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Novel high-efficiency protease activity detecting method

A technology for protease activity and detection method, applied in the field of novel and efficient protease activity detection, can solve the problems of lack of universal applicability, high cost, limited sensitivity, etc., and achieve the effects of high economical practicability, low cost and high sensitivity

Inactive Publication Date: 2013-06-12
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to provide a new and efficient protease activity detection method, which aims to solve the problems of limited sensitivity, high cost and lack of universal applicability of current protease detection methods

Method used

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  • Novel high-efficiency protease activity detecting method
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  • Novel high-efficiency protease activity detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: A novel and efficient protease activity detection method

[0048] The protease activity detection method flow chart can be found in figure 1 shown. This example is a method for detecting trypsin (English name: trypsin) activity.

[0049] first part:

[0050] The correlation between trypsin activity and the peak intensity of the fluorescent spectrum of the quantum dot synthesized by the corresponding product is established in advance, and the establishment of the correlation is composed of the following steps:

[0051] In the first step, for trypsin, a polypeptide substrate containing a specific cleavage site for the protease is designed, the polypeptide substrate includes a peptide chain composed of 4 amino acids, and the peptide chain contains Specifically cleaved peptide bonds and two cysteines (symbol: C) with sulfhydryl groups, the specific enzyme cleavage site is the amino terminal (chemical formula: NH 2 -) is a peptide bond between lysine (symbol: ...

Embodiment 2

[0071] Example 2: A novel and efficient protease activity detection method

[0072] The protease activity detection method flow chart can be found in figure 1 shown. This example is a method for detecting activity of collagenase (English name: collagenase).

[0073] first part:

[0074] The correlation between collagenase activity and the fluorescence spectrum peak intensity of the quantum dot synthesized by the corresponding product is established in advance, and the establishment of the correlation is composed of the following steps:

[0075] In the first step, for collagenase, a polypeptide substrate containing a specific cleavage site for the protease is designed, and the polypeptide substrate includes a peptide chain composed of 4 amino acids, and the peptide chain contains Specifically cleaved peptide bonds and two cysteines with sulfhydryl groups (symbol: C), the specific enzyme cleavage site is proline-cysteine-glycine-proline (symbol: The peptide bond after cystei...

Embodiment 3

[0089] Example 3: A novel and efficient protease activity detection method

[0090] This example is a method for detecting thrombin (English name: thrombin) activity.

[0091] first part:

[0092] The correlation between thrombin activity and the peak intensity of the fluorescent spectrum of the quantum dot synthesized by the corresponding product is established in advance, and the establishment of the correlation is composed of the following steps:

[0093] In the first step, for thrombin, a polypeptide substrate containing a specific cleavage site for the protease is designed, the polypeptide substrate includes a peptide chain composed of 4 amino acids, and the peptide chain contains Peptide bond for specific cleavage and two cysteines (symbol: C) with sulfhydryl groups, the specific enzyme cleavage site is the amino terminus (NH 2 -) is the peptide bond between arginine (R), and the carboxyl terminal (COOH-) is glycine (G). The two ends of the peptide chain are respective...

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Abstract

The invention discloses a novel high-efficiency protease activity detecting method. The novel high-efficiency protease activity detecting method is characterized in that establishing correlativity between to-be-detected protease activity and fluorescence spectrum peak intensity of quantum dots synthesized by corresponding products in advance, designing polypeptide substrate including a special cleavage site of the protease according to the category of the to-be-detected protease, according to an optimal temperature and optimal potential of hydrogen (pH) of the protease, putting into protease content with unknown concentration, conducting enzyme digestion reaction on the same amount of polypeptide substrate, putting the same amount of precursors of synthesized quantum dots to enzyme-digested products of the polypeptide substrate, enabling the enzyme digestion reaction to generate the quantum dots under an appropriate temperature, after the reaction ends up, detecting the fluorescence spectrum of the synthesized quantum dots, and comparing the fluorescence spectrum peak intensity of quantum dots synthesized by the enzyme-digested products of the protease with unknown concentration with the correlativity in order to obtain the protease activity of the protease with concentration unknown. The novel high-efficiency protease activity detecting method is convenient to achieve, quick, high in sensitivity and low in cost. The novel high-efficiency protease activity detecting method also has general applicability.

Description

technical field [0001] The present invention relates to the field of functional nanomaterials and biochemistry, in particular to a novel and efficient detection method for protease activity. The present invention uses the enzymatic cleavage reaction of protease to polypeptide substrates, and uses the obtained enzymatic cleavage products as surface ligands to synthesize a protease with fluorescent Effect nanomaterial quantum dots, and then according to the change of the fluorescent effect of the synthesized quantum dots, to reflect the change of the product after the enzyme cleavage reaction, so as to reflect the change of protease activity. Background technique [0002] Proteases specifically catalyze the hydrolysis of protein and polypeptide substrates, which play an extremely important role in the normal life activities of organisms. It is difficult to directly express the amount of enzyme (such as mass, volume, concentration) to detect the content and existence of enzyme,...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 马楠何学文
Owner SUZHOU UNIV
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