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

Abstract

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 <i>Staphylococcus aureus</i>. The Co₃O₄ MNE was conjugated to <i>S. aureus</i>-specific fusion-pVIII (Co₃O₄ MNE@fusion-pVIII), screened from the <i>S. aureus</i>-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 <i>S. aureus</i> in sterile milk, which were then isolated from milk magnetically. Assisted by polyethylene glycol, the Co₃O₄ MNE@fusion-pVIII@<i>S. aureus</i> 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@<i>S. aureus</i> 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 <i>S. aureus</i> 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.