Engineering Research Center of Optical Instrument and System, Key Lab of Optical Instruments and Equipment for Medical Engineering, Ministry of Education, Shanghai Key Lab of Modern Optical System, Shanghai Environmental Biosafety Instruments and Equipment Engineering Technology Research Center, University of Shanghai for Science and Technology, Shanghai, 200093, China.
State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai, 200050, China; Department of Clinical Laboratory, Third Affiliated Hospital of Guangzhou Medical University, Guangdong, 510150, China.
Anal Chim Acta. 2023 Apr 22;1251:340995. doi: 10.1016/j.aca.2023.340995. Epub 2023 Feb 24.
Rapid diagnosis of harmful microorganisms demonstrated its great importance for social health. Continuous flow PCR (CF-PCR) can realize rapid amplification of target genes by placing the microfluidic chip on heaters with different temperature. However, bubbles and evaporation always arise from heating, which makes the amplification not stable. Water-in-oil droplets running in CF-PCR microfluidic chip with uniform height takes long time because of the high resistance induced by long meandering microchannel. To overcome those drawbacks, we proposed a double-layer droplet CF-PCR microfluidic chip to reduce the fluidic resistance, and meanwhile nanoliter droplets were generated to minimize the bubbles and evaporation.
Experiments showed that (1) fluidic resistance could be reduced with the increase of the height of the serpentine microchannel if the height of the T-junction part was certain. (2) Running speed, the size and the number of generated droplets were positively correlated with the cross-sectional area of the T-junction and water pressure. (3) Droplet fusion happened at higher water pressure if other experimental conditions were the same. (4) 0.032 nL droplet was created if the cross-sectional area of T-junction and water pressure were 1600 μm (40 × 40 μm) and 7 kPa, respectively. Finally, we successfully amplified the target genes of Porphyromonas gingivalis within 11'16″ and observed the fluorescence from droplets.
Such a microfluidic chip can effectively reduce the high resistance induced by long meandering microchannel, and greatly save time required for droplets CF-PCR. It offers a new way for the rapid detection of bacterial.
有害微生物的快速诊断对社会健康具有重要意义。连续流动 PCR（CF-PCR）通过将微流控芯片放置在具有不同温度的加热器上，可以实现目标基因的快速扩增。然而，加热时总会产生气泡和蒸发，这使得扩增不稳定。由于蜿蜒微通道引起的高阻力，在 CF-PCR 微流控芯片中运行的具有均匀高度的油包水乳状液需要很长时间。为了克服这些缺点，我们提出了一种双层液滴 CF-PCR 微流控芯片，以降低流体阻力，同时生成纳升级液滴，以最小化气泡和蒸发。
实验表明：（1）如果 T 型接头部分的高度确定，则增加蛇形微通道的高度可以降低流体阻力。（2）运行速度、生成液滴的大小和数量与 T 型接头的横截面面积和水压成正比。（3）如果其他实验条件相同，则在较高水压下会发生液滴融合。（4）如果 T 型接头的横截面面积和水压分别为 1600μm（40×40μm）和 7kPa，则可以生成 0.032nL 的液滴。最后，我们成功地在 11'16" 内扩增了牙龈卟啉单胞菌的靶基因，并观察到了来自液滴的荧光。
这种微流控芯片可以有效地降低蜿蜒微通道引起的高阻力，大大节省液滴 CF-PCR 所需的时间。它为细菌的快速检测提供了一种新方法。
