Rare Cell Sorting And Single-Cell Proteomic Analysis Based On Squential Operation Droplet Array Technique

In recent years,single-cell analysis is becoming a research hotspot with the deeply understanding of cellular heterogeneity.The analysis of rare cells,such as circulating tumor cells,has received much attention,because they have extremely low abundance of less than 0.01% but play important roles in disease development.The efficient identification,sorting and analysis of rare single cells has always been a clinically meaningful medical challenge.The object of this work is to develop an automated platform capable of sorting single rare cells from human blood samples,and to develop a proteomic analysis method for single cell samples.In Chapter 1,the current development of rare cell sorting technologies and shotgunbased single-cell proteomic analysis methods are reviewed.Among them,microfluidic technology has shown its unique advantages in many single-cell sorting and analysis technologies.It can not only be applied to a variety of rare cell sorting scenarios by virtue of its flexible manipulation ability of tiny fluids,but also can effectively improve the identification depth of single-cell proteomics analysis because of its high reaction efficiency and low sample adsorption loss in micro-volumes.In Chapter 2,by combing machine vision-based image recognition,liquid handling robot and droplet-based microfluidic techniques,we developed an automated platform for rare cell sorting capable of achieving the detection,identification,capture,and droplet generation of target cells from samples containing large numbers of normal cells.We proposed a novel “gold panning” strategy for large scale target-cell sorting,which can significantly improve the cell sorting efficiency by about 250 times when the sorting purity is over 90%.With this platform,approximately 1,000 extremely rare circulating endothelial progenitor cells were successfully identified from over3,000,000 cells in the peripheral blood of patients with coronary heart disease,with scanning speed up to 4,000 cells/s,and 20 25 n L droplets containing single target cells were generated.In Chapter 3,we further expanded the ability of the single-cell platform to operate on nanoliter-scale liquids and developed a whole-process solution for single-cell proteomics analysis based on the pick-up operation mode.In response to the characteristics and demands of single-cell proteomics analysis,we developed a“hamburger” nanoliter-scale microreacter based on an evaporation-compensated moisturizing strategy,which significantly improved the sample reaction efficiency and reduced the adsorption loss on the reactor surface.Based on this method,more than1,000 protein groups were quantitatively identified from single He La cells with labelfree mass spectrometry approach,achieving deep-coverage proteomic analysis at the single-cell level.