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Vesicle for detecting phospholipase a2 activity, preparation method thereof and method for detecting phospholipase a2 activity with fluorescent probe

A fluorescent probe, phospholipase technology, applied in fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of low sensitivity of titration method, expensive probe sensitivity, insufficient sensitivity, etc., and achieve molecular simplicity, life span and enzyme activity correlation Good, accurate results

Inactive Publication Date: 2018-05-18
EAST CHINA UNIV OF SCI & TECH
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Problems solved by technology

[0005] Each of the above methods has different problems: the titration method is not very sensitive; the radiolabeling method is very sensitive, but requires phospholipids labeled with radioactive elements, which are usually very expensive; spectrophotometry and fluorescence analysis methods are the most widely used, but usually The labels and probes used are expensive or not sensitive enough

Method used

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  • Vesicle for detecting phospholipase a2 activity, preparation method thereof and method for detecting phospholipase a2 activity with fluorescent probe
  • Vesicle for detecting phospholipase a2 activity, preparation method thereof and method for detecting phospholipase a2 activity with fluorescent probe
  • Vesicle for detecting phospholipase a2 activity, preparation method thereof and method for detecting phospholipase a2 activity with fluorescent probe

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Embodiment 1

[0028] The drawing of embodiment 1 standard curve

[0029] Lecithin and cholesterol that have been accurately weighed to calculate the weight are mixed and dissolved in the mixed solution of chloroform-methanol in a mass ratio of 3:1 (the volume ratio of chloroform and methanol is 2:1), and then use a rotary evaporator at 40 °C rotary evaporation in a constant temperature water bath. After the solvent is completely evaporated, take it out and put it in a vacuum desiccator to evacuate, so as to evaporate the solvent as much as possible. Then, 10 mL of Tris-HCl buffer containing 1-naphthol at a concentration of 4 μmol / L was added to make the total lipid concentration 3%, to obtain a pale yellow solution. Stir to make the membrane fall off, and incubate at 60°C for 1 hour to optimize the vesicle structure and ensure the balanced distribution of probe molecules in the aqueous phase and vesicles. Then, measure its spectrum after it stabilizes. When measuring the steady-state spec...

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Abstract

The present invention provides a vesicle for detecting the activity of phospholipase A2, a preparation method thereof, and a method for detecting the activity of phospholipase A2 by using the vesicle. Using 1-naphthol as a British crown probe, the fluorescence intensities at 450 nm and 360 nm are respectively measured And obtain the ratio of the two, and read the corresponding enzyme activity degree in the standard curve. The probe molecule used in the present invention is simple and inexpensive, and its special properties are very suitable for the determination of the vesicle type substrate structure and enzyme-catalyzed reaction with phospholipids. At the same time, the detection method of the present invention has a good correlation between steady-state spectrum, lifetime and enzyme activity, and the results obtained by the two methods are relatively matched.

Description

technical field [0001] The present invention relates to phospholipase A 2 Activity detection, specifically a detection of phospholipase A 2 Active vesicle and its preparation method and application of the vesicle fluorescent probe to detect phospholipase A 2 active method. Background technique [0002] Phospholipids are widely found in all animal and plant organisms in nature and are the main components of biofilms. The research on the metabolic process of phospholipids, such as the hydrolysis of phospholipases, plays an important role in the field of biochemistry. [0003] Phospholipase A 2 (PLA 2 ) is a family of enzymes that catalyze the hydrolysis of phospholipid diacyl (Sn2), which is widely found in tissues, cells and secretions of bacteria, plants and mammals, and has the ability to produce eicosanic acid inflammatory mediators and participate in phospholipid remodeling , alveolar surfactant metabolism, cell signal transmission, host response, promotion of blood...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
Inventor 安学勤牟尧李素军
Owner EAST CHINA UNIV OF SCI & TECH
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