Polymer microsphere and preparation method thereof

Abstract

The invention provides a polymer microsphere and a preparation method thereof, and relates to the field of biomedicine.The polymer microsphere comprises a core, a shell wrapping the core and a filtering layer located on the side, away from the core, of the shell; the core body comprises at least one magnetic particle, and the shell comprises a modification layer; wherein the filtering layer comprises a plurality of pores, and the average size of the pores is larger than or equal to the size of an object to be detected and smaller than the size of pollutants. When the polymer microspheres are applied to nucleic acid extraction, the extraction purity of nucleic acid can be improved to a great extent.

Description

Technical Field

[0001] The present application relates to the biomedical field, and in particular to a polymer microsphere and a preparation method thereof. Background Art

[0002] Magnetic nano- or micro-composite materials, such as magnetic microspheres, have attracted wide attention due to their potential applications in the biomedical field. The magnetic microspheres in the related art are easily interfered by residual proteins during nucleic acid extraction, thereby reducing the purity of nucleic acid extraction. At present, it is urgent to provide a new polymer microsphere to solve the above problems. Summary of the invention

[0003] The embodiments of the present application provide a polymer microsphere and a preparation method thereof. When the polymer microsphere is used for nucleic acid extraction, the extraction purity of nucleic acid can be greatly improved.

[0004] The embodiments of the present application adopt the following technical solutions:

[0005] In a first aspect, an embodiment of the present application provides a polymer microsphere, wherein the polymer microsphere comprises:

[0006] a core body, the core body comprising at least one magnetic particle;

[0007] A shell encapsulating the core, wherein the shell comprises a modification layer;

[0008] A filtration layer is located on a side of the shell away from the core; wherein the filtration layer comprises a plurality of pores, and an average size of the pores is greater than or equal to the size of the object to be detected and smaller than the size of the pollutant.

[0009] In at least one embodiment of the present application, the analyte includes nucleic acid molecule fragments, and the contaminants include at least protein molecules in nucleoprotein; the size of the nucleic acid molecule fragments is nanometer order, and the size of the nucleoprotein molecules is nanometer order or micrometer order.

[0010] In at least one embodiment of the present application, the material of the filtration layer includes a high molecular polymer having a network cross-linked structure formed by a first component, a second component and an initiator.

[0011] In at least one embodiment of the present application, the first component includes at least one of acrylamide, methacrylic acid, and N-isopropylacrylamide, the second component includes at least one of N,N-methylenebisacrylamide, styrene, and divinylbenzene, the initiator includes a first initiator and a second initiator, the first initiator includes at least one of tetramethylethylenediamine and azobisisobutyronitrile, and the second initiator includes at least one of ammonium persulfate, potassium persulfate, and sodium persulfate.

[0012] In at least one embodiment of the present application, the material of the modification layer includes at least one group selected from the group consisting of carboxyl, hydroxyl, and silanol.

[0013] In at least one embodiment of the present application, some groups in the material of the modification layer extend into the pores.

[0014] In at least one embodiment of the present application, the size of the pores ranges from 50 nm to 500 nm.

[0015] In at least one embodiment of the present application, the first component includes acrylamide, the second component includes N,N-methylenebisacrylamide, and the molar ratio of the first component to the second component ranges from 5:1 to 1:2.

[0016] In at least one embodiment of the present application, the second component includes N,N-methylenebisacrylamide, and the molar ratio of the second component to the magnetic particles is in the range of 1:8 to 1:2.

[0017] In at least one embodiment of the present application, the second component includes N,N-methylenebisacrylamide, the first initiator includes tetramethylethylenediamine, and the second initiator includes ammonium persulfate;

[0018] The molar ratio of the second component, the second initiator and the first initiator is in the range of 2:2:1 to 5:4:1.

[0019] In at least one embodiment of the present application, the magnetic particles include a combination of one or more of iron oxide, cobalt oxide, and nickel oxide, and the size of the magnetic particles ranges from 50 nm to 500 nm.

[0020] In at least one embodiment of the present application, hydrogen bonds exist in a partial region between the material of the modification layer and the material of the filtration layer.

[0021] In at least one embodiment of the present application, the size of the polymer microspheres ranges from 1 μm to 2 μm.

[0022] In a second aspect, an embodiment of the present application provides a method for preparing polymer microspheres, which is used to prepare the polymer microspheres as described in any one of the first aspects, and the method comprises:

[0023] forming magnetic particles;

[0024] forming a modified layer to obtain an intermediate particle; wherein the modified layer wraps at least one of the magnetic particles;

[0025] A filtration layer is formed to obtain the polymer microspheres; wherein the filtration layer wraps the intermediate particles, the material of the filtration layer is a polymer with a network cross-linked structure, the filtration layer includes a plurality of pores, and the average size of the pores is greater than or equal to the size of the object to be detected and smaller than the size of the pollutant.

[0026] In at least one embodiment of the present application, the step of forming a filtration layer to obtain the polymer microspheres comprises:

[0027] Dissolving the intermediate particles in a first buffer solution and dispersing them evenly to obtain an intermediate particle solution;

[0028] dissolving the first component and the second component in a second buffer solution and prepolymerizing the components to obtain a prepolymer solution;

[0029] The prepolymer solution and the intermediate particle solution are mixed together, an initiator is added, and the mixture is stirred at room temperature for a preset time to obtain the polymer microsphere mixed solution having the filter layer.

[0030] In at least one embodiment of the present application, after the step of mixing the prepolymer solution and the intermediate particle solution together, adding an initiator, and stirring the reaction at room temperature for a preset time to obtain the polymer microsphere mixed solution having the filter layer, the method further comprises:

[0031] Collecting the polymer microspheres in the polymer microsphere mixture by magnetic attraction;

[0032] Using a mixture of methanol and acetic acid to wash the polymer microspheres to remove impurities and break the hydrogen bonds between the modification layer and the filter layer material;

[0033] Freeze-dry and store.

[0034] An embodiment of the present application provides a polymer microsphere and a preparation method thereof, wherein the polymer microsphere comprises a core, a shell encapsulating the core, and a filtration layer located on a side of the shell away from the core; the core comprises at least one magnetic particle, and the shell comprises a modified layer; wherein the filtration layer comprises a plurality of pores, and the average size of the pores is greater than or equal to the size of the object to be detected and smaller than the size of the pollutant.

[0035] In an embodiment of the present application, a filtration layer is provided on the outside of the polymer microspheres. Since the filtration layer includes a plurality of pores, the average size of the pores is greater than or equal to the size of the analyte and smaller than the size of the contaminants. When the polymer microspheres are used for nucleic acid purification, the modification layer can adsorb the analyte, such as nucleic acid, on the polymer microspheres through the pores, while the filtration layer excludes contaminants, such as residual protein molecules in the nucleic acid, from the polymer microspheres, thereby improving the extraction purity and extraction efficiency of the nucleic acid and solving the problems of residual protein contamination and low nucleic acid purity during nucleic acid extraction.

[0036] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application or related technologies, the drawings required for use in the embodiments or descriptions of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1-Figure 3 Schematic diagram of the structures of three polymer microspheres provided in the embodiments of the present application;

[0039] Figure 4 A flow chart of a method for preparing polymer microspheres provided in an embodiment of the present application;

[0040] Figure 5 A schematic diagram illustrating the principle of prepolymerization of a first component and a second component provided in an embodiment of the present application;

[0041] Figure 6 A schematic diagram of the formation of hydrogen bonds between a material of a filtration layer and a raw material of a modification layer provided in an embodiment of the present application;

[0042] Figure 7 A schematic diagram of the breaking of hydrogen bonds between a material of a filtration layer and a material of a modification layer provided in an embodiment of the present application. Specific embodiments

[0043] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0044] Unless the context requires otherwise, throughout the specification and claims, the term "including" is to be interpreted as an open, inclusive meaning, that is, "including, but not limited to". In the description of the specification, the terms "one embodiment", "some embodiments", "exemplary embodiment", "example", "specific example" or "some examples" and the like are intended to indicate that specific features, structures, materials or characteristics associated with the embodiment or example are included in at least one embodiment or example of the present application. The schematic representation of the above terms does not necessarily refer to the same embodiment or example. In addition, the specific features, structures, materials or characteristics may be included in any one or more embodiments or examples in any appropriate manner.

[0045] In the embodiments of the present application, words such as "first" and "second" are used to indicate parts of identical or similar items having substantially the same functions and effects. This is only for the purpose of clearly describing the technical solutions of the embodiments of the present application and shall not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features.

[0046] As one of the most basic experiments in molecular experiments, nucleic acid extraction is the basis of almost all experiments. No matter the subsequent cloning, PCR (Polymerase Chain Reaction), DPCR (Digital PCR), QPCR (Real-time Quantitative PCR Detecting System), library construction and sequencing, nucleic acids are needed to proceed smoothly. With the rapid development of genetic diagnosis, genetically modified food testing, personalized medicine, etc., the use of magnetic beads to extract nucleic acids is becoming more and more popular among scientific researchers due to its safety, non-toxicity, simple operation, short time, and the ability to realize automation and large-scale operation. However, in actual application, it is difficult to completely remove the protein in the nucleic acid sample, and some residual proteins will be adsorbed on the magnetic beads (magnetic microspheres), resulting in problems such as low purity of the obtained nucleic acid product.

[0047] Hydrogels are polymers with three-dimensional cross-linked networks, and their cross-linked networks are established through covalent or non-covalent interactions. Non-covalent interactions mainly include physical entanglement, hydrogen bonds, hydrophobic interactions, supramolecular interactions, and electrostatic interactions. Covalent interactions are mainly formed by some stable molecules forming covalent bonds between polymer chains. With the continuous deepening of research on hydrogels, the physical and chemical properties of hydrogels, such as strength, toughness, and shape memory, can be well regulated by external stimuli (light, electricity, heat, magnetism, chemistry, etc.). At present, a series of natural hydrogels (such as agarose, cellulose, hyaluronic acid, sodium alginate, etc.) and synthetic hydrogels (such as polyacrylamide, polyvinyl alcohol, polyacrylic acid, etc.) have been widely used in various fields of production and life. Among them, permanent covalent cross-linking is a preparation method for synthetic hydrogels. This method can form a permanent hydrogel network structure with a certain rigidity. The hydrogel can have multiple pores, and the size of the pores can be adjusted by adjusting the concentration of the reaction monomer.

[0048] Based on this, an embodiment of the present application provides a polymer microsphere and a preparation method thereof, wherein the polymer microsphere includes a core body, a shell that wraps the core body, and a filtration layer located on the side of the shell away from the core body; the core body includes at least one magnetic particle, and the shell includes a modification layer; wherein the filtration layer includes a plurality of pores, and the average size of the pores is greater than or equal to the size of the object to be detected and smaller than the size of the pollutant. In this way, by arranging a filtration layer on the outside of the polymer microsphere, since the filtration layer includes a plurality of pores, and the average size of the pores is greater than or equal to the size of the object to be detected and smaller than the size of the pollutant, when the polymer microsphere is applied to nucleic acid purification, the modification layer can adsorb the object to be detected, such as nucleic acid, on the polymer microsphere through the pores, and the filtration layer excludes pollutants, such as residual protein molecules in nucleic acid, from the polymer microsphere, thereby improving the extraction purity and extraction efficiency of nucleic acid, and solving the problems of residual protein contamination and low nucleic acid purification during nucleic acid extraction. wherein, the material of the above-mentioned filtration layer can be a hydrogel network structure.

[0049] The polymer microspheres and the preparation method thereof provided in the embodiments of the present application will be specifically introduced below with reference to the accompanying drawings.

[0050] The embodiment of the present application provides a polymer microsphere, combined with Figure 1 , Figure 2 and Figure 3 As shown, the polymer microspheres include:

[0051] A core body H, the core body H comprises at least one magnetic particle 1;

[0052] A shell K encapsulating the core H, wherein the shell K comprises a modification layer 2;

[0053] The filter layer 3 is located on the side of the shell K away from the core H; wherein, Figure 1 As shown, the filter layer 3 includes a plurality of pores X, and the average size of the pores X is greater than or equal to the size of the object to be detected and smaller than the size of the pollutant.

[0054] The material of the magnetic particles 1 is not limited here. For example, the material of the magnetic particles HC may include a magnetic material;

[0055] For example, the magnetic material can be an alloy material, and the main varieties of alloys include AlNi(Co), FeCr(Co), FeCrMo, FeAlC, FeCo(V)(W), Re-Co (Re represents a rare earth element), Re-Fe, AlNi(Co), FeCrCo, FeCrCo, PtCo, MnAlC, CuNiFe and AlMnAg, etc.;

[0056] For another example, the magnetic material may include a ferrite material, the main component of which is MO·6Fe2O3, where M represents a composite component such as Ba, Sr, Pb or SrCa, LaCa, etc.;

[0057] For another example, the magnetic material may be an intermetallic compound, mainly represented by MnBi.

[0058] The magnetic particles 1 provided in the embodiments of the present application are described by taking as an example a magnetic particle whose main component is iron oxide (Fe3O4) and / or ferrous oxide (Fe2O3).

[0059] In an exemplary embodiment, if Figure 2 or Figure 3 As shown, the modification layer 2 contains functional groups, such as at least one of carboxyl (-COOH), hydroxyl (-OH), and silanol (-Si-OH), so that the surface of the polymer microsphere carries at least one of these functional groups.

[0060] In an exemplary embodiment, the analyte includes but is not limited to nucleic acid molecules, and the contaminants include but are not limited to proteins, red blood cells, white blood cells, and the like.

[0061] In at least one embodiment of the present application, the polymer microspheres can be used for extraction and purification of nucleic acids.

[0062] In an exemplary embodiment, when the modification layer 2 includes a carboxyl group, the polymer microspheres can be used for the extraction and purification of DNA; when the modification layer 2 includes a hydroxyl group or a silanol group, the polymer microspheres can be used for the extraction and purification of RNA.

[0063] This does not limit the specific material of the above-mentioned filtration layer 3, but only imposes requirements on the size of the pores X on the filtration layer 3. When the pores X that meet the requirements can be prepared on the filtration layer 3, the material of the filtration layer 3 can be any material.

[0064] Exemplarily, the material of the filter layer 3 may be graphene porous gel.

[0065] Exemplarily, the material of the filtration layer 3 may be a polymer porous hydrogel.

[0066] In an exemplary embodiment, when the analyte is a nucleic acid, the size of the analyte includes the length of the nucleic acid molecule and the helical diameter of the nucleic acid molecule. The helical diameter of the nucleic acid molecule is approximately less than or equal to 2 nm. The nucleic acid molecule has different lengths depending on the number of nucleotides in the nucleic acid molecule.

[0067] The nucleic acid molecule is a helical molecule having a single strand or a double strand. Generally, the helical radius of a double-stranded nucleic acid molecule is about 2 nm.

[0068] In practical applications, nucleic acids in organisms often combine with proteins to form nucleoproteins. During the extraction and purification of nucleic acids, this type of protein is the main interfering factor that reduces the purity of nucleic acid extraction.

[0069] The size of the above-mentioned pollutants includes but is not limited to the volume of the pollutants, the length of the pollutants, the side length, diagonal, radius, etc. of the plane figure of the pollutants, which are specifically related to the shape of the pollutants; no matter what shape the pollutants are, since the size of the pollutants is smaller than the average size of pore X, the pollutants cannot enter pore X.

[0070] In at least one embodiment of the present application, the size of the pore X ranges from 50 nm to 500 nm.

[0071] Exemplarily, the size of the pore X can be 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 350nm, 380nm, 400nm, 430nm, 450nm, 480nm, 500nm.

[0072] It should be noted that the dimensions in this specification are all average values, and fluctuations of 3% to 5% around the average values ​​fall within the protection scope of this application.

[0073] The size of the pore X refers to the diameter, side length, diagonal line or length along a preset direction of the plane figure of the pore, rather than the depth of the pore X. The depth of the pore X can be determined according to the thickness of the filter layer 3.

[0074] When the size of the pore X is greater than or equal to the helical diameter of the nucleic acid molecule and less than the length of the nucleic acid molecule, the groups on the modified layer 2 can adsorb one end of the nucleic acid molecule onto the polymer microsphere through the pore X.

[0075] When the size of the pore X is larger than the length of the nucleic acid molecule, the groups on the modified layer 2 can adsorb the entire nucleic acid molecule onto the polymer microsphere through the pore X.

[0076] The plane shape of the pore X is not limited here. For example, the plane shape of the pore X can be a polygon, an arc, or a shape formed by a combination of a polygon and an arc.

[0077] For example, the plane figure of the pore X may be a closed figure formed by lines with uneven edges.

[0078] The embodiments of the present application provide Figure 1 In the figure, the plane figure of the pore X is drawn as a quadrilateral as an example.

[0079] Exemplarily, when the plane figure of the pore X is approximately a polygon, the size of the pore X refers to the side length or diagonal of the polygon.

[0080] Exemplarily, when the plane shape of the pore X is close to an arc (including but not limited to a circle), the size of the pore X refers to the diameter or the length along a preset direction; wherein the preset direction may include any direction.

[0081] In the embodiment of the present application, by setting a filtration layer 3 on the outside of the polymer microsphere, since the filtration layer 3 includes a plurality of pores X, the average size of the pores X is greater than or equal to the size of the analyte and smaller than the size of the pollutant, when the polymer microsphere is used for nucleic acid purification, the modification layer 2 can adsorb the nucleic acid on the polymer microsphere through the pores X, and the filtration layer 3 excludes the residual pollutants in the nucleic acid from the polymer microsphere, thereby improving the extraction purity and extraction efficiency of the nucleic acid, and solving the problems of residual pollutant contamination and low nucleic acid purity during nucleic acid extraction.

[0082] In at least one embodiment of the present application, the analyte includes nucleic acid molecule fragments, and the contaminants include at least protein molecules in nucleoprotein; the size of the nucleic acid molecule fragments is nanometer-order, and the size of the nucleoprotein molecules is nanometer-order or micrometer-order.

[0083] According to different chemical compositions, nucleic acids can be divided into ribonucleic acid (RNA for short) and deoxyribonucleic acid (DNA for short). DNA is the main material basis for storing, replicating and transmitting genetic information. RNA plays an important role in the protein synthesis process. Among them, transfer RNA, referred to as tRNA, plays the role of carrying and transferring activated amino acids; messenger RNA, referred to as mRNA, is the template for protein synthesis; ribosomal RNA, referred to as rRNA, is the main site for cell protein synthesis.

[0084] Exemplarily, the nucleic acid molecule fragment may include a nucleic acid molecule in a single-stranded state.

[0085] Nucleic acids in organisms often combine with proteins to form nucleoproteins. During the extraction and purification of nucleic acids, this type of protein is the main interfering factor that reduces the purity of nucleic acid extraction.

[0086] For example, the pollutants may also include red blood cells, white blood cells and other types of protein molecules in the blood of the object. It should be noted that in practical applications, nanometer-sized red blood cells, white blood cells and most protein molecules are mainly dissolved and separated by lysis solution. The relevant introduction of lysis solution can refer to the introduction in the relevant technology.

[0087] Exemplarily, the size of the nucleic acid molecule fragment ranges from 300 nm to 800 nm, wherein the size of the nucleic acid molecule fragment refers to the length of the nucleic acid molecule fragment.

[0088] For example, the length of the nucleic acid molecule fragment is 330nm±3nm, 350nm±3nm, 380nm±3nm, 400nm±3nm, 420nm±3nm, 430nm±3nm, 450nm±3nm, 465nm±3nm, 480nm±3nm, 498nm±3nm, 520nm±3nm, 550nm±3nm, 580nm±3nm, 600nm±3nm, 660nm±3nm, 690nm±3nm, 720nm±3nm, 750nm±3nm, and 780nm±3nm.

[0089] Exemplarily, the size of the nucleoprotein molecule is greater than or equal to 500 nm.

[0090] For example, the sizes of nucleoprotein molecules are 600nm, 800nm, 1000nm, 1200nm.

[0091] In the embodiments of the present application, the analyte includes nucleic acid molecule fragments, and the contaminants include at least protein molecules in nucleoprotein; the size of the nucleic acid molecule fragments is of the order of nanometers, and the size of the nucleoprotein molecules is of the order of nanometers or micrometers; when the polymer microspheres are used for nucleic acid purification, the modification layer 2 can adsorb the nucleic acid on the polymer microspheres through the pores X, and the filtration layer 3 excludes the residual contaminants in the nucleic acid from the polymer microspheres, thereby improving the extraction purity and extraction efficiency of the nucleic acid, and solving the problems of residual contaminants and low nucleic acid purity during nucleic acid extraction.

[0092] In at least one embodiment of the present application, the material of the filtration layer 3 includes a high molecular polymer having a network cross-linked structure formed by a first component, a second component and an initiator.

[0093] Crosslinking refers to the process of connecting linear or branched polymer chains to form a network polymer by covalent bonds. It is divided into chemical crosslinking and physical crosslinking. Chemical crosslinking is generally achieved through polycondensation and polyaddition reactions; physical crosslinking uses radiation such as light and heat to crosslink linear polymers. In the embodiment of the present application, the material of the filter layer 3 is a three-dimensional network polymer obtained after crosslinking, also known as a crosslinked polymer, a crosslinked polymer or a bulk polymer.

[0094] In an exemplary embodiment, the high molecular polymer having a network cross-linked structure may be a product of condensation polymerization (polycondensation reaction); or, the high molecular polymer having a network cross-linked structure may be a product of addition polymerization (polyaddition reaction).

[0095] Among them, addition polymerization refers to the reaction in which olefin monomers are polymerized by addition. There are no by-products in addition polymerization, and the monomers react with each other to form a polymer compound; condensation polymerization is the process of forming a polymer by repeated condensation reactions. It has the dual meanings of condensing low molecules and polymerizing high molecules. The reaction product is called a condensation polymer.

[0096] In an exemplary embodiment, the polymer having a network cross-linked structure is a copolymer, also known as an interpolymer. A copolymer is a polymer obtained by addition polymerization of two or more different monomers. According to the different arrangements of various monomers in the copolymer molecular chain, copolymers can be divided into random copolymers, alternating copolymers, block copolymers and graft copolymers. The type of copolymer is not limited in this application.

[0097] In an exemplary embodiment, the high molecular polymer having a network cross-linked structure is a condensation polymer, wherein a polymer formed by a condensation polymerization reaction of two monomers containing different groups is called a condensation polymer. In this case, both the first component and the second component can be olefin monomers.

[0098] Copolymers are polymers obtained by addition polymerization, and condensation polymers are polymers formed by condensation polymerization. The reaction mechanisms of the two are different. The specific differences between addition polymerization and condensation polymerization can be referred to in the introduction and related technologies in the previous article, and will not be repeated here.

[0099] In at least one embodiment of the present application, the first component includes at least one of acrylamide, methacrylic acid, and N-isopropylacrylamide, the second component includes at least one of N,N-methylenebisacrylamide, styrene, and divinylbenzene, the initiator includes a first initiator and a second initiator, the first initiator includes at least one of tetramethylethylenediamine and azobisisobutyronitrile, and the second initiator includes at least one of ammonium persulfate, potassium persulfate, and sodium persulfate.

[0100] Exemplarily, the first component is used as a functional monomer, and the second component is used as a cross-linking agent. Under the action of an initiator, the functional monomer and the cross-linking agent react to generate a high molecular polymer having a network cross-linking structure.

[0101] Among them, the structural formula of acrylamide is: The structural formula of methacrylic acid is The structural formula of N-isopropylacrylamide is:

[0102] Among them, the structural formula of N,N-methylenebisacrylamide is: The structural formula of styrene is The structural formula of divinylbenzene is

[0103] There is no limitation on the quantity of the first component and the second component. For example, in order to facilitate the polymerization reaction and simplify the polymerization preparation process, the first component may include a functional monomer and the second component may include a cross-linking agent.

[0104] For example, the first component may include acrylamide, the second component may include N,N-methylenebisacrylamide, and the initiator may include tetramethylethylenediamine and ammonium persulfate.

[0105] In at least one embodiment of the present application, the material of the modification layer 2 includes at least one group selected from the group consisting of carboxyl (—COOH), hydroxyl (—OH), and silanol (—Si—OH).

[0106] In an exemplary embodiment, when the material of the modified layer 2 includes a carboxyl group (-COOH), the polymer microspheres can be used for the extraction and purification of DNA; when the material of the modified layer 2 includes a hydroxyl group (-OH) or a silanol group (-Si-OH), the polymer microspheres can be used for the extraction and purification of RNA.

[0107] It should be noted that, when carboxyl (-COOH) and hydroxyl (-OH) exist in the solution at the same time, acid-base neutralization will occur between the two. Therefore, when the material of the modification layer 2 includes hydroxyl (-OH), the first component in the above text avoids using functional monomers having carboxyl (-COOH) such as methacrylic acid. Similarly, the second component in the above text also avoids using crosslinking agents having carboxyl (-COOH).

[0108] In at least one embodiment of the present application, some groups in the material of the modification layer 2 extend into the pores X.

[0109] In an exemplary embodiment, the groups in the material of the modified layer 2 (especially the groups located on the surface of the modified layer 2) extend outward and are located in the space within the filtration layer 3. When the position in the filtration layer 3 corresponding to the groups in the material of the modified layer 2 extending outward is pore X, the groups can extend into the pore X.

[0110] It should be noted that the extension of the group into the pore X does not mean that the group is completely inside the pore X, and may also include the situation where some atoms in a group extend into the pore X. When the number of pores X in the filtration layer 3 is large and the size is relatively large, as many groups in the material of the modification layer 2 as possible can extend into the pore X, which is more conducive to the adsorption of more nucleic acid molecules, thereby improving the efficiency of nucleic acid purification.

[0111] In at least one embodiment of the present application, the first component includes acrylamide, the second component includes N,N-methylenebisacrylamide, and the molar ratio of the first component to the second component ranges from 5:1 to 1:2. The molar amount is actually the abbreviation of molar mass. The mass of each mole of a substance is called "molar mass".

[0112] Illustratively, the molar ratio of the first component to the second component can be 5:1, 4.5:1, 4.3:1, 4:1, 3.8:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.8:1, 1.5:1, 1.2:1, 1:1, 1:1.3, 1:1.5, 1:1.8, or 1:2.

[0113] In the embodiment of the present application, the first component is a functional monomer, and the second component is a cross-linking agent. The larger the proportion of the cross-linking agent, the smaller the size of the pore X. By setting the first component to include acrylamide and the second component to include N,N-methylenebisacrylamide, the molar ratio of the first component to the second component is in the range of 5:1 to 1:2, the size range of the pore X can be effectively controlled, so that the average size of the pore X is greater than or equal to the helical diameter of the nucleic acid molecule and smaller than the size of the protein molecule. In this way, when the polymer microsphere is applied to nucleic acid purification, the modification layer 2 can adsorb the nucleic acid on the polymer microsphere through the pore X, and the filter layer 3 excludes the contaminants in the nucleic acid from the polymer microsphere, thereby improving the extraction purity and extraction efficiency of the nucleic acid, and solving the problems of residual contamination and low nucleic acid purity during nucleic acid extraction. .

[0114] In an exemplary embodiment, in order to ensure that the polymer microspheres have stable and accurate magnetic responsiveness, requirements are set on the quality of the magnetic particles 1 .

[0115] In at least one embodiment of the present application, the second component includes N,N-methylenebisacrylamide, and the molar ratio of the second component to the magnetic particles 1 is in the range of 1:8 to 1:2.

[0116] Exemplarily, the molar ratio of the second component to the magnetic particles 1 can be 1:8, 1:7.5, 1:7.3, 1:7, 1:6.8, 1:6.5, 1:6.3, 1:6, 1:5.8, 1:5.5, 1:5.3, 1:5, 1:4.8, 1:4.5, 1:4.3, 1:4, 1:3.8, 1:3.5, 1:3.3, 1:3, 1:2.8, 1:2.5, 1:2.3, 1:2. The smaller the ratio, the higher the proportion of magnetic particles 1 in the polymer microspheres, and the better the magnetic response effect of the polymer microspheres.

[0117] It should be noted that the greater the molar ratio of the second component to the magnetic particles 1, the greater the thickness of the filter layer 3. When the molar ratio of the second component to the magnetic particles 1 is greater than 1:2, the thickness of the filter layer 3 is too large, and the magnetic responsiveness of the polymer microspheres may decrease, thereby reducing the detection accuracy of the polymer microspheres in the biomedical field and reducing their use efficiency. When the molar ratio of the second component to the magnetic particles 1 is less than 1:8, the thickness of the filter layer 3 is too small, and the filter layer 3 is insufficient to exclude the residual protein molecules in the nucleic acid from the polymer microspheres, making it difficult to improve the extraction purity and extraction efficiency of the nucleic acid.

[0118] In at least one embodiment of the present application, the second component includes N,N-methylenebisacrylamide, the first initiator includes tetramethylethylenediamine, and the second initiator includes ammonium persulfate; the molar ratio of the second component, the second initiator and the first initiator ranges from 2:2:1 to 5:4:1.

[0119] Illustratively, the molar ratio of the second component, the second initiator, and the first initiator can be 2:2:1, 2.5:2:1, 2.5:2.5:1, 3:2:1, 3:2.5:1, 3:3:1, 4.5:3:1, 4.5:3.5:1, 4.5:4:1, or 5:4:1.

[0120] In an embodiment of the present application, when the second component includes N,N-methylenebisacrylamide, the first initiator includes tetramethylethylenediamine, and the second initiator includes ammonium persulfate, by setting the molar ratio of the second component, the second initiator and the first initiator to a range of 2:2:1 to 5:4:1, the polymerization reaction has better initiation efficiency and cross-linking effect, and the efficiency of the polymerization reaction can be improved.

[0121] In at least one embodiment of the present application, the magnetic particles 1 include one or more combinations of iron oxide, cobalt oxide, and nickel oxide, and the size of the magnetic particles 1 ranges from 50 nm to 500 nm.

[0122] Exemplarily, the magnetic particles 1 may include ferrite materials, the main component of which is MO·6Fe2O3, where M represents a composite component such as Ba, Sr, Pb or SrCa, LaCa, etc.

[0123] Illustratively, the magnetic particles 1 may include at least one of ferroferric oxide and ferrous oxide.

[0124] Exemplarily, the size of the magnetic particle 1 can be 60nm, 70nm, 80nm, 90nm, 100nm, 150nm, 180nm, 200nm, 220nm, 250nm, 280nm, 300nm, 330nm, 350nm, 380nm, 400nm, 430nm, 450nm, or 480nm.

[0125] Exemplarily, the size of the magnetic particles 1 is larger than the size of the pores X on the filtration layer 3 to prevent the magnetic particles 1 from leaking out of the pores X of the filtration layer 3 when the filtration layer wraps a plurality of magnetic particles 1 .

[0126] In at least one embodiment of the present application, the size of the polymer microspheres may be 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, or 1.9 μm.

[0127] It should be noted that the dimensions in this specification are all average values, and fluctuations of 3% to 5% around the average values ​​fall within the protection scope of this application.

[0128] In at least one embodiment of the present application, hydrogen bonds exist in a partial region between the material of the modification layer 2 and the material of the filtration layer 3;

[0129] Alternatively, methanol and / or acetic acid are present in the mixture of polymer microspheres.

[0130] In an exemplary embodiment, since the material of the modification layer 2 includes one of the groups of carboxyl, hydroxyl, and silanol, and the material of the filtration layer 3 may include acrylamide and N,N-methylenebisacrylamide, the -NH- group in acrylamide and N,N-methylenebisacrylamide forms hydrogen bonds with -COOH, -OH, and Si-OH in the material of the modification layer 2, for example, the following hydrogen bonds -OH...NH-, -COOH...NH-, -Si-OH...NH- are formed. Before the mixture of polymer microspheres is used, in order to release the carboxyl, hydroxyl, and silanol related groups on the modification layer 2, it is necessary to break the hydrogen bonds between the material of the modification layer 2 and the material of the filtration layer 3 through treatment to prevent the groups on the material of the modification layer 2 from being occupied by hydrogen bonds and unable to adsorb nucleic acid molecules. However, in the actual product, some of the above hydrogen bonds may still exist and have not been broken.

[0131] In addition, in some embodiments, since the polymer microsphere mixture is cleaned with methanol and acetic acid solution, most of its hydrogen bonds are broken. When the subsequent drying time is short, some methanol and / or acetic acid may remain in the polymer microsphere mixture.

[0132] In at least one embodiment of the present application, the hydrogen bonds between the material of the modification layer 2 and the material of the filtration layer 3 are configured to be disconnected.

[0133] Since the material of the modification layer 2 includes at least one of carboxyl, hydroxyl, and silanol groups, and the material of the filtration layer 3 may include acrylamide and N,N-methylenebisacrylamide, when hydrogen bonds exist between the material of the modification layer 2 and the material of the filtration layer 3, hydrogen bonds are formed between the material of the filtration layer 3 and the material of the modification layer 2, thereby reducing the effective number of groups on the modification layer 2 that can adsorb nucleic acids; in order to improve the adsorption efficiency of nucleic acids by the groups on the material of the modification layer 2, the polymer microspheres need to be treated to break the hydrogen bonds between the material of the modification layer 2 and the material of the filtration layer 3, avoid the formation of hydrogen bonds between the material of the filtration layer 3 and the material of the modification layer 2, and reduce the effective number of groups on the modification layer 2 that can adsorb nucleic acids, thereby avoiding reducing the purification effect of the polymer microspheres on nucleic acids.

[0134] Next, the preparation method of polymer microspheres is introduced.

[0135] In practical applications, magnetic microspheres are usually prepared by first preparing magnetic particles and then coating the magnetic particles with a silicon dioxide layer. The methods for preparing magnetic particles in related technologies include: solvent gel method, coprecipitation method, hydrothermal method, high temperature organic phase thermal decomposition method, etc. The specific process of the above methods can be referred to the introduction in the related technology, which will not be repeated here.

[0136] It is difficult for the magnetic microspheres prepared by the preparation method in the related art to completely remove the proteins in the nucleic acid sample. Some residual proteins will be adsorbed on the magnetic microspheres, resulting in problems such as low purity of the obtained nucleic acid product.

[0137] Based on this, an embodiment of the present application provides a method for preparing polymer microspheres, which is applied to prepare the polymer microspheres as described above, and the method comprises:

[0138] S801, forming magnetic particles 1;

[0139] Exemplarily, the magnetic particles 1 include a combination of one or more of iron oxide, cobalt oxide, and nickel oxide.

[0140] For example, the magnetic particles 1 may include Fe3O4 particles.

[0141] The following takes the magnetic particles 1 as Fe3O4 particles as an example to illustrate the specific preparation method:

[0142] Dissolve 0.7g FeCl3·6H2O and 1.8g anhydrous sodium acetate in 20mL ethylene glycol, add 0.5mL ethylene glycol ether and 0.5mL water, stir magnetically for 30min, mix evenly; put into a high temperature reaction box, react at 200℃ for 12h; after the reaction, cool to room temperature, wash alternately with water and ethanol until the supernatant is clear. Put into a vacuum drying box, dry at 80℃ for 12h, and obtain Fe3O4 magnetic particles.

[0143] S802, forming a modified layer 2 to obtain an intermediate particle; the modified layer 2 wraps at least one magnetic particle 1;

[0144] The following takes the group included in the modification layer 2 as a carboxyl group (-COOH) as an example to illustrate the specific preparation method of the modification layer 2:

[0145] 1) SiO2 coating: Disperse 100 mg Fe3O4 in 30 mL deionized water, transfer to a 250 mL round-bottom flask after ultrasonic dispersion for 30 min, add 120 mL anhydrous ethanol, 3.0 mL 25% ammonia water, dropwise add 2.0 mL tetraethyl orthosilicate (TEOS), and mechanically stir overnight at 30°C; the solution color changes from dark black to light gray, indicating that the SiO2 coating is successful; wash with water and ethanol alternately until the supernatant is clear; place in a vacuum drying oven and dry at 80°C for 12 h to obtain SiO2-coated Fe3O4 magnetic particles;

[0146] 2) Surface amination treatment: After the surface of the SiO2-coated Fe3O4 magnetic particles was electrostatically treated with plasma, 200 mg of the SiO2-coated Fe3O4 magnetic particles were added to 100 mL of anhydrous toluene, and then 8.0 mL of 3-aminopropyltriethoxysilane (APTES) was added under magnetic stirring, and the mixture was heated to reflux in an oil bath at 110°C for 18 h; after natural cooling, the mixture was ultrasonically washed with anhydrous ethanol for 3 times, and then separated by a magnet; the mixture was placed in a vacuum drying oven and dried at 80°C for 12 h to obtain SiO2-coated Fe3O4 magnetic particles with amino groups (-NH2) on the surface;

[0147] 3) Surface carboxylation: 150 mg of aminated SiO2-coated Fe3O4 magnetic particles were transferred to a three-necked flask, and 150 mL of N,N-dimethylformamide (DMF) was added; 300 mg of succinic anhydride was weighed and dissolved in DMF, and slowly dripped into the reaction system under mechanical stirring, at 25°C, the dripping speed was controlled to be about 60 d / min, and the reaction was stirred for 24 hours after the dripping was completed. Wash with anhydrous ethanol several times, filter; place in a vacuum drying oven, and dry at 80°C for 12 hours to obtain surface carboxylated SiO2-coated Fe3O4 magnetic particles, wherein the surface of the magnetic particles has SiO2-NH2-COOH groups.

[0148] The intermediate particles may include magnetic particles of Fe3O4 coated with SiO2 having a carboxylated surface.

[0149] S803, forming a filtration layer 3 to obtain polymer microspheres; wherein the filtration layer 3 wraps the intermediate particles, the material of the filtration layer 3 is a polymer with a network cross-linked structure, the filtration layer 3 includes a plurality of pores X, and the average size of the pores X is greater than or equal to the size of the nucleic acid molecule and smaller than the size of the protein molecule.

[0150] This does not limit the specific material of the above-mentioned filtration layer 3, but only imposes requirements on the size of the pores X on the filtration layer 3. When the pores X that meet the requirements can be prepared on the filtration layer 3, the material of the filtration layer 3 can be any material.

[0151] Exemplarily, the material of the filter layer 3 may be graphene porous gel.

[0152] Exemplarily, the material of the filtration layer 3 may be a polymer porous hydrogel.

[0153] In practical applications, nucleic acids in organisms often combine with proteins to form nucleoproteins. In the process of nucleic acid extraction and purification, this type of protein is the main interfering factor that reduces the purity of nucleic acid extraction. The protein molecules in the above "the average size of pores X is greater than or equal to the helical diameter of nucleic acid molecules and smaller than the size of protein molecules" mainly include this type of protein that combines with nucleic acids.

[0154] In at least one embodiment of the present application, the size of the pore X ranges from 50 nm to 500 nm.

[0155] The size of the pore X refers to the diameter, side length, diagonal line or length along a preset direction of the plane figure of the pore, rather than the depth of the pore X. The depth of the pore X can be determined according to the thickness of the filter layer 3.

[0156] In an embodiment of the present application, the polymer microspheres prepared by the above method have a filtration layer 3. Since the filtration layer 3 includes a plurality of pores X, the average size of the pores X is greater than or equal to the spiral diameter of the nucleic acid molecules and smaller than the size of the protein molecules. When the polymer microspheres are applied to nucleic acid purification, the modified layer 2 can adsorb the nucleic acid on the polymer microspheres through the pores X, and the filtration layer 3 excludes the residual protein molecules in the nucleic acid from the polymer microspheres, thereby improving the extraction purity and extraction efficiency of the nucleic acid, and solving the problems of residual protein contamination and low nucleic acid purity during nucleic acid extraction.

[0157] In at least one embodiment of the present application, the step of forming the filtration layer 3 in S803 to obtain polymer microspheres includes:

[0158] S8031, dissolving the intermediate particles in the first buffer solution and dispersing them evenly to obtain an intermediate particle solution;

[0159] Exemplarily, the first buffer may include a citrate buffer.

[0160] Exemplarily, the pH value of the citrate buffer is in the range of 6.2 to 7.2.

[0161] Exemplarily, ultrasonic treatment can be used for dispersion. In practical applications, the ultrasonic time can be appropriately adjusted according to the power of the ultrasonic equipment, such as 10 minutes or 15 minutes, to help disperse the intermediate particles.

[0162] S8032, dissolving the first component and the second component in a second buffer solution and prepolymerizing to obtain a prepolymer solution;

[0163] The prepolymer solution includes the prepolymer, an unreacted first component (functional monomer) and an unreacted second component (crosslinking agent).

[0164] Exemplarily, the second buffer may include a citrate buffer.

[0165] Exemplarily, the pH value of the citrate buffer is in the range of 6.2 to 7.2.

[0166] Exemplarily, the first component includes at least one of acrylamide, methacrylic acid, and N-isopropylacrylamide, and the second component includes at least one of N,N-methylenebisacrylamide, styrene, and divinylbenzene.

[0167] The following takes acrylamide as the first component and N,N-methylenebisacrylamide as the second component as an example to illustrate the prepolymerization reaction of the first component and the second component;

[0168] The functional monomer acrylamide (AAm) is dissolved in a citric acid buffer solution, and then N,N-methylenebisacrylamide (MBA) is added to obtain a mixed solution after sufficient dissolution, and the mixed solution is prepolymerized at room temperature for 0.5h to 2h, wherein the molar ratio of acrylamide (AAm) to N,N-methylenebisacrylamide (MBA) ranges from 5:1 to 1:2.

[0169] For example, Figure 5 As shown, the structure of the prepolymer obtained by the prepolymerization reaction of the first component and the second component can be

[0170] In the embodiments of the present application, by dissolving the first component and the second component in the second buffer solution and performing prepolymerization, the polymerization reaction speed and the conversion rate of the product can be increased.

[0171] S8033, mixing the prepolymer solution and the intermediate particle solution together, adding an initiator, stirring and reacting at room temperature for a preset time, and obtaining a polymer microsphere mixed solution having a filtration layer 3.

[0172] The initiator includes a first initiator and a second initiator, the first initiator includes at least one of tetramethylethylenediamine and azobisisobutyronitrile, and the second initiator includes at least one of ammonium persulfate, potassium persulfate and sodium persulfate.

[0173] Specifically, the prepolymer solution and the intermediate particle solution are mixed together, and 10% by mass of ammonium persulfate (APS) solution and tetramethylethylenediamine (TEMED) are added in sequence, and the mixture is mechanically stirred at room temperature for 24 hours to obtain a polymer microsphere mixed solution having a filter layer 3.

[0174] It should be noted that after the prepolymer solution and the intermediate particle solution are mixed together, Figure 6 As shown, the prepolymer, functional monomer and cross-linking agent can also form hydrogen bonds with the material of the modified layer 2 on the surface of the intermediate particle; on this basis, further polymerization reaction is carried out to obtain a polymer microsphere mixed liquid with a filtration layer 3. At this time, the hydrogen bonds between the material of the filtration layer 3 and the material of the modified layer 2 on the surface of the intermediate particle still exist. Figure 6 Only the hydrogen bonds between the functional monomer and the cross-linking agent and the materials of the modification layer 2 on the surface of the intermediate particle are schematically shown.

[0175] In practical applications, it is necessary to break the hydrogen bonds between the modification layer 2 and the filtration layer 3 materials.

[0176] When the second component includes N,N-methylenebisacrylamide, the first initiator includes tetramethylethylenediamine, and the second initiator includes ammonium persulfate, the molar ratio of the second component, the second initiator and the first initiator is in the range of 2:2:1 to 5:4:1, so that the polymerization reaction has better initiation efficiency and cross-linking effect, and can improve the efficiency of the polymerization reaction.

[0177] In at least one embodiment of the present application, after the step of mixing the prepolymer solution and the intermediate particle solution, adding an initiator, stirring the reaction at room temperature for a preset time, and obtaining a polymer microsphere mixed solution having a filtration layer 3, the method further comprises:

[0178] S8034, collecting polymer microspheres in the polymer microsphere mixture by magnetic attraction;

[0179] S8035, using a mixture of methanol and acetic acid to wash the polymer microspheres to remove impurities and break the hydrogen bonds between the modification layer 2 and the filter layer 3 materials;

[0180] Exemplarily, the washing time ranges from 3 h to 8 h, for example, 4 h, 5 h, 6 h, 7 h.

[0181] Figure 7 FIG. 2 shows a schematic diagram of the breaking of the hydrogen bond between the material of the filter layer 3 and the material of the modification layer 2 after washing in a mixture of methanol and acetic acid when the material of the modification layer 2 includes a carboxyl group. After the hydrogen bond between the material of the filter layer 3 and the material of the modification layer 2 is broken, the functional groups such as the carboxyl group (-COOH) occupied by the hydrogen bond on the material of the modification layer 2 can be released, so that the functional groups can adsorb nucleic acids.

[0182] In the embodiment of the present application, since the material of the modification layer 2 includes at least one of carboxyl, hydroxyl, and silanol groups, and the material of the filtration layer 3 may include acrylamide and N,N-methylenebisacrylamide, when hydrogen bonds exist between the material of the modification layer 2 and the material of the filtration layer 3, hydrogen bonds are formed between the material of the filtration layer 3 and the material of the modification layer 2, thereby reducing the effective number of groups on the modification layer 2 that can adsorb nucleic acids; in order to improve the adsorption efficiency of nucleic acids by the groups on the material of the modification layer 2, the polymer microspheres need to be treated to break the hydrogen bonds between the material of the modification layer 2 and the material of the filtration layer 3, to avoid the formation of hydrogen bonds between the material of the filtration layer 3 and the material of the modification layer 2, thereby reducing the effective number of groups on the modification layer 2 that can adsorb nucleic acids, thereby avoiding reducing the purification effect of the polymer microspheres on nucleic acids.

[0183] S8036, freeze-dried and stored.

[0184] Specifically, the eluted product was placed in a refrigerator for 20 minutes, transferred to a vacuum freeze dryer for freeze drying for 5 to 7 hours, and then stored in a refrigerator at 4°C ± 2°C for later use.

[0185] The freezing temperature may be -4°C to -24°C, for example, -10°C, -15°C, or -20°C.

[0186] In at least one embodiment of the present application, the first buffer solution and the second buffer solution both include a citric acid buffer solution, and the pH range of the citric acid buffer solution is 6 to 7.5.

[0187] Exemplarily, the pH range of the citrate buffer is 6.2-7.2, for example, the pH is 6.5, 6.8, or 7.

[0188] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A polymer microsphere, characterized in that: The polymer microspheres include: a core body, the core body comprising at least one magnetic particle; A shell encapsulating the core, wherein the shell comprises a modification layer; A filtration layer is located on a side of the shell away from the core; wherein the filtration layer comprises a plurality of pores, the average size of the pores being greater than or equal to the size of the object to be detected and smaller than the size of the pollutant.

2. The polymer microsphere according to claim 1, characterized in that The analyte includes nucleic acid molecule fragments, and the pollutants include at least protein molecules in nucleoprotein; the size of the nucleic acid molecule fragments is nanometer order, and the size of the nucleoprotein molecules is nanometer order or micrometer order.

3. The polymer microsphere according to claim 2, characterized in that The material of the filtration layer includes a high molecular polymer with a network cross-linking structure formed by a first component, a second component and an initiator.

4. The polymer microsphere according to claim 3, characterized in that: The first component includes at least one of acrylamide, methacrylic acid, and N-isopropylacrylamide, and the second component includes at least one of N,N-methylenebisacrylamide, styrene, and divinylbenzene; the initiator includes a first initiator and a second initiator, the first initiator includes at least one of tetramethylethylenediamine and azobisisobutyronitrile, and the second initiator includes at least one of ammonium persulfate, potassium persulfate, and sodium persulfate.

5. The polymer microsphere according to claim 2, characterized in that: The material of the modification layer includes at least one group selected from the group consisting of carboxyl, hydroxyl and silanol.

6. The polymer microsphere according to claim 5, characterized in that Some groups in the material of the modification layer extend into the pores.

7. The polymer microsphere according to claim 2, characterized in that: The size of the pores ranges from 50 nm to 500 nm.

8. The polymer microsphere according to claim 4, characterized in that The first component includes acrylamide, the second component includes N,N-methylenebisacrylamide, and the molar ratio of the first component to the second component ranges from 5:1 to 1:

2.

9. The polymer microsphere according to claim 4, characterized in that The second component includes N,N-methylenebisacrylamide, and the molar ratio of the second component to the magnetic particles is in the range of 1:8 to 1:

2.

10. The polymer microsphere according to claim 4, characterized in that: The second component includes N,N-methylenebisacrylamide, the first initiator includes tetramethylethylenediamine, and the second initiator includes ammonium persulfate; The molar ratio of the second component, the second initiator and the first initiator is in the range of 2:2:1 to 5:4:

1.

11. The polymer microsphere according to claim 1, characterized in that: The magnetic particles include one or more of iron oxide, cobalt oxide, and nickel oxide, and the size of the magnetic particles ranges from 50 nm to 500 nm.

12. The polymer microsphere according to any one of claims 1 to 11, characterized in that Hydrogen bonds exist in a partial region between the material of the modification layer and the material of the filtration layer.

13. The polymer microsphere according to any one of claims 1 to 11, characterized in that The size of the polymer microspheres ranges from 1 μm to 2 μm.

14. A method for preparing polymer microspheres, characterized in that: Applicable to preparing polymer microspheres according to any one of claims 1 to 13, the method comprising: forming magnetic particles; forming a modified layer to obtain an intermediate particle; the modified layer wraps at least one of the magnetic particles; A filtration layer is formed to obtain the polymer microspheres; wherein the filtration layer wraps the intermediate particles, the material of the filtration layer is a polymer with a network cross-linked structure, the filtration layer includes a plurality of pores, and the average size of the pores is greater than or equal to the size of the object to be detected and smaller than the size of the pollutant.

15. The preparation method according to claim 14, characterized in that: The step of forming a filtration layer to obtain the polymer microspheres comprises: Dissolving the intermediate particles in a first buffer solution and dispersing them evenly to obtain an intermediate particle solution; dissolving the first component and the second component in a second buffer solution and prepolymerizing the components to obtain a prepolymer solution; The prepolymer solution and the intermediate particle solution are mixed together, an initiator is added, and the mixture is stirred at room temperature for a preset time to obtain the polymer microsphere mixed solution having the filter layer.

16. The preparation method according to claim 15, characterized in that: After the step of mixing the prepolymer solution and the intermediate particle solution together, adding an initiator, and stirring the reaction at room temperature for a preset time to obtain the polymer microsphere mixed solution having the filter layer, the method further comprises: Collecting the polymer microspheres in the polymer microsphere mixture by magnetic attraction; Using a mixture of methanol and acetic acid to wash the polymer microspheres to remove impurities and break the hydrogen bonds between the modification layer and the filter layer material; Freeze-dry and store.

Images