Simultaneous detection of multispecies foodborne pathogens using multiple phage-functionalized nanozyme

Herein, a novel colorimetric strategy for the multiplex detection of foodborne pathogens utilizing multiple phage-functionalized nanozyme integrating phage-driven specific recognition with nanozyme-catalyzed signal amplification was proposed. Initially, oxidase-like FeCoCe nanoflower (FeCoCe NF) was prepared, which can catalyze generation of superoxide anion and singlet oxygen, triggering 3, 3′, 5, 5′-tetramethylbenzidine (TMB) chromogenic reaction. For detection of individual pathogenic bacteria, EhpYZU20, SalmpYZU54, and SapYZUH5 were individually immobilized on the FeCoCe NF, thereby forming EhpYZU20@FeCoCe NF, SalmpYZU54@FeCoCe NF, and SapYZUH5@FeCoCe NF, respectively. They demonstrate high sensitivity, achieving limits of detection of 56.34 CFU mL −1 ( Enterobacter hormaechei ), 39.14 CFU mL −1 ( Salmonella enteritidis ), and 74.36 CFU mL −1 ( Staphylococcus aureus ), along with specific recognition of target pathogens and strong anti-interference capability. For detection of multiple pathogenic bacteria, EhpYZU20, SalmpYZU54, and SapYZUH5 were immobilized on the FeCoCe NF, thereby forming phages@FeCoCe NF. It enabled simultaneous quantification of all three pathogens, achieving LODs, as low as 21.67 ∼ 31.84 CFU mL −1 . In addition, principal component analysis (PCA) and hierarchical cluster analysis (HCA) further enabled precise differentiation of E. hormaechei , S. enteritidis , and S. aureus based on phages@FeCoCe NF sensor assay. This work provides a robust and versatile tool for food safety surveillance, effectively addressing the challenges posed by complex food matrices and multi-pathogen contamination.