A Microfluidic Multi-step Droplet Splitting System For Single-cell Encapsulation

Single cell analysis is of great significance to understand the physiological activity of organisms, diagnosis and treatment of diseases, drug development, etc. Microfluidic droplet technology is an important analytical platform for single-cell analysis. The common method based on single droplet generation structure and random encapsulation mode is low in droplet generation frequency and cell encapsulation efficiency. In this paper, flow focusing structure and multi-step splitting structure were integrated to form droplets with high frequency, and whole blood cells were encapsulated in droplets with improved single cells encapsulation efficinecy.In chapter one, microfluidic droplet formation and manipulation, as well as the research progress of encapsulation of (single) cells in droplets were mainly overviewed. A variety of cell encapsulation methods were introduced in detail, including the advantages and limitations of these methods. The purpose and design of this paper were proposed in the end.In chapter two, a microfluidic chip-based multi-step droplet splitting system was developed. Flow focusing structure was selected for droplet formation. Each droplet generated was split equally into branch channels passing through multi-level Y-shaped structure. The number of channels increased twice and the width of them decreased with the increment of the step. The experiment results indicated that droplet size and generation frequency could be changed by adjusting the flow rates of the two phases. With a given flow rate of aqueous phase, the faster the oil phase flowed, the smaller size and the higher formation frequency were obtained. Droplets with good monodispersity were produced with aqueous phase flow rate of 1 μL/min and the oil phase flow rate in a range of 0.5-5 μL/min. RSD of the droplet size in a single channel was 2%-5%, while in the 8 channels was 3%-6%. Droplet generation frequency reached 1021 Hz with the aqueous phase flow rate of 1.0 μL/min and the oil phase flow rate of 15 μL/min. The droplet generation system features simple structure, good monodispersity and high throughput.In chapter three, whole blood cells were encapsulated using the microfluidic multi-step droplet splitting system. By designing winding channels in the cell entrance channel and in front of the Y-shaped branching channels, the uneven distribution of cells was effectively improved. By using OptiprepTM diluent, the cell sedimentation phenomenon was improved. The influence of cell concentrations on cell encapsulation was investigated. A single-cell encapsulation efficiency of 31% was obtained with the theoretical blood cell concentration of 2.5×10O4/μL, and the multicellular droplet percentage was only 1.3%. We assessed the encapsulation of HeLa cells, and the encapsulation was not satisfactory, which might be improved by enhancing mixing.In chapter four, the multi-step droplet splitting system and its application in single cell encapsulation were summarized and predicted.