Development Of Microfluidic Chips For Pairwise Capture Of Cells And Beads And Isolated Lysis

Single cell transcriptome sequencing technology can perform high-throughput sequencing at the single cell level to reveal the heterogeneity among cells,which has profound significance for the research of basic life science and diagnosis of major disease.The difficulty of single cell transcriptome sequencing lies in the one-to-one pairing of cell and bead containing unique molecular identifiers,resulting in two mainstream microfluidic systems,namely droplet-based and microwell-based microfluidic systems.The droplet-based microfluidic systems encapsulate single cell and single bead in a droplet.This system follows a random capture approach and has a great number of unused droplets,resulting in waste of reagents and cells.In contrast,microwell-based microfluidic systems are cheaper and more portable,using the size-selection principle of microwells to restrict the pairing of single cell and single bead,but this method still uses a random capture mechanism,resulting in low capture efficiency;the improved microwell-based microfluidic systems utilize the active capture mechanism of dielectrophoresis(DEP),but existing devices have shortcomings such as complicated operation.To solve these problems,this thesis designs two microwell-based microfluidic chip,namely,new microwell-based microfluidic chip and wall-electrode microfluidic chip,when the chip is powered up,the electrode with a two-layer three-dimensional structure creates an unequal vertical electrical field used to actively capture beads and cells.The size-selection principle of microwells ensures that single cell and single bead are paired,and the capture efficiency of the cells and beads exceeds the limit of Poisson distribution and can process samples with high-throughput;the difference between the two chips is the structure of electrodes and wells,using new microwell-based microfluidic chip to capture and pair beads and cells,the capture efficiency of beads is 99.28±0.69%,and the highest reaches 100%,the capture efficiency of HL-60 cells and K562 cells is 96.06±3.25% and91.61±1.67% respectively,the pairing efficiency of bead and HL-60 cell is71.13 ± 4.84%,and the pairing efficiency of bead and K562 cell is 79.77 ±0.64%,achieving high capture and pairing efficiency.For the steps of cell lysis,this thesis discusses the different formulation of lysis buffer and the effect of different dilution ratios of lysis buffer on cell lysis,determines the optimal dilution ratio of lysis buffer,and conducts experiments on HL-60 cells and K562 cells,with the lysis efficiency of91.23±1.67% and 90.46±0.63%,respectively.Fluorescent dyes are used to verify that the chip is beneficial for rinsing to remove free mRNA,and injecting mineral oil can seal the microwell.In particular,in order to improve the viability of cells in the wells of the new microwell-based microfluidic chip to obtain more useful data,this thesis designs a microfluidic chip for cell sorting.The chip uses the active separation mechanism of dielectrophoresis,and the cell sorting efficiency reaches 94.82±1.72%.