Adsorption carrier for extracting free DNA in sample, preparation method and method for extracting free DNA in sample

Abstract

The embodiment of the invention provides an adsorption carrier for extracting free DNA in a sample, a preparation method of the adsorption carrier and a method for extracting the free DNA in the sample. The adsorption carrier comprises a carrier matrix and a molecularly imprinted polymer layer located on at least part of the surface of the carrier matrix, the molecularly imprinted polymer layer has a nucleosome specific binding site; the nucleosome specific binding site has a shape matched with that of a nucleosome histone. Compared with the prior art, the method has at least one of the following beneficial effects: the method can be specifically combined with nucleosome histone, the extraction efficiency of free DNA is improved, the pollution of genome DNA to free DNA is reduced, the method can be repeatedly utilized, and the cost is saved.

Description

Technical Field

[0001] The present application relates to the field of nucleic acid extraction, and in particular to an adsorption carrier for extracting free DNA from a sample, a preparation method thereof, and a method for extracting free DNA from a sample. Background Art

[0002] Cell-free DNA (cfDNA) is a type of DNA that is free from cells in the blood or other body fluids, and often exists in the form of nucleosomes in the blood or other body fluids. The source of free DNA in body fluids is related to cell apoptosis, necrosis, and active secretion of DNA by cells. Under physiological conditions, free DNA in the blood mainly comes from necrosis and apoptosis of white blood cells. In certain diseases and special conditions, other cells will also release free DNA, including circulating tumor DNA (ctDNA) from tumor cells and free DNA from the fetus. With the continuous emergence of new research results, the potential value of free DNA in early diagnosis of diseases, prognosis assessment, and efficacy monitoring has received more and more attention. Detection of free DNA in plasma or serum can be regarded as a "liquid biopsy", which is conducive to various related diagnoses and can reduce invasive tissue biopsies.

[0003] However, current methods for extracting free DNA from samples still need to be improved. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide an adsorption carrier for extracting free DNA in a sample, a preparation method and a method for extracting free DNA in a sample, so as to at least alleviate or even solve at least one of the problems raised in the above background technology to a certain extent.

[0005] In one aspect of the present invention, the present invention provides an adsorption carrier for extracting free DNA in a sample, the adsorption carrier comprising a carrier matrix, and a molecular imprinting polymer layer located on at least a portion of the surface of the carrier matrix; the molecular imprinting polymer layer has a nucleosome-specific binding site; the nucleosome-specific binding site has a morphology that matches that of a nucleosome histone.

[0006] Furthermore, the molecular imprinting polymer layer covers the surface of the carrier matrix, and the nucleosome histones include core histones.

[0007] Furthermore, the molecular imprinting polymer layer includes a functional polymer, and the functional polymer includes an acrylic copolymer.

[0008] Furthermore, the molecular imprinting polymer layer is formed by polymerizing the functional monomers in a medium containing a template protein and then eluting the template protein.

[0009] Furthermore, the carrier matrix includes magnetic particles, and the surface of the magnetic particles has at least one of a protecting group, a protein binding group and a functional polymer binding group.

[0010] Furthermore, the protecting group includes a siloxane group, the protein binding group includes at least one of a hydroxyl group, a carboxyl group and an amino group, and the functional polymer binding group includes an olefinic group.

[0011] In another aspect of the present invention, the present invention provides a method for extracting free DNA from a sample, the method comprising: using the above-mentioned adsorption carrier to adsorb the sample to obtain a nucleosome-adsorption carrier conjugate; extracting the nucleosome-adsorption carrier conjugate to obtain the free DNA.

[0012] Furthermore, the extraction process includes: eluting the nucleosome-adsorption carrier conjugate to obtain a nucleosome elution solution; performing protein degradation on the nucleosome elution solution to obtain a protein degradation solution containing the free DNA; and performing DNA separation on the protein degradation solution to obtain the free DNA.

[0013] Furthermore, the adsorption treatment includes adding the adsorption carrier to the sample and reacting at a temperature of 4-60° C. for 0.5-24 hours.

[0014] Furthermore, the elution treatment includes treating the collected nucleosome-adsorption carrier complex with an elution reagent to elute the nucleosome.

[0015] Furthermore, the treatment temperature of the elution treatment is 4-60°C, and the treatment time is 0.5-24h.

[0016] Furthermore, the protein degradation treatment includes treating the nucleosome eluate with a degradation agent to degrade histones in the nucleosomes and release the free DNA.

[0017] Furthermore, the protein degradation treatment has a treatment temperature of 25-70°C and a treatment time of 5-60 minutes.

[0018] In another aspect of the present invention, the present invention provides a method for preparing the above-mentioned adsorption carrier, the method comprising the steps of preparing a carrier matrix and preparing a molecular imprinting polymer layer.

[0019] Furthermore, the preparation of the carrier matrix includes the steps of preparing magnetic particles and optionally introducing at least one of a protecting group, a protein binding group and a functional polymer binding group on the surface of the magnetic particles.

[0020] Furthermore, the protecting group includes a siloxane group; the protein binding group includes at least one of a hydroxyl group, a carboxyl group and an amino group; and the functional polymer binding group includes an alkenyl group.

[0021] Furthermore, the step of preparing the molecularly imprinted polymer layer includes: adding functional monomers to the carrier matrix and adding template proteins to mix, allowing the functional monomers to undergo polymerization reaction, and forming a pre-assembled layer containing the template protein on the surface of the carrier matrix; performing a second elution treatment on the pre-assembled layer to remove the template protein, and obtaining the molecularly imprinted polymer layer;

[0022] Furthermore, the template protein includes nucleosome histones, and causing the functional monomers to undergo polymerization reaction includes causing the functional monomers to undergo polymerization in the presence of a cross-linking agent and an initiator.

[0023] Furthermore, the functional monomer includes at least one of an acrylic compound and an unsaturated dibasic organic acid.

[0024] Furthermore, the cross-linking agent includes at least one of N,N'-methylenebisacrylamide, vinyl dimethacrylate and ethylene glycol dimethacrylate.

[0025] Furthermore, the initiator includes at least one of ammonium persulfate and tetramethylethylenediamine.

[0026] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present application will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the multiple drawings represent the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings only depict some embodiments disclosed in the present application and should not be regarded as limiting the scope of the present application.

[0028] Figure 1 This is a schematic diagram of the structure of an adsorption carrier for extracting free DNA from a sample according to an embodiment of the present invention;

[0029] Figure 2 A flow chart of a method for extracting free DNA from a sample according to an embodiment of the present invention;

[0030] Figure 3 A flow chart of a method for preparing an adsorption carrier according to an embodiment of the present invention;

[0031] Figure 4 A schematic diagram of introducing protective groups, protein binding groups and functional polymer binding groups on the surface of magnetic particles according to an embodiment of the present invention;

[0032] Figure 5 A schematic diagram of preparing a molecular imprinting polymer layer on the surface of a carrier substrate according to an embodiment of the present invention;

[0033] Figure 6 This is a result diagram of the performance of the adsorption carrier in different environments in Example 1 of the present invention;

[0034] Figure 7 This is a comparison diagram of the extraction effect of free DNA by the adsorption carrier of Example 2 of the present invention;

[0035] Figure 8 This is a graph showing the variation of the amount of nucleosome histone adsorption by the adsorption carrier of Example 3 of the present invention over time.

[0036] Reference numerals: 100 - carrier matrix, 200 - molecularly imprinted polymer layer, 210 - nucleosome-specific binding site. DETAILED DESCRIPTION

[0037] In order to more clearly understand the technical features, purposes and beneficial effects of the present invention, the technical scheme of the present invention is now further described in detail. Hereinafter, only some exemplary embodiments are simply described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present application. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0038] In one aspect of the present invention, the present invention provides an adsorption carrier for extracting free DNA from a sample. Figure 1 The adsorption carrier includes a carrier matrix 100 and a molecular imprinting polymer layer 200 located on at least part of the surface of the carrier matrix 100. The molecular imprinting polymer layer 200 has a nucleosome-specific binding site 210, and the nucleosome-specific binding site 210 has a morphology that matches the nucleosome histone. Specifically, the adsorption carrier has a nucleosome-specific binding site 210 whose morphology, size, and functional groups match those of the nucleosome histone, and can specifically adsorb histones, thereby extracting free DNA from the sample. The adsorption carrier has at least one of the following beneficial effects: it can specifically bind to nucleosome histones, improve the efficiency of free DNA extraction, reduce the contamination of free DNA by genomic DNA, is reusable, and saves costs.

[0039] For ease of understanding, the following first briefly explains the principle by which the adsorption carrier can achieve the above beneficial effects:

[0040] As mentioned above, free DNA often exists in body fluids in the form of nucleosomes. Nucleosomes are the basic components of chromosomes, consisting of 8 core histones (including diploid histones H2A, H2B, H3 and H4) and about 146 bp of DNA wrapped around them. Another histone H1 is located outside the core histones and connects about 20 bp of DNA, so a nucleosome contains about 166 bp of DNA. Since the free DNA in blood or other body fluids is very small, different sampling methods and DNA degradation are one of the main obstacles to current research. The current extraction method cannot effectively obtain trace amounts of free DNA, the collection operation is difficult, the loss is large, and the test sensitivity is low. Taking blood samples as an example, ordinary EDTA tubes for blood collection or improper processing methods are prone to hemolysis or white blood cell residues, which may cause genomic DNA to contaminate free DNA and interfere with subsequent detection. The current solution to deal with genomic DNA contamination in free DNA is to use purified magnetic beads for secondary sorting to separate and select small fragments of DNA. However, this method has a large loss of free DNA, a low recovery rate, and is not conducive to downstream experimental operations.

[0041] The adsorption carrier of the present application uses nucleosome histone as a template, forms a molecular imprinting polymer layer 200 having a nucleosome histone structure on at least part of the surface of the carrier matrix 100, and obtains many "holes" whose three-dimensional shapes, sizes and functional groups match those of the template protein nucleosome histone, namely nucleosome-specific binding sites 210. The sites can selectively adsorb free nucleosomes, thereby separating free DNA and genomic DNA.

[0042] According to an embodiment of the present invention, in the adsorption carrier, the shape of the carrier matrix 100 is not particularly limited, and can be a plane geometry, a curved geometry or a combination thereof. The thickness and shape of the molecular imprinting polymer layer 200 are also not particularly limited, the local thickness can be the same or different, and the position and number of nucleosome-specific binding sites 210 on the molecular imprinting polymer layer 200 are not particularly limited. In a specific embodiment, the molecular imprinting polymer layer 200 can cover most of the surface of the carrier matrix 100 and wrap the carrier matrix 100 inside it. Therefore, the shape of the carrier matrix 100 is also not particularly limited, because its shape is determined by both the shape of the carrier matrix 100 and the shape of the molecular imprinting polymer layer 200. In other words, the shape of the adsorption carrier of the present invention includes but is not limited to Figure 1 The shape shown in .

[0043] In the present application, the term "covering most of the surface of the carrier substrate 100" should be understood in a broad sense. Most of the surface may be covering more than 50% of the surface of the carrier substrate 100, such as more than 50%, more than 60%, more than 70%, more than 80%, or even more than 90%. That is, the surface area of ​​the carrier substrate 100 covered by the molecularly imprinted polymer layer 200 may account for a proportion of the surface area of ​​the carrier substrate 100 that is greater than the above values.

[0044] According to an embodiment of the present invention, the nucleosome-specific binding site 210 refers to a site that specifically binds to nucleosome histones. The nucleosome histones referred to in the present application include core histones, such as core histones H2A, H2B, H3, H4, and at least one of histones H1 outside the core histones. For example, it may specifically include a core histone octamer formed by H2A diploid, H2B diploid, H3 diploid, and H4 diploid. Thus, the formed nucleosome-specific binding site 210 can better bind to nucleosome histones.

[0045] According to an embodiment of the present invention, the molecular imprinting polymer layer 200 includes a functional polymer. The functional polymer can be formed by polymerizing a functional monomer. In a specific embodiment, the type of the functional polymer is not particularly limited, for example, it can include a copolymer formed by polymerizing a functional monomer, wherein the functional monomer can include at least one of an acrylic compound and an unsaturated dibasic organic acid. Thus, on the one hand, a functional polymer with a stable structure can be formed to improve the stability of the molecular imprinting polymer layer 200, and on the other hand, the template protein can be easily embedded in it while forming the functional polymer, so as to form the aforementioned nucleosome-specific binding site 210 through subsequent operations. Specifically, in the process of forming the molecular imprinting polymer layer 200, the functional monomer can first be polymerized in a medium containing the template protein, and the template protein can be wrapped in it during the process of forming the functional polymer, and then the template protein can be washed off to form many specific binding sites whose three-dimensional shapes, sizes and functional groups match those of the template protein, and finally a molecular imprinting polymer layer 200 that can specifically bind to nucleosome histones is obtained.

[0046] According to an embodiment of the present invention, the type of the carrier matrix 100 is not particularly limited, for example, it includes magnetic particles. The carrier matrix 100 formed by magnetic particles can be more convenient to enrich the adsorbed carrier by magnetism. In a specific embodiment, the surface of the magnetic particles may have at least one of a protective group, a protein binding group and a functional polymer binding group. Among them, the type of the protective group is not particularly limited, as long as the magnetic particles can be protected so that they are not easy to agglomerate with each other and prevent the magnetic particles from being oxidized. For example, the protective group includes a siloxane group, more specifically, an O-Si-O group. The type of the protein binding group is not particularly limited, as long as it can react with hydroxyl, carboxyl, amino and other active groups on the surface of the protein to form hydrogen bonds. For example, the protein binding group includes at least one of a hydroxyl, carboxyl and amino group. In addition, the functional polymer binding group is also not particularly limited, as long as it can be combined with the functional polymer in the molecular imprinting polymer layer 200 to form a stable covalent bond, for example, it can include an unsaturated group, for example, including but not limited to an olefin group.

[0047] That is to say, the carrier matrix 100 of the present invention utilizes the protective groups on its surface to protect the magnetic particles, making them less likely to agglomerate with each other and preventing the magnetic particles from being oxidized; utilizes the protein binding groups on its surface to form hydrogen bonds with the active groups on the protein surface and connect to the protein template to facilitate the subsequent elution of the protein template; and utilizes the functional polymer binding groups to react with the functional polymer to form covalent bonds, thereby forming a molecular imprinting polymerization layer 200 around the protein template that matches its structure and morphology.

[0048] According to an embodiment of the present invention, the adsorption carrier can be used for, including but not limited to, extracting free DNA in a sample, and can also be used to extract other proteins or protein conjugates in the sample. For example, other types of proteins can be used as template proteins to form a molecular imprinting polymer layer 200 that can specifically bind to the protein, thereby achieving the extraction of the protein.

[0049] In another aspect of the present invention, the present invention provides a method for extracting free DNA from a sample. Figure 2 , the method comprises: a step of performing adsorption treatment on the sample using an adsorption carrier to obtain a nucleosome-adsorption carrier conjugate; and a step of performing extraction treatment on the nucleosome-adsorption carrier conjugate to obtain free DNA. The method uses the adsorption carrier of the present invention to perform specific adsorption on the nucleosomes in the sample, achieves the enrichment of nucleosomes, obtains the nucleosome-adsorption carrier conjugate, thereby achieving the enrichment of free DNA, and fully separating the free DNA from the genomic DNA in the sample; free DNA can be extracted by treating the nucleosome-adsorption carrier conjugate, thereby reducing the contamination of free DNA by genomic DNA and improving the efficiency of extracting free DNA.

[0050] According to an embodiment of the present invention, the type of "sample" mentioned in the present application is not particularly limited, as long as it contains free DNA. In a specific embodiment, the "sample" may specifically include body fluid samples, such as but not limited to plasma, cerebrospinal fluid, gastric juice, digestive juice, semen, saliva, tears, sweat, urine and other body fluid samples. And in a specific embodiment, in order to improve the extraction efficiency of free DNA in the sample, the sample can be appropriately diluted according to its initial concentration so that its concentration is maintained at a level suitable for free DNA extraction. And the solution used for dilution is not particularly limited, and a buffer suitable for dilution can be selected specifically according to the type of sample, such as but not limited to at least one of PBS buffer, MES buffer and Tris buffer.

[0051] According to an embodiment of the present invention, in the method of the present invention, the extraction treatment includes firstly eluting the nucleosome-adsorption carrier conjugate to destroy the hydrogen bond formed between the nucleosome histone and the adsorption carrier, so that the nucleosome is separated from the adsorption carrier, thereby obtaining a nucleosome eluate rich in nucleosomes. Since the nucleosome includes histones and free DNA wrapped around the histones, after obtaining the nucleosome eluate, it further includes protein degradation treatment to degrade the nucleosome histones to release the free DNA and obtain a protein degradation solution containing free DNA; finally, the obtained protein degradation solution is subjected to DNA separation treatment to obtain free DNA.

[0052] According to an embodiment of the present invention, the adsorption treatment includes adding the adsorption carrier of the present invention to the sample and mixing it evenly, and adsorbing the nucleosome histone. The temperature of the adsorption treatment is not subject to special restrictions, for example, it can be 4-35°C; the treatment time is not subject to special restrictions, for example, it can be not less than 1h, more specifically 1-5h. In addition, the content of the adsorption carrier and the sample content used for the adsorption treatment are not subject to special restrictions, for example, the ratio of the mass of the adsorption carrier to the sample volume can be (2-8) mg: (0-3) mL; and in a specific embodiment, after the adsorption carrier is added to the sample, the two can be fully mixed by vortexing to obtain an adsorption reaction liquid, and then the adsorption treatment is carried out under oscillation conditions to increase the adsorption efficiency.

[0053] According to an embodiment of the present invention, the method of the present invention further includes a step of enriching the nucleosome-adsorption carrier conjugate obtained by the above-mentioned adsorption treatment. Specifically, after the adsorption treatment is completed, the adsorption reaction liquid can be placed in an external magnetic field, and the nucleosome-adsorption carrier conjugate can be aggregated together by magnetic attraction, and the supernatant can be discarded, and the enriched nucleosome-adsorption carrier conjugate can be taken out from the external magnetic field, and a cleaning reagent is added thereto for cleaning treatment, and then the residual reaction liquid is removed by using the external magnetic field to obtain a clean nucleosome-adsorption carrier conjugate. Among them, the type of cleaning reagent is not particularly limited, and preferably includes an inorganic salt solution, such as at least one of sodium salt, potassium salt, calcium salt, phosphate salt and magnesium salt. And during the cleaning process, the number of treatments is not particularly limited, and can be performed once or multiple times. The amount of cleaning reagent added in each cleaning treatment is also not particularly limited, as long as the nucleosome-adsorption carrier conjugate is immersed.

[0054] According to an embodiment of the present invention, the method further includes the step of eluting the clean nucleosome-adsorption carrier conjugate obtained above to elute the nucleosomes. Specifically, the elution treatment includes treatment with an elution reagent, wherein the treatment time and temperature are not particularly limited, for example, the treatment temperature can be 4-60°C, and the treatment time can be 0.5-24h. In a specific embodiment, after adding the elution reagent to the clean nucleosome-adsorption carrier conjugate, the two can be mixed evenly by vortexing to obtain an elution reaction liquid, and then the elution treatment is performed under oscillation. After the elution treatment is completed, the elution reaction liquid can be placed in an external magnetic field to absorb and retain the nucleosome elution liquid containing nucleosomes. Among them, the components of the elution reagent are not particularly limited, as long as there are compounds that can break intramolecular and intermolecular hydrogen bonds, cause the molecules to unfold and destroy the secondary structure and tertiary structure of the protein molecule. For example, the elution reagent can include at least one of sodium dodecyl sulfate, lithium dodecyl sulfate and other protein denaturants. Furthermore, during the elution process, the number of treatments is not particularly limited and can be performed once or multiple times. The amount of elution reagent added in each elution treatment is also not particularly limited, as long as the nucleosome-adsorption carrier complex is immersed.

[0055] According to an embodiment of the present invention, the method further includes performing protein degradation treatment on the nucleosome eluate obtained by the above elution treatment to degrade the histones in the nucleosomes and release the free DNA. Specifically, the protein degradation treatment includes treatment with a degradation reagent, wherein the temperature and time of the treatment are not particularly limited, for example, the treatment temperature can be 25-70°C, more specifically 30-60°C; the treatment time can be 5-60min, more specifically 20-40min. In a specific embodiment, the degradation reagent can be added to the nucleosome eluate, and the two can be mixed evenly by vortexing to obtain a degradation reaction solution, and the mixture can be inverted and mixed several times during the treatment process. After the reaction is completed, the reaction solution can be placed at room temperature to restore its temperature to room temperature to obtain a protein degradation solution containing free DNA. Among them, the components of the degradation reagent are not particularly limited, as long as they have components that can degrade proteins. For example, it can include proteases, such as proteinases; more specifically, it includes proteinase K. In addition, during the protein degradation treatment, the number of treatments is not particularly limited, and can be performed once or multiple times, for example, once. The amount of degradation reagent added in each treatment is not particularly limited and can be determined based on the volume of the obtained nucleosome eluate. Specifically, the ratio of the volume of the nucleosome eluate to the volume of the degradation reagent can be (80-120):(3-8).

[0056] According to an embodiment of the present invention, the method further comprises a step of carrying out DNA separation treatment on the protein degradation solution, and separating the free DNA. Specifically, the method of DNA separation treatment is not particularly limited, and any method of separating DNA in the prior art can be used, for example, including but not limited to at least one of boiling method, salting-out method, phenol-chloroform method, isopropanol precipitation method, centrifugal column method and magnetic bead method. And the specific operation steps of the above method are not particularly limited, and can be carried out using commercially available kits. In addition, since the method of the present application is to separate free DNA from protein lysate, in a specific embodiment, preferably magnetic bead method is used to separate free DNA, to reduce the introduction of other chemical reagents, save costs and improve the separation efficiency of free DNA.

[0057] In another aspect of the present invention, the present invention also provides a method for preparing the above-mentioned adsorption carrier. Figure 3 The method includes the steps of preparing a carrier matrix and preparing a molecular imprinting polymer layer on the surface of the carrier matrix. The adsorption carrier prepared by the method has a specific binding site for nucleosome histones on the molecular imprinting polymer layer, which can specifically extract nucleosomes, thereby extracting trace amounts of free DNA in the sample and reducing genomic DNA contamination.

[0058] According to an embodiment of the present invention, the preparation of the carrier matrix includes the step of preparing magnetic particles so as to enrich the carrier matrix by magnetic attraction, and preferably the step of introducing protective groups, protein binding groups, functional polymer binding groups or a combination thereof on the surface of the prepared magnetic particles so as to enable the protein and the functional monomer to be connected to the carrier matrix. Specifically, the type of magnetic particles is not particularly limited, as long as they are magnetic, and the magnetic particles include magnetic iron oxide particles, such as ferroferric oxide (Fe3O4). In addition, the particle size, shape and composition of the magnetic particles are not particularly limited, and can be determined specifically according to the type of reagents used in the preparation process, pH value, ionic strength, etc. In addition, the method for preparing magnetic particles is not particularly limited, and the specific preparation methods that can be used include but are not limited to at least one of coprecipitation synthesis method, microemulsion method and thermal decomposition method. For example, the coprecipitation synthesis method can be used to prepare magnetic particles.

[0059] In some specific embodiments, in order to prevent oxidation of the magnetic particles, the preparation process may be carried out under anaerobic conditions, and an inert gas may be introduced during the reaction to reduce the probability of oxidation reaction.

[0060] Specifically, the coprecipitation synthesis method includes 2+ and Fe 3+ Adding alkali solution to the salt solution causes the two to coprecipitate and synthesize iron oxide (Fe3O4, γ-Fe2O3 or a combination thereof). The particle size, shape and composition of the magnetic particles obtained by this method depend on the type of salt used (e.g. chloride, sulfate, nitrate, perchlorate, etc.). 2+ and Fe 3+ In a specific embodiment, the particle size, shape and composition of the magnetic particles do not affect the function of the carrier matrix prepared by the present invention. However, in a specific embodiment, the Fe 2+ and Fe 3+ The molar ratio is (0.5-1.5): (0.5-3), thereby synthesizing particles with better particle size and magnetic properties. In addition, the type of alkali solution used is not particularly limited, and can be at least one of a weak base or a strong base. And in a specific embodiment, ammonia water can be used as an alkali solution to adjust the pH value of the reaction material to a pH value greater than 7, preferably to a pH value of 9-11.

[0061] In one embodiment of the present invention, 0.7-1g FeSO4·7H2O and 0.9-1.5g FeCl3·6H2O can be weighed, 150-300mL solvent is added thereto, the reaction is stirred at 50-75°C for 15-60min, and an inert gas is continuously introduced during the reaction to remove oxygen in the reaction mass. The type of inert gas is subject to special restrictions, including but not limited to at least one of helium, neon, argon, and nitrogen. Then, alkali solution is added to the reaction mass to adjust the pH value of the reaction mass to greater than 7 and stop, to obtain magnetic particles. Among them, the type of solvent used is not particularly limited, and can be at least one of anhydrous ethanol and deionized water. For example, the solvent can include a mixture of anhydrous ethanol and deionized water. And the stirring method is not particularly limited, and can be manually stirred or stirred using a stirrer, and the stirring speed is not particularly limited, and can be 300-1200rpm / min.

[0062] The types of protecting groups, protein binding groups, and functional polymer binding groups have been described in detail above and will not be repeated here.

[0063] According to an embodiment of the present invention, the step of introducing a protective group, a protein binding group, a functional polymer binding group or a combination thereof on the surface of the prepared magnetic particles comprises adding a compound containing a protective group, a compound containing a protein binding group, a compound containing a functional polymer binding group or a combination thereof to the magnetic particles prepared above, and the order and amount of addition of each compound are not particularly limited. In a specific embodiment, the compound containing a protective group includes but is not limited to a compound containing a siloxane group, specifically including tetraethyl orthosilicate; the compound containing a protein binding group includes but is not limited to a compound containing a hydroxyl group, a carboxyl group, an amino group or a combination thereof, specifically including olefinic acid, such as acrylic acid; the compound containing a functional polymer binding group includes but is not limited to a compound containing an olefinic group, specifically including olefinic acid, such as acrylic acid. Therefore, in a specific embodiment, a compound having both a protein binding group and a functional polymer binding group can be used, so that the protein binding group and the functional polymer binding group can be introduced simultaneously by adding one compound.

[0064] In a specific example, refer to Figure 4, showing the steps of introducing protective groups, protein binding groups and functional polymer binding groups on the surface of magnetic particles. Specifically, by adding tetraethyl orthosilicate (TEOS), O-Si-O groups are introduced on the surface of magnetic particles to protect the magnetic particles, making them less likely to agglomerate with each other and preventing the particles from oxidizing. By adding acrylic acid (AA), vinyl and carboxyl groups are introduced at the same time to provide binding sites for subsequent reactions with functional monomers and template proteins, so that when the template protein is added for pre-assembly, the active groups such as -OH, -COOH and -NH2 on the surface of the protein form hydrogen bonds with -OH and -COOH on the surface of the magnetic particles, and are temporarily fixed on the surface of the magnetic particles.

[0065] In one embodiment of the present invention, after the magnetic particles are prepared, a compound containing a protective group can be first added thereto, and the reaction can be carried out under stirring conditions, the reaction time includes 1-5h, and the stirring speed is not particularly limited, and can be 300-1200rpm / min; then the above reaction solution is placed in an external magnetic field, the supernatant is discarded, the magnetic particles are taken out from the external magnetic field, and the obtained magnetic particles are washed once or multiple times with deionized water; then the compound containing a protein binding group and a functional polymer binding group are added to the washed magnetic particles, and the reaction is stirred for 1-5h at a temperature of 20-60°C, and an inert gas is continuously introduced during the reaction, and the stirring speed is not particularly limited, and can be 300-1200rpm / min. In a specific embodiment, after the above reactions are completed, the obtained magnetic particles can be washed once or multiple times with at least one of deionized water and anhydrous ethanol until the pH value of the supernatant in the solution is neutral. In addition, it is more preferred that the finally obtained magnetic particles are vacuum concentrated and dried to obtain a carrier matrix that can be used for subsequent treatment.

[0066] According to an embodiment of the present invention, the step of preparing a molecular imprinting polymer layer on the surface of a carrier matrix includes adding a functional monomer to the carrier matrix prepared above, adding a template protein to mix, allowing the template protein to react with the protein binding group on the carrier matrix and connect through hydrogen bonds; allowing a covalent bond to form between the functional monomer and the functional polymer binding group on the carrier matrix, and a polymerization reaction also occurs between the functional monomers to form a functional polymer. While forming the functional polymer, the template protein is wrapped inside, and finally a pre-assembled layer containing the template protein is formed on the surface of the carrier matrix; the pre-assembled layer is subjected to a second elution treatment to elute the template protein connected to the carrier matrix, thereby obtaining a molecular imprinting polymer layer.

[0067] According to an embodiment of the present invention, the template protein used in preparing the carrier matrix includes nucleosome histones, so that the prepared molecular imprinting polymer layer has nucleosome specific binding sites that can specifically match the nucleosome histones.

[0068] In addition, in a specific embodiment, the polymerization reaction between the functional monomers includes the polymerization reaction in the presence of a crosslinking agent and an initiator. That is to say, when the functional monomer and the template protein are added to the carrier matrix, the functional monomer and the template protein can be connected to the carrier matrix, but the polymerization reaction may not occur. Then, after adding the crosslinking agent and the initiator to the reactant, polymerization can be ordered between the functional monomers under the catalysis of the two to form a stable functional polymer, and the template protein is wrapped in the formation process. And the order of adding the functional monomer, the template protein, the crosslinking agent and the initiator is not particularly limited, but in a specific embodiment, the occurrence of the polymerization reaction can be controlled by adding the initiator at the end. In addition, the types of the functional monomers, the crosslinking agent and the initiator in the present invention are not particularly limited. Specifically, the type of the functional monomer can be determined according to the type of the group introduced on the surface of the carrier matrix, specifically including at least one of an acrylic compound and an unsaturated dibasic organic acid, wherein the acrylic compound includes methacrylic acid, and the dibasic organic acid includes itaconic acid. The type of crosslinking agent specifically includes at least one of N,N'-methylenebisacrylamide, vinyl dimethacrylate and ethylene glycol dimethacrylate, and more specifically includes N,N'-methylenebisacrylamide. The type of initiator can be determined according to the type of reaction between the selected reactive monomer and the crosslinking agent, and specifically includes at least one of ammonium persulfate and tetramethylethylenediamine. In a specific embodiment, the amount of functional monomer, crosslinking agent, template protein and initiator added is not particularly limited. In some examples, the mass ratio of the carrier matrix: template protein can be (30-100): (5-20). When the mass ratio of the carrier matrix and the template protein is within the above range, the number of nucleosome-specific binding sites in the synthesized molecular imprinting polymer layer is moderate, the molecular imprinting polymer layer can cover most of the surface of the carrier matrix, and a relatively stable binding force can be formed between the molecular imprinting polymer layer and the carrier matrix.

[0069] refer to Figure 5, showing the steps of preparing a molecular imprinting polymer layer on the surface of a carrier matrix. Specifically, the surface of the carrier matrix can be first modified to form a protecting group, a protein binding group and a functional polymer binding group, and then the template protein is added to the above-mentioned carrier matrix. After adding a functional monomer, a cross-linking agent and an initiator to the system, the functional monomer undergoes a polymerization reaction, and the reaction that occurs simultaneously also includes the reaction of the functional monomer, the template protein and the unsaturated groups on the surface of the carrier matrix. As the polymerization reaction of the functional protein and the reaction of the template protein and the surface of the carrier matrix proceed, the template protein can be wrapped inside the polymer formed by the functional monomer, and a pre-assembled layer containing the template protein is formed on the surface of the carrier matrix. After the template protein in the pre-assembled layer is washed clean with an eluent, many holes whose three-dimensional shape, size and functional groups match those of the template protein are obtained, thereby obtaining a molecular imprinting polymer layer.

[0070] In a specific embodiment, the functional monomer and the cross-linking agent can be first added to the carrier matrix prepared above, and the PBS buffer is used as the reaction medium, and the above reactants are fully shaken and mixed. Specifically, ultrasonic oscillation can be used to mix for 5-20 minutes. Then, the template protein is added to the above mixture and mixed evenly, and the reaction is stirred and shaken at a temperature of 20-35°C for 0.5-3h. The stirring speed in this step is not particularly limited, and can be 100-600rpm / min, and an inert gas is continuously introduced during the reaction. After the reaction is completed, an initiator is added and stirred and shaken at a temperature of 15-35°C overnight. The stirring speed is not particularly limited and can be 100-600rpm / min. Then, the above-mentioned overnight reaction material is placed in an external magnetic field, the supernatant is discarded, and sodium dodecyl sulfate (SDS) solution is used as an eluent to shake and elute the template protein on the carrier matrix. The end point of elution can be determined according to actual needs, for example, until the supernatant has no characteristic absorption detected at 350-480nm, specifically no characteristic absorption peak detected at 390-430nm. Then, the reaction solution is placed in an external magnetic field, the supernatant is discarded, and the adsorption carrier of the present invention is obtained after drying at room temperature.

[0071] In some examples, the functional monomer may be a mixture of methacrylic acid and itaconic acid, and the ratio of methacrylic acid to itaconic acid is preferably (5-10) μL: (1-5) mg. The crosslinking agent may be N,N'-methylenebisacrylamide; the initiator may include ammonium persulfate and tetramethylethylenediamine, and the ratio of ammonium persulfate to tetramethylethylenediamine is (5-8) μL: (1-2) μL. When the ratio of ammonium sulfate to tetramethylethylenediamine is within the above range, the polymerization reaction can be carried out more completely.

[0072] In addition, in a specific embodiment, the adsorption carrier obtained after drying at room temperature can be added to a storage buffer for storage. And the concentration of the adsorption carrier in the mixture of the adsorption carrier and the storage buffer can be calculated by weighing the adsorption carrier, adding the adsorption carrier with a known weight to the storage buffer with a known volume, so as to facilitate weighing the adsorption carrier for subsequent experiments. In a specific embodiment, the components of the storage buffer are not particularly limited, as long as they can isolate external oxygen and prevent the adsorption carrier from reacting. For example, the storage buffer components include Tris buffer and TritonX-100.

[0073] Example

[0074] The method proposed in the present invention is described in detail below through specific examples. The methods used in the following examples are conventional methods unless otherwise specified, and the reagents used are commercially available reagents unless otherwise specified.

[0075] Example 1

[0076] This embodiment uses the method of the present invention to prepare an adsorption carrier, which specifically includes preparing a carrier matrix and preparing a molecular imprinting polymer layer on the carrier matrix to obtain an adsorption carrier that can be used to specifically adsorb nucleosome histones.

[0077] 1. Preparation of carrier matrix:

[0078] (1) Weigh 0.8 g of FeSO4·7H2O and 1.16 g of FeCl3·6H2O into a four-necked flask, add 200 mL of deionized water and 40 mL of anhydrous ethanol, and react at 900 r / min in a 60°C water bath with a mechanical stirrer for 30 min. During the reaction, nitrogen was continuously introduced to remove oxygen from the reaction solution.

[0079] (2) Add 25% ammonia water to adjust the pH of the reaction solution to 10-11.

[0080] (3) The water bath temperature was adjusted to 30° C., 20 mL of 5% tetraethyl orthosilicate (TEOS)-ethanol solution was added dropwise, the stirring speed was maintained at 900 r / min, and the reaction was carried out for 2 hours.

[0081] (4) The reaction solution was placed in an external magnetic field, the supernatant was discarded, the magnetic particles were washed twice with deionized water, 160 mL of ethanol and 5 mL of acrylic acid (AA) were added, and the mixture was reacted at 900 r / min in a 30°C water bath for 2 hours using a mechanical stirrer. Nitrogen was continuously introduced during the reaction.

[0082] (5) Place the reaction solution in an external magnetic field, discard the supernatant, and wash the magnetic particles three times with deionized water and ethanol respectively until the pH of the supernatant solution is neutral. Concentrate the magnetic particles under vacuum and dry them for later use.

[0083] 2. Preparation of molecularly imprinted polymer layer on the carrier matrix

[0084] (1) Take 50 mg of the prepared magnetic particles, add 70 μL of methacrylic acid (MAA) and 25 mg of itaconic acid (IA) as functional monomers, 16 mg of methylenebisacrylamide (N'N-MBA) as a cross-linking agent, and 20 mL of 0.02 mol / L, pH = 7 phosphate buffer solution (PBS) as a reaction medium, and mix by ultrasonic oscillation for 10 minutes.

[0085] (2) Add 10 mg of nucleosomal histone as template protein and mix well. Oscillate at 300 rpm and 25°C for 1 hour. Nitrogen is continuously introduced during the reaction.

[0086] (3) 70 μL of 20% ammonium persulfate (APS) and 14 μL of tetramethylethylenediamine (TEMED) were added as initiators, and the mixture was reacted at 25° C. and 300 rpm overnight.

[0087] (4) The reaction solution was placed in an external magnetic field, the supernatant was discarded, and 2% sodium dodecyl sulfate (SDS) solution was used as an eluent to shake and elute the imprinted template on the molecular imprinting-magnetic particles until no characteristic absorption was detected at 406 nm in the supernatant.

[0088] (5) The reaction solution was placed in an external magnetic field, the supernatant was discarded, and after drying at room temperature, a storage buffer (containing 50 mM Tris-HCl and 1% Triton X-100, pH 7.4) was added to obtain an adsorption carrier.

[0089] 3. Characterization of Adsorption Support

[0090] (1) Fourier transform infrared spectroscopy characterization: The adsorption carrier and potassium bromide powder were mixed in a mass ratio of 1:100 and then fully ground. The mixture was pressed into a transparent and uniform thin sheet at a pressure of 26 MPa. The sheet was scanned by infrared spectrum in the wave number range of 4000-400 cm-1. Functional modifications of -OH, -COOH and olefin groups were detected on the surface of the adsorption carrier.

[0091] (2) Transmission electron microscopy characterization: The adsorption carrier was ultrasonically dispersed in pure water, and the dispersion was dropped on aluminum foil. After the solvent evaporated, the adsorption carrier was observed to have a uniform morphology and a good dispersion state under a transmission electron microscope.

[0092] (3) Elemental analysis: Weigh about 2 mg (accurate to 0.001 mg) of the adsorption carrier and place it in a tin boat. After folding the tin boat to completely wrap the adsorption carrier, place it in the injection tube of the elemental analyzer. After high-temperature calcination, detect the content of C, H, N, and S elements in the adsorption carrier. The purity of the adsorption carrier is relatively high.

[0093] (4) The manifestation of adsorption carriers in different environments. Figure 6 , showing the behavior of the adsorbed carrier in the absence or presence of an external magnetic field. Figure 6 In (a), in the absence of an external magnetic field, the adsorbent carrier is dispersed in the solution. Figure 6 In (b), under the action of an external magnetic field, the adsorption carrier can be quickly aggregated and effectively separated from the aqueous phase.

[0094] Example 2

[0095] This embodiment uses the adsorption carrier and method of the present invention to extract free DNA from a plasma sample, specifically including nucleosome adsorption separation, free DNA extraction, and free DNA detection.

[0096] 1. Nucleosome adsorption and separation:

[0097] (1) The plasma sample was diluted to 2 mL with 0.02 mol / L, pH 7 PBS buffer, and 5 mg of the adsorption carrier prepared in Example 1 was added, vortexed and mixed, and adsorbed at 25° C. for 2 hours.

[0098] (2) Place the reaction solution in an external magnetic field, discard the supernatant, wash once with 2 mol / L NaCl solution, and retain the adsorption carrier.

[0099] (3) Add 2% SDS solution, vortex to mix, and shake at 25°C for 1 hour.

[0100] (4) The reaction solution is placed in an external magnetic field, and the supernatant is aspirated and retained to obtain the nucleosome eluate.

[0101] 2. Free DNA extraction:

[0102] The Magen IVD5435 kit was used to extract free DNA. The specific steps are as follows:

[0103] (1) Take 1 mL of nucleosome eluate, add 50 μL of proteinase K for protein degradation, vortex to mix, incubate at 55°C for 30 minutes, invert several times. Leave at room temperature until the digestion solution returns to room temperature. Add DNA binding solution and DNA binding magnetic beads, invert and mix at room temperature for 10 minutes.

[0104] (2) Place the reaction solution in an external magnetic field, discard the supernatant, retain the DNA-bound magnetic beads, and wash twice with washing buffers I and II, respectively.

[0105] (3) After a brief centrifugation, remove all residual liquid and dry at 37°C for 10-15 minutes.

[0106] (4) Add 50 μL of enzyme-free water and incubate at 37°C with shaking for 5 minutes to allow the DNA to fully dissolve.

[0107] (5) Place the reaction solution in an external magnetic field, aspirate the supernatant and store at -20°C.

[0108] 3. Free DNA detection:

[0109] (1) Concentration determination: The concentration was determined using the Thermo Fisher Q32854 kit. Specifically, the test solution was prepared by mixing the dye and Qubit dsDNA HS Master Mix at a ratio of 1:199. 190 μL of the test solution was added with 10 μL of the standard to draw a standard curve. 190 to 199 μL of the test solution was added with 1 to 10 μL of the sample to be tested to ensure that the final volume was 200 μL. The prepared standard and sample were allowed to react at room temperature for 3 minutes before testing.

[0110] (2) Fragment analysis: The Agilent 5067-5603 kit was used for fragment analysis. Specifically, the test buffer and sample solution were mixed at a ratio of 1:1, and 4 μL was taken for detection.

[0111] refer to Figure 7 , showing a comparison of the extraction effect of free DNA by adsorption carrier. In the plasma sample (a) treated with the adsorption carrier, the DNA fragments are mainly distributed in the range of 170-190 bp, and there are almost no DNA fragments >1000 bp, which is consistent with the fragment size of free DNA and no genomic DNA contamination. In the plasma sample (b) not treated with molecular imprinting-magnetic particles, there are more DNA fragments distributed at >1000 bp, proving that there is genomic DNA contamination. Note that since the mixed test buffer used for fragment analysis contains its own markers, the fragment distribution is shown at 25 and 1500.

[0112] Example 3

[0113] In this embodiment, the adsorption carrier of the present invention is used to detect the adsorption time of free DNA in the plasma sample.

[0114] In this example, 0.02 mol / L, pH=7 PBS buffer was used to prepare a nucleosome histone standard solution with an initial concentration of 1.0 mg / mL. After taking an equal amount of adsorption carrier (5 mg) and mixing with the standard solution, the adsorption was carried out at 25°C for different time periods (15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 120 minutes), and the absorbance of the supernatant before and after adsorption at 460 nm was measured to calculate the adsorbed protein concentration.

[0115] refer to Figure 8 , shows the change of the adsorption amount of nucleosome histones by the adsorption carrier over time. In the first 30 minutes of adsorption, the adsorption amount of nucleosome histones by the adsorption carrier increased rapidly, and then with the increase of time, the increase rate of the adsorption amount of nucleosome histones by the adsorption carrier slowed down. At about 90 minutes, the adsorption amount tended to be stable, and the adsorption of nucleosome histones by the adsorption carrier gradually reached equilibrium, and the equilibrium adsorption amount could reach 120.2 mg / g particles. Considering the efficiency factor, it was finally calculated that the optimal adsorption time was 1 hour.

[0116] In general, the present invention proposes an adsorption carrier for extracting free DNA from a sample, a preparation method and a use thereof. The invention uses nucleosome histone as a template, acrylamide and itaconic acid as functional monomers, and connects functional polymers on magnetic particles to prepare a spatially specific adsorption carrier, and selectively adsorbs free nucleosomes, thereby separating free DNA from genomic DNA. The nucleosomes are detached from the adsorption carrier by elution, the nucleosomes are recovered, the histones are digested with proteinase K to release the DNA, the DNA is adsorbed by DNA-bound magnetic beads, and the DNA is washed to remove proteins, inorganic salts and impurities, so as to finally obtain free DNA free from large genomic fragment contamination.

[0117] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive.

[0118] The disclosure above provides many different embodiments or examples to implement different structures of the present disclosure. In order to simplify the present disclosure, the components and settings of specific examples are described above. Of course, they are only examples, and the purpose is not to limit the present disclosure. In addition, the present disclosure can repeat reference numbers and / or reference letters in different examples, and this repetition is for the purpose of simplification and clarity, which itself does not indicate the relationship between the various embodiments and / or settings discussed.

[0119] The above are only specific implementation methods of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any technician familiar with the technical field can easily think of various changes or substitutions within the technical scope disclosed in the present disclosure, which should all be included in the protection scope of the present disclosure.

[0120] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. An adsorption carrier for extracting free DNA from a sample, characterized in that: It comprises a carrier matrix, and a molecular imprinting polymer layer located on at least a part of the surface of the carrier matrix; The molecularly imprinted polymer layer has a nucleosome-specific binding site; The nucleosome-specific binding site has a morphology that matches that of nucleosome histones.

2. The adsorption carrier according to claim 1, characterized in that: The molecular imprinting polymer layer covers the surface of the carrier substrate, The nucleosomal histones include core histones.

3. The adsorption carrier according to claim 2, characterized in that: The molecularly imprinted polymer layer includes a functional polymer, and the functional polymer includes an acrylic copolymer.

4. The adsorption carrier according to claim 3, characterized in that: The molecular imprinting polymer layer is formed by polymerizing the functional monomers in a medium containing a template protein and then eluting the template protein.

5. The adsorption carrier according to claim 2, characterized in that: The carrier matrix comprises magnetic particles, and the surface of the magnetic particles has at least one of a protective group, a protein binding group and a functional polymer binding group, The protective group includes a siloxane group, The protein binding group includes at least one of a hydroxyl group, a carboxyl group and an amino group, The functional polymer binding group includes an alkenyl group.

6. A method for extracting free DNA from a sample, characterized in that: The method comprises: Using the adsorption carrier according to any one of claims 1 to 5 to perform adsorption treatment on the sample to obtain a nucleosome-adsorption carrier conjugate; The nucleosome-adsorption carrier conjugate is subjected to extraction treatment to obtain the free DNA.

7. The method according to claim 6, characterized in that The extraction process comprises: The nucleosome-adsorption carrier conjugate is subjected to elution treatment to obtain a nucleosome elution solution; the nucleosome elution solution is subjected to protein degradation treatment to obtain a protein degradation solution containing the free DNA; and the protein degradation solution is subjected to DNA separation treatment to obtain the free DNA.

8. The method according to claim 7, characterized in that The method satisfies at least one of the following conditions: The adsorption treatment includes adding the adsorption carrier to the sample and reacting at a temperature of 4-60° C. for 0.5-24 hours; The elution treatment includes treating the collected nucleosome-adsorption carrier conjugate with an elution reagent to elute the nucleosome. Wherein, the treatment temperature of the elution treatment is 4-60°C, and the treatment time is 0.5-24h.

9. The method according to claim 7, characterized in that: The protein degradation treatment comprises treating the nucleosome eluate with a degradation agent to degrade the histones in the nucleosomes and release the free DNA; The protein degradation treatment is carried out at a temperature of 25-70°C and for a time of 5-60 minutes.

10. A method for preparing the adsorption carrier according to any one of claims 1 to 5, characterized in that: The method comprises the steps of preparing a carrier matrix and preparing a molecular imprinting polymer layer.

11. The method according to claim 10, characterized in that The preparation of the carrier matrix comprises the steps of preparing magnetic particles and optionally introducing at least one of a protecting group, a protein binding group and a functional polymer binding group on the surface of the magnetic particles. The protective group includes a siloxane group; The protein binding group includes at least one of a hydroxyl group, a carboxyl group and an amino group; The functional polymer binding group includes an alkenyl group.

12. The method according to claim 10, characterized in that The step of preparing the molecularly imprinted polymer layer comprises: Adding functional monomers to the carrier matrix and adding template proteins to mix, allowing the functional monomers to undergo polymerization reaction, and forming a pre-assembled layer containing the template proteins on the surface of the carrier matrix; performing a second elution treatment on the pre-assembled layer to remove the template protein and obtain the molecularly imprinted polymer layer; Wherein, the template protein includes nucleosome histone, and causing the functional monomers to undergo polymerization reaction includes causing the functional monomers to undergo polymerization in the presence of a cross-linking agent and an initiator.

13. The method according to claim 12, characterized in that The functional monomer includes at least one of an acrylic compound and an unsaturated dibasic organic acid; The cross-linking agent includes at least one of N,N'-methylenebisacrylamide, vinyl dimethacrylate and ethylene glycol dimethacrylate; The initiator includes at least one of ammonium persulfate and tetramethylethylenediamine.

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