fabricated sensor electrodes facilitates increased diffusion of the food colourant enabling detection and quantification of nanomolar quantities of said food colorant.
2. The sensor electrodes as claimed in claim 1 wherein, the said electrodes are used to detect and quantify nanomolar quantities of food colorant rhodamine B (RhB) in cotton candy.
3. The sensor electrodes as claimed in claim 1 wherein rhodamine B is detected and quantified through cyclic and differential pulse voltametric techniques.
4. The sensor electrodes as claimed in claim 1 wherein the sensor is fabricated by drop casting MoS2 and SA on a screen-printed carbon electrode.
5. The sensor electrodes as claimed in claim 1 wherein said screen printed carbon electrodes undergo hybrid modification of the overlaid SA and MoS2.
6. The sensor electrodes as claimed in claim 1 wherein the concentration of MoS2 and SA ranges from 0.8mM to 1.5mM.
7. The sensor electrodes as claimed in claim 1 wherein RhB can be detected at pH ranging from 4 to 9.
8. The sensor electrodes as claimed in claim 1 wherein the limit of detection (LOD) and limit of quantification (LOQ) of RhB is 0.141 nM and 0.471 nM, respectively, with the LOQ being as high as 10-70 µM.
9. The sensor electrodes as claimed in claim 1 wherein said fabricated sensor can detect and quantify nanomolar concentration of RhB even in the presence 21-fold higher concentration of other interferent species in food samples.
10. A method of fabrication of Sensor electrodes for selective detection and quantification of nanomolar quantities of food colorant as claimed in claim 1, the method comprising the steps of:
- preparing homogeneous suspension of 2 mM of molybdenum disulfide and 2 mM of silicic acid in 5 mL DMSO through ultrasonication, as stock,
- preparing equimolar concentrations of MoS2 and silicic acid ranging from 0.8 to 1.5 mM from the stock solution through the method of dilution and
- drop casting 5µl of the solutions to the working electrode surface of the Zensor screen printed carbon electrode,
- keeping the fabricated electrode for drying at room temperature (~ 28-30°C).