JP2011232072A - Silica nano-particle containing organic fluorescence dye, its manufacturing method and biological substance indicating agent - Google Patents
Silica nano-particle containing organic fluorescence dye, its manufacturing method and biological substance indicating agent Download PDFInfo
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- JP2011232072A JP2011232072A JP2010100697A JP2010100697A JP2011232072A JP 2011232072 A JP2011232072 A JP 2011232072A JP 2010100697 A JP2010100697 A JP 2010100697A JP 2010100697 A JP2010100697 A JP 2010100697A JP 2011232072 A JP2011232072 A JP 2011232072A
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- organic fluorescent
- fluorescent dye
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 title claims abstract description 254
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 124
- 239000000377 silicon dioxide Substances 0.000 title claims abstract description 119
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 102
- 239000000126 substance Substances 0.000 title claims abstract description 43
- 239000003795 chemical substances by application Substances 0.000 title claims abstract description 41
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 18
- 239000002245 particle Substances 0.000 claims abstract description 31
- 238000002372 labelling Methods 0.000 claims description 46
- 238000000034 method Methods 0.000 claims description 43
- -1 silane compound Chemical class 0.000 claims description 27
- 125000003277 amino group Chemical group 0.000 claims description 25
- 239000000975 dye Substances 0.000 claims description 24
- 125000000524 functional group Chemical group 0.000 claims description 22
- 239000012620 biological material Substances 0.000 claims description 19
- 125000003808 silyl group Chemical group [H][Si]([H])([H])[*] 0.000 claims description 19
- 238000010521 absorption reaction Methods 0.000 claims description 17
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 13
- 229910052710 silicon Inorganic materials 0.000 claims description 13
- 239000010703 silicon Substances 0.000 claims description 13
- 125000001424 substituent group Chemical group 0.000 claims description 13
- 230000008569 process Effects 0.000 claims description 9
- 238000006460 hydrolysis reaction Methods 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 5
- 229910000077 silane Inorganic materials 0.000 claims description 5
- 125000003545 alkoxy group Chemical group 0.000 claims description 3
- 125000004432 carbon atom Chemical group C* 0.000 claims description 3
- 150000004703 alkoxides Chemical class 0.000 claims description 2
- 239000007795 chemical reaction product Substances 0.000 claims description 2
- 239000000049 pigment Substances 0.000 claims description 2
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 64
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 57
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 49
- VYXSBFYARXAAKO-WTKGSRSZSA-N chembl402140 Chemical compound Cl.C1=2C=C(C)C(NCC)=CC=2OC2=C\C(=N/CC)C(C)=CC2=C1C1=CC=CC=C1C(=O)OCC VYXSBFYARXAAKO-WTKGSRSZSA-N 0.000 description 28
- 238000003756 stirring Methods 0.000 description 19
- 238000006243 chemical reaction Methods 0.000 description 17
- 239000000203 mixture Substances 0.000 description 17
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 13
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 description 12
- 239000012103 Alexa Fluor 488 Substances 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 238000005406 washing Methods 0.000 description 11
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical class OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 10
- 239000011541 reaction mixture Substances 0.000 description 10
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 8
- 239000013049 sediment Substances 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 7
- 239000003960 organic solvent Substances 0.000 description 7
- 238000012546 transfer Methods 0.000 description 7
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 6
- 125000000022 2-aminoethyl group Chemical group [H]C([*])([H])C([H])([H])N([H])[H] 0.000 description 5
- 239000006087 Silane Coupling Agent Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000006185 dispersion Substances 0.000 description 5
- 238000003018 immunoassay Methods 0.000 description 5
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 239000000090 biomarker Substances 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 4
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- BBEAQIROQSPTKN-UHFFFAOYSA-N pyrene Chemical compound C1=CC=C2C=CC3=CC=CC4=CC=C1C2=C43 BBEAQIROQSPTKN-UHFFFAOYSA-N 0.000 description 4
- VUFNRPJNRFOTGK-UHFFFAOYSA-M sodium;1-[4-[(2,5-dioxopyrrol-1-yl)methyl]cyclohexanecarbonyl]oxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)C1CCC(CN2C(C=CC2=O)=O)CC1 VUFNRPJNRFOTGK-UHFFFAOYSA-M 0.000 description 4
- 125000003396 thiol group Chemical group [H]S* 0.000 description 4
- 239000012112 Alexa Fluor 633 Substances 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical group ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 239000002585 base Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 125000004185 ester group Chemical group 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- IQPQWNKOIGAROB-UHFFFAOYSA-N isocyanate group Chemical group [N-]=C=O IQPQWNKOIGAROB-UHFFFAOYSA-N 0.000 description 3
- ZBKFYXZXZJPWNQ-UHFFFAOYSA-N isothiocyanate group Chemical group [N-]=C=S ZBKFYXZXZJPWNQ-UHFFFAOYSA-N 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 230000009257 reactivity Effects 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- DCQBZYNUSLHVJC-UHFFFAOYSA-N 3-triethoxysilylpropane-1-thiol Chemical compound CCO[Si](OCC)(OCC)CCCS DCQBZYNUSLHVJC-UHFFFAOYSA-N 0.000 description 2
- IKYJCHYORFJFRR-UHFFFAOYSA-N Alexa Fluor 350 Chemical compound O=C1OC=2C=C(N)C(S(O)(=O)=O)=CC=2C(C)=C1CC(=O)ON1C(=O)CCC1=O IKYJCHYORFJFRR-UHFFFAOYSA-N 0.000 description 2
- JLDSMZIBHYTPPR-UHFFFAOYSA-N Alexa Fluor 405 Chemical compound CC[NH+](CC)CC.CC[NH+](CC)CC.CC[NH+](CC)CC.C12=C3C=4C=CC2=C(S([O-])(=O)=O)C=C(S([O-])(=O)=O)C1=CC=C3C(S(=O)(=O)[O-])=CC=4OCC(=O)N(CC1)CCC1C(=O)ON1C(=O)CCC1=O JLDSMZIBHYTPPR-UHFFFAOYSA-N 0.000 description 2
- WEJVZSAYICGDCK-UHFFFAOYSA-N Alexa Fluor 430 Chemical compound CC[NH+](CC)CC.CC1(C)C=C(CS([O-])(=O)=O)C2=CC=3C(C(F)(F)F)=CC(=O)OC=3C=C2N1CCCCCC(=O)ON1C(=O)CCC1=O WEJVZSAYICGDCK-UHFFFAOYSA-N 0.000 description 2
- WHVNXSBKJGAXKU-UHFFFAOYSA-N Alexa Fluor 532 Chemical compound [H+].[H+].CC1(C)C(C)NC(C(=C2OC3=C(C=4C(C(C(C)N=4)(C)C)=CC3=3)S([O-])(=O)=O)S([O-])(=O)=O)=C1C=C2C=3C(C=C1)=CC=C1C(=O)ON1C(=O)CCC1=O WHVNXSBKJGAXKU-UHFFFAOYSA-N 0.000 description 2
- ZAINTDRBUHCDPZ-UHFFFAOYSA-M Alexa Fluor 546 Chemical compound [H+].[Na+].CC1CC(C)(C)NC(C(=C2OC3=C(C4=NC(C)(C)CC(C)C4=CC3=3)S([O-])(=O)=O)S([O-])(=O)=O)=C1C=C2C=3C(C(=C(Cl)C=1Cl)C(O)=O)=C(Cl)C=1SCC(=O)NCCCCCC(=O)ON1C(=O)CCC1=O ZAINTDRBUHCDPZ-UHFFFAOYSA-M 0.000 description 2
- IGAZHQIYONOHQN-UHFFFAOYSA-N Alexa Fluor 555 Chemical compound C=12C=CC(=N)C(S(O)(=O)=O)=C2OC2=C(S(O)(=O)=O)C(N)=CC=C2C=1C1=CC=C(C(O)=O)C=C1C(O)=O IGAZHQIYONOHQN-UHFFFAOYSA-N 0.000 description 2
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 239000000084 colloidal system Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 2
- GVEPBJHOBDJJJI-UHFFFAOYSA-N fluoranthrene Natural products C1=CC(C2=CC=CC=C22)=C3C2=CC=CC3=C1 GVEPBJHOBDJJJI-UHFFFAOYSA-N 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 229940127121 immunoconjugate Drugs 0.000 description 2
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- HQCYVSPJIOJEGA-UHFFFAOYSA-N methoxycoumarin Chemical compound C1=CC=C2OC(=O)C(OC)=CC2=C1 HQCYVSPJIOJEGA-UHFFFAOYSA-N 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 125000005372 silanol group Chemical group 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- YMYNYVDHGPDOCK-UHFFFAOYSA-N 3',6'-dihydroxy-3-oxospiro[2-benzofuran-1,9'-xanthene]-5,6-dicarboxylic acid Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=C1C=C(C(O)=O)C(C(=O)O)=C2 YMYNYVDHGPDOCK-UHFFFAOYSA-N 0.000 description 1
- ULRCHFVDUCOKTE-UHFFFAOYSA-N 3-[3-aminopropyl(diethoxy)silyl]oxybutan-1-amine Chemical compound NCCC[Si](OCC)(OCC)OC(C)CCN ULRCHFVDUCOKTE-UHFFFAOYSA-N 0.000 description 1
- NJYVEMPWNAYQQN-UHFFFAOYSA-N 5-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C21OC(=O)C1=CC(C(=O)O)=CC=C21 NJYVEMPWNAYQQN-UHFFFAOYSA-N 0.000 description 1
- IDLISIVVYLGCKO-UHFFFAOYSA-N 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein Chemical compound O1C(=O)C2=CC=C(C(O)=O)C=C2C21C1=CC(OC)=C(O)C(Cl)=C1OC1=C2C=C(OC)C(O)=C1Cl IDLISIVVYLGCKO-UHFFFAOYSA-N 0.000 description 1
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 1
- 239000012104 Alexa Fluor 500 Substances 0.000 description 1
- 239000012105 Alexa Fluor 514 Substances 0.000 description 1
- 239000012109 Alexa Fluor 568 Substances 0.000 description 1
- 239000012110 Alexa Fluor 594 Substances 0.000 description 1
- 239000012111 Alexa Fluor 610 Substances 0.000 description 1
- 239000012118 Alexa Fluor 750 Substances 0.000 description 1
- 239000012099 Alexa Fluor family Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 1
- IXQIUDNVFVTQLJ-UHFFFAOYSA-N Naphthofluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C(C=CC=1C3=CC=C(O)C=1)=C3OC1=C2C=CC2=CC(O)=CC=C21 IXQIUDNVFVTQLJ-UHFFFAOYSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 125000005370 alkoxysilyl group Chemical group 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229960000956 coumarin Drugs 0.000 description 1
- 235000001671 coumarin Nutrition 0.000 description 1
- 238000000635 electron micrograph Methods 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 125000003700 epoxy group Chemical group 0.000 description 1
- ZZRGHKUNLAYDTC-UHFFFAOYSA-N ethoxy(methyl)silane Chemical compound CCO[SiH2]C ZZRGHKUNLAYDTC-UHFFFAOYSA-N 0.000 description 1
- VYXSBFYARXAAKO-UHFFFAOYSA-N ethyl 2-[3-(ethylamino)-6-ethylimino-2,7-dimethylxanthen-9-yl]benzoate;hydron;chloride Chemical compound [Cl-].C1=2C=C(C)C(NCC)=CC=2OC2=CC(=[NH+]CC)C(C)=CC2=C1C1=CC=CC=C1C(=O)OCC VYXSBFYARXAAKO-UHFFFAOYSA-N 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 125000003827 glycol group Chemical group 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- BFXIKLCIZHOAAZ-UHFFFAOYSA-N methyltrimethoxysilane Chemical compound CO[Si](C)(OC)OC BFXIKLCIZHOAAZ-UHFFFAOYSA-N 0.000 description 1
- INJVFBCDVXYHGQ-UHFFFAOYSA-N n'-(3-triethoxysilylpropyl)ethane-1,2-diamine Chemical compound CCO[Si](OCC)(OCC)CCCNCCN INJVFBCDVXYHGQ-UHFFFAOYSA-N 0.000 description 1
- VLVOIQGDDTVQOB-UHFFFAOYSA-N n'-(3-triethoxysilylpropyl)hexane-1,6-diamine Chemical compound CCO[Si](OCC)(OCC)CCCNCCCCCCN VLVOIQGDDTVQOB-UHFFFAOYSA-N 0.000 description 1
- PHQOGHDTIVQXHL-UHFFFAOYSA-N n'-(3-trimethoxysilylpropyl)ethane-1,2-diamine Chemical compound CO[Si](OC)(OC)CCCNCCN PHQOGHDTIVQXHL-UHFFFAOYSA-N 0.000 description 1
- RRQTYXHHYIJDFB-UHFFFAOYSA-N n'-(triethoxysilylmethyl)hexane-1,6-diamine Chemical compound CCO[Si](OCC)(OCC)CNCCCCCCN RRQTYXHHYIJDFB-UHFFFAOYSA-N 0.000 description 1
- MQWFLKHKWJMCEN-UHFFFAOYSA-N n'-[3-[dimethoxy(methyl)silyl]propyl]ethane-1,2-diamine Chemical compound CO[Si](C)(OC)CCCNCCN MQWFLKHKWJMCEN-UHFFFAOYSA-N 0.000 description 1
- SJYNFBVQFBRSIB-UHFFFAOYSA-N norbornadiene Chemical compound C1=CC2C=CC1C2 SJYNFBVQFBRSIB-UHFFFAOYSA-N 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 230000001443 photoexcitation Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- WGYKZJWCGVVSQN-UHFFFAOYSA-N propylamine Chemical group CCCN WGYKZJWCGVVSQN-UHFFFAOYSA-N 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 239000001044 red dye Substances 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 229940043267 rhodamine b Drugs 0.000 description 1
- 239000001022 rhodamine dye Substances 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 150000004756 silanes Chemical class 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- LFQCEHFDDXELDD-UHFFFAOYSA-N tetramethyl orthosilicate Chemical compound CO[Si](OC)(OC)OC LFQCEHFDDXELDD-UHFFFAOYSA-N 0.000 description 1
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- JCVQKRGIASEUKR-UHFFFAOYSA-N triethoxy(phenyl)silane Chemical compound CCO[Si](OCC)(OCC)C1=CC=CC=C1 JCVQKRGIASEUKR-UHFFFAOYSA-N 0.000 description 1
- JXUKBNICSRJFAP-UHFFFAOYSA-N triethoxy-[3-(oxiran-2-ylmethoxy)propyl]silane Chemical compound CCO[Si](OCC)(OCC)CCCOCC1CO1 JXUKBNICSRJFAP-UHFFFAOYSA-N 0.000 description 1
- QQQSFSZALRVCSZ-UHFFFAOYSA-N triethoxysilane Chemical compound CCO[SiH](OCC)OCC QQQSFSZALRVCSZ-UHFFFAOYSA-N 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Silicon Compounds (AREA)
Abstract
Description
本発明は、シリカ粒子中に複数種の有機蛍光色素を内包したシリカナノ粒子、その製造方法、及びそれを用いた生体物質標識剤に関する。 The present invention relates to silica nanoparticles in which a plurality of types of organic fluorescent dyes are encapsulated in silica particles, a method for producing the same, and a biomaterial labeling agent using the same.
疾病の診断あるいは病態の把握を目的に、血液などに含まれる特定の生体分子（バイオマーカー）の検出が注目されている。 Detection of specific biomolecules (biomarkers) contained in blood or the like has been attracting attention for the purpose of diagnosing diseases or grasping disease states.
現在、生体分子の検出のために抗原抗体反応を利用した免疫分析（イムノアッセイ）が主に用いられている。酵素反応を利用した化学発光法が高感度とされ、現在主流である。 Currently, immunoassay (immunoassay) using antigen-antibody reaction is mainly used for detection of biomolecules. Chemiluminescence using an enzyme reaction is considered to be highly sensitive and is currently the mainstream.
しかしながら、生体物質である酵素を用いるため温度管理などを厳密にするなど煩雑な操作が要求され、より簡便なシステムが求められている。 However, since an enzyme that is a biological material is used, complicated operations such as strict temperature control are required, and a simpler system is required.
その一つとして予め蛍光物質標識された生体物質標識剤を用いる蛍光イムノアッセイ法が知られている。蛍光物質としては、有機色素や、量子ドットを使用することができる。化学発光法に比べ、簡便な操作で行えるものの、用いる蛍光物質の蛍光強度が非常に小さいため、検出感度が十分でなかった。 As one of them, a fluorescent immunoassay method using a biological substance labeling agent that has been previously labeled with a fluorescent substance is known. As the fluorescent material, organic dyes or quantum dots can be used. Although it can be performed by a simple operation as compared with the chemiluminescence method, the fluorescence intensity of the fluorescent substance used is very small, so that the detection sensitivity is not sufficient.
近年、より超早期での疾病の診断を行うため、すなわち、極微量のバイオマーカーの検出を行うため、より高蛍光強度を有する蛍光物質で標識された生体物質標識剤が求められている。その一つとして複数の蛍光物質を一つのシリカナノ粒子に内包させる技術が開示されている（例えば特許文献１及び２参照）。 In recent years, in order to diagnose a disease at an extremely early stage, that is, to detect a very small amount of a biomarker, a biological material labeling agent labeled with a fluorescent material having higher fluorescence intensity has been demanded. As one of the techniques, a technique for encapsulating a plurality of fluorescent substances in one silica nanoparticle is disclosed (for example, see Patent Documents 1 and 2).
蛍光物質として、有機蛍光色素を内包したシリカナノ粒子用いて蛍光イムノアッセイを行った場合、一般に有機蛍光色素が吸収極大波長と蛍光極大波長の差（以下「ストークスシフト」という。）が１０〜２０ｎｍと小さいので、蛍光検出器へ励起光の一部が入りこんでしまう。入りこんだ励起光強度（Ｎ）が大きいと、本来検出すべき蛍光強度（Ｓ）との比（Ｓ／Ｎ比）を小さくなってしまい、所望とする高感度検出の妨げとなる。 When fluorescent immunoassay is performed using silica nanoparticles encapsulating an organic fluorescent dye as a fluorescent substance, the organic fluorescent dye generally has a small difference between the absorption maximum wavelength and the fluorescence maximum wavelength (hereinafter referred to as “Stokes shift”) as small as 10 to 20 nm. Therefore, a part of the excitation light enters the fluorescence detector. If the excitation light intensity (N) that enters is large, the ratio (S / N ratio) to the fluorescence intensity (S) that should be detected becomes small, which hinders the desired high-sensitivity detection.
これを解決するため特許文献２には、蛍光波長の異なる色素を内包し、それらの蛍光共鳴エネルギー移動（Ｆｌｕｏｒｅｓｃｅｎｃｅ Ｒｅｓｏｎａｎｔ Ｅｎｅｒｇｙ Ｔｒａｎｓｆｅｒ；以下「ＦＲＥＴ」と略す。）の利用が開示されている。すなわち、蛍光波長の短い第１の色素を励起し、励起された第１の色素からより蛍光波長の長い第２の色素へエネルギーが移動、そして第２の色素から蛍光が発することで、励起光と蛍光の波長が大きく離れ、擬似的にストークスシフトが大きい色素を内包したシリカナノ粒子を作るものである。 In order to solve this problem, Patent Document 2 discloses the use of fluorescence resonance energy transfer (hereinafter abbreviated as “FRET”) including dyes having different fluorescence wavelengths. That is, the first dye having a short fluorescence wavelength is excited, energy is transferred from the excited first dye to the second dye having a longer fluorescence wavelength, and fluorescence is emitted from the second dye. Thus, silica nanoparticles encapsulating a dye having a large fluorescence wavelength and a large Stokes shift are produced.
ところが、本発明者らが、特許文献２に開示されている方法により、第１の色素としてＢＯＤＩＰＹ ＦＬ（インビトロジェン社登録商標）、第２の色素としてテトラメチルローダミンそれぞれを別個に添加する方法により、共内包したシリカナノ粒子を作製し、ＢＯＤＩＰＹ ＦＬを励起し、テトラメチルローダミンの蛍光の検出を試みたところ、テトラメチルローダミン由来の蛍光は微弱であった。このことは、第１の色素から第２の色素へのＦＲＥＴ効率が著しく低いことを意味しており、さらなる改善が必要であることが分かった。 However, by the method disclosed in Patent Document 2, the present inventors separately added BODIPY FL (registered trademark of Invitrogen) as the first dye and tetramethylrhodamine as the second dye, When co-encapsulated silica nanoparticles were prepared and BODIPY FL was excited to detect the fluorescence of tetramethylrhodamine, the fluorescence derived from tetramethylrhodamine was weak. This means that the FRET efficiency from the first dye to the second dye is significantly lower, and it has been found that further improvement is necessary.
本発明は、上記問題・状況にかんがみてなされたものであり、その解決課題は、ストークスシフトが大きく、かつ発光強度の高い有機蛍光色素内包シリカナノ粒子及びその製造方法を提供することである。また、それを用いた生体物質標識剤を提供することにある。 The present invention has been made in view of the above problems and situations, and a solution to that problem is to provide organic fluorescent dye-containing silica nanoparticles having a large Stokes shift and high emission intensity, and a method for producing the same. Another object is to provide a biological substance labeling agent using the same.
本発明者等は、上記課題を解決すべく鋭意検討の結果、第１有機蛍光色素と、第１有機蛍光色素より蛍光波長の長い第２有機蛍光色素とを同一分子に共有結合により結合させ、さらにその分子がシリカ粒子と共有結合により結合した有機蛍光色素内包シリカナノ粒子を用いた場合、ＦＲＥＴ効率が向上することにより、第２有機蛍光色素由来の蛍光強度が飛躍的に向上し、高感度検出が可能となることを見出し、本発明に至った。 As a result of intensive studies to solve the above-mentioned problems, the present inventors bound the first organic fluorescent dye and the second organic fluorescent dye having a longer fluorescence wavelength than the first organic fluorescent dye to the same molecule by a covalent bond, Furthermore, when organic fluorescent dye-encapsulated silica nanoparticles whose molecules are covalently bonded to silica particles are used, the FRET efficiency is improved, and the fluorescence intensity derived from the second organic fluorescent dye is dramatically improved, and high sensitivity detection is achieved. Has been found to be possible, leading to the present invention.
すなわち、本発明に係る上記課題は、以下の手段により解決される。 That is, the said subject which concerns on this invention is solved by the following means.
１．シリカ粒子中に、蛍光波長が相異する二種以上の有機蛍光色素分子同士を連結して形成された連結分子を内包した有機蛍光色素内包シリカナノ粒子であって、当該連結分子がシリカナノ粒子と結合していることを特徴とする有機蛍光色素内包シリカナノ粒子。 1. An organic fluorescent dye-encapsulated silica nanoparticle encapsulating a linking molecule formed by linking two or more organic fluorescent dye molecules having different fluorescence wavelengths in a silica particle, and the linking molecule binds to the silica nanoparticle Organic fluorescent dye-encapsulated silica nanoparticles, characterized in that
２．連結された前記二種以上の有機蛍光色素分子のうちの一種の有機蛍光色素部分（「第１有機蛍光色素部分」という。）が吸収した光エネルギーが、別種の有機蛍光色素部分（「第２有機蛍光色素部分」という。）に蛍光共鳴エネルギー移動により移動し、光吸収した当該第１有機蛍光色素部分とは異なる当該第２有機蛍光色素部分から蛍光が発することを特徴とする前記第１項に記載の有機蛍光色素内包シリカナノ粒子。 2. The light energy absorbed by a kind of organic fluorescent dye part (referred to as “first organic fluorescent dye part”) of the two or more kinds of linked organic fluorescent dye molecules is converted into another kind of organic fluorescent dye part (“second organic fluorescent dye part”). The first item is characterized in that the fluorescence is emitted from the second organic fluorescent dye part different from the first organic fluorescent dye part that has been moved and absorbed by the fluorescence resonance energy transfer to the organic fluorescent dye part. The organic fluorescent dye-encapsulated silica nanoparticles described in 1.
３．前記第１有機蛍光色素部分の吸収極大波長と第２有機蛍光色素部分の蛍光極大波長が、２０ｎｍ以上離れていることを特徴とする前記第２項に記載の有機蛍光色素内包シリカナノ粒子。 3. 3. The organic fluorescent dye-encapsulated silica nanoparticles according to item 2, wherein the absorption maximum wavelength of the first organic fluorescent dye part and the fluorescent maximum wavelength of the second organic fluorescent dye part are separated by 20 nm or more.
４．前記第１項から第３項までのいずれか一項に記載の有機蛍光色素内包シリカナノ粒子を製造する有機蛍光色素内包シリカナノ粒子の製造方法であって、下記工程（ａ）及び工程（ｂ）を含んでなることを特徴とする有機蛍光色素内包シリカナノ粒子の製造方法。
工程（ａ）：同一分子内に二種以上のアミノ基と、少なくとも一種の加水分解性置換基を有するシリル基を有する分子と、当該アミノ基と反応する官能基を有する有機蛍光色素とを反応させる工程、
工程（ｂ）：前記工程（ａ）で得られた反応生成物を含ケイ素アルコキシドと混合し、塩基性条件下加水分解反応を行う工程。
4). It is a manufacturing method of the organic fluorescent dye inclusion silica nanoparticle which manufactures the organic fluorescent dye inclusion silica nanoparticle as described in any one of said 1st term | claim-3rd, Comprising: The following process (a) and process (b) are carried out. A method for producing organic fluorescent dye-encapsulated silica nanoparticles, comprising:
Step (a): Reacting a molecule having two or more amino groups in the same molecule, a molecule having a silyl group having at least one hydrolyzable substituent, and an organic fluorescent dye having a functional group that reacts with the amino group The process of
Step (b): A step of mixing the reaction product obtained in the step (a) with a silicon-containing alkoxide and performing a hydrolysis reaction under basic conditions.
５．前記同一分子内に二種以上のアミノ基と、少なくとも一種の加水分解性置換基を有するシリル基を有する分子が下記一般式（１）であらわされるシラン化合物であることを特徴とする前記第４項に記載の有機蛍光色素内包シリカナノ粒子の製造方法。
一般式（１）：ＮＨ２−（ＣＨ２）ｎ−ＮＨ−（ＣＨ２）ｍ−ＳｉＲｌ（ＯＲ１）ｋ
（式中、ｎ及びｍは１〜１２の整数を表し、互いに同一でも異なっていてもよい。ｌ及びｋはｌ＋ｋ＝４を満たし、ｌは０〜３の整数、ｋは１〜４の整数を表す。Ｒ１は非加水分解性の置換基を表し、ＯＲ１は加水分解性の炭素数１〜６のアルコキシ基を表し、複数ある場合、互いに同一でも異なっていてもよい。）
６．前記第１項から第３項までのいずれか一項に記載の有機蛍光色素内包シリカナノ粒子を用いた生体物質標識剤であって、当該有機蛍光色素内包シリカナノ粒子と分子標識物質とが、有機分子を介して結合されていることを特徴とする生体物質標識剤。
5). Wherein the molecule having a silyl group having at least one amino group and at least one hydrolyzable substituent in the same molecule is a silane compound represented by the following general formula (1): The manufacturing method of the organic fluorescent dye inclusion | inner_cover silica nanoparticle of description to term.
Formula (1): NH 2 - ( CH 2) n -NH- (CH 2) m -SiR l (OR 1) k
(In the formula, n and m each represents an integer of 1 to 12, and may be the same or different. L and k satisfy l + k = 4, l is an integer of 0 to 3, and k is an integer of 1 to 4) R 1 represents a non-hydrolyzable substituent, OR 1 represents a hydrolyzable alkoxy group having 1 to 6 carbon atoms, and when there are a plurality of them, they may be the same or different from each other.
6). A biological substance labeling agent using the organic fluorescent dye-encapsulated silica nanoparticles according to any one of Items 1 to 3, wherein the organic fluorescent dye-encapsulated silica nanoparticles and the molecular labeling substance are organic molecules. Biological substance labeling agent characterized by being bonded via.
本発明により、ストークスシフトが大きく、かつ発光強度の高い有機蛍光色素内包シリカナノ粒子及びその製造方法を提供することができる。また、それを用いた生体物質標識剤を提供することができる。 According to the present invention, it is possible to provide organic fluorescent dye-encapsulated silica nanoparticles having a large Stokes shift and high emission intensity, and a method for producing the same. Moreover, the biological material labeling agent using the same can be provided.
従来法の有機蛍光色素内包シリカナノ粒子では、第１有機蛍光色素と第２有機蛍光色素をそれぞれ別個に添加することから、第１有機蛍光色素と第２有機蛍光色素を空間的に十分近づけることができないためＦＲＥＴ効率が低いと考えられる。これに対し、本発明では、第１有機蛍光色素と第２有機蛍光色素を空間的に十分近づけることができるためＦＲＥＴ効率が飛躍的に高いと考えられる。 In the conventional organic fluorescent dye-encapsulated silica nanoparticles, the first organic fluorescent dye and the second organic fluorescent dye are added separately, so that the first organic fluorescent dye and the second organic fluorescent dye can be spatially sufficiently close to each other. It is considered that FRET efficiency is low because it is not possible. In contrast, in the present invention, the first organic fluorescent dye and the second organic fluorescent dye can be spatially sufficiently close to each other, so that the FRET efficiency is considered to be remarkably high.
本発明の有機蛍光色素内包シリカナノ粒子は、シリカ粒子中に、蛍光波長が相異する二種以上の有機蛍光色素分子同士を連結して形成された連結分子を内包した有機蛍光色素内包シリカナノ粒子であって、当該連結分子がシリカナノ粒子と結合していることを特徴とする。この特徴は、請求項１から請求項６までの請求項に係る発明に共通する技術的特徴である。 The organic fluorescent dye-encapsulated silica nanoparticle of the present invention is an organic fluorescent dye-encapsulated silica nanoparticle encapsulating a linking molecule formed by linking two or more organic fluorescent dye molecules having different fluorescence wavelengths in the silica particle. The linking molecule is bonded to silica nanoparticles. This feature is a technical feature common to the inventions according to claims 1 to 6.
本発明の実施形態としては、本発明の効果発現の観点から、連結された前記二種以上の有機蛍光色素分子のうちの一種の有機蛍光色素部分（「第１有機蛍光色素部分」という。）が吸収した光エネルギーが、別種の有機蛍光色素部分（「第２有機蛍光色素部分」という。）に（蛍光共鳴エネルギー移動により）移動し、光吸収した当該第１有機蛍光色素部分とは異なる当該第２有機蛍光色素部分から蛍光が発することが好ましい。また、当該第１有機蛍光色素部分の吸収極大波長と当該第２有機蛍光色素部分の蛍光極大波長が、２０ｎｍ以上離れていることが好ましい。 As an embodiment of the present invention, from the viewpoint of manifestation of the effect of the present invention, a kind of organic fluorescent dye part (referred to as “first organic fluorescent dye part”) of the two or more kinds of linked organic fluorescent dye molecules. The light energy absorbed by is transferred to another type of organic fluorescent dye portion (referred to as “second organic fluorescent dye portion”) (by fluorescence resonance energy transfer), and is different from the first organic fluorescent dye portion that has absorbed the light. It is preferable that fluorescence is emitted from the second organic fluorescent dye portion. Moreover, it is preferable that the absorption maximum wavelength of the said 1st organic fluorescent dye part and the fluorescence maximum wavelength of the said 2nd organic fluorescent dye part are 20 nm or more apart.
本発明の有機蛍光色素内包シリカナノ粒子を製造する有機蛍光色素内包シリカナノ粒子の製造方法としては、前記工程（ａ）及び工程（ｂ）を含んでなる製造方法であることが好ましい。当該製造方法の場合、前記同一分子内に二種以上のアミノ基と、少なくとも一種の加水分解性置換基を有するシリル基を有する分子が前記一般式（１）であらわされるシラン化合物であることが好ましい。 As a manufacturing method of the organic fluorescent dye inclusion silica nanoparticle which manufactures the organic fluorescent dye inclusion silica nanoparticle of this invention, it is preferable that it is a manufacturing method which includes the said process (a) and a process (b). In the case of the production method, the molecule having a silyl group having two or more amino groups and at least one hydrolyzable substituent in the same molecule is a silane compound represented by the general formula (1). preferable.
本発明の有機蛍光色素内包シリカナノ粒子は、当該シリカナノ粒子と分子標識物質とが、有機分子を介して結合されてなる生体物質標識剤に好適に用いることができる。 The organic fluorescent dye-encapsulated silica nanoparticles of the present invention can be suitably used for a biological material labeling agent in which the silica nanoparticles and a molecular labeling substance are bonded via organic molecules.
以下、本発明とその構成要素、及び発明を実施するための形態について詳細な説明をする。 Hereinafter, the present invention, its components, and modes for carrying out the invention will be described in detail.
〔複数種の有機蛍光色素の連結〕
本発明の有機蛍光色素内包シリカナノ粒子は、シリカ粒子中に、蛍光波長が相異する二種以上の有機蛍光色素分子同士を連結して形成された連結分子を内包していることを特徴とする。
[Concatenation of multiple organic fluorescent dyes]
The organic fluorescent dye-encapsulated silica nanoparticles of the present invention are characterized in that the silica particles contain a linking molecule formed by linking two or more organic fluorescent dye molecules having different fluorescence wavelengths. .
複数種の有機蛍光色素をシリカナノ粒子に内包させるにあたり、両者の距離を近づけるために複数種の有機蛍光色素を同一分子内に含むこと必要である。すなわち、それぞれの有機蛍光色素同士は、共有結合、イオン結合、水素結合などを介し連結してひとつの連結分子を形成していることを要するが、化学的安定性から共有結合を介して結合していることが好ましい。 In order to enclose a plurality of types of organic fluorescent dyes in silica nanoparticles, it is necessary to include a plurality of types of organic fluorescent dyes in the same molecule in order to reduce the distance between them. In other words, each organic fluorescent dye must be linked via a covalent bond, an ionic bond, a hydrogen bond, etc. to form one linked molecule. It is preferable.
また、複数種の有機蛍光色素を連結させた分子が、当該シリカナノ粒子と結合していることが必要である。結合の方法は、共有結合、イオン結合、水素結合などがあげられるが、化学的安定性から共有結合であるのが好ましい。当該色素を連結させた分子がシリカ粒子と結合していないと、本発明で得られる有機蛍光色素内包シリカナノ粒子を水分散液として保存しているうちに、徐々に当該色素を連結させた分子が漏洩するおそれがあり、生体物質標識剤としての応用上好ましくない。 Moreover, the molecule | numerator which connected multiple types of organic fluorescent pigment | dye needs to couple | bond with the said silica nanoparticle. Examples of the bonding method include a covalent bond, an ionic bond, and a hydrogen bond, and a covalent bond is preferable from the viewpoint of chemical stability. When the molecule to which the dye is linked is not bonded to the silica particles, the organic fluorescent dye-encapsulated silica nanoparticles obtained in the present invention are stored as an aqueous dispersion while the molecule to which the dye is gradually linked is There is a possibility of leakage, which is not preferable for application as a biological material labeling agent.
本発明の有機蛍光色素内包シリカナノ粒子に用いられる有機蛍光色素としては、２００〜７００ｎｍの範囲内の波長の紫外〜近赤外光により励起されたときに、４００〜９００ｎｍの範囲内の波長の可視〜近赤外光の発光を示す態様の有機蛍光色素であることが好ましい。 The organic fluorescent dye used in the organic fluorescent dye-encapsulated silica nanoparticles of the present invention has a wavelength in the range of 400 to 900 nm when excited by ultraviolet to near infrared light having a wavelength in the range of 200 to 700 nm. -It is preferable that it is an organic fluorescent dye of the aspect which shows light emission of near-infrared light.
有機蛍光色素としては、フルオレセイン系色素分子、ローダミン系色素分子、Ａｌｅｘａ Ｆｌｕｏｒ（Ｉｎｖｉｔｒｏｇｅｎ社製）系色素分子、ＢＯＤＩＰＹ（Ｉｎｖｉｔｒｏｇｅｎ社製）系色素分子、カスケード系色素分子、クマリン系色素分子、エオジン系色素分子、ＮＢＤ系色素分子、ピレン系色素分子、Ｔｅｘａｓ Ｒｅｄ系色素分子、シアニン系色素分子等を挙げることができる。 Examples of organic fluorescent dyes include fluorescein dye molecules, rhodamine dye molecules, Alexa Fluor (Invitrogen) dye molecules, BODIPY (Invitrogen) dye molecules, cascade dye molecules, coumarin dye molecules, and eosin dyes. Examples include molecules, NBD dye molecules, pyrene dye molecules, Texas Red dye molecules, and cyanine dye molecules.
具体的には、５−カルボキシ−フルオレセイン、６−カルボキシ−フルオレセイン、５，６−ジカルボキシ−フルオレセイン、６−カルボキシ−２′，４，４′，５′，７，７′−ヘキサクロロフルオレセイン、６−カルボキシ−２′，４，７，７′−テトラクロロフルオレセイン、６−カルボキシ−４′，５′−ジクロロ−２′，７′−ジメトキシフルオレセイン、ナフトフルオレセイン、５−カルボキシ−ローダミン、６−カルボキシ−ローダミン、５，６−ジカルボキシ−ローダミン、ローダミン ６Ｇ、テトラメチルローダミン、Ｘ−ローダミン、及びＡｌｅｘａ Ｆｌｕｏｒ ３５０，Ａｌｅｘａ Ｆｌｕｏｒ ４０５、Ａｌｅｘａ Ｆｌｕｏｒ ４３０、Ａｌｅｘａ Ｆｌｕｏｒ ４８８、Ａｌｅｘａ Ｆｌｕｏｒ ５００、Ａｌｅｘａ Ｆｌｕｏｒ ５１４、Ａｌｅｘａ Ｆｌｕｏｒ ５３２、Ａｌｅｘａ Ｆｌｕｏｒ ５４６、Ａｌｅｘａ Ｆｌｕｏｒ ５５５、Ａｌｅｘａ Ｆｌｕｏｒ ５６８、Ａｌｅｘａ Ｆｌｕｏｒ ５９４、Ａｌｅｘａ Ｆｌｕｏｒ ６１０、Ａｌｅｘａ Ｆｌｕｏｒ ６３３、Ａｌｅｘａ Ｆｌｕｏｒ ６３５、Ａｌｅｘａ Ｆｌｕｏｒ ６４７、Ａｌｅｘａ Ｆｌｕｏｒ ６６０、Ａｌｅｘａ Ｆｌｕｏｒ ６８０、Ａｌｅｘａ Ｆｌｕｏｒ ７００、Ａｌｅｘａ Ｆｌｕｏｒ ７５０、ＢＯＤＩＰＹ ＦＬ，ＢＯＤＩＰＹ ＴＭＲ、ＢＯＤＩＰＹ ４９３／５０３、ＢＯＤＩＰＹ ５３０／５５０、ＢＯＤＩＰＹ ５５８／５６８、ＢＯＤＩＰＹ ５６４／５７０、ＢＯＤＩＰＹ ５７６／５８９、ＢＯＤＩＰＹ ５８１／５９１、ＢＯＤＩＰＹ ６３０／６５０、ＢＯＤＩＰＹ ６５０／６６５（以上Ｉｎｖｉｔｒｏｇｅｎ社製）、メトキシクマリン、エオジン、ＮＢＤ、ピレン、Ｃｙ５、Ｃｙ５．５、Ｃｙ７等を挙げることができる。 Specifically, 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein, 6-carboxy-2 ', 4,4', 5 ', 7,7'-hexachlorofluorescein, 6 -Carboxy-2 ', 4,7,7'-tetrachlorofluorescein, 6-carboxy-4', 5'-dichloro-2 ', 7'-dimethoxyfluorescein, naphthofluorescein, 5-carboxy-rhodamine, 6-carboxy Rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, and Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 633, Alexa Fluor 633 , Alexa Fluor 750, BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, 3030 ODIPY 650/665 (all manufactured by Invitrogen), methoxy coumarin, eosin, NBD, pyrene, Cy5, Cy5.5, can be mentioned Cy7 like.
複数種の有機蛍光色素の組み合わせを選択するにあたり、特定の有機蛍光色素が吸収した光エネルギーが、別の一種以上の有機蛍光色素間を蛍光共鳴エネルギー移動により移動し、光吸収したし有機蛍光色素とは異なる有機蛍光色素から蛍光が発することが好ましい。 In selecting a combination of multiple types of organic fluorescent dyes, the light energy absorbed by a specific organic fluorescent dye is transferred between other types of organic fluorescent dyes by transfer of fluorescence resonance energy, and the light is absorbed. It is preferable that fluorescence is emitted from an organic fluorescent dye different from the above.
以下、二種の有機蛍光色素の組み合わせを例に具体例を説明するが、三種以上の有機蛍光色素の組み合わせの場合も同様である。 Hereinafter, a specific example will be described taking a combination of two types of organic fluorescent dyes as an example, but the same applies to a combination of three or more types of organic fluorescent dyes.
第１の有機蛍光色素が吸収した光励起エネルギーが、第２の有機蛍光色素へ蛍光共鳴エネルギー移動により移動し、第２の有機蛍光色素から蛍光を発するように選択するのが好ましい。選択にあたり第１の有機蛍光色素の蛍光スペクトルと、第２の有機蛍光色素の吸収スペクトルを測定し、両者の重なりが十分大きいことが好ましい。さらに第１の有機蛍光色素の吸収極大波長と第２の有機蛍光色素の蛍光極大波長が、２０ｎｍ以上離れていることが好ましく、２０ｎｍ以上、９０ｎｍ以下離れていることがより好ましい。２０ｎｍ以上とすることで、従来の単一の有機蛍光色素のストークスシフトより大きくなり、本発明の効果の発現の点でより好ましい。 It is preferable that the photoexcitation energy absorbed by the first organic fluorescent dye is selected so as to move to the second organic fluorescent dye by fluorescence resonance energy transfer and emit fluorescence from the second organic fluorescent dye. In selection, it is preferable that the fluorescence spectrum of the first organic fluorescent dye and the absorption spectrum of the second organic fluorescent dye are measured, and the overlap between them is sufficiently large. Furthermore, the absorption maximum wavelength of the first organic fluorescent dye and the fluorescence maximum wavelength of the second organic fluorescent dye are preferably separated by 20 nm or more, more preferably 20 nm or more and 90 nm or less. By setting it to 20 nm or more, it becomes larger than the Stokes shift of the conventional single organic fluorescent dye, and is more preferable in terms of manifestation of the effect of the present invention.
具体的には、第１の有機蛍光色素として、蛍光極大波長が波長４９５ｎｍ付近にあるフルオレセインを選択した場合、蛍光共鳴エネルギー移動が可能な第２の有機蛍光色素としてはローダミン６Ｇが適用可能である。それぞれフルオレセイン、ローダミン６Ｇに限らず蛍光極大波長の近似した他の有機蛍光色素を用いてもよい。たとえば、フルオレセインの代わりに、ＢＯＤＩＰＹ ＦＬ、ＡＬＥＸＡ Ｆｌｕｏｒ ４８８、ＨｉＬｙｔｅ Ｆｌｕｏｒ ４８８、ＤｙＬｉｇｈｔ ４８８などが、ローダミン６Ｇの代わりには、ＴＡＭＲＡ、ローダミンＢなどがあげられる。 Specifically, when fluorescein having a fluorescence maximum wavelength near 495 nm is selected as the first organic fluorescent dye, rhodamine 6G is applicable as the second organic fluorescent dye capable of fluorescence resonance energy transfer. . In addition to fluorescein and rhodamine 6G, other organic fluorescent dyes having approximate fluorescence maximum wavelengths may be used. For example, BODIPY FL, ALEXA Fluor 488, HiLyte Fluor 488, DyLight 488, etc. are used instead of fluorescein, and TAMRA, Rhodamine B, etc. are mentioned instead of Rhodamine 6G.
また、第１の有機蛍光色素としてローダミン６Ｇを選択した場合、第２の有機蛍光色素としてＴＡＭＲＡが適用可能である。 When rhodamine 6G is selected as the first organic fluorescent dye, TAMRA can be applied as the second organic fluorescent dye.
同一分子内に複数種の有機蛍光色素が連結した分子を作製する方法は、複数の反応性官能基と少なくとも一種の加水分解性置換基を有するシリル基を有する分子と前記反応性官能基と反応しうる官能基をもつ有機蛍光色素との反応を用いることができる。 A method of preparing a molecule in which a plurality of types of organic fluorescent dyes are linked in the same molecule is a method of reacting a molecule having a plurality of reactive functional groups and a silyl group having at least one hydrolyzable substituent with the reactive functional group. Reaction with an organic fluorescent dye having a functional group that can be used can be used.
前記反応性官能基としては、アミノ基、メルカプト基、マレイミド基、イソシアネート基、イソチオシアネート基、カルボキシル基、Ｎ−ヒドロキシスクシンイミド基など活性エステル基があげられる。中でも安定性や反応性からアミノ基が好ましい。 Examples of the reactive functional group include active ester groups such as amino groups, mercapto groups, maleimide groups, isocyanate groups, isothiocyanate groups, carboxyl groups, and N-hydroxysuccinimide groups. Of these, amino groups are preferred from the standpoint of stability and reactivity.
複数のアミノ基と少なくとも一種の加水分解性置換基を有するシリル基を有する分子は、特に限定されるものではないが、二種の有機蛍光色素を用いる場合、下記一般式（１）であらわされる分子を用いるのが、有機蛍光色素間の距離、シリル基の加水分解反応性などの観点から好適である。 The molecule having a silyl group having a plurality of amino groups and at least one hydrolyzable substituent is not particularly limited, but when two organic fluorescent dyes are used, the molecule is represented by the following general formula (1). It is preferable to use molecules from the viewpoints of the distance between organic fluorescent dyes, the hydrolysis reactivity of silyl groups, and the like.
一般式（１）：ＮＨ２−（ＣＨ２）ｎ−ＮＨ−（ＣＨ２）ｍ−ＳｉＲｌ（ＯＲ１）ｋ
（式中、ｎ及びｍは１〜１２の整数を表し、互いに同一でも異なっていてもよい。ｌ及びｋはｌ＋ｋ＝４を満たし、ｌは０〜３の整数、ｋは１〜４の整数を表す。Ｒ１は非加水分解性の置換基を表し、ＯＲ１は加水分解性の炭素数１〜６のアルコキシ基を表し、複数ある場合、互いに同一でも異なっていてもよい。）
具体的には、Ｎ−（２−アミノエチル）−３−アミノプロピルメチルジメトキシシラン、Ｎ−（２−アミノエチル）−３−アミノプロピルトリエトキシシラン、Ｎ−（２−アミノエチル）−３−アミノプロピルトリメトキシシラン、Ｎ−（６−アミノヘキシル）アミノメチルトリエトキシシラン、Ｎ−（６−アミノヘキシル）アミノプロピルトリエトキシシラン、Ｎ−（２−アミノエチル）−１１−アミノウンデシルトリメトキシシラン（以上Ｇｅｌｅｓｔ社製）などをあげることができる。
Formula (1): NH 2 - ( CH 2) n -NH- (CH 2) m -SiR l (OR 1) k
(In the formula, n and m each represents an integer of 1 to 12, and may be the same or different. L and k satisfy l + k = 4, l is an integer of 0 to 3, and k is an integer of 1 to 4) R 1 represents a non-hydrolyzable substituent, OR 1 represents a hydrolyzable alkoxy group having 1 to 6 carbon atoms, and when there are a plurality of them, they may be the same or different from each other.
Specifically, N- (2-aminoethyl) -3-aminopropylmethyldimethoxysilane, N- (2-aminoethyl) -3-aminopropyltriethoxysilane, N- (2-aminoethyl) -3- Aminopropyltrimethoxysilane, N- (6-aminohexyl) aminomethyltriethoxysilane, N- (6-aminohexyl) aminopropyltriethoxysilane, N- (2-aminoethyl) -11-aminoundecyltrimethoxy Examples thereof include silane (manufactured by Gelest Co., Ltd.).
一方前記反応性官能基と反応しうる官能基をもつ有機蛍光色素としては、アミノ基、メルカプト基、マレイミド基、イソシアネート基、イソチオシアネート基、カルボキシル基、Ｎ−ヒドロキシスクシンイミド基など活性エステル基をもつものがあげられる。なかでも上述したようにアミノ基を用いた場合、有機蛍光色素もアミノ基と反応する官能基イソシアネート基、イソチオシアネート基、カルボキシル基、Ｎ−ヒドロキシスクシンイミド基など活性エステル基が好適である。 On the other hand, the organic fluorescent dye having a functional group capable of reacting with the reactive functional group has an active ester group such as amino group, mercapto group, maleimide group, isocyanate group, isothiocyanate group, carboxyl group, and N-hydroxysuccinimide group. Things can be given. In particular, when an amino group is used as described above, an active ester group such as a functional group isocyanate group, an isothiocyanate group, a carboxyl group, or an N-hydroxysuccinimide group that reacts with the amino group is preferable.
同一分子内に複数種の有機蛍光色素が連結した分子は、複数の反応性官能基と少なくとも一種の加水分解性置換基を有するシリル基を有する分子を有機溶媒に溶解せたところに、前記反応性官能基と反応しうる官能基をもつ有機蛍光色素複数種とを、順次添加、混合することで作製できる。複数種の有機蛍光色素を順次反応させるため、複数の反応性官能基と少なくとも一種の加水分解性置換基を有するシリル基を有する分子に対し、官能基のモル等量あたり、１等量ずつ前記反応性官能基と反応しうる官能基をもつ有機蛍光色素を添加する。 A molecule in which plural kinds of organic fluorescent dyes are linked in the same molecule is obtained by dissolving a molecule having a plurality of reactive functional groups and a silyl group having at least one hydrolyzable substituent in an organic solvent. It can be produced by sequentially adding and mixing a plurality of organic fluorescent dyes having a functional group capable of reacting with a functional functional group. In order to sequentially react a plurality of types of organic fluorescent dyes, the molecule having a silyl group having a plurality of reactive functional groups and at least one hydrolyzable substituent, one equivalent per mole equivalent of the functional groups An organic fluorescent dye having a functional group capable of reacting with a reactive functional group is added.
反応時には必要により反応を促進させる添加剤、触媒などを用いてもよい、たとえば前記反応性官能基がアミノ基、有機蛍光色素がカルボキシル基を持つ場合、縮合剤を用いてもよい。 In the reaction, an additive or a catalyst for promoting the reaction may be used if necessary. For example, when the reactive functional group has an amino group and the organic fluorescent dye has a carboxyl group, a condensing agent may be used.
用いられる有機溶媒としては、シリル基上の加水分解性基との反応性がないものであればよく、たとえば、テトラヒドロフラン、ジメチルスルホキシド、ジメチルホルムアミドなどをあげることができる。 The organic solvent used is not particularly limited as long as it has no reactivity with the hydrolyzable group on the silyl group, and examples thereof include tetrahydrofuran, dimethyl sulfoxide, dimethylformamide and the like.
反応温度は特に限定されるものではないが、−２０〜５０℃の間で行うことができる。反応温度を各段階でかえることで複数種有機蛍光色素を順次反応させることができる。 Although reaction temperature is not specifically limited, It can carry out between -20-50 degreeC. By changing the reaction temperature at each stage, a plurality of organic fluorescent dyes can be reacted sequentially.
反応時間は、１時間以上５０時間以下であることが好ましい、これより短いと反応は完結しておらず、収率が低下する。またこれより長いと反応が進行しすぎて、不溶物が形成することがある。 The reaction time is preferably 1 hour or more and 50 hours or less. If the reaction time is shorter than this, the reaction is not completed and the yield decreases. On the other hand, if the length is longer than this, the reaction proceeds too much, and insoluble matter may be formed.
反応終了後は、精製することなく次工程へ用いてもよい。必要に応じて、常法により再結晶、カラムクロマトグラフィーなどによる精製を行ってもよい。 After completion of the reaction, it may be used for the next step without purification. If necessary, purification by recrystallization, column chromatography or the like may be performed by a conventional method.
〔シリカナノ粒子の製造方法〕
例えば、ジャーナル・オブ・コロイドサイエンス ２６巻、６２ページ（１９６８年）に記載されている、アンモニア水などを用いたアルカリ性条件下でテトラエトキシシランなどの含ケイ素アルコキシド化合物の加水分解を行う「ストーバー法」と呼ばれる方法により製造することが好ましい。粒径は、添加する水、エタノール、アルカリ量などについて公知の反応条件を適用することで自在に調整でき、平均粒径３０〜８００ｎｍ程度にできる。また粒径のばらつきを示す変動係数は２０％以下とすることができる。
[Method for producing silica nanoparticles]
For example, as described in Journal of Colloid Science, Vol. 26, p. 62 (1968), a “Stober method for hydrolyzing a silicon-containing alkoxide compound such as tetraethoxysilane under an alkaline condition using aqueous ammonia or the like. It is preferable to manufacture by the method called. The particle size can be freely adjusted by applying known reaction conditions for the water, ethanol, alkali amount, etc. to be added, and the average particle size can be about 30 to 800 nm. The coefficient of variation indicating the variation in particle size can be 20% or less.
本発明において、平均粒径とは、走査型電子顕微鏡（ＳＥＭ）を用いて電子顕微鏡写真を撮影し十分な数の粒子について断面積を計測し、その計測値を相当する円の面積としたときの直径を粒径として求めた。本願においては、１０００個の粒子の粒径の算術平均を平均粒径とした。変動係数も、１０００個の粒子の粒径分布から算出した値とした。 In the present invention, the average particle diameter is obtained when an electron micrograph is taken using a scanning electron microscope (SEM), the cross-sectional area is measured for a sufficient number of particles, and the measured value is defined as the area of a corresponding circle. The diameter was determined as the particle size. In the present application, the arithmetic average of the particle sizes of 1000 particles is defined as the average particle size. The coefficient of variation was also a value calculated from the particle size distribution of 1000 particles.
〔有機蛍光色素内包シリカナノ粒子の製造方法〕
本発明の蛍光体内包シリカナノ粒子は、公知の方法例えば、非特許文献（ラングミュア ８巻、２９２１ページ（１９９２年））に記載されている方法を参考にすることができる。
[Method for producing organic fluorescent dye-encapsulated silica nanoparticles]
The phosphor-encapsulated silica nanoparticles of the present invention can be referred to a known method, for example, a method described in a non-patent document (Langmuir 8, Vol. 2921 (1992)).
工程（１）：複数の反応性官能基と少なくとも一種の加水分解性置換基を有するシリル基を有する分子と前記反応性官能基と反応しうる官能基をもつ有機蛍光色素とを混合反応する。 Step (1): A molecule having a silyl group having a plurality of reactive functional groups and at least one hydrolyzable substituent is mixed with an organic fluorescent dye having a functional group capable of reacting with the reactive functional group.
工程（２）：工程（１）で得られたものを、テトラエトキシシランなどの含ケイ素アルコキシド化合物を混合する。 Step (2): A silicon-containing alkoxide compound such as tetraethoxysilane is mixed with the product obtained in step (1).
工程（３）：エタノールなどの有機溶媒、水及び塩基を混合する。 Step (3): An organic solvent such as ethanol, water and a base are mixed.
工程（４）：工程（３）で作製した混合液を撹拌しているところに、工程（２）で得られた有機蛍光色素含有液を添加し、反応を進行させる。 Step (4): The organic fluorescent dye-containing liquid obtained in Step (2) is added to the stirring of the liquid mixture prepared in Step (3), and the reaction proceeds.
工程（５）：反応混合物から生成した有機蛍光色素内包シリカナノ粒子を、ろ過もしくは遠心分離により回収する。 Step (5): The organic fluorescent dye-containing silica nanoparticles produced from the reaction mixture are collected by filtration or centrifugation.
工程（６）：工程（５）で得られた有機蛍光色素内包シリカナノ粒子を分子標識物質と結合させ、生体物質標識剤を得る。 Step (6): The organic fluorescent dye-encapsulated silica nanoparticles obtained in the step (5) are combined with a molecular labeling substance to obtain a biological substance labeling agent.
なお、上記工程（２）で用いられる含ケイ素アルコキシド化合物としては、テトラエトキシシラン、テトラメトキシシランといったテトラアルコキシドシラン、メチルトリメトキシシラン、メチルエトキシシラン、フェニルトリエトキシシランなどのトリアルコキシシランなどをあげることができる。また有機官能基を有する含ケイ素アルコキシド化合物をあげることができる。具体的にはメルカプトプロピルトリエトキシシラン、アミノプロピルトリエトキシシランなどがあげられる。 Examples of the silicon-containing alkoxide compound used in the step (2) include tetraalkoxide silanes such as tetraethoxysilane and tetramethoxysilane, trialkoxysilanes such as methyltrimethoxysilane, methylethoxysilane, and phenyltriethoxysilane. be able to. Moreover, the silicon-containing alkoxide compound which has an organic functional group can be mention | raise | lifted. Specific examples include mercaptopropyltriethoxysilane and aminopropyltriethoxysilane.
含ケイ素アルコキシド化合物は上記の一種もしくは二種以上を併用することもできる。 One or more of the silicon-containing alkoxide compounds may be used in combination.
含ケイ素アルコキシド化合物と工程１で得られる有機蛍光色素結合分子の混合比に制限はないが、最終的に得られるシリカナノ粒子中に１×１０−６ｍｏｌ／Ｌ以上１×１０−２ｍｏｌ／Ｌ以下になるように混合することが好ましい。濃度を１×１０−６ｍｏｌ／Ｌ以上とすることで十分な蛍光が得られる。また１×１０−２ｍｏｌ／Ｌ以下とすることで、シリカ内で均一に分散でき、かつ濃度消光を防止できる点で好ましい。 The mixing ratio of the silicon-containing alkoxide compound and the organic fluorescent dye-binding molecule obtained in Step 1 is not limited, but the silica nanoparticles finally obtained have a mixing ratio of 1 × 10 −6 mol / L or more and 1 × 10 −2 mol / L. It is preferable to mix so that it may become the following. Sufficient fluorescence can be obtained by setting the concentration to 1 × 10 −6 mol / L or more. Moreover, it is preferable at 1 * 10 <-2 > mol / L or less at the point which can disperse | distribute uniformly in a silica and can prevent concentration quenching.
工程（３）で用いられる有機溶媒として、通常の含ケイ素アルコキシド化合物の加水分解反応で用いられるものであればよく、メタノール、エタノール、テトラヒドロフラン、ジメチルホルムアミド、ジメチルスルホキシドなどが用いられる。一種もしくは二種以上の混合としてもよい。 The organic solvent used in step (3) may be any organic solvent that can be used in the usual hydrolysis reaction of silicon-containing alkoxide compounds, and methanol, ethanol, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, and the like are used. One kind or a mixture of two or more kinds may be used.
また工程（３）で用いられる塩基としては、通常の含ケイ素アルコキシド化合物の加水分解反応で用いられるものであればよく、アンモニア、水酸化ナトリウム、水酸化カリウムなどを用いることができ、それぞれ水溶液として用いてもよい。 Moreover, as a base used at a process (3), what is necessary is just to be used by the hydrolysis reaction of a normal silicon-containing alkoxide compound, Ammonia, sodium hydroxide, potassium hydroxide etc. can be used, respectively, It may be used.
含ケイ素アルコキシド化合物としてテトラエトキシシラン、有機溶媒としてエタノール、塩基としてアンモニア水を用いた場合のそれぞれの仕込みモル比を以下にあげる。 The charged molar ratios of tetraethoxysilane as the silicon-containing alkoxide compound, ethanol as the organic solvent, and aqueous ammonia as the base are listed below.
テトラエトキシシランを１ｍｏｌとした場合、エタノールをａ ｍｏｌ、水をｒ ｍｏｌ、アンモニアをｂ ｍｏｌとすると、ａは２０以上４００以下、ｒは１０以上２００以下、ｂは１０以上４０以下で混合する。具体的には、ジャーナル・オブ・コロイドサイエンス ２６巻、６２ページ（１９６８年）に記載されている条件を適用することができる。 When tetraethoxysilane is 1 mol, a is 20 to 400, r is 10 to 200, and b is 10 to 40, assuming that ethanol is a mol, water is r mol, and ammonia is b mol. Specifically, the conditions described in Journal of Colloid Science, Vol. 26, page 62 (1968) can be applied.
工程（４）において、反応温度は通常の含ケイ素アルコキシド化合物の加水分解反応で適用される条件でよく、室温から５０℃の間で行うことができる。 In the step (4), the reaction temperature may be a condition applied in a normal hydrolysis reaction of a silicon-containing alkoxide compound, and can be performed between room temperature and 50 ° C.
蛍光色素含有液を添加する方法としては、限定されるものはなくシリンジポンプ、滴下ロートなど用いればよい。 The method for adding the fluorescent dye-containing liquid is not limited, and a syringe pump, a dropping funnel or the like may be used.
工程（４）における反応時間は、通常の含ケイ素アルコキシド化合物の加水分解反応で適用される条件でよく、１時間以上５０時間以下であることが好ましい、これより短いと反応は完結しておらず、収率が低下する。またこれより長いと反応が進行しすぎて、不溶物が形成することがある。 The reaction time in the step (4) may be the conditions applied in the usual hydrolysis reaction of the silicon-containing alkoxide compound, and is preferably 1 hour or more and 50 hours or less. If shorter than this, the reaction is not completed. Yield decreases. On the other hand, if the length is longer than this, the reaction proceeds too much, and insoluble matter may be formed.
工程（５）における反応混合物から生成した蛍光物質内包シリカナノ粒子の回収方法は、通常ナノ粒子の回収で行われるろ過、もしくは遠心分離などを用いることができる。回収した蛍光色素内包シリカナノ粒子は必要に応じて、未反応原料などを除くため、有機溶媒もしくは水による洗浄をしてもよい。 As a method for recovering the fluorescent substance-encapsulated silica nanoparticles generated from the reaction mixture in the step (5), filtration, centrifugation, or the like usually performed for recovering the nanoparticles can be used. The recovered fluorescent dye-encapsulated silica nanoparticles may be washed with an organic solvent or water in order to remove unreacted raw materials as necessary.
〔有機蛍光色素内包シリカナノ粒子と分子標識物質とを結合する有機分子〕
本発明に係る生体物質標識剤は、有機分子修飾された有機蛍光色素内包シリカナノ粒子と、分子標識物質とが有機分子により結合されている態様であることが好ましい。上記結合の態様としては特に限定されず、共有結合、イオン結合、水素結合、配位結合、物理吸着及び化学吸着等が挙げられる。結合の安定性から共有結合などの結合力の強い結合が好ましい。
[Organic molecule that binds organic fluorescent dye-encapsulated silica nanoparticles and molecular labeling substance]
The biological substance labeling agent according to the present invention is preferably in an embodiment in which organic fluorescent dye-encapsulated silica nanoparticles modified with an organic molecule and a molecular labeling substance are bound by organic molecules. The form of the bond is not particularly limited, and examples thereof include covalent bond, ionic bond, hydrogen bond, coordinate bond, physical adsorption, and chemical adsorption. A bond having a strong bonding force such as a covalent bond is preferable from the viewpoint of bond stability.
有機蛍光色素内包シリカナノ粒子の表面に結合し、分子標識物質とも結合しうる有機分子として、例えば、無機物と有機物を結合させるために広く用いられている化合物であるシランカップリング剤を用いることができる。このシランカップリング剤は、分子の一端に加水分解でシラノール基を与えるアルコキシシリル基を有し、他端に、カルボキシル基、アミノ基、エポキシ基、アルデヒド基などの官能基を有する化合物であり、上記シラノール基の酸素原子を介して無機物と結合する。具体的には、メルカプトプロピルトリエトキシシラン、グリシドキシプロピルトリエトキシシラン、アミノプロピルトリエトキシシランなどがあげられる。 As an organic molecule that binds to the surface of the organic fluorescent dye-encapsulated silica nanoparticles and can also bind to the molecular labeling substance, for example, a silane coupling agent, which is a compound widely used for binding an inorganic substance and an organic substance, can be used. . This silane coupling agent is a compound having an alkoxysilyl group that gives a silanol group by hydrolysis at one end of the molecule and a functional group such as a carboxyl group, an amino group, an epoxy group, an aldehyde group at the other end, Bonding with an inorganic substance through an oxygen atom of the silanol group. Specific examples include mercaptopropyltriethoxysilane, glycidoxypropyltriethoxysilane, and aminopropyltriethoxysilane.
また、後述する生体物質標識剤として用いる場合、生体物質との非特異的吸着を抑制するためポリエチレングリコール鎖をもつシランカップリング剤（例えば、Ｇｅｌｅｓｔ社製ＰＥＧ−ｓｉｌａｎｅ ｎｏ．ＳＩＭ６４９２．７）を用いることができる。 When used as a biological material labeling agent described later, a silane coupling agent having a polyethylene glycol chain (for example, PEG-silane no. SIM6492.7 manufactured by Gelest) is used to suppress nonspecific adsorption with biological materials. be able to.
シランカップリング剤を用いる場合、二種以上を併用してもよい。 When using a silane coupling agent, you may use 2 or more types together.
有機蛍光色素内包シリカナノ粒子とシランカップリング剤との反応手順は、公知の手法を用いることができる。例えば、得られた蛍光色素内包シリカナノ粒子を純水中に分散させ、アミノプロピルトリエトキシシランを添加し、室温で１２時間反応させる。反応終了後、遠心分離又はろ過により表面がアミノプロピル基で修飾された蛍光物質内包シリカナノ粒子を得ることができる。 A known procedure can be used for the reaction procedure of the organic fluorescent dye-encapsulated silica nanoparticles and the silane coupling agent. For example, the obtained fluorescent dye-encapsulated silica nanoparticles are dispersed in pure water, aminopropyltriethoxysilane is added, and the mixture is reacted at room temperature for 12 hours. After completion of the reaction, fluorescent substance-encapsulated silica nanoparticles whose surface is modified with an aminopropyl group can be obtained by centrifugation or filtration.
〔生体物質標識剤〕
本発明に係る生体物質標識剤は、上述した有機蛍光色素内包シリカナノ粒子と、分子標識物質と有機分子を介して結合させて得られる。
[Biological substance labeling agent]
The biological substance labeling agent according to the present invention is obtained by binding the above-described organic fluorescent dye-encapsulated silica nanoparticles, a molecular labeling substance and an organic molecule.
本発明に係る生体物質標識剤は分子標識物質が目的とする生体物質と特異的に結合及び／又は反応することにより、生体物質の標識が可能となる。 The biological substance labeling agent according to the present invention can label a biological substance by specifically binding and / or reacting with the target biological substance.
当該分子標識物質としては例えば、ヌクレオチド鎖、タンパク質、抗体等が挙げられる。 Examples of the molecular labeling substance include nucleotide chains, proteins, antibodies and the like.
具体例として、アミノプロピルトリエトキシシランで修飾した蛍光物質内包シリカナノ粒子のアミノ基と抗体中のカルボキシル基とを反応させることで、アミド結合を介し抗体を蛍光物質内包シリカナノ粒子と結合させることができる。必要に応じＥＤＣ（１−Ｅｔｈｙｌ−３−［３−Ｄｉｍｅｔｈｙｌａｍｉｎｏｐｒｏｐｙｌ］ｃａｒｂｏｄｉｉｍｉｄｅ Ｈｙｄｒｏｃｈｌｏｒｉｄｅ：Ｐｉｅｒｃｅ社製）のような縮合剤を用いることもできる。 As a specific example, by reacting the amino group of a fluorescent substance-encapsulated silica nanoparticle modified with aminopropyltriethoxysilane with a carboxyl group in the antibody, the antibody can be bound to the fluorescent substance-encapsulated silica nanoparticle via an amide bond. . If necessary, a condensing agent such as EDC (1-Ethyl-3- [3-Dimethylaminopropyl] carbohydrate Hydrochloride: Pierce) may be used.
必要により有機分子修飾された有機蛍光色素内包シリカナノ粒子と直接結合しうる部位と、分子標的物質と結合しうる部位とを有するリンカー化合物を用いることができる。具体例としてアミノ基と選択的に反応する部位とメルカプト基と選択的に反応する部位の両方をもつｓｕｌｆｏ−ＳＭＣＣ（Ｓｕｌｆｏｓｕｃｃｉｎｉｍｉｄｙｌ ４［Ｎ−ｍａｌｅｉｍｉｄｏｍｅｔｈｙｌ］−ｃｙｃｌｏｈｅｘａｎｅ−１−ｃａｒｂｏｘｙｌａｔｅ：Ｐｉｅｒｃｅ社製）を用いると、アミノプロピルトリエトキシシランで修飾した有機蛍光色素内包シリカナノ粒子のアミノ基と、抗体中のメルカプト基を結合させることで、抗体結合した有機蛍光色素内包シリカナノ粒子ができる。 If necessary, a linker compound having a site capable of directly binding to organic fluorescent dye-encapsulated silica nanoparticles modified with an organic molecule and a site capable of binding to a molecular target substance can be used. As a specific example, use is made of sulfo-SMCC (Sulfosuccinimidyl 4 [N-maleimidemethyl] -cyclohexane-1-carboxylate: manufactured by Pierce) having both a site selectively reacting with an amino group and a site selectively reacting with a mercapto group. By combining the amino group of the organic fluorescent dye-encapsulated silica nanoparticles modified with aminopropyltriethoxysilane and the mercapto group in the antibody, antibody-bound organic fluorescent dye-encapsulated silica nanoparticles can be produced.
以下、実施例を挙げて本発明を具体的に説明するが、本発明はこれらに限定されるものではない。なお、実施例において「部」あるいは「％」の表示を用いるが、特に断りがない限り「質量部」あるいは「質量％」を表す。 EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto. In addition, although the display of "part" or "%" is used in an Example, unless otherwise indicated, "part by mass" or "mass%" is represented.
《蛍光物質内包シリカナノ粒子》
〔二種有機蛍光色素内包シリカナノ粒子１の作製〕
（シリカナノ粒子１：ＢＯＤＩＰＹ−ＦＬ／ＴＡＭＲＡ共内包シリカナノ粒子）
ＢＯＤＩＰＹ−ＦＬ及びＴＡＭＲＡを同一分子内にもち、かつ加水分解性基をもつシリル基をもつ分子を用いて、下記工程（１）〜（５）の方法により、シリカナノ粒子１を作製した。
《Fluorescent substance-encapsulated silica nanoparticles》
[Production of two types of organic fluorescent dye-encapsulated silica nanoparticles 1]
(Silica nanoparticles 1: BODIPY-FL / TAMRA co-encapsulated silica nanoparticles)
Silica nanoparticles 1 were produced by the methods of the following steps (1) to (5) using a molecule having BODIPY-FL and TAMRA in the same molecule and having a hydrolyzable silyl group.
工程（１）：ＢＯＤＩＰＹ ＦＬのＮ−ヒドロキシスクシンイミドエステル誘導体（Ｉｎｖｉｔｒｏｇｅｎ社製 ＢＯＤＩＰＹ ＦＬ，ＳＥ）１．９ｍｇ（０．００４８ｍｍｏｌ）をジメチルホルムアミド０．８ｍＬに溶解させたところに、氷冷下Ｎ−（２−アミノエチル）−３−アミノプロピルトリエトキシシラン（Ｇｅｌｅｓｔ社製）１μＬ（０．００４８ｍｍｏｌ）添加し、３０分間撹拌した。 Step (1): When 1.9 mg (0.0048 mmol) of N-hydroxysuccinimide ester derivative of BODIPY FL (BODIPY FL, SE, manufactured by Invitrogen) was dissolved in 0.8 mL of dimethylformamide, N- ( 1 μL (0.0048 mmol) of 2-aminoethyl) -3-aminopropyltriethoxysilane (manufactured by Gelest) was added and stirred for 30 minutes.
ついで、室温下ＴＡＭＲＡのＮ−ヒドロキシスクシンイミドエステル誘導体（Ｉｎｖｉｔｒｏｇｅｎ社製５（６）−ＴＡＭＲＡ−ＮＨＳ，ＳＥ）２．６ｍｇ（０．００４８ｍｍｏｌ）をジメチルホルムアミド０．２ｍＬに溶解させたものを添加し、３０分間撹拌した。 Then, 2.6 mg (0.0048 mmol) of TAMRA N-hydroxysuccinimide ester derivative (Invitrogen 5 (6) -TAMRA-NHS, SE) dissolved in 0.2 mL of dimethylformamide at room temperature was added, Stir for 30 minutes.
工程（２）：工程（１）で得られたＤＭＦ溶液とテトラエトキシシラン４０μＬを混合した。 Step (2): The DMF solution obtained in step (1) and 40 μL of tetraethoxysilane were mixed.
工程（３）：エタノール４０ｍＬ、１４％アンモニア水１０ｍＬを混合した。 Step (3): Ethanol 40 mL and 14% aqueous ammonia 10 mL were mixed.
工程（４）：工程３で作製した混合液を室温下撹拌しているところに、工程（２）で作製した混合液を添加した。添加開始から１２時間撹拌を行った。 Step (4): The mixture prepared in Step (2) was added to the mixture prepared in Step 3 while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
工程（５）：反応混合物を１００００ｇで６０分遠心分離を行い、上澄みを除去した。エタノールを加え、沈降物を分散させ、再度遠心分離を行った。同様の手順でエタノールと純水による洗浄を一回ずつ行った。 Step (5): The reaction mixture was centrifuged at 10,000 g for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. In the same procedure, washing with ethanol and pure water was performed once.
得られたシリカナノ粒子１の走査型電子顕微鏡（ＳＥＭ；日立社製Ｓ−８００型）観察を行ったところ、平均粒径１１０ｎｍ、変動係数は１２％であった。なお、当該観察は、１０００個の粒子について断面積を計測し、その計測値を相当する円の面積としたときの直径を粒径として求め、１０００個の粒子の粒径の算術平均を平均粒径とした。また、変動係数も、１０００個の粒子の粒径分布から算出した値とした。 When the obtained silica nanoparticle 1 was observed with a scanning electron microscope (SEM; Hitachi S-800 type), the average particle size was 110 nm and the coefficient of variation was 12%. In this observation, the cross-sectional area is measured for 1000 particles, the diameter when the measured value is the area of the corresponding circle is obtained as the particle size, and the arithmetic average of the particle size of 1000 particles is the average particle size. The diameter. The coefficient of variation was also a value calculated from the particle size distribution of 1000 particles.
（シリカナノ粒子２：ＢＯＤＩＰＹ−ＦＬ／ＴＡＭＲＡ共内包シリカナノ粒子）
特許文献２にならい下記工程（１）〜（６）の方法により、ＢＯＤＩＰＹ ＦＬとＴＡＭＲＡをそれぞれ連結せずに内包した、シリカナノ粒子２を作製した。
(Silica nanoparticles 2: BODIPY-FL / TAMRA co-encapsulated silica nanoparticles)
Silica nanoparticles 2 encapsulating BODIPY FL and TAMRA without being connected to each other were produced by the method of the following steps (1) to (6) following Patent Document 2.
工程（１）：ＢＯＤＩＰＹ ＦＬのＮ−ヒドロキシスクシンイミドエステル誘導体（Ｉｎｖｉｔｒｏｇｅｎ社製 ＢＯＤＩＰＹ ＦＬ，ＳＥ）１．９ｍｇ（０．００４８ｍｍｏｌ）をジメチルホルムアミド０．５ｍＬに溶解させたところに、室温下３−アミノプロピルトリエトキシシラン（信越化学工業社製）１μＬ添加し、３０分撹拌した。 Step (1): When 1.9 mg (0.0048 mmol) of N-hydroxysuccinimide ester derivative of BODIPY FL (BODIPY FL, SE, manufactured by Invitrogen) was dissolved in 0.5 mL of dimethylformamide, 3-aminopropyl was obtained at room temperature. 1 μL of triethoxysilane (manufactured by Shin-Etsu Chemical Co., Ltd.) was added and stirred for 30 minutes.
工程（２）：ＴＡＭＲＡのＮ−ヒドロキシスクシンイミドエステル誘導体（Ｉｎｖｉｔｒｏｇｅｎ社製５（６）−ＴＡＭＲＡ−ＮＨＳ，ＳＥ）２．６ｍｇ（０．００４８ｍｍｏｌ）をジメチルホルムアミド０．５ｍＬに溶解させた溶解させたところに、室温下３−アミノプロピルトリエトキシシラン（信越化学工業社製）１μＬ添加し、３０分間撹拌した。 Step (2): N-hydroxysuccinimide ester derivative of TAMRA (Invitrogen 5 (6) -TAMRA-NHS, SE) 2.6 mg (0.0048 mmol) dissolved in 0.5 mL of dimethylformamide 1 μL of 3-aminopropyltriethoxysilane (manufactured by Shin-Etsu Chemical Co., Ltd.) was added thereto at room temperature and stirred for 30 minutes.
工程（３）：工程（１）で得られたＤＭＦ溶液、工程（２）で得られたＤＭＦ溶液及びテトラエトキシシラン４０μＬを混合した。 Step (3): The DMF solution obtained in Step (1), the DMF solution obtained in Step (2), and 40 μL of tetraethoxysilane were mixed.
工程（４）：エタノール４０ｍＬ、１４％アンモニア水１０ｍＬを混合した。 Step (4): Ethanol 40 mL and 14% aqueous ammonia 10 mL were mixed.
工程（５）：工程（４）で作製した混合液を室温下撹拌しているところに、工程（３）で作製した混合液を添加した。添加開始から１２時間撹拌を行った。 Step (5): The mixture prepared in Step (3) was added to the mixture prepared in Step (4) while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
工程（６）：反応混合物を１００００ｇで６０分遠心分離を行い、上澄みを除去した。エタノールを加え、沈降物を分散させ、再度遠心分離を行った。同様の手順でエタノールと純水による洗浄を一回ずつ行った。 Step (6): The reaction mixture was centrifuged at 10,000 g for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. In the same procedure, washing with ethanol and pure water was performed once.
得られたシリカナノ粒子２のＳＥＭ観察を行ったところ、平均粒径１００ｎｍ、変動係数は１０％であった。 When the SEM observation of the obtained silica nanoparticle 2 was performed, the average particle diameter was 100 nm and the variation coefficient was 10%.
（シリカナノ粒子３：ＢＯＤＩＰＹ−ＦＬ／ＴＡＭＲＡ共内包シリカナノ粒子）
ＢＯＤＩＰＹ−ＦＬ及びＴＡＭＲＡを同一分子内にもつが、加水分解性基をもつシリル基をもたない分子を用いて、下記工程（１）〜（５）の方法により、シリカナノ粒子３を作製した。
(Silica nanoparticles 3: BODIPY-FL / TAMRA co-encapsulated silica nanoparticles)
Silica nanoparticles 3 were produced by the methods of the following steps (1) to (5) using a molecule having BODIPY-FL and TAMRA in the same molecule but not having a silyl group having a hydrolyzable group.
工程（１）：ＢＯＤＩＰＹ ＦＬのＮ−ヒドロキシスクシンイミドエステル誘導体（Ｉｎｖｉｔｒｏｇｅｎ社製 ＢＯＤＩＰＹ ＦＬ，ＳＥ）１．９ｍｇ（０．００４８ｍｍｏｌ）をジメチルホルムアミド０．８ｍＬに溶解させたところに、氷冷下エチレンジアミン（和光純薬社製）０．３μＬ（（０．００４８ｍｍｏｌ）添加し、３０分間撹拌した。 Step (1): When 1.9 mg (0.0048 mmol) of N-hydroxysuccinimide ester derivative of BODIPY FL (BODIPY FL, SE, manufactured by Invitrogen) was dissolved in 0.8 mL of dimethylformamide, ethylenediamine (sum) 0.3 μL ((0.0048 mmol) manufactured by Kojun Pharmaceutical Co., Ltd.) was added and stirred for 30 minutes.
ついで、室温下ＴＡＭＲＡのＮ−ヒドロキシスクシンイミドエステル誘導体（Ｉｎｖｉｔｒｏｇｅｎ社製 ５（６）−ＴＡＭＲＡ−ＮＨＳ，ＳＥ）２．６ｍｇ（０．００４８ｍｍｏｌ）をジメチルホルムアミド０．２ｍＬに溶解させたものを添加し、３０分間撹拌した。 Next, N-hydroxysuccinimide ester derivative of TAMRA (Invitrogen 5 (6) -TAMRA-NHS, SE) 2.6 mg (0.0048 mmol) dissolved in 0.2 mL of dimethylformamide was added at room temperature, Stir for 30 minutes.
工程（２）：工程（１）で得られたＤＭＦ溶液とテトラエトキシシラン４０μＬを混合した。 Step (2): The DMF solution obtained in step (1) and 40 μL of tetraethoxysilane were mixed.
工程（３）：エタノール４０ｍＬ、１４％アンモニア水１０ｍＬを混合した。 Step (3): Ethanol 40 mL and 14% aqueous ammonia 10 mL were mixed.
工程（４）：工程（３）で作製した混合液を室温下撹拌しているところに、工程（２）で作製した混合液を添加した。添加開始から１２時間撹拌を行った。 Step (4): The mixture prepared in Step (2) was added to the mixture prepared in Step (3) while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
工程（５）：反応混合物を１００００ｇで６０分遠心分離を行い、上澄みを除去した。エタノールを加え、沈降物を分散させ、再度遠心分離を行った。同様の手順でエタノールと純水による洗浄を一回ずつ行った。 Step (5): The reaction mixture was centrifuged at 10,000 g for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. In the same procedure, washing with ethanol and pure water was performed once.
得られたシリカナノ粒子３のＳＥＭ観察を行ったところ、平均粒径１１５ｎｍ、変動係数は１１％であった。 When the SEM observation of the obtained silica nanoparticle 3 was performed, the average particle diameter was 115 nm and the coefficient of variation was 11%.
ＢＯＤＩＰＹ ＦＬの吸収極大波長は、５０５ｎｍ、ＴＡＭＲＡの吸収極大波長は、５４６ｎｍである。したがって、波長４８８ｎｍの光では、ＢＯＤＩＰＹ ＦＬのみが励起され、ＴＡＭＲＡは励起されない。 The absorption maximum wavelength of BODIPY FL is 505 nm, and the absorption maximum wavelength of TAMRA is 546 nm. Therefore, with light having a wavelength of 488 nm, only BODIPY FL is excited, and TAMRA is not excited.
また、ＢＯＤＩＰＹ ＦＬの蛍光極大波長は５１１ｎｍであり、ＴＡＭＲＡの蛍光極大波長は５７６ｎｍである。したがって、波長５８０ｎｍの蛍光はＴＡＭＲＡ由来の発光に帰属できる。 Moreover, the fluorescence maximum wavelength of BODIPY FL is 511 nm, and the fluorescence maximum wavelength of TAMRA is 576 nm. Therefore, fluorescence having a wavelength of 580 nm can be attributed to light emission derived from TAMRA.
波長を４８８ｎｍの光によりＢＯＤＩＰＹ ＦＬを励起したときに、波長５８０ｎｍに蛍光が観察された場合、ＢＯＤＩＰＹ ＦＬからＴＡＭＲＡへの蛍光共鳴エネルギー移動が起こったといえ、その蛍光強度が高いほどエネルギー移動効率が高いといえる。 When BODIPY FL is excited by light having a wavelength of 488 nm and fluorescence is observed at a wavelength of 580 nm, it can be said that fluorescence resonance energy transfer from BODIPY FL to TAMRA has occurred, and the higher the fluorescence intensity, the higher the energy transfer efficiency. It can be said.
（シリカナノ粒子４：Ａｌｅｘａ Ｆｌｕｏｒ４８８／ローダミン６Ｇシリカナノ粒子）
Ａｌｅｘａ Ｆｌｕｏｒ４８８及びローダミン６Ｇを同一分子内にもち、かつ加水分解性基をもつシリル基をもつ分子を用いて、下記工程（１）〜（５）の方法により、シリカナノ粒子４を作製した。
(Silica nanoparticles 4: Alexa Fluor 488 / Rhodamine 6G silica nanoparticles)
Silica nanoparticles 4 were prepared by the methods of the following steps (1) to (5) using Alexa Fluor 488 and rhodamine 6G in the same molecule and using a molecule having a hydrolyzable silyl group.
工程（１）：Ａｌｅｘａ Ｆｌｕｏｒ４８８のＮ−ヒドロキシスクシンイミドエステル誘導体（Ｉｎｖｉｔｒｏｇｅｎ社製）３．１ｍｇ（０．００４８ｍｍｏｌ）をジメチルホルムアミド０．８ｍＬに溶解させたところに、氷冷下Ｎ−（２−アミノエチル）−３−アミノプロピルトリエトキシシラン（Ｇｅｌｅｓｔ社製）１μＬ（０．００４８ｍｍｏｌ）添加し、３０分間撹拌した。 Step (1): 3.1 mg (0.0048 mmol) of N-hydroxysuccinimide ester derivative (manufactured by Invitrogen) of Alexa Fluor 488 was dissolved in 0.8 mL of dimethylformamide, and then N- (2-aminoethyl) was cooled with ice. ) -3-Aminopropyltriethoxysilane (manufactured by Gelest) 1 μL (0.0048 mmol) was added and stirred for 30 minutes.
ついで、室温下ローダミン６ＧのＮ−ヒドロキシスクシンイミドエステル誘導体（Ｉｎｖｉｔｒｏｇｅｎ社製５（６）ＣＲ６ＧＳＥ）２．７ｍｇ（０．００４８ｍｍｏｌ）をジメチルホルムアミド０．２ｍＬに溶解させたものを添加し、３０分間撹拌した。 Next, a solution of 2.7 mg (0.0048 mmol) of rhodamine 6G N-hydroxysuccinimide ester derivative (Invitrogen 5 (6) CR6GSE) dissolved in 0.2 mL of dimethylformamide at room temperature was added and stirred for 30 minutes. .
工程（２）：工程（１）で得られたＤＭＦ溶液とテトラエトキシシラン４０μＬを混合した。 Step (2): The DMF solution obtained in step (1) and 40 μL of tetraethoxysilane were mixed.
工程（３）：エタノール４０ｍＬ、１４％アンモニア水１０ｍＬを混合した。 Step (3): Ethanol 40 mL and 14% aqueous ammonia 10 mL were mixed.
工程（４）：工程３で作製した混合液を室温下撹拌しているところに、工程（２）で作製した混合液を添加した。添加開始から１２時間撹拌を行った。 Step (4): The mixture prepared in Step (2) was added to the mixture prepared in Step 3 while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
工程（５）：反応混合物を１００００ｇで６０分遠心分離を行い、上澄みを除去した。エタノールを加え、沈降物を分散させ、再度遠心分離を行った。同様の手順でエタノールと純水による洗浄を一回ずつ行った。 Step (5): The reaction mixture was centrifuged at 10,000 g for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. In the same procedure, washing with ethanol and pure water was performed once.
得られたシリカナノ粒子４のＳＥＭ観察を行ったところ、平均粒径１０５ｎｍ、変動係数は１２％であった。 When the SEM observation of the obtained silica nanoparticle 4 was performed, the average particle diameter was 105 nm and the coefficient of variation was 12%.
Ａｌｅｘａ Ｆｌｕｏｒ４８８の吸収極大波長は、４９５ｎｍ、ローダミン６Ｇの吸収極大波長は、５２４ｎｍである。したがって、波長４８８ｎｍの光では、Ａｌｅｘａ Ｆｌｕｏｒ４８８のみが励起され、ローダミン６Ｇは励起されない。 Alexa Fluor 488 has an absorption maximum wavelength of 495 nm, and Rhodamine 6G has an absorption maximum wavelength of 524 nm. Therefore, with light having a wavelength of 488 nm, only Alexa Fluor 488 is excited, and rhodamine 6G is not excited.
また、Ａｌｅｘａ Ｆｌｕｏｒ４８８の蛍光極大波長は５１９ｎｍであり、ローダミン６Ｇの蛍光極大波長は５５２ｎｍである。したがって、波長５８０ｎｍの蛍光はローダミン６Ｇ由来の発光に帰属できる。 Alexa Fluor 488 has a fluorescence maximum wavelength of 519 nm, and Rhodamine 6G has a fluorescence maximum wavelength of 552 nm. Therefore, fluorescence having a wavelength of 580 nm can be attributed to light emission derived from rhodamine 6G.
波長を４８８ｎｍの光によりＡｌｅｘａ Ｆｌｕｏｒ４８８を励起したときに、波長５８０ｎｍに蛍光が観察された場合、Ａｌｅｘａ Ｆｌｕｏｒ４８８からローダミン６Ｇへの蛍光共鳴エネルギー移動が起こったといえ、その蛍光強度が高いほどエネルギー移動効率が高いといえる。 When Alexa Fluor 488 is excited by light having a wavelength of 488 nm and fluorescence is observed at a wavelength of 580 nm, it can be said that fluorescence resonance energy transfer from Alexa Fluor 488 to rhodamine 6G has occurred, and the higher the fluorescence intensity, the higher the energy transfer efficiency. It can be said that it is expensive.
（シリカナノ粒子５：ＨｉＬｙｔｅ Ｆｌｕｏｒ４８８／ローダミン６Ｇシリカナノ粒子）
ＨｉＬｙｔｅ Ｆｌｕｏｒ４８８及びローダミン６Ｇを同一分子内にもち、かつ加水分解性基をもつシリル基をもつ分子を用いて、下記工程（１）〜（５）の方法により、シリカナノ粒子５を作製した。
(Silica nanoparticles 5: HiLyte Fluor488 / Rhodamine 6G silica nanoparticles)
Silica nanoparticles 5 were produced by the methods of the following steps (1) to (5) using a molecule having a silyl group having a hydrolyzable group and having HiLy Fluor 488 and rhodamine 6G in the same molecule.
工程（１）：ＨｉＬｙｔｅ Ｆｌｕｏｒ４８８のＮ−ヒドロキシスクシンイミドエステル誘導体（ＡｎａＳｐｅｃ社製ＨｉＬｙｔｅ Ｆｌｕｏｒ４８８ ａｃｉｄ ＳＥ）２．５ｍｇ（０．００４８ｍｍｏｌ）をジメチルホルムアミド０．８ｍＬに溶解させたところに、氷冷下Ｎ−（２−アミノエチル）−３−アミノプロピルトリエトキシシラン（Ｇｅｌｅｓｔ社製）１μＬ（０．００４８ｍｍｏｌ）添加し、３０分間撹拌した。 Step (1): N-hydroxysuccinimide ester derivative of HiLyte Fluor 488 (HiLy Fluor 488 acid SE, manufactured by AnaSpec) 2.5 mg (0.0048 mmol) was dissolved in 0.8 mL of dimethylformamide, and N- ( 1 μL (0.0048 mmol) of 2-aminoethyl) -3-aminopropyltriethoxysilane (manufactured by Gelest) was added and stirred for 30 minutes.
ついで、室温下ローダミン６ＧのＮ−ヒドロキシスクシンイミドエステル誘導体（Ｉｎｖｉｔｒｏｇｅｎ社製５（６）ＣＲ６ＧＳＥ）２．７ｍｇ（０．００４８ｍｍｏｌ）をジメチルホルムアミド０．２ｍＬに溶解させたものを添加し、３０分間撹拌した。 Next, a solution of 2.7 mg (0.0048 mmol) of rhodamine 6G N-hydroxysuccinimide ester derivative (Invitrogen 5 (6) CR6GSE) dissolved in 0.2 mL of dimethylformamide at room temperature was added and stirred for 30 minutes. .
工程（２）：工程（１）で得られたＤＭＦ溶液とテトラエトキシシラン４０μＬを混合した。 Step (2): The DMF solution obtained in step (1) and 40 μL of tetraethoxysilane were mixed.
工程（３）：エタノール４０ｍＬ、１４％アンモニア水１０ｍＬを混合した。 Step (3): Ethanol 40 mL and 14% aqueous ammonia 10 mL were mixed.
工程（４）：工程３で作製した混合液を室温下撹拌しているところに、工程（２）で作製した混合液を添加した。添加開始から１２時間撹拌を行った。 Step (4): The mixture prepared in Step (2) was added to the mixture prepared in Step 3 while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
工程（５）：反応混合物を１００００ｇで６０分遠心分離を行い、上澄みを除去した。エタノールを加え、沈降物を分散させ、再度遠心分離を行った。同様の手順でエタノールと純水による洗浄を一回ずつ行った。 Step (5): The reaction mixture was centrifuged at 10,000 g for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. In the same procedure, washing with ethanol and pure water was performed once.
得られたシリカナノ粒子５のＳＥＭ観察を行ったところ、平均粒径９８ｎｍ、変動係数は１６％であった。 When the SEM observation of the obtained silica nanoparticle 5 was performed, the average particle diameter was 98 nm, and the variation coefficient was 16%.
ＨｉＬｙｔｅ Ｆｌｕｏｒ４８８の吸収極大波長は、５０１ｎｍ、ローダミン６Ｇの吸収極大波長は、５２４ｎｍである。したがって、波長４８８ｎｍの光では、ＨｉＬｙｔｅ Ｆｌｕｏｒ４８８のみが励起され、ローダミン６Ｇは励起されない。 The absorption maximum wavelength of HiLyte Fluor 488 is 501 nm, and the absorption maximum wavelength of Rhodamine 6G is 524 nm. Therefore, in the light with a wavelength of 488 nm, only HiLyte Fluor 488 is excited and rhodamine 6G is not excited.
また、ＨｉＬｙｔｅ Ｆｌｕｏｒ４８８の蛍光極大波長は５２７ｎｍであり、ローダミン６Ｇの蛍光極大波長は５５２ｎｍである。したがって、波長５８０ｎｍの蛍光はローダミン６Ｇ由来の発光に帰属できる。 Moreover, the fluorescence maximum wavelength of HiLyte Fluor 488 is 527 nm, and the fluorescence maximum wavelength of rhodamine 6G is 552 nm. Therefore, fluorescence having a wavelength of 580 nm can be attributed to light emission derived from rhodamine 6G.
波長を４８８ｎｍの光によりＨｉＬｙｔｅ Ｆｌｕｏｒ４８８を励起したときに、波長５８０ｎｍに蛍光が観察された場合、ＨｉＬｙｔｅ Ｆｌｕｏｒ４８８からローダミン６Ｇへの蛍光共鳴エネルギー移動が起こったといえ、その蛍光強度が高いほどエネルギー移動効率が高いといえる。 When fluorescence is observed at a wavelength of 580 nm when HiLyte Fluor 488 is excited by light having a wavelength of 488 nm, it can be said that fluorescence resonance energy transfer from HiLyte Fluor 488 to rhodamine 6G has occurred, and the higher the fluorescence intensity, the higher the energy transfer efficiency. It can be said that it is expensive.
（シリカナノ粒子６：ＤｙＬｉｇｈｔ４８８／ＴＡＭＲＡ共内包シリカナノ粒子）
ＤｙＬｉｇｈｔ４８８及びＴＡＭＲＡを同一分子内にもち、かつ加水分解性基をもつシリル基をもつ分子を用いて、下記工程（１）〜（５）の方法により、シリカナノ粒子６を作製した。
(Silica nanoparticles 6: DyLight 488 / TAMRA co-encapsulated silica nanoparticles)
Silica nanoparticles 6 were produced by the methods of the following steps (1) to (5) using a molecule having DyLight 488 and TAMRA in the same molecule and having a hydrolyzable silyl group.
工程（１）：ＤｙＬｉｇｈｔ４８８のＮ−ヒドロキシスクシンイミドエステル誘導体（ＴｈｅｒｍｏＦｉｓｈｅｒ社製 ＤｙＬｉｇｈｔ４８８ ＮＨＳ Ｅｓｔｅｒ）１．９ｍｇ（０．００４８ｍｍｏｌ）をジメチルホルムアミド０．８ｍＬに溶解させたところに、氷冷下Ｎ−（２−アミノエチル）−３−アミノプロピルトリエトキシシラン（Ｇｅｌｅｓｔ社製）１μＬ（０．００４８ｍｍｏｌ）添加し、３０分間撹拌した。 Step (1): When 1.9 mg (0.0048 mmol) of DyLight 488 N-hydroxysuccinimide ester derivative (DyLight 488 NHS Ester manufactured by ThermoFisher) was dissolved in 0.8 mL of dimethylformamide, N- (2- 1 μL (0.0048 mmol) of aminoethyl) -3-aminopropyltriethoxysilane (Gelest) was added and stirred for 30 minutes.
ついで、室温下ＴＡＭＲＡのＮ−ヒドロキシスクシンイミドエステル誘導体（Ｉｎｖｉｔｒｏｇｅｎ社製 ５（６）−ＴＡＭＲＡ−ＮＨＳ，ＳＥ）２．６ｍｇ（０．００４８ｍｍｏｌ）をジメチルホルムアミド０．２ｍＬに溶解させたものを添加し、３０分間撹拌した。 Next, N-hydroxysuccinimide ester derivative of TAMRA (Invitrogen 5 (6) -TAMRA-NHS, SE) 2.6 mg (0.0048 mmol) dissolved in 0.2 mL of dimethylformamide was added at room temperature, Stir for 30 minutes.
工程（２）：工程（１）で得られたＤＭＦ溶液とテトラエトキシシラン４０μＬを混合した。 Step (2): The DMF solution obtained in step (1) and 40 μL of tetraethoxysilane were mixed.
工程（３）：エタノール４０ｍＬ、１４％アンモニア水１０ｍＬを混合した。 Step (3): Ethanol 40 mL and 14% aqueous ammonia 10 mL were mixed.
工程（４）：工程３で作製した混合液を室温下撹拌しているところに、工程（２）で作製した混合液を添加した。添加開始から１２時間撹拌を行った。 Step (4): The mixture prepared in Step (2) was added to the mixture prepared in Step 3 while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
工程（５）：反応混合物を１００００ｇで６０分遠心分離を行い、上澄みを除去した。エタノールを加え、沈降物を分散させ、再度遠心分離を行った。同様の手順でエタノールと純水による洗浄を一回ずつ行った。 Step (5): The reaction mixture was centrifuged at 10,000 g for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. In the same procedure, washing with ethanol and pure water was performed once.
得られたシリカナノ粒子６のＳＥＭ観察を行ったところ、平均粒径９８ｎｍ、変動係数は１６％であった。 When the SEM observation of the obtained silica nanoparticle 6 was performed, the average particle diameter was 98 nm, and the variation coefficient was 16%.
ＤｙＬｉｇｈｔ４８８の吸収極大波長は、４９３ｎｍ、ＴＡＭＲＡの吸収極大波長は、５４６ｎｍである。したがって、波長４８８ｎｍの光では、ＤｙＬｉｇｈｔ４８８のみが励起され、ＴＡＭＲＡは励起されない。 The absorption maximum wavelength of DyLight 488 is 493 nm, and the absorption maximum wavelength of TAMRA is 546 nm. Therefore, with light having a wavelength of 488 nm, only DyLight 488 is excited, and TAMRA is not excited.
また、ＤｙＬｉｇｈｔ４８８の蛍光極大波長は５１８ｎｍであり、ＴＡＭＲＡの蛍光極大波長は５７６ｎｍである。したがって、波長５８０ｎｍの蛍光はＴＡＭＲＡ由来の発光に帰属できる。 The fluorescent maximum wavelength of DyLight 488 is 518 nm, and the fluorescent maximum wavelength of TAMRA is 576 nm. Therefore, fluorescence having a wavelength of 580 nm can be attributed to light emission derived from TAMRA.
波長を４８８ｎｍの光によりＤｙＬｉｇｈｔ４８８を励起したときに、波長５８０ｎｍに蛍光が観察された場合、ＤｙＬｉｇｈｔ４８８からＴＡＭＲＡへの蛍光共鳴エネルギー移動が起こったといえ、その蛍光強度が高いほどエネルギー移動効率が高いといえる。 When fluorescence is observed at a wavelength of 580 nm when DyLight 488 is excited with light having a wavelength of 488 nm, it can be said that fluorescence resonance energy transfer from DyLight 488 to TAMRA has occurred, and the higher the fluorescence intensity, the higher the energy transfer efficiency. .
（シリカナノ粒子７：ローダミン６Ｇ／ＴＡＭＲＡ共内包シリカナノ粒子）
ローダミン６Ｇ及びＴＡＭＲＡを同一分子内にもち、かつ加水分解性基をもつシリル基をもつ分子を用いて、下記工程（１）〜（５）の方法により、シリカナノ粒子７を作製した。
(Silica nanoparticles 7: Rhodamine 6G / TAMRA co-encapsulated silica nanoparticles)
Silica nanoparticles 7 were produced by the methods of the following steps (1) to (5) using a molecule having rhodamine 6G and TAMRA in the same molecule and having a hydrolyzable silyl group.
工程（１）：ローダミン６ＧのＮ−ヒドロキシスクシンイミドエステル誘導体（Ｉｎｖｉｔｒｏｇｅｎ社製５（６）ＣＲ６ＧＳＥ）２．７ｍｇ（０．００４８ｍｍｏｌ）をジメチルホルムアミド０．８ｍＬに溶解させたところに、氷冷下Ｎ−（２−アミノエチル）−３−アミノプロピルトリエトキシシラン（Ｇｅｌｅｓｔ社製）１μＬ（０．００４８ｍｍｏｌ）添加し、３０分間撹拌した。 Step (1): When 2.7 mg (0.0048 mmol) of N-hydroxysuccinimide ester derivative of rhodamine 6G (Invitrogen 5 (6) CR6GSE) was dissolved in 0.8 mL of dimethylformamide, N— 1 μL (0.0048 mmol) of (2-aminoethyl) -3-aminopropyltriethoxysilane (Gelest) was added and stirred for 30 minutes.
ついで、室温下ＴＡＭＲＡのＮ−ヒドロキシスクシンイミドエステル誘導体（Ｉｎｖｉｔｒｏｇｅｎ社製５（６）−ＴＡＭＲＡ−ＮＨＳ，ＳＥ）２．６ｍｇ（０．００４８ｍｍｏｌ）をジメチルホルムアミド０．２ｍＬに溶解させたものを添加し、３０分間撹拌した。 Then, 2.6 mg (0.0048 mmol) of TAMRA N-hydroxysuccinimide ester derivative (Invitrogen 5 (6) -TAMRA-NHS, SE) dissolved in 0.2 mL of dimethylformamide at room temperature was added, Stir for 30 minutes.
工程（２）：工程（１）で得られたＤＭＦ溶液とテトラエトキシシラン４０μＬを混合した。 Step (2): The DMF solution obtained in step (1) and 40 μL of tetraethoxysilane were mixed.
工程（３）：エタノール４０ｍＬ、１４％アンモニア水１０ｍＬを混合した。 Step (3): Ethanol 40 mL and 14% aqueous ammonia 10 mL were mixed.
工程（４）：工程３で作製した混合液を室温下撹拌しているところに、工程（２）で作製した混合液を添加した。添加開始から１２時間撹拌を行った。 Step (4): The mixture prepared in Step (2) was added to the mixture prepared in Step 3 while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
工程（５）：反応混合物を１００００ｇで６０分遠心分離を行い、上澄みを除去した。エタノールを加え、沈降物を分散させ、再度遠心分離を行った。同様の手順でエタノールと純水による洗浄を一回ずつ行った。 Step (5): The reaction mixture was centrifuged at 10,000 g for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. In the same procedure, washing with ethanol and pure water was performed once.
得られたシリカナノ粒子７のＳＥＭ観察を行ったところ、平均粒径１００ｎｍ、変動係数は１１％であった。 When the SEM observation of the obtained silica nanoparticle 7 was performed, the average particle diameter was 100 nm and the variation coefficient was 11%.
ローダミン６Ｇの吸収極大波長は、５２４ｎｍ、ＴＡＭＲＡの吸収極大波長は、５４６ｎｍである。したがって、波長４８８ｎｍの光では、ローダミン６Ｇのみが励起され、ＴＡＭＲＡは励起されない。 Rhodamine 6G has an absorption maximum wavelength of 524 nm, and TAMRA has an absorption maximum wavelength of 546 nm. Therefore, in the light having a wavelength of 488 nm, only rhodamine 6G is excited, and TAMRA is not excited.
また、ローダミン６Ｇの蛍光極大波長は５５２ｎｍであり、ＴＡＭＲＡの蛍光極大波長は５７６ｎｍである。したがって、波長５８０ｎｍの蛍光はＴＡＭＲＡ由来の発光に帰属できる。 The fluorescence maximum wavelength of rhodamine 6G is 552 nm, and the fluorescence maximum wavelength of TAMRA is 576 nm. Therefore, fluorescence having a wavelength of 580 nm can be attributed to light emission derived from TAMRA.
波長を４８８ｎｍの光によりローダミン６Ｇを励起したときに、波長５８０ｎｍに蛍光が観察された場合、ＤｙＬｉｇｈｔからＴＡＭＲＡへの蛍光共鳴エネルギー移動が起こったといえ、その蛍光強度が高いほどエネルギー移動効率が高いといえる。 When rhodamine 6G is excited by light having a wavelength of 488 nm, when fluorescence is observed at a wavelength of 580 nm, it can be said that fluorescence resonance energy transfer from DyLight to TAMRA has occurred. The higher the fluorescence intensity, the higher the energy transfer efficiency. I can say that.
得られたシリカナノ粒子の１ｎＭＰＢＳ（リン酸緩衝生理食塩水）分散液をそれぞれ調製し、蛍光分光光度計Ｆ−７０００（商品名、日立ハイテクノロジーズ社製）を用いて、励起光波長を４８８ｎｍとし、波長５８０ｎｍの蛍光強度を測定した。作製したシリカナノ粒子１の５８０ｎｍでの蛍光強度を１００としたときの相対値として評価した。評価結果を表１に示す。 Each 1 nM PBS (phosphate buffered saline) dispersion of the silica nanoparticles obtained was prepared, and using a fluorescence spectrophotometer F-7000 (trade name, manufactured by Hitachi High-Technologies Corporation), the excitation light wavelength was 488 nm, The fluorescence intensity at a wavelength of 580 nm was measured. The produced silica nanoparticles 1 were evaluated as relative values when the fluorescence intensity at 580 nm was defined as 100. The evaluation results are shown in Table 1.
表１に示した結果から明らかなように、本発明のＢＯＤＩＰＹ ＦＬとＴＡＭＲＡを連結かつ、シリカ粒子へ結合させたものの５８０ｎｍでの蛍光強度は、ＢＯＤＩＰＹ ＦＬとＴＡＭＲＡを連結せず、それぞれ別個にシリカ粒子に内包、結合させる公知技術のものに比べ、著しく大きいことが分かる。このことはＢＯＤＩＰＹ ＦＬとＴＡＭＲＡとを同一分子内に連結させたことにより、両者の距離を最適に近付けることができたと考える。 As apparent from the results shown in Table 1, the BODIPY FL of the present invention was linked to TAMRA and bound to silica particles, but the fluorescence intensity at 580 nm was not linked to BODIPY FL and TAMRA. It can be seen that it is significantly larger than that of a known technique in which particles are encapsulated and bonded. This suggests that BODIPY FL and TAMRA can be optimally brought close to each other by linking BODIPY FL and TAMRA in the same molecule.
同様にＡｌｅｘａ Ｆｌｕｏｒ４８８とローダミン６Ｇとを連結させたもの、ＨｉＬｙｔｅ Ｆｌｕｏｒ４８８とローダミン６Ｇとを連結させたもの、ＤｙＬｉｇｈｔ４８８とＴＡＭＲＡとを連結させたものおよびローダミン６ＧとＴＡＭＲＡとを連結させたものをそれぞれシリカ粒子へ結合させたものも、高いエネルギー移動効率の結果として５８０ｎｍでの蛍光強度が極めて大きいことが分かる。 Similarly, silica particles obtained by linking Alexa Fluor 488 and rhodamine 6G, linking HiLy Fluor 488 and rhodamine 6G, linking DyLight 488 and TAMRA, and linking Rhodamine 6G and TAMRA, respectively. It can also be seen that the fluorescence intensity at 580 nm is also very high as a result of the high energy transfer efficiency.
また、ＢＯＤＩＰＹ ＦＬとＴＡＭＲＡと連結させた分子を用いても、その分子が加水分解性基をもつシリル基をもたない場合、５８０ｎｍでの蛍光強度は本発明に比べ大きく低下した。このことは容易にシリカ粒子から漏えいしてしまい、洗浄により除去されたためと考えられる。 Further, even when a molecule in which BODIPY FL and TAMRA were linked was used, when the molecule did not have a silyl group having a hydrolyzable group, the fluorescence intensity at 580 nm was greatly reduced as compared with the present invention. This is presumably because it was easily leaked from the silica particles and removed by washing.
《生体物質標識剤》
シリカナノ粒子１及び２を用い、分子修飾シリカナノ粒子Ａ及びＢを各々調製し、更にこれを用いて生体物質標識剤１、２及び３を調製して、生体物質標識剤の長期保存性を評価した。
<Biological substance labeling agent>
Using silica nanoparticles 1 and 2, molecularly modified silica nanoparticles A and B were prepared, respectively, and further, biomaterial labeling agents 1, 2, and 3 were prepared, and the long-term storage stability of the biomaterial labeling agent was evaluated. .
〔分子修飾シリカナノ粒子Ａの調製〕
（分子修飾シリカナノ粒子Ａ：アミノ基修飾したＢＯＤＩＰＹ−ＦＬ／ＴＡＭＲＡ連結内包シリカナノ粒子）
シリカナノ粒子１ １ｍｇを純水５ｍＬに分散させた。アミノプロピルトリエトキシシラン水分散液１００μＬを添加し、室温で１２時間撹拌した。
[Preparation of molecularly modified silica nanoparticles A]
(Molecular modified silica nanoparticles A: amino group modified BODIPY-FL / TAMRA linked silica nanoparticles)
1 mg of silica nanoparticles 1 was dispersed in 5 mL of pure water. 100 μL of aminopropyltriethoxysilane aqueous dispersion was added and stirred at room temperature for 12 hours.
反応混合物を１００００ｇで６０分遠心分離を行い、上澄みを除去した。エタノールを加え、沈降物を分散させ、再度遠心分離を行った。同様の手順でエタノールと純水による洗浄を行った。 The reaction mixture was centrifuged at 10,000 g for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. Washing with ethanol and pure water was performed in the same procedure.
得られたアミノ基修飾したシリカナノ粒子ＡのＦＴ−ＩＲ測定を行ったところ、アミノ基に由来する吸収が観測でき、アミノ基修飾できたことを確認できた。 When the FT-IR measurement of the obtained amino group-modified silica nanoparticles A was performed, absorption derived from the amino group was observed, and it was confirmed that the amino group was modified.
〔分子修飾シリカナノ粒子Ｂの調製〕
（分子修飾シリカナノ粒子Ｂ：アミノ基修飾したＢＯＤＩＰＹ−ＦＬ／ＴＡＭＲＡ共内包シリカナノ粒子）
シリカナノ粒子２について、分子修飾シリカナノ粒子Ａの調製と同様の手順で、アミノ基修飾を行った。
[Preparation of molecularly modified silica nanoparticles B]
(Molecular modified silica nanoparticles B: amino group-modified BODIPY-FL / TAMRA co-encapsulated silica nanoparticles)
About silica nanoparticle 2, amino group modification was performed in the same procedure as the preparation of molecular modified silica nanoparticle A.
得られたアミノ基修飾したシリカ被覆テトラメチルローダミン内包シリカナノ粒子のＦＴ−ＩＲ測定を行ったところ、アミノ基に由来する吸収が観測でき、アミノ基修飾できたことを確認できた。 When the FT-IR measurement of the silica-coated tetramethylrhodamine-encapsulated silica nanoparticles obtained by amino group modification was performed, absorption derived from amino groups could be observed, and it was confirmed that the amino groups could be modified.
〔生体物質標識剤１の調製〕
（生体物質標識剤１：アミノ基修飾ＢＯＤＩＰＹ−ＦＬ／ＴＡＭＲＡ連結内包シリカナノ粒子への抗体結合体）
分子修飾シリカナノ粒子Ａの調製で得られたアミノ基修飾ＢＯＤＩＰＹ−ＦＬ／ＴＡＭＲＡ連結内包シリカナノ粒子０．５ｍｇを純水０．５ｍＬに分散させたもの０．１ｍＬをＤＭＳＯ２ｍＬに添加した。そこへ、ｓｕｌｆｏ−ＳＭＣＣ（Ｐｉｅｒｃｅ社製）をいれ１時間反応させた。過剰のｓｕｌｆｏ−ＳＭＣＣなどを遠心分離により除去する、一方で、抗ｈＣＧ抗体を１Ｍジチオスレイトール（ＤＴＴ）で還元処理を行い、ゲルろ過カラムにより過剰のＤＴＴを除去した。
[Preparation of biological material labeling agent 1]
(Biological material labeling agent 1: antibody conjugate to amino group-modified BODIPY-FL / TAMRA-linked encapsulated silica nanoparticles)
0.1 mL of a dispersion of 0.5 mg of amino group-modified BODIPY-FL / TAMRA encapsulated silica nanoparticles obtained in the preparation of molecularly modified silica nanoparticles A in 0.5 mL of pure water was added to 2 mL of DMSO. Sulfo-SMCC (Pierce) was added thereto and reacted for 1 hour. Excess sulfo-SMCC and the like were removed by centrifugation, while anti-hCG antibody was reduced with 1M dithiothreitol (DTT), and excess DTT was removed by a gel filtration column.
ｓｕｌｆｏ−ＳＭＣＣ処理したＢＯＤＩＰＹ−ＦＬ／ＴＡＭＲＡ連結内包シリカナノ粒子と、ＤＴＴ処理した抗ｈＣＧ抗体を混合し、１時間反応させた。１０ｍＭメルカプトエタノールを添加し、反応を停止させた。ゲルろ過カラムにより未反応物を除去し、抗ｈＣＧ抗体が結合したＢＯＤＩＰＹ−ＦＬ／ＴＡＭＲＡ連結内包シリカナノ粒子（生体物質標識剤１）を得た。 Sulfo-SMCC-treated BODIPY-FL / TAMRA-linked encapsulated silica nanoparticles and DTT-treated anti-hCG antibody were mixed and reacted for 1 hour. 10 mM mercaptoethanol was added to stop the reaction. Unreacted substances were removed by a gel filtration column to obtain BODIPY-FL / TAMRA linked encapsulated silica nanoparticles (biological substance labeling agent 1) to which an anti-hCG antibody was bound.
〔生体物質標識剤２の調製〕
（生体物質標識剤２：アミノ基修飾ＢＯＤＩＰＹ−ＦＬ／ＴＡＭＲＡ共内包シリカナノ粒子への抗体結合体）
分子修飾シリカナノ粒子Ｂの調製で得られたアミノ基修飾シＢＯＤＩＰＹ−ＦＬ／ＴＡＭＲＡ共内包シリカナノ粒子について、生体物質標識剤１の調製と同様の手順で、抗ｈＣＧ抗体が結合したＢＯＤＩＰＹ−ＦＬ／ＴＡＭＲＡ共内包シリカナノ粒子（生体物質標識剤２）を得た。
[Preparation of biological material labeling agent 2]
(Biological substance labeling agent 2: antibody conjugate to amino group-modified BODIPY-FL / TAMRA co-encapsulated silica nanoparticles)
BODIPY-FL / TAMRA to which an anti-hCG antibody was bound in the same manner as in the preparation of biomaterial labeling agent 1 for amino group-modified silica BODIPY-FL / TAMRA co-encapsulated silica nanoparticles obtained in the preparation of molecularly modified silica nanoparticles B Co-encapsulated silica nanoparticles (biological substance labeling agent 2) were obtained.
生体物質標識剤１及び２を用いたイムノアッセイを下記の手順で行った。
１）マイクロプレート上ウェル内にアンチ−ｈαサブニットを固定化した。
２）抗原であるｈＣＧを各ウェルに濃度を変えて入れた。
３）過剰のｈＣＧを洗浄により除去後、各ウェルに生体物質標識剤分散液を入れた。
４）過剰の生体物質標識剤を洗浄により除去した。
５）マイクロプレートリーダーフルオロスキャンアセントＦＬ（商品名、サーモフィッシャーサイエンティフック社製）により各ウェルの蛍光強度を測定した。
An immunoassay using biological substance labeling agents 1 and 2 was carried out according to the following procedure.
1) Anti-hα subunit was immobilized in the well on the microplate.
2) The antigen hCG was added to each well at different concentrations.
3) After removing excess hCG by washing, a biological material labeling agent dispersion was added to each well.
4) Excess biological material labeling agent was removed by washing.
5) The fluorescence intensity of each well was measured with a microplate reader Fluoroscan Ascent FL (trade name, manufactured by Thermo Fisher Scientific).
生体物質標識剤１又は生体物質標識剤２を用いたところ、どちらも抗原濃度に応じて蛍光強度が上昇した。すなわち、本発明で得られた生体物質標識剤１及び生体物質標識剤２はいずれもＢ、抗原認識能を損なっていないことが言える。 When the biological material labeling agent 1 or the biological material labeling agent 2 was used, the fluorescence intensity increased in accordance with the antigen concentration. That is, it can be said that the biological substance labeling agent 1 and the biological substance labeling agent 2 obtained in the present invention are both B and do not impair antigen recognition ability.
生体標識剤１を用い、ｈＣＧ抗体濃度が１ｎｇ／ｍＬの時の蛍光強度を１００としたときの、それぞれの生体標識剤について各ｈＣＧ抗体濃度で測定した蛍光強度を表２に示す。 Table 2 shows the fluorescence intensity measured for each biomarker at each hCG antibody concentration when the biomarker 1 was used and the fluorescence intensity when the hCG antibody concentration was 1 ng / mL was taken as 100.
表２に示した結果から明らかなように、本発明で得られたＢＯＤＩＰＹ−ＦＬ／ＴＡＭＲＡ連結内包シリカナノ粒子からなる生体物質標識剤１は、公知技術で得られたＢＯＤＩＰＹ−ＦＬ／ＴＡＭＲＡ共内包シリカナノ粒子からなる生体物質標識剤２に比べ、より低濃度のｈＣＧ抗体濃度を検出できることが分かる。すなわち、この結果により、本発明により高感度検出が可能な生体物質標識剤を提供することができることが分かる。 As is clear from the results shown in Table 2, the biological substance labeling agent 1 composed of the BODIPY-FL / TAMRA linked inclusion silica nanoparticles obtained in the present invention is obtained from the BODIPY-FL / TAMRA co-encapsulation silica nanoparticle obtained by a known technique. It can be seen that a lower concentration of hCG antibody can be detected as compared with the biological substance labeling agent 2 made of particles. That is, it can be seen from this result that a biological substance labeling agent capable of highly sensitive detection can be provided by the present invention.
Claims (6)
工程（ａ）：同一分子内に二種以上のアミノ基と、少なくとも一種の加水分解性置換基を有するシリル基を有する分子と、当該アミノ基と反応する官能基を有する有機蛍光色素とを反応させる工程、
工程（ｂ）：前記工程（ａ）で得られた反応生成物を含ケイ素アルコキシドと混合し、塩基性条件下加水分解反応を行う工程。 It is a manufacturing method of the organic fluorescent dye inclusion silica nanoparticle which manufactures the organic fluorescent dye inclusion silica nanoparticle as described in any one of Claim 1- 3, Comprising: The following process (a) and process (b) are included. The manufacturing method of the organic fluorescent pigment | dye inclusion | inner_cover silica nanoparticle characterized by these.
Step (a): Reacting a molecule having two or more amino groups in the same molecule, a molecule having a silyl group having at least one hydrolyzable substituent, and an organic fluorescent dye having a functional group that reacts with the amino group The process of
Step (b): A step of mixing the reaction product obtained in the step (a) with a silicon-containing alkoxide and performing a hydrolysis reaction under basic conditions.
一般式（１）：ＮＨ２−（ＣＨ２）ｎ−ＮＨ−（ＣＨ２）ｍ−ＳｉＲｌ（ＯＲ１）ｋ
（式中、ｎ及びｍは１〜１２の整数を表し、互いに同一でも異なっていてもよい。ｌ及びｋはｌ＋ｋ＝４を満たし、ｌは０〜３の整数、ｋは１〜４の整数を表す。Ｒ１は非加水分解性の置換基を表し、ＯＲ１は加水分解性の炭素数１〜６のアルコキシ基を表し、複数ある場合、互いに同一でも異なっていてもよい。） 5. The molecule having a silyl group having two or more amino groups and at least one hydrolyzable substituent in the same molecule is a silane compound represented by the following general formula (1). The manufacturing method of the organic fluorescent dye inclusion | inner_cover silica nanoparticle of description.
Formula (1): NH 2 - ( CH 2) n -NH- (CH 2) m -SiR l (OR 1) k
(In the formula, n and m each represents an integer of 1 to 12, and may be the same or different. L and k satisfy l + k = 4, l is an integer of 0 to 3, and k is an integer of 1 to 4) R 1 represents a non-hydrolyzable substituent, OR 1 represents a hydrolyzable alkoxy group having 1 to 6 carbon atoms, and when there are a plurality of them, they may be the same or different from each other.
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| CN111484842A (en) * | 2020-03-13 | 2020-08-04 | 大连理工大学 | Fluorescent silica nanoparticles with nano hydrophobic cage structure and preparation method and application thereof |
| WO2021157475A1 (en) * | 2020-02-03 | 2021-08-12 | コニカミノルタ株式会社 | Fluorescent silica nanoparticles and method for manufacturing fluorescent silica nanoparticles |
| WO2023032306A1 (en) * | 2021-08-30 | 2023-03-09 | コニカミノルタ株式会社 | Light-emitting nanoparticles and light-emitting labeling material for pathological diagnosis use |
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| US11726000B2 (en) | 2020-12-22 | 2023-08-15 | Whirlpool Corporation | Hybrid fluorescent UV dye for refrigerant systems |
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| WO2023032306A1 (en) * | 2021-08-30 | 2023-03-09 | コニカミノルタ株式会社 | Light-emitting nanoparticles and light-emitting labeling material for pathological diagnosis use |
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