JP4604187B2 - 糖鎖を介したルシフェラーゼと有機蛍光色素間のエネルギー移動を利用した可視−近赤外光プローブ - Google Patents
糖鎖を介したルシフェラーゼと有機蛍光色素間のエネルギー移動を利用した可視−近赤外光プローブ Download PDFInfo
- Publication number
- JP4604187B2 JP4604187B2 JP2007211216A JP2007211216A JP4604187B2 JP 4604187 B2 JP4604187 B2 JP 4604187B2 JP 2007211216 A JP2007211216 A JP 2007211216A JP 2007211216 A JP2007211216 A JP 2007211216A JP 4604187 B2 JP4604187 B2 JP 4604187B2
- Authority
- JP
- Japan
- Prior art keywords
- luciferase
- fluorescent dye
- organic fluorescent
- sugar chain
- visible
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108060001084 Luciferase Proteins 0.000 title claims description 63
- 239000005089 Luciferase Substances 0.000 title claims description 62
- 239000007850 fluorescent dye Substances 0.000 title claims description 39
- 239000000523 sample Substances 0.000 title claims description 22
- 238000012546 transfer Methods 0.000 title claims description 10
- 108010031180 cypridina luciferase Proteins 0.000 claims description 21
- 239000000975 dye Substances 0.000 claims description 8
- 125000003277 amino group Chemical group 0.000 claims description 7
- 230000005284 excitation Effects 0.000 claims description 5
- 229960004657 indocyanine green Drugs 0.000 claims description 5
- GJCOSYZMQJWQCA-UHFFFAOYSA-N 9H-xanthene Chemical class C1=CC=C2CC3=CC=CC=C3OC2=C1 GJCOSYZMQJWQCA-UHFFFAOYSA-N 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 4
- MOFVSTNWEDAEEK-UHFFFAOYSA-M indocyanine green Chemical class [Na+].[O-]S(=O)(=O)CCCCN1C2=CC=C3C=CC=CC3=C2C(C)(C)C1=CC=CC=CC=CC1=[N+](CCCCS([O-])(=O)=O)C2=CC=C(C=CC=C3)C3=C2C1(C)C MOFVSTNWEDAEEK-UHFFFAOYSA-M 0.000 claims description 4
- QDLAGTHXVHQKRE-UHFFFAOYSA-N lichenxanthone Chemical class COC1=CC(O)=C2C(=O)C3=C(C)C=C(OC)C=C3OC2=C1 QDLAGTHXVHQKRE-UHFFFAOYSA-N 0.000 claims description 4
- 125000000717 hydrazino group Chemical group [H]N([*])N([H])[H] 0.000 claims description 3
- 239000013543 active substance Substances 0.000 description 23
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 238000003384 imaging method Methods 0.000 description 12
- 238000000034 method Methods 0.000 description 11
- 239000000243 solution Substances 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 239000011780 sodium chloride Substances 0.000 description 8
- 239000000427 antigen Substances 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 239000003431 cross linking reagent Substances 0.000 description 6
- -1 haptens Substances 0.000 description 6
- 241000872931 Myoporum sandwicense Species 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000000295 emission spectrum Methods 0.000 description 5
- 239000005556 hormone Substances 0.000 description 5
- 229940088597 hormone Drugs 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000002096 quantum dot Substances 0.000 description 5
- 102000004310 Ion Channels Human genes 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- 125000003275 alpha amino acid group Chemical group 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- GOLORTLGFDVFDW-UHFFFAOYSA-N 3-(1h-benzimidazol-2-yl)-7-(diethylamino)chromen-2-one Chemical compound C1=CC=C2NC(C3=CC4=CC=C(C=C4OC3=O)N(CC)CC)=NC2=C1 GOLORTLGFDVFDW-UHFFFAOYSA-N 0.000 description 3
- UJKPHYRXOLRVJJ-MLSVHJFASA-N CC(O)C1=C(C)/C2=C/C3=N/C(=C\C4=C(CCC(O)=O)C(C)=C(N4)/C=C4\N=C(\C=C\1/N\2)C(C)=C4C(C)O)/C(CCC(O)=O)=C3C Chemical compound CC(O)C1=C(C)/C2=C/C3=N/C(=C\C4=C(CCC(O)=O)C(C)=C(N4)/C=C4\N=C(\C=C\1/N\2)C(C)=C4C(C)O)/C(CCC(O)=O)=C3C UJKPHYRXOLRVJJ-MLSVHJFASA-N 0.000 description 3
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical class OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 3
- 125000003172 aldehyde group Chemical group 0.000 description 3
- 150000001299 aldehydes Chemical class 0.000 description 3
- 239000003638 chemical reducing agent Substances 0.000 description 3
- ZFKJVJIDPQDDFY-UHFFFAOYSA-N fluorescamine Chemical compound C12=CC=CC=C2C(=O)OC1(C1=O)OC=C1C1=CC=CC=C1 ZFKJVJIDPQDDFY-UHFFFAOYSA-N 0.000 description 3
- 229960003569 hematoporphyrin Drugs 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 238000004020 luminiscence type Methods 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 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 3
- 230000001988 toxicity Effects 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- ZWPWSXGBDMGKKS-ZDUSSCGKSA-N Cypridina luciferin Chemical compound C1=CC=C2C(C=3NC(CCCNC(N)=N)=C4N=C(C(N4C=3)=O)[C@@H](C)CC)=CNC2=C1 ZWPWSXGBDMGKKS-ZDUSSCGKSA-N 0.000 description 2
- JREHDCFHGRHVKG-ZDUSSCGKSA-N Cypridina luciferin Natural products CC[C@H](C)C1=NC2=C(CCCNC(=N)N)NC(=CN2C1=O)c3cc4ccccc4[nH]3 JREHDCFHGRHVKG-ZDUSSCGKSA-N 0.000 description 2
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 2
- 241000257465 Echinoidea Species 0.000 description 2
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 2
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- 238000012984 biological imaging Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 238000000101 transmission high energy electron diffraction Methods 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 description 1
- TYKASZBHFXBROF-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 2-(2,5-dioxopyrrol-1-yl)acetate Chemical compound O=C1CCC(=O)N1OC(=O)CN1C(=O)C=CC1=O TYKASZBHFXBROF-UHFFFAOYSA-N 0.000 description 1
- VLARLSIGSPVYHX-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-(2,5-dioxopyrrol-1-yl)hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCN1C(=O)C=CC1=O VLARLSIGSPVYHX-UHFFFAOYSA-N 0.000 description 1
- LTDQGCFMTVHZKP-UHFFFAOYSA-N (4-bromophenyl)-(4,6-dimethoxy-3-methyl-1-benzofuran-2-yl)methanone Chemical compound O1C2=CC(OC)=CC(OC)=C2C(C)=C1C(=O)C1=CC=C(Br)C=C1 LTDQGCFMTVHZKP-UHFFFAOYSA-N 0.000 description 1
- VOTJUWBJENROFB-UHFFFAOYSA-N 1-[3-[[3-(2,5-dioxo-3-sulfopyrrolidin-1-yl)oxy-3-oxopropyl]disulfanyl]propanoyloxy]-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCSSCCC(=O)ON1C(=O)C(S(O)(=O)=O)CC1=O VOTJUWBJENROFB-UHFFFAOYSA-N 0.000 description 1
- NQTSTBMCCAVWOS-UHFFFAOYSA-N 1-dimethoxyphosphoryl-3-phenoxypropan-2-one Chemical compound COP(=O)(OC)CC(=O)COC1=CC=CC=C1 NQTSTBMCCAVWOS-UHFFFAOYSA-N 0.000 description 1
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000254173 Coleoptera Species 0.000 description 1
- 241000035538 Cypridina Species 0.000 description 1
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 241000254158 Lampyridae Species 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 108010052090 Renilla Luciferases Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 125000004450 alkenylene group Chemical group 0.000 description 1
- 125000002947 alkylene group Chemical group 0.000 description 1
- NIGUVXFURDGQKZ-UQTBNESHSA-N alpha-Neup5Ac-(2->3)-beta-D-Galp-(1->4)-[alpha-L-Fucp-(1->3)]-beta-D-GlcpNAc Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@H]2[C@@H]([C@@H](O[C@]3(O[C@H]([C@H](NC(C)=O)[C@@H](O)C3)[C@H](O)[C@H](O)CO)C(O)=O)[C@@H](O)[C@@H](CO)O2)O)[C@@H](CO)O[C@@H](O)[C@@H]1NC(C)=O NIGUVXFURDGQKZ-UQTBNESHSA-N 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 125000000732 arylene group Chemical group 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 229940088623 biologically active substance Drugs 0.000 description 1
- 238000000225 bioluminescence resonance energy transfer Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910052793 cadmium Inorganic materials 0.000 description 1
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 150000002009 diols Chemical group 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 125000004185 ester group Chemical group 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- OAKJQQAXSVQMHS-UHFFFAOYSA-N hydrazine group Chemical group NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 125000000468 ketone group Chemical group 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 125000000896 monocarboxylic acid group Chemical group 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 229960002715 nicotine Drugs 0.000 description 1
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 238000002428 photodynamic therapy Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229940076279 serotonin Drugs 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical group O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 1
- 238000003786 synthesis reaction Methods 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
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0039—Coumarin dyes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
- A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0036—Porphyrins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
- A61K49/0043—Fluorescein, used in vivo
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0056—Peptides, proteins, polyamino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
- C12Y113/12—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12)
- C12Y113/12006—Cypridina-luciferin 2-monooxygenase (1.13.12.6), i.e. cypridina-luciferase
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Peptides Or Proteins (AREA)
Description
あり、個体イメージングに最も適する650-750nmの光エネルギーは極わずかである。また
、ウミホタルルシフェラーゼは、そのルシフェリン類縁体やナノ量子ドットの組み合わせにより紫外光に近い最大発光波長380nm(特許文献3)から個体深部からの透過効率の高
い赤色から近赤外光最大発光波長650 nm近くの光(特許文献4)を生み出すことができる、有用性の高い発光プローブであることは明らかである。特にウミホタルルシフェラーゼとナノ量子ドット融合体の間のエネルギー移動を活用した近赤外発光プローブは、個体透過性の高い光を生み出す有効な方法であることは明らかである。しかしながら、ナノ量子ドットはカドミウムなどの生体毒金属を用いる点、腎臓等で排出しにくい大きさである点など、個体イメージングに際して、安全毒性の問題や発光プローブの引き起こす生命情報かく乱の問題が指摘されている。併せて、ウミシイタケルシフェラーゼとナノ量子ドット融合体による近赤外発光プローブも報告されている(非特許文献1)が、上記の理由により安全性が懸念されている。
してなる、ルシフェラーゼ誘導体。
に蛍光極大を有する、項1又は2に記載の誘導体。
る色素である、項1〜3のいずれかに記載の誘導体。
標識化生理活性物質。
あり、ウミホタルルシフェラーゼと有機蛍光色素間のエネルギー移動による650-750nmの
範囲の蛍光極大を検出することを特徴とする、生体イメージング法。
コチン、グルタミン酸、セロトニンなどが挙げられる。糖鎖としては、シアリルルイスX又はその誘導体などの生体ないし細胞に認識され得る糖鎖が好ましい。
複合体及びルシフェリンによる発光は、例えばウミホタルホタルルシフェラーゼと量子ドットの組み合わせと比較して、2倍以上、好ましくは4倍以上、より好ましくは10倍以上
の発光量を有する。且つナノ金属ドットの有毒性を回避できる。
本発明で使用され得るウミホタルルシフェラーゼは公知である。本明細書および特許請求の範囲において、「ウミホタルルシフェラーゼ」とは、野生型ウミホタルルシフェラーゼあるいは任意のその改変体を広く包含する。野生型ウミホタルルシフェラーゼのアミノ酸配列はAAB86460, AAA30332, BAD08210などに記載されている。 ウミホタルルシフェラーゼ改変体は、1または複数個、好ましくは1または十数個(例えば15,14, 13,12,11,10,9,8,7,6,5,4,3,2個)のアミノ酸が置換、付加、欠失、挿入されていてもよく、ウミホ
タルルシフェリンを基質として発光させる活性を有している限り任意の改変体を包含する。該改変体は、上記の野生型ウミホタルルシフェラーゼとホモロジーが70%以上、好ましくは80%以上、より好ましくは90%以上、さらに好ましくは95%以上、特に好ましくは98%以上、最も好ましくは99%以上のホモロジーを有する。
〕などの方法により改変したり、リン酸トリエステル法やリン酸アミダイト法などの化学合成手段〔J. Am. Chem. Soc., 89, p4801 (1967);同91, p3350 (1969);Science, 150,p178 (1968) ;Tetrahedron Lett., 22, p1859 (1981);同24, p245 (1983)〕により変異させたDNAを合成したり、それらの組合せにより実施することができる。
じて使用して、有機蛍光色素が糖鎖部分で連結されたルシフェラーゼ誘導体を得ることができる。
導入する反応スキームの1例を以下に示す。
の溶液と混合し、4℃などの低温から室温で10分から3時間程度反応させ、必要に応じ
てカラムで精製するなどの後処理を行なうことで、アルデヒド基を有するルシフェラーゼとする。このルシフェラーゼを有機蛍光色素-NHNH2(当量又は過剰量または化学量論量未
満の量)と反応させることにより、有機蛍光色素が連結された目的とするルシフェラーゼ
誘導体を得ることができる。
溶液と反応させ、アルデヒド残基がルシフェラーゼあたり1個以上残存するように有機蛍光色素-NHNH2を反応させ、次いでアミノ基を有する生理活性物質を反応させ、NaBH3CNなどの還元剤を使用して、さらに生理活性物質が連結された有機蛍光色素−ルシフェラーゼ−生理活性物質複合体を得ることができる。或いは、有機蛍光色素が連結されたルシフェラーゼ誘導体を再度NaIO4の溶液と反応させてアルデヒド基を生成し、次いでアミ
ノ基を有する生理活性物質を反応させ、NaBH3CNなどの還元剤を使用して、さらに生理活性物質が連結された有機蛍光色素−ルシフェラーゼ−生理活性物質複合体を得ることができる。
ケニレン、ポリオキシエチレン、ポリオキシプロピレンなどの任意の二価又は多価の基で連結したものが挙げられる。具体的な架橋剤としては、AEDP,AMAS,APG,ASBA,BASED,BMB, BMDB,DTSSP,EMCA,EMCH,EMCS,HBVSKMUA,SADP,SAEDなどが例示でき、架橋剤は、生理活性物
質が有する反応性基(アミノ基、SH基、OH基、アルデヒド基またはケトン基など)の種類によって適宜選択することができる。架橋剤は、へテロ二官能性の架橋剤であって、ルシフェラーゼと生理活性物質を段階的に連結可能なものが好ましい。ルシフェラーゼのN
末とC末に挿入されるペプチド配列はHaloTag、AviTag、SNAPTag、抗体ならびにその断片
などが挙げられる。
ができる。エネルギー移動が生じるために、有機蛍光色素は400nm〜700nm程度、例えば400nm〜550nm程度、或いは420nm〜500nm程度、の蛍光極大λmaxを有する色素であるのがよ
い。
実施例1
1、ウミホタルルシフェラーゼの糖鎖に蛍光色素の導入
量の20 mM NaIO4の0.1M acetate buffer pH5.2溶液と混合し、4度で0.5時間ゆっくり
攪拌した。反応液をGE Health社PD-10カラムに載せ、100mM リン酸ナトリウム、150mM NaCl 溶液で溶出し、活性分画のみ回収し(約2mL)した。ミリポア社のBiomax100kで2mL液を約0.02mlまでに濃縮した。1mgのHilyte fluore647(NH2NH-インドシアニングリーン;Anaspec)を0.1Macetate buffer pH5.2溶液0.1mlで溶かした。ルシフェラーゼ液0.02mlと同量で、室温で2時間反応させた。反応液をGE Health社PD-10カラムに載せ、100mM リン酸ナトリウム、150mM NaCl 溶液で溶出し、活性分画のみ回収
(約2mL)した。
2、ウミホタルルシフェラーゼ・蛍光色素の発光スペクトル
0.001ml Cluc-dyeを0.1ml下記の緩衝液の一種類で溶かし、0.001 mLウミホタルルシフェリン溶液(0.001mM)と反応させ、発光スペクトルを測定した。結果を図1、図2に示す。
0.1 M Phosphate buffer pH7.4 /100 mM NaCl
0.1 M Tris-HCl buffer pH 8.0 / 100 mM NaCl
0.1 M Phosphate buffer (0.1 M) pH6.4 /500 mM NaCl
0.1 M Phosphate buffer (0.1 M) pH7.4 /500 mM NaCl
0.1 M Tris-HCl buffer pH 8.0 / 500 mM NaCl
結果、ルシフェラーゼの最大発光波長(460nm)と発光エネルギー移動による色素の最
大発光波長(670nm)が観測された。さらに、この発光スペクトルは塩やpHの条件の変化による影響を受けないことがわかった。
Claims (5)
- 糖鎖と連結可能なアミノ基又はヒドラジノ基(NH2NH-)を有し、かつ、インドシアニングリーン又はキサンテンの骨格を有する化合物である有機蛍光色素がルシフェラーゼ由来糖鎖を介して糖鎖含有ルシフェラーゼに結合してなる、ルシフェラーゼ誘導体。
- ルシフェラーゼがウミホタルルシフェラーゼである、請求項1に記載の誘導体。
- 有機蛍光色素とルシフェラーゼの間のエネルギー移動により、650-750nmの範囲に蛍光極
大を有する、請求項1又は2に記載の誘導体。 - 有機蛍光色素が外部励起光源によって400nm〜700nmの蛍光極大(λmax)を有する色素で
ある、請求項1〜3のいずれかに記載の誘導体。 - 請求項1〜4のいずれかに記載のルシフェラーゼ誘導体の可視−近赤外光プローブとしての使用。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2007211216A JP4604187B2 (ja) | 2007-08-14 | 2007-08-14 | 糖鎖を介したルシフェラーゼと有機蛍光色素間のエネルギー移動を利用した可視−近赤外光プローブ |
US12/222,356 US8143013B2 (en) | 2007-08-14 | 2008-08-07 | Visible to near-infrared light probe using energy transfer between luciferase and an organic dye via a sugar chain |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2007211216A JP4604187B2 (ja) | 2007-08-14 | 2007-08-14 | 糖鎖を介したルシフェラーゼと有機蛍光色素間のエネルギー移動を利用した可視−近赤外光プローブ |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2009044964A JP2009044964A (ja) | 2009-03-05 |
JP4604187B2 true JP4604187B2 (ja) | 2010-12-22 |
Family
ID=40363125
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2007211216A Expired - Fee Related JP4604187B2 (ja) | 2007-08-14 | 2007-08-14 | 糖鎖を介したルシフェラーゼと有機蛍光色素間のエネルギー移動を利用した可視−近赤外光プローブ |
Country Status (2)
Country | Link |
---|---|
US (1) | US8143013B2 (ja) |
JP (1) | JP4604187B2 (ja) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7255851B2 (en) * | 1994-07-01 | 2007-08-14 | The Board Of Trustees Of The Leland Stanford Junior University | Non-invasive localization of a light-emitting conjugate in a mammal |
EP1969374A4 (en) | 2006-01-04 | 2010-04-14 | Univ Stanford | SELF-LIGHTING QUANTUM POINT SYSTEMS AND APPLICATION METHOD THEREFOR |
US20080241865A1 (en) * | 2007-03-26 | 2008-10-02 | National Institute Of Advanced Industrial Science And Technology | Visible to near-infrared light probe comprising complex of cypridina luciferase and quantum dot |
US20110182823A1 (en) | 2009-09-18 | 2011-07-28 | Stephen Oldfield | Compositions and methods for in vivo evaluation of bioluminescence |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9417593D0 (en) * | 1994-09-01 | 1994-10-19 | Secr Defence | Luciferase labelling method |
US6342379B1 (en) * | 1995-06-07 | 2002-01-29 | The Regents Of The University Of California | Detection of transmembrane potentials by optical methods |
JPWO2006106752A1 (ja) * | 2005-03-30 | 2008-09-11 | 東洋紡績株式会社 | 非侵襲性解析方法 |
EP1956086A4 (en) | 2005-11-16 | 2009-04-01 | Toyo Boseki | OPTIMIZED LUCIFERASE GENE FOR USE IN THE PICTORIAL ILLUSTRATION OF INTRA-CELLULAR LUMINESCENCE |
US20080038686A1 (en) * | 2006-04-18 | 2008-02-14 | Shigemi Nagai | Methods and kits for early stage caries detection |
US20070254311A1 (en) * | 2006-04-26 | 2007-11-01 | Cardiogenics Inc. | Covalent modification and conjugation of luciferase |
-
2007
- 2007-08-14 JP JP2007211216A patent/JP4604187B2/ja not_active Expired - Fee Related
-
2008
- 2008-08-07 US US12/222,356 patent/US8143013B2/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
JP2009044964A (ja) | 2009-03-05 |
US8143013B2 (en) | 2012-03-27 |
US20090047219A1 (en) | 2009-02-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Yao et al. | Upconversion luminescence nanomaterials: A versatile platform for imaging, sensing, and therapy | |
Chen | Nanoparticle fluorescence based technology for biological applications | |
Li et al. | Cage the firefly luciferin!–a strategy for developing bioluminescent probes | |
Hermanson | Bioconjugate techniques | |
Wessels et al. | Advances in cellular, subcellular, and nanoscale imaging in vitro and in vivo | |
Bhuckory et al. | In vivo biosensing using resonance energy transfer | |
Eilon-Shaffer et al. | ortho-Chlorination of phenoxy 1, 2-dioxetane yields superior chemiluminescent probes for in vitro and in vivo imaging | |
Li et al. | A near-infrared frequency upconversion probe for nitroreductase detection and hypoxia tumor in vivo imaging | |
Rusanov et al. | Lifetime imaging of FRET between red fluorescent proteins | |
Li et al. | A GSH-responsive PET-based fluorescent probe for cancer cells imaging | |
JP4604187B2 (ja) | 糖鎖を介したルシフェラーゼと有機蛍光色素間のエネルギー移動を利用した可視−近赤外光プローブ | |
Solhi et al. | Recent advances on the biosensing and bioimaging based on polymer dots as advanced nanomaterial: Analytical approaches | |
CN105807064B (zh) | 一种荧光素酶互补量子点生物传感器及其构建方法及其应用 | |
Díaz et al. | Quantum dots as förster resonance energy transfer acceptors of lanthanides in time-resolved bioassays | |
WO2007081716A2 (en) | Self-illuminating quantum dot systems and methods of use thereof | |
US20080299592A1 (en) | Red-Shifted Luciferase | |
WO2003055379A2 (en) | Method using a surface-selective nonlinear optical technique for imaging of biological samples | |
JPH04504713A (ja) | 共役ポリペプチドとその調製および使用方法 | |
Isailovic et al. | Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits | |
CN108148012A (zh) | 近红外第二窗口发射小分子稀土配合物荧光探针及其制备方法 | |
US8021647B2 (en) | In vivo optical imaging | |
McIntyre et al. | Optical proteolytic beacons for in vivo detection of matrix metalloproteinase activity | |
US20080241865A1 (en) | Visible to near-infrared light probe comprising complex of cypridina luciferase and quantum dot | |
Jalilian et al. | CdTe quantum dots with green fluorescence generated by bioluminescence resonance energy transfer from aequorin | |
Stell et al. | Multimodality imaging: novel pharmacological applications of reporter systems |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20090715 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20100119 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20100223 |
|
A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20100330 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20100628 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20100701 |
|
A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20100723 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20100907 |
|
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20100908 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 4604187 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20131015 Year of fee payment: 3 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20131015 Year of fee payment: 3 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
S533 | Written request for registration of change of name |
Free format text: JAPANESE INTERMEDIATE CODE: R313533 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
LAPS | Cancellation because of no payment of annual fees |