A high-stability quantum dot fluorescent microsphere and its preparation method and application

Abstract

The present invention relates to the field of immunochromatography technology, and in particular to a high-stability quantum dot fluorescent microsphere and its preparation method and application. The preparation method comprises the following steps: first, preparing polystyrene nanospheres rich in aldehyde groups on the surface and oil-soluble quantum dots with surface amino groups by emulsion polymerization, condensing the aldehyde groups with amino groups to form imines by Schiff base reaction, and then coating the microsphere surface with a polystyrene protective layer by seed polymerization and performing functional group modification on the microsphere surface. The quantum dot fluorescent microspheres of the present invention have a simple preparation process, are suitable for large-scale production, and are bonded by C=N double bonds to avoid dye leakage. In addition, the microspheres have the characteristics of a microsphere particle size range of 100-400nm, uniform particle size, good monodispersity, etc., and are suitable for quantitative detection of objects to be detected in the field of immunochromatography.

Description

Technical Field

[0001] The present invention relates to the field of immunochromatography technology, and specifically relates to a high-stability quantum dot fluorescent microsphere and a preparation method and application thereof. Background Art

[0002] Immunochromatography (ICA) is a rapid detection technology that combines chromatography principles with antigen-antibody reactions. It has applications in a variety of areas, including veterinary drug residue detection, environmental monitoring, and disease diagnosis. Quantum dot fluorescent microsphere immunochromatography is an immunochromatographic technique based on quantum dot fluorescent microspheres as markers. Quantum dot fluorescent dyes have advantages such as a wide excitation wavelength range, a narrow and symmetrical emission wavelength range, high fluorescence intensity, excellent photostability, and a long fluorescence lifetime. Quantum dot fluorescent microspheres have great development potential in the field of immunochromatography.

[0003] Quantum dot fluorescent microspheres can be prepared by various methods, including swelling, adsorption, and embedding. However, these methods can lead to quantum dot leakage, which weakens the fluorescence intensity of the microspheres themselves and limits their application in biological detection. In recent years, some quantum dot fluorescent microspheres have been prepared by reacting carboxyl / amino groups on the microsphere surface with amino / carboxylated quantum dots in the presence of EDC / NHS via an amide condensation reaction. However, the reported methods are complex and result in large particle sizes, reaching the micron level, making them unsuitable for immunochromatography.

[0004] CN111088033A discloses a method for preparing monodisperse, high-performance quantum dot fluorescent microspheres. The method involves combining surface-amino-modified polymer microspheres with surface-carboxylated quantum dots through an amide condensation reaction, and then coating the microspheres with a layer of polymer to modify the microsphere surface. This method produces quantum dots with uniform fluorescent size, uniform fluorescence distribution, high fluorescence intensity, and good stability. This method improves the stability of the quantum dots while also improving their hydrophilicity and imparting functionalization. However, the quantum dot fluorescent microspheres prepared by this method are relatively large, at 0.5-1 μm, making them unsuitable for use in the immune system.

[0005] CN111117606A discloses a method for preparing a carbon quantum dot fluorescent microsphere standard substance, comprising the following steps: (1) preparing polystyrene microspheres; (2) preparing nitrated polystyrene microspheres; (3) preparing amino polystyrene microspheres; (4) preparing carboxylated graphene quantum dots; and (5) preparing a carbon quantum dot fluorescent microsphere standard substance. The quantum dot fluorescent microspheres prepared by this method have a large particle size, reaching the micron level, and the steps are cumbersome, making them difficult to industrialize and unsuitable for immunochromatography.

[0006] CN109233836BA discloses a method for preparing quantum dot fluorescent microspheres. The method involves preparing aqueous quantum dots with surface-modified active groups by ligand exchange, coating with amphiphilic polymers, or directly synthesizing them in an aqueous phase. The microspheres are then activated to obtain active microspheres with groups reactive with the surface active groups of the active quantum dots. The aqueous quantum dots with surface-modified active groups and the active microspheres are then covalently bonded to form the resulting quantum dot fluorescent microspheres. This method produces relatively large, micron-sized quantum dot fluorescent microspheres, but the steps involved are complex and difficult to use in immunochromatography.

[0007] In summary, the current preparation technology of quantum dot fluorescent microspheres still has the following shortcomings: (1) The swelling method of encapsulation technology is prone to dye leakage; (2) The microspheres are large in size, and the micron level is not suitable for immunochromatography; (3) The process is complicated and not suitable for industrial production; (4) The method of combining the microspheres and quantum dots through amide bonds through EDC / NHS activation is relatively simple. Therefore, how to develop quantum dot fluorescent microspheres with a simple preparation process, suitable for industrial production, suitable particle size, high dispersion, and no dye leakage has become one of the urgent problems to be solved in this field. Summary of the Invention

[0008] The purpose of the present invention is to provide a high-stability quantum dot fluorescent microsphere and a preparation method and application thereof.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] A method for preparing highly stable quantum dot fluorescent microspheres is characterized by: first, preparing polystyrene nano-microspheres with aldehyde groups on the surface and oil-soluble quantum dots with amino groups on the surface by emulsion polymerization; condensing the aldehyde groups with amino groups to form imines by Schiff base reaction; then, coating the microspheres with a polystyrene protective layer by seed polymerization and modifying the microspheres with functional groups on the surface; and obtaining microspheres with a particle size of 100-400 nm.

[0011] The surface amino-modified oil-soluble quantum dots are prepared by a solvent thermal method, and the specific steps are as follows:

[0012] (1) Weigh appropriate amounts of CdO, oleylamine, and liquid paraffin into a polytetrafluoroethylene reactor, place it in a high-temperature oven at 150-180°C, and take it out after 2 hours to obtain a Cd precursor solution;

[0013] (2) Weigh an appropriate amount of Se and liquid paraffin into a polytetrafluoroethylene reactor, place it in a high-temperature oven at 200-240°C, and take it out after 3 hours to obtain a Se precursor solution;

[0014] (3) Then take an appropriate amount of Cd precursor solution and oleylamine and add them to the Se precursor solution. After stirring evenly, place it in a high-temperature oven at 200-220℃ and react for 0.5-8h.

[0015] Furthermore, in the step (1), the mass ratio of CdO to oleylamine is 1:2-10, and the molar concentration of Cd (i.e., the molar concentration of Cd in oleylamine and paraffin) is 0.2-1 mol / L; in the step (2), the molar concentration of Se (i.e., the molar concentration of Se in liquid paraffin) is 0.02-0.1 mol / L; in the step (3), the molar ratio of the Cd precursor to the Se precursor is 2:1, and the volume ratio of the Cd precursor to oleylamine is 1:0.1-0.5.

[0016] The preparation method of polystyrene microspheres rich in aldehyde groups on the surface is as follows: pure water is added to a reaction container, nitrogen is added to remove oxygen in the system, a surfactant is added, stirred and dissolved, styrene and an aldehyde monomer with a hydrophilic group are added, added to a reaction bottle, stirred at high speed, an initiator is added at 60°C, and the reaction is carried out at 80°C for 4-20 hours;

[0017] The structural formula of the aldehyde monomer is as follows, wherein n is 0 to 10; preferably, n is 1 to 3;

[0018] .

[0019] Furthermore, the surfactant is an anionic surfactant, preferably sodium lauryl sulfate.

[0020] Furthermore, the initiator is a water-soluble initiator, preferably potassium persulfate.

[0021] Furthermore, the volume ratio of styrene to aldehyde monomer is 1:0.4-2, and the initiator is 0.2%-2% of the total monomer amount.

[0022] Among them, the specific method of condensing aldehyde groups with amino groups to form imines through the Schiff base reaction is to disperse polystyrene nanoparticles with aldehyde groups on the surface into ethanol, add oil-soluble quantum dots with amino groups on the surface, add a few drops of acetic acid as a catalyst, and react at room temperature for 2-6 hours; the mass volume ratio of the polystyrene nanoparticles to the oil-soluble quantum dots is 1:1-4.

[0023] Among them, the method of functional group modification is to disperse the microspheres combined with quantum dots in pure water and anhydrous ethanol, pass nitrogen to remove oxygen in the system, then add surfactant, dissolve it, add styrene, divinylbenzene and methacrylic acid, heat it to 60°C, add initiator, and react at 70-85°C for 3-20 hours.

[0024] Furthermore, the volume ratio of styrene to methacrylic acid is 1:0.02-0.2, and the mass ratio of the crosslinking agent to the total monomers is 1:0.01-0.05.

[0025] The high-stability quantum dot fluorescent microspheres prepared by the invention can be used in quantum dot fluorescent microsphere immunochromatography.

[0026] For example, the specific process of labeling antibodies (D-DimermAb) is:

[0027] (1) Take 50 μL of quantum dot fluorescent microspheres and add them to a centrifuge tube containing 1 mL of coupling buffer (50 mmol MES, pH 6.0) and mix thoroughly by ultrasonication;

[0028] (2) Add 5 μL each of freshly prepared EDC (100 mg / mL) and NHS (100 mg / mL) and vortex to mix. Activate at room temperature in the dark for 30 min.

[0029] (3) Centrifuge at 15,000 rpm for 20 min, remove the supernatant, add coupling buffer, and mix by ultrasonication;

[0030] (4) Add 80 μg of antibody, vortex to mix, and couple at room temperature in the dark for 2 h;

[0031] (5) Add 500 μL of microsphere blocking solution (20 mM boric acid buffer pH 8.0, 1% BSA, 0.2% ethanolamine), vortex mix, and block at room temperature in the dark for 1 h;

[0032] (6) Centrifuge at 15000 rpm for 15 min, discard the supernatant, add 500 μL microsphere preservation solution, disperse evenly by ultrasonication, and store at 2-8°C in the dark until use.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The high-stability quantum dot fluorescent microspheres prepared by this method are simple to manufacture and suitable for large-scale production. Furthermore, the C=N double bond prevents dye leakage. Furthermore, the microspheres have a uniform particle size range of 100-400 nm and good monodispersity, making them suitable for quantitative detection of targets in immunochromatography. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a schematic diagram of the principle of covalent bonding between polystyrene nanospheres with aldehyde-rich surfaces and amino-grouped oil-soluble quantum dots through Schiff base reaction to form -C=N-.

[0036] Figure 2This is a schematic diagram of the structure of the highly dispersed quantum dot fluorescent microspheres of the present invention. The interior is composed of polystyrene microspheres containing aldehyde groups, which are covalently bonded to quantum dots through a Schiff base reaction. The exterior is a polystyrene protective layer with carboxyl groups on the surface, which can bind antibodies to achieve antigen detection.

[0037] Figure 3 Correlation curve for the D-Dimer antigen calibrator test. DETAILED DESCRIPTION

[0038] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0039] Example 1 Preparation of Oil-Soluble CdSe Quantum Dots with Surface Amination

[0040] Preparation by solvent thermal method: 10 mmol CdO, 6 mL oleylamine and 20 mL liquid paraffin were weighed into a polytetrafluoroethylene reactor, placed in a high-temperature oven at 150°C, and taken out after 2 h to obtain a transparent Cd precursor solution; 1 mmol Se and 50 mL liquid paraffin were weighed into a polytetrafluoroethylene reactor, placed in a high-temperature oven at 220°C, and taken out after 3 h to obtain a transparent Se precursor solution; then 5.2 mL Cd precursor solution and 2 mL oleylamine were added to the Se precursor solution, stirred evenly, and placed in a high-temperature oven at 220°C for 4 h.

[0041] Example 2 Preparation of Formaldehyde-Based Polystyrene Microspheres PCH-1 (100 nm)

[0042] Using an emulsion polymerization method, 100 mL of pure water was added to a three-necked flask and nitrogen was purged for 20 minutes to remove oxygen from the system. 155 mg of the surfactant sodium dodecyl sulfate was added and stirred to dissolve. 10 mL of styrene and 10 mL of 2-[2-(4-formylphenoxy)ethoxy]ethyl 2-methacrylate were then added to the reaction flask and stirred at high speed for 30 minutes. At 60°C, 80 mg of KPS initiator was added, and the reaction was continued at 80°C for 16 hours.

[0043] Example 3 Preparation of Formaldehyde-Based Polystyrene Microspheres PCH-2 (300 nm)

[0044] Using an emulsion polymerization method, 100 mL of pure water was added to a three-necked flask and nitrogen was purged for 20 minutes to remove oxygen from the system. 70 mg of the surfactant sodium dodecyl sulfate was added and stirred to dissolve. Then, 10 mL of styrene and 10 mL of 2-[2-(4-formylphenoxy)ethoxy]ethyl 2-methacrylate were added and stirred at high speed for 30 minutes. At 60°C, 80 mg of KPS initiator was added, and the reaction was continued at 80°C for 16 hours.

[0045] Example 4 Preparation of Formaldehyde-Based Polystyrene Microspheres PCH-3 (400 nm)

[0046] Using an emulsion polymerization method, 100 mL of pure water was added to a three-necked flask and nitrogen was purged for 20 minutes to remove oxygen from the system. 50 mg of the surfactant sodium dodecyl sulfate was added and stirred to dissolve. Then, 10 mL of styrene and 10 mL of 2-[2-(4-formylphenoxy)ethoxy]ethyl 2-methacrylate were added and stirred at high speed for 30 minutes. At 60°C, 80 mg of KPS initiator was added, and the reaction was continued at 80°C for 16 hours.

[0047] Example 5 Schiff base reaction binding quantum dots (PCH / QD-1)

[0048] 500 mg of the formaldehyde polystyrene microspheres PCH-1 prepared in Example 2 were dispersed in 20 mL of ethanol, 1 mL of the CdSe quantum dots prepared in Example 1 was added, and a few drops of acetic acid were added as a catalyst. The mixture was reacted at room temperature for 3 h.

[0049] Example 6 Schiff base reaction binding quantum dots (PCH / QD-2)

[0050] 500 mg of the formaldehyde-based polystyrene microspheres prepared in Example 3 were dispersed in 20 mL of ethanol, 1 mL of the CdSe quantum dots prepared in Example 1 was added, and a few drops of acetic acid were added as a catalyst, and the reaction was carried out at room temperature for 3 h.

[0051] Example 7 Schiff base reaction binding quantum dots (PCH / QD-3)

[0052] 500 mg of the formaldehyde-based polystyrene microspheres prepared in Example 4 were dispersed in 20 mL of ethanol, 1 mL of the CdSe quantum dots prepared in Example 1 was added, and a few drops of acetic acid were added as a catalyst. The mixture was reacted at room temperature for 3 h.

[0053] Example 8 Surface carboxyl modification (PCH / QD / COOH-1)

[0054] 100 mg of the microspheres PCH / QD-1 in Example 5 were dispersed in 150 mL of pure water and 3 mL of anhydrous ethanol. Nitrogen was passed through the system for 20 min to remove oxygen. Then, 60 mg of the surfactant SDS was added and dissolved. 4 mL of St, 0.1 mL of DVB, and 0.3 mL of MAA were added. The temperature was raised to 60°C, 40 mg of the initiator KPS was added, and the reaction was incubated at 75°C for 7 h.

[0055] Example 9 Surface carboxyl modification (PCH / QD / COOH-2)

[0056] 100 mg of PCH / QD-2 microspheres in Example 6 were dispersed in 150 mL of pure water and 3 mL of anhydrous ethanol. Nitrogen was passed through the system for 20 min to remove oxygen. Then 40 mg of surfactant SDS was added and dissolved. 4 mL of St, 0.1 mL of DVB, and 0.3 mL of MAA were added. The temperature was raised to 60°C, 40 mg of initiator KPS was added, and the reaction was incubated at 75°C for 7 h.

[0057] Example 10 Surface carboxyl modification (PCH / QD / COOH-3)

[0058] 100 mg of PCH / QD-3 microspheres in Example 7 were dispersed in 150 mL of pure water and 3 mL of anhydrous ethanol. Nitrogen was passed through the system for 20 min to remove oxygen. Then 30 mg of surfactant SDS was added and dissolved. 4 mL of St, 0.1 mL of DVB, and 0.3 mL of MAA were added. The temperature was raised to 60°C, 40 mg of initiator KPS was added, and the reaction was incubated at 75°C for 7 h.

[0059] In order to highlight the beneficial effects of the present invention, the following comparative example experiments are given.

[0060] Comparative Example 1

[0061] Using an emulsion polymerization method, 100 mL of pure water was added to a three-necked flask and nitrogen was purged for 20 minutes to remove oxygen from the system. 155 mg of the surfactant sodium dodecyl sulfate was added and stirred to dissolve. 10 mL of styrene and 10 mL of 4-vinylbenzaldehyde were then added to the reaction flask and stirred at high speed for 30 minutes. At 60°C, 80 mg of KPS initiator was added, and the reaction was allowed to proceed at 80°C for 16 hours.

[0062] Comparative Example 2

[0063] 500 mg of the formaldehyde-based polystyrene microspheres prepared in Comparative Example 1 were dispersed in 20 mL of ethanol, 1 mL of the CdSe quantum dots prepared in Example 1 was added, and a few drops of acetic acid were added as a catalyst, and the mixture was reacted at room temperature for 3 h.

[0064] Comparative Example 3

[0065] 100 mg of the microspheres in Comparative Example 2 were dispersed in 150 mL of pure water and 3 mL of anhydrous ethanol. Nitrogen was passed through the system for 20 min to remove oxygen. Then, 60 mg of the surfactant SDS was added and dissolved. 4 mL of St, 0.1 mL of DVB, and 0.3 mL of MAA were added. The temperature was raised to 60°C, 40 mg of the initiator KPS was added, and the reaction was incubated at 75°C for 7 h.

[0066] Example 11 Labeled Antibody (Taking D-Dimer mAb as an example, purchased from Nanjing Liding Medical Technology Co., Ltd.)

[0067] (1) Take 50 μL of the microspheres in Examples 8, 9, 10 and Comparative Example 3 and add them to a centrifuge tube containing 1 mL of coupling buffer (50 mmol MES, pH 6.0) and mix them by ultrasonication;

[0068] (2) Add 5 μL each of freshly prepared EDC (100 mg / mL) and NHS (100 mg / mL) and vortex to mix. Activate at room temperature in the dark for 30 min.

[0069] (3) Centrifuge at 15,000 rpm for 20 min, remove the supernatant, add coupling buffer, and mix by ultrasonication;

[0070] (4) Add 80 μg of antibody, vortex to mix, and couple at room temperature in the dark for 2 h;

[0071] (5) Add 500 μL of microsphere blocking solution (20 mM boric acid buffer pH 8.0, 1% BSA, 0.2% ethanolamine), vortex mix, and block at room temperature in the dark for 1 h;

[0072] (6) Centrifuge at 15000 rpm for 15 min, discard the supernatant, add 500 μL microsphere preservation solution, disperse evenly by ultrasonication, and store at 2-8°C in the dark until use.

[0073] Example 12 Test

[0074] (1) Particle size test

[0075] The Otsuka nanoSAQLA particle size analyzer was used for the test. Each sample was tested three times and the average value was taken.

[0076] (2) Calibrator testing

[0077] The test was performed using a Lanbo AFS-1000 dry-type fluorescent immunoassay analyzer with an excitation wavelength of 365 nm and an emission wavelength of 620 nm to test the luminescence intensity of four calibrators.

[0078] Table 1 D-Dimer calibrator test

[0079]

[0080] In Table 1, the T value and C value of the calibrator detected after coating with D-Dimer antibody in Comparative Example 3 are significantly lower than those of PCH / QD / COOH-1, PCH / QD / COOH-2, and PCH / QD / COOH-3. This is because the monomer used in the quantum dot fluorescent microspheres prepared in Comparative Example 3 is 4-vinylbenzaldehyde, which is a lipophilic group. During the polymerization process, most of it may be distributed inside the microspheres, resulting in a low efficiency of binding to quantum dots, and ultimately resulting in low fluorescence intensity of the T line and C line of the calibrator. The aldehyde group used in Examples 8, 9, and 10 introduces a hydrophilic long-chain structure containing ester and ether bond hydrophilic functional groups. During the polymerization process, it tends to be distributed on the surface of the microspheres, greatly increasing the content of aldehyde groups on the surface of the microspheres, improving the binding efficiency of the microspheres and quantum dots, and thus increasing the fluorescence intensity of the T line and C line during the detection of the calibrator, thereby improving sensitivity.

[0081] Table 1 and Figure 3 The results of the quantum dot fluorescent microspheres prepared in Examples 8, 9, and 10 and the detection of D-Dimer Ag calibrants after combining with D-Dimer mAb are given in FIG. 2 >0.99, indicating a good correlation.

[0082] Table 2 Microsphere particle size and dispersion index

[0083]

[0084] Table 2 shows the particle size and dispersion index of the microspheres. The particle sizes of formaldehyde polystyrene PCH-1, PCH-2, and PCH-3 are 101 nm, 282 nm, and 385 nm, respectively, and the dispersion index (PDI) is less than 0.05, which shows good monodispersity and particle size uniformity. The particle sizes of quantum dot fluorescent microspheres PCH / QD / COOH-1, PCH / QD / COOH-2, and PCH / QD / COOH-3 are 120 nm, 305 nm, and 402 nm, respectively, and the PDI is less than 0.05, which shows good monodispersity and particle size uniformity.

[0085] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing highly stable quantum dot fluorescent microspheres, characterized by: First, polystyrene nanoparticles with aldehyde-rich surfaces and amino-modified oil-soluble quantum dots were prepared by emulsion polymerization. The aldehyde groups were condensed with amino groups to form imines via a Schiff base reaction. Then, a polystyrene protective layer was coated on the surface of the microspheres by seed polymerization and the surface of the microspheres was modified with functional groups. The resulting microspheres had a particle size of 100-400 nm. The preparation method of polystyrene microspheres rich in aldehyde groups on the surface is as follows: pure water is added to a reaction container, nitrogen is added to remove oxygen in the system, a surfactant is added, stirred and dissolved, styrene and an aldehyde monomer with a hydrophilic group are added, added to a reaction bottle, stirred at high speed, an initiator is added at 60°C, and the reaction is carried out at 80°C for 4-20 hours; The structural formula of the aldehyde monomer is as follows, wherein n is 0 to 10; 。 2. The method for preparing high-stability quantum dot fluorescent microspheres according to claim 1, wherein: The surface amino-modified oil-soluble quantum dots are prepared by a solvothermal method, specifically by the following steps: (1) Weigh appropriate amounts of CdO, oleylamine, and liquid paraffin into a polytetrafluoroethylene reactor, place it in a high-temperature oven at 150-180°C, and take it out after 2 hours to obtain a Cd precursor solution; (2) Weigh an appropriate amount of Se and liquid paraffin into a polytetrafluoroethylene reactor, place it in a high-temperature oven at 200-240°C, and take it out after 3 hours to obtain a Se precursor solution; (3) Then take an appropriate amount of Cd precursor solution and oleylamine and add them to the Se precursor solution. After stirring evenly, place it in a high-temperature oven at 200-220℃ and react for 0.5-8h.

3. The method for preparing high-stability quantum dot fluorescent microspheres according to claim 2, wherein: In the step (1), the mass ratio of CdO to oleylamine is 1:2-10, and the molar concentration of Cd is 0.2-1 mol / L; in the step (2), the molar concentration of Se is 0.02-0.1 mol / L; in the step (3), the molar ratio of the Cd precursor to the Se precursor is 2:1, and the volume ratio of the Cd precursor to oleylamine is 1:0.1-0.

5.

4. The method for preparing high-stability quantum dot fluorescent microspheres according to claim 1, wherein: The n is 1~3.

5. The method for preparing high-stability quantum dot fluorescent microspheres according to claim 1, wherein: The surfactant is sodium lauryl sulfate, and the initiator is potassium persulfate.

6. The method for preparing high-stability quantum dot fluorescent microspheres according to claim 5, characterized in that: The volume ratio of styrene to aldehyde monomer is 1:0.4-2, and the initiator is 0.2%-2% of the total monomer amount.

7. The method for preparing highly stable quantum dot fluorescent microspheres according to claim 1, wherein: The specific method for condensing aldehyde groups with amino groups to generate imines through the Schiff base reaction is as follows: polystyrene nanoparticles with surface-rich aldehyde groups are dispersed in ethanol, oil-soluble quantum dots with surface amino groups are added, acetic acid is dripped as a catalyst, and the reaction is carried out at room temperature for 2-6 hours; the mass-to-volume ratio of the polystyrene nanoparticles to the oil-soluble quantum dots is 1:1-4.

8. The method for preparing high-stability quantum dot fluorescent microspheres according to claim 1, wherein: The functional group modification method comprises dispersing quantum dot-bound microspheres in pure water and anhydrous ethanol, passing nitrogen to remove oxygen in the system, adding a surfactant, and dissolving the resulting solution. Then, styrene, divinylbenzene, and methacrylic acid are added, the temperature is raised to 60° C., an initiator is added, and the reaction is carried out at 70-85° C. for 3-20 hours. The volume ratio of styrene to methacrylic acid is 1:0.02-0.2, and the mass ratio of the crosslinker to the total monomers is 1:0.01-0.

05.

9. High-stability quantum dot fluorescent microspheres prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the high-stability quantum dot fluorescent microspheres according to claim 9 in quantum dot fluorescent microsphere immunochromatography.

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