Magloci composite microspheres and applications thereof

Abstract

The application discloses a kind of MagLOCI composite microspheres and its application.The application discloses a kind of detection kit based on photochemical chemiluminescence, the detection kit includes magnetic composite microspheres, namely MagLOCI composite microspheres;Wherein, the magnetic composite microspheres include main body magnetic microspheres and guest nanometer microspheres covalently connected on the surface of the main body magnetic microspheres, and the guest nanometer microspheres are used to generate singlet oxygen or convert the energy of singlet oxygen into fluorescence.The application can make up for the deficiency of existing LOCI technology, and expand the application field of LOCI.Compared with traditional LOCI technology, MagLOCI technology is a higher performance, general, flexible photochemical chemiluminescence detection platform.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a MagLOCI composite microsphere and its application. Background Technology

[0002] Immunodiagnostics has surpassed biochemical diagnostics to become the largest segment in my country's in vitro diagnostics industry. Chemiluminescence immunoassay, in particular, has gradually become the mainstream immunoassay technology due to its excellent sensitivity, dynamic range, fully automated operation, and high-throughput testing speed. In developed countries in Europe and America, chemiluminescence immunoassay has essentially replaced enzyme-linked immunosorbent assay (ELISA), accounting for over 90% of the immunodiagnostics market share. In 2021, chemiluminescence accounted for approximately 84% of the market in my country, with a market size reaching 28.4 billion yuan.

[0003] Chemiluminescence technology can be divided into heterogeneous chemiluminescence immunoassay systems and homogeneous chemiluminescence immunoassay systems based on the state of the immune response. The former is currently the mainstream chemiluminescence system, mainly including enzyme-catalyzed chemiluminescence, direct chemiluminescence, and electrochemiluminescence. Traditional heterogeneous chemiluminescence platforms require multiple repeated washing steps to completely eliminate the influence of free labeled molecules on the background signal. However, multiple washings lead to uncontrollable microsphere loss in the sample, increasing detection time and introducing significant results errors, thus affecting detection precision. Homogeneous chemiluminescence immunoassay systems do not require a separation system during detection, and its representative technology is photoluminescence. Photoluminescence, also known as luminescent oxygen channeling immunoassay (LOCI), was invented in 1994 by Ullman EF et al. [Proc. Natl. Acad. Sci. US A 1994, 91, 5426.] and later developed into commercial products by PerkinElmer and Siemens. The LOCI detection system consists of donor spheres (DB) and acceptor spheres (AB). DB spheres are doped with a photosensitizer, which generates singlet oxygen upon photoexcitation. AB spheres are doped with dimethylthiophene and lanthanide metal complexes. Dimethylthiophene converts the energy of the singlet oxygen into 360nm emission light, exciting the lanthanide metal complexes to produce fluorescence. Upon irradiation with a 680nm laser, DB activates oxygen in the surrounding environment to convert into singlet oxygen, with a survival time of only 4 microseconds. This short survival time results in a very small propagation diameter for the ionic oxygen (approximately 200nm). When the analyte is present, it forms an immune complex with AB (conjugated with a capture antibody) and DB (conjugated with a detection antibody) through a double-antibody sandwich mechanism. At this point, the distance between AB and DB is less than 200nm, and AB receives the singlet oxygen generated by DB and is excited to emit a 615nm light signal. However, when no analyte is present, the distance between AB and DB is too great to excite the AB's light signal.

[0004] PerkinElmer has introduced the AlphaPlex™ system [Publication No.: US8486719], which utilizes lanthanide metal complexes doped with different emission wavelengths (615nm, 545nm, 645nm) within AB spheres to detect different analytes, achieving multiplex detection. However, because the detection requires the addition of three detection antibodies and three types of AB spheres, and the serum / plasma sample matrix has complex compositions, the risk of interference in multiplex detection is significantly increased. Furthermore, optimizing the detection performance of three indicators within a single test well is significantly more difficult and costly.

[0005] LOCI technology offers advantages such as being wash-free, having low background, high sensitivity, good repeatability, and being simple and quick to operate. Although the market size of LOCI-based photochemiluminescence has maintained rapid growth in recent years, it still accounts for a small proportion of the overall chemiluminescence market. This may be because the non-washable nature of existing LOCI technology leads to poor performance in certain situations (severe matrix effect problems, poor performance of indirect antibody detection, severe hook effect, etc.), thus limiting its wider application. Therefore, the photochemiluminescence market has significant growth potential.

[0006] To address the matrix interference issue caused by the inability to wash the matrix in LOCI technology, Komei Diagnostics' patent [Patent No.: CN109709317B] uses avidin-modified magnetic beads to achieve matrix separation. However, this method involves adding DB to label the remaining sites on the avidin microspheres that have bound the immune complex and AB after the magnetic beads and AB have formed an immune complex. The labeling efficiency is affected by steric hindrance and the limited number of biotin-avidin sites, resulting in less than ideal performance.

[0007] The existing LOCI technology still has several drawbacks that limit its clinical application, mainly in the following aspects:

[0008] 1. Detection performance is affected by the sample matrix: 1) Indirect methods have poor antibody detection performance. Methods for detecting antibodies through immunoassay include indirect methods, double-antigen sandwich methods, competitive methods, and neutralization methods. Double-antigen sandwich methods are limited by steric hindrance, restricting the molecular weight of the antigen used and thus limiting the detectable indicators. Competitive methods are suitable for small molecules or indicators with only one antigenic epitope; the reliability of the test results is affected by the specificity and affinity of the competing antibody. Double-antigen sandwich or competitive methods have high requirements for the antigen or competing antibody coated on the solid-phase carrier and cannot distinguish the type of immunoglobulin being tested, such as IgG and IgM. Classical indirect methods generally involve two steps: first, the known antigen coated on the solid-phase carrier binds to the antibody to be tested; second, a labeled anti-antibody (secondary antibody) binds to the antibody to be tested. Indirect methods can distinguish the type of immunoglobulin being tested by the anti-antibody, thus determining recent infection (IgM indicator) and detecting population antibody (immunity) levels (IgG indicator), which is of great value in determining the course of infectious diseases. However, in the indirect method, non-specific antibodies need to be washed away between the two steps of the reaction; otherwise, non-specific antibodies will bind to the second antibody, causing false negative results, or non-specific antibodies will adsorb onto the surface of the solid-phase carrier, causing false positive results. Therefore, the existing LOCI technology is not suitable for simple, quantitative detection of antibodies using the indirect method. 2) It cannot detect special samples and has low sensitivity for detecting clinical samples. Although the LOCI technology does not require a separation and washing step, due to the matrix effect, serum and plasma samples often contain endogenous interfering proteins, leading to false positive and false negative results in immunoassay. These include substances that cause interference, such as complement, lysozyme, fibrin, autoantibodies, rheumatoid factor, and heterophilic antibodies. In addition, the LOCI technology cannot accurately detect samples containing biotin. Biotin, or vitamin B7, has a normal plasma concentration of approximately 0.12-0.36 nmol / L. When a large dose of biotin is ingested, the concentration of free biotin and its metabolites in the blood can reach hundreds to thousands of times the normal concentration. Serum / plasma samples containing biotin can cause streptavidin to bind to the surface of donor microspheres, reducing the detection signal and leading to false negative results. To address biotin interference, a patent application filed by Komei Diagnostics [Patent No.: CN111122844A] utilizes porous microspheres with surface-modified avidin as an anti-interference agent. This agent eliminates biotin molecules by co-incubating the microspheres with the biotin-containing sample. However, the preparation process of this anti-interference agent is cumbersome and requires an additional incubation step to eliminate biotin molecules, making its feasibility low.

[0009] 2. Hook effect exists.

[0010] Because the free antigen or antibody to be tested remains in the reaction system, the hook effect of LOCI is severe, making it impossible to directly detect high concentrations of the test sample and resulting in a narrow linear range. In response, a patent CN108204959B by Boyang Biotechnology determines the presence of the hook effect by comparing the increase in the difference between two signal readings of the test sample, but this does not fundamentally solve the problem of the hook effect. Summary of the Invention

[0011] The technical problem this invention aims to solve is to overcome the shortcomings of existing LOCI technology, which, due to its inability to be washed, performs poorly in certain situations (severe matrix effect problems, inability to detect antibody indicators via indirect methods, severe hook effect, etc.). This invention provides a MagLOCI composite microsphere and its applications. By introducing a complex of receptor microspheres and magnetic microspheres, this invention endows the MagLOCI platform with the washing function of LOCI technology, thus overcoming the deficiencies of existing LOCI technology and expanding its application areas. Compared to traditional LOCI technology, MagLOCI technology is a higher-performance, more versatile, and more flexible photochemiluminescence detection platform.

[0012] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0013] A first aspect of the present invention provides a detection kit based on photo-induced chemiluminescence, the detection kit comprising magnetic composite microspheres; wherein the magnetic composite microspheres comprise a host magnetic microsphere and guest nanospheres covalently connected to the surface of the host magnetic microsphere, the guest nanospheres being used to generate singlet oxygen or convert the energy of singlet oxygen into fluorescence.

[0014] In some embodiments of the present invention, the surface of the host magnetic microsphere or guest nanosphere is modified with monomers or polymers containing amino, carboxyl or aldehyde groups.

[0015] In some preferred embodiments of the present invention, the surface of the host magnetic microsphere or guest nanosphere is modified with an amino group or a polymer thereof, and correspondingly the surface of the guest nanosphere or host magnetic microsphere is modified with a monomer or polymer thereof containing a carboxyl group or an aldehyde group.

[0016] In some specific embodiments of the present invention, the surface of the host magnetic microsphere is modified with an amino group or its polymer, and the surface of the guest nanosphere is modified with a monomer or its polymer containing a carboxyl or aldehyde group.

[0017] In some embodiments of the present invention, the density of the monomer or polymer thereof containing amino, carboxyl or aldehyde groups is at least 100 μmol / mg for guest nanospheres or host magnetic microspheres.

[0018] In this invention, the monomers or polymers containing amino, carboxyl, or aldehyde groups can be conventional in the art, and the amino-containing monomer polymers are, for example, polyethyleneimine.

[0019] In some embodiments of the present invention, the magnetic composite microspheres have a magnetic content of 4 to 75 wt.%, for example, 4 wt.%, 8 wt.%, 12 wt.%, 16 wt.%, 20 wt.%, 24 wt.%, 28 wt.%, 32 wt.%, 36 wt.%, 40 wt.%, 44 wt.%, 48 wt.%, 52 wt.%, 56 wt.%, 60 wt.%, 64 wt.%, 68 wt.%, 72 wt.%, 75 wt.%, or any range between two of the above magnetic contents.

[0020] In some embodiments of the present invention, the magnetic content of the magnetic composite microspheres is 5 to 50 wt.%, for example, 5 wt.%, 10 wt.%, 15 wt.%, 22 wt.%, 25 wt.%, 30 wt.%, 35 wt.%, 40 wt.%, 45 wt.%, 50 wt.%, or any range between two of the above magnetic contents.

[0021] In some embodiments of the present invention, the magnetic content of the magnetic composite microspheres is 30-35 wt.%, for example, 31 wt.%, 32 wt.%, 33 wt.%, 34 wt.%, or 35 wt.%.

[0022] In some embodiments of the present invention, in the detection kit, the surface of the guest nanospheres is further coupled with biocapture molecules, which are used to bind the analyte molecules.

[0023] In this invention, the biocapture molecule can be conventional in the art, such as an antibody molecule or an antigen molecule.

[0024] In some embodiments of the present invention, the detection kit further includes matching microspheres, which are used to convert the energy of singlet oxygen into fluorescence when the guest nanospheres generate singlet oxygen; and the matching microspheres are used to generate singlet oxygen when the guest nanospheres are used to convert the energy of singlet oxygen into fluorescence.

[0025] In some embodiments of the present invention, the detection kit further includes a biodetector molecule for binding to the analyte molecule, and the biodetector molecule and the biocapture molecule bind to different sites of the analyte molecule; wherein the biodetector molecule specifically binds to the matching microspheres.

[0026] In this invention, the term "specific binding" refers to the binding characteristics of paired molecules such as enzyme-substrate, antigen-antibody, and ligand-receptor, which can be selected by those skilled in the art based on common knowledge as needed. An example of a ligand-receptor capable of specific binding is the biotin-avidin system, such as biotin-streptavidin.

[0027] In some specific embodiments of the present invention, the biodetector molecule specifically binds to the matching microspheres via a biotin-avidin system.

[0028] In some embodiments of the present invention, the biodetector molecule or the analyte is a biotin-modified biodetector molecule or the analyte, and the matching microspheres are streptavidin-modified matching microspheres.

[0029] In other embodiments of the present invention, the biodetector molecule or analyte is a streptavidin-modified biodetector molecule or analyte, and the matching microspheres are biotin-modified matching microspheres.

[0030] In some embodiments of the present invention, the matched microspheres or guest nanospheres that generate singlet oxygen contain a photosensitizer that generates singlet oxygen upon receiving excitation light; the guest nanospheres or matched microspheres that convert the energy of the singlet oxygen contain dimethylthiophene and a lanthanide metal complex, wherein the dimethylthiophene converts the energy of the singlet oxygen into emitted light and generates fluorescence through the lanthanide metal complex.

[0031] In some embodiments of the present invention, the lanthanide metal complex is selected from one or more of europium, terbium, and samarium.

[0032] In this invention, when the lanthanide metal complex is one of europium, terbium, and samarium, the detection kit can perform single sample detection; when the lanthanide metal complex is selected from two or three of europium, terbium, and samarium, the detection kit can perform multiple detection.

[0033] A second aspect of the present invention provides a method for detecting biomolecules, the method comprising:

[0034] (1) When the biodetector molecule is present or absent, the magnetic composite microspheres and the sample containing the analyte molecule are mixed and incubated, and the matrix reaction solution is removed;

[0035] (2) Add the microspheres obtained in (1) to a solution containing matching microspheres, mix and incubate, and detect and collect the luminescence signal;

[0036] In (1), when the biodetector molecule is not present, (2) further includes adding the microspheres obtained in (1) to a solution containing the biodetector molecule, mixing and incubating, and removing the matrix reaction solution;

[0037] The magnetic composite microspheres, the biodetector molecules, and the matching microspheres are as described in the first aspect.

[0038] In some embodiments of the present invention, the method includes:

[0039] (1) Mix and incubate the magnetic composite microspheres, biodetector molecules and sample containing the analyte molecules, and remove the matrix reaction solution;

[0040] (2) Add the microspheres obtained in (1) to a solution containing matching microspheres, mix and incubate, and detect and collect the luminescence signal.

[0041] In the above implementation scheme, (1) washing may or may not be performed after removing the matrix reaction solution;

[0042] (2) Washing may or may not be performed after the mixed incubation.

[0043] In other embodiments of the present invention, the method includes:

[0044] (1) Mix and incubate the magnetic composite microspheres with the sample containing the analyte molecule, and remove the matrix reaction solution;

[0045] (2) Add the microspheres obtained in (1) to a solution containing biodetection molecules, mix and incubate, and remove free biodetection molecules;

[0046] (3) Add the microspheres obtained in (2) to the solution of matching microspheres, mix and incubate, and detect and collect the luminescence signal.

[0047] In the above implementation scheme, (1) washing may or may not be performed after removing the matrix reaction solution;

[0048] (2) In the mixed incubation, washing may or may not be performed;

[0049] (3) In the process of mixing and incubating, washing may or may not be performed.

[0050] In this invention, the matrix removal reaction solution is preferably removed by magnetic adsorption.

[0051] In this invention, the term "matrix" refers to non-target molecules and unbound target molecules in the reaction system, excluding the buffer solution, that may interfere with the immune response. For example, in a reaction system using PBS as the buffer, the matrix refers to the unbound test sample or antibody; in a reaction system containing human serum or plasma, the matrix also includes components in the human serum / plasma that may interfere with detection, such as non-specific human IgG antibodies that affect the indirect method, biotin molecules that affect the biotin-avidin reaction system, and substances in the serum / plasma sample such as complement, lysozyme, fibrin, autoantibodies, rheumatoid factor, heterophilic antibodies, etc.

[0052] In this invention, the molecules to be tested include, but are not limited to, nucleic acids and their derivatives, amino acids and their derivatives, polypeptides, proteins, polysaccharides, glycoproteins, fatty acids and their derivatives, and small molecule compounds.

[0053] In this invention, the sample can be conventional in the art, such as serum or plasma, or bodily fluids such as urine, semen, saliva, tears, etc.

[0054] In this invention, the mixing and incubation can be carried out in a buffer solution; the buffer solution can be conventional in the art, such as PBS buffer, HEPES buffer, Tris-HCl buffer, or MES buffer.

[0055] In some specific embodiments of the present invention, the buffer is a PBS buffer containing 1% (v / v) BSA and 0.05% (v / v) Tween-20.

[0056] In this invention, the amount of magnetic composite microspheres fed can be 50-250 μg / mL, for example, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL or any two of the above feeding amounts.

[0057] In some embodiments of the present invention, the reagent used for washing is PBST buffer; the number of washing cycles is 1 to 3.

[0058] In some embodiments of the present invention, the incubation conditions are 37°C with shaking for 15 to 30 minutes.

[0059] In some embodiments of the present invention, the light emission signal is acquired by a photodetector or an imaging system.

[0060] In this invention, the photodetector can be conventional in the art, such as a PMT, PD or APD, with an excitation wavelength of 680 nm and a measurement wavelength of 615 nm.

[0061] A third aspect of the present invention provides a method for preparing magnetic composite microspheres, the method comprising mixing a host magnetic microsphere and a guest nanosphere under liquid phase conditions, and performing a light-shielded reaction to covalently link the host magnetic microsphere and the guest nanosphere; the guest nanosphere is used to generate singlet oxygen or convert the energy of singlet oxygen into fluorescence; the feed ratio of the host magnetic microsphere and the guest nanosphere is (1-10):1.

[0062] In some embodiments of the present invention, the feeding ratio of the main magnetic microspheres and the guest nanospheres is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

[0063] In some embodiments of the present invention, the feeding ratio of the main magnetic microspheres to the guest nanospheres is (2-5):1, for example, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.

[0064] In some embodiments of the present invention, the surface of the host magnetic microsphere or guest nanosphere is modified with monomers or polymers containing amino, carboxyl or aldehyde groups.

[0065] In some preferred embodiments of the present invention, the surface of the host magnetic microsphere or guest nanosphere is modified with an amino group or a polymer thereof, and correspondingly the surface of the guest nanosphere or host magnetic microsphere is modified with a monomer or polymer thereof containing a carboxyl group or an aldehyde group; the amino-containing monomer polymer is, for example, polyethyleneimine.

[0066] In some embodiments of the present invention, the liquid phase condition is a MEST solution.

[0067] In some specific embodiments of the present invention, the mixed matrix magnetic microsphere MEST solution is dropped into the guest nanosphere MEST solution.

[0068] A fourth aspect of the present invention provides the application of the detection kit as described in the first aspect in the detection of biomolecules.

[0069] In some embodiments of the present invention, the biomolecule is a protein molecule.

[0070] Because MagLOCI inherently possesses the characteristics of LOCI technology, the "washing" process of MagLOCI only needs to remove most of the interfering substances. Therefore, compared to traditional heterogeneous chemiluminescence technology, MagLOCI can significantly improve the performance of the original LOCI technology with just a simple wash or separation of the reaction solution.

[0071] Meanwhile, the MagLOCI technology of this invention, as a novel chemiluminescence platform, is highly versatile and flexible. By adjusting its reaction steps, number of washes, and washing time, it can be matched to different application scenarios. The two-step, non-washing reaction mode can handle detection projects that do not require high sensitivity but need rapid results. The two-step washing reaction mode can handle the detection of clinical samples with higher sensitivity requirements and some projects severely affected by specific components in the matrix (such as biotin and heterophilic antibodies), and the number of washes can be adjusted according to the concentration of interfering substances. The three-step washing reaction mode can eliminate the hook effect and detect antibodies indirectly.

[0072] The MagLOCI three-step washing method can greatly reduce the risk of interference in multiplex detection, and can easily and quickly develop multiplex detection reagents with the same detection performance as single-detection reagents.

[0073] This invention assembles LOCI acceptor spheres onto the surface of magnetic microspheres to obtain MagLOCI composite microspheres with a host-guest structure. By introducing magnetic spheres, the previously unwashable LOCI technology can achieve the function of magnetic separation and washing of interfering substances, resulting in detection performance superior to traditional LOCI technology.

[0074] Optimized detection performance was achieved by adjusting the size, quantity, and magnetic content parameters of MagLOCI composite microspheres.

[0075] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0076] The reagents and raw materials used in this invention are all commercially available.

[0077] The positive and progressive effects of this invention are as follows:

[0078] The MagLOCI composite microspheres of this invention uniformly assemble LOCI acceptor spheres on the surface of magnetic microspheres to obtain a host-guest structure, achieving the function of magnetic separation and washing of interfering substances, thus improving detection performance. The detection kit of this invention is highly versatile, allowing for adjustment of dosage and washing cycles as needed to adapt to the detection requirements of multiple platforms. Attached Figure Description

[0079] Figure 1This is a schematic diagram illustrating the detection process and applicable scenarios for MagLOCI.

[0080] Figure 2A This is a schematic diagram illustrating the effect of microspheres with different magnetic contents on the detection signal (signal value).

[0081] Figure 2B This is a schematic diagram showing the effect of microspheres with different magnetic contents on the detection signal (P / N value).

[0082] Figure 3A This is a schematic diagram showing the effect of different amounts of microspheres added on the detection signal (signal value).

[0083] Figure 3B A schematic diagram showing the effect of different microsphere addition amounts on the detection signal (P / N value).

[0084] Figure 4A This is a schematic diagram comparing the detection performance (signal values) of MagLOCI and Multi-LOCI.

[0085] Figure 4B This is a schematic diagram comparing the detection performance (P / N value) of MagLOCI and Multi-LOCI.

[0086] Figure 5A This is a schematic diagram of the standard curves for detecting novel coronavirus IgG using MagLOCI washing, no washing, and the LOCI method via indirect detection.

[0087] Figure 5B This diagram illustrates the positive / negative signal (P / N) ratios for detecting novel coronavirus IgG via indirect methods using MagLOCI washing, no washing, and the LOCI method.

[0088] Figure 6A This diagram illustrates the results (signal values) of PCT clinical samples detected using MagLOCI washing, no washing, and the LOCI method.

[0089] Figure 6B This diagram illustrates the results (P / N values) of PCT clinical samples detected using MagLOCI washing, no washing, and the LOCI method.

[0090] Figure 7 This is a schematic diagram showing the results of eliminating the hook effect using MagLOCI washing.

[0091] Figure 8 This is a schematic diagram of the results of combined detection of PCT / IL-6 / IFN-γ using MagLOCI technology. Detailed Implementation

[0092] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0093] Example 1: Preparation method of host-guest structured MagLOCI microspheres

[0094] The host microspheres used are superparamagnetic magnetic spheres with a size of 0.6–10 μm. They are amino microspheres with 10k–750k PEI modification on their surface and a magnetic content of 20–75 wt.%. The guest nanospheres are nanospheres internally doped with dimethylthiophene and lanthanide metal complexes, purchased from PerkinElmer, with a particle size of 200 nm and surface groups of carboxyl or aldehyde groups. The amino groups on the surface of the host magnetic microspheres are covalently coupled with the carboxyl or aldehyde groups on the surface of the AB spheres to obtain MagLOCI composite microspheres with a host-guest structure. The AB spheres can react with singlet oxygen to generate a detectable signal.

[0095] Example 1-1 (Assembly of magnetic spheres and surface carboxyl-based AB spheres):

[0096] Step 1: Using 5.5 μm diameter magnetic microspheres as the core matrix, 1.3 mg of magnetic microspheres were magnetically separated to remove the supernatant. The microspheres were washed three times with 400 μL MEST (100 mM MES, 0.05% Tween 20, pH 5.0) and finally dispersed in 150 μL MEST solution to obtain a magnetic microsphere dispersion. 0.6 mg of 200 nm AB spheres was taken, centrifuged to remove the supernatant, and then ultrasonically dispersed in 150 μL MEST solution to obtain a guest AB sphere dispersion.

[0097] Step 2: The magnetic microsphere dispersion from Step 1 was added dropwise to the guest AB sphere dispersion under ultrasonic conditions. After ultrasonic mixing, the mixture was placed on a mixer and mixed for 30 min. A 50 mg / mL solution of carbodiimide (EDC) and N-hydroxysuccinimide (NHS) was prepared. The EDC / NHS solution was then added to the mixture of magnetic microspheres and AB spheres, vortexed until homogeneous, and reacted in the dark for 3 h. After the reaction was completed, the mixture was washed three times with 2.5 mM sodium hydroxide (NaOH), then washed three times with ultrapure water, and finally dispersed in 150 μL of ultrapure water to obtain the host-guest MagLOCI microspheres.

[0098] When using magnetic microspheres with different diameters and magnetic contents, the assembly method of the magnetic microspheres and the guest AB spheres is basically the same as the method described above. Only the amount of guest AB spheres and the amount of related reactants need to be slightly adjusted according to the change in the surface area of ​​the microspheres.

[0099] Examples 1-2 (Assembly of magnetic spheres and surface aldehyde-based AB spheres):

[0100] Step 1: Using 1.7 μm diameter magnetic microspheres as the core matrix, 0.56 mg of magnetic microspheres were magnetically separated to remove the supernatant, washed three times with 400 μL of MEST solution, and finally dispersed in 150 μL of MEST solution to obtain a magnetic microsphere dispersion. 0.6 mg of 200 nm AB spheres were taken, centrifuged to remove the supernatant, and then ultrasonically dispersed in 150 μL of MEST solution to obtain a guest AB sphere dispersion. 15 μL of NaCNBH3 (25 mg / mL) was then added.

[0101] Step 2: The magnetic microsphere solution from Step 1 was added dropwise to the guest AB microsphere solution from Step 1 under ultrasonic conditions. After the addition was complete, the mixture was vortexed and placed in a 37°C incubator on a rotary mixer in the dark for 48 hours. After the reaction, the supernatant was removed by magnetic separation, and the mixture was washed three times with 2.5 mM NaOH and three times with ultrapure water. The resulting microspheres were dispersed in 150 μL of ultrapure water to obtain the host-guest MagLOCI microspheres.

[0102] When using magnetic microspheres with different diameters and magnetic contents, the assembly method of the magnetic microspheres and the guest AB spheres is basically the same as the method described above. Only the amount of guest AB spheres and the amount of related reactants need to be slightly adjusted according to the change in the surface area of ​​the microspheres.

[0103] Example 2: Bioconjugation on the surface of MagLOCI composite microspheres

[0104] Example 2-1 (Surface carboxyl group AB globule coupling to capture antibody molecules):

[0105] Step 1: Take 1 mg of 5.9 μm diameter host-guest structure magnetic microspheres into a 2 mL centrifuge tube, magnetically separate the supernatant, and wash three times with 400 μL of 10 mM MEST solution. Add 250 μL of a 50 mg / mL EDC and NHS solution, disperse the magnetic microspheres evenly, and react at room temperature for 20 min on a rotary mixer.

[0106] Step 2: Add 400 μL of 0.25 mg / mL capture antibody (Anti-PCT McAb, purchased from Shanghai Lingchao Biotechnology Co., Ltd., catalog number: L3C00901) solution, mix well, and place in a 37℃ incubator for 2 hours of rotation reaction.

[0107] Step 3: After the reaction is complete, wash the microspheres three times with blocking buffer (PBS buffer containing 0.3% glycine and 0.5% BSA), then add 400 μL of blocking buffer and block overnight at 4°C.

[0108] Step 4: After blocking the magnetic beads, wash them three times with storage solution (PBST buffer containing 0.1% BSA), then add 25 μL of storage solution to store the microspheres to obtain magnetic beads conjugated with antibody molecules.

[0109] Example 2-2 (Surface carboxyl AB globule-coupled antigen molecule):

[0110] Step 1: Take 1.25 mg of host-guest structured magnetic microspheres with a diameter of 5.9 μm into a 2 mL centrifuge tube, magnetically separate the supernatant, and wash three times with 400 μL of 10 mM MEST solution. Add 100 μL of 2 mg / mL EDC and 4 mg / mL NHS solution, disperse the magnetic microspheres evenly, and react at room temperature for 20 min on a rotary mixer.

[0111] Step 2: Add 12 μg of novel coronavirus antigen N protein (SARS-CoV-2NP-His, purchased from LONGi Biotechnology Co., Ltd., catalog number: AI04504), mix well, and place in a 37℃ constant temperature incubator for 2 hours by rotation reaction.

[0112] Step 3: After the reaction is complete, wash the microspheres three times with blocking solution, then add 400 μL of blocking solution and seal overnight in a 4°C refrigerator;

[0113] Step 4: After the magnetic beads are sealed, wash them three times with the storage solution, then add 25 μL of storage solution to store the microspheres, and obtain magnetic beads coupled with antigen molecules.

[0114] MagLOCI's detection process and applicable scenarios, such as Figure 1 As shown.

[0115] Example 3: The Influence of Physical Properties of MagLOCI Composite Microspheres on Detection Performance

[0116] Example 3-1 (MagLOCI detection procedure using a double-antibody sandwich method)

[0117] The MagLOCI technology described in this embodiment uses a double-antibody sandwich method to detect antigen samples through a two-step washing process. The following reagents are required:

[0118] (a) MagLOCI composite microspheres conjugated with antibody molecules; the microsphere preparation method is the same as in Example 2-1, except that each example uses amino-based microspheres with different particle sizes and / or magnetic contents.

[0119] (b) A biotin-modified detection antibody (Ab2) solution, which can form a double antibody sandwich immune complex with the capture antibody on the surface of MagLOCI complex microspheres through an immune reaction.

[0120] (c) A streptavidin-labeled donor microsphere (DB-SA) solution, wherein the DB-SA solution is capable of generating singlet oxygen in an excited state.

[0121] Specifically, the procedure for detecting procalcitonin (PCT) antigen samples using MagLOCI technology is as follows:

[0122] The reaction was carried out in PBS buffer containing 1% BSA and 0.05% Tween-20;

[0123] Step 1: Take 10 μL of MagLOCI composite microsphere solution conjugated with PCT capture antibody molecule (Anti-PCT McAb, purchased from Shanghai Lingchao Biotechnology Co., Ltd., catalog number: L3C00901) into each well of a 96-well plate; 10 μL of Ab2 (Anti-PCTMcAb, purchased from Shanghai Lingchao Biotechnology Co., Ltd., catalog number: L3C00904) solution; and 16 μL of serially diluted PCT recombinant antigen test sample (purchased from Shanghai Lingchao Biotechnology Co., Ltd., catalog number: L4C00901). After mixing thoroughly, incubate at 37°C with shaking for 30 min.

[0124] Step 2: Use a magnetic adsorption plate to fully adsorb the MagLOCI composite microspheres, remove the reaction liquid containing the matrix, add 200 μL of PBST buffer to wash the microspheres, and remove the MagLOCI composite microspheres by adsorbing the washing solution through the magnetic adsorption plate. The number of times the microspheres are washed depends on the content of interfering substances in the matrix. The higher the content of interfering substances, the more times they need to be washed.

[0125] Step 3: Add 180 μL of 20 μg / mL DB-SA solution to the cleaned microspheres and incubate with shaking at 37 °C for 15 min.

[0126] Step 4: Place the solution directly on the instrument to read the chemiluminescence signal value of the solution (excitation wavelength 680nm, measurement wavelength 615nm).

[0127] Example 3-2 (The Influence of Microsphere Magnetic Content on Detection Results)

[0128] The higher the magnetic content of MagLOCI composite microspheres, the faster the magnetic response speed of the microspheres and the smaller the error caused by washing. However, the higher the magnetic content, the stronger the absorption of LOCI fluorescence signal by the microspheres. This example compares the effect of microspheres with different magnetic contents on the detection results. The operation procedure is the same as in Example 3-1. MagLOCI microspheres with magnetic contents of 20 wt.%, 32 wt.%, 55 wt.%, and 75 wt.% were added to detect serially diluted PCT recombinant antigen. Washing was performed once between steps 2 and 3. The test results are as follows: Figure 2A and Figure 2B As shown.

[0129] As shown in the figure, microspheres with higher magnetic content exhibit stronger absorption of the detection signal and more significant signal reduction in high-concentration samples, while having little effect on the detection signal of low-concentration samples. Considering both detection performance and microsphere washing loss rate, the optimal magnetic content for microspheres is 10–30 wt.%.

[0130] Example 3-3 (Influence of Microsphere Feed Rate on Detection Results)

[0131] The amount of MagLOCI composite microspheres added affects the number of AB spheres in the reaction solution; more AB spheres produce a greater fluorescence signal. This example compares the effect of different numbers of magnetic spheres on the detection results. The procedure is the same as in Example 3-1: 10, 50, 250, and 1250 μg / mL of 5.9 μm MagLOCI microspheres were added to detect serially diluted PCT recombinant antigen. A wash was performed between steps 2 and 3. The test results are as follows: Figure 3A and Figure 3B As shown.

[0132] As shown in the figure, with the increase of the number of MagLOCI composite microspheres, the number of AB spheres also increases, thus increasing the detection signal value. However, the more magnetic spheres added, the higher the background signal. At low antigen concentrations, the more magnetic spheres added, the lower the P / N value. Considering both high and low concentration detection signals, the optimal magnetic sphere dosage is 50-250 μg / mL.

[0133] Comparative Example 1

[0134] This invention, by reading the overall fluorescence signal of the microsphere solution, has a detection performance superior to the multi-LOCI technology proposed in CN113376146A, which uses a fluorescence microscope to collect the light signal on a single microsphere.

[0135] The detection results using the same reaction conditions (same as Example 3-1) and two methods (the difference being the different detection methods) are as follows: Figure 4A and Figure 4B As shown, the detection limit of the MagLOCI technology of this invention is 3.3 pg / mL, which is much lower than the detection limit of 101.3 pg / mL of the Multi-LOCI technology, indicating a significant improvement in sensitivity. Furthermore, the dynamic range of the MagLOCI technology of this invention is four orders of magnitude, significantly higher than the two orders of magnitude of the Multi-LOCI technology.

[0136] Example 4: Magnetic Separation Anti-Matrix Interference Function

[0137] The MagLOCI technology proposed in this invention, through the introduction of magnetic microspheres, immobilizes receptor microspheres from photo-induced chemiluminescence (LOCI) technology onto the surface of the magnetic microspheres in a host-guest structure, thus endowing LOCI technology with a washing function. This satisfies certain clinical testing scenarios that require washing due to matrix interference, such as indirect antibody detection, detection of biotin-containing samples, and detection of clinical samples with high sensitivity requirements. Specific examples are as follows.

[0138] Example 4-1 (Indirect method for detecting COVID-19 IgG antibodies)

[0139] Homogeneous photochemiluminescence technology, due to its wash-free nature, cannot remove the large amounts of non-specific and free antibodies present in serum and plasma samples. This results in poor performance for indirect antibody detection and an inability to distinguish the type of immunoglobulin being detected. This invention utilizes magnetic separation to remove interfering antibodies from the sample, thus enabling indirect antibody detection. Taking the detection of COVID-19 IgG antibodies as an example, this invention demonstrates the function of detecting antibodies using an indirect, three-step washing method. Specifically, the operation procedure is as follows:

[0140] The reaction was carried out in PBS buffer containing 1% BSA and 0.05% Tween-20;

[0141] Step 1: Dilute the negative serum of healthy individuals 50 times with PBS buffer to use as the negative sample for testing. Use the diluted negative serum to serially dilute the COVID-19 IgG antibody strong positive control sample to use as the positive sample for testing.

[0142] Step 2: Add 2.5 μg of MagLOCI composite microsphere solution with 5.9 μm conjugated antigen molecules to a 96-well plate (microsphere preparation method is the same as in Example 2-1), then add 100 μL of serially diluted COVID-19 IgG antibody strong positive control sample, and incubate at 37°C with shaking for 30 min.

[0143] Step 3: Use a magnetic adsorption plate to fully adsorb MagLOCI composite microspheres, remove the reaction liquid containing interfering antibodies, add 200 μL of PBST buffer to wash the microspheres once, and then use the magnetic adsorption plate to adsorb MagLOCI composite microspheres to remove the washing solution.

[0144] Step 4: Add 190 μL of 4 μg / mL biotin-labeled secondary antibody (Mouse anti-human IgG, purchased from LONGi Biotechnology Co., Ltd., catalog number: MS00601) to the cleaned microspheres and incubate with shaking at 37°C for 30 min.

[0145] Step 5: Use a magnetic adsorption plate to fully adsorb MagLOCI composite microspheres, remove free secondary antibody, add 200 μL PBST buffer to wash the microspheres once, and then use the magnetic adsorption plate to adsorb MagLOCI composite microspheres to remove the washing solution.

[0146] Step 6: Add 200 μL of 20 μg / mL DB-SA solution to the cleaned microspheres and incubate with shaking at 37 °C for 15 min.

[0147] Step 7: Place the solution directly on the instrument to read the chemiluminescence signal value of the solution (excitation wavelength 680nm, measurement wavelength 615nm).

[0148] The test measured the positive signal values ​​of quality control samples containing IgG antibodies for COVID-19 testing at serial dilutions, as well as the negative signal values ​​of negative serum. The ratio (P / N) of the positive to negative sample signal values ​​at different dilutions can be used to determine the discriminative power of the detection signal at different IgG antibody concentrations. The test results are shown below. Figure 5A and Figure 5B .

[0149] Depend on Figure 5A and Figure 5B It can be seen that the MagLOCI indirect method of the present invention, which uses washing to detect novel coronavirus IgG antibodies, exhibits a good linear range and can accurately detect the novel coronavirus IgG antibodies in serum. However, the performance of the MagLOCI method without washing and the indirect method of photo-induced chemiluminescence for antibody determination is poor.

[0150] Example 4-2 (Detection of samples containing biotin)

[0151] Serum / plasma samples containing biotin can interfere with biotin-avidin binding, leading to false negative results. MagLOCI removes free biotin from the sample through magnetic separation, thereby eliminating the influence of biotin on the binding of Ab2 to donor spheres. This application demonstrates the results of detecting biotin-containing procalcitonin (PCT) samples using a double-antibody sandwich method. The procedure is the same as in Example 3-1, using a gradient of 0.1-10 μg / mL biotin (Biotin, purchased from Sigma, catalog number: V900418-1G) solutions prepared with PBS buffer. 1 ng / mL of PCT recombinant antigen was diluted with the biotin solution, and the serially diluted biotin was used as the test sample. To ensure thorough washing of biotin, the microspheres were washed three times in step two. The MagLOCI test results are shown in Table 1, and the LOCI technology was also compared as a control; the test results are shown in Table 2. As can be seen, the detection signal of the wash-free LOCI technology was significantly suppressed at biotin solution concentrations of 0.1, 1, and 10 μg / mL, with a signal-to-weight ratio reduction of 79.5% compared to the sample without biotin. However, the MagLOCI technology, after washing, showed a significantly reduced decrease in detection signal at biotin solution concentrations of 0.1, 1, and 10 μg / mL, with signal-to-weight ratio reductions of 18.7%, 27.1%, and 30.7% respectively compared to the sample without biotin.

[0152] Table 1

[0153] Biotin concentration pg / mL 10 1 0.1 0 1 ng / mL PCT sample signal value 2221 1979 2605 3099 0 ng / mL PCT sample signal value 193 163 193 187 Signal to background ratio 11.5 12.1 13.5 16.6

[0154] Table 2

[0155] Biotin concentration pg / mL 10 1 0.1 0 1 ng / mL PCT sample signal value 4554 4477 6190 12063 0 ng / mL PCT sample signal value 5480 4780 7663 3058 Signal to background ratio 0.8 0.9 0.8 3.9

[0156] Example 4-3 (Detection of clinical samples)

[0157] Serum and plasma samples often contain endogenous interfering proteins, which can lead to false positives and false negatives in immunoassays. These include substances such as complement, lysozyme, fibrin, autoantibodies, rheumatoid factor, and heterophile antibodies. Magnetic separation to remove the clinical sample matrix can eliminate interfering substances, improving the sensitivity and accuracy of sample detection. This invention demonstrates the results of detecting PCT clinical samples using a double-antibody sandwich method. The procedure is the same as in Example 3-1, except that the microspheres are washed only once in step two. The ratio of positive to negative sample signal values ​​(P / N) at different dilutions can be used to determine the discriminative power of the detection signal at different PCT clinical sample concentrations. The test results are shown in [Figure 1]. Figure 6A and Figure 6B .

[0158] Depend on Figure 6A and 6BIt can be seen that the double-antibody sandwich detection of PCT clinical samples in this invention shows good linearity and can accurately quantify the PCT antigen molecules to be detected in serum. In contrast, the photo-induced chemiluminescence method without washing has poor detection sensitivity due to matrix interference.

[0159] Example 5: Magnetic Separation to Eliminate Hook Effect

[0160] Example 5-1 (Magnetic Separation to Eliminate Hook Effect)

[0161] During immunoassay, excessively high concentrations of the analyte can reduce the formation of immune complexes, leading to lower test signal values ​​and false negatives, known as the hook effect. This invention, using a double-antibody sandwich assay for PCT recombinant antigen detection as an example, employs a three-step reaction process with magnetic separation capability. After the antigen molecules react with the detection antibody, free antigen molecules are washed away, thus eliminating the hook effect. Specifically, the operation procedure is as follows:

[0162] The reaction was carried out in PBS buffer containing 1% BSA and 0.05% Tween-20;

[0163] Step 1: Use PBS buffer to gradient dilute PCT recombinant antigen.

[0164] Step 2: Add 2.5 μg of MagLOCI composite microsphere solution with 5.9 μm PCT capture antibody to a 96-well plate (microsphere preparation method is the same as in Example 2-1), then add 16 μL of serially diluted PCT recombinant antigen, and incubate with shaking at 37°C for 30 min.

[0165] Step 3: Use a magnetic adsorption plate to fully adsorb MagLOCI composite microspheres, remove the reaction liquid containing interfering antibodies, add 200 μL of PBST buffer to wash the microspheres once, and then use the magnetic adsorption plate to adsorb MagLOCI composite microspheres to remove the washing solution.

[0166] Step 4: Add 200 μL of 0.2 μg / mL biotin-labeled detection antibody to the cleaned microspheres and incubate with shaking at 37°C for 30 min.

[0167] Step 5: Use a magnetic adsorption plate to fully adsorb MagLOCI composite microspheres, remove free secondary antibody, add 200 μL PBST buffer to wash the microspheres once, and then use the magnetic adsorption plate to adsorb MagLOCI composite microspheres to remove the washing solution.

[0168] Step 6: Add 200 μL of 20 μg / mL DB-SA solution to the cleaned microspheres and incubate with shaking at 37 °C for 15 min.

[0169] Step 7: Place the solution directly on the instrument and read the chemiluminescence signal value (excitation wavelength 680 nm, measurement wavelength 615 nm). See the test results below. Figure 7 .

[0170] Depend on Figure 7 It was found that unwashed microspheres exhibited a severe hook effect at high antigen concentrations (>100 ng / mL). The MagLOCI technique, employing a three-step reaction, eliminates this hook effect by washing the microspheres in the first two steps.

[0171] Example 6: Standard curve for multiple detection (triple standard curve for detecting PCT / IL-6 / IFN-γ)

[0172] The MagLOCI three-step washing method can significantly reduce the risk of interference in multiplex assays, enabling the simple and rapid development of multiplex assay reagents with performance consistent with single-analysis. Specifically, the operation procedure is as follows:

[0173] The reaction was carried out in PBS buffer containing 1% BSA and 0.05% Tween-20;

[0174] Step 1. Prepare MagLOCI composite microsphere solution conjugated with PCT, IL-6, and IFN-γ capture antibodies (IL-6 capture antibody, purchased from R&D, catalog number: MAB206-500; IFN-γ capture antibody, purchased from Biolegend, catalog number: 507502). The microsphere preparation method is the same as in Example 2-1. The PCT / IL-6 / IFN-γ indicators are obtained by using AB nanospheres doped with europium (615nm emission), terbium (545nm emission), and samarium (645nm emission) metal complexes as guest nanospheres.

[0175] Step 2. Preparation of biotin-modified detection antibody (Ab2) solution: Dilute the biotin-modified PCT, IL-6, and IFN-γ detection antibodies (IL-6 detection antibody, purchased from R&D, catalog number: BAF206; IFN-γ detection antibody, purchased from Biolegend, catalog number: 502503) to a concentration of 0.6 ug / mL and mix them.

[0176] Step 3. Use PBS buffer to serially dilute PCT, IL-6, and IFN-γ antigen standards (IL-6 recombinant antigen, purchased from R&D, catalog number: 206-IL-010; IFN-γ recombinant antigen, purchased from Biolegend, catalog number: 570209) to 300, 30, 3, 0.3, 0.03, and 0 ng / mL, and mix each standard at the same concentration.

[0177] Step 4. Add 2.5 μg of 5.9 μm to a 96-well plate, then add 16 μL of serially diluted PCT, IL-6, and IFN-γ recombinant antigen, and incubate with shaking at 37°C for 30 min.

[0178] Step 5. Use a magnetic adsorption plate to fully adsorb the MagLOCI composite microspheres, remove the reaction liquid containing interfering antibodies, add 200 μL of PBST buffer to wash the microspheres once, and then use the magnetic adsorption plate to adsorb the MagLOCI composite microspheres to remove the washing solution.

[0179] Step 6. After cleaning, add 200 μL of a mixture of biotin-labeled detection antibodies to the microspheres and incubate with shaking at 37°C for 30 min.

[0180] Step 7. Use a magnetic adsorption plate to fully adsorb MagLOCI composite microspheres, remove free secondary antibody, add 200 μL PBST buffer to wash the microspheres once, and then use the magnetic adsorption plate to adsorb MagLOCI composite microspheres to remove the washing solution.

[0181] Step 8. Add 200 μL of 20 μg / mL DB-SA solution to the cleaned microspheres and incubate with shaking at 37 °C for 15 min.

[0182] Step 9. Place the solution directly onto the instrument and read the signal values ​​from the three AB sphere sources in the solution using the multiple detection method described in Example 7. The test results are shown below. Figure 8 .

[0183] from Figure 8 The results show that the MagLOCI technique, using a three-step washing method for microspheres, is feasible for simultaneously detecting PCT / IL-6 / IFN-γ indices.

Claims

1. A method for detecting biomolecules, characterized in that, The method includes: (1) When the biodetector molecule is present or absent, the magnetic composite microspheres and the sample containing the analyte molecule are mixed and incubated, and the matrix reaction solution is removed; (2) Add the microspheres obtained in (1) to a solution containing matching microspheres, mix and incubate, and detect and collect the luminescence signal; In (1), when the biodetector molecules are not present, (2) further includes the steps of adding the microspheres obtained in (1) to a solution containing biodetector molecules, mixing and incubating, and removing the matrix reaction solution. The magnetic composite microspheres comprise a host magnetic microsphere and guest nanospheres covalently attached to the surface of the host magnetic microsphere. The guest nanospheres are used to generate singlet oxygen or convert the energy of singlet oxygen into fluorescence. Biocapture molecules are coupled to the surface of the guest nanospheres and are used to bind to analyte molecules. Biodetection molecules are used to bind to analyte molecules, and the biodetection molecules and the biocapture molecules bind to different sites on the analyte molecules. The biodetection molecules specifically bind to the matched microspheres. And, when the guest nanospheres generate singlet oxygen, the matching microspheres are used to convert the energy of the singlet oxygen into fluorescence; when the guest nanospheres are used to convert the energy of the singlet oxygen into fluorescence, the matching microspheres are used to generate singlet oxygen; The magnetic composite microspheres have a magnetic content of 10~30 wt.%; The amount of magnetic composite microspheres fed is 50-250 μg / mL; The surface of the host magnetic microsphere or guest nanosphere is modified with monomers or polymers containing amino, carboxyl or aldehyde groups. The surface of the host magnetic microsphere or guest nanosphere is modified with an amino group or its polymer, and correspondingly the surface of the guest nanosphere or host magnetic microsphere is modified with a monomer or its polymer containing a carboxyl group or aldehyde group; and / or, the density of the monomer or its polymer containing an amino group, carboxyl group or aldehyde group modified on the surface of the guest nanosphere or host magnetic microsphere is at least 100 μmol / mg. The biocapture molecule is an antibody molecule or an antigen molecule.

2. The method as described in claim 1, characterized in that, The surface of the host magnetic microsphere is modified with an amino group or its polymer, and the surface of the guest nanosphere is modified with a monomer or its polymer containing a carboxyl or aldehyde group; and / or, the amino-containing polymer is polyethyleneimine.

3. The method as described in claim 1, characterized in that, The matching microspheres or guest nanospheres that generate singlet oxygen contain a photosensitizer that generates singlet oxygen upon receiving excitation light; the guest nanospheres or matching microspheres that convert the energy of the singlet oxygen contain dimethylthiophene and a lanthanide metal complex, wherein the dimethylthiophene converts the energy of the singlet oxygen into emitted light and generates fluorescence through the lanthanide metal complex; and / or, the biodetector molecule specifically binds to the matching microspheres via a biotin-avidin system.

4. The method as described in claim 3, characterized in that, The biodetector molecule or analyte is a biotin-modified biodetector molecule or analyte, and the matching microspheres are streptavidin-modified matching microspheres; and / or, the lanthanide metal complex is selected from one or more of europium, terbium, and samarium.

5. The method as described in claim 1, characterized in that, The matrix removal reaction solution is performed via magnetic adsorption; And / or, in (1), the step of washing is further included after the removal of the matrix reaction solution; And / or, in (2), the mixing and incubation may optionally include a washing step.

6. The method as described in claim 5, characterized in that, In (1), the step of washing is further included after removing the matrix reaction solution; and / or, in (2), the step of washing is further included after mixing and incubation.

7. The method according to any one of claims 1 to 6, characterized in that, The mixing and incubation are carried out in a buffer solution; and / or, the luminescence signal is acquired by a photodetector or imaging system.

8. The method as described in claim 7, characterized in that, The photodetector is a PMT or PD, with an excitation wavelength of 680 nm and a measurement wavelength of 615 nm.

9. The method as described in claim 8, characterized in that, The photodetector is an APD.

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