Paratoluensulfonylated magnetic microspheres and preparation method thereof

Abstract

The invention belongs to the technical field of magnetic microspheres, and particularly relates to p-toluenesulfonylated magnetic microspheres and a preparation method thereof. The preparation method comprises the following steps: S1, forming ferroferric oxide magnetic particles from a ferric iron solution at a high temperature; s2, preparing magnetic microspheres after swelling; s3, the magnetic microspheres obtained after swelling in the step S2 are wrapped with 3-glycidyl ether oxypropyl trimethoxy silane, and the wrapped magnetic microspheres are obtained; s4, carrying out ring opening on the wrapped magnetic microspheres obtained in the step S3 under an alkaline condition, and carrying out a reaction with tris (hydroxymethyl) aminomethane to obtain hydroxylated magnetic microspheres; and S5, carrying out a reaction on the hydroxylated magnetic microspheres and paratoluensulfonyl chloride to obtain the paratoluensulfonylated magnetic microspheres. The paratoluensulfonylation magnetic spheres prepared by the method are uniform in particle size and good in magnetic responsiveness, and can meet clinical requirements.

Description

[0001] The invention belongs to the technical field of magnetic microspheres, and specifically relates to toluenesulfonyl magnetic microspheres and a preparation method thereof. Background Art

[0002] Polystyrene is widely used in many fields, including wastewater treatment, construction, medical devices and bioimmunotechnology, due to its low cost and chemical and thermodynamic stability. As a base material, it provides a solid attachment point for various functional groups such as hydroxyl, carboxyl, tosyl and so on. After adding magnetic materials, they play an important role in the directional delivery of drugs, nuclear magnetic resonance imaging and magnetic immunoseparation due to their magnetism. Its magnetism comes from the magnetic particles on the microspheres, which are generally prepared by coprecipitation, but their size is usually between 10 and 50 nanometers. The small size does not have strong magnetism, which hinders the detection efficiency. In addition, in the process of preparing magnetic microspheres, a common method is to introduce target functional groups on the surface of the microspheres, or to use magnetic particles as a base and gradually introduce functional groups to achieve the desired functions. At present, functional groups such as hydroxyl, carboxyl, and amino can be introduced stably, but for functional groups such as epoxy and tosyl and their mutual binding mechanism, the literature is still insufficient.

[0003] The common method of adding p-toluenesulfonyl groups is mainly to provide a hydroxyl base, which can react with the chlorine atom of p-toluenesulfonyl chloride to remove a molecule of HCl. However, the hydroxyl groups provided by this traditional method are limited, resulting in the p-toluenesulfonyl content being limited by the hydroxyl content, and the hydroxyl group needs to be further increased to obtain a better immune effect. In addition, in the coupling process of antibody protein with carboxyl or amino magnetic microspheres, a carboxyl and an agent are usually required, and this step is relatively complicated. However, for p-toluenesulfonyl-modified magnetic microspheres, due to the special chemical properties of their surface, chemical bonding with amino-containing molecules can be directly achieved without relying on carboxyl and an agent. Through simple operations, stable chemical bonds can be formed on the surface of the microspheres. Therefore, developing a method for preparing magnetic microspheres modified with p-toluenesulfonyl groups is crucial for simplifying the immunoassay process and improving detection efficiency. Summary of the invention

[0004] The invention provides a toluenesulfonyl magnetic microsphere and a preparation method thereof.

[0005] The specific technical solutions are as follows:

[0006] A method for preparing toluenesulfonated magnetic microspheres comprises the following steps:

[0007] S1, forming ferroferric oxide magnetic particles at high temperature from a ferric iron solution;

[0008] S2, adding polystyrene microspheres to isopropanol, and thoroughly mixing by ultrasound, wherein the mass ratio of polystyrene microspheres to isopropanol is 1:9, and then taking the magnetic particles obtained in step S1 and adding isopropanol, and thoroughly mixing the above two solutions by ultrasound, wherein the mass ratio of magnetic particles to isopropanol is 1:18, and reacting at a temperature of 75°C for 8 hours, and finally washing by ethanol magnetic separation twice to obtain swollen magnetic microspheres, wherein the mass ratio of polystyrene microspheres to magnetic particles is 2:1;

[0009] S3, wrapping the swollen magnetic microspheres in step S2 with 3-glycidyloxypropyltrimethoxysilane to obtain wrapped magnetic microspheres;

[0010] S4, the encapsulated magnetic microspheres obtained in step S3 are subjected to ring-opening under alkaline conditions and reacted with tris(hydroxymethyl)aminomethane to obtain hydroxylated magnetic microspheres, and polyhydroxyl reactive groups are used as reaction substrates to make the reaction in step 5 more thorough and introduce more p-toluenesulfonyl groups;

[0011] S5. reacting the hydroxylated magnetic microspheres with p-toluenesulfonyl chloride to obtain p-toluenesulfonylated magnetic microspheres.

[0012] Furthermore, the preparation of the ferroferric oxide magnetic particles in step S1 comprises the following steps:

[0013] S11, dissolving ferric chloride hexahydrate in ethylene glycol to obtain solution A, wherein the mass ratio of ferric chloride hexahydrate to ethylene glycol is 1:25;

[0014] S12, fully mixing anhydrous sodium acetate, sodium citrate and ethylene glycol in a water bath at 40° C. to obtain solution B, wherein the mass ratio of anhydrous sodium acetate, sodium citrate and ethylene glycol is 0.8:0.5:25;

[0015] S13, slowly pouring solution A into the stirring solution B, maintaining a water bath at 40°C and mixing thoroughly, to obtain solution C;

[0016] S14. Place solution C in a high-temperature reactor at 180°C for 2 hours, then heat to 220°C for 14 hours, then cool to 180°C for 2 hours, separate and wash with ethanol after cooling, and dry at 50°C to obtain ferroferric oxide magnetic powder. The high-temperature reaction process adopts a segmented program temperature control method, which has a better reaction effect. The prepared ferroferric oxide magnetic powder has higher purity, stronger adhesion and better quality.

[0017] Furthermore, the preparation of the encapsulated magnetic microspheres in step S3 includes the following steps: adding the swollen magnetic microspheres in step S2 to a mixed solution of ethanol and water, adjusting the pH value to 9 with ammonia water, placing in an ultrasonic reactor, and adding 3-glycidyloxypropyltrimethoxysilane (KH-560) dropwise for 5 minutes. After the addition is completed, react for 3 hours, and then use ethanol magnetic separation and washing, and then add ethanol and water mixed solution, and use acetic acid to adjust the pH value to maintain at 4, and then add 3-glycidyloxypropyltrimethoxysilane, shake for 3 hours, and then use ethanol magnetic separation and washing, and then use 0.1 mol / L sodium bicarbonate solution to magnetically wash to obtain the encapsulated magnetic microspheres, and the mass ratio of ethanol, water and 3-glycidyloxypropyltrimethoxysilane is 5:1:(1.5-6).

[0018] Furthermore, the preparation of hydroxylated magnetic microspheres in step S4 includes the following steps: adding the encapsulated magnetic microspheres prepared in step S3 to a 0.1 mol / L sodium bicarbonate aqueous solution, then adding tris(hydroxymethyl)aminomethane, reacting for 6 hours, and finally centrifugally washing with ethanol and sodium bicarbonate aqueous solution to obtain hydroxylated magnetic microspheres, wherein the mass of the added tris(hydroxymethyl)aminomethane is equal to the mass of the polystyrene microspheres in step S2, and the mass of the sodium bicarbonate aqueous solution is 24 times that of tris(hydroxymethyl)aminomethane.

[0019] Furthermore, the preparation of toluenesulfonyl magnetic microspheres in step S5 includes the following steps: adding the hydroxylated magnetic microspheres prepared in step S4 to acetone, then adding toluenesulfonyl chloride and triethylamine, adding them in batches during ultrasound with an interval of 20 minutes, then continuing ultrasound for 3 hours, and finally washing them once with acetone and then washing them with ethanol magnetic absorption to obtain toluenesulfonyl magnetic microspheres, wherein the added mass of toluenesulfonyl chloride is 6 times the mass of the polystyrene microspheres in step S2, the added mass of triethylamine is 3.2 times the mass of the polystyrene microspheres in step S2, and the added mass of acetone is 20 times the mass of the polystyrene microspheres in step S2.

[0020] At the same time, the present invention also provides toluenesulfonyl magnetic microspheres prepared by the above preparation method.

[0021] Furthermore, the magnetic microspheres need to be stored in an ethanol solution at 4°C.

[0022] The toluenesulfonyl magnetic spheres prepared by the method have good magnetic responsiveness and can meet clinical requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 SEM image of magnetic microspheres prepared in Example 1 DETAILED DESCRIPTION

[0024] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Example 1

[0026] A method for preparing toluenesulfonated magnetic microspheres, characterized in that it comprises the following steps:

[0027] S1, forming ferroferric oxide magnetic particles at high temperature from a trivalent iron solution, wherein the preparation of ferroferric oxide magnetic particles in step S1 comprises the following steps:

[0028] S11, taking 72g of ferric chloride hexahydrate and dissolving it in 1800g of ethylene glycol to obtain solution A;

[0029] S12, 57.6 g of anhydrous sodium acetate, 36 g of sodium citrate and 1800 g of ethylene glycol were fully mixed in a water bath at 40° C. to obtain a solution B, wherein the mass ratio of anhydrous sodium acetate, sodium citrate and ethylene glycol was 0.8:0.5:25;

[0030] S13, slowly pouring solution A into the stirring solution B, maintaining a water bath at 40°C and mixing thoroughly, to obtain solution C;

[0031] S14, placing solution C in a high temperature reactor at 180°C for reaction for 2h, then heating to 220°C for reaction for 14h, then cooling to 180°C for further reaction for 2h, and after cooling, performing magnetic separation and washing with ethanol, and drying at 50°C to obtain ferroferric oxide magnetic powder;

[0032] S2, adding 20g of polystyrene microspheres to 180g of isopropanol, and mixing them thoroughly by ultrasonication, then taking 10g of the magnetic particles obtained in step S1 and adding 180g of isopropanol, and mixing the two solutions thoroughly by ultrasonication, reacting at a temperature of 75°C for 8 hours, and finally washing them twice by ethanol magnetic separation to obtain swollen magnetic microspheres;

[0033] S3, the swollen magnetic microspheres in step S2 are wrapped with 3-glycidyloxypropyltrimethoxysilane to obtain wrapped magnetic microspheres, the preparation process is to add the swollen magnetic microspheres in step S2 to a mixed solution of 200g ethanol and 40g water, adjust the pH value to 9 with ammonia water, place it in an ultrasonic reactor, add 100g 3-glycidyloxypropyltrimethoxysilane dropwise, the dropwise addition time is 5min, after the dropwise addition is completed, react for 3h, and then use ethanol magnetic separation and washing; then add 200g ethanol and 40g mixed solution, and adjust the pH value to 4 with acetic acid, then add 100g 3-glycidyloxypropyltrimethoxysilane, shake for 3h, then use ethanol magnetic separation and washing, and then use 0.1mol / L sodium bicarbonate solution magnetic washing to obtain wrapped magnetic microspheres;

[0034] S4, the encapsulated magnetic microspheres obtained in step S3 are subjected to ring-opening and reaction with tris(hydroxymethyl)aminomethane under alkaline conditions to obtain hydroxylated magnetic microspheres, wherein the preparation process is to add the encapsulated magnetic microspheres prepared in step S3 to 480 g of 0.1 mol / L sodium bicarbonate aqueous solution, then add 20 g of tris(hydroxymethyl)aminomethane, react for 6 h, and finally centrifuge and wash with ethanol and sodium bicarbonate aqueous solution to obtain hydroxylated magnetic microspheres;

[0035] S5. The hydroxylated magnetic microspheres are reacted with p-toluenesulfonyl chloride to obtain p-toluenesulfonated magnetic microspheres. The preparation process is as follows: the hydroxylated magnetic microspheres prepared in step S4 are added to 400 g of acetone, and then 120 g of p-toluenesulfonyl chloride and 72 g of triethylamine are added in batches during ultrasound with an interval of 20 minutes, and then ultrasound is continued for 3 hours. Finally, the p-toluenesulfonated magnetic microspheres are washed once with acetone and then washed with ethanol magnetic absorption to obtain p-toluenesulfonated magnetic microspheres.

[0036] The present invention utilizes the above method to prepare microspheres of other embodiments. The preparation process and materials are the same as those of Example 1, and only the proportions of the components in the table are changed. See Table 1, wherein in step S2, the mass ratio of styrene magnetic microspheres to 3-glycidyloxypropyltrimethoxysilane is M, and the mass ratio of triethylamine to p-toluenesulfonyl chloride is N.

[0037] The magnetic balls prepared in all embodiments of the present invention were subjected to performance tests. The test results are shown in Table 1. The specific process is as follows:

[0038] Magnetic ball chemical immunofluorescence detection process: Add appropriate concentrations of antibodies, magnetic balls, ammonia water and coating solution to a 2 ml round-bottom centrifuge tube in sequence, and mix them to make the final concentration of magnetic balls reach 5 mg / mL. The total volume after mixing is 600 μL, and then mix by inversion at 37°C for 6 to 8 hours.

[0039] Magnetic ball washing and blocking: Use a magnetic rack to adsorb the magnetic balls for 2 minutes and remove the coating solution. Wash with magnetic ball diluent 4 times, each time at 37°C for 5 minutes. Block with magnetic ball blocking solution at 37°C for 4 hours.

[0040] Magnetic ball storage: dilute the coupled magnetic balls into 50 ml of magnetic ball diluent to a concentration of 120 μg / mL, and then store at 4°C for future use.

[0041] Reagent configuration: Protein standard solution: Prepare 25 mg / mL protein standard solution and store at 20°C. Dilute to 0.5 mg / mL protein standard solution and store at 20°C.

[0042] BCA working solution: Mix reagent A and reagent B in proportion to form BCA working solution, which can be stable at room temperature for 24 hours. The specific preparation of BCA reagent is as follows: ① Reagent A, 1L: Weigh 10g BCA (1%), 20g Na2CO3·H2O (2%), 1.6g Na2C4H4O6·2H2O (0.16%), 4g NaOH (0.4%), 9.5g NaHCO3 (0.95%), add water to 1L, and adjust the pH value to 11.25 with NaOH or solid NaHCO3. ② Reagent B, 50ml: Take 2g CuSO4·5H2O (4%) and add distilled water to 50ml. ③ BCA reagent: Take 50 parts of reagent A and 1 part of reagent B and mix them evenly.

[0043] Determination of protein concentration of magnetic ball supernatant: Standard preparation: Prepare protein standards of different concentrations to form a concentration gradient. Sample test: Add an appropriate volume of sample. Reagent reaction: Add 200 μL of BCA working solution to each well and place at 37°C for 24 hours. Detection and analysis: Use an immunofluorescence analyzer to obtain the fluorescence values ​​of different samples.

[0044] Table 1

[0045] Example M N Relative value of fluorescence intensity Example 1 0.5:5 3:5 1.78 Example 2 0.5:1.5 1:1.5 0.93 Example 3 0.5:2 1:1.5 1.3 Example 4 0.5:2.5 1:1.5 1.4 Example 5 0.5:3 1:1.5 1.7 Example 6 0.5:4 1:1.5 1.75 Example 7 0.5:6 1:1.5 2.0 Example 8 0.5:9 1:1.5 1.85 Example 9 0.5:6 1:2 1.54 Example 10 0.5:6 1:3 1.40

[0046] As can be seen from the data in the above table, the data of Examples 2 to 8 show that increasing the amount of 3-glycidyloxypropyltrimethoxysilane can achieve better immune effects, but there is still an upper limit. The data of Examples 7, 9, and 10 show that increasing the amount of triethylamine can improve the immune effect.

[0047] In summary, the toluenesulfonyl magnetic particles prepared by this method have uniform particle size and good magnetic responsiveness, and can meet clinical requirements.

[0048] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing toluenesulfonated magnetic microspheres, characterized in that: The steps include: S1, forming ferroferric oxide magnetic particles at high temperature from a ferric iron solution; S2, adding polystyrene microspheres to isopropanol, and thoroughly mixing by ultrasound, wherein the mass ratio of polystyrene microspheres to isopropanol is 1:9, and then taking the magnetic particles obtained in step S1 and adding isopropanol, and thoroughly mixing the above two solutions by ultrasound, wherein the mass ratio of magnetic particles to isopropanol is 1:18, and reacting at a temperature of 75°C for 8 hours, and finally washing by ethanol magnetic separation twice to obtain swollen magnetic microspheres, wherein the mass ratio of polystyrene microspheres to magnetic particles is 2:1; S3, wrapping the swollen magnetic microspheres in step S2 with 3-glycidyloxypropyltrimethoxysilane to obtain wrapped magnetic microspheres; S4, opening the encapsulated magnetic microspheres obtained in step S3 under alkaline conditions and reacting with tris(hydroxymethyl)aminomethane to obtain hydroxylated magnetic microspheres; S5. reacting the hydroxylated magnetic microspheres with p-toluenesulfonyl chloride to obtain p-toluenesulfonylated magnetic microspheres.

2. The method for preparing magnetic microspheres according to claim 1, characterized in that: The preparation of the ferroferric oxide magnetic particles in step S1 comprises the following steps: S11, dissolving ferric chloride hexahydrate in ethylene glycol to obtain solution A, wherein the mass ratio of ferric chloride hexahydrate to ethylene glycol is 1:25; S12, fully mixing anhydrous sodium acetate, sodium citrate and ethylene glycol in a water bath at 40° C. to obtain solution B, wherein the mass ratio of anhydrous sodium acetate, sodium citrate and ethylene glycol is 0.8:0.5:25; S13, slowly pouring solution A into the stirring solution B, maintaining a water bath at 40°C and mixing thoroughly, to obtain solution C; S14, placing solution C in a high temperature reactor at 180°C for 2 hours, then heating to 220°C for 14 hours, then cooling to 180°C for 2 hours, washing with ethanol by magnetic separation after cooling, and drying at 50°C to obtain ferroferric oxide magnetic powder.

3. The method for preparing magnetic microspheres according to claim 1, characterized in that: The preparation of the encapsulated magnetic microspheres in step S3 comprises the following steps: adding the swollen magnetic microspheres in step S2 to a mixed solution of ethanol and water, adjusting the pH value to 9 with ammonia water, placing in an ultrasonic reactor, and adding 3-glycidyloxypropyltrimethoxysilane dropwise for 5 minutes. After the addition is completed, reacting for 3 hours, and then using ethanol magnetic separation and washing, and then adding ethanol and water mixed solution, and adjusting the pH value to 4 with acetic acid, and then adding 3-glycidyloxypropyltrimethoxysilane, shaking for 3 hours, and then using ethanol magnetic separation and washing, and then using 0.1 mol / L sodium bicarbonate solution to magnetically wash to obtain the encapsulated magnetic microspheres, wherein the mass ratio of ethanol, water and 3-glycidyloxypropyltrimethoxysilane is 5:1:(1.5-6).

4. The method for preparing magnetic microspheres according to claim 1, characterized in that: The preparation of the hydroxylated magnetic microspheres in step S4 comprises the following steps: adding the encapsulated magnetic microspheres prepared in step S3 to a 0.1 mol / L sodium bicarbonate aqueous solution, then adding tris(hydroxymethyl)aminomethane, reacting for 6 hours, and finally centrifugally washing with ethanol and sodium bicarbonate aqueous solution to obtain the hydroxylated magnetic microspheres, wherein the mass of the added tris(hydroxymethyl)aminomethane is equal to the mass of the polystyrene microspheres in step S2, and the mass of the sodium bicarbonate aqueous solution is 24 times that of the tris(hydroxymethyl)aminomethane.

5. The method for preparing magnetic microspheres according to claim 1, characterized in that: The preparation of toluenesulfonyl magnetic microspheres in step S5 includes the following steps: adding the hydroxylated magnetic microspheres prepared in step S4 to acetone, then adding toluenesulfonyl chloride and triethylamine, adding them in batches during ultrasound with an interval of 20 minutes, then continuing ultrasound for 3 hours, and finally washing them once with acetone and then washing them with ethanol magnetic absorption to obtain toluenesulfonyl magnetic microspheres, wherein the added mass of toluenesulfonyl chloride is (4.8 to 9.6) times the mass of the polystyrene microspheres in step S2, the added mass of triethylamine is 3.2 times the mass of the polystyrene microspheres in step S2, and the added mass of acetone is 20 times the mass of the polystyrene microspheres in step S2.

6. Toluenesulfonyl magnetic microspheres prepared by the preparation method according to any one of claims 1 to 5.

7. The magnetic microspheres according to claim 6, characterized in that: It needs to be stored in ethanol solution at 4°C.

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