High-throughput Single-molecule Protein Detection And Single-cell 3D Culture And Analysis Based On Microfluidic Chip

The establishment and development of the miniaturized total analysis system(?-TAS)has enabled the rapid development of chip technology,making the chip more versatile in structure and functionality.This technology allows various complexities to be implemented on the chip.Cell manipulations such as cell culture,sorting,capture,lysis,separation,and detection have become the most promising research areas for single molecule detection,single cell manipulation,and single cell analysis.Among them,the microarray chip is a device having a high-density micro-hole array on a several square centimeters substrate,and the length and width of each micro-hole array can generally be from a dozen to several hundred micrometers,and the depth can be up to 100 micrometers,which is similar to mammalian cells' diameter.Therefore,microarrays are suitable for the analysis of single mammalian cell.And the microarray chip has a high-density microarray,as well as a network of two-dimensional or three-dimensional channels,so it is easy to manipulate single cell and can integrate a variety of operational analysis methods to meet the needs of high-throughput cell culture and analysis,and expected to be automated.Microfluidic technology has a very high application potential and development prospects in the study of single molecule/single cell.The main works of this thesis are listed as following:1.Microfluidic droplet based single-molecule matrix metalloproteinase-2 digital detectionMatrix metalloproteinases is closely related to the migration and metastasis of tumors,the study of matrix metalloproteinases molecules can help us understand the tumor cell biological mechanisms of invasion and metastasis,has profound application in clinical diagnosis.To solve the problem that the traditional method can not digital detect the enzyme activity concentration of matrix metalloproteinase,we proposed a digital quantitative detection of single molecule matrix metalloproteinase-2(MMP-2)based on droplet microfluidics.In this paper,a dual-channel flow focusing chip was used to inject MMP-2 and its specifically recognized fluorescent substrate(5-FAM/QXLTM520 fluorescent peptide),respectively,to generate droplets encapsulating MMP-2 and its specific substrate.In the droplet containing the MMP-2 enzyme,the substrate is enzymatically cleaved to generate a free fluorescent peptide,and the droplet fluoresces.Droplets of enzyme-free molecules do not have corresponding fluorescence.By statistically analyzing the number of fluorescent droplets and non-fluorescent droplets,according to the Poisson distribution,can absolute quantitatively detect the activity concentration of MMP-2 enzyme.Absolute quantitative detection of enzyme concentrations using microdroplets has the advantages of high sensitivity,high throughput,small size,small sample consumption,high precision,and enables the analysis of individual enzyme molecules.Compared with the traditional ELISA method,this method can digitally detects the enzyme concentration.We have developed a method based on droplet microfluidic technology to digitally detect the MMP-2 enzyme activity concentration by counting bright and dark droplets,and demonstrated that this method can be very reliable and reproducible.2.High-throughput Single-cell Capture and 3D Culture Based on Microfluidic Agarose Droplet ArrayIn vitro culture of single cells facilitates biological research by reducing the heterogeneity of population heterogeneity.However,there is still a lack of simple and high-throughput methods for single cell culture and analysis.In addition,most of the single-cell culture and analysis devices are based on 2D culture conditions,which can not simulate the real cell microenvironment in vivo.As a result,we develop a novel microfluidic array based a U-Fluidic trap chip for highly efficient single cell capture and 3D culture in micro-chamber.The method capitalizes on the unique thermo-responsive sol-gel switching property of agarose for single cell capture and long term 3D culture.By adjusting the size of the U-shaped structure of the chip array,the chip can capture cells with different diameters without affecting the single-cell capture efficiency(-90%).The design of the chip achieves high throughput single cell capture and is suitable for high throughput capture of different kinds and different number of cells.We have demonstrated that this chip can realize single-cell 3D culture and cell mass formation by using 293T cells as model cells,and observed the proliferation heterogeneity of the offspring of different single cells.In the single cell level,agarose droplet microarray can also be applied in the study of drug toxicity,evaluation on toxic drugs and chemical composition of cells,effectively reduce the drug side effect for drug design to provide important basis.