CN103289681A - Glutathione fluorescent probe and preparation method and application thereof - Google Patents
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 49
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 title claims abstract description 26
- 108010024636 Glutathione Proteins 0.000 title claims abstract description 13
- 229960003180 glutathione Drugs 0.000 title claims abstract description 13
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims abstract description 27
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- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 description 2
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- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a glutathione fluorescent probe and a preparation method and application thereof, wherein the structure of the probe is shown as a formula (1); the preparation method comprises the following steps: dissolving 2, 4-dinitrofluorobenzene, fluorescein and potassium carbonate in anhydrous DMF, and reacting to generate a glutathione fluorescent probe; the application is used for detecting the content of glutathione with non-diagnostic property. The fluorescent probe only performs a fluorescent reaction with glutathione, has no reaction to other amino acids, and has good selectivity and specificity; the probe preparation process is simple and feasible, and is easy for large-scale production.
Description
Technical field
The present invention relates to a kind of gsh fluorescent probe and its production and use.
Background technology
Mercaptan has important effect in the antioxidant system of cell, be numerous protein and micromolecular important component part in the organism.Small molecules mercaptan mainly comprises gsh (GSH), halfcystine (Cys), homocysteine (Hcy).A large amount of physiological phenomenons is considered to depend on the reaction that these thio-alcohol materials that comprise sulfydryl participate in the organism, thanks to reaction as sulfo-in redox reaction, Methyl transporters reaction and the organism.The concentrations of mercaptans that contains in the organism is considered to relevant with numerous disease.The shortage of mercaptan can cause many health problems in the organism, as cessation of growth cessation, and alopecia, drowsiness, liver injury, become thin and some dermatosis.Gsh is the highest small molecules mercaptan (1-10mM) of cell intensive amount, and there is the redox running balance of Sleep-promoting factor B (GSSG) and reduced form GSH in it.Great mass of data shows, gsh is being kept the redox running balance of cell, oxidative stress and played an important role in the growth of cell, function, and the level of gsh also has directly with numerous disease and cancer and contacts.Therefore, in the detection by quantitative organism mercaptan particularly the concentration of gsh is significant in biological chemistry and clinical chemistry.
Method for detection of gsh mainly is divided into four big classes at present: spectrophotometry, high performance liquid chromatography, enzyme transforming process and fluorescent probe method.
Spectrophotometry produces color based on sulfydryl and developer reaction mostly, so other amino acid that contains sulfydryl can seriously disturb the mensuration of GSH.In addition, the gained color and luster is very unstable after the color reaction, and colorimetric needs to finish in 30 seconds, otherwise its optical density(OD) can significantly reduce.
Though high performance liquid chromatography can be distinguished the amino acid derivative that GSH and kind more than 20 contain sulfydryl, its sensitivity is not high, and sample pretreatment process is consuming time, and employed chromatographic column is special, is difficult for obtaining.
The example of using enzyme transforming process detection gsh is also a lot, for example patent 201210568880.3 discloses a kind of use gsh oxydo-reductase and has detected the gsh method, it may further comprise the steps: 1) PCR clone gsh oxidoreductase gene, 2) make up the recombinant expressed bacterium of gsh oxydo-reductase, 3) the recombinant expressed bacterium abduction delivering of gsh oxydo-reductase, 4) obtain gsh oxydo-reductase enzyme liquid, 5) preparation test sample, 6) make up catalytic reaction system, 7) detect and calculating concentration.Though the sensitivity of enzyme transforming process is higher, its process is loaded down with trivial details, and especially the preparation of saccharase is very consuming time.
The fluorescent probe method is highly sensitive, response is quick, technology is simple, but because gsh (GSH), halfcystine (Cys), homocysteine (Hcy) are structurally very similar, the fluorescent probe of the present detection GSH that develops produces same or analogous influence to Cys and Hcy.Need badly and develop the fluorescent probe that highly selective detects GSH.
Summary of the invention
Technical problem to be solved by this invention is: overcome the problem that prior art exists, the fluorescent probe of a kind of highly selective and high-sensitivity detection gsh is provided, and preparation method and the purposes of this probe in gsh detects of this fluorescent probe are provided.
Technical conceive of the present invention is as follows: the applicant finds that after deliberation 2,4-dinitrophenyl fluorescein ether compound is alternative to react with gsh and the generation intense fluorescence.This reaction is not subjected to the interference of other compounds containing thiol groups, and the fluorescence intensity that produces and glutathione concentrations linear dependence.The inventor prepares 2,4-dinitrophenyl fluorescein ether first and uses it for the selectivity detection of gsh first, thereby solves the problems of the technologies described above.
The technical scheme that the present invention solves its technical problem is as follows:
A kind of gsh fluorescent probe is characterized in that, described probe be as the formula (1) 2,4-dinitrophenyl fluorescein ether:
A kind of preparation method of above-mentioned gsh fluorescent probe is characterized in that, may further comprise the steps:
2,4-dinitrofluorobenzene, fluorescein and salt of wormwood are dissolved in the dry DMF, 2 of reaction back generation, 4-dinitrophenyl fluorescein ether is the gsh fluorescent probe.
Preferably, reaction conditions is: reacted 2 hours in 75 C under the nitrogen protection.
Preferably, carry out purifying after the reaction: earlier reaction product is dried after with distilled water wash crude product, be eluent with the methylene dichloride then, behind purifying crude product on the silicagel column, namely get the pure product of gsh fluorescent probe.
The glutathione content that above-mentioned gsh fluorescent probe is used for non-diagnostic properties detects.
Beneficial effect of the present invention is as follows:
1) fluorescent probe of the present invention is colourless and does not have fluorescence in solution, shows yellow and sends strong green fluorescence with gsh reaction back.
2) adopt fluorescent probe of the present invention after, the detection sensitivity height can reach 10 to the detectability of gsh
-8M.
3) fluorescent probe of the present invention only with gsh generation fluorescent reaction, all reactionless to other amino acid, have good selectivity and specificity.In addition, the colour developing phenomenon is not subjected to the influence of pH value and has suitable fluorescent emission wavelength (520nm).
4) preparation is simple for fluorescent probe of the present invention, is easy to large-scale production.
Description of drawings
Fig. 1 is that the embodiment of the invention 1 makes the pure product of fluorescent probe
1H-NMR figure.
Fig. 2 makes the high resolution mass spectrum figure of the pure product of fluorescent probe for the embodiment of the invention 1.
Fig. 3 is the fluorescence emission spectrum of the embodiment of the invention 2 fluorescent probes and the reaction of each seed amino acid.
Fig. 4 is the ultraviolet-visible absorption spectroscopy of the embodiment of the invention 2 fluorescent probes and the reaction of each seed amino acid.
Fig. 5 is the fluorescence increment graph of the embodiment of the invention 3 fluorescent probes and gsh reaction.
Fig. 6 is that the embodiment of the invention 3 fluorescent probes are to the fluorescence intensity working curve of glutathione concentrations.
Fig. 7 is the embodiment of the invention 4 fluorescent probes and three kinds of colour-change that biological thiol reacts respectively.
Fig. 8 is the embodiment of the invention 4 fluorescent probes and three kinds of change in fluorescence that biological thiol reacts respectively.
Embodiment
With reference to the accompanying drawings and in conjunction with the embodiments the present invention is described in further detail.But the invention is not restricted to given example.
With 1.861g2,4-dinitrofluorobenzene, 1.994g fluorescein and 2.764g salt of wormwood are dissolved in the 20mL dry DMF; Under nitrogen protection, in 75 ° of C reactions 2 hours; After reaction finishes, add 20mL distilled water wash, filtration, with the solid that obtains in vacuum drying oven, dry crude product; Be eluent with the methylene dichloride, purifying crude product on silicagel column obtains being 2 of yellow solid, 4-dinitrophenyl fluorescein ether 1.214g, be the pure product of gsh fluorescent probe (
1H-NMR figure and high resolution mass spectrum figure see Fig. 1, Fig. 2).The pure product actual measurement of gained fluorescent probe molecular weight is 664.
The present embodiment operational path:
Take by weighing 6.6mg embodiment 1 and make the gsh fluorescent probe, being made into concentration is the 10mL acetonitrile solution of 1mM, as mother liquor.
Fluorescence spectrum test: the above-mentioned mother liquor of 15 μ L and 1.5mM bromohexadecane base Trimethylamine 99 are joined a certain amount of 20mM ethanol-PBS buffered soln (3:7v/v, pH7.4) in, add each seed amino acid then respectively: one of gsh, halfcystine, homocysteine, glycine, phenylalanine, methionine(Met), L-glutamic acid, glutamine, Methionin, arginine, Histidine, L-Ala, Serine and tyrosine, making the amino acid final concentration is 50 μ M, and the fluorescent probe final concentration is 5 μ M.Behind the reaction 30min, under the 485nm excitation wavelength, test its fluorescence emission spectrum under the normal temperature.Exciting with the slit width of launching is 1.5nm.The gained result as shown in Figure 3.
Absorption spectrum test: the above-mentioned mother liquor of 150 μ L and 1.5mM CTAB are joined a certain amount of 20mM ethanol-PBS buffered soln (3:7v/v, pH7.4) in, add each seed amino acid then respectively: one of gsh, halfcystine, homocysteine, glycine, phenylalanine, methionine(Met), L-glutamic acid, glutamine, Methionin, arginine, Histidine, L-Ala, Serine and tyrosine, making the amino acid final concentration is 500 μ M, and the fluorescent probe final concentration is 50 μ M.Behind the reaction 30min, test its absorption spectrum under the normal temperature.The gained result as shown in Figure 4.
Above result shows:
(1) embodiment 1 makes fluorescent probe this is colourless and does not have fluorescence in solution, but with the adding of gsh, this probe produces at 500nm place and absorbs, and at 520nm place generation green fluorescence.
(2) embodiment 1 makes fluorescent probe gsh is had selectivity and the specificity of height, and under these conditions, can distinguish gsh from three kinds of common biological thiols (gsh, halfcystine and homocysteine).
Embodiment 3 makes the spectral quality of gsh fluorescent probe and gsh reaction product
Mother liquor among the 3 μ L embodiment 2 and 1.5mM CTAB are joined a certain amount of 20mM ethanol-PBS buffered soln (3:7v/v, pH7.4) in, the glutathione solution that adds different equivalents then, the final concentration that makes fluorescent probe is 1 μ M, and the gsh final concentration is respectively 0 μ M, 1 μ M, 2 μ M, 3 μ M, 4 μ M, 5 μ M, 6 μ M, 7 μ M, 8 μ M, 9 μ M, 10 μ M.Behind the reaction 30min, measure its fluorescence emission spectrum under the normal temperature.When measuring, fluorescence emission spectrum excites with 485nm; Exciting with the slit width of launching is 3nm.Gained fluorescence increment graph is seen Fig. 5; Fluorescence intensity data with the 520nm place is made working curve, the results are shown in Figure 6.
This experimental result shows that reacted fluorescence intensity increases with the increase of glutathione concentrations; When adopting the fluorescent probe of final concentration 1 μ M, the glutathione concentrations in reaction back fluorescence intensity and the 0-10 μ M scope is linear, and the glutathione content that can be used for non-diagnostic properties detects.
Mother liquor among the 60 μ L embodiment 2 and 1.5mM CTAB are joined a certain amount of 20mM ethanol-PBS buffered soln (3:7v/v, pH7.4) in, add a kind of biological thiol solution (one of gsh, halfcystine, homocysteine) then respectively, making the amino acid final concentration is 20 μ M, and the fluorescent probe final concentration is 20 μ M; Use 20mM ethanol-PBS buffered soln constant volume to 3mL again.Behind the reaction 30min, observe its colour-change under the normal temperature, the results are shown in Figure 7, the sample hose that adds probe except gsh shows light green, and other sample hose is colourless; Use the 365nm ultra violet lamp simultaneously, observe its change in fluorescence, the results are shown in Figure 8, the sample hose that adds probe except gsh shows green fluorescence, and other sample hose does not all have fluorescence.
This experimental result shows that embodiment 1 makes fluorescent probe can distinguish gsh from three kinds of biological thiols (gsh, halfcystine, homocysteine), and its color, change in fluorescence naked eyes are as seen.
In addition to the implementation, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of requirement of the present invention.
Claims (5)
1. a gsh fluorescent probe is characterized in that, described probe be as the formula (1) 2,4-dinitrophenyl fluorescein ether:
2. the preparation method of the described gsh fluorescent probe of claim 1, it is characterized in that, may further comprise the steps: 2,4-dinitrofluorobenzene, fluorescein and salt of wormwood are dissolved in the dry DMF, 2 of reaction back generation, 4-dinitrophenyl fluorescein ether is the gsh fluorescent probe.
3. according to the described preparation method of claim 2, it is characterized in that reaction conditions is: reacted 2 hours in 75 ° of C under the nitrogen protection.
4. according to the described preparation method of claim 3, it is characterized in that, carry out purifying after the reaction: earlier reaction product is dried after with distilled water wash crude product, be eluent then with the methylene dichloride, behind purifying crude product on the silicagel column, namely get the pure product of gsh fluorescent probe.
5. the described gsh fluorescent probe of claim 1 is used for the glutathione content detection of non-diagnostic properties.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103601679A (en) * | 2013-12-04 | 2014-02-26 | 山东大学 | Reduced glutathione fluorescent probe with pyrazoline as maternal body |
| CN104710975A (en) * | 2015-01-06 | 2015-06-17 | 南京工业大学 | Gold ion probe and preparation method and application thereof |
| CN104974169A (en) * | 2014-04-09 | 2015-10-14 | 中国科学院大连化学物理研究所 | Fluorescein H2S fluorescent probe as well as preparation and application thereof |
| CN105601658A (en) * | 2016-01-15 | 2016-05-25 | 中南大学 | Application and preparation method of novel fluorescent probe capable of distinguishing biological mercaptans |
| CN105969337A (en) * | 2016-05-13 | 2016-09-28 | 南京理工大学 | GSH fluorescence sensor based on rhodamine derivates, preparation method and application |
| CN108101929A (en) * | 2017-12-15 | 2018-06-01 | 西安交通大学 | A kind of GSH fluorescent optical sensors and its preparation method and application |
| CN114853779A (en) * | 2022-06-09 | 2022-08-05 | 南京工业大学 | Fluorescent probe for detecting biological thiol in cells and organisms and quickly labeling thiol protein specifically, preparation method and application |
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| 苏转: "《含亲电基团的荧光探针的合成及其在生物分析中的应用研究》", 《中国优秀硕士学位论文全文数据库（西北大学硕士学位论文）》 * |
| 陈亭: "《荧光素钠互变异构体的表面增强共振喇曼散射光谱》", 《物理化学学报》 * |
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