Abstract: A highly sensitive and regenerable electrochemical biosensor was developed for detecting microRNA-133a-5p (miRNA-133a-5p) by integrating multi-legged DNA walker with host-guest recognition. The sensor featured a regenerable interface constructed by modifying the electrode surface with reduced graphene oxide-gold nanoparticle composites (rGO@AuNPs) and immobilizing abundant β-cyclodextrin (β-CD). Upon introduction of miRNA-133a-5p, the target triggered the self-assembly of three hairpin DNA probes into a three-legged DNA walker. Crucially, miRNA-133a-5p was displaced during this process, enabling its cyclic reuse and subsequent amplification of walker generation. The resulting walkers efficiently cleaved signal probes, yielding numerous ferrocene (Fc)-labeled single-stranded DNA fragments. These fragments were captured by β-CD on the electrode surface via host-guest interactions, generating measurable current signals for ultrasensitive miRNA-133a-5p detection. Benefiting from target recycling amplification and the high cleavage efficiency of the three-legged DNA walker, the sensor achieved a remarkably low detection limit of 19.7 fmol/L. Furthermore, electrochemical regulation of the host-guest interaction between Fc and β-CD facilitated six regeneration cycles. This work establishes a novel platform for myocardial infarction diagnosis and proposes an effective strategy for designing regenerable electrochemical biosensors.

Key words: Electrochemical biosensors; Acute myocardial infarction, DNA walker, miRNA-133a-5p, Regenerable