Abstract

Staphylococcus aureus (S. aureus), one of the most prevalent foodborne pathogens, poses a persistent global threat to public health by causing severe infections and food poisoning. We present β-cyclodextrin/surfactant inclusion-mediated nucleic acid enrichment strategy synergized with a ratiometric electrochemical biosensor for the robust determination of S. aureus. Unlike commercial kits limited by dilution effects, our method utilizes cetyltrimethylammonium bromide (CTAB) to induce the condensation of nucleic acids from large-volume lysates, followed by a competitive inclusion reaction where β-cyclodextrin encapsulates the hydrophobic alkyl chains of CTAB. This mechanism triggers the efficient release of DNA into a micro-volume, achieving a ~ 12-fold enrichment. The enriched target is subsequently quantified using a ratiometric electrochemical interface equipped with methylene blue (MB) and ferrocene (Fc), where the current ratio (IMB/IFc) serves as an intrinsic self-calibration parameter to eliminate the internal/external disturbances. Under optimized experimental conditions, the developed biosensor demonstrated a wide linear range spanning from 10¹ to 107 CFU/mL, with a low limit of detection of 8 CFU/mL. Moreover, the biosensor achieved reliable quantification of S. aureus in complex real samples, including peach juice and UHT skim milk, confirming its practical applicability. This work establishes a novel and costeffective electrochemical sensing strategy that couples DNA enrichment and release with ratiometric signal, providing a feasible technical approach for rapid pathogen detection.

Keywords： Staphylococcus aureus · Cetyltrimethylammonium bromide (CTAB) · DNA enrichment · β-Cyclodextrin · Ratiometric electrochemical biosensor · Square-wave voltammetry