Immunoassay reagent based on AIE magnetic encoded microspheres and AIE nano-microspheres, and preparation method therefor and use thereof

Abstract

An immunoassay reagent based on AIE magnetic encoded microspheres and AIE nano-microspheres, and a preparation method therefor and a use thereof. The immunoassay reagent based on AIE magnetic encoded microspheres and AIE nano-microspheres comprises antibody-1 labeled AIE magnetic encoded microspheres and antibody-2 labeled AIE nano-microspheres. The antibody-1 labeled AIE magnetic encoded microspheres and the antibody-2 labeled AIE nano-microspheres are added into a solution to be detected containing a corresponding antigen for reaction, magnetic separation is performed, an encoding peak and a fluorescence quantitative detection concentration are obtained by means of a flow cytometer, realizing long linear range detection in complex samples with multiple markers.

Description

An immunoassay reagent based on AIE magnetically encoded microspheres and AIE nanospheres, and its preparation method and application Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and in particular relates to an immunoassay reagent based on AIE magnetically encoded microspheres and AIE nanospheres, and a preparation method and application thereof. Background Art

[0002] The recent development and completion of the human genome and proteome has led to a tremendous demand for large-scale biomolecular analysis, driving the rapid development of liquid-phase suspension biochips centered around encoded microspheres. Liquid-phase suspension biochips, utilizing rapid, high-throughput flow cytometry and magnetic separation as analytical tools, are enabling rapid, high-throughput, multiplexed detection of markers such as proteins, nucleic acids, and small molecules, and are becoming a new target for precision diagnosis. However, developing encoded microspheres with high encoding capacity and stability remains a significant challenge. Suitable encoded microsphere designs require high photostability, excellent antimagnetic properties, a high number of encodings, and no reabsorption or crosstalk.

[0003] Spectral signals are widely used in liquid-phase biochip construction due to their flexible encoding and fast decoding speeds. Currently, commonly used spectral encoding materials include organic materials, quantum dots, upconversion dots, and photonic crystals. Quantum dots have become a popular encoding material due to their high brightness and narrow emission spectrum. However, their encoding capabilities are limited by reabsorption and cross-talk between quantum dot fluorescence signals, restricting the application of quantum dot-encoded microspheres.

[0004] Currently, commercial liquid-phase biochips such as the Luminex system and BD's cytometric bead arrays all use organic dyes for encoding. However, traditional organic materials exhibit fluorescence quenching when aggregated, limiting their encoding capacity and stability. The novel aggregation-induced emission (AIE) material, discovered by Academician Tang Benzhong's team in 2001, is a promising material for the preparation of encoded microspheres because it can achieve in situ enhancement of the fluorescence signal during the aggregation process. Aggregation-induced emission materials exhibit properties such as large Stokes shift, strong resistance to photobleaching, and antimagnetism, making them excellent materials for encoding microspheres.

[0005] Until recently, Li Wanwan used membrane emulsification technology to prepare AIE-encoded non-magnetic microspheres for the detection of multiple markers. However, the lack of magnetic materials has prevented high-throughput testing. Currently, there are few published reports on the efficient preparation of AIE magnetic fluorescent encoding using AIE materials. Therefore, the development of a technology for the efficient and direct generation of AIE magnetic-encoded microspheres is essential.

[0006] Summary of the Invention

[0007] In order to address the deficiencies in the prior art, the present invention aims to provide an immunoassay reagent based on AIE magnetically encoded microspheres and AIE nanospheres, as well as a preparation method and application thereof.

[0008] The technical solution adopted in the present invention is:

[0009] An immunoassay reagent based on AIE magnetically encoded microspheres and AIE nanospheres comprises AIE magnetically encoded microspheres labeled with antibody-1 and AIE nanospheres labeled with antibody-2.

[0010] Preferably, the mass amount of the AIE nanospheres labeled with antibody-2 is 0.001-0.01 wt% of the mass amount of the AIE magnetic encoding microspheres labeled with antibody-1;

[0011] Preferably, the particle size ratio of the AIE magnetically encoded microspheres to the AIE nanospheres is (10-350):1;

[0012] Further preferably, the particle size ratio of the AIE magnetically encoded microspheres to the AIE nanospheres is (100-350):1;

[0013] Further preferably, the particle size of the AIE magnetically encoded microspheres is in the range of 1.5 μm to 20 μm; the particle size of the AIE nanospheres is in the range of 10 nm to 1000 nm;

[0014] Preferably, the antibody-1 and antibody-2 can specifically bind to the same antigen.

[0015] Further preferably, the antigen is a cytokine marker, including IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α, and IFN-γ.

[0016] Further preferably, the antibody-1 is selected from at least one of IL-2 monoclonal antibody, IL-4 monoclonal antibody, IL-6 monoclonal antibody, IL-10 monoclonal antibody, IL-17 monoclonal antibody, TNF-α monoclonal antibody and IFN-γ monoclonal antibody; antibody-2 is selected from the anti-antibody of antibody-1;

[0017] More preferably, the antibody-1 and antibody-2 are mouse antibodies.

[0018] Preferably, the emission wavelength of the AIE nanospheres is in the range of 450 nm to 550 nm;

[0019] Preferably, the AIE nanospheres include carboxyl-modified blank polymer nanospheres and AIE-N molecules; the AIE-N molecules are embedded in the carboxyl-modified blank polymer nanospheres;

[0020] Further preferably, in the AIE nanospheres,

[0021] The carboxyl-modified blank polymer nanospheres are selected from at least one of carboxyl-modified polystyrene microspheres, carboxyl-modified polymethyl methacrylate microspheres, and carboxyl-modified polyglycidyl acrylate microspheres;

[0022] The AIE-N molecules are embedded in the interior of the blank polymer nanospheres modified with carboxyl groups by an active swelling method. The amount of the AIE-N molecules used in the active swelling method is 0.01-10 wt % of the amount of the blank polymer nanospheres modified with carboxyl groups.

[0023] The AIE-N molecules in the AIE nanospheres are selected from at least one of the following AIE-N1 to AIE-N10:

[0024] The alkyl chains in AIE-N8, AIE-N9, and AIE-N10 molecules are -C8H 17 Each is independently a branched or straight chain alkyl group.

[0025] More preferably, the active swelling method is to allow the AIE-N molecules to swell with the carboxyl-modified blank polymer nanospheres in a swelling agent so that the AIE-N molecules are embedded in the carboxyl-modified blank polymer nanospheres;

[0026] In the active swelling method, the swelling agent is selected from at least one of dichloromethane, tetrahydrofuran, chloroform, anisole, benzyl alcohol and toluene, and the particle size of the carboxyl-modified blank polymer nanospheres ranges from 10 nm to 500 nm; the carboxyl-modified blank polymer nanospheres are nanoscale microsphere matrices known in the art.

[0027] Preferably, the preparation method of the AIE nanospheres comprises the following steps:

[0028] (1) dispersing carboxyl-modified blank polymer nanospheres in water to obtain a nanosphere dispersion;

[0029] (2) dissolving the AIE-N molecules in a swelling agent to obtain an AIE swelling agent solution;

[0030] (3) adding the AIE swelling agent solution described in step (2) to the microsphere dispersion described in step (1), and after ultrasonic treatment, sealing the reactor to carry out a swelling reaction. After the reaction is completed, volatilizing and removing the swelling agent to obtain AIE nanospheres;

[0031] Further preferably, the preparation method of the AIE nanospheres comprises the following steps:

[0032] (1) dispersing carboxyl-modified blank polymer nanospheres in water to obtain a nanosphere dispersion;

[0033] (2) dissolving the AIE-N molecules in a swelling agent to obtain an AIE swelling agent solution;

[0034] (3) adding the AIE swelling agent solution described in step (2) to the microsphere dispersion described in step (1), ultrasonically treating the microsphere dispersion for 1 to 30 minutes at an ultrasonic power of 50 W to 400 W, and then sealing the reactor to carry out a swelling reaction for 0.5 to 4 hours. After the reaction is completed, the swelling agent is volatilized and removed to obtain AIE nanospheres;

[0035] Further preferably, in step (1), the mass amount of the carboxyl-modified blank polymer nanospheres in the microsphere dispersion is 0.1% to 20% of the mass amount of water;

[0036] Further preferably, in step (2), the mass amount of AIE-N molecules in the AIE swelling agent solution is 0.05% to 20% of the mass amount of the swelling agent; further preferably, the mass amount of AIE-N molecules in the AIE swelling agent solution is 0.1% to 5% of the mass amount of the swelling agent.

[0037] Further preferably, in step (3), the time for volatilizing and removing the swelling agent is 2 hours to 10 hours.

[0038] Preferably, the encoding capacity of the AIE magnetically encoded microspheres is ≥9, and the CV value of the flow fluorescence signal and the CV value of the flow FCS are both less than 10%;

[0039] Preferably, the encoding capacity of the AIE magnetically encoded microspheres is ≥24; the AIE magnetically encoded microspheres are dual-wavelength aggregation-induced luminescence magnetically encoded microspheres, comprising AIE polymer microspheres and magnetic particles; the AIE polymer microspheres are obtained by embedding AIE molecules inside blank polymer microspheres, and the magnetic particles are deposited on the surface of the AIE polymer microspheres; the AIE molecules include AIE-R molecules and AIE-IR molecules;

[0040] The difference between the maximum emission wavelength of the AIE-IR molecule and the maximum emission wavelength of the AIE-R molecule is in the range of 160nm to 290nm, and satisfies Δλ≥185. Where I / I0 is the average ratio of the fluorescence intensity of the AIE-IR molecule and the fluorescence intensity of the AIE-IR molecule before and after embedding, λ IR is the maximum emission wavelength of the AIE-IR molecule, λ R is the maximum emission wavelength of the AIE-R molecule.

[0041] More preferably,

[0042] In the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres,

[0043] The blank polymer microspheres are selected from at least one of polystyrene microspheres, polymethyl methacrylate microspheres and polyglycidyl acrylate microspheres; more preferably, the blank polymer microspheres are selected from polystyrene microspheres or polyglycidyl acrylate microspheres.

[0044] The magnetic particles are Fe3O4;

[0045] The AIE-R molecule is selected from at least one of the following AIE-R1 to AIE-R16 molecules:

[0046] The alkyl chain in the AIE-R1 molecule is -C8H 17 Each is independently a branched or straight chain alkyl group.

[0047] The alkyl chain in the AIE-R11 molecule is -C6H 13 Each is independently a branched or straight chain alkyl group.

[0048] The AIE-IR molecule is selected from at least one of the following AIE-IR1 to AIE-IR10 molecules:

[0049] The AIE molecules are embedded in the blank polymer microspheres by an active swelling method, wherein the amount of AIE molecules used in the active swelling method is 0.01-10 wt % of the amount of the blank polymer microspheres, wherein the mass amount of the AIE-R molecules accounts for 0.01% to 1000% of the mass amount of the AIE-IR molecules;

[0050] The magnetic particles are deposited on the surface of the AIE polymer microspheres by an in-situ precipitation method of iron salts, wherein the amount of iron salts used in the in-situ precipitation method is 0.1-25 wt % of the amount of blank polymer microspheres used.

[0051] More preferably,

[0052] The active swelling method is to allow the AIE molecules to swell with the blank polymer microspheres in a swelling agent so that the AIE molecules are embedded in the blank polymer microspheres.

[0053] In the active swelling method, the swelling agent is selected from at least one of dichloromethane, tetrahydrofuran, chloroform, anisole, benzyl alcohol and toluene, and the particle size of the blank polymer microspheres ranges from 1.5 μm to 20 μm.

[0054] The in-situ precipitation method is to add an alkaline solution and react with the iron salt chelated on the surface of the AIE polymer microspheres in situ to generate magnetic particles that are deposited on the surface of the AIE polymer microspheres;

[0055] In the in-situ precipitation method, the surface of the AIE polymer microspheres is modified by sulfonation, the alkaline solution is selected from at least one of sodium hydroxide solution, ammonia water, potassium hydroxide solution and sodium bicarbonate solution; the iron salt is selected from at least one of trivalent iron salt and divalent iron salt; the trivalent iron salt is selected from at least one of ferric chloride hexahydrate and anhydrous ferric chloride; the divalent iron salt is selected from at least one of ferrous chloride tetrahydrate, ferrous chloride dihydrate, ferrous sulfate and ferrous sulfate heptahydrate.

[0056] Taking into account the uniformity of magnetic particles deposited on the surface of the AIE polymer microspheres, the iron salt is a compound of a trivalent iron salt and a divalent iron salt, wherein the mass amount of the trivalent iron salt is 10% to 60% of the mass amount of the divalent iron salt.

[0057] Further preferably, the preparation method of the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres comprises the following steps:

[0058] S1. Preparation of AIE polymer microspheres by active swelling method

[0059] (1) dispersing blank polymer microspheres in water to obtain a microsphere dispersion;

[0060] (2) dissolving the AIE molecules in a swelling agent to obtain an AIE swelling agent solution;

[0061] (3) adding the AIE swelling agent solution described in step (2) to the microsphere dispersion described in step (1), and after ultrasonic treatment, closing the reactor to carry out a swelling reaction. After the reaction is completed, volatilizing and removing the swelling agent to obtain AIE polymer microspheres;

[0062] S2. Preparation of Dual-Wavelength Aggregation-Induced Luminescence Magnetic Encoded Microspheres by In Situ Precipitation

[0063] The AIE polymer microspheres prepared in step S1 were dissolved in a sulfuric acid solution, the temperature was raised to react, and the supernatant was removed by centrifugation to obtain AIE polymer microspheres with sulfonated surfaces. The AIE polymer microspheres were mixed with iron salt and water to react, and the supernatant was removed by centrifugation to obtain iron salt-chelated AIE polymer microspheres. An alkaline solution was added, the temperature was raised, and the iron salt reacted in situ to generate magnetic particles that were deposited on the surface of the AIE polymer microspheres. The supernatant was removed by centrifugation to prepare dual-wavelength aggregation-induced luminescence magnetically encoded microspheres.

[0064] More preferably, the preparation method of the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres comprises the following steps:

[0065] S1. Preparation of AIE polymer microspheres by active swelling method

[0066] (1) dispersing blank polymer microspheres in water to obtain a microsphere dispersion;

[0067] (2) dissolving the AIE molecules in a swelling agent to obtain an AIE swelling agent solution;

[0068] (3) adding the AIE swelling agent solution described in step (2) to the microsphere dispersion described in step (1), ultrasonically treating the microsphere dispersion at an ultrasonic power of 50 W to 400 W for 1 to 30 minutes, sealing the reactor to carry out a swelling reaction for 0.5 to 4 hours, and after the reaction is completed, volatilizing and removing the swelling agent to obtain AIE polymer microspheres;

[0069] S2. Preparation of Dual-Wavelength Aggregation-Induced Luminescence Magnetic Encoded Microspheres by In Situ Precipitation

[0070] The AIE polymer microspheres of step S1 are dissolved in a sulfuric acid solution, the temperature is raised to 30°C to 100°C and the reaction is carried out for 4h to 12h, and the supernatant is removed by centrifugation to obtain AIE polymer microspheres with sulfonation-modified surfaces. The AIE polymer microspheres are mixed with iron salt and water, soaked and reacted for 0.5h to 10h, and the supernatant is removed by centrifugation to obtain iron salt-chelated AIE polymer microspheres. An alkaline solution is added, the temperature is raised to 60°C to 120°C and the reaction is carried out for 0.5h to 4h, the iron salt reacts in situ to generate magnetic particles deposited on the surface of the AIE polymer microspheres, and the supernatant is removed by centrifugation to prepare aggregation-induced luminescence magnetically encoded microspheres.

[0071] More preferably, in step S1, the ultrasonic power in step (3) is 300W, the ultrasonic treatment time is 5 minutes, and the time for volatilizing and removing the swelling agent is 2 hours to 10 hours. Further preferably, the time for volatilizing and removing the swelling agent is 4 hours.

[0072] More preferably, in step S2, the soaking reaction time is 2 hours to 4 hours.

[0073] More preferably, in step S1, the mass amount of blank polymer microspheres in the microsphere dispersion is 0.1% to 20% of the mass amount of water; further preferably, the mass amount of blank polymer microspheres in the microsphere dispersion is 0.5% to 10% of the mass amount of water.

[0074] More preferably, in step S1, the mass amount of the AIE molecules in the AIE swelling agent solution is 0.1% to 50% of the mass amount of the swelling agent;

[0075] More preferably, in step S2, the mass concentration of the sulfuric acid solution is 70% to 99%; the mass amount of the AIE polymer microspheres is 1% to 30% of the mass amount of the sulfuric acid solution; further preferably, the mass concentration of the sulfuric acid solution is 98.3%; the mass amount of the AIE polymer microspheres is 5% to 20% of the mass amount of the sulfuric acid solution.

[0076] More preferably, in step S2, the mass amount of the iron salt is 0.0005% to 25% of the mass amount of water; the mass amount of the alkaline solution is 0.1% to 50% of the mass amount of water; the mass concentration of the alkaline solution is 1% to 10%;

[0077] More preferably, the water in step S1 and step S2 is ultrapure water; the centrifugal speed is 3000 rpm to 6000 rpm, the centrifugal time is 5 min to 60 min, and the number of centrifugation is three or more.

[0078] The preparation method of the above-mentioned immunoassay reagent based on AIE magnetically encoded microspheres and AIE nanospheres comprises the following steps:

[0079] (1) Carboxyl modification of AIE magnetically encoded microspheres, activation of the carboxyl groups, and coupling with antibody-1 to obtain antibody-1 labeled AIE magnetically encoded microspheres;

[0080] (2) activating the carboxyl groups of the AIE nanospheres and coupling them with antibody-2 to obtain antibody-2 labeled AIE nanospheres;

[0081] Immunodetection reagents based on AIE magnetically encoded microspheres and AIE nanospheres are obtained.

[0082] Preferably,

[0083] In step (1), the carboxyl modification comprises: mixing and reacting the AIE magnetically encoded microspheres, a base, TEOS and a silane coupling agent, magnetically separating, and adding a monomer containing a double bond and a carboxyl group to react to obtain the carboxyl-modified AIE magnetically encoded microspheres;

[0084] In step (1), the activation of the carboxyl group comprises: dispersing the carboxyl-modified AIE magnetic encoded microspheres in an acidic buffer, adding a carbodiimide condensation reagent, and stirring the reaction to activate the carboxyl group, thereby obtaining the carboxyl-activated AIE magnetic encoded microspheres;

[0085] In step (1), the antibody-1 coupling comprises: dispersing the AIE magnetically encoded microspheres with activated carboxyl groups in an alkaline buffer, adding the antibody-1, incubating with stirring, separating by magnetic attraction, adding a blocking solution and a preservation solution, and obtaining the AIE magnetically encoded microspheres labeled with the antibody-1;

[0086] In step (2), the activation of the carboxyl group comprises: dispersing the carboxyl-modified AIE nanospheres in an acidic buffer, adding a carbodiimide condensation reagent, and stirring the reaction to activate the carboxyl group, thereby obtaining the carboxyl-activated AIE nanospheres;

[0087] In step (2), the antibody-2 coupling includes: dispersing the activated carboxyl AIE nanospheres in an alkaline buffer, adding antibody-2, incubating with stirring, separating by magnetic attraction, adding blocking solution and preservation solution, and obtaining antibody-2 labeled AIE nanospheres.

[0088] More preferably,

[0089] In the carboxyl modification of step (1), the silane coupling agent is selected from at least one of the following: 3-vinyltrimethoxysilane, methacryloxypropyltrimethoxysilane, vinyltrimethoxysilane, and vinyltriethoxysilane;

[0090] In the carboxyl modification of step (1), the monomer containing a double bond and a carboxyl group is selected from at least one of the following: acrylic acid, methacrylic acid, and maleic anhydride;

[0091] In the carboxyl modification of step (1), the base is aqueous ammonia with a concentration of 20% to 28%;

[0092] In the carboxyl modification of step (1), the mass volume ratio of AIE magnetic encoded microspheres, base, TEOS, silane coupling agent and monomer containing double bonds and carboxyl groups is 0.1g:2~3mL:1~1.5mL:0.45~0.6mL:0.3~0.5g; the temperature of the mixed reaction is 25℃~55℃, and the time is 4h~12h; the temperature of the reaction of adding the monomer containing double bonds and carboxyl groups is 60℃~80℃, and the time is 6h~24h.

[0093] In the activated carboxyl groups in steps (1) and (2), the acidic buffer is a glycine-hydrochloric acid buffer, a phthalic acid-hydrochloric acid buffer, a disodium hydrogen phosphate-citric acid buffer, or a 2-(N-morpholino)ethanesulfonic acid buffer; the pH value of the acidic buffer is 6.0 to 6.5;

[0094] In the activated carboxyl groups of steps (1) and (2), the carbodiimide condensing agent is selected from at least one of N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0095] In the steps (1) and (2) of activating the carboxyl group, the activation time of the carboxyl group is 5 min to 3 h;

[0096] In the activated carboxyl group of step (1), the mass amount of the acidic buffer is 10 to 100 times the mass amount of the AIE magnetically encoded microspheres;

[0097] In the activated carboxyl group of step (1), the mass amount of the carbodiimide condensation reagent is 1% to 90% of the mass amount of the AIE magnetic encoded microspheres;

[0098] In the activated carboxyl group of step (2), the mass amount of the acidic buffer solution is 10 to 100 times the mass amount of the AIE nanospheres;

[0099] In the activated carboxyl group of step (2), the mass amount of the carbodiimide condensation reagent is 1% to 90% of the mass amount of the AIE nanospheres;

[0100] In the antibody-1 coupling in step (1) and the antibody-2 coupling in step (2), the alkaline buffer is tris-hydrochloric acid buffer, boric acid buffer or phosphate buffer saline; the pH value of the alkaline buffer is 7.0 to 9.0;

[0101] In the antibody-1 coupling in step (1), the mass amount of the alkaline buffer is 5 to 90 times the mass amount of the AIE magnetic encoded microspheres.

[0102] In the antibody-2 coupling in step (2), the mass amount of the alkaline buffer is 5 to 90 times the mass amount of the AIE nanospheres.

[0103] In the antibody-1 coupling in step (1), the mass amount of the antibody-1 is 0.005 to 0.02% of the mass amount of the AIE magnetically encoded microspheres.

[0104] In the antibody-1 coupling in step (1), the stirring incubation temperature is 25° C. to 35° C., and the time is 5 minutes to 3 hours.

[0105] In the antibody-2 coupling in step (2), the mass amount of the antibody-1 is 0.005 to 0.02% of the mass amount of the AIE nanospheres.

[0106] In the antibody-2 coupling in step (2), the stirring incubation temperature is 25° C. to 35° C., and the time is 5 minutes to 3 hours.

[0107] The application of the above-mentioned immunoassay reagent based on AIE magnetically encoded microspheres and AIE nanospheres in antigen immunoassay comprises the following steps:

[0108] Antibody-1 labeled AIE magnetic encoding microspheres and antibody-2 labeled AIE nanospheres were added to the test solution containing the corresponding antigen. After the reaction, they were separated by magnetic attraction and the encoding peak and fluorescence quantitative detection concentration were obtained by flow cytometry.

[0109] Preferably, the mass amount of the corresponding antigen is 0.01-1 wt% of the mass amount of the AIE nanoparticles labeled with antibody-2.

[0110] The present invention can use AIE magnetically encoded microspheres labeled with antibody-1 for multiple different antigens and AIE nanospheres labeled with antibody-2 for the corresponding antigens to detect the test solution respectively, thereby realizing the detection of multiple antigens in the test solution; that is, the AIE magnetically encoded microspheres labeled with antibody-1 for one antigen and the AIE nanospheres labeled with antibody-2 for the corresponding antigen are used to detect the test solution each time, and this process is repeated multiple times.

[0111] The present invention can use AIE magnetic encoded microspheres with different coding intensities respectively labeled with antibodies-1 for multiple different antigens and AIE nanospheres labeled with antibodies-2 for corresponding antigens to perform a single detection on the test solution, thereby realizing the detection of multiple antigens in the test solution; that is, the AIE magnetic encoded microspheres labeled with antibodies-1 for multiple different antigens include AIE magnetic encoded microspheres with different coding intensities respectively labeled with antibodies-1 for multiple different antigens, and the AIE nanospheres labeled with antibodies-2 include AIE nanospheres labeled with antibodies-2 corresponding to multiple different antigens; and the test solution is detected once.

[0112] The above-mentioned immunoassay reagents based on AIE magnetically encoded microspheres and AIE dyes are used in the field of in vitro non-disease diagnostic testing, such as food testing and public safety testing.

[0113] The present invention labels different types of cytokine antibodies-1 on the surface of AIE magnetically encoded microspheres, and labels corresponding antibodies-2 on the surface of carboxyl-modified AIE nanospheres as quantitative detection markers for analysis. Through sandwich immune reaction, the aggregation-induced luminescence property of AIE materials is utilized to achieve multi-item quantitative immunoassay of seven cytokine markers (IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α, and IFN-γ), with a detection limit of 100 pg·mL -1 ~mg·mL -1 Measure within the range.

[0114] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0115] (1) Compared with the fluorescent materials of traditional magnetically encoded microspheres, the aggregation-induced luminescence magnetically encoded microspheres of the present invention improve the discrimination of magnetically encoded microspheres and solve the fluorescence reabsorption effect of traditional fluorescent materials inside the microspheres. At the same time, the AIE material has high photostability, so that there is no obvious fluorescence attenuation after repeated irradiation.

[0116] (2) Compared with traditional ACQ molecules, the water-soluble AIE molecules of the present invention are embedded in the interior of the nanospheres, isolating the AIE molecules from the influence of the external environment. The signal amplification effect of the AIE nanospheres is utilized during the detection process, and trace detection in low-concentration test samples can be achieved even in complex samples with multiple markers.

[0117] (3) The AIE material of the present invention, as a coding element, exhibits a large Stoss shift and no reabsorption effect. Furthermore, the dipole moment interaction between the two AIE molecules is greater than that of the FRET process, resulting in a fluorescence enhancement effect within the microspheres. Furthermore, the AIE material exhibits typical aggregation-induced emission properties, with the two different AIE molecules embedded within the microspheres emitting strong fluorescence within the aggregate.

[0118] (4) The present invention provides a simple method that utilizes the high discrimination of AIE-encoded microspheres and the signal amplification effect of AIE nanospheres for quantitative detection to obtain a multi-item immunoassay technology based on AIE magnetic-encoded microspheres and AIE nanospheres, providing a new detection strategy for multiple marker immunoassays. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] FIG1 is a scanning electron microscope image of the dual-wavelength AIE magnetically encoded microspheres prepared in Example 1.

[0120] Figure 2 shows the emission spectra of AIE-R1 and AIE-IR1.

[0121] FIG3 is a schematic diagram of flow encoding of the dual-wavelength AIE magnetic encoding microspheres prepared in Example 1.

[0122] FIG4 is a coding peak diagram corresponding to the combined determination of seven cytokines in Example 1. DETAILED DESCRIPTION

[0123] The present invention will be further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0124] The blank polymer microspheres in the examples and comparative examples are polystyrene polymer microspheres with diameters of 1 μm, 5 μm, 6 μm, 8 μm, and 10 μm, which are from the AIE Institute and have product numbers of NWKPB-100, NWKPB-500, NWKPB-600, NWKPB-800, and NWKPB-1000.

[0125] The carboxyl-modified blank polymer nanospheres used in the Examples and Comparative Examples were carboxyl-modified polystyrene polymer microspheres with diameters of 1000 nm, 500 nm, 50 nm, 40 nm, and 30 nm, obtained from the AIE Institute (product numbers NAGPC-100, NAGPC-050, NAGPC-005, NAGPC-004, and NAGPC-003). Antibody-1, Antibody-2, and Antigen were obtained from Maichuan (Guangzhou) Biotechnology Co., Ltd. (product number AB10 series).

[0126] Example 1

[0127] 1. Preparation of Carboxyl-Modified AIE Magnetic Encoded Microspheres

[0128] Weigh 1 g of blank polymer microspheres (particle size 5 μm) and disperse them in 100 g of water to obtain a microsphere dispersion;

[0129] 5 mg of AIE-R1 molecules (alkyl chain -C8H 17 AIE polymer microspheres were prepared by dissolving 5 mg of AIE-IR1 molecules (a linear alkyl group) in 10 mL of tetrahydrofuran to obtain an AIE swelling agent solution. The two solutions were mixed (the total mass of the AIE molecules accounted for 1 wt% of the mass of the blank polymer microspheres) and sonicated at 100 W for 1 minute. After a closed swelling period of 2 hours, the solvent was allowed to evaporate open for 4 hours, and the supernatant was removed by centrifugation. The resulting AIE polymer microspheres were then added to 50 mL of concentrated sulfuric acid (98.3 wt%), heated to 80°C, and reacted for 8 hours. The microspheres were then centrifuged at 5000 rpm and resuspended in water. This process was repeated three times to obtain surface-sulfonated AIE polymer microspheres.

[0130] Ferrous sulfate heptahydrate and ferric chloride hexahydrate were mixed with 100 g of water in a mass ratio of 2:1 (the total mass of iron salt accounted for 5 wt% of the mass of blank polymer microspheres), and the AIE polymer microspheres with sulfonated surfaces obtained above were added. After soaking and reacting for 1 hour, the microspheres were centrifuged at 5000 rpm and resuspended in water. The reaction was repeated three times to obtain iron salt-chelated AIE polymer microspheres; 50 mL of 1 mol / L sodium hydroxide solution was added, the temperature was raised to 80°C, and the reaction was carried out for 2 hours. The iron salt reacted in situ to generate magnetic particles that were deposited on the surface of the AIE polymer microspheres; the microspheres were centrifuged at 5000 rpm and resuspended in water. After repeating three times, dual-wavelength aggregation-induced emission magnetic encoded microspheres were obtained. 3 mL of ammonia water (28%), 1.5 mL of TEOS and 0.6 mL of 3-vinyltrimethoxysilane were added to the above-mentioned aggregation-induced emission magnetically encoded microspheres (0.1 g). After reacting for 6 hours and magnetic separation, 0.5 g of acrylic acid monomer was added, the temperature was raised to 80°C, and after reacting for 8 hours and magnetic separation, carboxyl-modified AIE magnetically encoded microspheres were obtained.

[0131] The dosage of AIE-R1 molecules was changed to 0.001 mg, 0.01 mg, 0.025 mg, 0.05 mg, 0.1 mg, and 0.5 mg to obtain carboxyl-modified AIE magnetic encoding microspheres with different encoding intensities (other parameters remained unchanged).

[0132] 2. Antibody-1 labeled AIE magnetically encoded microspheres

[0133] The carboxyl-modified AIE magnetically encoded microspheres prepared above (AIE-R1 molecule dosage 5 mg) were added to 10 g MES buffer (pH = 6.5), ultrasonically dispersed, and then 0.0095 g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added. The mixture was placed in a mixer to activate the carboxyl groups at 300 rpm for 5 min. The supernatant was removed by magnetic attraction to obtain the activated AIE magnetically encoded microspheres. 3 g boric acid buffer (pH = 8) and 10 μg mouse antibody-1 for cytokine IL-2 were added, and the mixture was placed in a mixer and incubated for 30 min. The supernatant was removed by magnetic attraction, and 0.5 mL of blocking solution BSA (0.5%) and Tris (0.1%) microsphere storage solution was added. Ultrasonic dispersion was performed to obtain a solution of AIE magnetically encoded microspheres labeled with IL-2 antibody-1 (0.2 g / mL).

[0134] The preparation method of the AIE magnetic encoded microsphere solution labeled with antibody-1 corresponding to the cytokines IL-4, IL-6, IL-10, IL-17, TNF-α and IFN-γ is the same as above, except that the mouse antibody-1 and carboxyl-modified AIE magnetic encoded microspheres of the cytokine IL-2 are replaced by the mouse antibody-1 and carboxyl-modified AIE magnetic encoded microspheres of the cytokine IL-4 (the amount of AIE-R1 molecules is 0.001 mg), the mouse antibody-1 and carboxyl-modified AIE magnetic encoded microspheres of the cytokine IL-6 (the amount of AIE-R1 molecules is 0.01 mg), respectively. ), mouse antibody-1 for cytokine IL-10 and carboxyl-modified AIE magnetic encoded microspheres (AIE-R1 molecule dosage is 0.025 mg), mouse antibody-1 for cytokine IL-17 and carboxyl-modified AIE magnetic encoded microspheres (AIE-R1 molecule dosage is 0.05 mg), mouse antibody-1 for cytokine TNF-α and carboxyl-modified AIE magnetic encoded microspheres (AIE-R1 molecule dosage is 0.1 mg), mouse antibody-1 for cytokine IFN-γ and carboxyl-modified AIE magnetic encoded microspheres (AIE-R1 molecule dosage is 0.5 mg).

[0135] 3. Antibody-2 labeled AIE nanofluorescent microspheres

[0136] 1 mg of the detection AIE-N1 molecule was weighed, dissolved in 1 mL of tetrahydrofuran, and added to an aqueous solution of carboxyl-modified blank polymer nanospheres (particle size 500 nm) (the mass of the carboxyl-modified blank polymer nanospheres was 0.1 g and the mass of water was 10 g). The solution was ultrasonically treated at a power of 100 W for 1 min. After the swelling reaction was blocked for 2 h, the solvent was evaporated open for 4 h, and the supernatant was removed by centrifugation to obtain carboxyl-modified AIE nanofluorescent microspheres.

[0137] The obtained carboxyl-modified AIE nanofluorescent microspheres were activated, coupled with IL-2 antibody-2, and blocked according to the method of step (2) above to obtain an IL-2 antibody-2 labeled AIE nanofluorescent microsphere solution (0.1 μg / mL) (wherein IL-2 antibody-2 is a mouse antibody-2 of cytokine IL-2).

[0138] The preparation method of AIE nanofluorescent microsphere solution labeled with antibody-2 corresponding to cytokines IL-4, IL-6, IL-10, IL-17, TNF-α and IFN-γ is the same as above, with the only difference being that the mouse antibody-2 of cytokine IL-2 is replaced by the mouse antibody-2 of cytokine IL-4, IL-6, IL-10, IL-17, TNF-α or IFN-γ.

[0139] 4. Determination of Multiple Marker Immunoassays

[0140] 10 μL of each of the seven different types of AIE magnetic encoded microsphere solutions labeled with antibody-1 and 10 μL of each of the corresponding AIE nanofluorescent microsphere solutions labeled with antibody-2 were added to the same flow tube, and then 5 μL of the test sample (a mixed solution of seven cytokines IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α and IFN-γ) was added. After magnetic separation, the coding peaks were obtained through the coding channel of the flow cytometer, and the types of cytokines in the test solution were distinguished by the coding channel and intensity; the fluorescence intensity of the AIE nanofluorescent microspheres was detected to achieve quantitative determination of each cytokine in the test sample (Figure 4).

[0141] The morphology of AIE magnetically encoded microspheres was observed using a field emission SEM electron microscope. The microspheres showed a regular spherical structure with a statistical particle size of about 5 μm (as shown in Figure 1). The particle size of the antibody-2 labeled AIE nanofluorescent microspheres was tested using a DLS nanoparticle size analyzer, and the Z-average particle size was 500 nm. The fluorescence spectrum of the antibody-2 labeled AIE nanofluorescent microspheres was tested using a fluorescence spectrometer. The excitation wavelength of AIE was 400 nm and the emission wavelength was 550 nm. The minimum detection concentration of the seven cytokine markers was tested using an analytical flow cytometer. The minimum detection limit is shown in Table 1 below. The multiple marker analysis and detection effect is good.

[0142] Example 2

[0143] The 500 nm carboxyl-modified blank polymer nanospheres in step (iii) of Example 1 were replaced with 50 nm carboxyl-modified blank polymer nanospheres. All other conditions were the same as in Example 1. The minimum detection concentrations of seven cytokine markers were tested using an analytical flow cytometer. The minimum detection limits are shown in Table 1 below, demonstrating good detection performance for the multiple marker analysis.

[0144] Example 3

[0145] The 500 nm carboxyl-modified blank polymer nanospheres from step (iii) of Example 1 were replaced with 40 nm carboxyl-modified blank polymer nanospheres. All other conditions were the same as in Example 1. The minimum detection concentrations of seven cytokine markers were tested using an analytical flow cytometer. The minimum detection limits are shown in Table 1 below, demonstrating good detection performance for the multiple marker analysis.

[0146] Example 4

[0147] The 500 nm carboxyl-modified blank polymer nanospheres from step (iii) of Example 1 were replaced with 30 nm carboxyl-modified blank polymer nanospheres. All other conditions were the same as in Example 1. The minimum detection concentrations of seven cytokine markers were tested using an analytical flow cytometer. The minimum detection limits are shown in Table 1 below, demonstrating good detection performance for the multiple marker analysis.

[0148] Example 5

[0149] The 5 μm blank polymer microspheres in step (1) of Example 1 were replaced with 6 μm blank polymer microspheres, and the 500 nm carboxyl-modified blank polymer nanospheres in step (3) of Example 1 were replaced with 30 nm carboxyl-modified blank polymer nanospheres. All other conditions were the same as in Example 1. The minimum detection concentration of the seven cytokine markers was tested using an analytical flow cytometer. The minimum detection limit is shown in Table 1 below, indicating good detection results for the multiple marker analysis.

[0150] Example 6

[0151] The 5 μm blank polymer microspheres in step (1) of Example 1 were replaced with 8 μm blank polymer microspheres, and the 500 nm carboxyl-modified blank polymer nanospheres in step (3) of Example 1 were replaced with 30 nm carboxyl-modified blank polymer nanospheres. All other conditions were the same as in Example 1. The minimum detection concentration of the seven cytokine markers was tested using an analytical flow cytometer. The minimum detection limit is shown in Table 1 below, indicating good detection results for the multiple marker analysis.

[0152] Example 7

[0153] The 5 μm blank polymer microspheres in step (1) of Example 1 were replaced with 10 μm blank polymer microspheres, and the 500 nm carboxyl-modified blank polymer nanospheres in step (3) of Example 1 were replaced with 30 nm carboxyl-modified blank polymer nanospheres. All other conditions were the same as in Example 1. The minimum detection concentration of the seven cytokine markers was tested using an analytical flow cytometer. The minimum detection limit is shown in Table 1 below, indicating good detection results for the multiple marker analysis.

[0154] Table 1 Effects of AIE-encoded particle size, detection microsphere size, and emission wavelength on seven-fold cytokine marker immunoassay

[0155] Comparative Example 1

[0156] The 5 μm blank polymer microspheres in step (1) of Example 1 were replaced with 1 μm blank polymer microspheres, and the 500 nm carboxyl-modified blank polymer nanospheres in step (3) of Example 1 were replaced with 50 nm carboxyl-modified blank polymer nanospheres. All other steps were the same as in Example 1.

[0157] The morphology of the AIE magnetically encoded microspheres was observed using a field emission SEM electron microscope. The microspheres showed a regular spherical structure with a statistical particle size of approximately 1 μm. The particle size of the antibody-2 labeled AIE nanofluorescent microspheres was tested using a DLS nanoparticle size analyzer, and the Z-average particle size was 50 nm. The fluorescence spectrum of the antibody-2 labeled AIE nanofluorescent microspheres was tested using a fluorescence spectrometer. The excitation wavelength of AIE was 400 nm and the emission wavelength was 550 nm. Analytical flow cytometry was used for testing. Due to the small size of the encoded microspheres and the large noise points, the encoding failed and the detection effect was poor.

[0158] Comparative Example 2

[0159] The 500 nm carboxyl-modified blank polymer nanospheres in step (3) of Example 1 were replaced with 1000 nm carboxyl-modified blank polymer nanospheres. All other aspects were the same as in Example 1.

[0160] The morphology of AIE magnetically encoded microspheres was observed using a field emission SEM electron microscope. The microspheres showed a regular spherical structure with a statistical particle size of approximately 5 μm. The particle size of the antibody-2 labeled AIE nanofluorescent microspheres was tested using a DLS nanoparticle size analyzer, and the Z-average particle size was 1000 nm. The fluorescence spectrum of the antibody-2 labeled AIE nanofluorescent microspheres was tested using a fluorescence spectrometer. The excitation wavelength of AIE was 400 nm and the emission wavelength was 550 nm. Analytical flow cytometry was used for testing. Due to the large particle size of the AIE nanofluorescent microspheres and the weak antibody binding ability, the detection sensitivity was poor and the detection effect was poor.

[0161] Comparative Example 3

[0162] The 500 nm carboxyl-modified blank polymer nanospheres in step (iii) of Example 1 were replaced with 50 nm carboxyl-modified blank polymer nanospheres, and the AIE-N1 molecules in step (iii) of Example 1 were replaced with AIE-R1 molecules. All other steps were the same as in Example 1.

[0163] The morphology of AIE magnetically encoded microspheres was observed using a field emission SEM electron microscope. The microspheres showed a regular spherical structure with a statistical particle size of approximately 5μm. The particle size of the antibody-2 labeled AIE nanofluorescent microspheres was tested using a DLS nanoparticle size analyzer, and the Z-average particle size was 50nm. The fluorescence spectrum of the antibody-2 labeled AIE nanofluorescent microspheres was tested using a fluorescence spectrometer. The excitation wavelength of AIE was 400nm and the emission wavelength was 620nm. Analytical flow cytometry was used for testing. Since the emission wavelength of the AIE nanofluorescent microspheres was around 620nm, which overlapped with the AIE emission spectrum of the encoded microspheres, the quantitative detection of the cytokine concentration failed and the detection effect was poor.

[0164] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An immunoassay reagent based on AIE magnetically encoded microspheres and AIE nanospheres, characterized in that: It includes AIE magnetic encoded microspheres labeled with antibody-1 and AIE nanospheres labeled with antibody-2.

2. The immunoassay reagent based on AIE magnetically encoded microspheres and AIE nanospheres according to claim 1, characterized in that: The mass amount of the AIE nanoparticles labeled with antibody-2 is 0.001-0.01 wt% of the mass amount of the AIE magnetic encoding microspheres labeled with antibody-1; The encoding capacity of the AIE magnetically encoded microspheres is ≥4, and the CV value of the flow fluorescence signal and the CV value of the flow FCS are both less than 10%; The emission wavelength range of the AIE nanospheres is 450nm-550nm; The antibody-1 and antibody-2 can specifically bind to the same antigen.

3. The immunoassay reagent based on AIE magnetically encoded microspheres and AIE dyes according to claim 2, characterized in that: The antibody-1 is selected from at least one of IL-2 monoclonal antibody, IL-4 monoclonal antibody, IL-6 monoclonal antibody, IL-10 monoclonal antibody, IL-17 monoclonal antibody, TNF-α monoclonal antibody and IFN-γ monoclonal antibody; antibody-2 is selected from the anti-antibody of antibody-1; The antibody-1 and antibody-2 are mouse antibodies.

4. The immunoassay reagent based on AIE magnetically encoded microspheres and AIE nanospheres according to claim 2, characterized in that: The particle size of the AIE magnetically encoded microspheres ranges from 1.5 μm to 20 μm; the particle size of the AIE nanospheres ranges from 10 nm to 1000 nm; The AIE nanospheres include carboxyl-modified blank polymer nanospheres and AIE-N molecules; the AIE-N molecules are embedded in the carboxyl-modified blank polymer nanospheres; The encoding capacity of the AIE magnetically encoded microspheres is ≥24; the AIE magnetically encoded microspheres are dual-wavelength aggregation-induced luminescence magnetically encoded microspheres, including AIE polymer microspheres and magnetic particles; the AIE polymer microspheres are obtained by embedding AIE molecules inside blank polymer microspheres, and the magnetic particles are deposited on the surface of the AIE polymer microspheres; the AIE molecules include AIE-R molecules and AIE-IR molecules; The difference between the maximum emission wavelength of the AIE-IR molecule and the maximum emission wavelength of the AIE-R molecule is in the range of 160nm to 290nm, and satisfies Δλ≥185. Where I / I0 is the average ratio of the fluorescence intensity of the AIE-IR molecule and the fluorescence intensity of the AIE-IR molecule before and after embedding, λ IR is the maximum emission wavelength of the AIE-IR molecule, λ R is the maximum emission wavelength of the AIE-R molecule.

5. The immunoassay reagent based on AIE magnetically encoded microspheres and AIE nanospheres according to claim 4, characterized in that: In the AIE nanospheres, The carboxyl-modified blank polymer nanospheres are selected from at least one of carboxyl-modified polystyrene microspheres, carboxyl-modified polymethyl methacrylate microspheres, and carboxyl-modified polyglycidyl acrylate microspheres; The AIE-N molecules are embedded in the interior of the blank polymer nanospheres modified with carboxyl groups by an active swelling method. The amount of the AIE-N molecules used in the active swelling method is 0.01-10 wt % of the amount of the blank polymer nanospheres modified with carboxyl groups. The AIE-N molecules in the AIE nanospheres are selected from at least one of the following AIE-N1 to AIE-N10: In the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres, The blank polymer microspheres are selected from at least one of polystyrene microspheres, polymethyl methacrylate microspheres and polyglycidyl acrylate microspheres; The magnetic particles are Fe3O4; The AIE-R molecule is selected from at least one of the following AIE-R1 to AIE-R16 molecules: The AIE-IR molecule is selected from at least one of the following AIE-IR1 to AIE-IR10 molecules: The AIE molecules are embedded in the blank polymer microspheres by an active swelling method, wherein the amount of AIE molecules used in the active swelling method is 0.01-10 wt % of the amount of the blank polymer microspheres, wherein the mass amount of the AIE-R molecules accounts for 0.01% to 1000% of the mass amount of the AIE-IR molecules; The magnetic particles are deposited on the surface of the AIE polymer microspheres by an in-situ precipitation method of iron salts, wherein the amount of iron salts used in the in-situ precipitation method is 0.1-25 wt % of the amount of blank polymer microspheres used.

6. The immunoassay reagent based on AIE magnetically encoded microspheres and AIE nanospheres according to claim 5, characterized in that: In the AIE nanospheres, The active swelling method is to allow AIE-N molecules to swell with carboxyl-modified blank polymer microspheres in a swelling agent so that the AIE-N molecules are embedded in the interior of the carboxyl-modified blank polymer microspheres; In the active swelling method, the swelling agent is selected from at least one of dichloromethane, tetrahydrofuran, chloroform, anisole, benzyl alcohol and toluene, and the particle size of the carboxyl-modified blank polymer microspheres ranges from 10 nm to 500 nm; In the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres, The active swelling method is to allow the AIE molecules to swell with the blank polymer microspheres in a swelling agent so that the AIE molecules are embedded in the blank polymer microspheres. In the active swelling method, the swelling agent is selected from at least one of dichloromethane, tetrahydrofuran, chloroform, anisole, benzyl alcohol and toluene, and the particle size of the blank polymer microspheres ranges from 1.5 μm to 20 μm; The in-situ precipitation method is to add an alkaline solution and react with the iron salt chelated on the surface of the AIE polymer microspheres in situ to generate magnetic particles that are deposited on the surface of the AIE polymer microspheres; In the in-situ precipitation method, the surface of the AIE polymer microspheres is modified by sulfonation, the alkaline solution is selected from at least one of sodium hydroxide solution, ammonia water, potassium hydroxide solution and sodium bicarbonate solution; the iron salt is selected from at least one of trivalent iron salt and divalent iron salt; the trivalent iron salt is selected from at least one of ferric chloride hexahydrate and anhydrous ferric chloride; The divalent iron salt is at least one selected from ferrous chloride tetrahydrate, ferrous chloride dihydrate, ferrous sulfate and ferrous sulfate heptahydrate.

7. The method for preparing an immunoassay reagent based on AIE magnetically encoded microspheres and AIE nanospheres according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) Carboxyl modification of AIE magnetically encoded microspheres, activation of the carboxyl groups, and coupling with antibody-1 to obtain antibody-1 labeled AIE magnetically encoded microspheres; (2) activating the carboxyl groups of the AIE nanospheres and coupling them with antibody-2 to obtain antibody-2 labeled AIE nanospheres; Immunodetection reagents based on AIE magnetically encoded microspheres and AIE nanospheres are obtained.

8. The preparation method according to claim 7, characterized in that In step (1), the carboxyl modification comprises: mixing and reacting the AIE magnetically encoded microspheres, a base, TEOS and a silane coupling agent, magnetically separating, and adding a monomer containing a double bond and a carboxyl group to react to obtain the carboxyl-modified AIE magnetically encoded microspheres; In step (1), the activation of the carboxyl group comprises: dispersing the carboxyl-modified AIE magnetic encoded microspheres in an acidic buffer, adding a carbodiimide condensation reagent, and stirring the reaction to activate the carboxyl group, thereby obtaining the carboxyl-activated AIE magnetic encoded microspheres; In step (1), the antibody-1 coupling comprises: dispersing the AIE magnetically encoded microspheres with activated carboxyl groups in an alkaline buffer, adding the antibody-1, incubating with stirring, separating by magnetic attraction, adding a blocking solution and a preservation solution, and obtaining the AIE magnetically encoded microspheres labeled with the antibody-1; In step (2), the activation of the carboxyl group comprises: dispersing the carboxyl-modified AIE nanospheres in an acidic buffer, adding a carbodiimide condensation reagent, and stirring the reaction to activate the carboxyl group, thereby obtaining the carboxyl-activated AIE nanospheres; In step (2), the antibody-2 coupling includes: dispersing the activated carboxyl AIE nanospheres in an alkaline buffer, adding antibody-2, incubating with stirring, separating by magnetic attraction, adding blocking solution and preservation solution, and obtaining antibody-2 labeled AIE nanospheres.

9. Use of the immunoassay reagent based on AIE magnetically encoded microspheres and AIE nanospheres according to any one of claims 1 to 6 in antigen immunoassay, characterized in that: The following steps are involved: Antibody-1 labeled AIE magnetic encoding microspheres and antibody-2 labeled AIE nanospheres were added to the test solution containing the corresponding antigen. After the reaction, they were separated by magnetic attraction and the encoding peak and fluorescence quantitative detection concentration were obtained by flow cytometry.

10. The use according to claim 9, characterized in that The mass amount of the corresponding antigen is 0.01-1wt% of the mass amount of the AIE nanoparticles labeled with antibody-2; The antibody-1 labeled AIE magnetic encoding microspheres include AIE magnetic encoding microspheres with different encoding intensities labeled with antibodies-1 of multiple different antigens, and the antibody-2 labeled AIE nanospheres include AIE nanospheres labeled with antibodies-2 corresponding to multiple different antigens.

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