Controlled and covalent bioconjugation of materials with site-specifically functionalized nanobodies for advanced medical diagnostics

Abstract

Site-specific modification of proteins and controlled bioconjugation to complementary functionalized species is an important demand for developing advanced biomaterials with highly improved performances. This PhD research is aimed to design, develop and optimize methodologies for the site-specific modification of nanobodies with a unique and bio-orthogonal functionality (i.e. an alkyne) in order to subsequently conjugate them covalently and with a uniform orientation to materials. In this way, the active site of all conjugated nanobodies stays accessible, resulting in an increased antigen binding sensitivity, and paving the way to device miniaturization. The nanobody targeting vascular cell adhesion molecule-1 (i.e. NbVCAM1) is used as a workhorse towards enhanced detection capacity for VCAM1, an important biomarker for atherosclerosis as well as some types of cancer. For this purpose, two approaches are investigated: i) expressed protein ligation (EPL) followed by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and ii) sortase A- mediated protein ligation (SPL).