CN112552456A - Preparation method of carrier initial microspheres for uniform-particle-size DNA/RNA solid-phase synthesis - Google Patents
Preparation method of carrier initial microspheres for uniform-particle-size DNA/RNA solid-phase synthesis Download PDFInfo
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- 239000004005 microsphere Substances 0.000 title claims abstract description 115
- 238000010532 solid phase synthesis reaction Methods 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 239000002245 particle Substances 0.000 claims abstract description 54
- 229920000642 polymer Polymers 0.000 claims abstract description 42
- 239000012071 phase Substances 0.000 claims description 114
- 230000008961 swelling Effects 0.000 claims description 74
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 45
- 239000012498 ultrapure water Substances 0.000 claims description 45
- 239000000839 emulsion Substances 0.000 claims description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 27
- 238000002156 mixing Methods 0.000 claims description 26
- 238000010438 heat treatment Methods 0.000 claims description 25
- 238000003756 stirring Methods 0.000 claims description 21
- 238000000967 suction filtration Methods 0.000 claims description 20
- 238000001035 drying Methods 0.000 claims description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- 230000001804 emulsifying effect Effects 0.000 claims description 18
- 239000000178 monomer Substances 0.000 claims description 18
- 238000005406 washing Methods 0.000 claims description 18
- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical group C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 claims description 16
- 238000006116 polymerization reaction Methods 0.000 claims description 16
- 239000003995 emulsifying agent Substances 0.000 claims description 13
- 239000000463 material Substances 0.000 claims description 13
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims description 12
- LQZZUXJYWNFBMV-UHFFFAOYSA-N dodecan-1-ol Chemical compound CCCCCCCCCCCCO LQZZUXJYWNFBMV-UHFFFAOYSA-N 0.000 claims description 12
- 239000003381 stabilizer Substances 0.000 claims description 12
- 239000011148 porous material Substances 0.000 claims description 11
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 10
- 238000001816 cooling Methods 0.000 claims description 10
- 239000000706 filtrate Substances 0.000 claims description 10
- 230000007935 neutral effect Effects 0.000 claims description 10
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 9
- 239000003431 cross linking reagent Substances 0.000 claims description 9
- 239000003999 initiator Substances 0.000 claims description 9
- 238000006136 alcoholysis reaction Methods 0.000 claims description 8
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 claims description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- 230000003301 hydrolyzing effect Effects 0.000 claims description 7
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 6
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- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 6
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 5
- 230000007062 hydrolysis Effects 0.000 claims description 5
- 238000006460 hydrolysis reaction Methods 0.000 claims description 5
- 239000004342 Benzoyl peroxide Substances 0.000 claims description 4
- OMPJBNCRMGITSC-UHFFFAOYSA-N Benzoylperoxide Chemical compound C=1C=CC=CC=1C(=O)OOC(=O)C1=CC=CC=C1 OMPJBNCRMGITSC-UHFFFAOYSA-N 0.000 claims description 4
- 229920000858 Cyclodextrin Polymers 0.000 claims description 4
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- 150000007824 aliphatic compounds Chemical class 0.000 claims description 4
- 239000008346 aqueous phase Substances 0.000 claims description 4
- 235000019400 benzoyl peroxide Nutrition 0.000 claims description 4
- HPXRVTGHNJAIIH-UHFFFAOYSA-N cyclohexanol Chemical compound OC1CCCCC1 HPXRVTGHNJAIIH-UHFFFAOYSA-N 0.000 claims description 4
- ZSIAUFGUXNUGDI-UHFFFAOYSA-N hexan-1-ol Chemical compound CCCCCCO ZSIAUFGUXNUGDI-UHFFFAOYSA-N 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
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- 239000012875 nonionic emulsifier Substances 0.000 claims description 3
- 229920002134 Carboxymethyl cellulose Polymers 0.000 claims description 2
- 239000001116 FEMA 4028 Substances 0.000 claims description 2
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- 239000003513 alkali Substances 0.000 claims description 2
- 150000001491 aromatic compounds Chemical class 0.000 claims description 2
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims description 2
- 235000011175 beta-cyclodextrine Nutrition 0.000 claims description 2
- 229960004853 betadex Drugs 0.000 claims description 2
- 239000001768 carboxy methyl cellulose Substances 0.000 claims description 2
- 235000010948 carboxy methyl cellulose Nutrition 0.000 claims description 2
- 239000008112 carboxymethyl-cellulose Substances 0.000 claims description 2
- 229940105329 carboxymethylcellulose Drugs 0.000 claims description 2
- 239000004088 foaming agent Substances 0.000 claims description 2
- 150000002366 halogen compounds Chemical class 0.000 claims description 2
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- 229910052588 hydroxylapatite Inorganic materials 0.000 claims description 2
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- 229940031574 hydroxymethyl cellulose Drugs 0.000 claims description 2
- 229940071676 hydroxypropylcellulose Drugs 0.000 claims description 2
- 229940057995 liquid paraffin Drugs 0.000 claims description 2
- ZQMHJBXHRFJKOT-UHFFFAOYSA-N methyl 2-[(1-methoxy-2-methyl-1-oxopropan-2-yl)diazenyl]-2-methylpropanoate Chemical compound COC(=O)C(C)(C)N=NC(C)(C)C(=O)OC ZQMHJBXHRFJKOT-UHFFFAOYSA-N 0.000 claims description 2
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 29
- 238000006243 chemical reaction Methods 0.000 description 16
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- 238000004140 cleaning Methods 0.000 description 8
- -1 alkyl diphenyl ether Chemical compound 0.000 description 6
- 230000006872 improvement Effects 0.000 description 6
- HFQQZARZPUDIFP-UHFFFAOYSA-M sodium;2-dodecylbenzenesulfonate Chemical compound [Na+].CCCCCCCCCCCCC1=CC=CC=C1S([O-])(=O)=O HFQQZARZPUDIFP-UHFFFAOYSA-M 0.000 description 6
- 239000005289 controlled pore glass Substances 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 230000006820 DNA synthesis Effects 0.000 description 3
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 3
- 230000006819 RNA synthesis Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000004945 emulsification Methods 0.000 description 3
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 3
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- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
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- WNAHIZMDSQCWRP-UHFFFAOYSA-N dodecane-1-thiol Chemical compound CCCCCCCCCCCCS WNAHIZMDSQCWRP-UHFFFAOYSA-N 0.000 description 2
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
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- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 239000002952 polymeric resin Substances 0.000 description 2
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 2
- 238000010557 suspension polymerization reaction Methods 0.000 description 2
- 229920003002 synthetic resin Polymers 0.000 description 2
- 125000002103 4,4'-dimethoxytriphenylmethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)(C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H])C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H] 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
- 150000004996 alkyl benzenes Chemical group 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 1
- USIUVYZYUHIAEV-UHFFFAOYSA-N diphenyl ether Natural products C=1C=CC=CC=1OC1=CC=CC=C1 USIUVYZYUHIAEV-UHFFFAOYSA-N 0.000 description 1
- 150000002191 fatty alcohols Chemical class 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000010907 mechanical stirring Methods 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000001668 nucleic acid synthesis Methods 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
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- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
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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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/28—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a liquid phase from a macromolecular composition or article, e.g. drying of coagulum
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2351/00—Characterised by the use of graft polymers in which the grafted component is obtained by reactions only involving carbon-to-carbon unsaturated bonds; Derivatives of such polymers
Abstract
The invention relates to the technical field of preparation methods of polymer microspheres, and provides a preparation method of carrier initial microspheres for DNA/RNA solid phase synthesis with uniform particle size.
Description
Technical Field
The invention relates to the technical field of preparation methods of polymer microspheres, in particular to a preparation method of initial microspheres of a carrier for DNA/RNA solid phase synthesis with uniform particle size.
Background
Nucleic acid drugs are nucleotide fragments which are artificially synthesized and chemically modified, can be combined with target mRNA through self-designed specific fragments, interfere the production of pathogenic proteins at the gene level, and have become hot spots for drug research and development in recent years due to high selectivity and low side effect.
At the start of nucleotide polymerization, a molecule having a specific nucleotide sequence, which stimulates synthesis, is covalently linked to a reactant, and such a molecule is called a primer. The primers are typically synthetic oligonucleotide sequences, one complementary to one DNA/RNA template strand at one end of the target region and the other complementary to the other DNA/RNA template strand at the other end of the target region, and function as a point of initiation of nucleotide polymerization, from which 3' end a nucleic acid polymerase can begin synthesizing a new nucleic acid strand. Artificially designed primers are widely used in Polymerase Chain Reaction (PCR), sequencing, and gene probe synthesis. Nucleic acid drugs and primers are mainly synthesized by chemical methods, and in 1965, Letsinger and Mahadevan applied solid-phase synthesis methods to nucleic acid synthesis for the first time, and rapidly developed into the mainstream methods of nucleic acid. The current most commonly used DNA/RNA synthetic carrier is Controlled Pore Glass (CPG), CPG fixes a connecting arm on its surface by a siloxane bond, nucleoside 3 '-OH is covalently connected to the connecting arm on the CPG surface by succinate bond, 5' -OH is blocked by DMTr group. However, CPG as an inorganic siloxy carrier has the surface which is easy to be chemically derivatized, but the surface is rich in hydroxyl groups, so that the surface is strong in hydrophilicity, the coupling efficiency is affected and wrong sequences are generated due to the difficulty in completely removing water, and the hydroxyl groups are easy to fall off in the synthesis process after being protected, so that the synthesis purity and quality of DNA/RNA are easily affected.
The polystyrene-divinylbenzene microsphere carrier is rich in a large number of benzene rings, so that the polystyrene-divinylbenzene microsphere carrier is strong in hydrophobicity, can reduce the interference of moisture on DNA/RNA synthesis, has relatively few active sites after surface benzene ring derivatization, is not easy to generate wrong sequences in the DNA/RNA synthesis process, and is high in product purity, so that the polystyrene-divinylbenzene microsphere carrier is more suitable for the solid-phase synthesis of DNA/RNA. The Nissandong company launches a 90-micron polymer resin product with uneven particles in 2000, which is high in price and has potential risk of poor batch reproducibility; the GE company of America promoted a 30-micron polymer resin product with high price and small particle size, and the product is not easy to carry out post-treatment.
Disclosure of Invention
Therefore, in order to solve the above technical problems, the present invention provides a method for preparing carrier-initiated microspheres for DNA/RNA solid phase synthesis having a uniform particle size.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a preparation method of initial microspheres of carrier for DNA/RNA solid phase synthesis with uniform particle diameter is disclosed, which is characterized in that based on swellable seed polymer microspheres, specific functional monomer, pore-forming agent, cross-linking agent and initiator which can be polymerized are swelled into the seed microspheres, after polymerization reaction, hydrolysis or alcoholysis reaction is carried out to obtain the initial microspheres of carrier for DNA/RNA solid phase synthesis with monodisperse particle diameter, specific pore diameter and specific functionalized groups on the surface,
the monomer is M1 or M2, and the structure of M1 is
Wherein R is2Represents an aliphatic compound or an aromatic compound; the structure of the M2 is
Wherein R is1Represents a hydrogen atom, an aliphatic compound or a halogen compound.
A method for preparing initial microspheres of a carrier for DNA/RNA solid phase synthesis with uniform particle size comprises the following steps:
(1) preparation of oil phase:
mixing polymerizable monomer M1, pore-forming agent, cross-linking agent, emulsifier and ultrapure water according to the mass ratio of 5:1-5:1-3:0.02:10-20, and homogenizing and emulsifying for 2-15min to obtain an oil phase;
(2) swelling:
adding the oil phase emulsion obtained in the step (1) into seed polymer microspheres, wherein the mass ratio of the oil phase emulsion to the seed polymer microspheres is 20-64:1, the system temperature is controlled at 10-70 ℃, and the oil phase swelling material is obtained after stirring and swelling for 30-200 min;
mixing initiator, emulsifier and ultrapure water according to the mass ratio of 0.05-0.15:0.05-0.1:1-5, homogenizing and emulsifying for 2-15min, adding into the oil phase swelling matter with the addition amount of 0.8-4% of the oil phase swelling matter, and continuing swelling for 20-100 min;
(3) preparation of the aqueous phase:
adding a stabilizer into ultrapure water, heating to dissolve, and obtaining a water phase with the mass fraction of 0.8-2% after the stabilizer is completely dissolved;
(4) polymerization reaction:
adding the water phase obtained in the step (3) into the oil phase swelling matter, wherein the mass ratio of the water phase to the oil phase swelling matter is 5-10:1, uniformly stirring, heating to 60-90 ℃, and preserving heat for 6-24 hours; after the polymerization reaction is finished, cooling to room temperature, carrying out suction filtration, washing and drying to obtain polymer microspheres;
(5) hydrolysis:
adding the dried polymer microspheres into dilute acid or dilute alkali solution, heating to 60-100 ℃, and hydrolyzing for 4-10 h; and (3) carrying out suction filtration and water washing, washing until the filtrate is neutral, and drying to obtain the initial microspheres of the carrier for DNA/RNA solid phase synthesis with uniform particle size.
A method for preparing initial microspheres of a carrier for DNA/RNA solid phase synthesis with uniform particle size can also be prepared according to the following steps:
(1) preparation of oil phase:
mixing polymerizable monomer M2, pore-forming agent, cross-linking agent, emulsifier and ultrapure water according to the mass ratio of 5:1.5-5:1.2-3.6:0.05:10-18, and homogenizing and emulsifying for 2-15min to obtain an oil phase;
(2) swelling:
adding the oil phase emulsion obtained in the step (1) into seed polymer microspheres, wherein the mass ratio of the oil phase emulsion to the seed polymer microspheres is 20-64:1, the system temperature is controlled at 20-70 ℃, and the oil phase swelling material is obtained after stirring and swelling for 10-200 min;
mixing initiator, emulsifier and ultrapure water according to the mass ratio of 0.05-0.15:0.08-0.15:1-5, homogenizing and emulsifying for 2-15min, adding into the oil phase swelling matter with the addition amount of 1-3% of the oil phase swelling matter, and continuing swelling for 20-100 min;
(3) preparation of the aqueous phase:
adding a stabilizer into ultrapure water, heating to dissolve the stabilizer, and obtaining a water phase with the mass fraction of 1.5-3% after the stabilizer is completely dissolved;
(4) polymerization reaction:
adding the water phase obtained in the step (3) into the oil phase swelling matter, wherein the mass ratio of the water phase to the oil phase swelling matter is 5-10:1, uniformly stirring, heating to 55-90 ℃, and preserving heat for 6-24 hours; after the polymerization reaction is finished, cooling to room temperature, carrying out suction filtration, washing and drying to obtain polymer microspheres;
(5) alcoholysis:
adding the dried polymer microspheres into ethanolamine or ethanol, heating to 60-80 ℃, and hydrolyzing for 4-10 h; and (3) carrying out suction filtration and water washing, washing until the filtrate is neutral, and drying to obtain the initial microspheres of the carrier for DNA/RNA solid phase synthesis with uniform particle size.
The further improvement is that: the pore-foaming agent is a mixture consisting of one or more than two of toluene, isobutanol, hexanol, cyclohexanol, n-heptane, liquid paraffin and dodecanol.
The further improvement is that: the crosslinking agent is divinylbenzene.
The further improvement is that: the emulsifier is one or a mixture of ionic emulsifier and non-ionic emulsifier in any ratio. The ionic emulsifier is alkylbenzene sulfonate, alkyl succinate sulfonate or alkyl diphenyl ether sulfonate. The non-ionic emulsifier is alkylphenol polyoxyethylene, benzyl phenol polyoxyethylene, phenethyl phenol polyoxyethylene, fatty alcohol polyoxyethylene or fatty amine polyoxyethylene.
The further improvement is that: the initiator is any one of benzoyl peroxide, azobisisobutyronitrile, dimethyl azobisisobutyrate and azobisisobutylamidine.
The further improvement is that: the stabilizer is prepared by mixing any one or two of polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, beta-cyclodextrin, beta-methyl cyclodextrin and hydroxyapatite in any ratio.
The further improvement is that: the particle size of the microsphere is 80-100 μm; the pore diameter of the microspheres is
By adopting the technical scheme, the invention has the beneficial effects that:
the invention is based on swellable seed polymer microspheres, a polymerizable specific functional monomer, a pore-forming agent, a cross-linking agent and an initiator are swelled into the seed microspheres, and hydrolysis or alcoholysis reaction is carried out after polymerization reaction to prepare the uniform particle carrier which contains a large number of hydrophobic groups and a special functional group on the surface and can be used for initial microspheres for DNA/RNA solid phase synthesis. The synthesized uniform particle carrier of the microspheres for DNA/RNA solid phase synthesis has the advantages of uniform and controllable particle size, small coefficient of variation of particle size (less than 3.0%), controllable pore size, high carrying capacity, good universality, strong stability and the like.
The synthetic method can directly utilize the conventional industrial conventional equipment to carry out large-scale and batch production, and has strong batch reproducibility.
Drawings
FIG. 1 is an SEM photograph of carrier-initiated microspheres for solid-phase DNA/RNA synthesis prepared in the first example;
FIG. 2 is a graph showing the particle size of carrier-initiated microspheres for DNA/RNA solid phase synthesis prepared in example one;
FIG. 3 is a graph showing the pore size of carrier-initiated microspheres for DNA/RNA solid phase synthesis prepared in example one (mercury intrusion method).
Detailed Description
The following detailed description will be provided for the embodiments of the present invention with reference to specific embodiments, so that how to apply the technical means to solve the technical problems and achieve the technical effects can be fully understood and implemented.
Unless otherwise indicated, the techniques employed in the examples are conventional and well known to those skilled in the art, and the reagents and products employed are also commercially available. The source, trade name and if necessary the constituents of the reagents used are indicated at the first appearance.
The seed polymer microspheres are polystyrene microspheres, are prepared by a suspension polymerization method or a swelling method, the preparation method is determined according to the particle size of the needed seed polymer microspheres, and suspension polymerization is adopted when the particle size of the seed polymer microspheres is less than 5 mu m; when the particle size of the seed polymer microsphere is more than 5 mu m, a swelling method is adopted.
Suspension polymerization process
160mL of ethanol and 20mL of ultrapure water are weighed and added into a 500mL round-bottom flask, a reflux condenser tube, mechanical stirring, a nitrogen ventilating duct and a thermometer are arranged on a reaction bottle, 2.0g of dispersant PVP and 1.0g of auxiliary dispersant dodecanol are added, stirring and mixing are carried out uniformly, nitrogen is introduced into the flask, and the system is heated to 60 ℃; weighing 1.0g of dodecanethiol and 0.14g of azobisisobutyronitrile, dissolving in 20g of styrene, adding into the reaction flask, keeping the temperature for reaction for 20 hours to obtain polystyrene microspheres, and after cleaning, testing the molecular weight Mn of the seeds to be 3284 and the particle size to be 4.2 mu m.
Swelling seed preparation
Weighing 2.5g of polystyrene microspheres with the particle size of 4.2 mu m prepared by the method into a 250mL round-bottom flask; weighing 0.11g of azobisisobutyronitrile, 30g of styrene, 0.4g of dodecanethiol, 0.1g of SDS and 20mL of ultrapure water into a 100mL beaker, emulsifying to obtain an emulsion with the particle size of about 10 micrometers, adding the emulsion into the flask, heating to 60 ℃, keeping the temperature for reaction for 3 hours, adding 0.5g of PEG4000/40g of ultrapure water solution, reacting for 20 hours at 80 ℃ to obtain polystyrene microspheres, and washing to obtain the polystyrene microspheres, wherein the molecular weight Mn of the seeds is 10448 and the particle size is 28.8 micrometers.
Example one
The preparation process of initial microsphere as carrier for solid phase synthesis of DNA/RNA in homogeneous particle size includes the following steps:
mixing polymerizable monomer M1, isobutanol, divinyl benzene, SDS and ultrapure water according to the mass ratio of 5:1:1:0.02:10, and performing ultrasonic emulsification for 5min to obtain an oil phase emulsion;
wherein the knot of M1Is constructed as
R2is-CH2CH3；
Adding the oil phase emulsion into a reactor containing polystyrene microspheres (with the particle size of 28.8 microns), wherein the mass ratio of the oil phase emulsion to the polystyrene microspheres is 40:1, and stirring and swelling the oil phase emulsion for 200min at 10 ℃ to obtain an oil phase swelling matter; mixing azodiisobutyronitrile, SDS and ultrapure water according to the mass ratio of 0.05:0.05:1, ultrasonically emulsifying for 5min, adding into the oil phase swelling matter with the addition amount of 0.8% of the oil phase swelling matter, and continuing swelling for 20 min;
dissolving PVA in ultrapure water to prepare a water phase with the mass fraction of 0.8%, adding the water phase and an oil phase swelling material into the reactor, wherein the mass ratio of the water phase to the oil phase swelling material is 5:1, uniformly stirring and mixing, heating to 60 ℃, carrying out polymerization reaction for 24 hours, cooling to room temperature after the reaction is finished, carrying out suction filtration on a sand core, alternately cleaning with ethanol and ultrapure water for 3 times, and drying to obtain polymer microspheres;
adding the polymer microspheres into a NaOH solution with the concentration of 0.1mol/L, heating to 60 ℃, and hydrolyzing for 10 h; after the reaction is finished, carrying out suction filtration on a sand core, washing with ultrapure water until filtrate is neutral, and drying to obtain the initial microspheres of the carrier for DNA/RNA solid phase synthesis with uniform particle size, wherein the particle size of the microspheres is 90 mu m (C.V is 2.1%), and the pore diameter of the microspheres is
Example two
The preparation process of initial microsphere as carrier for solid phase synthesis of DNA/RNA in homogeneous particle size includes the following steps:
mixing polymerizable monomer M1, isobutanol, cyclohexanol, divinylbenzene, SDS and ultrapure water according to the mass ratio of 5:0.8:1.5:2:0.02:15, and performing ultrasonic emulsification for 10min to obtain an oil phase emulsion;
wherein M1 has the structure
R2is-CH2CH3；
Adding the oil phase emulsion into a reactor containing polystyrene microspheres (with the particle size of 28.8 microns), wherein the mass ratio of the oil phase emulsion to the polystyrene microspheres is 20:1, and stirring and swelling the oil phase emulsion for 100min at 40 ℃ to obtain an oil phase swelling matter; mixing benzoyl peroxide, SDS and ultrapure water according to the mass ratio of 0.1:0.07:3, ultrasonically emulsifying for 10min, adding into the oil phase swelling matter, adding 2.4% of the oil phase swelling matter, and continuing swelling for 60 min;
dissolving PVA in ultrapure water to prepare a water phase with the mass fraction of 1.5%, adding the water phase and an oil phase swelling material into the reactor, wherein the mass ratio of the water phase to the oil phase swelling material is 8:1, uniformly stirring and mixing, heating to 75 ℃, carrying out polymerization reaction for 15h, cooling to room temperature after the reaction is finished, carrying out suction filtration on a sand core, alternately cleaning with ethanol and ultrapure water for 3 times, and drying to obtain polymer microspheres;
adding the polymer microspheres into a NaOH solution with the concentration of 0.1mol/L, heating to 80 ℃, and hydrolyzing for 7 h; after the reaction is finished, carrying out suction filtration on a sand core, washing with ultrapure water until filtrate is neutral, and drying to obtain the initial microspheres of the carrier for DNA/RNA solid phase synthesis with uniform particle size, wherein the particle size of the microspheres is 95 mu m (C.V is 2.8%), and the pore diameter of the microspheres is
EXAMPLE III
The preparation process of initial microsphere as carrier for solid phase synthesis of DNA/RNA in homogeneous particle size includes the following steps:
mixing polymerizable monomer M1, cyclohexanol, n-heptane, divinylbenzene, Tritro X-405 and ultrapure water according to the mass ratio of 5:5:3:0.02:20, and performing ultrasonic emulsification for 15min to obtain an oil phase emulsion;
wherein M1 has the structure
R2is-CH2CH3；
Adding the oil phase emulsion into a reactor containing polystyrene microspheres (with the particle size of 28.8 microns), wherein the mass ratio of the oil phase emulsion to the seed polymer microspheres is 64:1, and stirring and swelling the oil phase emulsion at 70 ℃ for 30min to obtain an oil phase swelling matter; mixing benzoyl peroxide, Tritro X-405 and ultrapure water according to the mass ratio of 0.15:0.1:5, ultrasonically emulsifying for 15min, adding into the oil phase swelling matter with the addition amount of 4% of the oil phase swelling matter, and continuing swelling for 100 min;
dissolving hydroxypropyl cellulose in ultrapure water to prepare a water phase with the mass fraction of 2%, adding the water phase and an oil phase swelling material into the reactor, wherein the mass ratio of the water phase to the oil phase swelling material is 10:1, uniformly stirring and mixing, heating to 90 ℃, carrying out polymerization reaction for 6 hours, cooling to room temperature after the reaction is finished, carrying out suction filtration on a sand core, alternately cleaning with ethanol and ultrapure water for 3 times, and drying to obtain polymer microspheres;
adding the polymer microspheres into a NaOH solution with the concentration of 0.1mol/L, heating to 100 ℃, and hydrolyzing for 4 h; after the reaction is finished, carrying out suction filtration on a sand core, washing with ultrapure water until filtrate is neutral, and drying to obtain the initial microspheres of the carrier for DNA/RNA solid phase synthesis with uniform particle size, wherein the particle size of the microspheres is 100 mu m (C.V is 2.9%), and the pore diameter of the microspheres is 100 mu m
Example four
The preparation process of initial microsphere as carrier for solid phase synthesis of DNA/RNA in homogeneous particle size includes the following steps:
mixing polymerizable monomer M2, n-dodecanol, divinylbenzene, SDBS and ultrapure water according to the mass ratio of 5:1.5:1.2:0.05:10, and homogenizing and emulsifying for 5min to obtain an oil phase emulsion;
wherein the monomer M2 has the structure
R1Represents a hydrogen atom;
adding the oil phase emulsion into a reactor containing polystyrene microspheres (with the particle size of 28.8 microns), wherein the mass ratio of the oil phase emulsion to the seed polymer microspheres is 40:1, and stirring and swelling the oil phase emulsion and the seed polymer microspheres for 200min at 20 ℃ to obtain an oil phase swelling matter; mixing azodiisobutyronitrile, SDBS and ultrapure water according to the mass ratio of 0.05:0.08:1, homogenizing and emulsifying for 5min, adding into the oil phase swelling matter with the addition amount of 1% of the oil phase swelling matter, and continuing swelling for 20 min;
dissolving PVP in ultrapure water to prepare a water phase with the mass fraction of 1.5%, adding the water phase and an oil phase swelling material into the reactor, uniformly stirring, heating to 55 ℃, preserving heat for 24 hours, cooling to room temperature after the reaction is finished, performing suction filtration on a sand core, alternately cleaning ethanol and ultrapure water for 3 times, and drying to obtain polymer microspheres;
adding the polymer microspheres into an ethanolamine solution, heating to 60 ℃, and carrying out alcoholysis reaction for 10 hours; after the reaction is finished, carrying out suction filtration on a sand core, washing with ultrapure water until filtrate is neutral, and drying to obtain the initial microspheres of the carrier for DNA/RNA solid phase synthesis with uniform particle size, wherein the particle size of the microspheres is 90.5 mu m (C.V is 2.6%), and the pore diameter of the microspheres is uniform
EXAMPLE five
The preparation process of initial microsphere as carrier for solid phase synthesis of DNA/RNA in homogeneous particle size includes the following steps:
mixing polymerizable monomer M2, n-dodecanol, divinylbenzene, SDBS and ultrapure water according to the mass ratio of 5:3:2.4:0.05:15, and homogenizing and emulsifying for 10min to obtain an oil phase emulsion;
wherein the monomer M2 has the structure
R1Represents a hydrogen atom;
adding the oil phase emulsion into a reactor containing polystyrene microspheres (with the particle size of 28.8 microns), wherein the mass ratio of the oil phase emulsion to the seed polymer microspheres is 20:1, and stirring and swelling the oil phase emulsion at the temperature of 45 ℃ for 100min to obtain an oil phase swelling matter; mixing azodiisobutyronitrile, SDBS and ultrapure water according to the mass ratio of 0.1:0.12:3, homogenizing and emulsifying for 10min, adding into the oil phase swelling matter with the addition amount of 2% of the oil phase swelling matter, and continuing swelling for 60 min;
dissolving PVP in ultrapure water to prepare a water phase with the mass fraction of 2.5%, adding the water phase and an oil phase swelling material into the reactor at the mass ratio of 8:1, uniformly stirring, heating to 70 ℃, preserving heat for 15h, cooling to room temperature after the reaction is finished, performing suction filtration on a sand core, alternately cleaning ethanol and ultrapure water for 3 times, and drying to obtain polymer microspheres;
adding the polymer microspheres into an ethanolamine solution, heating to 70 ℃, and carrying out alcoholysis reaction for 7 hours; after the reaction is finished, carrying out suction filtration on a sand core, washing with ultrapure water until filtrate is neutral, and drying to obtain the initial microspheres of the carrier for DNA/RNA solid phase synthesis with uniform particle size, wherein the particle size of the microspheres is 92.1 mu m (C.V is 2.8%), and the pore diameter of the microspheres is
EXAMPLE six
The preparation process of initial microsphere as carrier for solid phase synthesis of DNA/RNA in homogeneous particle size includes the following steps:
mixing polymerizable monomer M2, n-dodecanol, divinylbenzene, SDBS and ultrapure water according to the mass ratio of 5:5:3.6:0.05:18, and homogenizing and emulsifying for 15min to obtain an oil phase emulsion;
wherein the monomer M2 has the structure
R1Represents a hydrogen atom;
adding the oil phase emulsion into a reactor containing polystyrene microspheres (with the particle size of 28.8 microns), wherein the mass ratio of the oil phase emulsion to the seed polymer microspheres is 64:1, and stirring and swelling the oil phase emulsion at 70 ℃ for 10min to obtain an oil phase swelling matter; mixing azodiisobutyronitrile, SDBS and ultrapure water according to the mass ratio of 0.15:0.15:5, homogenizing and emulsifying for 15min, adding into the oil phase swelling matter with the addition amount of 3% of the oil phase swelling matter, and continuing swelling for 100 min;
dissolving PVP in ultrapure water to prepare a water phase with the mass fraction of 3%, adding the water phase and an oil phase swelling material into the reactor at the mass ratio of 10:1, uniformly stirring, heating to 90 ℃, preserving heat for 6 hours, cooling to room temperature after the reaction is finished, performing suction filtration on a sand core, alternately cleaning ethanol and ultrapure water for 3 times, and drying to obtain polymer microspheres;
adding the polymer microspheres into an ethanolamine solution, heating to 80 ℃, and carrying out alcoholysis reaction for 4 hours; after the reaction is finished, carrying out suction filtration and ultrapure water cleaning on the sand coreDrying until the filtrate is neutral to obtain initial microspheres with uniform particle size of 98.6 μm (C.V ═ 2.9%) and pore diameter
The above description is only an embodiment utilizing the technical content of the present disclosure, and any modification and variation made by those skilled in the art can be covered by the claims of the present disclosure, and not limited to the embodiments disclosed.
Claims (9)
1. A method for preparing carrier initial microspheres for DNA/RNA solid phase synthesis with uniform particle size is characterized in that: based on the swellable seed polymer microsphere, a polymerizable specific functional monomer, a pore-forming agent, a cross-linking agent and an initiator are swelled into the seed microsphere, and then hydrolysis or alcoholysis reaction is carried out after polymerization reaction to obtain the initial microsphere of the carrier for DNA/RNA solid phase synthesis, which has monodisperse particle size, specific pore diameter and specific functionalized groups on the surface,
the monomer is M1 or M2, and the structure of M1 is
Wherein R is2Represents an aliphatic compound or an aromatic compound; the structure of the M2 is
Wherein R is1Represents a hydrogen atom, an aliphatic compound or a halogen compound.
2. The method for preparing carrier-initiated microspheres for DNA/RNA solid phase synthesis with uniform particle size according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:
(1) preparation of oil phase:
mixing polymerizable monomer M1, pore-forming agent, cross-linking agent, emulsifier and ultrapure water according to the mass ratio of 5:1-5:1-3:0.02:10-20, and homogenizing and emulsifying for 2-15min to obtain an oil phase;
(2) swelling:
adding the oil phase emulsion obtained in the step (1) into seed polymer microspheres, wherein the mass ratio of the oil phase emulsion to the seed polymer microspheres is 20-64:1, the system temperature is controlled at 10-70 ℃, and the oil phase swelling material is obtained after stirring and swelling for 30-200 min;
mixing initiator, emulsifier and ultrapure water according to the mass ratio of 0.05-0.15:0.05-0.1:1-5, homogenizing and emulsifying for 2-15min, adding into the oil phase swelling matter with the addition amount of 0.8-4% of the oil phase swelling matter, and continuing swelling for 20-100 min;
(3) preparation of the aqueous phase:
adding a stabilizer into ultrapure water, heating to dissolve, and obtaining a water phase with the mass fraction of 0.8-2% after the stabilizer is completely dissolved;
(4) polymerization reaction:
adding the water phase obtained in the step (3) into the oil phase swelling matter, wherein the mass ratio of the water phase to the oil phase swelling matter is 5-10:1, uniformly stirring, heating to 60-90 ℃, and preserving heat for 6-24 hours; after the polymerization reaction is finished, cooling to room temperature, carrying out suction filtration, washing and drying to obtain polymer microspheres;
(5) hydrolysis:
adding the dried polymer microspheres into dilute acid or dilute alkali solution, heating to 60-100 ℃, and hydrolyzing for 4-10 h; and (3) carrying out suction filtration and water washing, washing until the filtrate is neutral, and drying to obtain the initial microspheres of the carrier for DNA/RNA solid phase synthesis with uniform particle size.
3. The method for preparing carrier-initiated microspheres for DNA/RNA solid phase synthesis with uniform particle size according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:
(1) preparation of oil phase:
mixing polymerizable monomer M2, pore-forming agent, cross-linking agent, emulsifier and ultrapure water according to the mass ratio of 5:1.5-5:1.2-3.6:0.05:10-18, and homogenizing and emulsifying for 2-15min to obtain an oil phase;
(2) swelling:
adding the oil phase emulsion obtained in the step (1) into seed polymer microspheres, wherein the mass ratio of the oil phase emulsion to the seed polymer microspheres is 20-64:1, the system temperature is controlled at 20-70 ℃, and the oil phase swelling material is obtained after stirring and swelling for 10-200 min;
mixing initiator, emulsifier and ultrapure water according to the mass ratio of 0.05-0.15:0.08-0.15:1-5, homogenizing and emulsifying for 2-15min, adding into the oil phase swelling matter with the addition amount of 1-3% of the oil phase swelling matter, and continuing swelling for 20-100 min;
(3) preparation of the aqueous phase:
adding a stabilizer into ultrapure water, heating to dissolve the stabilizer, and obtaining a water phase with the mass fraction of 1.5-3% after the stabilizer is completely dissolved;
(4) polymerization reaction:
adding the water phase obtained in the step (3) into the oil phase swelling matter, wherein the mass ratio of the water phase to the oil phase swelling matter is 5-10:1, uniformly stirring, heating to 55-90 ℃, and preserving heat for 6-24 hours; after the polymerization reaction is finished, cooling to room temperature, carrying out suction filtration, washing and drying to obtain polymer microspheres;
(5) alcoholysis:
adding the dried polymer microspheres into ethanolamine or ethanol, heating to 60-80 ℃, and hydrolyzing for 4-10 h; and (3) carrying out suction filtration and water washing, washing until the filtrate is neutral, and drying to obtain the initial microspheres of the carrier for DNA/RNA solid phase synthesis with uniform particle size.
4. The method for preparing carrier-initiated microspheres for uniform-particle-size DNA/RNA solid-phase synthesis according to claim 2 or 3, wherein: the pore-foaming agent is a mixture consisting of one or more than two of toluene, isobutanol, hexanol, cyclohexanol, n-heptane, liquid paraffin and dodecanol.
5. The method for preparing carrier-initiated microspheres for uniform-particle-size DNA/RNA solid-phase synthesis according to claim 2 or 3, wherein: the crosslinking agent is divinylbenzene.
6. The method for preparing carrier-initiated microspheres for uniform-particle-size DNA/RNA solid-phase synthesis according to claim 2 or 3, wherein: the emulsifier is one or a mixture of ionic emulsifier and non-ionic emulsifier in any ratio.
7. The method for preparing carrier-initiated microspheres for uniform-particle-size DNA/RNA solid-phase synthesis according to claim 2 or 3, wherein: the initiator is any one of benzoyl peroxide, azobisisobutyronitrile, dimethyl azobisisobutyrate and azobisisobutylamidine.
8. The method for preparing carrier-initiated microspheres for uniform-particle-size DNA/RNA solid-phase synthesis according to claim 2 or 3, wherein: the stabilizer is prepared by mixing any one or two of polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, beta-cyclodextrin, beta-methyl cyclodextrin and hydroxyapatite in any ratio.
9. The method for preparing carrier-initiated microspheres for uniform-particle-size DNA/RNA solid-phase synthesis according to claim 2 or 3, wherein: the particle size of the microsphere is 80-100 μm; the aperture of the microsphere is 500-
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