Carboxyl modified magnetic microspheres as well as preparation method and application thereof

Abstract

The invention relates to the technical field of magnetic polymer microspheres, in particular to carboxyl modified magnetic microspheres and a preparation method and application thereof, and the preparation method comprises the following steps: 1) synthesizing an oleic acid modified Fe3O4 magnetic core, namely OA-Fe3O4, with uniform particle size by adopting a coprecipitation method; (2) preparing surface amino modified polystyrene microspheres PS-NH2 by a dispersion polymerization method; (3) embedding and adsorbing OA-Fe3O4 by a swelling method; and 4) carrying out secondary polymerization to fix the magnetic particles and carrying out magnetic coating. A secondary polymerization technology is adopted, the problem that magnetic particles are prone to leakage is solved, the outer portions of the microspheres are further coated with the polymer shell layers, the magnetic particles adsorbed to the surfaces of the microspheres are also embedded into the microspheres, and the magnetic content of the microspheres is greatly increased.

Description

Technical Field

[0001] The present invention relates to the technical field of magnetic polymer microspheres, and specifically to a carboxyl-modified magnetic microsphere, a preparation method and an application thereof. Background Art

[0002] As a new type of functional material, magnetic polymer microspheres are composite microspheres formed by combining inorganic magnetic particles with polymer materials. They have many characteristics of both polymer microspheres and magnetic particles. They can be dispersed evenly and stably in the absence of an external magnetic field, and can be rapidly separated under the condition of an external magnetic field. In addition, the surface of the microspheres can be modified to endow them with various reactive functional groups (such as carboxyl, amino, hydroxyl, sulfonic acid groups, etc.) on the surface, making them have good biocompatibility. Therefore, they have broad application prospects in biological fields such as cell separation, analysis and detection, and immunoassay. Currently, common magnetic polymer microspheres include polystyrene-based, silane-based, and polymethyl methacrylate-based, etc. Among them, polystyrene-based and silane-based polymer materials are commonly used in the field of chemiluminescence immunoassay, and the most studied are polystyrene-based magnetic microspheres.

[0003] The preparation methods of magnetic polystyrene microspheres include swelling method, dispersion polymerization method, seed polymerization method, etc. Among them, the swelling method usually obtains magnetic polystyrene microspheres by mixing polystyrene microspheres with oleic acid-modified Fe3O4. For example, CN111701544A discloses a preparation method of magnetic polystyrene microspheres, specifically: first prepare porous polystyrene microspheres, disperse the microspheres in an organic solution to obtain a mixed solution A, and the organic solution is dichloromethane / cyclohexanol or dichloromethane / xylene; then disperse oleic acid-modified magnetic iron oxide in a xylene solution to obtain a mixed solution B; then mix the mixed solution A and the mixed solution B, after swelling at room temperature, dry and wash to obtain magnetic polystyrene microspheres. However, this method usually has problems such as low embedding rate of magnetic materials and easy magnetic leakage, and this preparation method uses a variety of organic solvents harmful to the human body and is prone to environmental pollution. Summary of the Invention

[0004] In view of the problems in the prior art such as low embedding rate of magnetic materials, easy magnetic leakage, use of a large amount of organic solvents harmful to the human body and easy environmental pollution, the present invention provides a carboxyl-modified magnetic microsphere, a preparation method and an application thereof.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A preparation method of a carboxyl-modified magnetic microsphere, comprising the following steps:

[0007] (1) Synthesize uniformly sized oleic acid-modified Fe3O4 magnetic cores by the co-precipitation method: Dissolve FeCl3·6H2O and FeCl2·4H2O in pure water, then add ammonia water until the pH of the system is 9 - 11. A black precipitate of Fe3O4 is formed. Then add a little oleic acid to react to form oleic acid-modified Fe3O4, namely OA-Fe3O4. Among them, the mass ratio of oleic acid to the total iron salt is 1:0.5 - 5.

[0008] (2) Prepare surface amino-modified polystyrene microspheres PS-NH2 by dispersion polymerization: Add pure water and ethanol to the reaction vessel, purge with nitrogen for 30 min to remove oxygen, and then add an appropriate amount of polyvinylpyrrolidone. Measure styrene and N-isopropylacrylamide into an EP tube, add an oil-soluble initiator AIBN, dissolve by ultrasound, add it to the reaction vessel, set the temperature to 70 °C, react for 4 - 8 h, and store it in ethanol for later use.

[0009] (3) Embed and adsorb OA-Fe3O4 by the swelling method: Add the polystyrene microspheres prepared in step (2) and a swelling agent to the reaction vessel, disperse by ultrasound, and then add the OA-Fe3O4 prepared in step (1). React at 70 °C for 1 - 4 h. After the reaction, wash 3 times with ethanol and store in pure water.

[0010] (4) Secondary polymerization to fix magnetic particles and magnetic coating: Add the magnetic polystyrene microspheres prepared in step (3) and ethanol to the reaction vessel, purge with nitrogen for 30 min; then add polyvinyl alcohol, set the temperature to 60 °C; weigh the initiator AIBN, dissolve it in a little ethanol and add it to the reaction vessel; after 1 h, measure styrene and methacrylic acid in an ethanol solution, and slowly add it dropwise to the reaction vessel drop by drop, and react overnight.

[0011] Among them, in the step (1), the molar ratio of FeCl3·6H2O to FeCl2·4H2O is 1:0.5 - 1.

[0012] Among them, in the step (2), the volume ratio of water to ethanol is 1:4 - 9, the volume ratio of styrene to N-isopropylacrylamide is 1:0.02 - 0.2, and the initiator accounts for 0.5 - 5% of the total monomer; the mass ratio of polyvinylpyrrolidone to the monomer is 1:2 - 8.

[0013] Among them, in the step (3), the swelling agent is anhydrous ethanol. The volume ratio of the swelling agent to water is 4 - 8:1, and the mass ratio of PS-NH2 to OA-Fe3O4 is 5 - 20:1.

[0014] Among them, in the step (4), the initiator accounts for 0.5-5% of the total amount of monomers, the volume ratio of styrene to methacrylic acid is 1:0.02-0.1; the volume ratio of magnetic polystyrene microspheres to ethanol is 1:1-10; the mass concentration of polyvinyl alcohol in the system solvent is 0.1%-1%.

[0015] The carboxyl-modified magnetic microspheres prepared by the present invention can be used in chemiluminescence immunoassay.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] For the preparation method of the carboxyl-modified magnetic microspheres of the present invention, oleic acid-coated Fe3O4 magnetic particles are first prepared by the co-precipitation method, and then surface amino-modified polystyrene microspheres are prepared by dispersion polymerization. Then, the oleic acid-modified Fe3O4 is embedded into the microspheres by the swelling method. Usually, a part of the magnetic particles OA-Fe3O4 will swell into the microspheres, and another part will be adsorbed on the surface of the microspheres due to electrostatic adsorption and hydrogen bond action. In order to prevent the magnetic particles OA-Fe3O4 inside and on the surface of the microspheres from leaking, a little initiator is first added to the system to initiate the polymerization of the remaining monomers inside and on the surface of the microspheres, and then a little styrene and carboxylic acid monomers are slowly added to continue the polymerization, and a functionalized polymer shell layer is coated outside the microspheres, so as to achieve the purpose of fixing the magnetic particles inside the microspheres and embedding the magnetic particles on the surface of the microspheres.

[0018] The present invention adopts the secondary polymerization technology, which not only solves the problem of easy leakage of magnetic particles, but also coats a polymer shell layer outside the microspheres, embedding the magnetic particles adsorbed on the surface of the microspheres into the microspheres, greatly improving the magnetic content of the microspheres. Description of the Drawings

[0019] Figure 1 It is a flow chart of the preparation method of the carboxyl-modified magnetic microspheres of the present invention. Detailed Embodiments

[0020] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0021] Example 1

[0022] As Figure 1 shown, a preparation method of carboxyl-modified magnetic microspheres, the specific steps are as follows:

[0023] (1) Preparation of OA-Fe3O4 by co-precipitation method

[0024] Weigh 2.7 g of FeCl3·6H2O and 1.2 g of FeCl2·4H2O into a reaction flask, add 30 mL of pure water and stir to dissolve. Quickly add 10 mL of ammonia water, and react at 60 °C for 2 h. Add 2 mL of oleic acid, terminate the reaction after 1 h, wash with water three times, and finally store in a small amount of water, and adjust the solid content to 10%.

[0025] (2) Preparation of PS-NH2 by dispersion polymerization

[0026] Add 10 mL of pure water and 90 mL of ethanol to a three-necked flask, purge with nitrogen for 30 min to remove oxygen, and then add 2 g of polyvinylpyrrolidone to the reaction flask. Measure 5 mL of styrene and 0.4 mL of N-isopropylacrylamide into an EP tube, add 60 mg of initiator AIBN, dissolve by ultrasonic wave, add to the reaction flask, and set the temperature to 70 °C for reaction for 4 h. Wash with water three times and wash with ethanol three times, and finally store in 10 mL of pure water.

[0027] (3) Embedding and adsorbing OA-Fe3O4 by swelling method

[0028] Add 10 mL of the reaction product prepared in step (2) and 80 mL of anhydrous ethanol to a three-necked flask, disperse by ultrasonic wave, and then add 5 mL of the reaction product prepared in step (1), and react at 70 °C for 2 h. After the reaction, wash with ethanol three times, and finally store in 20 mL of pure water.

[0029] (4) Secondary polymerization to fix magnetic particles and magnetic coating

[0030] Add 20 mL of magnetic polystyrene microspheres and 60 mL of ethanol in step (3) to a three-necked flask, and purge with nitrogen for 30 min. Then add 0.5 g of polyvinyl alcohol, and set the temperature to 70 °C. Weigh another 60 mg of initiator AIBN, dissolve it in a little ethanol and add it to the reaction flask. After 1 h, measure 3 mL of styrene and 0.1 mL of methacrylic acid in 20 mL of ethanol solution, and slowly add it drop by drop to the reaction flask, and react overnight.

[0031] Example 2

[0032] As Figure 1 shown, a preparation method of carboxyl-modified magnetic microspheres, the specific steps are as follows:

[0033] (1) Preparation of OA-Fe3O4 by coprecipitation method

[0034] Weigh 2.7 g of FeCl3·6H2O and 1.2 g of FeCl2·4H2O into a reaction flask, add 30 mL of pure water and stir to dissolve. Quickly add 10 mL of ammonia water, and react at 60 °C for 2 h. Add 2.5 mL of oleic acid, terminate the reaction after 1 h, wash with water three times, and finally store in a small amount of water, and adjust the solid content to 10%.

[0035] (2) Preparation of PS-NH2 by dispersion polymerization

[0036] Add 10 mL of pure water and 80 mL of ethanol into a three-necked flask, purge with nitrogen for 30 min to remove oxygen, and then add 0.8 g of polyvinylpyrrolidone into the reaction flask. Measure 5 mL of styrene and 0.5 mL of N-isopropylacrylamide into an EP tube, add 60 mg of initiator AIBN, dissolve by ultrasound, add into the reaction flask, and set the temperature to 70 °C for reaction for 4 h. Wash three times with water and three times with ethanol, and finally store in 10 mL of pure water.

[0037] (3) Entrapment and adsorption of OA-Fe3O4 by swelling method

[0038] Add 10 mL of the reaction product prepared in step (2) and 80 mL of absolute ethanol into a three-necked flask, disperse by ultrasound, then add 5 mL of the reaction product prepared in step (1), and react at 70 °C for 2 h. After the reaction, wash three times with ethanol, and finally store in 20 mL of pure water.

[0039] (4) Secondary polymerization for fixing magnetic particles and magnetic coating

[0040] Add 20 mL of the magnetic polystyrene microspheres in step (3) and 60 mL of ethanol into a three-necked flask, purge with nitrogen for 30 min. Then add 0.3 g of polyvinyl alcohol, and set the temperature to 70 °C. Weigh another 60 mg of initiator AIBN, dissolve it in a little ethanol and add it into the reaction flask. After 1 h, measure 3 mL of styrene and 0.2 mL of methacrylic acid in 20 mL of ethanol solution, and slowly add them drop by drop into the reaction flask, and react overnight.

[0041] Example 3

[0042] As Figure 1 shown, a preparation method of carboxyl-modified magnetic microspheres, the specific steps are as follows:

[0043] (1) Preparation of OA-Fe3O4 by coprecipitation method

[0044] Weigh 2.7 g of FeCl3·6H2O and 1.2 g of FeCl2·4H2O into a reaction flask, add 30 mL of pure water and stir to dissolve. Quickly add 10 mL of ammonia water, and react at 60 °C for 2 h. Add 3 mL of oleic acid, terminate the reaction after 1 h, wash three times with water, and finally store in a small amount of water, and adjust the solid content to 10%.

[0045] (2) Preparation of PS-NH2 by dispersion polymerization

[0046] Add 10 mL of pure water and 60 mL of ethanol to a three-necked flask, purge with nitrogen for 30 min to remove oxygen, and then add 2 g of polyvinylpyrrolidone to the reaction flask. Measure 5 mL of styrene and 0.4 mL of N-isopropylacrylamide into an EP tube, add 80 mg of initiator AIBN, dissolve by ultrasound, add to the reaction flask, and set the temperature to 70 °C for reaction for 4 h. Wash three times with water and three times with ethanol, and finally store in 10 mL of pure water.

[0047] (3) Entrapment and adsorption of OA-Fe3O4 by swelling method

[0048] Add 10 mL of the reaction product prepared in step (2) and 80 mL of absolute ethanol to a three-necked flask, disperse by ultrasound, and then add 5 mL of the reaction product prepared in step (1), and react at 70 °C for 2 h. After the reaction, wash three times with ethanol, and finally store in 20 mL of pure water.

[0049] (4) Secondary polymerization for fixing magnetic particles and magnetic coating

[0050] Add 20 mL of the magnetic polystyrene microspheres and 80 mL of ethanol in step (3) to a three-necked flask, purge with nitrogen for 30 min. Then add 0.1 g of polyvinyl alcohol and set the temperature to 70 °C. Weigh another 80 mg of initiator AIBN, dissolve it in a little ethanol and add it to the reaction flask. After 1 h, measure 2 mL of styrene and 0.2 mL of methacrylic acid in 20 mL of ethanol solution, and slowly add them drop by drop to the reaction flask, and react overnight.

[0051] Example 4

[0052] As Figure 1 shown, a method for preparing carboxyl-modified magnetic microspheres, the specific steps are as follows:

[0053] (1) Preparation of OA-Fe3O4 by coprecipitation method

[0054] Weigh 2.7 g of FeCl3·6H2O and 1.2 g of FeCl2·4H2O into a reaction flask, add 30 mL of pure water and stir to dissolve. Quickly add 10 mL of ammonia water, and react at 60 °C for 2 h. Add 2 mL of oleic acid, terminate the reaction after 1 h, wash three times with water, and finally store in a small amount of water, and adjust the solid content to 10%.

[0055] (2) Preparation of PS-NH2 by dispersion polymerization

[0056] Add 10 mL of pure water and 80 mL of ethanol to a three-necked flask, purge with nitrogen for 30 min to remove oxygen, and then add 1 g of polyvinylpyrrolidone to the reaction flask. Measure 5 mL of styrene and 0.8 mL of N-isopropylacrylamide into an EP tube, add 100 mg of initiator AIBN, dissolve by ultrasonic treatment, add to the reaction flask, and set the temperature to 70 °C for reaction for 4 h. Wash three times with water and three times with ethanol, and finally store in 10 mL of pure water.

[0057] (3) Embedding and adsorbing OA-Fe3O4 by swelling method

[0058] Add 10 mL of the reaction product prepared in step (2) and 80 mL of absolute ethanol to a three-necked flask, disperse by ultrasonic treatment, and then add 5 mL of the reaction product prepared in step (1), and react at 70 °C for 2 h. After the reaction, wash three times with ethanol, and finally store in 20 mL of pure water.

[0059] (4) Secondary polymerization to fix magnetic particles and magnetic coating

[0060] Add 20 mL of the magnetic polystyrene microspheres and 80 mL of ethanol in step (3) to a three-necked flask, purge with nitrogen for 30 min. Then add 0.2 g of polyvinyl alcohol and set the temperature to 70 °C. Weigh another 100 mg of initiator AIBN, dissolve it in a little ethanol and add it to the reaction flask. After 1 h, measure 3 mL of styrene and 0.3 mL of methacrylic acid into 20 mL of ethanol solution, and slowly add it drop by drop to the reaction flask, and react overnight.

[0061] Example 5 Application example of magnetic carboxyl microspheres (taking myoglobin (MYO)-labeled antibody as an example)

[0062] (1) Dilution of microspheres

[0063] Take 2 mg of magnetic carboxyl microspheres in a centrifuge tube, perform magnetic separation to remove the microsphere preservation solution, and then add 1 mL of labeling buffer.

[0064] (2) Activation of magnetic carboxyl microspheres

[0065] Prepare 10 mg / mL NHS and EDC (prepare and use immediately); take 10 μL of NHS and add it to the washed microspheres, mix quickly; then take another 10 μL of EDC and add it to the microspheres, mix quickly; mix and incubate at room temperature for 20 min.

[0066] (3) Magnetic separation to remove residual EDC

[0067] Place the activated microspheres on a magnetic separation rack, perform magnetic separation to remove the supernatant, and resuspend the microspheres with 1 mL of labeling buffer; repeat twice for standby.

[0068] (4) Coupling antibody

[0069] Take 0.2 mg of the antibody in a 10 mL centrifuge tube, add the activated microspheres, mix quickly, and then mix well and incubate at room temperature for 2 h.

[0070] (5) Blocking

[0071] Prepare 10% BSA, that is, weigh 1 g of BSA and dissolve it in 10 mL of deionized water for later use; after the microspheres are labeled with the antibody, add 500 μL of the above blocking solution, mix well and incubate at room temperature for 30 min.

[0072] (6) Magnetic separation to remove unbound antibody

[0073] Remove the free antibody that is not bound to the microspheres by magnetic separation. Finally, resuspend the microspheres with 5 mL of the dilution solution to obtain the antibody-microsphere labeled complex, and store it at 4°C for later use. If stored for a long time, add a solution of 0.2% BSA and 0.02% NaN3 at the final concentration.

[0074] After the magnetic carboxyl microspheres in Examples 1-4 were labeled with the MYO antibody, the signal value results are shown in Table 1.

[0075] Table 1 Signal values after the magnetic color microspheres bind to the antibody

[0076]

[0077]

[0078] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing carboxyl-modified magnetic microspheres, characterized in that, It includes the following steps: (1) Synthesize oleic acid-modified Fe3O4 magnetic cores with uniform particle size by coprecipitation method, namely OA-Fe3O4; (2) Prepare surface amino-modified polystyrene microspheres PS-NH2 by dispersion polymerization; (3) Embed and adsorb OA-Fe3O4 by swelling method: Add the polystyrene microspheres prepared in step (2) and a swelling agent into the reaction vessel, ultrasonically disperse, then add the OA-Fe3O4 prepared in step (1), and react at 70 °C for 1-4 h; after the reaction, wash with ethanol and store in pure water; (4) Secondary polymerization to fix magnetic particles and magnetic coating: Add the magnetic polystyrene microspheres prepared in step (3) and ethanol into the reaction vessel, and fill with nitrogen; then add polyvinyl alcohol, and set the temperature to 60 °C; then weigh the initiator AIBN, dissolve it in a little ethanol and add it into the reaction vessel; after 1 h, measure styrene and methacrylic acid in an ethanol solution, and slowly add them drop by drop into the reaction vessel, and react overnight.

2. The preparation method of the carboxyl-modified magnetic microspheres according to claim 1, characterized in that: The specific operation of step (1) is to dissolve FeCl3·6H2O and FeCl2·4H2O in pure water, then add ammonia water, adjust the pH to 9-11, react to generate black precipitate Fe3O4, and then add oleic acid to react to generate oleic acid-modified Fe3O4, namely OA-Fe3O4; among them, the mass ratio of oleic acid to total iron salt is 1:0.5-5.

3. The preparation method of the carboxyl-modified magnetic microspheres according to claim 2, wherein: The molar ratio of FeCl3·6H2O to FeCl2·4H2O is 1:0.5-1.

4. The preparation method of the carboxyl-modified magnetic microspheres according to claim 1, wherein: The specific operation of step (2) is to add pure water and ethanol into the reaction vessel, purge with nitrogen to remove oxygen, and then add polyvinylpyrrolidone; measure styrene and N-isopropylacrylamide in an EP tube, add the oil-soluble initiator AIBN, ultrasonically dissolve, add it into the reaction vessel, set the temperature to 70 °C, react for 4-8 h, and store in ethanol for use.

5. The preparation method of the carboxyl-modified magnetic microspheres according to claim 4, characterized in that: In step (2), the volume ratio of water to ethanol is 1:4-9, the volume ratio of styrene to N-isopropylacrylamide is 1:0.02-0.2, the initiator accounts for 0.5-5% of the total monomer; the mass ratio of polyvinylpyrrolidone to monomer is 1:2-8.

6. The preparation method of the carboxyl-modified magnetic microspheres according to claim 1, characterized in that: In step (3), the swelling agent is anhydrous ethanol.

7. The preparation method of the carboxyl-modified magnetic microspheres according to claim 6, characterized in that: In step (3), the volume ratio of the swelling agent to water is 4-8:1, and the mass ratio of PS-NH2 to OA-Fe3O4 is 5-20:

1.

8. The preparation method of the carboxyl-modified magnetic microspheres according to claim 1, wherein: In step (4), the initiator accounts for 0.5-5% of the total monomer, the volume ratio of styrene to methacrylic acid is 1:0.02-0.1; the volume ratio of magnetic polystyrene microspheres to ethanol is 1:1-10; the mass concentration of polyvinyl alcohol in the system solvent is 0.1%-1%.

9. The carboxyl-modified magnetic microspheres prepared by the preparation method according to any one of claims 1-8.

10. The application of the carboxyl-modified magnetic microspheres according to claim 9 in chemiluminescence immunoassay.

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