Fast Thermocycling in Custom Microfluidic Cartridge for Rapid Single-Molecule Droplet PCR

The microfluidic droplet polymerase chain reaction (PCR), which enables simultaneous DNA amplification in numerous droplets, has led to the discovery of various applications that were previously deemed unattainable. Decades ago, it was demonstrated that the temperature holding periods at the denaturation and annealing stages in thermal cycles for PCR amplification could be essentially eliminated if a rapid change of temperature for an entire PCR mixture was achieved. Microfluidic devices facilitating the application of such fast thermocycling protocols have significantly reduced the time required for PCR. However, in microfluidic droplet PCR, ensuring successful amplification from single molecules within droplets has limited studies on accelerating assays through fast thermocycling. Our developed microfluidic cartridge, distinguished for its convenience in executing single-molecule droplet PCR with common laboratory equipment, features droplets positioned on a thin glass slide. We hypothesized that applying fast thermocycling to this cartridge would achieve single-molecule droplet PCR amplification. Indeed, the application of this fast protocol demonstrated successful amplification in just 22 min for 30 cycles (40 s/cycle). This breakthrough is noteworthy for its potential to expedite microfluidic droplet PCR assays, ensuring efficient single-molecule amplification within a remarkably short timeframe.