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A preparation method and application of a fluorescent molecular probe for detecting glutathione thiol transferase

A fluorescent molecular probe and sulfhydryl transferase technology, applied in the field of analytical chemistry, can solve the problem of inability to provide in-situ and real-time information of glutathione sulfhydryl transferase, achieve fast detection speed, high sensitivity, and improve detection sensitivity Effect

Active Publication Date: 2022-04-12
BINZHOU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These assays can be used to detect and track GST in cell lysates or in vitro, however, they cannot provide in situ and real-time information on GST in living cells and in vivo

Method used

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  • A preparation method and application of a fluorescent molecular probe for detecting glutathione thiol transferase
  • A preparation method and application of a fluorescent molecular probe for detecting glutathione thiol transferase
  • A preparation method and application of a fluorescent molecular probe for detecting glutathione thiol transferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Preparation of multifunctional ultrasensitive glutathione thiol transferase detection probe

[0057] (1) Dissolve 75 mmol 2,3,3-trimethyl-3H-benzindole and 75 mmol ethyl iodide in 40 mL of anhydrous acetonitrile, mix in a 250 mL round bottom flask, and then reflux the mixture Reacted for 12 hours, then turned off the heat and cooled to room temperature. The precipitate was filtered through a Buchner funnel, the solid product was washed with ether and dried in vacuo to give product 1;

[0058] (2) Put a solution of 40 mL of anhydrous N,N-dimethylformamide (DMF) and 40 mL of anhydrous dichloromethane in a 250 mL round bottom flask, cool the solution to -10°C, and stir for 20 minutes . Then, 37 mL of phosphorus oxychloride and 35 mL of anhydrous dichloromethane were added dropwise to the above solution through a constant pressure dropping funnel. Then 52.6 mmol cyclohexanone was added to the mixture in batches, and the solution changed from colorless to yello...

Embodiment 2

[0068] Implementation Example 2: The multifunctional ultrasensitive glutathione thiol transferase prepared in Example 1 detects the fluorescent molecular probe quantitative analysis of glutathione thiol transferase in the standard product

[0069] 1) Prepare the solution

[0070] Probe stock solution: Accurately weigh the multifunctional ultrasensitive glutathione sulfhydryl transferase detection fluorescent molecular probe and dissolve it in anhydrous DMSO to prepare a probe stock solution with a concentration of 100 μM;

[0071] Glutathione thiol transferase stock solution: Accurately weigh 1 mg of glutathione thiol transferase, the target substance to be tested, and dissolve it in 10 ml of distilled water to prepare a glutathione thiol transferase stock solution with a concentration of 100 μg / mL;

[0072] 2) Establish the linear equation of glutathione sulfhydryl transferase standard

[0073] Dilute the glutathione thiol transferase stock solution prepared in step 1) with ...

Embodiment 3

[0074] Implementation Example 3: Qualitative detection of glutathione sulfhydryl transferase in biological samples

[0075] Detection of glutathione sulfhydryl transferase in living cells, the method is: culture IMR-90 cells in the culture medium for 18-26 h, and the inoculation amount of living cells to be tested is 2×10 7 ~9×10 7Each / mL, then add multifunctional ultrasensitive glutathione sulfhydryl transferase to detect fluorescent molecular probes, the probe concentration is 1 μM, incubate at 37°C for 30 minutes, wash with PBS solution for several times, and then observe under a fluorescent microscope Cell imaging. The result of cell imaging is: the fluorescence is enhanced, so it can be judged that the IMR-90 cells contain glutathione sulfhydryl transferase.

[0076] Detection of glutathione sulfhydryltransferase in living mice as follows: C57BL / 6 mice (8 weeks) were housed on a 12-h light / dark cycle with free access to food and water. Pulmonary fibrosis animal models ...

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Abstract

The invention belongs to the field of analytical chemistry, and the fluorescent molecular probe for detecting glutathione thiol transferase is prepared by the following steps: dissolving 2,3,3-trimethyl-3H-benzindole and ethyl iodide React in anhydrous acetonitrile to obtain product 1; mix anhydrous N,N-dimethylformamide and anhydrous dichloromethane, add dropwise phosphorus oxychloride and anhydrous dichloromethane, add cyclohexanone, pour into Product 2 was collected on ice; product 1 and product 2 were dissolved in n-butanol and benzene solution to obtain product 3; product 3, methylamine hydrochloride and triethylamine were dissolved in anhydrous N,N-dimethylformamide 3,4-dinitrobenzoic acid and triphosgene were dissolved in anhydrous dichloromethane and mixed, N,N-diisopropylethylamine was added to react, and the obtained residue was dissolved in anhydrous Add N,N-diisopropylethylamine and 4-dimethylaminopyridine to dichloromethane water, dissolve the product 4 in dichloromethane and add the above mixture to obtain the molecular probe.

Description

technical field [0001] The invention belongs to the field of analytical chemistry, and relates to a preparation method and application of a fluorescent molecular probe for detecting glutathione sulfhydryl transferase. Background technique [0002] Glutathione sulfhydryltransferase is a group of phase II metabolic enzymes commonly found in various organisms. It is the main detoxification system in many organisms. It is related to the excretion of many physiological and heterologous substances. Its common substrate is 2 , 4-Dinitrochlorobenzene (CDNB). Glutathione sulfhydryltransferase can catalyze the sulfhydryl group (-SH) of glutathione and some insoluble substances in water, such as dyes, bile acids, heme, hormones and other foreign substances with strong hydrophobicity, etc. Combined with the metabolites of the source, the polarity is enhanced, it is easily soluble in water, and finally it is degraded and excreted. In addition, glutathione thiol transferase also plays a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D209/60C09K11/06A61K49/00G01N21/64
Inventor 吕长俊陈令新赫娜于法标黄严
Owner BINZHOU MEDICAL COLLEGE
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