Single-cell Sequencing Sample Preparation Based On High-throughput Droplet Sorting And Fusion

Single-cell sequencing technology refers to the analysis of cell transcriptomes and genomes at the single-cell level.It can reveal the heterogeneity between cells and is of great significance for cell subtype screening and pathogenesis research.Microfluidic technology is to process fluids at the micro-nano scale,which has the advantages of small volume,fast reaction speed,and small reagent consumption.Microdroplets are a very important branch among them,single-cell sequencing based on microdroplets is high-throughput and low degree of cross-contamination so that it's widely used in single-cell research.However,due to the dual Poisson distribution of the droplets during the co-encapsulation of the cells and microbeads,in order to ensure the high purity of the effective sample,the problem of sample loss and too low the ratio of effective samples appeared.In response to this specific problem,this paper designed and tested a high-throughput droplet sorting and merging scheme,which aims to solve the sample loss caused by the dual Poisson distribution:1)By analyzing the problems caused by the dual Poisson distribution in single cell sequencing and comparing the types of droplets generated by different ? values,it is proposed to change the dual Poisson distribution into two independent Poisson distributions,encapsulate the cells and beads,respectively.Then the purity of the two samples is improved by droplet sorting,and the two droplets are fused by electrocoalescence Based on this scheme,a set of microfluidic chips were designed and fabricated,which were used for droplet generation,sorting,merging,and array.2)By comparing different droplet sorting forces and detection targets,a fluorescent activted droplet sorting scheme based on dielectrophoresis was determined,and the flow field,electric field,and sorting process of the sorting chip were studied through COMSOL simulation to Optimize the flow channel structure,initial flow rate and voltage value.Based on an inverted microscope,an optical system for high-throughput fluorescence detection was built;using PMT as the interface,a data collection and control system for fluorescence detection was built.Finally,we enriches fluorescent droplets encapsulated 16?m fluorescentmicrospheres.3)In this paper,a new "active and passive droplet merging scheme" is designed.The groove structure is used as the reaction chamber.The combination of drag force change,dielectrophoretic force,and electrocoalescence can realize the capture,waiting,fusion and release of droplets.The droplet merging experiment was carried out with 50?m droplets as samples,and the experiment showed that the droplets could be merged with high throughput.4)In order to facilitate the observation and analysis of the changes of droplets before and after sorting and merging,this paper studied a large-scale microdroplet array chip,and arrayed 50?m droplets in the chip successfully.