Claims:WE CLAIM:
1. An electro chemical method for quantitative detection of nitrite comprises of employing characterized glassy carbon modified with polyaniline protected cellulose nanofibers PANI/CNF in predetermined concentration of a buffer solution of predetermined pH where in oxidation of nitrite occurred at predetermined Voltage with respect to reference electrode.

2. An electro chemical method for quantitative detection of nitrite comprises of employing characterized glassy carbon electrode modified with polyaniline protected cellulose nanofibers PANI/CNF in 0.1M of KCL containing PBS of 7.0pH wherein oxidation of nitrite occurred at +0.85V with respect to reference electrode.

3. The electro chemical method as claimed in claim 1&2 wherein the said characterized glassy carbon electrode modified with polyaniline protected cellulose nanofibers PANI/CNF is prepared by employing PANI/CNF as electrode modifier to deposit electrode surface as electron transfer mediator for sensing of nitrite.

4. The electro chemical method as claimed in claim 1&2 wherein the said PANI/CNF is prepared by surface modification of cellulose nanofibers with polyaniline through oxidative polymerization aniline using ammonium persulfate.

5. The process as claimed in claim 4 wherein the said cellulose nanofibers is isolated from sugarcane bagasse biomass by the acid hydrolysis method.

6. The electro chemical method as claimed in claim 1&2 wherein the said electro chemical method is cyclic voltammetry method.
7. The electro chemical method as claimed in claim 1&2 exhibits a linear range of detection between 0.5 mM to 6.5 mM with detection limit of 0.0722 ?M.

8. The electro chemical method as claimed in claim 1&2 exhibits a sensitivity of 62.2 ?A. ?M-1.

Dated this 27th day of May 2020

For UNIVERSITY OF MADRAS
By its Patent Agent

Dr.B.Deepa
, Description:Form 2

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

“A METHOD OF QUANTIFICATION DETECTION OF NITRITE IN FOOD SAMPLES EMPLOYING MODIFIED ELECTRODE POLYANILINE PROTECTED CELLULOSE NANOFIBERS DERIVED FROM SUGARCANE BAGASSE”

in the name of UNIVERSITY OF MADRAS an Indian National having address at Dept of Inorganic Chemistry, University of Madras, Guindy Campus, Chennai-600 025, 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 the field of electrochemical detection. More particularly the present invention relates to electrochemical method for quantitative detection of nitrite in food samples.

BACKGROUND OF THE INVENTION:

The determination of nitrite ion is an important aspect in the analysis of food samples. The determination of nitrite in water and food stuffs has attracted the scientific community significantly in recent years because of its harmful impact on human health. The permissible level of nitrite in drinking water is 1 ?g ml-1. Excessive contamination of nitrite in drinking water could be hazardous to health, especially for infants and pregnant women. Nitrite oxidizes iron in hemoglobin of red blood cells to form methemoglobin, which reduces the oxygen carrying ability which is known as methemoglobinemia (blue baby syndrome). In addition, the reaction between nitrite and secondary or tertiary amines can result in the formation of N-nitroso compounds which are known to be carcinogenic, teratogenic, and mutagenic. Due to these toxic effects, it is very important to develop new methods with high sensitivity and selectivity for its continuous monitoring in food products. There are reports available in the literature above the existence of various methods for detection of nitrites. However such methods pose various drawbacks which includes, lack of sensitivity or time consuming or expensive or tedious and cumbersome. Thus there exists a need in the state of art to develop a novel electrochemical detection method for the detection of nitrite at trace levels which is devoid of above said drawbacks.

OBJECTIVE OF THE INVENTION:
The main objective of the present invention is to develop a novel electrochemical method for quantitative detection of nitrite.
Another objective of the present invention is to employ polyaniline protected cellulose nanofibers derived from sugarcane bagasse modified electrode for electrochemical detection of nitrite.
Yet another objective of the present invention is to prepare cellulose nanofibers from sugarcane bagasse biomass through acid hydrolysis method.
Further objective of the present invention is to prepare polyaniline shell over coated cellulose nanofibre by the conventional oxidative polymerization of aniline in presence cellulose nanofibers as solid support
Hence objective of the present invention is to characterize the polyaniline shell on cellulose nanofibers by various instrumental methods like UV-Visible Spectroscopy, FT-IR, XRD, TGA, SEM and TEM
Yet another objective of the present invention is to employ the PANI/CNF as electrode modifier to deposite the electrode surface as electron transfer mediator for sensing of nitrite.
Yet another object of the present invention is to elevate the oxidation of nitrite using PANI/CNF modified glassy carbon under optimized experimental condition including pH of the medium, applied potential and influence of scan rate
Further object of the present invention is to exploit the use of the modified electrode for the quantitative detection of nitrite in food samples.

BRIEF DESCRIPTION OF DRAWINGS:
Figure 1 depicts the flowchart of the overview of present invention.
Figure 2 depicts the schematic diagram of PANI/CNF nanocomposite.
Figure 3 depicts the (A) UV-Vis Spectrum of a) PANI and b) PANI/CNF nanocomposite, (B) FT-IR Spectrum of a) CF and b) PANI/CNF nanocomposite, (C) X-Ray Diffraction of a) PANI, b) PANI/CNF nanocomposite, (D) TGA analysis of a) PANI b) PANI/CNF nanocomposite.
Figure 4 depicts the FE-SEM image of CNF (a&b), PANI/CNF (c&d)nanocomposite
Figure 5 depicts the TEM image of CNF (a&b), PANI/CNF (c&d)nanocomposite.
Figure 6 depicts the CV of PANI/CNF/GCE (Glassy Carbon Electrode) in presence of different electrolytes like 0.1M HCl, 0.1M PB and 0.1M KCl at a scan rates of 50mV/s. (A) The CV of bare GCE andbare with NO2 and PANI/CNF/GCE in presence of 0.1M NO2 at 0.1M KCl at a scan rates of 50mV/s.
Figure 7 depicts the (A) DPV of NO2- at PANI/CNF/GCE in 0.1 M KCL at various concentrations. (B) Calibration plot of peak current vs. concentration. (C) Amperometric response of PANI/CNF/GCE at an applied potential +0.85V to subsequent addition of different concentrations from (0.05 × 10-8M to 6.5 × 10-8M) of 1mM NO2- with 0.1M KCL containing PB (pH 7.0). (D) Calibration plot of Ipvs.conc of NO2-.

SUMMARY OF THE INVENTION:
The present invention discloses an electro chemical method for quantitative detection of nitrite. The detection method comprises of employing characterized glassy carbon electrode modified with polyaniline protected cellulose nanofibers PANI/CNF in predetermined concentration of a buffer solution of predetermined pH in which oxidation of nitrite occurred at predetermined Voltage with respect to reference electrode.

DETAILED DESCRIPTION OF THE INVESTICATION:
The present invention disclose an electro chemical method for quantitative detection of nitrite employing characterised polyaniline protected cellulose nanofibers derived from sugarcane bagassemodified electrode.
The plan of the invention is depicted in Figure 1
Initially isolation of cellulose nanofibers from sugarcane biomass was carried out by the acid hydrolysis method. Surface modification of cellulose nanofibers was performed with polyaniline through oxidative polymerization aniline using ammonium persulfate (figure 2). Next Characterization of PANI/CNF were carried out using various analytical tools like UV-Visible Spectroscopy, FT-IR, TGA, XRD, SEM and TEM techniques (Figure 3-5).
Figure 3 (A) shows the UV-Vis spectra of PANI a peak at 400 nm is attributed to the polaron p* transition in the emerladine salts (ES). The absorption spectrum of PANI shows bands around 355 nm - 450 nm corresponding to p – p* of benzenoid moieties which is due to the formation of ‘exciton’ transition, caused by inter-band charge transfer from benzenoid to quinoid moieties, respectively. The localized polaron peak around 750 nm indicates a compact coiled (tightly coiled chains) conformation of PANI.
Figure 3(B) depictedFT-IR spectrum of CNF and PANI/CNF are shown in a peak at 3452 cm-1 is due to O-H stretching vibrations, the other two peak at 2367 and 2078 cm-1 are assigned due to the aliphatic C-H stretching vibrations. Major peaks at 1639 and 1409 cm-1 are due to the asymmetric and symmetric stretching of the carboxylate group (-COO-), and a sharp peak at 1409 cm-1 is due to C-O-C stretching vibrations, respectively. In the case of PANI/CF three major peaks at 2835, 2367 and 1639 cm-1 (weak bands) for aliphatic C-H stretching vibrations and carbonyl stretching vibrations of CF respectively. From the above studies it is confirmed that the PANI was successfully grown over the surface of CNF.
Figure 3(C) XRD was used to further characterize the structure of PANI/CNF composite. Two peaks located at 2O = 21.6? and 24.5??were observed the PANI microstructures. These characteristics also confirmed that PANI is partially crystalline similar to the XRD curve of CNF. Two peaks at 15.24? and 22.4? appeared on the diffractive curves of the PANI/CNF composite.
Figure 3(D) Thermogravimetric analysis (TGA) was performed to study the thermal stability of PANI/CNF. The typical single step weight loss behaviour of PANI and PANI/CNF.However, the degradation temperature of PANI/CNF was observed at around 300?C, this may be due to the inclusion of PANI which weakens the inter-and intra-molecular hydrogen bonding in cellulose Nanofiber.
Figure 4 surface morphologies of PANI/CNF analysed by SEM analysis. From SEM the shape of fibers before and after modification of PANI. Illustrates the SEM image of pristine CNF (a&b) and illustrates the SEM image of Insitu polymerized PANI/CNF (c&d). By comparing both figures, it could be seen that during in situ polymerization to the PANI on the surface of the cellulose nanofibers, which resulted in the high conductivity of the samples.
Figure 5 Transmission electron micrograph of CNF and PANI-CF nanocomposite are shown in the nanocomposites were irregular in shape and varied from 40 to 80 nm in size. The nanocomposites were presented as in aggregates, and their surfaces are dark in colour because of polyaniline grown over the cellulose nanofiber surfaces. Polyaniline was present as aggregates and its sizes varied from 50 to 200 nm.

Consequently electrochemical oxidation of nitrite using PANI/CF modified electrode by cyclic voltammetry method was studied and parameters such as scan rate, pH etc are optimized.
Figure 6: It is inferred that Cyclic voltammogram of CF/PANI modified GCE in presence of different electrolytes like 0.1 MHCl, 0.1 M KCl and 0.1 M of PB are shown in Fig.6 (A). Two set of redox peaks were observed in the case PANI/CNF in 0.1M HCl medium. The redox behaviour is lost when the modified electrode was placed in phosphate buffer medium as well as KCl medium. In the case of KCl medium a decrease in current values is noted which is due decrease in conductivity of the film at neutral pH.
Figure 6(B) are shown in the electrochemical oxidation of nitrite was investigated using PANI/CNF modified GCE in 0.1 M KCl medium. In general, Nitrite ions undergoes a single oxidation reaction by the involvement of 2e- and 2H+ reaction. The electrochemical oxidation behaviour of Nitrite ion is confirmed by cyclic voltammetry studies. A well define oxidation peak was noted at +1.0V vs Ag/AgCl. In the case of PANI/CNF/GCE the oxidation peak potential was slightly shifted towards more negative values with enhance oxidation peak current values (almost ten times higher current response). The enhancement of peak highest is due to the catalytic oxidation behaviour of polyaniline present on the surface of CNF in 0.1M KCl medium at a scan rate of 50 mV/s.
Figure 7: The DPV method was used for the sensitive detection of NO2- using the PANI/CNF/GCE as shown in Fig 7 (A & B). A better sensitivity was achieved when the modified electrode system was modified with PANI/CNF instead of bare GCE. A sharp peak was observed at a peak potential of + 0.88 V vs. Ag/AgCl. This value is very close to the cyclic voltammetry peak potential. A calibration graph was plotted against the concentration of NO2- with respect to oxidation peak current values. These results strongly suggest the selective and sensitive determination of NO2- by PANI/CNF/GCE. Fig 7 (C & D)
Finally quantitative of nitrite in food samples by amperometry method using PANI/CNF detection modified electrode was done (Figure 7).

Determination of nitritein food samples.

To determination of NO2- in PANI/CNF/GCE was successfully applied for food samples such as Chips, Pickle and juice. Real samples were taken for analysis using PANI/CNF modified GCE under optimised experimental condition. To measure the unknown concentration of nitrite present in food samples, a standard addition method was followed. From the calibration graph the unknown concentration of nitrite can be calculated. The results were summarized and shown in Table 1. The results show a good consistency between the found values and also with the values determined on the DPV method. This demonstrated that the developed sensing method was potentially applicable for the determination of NO2- in food samples.
Table 1.Detection of NO2- in food Samples (n=3)
Food samples Added (?M) Actual found
(?M) RSD (%) Relative error (%)
Chips 10
20 9.40 ± 1.2
20.15 ± 3.2 3.1
2.7 94
100.75
Pickle 10
20 10.05 ± 1.8
19.39 ± 2.3 3.1
1.9 105
96.95
juice 10
20 8.6 ± 2.3
20.78 ± 2.7 3.1
2.4 98.6
103.9

Nitrite is extensively used in dye synthesis food industry and corrosion inhibitor the WHO has regulated that the amount the nitrite in raw water should not exceed 3mg/L therefore the simple and effective determination of nitrite is of great significance in public health
The inventiveness of the present invention lies the following:
a. A new method for preparing conducting polymer over coated cellulose nanofibers for electrochemical sensor applications is reported
b. Electrochemical method is convenient, inexpensive, sensitive and accurate detection of nitrite than other techniques like UV-Visible spectroscopy, fluorescence, ion chromatography and other technique
c. Extensive studies have been carried out to study the morphology, nature of bonding, and electrochemical behaviour of the PANI/CNF composite
d. First time reporting natural fiber isolated from sugar bagasse and surface functionalization of polyaniline protected cellulose nanofiber in a systematic approach
e. Detailed studies have been carried to develop an electrochemical sensor device in a simple approach.
f. The proposed method is simple, stable, reliable and reproducible.
g. Inexpensive and Robotic method to detection the concentration of Nitrite in food Samples using PANI/CF.
In one of the preferred embodiment, the present invention shall disclose an electro chemical method for quantitative detection of nitrite. The detection method comprises of employing characterized glassy carbon electrode modified with polyaniline protected cellulose nanofibers PANI/CNF in predetermined concentration of a buffer solution of predetermined pH in which oxidation of nitrite occurred at predetermined Voltage with respect reference electrode.
In another preferred embodiment, the present invention shall disclose an electro chemical method for quantitative detection of nitrite which comprises of employing characterized glassy carbon electrode modified with polyaniline protected cellulose nanofibers PANI/CNF in 0.1M KCL containing PBS of 7 pH in which oxidation of nitrite occurred at +0.85V with respect reference electrode.
According to the invention, in the electro chemical method, the characterized glassy carbon electrode modified with polyaniline protected cellulose nanofibers PANI/CNF is prepared by employing PANI/CNF as electrode modifier to deposit electrode surface as electron transfer mediator for sensing of nitrite.
As per the invention, in the electro chemical method, the PANI/CNF is prepared by surface modification of cellulose nanofibers with polyaniline through oxidative polymerization aniline using ammonium persulfate.
In accordance with the invention, in the process of preparation of PANI/CNF the cellulose nanofibers is isolated from sugarcane bagasse biomass by the acid hydrolysis method
As per the invention, the electrochemical method is cyclic voltammetry method.
According to theamperometry invention, the electrochemical method exhibits a linear range of detection between 0.5 ?M to 6.5 ?M with detection limit of 0.0722?M
In accordance with the invention, the electro chemical method exhibits a sensitivity of 62.2 ?A. ?M-1.
Although preferred embodiments have been depicted and described in detail here in, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the invention as defined in the claims which follow.