Journal
NANO LETTERS
Volume 18, Issue 5, Pages 3185-3192Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.nanolett.8b00875
Keywords
Total internal reflection fluorescence correlation spectroscopy (TIR-FCS); surface binding kinetics; binding rates; DNA hybridization; DNA-PAINT
Categories
Funding
- Gottfried Wilhelm Leibniz Prize by the German Research foundation
- DFG [DFG JU 2957/1-1]
- ERC [680241]
- Max Planck Society
- Max Planck Foundation
- Center for Nanoscience (CeNS)
- excellence cluster Nanosystems Initiative Munich
- [SFB 1032]
- European Research Council (ERC) [680241] Funding Source: European Research Council (ERC)
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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.
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