The Device And Method For High Throughput Single Cell Secreted Proteins Analysis

As cells are the basic units of organism,cell research is always the hot area people focusing on.Traditional cell research is based on the average result from bulk cells,which would cover the heterogeneity of cells and then miss some key information.As the phenomenon of cell heterogeneity is widely accepted,we need to get details form single cells and make a high-throughput analysis,which is favorable to understand the biology process from immunology to phymatology.Protein is one of the essential elements for life activities,protein analysis from single cell level can help us under the mechanism of cell reaction to drugs or external stimulation.There are two challenges for single cell analysis.One challenge is how to isolate single cell from cell populations.Infinite dilution method is commonly used in laboratory to pick up cells by capillary under microscope,but this method is not efficient,low throughput with large reagents consumption.The other challenge is how to detect trace protein with high sensitivity.The protein of cell single is low-abundant and easy-degraded.Therefore,it is important to develop a sensitive method to realize single cell protein analysis.Based on the above challenges,this thesis is focusing on developing a new cell capture platform for single cell isolation and secreted protein detection.The details are:On the basis of previously proposed method for single cell capture,which is based on the fluid mechanics and flow resistance principle,we redesigned the micro-channel structure of microfluidic chip.This newly designed chip can realize high-throughput single cell secreted protein analysis by using the immune analysis technique,which expands the application range of single cell analysis.This chip,sealed by home-made clamp,can realize single cell capture with high efficiency based on the principle of flow resistance.Fluids could be precisely controlled by the aid of micro-pump and micro-valve,which facilitates single cell incubation units isolation.By utilizing cell culture gas as isolation phase and combining with chip hydrophobicity modification and water saturation approaches,the isolation units can be preserved for a long time with high cell survivability.Based on the principle of immune analysis strategy,secreted protein with good specificity and high sensitivity could be achieved.All in all,we established a novel microfluidic system for single cell secreted protein analysis.This platform can realize single cell capture with high efficiency and secreted protein analysis with high sensitivity.This method solved the problem of micro-droplet evaporation under normal incubation temperature and kept high survival rate of single cells,which is favorable for secreted protein analysis.With this platform,macrophages and U87 could be cultured for a long time(4 h),and secreted protein could be detected in a rapid and sensitive way.