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Biosensors 2016, 6(3), 34; doi:10.3390/bios6030034

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

1
Biomolecule Design Group, Institute for Materials Research (IMO), Hasselt University, Diepenbeek BE-3590, Belgium
2
Faculty of Food Technology and Biotechnology, Can Tho University of Technology, Can Tho 900000, Vietnam
3
Immunology and Biochemistry, Biomedical Research Institute (Biomed) and School of Life Sciences, Transnationale Universiteit Limburg, Hasselt University, Diepenbeek BE-3590, Belgium
4
Maastricht Science Programme, Maastricht University, Maastricht 6200 MD, The Netherlands
5
Applied and Analytical Chemistry, Institute for Materials Research (IMO), Hasselt University, Diepenbeek BE-3590, Belgium
*
Author to whom correspondence should be addressed.
Academic Editor: Jeff D. Newman
Received: 31 May 2016 / Revised: 27 June 2016 / Accepted: 29 June 2016 / Published: 5 July 2016
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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. View Full-Text
Keywords: uniformly oriented bioconjugation; biosensor; CuAAC; expressed protein ligation; VCAM1-targeting nanobody uniformly oriented bioconjugation; biosensor; CuAAC; expressed protein ligation; VCAM1-targeting nanobody
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Ta, D.T.; Guedens, W.; Vranken, T.; Vanschoenbeek, K.; Steen Redeker, E.; Michiels, L.; Adriaensens, P. Enhanced Biosensor Platforms for Detecting the Atherosclerotic Biomarker VCAM1 Based on Bioconjugation with Uniformly Oriented VCAM1-Targeting Nanobodies. Biosensors 2016, 6, 34.

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