Developing An Ellipsometry-based Biosensor Platform via Covalent and Oriented Coupling of The Nanobody Targeting The Vascular Cell Adhesion Molecule-1 To A Silicon Wafer

Protein immobilization plays a key role in the development of all protein-based biosensors in order to achieve good performances. Conventional techniques such as physical adsorption or randomly chemical coupling usually lead to low activity and reproducibility. Therefore there is an essential need for uniformly oriented, covalent immobilization approaches. In this study, the nanobody targeting Vascular Cell Adhesion Molecule-1 (NbVCAM1) is engineered with a C-terminal alkyne function via the Intein-mediated Protein Ligation (IPL) technique. The alkynylated nanobody exhibits high binding capacity towards the human VCAM1. Moreover, it can be covalently coupled to an azidified silicon wafer via the Copper(I)-catalyzed azide-alkyne cycloaddition, hyphothetically with a unique orientation. The resulting nanobody-conjugated surfaces exhibit a remarkable increase in binding affinity and sensitivity when monitored by ellipsometry, as compared to those loaded with nanobodies via random couplings. Therefore, the results show an improved performance of the uniformly oriented nanobodyfunctionalized surfaces as a biosensor platform.