A rapid one-step electrochemical method based on cleat-equipped molecular walking machine

Various nucleic acid molecular machines have emerged in recent years. However, when the nucleic acid tracks are fully depleted, these walkers are highly susceptible to premature release or stalling in regions where the tracks are locally exhausted. In this work, a molecular walking machine with a cleat domain preventing dissociation from the track was explored for ultrasensitive detection of miRNA. It has been verified that the cleat design can enhance the signal amplification efficiency of molecular walking machines for electrochemical miRNA-141 detection. Notably, the single-step electrochemical biosensing platform utilizing the cleat-equipped molecular walking machine (CMWM) is exceptionally straightforward and rapid, concluding the reaction within 90 min and achieving a remarkable low detection limit of 0.26 fM. The proposed molecular walking machine with this specific cleat structure was utilized for the identification of miRNA-141 in cellular lysates, exhibiting remarkable selectivity and consistent reproducibility, showcasing its effective utility in bioanalysis. Therefore, the cleat walker developed in this study introduces an innovative method for constructing a miRNA electrochemical biosensing platform, offering new perspectives for its application in biomolecule detection and clinical disease diagnosis.