Engineering Multipedal DNA Walker on Paper for Sensitive Electrochemical Detection of Plant MicroRNA

Comprehensive Summary MicroRNAs (miRNAs) are vital regulators in both plants and animals. Therefore, it is highly desirable to develop portable and user‐friendly biosensors for convenient and sensitive detection of miRNAs. Herein, a novel paper‐based electrochemical biosensor was intelligently engineered for the detection of plant miRNA based on a smart DNA walking machine and λ‐exonuclease (λ‐Exo)‐assisted target recycling amplification. Using TaMIR5086 as a target plant miRNA, the presence of TaMIR5086 could initiate the target recycling process and activate the DNA walker to move along the track on the paper. Then, numerous electroactive molecules‐labeled single‐strand DNA (ssDNA) would be released and adsorbed onto the surface of screen‐printed electrode, generating a remarkably increased electrochemical signal. Benefitting from the dual amplification, the developed biosensor exhibits excellent analytical performance toward TaMIR5086 with a detection limit down to 0.37 pmol/L. Furthermore, the paper‐based biosensor could be applied for the analysis of target miRNA in complex biological samples, which found great potential in the fields of miRNA analysis and plant biology research.