Sensitive colorimetric immunoassay of Vibrio parahaemolyticus based on specific nonapeptide probe screening from a phage display library conjugated with MnO2 nanosheets with peroxidase-like activity

Abstract

Pathogen detection continues to receive significant attention due to the harmful effects of pathogens on public health. Herein, specific nonapeptide-fusion proteins pVIII (pVIII fusion) were isolated from phage VQTVQIGSD (designated by the sequence of a fused foreign peptide), which was specifically screened from the f8/9 landscape phage library against Vibrio parahaemolyticus (V. parahaemolyticus) in a high-throughput way. The as-prepared V. parahaemolyticus-specific recognition element is cheaper and more available than antibodies. Further, a highly sensitive colorimetric immunoassay for V. parahaemolyticus was established using pVIII fusion as capture probes coupled with protein-templated MnO₂ nanosheets (NSs) as signal probes. In the presence of a target bacterium, V. parahaemolyticus, a sandwich-type complex of pVIII fusion–V. parahaemolyticus–MnO₂ NS@pVIII fusion was formed through specific recognition of pVIII fusion and V. parahaemolyticus. The signal probes (MnO₂ NSs) could catalyze the reaction of 3,3′,5,5′-tetramethylbenzidine and H₂O₂ to generate a colorimetric change. The proposed V. parahaemolyticus detection method demonstrated a wide detection range (20–10⁴ colony-forming units (CFU) mL⁻¹), low limit of detection (15 CFU mL⁻¹), excellent selectivity, and high reliability for real marine samples, showing potential application in marine microbiological detection and control.