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Trypsin-chymotrypsin electrochemical synchronous detection method based on enzyme digestion

A technology of trypsin and chymotrypsin, which is applied in the field of electrochemical sensing, can solve the problem of simultaneous determination of multiple proteases, achieve high sensitivity and selectivity for simultaneous identification and detection, and have good application prospects

Inactive Publication Date: 2014-09-17
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods are mostly used for the detection of a single protease, and it is difficult to simultaneously measure multiple proteases in biological samples

Method used

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  • Trypsin-chymotrypsin electrochemical synchronous detection method based on enzyme digestion
  • Trypsin-chymotrypsin electrochemical synchronous detection method based on enzyme digestion
  • Trypsin-chymotrypsin electrochemical synchronous detection method based on enzyme digestion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Preparation of DNA-polypeptide complexes and electrochemical nanosignaling probes:

[0025] (1) Preparation of DNA-polypeptide complex: Mix 200 μL of 25 μM polypeptide, 200 μL of 5 μM DNA and 1 mg / mL streptavidin, and react at room temperature for 3 hours. Remove by ultrafiltration for 5 minutes at a speed of rotation per minute to obtain a DNA-polypeptide complex, which is stored at 4°C;

[0026] (2) Preparation of electrochemical nanosignal probe: 50 mL of 0.01% HAuCl 4 The solution was heated to boiling with constant stirring, then 1 mL of trisodium citrate with a mass concentration of 5% was added, continued to stir and kept boiling until the color of the solution changed from yellow to deep red, and continued to boil for 10 minutes. Cool to room temperature to make a gold nanoparticle solution. Mix 0.3 mL of 25 μM thiol DNA with 4.7 mL of 12 nM gold colloid for 24 hours, and the prepared DNA-AuNPs complex was stabilized with 1 M NaCl; 0.2 mL of 0.1 mM electron me...

Embodiment 2

[0030] Construction of trypsin and chymotrypsin electrochemical sensors based on enzymatic cleavage:

[0031] Dip the treated gold electrode into the mixed solution of DNA1-polypeptide 1 and DNA2-polypeptide 2 and incubate for 12 hours, block it with 1 mM mercaptohexanol for 1 hour, and then immerse it in the solution containing DNA1′-Au NPs-Thi and DNA2′-Au In the solution of NPs-Fc nano-signal probes, the two nano-signal probes are immobilized on the electrode surface through DNA hybridization reaction.

[0032] The preparation process of the sensor was characterized by electrochemical impedance spectroscopy, and the results were as follows: Figure 4 shown. The bare gold electrode (curve a) has a small semicircular area, indicating that the electron transfer speed is fast on the surface of the gold electrode. When the bare gold electrode was immersed in a solution containing two peptides (curve b), the impedance increased rapidly to 1358 Ω. After sealing the electrode wi...

Embodiment 3

[0035] (2) Detection of a single protease by the sensor

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Abstract

The invention discloses a trypsin-chymotrypsin electrochemical synchronous detection method based on enzyme digestion and belongs to the technical field of electrochemical sensing. Through gold-mercapto bond action, a DNA-polypeptide compound is fixed to the surface of a gold electrode, and through DNA hybridization reaction, a DNA-gold nanoparticle-electronic mediator nanometer signal probe is captured. When target protease exists, an arginine carboxyl site of one of polypeptides is cut by trypsin and a tyrosine carboxyl site of the other one of the polypeptides is cut by chymotrypsin so that the corresponding nanometer signal probes connected to the polypeptides are separated from the surface of the electrode and thus peak current of the corresponding electronic mediator is reduced. Therefore, the trypsin-chymotrypsin electrochemical synchronous detection method can realize synchronous detection of sensitivity and selectivity of trypsin and chymotrypsin.

Description

technical field [0001] The invention relates to an electrochemical detection method based on enzymatic cleavage and its application in the simultaneous detection of trypsin and chymotrypsin, belonging to the technical field of electrochemical sensing. Background technique [0002] Protease is a class of enzymes that can decompose proteins in organisms. Protease dysfunction will lead to diseases including cancer, viral infection, neurodegenerative diseases, etc., making protease a hot spot in clinical research. Calorimetry, electrochemical methods, fluorescence, inductively coupled plasma-mass spectrometry, surface Raman scattering, and electrophoresis are widely used in biological experiments of proteases. However, most of these methods are used for the detection of a single protease, and it is difficult to simultaneously measure multiple proteases in biological samples. Research has shown that proteases rarely work alone, but rather in a system called a "protease network."...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/26G01N27/327
Inventor 邱建丁田小翠梁汝萍
Owner NANCHANG UNIV
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