Multifunctional monolayer assemblies for reversible direct fluorescence transduction of protein-ligand interactions at surfaces

This paper describes a convenient new method for preparing functionalizable protein-resistant monolayers that can be used to incorporate ligands and protein-sensitive fluorescent reporter groups, and the use of these monolayers for the detection of protein-ligand interactions. BODIPY X-650/665, a diode laser compatible fluorophore, and biotin, a model ligand, have been used to transduce biospecific interactions between proteins and biotin at surfaces. Silicon wafers or quartz slides were coated with (3-aminopropyl)triethoxysilane, and treated with glutaraldehyde and then 2,2'-(ethylenedioxy)bis(ethylenediamine) The resultant surface layers are resistant to nonspecific protein adsorption and contain primary amine groups that are available for subsequent derivatization. Chemical modification of the amine-terminated monolayers thus obtained was accomplished using the N-hydroxysuccinimide active eater of BODIPY X-650/665 and biotin activated with Woodward's reagent K. Surfaces treated only with the BODIPY dye for long periods of time to produce a near monolayer coverage of the fluorophore exhibited a dramatic attenuation of the emission of the fluore upon nonspecific adsorption of protein (e.g., albumin). Nonspecific adsorption of proteins can be minimized by diluting the fluore on the surface. Incorporation of a biospecific ligand (i.e., biotin) and the BODIPY fluore in mixed monolayers by serial chemical modification of amine-terminated monolayers yielded surfaces that can be used for fluorescence transduction of biospecific protein adsorption. Specific binding of streptavidin and anti-biotin was detected by a decrease in both the intensity and excited-state lifetime of the fluorescence of the BODIPY dye. Binding of anti-biotin to these surfaces is reversible. No significant change in the intensity was observed upon exposure of these surfaces to solutions of biotin-blocked streptavidin and anti-human IgG. Only a slight change in intensity was observed upon exposure to bovine serum albumin. Phase angle measurements obtained at a single frequency (100 MHz) were used to detect the reversible binding of anti-biotin at the monolayer surface. These observations indicate that it is possible to construct architectures containing ligands and fluores that can be used to detect binding events using lifetime-based measurements. These assemblies should be generalizable to study a wide variety of protein- and cell-surface interactions in biotechnological applications.