Department of Applied Chemistry, Graduate School of Urban Environmental Sciences , Tokyo Metropolitan University , Minamiohsawa , Hachioji, Tokyo 192-0397 , Japan.
Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology , Tsinghua University , Beijing 100084 , China.
Anal Chem. 2018 Apr 17;90(8):5329-5334. doi: 10.1021/acs.analchem.8b00463. Epub 2018 Apr 3.
We report on the development of a novel and flexible online digital polymerase chain reaction (dPCR) system. The system was composed of three parts: an inkjet for generating the droplets, a coiled fused-silica capillary for thermal cycling, and a laser-induced fluorescence detector (LIFD) for positive droplet counting. Upon inkjet printing, monodisperse droplets were continuously generated in the oil phase and then introduced into the capillary in the form of a stable dispersion. The droplets containing one or zero molecules of target DNA passed through the helical capillary that was attached to a cylindrical thermal cycler for PCR amplification, resulting in the generation of fluorescence for the DNA-positive droplet. After 36 PCR cycles, the fluorescence signal intensity was detected by laser-induced fluorescence located at the downstream of the capillary, followed by a positive/negative counting. The present system was successfully applied to the absolute quantification of the HPV sequence in Caski cells with dynamic ranges spanning 4 orders of magnitude.
我们报告了一种新颖且灵活的在线数字聚合酶链反应 (dPCR) 系统的开发。该系统由三部分组成：用于生成液滴的喷墨装置、螺旋熔融硅毛细管用于热循环，以及用于正液滴计数的激光诱导荧光检测器 (LIFD)。在喷墨打印时，单分散液滴连续在油相中生成，然后以稳定分散体的形式引入毛细管。含有一个或零个靶 DNA 分子的液滴通过螺旋毛细管传递，该毛细管连接到圆柱形热循环仪进行 PCR 扩增，导致 DNA 阳性液滴产生荧光。经过 36 个 PCR 循环后，激光诱导荧光位于毛细管下游处检测荧光信号强度，随后进行阳性/阴性计数。该系统成功应用于 Caski 细胞中 HPV 序列的绝对定量，动态范围跨越 4 个数量级。
