Avi-tag mediated Phage@gold nanoprobe illuminates bacteria

• A hybrid particle probe of gold nanoparticles (GNPs) assembled on a phage capsid protein was constructed. • Phage@GNPs probe demonstrates excellent monodispersity and maintains a strong affinity for the host bacteria. • The Phage@GNPs probe allows for the visualization of host bacteria at a significantly low concentration using dark-field microscopy. The phage demonstrates specific recognition towards its host bacteria, while the gold nanoparticle (GNP) exhibits a strong surface plasmon resonance (SPR) effect and emits visible fluorescence with dark-field microscope (DFM). Therefore, phage modified with multiple GNPs (phage@GNPs) targeting bacteria enables the visualization of targets under DFM. However, a universal strategy for constructing a stable phage nanoprobe suitable for clinical detection remains a challenge. Herein, we developed a GNP-functionalized phage (phage@GNPs) by displaying an AVI-tag at the N-terminal region of the major capsid protein in S55 (a Salmonella phage). The S55 displaying AVI-tag exhibits equivalent infectivity towards various serotypes of Salmonella as the native S55 and can be further biotinylated through a reaction between lysine residues on the AVI-tag and COOH groups on D-biotin mediated. Consequently, biotinylated S55 was mixed with streptavidin functionalized GNPs to generate the S55@GNPs due to the strong interaction between biotin and streptavidin. Analysis results showed that the obtained S55@GNPs maintained native affinity to host bacteria and better monodispersity and stability than the other four modification and assembly methods for constructing phage@GNPs. Furthermore, the results of simulated and clinical Salmonella samples demonstrated that AVI-tag mediated S55@GNPs probes possess a qPCR-like sensitivity with a detection limit of 1 CFU/μL. Notably, the remarkable AVI-tag mediated phage@GNPs DFM strategy covers an extensive detection range spanning from 1 to 50,000 CFU/μL. Additionally, we employed the AVI-tag mediated assemble of phage@GNPs for phages of Vibrio and Klebsiella . The results showed similar data as S55 for illuminating host cells, suggesting that AVI-tag mediated phage@GNPs assembly is a universal strategy applicable to phage of any other bacteria for illuminating their respective host bacterial under DFM.