CN114316132A - Method for synthesizing functional polymer microspheres by emulsion polymerization - Google Patents
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Abstract
The invention relates to a method for synthesizing functional polymer microspheres by emulsion polymerization, in particular to a method for preparing functional polyacrylamide microspheres, which can realize the high-efficiency quantitative immobilization of oligonucleotides. According to the invention, the emulsion is prepared by using an emulsifying machine, wherein an aqueous solution of a water-soluble monomer acrylamide monomer, a cross-linking agent bisacrylamide monomer, a functional acrylamide monomer and an auxiliary additive is used as a water phase, and a mixed solution of an organic solvent and a surfactant is used as an oil phase. A free radical polymerization method is applied, polyacrylamide functional microspheres are formed by copolymerization and crosslinking in an emulsion polymerization mode, and quantitative immobilization of the oligonucleotide is realized through click reaction between azide groups carried by the microspheres and alkynyl groups in oligonucleotide molecules. The invention provides a preparation method for preparing functional polymer microspheres by emulsion polymerization, which is simple to operate, stable in process and suitable for large-scale production, and meanwhile, the microspheres have good biocompatibility and oligonucleotide quantitative immobilization.
Description
Technical Field
The invention discloses a method for synthesizing functional polymer microspheres by emulsion polymerization, and belongs to the field of gene sequencing.
Background
The bioactive supported Microsphere (Microsphere) is spherical particle with particle size of 50 nm-2 mm and has reactive group such as-NH 2, -COOH, -SH, etc. The microspheres have small size, so that the microspheres have obvious surface effects such as good affinity and biocompatibility of materials, easy absorption and migration in organisms and the like, and are widely applied to the fields of cytology, immunology, microbiology, molecular biology, clinical diagnosis and treatment, high-throughput gene detection and the like.
In the method of microsphere for high-throughput gene detection analysis of certain nucleic acid sequences, the size distribution of microspheres is about 1 μm, the microspheres are highly uniformly dispersed, and the loading of the microspheres on nucleic acid chains is relied on. The sequence of the loaded nucleic acid strand can then be determined by a number of different methods well known in the art. In some of the microspheres used in nucleic acid sequencing methods, the procedure involves loading the microspheres with a nucleic acid sample to be sequenced by rapidly and efficiently binding to their corresponding reactive groups by a number of methods well known in the art; nucleic acid-loaded microspheres are introduced into the channels of the flow cell and into the corresponding reaction micro-tunnels, and incubated for a fixed time to generate all downstream chemical processing steps that are consistently capable of supporting amplification and sequencing. The invention discloses a method for synthesizing functional polymer microspheres by emulsion polymerization, wherein the microspheres have stable internal structures and are particularly suitable for nucleic acid detection.
Disclosure of Invention
The invention discloses a method for synthesizing functional polymer microspheres by emulsion polymerization, which is characterized by comprising the following steps,
(1) Preparing an oil phase by the organic solvent and the surfactant according to different proportions;
(2) dissolving a water-soluble acrylamide monomer, a cross-linking agent bisacrylamide monomer, a functional acrylamide monomer and an auxiliary additive into water together to be used as a water phase;
(3) adding a water-soluble initiator into the water phase obtained in the step (2) to prepare a water phase containing the water-soluble initiator;
(4) mixing the oil phase in the step (1) and the water phase containing the water-soluble initiator in the step (3) according to different proportions, and preparing into emulsion containing the water-soluble initiator by using an emulsifying machine;
(5) after argon is used for removing oxygen in the emulsion containing the water-soluble initiator in the step (4), heating the emulsion to a set reaction temperature from room temperature, and carrying out free radical initiated polymerization to obtain functional polyacrylamide microspheres;
(6) under the co-catalysis of cuprous ions and ligands, the oligonucleotide can be efficiently and quantitatively immobilized through the click reaction between azide groups contained in the polyacrylamide microspheres and alkynyl groups on oligonucleotide molecules.
Method 2 step
(1) Preparing an oil phase by the organic solvent and the surfactant according to different proportions;
(2) dissolving a water-soluble acrylamide monomer, a cross-linking agent bisacrylamide monomer, a functional acrylamide monomer and an auxiliary additive into water together to be used as a water phase;
(3) dissolving an oil-soluble initiator in the oil phase obtained in the step (1) to prepare an initiator oil phase solution;
(4) mixing the oil phase in the step (1) and the water phase in the step (2) according to different proportions, and preparing into emulsion by using an emulsifying machine;
(5) adding a certain proportion of the initiator oil phase solution obtained in the step (3) into the emulsion obtained in the step (4) to prepare an emulsion containing an oil-soluble initiator, heating the emulsion to a set reaction temperature from room temperature after argon is deoxidized, and carrying out free radical initiated polymerization to obtain functional polyacrylamide microspheres;
(6) under the co-catalysis of cuprous ions and ligands, the oligonucleotide can be efficiently and quantitatively immobilized through the click reaction between azide groups contained in the polyacrylamide microspheres and alkynyl groups on oligonucleotide molecules.
According to a preferred embodiment, in step (1) of method 1, the oil phase is a mixed solution of an organic solvent and a surfactant. The mass ratio of the organic solvent to the surfactant is 20: 1-10: 1, the organic solvent is two or more of diethylhexyl carbonate, mineral oil, isopropyl palmitate, n-hexane, n-heptane and the like, and the surfactant is one or more of Abil WE09, EM90, EM180, Span80, Span60, Tween80, Tween20, Triton-X100 and the like.
According to a preferred embodiment, the water-soluble acrylamide-based monomer described in step (2) of method 1 is acrylamide, hydroxyethylacrylamide or methacrylamide; the cross-linking agent bisacrylamide monomer is N, N-methylene bisacrylamide, N' - (1, 2-dihydroxyethylene) bisacrylamide, diacrylamide polyethylene glycol and the like, and the functional acrylamide monomer is an acrylamide monomer for modifying an azide group and a biotin-like group and the like; the auxiliary additive comprises inorganic salt (chloride, nitrate, sulfate, phosphate and the like), the concentration of the inorganic salt is 0.05-1M, and polymer auxiliary materials (polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, alginic acid, agarose, glucan and the like) have the molecular weight of 5000-50000 and the weight ratio of the polymer auxiliary materials to the acrylamide monomer substances is 0.1-0.5; the quantity ratio of the functional acrylamide monomer to the acrylamide monomer is 0.01-0.1; the amount ratio of the acrylamide crosslinking agent to the acrylamide monomer substance is 0.01-0.1;
according to a preferred embodiment, the water-soluble radical initiator added in step (3) of method 1 is potassium persulfate, sodium persulfate, azobisisobutyramidine hydrochloride, ammonium persulfate or the like; the ratio of the amount of the free radical initiator substance to the amount of the acrylamide monomer substance is 0.005-0.02;
according to a preferable embodiment, in the step (4), the volume ratio of the water phase to the oil phase is 0.01-0.5. The emulsifying machine is a FLUKO FA25 high-shear disperser, a Silverson L5M-A high-shear disperser, an IKA ULTRA-TURRAX UTTD controlled test tube dispersing, homogenizing, a ball milling all-in-one machine, an SPG membrane emulsifying machine and the like, the emulsifying speed is controlled at 100-30000r/min, the emulsifying volume is 5-1000ml, and the emulsifying time is 0.1-24 h;
according to a preferred embodiment, the oxygen removal time in step (5) of method 1 is between 10 and 40 min; the polymerization temperature is 50-90 ℃, the polymerization reaction time is 0.5-6 hours, and the polymerization reaction stirring speed is 50-500 r/min; the heating mode comprises a gas bath shaking table, a heating magnetic stirrer, an oven and the like;
according to a preferred embodiment, in the method 1, the polymer microspheres are quantitatively combined with the oligonucleotides in the step (6) through a 'click' chemistry, wherein the 'click' chemistry refers to that the oligonucleotides are immobilized with high-efficiency and quantitative through a click reaction between azide groups carried by the microspheres and alkynyl groups in the oligonucleotide molecules.
According to a preferred embodiment, in step (1) of method 2, the oil phase is a mixed solution of an organic solvent and a surfactant. The mass ratio of the organic solvent to the surfactant is 20: 1-10: 1, the organic solvent is two or more of diethylhexyl carbonate, mineral oil, isopropyl palmitate, n-hexane, n-heptane and the like, and the surfactant is one or more of Abil WE09, EM90, EM180, Span80, Span60, Tween80, Tween20, Triton-X100 and the like.
According to a preferred embodiment, in step (2) of method 2, the water-soluble acrylamide-based monomer is acrylamide, hydroxyethyl acrylamide or methacrylamide; the cross-linking agent bisacrylamide monomer is N, N-methylene bisacrylamide, N' - (1, 2-dihydroxyethylene) bisacrylamide, diacrylamide polyethylene glycol and the like, and the functional acrylamide monomer is an acrylamide monomer for modifying an azide group and a biotin-like group and the like; the auxiliary additive comprises inorganic salt (chloride, nitrate, sulfate, phosphate and the like), the concentration of the inorganic salt is 0.05-1M, and polymer auxiliary materials (polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, alginic acid, agarose, glucan and the like) have the molecular weight of 5000-50000 and the weight ratio of the polymer auxiliary materials to the acrylamide monomer substances is 0.1-0.5; the quantity ratio of the functional acrylamide monomer to the acrylamide monomer is 0.01-0.1; the amount ratio of the acrylamide crosslinking agent to the acrylamide monomer substance is 0.01-0.1;
according to a preferred embodiment, an oil-soluble free radical initiator is added to step (3) of method 2; the oil-soluble free radical initiator is azobisisobutyronitrile, azobisisoheptonitrile, dimethyl azobisisobutyrate, benzoyl peroxide and the like, and the amount ratio of the initiator to the acrylamide monomer is 0.005-0.01;
according to a preferred embodiment, in the step (4) of the method 2, the volume ratio of the water phase to the oil phase is 0.01-0.5. The emulsifying machine is a FLUKO FA25 high-shear disperser, a Silverson L5M-A high-shear disperser, an IKA ULTRA-TURRAX UTTD controlled test tube dispersing, homogenizing, a ball milling all-in-one machine, an SPG membrane emulsifying machine and the like, the emulsifying speed is controlled at 100-30000r/min, the emulsifying volume is 5-1000ml, and the emulsifying time is 0.1-24 h;
according to a preferred embodiment, in step (5) of method 2, a certain proportion of initiator oil phase solution is added, wherein the volume ratio of the initiator oil phase solution to the emulsion is 0.02-1.0; the oxygen removing time is 10-40 min; the polymerization temperature is 50-90 ℃, the polymerization reaction time is 0.5-6 hours, and the polymerization reaction stirring speed is 50-500 r/min; the heating mode comprises a gas bath shaking table, a heating magnetic stirrer, an oven and the like;
according to a preferred embodiment, the polymer microspheres in step (6) of method 2 are quantitatively bound with oligonucleotides through "click" chemistry, wherein the "click" chemistry refers to the high-efficiency quantitative immobilization of oligonucleotides through the click reaction between azide groups carried by the microspheres and alkynyl groups in oligonucleotide molecules.
The invention provides an oligonucleotide-supported polymer microsphere, which is characterized by being prepared according to the method. The invention has the following remarkable advantages: (1) the emulsion polymerization microsphere has stable internal structure, mild storage condition and long storage period; (2) compared with other functional polymer microspheres, the functional polymer microspheres prepared by emulsion polymerization have low content of oligomers in the microspheres, the influence of the oligomers on subsequent experiments is reduced, and the application field is widened. (3) The emulsion polymerization operation is simple and easy, and the rapid mass preparation of the functional polymer microspheres is easy to realize; (4) the functional polymer microspheres prepared by emulsion polymerization have wide application range, and especially play an important role as raw materials in the field of in vitro detection (such as nucleic acid sequence detection and the like); (5) the single click reaction can prepare microspheres with various oligonucleotide sequences, and can be used for rapid capture detection of molecules with target nucleic acid sequences.
Drawings
Fig. 1. structural formula of compound a B C, wherein R ═ H or CH3 n ═ 2-10, m ═ 1-20, k ═ 10-100;
FIG. 2 is a scanning electron microscope image of the emulsion polymerization method of example 2 to prepare functional polymer microspheres;
FIG. 3 is a graph of the effect of dynamic light scattering test of functional polymer microspheres prepared by emulsion polymerization in example 2;
FIG. 4 is a fluorescence image of functional polymer microspheres immobilized with fluorescein modified oligonucleotide prepared by emulsion polymerization in example 3;
FIG. 5 is a graph showing the relationship between fluorescence intensity of the microsphere immobilized oligonucleotide and the concentration of the alkynyl-modified oligonucleotide in example 3.
Detailed Description
To further illustrate the core of the present invention, the following examples are given as illustrations of the invention. The examples are intended to further illustrate the summary of the invention and are not intended to limit the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The structural formulas of various different functional acrylamide monomers are shown in figure 1, a compound A is a structural diagram of different repeating methylene units with hydrophobic azide functional acrylamide monomers, a compound B is a structural diagram of different repeating ethylene glycol units with hydrophilic azide functional acrylamide monomers, and a structural diagram of different repeating ethylene glycol unit biotin functional acrylamide monomers of a compound C.
Example 1
Preparation of functional polymer microspheres by emulsion polymerization
The experimental process comprises the following steps:
1) preparing an oil phase: weighing 200ml of isopropyl palmitate and 18015 g of EM, and uniformly mixing for 30min by magnetic stirring;
2) preparing a water phase: weighing 600mg of acrylamide, 30mg of N, N-dimethyl bisacrylamide, 12mg of biotin-modified polyethylene glycol acrylamide, 150mg of functional acrylamide azide monomer and 27mg of sodium chloride 45mg of polyvinylpyrrolidone 150mg of potassium persulfate to dissolve in 5mL of ultrapure water;
3) 30mL of the oil phase and 5mL of the aqueous phase are added to a 50mL centrifuge tube and mixed. Emulsifying for 20min at FA-25C level by a high shear dispersion emulsifying machine;
4) and (3) deoxidizing: adding the emulsion into a 100ml single-mouth bottle with magnetons after emulsification, sealing a rubber plug, and introducing Ar for oxygen discharge for 15 min;
5) reaction: at the moment, the reaction system is in an argon protection state; the mixture was put into an oil bath at 90 ℃ and stirred at 400rpm for 2 hours. After the reaction is finished, directly putting the mixture into an ice bath to quench for 30 min;
6) demulsifying: 10mL of the emulsion was centrifuged rapidly and the supernatant discarded. Adding 20mL of demulsifier, mixing uniformly, centrifuging (15000rpm, 5min), and removing supernatant; adding 20mL of demulsifier, re-dispersing, uniformly mixing, centrifuging, discarding supernatant, and repeating for three times; and (3) determining the volume of 10mL by using pure water to obtain the functional polymer microspheres.
Example 2
Preparation of functional polymer microspheres by emulsion polymerization
The experimental process comprises the following steps:
1) preparing an oil phase: preparing 365ml TEGOSOFT DEC, 100ml mineral oil and 35g Abil WE09, and magnetically stirring and uniformly mixing for 30 min;
2) preparing a water phase: weighing 680mg of acrylamide, 33mg of N, N-dimethyl bisacrylamide, 14mg of biotin-modified polyethylene glycol acrylamide and 200mg of functional acrylamide azide monomer, 160mg of sodium chloride and 90mg of polyethylene glycol to dissolve in 5mL of ultrapure water;
3) preparing an initiator oil phase solution: weighing 500mg of azodiisobutyronitrile, dissolving in 50mL of oil phase to prepare an initiator oil phase solution;
4) 27mL of oil phase and 3mL of aqueous phase solution were added to a 50mL centrifuge tube, mixed well and placed in an ice bath for 5 min. Emulsifying for 10min in ice bath by a high-shear emulsifying machine D grade;
5) and (3) deoxidizing: after emulsification, adding 30mL of emulsion and 3mL of initiator oil phase solution into a 100mL single-mouth bottle with magnetons, sealing a rubber plug, and introducing argon into an ice bath to remove oxygen for 15 min;
6) reaction: at the moment, the reaction system is in an argon protection state; the mixture was put into an oil bath at 80 ℃ and stirred at 200rpm to react for 3 hours. After the reaction is finished, directly putting the mixture into an ice bath to quench for 30 min;
7) demulsifying: 10mL of the emulsion was centrifuged rapidly and the supernatant discarded. Adding 20mL of demulsifier, mixing uniformly, centrifuging (15000rpm, 5min), and removing supernatant; adding 20mL of demulsifier, re-dispersing, uniformly mixing, centrifuging, discarding supernatant, and repeating for three times; and (3) determining the volume of 10mL by using pure water to obtain the functional polymer microspheres.
8) The polymer microspheres are characterized by a scanning electron microscope and dynamic light scattering, and the characterization result is shown in figure 2 and figure 3. FIG. 2 shows that the functional polymer microspheres have uniform particle size distribution and average particle size of about 900nm in a scanning electron microscope, and FIG. 3 shows that the dynamic light scattering test results of the microspheres have particle size of about 850nm, and the dynamic light scattering test values are consistent with the particle size results of the scanning electron microscope.
Example 3
Preparation of functional polymer microspheres by emulsion polymerization
1) Preparing an oil phase: preparing 100mL of mineral oil, 4.5mL of tween80, 0.4mL of span80 and 100ul of TrixonX-100, and magnetically stirring and uniformly mixing for 30 min;
2) preparing a water phase: weighing 319mg of acrylamide, 15mg of N, N-dimethyl bisacrylamide, 16mg of biotin-modified polyethylene glycol acrylamide and 125mg of functional acrylamide azide monomer, 45mg of sodium chloride and 129mg of polyvinylpyrrolidone, and dissolving in 5mL of ultrapure water;
3) preparing an initiator oil phase solution: weighing 500mg of azodiisobutyronitrile, dissolving in 50mL of continuous phase matching to prepare an initiator oil phase solution;
4) adding 80mL of oil phase into a 100mL beaker, adding 5mL of water phase into an SPG membrane emulsifier, and emulsifying at an emulsifying speed of 300r/min under an emulsifying pressure of 200kPa for 10 h;
5) and (3) deoxidizing: after emulsification, adding 32mL of emulsion and 6.4mL of initiator oil phase solution into a 100mL single-mouth bottle with magnetons, sealing a rubber plug, and introducing argon into an ice bath to remove oxygen for 15 min;
6) reaction: at the moment, the reaction system is in an argon protection state; the mixture was put into a 70 ℃ gas bath shaker, stirred at 200rpm and reacted for 3 hours. After the reaction is finished, directly putting the mixture into an ice bath to quench for 30 min;
7) demulsifying: 10mL of the emulsion was centrifuged rapidly and the supernatant discarded. Adding 20mL of demulsifier, mixing uniformly, centrifuging (15000rpm, 5min), and removing supernatant; adding 20mL of demulsifier, re-dispersing, uniformly mixing, centrifuging, discarding supernatant, and repeating for three times; and (3) determining the volume of 10mL by using pure water to obtain the target functional polymer microsphere.
8) Click reaction: functional polymer microspheres prepared by emulsion polymerization were characterized by "click" reaction and oligonucleotide probes with fluorescein attached, and fluorescence microscopy, and the results are shown in FIG. 4 and FIG. 5. The fluorescence micrograph of figure 4 shows that the microspheres have uniform particle size, and the particle size of the microspheres is 1.2 microns calculated by fluorescence intensity half-peak width, and the particle size difference is 12%. Functional polymer microspheres prepared by emulsion polymerization and alkynyl oligonucleotide with different concentrations (0.8-2.0M) are subjected to click chemical reaction, fluorescence intensity of immobilized microspheres is measured by a fluorescence microscope (exposure condition: light intensity of blue light is 200, and exposure time is 0.5s), reaction concentration of oligonucleotide and fluorescence intensity of microsphere immobilized oligonucleotide show high linear correlation, a linear equation y is 724x, and a linear correlation coefficient R2 reaches 0.997.
The emulsion polymerization method provided by the invention can realize synthesis of functional polymer microspheres, the adjustment range of emulsion polymerization monomers is wide, the types of prepared microspheres are various, the adjustment factors are many, the emulsion polymerization microspheres have stable structures and mild storage conditions, and the effect period is long.
The invention has the following remarkable advantages: (1) the emulsion polymerization microsphere has stable internal structure, mild storage condition and long storage period; (2) compared with other functional polymer microspheres, the functional polymer microspheres prepared by emulsion polymerization have low content of oligomers in the microspheres, the influence of the oligomers on subsequent experiments is reduced, and the application field is widened. (3) The emulsion polymerization operation is simple and easy, and the rapid mass preparation of the functional polymer microspheres is easy to realize; (4) the functional polymer microspheres prepared by emulsion polymerization have wide application range, and especially play an important role as raw materials in the field of in vitro detection (such as nucleic acid sequence detection and the like); (5) the single click reaction can prepare microspheres with various oligonucleotide sequences, and can be used for rapid capture detection of molecules with target nucleic acid sequences.
Claims (10)
1. A method for synthesizing functional polymer microspheres by emulsion polymerization comprises the following steps:
(1) preparing an oil phase by the organic solvent and the surfactant according to different proportions;
(2) dissolving a water-soluble acrylamide monomer, a cross-linking agent bisacrylamide monomer, a functional acrylamide monomer and an auxiliary additive into water together to be used as a water phase;
(3) adding a water-soluble initiator into the water phase obtained in the step (2) to prepare a water phase containing the water-soluble initiator;
(4) mixing the oil phase in the step (1) and the water phase containing the water-soluble initiator in the step (3) according to different proportions, and preparing into emulsion containing the water-soluble initiator by using an emulsifying machine;
(5) after argon is used for removing oxygen in the emulsion containing the water-soluble initiator in the step (4), heating the emulsion to a set reaction temperature from room temperature, and carrying out free radical initiated polymerization to obtain functional polyacrylamide microspheres;
(6) under the co-catalysis of cuprous ions and ligands, the efficient quantitative immobilization of the oligonucleotide is realized through the click reaction between azide groups contained in the polyacrylamide microspheres and alkynyl groups on oligonucleotide molecules.
2. A method for synthesizing functional polymer microspheres by emulsion polymerization comprises the following steps:
(1) preparing an oil phase by the organic solvent and the surfactant according to different proportions;
(2) dissolving a water-soluble acrylamide monomer, a cross-linking agent bisacrylamide monomer, a functional acrylamide monomer and an auxiliary additive into water together to be used as a water phase;
(3) dissolving an oil-soluble initiator in the oil phase obtained in the step (1) to prepare an initiator oil phase solution;
(4) mixing the oil phase in the step (1) and the water phase in the step (2) according to different proportions, and preparing into emulsion by using an emulsifying machine;
(5) adding a certain proportion of the initiator oil phase solution obtained in the step (3) into the emulsion obtained in the step (4) to prepare an emulsion containing an oil-soluble initiator, heating the emulsion to a set reaction temperature from room temperature after argon is deoxidized, and carrying out free radical initiated polymerization to obtain functional polyacrylamide microspheres;
(6) under the co-catalysis of cuprous ions and ligands, the efficient quantitative immobilization of the oligonucleotide is realized through the click reaction between azide groups contained in the polyacrylamide microspheres and alkynyl groups on oligonucleotide molecules.
3. The method according to claim 1 or 2, characterized in that: in the step (1), the oil phase is a mixed solution of an organic solvent and a surfactant; the mass ratio of the organic solvent to the surfactant is 100: 1-10: 1, the organic solvent is one or more of diethylhexyl carbonate, mineral oil, isopropyl palmitate, n-heptane and the like, and the surfactant is one or more of Abil WE09, EM90, EM180, Span80, Span60, Tween80, Tween20 and Triton-X100.
4. The method according to claim 1 or 2, characterized in that: the water-soluble acrylamide monomer in the step (2) is acrylamide, hydroxyethyl acrylamide or methacrylamide; the cross-linking agent is one of N, N-methylene bisacrylamide, N' - (1, 2-dihydroxyethylene) bisacrylamide and diacrylamide-based polyethylene glycol as a bisacrylamide monomer; the functional acrylamide monomer is an acrylamide monomer for modifying an azide group and a biotin-like group; the auxiliary additive comprises inorganic salt with the concentration of 0.01-3M, the molecular weight of the polymer auxiliary material is 1000-100000, and the weight ratio of the polymer auxiliary material to the acrylamide monomer substance is 0.0-1.0; the quantity ratio of the functional acrylamide monomer to the acrylamide monomer is 0.01-0.25; the amount ratio of the acrylamide crosslinking agent to the acrylamide monomer substance is 0.01-0.1;
wherein the inorganic salt is one or more of chloride, nitrate, sulfate, carbonate and phosphate;
the polymer auxiliary materials are one or more of polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, alginic acid, agarose and glucan.
5. The method of claim 1, wherein: adding a water-soluble free radical initiator such as potassium persulfate, sodium persulfate, azobisisobutyramidine hydrochloride or ammonium persulfate and the like into the step (3); the amount of the free radical initiator such as potassium persulfate, sodium persulfate, azodiisobutylamine hydrochloride or ammonium persulfate and the like is 0.001-0.1 of the amount of the acrylamide monomer substance.
6. The method according to claim 1 or 2, characterized in that: in the step (4), the volume ratio of the water phase to the oil phase is 0.01-0.5; the emulsifying machine comprises a FLUKO FA25 high-shear disperser, a Silverson L5M-A high-shear disperser, an IKA ULTRA-TURRAX UTTD controlled test tube dispersing, homogenizing and ball milling integrated machine and an SPG membrane emulsifying machine; the emulsifying speed is controlled at 100-30000r/min, the emulsifying volume is 5-1000ml, and the emulsifying time is 0.1-24 h.
7. The method of claim 1, wherein: the oxygen removing time in the step (5) is 10-40 min; the polymerization temperature is 50-90 ℃, the polymerization reaction time is 0.5-6 hours, and the polymerization reaction stirring speed is 50-500 r/min; the heating mode comprises a gas bath shaking table, a heating magnetic stirrer, an oven and the like.
8. The method according to claim 1 or 2, characterized in that: the polymer microspheres are quantitatively combined with oligonucleotides through 'click' chemistry, wherein the 'click' chemistry refers to that the oligonucleotides are efficiently and quantitatively immobilized through click reaction between azide groups carried by the microspheres and alkynyl groups in oligonucleotide molecules.
9. The process of claim 2, wherein an oil-soluble free radical initiator is added to step (3); the oil-soluble free radical initiator is one of azobisisobutyronitrile, azobisisoheptonitrile, dimethyl azobisisobutyrate and benzoyl peroxide, and the amount ratio of the initiator substance to the acrylamide monomer substance is 0.0001-0.1.
10. The method of claim 2, wherein a proportion of initiator oil phase solution is added to step (5), wherein the initiator oil phase solution and the emulsion have a volume ratio of 0.02 to 1.0; the oxygen removing time is 10-40 min; the polymerization temperature is 50-90 ℃, the polymerization reaction time is 0.5-6 hours, and the polymerization reaction stirring speed is 50-500 r/min; the heating mode comprises a gas bath shaking table, a heating magnetic stirrer, an oven and the like.
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