Lateral flow chromatography and device based on nano-enzyme

Abstract

The invention provides a nano-enzyme based lateral flow chromatography kit, a nano-enzyme based lateral flow chromatography device and in-vitro detection, and the lateral flow chromatography uses p-phenylenediamine with the optimum concentration of 0.1 mM to 10 mM as a chromogenic substrate of aptamer coupled gold nano-enzyme with catalytic activity, so that the sensitivity is improved. The chromogenic substrate generates stable brown color after being oxidized by the catalytic active nanometer material. The brown color can be kept stable for several weeks and forms high contrast with the background. This overcomes the limitation that detection of analytes present at critical levels below micrograms / milliliters by ordinary LFAs would typically result in false negative results. A universal and high-sensitivity instant detection and diagnosis technology is provided for rapid screening of infectious disease antigens and detection of biomarkers of diseases (such as cancers, heart diseases and neurodegenerative diseases).

Description

Technical Field

[0001] This invention relates to the field of point-of-care tests. Specifically, this invention relates to a lateral flow chromatography apparatus for detecting target analytes in samples, a method for detecting target analytes in samples, and a kit thereof. The lateral flow chromatography apparatus, method, and kit are capable of detecting extremely low concentrations of target analytes and have the ability to quantify the analytes, thereby confirming the presence of pathogens or ailments / diseases. Background Technology

[0002] For effective patient management and timely disease control, it is necessary to screen for disease antigens and detect biomarkers for various diseases (such as cancer, infectious diseases, heart disease, and neurodegenerative diseases) in a timely manner. One diagnostic technique used for rapid screening of infectious disease antigens is Lateral Flow Assay (LFA). LFA is a common point-of-care diagnostic technique used in the medical diagnostics field, but it is mainly considered a preliminary screening test and requires secondary confirmatory laboratory tests, such as enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR), to confirm the disease.

[0003] A drawback of LFA is that the detection of analytes present at critical levels below micrograms per milliliter often leads to false negative results. Furthermore, due to its lower specificity compared to standard laboratory techniques, LFA is considered to limit its clinical translation. Quantitative detection of analytes in biological samples is a critical parameter for patient testing and clinical care. The weak signal output of LFA results in low sensitivity and an inability to quantify, further limiting its application in clinical practice.

[0004] To overcome this challenge and improve sensitivity and signal output, various signal amplification techniques have been integrated with LFA technology. One effective approach is nanozyme-based LFA, which utilizes the inherent catalytic properties of nanomaterials to generate a quantifiable, strong output signal. Therefore, nanozyme-based LFA has significantly impacted the development of point-of-care diagnostic technologies for detecting a wide range of bioanalytes in clinical samples at extremely low concentrations (nanoscale). The 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 early stages. Further research and development in this field could lead to the widespread use of LFA in clinical practice, thereby improving patient outcomes and disease control.

[0005] Nanozyme-based lateral flow chromatography utilizes catalytically active nanoparticles and chromogenic substrates to enhance the colorimetric signal and sensitivity of the detection line. Traditionally, TMB (3,3',5,5'-tetramethylbenzidine) and DAB (diaminobenzidine) are commonly used as chromogenic substrates, which produce colored products through oxidation by peroxidases such as horseradish peroxidase (HRP) and catalytically active nanoparticles.

[0006] EP3080272B1 discloses the use of aptamer-coupled mesoporous silica particles as catalytically active nanomaterials in LFA, utilizing TMB as a chromogenic substrate. However, TMB has limitations as a chromogenic substrate for catalytically active nanomaterials in LFA applications due to its low color contrast on the detection line, color degradation over time, and the need for slightly acidic conditions and a dark environment to produce the maximum amount of product.

[0007] Some of the most advanced techniques in this field include research papers and granted patents on catalytically active nanomaterials for developing LFA kits with higher sensitivity. For example, Kidwell et al. (US20180052153A1) developed colloidal palladium nanoparticles as catalytically active nanomaterials for LFA applications. Veli et al. (EP3080272B1) investigated aptamer-coupled and signal molecule-loaded silica particles as nanozymes to improve the sensitivity of lateral flow chromatography. 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 MA 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] used palladium and platinum core-shell nanostructures as signal-enhancing nanozymes in LFA for precise pathogen detection. However, only a very limited number of studies have focused on the chromogenic substrates that produce the greatest sensitivity in LFA.

[0008] Previous research in this field has focused on introducing novel catalytically active nanomaterials. Therefore, there is a need in this field to develop more sensitive and accurate LFA kits for the detection of various diseases.

[0009] Purpose of the invention One object of the present invention is to develop a highly sensitive nanozyme-based side-flow chromatography device for detecting target analytes in samples.

[0010] Another objective of this invention is to develop a highly sensitive method for detecting target analytes in samples.

[0011] Another objective of this invention is to develop a highly sensitive kit for detecting target analytes in samples.

[0012] Another object of the present invention is to use p-phenylenediamine (PPD) as a chromogenic substrate for the development of highly sensitive nanozyme-based LFA devices.

[0013] Another object of the present invention is to provide a universal point-of-care testing device that can rapidly screen for antigens / microorganisms and detect biomarkers for a variety of diseases, including cancer, heart disease and neurodegenerative diseases.

[0014] Another object of the present invention is to provide an LFA device that overcomes the limitations of detecting analytes present at critical levels below micrograms per milliliter, thereby avoiding false negative results.

[0015] One object of the present invention is to develop a nanozyme-based lateral flow chromatography device and kit with a quantifiable strong output signal.

[0016] Another object of the present invention is to provide a nanozyme-based lateral flow chromatography that uses catalytically active nanoparticles and chromogenic substrates to enhance the colorimetric signal and sensitivity of the detection line.

[0017] Another object of the present invention is to develop nanozyme-based LFAs that use chromogenic substrates, which are not limited in application by low color contrast, color degradation over time, and the need for conditions such as slightly acidic and dark environments to produce the maximum amount of product.

[0018] Another objective of this invention is to overcome the problems of currently available commercially available sideflow chromatography kits, which have low sensitivity to analytes.

[0019] These and other objects and advantages of this subject matter will be apparent to those skilled in the art when taken in conjunction with the accompanying drawings illustrating preferred embodiments of the subject matter and considering the following detailed description. Summary of the Invention

[0020] The following is a brief summary of the invention to provide a basic understanding of some aspects of it. This summary is not a comprehensive overview of the invention. Its purpose is not to identify key / essential elements of the invention or to describe its scope. Its sole purpose is to present some concepts of the invention in a simplified form as a prelude to the more detailed description of the invention that follows.

[0021] One aspect of the present invention provides a side-flow chromatography apparatus for detecting a target analyte (13) in a sample (14), the apparatus comprising a test strip (1) having a top surface (11) and a bottom surface (12); The test strip (1) includes: a) Sample pad (2), which is disposed on the top surface (11) of the test strip (1) and is adapted to receive the sample (14). b) A detection area (17), which is disposed on the top surface (11) of the test strip (1) and downstream of the sample pad (2), includes: (i) A binding pad (3) on the surface of which a nanozyme (15) for capturing a target analyte (13) is placed; the nanozyme (15) includes a first binding molecule (5) coupled to a detectable reporter molecule (4); (ii) A flow membrane (6) including a detection point (8) on which a second binding molecule (7) is immobilized; c) Absorbent pad (9), which is located on the top surface (11) of the test strip (1) and downstream of the test area (17), and is suitable for absorbing excess fluid; Among them, a flow path (10) extending from the sample pad (2) to the absorbent pad (9) is defined on the top surface (11) of the test strip (1). In this process, the target analyte (13) binds to the nanozyme (15) to form a first analyte-nanozyme complex, which is further captured by a second binding molecule, thereby forming a second analyte-nanozyme complex at the detection point (8). The second nanozyme complex reacts with p-phenylenediamine and activator (16) to produce a dark brown product at the detection point (8), thereby confirming the presence of the target analyte (13).

[0022] Another aspect of the invention includes an in vitro method for detecting a target analyte (13) in a sample (14), the method comprising the following steps: i. Add the sample (14) to the sample pad (2) of the side-flow chromatography apparatus; ii. Allow the sample to move along the flow path (10) of the device; iii. Add the chromogenic substrate and activator (16) together to the detection point (8); and iv. Observe the color intensity change at the detection point (8) after 5-10 minutes; If a dark brown color appears at the detection point (8), it indicates that the target analyte (13) is present in the sample (14). The chromogenic substrate is p-phenylenediamine, with a concentration ranging from 0.1 mM to 10 mM. The activator is hydrogen peroxide, with a concentration ranging from 10 mM to 100 mM.

[0023] Another aspect of the present invention provides a sideflow chromatography kit for detecting a target analyte in a sample, the kit comprising: a. Side-flow chromatography apparatus; b. p-phenylenediamine; c. Activator; and d. User manual.

[0024] These and other aspects of the disclosed subject matter, as well as additional novel features, will become apparent from the description provided herein. This summary is not intended to be a complete description of the claimed subject matter, but rather to provide a brief overview of some of its functions. Other systems, methods, features, and advantages provided herein will be apparent to those skilled in the art upon review of the following figures and detailed description. All such additional systems, methods, features, and advantages included in this specification are intended to be included within the scope of any claim. Attached Figure Description

[0025] However, it should be noted that the accompanying drawings only illustrate typical embodiments of the subject matter and should not be considered as limiting its scope of protection, as the invention allows for other equally effective embodiments. A detailed description is given with reference to the accompanying drawings. Some embodiments of the system or method according to the subject matter will now be described by way of example, with reference to the accompanying drawings, in which: Figure 1 The components of a side-flow chromatography (LFA) apparatus are depicted, which consists of a test strip (1) having a sample pad (2), a binding pad (3), a chromatography membrane (6), and an absorption pad (9).

[0026] Figure 2 (A) is a schematic diagram of the working mechanism of a side-flow chromatography apparatus, showing sample collection, the flow direction of the target analyte, and the low-contrast signal generated at the detection point by the nanozyme and the second binding molecule.

[0027] Figure 2(B) Describes the signal enhancement of the method for detecting the target analyte using a chromogenic substrate (PPD) and an activator, and the high-contrast signal that appears after 5 minutes.

[0028] Figure 3 The images show a comparison of the lateral flow chromatography apparatus before and after amplification when detecting Covid-19.

[0029] Figure 4 A comparison of the absorption spectra of gold nanoparticles (GNP), PPD, and combinations of GNP, PPD, and H2O2 is shown.

[0030] Figure 5 The optimization of the catalytic activity of gold nanoparticles at different concentrations (1 nM to 10 nM) is shown under fixed concentrations of PPD (5 mM) and H2O2 (50 mM).

[0031] Figure 6 The optimization of PPD concentration on nitrocellulose membranes is shown, and the inset shows a picture of the nitrocellulose membrane.

[0032] Figure 7 Images show the detection of β-amyloid 42 by lateral flow chromatography at different concentrations using PPD as a chromogenic substrate.

[0033] Figure 8 Images show the detection of β-amyloid 40 by lateral flow chromatography at different concentrations using PPD as a chromogenic substrate. Detailed Implementation

[0034] This document describes in detail various exemplary embodiments of the present disclosure. It should be noted that the description of the embodiments herein is very detailed in order to convey the content of this disclosure. However, the level of detail provided herein is not intended to limit the contemplated variations of the embodiments; rather, it is intended to cover all modifications, equivalents, and alternatives that fall within the spirit and scope of this disclosure.

[0035] Unless otherwise defined, all terms (technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the exemplary embodiments pertain. It should also be further understood that terms, such as those defined in common dictionaries, should be interpreted as having a meaning consistent with their meaning in the relevant technical context, and should not be interpreted as having an idealized or overly formal meaning unless explicitly defined herein.

[0036] The terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the exemplary implementations. As used herein, unless the context otherwise indicates, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well. It should also be understood that when the terms “comprises,” “comprising,” “includes,” and / or “including” are used herein, they specify the presence of the stated features, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or combinations thereof.

[0037] The term "further" is used in the embodiments and claims of this application. This term is a broadly accepted term used to narrow the scope of any key feature. Therefore, the scope of the term will be clearly understood by those skilled in the art within the context of this disclosure.

[0038] As used herein, the terms “aptamer,” “aptamer molecule,” and “capture aptamer molecule” refer to single-stranded oligonucleotide molecules, typically composed of DNA or RNA folded into a specific three-dimensional structure capable of binding target molecules with high affinity and specificity. As used herein, aptamers consist of 50 to 70 nucleotide base pairs and serve as biorecognition elements.

[0039] As used herein, the term "test strip" refers to a disposable device containing a binding molecule and at least one detection zone.

[0040] As used herein, the term "target analyte" refers to a specific substance or molecule of interest that is detected or measured in a sample. These can be antigens, nucleic acids, proteins, bacterial cells, viral particles, fungal cells, lipids, peptides, or carbohydrates.

[0041] As used herein, the term "chromogenic substrate" refers to a compound that reacts to produce a colored product. This colored product can then be detected visually or measured using instruments.

[0042] As used in this article, the term "nanozyme" refers to nanomaterials that exhibit enzyme-like activity. These materials can catalyze specific chemical reactions, mimicking the function of natural enzymes.

[0043] As used herein, the term “fixed” refers to molecules, particles, or components whose movement within a system is restricted by their attachment to a solid surface, such as a bead, film, or matrix surface.

[0044] As used herein, the term "oxidation" refers to a chemical reaction involving the loss of electrons from molecules, atoms, or ions. This loss of electrons is usually accompanied by the acquisition of oxygen from the surrounding environment. However, oxidation can also occur without the direct involvement of oxygen, provided that electron loss is present.

[0045] As used in this article, 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 structural and functional groups on the interacting molecules.

[0046] As used in this article, "gold nanoparticles" refers to a colloidal suspension of gold nanoparticles in water. Suspensions of spherical gold nanoparticles with a size less than 100 nm are typically wine-red in color.

[0047] As used herein, “sample” refers to any material that may be suspected of containing the target analyte. This may include, but is not limited to, blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid (CSF), synovial fluid, feces, water, soil, air filter, food extracts, nasal swabs, throat swabs, wound swabs, biopsy samples, surgical specimens, vaginal fluid, and semen.

[0048] The present invention relates to a side-flow chromatography apparatus for detecting a target analyte (13) in a sample (14), the apparatus comprising a test strip (1) having a top surface (11) and a bottom surface (12); The test strip (1) includes: a) Sample pad (2), which is disposed on the top surface (11) of the test strip (1) and is adapted to receive the sample (14). b) A detection area (17), which is disposed on the top surface (11) of the test strip (1) and downstream of the sample pad (2), includes: (i) A binding pad (3) on the surface of which a nanozyme (15) for capturing a target analyte (13) is disposed; the nanozyme (15) includes a first binding molecule (5) coupled to a detectable reporter molecule (4); (ii) A chromatography membrane (6) including a detection point (8) on which a second binding molecule (7) is immobilized; c) Absorbent pad (9), which is located on the top surface (11) of the test strip (1) and downstream of the test area (17), and is suitable for absorbing excess fluid; Among them, a flow path (10) extending from the sample pad (2) to the absorbent pad (9) is defined on the top surface (11) of the test strip (1). In this process, the target analyte (13) binds to the nanozyme (15) to form a first analyte-nanozyme complex, which is further captured by a second binding molecule, thereby forming a second analyte-nanozyme complex at the detection point (8). The second nanozyme complex reacts with p-phenylenediamine and activator (16) to produce a dark brown product at the detection point (8), thereby confirming the presence of the target analyte (13).

[0049] In an embodiment of the present invention, a side-flow chromatography apparatus is provided, wherein the activator is hydrogen peroxide.

[0050] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein the chromatography membrane (6) is made of nitrocellulose.

[0051] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein both the sample pad (2) and the absorbent pad (9) are made of cellulose fibers.

[0052] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein the binding pad (3) is made of glass fiber.

[0053] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein the first binding molecule (5) and the second binding molecule (7) are each independently selected from the group consisting of aptamers, antibodies, peptides, enzymes, nucleic acids, carbohydrates, lipids, hormones and proteins.

[0054] In an embodiment of the present invention, a side-flow chromatography apparatus is provided, wherein the first binding molecule (5) is selected from the group consisting of aptamers, antibodies, peptides, enzymes, nucleic acids, carbohydrates, lipids, hormones and proteins.

[0055] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein the second binding molecule (7) is selected from the group consisting of aptamers, antibodies, peptides, enzymes, nucleic acids, carbohydrates, lipids, hormones and proteins.

[0056] In another embodiment of the present invention, a side-flow chromatography apparatus is provided, wherein the first binding molecule (5) and the second binding molecule (7) are both aptamers.

[0057] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein the first binding molecule (5) and the second binding molecule (7) are both antibodies.

[0058] In an embodiment of the present invention, a side-flow chromatography apparatus is provided, wherein the first binding molecule (5) and the second binding molecule (7) are both peptides.

[0059] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein the first binding molecule (5) and the second binding molecule (7) are both enzymes.

[0060] In another embodiment of the present invention, a side-flow chromatography apparatus is provided, wherein the first binding molecule (5) and the second binding molecule (7) are both nucleic acids.

[0061] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein the first binding molecule (5) and the second binding molecule (7) are both carbohydrates.

[0062] In an embodiment of the present invention, a side-flow chromatography apparatus is provided, wherein the first binding molecule (5) and the second binding molecule (7) are both lipids.

[0063] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein the first binding molecule (5) and the second binding molecule (7) are both hormones.

[0064] In another embodiment of the present invention, a side-flow chromatography apparatus is provided, wherein the first binding molecule (5) and the second binding molecule (7) are both proteins.

[0065] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein the detectable reporter molecule (4) is selected from the group consisting of gold nanoparticles, carbon nanoparticles, composite nanoparticles, palladium nanoparticles, cellulose beads and quantum dots.

[0066] In an embodiment of the present invention, a side-flow chromatography apparatus is provided, wherein the detectable reporter molecule (4) is gold nanoparticles.

[0067] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein the target analyte (13) is selected from the group consisting of antigens, nucleic acids, proteins, bacterial cells, viral particles, fungal cells, lipids, and carbohydrates.

[0068] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein the target analyte (13) is an antigen.

[0069] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein the target analyte (13) is a nucleic acid.

[0070] In an embodiment of the present invention, a side-flow chromatography apparatus is provided, wherein the target analyte (13) is a protein.

[0071] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein the target analyte (13) is bacterial cells.

[0072] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein the target analyte (13) is a virus particle.

[0073] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein the target analyte (13) is fungal cells.

[0074] In an embodiment of the present invention, a side-flow chromatography apparatus is provided, wherein the target analyte (13) is lipid.

[0075] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein the target analyte (13) is a carbohydrate.

[0076] In another embodiment of the invention, a lateral flow chromatography apparatus is provided, wherein the sample (14) is selected from the group consisting of blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, synovial fluid, feces, water, soil, air filter, food extract, nasopharyngeal swab, pharyngeal swab, wound swab, tissue, tumor cells, vaginal fluid, and semen.

[0077] In an embodiment of the present invention, a side-flow chromatography apparatus is provided, wherein the sample (14) is a nasopharyngeal swab.

[0078] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein the sample (14) is plasma.

[0079] In another embodiment of the invention, a side-flow chromatography apparatus is provided, wherein the detection limit of the apparatus is 0.2 nanograms of target analyte (13) / 100 microliters of sample (14).

[0080] 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 following steps: i. Add the sample (14) to the sample pad (2) of the side-flow chromatography apparatus; ii. Allow the sample to move along the flow path (10) of the device; iii. Add the chromogenic substrate and activator (16) together to the detection point (8); and iv. Observe the color intensity change at the detection point (8) after 5-10 minutes; If a dark brown color appears at the detection point (8), it indicates that the target analyte (13) is present in the sample (14). The chromogenic substrate is p-phenylenediamine, with a concentration ranging from 0.1 mM to 10 mM. The activator is hydrogen peroxide, with a concentration ranging from 10 mM to 100 mM.

[0081] In an embodiment of the present invention, an in vitro method for detecting a target analyte (13) in a sample is provided, wherein the chromogenic substrate is p-phenylenediamine with a concentration in the range of 2.5 mM to 10 mM.

[0082] In another embodiment of the invention, an in vitro method for detecting a target analyte (13) in a sample is provided, wherein the chromogenic substrate is p-phenylenediamine at a concentration of 5 mM.

[0083] In another embodiment of the invention, an in vitro method for detecting a target analyte (13) in a sample is provided, wherein the activator is hydrogen peroxide at a concentration of 50 mM.

[0084] In another embodiment of the invention, an in vitro method is provided for detecting a target analyte (13) in a sample, wherein the target analyte (13) is selected from the group consisting of antigens, nucleic acids, proteins, bacterial cells, viral particles, fungal cells, lipids, and carbohydrates.

[0085] In another embodiment of the invention, an in vitro method is provided for detecting a target analyte (13) in a sample, wherein the sample (14) is selected from the group consisting of blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, synovial fluid, feces, water, soil, air filter, food extract, nasopharyngeal swab, throat swab, wound swab, tissue, tumor cells, vaginal fluid, and semen.

[0086] In an embodiment of the present invention, an in vitro method for detecting a target analyte (13) in a sample is provided, wherein the detection limit of the method is 0.2 nanograms of target analyte (13) / 100 microliters of sample (14).

[0087] 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 following steps: i. Add the sample (14) to the sample pad (2) of the side-flow chromatography apparatus; ii. Allow the sample to move along the flow path (10) of the device; iii. Add the chromogenic substrate and activator (16) together to the detection point (8); and iv. Observe the color intensity change at the detection point (8) after 5-10 minutes; If a dark brown color appears at the detection point (8), it indicates that the target analyte (13) is present in the sample (14). The chromogenic substrate is p-phenylenediamine, with a concentration ranging from 2.5 mM to 10 mM. The activator is hydrogen peroxide at a concentration of 50 mM.

[0088] 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 following steps: i. Add the sample (14) to the sample pad (2) of the side-flow chromatography apparatus; ii. Allow the sample to move along the flow path (10) of the device; iii. Add the chromogenic substrate and activator (16) together to the detection point (8); and iv. Observe the color intensity change at the detection point (8) after 3-6 minutes; If a dark brown color appears at the detection point (8), it indicates that the target analyte (13) is present in the sample (14). The chromogenic substrate was p-phenylenediamine at a concentration of 5 mM. The activator is hydrogen peroxide at a concentration of 50 mM.

[0089] Another embodiment of the present invention provides a sideflow chromatography kit for detecting a target analyte in a sample, the kit comprising: a. Side-flow chromatography apparatus; b. p-phenylenediamine; c. Activator; and d. User manual.

[0090] In another embodiment of the invention, a side-flow chromatography kit is provided, wherein the activator is hydrogen peroxide.

[0091] In another embodiment of the invention, a side-flow chromatography kit is provided, wherein the concentration of p-phenylenediamine is in the range of 0.1 mM to 10 mM.

[0092] In another embodiment of the invention, a side-flow chromatography kit is provided, wherein the concentration of p-phenylenediamine is 5 mM.

[0093] In an embodiment of the present invention, a side-flow chromatography kit is provided, wherein the concentration of the activator is in the range of 10 mM to 100 mM.

[0094] In another embodiment of the invention, a side-flow chromatography kit is provided, wherein the concentration of the activator is 50 mM.

[0095] In an embodiment of the present invention, a lateral flow chromatography kit is provided, wherein the target analyte is selected from the group consisting of antigens, nucleic acids, proteins, bacterial cells, viral particles, fungal cells, lipids, and carbohydrates.

[0096] In another embodiment of the invention, a lateral flow chromatography kit is provided, wherein the sample is selected from the group consisting of blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, synovial fluid, feces, water, soil, air filter, food extract, nasopharyngeal swab, throat swab, wound swab, tissue, tumor cells, vaginal fluid, and semen.

[0097] 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 following steps: i. Add the sample (14) to the sample pad (2) of the side-flow chromatography apparatus; ii. Allow the sample to move along the flow path (10) of the device; iii. Add the chromogenic substrate and activator (16) together to the detection point (8); and iv. Observe the color intensity change at the detection point (8) after 5-10 minutes; If a dark brown color appears at the detection point (8), it indicates that the target analyte (13) is present in the sample (14). The chromogenic substrate is p-phenylenediamine, with a concentration ranging from 0.1 mM to 10 mM. The activator is hydrogen peroxide, with a concentration ranging from 10 mM to 100 mM.

[0098] The side-flow chromatography apparatus consists of a test strip (1) having a top surface (11) and a bottom surface (12), the test strip (1) comprising: a) Sample pad (2), which is disposed on the top surface (11) of the test strip (1) and is adapted to receive the sample (14). b) A detection area (17), which is disposed on the top surface (11) of the test strip (1) and downstream of the sample pad (2), includes: (i) A binding pad (3) on the surface of which a nanozyme (15) for capturing a target analyte (13) is disposed; the nanozyme (15) includes a first binding molecule (5) coupled to a detectable reporter molecule (4); (ii) A chromatography membrane (6) including a detection point (8) on which a second binding molecule (7) is immobilized; c) Absorbent pad (9), which is located on the top surface (11) of the test strip (1) and downstream of the test area (17), and is suitable for absorbing excess fluid; Among them, a flow path (10) extending from the sample pad (2) to the absorbent pad (9) is defined on the top surface (11) of the test strip (1). In this process, the target analyte (13) binds to the nanozyme (15) to form a first analyte-nanozyme complex, which is further captured by a second binding molecule, thereby forming a second analyte-nanozyme complex at the detection point (8). The second nanozyme complex reacts with p-phenylenediamine and activator (16) to produce a dark brown product at the detection point (8), thereby confirming the presence of the target analyte (13).

[0099] According to the present invention, a lateral flow chromatography (LFA) device, method, and kit for point-of-care detection are provided, which utilize nanozymes to enhance the sensitivity of the device and the detection. The components of the LFA test strip broadly include: (i) a sample pad, (ii) a binding pad, (iii) a chromatographic membrane including a detection point, and (iv) an absorbent pad.

[0100] Figure 1 The components of a side-flow chromatography (LFA) device are depicted, which consists of a test strip having a sample pad, a binding pad, a chromatographic membrane, and an absorbent pad. The side-flow chromatography device consists of a test strip (1) having a top surface (11) and a bottom surface (12). The test strip (1) includes: a sample pad (2) disposed on the top surface (11), a detection zone (17) located downstream of the sample pad (2), and an absorbent pad (9) located downstream of the detection zone (17). The detection zone (17) includes a binding pad (3) and a chromatographic membrane (6). The binding pad includes a first binding molecule (5) [referred to as a nanozyme (15)] coupled to a detectable reporter molecule (4) placed on the surface of the pad; the chromatographic membrane (6) includes a second binding molecule (7) immobilized on a detection point (8).

[0101] Figure 2 (A) illustrates the working mechanism of the side-flow chromatography apparatus and demonstrates sample collection, the flow direction of the target analyte, and the low-contrast signal generated at the detection point by the nanozyme and the second binding molecule. The sample (14) separated from the subject may include the target analyte (13) to be detected. The sample (14) is added to the sample pad (2) and allowed to move along the flow path (10) of the test strip (1). The target analyte (13) present in the sample (14) first binds to the nanozyme (15) placed on the binding pad (3) to form a first analyte-nanozyme complex, which is further captured by the second binding molecule (7), thereby forming a second analyte-nanozyme complex at the detection point (8) on the chromatography membrane (6).

[0102] Figure 2(B) illustrates the signal enhancement of the method for detecting the target analyte using a chromogenic substrate (PPD) and an activator, as well as the generation of a high-contrast signal after 5 minutes. Once a second analyte-nanozyme complex is formed at the detection point (8) on the chromatography membrane (6), the chromogenic substrate is added together with the activator (16) to the detection point (8), where a second analyte-nanozyme complex bound to p-phenylenediamine and the activator (18) is formed. A stable brown product is generated at the detection point (8), and the color intensity generated at the detection point allows for quantification of the signal intensity.

[0103] This device uses p-phenylenediamine (PPD) as a chromogenic substrate and its activator, hydrogen peroxide, to enhance detection sensitivity. PPD is an organic compound that, when oxidized by peroxidase, produces (3E,6E)-3,6-bis[(4-aminophenyl)imino]cyclohexyl-1,4-diene-1,4-diamine (bandrosyl base), which is brown in color. The device utilizes a second nanoenzyme complex as a catalytically active nanomaterial to promote the oxidation of PPD to the colored product, while simultaneously acting as a biorecognition element to capture the target analyte.

[0104] According to the present invention, the method for detecting target analytes in a sample using the device and kit comprises two steps: initial sample movement; and enhancing sensitivity by using a PPD.

[0105] The target analyte can be a molecule selected from antigens, nucleic acids, proteins, lipids, carbohydrates, or microorganisms selected from bacterial cells, viral particles, or fungal cells, or cancer cells. Careful selection is made of two specific binding molecules that are specific to the target analyte. These binding molecules can be molecules selected from aptamers, antibodies, peptides, enzymes, nucleic acids, carbohydrates, lipids, hormones, or proteins. The two binding molecules can belong to the same category, such as two aptamers, two antibodies, two peptides, two enzymes, etc., with different sequences. The binding molecules can also be molecules of different categories, such as an antibody as the first binding molecule and a nucleic acid as the second. These binding molecules can have two different binding sites at opposite ends of the target analyte to facilitate sandwich lateral flow chromatography (LFA).

[0106] Depending on the properties of the binding molecule, a first binding molecule is coupled to gold nanoparticles using an EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide / N-hydroxysuccinimide) coupling method, or any such coupling method known in the art, to generate a catalytically active nanozyme and a biorecognition element. The first binding molecule-nanozyme conjugate is loaded onto a binding matrix pad, dried, and a second binding molecule is immobilized on the detection area to capture the complex formed by the analyte-first binding molecule-conjugated gold nanozyme. Subsequently, a test strip is assembled with a sample pad and an absorbent pad to facilitate detection. In the presence of a target analyte in the sample, the gold nanozyme conjugated to the first binding molecule captures the target analyte, which is further captured by the second binding molecule immobilized at the detection site.

[0107] The catalytic efficiency of nanozymes was evaluated using PPD as a chromogenic substrate and hydrogen peroxide as an electron donor to activate the reaction. A corresponding increase in absorption peak intensity was observed as the nanozyme concentration increased from 1 nM to 10 nM. Furthermore, the optimal concentration of PPD in the range of 0.1 mM to 10 mM was evaluated in the presence of nanozymes. The results showed that concentrations of PPD from 2.5 mM to 10 mM produced the most favorable conditions for nanozyme-based signal enhancement in LFA. To amplify the signal and improve sensitivity, 2 μL of 5 mM PPD and 50 mM hydrogen peroxide were added to the detection zone. The reaction between PPD and the target analyte-bound gold nanozyme at the detection point produced a stable brown product as a visual signal output. The color intensity produced at each detection point in the detection zone allowed for quantification of the signal intensity. The oxidation product of the chromogenic substrate and the catalytically active nanozyme was brown, which remained stable for several weeks, providing high contrast against the background and allowing for measurement at an absorbance of 460 nm.

[0108] The sideflow chromatography (LFA) kit for detecting target analytes in samples has the following main components, including: a. Side-flow chromatography apparatus; b. p-phenylenediamine (PPD) as a chromogenic substrate; and c. Hydrogen peroxide as an activator.

[0109] This kit is cost-effective and highly sensitive. The gold nanozyme lateral flow kit is designed to detect pathogens such as SARS-CoV-2 virus and human immunodeficiency virus, as well as biomarkers of diseases such as cancer, heart disease, and neurodegenerative diseases.

[0110] The chromogenic substrate used in the kit is p-phenylenediamine, with an optimal concentration ranging from 0.1 mM to 10 mM, and is combined with the activator hydrogen peroxide, with an optimal concentration ranging from 10 mM to 100 mM. The LFA kit has the potential to detect analytes present at critical levels below micrograms per milliliter.

[0111] The lateral flow chromatography LFA (kit) of this invention offers improved specificity while ensuring stability and cost-effectiveness. During evaluation, the nanozyme-based LFA kit demonstrated a detection limit of 0.2 nanograms per 100 microliters of sample. The developed nanozyme-based LFA kit achieves a sensitivity level comparable to standard laboratory techniques, such as ELISA (nanogram to picogram), while remaining cost-effective. The total run time for this assay is less than 30 minutes.

[0112] As one exemplary embodiment, for the detection of the target analyte using the apparatus and method of the present invention, “covid19 spike protein” (SEQ ID NO. 3) was selected as the target analyte. Two specific aptamers, aptamer 51 (SEQ ID NO. 1) and aptamer 67 (SEQ ID NO. 2), were carefully selected as the first binding molecule and the second binding molecule, respectively. These aptamers have binding sites at opposite ends of the target analyte, which facilitates the implementation of sandwich sideflow chromatography (LFA).

[0113] Aptamer 51 (the first binding molecule) was coupled to gold nanoparticles using an EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide / N-hydroxysuccinimide) coupling method, thereby generating catalytically active nanozymes and biorecognition elements. The catalytic efficiency of the nanozyme was evaluated using PPD as a chromogenic substrate and hydrogen peroxide as an electron donor to activate the reaction. The nanozyme conjugate was loaded onto a binding pad made of glass fiber and dried. Aptamer-67 (the second binding molecule) was immobilized at a detection point on a chromatographic membrane made of nitrocellulose to capture the complex formed by the analyte-aptamer-coupled gold nanozyme. Subsequently, a test strip was assembled with a sample pad and an absorbent pad made of fiber to facilitate detection.

[0114] In the presence of the COVID-19 spike protein (SEQ ID NO. 3), the aptamer-conjugated gold nanozyme captures the protein, which is further captured by aptamer-67 (SEQ ID NO. 2) immobilized at the detection site. To amplify the signal and improve sensitivity, 2 μL of 5 mM PPD and 50 mM hydrogen peroxide are added to the detection site. 5–10 minutes after the addition of PPD and hydrogen peroxide, the reaction between PPD and the aptamer-conjugated gold nanozyme at the detection site produces a stable brown product, which serves as the visual signal output. The color intensity generated at each detection site in the detection area allows for quantification of the signal intensity.

[0115] Example The following examples are given by way of illustration and should not be construed as limiting the scope of the invention.

[0116] Example 1 Preparation of a side-flow chromatography device For this experiment, the SARS-CoV-2 spike protein was selected as the target analyte. Two specific aptamer sequences, aptamer 51 (as shown in SEQ ID NO. 1) and aptamer 67 (as shown in SEQ ID NO. 2), were carefully selected as the first and second binding molecules, respectively. These aptamers have binding sites at opposite ends of the target analyte, which is the SARS-CoV-2 spike protein (as shown in SEQ ID NO. 3) [6VXX_I|chain A, B, C|spike glycoprotein|Severe Acute Respiratory Syndrome Coronavirus 2 (2697049)]. Table 1 provides the sequences of SEQ ID NO. 1-3.

[0117] Using an EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide / N-hydroxysuccinimide) coupling method, 10 μL of 10 μM aptamer 51 was coupled to 1 mL of synthesized gold nanoparticles (average size 15 nm) to generate a nanozyme, which serves as both a catalytically active nanozyme and a biorecognition element. The aptamer 51 and gold nanoparticle conjugate (nanozyme) was loaded onto a binding pad and dried. 1 μL of 10 μM aptamer 67 was immobilized at the detection point on the chromatography membrane.

[0118] A sideflow chromatography apparatus was assembled using a sample pad (made of cellulose fibers for receiving the sample), a binding pad (made of glass fibers coated with aptamer-51-coupled gold nanoparticles for capturing analytes present in the sample), a chromatography membrane (made of nitrocellulose immobilized with aptamer-67 for capturing the aptamer-51-coupled gold nanoparticle and analyte complex), and an absorbent pad (made of cellulose fibers for absorbing excess fluid). This configuration constitutes a sideflow chromatography apparatus.

[0119] Table 1

[0120]

[0121] Example 2 Detection procedures for detecting target analytes The detection procedure consists of two steps: (i) initial sample movement; followed by (ii) enhancing sensitivity through the use of PPD.

[0122] A 100 μL nasopharyngeal swab sample prepared in phosphate-buffered saline (containing 0.04% bovine serum albumin, 5% sucrose, and 0.01% Tween 20) was added to the sample pad of a side-flow chromatography apparatus and allowed to flow through the entire length of the apparatus. The SARS-CoV-2 spike protein was captured by aptamer-conjugated gold nanoparticles placed on the binding pad and further captured by aptamer 67 immobilized on the detection zone. To amplify the signal and improve sensitivity, 2 μL of 5 mM PPD and 50 mM hydrogen peroxide were added to the detection zone. The reaction between the PPD and the gold nanoparticles conjugated to aptamer 67 on the detection zone produced a stable brown product after 5 minutes, which served as a visual signal output, confirming the presence of the SARS-CoV-2 spike protein.

[0123] Table 2 summarizes the various aspects of the detection procedure.

[0124] Table 2

[0125] Example 3 Catalytic efficiency The catalytic efficiency of aptamer-coupled gold nanozymes was evaluated using PPD as a chromogenic substrate and hydrogen peroxide as an electron donor to activate the reaction. First, the absorption spectrum of individual gold nanoparticles (10 nM) was measured, showing a maximum absorption peak at 522 nm. The absorption spectrum of PPD (5 mM) did not show a significant absorption peak. However, when PPD (5 mM) and H2O2 (50 mM) were incubated together with gold nanoparticles (2 nM), the absorption peak shifted from 522 nm to 450 nm, indicating the formation of a brown bandrosi base product. This shift confirmed the catalytic efficiency of the gold nanoparticles in converting PPD to the brown bandrosi base product, which could be measured at 450 nm. Figure 4 ).

[0126] Example 4 catalytic activity The catalytic activity of gold nanozymes at different concentrations was investigated. With fixed concentrations of PPD (5 mM) and H2O2 (50 mM), a corresponding increase in absorption peak intensity was observed when the concentration of gold nanozymes increased from 1 nM to 10 nM, indicating that the catalytic properties are concentration-dependent. Figure 5 ).

[0127] Example 5 To evaluate the optimal concentration of PPD on nitrocellulose membranes for nanozyme-based signal enhancement in lateral flow chromatography (LFA).

[0128] To this end, 2 μL of a fixed concentration of aptamer-conjugated gold nanozyme (1 nM) was drop-coated onto a nitrocellulose membrane and air-dried for 30 minutes. The membrane was then incubated with different concentrations of PPD ranging from 0.1 mM to 12.5 mM. The results showed that, with a fixed H₂O₂ (50 mM) concentration, the color intensity gradually increased as the PPD concentration varied from 0.5 mM to 2.5 mM. However, increasing the concentration above 5 mM did not result in a significant increase in signal intensity. Therefore, a concentration range of 2.5 mM to 10 mM was selected as the optimal concentration for nanozyme-based signal enhancement in LFA. Figure 6 ).

[0129] Example 6 β-amyloid 42 (Aβ 42) was detected using a side-flow chromatography apparatus. β-amyloid 42 (Aβ 42) (a biomarker of interest in the diagnosis of Alzheimer's disease) was also used as the target analyte to test the nanozyme signal amplification method.

[0130] For this purpose, an EDC / NHS conjugation method was used to conjugate 10 μL of a 10 μM monoclonal antibody (Abcam, product ID-ab201060) specifically recognizing Aβ 42 onto 1 mL of gold nanoparticles with an average size of 15 nm. The conjugated antibody was then purified and dispersed in 50 μL of phosphate buffer containing 0.04% bovine serum albumin, 20% sucrose, and 0.1% Tween 20.

[0131] The conjugate was used as a nanozyme, loaded onto the conjugation pad in a volume of 10 μL and dried. Then, 1 μL of 10 μM polyclonal anti-Aβ 42 antibody (Thermo Fisher Scientific, product ID-44-344) was immobilized on the detection point on the chromatography membrane.

[0132] Aβ42 was diluted to different concentrations (0.1 ng / mL to 100 ng / mL) in 100 μL of human plasma (collected from individuals aged 60 years and older in Trivandrum, Kerala, India) and mixed with 100 μL of phosphate buffer (containing 0.04% bovine serum albumin, 5% sucrose, and 0.01% Tween 20) to prepare samples.

[0133] Add 100 μL of the prepared sample to the sample pad. Let the LFA strip stand for 10 minutes. Then, add 1 μL of 5 mM MPPD and 50 mM H2O2 to the detection area, and observe the results after incubation for 10 minutes. The results are as follows. Figure 7 As shown in the figure. The results indicate that Aβ42 (the target analyte) was detected, and the intensity of the brown product varied with the concentration of Aβ42.

[0134] Example 7 β-amyloid 40 (Aβ 40) was detected using a side-flow chromatography apparatus. The same side-flow chromatography apparatus was also used to detect another Alzheimer's disease-specific biomarker, β-amyloid 40 (Aβ 40), which is another target analyte.

[0135] For this purpose, an EDC / NHS conjugation method was used to conjugate 10 μL of a 10 μM monoclonal antibody (Abcam, product ID-ab20068) specifically recognizing Aβ 40 onto 1 mL of gold nanoparticles with an average size of 15 nm. The conjugated antibody was then purified and dispersed in 50 μL of phosphate buffer containing 0.04% bovine serum albumin, 20% sucrose, and 0.1% Tween 20.

[0136] The conjugate was used as a nanozyme, loaded onto the conjugation pad in a volume of 10 μL and dried. Then, 1 μL of 10 μM polyclonal anti-Aβ40 antibody (Thermo Fisher Scientific, product ID-44-136) was immobilized on the detection point on the chromatography membrane.

[0137] Different concentrations (0.1 ng / mL to 100 ng / mL) of Aβ40, prepared from human plasma (collected from individuals aged 60 years and older in Trivandrum, Kerala, India), were diluted 1:1 by volume in phosphate buffer (containing 0.04% bovine serum albumin, 5% sucrose, and 0.01% Tween 20) and added to the sample pad. After 10 minutes, 1 μL of 5 mM PPD and 50 mM H2O2 were added to the detection area, and after incubation for 10 minutes, the formation of a brown product was observed. The results are as follows: Figure 8 As shown. The results indicate that Aβ40 (the target analyte) was detected, and the intensity of the brown product varied with the concentration of Aβ40.

[0138] Although embodiments of this subject matter have been described in language specific to packaging features, it should be understood that this subject matter is not necessarily limited to the specific features described. Rather, specific features and methods are disclosed as embodiments of this subject matter. Many modifications and improvements to the inventive system / apparatus will be apparent to those skilled in the art, and therefore, the appended claims are intended to cover all such modifications and improvements falling within the scope of this subject matter.

[0139] It should also be understood that the function or structure of multiple components or steps can be combined into a single component or step, or the function or structure of a step or component can be decomposed into multiple steps or components. This invention covers all such combinations. Unless otherwise stated, the dimensions and geometries of the various structures described herein are not intended to limit the invention, and other dimensions or geometries are possible. Furthermore, while features of the invention may be described only in the context of one of the illustrated embodiments, such features may be combined with one or more other features of other embodiments for any given application. It should also be understood from the foregoing that the manufacture and operation of the unique structures described herein also constitute a method according to the invention. The invention also includes intermediate and final products resulting from the implementation of the methods described herein.

[0140] List of reference numerals 1- Test strip 2- Sample pad 3- Bonding pad 4- Detectable reporter molecules 5- First binding molecule 6- Chromatography membrane 7- Second binding molecule 8- Testing Point 9- Absorbent Pad 10- Flow path 11- Top surface 12- Bottom 13- Target Analyte 14- Sample 15-Nanozyme 16- Chromogenic substrate with activator (p-phenylenediamine) 17- Testing Area 18- A second analyte-nanozyme complex coupled with p-phenylenediamine and an activator. Claims (as amended under Article 19 of the Treaty) 1. A side-flow chromatography apparatus for detecting a target analyte (13) in a sample (14), the apparatus comprising a test strip (1) having a top surface (11) and a bottom surface (12); The test strip (1) includes: a) Sample pad (2), which is disposed on the top surface (11) of the test strip (1) and configured to receive the sample (14). b) A detection area (17), which is disposed on the top surface (11) of the test strip (1) and located downstream of the sample pad (2), comprising: (i) A binding pad (3) on the surface of which a nanozyme (15) for capturing a target analyte (13) is disposed; the nanozyme (15) includes a first binding molecule (5) coupled to a detectable reporter molecule (4); (ii) A chromatography membrane (6) comprising a detection point (8) on which a second binding molecule (7) is immobilized; c) Absorbent pad (9), which is located on the top surface (11) of the test strip (1) and downstream of the test area (17), and is designed to absorb excess fluid to maintain the consistency of sample flow. A flow path (10) extending from the sample pad (2) to the absorbent pad (9) is defined on the top surface (11) of the test strip (1). The target analyte (13) binds to the nanozyme (15) to form a primary analyte-nanozyme complex. The primary analyte-nanozyme complex is further captured by the second binding molecule, thereby forming a secondary nanozyme complex composed of the primary analyte-nanozyme complex and the second binding molecule at the detection point (8). The secondary nanozyme complex reacts with p-phenylenediamine and activator (16) to produce a dark brown product at the detection point (8), thereby confirming the presence of the target analyte (13). 2. The side-flow chromatography apparatus according to claim 1, wherein the activator is hydrogen peroxide; The chromatography membrane (6) mentioned above is made of nitrocellulose; The sample pad (2) and the absorbent pad (9) are both made of cellulose fibers; The bonding pad (3) mentioned therein is made of glass fiber. 3. The side-flow chromatography apparatus according to claim 1, wherein the first binding molecule (5) and the second binding molecule (7) are both selected from the group consisting of aptamers, antibodies, peptides, enzymes, nucleic acids, carbohydrates, lipids, hormones and proteins; The detectable reporter molecule (4) is selected from the group consisting of gold nanoparticles, carbon nanoparticles, composite nanoparticles, palladium nanoparticles, cellulose beads and quantum dots. 4. The side-flow chromatography apparatus according to claim 1, wherein the target analyte (13) is selected from the group consisting of antigens, nucleic acids, proteins, bacterial cells, viral particles, fungal cells, lipids, and carbohydrates; The sample (14) was selected from the group consisting of blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, synovial fluid, feces, water, soil, air filter, food extract, nasopharyngeal swab, pharyngeal swab, wound swab, tissue, tumor cells, vaginal fluid and semen. 5. The side-flow chromatography apparatus according to claim 1, wherein the detection limit of the apparatus is 0.2 nanograms of target analyte (13) / 100 microliters of sample (14). 6. An in vitro method for detecting a target analyte (13) in a sample (14), the method comprising the following steps: i. Add the sample (14) to the sample pad (2) of the side-flow chromatography apparatus according to claim 1; ii. Allow the sample to move along the flow path (10) of the device; iii. Add the chromogenic substrate and activator (16) together to the detection point (8); and iv. Observe the color intensity change at the detection point (8) after 5-10 minutes; If a dark brown color appears at the detection point (8), it indicates that the target analyte (13) is present in the sample (14). The chromogenic substrate is p-phenylenediamine, with a concentration in the range of 0.1 mM to 10 mM; The activator is hydrogen peroxide, with a concentration in the range of 10 mM to 100 mM. 7. The in vitro method for detecting a target analyte (13) according to claim 6, wherein the target analyte (13) is selected from the group consisting of antigens, nucleic acids, proteins, bacterial cells, viral particles, fungal cells, lipids, and carbohydrates; The sample (14) was selected from the group consisting of blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, synovial fluid, feces, water, soil, air filter, food extract, nasopharyngeal swab, pharyngeal swab, wound swab, tissue, tumor cells, vaginal fluid and semen. 8. The in vitro method for detecting the target analyte (13) according to claim 6, wherein the detection limit of the method is 0.2 nanograms of target analyte (13) / 100 microliters of sample (14). 9. A sideflow chromatography kit for detecting a target analyte in a sample, comprising: a. The side-flow chromatography apparatus as described in claim 1; b. p-phenylenediamine; c. Activator; and d. User manual. 10. The sideflow chromatography kit according to claim 9, wherein the activator is hydrogen peroxide; The target analytes are selected from the group consisting of antigens, nucleic acids, proteins, bacterial cells, viral particles, fungal cells, lipids, and carbohydrates. The samples selected are from the group consisting of blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, synovial fluid, feces, water, soil, air filters, food extracts, nasopharyngeal swabs, throat swabs, wound swabs, tissues, tumor cells, vaginal fluid, and semen.

Claims

1. A side-flow chromatography apparatus for detecting a target analyte (13) in a sample (14), the apparatus comprising a test strip (1) having a top surface (11) and a bottom surface (12); in, The test strip (1) includes: a) Sample pad (2), which is disposed on the top surface (11) of the test strip (1) and is adapted to receive the sample (14). b) A detection area (17), which is disposed on the top surface (11) of the test strip (1) and located downstream of the sample pad (2), comprising: (i) A binding pad (3) on the surface of which a nanozyme (15) for capturing the target analyte (13) is disposed; the nanozyme (15) includes a first binding molecule (5) coupled to a detectable reporter molecule (4). (ii) A chromatography membrane (6) comprising a detection point (8) on which a second binding molecule (7) is immobilized; c) Absorbent pad (9), which is located on the top surface (11) of the test strip (1) and downstream of the test area (17), and is suitable for absorbing excess fluid; A flow path (10) extending from the sample pad (2) to the absorbent pad (9) is defined on the top surface (11) of the test strip (1). The target analyte (13) binds to the nanozyme (15) to form a first analyte-nanozyme complex. The first analyte-nanozyme complex is further captured by the second binding molecule, thereby forming a second analyte-nanozyme complex at the detection point (8). The second nanozyme complex reacts with p-phenylenediamine and activator (16) to produce a dark brown product at the detection point (8), thereby confirming the presence of the target analyte (13).

2. The side-flow chromatography apparatus according to claim 1, wherein, The activator is hydrogen peroxide; The chromatography membrane (6) mentioned above is made of nitrocellulose; The sample pad (2) and the absorbent pad (9) are both made of cellulose fibers; The bonding pad (3) mentioned therein is made of glass fiber.

3. The side-flow chromatography apparatus according to claim 1, wherein, The first binding molecule (5) and the second binding molecule (7) are each independently selected from the group consisting of aptamers, antibodies, peptides, enzymes, nucleic acids, carbohydrates, lipids, hormones and proteins; The detectable reporter molecule (4) is selected from the group consisting of gold nanoparticles, carbon nanoparticles, composite nanoparticles, palladium nanoparticles, cellulose beads and quantum dots.

4. The side-flow chromatography apparatus according to claim 1, wherein, The target analyte (13) is selected from the group consisting of antigens, nucleic acids, proteins, bacterial cells, viral particles, fungal cells, lipids, and carbohydrates; The sample (14) was selected from the group consisting of blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, synovial fluid, feces, water, soil, air filter, food extract, nasopharyngeal swab, pharyngeal swab, wound swab, tissue, tumor cells, vaginal fluid and semen.

5. The side-flow chromatography apparatus according to claim 1, wherein, The detection limit of the device is 0.2 nanograms of target analyte (13) / 100 microliters of sample (14).

6. An in vitro method for detecting a target analyte (13) in a sample (14), the method comprising the following steps: i. Add the sample (14) to the sample pad (2) of the side-flow chromatography apparatus according to claim 1; ii. Allow the sample to move along the flow path (10) of the device; iii. Add the chromogenic substrate and activator (16) together to the detection point (8); and iv. Observe the color intensity change at the detection point (8) after 5-10 minutes; If a dark brown color appears at the detection point (8), it indicates that the target analyte (13) is present in the sample (14). The chromogenic substrate is p-phenylenediamine, with a concentration in the range of 0.1 mM to 10 mM; The activator is hydrogen peroxide, with a concentration in the range of 10 mM to 100 mM.

7. The in vitro method for detecting the target analyte (13) according to claim 6, wherein, The target analyte (13) is selected from the group consisting of antigens, nucleic acids, proteins, bacterial cells, viral particles, fungal cells, lipids, and carbohydrates; The sample (14) was selected from the group consisting of blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, synovial fluid, feces, water, soil, air filter, food extract, nasopharyngeal swab, pharyngeal swab, wound swab, tissue, tumor cells, vaginal fluid and semen.

8. The in vitro method for detecting the target analyte (13) according to claim 6, wherein, The detection limit of the method is 0.2 nanograms of target analyte (13) / 100 microliters of sample (14).

9. A sideflow chromatography kit for detecting a target analyte in a sample, comprising: a. The side-flow chromatography apparatus as described in claim 1; b. p-phenylenediamine; c. Activator; and d. User manual.

10. The sideflow chromatography kit according to claim 9, wherein, The activator is hydrogen peroxide; The target analytes are selected from the group consisting of antigens, nucleic acids, proteins, bacterial cells, viral particles, fungal cells, lipids, and carbohydrates. The samples selected are from the group consisting of blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, synovial fluid, feces, water, soil, air filters, food extracts, nasopharyngeal swabs, throat swabs, wound swabs, tissues, tumor cells, vaginal fluid, and semen.

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