Establishment and Validation of an Integrated Microfluidic Step Emulsification Chip Supporting Droplet Digital Nucleic Acid Analysis.

Uniform and stable droplet generation is critical for accurate and efficient digital nucleic acid analysis (dNAA). In this study, an integrated microfluidic step emulsification device with wide-range droplet generation capability, small device dimensions, convenient fabrication strategy, low contamination and high robustness was developed. A tree-shaped droplet generation nozzle distribution design was proposed to increase the uniformity of droplet generation by equating flow rates, and the flow field in the design was numerically simulated. Theoretical analysis and comparative experiments on droplet size were performed regarding the influences of nozzle dimensions and surface properties. With incubation and hydrophobic reagent treatment, droplets as small as 73.1 μm were generated with multiplex nozzles of 18 μm (h) × 80 μm (w). The droplets were then collected into a standard PCR tube and an on-chip monolayer droplet collection chamber, without manual transfer and sample contamination. The oil-to-sample volume ratio in the PCR tube was recorded during collection. In the end, the droplets generated and collected using the microfluidic device proved to be stable and uniform for nucleic acid amplification and detection. This study provides reliable characteristic information for the design and fabrication of a micro-droplet generation device, and represents a promising approach for the realization of a three-in-one dNAA device under a step emulsification method.