Leiden Academic Centre for Drug Research (LACDR), Division of Analytical Biosciences, Leiden University, Einsteinweg 55, 2333CC, Leiden, The Netherlands.
Lab Chip. 2013 Aug 7;13(15):2931-6. doi: 10.1039/c3lc50332a.
A microfluidic chip is presented for lysis and one-step RNA purification from bacteria. Bacteria are lysed by joule-heating followed by a gel electrophoresis step for clean-up and subsequent elution of small RNA. Bubble formation during electrophoresis at constant current is suppressed through the use of a silver chloride cathode and a silver anode. To prevent silver chloride sediment in the bulk solution, the anode was immersed in a saturated chloride solution. Salt bridges in the form of polyacrylamide gels are used that could be precisely patterned with the help of phaseguides. Bubble-free actuation could be performed for more than 20 min under a constant current. For longer actuation times, cathodic silver-chloride became depleted and a silver-chloride sediment formed in the anodic microchamber at increasing distance from the anode with time. The chip functioning was verified by extraction of transfer-messenger RNA from Escherichia coli and subsequent amplification using reverse transcription real-time PCR. Incorporation of salt bridges enables effective bubble free actuation of Ag/AgCl electrodes in a microfluidic chip. This opens up new possibilities in a surge towards fully integrated diagnostic cartridges that are miniaturized and disposable.
本文提出了一种用于从细菌中裂解和一步提取 RNA 的微流控芯片。细菌通过焦耳加热裂解，然后进行凝胶电泳步骤进行清洗，随后从小型 RNA 中洗脱。通过使用氯化银阴极和银阳极抑制恒流电泳过程中的气泡形成。为了防止在大量溶液中形成氯化银沉淀，将阳极浸入饱和氯化物溶液中。以聚丙烯酰胺凝胶的形式使用盐桥，借助相移法可以精确地对其进行图案化。在恒流下，无气泡的驱动可以持续 20 多分钟。对于更长的驱动时间，阴极的氯化银会耗尽，并且随着时间的推移，在阳极附近的距离增加，阳极微腔中会形成氯化银沉淀。通过从大肠杆菌中提取转移信使 RNA 并使用逆转录实时 PCR 进行后续扩增，验证了芯片的功能。盐桥的引入使得在微流控芯片中可以有效地进行无气泡的 Ag/AgCl 电极驱动。这为朝着完全集成的诊断盒方向迈进开辟了新的可能性，这些诊断盒具有微型化和一次性使用的特点。
