Easy-to-Operate Co-flow Step Emulsification Device for Droplet Digital Polymerase Chain Reaction.

Digital polymerase chain reaction (PCR) plays important roles in the detection and quantification of nucleic acid targets, while there still remain challenges including high cost, complex operation, and low integration of the instrumental system. Here, in this work, a novel microfluidic chip based on co-flow step emulsification is proposed for droplet digital PCR (ddPCR), which can achieve droplet generation, droplet array self-assembly, PCR amplification, and fluorescence detection on a single device. With the combination of single-layer lithography and punching operation, a step microstructure was constructed and it served as the key element to develop a Laplace pressure gradient at the Rayleigh-Plateau instability interface so as to achieve droplet generation. It is demonstrated that the fabrication of step microstructure is low cost, easy-to-operate, and reliable. In addition, the single droplet volume can be adjusted flexibly due to the co-flow design; thus, the ddPCR chip can get an ultrahigh upper limit of quantification to deal with DNA templates with high concentrations. Furthermore, the volume fraction of the resulting droplets in this ddPCR chip can be up to 72% and it results in closely spaced droplet arrays, makes the best of CCD camera for fluorescence detections, and is beneficial for the minimization of a ddPCR system. The quantitative capability of the ddPCR chip was evaluated by measuring template DNA at concentrations from 20 to 50 000 copies/μL. Owing to the characteristics of low cost, easy operation, excellent quantitative capability, and minimization, the proposed ddPCR chip meets the requirements of DNA molecule quantification and is expected to be applied in the point-of-care testing field.