Description:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patent Rules, 2003
COMPLETE SPECIFICATION
(See sections 10 & rule 13)
1. TITLE OF THE INVENTION
MXene GRAPHENE NANOHYBRID THIN FILM BASED ELECTROCHEMICAL BIOSENSORS FOR ANALYTE DETECTION, ITS METHOD OF PREPARATION AND APPLICATIONS THEREOF
2. APPLICANT (S)
NAME NATIONALITY ADDRESS
DIVYASAMPARK IHUB ROORKEE FOR DEVICES MATERIALS AND TECHNOLOGY FOUNDATION IN Indian Institute of Technology Roorkee, Roorkee- 247667, Uttarakhand, India.
3. PREAMBLE TO THE DESCRIPTION
COMPLETE SPECIFICATION

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

FIELD OF INVENTION:
[001] The present invention relates to the field of biosensor. The present invention in particular relates to MXene graphene nanohybrid thin film based electrochemical biosensors for analyte detection and applications thereof.
DESCRIPTION OF THE RELATED ART:
[002] In recent years, two-dimensional nanomaterials have attracted more and more attention as enzyme immobilization substrates. Among them, nanomaterials composed of carbon (such as graphene, carbon nanotubes, mesoporous carbon, etc.) have large specific surface area, good conductivity and biocompatibility, but they are not easy to disperse in the aqueous phase, resulting in electrical chemoenzyme biosensors have poor reproducibility, which limits their application development. As a new type of two-dimensional crystalline nanomaterial, MXene-Ti3C2 has a multilayer sheet structure similar to graphene, a large specific surface area, good biocompatibility, and good metal conductivity. In addition, MXene The -OH functional group on the surface of Ti3C2 makes it have good hydrophilicity and can be uniformly dispersed in water. These advantages make it a research hotspot in the field of materials in recent years, and it has been widely used in the fields of energy storage and hydrogen storage, catalysis, adsorption and biosensors.
[003] Reference may be made to the following:
[004] Publication No. CN110057882 relates to an electrochemical biosensor based on MXene-Ti<3>C<2> and a preparation method and application of the electrochemical biosensor. According to the preparation method, first, a pattern enzyme is fixedly carried on the surface of MXene-Ti<3>C<2>, wherein the surface is provided with -OH functional groups; and second, MXene-Ti<3>C<2> is mixed with a film-forming material to form a composite material, the composite material is fixed to the surface of a glassy carbon electrode, and the electrochemical biosensor is prepared. According to the electrochemical biosensor, the two-dimensional nanometer material MXene-Ti<3>C<2> is fully utilized to serve as an enzyme carrier of the sensor, and the electrochemical biosensor has a specific flaky structure, a large specific surface area, excellent metal conductivity and extremely good aqueous phase dispersity; the preparation process is simple, the prepared sensor has high sensitivity to a phenolic compound, a low detection limit and good reproducibility; a complicated sample pretreatment process is not needed in the detection process, response to a target compound is rapid, miniaturization is easy, and the electrochemical biosensor is suitable for on-site detection, continuous online monitoring, etc.
[005] In present invention Ti3C2-MXene and amine functionalized graphene (f-graphene@Ti3C2-MXene) nanohybrid based thin film has been deposited on ITO coated glass substrate via the air-brush spray coating technique. The mentioned patent has drop casted the MXene composite on glass carbon electrode. Present method of thin film fabrication provides the uniform, reproducible thin-film with customized thickness in a cost-effective manner whereas it is difficult to obtain the uniformity in the drop casting method.
[006] The mentioned patent is enzyme-based biosensor whereas present invention is immunosensor based on antigen-antibody interactions. Present process of fabricating BSA/anti-CEA/f-graphene@Ti3C2-MXene/ITO immunoelectrode is different from the mentioned patent.
[007] In present invention the antibody of targeted cancer biomarker is conjugated with the graphene and MXene nanohybrid using covalent binding wherein carboxylic groups of antibodies are immobilized to the amine groups of the graphene-MXene nanohybrid whereas the mentioned patent has fixed the enzyme with -OH groups of MXene. Both these approaches are totally different.
[008] Publication No. CN109975382 relates to a tyrosinase biosensor comprising a phosphorus-doped MXene modified electrode, and a preparation method and an application thereof, which belong to the technical field of electrochemical biosensors. The tyrosinase biosensor comprises a glassy carbon electrode, wherein the glassy carbon electrode is a glassy carbon electrode with tyrosinase/phosphorus-doped MXene/chitosan modified on the surface.
[009] In present Ti3C2-MXene and amine functionalized graphene (f-graphene@Ti3C2-MXene) nanohybrid based thin film has been deposited on ITO coated glass substrate via the air-brush spray coating technique. The mentioned patent has drop casted the phosphorous doped MXene/chitosan on glass carbon electrode. Our method of thin film fabrication provides the uniform, reproducible thin-film with customized thickness in a cost-effective manner whereas it is difficult to obtain the uniformity in the drop casting method.
[010] The mentioned patent is tyrosinase (enzyme) based biosensor whereas our work is carcinoembryonic antigen (CEA) antibody based immunosensor. Therefore, the process of fabricating the immunoelectrode (BSA/anti-CEA/f-graphene@Ti3C2-MXene/ITO) is different from the existing one.
[011] In present invention conjugated the antibody of targeted cancer biomarker is conjugated with the graphene and MXene nanohybrid using covalent binding wherein carboxylic groups of antibodies are immobilized to the amine groups of the graphene-MXene nanohybrid whereas the mentioned patent has fixed the enzyme with P-doped MXene.
[012] Patent No. US9676621 relates to a field-effect transistor (FET)-based biosensor and uses thereof. In particular, FET-based biosensors using thermally reduced graphene-based sheets as a conducting channel decorated with nanoparticle-biomolecule conjugates.
[013] The mentioned patent is a field-effect transistor (FET)-based sensor whereas the present invention is the thin-film based electrochemical biosensor. The working principle and the method of detection of these two approaches are different.
[014] The mentioned patent is based on reduced graphene oxide-gold nanoparticle whereas our work is based on graphene and MXene nanohybrid based thin films. Present nanohybrid thin-film based electrochemical biosensor is more sensitive and efficient than FET based biosensor.
[015] Publication No. US2013248380 relates to a biosensor comprising a graphene electrode linked to a biosensing element by a linker, the biosensing element bonded to a flexible substrate. The graphene electrode has a first end and a second end, such that the first end may be a positive terminal and the second end a negative terminal.
[016] In present invention Ti3C2-MXene and amine functionalized graphene (f-graphene@Ti3C2-MXene) nanohybrid based thin film has been deposited on ITO coated glass substrate via the air-brush spray coating technique. The mentioned patent has transferred the graphene onto flexible substrate. The present method of thin film fabrication provides the uniform, reproducible thin-film with customized thickness in a cost-effective manner.
[017] The mentioned patent is enzyme-based biosensor whereas present invention is immunosensor based on antigen-antibody interactions.
[018] In present invention the antibody of targeted cancer biomarker is conjugated with the graphene and MXene nanohybrid using covalent binding wherein carboxylic groups of antibodies are immobilized to the amine groups of the graphene-MXene nanohybrid whereas the mentioned patent has linked the enzyme graphene using an additional linker.
[019] Publication No. CN114354711 electrochemical biosensor for detecting lead. The DNA electrochemical biosensor is characterized in that a sensitive film is formed by GR5-DNAzyme and MXene nanosheets and is modified on the surface of a glassy carbon electrode; a DNA enzyme GR5-DNAzyme with specific reactivity to Pb < 2 + > is used as a reaction element, and the characteristic that a two-dimensional transition metal carbide new material MXene has different affinity to DNA single and double chains is utilized, so that the detection electrochemical biosensor with high sensitivity to Pb < 2 + > is constructed on a glassy carbon electrode GCE. The sensor is simple and rapid to operate, and sensitively and efficiently detects the lead content in animal tissues.
[020] In present invention Ti3C2-MXene and amine functionalized graphene (f-graphene@Ti3C2-MXene) nanohybrid based thin film has been deposited on ITO coated glass substrate nanohybrid based thin film has been deposited on ITO coated glass substrate via the air-brush spray coating technique. The mentioned patent has dropped casted the GR5-DNAzyme-MXene on glass carbon electrode. The present method of thin film fabrication provides uniform, reproducible thin-film with customized thickness in a cost-effective manner whereas it is difficult to obtain the uniformity in the drop casting method.
[021] The mentioned patent is DNA based biosensor for lead detection whereas present invention is immunosensor based on antigen-antibody specific interactions for cancer biomarker detection.
[022] In present invention the antibody of targeted cancer biomarker is conjugated with the graphene and MXene nanohybrid using covalent binding wherein carboxylic groups of antibodies are immobilized to the amine groups of the graphene-MXene nanohybrid whereas the mentioned patent has bound the double stranded DNA with MXene. Both these approaches are totally different.
[023] Publication No. CN113484385 relates to a biosensor for fixing cholesterol oxidase based on an MXene material and a detection method of the biosensor. In Present invention Ti3C2-MXene and amine functionalized graphene (f-graphene@Ti3C2-MXene) nanohybrid based thin film has been deposited on ITO coated glass substrate nanohybrid based thin film has been deposited on ITO coated glass substrate via the air-brush spray coating technique. The mentioned patent has dropped the MXene material/chitosan on working electrode. The present method of thin film fabrication provides the uniform, reproducible thin-film with customized thickness in a cost-effective manner.
[024] Herein, potassium ferricyanide/ potassium ferrocyanide solution containing potassium chloride has been used as the electrode, whereas uses hexaammineruthenium [Ru(NH3)6]3+ is used as the redox probe or electrolyte solution. During our experiments, we have observed that the oxidation current reflects instabilities after the initial cycle with a successive reduction in its intensity (Figure 1b). This might have occurred because of the formation of oxidized TiO2 layers on the surface of Ti3C2-MXene sheets at higher potential windows (-0.2 to 0.8 V). Ruthenium has a distinguished property of oxidation and reduction at the lower potential window (-0.8 to 0.2 V) without reacting with the surface of Ti3C2-MXene sheets. Hexaammineruthenium [Ru(NH3)6]+3 has been found to be a suitable redox probe for the electrolytic reaction at lower potentials and has been used as an alternative to [Fe(CN)6]-3/-4. In the presence of [Ru(NH3)6]3+ stable peaks are observed at desired voltage ranges for f-graphene@Ti3C2-MXene nanohybrid (Figure 1c).
[025] The mentioned patent is cholesterol oxidase enzyme-based biosensor for cholesterol detection whereas present is carcinoembryonic antigen (CEA) antibody based immunosensor based on antigen-antibody interactions.
[026] Publication No. CN111855777 relates to a glutamate oxidase biosensor as well as a preparation method and application thereof. The enzyme biosensor can be used for detecting the content of sodium glutamate (MSG) in food, and is high in sensitivity, low in detection limit and good in anti-interference performance.
[027] The mentioned patent is glutamate oxidase enzyme-based biosensor for sodium glutamate (MSG) detection in food samples whereas present invention is carcinoembryonic antigen (CEA) antibody based immunosensor based on antigen-antibody interactions for detecting CEA cancer biomarker in serum samples.
[028] In present invention Ti3C2-MXene and amine functionalized graphene (f-graphene@Ti3C2-MXene) nanohybrid based thin film has been deposited on ITO coated glass substrate via the air-brush spray coating technique. The mentioned patent has dropped casted the glutamate oxidase enzyme linked PtNP-coated MXene-Ti3C2Tx solution on glassy carbon electrode. Present method of thin film fabrication provides the uniform, reproducible thin-film with customized thickness in a cost-effective manner.
[029] In present invention the antibody of targeted cancer biomarker is conjugated with the graphene and MXene nanohybrid using covalent binding wherein carboxylic groups of antibodies are immobilized to the amine groups of the graphene-MXene nanohybrid whereas the mentioned patent has fixed the glutamate oxidase enzyme with camphene-Ti3C2Tx-chitosan solution using the layer-by-layer method.
[030] Reference may be made to an article entitled “Recent advances in graphene quantum dot-based optical and electrochemical (bio) analytical sensors” by Ashish Kalkal, Sachin Kadian, Rangadhar Pradhan, Gaurav Manik and Gopinath Packirisamy; Mater. Adv., 2, 5513-5541; 23rd March 2021 which enlightens the scope of GQDs in a variety of optical and electrochemical chemosensors as well as biosensors. Recent advancements pertaining to their synthesis methods, unique properties, and their regulation through heteroatom-doping and surface-functionalization strategies are discussed, along with the current challenges and future prospects. This explains the potential of GQDs in optical and electrochemical biosensor fabrication.
[031] Reference may be made to an article entitled “Recent advances in MXene-based electrochemical sensors” by Chirag Verma, Kamal Kishor Thakur; European Journal of Molecular & Clinical Medicine, Volume 07, Issue 07; 2020 which talks about research done on the electrochemical detecting properties of Titanium carbide MXene from year 2015- 2020, we have examined the electrochemical sensors fabricated by using Ti3C2Tx for biomedical application, biomolecule detection and environmental monitoring. This is for breast cancer biomarker Mucin1 detection. In this work MXene nanosheets (Ti3C2) were used for fabricating electrochemical biosensor. Ferrocene-labeled DNA was first bounded on MXene surface to fabricate cDNA-Fc/MXene probe. After that AuNPs were electrodeposited using the chronoamperometry process on GCE surface and then MUC1 aptamer was fixed on electrode surface through Au-S bonding to fabricate Apt/Au/GCE electrode. After that Apt/Au/GCE was immersed in cDNAFc/MXene solution and washed with PBS buffer solution. Thus, the developed electrochemical aptasensor cDNA-Fc/MXene/Apt/Au/GCE was utilized for MUC1 detection.
[032] Present invention reports results of the studies related to the development of Ti3C2-MXene and amine functionalized graphene (f-graphene@Ti3C2-MXene) nanohybrid based label-free electrochemical biosensing platform for CEA detection. The airbrush spray coating technique is employed for depositing the uniform thin film of f-graphene@Ti3C2-MXene nanohybrid on indium tin oxide (ITO) coated glass substrate.
[033] Reference may be made to an article entitled “Three-dimensional porous Ti3C2Tx MXene–graphene hybrid films for glucose biosensing” by Hui Gu, Yidan Xing, Ping Xiong, Huiling Tang, Chenchen Li, Shu Chen, Rongjin Zeng, Kai Han and Guoyue Shi; pubs.acs; September 25, 2019, which talks about a 3D porous Ti3C2Tx MXene–graphene (MG) hybrid film through a facile mixing–drying process. Ti3C2Tx MXene nanosheets (MNS) with hydrophilic groups on the rigid flakes endowed the MG hybrid film with open porous structure and a highly hydrophilic microenvironment. By simply controlling the content of Ti3C2Tx MNS and graphene sheets, the sizes of the internal pores were tunable accordingly. The 3D porous hybrid film, fabricated from Ti3C2Tx MNS and graphene sheets (weight ratios of 1:2 and 1:3), supplied more open structure to facilitate the glucose oxidase (GOx) entering the internal pores, which probably enhanced the stable immobilization and retaining of the GOx in the film. As a result, the as-proposed biosensor exhibited prominent electrochemical catalytic capability toward glucose biosensing, which was finally applied for glucose assay in sera. The preparation of the size-controlled 3D porous hybrid film provided a method for effectively binding enzymes/protein further to develop elegant biosensors.
[034] The mentioned patent is glucose oxidase enzyme-based biosensor for detection whereas our work is carcinoembryonic antigen (CEA) antibody based immunosensor based on antigen-antibody interactions for detecting CEA cancer biomarker.
[035] The present invention is the method of preparing BSA/anti-CEA/f-graphene@Ti3C2-MXene/ITO immunoelectrode.
[036] Hence, there is the requirement of a biosensor for early diagnosis of lung cancer to curb the menace of cancer.
[037] In order to overcome the above listed prior art, the present invention aims to provide MXene graphene nanohybrid thin film based electrochemical biosensors for analyte detection, its method of preparation and applications thereof.
OBJECTS OF THE INVENTION:
[038] The principal object of the present invention is to provide MXene graphene nanohybrid thin film based electrochemical biosensors for analyte detection, its method of preparation and applications thereof.
[039] Another object of the present invention is to provide air-brush spray coated amine functionalized graphene (f-graphene) and Ti3C2-MXene nanohybrid based immunosensing platform for detection of lung cancer biomarker.
[040] Yet another object of the present invention is to provide MXene graphene nanohybrid thin film based electrochemical biosensors for analyte detection for early detection of lung cancer.
SUMMARY OF THE INVENTION:
[041] The present invention relates to the MXene graphene nanohybrid thin film based electrochemical biosensors for analyte detection, its method of preparation and applications thereof. The present invention provides the fabrication of selective, sensitive, and label-free electrochemical immunosensing platform based on BSA/anti-CEA/f-graphene@Ti3C2-MXene/ITO immunoelectrode for CEA detection. The airbrush spray coating technique provided uniform thin-films of amine functionalized graphene and Ti3C2-MXene nanohybrid on ITO coated glass substrate. The developed platform detects CEA biomarker in the broad linear range which covers the whole physiological secretion range of CEA in healthy and cancer patients. Besides, good sensitivity with remarkable specificity, and lower limit of detection have been provided.
BREIF DESCRIPTION OF THE INVENTION
[042] It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered for limiting of its scope, for the invention may admit to other equally effective embodiments.
[043] Figure 1: Electrochemical response of fabricated biosensing platform in PBS with (a) Ferri-ferrocyanide redox couple [Fe(CN)6]-3/-4); (b) hexaammineruthenium [Ru(NH3)6]3+ redox probe (c) no redox (bare PBS).
[044] Figure 2: (a) pH study of BSA/anti-CEA/f-graphene@Ti3C2-MXene/ITO immunoelectrode; (b) Electrochemical response of bare ITO, f-graphene, Ti3C2-MXene, f-graphene@Ti3C2-MXene, anti-CEA/f-graphene@Ti3C2-MXene and BSA/anti-CEA/f-graphene@Ti3C2-MXene immunoelectrode carried out with CV.
[045] Figure 3: (a) Electrochemical response; (b) Randles-Sevcik plot of BSA/anti-CEA/f-graphene@Ti3C2-MXene/ITO immunoelectrode with different scan rate ranging from 10-150 mV/sec.
[046] Figure 4: (a) Electrochemical response of BSA/anti-CEA/f-graphene@Ti3C2-MXene/ITO immunoelectrode for different concentrations of CEA (0.01 pg mL-1 to 1000 ng mL-1); (b) Magnified view of obtained response; (c) Calibration plot between peak current and log of CEA concentration; (d) Interferent study of fabricated biosensing platform (BSA/anti-CEA/f-graphene@Ti3C2-MXene/ITO) with different analytes.
DETAILED DESCRIPTION OF THE INVENTION:
[047] The present invention provides MXene graphene nanohybrid thin film based electrochemical biosensors for analyte detection, its method of preparation and applications thereof. The present invention comprises air-brush spray coated amine functionalized graphene (f-graphene) and Ti3C2-MXene nanohybrid based immunosensing platform for detection of lung cancer biomarker, more specifically to the BSA/anti-CEA/f-graphene@Ti3C2-MXene/ITO immunoelectrode for carcinoembryonic antigen (CEA) detection.
[048] The f-graphene@Ti3C2-MXene nanohybrid thin-film based electrochemical biosensing platform has been developed for carcinoembryonic antigen (CEA) detection. The airbrush spray coating technique has been utilized for depositing uniform thin films of amine functionalized graphene (f-graphene) and Ti3C2-MXene nanohybrid on indium tin oxide (ITO) coated glass substrate. The EDC-NHS chemistry is employed to immobilize the deposited thin films with monoclonal anti-CEA antibodies followed by blocking of non-specific binding sites with bovine serum albumin (BSA). The electrochemical response of BSA/anti-CEA/f-graphene@Ti3C2-MXene/ITO immunoelectrode has been recorded with different concentrations of carcinoembryonic antigen (CEA) biomarker in standard samples using cyclic voltammetry (CV) technique.
[049] Fabrication of electrochemical biosensing platform for CEA detection:
[050] The airbrush spray coating technique is utilized to deposit the thin films of f-graphene@Ti3C2-MXene nanohybrid on indium tin oxide (ITO) coated glass substrate. The experimental setup was implemented inside a closed box to ensure well controlled environmental conditions and reduce cross contamination throughout the entire fabrication process. The ITO coated glass substrate was heated over the controlled temperature of 100 ºC for 10 min. Airbrush along with a pneumatic pump was used for the deposition of a fine, small-diameter spray of f-graphene@Ti3C2-MXene nanohybrid on pre-heated ITO coated glass substrate. When connected to an air compressor, the airbrush breaks the solution liquid into tiny droplets, called atomization. Atomization produces seamless blends, coatings, and gradient over the substrate. The Airbrush spray gun uses compressed air from a nozzle to atomize the desired solution into a controlled manner. The spray nozzle operates by impinging high-velocity turbulent air on the surface of filaments or films of liquid, causing them to collapse into droplets with a wide range of sizes. Airbrushes can be used with almost any solution, although highly viscous liquids may not produce quality results. The airflow was maintained at 7.5 L/min over a maximum air pressure of 25 psi. Further, to fabricate the biosensing platform, f-graphene@Ti3C2-MXene nanohybrid is biofunctionalized employing well-known EDC-NHS chemistry [12, 18]. The carboxyl groups present in monoclonal anti-CEA antibodies are animated by preparing a uniform solution having 0.05 M NHS, 50 µg mL–1 of anti-CEA, and 0.2 M EDC in 1:2:1 ratio. The obtained anti-CEA solution was added to same volume of f-graphene@Ti3C2-MXene nanohybrid solution. The resultant mixture was incubated under humid conditions for the next 2 h. The unbound antibodies are removed by washing with phosphate buffered saline (PBS) solution. Bovine serum albumin (BSA) is used to block the non-specific sites present in the fabricated platform, thereby proving BSA/anti-CEA/f-graphene@Ti3C2-MXene/ITO immunoelectrode.
[051] Optimization of biosensing parameters
[052] Electrochemical analysis of the deposited thin-films and fabricated biosensing platforms were conducted in phosphate buffered solution (PBS) (50 mM, 0.9% NaCl) using cyclic voltammetry (CV) with Metrohm Autolab potentiostat. In the bare PBS solution, a flat curve indicating the deceleration of ionic particles is observed (Figure 1a). To accelerate the movement of these particles, 5 mM ferri-ferrocyanide redox couple ([Fe(CN)6]-3/-4) is used in the PBS solution. The obtained results of the electrochemical reaction indicated that the oxidation current reflects instabilities after the initial cycle with a successive reduction in its intensity (Figure 1b). This might have occurred because of the formation of oxidized TiO2 layers on the surface of Ti3C2-MXene sheets at higher potential windows (-0.2 to 0.8 V). To overcome this limitation of a sudden drop in the current peaks, hexaammineruthenium [Ru(NH3)6]3+ is used as the redox probe. Ruthenium has a distinguished property of oxidation and reduction at the lower potential window (-0.8 to 0.2 V) without reacting with the surface of Ti3C2-MXene sheets. Hexaammineruthenium [Ru(NH3)6]+3 has been found to be a suitable redox probe for the electrolytic reaction at lower potentials and has been used as an alternative to [Fe(CN)6]-3/-4. In the presence of [Ru(NH3)6]3+ stable peaks are observed at desired voltage ranges for f-graphene@Ti3C2-MXene nanohybrid (Figure 1c).
[053] Extreme pH is known to cause conformational changes in biomolecules, impairing antibody-antigen interactions. Therefore, prior to electrochemical CEA detection, the effect of pH on the electrochemical behaviour of the fabricated immunoelectrode (BSA/anti-CEA/f-graphene@Ti3C2-MXene/ITO) is investigated using CV in PBS solution having 5 mM [Ru(NH3)6]+3 at pH (6.0-8.0). The obtained results indicate that as the pH increased, the magnitude of the peak current increased, and the maximum response is observed at pH 7.0. Whereas the peak current intensity was found to decrease in both acidic and basic pH. This maximum peak current was due to the presence of natural form of anti-CEA biomolecules at neutral pH, expressing highest activity. However, in both acidic and basic pH, the interaction between the H+ or OH- ions present in the buffer tends to denature biomolecules resulting in a decrease of the peak current and hinder their natural activity. Therefore, a standard pH of 7.0 was maintained throughout the further electrochemical experiment (Figure 2a).
[054] In accordance with the stages of development of the biosensing platforms, different electrodes have been prepared and their electrochemical behaviour have been carried out using CV. The obtained electrochemical results conducted on bare ITO, f-graphene, Ti3C2-MXene, f-graphene@Ti3C2-MXene, anti-CEA/f-graphene@Ti3C2-MXene and BSA/anti-CEA/f-graphene@Ti3C2-MXene immunoelectrode have been illustrated in Figure 2b. According to the results, it is evident that the magnitude of peak current for f-graphene@Ti3C2-MXene nanohybrid is greater as compared to the bare f-graphene and Ti3C2-MXene electrodes. It can be attributed to the synergistic effect of f-graphene@Ti3C2-MXene nanohybrid proving better electron transport pathways leading to enhanced electron transfer between the electrode and bulk electrolyte. On the other hand, a reduction in peak current is observed after antibody immobilization onto the f-graphene@Ti3C2-MXene/ITO electrode due to the insulating characteristics of antibodies wherein redox active sites are entrapped, inhibiting the electron transfer kinetics. After BSA immobilization onto the anti-CEA/f-graphene@Ti3C2-MXene/ITO electrode, further reduction in the magnitude of peak current is observed because of the blockage of non-specific active sites on the immunoelectrode surface which impede the electron transfer process.
[055] Further, the redox property and interfacial kinetics of fabricated BSA/anti-CEA/f-graphene@Ti3C2-MXene/ITO immunoelectrodes were evaluated using CV with different scan rate ranging from (10-150 mV/sec). The negative shift of anodic peak current (Ipa) and positive shift of cathodic peak current (Ipc) with increasing scan rate has been observed. In addition, both Ipa and Ipc were found to vary linearly with the square root of scan rate, revealing the diffusion controlled electrochemical reaction, described by linear Eq. 1 and Eq. 2.
Ipc = [0.072 µA (s/mV) x (scan rate [mV/s])1/2] -0.0163 µA; R2 = 0.99…………………. (1)
Ipa = [-0761 µA (s/mV) x (scan rate [mV/s])1/2] -0.05988 µA; R2 = 0.99………………….. (2)
[056] Electrochemical detection of CEA
[057] Under optimal parameters, electrochemical response studies were performed on the BSA/anti-CEA/f-graphene@Ti3C2-MXene/ITO immunoelectrode towards CEA biomolecule detection with an incubation time of 15 min, using CV at a potential range between -0.8 to 0.2 V, and scan rate of 50 mV/sec with increasing CEA concentration ranging from 0.01 pg mL-1 to 1000 ng mL-1 in a PBS solution consisting of 5 mM [Ru(NH3)6]+3 redox probe. The magnitude of the electrochemical current was found to be decreasing with increasing concentration of CEA, attributed to the CEA-anti-CEA (antigen–antibody) immunocomplex formation at the electrode-electrolyte interface (Figure 4a). The formed complex inhibited the transport of generated electrons via the redox probe, [Ru(NH3)6]3+ to electrode surface.
[058] Further, the calibration plot between the logarithm of CEA concentration and the magnitude of peak current was investigated (Figure 4c). Good linearity was observed in the CEA concentration of 0.01 pg mL-1 to 1000 ng mL-1, depicted by the linear Eq. (3).
I=-12.49 µA ?mL/ng log?_10??CEA (?ng mL?^(-1) )?+0.49 mA, R^2=0.98……….. (3)
[059] The lower limit of detection (LOD) was calculated using Eq. (4) with a triple signal-to-noise ratio (S/N = 3).
LOD=3s/S…………….. (4)
[060] where S denotes sensitivity of fabricated immunosesning platform (evaluated by the slope of the calibration curve) and s denotes the standard deviation. The LOD for anti-CEA/amine-GQDs/AuNPs@rGO immunosensing probe was found to be 0.33 pg mL-1. Further, the selectively of BSA/anti-CEA/f-graphene@Ti3C2-MXene/ITO immunoelectrode was investigated by recording the electrochemical response of fabricated platform with different interferents known to be present in human blood such as glucose, potassium chloride, other cancer biomarkers viz. neuron-specific enolase (NSE), endothelin-1 (ET-1), cardiac troponin-I (cTnI), cytokeratin-19 fragment (CYFRA-21-1), etc. and the mixture of all with CEA. It was observed that compared to the control sample the magnitude of peak current changes slightly with the addition of various interferents. However, the peak current intensity was found to decrease significantly with the addition of the target CEA antigen ascribed to the specific antigen-antibody interactions. In a similar manner, the peak current decreased considerably with the mixture of all interferants, demonstrating the high selectivity of designed sensing platform for CEA detection (Figure 4d).
[061] Numerous modifications and adaptations of the system of the present invention will be apparent to those skilled in the art, and thus it is intended by the appended claims to cover all such modifications and adaptations which fall within the true spirit and scope of this invention.

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, Claims:WE CLAIM:
1. MXene graphene nanohybrid thin film based electrochemical biosensors and its method of preparation comprises air-brush spray coated amine functionalized graphene (f-graphene) and Ti3C2-MXene nanohybrid on indium tin oxide (ITO) coated glass substrate based immunosensing platform for detection of lung cancer biomarker, and BSA/anti-CEA/f-graphene@Ti3C2-MXene/ITO immunoelectrode for carcinoembryonic antigen (CEA) detection wherein deposited thin films are immobilized with monoclonal anti-CEA antibodies followed by blocking of non-specific binding sites with bovine serum albumin (BSA).
2. The MXene graphene nanohybrid thin film based electrochemical biosensors, as claimed in claim 1, wherein the analyte can be a biomarker for cancer, viral infection, bacterial infection, neurological disease detection, organ monitoring, environmental toxin, food adulteration/toxins.
3. The MXene graphene nanohybrid thin film based electrochemical biosensors, as claimed in claim 1, wherein the deposited material can be metal oxide (Titanium oxide, zirconium oxide, hafnium oxide, molybdenum oxide), metal nanoparticles (Gold nanoparticles, silver nanoparticles, platinum nanoparticles), carbon nanomaterials (carbon nanotubes, graphene, graphene quantum dots, carbon dots), nanofibers, 2D materials (Ti3C2-MXene, graphene, molybdenum sulphide) and combinations/nanohybrid/nanocomposites thereof.
4. The MXene graphene nanohybrid thin film based electrochemical biosensors, as claimed in claim 1, wherein the substrate can be Indium tin oxide (ITO) coated glass, screen printed electrodes (SPE), Glassy carbon electrodes (GCE), Interdigitated electrodes (IDE), Flexible substrate (Kapton Polyimide films), Metal sheets/tape.
5. The MXene graphene nanohybrid thin film based electrochemical biosensors, as claimed in claim 1, wherein the antibodies are covalently immobilized to the uniform thin-films of the deposited nanomaterial.
6. The MXene graphene nanohybrid thin film based electrochemical biosensors, as claimed in claim 1, wherein the non-specific sites are blocked by bovine serum albumin (BSA).
7. The MXene graphene nanohybrid thin film based electrochemical biosensors, as claimed in claim 1, wherein the Fabrication of electrochemical biosensing platform for CEA detection includes following steps:
? deposit the thin films of f-graphene@Ti3C2-MXene nanohybrid on indium tin oxide (ITO) coated glass substrate using airbrush spray coating technique.
? The ITO coated glass substrate was heated over the controlled temperature of 100 ºC for 10 min.
? Airbrush along with a pneumatic pump used for the deposition of a fine, small-diameter spray of f-graphene@Ti3C2-MXene nanohybrid on pre-heated ITO coated glass substrate and when connected to an air compressor, the airbrush breaks the solution liquid into tiny droplets, called as atomization which produces seamless blends, coatings, and gradient over the substrate maintaining airflow at 7.5 L/min over a maximum air pressure of 25 psi.
? f-graphene@Ti3C2-MXene nanohybrid is biofunctionalized to fabricate the biosensing platform.
? The carboxyl groups present in monoclonal anti-CEA antibodies are animated by preparing a uniform solution having 0.05 M NHS, 50 µg mL–1 of anti-CEA, and 0.2 M EDC in 1:2:1 ratio.
? The obtained anti-CEA solution was added to same volume of f-graphene@Ti3C2-MXene nanohybrid solution.
? The resultant mixture was incubated under humid conditions for the next 2 h.
? The unbound antibodies are removed by washing with phosphate buffered saline (PBS) solution.
? Bovine serum albumin (BSA) is used to block the non-specific sites present in the fabricated platform, thereby proving BSA/anti-CEA/f-graphene@Ti3C2-MXene/ITO immunoelectrode.
8. The MXene graphene nanohybrid thin film based electrochemical biosensors, as claimed in claim 7, wherein the airbrush spray gun uses compressed air from a nozzle to atomize the desired solution into a controlled manner and the spray nozzle operates by impinging high-velocity turbulent air on the surface of filaments or films of liquid, causing them to collapse to droplets with a wide range of sizes.