Sortase-Mediated Dual-Display Phage Bioluminescent Immunoassay for Detection of <i>Vibrio parahemolyticus</i> Using Affinity-Enhanced Nanobody

Immunoassays are widely used for detecting foodborne pathogens such as Vibrio parahemolyticus due to their simplicity and effectiveness. However, current immunoassays for V. parahemolyticus still suffer from limitations including low specificity and insufficient sensitivity. To address these limitations, we developed a sortase-mediated dual-display phage bioluminescent immunoassay (DP-BLEIA) by integrating hotspot mutagenesis-derived affinity-enhanced nanobody (Nb) and sortase-mediated phage decoration for signal amplification. First, two specific Nbs targeting V. parahemolyticus, designated Nb-A1 and Nb-A8, were identified from the naive and synthetic Nb libraries, respectively. On this basis, two mutants of Nb-A1 (Nb-8D and Nb-8H) with enhanced affinity and antigen-binding activity were generated based on a novel hotspot mutagenesis strategy. To further enhance the detection sensitivity, the phage display system was engineered to produce phage particles that express Nb-8D on the pIII protein and fuse a short polyglycine peptide to the pVIII protein. This design enables the covalent conjugation of nanoluciferase (Nluc) to the phage particles via sortase A (SrtA). Therefore, a signal amplification probe, Nluc-modified phage-8D (Nluc@phage-8D), was prepared and utilized to develop a DP-BLEIA for V. parahemolyticus detection. Under optimized conditions, the DP-BLEIA achieved a detection limit of 5.54 × 103 CFU/mL, showing a 12.73-fold sensitivity enhancement compared to phage-mediated sandwich enzyme-linked immunoassay (P-ELISA). The proposed method demonstrated high specificity, showing no cross-reactivity with other common foodborne pathogens, and was successfully applied to spiked food samples. Overall, this study provides a robust strategy for developing affinity-enhanced recognition elements and signal amplification probes to significantly improve the sensitivity of immunoassays.