Covalent and uniformly oriented immobilization of the ‘clickable’ alkynated nanobody targeting the vascular cell adhesion molecule-1 for advanced biosensing applications

Abstract

The innovation of protein immobilization techniques has been gaining enormous attention to fabricate advanced biomaterials such as biochips and biosensors. Conventional immobilizing strategies such as physisorption or randomly oriented, covalent coupling usually result in fabricated surfaces with low activity and reproducibility. Recently the application of uniformly oriented and covalent protein coupling can circumvent these limitations. In the present study, the nanobody targeting Vascular Cell Adhesion Molecule-1 (NbVCAM1) was functionally expressed in the E. coli cytoplasm and was successfully appended with a C-terminal ‘clickable’ alkyne moiety using the Expressed Protein Ligation (EPL) technique. It was found that the alkynated nanobody retained its solubility, stability and high binding capacity towards human VCAM1 antigen (hVCAM1). The well-known Huisgen CuAAC-mediated immobilization was performed on different azide-functionalized surfaces including azidified silicon and a Biacore® gold-coated platform for hVCAM1 detection using ellipsometry and surface plasmon resonance (SPR), respectively. The CuAAC-mediated site-directed immobilization of the C-terminally alkynated nanobody resulted in an SPR-based biosensor platform which exerted a significant increase in binding affinity (up to 177 fold) and sensitivity (up to 57 fold) and an improved reproducibility as compared to the surfaces functionalized with nanobodies by random coupling methods such as physisorption and EDC/NHS chemistry. Although somewhat less, improvement was also achieved for the ellipsometry-based silicon platform. Conclusively, our findings have proven the advantages in coupling efficiency, nanobody orientation, antigen binding capacity and reproducibility of nanobody-functionalized biosensor surfaces developed by using a covalent and controlled immobilization strategy.