Simple and Sensitive Electrochemical DNA Detection of Primer Generation-Rolling Circle Amplification

A new electrochemical sequence-specific DNA detection platform based on primer generation-rolling circle amplification (PG-RCA), methylene blue (MB) redox indicator, and indium tin oxide (ITO) electrode is reported. In the presence of a specific target sequence, PG-RCA, an isothermal DNA amplification technique, produced large amounts of amplicons in an exponential manner. In addition to the standard components, the reaction mixture contained MB, which bound with the PG-RCA amplicons. End-point electrochemical measurement by differential pulse voltammetry (DPV) was performed using ITO electrode. The amplicon-bound MB resulted in a lower DPV signal than free MB due to a smaller diffusion coefficient as well as electrostatic repulsion between the negatively charged amplicon-bound MB and ITO electrode. With simple assay design (recognition probe) and instrumentation (operating temperature at 37°C and ITO electrode without the need for probe immobilization), this detection platform is well suited for point-of-care and on-site testing. Real-time measurement was also achieved by pretreating the ITO electrode with bovine serum albumin. KGaA, Weinheim.