Magnetic porous microsphere for in-vitro diagnosis and preparation method thereof

Abstract

The invention relates to the technical field of preparation of micro-nano adsorption materials, in particular to a magnetic porous microsphere for in-vitro diagnosis and a preparation method of the magnetic porous microsphere. The invention discloses a preparation method of magnetic porous microspheres for in-vitro diagnosis. The preparation method comprises the following steps: S1, preparing polystyrene-based spheres; s2, preparing porous polystyrene microspheres; s3, preparing magnetic composite microspheres; s4, constructing a porous silicon dioxide middle layer; and S5, introduction of a surface functionalized layer: polymerizing a monomer containing a functional group on the surface of the composite microsphere coated with a layer of porous silicon dioxide through a monomer swelling polymerization method, so as to introduce a functionalized polymer layer on the surface of the microsphere and in a porous channel, thereby obtaining the magnetic porous microsphere for in vitro diagnosis. The prepared magnetic porous microspheres are low in settling velocity, good in magnetic separation efficiency, good in monodispersity, uniform in particle size distribution, high in magnetic content, high in surface functional group content and wide in application scene.

Description

Technical Field

[0001] This application relates to the field of micro / nano adsorption material preparation technology, and in particular to a magnetic porous microsphere for in vitro diagnostics and its preparation method. Background Technology

[0002] In vitro diagnostics (IVD), a technology that obtains clinical diagnostic information by testing human samples outside the human body, offers advantages such as speed, convenience, and effectiveness. However, the domestic market share of upstream reagent raw materials is relatively small, and the quality of these raw materials determines the stability and sensitivity of the diagnostics. Micro- and nanoporous materials, due to their significantly larger specific surface area and unique pore structure compared to conventional macroscopic materials, can provide a large number of active sites. This not only facilitates surface molecular modification but also allows for the encapsulation of magnetic materials within the pores, thereby increasing magnetic content and stability, and fundamentally improving detection sensitivity. These micro- and nanoporous materials, with different surface modifications, can selectively detect small molecules, nucleic acids, proteins, and microorganisms, making them crucial in in vitro diagnostic applications such as bioanalysis and biosensors. Currently, commercially available magnetic microspheres are mostly embedded in polymer matrices using physical blending or in-situ precipitation methods. These methods have significant drawbacks: 1) Conflict between magnetic content and dispersibility: Increasing the magnetic particle content to improve magnetic responsiveness easily leads to microsphere aggregation, destroying monodispersity and affecting the uniformity and precision of detection; 2) Limited specific surface area: Most are solid or low-porosity structures with small specific surface area, resulting in fewer active sites for biomolecules (such as antigens, antibodies, and nucleic acid probes) to bind, thus limiting the amplification of detection signals; 3) Limited functionalization methods: Surface functional groups are usually introduced through post-modification grafting, resulting in low density, uneven distribution, and poor binding strength, making them prone to detachment in complex biological systems; 4) Complex preparation process: Traditional preparation methods for highly cross-linked porous microspheres have low production efficiency and are difficult to meet the needs of large-scale production. For example, patent CN 119819269 A discloses a magnetic porous microsphere for in vitro diagnostics and its preparation method. It describes the preparation of monodisperse polystyrene microspheres using styrene as the polymer monomer via dispersion polymerization. Ferric chloride and ferrous sulfate are selected as precursors, and magnetic nanoparticles are synthesized under alkaline conditions through co-precipitation. The initial loading amount is modified, and magnetic nanoparticles are loaded onto the surface and pores of the polymer porous microspheres via a multi-step seed growth method, synthesizing composite magnetic polystyrene microspheres with high magnetic content, good magnetic responsiveness, and superparamagnetism. Finally, functional groups are introduced onto the surface of the magnetic spheres via monomer swelling polymerization, resulting in magnetic porous microspheres. However, this method suffers from problems such as a small specific surface area of ​​the prepared magnetic porous microspheres, low content of introduced functional groups, and poor detection sensitivity.

[0003] Therefore, developing a magnetic porous microsphere with uniform particle size, high and stable magnetic content, high specific surface area, porous structure, flexible control of surface functional group density and type, and simple, efficient and environmentally friendly preparation process has significant industrial value. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this application provides a magnetic porous microsphere for in vitro diagnostics and its preparation method. The magnetic porous microspheres prepared in this application have excellent monodispersity, uniform particle size, large specific surface area, tunable surface functional groups, and rapid magnetic response, which can better bind to antigens and antibodies, thereby enabling in vitro diagnostic functional detection such as nucleic acid extraction and chemiluminescence.

[0005] In a first aspect, this application provides a method for preparing magnetic porous microspheres for in vitro diagnostics, employing the following technical solution: A method for preparing magnetic porous microspheres for in vitro diagnostics includes the following steps: S1. Preparation of polystyrene-based spheres: Monodisperse polystyrene-based spheres were prepared by dispersion polymerization using styrene as the polymerizing monomer. S2. Preparation of porous polystyrene microspheres: Monodisperse porous polystyrene microspheres are prepared by swelling monodisperse polystyrene-based spheres. S3. Preparation of magnetic composite microspheres: Porous polystyrene microspheres are dispersed in an emulsifier, and then a composite iron salt solution is added under a nitrogen atmosphere. The mixture is stirred at 15-35℃ for 1-2 hours. The pH of the reaction system is then adjusted to 10-11, and the reaction continues for 12-16 hours. Finally, the mixture is magnetically separated to obtain magnetic composite microspheres. S4. Construction of the porous silica intermediate layer: Silica is encapsulated on the surface of the magnetic composite microspheres to obtain composite microspheres with a porous silica coating on the surface. S5. Introduction of surface functionalized layer: Monomers containing functional groups are polymerized on the surface of composite microspheres coated with a layer of porous silica by monomer swelling polymerization, thereby introducing a functionalized polymer layer on the surface of the microspheres and in the porous channels to obtain magnetic porous microspheres for in vitro diagnostics.

[0006] Preferably, in step S1, the specific process for preparing polystyrene-based spheres is as follows: ammonium persulfate, polyvinylpyrrolidone, and styrene are dissolved in an alcohol-water mixed solution at a mass ratio of 0.1-0.3:0.05-0.1:10-12, then the mixture is heated to 55-60℃ and reacted for 7-8 hours. After centrifugation, washing, and vacuum drying at 83℃ for 6-8 hours, polystyrene-based spheres are obtained.

[0007] Preferably, in step S2, the specific process for preparing porous polystyrene microspheres is as follows: polystyrene-based spheres are dispersed in a surfactant, polyvinylpyrrolidone is added, and the mixture is homogeneous to form an aqueous phase; an initiator is added to the polymerizing monomer, a porogen, a comonomer, and a crosslinking agent are added, the mixture is homogeneous, and then added to the aqueous phase; ultrasonic emulsification is performed for 2-2.5 hours, followed by swelling at 30-35°C for 20-24 hours, followed by heating to 70-75°C and nitrogen-purified polymerization for 18-24 hours.

[0008] Preferably, the surfactant is selected from at least one of sodium dodecyl sulfate and sodium dodecylbenzene sulfonate, and the aqueous solution has a mass concentration of 0.5%, and the amount of surfactant used is 0.5-1 times the mass of the polystyrene spheres; the amount of polyvinylpyrrolidone used is 2-3 times the mass of the polystyrene spheres; the polymerizing monomer is methyl methacrylate, and its amount is 23-25 ​​times the mass of the polystyrene spheres; the initiator is selected from at least one of potassium persulfate, azobisisobutyronitrile, and sodium persulfate, and its amount is 2-4% of the mass of the polymerizing monomer; the porogen is selected from at least one of toluene, acetone, dichloromethane, n-hexane, and n-octane, and its amount is 23-26 times the mass of the polystyrene spheres; the comonomer is at least one of methacrylic acid and maleic anhydride, and its amount is 3-5 times the mass of the polystyrene spheres; the crosslinking agent is selected from at least one of ethylene glycol dimethacrylate and tert-butyl peroxide, and its amount is 13-15 times the mass of the polystyrene spheres.

[0009] Preferably, in step S3, the ratio of the porous polystyrene microspheres, emulsifier, and composite iron salt solution is 0.3-0.4g:12-20mL:7-8mL.

[0010] Preferably, in step S3, the emulsifier is prepared by dissolving 2-3g sodium dodecylbenzenesulfonate and 4-5g polyvinylpyrrolidone in 100mL of deionized water; the composite iron salt solution is prepared by dissolving 5-6g ferrous chloride tetrahydrate and 2-3g ferric chloride hexahydrate in 50mL of deionized water.

[0011] Preferably, in step S4, the specific process for constructing the porous silica intermediate layer is as follows: 2-3g of magnetic composite microspheres are added to 200-300mL of ethanol, stirred at 300-400rpm for 20-30 minutes under ultrasonication, 5-5.5mL of ammonia is added, and ultrasonication is continued for another 20-30 minutes. Then, 10-12mL of methyl orthosilicate is added, and the mixture is reacted in a water bath at 30-33℃ under stirring at 300-400rpm for 2-3 hours. The product is magnetically separated, and washed three times each with ethanol and water to obtain composite microspheres with a porous silica coating on the surface.

[0012] By employing the above technical solution, the prepared porous silica interlayer is not only a structural isolation layer but also a key functional component for achieving high performance in the final product. Its main functions and synergistic effects are reflected in the following aspects: 1) Physical isolation and anti-agglomeration: The porous silica interlayer effectively isolates the internal magnetic nanoparticles. This prevents the magnetic nanoparticles from agglomerating due to strong magnetic dipole interactions, thus significantly improving the stability of the entire microsphere colloidal system. This function synergizes with the goal of "combining more magnetic nanoparticles to increase magnetic content" in step S3. By preventing agglomeration, it ensures that the magnetic particles remain uniformly dispersed even under high loading, thereby maintaining the monodispersity and particle size uniformity of the microspheres while improving magnetic responsiveness and detection sensitivity. 2) Encapsulation and purification: As a physical barrier, the interlayer encapsulates the internal magnetic particles. This characteristic effectively avoids interference from free magnetic particles in subsequent detection processes, ensuring the accuracy and reliability of the detection. This function guarantees the effectiveness of the surface functionalization modification in step S5. A pure and stable core provides a reliable platform for the specific binding of outer functional molecules to target substances (such as antigens and antibodies), ensuring accurate transmission of diagnostic signals. In summary, the porous silica interlayer acts as a bridge within the entire magnetic porous microsphere. It stabilizes the high-magnetic-content core and provides a solid foundation for efficient external biosensor functions, making it crucial for achieving excellent overall performance.

[0013] Preferably, in step S5, the specific process for introducing the surface functionalized layer is as follows: 2-3g of composite microspheres coated with a layer of porous silica are dispersed in 200ml of ethanol, 2-3mL of ammonia and 2-3mL of γ-aminopropyltrimethoxysilane are added, and the mixture is stirred for 8-12 hours. After magnetic separation and washing with ethanol, amino-modified microspheres are obtained. Then, the amino-modified microspheres are dispersed in 200-250mL of N,N-dimethylformamide containing 16-20mg / mL of the functional monomer. The mixture is stirred for 12-14 hours, centrifuged, and the product is washed three times with ethanol and water, then dried to obtain magnetic porous microspheres. The functional monomer is prepared by mixing methacrylic acid and adipic anhydride in a mass ratio of 1:1-2.

[0014] By adopting the above technical solution, the introduction of the surface functionalized layer in step S5 is a decisive step in realizing the in vitro diagnostic application value of magnetic porous microspheres. This step, through precise chemical reactions, accurately anchors bioactive functional groups on the surface and internal channels of the microspheres. Its core functions and synergistic effects are as follows: 1) By introducing active functional groups such as carboxyl groups (-COOH), a chemical bridge is provided for subsequent covalent conjugation of biomolecules such as antibodies, antigens, and nucleic acid probes. This transforms the microspheres from an inert material into an active carrier capable of specifically capturing target molecules. 2) Improved biocompatibility and stability: The functionalized polymer layer covering the microsphere surface provides a more hydrophilic and biofriendly interface, reducing non-specific adsorption and improving dispersion stability in biological samples (such as serum and plasma). This is crucial for ensuring the accuracy of detection results. 3) Maximizing the utilization of specific surface area: Through the "monomer swelling polymerization method," functional monomers can not only modify the outer surface of the microspheres but also penetrate into the porous silica layer and the channels of the internal polystyrene core for polymerization. This functionalizes the large internal surface area of ​​the microspheres, significantly increasing the total number of sites available for binding biomolecules, thereby improving detection sensitivity and loading capacity. The process design of step S5 demonstrates a sophisticated synergistic effect, specifically: 1) Synergy between amino modification and preceding steps, and with step S4: The porous silica layer constructed in step S4 is an ideal substrate for amino modification. The methoxy group in γ-aminopropyltrimethoxysilane (APTMS) undergoes a hydrolytic condensation reaction with the silanol groups on the silica surface, forming a strong Si-O-Si covalent bond, thus stably introducing the amino group (-NH2) onto the microsphere surface. This step prepares a "chemical handle" for subsequent functional monomer grafting. 2) Synergy between functional monomers, specifically the synergy between methacrylic acid (MAA) and adipic anhydride (AAH): These two monomers work together to efficiently increase the carboxyl content of the microspheres. Methacrylic acid forms polymer chains through free radical polymerization, directly introducing carboxyl groups. Adipic anhydride undergoes a ring-opening amidation reaction with the amino groups introduced by APTMS on the microsphere surface, generating segments terminated with carboxyl groups. Carboxyl groups can be introduced in large quantities through different pathways and at different sites (surface and pores) on the microspheres, achieving synergy between methacrylic acid (MAA) and adipic anhydride (AAH), ensuring a high and accessible content of surface functional groups in the final product. 3) The synergy between the functionalized layer and the overall microsphere structure: the high content of carboxyl groups ensures efficient biomolecule immobilization, while the high magnetic content introduced in step S3 ensures rapid magnetic separation. The combination of these two aspects achieves a perfect balance between "efficient capture" and "rapid separation," which is precisely the core performance required for automated, high-throughput in vitro diagnostic reagents. The functionalized layer modifies the entire porous network, allowing the large specific surface area advantage of the microspheres to be fully utilized. More binding sites mean higher detection sensitivity and a wider linear range.In summary, the surface functionalization step S5 is a crucial step in transforming the meticulously constructed "well-structured microsphere carrier" into a "powerful diagnostic tool." Through amino modification and a bifunctional monomer strategy, it achieves a high-density, uniform introduction of carboxyl functional groups. These functional groups, combined with the monodispersity, high magnetic responsiveness, and large specific surface area of ​​the microspheres, create a synergistic amplification effect, ultimately contributing to the superior performance of this magnetic porous microsphere in in vitro diagnostic applications such as nucleic acid extraction and chemiluminescence.

[0015] Secondly, this application provides a magnetic porous microsphere for in vitro diagnostics, employing the following technical solution: As a general technical concept, this application also provides the above-mentioned magnetic porous microspheres for in vitro diagnostics, which are prepared by the above-mentioned method for preparing magnetic porous microspheres for in vitro diagnostics.

[0016] In summary, the beneficial technical effects of this application are as follows: 1. Core structural advantages: Using monodisperse polystyrene microspheres as a perfect template, and through multi-step precise control, the final product has excellent monodispersity and highly uniform particle size, which is the fundamental guarantee for achieving high-precision and high-throughput detection.

[0017] 2. Unique porous design: The pores of the microspheres are not only distributed on the surface of the shell, but also uniformly distributed in the inner core layer. This structure creates a huge specific surface area, which can efficiently load magnetic particles and facilitate the free diffusion of biomolecules, thereby improving binding capacity and reaction efficiency.

[0018] 3. High-performance magnetic core: The high content of magnetic nanoparticles generated in situ in the porous framework endows the microspheres with rapid magnetic response, enabling rapid liquid-solid separation within tens of seconds, greatly shortening the detection time.

[0019] 4. Key Stability Guarantee: The introduced porous silica interlayer plays a crucial role. It effectively isolates internal magnetic particles, preventing their aggregation and leakage, thus greatly improving the product's colloidal stability and reliability.

[0020] 5. Highly active functionalized surface: Through the synergistic effect of amino modification and methacrylic acid / adipic anhydride, a high density of carboxyl functional groups are introduced into the surface and channels of the microspheres, providing a solid foundation for efficient coupling of biomolecules such as antibodies and antigens, and ensuring high detection sensitivity.

[0021] 6. Comprehensive application value: The organic combination of the above characteristics (uniformity, porosity, strong magnetism, stability, and high activity) enables the microspheres to show significant advantages in high-end in vitro diagnostic fields such as nucleic acid extraction and chemiluminescence immunoassay, and their performance is comprehensively superior to traditional magnetic beads. Detailed Implementation

[0022] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this application. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0023] Example 1 A method for preparing magnetic porous microspheres for in vitro diagnostics includes the following steps: S1. Preparation of polystyrene-based spheres: Dissolve 0.1g ammonium persulfate, 0.05g polyvinylpyrrolidone and 10g styrene in a mixed solution consisting of 10mL deionized water and 40mL anhydrous ethanol, then heat to 55℃ and react for 8h. Centrifuge, wash, and vacuum dry at 83℃ for 6h to obtain polystyrene-based spheres. S2. Preparation of porous polystyrene microspheres: 2g of polystyrene-based microspheres were dispersed in 200mL of 0.5% sodium dodecyl sulfate solution, and 4g of polyvinylpyrrolidone were added. The mixture was stirred until homogeneous to form an aqueous phase. 0.92g of potassium persulfate was added to 46g of methyl methacrylate, followed by 46g of toluene, 26g of ethylene glycol dimethacrylate, and 6g of methacrylic acid. The mixture was stirred until homogeneous and then added to the aqueous phase. The mixture was ultrasonically emulsified for 2 hours, then swollen at 30℃ for 24 hours. The temperature was then raised to 70℃, and nitrogen gas was introduced for polymerization for 24 hours. The product was vacuum filtered and washed with anhydrous ethanol and ultrapure water by centrifugation to obtain porous polystyrene microspheres. S3. Preparation of magnetic composite microspheres: 3g of porous polystyrene microspheres were dispersed in 120mL of emulsifier, followed by the addition of 30mL of composite iron salt solution under a nitrogen atmosphere. The mixture was stirred at 15℃ for 2h, and the pH of the reaction system was adjusted to 10 with 25% ammonia solution. The reaction was continued for 12h, and finally magnetic separation was performed to obtain magnetic composite microspheres. The emulsifier was prepared by dissolving 4g of sodium dodecylbenzenesulfonate and 10g of polyvinylpyrrolidone in 200mL of deionized water. The composite iron salt solution was prepared by dissolving 5g of ferrous chloride tetrahydrate and 3g of ferric chloride hexahydrate in 50mL of deionized water. S4. Construction of the porous silica intermediate layer: 3g of magnetic composite microspheres were added to 200mL of ethanol and stirred at 300rpm for 30 minutes under ultrasonication. 5mL of ammonia water was added and ultrasonication was continued for 20 minutes. 10mL of methyl orthosilicate was added and reacted in a 30℃ water bath under stirring at 300rpm for 2 hours. The product was magnetically separated and washed 3 times each with ethanol and water to obtain composite microspheres with a porous silica layer on the surface. S5. Introduction of surface functionalized layer: 2g of composite microspheres coated with a layer of porous silica were dispersed in 200ml of ethanol, 2mL of ammonia and 2mL of γ-aminopropyltrimethoxysilane were added, and the mixture was stirred for 8 hours. After magnetic separation and washing with ethanol, amino-modified microspheres were obtained. Then, the amino-modified microspheres were dispersed in 200mL of N,N-dimethylformamide containing 16mg / mL of functional monomer, and the mixture was stirred for 12 hours. After centrifugation, the product was washed three times with ethanol and water and dried to obtain magnetic porous microspheres. The functional monomer was prepared by mixing methacrylic acid and adipic anhydride in a mass ratio of 1:1.

[0024] Example 2 A method for preparing magnetic porous microspheres for in vitro diagnostics includes the following steps: S1. Preparation of polystyrene-based spheres: Dissolve 0.3g ammonium persulfate, 0.1g polyvinylpyrrolidone and 12g styrene in a mixed solution consisting of 10mL deionized water and 40mL anhydrous ethanol, then heat to 60℃ and react for 7h. Centrifuge, wash, and vacuum dry at 83℃ for 8h to obtain polystyrene-based spheres. S2. Preparation of porous polystyrene microspheres: 2g of polystyrene-based microspheres were dispersed in 400mL of sodium dodecylbenzenesulfonate with a mass concentration of 0.5%, and 6g of polyvinylpyrrolidone was added. The mixture was stirred evenly to form an aqueous phase. 2g of azobisisobutyronitrile was added to 50g of methyl methacrylate, followed by 52g of acetone, 30g of tert-butyl peroxide, and 10g of maleic anhydride. The mixture was stirred evenly and then added to the aqueous phase. The mixture was ultrasonically emulsified for 2.5 hours, then swollen at 35℃ for 20 hours. The temperature was then raised to 75℃, and nitrogen gas was introduced for polymerization for 18 hours. The product was vacuum filtered and washed with anhydrous ethanol and ultrapure water by centrifugation to obtain porous polystyrene microspheres. S3. Preparation of magnetic composite microspheres: 4g of porous polystyrene microspheres were dispersed in 200mL of emulsifier, followed by the addition of 80mL of composite iron salt solution under a nitrogen atmosphere. The mixture was stirred at 35℃ for 1h, and the pH of the reaction system was adjusted to 11 with 25% ammonia solution. The reaction was continued for 16h, and finally, magnetic separation was performed to obtain magnetic composite microspheres. The emulsifier was prepared by dissolving 6g of sodium dodecylbenzenesulfonate and 8g of polyvinylpyrrolidone in 200mL of deionized water. The composite iron salt solution was prepared by dissolving 6g of ferrous chloride tetrahydrate and 2g of ferric chloride hexahydrate in 50mL of deionized water. S4. Construction of the porous silica intermediate layer: 3g of magnetic composite microspheres were added to 300mL of ethanol and stirred at 400rpm for 20 minutes under ultrasonication. 5.5mL of ammonia water was added and ultrasonication was continued for 30 minutes. Then, 12mL of methyl orthosilicate was added and reacted in a 33℃ water bath under stirring at 400rpm for 3 hours. The product was magnetically separated and washed 3 times each with ethanol and water to obtain composite microspheres with a porous silica layer on the surface. S5. Introduction of surface functionalized layer: 3g of composite microspheres coated with a layer of porous silica were dispersed in 200ml of ethanol, 3mL of ammonia and 3mL of γ-aminopropyltrimethoxysilane were added, and the mixture was stirred for 12 hours. After magnetic separation and washing with ethanol, amino-modified microspheres were obtained. Then, the amino-modified microspheres were dispersed in 250mL of N,N-dimethylformamide containing 20mg / mL of functional monomer, and the mixture was stirred for 14 hours. After centrifugation, the product was washed three times with ethanol and water and dried to obtain magnetic porous microspheres. The functional monomer was prepared by mixing methacrylic acid and adipic anhydride in a mass ratio of 1:2.

[0025] Example 3 A method for preparing magnetic porous microspheres for in vitro diagnostics includes the following steps: S1. Preparation of polystyrene-based spheres: Dissolve 0.2g ammonium persulfate, 0.08g polyvinylpyrrolidone and 11g styrene in a mixed solution consisting of 10mL deionized water and 40mL anhydrous ethanol, then heat to 57℃ and react for 7.8h. Centrifuge, wash, and vacuum dry at 83℃ for 7h to obtain polystyrene-based spheres. S2. Preparation of porous polystyrene microspheres: 2g of polystyrene-based microspheres were dispersed in 300mL of 0.5% sodium dodecyl sulfate, and 5g of polyvinylpyrrolidone were added. The mixture was stirred until homogeneous to form an aqueous phase. 1.3g of sodium persulfate was added to 48g of methyl methacrylate, followed by 50g of dichloromethane, 28g of ethylene glycol dimethacrylate, and 8g of methacrylic acid. The mixture was stirred until homogeneous and then added to the aqueous phase. The mixture was ultrasonically emulsified for 2.3 hours, then swollen at 32℃ for 22 hours. The temperature was then raised to 73℃, and nitrogen gas was introduced for polymerization for 21 hours. The product was vacuum filtered and washed with anhydrous ethanol and ultrapure water by centrifugation to obtain porous polystyrene microspheres. S3. Preparation of magnetic composite microspheres: 3.5 g of porous polystyrene microspheres were dispersed in 160 mL of emulsifier, and then 50 mL of composite iron salt solution was added under a nitrogen atmosphere. The mixture was stirred at 25 °C for 1.2 h. The pH of the reaction system was then adjusted to 10.5 with 25% ammonia water, and the reaction was continued for 14 h. Finally, magnetic separation was performed to obtain magnetic composite microspheres. The emulsifier was prepared by dissolving 4.8 g of sodium dodecylbenzenesulfonate and 9 g of polyvinylpyrrolidone in 200 mL of deionized water. The composite iron salt solution was prepared by dissolving 5.5 g of ferrous chloride tetrahydrate and 2.4 g of ferric chloride hexahydrate in 50 mL of deionized water. S4. Construction of the porous silica intermediate layer: 2.5g of magnetic composite microspheres were added to 250mL of ethanol and stirred at 350rpm for 25 minutes under sonication. 5.3mL of ammonia water was added and sonicated for another 25 minutes. Then, 11mL of methyl orthosilicate was added and reacted in a 32℃ water bath at 350rpm for 2.5 hours. The product was magnetically separated and washed three times each with ethanol and water to obtain composite microspheres with a porous silica layer on the surface. S5. Introduction of surface functionalized layer: 2.5g of composite microspheres coated with a layer of porous silica were dispersed in 200ml of ethanol, 2.5mL of ammonia and 2.4mL of γ-aminopropyltrimethoxysilane were added, and the mixture was stirred for 10 hours. After magnetic separation and washing with ethanol, amino-modified microspheres were obtained. Then, the amino-modified microspheres were dispersed in 230mL of N,N-dimethylformamide containing 18mg / mL of functional monomer, and the mixture was stirred for 13 hours. After centrifugation, the product was washed three times with ethanol and water and dried to obtain magnetic porous microspheres. The functional monomer was prepared by mixing methacrylic acid and adipic anhydride in a mass ratio of 1:1.5.

[0026] Comparative Example 1 Similar to Example 3, except that: the processes in step S4, the construction of the porous silica intermediate layer, and the amino modification process in step S5 were not performed; instead, the magnetic composite microspheres obtained in step S3 were directly dispersed in 230 mL of N,N-dimethylformamide containing 18 mg / mL of functional monomer, stirred for 13 hours, centrifuged, and the product was washed three times with ethanol and water and then dried to obtain magnetic porous microspheres. The functional monomer was prepared by mixing methacrylic acid and adipic anhydride in a mass ratio of 1:1.5.

[0027] Comparative Example 2 Same as Example 3, except that in step S5, the functional monomer is methacrylic acid.

[0028] Comparative Example 3 Same as Example 3, except that in step S5, the functional monomer is adipic anhydride.

[0029] 1. Performance Testing The magnetic porous microspheres prepared in Examples 1-3 and Comparative Examples 1-3 were subjected to the following performance tests. Three samples were taken from each group for testing, and the average value of the results was taken. The results are shown in Table 1.

[0030] The particle size of the above-mentioned magnetic porous microspheres was tested using a laser particle size analyzer (DLS). The specific surface area was measured using the Bestar Instruments BSD-BET400 fully automated nitrogen adsorption BET specific surface area tester. The carboxyl content of the above-mentioned magnetic porous microspheres was tested using a fully automated acid-base titrator. Magnetic separation efficiency test: Using the above-mentioned magnetic porous microspheres as samples, 1 mL of samples with the same concentration (1 mg / mL) were taken and magnetic adsorption separation test was performed using a magnet under a magnetic field of 0.5 T. Settling velocity test: Using the above-mentioned magnetic porous microspheres as samples, 1 mL of samples with the same concentration (1 mg / mL) were taken and the natural settling rate was observed and the settling time was recorded. Table 1 Test Results

[0031] Analyzing the data in Table 1, we can see that: 1) The magnetic porous microspheres for in vitro diagnostics prepared in Examples 1-3 have slow sedimentation speed, good magnetic separation efficiency, good monodispersity, uniform particle size distribution, and high surface carboxyl content, and have a wide range of applications.

[0032] 2) A comparative analysis of the performance of the magnetic porous microspheres for in vitro diagnostics prepared in Example 3 and Comparative Example 1 shows that the prepared porous silica interlayer is not only a structural isolation layer, but also a key functional component for achieving high performance in the final product. The porous silica interlayer effectively isolates the internal magnetic nanoparticles. This prevents the magnetic nanoparticles from agglomerating due to strong magnetic dipole interactions, thereby significantly improving the stability of the entire microsphere colloidal system. It stabilizes the high magnetic content core inside and provides a solid foundation for efficient external biodetection functions. Furthermore, subsequent amino modification can significantly increase the carboxyl content of the magnetic porous microspheres.

[0033] 3) A comparative analysis of the performance of the magnetic porous microspheres for in vitro diagnostics prepared in Example 3 and Comparative Examples 2-3 shows that the synergistic effect of methacrylic acid (MAA) and adipic anhydride (AAH) effectively increases the carboxyl content of the microspheres. Methacrylic acid forms polymer chains through free radical polymerization, directly introducing carboxyl groups. Adipic anhydride then undergoes a ring-opening amidation reaction with the amino groups introduced by APTMS on the surface of the microspheres, generating segments terminated with carboxyl groups. Carboxyl groups can be introduced in large quantities through different pathways and at different sites (surface and pores) of the microspheres, achieving the synergistic effect of methacrylic acid (MAA) and adipic anhydride (AAH), ensuring that the final product has a high and accessible content of surface functional groups.

[0034] 2. Specific adsorption properties Using the magnetic porous microspheres for in vitro diagnostics prepared in Example 3 and commercial control magnetic beads as samples, the magnetic porous microspheres without antibody conjugation and protein complex were incubated in a chemiluminescence immunoassay analyzer, washed, and the luminescence value was detected to conduct a specific adsorption test on the magnetic porous microspheres themselves and to observe the specific adsorption performance of the carboxyl magnetic beads. The results are shown in Table 2.

[0035] Table 2. Test results of specific adsorption performance of carboxyl magnetic beads

[0036] As can be seen from Table 2, the magnetic porous microspheres for in vitro diagnostics prepared in Example 3 have a lower background value (background value refers to the background value under natural conditions, including non-specific binding values ​​caused by non-antigen antibody specific binding, and the lower the value, the better) and less non-specific adsorption, indicating that the magnetic porous microspheres prepared in this application have excellent specific adsorption capacity.

[0037] 3. Nucleic acid extraction performance test: The in vitro diagnostic magnetic porous microspheres prepared in Example 3 and commercial control magnetic beads were used as samples for nucleic acid extraction performance testing. The specific steps included: (1) Add 100 μL of the sample to be tested to 250 μL of working solution, shake and mix well, and heat in a 55℃ dry bath for 5 min for lysis; (2) After instantaneous centrifugation for 5 s, place the centrifuge tube on a magnetic separator for 1 min, and then remove the supernatant; (3) Add 600 μL of extraction reagent II, cap the tube, shake and mix well for about 5 s, centrifuge briefly, place the centrifuge tube on a magnetic separator for 1 min, and then remove the supernatant; (4) After standing for 1 min, remove the liquid remaining at the bottom of the tube; (5) Add 100 μL of elution buffer, cap the tube, shake and mix well for about 5 s, and centrifuge briefly; (6) Place the centrifuge tube on an 80℃ dry bath and heat and wash for 2 min; (7) Place the centrifuge tube on a magnetic separator and take the supernatant for later use; (8) Take out each component from the nucleic acid extraction packaging box (purchased from Zhejiang Yiside Biotechnology Co., Ltd.), and shake the liquid that may adhere to the aluminum membrane and well wall of the 96-well plate to the bottom of the well, and let it stand for 5 min for later use; (9) Carefully tear open the aluminum membrane of the 96-well deep well plate, and add 15 μL of proteinase K and 200 μL of the sample to be tested to the AI1~HI and A7~H7 wells in sequence; (10) Use a nucleic acid extractor, set the program, and complete the nucleic acid extraction work. The extraction process takes about 10 min; the results are shown in Table 3.

[0038] Table 3 Comparison of Nucleic Acid Extraction Performance of Carboxyl Magnetic Beads

[0039] As can be seen from Table 3, the magnetic porous microspheres prepared using this application have good nucleic acid extraction performance.

[0040] The above embodiments are only used to explain the technical solutions of this application and are not intended to limit it. Although the above embodiments have provided specific descriptions of this application, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation methods of this application. Any modifications and equivalent substitutions that do not depart from the spirit and scope of this application should be covered within the protection scope of this application.

Claims

1. A method for preparing magnetic porous microspheres for in vitro diagnostics, characterized in that, Includes the following steps: S1. Preparation of polystyrene-based spheres: Monodisperse polystyrene-based spheres were prepared by dispersion polymerization using styrene as the polymerizing monomer. S2. Preparation of porous polystyrene microspheres: Monodisperse porous polystyrene microspheres are prepared by swelling monodisperse polystyrene-based spheres. S3. Preparation of magnetic composite microspheres: Porous polystyrene microspheres are dispersed in an emulsifier, and then a composite iron salt solution is added under a nitrogen atmosphere. The mixture is stirred at 15-35℃ for 1-2 hours. The pH of the reaction system is then adjusted to 10-11, and the reaction continues for 12-16 hours. Finally, the mixture is magnetically separated to obtain magnetic composite microspheres. S4. Construction of the porous silica intermediate layer: Silica is encapsulated on the surface of the magnetic composite microspheres to obtain composite microspheres with a porous silica coating on the surface. S5. Introduction of surface functionalized layer: 2-3g of composite microspheres coated with a layer of porous silica were dispersed in 200ml of ethanol, 2-3mL of ammonia and 2-3mL of γ-aminopropyltrimethoxysilane were added, and the mixture was stirred for 8-12 hours. After magnetic separation and washing with ethanol, amino-modified microspheres were obtained. Then, the amino-modified microspheres were dispersed in 200-250mL of N,N-dimethylformamide containing 16-20mg / mL of functional monomers. After stirring for 12-14 hours, the mixture was centrifuged, and the product was washed three times with ethanol and water and then dried to obtain magnetic porous microspheres.

2. The method for preparing magnetic porous microspheres for in vitro diagnostics according to claim 1, characterized in that, In step S1, the specific process for preparing polystyrene-based spheres is as follows: ammonium persulfate, polyvinylpyrrolidone, and styrene are dissolved in an alcohol-water mixed solution at a mass ratio of 0.1-0.3:0.05-0.1:10-12, then the mixture is heated to 55-60℃ and reacted for 7-8 hours. After centrifugation, washing, and vacuum drying at 83℃ for 6-8 hours, polystyrene-based spheres are obtained.

3. The method for preparing magnetic porous microspheres for in vitro diagnostics according to claim 1, characterized in that, In step S2, the specific process for preparing porous polystyrene microspheres is as follows: polystyrene-based spheres are dispersed in a surfactant, polyvinylpyrrolidone is added, and the mixture is homogeneous to form an aqueous phase; an initiator is added to the polymerizing monomer, a pore-forming agent, a comonomer, and a crosslinking agent are added, the mixture is homogeneous, and then added to the aqueous phase. The mixture is ultrasonically emulsified for 2-2.5 hours, then swollen at 30-35°C for 20-24 hours, and then heated to 70-75°C for a nitrogen-purified polymerization reaction for 18-24 hours.

4. The method for preparing magnetic porous microspheres for in vitro diagnostics according to claim 3, characterized in that, The surfactant is selected from at least one of sodium dodecyl sulfate and sodium dodecylbenzene sulfonate, and its aqueous solution has a mass concentration of 0.5%. The amount of surfactant used is 0.5-1 times the mass of the polystyrene spheres. The amount of polyvinylpyrrolidone used is 2-3 times the mass of the polystyrene spheres. The polymerizing monomer is methyl methacrylate, and its amount is 23-25 ​​times the mass of the polystyrene spheres. The initiator is selected from at least one of potassium persulfate, azobisisobutyronitrile, and sodium persulfate, and its amount is 2-4% of the mass of the polymerizing monomer. The porogen is selected from at least one of toluene, acetone, dichloromethane, n-hexane, and n-octane, and its amount is 23-26 times the mass of the polystyrene spheres. The comonomer is at least one of methacrylic acid and maleic anhydride, and its amount is 3-5 times the mass of the polystyrene spheres. The crosslinking agent is selected from at least one of ethylene glycol dimethacrylate and tert-butyl peroxide, and its amount is 13-15 times the mass of the polystyrene spheres.

5. The method for preparing magnetic porous microspheres for in vitro diagnostics according to claim 1, characterized in that, In step S3, the ratio of the porous polystyrene microspheres, emulsifier, and composite iron salt solution is 0.3-0.4g:12-20mL:7-8mL.

6. The method for preparing magnetic porous microspheres for in vitro diagnostics according to claim 1, characterized in that, In step S3, the emulsifier is prepared by dissolving 2-3g sodium dodecylbenzenesulfonate and 4-5g polyvinylpyrrolidone in 100mL of deionized water; the composite iron salt solution is prepared by dissolving 5-6g ferrous chloride tetrahydrate and 2-3g ferric chloride hexahydrate in 50mL of deionized water.

7. The method for preparing magnetic porous microspheres for in vitro diagnostics according to claim 1, characterized in that, In step S4, the specific process for constructing the porous silica intermediate layer is as follows: 2-3g of magnetic composite microspheres are added to 200-300mL of ethanol, stirred at 300-400rpm for 20-30 minutes under ultrasonication, 5-5.5mL of ammonia water is added, and ultrasonication is continued for another 20-30 minutes. Then, 10-12mL of methyl orthosilicate is added, and the mixture is reacted in a water bath at 30-33℃ under stirring at 300-400rpm for 2-3 hours. The product is magnetically separated, and washed three times each with ethanol and water to obtain composite microspheres with a porous silica coating on the surface.

8. The method for preparing magnetic porous microspheres for in vitro diagnostics according to claim 1, characterized in that, The functional monomer is prepared by mixing methacrylic acid and adipic anhydride in a mass ratio of 1:1-2.

9. A magnetic porous microsphere for in vitro diagnostics, characterized in that, The magnetic porous microspheres for in vitro diagnostics are prepared using the method described in any one of claims 1-8.

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