Characterisation of self-assembled engineered proteins on gold nanoparticles and their application to biosensing

PhD ThesisThe use of gold nanoparticles (AuNP) has a long and varied history, thought to cover several thousand years. More recently the unique properties of nanoscale materials have stimulated extensive work on nanoparticles and other nanomaterials leading to their use in novel technologies. AuNPs have been of particular interest for bioscience applications due to their biocompatibility and the ease with which biological molecules can be conjugated to their surface. In this study the assembly of engineered proteins, specifically the transmembrane domain of Escherichia coli outer membrane protein A (OmpATM), onto the surface of AuNPs was investigated both in solution and with the particles attached to a SiO2 substrate. AuNPs were adhered to SiO2 surfaces using a novel silane treatment developed by the industrial sponsor and were characterised using spectroscopy, electron and atomic force microscopy. The addition of a single cysteine residue to the OmpATM structure was shown, by UV-Vis and fluorescence spectroscopy, to increase protein binding at equilibrium and form higher stability protein-AuNP complexes in solution. Following this, engineered OmpATM proteins containing tandem antibody-binding domains from Streptococcal protein G were assembled on the AuNP surface and their structure interrogated using neutron and light scattering. This revealed an oriented protein layer where the functional domains extend away from the AuNP surface and are available to bind antibodies. OmpATM-AuNP conjugates were used to develop biosensing assays using both well-established methods, such as lateral flow assays, and novel spectroscopic methods, which use the unique optical properties of AuNPs. Detection of influenza A nucleoprotein, an antigen used to clinically diagnose influenza, was achieved using a bespoke anti-nucleoprotein single-chain antibody domain fused to OmpATM and assembled on 20 nm diameter AuNPs. The results demonstrate that engineered OmpATM proteins conjugated to AuNPs can be used to develop novel diagnostics using a range of read out technologies