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

Abstract

Zero mode waveguide (ZMW) nanoapertures efficiently confine the light down to the nanometer scale and overcome the diffraction limit in single molecule fluorescence analysis. However, unwanted adhesion of the fluorescent molecules on the ZMW surface can severely hamper the experiments. Therefore a proper surface passivation is required for ZMWs, but information is currently lacking on both the nature of the adhesion phenomenon and the optimization of the different passivation protocols. Here we monitor the influence of the fluorescent dye (Alexa Fluor 546 and 647, Atto 550 and 647N) on the non-specific adhesion of double stranded DNA molecule. We show that the nonspecific adhesion of DNA double strands onto the ZMW surface is directly mediated by the organic fluorescent dye being used, as Atto 550 and Atto 647N show a pronounced tendency to adhere to the ZMW while the Alexa Fluor 546 and 647 are remarkably free of this effect. Despite the small size of the fluorescent label, the surface charge and hydrophobicity of the dye appear to play a key role in promoting the DNA affinity for the ZMW surface. Next, different surface passivation methods (bovine serum albumin BSA, polyethylene glycol PEG, polyvinylphosphonic acid PVPA) are quantitatively benchmarked by fluorescence correlation spectroscopy to determine the most efficient approaches to prevent the adsorption of Atto 647N labeled DNA. Protocols using PVPA and PEG-silane of 1000 Da molar mass are found to drastically avoid the non-specific adsorption into ZMWs. Optimizing both the choice of the fluorescent dye and the surface passivation protocol are highly significant to expand the use of ZMWs for single molecule fluorescence applications.

Fluorolabeling of the PPTase-Related Chemical Tags: Comparative Study of Different Membrane Receptors and Different Fluorophores in the Labeling Reactions

“…We argue that, for the Atto550 and Atto633 cases, this unspecific signal is a direct consequence of the unspecific interaction of the fluorophore per se with cell membranes, as reported by other studies (Bosch et al, 2014;Hughes et al, 2014). This in turn may depend on the higher hydrophobicity in general displayed by Atto dyes when compared, e.g., to Alexa dyes [ Supplementary Table 1 and (Patra et al, 2020)]. Furthermore, these two fluorophores bring a net positive charge in physiological conditions ( Supplementary Table 1) which could also promote adhesion to cell membrane.…”

Recent advances in plasmonic nanocavities for single-molecule spectroscopy

“…Moreover, advanced nanofabrication approaches enable the preparation of hybrid devices that integrate both solid-state and biological elements (such as organic coatings or functional proteins). 79,80 Future development in nanocavities engineering might include the exploration of highindex dielectric materials for the fabrication of dimers, 81,82 and their integration with MNPs. 83 Other directions might include the combination of dimers and NPoM structures, so called dimers-on-lm, in order to obtain narrow resonances through the superposition of bright and dark modes.…”

Gold Ion Beam Milled Gold Zero-Mode Waveguides

“…The mPEG-SH is prepared at 20 μM concentration in 1× phosphate-buffered saline (PBS) pH 7.5 and was allowed to react with the gold surface for three hours at room temperature. The choice of 2 kD PEG was based on a recent study of passivation effects, where it was found that positively charged dyes such as Atto647 adhere to sample surfaces unless the surface is passivated with PEG longer than 0.5 kD and shorter than 5 kD [ 74 ]. The ZMW were rinsed with ultra pure water.…”