Fluorescent probe for detecting beta-galactosidase as well as preparation method and application of fluorescent probe

A galactosidase and fluorescent probe technology, applied in the preparation of sugar derivatives, biochemical equipment and methods, fluorescence/phosphorescence, etc., can solve the problems of high cost, complex probe synthesis, etc. Good performance and high sensitivity

Inactive Publication Date: 2020-10-13
WUHAN INSTITUTE OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many fluorescent probes for β-galactosidase have been reported, the synthesis of such probes is relatively complicated and the cost is relatively high, and the probes are mainly used for the detection of β-galactosidase at the cellular level or in vivo. horizontal diagnostic imaging
Probes for β-galactosidase inhibitor screening are rarely reported

Method used

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  • Fluorescent probe for detecting beta-galactosidase as well as preparation method and application of fluorescent probe
  • Fluorescent probe for detecting beta-galactosidase as well as preparation method and application of fluorescent probe
  • Fluorescent probe for detecting beta-galactosidase as well as preparation method and application of fluorescent probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] A fluorescent probe for detecting β-galactosidase, the preparation method of which comprises the following steps:

[0043] 1) Synthesis of compound a: Weigh 1g of 2,4-dihydroxybenzaldehyde (7.2mmol), place 1.57g of sodium propionate (16.3mmol) in a 25mL round-bottomed flask, measure 3.16mL of propionic anhydride ( 24.5mmol), 1.15mL of dry triethylamine (8.3mmol) were added to the above system, heated to 170°C and refluxed for 2h, the reaction was complete as monitored by TLC, after the reaction was completed, cooled to room temperature, stirred with water, at this time there were a large amount of white solid separated out, then extracted with ethyl acetate, washed the organic phase three times with water, anhydrous MgSO 4Drying, spin-drying, and then by column chromatography (ethyl acrylate:petroleum ether=1:6, v / v) to obtain a light yellow solid, then recrystallized with ethyl acrylate, after vacuum drying to obtain 1.1g of product a, the yield About 65%. product a ...

Embodiment 2

[0055] Measure the effect of the fluorescent probe obtained in Example 1 and β-galactosidase reaction:

[0056] Add 3mL of fluorescent probe molecules prepared in Example 1 (10 μM, dissolved in PBS buffer solution with a pH value of 7.4 at 37°C) to the cuvette as a control group, test its ultraviolet absorption spectrum and fluorescence spectrum, and then In another cuvette, add 3 mL of fluorescent probe molecules prepared in Example 1 (10 μM, dissolved in PBS buffer solution with a pH value of 7.4) and a mixture of β-galactosidase (0.10 U / mL) as The experimental group tested its ultraviolet absorption spectrum and fluorescence emission spectrum. Such as Figure 7 Shown is the high performance liquid chromatography and mass spectrometry of the fluorescent probe detecting β-galactosidase. It can be seen from the figure that β-galactosidase breaks the D-galactosidic bond and releases the fluorescent group, which illustrates the reaction mechanism of the probe Consistent with a...

Embodiment 3

[0058] Measure the change of the concentration of the fluorescent probe obtained in Example 1 and the β-galactosidase reaction intensity of different concentrations:

[0059] Add 3 mL of fluorescent probe molecules (10 μM, dissolved in PBS buffer solution with a pH value of 7.4 at 37° C.) and different concentrations of β-galactosidase (0 , 0.01, 0.03, 0.05, 0.07, 0.1, 0.15U / mL) mixed solution, after reacting for 15 minutes, test the fluorescence spectrum of each mixed solution. As the concentration of β-galactosidase increases (0-0.10U / mL), the fluorescence intensity increases gradually, and a red shift occurs at the same time. However, when a larger amount of β-galactosidase (0.15U / mL) was added, the fluorescence intensity no longer increased, which indicated that 0.10U / mL β-galactosidase could catalyze the hydrolysis of 10 μM fluorescent probe, different The titration test chart of the concentration of β-galactosidase to the fluorescent probe is as follows Figure 11 show...

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Abstract

The invention relates to a fluorescent probe for detecting beta-galactosidase as well as a preparation method and application of the fluorescent probe. The chemical structural formula of the fluorescent probe is shown as (I). The fluorescent probe provided by the invention is high in sensitivity, low in detection limit (0.168 mU / mL), good in selectivity, quick in response and capable of specifically detecting the activity and content of beta-galactosidase. A fluorescent method for screening a beta-galactosidase inhibitor is established for the first time on the basis of the fluorescent probe,the test result is consistent with the test result of a commercial substrate, and the method is reliable.

Description

technical field [0001] The invention belongs to the technical field of chemical analysis and detection, and relates to a fluorescence-enhanced probe of β-galactosidase, a preparation method thereof and an application in inhibitor screening. Background technique [0002] β-galactosidase is a hydrolase that hydrolyzes glycosidic bonds. It widely exists in various animals, plants and microorganisms, and plays an important role in maintaining normal life activities. Not only that, the activity and content of β-galactosidase are closely related to many diseases, such as ovarian cancer, renal tubular injury, Moore's syndrome, GM1 gangliosidosis and so on. Therefore, it is of great practical significance to develop a cheap, efficient and highly sensitive detection method for β-galactosidase activity detection and inhibitor screening. [0003] So far, traditional β-galactosidase detection techniques include magnetic resonance, colorimetry, chemiluminescence, electrochemical methods...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H17/075C07H1/00C09K11/06G01N21/64C12Q1/40
CPCC07H17/075C07H1/00C09K11/06G01N21/6428G01N21/6486C12Q1/40C09K2211/1088G01N2021/6432G01N2333/938
Inventor 孙琦
Owner WUHAN INSTITUTE OF TECHNOLOGY
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