Journal
ACS NANO
Volume 7, Issue 9, Pages 8158-8166Publisher
AMER CHEMICAL SOC
DOI: 10.1021/nn403447s
Keywords
zero-mode waveguide; nanoaperture; single-molecule fluorescence; microscopy; fluorescence resonance energy transfer; self-assembled monolayer; translation factor
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Funding
- ACS [RSG GMC-117152]
- NIH-NIGMS [R01 GM084288]
- CEPSR Clean Room at Columbia University
- Department of Energy Office of Science Graduate Fellowship Program (DOE SCGF) [DE-AC05-06OR23100]
- Columbia University's NIH Training Program in Molecular Biophysics [T32-GM008281]
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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 mu M background concentrations of ligand biomolecules, thereby rendering previously impracticable biological systems accessible to single-molecule fluorescence investigations.
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