Monodisperse functionalized polymer magnetic composite microspheres

Abstract

The application provides a monodisperse functionalized polymer magnetic composite microsphere and belongs to the technical field of magnetic materials. The monodisperse polystyrene microsphere is prepared by using a dispersion polymerization method, a two-step swelling method is used to obtain porous crosslinked polystyrene microspheres, then the porous crosslinked polystyrene microspheres are mixed with sulfuric acid to obtain porous polystyrene microspheres containing sulfonic acid groups on the surface, then the in-situ precipitation method is used to combine with ferrous ions, in an alkaline environment, the magnetic porous polystyrene microspheres are obtained, finally in acetonitrile solvent, various functional monomers are added to obtain the monodisperse functionalized magnetic composite microspheres which have good suspension performance and fast magnetic attraction rate.

Description

Technical Field

[0001] This invention relates to the field of magnetic materials technology, specifically to a monodisperse functionalized polymer magnetic composite microsphere. Background Technology

[0002] Magnetic nanoparticles or microparticles have gained increasing attention due to their vast applications in protein and enzyme immobilization, nucleic acid purification, separation of biochemical products, controlled drug release, separation and purification of proteins and cells, magnetic resonance imaging, biosensing, and the analysis, separation, purification, and detection of heavy and precious metals. These applications are primarily due to the unique properties of magnetic particles: narrow or monodisperse size distribution, varied morphologies, chemical and physical stability, hydrophilic surface, easy surface functionalization, and especially superparamagnetism. Because of their small size and high specific surface area, magnetic nanoparticles can be easily transported to target tissues via the bloodstream, showing great promise for in vivo applications such as drug delivery, contrast enhancement in magnetic resonance imaging, and thermotherapy.

[0003] Magnetic microparticles are favored in in vitro magnetic separation due to their advantages such as low magnetic field strength requirements, readily available and simple magnetic field equipment, and rapid separation capabilities. Currently, magnetic composite microspheres can be classified into four types according to their microsphere structure: core-shell type, anti-core-shell type, dispersed type, and sandwich type. There are three main methods for preparing these structural microspheres: The first method involves encapsulating magnetic inorganic particles with inorganic materials or polymers to obtain core-shell magnetic composite microspheres. For example, patent CN101256864A provides a method for preparing magnetic composite microspheres. The core of the magnetic composite microspheres prepared by this method is a submicron-sized magnetic particle, which is generally heavy and has poor suspension. The second method, which is currently the most widely used, uses inorganic magnetic particles as the core and polymerizes them to prepare magnetic polymer microspheres. For example, patent CN102775543A uses magnetic nanoparticles as the core and prepares magnetic composite microspheres by seed emulsion polymerization. The magnetic composite microspheres prepared by this method generally have small particle size and specific surface area, and the process control is difficult. The third method involves generating magnetic materials on or inside the polymer microspheres. Iron oxide particles are generated in situ inside the porous polymer microspheres. Although the magnetic particles in the magnetic composite microspheres prepared by this method are uniformly distributed, they still have disadvantages such as low saturation magnetization and buried surface functional groups, which limits their application range.

[0004] Therefore, there is an urgent need for a monodisperse functionalized polymer magnetic composite microsphere. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention prepares a monodisperse functionalized polymer magnetic composite microsphere with high magnetic content, high carboxyl content, and high antibody capture ability.

[0006] To achieve the above objectives, the present invention provides the following technical solution to address the technical problem: In a first aspect, the present invention provides a method for preparing monodisperse functionalized polymer magnetic composite microspheres, comprising the following steps: S1. Styrene, PVP, AIBN and ethanol are reacted, and after the reaction, the mixture is washed and dried to obtain polystyrene seed pellets; S2. Take the polystyrene seed balls obtained in step S1 and carry out a two-step swelling polymerization reaction to obtain porous cross-linked polystyrene microspheres; S3. Sulfonated porous polystyrene microspheres are obtained by mixing and reacting porous cross-linked polystyrene microspheres with acid solution; S4. After dispersing sulfonated porous cross-linked polystyrene microspheres in water, an aqueous solution of FeSO4·7H2O was added for the first reaction. After the reaction, the microspheres were washed, and concentrated ammonia was added for the second reaction. After the reaction was completed, the microspheres were washed and dried to obtain magnetic porous microspheres. S5. Magnetic porous microspheres are reacted with functional monomers by precipitation polymerization to obtain monodisperse functionalized magnetic composite microspheres.

[0007] In some embodiments, in step S1, the mass ratio of styrene, PVP, AIBN, and ethanol is 60:3.6:1.2:200; the reaction temperature is 70°C, the reaction time is 24 hours, and the stirring speed during the reaction is 300 r / min.

[0008] In some embodiments, in step S2, the two-step swelling polymerization reaction includes: first swelling polystyrene seed spheres, 0.25 wt% sodium dodecyl sulfate, and dibutyl phthalate to obtain solution A; preparing styrene monomer, divinylbenzene, and benzoyl peroxide into solution B, adding it to solution A for a second swelling to obtain solution C; then adding 5 wt% polyvinyl alcohol solution to carry out the polymerization reaction; after the reaction is completed, the product is washed and dried to obtain porous cross-linked polystyrene microspheres.

[0009] In some embodiments, the temperature for the first and second swelling is 30°C, the time is 24 hours, and the stirring rate is 120 r / min; the polymerization reaction temperature is 70°C, and the reaction time is 24 hours.

[0010] In some embodiments, the solid-liquid ratio of the polystyrene seed pellets, sodium dodecyl sulfate, and dibutyl phthalate is 0.5 g: 50 mL: 2 g; and the mass ratio of the styrene monomer, divinylbenzene, and benzoyl peroxide is 6: 6: 1.

[0011] In some embodiments, in step S3, the mass ratio of the porous cross-linked polystyrene microspheres to the acid solution is 3:200; the reaction is first ultrasonically dispersed at 300 r / min for 0.5 h, and then reacted at 300 r / min in a 50°C water bath for 2 h.

[0012] In some embodiments, in step S4, the first reaction is carried out with stirring at 300 r / min for 24 h; the second reaction is carried out at 80 °C and 300 r / min for 2 h.

[0013] In some embodiments, in step S4, the functional monomer is one or more of glycidyl methacrylate, divinylbenzene, acrylic acid, methacrylic acid, and ethylene glycol dimethacrylate.

[0014] Secondly, the present invention provides a monodisperse functionalized polymer magnetic composite microsphere, which is prepared by the above-described preparation method.

[0015] Thirdly, the present invention also provides the application of monodisperse functionalized polymer magnetic composite microspheres in nucleic acid extraction, protein extraction or protein purification.

[0016] In some embodiments, the monodisperse magnetic polystyrene microspheres are carboxylated, cyclocarboxylated, or epoxidized.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention first uses a dispersion polymerization method to prepare monodisperse polystyrene microspheres, then introduces a two-step swelling method to prepare porous cross-linked polystyrene microspheres, and then mixes them with an acid solution to obtain porous polystyrene microspheres with sulfonate groups on the surface and high sulfonation density. Then, magnetic porous polystyrene microspheres are obtained by in-situ precipitation, which have high specific surface area, low density, and high porosity, and can more fully attach magnetic nanoparticles, greatly improving the magnetic content of subsequent magnetic polymer microspheres. Moreover, most of the magnetic nanoparticles are attached to the pores on the surface of the porous polymer microspheres.

[0018] This invention uses the microsphere as the core, which has a controllable particle size and can improve the overall dispersibility of the microsphere. The porous cross-linked microsphere has high rigidity, high mechanical strength and chemical stability. The higher the degree of cross-linking, the less likely the polymer microsphere is to break during subsequent operations.

[0019] The method for preparing magnetic porous microspheres of the present invention is simple and convenient to operate, and the conditions are easier to control. It does not require strict nitrogen purging and oxygen removal. It can be operated under air flow conditions, which makes it easy to control reaction conditions and saves experimental costs.

[0020] The magnetic porous microspheres obtained in this invention do not require any modification and can be used directly with a polymer shell coated on their surface. Their good dispersibility results in high immunoassay capture efficiency, making them effective in chemiluminescent immunoassay. Furthermore, by using different functional monomers and crosslinking agents, functionalized magnetic composite microspheres carrying different functional groups and polymer shells with varying hydrophilic and hydrophobic properties can be obtained, leading to a wide range of applications. Attached Figure Description

[0021] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a flowchart illustrating the preparation process of monodisperse functionalized polymer magnetic composite microspheres in this invention.

[0023] Figure 2 This is a scanning electron microscope image of the polystyrene seed pellets in an embodiment of the present invention.

[0024] Figure 3 This is a scanning electron microscope image of porous cross-linked polystyrene microspheres in an embodiment of the present invention.

[0025] Figure 4 This is a scanning electron microscope image of the magnetic porous microspheres in an embodiment of the present invention.

[0026] Figure 5 This is a scanning electron microscope image of monodisperse epoxide polymer magnetic composite microspheres in an embodiment of the present invention.

[0027] Figure 6 This is a scanning electron microscope image of monodisperse carboxylated polymer magnetic composite microspheres in an embodiment of the present invention.

[0028] Figure 7 This is a graph showing the results of determining the carboxyl content of monodisperse carboxylated polymer magnetic composite microspheres by conductivity titration in an embodiment of the present invention.

[0029] Figure 8 This is a scanning electron microscope image of monodisperse carboxylated polymer magnetic composite microspheres in an embodiment of the present invention.

[0030] Figure 9 This is a graph showing the results of determining the carboxyl content of monodisperse carboxylated polymer magnetic composite microspheres by conductivity titration in an embodiment of the present invention.

[0031] Figure 10 This is a graph showing the magnetic response test results of different magnetic porous microspheres in the embodiments of the present invention.

[0032] Figure 11 This is a graph showing the test results of different functionalized polymer magnetic composite microspheres coupled with AFP antibodies to capture immune complexes in embodiments of the present invention. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Unless otherwise specified, the experimental methods used in this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0035] like Figure 1 As shown, this invention first uses dispersion polymerization to prepare monodisperse polystyrene microspheres, then uses a two-step swelling method to prepare porous cross-linked polystyrene microspheres, and then mixes them with sulfuric acid to obtain porous polystyrene microspheres with sulfonate groups on the surface. Then, through in-situ precipitation, they are combined with ferrous ions to obtain magnetic porous polystyrene microspheres in an alkaline environment. Finally, in acetonitrile solvent, various functional monomers are added through precipitation polymerization to obtain monodisperse functionalized magnetic composite microspheres.

[0036] Example 1 A monodisperse polymer magnetic composite microsphere is prepared by the following steps: 1. Preparation of polystyrene seed bulbs: Weigh out 60g of styrene, 3.6g of PVP K30 (polyvinylpyrrolidone K30), 1.2g of AIBN (azobisisobutyronitrile), and 200g of anhydrous ethanol into a flask. Place the flask in a water bath, install a condenser, and evacuate the system to a vacuum state using a syringe. Seal the reaction system with a nitrogen balloon, turn on the electric stirrer at 300 rpm, and simultaneously heat the water bath to 70°C. React at this temperature for 24 hours. After the reaction is complete, centrifuge to remove the supernatant, add anhydrous ethanol, and repeatedly centrifuge and wash 5 times. Then, dry the product under vacuum at 60°C to obtain 1μm polystyrene seed pellets. Figure 2 As shown.

[0037] 2. Preparation of porous cross-linked polystyrene microspheres: 0.5 g of seed microspheres were added to 50 mL of a 0.25% sodium dodecyl sulfate aqueous solution to prepare a homogeneous emulsion. 5 g of dibutyl phthalate was added, and the mixture was stirred at 200 rpm for 24 h at 35 °C to induce swelling, yielding a swollen mixed solution. A homogeneous solution of 5 g styrene monomer, 7 g divinylbenzene, and 0.5 g benzoyl peroxide was added to the swollen mixed solution, and the mixture was stirred at 200 rpm for another 24 h at 30 °C. Finally, 10 mL of a 5% polyvinyl alcohol solution was added, and the mixture was stirred at 200 rpm for 24 h at 80 °C to induce a thermopolymerization reaction. Anhydrous ethanol was added, and after the reaction, the product was repeatedly centrifuged and washed five times, then vacuum dried at 60 °C to obtain 2.8 μm porous cross-linked polystyrene microspheres. Figure 3 As shown.

[0038] 3. Sulfonation of porous cross-linked polystyrene microspheres: Add 3g of the above porous cross-linked polystyrene microspheres and 200g of concentrated sulfuric acid to a 100mL three-necked flask; after ultrasonic dispersion by stirring at 300r / min for 0.5h, react by stirring at 300r / min in a 50℃ water bath for 2h; after the reaction is completed, centrifuge the product to remove the supernatant, add deionized water, repeat centrifugation and washing 5 times, and then dry in a 60℃ vacuum oven to obtain sulfonated polystyrene microspheres with sulfonate groups on the surface.

[0039] 4. Preparation of monodisperse polymer magnetic composite microspheres: Take the above aqueous solution containing 1g of sulfonated polystyrene microspheres (solid-liquid ratio of polystyrene microspheres to deionized water is 1:200) and transfer it to a three-necked flask. Then add 5g of FeSO4·7H2O, i.e., the mass ratio of microspheres to FeSO4·7H2O is 1:5. Maintain a stirring speed of 300r / min and react for 24h. After the reaction is complete, pour out the product, centrifuge it repeatedly with deionized water more than 4 times, disperse the product in water, place it in an 80℃ double-hole water bath, turn on the electric stirrer at a stirring speed of 300r / min, add 10mL of ammonia solution, and react for 2h. After the reaction is complete, pour out the product, magnetically separate and wash it 4 times with deionized water, and dry it in a 60℃ vacuum oven to obtain magnetic porous microspheres, i.e., monodisperse polymer magnetic composite microspheres. Figure 4 As shown.

[0040] Example 2 The preparation of a monodisperse epoxidized polymer magnetic composite microsphere includes the following steps: The magnetic porous microspheres prepared in Example 1 were dispersed in 40g of acetonitrile and transferred to a three-necked flask. 0.15g of GMA (glycidyl methacrylate) and 0.1g of DVB (divinylbenzene) were added, followed by 0.005g of AIBN (azobisisobutyronitrile). The flask was placed in a water bath, a condenser was installed, and the system was evacuated to a vacuum using a syringe. The reaction system was sealed, and an electric stirrer was turned on at 300 rpm. Simultaneously, the water bath was heated to 80°C, and the reaction was maintained at this temperature for 24 hours. After the reaction, the product was magnetically separated and washed four times with acetonitrile to obtain monodisperse epoxidized polymer magnetic composite microspheres. Figure 5 As shown.

[0041] Example 3 The preparation of a monodisperse carboxylated magnetic composite microsphere includes the following steps: The magnetic porous microspheres obtained in Example 1 were dispersed in 20g of acetonitrile and transferred to a three-necked flask. 0.15g of AA (acrylic acid) and 0.1g of DVB (divinylbenzene) were added, followed by 0.005g of AIBN (azobisisobutyronitrile). The flask was placed in a water bath, a condenser was installed, and the system was evacuated to a vacuum using a syringe. The reaction system was sealed, and an electric stirrer was turned on at 300 rpm. Simultaneously, the water bath was heated to 70°C, and the reaction was maintained at this temperature for 24 hours. After the reaction, the product was washed four times with acetonitrile using magnetic separation to obtain monodisperse carboxylated magnetic composite microspheres. Figure 6 As shown. Figure 7 As shown, the carboxyl content is 574 μmol / g.

[0042] Example 4 The preparation of a monodisperse carboxylated magnetic composite microsphere includes the following steps:

[0043] The magnetic porous microspheres prepared in Example 1 were dispersed in 40g of acetonitrile and transferred to a three-necked flask. 0.15g of MAA (methacrylic acid) and 0.1g of EGDMA (ethylene glycol dimethacrylate) were added, followed by 0.005g of AIBN (azobisisobutyronitrile). The flask was placed in a water bath, a condenser was installed, and the system was evacuated to a vacuum using a syringe. The reaction system was sealed, and an electric stirrer was turned on at 300 rpm. Simultaneously, the water bath was heated to 70°C, and the reaction was maintained at this temperature for 24 hours. After the reaction, the product was washed four times with acetonitrile using magnetic separation to obtain monodisperse carboxylated polymer magnetic composite microspheres. Figure 8 As shown. Figure 9 As shown, the carboxyl content is 929 μmol / g.

[0044] Example 5 The preparation of a monodisperse cyclic hydroxylated magnetic composite microsphere includes the following steps: The magnetic porous microspheres obtained in Example 1 were dispersed in 20g of acetonitrile and transferred to a three-necked flask. 0.1g of HEMA (2-hydroxyethyl methacrylate) and 0.1g of MBA (N,N′-methylenebisacrylamide) were added, followed by 0.004g of AIBN (azobisisobutyronitrile). The flask was placed in a water bath, a condenser was installed, and the system was evacuated to a vacuum state using a syringe. The reaction system was sealed, and an electric stirrer was turned on at 300r / min. At the same time, the water bath was heated to 70℃ and reacted at a constant temperature for 24h. After the reaction was completed, the product was washed four times with acetonitrile magnetic separation to obtain monodisperse hydroxylated magnetic composite microspheres.

[0045] Test Example 1 Magnetic response test 1g of the magnetic porous microspheres (sulfonated porous microspheres) and nitrated porous microspheres prepared in Example 1 were dispersed in a beaker containing 50mL of deionized water, and a common magnet was placed at the bottom of the beaker to test the magnetic response.

[0046] Preparation of nitrated porous microspheres: Add 3g of the porous cross-linked polystyrene microspheres prepared in Example 1, 150mL of concentrated sulfuric acid, and 50mL of concentrated nitric acid to a 100mL three-necked flask. After stirring and ultrasonically dispersing at 300r / min for 0.5h, react with stirring at 300r / min in a 50℃ water bath for 2h. After the reaction is complete, centrifuge to remove the supernatant, add deionized water, repeat centrifugation and washing 5 times, and then dry in a 60℃ vacuum oven to obtain nitrated porous polystyrene microspheres.

[0047] The obtained nitrated porous polystyrene microspheres are then used to replace the sulfonated polystyrene microspheres in step 4 of Example 1, and the same steps are performed to prepare nitrated porous microspheres.

[0048] Table 1. Magnetic response test results of microspheres prepared under different conditions.

[0049] like Figure 10 As shown in Table 1, in the beaker containing sulfonated porous microspheres, all particles were adsorbed to the bottom of the beaker, and the liquid was clear. However, in the beaker containing nitrated porous microspheres, the particles remained suspended, and the liquid was turbid. This is because traditional nitration treatment makes the nitro groups on the surface and inside of the porous microspheres highly oxidizing, causing the ferrous ions in ferrous sulfate heptahydrate to be easily oxidized, ultimately generating porous microspheres with weak or no magnetic properties. After sulfonation treatment, the sulfonate groups on the surface and inside of the porous microspheres have no obvious oxidizing properties under normal conditions and can stably combine with ferrous ions. After the reaction, magnetic porous microspheres are generated.

[0050] Test Example 2 Assay for capturing immune complexes with AFP antibodies coupled to magnetic composite microspheres.

[0051] Test samples: Carboxylated microspheres prepared in Example 3 and commercially available carboxylated magnetic beads as a control.

[0052] The specific steps of the test include: 1. Take 1 mg of each microsphere sample into a centrifuge tube, separate magnetically and discard the supernatant.

[0053] 2. Add 1 mL to the centrifuge tube, mix the microspheres by pipetting, and discard the supernatant after magnetic separation.

[0054] 3. Add 1 mL of activation buffer to the centrifuge tube, mix at room temperature for 1 hour on a mixer, and then magnetically separate and discard the supernatant.

[0055] 4. Add 1 mL of buffer solution to the centrifuge tube and mix the magnetic beads by pipetting; discard the supernatant after magnetic separation.

[0056] 5. Add 980 μL of coupling buffer to the centrifuge tube, mix the magnetic beads by pipetting, sonicate, and then add 20 μL of AFP antibody.

[0057] 6. Place the centrifuge tube on a mixer and mix at room temperature. Allow the coupling reaction to proceed for 3 hours.

[0058] 7. After coupling is complete, add 200 μL of blocking solution to the centrifuge tube and mix at room temperature for 3 hours using a mixer.

[0059] 8. Magnetic separation of the magnetic beads in the centrifuge tube, discarding the supernatant, add 1 mL of chemiluminescent washing buffer to the centrifuge tube, mix the magnetic beads by pipetting, magnetic separation, and discarding the supernatant.

[0060] 9. Add 1 mL of magnetic bead storage buffer to the centrifuge tube to prepare a working solution of microspheres with a concentration of 2 mg / mL and store it at 4℃ for later use.

[0061] 10. Use a pipette to transfer 50 μL of the composite microspheres coupled with AFP capture antibody into a centrifuge tube.

[0062] 11. Add 10 μL of AFP antigen, vortex to mix, and incubate at 37°C.

[0063] 12. After the reaction is complete, place the centrifuge tube on a magnetic rack and magnetically separate for 5 minutes to make the supernatant completely clear. Discard the supernatant.

[0064] 13. Add 200 μL of chemiluminescent washing solution, mix horizontally with a mixer, and remove the washing solution using magnetic separation.

[0065] 14. Add 50 μL of ALP-AFP detection antibody to the centrifuge tube and incubate at 37°C on a shaker for 1 hour.

[0066] 15. After the reaction is complete, place the centrifuge tube on a magnetic rack and magnetically separate for 5 minutes to allow the supernatant to become completely clear. Discard the supernatant.

[0067] 16. Add 200 μL of chemiluminescent washing solution, mix horizontally with a mixer, and remove the washing solution using magnetic separation. Repeat the washing step 3 times.

[0068] 17. Add 100 μL of chemiluminescent substrate to the centrifuge tube and mix horizontally with a mixer.

[0069] 18. Use a pipette to transfer all the liquid in the tube to the strips of the chemiluminescence plate.

[0070] 19. Test the light emission signal on the machine and record the test results.

[0071] like Figure 11 As shown, the fluorescence signal value of the monodisperse carboxylated polymer magnetic composite microspheres prepared by the magnetic porous microspheres of the present invention is significantly higher than that of traditional polymer microspheres. This indicates that after surface functionalization modification, the magnetic porous microspheres of the present invention can couple more and more active AFP antibodies, exhibiting higher capture efficiency and demonstrating excellent antibody capture performance. Furthermore, the higher signal indicates less non-specific adsorption. In addition, the magnetic porous microspheres of the present invention have more optimized particle size, pore size, or porous structure, reducing the problem of decreased binding efficiency caused by steric hindrance.

[0072] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing monodisperse functionalized polymer magnetic composite microspheres, characterized in that, Includes the following steps: S1. Styrene, PVP, AIBN and ethanol are reacted, and after the reaction, the mixture is washed and dried to obtain polystyrene seed pellets; S2. Take the polystyrene seed balls obtained in step S1 and carry out a two-step swelling polymerization reaction to obtain porous cross-linked polystyrene microspheres; S3. Sulfonated porous polystyrene microspheres are obtained by mixing and reacting porous cross-linked polystyrene microspheres with acid solution; S4. After dispersing sulfonated porous cross-linked polystyrene microspheres in water, an aqueous solution of FeSO4·7H2O was added for the first reaction. After the reaction, the microspheres were washed, and concentrated ammonia was added for the second reaction. After the reaction was completed, the microspheres were washed and dried to obtain magnetic porous microspheres. S5. Magnetic porous microspheres are reacted with functional monomers by precipitation polymerization to obtain monodisperse functionalized magnetic composite microspheres.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of styrene, PVP, AIBN, and ethanol is 60:3.6:1.2:200; the reaction temperature is 70℃, the reaction time is 24h, and the stirring speed during the reaction is 300r / min.

3. The preparation method according to claim 1, characterized in that, In step S2, the two-step swelling polymerization reaction includes: first swelling of polystyrene seed balls, 0.25wt% sodium dodecyl sulfate, and dibutyl phthalate to obtain solution A; second swelling of styrene monomer, divinylbenzene, and benzoyl peroxide to obtain solution B, adding it to solution A to obtain solution C; then adding 5wt% polyvinyl alcohol solution to carry out the polymerization reaction; after the reaction is completed, the product is washed and dried to obtain porous cross-linked polystyrene microspheres.

4. The preparation method according to claim 3, characterized in that, The first and second swellings were performed at a temperature of 30°C for 24 hours, with a stirring rate of 120 r / min; the polymerization reaction was carried out at a temperature of 70°C for 24 hours.

5. The preparation method according to claim 3, characterized in that, The solid-liquid ratio of the polystyrene seed pellets, sodium dodecyl sulfate, and dibutyl phthalate is 0.5g:50mL:2g; the mass ratio of the styrene monomer, divinylbenzene, and benzoyl peroxide is 6:6:

1.

6. The preparation method according to claim 1, characterized in that, In step S3, the mass ratio of the porous cross-linked polystyrene microspheres to the acid solution is 3:200; the reaction is first ultrasonically dispersed at 300 r / min for 0.5 h, and then reacted at 300 r / min in a 50°C water bath for 2 h.

7. The preparation method according to claim 1, characterized in that, In step S4, the first reaction is carried out at 300 r / min for 24 h with stirring; the second reaction is carried out at 80°C and 300 r / min for 2 h.

8. The preparation method according to claim 1, characterized in that, In step S4, the functional monomer is one or more of glycidyl methacrylate, divinylbenzene, acrylic acid, methacrylic acid, and ethylene glycol dimethacrylate.

9. A monodisperse functionalized polymer magnetic composite microsphere, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 8.

10. The application of monodisperse functionalized polymer magnetic composite microspheres prepared by any one of claims 1 to 8 in nucleic acid extraction, protein extraction or protein purification.

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