Electrochemical Impedimetric Real-Time Polymerase Chain Reactions Using Anomalous Charge Transfer Enhancement

We developed a new electrochemical impedimetric method for the real-time detection of polymerase chain reactions (PCR) based on our recent discovery that the DNA intercalator, [Ru(bpy)₂DPPZ]²⁺, anomalously enhances charge transfer between redox mediators, K₄[Fe(CN)₆]/K₃[Fe(CN)₆], and a carbon electrode. Three mM [Fe(CN)₆]^3-/4- and 5 μM [Ru(bpy)₂DPPZ]²⁺ were added to the PCR solution, and electrochemical impedance spectroscopy (EIS) measurements were performed at each elongation heat cycle. The charge transfer resistance (Rct) was initially low due to the presence of [Ru(bpy)₂DPPZ]²⁺ in the solution. As PCR progressed, amplicon dsDNA was produced exponentially, and intercalated [Ru(bpy)₂DPPZ]²⁺ ions, which could be detected as a steep Rct, increased at specific heat cycles depending on the amount of template DNA. The Rct increase per heat cycle, ΔRct, showed a peak at the same heat cycle as optical detection, proving that PCR can be accurately monitored in real time by impedance measurement. This simple method will enable a cost-effective and portable PCR device.