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System for simultaneously detecting matrix metalloproteinase-9 and matrix metalloproteinase-2, preparation method thereof and application thereof

A matrix metal and protease technology, applied in the field of biomedical detection, can solve the problems of limited MMP-9 and MMP-2 detection, high detection cost, and complicated steps, and achieve the effects of saving sample volume, improving sensitivity, and low cost

Pending Publication Date: 2020-02-21
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Existing detection of MMP-9 or / and MMP-2 protein content and activity is mainly by extracting cell protein, tissue homogenate protein, application of western blotting, immunostaining, gel zymography and other methods to determine, but these methods generally have cumbersome steps , time-consuming, high detection cost, long-term trained technicians are required to operate, etc.
At present, the determination of MMP-9 or / and MMP-2 in serum or urine mainly relies on ELISA kits, which greatly limits the clinical detection of MMP-9 and MMP-2

Method used

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  • System for simultaneously detecting matrix metalloproteinase-9 and matrix metalloproteinase-2, preparation method thereof and application thereof
  • System for simultaneously detecting matrix metalloproteinase-9 and matrix metalloproteinase-2, preparation method thereof and application thereof
  • System for simultaneously detecting matrix metalloproteinase-9 and matrix metalloproteinase-2, preparation method thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Successful verification of PDANS synthesis

[0048] Prepare PDANS:

[0049] (1) Prepare 100mL 10mM Tris-HCl (pH 7.4) with ultrapure water, add Tris-HCl and isopropanol (40 mL) into the beaker, dilute with ammonia water, add a small amount to the beaker several times, and test with pH test paper. The pH value of the final solution was about 8.0, and the magnetic stirrer was stirred slowly for 30 minutes to fully mix the solution.

[0050] (2) Subsequently, 100 mg of dopamine hydrochloride was weighed and dissolved in 2 mL of ultrapure water, and the dissolved dopamine hydrochloride was slowly added into the solution, stirred for 30 h, and then stopped.

[0051] (3) Transfer the solution obtained in step (2) to a centrifuge tube, centrifuge at 10000rpm for 10min, discard the supernatant, dissolve the precipitate in ultrapure water, vortex mix, centrifuge, discard the supernatant, repeat several times, until the supernatant is clear.

[0052] (4) After centrifugation an...

Embodiment 2

[0058] Building PDANS-aptamers nanosensors

[0059] 1. Modified FAM fluorescein (MMP-9-aptamer-FAM) at the 5' end of MMP-9-aptamer, and modified Texas Red fluorescein (MMP-2-aptamer-Texas Red) at the 5' end of MMP-2-aptamer, The sequence was synthesized by Shanghai Sangong Technology Co., Ltd.

[0060] name Nucleotide sequence MMP-9-aptamer-FAM 5'-FAM-tacggccgcacgaaaaggtgccccataactcaatgccga-3' MMP-2-aptamer-Texas Red 5'-Texas Red-tcgccgtgtaggattaggccaggtatgggaacccggtaac-3'

[0061] Construction of the PDANS-aptamers nanosensor involves the following steps:

[0062] (1) Preparation of samples: Prepare MMP-9-aptamer-FAM and MMP-2-aptamer-TexasRed standard solutions of 1000 nM with ultrapure water for use; prepare PDANS standard solution with ultrapure water at a concentration of 2 mg / mL.

[0063] (2) Reaction: Add MMP-9-aptamer-FAM and MMP-2-aptamer-Texas Red standard solutions in sequence to make the final concentrations 100nM respectively; then ...

Embodiment 3

[0069] Investigate the detection sensitivity experiment of the whole detection system to MMP-9 and MMP-2

[0070] 1. The sequence required for this experiment (as in Example 2):

[0071] 2. Investigate the sensitivity of MMP-9 and MMP-2 standard solutions based on DNase-I amplification system,

[0072] Include the following steps:

[0073] (1) Preparation of samples: prepare MMP-9 concentration of 0.48~120ng / mL, so that the concentrations are 0.48 ng / mL, 0.96ng / mL, 2.4ng / mL, 4.8ng / mL, 12ng / mL, 24ng / mL, 60ng / mL, 120ng / mL; MMP-2 concentration 1.28~320ng / mL, so that the concentrations are 1.28ng / mL, 2.56ng / mL, 6.4ng / mL, 12.8ng / mL, 32ng / mL, 64ng / mL , 160ng / mL, 320ng / mL; prepare DNase-I enzyme 2U / μL in ultrapure water.

[0074] (2) Perform the reaction: add MMP-9-aptamer-FAM and MMP-2-aptamer-Texas Red standard solutions in sequence to make the final concentration 100nM respectively, then add PDANS to make the final concentration 0.3 mg / mL, buffer ion Concentration of 5mM CaCl 2...

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Abstract

The invention discloses a system for simultaneously detecting matrix metalloproteinase-9 and matrix metalloproteinase-2, a preparation method thereof and application thereof. The system comprises a polydopamine nano microsphere-fluorescein modified nucleic acid aptamer nano sensor and deoxyribonuclease, and the sensor is formed by adding MMP-9-aptamer-FAM and MMP-2-aptamer-Texas Red into polydopamine nano microspheres. According to the invention, polydopamine nano microspheres with an excellent quenching effect are prepared, a PDANS-modified fluorescein nucleic acid aptamer nano sensor is constructed through non-public valence bond combination, DNase-I is added, aptamer is hydrolysed, an object to be detected is dissociated, cyclic reaction is carried out, the fluorescence signal amplification is realized, and the sensitivity of a system is improved. The system is simple in preparation, the reaction condition is mild, the cost is low, and a new tool and a new method are provided for simultaneous and rapid detection of the matrix metalloproteinase-9 and the matrix metalloproteinase-2.

Description

technical field [0001] The invention belongs to the field of biomedical detection, and in particular relates to a system for simultaneously detecting matrix metalloproteinase-9 and 2, a preparation method and application thereof. Background technique [0002] Matrix metalloproteinases (MMPs) are a large class of enzymes that require zinc, including interstitial collagenase, matrix protease, gelatinase, etc., which synergistically degrade various protein components in the extracellular matrix, and are involved in various pathophysiological processes (including embryonic development). , inflammation, wound healing and fibrosis) and tissue remodeling. Among them, type IV collagenase is an important class, expressed in the form of matrix metalloproteinase-9 (MMP-9) and matrix metalloproteinase-2 (MMP-2), which have the ability to degrade type IV basement membrane collagen. During the development of renal fibrosis, the destruction of the integrity of the tubular basement membran...

Claims

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Application Information

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IPC IPC(8): G01N33/573G01N33/58C12Q1/44
CPCG01N33/573G01N33/582C12Q1/44G01N2333/922G01N2333/96494
Inventor 田蒋为余伯阳胡依婷
Owner CHINA PHARM UNIV
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