An Ago-mediated duplex biosensing platform with orthogonal electrochemical coding for microRNA detection based on TDN interfaces and UiO-66-NH2 nanocarriers.

Circulating microRNAs (miRNAs) are colorectal cancer (CRC) biomarkers, but their short length, high sequence homology, and low abundance make sensitive and multiplexed detection difficult. Electrochemical biosensing offers a route for miRNA analysis, yet duplex detection remains limited by the need for reporter pairs with well-separated signals and minimal crosstalk. Here, we report an Ago-mediated Duplex Orthogonal Readout for microRNA Electrochemical detection (ADORE) for simultaneous detection of CRC-related miRNAs, miR-21 and miR-92a. ADORE uses methylene blue and L-012 as an orthogonal electrochemical code pair, validating L-012, a luminol derivative used in chemiluminescence and electrochemiluminescence, as an electrochemical reporter. The sensing interface integrates tetrahedral DNA nanostructures with reporter-loaded UiO-66-NH2 carriers to stabilize probe organization and amplify electrochemical responses. Target miRNAs first initiate an exponential amplification reaction (EXPAR) to generate guide DNAs. These guides then program Clostridium butyricum Argonaute (CbAgo) for sequence-specific cleavage at the interface, producing target-dependent signal-off responses. ADORE achieves limits of detection of 1.85 aM for miR-21 and 0.51 aM for miR-92a. To improve analysis of serum samples, multidimensional differential pulse voltammetry-derived features were integrated into a support vector machine model, increasing diagnostic performance from an area under the receiver operating characteristic curve (AUC) of 0.88 using single-feature analysis to 0.94 using multifeature classification in serum discrimination. This work provides an integrated strategy combining Ago-mediated signal transduction, orthogonal electrochemical coding, nanostructured interface engineering, and machine learning for duplex serum miRNA analysis and CRC-related biomarker detection, supporting development of multiplexed electrochemical liquid-biopsy assays for CRC screening.