Modified immunomagnetic beads, preparation method and application thereof

Abstract

The present invention provides modified immunomagnetic beads, their preparation method, and applications. The immunomagnetic beads exhibit low nonspecific adsorption, a high density of surface functional groups, and excellent stability. The method for preparing the immunomagnetic beads comprises coating hydrophobic magnetic beads with an amphiphilic block copolymer to obtain amphiphilic magnetic beads, and coupling tosyl functional groups to the surface of the beads. After coupling the magnetic beads to target proteins or antibodies, they can be used for a variety of immune responses.

Description

Technical Field

[0001] The present invention belongs to the field of immunoassay technology; more specifically, the present invention relates to a modified immunomagnetic bead, a preparation method thereof and an application thereof. Background Art

[0002] Immunomagnetic beads use magnetic particles as the core, with an inorganic layer or polymer layer coated on the surface of the beads. Active molecules such as antibodies, proteins, and enzymes are then immobilized through functional groups on the surface of the beads (such as -amino, -carboxyl, -sulfhydryl, -epoxy, -NHS ester, and -toluenesulfonyl).

[0003] Immunomagnetic beads usually have a small particle size and a large specific surface area, so they are prone to agglomeration and reduce their own specific surface area by adsorbing other substances to achieve a stable state. Immunomagnetic beads are prone to non-specific adsorption. For example, patent CN111004329A discloses a weak interaction capture magnetic bead based on photoaffinity covalently linked protein molecules and its preparation method and application, wherein ThermoFisher's NHS magnetic bead solution (Article No. 88826) is used, and NH2-PEG-SH polymer is modified in the outer layer of NHS magnetic beads as a passivation layer, and at the same time, the magnetic beads and photoaffinity peptide probes are connected as intermediate layer connecting molecules, and the magnetic beads ensure high spatiotemporal resolution to capture weakly interacting proteins in order to reduce non-specific interactions. However, this method is relatively complicated, its effect needs to be further improved, and its application is also relatively limited.

[0004] Therefore, there is an urgent need to develop improved immunomagnetic beads in this field. Summary of the Invention

[0005] The purpose of the present invention is to provide a modified immunomagnetic bead, a preparation method and application thereof.

[0006] In a first aspect of the present invention, a method for preparing immunomagnetic beads is provided, the method comprising: (1) preparing hydrophobic magnetic beads; (2) coating the hydrophobic magnetic beads with an amphiphilic block copolymer to obtain modified amphiphilic magnetic beads; the amphiphilic block copolymer comprises a hydrophilic end and a hydrophobic end, the hydrophilic end is selected from PEG or PEG methyl ether, and the hydrophobic end is selected from PMPS (poly(3-methacryloxypropyltrimethoxysilane)) or PPDSM; the molecular weight (Mn) of the PEG is 2000 to 10000; (3) performing a tosylation reaction on the amphiphilic magnetic beads to obtain amphiphilic magnetic beads with tosyl functional groups on the surface.

[0007] In one or more embodiments, the amphiphilic block copolymer includes: PEG-b-PPDSM or PEG-b-PMPS.

[0008] In one or more embodiments, in (1), the hydrophobic magnetic beads are hydrophobic iron oxide magnetic beads.

[0009] In one or more embodiments, the method for preparing the hydrophobic magnetic beads includes: coating iron oxide (Fe3O4) with oleic acid to obtain oleic acid-coated hydrophobic magnetic beads (OA@Fe3O4).

[0010] In one or more embodiments, in (3), the surface of the amphiphilic magnetic beads is first modified with (3-aminopropyl)triethoxysilane (APTES) and then reacted with toluenesulfonyl chloride, so that the chlorine of toluenesulfonyl chloride directly reacts with the amino magnetic beads.

[0011] In one or more embodiments, the PEG-b-PMPS is prepared by mixing PEG-CTA with 3-(trimethoxysilyl)propyl methacrylate (MPS) and azobisisobutyronitrile (AIBN) (preferably using 1,4-dioxane as a solvent).

[0012] In one or more embodiments, the PEG-b-PPDSM is prepared by mixing PEG-CTA with PDSM and azobisisobutyronitrile (preferably using N,N-dimethylacetamide as solvent).

[0013] In one or more embodiments, the PEG-CTA is obtained by mixing polyethylene glycol methyl ether, TEA, 2-chloro-2,2-diphenylacetyl chloride (CTA), and tetrahydrofuran, and refluxing; removing the tetrahydrofuran, adding chloroform to form a solution; washing with a sodium bicarbonate solution, drying the organic phase, and removing the solvent.

[0014] In one or more embodiments, the molecular weight (Mn) of the PEG is 3000-9000, preferably 4000-8000, such as 5000, 6000, or 7000.

[0015] In one or more embodiments, after step (3), the method further includes: coupling the tosyl amphiphilic magnetic beads with capture molecules to obtain immunomagnetic beads coupled with capture molecules; preferably, the capture molecules are biomolecules carrying amino groups; preferably, the capture molecules include (but are not limited to): antibodies (such as IgG antibodies, HuR antibodies, etc.), ligands, proteins (such as Protein A, Protein G, Streptavidin, etc.).

[0016] In one or more embodiments, after coupling the tosyl amphiphilic magnetic beads to the capture molecules, the method further comprises blocking with a blocking solution containing polymethacryloyloxyethyl phosphorylcholine (PMPC); preferably, the amount of PMPC is 0.05-0.3% (mass ratio).

[0017] In one or more embodiments, blocking is performed with a blocking solution containing PMPC and BSA.

[0018] In one or more embodiments, the amount of PMPC is 0.06-0.25%, preferably 0.07-0.2%, more preferably 0.08-0.15%, and even more preferably 0.09-0.12%.

[0019] In one or more embodiments, the amount of BSA is 0.2-2%, preferably 0.3-1.5%, more preferably 0.35-1%, and even more preferably 0.4-0.8%.

[0020] In another aspect of the present invention, an immunomagnetic bead is provided, comprising: modified amphiphilic magnetic beads coated with an amphiphilic block copolymer, and having a tosyl functional group on its surface; wherein the amphiphilic block copolymer comprises a hydrophilic end and a hydrophobic end, the hydrophilic end is selected from PEG or PEG methyl ether, and the hydrophobic end is selected from PMPS or PPDSM; the molecular weight (Mn) of the PEG is 2000 to 10000.

[0021] In one or more embodiments, the immunomagnetic beads are further coupled with a capture molecule; preferably, the capture molecule is a biomolecule carrying an amino group; preferably, the capture molecule includes (but is not limited to): an antibody, a ligand, and a protein.

[0022] In one or more embodiments, the immunomagnetic beads are prepared by any of the methods described above.

[0023] In another aspect of the present invention, the use of the immunomagnetic beads in an immune response is provided; preferably, the immune response includes: protein sorting (such as through protein-protein interaction) or immobilization, nucleic acid sorting (such as through protein-nucleic acid interaction) or immobilization; preferably, the sorting is separated by immunoprecipitation (IP).

[0024] In another aspect of the present invention, a method for conducting an immune reaction using immunomagnetic beads is provided, comprising: (a) providing the immunomagnetic beads, which are coupled to capture molecules, and the capture molecules capture target molecules (such as by binding or adsorption); and (b) contacting the immunomagnetic beads of (a) with a sample to be tested to capture the target molecules in the sample to be tested.

[0025] In one or more embodiments, the immunoreaction method includes: protein sorting (such as through protein-protein interaction) or immobilization, nucleic acid sorting (such as through protein-nucleic acid interaction) or immobilization; preferably, the sorting is separated by immunoprecipitation (IP); preferably, the immunoprecipitation includes: chromatin immunoprecipitation (ChIP), RNA binding protein immunoprecipitation (RIP), and co-immunoprecipitation (Co-IP).

[0026] In another aspect of the present invention, a kit for immune response is provided, wherein the kit comprises the immunomagnetic beads.

[0027] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 , preparation process of amphiphilic magnetic beads.

[0029] Figure 2 , preparation process of tosyl amphiphilic magnetic beads.

[0030] Figure 3 , flow chart of amphiphilic magnetic beads coupled to antibodies / proteins.

[0031] Figure 4 、 Four The Western blotting results of protein samples after using the two types of magnetic beads in a ChIP experiment on HeLa cells. M2 shows the Western blotting results of a protein sample prepared using amphiphilic Protein A / G magnetic beads prepared by the present invention, bound to Histone H3 antibodies, and incubated with HeLa cell lysate for a ChIP experiment. The # position indicates the position of a nonspecific band. M1 shows the Western blotting results of a protein sample prepared using amphiphilic Protein A / G magnetic beads prepared by the present invention, bound to rabbit IgG, and incubated with HeLa cell lysate for a ChIP experiment. No target band was captured, indicating good anti-nonspecificity. P2 shows the results of capturing the target band in HeLa cell lysate after incubation with Histone H3 antibodies using control Protein A / G hydrophobic magnetic beads. P1 shows the presence of nonspecific bands after incubation with rabbit IgG using control Protein A / G hydrophobic magnetic beads and HeLa cell lysate, indicating strong nonspecificity.

[0032] Figure 5 、 FourThe experimental results of using the two magnetic beads for RIP of HeLa cells are shown in Figure 4. M4 shows the Western blotting results after incubation of the amphiphilic HuR magnetic beads prepared by the present invention with HeLa cell lysate, where the # indicates the position of a nonspecific band. M3 shows the Western blotting results after incubation of the amphiphilic rabbit IgG magnetic beads prepared by the present invention with HeLa cell lysate, which showed no capture of the target band, indicating good anti-nonspecificity. P4 shows the capture of the target band of the HeLa cell lysate by the control HuR hydrophobic magnetic beads. P3 shows the presence of nonspecific bands after incubation of the control rabbit IgG hydrophobic magnetic beads with HeLa cell lysate, indicating strong nonspecificity. DETAILED DESCRIPTION

[0033] After in-depth research, the inventors have revealed for the first time a type of immunomagnetic bead with improved performance, which has low non-specific adsorption. The method for preparing the immunomagnetic beads includes: using an amphiphilic block copolymer to coat hydrophobic magnetic beads to obtain amphiphilic magnetic beads (or polymer-modified magnetic beads), and then coupling tosyl functional groups to the surface of the magnetic beads. After coupling the functional magnetic beads with target proteins or antibodies, they can be used in a variety of immune reactions (such as immunoprecipitation experiments such as ChIP, RIP, IP, and Co-IP).

[0034] As used in the present invention, the terms “contain,” “include,” or “have” include “comprise,” “mainly consist of,” “substantially consist of,” and “consist of”; “mainly consist of,” “substantially consist of,” and “consist of” are subordinate concepts of “contain,” “have,” or “include.”

[0035] As used herein, "about," "approximately," "roughly," or "substantially" generally refers to a particular value or range, such as within 20%, preferably within 10%, and more preferably within 5%. The values used herein are approximate, meaning that unless expressly stated, the terms "about," "approximately," "roughly," or "substantially" can be inferred to apply.

[0036] As used in the present invention, the "main ingredient (component)" or "main active ingredient" or "active ingredient" refers to the necessary component that plays a role in maintaining protein stability, and the present invention mainly includes the following components: amphiphilic block polymers, ionic liquids and antimicrobial peptides, preferably also including sucrose; or consisting of them.

[0037] As used in the present invention, b(-b-) is the abbreviation of block, which represents block polymerization, a special polymer formed by polymer segments on both sides being connected together.

[0038] In the present invention, the structural formulas of PEG-b-PMPS (left) and PEG-b-PPDSM (right) are as follows:

[0039]

[0040] In the structural formula, n represents the number of repetitions of the repeating unit in PEG (degree of polymerization), corresponding to a molecular weight (Mn) of 2000 to 10000, and n is 45 to 227.

[0041] In the structural formula, x represents the number of repetitions (polymerization degree) of the repeating unit in PMPS, and x is a positive integer from 20 to 40, such as 22, 25, 28, 30, 32, 35, 38; in a preferred embodiment, x=25 in PMPS.

[0042] In the structural formula, y represents the number of repetitions (degree of polymerization) of the repeating unit in PPDSM, and y is a positive integer from 20 to 40, such as 22, 25, 28, 30, 32, 35, 38; in a preferred embodiment, y=24 in the embodiment PPDSM.

[0043] The magnetic beads of the present invention are coated with a preferred amphiphilic block copolymer, comprising a hydrophilic end and a hydrophobic end, wherein the hydrophilic end is selected from PEG or PEG methyl ether, and the hydrophobic end is selected from PMPS or PPDSM; the molecular weight (Mn) of the PEG is 2000 to 10000. In a preferred embodiment, the molecular weight (Mn) of the PEG is 6000±1500, more preferably 6000±1000, 6000±800, 6000±500, or 6000±300.

[0044] The use of the amphiphilic block copolymers of the present invention significantly improves the properties of magnetic beads. Compared to conventional hydrophobic magnetic beads not coated with the preferred amphiphilic block copolymers of the present invention, the magnetic beads of the present invention exhibit superior properties, including but not limited to: (1) better reduction of nonspecific adsorption of the hydrophobic magnetic beads themselves; and (2) amphiphilic magnetic beads can be pre-added with tosyl groups, thereby enabling subsequent coupling to proteins or antibodies with amino or thiol groups without the need for subsequent activation, making them more convenient to use.

[0045] Although tosyl modification on magnetic beads has been mentioned in the art, for example, by tosylating a hydrophilic polymer (e.g., using tosyl chloride) and then reacting it with amino magnetic microspheres to obtain hydrophilic tosyl magnetic beads, the tosyl groups on the magnetic beads do not need to be activated and coupled with amino biomolecules. However, this operation method first reacts the chlorine of tosyl chloride with the hydrophilic polymer, and then the tosyl groups react with the amino magnetic beads, resulting in the loss of some tosyl groups. In the present invention, the chlorine of tosyl chloride directly reacts with the amino magnetic beads, which is convenient and does not consume additional tosyl groups. This is more user-friendly for subsequent use, and the density of tosyl groups on the magnetic beads is also higher.

[0046] The present invention modifies magnetic beads using amphiphilic block copolymers, with polyethylene glycol or polyethylene glycol methyl ether (Mn 2000-10000) as the hydrophilic end and block polymerized methacrylate as the hydrophobic end. The modified surface of the hydrophobic magnetic beads effectively reduces nonspecific adsorption. The hydrophobic end of the magnetic beads reacts with an aminosilane coupling agent, and then the amino groups of the magnetic beads are modified with a tosyl functional group that reduces nonspecific adsorption. After coupling with a target protein or antibody (such as protein A / G, streptavidin, antibodies, etc.), the beads can be used in experiments such as ChIP, RIP, IP (immunoprecipitation), or Co-IP (co-immunoprecipitation) to significantly reduce nonspecific adsorption.

[0047] Protein-DNA interaction is the key to gene transcription regulation and is also the prerequisite for starting gene transcription. Protein-DNA interaction mainly includes histone (Histone), transcription factor, DNA methylase and chromatin remodeling complex etc. The method for studying protein-DNA interaction has a lot, among which chromatin immunoprecipitation (ChIP) is a standard method for detecting protein-DNA interaction within the whole genome, and the method combines, precipitates or pulls down (Pull-down) by Protein A / Protein G microspheres or magnetic beads by antibody-transcription factor-chromatin complex, detects the DNA sequence combined with the target protein by PCR or sequencing, and then studies the action site of these transcription factors in cell development or growth. When using common magnetic bead-coupled antibodies or Protein A / Protein G microspheres, magnetic beads-bound antibodies, non-specific adsorption is easily arranged, which has a great impact on the experiment. And the technical solution of the present invention overcomes this bottleneck problem to a large extent, and the non-specific adsorption of magnetic beads can be effectively reduced to ensure the authenticity, stability and repeatability of the experimental results.

[0048] RNA plays an important role in the process of organism genetic information expression, and its processing and maturation process are often relevant to gene transcription regulation. RNA binding proteins are of various types and diverse functions. They participate in multiple biological processes by being combined with RNA. Therefore, the identification and research of RNA-protein interactions are of great significance to the understanding of the complex physiological regulatory system in cells. RNA binding protein immunoprecipitation (RIP) is the most widely used research method. It is based on the antigen-antibody hybridization principle and can study the physical binding situation between single protein and RNA molecules under natural conditions in cells. It is a powerful tool for understanding the dynamic process of post-transcriptional regulatory network. The key to the RIP experiment is to avoid the selection of RNA degradation and immunomagnetic beads, and the immunomagnetic beads of the present invention have very low non-specific adsorption to ensure the success of the RIP experiment.

[0049] The technical solution of the present invention can cover applications in multiple fields such as cell sorting, immunoassay, separation and purification of proteins and nucleic acids, enzyme immobilization, protein-DNA interaction, protein-RNA interaction, etc.

[0050] The reagents used to obtain the magnetic beads of the present invention, or the magnetic beads prepared by the present invention, can be sub-packaged in containers, packaged, or made into a kit for use by those skilled in the art. In addition, the packaging or kit may also include instructions for use. Such packaging or kit is also encompassed within the scope of the present invention.

[0051] In addition to having low aggregation and low non-specific adsorption properties, the immunomagnetic beads of the present invention also have the characteristics of simple and convenient process, good stability, easy scale-up, and strong practicality.

[0052] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0053] Materials and Methods

[0054] Experimental materials, reagents and instruments

[0055] Ferric acetylacetonate (CAS:14024-18-1);

[0056] 1,2-Hexadecanediol (CAS: 6920-24-7);

[0057] Oleic acid (CAS: 112-80-1);

[0058] Benzyl ether (CAS: 103-50-4);

[0059] 3-(Trimethoxysilyl)propyl methacrylate (MPS) (CAS: 2530-85-0);

[0060] Polyethylene glycol methyl ether (M n 5000)(CAS:9004-74-4, Sigma, Cat. No. 81323);

[0061] Polyethylene glycol (Mn 4000-8000) (CAS: 25322-68-3);

[0062] Pyridyl disulfide ethyl methacrylate (PDSM) (CAS: 910128-59-5);

[0063] 2-Chloro-2,2-diphenylacetyl chloride (CTA) (CAS: 2902-98-9);

[0064] 4-Toluenesulfonyl chloride (CAS: 98-59-9);

[0065] (3-Aminopropyl)triethoxysilane (APTES) (CAS: 919-30-2);

[0066] Polymethacryloyloxyethyl phosphorylcholine (PMPC, Mn9000, Sigma; Cat. No. 922749);

[0067] Triethylamine (TEA) (CAS: 121-44-8);

[0068] Tetrahydrofuran (CAS: 109-99-9);

[0069] Chloroform (CAS: 67-66-3);

[0070] Sodium bicarbonate (CAS:144-55-8);

[0071] Anhydrous magnesium sulfate (CAS: 7487-88-9);

[0072] Diethyl ether (CAS: 60-29-7);

[0073] N,N-dimethylacetamide (CAS:127-19-5);

[0074] Azobisisobutyronitrile (AIBN) (CAS: 78-67-1);

[0075] 1,4-Dioxane (CAS: 123-91-1);

[0076] Isopropyl alcohol (CAS: 67-63-0);

[0077] 300 nm tosyl magnetic beads (BMJ2800-2);

[0078] BeyoRIP TM RIP Assay Kit (Protein A / G agarose) (Biyuntian, P1801);

[0079] Rabbit IgG (Biyuntian, A7016);

[0080] Histone H3 Rabbit Polyclonal Antibody (Biyuntian, AF7101);

[0081] Recombinant Streptavidin (Biyuntian, P5084);

[0082] Biotin-labeled horseradish peroxidase (Biyuntian, A0308);

[0083] BeyoRIP TM Antibody and Primer Pair Kit for HuR RIP Assay (Biyuntian, P1821);

[0084] RNAeasy TM Animal RNA extraction kit (spin column type) (Biyuntian, R0027);

[0085] BeyoRT TM III cDNA first-strand synthesis kit (Biyuntian, D7178);

[0086] BeyoFast TM SYBR Green qPCR Mix (2X) (Biyuntian, D7262);

[0087] BeyoGel TM Plus PAGE precast gel (Hepes, 4-15%, 10 wells) (Biyuntian, P0519);

[0088] BeyoGel TM Plus SDS-PAGE Hepes electrophoresis buffer (20X) (Biyuntian, P0552);

[0089] BeyoColor TM Color pre-stained protein molecular weight standard (6.5-270 kD) (Biyuntian, P0071);

[0090] SDS-PAGE protein loading buffer (5X, odorless) (Biyuntian, P0286);

[0091] BeyoMag TM Magnetic separation rack (Biyuntian, FMS008 / FMS154 / FMS504);

[0092] BeyoMag TM Magnetic separation rack (4-well, 50 ml, blue) (Biyuntian, FMS504);

[0093] 100ml polytetrafluoroethylene hydrothermal reactor (HT-SR-100T);

[0094] LCD digital control long axis rotary mixer (Biyuntian, E1505);

[0095] MiniProGel TM Protein gel preparation and electrophoresis system (Biyuntian, E6001);

[0096] Multifunctional microplate reader Varioskan LUX (ThermoFisher);

[0097] BeyoImager TM 600 chemiluminescence imaging system (Biyuntian, EI600);

[0098] BeyoGold TM Cell strainer (40 μm pore size, individually wrapped in paper and plastic, sterile) (Biyuntian, FSTR040).

[0099] Experimental methods

[0100] 1. Preparation of hydrophobic nanomagnetic beads

[0101] First, add 2mmol of ferric acetylacetonate, 12mmol of 1,2-hexadecanediol, 8mmol of oleic acid, and 24ml of benzyl ether to a 100ml three-necked flask and stir at 300rpm for half an hour to mix thoroughly. The mixture is then transferred to a 100ml polytetrafluoroethylene hydrothermal reactor and placed in an electric forced-air drying oven. The reaction is heated at 250°C for 8 hours. The oven is then closed and the reactor is allowed to cool to room temperature, forming oleic acid-coated hydrophobic magnetic beads. The reactor is opened, the reaction solution is poured out, and the beads are rinsed three times with anhydrous ethanol and then three times with deionized water to obtain oleic acid (OA)-coated hydrophobic magnetic beads (OA@Fe3O4). The water content is dried in a 37°C oven, and the bead concentration is measured before use.

[0102] 2. Preparation of PEG-CTA

[0103] 15 mmol of polyethylene glycol (molecular weight 6000), 45 mmol of TEA, and 30 mmol of 2-chloro-2,2-diphenylacetyl chloride (CTA) were added to a 500 ml round-bottom flask. Then, 300 ml of dry tetrahydrofuran was added and mixed thoroughly. The mixture was refluxed at atmospheric pressure for 2 days. After the tetrahydrofuran was removed, 300 ml of chloroform was added. The resulting solution was washed five times with saturated sodium bicarbonate solution. The resulting yellow organic phase was dried over anhydrous magnesium sulfate and then vacuum-dried to remove the solvent. The product was further purified by precipitation with glacial ether to obtain the ester of chlorophenylacetic acid and polyethylene glycol methyl ether, namely PEG-CTA. The CTA modification method for PEG 2000-10000 is similar.

[0104] 3. Synthesis of amphiphilic block copolymer PEG-b-PMPS

[0105] MPS (10 mmol), PEG-CTA (0.24 mmol), and AIBN (0.08 mmol) were added to 4 ml of 1,4-dioxane. The mixture was transferred to a round-bottom flask, degassed under vacuum, sealed under vacuum, and placed in a 70°C oil bath to initiate polymerization. After 17 hours of reaction, the flask was immersed in a dry ice / isopropanol mixture to terminate the polymerization. Polymer purification was performed in an argon-filled airbag. After three petroleum ether precipitations, the resulting polymer was vacuum-dried overnight. This yielded an amphiphilic PEG-b-PMPS (where x = 25) block copolymer. The product was weighed and stored for later use.

[0106] 4. Synthesis of amphiphilic block copolymer PEG-b-PPDSM

[0107] 6 mmol of PDSM, 0.24 mmol of PEG-CTA, and 0.06 mmol of AIBN were dissolved in 15 ml of N,N-dimethylacetamide. The mixture was transferred to a round-bottom flask and subjected to four freeze-pump-thaw cycles to remove oxygen. The flask was then placed in a 70°C oil bath to initiate polymerization. After 12 hours of polymerization, the flask was immersed in a dry ice / isopropanol mixture to terminate the polymerization. After thawing, the reactants were precipitated with diethyl ether three times and then vacuum-dried overnight to obtain the amphiphilic block copolymer PEG-b-PPDSM (where x = 24). The mixture was weighed and stored for later use.

[0108] 5. OA@Fe3O4 coated amphiphilic block copolymer

[0109] OA@Fe₃O₄ is unstable in water, so the solution was exchanged with anhydrous tetrahydrofuran on a magnetic rack to a concentration of 5 mg / ml. The amphiphilic block copolymer, PEG-b-PMPS for example, was dissolved in anhydrous tetrahydrofuran to a concentration of 50 mg / ml. 10 ml of OA@Fe₃O₄ and 10 ml of PEG-b-PMPS were mixed evenly. After aging the mixture for 4 days, the mixture was added dropwise to 200 ml of deionized water under mechanical stirring. The tetrahydrofuran in the solution was dialyzed against deionized water. The resulting solution was filtered through a 40 μm cell strainer to remove large aggregates, and the flow-through was collected on a magnetic rack. This process was repeated three times to further remove aggregates. The resulting amphiphilic block copolymer-coated magnetic beads exhibit reduced nonspecific adsorption.

[0110] 6. Tosylation reaction of magnetic beads

[0111] 20 ml of amphiphilic block copolymer-coated magnetic beads (10 mg / ml) were ultrasonically dispersed with isopropyl alcohol and 5 ml of APTES was added. The reaction mixture was tumbled at room temperature for two days, then washed on a magnetic rack to remove unreacted APTES. After washing, the beads were dispersed in acetone, and 49 mg of 4-toluenesulfonyl chloride was added. The reaction was tumbled at room temperature for two hours, and the beads were rinsed several times with water. The resulting magnetic beads had toluenesulfonyl groups on their surface, i.e., toluenesulfonated magnetic beads. These were used for testing purposes. Due to the optimized reaction pathway of the present method, the density of toluenesulfonyl groups was higher.

[0112] 7. Tosylation of magnetic beads coupled to Protein A / G

[0113] 20 mg of tosyl magnetic beads were washed three times with 0.1 M boric acid buffer (pH 9.5), then resuspended in 1 ml of 0.1 M boric acid buffer (pH 9.5). The beads were then aliquoted and halved, and 0.5 ml of 2 mg / ml Protein A and Protein G (dispersed in a mixture of 0.1 M boric acid buffer (pH 9.5) and 0.3 M (NH₄)₂SO₄) were added to the beads. The beads were incubated overnight at 37°C with rotation. After rotation, both beads were washed with 1 ml of PBS and then blocked with blocking buffer. The blocking buffer consisted of PBS containing 0.5% BSA and 0.1% PMPC, which effectively reduces nonspecific adsorption to the beads. The resulting Protein A and Protein G beads were mixed in a 1:1 ratio to produce Protein A / G magnetic beads.

[0114] 8. Preparation of control hydrophobic magnetic beads

[0115] Magnetic beads unmodified with amphiphilic polymers served as negative control beads (control hydrophobic beads). Their preparation method was as follows: First, prepare OA@Fe3O4 as described above. Take 20 ml of 10 mg / ml magnetic beads, ultrasonically disperse them with isopropanol, and add 5 ml of APTES. The reaction mixture was tumbled at room temperature for two days. Unreacted APTES was then washed on a magnetic rack. After washing, the beads were dispersed in acetone, and 49 mg of 4-toluenesulfonyl chloride was added. The reaction was tumbled at room temperature for two hours. The beads were then washed several times with water. The resulting beads had toluenesulfonyl groups on their surface, thus forming tosyl-grouped beads. This was used for testing. Protein A and Protein G were coupled according to step 7 to obtain the control Protein A / G hydrophobic magnetic beads.

[0116] 9. Tosylation of magnetic beads coupled to IgG and HuR antibodies

[0117] 20 mg of tosyl magnetic beads were washed three times with 0.1 M boric acid buffer (pH 9.5), and then resuspended in 1 ml of 0.1 M boric acid buffer (pH 9.5). The beads were divided into two equal parts and 0.5 ml of 2 mg / ml rabbit IgG antibody or HuR Rabbit mAb was added to each part. Rabbit IgG antibody and HuR Rabbit mAb (BeyoRIP TM Antibody and Primer Pair Kit for HuRRIP Assay) is prepared using a mixture of 0.1M boric acid buffer (pH9.5) and 0.3M (NH4)2SO4. After the antibody is added to the magnetic beads, mix them evenly and then flip them at 37°C overnight. After flipping, wash the two magnetic beads separately with 1ml PBS, and then block the magnetic beads with blocking solution. The blocking solution is PBS buffer, containing 0.5% BSA and 0.1% PMPC. PMPC can effectively reduce the nonspecific adsorption of magnetic beads. The amphiphilic IgG magnetic beads obtained in the present invention are recorded as M3, and the amphiphilic HuR magnetic beads are recorded as M4. The magnetic beads with unmodified amphiphilic polymers (control hydrophobic magnetic beads) are used as negative control magnetic beads. Except for the unmodified amphiphilic polymers, the other preparation methods are the same. The control IgG hydrophobic magnetic beads obtained are recorded as P3, and the control HuR hydrophobic magnetic beads are recorded as P4.

[0118] 10. Tosyl magnetic beads coupled with streptavidin

[0119] 10mg of amphiphilic tosyl magnetic beads, control tosyl magnetic beads, and comparable Biomag tosyl magnetic beads were washed three times with 0.1M boric acid buffer (pH 9.5). The beads were then resuspended in 0.5ml of 0.1M boric acid buffer (pH 9.5), and 0.5ml of 2mg / ml Streptavidin (Streptavidin is prepared using a mixture of 0.1M boric acid buffer (pH 9.5) and 0.3M (NH4)2SO4) was added to each sample. After adding Streptavidin to the beads, the mixture was mixed thoroughly and then incubated at 37°C overnight. After incubation, the beads were washed with 1ml of PBS and then blocked with blocking buffer. The blocking buffer consisted of PBS containing 0.5% BSA and 0.1% PMPC, which effectively reduces nonspecific adsorption to the beads. The amphiphilic Streptavidin magnetic beads obtained in the present invention are marked as S1, the control Streptavidin magnetic beads are marked as S2, and the Biomag Streptavidin magnetic beads are marked as S3.

[0120] 11. ChIP experiment

[0121] The amphiphilic Protein A / G magnetic beads prepared according to the present invention and control Protein A / G hydrophobic magnetic beads (used as control beads) were used in Chromatin Immunoprecipitation (ChIP) experiments on HeLa cells. After equilibration, the amphiphilic Protein A / G magnetic beads were bound to IgG and Histone H3 antibodies (denoted as M1 and M2 beads, respectively), while the control Protein A / G hydrophobic magnetic beads were bound to IgG and Histone H3 antibodies (denoted as P1 and P2 beads, respectively). Unbound antibodies were washed away and the cells were then used directly in ChIP experiments. At the time point when binding of the target protein to genomic DNA was expected, 270 μl of 37% formaldehyde was added directly to 10 ml of HeLa cell culture medium and gently mixed to a final concentration of 1%. The cells were then incubated at 37°C for 10 minutes to crosslink the target protein and the corresponding genomic DNA. 1.1 ml of Glycine Solution (10X) was added, gently mixed, and allowed to stand at room temperature for 5 minutes. Place the culture dish containing the cell sample on ice and aspirate the culture medium containing formaldehyde and glycine, ensuring minimal residual liquid. While the sample is incubating at room temperature for 5 minutes, add an appropriate amount of protease inhibitors or protease phosphatase inhibitors to ice-cold PBS. Add 5-10 ml of ice-cold PBS containing protease inhibitors to wash the cells, aspirating to ensure minimal residual liquid. Add another 5-10 ml of ice-cold PBS containing protease inhibitors to further wash the cells, aspirating to ensure minimal residual liquid. Add 1 ml of ice-cold PBS containing protease inhibitors and scrape the cells with a cell scraper or spatula, collecting them into a centrifuge tube. Centrifuge at 1,000 x g for 1-2 minutes at 4°C to fully pellet the cells. Prepare an appropriate amount of SDS Lysis Buffer containing protease inhibitors. Resuspend the 1 million cell pellet from the previous step in 0.2 ml of SDS Lysis Buffer containing protease inhibitors. Incubate on ice for 10 minutes to fully lyse the cells. Perform ultrasonic treatment to shear the genomic DNA, breaking most of the DNA into fragments of 200-1000 bp. It would be better if most of the fragments can be controlled within 400-800 bp. Add 8 μl of 5M NaCl to 0.2 ml of the ultrasonically treated sample and mix well. Heat at 65°C for 4 hours to remove crosslinks between the protein and the genomic DNA. Add an equal volume of Tris-equilibrated phenol, vortex vigorously mix, and then centrifuge at 4°C, 12,000 × g for 5 minutes. Pipette the supernatant into another centrifuge tube. Add an equal volume of chloroform, vortex vigorously mix, and then centrifuge at 4°C, 12,000 × g for 5 minutes. Pipette the supernatant into another centrifuge tube and place it in an ice bath. Prepare an appropriate amount of ChIP Dilution Buffer containing protease inhibitors.Dilute the sonicated sample with 1.8 ml of ChIP Dilution Buffer containing protease inhibitors to a final volume of 2 ml. Remove 20 μl (1%) of the sample as input for subsequent analysis. Divide the remaining approximately 2 ml of sample into four aliquots, add 50 μl of P1, P2, M1, and M2 magnetic beads, and mix thoroughly by gentle rotation or rocking at 4°C for 30 minutes. Then, separate the beads on a magnetic stand for 10 seconds and transfer the supernatant to a new 2 ml centrifuge tube. Wash the beads three times for 3-5 minutes each, then separate them on a magnetic stand for 10 seconds and carefully remove the supernatant. Elute the beads with 100 μl of elution buffer (1% SDS, 0.1 M NaHCO₃) by rotating at room temperature for 3-5 minutes. Separate the beads on a magnetic stand for 10 seconds and transfer the supernatant to a new centrifuge tube. Add another 100 μl of elution buffer to the pellet. Vortex to mix thoroughly and continue elution for 3-5 minutes by rotating at room temperature. Separate the beads on a magnetic stand for 10 seconds and remove the supernatant. Combine the supernatants of the two elutions. Add 10 μl of 5M NaCl to 200 μl of supernatant and mix well. Heat at 65°C for 4 hours to remove the crosslinks between the protein and genomic DNA. For the 20 μl sample used as input, add 1 μl of 5M NaCl, mix well, and heat at 65°C for 4 hours, which is also used to remove the crosslinks between the protein and genomic DNA. After this step is completed, PCR can be used to amplify the partial gene sequence of human GAPDH (primer sequence: 5'-TACTAGCGGTTTTACGGGCG-3' (SEQ ID NO: 1); 5'-TCGAACAGGAGGAGCAGAGAGCGA-3' (SEQ ID NO: 2)) using BeyoFast. TM SYBR Green qPCR Mix (2X) (D7262) was used for verification. ChIP products can also be detected by Western blotting.

[0122] 12. RIP experiment

[0123] The amphiphilic IgG magnetic beads (M3) and amphiphilic HuR magnetic beads (M4) prepared by the present invention, as well as the control IgG hydrophobic magnetic beads (P3) and the control HuR hydrophobic magnetic beads (P4), are used for the RIP (RNA immunoprecipitation) experiment of HeLa cells. After balancing the magnetic beads, they can be directly used for the RIP experiment. After the HeLa cells are digested, 300μl of lysis buffer is added to every 1.5 million cells, and the cells are blown several times with a gun to allow the lysis buffer and cells to fully contact. The cells are placed in an ice bath for 15 minutes to fully lyse the cells, and then centrifuged at 4°C and 16,000×g for 10 minutes to take the supernatant. After balancing the above magnetic beads, 30μl is taken from each of them, washed with NT2 Wash buffer, and the magnetic beads are resuspended with 100μl, and then 300μl of cell supernatant is added and incubated on a shaker at 4°C overnight. Wash the magnetic beads and then elute with 100μl Elution buffer at 55°C for 30 minutes. Use Biyuntian RNAeasy TM Animal RNA Extraction Kit (Spin Column) (R0027) purifies RNA, and the purified RNA is directly used for experiments or stored at -80℃. TM SYBR Green qPCR Mix (2X) (D7262) was used for verification.

[0124] 13. Streptavidin magnetic beads combined with Biotin-HRP

[0125] 50 μl of each of S1, S2, and S3 magnetic beads and the three types of magnetic beads corresponding to unbound Streptavidin (denoted as C1, C2, and C3) were washed three times with PBS. Then, 2 μg of Biotin-HRP was added, followed by 300 μl of Binding Buffer (PBS (pH 7.4), 0.05% Tween-20, with or without 0.01%-0.1% BSA). The beads were incubated at room temperature for 6 hours, and then washed three times with PBS. The beads were resuspended in 50 μl of PBS and diluted 10-fold to 10,000-fold. Using a 96-well plate, 20 μl of the mixture was added to each well, followed by 100 μl of TMB colorimetric solution (Biyuntian, P0209). The plates were incubated at room temperature in the dark for 0.5 hours, and the plates were read. Finally, the data from the 100-fold dilution were used.

[0126] Example 1, Preparation process of amphiphilic magnetic beads

[0127] 1. Preparation of amphiphilic block copolymers

[0128] First, PEG-b-PMPS is prepared, wherein the hydrophilic end PEG includes polyethylene glycol methyl ether or polyethylene glycol with a molecular weight of 2000-10000; the hydrophobic end includes PMPS or PPDSM. The hydrophilic and hydrophobic ends can be cross-prepared to obtain a group of amphiphilic block copolymers (including PEG-b-PMPS or PEG-b-PPDSM). The amphiphilic block copolymers can effectively reduce the nonspecific adsorption of magnetic beads.

[0129] OA-modified magnetic beads provide an appropriate hydrophobic core that can effectively bind to amphiphilic polymers to form a structure with the hydrophobic end inside and the hydrophilic end outside. The inventors' experiments have shown that without OA modification, the hydrophilic-hydrophobic structure cannot be effectively achieved.

[0130] Figure 1 The figure shows the preparation of amphiphilic magnetic beads by modifying hydrophobic magnetic beads with PEG-b-PMPS amphiphilic block copolymer.

[0131] 2. Preparation of tosyl amphiphilic magnetic beads

[0132] The surface of the obtained amphiphilic magnetic beads was first modified with an APTES silane coupling agent, that is, the hydrophobic layer of the amphiphilic magnetic beads reacted with APETS, and then the amino group of APTES reacted with toluenesulfonyl chloride to obtain toluenesulfonyl functional groups on the surface of the magnetic beads, thereby preparing toluenesulfonyl amphiphilic magnetic beads ( Figure 2 ).

[0133] 3. Amphiphilic magnetic beads coupled to antibodies or proteins

[0134] The tosyl amphiphilic magnetic beads prepared by the present invention can be coupled to target antibodies or proteins, such as HuR antibodies and IgG, Protein A and Protein G, overnight under mild conditions without activation. The process is as follows: Figure 3 .

[0135] Example 2: ChIP experimental analysis

[0136] The following magnetic beads were used for ChIP assay analysis:

[0137] P1 magnetic beads: control Protein A / G hydrophobic magnetic beads bound to IgG antibodies;

[0138] P2 magnetic beads: control Protein A / G hydrophobic magnetic beads bound to Histone H3 antibody;

[0139] M1 magnetic beads: amphiphilic (PEG at the hydrophilic end, PMPS at the hydrophobic end, PEG molecular weight 6000) Protein A / G magnetic beads prepared by the present invention bound to IgG antibodies;

[0140] M2 magnetic beads: amphiphilic (PEG at the hydrophilic end, PMPS at the hydrophobic end, PEG molecular weight 6000) Protein A / G magnetic beads prepared by the present invention are combined with Histone H3 antibody.

[0141] ChIP experiments showed that M2 magnetic beads had a high binding capacity, with the CT value of M2 magnetic beads being similar to that of P2 magnetic beads. Negative control magnetic beads, hydrophobic control beads, were used to verify the nonspecific adsorption properties of the magnetic beads themselves. The CT value of P1 magnetic beads was found to be approximately 3 lower than that of M1 magnetic beads, indicating that the nonspecific adsorption of P1 was approximately 10 times that of M1. The CT value of M2 indicates the binding capacity; a lower value indicates higher binding of the target protein and better efficacy. The CT values of M1 and P1 indicate nonspecificity; a higher value indicates lower nonspecificity.

[0142] The specific results of using the control Protein A / G hydrophobic magnetic beads and the amphiphilic Protein A / G magnetic beads of the present invention for ChIP PCR are shown in Table 1.

[0143] Table 1

[0144]

[0145]

[0146] The experimental results of four magnetic beads Western Blot are as follows Figure 4 The bands of M2 and P2 are similar in thickness, indicating similar binding to target proteins. However, the miscellaneous bands of P1 beads are significantly more numerous than those of M1 beads, indicating that the amphiphilic Protein A / G magnetic beads prepared in this invention are significantly superior to the control Protein A / G hydrophobic magnetic beads.

[0147] Example 3, RIP experimental analysis

[0148] The following magnetic beads were used for RIP assay analysis:

[0149] M3 magnetic beads: amphiphilic (PEG at the hydrophilic end, PMPS at the hydrophobic end, PEG molecular weight 6000) IgG magnetic beads prepared by the present invention;

[0150] M4 magnetic beads: amphiphilic (hydrophilic end PEG, hydrophobic end PMPS, PEG molecular weight 6000) HuR magnetic beads prepared by the present invention;

[0151] P3 magnetic beads: control IgG hydrophobic magnetic beads;

[0152] P4 magnetic beads: control HuR hydrophobic magnetic beads.

[0153] The experimental results of using the amphiphilic antibody magnetic beads of the present invention and the control hydrophobic antibody magnetic beads for RIP PCR are shown in Table 2. The RIP experiment showed that the binding capacity of the M4 magnetic beads was high, and the CT value of the M4 magnetic beads was close to the CT value of the P4 magnetic beads; the negative control magnetic beads (control hydrophobic magnetic beads) were used to verify the non-specific adsorption performance of the magnetic beads themselves, and it was found that the CT value of the P3 magnetic beads was about 3 less than the CT value of the M3 magnetic beads, that is, the non-specific adsorption of P3 was about 10 times that of the M3 non-specific adsorption. The specific results are shown in Table 2. The CT value of M4 represents the data of the binding amount. The lower the value, the higher the target protein bound and the better the effect. The CT values of M3 and P3 represent non-specific data. The larger the value, the lower the non-specificity.

[0154] Table 2

[0155]

[0156] The experimental results of four magnetic beads Western Blot are as follows Figure 3 The bands of M4 and P4 are consistent, and the bound target proteins are similar. However, the mixed bands of P3 magnetic beads are significantly more than those of M3 magnetic beads, so the amphiphilic magnetic beads prepared by the present invention are significantly superior to the control hydrophobic magnetic beads.

[0157] Example 4: Results of Streptavidin Magnetic Beads Binding to Biotin-HRP

[0158] The amphiphilic (hydrophilic end PEG, hydrophobic end PMPS, PEG molecular weight 6000) Streptavidin magnetic beads (S1), control Streptavidin magnetic beads (S2), Biomag Streptavidin magnetic beads (S3), and corresponding tosyl magnetic beads (corresponding to the magnetic beads not bound to Streptavidin are denoted as C1, C2, and C3), a total of six types of magnetic beads, were incubated with Biotin-HRP and the TMB colorimetric reading results were obtained.

[0159] The results of the amphiphilic Streptavidin magnetic beads, control Streptavidin magnetic beads, Biomag Streptavidin magnetic beads, and corresponding tosyl magnetic beads binding to Biotin-HRP are shown in Table 3 (reading values after 100-fold dilution).

[0160] Table 3

[0161] S1 S2 S3 C1 C2 C3 0.5896 0.5794 0.4997 0.0604 0.0898 0.0908

[0162] As can be seen, the hydrophilic magnetic beads of the present invention and the control magnetic beads bound more Biotin-HRP than the Biomag magnetic beads. This result indicates that the magnetic beads of the present invention have a higher density of tosyl groups, namely, that the beads are amino-grouped by APTES and then reacted with tosyl chloride to achieve a higher density of tosyl groups. Generally speaking, the hydrophilic tosyl magnetic beads of the present invention have significantly lower nonspecific adsorption, while the nonspecific adsorption of the control magnetic beads and Biomag magnetic beads is not much different.

[0163] Example 5: Analysis of Hydrophilic End Compounds of Block Copolymers

[0164] PEO and PEG differ in their groups (CH3O-) and molecular weight. The amphiphilic magnetic beads of the present invention (PEG-b-PMPS, with PEG6000 at the hydrophilic end) were prepared as described above. Also prepared were magnetic beads (PEO5000-b-PMPS) with PEO5000 replacing PEG6000 but otherwise identical in structure, and then analyzed and compared.

[0165] 1. ChIP experiment

[0166] PEO5000 magnetic beads bound to IgG (A1 beads) and Histone H3 antibody (A2 beads). Amphiphilic Protein A / G magnetic beads prepared in this invention bound to IgG (M1 beads) and Histone H3 (M2 beads) were used in ChIP experiments. In M1, M2, A1, and A2, 2 represents binding capacity, and 1 represents nonspecific adsorption.

[0167] Table 4 shows the experimental results of ChIPPCR using PEO5000-b-PMPS Protein A / G magnetic beads and the amphiphilic Protein A / G magnetic beads of the present invention. The results show that the M2 magnetic beads have a high binding capacity, and the CT value of the M2 magnetic beads is similar to that of the A2 magnetic beads. When PEO5000-modified magnetic beads were used as a control, the CT value of the A1 magnetic beads was found to be approximately 1 less than that of the M1 magnetic beads, indicating that the nonspecific adsorption of A1 is approximately three times that of M1.

[0168] Table 4

[0169]

[0170]

[0171] 2. RIP experiment

[0172] The amphiphilic IgG magnetic beads (M3 magnetic beads) and HuR magnetic beads (M4 magnetic beads) prepared by the present invention, and the IgG magnetic beads (A3 magnetic beads) and HuR magnetic beads (A4 magnetic beads) prepared by PEO5000-b-PMPS magnetic beads were used for RIP experiments.

[0173] The experimental results of using the amphiphilic antibody magnetic beads of the present invention and PEO5000-b-PMPS magnetic beads for RIP PCR are shown in Table 5. The results show that the binding capacity of M4 magnetic beads is high, and the CT value of M4 magnetic beads is close to the CT value of P4 magnetic beads. The CT value of A3 magnetic beads is approximately 1 less than that of M3 magnetic beads, indicating that the nonspecific adsorption of A3 is approximately three times that of M3.

[0174] Table 5

[0175]

[0176] Therefore, the non-specific adsorption of PEG6000-based immunomagnetic beads is lower.

[0177] Example 6: Analysis of PMPC in Reducing Nonspecific Adsorption

[0178] 1. ChIP PCR experiment

[0179] The magnetic beads of the present invention (PEG-b-PMPS, with PEG6000 at the hydrophilic end) were stored in blocking solution PBS + 0.5% BSA (blocking solution 1) and PBS + 0.5% BSA + 0.1% PMPC amphiphilic surfactant (blocking solution 2) to investigate the nonspecific adsorption of the magnetic beads.

[0180] The amphiphilic Protein A / G magnetic beads prepared by the present invention were stored in the above two blocking solutions for 24 hours and then used to bind IgG (M1 magnetic beads) and Histone H3 magnetic beads (M2 magnetic beads) for ChIP experiments. Those stored in blocking solution 1 were marked as B, and those stored in blocking solution 2 were normal M1 and M2.

[0181] Table 3 shows the experimental results of ChIP PCR using the amphiphilic Protein A / G magnetic beads of the present invention after being stored in two blocking solutions. The results show that the binding capacity of the M2 magnetic beads was high, and the CT value of the M2 magnetic beads was similar to that of the P2 magnetic beads. The CT value of the B1 magnetic beads was approximately 0.5 lower than that of the M1 magnetic beads, indicating that the nonspecific adsorption of A1 was approximately 1.5 times that of M1. Specific results are shown in Table 6 below.

[0182] Table 6

[0183]

[0184] Therefore, the blocking solution containing PMPC of the present invention has a better ability to reduce nonspecific adsorption.

[0185] 2. RIP experiment

[0186] The magnetic beads of the present invention (PEG-b-PMPS, with PEG6000 at the hydrophilic end) were stored in blocking solution PBS + 0.5% BSA (blocking solution 1) and PBS + 0.5% BSA + 0.1% PMPC amphiphilic surfactant (blocking solution 2), and the nonspecific adsorption of the magnetic beads was investigated.

[0187] The amphiphilic IgG magnetic beads (M3 magnetic beads) and HuR magnetic beads (M4 magnetic beads) prepared by the present invention are stored in blocking solution 1 and are marked as B magnetic beads. The prepared IgG magnetic beads (B3 magnetic beads) and HuR magnetic beads (B4 magnetic beads) are used for RIP experiments.

[0188] The experimental results of using the amphiphilic Protein A / G magnetic beads of the present invention in RIP PCR after being stored in two blocking solutions are shown in Table 7.

[0189] The M4 magnetic beads showed a high binding capacity, with the CT value of the M4 beads being close to that of the P4 magnetic beads. The CT value of the B3 magnetic beads was found to be approximately 0.5 lower than that of the M3 magnetic beads, indicating that the nonspecific adsorption of the B3 beads was approximately 1.5 times that of the M3 beads. Detailed results are shown in Table 7.

[0190] Table 7

[0191]

[0192] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the appended claims. At the same time, all documents mentioned in this application are cited as references in this application, just as if each document was cited as a reference individually.

Claims

1. A method for preparing immunomagnetic beads, characterized in that: The method comprises: (1) preparing hydrophobic magnetic beads; the hydrophobic magnetic beads are hydrophobic iron oxide magnetic beads (OA@Fe3O4); the preparation method of the hydrophobic magnetic beads comprises: coating iron oxide with oleic acid to obtain oleic acid-coated hydrophobic magnetic beads; (2) coating the hydrophobic magnetic beads with an amphiphilic block copolymer to obtain amphiphilic block copolymer-coated magnetic beads; The amphiphilic block copolymer is PEG-b-PMPS, which includes a hydrophilic end and a hydrophobic end, wherein the hydrophilic end is polyethylene glycol (PEG) and the hydrophobic end is poly(3-methacryloxypropyltrimethoxysilane) (PMPS); the molecular weight of the PEG is 6000 ± 500; the structure of the PEG-b-PMPS is as follows: ； wherein x is 20 to 30, and the value of n is consistent with the molecular weight of the PEG; PEG 6000 ± 500, triethylamine (TEA), and 2-chloro-2,2-diphenylacetyl chloride (CTA) are added to tetrahydrofuran, mixed, and refluxed at normal pressure; tetrahydrofuran is removed, and chloroform is added to form a solution; PEG-CTA is obtained by washing with a sodium bicarbonate solution, drying the organic phase, and removing the solvent; The PEG-b-PMPS was prepared by adding the PEG-CTA, 3-(trimethoxysilyl)propyl methacrylate (MPS), and azobisisobutyronitrile (AIBN) to 1,4-dioxane, mixing, vacuum sealing, polymerizing in an oil bath at 70° C., and precipitating with petroleum ether. The amphiphilic block copolymer-coated magnetic beads are prepared by dissolving the PEG-b-PMPS in anhydrous tetrahydrofuran, mixing the mixture with the OA@Fe3O4, aging the mixture, and adding the mixture dropwise to deionized water under stirring. The mixture is dialyzed with a 40 μm cell filter to remove large aggregates, and the flow-through is collected to obtain the amphiphilic block copolymer-coated magnetic beads. (3) performing a tosylation reaction on the magnetic beads coated with the amphiphilic block copolymer to obtain tosylated amphiphilic magnetic beads; The surface of the amphiphilic magnetic beads is first modified with (3-aminopropyl)triethoxysilane (APTES); the process includes ultrasonically dispersing the amphiphilic block copolymer-coated magnetic beads with isopropyl alcohol, adding APTES, washing, and then dispersing the magnetic beads in acetone; and then reacting with 4-toluenesulfonyl chloride at room temperature, whereby the chlorine of the toluenesulfonyl chloride directly reacts with the amino groups of the magnetic beads; (4) coupling and blocking the tosylating amphiphilic magnetic beads to obtain the immunomagnetic beads; The tosylated amphiphilic magnetic beads are coupled to the capture molecules and blocked with a blocking solution containing polymethacryloyloxyethylphosphorylcholine (PMPC) to obtain immunomagnetic beads coupled with the capture molecules; the blocking solution is a PBS buffer solution containing BSA and 0.05-0.3% PMPC.

2. The method according to claim 1, wherein The capture molecule is a biomolecule carrying an amino group.

3. The method according to claim 2, wherein The capture molecule is a protein.

4. The method according to claim 3, wherein The capture molecule is an antibody or a ligand.

5. The method according to claim 1, wherein The amount of PMPC is 0.1%.

6. Use of immunomagnetic beads in immune response, wherein the immunomagnetic beads are modified amphiphilic magnetic beads coated with amphiphilic block copolymers and having tosyl functional groups on their surfaces; wherein: The amphiphilic block copolymer includes a hydrophilic end and a hydrophobic end, the hydrophilic end is PEG, and the hydrophobic end is PMPS; the molecular weight of the PEG is 6000±500; the immunomagnetic beads are prepared by the method described in any one of claims 1 to 5; the application is non-disease diagnostic and non-disease therapeutic.

7. The use according to claim 6, characterized in that The immune response includes: protein sorting or immobilization, nucleic acid sorting or immobilization.

8. The use according to claim 7, characterized in that The sorting was performed by immunoprecipitation.

9. A method for conducting an immune response using immunomagnetic beads, characterized in that: include: (a) providing immunomagnetic beads prepared by the method according to any one of claims 1 to 5, wherein the immunomagnetic beads are coupled to capture molecules, and the capture molecules capture target molecules; (b) contacting the immunomagnetic beads of (a) with the sample to be tested to capture the target molecules in the sample to be tested; The method is a non-disease diagnostic and non-disease therapeutic method.

10. The method according to claim 9, wherein The immune response method includes: protein sorting or immobilization, nucleic acid sorting or immobilization.

11. The method according to claim 10, wherein The sorting was performed by immunoprecipitation.

12. The method according to claim 11, wherein The immunoprecipitation includes: chromatin immunoprecipitation and RNA binding protein immunoprecipitation.

13. A kit for immune response, characterized in that The kit comprises the immunomagnetic beads prepared by the method according to any one of claims 1 to 5.

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