Light-activated chemiluminescence detection method and application thereof

Abstract

The invention relates to a light-activated chemiluminescence detection method and application thereof. The method comprises the following steps: mixing and reacting a to-be-detected sample with acceptor microspheres and donor microsphere reagents to obtain a reaction solution, and adding tool molecules into the reaction solution; wherein the receptor microsphere is coated with two specific binding sites, the first specific binding site is selected from a first biomolecule capable of specifically recognizing and binding a target object to be detected, and the second specific binding site is selected from a first tag molecule capable of being specifically recognized and bound by a tool molecule; each tool molecule can be combined with at least two first tag molecules, and at least two acceptor microspheres are gathered through the combination of the tool molecules and the first tag molecules. According to the technical scheme, the light-activated chemiluminescence detection signal-to-noise ratio is increased by increasing the utilization rate of the singlet oxygen generated by the donor microspheres, the light-activated chemiluminescence detection performance such as the detection sensitivity and the detection result accuracy is improved, and the detection capacity of a low-value sample is improved.

Description

Technical Field

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

[0002] Chemiluminescence immunoassay is a method based on the specific immune reaction between antigens and antibodies, combined with chemiluminescence detection technology to quantitatively or qualitatively analyze target analytes. It is widely used in clinical diagnosis, disease monitoring, and biomedical research. Among these, photo-induced chemiluminescence analysis is a next-generation immunoassay technology based on nanoscale polymer particles. Its core principle utilizes two polymer particles to generate and transfer reactive oxygen species, achieving high sensitivity and rapid detection of the target analyte.

[0003] When the analyte is present in the detection system, the antibodies / antigens modified on the surfaces of the two microspheres react with the analyte to form an immune complex. Upon irradiation with excitation light, the donor microsphere in the complex generates singlet oxygen that diffuses to the acceptor microsphere. The chemiluminescent reagent in the acceptor microsphere captures the singlet oxygen and generates a light signal. Due to the high reactivity and short half-life of singlet oxygen, the singlet oxygen released by the donor microsphere can only effectively reach the acceptor microsphere and generate a light signal when the donor and acceptor microspheres are sufficiently close.

[0004] However, when the target analyte is absent from the system or its concentration is extremely low, the formed immune complexes are limited, and the singlet oxygen generated by the donor microspheres is rapidly quenched in the system, resulting in extremely low optical signals that are difficult to detect. Therefore, improving the signal-to-noise ratio (S / N) is of great significance for enhancing the detection capability of low-value samples. Summary of the Invention

[0005] To address or partially address the problems existing in related technologies, this application provides a photo-induced chemiluminescence detection method and its application, which can improve the utilization rate of singlet oxygen generated by donor microspheres, thereby improving the signal-to-noise ratio of photo-induced chemiluminescence detection and enhancing the detection performance of photo-induced chemiluminescence, such as detection sensitivity and accuracy of detection results.

[0006] The first aspect of this application provides a photo-induced chemiluminescence detection method, comprising: mixing a sample to be tested with receptor microspheres and donor microspheres to obtain a reaction solution, and adding a tool molecule to the reaction solution; wherein the receptor microspheres are coated with two specific binding sites, one of which is selected from a first biomolecule capable of specifically recognizing and binding the target analyte, and the other of which is selected from a first tag molecule capable of being specifically recognized and bound by the tool molecule; each tool molecule can bind to at least two of the first tag molecules.

[0007] In some embodiments, the number of moles of the receptor microspheres is greater than the number of moles of the target analyte in the sample to be tested.

[0008] In some embodiments, the tool molecule and the first tag molecule can specifically bind by forming a covalent bond or a non-covalent bond; preferably, the tool molecule and the first tag molecule are selected from Tag-Catcher, click chemistry, or base pairing systems; preferably, the first tag molecule is selected from one or more of Spy-Tag, His-Tag, HA-Tag, Snoop-Tag, Flag-Tag, Myc-Tag, and antibody Fc fragments.

[0009] In some embodiments, the molar amount of the added tool molecule is less than or equal to the molar amount of the first tag molecule in the reaction solution; preferably, the molar ratio of the tool molecule to the first tag molecule is 1:(1~16); more preferably, it is 1:(2~8).

[0010] In some embodiments, the degree of polymerization of the tool molecule is negatively correlated with its optimal molar amount; preferably, the product of the degree of polymerization of the tool molecule and its molar amount is 1 to 2.5 times the molar amount of the first tag molecule; preferably, the tool molecule is selected from multimeric Catcher protein with a degree of polymerization of not less than 2; preferably, it is octamer Catcher protein.

[0011] In some embodiments, the first tag molecule and the first biomolecule are co-expressed and then coated together onto the receptor microspheres; or, the first tag molecule and the first biomolecule are separately coated onto the receptor microspheres.

[0012] In some embodiments, the tool molecule is added and then incubated for 0-15 min; preferably 3-9 min; more preferably 5-7 min.

[0013] In some embodiments, the method includes: mixing and reacting a sample to be tested with a receptor microsphere reagent containing a first biomolecule and a first tag molecule, a second biomolecule reagent containing a second tag molecule, and a donor microsphere reagent containing a pairing molecule to obtain a reaction solution; The first and second biomolecules can specifically bind to the target analyte in the sample to be tested, respectively; the paired molecule can specifically recognize and bind to the second tag molecule.

[0014] In some embodiments, the second tag molecule and the pairing molecule do not react with each other, nor with the first tag molecule and the tool molecule.

[0015] In some embodiments, the second tag molecule and the pairing molecule are selected from the biotin-avidin system.

[0016] A second aspect of this application provides an application of the above method in detecting biomarkers in blood samples; the biomarkers include, but are not limited to, NfL, GFAP, UCH-L1, and p-tau 217.

[0017] It is worth noting that the above applications are for purposes other than disease diagnosis.

[0018] The technical solution provided in this application can include the following beneficial effects: By leveraging the ability of the tool molecules added to the reaction system to capture the first tag molecules on the receptor microspheres, at least two receptor microspheres coated with the first tag molecules can aggregate, thereby binding free receptor microspheres that are not bound to the analyte to the receptor microspheres in the "receptor microsphere-analyte-donor microsphere" sequence. This increases the amount of receptor microspheres capable of receiving singlet oxygen generated by the donor microspheres, improves singlet oxygen utilization, and thus enhances the detection signal value and photochemiluminescence detection performance, meeting the detection requirements for low-value samples. During the immune reaction, the receptor microspheres remain in their original state, with no change in particle size or mass, ensuring the rate of antigen-antibody immune reaction and avoiding a decrease in detection sensitivity due to a slowdown in the migration rate of receptor microspheres caused by increased particle size. The detection method is simple, easy to operate, low in cost, highly applicable, and has a wide range of applications, enabling its use in the detection of various biomarkers.

[0019] 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

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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 pathogens or animal tissues, or recombinant antigens with specific antigenic properties prepared through genetic engineering techniques. Where necessary, antigens can be further conjugated to other components, such as specific binding pairing members, for example, biotin or avidin.

[0026] The terms "combination," "connection," and "coupling" as 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.

[0027] 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.

[0028] 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.

[0029] The specific binding site described in this article refers to a specific region on or inside the surface of a biomolecule (such as a protein or nucleic acid) that can bind to a specific ligand (such as another molecule or ion) with high selectivity and affinity. For example, the binding of an antibody to an antigen occurs through a specific binding site on the antibody molecule that binds to a specific antigenic epitope.

[0030] The tool molecules described in this article refer to molecules capable of specifically recognizing target substances and binding to them via covalent or non-covalent bonds. Tool molecules are tools used to capture and enrich target substances. Each tool molecule possesses at least one specific binding site, enabling it to specifically bind to at least one target substance.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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+ .

[0037] 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.

[0038] 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.

[0039] 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.

[0040] II. Specific Implementation Plan This application will now be described in more detail.

[0041] In photocatalytic chemiluminescence analysis, donor microspheres (GG) coated with antibodies / antigens and receptor microspheres (FG) coated with antibodies / antigens specifically bind to the analyte (antigen / antibody) in the sample, forming a sandwich immune complex of "receptor microsphere-analyte-donor microsphere". When the donor microspheres are irradiated with light of a specific wavelength, they generate singlet oxygen. This singlet oxygen diffuses into the immune complex, exciting the luminescent composition on the receptor microspheres and generating a chemiluminescent signal. Qualitative or quantitative analysis of the analyte antigen / antibody can be achieved by detecting the intensity of this luminescent signal.

[0042] 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-concentration target analytes.

[0043] The photo-induced chemiluminescence detection method disclosed in this application can improve the detection signal value by increasing the utilization rate of singlet oxygen generated by the donor microspheres, thereby improving the signal-to-noise ratio of photo-induced chemiluminescence detection and enhancing the detection performance of photo-induced chemiluminescence, such as detection sensitivity and accuracy of detection results, so as to improve the detection capability of low-value samples.

[0044] The detection method of this application includes: mixing the sample to be tested with receptor microspheres and donor microspheres to obtain a reaction solution, and adding a tool molecule to the reaction solution. The receptor microspheres are coated with two specific binding sites: one specific binding site is selected from a first biomolecule capable of specifically recognizing and binding to the target analyte, and the other specific binding site is selected from a first tag molecule capable of being specifically recognized and bound by the tool molecule; each tool molecule can bind to at least two first tag molecules.

[0045] In this embodiment, by adding a tool molecule to the reaction system, its ability to capture the first tag molecule on the acceptor microspheres enables at least two acceptor microspheres coated with the first tag molecule to aggregate. This allows free acceptor microspheres not bound to the analyte in the reaction system to bind to the acceptor microsphere in the "acceptor microsphere-analyte-donor microsphere" chain (hereinafter referred to as the acceptor microsphere bound to the analyte as the host acceptor microsphere), thereby bringing more acceptor microspheres closer to the donor microsphere. When the donor microsphere is excited by light of a specific wavelength, the singlet oxygen generated can be captured by the host acceptor microsphere and at least one free acceptor microsphere, reducing the quenched singlet oxygen in the system, improving the utilization rate of singlet oxygen, and thus increasing the detection signal value. This enhances the photochemiluminescence detection performance and meets the detection requirements of low-value samples.

[0046] Furthermore, after the antigen-antibody immune reaction ends and a sandwich immune complex of "receptor microsphere-analyte-donor microsphere" is formed, the added tool molecule remains in its original state during the immune reaction, without changing its particle size or mass. It has a good particle migration rate, which can ensure the occurrence rate of the antigen-antibody immune reaction and avoid the reduction in detection sensitivity due to the slowing of the migration rate of the receptor microsphere caused by the increase in the particle size of the receptor microsphere. Therefore, the detection sensitivity can be improved overall through the method described in this application.

[0047] In some embodiments of this application, the molar number of receptor microspheres is greater than the molar number of the target analyte in the sample to be tested, thus meeting the detection requirements for low-value samples.

[0048] In some embodiments of this application, the tool molecule and the first tag molecule can achieve specific binding through the formation of covalent or non-covalent bonds. Specifically, the tool molecule and the first tag molecule can form a specific tag system, which can be selected from the Tag-Catcher system, click chemistry system, or base complementary pairing system.

[0049] For example, when selected from the Tag-Catcher system, the irreversible and stable binding of the tool molecule and the first tag molecule can be achieved through the isopeptide bonds specifically formed by the Tag and Catcher, thereby allowing the free acceptor microspheres to aggregate with the host acceptor microspheres to obtain a highly stable polymeric composition. Alternatively, click chemistry systems capable of forming specific carbon-heteroatom bonds, or base complementary pairing systems that selectively form hydrogen bonds, can also be selected.

[0050] In some specific embodiments of this application, the first tag molecule may 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 tool molecule is selected from the corresponding Spy-Catcher; when it is selected from His-Tag, the tool molecule is selected from the corresponding Anti-His (in this case, the Catcher is Anti-His); when it is selected from Snoop-Tag, the tool molecule is selected from the corresponding Snoop-Catcher, etc. Catchers and Tags have excellent specific recognition and binding capabilities, and using a tag with a small molecular weight as the first tag molecule to coat the receptor microspheres has no significant impact on the expression of the first biomolecule or the properties of the receptor microspheres.

[0051] In some embodiments of this application, the molar amount of the added tool molecule is less than or equal to the molar amount of the first tag molecule. Preferably, the molar ratio of the tool molecule to the first tag molecule is 1:(1~16); more preferably, it is 1:(2~8). The proportion of tool molecules directly affects the aggregation of receptor microspheres. If it is too low, the aggregation efficiency and degree will be low, and the effect on improving detection performance will not be obvious; if it is too high, it is easy to produce a "hook effect", making it difficult for receptor microspheres to aggregate, resulting in no significant improvement in detection performance, or even affecting the accuracy of detection results.

[0052] When the first tag molecule and the tool molecule are selected from the Tag-Catcher system, the higher the degree of polymerization of the tool molecule (Catcher), the lower its required optimal molar amount; that is, the degree of polymerization of the tool molecule is negatively correlated with its optimal molar amount. Preferably, when the product of the degree of polymerization of the tool 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 will affect the aggregation rate and degree of aggregation of the receptor microspheres, and the performance improvement will not be significant.

[0053] Furthermore, the tool molecule can be selected from the monomer or multimeric protein of the Catcher; preferably a multimeric protein with a degree of polymerization of not less than 2, and more preferably an octamer of the Catcher. The degree of polymerization of the Catcher directly affects the rate at which the Catcher captures the tag, and thus affects the aggregation rate of the receptor microspheres. The higher the degree of polymerization of the Catcher, the higher the tag capture rate, the higher the aggregation rate of the receptor microspheres, and the better the effect on enhancing the light signal intensity.

[0054] The degree of polymerization and amount of the tool molecule can be determined based on the amount of the first tag molecule. The higher the degree of polymerization of the tool 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 tool molecule, the higher the required optimal molar amount and the faster the aggregation rate.

[0055] In some embodiments of this application, the first tag molecule can be co-expressed with the first biomolecule and then coated onto the receptor microspheres. Alternatively, the first tag molecule and the first biomolecule can be coated onto the receptor microspheres separately or stepwise. This application does not limit the coupling order of the first tag molecule and the first biomolecule on the receptor microspheres.

[0056] The first biomolecule can be an antibody molecule, and the first tag molecule can bind to the constant region of the antibody molecule through co-expression to form 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 allows for a specific binding immune response to the target analyte. This is beneficial for increasing the effective antibody quantity and activity in the reaction system, thereby improving detection sensitivity.

[0057] In some embodiments of this application, the incubation time after adding the tool molecule can be 0-15 min. As the reaction time increases, the aggregation reaction of the receptor microspheres gradually increases and tends to equilibrium; therefore, the reaction time is preferably 3-9 min, more preferably 5-7 min.

[0058] Based on the aforementioned method for improving the detection capability of low-value samples in chemiluminescence detection, this application also relates to a chemiluminescence detection kit for low-value samples, specifically a photo-induced chemiluminescence detection kit. It includes: acceptor microspheres coated with a first biomolecule and a first tag molecule, a second biomolecule coupled with a second tag molecule, donor microspheres coated with a pairing molecule, and tool molecules. The first and second biomolecules can specifically recognize and bind to the analyte in the sample, and the pairing molecule can specifically recognize and bind to the second tag molecule.

[0059] Based on the above-described photo-induced chemiluminescence detection method, this detection method may specifically include the following steps: S1. The sample to be tested is mixed with a receptor microsphere reagent containing a first biomolecule and a first tag molecule, a second biomolecule reagent containing a second tag molecule, and a donor microsphere reagent containing a pairing molecule to obtain a reaction solution; S2. Add a reagent containing the above-mentioned tool molecules to the reaction solution and mix, then detect the light signal value; The first and second biomolecules can specifically bind to the target analyte in the sample, and each tool molecule can bind to at least two first tag molecules, while the paired molecule can specifically bind to the second tag molecule.

[0060] After the routine detection sequence of the sample to be tested described in step S1 is completed, the resulting reaction solution contains a sandwich immune complex of "receptor microspheres-target analyte-donor microspheres" and unreacted free receptor microspheres. The added tool molecules at this point can cause the free receptor microspheres to aggregate around the main receptor microspheres of the complex. The singlet oxygen generated by the donor microspheres can be received by multiple receptor microspheres (including the main receptor microsphere and at least one free receptor microsphere) and simultaneously generate a light signal, effectively improving the utilization rate of singlet oxygen, increasing the light signal intensity, and thus enhancing the detection sensitivity, meeting the photochemiluminescence detection requirements for low-value samples.

[0061] Furthermore, the timing of adding the tool molecule in this scheme after the immune reaction is complete does not alter the original detection process, making it highly applicable and broadly applicable. It represents a platform-type improvement and can be applied to the detection of various biomarkers. Moreover, this scheme does not interfere with the original immune reaction, significantly improving detection performance while maintaining the characteristics of a simple, easy-to-operate, and low-cost detection method.

[0062] To avoid interference from specific pairing reactions between the two sets of specific tagging systems, which could lead to receptor microsphere aggregation and affect the accuracy and stability of the detection results, the two sets of specific tagging systems, consisting of the first tag molecule and the tool molecule, and the second tag molecule and the pairing molecule, are selected from two sets of non-reactive systems.

[0063] In some embodiments of this application, the specific tagging system consisting of the second tag molecule and the pairing molecule may be selected from the biotin-avidin system.

[0064] For example, when the first tag molecule and tool molecule can be selected from the Tag-Catcher system, the second tag molecule and pairing molecule can be selected from the biotin-avidin system. The Tag, as the first tag molecule, coats the recipient microspheres; its small molecular weight does not significantly affect the properties of the luminescent microspheres. The Cater, as the tool molecule, can bind at least two first tag molecules, resulting in high capture efficiency and high aggregation effect and efficiency of the recipient microspheres. The second biomolecule is labeled with biotin, and avidin or streptavidin coats the donor microspheres.

[0065] In some embodiments of this application, the detection method further includes diluting each component in the kit to a suitable concentration before mixing the reagents with the sample to be tested. Therefore, the kit also includes a buffer solution or diluent for diluting the components.

[0066] Furthermore, the buffer solution may contain components such as a buffer solution and a stabilizer. 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. For example, the buffer solution may include PBS buffer, bovine serum albumin, dextran, and Tween 20.

[0067] Correspondingly, the detection method includes: S10. The components in the kit were diluted with a buffer solution to obtain reagent R1 containing receptor microspheres, reagent R2 containing a second biomolecule, reagent R3 containing donor microspheres, and reagent R4 containing a tool molecule. S11. Mix the sample to be tested with reagents R1 and R2, and incubate at 37°C to obtain the first reactant; S12. Add reagent R3 and incubate at 37°C to obtain the second reactant; S21. Add reagent R4 and incubate at 37°C to obtain the third reactant; S22. Irradiate the third reactant with light of a specific wavelength and detect the luminescence value.

[0068] The method described in this application can be applied to the detection of biomarkers in blood samples, and is particularly suitable for photo-induced chemiluminescence detection of low-value samples. The biomarkers detected include, but are not limited to, NfL, GFAP, UCH-L1, and p-tau217.

[0069] 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.

[0070] 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.

[0071] The detection method described in this application can significantly increase the amount of acceptor microspheres in the detection system that can be used to receive singlet oxygen generated by donor microspheres, thereby effectively improving the utilization rate of singlet oxygen, achieving the effect of increasing the intensity of detection light signal, and thus improving 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.

[0072] 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.

[0073] 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-acceptor microsphere mode on the photo-induced chemiluminescence detection performance of low-value samples.

[0074] Example 1: Preparation of antibody-coated receptor microspheres and biotin-labeled antibodies 1. The main experimental materials and equipment are shown in Table 1: Table 1

[0075] 2. Experimental Procedure 2.1 Luminescent microparticles coated with antibody Ab1 and the first 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℃ 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, wash it once with reconstitution solution (0.02M PBS + 0.5% BSA + 0.5% Tween 20 + 1% dextran), and then add 1mL of reconstitution solution and sonicate to reconstitute, thus obtaining receptor microspheres coated with Ab1 and Spy Tag.

[0076] 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.

[0077] 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).

[0078] 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.

[0079] 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.

[0080] Example 2: Effects of the degree of polymerization and proportion of tool proteins on photochemiluminescence detection performance 1. Experimental Procedure 1.1 Reagent Preparation Taking NfL antibody molecules as an example, receptor microspheres coated with NfL Ab1 and Spy Tag were diluted to 83.3 μg / mL with a reconstitution solution to obtain reagent R1. Bio-NfL Ab2 was diluted to 3.3 μg / mL with a reconstitution solution 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.

[0081] 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.

[0082] 2. Experimental Results 2.1 Effects of the Spy Catcher dimer reaction: Table 2 Signal Values

[0083] Table 3 Discrimination

[0084] 2.2 Spy Catcher pentamer reaction effect: Table 4 Signal Values

[0085] Table 5 Discrimination

[0086] 2.3 Spy Catcher Octamer Reaction Effect: Table 6 Signal Values

[0087] Table 7 Discrimination

[0088] 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 receptor microspheres that receive singlet oxygen. The higher the degree of polymerization of the tool molecule corresponding to the first tag molecule, the better the improvement effect. When the tool molecule is the SpyCatcher 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:(1~16), 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.

[0089] ③ Using Spy Tag as a tag and Spy Catcher as a pairing molecule significantly improves the positive signal and discrimination for both recombinant antigens and actual samples of the target analyte.

[0090] ④ The degree of polymerization of the tool molecule and its concentration depend on the molecular weight of the first tag. The molar amount of the tool molecule should be lower than or equal to the molar amount of the first tag molecule, and the degree of polymerization of the tool molecule is negatively correlated with the optimal molar amount. The results showed that when the product of the degree of polymerization of the tool molecule and its molar amount was 1 to 2.5 times the molar amount of the first tag molecule, the positive signal and discrimination were significantly improved.

[0091] Therefore, using a specific tagging system such as Spy Catcher-Tag to aggregate free receptor microspheres into a complex to form multi-receptor microspheres can improve positive signals and discrimination, significantly enhance photochemiluminescence detection sensitivity, and meet the detection needs of low-value samples.

[0092] Example 3: The effect of aggregation time on photochemiluminescence detection performance 1. Experimental Procedure 1.1 Reagent Preparation Taking NfL antibody molecules as an example, receptor microspheres coated with NfL Ab1 and Spy Tag were diluted to 83.3 μg / mL with a reconstitution solution to obtain reagent R1. Bio-NfL Ab2 was diluted to 3.3 μg / mL with a reconstitution solution 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 detected on a LiCA chemiluminescence detection system with the sample to be tested.

[0093] 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.

[0094] 2. Experimental Results Table 8 Signal Values

[0095] Table 9 Discrimination

[0096] 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 tool molecules), the positive signal and discrimination gradually improve and tend to reach equilibrium; the aggregation reaction of free receptor microspheres reaches equilibrium at about 6 minutes of reaction.

[0097] Therefore, in the process of using a specific tagging system such as Spy Catcher-Tag to aggregate free receptor microspheres into a complex to form polymeric receptor microspheres, from the perspective of balancing reaction time and performance, the reaction time after adding R4 reagent can be 0~15min, the preferred reaction time is 3~9min, and the optimal reaction time is 5~7min.

[0098] Example 4: Applicability of Specific Labeling Systems to Different Items 1. Experimental Procedure 1.1 Reagent Preparation Receptor 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.

[0099] 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.

[0100] 2. Experimental Results 2.1 Detection results of GFAP: Table 10 Signal Values

[0101] Table 11 Discrimination

[0102] 2.2 UCH-L1 detection results: Table 12 Signal Values

[0103] Table 13 Discrimination

[0104] 2.3 Detection results of p-tau 217: Table 14 Signal Values

[0105] Table 15 Discrimination

[0106] 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.

[0107] Therefore, the method for improving the detection capability of low-value samples using the specific labeling system described in this application is applicable to the detection of NfL, GFAP, UCH-L1, p-tau 217 and other items.

[0108] Example 5: Verification of the effect of different labeling systems on improving detection capabilities 1. Experimental Procedure 1.1 Reagent Preparation Following the method for coating antibody onto receptor microspheres as described in Example 1 above, receptor microspheres coated with NfL Ab1 bearing different first tag molecules (Spy-Tag, His-Tag, Azide) were prepared and diluted to 83.3 μg / mL with a reconstitution solution to obtain reagent R1. Bio-NfL Ab2 was diluted to 3.3 μg / mL with a reconstitution solution to obtain reagent R2. Tool proteins (octamers Spy-Catcher, Anti-His, and BSA-DBCO corresponding to the first tag molecules, with at least 8 DBCO tags on each BSA molecule) were diluted with a reconstitution solution to reagent R4 with a molar ratio of 1:16, 1:4, and 1:1 to the first tag molecules in reagent R1. These were then combined with the universal photosensitive microparticle solution reagent R3 and detected on a LiCA chemiluminescence detection system with the test sample.

[0109] 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.

[0110] 2. Experimental Results Table 18 Signal Values

[0111] Table 19 Discrimination

[0112] 3. Results Analysis Based on Tables 18-19, from the perspective of reagent performance, the use of different specific tagging 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.

[0113] Therefore, the specific tagging system described in this application has a good effect on the aggregation of free receptor microspheres into multi-receptor 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.

[0114] Example 6: The impact of the coating method of the first tag molecule on detection performance 1. Experimental Procedure 1.1 Reagent Preparation ① Following the method described above for coating antibody molecules and first tag molecules onto luminescent microparticles, NfL Ab1 without the first tag molecule was coated onto the luminescent microparticles. Then, the first tag molecule Spy-Tag was added and different gradient ratios were set to complete the coating process to obtain receptor microspheres. The microspheres were then diluted with a reconstitution solution to 83.3 μg / mL to obtain reagent R1.

[0115] ② Receptor microspheres coated with the antibody molecule NfL Ab1, fused with the first tag molecule Spy-Tag, in the same proportion were prepared. 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. The fractions that met 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. Receptor microspheres coated with the fusion tag NfL Ab1-Spy Tag were prepared according to the method of coating antibody molecules with luminescent microparticles in Example 1 above, and diluted to 83.3 μg / mL with reconstitution solution to obtain reagent R1.

[0116] ③ Dilute Bio-Ab2 to 3.3 μg / mL with a reconstitution solution to obtain reagent R2.

[0117] 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.

[0118] ⑤ Combine with photosensitive microparticle universal liquid reagent R3 and the sample to be tested on the LiCA chemiluminescence detection system.

[0119] 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.

[0120] 2. Experimental Results Table 20 Signal Values

[0121] Table 21 Discrimination

[0122] 3. Results Analysis Referring to Tables 20-21, from the perspective of reagent performance, both the batch-by-batch, stepwise coating method using the first tag molecule and antibody, and the method of first fusing the first tag molecule into the antibody and then coating it onto the receptor microspheres, showed aggregation effects on free receptor 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.

[0123] The first tag molecule, whether fused with an antibody molecule or individually coated onto receptor microspheres, can be captured by the tool protein, causing the free receptor microspheres to aggregate in the complex, and the reagent performance enhancement effect is consistent.

[0124] 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 method, characterized in that, include: The sample to be tested is mixed with acceptor microspheres, donor microspheres and reagents to obtain a reaction solution, and tool molecules are added to the reaction solution; The receptor microspheres are coated with two specific binding sites. Specific binding site one is selected from a first biomolecule that can specifically recognize and bind to the target analyte, and specific binding site two is selected from a first tag molecule that can be specifically recognized and bound by the tool molecule. Each tool molecule can bind to at least two of the first tag molecules.

2. The method according to claim 1, characterized in that, The number of moles of the receptor microspheres is greater than the number of moles of the target analyte in the sample to be tested.

3. The method according to claim 1, characterized in that, The tool molecule and the first tag molecule can bind specifically through covalent or non-covalent bonding. Preferably, the tool molecule and the first tag molecule are selected from Tag-Catcher, click chemistry, or base complementary pairing systems; Preferably, the first tag molecule is selected from one or more of Spy-Tag, His-Tag, HA-Tag, Snoop-Tag, Flag-Tag, Myc-Tag, and antibody Fc fragments.

4. The method according to claim 1, characterized in that, The molar amount of the added tool molecule is less than or equal to the molar amount of the first tag molecule in the reaction solution; Preferably, the molar ratio of the tool molecule to the first tag molecule is 1:(1~16); more preferably, it is 1:(2~8). Preferably, the degree of polymerization of the tool molecule is negatively correlated with its optimal molar amount; Preferably, the product of the degree of polymerization of the tool molecule and its molar amount is 1 to 2.5 times the molar amount of the first tag molecule; Preferably, the tool molecule is selected from multimeric Catcher protein with a degree of polymerization of not less than 2; preferably, it is octamer Catcher protein.

5. The method according to claim 1, characterized in that, The first tag molecule and the first biomolecule are co-expressed and then co-coated onto the receptor microspheres; or The first tag molecule and the first biomolecule are respectively coated onto the receptor microspheres.

6. The method according to claim 1, characterized in that, After adding the tool molecule, incubate for 0-15 min; preferably 3-9 min; more preferably 5-7 min.

7. The method according to claim 1, characterized in that, include: The sample to be tested is mixed with a receptor microsphere reagent containing a first biomolecule and a first tag molecule, a second biomolecule reagent containing a second tag molecule, and a donor microsphere reagent containing a pairing molecule to obtain a reaction solution. The first and second biomolecules can specifically bind to the target analyte in the sample to be tested, respectively; the paired molecule can specifically recognize and bind to the second tag molecule.

8. The method according to claim 7, characterized in that, The second tag molecule and its pairing molecule do not react with each other, nor with the first tag molecule and its tool molecule.

9. The method according to claim 7, characterized in that, The second tag molecule and pairing molecule are selected from the biotin-avidin system.

10. The application of the method of any one of claims 1 to 9 in detecting biomarkers in blood samples; said biomarkers include, but are not limited to, NfL, GFAP, UCH-L1, and p-tau 217.

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