Electrochemiluminescence Biosensor Based on a Self-protected DNAzyme Walker with a Circular Bulging DNA Shield for MicroRNA Detection

Herein, an electrochemiluminescence (ECL) biosensor is established for the ultrasensitive detection of microRNA (miRNA) by integrating a self-protected DNAzyme walker machine on a Au nanoparticle–modified electrode. Using Let-7a miRNA as the model target and by introducing a target-binding domain into the middle of the catalytic core, the catalytic core of the DNAzyme walker is separated by a target-binding domain that can inhibit the cleavage activity and serve as an arch-like protective shield, resulting in a self-protected DNAzyme walker. High-efficiency hybridization between the target Let-7a miRNA and the target-binding domain activates the DNAzyme walker machine, enabling high catalytic cleavage of its substrate without requiring additional energy input. Importantly, each step of the DNAzyme walker results in the cleavage of a substrate strand and the liberation of a Ru(bpy)₂(mcpbpy)²⁺ (Ru)-labeled DNA fragment, considerably reducing the ECL signal of Ru. Under optimized experimental conditions, the limit of detection of Let-7a miRNA is 51.4 aM within a wide linear range of 100 aM–100 pM. This proposed strategy is a bold innovation in the rapid and sensitive detection of low-abundance biomarkers, offering a promising application for early cancer diagnosis and relevant research.