DNAzyme walker-driven, electrode-surface label/wash-free ultrasensitive ECL miRNA detection via ssDNA-enhanced peroxidase activity of g-C3N4 nanosheets

Abstract

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 via 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.