Visual Countingof Influenza A Viruses with MagneticT4 Phage SPR Probe

Influenza A virus (IAV) represents a considerable threat to both animal and human health, while current detection methods encounter challenges related to the spectrum, rapidity, and sensitivity of viral identification. Herein, we describe the development of a magnetic T4 phage surface plasmon resonance probe for universal, rapid, highly sensitive, and visually detectable IAV detection under dark field microscopy (DFM). Briefly, we initially fused the Soc protein of the T4 phage with a single-chain variable fragment (scFv) antibody that exhibits broad-spectrum affinity toward the hemagglutinins of group 1 and group 2 influenza viruses, resulting in the generation of the recombinant Soc-scFv protein. Additionally, we generated another recombinant protein called AviTag-Hoc by fusing the Hoc capsid protein of T4 phage with biotin receptor peptides (AviTag). These two recombinant proteins were assembled on the head region of the T4 phage lacking both Soc and Hoc proteins. Subsequently, the resulting assembly was covalently modified with biotin using biotin-protein ligase, enabling conjugation with streptavidin-modified magnetic nanoparticles (SA@MNPs) to generate the magnetic T4 phage probe (T4@scFv@MNPs). Binding experiments demonstrated that this magnetic phage probe specifically binds to a wide range of IAVs of group 1 and group 2. Furthermore, in the presence of influenza viruses, the magnetic T4 phage probe and antibodies functionalized chip can form a sandwich complex that appears as a distinct bright golden yellow fluorescence spot visible to the naked eye under DFM. The number of viruses in samples can be automatically counted using artificial intelligence-assisted software. Assay results from both pure and real virus samples show that our magnetic phage-based DFM strategy is highly time efficient, taking approximately 30 min to complete. The method also showed excellent virus binding efficiency (>85%) in both high and low concentration samples and an extremely low detection limit (1 PFU/μL).