Programmable dual-fluorescence DNA aptasensor using cascade amplification and sequence-engineered AgNCs for ultrasensitive detection of Clostridioides difficile RNase H2

Abstract

Rapid and accurate detection of Clostridioides difficile remains challenging due to the limited sensitivity, complex protocols, and costly instrumentation required by current diagnostic assays. Here, we present a label-free, enzyme-free dual-fluorescence aptasensor that integrates entropy-driven catalysis (EDC) and catalytic hairpin assembly (CHA) with DNA-templated silver nanoclusters (DNA-AgNCs) for the sensitive and reliable detection of RNase H2, a highly specific biomarker of C. difficile. Aptamers immobilized on magnetic beads selectively bind RNase H2 and release complementary primers that trigger a cascaded EDC-CHA amplification network. The amplified primers modulate the conformation of AgNC-templating hairpins, enabling sequence-directed tuning of AgNC emission. By tuning the nucleation sequence, Two AgNC emitters respond inversely: red fluorescence is enhanced while yellow emission is simultaneously quenched. This ratiometric dual-signal mechanism provides intrinsic self-correction against environmental fluctuations and significantly improves quantitative accuracy. Under optimized conditions, the sensor exhibits a broad linear detection range (0.01–100 ng/mL) and an ultralow detection limit of 9.27 pg/mL, along with high specificity and accurate recovery in river water samples. This sequence-programmable DNA–AgNC platform establishes a robust, cost-effective strategy for rapid pathogen diagnostics.