A Porous Chitosan Film-Based Dual-Modal Biosensor Integrated with Catalytic Hairpin Assembly for Sensitive Detection of Escherichia coli

Abstract

Catalytic hairpin assembly (CHA), as an enzyme-free isothermal nucleic acid amplification technique, offers significant advantages in biosensing owing to its exponential signal gain. Integrating CHA with dual-modal signal output holds promise for further improving the sensitivity and reliability of pathogen detection in complex matrices through cross-validation of multi-dimensional information. However, when two signals are generated within the same reaction system, they are prone to crosstalk and competition, while the interfacial reaction is inherently constrained by steric hindrance and limited mass transfer. To address these issues, we report herein a CHA-based dual-modal detection strategy for Escherichia coli nucleic acid. In this design, the electrochemiluminescence (ECL) signal is localized at the electrode interface, whereas the fluorescence signal is released in the bulk solution, thereby circumventing energy competition and channel crosstalk between the two signals. Concurrently, a three-dimensional scaffold is constructed using the porous structure of phase-transformed chitosan, which provides a high specific surface area to enhance the interfacial loading of hairpin probes and offers unobstructed channels for efficient mass transfer of co-reactants and target molecules, thus alleviating steric hindrance and diffusion limitations. Upon recognition of the target E. coli nucleic acid, the CHA cascade is triggered, driving a programmed conformational transition of the hairpin probes and simultaneously modulating the fluorescence resonance energy transfer (FRET) between FAM-BHQ pairs as well as the electrochemiluminescence resonance energy transfer (ECL-RET) between CdS quantum dots and AuNPs, enabling synchronous output and mutual validation of the dual-modal signals. Under optimized conditions, the proposed strategy exhibits satisfactory sensitivity, stability, and selectivity. This work provides a feasible approach for the deep integration of CHA amplification with dual-modal sensing, offering a reliable tool for rapid and accurate screening of pathogenic microorganisms.