To deal with the general problem of biomolecule specific binding analysis, we have applied the technique of difference spectra to the surface plasmon resonance (SPR)-enhanced total internal reflection ellipsometry measurement. We suggest a three-step treatment of the SPR background that can easily be integrated with the usual measurement routine. First, making use of the difference spectrum in ellipsometric angle Δ, single peak footprints of the topmost layer are obtained that facilitate its sensitive detection during film growth. Subsequently, circumventing the need for explicit knowledge of the substrate properties, the difference spectra peaks can be used for the end-point analysis of a binding. Finally, tracking the binding effectivity of the analyte we determine the injection speed and analyte concentration windows needed for successful monitoring of the film growth. We demonstrate our approach on a comprehensive two-stage binding experiment involving two biologically relevant molecules: the C-terminal domain (CTD) of RNA polymerase II and CTD-interacting domain of one of its transcription factors, the Rtt103 protein.
Keywords: C-terminal domain; CTD; RNA polymerase II; Rtt103 protein; SPR; Spectroscopic ellipsometry; TIRE; surface plasmon resonance; total internal reflection ellipsometry.