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

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 p...

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Autores principales: Patra, Satyajit, Baibakov, Mikhail, Claude, Jean-Benoît, Wenger, Jérôme
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7089978/
https://www.ncbi.nlm.nih.gov/pubmed/32251328
http://dx.doi.org/10.1038/s41598-020-61856-9
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author Patra, Satyajit
Baibakov, Mikhail
Claude, Jean-Benoît
Wenger, Jérôme
author_facet Patra, Satyajit
Baibakov, Mikhail
Claude, Jean-Benoît
Wenger, Jérôme
author_sort Patra, Satyajit
collection PubMed
description 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.
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spelling pubmed-70899782020-03-26 Surface passivation of zero-mode waveguide nanostructures: benchmarking protocols and fluorescent labels Patra, Satyajit Baibakov, Mikhail Claude, Jean-Benoît Wenger, Jérôme Sci Rep Article 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. Nature Publishing Group UK 2020-03-23 /pmc/articles/PMC7089978/ /pubmed/32251328 http://dx.doi.org/10.1038/s41598-020-61856-9 Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Patra, Satyajit
Baibakov, Mikhail
Claude, Jean-Benoît
Wenger, Jérôme
Surface passivation of zero-mode waveguide nanostructures: benchmarking protocols and fluorescent labels
title Surface passivation of zero-mode waveguide nanostructures: benchmarking protocols and fluorescent labels
title_full Surface passivation of zero-mode waveguide nanostructures: benchmarking protocols and fluorescent labels
title_fullStr Surface passivation of zero-mode waveguide nanostructures: benchmarking protocols and fluorescent labels
title_full_unstemmed Surface passivation of zero-mode waveguide nanostructures: benchmarking protocols and fluorescent labels
title_short Surface passivation of zero-mode waveguide nanostructures: benchmarking protocols and fluorescent labels
title_sort surface passivation of zero-mode waveguide nanostructures: benchmarking protocols and fluorescent labels
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7089978/
https://www.ncbi.nlm.nih.gov/pubmed/32251328
http://dx.doi.org/10.1038/s41598-020-61856-9
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