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. This enzyme-assisted toehold E-DNA (etE-DNA) sensor achieves a satisfactory detection limit down to 42 fM, which is lower than that of the routine switchable E-DNA sensor by several orders of magnitude. In addition, the strategy shows high selectivity against a single-base mismatch and is capable of probing low abundant target DNA directly in human serum with minimal interference. By synergizing the toehold-based high selectivity, EATR, and one-step conformation-switchable signaling, this functional etE-DNA sensor appears to be a promising bioassay approach for clinical diagnostics.