CN111285833A - Detection ONOO-Ratiometric fluorescent molecular probe and preparation method and application thereof - Google Patents
Detection ONOO-Ratiometric fluorescent molecular probe and preparation method and application thereof Download PDFInfo
- Publication number
- CN111285833A CN111285833A CN202010089409.0A CN202010089409A CN111285833A CN 111285833 A CN111285833 A CN 111285833A CN 202010089409 A CN202010089409 A CN 202010089409A CN 111285833 A CN111285833 A CN 111285833A
- Authority
- CN
- China
- Prior art keywords
- onoo
- concentration
- cells
- probe
- solution
- 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.)
- Pending
Links
- 239000003068 molecular probe Substances 0.000 title claims abstract description 48
- 238000001514 detection method Methods 0.000 title claims description 34
- 238000002360 preparation method Methods 0.000 title claims description 17
- 239000000523 sample Substances 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 19
- 238000003384 imaging method Methods 0.000 claims abstract description 12
- CRTCYKPYJMUQHL-UHFFFAOYSA-N 2-[6-(diethylamino)-4-[(4-methylsulfanylphenyl)methylidene]-2,3-dihydro-1H-anthracen-9-yl]benzoic acid Chemical compound C(=O)(O)C1=C(C=CC=C1)C=1C2=CC=C(C=C2C=C2C(CCCC=12)=CC1=CC=C(C=C1)SC)N(CC)CC CRTCYKPYJMUQHL-UHFFFAOYSA-N 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 56
- 239000000243 solution Substances 0.000 claims description 33
- 238000012258 culturing Methods 0.000 claims description 32
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 30
- JHIVVAPYMSGYDF-UHFFFAOYSA-N cyclohexanone Chemical compound O=C1CCCCC1 JHIVVAPYMSGYDF-UHFFFAOYSA-N 0.000 claims description 24
- 239000001963 growth medium Substances 0.000 claims description 24
- 239000007853 buffer solution Substances 0.000 claims description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 15
- 239000002953 phosphate buffered saline Substances 0.000 claims description 15
- 239000012086 standard solution Substances 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 239000002158 endotoxin Substances 0.000 claims description 14
- 229920006008 lipopolysaccharide Polymers 0.000 claims description 14
- PHEDXBVPIONUQT-RGYGYFBISA-N phorbol 13-acetate 12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(OC(C)=O)C1(C)C PHEDXBVPIONUQT-RGYGYFBISA-N 0.000 claims description 14
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 10
- QRVYABWJVXXOTN-UHFFFAOYSA-N 4-methylsulfanylbenzaldehyde Chemical compound CSC1=CC=C(C=O)C=C1 QRVYABWJVXXOTN-UHFFFAOYSA-N 0.000 claims description 9
- 235000013311 vegetables Nutrition 0.000 claims description 9
- 239000013067 intermediate product Substances 0.000 claims description 8
- 230000035484 reaction time Effects 0.000 claims description 8
- BTPSFFBUMTZNAS-UHFFFAOYSA-N 2-[6-(diethylamino)-1,2,3,4-tetrahydroanthracen-9-yl]benzoic acid Chemical compound C(=O)(O)C1=C(C=CC=C1)C=1C2=CC=C(C=C2C=C2CCCCC12)N(CC)CC BTPSFFBUMTZNAS-UHFFFAOYSA-N 0.000 claims description 6
- 239000005711 Benzoic acid Substances 0.000 claims description 6
- 241000283690 Bos taurus Species 0.000 claims description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 6
- 235000010233 benzoic acid Nutrition 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 238000000799 fluorescence microscopy Methods 0.000 claims description 6
- 239000005457 ice water Substances 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 6
- 210000002540 macrophage Anatomy 0.000 claims description 6
- 210000001161 mammalian embryo Anatomy 0.000 claims description 6
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims description 6
- 210000002966 serum Anatomy 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000004113 cell culture Methods 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 5
- 230000005284 excitation Effects 0.000 claims description 5
- 235000021110 pickles Nutrition 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 230000008859 change Effects 0.000 claims description 4
- 230000003834 intracellular effect Effects 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- FQNKTJPBXAZUGC-UHFFFAOYSA-N 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid Chemical compound OC1=CC(N(CC)CC)=CC=C1C(=O)C1=CC=CC=C1C(O)=O FQNKTJPBXAZUGC-UHFFFAOYSA-N 0.000 claims description 3
- 239000003086 colorant Substances 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000000227 grinding Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000002504 physiological saline solution Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 238000000527 sonication Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000009210 therapy by ultrasound Methods 0.000 claims description 3
- 230000001413 cellular effect Effects 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 2
- 238000011068 loading method Methods 0.000 claims description 2
- 239000002644 phorbol ester Substances 0.000 claims description 2
- 239000007850 fluorescent dye Substances 0.000 abstract description 7
- 239000012472 biological sample Substances 0.000 abstract description 4
- 201000010099 disease Diseases 0.000 abstract description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 2
- 238000013399 early diagnosis Methods 0.000 abstract description 2
- 238000011503 in vivo imaging Methods 0.000 abstract description 2
- 238000006862 quantum yield reaction Methods 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 230000000007 visual effect Effects 0.000 abstract description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- CMFNMSMUKZHDEY-UHFFFAOYSA-M peroxynitrite Chemical compound [O-]ON=O CMFNMSMUKZHDEY-UHFFFAOYSA-M 0.000 description 6
- 150000002500 ions Chemical class 0.000 description 5
- 238000001228 spectrum Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 4
- 238000001215 fluorescent labelling Methods 0.000 description 4
- 230000003833 cell viability Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- GQPLMRYTRLFLPF-UHFFFAOYSA-N Nitrous Oxide Chemical compound [O-][N+]#N GQPLMRYTRLFLPF-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 235000002639 sodium chloride Nutrition 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- BJFTUPCKULNIPD-UHFFFAOYSA-N 4-methyl-2-(2H-tetrazol-5-yl)-1,3-thiazole Chemical compound CC=1N=C(SC=1)C1=NN=NN1 BJFTUPCKULNIPD-UHFFFAOYSA-N 0.000 description 1
- VCPWRJDXHZYSKV-UHFFFAOYSA-N C(=O)(O)C1=C(C=CC=C1)C1C2=CC=C(C=C2OC=2CCCCC1=2)N(CC)CC Chemical compound C(=O)(O)C1=C(C=CC=C1)C1C2=CC=C(C=C2OC=2CCCCC1=2)N(CC)CC VCPWRJDXHZYSKV-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 238000003570 cell viability assay Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001120 cytoprotective effect Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000001272 nitrous oxide Substances 0.000 description 1
- 238000012634 optical imaging Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 238000000954 titration curve Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/78—Ring systems having three or more relevant rings
- C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
- C07D311/82—Xanthenes
-
- 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
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
Landscapes
- 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)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention aims to provide a method for detecting ONOO‑The prepared molecular probe can be applied to the ratio type probe molecule for detecting a two-photon biological sample, 9- (2-carboxyphenyl) -6 (diethylamino) -4 (4-methylthio-benzylidene) -1,2,3, 4-tetrahydroanthracene is used as a fluorescent group, activated sulfydryl is used as a reactive group, and a fluorescent probe F510Has ultrahigh sensitivity, selectivity, light stability and non-invasive performance, and can realize visual observation of ONOO‑. The biological sample imaging is slightly influenced by external conditions, and the result is accurate. The probe also has the advantages of high fluorescence quantum yield, obvious phenomenon, easy observation and high accuracy, and the equipment is convenient to operate and strong in implementabilityIs suitable for in vivo imaging of living organisms and is expected to be used for early diagnosis of diseases.
Description
Technical Field
The invention belongs to the field of fluorescent probe detection, and particularly relates to a method for detecting ONOO-The ratio type fluorescent molecular probe and the preparation method and the application thereof.
Background
Nitrous oxide ion, abbreviated as ONOO-Their reaction with biomolecules can lead to cytotoxic and cytoprotective events, induction of cardiovascular and neurological diseases, cancer and aging, and the like. Excessive ONOO due to the difficulty of directly and accurately quantifying its content in vivo-Early lesions that are caused in the body are not readily detectable. Optical imaging has become the ONOO in living beings in recent years-The method is a powerful non-invasive method, but the ion-like detection is difficult, and an effective molecular probe is needed for detection.
Disclosure of Invention
Based on the existing problems, the invention aims to provide a method for detecting ONOO-The prepared molecular probe can be applied to the detection ratio type probe molecules of biological samples, 9- (2-carboxyphenyl) -6 (diethylamino) -4 (4-methylthio benzylidene) -1,2,3, 4-tetrahydroanthracene is used as a fluorescent group, activated sulfydryl is used as a reactive group, and a fluorescent probe F510Has ultrahigh sensitivity, selectivity, light stability and non-invasive performance, and can realize visual observation of ONOO-. The biological sample imaging is slightly influenced by external conditions, and the result is accurate. The probe also has the advantages of high fluorescence quantum yield, obvious phenomenon, easy observation and high accuracy, the equipment is convenient to operate and strong in implementability, is suitable for in vivo imaging of organisms, and is expected to be used for early diagnosis of diseases.
The technical scheme of the invention is as follows:
detection ONOO-The ratiometric fluorescent molecular probe takes 9- (2-carboxyphenyl) -6 (diethylamino) -4 (4-methylthio-benzylidene) -1,2,3, 4-tetrahydroanthracene as a fluorescent group and activated sulfydryl as a reactive group(ii) a The molecular probe is preferably 9- (2-carboxyphenyl) -6 (diethylamino) -1,2,3, 4-tetrahydroanthracene, and the chemical structural formula is as follows:
the invention also provides the ONOO detection method-The preparation method of the ratiometric fluorescent molecular probe comprises the following steps: preferably comprising the steps of:
1) placing concentrated sulfuric acid into a container, adding cyclohexanone, and finally adding 2- [4- (diethylamino) -2-hydroxybenzoyl]Benzoic acid reacts at the temperature of 70-90 ℃, after the reaction is finished, the solution is cooled to room temperature, poured into ice water and stirred evenly, and then perchloric acid is dripped into the mixed liquid to separate out solid, thus obtaining an intermediate product F376；
2) Intermediate product F376Dissolving with anhydrous ethanol, adding 4- (methyl mercapto) benzaldehyde, stirring and refluxing at 60-90 deg.C, cooling to room temperature, spin drying the filtrate, and separating with chromatography column to obtain target product F510。
The above detection ONOO-The preparation method of the fluorescent molecular probe comprises the following specific preferred processes:
in step (1)
The molar ratio of 2- [ (4-diethylamino) -2-hydroxybenzoyl ] benzoic acid to cyclohexanone is preferably 1:2,
the reaction time is preferably 2 hours,
the loading sequence is preferably: adding concentrated sulfuric acid, stirring in ice-water bath for 5 min, adding cyclohexanone, stirring at 0 deg.C for 5 min, and adding 2- [ (4-diethylamino) -2-hydroxybenzoyl ] benzoic acid at 70-90 deg.C.
In the step (2), the step (c),
the molar ratio of intermediate 9- (2-carboxyphenyl) -6- (diethylamino) -1,2,3, 4-tetrahydroanthracene to cyclohexanone to 4- (methylmercapto) benzaldehyde is preferably 1:2, the molar number of intermediate 1, and the molar number of 4- (methylmercapto) benzaldehyde 2.
Preferably, the temperature is increased to (60-90) DEG C and the reaction is stirred and refluxed for 30 min.
The invention also provides a pair ONOO-Use of responsive ratiometric fluorescent molecular probes as described above, in particular for the detection of ONOO-Total content of, detecting exogenous ONOO in cells-Concentration or cellular endogenous ONOO-Concentration and cell ONOO-And (4) fluorescence imaging.
Specifically, the endogenous concentration is induced by LPS + PMA drug, and the cells generate ONOO by themselves-Exogenous is the external addition of ONOO-Finally absorbed by cells, and fluorescence imaging is carried out on the ONOO in the cells-And (6) imaging.
Preferably, molecular probes F are established510And ONOO-The linear working curve step comprises:
(1) phosphate buffered saline (PBS buffer for short) with pH 7.40 and concentration of 10mM is prepared, and ONOO with concentration of 1mM is prepared-H of (A) to (B)2O Standard solution and Probe F at a concentration of 1mM510A DMSO solution of (1);
(2) respectively collecting 2.5 μ L, 5 μ L, 7.5 μ L, 10 μ L, 12.5 μ L, 15 μ L, 17.5 μ L, 20 μ L, 22.5 μ L and 25 μ L of ONOO with concentration of 1mM-The standard solution (2) was added to each fluorescence cuvette in a total of 10 parts, 100. mu.L of a 10mM PBS buffer solution was added, and 100. mu.L of a 0.05mM probe F was added510Finally, respectively adding physiological saline to a constant volume of 1mL, and uniformly mixing;
(3) measuring fluorescence intensity with a fluorescence spectrophotometer to obtain fluorescence intensity ratio, preferably detecting ONOO-The excitation wavelength of the fluorescence intensity is 500nm, and the reaction time is 30 min;
(4) by ONOO-The concentration of (A) is the abscissa and the fluorescence intensity ratio is the ordinate, obtaining the probe molecule pair ONOO-Linear equation of concentration and fluorescence intensity ratio.
Preferably, the fluorescent molecular probe is applied to detect ONOO-The effect judgment indexes are as follows:
detecting speed: the reaction is carried out immediately, and the reaction time is 0-10 seconds, and more preferably within 3 seconds;
color change: the appearance of the fluorescent lamp is changed from purple to none; the color changed from red to green under the ultraviolet lamp.
Peroxynitrite is a common salt for low-quality vendors to pickle vegetables, and can be decomposed into ONOO by microbial decomposition-If people take the salted vegetable pickled by peroxynitrite, the peroxynitrite can be induced in vivo, so the ONOO in the salted vegetable can be induced-Is necessary.
In some embodiments of the invention, fluorescent molecular probe F is used510The step of detecting the content of ONOO-in the salted vegetables preferably comprises:
(1) drying and grinding a sample to be detected, placing (500-700mg) into a container, adding deionized water, and performing ultrasonic treatment for 10 min; (2) two containers were used as control and experimental groups, and 2. mu.L, 10mM F was added510A standard solution of (4); (3) wherein 995uL ethanol and 995uLPBS buffer solution are added into the control group, and the volume is determined to be 2 mL; (4) 990uL ethanol and 990uLPBS buffer solution were added to the experimental group, and 10uL of the sample supernatant after sonication was added; (5) detecting to obtain fluorescence spectrograms of a control group and an experimental group, and obtaining the ONOO in the pickles by looking up a linear equation according to the ratio of the two peak intensities of the experimental group-The content of (a).
In other embodiments of the invention, fluorescent molecular probes F are used510Detection of exogenous ONOO in living cells-The concentration step of (a) preferably comprises:
(1) the ONOO with the concentration of 1mM is prepared-Water, probe F at a concentration of 1mM510The DMSO standard solution of (1) g/ml lipopolysaccharide aqueous solution (LPS solution for short) and 1 g/ml phorbol ester aqueous solution (PMA solution for short);
(2) cell culture: selecting mononuclear macrophage (RAW cell for short), culturing the recovered cells, wherein the culture medium comprises 10 wt% of bovine embryo serum, 1 wt% of double antibody and 89 wt% of DMEM culture medium, and culturing at 37 deg.C and 5% vol CO2Culturing for 24h in the environment to obtain cells with good growth vigor for later use;
(3) placing living cells of RAW cells into cultureCulturing in medium, culturing 5 groups respectively, wherein the inoculum size in each group of medium is 2 × 107～9×107one/mL, incubated for 24h, and 10. mu.L of 1mM concentration probe F added510Respectively adding 0. mu.L, 5. mu.L, 10. mu.L, 15. mu.L and 20. mu.L of ONOO with a concentration of 1mM-Co-incubating the standard solution at 37 ℃ for 2 h;
(4) then, the co-cultured cells are placed in a PBS buffer solution, the PBS buffer solution is placed under confocal observation imaging, the fluorescence intensities of the light channels with different colors are collected, and the fluorescence intensity ratio is carried out, so that the exogenous ONOO related to the cells is obtained-Is a linear equation of the ratio of the concentration of (a) to the fluorescence intensity.
In some other preferred embodiments of the present invention, probe F is used510Detection of endogenous ONOO in living cells-The concentration step of (a) preferably comprises:
(1) selecting mononuclear macrophage (RAW cell for short), culturing the recovered cells, wherein the culture medium comprises 10 wt% of bovine embryo serum, 1 wt% of double antibody and 89 wt% of DMEM culture medium, and culturing at 37 deg.C and 5% vol CO2Culturing for 24h in the environment to obtain cells with good growth vigor for later use;
(2) placing living cells of RAW cells into culture medium for culturing, and culturing 4 groups respectively, wherein the inoculum size in each group of culture medium is 2 × 107～9×107Culturing for 24 hours per mL;
(3) 2 groups of the culture medium were selected, and the first group was prepared without adding LPS solution and PMA solution, and 10. mu.L of 0.5mM probe F was added510DMSO solution of (g) as control;
(4) to the second group, 100. mu.L of LPS solution at a concentration of 1. mu.g/ml, 100. mu.L of PMA solution at a concentration of 1. mu.g/ml were added to stimulate cells to produce endogenous ONOO-Then, 10. mu.L of F with a concentration of 0.5mM was added510Culturing the DMSO solution, observing and imaging under confocal condition, collecting fluorescence intensity of different color light channels, and performing fluorescence intensity ratio according to intracellular ONOO-The linear equation of the ratio of the concentration to the fluorescence intensity of the exogenous ONOO-To the ratio of the concentration to the fluorescence intensity of the cells, thereby obtaining the endogenous ONOO of the cells-The concentration of (c).
The preparation method of the invention, the adding sequence of various materials and the specific reaction steps can be adjusted by persons skilled in the art, and the preparation method is not only suitable for small-scale preparation in a laboratory, but also suitable for industrial large-scale production in a chemical plant. In the case of industrial large-scale production, the specific reaction parameters can be determined experimentally by the person skilled in the art.
The invention has the following beneficial effects:
(1) the ratio type fluorescence labeling reagent of the invention uses 9- (2-carboxyphenyl) -6 (diethylamino) -1,2,3, 4-tetrahydroxanthene as a fluorescent group and activated sulfydryl as a reaction site to enable a probe pair ONOO-Has excellent selectivity and has obvious fluorescent signal read-out.
(2) The ratio-type fluorescence labeling reagent of the invention has sensitive response to ONOO-Within seconds.
(3) The rate-type fluorescently labeled reagents of the invention have low detection limits and are comparable to ONOO compared to commercial fluorescently labeled reagents-The detection limit of (2) is 1.5nm/L, which is far lower than the content of the two substances normally existing in cells, so that the excessive ONOO can be timely found and early diagnosed-。
(4) The ratio-type fluorescent labeling reagent of the present invention can greatly reduce the influence from the external environment and instruments, etc. compared with the switch-type fluorescent labeling reagent, based on the ratio of the fluorescence intensities rather than the direct fluorescence intensity.
(5) The invention is applied to the detection of living cells, and further promotes the research of the effect of the biological micromolecules in the living body.
(6) The change of the ultraviolet color after the reaction is obvious, so that the ultraviolet color can be detected by naked eyes, and the method is convenient and quick.
(7) Due to its long wavelength in the infrared part, ONOO-And is less harmful to living organisms without excessive damage.
Drawings
FIG. 1 shows the detection of ONOO in embodiment 1 of the present invention-The preparation method of the ratiometric fluorescent molecular probe and a reaction scheme diagram.
FIG. 2 is a diagram of detecting ONOO in embodiment 1 of the present invention-Mass spectrum of the ratiometric fluorescent molecular probe of (1);
FIG. 3 shows the detection of ONOO in embodiment 1 of the present invention-Nuclear magnetic H spectrum of the ratiometric fluorescent molecular probe of (a);
FIG. 4 shows the detection of ONOO in embodiment 1 of the present invention-Nuclear magnetic C spectrum of the ratiometric fluorescent molecular probe of (a);
FIG. 5 is a flowchart of the method for detecting ONOO in embodiment 2 of the present invention-In the application of the ratiometric fluorescent molecular probe of (1): a in vitro Probe molecule F510To ONOO-Fluorescence titration curve chart of (a); b in vitro probe molecule pair ONOO-A plot of fluorescence linear equations of (a);
FIG. 6 is a flowchart of the embodiment 3 of the present invention for detecting ONOO-In the application of the ratiometric fluorescent molecular probe of (1), molecular probe F510For ONOO in pickled salted vegetables-A fluorescence response map;
FIG. 7 is a flowchart of detecting ONOO in embodiment 4 of the present invention-In the application of the ratiometric fluorescent molecular probe of (1), molecular probe F510Detecting exogenetic related cell pattern, adding 0uLONOO from left to right-,5uLONOO-,10uLONOO-,15uLONOO-,20uLONOO-。
FIG. 8 is a flowchart of the method for detecting ONOO in embodiment 5 of the present invention-In the application of the ratiometric fluorescent molecular probe, the ratio intensity of fluorescence in cells to ONOO-Linear curve of content.
FIG. 9 shows the detection of ONOO in embodiment 5 of the present invention-In the application of the ratio-type fluorescent molecular probe, the relevant cell map of ONOO-is a blank group from left to right, and an experimental group of LPS + PMA;
FIG. 10 is a diagram of the detection of ONOO in accordance with the present invention-The ratio-type fluorescent molecular probe (A) is a fluorescence spectrum chart of selectivity of various interference ions, and (B) is a bar chart of selectivity of the probe to various interference ions;
FIG. 11 is a diagram of the present invention for detecting ONOO-Histogram of ratiometric fluorescent molecular probes against cell viability.
Detailed Description
The invention is further illustrated by the following specific examples, it being understood that the preparation of the examples is illustrative only and is not intended to be limiting; on the premise of the conception of the invention, simple modification of the preparation method of the invention belongs to the protection scope of the invention.
It should also be noted that various preferred features of the method of the present invention mentioned above and various specific features in the embodiments specifically described below can be combined together, and all combinations of these features falling within the scope of the present invention, including all numerical ranges whose upper and lower limits are specifically disclosed by the present invention, and so on.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
The materials and reagents used in the following examples can be obtained commercially or synthesized from commercially available raw materials, unless otherwise specified.
The following examples were performed by HPLC using an Agilent 1100 mass spectrometry system (Agilent, USA) equipped with a degasser, quaternary pump, autosampler, and HPLC using a Hypersil GOLD C18 column (2.1 mm. times.50 mm,1.8 μm i.d., Agilent, USA). The fluorescence detection is carried out by using a Hitachi F-4600 fluorescence spectrometer for ONOO-The detection excitation wavelength is 500nm, the excitation and emission slit widths are both 10.0nm, the voltage is 400V, and the scanning speed is 2400 nm/min. Fluorescence imaging observations were performed by Olympus Fluo View FV1000 (Japan) confocal, with 40-fold objective. The separation and purification of the compound are realized by adopting a thin-layer chromatography silica gel column, wherein the filler is 300-400 meshes.
Example 1:
detection ONOO-The ratiometric fluorescent molecular probe takes 9- (2-carboxyphenyl) -6 (diethylamino) -4 (4-methylthio-benzylidene) -1,2,3, 4-tetrahydroanthracene as a fluorescent group and activated sulfydryl as a reactive group; the molecular probe is preferably 9- (2-carboxyphenyl) -6 (diethylamino) -1,2,3, 4-tetrahydroanthracene, and the chemical structural formula is as follows:
the above detection ONOO-The preparation method of the ratiometric fluorescent molecular probe comprises the following steps: the method comprises the following steps:
1) placing concentrated sulfuric acid into a container, adding cyclohexanone, and finally adding 2- [4- (diethylamino) -2-hydroxybenzoyl]Benzoic acid reacts at the temperature of 70-90 ℃, after the reaction is finished, the solution is cooled to room temperature, poured into ice water and stirred evenly, and then perchloric acid is dripped into the mixed liquid to separate out solid, thus obtaining an intermediate product F376；
2) Intermediate product F376Dissolving with anhydrous ethanol, adding 4- (methyl mercapto) benzaldehyde, stirring and refluxing at 60 deg.C, cooling to room temperature, spin drying the filtrate, and separating with chromatographic column to obtain target product F510。
The above detection ONOO-The preparation method of the fluorescent molecular probe comprises the following specific processes:
in step (1)
The mol ratio of the 2- [ (4-diethylamino) -2-hydroxybenzoyl ] benzoic acid to the cyclohexanone is 1:2,
the reaction time is 2 hours,
the sample adding sequence is as follows: adding concentrated sulfuric acid, stirring in ice-water bath for 5 min, adding cyclohexanone, stirring at 0 deg.C for 5 min, adding 2- [ (4-diethylamino) -2-hydroxybenzoyl ] benzoic acid, and reacting at 70-90 deg.C.
In the step (2), the step (c),
the molar ratio of intermediate 9- (2-carboxyphenyl) -6- (diethylamino) -1,2,3, 4-tetrahydroanthracene to cyclohexanone to 4- (methylmercapto) benzaldehyde is preferably 1:2, the number of moles of intermediate 1 and the number of moles of 4- (methylmercapto) benzaldehyde 2. Heating to 60-90 deg.C, and reflux-reacting under stirring for 30 min.
The following description is for ONOO-The application, structure and preparation method of the responsive ratiometric fluorescent molecular probe are the same as those in example 1, and the ratiometric fluorescent molecular probe is respectively applied to the detection of ONOO-Total content of, detecting exogenous ONOO in cells-Concentration or intracellular endogenous concentration andfluorescence imaging thereof.
Specifically, the endogenous concentration is induced by LPS + PMA drug, and the cells generate ONOO by themselves-Exogenous is the external addition of ONOO-Finally absorbed by cells, and fluorescence imaging is carried out on the ONOO in the cells-And (6) imaging.
Example 2:
establishment of the molecular Probe F obtained in example 1510And ONOO-The detecting step comprises:
(1) PBS buffer solution with pH 7.40 and concentration of 10mM is prepared, and ONOO with concentration of 1mM is prepared-H of (A) to (B)2O Standard solution and Probe F at a concentration of 1mM510A DMSO solution of (1);
(2) respectively collecting 2.5 μ L, 5 μ L, 7.5 μ L, 10 μ L, 12.5 μ L, 15 μ L, 17.5 μ L, 20 μ L, 22.5 μ L and 25 μ L of ONOO with concentration of 1mM-The standard solution (2) was added to each fluorescence cuvette in a total of 10 parts, 100. mu.L of a 10mM PBS buffer solution was added, and 100. mu.L of a 0.05mM probe F was added510Finally, respectively adding physiological saline to a constant volume of 1mL, and uniformly mixing;
(3) after reacting for 30min, testing fluorescence intensity by a fluorescence spectrophotometer to obtain fluorescence intensity ratio, and detecting ONOO-The excitation wavelength of the fluorescence intensity is 500 nm;
(4) by ONOO-The concentration of (A) is the abscissa and the fluorescence intensity ratio is the ordinate, obtaining the probe molecule pair ONOO-Linear equation of concentration and fluorescence intensity ratio.
(5) Mixing the sample to be tested with the buffer solution according to the volume ratio of 1:4, taking 900mL, and adding 100 mu L of F with the concentration of 0.05mM510According to exogenous ONOO-The linear equation of the ratio of the concentration to the fluorescence intensity obtains ONOO-The concentration of (c).
FIG. 5 is F510To ONOO-The fluorescence intensity and the linear equation of (1), wherein A is 0-22.5. mu.M of ONOO-The fluorescence titration chart of (A) is for ONOO-Plot of the titrated fluorescence linear equation.
When the fluorescent molecular probe is applied, the ONOO is detected-The effect judgment indexes are as follows:
detecting speed: the reaction is carried out immediately, and the reaction time is 3 seconds;
color change: the appearance of the fluorescent lamp is changed from purple to none; the color changed from red to green under the ultraviolet lamp.
Example 3:
peroxynitrite is a common salt for low-quality vendors to pickle vegetables, and can be decomposed into ONOO by microbial decomposition-If people take the salted vegetable pickled by peroxynitrite, the peroxynitrite can be induced in vivo, so the ONOO in the salted vegetable can be induced-Is necessary.
In this example, a probe F was used510The method for detecting the content of ONOO-in the salted vegetables comprises the following steps:
(1) drying and grinding a sample to be detected, putting 600mg into a container, adding deionized water, and performing ultrasonic treatment for 10 min; (2) two containers were used as control and experimental groups, and 2. mu.L, 10mM F was added510A standard solution of (4); (3) wherein 995uL ethanol and 995uLPBS buffer solution are added into the control group, and the volume is determined to be 2 mL; (4) 990uL ethanol and 990uL PBS buffer solution were added to the experimental group, and 10uL of the sample supernatant after sonication was added; (5) detecting to obtain fluorescence spectrograms of a control group and an experimental group, and obtaining the ONOO in the pickles by looking up a linear equation according to the ratio of the two peak intensities of the experimental group-The content of (a).
The fluorescence curve is shown in FIG. 6.
Example 4:
in this example, a probe F was used510Detection of exogenous ONOO in living cells-The concentration step of (a) comprises:
(1) the ONOO with the concentration of 1mM is prepared-Water, probe F at a concentration of 1mM510The DMSO standard solution of (1), LPS solution with concentration of 1 mug/ml, PMA solution with concentration of 1 mug/ml;
(2) cell culture: selecting RAW cells, culturing the recovered cells, and packaging the culture mediumContains 10 wt% bovine embryo serum, 1 wt% diabody and 89 wt% DMEM culture medium, and 5% vol CO at 37 deg.C2Culturing for 24h in the environment to obtain cells with good growth vigor for later use;
(3) placing living cells of RAW cells into culture medium for culturing, and culturing 5 groups respectively, wherein the inoculum size in each group of culture medium is 2 × 107～9×107one/mL, incubated for 24h, and 10. mu.L of 1mM concentration probe F added510Respectively adding 0. mu.L, 5. mu.L, 10. mu.L, 15. mu.L and 20. mu.L of ONOO with a concentration of 1mM-Co-incubating the standard solution at 37 ℃ for 2 h;
(4) then, the co-cultured cells are placed in a PBS buffer solution, the PBS buffer solution is placed under confocal observation imaging, the fluorescence intensities of the light channels with different colors are collected, and the fluorescence intensity ratio is carried out, so that the exogenous ONOO related to the cells is obtained-Is a linear equation of the ratio of the concentration of (a) to the fluorescence intensity.
FIG. 7 is the exogenous addition of ONOO-FIG. 8 is a plot of fluorescence ratio intensity versus ONOO-Linear curve of the content in the cell.
Example 5:
in this example, a probe F was used510Detection of endogenous ONOO in living cells-The concentration step of (a) preferably comprises:
(1) selecting mononuclear macrophage (RAW cell for short), culturing the recovered cells, wherein the culture medium comprises 10 wt% of bovine embryo serum, 1 wt% of double antibody and 89 wt% of DMEM culture medium, and culturing at 37 deg.C and 5% vol CO2Culturing for 24h in the environment to obtain cells with good growth vigor for later use;
(2) placing living cells of RAW cells into culture medium for culturing, and culturing 4 groups respectively, wherein the inoculum size in each group of culture medium is 2 × 107～9×107Culturing for 24 hours per mL;
(3) 2 groups of the culture medium were selected, and the first group was prepared without adding LPS solution and PMA solution, and 10. mu.L of 0.5mM probe F was added510DMSO solution of (g) as control;
(4) to the second group, 100. mu.L of LPS solution at a concentration of 1. mu.g/ml, 100. mu.L of PMA solution at a concentration of 1. mu.g/ml were added to stimulate cells to produce endogenous ONOO-Then, 10. mu.L of F with a concentration of 0.5mM was added510Culturing the DMSO solution, observing and imaging under confocal condition, collecting fluorescence intensity of different color light channels, and performing fluorescence intensity ratio according to intracellular ONOO-The linear equation of the ratio of the concentration to the fluorescence intensity of the exogenous ONOO-To the ratio of the concentration to the fluorescence intensity of the cells, thereby obtaining the endogenous ONOO of the cells-The concentration of (c).
FIG. 9 shows the endogenous detection ONOO-The relevant cell map of (a).
Probe molecule F510Selective experiment of
To explore F510For ONOO-The present invention selects a representative number of ions, 1mM each of ONOO-,OH,O2 -,H2O2,HClO,GSH,Cys,Hcy,Tempo,NO3 -,NO2 -NO and1O2and preparing a Cu concentration of 30mM2+,Cu+And Fe3The ionic solution of + metal ions is described.
From FIG. 7, it can be seen that the ONOO is several times larger-Ions of concentration added to F510In 365nm ultraviolet lamp, when ONOO-The addition was found to result in disappearance of the red fluorescence and appearance of the green fluorescence.
The correlation spectrum is shown in FIG. 10, wherein A is a selective fluorescence titration spectrum showing the metal ion pair F510The selectivity influence is specifically the phenomenon of adding an interfering ion solution under a 365nm ultraviolet lamp, B is a selective columnar spectrum, and the selectivity research of some common anion pair probes is specifically the phenomenon of correspondingly adding the interfering ion solution under the 365nm ultraviolet lamp.
Cell viability assay:
cell viability experiments primary validation F510The effect of toxicity on cell life. Adding probes F with different concentrations into cell culture solution 5100M, 5M, 10M, 20M, 30M and 50M, 5% CO at 37 ℃2Was cultured in the incubator of (1) for 24 hours, followed by 25. mu.L of 4-methylthiazolyltetrazole MTT, 5 mg. multidot.mL-1And then added into the cell culture solution to be cultured for 4 hours. Results cell viability was assessed by the MTT cuvette method. To add no F510The group of cells survived 100%, with different concentrations of F510The data associated with the added experimental groups were plotted against a histogram and the results are shown in FIG. 11.
The foregoing is a description of the preferred embodiments of the present invention to enable one skilled in the art to make or use the invention, and certain modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the scope or spirit of the invention. Accordingly, the scope of the invention is not limited by the specific embodiments described above.
Claims (10)
1. Detection ONOO-The ratiometric fluorescent molecular probe is characterized in that 9- (2-carboxyphenyl) -6 (diethylamino) -4 (4-methylthio benzylidene) -1,2,3, 4-tetrahydroanthracene is used as a fluorescent group, and an activated sulfydryl group is used as a reactive group; the molecular probe is preferably 9- (2-carboxyphenyl) -6 (diethylamino) -1,2,3, 4-tetrahydroanthracene, and the chemical structural formula is as follows:
2. detection ONOO-The method for preparing a ratiometric fluorescent molecular probe of (1), wherein the fluorescent molecular probe has the structure of claim 1, and the preparation comprises the steps of:
1) placing concentrated sulfuric acid into a container, adding cyclohexanone, and finally adding 2- [4- (diethylamino) -2-hydroxybenzoyl]Benzoic acid reacts at the temperature of 70-90 ℃, after the reaction is finished, the solution is cooled to room temperature, poured into ice water and stirred evenly, and then perchloric acid is dripped into the mixed liquid to separate out solid, thus obtaining an intermediate product F376；
2) Intermediate product F376Dissolving in absolute ethanol, adding 4- (methyl mercapto) benzaldehyde, stirring at 60-90 deg.C, refluxing, and coolingCooling to room temperature, spin-drying the filtrate, and separating by chromatography column to obtain target product F510。
3. A method of detecting ONOO according to claim 2-The preparation method of the ratiometric fluorescent molecular probe is characterized by comprising the following steps: the above detection ONOO-The preparation method of the fluorescent molecular probe comprises the following specific processes:
in step (1)
The molar ratio of 2- [ (4-diethylamino) -2-hydroxybenzoyl ] benzoic acid to cyclohexanone is preferably 1:2,
the reaction time is preferably 2 hours,
the loading sequence is preferably: adding concentrated sulfuric acid, stirring for 5 min in ice-water bath, adding cyclohexanone, stirring for 5 min at 0 deg.C, and adding 2- [ (4-diethylamino) -2-hydroxybenzoyl ] benzoic acid at 70-90 deg.C;
in the step (2), the step (c),
the molar ratio of the intermediate product 9- (2-carboxyphenyl) -6- (diethylamino) -1,2,3, 4-tetrahydroanthracene to cyclohexanone to 4- (methylmercapto) benzaldehyde is preferably 1:2, the molar number of the intermediate product is 1, and the molar number of the 4- (methylmercapto) benzaldehyde is 2; preferably, the temperature is increased to (60-90) DEG C and the reaction is stirred and refluxed for 30 min.
4. To ONOO-Use of a responsive ratiometric fluorescent molecular probe, characterized by: fluorescent molecular probe having the structure of claim 1 or prepared by the method of claims 2-3 for use in the detection of ONOO-Total content of, detecting exogenous ONOO in cells-Concentration or cellular endogenous ONOO-Concentration and cell ONOO-And (4) fluorescence imaging.
5. Use according to claim 4, characterized in that: endogenous concentration is induced by LPS + PMA drug, and cells generate ONOO-Exogenous is the external addition of ONOO-Finally absorbed by cells, and fluorescence imaging is carried out on the ONOO in the cells-And (6) imaging.
6. Use according to claim 4, characterized in that: establishment of molecular Probe F510And ONOO-The linear working curve step comprises:
(1) phosphate buffered saline (pH 7.40) at a concentration of 10mM, and ONOO at a concentration of 1mM were prepared-H of (A) to (B)2O Standard solution and Probe F at a concentration of 1mM510A DMSO solution of (1);
(2) respectively collecting 2.5 μ L, 5 μ L, 7.5 μ L, 10 μ L, 12.5 μ L, 15 μ L, 17.5 μ L, 20 μ L, 22.5 μ L and 25 μ L of ONOO with concentration of 1mM-The standard solution (2) was added to each fluorescence cuvette in a total of 10 parts, 100. mu.L of a 10mM PBS buffer solution was added, and 100. mu.L of a 0.05mM probe F was added510Finally, respectively adding physiological saline to a constant volume of 1mL, and uniformly mixing;
(3) measuring fluorescence intensity with a fluorescence spectrophotometer to obtain fluorescence intensity ratio, preferably detecting ONOO-The excitation wavelength of the fluorescence intensity is 500nm, and the reaction time is 30 min;
(4) by ONOO-The concentration of (A) is the abscissa and the fluorescence intensity ratio is the ordinate, obtaining the probe molecule pair ONOO-Linear equation of concentration and fluorescence intensity ratio.
7. Use according to claim 4, characterized in that: the fluorescent molecular probe is applied to detect ONOO-The effect judgment indexes are as follows:
detecting speed: the reaction is carried out immediately, and the reaction time is 0-10 seconds, and more preferably within 3 seconds;
color change: the appearance of the fluorescent lamp is changed from purple to none; the color changed from red to green under the ultraviolet lamp.
8. Use according to any one of claims 4-7, characterized in that: using fluorescent molecular probes F510Detecting the content of ONOO-in the salted vegetables, comprising the following steps:
(1) drying and grinding the sample to be detected, taking (500-700mg) and placing the sample inAdding deionized water into a container, and performing ultrasonic treatment for 10 min; (2) two containers were used as control and experimental groups, and 2. mu.L, 10mM F was added510A standard solution of (4); (3) wherein 995uL ethanol and 995uLPBS buffer solution are added into the control group, and the volume is determined to be 2 mL; (4) 990uL ethanol and 990uL PBS buffer solution were added to the experimental group, and 10uL of the sample supernatant after sonication was added; (5) detecting to obtain fluorescence spectrograms of a control group and an experimental group, and obtaining the ONOO in the pickles by looking up a linear equation according to the ratio of the two peak intensities of the experimental group-The content of (a).
9. Use according to any one of claims 4-7, characterized in that: using fluorescent molecular probes F510Detection of exogenous ONOO in living cells-The concentration step of (a) preferably comprises:
(1) the ONOO with the concentration of 1mM is prepared-Water, probe F at a concentration of 1mM510The DMSO standard solution of (1) is lipopolysaccharide aqueous solution with the concentration of 1 mug/ml and phorbol ester aqueous solution with the concentration of 1 mug/ml; .
(2) Cell culture: selecting mononuclear macrophage, culturing the recovered cells in culture medium containing 10 wt% bovine embryo serum, 1 wt% double antibody and 89 wt% DMEM culture medium at 37 deg.C and 5% vol CO2Culturing for 24h in the environment to obtain cells with good growth vigor for later use;
(3) culturing the living cells of mononuclear macrophage in culture medium, and culturing 5 groups respectively, wherein the inoculum size in each group of culture medium is 2 × 107～9×107one/mL, incubated for 24h, and 10. mu.L of 1mM concentration probe F added510Respectively adding 0. mu.L, 5. mu.L, 10. mu.L, 15. mu.L and 20. mu.L of ONOO with a concentration of 1mM-Co-incubating the standard solution at 37 ℃ for 2 h;
(4) then, the co-cultured cells are placed in a PBS buffer solution, the PBS buffer solution is placed under confocal observation imaging, the fluorescence intensities of the light channels with different colors are collected, and the fluorescence intensity ratio is carried out, so that the exogenous ONOO related to the cells is obtained-Is a linear equation of the ratio of the concentration of (a) to the fluorescence intensity.
10. Use according to claim 9, characterized in that: using a probe F510Detection of endogenous ONOO in living cells-The concentration step of (a) preferably comprises:
(1) selecting mononuclear macrophage, culturing the recovered cells in culture medium containing 10 wt% bovine embryo serum, 1 wt% double antibody and 89 wt% DMEM culture medium at 37 deg.C and 5% vol CO2Culturing for 24h in the environment to obtain cells with good growth vigor for later use;
(2) culturing living cells of RAW cells in culture medium, and culturing 4 groups respectively, wherein the inoculum size in each group of culture medium is (2 × 10)7～9×107) Culturing for 24 hours per mL;
(3) 2 groups of the culture medium were selected, and the first group was prepared without adding LPS solution and PMA solution, and 10. mu.L of 0.5mM probe F was added510DMSO solution of (g) as control;
(4) to the second group, 100. mu.L of LPS solution at a concentration of 1. mu.g/ml, 100. mu.L of PMA solution at a concentration of 1. mu.g/ml were added to stimulate cells to produce endogenous ONOO-Then, 10. mu.L of F with a concentration of 0.5mM was added510Culturing the DMSO solution, observing and imaging under confocal condition, collecting fluorescence intensity of different color light channels, and performing fluorescence intensity ratio according to intracellular ONOO-The linear equation of the ratio of the concentration to the fluorescence intensity of the exogenous ONOO-To the ratio of the concentration to the fluorescence intensity of the cells, thereby obtaining the endogenous ONOO of the cells-The concentration of (c).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010089409.0A CN111285833A (en) | 2020-02-12 | 2020-02-12 | Detection ONOO-Ratiometric fluorescent molecular probe and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010089409.0A CN111285833A (en) | 2020-02-12 | 2020-02-12 | Detection ONOO-Ratiometric fluorescent molecular probe and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111285833A true CN111285833A (en) | 2020-06-16 |
Family
ID=71017660
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010089409.0A Pending CN111285833A (en) | 2020-02-12 | 2020-02-12 | Detection ONOO-Ratiometric fluorescent molecular probe and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111285833A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112358440A (en) * | 2020-11-10 | 2021-02-12 | 山东第一医科大学(山东省医学科学院) | ONOO-fluorescent probe, preparation method and application thereof |
| CN112457286A (en) * | 2020-12-02 | 2021-03-09 | 青岛科技大学 | Application of compound containing oxyanion in preparation of fluorescent molecular probe for detecting nitroso peroxide ion |
| CN113324984A (en) * | 2021-06-07 | 2021-08-31 | 江苏大学 | Method for detecting nitrite ions based on ratio type colorimetry |
| CN115232098A (en) * | 2022-07-18 | 2022-10-25 | 嘉兴学院 | Rhodol fluorescent probe for rapidly and sensitively detecting peroxynitrate and preparation method and application thereof |
| CN117126125A (en) * | 2023-06-21 | 2023-11-28 | 徐州医科大学 | Single-molecule fluorescent probe for ultra-high sensitivity detection of Nitric Oxide (NO), and preparation method and application thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344794A (en) * | 2010-07-30 | 2012-02-08 | 中国科学院大连化学物理研究所 | Fluorescent probe and its application in reversible detection of peroxy nitrosyl |
| CN104220438A (en) * | 2012-01-30 | 2014-12-17 | 香港大学 | Diarylamine-based fluorogenic probes for detection of peroxynitrite |
| US20170059573A1 (en) * | 2014-02-27 | 2017-03-02 | University Of Bath | Method of detecting peroxynitrite using a complex of a saccharide and an arylboronate-based fluorescent probe |
| CN106588846A (en) * | 2016-12-08 | 2017-04-26 | 曲阜师范大学 | Preparation method and application of double-rate-type multifunctional high-sensitivity florescent probe for carboxylesterase detection |
| CN108690042A (en) * | 2018-05-16 | 2018-10-23 | 山东师范大学 | It is a kind of to detect ONOO simultaneously-And H2The fluorescence probe and its synthetic method of S and application |
| CN108752377A (en) * | 2018-06-28 | 2018-11-06 | 湖南文理学院 | A kind of fluorescence probe, synthetic method and the application of detection peroxynitrite |
| CN110128435A (en) * | 2019-06-03 | 2019-08-16 | 湘潭大学 | A kind of preparation and application of peroxynitrite Ratiometric fluorescent probe |
| US20190310263A1 (en) * | 2018-04-05 | 2019-10-10 | The University Of Kansas | Fluorescent sensors of peroxynitrite targeted to the endoplasmic reticulum |
-
2020
- 2020-02-12 CN CN202010089409.0A patent/CN111285833A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344794A (en) * | 2010-07-30 | 2012-02-08 | 中国科学院大连化学物理研究所 | Fluorescent probe and its application in reversible detection of peroxy nitrosyl |
| CN104220438A (en) * | 2012-01-30 | 2014-12-17 | 香港大学 | Diarylamine-based fluorogenic probes for detection of peroxynitrite |
| US20170059573A1 (en) * | 2014-02-27 | 2017-03-02 | University Of Bath | Method of detecting peroxynitrite using a complex of a saccharide and an arylboronate-based fluorescent probe |
| CN106588846A (en) * | 2016-12-08 | 2017-04-26 | 曲阜师范大学 | Preparation method and application of double-rate-type multifunctional high-sensitivity florescent probe for carboxylesterase detection |
| US20190310263A1 (en) * | 2018-04-05 | 2019-10-10 | The University Of Kansas | Fluorescent sensors of peroxynitrite targeted to the endoplasmic reticulum |
| CN108690042A (en) * | 2018-05-16 | 2018-10-23 | 山东师范大学 | It is a kind of to detect ONOO simultaneously-And H2The fluorescence probe and its synthetic method of S and application |
| CN108752377A (en) * | 2018-06-28 | 2018-11-06 | 湖南文理学院 | A kind of fluorescence probe, synthetic method and the application of detection peroxynitrite |
| CN110128435A (en) * | 2019-06-03 | 2019-08-16 | 湘潭大学 | A kind of preparation and application of peroxynitrite Ratiometric fluorescent probe |
Non-Patent Citations (2)
| Title |
|---|
| WEI SHU等: "A mitochondria-targeted far red fluorescent probe for ratiometric imaging of endogenous peroxynitrite", 《DYES AND PIGMENTS》 * |
| XIONG-JIE ZHAO等: "A novel "turn-on" mitochondria-targeting near-infrared fluorescent probe for H2S detection and in living cells imaging", 《TALANTA》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112358440A (en) * | 2020-11-10 | 2021-02-12 | 山东第一医科大学(山东省医学科学院) | ONOO-fluorescent probe, preparation method and application thereof |
| CN112457286A (en) * | 2020-12-02 | 2021-03-09 | 青岛科技大学 | Application of compound containing oxyanion in preparation of fluorescent molecular probe for detecting nitroso peroxide ion |
| CN112457286B (en) * | 2020-12-02 | 2022-03-04 | 青岛科技大学 | Application of compound containing oxyanion in preparation of fluorescent molecular probe for detecting nitroso peroxide ion |
| CN113324984A (en) * | 2021-06-07 | 2021-08-31 | 江苏大学 | Method for detecting nitrite ions based on ratio type colorimetry |
| CN115232098A (en) * | 2022-07-18 | 2022-10-25 | 嘉兴学院 | Rhodol fluorescent probe for rapidly and sensitively detecting peroxynitrate and preparation method and application thereof |
| CN115232098B (en) * | 2022-07-18 | 2023-10-03 | 嘉兴学院 | Rhodol fluorescent probe for rapidly and sensitively detecting peroxynitrate as well as preparation method and application thereof |
| CN117126125A (en) * | 2023-06-21 | 2023-11-28 | 徐州医科大学 | Single-molecule fluorescent probe for ultra-high sensitivity detection of Nitric Oxide (NO), and preparation method and application thereof |
| CN117126125B (en) * | 2023-06-21 | 2024-03-01 | 徐州医科大学 | Single-molecule fluorescent probe for ultra-high sensitivity detection of Nitric Oxide (NO), and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111285833A (en) | Detection ONOO-Ratiometric fluorescent molecular probe and preparation method and application thereof | |
| CN109081836B (en) | Mercury ion near-infrared fluorescent probe based on hemicyanine structure and preparation method and application thereof | |
| CN111349070B (en) | Near-infrared fluorescent molecular probe for detecting biological cell viscosity and preparation method and application thereof | |
| CN108398409B (en) | Method for detecting hypochlorite by fluorescence ratio | |
| CN108003869B (en) | Fluorescent probe for detecting hypochlorite with high sensitivity and synthesis method and application thereof | |
| CN107056769A (en) | A kind of L cysteines fluorescence probe and preparation method thereof | |
| CN113999219B (en) | Double-site fluorescent probe and synthetic method and application thereof | |
| CN110229165A (en) | Up-conversion fluorescence probe Rhodamine Derivatives and its application | |
| CN112745287A (en) | Fluorescent probe HM and preparation method and application thereof | |
| CN110878085B (en) | Rapid high-selectivity hypobromous acid fluorescent probe, preparation method and application | |
| CN107286151B (en) | Carbazole-based two-photon fluorescent probe and preparation method and application thereof | |
| CN114276356B (en) | Mitochondria-targeted fluorescent probe and synthesis method and application thereof | |
| Wu et al. | A near-infrared fluorescent probe of dicyanoisophorone derivatives for selective detection and fluorescence cellular imaging of Palladium | |
| CN112794857A (en) | Preparation and application of novel fluorescent probe for high-selectivity detection of ferrous ions | |
| CN111362900A (en) | Responding to ONOO-Ratio type small molecule fluorescent probe and preparation method and application thereof | |
| CN111349071A (en) | For detecting ONOO-Xanthene derivative, and synthetic method and application thereof | |
| CN110964044B (en) | Peroxynitrite fluorescent probe based on dicoumarin derivative, preparation method and application | |
| CN110372681B (en) | Application of self-assembled nano fluorescent probe for selectively detecting human serum albumin | |
| CN110357896B (en) | Compound, preparation and application thereof in detecting divalent copper ions and strong acid pH | |
| CN115772096B (en) | Two-photon fluorescent probe for double-channel detection and preparation method and application thereof | |
| CN113402470B (en) | Multichannel reversible colorimetric mercury ion fluorescent probe, preparation method and application | |
| CN115141145A (en) | Fluorescence probe for detecting lysosome hypobromous acid, preparation method and application | |
| CN108299402A (en) | A kind of preparation method and application of multi-functional hypersensitive Zn2+ two-photons detection fluorescent molecular probe | |
| CN114249760A (en) | Synthesis and application of fluorescent probe capable of simultaneously distinguishing hypochlorous acid and hydrogen peroxide in three channels | |
| CN109134483B (en) | Hydrogen sulfide fluorescent probe and preparation method and application thereof |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200616 |
|
| RJ01 | Rejection of invention patent application after publication |