Field of the invention:
The present disclosure generally relates to the field of electrochemical sensing systems, and in specific relates to a label-free direct detection of ligand-receptor binding using electrochemical biosensor platform and the method of detection is suitable for both small and large ligand molecules therewith.
Background of the invention:
Electrochemical biosensors are the biosensors that transform biochemical information such as analyte concentrations into an analytically useful signal such as current or voltage. These electrochemical biosensors have played a critical role in the advancement of commercial point-of-care systems with detectors for glucose, uric acid, and cholesterol currently available as diagnostic devices.

In biochemistry, receptor-ligand kinetics is a branch of chemical kinetics in which the kinetic species are defined by different non-covalent bindings and/or conformations of the molecules involved, which are denoted as receptors and ligands. Receptor ligand binding kinetics also involves the on- and off-rates of binding.

The ligand is an ion or molecule (functional group) that binds to a central metal atom to form a coordination complex. The bonding with the metal generally involves a formal donation of one or more of the ligand's electron pairs.

Conventionally, Surface Plasmon Resonance (SPR) or inflammatory based platforms are used for evaluating ligand-receptor binding kinetics. This evaluation approach is suitable for macromolecular analytes (i.e., molecular weight > 5000 daltons) and can do direct measurements for large protein-protein interaction for association and disassociation. The direct detection of smaller molecules is possible but, the useful range of the essay is generally limited. In such cases, an indirect method (i.e., an association based method) must be needed for binding analysis of analyte to small ligand molecules. And, the currently available methods which are used for binding are very expensive.

Traditionally, electrochemical sensors are used for measuring target induced changes in current output. The chronoamperometry is carried out to measure current decay kinetics as the indicator of target binding and also aid in determining the electron transfer kinetics. The screen printed electrodes is used as a working electrode for performing chronoamperometric measurements and detect the small molecule target species. However, current electrochemical sensors use labeled detection for evaluation of ligand-receptor bindings and require an extra mediator for binding smaller ligand molecules.

In updated technology, an electrochemical apparatus is used for detecting binding between members of a biological binding pair i.e. a receptor protein or ligand binding fragment. The apparatus mainly comprises three electrodes such as the first electrode, reference electrode, and auxiliary electrode. Chronoamperometry is used as the electrochemical assay for measuring current responses. It aids in providing screening assays for detecting specific binding affinity between the biologic binding pair for use in drug development, biochemical analysis, and purification assays. These measurements are used for determining dissociation constants. However, an electrochemical biosensor platform with label-free detection of ligand-receptor bindings which is suitable for both small and large ligand molecules does not exist.

Therefore, there exists a need for an electrochemical biosensor platform for the evaluation of kinetics of ligand-receptor interaction using a screen-printed electrode with a label-free direct single detection platform. There is a need for a chronoamperometry method which is used to calculate rate of association, rate of dissociation and dissociation constants. There is a need to provide a highly sensitive, time dependent behavior and cost-effective electrochemical technique that can be suitable for both small as well as large ligand molecules. Such a biosensor must have the ability of being used for screening of drug molecules, protein-protein interaction affinity, protein-ligand (small molecules) affinity, and antibody-antigen or antibody-hormone interaction.

Objectives of the invention:
The primary objective of the invention is to provide an electrochemical biosensor platform for the evaluation of kinetics of ligand-receptor interaction.

Another objective of the invention is to evaluate kinetics of ligand-receptor interaction using screen-printed electrode based chip employing with a label-free direct single detection platform.

The other objective of the invention is to provide a method of fabrication of the biosensor platform on to screen printed electrodes that enables detection up to size of 200 daltons and with a lower detection limit of 0.63fm.

Yet another objective of the invention is to provide a chronoamperometry method which is used to calculate rate of association, rate of dissociation and dissociation constants.

Further objective of the invention is to provide a highly sensitive and time dependent behaviour of electrochemical technique that can be suitable for both small as well as large ligand molecules.

The other objective of the invention is to provide a cost-effective biosensor for screening of drug molecules, protein-protein interaction affinity, protein-ligand (small molecules) affinity, and antibody-antigen or antibody-hormone interaction.
Summary of the Invention:
The disclosure proposes an electrochemical biosensor based evaluation of ligand-receptor interaction. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a label-free direct detection of ligand-receptor binding using electrochemical biosensor platform and the method of detection is suitable for both small and large ligand molecules therewith.

According to an aspect, the invention provides a method of fabrication of the electrochemical biosensor platform for label-free evaluation of ligand-receptor interaction. The method includes the steps comprising of washing a screen printed electrode with water. In specific, the screen printed electrode is either a carbon screen printed electrode or a gold screen printed electrode. When the screen printed electrode is a carbon electrode modified using gold nanoparticles that is washed with 0.1 N H2SO4 before washing with water. when the screen printed electrode is a carbon electrode modified using graphene oxide-polypyrrole nanocomposite, after washing with water the working electrode surface of the screen printed electrode is functionalized by 5mM 3-Aminopropyltriethoxysilane (APTES) to get NH2 groups on the electrode surface and is then washed with water and incubated with graphene oxide-polypyrrole nanocomposite.

Then, a ligand is injected onto the working electrode of the screen printed electrode surface. Next, the ligand injected screen printed electrode is washed with ultrapure water for two to three times after two to three hours. The ligand injected to the screen printed electrode includes either cystamine dihydrochloride while using graphene oxide-polypyrrole nanocomposite as nanomaterial or cysteamine hydrochloride while using gold nanoparticles as nanomaterial.

Later, a nanomaterial is added and the screen printed electrode is left at room temperature for two to three hours for incubation. In specific, the nanomaterial added to the ligand injected screen printed electrode includes either gold nanoparticles or a graphene oxide-polypyrrole nanocomposite.

Then, a protein is immobilized over the screen printed electrode surface by incubating for 20 minutes under ambient conditions. Next, the screen printed electrode is washed with either PBS of 0.01 M at 7.4 pH after 20 minutes or adding 10?L 0.75 microgram) antibody and washed with PBS after leaving overnight at 4°C, to remove unbound protein. Later, a suitable blocking buffer is added if the sample is not pure. In specific, the suitable blocking buffer includes either 1% BSA or 0.5% gelatin or skimmed milk to avoid non-specific interactions. The method of fabrication provides a detection platform that is suitable for different types of electrodes with a lower detection limit of 0.63fm and detects up to a molecular size of 200 daltons.

When the screen printed electrode is a gold screen printed electrode modified using gold nanoparticles, the method includes adding 10 ?L of NTA (50 mM) prepared in glycine-NaOH buffer (pH: 9.5) and incubating for2-3 hrs followed by drying wash with water after adding a nanomaterial to fabricate the platform for His tagged proteins immobilization through His tag. After adding 10 ?L of NTA the method includes adding 10 ?L of NiSO4 or CoSO4or CoCl2(50 mM) solution prepared in alkaline water and drying for 2-3 hours followed by washing with water after blocking the electrode surface with 0.2% gelatine or BSA or skimmed milk blocking solution prepared in 0.01 M PBS (pH 7.4). To fabricate the platform for His tagged proteins immobilization through His tag the screen printed electrode is immobilized with 10 ?L of His tag protein for 15-20 minutes and then washed with 0.01 M PBS (pH 7.4) before immobilizing the gold screen printed electrodes modified using gold nanoparticles with a protein.

According to another aspect of the invention, the method for evaluating the kinetics of ligand-receptor interaction includes the steps comprising of adding 100 µL of electrolyte solution (i.e., 0.5mM Ferri/ferrocyanide in 0.01 M PBS (pH 7.4))to the electrode surface thereby covering all three working, reference, and auxiliary electrodes. Then, the chronoamperometric response was recorded at an applied potential of 0.1V. Initially, a rise in current response was observed which stabilizes after 15 to 20 seconds. Later, after current saturates to a fixed value, 10 µL of ligand is injected into the electrolyte solution covering the entire electrode surface. Then, the ligand binds with the protein in an electrolyte solution and immobilizes over the electrode surface that result in decreasing in the current response.

As time progresses more and more of ligand interacts with the protein (protein-ligand complex formation) and the current response keeps on decreasing. The current response was observed till saturation was achieved i.e., indicative of either no more free ligand is left in an electrolyte solution or the protein is saturated or no more ligand can bind. At saturation point, the electrode was washed by replacing electrolyte plus ligand solution present on the electrode surface with the electrolyte solution depending on the affinity of the ligand. The saturation current reaches certain value depends on the affinity of the ligand. The current response shows a noisy signal that stabilizes after a few seconds. Later, dissociation kinetics aid in dissociating the protein-ligand complex and then, 1 M of sodium citrate tri-hydrate and buffer pH 5.0 was injected into the electrolyte solution covering the electrode surface thereby increasing current response with time. Finally, output current attains saturation indicative of completion of the dissociation process.

Further, objects and advantages of the present invention will be apparent of the following portion of the specifications, the claims and the attached drawings.
Detailed description of drawings:
The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, serve to explain the principles of the invention.

FIG. 1 illustrates an exemplary method for label-free evaluation of the kinetics of ligand-receptor interaction interaction in accordance to an exemplary embodiment of the invention.

FIG. 2 illustrates an exemplary block diagram of a method of fabrication of biosensor platform onto carbon screen printed electrodes using gold nanoparticles in accordance to an exemplary embodiment of the invention.

FIG. 3 illustrates an exemplary block diagram of a method of fabrication of biosensor platform onto gold screen printed electrodes using gold nanoparticles in accordance to an exemplary embodiment of the invention.

FIG. 4 illustrates an exemplary block diagram of a method of fabrication of biosensor platform onto carbon screen printed electrodes using graphene oxide-polypyrrole nanocomposite in accordance to an exemplary embodiment of the invention.

FIG. 5 illustrates an exemplary block diagram of a method of fabrication of biosensor platform onto gold screen printed electrodes using gold nanoparticles for label-free evaluation of His tagged proteins immobilization through His tag ligand-receptor interaction in accordance to an exemplary embodiment of the invention.

FIG. 6 illustrates an exemplary block diagram of a biosensor for detection of protein-ligand interaction in accordance to an exemplary embodiment of the invention.

FIG. 7 illustrates an exemplary biosensor device for detection of protein-ligand interaction in accordance to an exemplary embodiment of the invention.

FIG. 8 illustrates an exemplary circuit diagram of the biosensor device in accordance to an exemplary embodiment of the invention.

FIG. 9 Illustrates the current response with time for protein immobilized on the surface binding with protein 1 and BSA as ligand (Negative control) in accordance to an exemplary embodiment of the invention.

FIG. 10 depicts the current response with time for protein immobilized on the surface binding with protein1 and protein 2 in accordance to an exemplary embodiment of the invention.

FIG. 11 depicts the current response with time for protein immobilized on the surface binding with protein 3 and small drug like molecule in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present disclosure to provide a label-free direct detection of ligand-receptor binding using electrochemical biosensor platform and the method of detection is suitable for both small and large ligand molecules therewith.

According to an exemplary embodiment of the invention, FIG. 1 refers to a method 100 for evaluating the kinetics of ligand-receptor interaction. The method 100 includes the steps comprising of adding 100 µL of electrolyte solution (i.e., 0.5mM Ferri/ferrocyanide in 0.01 M PBS (pH 7.4)) to the electrode surface thereby covering all three working, reference, and auxiliary electrodes at step 101. Then, the chronoamperometric response was recorded at an applied potential of 0.1V. Initially, a rise in current response was observed which stabilizes after 15 to 20 seconds. Later at a step 102, after current saturates to a fixed value, 10 µL of ligand is injected into the electrolyte solution covering the entire electrode surface. Then, the ligand interacts with the protein in an electrolyte solution and immobilizes over the electrode surface that result in decreasing in the current response.

As time progresses more and more of ligand interacts with the protein (protein-ligand complex formation) and the current response keeps on decreasing. The current response was observed till saturation was achieved i.e., indicative of either no more free ligand is left in an electrolyte solution or the protein is saturated or no more ligand can bind. At saturation point, the electrode was washed at step 103 by replacing electrolyte plus ligand solution present on the electrode surface with the electrolyte solution depending on the affinity of the ligand at step 104.

The saturation current reaches certain value depends on the affinity of the ligand. The current response shows a noisy signal that stabilizes after a few seconds. Later, the dissociation buffer is added at step 105, while dissociation kinetics aid in dissociating the protein-ligand complex and then, 1 M of sodium citrate tri-hydrate and buffer pH 5.0 are injected into the electrolyte solution covering the electrode surface thereby increasing current response with time at step. Finally, output current attains saturation indicative of completion of the dissociation process.

The kinetic parameters were obtained from analyzing the chronoamperometric curve. The association and dissociation rates such as kon and koff were calculated from the slope of the linear region of the curve showing association and the slope of dissociation curve divided by ligand concentration in the sample respectively. The dissociation constant (KD) was evaluated as
K_D=K_off/K_on
According to another exemplary embodiment of the invention, FIG. 2 refers to an exemplary block diagram of a method 200 of fabrication of biosensor platform onto carbon screen printed electrodes using gold nanoparticles. The method includes the steps comprising of washing the screen printed carbon electrode with 0.1 N H2SO4 by recording cyclic voltammogram from -0.1V to 0.1 V at a scan rate of 100mV/sec at step 201. Then at step 202, the electrode is washed with water 2-3 times followed by addition of 2.5 µL of ligand i.e., cysteamine hydrochloride (2 mg/mL) onto working electrode surface under ambient conditions in dark room. Next step 203, the electrode is washed 2-3 times with ultrapure water after 2-3 hours. In specific, the ultrapure water utilized is milliQ water. Later at step 204, nanomaterial i.e., 2 µL gold nanoparticles are added (15mg/mL) and left at room temperature for 2-3 hours.

Then at step 205, after incubation, the electrode is washed 2-3 times with ultrapure water. Next at step 206, 2.5 µL protein (5mg/mL) is immobilized over the electrode surface by incubating for 20 minutes under ambient conditions. Amine group presented on the protein forms amide bond with COO-groups of gold nanoparticles. After 20 minutes of immobilization, electrode is washed with PBS (0.01 M, pH 7.4) to remove unbound protein.

Optionally, add 10µL antibody (0.1 µg antibody (antibody is diluted in 100 mM bicarbonate/carbonate coating buffer at a pH of 9.6 instead of immobilization. At this high pH, antibody gets negatively charged and COO- group at the Fc region of antibody forms amide bond with NH2 group presented on the electrode surface. Leave overnight at 4°C followed by washing with PBS (0.01 M, pH 7.4) to remove unbound antibody at step 206.

Then at step 207, the electrode surface is blocked with suitable blocking buffer if sample is not pure sample. Blocking buffers depend on body fluid sample. Blocking buffers include 1% BSA or 0.5% gelatin or skimmed milk to avoid non-specific interactions. Generally, BSA is used for blocking.

According to another exemplary embodiment of the invention, FIG. 3 refers to an exemplary block diagram of a method 300 of fabrication of biosensor platform onto gold screen printed electrodes using gold nanoparticles. The method includes the steps comprising of washing the screen printed carbon electrode with water and sonicating at step 301. Then at step 302, 2.5 µL of ligand i.e., cysteamine hydrochloride (2 mg/mL) is added onto working electrode surface under ambient conditions in dark room. Next step 303, the electrode is incubated for 2-3 hours in dark and it is washed with ultrapure water. Later at step 304, nanomaterial i.e., 2 µL gold nanoparticles are added (15 mg/mL), incubated for 2 to 3 hours and washed with ultrapure water for 2-3 times.

Then at step 305, 2.5 µL protein (1 mg/mL) is immobilized over the electrode surface for 15 to 20 minutes under ambient conditions. Amine group presented on the protein forms amide bond with COO-groups of gold nanoparticles. After 20 minutes of immobilization, electrode is washed with PBS (0.01 M, pH 7.4) to remove unbound protein.

Optionally, we can add 10 µL antibody (0.1 µg antibody (antibody is diluted in 100 mM bicarbonate/carbonate coating buffer at a pH of 9.6 instead of immobilization at step 305. At this high pH, antibody gets negatively charged and COO- group at the Fc region of antibody forms amide bond with NH2 group presented on the electrode surface. The electrode is left overnight at 4°C followed by washing with PBS (0.01 M, pH 7.4) to remove unbound antibody.

Then at step 306, the electrode surface is blocked with suitable blocking buffer if sample is not pure sample. Blocking buffers are the same according to the method 200.

According to another exemplary embodiment of the invention, FIG. 4 refers to a method 400 of fabrication of biosensor platform onto carbon screen printed electrodes using graphene oxide-polypyrrole nanocomposite. The method 400 includes the steps comprising of selecting a screen printed electrode with carbon as working electrode and washing it with water at step 401. Then at step 402, the working electrode surface is functionalized by 5mM 3-Aminopropyltriethoxysilane (APTES) to get NH2 groups on the electrode surface.

Next at step 403, the functionalized electrode is washed with water and incubated with graphene oxide-polypyrrole nanocomposite. Later, the ligand i.e., cystamine dihydrochloride is immobilized on the electrode to get NH2 groups at the surface at step 404. Then at step 405, 10 ?L protein (1 mg/mL) is immobilized for 15-20 minutes under ambient conditions and is washed with 0.01 M PBS with a pH of 7.4.

Optionally, we can add 10?l of antibody (0.1 ?g antibody (antibody is diluted in 100 mM bicarbonate/carbonate coating buffer (pH 9.6). At this high pH, antibody gets negatively charged and COO- group at the Fc region of antibody forms amide bond with NH2 group presented on the electrode surface. Then, it is left overnight at 4°C followed by washing with PBS (0.01 M, pH 7.4) to remove unbound antibody instead of immobilization at step 405. Finally at step 406, the electrode surface is blocked with suitable blocking buffer if sample is not pure. Blocking buffers are the same according to the method 200.

According to another exemplary embodiment of the invention, FIG. 5 refers to a method 500 of fabrication of biosensor platform onto gold screen printed electrodes using gold nanoparticles for label-free evaluation of His tagged proteins immobilization through His tag ligand-receptor interaction. The method includes the steps comprising washing the electrode by sonicating in water for 5-10 minutes at step 501. Then, 10 ?L of ligand i.e., cysteamine hydrochloride (2 mg/mL) is added to the working electrode with a diameter of 4 mm at step 502. Next, the electrode is incubated for 2 to 3 hours in dark and is then washed with ultrapure water 2 to 3 times at step 503.

Later at step 504, 10 ?L of nanomaterial i.e., gold nanoparticles (15 mg/mL) are added and incubated for 2 to 3 hours followed by washing with ultrapure water for 2 to 3 times. Then at step 505, 10 ?L of NTA (50 mM) that is prepared in glycine-NaOH buffer with a pH of 9.5 is added. Next, the electrode is incubated and washed with ultrapure water after it is dried at step 506. Later, the electrode surface is blocked with 0.2% gelatin or BSA or skimmed milk blocking solution prepared in 0.01 M PBS with a pH of 7.4 at step 507.

Then at step 508, 10 ?L of NiSO4 or CoSO4 or CoCl2 (50 mM) solution prepared in alkaline water is added. Next, the electrode is dried for 2 to 3 hours and is washed with water at step 509.

Later, the electrode is immobilized with 10 ?L of His tag protein for 15-20 minutes and is washed with 0.01 M PBS with a pH of 7.4 at step 510. Then at step 511, 10 ?L protein (1 mg/mL) is immobilized for 15-20 minutes under ambient conditions and it is washed with 0.01 M PBS of pH 7.4.

Optionally, 10?l of antibody (0.1 ?g antibody (antibody is diluted in 100 mM bicarbonate or carbonate coating buffer (pH 9.6))). At this high pH, antibody gets negatively charged and COO- group at the Fc region of antibody forms amide bond with NH2 group presented on the electrode surface.) Leave overnight at 4?C followed by washing with PBS (0.01 M, pH 7.4) to remove unbound antibody instead of immobilization at step 511. Finally at step 512, the electrode surface is blocked with a suitable blocking buffer if the sample is not pure.

According to another exemplary embodiment of the invention, FIG. 6 refers to an exemplary block diagram 600 of a biosensor. The biosensor device comprises a screen printed electrode 602, a microcontroller 603 to process the blood sample 601, and a display unit 604 configured to display the test results.

According to another exemplary embodiment of the invention, FIG. 7 refers to an exemplary biosensor device 700. The device 700 is equipped with a display 701 on top, and a cartridge insertion slot 702. The cartridge insertion slot 702 enables insertion of testing cartridges 703 into the device 700. After the test is performed, the response is displayed in the display 701.

According to another exemplary embodiment of the invention, FIG. 8 refers to an exemplary circuit diagram of the biosensor device.

According to another exemplary embodiment of the invention, FIG. 9 depicts the current response with time for protein immobilized on the surface binding with protein 1 and BSA as ligand (Negative control). In specific, on the addition of BSA, there is an initial dip and then the current response increases continuously by saturating (i.e., no decrease in current response signature of binding between BSA and the immobilized protein).

Referring to FIG. 10, when the current response is recorded in the electrolyte solution, then there is an increase in current that saturates. On injecting ligand (another protein), there arises a spike and then the current decreases. The rate of decrease of current gives binding rate, and then later after some time the buffer gets exchanged with electrolyte solution thereby showing the fluctuations/spikes in the current response that eventually saturates. On the addition of dissociation buffer of pH 5.0, the ligand-protein starts dissociating from receptor protein and is marked by an increase in current that eventually saturates. The rate of increase of current gives rate of association.

Referring to FIG. 11, protein3 is immobilized to the electrode surface and its ligand i.e., a small drug-like molecule is injected in the electrolyte solution. This is marked by decreasing the current response after the initial spike and rate of decrease of current gives association rate. The buffer is exchanged with electrolyte solution and then, the dissociation buffer of pH 5.0 is added thereby increasing in current response due to the dissociation of quercetin.

Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, a label-free single direct detection of ligand-receptor binding using electrochemical biosensor platform and the method of detection is suitable for both small and large ligand molecules therewith. Further, the method of fabrication of the biosensor platform on to screen printed electrodes enables detection up to size of 200 daltons and with a lower detection limit of 0.63fm. The proposed chronoamperometry method is used to calculate rate of association, rate of dissociation and dissociation constants. The electrochemical technique provides a highly sensitive, cost-effective and time dependent behavior that can be suitable for both small as well as large ligand molecules. Such a biosensor is used for screening of drug molecules, protein-protein interaction affinity, protein-ligand (small molecules) affinity, and antibody-antigen or antibody-hormone interaction.

It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application.

CLAIMS:CLAIMS:
I / We Claim:
1. A method of fabrication of electrochemical biosensor platform for label-free evaluation of ligand-receptor interaction, comprising:
washing a screen printed electrode with water;
injecting a ligand onto working electrode of said screen printed electrode surface;
washing said ligand injected screen printed electrode with ultrapure water for two to three times after two to three hours;
adding a nanomaterial and leaving at room temperature for two to three hours for incubation;
immobilizing a protein over the screen printed electrode surface by incubating for 20 minutes under ambient conditions;
washing with either PBS of 0.01 M at 7.4 pH after 20 minutes or adding 10?L antibody and washing with said PBS after leaving overnight at 4°C, to remove unbound protein; and
adding a suitable blocking buffer if sample is not pure,
whereby said method of fabrication provides a detection platform that is suitable for different types of electrodes with a lower detection limit of 0.63fm and detects up to molecular size of 200 daltons.
2. The method of fabrication of electrochemical biosensor platform as recited in claim 1, wherein said screen printed electrode is either a carbon screen printed electrode or a gold screen printed electrode.
3. The method of fabrication of electrochemical biosensor platform as recited in claim 1, wherein when said screen printed electrode is a carbon electrode modified using gold nanoparticles that is washed with 0.1 N H2SO4 before washing with water.
4. The method of fabrication of electrochemical biosensor platform as recited in claim 1, wherein said nanomaterial added to the ligand injected screen printed electrode includes either gold nanoparticles or a graphene oxide-polypyrrole nanocomposite.
5. The method of fabrication of electrochemical biosensor platform as recited in claim 1, wherein said ligand injected to the screen printed electrode includes either cystamine dihydrochloride while using graphene oxide-polypyrrole nanocomposite as nanomaterial or cysteamine hydrochloride while using gold nanoparticles as nanomaterial.
6. The method of fabrication of electrochemical biosensor platform as recited in claim 1, wherein said suitable blocking buffer includes either 1% BSA or 0.5% gelatin or skimmed milk to avoid non-specific interactions.
7. The method of fabrication of electrochemical biosensor platform as recited in claim 1, wherein when said screen printed electrode is a carbon electrode modified using graphene oxide-polypyrrole nanocomposite, after washing with water the working electrode surface of said screen printed electrode is functionalized by 5mM 3-Aminopropyltriethoxysilane (APTES) to get NH2 groups on the electrode surface and is then washed with water and incubated with graphene oxide-polypyrrole nanocomposite.
8. The method of fabrication of electrochemical biosensor platform as recited in claim 1, wherein when said screen printed electrode is a gold screen printed electrode modified using gold nanoparticles said method includes adding 10 ?L of NTA (50 mM) prepared in glycine-NaOH buffer (pH: 9.5) and incubating for2-3 hrs followed by drying wash with water after adding a nanomaterial to fabricate the platform for His tagged proteins immobilization through His tag.
9. The method of fabrication of electrochemical biosensor platform as recited in claim 8, wherein after adding 10 ?L of NTA said method includes adding 10 ?L of NiSO4 or CoSO4or CoCl2(50 mM) solution prepared in alkaline water and drying for 2-3 hours followed by washing with water after blocking the electrode surface with 0.2% gelatine or BSA or skimmed milk blocking solution prepared in 0.01 M PBS (pH 7.4).
10. The method of fabrication of electrochemical biosensor platform as recited in claim 1, wherein to fabricate the platform for His tagged proteins immobilization through His tag said screen printed electrode is immobilized with 10 ?L of His tag protein for 15-20 minutes and then washed with 0.01 M PBS (pH 7.4) before immobilizing the gold screen printed electrodes modified using gold nanoparticles with a protein.