Plasmonic scattering imaging of single proteins and binding kinetics


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.


  1. 1.

    Santos, R. et al. A comprehensive map of molecular drug targets. Nat. Rev. Drug Discov. 16, 19–34 (2017).

    CAS  PubMed  Google Scholar 

  2. 2.

    Polanski, M. & Anderson, N. L. A list of candidate cancer biomarkers for targeted proteomics. Biomark. Insights 1, 1–48 (2007).

    PubMed  PubMed Central  Google Scholar 

  3. 3.

    Homola, J. Present and future of surface plasmon resonance biosensors. Anal. Bioanal. Chem. 377, 528–539 (2003).

    CAS  PubMed  Google Scholar 

  4. 4.

    Phillips, K. S. & Cheng, Q. Recent advances in surface plasmon resonance based techniques for bioanalysis. Anal. Bioanal. Chem. 387, 1831–1840 (2007).

    CAS  PubMed  Google Scholar 

  5. 5.

    Masson, J.-F. Surface plasmon resonance clinical biosensors for medical diagnostics. ACS Sens. 2, 16–30 (2017).

    CAS  PubMed  Google Scholar 

  6. 6.

    Fang, Y. et al. Plasmonic imaging of electrochemical reactions of single nanoparticles. Acc. Chem. Res. 49, 2614–2624 (2016).

    CAS  PubMed  Google Scholar 

  7. 7.

    Huang, B., Yu, F. & Zare, R. N. Surface plasmon resonance imaging using a high numerical aperture microscope objective. Anal. Chem. 79, 2979–2983 (2007).

    CAS  PubMed  Google Scholar 

  8. 8.

    Wang, W. et al. Single cells and intracellular processes studied by a plasmonic-based electrochemical impedance microscopy. Nat. Chem. 3, 249–255 (2011).

    PubMed  PubMed Central  Google Scholar 

  9. 9.

    Wang, W. et al. Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells. Nat. Chem. 4, 846–853 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Yang, Y. Z. et al. Label-free tracking of single organelle transportation in cells with nanometer precision using a plasmonic imaging technique. Small 24, 2878–2884 (2015).

    Google Scholar 

  11. 11.

    Wang, S. P. et al. Label-free imaging, detection, and mass measurement of single viruses by surface plasmon resonance. Proc. Natl Acad. Sci. USA 107, 16028–16032 (2010).

    CAS  PubMed  Google Scholar 

  12. 12.

    Shan, X. N. et al. Imaging the electrocatalytic activity of single nanoparticles. Nat. Nanotechnol. 7, 668–672 (2012).

    CAS  PubMed  Google Scholar 

  13. 13.

    Fang, Y. M. et al. Intermittent photocatalytic activity of single CdS nanoparticles. Proc. Natl Acad. Sci. USA 114, 10566–10571 (2017).

    CAS  PubMed  Google Scholar 

  14. 14.

    Yang, Y. T. et al. Interferometric plasmonic imaging and detection of single exosomes. Proc. Natl Acad. Sci. USA 115, 10275–10280 (2018).

    CAS  PubMed  Google Scholar 

  15. 15.

    Vollmer, F. & Arnold, S. Whispering-gallery-mode biosensing: label-free detection down to single molecules. Nat. Methods 5, 591–596 (2008).

    CAS  PubMed  Google Scholar 

  16. 16.

    Zijlstra, P., Paulo, P. M. R. & Orrit, M. Optical detection of single non-absorbing molecules using the surface plasmon resonance of a gold nanorod. Nat. Nanotechnol. 7, 379–382 (2012).

    CAS  PubMed  Google Scholar 

  17. 17.

    Baaske, M. D., Foreman, M. R. & Vollmer, F. Single-molecule nucleic acid interactions monitored on a label-free microcavity biosensor platform. Nat. Nanotechnol. 9, 933–939 (2014).

    CAS  PubMed  Google Scholar 

  18. 18.

    Mauranyapin, N. P., Madsen, L. S., Taylor, M. A., Waleed, M. & Bowen, W. P. Evanescent single-molecule biosensing with quantum-limited precision. Nat. Photonics 11, 477–481 (2017).

    CAS  Google Scholar 

  19. 19.

    Zheng, Y. H. et al. Reversible gating of smart plasmonic molecular traps using thermoresponsive polymers for single-molecule detection. Nat. Commun. 6, 8797 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Gaiduk, A., Yorulmaz, M., Ruijgrok, P. V. & Orrit, M. Room-temperature detection of a single molecule’s absorption by photothermal contrast. Science 330, 353–356 (2010).

    CAS  PubMed  Google Scholar 

  21. 21.

    Arroyo, J. O., Cole, D. & Kukura, P. Interferometric scattering microscopy and its combination with single-molecule fluorescence imaging. Nat. Protoc. 11, 617–633 (2016).

    Google Scholar 

  22. 22.

    Young, G. et al. Quantitative mass imaging of single biological macromolecules. Science 360, 423–427 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Liu, X. W. et al. Plasmonic-based electrochemical impedance imaging of electrical activities in single cells. Angew. Chem. 56, 8855–8859 (2017).

    CAS  Google Scholar 

  24. 24.

    Lu, J. & Li, J. H. Label-free imaging of dynamic and transient calcium signaling in single cells. Angew. Chem. 54, 13576–13580 (2015).

    CAS  Google Scholar 

  25. 25.

    Shan, X. N., Patel, U., Wang, S. P., Iglesias, R. & Tao, N. J. Imaging local electrochemical current via surface plasmon resonance. Science 327, 1363–1366 (2010).

    CAS  PubMed  Google Scholar 

  26. 26.

    Yu, H., Shan, X. N., Wang, S. P., Chen, H. Y. & Tao, N. J. Molecular scale origin of surface plasmon resonance biosensors. Anal. Chem. 86, 8992–8997 (2014).

    CAS  PubMed  Google Scholar 

  27. 27.

    Kretschmann, M. Decay of non radiative surface plasmons into light on rough silver films. Comparison of experimental and theoretical results. Opt. Commun. 6, 185–187 (1972).

    Google Scholar 

  28. 28.

    Bozhevolnyi, S. I. & Coello, V. Elastic scattering of surface plasmon polaritons: modeling and experiment. Phys. Rev. B 58, 10899–10910 (1998).

    CAS  Google Scholar 

  29. 29.

    Shchegrov, A. V., Novikov, I. V. & Maradudin, A. A. Scattering of surface plasmon polaritions by a circularly symmetric surface defect. Phys. Rev. Lett. 78, 4269–4272 (1997).

    CAS  Google Scholar 

  30. 30.

    Sirbuly, D. J., Tao, A., Law, M., Fan, R. & Yang, P. D. Multifunctional nanowire evanescent wave optical sensors. Adv. Mater. 19, 61–66 (2007).

    CAS  Google Scholar 

  31. 31.

    Agnarsson, B. et al. Evanescent light-scattering microscopy for label-free interfacial imaging: from single sub-100 nm vesicles to live cells. ACS Nano 9, 11849–11862 (2015).

    CAS  PubMed  Google Scholar 

  32. 32.

    Cole, D., Young, G., Weigel, A., Sebesta, A. & Kukura, P. Label-free single-molecule imaging with numerical-aperture-shaped interferometric scattering microscopy. ACS Photonics 4, 211–216 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Liebel, M., Hugall, J. T. & Hulst, N. F. Ultrasensitive label-free nanosensing and high-speed tracking of single proteins. Nano Lett. 17, 1277–1281 (2017).

    CAS  PubMed  Google Scholar 

  34. 34.

    Rita, S. Weighing single proteins with light. Nat. Methods 15, 477 (2018).

    Google Scholar 

  35. 35.

    Simpson, W. D. & Volkmar, H. Modulation of the drag forceexerted by microfluidic flow on laser-trapped particles: A new method to assess surface-binding kinetics, analyte size, and solution viscosity. Biophys. J. 114, 692a (2018).

    Google Scholar 

  36. 36.

    Huang, Y. et al. Coherent brightfield microscopy provides the spatiotemporal resolution to study early stage viral infection in live cells. ACS Nano 11, 2575–2585 (2017).

    CAS  PubMed  Google Scholar 

  37. 37.

    Ignatovich, F. V. & Novotny, L. Real-time and background-free detection of nanoscale particles. Phys. Rev. Lett. 96, 013901 (2006).

    PubMed  Google Scholar 

  38. 38.

    Deutsch, B., Beams, R. & Novotny, L. Nanoparticle detection using dual-phase interferometry. Appl. Opt. 49, 4921–4925 (2010).

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Özkumur, E. et al. Label-free and dynamic detection of biomolecular interactions for high-throughput microarray applications. Proc. Natl Acad. Sci. USA 105, 7988–7992 (2008).

    PubMed  Google Scholar 

  40. 40.

    Piliarik, M. & Sandoghdar, V. Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites. Nat. Commun. 5, 4495 (2014).

    CAS  PubMed  Google Scholar 

  41. 41.

    S Abramoff, M. D., Magalhaes, P. J. & Ram, S. J. Image processing with imageJ. Biophotonics Int. 11, 36–42 (2004).

    Google Scholar 

  42. 42.

    Tinevez, J.-Y. et al. TrackMate: an open and extensible platform for single-particle tracking. Methods 115, 80–90 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

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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.

Author information




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).

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