Description:FIELD OF INVENTION
[001] The present invention relates to an electrochemical sensor. Particularly, the present invention relates to a nanoparticle-based metal organic framework and a biosensor prepared therefrom. The present invention also relates to a method for electrochemical deposition of Thionine/reduced Graphene Oxide/Gold nanoparticles on an Indium-Tin -Oxide (ITO) glass electrode and a method of preparation of a biosensor therefrom.

BACKGROUND OF THE INVENTION
[002] Biosensors are devices comprising of biological and physicochemical components used to detect a chemical constituent of interest in analytical procedures by producing a signal which can be measured. Indium Tin Oxide (ITO) electrodes have been widely used in the electrochemical industry owing to its high conductivity and high optical transparency.

[003] Conventional deposition techniques on bio-sensors employ deposition of materials like graphene (a carbon nano-allotrope) and gold nano-particles using various technologies. Graphene based bio sensors are largely used owing to their high specific surface area, extraordinary electronic properties, electron transport capabilities and ultra-high flexibility.

[004] However, some of the properties of the graphene based bio-sensors cannot be considered reliable. The ultra-high sensitivity of graphene based bio-sensors can lead to overestimation of fluorescence signal, high cost and difficult workability. Further, the methods disclosed in various prior arts involve drop method for deposition of the material on carbon electrodes. The drop method suffers from shortcomings like issue in reproducing the end results and day by day deteriorations in detection of the electrochemical activity of the electrode.

[005] Patent application number CN112986350A discloses a method for detecting nitrite by using AuNPs/NiNFs/ITO electrode. The application discloses indium tin oxide conductive glass (ITO) being used as a substrate and the gold nano-nickel particles are deposited on the substrate by using an electrochemical deposition method to prepare a gold nano-nickel electrode. The application is directed towards a tri-electrode with, AuNPs/NiNFs/ITO glass electrode being used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, and a platinum wire is used as an auxiliary electrode. The independent claim of the application is directed towards the tri-electrode system along with the method of their preparation disclosing that ITO glass electrode of the application is electrochemically deposited with gold-nano particles and is used for detection of sodium nitrate and the procedure adapted is cyclic voltammetry electrodeposition. This prior art suffers from disadvantages that it is highly expensive and is an ecologically demanding process given its high energy demand especially during the manufacturing process.

[006] Further, the patent application KR101730766B1 discloses a preparation method of electro catalyst using reduced graphene oxide and gold dendritic nanostructures. The process disclosed involves forming an amine-functionalized silicate sol-gel matrix (TPDT) layer on an electrode, forming a -cyclodextrin (CD)-functionalized reduced graphene oxide (RFO-CD) sheet thereon, and growing gold dendrite nanostructures (Au-DNs) induced and controlled by CD thereon through electrodeposition. The application works towards realizing an increased electrochemical catalytic activity for electro-oxidation of nitrite and glucose and bio-sensing. The key features of this application are: Electrodeposition of gold on reduced graphene over ITO glass electrode and Silicate-sol gel matrix formulation functionalized with an ITO electrode. This prior art has disadvantages that it is highly expensive and leads to large volume of shrinkage thus leading to cracking.

[007] The non-patent literature titled “an ITO glass electrode Modified with Electrodeposited Graphene Oxide and Gold Nanoclusters for Detecting the Release of H2O2 From Bupivacaine-injured Neuroblastoma cells”, describes an amperometric sensor for hydrogen peroxide (H2O2) that uses an ITO glass electrode which was modified with a nanocomposite consisting of electrochemically reduced graphene oxide and gold nanoclusters (AuNCs). The method disclosed was applied to study to indicate bupivacaine (local anesthetic) induced cell damage and the protective effects of a-lipoic acid (anti-oxidant). The sensor disclosed can be easily fabricated and effective tool to study the interactions of drugs with cells.

[008] Another non-patent literature titled “Electrochemical Deposition of Gold Nanoparticles on Reduced Graphene Oxide by Fast Scan Cyclic Voltammetry for the Sensitive Determination of As(III)” describes an electrochemical sensor was fabricated by the electrochemical co-deposition of reduced graphene oxide (rGO) and gold nanoparticles on a glassy carbon electrode (rGO-Aunano/GCE) using cyclic voltammetry (CV). This method is used to trace As (III) by square wave anodic stripping voltammetry (SWASV). The method is dis-advantageous as the deposition on reduced graphene oxide may lead to fabrication of sensor which may not be ideal for conduction.

[009] The non-patent literature titled “Fabrication of gold/graphene nanostructures modified ITO glass electrode as highly sensitive electrochemical detection of Aflatoxin B1” describes the preparation of electrode based on layer –by-layer electrochemical deposition method. The Aspergillus flavus (AF’s) level is detected using spectroscopy and electrochemical techniques. The method was advantageous as it aids in a rapid probe, simple and economical detection of AFs level using Raman spectroscopy and electrochemical techniques. The technique is disadvantageous as it triggers non-uniformity of the distribution of particles on the electrode.

[0010] The non-patent literature titled “A paper-based electrochemical immunosensor with reduced graphene oxide(rGO)/thionine/gold nanoparticles nanocomposites modification for the detection of cancer antigen 125” describes an electrochemical immunosensor based on rGO/Thi/AuNPs nanocomposites was developed for the determination of cancer antigen 125. In this prior art, drop method was used to deposit rGO, thionine and gold nanoparticles on SPCE electrode.The above mentioned prior art has the disadvantage of leading to a non-uniform electrochemical deposition, limited sensing capability of the fabricated electrode, instability of the electrode and loss in chemical detection of the electrode with time.

[0011] Therefore, keeping in view the problems associated with the above state of the art, there is a need for method for preparation of highly conductive, stable, uniformly coated and highly sensitive fabricated ITO based electrodes and biosensors prepared therefrom.
OBJECTIVES OF THE INVENTION
[0012] The primary objective of the present invention is to provide a fabricated Indium-Tin-Oxide (ITO) glass electrode having high stability, uniform distribution of coating and time dependent stability.

[0013] Another objective of the invention is to provide a thionine/ reduced graphene oxide and gold nano-particles coated Indium-Tin-Oxide (ITO) glass electrode.

[0014] Another objective of the present invention is to provide a biosensor prepared from the thionine, reduced graphene oxide and gold nano-particles coated Indium-Tin-Oxide (ITO) glass electrode.

[0015] Another objective of the invention is to provide a method for electrochemically depositing thionine, reduced graphene oxide and.gold nano-particles on a Indium-Tin-Oxide (ITO) glass electrode.

[0016] Another objective of the present invention is to provide a method for uniform electrochemical deposition of thionine, reduced graphene oxide and.gold nano-particles on the ITO electrode.

[0017] Yet another objective of the present invention is to ensure that there is no loss in electrochemical detection of the electrode with time.

[0018] Other objects and advantages of the present invention will become apparent from the following description taken in connection with the accompanying drawings, wherein, by way of illustration and example, the aspects of the present invention are disclosed.
SUMMARY OF THE INVENTION
[0019] The present invention is a method for electrochemical deposition of thionine, reduced graphene oxide (rGO) and gold nano-particles (AuNP) on indium tin oxide glass electrode to obtain a coated ITOelectrode with enhanced stability and good conductive behavior and capable of being used as a biosensor. The method involves mixing thionine and rGO in a water-based salt solution to obtain a mixture and keeping said mixture on a test tube rotator for not limiting to12 hrs to obtain a conjugate of Thionine and rGO; followed by centrifuging the Thionine and rGO conjugate and washing said conjugate using a water-based salt solution to remove the unbound particles; adding gold-nanoparticle (AuNP) solution to the Thionine and rGO conjugate and mixing for 3 hrs on a gel rocker to obtain a solution; and electrochemically depositing the solution prepared in step (c) on a glass ITO by first using chronoamperometry at -1.5V for 500 secs followed by 25 cycles of cyclic voltammetry at 0.1 V/s scan rate and sweeping potential from -0.4 to 0.6V to obtain a uniform deposition of the solution obtained in step (c) on the glass ITO electrode.

BRIEF DESCRIPTION OF DRAWINGS
[0020] An understanding of the present invention may be obtained by reference to the accompanying drawings, when taken in conjunction with the description herein and in which:

[0021] Figure 1A illustrates flow diagram depicting a method for electrochemical deposition on an Indium-Tin-Oxide glass electrode.

[0022] Figure 1B illustrates a flow diagram depicting an Electrochemical Detection of Peroxide (Enzymatic Sensor).

[0023] Figure 2A illustrates Thionine, reduced graphene oxide (rGO) and gold nanoparticle’s (AuNP) electrochemical deposition on ITO glass electrode showing enhanced oxidation and reduction current (redox current) as compared to Thionine, rGO and Bare ITO electrode.

[0024] Figure 2B illustrates electrochemically deposited Thionine, rGO and AuNP electrode showing enhanced redox current or electrochemical window as compared to drop method.

[0025] Figure 3 illustrates a Thionine, rGO and AuNP deposited on ITO glass electrode via drop method showing a non-linear increase in current at different scan rate (20-200 mV).

[0026] Figure 4A-4B illustrate a Thionine, rGO and AuNP electrochemical deposition on ITO glass electrode showing linear increase in current at different scan rate (20-200mV).

[0027] Figure 5 illustrates Time dependent stability of Thionine, rGO and AuNP deposited on ITO glass electrode via drop method on first, fifth and fifteenth day showing drastic reduction of redox current.

[0028] Figure 6 illustrates Time dependent stability of Thionine, rGO and AuNP electrochemically deposited ITO glass electrode on first, fifth and fifteenth day showing no significant change in the redox current.

[0029] Figure 7A-7B illustrate Uniform and stable electrochemical deposition of Thionine, rGO and AuNP on ITO glass electrode.

[0030] Figure 8 illustrates electrochemical detection of H2O2 through electrochemically deposited Thionine, rGO and AuNP fabricated ITO glass electrode.

[0031] Figure 9A illustrates a bare ITO electrode obtained through a scanning electron microscopic (SEM).

[0032] Figure 9B illustrates the fabricated ITO electrode of the present invention through a SEM.

DETAILED DESCRIPTION OF THE INVENTION
[0033] The following description describes various features and functions of the disclosed system with reference to the accompanying figures. In the figures, similar symbols identify similar components, unless context dictates otherwise. The illustrative aspects described herein are not meant to be limiting. It may be readily understood that certain aspects of the disclosed system can be arranged and combined in a wide variety of different configurations, all of which have not been contemplated herein.

[0034] Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope of invention. In addition, descriptions of well-known functions and constructions are omitted for clarity and conciseness.

[0035] The terms and words used in the following description are not limited to the bibliographical meanings, but, are merely used to enable a clear and consistent understanding of the invention. Accordingly, it should be apparent to those skilled in the art that the following description of exemplary embodiments of the present invention are provided for illustrative purpose only and not for the purpose of limiting the invention.

[0036] It is to be understood that the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0037] It should be emphasized that the term “comprises/comprising” when used in this specification is taken to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. The equations used in the specification are only for computation purpose.

[0038] Accordingly, the present invention relates to electrochemical sensors. Particularly, the present invention relates to a nanoparticle-based metal organic framework and a biosensor prepared therefrom. The present invention also relates to a method for electrochemical deposition of Thionine/reduced Graphene Oxide/Gold nanoparticles on an Indium-Tin -Oxide (ITO) on glass electrode to obtain a coated ITO electrode with enhanced stability, good conductive behavior and a method of preparation of a biosensor therefrom. Theelectrochemical deposition of Thionine/ rGO / AuNP coated ITO glass electrode is done in a three electrode setup by first performing Chronoamperometry at -1.5V for 500s and 25 cycles of CV from -0.4 to 0.6V at a scan rate of 0.1V.

[0039] Thionine is an electroactive molecule, which has acceptable oxidation and reduction current response. The gold nano-particles are utilized due to their large specific surface area and good electrical amplification properties. Reduced graphene oxide has good electrical amplification properties. Reduced graphene oxide in combination with the metallic nanoparticles increase conductive performance of the fabricated electrode. A uniform deposition of the Thionine/rGO/AuNP on ITO glass electrode through the electrochemical deposition shows enhanced redox peak current response that indicates good conductivity of the fabricated electrode that a drop coated ITO glass electrode cannot achieve.

[0040] In an exemplary embodiment, the present invention provides a method comprising the steps of:

1. mixing thionine and rGO in a water-based salt solution to obtain a mixture;
2. rotating the mixture obtained in step (1) on a test tube rotator for at least 12 hrs to obtain a Thionine and rGO conjugate;
3. centrifuging the Thionine and rGO conjugate obtained in step (2) and washing said conjugate using a water-based salt solution to remove the unbound particles;
4. adding gold-nanoparticle (AuNP) solution to the Thionine and rGO conjugate and mixing for at least 3 hrs on a gel rocker to obtain a solution; and
5. depositing the solution prepared in step (4) on a glass ITO by first using chronoamperometry at -1.5V for 500 secs followed by 25 cycles of cyclic voltammetry at 0.1 V/s scan rate and sweeping potential from -0.4 to 0.6V to obtain a uniform deposition of the solution obtained in step (4) on the glass ITO electrode to obtain Thionine/ rGO / AuNP coated ITO glass electrode.

[0041] Further, the present invention provides a process of preparation of a biosensor comprising the steps of:
a. mixing a metallic enzyme with a solvent;
b. immobilizing the enzyme on the Thionine/ rGO / AuNP coated ITO glass electrode to be used as a biosensor.

[0042] The following examples are given by way of illustration of the working of the invention in actual practice and should not be constructed to limit the scope of the present invention in any way.

[0043] In an embodiment, as shown in Figure 1, the method for electrodeposition of thionine, reduced graphene oxide and gold nano-particles (AuNP) on ITO glass electrode (also may be referred to as a working electrode) comprises of the following steps:
a. mixing 0.01to 0.05M of the thionine which displays a fast electron transfer rate and 0.05 to 0.2mg/ml of the reduced graphene oxide in a water based solution and keeping on a test tube rotator for at least12 hrs;
b. centrifuging the thionine and reduced graphene oxide solution and washing said solution with water-based salt solution (at pH 6.5) to obtain the conjugate of thionine and reduced graphene oxide and removing the unbound particles. The water-based salt solution aids in maintaining constant pH of the solution in a constant environment;
c. adding 1-5ml of AuNP solution to thionine/reduced graphene oxide (rGO) and mixing for 3 hrs. on a gel rocker to obtain Thio/rGO/AuNP solution; and
d. depositing the solution prepared in step (c) on a glass ITO by first using chronoamperometry at -1.5V for 500 secs followed by 25 cycles of cyclic voltammetry at 0.1 V/s scan rate and sweeping potential from -0.4 to 0.6V to obtain a uniform deposition of the solution obtained in step (c) on the glass ITO electrode to obtain Thio/rGO/AuNP coated ITO electrode having surface area in the range of 6.25-25 mm2 (2.5x2.5 to 5x5 mm).

[0044] In one embodiment a potentiosat-galvanosat, preferably PGSTAT204 was used for the electrochemical deposition and sensing.

[0045] In the prepared solution, the molar ratio of thionine: gold nanoparticles in the final solution is in the range of 4:1 to 5:1. Specifically, for a thionine solution 45ml (18mM final concentration), AuNPs 5ml (4mM final concentration) is required. rGO is in the concentration of 0.1 mg per ml of the final solution. The final solution is used to coat an electrode whose effective surface area is calculated using Randles–Sevcik Equation. The electrode effective surface area of Thio/rGO/AuNP/ITO obtained was 0.879 cm2 which was 8-10 times more than the bare ITO electrode’s 0.0982 cm2 for the above solution.

[0046] In another embodiment, the Electrochemical Detection of enzyme (Enzymatic Sensor) may be performed by the following steps:
a. mixing an enzyme with 3-5% sulfonated tetrafluoroethylene based fluoropolymer-copolymer solution to obtain an enzyme solution having concentration range of 0.25-1U/mL;
b. immobilizing a 40µl of the enzyme on the Thionine\ rGO\ AuNP \ITO glass electrode in a two-step process where a 20 µl of enzyme was dropped on the fabricated ITO and was subjected to air drying for not limiting to 15 minutes. Said process was subjected to repetition to complete the two-step process; where the effective surface area of the electrode was calculated using Randles–Sevcik Equation. The electrode effective surface area of Thio/rGO/AuNP/ITO obtained was 0.879 cm2 which was 8-10 times more than the bare ITO electrode’s 0.0982 cm2;
c. using the prepared sensor as a working electrode alongside Silver/Silver Chloride (Ag/AgCl) which is used as a reference electrode and a wire used as a counter electrode to obtain a three-electrode system; and
d. dipping the three-electrode system obtained in step c in water-based salt solution (pH 6.5) -of 5mM peroxide; and
e. performing cyclic voltammetry at a sweeping cycle from -0.4 to 0.6V at a scan rate of 0.1V to detect peroxide.

[0047] In an exemplary embodiment, the counter electrode is selected from the group not limited to, platinum, palladium, rhodium, ruthenium, iridium, and osmium.

[0048] In another embodiment the materials for preparation of glass electrode include the procuring an Indium Tin Oxide coated glass.

[0049] In another embodiment the enzyme used in the electrochemical detection of Peroxide is Horse Radish Peroxidase (HRP). Peroxidase enzyme can be used to detect phenolic compounds such as hydroquinone, guaiacol and p-cresol. In an alternative embodiment of the present invention, peroxidase can be replaced with hematin, and ferrocene for peroxide sensing. The peroxide sensing platform in the present invention is aimed to provide biosensing application not limited to just peroxide sensing but can be extended to detect many other biomolecules, provided that specific enzyme or antibody must be used.

[0050] In an exemplary embodiment the Gold Nanoparticles (AuNPs) are synthesized using a modified Turkevich method where the synthesized particle size is in the range of 20-40 nm.

[0051] In an embodiment the AuNPs were characterized using a Nanoparticle Tracking Analyzer (NTA) and Transmission electron microscopy (TEM).

[0052] In an embodiment the reduced graphene oxide and an organic chloride salt is procured commercially.

[0053] In another exemplary embodiment the synthesized Gold Nano-particle is spherical and is in the size of 34 nm as measured through a particle size analyzer prepared via modified Turkevich method or Citrate reduction method.

[0054] In another exemplary embodiment the water-based salt solution to remove the unbound particles is a Phosphate buffer saline (PBS).

[0055] EXPERIMENTAL APPROACH

Materials and Method:
• Indium Tin Oxide coated glass was purchased from Techinstro
• Gold Nanoparticles (AuNPs) were synthesised using a modified Turkevich method. AuNPs were characterised using NTA and TEM.
• Reduced graphene oxide (rGO) was procured from Graphene Supermarket, USA.
• Thionine was purchased from Sigma Aldrich, USA
• The electrochemical deposition and sensing was carried utilizing the instrument Metrohm Autolab PGSTAT204.
EXAMPLE 1
The electrochemical process of deposition of Thionine, rGO and AuNP on ITO glass electrode:
The method was conducted in the following steps:
Prior to the process of deposition, synthesis and procuring of materials for deposition on bare ITO glass to form an electrode was done. Further, a step by step method for Electrodeposition of Thionine, rGO and AuNP on ITO electrode, and the finally an electrochemical detection of peroxide by performing Cyclic voltammetry at a predetermined voltage at a predetermined scan rate was done.

[0056] The method of Electrodeposition of Thionine, rGO and AuNP on ITO glass electrode involves the following steps:
a. 0.02M of Thionine (Thio) was mixed with 0.1mg/ml of reduced graphene oxide (rGO) nanomaterials in a phosphate buffer saline (PBS) and kept on a test tube rotator for not limiting to12 hrs.
b. The thionine and rGO solution was centrifuged and washed with phosphate buffer saline (pH 6.5) solution to obtain the Thio/rGO conjugate and remove unbound particles. The PBS helps in maintaining constant pH in a constant environment.
c. 5ml of AuNP solution was added to Thio/rGO conjugate and mixed for 3 hrs on a gel rocker to obtain Thio/rGO/AuNP solution; and
d. the solution prepared in step (c) was deposited on a glass ITO by first using chronoamperometry at -1.5V for 500 secs followed by 25 cycles of cyclic voltammetry at 0.1 V/s scan rate and sweeping potential from -0.4 to 0.6V to obtain a uniform deposition of the solution obtained in step (c) on the glass ITO electrode which is a prepared sensor.

[0057] Further, the process of Electrochemical Detection of Peroxide (Enzymatic Sensor) as depicted in figure 1B is performed in the following steps:
a. Horse Radish Peroxidase was mixed with 5% of 7H-Perfluoro-4-methyl-3,6-dioxaoctanesulfonicacid1,1,2,2-tetrafluoro-2-[1,1,1,2,3,3-hexafluoro-3-(1,2,2,2-tetrafluoroethoxy)propan-2-yl]oxyethanesulfonic acid solution;
b. 40µl of the enzyme was immobilized on the Thionine/rGO/AuNP/ITO glass electrode in a two-step process, where the 20 µl of enzyme was dropped on the fabricated ITO glass electrode and was subjected to air drying for at least 15 minutes. Said process was subjected to repetition to complete the two-step process to obtain a prepared sensor; where said prepared sensor is used as a working electrode alongside Ag/AgCl (reference electrode) and a Platinum wire to act as a counter electrode;
c. the three-electrode system obtained in step c was dipped in PBS (pH 6.5) -of 5mM peroxide concentration; and
d. performing the electrochemical detection of the Peroxide by performing Cyclic Voltametry (CV) at a sweeping cycle from -0.4 to 0.6V at a scan rate of 0.1V.

[0058] Figure 2A-2B, depict the electrochemical comparison via cyclic voltammetry between electrochemically deposited electrode and the electrode prepared by drop method. Figure 2A showed Thionine, rGO and AuNP electrochemical deposition on ITO glass electrode and clearly depicts an enhanced oxidation and reduction current (redox current) as compared to Thionine, Thionine /rGO and Bare ITO electrode. Figure 2B showed enhanced redox current or electrochemical window as compared to drop method in an electrochemically deposited Thionine, rGO and AuNP electrode.

[0059] Referring to Figure 3, the Thionine, rGO and AuNPs ITO glass electrode deposited via drop method depicting a non-linear increase in current at different scan rate (20-200 mV).

[0060] Figures 4A-4B are graphical representations depicting Thionine, rGO and AuNP electrochemical deposition on ITO glass electrode showing linear increase in current at different scan rate (20-200mV). This linear increase depicts the consistency in pattern in comparison to the conventional drop method.

[0061] Figure 5 illustrates time dependent stability of Thionine, rGO and AuNP ITO glass electrode deposited via drop method on first, fifth and fifteenth day of deposition showing drastic reduction of redox current.

[0062] Figure 6 illustrates the time dependent stability of Thionine, rGO and AuNP electrochemically deposited ITO glass electrode on first, fifth and fifteenth day showing no significant change in the redox current which is the current generated out of the redox reaction occurring on the electrode. The figure 6 further shows the stability of the electrode prepared according to the present invention where the same materials when coated with drop method (as shown in figure 5) deteriorated with time due to lack of uniform coating. The electrochemical analysis was repeated and as compared to the drop coating method the present invention was found to reproduce similar results even after a long period of storage. As per the figure, the first and fifteenth day cyclic voltammetry data showed no significant change in the redox current or electrochemical window of electrochemically deposited Thionine, rGO and AuNP electrode as compared to drop method used for deposition on electrode. The ITO glass electrode image showed uniform and stable coated surface of electrochemically deposited electrode.

[0063] Figures 7A-7B illustrate uniform and stable electrochemical deposition of Thionine, rGO and AuNP on ITO electrode. Coated matrix materials were gradually erased within 15 days in cases where the deposition was via drop coating method. This method is found to limit the sensing capability of fabricated electrode. The present invention was able to produce same electrochemical window and peaks even after 15 days of repeated analysis. This will save the cost of re-deposition of the materials.

[0064] Figure 8 illustrates electrochemical detection of H2O2 through electrochemically deposited Thionine, rGO and AuNP fabricated ITO electrode. The sensing of hydrogen peroxide was achieved by immobilising horseradish peroxidase enzyme on the fabricated surface of the electrode. In addition, different biomolecules are detectible by changing the type enzyme on freshly fabricated electrode that further makes the sensor matrix economically inexpensive.

[0065] Figure 9A illustrates the characterizing feature of a bare ITO electrode obtained through a scanning electron microscopic (SEM) which was found to be featureless and homogenous.

[0066] Figure 9B illustrates the characterization of Thionine-reduced graphene oxide-gold nanoparticles based ITO electrode. The characterization was carried out through SEM to illustrate the characteristic feature of the deposited electrode where the electrochemically deposited electrode showed circular like morphological structure with diameter in the range of 100-150 nm.

[0067] Thus, from the above experimental approach it can be stated that the present invention claims a biosensor having uniform surface, enhanced stability, reproducibility and economical sensing platform for the detection of biomolecules. The experimental results illustrated that in case of drop method, the deterioration in electrochemical deposition with time. The electrochemical deposition of the present invention is utilized to simultaneously coat thionine, rGO and AuNPs and improves their stability, reproducibility and economic advantage as compared to the existing prior art electrodes.

[0068] ADVANTAGES OF THE INVENTION

The electrochemical process disclosed in the present invention for the deposition of thionine, rGO and AuNP on ITO glass electrode exhibited the following advantages:
a) Enhanced redox current that clearly signifies the good conductive behavior of the working electrode as compared to electrodeposited electrodes.
b) Enhanced stability of the electrode.
c) Enhanced peak redox current response signifying good conductive behavior.
d) Uniform surface of the electrode in comparison to the electrodes prepared by drop method.
e) The fabricated electrode offers a reproducible economical sensing surface platform for the detection of biomolecules.
f) The electrode used is an ITO glass where the process of deposition and characterization can be carried out very easily unlike glassy carbon electrodes.

[0069] While the present invention has been described with reference to one or more preferred aspects, which aspects have been set forth in considerable detail for the purposes of making a disclosure of the invention, such aspects are merely exemplary and are not intended to be limiting or represent an exhaustive enumeration of all aspects of the invention. The scope of the invention, therefore, shall be defined solely by the claims.
, C , Claims:WE CLAIM:
1. A method for electrochemical deposition of thionine, reduced graphene oxide and gold nanoparticles on Indium Tin-Oxide coated glass electrode, comprising steps of:
a. mixing of thionine and reduced graphene oxide in a water-based salt solution to obtain a mixture and keeping the mixture on a test tube rotator for at least 12 hrs. to obtain a conjugate of thionine and reduced graphene oxide;
b. centrifuging the conjugate of thionine, reduced graphene oxide and washing said centrifuged conjugate using a water-based salt solution to remove the unbound particles;
c. adding gold-nanoparticles (AuNPs) solution to the conjugate of thionine\ reduced graphene oxide and mixing for 3 hrs on a gel rocker to obtain a solution;
d. depositing the solution prepared in step (c) on a glass ITO by using an analytical technique of chronoamperometry at -1.5V for 500 secs followed by 25 cycles of cyclic voltammetry at 0.1 V/s scan rate and sweeping potential from -0.4 to 0.6V to obtain a uniform deposition of the solution obtained in step (c) on the glass ITO electrode; and
e. obtaining a thionine/ reduced graphene oxide/gold nanoparticles on Indium Tin-Oxide coated glass electrode.

2. The method as claimed in claim 1, wherein the water-based salt solution is a phosphate buffered saline (PBS) solution at a pH of 5.5 to 8.5.
3. The method as claimed in claim 1, wherein the synthesized gold nano- particles is in the range of 20-40 nm.
4. The method as claimed in claim 1, wherein the Electrochemical Detection of Peroxide (Enzymatic Sensor) is performed in the steps comprising:
a. Mixing an enzyme with 5% of 7H-Perfluoro-4-methyl-3,6-dioxaoctanesulfonicacid1,1,2,2-tetrafluoro-2-[1,1,1,2,3,3-hexafluoro-3-(1,2,2,2-tetrafluoroethoxy)propan-2-yl]oxyethanesulfonic acid solution;
b. Immobilizing a 40µl of the enzyme on the Thionine\, reduced Graphene Oxide\ AuNPs on ITO glass electrode, where the 20 µl of enzyme was dropped on the fabricated ITO glass electrode and was subjected to air drying for not limiting to 15 minutes;
c. Repeating the step b to complete the two-step process to obtain a prepared sensor;
d. Using the prepared sensor obtained in step c as a working electrode alongside Ag/AgCl (used as a reference electrode) and not limiting to a Platinum wire to act as a counter electrode;
e. Dipping the three-electrode system obtained in step (c) in PBS of 6.5 pH with 5mM hydrogen peroxide; and
f. Performing the electrochemical detection of the peroxide by performing Cyclic Voltametry (CV) at a sweeping cycle from -0.4 to 0.6V at a scan rate of 0.1V.
5. The method as claimed in claim 5, wherein the analyte is deionized triple distilled water.
6. The method as claimed in claim 4, wherein the enzyme is selected from the group of but not limited to Horse Radish Peroxidase (HRP), hematin and ferrocene.

7. A thionine/reduced graphene oxide/gold nanoparticles coated Indium Tin-Oxide glass electrode prepared by a method as claimed in claim 1, characterized in that; the electrochemically deposited electrode showed circular morphological structure with diameter in the range of 100-150 nm.

8. A biosensor prepared from the thionine/reduced graphene oxide/gold nanoparticles coated Indium Tin-Oxide coated glass electrode prepared by a method as claimed in claim 4, wherein the biosensor is a peroxide sensing platform for detecting phenolic compounds such as but not limited to hydroquinone, guaiacol and p-cresol.