Toehold Mediated One-Step Conformation-Switchable “Signal-On” Electrochemical DNA Sensing Enhanced with Homogeneous Enzymatic Amplification

The development of highly sensitive and sequence-specific electrochemical DNA (E-DNA) sensors, featuring flexible, one-step, and “signal-on” design, is a long-lasting goal. Here, we present a single-step, toehold-triggered structure-switchable signaling design that is “signal-on” and compatible with homogeneous enzyme-assisted target recycling (EATR). In this design, a partially hybridized duplex is bifunctional, which consists of a signal probe having foldable hairpin sequence and a target recognition probe with exposed toehold domain. In the presence of both target and exonuclease, the toehold sequence rapidly fuels the strand displacement reaction, liberating the surface-confined toehold–target duplex into homogeneous solution for target recycling and meanwhile leaving the dehybridized signal probe to form a stem-loop structure for signaling. Through such an 1:N enzymatic catalysis, more and more unfolded probes self-hybridize to their original folded configuration, giving a remarkable signal gain. Thi...