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

Duy Tien Ta, Wanda Guedens, Tom Vranken, Katrijn Vanschoenbeek, Erik Steen Redeker, Luc Michiels, Peter Adriaensens*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

13 Citations (Web of Science)

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 (TM) 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.

Original languageEnglish
Article number34
Number of pages17
JournalBiosensors
Volume6
Issue number3
DOIs
Publication statusPublished - Sep 2016

Keywords

  • uniformly oriented bioconjugation
  • biosensor
  • CuAAC
  • expressed protein ligation
  • VCAM1-targeting nanobody
  • EXPRESSED PROTEIN LIGATION
  • CELL-ADHESION MOLECULE-1
  • SITE-SPECIFIC ATTACHMENT
  • ELECTRODE-THE BASE
  • ELECTROCHEMICAL BIOSENSOR
  • COLORECTAL-CANCER
  • TERMINAL ALKYNES
  • IMMUNE-SYSTEM
  • IMMOBILIZATION
  • ANTIBODIES

Cite this