A magnetic fluorescent microsphere, its preparation method and application

Abstract

This invention provides a magnetic fluorescent microsphere, its preparation method, and its application, belonging to the field of biodetection technology. The magnetic fluorescent microsphere utilizes samarium-cobalt-gold ternary composite magnetic nanoparticles as its core, significantly improving the magnetic response speed. Leveraging the long-lifetime, time-resolved fluorescence characteristics of europium complexes, it effectively eliminates background interference caused by autoluminescence in biological samples, greatly enhancing detection sensitivity and signal-to-noise ratio. Simultaneously, the polymer layer contains active functional groups, facilitating covalent coupling with biomolecules and reducing non-specific adsorption. This magnetic fluorescent microsphere can be widely applied in fields such as magnetic microparticle chemiluminescence detection, time-resolved fluorescence immunoassay, magnetic enzyme-linked immunosorbent assay (ELISA), and magnetic separation nucleic acid extraction. It can shorten the chemiluminescence detection reaction time to 5 minutes, and the luminescence intensity is nearly doubled compared to traditional labels, significantly improving detection throughput and sensitivity, and possessing significant clinical application value and market prospects.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to a magnetic fluorescent microsphere, its preparation method, and its application. Background Technology

[0002] Magnetic fluorescent microspheres are a class of composite nanomaterials possessing both superparamagnetic and fluorescent properties, with wide applications in biomedical detection, immunoassay, and nucleic acid separation. Their basic principle involves combining magnetic nanoparticles with fluorescent substances, enabling rapid enrichment and separation of target substances under an applied magnetic field, while simultaneously achieving quantitative detection via fluorescence signals. Currently, common magnetic fluorescent microspheres typically use iron(III) oxide (Fe3O4) as the magnetic core and organic fluorescent dyes or quantum dots as the fluorescence source, prepared via sol-gel methods, emulsion polymerization, or layer-by-layer self-assembly. In the field of in vitro diagnostics, magnetic microparticle chemiluminescence immunoassay has become a mainstream detection platform. This technology uses magnetic microspheres as a solid-phase carrier, generating detectable signals through a chemiluminescence system, offering advantages such as high automation and a wide detection range.

[0003] However, existing magnetic microspheres still have room for improvement in terms of magnetic response speed, fluorescence intensity, and stability. For example, the saturation magnetization of Fe3O4 magnetic cores is limited, resulting in a long magnetic separation time; common organic fluorescent dyes suffer from photobleaching and background fluorescence interference; and quantum dots may exhibit heavy metal toxicity. Furthermore, the fabrication processes of most magnetic fluorescent microspheres are complex, with significant batch-to-batch variations, and fluorescence leakage is common during long-term storage.

[0004] Therefore, developing a novel magnetic fluorescent microsphere with fast magnetic response, high fluorescence signal-to-noise ratio, and good stability is of great practical significance. Summary of the Invention

[0005] The purpose of this invention is to provide a magnetic fluorescent microsphere that solves the technical problems of slow magnetic response speed, high background interference in fluorescence detection, and poor long-term storage stability of existing magnetic fluorescent microspheres.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a magnetic fluorescent microsphere, characterized in that the magnetic fluorescent microsphere comprises samarium cobalt gold magnetic nanoparticles and a polymer layer coating the surface of the samarium cobalt gold magnetic nanoparticles, wherein the polymer layer is loaded with Eu. 3+ The complex, wherein the polymer layer is made of a cross-linked polymer containing active functional groups.

[0008] In this invention, the "Samarium Cobalt Gold Magnetic Nanoparticles" refer to magnetic nanoparticles with samarium, cobalt and gold as the main components, characterized by high saturation magnetization and fast magnetic response performance.

[0009] In this invention, the "polymer layer" refers to a layer of polymeric material coated on the surface of magnetic nanoparticles, which serves to fix europium complexes to the surface of magnetic particles and form a stable microsphere structure.

[0010] In this invention, the "active functional group" refers to a chemical group located on the surface of the polymer layer that can undergo covalent coupling reactions with biomolecules such as antibodies, antigens, or nucleic acids.

[0011] In this invention, the "Eu" 3+ "Coordination compounds" refer to coordination compounds formed by combining trivalent europium ions with organic ligands. They have long fluorescence lifetimes and can be used for time-resolved fluorescence detection.

[0012] In this invention, the "crosslinked polymer" refers to a polymeric material formed by the polymerization of multifunctional monomers, whose three-dimensional network structure makes the polymer layer insoluble in conventional solvents and has high mechanical strength.

[0013] Preferably, the active functional group is a carboxyl group.

[0014] Preferably, the crosslinked polymer is a copolymer of acrylic monomers, bifunctional acrylate monomers, and organosiloxane monomers.

[0015] This invention also provides a method for preparing the above-mentioned magnetic fluorescent microspheres, the method comprising the following steps: Samarium cobalt gold magnetic nanoparticles, Eu 3+ The complex is mixed with the crosslinking polymer raw material to carry out a crosslinking polymerization reaction, forming a loaded Eu on the surface of the samarium cobalt gold magnetic nanoparticles. 3+ The magnetic fluorescent microspheres are obtained by cross-linking a polymer layer of the complex.

[0016] Preferably, the samarium cobalt gold magnetic nanoparticles are prepared by a method comprising the following steps: Water, samarium trichloride hexahydrate, cobalt dichloride hexahydrate and tetrachloroauric acid are reacted under heating conditions for 5-15 minutes, and then trisodium citrate aqueous solution is added to continue the reaction for 15-25 minutes. The mass ratio of the tetrachloroauric acid to the volume of the water is 0.5~3.0g:800~1200mL; The mass ratio of samarium trichloride hexahydrate to tetrachloroauric acid is 1~4:0.5~3.0, and the mass ratio of cobalt dichloride hexahydrate to tetrachloroauric acid is 1~5:0.5~3.0; The mass percentage concentration of the trisodium citrate aqueous solution is 0.05%~0.5%, and the volume ratio of the trisodium citrate aqueous solution to the volume of water is 3~8:800~1200.

[0017] Preferably, the Eu 3+ The complex was prepared by a method comprising the following steps: dissolving EuCl3·6H2O in N-methylpyrrolidone, adding quinoline dimethacrylate, 1,10-o-phenanthroline and dibenzoylmethane, adjusting the pH to 6.2-7.2, reacting at 50-80°C for 7-10 h, cooling, filtering and washing with anhydrous ethanol to obtain the Eu... 3+ Coordination compounds; The mass-to-volume ratio of EuCl3·6H2O to N-methylpyrrolidone is 0.1~0.5g:80~120mL; The molar ratio of quinoline dimethacrylate, 1,10-phenanthroline and dibenzoylmethane is 1:2~8:3~6.

[0018] Preferably, the samarium cobalt gold magnetic nanoparticles and Eu are used. 3+ The process of mixing the complex with the crosslinking polymer raw material and carrying out the crosslinking polymerization reaction specifically includes the following steps: S1, a solution of samarium cobalt gold magnetic nanoparticles is mixed with hexadecyl ammonium bromide, D-glucose-6-phosphate disodium salt, polyoxyethylene lauroyl ether, and the aforementioned Eu. 3+ The complexes were mixed and stirred in a water bath at 70-90°C in the dark to obtain the first mixture. S2. Dissolve acrylic acid, ethylene glycol dimethacrylate and dimethylsiloxane in chloroform, add to the first mixture, add hexachloroplatinic acid and ethylenediamine, and stir in a water bath at 70~90℃ in the dark for 20~28h to obtain crude magnetic fluorescent microspheres. S3. Separate the crude product of the magnetic fluorescent microspheres using a magnetic separator to obtain the magnetic fluorescent microspheres.

[0019] Preferably, the volume ratio of the samarium cobalt gold magnetic nanoparticle solution to the mass ratio of the hexadecyl ammonium bromide is 800~1200mL:1~4g; The mass ratio of D-glucose-6-phosphate disodium to hexadecylammonium bromide is 2~6:1~4; The mass ratio of the polyoxyethylene lauroyl ether to the hexadecyl ammonium bromide is 3~5:1~4; The molar ratio of acrylic acid, ethylene glycol dimethacrylate and dimethylsiloxane is 1:1~3:1~2; The volume ratio of the trichloromethane to the mass ratio of the acrylic acid is 8-12 mL: 0.1-0.3 g. The mass ratio of hexachloroplatinic acid to acrylic acid is 0.1~0.4:0.1~0.3; The mass ratio of ethylenediamine to acrylic acid is 1~4:0.1~0.3.

[0020] The present invention also provides a detection kit comprising the above-mentioned magnetic fluorescent microspheres.

[0021] The present invention also provides the application of the above-mentioned magnetic fluorescent microspheres or magnetic fluorescent microspheres prepared by the above-mentioned preparation method in the preparation of magnetic microparticle chemiluminescence detection reagents, time-resolved fluorescence immunoassay reagents, magnetic enzyme-linked immunosorbent assay reagents or magnetic separation nucleic acid extraction reagents.

[0022] The beneficial effects of this invention are: This invention provides a magnetic fluorescent microsphere that uses samarium-cobalt-gold ternary composite magnetic nanoparticles as its core, significantly improving the magnetic response speed and enabling magnetic separation and enrichment within 1-5 seconds. The polymer layer is loaded with Eu. 3+ The complex utilizes the long lifetime time-resolved fluorescence properties of europium complexes to effectively eliminate background interference caused by the autoluminescence of biological samples, significantly improving detection sensitivity and signal-to-noise ratio. At the same time, the polymer layer contains active functional groups, which facilitates covalent coupling with biomolecules and reduces non-specific adsorption.

[0023] The preparation method provided by this invention is stable and low in cost. It uses an aqueous phase polymerization reaction to coat fluorescent dyes onto the surface of magnetic particles. The resulting magnetic fluorescent microspheres have uniform particle size, good dispersibility, a shelf life of up to 60 months, and excellent thermal stability, making them suitable for large-scale production and commercial applications.

[0024] The magnetic fluorescent microspheres of this invention can be widely used in fields such as magnetic microparticle chemiluminescence detection, time-resolved fluorescence immunoassay, magnetic enzyme-linked immunosorbent assay (ELISA), and magnetic separation nucleic acid extraction. They can shorten the reaction time of chemiluminescence detection to 5 minutes and increase the luminescence intensity by nearly double that of traditional labels, significantly improving detection throughput and sensitivity. They have important clinical application value and market prospects. Attached Figure Description

[0025] Figure 1 The images shown are electron microscope images of the samarium cobalt gold magnetic nanoparticles of this invention (a, b, and c in the figures are the observation results of products obtained from different batches). Figure 2 This is an image of the magnetic fluorescent microspheres of the present invention. Detailed Implementation

[0026] This invention provides a magnetic fluorescent microsphere comprising samarium cobalt gold magnetic nanoparticles and a polymer layer coating the surface of the samarium cobalt gold magnetic nanoparticles, wherein the polymer layer is loaded with Eu.3+ The complex, wherein the polymer layer is made of a cross-linked polymer containing active functional groups.

[0027] Preferably, the active functional group is a carboxyl group. The carboxyl group (-COOH) is one of the common active functional groups. It can form a stable amide bond with a primary amine group through a carbodiimide crosslinking agent (such as EDC, EDC·HCl), thereby covalently coupling biomolecules such as antibodies, antigens, and nucleic acid probes to the surface of microspheres. Besides the carboxyl group, other active functional groups such as amino, thiol, epoxy, and aldehyde groups can also be introduced according to actual application needs, but the present invention prefers the carboxyl group because of its mild coupling conditions, high efficiency, and low reagent cost.

[0028] Preferably, the crosslinked polymer is a copolymer of acrylic monomers, bifunctional acrylate monomers, and organosiloxane monomers. Examples of acrylic monomers include acrylic acid, methacrylic acid, and ethylacrylic acid, with acrylic acid being preferred. Examples of bifunctional acrylate monomers include ethylene glycol dimethacrylate, ethylene glycol diacrylate, and 1,4-butanediol dimethacrylate, with ethylene glycol dimethacrylate being preferred. Examples of organosiloxane monomers include dimethylsiloxane, diethylsiloxane, and methylphenylsiloxane, with dimethylsiloxane being preferred. The above three monomers are copolymerized via free radicals to form a polymer with a crosslinked network structure. This polymer combines the film-forming properties of polyacrylates with the flexibility of organosiloxanes, effectively encapsulating small molecule fluorescent complexes.

[0029] This invention also provides a method for preparing the above-mentioned magnetic fluorescent microspheres, the method comprising the following steps: mixing samarium cobalt gold magnetic nanoparticles and Eu... 3+ The complex is mixed with the crosslinking polymer raw material to carry out a crosslinking polymerization reaction, forming a loaded Eu on the surface of the samarium cobalt gold magnetic nanoparticles. 3+ The magnetic fluorescent microspheres are obtained by cross-linking a polymer layer of the complex.

[0030] In the above preparation method, as a preferred embodiment, the samarium cobalt gold magnetic nanoparticles are prepared by a method including the following steps: water, samarium trichloride hexahydrate, cobalt dichloride hexahydrate, and tetrachloroauric acid are reacted under heating conditions for 5-15 min, and then an aqueous solution of trisodium citrate is added to continue the reaction for 15-25 min. The heating conditions can be oil bath heating, electric heating mantle heating, or water bath heating, and the temperature is usually 95-100℃ to keep the water boiling. The reaction time can be selected as 6 min, 8 min, 10 min, 12 min, or 14 min, more preferably 10 min; the continued reaction time can be selected as 16 min, 18 min, 20 min, 22 min, or 24 min, more preferably 20 min. The mass ratio of the tetrachloroauric acid to the volume of the water is 0.5-3.0 g: 800-1200 mL, for example, 1.0 g: 1000 mL or 2.0 g: 1000 mL. The mass ratio of samarium trichloride hexahydrate to tetrachloroauric acid is 1-4:0.5-3.0, for example, 3:1 or 2:1; the mass ratio of cobalt dichloride hexahydrate to tetrachloroauric acid is 1-5:0.5-3.0, for example, 2:1 or 3:1. The mass percentage concentration of the trisodium citrate aqueous solution is 0.05%-0.5%, for example, 0.1%, 0.2%, or 0.3%; the volume ratio of the trisodium citrate aqueous solution to the volume of water is 3-8:800-1200, for example, 5:1000. Trisodium citrate acts as a reducing agent and stabilizer, reducing tetrachloroauric acid to nano-gold and simultaneously promoting the co-reduction of samarium and cobalt ions.

[0031] As another preferred option, the Eu 3+ The complex was prepared by a method comprising the following steps: dissolving EuCl3·6H2O in N-methylpyrrolidone, adding quinoline dimethacrylate, 1,10-o-phenanthroline and dibenzoylmethane, adjusting the pH to 6.2-7.2, reacting at 50-80°C for 7-10 h, cooling, filtering and washing with anhydrous ethanol to obtain the Eu... 3+ The complex is prepared with a pH value of 6.2, 6.5, 6.8, 7.0, or 7.2, preferably 7.2; a reaction temperature of 50℃, 60℃, 70℃, or 80℃, preferably 80℃; and a reaction time of 7h, 8h, 9h, or 10h, preferably 10h. The mass-to-volume ratio of EuCl3·6H2O to N-methylpyrrolidone is 0.1~0.5g:80~120mL, for example, 0.3g:100mL. The molar ratio of quinoline dimethacrylate, 1,10-o-phenanthroline, and dibenzoylmethane is 1:2~8:3~6, for example, 1:2:5 or 1:4:4. Quinoline dimethacrylate acts as the first ligand, 1,10-o-phenanthroline as the co-ligand, and dibenzoylmethane as the second ligand. The three compounds react with EuCl3·6H2O. 3+The ions form a stable ternary complex that emits characteristic red light under ultraviolet excitation and has a fluorescence lifetime ranging from microseconds to milliseconds, making it suitable for time-resolved fluorescence detection.

[0032] As another preferred embodiment, the samarium cobalt gold magnetic nanoparticles, Eu 3+ The specific steps involved in mixing the complex with the crosslinking polymer raw materials and carrying out the crosslinking polymerization reaction are as follows: S1, a solution of samarium cobalt gold magnetic nanoparticles is mixed with hexadecyl ammonium bromide, D-glucose-6-phosphate disodium salt, polyoxyethylene lauroyl ether, and the aforementioned Eu. 3+ The complexes are mixed and stirred in a water bath at 70-90°C in the dark to obtain a first mixture. The water bath temperature can be selected from 70°C, 75°C, 80°C, 85°C, and 90°C, with 80°C being preferred. Stirring in the dark is to prevent Eu from... 3+ The complex is photobleached under strong light.

[0033] S2. Dissolve acrylic acid, ethylene glycol dimethacrylate, and dimethylsiloxane in chloroform, add the solution to the first mixture, and add hexachloroplatinic acid and ethylenediamine. Stir and react in a water bath at 70-90°C in the dark for 20-28 hours to obtain crude magnetic fluorescent microspheres. The reaction time can be selected from 20h, 22h, 24h, 26h, and 28h, preferably 24h.

[0034] S3. Separate the crude product of the magnetic fluorescent microspheres using a magnetic separation frame to obtain the magnetic fluorescent microspheres. The magnetic separation frame can be a permanent magnet, with a magnetic field strength typically of 0.2~0.5T. The separation time is 1~5 seconds to adsorb the microspheres onto the container wall. After discarding the supernatant, purified microspheres are obtained.

[0035] In the above steps, as a more preferred parameter range, the volume ratio of the samarium cobalt gold magnetic nanoparticle solution to the mass ratio of the hexadecyl ammonium bromide is 800~1200mL:1~4g, for example 1000mL:2g or 1000mL:4g; the mass ratio of the D-glucose-6-phosphate disodium to the hexadecyl ammonium bromide is 2~6:1~4, for example 3:1 or 1.5:1; the mass ratio of the polyoxyethylene lauroyl ether to the hexadecyl ammonium bromide is 3~5:1~4, for example 1.25:1 or 1:1; The molar ratio of acrylic acid, ethylene glycol dimethacrylate, and dimethylsiloxane is 1:1 to 3:1 to 2, for example, 1:2:2 or 1:1:1; the volume ratio of chloroform to the mass ratio of acrylic acid is 8 to 12 mL: 0.1 to 0.3 g, for example, 10 mL: 0.15 g; the mass ratio of hexachloroplatinic acid to acrylic acid is 0.1 to 0.4: 0.1 to 0.3, for example, 0.2: 0.15; the mass ratio of ethylenediamine to acrylic acid is 1 to 4: 0.1 to 0.3, for example, 1: 0.15. Hexachloroplatinic acid, as an initiator of free radical polymerization, decomposes under heating conditions to generate free radicals, initiating the copolymerization of acrylic acid, ethylene glycol dimethacrylate, and dimethylsiloxane; ethylenediamine can act as both a crosslinking agent (reacting with the carboxyl groups of acrylic acid to form amide crosslinks) and a catalyst to promote the initiation efficiency of hexachloroplatinic acid.

[0036] This invention also provides a detection kit comprising the aforementioned magnetic fluorescent microspheres. The kit may further comprise one or more of the following components: buffer solution (e.g., phosphate buffer, Tris buffer, MES buffer), washing solution (e.g., buffer containing Tween-20), blocking solution (e.g., bovine serum albumin solution, skim milk powder solution), calibrators (standards containing different concentrations of target antigens or antibodies), quality controls, luminescent substrates (e.g., luminol, AMPPD, acridinium ester, etc.), and magnetic separation equipment (e.g., magnetic rack, magnetic separation module for a fully automated chemiluminescence analyzer). The kit can be packaged in different forms depending on the detection target, such as 96-well plate, tube, or microfluidic chip.

[0037] This invention also provides the application of the above-mentioned magnetic fluorescent microspheres, or magnetic fluorescent microspheres prepared by the above-mentioned method, in the preparation of magnetic microparticle chemiluminescence detection reagents, time-resolved fluorescence immunoassay reagents, magnetic enzyme-linked immunosorbent assay (ELISA) reagents, or magnetic separation nucleic acid extraction reagents. Magnetic microparticle chemiluminescence detection reagents utilize magnetic microspheres as a solid-phase carrier, coupling captured antibodies or antigens to the surface of the microspheres. After binding with the analyte in the sample, a chemiluminescent substrate is added for detection. These reagents are widely used in clinical testing for infectious diseases, tumor markers, hormones, and other items. Time-resolved fluorescence immunoassay reagents are characterized by the long-lived fluorescence of europium complexes. The time-resolved detection mode eliminates background fluorescence in the sample, significantly improving the signal-to-noise ratio, and is suitable for high-sensitivity detection of low-abundance markers. Magnetic enzyme-linked immunosorbent assay (ELISA) reagents combine enzyme-linked immunosorbent assay (ELISA) with magnetic separation technology. Magnetic separation allows for rapid washing, replacing the repeated centrifugation or aspiration steps of traditional ELISA, greatly shortening the operation time. Magnetic separation nucleic acid extraction reagents utilize magnetic microspheres to adsorb DNA / RNA under high-salt conditions, rapidly separating nucleic acids under an external magnetic field. Purified nucleic acids are then obtained through washing and elution, representing the mainstream nucleic acid extraction method in current high-throughput sequencing and PCR detection. All four types of reagents mentioned above can benefit from the magnetic fluorescent microspheres of this invention, achieving faster separation speeds, higher detection sensitivity, and longer shelf lives.

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1 This embodiment provides a method for preparing magnetic fluorescent microspheres: (1) Synthesis of samarium cobalt gold magnetic nanospheres by water boiling method: Heat 1000mL of water to boiling in an electric heater. Then add 3g of samarium trichloride hexahydrate, 2g of cobalt dichloride hexahydrate, and 1.0g of tetrachloroauric acid in sequence. Continue heating and boiling for 10 minutes. Then add 5mL of 0.1% trisodium citrate aqueous solution and continue heating and boiling for another 20 minutes. The whole solution turns dark red. Stop heating and let it cool for later use.

[0040] (2) Preparation of time-resolved fluorescent magnetic microspheres: Weigh 0.3g of EuCl3·6H2O and dissolve it in 100mL of N-methylpyrrolidone. Stir to dissolve, then add 5mL of quinoline dimethacrylate and stir until homogeneous. Finally, add 1g, 2g, and 5g of 1,10-o-phenanthroline and dibenzoylmethane, respectively; the molar ratio of quinoline dimethacrylate to 1,10-o-phenanthroline to dibenzoylmethane is 1:2:5. Adjust the pH to 7.2, heat the reaction solution to 80℃, and the heating reaction time is 10h. Cool to 20℃, let stand for 24h, filter, wash the filter cake several times with anhydrous ethanol, and filter again to obtain the time-resolved fluorescent dye Eu(III). Take 1000 mL of samarium cobalt gold magnetic nanosphere solution, add 4 g of cetyl ammonium bromide, 6 g of D-glucose-6-phosphate disodium salt, and 5 g of polyoxyethylene lauroyl ether and stir to dissolve. Then add the prepared time-resolved fluorescent dye Eu(III) and stir in an 80°C water bath in the dark. Subsequently, add 0.1 g of acrylic acid, 0.2 g of ethylene glycol dimethacrylate, and 0.2 g of dimethylsiloxane to 10 mL of chloroform and shake to dissolve. The molar ratio of acrylic acid: ethylene glycol dimethacrylate: dimethylsiloxane is 1:2:2. Slowly pour the dissolved solution into the solution being stirred. Weigh 0.2 g of hexachloroplatinum(IV) acid (H2PtCl6) and add it. Then add 1 g of ethylenediamine and stir in an 80°C water bath in the dark for 24 hours. After the reaction was completed, time-resolved fluorescent magnetic microspheres were separated using a magnetic separator. The microspheres were washed five times with MEST (MES buffer mixed with Tween 20) and the separated product was stored in 10 mL of MEST.

[0041] Electron microscopy images of the prepared samarium cobalt gold magnetic nanospheres are shown below. Figure 1 As shown; Photographs of the prepared time-resolved fluorescent magnetic microspheres are shown below. Figure 2 As shown, the magnetic microspheres obtained by this invention are uniformly dispersed in the solution, and the system is a dark black homogeneous suspension. After a magnet is placed next to the test tube, the black microspheres are quickly attracted by the magnet and gather on the side wall of the test tube, and the upper solution becomes clear and transparent, clearly proving that the microspheres have excellent magnetic responsiveness and can quickly separate solid and liquid under an external magnetic field.

[0042] In this embodiment, the performance of the prepared magnetic fluorescent microspheres was tested and compared with existing products (acrididine esters, Dynabeads), and the results are as follows.

[0043] Luminous intensity comparison With a fixed concentration gradient of the luminescent material, the fluorescence signal intensity of the present invention was evaluated by comparing it with that of the conventional luminescent label acridine ester. The results are shown in Table 1.

[0044] Table 1 Comparison of luminous intensity

[0045] As shown in Table 1, the luminescence intensity of the magnetic fluorescent microspheres of this invention is significantly higher than that of acridine ester at all test concentrations. At a concentration of 0.01 mM, the luminescence intensity of the magnetic fluorescent microspheres is 35,663,792 U, approximately 1.9 times that of acridine ester (18,715,246 U); at a lower concentration of 0.0005 mM, the luminescence intensity of the magnetic fluorescent microspheres is 1,027,751 U, approximately 3.4 times that of acridine ester (304,926 U). This indicates that the magnetic fluorescent microspheres of this invention have a stronger luminescence signal, which is beneficial for improving detection sensitivity.

[0046] Comparison of magnetic separation time The magnetic separation time of the magnetic fluorescent microspheres of this invention was compared with that of commercially available magnetic beads Dynabeads, and the magnetic response speed was evaluated through four repeated tests. The results are shown in Table 2.

[0047] Table 2. Time required for enrichment of fluorescent magnetic beads (unit: seconds, s)

[0048] As shown in Table 2, the magnetic separation time of the magnetic fluorescent microspheres of this invention is only 3-4 seconds, while that of Dynabeads is 15-17 seconds. The magnetic separation speed of the magnetic fluorescent microspheres of this invention is significantly better than that of the comparative products. The shorter magnetic separation time is beneficial for shortening the overall detection process and increasing the detection throughput.

[0049] Comparison of reaction times in chemiluminescence detection The magnetic fluorescent microspheres of this invention and Dynabeads were applied to the chemiluminescence detection of four viruses—swine fever, porcine reproductive and respiratory syndrome, African swine fever, and pseudorabies—using magnetic microparticles, respectively. The reaction times required to complete the project were compared. The results are shown in Table 3.

[0050] Table 3. Time required to complete the project process (unit: minutes)

[0051] As shown in Table 3, for the detection of four viruses, the reaction time of the magnetic fluorescent microspheres of this invention is 5 minutes, while the reaction time of Dynabeads is 15-20 minutes. The detection time of the magnetic fluorescent microspheres of this invention is shortened to 1 / 3 to 1 / 4 of the comparative products. This indicates that the magnetic fluorescent microspheres of this invention can significantly accelerate the detection reaction speed.

[0052] Long-term storage stability test The magnetic fluorescent microspheres of this invention were placed under storage conditions for different periods of time, and the changes in their luminescence intensity were tested to evaluate their long-term storage stability. The results are shown in Table 4.

[0053] Table 4 Retention period data

[0054] As shown in Table 4, the luminescence intensity of the magnetic fluorescent microspheres of the present invention remained at approximately 17.59 million U during the 60-month (5-year) storage period, with no significant fluctuations in the measured values ​​at each time point, indicating that the product has excellent long-term storage stability.

[0055] Thermal stability test The magnetic fluorescent microspheres of this invention were placed at 37°C for different numbers of days, and their luminescence intensity was tested to evaluate their thermal stability. The results are shown in Table 5.

[0056] Table 5 Thermal stability data at 37℃

[0057] As shown in Table 5, in the accelerated thermal stability test at 37℃, the luminescence intensity of the magnetic fluorescent microspheres of the present invention remained stable at about 17.61 million U after 28 days of storage, and the measured values ​​at each time point showed no significant decay, indicating that the product has good thermal stability and can be stored and transported at room temperature or higher temperatures.

[0058] Example 2 This embodiment provides a method for preparing magnetic fluorescent microspheres: (1) Synthesis of samarium cobalt gold magnetic nanospheres by water boiling method: Heat 900 mL of water to boiling in an electric heater. Then add 1.5 g of samarium trichloride hexahydrate, 4 g of cobalt dichloride hexahydrate, and 2.5 g of tetrachloroauric acid in sequence. Continue heating and boiling for 8 minutes. Then add 4 mL of 0.3% trisodium citrate aqueous solution and continue heating and boiling for 18 minutes. The whole solution turns dark red. Stop heating and let it cool for later use.

[0059] (2) Preparation of time-resolved fluorescent magnetic microspheres: Weigh 0.2 g of EuCl3·6H2O and dissolve it in 90 mL of N-methylpyrrolidone. Stir to dissolve, then add 4 mL of quinoline dimethacrylate and stir until homogeneous. Finally, add 1,10-o-phenanthroline and 3 g of dibenzoylmethane, respectively; the molar ratio of quinoline dimethacrylate:1,10-o-phenanthroline:dibenzoylmethane is 1:5:4. Adjust the pH to 6.8, heat the reaction solution to 65 °C, and react for 8 h. Cool to 25 °C, let stand for 24 h, filter, wash the filter cake several times with anhydrous ethanol, and filter again to obtain the time-resolved fluorescent dye Eu(III).

[0060] Take 900 mL of samarium cobalt gold magnetic nanosphere solution, add 2 g of cetyl ammonium bromide, 4 g of D-glucose-6-phosphate disodium salt, and 3.5 g of polyoxyethylene lauroyl ether and stir to dissolve. Then add the prepared time-resolved fluorescent dye Eu(III) and stir in a water bath at 75°C in the dark. Subsequently, add 0.2 g of acrylic acid, 0.4 g of ethylene glycol dimethacrylate, and 0.3 g of dimethylsiloxane to 9 mL of chloroform and shake to dissolve. The molar ratio of acrylic acid: ethylene glycol dimethacrylate: dimethylsiloxane is 1:2:1.5. Slowly pour the dissolved solution into the solution being stirred. Weigh 0.3 g of hexachloroplatinum(IV) acid (H2PtCl6) and add it. Then add 2.5 g of ethylenediamine and stir in a water bath at 75°C in the dark for 22 hours. After the reaction was completed, the time-resolved fluorescent magnetic microspheres were separated using a magnetic separator. The microspheres were washed five times with MEST (MES buffer with Tween 20) and the separated product was stored in 10 mL of MEST.

[0061] Example 3 This embodiment provides a method for preparing magnetic fluorescent microspheres: (1) Synthesis of samarium cobalt gold magnetic nanospheres by water boiling method: Heat 1100mL of water to boiling in an electric heater. Then add 4.5g of samarium trichloride hexahydrate, 1.5g of cobalt dichloride hexahydrate, and 0.8g of tetrachloroauric acid in sequence. Continue heating and boiling for 12 minutes. Then add 6mL of 0.45% trisodium citrate aqueous solution and continue heating and boiling for 22 minutes. The whole solution turns dark red. Stop heating and let it cool for later use.

[0062] (2) Preparation of time-resolved fluorescent magnetic microspheres: Weigh 0.4 g of EuCl3·6H2O and dissolve it in 110 mL of N-methylpyrrolidone. Stir to dissolve, then add 6 mL of quinoline dimethacrylate and stir until homogeneous. Finally, add 2.5 g of 1,10-o-phenanthroline and 4 g of dibenzoylmethane; the molar ratio of quinoline dimethacrylate to 1,10-o-phenanthroline to dibenzoylmethane is 1:7:5. Adjust the pH to 7.0, heat the reaction solution to 75 °C, and react for 9 h. Cool to 22 °C, let stand for 24 h, filter, wash the filter cake several times with anhydrous ethanol, and filter again to obtain the time-resolved fluorescent dye Eu(III).

[0063] Take 1100 mL of samarium cobalt gold magnetic nanosphere solution, add 3.5 g of cetyl ammonium bromide, 5 g of D-glucose-6-phosphate disodium, and 4.5 g of polyoxyethylene lauroyl ether and stir to dissolve. Then add the prepared time-resolved fluorescent dye Eu(III) and stir in an 85°C water bath in the dark. Subsequently, add 0.25 g of acrylic acid, 0.5 g of ethylene glycol dimethacrylate, and 0.15 g of dimethylsiloxane to 11 mL of chloroform and shake to dissolve. The molar ratio of acrylic acid: ethylene glycol dimethacrylate: dimethylsiloxane is 1:2.5:0.8. Slowly pour the dissolved solution into the stirring solution, weigh out 0.15 g of hexachloroplatinum(IV) acid (H2PtCl6) and add it, then add 3 g of ethylenediamine. Stir in an 85°C water bath in the dark for 26 hours. After the reaction was completed, the time-resolved fluorescent magnetic microspheres were separated using a magnetic separator. The microspheres were washed five times with MEST (MES buffer with Tween 20) and the separated product was stored in 10 mL of MEST.

[0064] As shown in the above embodiments, this invention provides a magnetic fluorescent microsphere and its preparation method. The microsphere has a well-defined core-shell structure; the magnetic core is composed of a ternary metal (smarium, cobalt, and gold), and the outer shell is a cross-linked polymer loaded with europium complexes. The samarium-cobalt-gold magnetic nanoparticles synthesized via an aqueous reduction method exhibit excellent magnetic response performance. Combined with an optimized polymerization coating process, the resulting microspheres significantly outperform existing products in terms of magnetic separation time, luminescence intensity, detection reaction time, shelf life, and thermal stability. Furthermore, these microspheres can be used for the chemiluminescence detection of various veterinary viral diseases, featuring short detection time, high specificity, and low background signal. This invention provides a reliable magnetic fluorescent microsphere platform for highly sensitive, rapid, and stable biological detection.

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A magnetic fluorescent microsphere, characterized in that, The magnetic fluorescent microspheres comprise samarium cobalt gold magnetic nanoparticles and a polymer layer coating the surface of the samarium cobalt gold magnetic nanoparticles, wherein the polymer layer is loaded with Eu. 3+ The complex, wherein the polymer layer is made of a cross-linked polymer containing active functional groups.

2. The magnetic fluorescent microspheres according to claim 1, characterized in that, The active functional group is a carboxyl group.

3. The magnetic fluorescent microspheres according to claim 1, characterized in that, The crosslinked polymer is a copolymer of acrylic monomers, bifunctional acrylate monomers, and organosiloxane monomers.

4. The method for preparing magnetic fluorescent microspheres according to any one of claims 1 to 3, characterized in that, The preparation method includes the following steps: Samarium cobalt gold magnetic nanoparticles, Eu 3+ The complex is mixed with the crosslinking polymer raw material to carry out a crosslinking polymerization reaction, forming a loaded Eu on the surface of the samarium cobalt gold magnetic nanoparticles. 3+ The magnetic fluorescent microspheres are obtained by cross-linking a polymer layer of the complex.

5. The preparation method according to claim 4, characterized in that, The samarium cobalt gold magnetic nanoparticles are prepared by a method including the following steps: Water, samarium trichloride hexahydrate, cobalt dichloride hexahydrate and tetrachloroauric acid are reacted under heating conditions for 5-15 minutes, and then trisodium citrate aqueous solution is added to continue the reaction for 15-25 minutes. The mass ratio of the tetrachloroauric acid to the volume of the water is 0.5~3.0g:800~1200mL; The mass ratio of samarium trichloride hexahydrate to tetrachloroauric acid is 1~4:0.5~3.0, and the mass ratio of cobalt dichloride hexahydrate to tetrachloroauric acid is 1~5:0.5~3.0; The mass percentage concentration of the trisodium citrate aqueous solution is 0.05%~0.5%, and the volume ratio of the trisodium citrate aqueous solution to the volume of water is 3~8:800~1200.

6. The preparation method according to claim 4, characterized in that, The Eu 3+ The complex was prepared by a method comprising the following steps: dissolving EuCl3·6H2O in N-methylpyrrolidone, adding quinoline dimethacrylate, 1,10-o-phenanthroline and dibenzoylmethane, adjusting the pH to 6.2-7.2, reacting at 50-80°C for 7-10 h, cooling, filtering and washing with anhydrous ethanol to obtain the Eu... 3+ Coordination compounds; The mass-to-volume ratio of EuCl3·6H2O to N-methylpyrrolidone is 0.1~0.5g:80~120mL; The molar ratio of quinoline dimethacrylate, 1,10-phenanthroline and dibenzoylmethane is 1:2~8:3~6.

7. The preparation method according to claim 4, characterized in that, The samarium cobalt gold magnetic nanoparticles, Eu 3+ The process of mixing the complex with the crosslinking polymer raw material and carrying out the crosslinking polymerization reaction specifically includes the following steps: S1. A solution of samarium cobalt gold magnetic nanoparticles is mixed with hexadecyl ammonium bromide, D-glucose-6-phosphate disodium salt, polyoxyethylene lauroyl ether, and the aforementioned Eu. 3+ The complexes were mixed and stirred in a water bath at 70-90°C in the dark to obtain the first mixture. S2. Dissolve acrylic acid, ethylene glycol dimethacrylate and dimethylsiloxane in chloroform, add to the first mixture, add hexachloroplatinic acid and ethylenediamine, and stir in a water bath at 70~90℃ in the dark for 20~28h to obtain crude magnetic fluorescent microspheres. S3. Separate the crude product of the magnetic fluorescent microspheres using a magnetic separator to obtain the magnetic fluorescent microspheres.

8. The preparation method according to claim 7, characterized in that, The volume ratio of the samarium cobalt gold magnetic nanoparticle solution to the mass ratio of the hexadecyl ammonium bromide is 800~1200mL:1~4g; The mass ratio of D-glucose-6-phosphate disodium to hexadecylammonium bromide is 2~6:1~4; The mass ratio of the polyoxyethylene lauroyl ether to the hexadecyl ammonium bromide is 3~5:1~4; The molar ratio of acrylic acid, ethylene glycol dimethacrylate and dimethylsiloxane is 1:1~3:1~2; The volume ratio of the trichloromethane to the mass ratio of the acrylic acid is 8-12 mL: 0.1-0.3 g. The mass ratio of hexachloroplatinic acid to acrylic acid is 0.1~0.4:0.1~0.3; The mass ratio of ethylenediamine to acrylic acid is 1~4:0.1~0.

3.

9. A test kit, characterized in that, It comprises the magnetic fluorescent microspheres according to any one of claims 1 to 3.

10. The use of the magnetic fluorescent microspheres according to any one of claims 1 to 3 or the magnetic fluorescent microspheres prepared by the preparation method according to any one of claims 4 to 8 in the preparation of magnetic microparticle chemiluminescence detection reagents, time-resolved fluorescence immunoassay reagents, magnetic enzyme-linked immunosorbent assay reagents or magnetic separation nucleic acid extraction reagents.

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