Colorimetric Assay of Bacterial Pathogens Based on Co3O4 Magnetic Nanozymes Conjugated with Specific Fusion Phage Proteins and Magnetophoretic Chromatography

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.