Nakayama Tsuyoshi, Kurosawa Yasunori, Furui Satoshi, Kerman Kagan, Kobayashi Masaaki, Rao S Ramachandra, Yonezawa Yuji, Nakano Kouichi, Hino Akihiro, Yamamura Shohei, Takamura Yuzuru, Tamiya Eiichi
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
Anal Bioanal Chem. 2006 Nov;386(5):1327-33. doi: 10.1007/s00216-006-0688-7. Epub 2006 Aug 8.
Polymerase chain reaction (PCR) is an essential part of research based on genomics or cell analysis. The development of a microfluidic device that would be suitable for high-temperature-based reactions therefore becomes an important contribution towards the integration of micro-total analysis systems (muTAS). However, problems associated with the generation of air bubbles in the microchannels before the introduction of the assay liquid, which we call the "initial start-up" in this study, made the flow irregular and unstable. In this report, we have tried to address these problems by adapting a novel liquid-flow method for high-temperature-based reactions. A PDMS-based microfluidic device was fabricated by soft-lithography techniques and placed on a cartridge heater. The generation of the air bubbles was prevented by introducing the fluorinated oil, an inert and highly viscous liquid, as the cap just before the introduction of the sample solutions into the microchannels. The technique was applied for continuous-flow PCR, which could perform PCR on-chip in a microfluidic system. For the evaluation of practical accuracy, plasmid DNA that serves as a reference molecule for the quantification of genetically modified (GM) maize was used as the template DNA for continuous-flow PCR. After PCR, the products were collected in a vial and analyzed by gel electrophoresis to confirm the accuracy of the results. Additionally, quantitative continuous-flow PCR was performed using TaqMan technology on our PCR device. A laser detection system was also used for the quantitative PCR method. We observed a linear relationship between the threshold cycle (Ct) and the initial DNA concentration. These results showed that it would be possible to quantify the initial copies of the template DNA on our microfluidic device. Accurate quantitative DNA analysis in microfluidic systems is required for the integration of PCR with muTAS, thus we anticipate that our device would have promising potential for applications in a wide range of research.
聚合酶链反应（PCR）是基于基因组学或细胞分析的研究的重要组成部分。因此，开发一种适用于高温反应的微流控装置对于微全分析系统（μTAS）的集成具有重要意义。然而，在引入检测液之前微通道中产生气泡的问题，在本研究中我们称之为“初始启动”，导致流动不规则且不稳定。在本报告中，我们尝试通过采用一种新颖的用于高温反应的液流方法来解决这些问题。通过软光刻技术制造了基于聚二甲基硅氧烷（PDMS）的微流控装置，并将其放置在盒式加热器上。在将样品溶液引入微通道之前，通过引入氟化油（一种惰性且高粘性的液体）作为覆盖层来防止气泡的产生。该技术应用于连续流动PCR，其可在微流控系统中进行芯片上的PCR。为了评估实际准确性，将用作转基因（GM）玉米定量参考分子的质粒DNA用作连续流动PCR的模板DNA。PCR后，将产物收集到小瓶中并通过凝胶电泳进行分析以确认结果的准确性。此外，在我们的PCR装置上使用TaqMan技术进行了定量连续流动PCR。激光检测系统也用于定量PCR方法。我们观察到阈值循环（Ct）与初始DNA浓度之间存在线性关系。这些结果表明，在我们的微流控装置上对模板DNA的初始拷贝进行定量是可能的。微流控系统中准确的定量DNA分析对于将PCR与μTAS集成是必需的，因此我们预计我们的装置在广泛的研究应用中具有广阔的前景。
