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Localized detection of ions and biomolecules with a force-controlled scanning nanopore microscope

Nature Nanotechnology volume 14, pages 791–798 (2019)Cite this article

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Abstract

Proteins, nucleic acids and ions secreted from single cells are the key signalling factors that determine the interaction of cells with their environment and the neighbouring cells. It is possible to study individual ion channels by pipette clamping, but it is difficult to dynamically monitor the activity of ion channels and transporters across the cellular membrane. Here we show that a solid-state nanopore integrated in an atomic force microscope can be used for the stochastic sensing of secreted molecules and the activity of ion channels in arbitrary locations both inside and outside a cell. The translocation of biomolecules and ions through the nanopore is observed in real time in live cells. The versatile nature of this approach allows us to detect specific biomolecules under controlled mechanical confinement and to monitor the ion-channel activities of single cells. Moreover, the nanopore microscope was used to image the surface of the nuclear membrane via high-resolution scanning ion conductance measurements.

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Fig. 1: Nanopore fabrication on the AFM cantilevers.
Fig. 2: Localized detection of proteins near the surface under mechanical confinement with a nanopore AFM.
Fig. 3: Extracellular recording from single cells.
Fig. 4: Extracellular recording from MEFs with and without Fn expression.
Fig. 5: Current maps for the cells with a controlled number of ion channels and the ratio of class II to class I events.
Fig. 6: Intracellular recording with nanopore AFM.

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All the data needed to evaluate the conclusions in the paper are present in the paper. Additional data and other findings of this study are available from the corresponding authors upon reasonable request.

References

  1. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. Molecular Biology of the Cell (Garland Science, 2009).

  2. Kollmannsberger, P., Bidan, C. M., Dunlop, J. W. C., Fratzl, P. & Vogel, V. Tensile forces drive a reversible fibroblast-to-myofibroblast transition during tissue growth in engineered clefts. Sci. Adv. 4, eaao4881 (2018).

    Article  Google Scholar 

  3. Catterall, W. A. From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 26, 13–25 (2000).

    Article  CAS  Google Scholar 

  4. Robert, C., Hue, I., McGraw, S., Gagné, D. & Sirard, M.-A. Quantification of cyclin B1 and p34(cdc2) in bovine cumulus–oocyte complexes and expression mapping of genes involved in the cell cycle by complementary DNA macroarrays. Biol. Reprod. 67, 1456–1464 (2002).

    Article  CAS  Google Scholar 

  5. McDonald, M. P. et al. Visualizing single-cell secretion dynamics with single-protein sensitivity. Nano Lett. 18, 513–519 (2018).

    Article  CAS  Google Scholar 

  6. Actis, P. et al. Compartmental genomics in living cells revealed by single-cell nanobiopsy. ACS Nano 8, 546–553 (2014).

    Article  CAS  Google Scholar 

  7. Gatterdam, V. et al. Focal molography is a new method for the in situ analysis of molecular interactions in biological samples. Nat. Nanotechnol. 12, 1089–1095 (2017).

    Article  CAS  Google Scholar 

  8. Li, X. et al. Label-free optofluidic nanobiosensor enables real-time analysis of single-cell cytokine secretion. Small 14, 1800698 (2018).

    Article  Google Scholar 

  9. Kennedy, E. et al. Method for dynamically detecting secretions from single cells using a nanopore. Nano Lett. 18, 4263–4272 (2018).

    Article  CAS  Google Scholar 

  10. Meister, A. et al. FluidFM: combining atomic force microscopy and nanofluidics in a universal liquid delivery system for single cell applications and beyond. Nano Lett. 9, 2501–2507 (2009).

    Article  CAS  Google Scholar 

  11. Dorwling-Carter, L., Aramesh, M., Han, H., Zambelli, T. & Momotenko, D. Combined ion conductance and atomic force microscope for fast simultaneous topographical and surface charge imaging. Anal. Chem. 90, 11453–11460 (2018).

    Article  CAS  Google Scholar 

  12. Li, J. et al. Ion-beam sculpting at nanometre length scales. Nature 412, 166–169 (2001).

    Article  CAS  Google Scholar 

  13. Aramesh, M. Ion-beam sculpting of nanowires. Phys. Status Solidi Rapid Res. Lett. 12, 1700333 (2018).

    Article  Google Scholar 

  14. Aramesh, M., Mayamei, Y., Wolff, A. & Ostrikov, K. Superplastic nanoscale pore shaping by ion irradiation. Nat. Commun. 9, 835 (2018).

    Article  Google Scholar 

  15. Ossola, D. et al. Simultaneous scanning ion conductance microscopy and atomic force microscopy with microchanneled cantilevers. Phys. Rev. Lett. 115, 238103 (2015).

    Article  Google Scholar 

  16. Lan, W. J., Kubeil, C., Xiong, J. W., Bund, A. & White, H. S. Effect of surface charge on the resistive pulse waveshape during particle translocation through glass nanopores. J. Phys. Chem. C 118, 2726–2734 (2014).

    Article  CAS  Google Scholar 

  17. Ivanov, A. P. et al. On-demand delivery of single DNA molecules using nanopipets. ACS Nano 9, 3587–3594 (2015).

    Article  CAS  Google Scholar 

  18. Klausen, L. H., Fuhs, T. & Dong, M. Mapping surface charge density of lipid bilayers by quantitative surface conductivity microscopy. Nat. Commun. 7, 12447 (2016).

    Article  CAS  Google Scholar 

  19. Perry, D., Al Botros, R., Momotenko, D., Kinnear, S. L. & Unwin, P. R. Simultaneous nanoscale surface charge and topographical mapping. ACS Nano 9, 7266–7276 (2015).

    Article  CAS  Google Scholar 

  20. Freedman, K. J. et al. Nanopore sensing at ultra-low concentrations using single-molecule dielectrophoretic trapping. Nat. Commun. 7, 10217 (2016).

    Article  CAS  Google Scholar 

  21. Steinbock, L. J., Steinbock, J. F. & Radenovic, A. Controllable shrinking and shaping of glass nanocapillaries under electron irradiation. Nano Lett. 13, 1717–1723 (2013).

    Article  CAS  Google Scholar 

  22. Dorwling-Carter, L. et al. Simultaneous scanning ion conductance and atomic force microscopy with a nanopore: effect of the aperture edge on the ion current images. J. Appl. Phys. 124, 174902 (2018).

    Article  Google Scholar 

  23. Nadappuram, B. P. et al. Nanoscale tweezers for single-cell biopsies. Nat. Nanotechnol. 14, 80 (2018).

    Article  Google Scholar 

  24. Pud, S. et al. Mechanical trapping of DNA in a double-nanopore system. Nano Lett. 16, 8021–8028 (2016).

    Article  CAS  Google Scholar 

  25. Wanunu, M., Morrison, W., Rabin, Y., Grosberg, A. Y. & Meller, A. Electrostatic focusing of unlabelled DNA into nanoscale pores using a salt gradient. Nat. Nanotechnol. 5, 160–165 (2010).

    Article  CAS  Google Scholar 

  26. Feng, J. et al. Identification of single nucleotides in MoS2 nanopores. Nat. Nanotechnol. 10, 1070–1076 (2015).

    Article  CAS  Google Scholar 

  27. Briggs, K. et al. DNA translocations through nanopores under nanoscale preconfinement. Nano Lett. 18, 660–668 (2018).

    Article  CAS  Google Scholar 

  28. Maglia, G., Restrepo, M. R., Mikhailova, E. & Bayley, H. Enhanced translocation of single DNA molecules through ɑ-hemolysin nanopores by manipulation of internal charge. Proc. Natl Acad. Sci. USA 105, 19720–19725 (2008).

    Article  CAS  Google Scholar 

  29. Kasianowicz, J. J., Brandin, E., Branton, D. & Deamer, D. W. Characterization of individual polynucleotide molecules using a membrane channel. Proc. Natl Acad. Sci. USA 93, 13770–13773 (1996).

    Article  CAS  Google Scholar 

  30. Meller, A., Nivon, L. & Branton, D. Voltage-driven DNA translocations through a nanopore. Phys. Rev. Lett. 86, 3435–3438 (2001).

    Article  CAS  Google Scholar 

  31. Balme, S. et al. Influence of adsorption on proteins and amyloid detection by silicon nitride nanopore. Langmuir 32, 8916–8925 (2016).

    Article  CAS  Google Scholar 

  32. Toyoda, Y. et al. Genome-scale single-cell mechanical phenotyping reveals disease-related genes involved in mitotic rounding. Nat. Commun. 8, 1266 (2017).

    Article  Google Scholar 

  33. Klotzsch, E., Stiegler, J., Ben-Ishay, E. & Gaus, K. Do mechanical forces contribute to nanoscale membrane organisation in T cells? Biochim. Biophys. Acta Mol. Cell Res. 1853, 822–829 (2015).

    Article  CAS  Google Scholar 

  34. Karner, A. et al. Tuning membrane protein mobility by confinement into nanodomains. Nat. Nanotechnol. 12, 260–266 (2017).

    Article  CAS  Google Scholar 

  35. Aramesh, M., Shimoni, O., Ostrikov, K., Prawer, S. & Cervenka, J. Surface charge effects in protein adsorption on nanodiamonds. Nanoscale 7, 5726–5736 (2015).

    Article  CAS  Google Scholar 

  36. Aramesh, M., Tran, P. A., Ostrikov, K. & Prawer, S. Conformal nanocarbon coating of alumina nanocrystals for biosensing and bioimaging. Carbon 122, 422–427 (2017).

    Article  CAS  Google Scholar 

  37. Konradi, R., Pidhatika, B., Mühlebach, A. & Textor, M. Poly-2-methyl-2-oxazoline: a peptide-like polymer for protein-repellent surfaces. Langmuir 24, 613–616 (2008).

    Article  CAS  Google Scholar 

  38. Weydert, S. et al. Easy to apply polyoxazoline-based coating for precise and long-term control of neural patterns. Langmuir 33, 8594–8605 (2017).

    Article  CAS  Google Scholar 

  39. 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 

  40. Blondel, V. D., Guillaume, Jean-Loup, Lambiotte, R. & Lefebvre, E. Fast unfolding of communities in large networks. J. Stat. Mech. Theory Exp. 2008, P10008 (2008).

    Article  Google Scholar 

  41. Sakai, T. et al. Plasma fibronectin supports neuronal survival and reduces brain injury following transient focal cerebral ischemia but is not essential for skin-wound healing and hemostasis. Nat. Med. 7, 324–330 (2001).

    Article  CAS  Google Scholar 

  42. Ruoslahti, E. Fibronectin and its receptors. Annu. Rev. Biochem. 57, 375–413 (1988).

    Article  CAS  Google Scholar 

  43. Gottlieb, P. A. in Current Topics in Membranes (ed. Gottlieb, P. A.) 1–36 (Current Topics in Membranes Vol. 79, Academic, 2017).

  44. Guillaume-Gentil, O. et al. Force-controlled fluidic injection into single cell nuclei. Small 9, 1904–1907 (2013).

    Article  CAS  Google Scholar 

  45. Larkin, J., Henley, R. Y., Muthukumar, M., Rosenstein, J. K. & Wanunu, M. High-bandwidth protein analysis using solid-state nanopores. Biophys. J. 106, 696–704 (2014).

    Article  CAS  Google Scholar 

  46. Wei, R., Gatterdam, V., Wieneke, R., Tampé, R. & Rant, U. Stochastic sensing of proteins with receptor-modified solid-state nanopores. Nat. Nanotechnol. 7, 257–263 (2012).

    Article  CAS  Google Scholar 

  47. 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 

  48. Ren, R. et al. Nanopore extended field-effect transistor for selective single-molecule biosensing. Nat. Commun. 8, 586 (2017).

    Article  Google Scholar 

  49. Guillaume-Gentil, O. et al. Tunable single-cell extraction for molecular analyses. Cell 166, 506–517 (2016).

    Article  CAS  Google Scholar 

  50. Misiunas, K., Ermann, N. & Keyser, U. F. QuipuNet: convolutional neural network for single-molecule nanopore sensing. Nano Lett. 18, 4040–4045 (2018).

    Article  CAS  Google Scholar 

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Acknowledgements

This project received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Individual Fellowship (project reference 706930) and the Career Seed Grant from ETH Zürich (project reference 0-20440-18) to M.A. The work was partially funded by the EUROSTARS project grant E!11644. D.M. is supported by the Swiss National Science Foundation Ambizione grant PZ00P2_174217/1. I.S. is thankful to Empa for financial support and to the Swiss National Science Foundation for support in equipment procurement (R’Equip 206021_133823). This work was supported by the Human Frontiers Science Program RGY0065/2017 to E.K.

The authors acknowledge the valuable and insightful discussions with C. Frei, V. Gatterdam, O. Guillaume-Gentil and V. Vogel. We are grateful to members of our laboratories for assistance during the project. We appreciate the technical assistance from S. Wheeler. The PAcrAm-g-(PMOXA, NH2, Si) polymer was a kind donation of SuSoS AG, Switzerland. We are grateful to the Martinac lab and M. Vassalli for valuable discussions and the provided materials. The authors acknowledge support of the Scientific Center for Optical and Electron Microscopy ScopeM of the Swiss Federal Institute of Technology ETHZ.

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Authors and Affiliations

  1. Laboratory of Biosensors and Bioelectronics, Institute for Biomedical Engineering, ETH Zürich, Zürich, Switzerland

    Morteza Aramesh, Csaba Forró, Livie Dorwling-Carter, Ines Lüchtefeld, Tilman Schlotter, Stephan J. Ihle, Dmitry Momotenko, Tomaso Zambelli & János Vörös

  2. Laboratory of Applied Mechanobiology, Department for Health Sciences and Technology, ETH Zürich, Zürich, Switzerland

    Morteza Aramesh, Vahid Hosseini & Enrico Klotzsch

  3. Laboratory for Transport at Nanoscale Interfaces, Swiss Federal Laboratories for Materials Science and Technology (Empa), Dübendorf, Switzerland

    Ivan Shorubalko

  4. Institute for Biology, Experimental Biophysics/ Mechanobiology, Humboldt University of Berlin, Berlin, Germany

    Enrico Klotzsch

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  1. Morteza Aramesh
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Contributions

M.A. and J.V. designed the experiments. M.A., C.F., L.D.-C., I.L., T.S., S.J.I., I.S. and V.H. performed the experiments. M.A., C.F. and S.J.I. performed the statistical analysis and coding with support from J.V. All the authors discussed the results and commented on the manuscript. M.A. wrote the manuscript with support from J.V.

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Correspondence to Morteza Aramesh or János Vörös.

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

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Aramesh, M., Forró, C., Dorwling-Carter, L. et al. Localized detection of ions and biomolecules with a force-controlled scanning nanopore microscope. Nat. Nanotechnol. 14, 791–798 (2019). https://doi.org/10.1038/s41565-019-0493-z

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