Plasmonic scattering imaging of single proteins and binding kinetics

Abstract

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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Fig. 1: Setup and principle of PSM.
Fig. 2: Validation and calibration of PSM with polystyrene nanoparticles of different diameters.
Fig. 3: Imaging single proteins with PSM.
Fig. 4: PSM identification of single proteins using antibodies.
Fig. 5: Single-molecule measurement of binding kinetics with PSM.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.

Code availability

MATLAB and ImageJ (Fiji) codes used for image processing are provided in Supplementary Notes 1419.

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Acknowledgements

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.

Author information

Affiliations

Authors

Contributions

P.Z. performed the experiments and data analysis. G.M. contributed to the protein studies. W.D. performed some of the preliminary experiments with nanoparticles. Z.W. prepared gold-coated glass slides and performed atomic force microscopy measurements. S.W. contributed to the design and construction of the optical setup. N.T. and S.W. conceived and supervised the project. P.Z., G.M., S.W. and N.T. wrote the manuscript. All authors reviewed and commented on the manuscript.

Corresponding author

Correspondence to Shaopeng Wang.

Ethics declarations

Competing interests

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.

Additional information

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 information

Supplementary Information

Supplementary Figs. 1–7, Tables 1 and 2 and Notes 1–19.

Reporting Summary

Supplementary Video 1

Dynamic binding of single 26-nm nanoparticles on bare gold.

Supplementary Video 2

Dynamic binding of single IgA and IgM proteins on bare gold.

Supplementary Video 3

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.

Supplementary Video 4

Differential video showing the on-off process of one IgA protein.

Supplementary Video 5

Three different behaviors of binding of individual IgA molecules.

Source data

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

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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

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