A nanozyme based lateral flow assay and device

Abstract

The present invention provides a kit, a device and an in-vitro assay based on a nanozyme-based lateral flow assay utilizing para-phenylenediamine of optimal concentration 0.1mM to 10mM as a chromogenic substrate for catalytically active aptamer-conjugated gold-nanozyme, which enhances sensitivity. The chromogenic substrate produces a robust brown color upon oxidation by catalytically active nanomaterial. The brown color remains stable for weeks and presents high contrast against the background. This overcomes the limitations of ordinary LFA in detecting analytes present at critical levels that are below microgram per milliliter, often resulting in false negative results. This provides a versatile and high-sensitive point-of-care diagnostic technique for rapid screening of infectious disease antigens and the detection of biomarkers for diseases such as cancer, cardiac and neurodegenerative diseases.

Description

[0001] A NANOZYME BASED LATERAL FLOW ASSAY AND DEVICE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of point-of-care tests. In particular, the present invention relates to a lateral flow assay device for detecting a target analyte in a sample, a method for detecting a target analyte in the sample and a kit thereof capable of detecting target analytes at very low concentrations and has the ability of quantification of analytes resulting in identification of presence of pathogens or an ailment / disease.

[0004] BACKGROUND OF THE INVENTION

[0005] Screening of disease antigens and the detection of biomarkers for various diseases, such as cancer, infectious diseases, cardiac diseases and neurodegenerative diseases needs to be done in a timely manner, for effective patient management and timely disease control. A diagnostic technique used for rapid screening of infectious disease antigens is Lateral Flow Assay (LFA). LFA is a versatile point-of-care diagnostic technique used in medical diagnostic field, but the LFA is mainly considered a primary screening test and requires secondary confirmatory laboratory tests such as Enzyme-linked immunosorbent assay (ELISA) or Polymerase Chain Reaction (PCR) to verify the disease.

[0006] A drawback of LFA is detection of analytes present at critical levels that are below microgram per milliliter, often resulting in false negative results. Further, LFA is considered to have limited clinical transition, due to its lower specificity compared to that of standard laboratory techniques. Quantitative detection of analytes in biological samples is a crucial parameter for patient detection and clinical care. The poor signal output from LFA makes it less sensitive and non-quantitative, further limiting its application in clinical practice.

[0007] To overcome this issue and to increase sensitivity and signal output, various signal amplification techniques have been integrated with the LFA technique. One of the effective method is a nanozyme-based LFA, which utilizes the inherent catalytic property of nanomaterials to produce a strong output signal that is quantifiable. Therefore, the nanozyme- based LFA has gained significant impact in the development of point-of-care diagnostic techniques for the detection of a wide range of bioanalytes present in very low concentrations (nanogram level) in clinical samples. Integration of signal amplification techniques such as nanozyme-based LFA can significantly enhance the sensitivity and signal output of LFA, making it a promising detection tool for detecting various diseases or analytes at an early stage. Further research and development in this area could lead to the widespread use of LFA in clinical practice, improving patient outcomes and disease control.

[0008] Nanozyme-based lateral flow assay employs catalytically active nanoparticles and a chromogenic substrate to enhance the colorimetric signal in the test line and sensitivity. Traditionally, TMB (3,3',5,5'-tetramethylbenzidine) and DAB (diaminobenzidine) have been used as common chromogenic substrates that generate a coloured product upon oxidation by peroxidase enzymes such as horseradish peroxidase (HRP) and catalytically active nanoparticles.

[0009] EP3080272B1 discloses use of aptamer-conjugated mesoporous silica particles as catalytically active nanomaterial in LFA, which utilizes TMB as a chromogenic substrate. However, TMB as a chromogenic substrate for catalytically active nanomaterials in LFA applications is limited due to low colour contrast onto the test line, degradation of colour over time, and requirement of slightly acidic conditions and a dark environment to yield maximum products.

[0010] Some of the state-of-the-art in this area include research papers and patents that have been granted on catalytically active nanomaterials for the development of LFA kits with higher sensitivity. For instance, Kidwell et al., (US20180052153A1) developed colloidal palladium nanoparticles as a catalytically active nanomaterial for LFA applications. Veli et al., (EP3080272B 1) worked on aptamer conjugated and signal molecule loaded silica particle used as a nanozyme to increase the lateral flow assay sensitivities. In another study, Cheng et al., [Nanozyme-Mediated Dual Immunoassay Integrated with Smartphone for Use in Simultaneous Detection of Pathogens., Nan Cheng, Yang Song, Mohamed M. A. Zeinhom, Yu-Chung Chang, Lina Sheng, Haolin Li, Dan Du, Lei Li, Mei-Jun Zhu, Yunbo Luo, Wentao Xu, and Yuehe Lin, ACS Appl. Mater. Interfaces 2017, 9, 46, 40671-40680] employed palladium and platinum core-shell nanostructures as a signal enhancement nanozyme in the LFA for the precious detection of pathogens. However, very limited studies have been conducted on chromogenic substrates to yield maximum sensitivity in LFA.

[0011] The previous studies in the art are focused on introducing new catalytically active nanomaterials. Therefore, there is a need in the art to develop more sensitive and accurate LFA kits for the detection of various diseases. OBJECTIVE OF THE INVENTION

[0012] An object of the present invention is to develop a highly sensitive nanozyme -based lateral flow assay device for detecting a target analyte in a sample.

[0013] Another object of the present invention is to develop a highly sensitive method for detecting a target analyte in a sample.

[0014] Yet another object of the present invention is to develop a highly sensitive kit for detecting a target analyte in a sample.

[0015] Still another object of the present invention is to employ para-phenylenediamine (PPD) as a chromogenic substrate for the development of highly sensitive nanozyme -based LFA device.

[0016] Another object of the present invention is to provide a versatile point-of-care device that can rapidly screen antigens / microbes and detect biomarkers for various diseases, including cancer, cardiac disease and neurodegenerative diseases.

[0017] Yet another object of the present invention is to provide a LFA device that overcomes the limitation of detecting analytes present at critical levels that are below microgram per millilitre, avoiding false negative results.

[0018] An object of the present invention is to develop a nanozyme-based lateral flow assay device and kit with strong output signal that is quantifiable.

[0019] Still another object of the present invention is to provide a nanozyme-based lateral flow assay that employs catalytically active nanoparticles and a chromogenic substrate for enhanced colorimetric signal in the test line and sensitivity.

[0020] Yet another object of the present invention is to develop a chromogenic substrate-utilising nanozyme-based LFA that is not limited in applications due to low contrast of colour, degradation of colour over time and the requirement of conditions such as slight acidity and dark environment to yield maximum products.

[0021] Another object of the present invention is to overcome the problems with the currently available commercial lateral flow assay kits which are less sensitive towards analytes.

[0022] These and other objects and advantages of the present subject matter will be apparent to a person skilled in the art after consideration of the following detailed description taking into consideration accompanying drawings in which preferred embodiments of the present subject matter are illustrated. SUMMARY OF THE INVENTION

[0023] The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the present invention. It is not intended to identify the key / critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concept of the invention in a simplified form as a prelude to a more detailed description of the invention presented later.

[0024] An aspect of the present invention provides a lateral flow assay device for detecting a target analyte (13) in a sample (14) consisting of a test strip (1) having a top surface (11) and a bottom surface (12), wherein the test strip (1) comprises: a) a sample pad (2) disposed on the top surface (11) of the test strip (1), adapted to receive said sample (14); b) a test zone (17) disposed on the top surface (11) of the test strip (1) and downstream from the sample pad (2) comprising:

[0025] (i) a conjugate pad (3) with a nanozyme (15) placed on the surface of the conjugate pad (3) for capturing the target analyte (13); said nanozyme (15) comprises a first binding molecule (5) conjugated with a detectable reporter molecule (4),

[0026] (ii) a flow membrane (6) comprising a test spot (8) with a second binding molecule (7) immobilized on the test spot (8); c) an absorbent pad (9) on the top surface (11) of the test strip (1), and downstream from the test zone (17) adapted to absorb excess fluid; wherein a flow path (10) is defined on the top surface (11) of the test strip (1) extending from the sample pad (2) to the absorbent pad (9), wherein the target analyte (13) binds with the nanozyme (15) to form a first analyte-nanozyme complex which is further captured by the second binding molecule to form a second analyte- nanozyme complex on the test spot (8), wherein the second-nanozyme complex reacts with para-phenylenediamine along with an activator (16) to give a dark brown-coloured product on the test spot (8) confirming the presence of target analyte (13).

[0027] Another aspect of the present invention comprises an in-vitro method for detecting a target analyte (13) in a sample (14), the method comprising the steps of: i. adding the sample (14) onto a sample pad (2) of the lateral flow assay device; ii. allowing the sample to migrate along the flow path (10) of the device; iii. adding a chromogenic substrate along with an activator (16) to the test spot (8); and iv. observing a change in intensity of the colour at the test spot (8) after 5-10 minutes; wherein appearance of a dark brown-colour at the test spot (8) indicates the presence of the target analyte (13) in the sample (14); wherein the chromogenic substrate is para-phenylenediamine at a concentration in the range of 0.1 mM to 10 mM, wherein the activator is hydrogen peroxide at a concentration in the range of 10 mM to 100 mM.

[0028] Yet another aspect of the present invention provides a lateral flow assay kit for detecting a target analyte in a sample comprising: a. the lateral flow assay device; b. para-phenylenediamine; c. an activator; and d. an instructions manual.

[0029] These and other aspects of the disclosed subject matter, as well as additional novel features, will be apparent from the description provided herein. The intent of this summary is not to be a comprehensive description of the claimed subject matter, but rather to provide a short overview of some of the subject matter's functionality. Other systems, methods, features and advantages here provided will become apparent to one with skill in the art upon examination of the following figures and detailed description. It is intended that all such additional systems, methods, features and advantages that are included within this description, be within the scope of any claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] It is to be noted, however, that the appended drawings illustrate only typical embodiments of the present subject matter and are therefore not to be considered for limiting its scope, for the invention may admit to other equally effective embodiments. The detailed description is described with reference to the accompanying figures. Some embodiments of system or methods in accordance with embodiments of the present subject matter are now described, by way of example, and with reference to the accompanying figures, in which:

[0031] Figure 1 depicts components of a lateral flow assay (LFA) device consisting a test strip(l) having a sample pad(2), conjugate pad(3), flow membrane(6) and an absorbent pad(9).

[0032] Figure 2(A) is a figure representing the working mechanism of the lateral flow assay device and demonstration of sample collection, flow direction of target analyte, the nanozyme and second binding molecule generating a low contrast signal in the test spot.

[0033] Figure 2(B) depicts the signal enhancement of the method for detecting a target analyte using a chromogenic substrate (PPD) with an activator and the generation of high-contrast signal after 5 minutes.

[0034] Figure 3 shows an image of comparison between lateral flow assay device before and after amplification while detecting Covid 19.

[0035] Figure 4 shows the comparison of absorbance spectra for gold nanoparticles (GNP), PPD, and the combination of GNP, PPD, and H2O2.

[0036] Figure 5 shows the optimization of catalytic activity of gold nanoparticles at various concentrations (1 to 10 nM) with a fixed concentration of PPD (5 mM) and H2O2 (50 mM).

[0037] Figure 6 shows the optimization of PPD concentration on a nitrocellulose membrane, with insets displaying photographs of the nitrocellulose membrane.

[0038] Figure 7 shows the photograph of Amyloid beta 42 detection in a lateral flow assay at various concentrations, using PPD as a chromogenic substrate.

[0039] Figure 8 shows the photograph of Amyloid beta 40 detection in a lateral flow assay at various concentrations, using PPD as a chromogenic substrate. DETAILED DESCRIPTION OF THE INVENTION

[0040] A detailed description of various exemplary embodiments of the disclosure is described herein. It should be noted that the embodiments are described herein in such details as to communicate the disclosure. However, the amount of details provided herein is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure.

[0041] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong. It will be further understood that terms, e.g., those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0042] The terminology used herein is to describe particular embodiments only and is not intended to be limiting to example embodiments. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context indicates otherwise. It will be further understood that the terms “comprise”, “comprising”, “includes” and / or “including” when used herein, specify the presence of stated features, steps, operations, elements and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components and / or groups thereof.

[0043] The term ‘further’ is used in the embodiments and claims of the present application. The said term is a well-accepted term to narrow down any principal feature. Therefore, the person skilled in the art would clearly understand the scope of the said term in the context of the present disclosure.

[0044] As used herein, the term ‘aptamer’, ‘aptamer molecule’, ‘capture aptamer molecule’ refers to a single- stranded oligonucleotide molecule, typically composed of DNA or RNA, that folds into a specific three-dimensional structure capable of binding to a target molecule with high affinity and specificity. The aptamer as used herein consists of 50 to 70 base pairs of nucleotides and serves as a biorecognition element.

[0045] As used herein, the term ‘test strip’ refers to a disposable device containing binding molecules and at least one test zone. As used herein, the term ‘target analyte’ refers to a specific substance or molecule of interest that is being detected or measured in a sample. They may be an antigen, a nucleic acid, a protein, a bacterial cell, a viral particle, a fungal cell, a lipid, a peptide, or a carbohydrate.

[0046] As used herein, the term ‘chromogenic substrate’ refers to a chemical compound that undergoes a reaction to produce a coloured product. This coloured product can then be detected visually or measured instrumentally.

[0047] As used herein, the term ‘nanozymes’ refers to nanomaterials with enzyme-like activity. These materials can catalyse specific chemical reactions, mimicking the function of natural enzymes.

[0048] As used herein, the term ‘immobilized’ refers to a molecule, particle, or component that is restricted in its movement within a system by attachment to a solid surface, such as a bead, membrane, or matrix.

[0049] As used herein, the term ‘oxidation’ refers to a chemical reaction that involves a loss of electrons from a molecule, atom, or ion. This loss of electrons is often accompanied by a gain of oxygen from the surrounding environment. However, oxidation can also occur without the direct involvement of oxygen, as long as there’s a loss of electrons.

[0050] As used herein, the term ‘biorecognition’ refers to the specific interaction between a biomolecule and a target molecule. This interaction involves highly specific recognition and binding mechanisms, typically mediated by complementary structures and functionalities on the interacting molecules.

[0051] As used herein, ‘gold nanoparticles’ refers to colloidal suspension of nanoparticles of gold in water. The suspension of spherical gold nanoparticles of size below 100 nm is usually wine red in colour.

[0052] As used herein, ‘sample’ as used herein refers to a broad range of materials that may be suspected of containing a target analyte. This can include, but is not limited to blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid (CSF), synovial fluid, fecal matter, water, soil, air filters, food extracts, nasal swabs, throat swabs, wound swabs, biopsies, surgical specimens, vaginal fluid and semen.

[0053] The present invention is directed towards a lateral flow assay device for detecting a target analyte (13) in a sample (14) consisting of a test strip (1) having a top surface (11) and a bottom surface (12), wherein the test strip (1) comprises: a) a sample pad (2) disposed on the top surface (11) of the test strip (1), adapted to receive said sample (14); b) a test zone (17) disposed on the top surface (11) of the test strip (1) and downstream from the sample pad (2) comprising:

[0054] (i) a conjugate pad (3) with a nanozyme (15) placed on the surface of the conjugate pad (3) for capturing the target analyte (13); said nanozyme (15) comprises a first binding molecule (5) conjugated with a detectable reporter molecule (4),

[0055] (ii) a flow membrane (6) comprising a test spot (8) with a second binding molecule (7) immobilized on the test spot (8); c) an absorbent pad (9) on the top surface (11) of the test strip (1), and downstream from the test zone (17) adapted to absorb excess fluid; wherein a flow path (10) is defined on the top surface (11) of the test strip (1) extending from the sample pad (2) to the absorbent pad (9), wherein the target analyte (13) binds with the nanozyme (15) to form a first analyte-nanozyme complex which is further captured by the second binding molecule to form a second analyte- nanozyme complex on the test spot (8), wherein the second-nanozyme complex reacts with para-phenylenediamine along with an activator (16) to give a dark brown-coloured product on the test spot (8) confirming the presence of target analyte (13).

[0056] In an embodiment of the present invention there is provided a lateral flow assay device, wherein the activator is hydrogen peroxide.

[0057] In another embodiment of the present invention there is provided a lateral flow assay device, wherein the flow membrane (6) is made of nitrocellulose.

[0058] In yet another embodiment of the present invention there is provided a lateral flow assay device, wherein the sample pad (2) and absorbent pad (9) are each made of cellulose fibers.

[0059] In still another embodiment of the present invention there is provided a lateral flow assay device, wherein the conjugated pad (3) is made of glass fibers.

[0060] In still another embodiment of the present invention there is provided a lateral flow assay device, wherein the first binding molecule (5) and the second binding molecule (7) are each independently selected from a group consisting of an aptamer, an antibody, a peptide, an enzyme, a nucleic acid, a carbohydrate, a lipid, a hormone and a protein.

[0061] In an embodiment of the present invention there is provided a lateral flow assay device, wherein the first binding molecule (5) is selected from a group consisting of an aptamer, an antibody, a peptide, an enzyme, a nucleic acid, a carbohydrate, a lipid, a hormone and a protein.

[0062] In another embodiment of the present invention there is provided a lateral flow assay device, wherein the second binding molecule (7) is selected from a group consisting of an aptamer, an antibody, a peptide, an enzyme, a nucleic acid, a carbohydrate, a lipid, a hormone and a protein.

[0063] In yet another embodiment of the present invention there is provided a lateral flow assay device, wherein the first binding molecule (5) and the second binding molecule (7) are each an aptamer.

[0064] In still another embodiment of the present invention there is provided a lateral flow assay device, wherein the first binding molecule (5) and the second binding molecule (7) are each an antibody.

[0065] In an embodiment of the present invention there is provided a lateral flow assay device, wherein the first binding molecule (5) and the second binding molecule (7) are each a peptide.

[0066] In another embodiment of the present invention there is provided a lateral flow assay device, wherein the first binding molecule (5) and the second binding molecule (7) are each an enzyme.

[0067] In yet another embodiment of the present invention there is provided a lateral flow assay device, wherein the first binding molecule (5) and the second binding molecule (7) are each a nucleic acid.

[0068] In still another embodiment of the present invention there is provided a lateral flow assay device, wherein the first binding molecule (5) and the second binding molecule (7) are each a carbohydrate.

[0069] In an embodiment of the present invention there is provided a lateral flow assay device, wherein the first binding molecule (5) and the second binding molecule (7) are each a lipid.

[0070] In another embodiment of the present invention there is provided a lateral flow assay device, wherein the first binding molecule (5) and the second binding molecule (7) are each a hormone.

[0071] In yet another embodiment of the present invention there is provided a lateral flow assay device, wherein the first binding molecule (5) and the second binding molecule (7) are each a protein. In still another embodiment of the present invention there is provided a lateral flow assay device, wherein the detectable reporter molecule (4) is selected from a group consisting of gold nanoparticle, carbon nanoparticle, composite nanoparticles, palladium nanoparticles, cellulose beads and quantum dots.

[0072] In an embodiment of the present invention there is provided a lateral flow assay device, wherein the detectable reporter molecule (4) is gold nanoparticle.

[0073] In another embodiment of the present invention there is provided a lateral flow assay device, wherein the target analyte (13) is selected from a group consisting of an antigen, a nucleic acid, a protein, a bacterial cell, viral particle, a fungal cell, a lipid and a carbohydrate.

[0074] In yet another embodiment of the present invention there is provided a lateral flow assay device, wherein the target analyte (13) is an antigen.

[0075] In still another embodiment of the present invention there is provided a lateral flow assay device, wherein the target analyte (13) is a nucleic acid.

[0076] In an embodiment of the present invention there is provided a lateral flow assay device, wherein the target analyte (13) is a protein.

[0077] In another embodiment of the present invention there is provided a lateral flow assay device, wherein the target analyte (13) is a bacterial cell.

[0078] In yet another embodiment of the present invention there is provided a lateral flow assay device, wherein the target analyte (13) is a viral particle.

[0079] In still another embodiment of the present invention there is provided a lateral flow assay device, wherein the target analyte (13) is a fungal cell.

[0080] In an embodiment of the present invention there is provided a lateral flow assay device, wherein the target analyte (13) is a lipid.

[0081] In another embodiment of the present invention there is provided a lateral flow assay device, wherein the target analyte (13) is a carbohydrate.

[0082] In yet another embodiment of the present invention there is provided a lateral flow assay device, wherein the sample (14) is selected from a group consisting of blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, synovial fluid, fecal matter, water, soil, air filter, food extract, nasopharyngeal swab, throat swab, wound swab, tissue, a tumor cell, vaginal fluid and semen. In an embodiment of the present invention there is provided a lateral flow assay device, wherein the sample (14) is nasopharyngeal swab.

[0083] In another embodiment of the present invention there is provided a lateral flow assay device, wherein the sample (14) is plasma.

[0084] In still another embodiment of the present invention there is provided a lateral flow assay device, wherein the device has a limit of detection of 0.2 nanograms of target analyte (13) per 100 microliters of the sample (14).

[0085] Another embodiment of the present invention provides an in-vitro method for detecting a target analyte (13) in a sample (14), the method comprising the steps of: i. adding the sample (14) onto a sample pad (2) of the lateral flow assay device; ii. allowing the sample to migrate along the flow path (10) of the device; iii. adding a chromogenic substrate along with an activator (16) to the test spot (8); and iv. observing a change in intensity of the colour at the test spot (8) after 5-10 minutes; wherein appearance of a dark brown-colour at the test spot (8) indicates the presence of the target analyte (13) in the sample (14); wherein the chromogenic substrate is para-phenylenediamine at a concentration in the range of 0.1 mM to 10 mM, wherein the activator is hydrogen peroxide at a concentration in the range of 10 mM to 100 mM.

[0086] In an embodiment of the present invention there is provided an in-vitro method for detecting a target analyte (13) in a sample, wherein the chromogenic substrate is para-phenylenediamine at a concentration in the range of 2.5 mM to 10 mM.

[0087] In still another embodiment of the present invention there is provided an in-vitro method for detecting a target analyte (13) in a sample, wherein the chromogenic substrate is para- phenylenediamine at a concentration of 5 mM.

[0088] In another embodiment of the present invention there is provided an in-vitro method for detecting a target analyte (13) in a sample, wherein the activator is hydrogen peroxide at a concentration of 50 mM. In yet another embodiment of the present invention there is provided an in-vitro method for detecting a target analyte (13) in a sample, wherein the target analyte (13) is selected from a group consisting of an antigen, a nucleic acid, a protein, a bacterial cell, viral particle, a fungal cell, a lipid and a carbohydrate.

[0089] In still another embodiment of the present invention there is provided an in-vitro method for detecting a target analyte (13) in a sample, wherein the sample (14) is selected from a group consisting of blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, synovial fluid, fecal matter, water, soil, air filters, food extracts, nasopharyngeal swab, throat swab, wound swab, tissue, a tumor cell, vaginal fluid and semen.

[0090] In an embodiment of the present invention there is provided an in-vitro method for detecting a target analyte (13) in a sample, wherein the method has a limit of detection of 0.2 nanograms of target analyte (13) per 100 microliters of the sample (14).

[0091] Yet another embodiment of the present invention provides an in-vitro method for detecting a target analyte (13) in a sample (14), the method comprising the steps of: i. adding the sample (14) onto a sample pad (2) of the lateral flow assay device; ii. allowing the sample to migrate along the flow path (10) of the device; iii. adding a chromogenic substrate along with an activator (16) to the test spot (8); and iv. observing a change in intensity of the colour at the test spot (8) after 5-10 minutes; wherein appearance of a dark brown-colour at the test spot (8) indicates the presence of the target analyte (13) in the sample (14); wherein the chromogenic substrate is para-phenylenediamine at a concentration in the range of 2.5 mM to 10 mM, wherein the activator is hydrogen peroxide at a concentration of 50 mM.

[0092] Another embodiment of the present invention provides an in-vitro method for detecting a target analyte (13) in a sample (14), the method comprising the steps of: i. adding the sample (14) onto a sample pad (2) of the lateral flow assay device; ii. allowing the sample to migrate along the flow path (10) of the device; iii. adding a chromogenic substrate along with an activator (16) to the test spot (8); and iv. observing a change in intensity of the color at the test spot (8) after 3-6 minutes; wherein appearance of a dark brown-color at the test spot (8) indicates the presence of the target analyte (13) in the sample (14); wherein the chromogenic substrate is para-phenylenediamine at a concentration of 5 mM, wherein the activator is hydrogen peroxide at a concentration of 50 mM.

[0093] Still another embodiment of the present invention provides a lateral flow assay kit for detecting a target analyte in a sample comprising: a. the lateral flow assay device; b. para-phenylenediamine; c. an activator; and d. an instructions manual.

[0094] In still another embodiment of the present invention there is provided a lateral flow assay kit, wherein the activator is hydrogen peroxide.

[0095] In another embodiment of the present invention there is provided a lateral flow assay kit, wherein para-phenylenediamine is at a concentration in the range of 0.1 mM to 10 mM.

[0096] In yet another embodiment of the present invention there is provided a lateral flow assay kit, wherein para-phenylenediamine is at a concentration of 5 mM.

[0097] In an embodiment of the present invention there is provided a lateral flow assay kit, wherein the activator is at a concentration in the range of 10 mM to 100 mM.

[0098] In another embodiment of the present invention there is provided a lateral flow assay kit, wherein the activator is at a concentration of 50 mM.

[0099] In an embodiment of the present invention there is provided a lateral flow assay kit, wherein the target analyte is selected from a group consisting of an antigen, a nucleic acid, a protein, a bacterial cell, viral particle, a fungal cell, a lipid and a carbohydrate.

[0100] In another embodiment of the present invention there is provided a lateral flow assay kit, wherein the sample is selected from a group consisting of blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, synovial fluid, fecal matter, water, soil, air filters, food extracts, nasopharyngeal swab, throat swab, wound swab, tissue, a tumor cell, vaginal fluid and semen. Still another embodiment of the present invention provides an in-vitro method for detecting a target analyte (13) in a sample (14), the method comprising the steps of: i. adding the sample (14) onto a sample pad (2) of the lateral flow assay device; ii. allowing the sample to migrate along the flow path (10) of the device; iii. adding a chromogenic substrate along with an activator (16) to the test spot (8); and iv. observing a change in intensity of the colour at the test spot (8) after 5-10 minutes; wherein appearance of a dark brown-colour at the test spot (8) indicates the presence of the target analyte (13) in the sample (14); wherein the chromogenic substrate is para-phenylenediamine at a concentration in the range of 0.1 mM to 10 mM, wherein the activator is hydrogen peroxide at a concentration in the range of 10 mM to 100 mM, wherein the lateral flow assay device consists of a test strip (1) having a top surface (11) and a bottom surface (12), comprising: a) a sample pad (2) disposed on the top surface (11) of the test strip (1), adapted to receive said sample (14); b) a test zone (17) disposed on the top surface (11) of the test strip (1) and downstream from the sample pad (2) comprising:

[0101] (i) a conjugate pad (3) with a nanozyme (15) placed on the surface of the conjugate pad (3) for capturing the target analyte (13); said nanozyme (15) comprises a first binding molecule (5) conjugated with a detectable reporter molecule (4),

[0102] (ii) a flow membrane (6) comprising a test spot (8) with a second binding molecule (7) immobilized on the test spot (8); c) an absorbent pad (9) on the top surface (11) of the test strip (1), and downstream from the test zone (17) adapted to absorb excess fluid; wherein a flow path (10) is defined on the top surface (11) of the test strip (1) extending from the sample pad (2) to the absorbent pad (9), wherein the target analyte (13) binds with the nanozyme (15) to form a first analyte - nanozyme complex which is further captured by the second binding molecule to form a second analyte-nanozyme complex on the test spot (8), wherein the second-nanozyme complex reacts with para-phenylenediamine along with an activator (16) to give a dark brown-coloured product on the test spot (8) confirming the presence of target analyte (13).

[0103] In accordance with the present invention, there is provided a point-of-care lateral flow assay (LFA) device, method and a kit utilizing nanozymes to enhance the sensitivity of the device and assay. The components of the LFA test strip broadly include (i) a sample pad, (ii) a conjugate pad, (iii) a flow membrane comprising a test spot, and (iv) an absorbent pad.

[0104] Figure 1 depicts components of a lateral flow assay (LFA) device consisting of a test strip having a sample pad, a conjugate pad, a flow membrane and an absorbent pad. The lateral flow assay device consists of a test strip (1) having a top surface (11) and a bottom surface (12). The test strip (1) comprises: a sample pad (2) disposed on the top surface (11), a test zone (17) downstream from the sample pad (2); and an absorbent pad (9) downstream from the test zone (17). The test zone (17) comprises a conjugate pad (3) and a flow membrane (6). The conjugate pad comprises a first binding molecule (5) conjugated with a detectable reporter molecule (4) [termed as nanozyme (15)] placed on the pad surface; and the flow membrane (6) comprises a second binding molecule (7) immobilized on a test spot (8).

[0105] Figure 2(A) represents the working mechanism of the lateral flow assay device and demonstration of sample collection, flow direction of target analyte, the nanozyme and second binding molecule generating a low contrast signal at the test spot. The sample (14) isolated from a subject may comprise the target analyte (13) which is to be detected. The sample (14) is added onto a sample pad (2) and allowed to run along the flow path (10) of the test strip (1). The target analyte (13) present in the sample (14) first binds with the nanozyme (15) placed on the conjugate pad (3) to form a first analyte-nanozyme complex which is further captured by the second binding molecule (7) to form a second analyte-nanozyme complex on the test spot (8) on the flow membrane (6).

[0106] Figure 2(B) depicts the signal enhancement of the method for detecting a target analyte using a chromogenic substrate (PPD) with an activator and the generation of high-contrast signal after 5 minutes. Once a second analyte-nanozyme complex is formed on the test spot (8) on the flow membrane (6), a chromogenic substrate along with an activator (16) is added to the test spot (8) where a second analyte-nanozyme complex conjugated with para-phenylenediamine and activator (18) is formed. A robust brown-coloured product is produced at the test spot (8) and the intensity of the colour produced on the test spot allows for quantification of the signal strength.

[0107] The device employs para-phenylenediamine (PPD) as a chromogenic substrate and its activator hydrogen peroxide to improve the sensitivity of the assay. PPD is an organic compound that, when oxidized by peroxidase enzymes, yields (3E,6E)-3,6-bis[(4-aminophenyl) imino] cyclohexa- l,4-diene-l,4-diamine (Bandrowski’s base), which is brown in colour. The device leverages second-nanozyme complex as catalytically active nanomaterial to facilitate the oxidation of PPD into a coloured product, while simultaneously acting as biorecognition elements to capture the target analyte.

[0108] In accordance with the present invention, the method for detecting a target analyte in a sample employing the device and kit comprises two steps: an initial sample running; and then sensitivity enhancement through the utilization of PPD.

[0109] The target analyte can be a molecule selected from an antigen, a nucleic acid, a protein, a lipid, a carbohydrate or a microorganism selected from a bacterial cell, viral particle, or a fungal cell or a cancerous cell. Two specific binding molecules are carefully chosen specific towards the target analyte. The binding molecules can be molecules selected from an aptamer, an antibody, a peptide, an enzyme, a nucleic acid, a carbohydrate, a lipid, a hormone or a protein. Both the binding molecules can be of the same category, for e.g. two aptamers having different sequences, two antibodies, two peptides, two enzymes, and such. The binding molecules can also be of different category of molecules, for e.g. first binding molecule being an antibody and the second being a nucleic acid, and such. These binding molecules may have two separate binding sites on opposite ends of the target analyte, to facilitate execution of a sandwich-type lateral flow assay (LFA).

[0110] The first binding molecule is conjugated to gold nanoparticles using the EDC / NHS (l-ethyl-3- (3-dimethylaminopropyl)-carbodiimide / N-hydroxysuccinimide) conjugation method, or any such method of conjugation known in the art depending on the nature of the binding molecule, creating a catalytically active nanozyme and a biorecognition element. The first binding molecule- nanozyme conjugates are loaded onto a conjugate matrix pad, dried, and the second binding molecule, is immobilized on the test zone to capture the complex formed by the analyte-first binding molecule-conjugated gold nanozyme. Subsequently, the test strip is assembled with a sample pad and an absorbent pad to facilitate the assay. In the presence of a target analyte in the sample, the first binding molecule -conjugated gold nanozyme captures the target analyte, which is further captured by the second binding molecule immobilized on the test spot.

[0111] The catalytic efficiency of the nanozyme is evaluated using PPD as a chromogenic substrate and hydrogen peroxide as an electron donor to activate the reaction. As the concentration of the nanozyme increases from 1 nM to 10 nM, a corresponding elevation in the intensity of the absorbance peak is observed. Additionally, the optimal concentration of PPD is evaluated in the presence of the nanozyme, with a range of 0.1 mM to 10 mM. Results indicate that a concentration of 2.5 mM to 10 mM of PPD yield the most favourable conditions for conducting nanozyme based signal enhancement in LFA. To amplify the signal and enhance sensitivity, 2 microliters of 5 mM PPD and 50 mM hydrogen peroxide is added onto the test zone. The reaction between PPD and the target analyte -binding molecule-conjugated gold nanozyme on the test spot generates a robust brown-coloured product as a visual signal output. The intensity of the colour produced on each test spot of the test zone allows for quantification of the signal strength. The oxidized product of the chromogenic substrate and catalytically active nanozymes is brown in colour, which remains stable for weeks, presents high contrast against the background and is measured at an absorbance of 460 nm.

[0112] The lateral flow assay (LFA) kit for detecting a target analyte in a sample has the following major components comprising: a. the lateral flow assay device; b. Para-phenylenediamine (PPD) as a chromogenic substrate; and c. hydrogen peroxide as an activator.

[0113] The kit is cost-effective and highly sensitive and the gold-nanozyme lateral flow kit is designed for detecting pathogens like SARS-CoV2 virus, Human Immunodeficiency virus and biomarkers for diseases such as cancer, cardiac disease, neurodegenerative disease etc.

[0114] The chromogenic substrate utilized in the kit is para-phenylenediamine at an optimal concentration in the range of 0.1 mM to 10 mM along with an activator, hydrogen peroxide at an optimal concentration in the range of 10 mM to 100 mM. The LFA kit has the potential for the detection of analytes present at critical levels that are below microgram per milliliter. The lateral flow assay LFA (kit) of the present invention has enhanced specificity and also ensures stability and cost effectiveness. The nanozyme-based LFA kit demonstrates a limit of detection of 0.2 nanograms per 100 microliters of samples during evaluation. The developed nanozyme based LFA kit enhances sensitivity levels comparable to standard laboratory techniques such as ELISA (nanogram to picogram) while being cost effective. The assay’s total running time is less than 30 minutes.

[0115] As one of the exemplary embodiments, in order to carry out the detection of a target analyte using the device and method of the present invention, the target analyte is chosen as a “covidl9 spike protein” (SEQ ID NO. 3). Two specific aptamers, namely aptamer 51 (SEQ ID NO. 1) and aptamer 67(SEQ ID NO. 2) are carefully chosen as the first binding molecule and the second binding molecule, respectively. These aptamers have binding sites on opposite ends of the target analyte, which facilitates the execution of a sandwich-type lateral flow assay (LFA).

[0116] Aptamer 51 (first binding molecule) is conjugated to gold nanoparticles using the EDC / NHS (l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide / N-hydroxysuccinimide) conjugation method, creating a catalytically active nanozyme and a biorecognition element. The catalytic efficiency of the nanozyme is evaluated using PPD as a chromogenic substrate and hydrogen peroxide as an electron donor to activate the reaction. The nanozyme conjugate is loaded onto conjugate pad made of glass fibres and dried. The aptamer-67 (the second binding molecule) is immobilized on a test spot on flow membrane made of nitrocellulose to capture the complex formed by the analyte- aptamer conjugated gold-nanozyme. Subsequently, the test strip is assembled with a sample pad and an absorbent pad made of fibres to facilitate the assay.

[0117] In the presence of covidl9 spike protein (SEQ ID no. 3), the aptamer-conjugated gold- nanozyme captures the protein, which is further captured by the aptamer-67 (SEQ ID NO. 2) immobilized on the test spot. To amplify the signal and enhance sensitivity, 2 microliters of 5 mM PPD and 50 mM hydrogen peroxide are added onto the test spot. The reaction between PPD and the aptamer-conjugated gold nanozyme on the test spot generates a robust browncoloured product as visual signal output after 5 -10 minutes of the addition of PPD and hydrogen peroxides. The intensity of the colour produced on each test spot of the test zone allows for quantification of the signal strength. EXAMPLES

[0118] Following examples are given by way of illustration, therefore, should not be construed to limit the scope of the invention.

[0119] EXAMPLE 1

[0120] PREPARATION OF LATERAL FLOW ASSAY DEVICE

[0121] For this experiment, the SARS-CoV-2 spike protein was selected as a target analyte. Two specific aptamer sequences, namely aptamer 51 (polynucleotide sequence as set forth in SEQ ID No. 1) and aptamer 67 (polynucleotide sequence as set forth in SEQ ID No. 2) were carefully chosen as the first binding molecule and the second binding molecule, respectively. These aptamers possess binding sites on opposite ends of the target analyte, i.e. SARS-CoV-2 spike protein (amino acid sequence as set forth in SEQ ID No. 3) [6VXX_l|Chains A, B, C|Spike glycoprotein] Severe acute respiratory syndrome coronavirus 2 (2697049]. Table 1 provides the sequences of SEQ ID Nos. 1-3.

[0122] 10 pL of lOpM aptamer 51 was conjugated to 1 ml of synthesized gold nanoparticles (having an average size of 15 nm) using the EDC / NHS (l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide / Nhydroxysuccinimide) conjugation method, creating a nanozyme, which served as a catalytically active nanozyme and biorecognition element. The aptamer-51 and gold- nanoparticle conjugate (nanozyme) were loaded onto the conjugate pad and dried. 1 pL of 10 pM aptamer-67 was immobilized at the test spot on the flow membrane.

[0123] The test strip was assembled with a sample pad made of cellulose fibres to receive the sample, a conjugated pad made of glass fibres coated with aptamer-51 conjugated gold nanoparticles to capture the analytes present in the sample, a flow membrane made of nitrocellulose immobilized with aptamer-67 to capture the aptamer-51 conjugated gold nanoparticle and analytes complex and an absorbent pad made of cellulose fibres to absorb the excess fluid to obtain the lateral flow assay device. This configuration constitutes the lateral flow assay device.

[0124] Table 1

[0125] EXAMPLE 2

[0126] ASSAY FOR DETECTION OF A TARGET ANALYTE

[0127] The assay procedure comprises two steps: (i) initial sample running; followed by (ii) sensitivity enhancement through the utilization of PPD.

[0128] 100 pL of nasopharyngeal swab sample prepared in phosphate buffered saline (containing 0.04% bovine serum albumin, 5 % sucrose, 0.01 % tween 20) was introduced on the sample pad of the lateral flow assay device and allowed to flow through the entire length of the device. The SARS-CoV-2 spike protein was captured by the aptamer-conjugated gold nanozyme placed at the conjugate pad, which was further captured by the aptamer 67 immobilized on the test zone. To amplify the signal and enhance sensitivity, 2 microliters of 5 mM PPD and 50 mM hydrogen peroxide were added onto the test zone. The reaction between PPD and the aptamer 67-conjugated gold nanoparticles on the test zone generated a robust brown-coloured product after 5 minutes as a visual signal output, which confirmed the presence of SARS-CoV- 2 spike protein.

[0129] Table 2 summarises the various aspects of the assay.

[0130] Table 2

[0131] EXAMPLE 3

[0132] CATALYTIC EFFICIENCY

[0133] The catalytic efficiency of the aptamer-conjugated gold nanozyme was assessed using PPD as a chromogenic substrate and hydrogen peroxide as an electron donor to activate the reaction. Initially, the absorbance spectrum of gold nanoparticles alone (10 nM) was measured, showing a maximum absorbance peak at 522 nm. The absorbance spectrum of PPD (5 mM) exhibited no significant absorbance peak. However, when PPD (5 mM) and H2O2 (50 mM) were incubated with gold nanoparticles (2 nM), the absorbance peak shifted from 522 nm to 450 nm, indicating the formation of the brown-colored Bandrowski’s base product. This shift confirms the catalytic efficiency of the gold nanoparticles in converting PPD into the brown-colored product Bandrowski’s base, which can be measured at 450 nm (Figure 4).

[0134] EXAMPLE 4

[0135] CATALYTIC ACTIVITY

[0136] The catalytic activity of the gold nanozyme was studied at various concentrations. As the concentration of the gold nanozyme increased from 1 nM to 10 nM with a fixed concentration of PPD (5 mM) and H2O2 (50 mM), a corresponding increase in the intensity of the absorbance peak was observed, indicating that the catalytic property is concentration-dependent (Figure 5).

[0137] EXAMPLE 5

[0138] To perform nanozyme-based signal enhancement in lateral flow assays (LFA), the optimal concentration of PPD on a nitrocellulose membrane was evaluated.

[0139] For this, 2 microliters of a fixed concentration of aptamer-conjugated gold nanozyme (1 nM) were drop-cast onto a nitrocellulose membrane and air-dried for 30 minutes. The membrane was then incubated with varying concentrations of PPD ranging from 0.1 mM to 12.5 mM. Results showed a gradual increase in colour intensity with PPD concentrations from 0.5 to 2.5 mM and a fixed concentration of H2O2 (50 mM). However, increasing the concentration above 5 mM did not result in a notable increase in signal intensity. Therefore, a concentration range of 2.5 to 10 mM was selected as optimal for conducting nanozyme-based signal enhancement in LFA (Figure 6).

[0140] EXAMPLE 6

[0141] DETECTION OF AMYLOID BETA 42 (Ap 42) USING LATERAL FLOW ASSAY DEVICE

[0142] The nanozymatic signal amplification method was also tested using Amyloid beta 42 (Ap 42), a biomarker of interest for Alzheimer’s disease diagnosis, as a target analyte.

[0143] For this, 10 pL of 10 pM monoclonal antibody specific to Ap 42 (Abeam, Product ID- ab201060) was conjugated to 1 mL of gold nanoparticles, with an average size of 15 nm, using the EDC / NHS conjugation method. The conjugated antibody was then purified and dispersed in 50 pL of phosphate -buffered saline containing 0.04% bovine serum albumin, 20% sucrose, and 0.1% Tween 20.

[0144] This conjugate, acting as a nanozyme, was loaded onto the conjugate pad in a volume of 10 micro litres and dried. Then, 1 pL of 10 pM polyclonal anti-Ap 42 antibody (Thermo Fisher Scientific, Product ID-44-344) was immobilized at the test spot on the flow membrane.

[0145] Ap 42 was diluted to different concentrations (0.1 to 100 ng / mL) in 100 pL of human blood plasma (collected from individuals aged over 60 from Thiruvananthapuram, Kerala, India) and mixed with 100 pL of phosphate-buffered saline (containing 0.04% bovine serum albumin, 5 % sucrose, 0.01 % tween 20) to prepare sample.

[0146] 100 pL of prepared sample was added onto the sample pad. The LFA strip was left undisturbed for 10 minutes. Subsequently, 1 pL of 5 mM PPD and 50 mM H2O2 was added to the test zone, and the results were visualized after 10 minutes of incubation. The results obtained are as provided in Figure 7. The results indicate detection of Ap 42 (target analyte) and the intensity of the brown-coloured product changed with respect to the concentration of Ap 42. EXAMPLE 7

[0147] DETECTION OF AMYLOID BETA 40 (Ap 40) USING LATERAL FLOW ASSAY

[0148] DEVICE

[0149] Another Alzheimer's disease-specific biomarker, Amyloid beta 40 (Ap 40), another target analyte, was also detected using this lateral flow assay device.

[0150] For this, 10 pL of 10 pM monoclonal antibody specific to Ap 40 (Abeam, Product ID-ab20068) was conjugated to 1 mL of gold nanoparticles, with an average size of 15 nm, using the EDC / NHS conjugation method. The conjugated antibody was then purified and dispersed in 50 pL of phosphate-buffered saline containing 0.04% bovine serum albumin, 20% sucrose, and 0.1% Tween 20.

[0151] This conjugate, acting as a nanozyme, was loaded onto the conjugate pad in a volume of 10 micro litres and dried. Then, 1 pL of 10 pM polyclonal anti-Ap 40 antibody (Thermo Fisher Scientific, Product ID-44-136) was immobilized at the test spot on the flow membrane.

[0152] Different concentrations (0.1 to 100 ng / mL) of Ap 40 prepared in human blood plasma (collected from individuals aged over 60 from Thiruvananthapuram, Kerala, India) were diluted in phosphate-buffered saline (containing 0.04% bovine serum albumin, 5 % sucrose, 0.01 % tween 20) in 1: 1 ratio by volume and added to the sample pad. After 10 minutes, 1 pL of 5 mM PPD and 50 mM H2O2 was added to the test zone, and the formation of the browncoloured product was visualized after 10 minutes of incubation. The results obtained are as provided in Figure 8. The results indicate detection of Ap 40 (target analyte) and the intensity of the brown-coloured product changed with respect to the concentration of Ap 40.

[0153] Although embodiments for the present subject matter have been described in language specific to package features, it is to be understood that the present subject matter is not necessarily limited to the specific features described. Rather, the specific features and methods are disclosed as embodiments for the present subject matter. Numerous modifications and adaptations of the system / device 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 scope of the present subject matter.

[0154] It will be further appreciated that functions or structures of a plurality of components or steps may be combined into a single component or step, or the functions or structures of one-step or component may be split among plural steps or components. The present invention contemplates all of these combinations. Unless stated otherwise, dimensions and geometries of the various structures depicted herein are not intended to be restrictive of the invention, and other dimensions or geometries are possible. In addition, while a feature of the present invention may have been described in the context of only one of the illustrated embodiments, such feature may be combined with one or more other features of other embodiments, for any given application. It will also be appreciated from the above that the fabrication of the unique structures herein and the operation thereof also constitute methods in accordance with the present invention. The present invention also encompasses intermediate and end products resulting from the practice of the methods herein.

[0155] LIST OF REFERENCE NUMERALS

[0156] 1- Test Strip

[0157] 2- Sample Pad

[0158] 3- Conjugate Pad

[0159] 4- Detectable reporter molecule

[0160] 5- First binding molecule

[0161] 6- Flow membrane

[0162] 7- Second binding molecule

[0163] 8- Test Spot

[0164] 9- Absorbent Pad

[0165] 10- Flow path

[0166] 11- Top Surface

[0167] 12- Bottom Surface

[0168] 13 - Target analyte

[0169] 14- Sample

[0170] 15- Nanozyme

[0171] 16- Chromogenic Substrate (para-phenylenediamine) with activator

[0172] 17- Test zone

[0173] 18- Second analyte-nanozyme complex conjugated with para-phenylenediamine and activator.

Claims

AMENDED CLAIMS received by the International Bureau on 11 February 2025 (11 .02.2025)WE CLAIM:

1. A lateral flow assay device for detecting a target analyte (13) in a sample (14) consisting of a test strip (1) having a top surface (11) and a bottom surface (12), wherein the test strip (1) comprises: a) a sample pad (2) disposed on the top surface (11) of the test strip (1), configured to receive said sample (14); b) a test zone (17) disposed on the top surface (11) of the test strip (1) and downstream from the sample pad (2) comprising:(i) a conjugate pad (3) with a nanozyme (15) placed on the surface of the conjugate pad (3) for capturing the target analyte (13); said nanozyme (15) comprises a first binding molecule (5) conjugated with a detectable reporter molecule (4),(ii) a flow membrane (6) comprising a test spot (8) with a second binding molecule (7) immobilized on the test spot (8); c) an absorbent pad (9) on the top surface (11) of the test strip (1), and downstream from the test zone (17) designed to absorb excess fluid to maintain consistent flow of the sample; wherein a flow path (10) is defined on the top surface (11) of the test strip(1) extending from the sample pad (2) to the absorbent pad (9), wherein the target analyte (13) binds with the nanozyme (15) to form a primary analyte-nanozyme complex which is further captured by the second binding molecule to form a secondary nanozyme complex consisting of the primary analyte-nanozyme complex and the second binding molecule on the test spot (8), wherein the secondary nanozyme complex rreeaaccttss wwiitthh paraphenylenediamine along with an activator (16) to give a dark brown-coloured product on the test spot (8) confirming the presence of target analyte (13).

2. The lateral flow assay device as claimed in claim 1, wherein the activator is hydrogen peroxide, wherein the flow membrane (6) is made of nitrocellulose,wherein the sample pad (2) and absorbent pad (9) are each made of cellulose fibers, wherein the conjugated pad (3) is made of glass fibers.

3. The lateral flow assay device as claimed in claim 1, wherein the first binding molecule (5) and the second binding molecule (7) are each selected from a group consisting of an aptamer, an antibody, a peptide, an enzyme, a nucleic acid, a carbohydrate, a lipid, a hormone and a protein; wherein the detectable reporter molecule (4) is selected from a group consisting of gold nanoparticle, carbon nanoparticle, composite nanoparticles, palladium nanoparticles, cellulose beads and quantum dots.

4. The lateral flow assay device as claimed in claim 1, wherein the target analyte (13) is selected from a group consisting of an antigen, a nucleic acid, a protein, a bacterial cell, viral particle, a fungal cell, a lipid and a carbohydrate, wherein the sample (14) is selected from a group consisting of blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, synovial fluid, fecal matter, water, soil, air filters, food extract, nasopharyngeal swab, throat swab, wound swab, tissue, a tumor cell, vaginal fluid and semen.

5. The lateral flow assay device as claimed in claim 1, wherein the device has a limit of detection of 0.2 nanograms of target analyte (13) per 100 microliters of the sample (14).

6. An in-vitro method for detecting a target analyte (13) in a sample (14), the method comprising the steps of: i. adding the sample (14) onto a sample pad (2) of the lateral flow assay device as claimed in claim 1 ; ii. allowing the sample to migrate along the flow path (10) of the device; iii. adding a chromogenic substrate along with an activator (16) to the test spot(8); and iv. observing a change in intensity of the colour at the test spot (8) after 5-10 minutes; wherein appearance of a dark brown-colour at the test spot (8) indicates the presence of the target analyte (13) in the sample (14);wherein the chromogenic substrate is para-phenylenediamine at a concentration in the range of 0.1 mM to 10 mM, wherein the activator is hydrogen peroxide at a concentration in the range of 10 mM to 100 mM.

7. The in-vitro method for detecting a target analyte (13) as claimed in claim 6, wherein the target analyte (13) is selected from a group consisting of an antigen, a nucleic acid, a protein, a bacterial cell, viral particle, a fungal cell, a lipid and a carbohydrate, wherein the sample (14) is selected from a group consisting of blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, synovial fluid, fecal matter, water, soil, air filters, food extract, nasopharyngeal swab, throat swab, wound swab, tissue, a tumor cell, vaginal fluid and semen.

8. The in-vitro method for detecting a target analyte (13) as claimed in claim 6, wherein the method has a limit of detection of 0.2 nanograms of target analyte (13) per 100 microliters of the sample (14).

9. A lateral flow assay kit for detecting a target analyte in a sample comprising: a. the lateral flow assay device as claimed in claim 1; b. para-phenylenediamine; c. an activator; and d. an instructions manual.

10. The lateral flow assay kit as claimed in claim 9, wherein the activator is hydrogen peroxide; wherein the target analyte is selected from a group consisting of an antigen, a nucleic acid, a protein, a bacterial cell, viral particle, a fungal cell, a lipid and a carbohydrate, wherein the sample is selected from a group consisting of blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, synovial fluid, fecal matter, water, soil, air filters, food extract, nasopharyngeal swab, throat swab, wound swab, tissue, a tumor cell, vaginal fluid and semen.STATEMENT UNDER ARTICLE 19(1)With reference to the Search Report and Written Opinion of the International Searching Authority [ISA / IN], we are suitably amending the claims to overcome the objection of clarity in the present invention.

1. Claim 1 has been amended to replace the term “adapted” with “configured” in (a) and with “designed” in (c). (c) in claim 1 has been further amended to specify that the absorbent pad is “designed to absorb excess fluid to maintain consistent flow of the sample” for more clarity. Claim 1 has also been amended to clarify first analyte - nanozyme complex as “primary analyte-nanozyme complex” and second analyte- nanozyme complex” as “secondary nanozyme complex consisting of the primary analyte-nanozyme complex and the second binding molecule”.

2. The term “independently” has been deleted from claim 3.

3. Any additions in the claims have been indicated by underline and any deletions in the claims have been indicated by strike through.The amended claims do not go beyond the disclosure of the international application as filed and have no impact on the description and drawings as filed.

Images