Nanobodies are an emerging class of proteins representing the single binding domain of heavy-chain antibodies that have demonstrated significant potential for use as therapeutics, antigen tracers, and biosensors. For biosensing applications these proteins are immobilized onto a surface, where performance is dependent upon achieving sufficient protein density with easily accessible binding sites and adequate analyte transport. While numerous methods have been developed to properly orient proteins on a surface, each technique either can only be used for specific classes of proteins or requires significant protein and/or surface pre-treatment. One potentially straightforward and effective technique for creating a dense array of properly oriented proteins is through utilization of block copolymer-like self-assembly. By conjugating a protein to a polymer, it has been demonstrated that these conjugates assemble into ordered nanostructures in solution similar to coil-coil diblock copolymers. Though protein-polymer conjugates offer many benefits over surface-immobilized protein biosensors, their use as biosensors has been largely unexplored.<br/><br/>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. By expanding nanobodies into 3D space above a surface, these conjugate biosensors display a significantly increased number of binding sites compared to surface-based sensors, decreasing device limit of detection. Small-angle X-ray scattering (SAXS) measurements indicate that the conjugates assemble into weakly-ordered lamellae with alternating domains of nanobody and polymer. Simple enzyme immunoassays (EIAs) are performed using thin films of the conjugates, displaying enhanced sensitivity compared to surface-immobilized nanobodies and establishing the capability of performing EIA assays within a thin film geometry. It is further demonstrated that tuning the polymer molecular weight serves as a method to control the size-based diffusion of molecules into the films.

Nanobody-polymer conjugate arrays for enhanced biosensor sensitivity and selectivity

256th National Meeting (2018)

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