| Abstract: | The reflection grating coupler for direct affinity sensing is characterized in detail. The performance of this device and its potential in affinity sensing application are investigated with two affinity-based systems: A self-assembling protein-multilayer system based on avidin-biotin interaction was used to compare the response of the device with theoretical expectations. The analytical performance was characterized by a pesticide immunoassay carried out in an indirect test format with a covalently immobilized triazine derivative. Experimentally determined parameters were in good agreement with model calculations. During the binding of 12 protein monolayers at the surface, the change in effective refractive index Dn(eff) detected for a single layer decreased from approximately 8 x 10(-4) to less than 4 x10(-5) by more than 95%, indicating a filling of the evanescent field. By comparison with bulk refractive-index measurements, a refractive index n(D) approximately 1.38 of the protein multilayer was estimated. Fitting of the model gave a refractive index n(D) = 1.377 of the protein multilayer and an average thickness of 11 nm for a single protein layer. An average noise of Dn(eff) = 8.5 x 10(-7) was detected, corresponding to approximately 1% of the maximum response for a protein monolayer. At a triazine derivative attached to the surface through dextran-based surface chemistry, a maximum antibody loading that corresponds to an Dn(eff) of 1.5 x 10(-3) was observed. In an indirect immunoassay of the herbicide simazine, a detection limit of 0.25 mug/1 of simazine was reached with polyclonal Fab fragments in a concentration of 1 mug/ml. |
