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.

Digitally encoded DNA nanostructures for multiplexed, single-molecule protein sensing with nanopores

Abstract

The simultaneous detection of a large number of different analytes is important in bionanotechnology research and in diagnostic applications. Nanopore sensing is an attractive method in this regard as the approach can be integrated into small, portable device architectures, and there is significant potential for detecting multiple sub-populations in a sample. Here, we show that highly multiplexed sensing of single molecules can be achieved with solid-state nanopores by using digitally encoded DNA nanostructures. Based on the principles of DNA origami, we designed a library of DNA nanostructures in which each member contains a unique barcode; each bit in the barcode is signalled by the presence or absence of multiple DNA dumbbell hairpins. We show that a 3-bit barcode can be assigned with 94% accuracy by electrophoretically driving the DNA structures through a solid-state nanopore. Select members of the library were then functionalized to detect a single, specific antibody through antigen presentation at designed positions on the DNA. This allows us to simultaneously detect four different antibodies of the same isotype at nanomolar concentration levels.

This is a preview of subscription content, access via your institution

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.

Figure 1: Signal for a single bit formed of dumbbell hairpins.
Figure 2: Design and nanopore measurement of DNA structures with multiple zones of protruding secondary structures.
Figure 3: Multiplexed barcode design and readout efficiency.
Figure 4: Binding-site presentation and analysis for the bound antibody in translocations.
Figure 5: Selective detection of multiple antibodies.

References

  1. Wanunu, M. Nanopores: A journey towards DNA sequencing. Phys. Life Rev. 9, 125–158 (2012).

    Article  Google Scholar 

  2. Li, W. et al. Single protein molecule detection by glass nanopores. ACS Nano 7, 4129–4134 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. 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 

  5. Bayley, H. & Martin, C. R. Resistive-pulse sensing from microbes to molecules. Chem. Rev. 100, 2575–2594 (2000).

    Article  CAS  Google Scholar 

  6. Bayley, H. & Cremer, P. S. Stochastic sensors inspired by biology. Nature 413, 226–230 (2001).

    Article  CAS  Google Scholar 

  7. Braha, O. et al. Designed protein pores as components for biosensors. Chem. Biol. 4, 497–505 (1997).

    Article  CAS  Google Scholar 

  8. Rotem, D., Jayasinghe, L., Salichou, M. & Bayley, H. Protein detection by nanopores equipped with aptamers. J. Am. Chem. Soc. 134, 2781–2787 (2012).

    Article  CAS  Google Scholar 

  9. Movileanu, L., Howorka, S., Braha, O. & Bayley, H. Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore. Nature Biotechnol. 18, 1091–1095 (2000).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Harrington, L., Cheley, S., Alexander, L. T., Knapp, S. & Bayley, H. Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate. Proc. Natl Acad. Sci. USA 110, E4417–E4426 (2013).

    Article  CAS  Google Scholar 

  12. Li, T., Liu, L., Li, Y., Xie, J. & Wu, H.-C. A universal strategy for aptamer-based nanopore sensing through host-guest interactions inside α-hemolysin. Angew. Chem. 127, 7678–7681 (2015).

    Article  Google Scholar 

  13. Kawano, R. et al. Rapid detection of a cocaine-binding aptamer using biological nanopores on a chip. J. Am. Chem. Soc. 133, 8474–8477 (2011).

    Article  CAS  Google Scholar 

  14. Kasianowicz, J. J., Henrickson, S. E., Weetall, H. H. & Robertson, B. Simultaneous multianalyte detection with a nanometer-scale pore. Anal. Chem. 73, 2268–2272 (2001).

    Article  CAS  Google Scholar 

  15. Bell, N. A. W. & Keyser, U. F. Specific protein detection using designed DNA carriers and nanopores. J. Am. Chem. Soc. 137, 2035–2041 (2015).

    Article  CAS  Google Scholar 

  16. Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006).

    Article  CAS  Google Scholar 

  17. Steinbock, L. J., Otto, O., Chimerel, C., Gornall, J. & Keyser, U. F. Detecting DNA folding with nanocapillaries. Nano Lett. 10, 2493–2497 (2010).

    Article  CAS  Google Scholar 

  18. Bell, N. A. W., Muthukumar, M. & Keyser, U. F. Translocation frequency of double-stranded DNA through a solid-state nanopore. Phys. Rev. E 93, 022401 (2016).

  19. Smeets, R. M. M., Keyser, U. F., Dekker, N. H. & Dekker, C. Noise in solid-state nanopores. Proc. Natl Acad. Sci. USA 105, 417–421 (2008).

    Article  CAS  Google Scholar 

  20. Tabard-Cossa, V., Trivedi, D., Wiggin, M., Jetha, N. N. & Marziali, A. Noise analysis and reduction in solid-state nanopores. Nanotechnology 18, 305505 (2007).

    Article  Google Scholar 

  21. Storm, A., Chen, J., Zandbergen, H. & Dekker, C. Translocation of double-strand DNA through a silicon oxide nanopore. Phys. Rev. E 71, 1–10 (2005).

    Article  Google Scholar 

  22. Muthukumar, M. Mechanism of DNA transport through pores. Annu. Rev. Biophys. Biomol. Struct. 36, 435–450 (2007).

    Article  CAS  Google Scholar 

  23. Chen, P. et al. Probing single DNA molecule transport using fabricated nanopores. Nano Lett. 4, 2293–2298 (2004).

    Article  CAS  Google Scholar 

  24. Lu, B., Albertorio, F., Hoogerheide, D. P. & Golovchenko, J. A. Origins and consequences of velocity fluctuations during DNA passage through a nanopore. Biophys. J. 101, 70–79 (2011).

    Article  CAS  Google Scholar 

  25. Saphire, E. O. et al. Contrasting IgG structures reveal extreme asymmetry and flexibility. J. Mol. Biol. 319, 9–18 (2002).

    Article  CAS  Google Scholar 

  26. Preiner, J. et al. IgGs are made for walking on bacterial and viral surfaces. Nature Commun. 5, 1–8 (2014).

    Article  Google Scholar 

  27. Mammen, M., Choi, S.-K. & Whitesides, G. M. Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew. Chem. Int. Ed. 37, 2754–2794 (1998).

    Article  Google Scholar 

  28. Kauert, D. J., Kurth, T., Liedl, T. & Seidel, R. Direct mechanical measurements reveal the material properties of 3D DNA-origami. Nano Lett. 11, 5558–5563 (2011).

    Article  CAS  Google Scholar 

  29. Castro, C. E., Su, H.-J., Marras, A. E., Zhou, L. & Johnson, J. Mechanical design of DNA nanostructures. Nanoscale 7, 5913–5921 (2015).

    Article  CAS  Google Scholar 

  30. McMullen, A., de Haan, H. W., Tang, J. X. & Stein, D. Stiff filamentous virus translocations through solid-state nanopores. Nature Commun. 5, 4171 (2014).

    Article  CAS  Google Scholar 

  31. Marchi, A. N., Saaem, I., Vogen, B. N., Brown, S. & LaBean, T. H. Towards larger DNA origami. Nano Lett 14, 5740–5747 (2014).

    Article  CAS  Google Scholar 

  32. Rosenstein, J. K., Wanunu, M., Merchant, C. A., Drndic, M. & Shepard, K. L. Integrated nanopore sensing platform with sub-microsecond temporal resolution. Nature Methods 9, 487–492 (2012).

    Article  CAS  Google Scholar 

  33. Rodriguez-Larrea, D. & Bayley, H. Multistep protein unfolding during nanopore translocation. Nature Nanotech. 8, 288–295 (2013).

    Article  CAS  Google Scholar 

  34. Rosen, C. B., Rodriguez-Larrea, D. & Bayley, H. Single-molecule site-specific detection of protein phosphorylation with a nanopore. Nature Biotechnol. 32, 179–181 (2014).

    Article  CAS  Google Scholar 

  35. Nivala, J., Marks, D. B. & Akeson, M. Unfoldase-mediated protein translocation through an α-hemolysin nanopore. Nature Biotechnol. 31, 247–250 (2013).

    Article  CAS  Google Scholar 

  36. Nivala, J., Mulroney, L., Li, G., Schreiber, J. & Akeson, M. Discrimination among protein variants using an unfoldase-coupled nanopore. ACS Nano 8, 12365–12375 (2014).

    Article  CAS  Google Scholar 

  37. Fu, J., Liu, M., Liu, Y. & Yan, H. Spatially-interactive biomolecular networks organized by nucleic acid nanostructures. Acc. Chem. Res. 45, 1215–1226 (2012).

    Article  CAS  Google Scholar 

  38. Pippig, D. A., Baumann, F., Strackharn, M., Aschenbrenner, D. & Gaub, H. E. Protein-DNA chimeras for nano assembly. ACS Nano 8, 6551–6555 (2014).

    Article  CAS  Google Scholar 

  39. Bell, N. A. W. et al. Multiplexed ionic current sensing with glass nanopores. Lab Chip 13, 1859–1862 (2013).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank J. Kong and V. Thacker for useful discussions. N.A.W.B. and U.F.K. acknowledge funding from an ERC starting grant (Passmembrane 261101) and an ERC consolidator grant (Designerpores 647144). N.A.W.B. also acknowledges funding from an EPSRC doctoral prize award.

Author information

Authors and Affiliations

Authors

Contributions

N.A.W.B. conceived the idea, N.A.W.B. and U.F.K. designed the experiments. N.A.W.B. performed the experiments and analysed the data, and N.A.W.B. and U.F.K. wrote the manuscript.

Corresponding authors

Correspondence to Nicholas A. W. Bell or Ulrich F. Keyser.

Ethics declarations

Competing interests

The authors have made a patent application related to this work.

Supplementary information

Supplementary information

Supplementary information (PDF 2345 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Bell, N., Keyser, U. Digitally encoded DNA nanostructures for multiplexed, single-molecule protein sensing with nanopores. Nature Nanotech 11, 645–651 (2016). https://doi.org/10.1038/nnano.2016.50

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nnano.2016.50

This article is cited by

Search

Quick links

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