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

Journal name:
Nature Nanotechnology
Year published:
Published online


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.

At a glance


  1. Optical detection of DNA by αHL in a DIB.
    Figure 1: Optical detection of DNA by αHL in a DIB.

    a, Right: schematic of a single DIB. A ×60 TIRF objective was used for both illumination and imaging. A voltage protocol was applied using Ag/AgCl electrodes present in the agarose substrate and in the droplet. Left: zoomed-in cartoon of the detection process. b, Representative fluorescence trace from a single DNA blockade cycle. Streptavidin-tethered ssDNA, which is anionic, is driven into the pore at +100 mV. Simultaneously, the cationic Ca2+ flows in the direction opposite to the DNA flow, and forms a complex with Fluo-8 to become fluorescent (I). Streptavidin (red squares) tethered ssDNA (yellow line) is driven into the pore, partially blocking the Ca2+ flux (II). At −50 mV, the trapped ssDNA is released (III). The fluorescence at −50 mV diminishes due to the near reversal of the Ca2+ flux at negative potentials. The applied bias is then returned to 0 mV (IV) and the cycle repeats. The fluorescence at 0 mV comes from diffusion of Ca2+ from the agarose substrate. The trace amplitude is normalized so that the mean intensities of (III) and (IV) are 0 and 1. The normalized fluorescence amplitude of (II) identifies the captured DNA. c, A sequence of nanopore blockades with a mixture of two types of DNA (X5, cyan, histogram level 3; C40, blue, histogram level 4). An additional lower fluorescence level is occasionally populated at −50 mV due to gating of the αHL pore at the negative potential.

  2. Amplitude resolution of oSCR for DNA identification.
    Figure 2: Amplitude resolution of oSCR for DNA identification.

    a, Current–voltage response from a single αHL nanopore in a PLB (cis: 1.32 M KCl, 8.8 mM HEPES, pH 7.0; trans: 0.66 M CaCl2, 8.8 mM HEPES, pH 7.0). b, The equivalent optical fluorescence–voltage relation for a single αHL in a DIB. c, Comparison of the response from optical and electrical recording for three different DNAs. Error bars for the residual fluorescence represent the standard deviation of 120 events (Supplementary Table 2). Error bars for the residual current represent the full-width at half-maximum (FWHM) of a Gaussian fit to the peak (Supplementary Table 4 and Supplementary Fig. 7). dg, Normalized fluorescence traces (Supplementary Methods 5) for different types of streptavidin-tethered ssDNA (C40 (d); X3 (e); X5 (f); C40 + X5 (g)). The fluorescence intensity is normalized so that the amplitude is 0 at −50 mV and 1 at 0 mV when the pore is open. Each blockade is fitted to the mean value of the corresponding data points and overlaid with colour-coded bars. All-points histograms are displayed on the right of each trace. The colour-coded dashed lines are assigned according to the mean values for each type of DNA blockade as shown in c. C40 and X5 are distinguished in g by a 50% threshold between the mean amplitudes corresponding to the two states. The total DNA concentration in the droplet for df is 267 nM, and for g is 133.5 nM each for C40 and X5.

  3. Optical discrimination of four nucleotides using the MspA M2 nanopore.
    Figure 3: Optical discrimination of four nucleotides using the MspA M2 nanopore.

    When streptavidin-tethered biotinylated DNA oligonucleotides block a pore, the ion flux is restricted and we observe a reversible stepwise change in the fluorescence intensity. ad, Normalized optical traces (Supplementary Methods 6) for the following 65-mers (Supplementary Table 1): C65 (5′-biotin-CCCCCCCCCCCC-CCC-C35-CCTGTCTCCCTGCCG-3′, a), T65 (5′-biotin-TTTTTTTTTTTT-TTT-T35-CCTGTCTCCCTGCCG-3′, b), A65 (5′-biotin-AAAAAAAAAAAA-AAA-A35-CCTGTCTCCCTGCCG-3′, c) or G3 in a background of A65 (5′-biotin-AAAAAAAAAAAA-GGG-A35-CCTGTCTCCCTGCCG-3′, d). The blockades are fitted to the mean value of the corresponding data points and represented with a coloured bar. Event histograms displaying the mean amplitudes are on the right of the traces for each DNA oligonucleotide (Supplementary Table 3). e, Normalized optical trace and corresponding event histogram for a mixture of C65 and A65.

  4. Detection of miRNA sequences by oSCR based on unzipping event duration.
    Figure 4: Detection of miRNA sequences by oSCR based on unzipping event duration.

    a, An example miRNA unzipping event (Plet7a/Let7a). miRNA (Let7a or Let7i) hybridizes with the DNA probe (Plet7a or Plet7i). Poly-C30 ssDNA tags on both ends of the probe are designed to enable pore threading and initiate unzipping. At +160 mV, an open nanopore (I) shows a decrease in fluorescence when the hybridized complex is captured and subsequently unzipped (II). Following unzipping, when the DNA probe has translocated through the pore the miRNA remains in the vestibule (III). The miRNA then translocates (IV) and the pore re-opens (V). b, A series of miRNA (Plet7a/Let7a) unzipping events at +160 mV. Magenta fitting lines (Supplementary Fig. 9) highlight capture/unzipping (II) events. c, Different probe/miRNA combinations show different capture/unzipping times. Matched miRNA and probe generate long events. miRNA without probe shows no capture/unzipping events (II). d, Histograms of capture/unzipping event (II) lifetimes for all probe/miRNA combinations fit with exponentials. The fitted rate constant for unzipping reflects the hybridization strength (Supplementary Section 2). e, Dependence of the unzipping rate constant on applied potential for Plet7a/Let7i (Supplementary Fig. 11).

  5. High-throughput and multi-sample oSCR.
    Figure 5: High-throughput and multi-sample oSCR.

    a, A frame containing multiple fluorescence spots representing open (white dashed circles) and blocked nanopores (pink dashed circles; DNA: streptavidin tethered C40). Scale bar, 10 µm. b, Parallel recordings of nine fluorescence traces simultaneously extracted from the same field of view. The fluorescence traces show spontaneous amplitude transitions due to consecutive miRNA (Plet7a/Let7i) capture/unzipping events (Supplementary Movie 4). oSCR results in a and b are both recorded in DIBs. c, A Fluo-8 containing hydrogel chip with cast pillar-array layer. Scale bar, 8 mm (Supplementary Fig. 13). The image inset in a black dashed square shows separation of the formed bilayers. After hydrogel-hydrogel bilayer array (HHBa) formation (Supplementary Movie 6), the pillar array is lifted slowly to separate the bilayers. Boundary lines delimiting the area containing formed bilayers are visualized (white arrows). Scale bar, 140 μm. Inset in blue dashed square: multiple fluorescence images have been stitched together to show an expanded view of an area of the chip containing various biological samples (1, –αHL, –DNA; 2, +αHL, –DNA; 3, +αHL, +DNA). Yellow dashed square: a single frame accommodates four bilayers at a time. Scale bar, 40 μm. d, Parallel single-molecule nanopore activity from a full-frame HHBa recording. Fluorescence traces are recorded simultaneously from nanopores in different bilayers of the array (see c, yellow dashed square). The unspotted HHBa shows constant fluorescence (trace 1). The HHBa loaded with αHL shows fluorescence changes synchronized with the voltage protocol (trace 2). DNA (C40) blockades (red points on the trace) are only detected in trace 3.


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Author information

  1. These authors contributed equally to this work

    • Shuo Huang &
    • Mercedes Romero-Ruiz


  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


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.

Competing financial 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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