Covalent-Bond-Based Immobilization Approaches for Single-Molecule Fluorescence

Abstract

The streptavidin-biotin bridge is commonly used in single-molecule studies to surface immobilize biomolecules onto microscope slides. However, the presence of tryptophanes impedes utilization of UV light and numerous fluorescent nucleotide analogs, such as 2-aminopurine. We present two new approaches to surface-immobilize nucleic acids for single molecule fluorescence experiments using covalent bonds and self-assembled monolayers instead of the traditional avidin-biotin linkage. The first approach takes advantage of a click-chemistry reaction between an azide and an alkyne to surface-immobilize nucleic acids through the resulting triazole linkage. The second approach uses disulfide bond bridges for immobilization. We have characterized the properties of the resulting surface-immobilized fluorophore-labeled DNA molecules and single-molecule fluorescence detection. We find that both approaches are specific and yield comparable surface densities and low fluorescence background to the avidin-biotin linkage, but offer new surface chemical properties that might be advantageous to prevent non-specific binding of biopolymers to the surface and to expand the range of fluorescent probes that can be employed in single molecule studies.