Graduate Institute of Biomedical Engineering, Chang Gung University, Taoyuan 333, Taiwan.
Department of Laboratory Medicine, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan.
Biosensors (Basel). 2022 May 6;12(5):303. doi: 10.3390/bios12050303.
Polymerase chain reaction (PCR) is limited by the long reaction time for point-of-care. Currently, commercial benchtop rapid PCR requires 30−40 min, and this time is limited by the absence of rapid and stable heating and cooling platforms rather than the biochemical reaction kinetics. This study develops an ultrafast PCR (<3 min) platform using flow-through microchannel chips. An actin gene amplicon with a length of 151 base-pairs in the whole genome was used to verify the ultrafast PCR microfluidic chip. The results demonstrated that the channel of 56 μm height can provide fast heat conduction and the channel length should not be short. Under certain denaturation and annealing/extension times, a short channel design will cause the sample to drive slowly in the microchannel with insufficient pressure in the channel, causing the fluid to generate bubbles in the high-temperature zone and subsequently destabilizing the flow. The chips used in the experiment can complete 40 thermal cycles within 160 s through a design with the 56 µm channel height and with each thermal circle measuring 4 cm long. The calculation shows that the DNA extension speed is ~60 base-pairs/s, which is consistent with the theoretical speed of the Klen Taq extension used, and the detection limit can reach 67 copies. The heat transfer time of the reagent on this platform is very short. The simple chip design and fabrication are suitable for the development of commercial ultrafast PCR chips.
聚合酶链反应（PCR）在即时检测中受到反应时间长的限制。目前，商用台式快速 PCR 需要 30-40 分钟，而这一时间的限制不是由于生化反应动力学，而是由于缺乏快速且稳定的加热和冷却平台。本研究使用流通式微流控芯片开发了一种超快 PCR（<3 分钟）平台。使用全长 151 个碱基对的肌动蛋白基因扩增子来验证超快 PCR 微流控芯片。结果表明，56μm 高度的通道可以提供快速的热传导，并且通道长度不应过短。在一定的变性和退火/延伸时间下，短通道设计会导致在通道中压力不足的情况下，样品在微通道中缓慢驱动，导致在高温区产生气泡，随后使流动不稳定。实验中使用的芯片通过 56 µm 通道高度的设计，在 160 秒内可完成 40 个热循环，每个热循环长 4 厘米。计算表明，DNA 延伸速度约为 60 个碱基对/s，与所用的 Klen Taq 延伸的理论速度一致，检测限可达 67 拷贝。该平台上试剂的传热时间非常短。这种简单的芯片设计和制造适合开发商用超快 PCR 芯片。
