Improved Glass Surface Passivation for Single-Molecule Nanoarrays

Abstract

Single-molecule fluorescence techniques provide a critical tool for probing biomolecular and cellular interactions with unprecedented resolution and precision. Unfortunately, many of these techniques are hindered by a common problem, namely the nonspecific adsorption of target biomolecules. This issue is mostly addressed by passivating the glass surfaces with a poly(ethylene glycol) (PEG) brush. This is effective only at low concentrations of the probe molecule, because there are defects inherent to polymer brushes formed on glass coverslips, due to the presence of surface impurities. Tween-20, a detergent, is a promising alternative that can improve surface passivation, but it is incompatible with living cells, and it also possesses limited selectivity for glass background over metallic nanoparticles, which are frequently used as anchors for the probe molecules. To address these issues, we have developed a more versatile method to improve the PEG passivation. A thin film of hydrogen silsesquioxane (HSQ) is spin-coated and thermally cured on glass coverslips in order to cover the surface impurities. This minimizes the formation of PEG defects and reduces nonspecific adsorption, resulting in an improvement comparable to Tween-20 treatment. This approach was applied to single-molecule nanoarrays of streptavidin bound to AuPd nanodots patterned by e-beam lithography (EBL). The fluorescence signal to background ratio (SBR) on HSQ coated glass was improved by ~ 4-fold as compared to PEG directly on glass. This improvement enables direct imaging of ordered arrays of single molecules anchored to lithographically patterned arrays of metallic nanodots.

Surface passivation of zero-mode waveguide nanostructures: benchmarking protocols and fluorescent labels

“…[41][42][43][44] However, all these applications require a proper surface passivation and/or functionalization of the ZMW in order to avoid the unwanted adsorption of the fluorescent molecules onto the ZMW surface that would impede the experiments. While extensive literature exists for passivating glass surfaces for single molecule fluorescence microscopy, [45][46][47][48][49][50][51][52] little is known about the surface passivation of ZMWs and the comparative analysis of different passivation strategies. For aluminum ZMWs, most studies reproduce the protocol based on polyvinylphosphonic acid (PVPA) as initially introduced by Korlach and coworkers in Ref.…”

Argonaute bypasses cellular obstacles without hindrance during target search

“…This is followed by assembly of microfluidic chambers with the use of double sided scotchtape. For a detailed protocol, we refer to 63 Further improvement of surface quality occurs through 15 min incubation of T50 and 5% Tween20 64 after which the channel is rinsed with 100 μL T50 buffer. Streptavidin (5 mg/mL) was diluted in T50 to 0.1 mg/mL.…”

Quantifying protein-protein interactions by molecular counting with mass photometry

“…As a result, MP affords real-time assessment of (dis)assembly completely label-free and entirely independent of immobilization, minimising any possible perturbations, as well as being intrinsically sensitive to binding stoichiometries and oligomerization. The current limitation to sub-µM affinities and concentration range can be addressed in the future by a combination of surface passivation approaches [32] , as well as improvements to hardware and software, with which we expect to reach the µM range in the near future. This will enable measurements up to 100 µM affinities, making MP a powerful approach for characterising biomolecular interactions without labels and single-molecule sensitivity in a minimally perturbative fashion.…”