Research ArticleAPPLIED SCIENCES AND ENGINEERING

Electrode-free nanopore sensing by DiffusiOptoPhysiology

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Science Advances  06 Sep 2019:
Vol. 5, no. 9, eaar3309
DOI: 10.1126/sciadv.aar3309
  • Fig. 1 DiffusiOptoPhysiology and its application in trimethyl-β-cyclodextrin sensing.

    (A to D) Schematics of ion transport through a nanopore in different measurement platforms. Electromigration and diffusion of ions are indicated by solid and dashed lines, respectively. (A) During electrophysiology recording, electrophoretic motion of K+ and Cl through a nanopore is observed when a transmembrane potential is applied via a pair of Ag/AgCl electrodes. (B) In the absence of electrodes, although thermal motion of ions across the nanopore exists in both directions, no net flow of ion transport should happen according to the rule of electroneutrality. (C) During optical single-channel recordings (oSCRs), directional motion of Ca2+, which is electrophoretically driven through a nanopore, establishes a steep Ca2+ concentration gradient. Upon binding with Fluo-8 in cis, the Fluo-8/Ca2+ complex around the pore vicinity emits strong fluorescence. (D) During DiffusiOptoPhysiology (DOP), a mild Ca2+ concentration gradient could be established around the pore vicinity due to the thermal motion of ions. Upon binding with Fluo-8, a weaker fluorescence emission than (C) is expected. (E) A cross-sectional view of the spatial distribution of the Fluo-8/Ca2+ complex around the pore. Dashed box: The zoomed-in view of the immediate vicinity area near the nanopore. (F) Top left: Corresponding image result from computer simulation. Top right: The simulated fluorescence intensity (FI) profile follows a Gaussian distribution. Bottom left: A representative frame acquired from DOP recording for a single wild-type (WT) α-hemolysin (α-HL) nanopore. Bottom right: The corresponding fluorescence intensity profile also follows a Gaussian distribution. Scale bars, 4 μm. a.u., arbitrary units. (G) Single-molecule sensing of trimethyl-β-cyclodextrin (TriM-β-CD) (75 mM) with an α-HL nanopore during DOP recording. Scale bars, 4 μm. (H) Plot of the reciprocals of the mean interevent intervals (1/τon) and mean dwell time (1/τoff) versus TriM-β-CD concentration. The mean and SD come from three independent experiments for each condition (n = 3). (I) Statistics of τoff and FP results acquired from DOP and electrophysiology recording at +20 mV, respectively. The DOP recordings (F to I) were performed with 1.5 M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0) in cis and 0.75 M CaCl2 and 10 mM HEPES (pH 7.0) in trans. The electrophysiology recordings were performed with 1.5 M KCl and 10 mM HEPES (pH 7.0) in both sides of the membrane. TriM-β-CD was added to the cis side with a final concentration of 4 mM.

  • Fig. 2 Enhanced sensing performance with adjusted osmosis during DOP recording.

    (A) Fluorescence traces acquired from DOP recording with different electrolyte combinations. DOP recordings were performed with α-HL nanopores. TriM-β-CD was added to cis with a final concentration of 15 mM. The interevent interval τon decreases when a larger osmosis gradient between cis and trans was established. (B) Plot of the osmotic pressure and 1/τon as a function of the KCl concentration in cis. The mean and SD are derived from three sets of independent experiments (n = 3). (C) The SBR analysis of fluorescent imaging results with different KCl concentrations in cis. Top: Representative images acquired from DOP recordings. The images from left to right were acquired by DOP recording with [KCl] in cis as 1.0, 1.5, 2.0, and 2.5 M, respectively. The value of SBR decreases when KCl concentration in cis increases. The mean and SD comes from five independent experiments (n = 5). Experiments in (A) to (C) were performed with 1 to 2.5 M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0) in cis and 0.75 M CaCl2 and 10 mM HEPES (pH 7.0) in trans. (D) Plot of simulated fluorescence intensity as a function of the KCl concentration in cis. Top: Corresponding two-dimensional (2D) profiles of fluorescence intensities from the simulation, which resembles the results acquired from DOP in (C). The simulations were performed with 1 to 2.5 M KCl in cis and 0.75 M CaCl2 in trans. (E) A cross-sectional view of the spatial distribution of Fluo-8 in the simulation space. The boundary condition of the simulation was set as 1.0 M KCl in cis and 0.75 M CaCl2 in trans. Sustained osmotic flow from cis to trans gives rise to an enriched distribution of Fluo-8 in the vicinity of the DIB.

  • Fig. 3 Enhanced SBR with increased Ca2+ flux during DOP recording.

    (A) Imaging results (top) and the corresponding 2D Gaussian fittings (bottom) acquired from DOP recording. The CaCl2 concentration in trans was increased when the KCl concentration in cis was adjusted so that the osmolarity concentrations were kept isotonic. The fluorescence spot, which corresponds to Ca2+ flux through a WT α-HL nanopore, becomes brighter with an increased Ca2+ flux. Scale bar, 4 μm. (B) The FWHM and SBR of the fluorescence imaging signals with different electrolyte osmolarity concentrations (n = 12). The DOP recordings in (A) and (B) were carried out with 0.75 to 2.25 M KCl, 400 μM EDTA, 40 μM Fluo-8, 10 mM HEPES, pH 7.0 in cis and 0.5–1.5 M CaCl2, 10 mM HEPES, pH 7.0 in trans. (C) A representative fluorescence trace shows PEG 1500 translocation signals through a WT α-HL nanopore, as acquired by DOP recording. PEG 1500 was added to the agarose substrate reaching a final concentration of 20 mM. The DOP recording was carried out with 2.25 M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0) in cis and 1.5 M CaCl2 and 10 mM HEPES (pH 7.0) in trans. (D) Simulated total fluorescence intensity as a function of osmolarity concentration, shown for four different pore sizes with a diameter of 2, 4, 6, and 8 nm, respectively. The electrolyte concentrations were kept isotonic to avoid the interference of osmosis in this demonstration. (E) Simultaneously imaging of WT α-HL and ClyA-RR nanopores in the same DIB. Because of a larger channel conductance, ClyA-RR appears as a larger and brighter spot in comparison with WT α-HL in the same field of view (yellow dashed circles, WT α-HL; red dashed circles, ClyA-RR). Scale bar, 20 μm. (F) Left: Simultaneous imaging of an α-HL and a ClyA-RR. Right: The fluorescence intensity profile along vertical lines as marked by location 1 and 2, respectively. The fluorescence intensity profile is fitted with a Gaussian distribution. Scale bar, 5 μm. (G) The FWHM and SBR of the fluorescence imaging signals of WT α-HL and ClyA-RR (n = 5). DOP recordings as demonstrated in (E) and (F) were carried out with 1.5 M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0) in cis and 1.5 M CaCl2 and 10 mM HEPES (pH 7.0) in trans.

  • Fig. 4 dsDNA sensing using ClyA-RR nanopores.

    (A) The schematic diagram of dsDNA sensing using ClyA-RR during DOP recording. (B) DOP imaging of a ClyA-RR nanopore and the corresponding fluorescence trace. No dsDNA was added in the droplet. (C) DOP imaging of ClyA-RR nanopore and the corresponding fluorescence trace when dsDNA was added in the droplet with a final concentration of 2 μM. Successive deep and long-residing fluorescence blockades were observed. Scale bar, 5 μm. The DOP recordings in (B) and (C) were performed with 2.25 M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0) in cis and 1.5 M CaCl2 and 10 mM HEPES (pH 7.0) in trans. dsDNA, which is of 78 bp, was add to cis with a final concentration of 2 μM. (D) Electrophysiology recording of dsDNA events recorded with ClyA-RR nanopore at +2 mV. With voltages as low as +2 mV, current blockages were still observable. The electrophysiology recordings were performed with 2.25 M KCl and 10 mM HEPES (pH 7.0) in cis and 1.5 M CaCl2 and 10 mM HEPES (pH 7.0) in trans. dsDNA, which is of 78 bp, was add to cis with a final concentration of 2 μM. (E) Histogram of the dwell time for dsDNA events extracted from electrophysiology recordings. (F) Histogram of the dwell time for dsDNA events extracted from DOP recordings. Because of a limited acquisition time (30 ms) of the electron-multiplying charge-coupled device in a large field of view, fast dsDNA translocations cannot be fully resolved. Data from DOP recordings (shown in olive color) were acquired with a frame rate of 30 ms. The electrophysiology trace (shown in black) were recorded with a +2-mV applied bias, sampled at 25 kHz, and low-pass–filtered at 1 kHz.

  • Fig. 5 Multiplex DOP recording with mini-DIBs.

    (A) Schematics of a mini-DIB array for multiplex DOP recordings. (B) A bright-field image of the mini-DIB array. (C) A frame of ClyA-RR nanopore inserted in a mini DIB. The diameter of the DIB is ~40 μm. This mini-DIB was established with 1.5 M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0) in cis and 1.5 M KCl and 10 mM HEPES (pH 7.0) in trans.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaar3309/DC1

    Section S1. The FEM simulation

    Section S2. 2D Gaussian fitting

    Section S3. SBR evaluation

    Table S1. 1/τon and 1/τoff of TriM-β-CD with different [TriM-β-CD].

    Table S2. 1/τon of TriM-β-CD with different [KCl] in cis.

    Table S3. FWHM and SBR with different [KCl].

    Table S4. FWHM and SBR with different [CaCl2].

    Table S5. FWHM and SBR of α-HL and ClyA-RR nanopores.

    Table S6. Nucleic acid abbreviations and sequences.

    Table S7. Blockade level of dsDNA events.

    Fig. S1. FEM model geometry.

    Fig. S2. The DIB device.

    Fig. S3. The schematic diagram of the setup.

    Fig. S4. Cyclodextrin binding kinetics.

    Fig. S5. Definition of signal and background during oSCR.

    Fig. S6. Demonstration of fluorescence trace normalization.

    Fig. S7. Event statistics derivation.

    Fig. S8. Baseline comparison during TriM-β-CD sensing.

    Fig. S9. FEM modeling of Fluo-8 distribution.

    Fig. S10. FEM modeling of the osmotic flow.

    Fig. S11. The preparation and characterization of ClyA-RR.

    Fig. S12. Observing dsDNA events with different dsDNA concentrations.

    Fig. S13. Statistics of dsDNA events acquired from ClyA-RR.

    Movie S1. Simultaneous imaging of α-HL and ClyA.

    Movie S2. dsDNA sensing by DOP.

    Movie S3. Parallel dsDNA sensing by DOP.

    Movie S4. A single ClyA-RR nanopore inserted in a miniaturized DIB.

  • Supplementary Materials

    The PDF file includes:

    • Section S1. The FEM simulation
    • Section S2. 2D Gaussian fitting
    • Section S3. SBR evaluation
    • Table S1. 1/τon and 1/τoff of TriM-β-CD with different TriM-β-CD.
    • Table S2. 1/τon of TriM-β-CD with different KCl in cis.
    • Table S3. FWHM and SBR with different KCl.
    • Table S4. FWHM and SBR with different CaCl2.
    • Table S5. FWHM and SBR of α-HL and ClyA-RR nanopores.
    • Table S6. Nucleic acid abbreviations and sequences.
    • Table S7. Blockade level of dsDNA events.
    • Fig. S1. FEM model geometry.
    • Fig. S2. The DIB device.
    • Fig. S3. The schematic diagram of the setup.
    • Fig. S4. Cyclodextrin binding kinetics.
    • Fig. S5. Definition of signal and background during oSCR.
    • Fig. S6. Demonstration of fluorescence trace normalization.
    • Fig. S7. Event statistics derivation.
    • Fig. S8. Baseline comparison during TriM-β-CD sensing.
    • Fig. S9. FEM modeling of Fluo-8 distribution.
    • Fig. S10. FEM modeling of the osmotic flow.
    • Fig. S11. The preparation and characterization of ClyA-RR.
    • Fig. S12. Observing dsDNA events with different dsDNA concentrations.
    • Fig. S13. Statistics of dsDNA events acquired from ClyA-RR.
    • Legends for movies S1 to S4

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Simultaneous imaging of α-HL and ClyA.
    • Movie S2 (.mp4 format). dsDNA sensing by DOP.
    • Movie S3 (.mp4 format). Parallel dsDNA sensing by DOP.
    • Movie S4 (.mp4 format). A single ClyA-RR nanopore inserted in a miniaturized DIB.

    Files in this Data Supplement:

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