Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University, 1-3 Tataramiyakodani, Kyotanabe, Kyoto 610-0321, Japan.
Anal Methods. 2021 Mar 4;13(8):974-985. doi: 10.1039/d0ay01779e.
We have previously established a cost-efficient in-house system for single-molecule droplet polymerase chain reaction (PCR) using a polydimethylsiloxane microfluidic cartridge and common laboratory equipment. However, the microfluidic cartridge was only capable of generating monodisperse water-in-oil droplets. Therefore, careful and time-consuming manual droplet handling using a micropipette was required to transfer droplets between the three discrete steps involved in the workflow of droplet PCR-i.e., (1) droplet generation; (2) PCR amplification; and (3) determination of the fluorescence intensity of the thermocycled droplets. In the current study, we developed a new microfluidic cartridge consisting of four layers, with a thin glass slide as the bottom layer. In this cartridge, droplets generated in the uppermost polydimethylsiloxane microfluidic layer are delivered to the glass slide in an online fashion. After the accumulation of many droplets on the glass slide, the cartridge is placed on the flatbed heat block of a thermocycler for PCR amplification. Direct fluorescence imaging of the thermocycled droplets on the glass slide is then carried out using a conventional fluorescence microscope. Efficient heat transfer from the heat block to the settled droplets through the thin glass slide was confirmed by successful PCR amplification inside the droplets, even from single template molecules. The new cartridge eliminates the need for manual droplet transfer between the major steps of droplet PCR analysis, allowing more convenient single-molecule droplet PCR than in our previous studies.
我们之前建立了一种使用聚二甲基硅氧烷微流控芯片和常见实验室设备的高效经济的单分子液滴聚合酶链反应（PCR）的内部系统。然而，微流控芯片只能产生单分散的油包水液滴。因此，需要使用微量移液器小心且耗时地手动处理液滴，以便在液滴 PCR 的三个离散步骤之间转移液滴，这三个步骤分别是：（1）液滴生成；（2）PCR 扩增；（3）循环后的液滴荧光强度的测定。在本研究中，我们开发了一种由四层组成的新型微流控芯片，其底层为薄玻璃片。在该芯片中，最上层的聚二甲基硅氧烷微流控层中生成的液滴以在线方式输送到玻璃片上。在玻璃片上积累了许多液滴之后，将该芯片放置在热循环仪的平板加热块上进行 PCR 扩增。然后使用常规荧光显微镜对玻璃片上的循环后的液滴进行直接荧光成像。通过薄玻璃片从加热块到沉降液滴的高效热传递得到了证实，即使是从单个模板分子中也可以成功进行 PCR 扩增。新的芯片消除了在液滴 PCR 分析的主要步骤之间手动转移液滴的需要，允许比我们之前的研究更方便地进行单分子液滴 PCR。
