Light-activated chemiluminescence detection reagent, detection method and application thereof

Abstract

The invention relates to a light-activated chemiluminescence detection reagent, a detection method and application thereof. The reagent comprises two groups of different specific capture systems, each specific capture system comprises a tag molecule and a capture molecule, and the capture molecule can specifically recognize and combine at least two tag molecules. According to the technical scheme, the detection signal value can be increased, the signal-to-noise ratio of light-excited chemiluminescence detection is increased, the detection sensitivity and specificity of light-excited chemiluminescence detection are further improved, and the accuracy of a detection result is improved.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, and in particular to a photo-induced chemiluminescence assay reagent, assay method and its application. Background Technology

[0002] Photo-induced chemiluminescence analysis is a next-generation immunoassay technique based on nanoscale polymer particles. Its core principle involves the generation and transfer of reactive oxygen species (ROS): photosensitive microspheres (GG) coated with antibodies / antigens and luminescent microspheres (FG) coated with antibodies / antigens can specifically bind to the target molecules (antigens / antibodies) in the sample, forming a sandwich immune complex. Then, irradiating the photosensitive microspheres with light of a specific wavelength causes them to generate ROS. These ROS diffuse onto the luminescent microspheres, exciting them to produce a chemiluminescence signal. The qualitative or quantitative analysis of the target antigen / antibody can then be achieved by detecting the intensity of this luminescence signal.

[0003] However, processing low-value samples in photochemiluminescence detection presents a significantly increased challenge. Because the concentration of the target analyte in low-value samples is extremely low, potentially close to or even below the sensitivity threshold of the detection platform, the detection signal is extremely weak and difficult to distinguish from background noise, thus affecting the accuracy and reliability of the detection results. Summary of the Invention

[0004] To address or partially address the problems existing in related technologies, this application provides a photochemiluminescence detection reagent, detection method, and its application, which can improve the detection signal value, thereby increasing the signal-to-noise ratio of photochemiluminescence detection, enhancing the detection sensitivity and specificity of photochemiluminescence detection, and improving the accuracy of detection results.

[0005] The first aspect of this application provides a photo-induced chemiluminescence detection reagent, which includes two different specific capture systems, wherein the specific capture system includes a tag molecule and a capture molecule, and the capture molecule is capable of specifically recognizing and binding to at least two tag molecules.

[0006] In some embodiments, the reagent further includes luminescent microspheres coated with a first antibody and a first tag molecule, wherein the first antibody is capable of specifically binding to the target molecule in the sample to form an immune complex.

[0007] In some embodiments, the tagged antibody formed by fusing the first antibody with the first tag molecule is coated onto the luminescent microspheres; in other embodiments, the first antibody and the first tag molecule are respectively coated onto the luminescent microspheres.

[0008] In some embodiments, the reagent further includes a second antibody and photosensitive microspheres; wherein the second antibody is capable of specifically binding to the target molecule in the sample to be tested, and the second antibody carries a second tag molecule; the photosensitive microspheres are coated with a second capture molecule.

[0009] In some embodiments, the two sets of specific capture systems are each independently selected from at least one of the Tag-Catcher system, click chemistry system, base pairing system, and biotin-avidin system; the two sets of specific capture systems do not react with each other; Preferably, the first tag molecule is selected from at least one of Spy-Tag, His-Tag, HA-Tag, Snoop-Tag, Flag-Tag, Myc-Tag, and antibody Fc fragment; Preferably, the first capturing molecule is selected from multimeric Catcher proteins with a degree of polymerization of not less than 2; more preferably, it is an octamer. Preferably, the second specific capture system is selected from the biotin-avidin system.

[0010] The second aspect of this application provides a photo-induced chemiluminescence detection method, in which the sample to be tested is reacted with a luminescent microsphere reagent containing a first antibody and a first tag molecule, a second antibody reagent containing a second tag molecule, and a photosensitive microsphere reagent containing a second capture molecule to form an immune complex, and then a reagent containing the first capture molecule is added to react and photo-induced chemiluminescence detection is performed.

[0011] In some implementations, the molar amount of the first captured molecule is less than or equal to the molar amount of the first tag molecule; Preferably, the molar ratio of the first capturing molecule to the first tag molecule is 1:(1~16); more preferably, it is 1:(2~8).

[0012] In some implementations, the degree of polymerization of the first captured molecule is negatively correlated with its optimal molar amount; Preferably, the product of the degree of polymerization of the first capturing molecule and its molar amount is 1 to 2.5 times the molar amount of the first tag molecule.

[0013] In some embodiments, the reaction time after adding the reagent containing the first capturing molecule is 0 to 15 min; preferably 3 to 9 min; more preferably 5 to 7 min.

[0014] A third aspect of this application provides an application of the above-described reagents or methods in detecting biomarkers in blood samples, wherein the biomarkers include, but are not limited to, NfL, GFAP, UCH-L1, and p-tau 217.

[0015] It is worth noting that the above applications are for purposes other than disease diagnosis.

[0016] The technical solution provided in this application may include the following beneficial effects: By utilizing two sets of specific capture systems in the reaction system, the specific recognition and binding ability between the captured molecules and the tag molecules can improve the utilization rate of singlet oxygen in photochemiluminescence detection, thereby increasing the detection signal value, improving the signal-to-noise ratio, and ultimately enhancing the detection sensitivity and specificity of photochemiluminescence detection, and improving the accuracy of the detection results. The detection method is simple, easy to operate, low in cost, highly applicable, and has a wide range of applications, capable of detecting various biomarkers. It can meet the detection needs of low-value samples.

[0017] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Detailed Implementation

[0018] To facilitate understanding of the present invention, it will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0019] Where numerical ranges are provided, it should be understood that every intermediate value between the upper and lower limits of the range and any other specified or intermediate value within the specified range is covered by this invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also covered by this invention, subject to any explicitly excluded limits within the specified range. Where a specified range includes one or two limits, the range excluding any or both of those included limits is also included by this invention.

[0020] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials, or equivalents thereof, may be used in the practice or testing of this invention, preferred methods and materials are now described.

[0021] I. Terminology The term "test sample" as used herein refers to a mixture that may contain the analyte, i.e., the target substance. The analyte includes, but is not limited to, proteins, hormones, antibodies, or antigens. Typical test samples that can be used in the methods disclosed in this invention include bodily fluids, such as blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid, and emphysema effusion. The test sample can be diluted with a diluent or buffer solution as needed before use. For example, to avoid the hook effect, the analyte can be diluted with a sample diluent before detection on the instrument. In this case, the diluted solution that may contain the analyte is collectively referred to as the test sample.

[0022] The term "antibody" as used herein is used in the broadest sense, including any isotype of antibody, antibody fragments that retain specific binding to antigens, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and non-antibody proteins. Where desired, antibodies may be further conjugated to other parts, such as specifically binding pairing members, for example, biotin or avidin.

[0023] The antigens described herein refer to substances capable of inducing antibody production, and can be classified into complete antigens and incomplete antigens (haptens). These antigens can be natural antigens extracted from pathogenic animal tissues or recombinant antigens with specific antigenic properties prepared through genetic engineering techniques. Where necessary, antigens can be further conjugated to other parts, such as specifically binding pairing members, for example, biotin or avidin.

[0024] The terms “combination,” “connection,” and “coupling” used in this article refer to the union between two substances caused by interactions such as covalent bonds, electrostatics, hydrophobicity, ions, hydrogen bonds, etc., or interactions including but not limited to salt bridges and water bridges.

[0025] The specific recognition described in this article refers to the ability of certain molecules in the immune system (such as antibodies or receptors) to recognize and distinguish specific antigens or epitopes based on the complementarity of their intermolecular structures.

[0026] The specific binding discussed in this article refers to the mutual recognition and selective binding reaction between two substances, which, from a stereostructural perspective, is the conformational correspondence between the reactants in the response. In the immune system, it specifically refers to the tight binding between certain molecules and the specific antigens or epitopes they recognize. This binding usually involves covalent or non-covalent bonds, such as hydrogen bonds, van der Waals forces, electrostatic interactions, etc., enabling the two substances to form a stable complex.

[0027] The specific capture system described in this article refers to two substances that have mutual recognition capabilities and can selectively bind through covalent or non-covalent bonds. The tag molecule and capture molecule described in this article are a group of substances that can specifically recognize and bind through covalent or non-covalent bonds. The tag molecule is a molecule that can fuse or bind to a target substance for the detection or purification of the target substance; the capture molecule is a molecule that can specifically recognize and bind to the tag molecule for the capture and enrichment of the tag molecule. The capture molecule has at least one binding site and is capable of capturing at least one target tag molecule.

[0028] The Tag-Catcher system described in this article is a protein covalent linking technology, comprising two parts: a Tag and a Catcher, each consisting of multiple amino acid residues. Covalent linking of proteins is achieved through a specific reaction between the Tag and the Catcher. The Tag-Catcher system can be a Spy Tag-Spy Catcher system developed based on the CnaB2 domain of the Streptococcus pyogenes fibronectin FbaB. The Spy Tag is a short peptide containing 13 amino acid residues, and the SpyCatcher is a protein containing 138 amino acid residues. During the linking process, the aspartic acid in the Spy Tag spontaneously reacts with the lysine residue on the SpyCatcher, catalyzed by the glutamate adjacent to the lysine residue, to form a heteropeptide covalent bond. The Catcher protein in the Tag-Catcher system can exist as a monomeric or multimeric protein. Monomeric proteins are protein molecules composed of a single polypeptide chain; multimeric proteins are proteins composed of two or more polypeptide chains, which may be the same or different, and are connected to each other by covalent bonds or non-covalent bonds (such as hydrogen bonds, hydrophobic interactions, van der Waals forces, etc.) to form multimeric macromolecular structural proteins.

[0029] The click chemistry system described in this article refers to a chemical synthesis technique in which a pair of functional groups can react rapidly and selectively to form new compounds. The click chemistry system can be a copper-catalyzed azide-alkyne cycloaddition reaction system, which can form a stable triazole ring structure; it can also be a thiol-ene reaction and a SuFEx (fluorine-sulfur exchange) reaction.

[0030] The base pairing system described in this article is a specific pairing mechanism between bases in nucleic acids (including DNA and RNA). Base pairing in DNA includes the pairing of adenine (A) with thymine (T), which forms two hydrogen bonds, and the pairing of guanine (G) with cytosine (C), which forms three hydrogen bonds. Base pairing in RNA includes the pairing of adenine (A) with uracil (U) and the pairing of guanine (G) with cytosine (C). The nucleic acid chain structures formed through this specific and selective pairing mechanism exhibit extremely high stability.

[0031] The biotin-avidin system described in this article is a bioreaction amplification system comprising two main components: biotin and either avidin or streptavidin. Biotin is a small molecule widely found in plant and animal tissues, possessing two ring structures: an imidazoline ring and a thiophene ring. The imidazoline ring is the primary binding site for avidin or streptavidin. Activated biotin can be coupled to almost all known biomolecules, including proteins, nucleic acids, polysaccharides, and lipids, mediated by protein cross-linking agents. Avidin is a protein secreted by Streptomyces. The streptavidin molecule consists of four identical peptide chains, each capable of binding one biotin, with a molecular weight of 65 kDa. Each antigen or antibody can simultaneously couple multiple biotin molecules, creating a "tentacle effect" with avidin to enhance analytical sensitivity. Where necessary, any reagent used in this invention, including antigens, antibodies, receptors or donors, may be conjugated to any of the specific binding pair members such as biotin-streptavidin, as required.

[0032] The reactive oxygen species (ROS) discussed in this article refer to a general term for oxygen-containing and reactive substances in the body or natural environment. They are primarily excited-state oxygen molecules, including the one-electron reduction product of oxygen (superoxide anion (O2·-), the two-electron reduction product of oxygen (hydrogen peroxide (H2O2), the three-electron reduction product of oxygen (hydroxyl radical (·OH), as well as nitric oxide and singlet oxygen). 1 O2), etc.

[0033] The luminescent microspheres described herein refer to polymeric microparticles filled with a luminescent composition, capable of reacting with reactive oxygen species to generate detectable light signals. Luminescent microspheres may also be called acceptor microspheres or luminescent microparticles. In some specific embodiments of the invention, the luminescent composition undergoes a chemical reaction with reactive oxygen species to form an unstable metastable intermediate, which can decompose and emit light simultaneously or subsequently. Typical examples of such substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan gum, 9-alkylidene-N-alkylacridinium, arylate ethers, diethylene oxide, dimethylthiophene, aromatic imidazoles, or gloss enhancers. In other specific embodiments of the invention, the luminescent composition may further comprise europium complexes; more preferably, the europium complex is MTTA-EU. 3+ .

[0034] The photosensitive microspheres described herein refer to polymeric microparticles filled with photosensitizers that can generate reactive oxygen species upon photoexcitation. These can also be called donor microspheres or photosensitive microparticles. Solutions containing such photosensitive microspheres can be called photosensitive solutions or universal solutions. The photosensitizers can be those known in the art, such as methylene blue, rose red, porphyrin, phthalocyanine, and chlorophyll, but are not limited to these. The photosensitive microspheres can also be filled with other sensitizers; non-limiting examples include certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Other examples of sensitizers include 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc. Heating these compounds or direct light absorption by these compounds releases reactive oxygen species.

[0035] The microparticles described herein can be of any size and shape, expandable or non-expandable, porous or non-porous, and have any density, but preferably close to that of water. They are preferably buoyant in water and are composed of transparent, partially transparent, or opaque materials. The microparticles can be solids (such as polymers, metals, glass, organic or inorganic substances such as minerals, salts, and diatoms), small oil droplets (such as hydrocarbons, fluorocarbons, and siliceous fluids), vesicles (such as synthetic phospholipids, or natural substances such as cells and organelles). A non-limiting example of microparticles suitable for use in this invention is carboxylated polystyrene latex microspheres.

[0036] Co-expression, as described in this article, refers to fusing proteins from two or more genes that require co-expression, enabling simultaneous expression. The co-expression linkage can be achieved by linking the coding sequences of two or more genes together using gene recombination technology to form a fusion gene. This fusion gene, upon expression, produces a fusion protein containing different functional domains encoded by multiple genes. Alternatively, the co-expression linkage can be achieved using technologies such as pClick (see "Synthesis of precision antibody conjugates using proximity-induced chemistry" (Theranostics. 2021 Aug 27; 11(18): 9107-9117. doi: 10.7150 / thno.62444.) to link two or more peptides, proteins, etc., with different or identical gene sequences, forming a fusion protein. Multiple genes in the fusion protein can be expressed simultaneously.

[0037] II. Specific Implementation Plan This application will now be described in more detail.

[0038] In photochemiluminescence analysis, various background interference factors, such as instrument noise and ambient light interference, can affect the signal-to-noise ratio (SNR). The SNR is the ratio of signal power to noise power. A higher SNR indicates a stronger detection signal, less background noise interference, and higher accuracy and reliability of the detection results. Improving the SNR can reduce false positives and false negatives, and enhance the specificity and sensitivity of the detection, especially important for detecting low concentrations of target molecules.

[0039] The photo-induced chemiluminescence detection reagent involved in this application improves the utilization rate of singlet oxygen generated by photosensitive microspheres by capturing the specific recognition and binding ability between the molecules and the tag molecules, thereby increasing the detection signal value, improving the signal-to-noise ratio of photo-induced chemiluminescence detection, enhancing detection sensitivity and specificity, and meeting the detection needs of low-value samples.

[0040] The photo-induced chemiluminescence detection reagent of this application includes two different specific capture systems. The specific capture system includes a tag molecule and a capture molecule. The capture molecule can specifically recognize and bind to at least two tag molecules.

[0041] By using two different specific capture systems, the ability of the capture molecules to capture the tagged molecules can be utilized to aggregate the components with tagged molecules and the capture molecules in the reaction system, thereby increasing the detection signal value in the reaction system, and thus improving the signal-to-noise ratio of photochemiluminescence detection, and enhancing detection sensitivity and specificity.

[0042] In some embodiments of this application, the reagent further includes luminescent microspheres coated with a first antibody and a first tag molecule, wherein the first antibody is capable of specifically binding to the target molecule in the sample to form an immune complex.

[0043] By coating luminescent microspheres with a first tag molecule, the ability of the corresponding capture molecule to capture the tag molecule enables at least two luminescent microspheres in the reaction system to aggregate into a multi-microsphere. When the target molecule is present in the sample, the first antibody specifically binds to the target molecule to form an immune complex. The luminescent microspheres linked to this immune complex can bind to at least one free luminescent microsphere in the system (i.e., luminescent microspheres in the reaction system that have not bound to the target molecule by the first antibody) through the tag molecule. During photochemiluminescence detection, more luminescent microspheres can receive singlet oxygen generated by the photosensitive microspheres, reducing quenched singlet oxygen in the system, improving singlet oxygen utilization, thereby increasing the detection signal value, enhancing the performance of photochemiluminescence detection, and meeting the detection needs of low-value samples.

[0044] In some embodiments of this application, the first tag molecule can be fused with the first antibody through co-expression to form a tag antibody, which is then coated onto the luminescent microspheres. Alternatively, the first tag molecule can be modified and linked to the first antibody via group coupling, fused into a tag antibody, and then coated onto the luminescent microspheres. The first antibody and the first tag molecule can also be coated onto the luminescent microspheres through separate, stepwise coupling. This application does not limit the coupling order of the first tag molecule and the first antibody on the luminescent microspheres.

[0045] When the first tag molecule binds to the first antibody via co-expression or group coupling, it can bind to the constant region of the antibody molecule, forming a tagged antibody molecule. The tagged antibody molecule obtained in this way can fully and effectively display the variable region or complementarity determining region (CDR) opposite to the constant region of the antibody, which is the site where a specific binding immune reaction occurs with the target analyte. This is beneficial for increasing the effective antibody quantity and activity in the reaction system, effectively improving detection sensitivity.

[0046] In some embodiments of this application, the reagent further includes a second antibody and photosensitive microspheres; the second antibody is capable of specifically binding to the target molecule in the sample to be tested, and the second antibody carries a second tag molecule; the photosensitive microspheres are coated with a second capture molecule capable of specifically binding to the second tag molecule.

[0047] During the use of this reagent, specifically in photochemiluminescence detection, both the first and second antibodies specifically recognize and bind to the target molecule in the sample, forming a sandwich immune complex. The specific binding of the second tag molecule on the second antibody to the second capture molecule on the photosensitive microsphere allows the photosensitive microsphere to also bind to this sandwich immune complex, bringing the photosensitive microsphere closer to the main luminescent microsphere. The main luminescent microsphere refers to the luminescent microsphere whose coating, the first antibody, binds to the target molecule in an immune reaction. Furthermore, the added first capture molecule binds to at least two first tag molecules, allowing free luminescent microspheres in the reaction system to bind to the main luminescent microsphere. During detection, both the main luminescent microsphere and the free luminescent microspheres bound to it can receive singlet oxygen generated by the photosensitive microsphere and emit light signals, significantly increasing the detection signal value and improving the signal-to-noise ratio, thus effectively enhancing the performance of photochemiluminescence detection.

[0048] It should be noted that the second tag molecule coupled to the photosensitive microspheres should be different from the first tag molecule on the luminescent microspheres. By using two different specific capture systems, the binding of the photosensitive microspheres to the second antibody and the aggregation of the free luminescent microspheres to the main luminescent microspheres in the reaction system can be ensured. This ensures that the singlet oxygen generated by the photosensitive microspheres in the reaction system can be effectively transferred to more luminescent microspheres, realizing the effective utilization of singlet oxygen and effectively improving the photo-induced chemiluminescence detection performance.

[0049] In some embodiments of this application, the two sets of specific capture systems may be independently selected from at least one of the Tag-Catcher system, click chemistry system, base complementary pairing system, and biotin-avidin system.

[0050] For example, when the capturing molecule and the tag molecule are selected from the Tag-Catcher system, irreversible and stable binding between the capturing molecule and the tag molecule can be achieved through the isopeptide bonds specifically formed by the Tag and Catcher. When the first capturing molecule and the first tag molecule are selected from the Tag-Catcher system, a highly stable polymer composition can be obtained after the free luminescent microspheres aggregate with the host luminescent microspheres. In addition, the capturing molecule and the tag molecule can also be selected from click chemistry systems that can form specific carbon-heteroatom bonds, base complementary pairing systems that selectively form hydrogen bonds, biotin-avidin systems that form stable amide bonds, etc.

[0051] Furthermore, to avoid interference between the capture molecules and tag molecules in the two sets of specific capture systems, which would affect the reaction and thus the accuracy and stability of the detection results, the two sets of specific capture systems, consisting of the first tag molecule and the first capture molecule, and the second tag molecule and the second capture molecule, are selected from two sets of systems that do not react with each other.

[0052] Preferably, the first capture molecule and the first tag molecule are selected from the Tag-Catcher system, and the second tag molecule and the second capture molecule are selected from the biotin-avidin system. More preferably, the first tag molecule is selected from a tag with a smaller molecular weight in the Tag-Catcher system, and the first capture molecule is selected from a Cater protein with a larger molecular weight in the corresponding Tag-Catcher system; the second tag molecule is selected from biotin in the biotin-avidin system, and biotin is used to label the second antibody to form a biotinylated antibody molecule; the second capture molecule is selected from the corresponding streptavidin.

[0053] In some specific embodiments of this application, the first tag molecule can be selected from one or more of Spy-Tag, His-Tag, HA-Tag, Snoop-Tag, Flag-Tag, Myc-Tag, and antibody Fc fragments. For example, when it is selected from Spy-Tag, the first capture molecule is selected from the corresponding Spy-Catcher; when it is selected from His-Tag, the first capture molecule is selected from the corresponding Anti-His (in this case, the Catcher is Anti-His); when it is selected from Snoop-Tag, the first capture molecule is selected from the corresponding Snoop-Catcher, etc. Catchers and Tags have excellent specific recognition and binding capabilities, and using a Tag tag with a small molecular weight as the first tag molecule to coat the luminescent microspheres has no significant impact on the expression of the first antibody or the properties of the luminescent microspheres.

[0054] Furthermore, when the first tag molecule is selected from Tag, the first capturing molecule can be selected from the monomer or multimeric protein of Catcher; preferably, it is a multimeric protein with a degree of polymerization of not less than 2, and preferably an octamer protein of Catcher. The degree of polymerization of Catcher directly affects the rate at which Catcher captures Tag, and thus affects the aggregation rate of luminescent microspheres. The higher the degree of polymerization of Catcher, the higher the Tag capture rate, the higher the aggregation rate of luminescent microspheres, and the better the effect on improving the light signal intensity.

[0055] The reagents described in the embodiments of this application can be used in a photo-induced chemiluminescence detection platform for photo-induced chemiluminescence detection. The specific detection method includes: reacting the sample to be tested with a luminescent microsphere reagent containing a first antibody and a first tag molecule, a second antibody reagent containing a second tag molecule, and a photosensitive microsphere reagent containing a second capture molecule to form an immune complex, and then adding a reagent containing the first capture molecule to react and perform photo-induced chemiluminescence detection.

[0056] After the test sample reacts with the luminescent microsphere reagent, the second antibody reagent, and the photosensitive microsphere reagent, the reaction system at this point contains a sandwich immune complex of "luminescent microsphere (first antibody) - target molecule - (second antibody) photosensitive microsphere" and unreacted free luminescent microspheres. The first trapping molecule reagent added in this state causes the free luminescent microspheres to aggregate around the main luminescent microsphere of the complex. The singlet oxygen generated by the photosensitive microsphere after receiving excitation at a specific wavelength can be simultaneously received by multiple luminescent microspheres (including the main luminescent microsphere and at least one free luminescent microsphere), generating a light signal. This effectively improves singlet oxygen utilization, increases light signal intensity, and thus enhances detection sensitivity and specificity.

[0057] Furthermore, the timing of adding the first capture molecule in this scheme after the immune reaction is complete does not alter the original detection process, making it highly applicable and versatile. It represents a platform-type improvement capable of detecting various biomarkers. Moreover, this scheme does not interfere with the original immune reaction. During the recognition and binding of the target molecule by the first antibody, the luminescent microspheres remain in the reaction system with their original mass and particle size, without altering the particle migration rate of the luminescent microspheres. This ensures a good immune reaction rate and avoids reduced detection sensitivity due to a slower migration rate caused by increased microsphere particle size. This method also features simplicity, ease of operation, and low cost.

[0058] In some embodiments of this application, the molar amount of the first capturing molecule should be less than or equal to the molar amount of the first tag molecule. Preferably, the molar ratio of the first capturing molecule to the first tag molecule is 1:(1~16); more preferably, it is 1:(2~8). The proportion of the first capturing molecule directly affects the aggregation of the luminescent microspheres. If it is too low, the aggregation efficiency and degree will be low, and the effect on improving the detection performance will not be obvious; if it is too high, it is easy to produce a "hook effect", which makes it difficult for the luminescent microspheres to aggregate, resulting in no significant improvement in detection performance, and even affecting the accuracy of the detection results.

[0059] When the first tag molecule and the first capture molecule are selected from the Tag-Catcher system, the higher the degree of polymerization of the first capture molecule (Catcher), the lower the molar ratio of the first capture molecule to the first tag molecule should be; that is, the degree of polymerization of the first capture molecule is negatively correlated with its optimal molar amount. Preferably, when the product of the degree of polymerization of the first capture molecule and its molar amount is 1 to 2.5 times the molar amount of the first tag molecule, the effect on improving the light signal intensity of the luminescent microspheres is significant. Excessive amounts can easily lead to the hook effect, affecting the detection results; insufficient amounts affect the aggregation rate and degree of aggregation of the luminescent microspheres, and the performance improvement is not significant.

[0060] The degree of polymerization and amount of the first capturing molecule can be determined based on the amount of the first tag molecule. The higher the degree of polymerization of the first capturing molecule, the lower the required optimal molar amount and the faster the reaction rate with the first tag molecule. The lower the degree of polymerization of the first capturing molecule, the higher the required optimal molar amount and the faster the aggregation rate.

[0061] In some embodiments of this application, the incubation time after adding the first capturing molecule can be 0-15 min. As the reaction time increases, the aggregation reaction of the luminescent microspheres gradually increases and tends to equilibrium; therefore, the reaction time is preferably 3-9 min, more preferably 5-7 min.

[0062] In some embodiments of this application, the detection method further includes diluting each component to a suitable concentration before mixing the components with the sample to be tested. The buffer solution or diluent for diluting the components may contain components such as buffer solutions and stabilizers. The buffer solution may be selected from at least one of PBS buffer, Tris-hydrochloric acid buffer, HEPES buffer, and MES buffer; the stabilizer may be selected from at least one of bovine serum albumin, dextran, sorbitol, glycerol, glycine, alanine, Tween 20, and Tween 80. In a specific example of this application, the buffer solution includes PBS buffer, bovine serum albumin, dextran, and Tween 20.

[0063] The reagents and detection methods described in this application are applicable to the detection of biomarkers in blood samples. They can also be applied to the detection of biomarkers in low-concentration samples. The biomarkers detected include, but are not limited to, NfL, GFAP, UCH-L1, and p-tau 217 in blood samples.

[0064] Neurological diseases are a class of illnesses affecting the central nervous system (brain and spinal cord) and peripheral nervous system. Common examples include Alzheimer's disease, Parkinson's disease, and Huntington's disease. Among the diagnostic methods for neurological diseases, imaging examinations such as positron emission tomography (PET) and magnetic resonance imaging (MRI) involve complex and expensive equipment; biomarker testing includes cerebrospinal fluid (CSF) analysis and blood biomarker analysis. Changes in CSF biomarkers, such as β-amyloid (Aβ) 42, total tau protein, and phosphorylated tau protein levels, can aid in the diagnosis of neurodegenerative diseases and differentiate them from other diseases. However, CSF testing requires lumbar puncture to collect a CSF sample, which is an invasive procedure and may cause discomfort and risks to the patient, such as headache and infection. Blood samples also contain some biomarkers associated with neurodegenerative diseases. For example, the levels of biomarkers such as tau protein and neurofilament light chains in the blood of Alzheimer's patients may change. Blood tests have the advantages of being non-invasive, convenient, and repeatable. However, due to the extremely low levels of biomarkers in blood samples, the requirements for testing equipment and reagents are extremely high. Current conventional testing methods are difficult to meet the requirements for detection sensitivity and specificity.

[0065] Among these, NfL (neurofiril light chain) is a biomarker of neuronal damage, and its levels are elevated in various neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis). GFAP (glial fibrillary acidic protein) is a marker of astrocyte activation, and its levels are elevated in brain injury and certain neurodegenerative diseases. For example, in the early diagnosis of Alzheimer's disease, plasma GFAP levels are significantly elevated even during the asymptomatic period. UCH-L1 (ubiquitin C-terminal hydrolase L1) is an enzyme associated with neurodegenerative processes, and its level changes can reflect the degree of neuronal damage; its levels may be elevated in brain injury and certain neurodegenerative diseases. p-tau 217 (phosphorylated tau protein at site 217) is an Alzheimer's disease-specific biomarker closely related to tau protein pathology in the brain; p-tau 217 levels are significantly elevated before the onset of Alzheimer's disease symptoms.

[0066] The reagents and detection methods described in this application can significantly increase the amount of luminescent microspheres in the detection system that can be used to receive singlet oxygen generated by photosensitive microspheres, thereby effectively improving the utilization rate of singlet oxygen, achieving the effect of increasing the intensity of the detection light signal, and thus improving the detection sensitivity, specificity and other detection performance, meeting the detection requirements of low-value samples such as blood samples for NfL, GFAP, UCH-L1 and p-tau 217.

[0067] III. Specific Implementation Examples To make the present invention easier to understand, the present application will be further described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not limited to the scope of application of the present application. Unless otherwise specified, the raw materials or components used in the present application can be obtained commercially or by conventional methods.

[0068] Next, taking the photo-induced chemiluminescence detection method as an example, we will compare the detection signal values ​​and discrimination between different experimental groups to determine the improvement effect of the multi-luminescent microsphere mode on the photo-induced chemiluminescence detection performance of low-value samples.

[0069] Example 1: Preparation of antibody-coated luminescent microspheres and biotin-labeled antibodies 1. The main experimental materials and equipment are shown in Table 1: Table 1

[0070] 2. Experimental Procedure 2.1 Luminescent microparticles coated with antibody Ab1 and tag molecule Spy-Tag ① Take 10 mg of luminescent microparticles into a centrifuge tube, wash twice with 0.05 M CB buffer and reconstitute; ② After dialyzing Ab1 with 0.05M CB buffer, the concentration was determined by the BCA method; ③ After thoroughly mixing the treated luminescent microparticles FG and Ab1 at a mass ratio of 10:0.75, react at 37℃ for 24 hours; ④ Take the Spy-Tag (obtained by constructing a plasmid using the Spy-Tag sequence AHIVMVDAYKPTK and inducing expression), dialyze it with 0.05M CB buffer, and then determine its concentration using the BCA method; ⑤ After thoroughly mixing the treated luminescent microparticles FG-Ab1 with Spy-Tag, react at 37°C for 24 hours; ⑥ Add 80 μL of glycine (75 mg / mL, 0.05 M CB) and 10 μL of NaBH4 (20 mg / mL, 0.05 M CB) to the reaction system, mix thoroughly, and react at 4 °C for 2 h to obtain the reaction solution; ⑦ After washing the reaction solution twice with PBST buffer, add the reconstitution solution (0.02M PBS + 0.5% BSA + 0.5% Tween 20 + 1% dextran) and wash once. Then add 1mL of the reconstitution solution and sonicate to reconstitute, thus obtaining luminescent microspheres coated with Ab1 and Spy Tag.

[0071] 2.2 Biotin-labeled antibody Ab2 ① Dialyze Ab2 with 0.05M NaHCO3 solution to remove impurities, and then determine the concentration using the BCA method; ② Add 30 molar amounts of Biotin-LC-LC-NHs to the dialyzed Ab2 solution, mix well, and react at 4℃ for 24 hours to obtain Bio-Ab2 solution. ③ After dialyzing with 0.02M PBS buffer to remove free biotin, the concentration was determined by BCA. Then, the reconstitution solution as described above was added to prepare a 3.3 μg / mL biotin-labeled antibody Bio-Ab2.

[0072] 2.3 Preparation and purification of Spy Catcher ① Construction of Catcher plasmid expression vector: The expression vector of Spy Catcher (E. coli vector-pet series expression plasmid) was constructed based on the sequence of Spy Catcher (GenBank accession number: JQ478411.1).

[0073] ②Catcher expression induced by *E. coli*: The *E. coli* expression vector induces the expression of Spy Catcher protein. Culture conditions: ① Self-induction medium (10g peptone, 5g yeast extract, 1x NPS, 1mM MgCl2, 1x 5052, pH=7.4), with 100nM ampicillin added; ② Induction conditions: overnight culture at 30℃ and 200rpm in a shaker.

[0074] ③Catcher purification: The cultured bacterial solution was centrifuged at 8000 rpm, and the bacterial pellet was collected. The bacterial cells were resuspended in 100 mL of purification loading buffer A (50 mM PBS, 150 mM NaCl, 20 mM ID, pH=7.4). The resuspended bacterial solution was centrifuged at 18000 rpm for 40 minutes, and the supernatant was collected and subjected to nucleic acid disruption, filtration, affinity column purification, elution, collection of protein, and reconstitution solution was added and sonicated to obtain Spy Catcher.

[0075] Example 2: The Influence of the Degree of Polymerization and Usage Ratio of Captured Molecules on the Photochemiluminescence Detection Performance of Low-Value Samples 1. Experimental Procedure 1.1 Reagent Preparation Taking NfL antibody molecules as an example, luminescent microspheres coated with NfL Ab1 and Spy Tag were diluted with a reconstitution solution to 83.3 μg / mL to obtain reagent R1. Bio-NfL Ab2 was diluted with a reconstitution solution to 3.3 μg / mL to obtain reagent R2. Dimers, pentamers, and octamers of Spy Catcher were prepared according to the method in Example 1, and then diluted with a reconstitution solution to obtain reagent R4 with a Tag molar ratio of 1:16, 1:8, 1:4, 1:2, 1:1, and 2:1, respectively. These were then combined with the photosensitive microparticle universal solution reagent R3 and detected with the test sample on a LiCA chemiluminescence detection system.

[0076] 1.2 Experimental Procedure Mix 30 μL of the test sample, 15 μL of reagent R1, and 15 μL of reagent R2, and incubate at 37 °C for 17 min. Add 175 μL of universal solution (containing reagent R3 with photosensitive microparticles) and incubate at 37 °C for 15 min. Add 15 μL of reagent R4, and add 15 μL of reconstituted solution to the control group, and incubate at 37 °C for 15 min respectively. After photoexcitation reaction, read the light signal value of each test group. The experimental data are shown below.

[0077] 2. Experimental Results 2.1 Effects of the Spy Catcher dimer reaction: Table 2 Signal Values

[0078] Table 3 Discrimination

[0079] 2.2 Spy Catcher pentamer reaction effect: Table 4 Signal Values

[0080] Table 5 Discrimination

[0081] 2.3 Spy Catcher Octamer Reaction Effect: Table 6 Signal Values

[0082] Table 7 Discrimination

[0083] 3. Results Analysis ① Referring to Table 2-7, within the current gradient range, the polymer particle size (degree of polymerization) does not affect the particle size of the luminescent microspheres that receive singlet oxygen. The higher the degree of polymerization of the first capturing molecule corresponding to the first tag molecule, the better the improvement effect. When the first capturing molecule is the Spy Catcher octamer, the positive signal and discrimination are significantly improved, and the detection performance of low-value samples is improved even more. ② When the molar ratio of Spy Catcher to Spy Tag in the R1 reagent is 1:(16~0.5), both the positive signal and the discrimination are improved; among them, the molar ratio of 1:4 (Spy Catcher octamer: Spy Tag) has the best effect, at which the positive signal and discrimination are significantly improved, and the detection performance of low value samples is improved even more.

[0084] ③ Using Spy Tag as a tag and Spy Catcher as a capture molecule significantly improves the positive signal and discrimination for both recombinant antigens and actual samples of the target analyte.

[0085] ④ The degree of polymerization and concentration of the first capturing molecule depend on the molecular weight of the first tag. The molar amount of the first capturing molecule should be lower than or equal to the molar amount of the first tag molecule. Furthermore, the degree of polymerization of the first capturing molecule is negatively correlated with the optimal molar amount; the higher the degree of polymerization, the lower the required optimal molar amount and the faster the reaction rate. The results show that when the product of the degree of polymerization and the molar amount of the first capturing molecule is 1 to 2.5 times the molar amount of the first tag molecule, both the positive signal and the discrimination are significantly improved.

[0086] Therefore, using a specific capture system such as Spy Catcher-Tag to aggregate free luminescent microspheres into a complex to form polymeric luminescent microspheres can improve positive signals and discrimination, significantly enhance the sensitivity of photo-induced chemiluminescence detection, and meet the detection needs of low-value samples.

[0087] Example 3: The effect of aggregation time on the detection performance of photochemiluminescence in low-value samples 1. Experimental Procedure 1.1 Reagent Preparation Taking NfL antibody molecules as an example, luminescent microspheres coated with NfL Ab1 and Spy Tag were diluted with a reconstitution solution to 83.3 μg / mL to obtain reagent R1. Bio-NfL Ab2 was diluted with a reconstitution solution to 3.3 μg / mL to obtain reagent R2. SpyCatcher octamer was diluted with a reconstitution solution to obtain reagent R4, which has a 1:4 molar ratio of Tag in reagent R1. These were then combined with the universal photosensitive microparticle solution reagent R3 and the sample to be tested on a LiCA chemiluminescence detection system.

[0088] 1.2 Experimental Procedure Mix 30 μL of the test sample, 15 μL of reagent R1, and 15 μL of reagent R2, and incubate at 37 °C for 17 min. Add 175 μL of universal solution (containing reagent R3 with photosensitive microparticles) and incubate at 37 °C for 15 min. Add 15 μL of reagent R4, and add 15 μL of reconstituted solution to the control group, and incubate at 37 °C for 3, 6, 9, 12, and 15 min, respectively. After photoexcitation, read the light signal value for each group. The experimental data are shown below.

[0089] 2. Experimental Results Table 8 Signal Values

[0090] Table 9 Discrimination

[0091] 3. Results Analysis Based on Tables 8-9, from the perspective of reagent performance, as the reaction time increases after the addition of reagent R4 (containing the first capture molecule), the positive signal and discrimination gradually improve and tend to reach equilibrium; the aggregation reaction of free luminescent microspheres reaches equilibrium at about 6 minutes of reaction.

[0092] Therefore, in the process of using a specific capture system such as Spy Catcher-Tag to aggregate free luminescent microspheres into a complex to form polymeric luminescent microspheres, from the perspective of balancing reaction time and performance, the reaction time after adding reagent R4 can be 0~15 min, the preferred reaction time is 3~9 min, and the optimal reaction time is 5~7 min.

[0093] Example 4: Applicability of the specific capture system to photochemiluminescence detection of low-value samples for different projects 1. Experimental Procedure 1.1 Reagent Preparation Luminescent microspheres coated with GFAP, UCH-L1, p-tau 217 antibody molecules Ab1 and Spy Tag were prepared and diluted to 83.3 μg / mL with a reconstitution solution to obtain reagent R1. Bio-Ab2 of the corresponding antibody molecule Ab2 was prepared and diluted to 3.3 μg / mL with a reconstitution solution to obtain reagent R2. Spy Catcher octamer was diluted with a reconstitution solution to obtain reagent R4, with a Tag molar ratio of 1:4 with reagent R1. These were then combined with the universal photosensitive microparticle solution reagent R3 and detected on a LiCA chemiluminescence detection system.

[0094] 1.2 Experimental Procedure Mix 30 μL of the test sample, 15 μL of reagent R1, and 15 μL of reagent R2, and incubate at 37 °C for 17 min. Add 175 μL of universal solution (containing reagent R3 with photosensitive microparticles) and incubate at 37 °C for 15 min. Add 15 μL of reagent R4, and add 15 μL of reconstituted solution to the control group, and incubate at 37 °C for 6 min respectively. After photoexcitation reaction, read the light signal value of each test group. The experimental data are shown below.

[0095] 2. Experimental Results 2.1 Detection results of GFAP: Table 10 Signal Values

[0096] Table 11 Discrimination

[0097] 2.2 UCH-L1 detection results: Table 12 Signal Values

[0098] Table 13 Discrimination

[0099] 2.3 Detection results of p-tau 217: Table 14 Signal Values

[0100] Table 15 Discrimination

[0101] 3. Results Analysis Based on Table 2-15, in terms of reagent performance, the use of Spy-Catcher / Spy-Tag can effectively improve the positive signal value and discrimination of low-value sample items such as NfL, GFAP, UCH-L1, and p-tau 217.

[0102] Therefore, the method of improving the detection capability of low-value samples using the specific capture system described in this application has a wide range of applications and can be used for the detection of a variety of biomarkers.

[0103] Example 5: Verification of the effect of different labeling systems on improving the detection capability of low-value samples 1. Experimental Procedure 1.1 Reagent Preparation ① Following the method of coating antibody onto luminescent microspheres in Example 1 above, prepare luminescent microspheres coated with NfL Ab1 carrying different tag molecules (Spy-Tag, His-Tag, Azide), and dilute them with a reconstitution solution to 83.3 μg / mL to obtain reagent R1.

[0104] ② Dilute Bio-NfL Ab2 with a reconstitution solution to 3.3 μg / mL to obtain reagent R2.

[0105] ③ The capture molecules (octamers Spy-Catcher, Anti-His, and BSA-DBCO corresponding to the tag molecules, with no less than 8 DBCO tags on each BSA molecule) are diluted with a reconstituted solution to R4 reagent at a molar ratio of 1:16, 1:4, and 1:1 with the tag molecules in reagent R1.

[0106] ④ Combine with photosensitive microparticle universal liquid reagent R3 and the sample to be tested on the LiCA chemiluminescence detection system.

[0107] 1.2 Experimental Procedure Mix 30 μL of the test sample, 15 μL of reagent R1, and 15 μL of reagent R2, and incubate at 37 °C for 17 min. Add 175 μL of universal solution (containing reagent R3 with photosensitive microparticles) and incubate at 37 °C for 15 min. Add 15 μL of reagent R4, and add 15 μL of reconstituted solution to the control group, and incubate at 37 °C for 15 min respectively. After photoexcitation reaction, read the light signal value of each test group. The experimental data are shown below.

[0108] 2. Experimental Results Table 18 Signal Values

[0109] Table 19 Discrimination

[0110] 3. Results Analysis Based on Tables 18-19, from the perspective of reagent performance, different specific capture systems such as Spy-Catcher / Spy-Tag, His-Tag / Anti His-Ab, and click chemistry can all improve the positive signal value and low-resolution of detection to varying degrees.

[0111] Therefore, the specific capture system described in this application has a good effect on the aggregation of free luminescent microspheres into polymeric luminescent microspheres to improve detection performance, and the photo-induced chemiluminescence detection sensitivity is effectively improved, which can meet the detection needs of low-value samples.

[0112] Example 6: The Influence of Tag Molecule Coating Method on Photochemiluminescence Detection Performance 1. Experimental Procedure 1.1 Reagent Preparation ① Following the method described above for coating antibody molecules and tag molecules onto luminescent microparticles, NfL Ab1 without tag molecules was coated onto luminescent microparticles. Then, the tag molecule Spy-Tag was added and different gradient ratios were set to complete the coating process to obtain luminescent microspheres. The microspheres were diluted with a reconstitution solution to 83.3 μg / mL to obtain reagent R1.

[0113] ② Prepare luminescent microspheres coated with the antibody molecule NfL Ab1 fused with the Spy-Tag in the same proportion. The Spy Tag (sequence AHIVMVDAYKPTK) was linked to antibody Ab1 using pClick technology to obtain the tag-linked antibody. The protein concentration of the collected fraction was determined by the BCA method, and the protein-containing fraction was then subjected to electrophoresis. Microspheres matching the molecular weight of the tag-antibody were collected and dialyzed into the buffer required for subsequent coupling reactions to obtain the co-expressed Ab1-SpyTag. Following the method of coating antibody molecules with luminescent microparticles in Example 1 above, luminescent microspheres coated with the fusion tag NfL Ab1-Spy Tag were prepared and diluted to 83.3 μg / mL with a reconstitution solution to obtain reagent R1.

[0114] ③ Dilute Bio-Ab2 to 3.3 μg / mL with a reconstitution solution to obtain reagent R2.

[0115] ④ Dilute the Spy Catcher octamer with a complex solution to R4 reagent, which has a molar ratio of 1:4 with the Tag in reagent R1.

[0116] ⑤ Combine with photosensitive microparticle universal liquid reagent R3 and the sample to be tested on the LiCA chemiluminescence detection system.

[0117] 1.2 Experimental Procedure Mix 30 μL of the test sample, 15 μL of reagent R1, and 15 μL of reagent R2, and incubate at 37 °C for 17 min. Add 175 μL of universal solution (containing reagent R3 with photosensitive microparticles) and incubate at 37 °C for 15 min. Add 15 μL of reagent R4, and add 15 μL of reconstituted solution to the control group, and incubate at 37 °C for 15 min respectively. After photoexcitation reaction, read the light signal value of each test group. The experimental data are shown below.

[0118] 2. Experimental Results Table 20 Signal Values

[0119] Table 21 Discrimination

[0120] 3. Results Analysis Referring to Tables 20-21, from the perspective of reagent performance, both the batch-by-batch, stepwise coating method and the method of first fusing the tag molecule to the antibody and then coating it onto the luminescent microspheres showed aggregation effects on the free luminescent microspheres. Under optimal conditions (Ab1:Spy Tag molar ratio of 1:2), there was no significant difference in the improvement of positive signal and discrimination between the batch-by-batch, stepwise coating method and the method of coating after fusion expression.

[0121] The first tag molecule, whether fused with the first antibody molecule and then coated onto the luminescent microspheres or coated independently, can be captured by the first capture molecule, causing the free luminescent microspheres to aggregate in the complex, and the reagent performance enhancement effect is consistent.

[0122] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A photo-induced chemiluminescence detection reagent, characterized in that, It includes two different sets of specific capture systems, each comprising a tag molecule and a capture molecule, the capture molecule being capable of specifically recognizing and binding to at least two tag molecules.

2. The reagent according to claim 1, characterized in that, The reagent also includes luminescent microspheres coated with a first antibody and a first tag molecule, wherein the first antibody can specifically bind to the target molecule in the sample to form an immune complex.

3. The reagent according to claim 2, characterized in that, The tagged antibody formed by fusing the first antibody with the first tag molecule is coated onto the luminescent microspheres; or The first antibody and the first tag molecule are respectively coated on the luminescent microspheres.

4. The reagent according to claim 1, characterized in that, The reagent also includes a second antibody and photosensitive microspheres; The second antibody is capable of specifically binding to the target molecule in the sample to be tested, and the second antibody carries a second tag molecule; the photosensitive microspheres are coated with a second capture molecule.

5. The reagent according to any one of claims 1 to 4, characterized in that, The two sets of specific capture systems are each independently selected from at least one of the Tag-Catcher system, click chemistry system, base pairing system, and biotin-avidin system; the two sets of specific capture systems do not react with each other; Preferably, the first tag molecule is selected from at least one of Spy-Tag, His-Tag, HA-Tag, Snoop-Tag, Flag-Tag, Myc-Tag, and antibody Fc fragment; Preferably, the first capturing molecule is selected from multimeric Catcher proteins with a degree of polymerization of not less than 2; more preferably, it is an octamer. Preferably, the second specific capture system is selected from the biotin-avidin system.

6. A photo-induced chemiluminescence detection method, characterized in that, The sample to be tested is reacted with a luminescent microsphere reagent containing a first antibody and a first tag molecule, a second antibody reagent containing a second tag molecule, and a photosensitive microsphere reagent containing a second capture molecule to form an immune complex. Then, a reagent containing the first capture molecule is added to react and photo-induced chemiluminescence detection is performed.

7. The method according to claim 6, characterized in that, The molar amount of the first captured molecule is less than or equal to the molar amount of the first tag molecule; Preferably, the molar ratio of the first capturing molecule to the first tag molecule is 1:(1~16); more preferably, it is 1:(2~8).

8. The method according to claim 7, characterized in that, The degree of polymerization of the first captured molecule is negatively correlated with its optimal molar amount; Preferably, the product of the degree of polymerization of the captured molecule and its molar amount is 1 to 2.5 times the molar amount of the first tag molecule.

9. The method according to claim 6, characterized in that, The reaction time after adding the reagent containing the first capturing molecule is 0-15 min; preferably 3-9 min; more preferably 5-7 min.

10. The application of the reagent as described in any one of claims 1 to 5, or the method as described in any one of claims 6 to 9, in the detection of biomarkers in blood samples, wherein the biomarkers include, but are not limited to, NfL, GFAP, UCH-L1, and p-tau 217.

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