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 p rotein-sensitive fluorescent reporter groups, and the use of these monolaye rs for the detection of protein-ligand interactions. BODIPY X-650/665, a di ode laser compatible fluorophore, and biotin, a model ligand, have been use d to transduce biospecific interactions between proteins and biotin at surf aces. Silicon wafers or quartz slides were coated with (3-aminopropyl)triet hoxysilane, and treated with glutaraldehyde and then 2,2'-(ethylenedioxy)bi s(ethylenediamine) The resultant surface layers are resistant to nonspecifi c protein adsorption and contain primary amine groups that are available fo r subsequent derivatization. Chemical modification of the amine-terminated monolayers thus obtained was accomplished using the N-hydroxysuccinimide ac tive 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 p roduce a near monolayer coverage of the fluorophore exhibited a dramatic at tenuation of the emission of the fluore upon nonspecific adsorption of prot ein (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 u sed for fluorescence transduction of biospecific protein adsorption. Specif ic binding of streptavidin and anti-biotin was detected by a decrease in bo th the intensity and excited-state lifetime of the fluorescence of the BODI PY dye. Binding of anti-biotin to these surfaces is reversible. No signific ant 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. Ph ase angle measurements obtained at a single frequency (100 MHz) were used t o detect the reversible binding of anti-biotin at the monolayer surface. Th ese observations indicate that it is possible to construct architectures co ntaining ligands and fluores that can be used to detect binding events usin g lifetime-based measurements. These assemblies should be generalizable to study a wide variety of protein- and cell-surface interactions in biotechno logical applications.