Optimization of Dual-Bacteriophage Magnetic Nanoprobe for the Detection of Staphylococcus aureus and Pseudomonas...

Due to the high host specificity of bacteriophages, a single-phage superparamagnetic nanoprobe can only detect one bacterial species at a time. In this study, we developed a series of dual-phage magnetic nanoprobes and performed a series of evaluations to identify the optimal formulation. Phages specific to Staphylococcus aureus and Pseudomonas aeruginosa were chemically conjugated to superparamagnetic iron oxide nanoparticles. The lytic activity of each phage was assessed individually and in combination. Six single-phage nanoprobes and nine dual-phage nanoprobes with different phage loading levels were prepared. The lowest detectable bacterial concentration and the number of bacteria captured by each probe were subsequently determined. We developed a dual-phage superparamagnetic nanoprobe with high sensitivity and a detection limit of 10¹ CFU/mL for either bacterium. The optimal probe contained 10⁷ PFU/mL of each phage. Increasing the phage loading beyond this level reduced bacterial capture efficiency. Additionally, the number of P-phages that could be conjugated to nanoparticles decreased when a high concentration of S-phages was present. No cross-infection between phages and non-target bacteria was observed, minimizing the risk of false-positive detection.