CN114316132B - Method for synthesizing functional polymer microsphere 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 efficient quantitative immobilization of oligonucleotides. The invention takes aqueous solution of water-soluble monomer acrylamide monomer, cross-linking agent bisacrylamide monomer, functional acrylamide monomer and auxiliary additive as water phase, takes mixed solution of organic solvent and surfactant as oil phase, and prepares emulsion by an emulsifying machine. And (3) copolymerizing and crosslinking in an emulsion polymerization mode by using a free radical polymerization method to form polyacrylamide functional microspheres, and realizing quantitative immobilization of the oligonucleotide by virtue of 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 has the advantages of simple operation, stable process and suitability 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, belonging to the field of gene sequencing.
Background
Bioactive supported microspheres (Microsphere) are spherical particles with a particle size between 50nm and 2mm, bearing reactive groups such as-NH 2, -COOH, -SH, etc. The microsphere has the characteristics of obvious surface effect such as good affinity and biocompatibility of materials, easy absorption and easy migration in organisms and the like due to the smaller size, and has been widely applied to a plurality of fields such as cytology, immunology, microbiology, molecular biology, clinical diagnosis and treatment, high-throughput gene detection and the like.
In the method of using microspheres for high throughput gene detection analysis of certain nucleic acid sequences, the size distribution of the microspheres is about 1 μm, highly uniform dispersion, and the loading of the microspheres to the nucleic acid strand is relied on. The sequence of the loaded nucleic acid strand can then be determined by a number of different methods known in the art. In certain microspheres for use in nucleic acid sequencing methods, the procedure involves rapid and efficient binding of the corresponding reactive groups of the microspheres to achieve loading by a number of methods well known in the art using a nucleic acid sample with sequencing; the nucleic acid-loaded microspheres are introduced into the channels of the flow cell and into the corresponding reaction micro-tunnels, incubated for a fixed time, yielding all downstream chemical processing steps that are always capable of supporting amplification and sequencing. The invention discloses a method for synthesizing functional polymer microspheres by emulsion polymerization, which has stable internal structure and is 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,
procedure 1
(1) Preparing an oil phase from an organic solvent and a surfactant according to different proportions;
(2) The water-soluble acrylamide monomer, the cross-linking agent bisacrylamide monomer, the functional acrylamide monomer and the auxiliary additive are dissolved in water together to be used as a water phase;
(3) Adding a water-soluble initiator into the water phase 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 emulsion containing the water-soluble initiator by using an emulsifying machine;
(5) After deoxidizing the emulsion containing the water-soluble initiator in the step (4) by argon, raising the temperature from room temperature to a set reaction temperature, and carrying out free radical initiated polymerization reaction to obtain the functional polyacrylamide microsphere;
(6) Under the co-catalysis of cuprous ions and ligands, the high-efficiency quantitative immobilization of the oligonucleotide can be realized through the click reaction between azide groups contained in the polyacrylamide microspheres and alkynyl groups on the oligonucleotide molecules.
Method 2 step
(1) Preparing an oil phase from an organic solvent and a surfactant according to different proportions;
(2) The water-soluble acrylamide monomer, the cross-linking agent bisacrylamide monomer, the functional acrylamide monomer and the auxiliary additive are dissolved in water together to be used as a water phase;
(3) The oil-soluble initiator is dissolved in the oil phase 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 emulsion by using an emulsifying machine;
(5) Adding a certain proportion of the initiator oil phase solution in the step (3) into the emulsion in the step (4) to prepare an oil-soluble initiator emulsion, raising the temperature from room temperature to a set reaction temperature after deoxidizing by argon, and carrying out free radical initiated polymerization reaction to obtain the functional polyacrylamide microsphere;
(6) Under the co-catalysis of cuprous ions and ligands, the high-efficiency quantitative immobilization of the oligonucleotide can be realized through the click reaction between azide groups contained in the polyacrylamide microspheres and alkynyl groups on the 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 diethyl hexyl 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 acrylamide, hydroxyethyl acrylamide or methacrylamide described in step (2) of method 1; the cross-linking agent bisacrylamide monomer is N, N-methylene bisacrylamide, N' - (1, 2-dihydroxyethylene) bisacrylamide, bisacrylamide polyethylene glycol and the like, and the functional acrylamide monomer is acrylamide monomer with modified azide groups, modified biotin similar groups and the like; the auxiliary additive comprises inorganic salt (chloride, nitrate, sulfate, phosphate and the like), wherein the concentration of the inorganic salt is 0.05-1M, and the molecular weight of the macromolecular auxiliary material is 5000-50000, and the mass ratio of the macromolecular auxiliary material to the acrylamide monomer substance is 0.1-0.5; the mass ratio of the functional acrylamide monomer to the acrylamide monomer substance is 0.01-0.1; the mass ratio of the acrylamide cross-linking agent to the acrylamide monomer substance is 0.01-0.1;
according to a preferred embodiment, the water-soluble free radical initiator added in step (3) of method 1 is potassium persulfate, sodium persulfate, azobisisobutyrimidine 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 preferred embodiment, in step (4), the volume ratio of the aqueous phase to the oil phase is between 0.01 and 0.5. theemulsifyingmachineisaFLUKOFA25high-sheardispersingmachine,aSilversonL5M-Ahigh-sheardispersingmachine,anIKAULTRA-TURRAXUTTDcontroltypetesttubedispersing,homogenizing,ballmillingintegratedmachine,SPGmembraneemulsifyingmachineandthelike,theemulsifyingrotatingspeediscontrolledtobe100-30000r/min,theemulsifyingvolumeis5-1000ml,andtheemulsifyingtimeis0.1-24h;
according to a preferred embodiment, the oxygen scavenging time in step (5) of method 1 is between 10 and 40min; the polymerization temperature is 50-90 ℃, the polymerization time is 0.5-6 hours, and the stirring speed of the polymerization reaction is 50-500 r/min; the heating mode comprises an air bath shaking table, a heating magnetic stirrer, a baking oven and the like;
according to a preferred embodiment, method 1, step (6) the polymeric microspheres are bound to the oligonucleotides quantitatively by "click" chemistry, which refers to the efficient quantitative immobilization of the oligonucleotides by a click reaction between the azide groups carried by the microspheres and the alkynyl groups within the oligonucleotide molecule.
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 diethyl hexyl 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 acrylamide, hydroxyethyl acrylamide or methacrylamide; the cross-linking agent bisacrylamide monomer is N, N-methylene bisacrylamide, N' - (1, 2-dihydroxyethylene) bisacrylamide, bisacrylamide polyethylene glycol and the like, and the functional acrylamide monomer is acrylamide monomer with modified azide groups, modified biotin similar groups and the like; the auxiliary additive comprises inorganic salt (chloride, nitrate, sulfate, phosphate and the like), wherein the concentration of the inorganic salt is 0.05-1M, and the molecular weight of the macromolecular auxiliary material is 5000-50000, and the mass ratio of the macromolecular auxiliary material to the acrylamide monomer substance is 0.1-0.5; the mass ratio of the functional acrylamide monomer to the acrylamide monomer substance is 0.01-0.1; the mass ratio of the acrylamide cross-linking 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 azodiisobutyronitrile, azodiisoheptonitrile, dimethyl azodiisobutyrate, benzoyl peroxide and the like, and the ratio of the amount of the initiator to the amount of the acrylamide monomer substance is 0.005-0.01;
according to a preferred embodiment, in step (4) of method 2, the volume ratio of the aqueous phase to the oil phase is between 0.01 and 0.5. theemulsifyingmachineisaFLUKOFA25high-sheardispersingmachine,aSilversonL5M-Ahigh-sheardispersingmachine,anIKAULTRA-TURRAXUTTDcontroltypetesttubedispersing,homogenizing,ballmillingintegratedmachine,SPGmembraneemulsifyingmachineandthelike,theemulsifyingrotatingspeediscontrolledtobe100-30000r/min,theemulsifyingvolumeis5-1000ml,andtheemulsifyingtimeis0.1-24h;
according to a preferred embodiment, in step (5) of method 2, a proportion of an initiator oil phase solution is added, the initiator oil phase solution and the emulsion volume ratio being between 0.02 and 1.0; the deoxidization time is 10-40min; the polymerization temperature is 50-90 ℃, the polymerization time is 0.5-6 hours, and the stirring speed of the polymerization reaction is 50-500 r/min; the heating mode comprises an air bath shaking table, a heating magnetic stirrer, a baking oven and the like;
according to a preferred embodiment, the polymeric microspheres in step (6) of method 2 are bound to the oligonucleotides quantitatively by "click" chemistry, which refers to the efficient quantitative immobilization of the oligonucleotides by a click reaction between the azide groups carried by the microspheres and the alkynyl groups within the oligonucleotide molecule.
The invention provides an oligonucleotide immobilized 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 microsphere prepared by emulsion polymerization has low content of oligomers, reduces the influence of the oligomers on subsequent experiments, and widens the application field. (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 emulsion polymerization prepared functional polymer microsphere has wide application range, and plays an important role as a raw material in the field of in vitro detection (such as nucleic acid sequence detection and the like); (5) A single click reaction can produce microspheres with a variety of different oligonucleotide sequences that can be used for rapid capture detection of molecules with target nucleic acid sequences.
Drawings
Fig. 1, structural formula of compound ab C, wherein r=h or ch3n=2-10, m=1-20, k=10-100;
FIG. 2 is a graph showing the effect of the emulsion polymerization process of example 2 on preparing functional polymer microspheres;
FIG. 3 is a graph showing the dynamic light scattering test effect of the functional polymer microspheres prepared by the emulsion polymerization method of example 2;
FIG. 4 is a fluorescent image of a functional polymer microsphere immobilized fluorescein-modified oligonucleotide prepared by emulsion polymerization in example 3;
FIG. 5 is a graph showing the relationship between fluorescence intensity of microsphere immobilized oligonucleotide and concentration of alkynyl modified oligonucleotide in example 3.
Detailed Description
To further illustrate the core of the present invention, the present invention will now be described by way of the following examples. The examples are provided to further illustrate the summary of the invention and are not intended to limit the invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The structural formula of various functional acrylamide monomers is shown in figure 1, the compound A is a structural diagram of acrylamide monomers with hydrophobic azide functional units in different repeated methylene units, the compound B is a structural diagram of acrylamide monomers with hydrophilic azide functional units in different repeated ethylene glycol units, and the compound C is a structural diagram of acrylamide monomers with biotin functional units in different repeated ethylene glycol units.
Example 1
Emulsion polymerization for preparing functional polymer microsphere
The experimental process comprises the following steps:
1) Preparing an oil phase: 200ml isopropyl palmitate and 180 g EM are weighed, and the mixture is stirred and mixed for 30min by magnetic force;
2) Preparing an aqueous phase: weighing 600mg of acrylamide, 30mg of N, N-dimethyl bisacrylamide, 12mg of biotin modified polyethylene glycol acrylamide, 150mg of functional acrylamide azide monomer, 45mg of sodium chloride, 150mg of polyvinylpyrrolidone and 27mg of potassium persulfate, and dissolving in 5mL of ultrapure water;
3) 30mL of the oil phase and 5mL of the water phase were added to a 50mL centrifuge tube and mixed well. Emulsifying for 20min in a high shear dispersing emulsifying machine FA-25C grade;
4) Deoxidizing: adding the emulsion into a 100ml single-port bottle with a magnet after emulsification, sealing a rubber plug, and introducing Ar for exhausting oxygen for 15min;
5) The reaction: at this time, the reaction system is in an argon protection state; put into an oil bath at 90 ℃ and stirred at 400rpm for 2 hours. Directly putting the mixture into an ice bath to quench for 30min after the reaction is finished;
6) Demulsification: 10mL of the emulsion was centrifuged rapidly and the supernatant discarded. Adding 20mL of demulsifier, mixing, centrifuging (15000 rpm, 5 min), and discarding supernatant; adding 20mL of demulsifier, re-dispersing, uniformly mixing, centrifuging, discarding the supernatant, and repeating for three times; and (5) fixing the volume to 10mL by using pure water to obtain the functional polymer microsphere.
Example 2
Emulsion polymerization for preparing functional polymer microsphere
The experimental process comprises the following steps:
1) Preparing an oil phase: 365ml of TEGOSOFT DEC, 100ml of minor oil and 35g of Abil WE09 are prepared and stirred magnetically for 30min;
2) Preparing an aqueous phase: weighing 680mg of acrylamide, 33mg of biotin-modified polyethylene glycol acrylamide, 14mg of N, N-dimethyl bisacrylamide, 160mg of functional acrylamide azide monomer, 90mg of sodium chloride and 200mg of polyethylene glycol into 5mL of ultrapure water;
3) Preparing an initiator oil phase solution: weighing 500mg of azodiisobutyronitrile, and dissolving in 50mL of oil phase to prepare an initiator oil phase solution;
4) 27mL of the oil phase and 3mL of the aqueous phase solution were added to a 50mL centrifuge tube, mixed well and placed in an ice bath for 5min. Emulsifying in ice bath for 10min in D grade with high shear emulsifying machine;
5) Deoxidizing: after emulsification, adding 30mL of emulsion and 3mL of initiator oil phase solution into a 100mL single-port bottle with a magnet, sealing a rubber plug, and introducing argon into an ice bath for discharging oxygen for 15min;
6) The reaction: at this time, the reaction system is in an argon protection state; put in an oil bath at 80 ℃ and stirred at 200rpm for reaction for 3 hours. Directly putting the mixture into an ice bath to quench for 30min after the reaction is finished;
7) Demulsification: 10mL of the emulsion was centrifuged rapidly and the supernatant discarded. Adding 20mL of demulsifier, mixing, centrifuging (15000 rpm, 5 min), and discarding supernatant; adding 20mL of demulsifier, re-dispersing, uniformly mixing, centrifuging, discarding the supernatant, and repeating for three times; and (5) fixing the volume to 10mL by using pure water to obtain the functional polymer microsphere.
8) The polymer microsphere is characterized by a scanning electron microscope and dynamic light scattering, and the characterization result is shown in fig. 2 and 3. The scanning electron microscope chart of fig. 2 shows that the functional polymer microsphere has uniform particle size distribution, the average particle size is about 900nm, and the dynamic light scattering test result of the microsphere has particle size of about 850nm, and the dynamic light scattering test value is consistent with the particle size result of the scanning electron microscope.
Example 3
Emulsion polymerization for preparing functional polymer microsphere
1) Preparing an oil phase: 100ml of mineral oil, 4.5ml of tween80, 0.4ml of span80 and 100ul of TrixonX-100 are prepared and stirred magnetically for 30min;
2) Preparing an aqueous phase: weighing 319mg of acrylamide, 15mg of N, N-dimethyl bisacrylamide, 16mg of biotin-modified polyethylene glycol acrylamide, 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, and preparing 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 200kPa for 10 hours at a speed of 300 r/min;
5) Deoxidizing: after emulsification, adding 32mL of emulsion and 6.4mL of initiator oil phase solution into a 100mL single-port bottle with a magnet, sealing a rubber plug, and introducing argon into an ice bath for discharging oxygen for 15min;
6) The reaction: at this time, the reaction system is in an argon protection state; put into an air bath shaker at 70 ℃ and stirred at 200rpm for reaction for 3 hours. Directly putting the mixture into an ice bath to quench for 30min after the reaction is finished;
7) Demulsification: 10mL of the emulsion was centrifuged rapidly and the supernatant discarded. Adding 20mL of demulsifier, mixing, centrifuging (15000 rpm, 5 min), and discarding supernatant; adding 20mL of demulsifier, re-dispersing, uniformly mixing, centrifuging, discarding the supernatant, and repeating for three times; and (5) fixing the volume to 10mL by using pure water to obtain the target functional polymer microsphere.
8) Click reaction: emulsion polymerization preparation functional Polymer microspheres were attached to fluorescein-bearing oligonucleotide probes by a "click" reaction, characterized by fluorescence microscopy, and the test results are shown in FIG. 4 and FIG. 5. Fluorescence micrograph fig. 4 shows that the microsphere size is uniform, the microsphere size is 1.2 microns and the difference in size is 12% as calculated by half-width of fluorescence intensity. The functional polymer microsphere prepared by emulsion polymerization is subjected to click chemistry reaction with alkynyl oligonucleotides with different concentrations (0.8-2.0M), the fluorescent intensity of the immobilized microsphere is measured by a fluorescent microscope (exposure condition: blue light intensity 200 exposure time 0.5 s), the oligonucleotide reaction concentration and the fluorescent intensity of the microsphere immobilized oligonucleotide show high linear correlation, a linear equation y=724 x, and a linear correlation coefficient R2 reaches 0.997.
The emulsion polymerization method introduced by the invention can realize the synthesis of functional polymer microspheres, has wide adjustment range of emulsion polymerization monomers, multiple types of prepared microspheres and multiple adjustment factors, and has stable structure, mild storage condition and long service life.
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 microsphere prepared by emulsion polymerization has low content of oligomers, reduces the influence of the oligomers on subsequent experiments, and widens the application field. (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 emulsion polymerization prepared functional polymer microsphere has wide application range, and plays an important role as a raw material in the field of in vitro detection (such as nucleic acid sequence detection and the like); (5) A single click reaction can produce microspheres with a variety of different oligonucleotide sequences that 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, comprising the following steps:
(1) Preparing an oil phase from an organic solvent and a surfactant according to different proportions;
(2) The water-soluble acrylamide monomer, the cross-linking agent bisacrylamide monomer, the functional acrylamide monomer and the auxiliary additive are dissolved in water together to be used as a water phase;
(3) Adding a water-soluble initiator into the water phase 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 emulsion containing the water-soluble initiator by using an emulsifying machine;
(5) After deoxidizing the emulsion containing the water-soluble initiator in the step (4) by argon, raising the temperature from room temperature to a set reaction temperature, and carrying out free radical initiated polymerization reaction to obtain the functional polyacrylamide microsphere;
(6) Under the co-catalysis of cuprous ions and ligands, the high-efficiency quantitative immobilization of the oligonucleotide is realized through the click reaction between azide groups contained in the polyacrylamide microspheres and alkynyl groups on the oligonucleotide molecules;
wherein 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 bisacrylamide polyethylene glycol; the functional acrylamide monomer is an acrylamide monomer with an azide group modified and a biotin group modified.
2. A method for synthesizing functional polymer microspheres by emulsion polymerization, comprising the following steps:
(1) Preparing an oil phase from an organic solvent and a surfactant according to different proportions;
(2) The water-soluble acrylamide monomer, the cross-linking agent bisacrylamide monomer, the functional acrylamide monomer and the auxiliary additive are dissolved in water together to be used as a water phase;
(3) The oil-soluble initiator is dissolved in the oil phase 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 emulsion by using an emulsifying machine;
(5) Adding a certain proportion of the initiator oil phase solution in the step (3) into the emulsion in the step (4) to prepare an oil-soluble initiator emulsion, raising the temperature from room temperature to a set reaction temperature after deoxidizing by argon, and carrying out free radical initiated polymerization reaction to obtain the functional polyacrylamide microsphere;
(6) Under the co-catalysis of cuprous ions and ligands, the high-efficiency quantitative immobilization of the oligonucleotide is realized through the click reaction between azide groups contained in the polyacrylamide microspheres and alkynyl groups on the oligonucleotide molecules;
wherein 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 bisacrylamide polyethylene glycol; the functional acrylamide monomer is an acrylamide monomer with an azide group modified and a biotin group modified.
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 diethyl hexyl carbonate, mineral oil, isopropyl palmitate, n-heptane and the like, and the surfactant is one or more of AbilWE09, EM90, EM180, span80, span60, tween80, tween20 and Triton-X100.
4. The method according to claim 1 or 2, characterized in that: the auxiliary additive in the step (2) comprises inorganic salt and high polymer auxiliary materials; wherein the concentration of the inorganic salt is 0.01-3M, the molecular weight of the high molecular auxiliary material is 1000-100000, and the mass ratio of the high molecular auxiliary material to the acrylamide monomer substance is 0.0-1.0; the mass ratio of the functional acrylamide monomer to the acrylamide monomer substance is 0.01-0.25; the mass ratio of the acrylamide cross-linking 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 material is one or more of polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, alginic acid, agarose and dextran.
5. The method according to claim 1, characterized in that: adding a water-soluble free radical initiator such as potassium persulfate, sodium persulfate, azobisisobutyrimidine hydrochloride or ammonium persulfate to the step (3); the ratio of the amount of the free radical initiator such as potassium persulfate, sodium persulfate, azo diisobutylamidine hydrochloride or ammonium persulfate and the like to the amount of the acrylamide monomer is 0.001-0.1;
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; theemulsifyingmachineisaFLUKOFA25high-sheardispersingmachine,aSilversonL5M-Ahigh-sheardispersingmachineandanSPGfilmemulsifyingmachine; the emulsifying rotating speed is controlled to be 100-30000r/min, the emulsifying volume is 5-1000ml, and the emulsifying time is 0.1-24h.
7. The method according to claim 1, characterized in that: the deoxidization time in the step (5) is 10-40min; the polymerization temperature is 50-90 ℃, the polymerization time is 0.5-6 hours, and the stirring speed of the polymerization reaction is 50-500 r/min; the heating mode comprises an air bath shaking table, a heating magnetic stirrer and an oven.
8. The method according to claim 1 or 2, characterized in that: the polymer microsphere is quantitatively combined with the oligonucleotide through a 'click' chemistry, wherein the 'click' chemistry refers to the realization of the efficient quantitative immobilization of the oligonucleotide through the click reaction between an azide group carried by the microsphere and an alkynyl group in an oligonucleotide molecule.
9. The process according to claim 2, wherein an oil-soluble free radical initiator is added to step (3); the oil-soluble free radical initiator is one of azodiisobutyronitrile, azodiisoheptonitrile, dimethyl azodiisobutyrate and benzoyl peroxide, and the ratio of the amount of the initiator substance to the amount of the acrylamide monomer substance is 0.0001-0.1.
10. The method of claim 2 wherein a ratio of initiator oil phase solution to emulsion volume ratio of 0.02 to 1.0 is added to step (5); the deoxidization time is 10-40min; the polymerization temperature is 50-90 ℃, the polymerization time is 0.5-6 hours, and the stirring speed of the polymerization reaction is 50-500 r/min; the heating mode comprises an air bath shaking table, a heating magnetic stirrer and an oven.
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| CN111171228B (en) * | 2020-01-20 | 2022-03-08 | 赛纳生物科技(北京)有限公司 | Preparation method of linear hydrogel containing azide functional groups |
| CN111171229B (en) * | 2020-02-12 | 2021-12-28 | 赛纳生物科技(北京)有限公司 | Preparation method of nucleic acid immobilized microspheres |
| FR3107454B1 (en) * | 2020-02-20 | 2023-02-10 | Soc Dexploitation De Produits Pour Les Industries Chimiques Seppic | Vaccine adjuvant comprising a reverse microlatex |
| CN111944105B (en) * | 2020-08-07 | 2022-07-08 | 武汉珈源同创科技有限公司 | Quantum dot fluorescent microsphere and preparation method thereof |
| CN112280827B (en) * | 2020-09-18 | 2022-12-06 | 赛纳生物科技(北京)有限公司 | Preparation method of core-shell nucleic acid immobilized microspheres |
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