A droplet digital PCR chip with passive bubble removal for absolute nucleic acid quantification

Droplet digital polymerase chain reaction (ddPCR) is an extremely sensitive method for the precisely determining the concentration of target nucleic acids. However, air bubbles between droplets during amplification can cause significant droplet loss and decreased accuracy in results. In the present study, an all-in-one microfluidic chip that integrates emulsification, passive bubble removal, droplet monolayer storage, on-chip nucleic acid amplification, and droplet fluorescence signal readout is proposed. The integrated passive bubble removal structures automatically complete the trapping and guiding of the bubbles, ensuring that the droplets do not touch the bubbles during amplification and thus is not lost. The ddPCR device with optimized key parameters proved to be effective and efficient by completely removing bubbles between droplets and having a dead volume of less than 1 %. The ability of the ddPCR chip to accurately quantify nucleic acids was evaluated by measuring plasmids with the SARS-CoV-2N gene at concentrations ranging from 10 to 50 000 copies/μL. The innovative ddPCR device satisfies the requirement for accurate nucleic acid quantification and is expected to accelerate the popularity of dPCR due to its low processing difficulty, ease of use and high robustness.