Controlling binding site densities on glass surfaces.

The density of surface-immobilized ligands or binding sites is an important issue for the development of sensors, array- or chip-based assays, and single-molecule detection methods. The goal of this research is to control the binding site density of reactive ligands on surfaces by diluting surface amine groups in self-assembled and cross-linked monolayers on glass prepared from solutions containing very low concentrations of (3-aminopropyl)triethoxysilane (APTES) and much higher concentrations of (2-cyanoethyl)triethoxysilane. The surface amine sites are suitable for attaching labels and ligands by reaction with succinimidyl ester reagents. Labeling the amine sites with fluorescent molecules and imaging the single molecules with fluorescence microscopy provides a means of determining the density of amine sites on the surface, which were incorporated into the self-assembled monolayer with micrometer spacings in proportion to the concentration of APTES in the synthesis. Biotin ligands were also bound to these surface amine sites using a succinimidyl ester linker, and the immobilized biotin was then reacted with either streptavidin-conjugated gold colloid particles or fluorescently labeled neutravidin. Imaging of these samples yields consistent amine and biotin site coverages, indicating that quantitative control and chemical conversion of binding sites can be achieved at very low (<10(-7)) fractions of a monolayer.