Multivalent nanobody engineering for enhanced physisorption and functional display on gold nanoparticles.

The ease of expression and engineering of single domain antibodies, known as nanobodies, make them attractive alternatives to conventional antibodies in point-of-care diagnostics such as lateral flow assays. In lateral flow assays, gold nanoparticle bioconjugates serve as labels which display affinity molecules on the gold surface. While examples of nanobody gold nanoparticle bioconjugates exist, few utilise the simple one-step approach of physisorption owing to undesirable nanoparticle aggregation and loss of functionality. Here we show that engineering nanobodies into multivalent structures can significantly enhance their functionality when physisorbed onto gold nanoparticles. This approach enables resulting bioconjugates to withstand multiple processing steps required for long-term nanoparticle storage within lateral flow assays. Specifically, we show that the trivalent version of VHHV nanobody (VHH3) against the S1 protein of SARS-CoV-2 can be immobilised onto gold nanoparticles through passive adsorption. Unlike its monovalent and bivalent nanobody counterparts, using VHHV3 preserves nanoparticle stability under salt stress, blocking, washing, and freeze-drying conditions while maintaining picomolar sensitivity to the S1 protein. We anticipate that this facile strategy is a significant advancement towards the integration of nanobodies in lateral flow assay development.