New Strategy Of Single Cell Analysis Based On Digital Microfluidics

Cell is the basic unit of life activities of the organism.Conventional bulk methods focusd on the statistical average results because of the lack of single-cell isolation or analysis methods and the limitation of the detection sensitivity,which masking the differences between individual cells.It would easily lead to the dilution or loss of cell information and affect the accuracy of the results.Single cell analysis is a method of studying the composition and morphology of cells at the single-cell level,which could provide more abundant and accurate individual information for life science research.However,the characteristics of low in content,difficult to obtain and easy to lose of single cell are the key issues restricting the development of single-cell research,which also to propose higher requirements on single-cell isolation and analysis methods in terms of high throughput,low damage and addressability.Digital micro fluidics is an emerging liquid-handling technology based on an array of microelectrodes for precise manipulation of large-scale discrete droplets with the characteristics of high parallelism,full automation and addressability.It can realize the real-time multiplex manipulation of droplets in picoliter to microliter volume,which is matched with cell size,demonstrating the high reaction efficiency while reducing the reagents consumption.Therefore,digital microfluidics is a forth promising platform in precise manipulation of single cell and its accurate analysis.In this thesis,we described a full-automated single-cell processing platform based on digital microfluidics.By constructing a multi-dimensional capture structure based on fluid dynamics and local-wetting characteristic on the chip,an efficient,fast,and nondestructive method for full-automated single cell isolation was achieved,conferring the benefits of high versatility,robustness,biocompatibility and automation capabilities.Based on this platform,we developed a novel single-cell whole-genome amplification and sequencing method which enables rapid and fully integrated single-cell wholegenome amplification with automatatic peocessing.Single-cell genomic fragment was limited to nanoliter volume,which overcame technical barriers of high bias and low efficiency of amplification due to the excessive volume,and improved sequencing coverage immensely.It enabled detection of copy number variation in an individual cell with significantly improved robustness and accuracy.In addition,we also combined digital microfluidics with digital proximity ligation assay for quantitative detection of single-cell protein.The information of single-cell protein with lowabundance was concentrated in nanoliter volume droplet,which was furthermore converted into nucleic acid for amplification and reading out,thus improving the detection sensitivity of single-cell protein.In summary,we developed a single-cell isolation and analysis method based on digital microfluidics,which can achieve the full-range of precise manipulation of discrete droplets with the advantages of automation,integration,small volume,and scalability,revealing the excellent application potential in cell heterogeneity and clinical research.