Silicon-coated colored fluorescent microspheres and their mild preparation method and application

Abstract

The present invention relates to the field of immunochromatography technology, in particular to a kind of silicon-coated colored fluorescent microspheres and their mild preparation method and application, the preparation method is as follows: 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, ammonia water is added, and a coupling agent, 3-aminopropyltriethoxysilane, is added successively, and the reaction is washed with pure water several times. The colored time-resolved fluorescent microspheres prepared by the present invention have a particle size range of 200-450nm, are prepared in a green and mild one-step method, do not use surfactants and dispersants, and have a surface amino-modified silica matrix. During the polymerization process, a high-luminescence intensity binuclear rare earth europium complex and an oil-soluble dye are embedded in the microspheres, and the dye is not easy to leak. The microspheres can be used for qualitative and quantitative dual immunoassays 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, a method developed in the late 20th century that combines immunoassays with chromatography, is a method characterized by specificity, ease of use, and rapidity. It is widely used in important fields such as clinical diagnosis, environmental monitoring, and food safety. Time-resolved fluorescence immunochromatography, a novel immunoassay technique, has evolved from this approach. It typically involves surface-carboxylated polystyrene microspheres embedded with rare earth complexes. These microspheres are chemically coupled to antibodies, and fluorescence intensity is used to quantitatively analyze the target substance.

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

[0004] CN116218516A discloses a time-resolved fluorescent microsphere and its preparation method and application, specifically: (1) styrene and carboxyl functional monomers are polymerized by soap-free emulsion polymerization to obtain surface carboxyl polystyrene microspheres; (2) Eu(L1)3(L2)1 rare earth mononuclear ternary complex is synthesized using europium chloride, 1,3-diphenyl-1,3-propanedione and 4,7-diphenyl-1,10-phenanthroline as raw materials; (3) the rare earth complex is embedded in the interior of the carboxyl polystyrene microspheres by a swelling method to obtain time-resolved fluorescent microspheres. This method is cumbersome and requires the preparation of surface carboxyl-coated polystyrene microspheres by emulsion polymerization. The polymerization temperature is high and the toxicity is high. Polystyrene is highly hydrophobic and easily aggregated. 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 the user needs to use an instrument to observe and read the value.

[0005] CN115466277A discloses a carboxyl-functionalized time-resolved fluorescent microsphere and a preparation method thereof, comprising the following steps: under acidic conditions, reacting a europium salt, a lanthanide metal salt, a β-diketone ligand, a cooperative ligand and a 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, a styrene monomer, an acrylic monomer, an initiator, polyvinyl pyrrolidone and an emulsifier to a dispersion polymerization reaction in the solvent to obtain the carboxyl-functionalized time-resolved fluorescent microsphere. Although this method can directly embed the dye inside 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 modified time-resolved fluorescent microspheres, specifically: (1) adding ultrapure water and nitrogen to a reactor, heating and stirring, adding distilled styrene, mixing and heating, 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 visually find abnormal test strips with the naked eye. However, the microspheres prepared by this method are actually prepared by encapsulating a mononuclear rare earth ternary complex and a solvent dye into the microspheres through a swelling method. The steps are cumbersome, the luminescence intensity of the mononuclear rare earth ternary complex is low, and the dye is easy to leak.

[0007] CN117186872A discloses a one-step method for preparing colored, time-resolved fluorescent microspheres. Specifically, nitrogen is passed through a mixture of ethanol and water in a three-necked flask to displace the air. A dispersant and europium chloride are then added to the system, stirred and dissolved. An alcoholic solution of the rare earth ligand 1,10-phenanthroline and ethyl 2-methylaminobenzoate is slowly added. The pH of the solution is adjusted to 6-8 with N₂OH. Styrene and a functional monomer, containing an initiator and dye, are then added to the reaction system. The reaction is carried out at 60-80°C for 6-16 hours to produce monodisperse colored, time-resolved fluorescent microspheres. This method is simple to prepare colored, time-resolved fluorescent microspheres and eliminates the use of organic solvents such as swelling agents. However, the synthesis of polystyrene microspheres involves high reaction temperatures and the use of a large amount of the dispersant, polyvinylpyrrolidone, which is difficult to remove, affecting the coupling efficiency between the microspheres and the antibody. Consequently, 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. The rare earth complex composite microspheres, an acrylate aqueous solution, a coupling agent, a dispersant, a defoaming agent, a cosolvent, a film-forming aid, a thickener, and a filler are prepared by preparing a mixture A, preparing a powder, mixing and grinding, and then filtering and stirring. The ink has a narrow luminescence spectrum, high color purity, good photostability, and is environmentally friendly. The rare earth complex composite microspheres contain SiO2 colloids containing the mononuclear rare earth ternary complex Eu(DBM)3Phen, with a particle size of approximately 20 nm, making them unsuitable for immunochromatography. Furthermore, the method first synthesizes the mononuclear rare earth ternary complex, which has low luminescence intensity, and then embeds it into silica microspheres in a system with a large amount of the toxic solvent acetone (approximately 2-4 times that of ethanol), ultimately yielding dry rare earth complex composite microspheres.

[0009] Based on the above analysis, 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 this field to develop a method for preparing color time-resolved fluorescent microspheres with simple reaction steps, green and mild conditions, no surfactants and dispersants, amino-modified silica matrix on the surface, and internally embedded binuclear rare earth complexes and oil-soluble dyes with high luminescence intensity for immunoassays in the field of immunochromatography. 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] A mild preparation method for silicon-coated colored time-resolved fluorescent microspheres comprises the following steps: first, 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 more ammonia water, and gradually adding a coupling agent, 3-aminopropyltriethoxysilane; and washing with pure water several times after the reaction is completed.

[0014] Furthermore, 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-naphthalenedicarboxylic acid (Nc), and react at room temperature for 2 h;

[0016] (2) Synthesis of silicon-coated 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 the coupling agent tetraethyl orthosilicate dropwise, and react overnight; then add an appropriate amount of ammonia water, stir evenly, add the 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] 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] In step (2), tetraethyl orthosilicate is used as the first coupling agent, and during the polymerization process, the time-resolved fluorescent dye and the oil-soluble dye are embedded in the silica spheres. 3-aminopropyltriethoxysilane is then used as the second coupling agent to modify the surface of the microspheres with amino groups. 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. 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 the oil-soluble dye 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 immunoassays 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 and are prepared by a one-step method with mild reaction conditions and low toxicity. No surfactants or dispersants are used. The surface amino-modified silica matrix is highly hydrophilic and the microspheres have good dispersibility. The polymerization process embeds a high-luminescence intensity binuclear rare earth europium complex and an oil-soluble dye into the microspheres, making the dye unlikely to leak. They can be used in the field of immunochromatography, achieving high-sensitivity quantitative detection of the object to be detected, and the test line and quality control line can be visually seen by the naked eye, allowing abnormal test strips to be quickly identified. BRIEF DESCRIPTION OF THE DRAWINGS

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

[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 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.

[0027] Example 1

[0028] like Figure 1As shown, 2 mmol of EuCl₃·6H₂O and 4 mmol of dibenzoylmethane (DBM) were weighed into a reaction flask and dissolved with 40 mL of ethanol. Then, 6 mmol of triethylamine was added and stirred thoroughly. Finally, 2 mmol of 1,10-phenanthroline (phen) and 1 mmol of 1,4-naphthalenedicarboxylic acid (Nc) were added and stirred at room temperature for 2 h.

[0029] Weigh 2g of Solvent Red into a reaction flask, add 40mL of anhydrous ethanol, 20mL of pure water, and 4mL of dichloromethane, and stir for 30 minutes to fully dissolve the dye. Quickly add 5mL of aqueous ammonia and stir thoroughly. Then, dropwise add 5mL of the coupling agent, tetraethyl orthosilicate, and allow to react overnight. Add another 0.8mL of aqueous ammonia and stir thoroughly. Then, dropwise add 800μL of the coupling agent, 3-aminopropyltriethoxysilane, in three portions. Let react at room temperature for two days. After the reaction, wash the mixture three times with pure water and store in pure water.

[0030] Example 2

[0031] like Figure 1 As shown, 2 mmol of EuCl₃·6H₂O and 4 mmol of dibenzoylmethane (DBM) were weighed into a reaction flask and dissolved with 40 mL of ethanol. Then, 6 mmol of triethylamine was added and stirred thoroughly. Finally, 2 mmol of 1,10-phenanthroline (phen) and 1 mmol of 1,4-naphthalenedicarboxylic acid (Nc) were added and stirred at room temperature for 2 h.

[0032] Weigh 2g of Solvent Red into a reaction flask, add 40mL of anhydrous ethanol, 20mL of pure water, and 4mL of dichloromethane, and stir for 30 minutes to fully dissolve the dye. Quickly add 6mL of aqueous ammonia and stir thoroughly. Then, dropwise add 6mL of tetraethyl orthosilicate (a coupling agent) and allow to react overnight. Add another 0.8mL of aqueous ammonia and stir thoroughly. Then, add 800μL of 3-aminopropyltriethoxysilane (a coupling agent) dropwise in three portions. Let react at room temperature for 2 days. After the reaction, wash the mixture three times with pure water and store in pure water.

[0033] Example 3

[0034] like Figure 1 As shown, 2 mmol of EuCl₃·6H₂O and 4 mmol of dibenzoylmethane (DBM) were weighed into a reaction flask and dissolved with 40 mL of ethanol. Then, 6 mmol of triethylamine was added and stirred thoroughly. Finally, 2 mmol of 1,10-phenanthroline (phen) and 1 mmol of 1,4-naphthalenedicarboxylic acid (Nc) were added and stirred at room temperature for 2 h.

[0035] Weigh 2g of Solvent Red into a reaction flask, add 40mL of anhydrous ethanol, 20mL of pure water, and 4mL of dichloromethane, and stir for 30 minutes to fully dissolve the dye. Quickly add 7mL of aqueous ammonia and stir thoroughly. Then, dropwise add 7mL of the coupling agent, tetraethyl orthosilicate, and allow to react overnight. Add an additional 0.8mL of aqueous ammonia and stir thoroughly. Then, add 800μL of the coupling agent, 3-aminopropyltriethoxysilane, dropwise in three portions. Let react at room temperature for two days. After the reaction, wash the mixture three times with pure water and store in pure water.

[0036] Example 4

[0037] like Figure 1 As shown, 2 mmol of EuCl₃·6H₂O and 4 mmol of dibenzoylmethane (DBM) were weighed into a reaction flask and dissolved with 40 mL of ethanol. Then, 6 mmol of triethylamine was added and stirred thoroughly. Finally, 2 mmol of 1,10-phenanthroline (phen) and 1 mmol of 1,4-naphthalenedicarboxylic acid (Nc) were added and stirred at room temperature for 2 h.

[0038] Weigh 2g of Solvent Red into a reaction flask, add 40mL of anhydrous ethanol, 20mL of pure water, and 4mL of dichloromethane, and stir for 30 minutes to fully dissolve the dye. Quickly add 8mL of aqueous ammonia and stir thoroughly. Then, dropwise add 8mL of tetraethyl orthosilicate (a coupling agent) and allow to react overnight. Add another 0.8mL of aqueous ammonia and stir thoroughly. Then, dropwise add 800μL of 3-aminopropyltriethoxysilane (a coupling agent) in three portions. Let react at room temperature for 2 days. After the reaction, wash the mixture three times with pure water and store in pure water.

[0039] Example 5

[0040] Weigh 2 mmol of EuCl₃·6H₂O and 4 mmol of dibenzoylmethane (DBM) into a reaction flask and add 40 mL of ethanol to dissolve. Then, add 6 mmol of triethylamine and stir until evenly mixed. Finally, add 2 mmol of 1,10-phenanthroline (phen) and 1 mmol of 1,4-naphthalenedicarboxylic acid (Nc) and stir at room temperature for 2 hours.

[0041] Weigh 2g of Solvent Red into a reaction flask, add 40mL of anhydrous ethanol, 20mL of pure water, and 4mL of dichloromethane, and stir for 30 minutes to fully dissolve the dye. Quickly add 9mL of aqueous ammonia and stir thoroughly. Then, dropwise add 9mL of the coupling agent, tetraethyl orthosilicate, and allow to react overnight. Add an additional 0.8mL of aqueous ammonia and stir thoroughly. Then, add 800μL of the coupling agent, 3-aminopropyltriethoxysilane, dropwise in three portions. Let react at room temperature for two days. After the reaction, wash the mixture three times with pure water and store in pure water.

[0042] Example 6

[0043] Weigh 2 mmol of EuCl₃·6H₂O and 4 mmol of dibenzoylmethane (DBM) into a reaction flask and add 40 mL of ethanol to dissolve. Then, add 6 mmol of triethylamine and stir until evenly mixed. Finally, add 2 mmol of 1,10-phenanthroline (phen) and 1 mmol of 1,4-naphthalenedicarboxylic acid (Nc) and stir at room temperature for 2 hours.

[0044] Weigh 5g of Solvent Red into a reaction flask, add 40mL of anhydrous ethanol, 20mL of pure water, and 4mL of dichloromethane, and stir for 30 minutes to fully dissolve the dye. Quickly add 10mL of aqueous ammonia and stir thoroughly. Then, dropwise add 10mL of tetraethyl orthosilicate (a coupling agent) and allow to react overnight. Add another 0.8mL of aqueous ammonia and stir thoroughly. Then, dropwise add 800μL of 3-aminopropyltriethoxysilane (a coupling agent) in three portions. Let react at room temperature for 2 days. After the reaction, wash the mixture three times with pure water and store in pure water.

[0045] Example 7

[0046] Weigh 2 mmol of EuCl₃·6H₂O and 4 mmol of dibenzoylmethane (DBM) into a reaction flask and add 40 mL of ethanol to dissolve. Then, add 6 mmol of triethylamine and stir until evenly mixed. Finally, add 2 mmol of 1,10-phenanthroline (phen) and 1 mmol of 1,4-naphthalenedicarboxylic acid (Nc) and stir at room temperature for 2 hours.

[0047] Weigh 5g of Solvent Red into a reaction flask, add 40mL of anhydrous ethanol, 20mL of pure water, and 4mL of dichloromethane, and stir for 30 minutes to fully dissolve the dye. Quickly add 10mL of aqueous ammonia and stir evenly. Then, dropwise add 10mL of tetraethyl orthosilicate (a coupling agent) and allow to react overnight. Add another 1mL of aqueous ammonia and stir evenly. Then, dropwise add 1mL of 3-aminopropyltriethoxysilane (a coupling agent) in three additions. Let react at room temperature for 2 days. After the reaction, wash the mixture three times with pure water and store in pure water.

[0048] Figure 2 Table 1 shows the particle size and dispersion index of the silicon-coated colored time-resolved fluorescent microspheres of Examples 1-7. The dispersion index PDI is <0.05, indicating 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 silicon-coated colored time-resolved fluorescent microspheres in immunochromatography

[0052] Taking the novel coronavirus antibody as an example, the process includes the following steps:

[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 well and resuspend for 1 h;

[0057] (3) Cleaning to remove residual glutaraldehyde

[0058] The activated microspheres were centrifuged at 15,000 rpm for 15 minutes (first pass); 2. The supernatant was removed and the microspheres were resuspended in 1 mL of labeling buffer; 3. The supernatant was centrifuged at 15,000 rpm for 15 minutes (second pass); 4. The supernatant was removed and the microspheres were resuspended in 0.2 mL of labeling buffer and set aside;

[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, 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, second time; 4. Remove the supernatant and resuspend the microspheres with 1mL of diluent, which is the antibody-microsphere labeled complex. Store at 4℃ for use. If stored for a long time, add a final concentration of 0.2% BSA and 0.02% NaN3 solution; 5. Wash several times if necessary.

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

[0064] Table 2 Detection of novel coronavirus antigen by microsphere-coupled novel coronavirus antibody in Example 1-7 (500-fold dilution)

[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 from left to right to the photos of color time-resolved fluorescent microspheres coupled to influenza A virus antibodies to detect influenza A virus antigen (diluted 500 times) in Examples 1-7. The quality control line and the test line can be seen with the naked eye.

[0067] 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 gentle method for preparing silicon-coated colored time-resolved fluorescent microspheres, characterized by: 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. 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 the coupling agent tetraethyl orthosilicate dropwise, and react overnight; then add an appropriate amount of ammonia water, stir evenly, and add the 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.

2. The gentle preparation method of silicon-encapsulated colored time-resolved fluorescent microspheres according to claim 1, characterized in that: In the step (1), the first ligand is dibenzoylmethane, the second ligand is 1,10-phenanthroline, and the bridging ligand is 1,4-naphthalene dicarboxylic acid.

3. The gentle preparation method of silicon-encapsulated colored time-resolved fluorescent microspheres according to claim 2, 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.

4. The gentle preparation method of silicon-encapsulated colored time-resolved fluorescent microspheres according to claim 1, 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.

5. The gentle preparation method of silicon-encapsulated colored time-resolved fluorescent microspheres according to claim 4, 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.

6. The gentle preparation method of silicon-encapsulated colored time-resolved fluorescent microspheres according to claim 5, 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.

7. Silicon-encapsulated colored time-resolved fluorescent microspheres prepared by the mild preparation method according to any one of claims 1 to 6.

8. Use of the silicon-coated colored time-resolved fluorescent microspheres according to claim 7 in qualitative and quantitative dual immunoassays in the field of immunochromatography.

Images