DNAzyme walker-driven, electrode-surface label/wash-free ultrasensitive ECL miRNA detection <i>via</i> ssDNA-enhanced peroxidase activity of g-C ₃ N ₄ nanosheets

The value of microRNAs (miRNAs) as clinical biomarkers lies in their differential expression in various diseases, enabling diagnosis and dynamic treatment response assessment. In this study, we developed a label/wash-free, ultrasensitive electrochemiluminescence (ECL) biosensor for miRNA detection based on a Mn²⁺-dependent DNAzyme walker and ssDNA-enhanced peroxidase-like activity of graphitic carbon nitride nanosheets (g-C₃N₄ NSs). Therein, the DNAzyme walker performed autonomous movement on Au@Fe₃O₄ nanocomposites, enabling continuous cleavage of substrate strands and generation of numerous single-stranded DNA (ssDNA) products in the presence of the target miRNA. These ssDNA products enhanced the peroxidase-like activity of g-C₃N₄ NSs <i>via</i> electrostatic attraction and π-π stacking, significantly amplifying the ECL signal of the luminol-H₂O₂ system. This strategy achieved a detection limit of 1 fM for miRNA-155, with excellent selectivity and reproducibility. Notably, the method eliminates the need for time-consuming chemical electrode modification and washing steps through label/wash-free ECL signal generation, facilitating relatively rapid and highly convenient operation. The biosensor was successfully applied to detect miRNA-155 in complex biological samples such as cell lysates, demonstrating its great potential for clinical molecular diagnosis.