Carboxyl-functionalized time-resolved fluorescent microspheres and a method for preparing the same

Abstract

The present application relates to a kind of carboxyl functional time-resolved fluorescent microspheres and its preparation method, belong to fluorescent microsphere technical field.The preparation method described in the present application includes the following steps, under acidic conditions, europium salt, lanthanide salt, beta-diketone ligand, synergistic ligand and bridging ligand occur in organic solvent, obtain binuclear rare earth complex;Binuclear rare earth complex, styrene monomer, acrylic monomer, initiator, polyvinylpyrrolidone, emulsifier occur in solvent dispersion polymerization reaction, obtain the carboxyl functional time-resolved fluorescent microsphere.The preparation method described in the present application directly coats dye in the microsphere interior in polymerization stage, need not consider time-resolved fluorescent dye steric effect, can select greater, but fluorescence intensity stronger, cannot be applied to swelling method binuclear rare earth complex as time-resolved fluorescent dye, so that the fluorescence intensity of time-resolved fluorescent microsphere is higher.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fluorescent microspheres, and particularly relates to a carboxyl-functionalized time-resolved fluorescent microsphere and a preparation method thereof. BACKGROUND

[0002] Traditional time-resolved fluorescent microspheres are mainly prepared by a swelling method, that is, first, time-resolved fluorescent dyes and carboxyl-functionalized polymer microspheres are prepared, then, an organic swelling agent solution of the time-resolved fluorescent dyes is added into a polymer organic dispersion system or a polymer water dispersion system containing a surfactant to swell and dye the microspheres, and finally, the swelling agent is removed by volatilization or washing.

[0003] However, in actual operation, the carboxyl-functionalized polymer microspheres are first prepared, and then the swelling method is used to dye the microspheres, so that the preparation process is complex and the operation steps are tedious. In addition, due to the effect of the swelling agent on the microspheres, problems such as uneven particle size distribution and surface corrosion of the microspheres often occur, and there is also a risk of dye leakage. At the same time, the steric hindrance effect of the dye entering the microspheres also needs to be considered, and a binuclear or multinuclear rare earth complex with higher fluorescence intensity cannot be used as a fluorescent dye.

[0004] Since the rare earth complex, especially the europium complex, has the advantages of strong fluorescence intensity, large stoke shift and long fluorescence lifetime, it has attracted widespread attention from scholars at home and abroad since it was reported, especially in the fields of clinical medical detection and time-resolved immunochromatographic detection.

[0005] At present, the europium complex used in time-resolved fluorescent microspheres is mainly a mononuclear europium complex with low fluorescence intensity. Therefore, it is necessary to explore a binuclear rare earth complex with higher fluorescence intensity and to prepare carboxyl-functionalized time-resolved fluorescent microspheres in a rapid and simple method. SUMMARY

[0006] Therefore, the technical problem to be solved by the present application is to overcome the problems in the prior art, such as complex preparation steps of time-resolved fluorescent microspheres, corrosion or deformation of the microspheres caused by the swelling agent used in the preparation process, and low fluorescence intensity.

[0007] To solve the above technical problems, the present application provides a carboxyl-functionalized time-resolved fluorescent microsphere and a preparation method thereof. Polystyrene is used as a base material, 2-phenyl acrylic acid is used as a carboxyl modifier to introduce carboxyl groups into the microspheres, and a rare earth complex is used as a time-resolved fluorescent dye. The carboxyl-functionalized time-resolved fluorescent microspheres are prepared by a one-step method.

[0008] The first object of the present application is to provide a preparation method of a carboxyl-functionalized time-resolved fluorescent microsphere, comprising the following steps,

[0009] (1) Under acidic conditions, europium salt, lanthanide metal salt, β-diketone ligand, cooperative ligand and bridging ligand react in an organic solvent to obtain a binuclear rare earth complex;

[0010] (2) The binuclear rare earth complex, styrene monomer, acrylic monomer, initiator, polyvinyl pyrrolidone and emulsifier described in step (1) undergo dispersion polymerization reaction in a solvent to obtain the carboxyl functionalized time-resolved fluorescent microspheres.

[0011] In one embodiment of the present invention, in step (1), the lanthanide metal is one or more of terbium, dysprosium, gadolinium, samarium, holmium, yttrium and erbium.

[0012] In one embodiment of the present invention, in step (1), the β-diketone ligand is 4,4,4-trifluoro-1-(4-trifluoromethylphenyl)-1,3-butanedione, 4,4,4-trifluoro-1-(p-tolyl)-1,3-butanedione, 1,4-bis(2-thienyl)-1,4-butanedione, 4,4,4-trifluoro-1-phenyl-1,3-butanedione, 1-(4-chlorophenyl)-4,4,4-trifluoro- One or more of 1,3-butanedione and 4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedione; the cooperative ligand is one or more of 4,7-dimethyl-1,10-phenanthroline, 2-nitrile-1,10-o-phenanthroline, 1,10-phenanthroline and 3,4,7,8-tetramethyl-1,10-phenanthroline; the bridging ligand is one or more of terephthalic acid, phthalic acid and isophthalic acid.

[0013] In one embodiment of the present invention, in step (1), a β-diketone ligand with a strong electron-donating group is used. Under the influence of this group, the diketone structure is more easily transformed into an alcohol-ene structure, making its coordination ability with rare earth ions stronger.

[0014] In one embodiment of the present invention, in step (1), a cooperating ligand with a larger planar structure and stronger rigidity is used to make the entire fluorescent molecule more rigid, thereby reducing the energy loss caused by intramolecular vibration and improving the fluorescence intensity.

[0015] In one embodiment of the present invention, in step (1), the molar ratio of europium ions to lanthanide metal ions is 10:0.5-1:2.

[0016] In one embodiment of the present invention, in step (1), the molar ratio of the europium salt, the β-diketone ligand, the cooperative ligand and the bridging ligand is 1:6-10:1-3:1-3.

[0017] In one embodiment of the present invention, in step (1), the pH of the acidic condition is 5-6; the pH regulator is an alcohol solution of NaOH or aqueous ammonia.

[0018] In one embodiment of the present invention, the alcohol solution of NaOH is a sodium hydroxide ethanol solution with a mass fraction of 50%.

[0019] In one embodiment of the present invention, in step (1), the organic solvent is one or more of toluene, dichloromethane, chloroform, and tetrahydrofuran.

[0020] In one embodiment of the present invention, in step (2), the acrylic monomer is one or more of 2-phenylacrylic acid monomer, acrylic acid and methacrylic acid; the initiator is one or more of azobisisobutyronitrile, dibenzoyl peroxide, ammonium persulfate and potassium persulfate; and the emulsifier is one or more of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate and polyvinyl alcohol.

[0021] In one embodiment of the present invention, in step (2), the mass ratio of the binuclear rare earth complex, styrene monomer, acrylic monomer, initiator, polyvinyl pyrrolidone, and emulsifier is 2-30:100:5-15:0.1-10:1-50:10-25.

[0022] In one embodiment of the present invention, in step (2), the acrylic monomer introduces carboxyl groups on the surface of the polystyrene microspheres, and the carboxyl content on the surface of the polystyrene microspheres is changed by changing the amount of the acrylic monomer added.

[0023] In one embodiment of the present invention, in step (2), the emulsifier can improve the dispersibility of the styrene particles in the reaction system.

[0024] In one embodiment of the present invention, in step (2), the reaction temperature is 65-75° C.; and the reaction time is 10-14 h.

[0025] In one embodiment of the present invention, in step (2), the solvent is water and / or ethanol.

[0026] The second object of the present invention is to provide carboxyl-functionalized time-resolved fluorescent microspheres prepared by the preparation method.

[0027] In one embodiment of the present invention, the structure of the carboxyl-functionalized time-resolved fluorescent microspheres is as follows:

[0028]

[0029] In one embodiment of the present invention, the particle size of the carboxyl functionalized time-resolved fluorescent microspheres is 50 nm to 5 μm. The particle size and particle size distribution of the microspheres can be adjusted by adjusting the initiator and styrene.

[0030] The third object of the present invention is to provide an application of the carboxyl-functionalized time-resolved fluorescent microspheres in immunochromatography.

[0031] The technical solution of the present invention has the following advantages over the prior art:

[0032] (1) The preparation method described in the present invention directly introduces the time-resolved fluorescent dye into the polymerization system, so that the time-resolved fluorescent dye directly enters the interior of the microspheres and is embedded in the polymer through polymerization.

[0033] (2) The preparation method described in the present invention directly encapsulates the dye inside the microspheres during the polymerization stage, without considering the steric hindrance effect of the time-resolved fluorescent dye. A binuclear rare earth complex that has a greater steric hindrance but a stronger fluorescence intensity and cannot be applied to the swelling method can be selected as the time-resolved fluorescent dye, thereby making the fluorescence intensity of the time-resolved fluorescent microspheres higher.

[0034] (3) The preparation method of the present invention eliminates the need for separate preparation of carboxyl polymer microspheres and the need for swelling the polymer microspheres with organic solvents, thereby simplifying the preparation steps and reducing the use of environmentally harmful organic solvents. Furthermore, since no organic solvent is required to swell the polymer microspheres, the risk of microsphere deformation is reduced, resulting in more uniform particle size and better surface morphology of the time-resolved fluorescent microspheres, which have excellent application in medical clinical examinations and time-resolved immunochromatography.

[0035] (4) The preparation method of the present invention has simplified steps, integrating the preparation of polystyrene microspheres and microsphere dyeing into one step, which is easy to scale up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0037] Figure 1 This is the scanning electron microscopy characterization in Test Example 1 of the present invention, wherein A is Example 1 and B is Comparative Example 2.

[0038] Figure 2 This is the scanning electron microscopy characterization in Test Example 2 of the present invention, wherein A is Example 2; B is Comparative Example 4.

[0039] Figure 3 This is the fluorescence spectrum diagram in Test Example 3 of the present invention.

[0040] Figure 4The fluorescence spectrum of Example 4 of the present application is tested. DETAILED DESCRIPTION

[0041] The present application will be further described in conjunction with the drawings and specific examples so that those skilled in the art can better understand and implement the present application, but the examples are not intended to limit the present application.

[0042] In the present application, unless otherwise specified, the preparation method of the binuclear rare earth complex is as follows:

[0043] (1) 0.183 g of europium chloride hexahydrate, 0.188 g of dysprosium chloride hexahydrate, 1.42 g of 4,4,4-trifluoro-1-(4-trifluoromethylphenyl)-1,3-butanedione, 0.416 g of 4,7-dimethyl-1,10-phenanthroline, and 0.187 g of p-terephthalic acid are weighed and dissolved in 50 mL of a mixed solution of ethanol and toluene in a volume ratio of 1:1;

[0044] (2) 50% sodium hydroxide solution is added dropwise, the pH is adjusted to about 5, and the reaction is stirred for 4 h;

[0045] (3) Centrifugal separation, washing, and drying are performed to obtain the dysprosium-europium binuclear rare earth complex.

[0046] In the present application, unless otherwise specified, the preparation method of the mononuclear rare earth complex is as follows:

[0047] (1) 0.366 g of europium chloride hexahydrate, 1.42 g of 4,4,4-trifluoro-1-(4-trifluoromethylphenyl)-1,3-butanedione, and 0.416 g of 4,7-dimethyl-1,10-phenanthroline are weighed and dissolved in 50 mL of a mixed solution of ethanol and toluene in a volume ratio of 1:1;

[0048] (2) 50% sodium hydroxide solution is added dropwise, the pH is adjusted to about 5, and the reaction is stirred for 4 h;

[0049] (3) Centrifugal separation, washing, and drying are performed to obtain the mononuclear europium complex.

[0050] Example 1

[0051] A 200 nm carboxyl-functionalized time-resolved fluorescence microsphere and a preparation method thereof, specifically comprising the following steps:

[0052] (1) 25 g of pure water and 25 g of anhydrous ethanol are mixed uniformly, and 0.5 g of PVP and 0.1 g of SDS are added and stirred uniformly;

[0053] (2) 0.08 g of the binuclear rare earth complex is added to 1 g of the purified styrene monomer, and is fully stirred and dissolved;

[0054] (3) Add the solution obtained in step (2) to the solution obtained in step (1) while stirring at 500 r / min, and simultaneously add 0.1 g of 2-phenylacrylic acid monomer, and stir for 5 min;

[0055] (4) The reaction system was heated to 40°C and reacted for 2 h under nitrogen purge to remove dichloromethane;

[0056] (5) Add 0.05 g of ammonium persulfate to the reaction system and heat to 70 °C for 12 h;

[0057] (6) The product was washed several times by centrifugation using alcohol / water and finally dispersed in pure water to obtain 200 nm carboxyl-functionalized time-resolved fluorescent microspheres.

[0058] Example 2

[0059] A 300 nm carboxyl-functionalized time-resolved fluorescent microsphere and a preparation method thereof, comprising the following steps:

[0060] Basically the same as Example 1, except that:

[0061] The amount of ammonium persulfate used in step (5) was adjusted from the original 0.05 g to 0.03 g.

[0062] Comparative Example 1

[0063] The swelling method is used to prepare 200 nm carboxyl-functionalized time-resolved fluorescent microspheres dyed with mononuclear rare earth complexes, which specifically includes the following steps:

[0064] 1) Preparation of carboxyl-functionalized polystyrene microspheres

[0065] (1) Weigh 25g of pure water and 25g of anhydrous ethanol, mix them evenly, add 0.5g of PVP and 0.1g of SDS and stir evenly;

[0066] (2) Add 1 g of styrene monomer and 0.1 g of 2-phenylacrylic acid monomer to the solution obtained in step (1) under stirring at 500 r / min, and stir for 5 min;

[0067] (3) Add 0.05 g of ammonium persulfate to the reaction system and heat to 70°C for 12 h;

[0068] (4) The product was washed several times by centrifugation using alcohol / water and finally dispersed in pure water.

[0069] 2) Preparation of carboxyl-functionalized time-resolved fluorescent microspheres by swelling method

[0070] (1) Disperse 1 g of carboxyl-functionalized polystyrene microspheres in 30 g of pure water, add 1 g of SDS and stir thoroughly to dissolve;

[0071] (2) Dissolve 0.08 g of binuclear rare earth complex fluorescent dye in 2 mL of dichloromethane;

[0072] (3) adding the solution obtained in step (2) dropwise to the solution obtained in step (1) at a stirring speed of 150 r / min;

[0073] (4) Ultrasonic oscillation for 5 minutes to fully emulsify;

[0074] (5) The microspheres were allowed to swell and dye for 1 h at a stirring speed of 150 r / min and a temperature of 30°C;

[0075] (6) The reaction system was heated to 60°C and reacted for 1 hour to evaporate and remove dichloromethane;

[0076] (7) After the reaction is completed, cool the reaction mixture for 1 h to room temperature, wash it by centrifugation, and then wash it with ethanol and pure water three times respectively.

[0077] Comparative Example 2

[0078] The method is basically the same as Comparative Example 1, except that the carboxyl functionalized time-resolved fluorescent microspheres were prepared by swelling method:

[0079] The binuclear rare earth complex used in step (2) is replaced by a mononuclear rare earth complex.

[0080] Comparative Example 3

[0081] Basically the same as Comparative Example 1, except that the carboxyl functionalized polystyrene microspheres were prepared as follows:

[0082] The amount of ammonium persulfate used in step (3) was adjusted from the original 0.05 g to 0.03 g.

[0083] Comparative Example 4

[0084] Basically the same as Comparative Example 1, except that:

[0085] Preparation of carboxyl-functionalized polystyrene microspheres: The amount of ammonium persulfate used in step (3) was adjusted from 0.05 g to 0.03 g;

[0086] Preparation of carboxyl functionalized time-resolved fluorescent microspheres by swelling method: the binuclear rare earth complex used in step (2) is replaced by a mononuclear rare earth complex.

[0087] Comparative Example 5

[0088] Basically the same as Example 1, except that:

[0089] The binuclear rare earth complex used in step (2) is replaced by a mononuclear rare earth complex.

[0090] Comparative Example 6

[0091] Basically the same as Example 1, except that:

[0092] The binuclear rare earth complex used in step (2) is replaced by a mononuclear rare earth complex;

[0093] The amount of ammonium persulfate used in step (5) was adjusted from the original 0.05 g to 0.03 g.

[0094] Test Example 1

[0095] The samples of Example 1 and Comparative Example 1 were characterized by scanning electron microscopy. Figure 1 As shown. Figure 1 As can be seen from A, the 200 nm time-resolved fluorescent microspheres prepared by the one-step method in Example 1 have a smooth surface, uniform particle size, and good monodispersity; Figure 1 As shown in Figure B, in the 200 nm time-resolved fluorescent microspheres prepared by the traditional two-step method, due to the erosion of the swelling agent during the second dyeing process, the particle size uniformity of the sample deteriorated and the particle size distribution became wider.

[0096] Test Example 2

[0097] The samples of Example 2 and Comparative Example 3 were characterized by scanning electron microscopy. Figure 2 As shown. Figure 2 A shows that the 300nm time-resolved fluorescent microsphere sample prepared by the one-step method has a smooth surface, uniform particle size, and good monodispersity; Figure 2 B shows that due to the erosion of the swelling agent during the preparation process, the particle size uniformity of the 300 nm time-resolved fluorescent microspheres prepared by the traditional two-step method deteriorates and the particle size distribution becomes wider.

[0098] Test Example 3

[0099] The fluorescence intensity of commercially available samples (purchased from Microbiology, 210nm Eu-time-resolved fluorescent microspheres), Example 1, Comparative Examples 1-2 and 5 were tested. The fluorescence intensity was measured using a fluorescence photometer under 365nm excitation conditions at 615nm. The results are as follows: Figure 3 As shown in Table 1:

[0100] Table 1

[0101] sample Commercially available Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 5 Fluorescence intensity 4000 11800 800 5150 8100

[0102] like Figure 3 As shown, the fluorescence intensity of the sample in Example 1 is much higher than that of the commercially available sample.

[0103] As shown in Table 1, Example 1 uses a binuclear rare earth complex with higher fluorescence intensity as the fluorescent dye, resulting in a higher fluorescence intensity compared to Comparative Example 5. However, due to the swelling method used in Comparative Example 1, the more sterically hindered binuclear rare earth complex exhibited poor dyeing performance and lower fluorescence intensity. Comparative Example 5, prepared using a one-step method, eliminated the risk of leakage by encapsulating the dye within the polystyrene microspheres. However, Comparative Example 2, prepared using a swelling method, experienced significant dye loss at the same dye dosage, with some dye leaking during the washing process. Consequently, the fluorescence value was lower than that of Comparative Example 5.

[0104] Test Example 4

[0105] The fluorescence intensity of commercially available samples (purchased from Microbiology, 316nm Eu-time-resolved fluorescent microspheres), Example 2, Comparative Examples 3-4 and 6 were tested. The fluorescence intensity was measured using a fluorescence photometer under 365nm excitation conditions at 615nm. The results are as follows: Figure 4 As shown in Table 2:

[0106] Table 2

[0107] sample Commercially available Example 2 Comparative Example 6 Comparative Example 3 Comparative Example 4 Fluorescence intensity 6000 16500 9240 840 6820

[0108] like Figure 4 As shown, the fluorescence intensity of the sample in Example 2 is much higher than that of the commercially available sample.

[0109] As shown in Table 1, Example 2 uses a binuclear rare earth complex with higher fluorescence intensity as the fluorescent dye, resulting in a higher fluorescence intensity compared to Comparative Example 6. However, Comparative Example 3, prepared using a swelling method, exhibits poor dyeing performance and a lower fluorescence intensity due to the greater steric hindrance of the binuclear rare earth complex. Comparative Example 6, prepared using a one-step method, eliminates the risk of leakage by encapsulating the dye within the polystyrene microspheres. However, Comparative Example 4, prepared using a swelling method, experiences significant dye loss at the same dye dosage, with some dye leaking during the washing process. Consequently, the fluorescence value is lower than that of Example 2.

[0110] Test Example 5

[0111] The commercially available samples (purchased from Microtest Biotechnology, 210 nm Eu-time-resolved fluorescent microspheres), the samples of Example 1, Comparative Examples 1-2 and 5 were subjected to immunochromatographic testing. The results are shown in Table 3:

[0112] Table 3

[0113]

[0114]

[0115] As shown in Table 3, compared with commercially available samples, the sample of Example 1 has excellent performance in immunochromatography applications, and its results are improved by nearly 2 times. Compared with Example 1, Comparative Example 5 uses a mononuclear rare earth complex as a dye, and its bare ball fluorescence value is low, which limits its application performance in immunochromatography. Compared with Example 1, the sample of Comparative Example 1 is prepared by a two-step method. Due to the steric effect of the binuclear complex, its dyeing effect is poor, the product fluorescence value is low, and the application result is poor. Compared with Example 1, the sample of Comparative Example 2 has a lower fluorescence value of the mononuclear rare earth complex used than the binuclear complex on the one hand, and the particle size of the microspheres is not uniform enough and the surface is corroded, resulting in its application performance being worse than that of Example 1.

[0116] Test Example 6

[0117] The commercially available samples (purchased from Microtest Biotechnology, 316 nm Eu-time-resolved fluorescent microspheres), the samples of Example 2, Comparative Examples 3-4 and 6 were subjected to immunochromatographic testing. The results are shown in Table 4:

[0118] Table 4

[0119]

[0120] As shown in Table 4, the results are similar to those of the 200nm time-resolved fluorescent microspheres. The sample in Example 2 uses a binuclear rare earth complex as a dye and is prepared by a one-step method. Its heroic performance is good and is much higher than that of commercially available samples. Comparative Example 6 uses a mononuclear rare earth complex as a dye. Its bare ball fluorescence value is low, which limits its application performance in immunochromatography. Compared with Example 2, the sample in Comparative Example 3 is prepared by a two-step method. Due to the steric effect of the binuclear complex, its dyeing effect is poor, the product fluorescence value is low, and the application results are poor. Compared with Example 2, the sample in Comparative Example 4 has a lower fluorescence value of the mononuclear rare earth complex used than the binuclear complex on the one hand, and the particle size of the microspheres is not uniform enough and the surface is corroded, resulting in its application performance being worse than that of Example 2.

[0121] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for preparing carboxyl-functionalized time-resolved fluorescent microspheres, characterized in that: The following steps are included: (1) Under acidic conditions, europium salt, lanthanide metal salt, β-diketone ligand, cooperative ligand and bridging ligand react in an organic solvent to obtain a binuclear rare earth complex; Wherein, the lanthanide metal of the lanthanide metal salt is dysprosium, the β-diketone ligand is 4,4,4-trifluoro-1-(4-trifluoromethylphenyl)-1,3-butanedione, the synergistic ligand is 4,7-dimethyl-1,10-phenanthroline, and the bridging ligand is one or more of terephthalic acid, phthalic acid and isophthalic acid; (2) The binuclear rare earth complex, styrene monomer, acrylic monomer, initiator, polyvinyl pyrrolidone and emulsifier described in step (1) undergo dispersion polymerization reaction in a solvent to obtain the carboxyl functionalized time-resolved fluorescent microspheres.

2. The method for preparing carboxyl functionalized time-resolved fluorescent microspheres according to claim 1, characterized in that: In step (1), the molar ratio of europium ions to lanthanide metal ions is 10:0.5-1:

2.

3. The method for preparing carboxyl functionalized time-resolved fluorescent microspheres according to claim 1, characterized in that: In step (1), the molar ratio of the europium salt, the β-diketone ligand, the cooperative ligand and the bridging ligand is 1:6-10:1-3:1-3.

4. The method for preparing carboxyl functionalized time-resolved fluorescent microspheres according to claim 1, characterized in that: In step (2), the acrylic monomer is one or more of 2-phenyl acrylic acid monomer, acrylic acid and methacrylic acid; the initiator is one or more of azobisisobutyronitrile, dibenzoyl peroxide, ammonium persulfate and potassium persulfate; and the emulsifier is one or more of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate and polyvinyl alcohol.

5. The method for preparing carboxyl functionalized time-resolved fluorescent microspheres according to claim 1, characterized in that: In step (2), the mass ratio of the binuclear rare earth complex, styrene monomer, acrylic monomer, initiator, polyvinyl pyrrolidone, and emulsifier is 2-30:100:5-15:0.1-10:1-50:10-25.

6. The method for preparing carboxyl-functionalized time-resolved fluorescent microspheres according to claim 1, characterized in that: In step (2), the reaction temperature is 65-75°C; the reaction time is 10-14 h.

7. Carboxyl-functionalized time-resolved fluorescent microspheres prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the carboxyl-functionalized time-resolved fluorescent microspheres according to claim 7 in immunochromatography.

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