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Quantifying Reversible Surface Binding via Surface-Integrated Fluorescence Correlation Spectroscopy

MPS-Authors
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Mücksch,  Jonas
Schwille, Petra / Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Max Planck Society;

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Blumhardt,  Philipp
Schwille, Petra / Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Max Planck Society;

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Strauss,  Maximilian T.
Jungmann, Ralf / Molecular Imaging and Bionanotechnology, Max Planck Institute of Biochemistry, Max Planck Society;

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Petrov,  Eugene P.
Schwille, Petra / Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Max Planck Society;

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Jungmann,  Ralf
Jungmann, Ralf / Molecular Imaging and Bionanotechnology, Max Planck Institute of Biochemistry, Max Planck Society;

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Schwille,  Petra
Schwille, Petra / Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Max Planck Society;

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Fulltext (public)

acs.nanolett.8b00875.pdf
(Publisher version), 2MB

Supplementary Material (public)

nl8b00875_si_001.pdf
(Supplementary material), 9MB

Citation

Mücksch, J., Blumhardt, P., Strauss, M. T., Petrov, E. P., Jungmann, R., & Schwille, P. (2018). Quantifying Reversible Surface Binding via Surface-Integrated Fluorescence Correlation Spectroscopy. Nano Letters, 18(5), 3185-3192. doi:10.1021/acs.nanolett.8b00875.


Cite as: https://hdl.handle.net/21.11116/0000-0002-C89A-F
Abstract
We present a simple and versatile single-molecule-based method for the accurate determination of binding rates to surfaces or surface bound receptors. To quantify the reversible surface attachment of fluorescently labeled molecules, we have modified previous schemes for fluorescence correlation spectroscopy with total internal reflection illumination (TIR-FCS) and camera-based detection. In contrast to most modern applications of TIR-FCS, we completely disregard spatial information in the lateral direction. Instead, we perform correlation analysis on a spatially integrated signal, effectively converting the illuminated surface area into the measurement volume. In addition to providing a high surface selectivity, our new approach resolves association and dissociation rates in equilibrium over a wide range of time scales. We chose the transient hybridization of fluorescently labeled single-stranded DNA to the complementary handles of surface-immobilized DNA origami structures as a reliable and well-characterized test system. We varied the number of base pairs in the duplex, yielding different binding times in the range of hundreds of milliseconds to tens of seconds, allowing us to quantify the respective surface affinities and binding rates.