Magnetic microspheres, preparation method thereof and application thereof in chemiluminescence detection

Abstract

The application discloses a kind of magnetic microspheres and its preparation method and application in detection, belong to the field of biological detection magnetic material.The present application takes charged polymer microspheres as core, through solvent-thermal method or electrostatic interaction to coat magnetic particle, then through Stober method to coat silicon layer and build charged interface, then composite nano emulsion microspheres on the surface of silicon layer, through high-temperature melting and flattening to form dense and uniform polymer outer layer, finally obtain multilayer sandwich structure magnetic polymer microspheres.The method of the present application simplifies process, and surface interface is easily controlled, and the microspheres prepared have the advantages of uniform particle size, controllable size, adjustable magnetism, high stability, low non-specific adsorption, etc., and are especially suitable for in vitro diagnostic detection field.

Description

Technical Field

[0001] This invention relates to the field of magnetic materials for biodetection, and more specifically to a method for preparing magnetic microspheres and their application in in vitro diagnostic detection. Background Technology

[0002] Magnetic microspheres combine the structural designability of polymer microspheres with the magnetic response characteristics of magnetic materials. They can achieve rapid separation and enrichment under an applied magnetic field, making them key functional materials in in vitro diagnostics, biopharmaceutical separation, and targeted drug delivery. Magnetic microspheres for in vitro diagnostic testing, in particular, have stringent requirements regarding particle size uniformity, controllable magnetic content, magnetic response rate, surface functional group density, system stability, and non-specific adsorption levels to ensure high sensitivity and specificity in the detection methods.

[0003] Currently, mainstream magnetic microspheres are mainly classified into three types based on their structure: core-shell, dispersed, and multilayer sandwich. Core-shell products, represented by Merck, typically use co-precipitation or solvothermal methods to prepare the magnetic core, followed by polymer encapsulation. These methods generally suffer from problems such as non-uniform magnetic core particle size, difficulty in size control, and easy agglomeration during encapsulation, making them unsuitable for high-sensitivity detection requirements. Dispersed structures, originally developed by Thermo Fisher Scientific, rely on porous microsphere preparation and ATRP polymer grafting coating processes, resulting in complex preparation processes, easy leakage of magnetic components, and poor system suspension. Multilayer structures, represented by JSR, require high-speed shearing equipment for magnetization and emulsion polymerization for polymer encapsulation. This process is not only cumbersome and equipment-dependent, but also prone to microsphere agglomeration during production, affecting batch-to-batch consistency. While the aforementioned dispersed and multilayer sandwich products currently dominate the market, they all have inherent shortcomings in terms of processing and performance.

[0004] Most domestic manufacturers and patented technologies have only imitated or made partial improvements to the two mainstream structures mentioned above, failing to fundamentally break through the core process bottlenecks. In the entire process of preparing magnetic microspheres, the magnetization step directly determines the magnetic response intensity and magnetic property stability, while the polymer encapsulation step determines the surface layer thickness, composition, and interface characteristics. These two steps together determine the product's detection sensitivity, specificity, suspension dispersibility, and batch-to-batch consistency, making them the most critical technical nodes affecting the final performance. However, existing processes generally fail to achieve precise, gentle, scalable, and controllable operation of these two steps, making it difficult to further improve the overall performance.

[0005] From a practical manufacturing perspective, clinical-grade magnetic microspheres require multiple steps, including seed preparation, magnetic loading, and coating protection. Existing technologies generally suffer from lengthy processes, stringent conditions, poor process controllability, and low consistency in large-scale production. Especially under ambient pressure and mild conditions, achieving controllable gradient temperature increases, uniform loading of magnetic components, and stable functional layer coating, while simultaneously simplifying the process and reducing equipment and production costs, remains a critical technical challenge that the industry urgently needs to address.

[0006] In summary, existing magnetic microsphere preparation technologies have significant shortcomings in terms of particle size monodispersity, functional group controllability, magnetic layer bonding stability, mild processing conditions, and mass production feasibility. Therefore, developing a simple, mild, structurally and performance-controllable magnetic microsphere preparation technology suitable for large-scale production for in vitro diagnostics has important application value and practical significance. Summary of the Invention

[0007] In view of the above-mentioned defects in the existing technology, the present invention provides a method for preparing magnetic microspheres, which aims to improve the two core links of magnetic coating process and polymer coating, and effectively solve the technical problems such as poor particle size uniformity of magnetic microspheres, uneven magnetic particle loading, complex outer coating process, and poor batch-to-batch consistency.

[0008] To achieve the above objectives, the present invention provides a magnetic microsphere, which is a polymer magnetic microsphere with a multi-layer sandwich structure. Its characteristic is that it uses a polymer microsphere carrying positive or negative charges as its core, coated with a layer of magnetic particles via a solvothermal method or electrostatic interaction, then coated with a silicon layer using the Stober method to construct a positively charged interface, subsequently composited with nano-latex microspheres on the silicon layer surface, and then melted and flattened at high temperature to form a dense and uniform polymer outer layer, ultimately obtaining a multi-layer sandwich structure polymer magnetic microsphere. The positive charge carried on the surface of the polymer microsphere as the core is formed by the amination modification of surface epoxy groups to form cationic sites; the negative charge is formed by the deprotonation of carboxyl-containing monomers introduced during polymerization to form anionic sites.

[0009] Negatively charged polymer microspheres are prepared by precipitation polymerization, with divinylbenzene and acrylic acid as monomers; positively charged polymer microspheres are prepared by dispersion polymerization, with styrene and glycidyl methacrylate as monomers; the core particle size of the polymer microspheres is between 0.5 and 5 μm.

[0010] The magnetic particle layer is coated onto the core surface by a solvothermal method or electrostatic interaction. The solvothermal method utilizes the interaction between carboxyl groups and iron ions on the core surface to achieve in-situ nucleation and deposition of magnetic particles on the surface through a one-pot reaction. The electrostatic method first prepares carboxyl magnetic particles using a solvothermal method, and then combines them with positively charged core polymer microspheres through electrostatic interaction. The carboxyl magnetic particles are obtained by adding sodium citrate during the preparation process.

[0011] The silicon source reagents used in the silicon layer are tetraethyl silicate and 3-aminopropyltriethoxysilane; the positive charge on the surface of the silicon layer is provided by the amino group carried by 3-aminopropyltriethoxysilane.

[0012] The nano-latex microspheres are hydrophilic and carry a negative charge on their surface, with a particle size ranging from 50 to 400 nm. The composition of the nano-latex microspheres includes at least one of styrene, methyl methacrylate, cyclohexyl methacrylate, tert-butyl methacrylate, glycidyl methacrylate, acrylic acid, methacrylic acid, hydroxyethyl methacrylate, hydroxyethyl acrylamide, and polyethylene glycol dimethacrylate.

[0013] The method for preparing the magnetic microspheres comprises the following steps: The first step is to prepare the core of the polymer microspheres: polymer microspheres with positive or negative charges on their surface are prepared by precipitation polymerization or dispersion polymerization. The second step is to prepare a magnetic particle layer: by adding the polymer microsphere core obtained in the first step as a template to the system by solvothermal method, the magnetic particle layer is nucleated and deposited on the core surface by means of the interaction between the carboxyl groups on the core surface and the iron ions in a one-pot reaction; or by first using the negatively charged carboxyl magnetic particles obtained by solvothermal method, and then using the electrostatic interaction between the positive charge and the negative charge magnetic particles on the surface of the polymer microsphere core to achieve the magnetic particle layer coating. The third step is to prepare a silicon layer: positive charges are introduced onto the surface of the silicon layer through chemical bonding reactions; The fourth step is to prepare the molten layer of nano-latex microspheres: nano-latex microspheres are prepared by emulsion polymerization or soap-free polymerization. The former is attached to the surface of the latter through electrostatic interaction or covalent bonding. Then, the nano-latex microspheres are melted by high temperature to form the molten layer of nano-latex microspheres.

[0014] The solvent used in the solvothermal method described in the second step is one of ethylene glycol, diethylene glycol, and triethylene glycol; the iron source reagent is ferric chloride; and the alkaline source is at least one of sodium acetate and sodium hydroxide.

[0015] The high-temperature heating in step four uses at least two of the following: methanol, ethanol, isopropanol, acetone, tetrahydrofuran, ethylene glycol, propylene glycol, butanediol, diethylene glycol, and glycerol; the high-temperature heating temperature range is between 6 and 250 degrees Celsius.

[0016] The application of the magnetic microspheres in chemiluminescence immunoassay.

[0017] Beneficial effects: ① Simplified production process. This invention adopts a core-magnetization-silicon coating-polymer coating technical route, which has significant process advantages compared to the traditional seed pellet-swelling-magnetization-polymer coating scheme: First, it uses a solid core, and the preparation process is mature and stable, with good microsphere particle size uniformity; second, it eliminates the need for swelling and pore-forming, and does not use a strong acid system, which improves process safety while effectively reducing material consumption and labor costs; third, compared with similar magnetization processes, the cleaning step after magnetic loading in this invention is simpler and more suitable for large-scale production; finally, in the polymer coating stage, the melt-spreading method is used instead of the traditional ATRP method or free radical polymerization method, which does not require strict anhydrous and oxygen-free reaction conditions, and the reaction process is mild and less prone to agglomeration, greatly simplifying the production process and significantly improving production efficiency and batch-to-batch consistency.

[0018] ② Significantly improved product stability. In traditional processes, magnetic particles are only coated with a single polymer layer a few nanometers to tens of nanometers thick, which easily leads to magnetic leakage, resulting in reduced antibody activity and attenuated luminescence signals. This invention employs a dual-layer coating structure of silicon layer + polymer layer, which can efficiently bind and stably isolate magnetic particles, fundamentally avoiding the magnetic leakage problem and significantly improving the stability and detection signal consistency of the magnetic microspheres coupled with biomacromolecules.

[0019] ③ Non-specific adsorption is significantly reduced. This invention employs a novel polymer coating process, which, compared to traditional surface functional group modification and free radical polymerization grafting modification methods, allows for flexible control of the polymer composition, coating thickness, functional group type, and functional group density on the microsphere surface. This facilitates the screening of optimal surface physicochemical properties, thereby effectively reducing non-specific adsorption. Furthermore, the polymer surface structure can be customized for different downstream detection projects, effectively shortening the development cycle of downstream reagent products. Attached Figure Description

[0020] Figure 1 This is a scanning electron microscope image of the polymer core obtained in Example 1.

[0021] Figure 2 The image shown is a scanning electron microscope image of the solvothermal magnetization obtained in Example 1.

[0022] Figure 3 The image shows a scanning electron microscope (SEM) image of the latex microspheres coated in Example 1.

[0023] Figure 4 This is a scanning electron microscope image of the final magnetic microspheres obtained in Example 1 after high-temperature melting and flattening.

[0024] Figure 5 This is a scanning electron microscope image of the polymer core obtained in Example 2.

[0025] Figure 6This is a scanning electron microscope image of electrostatic magnetization obtained in Example 2.

[0026] Figure 7 The image shows a scanning electron microscope (SEM) image of the latex microspheres obtained in Example 2.

[0027] Figure 8 This is a scanning electron microscope image of the final magnetic microspheres obtained in Example 2 after high-temperature melting and flattening. Detailed Implementation

[0028] To better explain and facilitate understanding of the present invention, specific embodiments are described below.

[0029] A method for preparing magnetic microspheres, comprising: Using charged polymer microspheres as the core, magnetic particles are coated by solvothermal method or electrostatic action, and then a silicon layer is coated by Stober method to construct a charged interface. Subsequently, nano-latex microspheres are composited on the surface of the silicon layer, and then melted and flattened at high temperature to form a dense and uniform polymer outer layer, finally obtaining a multi-layer sandwich structure magnetic polymer microsphere.

[0030] Furthermore, the polymer microspheres are prepared by precipitation polymerization or dispersion polymerization, and the particle size is between 0.5 and 5 μm.

[0031] Furthermore, the positively charged polymer microspheres have positive charges formed by the amination of surface epoxy groups to create cation sites.

[0032] Furthermore, the negatively charged polymer microspheres have negative charges formed by the deprotonation of carboxyl monomers to create anionic sites.

[0033] Furthermore, the epoxy group is provided by glycidyl methacrylate, and the carboxyl monomer is acrylic acid, with the two types of monomers accounting for 20-80% of each.

[0034] Furthermore, the positively charged polymer microspheres also include styrene, and the negatively charged polymer microspheres also include divinylbenzene.

[0035] Furthermore, in the solvothermal coating of magnetic particles, the solvent is one of ethylene glycol, diethylene glycol, or triethylene glycol; the iron source reagent is one of anhydrous ferric chloride or ferric chloride hexahydrate; and the alkaline source is at least one of sodium acetate or sodium hydroxide.

[0036] Furthermore, the electrostatically coated magnetic particles are prepared by a solvothermal method, and negatively charged modified magnetic particles are obtained by adding sodium citrate.

[0037] Furthermore, the silicon source reagents used in the silicon layer are tetraethyl silicate and 3-aminopropyltriethoxysilane; the positive charge on the surface of the silicon layer is provided by the amino group carried by 3-aminopropyltriethoxysilane.

[0038] Furthermore, the nano-latex microspheres can be prepared by emulsion polymerization or soap-free polymerization.

[0039] Furthermore, the nano-latex microspheres are hydrophilic and carry a negative charge on their surface, with a particle size ranging from 50 to 400 nm.

[0040] Furthermore, the composition of the nano-latex microspheres includes at least one of styrene, methyl methacrylate, cyclohexyl methacrylate, tert-butyl methacrylate, glycidyl methacrylate, acrylic acid, methacrylic acid, hydroxyethyl methacrylate, hydroxyethyl acrylamide, and polyethylene glycol dimethacrylate.

[0041] Furthermore, the electrostatic effect is achieved through the interaction between the positive charge on the silicon layer surface and the negative charge on the surface of the nano-latex microspheres, and the covalent bonding is achieved by the reaction of the carboxyl groups of the latex microspheres activated by EDC / NHS with the amino groups on the silicon layer surface.

[0042] Furthermore, the high-temperature heating uses at least two of the following: methanol, ethanol, isopropanol, acetone, tetrahydrofuran, ethylene glycol, propylene glycol, butanediol, diethylene glycol, and glycerol.

[0043] Furthermore, the temperature range for high-temperature heating is between 60 and 250 degrees Celsius.

[0044] According to a second aspect of the present invention, an application of magnetic microspheres in the field of in vitro diagnostic testing is provided.

[0045] The method provided by this invention is applicable to a variety of liquid and liquid-extracted samples, including but not limited to: water, urine, serum, plasma, whole blood, cerebrospinal fluid, saliva; soluble solid-liquid separations such as fecal extracts, tissue extracts, food extracts, soil extracts, and plant extracts; nucleic acids and nucleic acid amplification products; small molecule compounds such as T3 and T4 hormones and drug test substances; and other types of liquid test systems.

[0046] Example 1 The present invention provides the following steps for preparing magnetic microspheres by solvothermal magnetization: 1. Preparation of polymer core Weigh / measure 6g of divinylbenzene, 24g of acrylic acid, 2.4g of azobisisobutyronitrile, and 300mL of acetonitrile into a 500mL three-necked flask. After purging with nitrogen, heat to 80°C and stir for 6 hours. After returning to room temperature, wash several times with anhydrous ethanol by centrifugation, and dry overnight at 50°C to obtain positively charged polymer microspheres.

[0047] 2. Solvent thermal magnetization Take 10g of the above polymer microspheres and disperse them in 100mL of diethylene glycol. Add 3.5g of ferric chloride hexahydrate and 7g of sodium acetate. Stir at 60°C until fully dissolved. Transfer to a sealed reaction vessel and react at 180°C for 6 hours. Wash repeatedly with purified water and ethanol several times. Dry overnight at 60°C to obtain about 13g of magnetic microspheres.

[0048] 3. Coat the surface with a silicon layer and introduce charges. 13g of the above-mentioned magnetic microspheres were dispersed in 200mL of ethanol and aqueous solution (ethanol:water ratio 8:2). 2mL of ammonia solution was added, and the mixture was heated to 50°C. 2mL of TEOS was added dropwise while continuously stirring. After reacting for 2 hours, 3mL of KH550 was added dropwise, and stirring continued for another 3 hours. After the reaction was complete, the microspheres were repeatedly washed several times with ethanol and then stored in pure water via a gradient displacement process. Magnetic microspheres with a silicon-coated surface and a positive charge were obtained.

[0049] 4. Preparation of nano-latex microspheres Weigh / measure 3.5g styrene, 5.5g hydroxyethyl methacrylate, 1g methacrylic acid, and 100mL purified water into a 250mL three-necked flask. Sonicate the mixture for 5 minutes, purge with nitrogen, heat to 70°C, add 0.2g potassium persulfate, and stir for 12 hours. After the reaction is complete, wash repeatedly with purified water by centrifugation to obtain latex microspheres with a particle size of 100nm.

[0050] 5. Latex microspheres coated on the surface of silicon magnetic microspheres 0.25 g of the above latex microspheres were dispersed in 40 mL of 0.025 M pH 6.5 MES buffer. The temperature was raised to 40°C, and 5 g of magnetic microspheres with a silica-coated surface and positive charge, which were previously dispersed in 60 mL of 0.1 M pH 6.5 MES buffer, were added dropwise while stirring continuously. The mixture was stirred for 4 hours. After the reaction was completed, the microspheres were washed several times with purified water and finally transferred to 100 mL of ethylene glycol for storage. The magnetic microsphere concentration was approximately 5%.

[0051] 6. High-temperature heating to melt and spread out In the 100 mL ethylene glycol solution containing the magnetic microspheres, 10 mL of tetrahydrofuran was added while stirring. The temperature was gradually increased from 60°C to 150°C using a gradient heating method and maintained for 2 hours. Then, the temperature was gradually reduced to room temperature using a gradient cooling method. The solution was washed several times with ethanol and purified water and stored in purified water.

[0052] 7. Performance Testing Take 5 mg of the above magnetic microspheres and select JSR MS300 microspheres (particle size approximately 3 μM) as a control. Disperse them in pH 5.5 MES buffer, add EDC and Sulfo-NHS for activation, magnetically wash, redisperse in pH 7.2 PB buffer, add CKMB1 antibody for room temperature reaction, magnetically wash, block with blocking agent, and finally dilute and store with magnetic bead preservation solution.

[0053] The magnetic beads labeled with CKMB1 antibody were diluted to 0.5 mg / mL and used with acridine ester-labeled CKMB2 antibody. Samples of different concentrations were detected using a Cosmetic SMART-500s chemiluminescence analyzer, and the statistical results are as follows.

[0054] Table 1. Results of Chemiluminescence Signal Testing of CKMB Items using Magnetic Microspheres in Example 1

[0055] S0-S6 represent different concentrations, where 0 is water (background) and 1-6 represent different antigen concentrations (from low to high), with a concentration range between 0.3 ng / mL and 250 ng / mL.

[0056] The two columns of data represent chemiluminescence signal values. For example, 10814 and 11253 refer to the chemiluminescence signal values ​​of the S1 concentration antigen detected using the microsphere-labeled antibody of Example 1. The purpose of comparing Example 1 with the control group is to illustrate that the signal of the microspheres prepared by this method is slightly better than the imported method, especially in terms of water background and S1 / S0 signal-to-noise ratio, which is at least comparable to the imported method.

[0057] Example 2 The present invention provides the following steps for preparing magnetic microspheres by electrostatic magnetization: 1. Preparation of polymer core Weigh / measure 15g styrene, 15g glycidyl methacrylate, 1.2g azobisisobutyronitrile, 10g polyvinylpyrrolidone, 240mL ethanol, and 60mL purified water into a 500mL three-necked flask. After purging with nitrogen, heat to 70°C and stir for 15 hours. After returning to room temperature, wash several times with anhydrous ethanol by centrifugation, and dry overnight at 50°C to obtain polymer microspheres with surface-modified epoxy groups.

[0058] 2. Positive charge modification on the surface of polymer microspheres Take 5g of the above microspheres, disperse them ultrasonically with 100mL of anhydrous ethanol, add 10g of hexamethylenediamine, and stir at 60°C for 8 hours. After the reaction is complete, wash several times with anhydrous ethanol by centrifugation, and dry overnight at 50°C to obtain polymer microspheres with positively charged surfaces.

[0059] 3. Preparation of magnetic nanoparticles with negatively charged surfaces Weigh 4g of ferric chloride hexahydrate, 9g of sodium acetate, and 2g of sodium citrate into 100mL of triethylene glycol. Stir at 60°C until fully dissolved, then transfer to a sealed reaction vessel and react at 200°C for 6 hours. Wash repeatedly with purified water and ethanol, and dry overnight at 60°C to obtain magnetic nanoparticles with negatively charged surfaces.

[0060] 4. Electrostatic magnetization Take 5g of the positively charged polymer microspheres from step 2 above, sonicate them in PB buffer at pH 7.0, add 2g of the negatively charged magnetic nanoparticles from step 3, and stir at room temperature for 4 hours. Wash repeatedly with purified water and ethanol, and store in anhydrous ethanol to obtain approximately 6.5g of magnetic microspheres.

[0061] 5. Coat the surface with a silicon layer and introduce charges. 6.5 g of the magnetic microspheres were dispersed in 50 mL of ethanol and water in a 9:1 ratio. 1.5 mL of ammonia solution was added, and the mixture was heated to 50°C. While stirring continuously, 1 mL of tetraethyl orthosilicate (TEOS) was added dropwise. After reacting for 2 hours, 1.5 mL of KH550 was added dropwise, and stirring continued for 3 hours. After the reaction was complete, the microspheres were repeatedly washed with ethanol and then stored in pure water via a gradient displacement process. Magnetic microspheres with a silicon-coated surface and a positive charge were obtained.

[0062] 6. Preparation of nano-latex microspheres Weigh / measure 4g of methyl methacrylate, 5.2g of polyethylene glycol dimethacrylate, 0.8g of acrylic acid, 0.05g of sodium dodecyl sulfate, and 100mL of purified water into a 250mL three-necked flask. Sonicate the mixture for 5 minutes, purge with nitrogen, heat to 70°C, add 0.25g of potassium persulfate, and stir for 12 hours. After the reaction is complete, wash the mixture several times with purified water by centrifugation to obtain latex microspheres with a particle size of 250nm.

[0063] 7. Latex microspheres coated on the surface of silicon magnetic microspheres Disperse 0.1g of the above latex microspheres in 10mL of 0.05M pH 5.5 MES buffer, add appropriate amounts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxythiosuccinimide (Sulfo-NHS), activate at room temperature for 20 minutes, wash with 0.1M neutral PB buffer by centrifugation, and store for later use. Disperse 5g of positively charged magnetic microspheres in 200mL of 0.1M neutral PB buffer, rapidly add the activated latex microspheres while stirring at high speed, and continue stirring for 4 hours. After the reaction is complete, wash repeatedly with purified water several times, and finally transfer to 100mL of ethylene glycol for storage. The magnetic microsphere concentration is approximately 5%.

[0064] 8. High-temperature heating to melt and spread out In the above-mentioned 100 mL ethylene glycol solution containing magnetic microspheres, 15 mL of acetone was added while stirring. The temperature was gradually increased from 60°C to 150°C using a gradient heating method and maintained for 2 hours. Then, the temperature was gradually reduced to room temperature using a gradient cooling method. The solution was washed several times with ethanol and purified water and stored in purified water.

[0065] 9. Performance Testing Take 5 mg of the above magnetic microspheres and select JSR MS300 microspheres (particle size approximately 3 μM) as a control. Disperse them in pH 5.5 MES buffer, add EDC and Sulfo-NHS for activation, wash with magnetic adsorption, redisperse in pH 7.2 PB buffer, add D-dimer 1 antibody for room temperature reaction, wash with magnetic adsorption, block with blocking agent, and finally dilute and store with magnetic bead preservation solution.

[0066] The magnetic beads labeled with D-dimer 1 antibody were diluted to 0.5 mg / mL and used with acrid ester-labeled D-dimer 2 antibody. Samples of different concentrations were detected using a Cosmetic SMART-500s chemiluminescence analyzer, and the statistical results are as follows.

[0067] Table 2. Chemiluminescence signal results of D-dimer test in Example 2 of magnetic microspheres.

[0068] S0-S6 represent different concentrations, where 0 represents water (background) and 1-6 represent different antigen concentrations (from low to high), with a concentration range between 0.05 mg / L and 40 mg / L.

[0069] The S0 background (i.e., nonspecific adsorption) of Examples 1 and 2 was lower than that of the control group, and the signal-to-noise ratio S1 / S0 was also better than that of the control group.

Claims

1. A magnetic microsphere, which is a polymer magnetic microsphere with a multi-layer sandwich structure, characterized in that, The process involves using polymer microspheres carrying positive or negative charges as the core, coating a layer of magnetic particles using a solvothermal method or electrostatic interaction, then coating a silicon layer using the Stober method to construct a positively charged interface, subsequently assembling nano-latex microspheres on the silicon layer surface, and then melting and flattening at high temperature to form a dense and uniform polymer outer layer, ultimately obtaining a multi-layer sandwich structure polymer magnetic microsphere. The positive charge carried on the surface of the polymer microspheres, which serves as the core, is formed by the amination modification of surface epoxy groups to form cationic sites; the negative charge is formed by the deprotonation of carboxyl-containing monomers introduced during the polymerization process to form anionic sites.

2. The magnetic microspheres according to claim 1, characterized in that, Negatively charged polymer microspheres are prepared by precipitation polymerization, with divinylbenzene and acrylic acid as monomers; positively charged polymer microspheres are prepared by dispersion polymerization, with styrene and glycidyl methacrylate as monomers; the core particle size of the polymer microspheres is between 0.5 and 5 μm.

3. The magnetic microspheres according to claim 1, characterized in that, The magnetic particle layer is coated onto the core surface by a solvothermal method or electrostatic interaction. The solvothermal method utilizes the interaction between carboxyl groups and iron ions on the core surface to achieve in-situ nucleation and deposition of magnetic particles on the surface through a one-pot reaction. The electrostatic method first prepares carboxyl magnetic particles using a solvothermal method, and then combines them with positively charged core polymer microspheres through electrostatic interaction. The carboxyl magnetic particles are obtained by adding sodium citrate during the preparation process.

4. The magnetic microspheres according to claim 1, characterized in that, The silicon source reagents used in the silicon layer are tetraethyl silicate and 3-aminopropyltriethoxysilane; the positive charge on the surface of the silicon layer is provided by the amino group carried by 3-aminopropyltriethoxysilane.

5. The magnetic microspheres according to claim 1, characterized in that, The nano-latex microspheres are hydrophilic and carry a negative charge on their surface, with a particle size ranging from 50 to 400 nm. The composition of the nano-latex microspheres includes at least one of styrene, methyl methacrylate, cyclohexyl methacrylate, tert-butyl methacrylate, glycidyl methacrylate, acrylic acid, methacrylic acid, hydroxyethyl methacrylate, hydroxyethyl acrylamide, and polyethylene glycol dimethacrylate.

6. A method for preparing the magnetic microspheres according to any one of claims 1 to 5, characterized in that, The specific steps are as follows: The first step is to prepare the core of the polymer microspheres: polymer microspheres with positive or negative charges on their surface are prepared by precipitation polymerization or dispersion polymerization. The second step is to prepare a magnetic particle layer: by adding the polymer microsphere core obtained in the first step as a template to the system by solvothermal method, the magnetic particle layer is nucleated and deposited on the core surface by means of the interaction between the carboxyl groups on the core surface and the iron ions in a one-pot reaction; or by first using the negatively charged carboxyl magnetic particles obtained by solvothermal method, and then using the electrostatic interaction between the positive charge and the negative charge magnetic particles on the surface of the polymer microsphere core to achieve the magnetic particle layer coating. The third step is to prepare a silicon layer: positive charges are introduced onto the surface of the silicon layer through chemical bonding reactions; The fourth step is to prepare the molten layer of nano-latex microspheres: nano-latex microspheres are prepared by emulsion polymerization or soap-free polymerization. The former is attached to the surface of the latter through electrostatic interaction or covalent bonding. Then, the nano-latex microspheres are melted by high temperature to form the molten layer of nano-latex microspheres.

7. The method according to claim 6, characterized in that, The solvent used in the solvothermal method described in the second step is one of ethylene glycol, diethylene glycol, and triethylene glycol; the iron source reagent is ferric chloride; and the alkaline source is at least one of sodium acetate and sodium hydroxide.

8. The method according to claim 6, characterized in that, The high-temperature heating in step four uses at least two of the following: methanol, ethanol, isopropanol, acetone, tetrahydrofuran, ethylene glycol, propylene glycol, butanediol, diethylene glycol, and glycerol; the high-temperature heating temperature range is between 6 and 250 degrees Celsius.

9. The application of the magnetic microspheres according to any one of claims 1 to 5 in chemiluminescence immunoassay detection.

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