Magnetic microsphere as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a magnetic microsphere, a preparation method and application thereof and a reagent containing the magnetic microsphere. The preparation method comprises the following steps: firstly, preparing carboxyl-containing polymer microspheres by a one-step method, then carrying out heat treatment under an alkaline condition, and then carrying out THF oscillation to finally prepare porous polystyrene carboxyl microspheres with uniform pore channels, meanwhile, avoiding using divinyl benzene as a cross-linking agent, reducing the luminous background of the finally obtained magnetic microspheres, and improving the luminous efficiency of the magnetic microspheres. The method can be better applied to the field of chemiluminescence immunoassay. Due to the existence of a surface porous structure and carboxyl, the stability and the functionalization degree of the magnetic material are improved, the problems of magnetic leakage and magnetic shell layer falling are avoided, the surface of the porous ball is smoother and more uniform, and a solid foundation is laid for the subsequent coating process and reduction of non-specific adsorption of the surface of the magnetic ball; the magnetic ball is prepared by using a grinding method, and has the advantages of safety, no toxicity, easiness in operation, no pollution and easiness in industrial popularization.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to magnetic microspheres, their preparation methods, and applications. Background Technology

[0002] Magnetic microspheres, due to their advantages such as superparamagnetism, large specific surface area, and biocompatibility, have wide applications in the field of biomagnetic beads. The surface of magnetic microspheres can be chemically modified by grafting various functional groups (such as amino, carboxyl, and thiol groups), thereby coupling them with biomolecules with specific recognition functions, such as antibodies, aptamers, and peptides. This surface functionalization allows magnetic microspheres to specifically recognize and bind to target biomolecules or cells, achieving targeted separation, diagnosis, and treatment. Furthermore, magnetic microspheres can be combined with other functional materials (such as quantum dots and fluorescent dyes) to construct multimodal functional composite systems, meeting the diverse application needs of the biomedical field.

[0003] Carboxylated magnetic beads with a particle size of approximately 1 μm exhibit irreplaceable advantages in biomolecule separation and purification, immunoassay, and targeted drug delivery due to their moderate size, good suspension properties, abundant surface active sites, and sufficient magnetic responsiveness. However, the preparation process of these 1 μm carboxylated magnetic beads is quite complex. It is difficult to obtain magnetic nanoparticles with uniform particle size and good magnetic properties, as their particle size is largely limited by the size of polystyrene seed spheres. Furthermore, the raw materials used in the preparation process, such as magnetic nanoparticles, surface modifiers, and functionalizing reagents, are relatively expensive. Combined with the high energy consumption resulting from the complex process, the production cost of 1 μm carboxylated magnetic beads remains high, limiting their large-scale application.

[0004] Magnetic microspheres have been widely used in separation and purification, cell separation, nucleic acid extraction, targeted drug delivery, and magnetothermal therapy. In immunoassay applications, magnetic materials can serve as solid-phase carriers for detection and as signal markers. Among magnetic microspheres, superparamagnetic microspheres are the most widely used. Superparamagnetic microspheres exhibit magnetism under the influence of an external magnetic field, and once the external magnetic field is removed, the microspheres can be uniformly dispersed in the solution. This allows for rapid separation of impurities and antigen-antibody complexes without the need for centrifugation. Magnetic microspheres can also serve as solid-phase carriers for immunoassays, immobilizing coated antibodies. Furthermore, they can act as detection signals; by detecting the intensity of the magnetic signal, quantitative analysis of the sample can be performed.

[0005] Chinese invention application number CN 114907612 A discloses porous polystyrene microspheres prepared using styrene and divinylbenzene as functional monomers, and magnetizing the porous polymer microspheres to obtain magnetized porous polymer microspheres, ultimately preparing carboxyl magnetic spheres with a particle size of about 1 μm.

[0006] The existing technologies described above still have certain problems. Currently, most carboxylated magnetic microspheres use styrene as a monomer and potassium persulfate as an initiator, through in-situ growth of iron oxide (Fe3O4), followed by modification with acrylic acid to obtain carboxylated magnetic microspheres. The polystyrene obtained by this method is unstable and easily detaches. Regarding the patent in CN 114907612 A, which uses styrene and divinylbenzene as functional monomers to prepare porous polystyrene microspheres and then magnetizes them, the resulting magnetic material suffers from high luminescence background, which is unfavorable for high-sensitivity chemiluminescence detection. Furthermore, the issues of raw material cost and energy consumption have not been effectively resolved, making it difficult to reduce production costs and hindering large-scale application. Therefore, developing a simple, uniform, low-cost method for preparing carboxylated magnetic beads with a particle size of approximately 1 μm and good magnetic properties is of significant practical importance. Summary of the Invention

[0007] In view of this, the present invention provides magnetic microspheres, their preparation methods and applications, and reagents containing magnetic microspheres.

[0008] This invention provides a method for preparing magnetic microspheres, comprising:

[0009] S1: The polymer monomer, dispersant, crosslinking agent and carboxyl monomer are reacted in a dispersion medium, and the reaction is continued after the initiator is added to obtain carboxyl polymer microspheres;

[0010] S2. After mixing and reacting the carboxyl polymer microspheres with alkaline solution, centrifuge and wash until neutral, add 10%~70% THF solution, shake, centrifuge and wash again to obtain carboxylate modified microspheres.

[0011] S3. The carboxylate-modified microspheres are magnetized to obtain magnetic microspheres.

[0012] In step S1:

[0013] In some embodiments, the polymerizing monomer is styrene, and the resulting microspheres are carboxylated polystyrene microspheres.

[0014] In some embodiments, the polymeric monomers include styrene and methyl methacrylate. In some specific embodiments, the polymeric monomers are composed of styrene and methyl methacrylate, and the resulting polymer microspheres are poly(styrene-methyl methacrylate) microspheres. Further, the mass ratio of styrene to methyl methacrylate is (8~9):(1~2), specifically 9:1 or 8:2.

[0015] In some embodiments, the initiator is one or more of azobisisobutyronitrile, potassium persulfate (KPS), and 2,2'-azobisisobutyramidine hydrochloride (AIBA). In some specific embodiments, the initiator is azobisisobutyronitrile.

[0016] In some embodiments, the dispersant is one or more of polyvinylpyrrolidone, polyethylene glycol, and SDS. In some specific embodiments, the dispersant is polyvinylpyrrolidone.

[0017] In some embodiments, the crosslinking agent is one or more of ethylene glycol dimethacrylate and trimethylolpropane trimethacrylate. In some specific embodiments, the crosslinking agent is ethylene glycol dimethacrylate.

[0018] In some embodiments, the ratio of the polymerizing monomer, initiator, dispersant, crosslinking agent and carboxyl monomer is 15~36ml:1~1.8g:7~15g:2~3ml:1~2ml, specifically 15ml:1g:7g:2ml:1ml, 30ml:1.8g:15g:3ml:2ml, 33ml:1.8g:15g:3ml:2ml or 36ml:1.8g:15g:3ml:2ml.

[0019] In some embodiments, an initiator is added when the temperature of the reaction system reaches 65°C, and the temperature at which the reaction continues is 75°C.

[0020] In some embodiments, the particle size of the carboxyl polymer microspheres is 700-900 nm. In some specific embodiments, the particle size of the carboxyl polymer microspheres is 700 nm, 800 nm, or 900 nm.

[0021] In some embodiments, in step S2, the alkaline solution is at least one of sodium hydroxide solution, potassium hydroxide solution, and ammonia water, preferably a sodium hydroxide solution. In some embodiments, the concentration of the alkaline solution is 1M to 5M, preferably 1M. In some specific embodiments, the alkaline solution is a 1M sodium hydroxide solution.

[0022] This invention has found that excessively high THF solution concentrations (e.g., 100% pure THF) can cause PS microspheres to clump together and become unwashable, while excessively low concentrations (below 10%) can affect microsphere pore formation. To ensure PS microspheres swell and form pores while facilitating washing and preventing clumping, the concentration of the THF solution must be strictly controlled. In some embodiments, the THF mass concentration is 10%–70%, preferably 10%–30%, specifically 10%, 20%, or 30%; in some specific embodiments, the THF mass concentration is 10%. Experiments show that compared to other concentrations within the above range, treatment with a 10% THF solution yields the best results, specifically: the porous structure of the microspheres is more uniformly distributed, the pore size is more consistent, and there is no obvious adhesion between the microspheres; after centrifugation and washing, well-dispersed monodisperse microspheres can be obtained.

[0023] In this invention, the mass concentration of the THF solution is 10-70%, preferably 10-30%, specifically 10%, 20% or 30%, wherein 10% is preferred.

[0024] In some implementations, in step S2, the solution used for centrifugal washing and re-centrifugal washing is anhydrous ethanol and / or pure water.

[0025] In some embodiments, the reaction temperature in step S2 is 50-80°C. Existing techniques for treating PS microspheres at room temperature cannot achieve the desired loose, porous structure. In this application, controlling the reaction temperature to 50-80°C effectively enhances the porosity, resulting in a porous surface. Excessive temperature can lead to PS microsphere aggregation and slight corrosion. In some specific embodiments, the reaction temperature can be 50°C, 60°C, 70°C, or 80°C, preferably 70°C. In some embodiments, the reaction time is 2-10 hours, preferably 2 hours.

[0026] The shaking operation allows the THF solution to fully contact the carboxyl polymer microspheres, ensuring a uniform swelling process and resulting in a well-structured porous morphology. In some embodiments, the shaking time is 2 to 10 hours, preferably 2 to 5 hours, and more preferably 2 hours.

[0027] In some embodiments, step S3 includes: mixing the carboxylate-modified polymer microspheres, ferric chloride, and ferrous chloride, reacting them under a nitrogen atmosphere for 1 h, adding an ammonia solution, initially heating to 80 °C, and after reacting for 3 h, washing with water until neutral, drying, and obtaining magnetic microspheres.

[0028] To further optimize the surface morphology and dispersibility of the magnetic microspheres, the preparation method provided by this invention may further include a step of rotary ball milling the magnetic microspheres with zirconia beads of different diameters. By controlling the milling time and rotation speed, the mechanical force of the zirconia beads effectively removes burrs and agglomerated particles that may exist on the surface of the magnetic microspheres, making the surface of the microspheres smoother and more uniform, with a more concentrated particle size distribution, while avoiding damage to the internal structure and magnetic properties of the microspheres caused by severe mechanical action. In some embodiments, the particle size of the zirconia beads is 1.1-1.4 mm. The rotary ball milling time is 5 h to 8 h, specifically 5 h, 6 h / 7 h, or 8 h.

[0029] This invention also includes magnetic microspheres prepared by the method described above.

[0030] Experiments show that this invention, through heat treatment under alkaline conditions followed by THF oscillation, ultimately yields porous polystyrene carboxyl magnetic microspheres with uniform pore size, ranging from 0.8 to 1.2 μm. Simultaneously, it avoids the use of divinylbenzene as a crosslinking agent, reducing the luminescence background of the final magnetic microspheres and enabling better application in chemiluminescence immunoassay. The presence of a porous surface structure increases the stability and functionalization of the magnetic material, avoiding magnetic leakage and magnetic shell detachment issues. Furthermore, the smoother and more uniform surface of the porous spheres facilitates coating and reduces non-specific adsorption on the magnetic sphere surface.

[0031] The present invention also provides the magnetic microspheres described herein and acceptable additives.

[0032] This invention first prepares carboxyl polymer microspheres in a one-step process, then heat-treats them under alkaline conditions, followed by THF oscillation, ultimately yielding porous polystyrene carboxyl microspheres with uniform pores. This process avoids the use of divinylbenzene as a crosslinking agent, reducing the luminescence background of the final magnetic microspheres and enabling better application in chemiluminescence immunoassay. The presence of a porous surface structure increases the stability and functionalization of the magnetic material, avoiding magnetic leakage and magnetic shell detachment problems. Furthermore, the porous spheres have a smoother and more uniform surface, laying a solid foundation for subsequent coating processes and reducing non-specific adsorption on the magnetic sphere surface. This ball milling method is safe, non-toxic, easy to operate, pollution-free, and easily industrialized. Attached Figure Description

[0033] Figure 1 SEM image of the PS polymer microspheres prepared in Example 1;

[0034] Figure 2 The carboxylate-modified microspheres obtained in Example 1 were treated with hot alkali and THF.

[0035] Figure 3 The magnetic microspheres were prepared in Example 1. Detailed Implementation

[0036] This invention provides magnetic microspheres, their preparation methods and applications, and reagents containing magnetic microspheres. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0037] In this document, the terms "including", "comprising", and "having" describe both closed-loop technical solutions consisting of the listed features and open-loop technical solutions that include the listed features.

[0038] In this document, the term “and / or” as used includes any and all combinations of one or more of the related listed items.

[0039] In this document, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when a range refers to an integer, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0040] In this article, when referring to units for data ranges, if the unit is only followed by the right endpoint, it means that the units for the left and right endpoints are the same. For example, 50-80℃ means that the units for the left endpoint "50" and the right endpoint "80" are both in degrees Celsius (℃).

[0041] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0042] The present invention will be further illustrated below with reference to the embodiments:

[0043] Example 1

[0044] (1) Dissolve 15 g of polyvinylpyrrolidone (PVP) in 80% ethanol solution. Accurately measure 30 mL of styrene (St), add 3 mL of ethylene glycol dimethacrylate, add 2 mL of methacrylic acid, and mechanically stir. When the reaction reaches 65 °C, add 1.8 g of dissolved azobisisobutyronitrile (AIBN). Let the reaction solution react at 75 °C. Measure the particle size at intervals. When the DLS particle size is 700 nm, stop the reaction to obtain carboxylated polystyrene (Pt) microspheres. Figure 1 As shown.

[0045] (2) Carboxylated polystyrene microspheres were added to a three-necked flask, along with 1M sodium hydroxide. The mixture was reacted at 70°C for 2 hours, and then washed with anhydrous ethanol and pure water by centrifugation until neutral. 10% tetrahydrofuran (THF) was added, and the mixture was shaken for 2 hours. The microspheres were then washed with anhydrous ethanol by centrifugation to obtain the modified polystyrene microspheres. SEM images are shown below. Figure 2 As shown.

[0046] (3) Polystyrene microspheres, 12.36 g of dissolved ferric chloride, and 5.1 g of ferrous chloride were added to the reaction vessel in sequence. After reacting for 1 h under a nitrogen atmosphere, ammonia solution was added and the temperature was raised to 80 °C. After reacting for 3 h, the reaction was stopped and the mixture was washed with water until neutral. Finally, it was placed in a drying oven until constant weight was obtained to obtain solid polystyrene magnetic spheres (i.e., magnetic porous spheres).

[0047] The magnetic porous spheres prepared through steps (1) to (3) above have rough solid surfaces, and most of the iron oxide (Fe3O4) has not entered the pores of the spheres. To make the surface of the magnetic porous spheres smooth and increase the content of magnetic material in the pores, the magnetic porous spheres are ground using a three-roll mill. The details are as follows:

[0048] Magnetic microspheres were placed together with zirconia beads of different diameters in a ball mill jar, and the jar was placed on a three-roll mill for rotary grinding for 5 hours. After grinding, the microspheres were washed with anhydrous ethanol and stored in an ethanol solution. The magnetic microspheres showed a 300 nm increase in diameter compared to the polystyrene microspheres, as shown in the SEM images. Figure 3 As shown.

[0049] Example 2

[0050] (1) Take 15 g of polyvinylpyrrolidone (PVP) and dissolve it in 80% ethanol solution. Accurately measure 30 mL of styrene (St) + 3 mL of methyl methacrylate (MMA), add 3 mL of ethylene glycol dimethacrylate, add 2 mL of acrylic acid, and stir mechanically. When the reaction reaches 65 °C, add 1.8 g of dissolved azobisisobutyronitrile (AIBN) and let the reaction solution react at 75 °C. Measure the particle size every once in a while. When the particle size of DLS is 800 nm, stop the reaction to obtain carboxylated polystyrene (Pt) microspheres.

[0051] (2) Preparation of polystyrene (Pt) microspheres: Carboxylated polystyrene microspheres were added to a three-necked flask, 1M sodium hydroxide was added, and the mixture was reacted at 70°C for 5 hours. The mixture was then washed with anhydrous ethanol and pure water by centrifugation until neutral. 10% THF was added and shaken for 5 hours. The mixture was then washed with anhydrous ethanol by centrifugation to obtain fluffy and porous polystyrene-modified microspheres.

[0052] (3) Carboxylated polystyrene microspheres, 12.36 g of dissolved ferric chloride, and 5.1 g of ferrous chloride were added sequentially to the reaction vessel. After reacting for 1 h under a nitrogen atmosphere, an ammonia solution was added and the temperature was raised to 80 °C. After reacting for 3 h, the reaction was stopped and the mixture was washed with water until neutral. Finally, the mixture was placed in a drying oven until constant weight was obtained to obtain magnetic porous sphere solids.

[0053] (4) The magnetic porous sphere solid was placed together with zirconia beads of different diameters into a ball mill jar, and the ball mill jar was placed on a three-roll mill for rotary grinding for 10 hours. After grinding, it was washed with anhydrous ethanol and stored in an ethanol solution to obtain the final magnetic sphere DLS. The magnetic sphere DLS increased in size by 200 nm compared with polystyrene microspheres, resulting in 1 μm magnetic beads.

[0054] Example 3

[0055] (1) Take 15 g of polyvinylpyrrolidone (PVP) and dissolve it in 80% ethanol solution. Accurately measure 30 mL of styrene (St) + 6 mL of methyl methacrylate (MMA), add 3 mL of ethylene glycol dimethacrylate, 1 mL of methacrylic acid and 1 mL of acrylic acid, and stir mechanically. When the reaction reaches 65 °C, add 1.8 g of dissolved azobisisobutyronitrile (AIBN) and let the reaction solution react at 75 °C. Measure the particle size every once in a while. When the particle size of DLS is 900 nm, stop the reaction to obtain carboxylated polystyrene (Pt) microspheres.

[0056] (2) Carboxylated polystyrene microspheres were added to a three-necked flask, and 1M sodium hydroxide was added. The mixture was reacted at 70°C for 8 hours. The microspheres were then washed with anhydrous ethanol and pure water by centrifugation until neutral. 10% THF was added and the mixture was shaken for 8 hours. The microspheres were then washed with anhydrous ethanol by centrifugation to obtain the modified polystyrene microspheres.

[0057] (3) Carboxylated polystyrene microspheres, 12.36 g of dissolved ferric chloride, and 5.1 g of ferrous chloride were added sequentially to the reaction vessel. After reacting for 1 h under a nitrogen atmosphere, an ammonia solution was added, and the temperature was raised to 80 °C. After reacting for 3 h, the reaction was stopped, and the mixture was washed with water until neutral. Finally, the mixture was placed in a drying oven until constant weight was obtained to obtain magnetic porous spherical solids.

[0058] (4) The magnetic microsphere solid was placed together with zirconia beads of different diameters into a ball mill jar, and the ball mill jar was placed on a three-roll mill for rotational grinding for 15 h. After grinding, it was washed with anhydrous ethanol and stored in an ethanol solution. The magnetic microspheres DLS increased in size by 100 nm compared with polystyrene polymer microspheres, and 1 μm magnetic beads were obtained.

[0059] Test case

[0060] The magnetic sphere DLS prepared in Examples 1-3 were subjected to SA chemiluminescence testing, and the results are as follows:

[0061] The test results of magnetic ball DLS in Example 1 were as follows: background was 760, biotin-IgG binding capacity was 11.4 ug / mg, enzyme-catalyzed chemiluminescence background was 8249, and resolution was 21.

[0062] The test results of magnetic ball DLS in Example 2 were as follows: background was 684, biotin-IgG binding capacity was 13.6 ug / mg, enzyme-catalyzed chemiluminescence background was 7945, and resolution was 23.

[0063] The test results of magnetic ball DLS in Example 3 were as follows: background was 547, biotin-IgG binding capacity was 18.7 ug / mg, enzyme-catalyzed chemiluminescence background was 6432, and resolution was 47.

[0064] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing magnetic microspheres, characterized by, The preparation method comprises the following steps: S1: reacting a polymerization monomer, an initiator, a dispersing agent, a crosslinking agent and a carboxyl monomer in a dispersion medium, adding the initiator and continuing the reaction to obtain carboxyl polymer microspheres; S2: mixing and reacting the carboxyl polymer microspheres and an alkali solution, centrifuging and washing to neutral, adding a 10%-70% THF solution and oscillating, and centrifuging and washing again to obtain carboxylate modified microspheres; S3: magnetizing the carboxylate modified microspheres to obtain magnetic microspheres.

2. The production method according to claim 1, characterized by, In step S1, the polymerization monomer is styrene or a mixture of styrene and methacrylic acid, and / or, the initiator is one or more of azobisisobutyronitrile, potassium persulfate KPS and 2,2'-azobis isobutyl imidazole hydrochloride AIBA; and / or, the dispersing agent is one or more of polyvinylpyrrolidone, polyethylene glycol and SDS; and / or, the crosslinking agent is one or more of ethylene glycol dimethyl acrylate and trimethylolpropane trimethyl acrylate; and / or, the carboxyl monomer is acrylic acid and / or methacrylic acid; and / or, the ratio of the polymerization monomer, the initiator, the dispersing agent, the crosslinking agent and the carboxyl monomer is 15-36 ml: 1-1.8 g: 7-15 g: 2-3 ml: 1-2 ml.

3. The preparation method according to claim 1, characterized in that, In step S1, the initiator is added when the temperature of the reaction system of the reaction rises to 65°C, and the temperature of the continued reaction is 75°C.

4. The method of claims 1-3, wherein the compound is prepared by the method of claim 1. In step S1, the particle size of the carboxyl seed microspheres is 700-900 nm.

5. The preparation method according to claim 1, characterized in that, In step S2, the alkali solution is at least one of sodium hydroxide solution, potassium hydroxide solution and ammonia water; and the concentration of the alkali solution is 1M; and / or, the mass concentration of the THF is 10%; and / or, the solutions used in the centrifuging and washing and the centrifuging and washing again are anhydrous ethanol and / or pure water.

6. The method of claim 1, wherein, In step S2, the temperature of the reaction is 50-80°C; and the time of the reaction is 2 hours. and / or, the time of the oscillation is 2-10 hours.

7. The preparation method according to claim 1, characterized in that, In step S3, the carboxylate modified polymer microspheres, ferric chloride and ferrous chloride are mixed, reacted for 1 h under a nitrogen atmosphere, then an ammonia solution is added, the temperature is raised to 80°C and reacted for 1 h, washed with water to neutral, dried and magnetic microspheres are obtained.

8. The method of any one of claims 1 to 7, wherein the method further comprises the step of: The preparation method further comprises the step of rotating and grinding the magnetic microspheres and zirconium oxide beads. ​ 9. The magnetic microspheres prepared by the preparation method according to any one of claims 1-8.

10. An agent, characterized in that, The magnetic microspheres according to claim 9 and an acceptable adjuvant are included.

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