Circumventing air bubbles in microfluidic systems and quantitative continuous-flow PCR applications
Circumventing air bubbles in microfluidic systems and quantitative continuous-flow PCR applications
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DOI：
10.1007/s00216-006-0688-7
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发表时间：
2006-11-01
影响因子：
4.3
通讯作者：
Tamiya, Eiichi
中科院分区：
文献类型：
--
作者：
Nakayama, Tsuyoshi;Kurosawa, Yasunori;Tamiya, Eiichi
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，开发适合于基于高温的反应的微流体装置成为对微全分析系统（mu TAS）集成的重要贡献。然而，与在引入测定液体之前在微通道中产生气泡相关的问题，我们在本研究中称之为“初始启动”，使得流动不规则且不稳定。在本报告中，我们试图通过采用一种新型的液流方法来解决这些问题，以用于高温反应。基于PDMS的微流控器件通过软光刻技术制造，并放置在加热筒上。通过在即将将样品溶液引入微通道之前引入氟化油（一种惰性且高粘性的液体）作为盖来防止气泡的产生。该技术应用于连续流PCR，可以在微流控系统中进行芯片上PCR。为了评估实际的准确性，质粒DNA，作为一个参考分子的定量转基因（GM）玉米被用作模板DNA的连续流动PCR。PCR后，将产物收集在小瓶中并通过凝胶电泳分析以确认结果的准确性。此外，使用TaqMan技术在我们的PCR装置上进行定量连续流动PCR。激光检测系统也用于定量PCR方法。我们观察到的阈值循环（Ct）和初始DNA浓度之间的线性关系。这些结果表明，可以在我们的微流体装置上定量模板DNA的初始拷贝。在微流控系统中进行精确的定量DNA分析是将PCR与μ TAS相结合所必需的，因此我们预计我们的装置在广泛的研究中具有很好的应用潜力。
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 (mu TAS). 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 mu TAS, thus we anticipate that our device would have promising potential for applications in a wide range of research.