Rapid and direct detection of m⁶A methylation by DNAzyme-based and smartphone-assisted electrochemical biosensor

m⁶A methylation detection is crucial for understanding RNA functions, revealing disease mechanisms, guiding drug development and advancing epigenetics research. Nevertheless, high-throughput sequencing and liquid chromatography-based traditional methods still face challenges to rapid and direct detection of m⁶A methylation. Here we report a DNAzyme-based and smartphone-assisted electrochemical biosensor for rapid detection of m⁶A. We initially identified m⁶A methylation-sensitive DNAzyme mutants through site mutation screening. These mutants were then combined with tetrahedral DNA to modify the electrodes, creating a 3D sensing interface. The detection of m⁶A was accomplished by using DNAzyme to capture and cleave the m⁶A sequence. The electrochemical biosensor detected the m⁶A sequence at nanomolar concentrations with a low detection limit of 0.69 nM and a wide detection range from 10 to 10⁴ nM within 60 min. As a proof of concept, the 3'-UTR sequence of rice was selected as the m⁶A analyte. Combined with a smartphone, our biosensor shows good specificity, sensitivity, and easy-to-perform features, which indicates great prospects in the field of RNA modification detection and epigenetic analysis.