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 non-specific 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.

High‐throughput single‐molecule studies of protein–DNA interactions

“…For example, the reaction volume can be reduced by encapsulating the biochemical reaction of interest in a porous lipid vesicle [41][42][43][44][45], within a PDMS nanochannel [46], or within a confined volume induced by a convex lens and a coverslip [47]. Alternatively, the laser excitation can be confined to an attoliter volume within a zero-mode waveguide [48][49][50], or near plasmonic nano-structures that locally enhance the light excitation [51,52]. These methods are applicable for monitoring reactions that occur on short (<100 bp) nucleic acid substrates and are listed in Table 2.…”

smFRET studies of the ‘encounter’ complexes and subsequent intermediate states that regulate the selectivity of ligand binding

“…Because an acceptor will only be efficiently excited via FRET when it is within tens of Å of a donor that is directly excited by an excitation light source ( e.g., a laser), smFRET experiments in which a donor-labeled biomolecule of interest is tethered to the surface of the microfluidic, observation flowcell and an acceptor-labeled ligand is supplied in the imaging buffer greatly ameliorate the limitations imposed by the concentration barrier [28,106]. Despite this advantage of smFRET experiments, acceptors can still be directly, albeit inefficiently, excited by the laser that is used to directly excite the donor fluorophore – a low probability event known as excitation crosstalk [10].…”

“…More recently, an alternative passivation scheme has been reported that uses thiol-based self-assembled monolayers (SAMs) of PEG to robustly passivate gold-based nanoapertures [106]. Given the widespread success of PEG-based passivation schemes in single-molecule fluorescence studies, we anticipate that PEG-passivated nanoaperture arrays will provide a more general solution to the problem of non-specific adsorption of biomolecules to the nanoaperture surfaces and will thereby enable studies of a wide-range of biomolecular systems using nanoaperture arrays (Fig.…”

“…4A). Perhaps most excitingly, the use of gold-based nanoapertures such as the PEG-passivated, gold based nanoapertures described by Kinz-Thompson et al [106] also allows the surface-plasmon of the gold to be used for plasmon-based enhancement of the fluorescence signals at the bottom of the nanoapertures, an approach that has been recently demonstrated [149,150] and that could potentially facilitate the use of nanoaperture arrays for smFRET applications.…”

“…Figure adapted from ref. [106]. (B) Graphical model of the coupled Bayesian HMM used in ebFRET and vbFRET.…”