Measuring the binding kinetics of single proteins represents one of the most important and challenging tasks in protein analysis. Here we show that this is possible using a surface plasmon resonance (SPR) scattering technique. SPR is a popular label-free detection technology because of its extraordinary sensitivity, but it has never been used for imaging single proteins. We overcome this limitation by imaging scattering of surface plasmonic waves by proteins. This allows us to image single proteins, measure their sizes and identify them based on their specific binding to antibodies. We further show that it is possible to quantify protein binding kinetics by counting the binding of individual molecules, providing a digital method to measure binding kinetics and analyze heterogeneity of protein behavior. We anticipate that this imaging method will become an important tool for single protein analysis, especially for low volume samples, such as single cells.
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The data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.
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We are grateful for financial support from the Gordon and Betty Moore Foundation (N.T.) and the National Institute of General Medical Sciences of the National Institutes of Health grant R01GM107165 (S.W.). We acknowledge the use of facilities within the ASU NanoFab supported in part by NSF program NNCI-ECCS-1542160. The content is solely the responsibility of the authors and does not necessarily represent the official views of the sponsors.
A US provisional patent application (62/975,473) has been filed by Arizona Board of Regents on behalf of Arizona State University for single-molecule imaging based on an early draft of this article. Inventors are N.T., S.W. and P.Z.
Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figs. 1–7, Tables 1 and 2 and Notes 1–19.
Dynamic binding of single 26-nm nanoparticles on bare gold.
Dynamic binding of single IgA and IgM proteins on bare gold.
Identification of single IgA and IgM proteins on anti-IgA antibody coated gold, and identification of single anti-CaM antibody and IgA proteins on CaM-coated gold.
Differential video showing the on-off process of one IgA protein.
Three different behaviors of binding of individual IgA molecules.
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Zhang, P., Ma, G., Dong, W. et al. Plasmonic scattering imaging of single proteins and binding kinetics. Nat Methods 17, 1010–1017 (2020). https://doi.org/10.1038/s41592-020-0947-0