Advanced Liquid Logic Incorporated, Research Triangle Park, North Carolina, USA.
Anal Chem. 2010 Mar 15;82(6):2310-6. doi: 10.1021/ac902510u.
This paper details the development of a digital microfluidic platform for multiplexed real-time polymerase chain reactions (PCR). Liquid samples in discrete droplet format are programmably manipulated upon an electrode array by the use of electrowetting. Rapid PCR thermocycling is performed in a closed-loop flow-through format where for each cycle the reaction droplets are cyclically transported between different temperature zones within an oil-filled cartridge. The cartridge is fabricated using low-cost printed-circuit-board technology and is intended to be a single-use disposable device. The PCR system exhibited remarkable amplification efficiency of 94.7%. To test its potential application in infectious diseases, this novel PCR system reliably detected diagnostic DNA levels of methicillin-resistant Staphylococcus aureus (MRSA), Mycoplasma pneumoniae , and Candida albicans . Amplification of genomic DNA samples was consistently repeatable across multiple PCR loops both within and between cartridges. In addition, simultaneous real-time PCR amplification of both multiple different samples and multiple different targets on a single cartridge was demonstrated. A novel method of PCR speed optimization using variable cycle times has also been proposed and proven feasible. The versatile system includes magnetic bead handling capability, which was applied to the analysis of simulated clinical samples that were prepared from whole blood using a magnetic bead capture protocol. Other salient features of this versatile digital microfluidic PCR system are also discussed, including the configurability and scalability of microfluidic operations, instrument portability, and substrate-level integration with other pre- and post-PCR processes.
本文详细介绍了一种用于多重实时聚合酶链反应（PCR）的数字微流控平台的开发。通过使用电润湿，以离散液滴形式存在的液体样品可在电极阵列上进行可编程操作。快速 PCR 热循环以闭环流动形式进行，其中对于每个循环，反应液滴在充满油的盒内的不同温度区域之间周期性地运输。该盒使用低成本印刷电路板技术制造，旨在成为一次性使用的一次性设备。PCR 系统表现出显著的扩增效率为 94.7%。为了测试其在传染病中的潜在应用，该新型 PCR 系统可靠地检测了耐甲氧西林金黄色葡萄球菌（MRSA）、肺炎支原体和白色念珠菌的诊断 DNA 水平。在多个 PCR 循环内和之间，基因组 DNA 样本的扩增始终具有可重复性。此外，还证明了在单个盒上同时实时多重不同样本和多个不同靶标的 PCR 扩增是可行的。还提出并证明了一种使用可变循环时间的 PCR 速度优化的新方法是可行的。该多功能系统包括磁珠处理能力，已应用于使用磁珠捕获方案从全血制备的模拟临床样本的分析。还讨论了这种多功能数字微流控 PCR 系统的其他显著特点，包括微流控操作的可配置性和可扩展性、仪器的便携性以及与其他预 PCR 和后 PCR 过程的基板级集成。
