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.
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Data are available at https://doi.org/10.5281/zenodo.5059802.
Code for data analysis of nanopore recordings as described herein are available at https://doi.org/10.5281/zenodo.5059802.
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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.).
The authors declare no competing interests.
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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
Nature Communications (2022)
Nature Nanotechnology (2022)
Nature Nanotechnology (2021)