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A Simultaneous Electrochemical Detection Method of Trypsin and Chymotrypsin Based on Enzyme Digestion

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

Inactive Publication Date: 2016-05-11
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A Simultaneous Electrochemical Detection Method of Trypsin and Chymotrypsin Based on Enzyme Digestion
  • A Simultaneous Electrochemical Detection Method of Trypsin and Chymotrypsin Based on Enzyme Digestion
  • A Simultaneous Electrochemical Detection Method of Trypsin and Chymotrypsin Based on Enzyme Digestion

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Preparation of DNA-polypeptide complexes and electrochemical nanosignaling probes:

[0024] (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 filtration for 5 minutes to obtain the DNA-polypeptide complex, which is stored at 4°C;

[0025] (2) Preparation of electrochemical nano-signal probe: 50 mL of 0.01% HAuCl 4 Heat the solution to boiling with constant stirring, then add 1 mL of trisodium citrate with a mass concentration of 5%, continue to stir and keep boiling until the color of the solution changes from yellow to deep red, keep boiling for 10 minutes, and cool naturally to room temperature to make a gold nanoparticle solution. 0.3mL of 25μM thiol DNA was mixed with 4.7mL of 12nM gold colloid for 24 hours, and the prepared DNA-AuNPs complex was stabilized with 1M NaCl; 0.2mL of 0.1mM electron mediator was added to 0.2mL of DNA-AuNPs soluti...

Embodiment 2

[0029] Construction of trypsin and chymotrypsin electrochemical sensors based on enzymatic cleavage:

[0030] The treated gold electrode was immersed in the mixed solution of DNA1-polypeptide 1 and DNA2-polypeptide 2 and incubated for 12 hours, blocked with 1 mM mercaptohexanol for 1 hour, and then immersed in the In the solution of the nano-signal probe, the two nano-signal probes are immobilized on the electrode surface through a DNA hybridization reaction.

[0031] 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 with MCH (curve c), the impedance increases furth...

Embodiment 3

[0034] (2) Detection of a single protease by the sensor

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Abstract

The invention discloses an electrochemical simultaneous detection method of trypsin and chymotrypsin based on enzymatic cleavage, belonging to the technical field of electrochemical sensing. The DNA-polypeptide complex is immobilized on the surface of the gold electrode through the interaction of gold-sulfhydryl bonds, and then the DNA-gold nanoparticle-electron mediator nano-signal probe is captured by DNA hybridization reaction. When the target protease exists, trypsin cleaves the arginine carboxyl site on the polypeptide, and chymotrypsin cleaves the tyrosine carboxyl site on the other polypeptide, so that the corresponding nano signal probe attached to the polypeptide is detached from the electrode surface, leading to a decrease in the peak current of the corresponding electron mediator, and accordingly, the sensitivity and selectivity of trypsin and chymotrypsin can be detected simultaneously.

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 Patents(China)
IPC IPC(8): G01N27/26G01N27/327
Inventor 邱建丁田小翠梁汝萍
Owner NANCHANG UNIV
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