Surface passivation with a perfluoroalkane brush improves the precision of single-molecule measurements

Bueno-Alejo, C. J. et al. (2022) Surface passivation with a perfluoroalkane brush improves the precision of single-molecule measurements. ACS Applied Materials and Interfaces, 14(44), pp. 49604-49616. (doi: 10.1021/acsami.2c16647) (PMID:36306432) (PMCID:PMC9650645)

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Single-molecule imaging is invaluable for investigating the heterogeneous behavior and interactions of biological molecules. However, an impediment to precise sampling of single molecules is the irreversible adsorption of components onto the surfaces of cover glasses. This causes continuous changes in the concentrations of different molecules dissolved or suspended in the aqueous phase from the moment a sample is dispensed, which will shift, over time, the position of chemical equilibria between monomeric and multimeric components. Interferometric scattering microscopy (iSCAT) is a technique in the single-molecule toolkit that has the capability to detect unlabeled proteins and protein complexes both as they adsorb onto and desorb from a glass surface. Here, we examine the reversible and irreversible interactions between a number of different proteins and glass via analysis of the adsorption and desorption of protein at the single-molecule level. Furthermore, we present a method for surface passivation that virtually eliminates irreversible adsorption while still ensuring the residence time of molecules on surfaces is sufficient for detection of adsorption by iSCAT. By grafting high-density perfluoroalkane brushes on cover-glass surfaces, we observe approximately equal numbers of adsorption and desorption events for proteins at the measurement surface (±1%). The fluorous–aqueous interface also prevents the kinetic trapping of protein complexes and assists in establishing a thermodynamic equilibrium between monomeric and multimeric components. This surface passivation approach is valuable for in vitro single-molecule experiments using iSCAT microscopy because it allows for continuous monitoring of adsorption and desorption of protein without either a decline in detection events or a change in sample composition due to the irreversible binding of protein to surfaces.

Item Type:Articles
Additional Information:This work was supported by a Strategic Longer and Larger Grant: Frontier Bioscience from the Biotechnology and Biological Sciences Research Council (How do RNA-binding proteins control splice site selection? BB/T000627/1, awarded to I.C.E., A.J.H., A.W.C., G.A.B., and C.D.). A.W.C. is would also like to acknowledge support from the Leverhulme Trust (RPG-2018- 149), the Engineering and Physical Sciences Research Council (EP/V030515/1), and the Biotechnology and Biological Sciences Research Council (BB/N016734/1).
Glasgow Author(s) Enlighten ID:Santana Vega, Dr Marina and Clark, Professor Alasdair and Burley, Dr Glenn
Authors: Bueno-Alejo, C. J., Santana Vega, M., Chaplin, A. K., Farrow, C., Axer, A., Burley, G. A., Dominguez, C., Kara, H., Paschalis, V., Tubasum, S., Eperon, I. C., Clark, A. W., and Hudson, A. J.
College/School:College of Science and Engineering > School of Engineering > Biomedical Engineering
Journal Name:ACS Applied Materials and Interfaces
Publisher:American Chemical Society
ISSN (Online):1944-8252
Published Online:28 October 2022
Copyright Holders:Copyright © 2022 The Authors
First Published:First published in ACS Applied Materials and Interfaces 14(44): 49604-49616
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
301408Macromolecular construction of nucleic acid networks directed by the flourous effect: A new paradigm for the programmable assembly of molecular informationAlasdair ClarkLeverhulme Trust (LEVERHUL)RPG-2018-149/ 170977ENG - Biomedical Engineering
309846Decentralised water technologiesWilliam SloanEngineering and Physical Sciences Research Council (EPSRC)EP/V030515/1ENG - Infrastructure & Environment
172826DNA-directed construction of three-dimensional photosynthetic assembliesAlasdair ClarkBiotechnology and Biological Sciences Research Council (BBSRC)BB/N016734/1ENG - Biomedical Engineering