CN108484621A - A kind of probe dye and its preparation method and application of the unicellular metamorphosis of efficient tracing detection - Google Patents
A kind of probe dye and its preparation method and application of the unicellular metamorphosis of efficient tracing detection Download PDFInfo
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- CN108484621A CN108484621A CN201810421485.XA CN201810421485A CN108484621A CN 108484621 A CN108484621 A CN 108484621A CN 201810421485 A CN201810421485 A CN 201810421485A CN 108484621 A CN108484621 A CN 108484621A
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- Prior art keywords
- unicellular
- metamorphosis
- probe dye
- probe
- solvent
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Links
- 239000000523 sample Substances 0.000 title claims abstract description 76
- 238000001514 detection method Methods 0.000 title claims abstract description 31
- 230000029052 metamorphosis Effects 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- BQPIGGFYSBELGY-UHFFFAOYSA-N mercury(2+) Chemical compound [Hg+2] BQPIGGFYSBELGY-UHFFFAOYSA-N 0.000 claims abstract description 26
- 239000000243 solution Substances 0.000 claims description 41
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Natural products CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 34
- 239000002904 solvent Substances 0.000 claims description 29
- 235000019441 ethanol Nutrition 0.000 claims description 16
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 229940042795 hydrazides for tuberculosis treatment Drugs 0.000 claims description 7
- WVFVAVUHWLDTCE-UHFFFAOYSA-N n,n-diaminobenzamide Chemical compound NN(N)C(=O)C1=CC=CC=C1 WVFVAVUHWLDTCE-UHFFFAOYSA-N 0.000 claims description 7
- 238000001953 recrystallisation Methods 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical group CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- MGNCLNQXLYJVJD-UHFFFAOYSA-N cyanuric chloride Chemical compound ClC1=NC(Cl)=NC(Cl)=N1 MGNCLNQXLYJVJD-UHFFFAOYSA-N 0.000 claims description 6
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- QKFJKGMPGYROCL-UHFFFAOYSA-N phenyl isothiocyanate Chemical compound S=C=NC1=CC=CC=C1 QKFJKGMPGYROCL-UHFFFAOYSA-N 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 5
- 239000011550 stock solution Substances 0.000 claims description 5
- 238000004440 column chromatography Methods 0.000 claims description 4
- 238000003384 imaging method Methods 0.000 claims description 4
- 239000011261 inert gas Substances 0.000 claims description 4
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 229940117953 phenylisothiocyanate Drugs 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 2
- KBYOBAICCHNMNJ-UHFFFAOYSA-L diperchloryloxymercury Chemical group [Hg+2].[O-]Cl(=O)(=O)=O.[O-]Cl(=O)(=O)=O KBYOBAICCHNMNJ-UHFFFAOYSA-L 0.000 claims description 2
- 125000005909 ethyl alcohol group Chemical group 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 238000010792 warming Methods 0.000 claims description 2
- JMANVNJQNLATNU-UHFFFAOYSA-N oxalonitrile Chemical compound N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 claims 2
- 238000001704 evaporation Methods 0.000 claims 1
- 230000008020 evaporation Effects 0.000 claims 1
- YXCDJKQYFBEAOU-UHFFFAOYSA-N phenyl thiocyanate Chemical compound N#CSC1=CC=CC=C1 YXCDJKQYFBEAOU-UHFFFAOYSA-N 0.000 claims 1
- 229920001596 poly (chlorostyrenes) Polymers 0.000 claims 1
- 150000002500 ions Chemical class 0.000 abstract description 18
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical group [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 abstract description 13
- 229910052753 mercury Inorganic materials 0.000 abstract description 9
- 239000007787 solid Substances 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 5
- 239000000843 powder Substances 0.000 abstract description 4
- 238000003860 storage Methods 0.000 abstract description 3
- 238000010189 synthetic method Methods 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 42
- 239000000975 dye Substances 0.000 description 33
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 15
- 229910021645 metal ion Inorganic materials 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 7
- 239000007788 liquid Substances 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 5
- 230000001413 cellular effect Effects 0.000 description 5
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000013307 optical fiber Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 3
- 230000000536 complexating effect Effects 0.000 description 3
- 238000002189 fluorescence spectrum Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 2
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 2
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
- 102000004310 Ion Channels Human genes 0.000 description 2
- 208000005374 Poisoning Diseases 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 159000000009 barium salts Chemical class 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 150000001661 cadmium Chemical class 0.000 description 2
- 159000000007 calcium salts Chemical class 0.000 description 2
- 230000036952 cancer formation Effects 0.000 description 2
- 231100000504 carcinogenesis Toxicity 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
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- 239000010408 film Substances 0.000 description 2
- 239000003501 hydroponics Substances 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 2
- RVPVRDXYQKGNMQ-UHFFFAOYSA-N lead(2+) Chemical compound [Pb+2] RVPVRDXYQKGNMQ-UHFFFAOYSA-N 0.000 description 2
- 159000000003 magnesium salts Chemical class 0.000 description 2
- 150000002696 manganese Chemical class 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 150000002730 mercury Chemical class 0.000 description 2
- 238000000103 photoluminescence spectrum Methods 0.000 description 2
- 231100000572 poisoning Toxicity 0.000 description 2
- 230000000607 poisoning effect Effects 0.000 description 2
- 238000002390 rotary evaporation Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 159000000000 sodium salts Chemical class 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 description 2
- 150000003751 zinc Chemical class 0.000 description 2
- RLLPVAHGXHCWKJ-IEBWSBKVSA-N (3-phenoxyphenyl)methyl (1s,3s)-3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropane-1-carboxylate Chemical compound CC1(C)[C@H](C=C(Cl)Cl)[C@@H]1C(=O)OCC1=CC=CC(OC=2C=CC=CC=2)=C1 RLLPVAHGXHCWKJ-IEBWSBKVSA-N 0.000 description 1
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical compound C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 229920000049 Carbon (fiber) Polymers 0.000 description 1
- 208000002454 Nasopharyngeal Carcinoma Diseases 0.000 description 1
- 206010061306 Nasopharyngeal cancer Diseases 0.000 description 1
- 229920001609 Poly(3,4-ethylenedioxythiophene) Polymers 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 231100000570 acute poisoning Toxicity 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000004917 carbon fiber Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 150000001805 chlorine compounds Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000000921 elemental analysis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000000630 fibrocyte Anatomy 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 230000009246 food effect Effects 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N hydrogen thiocyanate Natural products SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- GRHBQAYDJPGGLF-UHFFFAOYSA-N isothiocyanic acid Chemical compound N=C=S GRHBQAYDJPGGLF-UHFFFAOYSA-N 0.000 description 1
- 150000003951 lactams Chemical class 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002063 nanoring Substances 0.000 description 1
- 201000011216 nasopharynx carcinoma Diseases 0.000 description 1
- -1 phenyl ester Chemical class 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920006254 polymer film Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000941 radioactive substance Substances 0.000 description 1
- 239000000985 reactive dye Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 230000005619 thermoelectricity Effects 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 150000003583 thiosemicarbazides Chemical class 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
- C07D491/10—Spiro-condensed systems
- C07D491/107—Spiro-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
-
- 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
- C09B57/00—Other synthetic dyes of known constitution
-
- 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
- G01N21/6486—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
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1014—Carbocyclic compounds bridged by heteroatoms, e.g. N, P, Si or B
-
- 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
- C09K2211/1059—Heterocyclic compounds characterised by ligands containing three 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
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Materials Engineering (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of probe dye and its preparation method and application of the efficient unicellular metamorphosis of tracing detection, the structural formula of the probe dye isThe unicellular metamorphosis polluted by mercury ion applied to tracing detection.The present invention has good tracking effect to the unicellular metamorphosis because of caused by ion concentration of mercury；Product form is solid powder, is easy to use storage, and synthetic method is simple, high income, at low cost, and application prospect is good.
Description
Technical field
The invention belongs to the unicellular field of tracing detection, more particularly to a kind of unicellular metamorphosis of efficient tracing detection
Probe dye and its preparation method and application.
Background technology
Heavy metal Hg is the substance for constituting the earth's crust, is distributed in nature very extensive.It is in each portion of natural environment
Point there is minimum content, will be to water body if its content is above standard, soil, human body cause immeasurable pollution with
Harm.The content of normal mercury is less than 0.01mg/L in human body, is the slow poisoning of mercury during 0.01mg/L-0.5mg/L, with
The content for cylinder accumulation mercury increases, and incidence can accelerate, and when reaching 0.5mg/L, above concentration is for acute poisoning, even
People can will be allowed to lose life within short a few houres.And accumulation of the slow poisoning with mercury ion in vivo, it can bring
Corresponding disease canceration etc..
Mercury element in environment, which enters human body, to be realized by the effect of food chain, mercury element of the people Jing Guo food intake
The mercury in soils constituent content with its location has a direct relation to content in blood, and the increase of mercury element content can cause it is a series of
Cell carcinogenesis, the content of especially liver cancer, nasopharyngeal carcinoma, breast cancer, leukaemia death rate of the onset and mercury element has direct relation,
And content is higher, and incidence is faster.And cancer is since the variation of human inner cell, the canceration of cell is to connect for a long time
Radioactive substance, or contact chemical substance are touched, or the certain viruses of infection are possible to that normal cell is made to be changed into cancer cell.Just
The canceration of normal cell one of maximum feature cancer cell form and normal morphology structure are changed, for example, normally at
Fibrocyte is in flat fusiformis, after this cell carcinogenesis, will become spherical.This variation is gradual, is normal
The form of cell slowly becomes cancer cell.
Chinese patent " 201110211911.5 " in the multiprobe of Single cell analysis for reporting：It is surveyed using temperature
Amount, film potential measure and ion channel detection, pH detections, ion concentration detection.The probe includes intracellular temperature-measuring thin film thermoelectricity
Three microelectrodes of even summation, described three microelectrodes are：Film potential measures and ion channel detects microelectrode, and pH value measures micro- electricity
Pole, ion concentration detect microelectrode；Three microelectrodes are cone, and the common measurement of institute's parameter may be implemented, help
In the structure and properties of preferably research cell level.
Chinese patent " 201310052402.1 " is based on the Single cell analysis instrument of nano optical fibers probe and its probe manufacturing side
Report a kind of Single cell analysis instrument based on nano optical fibers probe in method, including nano-probe, light source unit, micro- behaviour's system,
Point location detection unit, cellular localization system and photon detection unit, nano-probe innermost layer is optical fiber layer, in optical fiber layer outer wall packet
It is wrapped with nano-rings electrode layer, insulating layer is enclosed in its outer wall.The present invention has high sensitivity, and unicellular rank may be implemented
Detection, compared to traditional detection means for needing to crush ten hundreds of cells, the cell quantity needed greatly reduces, and improves
The success rate of getting up early disease detection.
A kind of point bit-type microelectrode sensors can be used for Single cell analysis of Chinese patent " 201710700839.X " and its
Using, the present invention relates to electrode sensor, specifically a kind of current potential microelectrode sensor can be used for Single cell analysis
And its application.Electric potential type microelectrode sensors are polymer film ion-selective microelectrode, by carbon fiber microelectrodes with micro pipette tips, PEDOT
Pass layer and polymer ions selective membrane composition.The invention has high sensitivity, simple for production, at low cost, is easy to minimize
The advantages that, while there is the advantages that microelectrode mass transfer rate is high, and current density is big, fast response time, while may be implemented to more
The detection of the different kinds of ions concentration variation of a variety of slender cellular surfaces under kind environmental stimulus.
Based on retrieve published patent it can be found that at present the method for Single cell analysis be all based on electrochemical probe come
It realizes, the operation of method generally existing is not simple enough, too complicated.Only the variation of the ion concentration of slender cellular surface is examined
It surveys rather than the variation of cellular morphology, but the most basic feature of the origin of the diseases such as cancer is the form with normal cell
It varies widely, just very faint variation will occur for the form of normal cell at first for morbidity.So to unicellular
Metamorphosis be detected be it is highly important, can be with the occurrence and development of predicted treatment disease.
Invention content
Technical problem to be solved by the invention is to provide a kind of spies of the dyestuff of efficient unicellular metamorphosis of tracing detection
Needle and its preparation method and application, the probe dye have the unicellular metamorphosis because of caused by ion concentration of mercury good
Tracking effect；Product form is solid powder, is easy to use storage, and synthetic method is simple, high income, at low cost, application prospect
Well.
The present invention provides a kind of probe dye of the efficient unicellular metamorphosis of tracing detection, the knots of the probe dye
Structure formula is
The present invention also provides a kind of preparation method of the probe dye of the efficient unicellular metamorphosis of tracing detection, packets
It includes：
(1) it takes para aminobenzoyl hydrazine to be dissolved in solvent, 40-50 DEG C of stirring and dissolving is heated under inert gas shielding and drips
Add phenyl isothiocyanate, flow back 1-2h, cooling, and recrystallization obtains intermediate；Wherein, para aminobenzoyl hydrazine and isothiocyanic acid
The molar ratio of phenyl ester is 1.0:1.0~1.1；
(2) intermediate that step (1) obtains is dissolved in solvent and obtains solution, temperature control is in 0-5 DEG C in ice bath；
Cyanuric Chloride is dissolved, the pH value of reaction system is controlled in 6.5-7.5, and 0-5 DEG C is stirred to react 5-7h, obtains mixed liquor；It will be glimmering
Light element hydrazides dissolves, and is warming up to 60-70 DEG C of stirring 1-1.5h, continues the pH value for controlling reaction system, after reacting 2-4h, filters,
Rotary evaporation, column chromatography for separation, recrystallization obtain required product；Wherein, intermediate, Cyanuric Chloride and fluorescein hydrazides rub
You are than being 1.0:1.0~1.1:1.0~1.1.
The solvent of para aminobenzoyl hydrazine is ethyl alcohol in the step (1)；Inert gas is nitrogen.
The solvent of intermediate is anhydrous tetrahydro furan in the step (2)；The solvent of Cyanuric Chloride is acetone；Fluorescein acyl
The solvent of hydrazine is anhydrous tetrahydro furan.
NaHCO is used in the step (2)3Solution controls pH value of reaction system.
Recrystallization in the step (1) and (2) is recrystallized using absolute ethyl alcohol.
The solvent that column chromatography for separation in the step (2) uses is volume ratio 28:1-32:1 chloroform and ethyl alcohol.
The present invention also provides a kind of application of the probe dye of the efficient unicellular metamorphosis of tracing detection, the dyestuffs
The application process of probe is as follows：
(1) probe dye is dissolved in solvent, is configured to 0.9 × 10-2M-1.1×10-2The probe dye stock solution of M；Claim
Mercury ion salt is taken to be dissolved in solvent, it is configured to mercury ion solution；
(2) it takes the probe dye stock solution in step (1) to dilute, adds BSA solution, it is molten to obtain probe dye for constant volume
Liquid；After cell culture is good, the mercury ion solution of various concentration is added in different orifice plates, after cultivating 4-6h, dyestuff is added and visits
Needle hydroponics 20-30min carries out inverted fluorescence microscope imaging, you can carries out Follow-up observation.
Solvent in the step (1) is absolute ethyl alcohol；Mercury ion salt is mercuric perchlorate.
A concentration of 0.9g/L-1.1g/L of BSA solution in the step (2)；Addition is to be intended that with concentration probe liquid
The 1.6%-1.8% of volume.
It is 100uL that the volume of the mercury ion solution of various concentration is added in the step (2).
It is added in the step (2) after probe dye hydroponics 20-30min and carries out cleaning 3-5 with PBS buffer solutions
It is secondary.
The principle of the present invention is as follows：
It uses Cyanuric Chloride as bridged group, thiocarbamide and fluorescein hydrazides is chained up, mercury ion and acyl in fluorescein
S-O keys complexing in the N-O keys and thiosemicarbazides of amine, leads to lactams open loop to make Fluorescence Increasing, forms " on-off " type
Probe dye.Then the probe dye is dissolved in ethyl alcohol and configures probe solution, it is same to configure mercury ion solution, profit after complexing
The fluorescence intensity for adjusting complex system is added dropwise with BSA (when bovine serum albumin is added in probe molecule, since protein is double
Hydrogen bond inducing action in helical structure enables probe molecule is regular to be arranged in the double-spiral structure of protein, aggregation
The effect of induced luminescence.Since protein has the ability of adion, so the two synergistic effect can speed probe molecule
To the response time of ion, and its fluorescence intensity can be improved, therefore after mercury ion enters intracellular, it can be with using such method
The variation of good detecting and tracking cell form caused by ion concentration of mercury difference), determine best ethyl alcohol and BSA volume ratios.
The present invention can be to be added the amount of various concentration same volume mercury ion, come degree of realization unicellular metamorphosis when applied culture cell
Tracking imaging.
Preparation route is as follows：
Advantageous effect
The present invention has good tracking effect to the unicellular metamorphosis because of caused by ion concentration of mercury；Product form
For solid powder, it is easy to use storage, synthetic method is simple, high income, at low cost, and application prospect is good.
Description of the drawings
Fig. 1 is that for probe dye to the Toxic test results of cell, what abscissa represented is that the dyestuff being added is visited in embodiment 3
The concentration of needle, ordinate are the survival rate of cell.
Fig. 2 be in embodiment 4 probe dye to the selectivity test of metal ion as a result, abscissa is the wave of fluorescence spectrum
Long, ordinate is fluorescence intensity.
Fig. 3 is influence of the coexisting ion to the fluorescence intensity of mercury ion probe dye system in embodiment 5；Abscissa is gold
Belong to ionic species, ordinate is fluorescence intensity；Black bar is the fluorescence intensity after probe and complexing of metal ion, gray bars
For probe dye/Hg2+The fluorescence intensity after different metal ions is added in system.
Fig. 4 a-d are for cell in embodiment 6 as ion concentration of mercury difference leads to the fluorogram of metamorphosis.
Specific implementation mode
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, people in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Embodiment 1
The synthesis of 2- benzyls-N- (phenyl) thiosemicarbazide
1.6g (10.6mmol) para aminobenzoyl hydrazine is taken to be dissolved in 30mL ethyl alcohol, heating stirring dissolves simultaneously under nitrogen protection
0.47mL (11.0mmol) phenyl isothiocyanate is slowly added dropwise.Reflux 1.5h is in yellow solution to solution, and mixture is cooled to
A large amount of white solids are precipitated in room temperature.Absolute ethyl alcohol repeated recrystallize can obtain sterling, and suction filtration obtains white powdery solids, obtains
Intermediate probe, yield 53%.mp:136-137℃.FTIR(KBr)：V=3440cm-1,3296cm-1(-NH2),2069cm-1
(C=S), 1541cm-1(- NH), 1609cm (C=O)1H NMR(600MHz,DMSO,298K,δ/ppm):3.84(s,-NH2,
2H),7.92-7.71(m,ArH,9H),10.38-10.35(d,-NH,3H).13C NMR(600MHz,DMSO,298K,δ/ppm):
δ179.7,166.2,144.4,131.6,130.2,125.1,130.2,125.1,122.9,113.1,52.3,51.6.Element point
Analyse (C14H14N4SO)：Theoretical value：C, 58.72%；H, 4.93%；N, 19.57%；Experiment value：C, 57.44%；H, 4.33%；N,
18.93%.
Embodiment 2
The synthesis of probe dye
1.144g (4.0mmol) intermediate probe is dissolved in 20mL tetrahydrofurans, so that temperature is reached 0-5 DEG C in ice bath,
Then it uses 15mL acetone to dissolve 99% Cyanuric Chlorides of 0.730g (4.0mmol), shift in constant pressure funnel and is slowly added dropwise
Into three-necked flask, after stirring 1h, with dropping funel by 0.330g (4.0mmol) NaHCO3Solution is added dropwise, and prevents solution
Become acid (pH value of reaction system is controlled in 6.5-7.5).Mixed liquor stirs at 0-5 DEG C, reacts 6h.It will with the THF of 10ml
1.396g (4.0mmol) fluorescein hydrazides dissolves, and pours into constant pressure funnel, and be slowly dropped in three-necked flask, heats up
To 65 DEG C, 1h is stirred, with constant pressure funnel by 0.34g (4mmol) NaHCO3Solution is added dropwise.After reacting 3h, it will obtain
Solidliquid mixture filtered, and the filtrate that rotary evaporation is collected into, through silica gel column chromatography separation (chloroform/ethyl alcohol=
30:1) target product is used in combination ethyl alcohol recrystallization to obtain pale yellow powder solid, yield 62%.mp:149-150℃.FTIR
(KBr):V=3441cm-1(OH),3123cm-1(-NH-),2085cm-1(C=S), 1708cm-1(C=O), 1607cm-1,
1543cm-1(Ar-H),1499cm-1,1435cm-1,1283cm-1(triazine)1H NMR(600MHz,DMSO,298K,δ/ppm):δ
=3.82 (s ,-NH-, 1H), 6.44 (m, ArH, 2H), 6.55 (m, ArH, 2H), 6.61 (m, ArH, 2H), 7.22 (m, ArH,
4H),7.51-7.94(m,ArH,10H),9.99(s,-OH,2H),10.45(s,-NH-,1H),10.91(s,-NH-,1H),
11.11(s,-NH-,1H).13C NMR(600MHz,DMSO,298K,δ/ppm):δ=102.55,123.66,124.75,
129.14,129.85,132.02,134.57,150.21,153.14,159.32,163.67,168.06,169.52,170.23
(C=S), 66.89 (season C)；Elemental analysis:(C37H26ClN9O5S):Theoretical value:C, 59.72%；H, 3.52%；N, 16.94%.
Experiment value:C, 60.90%；H, 3.62%；N, 16.25%.
Embodiment 3
The toxotest of probe dye
The test (Fig. 1) of cytotoxicity is carried out using mtt assay.Concrete operations are as follows：
1. cell suspending liquid is made by suitable culture medium is added by the cell of digestion, it is planted in cell orifice plate, makes
Cell density in each aperture is consistent 5 × 10 substantially5It is a, aperture is encoded according to the scheme made, edge
It is filled with sterile PBS in hole.
2. being put in 37 DEG C, 5% CO212h or more is trained in incubator in advance, is taken out after cell is completely adherent.With primary
Property suction pipe exhaustion orifice plate in culture medium, PBS cleaning orifices repeatedly are used in combination, under the conditions of being protected from light be added concentration gradient probe
(concentration is followed successively by 0M, 10-8M、10-7M、10-6M、10-5M and 10-4M), each gradient adds 5 holes.40 μ are added in every hole again
The culture medium of LMTT and same concentrations are positioned over 37 DEG C, 5% CO24h is cultivated in incubator.
3. taking out cell orifice plate, equally the solution in orifice plate is exhausted under the conditions of being protected from light, in 400 μ L of each aerial addition
DMSO, rock 30min in 37 DEG C of constant-temperature tables；
4. the solution in each hole is taken out successively, it is placed in 96 orifice plates, each of which hole is surveyed at 490nm with microplate reader
OD values.Measurement result is calculated the probe dye through processing and complies fully with primary standard to the toxicity of cell.
Embodiment 4
Selectivity of the probe dye to ion in ethyl alcohol/BSA
In volume ratio 85:In 15 ethyl alcohol and BSA solution systems, measures the probe dye and configuration metal ions Zn is being added2+,
Ca2+,Cd2+,Sn2+,Mg2+,Fe3+,Co2+,Ni2+,Ba2+,Pb2+,Al3+,Cr3+,Mn2+,Cu2+,Hg2+Front and back fluorescence spectrum.
Step 1：The probe dye that embodiment 2 synthesizes is dissolved in ethyl alcohol and BSA systems, constant volume obtains 1.0 × 10-3M's
Probe solution.
Step 2：Lead salt, molysite, cadmium salt, zinc salt, magnesium salts, chromic salts, calcium salt, barium salt, sodium salt, manganese salt, mercury salt are dissolved in second
In alcoholic solvent, using solvent in 100ml volumetric flasks constant volume, obtain a concentration of 1.0 × 10-3Each metal ion solution of M.
Step 3：0.1ml a concentration of 1.0 × 10 is pipetted respectively-3Each metal ion storing solution of M is added in 1ml steps 1 and obtains
The probe storing solution arrived, using etoh solvent, constant volume detects its fluorescence intensity after standing 2min in 10ml volumetric flasks.
Find that the probe dye has good response in fluorescence spectrum to lead ion by experimental result (Fig. 2).
Embodiment 5
Ethyl alcohol/BSA intermediate ion interference test
Biological related coexisting ion is probed into probe dye/Hg2+Ethyl alcohol/BSA (85：15) solution fluorescence at 530nm
The influence of intensity.Wherein, concentration：10 μM (probe dyes), 100 μM of (Hg2+), 100 μM (other ions), black bar is molten
Different metal ions are added in agent system, gray bars are in probe dye-Hg2+Different metal ions are added in system.
Step 1：The reactive dye fluorescence probe that embodiment 2 synthesizes is dissolved in etoh solvent/BSA (85/15, v/v), profit
With solvent in 100ml volumetric flasks constant volume, obtain a concentration of 1.0 × 10-3The probe storing solution of M；
Step 2：Lead salt, molysite, cadmium salt, zinc salt, magnesium salts, chromic salts, calcium salt, barium salt, sodium salt, manganese salt, mercury salt are dissolved in molten
In agent ethyl alcohol, using etoh solvent in 100ml volumetric flasks constant volume, obtain a concentration of 1.0 × 10-2Each metal ion deposit of M
Liquid；
Step 3：Pipette 0.1ml a concentration of 1.0 × 10-2The probe obtained in 1ml steps 1 is added in the mercury ion storing solution of M
Storing solution, using etoh solvent, constant volume detects its photoluminescence spectrum intensity after standing 2min in 10ml volumetric flasks；
Step 4：Pipette 0.1ml a concentration of 1.0 × 10-2The probe obtained in 1ml steps 1 is added in the mercury ion storing solution of M
Storing solution is separately separately added into 0.1ml a concentration of 1.0 × 10-2Each metal ion storing solution of M is held using etoh solvent in 10ml
Constant volume in measuring bottle detects its photoluminescence spectrum intensity after standing 2min；
As can be seen from Fig. 3, other ions influence it less, and therefore, which has good anti-interference.
Embodiment 6
Probe dye tracing detection cellular morphology changes
It is made 5 × 10 by suitable culture medium is added by the cell of digestion4The cell suspending liquid of a/ml, extraction is wherein
400 μ L suspension, be planted in cell orifice plate, be placed on 5%CO2In 37 DEG C of incubators of saturated humidity for 24 hours.Wait for that cell is complete
It is entirely adherent to be tested.
Step 1：Configuration 1.0 × 10-5The probe solution of M, solvent are ethyl alcohol/BSA (85：15) mercury ion salting liquid, is configured,
Concentration is respectively 1.0 × 10-5M, 1.0 × 10-4M, 1.0 × 10-3M。
Step 2：A, b, c and d are numbered to the aperture in orifice plate by the scheme made.It is added 100uL's in a
PBS buffer solutions are added the 1.0 × 10 of 100uL in b-5The mercury ion solution of M is added the 1.0 × 10 of 100uL in c-4M
Mercury ion solution is added the 1.0 × 10 of 100uL in d-3M mercury ion solution.Cultivate 30min.
Step 3：The total probe dye of the step 1 of 200uL is separately added into a, b, c and the d in step 2 orifice plate after culture
Solution continues to cultivate 20min.Then it is cleaned 3 times with PBS buffer solutions, carries out cell imaging.
Figure 4, it is seen that the cell in a orifice plates is normal fusiformis, and the cell in b orifice plates is also almost just
It is normal, but the most cells in c orifice plates become round by fusiformis, and to be circle cut the cell in d orifice plates has sent out
Raw apoptosis.Illustrate that the probe dye of the present invention can intuitive tracing detection cellular morphology variation.
Claims (10)
1. a kind of probe dye of the unicellular metamorphosis of efficient tracing detection, it is characterised in that：The structure of the probe dye
Formula is
2. a kind of preparation method of the probe dye of the unicellular metamorphosis of efficient tracing detection, including：
(1) it takes para aminobenzoyl hydrazine to be dissolved in solvent, 40-50 DEG C of stirring and dissolving is heated under inert gas shielding and is added dropwise different
Thiocyanic acid phenyl ester, flow back 1-2h, cooling, and recrystallization obtains intermediate；Wherein, para aminobenzoyl hydrazine and phenyl isothiocyanate
Molar ratio be 1：1.0~1.1；
(2) intermediate that step (1) obtains is dissolved in solvent and obtains solution, temperature control is in 0-5 DEG C in ice bath；By three
Polychlorostyrene cyanogen dissolves, and the pH value of reaction system is controlled in 6.5-7.5, and 0-5 DEG C is stirred to react 5-7h, obtains mixed liquor；By fluorescein
Hydrazides dissolves, and is warming up to 60-70 DEG C of stirring 1-1.5h, continues the pH value for controlling reaction system, after reacting 2-4h, filters, rotation
Evaporation, column chromatography for separation, recrystallization obtain required product；Wherein, the molar ratio of intermediate, Cyanuric Chloride and fluorescein hydrazides
It is 1.0：1.0~1.1：1.0~1.1.
3. a kind of preparation method of the probe dye of efficient unicellular metamorphosis of tracing detection according to claim 2,
It is characterized in that：The solvent of para aminobenzoyl hydrazine is ethyl alcohol in the step (1)；Inert gas is nitrogen.
4. a kind of preparation method of the probe dye of efficient unicellular metamorphosis of tracing detection according to claim 2,
It is characterized in that：The solvent of intermediate is anhydrous tetrahydro furan in the step (2)；The solvent of Cyanuric Chloride is acetone；Fluorescence
The solvent of plain hydrazides is anhydrous tetrahydro furan.
5. a kind of preparation method of the probe dye of efficient unicellular metamorphosis of tracing detection according to claim 2,
It is characterized in that：NaHCO is used in the step (2)3Solution controls pH value of reaction system 6.5~7.5.
6. a kind of preparation method of the probe dye of efficient unicellular metamorphosis of tracing detection according to claim 2,
It is characterized in that：Recrystallization in the step (1) and (2) is recrystallized using absolute ethyl alcohol.
7. a kind of preparation method of the probe dye of efficient unicellular metamorphosis of tracing detection according to claim 2,
It is characterized in that：The solvent that column chromatography for separation in the step (2) uses is volume ratio 28:1-32:1 chloroform and second
Alcohol.
8. a kind of application of the efficiently probe dye of the unicellular metamorphosis of tracing detection as described in claim 1, feature
It is：The application process of the probe dye is as follows：
(1) probe dye is dissolved in solvent, is configured to 0.9 × 10-2M-1.1×10-2The probe dye stock solution of M；Weigh mercury
Ion salt is dissolved in solvent, it is configured to mercury ion solution；
(2) it takes the probe dye stock solution in step (1) to dilute, adds BSA solution, constant volume obtains probe dye solution；Carefully
After born of the same parents cultivate, the mercury ion solution of various concentration is added in different orifice plates, after cultivating 4-6h, probe dye solution is added
20-30min is cultivated, inverted fluorescence microscope imaging is carried out, you can carries out Follow-up observation.
9. a kind of application of the probe dye of efficient unicellular metamorphosis of tracing detection according to claim 8, special
Sign is：Solvent in the step (1) is absolute ethyl alcohol；Mercury ion salt is mercuric perchlorate.
10. a kind of application of the probe dye of efficient unicellular metamorphosis of tracing detection according to claim 8, special
Sign is：A concentration of 0.9g/L-1.1g/L of BSA solution in the step (2)；Addition is to use concentration probe stock solution
The 1.6%-1.8% of volume.
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