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Novel mercapto fluorescence probe and uses thereof

A fluorescent probe and sulfhydryl technology, applied in the field of fluorescent probes, can solve the problems of unstable light, difficult fluorescence imaging, low signal-to-noise ratio, etc., and achieve the effects of high sulfhydryl determination sensitivity, rapid fluorescence change, and enhanced fluorescence intensity.

Active Publication Date: 2008-08-27
合肥硕健医药科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The reported sulfhydryl fluorescent probes include aryl halides, dansylaziridines, etc., which have good selectivity and sensitivity, and the detection limit of sulfhydryl is generally around nanomolar (nmol), but these probes themselves All have strong fluorescence, which will cause a low signal-to-noise ratio when used in live cell imaging; there is also a class such as benzofuransulfonyl halide, although its autofluorescence is weak, but it needs to be used in alkaline conditions and above room temperature Under derivatization with sulfhydryl groups, it cannot be applied to live cell imaging
The most commonly used reagents for derivatizing sulfhydryl groups are acetyl halide derivatives, mainly iodoacetamide derivatives, which react quickly with sulfhydryl groups and can be carried out at room temperature and physiological pH, but the products are prone to lose fluorophores and are unstable to light
These existing sulfhydryl fluorescent probes cannot well meet the requirements of bioluminescence detection and fluorescence imaging.
[0005] Maleimide fluorescent compounds can undergo stable addition to sulfhydryl groups, and their autofluorescence is weak. After addition to sulfhydryl groups, the fluorescence increases. This characteristic of fluorescence enhancement only after the reaction makes it unique. Superiority, but after the general fluorophore is combined with maleimide, the quantum yield is low, the fluorescence lifetime is short, and the Stokes shift is small, it is difficult to perform high-quality intracellular fluorescence imaging

Method used

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  • Novel mercapto fluorescence probe and uses thereof
  • Novel mercapto fluorescence probe and uses thereof
  • Novel mercapto fluorescence probe and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: the synthetic route of probe 1 and probe 2:

[0043](1) Preparation of arylamine diazonium salt: Weigh 3-6g of aniline and add it to 40mL of water, then add 15mL of concentrated hydrochloric acid to completely dissolve the aniline into hydrochloride, and cool to 0°C in an ice bath. Weigh 2.3-5g of sodium nitrite and dissolve it in a small amount of water to form a solution, cool with ice, slowly add to the above solution while stirring, and then the benzenediazonium chloride solution can be prepared. Use potassium iodide test paper to test whether it turns blue. If it turns blue, it means that the solution contains excess nitrous acid, which can be neutralized with urea until the potassium iodide test paper does not change color;

[0044] (2) Preparation of 5-azophenyl salicylaldehyde: Weigh 2-4g NaOH and dissolve it in 300mL water, add it into a three-necked bottle containing 5-10g salicylaldehyde, and dissolve it into salicylaldehyde sodium salt in wate...

Embodiment 2

[0057] Embodiment 2: Fluorescence spectrum change of probe 1 and probe 2 reacting with thiol compound:

[0058] Dissolve probe 1 and probe 2 in DMSO to a final concentration of 100 μM, take 200 μl and react with 1 mM thiol-containing compound L-cysteine ​​(L-Cys) for 1, 5 min, then measure the change in fluorescence intensity, The wavelength is 336nm. The results can be seen in Figure 1 and Figure 2, and all fluorescence intensities were normalized.

[0059] It can be seen from Figure 1 and Figure 2 that the background fluorescence intensity of these two probes is very low, and after reacting with the thiol compound L-Cys, the fluorescence intensity is significantly enhanced within 1 minute (probe 1: 11.4 times, probe 2: 8.9 times), the maximum emission wavelength of fluorescence is at 505nm. After five minutes of reaction, the fluorescence tended to be stable, the fluorescence intensity of probe 1 was enhanced by 19.5 times, and the fluorescence intensity of probe 2 was enh...

Embodiment 3

[0060] Embodiment 3: the selectivity of probe to thiol

[0061] Probe 1 and probe 2 were taken and dissolved in DMSO to a concentration of 1 mM. Add two kinds of probes to the 96-well plate, add hydrogen peroxide, sodium sulfide, nitrate, nitrite, ammonium salt, cystine, and L-Cys respectively, and then add deionized water to all samples, The final concentration of probes obtained in each well was 100 μM, and the molar amounts of hydrogen peroxide, sodium sulfide, nitrate, nitrite, ammonium salt, cystine, and L-Cys were 100 times that of the corresponding probes, and the total The volume is 150 μl. After reacting for 1 hour, measure the fluorescence intensity at 505nm on a microplate reader, and divide the fluorescence intensity after the reaction by the fluorescence intensity of the original probe to obtain the fluorescence enhancement multiple after the reaction, and then determine the pairing of probe 1 and probe 2. Specificity of sulfhydryl reaction. The results are sho...

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Abstract

The invention belongs to the technical field of biological detection, which particularly relates to a fluorescent probe which can be applied in measuring active mercapto image in living cells and the application. The fluorescent probe is 2-(2-hydroxyphenyl) hydroxyphenyl compound which is decorated by maleimide or maleimide acid, the structure of the compound is N-{3-(2- benzimidazolyl)-4-hydroxyphenyl}maleimide and N-{3-(2- benzimidazolyl)-4-hydroxyphenyl}malemide acid. The compound can simply and rapidly get into living cells, which has a reaction with free active mercapto group in cells, and fluorescence intensity of the compound is led to be obviously enhancing, thereby the fluorescent probe can be used in fluorescent measurement or fluorescent image of fluactive mercapto group (such as glutathione and the like) in living cells.

Description

a technical field [0001] The invention belongs to the technical field of biological detection, specifically relates to a technique for measuring sulfhydryl content in living cells or living organisms, and is a fluorescent probe that can be used for imaging and measuring sulfhydryl in living cells and its application. Two background technology [0002] Sulfhydryl (-SH) is the most chemically active group in cells. In proteins, the sulfhydryl moiety is the most reactive functional group associated with enzymatic activity. The sulfhydryl group in cells, especially glutathione (GSH), as a nucleophilic and reducing agent in cells, can protect cells from hypoxia, toxins, mutagenesis, radiation and many carcinogens. With the gradual recognition of the important role of thiol functional groups in biological systems, methods for the identification and quantitative determination of biothiol compounds have attracted increasing attention. Traditional detection methods include nitropru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06C07D403/10C07D235/18G01N21/64
Inventor 张峻峰欧阳杰洪浩陈江宁沈超赵勇
Owner 合肥硕健医药科技有限公司
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