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Fabrication of electron tunneling probes for measuring single-protein conductance

Nature Protocols volume 18, pages 2579–2599 (2023)Cite this article

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Abstract

Studying the electrical properties of individual proteins is a prominent research area in the field of bioelectronics. Electron tunnelling or quantum mechanical tunnelling (QMT) probes can act as powerful tools for investigating the electrical properties of proteins. However, current fabrication methods for these probes often have limited reproducibility, unreliable contact or inadequate binding of proteins onto the electrodes, so better solutions are required. Here, we detail a generalizable and straightforward set of instructions for fabricating simple, nanopipette-based, tunnelling probes, suitable for measuring conductance in single proteins. Our QMT probe is based on a high-aspect-ratio dual-channel nanopipette that integrates a pair of gold tunneling electrodes with a gap of less than 5 nm, fabricated via the pyrolytic deposition of carbon followed by the electrochemical deposition of gold. The gold tunneling electrodes can be functionalized using an extensive library of available surface modifications to achieve single-protein–electrode contact. We use a biotinylated thiol modification, in which a biotin–streptavidin–biotin bridge is used to form the single-protein junction. The resulting protein-coupled QMT probes enable the stable electrical measurement of the same single protein in solution for up to several hours. We also describe the analysis method used to interpret time-dependent single-protein conductance measurements, which can provide essential information for understanding electron transport and exploring protein dynamics. The total time required to complete the protocol is ~33 h and it can be carried out by users trained in less than 24 h.

Key points

  • The single protein-coupled electron tunneling probe fabrication includes laser-assisted capillary pulling, pyrolytic carbon deposition, electrochemical carbon etching, gold deposition, tunneling feedback-controlled electrodeposition, probe stabilization and protein coupling via ligand modification and single-protein junction formation.

  • The probes are characterized using scanning electron microscopy, scanning transmission electron microscopy–energy dispersive spectroscopy, dark-field microscopy, current voltage and chronoamperometry measurements. Single-protein conductance is then achieved via time-dependent single-molecule electrical measurements and their data analysis.

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Fig. 1: Schematic of a protein-QMT coupled probe.
Fig. 2: Fabrication procedure of a protein-QMT probe.
Fig. 3: Schematic of setup used for pyrolytic carbon deposition.
Fig. 4: Electrochemical etching of carbon nanoelectrodes.
Fig. 5: Electrochemical initial deposition of gold on the carbon nanoelectrodes.
Fig. 6: Electrochemical fabrication of the QMT probes.
Fig. 7: Electrical characterization of QMT probes.
Fig. 8: Characterization of the QMT probe.
Fig. 9: Formation of a single SA protein junction with a QMT probe.
Fig. 10: Electrical characterization of the SA-coupled QMT probes.
Fig. 11: Statistical analysis of stochastic conductance switching for single SA-coupled QMT probes at a given bias.
Fig. 12: Statistical analysis of stochastic conductance switching for single SA-coupled QMT probes.
Fig. 13: Protein detection with bare QMT probes.

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Data availability

The main data discussed in this protocol are available in the supporting primary research papers44,45. The raw datasets are too large to be publicly shared but are available for research purposes from the corresponding authors upon reasonable request54,55.

References

  1. Lindsay, S. Ubiquitous electron transport in non-electron transfer proteins. Life 10, 72 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Bostick, C. D. et al. Protein bioelectronics: a review of what we do and do not know. Rep. Prog. Phys. 81, 026601 (2018).

    Article  PubMed  Google Scholar 

  3. Xiang, D., Wang, X., Jia, C., Lee, T. & Guo, X. Molecular-scale electronics: from concept to function. Chem. Rev. 116, 4318–4440 (2016).

    Article  CAS  PubMed  Google Scholar 

  4. Vilan, A., Aswal, D. & Cahen, D. Large-area, ensemble molecular electronics: motivation and challenges. Chem. Rev. 117, 4248–4286 (2017).

    Article  CAS  PubMed  Google Scholar 

  5. Amdursky, N. et al. Electronic transport via proteins. Adv. Mater. 26, 7142–7161 (2014).

    Article  CAS  PubMed  Google Scholar 

  6. Di Ventra, M. & Taniguchi, M. Decoding DNA, RNA and peptides with quantum tunnelling. Nat. Nanotechnol. 11, 117–126 (2016).

    Article  PubMed  Google Scholar 

  7. Choi, Y. et al. Single molecule lysozyme dynamics monitored by an electronic circuit. Science 335, 319–324 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Schmid, S., Stömmer, P., Dietz, H. & Dekker, C. Nanopore electro-osmotic trap for the label-free study of single proteins and their conformations. Nat. Nanotechnol. 16, 1244–1250 (2021).

    Article  CAS  PubMed  Google Scholar 

  9. Guo, C. et al. Tuning electronic transport via hepta-alanine peptides junction by tryptophan doping. Proc. Natl Acad. Sci. USA 39, 10785–10790 (2016).

    Article  Google Scholar 

  10. Greenwood, J. et al. Covalent modification of graphene and graphite using diazonium chemistry: tunable grafting and nanomanipulation. ACS Nano 9, 5520–5535 (2015).

    Article  CAS  PubMed  Google Scholar 

  11. Morpurgo, A. F., Marcus, C. M. & Robinson, D. B. Controlled fabrication of metallic electrodes with atomic separation. Appl. Phys. Lett. 74, 2084–2086 (1999).

    Article  CAS  Google Scholar 

  12. Kervennic, Y. V., Van der Zant, H. S. J., Morpurgo, A. F., Gurevich, L. & Kouwenhoven, L. P. Nanometer-spaced electrodes with calibrated separation. Appl. Phys. Lett. 80, 321–323 (2002).

    Article  CAS  Google Scholar 

  13. Kervennic, Y. V., Vanmaekelbergh, D., Kouwenhoven, L. P. & Van der Zant, H. S. J. Planar nanocontacts with atomically controlled separation. Appl. Phys. Lett. 83, 3782–3784 (2003).

    Article  CAS  Google Scholar 

  14. Bitton, O., Gutman, D. B., Berkovits, R. & Frydman, A. Multiple periodicity in a nanoparticle-based single-electron transistor. Nat. Commun. 8, 402 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Xin, N. et al. Concepts in the design and engineering of single-molecule electronic devices. Nat. Rev. Phys. 1, 211–230 (2019).

    Article  Google Scholar 

  16. Fereiro, J. A. et al. Tunnelling explains efficient electron transport via protein junctions. Proc. Natl Acad. Sci. USA 115, E4577–E4583 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Fereiro, J. A. et al. Protein electronics: chemical modulation of contacts control energy level alignment in gold-azurin-gold junctions. J. Am. Chem. Soc. 140, 13317–13326 (2018).

    Article  CAS  PubMed  Google Scholar 

  18. Zhang, B. & Lindsay, S. Electronic decay length in a protein molecule. Nano Lett. 19, 4017–4022 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhang, B., Ryan, E., Wang, X. & Lindsay, S. Probing bioelectronic connections using streptavidin molecules with modified valency. J. Am. Chem. Soc. 143, 15139–15144 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Albrecht, T. Electrochemical tunnelling sensors and their potential applications. Nat. Commun. 3, 829 (2012).

    Article  CAS  PubMed  Google Scholar 

  21. Fanget, A. et al. Nanopore integrated nanogaps for DNA detection. Nano Lett. 14, 244–249 (2014).

    Article  CAS  PubMed  Google Scholar 

  22. Sepunaru, L., Friedman, N., Pecht, I., Sheves, M. & Cahen, D. Temperature-dependent solid-state electron transport through bacteriorhodopsin: experimental evidence for multiple transport paths through proteins. J. Am. Chem. Soc. 134, 4169–4176 (2012).

    Article  CAS  PubMed  Google Scholar 

  23. Fereiro, J. A., Bendikov, T., Pecht, I., Sheves, M. & Cahen, D. Protein binding and orientation matter: bias-induced conductance switching in a mutated azurin junction. J. Am. Chem. Soc. 142, 19217–19225 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zhang, B. et al. Observation of giant conductance fluctuations in a protein. Nano Futures 1, 035002 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Simmons, J. G. Generalised formula for the electric tunnel effect between similar electrodes separated by a thin insulating film. J. Appl. Phys. 34, 1793–1803 (1963).

    Article  Google Scholar 

  26. Fereiro, J. A. et al. A solid-state protein junction serves as a bias-induced current switch. Angew. Chem. Int. Ed. 58, 11852–11859 (2019).

    Article  CAS  Google Scholar 

  27. Kayser, B. et al. Solid-state electron transport via the protein azurin is temperature-independent down to 4 K. J. Phys. Chem. Lett. 11, 144–151 (2020).

    Article  CAS  PubMed  Google Scholar 

  28. Castaneda Ocampo, O. E. et al. Mechanism of orientation-dependent asymmetric charge transport in tunnelling junctions comprising photosystem I. J. Am. Chem. Soc. 137, 8419–8427 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Mezei, G., Balogh, Z., Magyarkuti, A. & Halbritter, A. Voltage-controlled binary conductance switching in gold-4,4‘-bipyridine-gold single-molecule nanowires. J. Phys. Chem. Lett. 11, 8053–8059 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Zhang, B. et al. Engineering an enzyme for direct electrical monitoring of activity. ACS Nano 14, 1360–1368 (2020).

    Article  CAS  PubMed  Google Scholar 

  31. Zhang, B. et al. Role of contacts in long-range protein conductance. Proc. Natl Acad. Sci. USA 116, 5886–5891 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Tsutsui, M., Shoji, K., Taniguchi, M. & Kawai, T. Formation and self-breaking mechanism of stable atom-sized junctions. Nano Lett. 8, 345–349 (2008).

    Article  CAS  PubMed  Google Scholar 

  33. Dubois, V. et al. Massively parallel fabrication of crack-defined gold break junctions featuring sub-3 nm gaps for molecular devices. Nat. Commun. 9, 3433 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Ivanov, A. P., Freedman, K. J., Kim, M. J., Albrecht, T. & Edel, J. B. High precision fabrication and positioning of nanoelectrodes in a nanopore. ACS Nano 8, 1940–1948 (2014).

    Article  CAS  PubMed  Google Scholar 

  35. Ivanov, A. P. et al. DNA tunnelling detector embedded in a nanopore. Nano Lett. 11, 279–285 (2011).

    Article  CAS  PubMed  Google Scholar 

  36. Jia, C. et al. Covalently bonded single-molecule junctions with stable and reversible photoswitched conductivity. Science 352, 1443–1445 (2016).

    Article  CAS  PubMed  Google Scholar 

  37. Chang, T. H. P. Proximity effect in electron-beam lithography. J. Vac. Sci. Technol. 12, 1271–1275 (1975).

    Article  Google Scholar 

  38. Manheller, M., Trellenkamp, S., Wase, R. & Karthäuser, S. Reliable fabrication of 3 nm gaps between nanoelectrodes by electron-beam lithography. Nanotechnology 23, 125302 (2012).

    Article  PubMed  Google Scholar 

  39. Li, H., Wani, I. H., Hayat, A., Jafri, S. H. M. & Leifer, K. Fabrication of reproducible sub-5 nm nanogaps by a focused ion beam and observation of Fowler–Nordheim tunnelling. Appl. Phys. Lett. 107, 103108–103110 (2015).

    Article  Google Scholar 

  40. Nagase, T., Gamo, K., Kubota, T. & Mashiko, S. Direct fabrication of nano-gap electrodes by focused ion beam etching. Thin Solid Films 499, 279–284 (2006).

    Article  CAS  Google Scholar 

  41. Fereiro, J. A. et al. A solid-state protein junction serves as a bias-induced current switch. Angew. Chem. 131, 11978–11985 (2019).

    Article  Google Scholar 

  42. Guo, X. et al. Covalently bridging gaps in single-walled carbon nanotubes with conducting molecules. Science 311, 356–359 (2006).

    Article  CAS  PubMed  Google Scholar 

  43. Ruiz, M. P. et al. Bioengineering a single-protein junction. J. Am. Chem. Soc. 139, 15337–15346 (2017).

    Article  CAS  PubMed  Google Scholar 

  44. 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  PubMed  PubMed Central  Google Scholar 

  45. Tang, L. et al. Measuring conductance switching in single proteins using quantum tunneling. Sci. Adv. 8, eabm8149 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Nadappuram, B. P. et al. Nanoscale tweezers for single-cell biopsies. Nat. Nanotechnol. 14, 80–88 (2019).

    Article  CAS  PubMed  Google Scholar 

  47. Cadinu, P. et al. Single molecule trapping and sensing using dual nanopores separated by a zeptoliter nanobridge. Nano Lett. 17, 6376–6384 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Xiang, J., Liu, B., Liu, B., Ren, B. & Tian, Z.-Q. A self-terminated electrochemical fabrication of electrode pairs with angstrom-sized gaps. Electrochem. Commun. 8, 577–580 (2006).

    Article  CAS  Google Scholar 

  49. Xiang, J. et al. A controllable electrochemical fabrication of metallic electrodes with a nanometer/angstrom-sized gap using an electric double layer as feedback. Angew. Chem. Int. Ed. Engl. 44, 1265–1268 (2005).

    Article  CAS  PubMed  Google Scholar 

  50. Xia, Y., Xiong, Y., Lim, B. & Skrabalak, S. E. Shape-controlled synthesis of metal nanocrystals: simple chemistry meets complex physics? Angew. Chem. Int. Ed. 48, 60–103 (2009).

    Article  CAS  Google Scholar 

  51. Clausmeyer, J., Actis, P., Córdoba, A. L., Korchev, Y. & Schuhmann, W. Nanosensors for the detection of hydrogen peroxide. Electrochem. Commun. 40, 28–30 (2014).

    Article  CAS  Google Scholar 

  52. Li, C., He, H. & Tao, N. Quantised tunneling current in the metallic nanogaps formed by electrodeposition and etching. Appl. Phys. Lett. 77, 3995–3997 (2000).

    Article  CAS  Google Scholar 

  53. Wang, W., Lee, T. & Reed, M. A. in Nano and Molecular Electronics Handbook (eds Lyshevski, S. E.) (CRC Press, 2007).

  54. Tang, L., Jiang, T. A preparation method for forming a pair of nano-gap electrodes. Chinese patent: CN113406162B,2022-10-21.

  55. Tang, L., Jiang, T. A molecular tunneling detection device with integrated nanopore. Chinese patent: CN113390940B, 2022-10-21.

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Acknowledgements

This research was supported by the National Natural Science Foundation of China (grant nos. 62127818 and 21874119), the Natural Science Foundation of Zhejiang Province (grant no. LR22F050003) and the Fundamental Research Funds for the Central Universities. A.P.I. and J.B.E. acknowledge support from EPSRC grant EP/V049070/1, and Analytical Chemistry Trust Fund grant 600322/05. This project has also received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement nos. 724300 and 875525).

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Authors and Affiliations

  1. State Key Laboratory of Modern Optical Instrumentation, Institute of Quantum Sensing, Interdisciplinary Centre for Quantum Information, College of Optical Science and Engineering, International Research Center for Advanced Photonics, Zhejiang University, Hangzhou, China

    Tao Jiang, Xu Liu & Longhua Tang

  2. Department of Chemistry, Molecular Science Research Hub, Imperial College London, London, UK

    Long Yi, Aleksandar P. Ivanov & Joshua B. Edel

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Contributions

L.T., X.L., L.Y., A.P.I. and J.B.E designed and supervised the research. A.P.I., J.B.E, L.H.T. and T.J. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Longhua Tang.

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Key references using this protocol

Tang, L. et al. Nat. Commun. 12, 913 (2021): https://doi.org/10.1038/s41467-021-21101-x

Tang, L. et al. Sci. Adv. 8, eabm8149 (2022): https://doi.org/10.1126/sciadv.abm8149

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Supplementary Tables 1 and 2 and Figs. 1–5.

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Jiang, T., Yi, L., Liu, X. et al. Fabrication of electron tunneling probes for measuring single-protein conductance. Nat Protoc 18, 2579–2599 (2023). https://doi.org/10.1038/s41596-023-00846-3

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