Multiplex Real-Time Loop-Mediated Isothermal Amplification Using an Electrochemical DNA Chip Consisting of a Single Liquid-Flow Channel.

We developed a multiplex system capable of simultaneously quantifying different target sequences by applying an electrochemical DNA chip that consists of single liquid-flow channel with primers designed for loop-mediated isothermal amplification (LAMP). We applied this system for detecting mature microRNAs (miRNAs). miRNAs extracted from serum were enzymatically lengthened to about 100 base pairs by reverse-transcription and elongation reactions. The LAMP primers for amplifying the lengthened miRNAs were adsorbed and immobilized on the surface of the liquid-flow channel at five different positions. A LAMP solution containing the lengthened miRNAs, Tin DNA polymerase, and ruthenium hexaamine (RuHex) as a redox compound was injected into the DNA chip. The electrochemical reaction of RuHex in the LAMP solution was then measured continuously via linear-sweep voltammetry at 65 °C. The LAMP reaction of the positive control revealed that the cathodic peak current of RuHex increased. Additionally, the initial number of miRNA copies was correlated with the time when the cathodic current began to increase. Five miRNAs were simultaneously detected at 103-106 copies per 50 μL within 2 h. We expect these results will be useful for developing a simple and stable electrochemical-based method for the real-time monitoring of miRNAs, while also facilitating the implementation of electrochemical DNA chips for molecular analyses.