Folding-Upon-Binding and Signal-On Electrochemical DNA Sensor with High Affinity and Specificity

[Image: see text] Here we investigate a novel signal-on electrochemical DNA sensor based on the use of a clamp-like DNA probe that binds a complementary target sequence through two distinct and sequential events, which lead to the formation of a triplex DNA structure. We demonstrate that this target-binding mechanism can improve both the affinity and specificity of recognition as opposed to classic probes solely based on Watson–Crick recognition. By using electrochemical signaling to report the conformational change, we demonstrate a signal-on E-DNA sensor with up to 400% signal gain upon target binding. Moreover, we were able to detect with nanomolar affinity a perfectly matched target as short as 10 bases (K(D) = 0.39 nM). Finally, thanks to the molecular “double-check” provided by the concomitant Watson–Crick and Hoogsteen base pairings involved in target recognition, our sensor provides excellent discrimination efficiency toward a single-base mismatched target.