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

Abstract

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) 2 DPPZ] 2+ , anomalously enhances charge transfer between redox mediators, K 4 [Fe(CN) 6 ]/K 3 [Fe(CN) 6 ], and a carbon electrode. Three mM [Fe(CN) 6 ] 3−/4− and 5 μM [Ru-(bpy) 2 DPPZ] 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) 2 DPPZ] 2+ in the solution. As PCR progressed, amplicon dsDNA was produced exponentially, and intercalated [Ru-(bpy) 2 DPPZ] 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.

On-Demand Ligand-Base DNA Sensor with Electrochemical Impedance Spectroscopy

“…Therefore, to evaluate the charge transfer change quantitatively, we introduced a new parameter, Rct increase ratio (RI), which expressed how many times the Rct had increased relative to the initial Rct (eq ). This RI was highly reproducible, and its usefulness as a sensor output was shown in previous our papers …”

“…This RI was highly reproducible, and its usefulness as a sensor output was shown in previous our papers. 36 = RI (Rct increase ratio) Rct /Rct (after reactions) (initial)…”