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Method for detecting protease activity and inhibitor activity thereof based on chemiluminescence

A protease inhibitor, chemiluminescence technology, used in chemiluminescence/bioluminescence, analysis by chemical reaction of materials, etc., can solve the problems of cumbersome operation, high cost, low detection sensitivity, etc., to protect the environment, reduce Pollution, simple operation effect

Active Publication Date: 2013-04-24
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve the problems of complex flow path design, cumbersome operation, low detection sensitivity and high cost in existing methods for detecting enzyme activity, the present invention provides a simple, fast, highly sensitive and low-cost method for detecting protease activity based on chemiluminescence and methods for its inhibitory activity

Method used

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  • Method for detecting protease activity and inhibitor activity thereof based on chemiluminescence
  • Method for detecting protease activity and inhibitor activity thereof based on chemiluminescence
  • Method for detecting protease activity and inhibitor activity thereof based on chemiluminescence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1 Detection of trypsin activity

[0095] Step 1. Take nine 3 mL sample tubes and add cytochrome C with a final concentration of 50 nM respectively. Cytochrome C is mixed with Tris-HCl buffer solution (1 mM CaCl 2 ) diluted, pre-incubated in a 37°C water bath for 5 minutes;

[0096]Step 2: Add trypsin solutions with final concentrations of 0, 2, 5, 10, 20, 50, 100, 150, and 200 ng / mL to the 9 sample tubes in step 1, and mix them evenly immediately. The volume is 3mL to obtain a hydrolyzed solution;

[0097] Step 3: Take 200 μL of the hydrolysis solution in Step 2 and inject it into the flow injection chemiluminescence flow path, and undergo a chemiluminescent reaction with the luminol-hydrogen peroxide detection system. As the sample and the detection system flow through the detector, the generated chemical The luminescence is detected by the detector, collected and displayed on the display by the data acquisition system, and the chemiluminescence intensity val...

Embodiment 2

[0099] Example 2 Detection of proteinase K activity

[0100] Step 1. Take nine 3 mL sample tubes and add cytochrome C with a final concentration of 50 nM respectively. Cytochrome C is mixed with Tris-HCl buffer solution (1 mM CaCl 2 ) diluted, pre-incubated in a 35°C water bath for 10 minutes;

[0101] Step 2: Add proteinase K solution with a final concentration of 0, 2, 5, 10, 20, 50, 100, 150, and 200 ng / mL to the 9 sample tubes in step 1 in sequence, and mix them evenly immediately. The volume is 3mL to obtain a hydrolyzed solution;

[0102] Step 3: Take 180 μL of the hydrolysis solution in Step 2 and inject it into the flow injection chemiluminescence flow path, and undergo a chemiluminescence reaction with the luminol-hydrogen peroxide detection system. As the sample and the detection system flow through the detector, the generated chemical The luminescence is detected by the detector, collected and displayed on the display by the data acquisition system, the chemilumin...

Embodiment 3

[0104] Example 3 The detection of papain activity

[0105] Step 1. Take eight 3 mL sample tubes and add cytochrome C with a final concentration of 50 nM respectively. Cytochrome C is mixed with 50 mM Tris-HCl buffer solution (1mM CaCl 2 ) diluted, pre-incubated in a water bath at 40°C for 3 minutes;

[0106] Step 2: Add the papain solution with a final concentration of 0, 5, 10, 20, 50, 100, 200, 1000 ng / mL to the 8 sample tubes in step 1, mix immediately, and the total volume of the sample is 3mL to obtain a hydrolyzed solution;

[0107] Step 3: Inject 150 μL of the hydrolyzed solution in Step 2 into the flow injection chemiluminescent flow path, and undergo a chemiluminescent reaction with the luminol-hydrogen peroxide detection system. As the sample and the detection system flow through the detector, the generated chemical The luminescence is detected by the detector and collected and displayed on the display by the data acquisition system. The chemiluminescence intensity...

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Abstract

The invention provides a method for detecting protease activity and inhibitor activity thereof based on chemiluminescence, and relates to the technical field of biological analytical chemistry. The method for detecting the protease activity and the inhibitor activity thereof based on the chemiluminescence solves the problems that an existing method for detecting the enzyme activity is complex in flow design, complicated in operation, low in detection sensitivity, and high in cost. The method for detecting the protease activity and the inhibitor activity thereof based on the chemiluminescence comprises a first step of dissolving heme proteins into Tris - HCl buffer solution to achieve heme protein solution, a second step of adding protease with different concentrations into the heme protein solution of the first step to form a reaction system, and a third step of injecting the reaction system of the second step into a flow-injected chemiluminescence flow, and reacting a chemiluminescence reaction with a luminol hydrogen peroxide detecting system so as to detect the protease activity. The method for detecting the protease activity and the inhibitor activity thereof based on the chemiluminescence has the advantages of being simple, quick and high in sensitivity, low in cost, and low in detection limit, and the detection limit of trypsin can achieve to 1 ng / mL.

Description

technical field [0001] The invention relates to the technical field of bioanalytical chemistry, in particular to a method for detecting protease activity and its inhibitor activity based on a chemiluminescent method. Background technique [0002] Flow injection analysis technology is a new type of sample analysis technology developed since the 1980s. It uses a peristaltic pump to drive reagents and samples. When the reagents and samples are mixed and flow through the detector, the signal is detected. The basic flow road system such as figure 1 shown. Flow injection analysis technology has wide applicability and can be used in combination with various detection methods, and can be used for sample injection, online sample processing, automatic monitoring and process analysis, etc.; it has the advantage of high efficiency, and its sampling frequency can reach 40-300 / h; The sample consumption is low, generally 10-100 μL; the equipment is simple and cheap. [0003] Chemilumin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76
Inventor 于聪张青峰李文英陈健王方远王燕
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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