Colorimetric Assay of Bacterial Pathogen Based on Co3O4 Magnetic Nanozyme Conjugated with Specific Fusion Phage Protein 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 bovine serum albumin templated Co3O4 magnetic nanozyme (Co3O4 MNE) with a novel specific fusion phage protein and magnetophoretic chromatography to detect Staphylococcus aureus (S. 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 external magnetic field. After tansferring the isolated Co3O4 MNE@fusion-pVIII@S. aureus complexes into a 96-well plate, 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) and H2O2 were added to develop color due to the peroxidase mimetics activity of Co3O4 MNE. The S. aureus concentration within 10-10000 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 diseases outbreaks induced by pathogens in developing countries.