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Rapid and improved surface passivation method for Single-Molecule experiments.

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Title: Rapid and improved surface passivation method for Single-Molecule experiments.
Authors: Gonneville AN; Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA., Ward AE; Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA., Naidoo NR; Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA., Barrera FN; Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA., Lamichhane R; Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA. Electronic address: rajan@utk.edu.
Source: Methods (San Diego, Calif.) [Methods] 2026 Mar; Vol. 247, pp. 95-106. Date of Electronic Publication: 2026 Jan 07.
Publication Type: Journal Article
Language: English
Journal Info: Publisher: Academic Press Country of Publication: United States NLM ID: 9426302 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1095-9130 (Electronic) Linking ISSN: 10462023 NLM ISO Abbreviation: Methods Subsets: MEDLINE
Imprint Name(s): Publication: Duluth, MN : Academic Press
Original Publication: San Diego : Academic Press, c1990-
MeSH Terms: Polyethylene Glycols*/chemistry , Fluorescence Resonance Energy Transfer*/methods , Single Molecule Imaging*/methods, Silanes/chemistry ; Fluorescent Dyes/chemistry ; Microscopy, Fluorescence/methods ; Photobleaching ; Surface Properties
Abstract: Single-molecule fluorescence experiments are a powerful tool for studying biomolecular interactions, including protein dynamics and oligomerization, protein-protein interactions, and protein-nucleic acid interactions. Biomolecules are commonly immobilized on the microscope surface to extend the observation time. However, non-specific interactions between biomolecules and the surface present a major challenge. The first critical step in these experiments is preparing the surface using polyethylene glycol (PEG) coated slides, which facilitate biomolecule immobilization while minimizing non-specific interactions. The surface treatment typically uses PEG-SVA (Succinimidyl Valerate) coated slides, and the protocol for the treatment is lengthy and time-consuming. To overcome this issue, we have developed a process that uses PEG-Silane to improve efficiency while maintaining reproducibility. Here, we present a one-step, rapid PEGylation methodology that can be completed in minutes rather than hours. We demonstrate its validity and feasibility through single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule photobleaching experiments across various biological samples.
(Copyright © 2026 The Authors. Published by Elsevier Inc. All rights reserved.)
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Grant Information: R35 GM142946 United States GM NIGMS NIH HHS
Contributed Indexing: Keywords: FRET; PEG-Silane; PEGylation; Photobleaching; Single-molecule; TIRF microscopy
Substance Nomenclature: 3WJQ0SDW1A (Polyethylene Glycols)
0 (Silanes)
0 (Fluorescent Dyes)
Entry Date(s): Date Created: 20260109 Date Completed: 20260625 Latest Revision: 20260625
Update Code: 20260626
PubMed Central ID: PMC12933383
DOI: 10.1016/j.ymeth.2026.01.003
PMID: 41512980
Database: MEDLINE
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  Data: Rapid and improved surface passivation method for Single-Molecule experiments.
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  Data: <searchLink fieldCode="AU" term="%22Gonneville+AN%22">Gonneville AN</searchLink>; Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA.<br /><searchLink fieldCode="AU" term="%22Ward+AE%22">Ward AE</searchLink>; Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA.<br /><searchLink fieldCode="AU" term="%22Naidoo+NR%22">Naidoo NR</searchLink>; Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA.<br /><searchLink fieldCode="AU" term="%22Barrera+FN%22">Barrera FN</searchLink>; Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA.<br /><searchLink fieldCode="AU" term="%22Lamichhane+R%22">Lamichhane R</searchLink>; Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA. Electronic address: rajan@utk.edu.
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  Data: <searchLink fieldCode="JN" term="%229426302%22">Methods (San Diego, Calif.)</searchLink> [Methods] 2026 Mar; Vol. 247, pp. 95-106. <i>Date of Electronic Publication: </i>2026 Jan 07.
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  Data: <searchLink fieldCode="MM" term="%22Polyethylene+Glycols%22">Polyethylene Glycols*</searchLink>/<searchLink fieldCode="MM" term="%22Polyethylene+Glycols+chemistry%22">chemistry</searchLink> <br /><searchLink fieldCode="MM" term="%22Fluorescence+Resonance+Energy+Transfer%22">Fluorescence Resonance Energy Transfer*</searchLink>/<searchLink fieldCode="MM" term="%22Fluorescence+Resonance+Energy+Transfer+methods%22">methods</searchLink> <br /><searchLink fieldCode="MM" term="%22Single+Molecule+Imaging%22">Single Molecule Imaging*</searchLink>/<searchLink fieldCode="MM" term="%22Single+Molecule+Imaging+methods%22">methods</searchLink><br /><searchLink fieldCode="MH" term="%22Silanes%22">Silanes</searchLink>/<searchLink fieldCode="MH" term="%22Silanes+chemistry%22">chemistry</searchLink> ; <searchLink fieldCode="MH" term="%22Fluorescent+Dyes%22">Fluorescent Dyes</searchLink>/<searchLink fieldCode="MH" term="%22Fluorescent+Dyes+chemistry%22">chemistry</searchLink> ; <searchLink fieldCode="MH" term="%22Microscopy%2C+Fluorescence%22">Microscopy, Fluorescence</searchLink>/<searchLink fieldCode="MH" term="%22Microscopy%2C+Fluorescence+methods%22">methods</searchLink> ; <searchLink fieldCode="MH" term="%22Photobleaching%22">Photobleaching</searchLink> ; <searchLink fieldCode="MH" term="%22Surface+Properties%22">Surface Properties</searchLink>
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  Data: Single-molecule fluorescence experiments are a powerful tool for studying biomolecular interactions, including protein dynamics and oligomerization, protein-protein interactions, and protein-nucleic acid interactions. Biomolecules are commonly immobilized on the microscope surface to extend the observation time. However, non-specific interactions between biomolecules and the surface present a major challenge. The first critical step in these experiments is preparing the surface using polyethylene glycol (PEG) coated slides, which facilitate biomolecule immobilization while minimizing non-specific interactions. The surface treatment typically uses PEG-SVA (Succinimidyl Valerate) coated slides, and the protocol for the treatment is lengthy and time-consuming. To overcome this issue, we have developed a process that uses PEG-Silane to improve efficiency while maintaining reproducibility. Here, we present a one-step, rapid PEGylation methodology that can be completed in minutes rather than hours. We demonstrate its validity and feasibility through single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule photobleaching experiments across various biological samples.<br /> (Copyright © 2026 The Authors. Published by Elsevier Inc. All rights reserved.)
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  Data: <i>Keywords: </i>FRET; PEG-Silane; PEGylation; Photobleaching; Single-molecule; TIRF microscopy
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        Text: English
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      – SubjectFull: Fluorescent Dyes chemistry
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              Text: 2026 Mar
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