Laboratory for Sensors, Department of Microsystems Engineering (IMTEK), Albert-Ludwigs-Universität Freiburg, Georges-Köhler-Allee 103, Freiburg, Germany.
Lab Chip. 2010 Mar 7;10(5):610-6. doi: 10.1039/b913481f. Epub 2009 Dec 9.
The lack of sample pre-treatment concepts that are easily automatable, miniaturized and highly efficient for both small volumes and low target concentrations, is one of the key issues that block the road towards effective miniaturized diagnostic instruments. This paper presents a novel, highly efficient and simple method for low-molecular weight RNA extraction using electricity only. Cells are lysed by thermo-electric lysis and RNA is purified using a gel-electrophoretic purification step. The combination of the two steps in one integrated cartridge reduces the time frame between the two steps, thus protecting RNA from enzymatic degradation. A disposable chip solution is proposed using a novel dry film resist laminate technology that allows cheap, large-scale fabrication. The chip contains crucial microfluidic innovations that allow for a simple user interface, reproducible functioning and precise quantification. Phaseguides are invented that allow controlled spatial injection of gel, injection of sample and recovery of extracted RNA. A precise sample volume can be defined by integrating electrophoretic actuation electrodes in the microfluidic chamber. Electrolytic gas bubbles that are the result of constant-current actuation are driven out from the chip by the novel introduction of capillary bubble-expulsion techniques. The extraction approach and the functionality of the chip are demonstrated for Escherichia coli and Streptococcus thermophilus bacteria. Linear extraction behavior is obtained for transfer-messenger RNA down to one colony-forming unit per microlitre, or five colony-forming units per chip. The latter is an increase in extraction efficiency of a factor of 1000 with respect to the commercial extraction kit Ambion Ribopure. The chip shows particularly good performance for extraction of low-molecular weight RNA, thereby eliminating the need for large ribosomal RNA and DNA removal. RNA can be extracted in less than 11 min, being a speed-up of more than a factor of 20 with respect to commercial extraction kits. The presented solution may find broad acceptance and application in drug discovery and clinical diagnostics.
缺乏易于自动化、小型化和高效的样本预处理概念，这是阻碍小型化诊断仪器发展的关键问题之一。本文提出了一种新颖、高效且简单的方法，仅用电即可提取低分子量 RNA。细胞通过热电裂解裂解，然后通过凝胶电泳纯化步骤纯化 RNA。这两个步骤的组合在一个集成的试剂盒中完成，从而减少了两个步骤之间的时间间隔，从而保护 RNA 免受酶的降解。本文提出了一种使用新型干膜阻焊层技术的一次性芯片解决方案，这种技术可以实现廉价的大规模制造。该芯片包含了关键的微流控创新，可以实现简单的用户界面、可重复的功能和精确的定量。发明了相位引导器，允许控制凝胶的空间注入、样品的注入和提取的 RNA 的回收。通过在微流控室中集成电泳致动电极，可以精确定义样品体积。由于恒流致动而产生的电解气体气泡通过新颖的毛细气泡排出技术从芯片中排出。该提取方法和芯片的功能已通过大肠杆菌和嗜热链球菌细菌得到验证。对于转移信使 RNA，线性提取性能可达到每微升一个集落形成单位，或每芯片五个集落形成单位。与商业提取试剂盒 Ambion Ribopure 相比，这是提取效率提高了 1000 倍。该芯片在提取低分子量 RNA 方面表现尤其出色，因此无需去除大量核糖体 RNA 和 DNA。提取过程可在不到 11 分钟内完成，与商业提取试剂盒相比，速度提高了 20 多倍。该解决方案可能会在药物发现和临床诊断中得到广泛接受和应用。
