Suchergebnisse

Surface bioconjugation of biomolecules has gained enormous attention for developing advanced biomaterials including biosensors. While conventional immobilization (by physisorption or covalent couplings using the functional groups of the endogenous amino acids) usually results in surfaces with low activity, reproducibility and reusability, the application of methods that allow for a covalent and uniformly oriented coupling can circumvent these limitations. In this study, the nanobody targeting Vascular Cell Adhesion Molecule-1 (NbVCAM1), an atherosclerotic biomarker, is engineered with a C-terminal alkyne function via Expressed Protein Ligation (EPL). Conjugation of this nanobody to azidified silicon wafers and Biacore™ C1 sensor chips is achieved via Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) "click" chemistry to detect VCAM1 binding via ellipsometry and surface plasmon resonance (SPR), respectively. The resulting surfaces, covered with uniformly oriented nanobodies, clearly show an increased antigen binding affinity, sensitivity, detection limit, quantitation limit and reusability as compared to surfaces prepared by random conjugation. These findings demonstrate the added value of a combined EPL and CuAAC approach as it results in strong control over the surface orientation of the nanobodies and an improved detecting power of their targets—a must for the development of advanced miniaturized, multi-biomarker biosensor platforms. ; This research is funded by the FWO project G.0581.12N. The authors gratefully thank S. Muyldermans and N. Devoogdt (Vrije Universiteit Brussel, Belgium) for the vector pHEN6(c):pelB-NbVCAM1-His6, and J.-P. Noben and E. Rooyackers for the MS measurements. We further acknowledge the Hercules Foundation for the project "LC-MS@UHasselt: Linear Trap Quadrupole-Orbitrap mass spectrometer", and the Interreg IV-A project "BioMiMedics" which is financed by the EU and the province of Limburg (Belgium). We further thank the Interuniversity Attraction Poles program (P7/05) initiated by the Belgian Science Policy Office (BELSPO).