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Synthesis, lipid membrane incorporation, and ion permeability testing of carbon nanotube porins

Nature Protocols volume 11, pages 2029–2047 (2016)Cite this article

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Abstract

Carbon nanotube porins (CNTPs) are 10- to 20-nm-long segments of lipid-stabilized single-walled carbon nanotubes (CNTs) that can be inserted into phospholipid membranes to form nanometer-scale-diameter pores that approximate the geometry and many key transport characteristics of biological membrane channels. We describe protocols for CNTP synthesis by ultrasound-assisted cutting of long CNTs in the presence of lipid amphiphiles, and for validation of CNTP incorporation into a lipid membrane using a proton permeability assay. In addition, we describe protocols for measuring conductance of individual CNTPs in planar lipid bilayers and plasma membranes of live cells. The protocol for the preparation and testing of the CNTPs in vesicle systems takes 3 d, and single CNTP conductance measurements take 2–5 h. The CNTPs produced by this cutting protocol remain stable and active for at least 10–12 weeks.

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Figure 1: Schematic illustrating the steps required for the CNTP synthesis and self-assembly into liposomes for ion transport studies.
Figure 2: Customized ultrasonicator setup for cutting carbon nanotubes (Steps 12–21).
Figure 3: TEM and cryo-TEM imaging of CNTPs.
Figure 4: Conductance measurements of CNTP incorporation into synthetic lipid membranes.
Figure 5: Conductance measurements of CNTP incorporation into plasma membranes of live cells.
Figure 6: Changes of membrane current and conductance produced by CNTP incorporation.

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Acknowledgements

We thank K. Kim, J. Zhang, and J. Geng for developing some of the initial CNTP synthesis and testing protocols; L. Comolli, F.I. Allen, and Y. Wang for acquiring the cryo-TEM and HR-TEM images of CNTPs; and A.V. Shnyrova for assistance in the development of CNTP-in-BLM protocols and critical reading of the manuscript. Parts of this work were supported by the US Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering (characterization and transport studies), and the LDRD program at LLNL, 12-ERD-073 (synthesis). Work at LLNL was performed under the auspices of the US Department of Energy under contract DE-AC52-07NA27344. Work at the Molecular Foundry was supported by the Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under contract DE-AC02-05CH11231. Work at the Biofisika Institute (CSIC, UPV/EHU) was supported by the Spanish Ministry of Economy and Competitiveness (grant BIO2013-49843-EXP).

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Authors and Affiliations

  1. Biosciences and Biotechnology Division, Physics and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California, USA

    Ramya H Tunuguntla & Aleksandr Noy

  2. Biofisika Institute (CSIC, UPV/EHU), University of the Basque Country, Leioa, Spain

    Artur Escalada & Vadim A Frolov

  3. Department of Biochemistry and Molecular Biology, University of the Basque Country, Leioa, Spain

    Artur Escalada & Vadim A Frolov

  4. Ikerbasque, Basque Foundation for Science, Bilbao, Spain

    Vadim A Frolov

  5. School of Natural Sciences, University of California Merced, Merced, California, USA

    Aleksandr Noy

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  1. Ramya H Tunuguntla
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Contributions

R.H.T. and A.N. developed the protocol for optimized CNTP preparation procedure and assays for CNTP characterization. A.E. and V.A.F. developed the protocols for optimized single-CNTP conductance measurement assays and CNTP incorporation into live cells. All authors contributed to data analysis, discussion, and manuscript preparation.

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Correspondence to Aleksandr Noy.

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Tunuguntla, R., Escalada, A., A Frolov, V. et al. Synthesis, lipid membrane incorporation, and ion permeability testing of carbon nanotube porins. Nat Protoc 11, 2029–2047 (2016). https://doi.org/10.1038/nprot.2016.119

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