Department of Applied Chemistry, Graduate School of Engineering, Okayama University of Science, Ridai-cho, Kita-ku, Japan.
Analyst. 2011 Dec 21;136(24):5143-50. doi: 10.1039/c1an15638a. Epub 2011 Oct 19.
In this paper, the semi-real time electrochemical monitoring method using a screen-printed electrode, which employs reverse transcription loop-mediated isothermal amplification (RT-LAMP) for influenza virus RNA, is presented. The amplified DNA combined with methylene blue (MB), which was used as an electroactive DNA intercalator, and the electrochemical signal was monitored using square wave voltammetry in the presence of RT-LAMP reagent components. MB molecules binding to amplified DNA caused the reduction of the peak current due to the slow diffusion of MB-amplified DNA complex to the electrode surface. We successfully monitored the amplification process of DNA on the basis of RT-LAMP by measuring and analyzing the electrochemical signal of MB with only one screen-printed electrode that connected with a USB powered portable potentiostat. The peak height of the current was related to the extent of amplification of DNA and the amount of input RNA. Since laborious probe immobilization is not required and both the amplification and the monitoring are possible in a single tube, our method does not suffer from potential cross-contamination. Furthermore, our method provides a new rote for the development of electrochemical hand held biosensors.
本文提出了一种基于电化学监测的半实时检测方法，该方法使用丝网印刷电极，采用逆转录环介导等温扩增（RT-LAMP）对流感病毒 RNA 进行检测。扩增后的 DNA 与亚甲蓝（MB）结合，MB 作为一种电化学活性 DNA 嵌入剂，在 RT-LAMP 试剂成分存在的情况下，通过方波伏安法监测电化学信号。由于 MB-扩增 DNA 复合物向电极表面的缓慢扩散，MB 分子与扩增 DNA 的结合导致峰电流降低。我们成功地通过测量和分析仅用一个与 USB 供电便携式电化学工作站相连的丝网印刷电极的 MB 的电化学信号，在基于 RT-LAMP 的基础上监测了 DNA 的扩增过程。电流的峰值高度与 DNA 的扩增程度和输入 RNA 的量有关。由于不需要繁琐的探针固定，并且扩增和监测都可以在单个管中进行，因此我们的方法不会发生潜在的交叉污染。此外，我们的方法为电化学手持式生物传感器的发展提供了一种新的途径。
