A novel electrochemical biosensor for DNA detection based on exonuclease III-assisted target recycling and rolling circle amplification

Abstract

We developed a novel dual-amplification strategy for ultrasensitive electrochemical detection of DNA based on exonuclease III-assisted target recycling and rolling circle amplification (RCA). In this assay, a gold electrode was used to immobilize a molecular beacon (MB) with a 3′ overhang as recognition probe and perform the dual signal amplification procedure. In the presence of target DNA, MB hybridized with the target DNA to form a duplex region and created a 3′-blunt end for Exo III to initiate the target DNA recycling amplification process to cleave numerous MB probes. Then, the remaining MB fragments hybridized as primers with the RCA template to initiate the RCA process. Subsequently, the detection probe's modified Ag NPs hybridized with the long, amplified DNA products, resulting in the multiplication of Ag NPs on the electrode surface, which were used for subsequent electrochemical strip analysis of silver. This novel signal amplification strategy could detect target DNA down to 6.4 amol L⁻¹, with a dynamic range spanning five orders of magnitude. In addition, this method avoids the de-oxygenation procedure in standard electrochemical detection, thus presenting a promising application in clinical diagnosis.