Dual-wavelength aggregation-induced emission magnetically encoded micro-sphere, and preparation method therefor and application thereof

Abstract

Disclosed in the present invention are a dual-wavelength aggregation-induced emission magnetically encoded micro-sphere, and a preparation method therefore and an application thereof. The dual-wavelength aggregation-induced emission magnetically encoded micro-sphere comprises an AIE polymer micro-sphere and magnetic particles, wherein the AIE polymer micro-sphere is obtained by means of embedding AIE molecules into a blank polymer micro-sphere, and the magnetic particles are deposited on the surface of the AIE polymer micro-sphere; the AIE molecules comprise AIE-R molecules and AIE-IR molecules; and the difference between the maximum emission wavelength of the AIE-IR molecules and the maximum emission wavelength of the AIE-R molecules ranges from 160 nm to 290 nm, and satisfies Δλ≥185, [Equation 1]. The dual-wavelength AIE magnetically encoded micro-sphere of the present invention has an encoding capacity which is greater than or equal to 24 types, and both the CV values of a flow fluorescence signal and a flow FCS thereof are less than 10%. An important fluorescent material is added to encoding elements.

Description

Dual-wavelength aggregation-induced luminescence magnetically encoded microspheres and their preparation method and application Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and in particular relates to a dual-wavelength aggregation-induced luminescence magnetically encoded microsphere 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 biochip Luminex systems and BD's cytometric bead arrays all use organic dyes for encoding, but traditional organic materials exhibit fluorescence quenching in an aggregated state, limiting their encoding capacity and stability. In 2001, the team of Academician Tang Benzhong discovered a new type of aggregation-induced emission (AIE) material, which is a promising encoding material for the preparation of coded microspheres, because AIE materials can achieve in situ enhancement of fluorescence signals during the aggregation process. Aggregation-induced emission materials have properties such as large Stokes shift, strong resistance to photobleaching, and anti-magnetism, making them excellent encoding microsphere materials. However, there are few public reports on the efficient preparation of magnetic fluorescent codes using AIE materials. Therefore, it is very necessary to develop a technology that can efficiently and directly generate aggregation-induced emission magnetic coded microspheres.

[0005] Summary of the Invention

[0006] In order to address the shortcomings of the existing technology, the present invention aims to provide a dual-wavelength aggregation-induced luminescence magnetically encoded microsphere and its preparation method and application. The prepared dual-wavelength aggregation-induced luminescence magnetically encoded microsphere has the characteristics of high encoding capacity, no cross-color effect and high fluorescence intensity.

[0007] The technical solution adopted in the present invention is:

[0008] A dual-wavelength aggregation-induced luminescence magnetically encoded microsphere comprises an AIE polymer microsphere and magnetic particles; the AIE polymer microsphere is obtained by embedding AIE molecules inside a blank polymer microsphere, and the magnetic particles are deposited on the surface of the AIE polymer microsphere; the AIE molecules include AIE-R molecules and AIE-IR molecules;

[0009] 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.

[0010] Preferably, the blank polymer microspheres are selected from at least one of polystyrene microspheres, polymethyl methacrylate microspheres and polyglycidyl acrylate microspheres;

[0011] Further preferably, the blank polymer microspheres are selected from polystyrene microspheres or polyglycidyl acrylate microspheres.

[0012] Preferably, the magnetic particles are Fe3O4;

[0013] Preferably, the AIE-R molecule is selected from at least one of the following AIE-R1 to AIE-R16 molecules:

[0014] Alkyl chain in AIE-R 1 molecule -C8H 17 Each is independently a branched or straight chain alkyl group.

[0015] Alkyl chain in AIE-R 11 molecule -C6H 13 Each is independently a branched or straight chain alkyl group.

[0016] The AIE-IR molecule is selected from at least one of the following AIE-IR1 to AIE-IR10 molecules:

[0017] Preferably, 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;

[0018] Further preferably, the active swelling method is to allow the AIE molecules to swell with the blank polymer microspheres via a swelling agent so that the AIE molecules are embedded inside the blank polymer microspheres;

[0019] More preferably, 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 0.03 μm to 50 μm;

[0020] More preferably, the particle size of the blank polymer microspheres ranges from 0.5 μm to 10 μm.

[0021] Preferably, 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.

[0022] Further preferably, 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.

[0023] More preferably, 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 a trivalent iron salt and a 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.

[0024] More preferably, considering the uniformity of magnetic particles deposited on the surface of the AIE polymer microspheres, the iron salt is a compound of trivalent iron salt and 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.

[0025] Preferably, the encoding capacity of the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres is ≥24, and the CV value of the flow fluorescence signal and the CV value of the flow FCS are both less than 10%.

[0026] The preparation method of the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres comprises the following steps:

[0027] S1. Preparation of AIE polymer microspheres by active swelling method

[0028] (1) dispersing blank polymer microspheres in water to obtain a microsphere dispersion;

[0029] (2) dissolving the AIE 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, 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;

[0031] S2. Preparation of Dual-Wavelength Aggregation-Induced Luminescence Magnetic Encoded Microspheres by In Situ Precipitation

[0032] 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.

[0033] Preferably, the preparation method of the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres comprises the following steps:

[0034] S1. Preparation of AIE polymer microspheres by active swelling method

[0035] (1) dispersing blank polymer microspheres in water to obtain a microsphere dispersion;

[0036] (2) dissolving the AIE molecules in a swelling agent to obtain an AIE swelling agent solution;

[0037] (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;

[0038] S2. Preparation of Dual-Wavelength Aggregation-Induced Luminescence Magnetic Encoded Microspheres by In Situ Precipitation

[0039] 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 dual-wavelength aggregation-induced luminescence magnetically encoded microspheres.

[0040] Further 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. More preferably, the time for volatilizing and removing the swelling agent is 4 hours.

[0041] Further preferably, in step S2, the soaking reaction time is 2 hours to 4 hours.

[0042] 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;

[0043] Further preferably, the mass amount of blank polymer microspheres in the microsphere dispersion is 0.5% to 10% of the mass amount of water.

[0044] 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;

[0045] 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;

[0046] Further preferably, the mass concentration of the sulfuric acid solution is 98.3%; and the mass amount of the AIE polymer microspheres is 5% to 20% of the mass amount of the sulfuric acid solution.

[0047] 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%;

[0048] 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.

[0049] Application of the above-mentioned dual-wavelength aggregation-induced luminescence magnetically encoded microspheres in in vitro non-disease diagnostic testing.

[0050] This paper proposes a new technology that combines active swelling and in situ generation to efficiently prepare dual-wavelength aggregation-induced emission (AIE) magnetically encoded microspheres. Two AIE molecules of different wavelengths are actively swollen into the microsphere matrix through hydrophobic interaction and concentration gradient, and magnetic particles are generated on the microsphere surface using an in situ generation method using iron salts. By simultaneously selecting two AIE molecules of different wavelengths, dual-wavelength aggregation-induced emission magnetically encoded microspheres with uniform particle size and high encoding capacity are obtained. These microspheres have excellent detection stability and can achieve 24 or more encodings.

[0051] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0052] (1) Compared with the fluorescent materials of traditional magnetic encoding microspheres, the dual-wavelength aggregation-induced luminescence magnetic encoding microspheres of the present invention improve the encoding capacity of magnetic encoding microspheres and solve the fluorescence reabsorption effect of traditional fluorescent materials inside the microspheres. Even in high magnetic content microspheres, the dual-wavelength AIE encoding capacity can reach 24-fold capacity.

[0053] (2) The present invention uses a simple swelling volatilization and in situ generation technology to encapsulate high-concentration dual-wavelength AIE molecules inside the microspheres, and magnetic particles are in situ deposited on the surface of the microspheres. By regulating the content of AIE molecules of different wavelengths and the mass content of iron salts, AIE magnetic fluorescent encoded microspheres with optimal performance are obtained.

[0054] (3) Generally speaking, traditional fluorescent materials experience fluorescence attenuation during the FRET process. However, the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres of the present invention exhibit fluorescence enhancement within the microspheres due to the dipole moment interaction between the two AIE molecules being greater than the FRET process. Furthermore, AIE materials exhibit typical aggregation-induced luminescence properties, with the two different AIE molecules embedded within the microspheres emitting strong fluorescence within the aggregate.

[0055] (4) The preparation method of the dual-wavelength aggregation-induced luminescence magnetic encoded microspheres of the present invention is simple. The synergistic effect of the aggregation-induced luminescence of AIE molecules and the intermolecular dipole moment effect is utilized to obtain dual-wavelength AIE magnetic encoded microspheres with high encoding capacity, realizing the effective application of AIE molecules in the preparation of encoded microspheres, adding an important fluorescent material to the encoding element, and providing a new encoding strategy for the core raw materials of protein biochips. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIG1 is a scanning electron microscope image of the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres prepared in Example 1.

[0057] FIG2 is an emission spectrum of AIE-R1 and AIE-IR1 in the dual-wavelength aggregation-induced emission magnetically encoded microspheres prepared in Example 1.

[0058] FIG3 is a schematic diagram of flow encoding of the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres prepared in Example 1. DETAILED DESCRIPTION

[0059] 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.

[0060] The blank polymer microspheres in the examples and comparative examples are polystyrene polymer microspheres with a diameter of 5 μm, which are from AIE Research Institute and have a product number of NWKPB-500.

[0061] Example 1

[0062] Weigh 1 g of blank polymer microspheres and disperse them in 100 g of water to obtain a microsphere dispersion;

[0063] 5 mg of AIE-R1 molecules (alkyl chain-C8H 17 A 5-mg AIE-IR1 molecule (a linear alkyl group) and 5 mg of AIE-IR1 molecules were dissolved 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 evaporated open for 4 hours, and the supernatant was removed by centrifugation to obtain AIE polymer microspheres. The resulting AIE polymer microspheres were added to 50 mL of concentrated sulfuric acid (98.3 wt%), heated to 80°C, and reacted for 8 hours. The microspheres were centrifuged at 5000 rpm and resuspended in water. This process was repeated three times to obtain surface-sulfonated AIE polymer microspheres.

[0064] 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.

[0065] The morphology of the dual-wavelength aggregation-induced luminescence 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 (Figure 1). The fluorescence spectrum of the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres was tested using a fluorescence spectrometer. The emission wavelength of AIE-R1 was 620 nm, and the ratio of the fluorescence intensity before and after AIE-R1 coating was 1300. The emission wavelength of AIE-IR1 was 780 nm, and the ratio of the fluorescence intensity before and after AIE-IR1 coating was 1200. The difference in central wavelength was 160 nm (as shown in Figure 2). The encoding data of the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres was tested using an analytical flow cytometer. The CV value of the flow fluorescence signal was 4.6%, the CV value of the flow FCS was 5.7%, the encoding peak was 24-fold (as shown in Figure 3), and the encoding was successful.

[0066] Comparative Example 1

[0067] Weigh 1 g of blank polymer microspheres and disperse them in 100 g of water to obtain a microsphere dispersion;

[0068] 5mg of AIE-R4 and 5mg of AIE-IR1 molecules were dissolved in 10mL of tetrahydrofuran to obtain an AIE swelling agent solution. The two solutions were mixed (the total mass of AIE molecules relative to the mass of blank polymer microspheres was 1wt%) and sonicated at 100W for 1 minute. After a closed swelling period of 2h, the solvent was allowed to evaporate open for 4h, and the supernatant was removed by centrifugation to obtain AIE polymer microspheres. The resulting AIE polymer microspheres were added to 50mL of concentrated sulfuric acid (98.3wt%), heated to 80°C, and reacted for 8h. The microspheres were centrifuged at 5000rpm and resuspended in water. This process was repeated three times to obtain surface-sulfonated AIE polymer microspheres.

[0069] 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.

[0070] The morphology of the dual-wavelength aggregation-induced luminescence 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 fluorescence spectrum of the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres was tested using a fluorescence spectrometer. The emission wavelength of AIE-R4 was 650nm, and the ratio of the fluorescence intensity before and after AIE-R4 coating was 1200. The emission wavelength of AIE-IR1 was 780nm, and the ratio of the fluorescence intensity before and after AIE-IR1 coating was 1300. The difference in central wavelength was 130nm. The encoding data of the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres was tested using an analytical flow cytometer. The CV value of the flow fluorescence signal was 14.6%, and the CV value of the flow FCS was 25.7%, indicating encoding failure.

[0071] Comparative Example 2

[0072] Weigh 1 g of blank polymer microspheres and disperse them in 100 g of water to obtain a microsphere dispersion;

[0073] 5 mg of AIE-R1 molecules (alkyl chain -C8H 17 A 5-mg AIE-IR2 molecule (a linear alkyl group) and 5 mg of AIE-IR2 molecules were dissolved 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 evaporated open for 4 hours, and the supernatant was removed by centrifugation to obtain AIE polymer microspheres. The resulting AIE polymer microspheres were added to 50 mL of concentrated sulfuric acid (98.3 wt%), heated to 80°C, and reacted for 8 hours. The microspheres were centrifuged at 5000 rpm and resuspended in water. This process was repeated three times to obtain surface-sulfonated AIE polymer microspheres.

[0074] 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.

[0075] The morphology of the dual-wavelength aggregation-induced luminescence 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 fluorescence spectrum of the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres was tested using a fluorescence spectrometer. The emission wavelength of AIE-R1 was 620nm, and the ratio of the fluorescence intensity before and after AIE-R1 coating was 1100. The emission wavelength of AIE-IR2 was 1000nm, and the ratio of the fluorescence intensity before and after AIE-IR2 coating was 1500. The difference in center wavelength was 380nm. The encoding data of the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres was tested using an analytical flow cytometer. The CV value of the flow fluorescence signal was 24.6%, and the CV value of the flow FCS was 19.7%, indicating encoding failure.

[0076] Comparative Example 3

[0077] Weigh 1 g of blank polymer microspheres and disperse them in 100 g of water to obtain a microsphere dispersion;

[0078] 5 mg of fluorescein-1 (FITC) and 5 mg of fluorescein-2 (CY5) were dissolved in 10 mL of tetrahydrofuran to obtain an AIE swelling agent solution. The two solutions were mixed (the total mass of fluorescein molecules relative to the mass of the blank polymer microspheres was 1 wt%) and sonicated at 100 W for 1 minute. After 2 hours of blocked swelling, the supernatant was removed by centrifugation and the solvent was evaporated for 4 hours to produce fluorescein polymer microspheres. The resulting fluorescein polymer microspheres were added to 50 mL of concentrated sulfuric acid (98.3 wt%), heated to 80°C, and reacted for 8 hours. The microspheres were centrifuged at 5000 rpm and resuspended in water. This process was repeated three times to obtain fluorescein polymer microspheres with surface sulfonation modification.

[0079] 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 above-obtained sulfonated surface-modified fluorescein polymer microspheres were added. After soaking and reacting for 1 hour, the microspheres were centrifuged at 5000 rpm and resuspended in water. The process was repeated three times to obtain iron salt-chelated fluorescein 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 fluorescein polymer microspheres. The microspheres were centrifuged at 5000 rpm and resuspended in water. The process was repeated three times to obtain dual-wavelength fluorescein magnetically encoded microspheres.

[0080] The morphology of the dual-wavelength fluorescein 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 fluorescence spectrum of the dual-wavelength fluorescein magnetically encoded microspheres was tested using a fluorescence spectrometer. The emission wavelength of fluorescein-1 was 520nm, and the ratio of the fluorescence intensity before and after fluorescein-1 coating was 1.12. The emission wavelength of fluorescein-2 was 664nm, and the ratio of the fluorescence intensity before and after fluorescein-2 coating was 0.95. The difference in center wavelength was 144nm. The encoding data of the dual-wavelength fluorescein magnetically encoded microspheres was tested using an analytical flow cytometer. The CV value of the flow fluorescence signal was 24.6%, and the CV value of the flow FCS was 19.7%, indicating encoding failure.

[0081] Comparative Example 4

[0082] Weigh 1 g of blank polymer microspheres and disperse them in 100 g of water to obtain a microsphere dispersion;

[0083] 5 mg of quantum dot-1 (CdSe / ZnS) and 5 mg of quantum dot-2 (CdSe / CdS) were dissolved in 10 mL of tetrahydrofuran to obtain a quantum dot swelling agent solution. The two solutions were mixed (the total mass of the quantum dots 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 to obtain quantum dot polymer microspheres. The resulting quantum dot polymer microspheres were added to 50 mL of concentrated sulfuric acid (98.3 wt%), heated to 80°C, and reacted for 8 hours. The microspheres were centrifuged at 5000 rpm and resuspended in water. This process was repeated three times to obtain quantum dot polymer microspheres with sulfonated surfaces.

[0084] Ferrous sulfate heptahydrate and ferric chloride hexahydrate were mixed with 100g of water in a mass ratio of 2:1 (the total mass of iron salt accounted for 5wt% of the mass of blank polymer microspheres), and the above-obtained quantum dot polymer microspheres with sulfonated surfaces were added. After soaking and reacting for 1h, the microspheres were centrifuged at 5000rpm and resuspended in water. The process was repeated three times to obtain iron salt-chelated quantum dot polymer microspheres. 50mL of 1mol / L sodium hydroxide solution was added, the temperature was raised to 80℃, and the reaction was carried out for 2h. The iron salt reacted in situ to generate magnetic particles that were deposited on the surface of the quantum dot polymer microspheres. The microspheres were centrifuged at 5000rpm and resuspended in water. The process was repeated three times to obtain dual-wavelength quantum dot magnetically encoded microspheres.

[0085] The morphology of the dual-wavelength quantum dot 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 fluorescence spectrum of the dual-wavelength quantum dot magnetically encoded microspheres was tested using a fluorescence spectrometer. The emission wavelength of quantum dot-1 was 610nm, and the ratio of the fluorescence intensity of quantum dot-1 before and after coating was 1.13. The emission wavelength of quantum dot-2 was 780nm, and the ratio of the fluorescence intensity of quantum dot-2 before and after coating was 1.11. The difference in center wavelength was 170nm. The encoding data of the dual-wavelength quantum dot magnetically encoded microspheres was tested using an analytical flow cytometer. The CV value of the flow fluorescence signal was 20.6%, and the CV value of the flow FCS was 17.7%, indicating encoding failure.

[0086] The results of the microspheres encoded with different fluorescent materials in Example 1 and Comparative Examples 1-4 are shown in Table 1.

[0087] Table 1 Effects of different wavelengths of AIE fluorescent microspheres, fluorescein microspheres, and quantum dot microspheres on flow cytometry encoding results

[0088] Example 2

[0089] Weigh 0.5 g of blank polymer microspheres and disperse them in 50 g of water to obtain a microsphere dispersion;

[0090] 5 mg of AIE-R1 molecules (alkyl chain -C8H 17 AIE polymer microspheres were prepared by dissolving 5 mg of AIE-IR3 molecules (a linear alkyl group) in 5 mL of dichloromethane to obtain an AIE swelling agent solution. The two solutions were mixed and sonicated at 100 W for 1 minute. After a closed swelling period of 2 hours, the solvent was evaporated open for 4 hours, and the supernatant was removed by centrifugation. The resulting AIE polymer microspheres were added to 30 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.

[0091] A total mass of 0.05 g of ferrous sulfate heptahydrate and ferric chloride hexahydrate was mixed with 50 g of water in a mass ratio of 2:1, 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. This process was repeated three times to obtain iron salt-chelated AIE polymer microspheres. 30 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. This process was repeated three times to obtain dual-wavelength aggregation-induced emission magnetically encoded microspheres.

[0092] Fluorescence spectra of the dual-wavelength aggregation-induced emission magnetically encoded microspheres were measured using a fluorescence spectrometer. The emission wavelength of AIE-R1 was 620 nm, with a ratio of fluorescence intensity before and after AIE-R1 coating of 1300. The emission wavelength of AIE-IR3 was 790 nm, with a ratio of fluorescence intensity before and after AIE-IR3 coating of 1400. The difference in center wavelengths was 170 nm. Analytical flow cytometry was used to analyze the encoding data of the dual-wavelength aggregation-induced emission magnetically encoded microspheres. The flow fluorescence signal CV value was 3.6%, and the flow FCS CV value was 2.7%. The encoding peak was 24-plex, indicating successful encoding.

[0093] Example 3

[0094] Weigh 1 g of blank polymer microspheres and disperse them in 75 g of water to obtain a microsphere dispersion;

[0095] 25 mg of AIE-R3 and 5 mg of AIE-IR4 molecules were dissolved in 7.5 mL of dichloromethane to obtain an AIE swelling agent solution. The two solutions were mixed and sonicated at 100 W for 5 minutes. After a closed swelling period of 2 hours, the solvent was evaporated open for 4 hours, and the supernatant was removed by centrifugation to produce AIE polymer microspheres. The resulting AIE polymer microspheres were added to 75 mL of concentrated sulfuric acid (98.3 wt%), heated to 80°C, and reacted for 8 hours. The microspheres were centrifuged at 5000 rpm and resuspended in water. This process was repeated three times to obtain surface-sulfonated AIE polymer microspheres.

[0096] A total mass of 0.5 g of ferrous sulfate heptahydrate and ferric chloride hexahydrate was mixed with 100 g of water in a mass ratio of 2:1, 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 process was repeated three times to obtain iron salt-chelated AIE polymer microspheres. 75 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. The process was repeated three times to obtain dual-wavelength aggregation-induced emission magnetically encoded microspheres.

[0097] Fluorescence spectra of the dual-wavelength aggregation-induced emission magnetically encoded microspheres were measured using a fluorescence spectrometer. The emission wavelength of AIE-R3 was 630 nm, with a ratio of fluorescence intensity before and after AIE-R3 encapsulation of 1250. The emission wavelength of AIE-IR4 was 900 nm, with a ratio of fluorescence intensity before and after AIE-IR4 encapsulation of 1250. The difference in center wavelength was 270 nm. Analytical flow cytometry was used to analyze the encoding data of the dual-wavelength aggregation-induced emission magnetically encoded microspheres. The flow fluorescence signal CV value was 2.6%, and the flow FCS CV value was 4.3%. The encoding peak was 24-plex, indicating successful encoding.

[0098] Example 4

[0099] Weigh 2.5 g of blank polymer microspheres and disperse them in 150 g of water to obtain a microsphere dispersion;

[0100] 50 mg of AIE-R6 and 50 mg of AIE-IR1 molecules were dissolved in 15 mL of tetrahydrofuran to obtain an AIE swelling agent solution. The two solutions were mixed and sonicated at 100 W for 5 minutes. After a closed swelling period of 2 hours, the solvent was allowed to evaporate for 6 hours, and the supernatant was removed by centrifugation to produce AIE polymer microspheres. The resulting AIE polymer microspheres were added to 50 mL of concentrated sulfuric acid (98.3 wt%), heated to 80°C, and reacted for 8 hours. The microspheres were centrifuged at 5000 rpm and resuspended in water. This process was repeated three times to obtain surface-sulfonated AIE polymer microspheres.

[0101] A total mass of 0.5 g of ferrous chloride heptahydrate and ferric chloride hexahydrate was mixed with 75 g of water in a mass ratio of 2:1, 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. This process 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. This process was repeated three times to obtain dual-wavelength aggregation-induced emission magnetically encoded microspheres.

[0102] Fluorescence spectra of the dual-wavelength aggregation-induced emission magnetically encoded microspheres were measured using a fluorescence spectrometer. The emission wavelength of AIE-R6 was 620 nm, and the ratio of fluorescence intensity before and after AIE-R6 encapsulation was 1350. The emission wavelength of AIE-IR1 was 780 nm, and the ratio of fluorescence intensity before and after AIE-IR1 encapsulation was 1150. The difference in center wavelength was 160 nm. Analytical flow cytometry was used to analyze the encoding data of the dual-wavelength aggregation-induced emission magnetically encoded microspheres. The flow fluorescence signal CV value was 5.6%, and the flow FCS CV value was 6.3%. The encoding peak was 24-plex, indicating successful encoding.

[0103] Example 5

[0104] Weigh 10 g of blank polymer microspheres and disperse them in 500 g of water to obtain a microsphere dispersion;

[0105] 150 mg of AIE-R8 and 5 mg of AIE-IR7 molecules were dissolved in 25 mL of dichloromethane to obtain an AIE swelling agent solution. The two solutions were mixed and sonicated at 100 W for 10 minutes. After a closed swelling period of 2 hours, the solvent was evaporated without stirring for 10 hours, and the supernatant was removed by centrifugation to produce AIE polymer microspheres. The resulting AIE polymer microspheres were added to 100 mL of concentrated sulfuric acid (98.3 wt%), heated to 80°C, and reacted for 8 hours. The microspheres were centrifuged at 5000 rpm and resuspended in water. This process was repeated three times to obtain surface-sulfonated AIE polymer microspheres.

[0106] A total mass of 1g of ferrous chloride heptahydrate and ferric chloride hexahydrate was mixed with 50g of water in a mass ratio of 2:1, and the AIE polymer microspheres with sulfonated surfaces obtained above were added. After soaking and reacting for 1h, the microspheres were centrifuged at 5000rpm and resuspended in water. The process was repeated three times to obtain iron salt-chelated AIE polymer microspheres. 100mL of 1mol / L sodium hydroxide solution was added, the temperature was raised to 80℃, and the reaction was carried out for 2h. 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 5000rpm and resuspended in water. The process was repeated three times to obtain dual-wavelength aggregation-induced emission magnetically encoded microspheres.

[0107] Fluorescence spectra of the dual-wavelength aggregation-induced emission magnetically encoded microspheres were measured using a fluorescence spectrometer. The emission wavelength of AIE-R8 was 660 nm, with a ratio of fluorescence intensity before and after AIE-R8 coating of 1550. The emission wavelength of AIE-IR7 was 890 nm, with a ratio of fluorescence intensity before and after AIE-IR7 coating of 1150. The difference in center wavelength was 230 nm. Analytical flow cytometry was used to analyze the encoding data of the dual-wavelength aggregation-induced emission magnetically encoded microspheres, and the flow fluorescence signal CV value was 7.6%, and the flow FCS CV value was 3.2%. The encoding peak was 24-plex, indicating successful encoding.

[0108] Example 6

[0109] Weigh 0.1 g of blank polymer microspheres and disperse them in 12.5 g of water to obtain a microsphere dispersion;

[0110] 1 mg of AIE-R11 molecules (alkyl chain -C6H 13 AIE polymer microspheres were prepared by dissolving 1 mg of AIE-IR6 molecules (a linear alkyl group) in 2 mL of chloroform. The two solutions were mixed and sonicated at 100 W for 1 minute. After 2 hours of closed swelling, the solvent was evaporated by centrifugation for 4 hours. The supernatant was removed by centrifugation to obtain AIE polymer microspheres. The resulting AIE polymer microspheres were added to 15 mL of concentrated sulfuric acid (98.3 wt%), heated to 80°C, and reacted for 8 hours. The microspheres were centrifuged at 5000 rpm and resuspended in water. This process was repeated three times to obtain surface-sulfonated AIE polymer microspheres.

[0111] A total mass of 0.02 g of ferrous sulfate heptahydrate and ferric chloride hexahydrate was mixed with 20 g of water in a mass ratio of 2:1, 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. This process was repeated three times to obtain iron salt-chelated AIE polymer microspheres. 15 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. This process was repeated three times to obtain dual-wavelength aggregation-induced emission magnetically encoded microspheres.

[0112] Fluorescence spectra of the dual-wavelength aggregation-induced emission magnetically encoded microspheres were measured using a fluorescence spectrometer. The emission wavelength of AIE-R11 was 635 nm, with a ratio of fluorescence intensity before and after AIE-R11 coating of 1350. The emission wavelength of AIE-IR6 was 795 nm, with a ratio of fluorescence intensity before and after AIE-IR6 coating of 1450. The difference in center wavelengths was 160 nm. Analytical flow cytometry was used to analyze the encoding data of the dual-wavelength aggregation-induced emission magnetically encoded microspheres, and the flow fluorescence signal CV value was 4.6%, and the flow FCS CV value was 5.2%. The encoding peak was 24-plex, indicating successful encoding.

[0113] Example 7

[0114] Weigh 1 g of blank polymer microspheres and disperse them in 125 g of water to obtain a microsphere dispersion;

[0115] 50 mg of AIE-R15 and 50 mg of AIE-IR8 molecules were dissolved in 5 mL of dichloromethane to obtain an AIE swelling agent solution. The two solutions were mixed and sonicated at 100 W for 1 minute. After a closed swelling period of 2 hours, the solvent was allowed to evaporate for 4 hours, and the supernatant was removed by centrifugation to produce AIE polymer microspheres. The resulting AIE polymer microspheres were added to 50 mL of concentrated sulfuric acid (98.3 wt%), heated to 80°C, and reacted for 8 hours. The microspheres were centrifuged at 5000 rpm and resuspended in water. This process was repeated three times to obtain surface-sulfonated AIE polymer microspheres.

[0116] A total mass of 0.2 g of ferrous sulfate heptahydrate and ferric chloride hexahydrate was mixed with 100 g of water in a mass ratio of 2:1, 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 process 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. The process was repeated three times to obtain dual-wavelength aggregation-induced emission magnetically encoded microspheres.

[0117] Fluorescence spectra of the dual-wavelength aggregation-induced emission magnetically encoded microspheres were measured using a fluorescence spectrometer. The emission wavelength of AIE-R15 was 625 nm, with a ratio of fluorescence intensity before and after AIE-R15 coating of 1250. The emission wavelength of AIE-IR8 was 785 nm, with a ratio of fluorescence intensity before and after AIE-IR8 coating of 1550. The difference in center wavelength was 160 nm. Analytical flow cytometry was used to analyze the encoding data of the dual-wavelength aggregation-induced emission magnetically encoded microspheres, and the flow fluorescence signal CV value was 5.3%, and the flow FCS CV value was 3.2%. The encoding peak was 24-plex, indicating successful encoding.

[0118] 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. A dual-wavelength aggregation-induced luminescence magnetically encoded microsphere, characterized in that: The method comprises 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.

2. The dual-wavelength aggregation-induced luminescence magnetically encoded microspheres according to claim 1, characterized in that: 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:

3. The dual-wavelength aggregation-induced luminescence magnetically encoded microspheres according to claim 1, characterized in that: 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.

4. The dual-wavelength aggregation-induced luminescence magnetically encoded microspheres according to claim 3, characterized in that: The active swelling method is to allow the AIE molecules to swell with the blank polymer microspheres through a swelling agent so that the AIE molecules are embedded inside the blank polymer microspheres. The in-situ precipitation method is to add an alkaline solution to 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.

5. The dual-wavelength aggregation-induced luminescence magnetically encoded microspheres according to claim 4, characterized in that: 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 0.03 μm to 50 μm; 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.

6. The dual-wavelength aggregation-induced luminescence magnetically encoded microspheres according to claim 1, characterized in that: The encoding capacity of the dual-wavelength aggregation-induced luminescence magnetic encoding microspheres is ≥24, and the CV value of the flow fluorescence signal and the CV value of the flow FCS are both less than 10%.

7. The method for preparing dual-wavelength aggregation-induced luminescence magnetically encoded microspheres according to any one of claims 1 to 6, characterized in that: The steps include: S1. Preparation of AIE polymer microspheres by active swelling method (1) dispersing blank polymer microspheres in water to obtain a microsphere dispersion; (2) dissolving the AIE molecules in a swelling agent to obtain an AIE swelling agent solution; (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; S2. Preparation of Dual-Wavelength Aggregation-Induced Luminescence Magnetic Encoded Microspheres by In Situ Precipitation 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.

8. The method for preparing dual-wavelength aggregation-induced luminescence magnetically encoded microspheres according to claim 7, characterized in that: The following steps are involved: S1. Preparation of AIE polymer microspheres by active swelling method (1) dispersing blank polymer microspheres in water to obtain a microsphere dispersion; (2) dissolving the AIE molecules in a swelling agent to obtain an AIE swelling agent solution; (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; S2. Preparation of Dual-Wavelength Aggregation-Induced Luminescence Magnetic Encoded Microspheres by In Situ Precipitation 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 dual-wavelength aggregation-induced luminescence magnetically encoded microspheres.

9. The method for preparing dual-wavelength aggregation-induced luminescence magnetically encoded microspheres according to claim 8, characterized in that: 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; 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; 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; 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; and the mass concentration of the alkaline solution is 1% to 10%; 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 more than three times.

10. Use of the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres according to any one of claims 1 to 6 in in vitro non-disease diagnostic testing.

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