CN105505377A - Two-photon reversible type fluorescent probe FO-PSe for hypochlorous acid detection, and preparation method and application thereof - Google Patents
Two-photon reversible type fluorescent probe FO-PSe for hypochlorous acid detection, and preparation method and application thereof Download PDFInfo
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- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 title claims abstract description 57
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 27
- 230000002441 reversible effect Effects 0.000 title claims abstract description 15
- 238000001514 detection method Methods 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title claims abstract description 6
- 239000000523 sample Substances 0.000 claims abstract description 69
- 238000003384 imaging method Methods 0.000 claims abstract description 15
- 239000002994 raw material Substances 0.000 claims abstract description 10
- 241000252212 Danio rerio Species 0.000 claims description 23
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 16
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 5
- 238000010226 confocal imaging Methods 0.000 claims description 4
- 235000010290 biphenyl Nutrition 0.000 claims description 2
- 239000004305 biphenyl Substances 0.000 claims description 2
- 125000006267 biphenyl group Chemical group 0.000 claims description 2
- 239000003480 eluent Substances 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims description 2
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N phenylbenzene Natural products C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 19
- 238000003786 synthesis reaction Methods 0.000 abstract description 12
- 238000013461 design Methods 0.000 abstract description 4
- 210000000683 abdominal cavity Anatomy 0.000 abstract description 3
- 238000004458 analytical method Methods 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 3
- 150000001875 compounds Chemical class 0.000 abstract 1
- 238000000746 purification Methods 0.000 abstract 1
- 238000002474 experimental method Methods 0.000 description 17
- 239000000243 solution Substances 0.000 description 16
- 238000006243 chemical reaction Methods 0.000 description 15
- 238000000799 fluorescence microscopy Methods 0.000 description 15
- 230000005284 excitation Effects 0.000 description 13
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 12
- 239000007995 HEPES buffer Substances 0.000 description 12
- 230000015572 biosynthetic process Effects 0.000 description 11
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 8
- 238000010186 staining Methods 0.000 description 6
- 235000015097 nutrients Nutrition 0.000 description 5
- 239000002516 radical scavenger Substances 0.000 description 5
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 229910052786 argon Inorganic materials 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 229940125904 compound 1 Drugs 0.000 description 4
- 229940125782 compound 2 Drugs 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
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- 239000007787 solid Substances 0.000 description 4
- 230000003595 spectral effect Effects 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000000295 emission spectrum Methods 0.000 description 2
- 238000000695 excitation spectrum Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Chemical compound Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- XDFNWJDGWJVGGN-UHFFFAOYSA-N 2-(2,7-dichloro-3,6-dihydroxy-9h-xanthen-9-yl)benzoic acid Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC(Cl)=C(O)C=C2OC2=CC(O)=C(Cl)C=C21 XDFNWJDGWJVGGN-UHFFFAOYSA-N 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 241000370738 Chlorion Species 0.000 description 1
- 102000003896 Myeloperoxidases Human genes 0.000 description 1
- 108090000235 Myeloperoxidases Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010039361 Sacroiliitis Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
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- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
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- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 238000000482 two photon fluorescence microscopy Methods 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
Classifications
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- 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
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
-
- 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
- A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C391/00—Compounds containing selenium
- C07C391/02—Compounds containing selenium having selenium atoms bound to carbon atoms of six-membered aromatic rings
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- 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"
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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- C09K2211/1007—Non-condensed systems
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
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- C09K2211/1011—Condensed systems
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1014—Carbocyclic compounds bridged by heteroatoms, e.g. N, P, Si or B
Abstract
The invention designs and synthesizes a two-photon reversible type fluorescent probe FO-PSe for hypochlorous acid detection, and provides a preparation method and application of the two-photon reversible type fluorescent probe FO-PSe. Two kinds of easy-to-obtain synthesized compounds are used as initial raw materials to prepare the probe through one step of simple and efficient synthesis reaction; in addition, the post-treatment is easy; the purification is simple and convenient. The fluorescent probe has two-photon properties; the background fluorescence interference can be effectively reduced; in addition, the reversible performance is realized on hypochlorous acid; the concentration of the hypochlorous acid can be dynamically and reversibly detected. A good effect is achieved when the probe is used for live cell and living body imaging; in addition, the imaging analysis of the hypochlorous acid in the mouse abdominal cavity at different depth is realized. The probe has the advantages that the structure is novel, and the photophysical property is good. A good fluorescent tool is provided for studying the function and the biological significance of the hypochlorous acid in live cells and living bodies.
Description
Technical field:
The invention belongs to synthesis field, particularly a kind of two-photon reversible fluorescent probe FO-PSe for hypochlorous acid detection and its preparation method and application.
Background technology:
Hypochlorous acid is one of important active oxygen in human body, and it all plays vital effect in the physiology and pathology etc. of organism.Can there is chemical reaction in hydrogen peroxide and chlorion in organism under the katalysis of myeloperoxidase, and product hypochlorite and protonated form thereof and hypochlorous acid are present in organism in physiological conditions.Hypochlorous acid be a kind of have strong nucleophilie nucleus ability oxidizing substance, it can with the different kinds of molecules in organism as RNA, DNA, and lipid acid, cholesterol and multiple proteins react.It is its strong germ resistance that hypochlorous acid acts on the most significantly, and it is by required oxydasis in the reaction of certain viruses contact is fallen, thus reaches the antibacterial object of sterilization.If hypochlorous concentration occurs abnormal, will cause great destruction, cause the disease that some are serious simultaneously to life system, as sacroiliitis, arteriosclerosis and certain cancers etc., therefore development is used for the instrument of hypochlorous acid detection is highly significant.
In recent years, reported HClO fluorescent probe several, if DCFH, DHR are (see document: A.Bizyukin, etal, Bull.Exp.Biol.Med.1995,119,347.), APF (document: T.Nagano, J.Biol.Chem.2003,278,3170.), MitoAR (document: T.Nagano.J.Am.Chem.Soc.2007,129,10324.) etc., but above probe is except detecting HClO, also has fluorescence response in various degree to other active oxygen species.Because often kind of active oxygen has its physiology unique separately or pathological effect, therefore the demand of the strong HClO fluorescent probe of specificity is seemed particularly urgent.In the other HClO probe reported, what have need at the operational condition of the harshness of pH12 (document: H.M.Ma, Chem.Eur.J.2008,14,4719.), or poorly water-soluble (document: W.Tan, etal, Chem.Eur.J.2009,15,2305.), cannot really be applied in living things system.At present, really can be applied to the fluorescent probe that in cell, HClO detects few, need multi-step high cost prepare and obtain (document: T.Nagano, J.Am.Chem.Soc.2007,129,7313. under loaded down with trivial details exacting terms; Document: J.Tae, Org.Lett.2009,11, (4), 859.; Document: R.Weissleder, P.Libby, Chem.Biol.2007,14,1221.).
Fluorescent method has the advantages such as highly sensitive, highly selective, simple and convenient, real-time in-situ detection, makes it have application prospect quite widely at life and the field such as environmental science, analytical chemistry.Therefore design highly sensitive, highly selective, the novel hypochlorous acid fluorescent probe that identifies fast obtain the more next many attention of people, also become a very important problem simultaneously.Two-photon fluorescence probe is widely used in bio-imaging field, due to the advantage such as light injury, darker tissue penetration, lower biological context fluorescence interference that it is lower to biological tissue samples.Therefore, synthesis and the development of novel two-photon fluorescence probe are very important, especially the two-photon of reversible design, synthesis still very challenging.
Summary of the invention
During the object of the invention is that making up current hypochlorous acid detects, the situation that two-photon reversible fluorescent probe is less, simple and easy, synthesized a kind of two-photon reversible fluorescent probe (FO-PSe) efficiently, reversible refer to fluorescent probe (FO-PSe) can with hypochlorous acid generation oxidizing reaction, Fluorescence Increasing; With GSH, reduction reaction can occur, fluorescent weakening again afterwards, so circulation repeatedly.Be successfully applied to hypochlorous imaging analysis in viable cell and live body, and different depths hypochlorous imaging analysis in position, abdominal cavity in live body can be realized, substantially without background fluorescence, respond well.
For the two-photon reversible fluorescent probe FO-PSe that hypochlorous acid detects, its structural formula is:
The preparation method of above-mentioned probe FO-PSe, comprises the steps:
Get raw material 1 and 2 and be dissolved in DMF solution, add appropriate dithiothreitol (DTT) and 1,8-diazabicyclo [5.4.0] 11 carbon-7-alkene, react 25-40h in 75-100 DEG C, extraction, drying, secondary dissolve, cross post separation, to obtain final product;
The structural formula of described raw material 1 is:
Described raw material 2 is diphenyl disenenide.
Preferably, the ratio of above-mentioned raw materials 1 and 2, dithiothreitol (DTT), 1,8-diazabicyclo [5.4.0] 11 carbon-7-alkene, DMF amount of substance is 1-2:3-6:5-10:10-15.
Preferably, above-mentioned mistake post is separated eluent volume ratio used is sherwood oil: methylene dichloride 5-9:1-2 (V/V).
Above-mentioned probe FO-PSe can be used for detecting the hypochlorous acid content in or beyond organism.
Above-mentioned probe FO-PSe can be used for detection by quantitative hypochlorous acid.
Above-mentioned probe FO-PSe can be used for the living imaging of viable cell and zebra fish and mouse.
Above-mentioned probe FO-PSe can be used for laser confocal imaging.
Present invention also offers a kind of two-photon laser detection system detected for hypochlorous acid, comprise two-photon laser Laser Scanning Confocal Microscope and above-mentioned probe FO-PSe.
The present invention also finds: when probe FO-PSe detects the hypochlorous acid in or beyond organism, the wavelength of preferred exciting light is 425-450nm or 800 ~ 825nm.
A kind of two-photon reversible fluorescent probe detected for hypochlorous acid of design and synthesis of the present invention, called after FO-PSe, it has two-phpton property, and is the fluorescent probe of reversible.We are successfully applied to viable cell and in vivo hypochlorous research, achieve good effect.Its simple and easy, efficient synthesis step and excellent photophysical property belong to synthesis and technical field.
Usually, dye molecule can be dissolved in damping fluid or by the water-miscible organic solvents such as DMSO, then add suitable damping fluid and other organic reagents are tested.We have studied probe FO-PSe respectively at pH=7.4 aqueous buffer solution and variously common are the photophysical property in machine reagent and use it for viable cell and zebra fish and the experiment of mouse living imaging.The dyeing process of viable cell and zebra fish live body is added by probe in cultured viable cell or zebra fish nutrient solution, hatches the regular hour to remove Incubating Solution, and with PBS buffer solution 3 times, then carry out laser confocal imaging).The method of mouse vital staining is, with syringe, probe molecule is expelled to position, mouse abdominal cavity, then carries out laser confocal imaging.
Existing raw material midbody 1 and 2 before the present invention utilizes, simple and easy by a step, efficiently synthesize, eventually pass thin-layer chromatography post and be separated and obtain probe FO-PSe.
The invention has the beneficial effects as follows:
(1) this probe molecule has two-phpton property, significantly reduces self-absorption, improves imaging accuracy.
(2) this probe has reversibility to hypochlorous acid, can hypochlorous change in concentration in the detection living things system of dynamic reversible.
(3) viable cell and zebra fish and mouse Color good.Dyeing time shorter (10min), staining efficiency is higher.
(4) relatively simple, higher, the easy purifying of productive rate of synthesis step.
Accompanying drawing explanation
Fig. 1 is excitation spectrum and the emmission spectrum spectrogram of novel fluorescence probe of the present invention, and wherein a is excitation spectrum, and excitation wavelength is 425nm, b is emmission spectrum, and emission wavelength is 530nm; X-coordinate is wavelength (nm), and ordinate zou is fluorescence intensity.
Fig. 2 is that the two-photon fluorescence before and after novel fluorescence probe of the present invention and hypochlorous acid effect launches spectrogram, and wherein a is the two photon emission spectrum before fluorescent probe and hypochlorous acid effect, and b is the two photon emission spectrum after fluorescent probe and hypochlorous acid effect; X-coordinate is wavelength (nm), and ordinate zou is fluorescent emission intensity.
Fig. 3 is novel fluorescence probe of the present invention (10 μMs) and the hypochlorous acid (0-100 μM) adding different concns, single photon fluorescence spectrum.Inset: the fluorescence intensity of probe and the linear dependence of hypochlorous acid concentration.
Fig. 4 is novel fluorescence probe cell imaging figure of the present invention, namely with the imaging of scavenger cell single photon fluorescence and two-photon fluorescence imaging figure.
Fig. 5 is the fluorescence imaging figure of novel fluorescence probe of the present invention zebra fish.
Fig. 6 is the fluorescence imaging figure of novel fluorescence probe of the present invention at mouse peritoneal position.
Embodiment
The present invention is further illustrated below in conjunction with embodiment.
Embodiment 1: the synthesis of fluorescent probe
Under argon shield; compound 1 (0.374g, 1.2mmol) and dithiothreitol (DTT) (0.308g, 2.0mmol) are dissolved in N; in dinethylformamide 10-15mL; add compound 2 (0.135g, 0.4mmol) after 75-100 DEG C of reaction 30min, continue to stir 20min; add 1; 8-diazabicyclo [5.4.0] 11 carbon-7-alkene (0.75ml, 5.0mmol), 75-100 DEG C of reaction 25-40h.After completion of the reaction, by mixed solution respectively with methylene dichloride and saturated sodium bicarbonate extraction, by organic phase anhydrous magnesium sulfate drying.Then again dissolve post to be separated, obtain Orange red solid (40%-80%).
Nuclear-magnetism and mass spectral characteristi:
1HNMR(400MHz,CDCl
3):δ7.594(s,2H；ArH),7.528(d,2H；ArH),7.456(d,2H；ArH),7.180-7.248(m,10H；ArH).
13CNMR(100MHz,CDCl
3)δ191.54,141.72,137.18,132.77,132.53,130.59,128.60,127.17,127.06,126.69,119.94.
77SeNMR(150MHz,CDCl
3):δ431.4.MSdata,m/z:492.9619(M+H)
Effect experimental:
The one-photon excitation emission experiment of probe and two-photon fluorescence emission experiment:
Its one-photon excitation emission experiment of FO-PSe probe measurement, one-photon excitation and transmitting spectrogram are shown in Fig. 1.Fig. 1 X-coordinate is wavelength (nm), and ordinate zou represents fluorescence intensity.Two-photon fluorescence before and after fluorescent probe and hypochlorous acid effect is launched spectrogram and is shown in Fig. 2; X-coordinate is wavelength (nm), and ordinate zou is fluorescent emission intensity.What wherein two-photon was tested excites as 800nm, effectively can avoid autofluorescence, thus improves the sensitivity detected.The hypochlorous acid of probe FO-PSe and different concns reacts the photoluminescent property caused to be changed as shown in Figure 3.Along with the increase of hypochlorous acid concentration, the fluorescence intensity of probe strengthens gradually.Hypochlorous acid concentration is 5.0 × 10
-6~ 1.0 × 10
-4in the scope of M, there is good linear relationship in the fluorescence intensity of FO-PSe probe and hypochlorous acid concentration, and linearly dependent coefficient is 0.992, detects and be limited to 0.35 μM (n=11andS/N=3).
Probe is to viable cell and zebra fish and mouse vital staining imaging experiment:
Scavenger cell is cultivated by the DMEM nutrient solution of high sugar, add the probe HEPES aqueous solution of 10 μMs respectively, 10 minutes are hatched in 37 DEG C, then cell is carried out laser co-focusing fluorescence imaging, can find out that it is better to viable cell Color, due to its basic unstressed configuration in water, therefore cellular context lower (Fig. 3).
Zebra fish is added 20 μMs of probe HEPES solution, hatch 0.5 hour, then zebra fish is carried out laser co-focusing fluorescence imaging, can find out that it is better to zebra fish Color, substantially without background fluorescence (Fig. 4).
Add 20 μMs of probe HEPES solution at mouse peritoneal position, then laser co-focusing fluorescence imaging is carried out at mouse peritoneal position, can find out that it is better to mouse peritoneal image areas effect, substantially without background fluorescence (Fig. 5).
Embodiment 2: the synthesis of fluorescent probe
Under argon shield; compound 1 (0.374g, 1.2mmol) and dithiothreitol (DTT) (0.308g, 2.0mmol) are dissolved in N; in dinethylformamide 1mL; add compound 2 (0.135g, 0.4mmol) after 75 DEG C of reaction 30min, continue to stir 20min; add 1; 8-diazabicyclo [5.4.0] 11 carbon-7-alkene (0.75ml, 5.0mmol), 75 DEG C of reaction 25h.After completion of the reaction, by mixed solution respectively with methylene dichloride and saturated sodium bicarbonate extraction, by organic phase anhydrous magnesium sulfate drying.Then again dissolve post to be separated, obtain Orange red solid (40%).
Nuclear-magnetism and mass spectral characteristi:
1HNMR(400MHz,CDCl
3):δ7.594(s,2H；ArH),7.528(d,2H；ArH),7.456(d,2H；ArH),7.180-7.248(m,10H；ArH).
13CNMR(100MHz,CDCl
3)δ191.54,141.72,137.18,132.77,132.53,130.59,128.60,127.17,127.06,126.69,119.94.
77SeNMR(150MHz,CDCl
3):δ431.4.MSdata,m/z:492.9619(M+H)
Effect experimental:
The one-photon excitation emission experiment of probe and two-photon fluorescence emission experiment:
Its one-photon excitation emission experiment of FO-PSe probe measurement, one-photon excitation and transmitting spectrogram are shown in Fig. 1.Fig. 1 X-coordinate is wavelength (nm), and ordinate zou represents fluorescence intensity.Two-photon fluorescence before and after fluorescent probe and hypochlorous acid effect is launched spectrogram and is shown in Fig. 2; X-coordinate is wavelength (nm), and ordinate zou is fluorescent emission intensity.What wherein two-photon was tested excites as 800nm, effectively can avoid autofluorescence, thus improves the sensitivity detected.The hypochlorous acid of probe FO-PSe and different concns reacts the photoluminescent property caused to be changed as shown in Figure 3.Along with the increase of hypochlorous acid concentration, the fluorescence intensity of probe strengthens gradually.Hypochlorous acid concentration is 5.0 × 10
-6~ 1.0 × 10
-4in the scope of M, there is good linear relationship in the fluorescence intensity of FO-PSe probe and hypochlorous acid concentration, and linearly dependent coefficient is 0.992, detects and be limited to 0.35 μM (n=11andS/N=3).
Probe is to viable cell and zebra fish and mouse vital staining imaging experiment:
Scavenger cell is cultivated by the DMEM nutrient solution of high sugar, add the probe HEPES aqueous solution of 10 μMs respectively, 10 minutes are hatched in 37 DEG C, then cell is carried out laser co-focusing fluorescence imaging, can find out that it is better to viable cell Color, due to its basic unstressed configuration in water, therefore cellular context lower (Fig. 3).
Zebra fish is added 20 μMs of probe HEPES solution, hatch 0.5 hour, then zebra fish is carried out laser co-focusing fluorescence imaging, can find out that it is better to zebra fish Color, substantially without background fluorescence (Fig. 4).
Add 20 μMs of probe HEPES solution at mouse peritoneal position, then laser co-focusing fluorescence imaging is carried out at mouse peritoneal position, can find out that it is better to mouse peritoneal image areas effect, substantially without background fluorescence (Fig. 5).
Embodiment 3: the synthesis of fluorescent probe
Under argon shield; compound 1 (0.374g, 1.2mmol) and dithiothreitol (DTT) (0.308g, 2.0mmol) are dissolved in N; in dinethylformamide 15mL; add compound 2 (0.135g, 0.4mmol) after 100 DEG C of reaction 30min, continue to stir 20min; add 1; 8-diazabicyclo [5.4.0] 11 carbon-7-alkene (0.75ml, 5.0mmol), 100 DEG C of reaction 40h.After completion of the reaction, by mixed solution respectively with methylene dichloride and saturated sodium bicarbonate extraction, by organic phase anhydrous magnesium sulfate drying.Then again dissolve post to be separated, obtain Orange red solid (80%).
Nuclear-magnetism and mass spectral characteristi:
1HNMR(400MHz,CDCl
3):δ7.594(s,2H；ArH),7.528(d,2H；ArH),7.456(d,2H；ArH),7.180-7.248(m,10H；ArH).
13CNMR(100MHz,CDCl
3)δ191.54,141.72,137.18,132.77,132.53,130.59,128.60,127.17,127.06,126.69,119.94.
77SeNMR(150MHz,CDCl
3):δ431.4.MSdata,m/z:492.9619(M+H)
Effect experimental:
The one-photon excitation emission experiment of probe and two-photon fluorescence emission experiment:
Its one-photon excitation emission experiment of FO-PSe probe measurement, one-photon excitation and transmitting spectrogram are shown in Fig. 1.Fig. 1 X-coordinate is wavelength (nm), and ordinate zou represents fluorescence intensity.Two-photon fluorescence before and after fluorescent probe and hypochlorous acid effect is launched spectrogram and is shown in Fig. 2; X-coordinate is wavelength (nm), and ordinate zou is fluorescent emission intensity.What wherein two-photon was tested excites as 800nm, effectively can avoid autofluorescence, thus improves the sensitivity detected.The hypochlorous acid of probe FO-PSe and different concns reacts the photoluminescent property caused to be changed as shown in Figure 3.Along with the increase of hypochlorous acid concentration, the fluorescence intensity of probe strengthens gradually.Hypochlorous acid concentration is 5.0 × 10
-6~ 1.0 × 10
-4in the scope of M, there is good linear relationship in the fluorescence intensity of FO-PSe probe and hypochlorous acid concentration, and linearly dependent coefficient is 0.992, detects and be limited to 0.35 μM (n=11andS/N=3).
Probe is to viable cell and zebra fish and mouse vital staining imaging experiment:
Scavenger cell is cultivated by the DMEM nutrient solution of high sugar, add the probe HEPES aqueous solution of 10 μMs respectively, 10 minutes are hatched in 37 DEG C, then cell is carried out laser co-focusing fluorescence imaging, can find out that it is better to viable cell Color, due to its basic unstressed configuration in water, therefore cellular context lower (Fig. 3).
Zebra fish is added 20 μMs of probe HEPES solution, hatch 0.5 hour, then zebra fish is carried out laser co-focusing fluorescence imaging, can find out that it is better to zebra fish Color, substantially without background fluorescence (Fig. 4).
Add 20 μMs of probe HEPES solution at mouse peritoneal position, then laser co-focusing fluorescence imaging is carried out at mouse peritoneal position, can find out that it is better to mouse peritoneal image areas effect, substantially without background fluorescence (Fig. 5).
Embodiment 4: the synthesis of fluorescent probe
Under argon shield; compound 1 (0.374g, 1.2mmol) and dithiothreitol (DTT) (0.308g, 2.0mmol) are dissolved in N; in dinethylformamide 12mL; add compound 2 (0.135g, 0.4mmol) after 85 DEG C of reaction 30min, continue to stir 20min; add 1; 8-diazabicyclo [5.4.0] 11 carbon-7-alkene (0.75ml, 5.0mmol), 85 DEG C of reaction 36h.After completion of the reaction, by mixed solution respectively with methylene dichloride and saturated sodium bicarbonate extraction, by organic phase anhydrous magnesium sulfate drying.Then again dissolve post to be separated, obtain Orange red solid (60%).
Nuclear-magnetism and mass spectral characteristi:
1HNMR(400MHz,CDCl
3):δ7.594(s,2H；ArH),7.528(d,2H；ArH),7.456(d,2H；ArH),7.180-7.248(m,10H；ArH).
13CNMR(100MHz,CDCl
3)δ191.54,141.72,137.18,132.77,132.53,130.59,128.60,127.17,127.06,126.69,119.94.
77SeNMR(150MHz,CDCl
3):δ431.4.MSdata,m/z:492.9619(M+H)
Effect experimental:
The one-photon excitation emission experiment of probe and two-photon fluorescence emission experiment:
Its one-photon excitation emission experiment of FO-PSe probe measurement, one-photon excitation and transmitting spectrogram are shown in Fig. 1.Fig. 1 X-coordinate is wavelength (nm), and ordinate zou represents fluorescence intensity.Two-photon fluorescence before and after fluorescent probe and hypochlorous acid effect is launched spectrogram and is shown in Fig. 2; X-coordinate is wavelength (nm), and ordinate zou is fluorescent emission intensity.What wherein two-photon was tested excites as 800nm, effectively can avoid autofluorescence, thus improves the sensitivity detected.The hypochlorous acid of probe FO-PSe and different concns reacts the photoluminescent property caused to be changed as shown in Figure 3.Along with the increase of hypochlorous acid concentration, the fluorescence intensity of probe strengthens gradually.Hypochlorous acid concentration is 5.0 × 10
-6~ 1.0 × 10
-4in the scope of M, there is good linear relationship in the fluorescence intensity of FO-PSe probe and hypochlorous acid concentration, and linearly dependent coefficient is 0.992, detects and be limited to 0.35 μM (n=11andS/N=3).
Probe is to viable cell and zebra fish and mouse vital staining imaging experiment:
Scavenger cell is cultivated by the DMEM nutrient solution of high sugar, add the probe HEPES aqueous solution of 10 μMs respectively, 10 minutes are hatched in 37 DEG C, then cell is carried out laser co-focusing fluorescence imaging, can find out that it is better to viable cell Color, due to its basic unstressed configuration in water, therefore cellular context lower (Fig. 3).
Zebra fish is added 20 μMs of probe HEPES solution, hatch 0.5 hour, then zebra fish is carried out laser co-focusing fluorescence imaging, can find out that it is better to zebra fish Color, substantially without background fluorescence (Fig. 4).
Add 20 μMs of probe HEPES solution at mouse peritoneal position, then laser co-focusing fluorescence imaging is carried out at mouse peritoneal position, can find out that it is better to mouse peritoneal image areas effect, substantially without background fluorescence (Fig. 5).
As mentioned above, embodiments of the invention are explained, but as long as do not depart from inventive point of the present invention in fact and effect can have a lot of distortion, this will be readily apparent to persons skilled in the art.Therefore, such variation is also all included within protection scope of the present invention.
Claims (10)
1., for the two-photon reversible fluorescent probe FO-PSe that hypochlorous acid detects, it is characterized in that, structural formula is:
2. the preparation method of probe FO-PSe according to claim 1, is characterized in that, comprise the steps:
Get raw material 1 and 2 and be dissolved in DMF solution, add dithiothreitol (DTT) and 1,8-diazabicyclo [5.4.0] 11 carbon-7-alkene, react 25-40h in 75-100 DEG C, extraction, drying, secondary dissolve, cross post separation, to obtain final product;
The structural formula of described raw material 1 is:
Described raw material 2 is diphenyl disenenide.
3. method as claimed in claim 2, it is characterized in that, the ratio of described raw material 1 and 2, dithiothreitol (DTT), 1,8-diazabicyclo [5.4.0] 11 carbon-7-alkene, DMF amount of substance is 1-2:3-6:5-10:10-15.
4. method as claimed in claim 2, is characterized in that, it is sherwood oil that described mistake post is separated eluent volume ratio used: methylene dichloride=5-9:1-2.
5. probe FO-PSe according to claim 1 is detecting the application in the hypochlorous acid in or beyond organism.
6. probe FO-PSe according to claim 1 carries out the application in detection by quantitative at hypochlorous acid.
7. the application of probe FO-PSe according to claim 1 in viable cell and zebra fish and mouse living imaging.
8. the application of probe FO-PSe according to claim 1 in laser confocal imaging.
9., for the two-photon laser detection system that hypochlorous acid detects, it is characterized in that, comprise two-photon laser Laser Scanning Confocal Microscope and probe FO-PSe according to claim 1.
10. two-photon laser detection system according to claim 9, is characterized in that, determined wavelength is 425-450nm or 800 ~ 825nm.
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Citations (5)
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|---|---|---|---|---|
| US20040198693A1 (en) * | 1999-09-30 | 2004-10-07 | Deninno Michael P. | Compounds for the treatment of ischemia |
| CN103571457A (en) * | 2012-08-02 | 2014-02-12 | 中国科学院大连化学物理研究所 | Fluorescent probe and application thereof in dynamic detection of mercaptan |
| CN103571458A (en) * | 2012-08-02 | 2014-02-12 | 中国科学院大连化学物理研究所 | Selenium-containing fluorine-boron dye fluorescent probe and application thereof in ClO<-> detection |
| CN103848787A (en) * | 2012-12-06 | 2014-06-11 | 中国科学院大连化学物理研究所 | Fluorescence probe and application thereof in reversible detection of hypochlorous acid |
| WO2014136781A1 (en) * | 2013-03-04 | 2014-09-12 | 国立大学法人 東京大学 | Fluorescent probe |
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2015
- 2015-12-10 CN CN201510925577.8A patent/CN105505377A/en active Pending
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| US20040198693A1 (en) * | 1999-09-30 | 2004-10-07 | Deninno Michael P. | Compounds for the treatment of ischemia |
| CN103571457A (en) * | 2012-08-02 | 2014-02-12 | 中国科学院大连化学物理研究所 | Fluorescent probe and application thereof in dynamic detection of mercaptan |
| CN103571458A (en) * | 2012-08-02 | 2014-02-12 | 中国科学院大连化学物理研究所 | Selenium-containing fluorine-boron dye fluorescent probe and application thereof in ClO<-> detection |
| CN103848787A (en) * | 2012-12-06 | 2014-06-11 | 中国科学院大连化学物理研究所 | Fluorescence probe and application thereof in reversible detection of hypochlorous acid |
| WO2014136781A1 (en) * | 2013-03-04 | 2014-09-12 | 国立大学法人 東京大学 | Fluorescent probe |
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