Ordered Nanobody Arrays for Enhanced Biosensor Sensitivity and Selectivity

Nanobodies are an emerging class of proteins composed of a single antibody domain that have demonstrated significant potential for use as therapeutics, antigen tracers, and biosensors. For biosensing applications these proteins are incorporated onto a surface, where performance is dependent upon achieving sufficient protein density with easily accessible binding sites and adequate analyte transport. Here, we demonstrate the self-assembly of nanobody-polymer conjugates into domains of densely-packed, well-oriented nanobodies and domains of polymer meshes that can exclude molecules from the block copolymer assembly based on particle size. Small-angle X-ray scattering (SAXS) measurements indicate that the conjugates assemble into lamellae with alternating domains of nanobody and polymer. Enzyme immunoassays (EIAs) are performed using thin films of the conjugates, displaying orders of magnitude decrease in limit of detection compared to surface-immobilized nanobodies. It is further demonstrated that tuning the polymer molecular weight serves as a method to control the diffusion of molecules into the films.