Electrochemical Biosensors Combined with Isothermal Amplification for Quantitative Detection of Nucleic Acids

Tabata, Miyuki; Yao, Bo; Seichi, Ayaka; Suzuki, Koji; Miyahara, Yuji

In recent years, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). The integration of isothermal amplification with electrical or electrochemical devices has enabled high-throughput nucleic acid-based assays with high sensitivity. We performed solid-phase rolling circle amplification (RCA) on the surface of a Au electrode, and detected RCA products in situ using chronocoulometry (CC) with [Ru (NH(3))(6)](3+) as the signaling molecule. Detection sensitivity for DNA and a microRNA (miR-143) was 100 fM and 1 pM, respectively. Furthermore, we conducted potentiometric DNA detection using an ethidium ion (Et(+))-selective electrode (Et(+)ISE) for real-time monitoring of isothermal DNA amplification by primer-generation RCA (PG-RCA). The Et(+)ISE potential enabled real-time monitoring of the PG-RCA reaction in the range of 10 nM–1 μM of initial target DNA. Devices based on these electrochemical techniques represent a new strategy for replacing conventional PCR for on-site detection of nucleic acids of viruses or microorganisms.