Enhanced Biosensor Platforms for Detecting the Atherosclerotic Biomarker VCAM1 Based on Bioconjugation with Uniformly Oriented VCAM1-Targeting Nanobodies

Biosensors

Wanda Guedens¹  Tom Vranken¹  Duy Tien Ta¹  Peter Adriaensens¹  Luc Michiels²  Katrijn Vanschoenbeek²  Erik Steen Redeker³ 

[1] Biomolecule Design Group, Institute for Materials Research (IMO), Hasselt University, Diepenbeek BE-3590, Belgium;Immunology and Biochemistry, Biomedical Research Institute (Biomed) and School of Life Sciences, Transnationale Universiteit Limburg, Hasselt University, Diepenbeek BE-3590, Belgium;Maastricht Science Programme, Maastricht University, Maastricht 6200 MD, The Netherlands;

关键词: uniformly oriented bioconjugation;    biosensor;    CuAAC;    expressed protein ligation;    VCAM1-targeting nanobody;   

DOI  :  10.3390/bios6030034

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown