Surface plasmon resonance study for a reliable determination of the affinity constant of multivalent grafted beads

In this study, galactose-grafted beads were prepared using the main design principle of the cluster effect. Galactose was chosen as the sugar for investigation because it acts as the main building block of long glycan chains and because a simple and fast protocol is still required for its immobilization. For the analysis, the lectin, ligand of the galactose, was immobilized on a gold plasmonic substrate. After preliminary characterization of the galactose-grafted beads, the investigation of the surface plasmon surface behavior of the system was carried out, for studying the affinity constant of the multivalent beads. The results of steady-state and of the kinetics analysis evidenced a higher affinity of the galactose-grafted beads over the beadless galactose solution. For the association kinetics analysis, a Langmuir isotherm was applied to the data. The analysis of the rate of dissociation evidenced the most important differences between the two samples, based on the more difficult release of the galactose-grafted beads during washing. To confirm the influence of the glycoside cluster effect, a low-density lectin substrate was tested, and the results evidenced that the characteristic size of the molecules determines a threshold for the cluster density. The calculated detection limit and dissociation constants were 3.5 μM and 40.2 μM, respectively. Considering those results, the evaluation of the affinities toward the receptors depends on the cluster density and then, it should be designed for mimicking the biological samples.