Development Of Cell-bead Capture Microfluidics Forsingle-cell RNA Sequencing

Cellular heterogeneity plays an important role in interpretation of the occurrence and development of diseases.Analysis of heterogeneity at the single-cell level is critical for disease diagnosis and treatment.Ribonucleic Acid(RNA)is an significant role in cellular protein synthesis and gene regulation.Because single-cell RNA sequencing has the characteristics of high throughput and sensitivity,it is suitable for the analysis of cell heterogeneity.Single-cell capture is a crucial and fundamental step for RNA sequencing,but it is very difficult to isolate and capture single cells in the sample which using traditional technical means.Microfluidics,as a technology for manipulating and controlling microfluidics in micron-scale channels,perfectly fits the technical requirements of single-cell capture.Most of the existing micropore-based single-cell capture microfluidic chips use random drop for passive capture,which has problems such as low capture rate and complex processing technology.In order to solve the above problems,in this study,the microelectrode is combined with the microwell structure,and the cells and microbeads are actively captured by the dielectrophoretic force generated by the structure of the microelectrode.The electrode flow channel integrated molding is realized by using silver-polydimethylsiloxane(AgPDMS)conductive polymer as the electrode material.In order to combine the electrode structure with the microwell structure package,a set of high-precision alignment platform was built to quickly bond the electrode layer chip and the microwell layer chip.In this thesis,the microbead capture was firstly tested,and an automatic counting and statistical program was developed based on digital image technology.In the microbead capture experiment,by optimizing the size and depth of the micropores,the capture rate of more than 95% of the microbeads in 700 microwell was achieved.In the cell capture experiment,a capture rate of more than 86% of human promyelocytic acute leukemia cells(HL-60)was achieved;by using collagenase D to improve the aggregation of mouse fibroblasts(3T3)cells,the capture rate of 3T3 cells is more than 80%;finally,the capture pairing rate of HL-60 cells and microbeads has reached more than 80%,and the capture rate of 3T3 cells and microbeads has reached more than 70%.Capture pairing rate.