Tosyl-modified magnetic time-resolved fluorescent microspheres as well as preparation method and application thereof

Abstract

The invention relates to the technical field of immunochromatography, in particular to a TSYl modified magnetic time-resolved fluorescent microsphere as well as a preparation method and application thereof, and the preparation method comprises the following steps: 1) preparing a surface aminated Fe3O4 magnetic core by a hydrothermal synthesis method; (2) carrying out double-bond modification on the surface of Fe3O4 through nucleophilic substitution reaction; 3) preparing surface hydroxyl modified polymer magnetic microspheres by a seed emulsion polymerization method; 4) through nucleophilic substitution reaction, performing tosyl modification on the surface of the magnetic microsphere; 5) preparing a time-resolved fluorescent dye Eu (III) complex; and 6) embedding a rare earth complex into the microspheres by a swelling method to prepare the surface tosyl modified magnetic time-resolved fluorescent microspheres. The TSYl modified magnetic time-resolved fluorescent microspheres prepared by the method have the advantages that the particle size range is 200-400nm, the particle size is uniform, and the monodispersity is good; the antibody can be directly combined with protein / antibody without activation; good magnetic responsiveness and fluorescence intensity are realized.

Description

Technical Field

[0001] The invention relates to the technical field of immunochromatography, and specifically relates to a Tosyl-modified magnetic time-resolved fluorescent microsphere, a preparation method and an application thereof. Background Art

[0002] As the key raw material of time-resolved fluorescent immunochromatography technology, time-resolved fluorescent microspheres have very important scientific research value. Time-resolved immunochromatography technology uses time-resolved fluorescent microspheres as markers, and each microsphere contains thousands of fluorescent molecules, which greatly improves the labeling efficiency and detection sensitivity.

[0003] Commonly used time-resolved fluorescent microspheres are polystyrene microspheres with appropriate carboxyl groups on the surface, which are used for covalent coupling with proteins or antibodies to improve the stability of the markers. Magnetic nanomaterials have very wide application potential in the biomedical field due to their unique properties. Therefore, time-resolved fluorescent microspheres are combined with magnetic nanoparticles to prepare magnetic time-resolved fluorescent microspheres with magnetic-optical dual functions, thereby expanding the application of nanospheres in the biological field.

[0004] At present, the literature reports on magnetic time-resolved fluorescent microspheres include integrating magnetic spheres with rare earth complexes by covalent bonding; or preparing magnetic fluorescent complexes by coprecipitation, and then embedding magnetic cores and rare earth complexes into microspheres by swelling method; or coating small-size magnetic nanoparticles by simple emulsion method; or preparing magnetic cores by solvothermal method, and then modifying the surface of magnetic core Fe3O4@silicon layer with olefin bonds, and then coating polymers by precipitation polymerization, and finally embedding rare earth complexes into microspheres by swelling method. However, these microspheres are all surface carboxyl modified, and need to be activated before coating antibodies. The steps are cumbersome, and there are problems such as complex preparation process, poor magnetic responsiveness, and low fluorescence intensity, which make it difficult to meet application requirements.

[0005] Therefore, it is necessary to develop a time-resolved fluorescent microsphere that does not require activation, has a simple preparation process, has strong magnetic responsiveness, and has high fluorescence intensity. Summary of the invention

[0006] The purpose of the present invention is to provide a Tosyl-modified magnetic time-resolved fluorescent microsphere and a preparation method and application thereof.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for preparing Tosyl-modified magnetic time-resolved fluorescent microspheres, comprising the following steps: a first step, preparing a surface-amino Fe3O4 magnetic core by a hydrothermal synthesis method; a second step, modifying the Fe3O4 surface double bonds by a nucleophilic substitution reaction; a third step, preparing a surface hydroxyl-modified polymer magnetic microsphere by a seed emulsion polymerization method; a fourth step, modifying the surface of the magnetic microspheres by tosyl by a nucleophilic substitution reaction; a fifth step, preparing a time-resolved fluorescent dye Eu(III) complex; and a sixth step, embedding the rare earth complex into the microspheres by a swelling method to prepare a surface-tosyl-modified magnetic time-resolved fluorescent microsphere.

[0009] The specific steps are:

[0010] (1) A hydrothermal synthesis method is used to prepare Fe3O4 magnetic cores with surface amino groups: FeCl3·6H2O and 1,6-hexanediamine are dispersed in ethylene glycol. After dissolution, anhydrous sodium acetate is added to the above dispersion and reacted overnight. After magnetic separation and washing, Fe3O4 magnetic particles with a particle size of 100-200 nm are obtained;

[0011] (2) introducing double bonds on the surface of Fe3O4 magnetic cores by nucleophilic substitution reaction, specifically, adding Fe3O4 magnetic cores with surface amino groups to acetonitrile solution, adding excess methacryloyl chloride under the action of triethylamine, and reacting at 0°C for 2h to obtain Fe3O4@double bonds;

[0012] (3) The polystyrene shell layer was coated by seed emulsion polymerization and the surface was modified by hydroxylation, specifically: Fe3O4@ double bond was used as a seed, dispersed in pure water, nitrogen was passed for 20 minutes, anionic surfactant sodium dodecyl sulfate was added to dissolve, monomer styrene and functional monomer hydroxyethyl methacrylate were added, initiator potassium persulfate or ammonium persulfate was added at 60°C, and the reaction was carried out at 85°C overnight to obtain Fe3O4@PS-OH;

[0013] (4) Tosyl modification of the microsphere surface by nucleophilic substitution reaction: Fe3O4@PS-OH was added to acetonitrile solution, and under the action of triethylamine, an excess of p-toluenesulfonyl chloride in acetonitrile solution was added, and the reaction was carried out at 0°C for 2 h to obtain Fe3O4@PS-tosyl;

[0014] (5) Preparation of Eu(III): Dissolve o-phenanthroline and dibenzoylmethane in an appropriate amount of ethanol, then add triethylamine, and finally add an ethanol solution of EuCl3·6H2O. Set the temperature to 60°C, stop the reaction after 2 hours, wash with anhydrous ethanol several times, and filter again to obtain the time-resolved fluorescent dye Eu(III);

[0015] (6) The time-resolved fluorescent dye was embedded into the microspheres by the swelling method: an appropriate amount of surfactant was added to the Fe3O4@PS-tosyl emulsion, and ultrasonic dispersion was performed to prepare a microsphere emulsion; an appropriate amount of time-resolved fluorescent dye Eu(Ⅲ) was taken into an EP tube, and a small amount of dichloromethane was added to fully dissolve it to prepare a dye solution; the dye solution was poured into the microsphere emulsion, and the microspheres were placed in a constant temperature shaker with the speed set to 200 rpm, protected from light, and reacted at 30°C for three days to prepare Fe3O4@PS-tosyl@Eu(Ⅲ).

[0016] Wherein, in the step (1), the mass ratio of FeCl3·6H2O, CH3COONa, 1,6-hexanediamine and ethylene glycol is 1:1-10:1-5:30-60.

[0017] Wherein, in the step (2), the mass ratio of the surface aminated Fe3O4, triethylamine and methacryloyl chloride is 1:1-3:0.5-1.

[0018] Wherein, in the step (3), the mass ratio of Fe3O4@ double bond to styrene is 1:8-20, and the mass ratio of styrene to hydroxyethyl methacrylate is 1:0.04-0.2.

[0019] Wherein, in the step (4), the mass ratio of Fe3O4@PS-OH, triethylamine and p-toluenesulfonyl chloride is 1:1-3:0.5-1.5.

[0020] Wherein, in the step (5), the molar ratio of EuCl3·6H2O, dibenzoylmethane and o-phenanthroline is 1:3-3.3:1-1.1.

[0021] Wherein, in the step (6), the mass ratio of Fe3O4@PS-tosyl to Eu(III) is 1:0.3-1; and the volume ratio of pure water to dichloromethane in the entire reaction system is 1:0.1-0.5.

[0022] The particle size of the Tosyl modified magnetic time-resolved fluorescent microspheres prepared by the invention is 200-400nm.

[0023] The Tosyl-modified magnetic time-resolved fluorescent microspheres of the present invention can be used for immunofluorescence analysis, and the specific antibody labeling process is as follows:

[0024] (1) Take 20 μL of magnetic gold nanoparticles with a solid content of 1% in a 2 mL EP tube, add 1 mL of labeling buffer, perform magnetic separation, remove the supernatant, add 1 mL of labeling buffer, and perform ultrasonic dispersion. Repeat three times and finally store in 1 mL of labeling buffer.

[0025] (2) Take 0.1 mg of antibody into the above 2 mL EP tube, mix quickly, and incubate at room temperature for 2 h;

[0026] (3) Add 100 μL of 10% BSA blocking solution to the above EP tube, mix and incubate at room temperature for 30 min;

[0027] (4) Magnetic separation: remove the supernatant, add 1 mL of preservation solution and perform ultrasonic dispersion. Repeat this process three times and finally store the cells in 1 mL of preservation solution.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The Tosyl-modified magnetic time-resolved fluorescent microspheres prepared by the method of the present invention have a particle size range of 200-400nm, uniform particle size, and good monodispersity; the surface is tosyl-modified, and can be directly combined with proteins / antibodies without activation, and have good magnetic responsiveness and fluorescence intensity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the synthetic process roadmap for Tosyl-modified magnetic time-resolved fluorescent microspheres.

[0031] Figure 2 This is the SEM image of Fe3O4@PS-tosyl@Eu in Example 2. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work are within the scope of protection of the present invention.

[0033] Example 1

[0034] like Figure 1 As shown, a method for preparing Tosyl-modified magnetic time-resolved fluorescent microspheres comprises the following specific steps:

[0035] (1) Solvothermal preparation of surface-aminated Fe3O4 nanoparticles

[0036] Weigh 2g of FeCl3·6H2O and 6g of 1,6-hexanediamine, dissolve in 100mL of ethylene glycol, and stir to dissolve. Then weigh 3g of anhydrous sodium acetate and add it to the above solution, stir until completely dissolved; transfer the uniform viscous liquid after dissolution to a 250mL high-temperature reactor, tighten the lid, and place it in a 200℃ oven to heat overnight; after the reactor temperature cools naturally, pour out the black reaction liquid and separate it by magnetic separation; first wash it three times with anhydrous ethanol, then wash it three times with pure water, store it in pure water, determine the Fe3O4 content, and store it with nitrogen to deoxygenate and avoid oxidation.

[0037] (2)Fe3O4@Double bond

[0038] Place 0.5g of Fe3O4 in step (2) in a 50mL EP tube, wash it with acetonitrile 5 times, and finally disperse it in 20mL acetonitrile solution. Add an appropriate amount of molecular sieves, let it stand for 6 hours, pour the Fe3O4 dispersion stored in acetonitrile into a three-necked flask, add 1mL of triethylamine at 0℃, and stir for 10min. Then dissolve 0.3mL of methacryloyl chloride in 2mL acetonitrile solution, add it dropwise to the reaction bottle, and stop the reaction after 2h. Wash it with water 3 times, and finally store it in 10mL of pure water.

[0039] (3)Fe3O4@PS-OH

[0040] Disperse the double-bond modified magnetic core in step (2) in 50 mL pure water, and pass nitrogen for 20 min to remove oxygen in the system. Add 0.1 g sodium dodecyl sulfate (SDS) and stir for 10 min to fully dissolve it. Add 5 mL styrene and 0.5 mL hydroxyethyl methacrylate, add 50 mg initiator potassium persulfate (dissolved in 1 mL pure water) at 60°C, and react at 85°C for 8 h. Wash with water 3 times and with anhydrous ethanol 3 times.

[0041] (4)Fe3O4@PS-tosyl

[0042] Disperse the magnetic microspheres modified with surface hydroxyl groups in step (3) in a 50mL EP tube, wash with acetonitrile 5 times, and finally disperse in 20mL acetonitrile solution. Add an appropriate amount of molecular sieves, let stand for 6 hours, pour the microsphere dispersion stored in acetonitrile into a three-necked flask, add 1mL of triethylamine at 0°C, and stir for 10 minutes. Then dissolve 500mg of p-toluenesulfonyl chloride in 5mL acetonitrile solution, add dropwise to the reaction bottle, and stop the reaction after 2 hours. Wash with ethanol 3 times, wash with water 3 times, and finally store in 50mL pure water.

[0043] (5) Preparation of Eu(III)

[0044] 182.2 mg of o-phenanthroline (phen) and 673 mg of dibenzoylmethane (DBM) were dissolved in 20 mL of anhydrous ethanol, the temperature was set to 60°C, and then 0.4 mL of triethylamine was added. Finally, 366 mg of EuCl3·6H2O dissolved in 5 ml of ethanol was added dropwise. After observing the formation of precipitation, the precipitate was stirred for 2 hours and filtered, washed with anhydrous ethanol several times and filtered again to obtain a yellow powder, which was dried with a yield of 70%.

[0045] (6)Fe3O4@PS-tosyl@Eu

[0046] Take 10mL of Fe3O4@PS-tosyl magnetic microspheres in step (4), add 50mg PEG6000, and dissolve by ultrasonication. Take 50mg Eu(Ⅲ) in an EP tube and add 1mL DCM to dissolve. Mix the two solvents, place in a constant temperature shaker, 30℃-200rpm, avoid light, and react for 3 days. Wash with ethanol 3 times, wash with water 3 times, and finally store in pure water, and adjust the solid content to 1%.

[0047] Example 2

[0048] like Figure 1 As shown, a method for preparing Tosyl-modified magnetic time-resolved fluorescent microspheres comprises the following specific steps:

[0049] (1) Solvothermal preparation of surface-aminated Fe3O4 nanoparticles

[0050] Weigh 2.5g of FeCl3·6H2O and 6g of 1,6-hexanediamine, dissolve in 100mL of ethylene glycol, and stir to dissolve. Then weigh 3g of anhydrous sodium acetate and add it to the above solution, stir until completely dissolved; transfer the uniform viscous liquid after dissolution to a 250mL high-temperature reactor, tighten the lid, and place it in a 200℃ oven to heat overnight; after the reactor temperature cools naturally, pour out the black reaction liquid and separate it by magnetic separation; first wash it three times with anhydrous ethanol, then wash it three times with pure water, store it in pure water, determine the Fe3O4 content, and store it with nitrogen to deoxygenate and avoid oxidation.

[0051] (2)Fe3O4@Double bond

[0052] Place 0.5g of Fe3O4 in step (1) in a 50mL EP tube, wash it with acetonitrile 5 times, and finally disperse it in 20mL acetonitrile solution. Add an appropriate amount of molecular sieves, let it stand for 6 hours, pour the Fe3O4 dispersion stored in acetonitrile into a three-necked flask, add 1mL of triethylamine at 0℃, and stir for 10 minutes. Then dissolve 0.3mL of methacryloyl chloride in 2mL acetonitrile solution, add it dropwise to the reaction bottle, and stop the reaction after 2 hours. Wash it with water 3 times, and finally store it in 10mL of pure water.

[0053] (3)Fe3O4@PS-OH

[0054] Disperse the double-bond modified magnetic core in step (2) in 50 mL pure water, and pass nitrogen for 20 min to remove oxygen in the system. Add 0.05 g SDS and stir for 10 min to fully dissolve it. Add 7 mL styrene and 0.7 mL hydroxyethyl methacrylate, add 50 mg initiator potassium persulfate (dissolved in 1 mL pure water) at 60°C, and react at 85°C for 8 h. Wash with water 3 times and with anhydrous ethanol 3 times.

[0055] (4)Fe3O4@PS-tosyl

[0056] Disperse the magnetic microspheres modified with surface hydroxyl groups in step (3) in a 50mL EP tube, wash with acetonitrile 5 times, and finally disperse in 20mL acetonitrile solution. Add an appropriate amount of molecular sieves, let stand for 6 hours, pour the microsphere dispersion stored in acetonitrile into a three-necked flask, add 1mL of triethylamine at 0°C, and stir for 10 minutes. Then dissolve 500mg of p-toluenesulfonyl chloride in 5mL acetonitrile solution, add dropwise to the reaction bottle, and stop the reaction after 2 hours. Wash with ethanol 3 times, wash with water 3 times, and finally store in 50mL pure water.

[0057] (5) Preparation of Eu(III)

[0058] 182.2 mg of phen and 673 mg of DBM were dissolved in 20 mL of anhydrous ethanol, the temperature was set to 60°C, and then 0.4 mL of triethylamine was added. Finally, 366 mg of EuCl3·6H2O dissolved in 5 ml of ethanol was added dropwise. After observing the formation of precipitation, the precipitate was stirred for 2 hours and filtered, washed with anhydrous ethanol several times and filtered again to obtain a yellow powder. It was dried with a yield of 70%.

[0059] (6)Fe3O4@PS-tosyl@Eu

[0060] Take 10mL of Fe3O4@PS-tosyl magnetic microspheres in step (4), add 50mg PEG6000, and dissolve by ultrasonication. Take 50mg Eu(Ⅲ) in an EP tube and add 1mL DCM to dissolve. Mix the two solvents, place in a constant temperature shaker, 30℃-200rpm, avoid light, and react for 3 days. Wash with ethanol 3 times, wash with water 3 times, and finally store in pure water, and adjust the solid content to 1%.

[0061] Example 3

[0062] like Figure 1 As shown, a method for preparing Tosyl-modified magnetic time-resolved fluorescent microspheres comprises the following specific steps:

[0063] (1) Solvothermal preparation of surface-aminated Fe3O4 nanoparticles

[0064] Weigh 3g of FeCl3·6H2O and 6g of 1,6-hexanediamine, dissolve in 100mL of ethylene glycol, and stir to dissolve. Then weigh 3g of anhydrous sodium acetate and add it to the above solution, stir until completely dissolved; transfer the uniform viscous liquid after dissolution to a 250mL high-temperature reactor, tighten the lid, and place it in a 200℃ oven to heat overnight; after the reactor temperature cools naturally, pour out the black reaction liquid and separate it by magnetic separation; first wash it three times with anhydrous ethanol, then wash it three times with pure water, store it in pure water, determine the Fe3O4 content, and store it with nitrogen to deoxygenate and avoid oxidation.

[0065] (2)Fe3O4@Double bond

[0066] Place 0.5g of Fe3O4 in step (1) in a 50mL EP tube, wash it with acetonitrile 5 times, and finally disperse it in 20mL acetonitrile solution. Add an appropriate amount of molecular sieves, let it stand for 6 hours, pour the Fe3O4 dispersion stored in acetonitrile into a three-necked flask, add 1mL of triethylamine at 0℃, and stir for 10 minutes. Then dissolve 0.3mL of methacryloyl chloride in 2mL acetonitrile solution, add it dropwise to the reaction bottle, and stop the reaction after 2 hours. Wash it with water 3 times, and finally store it in 10mL of pure water.

[0067] (3)Fe3O4@PS-OH

[0068] Disperse the double-bond modified magnetic core in step (2) in 50 mL pure water, pass nitrogen for 20 min to remove oxygen in the system. Add 0.03 g SDS and stir for 10 min to fully dissolve it. Add 10 mL styrene and 1 mL hydroxyethyl methacrylate, add 50 mg initiator potassium persulfate (dissolved in 1 mL pure water) at 60°C, and react at 85°C for 8 h. Wash with water 3 times and with anhydrous ethanol 3 times.

[0069] (4)Fe3O4@PS-tosyl

[0070] Disperse the magnetic microspheres modified with surface hydroxyl groups in step (3) in a 50mL EP tube, wash with acetonitrile 5 times, and finally disperse in 20mL acetonitrile solution. Add an appropriate amount of molecular sieves, let stand for 6 hours, pour the microsphere dispersion stored in acetonitrile into a three-necked flask, add 1mL of triethylamine at 0°C, and stir for 10 minutes. Then dissolve 500mg of p-toluenesulfonyl chloride in 5mL acetonitrile solution, add dropwise to the reaction bottle, and stop the reaction after 2 hours. Wash with ethanol 3 times, wash with water 3 times, and finally store in 50mL pure water.

[0071] (5) Preparation of Eu(III)

[0072] 182.2 mg of phen and 673 mg of DBM were dissolved in 20 mL of anhydrous ethanol, the temperature was set to 60°C, and then 0.4 mL of triethylamine was added. Finally, 366 mg of EuCl3·6H2O dissolved in 5 ml of ethanol was added dropwise. After observing the formation of precipitation, the precipitate was stirred for 2 hours and filtered, washed with anhydrous ethanol several times and filtered again to obtain a yellow powder. It was dried with a yield of 70%.

[0073] (6)Fe3O4@PS-tosyl@Eu

[0074] Take 10mL of Fe3O4@PS-tosyl magnetic microspheres in step (4), add 50mg PEG6000, and dissolve by ultrasonication. Take 50mg Eu(Ⅲ) in an EP tube and add 1mL DCM to dissolve. Mix the two solvents, place in a constant temperature shaker, 30℃-200rpm, avoid light, and react for 3 days. Wash with ethanol 3 times, wash with water 3 times, and finally store in pure water, and adjust the solid content to 1%.

[0075] Embodiment 4 Application Example

[0076] Application examples of Fe3O4@PS-OH@Eu prepared in Examples 1-3 (taking influenza A virus (Flu A) detection as an example).

[0077] (1) Take 20 μL of Fe3O4@PS-OH@Eu with a solid content of 1% in a 2 mL EP tube, add 1 mL of labeling buffer, perform magnetic separation, remove the supernatant, add 1 mL of labeling buffer, and perform ultrasonic dispersion. Repeat three times and store in 1 mL of labeling buffer.

[0078] (2) Take 0.1 mg of antibody into the above 2 mL EP tube, mix quickly, and incubate at room temperature for 2 h;

[0079] (3) Add 100 μL of 10% BSA blocking solution to the above EP tube, mix and incubate at room temperature for 30 min;

[0080] (4) Magnetic separation: remove the supernatant, add 1 mL of preservation solution and perform ultrasonic dispersion. Repeat this process three times and finally store the cells in 1 mL of preservation solution.

[0081] The experimental results are as follows:

[0082] Table 1 Particle size of Fe3O4@PS-tosyl@Eu prepared in Examples 1-3

[0083] name Example 1 Example 2 Example 3 Particle size (nm) 212.3 303.5 397.6 Dispersion Index 0.03 0.012 0.022

[0084] Table 1 shows the particle sizes of Fe3O4@PS-tosyl@Eu in Examples 1-3, which are 212.3 nm, 303.5 nm, and 397.6 nm, respectively. The dispersion index is less than 0.05, indicating that the microspheres have good monodispersity and uniform particle size.

[0085] Figure 2 This is the SEM image of Fe3O4@PS-tosyl@Eu in Example 2. It can be seen from the image that the particle size of the microspheres is uniform.

[0086] Table 2 shows the detection of Flu A virus antigen after the Fe3O4@PS-tosyl@Eu prepared in Examples 1-3 was coupled with Flu A antibody. The blank was used as the control group, and the fluorescence intensity of the low-value and high-value samples was tested respectively. Among them, the Fe3O4@PS-tosyl@Eu with a particle size of 397.6 nm prepared in Example 3 had the highest fluorescence intensity.

[0087] Table 2 Fe3O4@PS-tosyl@Eu labeled antibody detection of Flu A antigen in Examples 1-3

[0088] sample Example 1 Example 2 Example 3 blank 433 577 662 Low value 754 1464 1727 High value 89667 119920 165503

[0089] Although 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 the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing Tosyl-modified magnetic time-resolved fluorescent microspheres, characterized in that: The following steps are involved: (1) The surface amino-treated Fe3O4 magnetic core was prepared by hydrothermal synthesis; (2) Double bonds on the Fe3O4 surface are modified through nucleophilic substitution reaction; (3) Preparation of surface hydroxyl-modified polymer magnetic microspheres by seed emulsion polymerization; (4) Tosyl modification of the surface of magnetic microspheres through nucleophilic substitution reaction; (5) Preparation of time-resolved fluorescent dye Eu(III) complex; (6) The rare earth complex was embedded into the microspheres by the swelling method to prepare surface tosyl-modified magnetic time-resolved fluorescent microspheres.

2. The method for preparing Tosyl-modified magnetic time-resolved fluorescent microspheres according to claim 1, characterized in that: The specific steps are: (1) A hydrothermal synthesis method is used to prepare Fe3O4 magnetic cores with surface amino groups: FeCl3·6H2O and 1,6-hexanediamine are dispersed in ethylene glycol. After dissolution, anhydrous sodium acetate is added to the above dispersion and reacted overnight. After magnetic separation and washing, Fe3O4 magnetic particles with a particle size of 100-200 nm are obtained; (2) introducing double bonds on the surface of Fe3O4 magnetic cores by nucleophilic substitution reaction, specifically, adding Fe3O4 magnetic cores with surface amino groups to acetonitrile solution, adding excess methacryloyl chloride under the action of triethylamine, and reacting at 0°C for 2h to obtain Fe3O4@double bonds; (3) The polystyrene shell layer was coated by seed emulsion polymerization and the surface was modified by hydroxylation, specifically: Fe3O4@ double bond was used as a seed, dispersed in pure water, nitrogen was passed for 20 minutes, anionic surfactant sodium dodecyl sulfate was added to dissolve, monomer styrene and functional monomer hydroxyethyl methacrylate were added, initiator potassium persulfate or ammonium persulfate was added at 60°C, and the reaction was carried out at 85°C overnight to obtain Fe3O4@PS-OH; (4) Tosyl modification of the microsphere surface by nucleophilic substitution reaction: Fe3O4@PS-OH was added to acetonitrile solution, and under the action of triethylamine, an excess of p-toluenesulfonyl chloride in acetonitrile solution was added, and the reaction was carried out at 0°C for 2 h to obtain Fe3O4@PS-tosyl; (5) Preparation of Eu(III): Dissolve o-phenanthroline and dibenzoylmethane in an appropriate amount of ethanol, then add triethylamine, and finally add an ethanol solution of EuCl3·6H2O. Set the temperature to 60°C, stop the reaction after 2 hours, wash with anhydrous ethanol several times, and filter again to obtain the time-resolved fluorescent dye Eu(III); (6) The time-resolved fluorescent dye was embedded into the microspheres by the swelling method: an appropriate amount of surfactant was added to the Fe3O4@PS-tosyl emulsion, and ultrasonic dispersion was performed to prepare a microsphere emulsion; an appropriate amount of time-resolved fluorescent dye Eu(Ⅲ) was taken into an EP tube, and a small amount of dichloromethane was added to fully dissolve it to prepare a dye solution; the dye solution was poured into the microsphere emulsion, and the microspheres were placed in a constant temperature shaker with the speed set to 200 rpm, protected from light, and reacted at 30°C for three days to prepare Fe3O4@PS-tosyl@Eu(Ⅲ).

3. The method for preparing Tosyl-modified magnetic time-resolved fluorescent microspheres according to claim 2, characterized in that: In the step (1), the mass ratio of FeCl3·6H2O, CH3COONa, 1,6-hexanediamine and ethylene glycol is 1:1-10:1-5:30-60.

4. The method for preparing Tosyl-modified magnetic time-resolved fluorescent microspheres according to claim 3, characterized in that: In the step (2), the mass ratio of the surface-aminated Fe3O4, triethylamine and methacryloyl chloride is 1:1-3:0.5-1.

5. The method for preparing Tosyl-modified magnetic time-resolved fluorescent microspheres according to claim 4, characterized in that: In the step (3), the mass ratio of Fe3O4@ double bond to styrene is 1:8-20, and the mass ratio of styrene to hydroxyethyl methacrylate is 1:0.04-0.

2.

6. The method for preparing Tosyl-modified magnetic time-resolved fluorescent microspheres according to claim 5, characterized in that: In the step (4), the mass ratio of Fe3O4@PS-OH, triethylamine and p-toluenesulfonyl chloride is 1:1-3:0.5-1.

5.

7. The method for preparing Tosyl-modified magnetic time-resolved fluorescent microspheres according to claim 6, characterized in that: In the step (5), the molar ratio of EuCl3·6H2O, dibenzoylmethane and o-phenanthroline is 1:3-3.3:1-1.

1.

8. The method for preparing Tosyl-modified magnetic time-resolved fluorescent microspheres according to claim 7, characterized in that: In the step (6), the mass ratio of Fe3O4@PS-tosyl to Eu(III) is 1:0.3-1; the volume ratio of pure water to dichloromethane in the entire reaction system is 1:0.1-0.

5.

9. Tosyl-modified magnetic time-resolved fluorescent microspheres prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The particle size of the microspheres is 200-400nm.

10. Use of the Tosyl-modified magnetic time-resolved fluorescent microspheres according to claim 9 in immunofluorescence analysis.

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