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High-throughput optical sensing of nucleic acids in a nanopore array

Nature Nanotechnology volume 10, pages 986–991 (2015)Cite this article

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Abstract

Protein nanopores such as α-haemolysin and Mycobacterium smegmatis porin A (MspA) can be used to sequence long strands of DNA at low cost. To provide high-speed sequencing, large arrays of nanopores are required, but current nanopore sequencing methods rely on ionic current measurements from individually addressed pores and such methods are likely to prove difficult to scale up. Here we show that, by optically encoding the ionic flux through protein nanopores, the discrimination of nucleic acid sequences and the detection of sequence-specific nucleic acid hybridization events can be parallelized. We make optical recordings at a density of ∼104 nanopores per mm2 in a single droplet interface bilayer. Nanopore blockades can discriminate between DNAs with sub-picoampere equivalent resolution, and specific miRNA sequences can be identified by differences in unzipping kinetics. By creating an array of 2,500 bilayers with a micropatterned hydrogel chip, we are also able to load different samples into specific bilayers suitable for high-throughput nanopore recording.

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Figure 1: Optical detection of DNA by αHL in a DIB.
Figure 2: Amplitude resolution of oSCR for DNA identification.
Figure 3: Optical discrimination of four nucleotides using the MspA M2 nanopore.
Figure 4: Detection of miRNA sequences by oSCR based on unzipping event duration.
Figure 5: High-throughput and multi-sample oSCR.

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Acknowledgements

The authors thank D. Aarts for cleanroom access, T. Sapra for cleanroom training and E. Mikhailova for protein preparation. This work was supported by funding from the US National Human Genome Research Institute (NHGRI), ‘$1000 Genome’ research grant R01 HG003709, ERC StG 106913, and Oxford Nanopore Technologies Ltd.

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Author notes

  1. Shuo Huang and Mercedes Romero-Ruiz: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Chemistry, University of Oxford, Oxford, OX1 3TA, UK

    Shuo Huang, Mercedes Romero-Ruiz, Oliver K. Castell, Hagan Bayley & Mark I. Wallace

  2. School of Chemistry and Chemical Engineering, Nanjing University, 210093, China

    Shuo Huang

  3. School of Pharmacy and Pharmaceutical Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, CF10 3NB, UK

    Oliver K. Castell

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  1. Shuo Huang
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Contributions

S.H., M.R.R., O.C., H.B. and M.I.W. designed the experiments. S.H., M.R.R. and O.C. performed the experiments. S.H. wrote the data analysis package program for the measurements with αHL, and performed the related data analysis. M.I.W. designed the data analysis methodology for the measurements with MspA. M.R.R. performed the MspA-related data analysis. S.H. designed and wrote the spotting robot program for the hydrogel array. S.H., M.R.R., O.C., H.B. and M.I.W. wrote the paper.

Corresponding author

Correspondence to Mark I. Wallace.

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Competing interests

H.B. is the founder, a director and a shareholder of Oxford Nanopore Technologies, a company engaged in the development of nanopore sensing and sequencing technologies. The work in this Article was supported in part by Oxford Nanopore Technologies. Methods developed by M.I.W. have been licensed by Oxford Nanopore Technologies.

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Huang, S., Romero-Ruiz, M., Castell, O. et al. High-throughput optical sensing of nucleic acids in a nanopore array. Nature Nanotech 10, 986–991 (2015). https://doi.org/10.1038/nnano.2015.189

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