A single-cell encapsulation method based on a microfluidic multi-step droplet splitting system

Single cell analysis is of great significance to understand the physiological activity of organisms. Microfluidic droplet is an ideal analytical platform for single-cell analysis. We developed a microfluidic droplet splitting system integrated with a flow-focusing structure and multi-step splitting structures to form 8-line droplets and encapsulate single cells in the droplets. Droplet generation frequency reached 1021 Hz with the aqueous phase flow rate of 1 μL/min and the oil phase flow rate of 15 μL/min. Relative standard deviation of the droplet size was less than 5% in a single channel, while less than 6% in all the 8 channels. The system was used for encapsulating human whole blood cells. A single-cell encapsulation efficiency of 31% was obtained with the blood cell concentration of 2.5×10⁴ cells/μL, and the multicellular droplet percentage was only 1.3%. The multi-step droplet splitting system for single cell encapsulation featured simple structure and high throughput.