Integrated nanopore sensing platform with sub-microsecond temporal resolution

Journal name:
Nature Methods
Volume:
9,
Pages:
487–492
Year published:
DOI:
doi:10.1038/nmeth.1932
Received
Accepted
Published online

Abstract

Nanopore sensors have attracted considerable interest for high-throughput sensing of individual nucleic acids and proteins without the need for chemical labels or complex optics. A prevailing problem in nanopore applications is that the transport kinetics of single biomolecules are often faster than the measurement time resolution. Methods to slow down biomolecular transport can be troublesome and are at odds with the natural goal of high-throughput sensing. Here we introduce a low-noise measurement platform that integrates a complementary metal-oxide semiconductor (CMOS) preamplifier with solid-state nanopores in thin silicon nitride membranes. With this platform we achieved a signal-to-noise ratio exceeding five at a bandwidth of 1 MHz, which to our knowledge is the highest bandwidth nanopore recording to date. We demonstrate transient signals as brief as 1 μs from short DNA molecules as well as current signatures during molecular passage events that shed light on submolecular DNA configurations in small nanopores.

At a glance

Figures

  1. The CNP.
    Figure 1: The CNP.

    (a) Schematic of the measurement setup. (b) Cross-section schematic of the low-capacitance thin-membrane chip. (c) Optical micrograph of the 8-channel CMOS voltage-clamp current preamplifier. (d) Magnified image of one preamplifier channel. (e) Optical image of a solid-state silicon nitride membrane chip mounted in the fluid cell. (f) Transmission electron microscope image of a 4-nm-diameter silicon nitride nanopore.

  2. Electrical modeling of a nanopore measurement.
    Figure 2: Electrical modeling of a nanopore measurement.

    (a) An illustration of the electronic impedances of a solid-state nanopore chip. (b) Simplified circuit schematic of the voltage-clamp current preamplifier. (c) Circuit design of the low-noise current source that substitutes for a feedback resistance (RF). (d) An example transient current pulse, characteristic of a single-molecule event such as the one illustrated. (e) Dominant sources of noise power spectral density, illustrated as a function of frequency.

  3. Noise measurements.
    Figure 3: Noise measurements.

    (a) Input-referred baseline current noise spectrum for CF = 0.15 pF, fs = 4 MS s−1. Also shown is the measured open-headstage of an Axopatch 200B in whole-cell mode. (b) Baseline RMS current noise as a function of bandwidth. (c) Baseline current noise traces corresponding to points in b. (d) Measured noise floor of the new amplifier with pore A. A polynomial fit is also shown to Sn(f) = Af −1 + B + Cf + Df 2, where AD are fitting parameters. (e,f) RMS current noise and traces with a pore present, corresponding to data in d. (g,h) SNR as a function of bandwidth for two nanopores measured with the CNP. For pore A, CM = 6 pF and ΔI = 882 pA (400-bp DNA, 400 mV, n = 430 events). For pore B, CM = 25 pF and ΔI = 840 pA (50-bp DNA, 400 mV, n = 2,974 events). As event durations varied, the SNR is shown for a range of 1 μs < τ < ∞. (i) Maximum achievable bandwidth for SNR > 5 as a function of signal amplitude (ΔI) for Pore A, Pore B, and the CNP baseline (CM = 0).

  4. Fast single-molecule events.
    Figure 4: Fast single-molecule events.

    A continuous trace recorded with 25-bp dsDNA fragments and pore B using the CNP platform, at a bias of 600 mV. The traces are recorded at 2.5 MS s−1 and digitally filtered to both B = 500 kHz and 100 kHz. Insets of the 29 translocation events in this 500-ms trace are displayed.

  5. Fast nanopore event statistics (50 bp dsDNA and pore B).
    Figure 5: Fast nanopore event statistics (50 bp dsDNA and pore B).

    (ac) The CNP output was sampled at 2.5 MS s−1 and then digitally filtered to B = 400 kHz, 100 kHz and 10 kHz signal bandwidths. Event rate as a function of applied bias, for a detection threshold of 5σ (a). Characteristic dwell time τ1 at 400 kHz and 100 kHz as a function of applied potential (b). τ1 was calculated from the width distribution P(τ) = A exp(–τ/τ1) + B exp(–τ/τ2), where τ1 is the shorter time constant18. Error bars, s.e.m. of the fitted parameter (n > 500). Histograms of event widths at applied potentials of 250–450 mV (c). (The listed event count n is for the 400 kHz data. In the 250–300 mV data the heights of the 10 kHz bins were reduced by half for visual clarity.) (d) A scatter plot of events from a subset of the 450 mV data at the three bandwidths. Brief events were severely distorted and attenuated by lower bandwidths. Inset, representative event in the 450 mV dataset. The event was notably distorted at 100 kHz, and undetected at 10 kHz.

  6. Intra-event structure.
    Figure 6: Intra-event structure.

    (a) An illustration of the sequential processes of translocation for short oligomers and small nanopores. (b) Typical signals observed for 50-bp dsDNA fragments with pore B (d = 3.5 nm) at 500 mV bias. (c) A histogram of event depths (ΔI) for 50-bp DNA at 500 mV bias (n = 3,955 events). The depth of the whole events and the depth of the last 2 μs of each event have distinct distributions. (d) A plot of the mean depths of the whole events, along with the mean depths of the first and last 2 μs of each event (error bars, s.e.m, n > 500). The depth of the last 2 μs retained a linear relationship with voltage up to 500 mV, whereas earlier portions of the events became shallower at high bias. This may be indicative of molecular dynamics at high electric fields which suppress polymer diffusion and extend the duration of the 'capture' phase relative to the 'threading' phase.

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Author information

Affiliations

  1. Department of Electrical Engineering, Columbia University, New York, New York, USA.

    • Jacob K Rosenstein &
    • Kenneth L Shepard
  2. Departments of Physics and Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts, USA.

    • Meni Wanunu
  3. Department of Physics, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Meni Wanunu,
    • Christopher A Merchant &
    • Marija Drndic

Contributions

J.K.R., K.L.S. and M.D. developed the platform concept. J.K.R. designed the amplifier and measurement system. J.K.R., K.L.S., M.W. and M.D. planned experiments. M.W. and C.A.M. fabricated nanopores. J.K.R. and M.W. performed nanopore experiments and analyzed data. J.K.R. and K.L.S. wrote the manuscript. All authors edited the manuscript.

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The authors declare no competing financial interests.

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