Narahari Tanya, Dahmer Joshua, Sklavounos Alexandros, Kim Taehyeong, Satkauskas Monika, Clotea Ioana, Ho Man, Lamanna Julian, Dixon Christopher, Rackus Darius G, Silva Severino Jefferson Ribeiro da, Pena Lindomar, Pardee Keith, Wheeler Aaron R
Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario, M5S 3H6, Canada.
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario, M5S 3E1, Canada.
Lab Chip. 2022 May 3;22(9):1748-1763. doi: 10.1039/d1lc01068a.
This paper introduces a digital microfluidic (DMF) platform for portable, automated, and integrated Zika viral RNA extraction and amplification. The platform features reconfigurable DMF cartridges offering a closed, humidified environment for sample processing at elevated temperatures, as well as programmable control instrumentation with a novel thermal cycling unit regulated using a proportional integral derivative (PID) feedback loop. The system operates on 12 V DC power, which can be supplied by rechargeable battery packs for remote testing. The DMF system was optimized for an RNA processing pipeline consisting of the following steps: 1) magnetic-bead based RNA extraction from lysed plasma samples, 2) RNA clean-up, and 3) integrated, isothermal amplification of Zika RNA. The DMF pipeline was coupled to a paper-based, colorimetric cell-free protein expression assay for amplified Zika RNA mediated by toehold switch-based sensors. Blinded laboratory evaluation of Zika RNA spiked in human plasma yielded a sensitivity and specificity of 100% and 75% respectively. The platform was then transported to Recife, Brazil for evaluation with infectious Zika viruses, which were detected at the 100 PFU mL level from a 5 μL sample (equivalent to an RT-qPCR cycle threshold value of 32.0), demonstrating its potential as a sample processing platform for miniaturized diagnostic testing.
本文介绍了一种用于便携式、自动化和集成式寨卡病毒RNA提取与扩增的数字微流控（DMF）平台。该平台的特点包括可重新配置的DMF盒，其为高温下的样品处理提供了一个封闭、湿润的环境，以及带有新型热循环单元的可编程控制仪器，该热循环单元采用比例积分微分（PID）反馈回路进行调节。该系统采用12 V直流电源运行，可由可充电电池组供电以进行远程测试。DMF系统针对由以下步骤组成的RNA处理流程进行了优化：1）从裂解的血浆样品中基于磁珠提取RNA，2）RNA纯化，以及3）寨卡病毒RNA的集成等温扩增。DMF流程与基于纸的比色无细胞蛋白质表达检测相结合，用于由基于toehold开关的传感器介导的扩增寨卡病毒RNA检测。对加标于人类血浆中的寨卡病毒RNA进行的盲法实验室评估，其灵敏度和特异性分别为100%和75%。然后，该平台被运往巴西累西腓，用于对感染性寨卡病毒进行评估，从5 μL样品中在100 PFU/mL水平检测到了寨卡病毒（相当于RT-qPCR循环阈值为32.0），证明了其作为小型化诊断测试样品处理平台的潜力。
