A microsphere composition for homogeneous chemiluminescent detection

Abstract

The present application provides a kind of microsphere composition for homogeneous chemiluminescence detection, it includes: luminescent latex microspheres, which can generate chemiluminescence after interacting with singlet oxygen, the luminescent latex microspheres contain optical reaction substance, and surface is connected with bioactive substance;Photosensitive latex microspheres, which can generate singlet oxygen after being excited by light, the photosensitive latex microspheres contain photosensitizer, and surface is connected with avidin;And blank latex microspheres, which do not contain any optical reaction substance, and surface is not connected with bioactive substance;Wherein, the material of the luminescent latex microspheres, photosensitive latex microspheres and blank latex microspheres is same, and all are polystyrene latex microspheres.The microsphere composition provided by the present application can eliminate the interference effect of the sample to be measured in the photochemical chemiluminescence method, avoid generating non-specific signal, and improve the accuracy of detection.

Description

Technical Field

[0001] This invention belongs to the field of photo-induced chemiluminescence analysis, and specifically relates to a microsphere composition for homogeneous chemiluminescence detection. Background Technology

[0002] Oropharyngeal or nasopharyngeal swab samples are typically collected from the oropharynx or nasopharynx using a relatively long cotton swab. These secretions are then placed in a pathogen extract solution. This type of sample is used clinically to test for pathogens, commonly used to detect respiratory pathogens such as influenza virus and coronavirus. Saliva is a digestive fluid, mostly water, but also contains mucin and salivary amylase. Nasal aspirate is secretion extracted from the nasal cavity using a special device. These samples contain components such as mucin secreted by epithelial cells. When using photoluminescence immunoassay (PLIA) to detect these sample types, mucin can strongly interfere with the detection system. This is because mucin carries a charge, while the microspheres used in PLATIC carry the opposite charge. Within the reaction system, the mucin attracts the two types of microspheres, directly linking them together to generate a signal. Therefore, it is necessary to address sample interference when using PLATIC to detect these types of samples. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a microsphere composition for homogeneous chemiluminescence detection to address the shortcomings of the prior art and solve the problem of non-characteristic signals generated when samples such as pharyngeal swabs are analyzed using photo-induced chemiluminescence analysis technology.

[0004] Therefore, a first aspect of the present invention provides a microsphere composition for homogeneous chemiluminescence detection, the microsphere composition comprising:

[0005] Luminescent latex microspheres, which can produce chemiluminescence after reacting with singlet oxygen, contain optically reactive substances and have bioactive substances attached to their surface;

[0006] Photosensitive latex microspheres, which generate singlet oxygen upon photoexcitation, contain a photosensitizer and have avidin attached to their surface; and

[0007] Blank latex microspheres contain no optically reactive substances and have no bioactive substances attached to their surface.

[0008] The luminescent latex microspheres, photosensitive latex microspheres, and blank latex microspheres are all made of the same material, and are all polystyrene latex microspheres.

[0009] According to some embodiments of the present invention, the surface of the blank latex microspheres is modified with chemical groups, wherein the chemical groups are selected from at least one of epoxy, chloromethyl, mercapto, amino, hydroxy, maleamine, sulfonic acid, carboxyl and aldehyde groups.

[0010] According to some embodiments of the present invention, the chemical group is selected from at least one of sulfonic acid group, carboxyl group and aldehyde group.

[0011] According to some embodiments of the present invention, the chemical group is a carboxyl group.

[0012] According to some embodiments of the present invention, the blank latex microspheres are prepared by an emulsion polymerization method without emulsifiers.

[0013] According to some embodiments of the present invention, the average particle size of the blank latex microspheres is 60-1000 nm, for example, it can be 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, or 900 nm.

[0014] According to a preferred embodiment of the present invention, the average particle size of the blank latex microspheres is 100-300 nm.

[0015] According to some embodiments of the present invention, the particle size of the blank latex microspheres is smaller than that of the luminescent latex microspheres.

[0016] According to some embodiments of the present invention, the particle size of the blank latex microspheres is smaller than that of the photosensitive latex microspheres.

[0017] According to some embodiments of the present invention, the surface of the blank latex microspheres carries a negative charge.

[0018] According to some embodiments of the present invention, the zeta potential of the blank latex microspheres is -10 to -50 mV, for example, it can be -15 mV, -20 mV, -25 mV, -30 mV, -35 mV, -40 mV, -45 mV, or -50 mV.

[0019] According to some embodiments of the present invention, the zeta potential of the blank latex microspheres is -15 to -30 mV.

[0020] According to some embodiments of the present invention, the blank latex microspheres and the luminescent latex microspheres carry the same type of charge.

[0021] A second aspect of the present invention provides the application of the microsphere composition according to the first aspect in the detection of analytes in oropharyngeal swabs, nasopharyngeal swabs, saliva or nasal aspirate samples.

[0022] According to some embodiments of the present invention, the method for detecting the analyte in the above-mentioned sample using the microsphere composition includes the following steps:

[0023] S1: The sample to be tested is mixed with blank latex microspheres and luminescent latex microspheres to obtain a first mixture;

[0024] S2: The first mixture is mixed with photosensitive latex microspheres to obtain a second mixture;

[0025] S3: Irradiate the second mixture with a laser and detect the chemiluminescence signal.

[0026] According to some embodiments of the present invention, in step S1, the temperature of the first mixing is 30-45°C and the time is 10-30 minutes.

[0027] According to a preferred embodiment of the present invention, in step S2, the temperature of the second mixing is 30-45°C and the time is 5-30 minutes.

[0028] According to some embodiments of the present invention, in step S3, the excitation wavelength of the laser irradiation is 650-700nm, and the detection wavelength is 550-650nm.

[0029] According to a preferred embodiment of the present invention, in step S3, the excitation wavelength of the laser irradiation is 675-685nm, and the detection wavelength is 610-620nm.

[0030] In this invention, when detecting antigens using photoluminescence chemiluminescence, a certain amount of blank latex microspheres are added to the reaction system. These blank latex microspheres carry the same charge as the microspheres used in photoluminescence chemiluminescence. By adsorbing interfering substances such as mucins through the blank latex microspheres, the interference caused by samples such as oropharyngeal swabs, nasopharyngeal swabs, saliva, and nasal aspirate in the photoluminescence method is eliminated. This solves the problem of non-specific signals generated by such samples when using photoluminescence analysis technology, thereby improving the accuracy of detection. Detailed Implementation

[0031] 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.

[0032] 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.

[0033] 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 similar to or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are now described.

[0034] The invention will now be described in more detail.

[0035] The technical principle of photo-induced chemiluminescence analysis is as follows: Under laser irradiation, the photosensitizer can excite oxygen molecules in the surrounding environment into singlet oxygen molecules. The singlet oxygen molecules can react with optical reactive substances about 200 nm away to generate a light signal of a certain wavelength. When the sample contains the antigen or antibody to be tested, the immune reaction of the antigen and antibody can cause the photosensitive latex microspheres containing the photosensitizer to bind with the luminescent latex microspheres containing the optical reactive substances, thereby generating a light signal of a specific wavelength. The content of the antigen or antibody to be tested can be detected by detecting this light signal.

[0036] "Sample to be tested" refers to a mixture that may contain the analyte. Typical samples to be tested that can be used in the methods disclosed in this invention include, but are not limited to, oropharyngeal swabs, nasopharyngeal swabs, saliva, nasal aspirate, serum, plasma, ascites, and pleural effusion. Samples to be tested typically contain mucin and / or other protein components carrying the same type of charge as mucin. These components are charged, while the microspheres used in the photo-induced chemiluminescence method carry the opposite charge. Within the reaction system, mucin can bind to the microspheres through the attraction between positive and negative charges, thereby achieving the function of generating a signal through photo-induced chemiluminescence, which can strongly interfere with the detection system.

[0037] Therefore, when using photoluminescence to detect samples that may contain analytes, this invention adds a certain amount of blank latex microspheres to the reaction system. These blank latex microspheres carry the same charge as the microspheres used in photoluminescence. By adsorbing interfering substances such as mucins through the blank latex microspheres, the interference caused by samples such as oropharyngeal swabs, nasopharyngeal swabs, saliva, and nasal aspirate in the photoluminescence method is eliminated. This solves the problem of non-specific signals generated by such samples when using photoluminescence analysis technology, and improves the accuracy of detection.

[0038] Therefore, a first aspect of the present invention provides a microsphere composition for homogeneous chemiluminescence detection, the microsphere composition comprising:

[0039] Luminescent latex microspheres, which can produce chemiluminescence after reacting with singlet oxygen, contain optically reactive substances and have bioactive substances attached to their surface;

[0040] Photosensitive latex microspheres, which can generate singlet oxygen when excited by light, contain photosensitizers and have avidin attached to their surfaces;

[0041] Blank latex microspheres contain no optically reactive substances and have no bioactive substances attached to their surface; wherein, the luminescent latex microspheres, photosensitive latex microspheres and blank latex microspheres are made of the same material and are all polystyrene latex microspheres.

[0042] The term "bioactive substance" as used in this invention refers to a biologically active compound, typically a member of a biologically active "specific binding pair." A "specific binding pair" is a pair of molecules that can specifically bind to each other, such as an enzyme-substrate pair, an antigen-antibody pair, or a ligand-receptor pair. A specific example of a specific binding pair is the biotin-avidin system, where biotin, widely found in plant and animal tissues, has two ring structures: an imidazoline ring and a thiophene ring, with the imidazoline ring being the primary binding site for avidin. Activated biotin can couple to almost all known biomolecules, including proteins, nucleic acids, polysaccharides, and lipids, mediated by protein cross-linking agents; while avidin is a protein secreted by Streptomyces with a molecular weight of 65 kDa. The avidin molecule consists of four identical peptide chains, each capable of binding one biotin. Therefore, each antigen or antibody can simultaneously couple multiple biotin molecules, creating a "tentacle effect" that enhances analytical sensitivity. The avidin described in this invention is selected from egg avidin, streptavidin, egg yolk avidin, neutral avidin and avidin-like substances, preferably selected from neutral avidin and / or streptavidin.

[0043] As used in this invention, the term "optically reactive substance" refers to a substance capable of participating in optical reactions, including substances capable of absorbing or emitting light of a specific wavelength. According to some embodiments of the invention, the "optically reactive substance" comprises a chemiluminescent compound and a metal chelate. Further, the chemiluminescent compound is selected from olefin compounds, preferably from dimethylthiophene, dibutyl ketone compounds, dioxane, enol ethers, enamines, 9-alkylene-xanthane, 9-alkylene-N-9,10-dihydroacridine, arylethyl ethers, aryl imidazoles, and luster compounds, and their derivatives, more preferably from dimethylthiophene and its derivatives. The metal in the metal chelate is a rare earth metal or a Group VIII metal, preferably selected from europium, terbium, dysprosium, samarium, osmium, and ruthenium, more preferably europium. The metal chelate comprises a chelating agent selected from the following: NHA, BHHT, BHHCT, DPP, TTA, NPPTA, NTA, TOPO, TPPO, BFTA, 2,2-dimethyl-4-perfluorobutyryl-3-butanone, 2,2'-bipyridine, bipyridylcarboxylic acid, azirocrown ethers, azirocavitary ligands, and trioctylphosphine oxide and their derivatives.

[0044] As used in this invention, the term "photosensitizer" refers to an active intermediate, such as singlet oxygen, that can generate a reaction with a receptor upon photoactivation. The photosensitizer can be any photosensitizer known in the art, preferably a relatively photostable compound that does not react effectively with singlet oxygen. Non-limiting examples include, for instance, compounds such as methylene blue, rose red, porphyrin, phthalocyanine, and chlorophyll disclosed in U.S. Patent 5709994 (which is incorporated herein by reference in its entirety), and derivatives of these compounds having 1-50 substituents, said substituents serving to make these compounds more lipophilic or more hydrophilic, and / or as linking groups to specifically binding pairing members. Other examples of photosensitizers known to those skilled in the art may also be used in this invention, such as those described in U.S. Patent 6406913, which is incorporated herein by reference.

[0045] The three types of latex microspheres described in this invention are made of the same material, differing mainly in the substances they contain and the substances they are modified on the surface, thus giving them different functions. The luminescent latex microspheres and photosensitive latex microspheres both contain optically reactive substances and have bioactive substances attached to their surfaces, while the blank latex microspheres do not contain either of these substances.

[0046] According to some embodiments of the present invention, the surface of the blank latex microspheres is modified with chemical groups, wherein the chemical groups are selected from at least one of epoxy, chloromethyl, mercapto, amino, hydroxy, maleamine, sulfonic acid, carboxyl and aldehyde groups.

[0047] According to some embodiments of the present invention, the chemical group is selected from at least one of sulfonic acid group, carboxyl group and aldehyde group.

[0048] According to some embodiments of the present invention, the chemical group is a carboxyl group.

[0049] According to some embodiments of the present invention, the blank latex microspheres are prepared by an emulsion polymerization method without emulsifiers.

[0050] In some embodiments of the present invention, the method for preparing the blank latex microspheres includes: reacting deionized water, styrene, hydroxyethyl methacrylate, and a small amount of divinylbenzene in the presence of potassium persulfate initiator, and obtaining microspheres with a clean surface and hydroxyl groups, with a diameter of approximately 60-1000 nm. Preferably, the reaction conditions include: polymerization reaction at 60-90°C, for example, 70°C, under a nitrogen atmosphere for 5-6 hours.

[0051] According to some embodiments of the present invention, the average particle size of the blank latex microspheres is 60-1000 nm, for example, it can be 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, or 900 nm.

[0052] According to a preferred embodiment of the present invention, the average particle size of the blank latex microspheres is 100-300 nm.

[0053] According to some embodiments of the present invention, the particle size of the blank latex microspheres is smaller than that of the luminescent latex microspheres.

[0054] According to some embodiments of the present invention, the particle size of the blank latex microspheres is smaller than that of the photosensitive latex microspheres.

[0055] According to some embodiments of the present invention, the surface of the blank latex microspheres carries a negative charge.

[0056] According to some embodiments of the present invention, the Zeta potential of the blank latex microspheres is -10 to -50 mV, for example, it can be -15 mV, -20 mV, -25 mV, -30 mV, -35 mV, -40 mV, -45 mV, or -50 mV. According to some embodiments of the present invention, the Zeta potential of the blank latex microspheres is -15 to -30 mV.

[0057] The ZETA potential value described in this invention refers to the potential value of blank latex microspheres in a dispersion system with a pH of 6–9. The ZETA potential of a particle refers to the potential at the shear plane; that is, the potential difference between the continuous phase and the fluid-stabilized layer attached to the microsphere. Because the dispersed particles carry a charge on their surface, they attract surrounding anti-charge ions. These anti-charge ions are distributed in a diffuse state at the interface between the two phases, forming a diffuse electric double layer. According to the Stern double layer theory, the double layer can be divided into two parts: the Stern layer and the diffuse layer. The Stern layer is defined as a planar layer composed of a layer of ion (IHP or OHP) charge centers adsorbed on the electrode surface. The potential of this planar layer relative to a point in the fluid far from the interface is called the Stern potential. The interface between the stationary layer (including the stern layer and the portion of the diffusion layer within the slipping plane) and the dispersion medium within the diffusion layer, where relative movement occurs, is called the slipping plane. The potential at this point relative to a point in the fluid far from the interface is called the Zeta potential or zeta potential. In other words, the Zeta potential is the potential difference between the continuous phase and the fluid stationary layer attached to the dispersed particles. It can be directly measured through electrokinetic phenomena. Currently, the main methods for measuring the Zeta potential include electrophoresis, electroosmosis, flow potential analysis, and ultrasound, with electrophoresis being the most widely used.

[0058] According to some embodiments of the present invention, the blank latex microspheres and the luminescent latex microspheres carry the same type of charge.

[0059] In a second aspect, the present invention provides the application of the microsphere composition according to the first aspect in the detection of analytes in oropharyngeal swabs, nasopharyngeal swabs, saliva or nasal aspirate samples.

[0060] According to some embodiments of the present invention, the method for detecting the analyte in the above-mentioned sample using the microsphere composition includes the following steps:

[0061] S1: The sample to be tested is mixed with blank latex microspheres and luminescent latex microspheres to obtain a first mixture;

[0062] S2: The first mixture is mixed with photosensitive latex microspheres to obtain a second mixture;

[0063] S3: Irradiate the second mixture with a laser and detect the chemiluminescence signal.

[0064] According to some embodiments of the present invention, in step S1, the temperature of the first mixing is 30-45°C and the time is 10-30 minutes.

[0065] According to a preferred embodiment of the present invention, in step S2, the temperature of the second mixing is 30-45°C and the time is 5-30 minutes.

[0066] According to some embodiments of the present invention, in step S3, the excitation wavelength of the laser irradiation is 650-700nm, and the detection wavelength is 550-650nm.

[0067] According to a preferred embodiment of the present invention, in step S3, the excitation wavelength of the laser irradiation is 675-685nm, and the detection wavelength is 610-620nm. Specific Implementation

[0069] To make the present invention easier to understand, specific examples of influenza A virus antigen detection and influenza B virus antigen detection are provided below to further illustrate the invention in detail. These examples are for illustrative purposes only and are not intended to limit the scope of application of the invention. Unless otherwise specified, all raw materials or components used in the present invention can be obtained commercially or by conventional methods.

[0070] Example 1

[0071] (I) Main testing reagents:

[0072] (1) Luminescent latex microsphere solution: luminescent latex microspheres coated with antibody 1

[0073] (2) Biotin-labeled antibody 2 solution

[0074] (3) Sample diluent

[0075] (4) Photosensitive latex microsphere solution: Photosensitive latex microspheres containing streptavidin-coated surface

[0076] (II) Preparation of blank latex microspheres:

[0077] The microspheres are prepared by emulsion polymerization without emulsifiers. Specifically, deionized water, styrene, hydroxyethyl methacrylate, and a small amount of divinylbenzene are mixed, and potassium persulfate is added to initiate the reaction. The polymerization reaction is carried out at 60-90℃, for example, 70℃, under a nitrogen atmosphere for 5-6 hours. After purification, microspheres with a clean surface and hydroxyl groups and a diameter of approximately 60-1000 nm are obtained. The diameter of the microspheres can be screened, preferably 100-300 nm. The blank latex microspheres obtained are made of polystyrene and can be modified with chemical groups. The chemical groups used for modification can be selected from epoxy, chloromethyl, mercapto, amino, hydroxyl, maleamine, sulfonic acid, carboxyl, and aldehyde groups.

[0078] Blank latex microspheres can be added to any one, two, or three of the following solutions: luminescent latex microsphere solution, biotin-labeled antibody 2 solution, and sample diluent, and their concentration can be adjusted as needed.

[0079] The zeta potential of blank latex microspheres was determined using Doppler electrophoresis (ELS) with a NICOMP 380Z3000, ranging from -10 to -50 mV.

[0080] (III) Testing process:

[0081] 1. Add 25 μL each of sample diluent, sample to be tested, luminescent latex microsphere solution and biotin-labeled antibody solution to the detection well and mix well to obtain the first mixture. Incubate at 37°C for 15 minutes.

[0082] 2. Add 175 μL of photosensitive latex microsphere solution to obtain the second mixture, and incubate at 37°C for another 15 minutes.

[0083] 3. After the incubation period, the light signal intensity is detected using a photo-induced chemiluminescence analyzer (excitation wavelength 680nm, detection wavelength 615nm). The sample is considered positive if the light signal intensity is ≥ the critical value (S / CO≥1).

[0084] Simultaneously with sample testing, a pre-calibrated critical value control standard is added to the reaction microwells according to the same detection mode and detected at the same time. The intensity of its luminescence signal is used as the critical value for antigen-positive results.

[0085] The above testing process can be performed at any time. The testing is completed automatically on the platform.

[0086] Example 2

[0087] (I) Sample Testing

[0088] 10 positive samples and 35 negative samples were tested.

[0089] (II) Main Reagents

[0090] (1) Luminescent latex microsphere solution: Luminescent latex microspheres coated with anti-influenza A virus antibody 1, the microspheres are made of polystyrene, negatively charged, with a diameter of 200±5nm and a concentration of 25mg / mL.

[0091] (2) Biotin-labeled anti-influenza A virus antibody 2 solution: antibody concentration was 0.5 μg / mL

[0092] (3) Sample diluent 1

[0093] (4) Sample diluent 2 (containing blank latex microspheres): Add blank latex microspheres to the sample diluent in (3). The microspheres are made of polystyrene, modified with carboxyl groups, with a diameter of 120±5nm, a Zeta potential of -25mV, and a concentration of 0.25mg / mL.

[0094] (5) Photosensitive latex microsphere solution: Photosensitive latex microspheres containing streptavidin coated on the surface, the microspheres are made of polystyrene, with a diameter of 200±5nm and a concentration of 40μg / mL.

[0095] (III) Detection process: See Example 1

[0096] (IV) Results Analysis

[0097] The test results are shown in Table 1 below:

[0098] Table 1

[0099]

[0100]

[0101] As can be seen from Table 1, using the microsphere composition of the present invention to detect influenza A virus antigen can reduce the false positive rate from 37.14% to 0%, which is very effective; and the detection results are consistent with those of third parties.

[0102] Example 3

[0103] (I) Sample Testing

[0104] 10 positive samples and 20 negative samples

[0105] (II) Main Reagents

[0106] (1) Luminescent latex microsphere solution 1: Luminescent latex microspheres coated with anti-influenza A virus antibody 1, the microspheres are made of polystyrene, negatively charged, with a diameter of 200±5nm and a concentration of 25mg / mL.

[0107] (2) Luminescent latex microsphere solution 2 (containing blank latex microspheres): Blank latex microspheres were added to the luminescent latex microsphere solution in (1). The microspheres were made of polystyrene and prepared by emulsion polymerization without emulsifiers. After carboxyl modification, the microspheres had a diameter of 120±5nm, a Zeta potential of -30mV, and a concentration of 0.25mg / mL.

[0108] (3) Luminescent latex microsphere solution 3 (containing blank latex microspheres): Blank latex microspheres were added to the luminescent latex microsphere solution in (1). The microspheres were made of polystyrene and prepared by emulsion polymerization without emulsifiers. After carboxyl modification, the microspheres had a diameter of 120±5nm, a Zeta potential of -15mV, and a concentration of 0.25mg / mL.

[0109] (4) Biotin-labeled anti-influenza A virus antibody 2 solution: concentration 0.5 μg / mL

[0110] (5) Sample diluent

[0111] (6) Photosensitive latex microsphere solution: Photosensitive latex microspheres containing streptavidin coated on the surface, the microspheres are made of polystyrene, with a diameter of 200±5nm and a concentration of 40μg / mL.

[0112] (III) Detection process: See Example 1

[0113] (IV) Results Analysis

[0114] The results of the clinical sample tests are shown in Table 2 below:

[0115] Table 2 Results of clinical samples with different blank latex microspheres

[0116]

[0117]

[0118] As can be seen from Table 2, when using the microsphere composition of the present invention to detect influenza A virus antigen, the Zeta potential of the composition containing blank latex microspheres is different, ranging from -15 to -30 mV, and the detection results are consistent with those of third parties.

[0119] Example 4

[0120] (I) Sample Testing

[0121] 10 positive samples and 35 negative samples

[0122] (II) Main Reagents

[0123] (1) Luminescent latex microsphere solution: Luminescent latex microspheres coated with anti-influenza B virus antibody 1, the microspheres are made of polystyrene, negatively charged, with a diameter of 200±5nm and a concentration of 25mg / mL.

[0124] (2) Biotin-labeled anti-influenza B virus antibody solution 2: concentration 0.5 μg / mL

[0125] (3) Biotin-labeled anti-influenza B virus antibody 2 solution 2 (containing blank latex microspheres): Blank latex microspheres were added to the biotin-labeled anti-influenza B virus antibody 2 solution in (2). The microspheres were made of polystyrene and prepared by emulsion polymerization without emulsifiers. After carboxyl modification, the microspheres had a diameter of 150±5nm, a Zeta potential of -20mV, and a concentration of 5mg / mL.

[0126] (4) Photosensitive latex microsphere solution: Photosensitive latex microspheres containing streptavidin coated on the surface, the microspheres are made of polystyrene, with a diameter of 200±5nm and a concentration of 40μg / mL.

[0127] (5) Sample diluent

[0128] (III) Detection process: See Example 1

[0129] (IV) Results Analysis

[0130] The results of clinical sample testing are shown in Table 3 below:

[0131] Table 3

[0132]

[0133]

[0134]

[0135] As can be seen from Table 3, the use of the microsphere composition of the present invention reduced the false positive rate of influenza B virus antigen detection from 34.29% to 0%, which is very effective and the detection results are consistent with those of third parties.

[0136] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A microsphere composition for homogeneous chemiluminescence detection, characterized in that, The microsphere composition comprises: Luminescent latex microspheres, which can produce chemiluminescence after reacting with singlet oxygen, contain optically reactive substances and have bioactive substances attached to their surface; Photosensitive latex microspheres, which generate singlet oxygen upon photoexcitation, contain a photosensitizer and have avidin attached to their surface; and Blank latex microspheres contain no optically reactive substances and have no bioactive substances attached to their surface. The luminescent latex microspheres, photosensitive latex microspheres, and blank latex microspheres are all made of the same material, and are all polystyrene latex microspheres; the blank latex microspheres and the luminescent latex microspheres carry the same type of charge.

2. The microsphere composition according to claim 1, characterized in that, The surface of the blank latex microspheres carries a negative charge.

3. The microsphere composition according to claim 2, characterized in that, The zeta potential of the blank latex microspheres is -10 to -50 mV.

4. The microsphere composition according to claim 2, characterized in that, The zeta potential of the blank latex microspheres is -15 to -30 mV.

5. The microsphere composition according to claim 1, characterized in that, The surface of the blank latex microspheres is modified with chemical groups, which are selected from at least one of epoxy, chloromethyl, mercapto, amino, hydroxy, maleamine, sulfonic acid, carboxyl, and aldehyde groups.

6. The microsphere composition according to any one of claims 1-4, characterized in that, The surface of the blank latex microspheres is modified with chemical groups, which are selected from at least one of sulfonic acid groups, carboxyl groups, and aldehyde groups.

7. The microsphere composition according to any one of claims 1-4, characterized in that, The surface of the blank latex microspheres is modified with chemical groups, namely carboxyl groups.

8. The microsphere composition according to any one of claims 1-5, characterized in that, The blank latex microspheres were prepared by emulsion polymerization without emulsifiers.

9. The microsphere composition according to any one of claims 1-5, characterized in that, The particle size of the blank latex microspheres is 60-1000 nm.

10. The microsphere composition according to any one of claims 1-5, characterized in that, The particle size of the blank latex microspheres is 100-300 nm.

11. The microsphere composition according to any one of claims 1-5, characterized in that, The particle size of the blank latex microspheres is smaller than that of the luminescent latex microspheres.

12. The microsphere composition according to any one of claims 1-5, characterized in that, The particle size of the blank latex microspheres is smaller than that of the photosensitive latex microspheres.

13. The use of the microsphere composition of any one of claims 1-12 for non-diagnostic purposes in detecting analytes in oropharyngeal swabs, nasopharyngeal swabs, saliva or nasal aspirate samples.

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