Homogeneity and Protein-Binding Capacity of APTES Coatings: Quantitative In Situ Characterization by a Label-Free Imaging Biosensor

The morphology of the functional coating (thickness, density, homogeneity) determines the accuracy and reproducibility ofabel-free biosensor analysis, yet the quality of the coating is usually assessed indirectly, outside the conditions of the analysis. We describe an in situ method for functional assessment of the coating by the biosensor’s own response, based on surface waves in a one-dimensional photonic crystal with two-dimensional imaging. In one cycle of glutaraldehyde activation and protein immobilization, we determine sorption capacity, interchannel homogeneity, flow stability and surface density. At a manyfold excess of reactive centers, the saturated binding signal is insensitive to theocal density of functional groups. On a series of 3-aminopropyltriethoxysilane (APTES) coatings, the sorption capacity and homogeneity depend critically on the solvent composition, and the solution-front delay maps separate the coating nonuniformity from mass transfer. For the optimized coating, formed in a 40% solution of acetic acid in ethanol, where theow water content and acidity suppress the self-condensation of APTES, the surface concentration of bovine serum albumin reaches 1.98 mg/m2, a dense side-on monolayer with a coefficient of variation of 1.2%. The results are confirmed by IgG immobilization and streptavidin–biotin binding. The approach enables noninvasive control of biofunctionalization.