Colorimetric Assay of Bacterial Pathogens Based on Co₃O₄ Magnetic Nanozymes Conjugated with Specific Fusion Phage...

It is important to detect pathogens rapidly, sensitively, and selectively for clinical medicine, homeland security, food safety, and environmental control. We report here a specific and sensitive colorimetric assay that incorporated a bovine serum albumin-templated Co₃O₄ magnetic nanozyme (Co₃O₄ MNE) with a novel specific fusion phage protein and magnetophoretic chromatography to detect Staphylococcus aureus. The Co₃O₄ MNE was conjugated to S. aureus-specific fusion-pVIII (Co₃O₄ MNE@fusion-pVIII), screened from the S. aureus-specific phage AQTFLGEQD (the phage monoclone is denoted by the peptide sequence). The as-prepared triple-functional Co₃O₄ MNE@fusion-pVIII particles were capable of capturing S. aureus in sterile milk, which were then isolated from milk magnetically. Assisted by polyethylene glycol, the Co₃O₄ MNE@fusion-pVIII@S. aureus complex was separated from the free Co₃O₄ MNE@fusion-pVIII by magnetophoretic chromatography in an external magnetic field. After transferring the isolated Co₃O₄ MNE@fusion-pVIII@S. aureus complexes into a 96-well plate, diammonium salt of 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) and H₂O₂ were added to develop color because of the peroxidase mimetics activity of the Co₃O₄ MNE. A S. aureus concentration within 10-10,000 cfu/mL in milk can be detected (detection limit: 8 cfu/mL). The as-developed method is simple, cost-efficient, and sensitive, which is useful for rapidly diagnosing pathogenic bacteria and helpful to prevent disease outbreaks induced by pathogens in developing countries.