A Spatial Confinement Effect-Based “Swing Probe” In Situ Entropy-Driven Assembly for the Sensitive Ratiometric Electrochemical Detection of Exosomal miRNA

Electrochemical biosensors based on DNA assembly signal amplification have emerged as powerful tools for exosomal miRNA detection, demonstrating broad application potential in accurate early cancer diagnosis. However, due to the passivation of the electrode by DNA nanostructures and inherent limitations in assembly reaction efficiency, electrochemical biosensors still face challenges in the sensitive and rapid detection of exosomal miRNA. Herein, we propose a novel spatial confinement effect based "swing probe" in situ entropy-driven assembly strategy. By constraining two auxiliary probes in a specific space with a T-type structure, the target miRNA can induce rapid in situ swinging of the probe at the electrode interface, facilitating the assembly of the auxiliary probes and the dissociation of labeled methylene blue (MB) signal probes. Therefore, the rapid and sensitive detection of miRNA can be achieved by analyzing the signal ratio between MB and ferrocene (Fc). The [Fe(CN)₆]^3--mediated electrocatalytic signal amplification reaction was further employed to realize significant signal response by utilizing the DNA nanostructures' passivation effect on the electrode. The developed biosensor exhibited an ultralow LOD of 4.08 fM and successfully distinguished hepatocellular carcinoma (HCC) patients from healthy donors. Overall, this study provides a novel approach for exosomal miRNA detection and holds promise for advancing miRNA-based liquid biopsy in early cancer diagnosis.