Quality of bioactive surface is crucial for achieving the required sensitivity and selectivity of biosensing systems. Numerous methods are available for the characterization of metal-coated surfaces, but only a few to test the efficacy of biomaterial immobilization and the level of non-specific binding. Herewith we propose to use total internal reflection fluorescence (TIRF) microscopy for the characterization of the surface analyte recognition capacity. Biomolecules were bound onto titanium/gold covered glass using three different self-assembled monolayers (SAM). The surfaces with attached antibodies were evaluated using the specific binding of fluorophore-labeled secondary antibodies and visualized with TIRF. Among studied SAMs, aminothiol layers with glutaraldehyde coupling demonstrated high binding capacity along with excellent homogeneity indicating their suitability for applications in biosensors.
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