JP4945959B2 - Thermally responsive magnetic fine particles, production method thereof, and adsorbent using the fine particles - Google Patents
Thermally responsive magnetic fine particles, production method thereof, and adsorbent using the fine particles Download PDFInfo
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- JP4945959B2 JP4945959B2 JP2005240999A JP2005240999A JP4945959B2 JP 4945959 B2 JP4945959 B2 JP 4945959B2 JP 2005240999 A JP2005240999 A JP 2005240999A JP 2005240999 A JP2005240999 A JP 2005240999A JP 4945959 B2 JP4945959 B2 JP 4945959B2
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- fine particles
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- 239000010419 fine particle Substances 0.000 title claims description 103
- 239000003463 adsorbent Substances 0.000 title claims description 30
- 238000004519 manufacturing process Methods 0.000 title claims description 5
- 229920000642 polymer Polymers 0.000 claims description 54
- 239000000178 monomer Substances 0.000 claims description 32
- 229920000208 temperature-responsive polymer Polymers 0.000 claims description 29
- -1 trimethylene amide Chemical class 0.000 claims description 28
- 239000011557 critical solution Substances 0.000 claims description 27
- 239000007864 aqueous solution Substances 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 22
- 239000013076 target substance Substances 0.000 claims description 20
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 16
- 125000000524 functional group Chemical group 0.000 claims description 16
- 239000011259 mixed solution Substances 0.000 claims description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 229920001577 copolymer Polymers 0.000 claims description 15
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims description 14
- QNILTEGFHQSKFF-UHFFFAOYSA-N n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C=C QNILTEGFHQSKFF-UHFFFAOYSA-N 0.000 claims description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 12
- 229910052757 nitrogen Inorganic materials 0.000 claims description 11
- 238000010526 radical polymerization reaction Methods 0.000 claims description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- 239000002245 particle Substances 0.000 claims description 10
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 claims description 10
- 229920001400 block copolymer Polymers 0.000 claims description 9
- 230000001747 exhibiting effect Effects 0.000 claims description 9
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N N-methylaminoacetic acid Natural products C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 claims description 8
- 108010077895 Sarcosine Proteins 0.000 claims description 8
- 239000011616 biotin Substances 0.000 claims description 8
- 229960002685 biotin Drugs 0.000 claims description 8
- 235000020958 biotin Nutrition 0.000 claims description 8
- 229920002189 poly(glycerol 1-O-monomethacrylate) polymer Polymers 0.000 claims description 8
- 229940043230 sarcosine Drugs 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- QRIMLDXJAPZHJE-UHFFFAOYSA-N 2,3-dihydroxypropyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCC(O)CO QRIMLDXJAPZHJE-UHFFFAOYSA-N 0.000 claims description 7
- 108090001008 Avidin Proteins 0.000 claims description 7
- 108010024636 Glutathione Proteins 0.000 claims description 7
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 7
- 238000007334 copolymerization reaction Methods 0.000 claims description 7
- 229960002089 ferrous chloride Drugs 0.000 claims description 7
- 229960003180 glutathione Drugs 0.000 claims description 7
- 150000004687 hexahydrates Chemical class 0.000 claims description 7
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 7
- 239000003505 polymerization initiator Substances 0.000 claims description 7
- 238000006116 polymerization reaction Methods 0.000 claims description 7
- 239000000126 substance Substances 0.000 claims description 7
- 150000004685 tetrahydrates Chemical class 0.000 claims description 7
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 claims description 6
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims description 5
- RGIKRHKHRAAZIO-CIUDSAMLSA-N (3as,4s,6ar)-4-(5-hydroxypentyl)-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-2-one Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCCO)SC[C@@H]21 RGIKRHKHRAAZIO-CIUDSAMLSA-N 0.000 claims description 4
- XLPJNCYCZORXHG-UHFFFAOYSA-N 1-morpholin-4-ylprop-2-en-1-one Chemical compound C=CC(=O)N1CCOCC1 XLPJNCYCZORXHG-UHFFFAOYSA-N 0.000 claims description 4
- RESPXSHDJQUNTN-UHFFFAOYSA-N 1-piperidin-1-ylprop-2-en-1-one Chemical compound C=CC(=O)N1CCCCC1 RESPXSHDJQUNTN-UHFFFAOYSA-N 0.000 claims description 4
- WLPAQAXAZQUXBG-UHFFFAOYSA-N 1-pyrrolidin-1-ylprop-2-en-1-one Chemical compound C=CC(=O)N1CCCC1 WLPAQAXAZQUXBG-UHFFFAOYSA-N 0.000 claims description 4
- AKVUWTYSNLGBJY-UHFFFAOYSA-N 2-methyl-1-morpholin-4-ylprop-2-en-1-one Chemical compound CC(=C)C(=O)N1CCOCC1 AKVUWTYSNLGBJY-UHFFFAOYSA-N 0.000 claims description 4
- RASDUGQQSMMINZ-UHFFFAOYSA-N 2-methyl-1-piperidin-1-ylprop-2-en-1-one Chemical compound CC(=C)C(=O)N1CCCCC1 RASDUGQQSMMINZ-UHFFFAOYSA-N 0.000 claims description 4
- LVCMKNCJDCTPIB-UHFFFAOYSA-N 2-methyl-1-pyrrolidin-1-ylprop-2-en-1-one Chemical compound CC(=C)C(=O)N1CCCC1 LVCMKNCJDCTPIB-UHFFFAOYSA-N 0.000 claims description 4
- YQIGLEFUZMIVHU-UHFFFAOYSA-N 2-methyl-n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C(C)=C YQIGLEFUZMIVHU-UHFFFAOYSA-N 0.000 claims description 4
- LPSFJOYQZGJDFZ-UHFFFAOYSA-N 2-methylidene-3-oxobutanamide Chemical compound CC(=O)C(=C)C(N)=O LPSFJOYQZGJDFZ-UHFFFAOYSA-N 0.000 claims description 4
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 claims description 4
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 4
- QRWZCJXEAOZAAW-UHFFFAOYSA-N n,n,2-trimethylprop-2-enamide Chemical compound CN(C)C(=O)C(C)=C QRWZCJXEAOZAAW-UHFFFAOYSA-N 0.000 claims description 4
- ZIWDVJPPVMGJGR-UHFFFAOYSA-N n-ethyl-2-methylprop-2-enamide Chemical compound CCNC(=O)C(C)=C ZIWDVJPPVMGJGR-UHFFFAOYSA-N 0.000 claims description 4
- SWPMNMYLORDLJE-UHFFFAOYSA-N n-ethylprop-2-enamide Chemical compound CCNC(=O)C=C SWPMNMYLORDLJE-UHFFFAOYSA-N 0.000 claims description 4
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 4
- IVBZNGISIGLAAT-UHFFFAOYSA-N 5-(2-oxobut-3-enyl)-1H-pyrimidine-2,4-dione Chemical compound C(=O)(C=C)CC=1C(NC(NC=1)=O)=O IVBZNGISIGLAAT-UHFFFAOYSA-N 0.000 claims description 3
- 102000004856 Lectins Human genes 0.000 claims description 3
- 108090001090 Lectins Proteins 0.000 claims description 3
- 229920002845 Poly(methacrylic acid) Polymers 0.000 claims description 3
- 229920002125 Sokalan® Polymers 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 239000002523 lectin Substances 0.000 claims description 3
- 229940088644 n,n-dimethylacrylamide Drugs 0.000 claims description 3
- YLGYACDQVQQZSW-UHFFFAOYSA-N n,n-dimethylprop-2-enamide Chemical compound CN(C)C(=O)C=C YLGYACDQVQQZSW-UHFFFAOYSA-N 0.000 claims description 3
- 230000003472 neutralizing effect Effects 0.000 claims description 3
- 239000004584 polyacrylic acid Substances 0.000 claims description 3
- 229920000137 polyphosphoric acid Polymers 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 2
- 229920000223 polyglycerol Polymers 0.000 claims description 2
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 claims 2
- CCIDRBFZPRURMU-UHFFFAOYSA-N 2-methyl-n-propylprop-2-enamide Chemical compound CCCNC(=O)C(C)=C CCIDRBFZPRURMU-UHFFFAOYSA-N 0.000 claims 1
- BVOCPVIXARZNQN-UHFFFAOYSA-N nipecotamide Chemical compound NC(=O)C1CCCNC1 BVOCPVIXARZNQN-UHFFFAOYSA-N 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 description 18
- 108090000623 proteins and genes Proteins 0.000 description 18
- 239000000047 product Substances 0.000 description 8
- 239000000725 suspension Substances 0.000 description 7
- 230000004044 response Effects 0.000 description 6
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 5
- 230000002776 aggregation Effects 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 229940032296 ferric chloride Drugs 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- ZUVVLBGWTRIOFH-UHFFFAOYSA-N methyl 4-methyl-2-[(4-methylphenyl)sulfonylamino]pentanoate Chemical compound COC(=O)C(CC(C)C)NS(=O)(=O)C1=CC=C(C)C=C1 ZUVVLBGWTRIOFH-UHFFFAOYSA-N 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 238000004220 aggregation Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 230000004043 responsiveness Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 229920002307 Dextran Polymers 0.000 description 3
- 150000001252 acrylic acid derivatives Chemical class 0.000 description 3
- 229920001519 homopolymer Polymers 0.000 description 3
- 229910021645 metal ion Inorganic materials 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 150000005846 sugar alcohols Polymers 0.000 description 3
- URBHUGQFAFSSRQ-UHFFFAOYSA-N 1-prop-2-enoylpiperidine-3-carboxamide Chemical compound NC(=O)C1CCCN(C(=O)C=C)C1 URBHUGQFAFSSRQ-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 108010070675 Glutathione transferase Proteins 0.000 description 2
- 102000005720 Glutathione transferase Human genes 0.000 description 2
- 229910000831 Steel Inorganic materials 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000000919 ceramic Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000011246 composite particle Substances 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- QBEGYEWDTSUVHH-UHFFFAOYSA-P diazanium;cerium(3+);pentanitrate Chemical compound [NH4+].[NH4+].[Ce+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O QBEGYEWDTSUVHH-UHFFFAOYSA-P 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 238000010559 graft polymerization reaction Methods 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000003100 immobilizing effect Effects 0.000 description 2
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000010959 steel Substances 0.000 description 2
- FFJCNSLCJOQHKM-CLFAGFIQSA-N (z)-1-[(z)-octadec-9-enoxy]octadec-9-ene Chemical compound CCCCCCCC\C=C/CCCCCCCCOCCCCCCCC\C=C/CCCCCCCC FFJCNSLCJOQHKM-CLFAGFIQSA-N 0.000 description 1
- OEKNWSKXAFSTPN-UHFFFAOYSA-N 5-methyl-6-prop-2-enoyl-1H-pyrimidine-2,4-dione Chemical compound C(=O)(C=C)C1=C(C(NC(N1)=O)=O)C OEKNWSKXAFSTPN-UHFFFAOYSA-N 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 229910000975 Carbon steel Inorganic materials 0.000 description 1
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 229920001214 Polysorbate 60 Polymers 0.000 description 1
- 235000002597 Solanum melongena Nutrition 0.000 description 1
- LWZFANDGMFTDAV-BURFUSLBSA-N [(2r)-2-[(2r,3r,4s)-3,4-dihydroxyoxolan-2-yl]-2-hydroxyethyl] dodecanoate Chemical compound CCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O LWZFANDGMFTDAV-BURFUSLBSA-N 0.000 description 1
- 150000003926 acrylamides Chemical class 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 230000000274 adsorptive effect Effects 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 150000003973 alkyl amines Chemical class 0.000 description 1
- 150000005215 alkyl ethers Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 108091006004 biotinylated proteins Proteins 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 238000012661 block copolymerization Methods 0.000 description 1
- 239000010962 carbon steel Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- QRXDDLFGCDQOTA-UHFFFAOYSA-N cobalt(2+) iron(2+) oxygen(2-) Chemical compound [O-2].[Fe+2].[Co+2].[O-2] QRXDDLFGCDQOTA-UHFFFAOYSA-N 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000000498 cooling water Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 229920000359 diblock copolymer Polymers 0.000 description 1
- 125000003700 epoxy group Chemical group 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- 229940044631 ferric chloride hexahydrate Drugs 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229910052595 hematite Inorganic materials 0.000 description 1
- 239000011019 hematite Substances 0.000 description 1
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 1
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 1
- 229940027941 immunoglobulin g Drugs 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- WSSMOXHYUFMBLS-UHFFFAOYSA-L iron dichloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Fe+2] WSSMOXHYUFMBLS-UHFFFAOYSA-L 0.000 description 1
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 description 1
- LIKBJVNGSGBSGK-UHFFFAOYSA-N iron(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[Fe+3].[Fe+3] LIKBJVNGSGBSGK-UHFFFAOYSA-N 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 125000005641 methacryl group Chemical group 0.000 description 1
- WDWDWGRYHDPSDS-UHFFFAOYSA-N methanimine Chemical compound N=C WDWDWGRYHDPSDS-UHFFFAOYSA-N 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 229910000480 nickel oxide Inorganic materials 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 229920002114 octoxynol-9 Polymers 0.000 description 1
- AJCDFVKYMIUXCR-UHFFFAOYSA-N oxobarium;oxo(oxoferriooxy)iron Chemical compound [Ba]=O.O=[Fe]O[Fe]=O.O=[Fe]O[Fe]=O.O=[Fe]O[Fe]=O.O=[Fe]O[Fe]=O.O=[Fe]O[Fe]=O.O=[Fe]O[Fe]=O AJCDFVKYMIUXCR-UHFFFAOYSA-N 0.000 description 1
- GNRSAWUEBMWBQH-UHFFFAOYSA-N oxonickel Chemical compound [Ni]=O GNRSAWUEBMWBQH-UHFFFAOYSA-N 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 229920002432 poly(vinyl methyl ether) polymer Polymers 0.000 description 1
- 229920000259 polyoxyethylene lauryl ether Polymers 0.000 description 1
- 229920002503 polyoxyethylene-polyoxypropylene Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 229910052761 rare earth metal Inorganic materials 0.000 description 1
- 150000002910 rare earth metals Chemical class 0.000 description 1
- FDRCDNZGSXJAFP-UHFFFAOYSA-M sodium chloroacetate Chemical compound [Na+].[O-]C(=O)CCl FDRCDNZGSXJAFP-UHFFFAOYSA-M 0.000 description 1
- 229950006451 sorbitan laurate Drugs 0.000 description 1
- 235000011067 sorbitan monolaureate Nutrition 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
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- 239000006228 supernatant Substances 0.000 description 1
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- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 description 1
- 229910052721 tungsten Inorganic materials 0.000 description 1
- 239000010937 tungsten Substances 0.000 description 1
- 229910000859 α-Fe Inorganic materials 0.000 description 1
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- Compounds Of Iron (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Peptides Or Proteins (AREA)
- Graft Or Block Polymers (AREA)
Description
本発明は、熱応答性磁性微粒子、その製造方法及び該微粒子を用いた吸着材に関する。 The present invention relates to a thermoresponsive magnetic fine particle, a method for producing the same, and an adsorbent using the fine particle.
リガンドが固定された微粒子を混合液に添加し、目的物質を吸着した後、微粒子を回収し、目的物質を微粒子から分離、回収する方法が知られている。具体的には、ビオチン及びアビジンから選ばれた1種以上が下限臨界溶液温度(以下、「LCST」と略す。)を有するポリマーを介して磁性微粒子に固定された熱応答性磁性微粒子と磁石の磁力を用いた生体物質の分離方法が開発されている(例えば、特許文献1参照)。しかし、この方法で用いられているポリマーは、単に磁性微粒子上に巻き付いているだけで固着されておらず、目的物質の分離回収前後に磁性微粒子からポリマーが剥がれることが多く、効率のよい目的物質の回収ができていなかった。また、熱等の刺激応答により磁性微粒子の凝集分散を繰り返すと、次第に磁性微粒子同士の2次凝集が生じ、粒子の分散性が損なわれる場合があった。 There is known a method in which fine particles having a ligand immobilized are added to a mixed solution, the target substance is adsorbed, the fine particles are collected, and the target substance is separated and collected from the fine particles. Specifically, one or more types selected from biotin and avidin are thermally responsive magnetic fine particles and magnets fixed to magnetic fine particles via a polymer having a lower critical solution temperature (hereinafter abbreviated as “LCST”). A biological material separation method using magnetic force has been developed (see, for example, Patent Document 1). However, the polymer used in this method is simply wound around the magnetic fine particles and is not fixed, and the polymer is often peeled off from the magnetic fine particles before and after the separation and recovery of the target substance. Could not be recovered. Further, when the aggregation and dispersion of magnetic fine particles are repeated by a stimulus response such as heat, secondary aggregation of the magnetic fine particles gradually occurs, and the dispersibility of the particles may be impaired.
これに対して、磁性微粒子と刺激応答性ポリマーを多価アルコールを介して結合し、固定化することでポリマーの磁性微粒子からの脱離と分散性を改善した方法が開発されている(例えば、特許文献2参照)。
しかしながら、この方法では、磁性微粒子と刺激応答性ポリマーを多価アルコール上の官能基とのグラフト重合により結合、あるいはポリマー官能基を介して結合させる際に、フリーポリマーの生成やポリマー間の立体障害により、磁性微粒子表面へのポリマーの固定化が十分に行われず、安定した凝集力が得られないという課題があった。 However, in this method, when the magnetic fine particles and the stimuli-responsive polymer are bonded by graft polymerization with a functional group on a polyhydric alcohol or bonded via a polymer functional group, free polymer formation or steric hindrance between the polymers is caused. Thus, there is a problem that the polymer is not sufficiently immobilized on the surface of the magnetic fine particles and a stable cohesive force cannot be obtained.
そこで、本発明の課題は、水溶液中での分散性が高く、また、熱応答による凝集分散を繰り返しても、2次凝集を起こすことが殆どなく、更に目的物質の分離回収前後に磁性微粒子から熱応答性ポリマーが剥がれることが少ない、熱応答性磁性微粒子及びこれを用いた吸着材を提供することである。また、本発明の更なる課題は、容易に確実に磁性微粒子に熱応答性ポリマーを固定化でき、効率よく選択的にタンパク質等の目的物質(以下、「目的物」ということがある。)の回収が可能な熱応答性磁性微粒子及びこれを用いた吸着材を提供することである。 Therefore, the problem of the present invention is that the dispersibility in an aqueous solution is high, and even when aggregation and dispersion due to thermal response are repeated, the secondary aggregation hardly occurs. It is an object to provide a thermoresponsive magnetic fine particle and an adsorbent using the same, in which the thermoresponsive polymer is less likely to be peeled off. In addition, a further problem of the present invention is that a thermoresponsive polymer can be easily and reliably immobilized on magnetic fine particles, and a target substance such as protein (hereinafter sometimes referred to as “target object”) can be efficiently and selectively selected. A heat-responsive magnetic fine particle that can be recovered and an adsorbent using the same.
本発明者らは、上記課題を解決するために鋭意検討を重ねた。その結果、以下の構成を採用することにより、本発明の課題を解決することを見出し、この知見に基づいて本発明を完成させた。 The present inventors have made extensive studies to solve the above problems. As a result, the inventors have found that the problems of the present invention can be solved by adopting the following configurations, and have completed the present invention based on this finding.
本発明は以下の構成を有する。
[1]熱応答性ポリマーを含有する溶液中で、塩化第二鉄・六水和物及び塩化第一鉄・四水和物の混合溶液を添加した後、アルカリ水溶液を添加し攪拌した後、酸で中和して磁性微粒子を製造することで得られる熱応答性磁性微粒子であって、熱応答性ポリマーが、磁性微粒子と親和性を有する官能基を含む単量体を共重合成分として含有する熱応答性共重合体であることを特徴とする、熱応答性磁性微粒子。また、前記熱応答性共重合体が、磁性微粒子と親和性を有する官能基を含む単量体をブロック共重合成分として含有するブロック共重合体である熱応答性磁性微粒子。
[2] 磁性微粒子と親和性を有する官能基を含む単量体が、ポリグリセロールモノメタクリレート、ポリアクリル酸、ポリメタクリル酸、ポリビニルアルコール、ポリリン酸からなる群から選ばれる少なくとも一種のポリマーの単量体であることを特徴とする前記[1]項記載の熱応答性微粒子。
The present invention has the following configuration.
[1] In a solution containing a thermoresponsive polymer, after adding a mixed solution of ferric chloride / hexahydrate and ferrous chloride / tetrahydrate, adding an alkaline aqueous solution and stirring, Thermally responsive magnetic fine particles obtained by neutralizing with acid to produce magnetic fine particles, wherein the thermoresponsive polymer contains a monomer containing a functional group having an affinity for magnetic fine particles as a copolymerization component A heat-responsive magnetic fine particle characterized by being a heat-responsive copolymer. Moreover, the thermoresponsive magnetic fine particle which is a block copolymer in which the said thermoresponsive copolymer contains the monomer containing the functional group which has affinity with a magnetic fine particle as a block copolymerization component.
[2] Monomer of at least one polymer selected from the group consisting of polyglycerol monomethacrylate, polyacrylic acid, polymethacrylic acid, polyvinyl alcohol, and polyphosphoric acid as the monomer containing a functional group having an affinity for magnetic fine particles The heat-responsive fine particles according to item [1], wherein the fine particles are a body.
[3] 熱応答性ポリマーが、下限臨界溶液温度を有するポリマーである前記[1]または[2]項記載の熱応答性磁性微粒子。
[4] 下限臨界溶液温度を有するポリマーが、N−n−プロピルアクリルアミド、N−イソプロピルアクリルアミド、N−エチルアクリルアミド、N,N−ジメチルアクリルアミド、N−アクリロイルピロリジン、N−アクリロイルピペリジン、N−アクリロイルモルホリン、N−n−プロピルメタクリルアミド、N−イソプロピルメタクリルアミド、N−エチルメタクリルアミド、N,N−ジメチルメタクリルアミド、N−メタクリロイルピロリジン、N−メタクリロイルピペリジン及びN−メタクリロイルモルホリンからなる群から選ばれる少なくとも1種の単量体を、熱応答性を示す共重合成分として含有することを特徴とする前記[3]項記載の熱応答性微粒子。
[3] The thermoresponsive magnetic fine particles according to the above [1] or [2], wherein the thermoresponsive polymer is a polymer having a lower critical solution temperature.
[4] A polymer having a lower critical solution temperature is Nn-propylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-acryloylpyrrolidine, N-acryloylpiperidine, N-acryloylmorpholine. At least selected from the group consisting of Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-ethylmethacrylamide, N, N-dimethylmethacrylamide, N-methacryloylpyrrolidine, N-methacryloylpiperidine and N-methacryloylmorpholine. The heat-responsive fine particles according to item [3], wherein one kind of monomer is contained as a copolymerization component exhibiting heat-responsiveness.
[5] 熱応答性ポリマーが、上限臨界溶液温度を有するポリマーである前記[1]または[2]項記載の熱応答性磁性微粒子。
[6] 上限臨界溶液温度を有するポリマーが、アクリルアミド、アセチルアクリルアミド、ビオチノールアクリレート、N−ビオチニル−N′−メタクロイルトリメチレンアミド、アクロイルグリシンアミド、アクロイルザルコシンアミド、メタクリルザルコシンアミド、アクロイルニペコタミド及びアクロイルメチルウラシルからなる群から選ばれる少なくとも1種の単量体を、熱応答性を示す共重合成分として含有することを特徴とする前記[5]項記載の熱応答性磁性微粒子。
[5] The thermoresponsive magnetic fine particles according to the above [1] or [2], wherein the thermoresponsive polymer is a polymer having an upper critical solution temperature.
[6] Polymer having upper critical solution temperature is acrylamide, acetylacrylamide, biotinol acrylate, N-biotinyl-N′-methacryloyl trimethylene amide, acroyl glycinamide, acroyl sarcosine amide, methacryl sarcosine amide The heat according to item [5], comprising at least one monomer selected from the group consisting of acroylnipecotamide and acroylmethyluracil as a copolymerization component exhibiting thermal responsiveness. Responsive magnetic fine particles.
[7] 熱応答性ポリマーが、ポリグリセロールモノメタクリレートと、ポリN−イソプロピルアクリルアミドとのジ・ブロック共重合体である、前記[1]〜[4]のいずれか1項記載の熱応答性磁性微粒子。
[8] 熱応答性ポリマーが、N,N−ジエチルジチオカルバミド酢酸を重合開始剤とし、グリセロールモノメタクリレートを重合用モノマーとし、UV照射によってリビングラジカル重合したポリグリセロールメタクリレートと、N−イソプロピルアクリルアミドとが、UV照射によるリビングラジカル重合により得られることを特徴とする、前記[1]項または前記[2]項記載の熱応答性磁性微粒子。
[9] 熱応答性磁性微粒子の平均粒径が、10〜1000nmであることを特徴とする、前記[1]〜[8]のいずれか1項記載の熱応答性磁性微粒子。
[7] The thermoresponsive magnet according to any one of [1] to [4], wherein the thermoresponsive polymer is a diblock copolymer of polyglycerol monomethacrylate and poly N-isopropylacrylamide. Fine particles.
[8] The heat-responsive polymer comprises N, N-diethyldithiocarbamide acetic acid as a polymerization initiator, glycerol monomethacrylate as a polymerization monomer, and living radical polymerization polyglycerol methacrylate by UV irradiation and N-isopropylacrylamide. The heat-responsive magnetic fine particles according to [1] or [2], which are obtained by living radical polymerization by UV irradiation.
[9] The heat-responsive magnetic fine particles according to any one of [1] to [8], wherein the heat-responsive magnetic fine particles have an average particle diameter of 10 to 1000 nm.
[10] 前記[1]〜[9]のいずれか1項記載の熱応答性磁性微粒子と、目的物と相互に特異的吸着作用する物質とからなる吸着材。
[11] 目的物と相互に特異的吸着作用する物質が、ビオチン、アビジン、グルタチオン、レクチン及び抗体からなる群から選ばれる少なくとも1種であることを特徴とする前記[10]項記載の吸着材。
[10] An adsorbent comprising the heat-responsive magnetic fine particles according to any one of [1] to [9] and a substance having a specific adsorptive action on the target product.
[11] The adsorbent according to [10], wherein the substance that specifically adsorbs to the target substance is at least one selected from the group consisting of biotin, avidin, glutathione, lectin, and an antibody. .
[12] 少なくとも下記の工程を有することを特徴とする、前記[10]項または前記[11]項記載の吸着材を用いた目的物質の分離方法。
1)目的物質を含む水溶液と吸着材との混合液を調整し、目的物質と吸着材との結合体を生成させる工程、
2)前記混合液の温度を下限臨界溶液温度または上限臨界溶液温度に設定し、前記結合体を凝集させる工程、
3)凝集した結合体を磁石で分離し、回収する工程、
4)回収した結合体から目的物質を分離し、回収する工程。
[12] A method for separating a target substance using the adsorbent according to [10] or [11] above, comprising at least the following steps.
1) a step of adjusting a mixed solution of an aqueous solution containing a target substance and an adsorbent to generate a combined body of the target substance and the adsorbent;
2) setting the temperature of the mixed solution to a lower critical solution temperature or an upper critical solution temperature, and agglomerating the conjugate;
3) Separating and recovering the aggregated aggregate with a magnet,
4) A step of separating and recovering the target substance from the recovered conjugate.
[13] 少なくとも下記の工程を有することを特徴とする熱応答性磁性微粒子の製造方法。
1) N,N−ジエチルジチオカルバミド酢酸を重合開始剤とし、グリセロールモノメタクリレートをUV照射でリビングラジカル重合する工程、
2) 1)で得られたポリグリセロールモノメタクリレートと、N−イソプロピルアクリルアミドとを混合し、UV照射でリビングラジカル重合する工程、
3) 2)で得られたブロックポリマーを含む水溶液に、塩化第二鉄・六水和物、塩化第一鉄・四水和物の混合溶液を添加、攪拌し、水酸化ナトリウム水溶液を添加、攪拌した後、塩酸で中和する工程、及び
4)重合未反応物を除去し、熱応答性磁性微粒子を分離する工程。
[13] A method for producing heat-responsive magnetic fine particles, comprising at least the following steps.
1) A step of living radical polymerization of N, N-diethyldithiocarbamidoacetic acid as a polymerization initiator and glycerol monomethacrylate by UV irradiation,
2) A step of mixing the polyglycerol monomethacrylate obtained in 1) with N-isopropylacrylamide and subjecting it to living radical polymerization by UV irradiation.
3) To the aqueous solution containing the block polymer obtained in 2), a mixed solution of ferric chloride / hexahydrate and ferrous chloride / tetrahydrate is added and stirred, and an aqueous sodium hydroxide solution is added, A step of neutralizing with hydrochloric acid after stirring, and 4) a step of removing unreacted polymerization products and separating heat-responsive magnetic fine particles.
本発明の熱応答性磁性微粒子は、磁性微粒子への熱応答性ポリマーの導入が容易に確実に行われ、水溶液中での分散性が高く、熱刺激応答による凝集分散を繰り返しても、2次凝集を起こすことが殆どないため、これを用いた吸着材は、目的物を効率よく吸着することができる。
本発明の熱応答性磁性微粒子及びこれを用いた吸着材は、目的物の分離回収前後に磁性微粒子から熱応答性ポリマーが殆ど剥がれ落ちない。そのため、本発明の吸着材を用いることで、細胞破砕懸濁液等のタンパク質混合液から目的タンパク質を効率よく選択的に回収することができる。
The heat-responsive magnetic fine particles of the present invention are easily and surely introduced with a heat-responsive polymer into the magnetic fine particles, have high dispersibility in an aqueous solution, and are secondary even if they are repeatedly aggregated and dispersed by a thermal stimulus response. Since the agglomeration hardly occurs, the adsorbent using this can adsorb the target product efficiently.
In the heat-responsive magnetic fine particles and the adsorbent using the same according to the present invention, the heat-responsive polymer hardly peels off from the magnetic fine particles before and after separation and recovery of the target product. Therefore, by using the adsorbent of the present invention, the target protein can be efficiently and selectively recovered from a protein mixture such as a cell disruption suspension.
以下、本発明について詳細に説明する。
本発明の熱応答性磁性微粒子は、磁性微粒子と熱応答性ポリマーとを用いて得られる。熱応答性ポリマーは、磁性微粒子と結合する部位、つまり磁性微粒子の成分である鉄イオン等の金属イオンと親和性の高い官能基、例えば、水酸基、カルボキシル基、リン酸基等を含有する単量体成分と熱応答性を示す単量体成分との共重合体、好ましくはブロック共重合体である。この熱応答性ポリマーは、磁性微粒子と結合するポリマー部位、および熱応答性ポリマー部位が少なくともそれぞれ一種類以上存在している共重合体であれば制限無く利用することができる。ジ・ブロック共重合体またはトリ・ブロック共重合体が好ましく利用でき、特に、調製の容易さの面からジ・ブロック共重合体が好ましく利用できる。
Hereinafter, the present invention will be described in detail.
The thermoresponsive magnetic fine particles of the present invention are obtained using magnetic fine particles and a thermoresponsive polymer. The thermoresponsive polymer is a single unit containing a functional group having a high affinity with a metal ion such as an iron ion, which is a component of the magnetic fine particle, for example, a hydroxyl group, a carboxyl group, a phosphate group, etc. A copolymer of a body component and a monomer component exhibiting thermal responsiveness, preferably a block copolymer. The thermoresponsive polymer can be used without limitation as long as it is a copolymer in which at least one polymer part bonded to magnetic fine particles and at least one thermoresponsive polymer part are present. A di-block copolymer or a tri-block copolymer can be preferably used. In particular, a di-block copolymer can be preferably used from the viewpoint of ease of preparation.
本発明に用いられる磁性微粒子との結合部位となる共重合成分としては、構成単位に前記の官能基を有する単量体であれば特に制限は無く、例えば、ポリグリセロールモノメタクリレート、ポリアクリル酸、ポリメタクリル酸、ポリビニルアルコール、ポリリン酸が挙げられる。本発明において、磁性微粒子上の金属イオンと、熱応答性ポリマーを構成する金属イオンと親和性を有するポリマー部位の有する官能基との結合により、磁性微粒子と熱応答性ポリマーとが結合される。熱応答性ブロックポリマーの有する官能基は、磁性微粒子との結合に利用されるだけでなく、磁性微粒子の水溶液中での分散性の向上にも効果があると推定される。 There are no particular limitations on the copolymer component used as a binding site for the magnetic fine particles used in the present invention, as long as it is a monomer having the above functional group as a structural unit. For example, polyglycerol monomethacrylate, polyacrylic acid, Examples include polymethacrylic acid, polyvinyl alcohol, and polyphosphoric acid. In the present invention, the magnetic fine particles and the thermoresponsive polymer are bonded by the bond between the metal ion on the magnetic fine particle and the functional group of the polymer portion having an affinity for the metal ion constituting the thermoresponsive polymer. It is presumed that the functional group of the thermoresponsive block polymer is not only used for bonding with the magnetic fine particles but also effective in improving the dispersibility of the magnetic fine particles in an aqueous solution.
本発明で用いられる熱応答性ポリマーは、下限臨界溶液温度を有するポリマー及び上限臨界溶液温度を有するポリマーに区別される。
本発明の下限臨界溶液温度を有するポリマーは、熱応答性を示すポリマー部位(共重合成分)として、例えば、N−n−プロピルアクリルアミド、N−イソプロピルアクリルアミド、N−エチルアクリルアミド、N,N−ジメチルアクリルアミド、N−アクリロイルピロリジン、N−アクリロイルピペリジン、N−アクリロイルモルホリン、N−n−プロピルメタクリルアミド、N−イソプロピルメタクリルアミド、N−エチルメタクリルアミド、N,N−ジメチルメタクリルアミド、N−メタクリロイルピロリジン、N−メタクリロイルピペリジン、N−メタクリロイルモルホリン等のN置換(メタ)アクリルアミド誘導体からなるポリマー;ヒドロキシプロピルセルロース、ポリビニルアルコール部分酢化物、ポリビニルメチルエーテル、(ポリオキシエチレン−ポリオキシプロピレン)ブロックコポリマー、ポリオキシエチレンラウリルアミン等のポリオキシエチレンアルキルアミン誘導体;ポリオキシエチレンソルビタンラウレート等のポリオキシエチレンソルビタンエステル誘導体;(ポリオキシエチレンノニルフェニルエーテル)アクリレート、(ポリオキシエチレンオクチルフェニルエーテル)メタクリレート等の(ポリオキシエチレンアルキルフェニルエーテル)(メタ)アクリレート類;及び(ポリオキシエチレンラウリルエーテル)アクリレート、(ポリオキシエチレンオレイルエーテル)メタクリレート等の(ポリオキシエチレンアルキルエーテル)(メタ)アクリレート類等のポリオキシエチレン(メタ)アクリル酸エステル誘導体等を含有するポリマーが挙げられる。これらの下限臨界溶液温度を有するポリマー部位は、これらの単量体からなる単独重合体及びこれらの少なくとも2種の単量体からなる共重合体のいずれでもよい。これらのなかでも、下限臨界溶液温度を有するポリマー部位として、N−n−プロピルアクリルアミド、N−イソプロピルアクリルアミド、N−エチルアクリルアミド、N,N−ジメチルアクリルアミド、N−アクリロイルピロリジン、N−アクリロイルピペリジン、N−アクリロイルモルホリン、N−n−プロピルメタクリルアミド、N−イソプロピルメタクリルアミド、N−エチルメタクリルアミド、N,N−ジメチルメタクリルアミド、N−メタクリロイルピロリジン、N−メタクリロイルピペリジン、N−メタクリロイルモルホリンからなる群から選ばれる少なくとも1種の単量体を含有する単独重合体または共重合体が好ましく利用できる。
The thermoresponsive polymer used in the present invention is classified into a polymer having a lower critical solution temperature and a polymer having an upper critical solution temperature.
The polymer having the lower critical solution temperature of the present invention has, for example, Nn-propylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N, N-dimethyl as a polymer portion (copolymerization component) exhibiting thermal response. Acrylamide, N-acryloylpyrrolidine, N-acryloylpiperidine, N-acryloylmorpholine, Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-ethylmethacrylamide, N, N-dimethylmethacrylamide, N-methacryloylpyrrolidine, Polymers composed of N-substituted (meth) acrylamide derivatives such as N-methacryloylpiperidine and N-methacryloylmorpholine; hydroxypropyl cellulose, polyvinyl alcohol partially acetylated product, polyvinyl methyl ether (Polyoxyethylene-polyoxypropylene) block copolymers, polyoxyethylene alkylamine derivatives such as polyoxyethylene laurylamine; polyoxyethylene sorbitan ester derivatives such as polyoxyethylene sorbitan laurate; (polyoxyethylene nonylphenyl ether) acrylate (Polyoxyethylene alkylphenyl ether) (meth) acrylates such as (polyoxyethylene octylphenyl ether) methacrylate; and (polyoxyethylene lauryl ether) acrylate and (polyoxyethylene oleyl ether) methacrylate (polyoxyethylene) Polymers containing polyoxyethylene (meth) acrylate derivatives such as alkyl ethers (meth) acrylates And the like. The polymer portion having these lower critical solution temperatures may be either a homopolymer composed of these monomers or a copolymer composed of at least two of these monomers. Among these, Nn-propylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-acryloylpyrrolidine, N-acryloylpiperidine, N, are polymer sites having a lower critical solution temperature. -From the group consisting of acryloylmorpholine, Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-ethylmethacrylamide, N, N-dimethylmethacrylamide, N-methacryloylpyrrolidine, N-methacryloylpiperidine, N-methacryloylmorpholine A homopolymer or copolymer containing at least one selected monomer can be preferably used.
また、本発明の上限臨界溶液温度を有するポリマーは、熱応答性を示すポリマー部位(共重合成分)として、例えば、アクリルアミド、アセチルアクリルアミド、ビオチノールアクリレート、N−ビオチニル−N′−メタクロイルトリメチレンアミド、アクロイルグリシンアミド、アクロイルザルコシンアミド、メタクリルザルコシンアミド、アクロイルニペコタミド及びアクロイルメチルウラシル等からなる群から選ばれる少なくとも1種の単量体を含有するポリマーが挙げられる。これらの上限臨界溶液温度を有するポリマー部位は、これらの単量体からなる単独重合体およびこれらの少なくとも2種の単量体からなる共重合体のいずれでもよい。これらのなかでも、上限臨界溶液温度を有するポリマー部位として、アクリルアミド、アセチルアクリルアミド、ビオチノールアクリレート、N−ビオチニル−N′−メタクロイルトリメチレンアミド、アクロイルグリシンアミド、アクロイルザルコシンアミド、メタクリルザルコシンアミド、アクロイルニペコタミド及びアクロイルメチルウラシルからなる群から選ばれる少なくとも1種の単量体を含有する重合体または共重合体が好ましく利用できる。 In addition, the polymer having the upper critical solution temperature of the present invention is, for example, acrylamide, acetylacrylamide, biotinol acrylate, N-biotinyl-N′-methacryloyl tri, as a polymer portion (copolymerization component) exhibiting heat response. And a polymer containing at least one monomer selected from the group consisting of methylene amide, acroyl glycinamide, acroyl sarcosine amide, methacryl sarcosine amide, acroyl nipecotamide, acroyl methyl uracil and the like. . The polymer portion having these upper critical solution temperatures may be either a homopolymer composed of these monomers or a copolymer composed of at least two of these monomers. Among these, acrylamide, acetylacrylamide, biotinol acrylate, N-biotinyl-N′-methacryloyl trimethylene amide, acroyl glycinamide, acroyl sarcosine amide, methacrylic acid are polymer sites having an upper critical solution temperature. A polymer or copolymer containing at least one monomer selected from the group consisting of sarcosine amide, acroylnipecotamide and acroylmethyluracil can be preferably used.
本発明に用いる磁性微粒子は、特に制限はないが、平均粒径が、10nm以上、1000nm未満であることが好ましく、目的物の認識性を高めるためには、平均粒径が、50nm以上、200nm未満であることが特に好ましい。磁性微粒子の素材は、例えば、マグネタイト、酸化ニッケル、フェライト、コバルト鉄酸化物、バリウムフェライト、炭素鋼、タングステン鋼、KS鋼、希土類コバルト磁石及びヘマタイト等の微粒子が挙げられる。 The magnetic fine particles used in the present invention are not particularly limited, but the average particle diameter is preferably 10 nm or more and less than 1000 nm. In order to improve the recognition of the target product, the average particle diameter is 50 nm or more and 200 nm. It is particularly preferred that it is less than. Examples of the magnetic fine particle material include fine particles such as magnetite, nickel oxide, ferrite, cobalt iron oxide, barium ferrite, carbon steel, tungsten steel, KS steel, rare earth cobalt magnet, and hematite.
これら磁性微粒子と、熱応答性ポリマーとの結合体、詳しくは熱応答性ポリマー成分である磁性微粒子と親和性の高い官能基を有するポリマー部位と磁性微粒子との結合体は、当技術分野における周知の方法によって調製することができる。例えば、米国特許第4452773号に記載されているように、ポリマー構成単位中に複数の水酸基を有する高分子であるデキストランの50重量%水溶液(10ml)中に、塩化第二鉄・六水和物(1.51g)及び塩化第一鉄・四水和物(0.64g)混合水溶液(10ml)を加えて攪拌し、60〜65℃に水浴中で、7.4(V/V)%アンモニア水溶液をpH10〜11程度になるように滴下しながら、加熱し、15分反応させる方法で得ることができる。 Conjugates of these magnetic fine particles and thermoresponsive polymers, and more specifically, conjugates of magnetic fine particles that are functional components having high affinity with magnetic fine particles that are thermoresponsive polymer components, are well known in the art. It can be prepared by the method. For example, as described in U.S. Pat. No. 4,452,773, ferric chloride hexahydrate in a 50% by weight aqueous solution (10 ml) of dextran, which is a polymer having a plurality of hydroxyl groups in the polymer structural unit. (1.51 g) and ferrous chloride-tetrahydrate (0.64 g) mixed aqueous solution (10 ml) were added and stirred, and 7.4 (V / V)% ammonia in a water bath at 60 to 65 ° C. It can be obtained by a method of heating and reacting for 15 minutes while dropping the aqueous solution to a pH of about 10-11.
本発明では、磁性微粒子と親和性を有する官能基を含有する単量体と熱応答性を示す単量体とを共重合させ、得られた共重合体を含有する溶液に、上記の従来公知の磁性微粒子を製造する方法を適宜用いることにより、磁性微粒子と熱応答性ポリマーとの結合体を製造することができる。特に、熱応答性ポリマーとしてブロック共重合体を用いる場合には、上記の一方の共重合成分を構成する単量体を重合開始剤を用いてリビングラジカル重合し、更にもう一方の共重合成分を構成する単量体をリビングラジカル重合させ、得られたブロック共重合体を上記の従来公知の磁性微粒子の製造法を適宜用いることにより、本発明の熱応答性磁性微粒子を得ることができる。 In the present invention, a monomer containing a functional group having an affinity for magnetic fine particles and a monomer exhibiting heat responsiveness are copolymerized, and the solution containing the obtained copolymer is added to the above-mentioned conventionally known solution. By appropriately using the method for producing magnetic fine particles, a conjugate of magnetic fine particles and a thermoresponsive polymer can be produced. In particular, when a block copolymer is used as the thermoresponsive polymer, the monomer constituting one of the copolymer components is subjected to living radical polymerization using a polymerization initiator, and the other copolymer component is further added. The thermoresponsive magnetic fine particles of the present invention can be obtained by subjecting the constituent monomers to living radical polymerization, and appropriately using the obtained block copolymer by the above-described conventionally known methods for producing magnetic fine particles.
より具体的には、少なくとも下記の工程を用いることにより、本発明の熱応答性磁性微粒子を容易に得ることができる。
ここでは、磁性微粒子と親和性を有する官能基を含む単量体としてグリセロールモノメタクリレート、熱応答性を示す共重合成分を構成する単量体としてN−イソプロピルアクリルアミド、及び磁性微粒子としてマグネタイトを用いた例を示したが、下記方法はこれらに限定されるものではない。
More specifically, the thermoresponsive magnetic fine particles of the present invention can be easily obtained by using at least the following steps.
Here, glycerol monomethacrylate was used as a monomer containing a functional group having an affinity for magnetic fine particles, N-isopropylacrylamide was used as a monomer constituting a copolymer component exhibiting thermal response, and magnetite was used as magnetic fine particles. Although examples are shown, the following methods are not limited to these.
1) N,N−ジエチルジチオカルバミド酢酸を重合開始剤とし、グリセロールモノメタクリレートをUV照射でリビングラジカル重合する。
2) 1)で得られたポリグリセロールモノメタクリレートと、N−イソプロピルアクリルアミドとを混合し、UV照射でリビングラジカル重合する。
3) 2)で得られたブロックポリマーを含む水溶液に、塩化第二鉄・六水和物、塩化第一鉄・四水和物の混合溶液を添加、攪拌し、水酸化ナトリウム水溶液を添加、攪拌した後、塩酸で中和する。
4)重合未反応物を除去し、熱応答性磁性微粒子を分離する。
1) Living radical polymerization of glycerol monomethacrylate by UV irradiation using N, N-diethyldithiocarbamide acetic acid as a polymerization initiator.
2) The polyglycerol monomethacrylate obtained in 1) and N-isopropylacrylamide are mixed and subjected to living radical polymerization by UV irradiation.
3) To the aqueous solution containing the block polymer obtained in 2), a mixed solution of ferric chloride / hexahydrate and ferrous chloride / tetrahydrate is added and stirred, and an aqueous sodium hydroxide solution is added, After stirring, neutralize with hydrochloric acid.
4) Remove unreacted polymer and separate thermally responsive magnetic fine particles.
本発明で磁性微粒子、熱応答性磁性微粒子及び吸着材の回収に用いる磁石は、用いる磁性微粒子の有する磁力の大きさによって異なるが、前記磁性微粒子の素材であれば、マグナ社製ネオジ磁石が利用できる。このように本発明では、磁石の磁力によって、熱応答性磁性微粒子及び吸着材等を回収するが、磁性微粒子の表面に熱応答性ポリマーが固定されていることで、分散状態では回収困難なナノサイズの磁性微粒子を意図的に凝集させて、回収率を高めることが可能になる。 The magnet used for collecting the magnetic fine particles, the thermoresponsive magnetic fine particles and the adsorbent in the present invention varies depending on the magnitude of the magnetic force of the magnetic fine particles used. it can. As described above, in the present invention, the heat-responsive magnetic fine particles and the adsorbent are collected by the magnetic force of the magnet. However, since the thermo-responsive polymer is fixed on the surface of the magnetic fine particles, it is difficult to collect the nano particles in a dispersed state. It is possible to intentionally agglomerate magnetic particles of a size and increase the recovery rate.
本発明では、目的物と相互的に特異的吸着作用を有する部位を磁性微粒子に導入し、固定することで、その部位と親和性を有する物質が特異的に結合できる。目的物がタンパク質の場合には、目的物と相互に特異的吸着作用する部位として、ビオチン、アビジン、グルタチオン、レクチン及び抗体等を熱応答性磁性微粒子に固定することで、それらに親和性を有するタンパク質と特異的に結合できる。 In the present invention, a part having a specific adsorption action with the target substance is introduced into the magnetic fine particles and fixed, whereby a substance having affinity with the part can be specifically bound. When the target substance is a protein, biotin, avidin, glutathione, lectin, antibody, etc. are immobilized on heat-responsive magnetic microparticles as sites that specifically adsorb to each other and have affinity for them. It can bind specifically to proteins.
ビオチンの場合は、アビジンとの特異的な結合を介してビオチン化された目的タンパク質と、またビオチン化された抗体を用いてそれらの抗原である種々のタンパク質と更に結合することが可能である。
本発明では、市販されているアビジン、ビオチン化タンパクが利用でき、ビオチン化は、当技術分野で周知の方法に従えばよい。
In the case of biotin, it is possible to further bind to a target protein that has been biotinylated through specific binding to avidin, and to various proteins that are antigens thereof using a biotinylated antibody.
In the present invention, commercially available avidin and biotinylated protein can be used, and biotinylation may be performed according to a method well known in the art.
グルタチオンの場合は、グルタチオン−S−トランスフェラーゼ(以下、「GST」という。)を含有するタンパク質と特異的に結合できる。このようなGST含有タンパク質の調製は当技術分野で周知の方法に従えばよい。磁性微粒子上の熱応答性ポリマーに、ビオチンまたはグルタチオンを固定する方法は、例えば、国際公開第01/09141号パンフレットに記載されているように、ビオチンをメタクリル基やアクリル基等の重合性の官能基と結合させて付加重合性単量体とし、他の単量体成分と共重合することにより達成される。この方法は、重合性を持つようにモノマー化されたリガンドを用いて、ポリマーの重合時に共重合させ、リガンドを固定する方法である。この方法を利用して熱応答性ポリマーにグルタチオンを固定することができる。また、ポリマーの重合時にカルボン酸、アミノ基またはエポキシ基等の官能基を持つモノマーを他のモノマーと共重合させ、当技術分野で周知の方法に従い、この官能基を介して、リガンドをポリマー上に固定する方法が利用できる。これにより、熱応答性磁性微粒子と、リガンドとからなる吸着材が得られる。 In the case of glutathione, it can specifically bind to a protein containing glutathione-S-transferase (hereinafter referred to as “GST”). Such a GST-containing protein may be prepared by methods well known in the art. As a method for immobilizing biotin or glutathione to a thermoresponsive polymer on magnetic fine particles, for example, as described in WO 01/09141, biotin can be polymerized with a polymerizable functional group such as a methacryl group or an acrylic group. This is achieved by combining with a group to form an addition polymerizable monomer and copolymerizing with other monomer components. This method is a method in which a ligand that is monomerized so as to have polymerizability is copolymerized at the time of polymerization of the polymer to fix the ligand. This method can be used to fix glutathione to a thermoresponsive polymer. In addition, a monomer having a functional group such as a carboxylic acid, an amino group, or an epoxy group is copolymerized with another monomer during polymerization of the polymer, and the ligand is attached to the polymer via the functional group according to a method well known in the art. The method of fixing to can be used. As a result, an adsorbent composed of thermoresponsive magnetic fine particles and a ligand is obtained.
本発明において、吸着材は、目的物質を回収するために用いることができる。その方法は、以下の通りである。
1)目的物質を含む水溶液と吸着材との混合液を調整し、目的物質と吸着材との結合体を生成させる。
2)前記混合液の温度を下限臨界溶液温度または上限臨界溶液温度に設定し、前記結合体を凝集させる。
3)凝集した結合体を磁石で分離し、回収する。
4)回収した結合体から目的物質を分離し、回収する。
In the present invention, the adsorbent can be used to recover the target substance. The method is as follows.
1) A mixed liquid of an aqueous solution containing a target substance and an adsorbent is prepared, and a combined body of the target substance and the adsorbent is generated.
2) The temperature of the mixed solution is set to the lower critical solution temperature or the upper critical solution temperature, and the combined body is aggregated.
3) Separate and collect the aggregated aggregates with a magnet.
4) Separate and recover the target substance from the recovered conjugate.
本発明では、少なくとも下記工程を有する方法により、微生物の細胞破砕懸濁液から目的タンパク質を分離することができる。なお、このとき用いる吸着材は、熱応答性磁性微粒子とビオチンとが熱応答性ポリマーを介して固定された構造であり、吸着材の平均粒径は、10〜1000nmであり、30〜200nmであることが好ましい。 In the present invention, the target protein can be separated from the cell disruption suspension of the microorganism by a method having at least the following steps. The adsorbent used at this time has a structure in which thermoresponsive magnetic fine particles and biotin are fixed via a thermoresponsive polymer, and the average particle diameter of the adsorbent is 10 to 1000 nm, and 30 to 200 nm. Preferably there is.
1)目的タンパク質の生産菌を培養し、得られた細胞懸濁液を破砕し、細胞破砕懸濁液を調製する。
2)目的タンパク質のポリクローナル抗体またはモノクローナル抗体を調製する。
3)上記2)の抗体、該抗体を抗原とするビオチン化イムノグロブリンG、アビジン及び吸着材を混合し、ここに目的タンパク質を含む細胞破砕懸濁液を加え、混合液とし、吸着材と目的タンパク質との結合体を生成させる。
4)前記混合液の温度を下限臨界溶液温度または上限臨界溶液温度に設定し、前記結合体を凝集させる。
5)凝集した結合体を磁石で分離し、回収する。
6)結合体からタンパク質を分離する。
1) Culturing the target protein producing bacteria, crushing the obtained cell suspension to prepare a cell disruption suspension.
2) Prepare a polyclonal antibody or monoclonal antibody of the target protein.
3) The antibody of 2) above, biotinylated immunoglobulin G using the antibody as an antigen, avidin, and an adsorbent are mixed, and a cell disruption suspension containing the target protein is added thereto to obtain a mixed solution. Generate a conjugate with the protein.
4) The temperature of the mixed solution is set to the lower critical solution temperature or the upper critical solution temperature, and the combined body is aggregated.
5) Separate and collect the aggregated aggregate with a magnet.
6) Separate the protein from the conjugate.
本発明では、少なくとも下記工程を有する方法により、微生物の細胞破砕懸濁液から目的タンパク質を分離することができる。なお、このとき用いる吸着材は、熱応答性磁性微粒子とグルタチオンとが熱応答性ポリマーを介して固定された構造であり、吸着材の平均粒径は、10〜1000nmであり、30〜200nmであることが好ましい。 In the present invention, the target protein can be separated from the cell disruption suspension of the microorganism by a method having at least the following steps. The adsorbent used at this time has a structure in which thermoresponsive magnetic fine particles and glutathione are fixed via a thermoresponsive polymer. The average particle diameter of the adsorbent is 10 to 1000 nm, and 30 to 200 nm. Preferably there is.
1)目的タンパク質とグルタチオン−S−トランスフェラーゼとの融合タンパク質を生産する、微生物を培養し、得られた細胞懸濁液を破砕し、細胞破砕懸濁液を調製する。
2)細胞破砕懸濁液に吸着材を加え、混合液とし、融合タンパク質と吸着材との結合体を生成させる。
3)前記混合液の温度を下限臨界溶液温度または上限臨界溶液温度に設定し、結合体を凝集させる。
4)凝集した結合体を磁石で分離し、回収する。
5)結合体からタンパク質を分離する。
1) A microorganism producing a fusion protein of a target protein and glutathione-S-transferase is cultured, and the resulting cell suspension is disrupted to prepare a cell disruption suspension.
2) An adsorbent is added to the cell disruption suspension to form a mixed solution, and a conjugate of the fusion protein and the adsorbent is generated.
3) The temperature of the mixed solution is set to the lower critical solution temperature or the upper critical solution temperature, and the combined body is aggregated.
4) Separate and collect the aggregated aggregates with a magnet.
5) Separate the protein from the conjugate.
以下、実施例によって本発明を具体的に説明するが、本発明はこれらによって何ら限定されることはない。また、実施例、比較例中における物性の測定方法、用いた材料の組成は以下の通りである。 EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited at all by these. Moreover, the measurement methods of physical properties in Examples and Comparative Examples, and the compositions of the materials used are as follows.
実施例1
<重合開始剤の調製>
N,N−ジエチルジチオカルバミド三水和物10g、クロロ酢酸ナトリウム5.17g、水100gを300mlナスフラスコ中、室温にて、マグネティックスターラーを用いて、2日間撹拌した。塩酸を添加してpH4にして生成物を沈殿させた後、ろ過、減圧乾燥し、回収した。アセトンにより再結晶させた。
Example 1
<Preparation of polymerization initiator>
10 g of N, N-diethyldithiocarbamide trihydrate, 5.17 g of sodium chloroacetate, and 100 g of water were stirred in a 300 ml eggplant flask at room temperature using a magnetic stirrer for 2 days. Hydrochloric acid was added to adjust the pH to 4, and the product was precipitated, filtered, dried under reduced pressure, and collected. Recrystallized with acetone.
<ブロックポリマーの調製>
グリセロールモノメタクリレート4.51g、N,N−ジエチルジチオカルバミド酢酸0.05g、1N水酸化ナトリウム水溶液1.5ml、水200mlを反応容器(理工科学産業株式会社製 UVL-400HA-200Pa 平底)に入れ、UVランプ、セラムラバーを装着した。冷却水で容器内温度を20℃に保ち、マグネティックスターラーで系内を撹拌しながら30分窒素置換を行い、60分UVを照射した。反応溶液を3日間透析し、ロータリーエバポレーターを用いて200gに調整した後、再び反応容器に入れ、N−イソプロピルアクリルアミド3.18gを添加して同様に90分間重合を行った。透析により精製し、凍結乾燥により生成物を回収した。得られたブロックポリマーはGPC測定で平均分子量Mn=7410、Mw=16300であった。
<Preparation of block polymer>
Put 4.51 g of glycerol monomethacrylate, 0.05 g of N, N-diethyldithiocarbamide acetic acid, 1.5 ml of 1N sodium hydroxide aqueous solution, and 200 ml of water in a reaction vessel (UVL-400HA-200Pa flat bottom manufactured by Riko Kagaku Sangyo Co., Ltd.) A UV lamp and a ceramic rubber were installed. The temperature inside the container was kept at 20 ° C. with cooling water, and nitrogen substitution was performed for 30 minutes while stirring the system with a magnetic stirrer, and UV irradiation was performed for 60 minutes. The reaction solution was dialyzed for 3 days, adjusted to 200 g using a rotary evaporator, then placed in the reaction vessel again, and 3.18 g of N-isopropylacrylamide was added, and polymerization was performed in the same manner for 90 minutes. The product was recovered by dialysis and lyophilized. The obtained block polymer was the average molecular weight Mn = 7410 and Mw = 16300 by GPC measurement.
<ブロックポリマー磁性体複合粒子の調製>
100ml容の4口フラスコ中に上記ポリマーを2mg、水を3ml入れ溶解し、撹拌シール、撹拌棒、セラムラバーを装着し、系内を30分窒素置換した。0.2M塩化第一鉄・四水和物水溶液:0.2M塩化第二鉄・六水和物水溶液(=1:2)水溶液43μlを添加して速度200rpmで30分撹拌後、0.5N水酸化ナトリウム水溶液を添加してpH12にし、60分反応を行った。塩酸を添加して系を中性にして反応を終了させた。その後、透析により精製した。その後、15000rpmで20分遠心分離を行い、上澄みを70000rpmで20分超遠心して得られる沈殿物を再分散させた。得られたブロックポリマー磁性体複合粒子の平均粒径は、光散乱光度計により約20nmであることがわかった。またこの微粒子はLCSTを34℃に有し、LCST未満の水溶液中では完全に分散し、磁石での回収は困難であったが、溶液をLCST以上にすると直ちに凝集し、磁石で回収することが可能であった。
<Preparation of block polymer magnetic composite particles>
In a 100 ml four-necked flask, 2 mg of the above polymer and 3 ml of water were placed and dissolved. A stir seal, a stir bar, and a ceramic rubber were attached, and the system was purged with nitrogen for 30 minutes. 0.2M ferrous chloride / tetrahydrate aqueous solution: 0.2M ferric chloride / hexahydrate aqueous solution (= 1: 2) aqueous solution 43 μl was added and stirred at a speed of 200 rpm for 30 minutes. A sodium hydroxide aqueous solution was added to adjust the pH to 12, and the reaction was performed for 60 minutes. Hydrochloric acid was added to neutralize the system and the reaction was terminated. Then, it refine | purified by dialysis. Then, centrifugation was performed at 15000 rpm for 20 minutes, and the precipitate obtained by centrifuging the supernatant at 70,000 rpm for 20 minutes was redispersed. The average particle diameter of the obtained block polymer magnetic composite particles was found to be about 20 nm by a light scattering photometer. This fine particle has LCST at 34 ° C. and is completely dispersed in an aqueous solution lower than LCST, and recovery with a magnet is difficult. However, when the solution is made higher than LCST, it immediately aggregates and can be recovered with a magnet. It was possible.
比較例1
特開2005−82538号公報記載の実施例と同様に熱応答性磁性微粒子を調製した。
<磁性微粒子の調製>
100ml容のフラスコに、塩化第二鉄・六水和物(1.0mol)及び塩化第一鉄・四水和物(0.5mol)混合水溶液を4ml、多価アルコールであるデキストラン(和光純薬社製、分子量32000〜40000)の10重量%水溶液60mlを入れ、メカニカルスターラーで攪拌し、この混合溶液を50℃に昇温した後、これに25重量%アンモニア溶液5.0mlを滴下し、1時間程度攪拌した。この操作で、平均粒径が約40nmのデキストランが固定された磁性微粒子が得られた。この磁性微粒子溶液3ml(磁性体濃度0.02wt%)中に系中濃度0.067(W/W)%になるようにN−イソプロピルアクリルアミドを添加し、20℃で窒素で十分溶液を脱気しながら30分攪拌した。更に0.2M硝酸二アンモニウムセリウム(IV)硝酸溶液5μlを添加し、2時間攪拌し、磁性微粒子上にグラフト重合反応を進行させた。得られた粒子を34℃以上に加熱したが、磁性微粒子液がわずかに濁りを生じるだけで磁石での回収はできなかった。
Comparative Example 1
Thermally responsive magnetic fine particles were prepared in the same manner as in the examples described in JP-A-2005-82538.
<Preparation of magnetic fine particles>
In a 100 ml flask, 4 ml of a mixed aqueous solution of ferric chloride / hexahydrate (1.0 mol) and ferrous chloride / tetrahydrate (0.5 mol), dextran, a polyhydric alcohol (Wako Pure Chemical) 60 ml of a 10% by weight aqueous solution having a molecular weight of 32,000 to 40,000) was added and stirred with a mechanical stirrer. After the temperature of this mixed solution was raised to 50 ° C., 5.0 ml of 25% by weight ammonia solution was added dropwise thereto. Stir for about an hour. By this operation, magnetic fine particles in which dextran having an average particle diameter of about 40 nm was fixed were obtained. N-isopropylacrylamide was added to 3 ml of this magnetic fine particle solution (magnetic substance concentration 0.02 wt%) so that the concentration in the system was 0.067 (W / W)%, and the solution was sufficiently degassed with nitrogen at 20 ° C. The mixture was stirred for 30 minutes. Further, 5 μl of 0.2M diammonium cerium nitrate (IV) nitric acid solution was added and stirred for 2 hours to allow the graft polymerization reaction to proceed on the magnetic fine particles. The obtained particles were heated to 34 ° C. or higher, but the magnetic fine particle liquid was slightly turbid and could not be recovered with a magnet.
Claims (13)
1)目的物質を含む水溶液と吸着材との混合液を調整し、目的物質と吸着材との結合体を生成させる工程、
2)前記混合液の温度を下限臨界溶液温度または上限臨界溶液温度に設定し、前記結合体を凝集させる工程、
3)凝集した結合体を磁石で分離し、回収する工程、
4)回収した結合体から目的物質を分離し、回収する工程。 The method for separating a target substance using an adsorbent according to claim 10 or 11, wherein the method comprises at least the following steps.
1) a step of adjusting a mixed solution of an aqueous solution containing a target substance and an adsorbent to generate a combined body of the target substance and the adsorbent;
2) setting the temperature of the mixed solution to a lower critical solution temperature or an upper critical solution temperature, and agglomerating the conjugate;
3) Separating and recovering the aggregated aggregate with a magnet,
4) A step of separating and recovering the target substance from the recovered conjugate.
1) N,N−ジエチルジチオカルバミド酢酸を重合開始剤とし、グリセロールモノメタクリレートをUV照射でリビングラジカル重合する工程、
2) 1)で得られたポリグリセロールモノメタクリレートと、N−イソプロピルアクリルアミドとを混合し、UV照射でリビングラジカル重合する工程、
3) 2)で得られたブロックポリマーを含む水溶液に、塩化第二鉄・六水和物、塩化第一鉄・四水和物の混合溶液を添加、攪拌し、水酸化ナトリウム水溶液を添加、攪拌した後、塩酸で中和する工程、及び
4)重合未反応物を除去し、熱応答性磁性微粒子を分離する工程。 A method for producing heat-responsive magnetic fine particles, comprising at least the following steps.
1) A step of living radical polymerization of N, N-diethyldithiocarbamidoacetic acid as a polymerization initiator and glycerol monomethacrylate by UV irradiation,
2) A step of mixing the polyglycerol monomethacrylate obtained in 1) with N-isopropylacrylamide and subjecting it to living radical polymerization by UV irradiation.
3) To the aqueous solution containing the block polymer obtained in 2), a mixed solution of ferric chloride / hexahydrate and ferrous chloride / tetrahydrate is added and stirred, and an aqueous sodium hydroxide solution is added, A step of neutralizing with hydrochloric acid after stirring, and 4) a step of removing unreacted polymerization products and separating heat-responsive magnetic fine particles.
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