Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University, 1-3 Tataramiyakodani, Kyotanabe 610-0321, Kyoto, Japan.
Sensors (Basel). 2023 Dec 17;23(24):9884. doi: 10.3390/s23249884.
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.
微流控液滴聚合酶链反应（PCR）可实现众多液滴中同时进行 DNA 扩增，由此带来了各种应用的发现，这些应用在以前被认为是无法实现的。几十年前，人们已经证明，如果能够实现整个 PCR 混合物的快速温度变化，那么在 PCR 扩增的热循环中，变性和退火阶段的温度保持时间基本上可以消除。促进这种快速热循环协议应用的微流控设备显著缩短了 PCR 所需的时间。然而，在微流控液滴 PCR 中，确保液滴内的单个分子成功扩增，限制了通过快速热循环加速检测的研究。我们开发的微流控盒，以其在普通实验室设备上执行单分子液滴 PCR 的便利性而著称，其特点是将液滴定位在薄玻璃载玻片上。我们假设将快速热循环应用于该微流控盒，将实现单分子液滴 PCR 扩增。实际上，该快速方案的应用仅在 30 个循环（40 秒/循环）中就实现了 22 分钟的成功扩增。这一突破意义重大，因为它有可能加速微流控液滴 PCR 检测，在极短的时间内实现高效的单分子扩增。
