CN106675553A - Application of cyanine compound in preparing biological dye - Google Patents
Application of cyanine compound in preparing biological dye Download PDFInfo
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- CN106675553A CN106675553A CN201611253536.XA CN201611253536A CN106675553A CN 106675553 A CN106675553 A CN 106675553A CN 201611253536 A CN201611253536 A CN 201611253536A CN 106675553 A CN106675553 A CN 106675553A
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- compound
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- dye
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- -1 cyanine compound Chemical class 0.000 title claims abstract description 26
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 5
- 229910052717 sulfur Inorganic materials 0.000 claims abstract description 5
- 229910052711 selenium Inorganic materials 0.000 claims abstract description 3
- 229910052740 iodine Inorganic materials 0.000 claims description 4
- 229910001914 chlorine tetroxide Inorganic materials 0.000 claims description 3
- VLTRZXGMWDSKGL-UHFFFAOYSA-M perchlorate Chemical compound [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 239000012128 staining reagent Substances 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 abstract description 72
- IPZJQDSFZGZEOY-UHFFFAOYSA-N dimethylmethylene Chemical group C[C]C IPZJQDSFZGZEOY-UHFFFAOYSA-N 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 72
- 239000000975 dye Substances 0.000 description 44
- VZLUMFQKUQQRKH-UHFFFAOYSA-N [I].N1=CC=CC2=CC=CC=C21 Chemical compound [I].N1=CC=CC2=CC=CC=C21 VZLUMFQKUQQRKH-UHFFFAOYSA-N 0.000 description 23
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 16
- 238000011282 treatment Methods 0.000 description 13
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 12
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 10
- 239000007850 fluorescent dye Substances 0.000 description 10
- 238000007334 copolymerization reaction Methods 0.000 description 9
- 210000004940 nucleus Anatomy 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 7
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 7
- 102000016911 Deoxyribonucleases Human genes 0.000 description 6
- 108010053770 Deoxyribonucleases Proteins 0.000 description 6
- 102000006382 Ribonucleases Human genes 0.000 description 6
- 108010083644 Ribonucleases Proteins 0.000 description 6
- 230000029087 digestion Effects 0.000 description 6
- 229910000397 disodium phosphate Inorganic materials 0.000 description 6
- 238000010494 dissociation reaction Methods 0.000 description 6
- 230000005593 dissociations Effects 0.000 description 6
- 238000004043 dyeing Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 4
- 108010089610 Nuclear Proteins Proteins 0.000 description 4
- 102000007999 Nuclear Proteins Human genes 0.000 description 4
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 4
- 239000005030 aluminium foil Substances 0.000 description 4
- 239000003054 catalyst Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000012043 crude product Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000000799 fluorescence microscopy Methods 0.000 description 4
- 238000012632 fluorescent imaging Methods 0.000 description 4
- 239000000376 reactant Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000010898 silica gel chromatography Methods 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000012472 biological sample Substances 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- IOJUPLGTWVMSFF-UHFFFAOYSA-N cyclobenzothiazole Natural products C1=CC=C2SC=NC2=C1 IOJUPLGTWVMSFF-UHFFFAOYSA-N 0.000 description 3
- 102000038379 digestive enzymes Human genes 0.000 description 3
- 108091007734 digestive enzymes Proteins 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 238000010926 purge Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 238000004621 scanning probe microscopy Methods 0.000 description 3
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 3
- 229920002554 vinyl polymer Polymers 0.000 description 3
- QGKMIGUHVLGJBR-UHFFFAOYSA-M (4z)-1-(3-methylbutyl)-4-[[1-(3-methylbutyl)quinolin-1-ium-4-yl]methylidene]quinoline;iodide Chemical compound [I-].C12=CC=CC=C2N(CCC(C)C)C=CC1=CC1=CC=[N+](CCC(C)C)C2=CC=CC=C12 QGKMIGUHVLGJBR-UHFFFAOYSA-M 0.000 description 2
- IITIZHOBOIBGBW-UHFFFAOYSA-N 3-ethyl-2h-1,3-benzothiazole Chemical compound C1=CC=C2N(CC)CSC2=C1 IITIZHOBOIBGBW-UHFFFAOYSA-N 0.000 description 2
- 241001597008 Nomeidae Species 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 210000003850 cellular structure Anatomy 0.000 description 2
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 238000006862 quantum yield reaction Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 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 1
- PXDAXYDMZCYZNH-UHFFFAOYSA-N 3-methyl-2h-1,3-benzothiazole Chemical compound C1=CC=C2N(C)CSC2=C1 PXDAXYDMZCYZNH-UHFFFAOYSA-N 0.000 description 1
- VCVACUXPDLCBHF-UHFFFAOYSA-N 3-methyl-2h-1,3-benzoxazole Chemical compound C1=CC=C2N(C)COC2=C1 VCVACUXPDLCBHF-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N DMSO Substances CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 1
- AYDQIZKZTQHYIY-UHFFFAOYSA-N OC(=O)C1(C)CC(C(O)=O)=CC=C1 Chemical compound OC(=O)C1(C)CC(C(O)=O)=CC=C1 AYDQIZKZTQHYIY-UHFFFAOYSA-N 0.000 description 1
- ZCQWOFVYLHDMMC-UHFFFAOYSA-N Oxazole Chemical compound C1=COC=N1 ZCQWOFVYLHDMMC-UHFFFAOYSA-N 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- MZZINWWGSYUHGU-UHFFFAOYSA-J ToTo-1 Chemical compound [I-].[I-].[I-].[I-].C12=CC=CC=C2C(C=C2N(C3=CC=CC=C3S2)C)=CC=[N+]1CCC[N+](C)(C)CCC[N+](C)(C)CCC[N+](C1=CC=CC=C11)=CC=C1C=C1N(C)C2=CC=CC=C2S1 MZZINWWGSYUHGU-UHFFFAOYSA-J 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 150000001449 anionic compounds Chemical class 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 229940111121 antirheumatic drug quinolines Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000005605 benzo group Chemical group 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000014789 establishment of RNA localization Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000010304 firing Methods 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229910001412 inorganic anion Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 238000001921 nucleic acid quantification Methods 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 150000005053 phenanthridines Chemical class 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 150000003248 quinolines Chemical class 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
- C09B23/06—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups three >CH- groups, e.g. carbocyanines
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
-
- G01N2015/1028—
Abstract
The invention discloses application of a cyanine compound in preparing a biological dye. The cyanine compound has a structure as shown in a general formula I, wherein in the general formula I, X is C(CH3)2, O, S or Se; Y<-> is selected from halide ion, CIO4<->, PF6<->, BF4<->, CH3COO<-> or OTs<->. The compound provided by the invention can specifically recognize and mark RNA.
Description
Technical field
The present invention relates to new application of the class cyanine compound in terms of biological stain.
Background technology
Fluorescent dye is used widely in every field of science and technology as functional pigmented, especially in life section
The research of the aspects such as, clinical treatment diagnosis, immunoassay detection gets most of the attention in the whole world.At present, phenanthridines class (EB, PI),
The commercial fluorescence dyestuffs such as acridine (AO), imidazoles (Hoechst, DAPI) and the Hua Jingjia same clans (Cy, TOTO, SYTO) are in base
Because all playing an important role in the fields such as omics technology, nucleic acid quantification detection, cell analysis.However, these dyestuffs are all each
From the limitation that there is application.For example, the exciting light for having quite a few fluorescent dye is in ultraviolet region, due to ultraviolet light
Serious damage, therefore use of this kind of fluorescence in fluorescence microscopy can be caused to components such as intracellular nucleic acid, albumen
(Davis SK, Bardeen CJ.Photochem Photobiol 2003 are limited by light firing time；77:675–
679).Additionally, when ultra-violet (UV) band carries out fluoroscopic examination, biological sample makes light enter inside biological tissue in the absorption in this interval
Become difficult, while the autofluorescence of some compositions forms very strong ambient interferences in biological sample, detection efficiency is dropped significantly
It is low.Therefore, research and develop with good fluorescence spectrum property, to special cells and there is the novel fluorescence of selectivity, small toxicity
Dyestuff remains the crucial and core for promoting the development of the field such as fluorescence analysis and life sciences.
In the fluorescent dye compound of numerous kinds, cyanine fluorochrome is with its wave-length coverage width, molar extinction coefficient
Greatly, the advantages of fluorescence quantum yield is moderate.Wherein quinolines asymmetric cyanine fluorochrome, by the length for changing polymethylene chain
The structure of the fragrant parent nucleus (thiazole, oxazole, quinoline, pyridine and indoline) at degree and two ends can obtain different heterodimer classes
Like thing and derivant.This kind of dyestuff almost unstressed configuration in the solution, reduces the fluorescence background interference in detection process, with nucleic acid
With reference to rear Fluorescence Increasing.Patent ZL201010022414.6, US8298766 and JP5671525 etc. describe a class and excite and send out
Ejected wave length more than 630 nanometers of nir dye, this kind of dyestuff can be used for nucleic acid fluorescent identification and in living cells it is glimmering to nucleic acid
Photoimaging.But cell is made up of many components, such as protein, nucleic acid, small-molecule substance.In addition, cell is in different growth ranks
Under section, varying environment, under the conditions of different lesions, can all there are different states.But with this kind of complex condition, to living cells,
Dead cell stain, the cyanines class nir dye (excite with launch wavelength be more than 630 nanometers) of abnormal cell dyeing it is little.
The content of the invention
It is an object of the invention to provide application of the class cyanine compound in biological dye is prepared, described cyanines class
Compound has the structure of formula I：
In formula I,
X is C (CH3)2, O, S or Se；
Y-Selected from halide ion, ClO4 -、PF6 -、BF4 -、CH3COO-Or OTs-。
The structure and its synthetic method of the compounds of formula I described in the invention described above has been reported in the prior art,
Those skilled in the art can according to prior art information acquisition structure compound.The present invention provides one of synthetic route
As specific embodiment：
With regard to above-mentioned compounds of formula I, the cognition of prior art is limited to its structure, synthesis and limited function.Can be with
Confirm, those skilled in the art is simultaneously unaware that such compound has the function of special RNA positioning.Conversely, having class
Like the compound of structure, ethylated compound E-TO3 is such as wrapped in structure, the identification object of targeted property is DNA rather than RNA.
The structure of compound E-TO3 is as follows：
In consideration of it, present invention particularly provides as compounds of formula I is preparing biological dye, especially cell dyeing
Agent, the more specifically application in RNA specific stains reagent.
Involved compounds of formula I in the invention described above, in, described X is C (CH3)2, O or S；It is preferred that C (CH3)2
Or S；More preferably X is S.
On the other hand, in formula I, described Y-For halide ion；It is preferred that Y-For I-。
Further, in our invention, it is experimentally confirmed, compounds of formula I of the present invention is special as RNA
The opposite sex identification fluorescent dye, also with it is following all many-sided the characteristics of：
(1) fluorescence quantum yield increase, improves detection sensitivity and after the combination of nucleic acid.
(2) with good permeability of cell membrane, range of application increase.
(3) compound launch wavelength wide ranges, up to the near infrared region of 630nm~900nm, can avoid the glimmering of biology itself
Light ambient interferences.
These features of the invention and advantage and other feature and advantage are concrete with the present invention with reference to the following drawings
Will become clear from after embodiment.
Description of the drawings
The width of accompanying drawing of the present invention 7:
Fig. 1 is compound TO-3 and commercialization RNA dyestuff SYTO RNA SelectTMLiving cells positioning, dyestuff TO-3 makes
Excited with Cy5 (633nm) passage, 645-695nm wave bands, SYTO RNA Select are collected in transmittingTMIt is logical using FITC (488nm)
Road is excited, and 495-545nm wave bands are collected in transmitting, as a result shows compound TO-3 dyeing effects identical with commercialization RNA dyestuff presentations
Really.
Fig. 2 is that the living cells of compound TO-3 and compound E-TO3 are positioned, and is excited using Cy5 (633nm) passage, is launched
Collect 645-695nm wave bands.As a result show, compound TO-3 is positioned with significant difference, compound with compound E-TO3 living cells
TO-3 mainly positions the RNA in entoblast, and compound E-TO3 cannot position entoblast RNA.
Fig. 3 is compound E-TO3 and commercialization RNA dyestuff SYTO RNA SelectTMLiving cells positioning, dyestuff E-TO3
Excited using Cy5 (633nm) passage, 645-695nm wave bands, SYTO RNA Select are collected in transmittingTMUsing FITC (488nm)
Passage is excited, and 495-545nm wave bands are collected in transmitting, as a result shows compound E-TO3 and commercialization RNA dyestuff SYTO RNA
SelectTMColor is different from Cytoplasm and nucleus, compound E-TO3 cannot selectivity positioning it is intracellular
RNA。
Fig. 4 is compound OO-3, TO-3, IO-3 and commercialization RNA dyestuff SYTO RNA SelectTMLiving cells positioning,
Dyestuff TO-3 is excited using Cy5 (633nm) passage, and 645-695nm wave bands are collected in transmitting, and compound OO-3, IO-3 uses 559nm
Passage is excited, and transmitting is collected as 600-650nm wave bands, SYTO RNA SelectTMExcited using FITC (488nm) passage, launched
495-545nm wave bands are collected, compound OO-3, TO-3, IO-3 positioning effect identical with commercialization RNA dyestuff presentations is as a result shown
Really.
Fig. 5 is the cell dissociation enzyme experiment of compound TO-3, is excited using Cy5 (633nm) passage, and 645- is collected in transmitting
695nm wave bands, as a result show, compound EO-3 can be positioned in dead cell core, and mainly in left figure matched group (control)
Kernel region is concentrated on, in DNase figures, the cell crossed of ferment treatment is digested by DNA, the fluorescence in its nucleus especially kernel region
Intensity is compared with matched group without significant change；And in RNase figures, the Jing RNA digestion cells crossed of ferment treatment, its kernel region it is glimmering
Light intensity is substantially reduced.
Fig. 6 is the cell dissociation enzyme experiment of compound OO-3, is excited using 559nm passages, and transmitting is collected as 600-650nm
Wave band.As a result show, compound OO-3 can be positioned in dead cell core in left figure matched group (control), and main concentration
In kernel region, DNase figures, the cell crossed of ferment treatment is digested by DNA, the fluorescence intensity in its nucleus especially kernel region
Without significant change compared with matched group；And in RNase figures, the cell that Jing RNA digestion ferment treatments are crossed, the fluorescence in its kernel region is strong
Degree is substantially reduced.
Fig. 7 is the cell dissociation enzyme experiment of compound IO-3, is excited using 559nm passages, and transmitting is collected as 600-650nm
Wave band.As a result show, compound IO-3 can be positioned in dead cell core in left figure matched group (control), and main concentration
In kernel region, DNase figures, the cell crossed of ferment treatment is digested by DNA, the fluorescence intensity in its nucleus especially kernel region
Without significant change compared with matched group；And in RNase figures, the cell that Jing RNA digestion ferment treatments are crossed, the fluorescence in its kernel region is strong
Degree is substantially reduced.
Specific embodiment
Unless otherwise indicated, term used herein has following meanings.
Term " halogen " used herein includes fluorine, chlorine, bromine and iodine.
Y used herein-Anion is represented, it can be any suitable anion, including inorganic anion and organic negative
Ion, can illustrate but be not limited to halide ion, ClO4 -、PF6 -、BF4 -、CH3COO-Or OTs-。
The present invention is intended to provide such as application of the cyanine compound of formula I in biological dye is prepared.It is wherein described
Biological dye can be the salt of the cyanine compound of formula I or be the derivant of type I compound, or including this
The compositionss of a little compounds.
Involved biological dye is typically used in fluorescence-activated cell sorter (FACS) in the present invention.Can be with this
The molecule that the involved biological dye of invention is conjugated can be the molecule specifically bound with cell or cell component, including but not
It is limited to antibody, antigen, receptor, part, enzyme, substrate, coenzyme etc..Generally, test sample incubates a period of time with biological dye,
So that some of fluorescent dye and test sample cell or cell component specific binding, fluorescent dye and cell or cell into
The combination for dividing also referred to as is dyeed.The staining procedure can be carried out successively repeatedly, or carry out various dyeing simultaneously with various dyestuffs.
After the completion of dyeing, sample is analyzed in fluorescence-activated cell sorter or fluorescence determination device, and wherein excitation source is excited
Fluorescent dye of the present invention, and determine device and determine the launching light produced by the fluorescent dye for exciting.The present invention is usually used and partly leads
Body laser.
The biological dye can be the compositionss comprising type I compound, and biological sample can be also included in said composition
Other components required for dyeing, such as solvent, osmotic pressure regulator, pH adjusting agent, surfactant etc..Biological dye contaminates
Toner can exist as aqueous solution form, or can exist as other suitable forms for being formulated as solution with water before use.
In order to the compound for illustrating the present invention is introduced in the structure to the Optimal improvements of dyestuff performance after Me, implement
Know that compound E-TO3 is reference.
Embodiment 1
Compound 1- ethyl -4- [3- (3- ethyl-benzothiazole -2- subunits) acrylic] quinoline iodine (E-TO3) structural formula
For：
Compound E-TO3 synthesizes according to following routes：
In the round-bottomed flask of 50mL, by hemicyanine dye intermediate 1- ethyl -2- [(2- anilino-s) vinyl] benzo thiophene
Azoles iodine salt (2.0mmol) and 1- ethyls -4- methylquinoline quaternary ammonium salts (2mmol) are dissolved in the 1 of 20mL:1(v/v)CH2Cl2/CH3OH
In, respectively the triethylamine and acetic anhydride of Deca 2mL makees catalyst.Flask wraps up lucifuge with aluminium-foil paper, is placed in oil bath and slowly heats,
Persistently stir room temperature reaction 1.5h.Reactant liquor is poured in ether, crude product is separated out, is fully washed with ether, then silicagel column
Chromatography is further purified, and obtains blue solid.HR-TOF-MS m/z Found:359.1595C23H23N2S(M+):requires
M,359.1576.1H NMR(400MHz,DMSO-d6):δ=1.33 (t, 3H, CH3, J=6.8Hz)；1.45 (t, 3H, CH3, J=
8.0Hz)；4.30(q,2H,CH2, J=8.0Hz)；4.61(q,2H,CH2, J=7.2Hz)；6.53 (d, 1H, CH, J=12Hz)；
7.14 (d, 1H, CH, J=11.2Hz)；7.32 (t, 1H, ArH, J=7.8Hz)；7.50 (t, 1H, ArH, J=7.8Hz)；7.61
(d, 1H, ArH, J=7.8Hz)；7.71 (t, 1H, ArH, J=7.8Hz)；7.88-7.90(m,2H,ArH)；7.97(t,1H,
ArH, J=7.8Hz)；8.10 (d, 1H, ArH, J=7.8Hz), 8.17 (t, 1H, CH, J=11.2Hz), 8.44-8.49 (m, 2H,
ArH).13C NMR(100MHz,DMSO-d6):δ=12.8,15.2,41.2,49.8,98.8,109.9,110.4,112.7,
118.3,123.2,124.6,124.8,125.3,125.7,127.2,128.2,133.9,138.2,141.5,142.7,
144.5,150.9,161.1.
Embodiment 2
Compound 1- methyl -4- [3- (3- methylbenzothiazole -2- subunits) acrylic] quinoline iodine (TO-3) structural formula is：
Compound TO-3 synthesizes according to following routes：
In the round-bottomed flask of 50mL, by hemicyanine dye intermediate 1- methyl -2- [(2- anilino-s) vinyl] benzo thiophene
Azoles iodine salt (2.0mmol) and 1- methyl -4- methylquinoline quaternary ammonium salts (2mmol) are dissolved in the 1 of 20mL:1(v/v)CH2Cl2/CH3OH
In, respectively the triethylamine and acetic anhydride of Deca 2mL makees catalyst.Flask wraps up lucifuge with aluminium-foil paper, is placed in oil bath and slowly heats,
Question response liquid color is changed into green post-heating and stops, and persistently stirs room temperature reaction 1.5h.Reactant liquor is poured in ether, is separated out dark
Purple little particle, and fully washed with ether.Crude product silica gel column chromatography, CH2Cl2/CH3OH mixed solvent gradient elutions.Receive
Collection blue portion, obtains blue solid, HR-TOF-MS m/z Found:331.1273C21H19N2S+(M+):requires
M,331.1269.1H-NMR(400MHz,DMSO-d6):δ=3.72 (s, 3H, CH3)；4.13(s,3H,CH3)；6.47(d,1H,
CH, J=12.4Hz)；7.12 (d, 1H, CH, J=13.2Hz)；7.31 (t, 1H, ArH, J=7.6Hz)；7.49(t,1H,ArH,J
=7.6Hz)；7.58 (d, 1H, ArH, J=8.8Hz)；7.74 (t, 1H, ArH, J=6Hz)；7.87 (d, 2H, ArH, J=
7.6Hz)；7.99(m,2H,ArH)；8.15 (t, 1H, CH, J=13.2Hz)；8.41 (d, 1H, ArH, J=7.6Hz)；8.48(d,
1H, ArH, J=8.8Hz) .13C NMR (100MHz, MeOD-d4):δ=32.8,42.2,98.5,109.2,109.5,112.4,
118.0,122.4,124.0,124.5,124.9,126.8,127.6,133.3,138.8,142.0,143.2,143.5,
150.4.
Embodiment 3
Compound 1- methyl -4- [3- (3- methylbenzoxazole -2- subunits) acrylic] quinoline iodine (OO-3) structural formula is：
Compound OO-3 synthesizes according to following routes：
In the round-bottomed flask of 50mL, hemicyanine dye intermediate 1- methyl -2- [(2- anilino-s) vinyl] benzo is disliked
Azoles iodine salt (2.0mmol) and 1- methyl -4- methylquinoline quaternary ammonium salts (2mmol) are dissolved in the 1 of 20mL:1(v/v)CH2Cl2/CH3OH
In, respectively the triethylamine and acetic anhydride of Deca 2mL makees catalyst.Flask wraps up lucifuge with aluminium-foil paper, is placed in oil bath and slowly heats,
Persistently stir room temperature reaction 1.5h.Reactant liquor is poured in ether, crude product is separated out, is fully washed with ether, then silicagel column
Chromatography is further purified, and obtains blue solid.HR-TOF-MS m/z Found:315.1506C21H19N2O+(M+):requires
M, 315.1492.1H NMR (500MHz, DMSO-d6) δ=8.46 (d, J=8.5Hz, 1H), 8.37 (d, 1H), 7.95 (t, J=
6.7Hz, 2H), 7.80 (d, 1H), 7.74 (t, J=5.5Hz, 1H), 7.59 (d, J=8.2Hz, 1H), 7.53 (t, 2H), 7.48
(d, 2H), 7.39 (t, J=14.5,6.7Hz, 1H), 7.30 (t, 1H), 7.06 (d, J=13.7Hz, 1H), 5.88 (d, 1H),
4.11(s,3H),3.66(s,3H)..13C NMR(500MHz,DMSO-d6):δ=30.0,42.0,107.2,108.8,
110.3,112.1,117.2,118.0,123.8,124.0,124.8,125.4,126.6,129.8,132.3,133.2,
138.8,143.0,146.3,150.9,160.9.
Embodiment 4
Compound 1,3,3- trimethyl -2- [3- (1- methyl isophthalic acid H- quinoline -4- subunits)-acrylic] -3H- indole iodine (IO-
3) structural formula is：
Compound IO-3 synthesizes according to following routes：
In the round-bottomed flask of 50mL, by trimethyl -2- (the 2- phenyl aminos-ethylene of hemicyanine dye intermediate 1,3,3-
Base) -3H- indole iodine salt (2.0mmol) and 1- methyl -4- methylquinoline quaternary ammonium salts (2mmol) is dissolved in the 1 of 20mL:1(v/v)
CH2Cl2/CH3In OH, respectively the triethylamine and acetic anhydride of Deca 2mL makees catalyst.Flask wraps up lucifuge with aluminium-foil paper, is placed in oil bath
In slowly heat, persistently stir room temperature reaction 1.5h.Reactant liquor is poured in ether, crude product is separated out, is fully washed with ether,
Then silica gel column chromatography is further purified, and obtains blue solid.HR-TOF-MS m/z Found:341.2010,C24H25N2 +(M+):
requires M,341.2021.1H NMR (500MHz, DMSO-d6) δ=8.64 (2H, m), 8.33 (1H, d, J=12.0Hz),
8.13 (3H, m), 7.81 (1H, m), 7.51 (1H, d, J=6.0Hz), 7.33 (2H, m), 7.18 (1H, d, J=6.0Hz), 7.08
(1H, t), 6.21 (1H, d, J=12.0Hz), 4.28 (3H, s), 3.46 (3H, s), 1.69 (6H, s)；13C NMR(500MHz,
D6-DMSO) δ=168.1,151.8,144.4,143.5,142.8,140.0,138.9,133.7,128.2,12 7.4,125.3,
124.5,122.5,122.1,118.6,112.5,111.4,109.2,100.1,62.1,47.5,43.0,30.1,28.2,
25.5.
Embodiment 5
Compound TO-3 and commercialization RNA dyestuff SYTO RNA SelectTMContrast test
(1) compound 1- ethyls -4- [3- (3- ethyl-benzothiazole -2- subunits) acrylic] quinoline iodine (TO-3) and business
Change RNA dyestuff SYTO RNA SelectTM(known to fluorescence data) spectrum in PH7.4, concentration 10mM Tris-HCl buffer
With living cells location data.Uv absorption and fluorescence emission spectrum are glimmering in HP-8453 ultraviolet spectrophotometers and FP-6500 respectively
Measure on light spectrophotometer, sample quality adopts BS-210S a ten thousandth electronic balance weighings.Testing result such as table 1.
Table 1
(2) compound TO-3 and SYTO RNA SelectTMLive cell fluorescent imaging results as shown in Figure 1：Right figure is
SYTO RNA SelectTMWith the superposition of the live cell fluorescent image of TO-3, dyestuff TO-3 using Cy5 (633nm) passage swash
Send out, 645-695nm wave bands, SYTO RNA Select are collected in transmittingTMExcited using FITC (488nm) passage, 495- is collected in transmitting
545nm wave bands, confocal laser scanning microscope, CLSM model：TCS-SP2, object lens magnification is 60 times, is as a result shown, dyestuff
TO-3 and commercialization RNA dyestuff SYTO RNA SelectTMLiving cells locating effect it is identical.
Embodiment 5
The living cells common location of compound TO-3, E-TO3
HeLa cells living TO-3, E-TO3 first respectively with 4 μM is according to dying operation incubation 30min before.With
PBS phosphate buffers (the NaH of 0.2M2PO4-Na2HPO4, pH=7.0) cell is washed twice after, add fresh culture medium
In the burnt special ware of copolymerization.Using TCS-SP2 confocal laser scanning microscope, CLSMs, object lens magnification is for live cell fluorescent imaging
100 times.Excited using Cy5 (633nm) passage, 645-695nm wave bands are collected in transmitting.
Two compounds chromatin dna (being enriched in nucleus) specifically can be clearly depicted and RNA (is enriched in core
Core) distribution in living cells.From figure 2 it can be seen that TO-3 is in strong clear dyeing to nucleolar RNA.It should be noted that
N- ethyls replace benzo thiazole heterocycle to replace the difference of benzo thiazole heterocycle with N- methyl, cause E-TO3 that stronger cell is presented
Core DNA is dyeed, and nucleolar RNA is less, illustrates in living cells, and E-TO3 is better than to RNA responses to DNA responses, and TO-3 then exists
Living cells have more preferable selectivity to RNA.
Embodiment 5
Compound E-TO3 and commercialization RNA dyestuff SYTO RNA SelectTM'sLiving cells common location
MCF-7 cells living with the dying operation of 4 μM OO-3, TO-3, IO-3 are incubated 30min respectively first.Use PBS phosphorus
Acid buffer (the NaH of 0.2M2PO4-Na2HPO4, pH=7.0) cell is washed twice after, add fresh culture to be based on copolymerization
In burnt special ware.In order to investigate the common location of compound and RNA, RNA selective dye SYTO RNA are added in culture dish
SelectTM, dye it is final concentration of 4 μM, in 37 DEG C, 5%CO2Under the conditions of be incubated 30min again.Subsequently, culture medium is sopped up, uses PBS
Phosphate buffer washs cell three times, in case carrying out Fluirescence observation under laser confocal scanning microscope.Live cell fluorescent
Imaging uses TCS-SP2 confocal laser scanning microscope, CLSMs, and object lens magnification is 60 times.Compound E-TO3 uses Cy5
(633nm) passage is excited, and 645-695nm wave bands, SYTO RNA Select are collected in transmittingTMSwashed using FITC (488nm) passage
Send out, 495-545nm wave bands are collected in transmitting.As a result as shown in figure 3, N- ethyls replace benzothiazole heterocyclic compound E-TO3 thin
With commercialization RNA dyestuff SYTO RNA Select in karyon and CytoplasmTMDifferent positioning results are presented.
Embodiment 6
The living cells common location of compound OO-3, TO-3, IO-3 and business RNA dyestuff
MCF-7 cells living with the dying operation of 4 μM OO-3, TO-3, IO-3 are incubated 30min respectively first.Use PBS phosphorus
Acid buffer (the NaH of 0.2M2PO4-Na2HPO4, pH=7.0) cell is washed twice after, add fresh culture to be based on copolymerization
In burnt special ware.In order to investigate the common location of compound and RNA, RNA selective dye SYTO RNA are added in culture dish
SelectTM, dye it is final concentration of 4 μM, in 37 DEG C, 5%CO2Under the conditions of be incubated 30min again.Subsequently, culture medium is sopped up, uses PBS
Phosphate buffer washs cell three times, in case carrying out Fluirescence observation under laser confocal scanning microscope.Live cell fluorescent
Imaging uses TCS-SP2 confocal laser scanning microscope, CLSMs, and object lens magnification is 60 times.Compound TO-3 uses Cy5
(633nm) passage is excited, and 645-695nm wave bands are collected in transmitting, and compound OO-3, IO-3 are excited using 559nm passages, and transmitting is received
Collection 600-650 wave bands.SYTO RNA SelectTMExcited using FITC (488nm) passage, 495-545nm wave bands are collected in transmitting.
As a result it is as shown in Figure 4.Three compounds and commercialization RNA selective dye SYTO RNA SelectTMIdentical positioning effect is presented
Really.
Embodiment 7
The cell dissociation enzyme experiment of compound TO-3
The MCF-7 cells being incubated in 37 DEG C of cell culture incubators are taken out, old culture medium is removed, PBS be (0.2M's
NaH2PO4-Na2HPO4, pH=7.0) clean twice, with 2mL ice ethanol is added after -20 DEG C of ice ethanol purge, it is placed in -20 DEG C
5min in refrigerator.After taking-up with PBS twice after, add 2mL PBS, by copolymerization Jiao ware be placed in 4 DEG C of refrigerators preserve.To two
DNA and RNA digestive enzyme is separately added in group copolymerization Jiao's ware, another set is used as controlled trial.Original PBS is sucked after 2h, then is used
PBS is washed twice, and the TO-3 for adding 4 μM is incubated altogether 30min, then removes PBS, then is washed with PBS three times, in case common in laser
Fluirescence observation is carried out under confocal scanning microscopy.Cell fluorescence imaging uses TCS-SP2 confocal laser scanning microscope, CLSMs, object lens
Amplification is 60 times.Compound TO-3 is excited using Cy5 (633nm) passage, and transmitting is collected as 645-695nm wave bands.From Fig. 5
As can be seen that compound TO-3 can be positioned in dead cell core in left figure matched group (control), and it is concentrated mainly on kernel
Region, in DNase figures, by DNA the cell crossed of ferment treatment is digested, its nucleus especially the fluorescence intensity in kernel region with compare
Group is compared without significant change；And in RNase figures, the cell that Jing RNA digestion ferment treatments are crossed, the fluorescence intensity in its kernel region is obvious
Reduce.
Embodiment 7
The cell dissociation enzyme experiment of compound OO-3
The MCF-7 cells being incubated in 37 DEG C of cell culture incubators are taken out, old culture medium is removed, PBS be (0.2M's
NaH2PO4-Na2HPO4, pH=7.0) clean twice, with 2mL ice ethanol is added after -20 DEG C of ice ethanol purge, it is placed in -20 DEG C
5min in refrigerator.After taking-up with PBS twice after, add 2mL PBS, by copolymerization Jiao ware be placed in 4 DEG C of refrigerators preserve.To two
DNA and RNA digestive enzyme is separately added in group copolymerization Jiao's ware, another set is used as controlled trial.Original PBS is sucked after 2h, then is used
PBS is washed twice, and the OO-3 for adding 4 μM is incubated altogether 30min, then removes PBS, then is washed with PBS three times, in case common in laser
Fluirescence observation is carried out under confocal scanning microscopy.Cell fluorescence imaging uses TCS-SP2 confocal laser scanning microscope, CLSMs, object lens
Amplification is 60 times.Compound OO-3 is excited using 559nm passages, and transmitting is collected as 600-650nm wave bands.Can be with from Fig. 6
Find out, compound OO-3 can be positioned in dead cell core in left figure matched group (control), and is concentrated mainly on nucleolar zone
Domain, in DNase figures, by DNA the cell crossed of ferment treatment is digested, the fluorescence intensity and matched group in its nucleus especially kernel region
Compare without significant change；And in RNase figures, the cell that Jing RNA digestion ferment treatments are crossed, the fluorescence intensity in its kernel region substantially drops
It is low.
Embodiment 7
The cell dissociation enzyme experiment of compound IO-3
The MCF-7 cells being incubated in 37 DEG C of cell culture incubators are taken out, old culture medium is removed, PBS be (0.2M's
NaH2PO4-Na2HPO4, pH=7.0) clean twice, with 2mL ice ethanol is added after -20 DEG C of ice ethanol purge, it is placed in -20 DEG C
5min in refrigerator.After taking-up with PBS twice after, add 2mL PBS, by copolymerization Jiao ware be placed in 4 DEG C of refrigerators preserve.To two
DNA and RNA digestive enzyme is separately added in group copolymerization Jiao's ware, another set is used as controlled trial.Original PBS is sucked after 2h, then is used
PBS is washed twice, and the IO-3 for adding 4 μM is incubated altogether 30min, then removes PBS, then is washed with PBS three times, in case common in laser
Fluirescence observation is carried out under confocal scanning microscopy.Cell fluorescence imaging uses TCS-SP2 confocal laser scanning microscope, CLSMs, object lens
Amplification is 60 times.Compound IO-3 is excited using 559nm passages, and transmitting is collected as 600-650nm wave bands.Can be with from Fig. 7
Find out, compound IO-3 can be positioned in dead cell core in left figure matched group (control), and is concentrated mainly on nucleolar zone
Domain, in DNase figures, by DNA the cell crossed of ferment treatment is digested, the fluorescence intensity and matched group in its nucleus especially kernel region
Compare without significant change；And in RNase figures, the cell that Jing RNA digestion ferment treatments are crossed, the fluorescence intensity in its kernel region substantially drops
It is low.
Above content is to combine specific preferred implementation further description made for the present invention, it is impossible to assert
The present invention be embodied as be confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of without departing from present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to the present invention's
Protection domain.It is a kind of purposes of noval chemical compound of the present invention as fluorescent dye, it is impossible to which the compound for assert the present invention is only used for
Fluorescent dye, for general technical staff of the technical field of the invention, based on the compounds of this invention fluorescence is being used as
Under the consideration of the identical mechanism of action of dyestuff, some simple inferences can also be made, draw the present invention compound other should
With purposes, protection scope of the present invention should be all considered as belonging to.
Claims (8)
1. application of the cyanine compound in biological dye is prepared, described cyanine compound has the structure of formula I：
In formula I,
X is C (CH3)2, O, S or Se；
Y-Selected from halide ion, ClO4 -、PF6 -、BF4 -、CH3COO-Or OTs-。
2. application according to claim 1, it is characterised in that in described formula I, X is C (CH3)2, O or S.
3. application according to claim 2, it is characterised in that in described formula I, X is C (CH3)2Or S.
4. application according to claim 3, it is characterised in that in described formula I, X is S.
5. application according to claim 1, it is characterised in that in described formula I, Y-For halide ion.
6. application according to claim 5, it is characterised in that in described formula I, Y-For I-。
7. application according to claim 1, it is characterised in that described biological dye is cell staining reagent.
8. application according to claim 1, it is characterised in that described biological dye is RNA specific stain reagents.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| Title |
|---|
| 吴彤: "噻唑橙类不对称菁染料的合成与生物应用", 《中国博士学位论文全文数据库 工程科技I辑》 * |
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Application publication date: 20170517 |