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 num...