title: Enhanced Biosensor Platforms for Detecting the Atherosclerotic Biomarker VCAM1 Based on Bioconjugation with Uniformly Oriented VCAM1-Targeting Nanobodies.
abstract: 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.
authors.affiliation: Biomolecule Design Group, Institute for Materials Research (IMO), Hasselt University, Diepenbeek BE-3590, Belgium. duytien.ta@uhasselt.be.|Faculty of Food Technology and Biotechnology, Can Tho University of Technology, Can Tho 900000, Vietnam. duytien.ta@uhasselt.be.
authors.forename: Duy Tien
authors.initials: DT
authors.lastname: Ta
authors.affiliation: Biomolecule Design Group, Institute for Materials Research (IMO), Hasselt University, Diepenbeek BE-3590, Belgium. tdtien@ctuet.edu.vn.
authors.forename: Wanda
authors.initials: W
authors.lastname: Guedens
authors.affiliation: Biomolecule Design Group, Institute for Materials Research (IMO), Hasselt University, Diepenbeek BE-3590, Belgium. wanda.guedens@uhasselt.be.
authors.forename: Tom
authors.initials: T
authors.lastname: Vranken
authors.affiliation: Immunology and Biochemistry, Biomedical Research Institute (Biomed) and School of Life Sciences, Transnationale Universiteit Limburg, Hasselt University, Diepenbeek BE-3590, Belgium. katrijn.vanschoenbeek@uhasselt.be.
authors.forename: Katrijn
authors.initials: K
authors.lastname: Vanschoenbeek
authors.affiliation: Maastricht Science Programme, Maastricht University, Maastricht 6200 MD, The Netherlands. erik.steenredeker@maastrichtuniversity.nl.
authors.forename: Erik
authors.initials: E
authors.lastname: Steen Redeker
authors.affiliation: Immunology and Biochemistry, Biomedical Research Institute (Biomed) and School of Life Sciences, Transnationale Universiteit Limburg, Hasselt University, Diepenbeek BE-3590, Belgium. luc.michiels@uhasselt.be.
authors.forename: Luc
authors.initials: L
authors.lastname: Michiels
authors.affiliation: Biomolecule Design Group, Institute for Materials Research (IMO), Hasselt University, Diepenbeek BE-3590, Belgium. peter.adriaensens@uhasselt.be.|Applied and Analytical Chemistry, Institute for Materials Research (IMO), Hasselt University, Diepenbeek BE-3590, Belgium. peter.adriaensens@uhasselt.be.
authors.forename: Peter
authors.initials: P
authors.lastname: Adriaensens
mesh_terms: D000941:Antigens; D050197:Atherosclerosis / Q000378:metabolism; D015415:Biomarkers; D015374:Biosensing Techniques; D002021:Buffers; D006801:Humans; D011485:Protein Binding; D012825:Silicon; D061905:Single-Domain Antibodies; D020349:Surface Plasmon Resonance; D019010:Vascular Cell Adhesion Molecule-1
chemical_list: D000941:Antigens; D015415:Biomarkers; D002021:Buffers; D061905:Single-Domain Antibodies; D019010:Vascular Cell Adhesion Molecule-1; D012825:Silicon
keywords: CuAAC; VCAM1-targeting nanobody; biosensor; expressed protein ligation; uniformly oriented bioconjugation