Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Nanopore electro-osmotic trap for the label-free study of single proteins and their conformations

Abstract

Many strategies have been pursued to trap and monitor single proteins over time to detect the molecular mechanisms of these essential nanomachines. Single-protein sensing with nanopores is particularly attractive because it allows label-free high-bandwidth detection on the basis of ion currents. Here we present the nanopore electro-osmotic trap (NEOtrap) that allows trapping and observing single proteins for hours with submillisecond time resolution. The NEOtrap is formed by docking a DNA-origami sphere onto a passivated solid-state nanopore, which seals off a nanocavity of a user-defined size and creates an electro-osmotic flow that traps nearby particles irrespective of their charge. We demonstrate the NEOtrap’s ability to sensitively distinguish proteins on the basis of size and shape, and discriminate between nucleotide-dependent protein conformations, as exemplified by the chaperone protein Hsp90. Given the experimental simplicity and capacity for label-free single-protein detection over the broad bio-relevant time range, the NEOtrap opens new avenues to study the molecular kinetics underlying protein function.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Working principle of the NEOtrap.
Fig. 2: Mass- and shape-dependent single-protein identification with the NEOtrap.
Fig. 3: Pore size-dependence of NEOtrap signals.
Fig. 4: NEOtrap characteristics as a function of voltage and ionic strength.
Fig. 5: Label-free NEOtrap detection of nucleotide-dependent conformational shifts of the chaperone protein Hsp90.

Data availability

Data are available at https://doi.org/10.5281/zenodo.5059802.

Code availability

Code for data analysis of nanopore recordings as described herein are available at https://doi.org/10.5281/zenodo.5059802.

References

  1. Wang, Q., Goldsmith, R. H., Jiang, Y., Bockenhauer, S. D. & Moerner, W. E. Probing single biomolecules in solution using the anti-Brownian electrokinetic (ABEL) trap. Acc. Chem. Res. 45, 1955–1964 (2012).

    Article  CAS  Google Scholar 

  2. Chen, Z. et al. Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments. Proc. Natl Acad. Sci. USA 117, 21328 LP–21321335 (2020).

    Article  CAS  Google Scholar 

  3. Ruggeri, F. et al. Single-molecule electrometry. Nat. Nanotechnol. 12, 488–495 (2017).

    Article  CAS  Google Scholar 

  4. Pang, Y. & Gordon, R. Optical trapping of a single protein. Nano Lett. 12, 402–406 (2012).

    Article  CAS  Google Scholar 

  5. Verschueren, D. V. et al. Label-free optical detection of DNA translocations through plasmonic nanopores. ACS Nano 13, 61–70 (2019).

    Article  CAS  Google Scholar 

  6. Barik, A. et al. Graphene-edge dielectrophoretic tweezers for trapping of biomolecules. Nat. Commun. 8, 1867 (2017).

    Article  CAS  Google Scholar 

  7. Tang, L. et al. Combined quantum tunnelling and dielectrophoretic trapping for molecular analysis at ultra-low analyte concentrations. Nat. Commun. 12, 913 (2021).

    Article  CAS  Google Scholar 

  8. van den Hout, M. et al. Controlling nanopore size, shape and stability. Nanotechnology 21, 115304 (2010).

    Article  CAS  Google Scholar 

  9. Yusko, E. C. et al. Controlling protein translocation through nanopores with bio-inspired fluid walls. Nat. Nano 6, 253–260 (2011).

    Article  CAS  Google Scholar 

  10. Kopatz, I. et al. Packaging of DNA origami in viral capsids. Nanoscale 11, 10160–10166 (2019).

    Article  CAS  Google Scholar 

  11. Plesa, C. et al. Ionic permeability and mechanical properties of DNA origami nanoplates on solid-state nanopores. ACS Nano 8, 35–43 (2014).

    Article  CAS  Google Scholar 

  12. Bell, N. A. W. & Keyser, U. F. Digitally encoded DNA nanostructures for multiplexed, single-molecule protein sensing with nanopores. Nat. Nanotechnol. 11, 645–651 (2016).

    Article  CAS  Google Scholar 

  13. Alibakhshi, M. A. et al. Picomolar fingerprinting of nucleic acid nanoparticles using solid-state nanopores. ACS Nano 11, 9701–9710 (2017).

    Article  CAS  Google Scholar 

  14. Li, C.-Y. et al. Ionic conductivity, structural deformation, and programmable anisotropy of DNA origami in electric field. ACS Nano 9, 1420–1433 (2015).

    Article  CAS  Google Scholar 

  15. Wong, C. T. A. & Muthukumar, M. Polymer capture by electro-osmotic flow of oppositely charged nanopores. J. Chem. Phys. 126, 164903 (2007).

    Article  CAS  Google Scholar 

  16. Firnkes, M., Pedone, D., Knezevic, J., Döblinger, M. & Rant, U. Electrically facilitated translocations of proteins through silicon nitride nanopores: conjoint and competitive action of diffusion, electrophoresis, and electroosmosis. Nano Lett. 10, 2162–2167 (2010).

    Article  CAS  Google Scholar 

  17. Willems, K. et al. Accurate modeling of a biological nanopore with an extended continuum framework. Nanoscale 12, 16775–16795 (2020).

    Article  CAS  Google Scholar 

  18. Mc Hugh, J., Andresen, K. & Keyser, U. F. Cation dependent electroosmotic flow in glass nanopores. Appl. Phys. Lett. 115, 113702 (2019).

    Article  CAS  Google Scholar 

  19. Yusko, E. C. et al. Real-time shape approximation and fingerprinting of single proteins using a nanopore. Nat. Nanotechnol. 12, 360–367 (2017).

    Article  CAS  Google Scholar 

  20. Kowalczyk, S. W., Grosberg, A. Y., Rabin, Y. & Dekker, C. Modeling the conductance and DNA blockade of solid-state nanopores. Nanotechnology 22, 315101 (2011).

    Article  CAS  Google Scholar 

  21. Schopf, F. H., Biebl, M. M. & Buchner, J. The HSP90 chaperone machinery. Nat. Rev. Mol. Cell Biol. 18, 345–360 (2017).

    Article  CAS  Google Scholar 

  22. Taipale, M., Jarosz, D. F. & Lindquist, S. HSP90 at the hub of protein homeostasis: emerging mechanistic insights. Nat. Rev. Mol. Cell Biol. 11, 515–528 (2010).

    Article  CAS  Google Scholar 

  23. Jaeger, A. M. & Whitesell, L. HSP90: enabler of cancer adaptation. Annu. Rev. Cancer Biol. 3, 275–297 (2019).

    Article  Google Scholar 

  24. Ali, M. M. U. et al. Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex. Nature 440, 1013–1017 (2006).

    Article  CAS  Google Scholar 

  25. Schmid, S., Götz, M. & Hugel, T. Single-molecule analysis beyond dwell times: demonstration and assessment in and out of equilibrium. Biophys. J. 111, 1375–1384 (2016).

    Article  CAS  Google Scholar 

  26. Hellenkamp, B., Wortmann, P., Kandzia, F., Zacharias, M. & Hugel, T. Multidomain structure and correlated dynamics determined by self-consistent FRET networks. Nat. Meth 14, 174–180 (2017).

    Article  CAS  Google Scholar 

  27. Southworth, D. R. & Agard, D. A. Species-dependent ensembles of conserved conformational states define the Hsp90 chaperone ATPase cycle. Mol. Cell 32, 631–640 (2008).

    Article  CAS  Google Scholar 

  28. Hessling, M., Richter, K. & Buchner, J. Dissection of the ATP-induced conformational cycle of the molecular chaperone Hsp90. Nat. Struct. Mol. Biol. 16, 287–293 (2009).

    Article  CAS  Google Scholar 

  29. Plesa, C. et al. Fast translocation of proteins through solid state nanopores. Nano Lett. 13, 658–663 (2013).

    Article  CAS  Google Scholar 

  30. Rothman, J. S. & Silver, R. A. NeuroMatic: an integrated open-source software toolkit for acquisition, analysis and simulation of electrophysiological data. Front. Neuroinform. 12, 14 (2018).

    Article  Google Scholar 

  31. Wagenbauer, K. F. et al. How we make DNA origami. Chem. Bio. Chem. 18, 1873–1885 (2017).

    Article  CAS  Google Scholar 

  32. van Ginkel, J. et al. Single-molecule peptide fingerprinting. Proc. Natl Acad. Sci. USA 115, 3338 (2018).

    Article  CAS  Google Scholar 

  33. Fairhead, M., Krndija, D., Lowe, E. D. & Howarth, M. Plug-and-play pairing via defined divalent streptavidins. J. Mol. Biol. 426, 199–214 (2014).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Hsp90 was a gift from B. Hermann and T. Hugel. Avidin was a gift from M. Howarth. ClpP and ClpX plasmids were a gift from C. Joo. We thank X. Shi and A. Fragasso for discussions, E. van der Sluis for discussions and protein purification, M.-Y. Wu and F. Tichelaar for TEM drilling. The work was funded by NWO-I680 (SMPS) and supported by the NWO/OCW Gravitation program NanoFront and the European Research Council Advanced grant no. 883684. S.S. acknowledges the Postdoc.Mobility fellowship no. P400PB_180889 by the Swiss National Science Foundation. This work was supported by a European Research Council Consolidator grant to H.D. (grant agreement no. 724261), the Deutsche Forschungsgemeinschaft through grants provided within the Gottfried-Wilhelm-Leibniz Program (to H.D.).

Author information

Authors and Affiliations

Authors

Contributions

S.S. and C.D. conceived the project. S.S. performed all nanopore experiments, analysed the data and purified proteins. H.D. and P.S. advised on DNA origami, and P.S. folded and characterized it. S.S. wrote the paper with C.D. All authors discussed the results and commented on the paper.

Corresponding author

Correspondence to Cees Dekker.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Nanotechnology thanks Adam Hall and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs 1–9, Table 1, Notes 1–4 and References.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Schmid, S., Stömmer, P., Dietz, H. et al. Nanopore electro-osmotic trap for the label-free study of single proteins and their conformations. Nat. Nanotechnol. 16, 1244–1250 (2021). https://doi.org/10.1038/s41565-021-00958-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41565-021-00958-5

Further reading

Search

Quick links

Find nanotechnology articles, nanomaterial data and patents all in one place. Visit Nano by Nature Research