Amplification-free CRISPR/Cas13a-based electrochemical biosensor for the detection of circulating tumor RNA using RNA-3Ag⁺ probes on Au porous-lattice nanoelectrode.

Circulating tumor RNA (ctRNA) is a sensitive biomarker for early cancer diagnosis, offering real-time gene expression profiles and tumor-specific signatures missed by circulating tumor DNA. Despite its potential, a simple, rapid, and sensitive ctRNA detection sensor has not yet been developed. For the first time, we present an amplification-free electrochemical biosensor for ctRNA detection by integrating the CRISPR/Cas13a system with a silver ion (Ag⁺)-mediated RNA probe and an Au porous-lattice nanoelectrode (APLNE). A three-cytosine-cytosine (C–C) mismatched RNA duplex was employed as a signal probe, allowing site-specific Ag⁺ intercalation to form stable C–Ag⁺–C coordination complexes (RNA-3Ag⁺) that generate strong redox peaks. To further enhance performance, the APLNE, featuring a highly aligned porous gold nanostructure, was used to increase the effective surface area, improving probe immobilization and electrochemical signals. Upon target recognition, CRISPR/Cas13a cleaved the RNA-3Ag⁺ probe, resulting in a significant signal reduction. This biosensor detects KRAS G12D ctRNA, a key pancreatic cancer mutation, with ultrahigh sensitivity (LOD = 0.5 fM) in just 20 min and demonstrates excellent specificity in complex biological samples. Operating without nucleic acid amplification or toxic redox reagents, this simple, cost-effective, and eco-friendly platform shows strong potential for liquid biopsy-based diagnostics and point-of-care cancer screening.