DNA logic-gated electrochemiluminescence biosensor for sensitive detection of dual-miRNA in bladder cancer

Early diagnosis of bladder cancer requires highly sensitive and specific biosensors for detecting trace biomarkers in complex biological samples. However, single- biomarker detection is often prone to false-positive results due to biological variability and nonspecific fluctuations. Herein, we present a dual-target electrochemiluminescence (ECL) biosensor that integrates a DNA “AND” logic gate with toehold-mediated strand displacement (TMSD) circuits for parallel detection of miR-183–5p and miR-96–5p in human urine. A high-surface-area UIO-66@AuNPs composite served as the electrode substrate, increasing the loading of DNA logic gates and thus the Ru(bpy) 3 2 + - labeled signal probes, which significantly enhanced ECL response. The constructed DNA “AND” logic gate, consisting of a Ru(bpy) 3 2+ -labeled signal strand, a ferrocene-quenched strand, and a hairpin probe, restored the ECL signal only when both target miRNAs triggered parallel TMSD reactions, thereby achieving “AND” logic operation. This dual-target strategy effectively suppresses false-positive signals arising from single-biomarker fluctuations, thereby improving diagnostic accuracy compared with conventional single-target assays. Benefiting from TMSD-mediated circuits, the biosensor achieved detection limits as low as 0.45 fM for miR-183–5p and 0.85 fM for miR-96–5p over a broad linear range of 1 fM to100 pM. Urine analysis revealed significantly elevated levels of these miRNAs in bladder cancer patients. Furthermore, the quantified miRNA concentrations were incorporated into a machine learning-based classification model, enabling accurate discrimination between healthy individuals and bladder cancer patients. This integration of logic-gated biosensing with data-driven analysis not only improves diagnostic reliability but also demonstrates a promising strategy for intelligent biosensing in biological analysis and clinical auxiliary diagnosis.