Design of M13 phage systems dual-displaying two functional proteins for developing a rapid biosensing probe

M13 phage dual-display systems, which co-present targeting and reporter elements, hold great promise for developing rapid biosensors. However, the efficiency and performance of different display-site combinations remain poorly characterized. This study systematically evaluates the dual-display capacity of M13 phage by co-displaying a Vibrio parahaemolyticus-specific nanobody (Nb20) on pIII protein and NanoLuc luciferase (Nluc) on either pVI, pVII, pVIII, or pIX capsid proteins under both monovalent and multivalent strategies. We found that display efficiency varied significantly with the coat protein site and infection time of helper phage. The pVIII-based monovalent display achieved the highest specific luminescent activity and a functional co-display proportion of up to 2.20%. Furthermore, the optimized mpVIII probe demonstrated high specificity, sensitivity (detection limit of 10² cfu/mL), and robust anti-interference capability in detecting V. parahaemolyticus in spiked shrimp samples. This work provides fundamental insights and a practical framework for constructing high-performance dual-display phage probes with one-step biosensing.