FIELD OF INVENTION:
The present invention relates to the field of molecularly imprinted polymers (MIPs)- based electrochemical based biosensors. The present invention in particular relates to an Ultra-sensitive and selective nano-molecular imprinting polymer based electrochemical sensor for Follicle-stimulating hormone (FSH) detection.
BACKGROUND OF THE INVENTION
Polycystic ovarian syndrome (PCOS) is considered to be one of the commonly occurring disease, which affects around 10% of reproductive aged women and sums up to 75%of anovutatory infertility. This is a complex disorder and continuum of metabolic disorders, endrocrine, reproductive and is distinguished by suppressing granulosa cell multiplication, early antral focille growth arrest, chronic anovolution, hyperandrogenemia and insulin resistance. The symptoms in women with PCOS are common, acne and/or oily skin, abnormal growth of hair on a woman's face and bod, irregular bleeding and infertility. And also have a high risk of long term morbidities which involves diabetes mellitus type 2, hypertension, cardiovascular diseases and also risk of different cancers. The family of gonadotropin hormones consists of includes human chorionic gonadotropin (hCG), luteinizing hormone (LH) and follicle stimulating hormone (FSH). Reference may be made to the following:
A literature "Electrochemical Sensing Platform Based on a Molecularly Imprinted Polymer Decorated 3D Nanoporous Nickel Skeleton for Ultrasensitive and Selective Determination of Metronidazole" relates to an

electrochemical sensor has been developed by using a composite element of three-dimensional (3D) nanoporous nickel (NPNi) and molecularly imprinted polymer (MIP). NPNi is introduced in order to enhance the electron-transport ability and surface area of the sensor, while the electrosynthesized MIP layer affords simultaneous identification and quantification of the target molecule by employing Fe(CN)63-/4- as the probe to indicate the current intensity. The morphology of the hybrid film was observed by scanning electron microscopy, and the properties of the sensor were examined by cyclic voltammetry and electrochemical impedance spectroscopy. By using metronidazole (MNZ) as a model analyte, the sensor based on the MIP/NPNi hybrid exhibits great features such as a remarkably low detection limit of 2 * 10-14 M (S/N = 3), superb selectivity in discriminating MNZ from its structural analogues, and good antiinterference ability toward several coexisting substances. Moreover, the proposed method also demonstrates excellent repeatability and stability, with relative standard deviations of less than 1.12% and 1.4%, respectively. Analysis of MNZ in pharmaceutical dosage form and fish tissue is successfully carried out without assistance of complicated pretreatment. The MIP/NPNi composite presented here with admirable merits makes it a promising candidate for developing electrochemical sensor devices and plays a role in widespread fields. Another literature "A highly sensitive and selective electrochemical sensor based on polydopamine funotionalized graphene and molecularly imprinted polymer for the 2,4-dichlorophenol recognition and detection" relates to a sensitive and selective electrochemical sensor based on

polydopamine-reduced graphene oxide (PDA-rGO) and molecular imprinted polymers (MIP) modified glassy carbon electrode for detection of 2,4-DCP was fabricated. The PDA-rGO was obtained through the auto polymerization of dopamine in graphene oxide solution at an alkaline environment. The MIP film was performed on the surface of the PDA-rGO modified electrode by electropolymerization the monomer of o-phenylenediamine (o-PD) with the template of 2,4-dichlorophenol (2,4-DCP). The bare hydroxyl and benzene rings of PDA-rGO could attract positive charged o-PD and provide TT-TT stacking effect with o-PD and 2,4-dichlorophenol, which made the compact imprinted film and more imprinted sites. The Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to character the MIP and NIP modified electrode in the presence of [Fe(CN)6]3-/[Fe(CN)6]4- as a probe for signal transduction. The relative current intensity of ferro/ferricyanide decreased linearly with increasing concentration of 2,4-DCP with a detection limit of 0.8 nM (S/N = 3). The MIP sensor had a much higher affinity towards 2,4-DCP than other analogues. The proposed method was successfully applied for the determination of 2,4-DCP in real water samples. Furthermore, FSH is produced by pituitary gland and is considered an important indicator of ovarian reserve function. Since women with PCOS have low levels of FSH. This is because, determination of FSH is important as inadequate FSH impairs ovarian follicle development, thus preventing ovulation. Eventually, the multiple small cysts formed in the ovary from follicles that failed to mature and ovulate result in the PCOS appearance.

Thus till date there is no invention deals with developing a sensor and method which is having molecularly imprinted polymers (MIPs)- based electrochemical based biosensors with their physical and chemical robustness, high selectivity, sensitivity and stability, low limit of detection, low cost, and reusability are not available in the prior-art. In order to overcome above listed prior art, the present invention have exploited the use of an electrochemical sensor is developed for the detection of FSH in PCOS women using NiCo2S4/rGo nanocomposite with MIP decorated onto an Indium tin oxide (ITO) electrode which is not being reported in any study yet.
SUMMARY OF THE INVENTION:
The present invention replaced the conventional techniques, devices systems and methods, in which the electrochemical sensor is developed for the detection of FSH in PCOS women using NiCo2S4/rGo nanocomposite with MIP decorated onto an Indium tin oxide (ITO) electrode in a relatively easier, less tedious and labour intensive manner than the conventional techniques.
In accordance with another important aspect of the present invention, based on FSH-imprinted MIP, resulting in increased electrochemical response and low detection limit. This developed sensing platform has the potential for point-of-care FSH detection.
In this respect, before explaining at least one object of the invention in detail, it is to be understood that the invention is not limited in its application to the details of set of rules and to the arrangements of the various models set forth in the following description or illustrated in the

drawings. The invention is capable of other objects and of being practiced and carried out in various ways, according to the need of that industry. Also, it is to be understood that the phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting.
These together with other objects of the invention, along with the various features of novelty which characterize the invention, are pointed out with particularity in the disclosure. For a better understanding of the invention, its operating advantages and the specific objects attained by its uses, reference should be made to the accompanying drawings and descriptive matter in which there are illustrated preferred embodiments of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be better understood and objects other than those set
forth above will become apparent when consideration is given to the
following detailed description thereof. Such description makes reference to
the annexed drawings wherein:
FIG. 1 depicts a schematic representation of the template molecule with
functional monomers and polymerize as cross-linker, in accordance with
an embodiment of the present invention;
FIG. 2 depicts the MIP electropolymerization schematic representation, in
accordance with an embodiment of the present invention;
FIG. 3 depicts Nyquist graph performed using a conventional three
electrode setup, in accordance with an embodiment of the present
invention;

FIG. 4 depicts a graphical representation of cyclic voltammetry (CV), in accordance with an embodiment of the present invention; FIG. 5 depicts Nyquist graphs, in accordance with an embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
In the following detailed description, reference is made to the accompanying drawings which form a part thereof, and which is shown by way of illustration specific embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that the embodiments may be combined, or that other embodiments may be utilized and that structural and logical changes may be made without departing from the spirit and scope of the present invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined by the appended claims and their equivalents.
The present invention discloses an Ultra-sensitive and selective nano-molecular imprinting polymer based electrochemical sensor for Follicle-stimulating hormone (FSH) detection. In the present invention, an electrochemical sensor is developed for the detection of FSH in PCOS women using NiCo2S4/rGo nanocomposite with MIP decorated onto an Indium tin oxide (ITO) electrode. The proposed hypothesis is based on FSH-imprinted MIP, resulting in increased electrochemical response and low detection limit. This developed sensing platform has the potential for point-of-care FSH detection.

Synthesis of nanomaterials
Synthesis of NiCo2S4/rGO composite was done by simplistic two-step hydrothermal reaction. For this, GO was synthesized by improved Hummer's method. Then, 3 mmol NiCI2»6 H20 (7 mg in 10 mL), 6 mmol CoCI2»6 H20 (14 mg in 10 mL), along with 10 mmol urea (6 mg in 10 mL) was dissolved in GO (10 mL) suspension. This final obtained solution (40 mL) was then ultrasonicated in a water bath for 30 min and afterward, it was shifted to Teflon-lined stainless-steel autoclave for 2000 C for 12 hrs. The obtained product was cooled at RT and the black precipitate was accumulated and washed thrice using water and ethanol. Then the resultant powder was calcinated at 1500 C for 2 hrs. at room temperature to obtain NiCo204/rGO composite. Fabrication of working electrode
FSH-based MIP was fabricated onto the ITO substrates. Significantly, ITO is used in biosensing devices due to their single pretreatment process rather than the monotonous cleaning step followed by pretreatment cycles of regular strong state electrodes (like lustrous carbon, gold, and platinum electrodes). Their unique properties like tunable electrochemical behavior of redox species (electrolyte solution), high chemical stability, low surface resistance, inertness, and broad potential window make them an excellent platform in sensors. Therefore, ITO was used as the working electrode. The ITO was diced with a diamond blade dicing saw into a size of 4.5 cm * 4 cm for electrochemical sensing. For cleaning of ITO, we used water bath ultrasonicator UCB-1, of company Relitech, with specifications: frequency-40-45 kHz, power- 500 W. The ratio of 1:1 acetone and water was used

for cleaning the surface of the ITO electrode for 10 mins and latterly wiped
with a tissue before use.
Optimization of the modified working electrode
Different concentrations of FSH (0.1 pM, 1pM, 10pM, 100pM, 1 nM, 10 nM, 100 nM, and 1 uM) on MIP surface using EIS technique (100mHz-100 kHz at an amplitude of 100 uA) in a 0.1 M electrolyte solution via Ret value were evaluated to check the analytical performance of the NiCo204/rGO/MIP-modified ITO electrode. Optimization of the MlP-based sensor was done for various pH (6.5, 7.0, 7.5, 8.0, 8.5, 9.0 phosphate buffers) each at 0.1 M. Electrochemical measurements of these buffers were carried out in the presence of and K3Fe(CN)6/K4Fe(CN)6 electrolyte solution. Likewise, electrochemical evaluation of developed biosensor were performed with moderately decreasing temperature at a difference of 10oCfrom90°to30°C.
The nanocomposite was electrodeposited using the CV technique within a potential range from - 1.0 to + 1.0 V (at a scan rate of 50 mV/s). For electrodeposition, a solution was prepared with 50 mg nanocomposite (NiCo2S4/rGO) in 10 mL DW. Further, a MIP solution was prepared that consist of a template: FSH (1 mM) and crosslinker: EGDMA (2 mM) were mixed well, and then, monomer: MAA (2 mM) was added to the mixture and sonicated properly for 45 mins. Afterward, onto the modified electrode, electro-polymerization with different molar ratios (1:2:2, 1:3:3, 1:4:4, 1:5:5) of an FSH (template), EGDMA (crosslinker), and MMA (monomer) was performed with CV (20 cycles) at the scan rate of 50 mV/s in a potential range between - 0.2 V to + 0.8 V instantly. Later, the

washing step was performed to remove the template (FSH) from the electrode surface to obtain the imprints of the biological compound. The working electrode was washed by immersing in 50% (v/v) aqueous ethanol for 6 mins to remove the FSH from the polymer cavities. For obtaining the control electrode to ensure effects seen are because of imprinting features, the same procedure was used, but template molecule was not used. This is referred to as non-imprinting polymer (NIP) which is derived from the interactions between crosslinker EGDMA and monomer MMA which are present in the polymerization complex. FIG. 1 illustrates a schematic representation of the template molecule with functional monomers and polymerize as cross-linker. Furthermore, the template is removed for the imprinted cavities.
In accordance with an embodiment of the present invention, the MIP electropolymerization schematic representation is shown in FIG. 2. ITO was used as working electrode onto which NiCo2S4/rGo nanocomposite was deposited. Onto this modified ITO electrode, FSH-MIP was electropolymerized by cyclic voltammetry (CV). And then MIP was washed. Electrochemical studies, including CV, electrochemical impedance spectroscopy (EIS) at each step.
In accordance with an embodiment of the present invention, further, FIG. 3 shows a nyquist graph performed using a conventional three electrode setup within a frequency range of 100mHz-100KHz at an amplitude of 100 uA in a 0.1M K3Fe(CN)6/K4Fe(CN)6 solution of the modified ITO electrode.

CV and EIS analysis were done at each step of modified electrode surface as shown in figure 1a. Bare ITO electrode showed a well-defined reduction and oxidation peak currents (Ipa = 0.385mA, Ipc = 0.039mA) of the redox pair being approx. equivalent and reversible. As represented in figure 3 NiCo204/rGO modified ITO illustrates a larger peak current (Ipa = 0.450mA, Ipc = 0.0435mA) in comparison with bare electrode. The results explained that the deposition of nanomaterials was sequential improving the conductivity of the sensing platform. After electropolymenzation of FSH-based MIP the current decreased and maximum current response (Ipa = 0.240mA, Ipc = 0.251mA) was seen after removal of the FSH (template), revealing that electropolymerizaton of FSH based MIP electrode as electroactive molecules diffusion. The current was higher after washing (Ipa = 0.498mA, Ipc = 0.053mA).
Similar results were obtained from the Galvanostatic electrochemical impedance spectroscopy (GEIS) Nyquist plot in which after template removal showed low (3.16kQ) Ret value and with template (FSH) high (33.96kQ) Ret value as shown in figure 4.
Cyclic voltammetry (CV) graph in FIG. 4 shows the potential range of -1.0 to 1.0 V at a scan rate of 50 mV/s in a 0.1M K3Fe(CN)6/K4Fe(CN)6 solution K3Fe(CN)6/K4Fe(CN)6 using a conventional three electrode setup. In addition, the calibration data relating to the EIS response of the MIP electrode towards increasing concentration of FSH are also shown in FIG. 5.
As shown in FIG. 1 & 2, which illustrates a molecular imprinting schematic representation. The template molecule assembles with the functional

monomers and polymerize with the cross-linker. The template is removed from the developed polymers to free the imprinted cavities, and a graphical representation of MIP by electropolymerization. In accordance with an embodiment of the present invention, referring to FIG. 3, which illustrates GEIS (frequency range of 100mHz-100KHz at an amplitude of 100 uA) in a 0.1 M K3Fe(CN)6/K4Fe(CN)6 solution. Now coming to FIG. 4, which illustrates Cyclic voltammetry (CV) (potential range of -1.0 to 1.0 V at a scan rate of 50 mV/s) in a 0.1M K3Fe(CN)6/K4Fe(CN)6 solution, and referring to figure 5, which illustrates Nyquist graphs (frequency range of 100mHz-100KHz at an amplitude of 100 uA) in a 0.1M K3Fe(CN)6/K4Fe(CN)6 solution at different concentrations of FSH.
Consequently, results of both CV and EIS were in line with each other confirming a desirable modification.
Figure 5 demonstrates the Nyquist plots of different FSH concentrations in which a linear change in the Ret values were obtained from the range between 0.1pm to 1um, wherein the Ret values were gradually increased concentrations from 0.1pm to 1um. This decrement led to the explanation of the supposition of enhanced conductivity when increase in concentration of FSH. Although 0.1pm showed the highest Ret value among all the FSH concentrations observed. The calibration curve showed a linear range between the various concentrations of FSH and Ret values as seen in figure 6. The FSH based MIP sensor showed a limit of detection of 0.1 pm and sensitivity of 0.72 mA/pm.

The above-mentioned invention is provided with the preciseness in its real-world applications to provide an ultra-sensitive and selective nano-molecular imprinting polymer based electrochemical sensor for Follicle-stimulating hormone (FSH) detection.
It is to be understood that the above description is intended to be illustrative, and not restrictive. For example, the above-discussed embodiments may be used in combination with each other. Many other embodiments will be apparent to those of skill in the art upon reviewing the above description.
The benefits and advantages which may be provided by the present invention have been described above with regard to specific embodiments. These benefits and advantages, and any elements or limitations that may cause them to occur or to become more pronounced are not to be construed as critical, required, or essential features of any or all of the embodiments.
While the present invention has been described with reference to particular embodiments, it should be understood that the embodiments are illustrative and that the scope of the invention is not limited to these embodiments. Many variations, modifications, additions and improvements to the embodiments described above are possible. It is contemplated that these variations, modifications, additions and improvements fall within the scope of the invention.

We Claim:
1. An Ultra-sensitive and selective nano- molecular imprinting polymer based electrochemical sensor for Follicle-stimulating hormone (FSH) detection comprising NiCo2S4/rGo nanocomposite with molecularly imprinted polymer (MIP) electrodeposited onto an Indium tin oxide (ITO) electrode with methyl methacrylate (MMA) as monomer, ethylene glycol dimethylacrylate (EGDMA) as crosslinker, and acetic acid.
2. The sensor as claimed in claim 1, wherein the preparation method comprises the steps of:

a) Dissolving 3 mmol NiCl2*6 H2O (7 mg in 10 ml_), 6 mmol CoCl2»6 H2O (14 mg in 10 ml_), along with 10 mmol urea (6 mg in 10 ml_) in GO (10 ml_) suspension.
b) ultrasonicating obtained solution (40 ml_) in a water bath for 30 min and afterward, it was shifted to Teflon-lined stainless-steel autoclave for 200° C for 12 hrs.
c) cooling the above solution at RT and accumulating the black precipitate with washing thrice using water and ethanol.
d) Calcinating the resultant powder at 150° C for 2 hrs. at room temperature to obtain NiCo204/rGO composite.
e) preparing a solution with 50 mg nanocomposite (NiCo2S4/rGO) in 10 mL DWfor electrodeposition.
f) preparing a MIP solution further comprising a template: FSH (1 mM) and crosslinker: EGDMA (2 mM) are mixed well, and then, monomer: MAA (2 mM) is added to the mixture and sonicated properly for 45 mins.
g) Afterward, onto the modified electrode, electro-polymerization with different molar ratios (1:2:2, 1:3:3, 1:4:4, 1:5:5) of an FSH, EGDMA (crosslinker), and MMA (monomer) is performed with CV (20 cycles) at the scan rate of 50 mV/s in a potential range between - 0.2V to + 0.8V instantly.

h) The working electrode is washed by immersing in 50% (v/v) aqueous ethanol for 6 mins to remove the FSH from the polymer cavities.
3. The sensor as claimed in claim 1, wherein The ITO is diced with a diamond blade dicing saw into a size of 4.5 cm* 4 cm for electrochemical sensing.
4. The sensor as claimed in claim 1, wherein the ratio of 1:1 acetone and water is used for cleaning the surface of the ITO electrode for 10 mins and latterly wiped with a tissue before use.
5. The sensor as claimed in claim 1, wherein Optimization of the MIP-based sensor is done for pH (6.5, 7.0, 7.5, 8.0, 8.5, 9.0 phosphate buffers) each at 0.1 M and electrochemical measurements of these buffers are carried out in the presence of and K3Fe(CN)6/K4Fe(CN)e electrolyte solution.
6. The sensor as claimed in claim 1, wherein the electrochemical evaluation of developed biosensor is performed with moderately decreasing temperature at a difference of 10 °C from 90° to 30°C.