Dark-field intelligent detection of V. parahaemolyticus using T4 bacteriophage displaying tail spike proteins and gold nanoparticles.

Vibrio parahaemolyticus, a significant seafood-borne pathogen, necessitates rapid and precise detection to guarantee food safety. Herein, we developed a visualization assay method by engineering T4 bacteriophage (T4) to display approximately 870 tail spike proteins (TSPs) derived from a lytic bacteriophage specific to target bacteria, along with about 30 gold nanoparticles (GNPs) of 5 nm at distinct capsid domains. Specifically, we fused the small outer capsid protein (Soc) of T4 with a TSP to obtain a fusion protein Soc-VP-TSP. In parallel, we fused the highly antigenic outer capsid protein (Hoc) with a biotin acceptor peptide (Avi-tag), generating the fusion protein Avi-Hoc. These two fusion proteins were co-assembled onto a T4ΔHS bacteriophage (T4ΔHS) capsid deficient in both native Hoc and Soc proteins, yielding a dual-functionalized phage (T4@TSPs@Avi) that simultaneously displays TSPs and Avi-tags. After covalent biotinylation via BirA biotin-protein ligase, the phages were conjugated to streptavidin-coated GNPs, forming a final detection probe termed T4@TSPs@GNPs. This dual-display T4 allows targeting bacteria to appear as visual golden rod-shaped structures under dark-field microscopy. To enable intelligent and efficient detection, we employed a DenseNet169 model combining saliency-guided region-of-interest extraction, multi-scale feature fusion, and Grad-CAM to accurately distinguish target bacteria from background noise. The entire detection process can be completed within 30 min, achieving a detection limit of 4 CFU/μL and an accuracy that is completely consistent with the gold standard. This integrated strategy provides a powerful and adaptable tool for intelligent pathogen detection in food safety, clinical diagnostics, and environmental monitoring.