LAUSR

Digital PCR (dPCR) is a powerful technique capable of absolute quantification of nucleic acids with good accuracy. Droplet-based dPCR (ddPCR), among others, is one of the most important dPCR techniques. However, the surface tension-controlled droplets may suffer from fusion/fission due to the vigorous temperature change in PCR thermal cycling. Besides, the free movement of droplets makes them unsuitable for real-time fluorescence monitoring. In this paper, we first developed a photo-immobilized planar droplet array (PIPDA) by using a photocurable polyurethane as the continuous oil phase. It is found that uniform water-in-oil droplets of various sizes can be readily generated, and more importantly, the oil phase can be rapidly solidified in just a few seconds upon exposure to UV irradiation. This process will leave the droplets immobilized in the accommodation chamber as a stable planar array, and thus effectively prevent the movement, coalescence and breakup of droplets. In addition, a novel multi-layered chip design has been proposed, which can thoroughly overcome the evaporation issue that commonly exists in polydimethylsiloxane (PDMS)-based dPCR chips. With these two innovations, the ddPCR experiment could be performed in a robust manner, and shows a promising potential in the development of real-time ddPCR technique. These features may therefore enable the wide application of PIPDA-based ddPCR in various fields.