Abstract

Effective detection of foodborne pathogenic bacteria is a crucial demand for global food security, yet conventional approaches for detection are often constrained by their reliance on species-specific targets, limiting their capability for broad-spectrum pathogenic bacteria recognition. This study aimed to introduce a novel electrochemical biosensor designed for conserved virulence factor-based broad-spectrum pathogenic bacteria recognition. We combined a prefabricated ready-to-use NaAlg@paper scaffold with a four-channel electrode device including an integrated culture chamber. This setup allowed for rapid cell immobilization on the electrode surface and supported long-term three-dimensional cultivation. The human NLR family apoptosis inhibitory protein–NLR family CARD domain-containing protein 4 (hNAIP–NLRC4) complex was expressed in HEK293 cells to construct HEK293/hNAIP–NLRC4 bioengineered cells as the sensing element, thereby enabling the specific detection of the type III secretion system (T3SS), which is a highly conserved virulence factor. This design effectively eliminated the interference caused by the complex receptor profiles of native immune cells. Based on this strategy, the sensor exhibited a linear range of 2.8 × 10² to 2.8 × 10⁷ CFU/mL for detecting Salmonella typhimurium with a detection limit of 1.7 × 10² CFU/mL. Moreover, this method successfully distinguished pathogenic from nonpathogenic Escherichia coli and specifically detected a broad spectrum of pathogenic bacteria carrying T3SS. These findings demonstrate the potential of conserved virulence factor-oriented biosensing for broad-spectrum recognition of pathogenic bacteria and offer a reliable technical solution to enhance food safety.