Immunodiagnosis magnetic microsphere with protein nonspecific adsorption resistance and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine micro-nano materials, and particularly relates to a magnetic microsphere with protein nonspecific adsorption resistance for immunodiagnosis as well as a preparation method and application of the magnetic microsphere. The preparation method comprises the following steps: S01, carrying out free radical copolymerization reaction on magnetic microspheres and a cross-linked copolymer material under the action of an initiator to introduce carboxyl to obtain first magnetic microspheres; s02, carrying out covalent coupling on the first magnetic microspheres, micromolecules or polymers with polyamino functional groups and micromolecules or polymers with polycarboxyl functional groups to obtain second magnetic microspheres; S03, adding zwitterions into the reaction system, and obtaining third magnetic microspheres on the basis of the second magnetic microspheres; and S04, adding a functional monomer with a carboxyl group or an epoxy group into the reaction system, and obtaining a final compound microsphere on the basis of the third magnetic microsphere. The composite magnetic microspheres provided by the invention can be used for immunodetection, and have higher signals and lower background.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical micro-nano materials, and specifically relates to a magnetic microsphere for immunodiagnosis with resistance to nonspecific protein adsorption, and a preparation method and application thereof. Background Art

[0002] Magnetic microspheres are a type of micro-nanoparticles with superparamagnetic properties and a specifically modified shell. They can be widely used in the biomedical field to achieve a variety of complex technologies such as cell sorting, immune recognition, nucleic acid separation and protein purification by coupling with different ligands. Magnetic microspheres used in the field of immunodiagnosis usually need to have the characteristics of high test signal and low background. Currently, this is usually achieved by functional modification of the microsphere surface and anti-nonspecific adsorption treatment.

[0003] There are several methods in the prior art to perform functional modification on the surface of micro-nano microspheres: (1) Through copolymerization, functional monomers and skeleton structure monomers or polymers with active functional groups are coated on the outside of the microspheres, thereby introducing the required active functional groups on the surface of the magnetic microspheres. However, this type of modification will inevitably lead to some functional groups being coated inside the microspheres, and there are fewer functional groups on the surface of the microspheres. Therefore, the number of active groups of the obtained magnetic microspheres for biocoupling is small, resulting in low subsequent coupling efficiency. (2) Through chemical modification methods such as Friedel-Crafts reaction, esterification, and amidation, specific functional groups are introduced on the surface of polymer microspheres under specific chemical reagents and reaction conditions, and then functional coatings and active reaction groups are gradually constructed on the introduced functional groups. Document WO2017211145A1 discloses a microsphere product covalently modified by a hydrophilic substance, which has lower nonspecific adsorption of proteins, nucleic acids and other substances than the micro-nano material itself, while retaining the activity of specific biological ligands. However, the modification process is cumbersome and requires the use of toxic or dangerous chemical reagents. It has high requirements on the safety of the environment and operators. At the same time, the reaction process control is complex, which is not conducive to industrial scale-up. Document CN116272706A discloses a method for modifying the surface of magnetic polymer microspheres. By chelating carboxyl hydrophobic long-chain molecules, and then adsorbing and cross-linking with amino-functional hydrophilic polymer molecules, amino-functionalized hydrophilic magnetic polymer microspheres are formed. Such microspheres have a high content of functionalized groups on the surface, and due to the introduction of hydrophilic groups, the nonspecific adsorption of proteins, etc. can be reduced to a certain extent. However, since such modification cannot achieve complete balance of surface charge, biomacromolecules will still be adsorbed due to electrostatic effects. (3) The functionalization of the microsphere surface can be achieved by using controlled free radical polymerization technology such as atom transfer radical polymerization (ATRP), which can accurately control the types of functional molecules, polymer chain length and distribution on the microsphere surface. Document CN101798372A discloses a method for grafting ATRP initiators on the microsphere surface using Friedel-Crafts acylation reaction, and then constructing multi-epoxy group modification sites by ATRP reaction. However, the initiator sites of this strategy are uncontrollable and may be inside the polymer, and the density is also difficult to control. (4) Connecting functional small molecules or polymers to the surface of microspheres by "grafting". Document CN114395135B discloses a modification method for grafting hyperbranched polyglycidyl ethers onto the surface of polymer microspheres, using this hydrophilic group as an effective component to resist nonspecific protein adsorption, and also as an active site to couple other biological molecules through activation. However, this type of grafting involves the reaction of macromolecules at the interface of micro-nano materials, and its reaction activity is not high and is difficult to monitor, making it difficult to achieve industrial scale-up.

[0004] In summary, it is necessary to propose improvements to the shortcomings of the existing technology. Summary of the invention

[0005] The purpose of the present invention is to provide a magnetic microsphere for immunodiagnosis with resistance to nonspecific protein adsorption and a preparation method and application thereof, so as to partially solve or alleviate the above-mentioned deficiencies in the prior art. The present invention specifically adopts the following technical solutions.

[0006] A method for preparing composite magnetic microspheres with resistance to nonspecific protein adsorption comprises the following steps: S01: introducing a first functional group on the magnetic microsphere by free radical copolymerization with a cross-linked copolymer material under the action of a first initiator to obtain a first magnetic microsphere; the cross-linked copolymer material comprises styrene and its derivative monomers, a cross-linking monomer and a first functional compound; the first functional group comprises a carboxyl group or an epoxy group; Wherein, the first initiator is a free radical copolymerization initiator; the first functional compound is a compound having a carboxyl group or an epoxy group (or also called a functional monomer having a carboxyl group or an epoxy group); Wherein, the mass ratio of the magnetic microspheres to the cross-linked copolymer material is 1: (1-100); the mass ratio of the first initiator to the styrene and its derivative monomers is (0.1-10): 100; S02: the first magnetic microsphere is coupled with the second functional compound through a ring-opening reaction or an amide reaction, and then covalently coupled with the third functional compound and the second initiator through an amide reaction to obtain the second magnetic microsphere; Wherein, the second functional compound is a small molecule or polymer having multiple amino functional groups; the third functional compound is a small molecule or polymer having multiple carboxyl functional groups; Wherein, the second initiator is an atom transfer radical polymerization initiator, including BMC or a BMC derivative; Wherein, the mass ratio of the first magnetic microsphere to the second functional compound is 1:(0.1-10); the mass ratio of the first magnetic microsphere to the third functional compound is 1:(0.1-10); the mass ratio of the first magnetic microsphere to the second initiator is 1:(0.1-10); S03: adding zwitterions to the reaction system, and the second initiator on the second magnetic microsphere initiates an atom transfer radical polymerization reaction of the zwitterion monomer to obtain a third magnetic microsphere, wherein the third magnetic microsphere has an active initiating end; Wherein, the mass ratio of the second magnetic microsphere to the zwitterion is 1:(0.1-5); Wherein, the zwitterionic monomer is selected from one or more of TMAO, CBMA or SBMA; S04: adding a fourth functional compound having a carboxyl group or an epoxy group (also called a functional monomer having a carboxyl group or an epoxy group) into the reaction system, and the active initiating end on the third magnetic microsphere undergoes a transfer radical polymerization reaction with the fourth functional compound to obtain the composite magnetic microsphere; Wherein, the mass ratio of the third magnetic microsphere to the fourth functional compound is 1:(0.1-5); Wherein, the fourth functional compound is selected from one or more of CBMA and OEG.

[0007] Furthermore, the chemical formula of the compound involved in the above preparation method is as follows.

[0008] BMC; BMC derivatives; TMAO; CBMA; SBMA; OEG.

[0009] Furthermore, the magnetic microspheres used in S01 are common commercially available magnetic microspheres. In some preferred embodiments, the particle size of the magnetic microspheres is 1 μm-5 μm.

[0010] Further, in S01, the styrene and its derivative monomers include one or more materials selected from the group consisting of styrene, 4-methylstyrene, 3-methylstyrene and 4-tert-butylstyrene; or, the cross-linking monomer includes one or more materials selected from the group consisting of divinylbenzene, ethylene glycol dimethacrylate, bisphenol A dimethacrylate, poly (ethylene glycol) diacrylate and N,N'-methylenebis (acrylamide); or, the first functional compound includes one or more materials selected from the group consisting of allyl glycidyl ether, glycidyl methacrylate, glycidyl acrylate, diglycidyl phthalate, diglycidyl terephthalate, diglycidyl adipate, methacrylic acid, and acrylic acid.

[0011] Furthermore, in S01, the mass ratio of the magnetic microspheres to the styrene and its derivative monomers is 1:(1-10); or, the mass ratio of the styrene and its derivative monomers to the cross-linking monomer is 1:(0.01-1); or, the mass ratio of the styrene and its derivative monomers to the first functional compound is 1:(0.1-10).

[0012] Further, the first initiator includes one or more materials selected from the group consisting of dibenzoyl peroxide, dicumyl peroxide, dodecyl peroxide, di-tert-butyl peroxide, azobisisobutyronitrile, azobisisoheptanenitrile and dimethyl azobisisobutyrate.

[0013] Further, in S02, the second functional compound includes one or more materials selected from the group consisting of ethylenediamine, propylenediamine, butylenediamine, spermine, spermidine, polyethyleneimine and polyacrylamine; or, the third functional compound includes one or more materials selected from the group consisting of oxalic acid, malonic acid, succinic acid, citric acid, tartaric acid, fumaric acid, maleic acid, polymethacrylic acid, polyacrylic acid and polymaleic acid.

[0014] Furthermore, R1 in S02 is selected from amino, bromine, chlorine and / or N-hydroxysuccinimide; R2, R3 and R4 respectively represent an alkyl group with a carbon chain length of 1 to 6. R2, R3 and R4 may be the same or different.

[0015] Furthermore, in S03, M represents an oxygen atom or a nitrogen atom; R5 represents an alkyl group with a carbon chain length of 2-6; and R6 represents an alkyl group with a carbon chain length of 2-6.

[0016] Furthermore, in S04, M represents an oxygen atom or a nitrogen atom; R5 represents an alkyl group with a carbon chain length of 2-6; R6 represents an alkyl group with a carbon chain length of 2-6; n=1-200, preferably n=10-200.

[0017] Furthermore, the atom transfer radical polymerization reaction described in S03 and S04 also includes a ligand and a catalyst system; the catalyst system includes one or more materials selected from the group consisting of cupric chloride, cuprous chloride, cupric bromide, cuprous bromide and sodium ascorbate.

[0018] As a preferred embodiment, the composite magnetic microspheres may be further coupled with streptavidin (SA), avidin or biotin.

[0019] The composite magnetic microspheres with resistance to nonspecific protein adsorption are prepared by the preparation method.

[0020] The composite magnetic microspheres with resistance to nonspecific protein adsorption prepared by the preparation method are used in the preparation of in vitro diagnostic reagents.

[0021] The composite magnetic microspheres with resistance to non-specific protein adsorption are used in the preparation of in vitro diagnostic reagents.

[0022] Beneficial technical effects: The present invention first provides a composite magnetic microsphere with dual functions, by first introducing a first functional group with an anchoring layer function on the surface of the basic magnetic microsphere; then constructing a signal amplification system, which can increase the initiation site density of controllable active polymerization; further utilizing the ATRP reaction to construct an anti-nonspecific adsorption coating based on zwitterions (derivatives); finally utilizing the ATRP reaction to construct multi-site active groups, greatly increasing the coupling rate of proteins or antibodies. Specifically, the composite magnetic microspheres described in the present invention have at least two functions: the signal amplification system increases the density of groups that can participate in coupling on the surface of the magnetic microspheres, thereby significantly increasing the amount of antibody, antigen or protein coupling and improving reactivity; the zwitterion anti-nonspecific adsorption modification layer can significantly reduce the nonspecific adsorption of antibodies, antigens or proteins on the surface of the magnetic microspheres, thereby reducing the background, ensuring that the magnetic microspheres have high reactivity and extremely low background; therefore, the composite magnetic microspheres described in the present invention have at least higher reactivity and better anti-nonspecific adsorption performance. In addition, the composite magnetic microspheres of the present invention are modified with multiple functional layers, but there is almost no adverse interference between the modified layers. The experiments of the present invention prove that the composite magnetic microspheres have higher stability (linear width) than imported reagents. Based on this, the composite magnetic microspheres of the present invention can be used to develop immunodiagnostic reagents, which have higher signals and lower background than comparative reagents and have higher commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0024] Figure 1 The schematic diagram of the synthesis of magnetic microspheres with resistance to nonspecific adsorption of the present invention; Figure 2 This is a scanning electron microscope image of magnetic microspheres S3 prepared in one embodiment of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0027] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0028] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0029] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0030] Definition of noun: The "anti-nonspecific adsorption" mentioned in the present invention means that the composite magnetic microspheres prepared by the present invention can significantly reduce the nonspecific adsorption of antibodies, antigens or protein couplings, thereby reducing the background. Those skilled in the art can understand that "background" is a measure of the ability of magnetic beads to specifically bind to antibodies, antigens or proteins. The lower the background reading, the better the anti-nonspecific binding (anti-nonspecific) ability of the magnetic beads.

[0031] The "signal amplification" mentioned in the present invention refers to increasing the density of groups on the surface of the magnetic microspheres that can participate in coupling.

[0032] Embodiment 1 Preparation of magnetic microspheres S1.

[0033] Based on commercially available magnetic microspheres with a particle size range of 1 μm-5 μm, free radical copolymerization occurs with a cross-linked copolymer material to perform a first layer modification on the magnetic microspheres, and the first modification layer can be called an anchoring layer.

[0034] The cross-linked copolymer material includes styrene and its derivative monomers, cross-linked monomers, and the first functional compound, which are formed by free radical copolymerization with the outer double bonds of the magnetic microspheres.

[0035] Wherein, the styrene and its derivative monomers are selected from one or more of the following monomers: styrene, 4-methylstyrene, 3-methylstyrene and 4-tert-butylstyrene.

[0036] The cross-linking monomer is selected from one or more of the following monomers: divinylbenzene, ethylene glycol dimethacrylate, bisphenol A dimethacrylate, poly(ethylene glycol) diacrylate, and N,N'-methylenebis(acrylamide).

[0037] The first functional compound has a carboxyl group or an epoxy group.

[0038] Among them, the first functional compound is selected from one or more of the following monomers: methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, glycidyl methacrylate, diglycidyl phthalate, diglycidyl terephthalate, diglycidyl adipate, methacrylic acid, and acrylic acid.

[0039] The free radical copolymerization reaction involved in this embodiment needs to be carried out under at least one of the following conditions.

[0040] (1) The mass ratio of the magnetic microspheres to the styrene and its derivative monomers is 1:(1-10).

[0041] (2) The mass ratio of the styrene and its derivative monomers to the crosslinking monomer is 1:(0.01 - 1).

[0042] (3) The mass ratio of the styrene and its derivative monomers to the first functional compound is 1:(0.1 - 10).

[0043] (4) The free radical copolymerization reaction is initiated by free radical copolymerization, wherein the mass ratio of the first initiator to the styrene and its derivative monomers used in the reaction is (0.1-10):100.

[0044] Among them, the first initiator described in this embodiment includes one or more of dibenzoyl peroxide, diisopropylbenzene peroxide, dodecyl peroxide, di-tert-butyl peroxide, azobisisobutyronitrile, azobisisoheptanenitrile and dimethyl azobisisobutyrate.

[0045] (5) The copolymerization reaction is carried out in a solution environment, and the reaction solvent used includes one or more of anhydrous ethanol, tetrahydrofuran, ethyl acetate, isopropanol, methanol, n-hexane and dioxane.

[0046] (6) The reaction temperature of the free radical copolymerization reaction is 60-85°C, and the reaction time is 2-24h.

[0047] Two specific examples are given below.

[0048] Example 1: Take 20g of magnetic microspheres with a particle size of 2μm and replace them with anhydrous ethanol three times, then disperse them in 1000mL of anhydrous ethanol and ultrasonicate for 5min. Transfer the dispersed magnetic microsphere solution into a three-necked flask, add 52.5g of styrene, 5.25g of ethylene glycol dimethacrylate and 15.75g of methacrylic acid in turn, replace them with nitrogen three times, then add 0.26g of dibenzoyl peroxide, replace them with nitrogen again three times, then heat to 80℃ and reflux, stir for 16h. After the reaction is completed, cool to room temperature, separate with a magnetic stand, pour off the clear liquid, wash with ethyl acetate three times, then wash with anhydrous ethanol three times, and finally wash with purified water three times, and finally disperse in pure water to obtain magnetic microspheres S1 with the first carboxyl group modified on the surface, referred to as magnetic microspheres S1, and the surface carboxyl content is measured to be 17μmol / g. The carboxyl groups on the magnetic microspheres S1 are connected to the magnetic microspheres by copolymerization.

[0049] Example 2: Take 20g of magnetic microspheres with a particle size of 2μm and replace them with anhydrous ethanol three times, then disperse them in 1000mL of anhydrous ethanol and ultrasonicate for 5min. Transfer the dispersed magnetic microsphere solution into a three-necked flask, add 52.5g of styrene, 2.63g of divinylbenzene and 21.0g of glycidyl methacrylate in sequence, replace them with nitrogen three times, then add 0.31g of dibenzoyl peroxide, replace them with nitrogen again three times, then heat to 80℃ and reflux, stir for 16h. After the reaction is completed, cool to room temperature, separate with a magnetic stand, pour off the clear liquid, wash with ethyl acetate three times, then wash with anhydrous ethanol three times, and finally wash with purified water three times, finally disperse in pure water to obtain surface epoxy-modified magnetic microspheres S1', referred to as magnetic microspheres S1'. The epoxy groups on the magnetic microspheres S1' are connected to the magnetic microspheres by copolymerization.

[0050] Embodiment 2 Preparation of magnetic microspheres S2.

[0051] On the basis of S1 or S1', a second modification layer is constructed. The second modification layer is mainly a signal amplification system constructed based on covalent bonding. Functional molecules are further grafted on the first modification layer, and the functional molecules include small molecules or polymers with multiple amino functional groups (second functional compounds) and small molecules or polymers with multiple carboxyl functional groups (third functional compounds).

[0052] The small molecules or polymers with multiple amino functional groups and the small molecules or polymers with multiple carboxyl functional groups are connected by covalent coupling; the amino-carboxyl polymer generated by covalent coupling is further connected to the carboxyl or epoxy group on the first modified layer. In addition, the second modified layer further introduces an atom transfer radical polymerization initiator molecule (the second initiator) for subsequent reaction preparation, and the atom transfer radical polymerization initiator molecule is based on the coupling of the amino or carboxyl group and BMC by chemical bonds.

[0053] Wherein, the small molecule or polymer having multiple amino functional groups is selected from one or more of the following molecules: ethylenediamine, propylenediamine, butylenediamine, spermine, spermidine, polyethyleneimine, and polypropyleneamine.

[0054] The small molecule or polymer having multiple carboxyl functional groups is selected from one or more of the following molecules: oxalic acid, malonic acid, succinic acid, citric acid, tartaric acid, fumaric acid, maleic acid, polymethacrylic acid, polyacrylic acid, and polyethylene polymaleic acid.

[0055] Among them, the second initiator includes BMC and its derivatives, and the structure is as follows: ; .

[0056] Among them, R1 can be selected from active groups such as hydroxyl, bromine, chlorine, N-hydroxysuccinimide, etc., preferably N-hydroxysuccinimide.

[0057] R2, R3 and R4 are alkyl groups with a carbon chain length of C1-C6. R2, R3 and R4 may be the same or different, and are preferably ethyl or propyl.

[0058] Wherein, the covalent coupling is carried out as follows: In the first step, the first functional group anchored on the first modified layer is coupled with a small molecule or polymer having multiple amino functional groups through a ring-opening reaction or an amide reaction.

[0059] In the second step, a small molecule or polymer having multiple carboxyl functional groups is further covalently coupled with the amino functional groups on the product of the first step reaction through an amide reaction.

[0060] Optionally, the above covalent coupling method can be constructed once or multiple times to effectively adjust the signal amplification degree of the second modification layer.

[0061] A specific example is given below.

[0062] Preparation of MEST solution: Add 9.76 g of morpholineethanesulfonic acid (MEST) and 5 L of purified water into a beaker, stir to mix, adjust the pH to 5.0 with 0.1 M sodium hydroxide aqueous solution, add 2.5 g of Tween 20 and continue stirring for 30 min to obtain MEST solution.

[0063] Preparation of PB solution: Disodium dihydrogen phosphate monohydrate (27.6 g, 0.2 mol) was dissolved in purified water and diluted to 1000 mL to obtain solution A; disodium hydrogen phosphate heptahydrate (53.6 g, 0.2 mol) was dissolved in purified water and diluted to 1000 mL to obtain solution B. 53 mL of solution A and 94.7 mL of solution B were mixed evenly, and finally the mixed solution was diluted to 2000 mL with purified water and mixed evenly to obtain PB solution.

[0064] Preparation Example 1 of Amino Magnetic Microspheres: Weigh 15g (dry weight) of magnetic microspheres S1 (carboxyl-magnetic microspheres) in a beaker, replace with MEST solution 3 times, then transfer to the reaction bottle with 450mL of MEST solution, and stir ultrasonically for 10min. Use a beaker to weigh 50mL of MEST solution and 15g of PEI, stir and mix, add to the reaction bottle at one time, and continue ultrasonic stirring for 10min. Stop ultrasound, weigh 50mL of MEST solution and 15g of EDCI in a beaker, ultrasonically dissolve, and then slowly add to the reaction bottle through a constant pressure dropping funnel, controlling the drop rate to about 1-2 drops / s. After the drop is complete, control the internal temperature at 25℃ and stir for 3h. After stirring, transfer to a magnetic stand for separation with a beaker, pour off the clear liquid, wash with purified water 5 times, and finally disperse in pure water to obtain amino-modified magnetic microspheres S2'.

[0065] Preparation Example 2 of Amino Magnetic Microspheres: Weigh 15g (dry weight) of magnetic microspheres S1' (epoxy-magnetic microspheres) in a beaker, replace with 1,4-dioxane solution 3 times, then transfer to the reaction bottle with 1500mL of 1,4-dioxane solution, and ultrasonically stir for 10min. Use a beaker to weigh 150mL of 1,4-dioxane solution and 15g of polyethyleneimine, stir and mix, then add to the reaction bottle at one time, and continue ultrasonic stirring for 10min. Heat to 70℃, control the internal temperature at 70℃, and stir for 12-16h. After stirring, transfer to a magnetic stand for separation with a beaker, pour off the clear liquid, wash with purified water 5 times, and finally disperse in pure water to obtain amino-modified magnetic microspheres S2''.

[0066] Preparation of magnetic microspheres S2: Weigh 10g (dry weight) of microspheres (magnetic microspheres S2' or magnetic microspheres S2'') in a beaker, replace with PB solution 3 times, then transfer to the reaction bottle with 400mL of PB solution, and stir ultrasonically for 10min. Then weigh 10g of BMC and 50g of PB solution in a beaker, add them to the reaction bottle at one time after ultrasonic dispersion. After the addition is complete, control the internal temperature at 45℃ and stir for 3h. After stirring, transfer to a magnetic stand for separation with a beaker, pour off the clear liquid, wash with purified water 5 times, and finally disperse in pure water to obtain initiator magnetic microspheres S2, referred to as magnetic microspheres S2.

[0067] It can be understood that a signal amplification system is constructed on the magnetic microsphere S2.

[0068] Embodiment 3 Preparation of magnetic microspheres S3.

[0069] The third modified layer is constructed, which is a coating layer for resisting non-specific adsorption. The coating layer for resisting non-specific adsorption is mainly constructed by the initiator molecules on the second modified layer, which trigger the atom transfer radical polymerization reaction of zwitterionic monomers.

[0070] Wherein, the zwitterionic monomer is selected from one or more of TMAO, CBMA, and SBMA, and the structural formula is as follows.

[0071] ; ; .

[0072] Wherein, M can be oxygen or nitrogen, preferably nitrogen.

[0073] R5 and R6 are alkyl groups with a carbon chain length of C2-C6. R5 and R6 may be the same or different, and are preferably ethyl or propyl.

[0074] The atom transfer radical polymerization reaction involved in this embodiment needs to be carried out under at least one of the following conditions: (1) The mass ratio of magnetic microspheres to TMAO / CBMA / SBMA and their derivatives or their combination is 1:(0.1-5).

[0075] (2) The atom transfer radical polymerization reaction includes zwitterionic monomers and derivatives or their combination, initiator, ligand and catalyst. The catalyst is selected from cupric chloride, cuprous chloride, cupric bromide, cuprous bromide and sodium ascorbate or a combination thereof. In some specific embodiments, the mass ratio of zwitterionic monomers and derivatives or their combination, initiator, ligand, cupric chloride and sodium ascorbate is (5-100): 10: 2.7: 1: 0.2.

[0076] (3) The polymerization time is 0.5h - 24h. Preferably, the polymerization time is 0.5h - 8h; more preferably, the polymerization time is 0.5h - 3h.

[0077] (4) The reaction temperature is 0 - 70°C; preferably, the reaction temperature is 20 - 50°C.

[0078] (5) The reaction is carried out in an aqueous solution or an alcohol solution; preferably, in an alcohol solution such as methanol, ethanol, isopropanol, etc.; more preferably, in ethanol.

[0079] A specific example is given below.

[0080] Preparation of deoxygenated ethanol: Add 2000 mL of anhydrous ethanol into a three-necked flask and bubble nitrogen to deoxygenate for about 1 hour.

[0081] Catalyst preparation: 1 g of chlorinated ketone, 0.2 g of sodium ascorbate and 100 mL of deoxygenated ethanol were added to a three-necked flask in sequence, stirring was started, and then 2.7 g of tris-(2-dimethylaminoethyl)amine was added. Under nitrogen protection, stirring was carried out at room temperature for 5 min.

[0082] Preparation of magnetic microspheres S3: Weigh 10g (dry weight) of magnetic microspheres S2 in a beaker, replace it with anhydrous ethanol twice, then replace it with deoxygenated ethanol once, and finally transfer it to a three-necked flask with 400mL of deoxygenated ethanol and start stirring. Then take 20g of SBMA in a beaker, ultrasonically dissolve it with 100mL of deoxygenated ethanol, and then add it to the reaction bottle all at once. After the addition, under nitrogen protection, add the catalyst to the reaction bottle all at once. After the addition, replace it with nitrogen three times, raise the temperature to 35°C, and stir for 3h. After stirring, transfer it to a magnetic stand for separation with a beaker, pour out the clear liquid, wash it with anhydrous ethanol three times, and then wash it with purified water three times. Finally, disperse it in pure water to obtain zwitterion-modified magnetic microspheres S3, referred to as magnetic microspheres S3. The scanning electron microscope image is as shown Figure 2 shown.

[0083] It can be understood that a zwitterionic polymer structure with a brush-like morphology is constructed on the magnetic microsphere S3, which has a non-specific adsorption function against polypeptides, proteins or antibodies.

[0084] Embodiment 4 Magnetic microspheres S4, S5 and S6 with coupling modification layers were prepared.

[0085] The construction of the coupling modification layer introduces reactive groups, such as carboxyl, p-toluenesulfonyl, etc., for coupling proteins or antibodies. It is constructed by the atom transfer radical polymerization reaction (ATRP reaction) of functional monomers with active groups initiated by the active initiating end on the third modification layer.

[0086] Among them, the functional monomer with an active group (the fourth functional compound) is selected from one or more of CBMA and OEG molecules, and the structural formula is as follows.

[0087] ; .

[0088] Wherein, M can be oxygen or nitrogen, preferably nitrogen.

[0089] R5 and R6 are alkyl groups with a carbon chain length of C2-C6. R5 and R6 may be the same or different, and are preferably ethyl or propyl.

[0090] The degree of polymerization n in OEG ranges from 10 to 200.

[0091] The atom transfer radical polymerization reaction involved in this embodiment needs to be carried out under at least one of the following conditions: (1) The mass ratio of zwitterion-modified magnetic microspheres to CBMA or OEG and their combination is 1:(0.1-5).

[0092] (2) Atom transfer radical polymerization reaction includes CBMA or OEG and their combination, initiator, ligand and catalyst. In some embodiments, the mass ratio of CBMA or OEG and their combination, initiator, ligand, copper chloride and sodium ascorbate is (5-100): 10: 2.7: 1: 0.2.

[0093] (3) The polymerization time is 0.5h - 24h; preferably, the polymerization time is 8h - 24h; more preferably, the polymerization time is 10h - 16h.

[0094] (4) The reaction temperature is 0 - 70°C; preferably, the reaction temperature is 40 - 70°C.

[0095] (5) The reaction is carried out in an alcohol solution such as methanol, ethanol, isopropanol or an aqueous solution; preferably, the reaction is carried out in an aqueous solution.

[0096] Three specific examples are given below.

[0097] Preparation of low carboxyl content coupled modified layer magnetic microspheres S4.

[0098] Preparation of deoxygenated water: Add 2000 mL of purified water into a three-necked flask and bubble nitrogen to deoxygenate for about 1 hour.

[0099] Catalyst preparation: Add 1 g of chlorinated ketone, 0.2 g of sodium ascorbate and 100 mL of deoxygenated water into a three-necked flask in sequence, start stirring, then add 2.7 g of tris-(2-dimethylaminoethyl)amine, and stir at room temperature for 5 min under nitrogen protection.

[0100] Preparation of magnetic microspheres S4: Weigh 10g (dry weight) of magnetic microspheres S3 in a beaker, replace with deoxygenated water twice, then transfer to a three-necked flask with 400mL of deoxygenated water, and start stirring. Then take 1g of CBMA in a beaker, dissolve it with 5mL of deoxygenated water by ultrasonication, and then add it to the reaction bottle at one time. After the addition, under nitrogen protection, add the catalyst to the reaction bottle at one time, replace with nitrogen 3 times, heat to 45°C, and stir for 16h. After stirring, transfer to a magnetic stand for separation with a beaker, pour off the clear liquid, wash with anhydrous ethanol 3 times, and then wash with purified water 5 times, and finally disperse in pure water to obtain low carboxyl content coupling modification layer magnetic microspheres S4, referred to as magnetic microspheres S4, and the surface carboxyl content was measured: 141μmol / g.

[0101] Preparation of p-toluenesulfonyl coupling modified layer magnetic microspheres S5.

[0102] The p-toluenesulfonyl coupling modification layer prepared in this example can be regarded as an alternative to the carboxyl coupling modification layer.

[0103] Preparation of deoxygenated water: Add 2000 mL of purified water into a three-necked flask and bubble nitrogen to deoxygenate for about 1 hour.

[0104] Catalyst preparation: Add 1 g of chlorinated ketone, 0.2 g of sodium ascorbate and 100 mL of deoxygenated water into a three-necked flask in sequence, start stirring, then add 2.7 g of tris-(2-dimethylaminoethyl)amine, and stir at room temperature for 5 min under nitrogen protection.

[0105] Preparation of magnetic microspheres S5: Weigh 10g (dry weight) of magnetic microspheres S3 in a beaker, replace with deoxygenated water twice, and finally transfer to a three-necked flask with 400mL of deoxygenated water, and start stirring; take 10g of poly (ethylene glycol) methacrylate in a beaker, dissolve it with 50mL of deoxygenated water by ultrasonication, and then add it to the reaction bottle at one time; after addition, under nitrogen protection, add the catalyst to the reaction bottle at one time, after addition, replace with nitrogen 3 times, heat to 65℃, and stir overnight. After stirring, transfer to a magnetic stand for separation with a beaker, pour out the clear liquid, replace with N,N-dimethylformamide 5 times, and then rinse the magnetic beads with 190mL of N,N-dimethylformamide into the reaction bottle. Start stirring, add 3g of triethylamine, and then weigh 3g of p-toluenesulfonyl chloride and 10mL of N,N-dimethylformamide in a centrifuge tube, dissolve and add to the reaction bottle at one time. After addition, stir at room temperature overnight. After stirring, the mixture was transferred to a magnetic stand for separation using a beaker, the clear liquid was discarded, the mixture was washed with anhydrous ethanol for 3 times, and then washed with purified water for 5 times, and finally dispersed in pure water to obtain magnetic microspheres S5 with a toluenesulfonyl coupling modification layer, referred to as magnetic microspheres S5.

[0106] Preparation of high carboxyl content coupled modified layer magnetic microspheres S6.

[0107] Preparation of magnetic microspheres S6: Weigh 10g (dry weight) of magnetic microspheres S3 in a beaker, replace with deoxygenated water twice, and finally transfer to a three-necked flask with 400mL of deoxygenated water and start stirring. Take 10g of CBMA in a beaker, dissolve it with 50mL of deoxygenated water by ultrasonication, and then add it to the reaction bottle at one time. After the addition, under nitrogen protection, add the catalyst to the reaction bottle at one time; after the addition, replace with nitrogen 3 times, heat to 45°C, and stir for 16h. After stirring, transfer to a magnetic stand for separation with a beaker, pour off the clear liquid, wash with anhydrous ethanol 3 times, and then wash with purified water 5 times, and finally disperse in pure water to obtain high carboxyl content coupling modification layer magnetic microspheres S6, referred to as magnetic microspheres S6, and the surface carboxyl content was measured: 198μmol / g.

[0108] The following table shows the groups / functional groups modified on the magnetic microspheres prepared in Examples 1 to 4.

[0109] Table 1 It can be understood that the above-mentioned signal amplification system amplifies an effective means to increase the density of groups on the surface of the magnetic microspheres that can participate in coupling, thereby significantly increasing the amount of antibody, antigen or protein coupling and improving reactivity; and the zwitterionic anti-nonspecific adsorption modification layer can significantly reduce the nonspecific adsorption of antibodies, antigens or proteins on the surface of the magnetic microspheres, thereby reducing the background and ensuring that the magnetic microspheres have high reactivity while having extremely low background.

[0110] Embodiment 5 This example provides verification of the anti-nonspecific adsorption performance of the magnetic microspheres prepared in the above examples.

[0111] The method for verifying the anti-nonspecific adsorption performance adopts the conventional enzyme-labeled detection method in this field. The better the anti-nonspecific adsorption performance, the less the labeled antibody or protein bound, and the lower the background reading. According to the sensitivity of different items to the background, high-sensitivity, medium-sensitivity and low-sensitivity items were selected as enzyme-labeled test items, namely: ALP, NSE, MYO, Ferr, iPTH, Rennie, AMH, hs-cTnI, CK-MB, CP.

[0112] 1 mg of each of the carboxyl-modified magnetic microspheres S1, S4, S5, and S6 was placed in a 1.5 ml EP tube, and the volume was filled to 1 ml with universal magnetic bead storage solution to prepare a 1 mg / ml magnetic bead solution, which was transferred to the reagent compartment for enzyme-labeled test. The results of the enzyme-labeled background test are shown in Table 2.

[0113] The results showed that the background readings of magnetic microsphere S4 in the 10 items tested were significantly lower than those of magnetic microsphere S1 modified with only carboxyl groups. This is because the modification with zwitterions significantly reduced the nonspecific adsorption of magnetic microsphere S4 to antibodies, that is, the anti-nonspecific adsorption performance of magnetic beads was significantly improved. (2) The background readings of magnetic microsphere S6 with higher carboxyl groups were further reduced compared with those of magnetic microsphere S4 with lower carboxyl groups, indicating that the surface carboxyl density of S6 increased, the surface hydrophilicity increased, the nonspecific adsorption of antibodies was further reduced, and the anti-nonspecific performance of magnetic beads was further improved. (3) The background readings of magnetic microsphere S5 modified with p-toluenesulfonyl groups were basically the same as those of magnetic microsphere S6 modified with carboxyl groups in the 10 items, indicating that the zwitterion modification layer also had a significant anti-nonspecific adsorption effect on p-toluenesulfonyl modified magnetic beads.

[0114] Table 2 Embodiment 6 Streptavidin magnetic beads are widely used in the field of in vitro diagnostics because of their high binding efficiency with biotin-labeled ligands, such as biotin-labeled nucleic acids, antibodies or cell surface proteins.

[0115] This example provides performance verification of streptavidin microspheres (SA-magnetic beads for short) further prepared based on the composite magnetic microspheres prepared in the present invention.

[0116] Take the magnetic microsphere S6 stock solution in an EP tube, place it on a magnetic rack, remove the preservation solution; add 100mM pH4.8 MEST buffer to wash the magnetic beads. Prepare an activator with EDC, NHS and MEST buffer, add it to the EP tube, vortex and mix it, and place it at 37℃ for 1 hour to activate the carboxyl groups on the surface of the magnetic beads. After the incubation is completed, place it on a magnetic rack, remove the supernatant, and add 10mM PB buffer to wash the magnetic beads twice. After removing the activator, add 10mM PB buffer to couple with streptavidin. Incubate at 37℃ for 3 hours. After the coupling reaction is completed, place the EP tube on a magnetic separation rack, remove the supernatant, add 3M Ethanolamine blocking solution (containing BSA and T-20), and place it at 37℃ for 1 hour. After blocking, the EP tube was placed on a magnetic separation rack, the supernatant was removed, and the tube was washed twice with 0.05% proclin-300 PBS buffer. Then, the concentration of the magnetic beads was adjusted to the target concentration with PBS buffer and stored to obtain the labeled SA magnetic beads, referred to as SA1-magnetic beads.

[0117] The SA1-magnetic beads were compared with the industry-recognized international benchmark SA magnetic beads (referred to as SA2-magnetic beads; manufacturer: Thermo Fisher, model: Dynabeads M-280) in terms of background reading, sensitivity, linearity and other performance in five projects including CA125, AFP, CEA, t-PSA and INS.

[0118] The results show that the overall performance of SA1-magnetic beads is better than that of the international benchmark SA2-magnetic beads. In terms of background readings, in the five projects, the background of SA1-magnetic beads is lower than that of the imported international benchmark SA2-magnetic beads, especially in the two projects of CEA and t-PSA. The background readings are reduced by about 50% compared with the international benchmark SA2-magnetic beads. For detailed data, see Table 3. In terms of sensitivity, in the five projects, the sensitivity of SA1-magnetic beads is higher than that of the international benchmark SA2-magnetic beads, especially in the CA125 and t-PSA projects, the sensitivity of SA1-magnetic beads is increased by more than 40% compared with the international benchmark SA2-magnetic beads. For detailed data, see Table 4. In terms of linear width, in the five projects, the linear width of SA1-magnetic beads is greater than that of the international benchmark SA2-magnetic beads, especially in the CA125 and t-PSA projects, the linear width of SA1-magnetic beads is increased by more than 20% compared with the international benchmark SA2-magnetic beads. For detailed data, see Table 5.

[0119] Sensitivity and linear width are important indicators of the quality and performance of in vitro diagnostic reagents; sensitivity represents the minimum concentration or content of the target substance that can be detected under specific experimental conditions. The higher the sensitivity, the lower the concentration of the target substance that can be detected, and the stronger the detection ability of the target substance. The linear width represents the concentration range in which the reagent can maintain a linear relationship during the detection process. The wider the linear width, the more reliable the detection results of the reagent at different concentrations, and the more diverse the detection needs can be met.

[0120] Table 3 Comparison of background of SA1-magnetic beads and international benchmark SA2-magnetic beads Table 4 Comparison of sensitivity between SA1-magnetic beads and international benchmark SA2-magnetic beads Table 5 Comparison of linear width between SA1-beads and international benchmark SA2-beads It is understandable that the composite magnetic microspheres of this embodiment can also be coupled with avidin or biotin.

[0121] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0122] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A method for preparing composite magnetic microspheres having resistance to nonspecific protein adsorption, characterized in that: The following steps are involved: S01: introducing a first functional group on the magnetic microsphere by free radical copolymerization with a cross-linked copolymer material under the action of a first initiator to obtain a first magnetic microsphere; the cross-linked copolymer material comprises styrene and its derivative monomers, a cross-linking monomer and a first functional compound; the first functional group comprises a carboxyl group or an epoxy group; Wherein, the first initiator is a free radical copolymerization initiator; the first functional compound is a compound having a carboxyl group or an epoxy group; Wherein, the mass ratio of the magnetic microspheres to the cross-linked copolymer material is 1: (1-100); the mass ratio of the first initiator to the styrene and its derivative monomers is (0.1-10): 100; S02: the first magnetic microsphere is coupled with the second functional compound through a ring-opening reaction or an amide reaction, and then covalently coupled with the third functional compound and the second initiator through an amide reaction to obtain the second magnetic microsphere; Wherein, the second functional compound is a small molecule or polymer having multiple amino functional groups; the third functional compound is a small molecule or polymer having multiple carboxyl functional groups; Wherein, the second initiator is an atom transfer radical polymerization initiator, including BMC or a BMC derivative, and the structural formula is as follows: BMC, BMC derivatives; Wherein, the mass ratio of the first magnetic microsphere to the second functional compound is 1:(0.1-10); the mass ratio of the first magnetic microsphere to the third functional compound is 1:(0.1-10); the mass ratio of the first magnetic microsphere to the second initiator is 1:(0.1-10); S03: adding zwitterions to the reaction system, and the second initiator on the second magnetic microsphere initiates an atom transfer radical polymerization reaction of the zwitterion monomer to obtain a third magnetic microsphere, wherein the third magnetic microsphere has an active initiating end; Wherein, the mass ratio of the second magnetic microsphere to the zwitterion is 1:(0.1-5); Wherein, the zwitterionic monomer is selected from one or more of the following molecules: TMAO, CBMA, SBMA; S04: adding a fourth functional compound having a carboxyl group or a hydroxyl group to the reaction system, and the active initiating end on the third magnetic microsphere undergoes an atom transfer radical polymerization reaction with the fourth functional compound to obtain the composite magnetic microsphere; Wherein, the mass ratio of the third magnetic microsphere to the fourth functional compound is 1:(0.1-5); Wherein, the fourth functional compound is selected from one or more of the following molecules: CBMA, OEG; Wherein, R1 is selected from amino, bromine, chlorine and / or N-hydroxysuccinimide; R2, R3 and R4 respectively represent alkyl groups with carbon chain lengths of 1-6; M represents an oxygen atom or a nitrogen atom; R5 represents an alkyl group with a carbon chain length of 2-6; R6 represents an alkyl group with a carbon chain length of 2-6; and n=1-200.

2. The preparation method according to claim 1, characterized in that In S01, the styrene and its derivative monomers include one or more materials selected from the group consisting of styrene, 4-methylstyrene, 3-methylstyrene and 4-tert-butylstyrene; or, the cross-linking monomer includes one or more materials selected from the group consisting of divinylbenzene, ethylene glycol dimethacrylate, bisphenol A dimethacrylate, poly(ethylene glycol) diacrylate and N,N'-methylenebis(acrylamide); or, the first functional compound includes one or more materials selected from the group consisting of allyl glycidyl ether, glycidyl methacrylate, glycidyl acrylate, diglycidyl phthalate, diglycidyl terephthalate, diglycidyl adipate, methacrylic acid and acrylic acid.

3. The preparation method according to claim 1, characterized in that: In S01, the mass ratio of the magnetic microspheres to the styrene and its derivative monomers is 1:(1-10); or, the mass ratio of the styrene and its derivative monomers to the cross-linking monomer is 1:(0.01-1); or, the mass ratio of the styrene and its derivative monomers to the first functional compound is 1:(0.1-10).

4. The preparation method according to claim 1, characterized in that: The first initiator includes one or more materials selected from the group consisting of dibenzoyl peroxide, dicumyl peroxide, dodecyl peroxide, di-tert-butyl peroxide, azobisisobutyronitrile, azobisisoheptanenitrile and dimethyl azobisisobutyrate.

5. The preparation method according to claim 1, characterized in that: In S02, the second functional compound includes one or more materials selected from the group consisting of ethylenediamine, propylenediamine, butylenediamine, spermine, spermidine, polyethyleneimine and polyacrylamine; or, the third functional compound includes one or more materials selected from the group consisting of oxalic acid, malonic acid, succinic acid, citric acid, tartaric acid, fumaric acid, maleic acid, polymethacrylic acid, polyacrylic acid and polymaleic acid.

6. The preparation method according to claim 1, characterized in that: n=10-200。 7. The preparation method according to claim 1, characterized in that: The atom transfer radical polymerization reaction described in S03 and S04 further includes a ligand and a catalyst system; the catalyst system includes one or more materials selected from the group consisting of cupric chloride, cuprous chloride, cupric bromide, cuprous bromide and sodium ascorbate.

8. Composite magnetic microspheres with resistance to nonspecific protein adsorption prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the composite magnetic microspheres with resistance to nonspecific protein adsorption prepared by the preparation method according to any one of claims 1 to 7, or the composite magnetic microspheres with resistance to nonspecific protein adsorption according to claim 8 in the preparation of in vitro diagnostic reagents.

10. The use according to claim 9, characterized in that The surface of the composite magnetic microsphere is coupled with streptavidin, avidin or biotin.

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