Ayrton, John-Paul; (2025) Engineering nanobody fusion proteins for nanoparticle stabilisation and application in biosensing. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The development of point-of-care diagnostics, crucial for containing infectious diseases, is often hindered by the time-consuming search for suitable antibody pairs. Nanobodies, with their facile production and design flexibility, present a promising alternative for lateral flow assays (LFAs). This work explores protein engineering strategies to optimise nanobodies for these applications, enhancing their stability and functionality upon integration into LFAs A key challenge addressed in this thesis is the inherent instability of nanobody-gold nanoparticle bioconjugates formed through physisorption. This instability, particularly relevant in the context of nanoparticle functionalisation, poses a significant hurdle in LFA development, where bioconjugates must withstand harsh processing steps like drying and exposure to complex biological samples. A trivalent nanobody is developed through innovative protein engineering that demonstrates efficient and stable bioconjugation through physisorption. Compared to its monovalent and divalent counterparts, these engineered bioconjugates exhibit enhanced stability, retaining picomolar sensitivity to the target antigen and withstanding storage processes like freeze-drying, making them ideal candidates for real-world diagnostic applications. Current LFA manufacturing and deployment can be subject to manufacturing bottlenecks and disruptions in supply chains, especially during global emergencies. This vulnerability underscores the need for robust and adaptable diagnostics. Leveraging the versatility of nanobodies, a modular display LFA based on in vivo biotinylation is also established. Nanoparticle and nitrocellulose functionalisation with streptavidin enables the simultaneous display of biotinylated nanobodies on the nanoparticle and test line. The system's design is validated through mechanistic mathematical modelling. The "plug-and-play" integration of nanobodies into prefabricated cassettes promises to accelerate LFA development significantly. This modular approach, coupled with rapid nanobody development and the exceptional streptavidin-biotin interaction, would be less affected by manufacturing bottlenecks and supply chain disruptions. This could enable a quicker response to infectious diseases, leading to improved patient outcomes and more effective disease control.

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