Exploring bi-recycled strand displacement to cascade mismatch-mediated hairpin assembly for amplified electrochemical biosensing

Exploring a responsive cascaded amplifier of bi-recycled strand displacement ( b SD) and mismatch-mediated hairpin assembly ( m HA) might be intriguing for constructing sensitive electrochemical biosensor. The proof-of-concept example for operating b SD started with the design of a recognizable hairpin encoding three functional modules, including specific complement, contiguous Nt.BbvCI site, and locked primer-matchable fragment. Upon introducing a targeted short-stranded oligonucleotide segment, b SD events were activated to execute repetitive strand elongation and nicking-mediated displacement reaction, generating a target analogue to implement similar affinity recognition for cooperative autocatalysis. This progress efficiently guided the production of numerous analogues for unfolding a metastable double-stem hairpin structure, and rationally induced progressive m HA via cross-opening and sequential hybridization between two functional hairpins, in one of which the inclusion of a mismatched base was beneficial for driving b SD- m HA cascade faster forward. As such, the labeled electroactive ferrocene (Fc) tags were orderly anchored in the resulting long linear product, thereby achieving significant amplification of current signal and highly sensitive detection down to femtomole. Compared with conventional hairpin assembly, our b SD- m HA-based logic operation substantially improved the transduction reaction kinetics, showing about 4.6-fold increment of reaction rate, while higher amplification yield emerged at lower trigger was more superior than b SD or m HA alone. Thus, this cascade-amplifying strategy would suggest a new paradigm to create robust and cost-effective electrochemical biosensors for applicable biosensing and bioassay.