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

<i>Staphylococcus aureus</i> 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 <i>S. aureus</i> DNA, the complex dissociates, resulting in fluorescence intensity decay. The fluorescence intensity is negatively correlated with the concentration of <i>Staphylococcus aureus</i>. 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.