Prakash R, Pabbaraju K, Wong S, Wong A, Tellier R, Kaler K V I S
Biosystems Research and Applications Group, Schulich School of Engineering, University of Calgary, Calgary, Alberta AB T2N 1N4, Canada.
Provincial Laboratory for Public Health of Alberta, ProvLAB, Calgary AB T2N4W4, Canada.
J Electrochem Soc. 2014 Jan;161(2):B3083-B3093. doi: 10.1149/2.013402jes. Epub 2013 Dec 27.
Miniaturized bio-diagnostic devices have the potential to allow for rapid pathogen screening in clinical patient samples, as a low cost and portable alternative to conventional bench-top equipment. Miniaturization of key bio-diagnostic techniques, such as: nucleic acid detection and quantification, polymerase chain reaction (PCR), DNA fingerprinting, enzyme linked immunosorbent assay (ELISA), results in substantial reduction of reaction volumes (expensive samples/reagents) and shorter reaction times. Droplet microfluidics (DMF) is one of several miniaturized bio-sample handling techniques available for manipulating clinical samples and reagents in microliter (10 L) to picoliter (10 L) volume regime. Electro-actuation of sample and reagent in the form of droplets in the aforementioned volume regime, using dielectrophoresis (DEP) and/or Electrowetting (EW) are achieved by means of patterned, insulated metal electrodes on one or more substrates. In this work, we have utilized electro-actuation based DMF technology, integrated with suitably tailored resistive micro-heaters and temperature sensors, to achieve chip based real-time, quantitative PCR (qRT-PCR). This qRT-PCR micro-device was utilized to detect and quantify the presence of influenza A and C virus nucleic acids, using in-vitro synthesized viral RNA segments. The experimental analysis of the DMF micro-device confirms its capabilities in qRT-PCR based detection and quantification of pathogen samples, with accuracy levels comparable to established commercial bench-top equipment (PCR efficiency ∼95%). The limit of detection (LOD) of the chip based qRT-PCR technique was estimated to be ∼5 copies of template RNA per PCR reaction.
小型化生物诊断设备有潜力在临床患者样本中实现快速病原体筛查，作为传统台式设备的低成本、便携式替代方案。关键生物诊断技术的小型化，如核酸检测与定量、聚合酶链反应（PCR）、DNA指纹识别、酶联免疫吸附测定（ELISA），可大幅减少反应体积（昂贵的样本/试剂）并缩短反应时间。液滴微流控（DMF）是几种可用于在微升（10⁻⁶升）至皮升（10⁻¹²升）体积范围内处理临床样本和试剂的小型化生物样本处理技术之一。通过在一个或多个基板上的图案化绝缘金属电极，以上述体积范围内的液滴形式对样本和试剂进行电驱动，利用介电泳（DEP）和/或电润湿（EW）来实现。在这项工作中，我们利用基于电驱动的DMF技术，集成了经过适当定制的电阻微加热器和温度传感器，以实现基于芯片的实时定量PCR（qRT-PCR）。该qRT-PCR微型设备用于使用体外合成的病毒RNA片段检测和定量甲型和丙型流感病毒核酸的存在。对DMF微型设备的实验分析证实了其在基于qRT-PCR的病原体样本检测和定量方面的能力，其准确度水平与成熟的商业台式设备相当（PCR效率约为95%）。基于芯片的qRT-PCR技术的检测限估计为每个PCR反应约5个模板RNA拷贝。
