Controlling Binding Site Densities on Glass Surfaces

Abstract

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.

Multimode Surface Functional Group Determination: Combining Steady-State and Time-Resolved Fluorescence with X-ray Photoelectron Spectroscopy and Absorption Measurements for Absolute Quantification

“…Such quantitative labeling, however, is only achievable in rare cases of well-isolated functional groups on surfaces and high-conversion reactions. 20 This difficulty can be circumvented by claiming only a quantification of accessible surface functional groups rather than the total amount. 21 The meaning of this value is yet debatable if a small-molecule dye is used for quantification of functional groups on surfaces which later on are intended for assays involving the binding of larger entities like DNA strands or proteins to the substrate.…”

Direct DNA Methylation Profiling Using Methyl Binding Domain Proteins

“…In the case of a fluorescence microarray surface, the density of a reactive silane (e.g. APTES or GPS) can be varied by dilution with another monofunctional “spacer” silane;48 this in turn affects the density of the probe through its covalent linkage to the reactive silane.…”

Surface Modification with Control over Ligand Density for the Study of Multivalent Biological Systems

“…by mixing a low concentration of (3‐aminopropyl)triethoxysilane (APTES) and a much larger concentration of (2‐cyanoethyl)triethoxysilane (CETES). The low amino group density on the surface due to the presence of APTES, allowed the modification of surfaces with a very low fraction of biotin moieties (<10 −7 ) …”

“…The low amino group density on the surface due to the presence of APTES, allowed the modification of surfaces with a very low fraction of biotin moieties (< 10 À 7 ). [42] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57…”