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Vesicle for detecting phospholipase A2 activity, preparation method of vesicle and fluorescence probe detection method of phospholipase A2 activity

A technology of fluorescent probes and phospholipases, applied in the fields of fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of low sensitivity of titration, expensive probes, sensitivity, and high cost, and achieve simple molecules, correlation between lifespan and enzyme activity Good, result-matching effect

Inactive Publication Date: 2015-11-25
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 of vesicle and fluorescence probe detection method of phospholipase A2 activity
  • Vesicle for detecting phospholipase A2 activity, preparation method of vesicle and fluorescence probe detection method of phospholipase A2 activity
  • Vesicle for detecting phospholipase A2 activity, preparation method of vesicle and fluorescence probe detection method of phospholipase A2 activity

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Embodiment 1

[0028] The drawing of embodiment 1 standard curve

[0029] Mix the lecithin and cholesterol that have been accurately weighed and calculated in a mass ratio of 3:1 and dissolve in a mixed solution of chloroform-methanol (the volume ratio of chloroform to 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. Next, 10 mL of Tris-HCl buffer solution containing 1-naphthol at a concentration of 4 μmol / L was added to make the total lipid concentration reach 3%, and a pale yellow solution was obtained. 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 water phase and vesicles. Then, measure its spectrum after it stabilizes. When measuring the steady-sta...

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Abstract

The invention provides a vesicle for detecting phospholipase A2 activity, a preparation method of the vesicle and a method for detecting phospholipase A2 activity with the application of the vesicle. 1-naphthol is used as a Yingguan probe to respectively measure fluorescence intensities at 450 nm and at 360 nm and obtain a ratio of the two fluorescence intensities, and a corresponding enzymatic activity degree is read from a standard curve. The probe molecule used in the invention is simple and low-cost, and special properties of the probe molecule enable the probe molecule to be very suitable for measuring a phospholipid-participated vesicle type substrate structure and an enzymic catalytic reaction. Meanwhile, the detection method has good steady-state spectrum, long life and good enzyme activity relationship. Results of the two methods match each other.

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 groups (Sn2). , alveolar surfactant metabolism, cell signal transmission, host response, promotion of blood coagulation, etc. Studies have shown that phospholipase A 2 In acute and chronic inflammatory diseases of the lung, cardiovascular, gastrointestinal tract, skin, bone and other systems, phos...

Claims

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Application Information

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Patent Type & Authority Applications(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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