Massive droplet generation for digital PCR via a smart step emulsification chip integrated in a reaction tube.

Step emulsification (SE) devices coupled with parallel generation nozzles are widely used in the production of large-scale monodisperse droplets, especially for droplet-based digital polymerase chain reaction (ddPCR) analysis. Although current ddPCR systems based on the SE method can provide a fully enclosed ddPCR scheme, high demands on chip fabrication and system control will increase testing costs and reduce its flexibility in ddPCR analysis. In this study, a compact SE device, integrating a smart SE chip into a reaction tube, was developed to prepare large-scale water-in-fluorinated-oil droplets for ddPCR analysis. The SE chip contained dozens of droplet-generation nozzles. By adjusting the nozzle height of the SE chip, monodisperse droplets in a picolitre to nanolitre vloume could be prepared at a production rate of tens to hundreds of microlitres per minute. Subsequently, we utilized such an integrated SE device to prepare monodisperse droplets for ddPCR experiments. The volume of PCR reagent and the number of droplets could be flexibly adjusted according to the requirements of the ddPCR analysis. The quantitative results showed that emulsions prepared by the SE device could achieve ddPCR detection with high accuracy, good repeatability, and an adaptive dynamic range, which also demonstrated the robustness and reliability of such devices in the droplet preparation. Thus, this compact SE device provides an inexpensive, flexible, and simplified droplet preparation method for digital PCR quantitative analysis.