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| - Real-Time Two-Wavelength Surface Plasmon Resonance asa Tool for the Vertical Resolution of Binding Processes inBiosensing Hydrogels
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| - In this paper, we describe a new setup for simultaneous measurements of surface plasmon resonancewith two wavelengths in real time. We have chosen a frequently used wavelength (λ = 784 nm) withmedium penetration depth and another one (λ = 600 nm) with little penetration depth. The setup is apowerful tool to obtain information about binding processes in hydrogels with spatial resolution normalto the surface. Its ability to provide this spatial resolution was tested in interaction experiments employinga dextran hydrogel to which streptavidin was immobilized: a multilayer structure was formed on top ofthe hydrogel by successive incubation with biotinylated liposomes and streptavidin, followed by experimentswith sodium dodecyl sulfate and biotinylated protein A giving rise to binding events inside this swollenpolymer layer. We developed a relative uncomplicated three-zone model, which can elegantly explain thetime behavior of the two SPR signals and gives suggestions for the mechanisms of binding processes andpreferred binding regions in the hydrogel. The data suggest that the hydrogel expands during immobilizationof streptavidin and that the binding process starts at the topmost reactive sites and then advances intodeeper zones of the hydrogel. Since streptavidin is positively charged during immobilization, we proposeprimarily 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 asanalyte seems not to change the hydrogel extension and seems to occur only in an upper hydrogel zonewith limited extension, which remains constant in time.
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