CN109180716A - 一种多信号比率型区分检测h2o2和h2s的荧光探针的设计、合成及应用 - Google Patents
一种多信号比率型区分检测h2o2和h2s的荧光探针的设计、合成及应用 Download PDFInfo
- Publication number
- CN109180716A CN109180716A CN201811090636.4A CN201811090636A CN109180716A CN 109180716 A CN109180716 A CN 109180716A CN 201811090636 A CN201811090636 A CN 201811090636A CN 109180716 A CN109180716 A CN 109180716A
- Authority
- CN
- China
- Prior art keywords
- probe
- fluorescence
- detection
- tcab
- ratio
- 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.)
- Granted
Links
- 239000000523 sample Substances 0.000 title claims abstract description 68
- 238000001514 detection method Methods 0.000 title claims abstract description 20
- 230000004069 differentiation Effects 0.000 title abstract description 5
- 230000015572 biosynthetic process Effects 0.000 title description 2
- 238000003786 synthesis reaction Methods 0.000 title description 2
- 238000001917 fluorescence detection Methods 0.000 claims abstract description 3
- 239000012472 biological sample Substances 0.000 claims 1
- 238000012921 fluorescence analysis Methods 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 13
- 150000001875 compounds Chemical class 0.000 abstract description 8
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 3
- 239000000975 dye Substances 0.000 abstract description 3
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract 1
- 230000005622 photoelectricity Effects 0.000 abstract 1
- SOBGIMQKWDUEPY-UHFFFAOYSA-N bis(3,4-dichlorophenyl)diazene Chemical compound C1=C(Cl)C(Cl)=CC=C1N=NC1=CC=C(Cl)C(Cl)=C1 SOBGIMQKWDUEPY-UHFFFAOYSA-N 0.000 description 33
- 239000000243 solution Substances 0.000 description 15
- 230000005284 excitation Effects 0.000 description 10
- 238000011534 incubation Methods 0.000 description 10
- 239000007853 buffer solution Substances 0.000 description 9
- 230000004044 response Effects 0.000 description 9
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 7
- 238000000799 fluorescence microscopy Methods 0.000 description 7
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 6
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 239000007995 HEPES buffer Substances 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- -1 Borate ester Chemical class 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 4
- 229960004424 carbon dioxide Drugs 0.000 description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 4
- 239000001569 carbon dioxide Substances 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 125000001820 oxy group Chemical group [*:1]O[*:2] 0.000 description 3
- 238000010189 synthetic method Methods 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 2
- 229920001651 Cyanoacrylate Polymers 0.000 description 2
- 238000010719 annulation reaction Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 229960003180 glutathione Drugs 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical group N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 230000036542 oxidative stress Effects 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 238000005292 vacuum distillation Methods 0.000 description 2
- QGVLYPPODPLXMB-UBTYZVCOSA-N (1aR,1bS,4aR,7aS,7bS,8R,9R,9aS)-4a,7b,9,9a-tetrahydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-1,1a,1b,4,4a,7a,7b,8,9,9a-decahydro-5H-cyclopropa[3,4]benzo[1,2-e]azulen-5-one Chemical compound C1=C(CO)C[C@]2(O)C(=O)C(C)=C[C@H]2[C@@]2(O)[C@H](C)[C@@H](O)[C@@]3(O)C(C)(C)[C@H]3[C@@H]21 QGVLYPPODPLXMB-UBTYZVCOSA-N 0.000 description 1
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- BMIBJCFFZPYJHF-UHFFFAOYSA-N 2-methoxy-5-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine Chemical compound COC1=NC=C(C)C=C1B1OC(C)(C)C(C)(C)O1 BMIBJCFFZPYJHF-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- KWGACYZYFZTYRN-UHFFFAOYSA-N OC(C)(C)C(C)(C)O.B(O)(O)O.BrCC1=CC=CC=C1 Chemical compound OC(C)(C)C(C)(C)O.B(O)(O)O.BrCC1=CC=CC=C1 KWGACYZYFZTYRN-UHFFFAOYSA-N 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- PYZJDLXOJFTXJU-QNKGDIEWSA-N [2-oxo-7-[[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]methoxy]chromen-4-yl]methyl (E)-3-[7-[(4-azidophenyl)methoxy]-1,4-diethyl-2,3-dihydroquinoxalin-6-yl]-2-cyanoprop-2-enoate Chemical compound B1(OC(C(O1)(C)C)(C)C)C2=CC=C(C=C2)COC3=CC4=C(C=C3)C(=CC(=O)O4)COC(=O)/C(=C/C5=CC6=C(C=C5OCC7=CC=C(C=C7)N=[N+]=[N-])N(CCN6CC)CC)/C#N PYZJDLXOJFTXJU-QNKGDIEWSA-N 0.000 description 1
- WITZXUPUMRPBJS-UHFFFAOYSA-N [B].BrCC1=CC=CC=C1 Chemical compound [B].BrCC1=CC=CC=C1 WITZXUPUMRPBJS-UHFFFAOYSA-N 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910002090 carbon oxide Inorganic materials 0.000 description 1
- 201000011529 cardiovascular cancer Diseases 0.000 description 1
- 230000004715 cellular signal transduction Effects 0.000 description 1
- 208000019065 cervical carcinoma Diseases 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 235000003969 glutathione Nutrition 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 125000001967 indiganyl group Chemical group [H][In]([H])[*] 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- QGVLYPPODPLXMB-QXYKVGAMSA-N phorbol Natural products C[C@@H]1[C@@H](O)[C@]2(O)[C@H]([C@H]3C=C(CO)C[C@@]4(O)[C@H](C=C(C)C4=O)[C@@]13O)C2(C)C QGVLYPPODPLXMB-QXYKVGAMSA-N 0.000 description 1
- 238000005375 photometry Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000001044 red dye Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- CIHOLLKRGTVIJN-UHFFFAOYSA-N tert‐butyl hydroperoxide Chemical compound CC(C)(C)OO CIHOLLKRGTVIJN-UHFFFAOYSA-N 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- BQMKAHQKDSZAIQ-UHFFFAOYSA-N tetrasodium;iron(3+);nitroxyl anion;pentacyanide Chemical compound [Na+].[Na+].[Na+].[Na+].[Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].O=[N-] BQMKAHQKDSZAIQ-UHFFFAOYSA-N 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- ORHBXUUXSCNDEV-UHFFFAOYSA-N umbelliferone Chemical compound C1=CC(=O)OC2=CC(O)=CC=C21 ORHBXUUXSCNDEV-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/04—Esters of boric acids
-
- 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
-
- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
-
- 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
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
-
- 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
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
-
- 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
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only 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
- C09K2211/1096—Heterocyclic compounds characterised by ligands containing other heteroatoms
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Optics & Photonics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Materials Engineering (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
本发明涉及的是化学分析检测技术领域，具体涉及一种多多信号比率型区分检测H2O2和H2S的荧光探针的制备方法以及该荧光探针在体外和活细胞内检测H2O2和H2S方面的应用。该探针化合物的结构式如式(I)所示。我们将两种识别基团通过染料的桥连构建到一个分子上，比率型荧光信号通过两个荧光信号的比值作为输出信号，探针分子本身的自校准能够克服诸如探针浓度、仪器灵敏度和环境因素带来的影响，提高荧光检测的准确性。通过探针与H2S和H2O2反应后获得不同组合的荧光信号来实现对H2S和H2O2的区分检测。此外该系列化合物有望在生物医药，光电以及环境保护领域有着较好的应用前景。
Description
技术领域
本发明涉及的是化学分析检测技术领域，具体涉及一种多信号比率型区分检测H2O2和H2S的荧光探针的制备方法以及该荧光探针在体外和活细胞内检测H2O2和H2S方面的应用。
背景技术
H2O2是其他活性氧的前体分子，在宿主防御，免疫应答和细胞信号传导中起着重要的作用。作为氧化应激的标志物，越来越多的研究表明，体内H2O2浓度的增加会诱导细胞蛋白的氧化损伤，导致神经退行性疾病，心血管疾病，癌症和阿尔兹海默症等。H2S作为体内的一种信号传递分子，在神经调节，细胞凋亡，抗炎症，抗氧化和抑制胰岛素信号传导等生理过程中起着广泛而重要的作用。研究发现，H2S通过提高细胞内谷胱甘肽的水平、调节抗氧化蛋白的高表达等途径，来保护细胞免受氧化应激的损伤。H2S本身具有的还原性也会直接清除H2O2等活性氧物质。H2S和H2O2在生理过程中有如此重要的作用，同时彼此互相影响，开发一种有效的检测方法是至关重要的，特别是对细胞内H2S和H2O2的浓度的监测，对生物学研究和临床诊断至关重要。
目前对H2S和H2O2的检测方法主要有比色法、电化学分析法、气相色谱法、滴定法、分光光度法和荧光法等。在这些方法中，荧光探针成像技术由于操作简单，灵敏度高，选择性好，实时成像等优点，特别是对组织的非破坏性而受到特别的关注。近年来开发了大量的荧光探针来检测H2S和H2O2，但他们都是独立的对H2S或H2O2进行选择性检测，还没有一个探针分子可以同时区分检测H2S和H2O2。
发明内容
本发明目的之一是提供一种合成路线简单、反应条件温和、成本较低的荧光探针合成方法；目的之二是提供一种灵敏度高、选择性好，抗干扰能力强，斯托克斯位移大，发射波长在近红外，能够对体外或者活细胞内进行监测或者细胞成像的荧光探针。比率型荧光信号通过两个荧光信号的比值作为输出信号，探针分子本身的自校准能够克服诸如探针浓度、仪器灵敏度和环境因素带来的影响，提高荧光检测的准确性。通过探针与H2S和H2O2反应后获得不同组合的荧光信号来实现对H2S和H2O2的区分检测。
本发明解决问题采取的技术方案为，一种多信号比率型区分检测H2O2和H2S的荧光探针，其分子结构式如下：合成路线如下：
具体合成方法如下：将化合物(7-羟基-2-氧代-2H-色烯-4-基)甲基(E)-3-(7-((4- 叠氮苯基)氧基)-1，4-二乙基-1，2，3，4-四氢喹喔啉-6-基)-2-氰基丙烯酸酯和4-溴甲基苯硼酸频哪醇酯溶于丙酮，置于圆底烧瓶中，加入碳酸钾60℃搅拌回流反应。TLC监测反应完成后，停止反应，减压蒸馏除去溶剂，剩余物用柱层析分离纯化，二氯甲烷/乙酸乙酯(v:v ＝20:1,含1％三乙胺)作为洗脱剂。干燥得到红色固体
本发明的荧光探针测试方法如下，将探针分子溶于二甲基亚砜(DMSO)中，配制成1.0×10-3mol/L的溶液，室温下进行测试。并且对低浓度的H2S和H2O2可以进行定量检测，具体实施方法在实施实例中详细介绍。
本发明的荧光探针的作用机理如下，在探针分子中，探针本身发蓝色荧光，且有两个反应位点。当探针与H2S反应时，位点1的叠氮基被还原，随后醚键被切断，经过快速的分子内成环反应得到发蓝色荧光的HCB和发红色荧光的TQC。当探针TCAB与H2O2反应时，位点2的醚键被切断，释放出发蓝绿色荧光的化合物TCA，探针的荧光发生了从蓝光到蓝绿色的比值变化。通过探针TCAB与H2S和H2O2反应得到不同的荧光信号：H2S(蓝光+红光)， H2O2(蓝光→蓝绿光)，可以实现对H2S和H2O2的区分检测。随后，我们进一步研究了探针与两种响应物叠加响应的情况。当向探针TCAB与H2S响应后的溶液中继续加入H2O2时，前一步的产物HCB上的苯硼酸酯会继续被H2O2切断，从而释放出蓝绿色的荧光染料HC，同时之前得到的红光染料TQC不受影响。同样的，向探针TCAB与H2O2响应后的溶液中继续加入H2S时，TCA上的叠氮基会继续被H2S还原，醚键断裂，再经过快速的分子内成环反应得到发蓝绿色荧光的染料HC和发红色荧光的染料TQC。由此可见，探针TCAB可以实现对H2S和H2O2的连续检测。
探针分子的响应过程：
本发明的荧光探针探针TCAB与H2S和H2O2的荧光响应测试在HEPES缓冲液(20 mM,1.0mM CTAB,pH＝7.4)中进行。探针本身在413nm处发蓝色荧光。当探针TCAB的溶液中加入H2O2时，413nm处的荧光下降，同时在486nm处有新的蓝绿色荧光生成(激发波长为325nm)。荧光滴定实验结果显示，随着H2O2浓度的增大，486nm处的荧光强度(F486nm) 与413nm处的荧光强度(F413nm)的比值显著增大，当H2O2浓度达到400μM时，F486nm/F413nm的值达到最大。并且F486nm/F413nm的值与H2O2的浓度在20-100μM范围内有很好的线性关系，线性相关系数为0.9968。另外，当探针TCAB与H2S响应时，反应液在413nm处的蓝色荧光增强(激发波长为325nm)，同时在627nm处有新的红色荧光生成(激发波长为475 nm)。随着H2S浓度的增加，反应体系在413nm和627nm处的荧光强度逐渐增强，在350μM 的H2S加入时，荧光强度达到最大值。并且探针TCAB在413nm和627nm处的荧光强度与 H2S在浓度范围为0-150μM时具有很好的线性关系，线性相关系数分别为0.9905和0.9901。根据信噪比S/N＝3，计算出探针TCAB对H2S和H2O2的检测限分别为0.058和0.044μM。随后，我们还测定了探针TCAB与H2S和H2O2响应前后的紫外光谱变化情况
本发明所述的探针分子合成路线简单，成本较低，对H2S和H2O2的选择性好、抗干扰能力强，斯托克位移大，该荧光探针在生物化学，环境科学等领域具有实际的应用价值。
附图说明
图1为本发明荧光探针TCAB(10.0μM)在HEPES缓冲液(20mM,1.0mM CTAB, pH＝7.4)中与H2O2(400μM)和H2S(350μM)响应前后的紫外吸收光谱。
图2(A-B)探针TCAB(10.0μM)与H2O2完全反应后的溶液随H2S浓度增加的荧光光谱变化。(C-D)探针TCAB(10.0μM)与H2S完全反应后的溶液随H2O2浓度增加的荧光光谱变化。激发波长：第一列325nm；第二列475nm。激发和发射狭缝宽度为5nm/5nm。
图3为本发明的荧光探针TCAB(10.0μM)在HEPES缓冲液(20mM,1.0mM CTAB, pH＝7.4)中与H2O2(400μM)和H2S(350μM)响应时，F486nm/F413nm的比值以及413nm和627 nm处的荧光强度随时间的变化。激发波长：(A)325nm,(B)325nm,(C)475nm。
图4为本发明的荧光探针TCAB(10.0μM)在HEPES缓冲液(20mM,1.0mM CTAB, pH＝7.4)中与相关分析物响应后，F486nm/F413nm的比值以及413nm和627nm处的荧光强度变化。分析物包括：(1)ROO.,(2)NO.,(3)ClO-,(4)O2 -,(5)TBHP,(6)TBO.,(7)ONOO-,(8)1O2,(9) OH-,(10)SO4 2-,(11)NO3 -,(12)SCN-,(13)Cl-,(14)CO3 2-,(15)SO3 2-,(16)N3 -,(17)AcO-,(18) Na+,(19)Mg2+,(20)Ca2+,(21)K+,(22)Fe3+,(23)Cys,(24)GSH,(25)H2O2,(26)H2S。浓度： (1)-(25)400μM,(26)350μM，激发波长：(A)325nm,(B)325nm,(C)475nm。
图5为高效液相色谱：(a)探针TCAB(50.0μM)；(b,c,d)探针TCAB(50.0μM)分别与10.0,20.0和40.0equiv.的H2O2在HEPES缓冲液(20mM,1.0mM CTAB,pH＝7.4)中响应 120分钟；(e)化合物TCA(50.0μM).流动相：H2O/CH3CN(v/v,2/8)；流速：0.5mL/min；温度： 25℃；检测波长：350nm。
图6高效液相色谱：(a)探针TCAB(50.0μM)；(b,c,d)探针TCAB(50.0μM)分别与10.0,20.0和40.0equiv.的H2S在HEPES缓冲液(20mM,1.0mM CTAB,pH＝7.4)中响应80 分钟；(e)化合物TQC(50.0μM)；(f)化合物HCB(50.0μM).流动相：H2O/CH3CN(v/v,2/8)；流速：0.5mL/min；温度：25℃；检测波长：350nm。
图7为本发明的荧光探针TCAB(10.0μM)在不同pH环境下与H2S和H2O2的响应情况。荧光强度：(a)F486nm/F413nm,(b)413nm,(c)627nm；激发波长：(a)325nm,(b)325nm,(c)475nm。
图8不同浓度的探针TCAB对HeLa细胞毒性的分析。
图9探针TCAB在HeLa细胞内对外源性H2O2和H2S的共聚焦荧光成像。(A)细胞先与500μM H2O2孵育30分钟，再与10μM探针TCAB孵育30分钟。(B)细胞先与500μM H2S孵育30分钟，再与10μM探针TCAB孵育30分钟。(C)细胞先与10μM探针TCAB 孵育30分钟，再与500μMH2O2孵育30分钟，最后与500μM H2S孵育30分钟。(D)细胞与10μM探针TCAB孵育30分钟。第一列：蓝光通道，激发波长405nm，收集范围420-450 nm；第二列：蓝绿光通道，激发波长405nm，收集范围460-510nm；第三列：红光通道，激发波长488nm，收集范围600-650nm；第四列：合并蓝光，蓝绿光和红光通道。
具体实施实例
本发明的探针分子结构式如下：
本发明的探针合成路线如下所示；
实施例1：探针的合成；
英文名称：
(2-oxo-7-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)-2H-chromen-4-yl)methyl (E)-3-(7-((4-azidobenzyl)oxy)-1,4-diethyl-1,2,3,4-tetrahydroquinoxalin-6-yl)-2-cyanoacrylate
中文名称：(2-氧代-7-((4-(4，4，5，5-四甲基-1，3，2-二氧杂环戊烷-2-基)苄基)氧基)-2H-色烯-4-基)甲基(E)-3-(7-((4-叠氮苯甲酰)氧基)-1，4-二乙基-1，2，3， 4-四氢喹喔啉-6-基)-2-氰基丙烯酸酯
结构式：
合成方法：将化合物TCA(60mg,0.1mmol)和4-溴甲基苯硼酸频哪醇酯(60mg,0.2mmol)溶于10mL丙酮，置于25mL圆底烧瓶中，加入碳酸钾(28mg,0.2mmol)，60℃搅拌回流反应3h。TLC监测反应完成后，停止反应，减压蒸馏除去溶剂，剩余物用柱层析分离纯化，二氯甲烷/乙酸乙酯(v:v＝20:1,含1％三乙胺)作为洗脱剂。干燥得到红色固体35mg，产率为43％。表征数据：HRMS(ESI)m/z:[M]calcd for C46H47BN6O8,822.3548；found,822.3605. 1HNMR(400MHz,CDCl3)δ8.69(s,1H),7.76(s,1H),7.49(d,J＝8.8Hz,1H),7.45–7.39(m, 4H),7.37(d,J＝7.8Hz,2H),7.03(d,J＝8.4Hz,2H),6.94(dd,J＝8.8,2.4Hz,1H),6.89(d,J＝2.3Hz,1H),6.42(s,1H),5.99(s,1H),5.41(s,2H),5.12(d,J＝23.9Hz,4H),3.56(s,2H),3.36(d, J＝6.2Hz,4H),3.21(s,2H),1.35(t,12H),1.26(q,J＝3.2Hz,6H).13C NMR(100MHz,CDCl3)δ 164.6,161.8,160.8,156.7,155.5,149.2,147.8,144.4,144.0,140.0,138.8,135.1,133.3,128.7, 126.6,126.1,124.7,119.4,118.4,113.2,111.0,110.4,109.9,102.4,94.2,83.9,70.8,70.4,65.3, 61.9,53.4,48.0,45.4,29.7,24.9,10.9,9.8.
实施例2：细胞培养和荧光成像；
HeLa细胞(人***细胞)来源于湖南大学化学生物传感与计量学国家重点实验室。 HeLa细胞培养在含10％的胎牛血清和1％的盘尼西林的DMEM培养液中，在37℃和5％二氧化碳条件下培养24h。然后接种到激光共聚焦培养皿中，继续孵育12h，待细胞贴壁后可进行荧光成像实验。
外源性H2S和H2O2的检测。实验组一和二：首先移除培养基，PBS缓冲液漂洗细胞三次，向培养皿中加入500μM的Na2S或H2O2，细胞在37℃和5％二氧化碳条件下培养 30分钟，然后用PBS缓冲液漂洗细胞三次，将探针TCAB(10μM)加入到培养皿中，与细胞孵育30分钟，PBS漂洗后进行荧光成像；实验组三：移除培养基，PBS缓冲液漂洗细胞三次，将探针TCAB(10μM)加入到培养皿中，与细胞孵育30分钟，PBS缓冲液漂洗细胞三次，然后向培养皿中加入500μM的Na2S，与细胞孵育30分钟，用PBS缓冲液漂洗细胞三次，然后向培养皿中加入500μM的H2O2，继续与细胞孵育30分钟，PBS漂洗后进行荧光成像。对照组：移除培养基，PBS缓冲液漂洗细胞三次，将探针TCAB(10μM)加入到培养皿中，在 37℃和5％二氧化碳条件下与细胞孵育30分钟，PBS漂洗后进行荧光成像。
内源性H2S和H2O2的检测。实验组一：移除培养基，PBS缓冲液漂洗细胞三次，向培养皿中加入佛波酯(PMA,1μg/mL)，细胞孵育30分钟后用PBS缓冲液漂洗三次，将探针 TCAB(10μM)加入到培养皿中，与细胞孵育30分钟，PBS漂洗后进行荧光成像；实验组二：移除培养基，PBS缓冲液漂洗细胞三次，向培养皿中加入硝普钠(SNP,100μM)，细胞孵育 30分钟后用PBS缓冲液漂洗三次，将探针TCAB(10μM)加入到培养皿中，与细胞孵育30 分钟，PBS漂洗后进行荧光成像；对照组：移除培养基，PBS缓冲液漂洗细胞三次，将探针 TCAB(10μM)加入到培养皿中，在37℃和5％二氧化碳条件下与细胞孵育30分钟，PBS 漂洗后进行荧光成像。
Claims (1)
1.一种多信号比率型区分检测H2O2和H2S的荧光探针，其结构如式(I)所示；
其特征在于该荧光探针用于环境或生物样品中的H2O2和H2S的荧光检测和分析。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811090636.4A CN109180716B (zh) | 2018-09-19 | 2018-09-19 | 一种多信号比率型区分检测h2o2和h2s的荧光探针的设计、合成及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811090636.4A CN109180716B (zh) | 2018-09-19 | 2018-09-19 | 一种多信号比率型区分检测h2o2和h2s的荧光探针的设计、合成及应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109180716A true CN109180716A (zh) | 2019-01-11 |
| CN109180716B CN109180716B (zh) | 2020-11-13 |
Family
ID=64908521
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811090636.4A Active CN109180716B (zh) | 2018-09-19 | 2018-09-19 | 一种多信号比率型区分检测h2o2和h2s的荧光探针的设计、合成及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109180716B (zh) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109970777A (zh) * | 2019-04-23 | 2019-07-05 | 湘潭大学 | 一种具有检测过氧化氢功能的荧光探针及其制备方法和应用 |
| CN110818734A (zh) * | 2019-10-23 | 2020-02-21 | 中南大学 | 一种具有双比值识别过氧化氢和次氯酸的荧光探针 |
| CN113896739A (zh) * | 2021-08-26 | 2022-01-07 | 福建师范大学 | 一种反应性罗丹明b衍生物荧光探针及其制备方法和应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107603603A (zh) * | 2017-11-02 | 2018-01-19 | 中南大学 | 一种识别过氧化氢的荧光探针 |
| CN107840829A (zh) * | 2017-11-02 | 2018-03-27 | 中南大学 | 一种特异性识别硫化氢的荧光探针 |
| CN108358906A (zh) * | 2018-03-14 | 2018-08-03 | 中南大学 | 一种特异性区分不同硫醇的荧光探针 |
-
2018
- 2018-09-19 CN CN201811090636.4A patent/CN109180716B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107603603A (zh) * | 2017-11-02 | 2018-01-19 | 中南大学 | 一种识别过氧化氢的荧光探针 |
| CN107840829A (zh) * | 2017-11-02 | 2018-03-27 | 中南大学 | 一种特异性识别硫化氢的荧光探针 |
| CN108358906A (zh) * | 2018-03-14 | 2018-08-03 | 中南大学 | 一种特异性区分不同硫醇的荧光探针 |
Non-Patent Citations (1)
| Title |
|---|
| LONG YI等: "A Dual-Response Fluorescent Probe Reveals the H2O2-Induced H2S Biogenesis through a Cystathionine β-Synthase Pathway", 《CHEM. EUR.J.》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109970777A (zh) * | 2019-04-23 | 2019-07-05 | 湘潭大学 | 一种具有检测过氧化氢功能的荧光探针及其制备方法和应用 |
| CN110818734A (zh) * | 2019-10-23 | 2020-02-21 | 中南大学 | 一种具有双比值识别过氧化氢和次氯酸的荧光探针 |
| CN113896739A (zh) * | 2021-08-26 | 2022-01-07 | 福建师范大学 | 一种反应性罗丹明b衍生物荧光探针及其制备方法和应用 |
| CN113896739B (zh) * | 2021-08-26 | 2023-09-22 | 福建师范大学 | 一种反应性罗丹明b衍生物荧光探针及其制备方法和应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109180716B (zh) | 2020-11-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105524612B (zh) | 一种异佛尔酮类荧光探针及其制备与应用 | |
| Gong et al. | A novel two-photon fluorescent probe with long-wavelength emission for monitoring HClO in living cells and tissues | |
| CN110746410B (zh) | 一种亮氨酸氨肽酶和单胺氧化酶激活的近红外荧光探针、合成方法及生物应用 | |
| CN105038762B (zh) | 一种检测过氧化氢的比率型荧光探针及其应用 | |
| CN106967102B (zh) | 一种基于罗丹明衍生物的过氧化氢增强型荧光探针 | |
| CN109593087A (zh) | 一种多通道同时区分检测Cys/Hcy，GSH和H2S的荧光探针的设计、合成及应用 | |
| CN109180716A (zh) | 一种多信号比率型区分检测h2o2和h2s的荧光探针的设计、合成及应用 | |
| CN104673278A (zh) | 一种检测谷胱甘肽的荧光探针及其制备方法与使用方法 | |
| Zhu et al. | Construction of a fluorine substituted chromenylium-cyanine near-infrared fluorophore for ratiometric sensing | |
| CN105622624A (zh) | 一种罗丹明b衍生物、制备方法及其作为荧光探针的应用 | |
| Zhou et al. | A mitochondria targetable two-photon excited near-infrared fluorescent probe for imaging of H2O2 in live cells and tissues | |
| CN103320120A (zh) | 一种含半胱氨酸乙酯结构的罗丹明B类靶向溶酶体pH荧光探针及应用 | |
| Deng et al. | A ratiometric fluorescent probe based on phenothiazine and HBI motif for rapid detection of HClO and its applications | |
| CN108641710B (zh) | 一种检测蛋白质硫巯基化的荧光探针及其制备方法和应用 | |
| CN108727265B (zh) | 一种检测甲醛和pH的双功能荧光探针及其制备方法和应用 | |
| CN114539183A (zh) | 一种癌细胞组织诊断的脂滴靶向和生物硫醇敏感的荧光探针及制备和应用 | |
| Zhu et al. | A Mitochondrial-targeted curcumin-based fluorescent probe for real-time tracking endogenous hydrogen peroxide in living cells | |
| CN108752275A (zh) | 一种pH荧光探针及其制备方法和应用 | |
| CN110642857B (zh) | 一种用于检测粘度和pH的双功能荧光探针及其制备与应用 | |
| Gu et al. | Development of a highly selective H 2 S fluorescent probe and its application to evaluate CSE inhibitors | |
| CN113637048A (zh) | 一种γ-谷氨酰转肽酶的双光子荧光探针及其制备方法和应用 | |
| CN114249760B (zh) | 三通道同时区分次氯酸和过氧化氢的荧光探针的合成与应用 | |
| CN110229203B (zh) | 一种氨基己糖酶荧光探针及其制备方法和应用 | |
| CN105884787A (zh) | 一种罗丹明6g衍生物及其制备方法和应用 | |
| CN115141145A (zh) | 一种检测溶酶体次溴酸荧光探针、制备方法与应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |