Target-triggered hybrid chain amplified fluorescence aptasensor based on label-free dye and MnO2 nanosheets system for Escherichia coli detection.

Bacterial contamination poses significant threats to public health through food safety issues, creating a critical need for rapid and sensitive bacterial detection platforms. Herein, we developed a novel label-free fluorescent detection system leveraging target-triggered hybridization chain reaction (HCR) amplification, using Escherichia coli (E. coli) as a proof of concept. We engineered a hairpin-structured capture probe integrating an E. coli-specific aptamer with an HCR trigger sequence, achieving fluorescence amplification through three synergistic mechanisms: (1) HCR-driven DNA duplex assembly; (2) enhanced SYBR Green I (SG-I, a cost-effective non-labeled dye) intercalation into HCR-generated DNA duplexes; and (3) target-selective fluorescence modulation via MnO2 nanosheets that quench background fluorescence through preferential adsorption of single-stranded DNA (ssDNA)-bound SG-I over duplex DNA-intercalated dye, exploiting the differential binding affinity of MnO2 for ssDNA versus double-stranded DNA. This strategy enabled a fluorescence sensor with high sensitivity (detection limit: 17 CFU/mL), excellent specificity, and broad dynamic range (5.0 × 101 ~ 5.0 × 107 CFU/mL), demonstrating robust performance in complex food matrices through successful E. coli detection in spiked milk and lettuce samples with recoveries of 98.81 to 104.26%, thereby underscoring the method's reliability and practical utility for on-site food safety monitoring in real-world scenarios.