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Real-time profiling Anti-EpCAM Based Immune capture, from Molecules to Cells using MP-SPR
摘要： Antibodies of epithelial cell-adhesion-molecule (anti-EpCAM)-based interfaces have proven to be highly efficient at capturing circulating tumor cells (CTCs). To achieve the bonding of anti-EpCAM to the interface, biotin and streptavidin are used to modify the surface. These processes are critical to subsequent cell-capture efficiencies. However, quantitative research on the interactions between biotin, streptavidin and biotinylated anti-EpCAM on the interface is lacking. In this work, the thermodynamics and kinetics of biomolecular interactions were determined by using surface plasmon resonance. The equilibrium binding affinities for biotinylated anti-EpCAM to streptavidin and streptavidin to biotin (illustrated by biotin-PEG400-thiol) were found to be 2.75×10^6 M^-1 and 8.82×10^6 M^-1, respectively. Each streptavidin can bind up to 2.30 biotinylated anti-EpCAM under thermodynamic equilibrium. The findings provide useful information to optimize the modification of anti-EpCAM and improve the capture efficiency of CTCs.
关键词： surface plasmon resonance,antibodies of epithelial cell-adhesion-molecule,cell capture,biotin–streptavidin interaction,thermodynamics,kinetics
更新于2025-09-23 15:23:52
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A Plasmonic Approach to Study Protein Interaction Kinetics through the Dimerization of Functionalized Ag Nanoparticles
摘要： Understanding the kinetics of protein interactions plays a key role in biology with significant implications for the design of analytical methods for disease monitoring and diagnosis in medical care, research and industrial applications. Herein, we introduce a novel plasmonic approach to study the binding kinetics of protein-ligand interactions following the formation of silver nanoparticles (Ag nps) dimers by UV-Vis spectroscopy that can be used as probes for antigen detection and quantification. to illustrate and test the method, the kinetics of the prototype biotin-streptavidin (Biot-StV) pair interaction was studied. controlled aggregates (dimers) of StV functionalized Ag nps were produced by adding stoichiometric quantities of gliadin-specific biotinylated antibodies (IgG-Biot). The dimerization kinetics was studied in a systematic way as a function of Ag NPs size and at different concentrations of IgG-Biot. The kinetics data have shown to be consistent with a complex reaction mechanism in which only the Ag NPs attached to the IgG-Biot located in a specific STV site are able to form dimers. These results help in elucidating a complex reaction mechanism involved in the dimerization kinetics of functionalized Ag nps, which can serve as probes in surface plasmon resonance-based bioassays for the detection and quantification of different biomarkers or analytes of interest.
关键词： biotin-streptavidin interaction,silver nanoparticles,dimerization,protein interaction kinetics,plasmonic approach,surface plasmon resonance,UV-Vis spectroscopy
更新于2025-09-11 14:15:04