Tetrahedral DNA nanostructure–gated solution-phase CRISPR/Cas12a system for probe immobilization-free, reagent-efficient, and dual-phase electrochemical detection of circulating microRNAs in liquid biopsy

Abstract

Liquid-biopsy nucleic acid biomarkers hold strong promise for non-invasive cancer diagnostics, yet their ultralow abundance presents substantial analytical challenges. Conventional electrochemical biosensors rely on complex probe immobilization, whereas emerging homogeneous strategies remain susceptible to matrix interference and require large reaction volumes. Here, we report a probe immobilization-free, reagent-efficient, dual-phase electrochemical platform that integrates solution-phase CRISPR/Cas12a reactions with a tetrahedral DNA nanostructure (TDN)–based interfacial gating mechanism for detecting circulating microRNAs. We found that TDNs extended with a thiolated blocking fragment (BF) impose a pronounced steric gating effect at the electrode–solution interface: the large, rigid TDN architecture prevents intact BF-linked structures from approaching the gold electrode, whereas smaller Cas12a-generated thiolated fragments readily assemble via Au–S interactions. This size-dependent gating transition switches the electrode from an HRP-accessible to an HRP-blocking state, producing a sharply resolved signal decrease that faithfully maps solution-phase cleavage onto an electrochemical readout. Using a target-competitive binding strategy to generate the activator DNA, this interfacial gating mechanism was developed into a robust dual-phase sensing platform driven by CRISPR/Cas12a. Because target recognition and Cas12a-mediated cleavage occur entirely in solution and only the final reaction products contact the electrode, the architecture retains high homogeneous reaction efficiency while avoiding diffusion-governed limitations and eliminating probe immobilization. The 10 µL reaction volume enables substantial reagent savings and strong compatibility with complex matrices. The assay achieves a detection limit of 4.1 aM and accurately quantifies renal cancer–associated circulating miRNA-21 in peripheral blood, yielding results fully consistent with RT-qPCR.