Fabrication of Low Noise Borosilicate Glass Nanopores for Single Molecule Sensing.

We show low-cost fabrication and characterization of borosilicate glass nanopores for single molecule sensing. Nanopores with diameters of ~100 nm were fabricated in borosilicate glass capillaries using laser assisted glass puller. We further achieve controlled reduction and nanometer-size control in pore diameter by sculpting them under constant electron beam exposure. We successfully fabricate pore diameters down to 6 nm. We next show electrical characterization and low-noise behavior of these borosilicate nanopores and compare their taper geometries. We show, for the first time, a comprehensive characterization of glass nanopore conductance across six-orders of magnitude (1M-1μM) of salt conditions, highlighting the role of buffer conditions. Finally, we demonstrate single molecule sensing capabilities of these devices with real-time translocation experiments of individual λ-DNA molecules. We observe distinct current blockage signatures of linear as well as folded DNA molecules as they undergo voltage-driven translocation through the glass nanopores. We find increased signal to noise for single molecule detection for higher trans-nanopore driving voltages. We propose these nanopores will expand the realm of applications for nanopore platform

DNA translocation through 20nm nanocapillary.

<p><b>A)</b> IV characteristic of 20nm nanopore with 0.5M KCl in TE buffer. <b>B)</b> Raw trace (mean subtracted) of nanopore conductance showing real-time detection of λ DNA translocation at +0.3V in 0.5M KCl concentration. <b>C)</b> Representative events from the raw trace showing DNA translocation events with different folding states shown above the events. In these sketches, the pore is assumed to be on the left side of the DNA. The event at far right is interpreted as simultaneous translocation of two DNA molecules. Such events are extremely rare events. <b>D)</b> ΔG histogram (conductance drops) of translocation events (n = 510 events) with Gaussian fit (solid line) showing first peak at 460±40 pS representing a single DNA inside the pore and the second peak at 780 ± 53 pS corresponding to folded DNA. <b>E)</b> Δt histogram (dwell times) of translocation events (n = 510 events) with most probable value from the log normal fit (solid line) to be 0.74 ± 0.14 ms. <b>F)</b> Scatter plot of ΔG vs Δt with two distinct population representing single and folded DNA states during translocation.</p

Pulling parameters for shorter taper capillaries.

<p>Pulling parameters for shorter taper capillaries.</p

Conductance and noise characterization of nanocapillaries.

<p><b>A)</b> I-V characteristics of a 77 nm borosilicate glass nanopore for the salt range from 1M-0.1M. <b>B)</b> Plot of Conductance vs Salt concentration of 77 nm pore from 1M-0.1M. Solid black line is the plot of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157399#pone.0157399.e001" target="_blank">eq 1</a>.All measurements are done in triplicate and mean and error bars are calculated. <b>C)</b> Plot of Conductance vs Salt concentration of 77 nm pore for the entire salt range of 1M-1μM. Solid black line is the plot of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157399#pone.0157399.e002" target="_blank">eq 2</a>. Inset is the side-on SEM image of a typical capillary showing the unshrunk long taper (scale bar 300nm). <b>D)</b> Plot of Conductance vs Salt concentration for 88 nm pore. The squares and triangles represent data for salt conductance measurements made in TE buffer and milliQ, respectively. Solid lines are the truncated model and full model (see main text) plots using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157399#pone.0157399.e002" target="_blank">eq 2</a>. Pore conductance, <b>E),</b> and I_RMS noise, <b>F),</b> as a function of salt concentration is compared for shrunk and unshrunk pores as well as across different taper geometries of glass nanopipettes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157399#pone.0157399.g001" target="_blank">Fig 1D–1F</a>). I_RMS Noise is average of three measurements taken at 0 mV and ± 100 mV.</p

Pulling parameters for long taper capillaries.

<p>Pulling parameters for long taper capillaries.</p

Pulling parameters for short taper capillaries.

<p>Pulling parameters for short taper capillaries.</p

Fabrication of nanocapillaries.

<p><b>A) [i-iv]</b>Snapshots of in-Lens SEM images of 134 nm borosilicate glass nanopore (scale bar of 200 nm), being sculpted down to 70 nm under constant 5kV electron beam exposure. The intermediate pore diameters are [ii-iv] 104 nm, 86 nm and 70 nm respectively. <b>B)</b> Shrunken borosilicate nanopore with pore diameter 6 nm (scale bar is 50 nm), from initial diameter of 130 nm. <b>C)</b> SEM image of a borosilicate glass nanopore with pore diameter of 20 nm (scale bar is 50 nm) which was shrunk from 170 nm, the taper geometry of the pore can be seen in Fig 1E<b>. D-F)</b> Taper images of borosilicate glass nanocapillary with ~150 nm pore diameter pulled with puller programs shown in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157399#pone.0157399.t001" target="_blank">1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157399#pone.0157399.t003" target="_blank">3</a> respectively. Scale bar is 1mm. <b>G)</b> Experimental scheme consisting of teflon sample cell with two fluid chambers with glass nanocapillary glued between the two reservoirs. The nanopore resides in the fluid chamber with negative electrode where the DNA sample is introduced.</p

Comparing linear and folded DNA translocations for different voltages.

<p>Comparison of typical DNA translocation through 20 nm pore at the applied driving potentials of <b>(A)</b> 300 mV, <b>(B)</b> 500 mV and <b>(C)</b> 700 mV. Increase in DNA translocation signal with increasing trans-pore potential is clearly seen. Statistical comparison of DNA translocation events acquired at different voltages is shown. <b>D)</b> ΔG histogram of DNA translocations at different potential shows a clear shift in increased conductance drops as a function of increase in applied potential, inset is the plot of the first ΔG peak positions (correspondingto a single DNA inside the pore) at different potential. <b>E)</b> Δt histogram at different potential shows a clear shift in the dwell time of the DNA inside the nanopore, inset is the plot of most probable dwell time vs applied positive potential. <b>F)</b> Scatter plot of ΔG vs Δt shows clear change in the conductance as well in the dwell time of the DNA inside the nanopore for different potentials.</p

Summary of ΔG (both first and second peaks) and most probable Δt values measured at the three voltages.

<p>Summary of ΔG (both first and second peaks) and most probable Δt values measured at the three voltages.</p

Small Molecule Permeation Across Membrane Channels: Chemical Modification to Quantify Transport Across OmpF

Biological channels facilitate the exchange of small molecules across membranes, but surprisingly there is a lack of general tools for the identification and quantification of transport (i.e., translocation and binding). Analyzing the ion current fluctuation of a typical channel with its constriction region in the middle does not allow a direct conclusion on successful transport. For this, we created an additional barrier acting as a molecular counter at the exit of the channel. To identify permeation, we mainly read the molecule residence time in the channel lumen as the indicator whether the molecule reached the exit of the channel. As an example, here we use the well-studied porin, OmpF, an outer membrane channel from E. coli. Inspection of the channel structure suggests that aspartic acid at position 181 is located below the constriction region (CR) and we subsequently mutated this residue to cysteine, where else cysteine free and functionalized it by covalent binding with 2-sulfonatoethyl methanethiosulfonate (MTSES) or the larger glutathione (GLT) blockers. Using the dwell time as the signal for transport, we found that both mono-arginine and tri-arginine permeation process is prolonged by 20% and 50% respectively through OmpFE181CMTSES, while the larger sized blocker modification OmpFE181CGLT drastically decreased the permeation of mono-arginine by 9-fold and even blocked the pathway of the tri-arginine. In case of the hepta-arginine as substrate, both chemical modifications led to an identical ‘blocked’ pattern observed by the dwell time of ion current fluctuation of the OmpFwt. As an instance for antibiotic permeation, we analyzed norfloxacin, a fluoroquinolone antimicrobial agent. The modulation of the interaction dwell time suggests possible successful permeation of norfloxacin across OmpFwt. This approach may discriminate blockages from translocation events for a wide range of substrates. A potential application could be screening for scaffolds to improve the permeability of antibiotics