WO2022257560A1 - 乙烯基单体-多乙烯基交联剂共聚物无孔微球及其制备方法和应用 - Google Patents

乙烯基单体-多乙烯基交联剂共聚物无孔微球及其制备方法和应用 Download PDF

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WO2022257560A1
WO2022257560A1 PCT/CN2022/083708 CN2022083708W WO2022257560A1 WO 2022257560 A1 WO2022257560 A1 WO 2022257560A1 CN 2022083708 W CN2022083708 W CN 2022083708W WO 2022257560 A1 WO2022257560 A1 WO 2022257560A1
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vinyl monomer
crosslinking agent
polyvinyl
agent copolymer
microspheres
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PCT/CN2022/083708
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English (en)
French (fr)
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丁忠
刘先成
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深圳普门科技股份有限公司
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Priority to EP22819165.6A priority Critical patent/EP4275787A1/en
Publication of WO2022257560A1 publication Critical patent/WO2022257560A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F257/00—Macromolecular compounds obtained by polymerising monomers on to polymers of aromatic monomers as defined in group C08F12/00
    • C08F257/02—Macromolecular compounds obtained by polymerising monomers on to polymers of aromatic monomers as defined in group C08F12/00 on to polymers of styrene or alkyl-substituted styrenes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
    • B01J20/267—Cross-linked polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • C—CHEMISTRY; METALLURGY
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    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/08—Epoxidation
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/52—Sorbents specially adapted for preparative chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F212/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring
    • C08F212/02—Monomers containing only one unsaturated aliphatic radical
    • C08F212/04—Monomers containing only one unsaturated aliphatic radical containing one ring
    • C08F212/14—Monomers containing only one unsaturated aliphatic radical containing one ring substituted by heteroatoms or groups containing heteroatoms
    • C08F212/22—Oxygen

Definitions

  • the invention relates to the technical field of polymer microspheres, in particular to a vinyl monomer-polyvinyl crosslinking agent copolymer non-porous microsphere and its preparation method and application.
  • Polymer microspheres usually have the characteristics of good spherical shape, small size, large specific surface area, strong adsorption performance and functional modification, so they are widely used as functional materials in various industries.
  • HPLC high-performance liquid chromatography
  • the ion-exchange packing for filling the liquid chromatography column is divided into porous packing and non-porous packing.
  • the non-porous packing has the advantages of greater mass transfer and smaller lateral diffusion, which can make the sample get faster , Efficient separation.
  • the non-porous microspheres have the defect of low specific surface area, resulting in low separation capacity, which limits their many applications in protein separation.
  • chromatographic packing materials based on natural polymers usually exhibit poor rigidity and insufficient pressure resistance, and are mainly used for the purification and separation of biopharmaceuticals.
  • the present invention provides a vinyl monomer-polyvinyl crosslinking agent copolymer non-porous microsphere, its preparation method and application.
  • the vinyl monomer-polyvinyl cross-linking agent copolymer non-porous microsphere has good chemical and mechanical stability, has excellent ion exchange performance, and has low non-specific adsorption to biological components (such as protein molecules).
  • One aspect of the present invention provides a non-porous microsphere of a vinyl monomer-polyvinyl crosslinking agent copolymer, comprising:
  • Vinyl monomer-multi-vinyl cross-linking agent copolymer cue ball the vinyl monomer-poly-vinyl cross-linking agent copolymer cue ball is modified with hydroxyl groups;
  • the epoxy compound grafted to the vinyl monomer-polyvinyl crosslinking agent copolymer cue ball via the hydroxyl group includes at least two epoxy groups.
  • the epoxy compound is selected from the group consisting of sorbitol tetraglycidyl ether, pentaerythritol tetraglycidyl ether, polyglycerol polyglycidyl ether, diglycerol triglycidyl ether, 1,4-butyl Glycol diglycidyl ether, glycerol diglycidyl ether, glycerol triglycidyl ether, ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, 2 , at least one of 2-bis(4-(2,3-epoxypropoxy)phenyl) propane; as preferably, the epoxy compound is selected from sorbitol tetraglycidyl ether and polyglycerol polyglycidyl ether.
  • the vinyl monomer-polyvinyl crosslinking agent copolymer cue ball includes:
  • Vinyl monomer-polyvinyl cross-linking agent copolymer particles the vinyl monomer-polyvinyl cross-linking agent copolymer particles are modified with epoxy groups; the vinyl monomer-polyvinyl cross-linking agent The agent copolymer particles are formed by crosslinking polystyrene seed microspheres and vinyl monomers through polyvinyl crosslinking agents; the vinyl monomers contain the epoxy groups;
  • the degree of crosslinking of the vinyl monomer-polyvinyl crosslinking agent copolymer cue ball is 50% to 80%.
  • the vinyl monomer-polyvinyl crosslinking agent The cross-linking degree of the agent copolymer cue ball is 65%-75%.
  • the vinyl monomer is selected from at least one of 4-vinylphenyl glycidyl ether, 4-vinylbenzyl glycidyl ether and glycidyl methacrylate.
  • the average particle diameter D50 of the polystyrene seed microspheres is 0.3 ⁇ m ⁇ 5 ⁇ m; preferably, the average particle diameter D50 of the polystyrene seed microspheres is 0.5 ⁇ m ⁇ 3 ⁇ m.
  • the hydroxyl compound is selected from at least one of ethylene glycol, 1,3-propanediol and 1,4-butanediol.
  • the polyvinyl crosslinking agent is selected from divinylbenzene, divinyltoluene, trivinylbenzene, ethylene glycol dimethacrylate, polyethylene glycol dimethacrylate , at least one of divinylnaphthalene and trivinylnaphthalene; preferably, the polyvinyl crosslinking agent is divinylbenzene.
  • the amount of the vinyl monomer is 5% to 40% of the total weight of the vinyl monomer and the polyvinyl crosslinking agent; preferably, the vinyl monomer The amount used is 10% to 30% of the total weight of the vinyl monomer and the polyvinyl crosslinking agent; more preferably, the amount of the vinyl monomer is the amount of the vinyl monomer and the polyvinyl crosslinking agent. 10% to 20% of the total weight of polyvinyl crosslinking agent.
  • the molar ratio of the epoxy compound to the epoxy group modified on the vinyl monomer-polyvinyl crosslinking agent copolymer particle is (1 ⁇ 20):1; as a preferred Preferably, the molar ratio of the epoxy compound to the epoxy group modified on the vinyl monomer-polyvinyl crosslinking agent copolymer particles is (3 ⁇ 10):1; more preferably, the The molar ratio of the epoxy compound to the epoxy group modified on the vinyl monomer-polyvinyl crosslinking agent copolymer particles is (4-6):1.
  • the vinyl monomer-polyvinyl crosslinking agent copolymer non-porous microspheres further include active groups, and the active groups are grafted to the epoxy compound.
  • the active group may be, for example, a group containing a functional group.
  • the active group includes NTA group.
  • NTA group is the aminotricarboxymethyl structural group.
  • the NTA group comprises an active end group; the active end group is selected from aspartic acid end group, lysine end group, cysteine end group and glutamic acid end group, etc. at least one of the
  • the average particle diameter D50 of the non-porous microspheres of the vinyl monomer-polyvinyl crosslinking agent copolymer is 2 ⁇ m to 10 ⁇ m; preferably, the vinyl monomer-polyvinyl crosslinking agent The average particle diameter D50 of the non-porous microspheres of the crosslinking agent copolymer is 3 ⁇ m to 6 ⁇ m.
  • Another aspect of the present invention provides a method for preparing non-porous microspheres of vinyl monomer-polyvinyl crosslinking agent copolymer, which is characterized in that it comprises the following steps:
  • An epoxy compound is grafted onto the vinyl monomer-polyvinyl crosslinking agent copolymer cue ball through the hydroxyl reaction, and the epoxy compound contains at least two epoxy groups.
  • the epoxy compound is selected from the group consisting of sorbitol tetraglycidyl ether, pentaerythritol tetraglycidyl ether, polyglycerol polyglycidyl ether, diglycerol triglycidyl ether, 1,4-butyl Glycol diglycidyl ether, glycerol diglycidyl ether, glycerol triglycidyl ether, ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, 2 , at least one of 2-bis(4-(2,3-epoxypropoxy)phenyl)propane, etc.; preferably, the epoxy compound is selected from sorbitol tetraglycidyl ether and polypropylene triglyceride At least one of alcohol polyglycidyl ethers.
  • the method for preparing the vinyl monomer-polyvinyl crosslinking agent copolymer cue ball comprises the following steps:
  • vinyl monomer-multi-vinyl cross-linking agent copolymer particles the vinyl monomer-poly-vinyl cross-linking agent copolymer particles are modified with epoxy groups
  • preparation steps include: using polystyrene seed microspheres As seeds, together with vinyl monomers and polyvinyl cross-linking agents, under the conditions of the presence of stabilizers and emulsifiers, polymerization is carried out in a medium by seed swelling method; said vinyl monomers contain said epoxy groups ;
  • a hydroxyl compound is grafted onto the vinyl monomer-polyvinyl crosslinking agent copolymer particles via the epoxy group reaction, the hydroxyl compound comprising the hydroxyl group.
  • the vinyl monomer is selected from at least one of 4-vinylphenyl glycidyl ether, 4-vinylbenzyl glycidyl ether and glycidyl methacrylate.
  • the average particle diameter D50 of the polystyrene seed microspheres is 0.3 ⁇ m ⁇ 5 ⁇ m; preferably, the average particle diameter D50 of the polystyrene seed microspheres is 0.5 ⁇ m ⁇ 3 ⁇ m.
  • the hydroxyl compound is selected from at least one of ethylene glycol, 1,3-propanediol and 1,4-butanediol.
  • the polyvinyl crosslinking agent is selected from divinylbenzene, divinyltoluene, trivinylbenzene, ethylene glycol dimethacrylate, polyethylene glycol dimethacrylate , at least one of divinylnaphthalene and trivinylnaphthalene; preferably, the polyvinyl crosslinking agent is divinylbenzene.
  • the amount of the vinyl monomer is 5% to 40% of the total weight of the vinyl monomer and the polyvinyl crosslinking agent; preferably, the vinyl monomer The amount used is 10% to 30% of the total weight of the vinyl monomer and the polyvinyl crosslinking agent; more preferably, the amount of the vinyl monomer is the amount of the vinyl monomer and the polyvinyl crosslinking agent. 10% to 20% of the total weight of polyvinyl crosslinking agent.
  • the molar ratio of the epoxy compound to the epoxy group modified on the vinyl monomer-polyvinyl crosslinking agent copolymer particle is (1 ⁇ 20):1; as a preferred Preferably, the molar ratio of the epoxy compound to the epoxy group modified on the vinyl monomer-polyvinyl crosslinking agent copolymer particles is (3 ⁇ 10):1; more preferably, the The molar ratio of the epoxy compound to the epoxy group modified on the vinyl monomer-polyvinyl crosslinking agent copolymer particles is (4-6):1.
  • the initiator used in the polymerization is an oily initiator; preferably, it is one of oil-soluble peroxides and oil-soluble azo compounds.
  • the amount of the initiator is 0.1% to 10% of the weight of the vinyl monomer; preferably, the amount of the initiator is 0.5% to 10% of the weight of the vinyl monomer. 5%.
  • the stabilizer is a water-soluble polymer; preferably, the stabilizer is at least one of polyvinyl alcohol, polyvinylpyrrolidone and modified cellulose.
  • the amount of the stabilizer is 0.5% to 15% of the weight of the vinyl monomer; preferably, the amount of the stabilizer is 1% to 15% of the weight of the vinyl monomer. 8%.
  • the emulsifier is selected from at least one of cationic surfactants, nonionic surfactants, amphoteric surfactants and anionic surfactants.
  • the cationic surfactant is selected from at least one of quaternary ammonium salts, alkylpyridinium salts and amine salts.
  • the nonionic surfactant is selected from at least one of alkyl ether polymers and alkylphenol polyalkoxylates.
  • amphoteric surfactant is selected from at least one of amino acid type and betaine type.
  • the anionic surfactant is selected from at least one of carboxylate surfactants, sulfonate surfactants, sulfate surfactants and phosphate surfactants.
  • the amount of the emulsifier is 0.05% to 10% of the weight of the vinyl monomer; preferably, the amount of the emulsifier is 0.1% to 10% of the weight of the vinyl monomer. 5%.
  • the preparation method of the vinyl monomer-polyvinyl crosslinking agent copolymer non-porous microspheres further includes: reacting and grafting on the epoxy compound to form active groups.
  • the active group can be, for example, a group containing a functional group.
  • reactive groups include NTA groups.
  • the method for forming the NTA group is selected from the N,N-dicarboxymethyllysine method, the active terminal amino acid method, the carboxylation method of dicarboxylic acid amino acids, and the construction of the aldehyde group first and then the ⁇ - One of the amino acid methods.
  • Another aspect of the present invention provides the non-porous microspheres of the vinyl monomer-polyvinyl crosslinking agent copolymer, and the vinyl monomer-polyvinyl crosslinking agent prepared by the preparation method.
  • Another aspect of the present invention provides the non-porous microspheres of the vinyl monomer-polyvinyl crosslinking agent copolymer, and the vinyl monomer-polyvinyl crosslinking agent prepared by the preparation method. Application of one of the agent copolymer nonporous microspheres in the detection of biological components.
  • the biological components are hemoglobin-like biological components.
  • the present invention has the following beneficial effects:
  • the vinyl monomer-polyvinyl cross-linking agent copolymer non-porous microspheres of the present invention are grafted on the basis of the vinyl monomer-polyvinyl cross-linking agent copolymer cue ball modified with hydroxyl groups.
  • Linking epoxy-based compounds containing at least two epoxy groups allows for more epoxy groups on the microspheres.
  • the method of grafting multiple epoxy groups can make up for the shortcomings of non-porous (ion-exchange) filler microspheres with low specific surface area and low ion-exchange capacity, so that the specific adsorption capacity of non-porous fillers for target biological components can be comparable to that of porous fillers. s level.
  • non-porous packings have the advantages of maximum mass transfer and minimum lateral diffusion in chromatographic separations, thus enabling rapid and efficient separation of biological samples.
  • the vinyl monomer-polyvinyl crosslinking agent copolymer non-porous microspheres can take into account the two advantages of high loading and high separation efficiency.
  • the chemical properties and mechanical stability (including rigidity) of the non-porous microspheres of the vinyl monomer-polyvinyl crosslinking agent copolymer using epoxy groups are good, and at the same time, under a small specific surface area, the surface Able to gather more ion exchange groups (such as NTA structure, to achieve multi-carboxylation).
  • the surface of the microsphere After the surface of the microsphere is modified with epoxy group, it can be used as a chromatographic filler, which shows excellent performance in the analysis, separation and purification of biomacromolecules, and eliminates the non-specific adsorption of hydrophobic structures to biomolecules.
  • the separation effect in the detection of hemoglobin biological components is remarkable, not only can quantitative analysis of HbA1c, but also Separation and analysis of 16 hemoglobin-like biological components, qualitative and quantitative analysis of common hemoglobin variants in the world, and high specific separation ability for hemoglobin and its variants.
  • the H9 glycosylated hemoglobin instrument produced by Shenzhen Pumen Technology Co., Ltd. can be used to separate and analyze hemoglobin and its variants, and it has high separation ability (analysis spectrum) and low protein non-specificity. Adsorption (long column life), high pressure performance (16MPa) and other advantages.
  • the vinyl monomer-polyvinyl crosslinking agent copolymer non-porous microspheres also have the advantages of monodispersity, good dispersibility, uniform and controllable particle size.
  • Fig. 1 is the PST seed microsphere scanning electron micrograph that embodiment 3 prepares
  • Fig. 2 is the particle size distribution figure of the PST seed microsphere prepared by embodiment 3;
  • Fig. 3 is the PSDVB-Epoxy microsphere scanning electron micrograph that embodiment 4 prepares;
  • Fig. 4 is the particle size distribution figure of PSDVB-Epoxy microspheres prepared by embodiment 4;
  • Fig. 5 is the fitting curve of flow velocity and column pressure in the range of 0.5mL/min ⁇ 5.25mL/min for the chromatographic column made of the PSDVB-Epoxy microspheres of Example 4 to Example 7 in Test Example 1;
  • Fig. 6 is the peak area statistical diagram of 20 injections of No. 1 chromatographic column in Test Example 2;
  • Fig. 7 is the peak area statistical diagram of 20 injections of No. 2 chromatographic column in test example 2;
  • Fig. 8 is the peak area statistical diagram of 20 injections of No. 3 chromatographic column in Test Example 2;
  • Fig. 9 is the chromatogram of the PSDVB-NTA microsphere of embodiment 11 in test example 3 as chromatographic filler;
  • Fig. 10 is the chromatogram of the PSDVB-NTA microsphere of embodiment 12 in test example 3 as chromatographic filler;
  • Fig. 11 is the chromatogram of the PSDVB-NTA microsphere of embodiment 13 in test example 3 as chromatographic filler;
  • Fig. 12 is the chromatogram of the PSDVB-NTA microsphere of embodiment 14 in test example 3 as chromatographic filler;
  • Fig. 13 is the chromatogram of the PSDVB-NTA microsphere of Comparative Example 1 in Test Example 3 as a chromatographic filler;
  • Fig. 14 is the chromatogram of the PSDVB-NTA microsphere of comparative example 2 in test example 3 as chromatographic filler
  • Fig. 15 is the liquid phase chromatogram of test sample and quality control product in test example 4.
  • Fig. 16 is the liquid chromatogram of the sample to be tested in test example 5.
  • Fig. 17 is the repeatability test result figure of test example 6.
  • Fig. 18 is the correlation test result figure of test example 7.
  • Fig. 19 is the quality control chart of test example 8.
  • Fig. 20 is the detection chromatogram of sample 1 ⁇ sample 5 in test example 9;
  • Fig. 21 is the liquid chromatogram of the Hb J-Bangkok whole blood sample of sample 6 in test example 9;
  • Figure 22 is the liquid chromatogram of the HbG-Taipei whole blood sample of sample 7 in Test Example 9;
  • non-porous microspheres of the vinyl monomer-polyvinyl crosslinking agent copolymer of the present invention and its preparation method and application will be further described in detail below in conjunction with specific examples.
  • the present invention can be embodied in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.
  • a polyvinyl crosslinker refers to a crosslinker that contains at least two vinyl groups.
  • the invention provides a non-porous microsphere of a vinyl monomer-polyvinyl crosslinking agent copolymer, comprising:
  • the epoxy compound is grafted to the vinyl monomer-polyvinyl crosslinking agent copolymer mother ball through the hydroxyl group, and the epoxy compound contains at least two epoxy groups.
  • vinyl monomers containing epoxy groups are used to replace styrene monomers and polyvinyl crosslinking agents for polymerization to form vinyl monomer-polyvinyl crosslinking agent copolymer particles modified with epoxy groups (For example: PSDVB-Epoxy microspheres), and then the epoxy groups on the vinyl monomer-polyvinyl crosslinking agent copolymer particles modified with epoxy groups are hydrolyzed under the action of hydroxyl compounds to form hydroxyl groups, which is convenient for Subsequent coupling of polyepoxides.
  • vinyl monomers containing epoxy groups are used to replace styrene monomers and polyvinyl crosslinking agents for polymerization, and the polymerization and modification can be completed through one-step reaction to obtain vinyl monomers modified with epoxy groups.
  • Body-polyvinyl cross-linking agent copolymer particles can avoid the problem that some modification sites are wrapped in the inner core of the polymer cue ball and are covered, and improve the lead to vinyl monomer-polyvinyl polymers (especially strong hydrophobic polymerization) PSDVB benzene ring) hydrophilization (coupling hydroxyl or epoxy group) conversion rate, increase the bonding amount of epoxy group.
  • vinyl monomer-polyvinyl cross-linking agent copolymer particles modified with epoxy groups as the cue ball can also endow the vinyl monomer-polyvinyl cross-linking agent copolymer non-porous microspheres with excellent resistance. oppressive.
  • the vinyl monomer-polyvinyl crosslinking agent copolymer cue ball includes:
  • Vinyl monomer-polyvinyl crosslinking agent copolymer particles vinyl monomer-polyvinyl crosslinking agent copolymer particles are modified with epoxy groups; vinyl monomer-polyvinyl crosslinking agent copolymer particles It is formed by cross-linking polystyrene seed microspheres and vinyl monomers through polyvinyl cross-linking agents; the vinyl monomers contain epoxy groups; and
  • the hydroxyl compound grafted on the vinyl monomer-polyvinyl crosslinking agent copolymer particle through the epoxy group, the hydroxyl compound contains a hydroxyl group.
  • the crosslinking degree of the vinyl monomer-polyvinyl crosslinking agent copolymer cue ball is 50%-80%, preferably 65%-75%.
  • the average particle diameter D50 of the polystyrene seed microspheres is 0.3 ⁇ m ⁇ 5 ⁇ m; preferably, the average particle diameter D50 of the polystyrene seed microspheres is 0.5 ⁇ m ⁇ 3 ⁇ m.
  • the present invention adopts the above-mentioned polystyrene seed microspheres of monodisperse particle size, which is beneficial to obtain non-porous microspheres of monodisperse vinyl monomer-polyvinyl crosslinking agent copolymer with controllable and uniform particle size, and improves the efficiency of vinyl monomer -The recognition ability of polyvinyl cross-linking agent copolymer non-porous microspheres to substances such as hemoglobin variants.
  • the average particle diameter D50 of the vinyl monomer-polyvinyl crosslinking agent copolymer non-porous microspheres is 2 ⁇ m to 10 ⁇ m; preferably, the vinyl monomer-polyvinyl crosslinking agent copolymer
  • the average particle diameter D50 of the non-porous microspheres is 3 ⁇ m to 6 ⁇ m.
  • the appearance and average particle size of the aforementioned microspheres are determined by a scanning electron microscope (SEM) and a laser particle size analyzer.
  • the polystyrene seed microspheres are non-crosslinked linear polymers prepared by dispersion polymerization. The use of polystyrene seed microspheres of non-crosslinked linear polymers is conducive to obtaining non-porous microspheres of monodisperse vinyl monomer-polyvinyl crosslinking agent copolymers with uniform particle size.
  • the vinyl monomer is selected from at least one of 4-vinylphenyl glycidyl ether, 4-vinylbenzyl glycidyl ether and glycidyl methacrylate.
  • the vinyl monomer is selected from at least one of 4-vinylphenyl glycidyl ether and 4-vinylbenzyl glycidyl ether.
  • 4-vinylphenyl glycidyl ether is prepared from 4-hydroxystyrene and epichlorohydrin;
  • 4-vinylbenzyl glycidyl ether is prepared from 4-chloromethylstyrene and glycidol .
  • the polyvinyl crosslinking agent is selected from at least one of divinylbenzene, divinyltoluene, trivinylbenzene, ethylene glycol dimethacrylate, divinylnaphthalene and trivinylnaphthalene.
  • polyvinyl crosslinking agent is divinylbenzene.
  • the amount of vinyl monomers is 5% to 40% of the total weight of vinyl monomers and polyvinyl crosslinking agents; preferably, the amount of vinyl monomers is 10%-30% of the total weight of the vinyl crosslinking agent; more preferably, the amount of the vinyl monomer is 10%-20% of the total weight of the vinyl monomer and the polyvinyl crosslinking agent.
  • the hydroxyl compound is selected from at least one of ethylene glycol, 1,3-propanediol and 1,4-butanediol.
  • the coupling of the polyepoxy compound is carried out, thereby introducing the polyepoxy group into the structure structure, to obtain non-porous microspheres of vinyl monomer-polyvinyl crosslinking agent copolymer with multiple epoxy groups.
  • the epoxy compound is selected from the group consisting of sorbitol tetraglycidyl ether, pentaerythritol tetraglycidyl ether, polyglycerol polyglycidyl ether, diglycerol triglycidyl ether, 1,4-butanediol di Glycidyl ether, glycerol diglycidyl ether, glycerol triglycidyl ether, ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether (molecular weight can be 200-2000), neopentyl glycol At least one of diglycidyl ether, 2,2-bis(4-(2,3-epoxypropoxy)phenyl)propane; preferably, the epoxy compound is selected from sorbitol tetraglycidyl ether and at least one of polyglycidyl ethers of polyglycerol.
  • the molar ratio of the epoxy compound to the epoxy group modified on the vinyl monomer-polyvinyl crosslinking agent copolymer particles is (1-20):1; preferably, the epoxy group
  • the molar ratio of the epoxy group modified on the compound to the vinyl monomer-polyvinyl crosslinking agent copolymer particle is (3 ⁇ 10):1; more preferably, the epoxy compound and the vinyl monomer-polyvinyl crosslinking agent
  • the molar ratio of epoxy groups modified on the vinyl crosslinking agent copolymer particles is (4-6):1. Most preferably, the molar ratio of the epoxy compound to the epoxy group modified on the vinyl monomer-polyvinyl crosslinking agent copolymer particles is 5:1.
  • active groups are constructed, and the active groups optionally include NTA groups to obtain polycarboxyl-rich Collected microspheres (for example: PSDVB-NTA), which can be used as a weak cation exchange packing for liquid chromatography.
  • NTA groups are grafted onto epoxy compounds.
  • the active group is not limited to the NTA group, and can also be other active groups, such as active groups containing functional groups such as -SO 3 H or -NH 2 , and can be used with polyepoxy groups as required.
  • the vinyl monomer-polyvinyl monomer microspheres incorporate the desired reactive groups.
  • the NTA group includes an active end group; the active end group is selected from at least one of an aspartic acid end group, a lysine end group, a cysteine end group and a glutamic acid end group.
  • the methods for constructing the NTA group include active terminal amino acid method, N,N-dicarboxymethyl lysine method, carboxylation of dicarboxylic acid structural amino acid (the dicarboxylic acid structural amino acid is preferably acidic Amino Acids), after the surface aldehydes of the microspheres are formed to construct ⁇ -amino acids (for details, please refer to the patent CN109663384A), and then generate NTA groups and the like.
  • the present invention also provides a method for preparing non-porous microspheres of vinyl monomer-polyvinyl crosslinking agent copolymer, comprising the following steps:
  • the vinyl monomer-polyvinyl crosslinking agent copolymer cue balls are modified with hydroxyl groups;
  • the epoxy compound is grafted onto the vinyl monomer-polyvinyl crosslinking agent copolymer cue ball through hydroxyl reaction, and the epoxy compound contains at least two epoxy groups.
  • the method for preparing vinyl monomer-multi-vinyl crosslinking agent copolymer cue ball comprises the steps:
  • the preparation steps include: using polystyrene seed microspheres as seeds , together with vinyl monomers and polyvinyl crosslinking agents, under the condition of the presence of stabilizers and emulsifiers, the polymerization is carried out in the medium by seed swelling method; the vinyl monomers contain epoxy groups;
  • the epoxy compound is grafted to the vinyl monomer-polyvinyl crosslinking agent copolymer particle through epoxy reaction, and the hydroxyl compound contains hydroxyl.
  • polystyrene and polyvinylbenzene aromatic compounds can be first polymerized to form vinyl monomer-polyvinyl polymers, and then the vinyl monomer-polyvinyl polymers can be polymerized with compounds containing epoxy groups. Modification so that vinyl monomer-polyvinyl polymers have epoxy groups bonded thereto.
  • This method first forms the polymer and then modifies it, so that part of the modification site is wrapped into the inner core of the polymer cue ball and covered, resulting in vinyl monomer-polyvinyl polymers (especially the strong hydrophobic polymer PSDVB benzene ring Above)
  • the conversion rate of hydrophilization is low, and the bonding amount of epoxy group is low.
  • polystyrene seed microspheres are used as seeds, and the vinyl monomers modified with epoxy groups and polyvinyl crosslinking agents are polymerized under the conditions of the presence of stabilizers and emulsifiers, which can be achieved through one-step reaction That is, the polymerization and modification are completed, and vinyl monomer-polyvinyl crosslinking agent copolymer particles modified with epoxy groups are obtained, which can avoid the problem that part of the modification sites are wrapped into the inner core of the polymer cue ball and are covered, and improve the vinyl content.
  • the monomer-polyvinyl polymer (especially on the benzene ring of the strongly hydrophobic polymer PSDVB) is hydrophilized (coupling hydroxyl or epoxy group) conversion rate, and the bonding amount of epoxy group is increased.
  • the initiator used in the polymerization is an oily initiator; preferably, one of oil-soluble peroxides and oil-soluble azo compounds.
  • oil-soluble peroxides can be exemplified by dibenzoyl peroxide, dioctanoyl peroxide, lauroyl peroxide, etc.
  • oil-soluble azo compounds can be exemplified by 2,2'-azobisisobutyronitrile, 2,2'-azobisisocapronitrile, etc.
  • the amount of the initiator is 0.1%-10% of the weight of the vinyl monomer; preferably, the amount of the initiator is 0.5%-5% of the weight of the vinyl monomer.
  • the stabilizer is a water-soluble polymer; preferably, the stabilizer is at least one of polyvinyl alcohol, polyvinylpyrrolidone and modified cellulose.
  • the stabilizer is used in an amount of 0.5% to 15% by weight of the vinyl monomer; preferably, the stabilizer is used in an amount of 1% to 8% by weight of the vinyl monomer.
  • the emulsifier refers to a suitable surfactant, such as at least one of cationic surfactant, nonionic surfactant, amphoteric surfactant and anionic surfactant.
  • the cationic surfactant is selected from at least one of quaternary ammonium salts, alkylpyridinium salts and amine salts;
  • the nonionic surfactant is selected from alkyl ether polymers and alkylphenol polyalkoxy compounds (such as alkyl At least one of base phenol polyethoxylates);
  • the amphoteric surfactant is selected from amino acid-type amphoteric surfactants (such as dodecylaminopropionic acid), betaine-type amphoteric surfactants (such as thiobetaine ) at least one;
  • the anionic surfactant is selected from at least one of carboxylate surfactants, sulfonate surfactants, sulfate surfactants and phosphate surfactants.
  • the emulsifier is used in an amount of 0.05% to 10% by weight of the vinyl monomer; preferably, the emulsifier is used in an amount of 0.1% to 5% by weight of the vinyl monomer.
  • the preparation process is as follows:
  • the vinyl monomer-polyvinyl cross-linking agent copolymer particle For example: PSDVB-Epoxy.
  • the PST seed is used as a template, and 4-vinylbenzyl glycidyl ether (ST-Epoxy) is used as a vinyl monomer with an epoxy group.
  • ST-Epoxy 4-vinylbenzyl glycidyl ether
  • the method for the cross-linked polyvinyl monomer-polyvinyl cross-linking agent copolymer particles for example: PSDVB-Epoxy
  • epoxy groups on the surface is:
  • PST seeds sodium dodecylsulfonate (SDBS)/water mixed phase, 4-vinylbenzyl glycidyl ether (ST-Epoxy) and divinylbenzene (DVB)/oil mixed phase were reacted to obtain Oxygen-based cross-linked polyvinyl monomer-polyvinyl cross-linker copolymer particles (for example: PSDVB-Epoxy).
  • SDBS sodium dodecylsulfonate
  • ST-Epoxy 4-vinylbenzyl glycidyl ether
  • DVD divinylbenzene
  • the polyepoxy compound is sorbitol tetraglycidyl ether.
  • the epoxy group on the surface of the cross-linked polyvinyl monomer-polyvinyl cross-linking agent copolymer particles (for example: PSDVB-Epoxy) with epoxy groups on the surface is hydrolyzed and coupled with polyepoxides
  • the method is: use ethylene glycol, epichlorohydrin and catalyst BF 3 ⁇ (CH 3 CH 2 ) on the cross-linked polyvinyl monomer-polyvinyl cross-linking agent copolymer particles with epoxy groups on the surface.
  • polyepoxy groups on the surface of vinyl monomer-polyvinyl crosslinking agent copolymer microspheres for example: PSDVB-mEpoxy
  • polyepoxy groups on the surface are converted into tricarboxylic acid groups, and the NTA structure is constructed to obtain Polycarboxy-enriched microspheres (eg PSDVB-NTA).
  • polycarboxy-rich microspheres are synthesized by the active terminal amino acid method.
  • the specific method is: the surface epoxy group of the vinyl monomer-polyvinyl crosslinking agent copolymer microsphere (for example: PSDVB-mEpoxy) with many epoxy groups on the surface is under alkaline conditions (such as 8.4 ⁇ pH ⁇ 9.0), introduce an amino acid with an active end group (preferably, the active end group amino acid is lysine, cysteine) to react, and directly construct an ⁇ -amino acid on the surface of the microsphere (the molar ratio can be 1:1), Then, bromoacetic acid was added dropwise step by step under alkaline conditions to construct the surface NTA structure and obtain multi-carboxyl-rich microspheres (for example: PSDVB-NTA).
  • the reaction mechanism is shown in formula (6):
  • the above preparation method introduces polyepoxy groups in the microsphere preparation process, which ensures good chemical properties and mechanical stability (including rigidity). At the same time, under a small specific surface area, the surface can accumulate more ion exchange Group, the separation effect is remarkable in the detection of hemoglobin.
  • the multi-epoxy group coupling active terminal amino acid method on the surface of the microsphere, the polycarboxylation process is simpler and easier. It has excellent performance in the analysis and separation and purification of biological macromolecules, especially in the detection of hemoglobin biomacromolecular components. It can not only quantitatively analyze HbA1c, but also separate and analyze 16 kinds of hemoglobin biomacromolecular components. The common hemoglobin variants can be analyzed qualitatively and quantitatively.
  • the present invention also provides non-porous microspheres of vinyl monomer-polyvinyl cross-linking agent copolymer as above, and the non-porous microspheres of vinyl monomer-polyvinyl cross-linking agent copolymer prepared by the above preparation method in chromatographic packing in the application.
  • the chromatographic filler refers to a weak cation exchange filler for liquid chromatography, which can be used for in vitro diagnostic testing of medical devices, especially for the detection of hemoglobin biomacromolecular components. For example, the determination of the precise content of glycosylated hemoglobin (HbA1c) in blood by ion-exchange high-performance liquid chromatography is recognized internationally as the gold standard for diabetes diagnosis.
  • the glycated hemoglobin analyzer is a high-performance liquid chromatography (HPLC) type medical device used for blood sugar detection of diabetic patients.
  • HPLC high-performance liquid chromatography
  • the liquid chromatography column packed with ion-exchange chromatography microspheres is one of the core hardware of the glycated hemoglobin analyzer.
  • non-porous filler microspheres such as a non-porous microsphere surface grafted polyacrylamide hydrophilic modified weak cation exchange filler
  • a non-porous microsphere surface grafted polyacrylamide hydrophilic modified weak cation exchange filler It can be used for the detection of hemoglobin, but the effect of this filler on the separation of hemoglobin variants is not good;
  • another example is a kind of polydisperse sulfonic acid type cation exchange polymer microspheres that can be used for the determination of hemoglobin, which has the advantage of low equipment requirements.
  • the pressure of the HPLC system is also relatively low, which can quickly detect glycosylated hemoglobin, but due to its uneven particle size distribution and low resolution, the ability to recognize hemoglobin variants is relatively poor.
  • vinyl monomer-polyvinyl crosslinking agent copolymer non-porous microsphere of the present invention is used as the chromatographic column filler of glycated hemoglobin analyzer, can directly measure the HbA1c in the whole blood, its intra-assay and inter-assay coefficient of variation
  • the CV can be less than 3%, the result is accurate, and the HbA1c detection result is not affected by the existing variant hemoglobin and its derivatives, which is especially suitable for the diagnosis of diabetics and the monitoring of blood sugar indicators.
  • it has a high specific separation ability for hemoglobin and its variants, and can separate and analyze 16 kinds of hemoglobin biological components.
  • the vinyl monomer-polyvinyl crosslinking agent copolymer non-porous microspheres also have the advantages of monodispersity, good dispersibility, uniform and controllable particle size.
  • the present invention also provides the non-porous microspheres of the vinyl monomer-polyvinyl cross-linking agent copolymer as above, and the non-porous microspheres of the vinyl monomer-polyvinyl cross-linking agent copolymer prepared by the above preparation method in hemoglobin Applications for the detection of biomacromolecular components. It can be understood that the detection of hemoglobin-like biological components is an in vitro detection method, and the direct purpose is not to obtain diagnostic results or health status, but to detect tissues and body fluids that have been separated from the human or animal body to obtain information as an intermediate result Methods.
  • the samples to be tested in the following examples are all fresh blood samples, taken from Shenzhen Hospital of Peking University.
  • the detection equipment for hemoglobin substances is a commercially available high performance liquid chromatograph.
  • the diluent is deionized water.
  • HbA1a (Hemoglobin-A1a) is lactosylated hemoglobin
  • HbA1b (Hemoglobin-A1b) is fructosylated hemoglobin
  • HbA1c (Stable Glycated Hemoglobin) is a stable glycosylated hemoglobin, and the content of HbA1c can represent the average blood sugar level in the past 2 to 3 months;
  • LA1c (Labile Glycated Hemoglobin) is an unstable glycosylated hemoglobin, and the intermediate product Schiff base in the glycosylation process can be degraded into glucose and hemoglobin;
  • HbA0 Non-glycated Hemoglobin
  • HbA0 Non-glycated Hemoglobin
  • HbF Fetal Hemoglobin
  • HbA2 (A2 Hemoglobin) is hemoglobin HbA2, the increase of its content is mainly seen in mild ⁇ -thalassemia anemia, and it is ⁇ -thalassemia factor;
  • P3 and P4 are the degradation peaks of hemoglobin HbA0, usually the content is less than 5%. If the blood sample is not fresh or other variant hemoglobin appears, the content of P3 and P4 will increase;
  • HbE HbE Variant Hemoglobin
  • Glu glutamic acid
  • Lys lysine
  • HbE is the most common type of abnormal hemoglobin in the globin ⁇ chain.
  • HbE is mainly distributed in Southeast Asian countries. In my country, HbE mainly exists in the south of the Yangtze River and coastal areas;
  • HbS HbS Variant Hemoglobin
  • Glu glutamic acid
  • Val Val
  • HbC HbC Variant Hemoglobin
  • Glu glutamic acid
  • Lys lysine
  • HbD HbD Variant Hemoglobin
  • HbD HbD Variant Hemoglobin
  • HbJ-Bangkok J-Bangkok Variant Hemoglobin
  • Gly glycine
  • Asp aspartic acid
  • Hb G-Taipei (G-Taipei Variant Hemoglobin) is an abnormal hemoglobin formed by replacing Glu (glutamic acid) at the 22nd position of the globin ⁇ chain with Gly (glycine), which occurs frequently in northern my country;
  • Hb Q-Thailand Q-Thailand Variant Hemoglobin
  • Asp aspartic acid
  • His histidine
  • Perchloric acid titration was used to measure the epoxy value of microspheres, and the specific operation was as follows: 0.05 g of microspheres were immersed in 10 mL of chloroform for 1 hour, and ultrasonically dispersed in a conical flask. Add 10 mL of tetraethylammonium bromide solution and 2 drops of crystal violet indicator into the Erlenmeyer flask. Titrate the solution in the Erlenmeyer flask with V mL of perchloric acid standard solution (0.1M) until the color of the solution changes from blue to bright green and remains unchanged for 2 hours. Do blank control experiment simultaneously, consume V0mL perchloric acid standard solution.
  • the formula for calculating the epoxy value of microspheres is as follows:
  • microsphere ion exchange capacity (IEC) detection is: add 11g microspheres and 150mL sulfuric acid solution (0.5mol/L) into the flask, stir and soak overnight. The soaked microspheres were washed several times with deionized water until neutral, dried and left to stand. Put 1 g of dried microspheres into a conical flask, add 0.08 L of standard NaOH solution (0.05 mol/L) into the conical flask at 35°C, shake slightly evenly, and place in a water bath at 35°C for one hour. Filtrate A was obtained after filtering off the microspheres. Wash the microspheres with 20 mL of sodium chloride solution four times in sequence, and obtain filtrate B after filtering out the microspheres.
  • IEC microsphere ion exchange capacity
  • the filtrates A and B were thoroughly mixed to obtain a mixed solution C, and then 20 mL of the mixed solution was poured into an Erlenmeyer flask. Put 2 drops of phenolphthalein indicator into the flask, add 0.05mol/L standard hydrochloric acid solution and titrate until the solution changes from purple to colorless, and does not change color within 30s, that is, the end point is reached, and the volume V of 0.05mol/L standard hydrochloric acid consumed is recorded HCl .
  • the calculation formula for microsphere ion exchange capacity (IEC) detection is as follows:
  • IEC (0.05 ⁇ 0.08-8 ⁇ 0.05 ⁇ V HCl )/V(eq/L).
  • Refining Add 10 mg of hydroquinone inhibitor to the crude product, and carry out high vacuum distillation at 83°C to obtain 4-vinylphenyl glycidyl ether after distillation, which is a light yellow oil with a small amount of impurities .
  • 4-vinylphenyl glycidyl ether was recrystallized with 200mL of n-hexane at -20°C, and the final product was dried at 35°C under vacuum to refine 4-vinylphenyl glycidyl ether to 5.730 g, content 98.0%, yield 54%.
  • Example 4 The synthesis of crosslinked polystyrene-divinylbenzene microspheres (PSDVB-Epoxy) with epoxy groups on the surface of 70% crosslinking degree
  • mixed phase 1 water phase
  • Example 5 The synthesis of crosslinked polystyrene-divinylbenzene microspheres (PSDVB-Epoxy) with epoxy groups on the surface of a 70% degree of crosslinking
  • Example 6 The synthesis of crosslinked polystyrene-divinylbenzene microspheres (PSDVB-Epoxy) with epoxy groups on the surface of 80% crosslinking degree
  • mixed phase 1 water phase
  • mixed phase 1 water phase
  • mixed phase 2 oil phase
  • control stirring speed 100rpm
  • swelling Time 24h
  • After swelling add 243g of 2% PVA solution, stir at 150rpm, stir for 30min, under N2 protection condition, increase temperature: 75°C, keep warm for 12h.
  • use qualitative filter paper to filter the reaction solution in a Buchner funnel, then wash three times with 500mL deionized water, three times with 300mL ethanol, three times with 300mL acetone to obtain PSDVB-Epoxy with a crosslinking degree of 80%.
  • Monodisperse non-porous cross-linked microspheres the particle size D50 measured by a scanning electron microscope (SEM) is 5.25 ⁇ m. Microsphere epoxy value: 0.47mmol/g.
  • Example 7 The synthesis of crosslinked polystyrene-divinylbenzene microspheres (PSDVB-Epoxy) with epoxy groups on the surface of 50% crosslinking degree
  • mixed phase 1 water phase
  • Embodiment 9 Sorbitol tetraglycidyl ether prepares cross-linked polystyrene-divinylbenzene microspheres (PSDVB-mEpoxy) with polyepoxy groups on the surface for polyepoxy compounds
  • Example 10 Polyglycerol polyglycidyl ether prepares cross-linked polystyrene-divinylbenzene microspheres (PSDVB-mEpoxy) with polyepoxy groups on the surface for polyepoxy compounds
  • Example 13 Lysine is used as an active terminal amino acid to prepare polystyrene-divinylbenzene microspheres (PSDVB-NTA) enriched in polycarboxylates
  • Example 14 Lysine is used as an active terminal amino acid to prepare polystyrene-divinylbenzene microspheres (PSDVB-NTA) enriched in polycarboxylates
  • the polystyrene-divinylbenzene microspheres (PSDVB-Epoxy) prepared in Examples 4 to 7 are homogenized with ethanol and filled into 4.0mm (inner diameter) ⁇ 30mm (length) respectively. column.
  • a high-performance liquid chromatograph (Agilent 1260 Infinity II HPLC) was used to carry out the microsphere withstand pressure test on each of the above-mentioned chromatographic columns. 50mM, pH6.0 PB buffer system was used as the eluent, the flow rate was increased from 0.5mL/min to 5.00mL/min in steps of 0.25mL/min, each flow rate was maintained for 3min, and the column temperature was 25°C.
  • Table 1 shows the column pressure of the chromatographic column made of the PSDVB-Epoxy microspheres of Examples 4 to 7 at a flow rate of 0.5mL/min to 5.25mL/min.
  • Table 2 shows the fitting curve equation of the flow rate and the column pressure of the chromatographic column made of PSDVB-Epoxy microspheres of Examples 4 to 7 in the corresponding flow rate range, wherein, Y is the column pressure, and x is the flow rate.
  • Fig. 5 is a fitting curve of flow velocity and column pressure in the range of flow velocity 0.5mL/min-5.25mL/min for chromatographic columns made of PSDVB-Epoxy microspheres of Examples 4 to 7.
  • PSDVB-Epoxy microspheres the higher the epoxy value of PSDVB-Epoxy microspheres, the more active groups that can be attached.
  • the PSDVB-Epoxy epoxy value of embodiment 4 and embodiment 5 is substantially equivalent;
  • the PSDVB-epoxy epoxy value of embodiment 4 and embodiment 5 is higher than embodiment 6, and slightly lower than embodiment 7.
  • the packing solvent is ultrapure water with pH 7.0.
  • the cross-linked polystyrene-divinylbenzene microspheres (PSDVB-mEpoxy) prepared in Example 9 with multiple epoxy groups are packed after hydrolysis;
  • the filler of the third chromatographic column (hereinafter referred to as No. 3 chromatographic column) is The polycarboxyl-rich polystyrene-divinylbenzene microspheres (PSDVB-NTA) prepared in Example 11 were obtained after filling.
  • BSA bovine serum albumin
  • the above-mentioned chromatographic columns are used to detect the detection samples by high-performance liquid chromatography (Agilent 1260 Infinity II HPLC), and the mobile phase is 10mM phosphate buffered saline Solution (containing 0.5M NaCl, pH 7.0), the flow rate is 0.5mL/min, the detector is UV 214nm, the column temperature is room temperature, the injection volume is 2 ⁇ L/needle, and each chromatographic column is continuously injected with 20 needles (that is, 20 times ).
  • Curve drawing Draw a scatter diagram with the number of injections as the abscissa and the BSA peak area as the ordinate.
  • the BSA recovery rate is 100%, and it is assumed that there is non-specific adsorption when the BSA recovery rate is less than 98%.
  • the calculation formula of the non-specific adsorption amount of BSA (unit: ⁇ g/mL) is as follows formula (1):
  • Non-specific adsorption amount of BSA (2N ⁇ 0.25)/(0.20 2 ⁇ 15) Formula (1)
  • N is the maximum number of injections when the BSA recovery rate is less than 98%.
  • Fig. 6 is the peak area statistical diagram of 20 injections of No. 1 chromatographic column in test example 2
  • Fig. 7 is the peak area statistical diagram of 20 injections of No. 2 chromatographic column in test example 2
  • Fig. 8 is test The peak area statistical chart of 20 injections of No. 3 chromatographic column in Example 2.
  • the non-specific adsorption capacity of No. 2 chromatographic column and No. 3 chromatographic column is all less than No. 1 chromatographic column, illustrates that the PSDVB-mEpoxy microsphere that the preparation of embodiment 9 obtains, embodiment 11
  • the non-specific adsorption capacity of the prepared polycarboxyl-rich polystyrene-divinylbenzene microspheres is less than that of the PSDVB-Epoxy microspheres prepared in Example 4, which further shows that the use of hydrophilic glycidol on the surface of the microspheres After ether modification, the non-specific adsorption of the microspheres can be reduced, the service life of the chromatographic column can be improved, and the separation effect of the target protein can be guaranteed.
  • PSDVB-NTA polycarboxyl-rich polystyrene-divinylbenzene microspheres
  • Examples 11 to 14 and Comparative Examples 1 to 2 were respectively used as chromatographic fillers, and after homogenizing the fillers with ultrapure water, use A high-pressure pneumatic pump packs it into a 4.6*30mm (column volume: 0.498mL) chromatographic column.
  • the separation performance of the standard protein cytochrome C and lysozyme was tested by high performance liquid chromatography (Agilent 1260 Infinity II HPLC).
  • buffer A 50mmol
  • PB Phosphate Buffer, phosphate buffer
  • buffer B phase A (that is, buffer A )+1MNaCl (pH6.0) as the eluent for gradient elution
  • the elution gradient is 0min: 100% (v/v) A
  • 10min 25% (v/v) A
  • Detection is carried out with a UV detector (wavelength 214 nm).
  • 0min: 100% (v/v) A, 10min: 25% (v/v) A and 75% (v/v) B means from 0min to 10min, buffer A and buffer in the eluate The proportion of B was varied from 100% (v/v) A and 0% (v/v) B to 25% (v/v) A and 75% (v/v) B.
  • the test results are shown in Table 4 and Figures 9 to 14 for details.
  • the separation degree in Table 4 is the separation degree of the chromatographic peak of lysozyme and the chromatographic peak of cytochrome C.
  • Fig. 9 is the chromatogram of the PSDVB-NTA microsphere of embodiment 11 in test example 3 as chromatographic filler; Fig.
  • FIG. 10 is the chromatogram of the PSDVB-NTA microsphere of embodiment 12 in test example 3 as chromatographic filler;
  • Fig. 11 is test The PSDVB-NTA microsphere of embodiment 13 in example 3 is used as the chromatogram of chromatographic filler;
  • Fig. 12 is the chromatogram of the PSDVB-NTA microsphere of embodiment 14 in test example 3 as chromatographic filler;
  • Fig. 13 is the chromatogram of test example 3
  • FIG. 14 is the chromatogram of the PSDVB-NTA microspheres of Comparative Example 2 in Test Example 3 as a chromatographic filler.
  • the chromatographic columns of Examples 11 to 14 all have good separation effects on lysozyme and cytochrome C.
  • the column efficiency and the degree of separation of the chromatographic peak of the lysozyme of embodiment 11 and embodiment 12 and the chromatographic peak of cytochrome C are all higher than embodiment 13 and embodiment 14, illustrate adopting aspartic acid to make more
  • the carboxyl-rich polystyrene-divinylbenzene microspheres are used as the packing of the chromatographic column, which is more conducive to improving the column efficiency and resolution of the ion-exchange chromatographic column for separating proteins.
  • the chromatographic columns of Comparative Examples 1-2 have short retention time for lysozyme and cytochrome C, low column efficiency, and small separation degree, and the separation effect on protein is significantly different from that of the chromatographic columns of Examples 11 to 14.
  • the polycarboxy-enriched polystyrene-divinylbenzene microspheres (PSDVB-NTA) prepared in Example 11 were used as chromatographic fillers, and the H9 glycated hemoglobin meter produced by Shenzhen Pumen Technology Co., Ltd. (Guangdong Machinery Note 20152401315) was used. In the mode of glycated hemoglobin, the blood sample to be tested is analyzed for glycated hemoglobin and variant hemoglobin.
  • the succinic acid buffer system was used as the eluent (Glycated hemoglobin (HbA1c) assay kit (high performance liquid chromatography) medical device registration certificate number: Guangdong Machinery Note 20152401314), the glycated hemoglobin control product of Pumen Technology (Guangdong Mechanical Note 20162400488) and calibrator (Guangdong Mechanical Note 20162400487), with a flow rate of 1.5mL/min, column temperature at 40°C, and a UV detector (wavelength 415nm) for detection.
  • the isocratic elution condition is 50s (phase A)-30s (phase C)-50s (phase B), according to the standard operating procedures of Pumen Technology H9 glycosylated hemoglobin instrument in the 130-second mode, the glycosylated hemoglobin was analyzed. Quantitative analysis and identification of common variants. "50s (Phase A)-30s (Phase C)-50s (Phase B)" means that the elution is 50 seconds with the A phase, followed by 30 seconds with the C phase, and then 50 seconds with the B phase; Similar descriptions are deduced in the same way, so no more details are given. And, according to the same test conditions, the quality control products were tested.
  • the blood sample to be tested is a whole blood sample, and the mass percentage of HbA1c in the whole blood sample is known to be 7.6%.
  • Quality control products include low-value quality control products (mass percentage content of HbA1c is 5.3%) and high-value quality control products (mass percentage content of HbA1c is 9.9%).
  • Table 5 shows the statistical table of test results of Test Example 4, and "%" in Table 5 represents the mass percentage.
  • FIG. 15 is a liquid chromatogram of the sample to be tested and the quality control in Test Example 4.
  • HbA1c-NGSP 0.9148HbA1c-IFCC+2.152
  • NGSP is the American National Glycated Hemoglobin Standardization Program
  • HbA1c-IFCC (10.93 ⁇ HbA1c-NGSP)-23.50
  • IFCC is the International Federation of Clinical Chemistry and Laboratory Medicine
  • the average Blood glucose concentration eAG (mmol/L) 1.59 ⁇ HbA1c-NGSP-2.59.
  • polycarboxy-enriched polystyrene-divinylbenzene microspheres (PSDVB-NTA) of Example 11, as a chromatographic filler, can separate and quantitatively detect glycosylated hemoglobin in whole blood samples, It can also separate and quantitatively detect non-glycosylated hemoglobin such as HbA0, HbA2 and HbF in whole blood samples.
  • the polycarboxy-enriched polystyrene-divinylbenzene microspheres (PSDVB-NTA) prepared in Example 11 were used as chromatographic fillers, and the H9 glycated hemoglobin meter produced by Shenzhen Pumen Technology Co., Ltd. (Guangdong Machinery Note 20152401315) was used.
  • the blood sample to be tested is analyzed for glycated hemoglobin and variant hemoglobin.
  • the specific test conditions are the same as those of Test Example 4, see above for details, and will not be repeated here.
  • sample 1 to sample 4 there are four blood samples to be tested, namely sample 1 to sample 4, sample 1 is a whole blood sample known to contain the variant HbE, sample 2 is a whole blood sample known to contain the variant HbD, and sample 3 is a known A whole blood sample containing the variant HbS, and sample 4 is a whole blood sample known to contain the variant HbC.
  • the measurement results are shown in Table 6 and Figure 16 for details.
  • Table 6 shows the statistical table of test results of Test Example 5.
  • FIG. 14 is a liquid chromatogram of the sample to be tested in Test Example 5.
  • E represents HbE
  • D represents HbD
  • S represents HbS
  • C represents HbC.
  • polycarboxy-enriched polystyrene-divinylbenzene microspheres (PSDVB-NTA) of Example 11, as a chromatographic filler, can also identify and detect HbE and HbD in the sample to be tested. , HbS and HbC and other variants, among which, the peak times of HbD and HbS are relatively close.
  • the polycarboxy-enriched polystyrene-divinylbenzene microspheres (PSDVB-NTA) prepared in Example 11 were used as chromatographic fillers, and the H9 glycated hemoglobin meter produced by Shenzhen Pumen Technology Co., Ltd. (Guangdong Machinery Note 20152401315) was used. In the middle glycated hemoglobin mode, the glycated hemoglobin (HbA1c) content (mass percentage) of the blood sample to be tested is detected.
  • the specific test conditions are the same as those of Test Example 4, see above for details, and will not be repeated here.
  • Table 7 is a statistical table of test results of Test Example 6.
  • FIG. 17 is a diagram of the repeatability test results of Test Example 6.
  • sample 1 to sample 3 there are three blood samples to be tested, namely sample 1 to sample 3; sample 1 is a clinical whole blood sample, and the mass percentage of HbA1c is 5%-7%; sample 2 is a clinical whole blood sample, and the mass percentage of HbA1c is content is 7%-9%); sample 3 is a clinical whole blood sample, and the mass percentage content of HbA1c is 9%-12%; each of the three blood samples is repeatedly detected 20 times, and the average value M and Standard deviation SD, coefficient of variation (CV) was obtained according to the following formula.
  • CV coefficient of variation
  • SD standard deviation of 20 measurements
  • M average of 20 measurements.
  • the coefficient of variation (CV) of repeated measurement results should not be greater than 3.0%.
  • the polycarboxy-enriched polystyrene-divinylbenzene microspheres (PSDVB-NTA) of Example 11 is used as a chromatographic filler, and the CV detected for sample 1 is 0.92%, and for sample 2 The CV detected is 0.45%, and the CV detected for sample 3 is 0.64%, which shows that the polycarboxy-enriched polystyrene-divinylbenzene microspheres (PSDVB-NTA) of Example 11 has better repeatability as a chromatographic filler, Meet repeatability requirements.
  • the polycarboxy-enriched polystyrene-divinylbenzene microspheres (PSDVB-NTA) prepared in Example 11 were used as chromatographic fillers, and the H9 glycated hemoglobin meter produced by Shenzhen Pumen Technology Co., Ltd. (Guangdong Machinery Note 20152401315) was used. In the middle glycated hemoglobin mode, the glycated hemoglobin (HbA1c) content (mass percentage) of the blood sample to be tested is detected.
  • the specific test conditions are the same as those of test example 4. See above for details, and will not be repeated here.
  • Test comparative example Use the HLC-723G8 automatic glycosylated hemoglobin analyzer from Tosoh Corporation of Japan and the matching glycosylated hemoglobin determination kit (high performance liquid chromatography) to perform glycosylated hemoglobin (HA) on the same blood sample to be tested above according to the operation instructions of the kit. , HbA1c) content (mass percentage) detection.
  • the blood samples to be tested were 40 clinical samples, including 12 normal physical examination samples and 28 diabetic samples.
  • the measurement results are shown in Table 8 and Figure 18 in detail.
  • Table 8 is a statistical table of test results of Test Example 7.
  • the relative deviation is the relative deviation of this study (the polycarboxy-enriched polystyrene-divinylbenzene microspheres (PSDVB-NTA) prepared in Example 11 as a chromatographic filler) and the comparative example for the detection results of the same sample .
  • FIG. 18 is a diagram of the correlation test results of Test Example 7.
  • the correlation R2 of the test results should not be lower than 0.99, and the relative deviation between the measured value of more than 90% of the samples and the target value is within ⁇ 6%. It can be seen from Table 8 and Figure 18 that the relative deviation between the detection results of the chromatographic system in this study and the detection results of Tosoh’s chromatographic system is within ⁇ 6 %, and the correlation R2 is 0.9978, which can meet the requirements.
  • the polycarboxy-enriched polystyrene-divinylbenzene microspheres (PSDVB-NTA) prepared in Example 11 were used as chromatographic fillers, and the H9 glycated hemoglobin meter produced by Shenzhen Pumen Technology Co., Ltd. (Guangdong Machinery Note 20152401315) was used.
  • the HbA1c content (mass percentage) of fresh whole blood samples of diabetic patients was detected in the mode of glycated hemoglobin.
  • the specific test conditions are the same as those of Test Example 4, see above for details, and will not be repeated here. Turn on and off the machine every day for quality control, test about 600T (that is, 600 times) per day, and test for about 5 days.
  • the polycarboxy-enriched polystyrene-divinylbenzene microspheres (PSDVB-NTA) prepared in Example 11 were used as chromatographic fillers, and the blood samples to be tested were analyzed using the H9 hemoglobin meter thalassemia analysis mode produced by Shenzhen Pumen Technology Co., Ltd. Variant hemoglobin analysis.
  • the succinic acid buffer system is used as the eluent, with a flow rate of 1.5mL/min, a column temperature of 30°C, and a UV detector (wavelength 415nm) for detection.
  • the analysis time is 380s, and the sample volume of the whole blood sample is 7 ⁇ L , after diluting evenly with 1.5mL ultrapure water diluent, the injection volume is 5 ⁇ L, isocratic elution conditions: 60S(A2 phase)-50S(B2 phase)-50S(A2 phase)-40S(B2 phase)-140S( C2 phase)-40S (A2 phase), A2 phase is 45mM succinate buffer (pH5.4), B2 phase is 100mM succinate buffer (pH5.4), C2 phase is 530mM succinate Buffer (pH5.4).
  • sample 1 is a whole blood sample containing HbA2
  • sample 2 is a whole blood sample containing HbE
  • sample 3 is a whole blood sample containing HbD
  • sample 4 is a whole blood sample containing HbS
  • sample 5 is a whole blood sample containing HbC
  • sample 6 is a whole blood sample containing HbJ-Bangkok
  • sample 7 is a whole blood sample containing Hb G-Taipei
  • sample 8 is a whole blood sample containing Hb Q-Thailand. See Table 10 to Table 11, Figure 20 to Figure 23 for details of the measurement results.
  • Table 10 shows the retention time range of conventional hemoglobin
  • Table 11 shows the retention time of variant hemoglobin in samples 1-8.
  • FIG. 20 is the detection chromatograms of samples 1 to 5 in test example 9.
  • Figure 21 is a liquid chromatogram of the Hb J-Bangkok whole blood sample of sample 6 in Test Example 9.
  • 22 is a liquid chromatogram of the HbG-Taipei whole blood sample of sample 7 in Test Example 9.
  • 23 is a liquid chromatogram of the Hb Q-Thailand whole blood sample of sample 8 in Test Example 9.
  • the PSDVB-NTA ion-exchange filler microspheres of the present application can separate HbA1a, HbA1b, LA1c, HbF, HbA1c, P3, P4, HbA0, HbA2, HbE, HbD, HbS, HbC, etc. 13 A hemoglobin substance. It can be seen from Table 11 and Figure 21 to Figure 23 that the PSDVB-NTA ion-exchange filler microspheres of the present application can also separate and detect common hemoglobin variants such as HbJ-Bangkok, HbG-Taipei, and HbQ-Thailand in China .
  • the PSDVB-NTA ion-exchange filler microspheres of the present invention can separate and quantify glycosylated hemoglobin within 130 seconds at most and can identify hemoglobin variants, and can separate glycosylated hemoglobin and various hemoglobin within 380 seconds at most.
  • 16 kinds of hemoglobin substances including variants have excellent ion exchange performance, have high specific separation ability for hemoglobin and its variants, and have high detection speed.
  • the PSDVB-NTA ion-exchange filler microspheres of the present invention can withstand a pressure of more than 20 MPa, and have the advantages of high separation ability, low protein non-specific adsorption, high pressure resistance, good dispersion, uniform and controllable particle size, and the like.

Abstract

本发明涉及一种乙烯基单体-多乙烯基交联剂共聚物无孔微球及其制备方法和应用。乙烯基单体-多乙烯基交联剂共聚物无孔微球包括：乙烯基单体-多乙烯基交联剂共聚物母球，乙烯基单体-多乙烯基交联剂共聚物母球修饰有羟基；经羟基接枝于乙烯基单体-多乙烯基交联剂共聚物母球的环氧基化合物，环氧基化合物包含至少两个环氧基。该乙烯基单体-多乙烯基交联剂共聚物无孔微球的化学和机械稳定性良好，且具有优良的离子交换性能，对血红蛋白及其变异体具有较高的特异性分离能力。

Description

乙烯基单体-多乙烯基交联剂共聚物无孔微球及其制备方法和应用 技术领域
本发明涉及聚合物微球技术领域，特别是涉及一种乙烯基单体-多乙烯基交联剂共聚物无孔微球及其制备方法和应用。
背景技术
聚合物微球通常具有球型好、尺寸小、比表面积大、吸附性能强和可进行功能化修饰等特性，因此，其作为功能性材料在各行业均得到广泛的应用。近年来，利用离子交换高效液相色谱法(HPLC)进行生物组分的测定日趋获得人们的关注，而离子交换色谱微球装填的液相色谱柱是利用离子交换高效液相色谱法进行生物组分的分离检测的核心硬件之一。
装填液相色谱柱的离子交换填料分为有孔填料和无孔填料，与有孔填料相比，无孔填料具有更大的传质和更小的横向扩散的优点，可以使得样品得到更快速、高效的分离。但是无孔填料的微球存在比表面积低的缺陷，导致分离容量不高，从而限制了其在蛋白分离中的许多应用。另外，基于天然聚合物的色谱填料通常表现出较差的刚性，不够耐压，主要用于生物药物的纯化分离。
传统的乙烯基单体-乙烯基交联剂共聚物无孔微球作为色谱填料，主要是甲基丙烯酸酯类共聚物和苯乙烯-二乙烯基苯类共聚物，这两种无孔微球具有良好的刚性，但就目前而言，这类无孔微球的离子交换性能较差，对生物组分，如蛋白分子载量较低，具有很强的非特异性吸附，影响色谱填料的使用寿命。
发明内容
基于此，本发明提供一种乙烯基单体-多乙烯基交联剂共聚物无孔微球及其制备方法和应用。该乙烯基单体-多乙烯基交联剂共聚物无孔微球的化学和机械稳定性良好，且具有优良的离子交换性能，对生物组分(如蛋白分子)的非特异性吸附较低。
具体技术方案如下：
本发明的一方面，提供一种乙烯基单体-多乙烯基交联剂共聚物无孔微球，包括：
乙烯基单体-多乙烯基交联剂共聚物母球，所述乙烯基单体-多乙烯基交联剂共聚物母球修饰有羟基；
经所述羟基接枝于所述乙烯基单体-多乙烯基交联剂共聚物母球的环氧基化合物，所述环氧基化合物包含至少两个环氧基。
在其中一个实施例中，所述环氧基化合物选自山梨醇四缩水甘油醚、季戊四醇四缩水甘油醚、聚丙三醇多缩水甘油醚、二丙三醇三缩水甘油醚、1,4-丁二醇二缩水甘油醚、丙三醇二缩水甘油醚、丙三醇三缩水甘油醚、乙二醇二缩水甘油醚、聚乙二醇二缩水甘油醚、新戊二 醇二缩水甘油醚、2,2-双(4-(2,3-环氧丙氧基)苯基)丙烷中的至少一种；作为优选地，所述环氧基化合物选自山梨醇四缩水甘油醚和聚丙三醇多缩水甘油醚。
在其中一个实施例中，所述乙烯基单体-多乙烯基交联剂共聚物母球包括：
乙烯基单体-多乙烯基交联剂共聚物颗粒，所述乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰有环氧基；所述乙烯基单体-多乙烯基交联剂共聚物颗粒由聚苯乙烯种子微球与乙烯基单体经多乙烯基交联剂交联而成；所述乙烯基单体中包含所述环氧基；
经所述环氧基接枝于所述乙烯基单体-多乙烯基交联剂共聚物颗粒的羟基化合物，所述羟基化合物包含所述羟基。
在其中一个实施例中，所述乙烯基单体-多乙烯基交联剂共聚物母球的交联度为50％～80％，优选地，所述乙烯基单体-多乙烯基交联剂共聚物母球的交联度为65％～75％。
在其中一个实施例中，所述乙烯基单体选自4-乙烯苯基缩水甘油醚、4-乙烯苄基缩水甘油醚和甲基丙烯酸缩水甘油醚中的至少一种。
在其中一个实施例中，所述聚苯乙烯种子微球的平均粒径D50为0.3μm～5μm；作为优选地，所述聚苯乙烯种子微球的平均粒径D50为0.5μm～3μm。
在其中一个实施例中，所述羟基化合物选自乙二醇、1,3-丙二醇及1,4-丁二醇中的至少一种。
在其中一个实施例中，所述多乙烯基交联剂选自二乙烯基苯、二乙烯基甲苯、三乙烯基苯、二甲基丙烯酸乙二醇酯、聚乙二醇二甲基丙烯酸酯、二乙烯基萘和三乙烯基萘等中的至少一种；作为优选地，所述多乙烯基交联剂为二乙烯基苯。
在其中一个实施例中，所述乙烯基单体的用量为所述乙烯基单体和所述多乙烯基交联剂总重量的5％～40％；作为优选地，所述乙烯基单体的用量为所述乙烯基单体和所述多乙烯基交联剂总重量的10％～30％；更为优选地，所述乙烯基单体的用量为所述乙烯基单体和所述多乙烯基交联剂总重量的10％～20％。
在其中一个实施例中，所述环氧基化合物与所述乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰的环氧基的摩尔比为(1～20):1；作为优选地，所述环氧基化合物与所述乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰的环氧基的摩尔比为(3～10):1；更为优选地，所述环氧基化合物与所述乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰的环氧基的摩尔比为(4～6):1。
在其中一个实施例中，所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球还包括活性基团，所述活性基团接枝于所述环氧基化合物。其中，活性基团例如可以为含有官能团的基团。
在其中一个实施例中，所述活性基团包括NTA基团。如无特别说明，以下内容中，NTA基团即为氨基三羧甲基结构基团。
在其中一个实施例中，所述NTA基团包含活性端基；所述活性端基选自天门冬氨酸端基、赖氨酸端基、半胱氨酸端基和谷氨酸端基等中的至少一种。
在其中一个实施例中，所述乙烯基单体-多乙烯基交联剂共聚物无孔微球的平均粒径D50为2μm～10μm；作为优选地，所述乙烯基单体-多乙烯基交联剂共聚物无孔微球的平均粒径D50为3μm～6μm。
本发明的又一方面，提供一种乙烯基单体-多乙烯基交联剂共聚物无孔微球的制备方法，其特征在于，包括如下步骤：
制备乙烯基单体-多乙烯基交联剂共聚物母球，所述乙烯基单体-多乙烯基交联剂共聚物母球修饰有羟基；
将环氧基化合物经所述羟基反应接枝至所述乙烯基单体-多乙烯基交联剂共聚物母球，所述环氧基化合物包含至少两个环氧基。
在其中一个实施例中，所述环氧基化合物选自山梨醇四缩水甘油醚、季戊四醇四缩水甘油醚、聚丙三醇多缩水甘油醚、二丙三醇三缩水甘油醚、1,4-丁二醇二缩水甘油醚、丙三醇二缩水甘油醚、丙三醇三缩水甘油醚、乙二醇二缩水甘油醚、聚乙二醇二缩水甘油醚、新戊二醇二缩水甘油醚、2,2-双(4-(2,3-环氧丙氧基)苯基)丙烷等中的至少一种；作为优选地，所述环氧基化合物选自山梨醇四缩水甘油醚和聚丙三醇多缩水甘油醚中的至少一种。
在其中一个实施例中，所述制备所述乙烯基单体-多乙烯基交联剂共聚物母球的方法包括如下步骤：
制备乙烯基单体-多乙烯基交联剂共聚物颗粒，所述乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰有环氧基，制备步骤包括：以聚苯乙烯种子微球为种子，与乙烯基单体和多乙烯基交联剂共同在稳定剂和乳化剂存在的条件下，通过种子溶胀法在介质中进行聚合；所述乙烯基单体中包含所述环氧基；
将羟基化合物经所述环氧基反应接枝至所述乙烯基单体-多乙烯基交联剂共聚物颗粒，所述羟基化合物包含所述羟基。
在其中一个实施例中，所述乙烯基单体选自4-乙烯苯基缩水甘油醚、4-乙烯苄基缩水甘油醚和甲基丙烯酸缩水甘油醚中的至少一种。
在其中一个实施例中，所述聚苯乙烯种子微球的平均粒径D50为0.3μm～5μm；作为优选地，所述聚苯乙烯种子微球的平均粒径D50为0.5μm～3μm。
在其中一个实施例中，所述羟基化合物选自乙二醇、1,3-丙二醇及1,4-丁二醇中的至少一种。
在其中一个实施例中，所述多乙烯基交联剂选自二乙烯基苯、二乙烯基甲苯、三乙烯基 苯、二甲基丙烯酸乙二醇酯、聚乙二醇二甲基丙烯酸酯、二乙烯基萘和三乙烯基萘中的至少一种；作为优选地，所述多乙烯基交联剂为二乙烯基苯。
在其中一个实施例中，所述乙烯基单体的用量为所述乙烯基单体和所述多乙烯基交联剂总重量的5％～40％；作为优选地，所述乙烯基单体的用量为所述乙烯基单体和所述多乙烯基交联剂总重量的10％～30％；更为优选地，所述乙烯基单体的用量为所述乙烯基单体和所述多乙烯基交联剂总重量的10％～20％。
在其中一个实施例中，所述环氧基化合物与所述乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰的环氧基的摩尔比为(1～20):1；作为优选地，所述环氧基化合物与所述乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰的环氧基的摩尔比为(3～10):1；更为优选地，所述环氧基化合物与所述乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰的环氧基的摩尔比为(4～6):1。
在其中一个实施例中，所述聚合采用的引发剂为油性引发剂；作为优选地，油溶性过氧化物和油溶性偶氮化合物中的一种。
在其中一个实施例中，所述引发剂的用量为所述乙烯基单体重量的0.1％～10％；作为优选地，所述引发剂的用量为所述乙烯基单体重量的0.5％～5％。
在其中一个实施例中，所述稳定剂为水溶性聚合物；作为优选地，所述稳定剂为聚乙烯醇、聚乙烯吡咯烷酮和改性纤维素中的至少一种。
在其中一个实施例中，所述稳定剂的用量为所述乙烯基单体重量的0.5％～15％；作为优选地，所述稳定剂的用量为所述乙烯基单体重量的1％～8％。
在其中一个实施例中，所述乳化剂选自阳离子表面活性剂、非离子表面活性剂、两性表面活性剂和阴离子表面活性剂中的至少一种。
在其中一个实施例中，所述阳离子表面活性剂选自季铵盐、烷基吡啶盐和胺盐中的至少一种。
在其中一个实施例中，所述非离子表面活性剂选自烷基醚聚合物和烷基酚聚烷氧基化合物中的至少一种。
在其中一个实施例中，所述两性表面活性剂选自氨基酸型、甜菜碱型中的至少一种。
在其中一个实施例中，所述阴离子表面活性剂选自羧基盐表面活性剂、磺酸盐表面活性剂、硫酸盐表面活性剂和磷酸盐表面活性剂中的至少一种。
在其中一个实施例中，所述乳化剂的用量为所述乙烯基单体重量的0.05％～10％；作为优选地，所述乳化剂的用量为所述乙烯基单体重量的0.1％～5％。
在其中一个实施例中，所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球的制备方法还包括：于所述环氧基化合物之上反应接枝形成活性基团。其中，活性基团例如可以为含有官 能团的基团。可选地，活性基团包括NTA基团。
在其中一个实施例中，形成NTA基团的方法选自N,N-二羧甲基赖氨酸法、活性端基氨基酸法、二羧酸结构氨基酸羧基化法、先构建醛基再构建α-氨基酸法中的一种。
本发明的又一方面，提供所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球，以及所述的所述制备方法制备得到的乙烯基单体-多乙烯基交联剂共聚物无孔微球之一在色谱填料中的应用。
本发明的又一方面，提供所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球，以及所述的所述制备方法制备得到的乙烯基单体-多乙烯基交联剂共聚物无孔微球之一在生物组分检测中的应用。
在其中一个实施例中，所述生物组分为血红蛋白类生物组分。
与现有技术相比较，本发明具有如下有益效果：
本发明所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球，在修饰有羟基的乙烯基单体-多乙烯基交联剂共聚物母球的基础上，接枝偶联包含至少两个环氧基的环氧基化合物，使微球上带有更多的环氧基。利用接枝多环氧基的方法弥补无孔(离子交换)填料微球比表面积低，离子交换容量低的缺点，使无孔填料对目标生物组分的特异性吸附能力达到与有孔填料相当的水平。另外，无孔填料在色谱分离中具有最大传质和最小横向扩散的优点，因此可使生物样品得到快速、高效的分离。综上，所述乙烯基单体-多乙烯基交联剂共聚物无孔微球能够兼顾高载量和高分离效率两个优点。
同时，采用环氧基的所述乙烯基单体-多乙烯基交联剂共聚物无孔微球的化学性能和机械稳定性(包括刚性)良好，同时，在较小的比表面积下，表面能够集聚更多的离子交换基团(如NTA结构，实现多羧基化)。微球表面采用环氧基修饰改性后，作为色谱填料，在生物大分子的分析、分离纯化中表现出优异的性能，消除了疏水性结构对生物分子的非特异性吸附。
进一步地，在对所述乙烯基单体-多乙烯基交联剂共聚物无孔微球进行NTA结构修饰后，在血红蛋白类生物组分检测中分离效果显著，不仅能定量分析HbA1c，还能分离分析16种血红蛋白类生物组分，对国际上常见的血红蛋白变异体可以进行定性和定量的分析，对血红蛋白及其变异体具有较高的特异性分离能力。在体外诊断测试中采用如深圳普门科技有限公司生产的H9糖化血红蛋白仪即可实现对血红蛋白及其变异体的分离和分析，且综合具有高的分离能力(分析谱图)、低蛋白非特异性吸附(色谱柱寿命长)、高的耐压性能(16MPa)等优点。
另外，所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球还具有单分散性、分散性良好， 粒径大小均一、可控的优点。
附图说明
图1为实施例3制备的PST种子微球扫描电镜图；
图2为实施例3制备的PST种子微球粒径分布图；
图3为实施例4制备的PSDVB-Epoxy微球扫描电镜图；
图4为实施例4制备的PSDVB-Epoxy微球粒径分布图；
图5为测试例1中实施例4至实施例7的PSDVB-Epoxy微球制成的色谱柱在流速为0.5mL/min～5.25mL/min范围内流速与柱压的拟合曲线；
图6为测试例2中1号色谱柱的20次进样检测的峰面积统计图；
图7为测试例2中2号色谱柱的20次进样检测的峰面积统计图；
图8为测试例2中3号色谱柱的20次进样检测的峰面积统计图；
图9为测试例3中实施例11的PSDVB-NTA微球作为色谱填料的色谱图；
图10为测试例3中实施例12的PSDVB-NTA微球作为色谱填料的色谱图；
图11为测试例3中实施例13的PSDVB-NTA微球作为色谱填料的色谱图；
图12为测试例3中实施例14的PSDVB-NTA微球作为色谱填料的色谱图；
图13为测试例3中对比例1的PSDVB-NTA微球作为色谱填料的色谱图；
图14为测试例3中对比例2的PSDVB-NTA微球作为色谱填料的色谱图
图15为测试例4中待测样本和质控品的液相色谱图；
图16为测试例5中待测样本的液相色谱图；
图17为测试例6的重复性测试结果图；
图18为测试例7的相关性测试结果图；
图19为测试例8的质控图；
图20为测试例9中样本1～样本5到的检测色谱图；
图21为测试例9中样本6的Hb J-Bangkok全血样品的液相色谱图；
图22为测试例9中样本7的Hb G-Taipei全血样品的液相色谱图；
图23为测试例9中样本8的Hb Q-Thailand全血样品的液相色谱图。
具体实施方式
以下结合具体实施例对本发明的乙烯基单体-多乙烯基交联剂共聚物无孔微球及其制备方法和应用作进一步详细的说明。本发明可以以许多不同的形式来实现，并不限于本文所描述的实施方式。相反地，提供这些实施方式的目的是使对本发明公开内容理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人 员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。
多乙烯基交联剂是指含有至少两个乙烯基的交联剂。
本发明提供一种乙烯基单体-多乙烯基交联剂共聚物无孔微球，包括：
乙烯基单体-多乙烯基交联剂共聚物母球，乙烯基单体-多乙烯基交联剂共聚物母球修饰有羟基；
经羟基接枝于乙烯基单体-多乙烯基交联剂共聚物母球的环氧基化合物，环氧基化合物包含至少两个环氧基。
进一步地，采用包含环氧基团的乙烯基单体替代苯乙烯单体和多乙烯基交联剂进行聚合，形成修饰有环氧基的乙烯基单体-多乙烯基交联剂共聚物颗粒(例如：PSDVB-Epoxy微球)，然后修饰有环氧基的乙烯基单体-多乙烯基交联剂共聚物颗粒上的环氧基在羟基化合物的作用下进行水解，形成羟基，便于进行后续多环氧基化合物的偶联。
本研究发现，采用乙烯基单体和多乙烯交联剂聚合形成乙烯基单体-多乙烯基聚合物，然后采用含有环氧基的化合物对乙烯基单体-多乙烯基聚合物进行修饰，以使乙烯基单体-多乙烯基聚合物上接合有环氧基。此种通过先形成聚合物再进行修饰，使得部分修饰位点裹入聚合物母球内核而被遮蔽，导致乙烯基单体-多乙烯基聚合物(尤其是强疏水性聚合物PSDVB苯环)亲水化(即偶联羟基或环氧基)转化率低，环氧基的接合量较低。本发明中，采用包含环氧基团的乙烯基单体替代苯乙烯单体和多乙烯基交联剂进行聚合，能够通过一步反应即完成聚合与修饰，得到修饰有环氧基的乙烯基单体-多乙烯基交联剂共聚物颗粒，能够避免部分修饰位点裹入聚合物母球内核而被遮蔽的问题，提高导致乙烯基单体-多乙烯基聚合物(尤其是强疏水性聚合物PSDVB苯环)亲水化(偶联羟基或环氧基)转化率，提高环氧基的接合量。同时，以该修饰有环氧基的乙烯基单体-多乙烯基交联剂共聚物颗粒作为母球还能够赋予乙烯基单体-多乙烯基交联剂共聚物无孔微球优异的抗压性。
具体地，乙烯基单体-多乙烯基交联剂共聚物母球包括：
乙烯基单体-多乙烯基交联剂共聚物颗粒，乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰有环氧基；乙烯基单体-多乙烯基交联剂共聚物颗粒由聚苯乙烯种子微球与乙烯基单体经多乙烯基交联剂交联而成；乙烯基单体中包含环氧基；以及
经环氧基接枝于乙烯基单体-多乙烯基交联剂共聚物颗粒的羟基化合物，羟基化合物包含羟基。
在其中一个示例中，乙烯基单体-多乙烯基交联剂共聚物母球的交联度为50％～80％，优选为65％～75％。
在其中一个示例中，聚苯乙烯种子微球的平均粒径D50为0.3μm～5μm；作为优选地，聚苯乙烯种子微球的平均粒径D50为0.5μm～3μm。本发明采用上述单分散粒径的聚苯乙烯种子微球，有利于得到粒径可控均一的单分散乙烯基单体-多乙烯基交联剂共聚物无孔微球，提高乙烯基单体-多乙烯基交联剂共聚物无孔微球对血红蛋白变异体等物质的识别能力。在其中一个示例中，乙烯基单体-多乙烯基交联剂共聚物无孔微球的平均粒径D50为2μm～10μm；作为优选地，乙烯基单体-多乙烯基交联剂共聚物无孔微球的平均粒径D50为3μm～6μm。前述微球外观和平均粒径由扫描电镜SEM和激光粒度仪测定。作为优选地，聚苯乙烯种子微球为非交联线性聚合物，由分散聚合法制备而成。采用非交联线性聚合物的聚苯乙烯种子微球，有利于得到单分散的、粒径均一的乙烯基单体-多乙烯基交联剂共聚物无孔微球。
在其中一个示例中，乙烯基单体选自4-乙烯苯基缩水甘油醚、4-乙烯苄基缩水甘油醚和甲基丙烯酸缩水甘油醚中的至少一种。作为优选地，乙烯基单体选自4-乙烯苯基缩水甘油醚和4-乙烯苄基缩水甘油醚中的至少一种。
具体地，4-乙烯苯基缩水甘油醚是由4-羟基苯乙烯和环氧氯丙烷制备而成；4-乙烯苄基缩水甘油醚是由4-氯甲基苯乙烯和缩水甘油制备而成。
在其中一个示例中，多乙烯基交联剂选自二乙烯基苯、二乙烯基甲苯、三乙烯基苯、二甲基丙烯酸乙二醇酯、二乙烯基萘和三乙烯基萘中的至少一种；作为优选地，多乙烯基交联剂为二乙烯基苯。
在其中一个示例中，乙烯基单体的用量为乙烯基单体和多乙烯基交联剂总重量的5％～40％；作为优选地，乙烯基单体的用量为乙烯基单体和多乙烯基交联剂总重量的10％～30％；更为优选地，乙烯基单体的用量为乙烯基单体和多乙烯基交联剂总重量的10％～20％。
在其中一个示例中，羟基化合物选自乙二醇、1,3-丙二醇及1,4-丁二醇中的至少一种。
进一步地，乙烯基单体-多乙烯基交联剂共聚物颗粒的环氧基在羟基化合物的作用下水解后，进行多环氧基化合物的偶联，由此在结构中导入多环氧基结构，得到带有多环氧基的乙烯基单体-多乙烯基交联剂共聚物无孔微球。
在其中一个示例中，环氧基化合物选自山梨醇四缩水甘油醚、季戊四醇四缩水甘油醚、聚丙三醇多缩水甘油醚、二丙三醇三缩水甘油醚、1,4-丁二醇二缩水甘油醚、丙三醇二缩水甘油醚、丙三醇三缩水甘油醚、乙二醇二缩水甘油醚、聚乙二醇二缩水甘油醚(其分子量可为200～2000)、新戊二醇二缩水甘油醚、2,2-双(4-(2,3-环氧丙氧基)苯基)丙烷中的至少一种；作为优选地，环氧基化合物选自山梨醇四缩水甘油醚和聚丙三醇多缩水甘油醚中的至少一种。
在其中一个示例中，环氧基化合物与乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰的环 氧基的摩尔比为(1～20):1；作为优选地，环氧基化合物与乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰的环氧基的摩尔比为(3～10):1；更为优选地，环氧基化合物与乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰的环氧基的摩尔比为(4～6):1。最为优选地，环氧基化合物与乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰的环氧基的摩尔比为5:1。
进一步地，在带有多环氧基的乙烯基单体-多乙烯基共聚物颗粒(例如：PSDVB-mEpoxy)上构建活性基团，活性基团可选地包括NTA基团，得到多羧基富集的微球(例如：PSDVB-NTA)，由此能够作为液相色谱用弱阳离子交换填料。具体地，NTA基团接枝于环氧基化合物之上。需要说明的是，活性基团不限于为NTA基团，也可以为其他活性基团，例如含有-SO 3H或者-NH 2等官能团的活性基团，可以根据需要在带有多环氧基的乙烯基单体-多乙烯基单体微球上结合所需的活性基团。
在其中一个示例中，NTA基团包含活性端基；活性端基选自天门冬氨酸端基、赖氨酸端基、半胱氨酸端基和谷氨酸端基中的至少一种。
在其中一个示例中，构建NTA基团的方法有活性端基氨基酸法、N,N-二羧甲基赖氨酸法、二羧酸结构氨基酸羧基化(所述二羧酸结构氨基酸优选为酸性氨基酸)、微球表面醛基化后再构建α-氨基酸(具体可以参见专利CN109663384A)后，然后生成NTA基团等。
本发明还提供一种乙烯基单体-多乙烯基交联剂共聚物无孔微球的制备方法，包括如下步骤：
制备乙烯基单体-多乙烯基交联剂共聚物母球，乙烯基单体-多乙烯基交联剂共聚物母球修饰有羟基；
将环氧基化合物经羟基反应接枝至乙烯基单体-多乙烯基交联剂共聚物母球，环氧基化合物包含至少两个环氧基。
进一步地，制备乙烯基单体-多乙烯基交联剂共聚物母球的方法包括如下步骤：
制备乙烯基单体-多乙烯基交联剂共聚物颗粒，乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰有环氧基，制备步骤包括：以聚苯乙烯种子微球为种子，与乙烯基单体和多乙烯基交联剂共同在稳定剂和乳化剂存在的条件下，通过种子溶胀法在介质中进行聚合；乙烯基单体中包含环氧基；
以及将环氧基化合物经环氧基反应接枝至乙烯基单体-多乙烯基交联剂共聚物颗粒，羟基化合物包含羟基。
本研究发现，可以先采用聚苯乙烯和多乙烯苯芳香族化合物聚合形成乙烯基单体-多乙烯基聚合物，然后采用含有环氧基的化合物对乙烯基单体-多乙烯基聚合物进行修饰，以使乙烯基单体-多乙烯基聚合物上接合有环氧基。此种方法通过先形成聚合物再进行修饰，使得部分 修饰位点裹入聚合物母球内核而被遮蔽，导致乙烯基单体-多乙烯基聚合物(尤其是强疏水性聚合物PSDVB苯环上)亲水化(偶联羟基或环氧基)转化率低，环氧基的接合量较低。本发明中，以聚苯乙烯种子微球为种子，与修饰有环氧基的乙烯基单体和多乙烯基交联剂共同在稳定剂和乳化剂存在的条件下进行聚合，能够通过一步反应即完成聚合与修饰，得到修饰有环氧基的乙烯基单体-多乙烯基交联剂共聚物颗粒，能够避免部分修饰位点裹入聚合物母球内核而被遮蔽的问题，提高乙烯基单体-多乙烯基聚合物(尤其是强疏水性聚合物PSDVB苯环上)亲水化(偶联羟基或环氧基)转化率，提高环氧基的接合量。
在其中一个示例中，聚合采用的引发剂为油性引发剂；作为优选地，油溶性过氧化物和油溶性偶氮化合物中的一种。具体地，油溶性过氧化物可以举例例如过氧化二苯甲酰、过氧化二辛酰、过氧化月桂酰等；油溶性偶氮化合物可以举例例如2,2’-偶氮二异丁腈、2,2’-偶氮二异己腈等。
在其中一个示例中，引发剂的用量为乙烯基单体重量的0.1％～10％；作为优选地，引发剂的用量为乙烯基单体重量的0.5％～5％。
在其中一个示例中，稳定剂为水溶性聚合物；作为优选地，稳定剂为聚乙烯醇、聚乙烯吡咯烷酮和改性纤维素中的至少一种。
在其中一个示例中，稳定剂的用量为乙烯基单体重量的0.5％～15％；作为优选地，稳定剂的用量为乙烯基单体重量的1％～8％。
在其中一个示例中，乳化剂是指合适的表面活性剂，如阳离子表面活性剂、非离子表面活性剂、两性表面活性剂和阴离子表面活性剂中的至少一种。
具体地，阳离子表面活性剂选自季铵盐、烷基吡啶盐和胺盐中的至少一种；非离子表面活性剂选自烷基醚聚合物和烷基酚聚烷氧基化合物(如烷基酚聚乙氧基化物)中的至少一种；两性表面活性剂选自氨基酸型两性表面活性剂(如十二烷基氨基丙酸)、甜菜碱型两性表面活性剂(如硫代甜菜碱)中的至少一种；阴离子表面活性剂选自羧基盐表面活性剂、磺酸盐表面活性剂、硫酸盐表面活性剂和磷酸盐表面活性剂中的至少一种。
在其中一个示例中，乳化剂的用量为乙烯基单体重量的0.05％～10％；作为优选地，乳化剂的用量为乙烯基单体重量的0.1％～5％。
为了更好地说明上述乙烯基单体-多乙烯基交联剂共聚物无孔微球的制备方法的过程和机理，不作限定地，制备过程如下：
S1、4-乙烯苯基缩水甘油醚作为包含环氧基的乙烯基单体的合成
4-乙烯苯基缩水甘油醚的合成方法为：4-羟基苯乙烯和环氧氯丙烷在碱性环境中合成得到。反应机理如式(1)所示：
Figure PCTCN2022083708-appb-000001
S2、4-乙烯苄基缩水甘油醚作为包含环氧基的乙烯基单体的合成
4-乙烯苄基缩水甘油醚的合成方法为：1-(氯甲基)-4-乙烯基苯和缩水甘油在碱性环境中合成而成。反应机理如式(2)所示：
Figure PCTCN2022083708-appb-000002
S3、聚苯乙烯(PST)种子的合成方法为：苯乙烯(St)单体聚合而成，反应机理如式(3)所示：
Figure PCTCN2022083708-appb-000003
S4、表面带有环氧基的乙烯基单体-多乙烯基交联剂共聚物颗粒(例如：PSDVB-Epoxy)的合成
在PST种子为模板，将包含环氧基的乙烯基单体和多乙烯基交联剂利用种子溶胀法合成表面带有环氧基的乙烯基单体-多乙烯基交联剂共聚物颗粒(例如：PSDVB-Epoxy)。
优选地，PST种子为模板，配合采用4-乙烯苄基缩水甘油醚(ST-Epoxy)为带有环氧基团的乙烯基单体。
优选地，表面带有环氧基的交联聚乙烯基单体-多乙烯基交联剂共聚物颗粒(例如：PSDVB-Epoxy)的方法为：
将PST种子、十二烷基磺酸钠(SDBS)/水混合相、4-乙烯苄基缩水甘油醚(ST-Epoxy)和二乙烯基苯(DVB)/油混合相反应得到表面带有环氧基的交联聚乙烯基单体-多乙烯基交联剂共聚物颗粒(例如：PSDVB-Epoxy)。反应机理如式(4)所示：
Figure PCTCN2022083708-appb-000004
S5、表面带有多环氧基的乙烯基单体-多乙烯交联剂共聚物微球(例如：PSDVB-mEpoxy) 的合成
对表面带有环氧基的交联聚乙烯基单体-多乙烯基交联剂共聚物颗粒(例如：PSDVB-Epoxy)表面的环氧基进行水解并偶联多环氧基化合物，得到表面带有多环氧基的乙烯基单体-多乙烯基交联剂共聚物微球(例如：PSDVB-mEpoxy)。
优选地，多环氧基化合物为山梨醇四缩水甘油醚。
优选地，对表面带有环氧基的交联聚乙烯基单体-多乙烯基交联剂共聚物颗粒(例如：PSDVB-Epoxy)表面的环氧基进行水解并偶联多环氧基化合物的方法为：将表面带有环氧基的交联聚乙烯基单体-多乙烯基交联剂共聚物颗粒先用乙二醇、环氧氯丙烷与催化剂BF 3·(CH 3CH 2) 2O在二氧六环溶剂中反应进行亲水性修饰，增加表面羟基和链长，再和山梨醇四缩水甘油醚的乙醇溶液在碱性条件下直接反应或加入适量的氰基硼氢化钠得到表面带有多环氧基的乙烯基单体-多乙烯基交联剂共聚物微球(例如：PSDVB-mEpoxy)。反应机理如式(5)所示：
Figure PCTCN2022083708-appb-000005
S6、多羧基富集的微球(例如：PSDVB-NTA)的合成
将表面带有多环氧基的乙烯基单体-多乙烯基交联剂共聚物微球(例如：PSDVB-mEpoxy)表面的多环氧基转化为三羧酸基团，构建NTA结构，得到多羧基富集的微球(例如：PSDVB-NTA)。
优选地，采用活性端基氨基酸法合成多羧基富集的微球(例如：PSDVB-NTA)。具体方法为：表面带有多环氧基的乙烯基单体-多乙烯基交联剂共聚物微球(例如：PSDVB-mEpoxy)的表面环氧基团在碱性条件(如8.4≤pH≤9.0)下，导入具有活性端基氨基酸(优选地，活性端基氨基酸为赖氨酸、半胱氨酸)进行反应，直接在微球表面构建α-氨基酸(摩尔比可采用1:1)，然后再在碱性条件下分步滴加溴乙酸，构建表面NTA结构，得到多羧基富集的微球(例如：PSDVB-NTA)。反应机理如式(6)所示：
Figure PCTCN2022083708-appb-000006
上述制备方法，一方面在微球制备过程中引入多环氧基，保证了良好的化学性能和机械稳定性(包括刚性)同时，在较小的比表面积下，表面能够集聚更多的离子交换基团，在血红蛋白类检测中分离效果显著。另一方面微球表面多环氧基偶联活性端基氨基酸法，多羧酸化过程更加简单易行。在生物大分子的分析、分离纯化中性能优异，尤其在血红蛋白类生物大分子组分检测中分离效果显著，不仅能定量分析HbA1c，还能分离分析16种血红蛋白类生物大分子组分，对国际上常见的血红蛋白变异体可以进行定性和定量的分析。
本发明还提供如上乙烯基单体-多乙烯基交联剂共聚物无孔微球，以及如上制备方法制备得到的乙烯基单体-多乙烯基交联剂共聚物无孔微球在色谱填料中的应用。进一步地，色谱填料是指液相色谱用弱阳离子交换填料，可以用于医疗器械体外诊断检测，特别是血红蛋白类生物大分子组分检测。举例而言，如利用离子交换高效液相色谱法测定血液中的糖化血红蛋白(HbA1c)的精确含量被国际间认定是糖尿病诊断的金标准。糖化血红蛋白分析仪是一种用于糖尿病人血糖检测的高效液相色谱法(HPLC)类型的医疗器械，采用离子交换色谱微球装填的液相色谱柱是糖化血红蛋白分析仪的核心硬件之一。
在针对血红蛋白类生物组分进行分离时，有一些研究工作者对无孔填料的微球进行表面处理，如一种无孔微球表面接枝聚丙烯酰胺亲水性改性的弱阳离子交换填料，可以用于血红蛋白检测，但是这种填料对血红蛋白的变异体分离效果不好；又如一种可用于血红蛋白类测定的多分散磺酸型阳离子交换聚合物微球，其优点是对设备要求低，在比较高速的条件下，HPLC系统压力也比较低，可以快速检测糖化血红蛋白，但是由于其粒径分布不均一、分辨率低，因此对血红蛋白的变异体识别能力比较差。
而将本发明的乙烯基单体-多乙烯基交联剂共聚物无孔微球用作糖化血红蛋白分析仪的色谱柱填料，可以直接测定全血中的HbA1c，其批内和批间变异系数CV均可以小于3％，结 果精确，且HbA1c检测结果不受存在的变异型血红蛋白及其衍生物的影响，特别适合于糖尿病人的诊断与血糖指标的监控。同时对血红蛋白及其变异体具有较高的特异性分离能力，能分离分析16种血红蛋白类生物组分。
另外，乙烯基单体-多乙烯基交联剂共聚物无孔微球还具有单分散性、分散性良好，粒径大小均一、可控的优点。
本发明还提供如上乙烯基单体-多乙烯基交联剂共聚物无孔微球，以及如上制备方法制备得到的乙烯基单体-多乙烯基交联剂共聚物无孔微球在血红蛋白类生物大分子组分检测的应用。可以理解地，该血红蛋白类生物组分检测为一种体外检测方法，直接目的不是获得诊断结果或健康状况，而是对已经脱离人体或动物体的组织、体液进行检测以获得作为中间结果的信息的方法。
以下为具体的实施例。
以下实施的测试例如未特殊说明，则不包括除不可避免的杂质外的其他组分。实施例中采用化学品和仪器如非特别说明，均为本领域传统选择。实施例中未注明具体条件的实验方法，按照传统条件，例如文献、书本中所述的条件或者生产厂家推荐的方法实现。
如无特别说明，以下实施例中的待测样品均为新鲜血液样品，取自于北京大学深圳医院。血红蛋白类物质的检测设备为市售高效液相色谱仪。稀释液为去离子水。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。
如无特别说明，以下内容中：
HbA1a(Hemoglobin-A1a)是乳糖化血红蛋白；
HbA1b(Hemoglobin-A1b)是果糖化血红蛋白；
HbA1c(Stable Glycated Hemoglobin)是稳定的葡萄糖化血红蛋白，HbA1c的含量能够表征过去2～3个月的平均血糖水平；
LA1c(Labile Glycated Hemoglobin)是不稳定的糖化血红蛋白，糖化过程中间产物Schiff碱可降解为葡萄糖和血红蛋白；
HbA0(Non-glycated Hemoglobin)是未糖化的血红蛋白，其含量占正常人的80％以上；
HbF(Fetal Hemoglobin)是胎儿血红蛋白；
HbA2(A2 Hemoglobin)即血红蛋白HbA2，其含量增多主要见于轻型β珠蛋白生成障碍性贫血，是β地贫因子；
P3(Peak3)和P4(Peak4)是血红蛋白HbA0的降解峰，通常含量小于5％。如果血液 样品不新鲜或出现其他变异血红蛋白，P3和P4含量将增加；
HbE(HbE Variant Hemoglobin)是珠蛋白β链第26位的Glu(谷氨酸)被Lys(赖氨酸)替代所形成的异常血红蛋白，HbE是珠蛋白β链异常血红蛋白中最常见的一种，HbE主要分布在东南亚国家。在我国，HbE主要存在于长江以南及沿海地区；
HbS(HbS Variant Hemoglobin)是珠蛋白β链第6位的Glu(谷氨酸)被Val(缬氨酸)替代所形成的异常血红蛋白，纯合子HbS称为镰刀细胞病，HbS最常见于北非及北美；
HbC(HbC Variant Hemoglobin)是珠蛋白β链第6位的Glu(谷氨酸)被Lys(赖氨酸)替代所形成的异常血红蛋白，主要分布于西非；
HbD(HbD Variant Hemoglobin)分布于印度西北部及南北美洲，其中，印度Panjab地区及印第安人携带HbD的人居多；
HbJ-Bangkok(J-BangkokVariant Hemoglobin)是珠蛋白β链第56位Gly(甘氨酸)被Asp(天冬氨酸)替代所形成的异常血红蛋白，在我国南方地区高发；
Hb G-Taipei(G-Taipei Variant Hemoglobin)是珠蛋白β链第22位Glu(谷氨酸)被Gly(甘氨酸)取代所形成的异常血红蛋白，在我国北方地区高发；
Hb Q-Thailand(Q-Thailand Variant Hemoglobin)是珠蛋白α链第74位Asp(天冬氨酸)被His(组氨酸)取代所形成的异常血红蛋白，在我国北方地区高发。
实施例中涉及的表征方式说明如下：
(1)微球表面形貌表征：采用扫描电子显微镜(SEM)进行微球的表面形貌的表征；
(2)微球粒径测定：使用市售激光粒度仪进行微球粒径测定；
(3)微球环氧值的测定。
采用高氯酸滴定法进行微球环氧值的测定，具体操作如下：将0.05g微球浸入10mL氯仿中1小时，超声分散在锥形瓶中。将10mL四乙基溴化铵溶液和2滴结晶紫指示剂加入锥形瓶中。用V mL校准高氯酸标准溶液(0.1M)滴定锥形瓶中的溶液，直到溶液颜色从蓝色转变为亮绿色并保持2h不变色。同时做空白对照实验，消耗V0mL高氯酸标准溶液。微球环氧值的计算公式如下：
E＝(V-V 0)*0.1/10。
(4)微球离子交换容量(IEC)检测
微球离子交换容量(IEC)检测的具体操作为：将11g微球和150mL硫酸溶液(0.5mol/L)加入烧瓶中，搅拌后浸泡过夜。将浸泡后的微球用去离子水洗涤几次至中性，干燥并静置。将干燥后的1g微球放入锥形瓶中，将0.08L标准NaOH溶液(0.05mol/L)于35℃加入锥形瓶中，轻微摇晃均匀，在35℃水浴一小时。滤出微球后得到滤液A。用20mL氯化钠溶液依次洗涤微 球4次，滤出微球后得到滤液B。将滤液A和B充分混合以获得混合液C，然后将20mL混合液倒入锥形烧瓶中。将2滴酚酞指示剂放入烧瓶中，加入0.05mol/L标准盐酸溶液滴定至溶液由紫红色变为无色，并30s内不变色，即达到终点，记录消耗0.05mol/L标准盐酸体积V HCl。微球离子交换容量(IEC)检测的计算公式如下：
IEC＝(0.05×0.08-8×0.05×V HCl)/V(eq/L)。
实施例1 4-乙烯苯基缩水甘油醚的合成
在250mL三颈烧瓶中加入4-羟基基苯乙烯(7.22g，60.1mmol)和乙醇(约10mL)中搅拌溶解备用；在配有磁力搅拌棒和的100mL烧杯中进一步用无水乙醇(50mL)溶解NaOH(3.12g，78.0mmol，1.3倍摩尔比)搅拌30分钟，加入上述三颈烧瓶。然后使用滴液漏斗将表氯醇(14.1mL，180mmol，3倍1.3倍摩尔比)快速添加至三颈烧瓶。在室温条件下，以150rpm转速搅拌，反应液逐渐变得浑浊，有氯化钠缓慢沉淀析出。继续反应16小时后，加入100mL水搅拌溶解析出的氯化钠，并使用分液漏斗用己烷(2×100mL)萃取混合物。有机相合并再用无水MgSO 4干燥，油相在真空下通过在35℃下旋转蒸发除去溶剂，得到4-乙烯基苯基缩水甘油醚粗粗品(10.71g)。
精制：将10mg对苯二酚阻聚剂添加到粗产物中，并在83℃下进行高真空蒸馏，得到蒸馏后4-乙烯基苯基缩水甘油醚，为淡黄色油状物，带有少量杂质。继续通过FLASH硅胶柱进行提纯，使用正己烷/二氯甲烷(80/20，V/V)作为洗脱液，可以除去大多数极性相对高的杂质。过柱提纯后4-乙烯基苯基缩水甘油醚再用200mL正己烷在-20℃下重结晶，最终产物在真空下通过在35℃下干燥后精制4-乙烯基苯基缩水甘油醚为5.730g，含量98.0％，产率为54％。
实施例2 4-乙烯苄基缩水甘油醚的合成
500mL三颈烧瓶通入氩气，在氩气气氛下，将氢化钠(60％在矿物油中，13.1g，327.60mmol，2.00倍摩尔比)加入DMF(300mL)中，并在0℃滴加缩水甘油(27.15mL，459.1mmol，2.50倍摩尔比)。将反应混合物搅拌30分钟后，控制搅拌速度150rpm，滴加1-(氯甲基)-4-乙烯基苯(23.3mL，163.80mmol，1.00倍摩尔比)，滴加结束后继续在室温下搅拌反应4小时。将混合物用乙酸乙酯(400mL)萃取。加入饱和NH 4Cl水溶液(200mL)以淬灭反应，水层继续用乙酸乙酯(3×100mL)萃取。合并有机层，用无水Na 2SO 4干燥，油相在真空下通过在35℃下旋转蒸发除去溶剂。残余物通过FLASH柱纯化，使用正己烷/二氯甲烷(80/20，V/V)作为洗脱液，可以除去大多数极性相对高的杂质。过柱提纯后在真空下通过在35℃下旋转蒸发除去溶剂得到4-乙烯基苄基缩水甘油醚27g，含量95.5％，收率86％。
实施例3 PST种子微球的合成
在1000mL烧杯加入异丙醇500g、PVP 10.0g溶解完全后，再加入琥珀酸二异辛酯磺酸钠3.65g，水85mL，室温搅拌溶解后制得溶液一。将AIBN 1.0g和St(苯乙烯)50g，搅拌溶解制得溶液二。于2000mL三口烧瓶通氮气，将溶液二加入溶液一中继续搅拌混合，将混合均匀的溶液加入反应瓶中。于搅拌速度250rpm、氮气保护的条件下，升温至65℃，继续保温反应6h。反应结束后，倒出反应液，离心，转速5000rpm，离心时间约10min，弃去上层反应液后用200mL乙醇洗涤3次；继续用200mL超纯水洗涤6次，离心，转速5000rpm，得到PST种子，用扫描电镜(SEM)测试粒径D50为1.50μm，如图1和2所示。
实施例4交联度为70％的表面带有环氧基的交联聚苯乙烯-二乙烯基苯微球(PSDVB-Epoxy)的合成
将1.8g十二烷基苯磺酸钠(SDBS)溶解于600g纯水中，搅拌溶解完全得到混合相1(水相)。在500mL烧杯称取18.75g 4-乙烯苄基缩水甘油醚(实施例2获得的单体)和131.25g二乙烯基苯(DVB)，加入引发剂3.75g过氧化二辛酰，磁力搅拌溶解完全得到混合相2(油相)。将混合相1和混合相2加入烧杯后超声乳化成0.5μm～1μm左右乳液。将实施例3获得的PST种子4.1g、混合相1(水相)和混合相2(油相)加入烧杯放入磁子磁力搅拌混合后，于室温条件下溶胀，控制搅拌转速：100rpm，溶胀时间：24h。溶胀结束后，加入2％PVA溶液243g，搅拌转速150rpm，搅拌30min，N 2保护条件下，升高温度：75℃，保温反应时间：12h。反应结束后，采用定性滤纸在布氏漏斗抽滤反应液，再用去500mL去离子水洗涤三次、300mL乙醇洗涤抽滤三次、300mL丙酮抽滤洗涤三次得到交联度为70％的PSDVB-Epoxy单分散无孔交联微球，用扫描电镜(SEM)测试粒径D50为5.01μm，如图3和4所示。微球环氧值：0.65mmol/g。
实施例5交联度为70％的表面带有环氧基的交联聚苯乙烯-二乙烯基苯微球(PSDVB-Epoxy)的合成
将1.8g十二烷基苯磺酸钠(SDBS)溶解于600g纯水中，搅拌溶解完全得到混合相1(水相)。在500mL烧杯称取18.75g 4-乙烯基苯基缩水甘油醚(实施例1获得的单体)和131.25g二乙烯基苯(DVB)，加入引发剂3.75g过氧化二辛酰，磁力搅拌溶解完全得到混合相2(油相)。将混合相1和混合相2加入烧杯后超声乳化成0.5μm～1μm左右乳液。将实施例3获得的PST种子4.1g、混合相1(水相)和混合相2(油相)加入烧杯放入磁子磁力搅拌混合后，于室温条件下溶胀，控制搅拌转速：100rpm，溶胀时间：24h。溶胀结束后，加入2％PVA溶液243g，搅拌转速150rpm，搅拌30min，N 2保护条件下，升高温度：75℃，保温反应时间：12h。反应结束后，采用定性滤纸在布氏漏斗抽滤反应液，再用去500mL去离子水洗涤三次、300mL乙醇洗涤抽滤三次、300mL丙酮抽滤洗涤三次得到交联度为70％的PSDVB-Epoxy单分散无孔交联微球，用扫描电镜(SEM)测试粒径D50为5.1μm。微球环氧值：0.62mmol/g。
实施例6交联度为80％的表面带有环氧基的交联聚苯乙烯-二乙烯基苯微球(PSDVB-Epoxy)的合成
将1.8g十二烷基苯磺酸钠(SDBS)溶解于600g纯水中，搅拌溶解完全得到混合相1(水相)。在500mL烧杯称取30.0g 4-乙烯苄基缩水甘油醚(实施例2获得的单体)和120.0g二乙烯基苯(DVB)，加入引发剂3.75g过氧化二辛酰，磁力搅拌溶解完全得到混合相2(油相)。将混合相1和混合相2加入烧杯后超声乳化成0.5μm～1μm左右乳液。将实施例3获得的PST种子3.9g、混合相1(水相)和混合相2(油相)加入烧杯放入磁子磁力搅拌混合后，于室温条件下溶胀，控制搅拌转速：100rpm，溶胀时间：24h。溶胀结束后，加入2％PVA溶液243g，搅拌转速150rpm，搅拌30min，N 2保护条件下，升高温度：75℃，保温反应时间：12h。反应结束后，采用定性滤纸在布氏漏斗抽滤反应液，再用去500mL去离子水洗涤三次、300mL乙醇洗涤抽滤三次、300mL丙酮抽滤洗涤三次得到交联度为80％的PSDVB-Epoxy单分散无孔交联微球，用扫描电镜(SEM)测试粒径D50为5.25μm。微球环氧值：0.47mmol/g。
实施例7交联度为50％的表面带有环氧基的交联聚苯乙烯-二乙烯基苯微球(PSDVB-Epoxy)的合成
将1.8g十二烷基苯磺酸钠(SDBS)溶解于600g纯水中，搅拌溶解完全得到混合相1(水相)。在500mL烧杯称取56.25g 4-乙烯苄基缩水甘油醚(实施例2获得的单体)和93.75g二乙烯基苯(DVB)，加入引发剂3.75g过氧化二辛酰，磁力搅拌溶解完全得到混合相2(油相)。将混合相1和混合相2加入烧杯后超声乳化成0.5μm～1μm左右乳液。将实施例3获得的PST种子3.8g、混合相1(水相)和混合相2(油相)加入烧杯放入磁子磁力搅拌混合后，于室温条件下溶胀，控制搅拌转速：100rpm，溶胀时间：24h。溶胀结束后，加入2％PVA溶液243g，搅拌转速150rpm，搅拌30min，N 2保护条件下，升高温度：75℃，保温反应时间：12h。反应结束后，采用定性滤纸在布氏漏斗抽滤反应液，再用去500mL去离子水洗涤三次、300mL乙醇洗涤抽滤三次、300mL丙酮抽滤洗涤三次得到交联度为50％的PSDVB-Epoxy单分散无孔交联微球，用扫描电镜(SEM)测试粒径D50为5.33μm。微球环氧值：0.79mmol/g。
实施例8 PSDVB-OH微球亲水化修饰
在500mL三颈烧瓶中，将50mL加入实施例4中制备的(PSDVB-Epoxy)微球分散在200mL二氧六环溶剂中，然后将30mL乙二醇和30g环氧氯丙烷加入到烧瓶中，搅拌均匀后，再加入三氟化硼乙醚络合物2mL。在氮气保护下将反应液加热至80℃，控制搅拌转速150rpm，持续反应8h。将反应液降温至室温后，用去离子水和无水乙醇洗涤抽滤(水洗涤至PH:7.0)。干燥微球残留的环氧值≤0.25mmol/g。
实施例9山梨醇四缩水甘油醚为多环氧基化合物制备表面带有多环氧基的交联聚苯乙 烯-二乙烯基苯微球(PSDVB-mEpoxy)
加入50g实施例8中制备的PSDVB-OH于250mL三口烧瓶中，继续加入50.0g 0.5M NaOH溶液，安装机械搅拌装置，40℃、150rpm搅拌反应1h，然后加入16g山梨醇四缩水甘油醚(溶解在20mL二氧六环中)，再加入2.0g氰基硼氢化钠，在40℃、150rpm搅拌继续反应3h，反应结束后用去离子水抽滤洗涤，直至滤液呈中性(pH：7.0)，获得多环氧基团化微球。干燥微球的环氧值为1.56mmol/g。
实施例10聚丙三醇多缩水甘油醚为多环氧基化合物制备表面带有多环氧基的交联聚苯乙烯-二乙烯基苯微球(PSDVB-mEpoxy)
加入50g实施例8中制备的PSDVB-OH于250mL三口烧瓶中，继续加入50.0g 1.5MNaOH溶液(50.04g去离子水中加入3.0gNaOH并溶解)，安装机械搅拌装置，60℃、150rpm搅拌反应1h，然后加入60g聚丙三醇多缩水甘油醚(溶解在60mL二氧六环中)，在60℃、150rpm搅拌继续反应3h，反应结束后用去离子水抽滤洗涤，直至滤液呈中性(pH：7.0)，获得多环氧基团化微球。干燥的PSDVB-mEpoxy微球的环氧值为1.68mmol/g。
实施例11天门冬氨酸采用二羧酸结构氨基酸羧基化法制备多羧基富集的聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)
将实施例9的PSDVB-mEpoxy微球30mL和超纯水30mL移入50mL三口圆底烧瓶中。在冰水浴下配置由0.536g NaOH、15.2mL H 2O、1.150g天门冬氨酸组成的溶液，再用Na 2CO 3调节pH值为10，并将其加入上述50mL三口圆底烧瓶中，在冷凝管水冷下用水浴锅加热，温度升至80℃，保温4h后冷却至室温，然后在抽滤漏斗上滤干，再用超纯水洗10次(每次30mL)，最后再用30mL质量比10％的Na 2CO 3溶液洗一次，用漏斗过滤，抽干，即得到连接上天门冬氨酸的微球。将5.4g的溴乙酸添加到20mL的1.8M NaOH溶液中以制备溶液。将溶液在30℃以300rpm搅拌下滴加到烧瓶中，继续搅拌反应20h。带有三羧酸结构的微球用去离子水洗涤几次至中性，得到多羧基富集的微球(PSDVB-NTA)。干燥微球的离子交换容量为25meg/L。
实施例12天门冬氨酸采用二羧酸结构氨基酸羧基化法制备多羧基富集的聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)
将实施例10的PSDVB-mEpoxy微球30mL和超纯水30mL移入50mL三口圆底烧瓶中。在冰水浴下配置由0.536g NaOH、15.2mL H 2O、1.150g天门冬氨酸组成的溶液，再用Na 2CO 3调节pH值为10，并将其加入上述50mL三口圆底烧瓶中，在冷凝管水冷下用水浴锅加热，温度升至80℃，保温4h后冷却至室温，然后在抽滤漏斗上滤干，再用超纯水洗10次(每次30mL)，最后再用30mL质量比10％的Na 2CO 3溶液洗一次，用漏斗过滤，抽干，即得到连接上天门冬氨酸的微球。将5.4g的溴乙酸添加到20mL的1.8M NaOH溶液中以制备溶液。将溶液在30℃以 300rpm搅拌下滴加到烧瓶中，继续搅拌反应20h。带有三羧酸结构的微球用去离子水洗涤几次至中性，得到多羧基富集的微球(PSDVB-NTA)。干燥微球的离子交换容量为22meg/L。
实施例13赖氨酸作为活性端基氨基酸制备多羧基富集的聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)
将20g实施例9的PSDVB-mEpoxy微球溶于烧杯中的20mL碳酸钠-碳酸氢钠缓冲液(0.15M)中，以将溶液的pH值调节至8.5。然后将18.0g赖氨酸溶解在20mL二氧六环中，加入上述反应液。该系统在50℃下以150rpm持续16h，获得含PSDVB-α-氨基羧酸微球。将20g PSDVB-α-氨基羧酸微球放入烧瓶中。将20mL的1.8M NaOH溶液在0℃以300rpm搅拌下倒入烧瓶中。将5.4g的溴乙酸添加到20mL的1.8M NaOH溶液中以制备溶液。将溶液在0℃以300rpm搅拌下滴加到烧瓶中2小时。然后将系统在室温下以150rpm搅拌16h。带有NTA的微球用去离子水洗涤几次至中性，然后于35℃真空干燥，得到多羧基富集的微球(PSDVB-NTA)。干燥微球的离子交换容量为20meg/L。
实施例14赖氨酸作为活性端基氨基酸制备多羧基富集的聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)
将20g实施例10的PSDVB-mEpoxy微球溶于烧杯中的20mL碳酸钠-碳酸氢钠缓冲液(0.15M)中，以将溶液的pH值调节至8.5。然后将18.0g赖氨酸溶解在20mL二氧六环中，加入上述反应液。该系统在50℃下以150rpm持续16h，获得含PSDVB-α-氨基羧酸微球。将20g PSDVB-α-氨基羧酸微球放入烧瓶中。将20mL的1.8M NaOH溶液在0℃以300rpm搅拌下倒入烧瓶中。将5.4g的溴乙酸添加到20mL的1.8M NaOH溶液中以制备溶液。将溶液在0℃以300rpm搅拌下滴加到烧瓶中2小时。然后将系统在室温下以150rpm搅拌16h。带有NTA的微球用去离子水洗涤几次至中性，然后于35℃真空干燥，得到多羧基富集的微球(PSDVB-NTA)。干燥微球的离子交换容量为18meg/L。
对比例1天门冬氨酸采用二羧酸结构氨基酸羧基化法制备多羧基富集的聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)
将实施例4的PSDVB-Epoxy微球30mL和超纯水30mL移入50mL三口圆底烧瓶中。在冰水浴下配置由0.536g NaOH、15.2mL H 2O、1.150g天门冬氨酸组成的溶液，再用Na 2CO 3调节pH值为10，并将其加入上述50mL三口圆底烧瓶中，在冷凝管水冷下用水浴锅加热，温度升至80℃，保温4h后冷却至室温，然后在抽滤漏斗上滤干，再用超纯水洗10次(每次30mL)，最后再用30mL质量比10％的Na 2CO 3溶液洗一次，用漏斗过滤，抽干，即得到连接上天门冬氨酸的微球。将5.4g的溴乙酸添加到20mL的1.8M NaOH溶液中以制备溶液。将溶液在30℃以 300rpm搅拌下滴加到烧瓶中，继续搅拌反应20h。带有三羧酸结构的微球用去离子水洗涤几次至中性，得到多羧基富集的微球(PSDVB-NTA)。干燥微球的离子交换容量为5.6meg/L。
对比例2赖氨酸作为活性端基氨基酸制备多羧基富集的聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)
将20g实施例4的PSDVB-Epoxy微球溶于烧杯中的20mL碳酸钠-碳酸氢钠缓冲液(0.15M)中，以将溶液的pH值调节至8.5。然后将18.0g赖氨酸溶解在20mL二氧六环中，加入上述反应液。该系统在50℃下以150rpm持续16h，获得含PSDVB-α-氨基羧酸微球。将20g PSDVB-α-氨基羧酸微球放入烧瓶中。将20mL的1.8M NaOH溶液在0℃以300rpm搅拌下倒入烧瓶中。将5.4g的溴乙酸添加到20mL的1.8M NaOH溶液中以制备溶液。将溶液在0℃以300rpm搅拌下滴加到烧瓶中2小时。然后将系统在室温下以150rpm搅拌16h。带有NTA的微球用去离子水洗涤几次至中性，然后于35℃真空干燥，得到多羧基富集的微球(PSDVB-NTA)。干燥微球的离子交换容量为4.3meg/L。
测试例1微球耐压性能测试
具体操作为：将实施例4至实施例7制备得到的聚苯乙烯-二乙烯基苯微球(PSDVB-Epoxy)分别用乙醇匀浆并分别装填成4.0mm(内径)×30mm(长度)的色谱柱。采用高效液相色谱仪(Agilent 1260Infinity II HPLC)对上述各色谱柱进行微球耐压测试。50mM、pH6.0的PB缓冲液体系作为洗脱液，流速由0.5mL/min以0.25mL/min为步长递增至5.00mL/min，每个流速维持3min，柱温为25℃，测定上述各色谱柱的柱压，并对一定流速范围内的检测数据进行线性拟合。测定结果详见表1、表2及图5。表1表示实施例4至实施例7的PSDVB-Epoxy微球制成的色谱柱在流速为0.5mL/min～5.25mL/min下的柱压。表2表示实施例4至实施例7的PSDVB-Epoxy微球制成的色谱柱在对应流速范围下的流速与柱压的拟合曲线方程，其中，Y为柱压，x为流速。图5为实施例4至实施例7的PSDVB-Epoxy微球制成的色谱柱在流速为0.5mL/min～5.25mL/min范围内流速与柱压的拟合曲线。
表1
Figure PCTCN2022083708-appb-000007
Figure PCTCN2022083708-appb-000008
表2
Figure PCTCN2022083708-appb-000009
需要说明的是，PSDVB-Epoxy的耐压越强，其形成的PSDVB-mEpoxy和PSDVB-NTA的耐压强度越高。与PSDVB-Epoxy微球相比，多环氧基和NTA基团的分子量较小，多环氧基的修饰及NTA基团的修饰对微球的粒径和耐压性能的基本可忽略。
从表1、表2和图5可以看出，在流速为0.5mL/min～4.5mL/min的范围内，实施例4的色谱柱的压力与流速具有较好的相关性(R 2＝0.9944)，线性较好，说明实施例4的PSDVB-Epoxy微球能够耐受21.1MPa的压力，采用实施例4的PSDVB-Epoxy微球制成的PSDVB-NTA微球能够耐受21.1MPa以上的压力，具有较优耐压性能；实施例5的PSDVB-Epoxy微球的耐压性能与实施例4的PSDVB-Epoxy微球的耐压性能接近；实施例6的色谱柱在0.5mL/min～5.25mL/min范围内，柱压与流速具有较好的相关性(R 2＝0.9991)，说明实施例6的PSDVB-Epoxy微球能够耐受25MPa的压力，采用实施例6的PSDVB-Epoxy微球制成的PSDVB-NTA微球能够耐受25MPa以上的压力，具有较优耐压性能；实施例7的色谱柱在0.5mL/min～2.5mL/min范围内的柱压与流速具有一定的相关性，说明实施例7的PSDVB-Epoxy微球能够耐受10MPa的压力，采用实施例7的PSDVB-Epoxy微球制成的PSDVB-NTA微球能够耐受10MPa的压力。由此可见，实施例4的PSDVB-Epoxy的耐压性能略低于实施例6，但远远高于实施例7。
再者，PSDVB-Epoxy微球环氧值越高，能够接上的活性基团的量越多。实施例4和实施例5的PSDVB-Epoxy环氧值基本相当；实施例4和实施例5的PSDVB-Epoxy环氧值高于实施例6，且略低于实施例7。
综上，从测试例1的测试结果可以看出，PSDVB-Epoxy微球的交联度越高，其耐压性能越强，而PSDVB-Epoxy微球环氧值也越低，能够接上的活性基团的量越少。为了兼具较强的耐压性能及较高的PSDVB-Epoxy微球环氧值，选择实施例4的PSDVB-Epoxy微球进行后续的实验。
测试例2非特异性吸附检测
1、将填料制成4.0mm×150mm的色谱柱。装柱溶剂为pH7.0的超纯水。色谱柱共有三种：第一种色谱柱(以下简称1号色谱柱)为实施例4制备的PSDVB-Epoxy微球水解后装填获得；第二种色谱柱(以下简称2号色谱柱)为实施例9制备得到的带有多环氧基的交联聚苯乙烯-二乙烯基苯微球(PSDVB-mEpoxy)水解后装填获得；第三种色谱柱(以下简称3号色谱柱)的填料为实施例11制备得到的多羧基富集聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)装填后获得。
2、测试方法：
以0.25mg/mL的BSA(牛血清白蛋白)作为检测样本，以上述各色谱柱并采用高效液相色谱仪(Agilent 1260 Infinity II HPLC)分别对检测样本进行检测，流动相为10mM磷酸盐缓冲溶液(含有0.5M NaCl，pH 7.0)，流速为0.5mL/min，检测器为UV 214nm，柱温为室温，进样量为2μL/针，每个色谱柱连续进样20针(即20次)。
3、曲线绘制：以进样次数为横坐标，BSA峰面积为纵坐标绘制散点图。
4、非特异性吸附量的计算：
以第20次检测计为BSA回收率为100％，并设定BSA回收率小于98％时存在非特异性吸附。BSA的非特异吸附量(单位为：μg/mL)的计算公式如下公式(1)：
BSA的非特异吸附量＝(2N×0.25)/(0.20 2×π×15)  公式(1)
其中，N为在BSA回收率小于98％下最大进样次数。
采用公式(1)计算各色谱柱的BSA的非特异吸附量，测定结果详见表3、图6至图8。图6为测试例2中1号色谱柱的20次进样检测的峰面积统计图；图7为测试例2中2号色谱柱的20次进样检测的峰面积统计图；图8为测试例2中3号色谱柱的20次进样检测的峰面积统计图。
表3各色谱柱的N及BSA的非特异吸附量
  1号色谱柱 2号色谱柱 3号色谱柱
N(次) 9 2 5
BSA的非特异吸附量(μg/mL) 2.39 0.53 1.33
从表3及图6至图8可以看出，2号色谱柱和3号色谱柱的非特异吸附量均小于1号色谱柱，说明实施例9的制备得到的PSDVB-mEpoxy微球、实施例11制备得到的多羧基富集 聚苯乙烯-二乙烯基苯微球的非特异性吸附量均小于实施例4制备得到的PSDVB-Epoxy微球，进而说明对微球表面采用亲水性的缩水甘油醚改性后能够降低微球的非特异性吸附，提高色谱柱的使用寿命，保证目标蛋白的分离效果。
测试例3
将实施例11～14及对比例1～2制备得到的多羧基富集聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)分别作为色谱填料，用超纯水将填料均化后，用高压气动泵将其装填到4.6*30mm(柱体积为0.498mL)的色谱柱中。通过高效液相色谱仪(Agilent 1260 Infinity II HPLC)对标准蛋白细胞色素C和溶菌酶进行分离性能测试。以1.5mL/min的流速，进样量为2μL，在室温下，应用缓冲液A：50mmol、pH6.0的PB(Phosphate Buffer，磷酸缓冲液)和缓冲液B：A相(即缓冲液A)+1MNaCl(pH6.0)作为洗脱液进行梯度洗脱，洗脱梯度为0min：100％(v/v)A，10min：25％(v/v)A及75％(v/v)B，采用紫外检测器(波长214nm)进行检测。“0min：100％(v/v)A，10min：25％(v/v)A及75％(v/v)B”表示从0min起至10min止，洗脱液中缓冲液A及缓冲液B的比例从100％(v/v)A及0％(v/v)B变化至25％(v/v)A及75％(v/v)B。检测结果详见表4及图9至图14。表4中分离度为溶菌酶的色谱峰与细胞色素C的色谱峰的分离度。图9为测试例3中实施例11的PSDVB-NTA微球作为色谱填料的色谱图；图10为测试例3中实施例12的PSDVB-NTA微球作为色谱填料的色谱图；图11为测试例3中实施例13的PSDVB-NTA微球作为色谱填料的色谱图；图12为测试例3中实施例14的PSDVB-NTA微球作为色谱填料的色谱图；图13为测试例3中对比例1的PSDVB-NTA微球作为色谱填料的色谱图；图14为测试例3中对比例2的PSDVB-NTA微球作为色谱填料的色谱图。
表4实施例11～14及比较例1～2的色谱柱的检测结果
Figure PCTCN2022083708-appb-000010
从表4及图9至图12可以看出，实施例11至实施例14的色谱柱均对溶菌酶与细胞色素C具有较好的分离效果。其中，实施例11及实施例12的溶菌酶的色谱峰与细胞色素C的色谱峰的柱效和分离度均比实施例13及实施例14的高，说明采用天门冬氨酸制成的多羧基富集的聚苯乙烯-二乙烯基苯微球作为色谱柱的填料，更有利于提高离子交换色谱柱分离蛋白的柱效和分离度。对比例1～2的色谱柱对溶菌酶和细胞色素C的保留时间短、柱效低、分离度小，对蛋白的分离效果与实施例11至实施例14的色谱柱有明显的差异。
测试例4
将实施例11制备得到的多羧基富集聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)作为色谱填料，采用深圳普门科技有限公司生产的H9糖化血红蛋白仪(粤械注准20152401315)中糖化血红蛋白模式对待测血样进行糖化血红蛋白及变异血红蛋白分析。丁二酸缓冲液体系作为洗脱液(糖化血红蛋白(HbA1c)测定试剂盒(高效液相色谱法)医疗器械注册证号：粤械注准20152401314)，普门科技的糖化血红蛋白质控品(粤械注准20162400488)和校准品(粤械注准20162400487)，以1.5mL/min的流速，柱温于40℃条件下，采用紫外检测器(波长415nm)进行检测。在130s分析时间，等度洗脱条件为50s(A相)-30s(C相)-50s(B相)，按照普门科技H9糖化血红蛋白仪130秒模式的标准操作规程要求，对糖化血红蛋白进行定量分析，并对常见变异体进行识别。“50s(A相)-30s(C相)-50s(B相)”表示以A相等度洗脱50秒，接着以C相等度洗脱30秒，然后以B相等度洗脱50秒；后续类似描述以此类推，不再赘述。并且，按照相同的测试条件对质控品进行检测。
其中，待测血样为全血样本，且全血样本中HbA1c的质量百分含量已知为7.6％。质控品包括低值质控品(HbA1c的质量百分含量为5.3％)和高值质控品(HbA1c的质量百分含量为9.9％)。测定结果详见表5和图15。表5表示测试例4的检测结果统计表，表5中“％”表示质量百分含量。图15为测试例4中待测样本和质控品的液相色谱图。表5中，HbA1c-NGSP＝0.9148HbA1c-IFCC+2.152，NGSP是美国国家糖化血红蛋白标准化计划；HbA1c-IFCC＝(10.93×HbA1c-NGSP)-23.50；IFCC是国际临床化学和实验室医学联盟；平均血糖浓度eAG(mmol/L)＝1.59×HbA1c-NGSP-2.59。
表5
Figure PCTCN2022083708-appb-000011
Figure PCTCN2022083708-appb-000012
从表5及图15可以看出，实施例11的多羧基富集聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)作为色谱填料，能够分离并定量检测全血样本中的糖化血红蛋白，还能够分离并定量检测全血样本中的HbA0、HbA2及HbF等非糖化血红蛋白。
测试例5
将实施例11制备得到的多羧基富集聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)作为色谱填料，采用深圳普门科技有限公司生产的H9糖化血红蛋白仪(粤械注准20152401315)中糖化血红蛋白模式对待测血样进行糖化血红蛋白及变异血红蛋白分析。具体测试条件与测试例4相同，详见上文，此处不再赘述。
其中，待测血样有四个，分别为样本1至样本4，样本1为已知含有变异体HbE的全血样本，样本2为已知含有变异体HbD的全血样本，样本3为已知含有变异体HbS的全血样本，样本4为已知含有变异体HbC的全血样本。测定结果详见表6及图16。表6表示测试例5的检测结果统计表。图14为测试例5中待测样本的液相色谱图。图16中E表示HbE，D表示HbD，S表示HbS，C表示HbC。
表6
  变异体HbE 变异体HbD 变异体HbS 变异体HbC
保留时间(秒，s) 87.0 95.2 96.2 98.4
从表6及图16可以看出，实施例11的多羧基富集聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)作为色谱填料，还能够识别并检测待测样本中的HbE、HbD、HbS和HbC等变异体，其中，HbD和HbS的出峰时间较为接近。
测试例6重复性测试
将实施例11制备得到的多羧基富集聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)作为色谱填料，采用深圳普门科技有限公司生产的H9糖化血红蛋白仪(粤械注准20152401315)中糖化血红蛋白模式对待测血样进行糖化血红蛋白(HbA1c)含量(质量百分含量)检测。具体测试条件与测试例4相同，详见上文，此处不再赘述。测定结果详见表7和图17。表7为测试例6的检测结果统计表。图17为测试例6的重复性测试结果图。
其中，待测血样有三个，分别为样本1至样本3；样本1为临床全血样本，HbA1c的质 量百分含量为5％-7％；样本2为临床全血样本，HbA1c的质量百分含量为7％-9％)；样本3为临床全血样本，HbA1c的质量百分含量为9％-12％；三个血液样本各重复检测20次，计算20次测量结果的平均值M和标准差SD，根据以下公式得出变异系数(CV)。
Figure PCTCN2022083708-appb-000013
式中：CV—变异系数；SD—20次测量结果的标准差；M—20次测量结果的平均值。
表7
序号 样本1 样本2 样本3
1 6.6％ 8.2％ 9.1％
2 6.6％ 8.2％ 9.1％
3 6.6％ 8.2％ 9.1％
4 6.6％ 8.2％ 9.1％
5 6.6％ 8.2％ 9.1％
6 6.6％ 8.2％ 9.1％
7 6.6％ 8.2％ 9.1％
8 6.7％ 8.2％ 9.0％
9 6.7％ 8.2％ 9.1％
10 6.6％ 8.3％ 9.2％
11 6.6％ 8.2％ 9.0％
12 6.5％ 8.3％ 9.2％
13 6.6％ 8.2％ 9.2％
14 6.5％ 8.2％ 9.1％
15 6.6％ 8.3％ 9.2％
16 6.7％ 8.2％ 9.1％
17 6.6％ 8.2％ 9.1％
18 6.5％ 8.2％ 9.2％
19 6.6％ 8.2％ 9.1％
20 6.5％ 8.2％ 9.1％
M 6.6％ 8.2％ 9.1％
SD 0.0605％ 0.0366％ 0.0587％
CV 0.92％ 0.45％ 0.64％
一般地，检测不同样本浓度为的样本，重复测量结果变异系数CV应不大于3.0％。从表7及图17可以看出，实施例11的多羧基富集聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)作为色谱填料，对样本1检测的CV为0.92％，对样本2检测的CV为0.45％，对样本3检测的CV为0.64％，说明实施例11的多羧基富集聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)作为色谱填料的重复性较好，满足重复性要求。
测试例7临床样本的相关性
将实施例11制备得到的多羧基富集聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)作为色谱 填料，采用深圳普门科技有限公司生产的H9糖化血红蛋白仪(粤械注准20152401315)中糖化血红蛋白模式对待测血样进行糖化血红蛋白(HbA1c)含量(质量百分含量)检测，具体测试条件与测试例4相同，详见上文，此处不再赘述。
测试对比例：采用日本东曹公司HLC-723G8全自动糖化血红蛋白分析仪以及配套的糖化血红蛋白测定试剂盒(高效液相色谱法)按照试剂盒的操作说明对上述相同待测血样进行糖化血红蛋白(HA，HbA1c)含量(质量百分含量)检测。
其中，待测血样为40例临床样本，包括12例正常体检样本和28例糖尿病样本。测定结果详见表8和图18。表8为测试例7的检测结果统计表。表8中，相对偏差即本研究(实施例11制备得到的多羧基富集聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)作为色谱填料)与对比例针对同一样本检测结果的相对偏差。图18为测试例7的相关性测试结果图。
表8
Figure PCTCN2022083708-appb-000014
Figure PCTCN2022083708-appb-000015
一般地，测试结果相关性R 2应不低于0.99，90％以上样本测值与靶值相对偏差在±6％以内。从表8和图18可以看出，本研究色谱系统的检测结果与东曹公司色谱系统的检测结果的相对偏差均在±6％以内，相关性R 2为0.9978，能够满足要求。
测试例8日间稳定性和层析柱寿命测试
将实施例11制备得到的多羧基富集聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)作为色谱填料，采用深圳普门科技有限公司生产的H9糖化血红蛋白仪(粤械注准20152401315)中糖化血红蛋白模式对新鲜的糖尿病患者全血样本进行糖化血红蛋白(HbA1c)含量(质量百分含量)检测。具体测试条件与测试例4相同，详见上文，此处不再赘述。每日开关机做质控，每日约600T(即600次)测试，测试5天左右，在层析柱3000T(即3000次)寿命范围内，计算日间质控的变异系数，150T(即150次)左右进行质控品测试，质控品测值变异系数应满足CV<3％，且峰型无展宽、拖尾、肩峰。质控品包括低值质控品(HbA1c的质量百分含量为5.3％)和高值质控品(HbA1c的质量百分含量为9.9％)。测定结果详见表9和图19。表9为测试例8的检测结果统计表。图19为测试例8的质控图。
表9
Figure PCTCN2022083708-appb-000016
Figure PCTCN2022083708-appb-000017
从表9和图19可以看出，同一根色谱柱经过3000T测试后质控的峰型无异常，保留时间稳定，质控CV<3％，说明采用本研究的PSDVB-NTA微球能够用于制备日间稳定性较高和寿命较长的色谱柱。
测试例9
将实施例11制备得到的多羧基富集聚苯乙烯-二乙烯基苯微球(PSDVB-NTA)作为色谱填料，采用深圳普门科技有限公司生产的H9血红蛋白仪地中海贫血分析模式对待测血样进行变异血红蛋白分析。丁二酸缓冲液体系作为洗脱液，以1.5mL/min的流速，柱温于30℃条件下，采用紫外检测器(波长415nm)进行检测，分析时间为380s，全血样本吸样量7μL，用超纯水稀释液1.5mL稀释均匀后，进样量5μL，等度洗脱条件：60S(A2相)-50S(B2相)-50S(A2相)-40S(B2相)-140S(C2相)-40S(A2相)，A2相为45mM丁二酸盐缓冲液(pH5.4)，B2相为100mM丁二酸盐缓冲液(pH5.4)，C2相为530mM丁二酸盐缓冲液(pH5.4)。待测血样分别为样本1～样本8，样本1为含有HbA2的全血样本，样本2为含有HbE的全血样本，样本3为含有HbD的全血样本，样本4为含有HbS的全血样本，样本5为含有HbC的全血样本，样本6为含有HbJ-Bangkok的全血样本，样本7为含有Hb G-Taipei的全血样本，样本8为含有Hb Q-Thailand的全血样本。测定结果详见表10至表11、图20至图23。表10是常规血红蛋白保留时间范围，表11是1～8号样本中变异血红蛋白保留时间。图20为测试例9中样本1～样本5到的检测色谱图。图21为测试例9中样本6的Hb J-Bangkok全血样品的液相色谱图。图22为测试例9中样本7的Hb G-Taipei全血样品的液相色谱图。图23为测试例9中样本8的Hb Q-Thailand全血样品的液相色谱图。
表10
色谱峰名称 保留时间及出峰范围(s)
HbA1a 13.8(12.0-15.0)
HbA1b 21.0(17.0-20.0)
HbF 30.1(27.0-34.0)
LA1c 38.8(36.0-40.0)
HbA1c 54.3(50.0-58.0)
P3 82.0(78.0-89.0)
P4 100.7(95.0-110.0)
HbA0 125.9(120.0-135.0)
表11
色谱峰名称 保留时间(s)
HbA2 186.0
HbE 230.7
HbD 245.3
HbS 270.5
HbC 310.7
HbJ-Bangkok 105.3
HbG-Taipei 226.8
HbQ-Thailand 299.7
从表10及图20可以看出，本申请的PSDVB-NTA离子交换填料微球能够分离HbA1a、HbA1b、LA1c、HbF、HbA1c、P3、P4、HbA0、HbA2、HbE、HbD、HbS、HbC等13种血红蛋白类物质。从表11、图21至图23可以看出，本申请的PSDVB-NTA离子交换填料微球还能够分离和检测出中国常见的HbJ-Bangkok、Hb G-Taipei和Hb Q-Thailand等血红蛋白变异体。
综上所述，本发明的PSDVB-NTA离子交换填料微球在至多130秒内即可分离和定量糖化血红蛋白并且能够识别血红蛋白变异体，在至多380秒内即可分离包括糖化血红蛋白及各种血红蛋白变异体在内的16种血红蛋白类物质，离子交换性能优良，对血红蛋白及其变异体具有较高的特异性分离能力，检测速度较高。本发明的PSDVB-NTA离子交换填料微球能够耐受20MPa以上的压力，具有高的分离能力、低蛋白非特异性吸附、高的耐压性能、分散性良好，粒径大小均一可控等优点。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (22)

  1. 一种乙烯基单体-多乙烯基交联剂共聚物无孔微球，其特征在于，包括：
    乙烯基单体-多乙烯基交联剂共聚物母球，所述乙烯基单体-多乙烯基交联剂共聚物母球修饰有羟基；
    经所述羟基接枝于所述乙烯基单体-多乙烯基交联剂共聚物母球的环氧基化合物，所述环氧基化合物包含至少两个环氧基。
  2. 根据权利要求1所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球，其特征在于，所述环氧基化合物选自山梨醇四缩水甘油醚、季戊四醇四缩水甘油醚、聚丙三醇多缩水甘油醚、二丙三醇三缩水甘油醚、1,4-丁二醇二缩水甘油醚、丙三醇二缩水甘油醚、丙三醇三缩水甘油醚、乙二醇二缩水甘油醚、聚乙二醇二缩水甘油醚、新戊二醇二缩水甘油醚、2,2-双(4-(2,3-环氧丙氧基)苯基)丙烷中的至少一种；作为优选地，所述环氧基化合物选自山梨醇四缩水甘油醚和聚丙三醇多缩水甘油醚中的至少一种。
  3. 根据权利要求1所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球，其特征在于，所述乙烯基单体-多乙烯基交联剂共聚物母球包括：
    乙烯基单体-多乙烯基交联剂共聚物颗粒，所述乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰有环氧基；所述乙烯基单体-多乙烯基交联剂共聚物颗粒由聚苯乙烯种子微球与乙烯基单体经多乙烯基交联剂交联而成；所述乙烯基单体中包含所述环氧基；
    经所述环氧基接枝于所述乙烯基单体-多乙烯基交联剂共聚物颗粒的羟基化合物，所述羟基化合物包含所述羟基。
  4. 根据权利要求3所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球，其特征在于，所述乙烯基单体-多乙烯基交联剂共聚物母球的交联度为50％～80％，优选地，所述乙烯基单体-多乙烯基交联剂共聚物母球的交联度为65％～75％。
  5. 根据权利要求3所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球，其特征在于，所述乙烯基单体选自4-乙烯苯基缩水甘油醚、4-乙烯苄基缩水甘油醚和甲基丙烯酸缩水甘油醚中的至少一种；
    及/或，所述聚苯乙烯种子微球的平均粒径D50为0.3μm～5μm；作为优选地，所述聚苯乙烯种子微球的平均粒径D50为0.5μm～3μm；
    及/或，所述羟基化合物选自乙二醇、1,3-丙二醇及1,4-丁二醇中的至少一种；
    及/或，所述多乙烯基交联剂选自二乙烯基苯、二乙烯基甲苯、二甲基丙烯酸乙二醇酯、聚乙二醇二甲基丙烯酸酯、三乙烯基苯、二乙烯基萘和三乙烯基萘中的至少一种；作为优选地，所述多乙烯基交联剂为二乙烯基苯。
  6. 根据权利要求3所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球，其特征在于，所 述乙烯基单体的用量为所述乙烯基单体和所述多乙烯基交联剂总重量的5％～40％；作为优选地，所述乙烯基单体的用量为所述乙烯基单体和所述多乙烯基交联剂总重量的10％～30％；更为优选地，所述乙烯基单体的用量为所述乙烯基单体和所述多乙烯基交联剂总重量的10％～20％。
  7. 根据权利要求3所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球，其特征在于，所述环氧基化合物与所述乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰的环氧基的摩尔比为(1～20):1；作为优选地，所述环氧基化合物与所述乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰的环氧基的摩尔比为(3～10):1；更为优选地，所述环氧基化合物与所述乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰的环氧基的摩尔比为(4～6):1。
  8. 根据权利要求1～7任一项所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球，其特征在于，还包括活性基团，所述活性基团接枝于所述环氧基化合物；
    可选地，所述活性基团包括NTA基团。
  9. 根据权利要求8所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球，其特征在于，所述活性基团包括NTA基团，所述NTA基团包含活性端基；所述活性端基选自天门冬氨酸端基、赖氨酸端基、半胱氨酸端基和谷氨酸端基中的至少一种。
  10. 根据权利要求1～7及9任一项所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球，其特征在于，所述乙烯基单体-多乙烯基交联剂共聚物无孔微球的平均粒径D50为2μm～10μm；作为优选地，所述乙烯基单体-多乙烯基交联剂共聚物无孔微球的平均粒径D50为3μm～6μm。
  11. 一种乙烯基单体-多乙烯基交联剂共聚物无孔微球的制备方法，其特征在于，包括如下步骤：
    制备乙烯基单体-多乙烯基交联剂共聚物母球，所述乙烯基单体-多乙烯基交联剂共聚物母球修饰有羟基；
    将环氧基化合物经所述羟基反应接枝至所述乙烯基单体-多乙烯基交联剂共聚物母球，所述环氧基化合物包含至少两个环氧基。
  12. 根据权利要求11所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球的制备方法，其特征在于，所述环氧基化合物选自山梨醇四缩水甘油醚、季戊四醇四缩水甘油醚、聚丙三醇多缩水甘油醚、二丙三醇三缩水甘油醚、1,4-丁二醇二缩水甘油醚、丙三醇二缩水甘油醚、丙三醇三缩水甘油醚、乙二醇二缩水甘油醚、聚乙二醇二缩水甘油醚、新戊二醇二缩水甘油醚、2,2-双(4-(2,3-环氧丙氧基)苯基)丙烷中的至少一种；作为优选地，所述环氧基化合物选自山梨醇四缩水甘油醚和聚丙三醇多缩水甘油醚中的至少一种。
  13. 根据权利要求12所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球的制备方法，其 特征在于，所述制备所述乙烯基单体-多乙烯基交联剂共聚物母球的方法包括如下步骤：
    制备乙烯基单体-多乙烯基交联剂共聚物颗粒，所述乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰有环氧基，制备步骤包括：以聚苯乙烯种子微球为种子，与乙烯基单体和多乙烯基交联剂共同在稳定剂和乳化剂存在的条件下，通过种子溶胀法在介质中进行聚合；所述乙烯基单体中包含所述环氧基；
    将羟基化合物经所述环氧基反应接枝至所述乙烯基单体-多乙烯基交联剂共聚物颗粒，所述羟基化合物包含所述羟基。
  14. 根据权利要求13所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球的制备方法，其特征在于，所述乙烯基单体选自4-乙烯苯基缩水甘油醚、4-乙烯苄基缩水甘油醚和甲基丙烯酸缩水甘油醚中的至少一种；
    及/或，所述聚苯乙烯种子微球的平均粒径D50为0.3μm～5μm；作为优选地，所述聚苯乙烯种子微球的平均粒径D50为0.5μm～3μm；
    及/或，所述羟基化合物选自乙二醇、1,3-丙二醇及1,4-丁二醇中的至少一种；
    及/或，所述多乙烯基交联剂选自二乙烯基苯、二乙烯基甲苯、三乙烯基苯、二甲基丙烯酸乙二醇酯、聚乙二醇二甲基丙烯酸酯、二乙烯基萘和三乙烯基萘中的至少一种；作为优选地，所述多乙烯基交联剂为二乙烯基苯。
  15. 根据权利要求13所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球的制备方法，其特征在于，所述乙烯基单体的用量为所述乙烯基单体和所述多乙烯基交联剂总重量的5％～40％；作为优选地，所述乙烯基单体的用量为所述乙烯基单体和所述多乙烯基交联剂总重量的10％～30％；更为优选地，所述乙烯基单体的用量为所述乙烯基单体和所述多乙烯基交联剂总重量的10％～20％。
  16. 根据权利要求13所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球的制备方法，其特征在于，所述环氧基化合物与所述乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰的环氧基的摩尔比为(1～20):1；作为优选地，所述环氧基化合物与所述乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰的环氧基的摩尔比为(3～10):1；更为优选地，所述环氧基化合物与所述乙烯基单体-多乙烯基交联剂共聚物颗粒上修饰的环氧基的摩尔比为(4～6):1。
  17. 根据权利要求13所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球的制备方法，其特征在于，所述聚合采用的引发剂为油性引发剂；作为优选地，油溶性过氧化物和油溶性偶氮化合物中的一种。
  18. 根据权利要求13所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球的制备方法，其特征在于，所述稳定剂为水溶性聚合物；作为优选地，所述稳定剂为聚乙烯醇、聚乙烯吡咯 烷酮和改性纤维素中的至少一种。
  19. 根据权利要求11～18任一项所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球的制备方法，其特征在于，还包括：于所述环氧基化合物之上反应接枝形成活性基团；
    可选地，所述活性基团包括NTA基团；
    进一步可选地，形成NTA基团的方法选自N,N-二羧甲基赖氨酸法、活性端基氨基酸法、二羧酸结构氨基酸羧基化法、先构建醛基再构建α-氨基酸法中的一种。
  20. 权利要求1～10任一项所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球，以及权利要求11～19任一项所述的所述制备方法制备得到的乙烯基单体-多乙烯基交联剂共聚物无孔微球之一在色谱填料中的应用。
  21. 权利要求1～10任一项所述的乙烯基单体-多乙烯基交联剂共聚物无孔微球，以及权利要求11～19任一项所述的所述制备方法制备得到的乙烯基单体-多乙烯基交联剂共聚物无孔微球之一在生物组分检测中的应用。
  22. 根据权利要求21所述的应用，其特征在于，所述生物组分为血红蛋白类生物组分。
PCT/CN2022/083708 2021-06-09 2022-03-29 乙烯基单体-多乙烯基交联剂共聚物无孔微球及其制备方法和应用 WO2022257560A1 (zh)

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