Silicon-coated color fluorescent microsphere as well as mild preparation method and application thereof

Abstract

The invention relates to the technical field of immunochromatography, in particular to a silicon-coated color fluorescent microsphere and a mild preparation method and application thereof.The preparation method comprises the steps that firstly, time-resolved fluorescent dye is synthesized in an ethanol system, then oil-soluble dye, an ethanol water solution and a swelling agent dichloromethane are added, stirring is conducted at the room temperature to enable the dye to be dissolved, and then the silicon-coated color fluorescent microsphere is obtained; and adding ammonia water and a silane coupling agent tetraethyl orthosilicate, reacting overnight, supplementing the ammonia water, gradually adding a coupling agent 3-aminopropyltriethoxysilane, and washing with pure water for several times after the reaction is finished. The particle size range of the prepared color time-resolved fluorescent microspheres is 200-450 nm, the color time-resolved fluorescent microspheres are prepared in a green and mild mode through a one-step method, a surface active agent, a dispersing agent and a surface amino modified silicon dioxide matrix are not used, the high-luminous-intensity binuclear rare earth europium complex and the oil-soluble dye are embedded into the microspheres in the polymerization process, the dye is not prone to leakage, and the color time-resolved fluorescent microspheres are environmentally friendly and environmentally friendly. The method can be used for qualitative and quantitative dual immunodetection in the field of immunochromatography.

Description

Technical Field

[0001] The present invention relates to the field of immunochromatography technology, and specifically relates to a silicon-coated colored fluorescent microsphere and a mild preparation method and application thereof. Background Art

[0002] Immunochromatography is an analytical method developed at the end of the 20th century that combines immunoassay and chromatography. This method is specific, simple to operate, and rapid, and is widely used in important fields such as clinical diagnosis, environmental monitoring, and food safety. As a new type of immunoassay technology, time-resolved fluorescence immunochromatography is developed based on fluorescence immunochromatography. It is usually a surface carboxylated polystyrene microsphere with rare earth complexes embedded inside. It is coupled with antibodies through chemical binding, and the fluorescence intensity is used to achieve the purpose of quantitative analysis of the object to be detected.

[0003] Time-resolved fluorescent microspheres are the key raw materials for time-resolved immunochromatography. The traditional preparation method is mainly the swelling method. The preparation method is as follows: first, prepare surface carboxyl-coated polystyrene microspheres; second, generally select rare earth element compounds such as Eu2O3, EuCl3·6H2O, Yb2O3, etc., and react with diketone ligands and phenanthroline to prepare mononuclear rare earth ternary complexes, and then embed the time-resolved fluorescent dye and carboxylated polystyrene microspheres into the microspheres in the presence of swelling agents and surfactants.

[0004] CN116218516A discloses a time-resolved fluorescent microsphere and a preparation method and application thereof, which specifically comprises the following steps: (1) polymerizing styrene and carboxyl functional monomers by soap-free emulsion polymerization to obtain surface carboxyl polystyrene microspheres; (2) synthesizing Eu(L1)3(L2)1 rare earth mononuclear ternary complex using europium chloride, 1,3-diphenyl-1,3-propanedione and 4,7-diphenyl-1,10-phenanthroline as raw materials; and (3) embedding the rare earth complex into the interior of carboxyl polystyrene microspheres by swelling method to obtain time-resolved fluorescent microspheres. This method has complicated steps. It is necessary to first prepare surface carboxyl polystyrene microspheres by emulsion polymerization. The polymerization temperature is high and the toxicity is high. Polystyrene is highly hydrophobic and easy to agglomerate. The luminescence intensity of the mononuclear rare earth ternary complex is low. The time-resolved fluorescent microspheres prepared by the swelling method are prone to leakage of the internal dye of the microspheres. In addition, in immunochromatography experiments and applications, the white or light yellow color of the microspheres themselves makes it difficult for users to observe the test line and quality control line with the naked eye, and they need to use instruments to observe and read the values.

[0005] CN115466277A discloses a carboxyl-functionalized time-resolved fluorescent microsphere and a preparation method thereof, comprising the following steps: under acidic conditions, reacting europium salt, lanthanide metal salt, β-diketone ligand, synergistic ligand and bridging ligand in an organic solvent to obtain a binuclear rare earth multi-component complex Eu(Ln)(L1)4(L2)2(L3)1; then subjecting the binuclear rare earth multi-component complex, styrene monomer, acrylic monomer, initiator, polyvinyl pyrrolidone and emulsifier to a dispersed polymerization reaction in a solvent to obtain a carboxyl-functionalized time-resolved fluorescent microsphere. Although this method can embed the dye directly into the microspheres during the polymerization stage, and the luminescence intensity of the binuclear rare earth five-membered complex with large steric hindrance is stronger, the preparation process of this method is complicated. The polystyrene material is prepared by dispersion polymerization, which is highly hydrophobic and has a high polymerization reaction temperature. The emulsifier and dispersant polyvinyl pyrrolidone used in the preparation of the polymer is difficult to remove completely. The residual emulsifier and polyvinyl pyrrolidone in the microspheres will affect the coupling efficiency of the microspheres and antibodies and affect the detection sensitivity. Moreover, in immunochromatography experiments and applications, the white or light yellow color of the microspheres themselves makes it difficult for users to observe the test line and quality control line with the naked eye, and they need to use instruments to observe and read the values.

[0006] CN111218270A discloses a method for preparing a modified time-resolved fluorescent microsphere, specifically: (1) adding ultrapure water and nitrogen to a reactor, stirring while heating, adding distilled styrene, mixing and heating together, and then adding an initiator to prepare white polystyrene latex microspheres; (2) weighing a solvent dye, 2-thenoyltrifluoroacetone, o-phenanthroline hydrochloride, europium chloride and an electron mediator and dissolving them in petroleum ether to form a mixture; slowly adding the above mixture dropwise to the white latex microsphere solution prepared in step (1) and stirring evenly to obtain dyed time-resolved fluorescent microspheres. The microspheres prepared by this method have bright colors and do not affect the intensity of their own fluorescence. Users can intuitively find abnormal test strips with the naked eye. However, the microspheres prepared by this method are actually formed by encapsulating the mononuclear rare earth ternary complex and the solvent dye into the microspheres by a swelling method, which has complicated steps, low luminescence intensity of the mononuclear rare earth ternary complex, and easy leakage of the dye.

[0007] CN117186872A discloses a one-step method for preparing colored time-resolved fluorescent microspheres, specifically: nitrogen is passed through a mixed solution of ethanol and water in a three-necked flask to replace air, a dispersant and europium chloride are added to the system and stirred to dissolve, an alcohol solution of rare earth ligand 1,10-phenanthroline and ethyl 2-aminobenzoate is slowly added, the pH of the solution is adjusted to 6-8 with NcOH, styrene and functional monomers dissolved with initiator and dye are added to the reaction system, and the reaction is carried out at 60-80°C for 6-16h to obtain monodispersed colored time-resolved fluorescent microspheres. The colored time-resolved fluorescent microspheres prepared by this method have simple steps and are not suitable for organic solvents such as swelling agents, but the reaction temperature is high for synthesizing polystyrene microspheres, and a large amount of dispersant polyvinyl pyrrolidone is used, which is difficult to remove cleanly, affecting the coupling efficiency of the microspheres with antibodies, and the luminescence intensity of the mononuclear rare earth ternary complex is low.

[0008] CN106243835A discloses a water-based rare earth complex composite microsphere fluorescent ink, wherein rare earth complex composite microsphere, acrylate aqueous solution, coupling agent, dispersant, defoamer, cosolvent, film-forming aid, thickener, and filler are prepared by preparing mixture A → preparing powder → mixing and grinding → filtering and stirring to prepare ink with narrow luminescence spectrum, high color purity, good light stability, and green environmental protection. The rare earth complex composite microsphere contains SiO2 colloid of mononuclear rare earth ternary complex Eu(DBM)3Phen, with a particle size of about 20nm, which is not suitable for the field of immunochromatography; moreover, the method first synthesizes the mononuclear rare earth ternary complex, which has low luminescence intensity, and is embedded in the interior of the silica microsphere in a system with a large amount of toxic solvent acetone (about 2-4 times that of ethanol), and finally obtains dry rare earth complex composite microspheres.

[0009] In summary, the current technology for preparing color time-resolved fluorescent microspheres has the following shortcomings: (1) The steps are cumbersome; (2) Most of them are prepared by the swelling method, the dye is easy to leak, and the microsphere structure is easily destroyed; (3) The polystyrene matrix is ​​highly hydrophobic and easy to agglomerate; (4) The polymerization temperature is high; (5) The toxicity is high; (6) The surfactants and dispersants are not easy to remove completely; (7) Most of them are mononuclear rare earth ternary complex fluorescent dyes with low luminescence intensity; (8) The microspheres are mostly white or light yellow, making it difficult for users to observe the test line and quality control line with the naked eye, and they need to use instruments to observe and read the values.

[0010] Based on this, there is an urgent need in the art to develop a method for preparing colored time-resolved fluorescent microspheres with simple reaction steps, green and mild conditions, no surfactants and dispersants, surface amino-modified silica matrix, internally embedded binuclear rare earth complexes with high luminescence intensity and oil-soluble dyes, for use in immunochromatography immunoassays. Summary of the invention

[0011] The purpose of the present invention is to provide a silicon-encapsulated colorful fluorescent microsphere and a mild preparation method and application thereof.

[0012] To achieve the above object, the present invention provides the following technical solutions:

[0013] The invention discloses a mild preparation method of silicon-coated colored time-resolved fluorescent microspheres. The method comprises the following steps: firstly, synthesizing a time-resolved fluorescent dye in an ethanol system, then adding an oil-soluble dye, an ethanol aqueous solution and a swelling agent, dichloromethane, stirring at room temperature to dissolve the dye, then adding ammonia water and a silane coupling agent, tetraethyl orthosilicate, reacting overnight, adding ammonia water, and gradually adding a coupling agent, 3-aminopropyltriethoxysilane, and washing with pure water for several times after the reaction is completed.

[0014] Further, the method specifically includes the following steps:

[0015] (1) Synthesis of binuclear time-resolved fluorescent dye in ethanol system: Weigh EuCl3·6H2O and dibenzoylmethane (DBM) and dissolve them in an appropriate amount of ethanol, then add triethylamine and stir evenly, finally add 1,10-phenanthroline (phen) and 1,4-naphthalene dicarboxylic acid (Nc), and react at room temperature for 2 h;

[0016] (2) Synthesis of silica-encapsulated colored time-resolved amino-modified nanospheres by sol-gel method: After stirring at room temperature, weigh an appropriate amount of oil-soluble dye into a reaction bottle, add an appropriate amount of ethanol aqueous solution and dichloromethane to dissolve the dye; quickly add an appropriate amount of ammonia water, stir evenly, then add coupling agent tetraethyl orthosilicate dropwise, and react overnight; then add an appropriate amount of ammonia water, stir evenly, add coupling agent 3-aminopropyltriethoxysilane dropwise three times, and react at room temperature for 2 days; after the reaction, wash with pure water three times and store in pure water.

[0017] Wherein, in the step (1), the first ligand is dibenzoylmethane, the second ligand is 1,10-phenanthroline, the bridging ligand is a conjugated dicarboxylic acid, and the conjugated dicarboxylic acid is 1,4-naphthalene dicarboxylic acid.

[0018] Among them, the molar ratio of EuCl3·6H2O to the first ligand is 1:2, the molar ratio of EuCl3·6H2O to the second ligand is 1:1, and the molar ratio of EuCl3·6H2O to the bridging ligand is 1:0.5.

[0019] Wherein, in the step (2), tetraethyl orthosilicate is used as the first coupling agent, and the time-resolved fluorescent dye and the oil-soluble dye are embedded in the silica sphere during the polymerization process, and then 3-aminopropyltriethoxysilane is used as the second coupling agent to modify the surface of the microsphere with amino groups. The volume ratio of ethanol aqueous solution to tetraethyl orthosilicate is 1:0.05-0.1, and the volume ratio of ethanol aqueous solution to 3-aminopropyltriethoxysilane is 1:0.006-0.015. The volume ratio of ammonia water to all coupling agents is 1:0.8-1.2, and the mass ratio of all coupling agents to oil-soluble dyes is 1:0.2-1.

[0020] The silica-coated colored time-resolved fluorescent microspheres prepared by the mild preparation method of the present invention can be used for qualitative and quantitative dual immunoassay in the field of immunochromatography.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The color time-resolved fluorescent microspheres prepared by the present invention have a particle size range of 200-450nm, are prepared by a one-step method, have mild reaction conditions and low toxicity, do not use surfactants and dispersants, have a surface amino-modified silica matrix, have strong hydrophilicity, and have good microsphere dispersibility, and embed high-luminescence intensity binuclear rare earth europium complexes and oil-soluble dyes into the microspheres during the polymerization process, and the dyes are not easy to leak, and can be used in the field of immunochromatography, can achieve high-sensitivity quantitative detection of the object to be detected, and can also visually see the detection line and quality control line through the naked eye, so as to quickly find abnormal test strips. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The present invention is a schematic diagram of the reaction process for preparing silicon-coated colored time-resolved fluorescent microspheres.

[0024] Figure 2 The figure is a chemical structure diagram of the binuclear rare earth europium complex of the present invention.

[0025] Figure 3 This is a photograph of the color time-resolved fluorescent microspheres prepared in Example 1-7 coupled with influenza A virus antibodies to detect influenza A virus antigen (diluted 500 times). DETAILED DESCRIPTION

[0026] 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.

[0027] Example 1

[0028] like Figure 1As shown, 2mmol EuCl3·6H2O and 4mmol dibenzoylmethane (DBM) were weighed into a reaction bottle, and 40mL ethanol was added to stir and dissolve. Then 6mmol triethylamine was added and stirred evenly, and finally 2mmol 1,10-phenanthroline (phen) and 1mmol 1,4-naphthalene dicarboxylic acid (Nc) were added and stirred at room temperature for 2h.

[0029] Weigh 2g of solvent red into a reaction bottle, add 40mL of anhydrous ethanol, 20mL of pure water and 4mL of dichloromethane, and stir for 30min to fully dissolve the dye. Quickly add 5mL of ammonia water, stir evenly, then add 5mL of coupling agent tetraethyl orthosilicate dropwise, and react overnight. Add 0.8mL of ammonia water, stir evenly, add 800μL of coupling agent 3-aminopropyltriethoxysilane dropwise three times, and react at room temperature for 2 days. After the reaction is completed, wash with pure water 3 times and store in pure water.

[0030] Example 2

[0031] like Figure 1 As shown, 2mmol EuCl3·6H2O and 4mmol dibenzoylmethane (DBM) were weighed into a reaction bottle, and 40mL ethanol was added to stir and dissolve. Then 6mmol triethylamine was added and stirred evenly, and finally 2mmol 1,10-phenanthroline (phen) and 1mmol 1,4-naphthalene dicarboxylic acid (Nc) were added and stirred at room temperature for 2h.

[0032] Weigh 2g of solvent red into a reaction bottle, add 40mL of anhydrous ethanol, 20mL of pure water and 4mL of dichloromethane, and stir for 30min to fully dissolve the dye. Quickly add 6mL of ammonia water, stir evenly, then add 6mL of coupling agent tetraethyl orthosilicate dropwise, and react overnight. Add 0.8mL of ammonia water, stir evenly, add 800μL of coupling agent 3-aminopropyltriethoxysilane dropwise three times, and react at room temperature for 2 days. After the reaction is completed, wash with pure water 3 times and store in pure water.

[0033] Example 3

[0034] like Figure 1 As shown, 2mmol EuCl3·6H2O and 4mmol dibenzoylmethane (DBM) were weighed into a reaction bottle, and 40mL ethanol was added to stir and dissolve. Then 6mmol triethylamine was added and stirred evenly, and finally 2mmol 1,10-phenanthroline (phen) and 1mmol 1,4-naphthalene dicarboxylic acid (Nc) were added and stirred at room temperature for 2h.

[0035] Weigh 2g of solvent red into a reaction bottle, add 40mL of anhydrous ethanol, 20mL of pure water and 4mL of dichloromethane, and stir for 30min to fully dissolve the dye. Quickly add 7mL of ammonia water, stir evenly, then add 7mL of coupling agent tetraethyl orthosilicate dropwise, and react overnight. Add 0.8mL of ammonia water, stir evenly, add 800μL of coupling agent 3-aminopropyltriethoxysilane dropwise three times, and react at room temperature for 2 days. After the reaction, wash with pure water 3 times and store in pure water.

[0036] Example 4

[0037] like Figure 1 As shown, 2mmol EuCl3·6H2O and 4mmol dibenzoylmethane (DBM) were weighed into a reaction bottle, and 40mL ethanol was added to stir and dissolve. Then 6mmol triethylamine was added and stirred evenly, and finally 2mmol 1,10-phenanthroline (phen) and 1mmol 1,4-naphthalene dicarboxylic acid (Nc) were added and stirred at room temperature for 2h.

[0038] Weigh 2g of solvent red into a reaction bottle, add 40mL of anhydrous ethanol, 20mL of pure water and 4mL of dichloromethane, and stir for 30min to fully dissolve the dye. Quickly add 8mL of ammonia water, stir evenly, then add 8mL of coupling agent tetraethyl orthosilicate dropwise, and react overnight. Add 0.8mL of ammonia water, stir evenly, add 800μL of coupling agent 3-aminopropyltriethoxysilane dropwise three times, and react at room temperature for 2 days. After the reaction, wash with pure water 3 times and store in pure water.

[0039] Example 5

[0040] Weigh 2mmol EuCl3·6H2O and 4mmol dibenzoylmethane (DBM) in a reaction bottle, add 40mL ethanol and stir to dissolve. Then add 6mmol triethylamine and stir evenly, and finally add 2mmol 1,10-phenanthroline (phen) and 1mmol 1,4-naphthalene dicarboxylic acid (Nc), and stir at room temperature for 2h.

[0041] Weigh 2g of solvent red into a reaction bottle, add 40mL of anhydrous ethanol, 20mL of pure water and 4mL of dichloromethane, and stir for 30min to fully dissolve the dye. Quickly add 9mL of ammonia water, stir evenly, then add 9mL of coupling agent tetraethyl orthosilicate dropwise, and react overnight. Add 0.8mL of ammonia water, stir evenly, add 800μL of coupling agent 3-aminopropyltriethoxysilane dropwise three times, and react at room temperature for 2 days. After the reaction, wash with pure water 3 times and store in pure water.

[0042] Example 6

[0043] Weigh 2mmol EuCl3·6H2O and 4mmol dibenzoylmethane (DBM) in a reaction bottle, add 40mL ethanol and stir to dissolve. Then add 6mmol triethylamine and stir evenly, and finally add 2mmol 1,10-phenanthroline (phen) and 1mmol 1,4-naphthalene dicarboxylic acid (Nc), and stir at room temperature for 2h.

[0044] Weigh 5g of solvent red into a reaction bottle, add 40mL of anhydrous ethanol, 20mL of pure water and 4mL of dichloromethane, and stir for 30min to fully dissolve the dye. Quickly add 10mL of ammonia water, stir evenly, then add 10mL of coupling agent tetraethyl orthosilicate dropwise, and react overnight. Add 0.8mL of ammonia water, stir evenly, add 800μL of coupling agent 3-aminopropyltriethoxysilane dropwise three times, and react at room temperature for 2 days. After the reaction is completed, wash with pure water 3 times and store in pure water.

[0045] Example 7

[0046] Weigh 2mmol EuCl3·6H2O and 4mmol dibenzoylmethane (DBM) in a reaction bottle, add 40mL ethanol and stir to dissolve. Then add 6mmol triethylamine and stir evenly, and finally add 2mmol 1,10-phenanthroline (phen) and 1mmol 1,4-naphthalene dicarboxylic acid (Nc), and stir at room temperature for 2h.

[0047] Weigh 5g of solvent red into a reaction bottle, add 40mL of anhydrous ethanol, 20mL of pure water and 4mL of dichloromethane, and stir for 30min to fully dissolve the dye. Quickly add 10mL of ammonia water, stir evenly, then add 10mL of coupling agent tetraethyl orthosilicate dropwise, and react overnight. Add 1mL of ammonia water, stir evenly, add 1mL of coupling agent 3-aminopropyltriethoxysilane dropwise three times, and react at room temperature for 2 days. After the reaction is completed, wash with pure water 3 times and store in pure water.

[0048] Figure 2 The chemical structure diagram of the binuclear rare earth europium complex prepared in the above example. Table 1 shows the particle size and dispersion index of the silicon-encapsulated color time-resolved fluorescent microspheres in Examples 1-7, with a dispersion index PDI < 0.05, uniform particle size, and good monodispersity.

[0049] Table 1 Microsphere particle size and dispersion index of Examples 1-7

[0050]

[0051] Application example: Application of silica-coated colored time-resolved fluorescent microspheres in immunochromatography

[0052] Taking the novel coronavirus antibody as an example, the following steps are included:

[0053] (1) Dilution of microspheres

[0054] Take 25 μL of microspheres (solid content 4%) in an EP tube, add 975 μL of labeling buffer (0.01 M PB, pH 7.0), and vortex to mix;

[0055] (2) Activation of microspheres

[0056] Add 25 μL of glutaraldehyde, mix and resuspend for 1 h;

[0057] (3) Cleaning to remove residual glutaraldehyde

[0058] The activated microspheres were: 1. centrifuged at 15000rpm for 15min, first time; 2. the supernatant was removed and the microspheres were resuspended in 1mL of labeling buffer; 3. the supernatant was removed and the microspheres were resuspended in 0.2mL of labeling buffer for later use;

[0059] (4) Conjugated Antibodies

[0060] Take 0.04 mg of antibody in a 2 mL centrifuge tube, add the activated microspheres in (3), mix quickly, and incubate at room temperature for 2 h;

[0061] (5) Removal of unbound antibodies

[0062] The purpose of this step is to remove free antibodies that are not bound to the microspheres by high-speed centrifugation. The specific process is as follows: 1. 15000rpm, centrifugation for 15min, the first time; 2. Remove the supernatant and resuspend the microspheres with 1mL of diluent (50mM PBS, pH 7.4 or 50mMTris, pH 8.0); 3. 15000rpm, centrifugation for 15min, the second time; 4. Remove the supernatant and resuspend the microspheres with 1mL of diluent, which is the antibody-microsphere labeled complex, and place it at 4℃ for use. If it is stored for a long time, add a final concentration of 0.2% BSA and 0.02% NaN3 solution; 5. If necessary, wash several times.

[0063] Table 2 shows the detection results of the new coronavirus antigen (diluted 500 times) after the silicon-coated color time-resolved fluorescent microspheres of Example 1-7 were coupled to the new coronavirus antibody. The excitation wavelength was 365nm and the emission wavelength was 610nm. From the fluorescence intensity analysis, the new coronavirus antigen diluted 500 times can be detected.

[0064] Table 2 Example 1-7 Microspheres coupled with novel coronavirus antibodies to detect novel coronavirus antigens (diluted 500 times)

[0065] serial number Microspheres Excitation wavelength (nm) Emission wavelength (nm) Fluorescence intensity 1 Example 1 365 610 64875.3 2 Example 2 365 610 72366.1 3 Example 3 365 610 74263.8 4 Example 4 365 610 78244.3 5 Example 5 365 610 75367.8 6 Example 6 365 610 82677.5 7 Example 7 365 610 86464.2

[0066] Figure 3 1 is the corresponding test card, which corresponds to the photos of the color time-resolved fluorescent microspheres coupled to influenza A virus antibodies to detect influenza A virus antigen (diluted 500 times) of Examples 1-7 from left to right. The quality control line and the test line can be seen by naked eyes.

[0067] 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 gentle preparation method of silicon-encapsulated colored time-resolved fluorescent microspheres, characterized in that: First, a time-resolved fluorescent dye is synthesized in an ethanol system. Then, an oil-soluble dye, an ethanol aqueous solution and a swelling agent, dichloromethane, are added. The dye is dissolved by stirring at room temperature. Ammonia water and a silane coupling agent, tetraethyl orthosilicate, are added. The reaction is allowed to proceed overnight, and then additional ammonia water is added. The coupling agent 3-aminopropyltriethoxysilane is added gradually. After the reaction is completed, the solution is washed with pure water several times.

2. The gentle preparation method of silicon-encapsulated colored time-resolved fluorescent microspheres according to claim 1, characterized in that: The following steps are involved: (1) Weigh EuCl3·6H2O and dibenzoylmethane and dissolve them in an appropriate amount of ethanol, then add triethylamine and stir evenly, and finally add 1,10-phenanthroline and 1,4-naphthalene dicarboxylic acid; (2) After stirring at room temperature, weigh an appropriate amount of oil-soluble dye into a reaction bottle, add an appropriate amount of ethanol aqueous solution and dichloromethane to dissolve the dye; quickly add an appropriate amount of ammonia water, stir evenly, then add coupling agent tetraethyl orthosilicate dropwise, and react overnight; then add an appropriate amount of ammonia water, stir evenly, and add coupling agent 3-aminopropyltriethoxysilane dropwise three times, and react at room temperature; after the reaction is completed, wash with pure water and store in pure water.

3. The gentle preparation method of silicon-encapsulated colored time-resolved fluorescent microspheres according to claim 2, characterized in that: In the step (1), the first ligand is dibenzoylmethane, the second ligand is 1,10-phenanthroline, and the bridging ligand is a conjugated dicarboxylic acid.

4. The gentle preparation method of silicon-encapsulated colored time-resolved fluorescent microspheres according to claim 3, characterized in that: The conjugated dicarboxylic acid is 1,4-naphthalene dicarboxylic acid.

5. The gentle preparation method of silicon-encapsulated colored time-resolved fluorescent microspheres according to claim 4, characterized in that: The molar ratio of EuCl3·6H2O to the first ligand is 1:2, the molar ratio of EuCl3·6H2O to the second ligand is 1:1, and the molar ratio of EuCl3·6H2O to the bridging ligand is 1:0.

5.

6. The gentle preparation method of silicon-encapsulated colored time-resolved fluorescent microspheres according to claim 2, characterized in that: In the step (2), tetraethyl orthosilicate is used as the first coupling agent, and the time-resolved fluorescent dye and the oil-soluble dye are embedded in the silica spheres during the polymerization process, and then 3-aminopropyltriethoxysilane is used as the second coupling agent to modify the surface of the microspheres with amino groups.

7. The gentle preparation method of silicon-encapsulated colored time-resolved fluorescent microspheres according to claim 6, characterized in that: In the step (2), the volume ratio of the ethanol aqueous solution to tetraethyl orthosilicate is 1:0.05-0.1, and the volume ratio of the ethanol aqueous solution to 3-aminopropyltriethoxysilane is 1:0.006-0.

015.

8. The gentle preparation method of silicon-encapsulated colored time-resolved fluorescent microspheres according to claim 7, characterized in that: In the step (2), the volume ratio of ammonia water to all coupling agents is 1:0.8-1.2, and the mass ratio of all coupling agents to oil-soluble dyes is 1:0.2-1.

9. Silicon-encapsulated colored time-resolved fluorescent microspheres prepared by the mild preparation method according to any one of claims 1 to 8.

10. Use of the silica-coated colored time-resolved fluorescent microspheres according to claim 9 in qualitative and quantitative dual immunoassays in the field of immunochromatography.

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