title: Robustly passivated, gold nanoaperture arrays for single-molecule fluorescence microscopy.
abstract: The optical confinement generated by metal-based nanoapertures fabricated on a silica substrate has recently enabled single-molecule fluorescence measurements to be performed at physiologically relevant background concentrations of fluorophore-labeled biomolecules. Nonspecific adsorption of fluorophore-labeled biomolecules to the metallic cladding and silica bottoms of nanoapertures, however, remains a critical limitation. To overcome this limitation, we have developed a selective functionalization chemistry whereby the metallic cladding of gold nanoaperture arrays is passivated with methoxy-terminated, thiol-derivatized polyethylene glycol (PEG), and the silica bottoms of those arrays are functionalized with a binary mixture of methoxy- and biotin-terminated, silane-derivatized PEG. This functionalization scheme enables biotinylated target biomolecules to be selectively tethered to the silica nanoaperture bottoms via biotin-streptavidin interactions and reduces the nonspecific adsorption of fluorophore-labeled ligand biomolecules. This, in turn, enables the observation of ligand biomolecules binding to their target biomolecules even under greater than 1 μM background concentrations of ligand biomolecules, thereby rendering previously impracticable biological systems accessible to single-molecule fluorescence investigations.
authors.affiliation: Department of Chemistry, Columbia University , New York, New York 10027, United States.
authors.forename: Colin D
authors.initials: CD
authors.lastname: Kinz-Thompson
authors.forename: Matteo
authors.initials: M
authors.lastname: Palma
authors.forename: Dileep K
authors.initials: DK
authors.lastname: Pulukkunat
authors.forename: Daniel
authors.initials: D
authors.lastname: Chenet
authors.forename: James
authors.initials: J
authors.lastname: Hone
authors.forename: Shalom J
authors.initials: SJ
authors.lastname: Wind
authors.forename: Ruben L
authors.initials: RL
authors.lastname: Gonzalez
mesh_terms: D001704:Biopolymers / Q000032:analysis*; D015374:Biosensing Techniques / Q000379:methods*; D006046:Gold / Q000737:chemistry*; D007089:Image Enhancement / Q000379:methods*; D053768:Metal Nanoparticles / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D012031:Refractometry / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D001704:Biopolymers; D006046:Gold