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Biosensor for detecting matrix metalloproteinase and application of biosensor

A biosensor and matrix metal technology, applied in the field of protease detection, can solve the problems of lack of sensitivity and achieve high sensitivity, good specificity and signal amplification effect

Active Publication Date: 2020-12-15
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods exhibit good selectivity due to the high specificity of enzymatic cleavage, they lack sensitivity

Method used

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  • Biosensor for detecting matrix metalloproteinase and application of biosensor
  • Biosensor for detecting matrix metalloproteinase and application of biosensor
  • Biosensor for detecting matrix metalloproteinase and application of biosensor

Examples

Experimental program
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Effect test

Embodiment 1

[0061] In the present embodiment, a kind of matrix metalloproteinase biological sensor and the method for detecting matrix metalloproteinase MMP-2 and MMP-7 by described sensor are provided, and described detection method comprises the following steps:

[0062] (1) Matrix metalloproteinases (MMPs) reaction and magnetic separation

[0063] First, prepare the reaction liquid of matrix metalloproteinase, in described 20 μ L reaction liquid, comprise the DNA-peptide conjugate-1 of 4 μ mol / L and the DNA-peptide conjugate-2 of 2 μ mol / L, 1×TCNB buffer solution ( 50mmol / L Tris-HCl, 10mmol / L CaCl 2 , 150mmol / LNaCl, 0.05% Brij-35, pH7.5).

[0064] Add different concentrations of MMP-2 and MMP-7 to the above reaction system, react at 37°C for 2 hours, then heat up to 95°C to terminate the reaction. Take 4 μL of the reaction product, add 15 μL of streptavidin-coated magnetic beads (concentration: 10 μg / μL) and 11 μL of 1×PBS buffer (pH 7.5) and incubate on a mixer for 30 minutes at roo...

Embodiment 2

[0088] In the present embodiment, the application of the biosensor of matrix metalloproteinases described in Example 1 in screening matrix metalloproteinase inhibitors is provided, and the experimental steps are as follows:

[0089] Different concentrations of Marimastat (BB-2516) with 30nmol / L MMP-2, 14.2nmol / LMMP-7, 4μmol / L DNA-peptide conjugate-1, 2μmol / L DNA-peptide Conjugate-1, and 1×TCNB buffer (50mmol / L Tris-HCl), 10mmol / LCaCl 2 , 150mmol / LNaCl, 0.05% Brij-35, pH 7.5) were reacted at 37°C for 2 hours, and Cy3 and Cy5 were counted.

[0090] In this example, Marimastat (BB-2516) was used as a model inhibitor to verify the feasibility of using the protease sensor described in Example 1 for an inhibition test. The relative activity of MMP-2 and MMP-7 decreased with the increase of Marimastat concentration ( Image 6 ). This embodiment uses the half maximal inhibitory concentration (IC 50 , the inhibitor concentration used when the D enzyme activity was inhibited to 50% ...

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Abstract

The present invention particularly relates to a biosensor for detecting matrix metalloproteinases and an application of the biosensor. MMP2 and MMP7 are closely related to physiological activities ofvarious diseases, and a protease detection method commonly used at present has defects of high cost, insufficient detection sensitivity, etc. The present invention aims to provide the biosensor for detecting multiple matrix metalloproteases (MMPs) with high sensitivity, through a protease specific cracking reaction and an nickase assisted signal amplification (NESA) technology, specific and high-sensitivity simultaneous detection of the multiple matrix metalloproteases is realized, and the biosensor can also be applied to screening inhibitors of the matrix metalloproteases, etc.

Description

technical field [0001] The invention belongs to the technical field of protease detection, and in particular relates to a biosensor for detecting matrix metalloproteinases, a method for detecting proteases based on the biosensor, and an application thereof. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Proteases play important roles in a variety of fundamental biological processes and may catalyze specific cleavage reactions. The imbalance of protease is closely related to various physiological activities. Therefore, proteases are attractive targets for drug design, and several protease inhibitors have been approved for drug development, e.g., Prinomastat (AG3340), Tan...

Claims

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Application Information

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IPC IPC(8): C12Q1/682C12Q1/37C12Q1/00
CPCC12Q1/682C12Q1/37C12Q1/001C12Q2563/107C12Q2525/131C12Q2521/537C12Q2521/301C12Q2565/607Y02A50/30
Inventor 张春阳胡娟李玥颖刘雯
Owner SHANDONG NORMAL UNIV
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