A label-free fluorescent aptasensor based on DNA-templated silver nanoclusters and catalytic hairpin assembly for highly specific detection of Escherichia coli O111:B4 lipopolysaccharide.

BACKGROUND: Escherichia coli O111:B4 is an important diarrheagenic serotype and an indicator of fecal contamination in water quality monitoring. Its specific detection remains challenging because of the high structural similarity among E. coli serotypes and limitations in conventional assays. Serotype-specific lipopolysaccharide (LPS) is a stable and discriminatory biomarker for selective detection.

RESULTS: We report an enzyme-free, label-free fluorescent aptasensor that integrates an O111:B4 LPS-specific DNA aptamer, catalytic hairpin assembly (CHA) amplification, and DNA-templated silver nanoclusters (DNA-AgNCs) reporting. Target binding releases a complementary strand to trigger the CHA reaction, leading to a programmed DNA rearrangement that brings guanine-rich motifs close to AgNCs and enhances fluorescence. Importantly, by changing only the AgNC nucleation sequence, the optical output can be reprogrammed to yield two emission channels (570 and 670 nm) under the same recognition and amplification workflow, enabling a two-channel consistency check for quantitative validation. The aptasensor exhibited a broad linear range of 2-1000 ng mL-1 with a detection limit of 1.58 ng mL-1 and demonstrated high selectivity against non-target bacteria and common environmental interferents. Spike-and-recovery tests in different samples achieved recoveries of 92-108%, indicating robustness in complex matrices.

SIGNIFICANCE: This work presents a sequence-programmable, dual-channel reporting strategy for LPS sensing, where target recognition and signal amplification are decoupled from optical output reconfiguration. The internal two-channel consistency validation and strong performance in various matrices support practical monitoring and provide a modular route toward adaptable and multiplexed biosensing platforms.