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Fabrication of nanopores with ultrashort single-walled carbon nanotubes inserted in a lipid bilayer

Nature Protocols volume 10, pages 1670–1678 (2015)Cite this article

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Abstract

We describe a protocol for the insertion of ultrashort single-walled carbon nanotubes (SWCNTs) to form nanopores in a Montal-Mueller lipid bilayer. The SWCNTs are designed to bind to a specific analyte of interest; binding will result in the reduction of current in single-channel recording experiments. The first stage of the PROCEDURE is to cut and separate the SWCNTs. We cut long, purified SWCNTs with sonication in concentrated sulfuric acid/nitric acid (3/1). Isolation of ultrashort SWCNTs is carried out by size-exclusion HPLC separation. The second stage is to insert these short SWCNTs into the lipid bilayer. This step requires a microinjection probe made from a glass capillary. The setup for protein nanopore research can be adopted for the single-channel recording experiments without any special treatment. The obtained current traces are of very high quality, showing stable baselines and little background noise. Example procedures are shown for investigating ion transport and DNA translocation through these SWCNT nanopores. This nanopore has potential applications in molecular sensing, nanopore DNA sequencing and early disease diagnosis. For example, we have selectively detected modified 5-hydroxymethylcytosine in single-stranded DNA (ssDNA), which may have implications in screening specific genomic DNA sequences. The protocol takes ∼15 d, including SWCNT purification, cutting and separation, as well as the formation of SWCNT nanopores for DNA analyses.

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Figure 1: Microinjection probe.
Figure 2: HPLC chromatograms of the separation of DNA segments and ultrashort SWCNTs.
Figure 3: Representation of single-channel recording traces and I–V curves.
Figure 4: Conductance distribution of SWCNT nanopores.
Figure 5: ssDNA translocation through SWCNT nanopore and detection of 5hmC site in ssDNA.

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Acknowledgements

This work was funded by the National Basic Research Program of China (973 program, grant no. 2013CB932800), the National Natural Science Foundation of China (grant nos. 21175135, 21375130, 21205119 and 21475132) and the Chinese Academy of Sciences Hundred Talents Program.

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Authors and Affiliations

  1. Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China

    Lei Liu, Jiani Xie, Ting Li & Hai-Chen Wu

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  1. Lei Liu
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  2. Jiani Xie
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Contributions

L.L. and H.-C.W. conceived the project; L.L., J.X. and T.L. performed the experiments; L.L., T.L. and H.-C.W. performed data analysis; and L.L., J.X. and H.-C.W. wrote the paper.

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Correspondence to Hai-Chen Wu.

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The authors declare no competing financial interests.

Supplementary information

Micro-injection probe interaction with the aperture

This movie illustrates how the micro-injection probe is controlled to interact with the PTFE aperture in the absence of buffer solutions. The round aperture is about 100 μm in diameter over which the lipid bilayer forms. The distance between the probe tip and the aperture can be precisely controlled. Starting from 40 s, the micro-injector is shown to inject solutions from the probe syringe. (MOV 3338 kb)

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Liu, L., Xie, J., Li, T. et al. Fabrication of nanopores with ultrashort single-walled carbon nanotubes inserted in a lipid bilayer. Nat Protoc 10, 1670–1678 (2015). https://doi.org/10.1038/nprot.2015.112

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