Preparation method of magnetic microspheres and amino and / or carboxyl magnetic microspheres

Abstract

The invention relates to the field of biological materials, in particular to a preparation method of magnetic microspheres and amino and / or carboxyl magnetic microspheres. The invention provides a preparation method of magnetic microspheres, which comprises the following steps: carrying out first polymerization reaction on styrene in the presence of an initiator to obtain polystyrene seed microspheres; swelling the seed microspheres in the presence of a pore-foaming agent, a monomer and an initiator to generate a second polymerization reaction; extracting and washing to obtain porous microspheres; modifying carboxyl on the inner and outer surfaces of the porous microspheres through a third polymerization reaction, mixing with iron ions, and coprecipitating to obtain the monodisperse superparamagnetic microspheres. The magnetic microspheres provided by the invention have monodispersity and superparamagnetism, and the particle size and the magnetic content of the magnetic microspheres can be regulated and controlled as required; the surface of the microsphere is rich in amino or carboxyl functional groups, and the content range of amino and carboxyl can be regulated and controlled according to the requirements of coupled protein; the method can be widely applied to the fields of in-vitro detection, biomedical research, protein separation and purification, cell sorting and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, in particular to a preparation method of magnetic microspheres and amino and / or carboxyl magnetic microspheres. Background Art

[0002] The particle size, morphology, pore structure, matrix material composition, and surface functionalization of microspheres all have a significant impact on their performance, and the barriers to industrial production are even higher. Immunomagnetic beads are a type of microsphere.

[0003] Immunomagnetic beads have excellent properties such as large specific surface area, monodispersity, material stability, superparamagnetic effect (facilitating separation and magnetic guidance), functional group characteristics (-OH, -COOH, -NH2, etc.), and can be endowed with a variety of specific reactive functional groups on their surface through copolymerization and surface modification according to different needs, and then combined with various functional substances. They are widely used in cell sorting, protein / antibody separation and purification, nucleic acid separation and purification, and immunoassay.

[0004] Magnetic microspheres are primarily categorized by structure: core-shell, sandwich, and diffuse. Core-shell structures are widely used in nucleic acid extraction magnetic beads for molecular diagnostics, while sandwich and diffuse structures are often used in immunomagnetic beads. Microspheres can also be categorized by material as inorganic or organic. Organic microspheres are primarily formed by the polymerization of organic polymers, such as polystyrene microspheres; inorganic microspheres are primarily formed by the polymerization of inorganic substances, such as silica magnetic beads.

[0005] The prior art discloses a method for preparing monodisperse microspheres by a seed swelling method, wherein glycidyl methacrylate seed microspheres are first prepared, and then the seed microspheres are swelled and polymerized into larger porous glycidyl methacrylate microspheres. The crosslinking agent is ethylene glycol dimethacrylate, and superparamagnetic nanoparticles are introduced after in-situ deposition of iron ions. Finally, the surface of the magnetic microspheres is modified with carboxylic acid by distillation precipitation polymerization. This patent introduces amino groups by opening the epoxy ring, and uses the amino groups to adsorb iron ions. However, the amino groups do not have the strong coordination ability with iron ions as carboxyl groups. The co-precipitation preparation of magnetic microspheres is a conventional process, which has very demanding requirements on the magnetic deposition process. It is difficult to obtain a high magnetic content, and the regularity of the microsphere morphology is generally average. The surface modification of the microspheres uses methacrylic acid or acrylic acid to introduce carboxyl groups. The carboxyl chains introduced by such monomers are short and not suitable for protein coating and coupling, and the efficiency is low.

[0006] In summary, there are still many problems in the process of preparing magnetic microspheres in the existing solutions, so it is now necessary to provide a reliable solution to solve this problem. Summary of the Invention

[0007] In light of this, the present invention provides methods for preparing magnetic microspheres and amino- and / or carboxyl-containing magnetic microspheres. The magnetic microspheres provided by the present invention are monodisperse and superparamagnetic, and their particle size and magnetic content can be regulated as needed. The microspheres are rich in amino or carboxyl functional groups on their surfaces, and the amino and carboxyl group content can be adjusted based on the needs of the coupled protein. These microspheres are widely used in in vitro testing, biomedical research, protein separation and purification, cell sorting, and other fields.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for preparing magnetic microspheres, comprising the following steps:

[0010] S1: Styrene undergoes a first polymerization reaction in the presence of an initiator to obtain polystyrene seed microspheres;

[0011] S2: The polystyrene seed microspheres swell in the presence of a porogen, a monomer, and an initiator, undergo a second polymerization reaction, extract, and wash to obtain porous microspheres;

[0012] S3: Modifying all surfaces of the porous microspheres with carboxyl groups, mixing with iron ions, and co-precipitating to obtain monodisperse superparamagnetic microspheres.

[0013] In some embodiments of the present invention, the molar ratio of the initiator to the styrene in the preparation method S1 is 1:(5-10).

[0014] In some embodiments of the present invention, the molar ratio of the initiator to the styrene in the preparation method S1 is 1:5, 1:7 or 1:10.

[0015] In some embodiments of the present invention, the molar ratio of the initiator to the styrene in the preparation method S1 is 1:7.

[0016] In some embodiments of the present invention, in the above preparation method, the initiator includes: azobisisobutyronitrile (AIBN) and / or benzoyl oxide (BPO).

[0017] In some embodiments of the present invention, the time of the first polymerization reaction in the above preparation method S1 is 2 to 16 hours.

[0018] In some embodiments of the present invention, the time of the first polymerization reaction in the above preparation method S1 is 2 hours, 6 hours or 16 hours.

[0019] In some embodiments of the present invention, the time of the first polymerization reaction in the above preparation method S1 is 16 hours.

[0020] In some embodiments of the present invention, the monomers in the preparation method S2 include: divinylbenzene (DVB) and styrene (St); the volume proportion of styrene does not exceed 20%.

[0021] In some embodiments of the present invention, the volume proportion of styrene in the above preparation method S2 is 10% or 20%.

[0022] In some embodiments of the present invention, the reaction time of the second polymerization in the above preparation method S2 is 16 hours.

[0023] In some embodiments of the present invention, the extraction in the above preparation method S2 uses dichloromethane and toluene; and the extraction time is 12 to 16 hours.

[0024] In some embodiments of the present invention, the step of surface modification with carboxyl groups in the above-mentioned preparation method S3 includes: modifying all surfaces of the porous microspheres with carboxyl groups through a third polymerization reaction of a carboxyl functional monomer; the carboxyl functional monomer includes: acrylic acid (AA) and / or methacrylic acid (MAA).

[0025] In some embodiments of the present invention, the third polymerization reaction in the preparation method S3 further comprises the steps of mixing with an initiator and passing nitrogen.

[0026] In some embodiments of the present invention, the preparation method S3 further comprises a step of mixing with oleic acid after the coprecipitation.

[0027] In some embodiments of the present invention, the co-precipitation in the above preparation method S3 includes the steps of mixing with concentrated aqueous ammonia and heating to 60°C.

[0028] The present invention provides a method for preparing magnetic microspheres, comprising the following steps:

[0029] S1: 100 mL of anhydrous ethanol, 50 mL of purified water, and 8 g of PVP were added to a 500 mL four-necked flask equipped with a mechanical stirrer and mechanically stirred to dissolve. 2 g of initiator AIBN and 10 mL of purified St monomer (molar ratio AIBN:St ≈ 1:7) were added, and nitrogen was introduced with stirring at 300 rpm for 30 min. The oil bath was heated to 70°C and the polymerization reaction was carried out for 16 h. Seed microspheres were obtained by centrifugation.

[0030] S2: 15 mL of toluene, 15 mL of DVB and St monomers (10% St, 1.5 mL), and about 0.2 g of initiator BPO were added to a 500 mL four-necked flask containing 200 mL of 0.2% SDS aqueous solution. The mixture was stirred at 300 rpm and controlled at 30°C for 16 h for emulsification. 2 g of seed microspheres obtained in S1 were then added, the mixture was stirred at 300 rpm and controlled at 30°C for 16 h for swelling. 100 mL of 2% PVP solution was then added and mixed thoroughly. The mixture was heated to 70°C and the reaction was continued for 16 h.

[0031] S3: Collect the porous microspheres from S2 by centrifugation and wash them once with industrial alcohol. Add an extractant (dichloromethane:toluene = 1:1) and extract overnight (12-16 hours). Remove the extractant by centrifugation and wash twice with ethanol. Disperse the porous microspheres in water and measure the solid content.

[0032] S4: 5 g of porous microspheres were dispersed in a 500 mL four-necked flask containing 200 mL of anhydrous ethanol and stirred thoroughly. 0.2 g of initiator AIBN, 3 mL of MAA monomer, and 0.5 mL of DVB monomer were then added. The mixture was stirred at 300 rpm and nitrogen was introduced for 30 min. The mixture was then stirred at room temperature for 2 h for adsorption. The water bath was gradually heated to 70°C and the reaction was continued for 24 h. The carboxylated porous microspheres were collected by centrifugation and then washed alternately with water and ethanol to remove excess MAA. The solid content was then determined.

[0033] S5: Take 5g of carboxylated porous spheres and disperse them in 150mL of water, add 1mL of concentrated hydrochloric acid, and stir at 200rpm with nitrogen for 30min; fully dissolve 9.6g of FeCl3·6H2O (g) and 5.4g of FeSO4·7H2O in 40mL of deoxygenated purified water, add them to the components containing carboxylated porous spheres, and continue to ventilate for 30min; after the ventilation is completed, adsorb at room temperature for 2h, add 30mL of ammonia water and heat to 60℃, quickly add 1mL of oleic acid, control the speed to 250rpm and react for 5h; cool to room temperature, enrich the magnetic microspheres with a magnet, remove excess ammonia water by washing with purified water, and make up the volume in ethanol solution to obtain the monodisperse superparamagnetic microspheres.

[0034] The present invention also provides monodisperse superparamagnetic microspheres obtained by the above preparation method.

[0035] The present invention also provides a method for preparing amino and / or carboxyl magnetic microspheres, comprising the following steps:

[0036] S1: mixing the monodisperse superparamagnetic microspheres with functional monomers and copolymerizing them to obtain monodisperse superparamagnetic microspheres with epoxy groups;

[0037] S2: subjecting the monodisperse superparamagnetic microspheres with epoxy groups described in S1 to a ring-opening reaction with an amination reagent to obtain magnetic microspheres with amino groups; or

[0038] S2: The monodisperse superparamagnetic microspheres with epoxy groups described in S1 are subjected to ring opening by introducing amino groups through an amination reagent, and then reacted with an acid anhydride to introduce carboxyl groups to obtain magnetic microspheres with carboxyl groups.

[0039] In some embodiments of the present invention, the functional monomers in the preparation method S1 include: glycidyl methacrylate and / or allyl alcohol glycidyl ether.

[0040] In some embodiments of the present invention, the ring-opening reaction described in the above-mentioned preparation method S2 can use a diamino reagent such as ethylenediamine, 1,6-hexanediamine, or a monoamino reagent such as ethanolamine. After the ring opening, ethylenediamine and the like can introduce an amino group, and after the ring opening, ethanolamine and the like can introduce a hydroxyl group or other non-amino functional group. The content of amino groups on the surface of the microspheres can be regulated by adjusting the ratio of ethanolamine and ethylenediamine (hexanediamine).

[0041] In some embodiments of the present invention, the anhydride reaction described in the above-mentioned preparation method S2 can use succinic anhydride, glutaric anhydride or acetic anhydride, propionic anhydride, etc., and carboxyl groups can be introduced after the reaction of succinic anhydride, glutaric anhydride and amino groups. After the reaction of acetic anhydride, propionic anhydride and amino groups, an amide bond is formed to introduce a methyl or ethyl group. The content of carboxyl groups on the surface of the microspheres can be regulated by adjusting the ratio of acetic anhydride (propionic anhydride) and succinic anhydride (or glutaric anhydride) and glutaric anhydride.

[0042] The present invention also provides amino and / or carboxyl magnetic microspheres obtained by the above preparation method.

[0043] The magnetic microspheres prepared by the present invention are monodisperse and superparamagnetic, and their particle size and magnetic content can be regulated as needed; the surface of the microspheres is rich in amino or carboxyl functional groups, and the content range of amino and carboxyl groups can be adjusted according to the needs of the coupled protein; they can be widely used in in vitro detection, biomedical research, protein separation and purification, cell sorting and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0045] Figure 1 shows a scanning electron micrograph of monodisperse polystyrene seed microspheres prepared by dispersion polymerization in Example 1;

[0046] Figure 2 The following are scanning electron micrographs of the porous microspheres prepared in Example 1; wherein: A shows the original porous microspheres; B shows the crushed porous microspheres;

[0047] Figure 3 The scanning electron microscope image of the porous spheres co-precipitated with Fe3O4 (oleic acid process) prepared in Example 1 is shown;

[0048] Figure 4 1 shows the hysteresis loop of the monodisperse superparamagnetic microspheres prepared in Example 1;

[0049] Figure 5 The finished monodisperse superparamagnetic carboxylated magnetic beads prepared in Example 1 are shown;

[0050] Figure 6 The seed microspheres prepared at different times in Example 2 are shown; A represents 2 hours; B represents 6 hours; C represents 16 hours;

[0051] Figure 7 The seed microspheres prepared at different times in Example 3 are shown; A represents 2 hours; B represents 6 hours; C represents 16 hours;

[0052] Figure 8 The effect of the swelling monomer composition on the morphology of porous microspheres in Example 4 is shown; among them: A shows 0% St; B shows 5% St; C shows 10% St; D shows 30% St. A high St content facilitates self-nucleation, while microspheres without St are easily broken;

[0053] Figure 9 FIG2 shows a scanning electron micrograph of the microspheres after magnetic deposition (non-oleic acid process) prepared in Example 5;

[0054] Figure 10 The scanning electron microscope image of the porous microspheres prepared in Example 6 after crushing shows that the proportion of seed initiator is low, and the microspheres form a hollow structure after swelling. DETAILED DESCRIPTION

[0055] The invention discloses a preparation method of magnetic microspheres and amino and / or carboxyl magnetic microspheres.

[0056] It should be understood that the expression "one or more of" includes individually each of the items recited after the expression and various combinations of two or more of the recited items, unless otherwise apparent from the context and usage. The expression "and / or" in conjunction with three or more recited items should be understood to have the same meaning, unless otherwise apparent from the context.

[0057] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0058] It should be understood that the order of steps or the order in which certain actions are performed are not important as long as the present invention remains operable. Additionally, two or more steps or actions may be performed simultaneously.

[0059] The use of any and all examples or exemplary language, such as "such as" or "including," herein is intended merely to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0060] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant numerical values ​​in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, amounts, values, and percentages used in this disclosure are modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0061] The preparation method of the magnetic microspheres and amino and / or carboxyl magnetic microspheres of the present invention mainly comprises the following steps:

[0062] (1) Monodisperse polystyrene (PS) seed microspheres were prepared by free radical polymerization of styrene in the presence of initiator azobisisobutyronitrile (AIBN) and an emulsifier or dispersant;

[0063] (2) PS microspheres were used as seed microspheres, toluene was selected as the porogen, divinylbenzene (DVB) and styrene (St) were selected as the monomers, and benzoyl peroxide (BPO) was selected as the initiator. After swelling, polymerization reaction was carried out;

[0064] (3) Extraction with dichloromethane and toluene, followed by washing with ethanol and purified water, can obtain larger monodisperse porous (St-DVB) microspheres;

[0065] (4) A carboxyl-containing functional monomer and a cross-linking agent are copolymerized to modify all surfaces of the porous microspheres (including the inner pores and the outer surface of the spheres) with a carboxyl-containing modification layer to facilitate the subsequent adsorption of iron ions;

[0066] (5) Add appropriate amount of Fe 2+ and Fe 3+ Ions and iron ions are bound to the carboxyl groups of the porous microspheres through coordination, concentrated ammonia is added and the temperature is gradually raised to 60°C for co-precipitation, and after the temperature reaches 60°C, oleic acid is quickly added to continue the reaction to obtain monodisperse superparamagnetic microspheres;

[0067] (6) A modified layer is formed on the surface of the magnetic microspheres by copolymerization of two or three monomers, epoxy groups are introduced, and then amino groups are introduced by ring opening with an amination reagent (this step can prepare magnetic microspheres with amino groups), and carboxyl groups are introduced after the reaction of acid anhydride and amino groups (to prepare magnetic microspheres with carboxyl groups).

[0068] In the preparation methods of the above-mentioned magnetic microspheres and amino and / or carboxyl magnetic microspheres, the PS seed microspheres adopt a free radical polymerization reaction, which can be emulsion polymerization or dispersion polymerization. By regulating the system parameters, the microsphere size can be controlled to be 0.3~1.2μm; specifically: the molar ratio of initiator to styrene is controlled between 1:5 and 1:10, and the polymerization reaction is carried out for 2~16 hours to obtain PS seed microspheres, and the initiator is AIBN.

[0069] The monomers used for the swelling are divinylbenzene (DVB) and a small amount of styrene (St), wherein the proportion of St is ≤20%. The main purpose of adding St is to reduce the rigidity of pure DVB porous microspheres and reduce the risk of breakage. The higher the proportion of St, the easier it is to form self-nucleating microspheres during the swelling polymerization process.

[0070] The method for uniformly coating the inner and outer surfaces of the porous microspheres with a carboxyl modification layer specifically comprises: depositing a carboxyl functional monomer and a cross-linking agent on the surface through a free radical polymerization reaction. The carboxyl functional monomer can be acrylic acid (AA), methacrylic acid (MAA), etc., the cross-linking agent can be divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), N,N'-methylenebisacrylamide (MBA), etc., and the initiator can be AIBN or BPO, etc.

[0071] In the alkaline coprecipitation method of iron ions, after adding concentrated ammonia water and heating to 60° C., it is necessary to immediately add an appropriate amount of oleic acid to facilitate the dispersion of the magnetic microspheres and make the morphology of the magnetic microspheres smoother.

[0072] The functional monomer used for the polymerization of the modification layer is glycidyl methacrylate (GMA) or allyl alcohol glycidyl ether (AGE), the comonomers are styrene (St), methyl methacrylate (MMA), acrylamide (AM), etc., the initiators are AIBN, BPO, etc., and the cross-linking agents can be divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), N,N'-methylenebisacrylamide (MBA), etc. The modifiable sites on the surface of the microspheres can be controlled by regulating the amount of glycidyl methacrylate.

[0073] The method for preparing surface-functionalized monodisperse superparamagnetic microspheres is characterized in that: the epoxy ring opening can be performed using a diamino reagent such as ethylenediamine, hexamethylenediamine, or a monoamino reagent such as ethanolamine. After the ring opening of ethylenediamine, an amino group can be introduced, and after the ring opening of ethanolamine, a hydroxyl group or other non-amino functional group can be introduced. The content of amino groups on the surface of the microspheres can be controlled by adjusting the ratio of ethanolamine and ethylenediamine (hexamethylenediamine).

[0074] The anhydride reaction can be carried out using succinic anhydride, glutaric anhydride, acetic anhydride, propionic anhydride, etc. The reaction of succinic anhydride, glutaric anhydride and amino groups can introduce carboxyl groups. The reaction of acetic anhydride, propionic anhydride and amino groups forms an amide bond to introduce a methyl or ethyl group. The content of carboxyl groups on the surface of the microspheres can be controlled by adjusting the ratio of acetic anhydride (propionic anhydride) to succinic anhydride (or glutaric anhydride) and glutaric anhydride.

[0075] The magnetic microspheres prepared by the present invention are monodisperse and superparamagnetic, and their particle size and magnetic content can be regulated as needed; the surface of the microspheres is rich in amino or carboxyl functional groups, and the content range of amino and carboxyl groups can be adjusted according to the needs of the coupled protein; they can be widely used in in vitro detection, biomedical research, protein separation and purification, cell sorting and other fields.

[0076] In Examples 1 to 7 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0077] The present invention will be further described below in conjunction with the embodiments:

[0078] Example 1

[0079] (1) 100 mL of anhydrous ethanol, 50 mL of purified water, and 8 g of PVP were added to a 500 mL four-necked flask equipped with a mechanical stirrer and mechanically stirred to dissolve them; 2 g of initiator AIBN and 10 mL of purified St monomer (molar ratio AIBN:St≈1:7) were added, and nitrogen was passed through the mixture at 300 rpm for 30 min; the oil bath was heated to 70 °C and the polymerization reaction was carried out for 16 h; and seed microspheres were obtained by centrifugation.

[0080] (2) Measure 15 mL of toluene, 15 mL of DVB and St monomers (10% of St, 1.5 mL), and about 0.2 g of initiator BPO, add them to a 500 mL four-necked flask containing 200 mL of 0.2% SDS aqueous solution, control the temperature at 30 ° C, and stir at 300 rpm for 16 h for emulsification; then measure 2 g of the seed microspheres prepared in step (1), control the temperature at 30 ° C, and stir at 300 rpm for 16 h for swelling; then add 100 mL of 2% PVP solution and mix thoroughly, raise the temperature to 70 ° C and continue the reaction for 16 h.

[0081] (3) The porous microspheres collected in step (2) were centrifuged and washed once with industrial alcohol; an extractant (dichloromethane: toluene = 1:1) was added and extracted overnight (12-16 h); the extractant was removed by centrifugation, and the porous microspheres were washed twice with ethanol, and then dispersed in water to determine the solid content.

[0082] (4) 5 g of porous microspheres were dispersed in a 500 mL four-necked flask containing 200 mL of anhydrous ethanol, and the mixture was thoroughly stirred. Then, 0.2 g of initiator AIBN, 3 mL of MAA monomer, and 0.5 mL of DVB monomer were added. The mixture was stirred at 300 rpm and nitrogen was passed through for 30 min. The mixture was then stirred at room temperature for 2 h for adsorption. The water bath was gradually heated to 70 °C and the reaction was continued for 24 h. The carboxylated porous microspheres were collected by centrifugation and then washed alternately with water and ethanol to remove excess MAA. The solid content was then determined.

[0083] (5) Take 5g of carboxylated porous spheres and disperse them in 150mL of water. Add 1mL of concentrated hydrochloric acid and stir at 200rpm with nitrogen for 30min. Dissolve 9.6g of FeCl3·6H2O (g) and 5.4g of FeSO4·7H2O in 40mL of deoxygenated purified water and add them to the component containing carboxylated porous spheres. Continue to aerate for 30min. After aeration, adsorb at room temperature for 2h. Add 30mL of ammonia water and heat to 60℃. Quickly add 1mL of oleic acid and control the speed to 250rpm to react for 5h. Cool to room temperature, collect the magnetic microspheres with a magnet, wash with purified water to remove excess ammonia water, dilute to volume in ethanol solution, and determine the solid content.

[0084] (6) 5 g of magnetic microspheres were weighed and dispersed in a 500 mL four-necked flask containing 200 mL of ethanol solution, and stirred at 300 rpm to disperse them evenly; 3 mL of GMA monomer, 2 mL of St monomer, 5 mL of DVB monomer, and 1 g of initiator AIBN were added, and the mixture was ventilated at 300 rpm for 30 min, then heated to 70 °C and reacted for 16 h; amino groups were introduced by ring-opening reaction of epoxy groups with ethylenediamine to obtain amino-grouped magnetic microspheres, and carboxyl groups were introduced by ring-opening reaction of succinic anhydride with amino groups to obtain carboxyl magnetic microspheres.

[0085] Example 2 Polymerization reaction time verification 1

[0086] 100 mL of methanol, 50 mL of purified water, and 5 g of polyvinylpyrrolidone (PVP) were added to a 500 mL four-necked flask equipped with a mechanical stirrer and mechanically stirred to dissolve them; 2.5 g of recrystallized initiator AIBN and 8 mL of purified St monomer (molar ratio AIBN:St≈1:5) were added thereto, and nitrogen was passed through at 300 rpm for 30 minutes; the oil bath was heated to 70°C for polymerization reaction, and samples were taken after 2 h, 6 h, and 16 h of reaction, respectively. Seed microspheres were obtained by centrifugation and washing. Electron microscopy characterization showed that the polymerization reaction time had little effect on the particle size of the seed microspheres.

[0087] Example 3 Polymerization reaction time verification 2

[0088] 110 mL of ethanol, 40 mL of purified water, and 5 g of polyvinylpyrrolidone (PVP) were added to a 500 mL four-necked flask equipped with a mechanical stirrer and mechanically stirred to dissolve them; 1.7 g of recrystallized initiator AIBN and 12 mL of purified St monomer (molar ratio AIBN:St≈1:10) were added thereto, and nitrogen was passed through at 300 rpm for 30 min; the oil bath was heated to 70°C for polymerization reaction, and samples were taken after the reaction for 2 h, 6 h, and 16 h, respectively. Seed microspheres were obtained by centrifugation and washing. Electron microscopy showed that the polymerization reaction time had little effect on the particle size of the seed microspheres.

[0089] Example 4 Effect of Swelling Monomer Composition on Microsphere Morphology

[0090] (1) 100 mL of anhydrous ethanol, 50 mL of purified water, and 8 g of PVP were added to a 500 mL four-necked flask equipped with a mechanical stirrer and mechanically stirred to dissolve them; 2 g of initiator AIBN and 10 mL of purified St monomer (molar ratio AIBN:St≈1:7) were added, and nitrogen was passed through the mixture at 300 rpm for 30 min; the oil bath was heated to 70 °C and the polymerization reaction was carried out for 16 h; and seed microspheres were obtained by centrifugation.

[0091] (2) Take four 500mL four-necked flasks equipped with mechanical stirring, add 15mL of toluene and 15mL of DVB and St mixed solution respectively, among which the DVB and St mixed solution is set to 4 gradients: ①DVB 15mL, ②DVB 13.5mL, St 1.5mL (10%), ③DVB13mL, St 3 mL (20%), ④DVB 11.5mL, St 4.5mL (30%); add about 0.2g of initiator BPO and 200mL of 0.2% SDS aqueous solution respectively, control the temperature at 30℃, stir at 300rpm for 16h for emulsification; then measure 2g of the seed microspheres prepared in step (1), control the temperature at 30℃, stir at 300rpm for 16h for swelling; then add 100ml of 2% PVP solution and mix thoroughly, raise the temperature to 70℃ and continue the reaction for 16h.

[0092] (3) The porous microspheres collected in step (2) were centrifuged and washed once with industrial alcohol; an extractant (dichloromethane: toluene = 1:1) was added and extracted overnight (12-16 h); the extractant was removed by centrifugation, and the porous microspheres were washed twice with ethanol, and then dispersed in water to determine the solid content.

[0093] Example 5 Magnetic Sedimentation Process without Oleic Acid

[0094] (1) 100 mL of anhydrous ethanol, 50 mL of purified water, and 8 g of dispersant PVP were added to a 500 mL four-necked flask equipped with a mechanical stirrer and mechanically stirred to dissolve them; 2 g of initiator AIBN and 10 mL of purified St monomer (molar ratio AIBN:St≈1:7) were added thereto, and nitrogen was passed through the mixture at 300 rpm for 30 min; the oil bath was heated to 70 °C and the polymerization reaction was carried out for 6 h; and seed microspheres were obtained by centrifugation.

[0095] (2) 15 mL of toluene, 15 mL of DVB and St monomers (10% of St, 1.5 mL), and 0.2 g of initiator BPO were added to a 500 mL four-necked flask containing 200 mL of 0.2% SDS aqueous solution, and the mixture was emulsified at 30 °C and stirred at 300 rpm for 16 h. 2 g of seed microspheres prepared in step (1) were then added, the mixture was swollen at 30 °C and stirred at 300 rpm for 16 h. 100 mL of 2% PVP solution was then added and mixed thoroughly. The mixture was heated to 70 °C and the reaction was continued for 16 h.

[0096] (3) The porous microspheres collected in step (2) were centrifuged and washed once with industrial alcohol; an extractant (dichloromethane: toluene = 1:1) was added and extracted overnight (12-16 h); the extractant was removed by centrifugation, and the porous microspheres were washed twice with ethanol, and then dispersed in water to determine the solid content.

[0097] (4) 5 g of porous microspheres were dispersed in a 500 mL four-necked flask containing 200 mL of anhydrous ethanol, and the mixture was thoroughly stirred. Then, 0.2 g of initiator AIBN, 3 mL of MAA monomer, and 0.5 ml of DVB monomer were added. The mixture was stirred at 300 rpm and nitrogen was passed through for 30 min. The mixture was then stirred at room temperature for 2 h for adsorption. The water bath was gradually heated to 70 °C and the reaction was continued for 24 h. The carboxylated porous microspheres were collected by centrifugation and then washed alternately with water and ethanol to remove excess MAA. The solid content was determined.

[0098] (5) Disperse 5 g of carboxylated porous spheres in 150 mL of water, add 1 mL of concentrated hydrochloric acid, and aerate with nitrogen at 200 rpm for 30 min. Dissolve 9.6 g of FeCl3·6H2O (g) and 5.4 g of FeSO4·7H2O in 40 mL of deoxygenated purified water, add them to the component containing carboxylated porous spheres, and continue aeration for 30 min. After aeration, adsorb at room temperature for 2 h, add 30 mL of ammonia water, continue heating to 60 °C and react for 5 h, controlling the speed at 250 rpm. Cool to room temperature, collect the magnetic microspheres with a magnet, remove excess ammonia water by washing with purified water, dilute to the volume in ethanol solution, and send for scanning electron microscopy analysis.

[0099] Example 6 Morphology of porous spheres after swelling when the initiator ratio is low

[0100] (1) 100 mL of anhydrous ethanol, 50 mL of purified water, and 8 g of PVP were added to a 500 mL four-necked flask equipped with a mechanical stirrer and mechanically stirred to dissolve them; 0.85 g of initiator AIBN and 12 mL of purified St monomer (molar ratio AIBN:St≈1:20) were added thereto, and nitrogen was passed through the mixture at 300 rpm for 30 min; the oil bath was heated to 70 °C and the polymerization reaction was carried out for 2 h; and seed microspheres were obtained by centrifugation.

[0101] (2) 15 mL of toluene, 15 mL of DVB and St monomers (10% of St, 1.5 mL), and 0.2 g of initiator BPO were added to a 500 mL four-necked flask containing 200 mL of 0.2% SDS aqueous solution, and the mixture was emulsified at 30 °C and stirred at 300 rpm for 16 h. 2 g of seed microspheres prepared in step (1) were then added, the mixture was swollen at 30 °C and stirred at 300 rpm for 16 h. 100 mL of 2% PVP solution was then added and mixed thoroughly. The mixture was heated to 70 °C and the reaction was continued for 16 h.

[0102] (3) Collect the porous microspheres from step (2) by centrifugation, wash them once with industrial alcohol; add an extractant (dichloromethane: toluene = 1:1) and extract overnight (12-16 hours); remove the extractant by centrifugation, wash them twice with ethanol, disperse the porous microspheres in water, and send them for SEM analysis.

[0103] Example 7 Modification of Monomers to Replace GMA with MAA

[0104] (1) 100 mL of anhydrous ethanol, 50 mL of purified water, and 8 g of PVP were added to a 500 mL four-necked flask equipped with a mechanical stirrer and mechanically stirred to dissolve them; 2 g of initiator AIBN and 10 mL of purified St monomer (molar ratio AIBN:St≈1:7) were added, and nitrogen was passed through the mixture at 300 rpm for 30 min; the oil bath was heated to 70 °C and the polymerization reaction was carried out for 16 h; and seed microspheres were obtained by centrifugation.

[0105] (2) 15 mL of toluene, 13 mL of DVB monomer, 2 mL of St monomer, and 0.2 g of initiator BPO were added to a 500 mL four-necked flask containing 200 mL of 0.2% SDS aqueous solution, and the mixture was emulsified at 30°C and stirred at 300 rpm for 16 h. 2 g of the seed microspheres prepared in step (1) were then added, the mixture was swelled at 30°C and stirred at 300 rpm for 16 h. 100 mL of 2% PVP solution was then added and mixed thoroughly. The mixture was heated to 70°C and the reaction was continued for 16 h.

[0106] (3) The porous microspheres collected in step (2) were centrifuged and washed once with industrial alcohol; an extractant (dichloromethane: toluene = 1:1) was added and extracted overnight (12-16 h); the extractant was removed by centrifugation, and the porous microspheres were washed twice with ethanol, and then dispersed in water to determine the solid content.

[0107] (4) 5 g of porous microspheres were dispersed in a 500 mL four-necked flask containing 200 mL of anhydrous ethanol, and the mixture was thoroughly stirred. Then, 0.2 g of initiator AIBN, 3 mL of MAA monomer, and 0.5 mL of DVB monomer were added. The mixture was stirred at 300 rpm and nitrogen was passed through for 30 min. The mixture was then stirred at room temperature for 2 h for adsorption. The water bath was gradually heated to 70 °C and the reaction was continued for 24 h. The carboxylated porous microspheres were collected by centrifugation and then washed alternately with water and ethanol to remove excess MAA. The solid content was determined.

[0108] (5) Take 5g of carboxylated porous spheres and disperse them in 150mL of water. Add 1mL of concentrated hydrochloric acid and stir at 200rpm with nitrogen for 30min. Dissolve 9.6g of FeCl3·6H2O (g) and 5.4g of FeSO4·7H2O in 40mL of deoxygenated purified water and add them to the component containing carboxylated porous spheres. Continue to aerate for 30min. After aeration, adsorb at room temperature for 2h. Add 30mL of ammonia water and heat to 60℃. Quickly add 1mL of oleic acid and control the speed to 250rpm to react for 5h. Cool to room temperature, collect the magnetic microspheres by magnet, remove excess ammonia water by washing with purified water, wash with 95% ethanol and dilute to ethanol solution for solid content determination.

[0109] (6) 5 g of magnetic microspheres were measured and dispersed in a 500 mL four-necked flask containing 200 mL of ethanol solution. The mixture was stirred at 300 rpm until uniform dispersion was achieved. 3 mL of MAA monomer, 2 mL of St monomer, 5 mL of DVB monomer, and 1 g of initiator AIBN were added. The mixture was ventilated at 300 rpm for 30 min and then heated to 70 °C for 16 h. Carboxyl magnetic microspheres were obtained.

[0110] Table 1

[0111]

[0112] Table 1: Comparison of the detection signal values ​​in the products of magnetic beads A prepared by introducing carboxyl groups by ring-opening of GMA in Example 1, magnetic beads B prepared by directly introducing carboxyl groups by polymerization of MAA in Example 7, and carboxyl magnetic beads C prepared by extending C by 6 on the basis of Example 7.

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing magnetic microspheres, characterized in that: The steps include: S1: Styrene undergoes a first polymerization reaction in the presence of an initiator to obtain polystyrene seed microspheres; S2: The polystyrene seed microspheres swell in the presence of a porogen, a monomer, and an initiator, undergo a second polymerization reaction, extract, and wash to obtain porous microspheres; S3: Modifying all surfaces of the porous microspheres with carboxyl groups, mixing with iron ions, and co-precipitating to obtain monodisperse superparamagnetic microspheres.

2. The preparation method according to claim 1, wherein The molar ratio of the initiator to the styrene in S1 is 1:(5~10).

3. The preparation method according to claim 1 or 2, wherein The time of the first polymerization reaction in S1 is 2 to 16 hours.

4. The preparation method according to any one of claims 1 to 3, characterized in that The monomers described in S2 include: divinylbenzene and styrene; the volume proportion of the styrene does not exceed 20%.

5. The preparation method according to any one of claims 1 to 4, characterized in that The step of modifying the surface with carboxyl groups in S3 includes: modifying all surfaces of the porous microspheres with carboxyl groups through a third polymerization reaction of carboxyl functional monomers; the carboxyl functional monomers include: acrylic acid and / or methacrylic acid.

6. The preparation method according to any one of claims 1 to 5, characterized in that The coprecipitation in S3 further includes a step of mixing with oleic acid.

7. Monodisperse superparamagnetic microspheres obtained by the preparation method according to any one of claims 1 to 6.

8. A method for preparing amino and / or carboxyl magnetic microspheres, characterized in that: The steps include: S1: mixing the monodisperse superparamagnetic microspheres according to claim 7 with a functional monomer containing an epoxy group, and performing copolymerization reaction to obtain monodisperse superparamagnetic microspheres with epoxy groups; S2: subjecting the monodisperse superparamagnetic microspheres with epoxy groups described in S1 to a ring-opening reaction with an amination reagent to introduce amino groups to obtain magnetic microspheres with amino groups; or S2: The monodisperse superparamagnetic microspheres with epoxy groups described in S1 are subjected to a ring-opening reaction with an amination reagent to introduce amino groups, and then reacted with an acid anhydride to introduce carboxyl groups to obtain magnetic microspheres with carboxyl groups.

9. The preparation method according to claim 8, wherein The functional monomers in S1 include glycidyl methacrylate and / or allyl alcohol glycidyl ether.

10. Amino and / or carboxyl magnetic microspheres obtained by the preparation method according to claim 8 or 9.

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