Description:Form 2

THE PATENT ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

“A PROCESS FOR PREPARING MYOGLOBIN COATED TITANIUM OXIDE NANOTUBE TO DETECT SUDAN-I IN FOOD PRODUCTS”

in the name of UNIVERSITY OF MADRAS an Indian national having address at UNIVERSITY OF MADRAS, GUINDY CAMPUS, CHENNAI, CHENNAI – 600025, TAMIL NADU, INDIA.

The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION:

The present invention relates to electrochemical detection method. More particularly the present invention relates to a method for preparation of Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode for rapid electrochemical screening of Sudan I based on Differential pulse voltammetry (DPV) detection method and amperometric detection method.

BACKGROUND OF THE INVENTION:

Due to urbanization and raise in population in the developing countries, there is huge demand in production of food grains and spices. Some of the Asian countries and Europe are widely usedchili powder in the food products as spice and taste bearing agent. In these food samples Sudan dyes have been added for the red coloring agent because of its cheaper cost which is considered as one of toxic food additives[Y. Shen et al., 2022]. Due to these reasons Sudan I is banned in western countries because of its toxicity[M. Elyasiet al., 2013]. Sudan I is a lipophilic soluble diazo- conjugate dye with the chemical formula 1- phenyl azo-2-naphthol with the commercial name CI Solvent Yellow 14. In the food industry, Sudan I is used as coloring agent alternate forchili powder inketchup, olive oil, and chicken eggs[L. Zhang et al., 2013, H.K. Maleh et al., 2014, L. Wang et al., 2015, Y. Mao et al., 2014]. Because of their toxic nature International Agency for research on Cancer has classified Sudan dyes as a category III carcinogen in 1975[M. Najafi et al., 2014]. Also Food and Drug Administration (FDA) and European Union (EU) have coined Sudan I dye as an illegal food additive since 2003[S. Tajik et al., 2021]. Despite all those health related issues, Sudan I is still being used in foodstuffs illegally. Various analytical techniques have been employed for the quantitative of Sudan I in food products. Mostly UV- Visible spectrophotometry[Z. A. Alothman et al., 2012],HPLC [C. Long et al., 2011], Chemiluminescence[Y. Zhang et al., 2006], SERS[M. I. Lopez et al., 2013],etc have been used for the detection of Sudan I. Alternatively electrochemical method can be employed for the sensitive detection of Sudan I at trace level for various reasons including low cost, simple and more accurate and fast screening method[Z. Mo et al., 2010].In the literature various chemically modified electrode have been employed to avoid electrode fouling and enhanced detection limit. Recent days metal and metal oxide nanoparticles and graphene based electrode have been used for the enhanced fast detection of Sudan I.Graphene/β-CD/PtNPs/GCE[S. Palanisamy, et al., 2017], MIP/AuNPs/GCE[M. Chao, et al., 2012], MWCNT/GCE[D. Yang, et al., 2010],AuNPs/RGO/GCE[J. Li, et al., 2015], Fe3O4/GCE[H. Yin, et al., 2011], OMC/GCE[D. Yang et al., 2009], GN-TiO2[T. Gan, et al., 2014], AgNPs@GO/GCE [E. Prabakaran, et al., 2015], Bi2WO6/GCE[V. Vinothkumar, et al., 2020], MMT-Ca/CPE[H. Lin, et al., 2008], Pt/GCE [L. Yu, et al., 2014] have been used. As in the modified electrodes the modifiers plays a key factor that influences the sensitivity and selectivity parameters. Hence an attempt has been made to fabricate a modified working electrode with enhanced sensitivity and selectivity parameters.

OBJECT OF THE INVENTION:

The main object of the present invention is to develop a method for preparation of Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode for rapid electrochemical screening of Sudan I.

Another object of the present invention is to develop a Differential pulse voltammetry (DPV) detection method of Sudan I employing Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode.
Yet another object of the present invention is to develop an amperometric detection method of Sudan I employing Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode.

Further object of the present invention is to employ the developed methods for rapid electrochemical screening of sudan I as toxic colorant in food products.

BRIEF DESCRIPTION OF DRAWINGS:

Figure 1 depicts the SEM images of TNT (A) and Myoglobin (Mb) modified TNT (B) and EDX spectra of Mb/TNT (C) at an operating voltage of 15 kV

Figure 2 depicts the XRD pattern of TNT (a) Mb/TNT (b) (A), Raman spectra of TNT (a) and Mb/TNT (b) (B)

Figure 3 depicts the Nyquist plots of bare GCE (a), TNT/GCE (b) and Mb/ TNT/GCE (c), in the presence of 10 mM [Fe(CN)6]3-/4- containing 0.1 M KCl as the supporting electrolyte. AC Amplitude: 5 mV; Frequency range: 0.01 Hz to 100 kHz(A) CV of bare GCE (a), TNT/GCE (b) and Mb/TNT/GCE (c) in the presence of 0.1 M KCl containing 10 mM [Fe(CN)6]3-/4- at a scan rate of 50 mVs-1(B). CV of bare GCE (a) and Mb/TNT/GCE (b) at scan rate of 50 mV/s in 0.1 M phosphate buffer (pH 7.0) (C) Cyclic voltammograms of bare GCE (a) , 3.3 × 10-4 M of Sudan I on GCE (b), 3.3 × 10-4 M of Sudan I on TNT modified GCE (c), and Mb/TNT in presence of 0.1 M of Sudan I (d) at scan rate of 50 mV/s in 0.1 M phosphate buffer (pH 7.0) (Insert : Histogram representation of different electrodes towards Sudan I sensing (3.3× 10-4 M))(D)

Figure 4 depicts the Effect of pH on cyclic voltammograms of 0.1 mM of Sudan I at Mb/TNT/GCE in presence of 0.1 M KCl containing phosphate buffer solution (pH 7.0) with various pH values (pH 1, 3, 5, 7, 9, and 11) at scan rate of 50 mV.s-1 (A). Variation of anodic peak current vs. pH, and Plot of Epa/mV vs. pH (B). CV of Sudan I at different scan rates 10- 190 mV/s in presence of 0.1 M phosphate buffer (pH 7.0) (C), Plot of Ipa vs. 1/2 (D).

Figure 5 depicts the DPV of Mb/TNT modified GCE in different concentrations of (0.6 x 10-6 to 14.1 x 10-5 M) Sudan I in 0.1 M phosphate buffer (pH 7.0) (A), Calibration plot of Ipa vs. conc. Sudan I (B), Amperometric response of Mb/TNT/GCE at an applied potential + 0.9 V subsequent addition of different concentrations ranges from of 0.6 x 10-6 to 20.6 x 10-5 M of Sudan I in the presence of 0.1 M KCl containing PBS (pH 7.0) (C), Calibration plot of Ip vs. conc of Sudan I (D).

Figure 6 depicts the CV response of reproducibility of modified electrodes (A) and storage stability of Mb/TNT modified GCE for electro-oxidation of Sudan I response after being kept for 70 days (B). All measurements were recorded in 3.3x10-3M Sudan I in 0.1 M PBS.

Figure 7 depicts the Amperometric response of Mb/TNT/GCE upon successive addition of 10 mM of HZ and other interfering chemicals to 0.1 M KCl containing PBS (pH 7.0) at the applied potential of +0.9 V, respectively.

Figure 8 depicts the scheme of surface modification process and electrochemical oxidation of Sudan I.

SUMMARY OF THE INVENTION:

The present invention presents a myoglobin based as a redox active metalloprotein immobilized titanium oxide nanotube can be used for the enhanced electrochemical oxidation of Sudan I in various food products. The nanotubular TiO2 was synthesized by template assisted method in which jute fiber was used as a hard template. Some of the analytical studies have proved that effective binding of myoglobin on TNT. The basic electron transfer behavior of the system tested by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques. The oxidation potential of Sudan I was observed at +0.9 V vs. Ag/AgCl in 0.1M phosphate buffer medium with enhanced oxidation peak current value than bare glassy carbon electrode. Differential pulse voltammetry and amperometry methods were employed for the electrochemical detection of Sudan I in various food samples. A calibration plot was obtained by plotting the peak current against concentration of Sudan I in the range of 0.10 to 25 µM and the detection limit 25 nm. The as prepared probe can be used for the real sample analysis with reproducible results.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention discloses a method for preparation of Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode for rapid electrochemical screening of Sudan I based on Differential pulse voltammetry (DPV) detection method and amperometric detection method.

Chemicals employed in the present invention: Titanium tetrafluride (TiF4), myoglobin(Mb),sodium phosphate monobasic dehydrate (Na2HPO4), sodium phosphate dibasic dehydrate (NaH2PO4), potassium chloride(KCl) were purchased from Sigma-Aldrich(Vijaya Scientific, Chennai, India. Sudan I and Potassium hydoxidewas purchased from SRL. All solvents used are of analytical grade and were used without any further purification. Jute fibers was kindly supplied by Prof. Grider, Department of Textile, Anna University, Chennai

The phosphate buffer solution in pH 7 is used as an electrolyte. The pH of the solution was tested using Elico-pH meter at room temperature. 20 mL of 0.1 mM Sudan I (stock solution) was freshly prepared with Milli-Q water and stored under dark condition until further use.

Instrument Techniques employed in the present invention: The structural and morphological interpretations have been carried out using field emission scanning electron microscopy (FE-SEM, SU6600, Hitachi, Japan). The elemental composition of the nanocomposite was studied using an energy dispersive X-ray spectrometer (EDAX, 8121-H, Japan)at an operating voltage of 20 kV. The UV-Visible absorption spectrum was recorded at RT using UV-Vis spectroscopy on a Schimadzu UV-Visible Spectrometer (model UV1800) dual-beam spectrometer operated at a resolution of 2 nm. XRD patterns of this powdered samples were also documented at RT by X” PERT-PRO diffractometer with a Cu Kα Radiation (λ = 1.5406 Å). Raman analysis was recorded using confocal Raman spectroscopy model 11I, Nanophoton, Japan, with a Ne – Ar laser source of 532.9 nm wavelength, grating 600/mm. Electrochemical experimentations were performed with CHI 660A electrochemical instrument (Texas, USA). A single cell with a 3-electrodes configuration setup was used. Hereglassy carbon electrode - GCE (3 mm in diameter) is used as a working electrode andPlatinum wire (0.5 mm in diameter) is functioning as counter electrode and the Ag/AgCl (3 M KCl) is act as reference electrodes.Both DPV (Scan rate: 20 mV.s-1, Pulse width: 50 mV, Pulse amplitude: 25 mV) and amperometry (i-t) experiment for sensing of Sudan I was carried at applied potential of + 0.8 V vs. Ag/AgCl (3 M KCl).
The electrochemical impedance studies were carried outusing the CHI-660B electrochemical system with an applied alternating current voltage of 5 mV amplitude at a frequency ranging from 0.01-100 Hz.The redox probe was prepared by using 5mM [Fe(CN)6]3–/4– containing 0.1 M KCl in a 100 mL standard flask using DD water. Prior to starting the electrochemical experiments, all the solutions were purged with high purity N2gasto scrap oxygen from the solutions for 15 minutes and each of the experiments was carried out at 25 + 5˚ C under a nitrogen atmosphere.

Experimental procedure: Synthesis of TNT: Jute fibers were finely chopped and pretreated with 1M alcoholic KOH solution under ultrasonic irradiation to remove all organic impurities, and then the fibers were washed with DI water and acetone. To deposit TiO2 thin-film onto the finely chopped fibers, 0.04 M of TiF4 was dissolved in 100 ml DI water containing chopped fibers and placed in Teflon beaker(pH of the solution was adjusted to 2 using 1% NH3). The temperature of the medium was fixed at 60oC for 24 h. TheTiO2 modified fibers were isolated by filtration, dried under a nitrogen atmosphere and finally calcined at 500oC for 1 h to remove the template.

Synthesis of Mb modified TNT: Myoglobin (Mb) modified TNT nanocomposite was prepared in a simple self-assembly method, about 0.15 g of myoglobin was dissolved in 25 ml of 0.1 phosphate buffer and then mixed with 0.25 g of prepared TNT. This reaction mixture was kept under static conditions andstored at 5oC for overnight and then centrifuged. The resultant product was isolated and dried in N2 atmosphere. The obtained product Mb/TNT was stored in the dark at 5oC until further use.

Fabrication of Mb/TNT modified GCE: TheMb/TNT nanocomposites modified electrode was fabricated by the following procedure. The surface of GCE was pre-cleaned mechanically by polishing with alumina (0.5 µm powder) and rinsed thoroughly with MiliQ-Pure water, washed with 1:1 v/v ratio of nitric acid and MiliQ-Pure water, dried at room temperature. About 1 mg of Mb/TNT nanocomposites was dispersed in 1 mL of 0.1M phosphate buffer and then sonicated for 10 min. The colloidal suspension ofMb/TNT (5 μL)was drop cast onto the surface of GCE and then allowed to dry at room temperature. The surface modification process and electrochemical oxidation of Sudan I is shown in Figure 8.

The structure and morphology of the prepared nanocomposite were examined using FE-SEM. Fig.1A-Bshows that the prepared TiO2 is tubular in structure. A rough surface on the tubular structure was noted inwhich is due to the modification of myoglobin on the TNT surface. On employing the composite to EDAX analysis the distinct signals for the elements Fe, Ti, and Oshows the presence of myoglobin and TiO2nanotube (Fig1C). Further, the adsorption of Mb onto the TNT surface was confirmed using UV-Visible spectrum studies by measuring the decreasing of soret bond intensity.The XRD analysis of the as-synthesized sample was done to identify the crystallographic phase of the sample. Fig. 2A,shows the X-ray diffraction (XRD) pattern of the annealed samples at 500 °C suggest that TiO2 nanotubes are showing the anatase phase of TNT (JCPDS Card 89-4921) and the appearance of well-defined sharp peaks are due to the anatase form of TNT obtained by this preparation method. The Mb modified TNT nanotubes show a slight shift in the peak position and the peak intensity also decreases as the surface of the TiO2 structure is being modified with Mb displayed. Raman spectral data of titanium oxide nanotubes are shown before and after treating with Mb. A well-defined six Raman peaks were observed from 50 to 1000 cm-1ranges. The peak positions are assigned as A1g + 2B1g + 3Eg mode of phonon vibration of TNT rhombohedron lattice vibration.This Raman pattern is found to be in correlation with the previously published results [A. Leon,et al.,2017,X. Xue, et al., 2012]. On treating TNT with Mb molecules, the intensity of Raman peaks was decreases further without changing their peak positions which is due to the surface modification(Fig.2B).
Electrochemical impedance spectroscopy (EIS): The interfacial electron transport property of the modified electrode was examined by electrochemical impedance spectroscopy for analysis changes in the surface features of the modified electrodes. The Randles circuit was mainly used as a fitting model in EIS analysis for interpretation of the Nyquist plot. Fig.3Adisplays the EIS curve of a) bare GCE, b) TNT/GCE and c) Mb/TNT/GCE in the supporting electrolyte of 0.1 M KCl containing 1 mM [Fe(CN)6]3-/4- at an applied frequency range from 0.1 Hz to 100 kHz with a constant amplitude of 10 mV. For a bare electrode, the semicircle was is higher than Mb/TNT/GCE which is due to the higher interfacial resistance of the bare/GCE and the Rct value was obtained at 50.47 k. After that, TNT/GCE showed a low resistance value (10.1 k) which is clearly implied that the presence of halloysite improved the electron transport. After TNT was composite with Hemin, the Rct value was further decreased to 4 k, probably because the Mb/TNT/GCE could generate an excellent electron transfer rate and increased surface area.

Electrochemical behavior of Mb/TNT modified electrode: The CV behaviour of Mb/TNT modified electrode and GCE was examined usingpotassium ferricyanide in phosphate buffer medium as an electrochemical probe. Fig.3B, shows CV curves of a) bare GCE, b) TNT/GCE, and c) Mb/TNT/GCE in the presence of 5mM K3[Fe(CN)6]3-/4-containing 0.1 KNO3 at a scan rate of 50 mV.s-1 at various modified electrodes. The CV of different modified electrodes exhibited a pair of well-defined redox peaks corresponding to K3[Fe(CN)6]3-/4- redox couple. Further, the modified electrodes exhibited higher peak current and lower peak potential separation value on compared with bare GCE. The anodic to cathodic peak potential separation at these modified electrodes was less on comparing to that of bare GCE, showing that the modified electrodes significantly increased the electrochemical behavior which is due to the higher surface area. The Mb/TNT modified electrode showed more improved electrochemical behavior with a 15 and 20 mV lower peak separation value than that of TNT modified electrode and bare GCEwhich is due to the redox-active center in the Mb and the fast electron-transfer kinetics.The peak potential separation (Ep) was estimated from the 1e-transfer of Fe3+/Fe2+ redox reaction on Mb/TNT modified electrode (Ep = 75 mV), TNT/GCE (Ep = 80 mV) and for bare GCE as (Ep = 95 mV).The Cyclic voltammogram of Mb/TNT modified GCE shows a well-defined redox peak measured at a scan rate of 50 mV/s in 0.1 M phosphate buffer (pH 7.0)which clearly shows that the myoglobin redox molecule is strongly bounded onto the surface of TNT(Fig 3 C).

Electrocatalytic oxidation of Sudan I at Mb/TNT modified electrode: To investigate the electrochemical behavior of Sudan I, cyclic voltammetry was employed for the detection of 3.3 × 10-4 M of Sudan I on different modified electrodes. Fig 3Dshows the CV graphs on bare GCE and modified electrodes like TNT/GCE and Mb/TNT on GCE in 3.3 × 10-4 M of Sudan I in the presence of 0.1 M KCl containing PBS ( pH 7) at a scan rate of 50 mV/s. From the graph, it is seen that Mb/TNT shows a maximum current response on comparing to that of TNT modified electrode and GCE with a slight shift at an oxidative potential of + 0.9 V. The bar diagram in (Fig 3D insert)clearly explains the oxidation peak current response of different modified and unmodified electrodes.

The influence of the pH of electrolyte solution on the electrochemical oxidation of Sudan I at the Mb/TNT modified glassy carbon electrodes was studied in the pH range from 1.0 to 11.0. From the Fig 4A, CV graph,it is found that pH 7.0 is found to be the optimum pH as a better current response was found at this pH for 0.1 mM of Sudan I at a scan rate of 50 mV.s-1.At lower pH ranges the anodic peak current was less significant which then shows a maximum peak current at pH 7.0 then again reduces on increasing the pH. Thus this is pH 7.0 was chosen for the subsequent analytical experiments. From the pH graph, it is studied that the peak potentials of Sudan I shifted linearly almost to the negative side on increasing the pH, confirming that the protons are directly involved in the electrode reaction process of the analyte.From Fig 4Bthe relationship between anodic peak current and thepH of the solution is expressed in the following regression equation can be expressed as Ep (V) = -0.0526 x + 0.9362 with a correlation coefficient of 0.9985. During the electrochemical oxidation process Sudan I is converted into oxidized form of Sudan I with the two-electron transfer process. Additionally, the effect of scan rate was investigated by CV. From Fig 4C a linear raise in the anodic peak current for 0.6 x 10-4 M Sudan I was noted with a slight shift in the oxidative peak potential on increasing the scan rate from 10- 190 mV/sin presence of 0.1 M phosphate buffer (pH 7.0). From Fig 4Dit is observed that the anodic peak current shows a linear relationship with the square root of scan rate and the regression equation is given asIpa = 6.3041 x – 17.2799, with a correlation coefficient of 0.9984, indicating that the electrochemical oxidation process of Sudan I at Mb/TNT modified GCE is a typical adsorption controlled process.

Electrochemical detection of Sudan I: Differential pulse voltammetry (DPV) method was used in the determination of Sudan I on account of its high sensitivity.The electrochemical oxidation of Sudan I was shown in the form calibration curve. Fig 5Ashows the calibration curve of Sudan I at Mb/TNT/GCE at different concentration in in 0.1 M phosphate buffer (pH 7.0). Under the optimized experimental conditions (0.8 V pulse potential, 0.05 V as pulse amplitude and 0.5 s as pulse width), the oxidation peak current increased linearly on increasing the concentration of Sudan I in the consecutive addition method. The peak currents were linearly proportional to Sudan Iconcentrations in the range of 0.6× 10-6 to 14.1 × 10-5 M and the linear regression equation of Ip(µM) = 0.4290x + 0.0310 was obtainedwith a correlation coefficient of R2 = 0.9994 as shown in Fig.5B. The detection limit value (3σ/slope where σ = standard deviation) was found to be25 nMat a signal to noise (S/N) ratio of 3. The lowest LOD obtained with the present sensor as compared to other reported methods (Table 1), indicates evidently that the Mb/TNT modified GCE provide a good platform for the effective detection of Sudan I in food samples at lower concentration ranges.

Amperometric studies: The amperometric technique (i-t) isfound to be the most sensitive and reliable method compared to the DPV technique to evaluate the electro-oxidative behavior of Sudan I at Mb/TNT/GCE. Fig.5C shows a typical i-t plot for the Mb/TNT/GCE under optimized experimental conditions with the successive addition of various concentrations of Sudan I in a constantly stirred PB solution at consecutive time intervals. The working potential was set as + 0.8 V vs Ag/AgCl for Sudan I. A considerable increase in current signal was noted on successive addition of Sudan I into the continuously stirred electrolyte solution. This modified glassy carbon electrode responded in seconds toeach addition of analyte and reached the steady-state within 5s. From the figure clearly shows the oxidation current increase on increasing the concentration of Sudan I in the linear range from 0.6 x 10-6 M to 20.6 x 10-4 Mrespectively. A linear calibration plot was obtained for the i-t curve and the concentration of Sudan I, the corresponding regression equation obtained is Ipa = 0.4304 (C/µM) - 0.3036 with a correlation coefficient of (R2 = 0.9990)(Fig 5D). The limit of detectionof Sudan I was calculated from the calibration graph as 22nMbased on the signal-to-noise ratio (S/N = 3) respectively and the current sensitivity was found to be 1.30436 µA µM-1 cm-2. The present work was compared with previously reported of Sudan I sensors that are listed in Table I. The developed sensor shows a low LOD, widest linear range, relatively high sensitivity than some other modified electrodes, justifying the feasibility and practical applicability of Mb/TNT/GCE for detection of food adulterant.

Table 1: Determination of Sudan I with various different electrodes and Mb/TNT modified GCE

Electrode Method Linear range
(µmol/L) LOD (µmol/L) Real Samples Ref
CuO/3DNPC/GCE DPV 2.5- 100 0.84 Ketchup & Chili sauces Q. Ye, et al., 2019
PVP/ZnSe-CTAB-GR/GCE DPV 0.004-15.0 0.5 - 41
La-Co3O4/SPE DPV 0.3-300 0.05 Tomato paste
Ketchup sauce &
Chilli powder H. M. Moghaddam, et al., 2019
NiFe2O4/SPE DPV 0-100 0.05 Tomato paste
Ketchup sauce &
Chilli powder H. Beitollahi, et al., 2018
ZnO-CuOnanoplates/ SPE DPV 0.6- 600.0 0.18 Chilli powder,
Ketchup sauce,&Tomato paste S. Tajik, et al.,2020
AgNPs/rGO DPV 0.041 Chilli powder F. Meng, et al.,2022
Fe3O4-ZIF-67/ILCPE DPV 0.5- 560 0.1 Chilli powder, &
Ketchup sauce M. Shahsavari, et al.,2022
Mb/TNT/GCE DPV 0.6- 141.0 0.025 Chili powder & Chili Sauce Present Invention

Reproducibility, Repeatability and Interference: The reproducibility of the proposed sensor was determined by involving the five different electrodes of the same group in the detection of 2.0x10-6 M of Sudan I (Fig 6A). The relative standard deviation of the current response of Sudan I was calculated to be below 3.0 %. Henceforth these results show that the reproducibility of the proposed Mb/TNT sensor towards Sudan I detection is withinthe acceptable range. The stability of the sensor was studied by sweeping the electrode for 200 cycles, about a 2.5 % decrease in the initial current response was observed for Sudan I indicating that the modified electrode shows better stability. Furthermore, the storage stability of the proposed sensor was evaluated for the detection of 2.0 x10-6 M of Sudan I with no major changes in the current response was noted for the first 15 days then a 5% decrease in current response was observed in response after the storage on a month period of duration. About 90% of current responses were found to retain after 60 to 70 days of storage as shown in Fig 6B. Henceforth, the long-term stability of the proposed electrode was good enough for the ceaseless and constant operation.

The selectivity of the Mb/TNT/GCE was investigated by sensing the response of this modified electrode towards Sudan I detection in the presence of various interfering species at different fold concentrations. The common potential interference for Sudan I in chili powder, sauce and ketchup were natural pigments like β- carotene, lycopene, and zeaxanthin. Hence most of them are liposoluble which isn’t dissolved in water or ethanol, and their peak potential is different from the potential of Sudan I. Hence some other potential interferences such as Mg2+, Ca2+, Zn2+in 300 folds, 150 folds of Cu2+, Cl-, ascorbic acid, phenylalanine, and 500 folds of Glucose, β- carotene, tryptophan were added along with Sudan I but none of these elements caused any interference towards the determination of Sudan I. Therefore, this Mb/TNT/GCE sensor shows better selectivity towards the detection of the basic food adulterant Sudan I.Amperometric response of Mb/TNT/GCE upon successive addition of 10 mM of Sudan I and other interfering chemicals to 0.1 M KCl containing PBS (pH 7.0) at the applied potential of + 0.9 V, is show in Fig 7.

Real sample analysis: Some commonly and regularly used food samples in human daily routine like chilli powder and chili sauce were employed to evaluate the practical application of the proposed Mb/TNT/GCE sensor for the detection of Sudan I. Since there was no electrochemical response for the real samples when employed in the detection a known amount of Sudan I solution was added to the real samples and the total content of Sudan I was then determined and the recovery has been calculated which is shown in table 2. The recovery was obtained in the range of 100.02%, indicating that the proposed Mb/TNT/GCE sensor is effective and accurate in Sudan I sensing.

Table 2: Determination of Sudan I in food samples (n=3) -Real sample analysis

Sample Original (M) Added (M) Found (M) Found by HPLC method(M) Recovery (%) RSD (%)

Chilli powder 0.0 5 4.98 3.5 ± 0.10 100.2 3.0± 0.149
0.0 10 9.80 7.1 ± 0.20 98.8 1.9± 0.151
0.0 15 14.99 12.1 ± 0.46 99.8 3.2± 0.480

Chilli sauce 0.0 4 3.86 2.1 ± 0.30 96.5 2.9 ± 0.045
0.0 8 7.90 6.5 ± 0.15 98.7 2.5± 0.197
0.0 12 11.99 10.9± 0.15 99.9 3.2± 0.383

Thus the present invention provides an interesting result for electrochemical detection of Sudan I at Mb/TNT modified GCE. An enhanced oxidation peak current was noted at +0.90V vs. Ag/AgCl and observed three fold enhanced current response than the bare GCE. For the sensitive detection of Sudan I. DPV and amperometry methods were employed. The proposed sensor is stable and reproducible for repeated usages.

In one of the preferred embodiment, the present invention shall discloses a method for preparation of Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode for rapid electrochemical screening of Sudan I. The process comprises of

a. Synthesis of TNT comprises of finely chopping Jute fibers and pretreating with alcoholic KOH solution under ultrasonic irradiation followed by washing with deionised water and acetone followed by adding TiF4 dissolved in deionised water adjusting the pH to 2 using 1% NH3 and maintaining the temperature at 60oC for 24 h followed by filtration, drying under a nitrogen atmosphere and calcinations at 500oC for 1 h to form TNT;

b. Synthesis of Mb modified TNT nanocomposites comprises of dissolving myoglobin in 0.1 phosphate buffer and mixing with the prepared TNT followed by allowing to keep under static conditions and storing at 5oC for overnight followed by centrifugation and isolating residue and drying in N2 atmosphere to form Mb/TNT nanocomposites;

c. Fabrication of Mb/TNT modified GCE comprises of
i. pre-cleaning surface of GCE mechanically by polishing with alumina powder and rinsing thoroughly with MiliQ-Pure water followed by washed with nitric acid and MiliQ-Pure water and drying at room temperature to form surface modified GCE;
ii. dispersing prepared Mb/TNT nanocomposites in phosphate buffer and sonicating to form colloidal suspension ofMb/TNT and drop casting the colloidal suspension onto the surface of surface modified GCE and allowing to dry at room temperature to form Mb/TNT modified GCE.

In another preferred embodiment, the present invention shall discloses a method for preparation of Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode for rapid electrochemical screening of Sudan I. The process comprises of

a. Synthesis of TNT comprises of finely chopping Jute fibers and pretreating with 1M alcoholic KOH solution under ultrasonic irradiation followed by washing with deionised water and acetone followed by adding 0.04 M of TiF4 dissolved in 100 ml deionised water adjusting the pH to 2 using 1% NH3 and maintaining the temperature at 60oC for 24 h followed by filtration, drying under a nitrogen atmosphere and calcinations at 500oC for 1 h to form TNT;
b. Synthesis of Mb modified TNT nanocomposites comprises of dissolving 0.15 g of myoglobin in 25 ml of 0.1 phosphate buffer and mixing with 0.25 g of the prepared TNT followed by allowing to keep under static conditions and storing at 5oC for overnight followed by centrifugation and isolating residue and drying in N2 atmosphere to form Mb/TNT nanocomposites;
c. Fabrication of Mb/TNT modified GCE comprises of
i. pre-cleaning surface of GCE mechanically by polishing with 0.5 µm alumina powder and rinsing thoroughly with MiliQ-Pure water followed by washed with 1:1 v/v ratio of nitric acid and MiliQ-Pure water and drying at room temperature to form surface modified GCE;
ii. dispersing 1 mg of prepared Mb/TNT nanocomposites in 1 mL of 0.1M phosphate buffer and sonicating for 10 min to form colloidal suspension ofMb/TNT and drop casting 5 μL of the colloidal suspension onto the surface of surface modified GCE and allowing to dry at room temperature to form Mb/TNT modified GCE.

In yet another preferred embodiment, the present invention shall disclose a Differential pulse voltammetry (DPV) detection method of Sudan I employing Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode. The method comprises of

a. preparing a Sudan I sample solution,
b. setting up an electrochemical cell with Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode, Ag/AgCl as reference electrode, and platinum wire as counter electrode;
c. applying a controlled potential of 0.8 V pulse potential, 0.05 V as pulse amplitude and 0.5 s as pulse width to the said working electrode, measuring the resulting current generated by Sudan I redox reaction at the electrode surface, and relating measured current to the Sudan I concentration using a calibration curve to determine the Sudan I concentration.

In further preferred embodiment, the present invention shall discloses an amperometric detection method of Sudan I employing Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode. The method comprises of

a. preparing a Sudan I sample solution,
b. setting up an electrochemical cell with Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode and Ag/AgCl as reference electrode;
c. applying a controlled potential of as + 0.8 V vs Ag/AgCl, and measuring the resulting current generated by Sudan I redox reaction at the electrode surface, and relating measured current to the Sudan I concentration using a calibration curve to determine the Sudan I concentration.

WORKING EXAMPLE:

a method for preparation of Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode for rapid electrochemical screening of Sudan I. The process comprises of

a. Synthesis of TNT comprises of finely chopping Jute fibers and pretreating with 1M alcoholic KOH solution under ultrasonic irradiation followed by washing with deionised water and acetone followed by adding 0.04 M of TiF4 dissolved in 100 ml deionised water adjusting the pH to 2 using 1% NH3 and maintaining the temperature at 60oC for 24 h followed by filtration, drying under a nitrogen atmosphere and calcinations at 500oC for 1 h to form TNT;

b. Synthesis of Mb modified TNT nanocomposites comprises of dissolving 0.15 g of myoglobin in 25 ml of 0.1 phosphate buffer and mixing with 0.25 g of the prepared TNT followed by allowing to keep under static conditions and storing at 5oC for overnight followed by centrifugation and isolating residue and drying in N2 atmosphere to form Mb/TNT nanocomposites;

c. Fabrication of Mb/TNT modified GCE comprises of
i. pre-cleaning surface of GCE mechanically by polishing with 0.5 µm alumina powder and rinsing thoroughly with MiliQ-Pure water followed by washed with 1:1 v/v ratio of nitric acid and MiliQ-Pure water and drying at room temperature to form surface modified GCE;
ii. dispersing 1 mg of prepared Mb/TNT nanocomposites in 1 mL of 0.1M phosphate buffer and sonicating for 10 min to form colloidal suspension ofMb/TNT and drop casting 5 μL of the colloidal suspension onto the surface of surface modified GCE and allowing to dry at room temperature to form Mb/TNT modified GCE.

A Differential pulse voltammetry (DPV) detection method of Sudan I employing Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode. The method comprises of

a. preparing a Sudan I sample solution,

b. setting up an electrochemical cell with Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode, Ag/AgCl as reference electrode, and platinum wire as counter electrode;

c. applying a controlled potential of 0.8 V pulse potential, 0.05 V as pulse amplitude and 0.5 s as pulse width to the said working electrode, measuring the resulting current generated by Sudan I redox reaction at the electrode surface, and relating measured current to the Sudan I concentration using a calibration curve to determine the Sudan I concentration.

An amperometric detection method of Sudan I employing Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode. The method comprises of

a. preparing a Sudan I sample solution,

b. setting up an electrochemical cell with Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode and Ag/AgCl as reference electrode;

c. applying a controlled potential of as + 0.8 V vs Ag/AgCl, and measuring the resulting current generated by Sudan I redox reaction at the electrode surface, and relating measured current to the Sudan I concentration using a calibration curve to determine the Sudan I concentration.

Although the invention has now been described in terms of certain preferred embodiments and exemplified with respect thereto, one skilled in art can readily appreciate that various modifications, changes, omissions and substitutions may be made without departing from the scope thereof. It is intended therefore that the present invention be limited solely by the scope of the following claims.
, Claims:WE CLAIM:

1. A method for preparation of Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode for rapid electrochemical screening of Sudan I, the claimed process comprises of

a. Synthesis of TNT comprises of finely chopping Jute fibers and pretreating with alcoholic KOH solution under ultrasonic irradiation followed by washing with deionised water and acetone followed by adding TiF4 dissolved in deionised water adjusting the pH to 2 using 1% NH3 and maintaining the temperature at 60oC for 24 h followed by filtration, drying under a nitrogen atmosphere and calcinations at 500oC for 1 h to form TNT;
b. Synthesis of Mb modified TNT nanocomposites comprises of dissolving myoglobin in 0.1 phosphate buffer and mixing with the prepared TNT followed by allowing to keep under static conditions and storing at 5oC for overnight followed by centrifugation and isolating residue and drying in N2 atmosphere to form Mb/TNT nanocomposites;
c. Fabrication of Mb/TNT modified GCE comprises of

i. pre-cleaning surface of GCE mechanically by polishing with alumina powder and rinsing thoroughly with MiliQ-Pure water followed by washed with nitric acid and MiliQ-Pure water and drying at room temperature to form surface modified GCE;
ii. dispersing prepared Mb/TNT nanocomposites in phosphate buffer and sonicating to form colloidal suspension ofMb/TNT and drop casting the said colloidal suspension onto the surface of surface modified GCE and allowing to dry at room temperature to form Mb/TNT modified GCE.

2. A method for preparation of Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode for rapid electrochemical screening of Sudan I, the claimed process comprises of

a. Synthesis of TNT comprises of finely chopping Jute fibers and pretreating with 1M alcoholic KOH solution under ultrasonic irradiation followed by washing with deionised water and acetone followed by adding 0.04 M of TiF4 dissolved in 100 ml deionised water adjusting the pH to 2 using 1% NH3 and maintaining the temperature at 60oC for 24 h followed by filtration, drying under a nitrogen atmosphere and calcinations at 500oC for 1 h to form TNT;
b. Synthesis of Mb modified TNT nanocomposites comprises of dissolving 0.15 g of myoglobin in 25 ml of 0.1 phosphate buffer and mixing with 0.25 g of the prepared TNT followed by allowing to keep under static conditions and storing at 5oC for overnight followed by centrifugation and isolating residue and drying in N2 atmosphere to form Mb/TNT nanocomposites;
c. Fabrication of Mb/TNT modified GCE comprises of

i. pre-cleaning surface of GCE mechanically by polishing with 0.5 µm alumina powder and rinsing thoroughly with MiliQ-Pure water followed by washed with 1:1 v/v ratio of nitric acid and MiliQ-Pure water and drying at room temperature to form surface modified GCE;
ii. dispersing 1 mg of prepared Mb/TNT nanocomposites in 1 mL of 0.1M phosphate buffer and sonicating for 10 min to form colloidal suspension ofMb/TNT and drop casting 5 μL of the said colloidal suspension onto the surface of surface modified GCE and allowing to dry at room temperature to form Mb/TNT modified GCE.

3. A Differential pulse voltammetry (DPV) detection method of Sudan I employing Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) of Claim 1 or 2 as working electrode, the claimed method comprises of

a. preparing a Sudan I sample solution,
b. setting up an electrochemical cell with Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode, Ag/AgCl as reference electrode, and platinum wire ascounter electrode;
c. applying a controlled potential of 0.8 V pulse potential, 0.05 V as pulse amplitude and 0.5 s as pulse width to the said working electrode, measuring the resulting current generated by Sudan I redox reaction at the electrode surface, and relating measured current to the Sudan I concentration using a calibration curve to determine the Sudan I concentration.
4. A amperometric detection method of Sudan I employing Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) of Claim 1or 2 as working electrode, the claimed method comprises of

a. preparing a Sudan I sample solution,
b. setting up an electrochemical cell with Myoglobin(Mb)/nanotubular TiO2 (TNT) modified glassy carbon electrodes(GCE) as working electrode and Ag/AgCl as reference electrode;
c. applying a controlled potential of as + 0.8 V vs Ag/AgCl, and measuring the resulting current generated by Sudan I redox reaction at the electrode surface, and relating measured current to the Sudan I concentration using a calibration curve to determine the Sudan I concentration.

Dated this 12th day of FEB 2025

For UNIVERSITY OF MADRAS
By its Patent Agent

Dr.B.Deepa
IN/PA 1477