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Method for detecting trypsinase activity

A trypsin and activity detection technology, which is applied in the field of trypsin activity detection, can solve the problems of not being able to know the size of the enzyme activity, cumbersome and time-consuming, etc., and achieve the effect of simple detection of trypsin, improving sensitivity and reducing impedance

Inactive Publication Date: 2018-05-04
CHANGZHOU AMANTE CHEM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention: Aiming at the current cumbersome and time-consuming operation of detecting trypsin, and some need to use radioactive elements, which can only be used to determine the amount of trypsin, but the activity of the enzyme cannot be known, a method is provided. A kind of method for trypsin activity detection

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] HEPES buffer solution: in parts by mass, take 100 parts of 4-hydroxyethylpiperazineethanesulfonic acid, 50 parts of tris(2-carboxyethyl)phosphine, and 400 parts of distilled water, and adjust the pH to 6.5.

[0035]Washing buffer: in parts by mass, mix 100 parts of 4-hydroxyethylpiperazineethanesulfonic acid, 10 parts of Tween-20, and 400 parts of distilled water to adjust the pH to 6.0.

[0036] Substrate polypeptide: synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., the specific sequence is HHHKSAGTGC.

[0037] A method for trypsin activity detection, comprising the steps of:

[0038] (1) Sonicate graphene oxide at -2°C for 30 minutes, add thionine at a mass ratio of 5:3 and mix evenly, stir at 20°C for 1 hour, then add ultrapure water with 60% graphene quality after ultrasonication, mix and stir, and filter , take the filter residue, dry to obtain the compound, and set aside;

[0039] (2) Mix sulfuric acid solution with a mass fraction of 80% and hydrogen ...

Embodiment 2

[0043] HEPES buffer solution: in parts by mass, take 110 parts of 4-hydroxyethylpiperazineethanesulfonic acid, 55 parts of tris(2-carboxyethyl)phosphine, and 400 parts of distilled water, and adjust the pH to 6.7.

[0044] Washing buffer: in parts by mass, mix 105 parts of 4-hydroxyethylpiperazineethanesulfonic acid, 12 parts of Tween-20, and 400 parts of distilled water to adjust the pH to 6.2.

[0045] Substrate polypeptide: synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., the specific sequence is HHHKSAGTGC.

[0046] A method for trypsin activity detection, comprising the steps of:

[0047] (1) Sonicate graphene oxide at -3°C for 35 minutes, add thionine at a mass ratio of 5:3 and mix evenly, stir at 22°C for 1 hour, then add ultrapure water with 65% graphene mass after ultrasonication, mix and stir, and filter , take the filter residue, dry to obtain the compound, and set aside;

[0048] (2) Mix sulfuric acid solution with a mass fraction of 80% and hydrogen pe...

Embodiment 3

[0052] HEPES buffer solution: in parts by mass, take 120 parts of 4-hydroxyethylpiperazineethanesulfonic acid, 60 parts of tris(2-carboxyethyl)phosphine, and 400 parts of distilled water, and adjust the pH to 6.8.

[0053] Washing buffer: in parts by mass, 110 parts of 4-hydroxyethylpiperazineethanesulfonic acid, 15 parts of Tween-20, and 400 parts of distilled water were mixed, and the pH was adjusted to 0.8.

[0054] Substrate polypeptide: synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., the specific sequence is HHHKSAGTGC.

[0055] A method for trypsin activity detection, comprising the steps of:

[0056] (1) Sonicate graphene oxide at -4°C for 40 minutes, add thionine at a mass ratio of 5:3 and mix evenly, stir at 25°C for 2 hours, then add ultrapure water with 70% graphene quality after ultrasonication, mix and stir, and filter , take the filter residue, dry to obtain the compound, and set aside;

[0057] (2) Mix sulfuric acid solution with a mass fraction of ...

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Abstract

The invention belongs to the field of biological, and particularly relates to a method for detecting trypsinase activity. A simple and convenient method for detecting the trypsinase activity is builtby using the mutual action differences of polypeptides before and after the trypsinase enzyme digestion and graphene oxide loaded with electrochemical active molecule thionine. The graphene oxide haslarger specific surface area, and can load a great amount of thionine; the effects of amplifying electrochemical signals and improving the detection sensitivity are achieved. The gold electrode surface is modified by gold sulfhydryl bond covalent formed by gold and sulfhydryl of tail end cysteine via substrate polypeptides; the sulfhydryl-containing polypeptides are subjected to covalent modification onto the gold surface; then, 6-mercapto-l-hexanol is used for space occupation; non-specific adsorption polypeptides are removed; the electrode surface blank position is sealed; the N tail end ofthe substrate polypeptide sequence is provided with histidine; the histidine has positive charges; graphene oxide containing rich carboxyls have negative charges after dissociation; stronger static electricity mutual action exists between the histidine and the graphene oxide, so that a prepared compound is adsorbed to the final modified electrode product.

Description

technical field [0001] The invention belongs to the field of biology, and in particular relates to a method for detecting trypsin activity. Background technique [0002] Trypsin is a widely used proteolytic enzyme. It specifically hydrolyzes the peptide bond at its carboxyl end by recognizing the positive charge on the side chain of arginine or lysine. It is an endopeptidase. According to the different properties of the spots, it can be divided into cationic trypsin, anionic trypsin and neutral trypsin. Trypsin is mainly used for the activation of prochymotrypsin, procarboxypeptidase, etc. Trypsin exists in the pancreas of animals in the form of inactive zymogen. + In the presence of enterokinase or active trypsin, it is activated by itself, and the peptide bond between the N-terminal lysine and isoleucine residues of the peptide chain is broken, and a hexapeptide is lost. After a certain change in the molecular conformation into active trypsin. [0003] The traditional ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/26G01N27/327
CPCG01N27/26G01N27/327
Inventor 吴昌财郑薇林凡
Owner CHANGZHOU AMANTE CHEM CO LTD
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