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Method for single cell level detection of MMP enzyme activity

A level detection, enzyme activity technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of long process, cumbersome, poor experimental reproducibility, etc., to achieve simple construction, low cost, uniform size Effect

Inactive Publication Date: 2016-08-31
SUZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The above-mentioned traditional methods have at least the following defects: 1. Experiments often have poor reproducibility, and the process is lengthy and cumbersome
However, such population cell detection results often ignore the differences in expression between individual cells

Method used

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  • Method for single cell level detection of MMP enzyme activity
  • Method for single cell level detection of MMP enzyme activity
  • Method for single cell level detection of MMP enzyme activity

Examples

Experimental program
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Effect test

Embodiment 1

[0048] 1. Culturing tumor cell U937.

[0049] U937 cells were grown in suspension in RPMI-1640 medium containing 10% fetal bovine serum, and placed in 5% CO 2 , Cultured in a cell culture incubator at 37°C and set aside.

[0050] 2. Prepare PEGDA mother liquor, mineral oil solution (continuous phase), cell solution (dispersion Phase A) and polypeptide solution (dispersed phase B).

[0051] Weigh 9.5 mg of ammonium sulfate (initiator), 30 mg of D-sorbitol (crosslinker) and 1.24 g of PEGDA (monomer), and dissolve them in 3.8 mL of PBS solution to prepare PEGDA mother liquor.

[0052] When the cells grew well, the cells were taken out and counted by a cell counter, and then the cells were centrifuged, washed once with PBS, and then resuspended in serum-free, phenol red-free 1640 medium. Take a certain volume of PEGDA mother solution and mix with the counted cell solution to make the final concentration of cells 6×10 5 per mL, prepared as dispersed phase A.

[0053] A ce...

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Abstract

The invention provides a method for single cell level detection of MMP enzyme activity. The method comprises a, through a microfluidic device, converging a cell solution and a polypeptide solution through two channels, further forming micro-droplets packaging single cells and polypeptide and discharging the micro-droplets under driving of a continuous phase, wherein the polypeptide solution contains a molecule for induced improvement of enzyme secretion, a polypeptide sequence comprises a specific digestion fragment and head and tail parts of the polypeptide sequence are respectively connected to a fluorophore and a quenching group, b, collecting the micro-droplets and detecting a signal through a fluorescence microscope and c, according to a fluorescence signal, determining MMP enzyme activity. The method realizes efficient, sensitive and low cost detection of activity of an enzyme related to tumor grade malignancy, virulence and a cancerometastasis capacity in a single cell level, is used for tumor diagnosis and treatment and has a good application prospect.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a method for detecting MMP enzyme activity at the single cell level, which can be further used to judge the degree of tumor cell proliferation. Background technique [0002] Malignant tumor cells refer to the tumor cells that diffuse from the primary tumor into some circulatory systems of the human body during the metabolic process of the human body. Their appearance marks that the tumor cells have begun to spread from the lesion to the surroundings, resulting in more organs and tissues. produce lesions. Studies have shown that about 90% of cancer deaths are related to the spread of tumor cells. In the process of tumor metastasis, tumor cells must break through the physiological barriers in their migration process, and tumor cells secrete matrix metalloproteinases (MMPs) that can hydrolyze the extracellular matrix (ECM), this feature plays a role in the process of tumor sprea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/37
CPCC12Q1/37
Inventor 刘坚余钊
Owner SUZHOU UNIV
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