Avidin-biotin complex-based capture coating platform for universal Influenza virus immobilization and characterization

Abstract

Influenza virus mutates quickly and unpredictably creating emerging pathogenic strains that are difficult to detect, diagnose, and characterize. Conventional tools to study and characterize virus, such as next generation sequencing, genome amplification (RT-PCR), and serological antibody testing, are not adequately suited to rapidly mutating pathogens like Influenza virus where the success of infection heavily depends on the phenotypic expression of surface glycoproteins. Bridging the gap between genome and pathogenic expression remains a challenge. Using sialic acid as a universal Influenza virus binding receptor, a novel virus avidin-biotin complex-based capture coating was developed and characterized that may be used to create future diagnostic and interrogation platforms for viable whole Influenza virus. First, fluorescent FITC probe studies were used to optimize coating component concentrations. Then atomic force microscopy (AFM) was used to profile the surface characteristics of the novel capture coating, acquire topographical imaging of Influenza particles immobilized by the coating, and calculate the capture efficiency of the coating (over 90%) for all four representative human Influenza virus strains tested.

Fig 1. Capture coating schematic depicting the layers of the custom avidin-biotin complex adsorbed to optically polished, c-cut sapphire slide windows.

The hemagglutinin (HA) glycoproteins on the envelope of the Influenza virus bind sialic acid of the functionalized biotin-PEG linker (structural formula shown, insert, was provided by the supplier, Nanocs, Inc.).

Fig 2. 3D AFM topography images with corresponding 1D profiles through dashed lines as shown.

A) 460 μm sapphire c-cut slide. B) Capture coating on sapphire slide. C) Influenza A H3N2 virus cluster immobilized in capture coating on sapphire slide. The tendrils of the capture coating bPEG_2kSA receptor tethers are clearly seen. D) A 2D example image of one of many single virions (this one Influenza A H3N2) immobilized in capture coating. 2DFFT analysis for all four AFM scans shown in S2 Fig.