CN109574980A - Nitric oxide production fluorescent probe molecule, preparation and purposes are detected based on Rhodamine Derivatives - Google Patents
Nitric oxide production fluorescent probe molecule, preparation and purposes are detected based on Rhodamine Derivatives Download PDFInfo
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- CN109574980A CN109574980A CN201811434365.XA CN201811434365A CN109574980A CN 109574980 A CN109574980 A CN 109574980A CN 201811434365 A CN201811434365 A CN 201811434365A CN 109574980 A CN109574980 A CN 109574980A
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- nitric oxide
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- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 title claims abstract description 211
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 62
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical class [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 title claims abstract description 33
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 238000001514 detection method Methods 0.000 claims abstract description 28
- 241000244206 Nematoda Species 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 230000003834 intracellular effect Effects 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 42
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 27
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 24
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- -1 ethyl m-aminophenol Chemical compound 0.000 claims description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 235000019441 ethanol Nutrition 0.000 claims description 12
- 229910052757 nitrogen Inorganic materials 0.000 claims description 11
- 229940125782 compound 2 Drugs 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 238000010898 silica gel chromatography Methods 0.000 claims description 6
- ZRYZBQLXDKPBDU-UHFFFAOYSA-N 4-bromobenzaldehyde Chemical compound BrC1=CC=C(C=O)C=C1 ZRYZBQLXDKPBDU-UHFFFAOYSA-N 0.000 claims description 5
- 229910021529 ammonia Inorganic materials 0.000 claims description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- 239000003480 eluent Substances 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 230000003647 oxidation Effects 0.000 claims description 4
- 238000007254 oxidation reaction Methods 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- WAVOOWVINKGEHS-UHFFFAOYSA-N 3-(diethylamino)phenol Chemical compound CCN(CC)C1=CC=CC(O)=C1 WAVOOWVINKGEHS-UHFFFAOYSA-N 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 239000008247 solid mixture Substances 0.000 claims description 3
- TXUICONDJPYNPY-UHFFFAOYSA-N (1,10,13-trimethyl-3-oxo-4,5,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-17-yl) heptanoate Chemical compound C1CC2CC(=O)C=C(C)C2(C)C2C1C1CCC(OC(=O)CCCCCC)C1(C)CC2 TXUICONDJPYNPY-UHFFFAOYSA-N 0.000 claims description 2
- 229940018563 3-aminophenol Drugs 0.000 claims description 2
- 229910021595 Copper(I) iodide Inorganic materials 0.000 claims description 2
- 229910021626 Tin(II) chloride Inorganic materials 0.000 claims description 2
- LSXDOTMGLUJQCM-UHFFFAOYSA-M copper(i) iodide Chemical compound I[Cu] LSXDOTMGLUJQCM-UHFFFAOYSA-M 0.000 claims description 2
- CWLKGDAVCFYWJK-UHFFFAOYSA-N m-Aminophenol Natural products NC1=CC=CC(O)=C1 CWLKGDAVCFYWJK-UHFFFAOYSA-N 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 239000012074 organic phase Substances 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims description 2
- 239000001119 stannous chloride Substances 0.000 claims description 2
- 235000011150 stannous chloride Nutrition 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 239000012491 analyte Substances 0.000 claims 1
- 238000004821 distillation Methods 0.000 claims 1
- 238000000605 extraction Methods 0.000 claims 1
- 239000000047 product Substances 0.000 claims 1
- 239000000523 sample Substances 0.000 description 18
- 210000004027 cell Anatomy 0.000 description 14
- 238000002474 experimental method Methods 0.000 description 9
- 239000007789 gas Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 7
- 239000007864 aqueous solution Substances 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- 238000002189 fluorescence spectrum Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000000799 fluorescence microscopy Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000010453 quartz Substances 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- 238000002211 ultraviolet spectrum Methods 0.000 description 2
- 238000005292 vacuum distillation Methods 0.000 description 2
- KZMAWJRXKGLWGS-UHFFFAOYSA-N 2-chloro-n-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-n-(3-methoxypropyl)acetamide Chemical compound S1C(N(C(=O)CCl)CCCOC)=NC(C=2C=CC(OC)=CC=2)=C1 KZMAWJRXKGLWGS-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 1
- 241000482313 Globodera ellingtonae Species 0.000 description 1
- 208000037309 Hypomyelination of early myelinating structures Diseases 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000001723 carbon free-radicals Chemical class 0.000 description 1
- 229940125904 compound 1 Drugs 0.000 description 1
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 125000000879 imine group Chemical group 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 230000009965 odorless effect Effects 0.000 description 1
- 125000001820 oxy group Chemical group [*:1]O[*:2] 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 239000002341 toxic gas Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
-
- 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
- C07D311/90—Xanthenes with hydrocarbon radicals, substituted by amino radicals, directly attached in position 9
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
-
- 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
Abstract
The present invention relates to one kind to detect nitric oxide production fluorescent probe molecule, Preparation method and use based on Rhodamine Derivatives.The fluorescent probe molecule structure is as shown below:The invention also discloses the preparation methods that nitric oxide production fluorescent probe molecule is detected based on Rhodamine Derivatives, and detection water body example, intracellular, in nematode body etc. nitric oxide purposes.
Description
Technical field
The invention belongs to organic functional material, it is related to detecting fluorescent probe molecule, preparation method and the use of micro substance
On the way.
Background technique
Nitric oxide (NO) is a kind of toxic gas of colorless and odorless, and the endogenic nitric oxide of organism is a kind of heavy
The gas messenger molecule wanted, it takes part in a large amount of Physiological effect activity, plays crucial effect in vivo.Research
Show that the human body diseases of many such as nitric oxide and cardiovascular disease, diabetes are closely related.Therefore, the one of organism is detected
Nitrogen oxide, and study and evaluate its with Physiological effect contact and act on life science and medical domain is of great significance.
NO is gas radicals in vivo, and diffusive migration speed quickly, is extremely difficult to detect；And active oxygen, activity
The other materials interference such as nitrogen and active carbon radicals, reduces the effect of the single-minded identification NO gas of fluorescence probe；Separately there are some inspections
Perhaps fluorescence is easy to be quenched the fluorescence probe of survey NO or fluorescence intensity is weak, similarly reduces detection effect.Therefore, it prepares
Fluorescence quantum efficiency with higher, excitation wavelength is in the near infrared region and the fluorescence probe of the single-minded identification NO of energy is with important
Theory and application value.
Summary of the invention
It is an object of the present invention to provide a kind of sensitivitys, and high, the good Rhodamine Derivatives that are based on of selectivity detect an oxidation
Fluorescent probe molecule, the Preparation method and use of nitrogen.
Above-mentioned purpose that the invention is realized by the following technical scheme, comprising:
(1) nitric oxide production fluorescent probe molecule is detected based on Rhodamine Derivatives
It has the following structure:
(2) method for detecting nitric oxide production fluorescent probe molecule based on Rhodamine Derivatives is prepared
The following steps are included:
(1) by p-bromobenzaldehyde and N, N- diethyl m-aminophenol is dissolved in the sulfuric acid that concentration is 60%, and 160 DEG C
Reaction 24 hours is adjusted to pH=7 with potassium hydroxide under ice bath, filters violet precipitate, washing, drying, by silica gel chromatography
Column, elution, separation, purification, obtains compound 2；
(2) compound 2, potassium hydroxide and stannous chloride described in step (1) are dissolved in 2- (2- ammonia ethyoxyl) ethyl alcohol
In, 90 DEG C are reacted 24 hours, and the mixed solution of reaction solution sodium chloride solution and methylene chloride extracts three times, dry organic phase,
Solid mixture is collected in filtering, vacuum distillation, and by silica gel chromatography column, elution, separation, purification, obtained solids is base
Nitric oxide production fluorescent probe molecule is detected in Rhodamine Derivatives.
Preferably, the mass ratio of p-bromobenzaldehyde and N in the step (1), N- diethyl m-aminophenol are 1.34g:
2g；The eluant, eluent is the mixture of methylene chloride and methanol, volume ratio 10:1.
Preferably, the mass ratio of compound 2, potassium hydroxide and cuprous iodide three is 0.2385g in the step (2):
0.056g:0.0049g；2- (2- ammonia ethyoxyl) ethyl alcohol volume used is 1.00mL；The eluant, eluent is methylene chloride and methanol
Mixture, volume ratio 10:1.
(3) purposes of nitric oxide production fluorescent probe molecule is detected based on Rhodamine Derivatives
Preferably, the purposes is detection water body example, intracellular, the intracorporal nitric oxide of nematode.
In the present invention, we provide the preparation method and applications based on Rhodamine Derivatives fluorescent probe molecule, have
Beneficial effect is:
1, fluorescent probe molecule synthetic method of the present invention is two steps, is easily-synthesized, high income.The fluorescent probe molecule
With with specific gas molecule --- nitric oxide, the imine group to react is that one kind is clear in structure, light property is steady
Fixed Rhodamine Derivatives.
2, fluorescent probe molecule of the present invention can very well select nitric oxide (Fig. 5), when the Asia of fluorescent probe molecule
After amine groups combination nitric oxide, the fluorescence of fluorescent probe molecule is changed, thus the oxygen in identification and detection liquid-phase system
Change nitrogen.(Fig. 6) is compared by the column diagram of each ion 590nm fluorescence intensity level, the fluorescent probe molecule is only at 590nm
Significantly enhance NO fluorescence (about enhancing 5 times), shows the fluorescence selectivity of height；And active oxygen, activity are distinguished significantly
The other materials such as nitrogen avoid its Interference Detection, have anti-interference.
3, fluorescent probe molecule physicochemical properties stability of the present invention is good.
4, fluorescent probe molecule of the present invention reacts front and back with nitric oxide, and fluorescent emission is in aqueous solution, cell, nematode
In vivo increase about 5 times, detect entire identification process change in fluorescence high sensitivity, be able to detect the nitric oxide in aqueous solution with
And fluorescence imaging cell and the intracorporal nitric oxide of nematode.
5, the good penetrability of fluorescent probe molecule penetrating cell of the present invention, can fluorescence detection cell, an oxidation in nematode body
Nitrogen.
Based on the above advantages, which can be applied to fluorescence identifying and detection environment water body example, living body are dynamic
Nitric oxide in object etc., application prospect are good.
Detailed description of the invention
Fig. 1 is the synthesis road according to the present invention that fluorescent probe molecule is detected based on Rhodamine Derivatives nitric oxide
Line.
Fig. 2 is according to the present invention based on Rhodamine Derivatives nitric oxide detection fluorescent probe molecule1H-NMR
Spectrogram.
Fig. 3 is according to the present invention based on Rhodamine Derivatives nitric oxide detection fluorescent probe molecule13C-NMR
Spectrogram.
Fig. 4 is the HRMS according to the present invention that fluorescent probe molecule is detected based on Rhodamine Derivatives nitric oxide
(ESI) spectrogram.
Fig. 5 is that the Rhodamine Derivatives nitric oxide according to the present invention that is based on detects fluorescent probe molecule in aqueous solution
To the fluorescence spectrum variation diagram of nitric oxide response in system.
Fig. 6 is that the Rhodamine Derivatives nitric oxide according to the present invention that is based on detects fluorescent probe molecule in aqueous solution
In system to nitric oxide and disturbance ion the fluorescence intensity histogram at 590nm.
Fig. 7 is that the Rhodamine Derivatives nitric oxide according to the present invention that is based on detects fluorescent probe molecule in aqueous solution
To the nitric oxide production ultraviolet spectra variation diagram of various concentration in system.
Fig. 8 is that the Rhodamine Derivatives nitric oxide according to the present invention that is based on detects fluorescent probe molecule in aqueous solution
To the nitric oxide production fluorescence spectrum variation diagram of various concentration in system.
Fig. 9 is that the Rhodamine Derivatives nitric oxide according to the present invention that is based on detects fluorescent probe molecule at 590 nm
Fluorescence intensity and corresponding nitric oxide concentration matched curve figure.
Figure 10 is that the Rhodamine Derivatives nitric oxide according to the present invention that is based on detects fluorescent probe molecule in the cell
Exogenous nitric oxide fluorescence identifying figure.Wherein, (a), (b), (c) it is light field, (f) is the channel DAPI, (g) (e) (d)
To be only added 1.0 × 10-5The fluorescence picture of the probe compound of mol/L；It (h) is addition 1.0 × 10-5The probe of mol/L
Close object and 4 × 10-4The fluorescence picture of the NO processing of mol/L；It (i) is addition 1.0 × 10-5The probe compound of mol/L with
And 8 × 10-4The fluorescence picture of the NO processing of mol/L.
Figure 11 is according to the present invention based on the Rhodamine Derivatives nitric oxide detection online polypide of fluorescent probe molecule
It is interior to nitric oxide fluorescence identifying figure.Wherein, a-c is light field；It (d) is only addition 1.0 × 10-5The probe compound of mol/L
Fluorescence picture；It (e) is addition 1.0 × 10-5The probe compound of mol/L and 4 × 10-4The fluorogram of the NO processing of mol/L
Piece；It (f) is addition 1.0 × 10-5The probe compound of mol/L and 8 × 10-4The fluorescence picture of the NO processing of mol/L.
Specific embodiment
Embodiment 1: the synthesis of the nitric oxide detection fluorescent probe molecule based on Rhodamine Derivatives
Specific synthetic route is shown in Fig. 1.
(1) preparation of compound 2: preparing 60% sulfuric acid of 20mL, be added 1.34g (7.28mmol) p-bromobenzaldehyde and
2.0g (14.58mmol) N, N- diethyl m-aminophenol, stirring make it dissolve for 20 minutes, after be slowly heated to 160 DEG C, instead
It answers 24 hours.After reaction, pH to 7 is adjusted with KOH, obtained purple is precipitated and is filtered, washed, drying, gained crude product
By silica gel chromatography column, with methylene chloride: methanol=10:1 is eluted, and concentrate eluant obtains 370mg compound 2, yield 12%.
(2) synthesis of the nitric oxide detection fluorescent probe molecule 1 of Rhodamine Derivatives: by 0.2385g (0.5 mmol)
Compound 1, the CuCl of the KOH of 0.056g (1mmol), 0.0049g (0.05mmol) is added to the 2- of 1.00mL (2- ammonia second
Oxygroup) in ethyl alcohol, reacted 24 hours at 90 DEG C.It is extracted three times with salt water and methylene chloride respectively after reaction, what is obtained has
For machine mutually with filtering after anhydrous sodium sulfate drying, vacuum distillation obtains solid mixture, by silica gel chromatography column, with methylene chloride: first
Alcohol=10:1 elution, concentrate eluant obtain 25mg solid chemical compound, yield 10%.1H-NMR(500MHz,CDCl3):δ(ppm)
7.60 (d, J=12.0 Hz, 2H), 7.25 (d, J=10.5Hz, 2H), 6.91 (d, J=10.5Hz, 2H), 6.82 (d, J=
10.0Hz, 2H), 6.65 (d, J=2.5Hz, 2H), 5.23 (s, 1H), 3.77 (m, 4H), 3.63 (t, J=5.5Hz, 2H),
3.55 (q, J=8.5Hz, 8H), 3.38 (t, J=6.0Hz, 2H), 1.25 (t, J=8.5Hz, 12H)13C-NMR(500MHz,
CDCl3):δ(ppm)158.74,158.03,156.89,153.83,150.80,131.99,131.23,129.16, 118.64,
117.42,112.33,111.82,111.61,95.18,71.59,68.19,60.53,44.89,43.98, 42.24,32.15,
30.90,21.67,13.10,11.66,11.51.HEMS(ESI):calcd for C31H40N3O3[M+H]+=502.3056,
Found m/z 502.3064. wherein, CDCl3For deuterated chloroform.Related spectrogram is shown in Fig. 2~4.
Embodiment 2: the nitric oxide detection fluorescent probe molecule based on Rhodamine Derivatives is to nitric oxide fluorescence detection
Selectivity
Using ethyl alcohol: PBS (0.01mol/L, pH=7.4)=1:99 (v:v) solution controls experiment condition.
Nitric oxide based on Rhodamine Derivatives is detected into fluorescent probe molecule 10mg, alcohol solvent is added and is settled to
20mL, being configured to fluorescent probe molecule concentration is 1 × 10-3The solution of mol/L.
Sample bottle is divided into 12 groups, it is 1 × 10 that every group of each sample bottle, which is separately added into 30 μ L concentration,-3Mol/L based on Luo Dan
The nitric oxide of bright derivative detects fluorescent probe molecule solution, and first bottle of solution is separately added into as blank group, other 11 groups
Different amounts of H2O2, NO2 -, NO3 -, ClO-, O2 ·-,1O2, NO2 ·, ONOO-,·OH, ROO-, NO solution, and PBS solution is supplemented to total
Volume is 3mL, makes H therein2O2, NO2 -, NO3 -, ClO-, O2 ·-,1O2, NO2 ·, ONOO-,·OH, ROO-, NO concentration is all 8 ×
10-4mol/L.After having prepared prepare liquid at room temperature, each test job liquid is transferred to the standard quartz cuvette of 1cm × 1cm
In, measure its fluorescence spectrum.Excitation wavelength is 520nm, launch wavelength 590nm.An oxidation based on Rhodamine Derivatives
Nitrogen detects fluorescent probe molecule and detects as shown in Figure 5 to nitric oxide fluorescence selectivity.It can be seen that one based on Rhodamine Derivatives
Nitrogen oxide, which detects fluorescent probe molecule, only has apparent Enhancement of Fluorescence (about 5 times of enhancings) to NO at 590nm, shows this
Nitric oxide detection fluorescent probe molecule involved in invention based on Rhodamine Derivatives shows height to nitric oxide
Fluorescence selectivity.
We have selected the fluorescence intensity level of each ion at 590nm to do a column diagram (Fig. 6), and Fig. 6 can be straight
It sees ground and finds out that the fluorescence selectivity of probe is very good.
Embodiment 3: the nitric oxide detection fluorescent probe molecule based on Rhodamine Derivatives is to nitric oxide production quantitative purple
Outer detection
Using ethyl alcohol: PBS (0.01mol/L, pH=7.4)=1:99 (v:v) solution controls experiment condition.
Nitric oxide based on Rhodamine Derivatives is detected into fluorescent probe molecule 10mg, alcohol solvent is added and is settled to
20mL, being configured to fluorescent probe molecule concentration is 1 × 10-3The solution of mol/L.
It is passed through nitrogen in the PBS solution of 100mL 30 minutes, then is passed through NO gas 30 minutes, is then considered as NO gas and exists
Reach saturation in PBS solution, NO concentration is 1.9mmol/L in the solution at this time, uses the solution as the source of NO.
Sample bottle is divided into 13 groups, it is 1 × 10 that every group of each sample bottle, which is separately added into 30 microlitres of concentration,-3Mol/L based on sieve
The nitric oxide of red bright derivative detects fluorescent probe molecule solution, then is separately added into the NO that different volumes are dissolved in PBS solution
Solution, and supplementing PBS solution to total volume is 3mL, makes nitric oxide concentration 0mol/L~8 × 10 in test system-4mol/
L.After having prepared prepare liquid at room temperature, each test job liquid is transferred in the standard quartz cuvette of 1cm × 1cm, is measured
Its ultraviolet spectra.
Fig. 7 is that the nitric oxide according to the present invention based on Rhodamine Derivatives detects fluorescent probe molecule water-soluble
The ultraviolet spectrogram changed in liquid system with nitric oxide concentration shows according to the present invention based on Rhodamine Derivatives
Nitric oxide detects fluorescent probe molecule can be with quantitative detection nitric oxide concentration in water solution system.
Embodiment 4: the nitric oxide detection fluorescent probe molecule based on Rhodamine Derivatives is to nitric oxide production quantitative glimmering
Light detection
Using ethyl alcohol: PBS (0.01mol/L, pH=7.4)=1:99 (v:v) solution controls experiment condition.
Nitric oxide based on Rhodamine Derivatives is detected into fluorescent probe molecule 10mg, alcohol solvent is added and is settled to
20mL, being configured to fluorescent probe molecule concentration is 1 × 10-3The solution of mol/L.
It is passed through nitrogen in the PBS solution of 100mL 30 minutes, then is passed through NO gas 30 minutes, is then considered as NO gas and exists
Reach saturation in PBS solution, NO concentration is 1.9mmol/L in the solution at this time, uses the solution as the source of NO.
Sample bottle is divided into 13 groups, it is 1 × 10 that every group of each sample bottle, which is separately added into 30 μ L concentration,-3Mol/L based on Luo Dan
The nitric oxide of bright derivative detects fluorescent probe molecule solution, then be separately added into different volumes be dissolved in PBS solution NO it is molten
Liquid, and supplementing PBS solution to total volume is 3mL, makes nitric oxide concentration 0mol/L~8 × 10 in test system-4mol/L。
After having prepared prepare liquid at room temperature, each test job liquid is transferred in the standard quartz cuvette of 1cm × 1cm, it is measured
Fluorescence spectrum.Fluorometric investigation grating gap size is 5nm × 5nm.Fig. 8 is according to the present invention based on Rhodamine Derivatives
Nitric oxide detection fluorescent probe molecule in water solution system with nitric oxide concentration change fluorescence spectra.It will be glimmering
Fluorescence intensity at light spectrum 590nm is fitted with corresponding nitric oxide concentration, is 0mol/L~8 in nitric oxide concentration
×10-4A matched curve (Fig. 9) is obtained within the scope of mol/L, shows one based on Rhodamine Derivatives according to the present invention
Nitrogen oxide detects fluorescent probe molecule can be with quantitative detection nitric oxide concentration in water solution system.
Embodiment 5: the nitric oxide based on Rhodamine Derivatives detects fluorescent probe molecule exogenous one in the cell
The fluorescence identifying of nitrogen oxide
Three culture dish Hi-5 cells are taken, the Hi-5 cell PBS buffer solution in culture dish (is contained in every liter of buffer
2.90 gram Na2HPO4·12H2O；0.30 gram of NaH2PO4·2H2O it) cleans three times, to remove culture solution.Again to this three culture dish
1mL PBS solution is added in Hi-5 cell, it is small then to continue incubation 2 after 10 microlitre of 1 mmol/L nuclei dyeing toner DAPI of addition
When.Hi-5 cell is washed 3 times with PBS buffer solution, is separately added into the NO solution of various concentration being dissolved in PBS, is being cultivated NO
Concentration in ware is respectively 0mol/L, and 40 × 10-5Mol/L and 8 × 10-4Mol/L, and ware is put in sterile culture case 37
It is incubated for 2 hours at DEG C.After Hi-5 cell washs 3 times with PBS solution again, it is 1 × 10 that 10 microlitres of concentration, which are added,-3Mol/L's
Continue to be incubated for 2 hours after nitric oxide detection fluorescent probe molecule solution and 1mL PBS solution based on Rhodamine Derivatives,
Then above-mentioned solution is sucked out, cleans cell three times with PBS buffer solution, cell is placed under confocal microscope and is carried out
Fluorescence imaging experiments.The result is shown in Figure 10.It can be seen that the nitric oxide detection fluorescence based on Rhodamine Derivatives is visited in Figure 10 g
Needle molecule is from unstressed configuration transmitting basic in cytoplasm, when nitric oxide concentration reaches 8 × 10-4It is intracellular to send out when mol/L
It projects red fluorescence (Figure 10 i).The experiment shows that the nitric oxide detection according to the present invention based on Rhodamine Derivatives is glimmering
Light probe molecule can in the cell exogenous nitric oxide carry out fluorescence identifying.Experiment instrument is Olympus
IX71 fluorescence microscope.
Embodiment 6: it is aoxidized in the nitric oxide detection online polypide of fluorescent probe molecule based on Rhodamine Derivatives to one
The fluorescence identifying of nitrogen
Nematode in culture dish M9 buffer (is contained into 15.12 grams of Na in every liter of buffer2HPO4·12H2O；3 grams
KH2PO4；5 grams of NaCl；0.25 gram of MgSO4·7H2O it) comes out, divides three to be assembled into centrifuge tube.Again into this three groups of centrifuge tubes
1mL M9 solution is added, it is 1 × 10 that concentration, which is added,-3The nitric oxide based on Rhodamine Derivatives of mol/L detects fluorescence probe
10 μ L of molecule is incubated for 2 hours at room temperature.Nematode is washed 3 times with M9 buffer, is centrifuged 2 under 3000r/min speed
Minute, it is then respectively adding the NO solution of various concentration being dissolved in PBS, making concentration of the NO in culture dish is respectively 0mol/
L, 40 × 10-5Mol/L and 8 × 10-4Mol/L, and be incubated for 2 hours at 20 DEG C.Nematode is washed 3 times with M9 solution again,
It is transferred on glass slide after being centrifuged 2 minutes under 3000r/min speed and carries out fluorescence imaging experiments.The result is shown in Figure 11.In Figure 11 d
It can be seen that the basic unstressed configuration hair in nematode of the nitric oxide detection fluorescent probe molecule itself based on Rhodamine Derivatives
It penetrates；When nitric oxide concentration reaches 8 × 10-4When mol/L, red fluorescence (Figure 11 f) is launched in nematode body.The experiment shows
Nitric oxide detection fluorescent probe molecule according to the present invention based on Rhodamine Derivatives can be in online polypide to an oxygen
Change nitrogen and carries out fluorescence identifying.Experiment instrument is Olympus BX51 fluorescence microscope.
Claims (6)
1. detecting nitric oxide production fluorescent probe molecule based on Rhodamine Derivatives, have the following structure:
。
2. the method for preparing fluorescent probe molecule as described in claim 1, comprising the following steps:
(1) by p-bromobenzaldehyde and N, N- diethyl m-aminophenol is dissolved in the sulfuric acid that concentration is 60%, 160 DEG C of reactions 24
Hour, pH=7 are adjusted to potassium hydroxide under ice bath, filter violet precipitate, washing is dried, by silica gel chromatography column, elution,
Separation, purification, obtain compound 2；
Compound 2, potassium hydroxide and stannous chloride described in step (1) are dissolved in 2- (2- ammonia ethyoxyl) ethyl alcohol, 90 DEG C
Three times, dry organic phase, filtering subtracts for the mixed solution extraction of reaction 24 hours, reaction solution sodium chloride solution and methylene chloride
Solid mixture is collected in pressure distillation, and by silica gel chromatography column, elution, separation, purification, obtained solids is to be based on rhodamine
The derivative nitric oxide production fluorescent probe molecule of analyte detection.
3. preparation method according to claim 2, it is characterised in that: p-bromobenzaldehyde and N in the step (1), N- bis-
The mass ratio of ethyl m-aminophenol is 1.34 g:2 g；The eluant, eluent is the mixture of methylene chloride and methanol, volume
Than for 10:1.
4. preparation method according to claim 2 or 3, it is characterised in that: compound 2, potassium hydroxide in the step (2)
And the mass ratio of cuprous iodide three is 0.2385 g:0.056 g:0.0049 g；2- (2- ammonia ethyoxyl) ethyl alcohol body used
Product is 1.00 mL；The eluant, eluent is the mixture of methylene chloride and methanol, volume ratio 10:1.
5. fluorescent probe molecule as described in claim 1 detects nitric oxide production purposes.
6. purposes according to claim 5, it is characterised in that: detection water body example, intracellular, the intracorporal oxidation of nematode
Nitrogen.
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| CN111393401A (en) * | 2020-04-13 | 2020-07-10 | 云南大学 | Rhodamine derivative-based fluorescent probe molecule for detecting diaphorase, preparation method and application |
| CN111647401A (en) * | 2020-07-06 | 2020-09-11 | 山西大学 | Orange fluorescent carbon dot and application thereof in detection of peroxynitrite ions |
| CN113121488A (en) * | 2021-04-21 | 2021-07-16 | 云南大学 | Fluorescent probe molecule for detecting azo reductase based on coumarin derivative and preparation method and application thereof |
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| CN113121488A (en) * | 2021-04-21 | 2021-07-16 | 云南大学 | Fluorescent probe molecule for detecting azo reductase based on coumarin derivative and preparation method and application thereof |
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