Rapid Detection of Staphylococcus aureus in Milk Samples by DNA Nanodendrimer-Based Fluorescent Biosensor

Staphylococcus aureus is the primary pathogen responsible for mastitis in dairy cows and foodborne illnesses, posing a significant threat to public health and food safety. Here, we developed an enhanced sensor based on solid-phase separation using gold-magnetic nanoparticles (Au@Fe3O4) and signal amplification via dendritic DNA nanostructures. The substrate chain was specifically immobilized using thiol–gold coordination, and a three-dimensional dendritic structure was constructed through sequential hybridization of DNAzymes, L chains, and Y chains, resulting in a 2.8-fold increase in initial fluorescence intensity. Upon specific cleavage of the substrate chain at the rA site by S. aureus DNA, the complex dissociates, resulting in fluorescence intensity decay. The fluorescence intensity is negatively correlated with the concentration of Staphylococcus aureus. After optimization, the biosensor maintains a detection limit of 1 CFU/mL within 3 min, with a linear range extended to 1–107 CFU/mL (R2 = 0.998) and recovery rates of 85.6–102.1%, significantly enhancing resistance to matrix interference. This provides an innovative solution for rapid on-site detection of foodborne pathogens.