CN112358508B - 一种通过光调控精确检测生物体内h2o2的荧光探针及其制备方法和应用 - Google Patents
一种通过光调控精确检测生物体内h2o2的荧光探针及其制备方法和应用 Download PDFInfo
- Publication number
- CN112358508B CN112358508B CN202011378243.0A CN202011378243A CN112358508B CN 112358508 B CN112358508 B CN 112358508B CN 202011378243 A CN202011378243 A CN 202011378243A CN 112358508 B CN112358508 B CN 112358508B
- Authority
- CN
- China
- Prior art keywords
- fluorescent probe
- ufps
- probe
- vivo
- accurate detection
- 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.)
- Active
Links
- 239000000523 sample Substances 0.000 title claims abstract description 26
- 238000001514 detection method Methods 0.000 title claims description 19
- 238000001727 in vivo Methods 0.000 title claims description 14
- 238000002360 preparation method Methods 0.000 title claims description 9
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 13
- 239000000975 dye Substances 0.000 claims abstract description 11
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 claims abstract description 9
- 150000001413 amino acids Chemical class 0.000 claims abstract description 7
- 238000006243 chemical reaction Methods 0.000 claims description 15
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 10
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 claims description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000012074 organic phase Substances 0.000 claims description 8
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- GZZBZWITJNATOD-UHFFFAOYSA-N [4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]methanol Chemical compound O1C(C)(C)C(C)(C)OB1C1=CC=C(CO)C=C1 GZZBZWITJNATOD-UHFFFAOYSA-N 0.000 claims description 5
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 5
- 238000010898 silica gel chromatography Methods 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 150000002500 ions Chemical class 0.000 claims description 4
- 238000010791 quenching Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 230000000171 quenching effect Effects 0.000 claims 1
- 238000002474 experimental method Methods 0.000 abstract description 4
- 230000003287 optical effect Effects 0.000 abstract description 2
- 230000035699 permeability Effects 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 238000010226 confocal imaging Methods 0.000 abstract 1
- 230000002900 effect on cell Effects 0.000 abstract 1
- 230000000214 effect on organisms Effects 0.000 abstract 1
- 231100000331 toxic Toxicity 0.000 abstract 1
- 230000002588 toxic effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 30
- 239000003642 reactive oxygen metabolite Substances 0.000 description 12
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 8
- 208000018737 Parkinson disease Diseases 0.000 description 8
- 238000003384 imaging method Methods 0.000 description 7
- 210000005013 brain tissue Anatomy 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 238000011534 incubation Methods 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 238000000692 Student's t-test Methods 0.000 description 4
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 4
- 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 4
- 239000007787 solid Substances 0.000 description 4
- 238000012353 t test Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000036542 oxidative stress Effects 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- BGNGWHSBYQYVRX-UHFFFAOYSA-N 4-(dimethylamino)benzaldehyde Chemical compound CN(C)C1=CC=C(C=O)C=C1 BGNGWHSBYQYVRX-UHFFFAOYSA-N 0.000 description 2
- 208000024827 Alzheimer disease Diseases 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- 241000255925 Diptera Species 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 229960004308 acetylcysteine Drugs 0.000 description 2
- SWLVFNYSXGMGBS-UHFFFAOYSA-N ammonium bromide Chemical compound [NH4+].[Br-] SWLVFNYSXGMGBS-UHFFFAOYSA-N 0.000 description 2
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 210000004720 cerebrum Anatomy 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 229960003180 glutathione Drugs 0.000 description 2
- 239000005457 ice water Substances 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 210000003470 mitochondria Anatomy 0.000 description 2
- 230000004770 neurodegeneration Effects 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 230000004792 oxidative damage Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- FTZIQBGFCYJWKA-UHFFFAOYSA-N 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Chemical compound S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 FTZIQBGFCYJWKA-UHFFFAOYSA-N 0.000 description 1
- MCSXGCZMEPXKIW-UHFFFAOYSA-N 3-hydroxy-4-[(4-methyl-2-nitrophenyl)diazenyl]-N-(3-nitrophenyl)naphthalene-2-carboxamide Chemical compound Cc1ccc(N=Nc2c(O)c(cc3ccccc23)C(=O)Nc2cccc(c2)[N+]([O-])=O)c(c1)[N+]([O-])=O MCSXGCZMEPXKIW-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000255601 Drosophila melanogaster Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 101000619542 Homo sapiens E3 ubiquitin-protein ligase parkin Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000008589 Obesity Diseases 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
- 208000006011 Stroke Diseases 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 238000002737 cell proliferation kit Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene chloride Substances ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 238000002610 neuroimaging Methods 0.000 description 1
- 230000016273 neuron death Effects 0.000 description 1
- 230000006764 neuronal dysfunction Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 238000012261 overproduction Methods 0.000 description 1
- 230000036284 oxygen consumption Effects 0.000 description 1
- 230000008557 oxygen metabolism Effects 0.000 description 1
- 102000045222 parkin Human genes 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000002824 peroxisome Anatomy 0.000 description 1
- 230000008832 photodamage Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 239000007845 reactive nitrogen species Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 229940080817 rotenone Drugs 0.000 description 1
- JUVIOZPCNVVQFO-UHFFFAOYSA-N rotenone Natural products O1C2=C3CC(C(C)=C)OC3=CC=C2C(=O)C2C1COC1=C2C=C(OC)C(OC)=C1 JUVIOZPCNVVQFO-UHFFFAOYSA-N 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
Images
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
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6596—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having atoms other than oxygen, sulfur, selenium, tellurium, nitrogen or phosphorus as ring hetero atoms
-
- 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"
-
- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- 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
- C09K2211/1096—Heterocyclic compounds characterised by ligands containing other heteroatoms
-
- 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
- G01N2021/6417—Spectrofluorimetric devices
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明涉及一种可通过光调控精确检测生物体内H2O2的荧光探针及其制备方法和应用，属于有机荧光探针领域。所述的荧光探针基于嘧啶基染料，为UFPS‑1荧光探针，UFPS‑1荧光探针的结构式如图(Ⅰ)所示。本发明的荧光探针能够精确检测生物体内的H2O2，避免细胞内其他离子氨基酸的干扰。该探针具有较大的斯托克斯位移，有较好的化学稳定性、生物兼容性和选择性等。荧光共聚焦成像实验表明该探针具有较好的细胞通透性，对细胞和生物体无毒副作用。
Description
技术领域
本发明涉及一种基于嘧啶基染料，可通过光调控精确检测生物体 内H2O2的荧光探针，属于有机荧光探针领域。
背景技术
过氧化氢(H2O2)是活性氧(ROS)中一种非常典型的物质，几 乎存在于所有细胞中，是氧代谢的重要产物，在调节多种细胞功能中 起着至关重要的作用。与ROS家族的其他物质(如超氧物或羟基自由 基)不同，H2O2可以自由扩散，结构相对稳定，寿命长。生物体内的H2O2通常来自线粒体、过氧化物酶体和胞浆酶，其中线粒体是H2O2的主要生理来源之一。
活性氧物种(ROS)的大量增加会导致各种生物分子的结构性氧 化损伤，这些生物分子对细胞的结构完整性至关重要，包括脂质、DNA 和蛋白质。活性氧引起的这种氧化损伤被称为“氧化应激”，并已被证 明与多种生物分子有关在癌症、糖尿病、肥胖症和中风等疾病中。中 枢神经系统(CNS)因其高耗氧量而对氧化应激特别敏感，过量产生 ROS可能导致神经元功能障碍或死亡，如阿尔茨海默氏症(AD)、帕 金森氏病(PD)和肌萎缩性侧索硬化症(ALS)和其他神经退行性疾 病。
因此，开发实时、无创的ROS特异性检测工具对于研究氧化应激 相关疾病的发病机制尤为重要。为了解决这一问题，亟需研发一种可 以高效、精准检测H2O2的荧光探针。
发明内容
本发明利用嘧啶基染料DMe的光学及生物性质，在其结构上引 入了H2O2特异性识别基团硼酸酯结构，进而达到精准检测活细胞内 H2O2的目的，亦可用于检测野生型及帕金森病(Parkinson’s disease, PD)模型果蝇脑组织中的H2O2。
本发明采用的技术方案是：一种通过光调控精确检测生物体内 H2O2的荧光探针，所述的荧光探针为UFPS-1探针，UFPS-1荧光探针 结构如图(Ⅰ)所示：
优选的，取25mL的圆底烧瓶，将嘧啶基染料DMe用10mL吡 啶溶解，逐滴加入三氯氧磷，搅拌2小时直至溶液澄清，随后放入冷 井-40℃环境，使用吡啶溶解4-(羟甲基)苯硼酸频哪醇酯，随后取出 并常温搅拌3小时，将反应体系放入-20℃的环境，用水淬灭反应， 乙酸乙酯萃取，洗涤有机相并用无水硫酸钠干燥，通过硅胶层析法分 离纯化得到目标产物UFPS-1荧光探针。
优选的，所述的嘧啶基染料DMe、三氯氧磷与4-(羟甲基)苯硼酸 频哪醇酯的摩尔比为1：3：1。
优选的，所述的嘧啶基染料DMe的制备方法如下：50mL圆底 烧瓶加入20mL乙醇和对二甲氨基苯甲醛，超声溶解固体，搅拌加 入浓盐酸5.6mL，95℃回流24h。反应结束后将溶液倒入冰水中， 加入碳酸氢钠中和；用CH2Cl2萃取两次后无水硫酸钠干燥有机相，除 去无水硫酸钠和有机溶剂，用硅胶层析法得到染料DMe。
优选的，本发明精确检测生物体内H2O2的荧光探针的制备反应 式如下：
本发明采用的另一技术方案是：所述的通过光调控精确检测生物 体内H2O2的荧光探针的应用，用于生理体系中精确检测生物体内的 H2O2的试剂制备。
优选的，所述试剂在细胞体系中通过光调控精确检测生物体内的 H2O2而避免来自细胞中其他离子和氨基酸的干扰的应用。
优选的，所述生物体内包括活细胞及动物组织为HepG2细胞株、 LO2细胞株、Parkin Null型及Wild type型果蝇大脑。
有益效果：
本发明的荧光探针具有近红外发射的优点，因此具有组织穿透能 力强、光损伤弱和受组织自发荧光影响弱等优点，且反应后荧光量子 产率显著增高。体外实验研究表明，探针与H2O2反应速度快。在生 物条件(pH 7.4)下,探针稳定，且对H2O2具有较高的灵敏度和选择 性，不受其他离子和氨基酸的干扰。生物实验研究表明，探针对癌细 胞及普通体细胞毒性均较低，且可以检测细胞中外源和内源性H2O2的水平。进一步将探针应用于PD果蝇脑模型中，实验证明探针可以 直观地显示出PD模型中内源性H2O2水平。
附图说明
图1-a,1-b,1-c分别是探针UFPS-1的氢谱、碳谱、质谱数据。
图2为2μM探针UFPS-1在PBS溶液(含0.2％DMSO和0.02％Triton) 中，与0-200倍当量的H2O2反应2小时的吸收光谱图。
图3为2μM探针UFPS-1在PBS溶液(含0.2％DMSO和0.02％Triton) 中，与0-200倍当量的H2O2反应2小时的发射光谱图。
图4为常见活性氧/活性氮物质在120分钟内对探针UFPS-1的影响(纵 坐标为实时荧光强度与初始荧光强度的比值)。
图5为探针UFPS-1对42种不同离子及氨基酸的选择性。
图6为不同pH条件对探针UFPS-1的影响。
图7为不同浓度UFPS-1作用24h后，HepG-2细胞存活率(MTT法)。
图8为不同浓度UFPS-1作用24h后，LO2细胞存活率(MTT法)。
图9-a为UFPS-1对HepG2细胞外源性/内源性H2O2水平的荧光成像。 比例尺＝10μm；图9-b为图9-a的相对荧光强度(1-5)。统计分析采 用t检验。***P<0.001。
图10-a为UFPS-1在室温下对野生型(3)和Parkin-Null(4)果蝇大 脑进行共焦荧光显微镜成像。图10-b为图10-a相对荧光强度(1-6)。 统计分析采用t检验。***P<0.001，**P<0.01。
具体实施方式
实施例1
一种通过光调控精确检测生物体内H2O2的荧光探针的制备方法 如下：
1、嘧啶基染料DMe的制备
50mL圆底烧瓶加入20mL乙醇和对二甲氨基苯甲醛(1.83g， 12.25mmol)超声溶解固体，搅拌加入浓盐酸5.6mL，95℃回流24h。 反应结束后将溶液倒入冰水中，加入碳酸氢钠中和。CH2Cl2萃取两次 后无水硫酸钠干燥有机相，除去无水硫酸钠和有机溶剂，用硅胶层析法得到深红色固体DMe。
1H NMR(500MHz,DMSO-d6,ppm):δ＝7.74(d,J＝16.25Hz,2H), 7.49(d,J＝8.35Hz,4H),6.77(d,J＝8.55Hz,4H),6.74(s,1H),6.71(d, J＝16.2Hz,2H),2.99(s,12H).
2、探针UFPS-1的制备
取25mL的圆底烧瓶，加入10mL吡啶溶解DMe(116mg，0.3 mmol)，逐滴加入三氯氧磷(85μL，0.9mmol)，滴加完毕后搅拌2 小时直至溶液澄清，随后烧瓶放入冷井-40℃环境，使用吡啶溶解 4-(羟甲基)苯硼酸频哪醇酯(0.69g，3mmol)缓慢滴加入反应体系， 随后取出烧瓶常温搅拌3个小时，随后将反应体系放入-20℃度的环 境，随后加入2mL水淬灭反应，乙酸乙酯萃取，水洗涤有机相6次， 无水硫酸钠干燥有机相，真空旋干有机相，通过硅胶层析法分离纯化， 得到橙黄色固体UFPS-1。
1H NMR(500MHz,CDCl3,ppm)δ＝7.82(m,6H),7.45(d,J＝8.75 Hz,4H),7.39(d,J＝7.85Hz,4H),6.98(s,1H),6.80(d,J＝15.8Hz,2H), 6.69(d,J＝8.75Hz,4H),5.39(d,J＝7.25Hz,4H),3.01(s,12H),1.33(s, 24H).
13C NMR(125MHz,CDCl3,ppm)δ＝166.29,151.25,138.95, 138.88,138.40,134.98,129.36,126.80,123.58,120.14,112.23,112.02, 83.80,70.02,40.21,24.87.
实施例2
一种通过光调控精确检测生物体内H2O2的荧光探针对H2O2的体 外响应检测：
1、UFPS-1对不同H2O2浓度的荧光响应(附图2、3)：
首先配制浓度10mM的PBS缓冲溶液(pH＝7.4，含0.02％Triton), 用缓冲溶液配制2μM UFPS-1探针，加入0-200当量的H2O2，用荧光 分光光度法测试，并绘制UFPS-1探针对不同当量H2O2的荧光光谱图。
2、UFPS-1对不同ROS及常见离子氨基酸的荧光响应(附图4、 5)：
首先配制浓度10mM的PBS缓冲溶液(pH＝7.4，含0.02％Triton), 用缓冲溶液配制2μM UFPS-1探针，加入7种ROS/RNS或43种离子 氨基酸(200μM)，在37℃的摇床上震荡2小时，然后在640nm处测 量反应溶液的发射值。并绘制UFPS-1探针对不同分析物的荧光光谱图。
3、UFPS-1对H2O2的检测限测定
通过检测UFPS-1与从0-20μM浓度梯度的H2O2响应后的荧光变 化，线性拟合640nm处的荧光强度以获得校准曲线。根据检测限计 算公式(3σ/S)计算出UFPS-1对H2O2的检出限为92nM。
实施例3
一种通过光调控精确检测生物体内H2O2的荧光探针对H2O2的体 内响应检测：
1、UFPS-1在不同细胞中的毒性测试(附图7、8)：
HepG2/LO2细胞在含有10％胎牛血清、100.0mg/mL链霉素和100 IU/mL青霉素的Dulbecco改良Eagle培养基中培养。细胞保持在37℃ 下5％二氧化碳的增湿空气中。
使用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2-H-四唑溴化铵(MTT) 比色细胞增殖试剂盒(Roche)测定UFPS-1的细胞毒性。HepG2/LO2 细胞在96孔板中生长到70-80％后收集，吸取培养基，然后用含有不 同浓度UFPS-1(0.1、0.5、1、5、10、20、30、40和50μM)的培养基代替。
培养24h后，向细胞中加入10μL MTT溶液(5mg/mL)。在37℃、 黑暗环境中进一步孵育4h后，用100μL二甲基亚砜代替溶液溶解产 生的晶体。10分钟后，用酶标仪在560nm处测量吸光度，并绘制细 胞活性图表。
与50μM UFPS-1孵育24h后，HepG2与LO2细胞均有90％以 上保持活力，这表明UFPS-1在工作浓度下具有较低的细胞毒性。
2、UFPS-1在HepG2细胞中的成像测试(附图9)：
将HepG2细胞以5×105个细胞的密度接种在35mm共焦激光成 像皿中，并生长24小时。细胞培养用UFPS-1探针在5μM浓度下孵 育，在37℃的5％CO2气氛中保持3h，然后用PBS(每盘3×2ml) 洗涤细胞。(附图9-a2)
对于外源性对照实验，细胞在37℃下用20μM H2O2孵育3小时， 然后用PBS洗涤3次后，细胞继续用5μM探针UFPS-1孵育3h。(附 图9-a3)
内源性H2O2实验的细胞用10μM鱼藤酮(ROS抑制剂)在37℃ 培养3小时，用PBS溶液洗涤3次后，用5μM探针UFPS-1进一步 孵育3小时。(附图9-a4)
为了证实荧光增强归因于细胞内H2O2，我们应用H2O2清除剂 ——N-乙酰半胱氨酸(NAC)作为对照。(附图9-a5)
所有细胞在成像前用PBS冲洗3次。最后，用共聚焦激光扫描显 微镜(蔡司LSM880)和单光子激光(63×水浸物镜)对细胞进行成 像，激发波长为543nm，记录的发射波长为570-700nm。
用Image J进行细胞成像图的荧光强度定量，各取每张图的平均 荧光强度数值，并在数值间进行t检验。(附图9-b)
实验结果表明，UFPS-1具有良好的细胞通透性。证明了通过光 调控的方法控制探针和H2O2的反应是非常成功的，实现了精确检测 细胞内的H2O2而不受细胞质内其他物质的干扰。
3、UFPS-1在野生型及PD模型果蝇脑组织中的成像测试(附图 10)：
野生型和PD型果蝇来自新加坡国家神经科学研究所的神经变性 研究实验室。
所有果蝇均在玉米粉-糖蜜培养基上生长并保持在25℃，分别将 PD和野生型果蝇记为实验组(附图10-a3、图10-a4)和对照组(附 图10-a1、图10-a2)，将添加谷胱甘肽(H2O2抑制剂)的果蝇作为抑 制组(附图10-a5、图10-a6)。解剖后，将实验组的果蝇脑组织放入含25μM UFPS-1的PBS(pH 7.4)缓冲液中。将抑制组的果蝇脑组 织浸入含UFPS-1(25μM)和GSH(250μM)的PBS缓冲液中，在37℃下孵育5小时。随后，将每组脑组织用PBS清洗3次。
最后，用共聚焦激光扫描显微镜(蔡司LSM 880)和单光子激光 (63×水浸物镜)对果蝇脑组织进行成像，激发波长为543nm，记录 的发射波长为570-700nm。
用Image J进行果蝇脑成像图的荧光强度定量，各取每张图的平 均荧光强度数值，并在数值间进行t检验。(附图10-b)
实验结果表明，UFPS-1具有良好的组织成像性能。本发明的不 局限于上述实施例所述的具体技术方案，凡采用等同替换形成的技术 方案均为本发明要求的保护范围。
Claims (6)
1.一种通过光调控精确检测生物体内H2O2的荧光探针，其特征在于：所述的荧光探针为UFPS-1探针，UFPS-1荧光探针结构如式(Ⅰ)所示：
2.根据权利要求1所述的通过光调控精确检测生物体内H2O2的荧光探针的制备方法，包括如下步骤：将嘧啶基染料DMe用10mL吡啶溶解，逐滴加入三氯氧磷，搅拌2小时直至溶液澄清，随后放入冷井-40℃环境，使用吡啶溶解4-(羟甲基)苯硼酸频哪醇酯，随后取出并常温搅拌3小时，将反应体系放入-20℃的环境，用水淬灭反应，乙酸乙酯萃取，洗涤有机相并用无水硫酸钠干燥，通过硅胶层析法分离纯化得到目标产物UFPS-1荧光探针。
3.根据权利要求2所述的通过光调控精确检测生物体内H2O2的荧光探针的制备方法，其特征在于：所述的嘧啶基染料DMe、三氯氧磷与4-(羟甲基)苯硼酸频哪醇酯的摩尔比为1：3：1。
4.根据权利要求2所述的通过光调控精确检测生物体内H2O2的荧光探针的制备方法，其特征在于：所述精确检测生物体内H2O2的荧光探针的制备反应式如下：
5.根据权利要求1所述的通过光调控精确检测生物体内H2O2的荧光探针的应用，其特征在于：用于生理体系中精确检测生物体内的H2O2的试剂制备。
6.根据权利要求5所述的通过光调控精确检测生物体内H2O2的荧光探针应用，其特征在于：所述试剂在细胞体系中通过光调控精确检测生物体内的H2O2而避免来自细胞中其他离子和氨基酸的干扰的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011378243.0A CN112358508B (zh) | 2020-12-01 | 2020-12-01 | 一种通过光调控精确检测生物体内h2o2的荧光探针及其制备方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011378243.0A CN112358508B (zh) | 2020-12-01 | 2020-12-01 | 一种通过光调控精确检测生物体内h2o2的荧光探针及其制备方法和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112358508A CN112358508A (zh) | 2021-02-12 |
| CN112358508B true CN112358508B (zh) | 2021-09-14 |
Family
ID=74535706
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011378243.0A Active CN112358508B (zh) | 2020-12-01 | 2020-12-01 | 一种通过光调控精确检测生物体内h2o2的荧光探针及其制备方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112358508B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113651759B (zh) * | 2021-07-27 | 2023-02-10 | 南京工业大学 | 一种检测生物体内Fe2+的双光子荧光探针及其制备方法和应用 |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106749359B (zh) * | 2016-11-30 | 2018-06-05 | 中南大学 | 一种检测过氧化氢新型荧光探针的合成与应用 |
| CN106866738B (zh) * | 2017-03-02 | 2018-11-13 | 南京工业大学 | 一种通过光调控精确检测线粒体内h2s2的荧光探针及其制备方法和应用 |
| 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 |
| CN108129500A (zh) * | 2018-01-11 | 2018-06-08 | 中南大学 | 一种识别过氧化氢的比值型荧光探针的合成与应用 |
| CN110540837B (zh) * | 2019-09-23 | 2022-12-13 | 湘潭大学 | 一种过氧化氢近红外荧光探针的制备和应用 |
-
2020
- 2020-12-01 CN CN202011378243.0A patent/CN112358508B/zh active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN112358508A (zh) | 2021-02-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106518860B (zh) | 一种基于荧光共振能量转移机理的靶向线粒体的次氯酸比例荧光探针及其应用 | |
| CN103173212B (zh) | 一种检测生物硫化氢的荧光探针及其制备与应用 | |
| CN107235946A (zh) | 一种谷胱甘肽荧光探针及其制备方法和应用 | |
| CN103773361B (zh) | 一种以香豆素为母核的半胱氨酸荧光探针及其应用 | |
| CN105924394A (zh) | 一种双光子甲醛荧光探针及其制备与应用 | |
| CN108484622B (zh) | 多信号荧光探针的合成及其同时区分检测Hcy、Cys和GSH的应用 | |
| CN106866689A (zh) | 一种检测血液中谷胱甘肽的荧光探针及其合成方法与应用 | |
| CN109776564A (zh) | 一种氧杂蒽结构的亚铁离子荧光探针及其合成方法和应用 | |
| CN112358508B (zh) | 一种通过光调控精确检测生物体内h2o2的荧光探针及其制备方法和应用 | |
| CN107286173B (zh) | Rhodol类衍生物及其制备方法和应用 | |
| CN106932371B (zh) | 一种细胞内谷胱甘肽的荧光成像方法 | |
| CN112062755A (zh) | 用于检测天冬氨酰氨肽酶的近红外荧光分子探针、制备方法及用途 | |
| CN108752373B (zh) | 一种基于苯硼酯识别过氧化氢的荧光探针 | |
| CN109180716B (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 | |
| CN107325062B (zh) | 一种检测过氧化氢活性的荧光探针及制备和应用 | |
| CN108383774A (zh) | 一种基于端基炔酮的半胱氨酸荧光探针及其制备和应用 | |
| CN110357817B (zh) | 一类可逆性检测丙酮醛和乙二醛荧光探针及其制备方法和应用 | |
| CN114763353A (zh) | 一种基于咪唑并吡啶母核的荧光检测试剂及其对信号分子h2s的检测技术 | |
| CN108191848B (zh) | 制备用于检测半胱氨酸的试剂盒的方法 | |
| CN111533730A (zh) | 一种免洗型细胞膜靶向荧光探针及其制备方法和应用 | |
| CN108148057B (zh) | 制备用于检测半胱氨酸的荧光探针的方法 | |
| CN101439191B (zh) | 一类可在细胞/组织/活体中应用的可见光激发的锌离子荧光造影试剂的合成方法 | |
| CN113999159B (zh) | 一种粘度敏感的荧光探针，其制备方法及应用 | |
| EP3551637A1 (en) | A compound for the detection of hno in biological systems | |
| CN113651759B (zh) | 一种检测生物体内Fe2+的双光子荧光探针及其制备方法和应用 |
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 |