Hügle M, Dame G, Behrmann O, Rietzel R, Karthe D, Hufert F T, Urban G A
Laboratory for Sensors, Department of Microsystems Engineering (IMTEK), University of Freiburg Georges-Köhler-Allee 103 Freiburg Germany.
Institute of Microbiology and Virology, Brandenburg Medical School Theodor Fontane Neuruppin Germany.
RSC Adv. 2018 Jun 1;8(36):20124-20130. doi: 10.1039/c8ra02177e. eCollection 2018 May 30.
To improve detection sensitivity, molecular diagnostics require preconcentration of low concentrated samples followed by rapid nucleic acid extraction. This is usually achieved by multiple centrifugation, lysis and purification steps, for instance, using chemical reagents, spin columns or magnetic beads. These require extensive infrastructure as well as time consuming manual handling steps and are thus not suitable for point of care testing (POCT). To overcome these challenges, we developed a microfluidic chip combining free-flow electrophoretic (FFE) preconcentration (1 ml down to 5 μl) and thermoelectric lysis of bacteria as well as purification of nucleic acids by gel-electrophoresis. The integration of these techniques in a single chip is unique and enables fast, easy and space-saving sample pretreatment without the need for laboratory facilities, making it ideal for the integration into small POCT devices. A preconcentration efficiency of nearly 100% and a lysis/gel-electrophoresis efficiency of about 65% were achieved for the detection of . The genetic material was analyzed by RT-qPCR targeting the superfolder Green Fluorescent Protein (sfGFP) transcripts to quantify mRNA recovery and qPCR to determine DNA background.
为提高检测灵敏度，分子诊断需要对低浓度样本进行预浓缩，然后进行快速核酸提取。这通常通过多次离心、裂解和纯化步骤来实现，例如使用化学试剂、离心柱或磁珠。这些方法需要大量的基础设施以及耗时的手动操作步骤，因此不适用于即时检测（POCT）。为克服这些挑战，我们开发了一种微流控芯片，该芯片结合了自由流电泳（FFE）预浓缩（从1 ml浓缩至5 μl）、细菌的热电裂解以及通过凝胶电泳纯化核酸。将这些技术集成在一个芯片中是独一无二的，能够实现快速、简便且节省空间的样品预处理，无需实验室设施，使其非常适合集成到小型POCT设备中。对于[此处原文缺失检测对象]的检测，实现了近100%的预浓缩效率和约65%的裂解/凝胶电泳效率。通过针对超折叠绿色荧光蛋白（sfGFP）转录本的逆转录定量聚合酶链反应（RT-qPCR）分析遗传物质，以量化mRNA回收率，并通过定量聚合酶链反应（qPCR）确定DNA背景。
