Carboxyl magnetic silica microspheres with controllable particle size, one-step preparation method and application thereof

Abstract

This invention relates to a carboxyl-based magnetic silica microsphere with controllable particle size, its one-step preparation method, and its applications, belonging to the field of magnetic material preparation technology. This invention uses Fe3O4 magnetic particles as the magnetic core, with a silica layer coated on the core surface. Carboxyl groups are grafted onto the surface using aminosilane compounds and diacid compounds as functionalizing agents to obtain Fe3O4@SiO2-COOH composite microspheres. The particle size of the carboxyl-based magnetic silica microspheres is 0.5 μm-10 μm. The preparation method of this invention is simple, highly controllable, and time-efficient. The particle size of the prepared magnetic silica microspheres can be controlled between the nanometer and micrometer levels, and it can also be used for large-scale production. The prepared microspheres have good dispersibility, high magnetic content, and high carboxyl content, and can specifically bind to proteins, which can be further used for the separation of proteins and other biomolecules.

Description

Technical Field

[0001] This invention belongs to the field of magnetic material preparation technology, specifically relating to a carboxyl magnetic silica microsphere with controllable particle size, its one-step preparation method, and its application. Background Technology

[0002] Magnetic microspheres are a novel material with advantages such as easy magnetic separation and good adsorption performance. They have wide applications in magnetic physics, chemical engineering, environmental monitoring, immobilized enzymes, protein separation, cell separation, targeted drug delivery, and immunoassay. Magnetic microspheres must possess narrow particle size distribution, high magnetic responsiveness, and high surface functional group density. Functionalized magnetic microspheres firstly exhibit strong paramagnetism, enabling rapid aggregation in a magnetic field and uniform dispersion upon removal from the magnetic field. Secondly, they possess a suitable and uniform particle size, ensuring sufficient magnetic responsiveness and good dispersibility. Thirdly, they possess abundant surface-active groups, allowing coupling with biochemical substances and separation from the sample under the influence of an external magnetic field. Generally, functionalized magnetic microspheres for application in the biological field need to meet several conditions: ① high magnetic responsiveness; ② high surface functional group content; ③ controllable particle size to meet different needs; ④ specific binding to biomolecules such as proteins and nucleic acids, exhibiting good biocompatibility.

[0003] Many functionalized magnetic microspheres have been reported, but none can simultaneously meet multiple requirements such as superparamagnetism, stable dispersion in a medium, high surface functional group content, tunable particle size between nanometer and micrometer levels, and specific adsorption of proteins. In existing carboxylated magnetic microsphere preparations, most are limited by particle size (not reaching the micrometer level) and low carboxyl content, resulting in low specific adsorption protein content, which greatly restricts their applications. Therefore, this application provides a carboxylated magnetic microsphere with tunable particle size, capable of one-step preparation of Fe3O4@SiO2-COOH composite microspheres with a particle size between 0.5 μm and 10 μm. These low-micrometer-scale carboxylated magnetic silica microspheres can be used for immunoglobulin preparation to overcome the aforementioned shortcomings. Furthermore, the preparation method is simple and can be scaled up for production. Summary of the Invention

[0004] To address the shortcomings of the existing technology, the present invention aims to design and provide a carboxyl magnetic silica microsphere with controllable particle size, a one-step preparation method thereof, and its application.

[0005] This invention uses Fe3O4 magnetic particles as the magnetic core, coats them with a layer of silicon dioxide, and uses aminosilane compounds and diacid compounds as functionalizing agents. While grafting functional groups onto the surface, an amidation reaction of amino and carboxyl groups occurs, and Fe3O4@SiO2-COOH composite microspheres are obtained in a one-step reaction.

[0006] To achieve the above objectives, the following technical solution is adopted:

[0007] On one hand, the present invention provides a carboxyl magnetic silica microsphere with controllable particle size, using Fe3O4 magnetic particles as the magnetic core, with a silica layer coated on the surface of the magnetic core, and using aminosilane compounds and diacid compounds as functionalizing reagents to graft carboxyl groups on the surface to obtain Fe3O4@SiO2-COOH composite microspheres.

[0008] The aforementioned carboxyl magnetic silica microspheres have a particle size controllable, wherein the particle size of the carboxyl magnetic silica microspheres is 0.5 μm-10 μm.

[0009] Secondly, the present invention provides a one-step preparation method for the aforementioned carboxyl magnetic silica microspheres with controllable particle size, comprising the following steps:

[0010] (1) Weigh out the oil phase alkane solvent, alkylphenol and ethylene oxide condensate, and sodium silicate aqueous solution, mix them and then ultrasonically disperse them to form a W / O emulsion system (i.e., water-in-oil emulsion system). Then add Fe3O4 magnetic particles and ultrasonically disperse them in the W / O emulsion system and stir evenly.

[0011] (2) Add long-chain fatty acid compounds dissolved in oil-phase alkane solvent dropwise, react at room temperature for 2-6 hours under nitrogen protection, add diacid compounds dissolved in deionized water by ultrasonication to form diacid compound solution, heat to 80℃, add aminosilane compounds dropwise, stir for 2-6 hours, and after washing with alternating ethanol and water, obtain Fe3O4@SiO2-COOH composite microspheres.

[0012] In the preparation method described above, the oil phase alkane solvent includes hexane, heptane, octane, hexadecane, and octadecane;

[0013] The alkylphenol-ethylene oxide condensate includes OP-4, OP-7, OP-9, and OP-10.

[0014] In the preparation method described above, the volume ratio of the sodium silicate aqueous solution to the oil phase alkane solvent added in step (1) is 1:25 to 3:10.

[0015] The volume ratio of the alkylphenol to ethylene oxide condensate and the oil phase alkane solvent added in step (1) is 1:25 to 2:5;

[0016] The mass ratio of the Fe3O4 magnetic particles to the oil phase alkane solvent added in step (1) is 0.2:79 to 4:79;

[0017] The concentration of the sodium silicate solution is 0.5-5M.

[0018] In the preparation method described above, the concentration of the diacid compound solution is 2-20 wt%.

[0019] The mass of the aminosilane compound is 2 to 27 wt% of the mass of the oil phase alkane solvent added in step (1).

[0020] The preparation method described herein includes long-chain fatty acid compounds that are soluble in oil-phase alkane solvents, preferably including tetradecanoic acid, octadecanoic acid, and oleic acid.

[0021] The preparation method described herein, wherein the diacid compounds include compounds containing two carboxyl groups or diacid anhydrides, preferably including maleic acid, succinic acid, maleic anhydride, and succinic anhydride;

[0022] The aminosilane compounds include 3-aminopropyltriethoxysilane (KH550) and 3-aminopropyltrimethoxysilane (KH540).

[0023] The preparation method described above, wherein the Fe3O4 magnetic particles are prepared by:

[0024] Dissolve FeCl3 6H2O and Fe(NH4)2(SO4)2 7H2O in deionized water, stir, and heat to 50-60℃. Add PEG8000 and react for 10 min. Add ammonia water, heat to 80℃, react for 15 min, stop stirring, let it settle naturally for 1 h, cool to room temperature, and wash.

[0025] Preferably, the mass ratio of FeCl3 6H2O to Fe(NH4)2(SO4)2 7H2O is 0.27:1 to 0.9:1.

[0026] Thirdly, the present invention provides the application of the aforementioned carboxyl magnetic silica microspheres with controllable particle size as immune microspheres.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The particle size of the composite microspheres of this invention can be changed by altering the ratio of sodium silicate and surfactant, changing the rotation speed, etc., thereby achieving particle size control between 0.5-10 μm, which can meet most needs. It also has superparamagnetic properties and good dispersibility. After activation, the Fe3O4@SiO2-COOH composite microspheres have a high bovine serum albumin adsorption rate.

[0029] The preparation method of this invention is simple, highly controllable, and time-saving. The particle size of the prepared magnetic silica microspheres can be adjusted between the nanometer and micrometer levels. It can also be used for large-scale production. The prepared microspheres have good dispersibility, high magnetic content, and high carboxyl content. They can specifically bind to proteins and have a relatively high protein coupling capacity, which can be further used for the separation of proteins and other biological macromolecules. Attached Figure Description

[0030] Figure 1 A scanning electron microscope (SEM) schematic diagram of the Fe3O4 magnetic particles prepared in Example 1;

[0031] Figure 2 X-ray diffraction diagram of the Fe3O4 magnetic particles prepared in Example 1;

[0032] Figure 3 A scanning electron microscope (SEM) schematic diagram of the Fe3O4@SiO2-COOH composite microspheres prepared in Example 1;

[0033] Figure 4 A schematic diagram of the Raman spectrum of the Fe3O4@SiO2-COOH composite microspheres prepared in Example 1;

[0034] Figure 5 This is a schematic diagram of the infrared spectrum of the Fe3O4@SiO2-COOH composite microspheres prepared in Example 1.

[0035] Figure 6 This is a transmission electron microscope (TEM) schematic diagram of the Fe3O4@SiO2-COOH composite microspheres prepared in Example 2;

[0036] Figure 7 This is a transmission electron microscope (TEM) schematic diagram of the Fe3O4@SiO2-COOH composite microspheres prepared in Example 3;

[0037] Figure 8 This is a transmission electron microscope (TEM) schematic diagram of the Fe3O4@SiO2-COOH composite microspheres prepared in Example 4. Detailed Implementation

[0038] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. However, those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0039] Example 1:

[0040] This embodiment 1 provides a one-step method for preparing carboxyl-based magnetic silica microspheres with controllable particle size. The preparation method includes:

[0041] (1) Preparation of Fe3O4 magnetic particles: 2g FeCl3·6H2O and 2.5g Fe(NH4)2(SO4)2·7H2O were weighed and dissolved in 50mL of water. The mixture was heated to 56℃, and 0.2g PEG-8000 was added. The mixture was stirred at 330rpm for 10min, and then 30mL of ammonia water was added. The temperature was raised to 80℃ and the mixture was reacted for 15min. The stirring was stopped, and the mixture was allowed to stand for 1.5h. After the reaction was completed, the particles were magnetically separated using a magnet. The particles were washed repeatedly with deionized water and ethanol to remove residual components and byproducts. The particles were stored in deionized water to obtain superparamagnetic Fe3O4 magnetic particles. The scanning electron microscope image is shown below. Figure 1 As shown, the X-ray diffraction pattern is as follows: Figure 2 As shown.

[0042] (2) Preparation of Fe3O4@SiO2-COOH composite microspheres: 4 mL of sodium silicate (1.5 M) solution, 100 mL of hexane and 12 mL of OP-10 were weighed into a beaker and sonicated for 30 min. Then, 1 g of the Fe3O4 magnetic particles prepared in step (1) were weighed and mixed with the above solution and sonicated for 30 min. The mixture was placed in a 500 mL two-necked flask and stirred at 250 rpm for 10 min. 1.375 g of octadecanoic acid was weighed and dissolved in 20 mL of hexane and added dropwise to the above solution for 15 min. The reaction was allowed to proceed for 2 h. Then, 5 g of maleic acid was weighed and sonicated and dissolved in 25 mL of deionized water and added to the above solution. The temperature was raised to 80 °C. 13 mL of the mixture was then weighed and added to the solution. 3-Aminopropyltriethoxysilane (KH550) was slowly added dropwise to a two-necked flask over 10 minutes, and stirring was continued for 2 hours. After the reaction was complete, the flask was placed on a magnetic rack for magnetic separation. The microspheres were washed several times with alternating ethanol and water to remove any reaction residue. After washing, the microspheres were dried in an oven for 24 hours to obtain Fe3O4@SiO2-COOH composite microspheres with a particle size of approximately 10 μm and good dispersibility. Scanning electron microscopy results are shown below. Figure 3 Raman spectroscopy, such as Figure 4 Infrared spectrum, such as Figure 5 .

[0043] Example 2:

[0044] This embodiment 2 provides a one-step method for preparing carboxyl-based magnetic silica microspheres with controllable particle size. The preparation method includes:

[0045] (1) Preparation of Fe3O4 magnetic particles: Weigh 2g FeCl3·6H2O and 2.5g Fe(NH4)2(SO4)2·7H2O and dissolve them in 50mL of water. Heat to 56℃, add 0.2g PEG-8000, stir at 330rpm for 10min, then add 30mL of ammonia water, heat to 80℃ and react for 15min. Stop stirring and let stand for 1.5h. After the reaction is complete, use a magnet for magnetic separation, wash with deionized water and ethanol alternately several times to remove reaction residues and byproducts, and store in deionized water to obtain superparamagnetic Fe3O4 magnetic particles.

[0046] (2) Preparation of Fe3O4@SiO2-COOH composite microspheres: 16 mL of sodium silicate (1.5 M) solution, 100 mL of hexane and 15 mL of... OP-10 was placed in a beaker and sonicated for 30 min. Then, 1.1 g of the Fe3O4 magnetic particles prepared in step (1) was weighed and mixed with the above mixture, and sonicated for 30 min. The mixture was placed in a 500 mL two-necked flask and stirred at 400 rpm for 10 min. 2 g of octadecanoic acid was weighed and dissolved in 20 mL of hexane, and added dropwise to the above solution for 15 min. The reaction was allowed to proceed for 4 h. Then, 5 g of maleic acid was weighed and sonicated and dissolved in 25 mL of deionized water, and added to the above solution. The temperature was raised to 80 °C. 13 mL of 3-aminopropyltriethoxysilane (KH550) was measured and slowly added dropwise to the two-necked flask over 10 min. The mixture was stirred for 2 h. After the reaction was completed, the mixture was placed on a magnetic rack for magnetic separation. The mixture was washed several times with alternating ethanol and water to remove the reaction residue. After washing, the mixture was placed in an oven and dried for 24 h to obtain Fe3O4@SiO2-COOH composite microspheres with a particle size of about 1.5 μm. Transmission electron microscopy was performed as follows. Figure 6 .

[0047] Example 3:

[0048] This embodiment provides a one-step method for preparing carboxyl-based magnetic silica microspheres with controllable particle size. The preparation method includes:

[0049] (1) Preparation of Fe3O4 magnetic particles: Weigh 2g FeCl3·6H2O and 2.5g Fe(NH4)2(SO4)2·7H2O and dissolve them in 50mL of water. Heat to 56℃, add 0.2g PEG-8000, stir at 330rpm for 10min, then add 30mL of ammonia water, heat to 80℃ and react for 15min. Stop stirring and let stand for 1.5h. After the reaction is complete, use a magnet for magnetic separation, wash with deionized water and ethanol alternately several times to remove reaction residues and byproducts, and store in deionized water to obtain superparamagnetic Fe3O4 magnetic particles.

[0050] (2) Preparation of Fe3O4@SiO2-COOH composite microspheres: 8 mL of sodium silicate (1.5 M) solution, 100 mL of hexane and 15 mL of OP-10 were weighed into a beaker and sonicated for 30 min. Then, 1 g of the Fe3O4 magnetic particles prepared in step (1) were weighed into the above mixture and sonicated for 30 min. The mixture was placed in a 500 mL two-necked flask and stirred at 500 rpm for 10 min. 2 g of octadecanoic acid was weighed and dissolved in 20 mL of hexane and added dropwise to the above solution for 15 min. The reaction was allowed to proceed for 3 h. Then, 5 g of maleic acid was weighed and sonicated into 25 mL of deionized water and added to the above solution. The temperature was raised to 80 °C. 13 mL of the mixture was then weighed into the solution and added to the solution. 3-Aminopropyltriethoxysilane (KH550) was slowly added dropwise to a two-necked flask over 10 minutes, and stirring was continued for 2 hours. After the reaction was complete, the flask was placed on a magnetic rack for magnetic separation. The mixture was washed several times with alternating ethanol and water to remove any reaction residue. After washing, the flask was dried in an oven for 24 hours to obtain Fe3O4@SiO2-COOH composite microspheres with a particle size of approximately 1 μm. Transmission electron microscopy (TEM) results are shown below. Figure 7 .

[0051] Example 4:

[0052] This embodiment provides a one-step method for preparing carboxyl-based magnetic silica microspheres with controllable particle size. The preparation method includes:

[0053] (1) Preparation of Fe3O4 magnetic particles: Weigh 2g FeCl3·6H2O and 2.5g Fe(NH4)2(SO4)2·7H2O and dissolve them in 50mL of water. Heat to 56℃, add 0.2g PEG-8000, stir at 330rpm for 10min, then add 30mL of ammonia water, heat to 80℃ and react for 15min. Stop stirring and let stand for 1.5h. After the reaction is complete, use a magnet for magnetic separation, wash with deionized water and ethanol alternately several times to remove reaction residues and byproducts, and store in deionized water to obtain superparamagnetic Fe3O4 magnetic particles.

[0054] (2) Preparation of Fe3O4@SiO2-COOH composite microspheres: 10 mL of sodium silicate (1.5 M) solution, 100 mL of hexane and 20 mL of... OP-10 was placed in a beaker and sonicated for 30 min. Then, 1.1 g of Fe3O4 magnetic particles prepared in step (1) was weighed and mixed with the above mixture, and sonicated for 30 min. The mixture was placed in a 500 mL two-necked flask and stirred for 10 min at a stirring rate of 300 rpm. 2 g of octadecanoic acid was weighed and dissolved in 20 mL of hexane and added dropwise to the above solution for 15 min. The reaction was carried out for 2 h. Then, 5 g of maleic anhydride was weighed and sonicated and dissolved in 25 mL of deionized hot water and added to the above solution. The temperature was raised to 80 °C. Then, 13 mL of 3-aminopropyltriethoxysilane (KH550) was measured and slowly added dropwise to the two-necked flask over 10 min. The stirring was continued for 2 h. After the reaction was completed, the mixture was placed on a magnetic rack for magnetic separation. The mixture was washed several times with alternating ethanol and water to remove the reaction residue. After washing, the mixture was placed in an oven and dried for 24 h to obtain Fe3O4@SiO2-COOH composite microspheres with a particle size of about 500 nm. Transmission electron microscopy, such as Figure 8 .

[0055] Example 5:

[0056] This embodiment provides a one-step method for preparing carboxyl-based magnetic silica microspheres with controllable particle size. The preparation method includes:

[0057] (1) Preparation of Fe3O4 magnetic particles: Weigh 2g FeCl3·6H2O and 2.5g Fe(NH4)2(SO4)2·7H2O and dissolve them in 50mL of water. Heat to 56℃, add 0.2g PEG-8000, stir at 330rpm for 10min, then add 30mL of ammonia water, heat to 80℃ and react for 15min. Stop stirring and let stand for 1.5h. After the reaction is complete, use a magnet for magnetic separation, wash with deionized water and ethanol alternately several times to remove reaction residues and byproducts, and store in deionized water to obtain superparamagnetic Fe3O4 magnetic particles.

[0058] (2) Preparation of Fe3O4@SiO2-COOH composite microspheres: Measure 4 mL of sodium silicate (2.5M) solution, 100 mL of octane and 12 mL of OP-10 into a beaker, sonicate for 30 min, then weigh 1.1 g of the Fe3O4 magnetic particles prepared in step (1) and the above mixture, sonicate for 30 min, place the mixture in a 500 mL two-necked flask, stir at 300 rpm for 10 min, weigh 1.375 g of tetradecanoic acid and dissolve it in 20 mL of octane, add it dropwise to the above solution for 15 min, react for 2 h, then weigh 5 g of succinic anhydride and sonicate it in 25 mL of deionized hot water, add it to the above solution, heat to 80 °C, and then measure 13 mL of... 3-Aminopropyltrimethoxysilane (KH540) was slowly added dropwise to a two-necked flask over 10 min, and stirring was continued for 2 h. After the reaction was completed, the flask was placed on a magnetic rack for magnetic separation. The flask was washed several times with alternating ethanol and water to remove the reaction residue. After washing, the flask was placed in an oven and dried for 24 h to obtain Fe3O4@SiO2-COOH composite microspheres with a particle size of about 500 nm.

[0059] Example 6:

[0060] This embodiment provides a one-step method for preparing carboxyl-based magnetic silica microspheres with controllable particle size. The preparation method includes:

[0061] (1) Preparation of Fe3O4 magnetic particles: Weigh 2g FeCl3·6H2O and 2.5g Fe(NH4)2(SO4)2·7H2O and dissolve them in 50mL of water. Heat to 56℃, add 0.2g PEG-8000, stir at 330rpm for 10min, then add 30mL of ammonia water, heat to 80℃ and react for 15min. Stop stirring and let stand for 1.5h. After the reaction is complete, use a magnet for magnetic separation, wash with deionized water and ethanol alternately several times to remove reaction residues and byproducts, and store in deionized water to obtain superparamagnetic Fe3O4 magnetic particles.

[0062] (2) Preparation of Fe3O4@SiO2-COOH composite microspheres: 16 mL of sodium silicate (2M) solution, 100 mL of heptane and 15 mL of OP-9 were weighed into a beaker and sonicated for 30 min. Then, 1.1 g of the Fe3O4 magnetic particles prepared in step (1) were weighed into the above mixture and sonicated for 30 min. The mixture was placed in a 500 mL two-necked flask and stirred at 400 rpm for 10 min. 2.5 g of octadecanoic acid was weighed and dissolved in 20 mL of heptane and added dropwise to the above solution for 15 min. The reaction was allowed to proceed for 2 h. Then, 8 g of succinic anhydride was weighed and sonicated into 25 mL of deionized hot water and added to the above solution. The temperature was raised to 80 °C. 13 mL of the mixture was then weighed into the above solution and added dropwise to the solution. 3-Aminopropyltriethoxysilane (KH550) was slowly added dropwise to a two-necked flask over 10 min, and stirring was continued for 2 h. After the reaction was completed, the flask was placed on a magnetic rack for magnetic separation. The flask was washed several times with alternating ethanol and water to remove the reaction residue. After washing, the flask was placed in an oven and dried for 24 h to obtain Fe3O4@SiO2-COOH composite microspheres with a particle size of about 500 nm.

[0063] Example 7:

[0064] Method for activating magnetic beads: Place 20 μl of well-mixed magnetic beads in a 2 mL centrifuge tube, wash three times with 200 μl of MES (2-morpholinoethanesulfonic acid) solution, remove the waste liquid, add 100 μl of freshly prepared 50 mg / mL EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) solution and 100 μl of freshly prepared NHS (N-hydroxysuccinimide) solution in ultrapure water, mix well, and rotate at low speed for 30 minutes at room temperature. After the process, place the tube on a magnetic rack for adsorption for 3 minutes, discard the supernatant, and wash three more times with 200 μl of MES solution.

[0065] The performance of the Fe3O4@SiO2-COOH composite particles provided in Example 1 of this invention was tested and compared with that of carboxyl magnetic beads (item number: 10004D) from a well-known foreign company. The results are shown in Table 1.

[0066] Table 1. Performance Comparison of Carboxyl Magnetic Silica Microspheres

[0067] This application (Example 1) well-known foreign companies Magnetic separation time Less than 10s Greater than 20s Magnetic saturation 35-50 emu / g 30-50 emu / g Bovine serum albumin conjugates 10-15mg / g 5mg / g Particle size selection range 10μm 2.8μm

[0068] As shown in Table 1, compared with carboxyl magnetic microspheres provided by well-known foreign companies, the Fe3O4@SiO2-COOH composite microspheres provided in this application offer a wider range of particle size options, shorter magnetic separation time (i.e., higher magnetic content), and higher coupling protein efficiency (i.e., higher carboxyl content). This indicates that the Fe3O4@SiO2-COOH composite microspheres prepared in this application have superior performance, shorter magnetic separation time, are more conducive to operation and large-scale use, and provide better separation results.

[0069] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A one-step preparation method for carboxyl-based magnetic silica microspheres with controllable particle size, characterized in that, The carboxyl magnetic silica microspheres with controllable particle size use Fe3O4 magnetic particles as the magnetic core, with a silica layer wrapped around the surface of the magnetic core. Carboxyl groups are grafted onto the surface using aminosilane compounds and diacid compounds as functionalizing agents to obtain Fe3O4@SiO2-COOH composite microspheres. The preparation method includes the following steps: (1) Weigh out the oil phase alkane solvent, alkylphenol and ethylene oxide condensate, and sodium silicate aqueous solution, mix them and then ultrasonically disperse them to form a W / O emulsion system. Then add Fe3O4 magnetic particles and ultrasonically disperse them in the W / O emulsion system and stir evenly. (2) Add long-chain fatty acid compounds dissolved in oil-phase alkane solvent dropwise, react at room temperature for 2-6 hours under nitrogen protection, heat to 80°C, add diacid compounds dissolved in deionized water by ultrasonication to form diacid compound solution, add aminosilane compounds dropwise, stir for 2-6 hours, wash with ethanol and water alternately to obtain Fe3O4@SiO2-COOH composite microspheres; The preparation method of the Fe3O4 magnetic particles includes: FeCl3 6H2O and Fe(NH4)2(SO4)2 7H2O were dissolved in deionized water and stirred. PEG8000 was added while the mixture was heated to 50-60℃. After reacting for 10 min, ammonia was added, and the temperature was raised to 80℃. The mixture was reacted for 15 min, and stirring was stopped. After natural sedimentation for 1-1.5 h, the mixture was cooled to room temperature, and magnetic separation and washing were performed to obtain Fe3O4 magnetic particles.

2. The one-step preparation method according to claim 1, characterized in that, The particle size of the carboxyl magnetic silica microspheres is 0.5 μm-10 μm.

3. The one-step preparation method as described in claim 1, characterized in that, The oil phase alkane solvent includes hexane, heptane, octane, hexadecane, and octadecane; The alkylphenol-ethylene oxide condensate includes OP-4, OP-7, OP-9, and OP-10.

4. The one-step preparation method according to claim 1, characterized in that, The volume ratio of the sodium silicate aqueous solution to the oil phase alkane solvent added in step (1) is 1:25 to 3:10; The volume ratio of the alkylphenol and ethylene oxide condensate to the oil phase alkane solvent added in step (1) is 1:25 to 2:5; the mass ratio of the Fe3O4 magnetic particles to the oil phase alkane solvent added in step (1) is 0.2:79 to 4:

79. The concentration of the sodium silicate aqueous solution is 0.5-5M.

5. The one-step preparation method according to claim 1, characterized in that, The concentration of the diacid compound solution is 2–20 wt%; The mass of the aminosilane compound is 2 to 27 wt% of the mass of the oil phase alkane solvent added in step (1).

6. The one-step preparation method according to claim 1, characterized in that, The long-chain fatty acid compounds include long-chain fatty acid acids that are soluble in oil-phase alkane solvents.

7. The one-step preparation method according to claim 6, characterized in that, The long-chain fatty acid compounds include tetradecanoic acid, octadecanoic acid, and oleic acid.

8. The one-step preparation method according to claim 1, characterized in that, The diacid compounds include compounds containing two carboxyl groups or diacid anhydrides.

9. The one-step preparation method as described in claim 8, characterized in that, The diacid compounds include maleic acid, succinic acid, maleic anhydride, and succinic anhydride; The aminosilane compounds include 3-aminopropyltriethoxysilane and 3-aminopropyltrimethoxysilane.

10. The one-step preparation method according to claim 1, characterized in that, The mass ratio of FeCl3 6H2O to Fe(NH4)2(SO4)2 7H2O is 0.27:1 to 0.9:

1.

11. The application of the one-step preparation method according to any one of claims 1-10 in the preparation of immune microspheres.

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