Real-time two-wavelength surface plasmon resonance as a tool for the vertical resolution of binding processes in biosensing hydrogels

In this paper, we describe a new setup for simultaneous measurements of surface plasmon resonance with two wavelengths in real time. We have chosen a frequently used wavelength (lambda = 784 nm) with medium penetration depth and another one (lambda = 600 nm) with little penetration depth, The setup is a powerful tool to obtain information about binding processes in hydrogels with spatial resolution normal to the surface. Its ability to provide this spatial resolution was tested in interaction experiments employing a dextran hydrogel to which streptavidin was immobilized: a multilayer structure was formed on top of the hydrogel by successive incubation with biotinylated liposomes and streptavidin, followed by experiments with sodium dodecyl sulfate and biotinylated protein A giving rise to binding events inside this swollen polymer layer. We developed a relative uncomplicated three-zone model, which can elegantly explain the time behavior of the two SPR signals and gives suggestions for the mechanisms of binding processes and preferred binding regions in the hydrogel. The data suggest that the hydrogel expands during immobilization of streptavidin and that the binding process starts at the topmost reactive sites and then advances into deeper zones of the hydrogel. Since streptavidin is positively charged during immobilization, we propose primarily electrostatic repulsive forces between streptavidin molecules to be responsible for the expansion. In contrast to this result, the interaction of the immobilized streptavidin with biotinylated protein A as analyte seems not to change the hydrogel extension and seems to occur only in an upper hydrogel zone with limited extension, which remains constant in time.