A novel voltammetric approach for real-time electrochemical detection of targeted nucleic acid sequences using LAMP.

We have developed a novel voltammetric DNA chip for real-time electrochemical detection of targeted nucleic acid sequences using loop-mediated isothermal amplification (LAMP) and ruthenium hexaamine (RuHex) as the intercalative redox compound. A GspSSD DNA polymerase was used for LAMP owing to its tolerance of the intercalative redox compound. The electrochemical reaction of 1 mM RuHex in the LAMP solution was measured continuously by linear sweep voltammetry at 65 °C using an electrochemical DNA chip. According to the LAMP reaction of the positive sample, the cathodic peak current of RuHex increased and the cathodic peak potential of RuHex shifted to negative voltage. The initial number of copies of the targeted nucleic acid was correlated with both the time when the cathodic current began to increase and the time when the cathodic potential began to shift rapidly. 103 to 106 copies/50 μL of the targeted nucleic acid were detected quantitatively and the detection limit was 101 copies within an hour. We expect these results to lead to the realization of simple and stable electrochemical real-time monitoring of targeted nucleic acids while also facilitating the implementation of electrochemical DNA chips in molecular testing.