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Mechanistic Study of the Conductance and Enhanced Single-Molecule Detection in a Polymer–Electrolyte Nanopore
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Cite this: ACS Nanosci. Au 2023, 3, 2, 172–181
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https://doi.org/10.1021/acsnanoscienceau.2c00050
Published January 10, 2023

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Abstract

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Solid-state nanopores have been widely employed in the detection of biomolecules, but low signal-to-noise ratios still represent a major obstacle in the discrimination of nucleic acid and protein sequences substantially smaller than the nanopore diameter. The addition of 50% poly(ethylene) glycol (PEG) to the external solution is a simple way to enhance the detection of such biomolecules. Here, we demonstrate with finite-element modeling and experiments that the addition of PEG to the external solution introduces a strong imbalance in the transport properties of cations and anions, drastically affecting the current response of the nanopore. We further show that the strong asymmetric current response is due to a polarity-dependent ion distribution and transport at the nanopipette tip region, leading to either ion depletion or enrichment for few tens of nanometers across its aperture. We provide evidence that a combination of the decreased/increased diffusion coefficients of cations/anions in the bath outside the nanopore and the interaction between a translocating molecule and the nanopore–bath interface is responsible for the increase in the translocation signals. We expect this new mechanism to contribute to further developments in nanopore sensing by suggesting that tuning the diffusion coefficients of ions could enhance the sensitivity of the system.

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Introduction

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Nanopore sensing is one of the leading label-free techniques for the analysis and manipulation of single molecules due to its high throughput and sensitivity. (1−4) In nanopore measurements, an ionic current is generated by applying a potential between two electrodes placed in two reservoirs separated by a small orifice. In general, the translocation of an analyte through a nanopore causes a decrease in magnitude of the ionic current due to the restricted transport of ions through the orifice (resistive-pulse event). (1) The amplitude, duration, and shape of the translocation event provide important information about the physicochemical properties of the molecule, such as size, charge, and shape. (1,5,6) Under low electrolyte concentrations, charged molecules, such as double-stranded DNA (dsDNA), can lead to a local ion enrichment, resulting in a current enhancement (conductive-pulse event). (1,7) The origin of this current enhancement was initially attributed to the additional charge carried by the counterion cloud around the dsDNA molecule. (8) However, conductive events are also observed at high salt concentrations, (9,10) suggesting that the total ionic current can increase or decrease depending on the concentration and transport properties of ions in and around the nanopore. (7,11,12)
Despite the developments in the field over the past decades, (13) the application of solid-state nanopores for the detection of proteins and short nucleic acids still remains challenging, requiring nanopores of comparable size to the molecules (<10 nm diameter), which are difficult to fabricate reproducibly. (14) Furthermore, the nanopore system needs to have a high signal-to-noise ratio (SNR) (15) to detect small perturbations to the ion current caused by the translocation of molecules and high-bandwidth electronics to characterize rapid translocations with sufficient temporal resolution. (16,17) So far, finite-element modeling has been extensively used to examine electrokinetic phenomena in nanopores. (10,18−21) In such systems, the ion current is due to the transport of ionic species under the influence of an electric field, and its physics can be considerably more complex than the one regulating simple ohmic conductors. (22) For example, the charge on the nanopore wall induces an electric double layer, leading to non-uniform ion concentration distributions, and the interacting physics of ion transport, electric fields, and fluid flows result in a wide range of non-linear behavior. (19,23,24)
We have recently reported the enhanced single-molecule detection by a nanopore when 50% poly(ethylene) glycol (PEG) is added to the external solution, (25) and we further characterized the system showing that the polymer–electrolyte interactions in the external solution govern the translocation dynamics of the analyte that is correlated to the properties of the electrolyte only in the external solution. (26) Here, we present a mechanism explaining this enhancement by using a combination of experiments and multiphysics modeling. We developed a finite-element model by coupling Nernst–Planck and Poisson equations to describe the physics of ion transport under an applied electric field when a nanopore sensing experiment is carried out in the presence of 50% PEG. Based on the cation-binding properties of PEG that have been previously reported in the literature, our model assumes a decrease in the diffusion coefficients of cations relative to anions in the external solution. (27−31) The model reproduces the experimental current–voltage responses in the presence and absence of PEG and provides an insight into the ion concentrations and transport rates responsible for the observed behavior. We then prove that the increase in the translocation signal is a combination of the unequal diffusion coefficients of ions in the bath outside the nanopore and the interaction between a translocating molecule and the nanopore–PEG interface. We expect this new mechanism to inform further developments in nanopore sensing by suggesting that approaches that affect the diffusion coefficients of ions in the external bath could be used to enhance the sensitivity of the system.

Results and Discussion

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Figure 1a shows the experimental setup used throughout this work in which a model solid-state nanopore based on a quartz nanopipette (aperture 25 nm in diameter) filled with a 0.1 M KCl solution is immersed into a bath containing 0.1 M KCl with or without 50% (w/v) PEG. In nanopore measurements, the current–voltage (i–V) response characterizes the ion transport, indirectly providing information about the physical properties of the nanopore (size, shape, and surface charge). The gray line in Figure 1b shows the current–voltage response of a nanopipette filled with a 0.1 M KCl solution and immersed in a bath containing 0.1 M KCl (no PEG). The slightly higher conductivity observed at a negative bias applied versus a positive bias is termed ion-current rectification (ICR) and arises from the negatively charged glass wall of the nanopipette, which makes the aperture region permselective to cations; this effect has been extensively described in the literature. (24,32−34)

Figure 1

Figure 1. Schematic and representative data for current-voltage and conductive-pulse measurements of dsDNA translocation through a nanopipette. (a) Nanopipette (25 nm pore diameter), filled with a 0.3 nM solution of 4.8 kbp dsDNA in 0.1 M KCl, is immersed in a solution of the same electrolyte with and without the presence of 50% (w/v) PEG 35K. The application of a negative potential to a Ag/AgCl quasi-reference electrode inside the nanopipette with respect to a ground electrode in the external solution causes outbound migration of DNA molecules. (b) Experimental (curves) and simulated (points) voltammograms of the nanopipette in the presence (orange) and absence (gray) of PEG in the outside solution. (c) Representative current trace recorded upon translocation of a dsDNA molecule through the nanopipette aperture with (orange trace) and without (gray trace) the presence of PEG in the external solution. Further examples of voltammetry and current traces from this and other nanopipettes are included in the Supporting Information (Section S4).

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When the same nanopipette is immersed in a bath of 0.1 M KCl with 50% PEG, a dramatic reversal in the rectification is observed in the i–V curve (orange line). The PEG solution is ∼9 times less conductive than 0.1 M KCl (Table ST1.2, Supporting Information), and, counterintuitively, the ion current observed at +500 mV is greater than the one measured in a PEG-free bath (above-bulk conductivity). Also, under a negative bias, the ion current is ∼4 times lower than observed without PEG in the external solution. This response cannot be explained only considering the difference in conductivity between the two solutions, or as a rectification effect induced by the surface charge on the nanopore wall, indicating that a different mechanism is responsible for the observed i–V response. We also demonstrated that the viscosity of the solution is not responsible for this observed phenomenon, as the i–V response obtained with PEG cannot be reproduced with other viscous solutions such as 50% (v/v) glycerol (Section S3, Supporting Information). In the following section, we describe a numerical model to calculate ionic currents (points in Figure 1b) in the PEG system and to explain this anomalous current–voltage behavior. The reproducibility of the experimental voltammograms in the presence and absence of PEG in the external solution was determined by testing six nanopipettes (Figures SF4.1 and SF4.2, Supporting Information).
As we have previously reported, (25) the presence of PEG in the external solution leads to a 4-fold enhancement of the ion current observed when a single molecule translocates through the nanopore (Figures 1c and SF4.3, Supporting Information). In particular, the SNR in the presence of PEG in the external solution was found to be approximately 2.2 times larger than the SNR in the absence of PEG with values SNRmeanPEG = 26.4 ± 3.9 and SNRmeanno PEG = 11.9 ± 3.9 (Figure SF4.4, Section S4.4, Supporting Information). Interestingly, the signal in the presence of PEG does not quickly return to the original baseline level after the translocation peak, suggesting a different kinetics regulating the translocation dynamics. The same signals plotted over an extended period are shown in Figure SF4.8 (Section S4.7, Supporting Information). It is worth noting that as the two current traces displayed in Figure 1c were both recorded using the same nanopipette tip aperture (d= 25 nm), applied voltage (−500 mV), and composition of the inner solution (0.1 M KCl and 0.3 nM 4.8 kbp dsDNA), the observed enhancement is only driven by the presence of PEG in the external solution. Moreover, these traces are representative of a large (>1000) number of peaks recorded from a single measurement, and small fluctuations in the recorded values (i.e., current peak maximum and frequency of events) are expected due to potential variability in the electrolyte or nanopipette properties (i.e., temperature, concentration, pore geometry, and aperture size). (35) For the PEG condition, individual translocation events randomly selected from recordings using three nanopipettes are shown in Figure SF4.3 (Supporting Information).
On another note, this experiment was repeated without adding any analyte to the nanopipette solution to check whether the conductive events were generated by the PEG molecules translocating through the pore. No translocation events were detected (Section S4.5, Figure SF4.5, Supporting Information), confirming that the conductive events were generated by the dsDNA molecules. To check whether the presence of PEG could change the properties of translocation events over time, a continuous 20 min measurement of dsDNA translocation was carried out. The analysis (Section S4.6, Supporting Information) shows that there are no significant changes over time in both the number of translocation events (Figure SF4.6, Supporting Information) and the translocation peak characteristics (Figure SF4.7, Supporting Information). During a conventional dsDNA translocation measurement through a nanopore where the solution is identical in both reservoirs, the conductive events are generally attributed to the presence of the counterion cloud carried by the dsDNA molecule and altered ion transport at the nanopipette tip, which result in a temporary increase in the ion concentration in this region. (7,10,11,36,37) The following sections present a new mechanism in the nanopore systems that explains not only the anomalous i–V response related to PEG but also the enhanced single-molecule sensitivity.

Finite-Element Simulations

We developed a finite-element model that coupled ion transport (diffusion and electromigration) and electrostatics at different applied potentials. A detailed description of the model is given in the Supporting Information. Briefly, a two-dimensional axisymmetric model simulates the geometry of a nanopipette as a simplified truncated hollow cone immersed in a spherical bath (Figure SF1.1, Supporting Information). The model was informed by experimental measurements (scanning electron microscopy graphs of the nanopipette tip geometry in Figure SF2.1, bulk conductivities and viscosities of the solutions in Table ST1.2, Supporting Information). The inner half-cone angle (θ), surface charge of the quartz glass (σ), and diffusion coefficients of the ions in the bath containing 50% PEG 35K (DK+, DCl–) could not be directly measured experimentally. The inner half-cone angle (θ = 7°) was determined by comparing experiments with an analytical expression for the resistance of the nanopipette immersed in a 0.1 M KCl solution (see Section S2.2, Supporting Information for more details). Similarly, the surface charge at the nanopipette quartz wall (σ=−12mCm2) was estimated using the closest fit to the experimental current rectification data (Section S2.4, Supporting Information).
In our system, charge is carried by ions migrating due to the presence of an electric field (electromigration) and concentration gradient (diffusion). (23) In 0.1 M KCl, the ion flux generated by electromigration depends on the sum of the diffusion coefficients of ions in solution (Section S2.3, Supporting Information), which defines the solution conductivity (κ) according to the Nernst−Einstein equation:
(DK++DCl−)RTF2cb=κ
(1)
where DK+ and DCl– are the diffusion coefficients of potassium and chloride, respectively, cb is the bulk concentration, F is the Faraday constant, R is the natural gas constant, and T is the temperature. For KCl, in normal conditions (no PEG), the ratio between the diffusion coefficients of the two species is very close to unity (DK+DCl−∼1), meaning that the contribution of potassium and chloride to the total conductivity κ is approximately the same.
Evidence in the literature has shown that PEG associates with cations in solution. (28−31,38) Zhang et al. (27) developed a molecular dynamics model and proved the interaction between cations and PEG, finding that the trapping time of the ion in the polymer chain is highly dependent on the ion radius with longer trapping times for larger radii. These findings clearly indicate that the diffusion properties of cations in solution are affected by the presence of PEG. In the simulations, we considered this effect by assuming a change in the diffusion coefficients of the two ion species in the external solution. The properties of the 0.1 M KCl electrolyte inside the nanopipette were kept constant as described above.
We performed a parametric study to determine the ratio of the diffusion coefficients by decreasing the contribution of the potassium ion and increasing that of chloride (DK+DCl−<1) to the total conductivity κPEG (Section S2.3, Supporting Information) to describe the experimental i–V of the nanopipette in the presence of PEG, as shown in Figure 1b (orange curve). The study revealed that the lower the ratio of diffusion coefficients, the more asymmetric the i–V response will be (Figure SF2.3, Supporting Information), which supports our hypothesis that the polymer–cation interactions are responsible for the distinctive current response in the presence of PEG. (25,26) We obtained the closest fit to the experimental data (orange square points, Figure 1b) by selecting a diffusion coefficient ratio of DK+DCl− = 0.54, meaning a 35% contribution from the cations and 65% from the anions to the total conductivity of the PEG solution. The simulated currents shown in Figure 1b (orange data points) quantitatively reproduce the experimentally observed i–V response (orange curve).
It is worth clarifying that all input parameters, with or without PEG in the external solution, were either measured experimentally (electrical conductivity, fluid viscosity, and electrolyte concentration) (Table ST1.2, Supporting Information) or found in the literature. In addition, we found that the nanopipette surface charge and any fluid flow in the system minimally influence the simulated i–V response in the presence of PEG in the external solution (Section 2.5 and Table ST2.1, Supporting Information); thus, all modeling results related to PEG presented below were obtained without considering these factors.

Average Ion Concentration at the Tip Region

Figure 2 shows the average ion concentration Cavg=[K+]+[Cl−]2 obtained with finite-element modeling under two opposite voltages applied (V = ±500 mV) in the presence (Figure 2a,b) and absence (Figure 2c,d) of PEG in the external solution (Section S5, Supporting Information). In the presence of PEG, a pronounced ion depletion is observed for V = –500 mV (Figure 2a), while ion enrichment is noticeable when V = +500 mV (Figure 2b), with a 20-fold increase in the ion concentration compared to when a negative bias is applied. This observation is the origin of the asymmetric current response observed in the presence of PEG (Figure 1b). In the absence of PEG in the external solution, a slightly higher ion concentration can be observed within the pore region under V = –500 mV (Figure 2c) compared to the case with V = +500 mV (Figure 2d). This explains the slightly asymmetric curve (ICR) for the no PEG case (gray curve) shown in Figure 1b.24Figure 2e plots the average ion concentration along the symmetry axis of the nanopipette (dashed red line, Figure 2a), allowing for quantitative comparison of the simulations. The average concentration for the PEG (orange curve) and no PEG (gray curve) cases is plotted for V = –500 mV (dashed line) and V = +500 mV (solid line). The average concentration under all the simulated applied potentials is shown in Figure SF5.2 (Section S5.2, Supporting Information). In our reference system, the interface between the nanopipette and the external solution is positioned at z = 0 nm, while z > 0 nm corresponds to the axis of symmetry inside the nanopipette and z < 0 nm to the external solution (Figure SF1.1, Supporting Information). Interestingly, the maximum ion concentration for V = +500 mV in the presence of PEG (orange solid line) is approximately four times higher than the corresponding case with no PEG (gray solid line). This observation indicates that the above-bulk conductivity arises from a dramatic increase in the ion concentration at the tip region of the nanopipette, despite the external solution in the presence of PEG being nine times less conductive (Table ST1.2, Supporting Information). It is worth noting that both the ion enrichment and depletion peaks at z = 0 nm approach Cavg = 100 mM when lower voltages are applied (±400, ±300, ±200, and ±100 mV), as illustrated in Figure SF5.2 (Section S5.2, Supporting Information).

Figure 2

Figure 2. Simulated ion distributions close to the nanopipette tip at ±500 mV in the presence and absence of PEG in the external solution. Average concentration (Cavg = 1/2([K+] + [Cl–])) with (a, b) and without (c, d) PEG in the external solution for an applied voltage of (a, c) −500 mV and (b, d) 500 mV. (e) Average ion concentrations along the nanopipette axis of symmetry (red dashed line in panel a) in the presence (orange) and absence (gray) of PEG for negative (dashed curves) and positive (solid curves) bias applied. Note that average ion concentrations under different applied potentials and individual cation and anion concentration distributions are included in the Supporting Information (Section S5).

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Experimentally, a similar increase in conductivity is observed upon the translocation of a single dsDNA molecule in the presence of PEG in the external solution, as shown in Figure 1c, suggesting that the signal amplification is related to the number of ions in the sensing region of the nanopipette. The vast difference in the ion concentration between the positive and negative bias is similar to the behavior of nanofluidic diodes (39−43) for ultrashort conical nanopores. In these studies, nanofluidic diodes were developed by introducing a surface charge discontinuity on a nanochannel, which forms a junction similar to bipolar semiconductors. In our case, we achieve a similar behavior by introducing an interface between a region where the values for the diffusion coefficients for cations and anions are approximately the same (i.e., the inner solution) and a region where the diffusion coefficient for the cations is much smaller than the one for anions due to the presence of PEG (i.e., the external solution). This discontinuity not only affects the ion distribution but also ion transport, as we describe in the next section.

Ion Transport at the Tip Region

The origin of the significant differences in the ion concentration (Cavg) in the presence of PEG can be understood by a careful analysis of the ion transport (NK+, NCl–) across the interface close to the nanopipette tip aperture, which represents the most sensitive region of our system (44) (Section S6, Supporting Information). We define the “sensing region” as a region between two equipotential lines (dashed lines I and IV, Figure 3) where a 50% drop of the applied voltage is observed. In the case of −500 mV being applied, the voltage drop across the sensing region (ΔVsens) is equal to 250 mV. In the presence of PEG and under −500 mV, we found that this region is about 40 nm in length along the z axis (from z = –20 to z = 20 nm with the interface between the inner and external solutions set at z = 0) (Figures SF6.3 and SF6.4, Supporting Information).

Figure 3

Figure 3. Visualization of the relative contributions of different physical processes to the transport rates of K+ and Cl– at −500 mV (a) and +500 mV (b) with PEG in the outer solution. The lengths of the arrows represent the magnitude of the total transport rate (black) across the respective equipotential line (dashed gray: I, II, III, and IV), which is the sum of electrophoretic (red) and diffusive (blue) contributions. In addition, the arrows being parallel to the z axis and the ion positions were selected for illustration purposes only. Arrows for negligible diffusive contributions are not shown in the plot for ease of representation. The color map in the background represents the average ion concentration, and the dotted line at the nanopipette aperture represents the interface between the inner and the external solution. Further details on the transport calculations and the individual values for the transport rate of each ion for each boundary are included in the Supporting Information (Section S6).

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This clearly indicates a highly resistive region positioned at the nanopipette tip, which leads to a significant drop in the measured current magnitude (baseline current), as shown in Figure 1b (orange curves and square points).
In any enclosed volume, the flux of ions through the surface surrounding the volume is equal to the rate of change in the number of ions (mass and charge conservation). (45) The transport rate for each ion species (Ni) was calculated by integrating the total flux of K+ and Cl– separately, along the equipotential lines (dashed gray lines I, II, III, and IV, Figure 3) selected around the nanopipette tip. An extensive description of these calculations is provided in Section S6 of the Supporting Information. In a nutshell, for 0.1 M KCl, where both ion species have a valence of zi = 1, the difference between the number of charges (ions) entering and exiting each dashed line over time is proportional to the current.
Since no convection was considered for this simulation, the total ion transport rate (black arrow, Figure 3) can be broken down into two components, the electrophoretic (Nim) and diffusive (Nid) (red and blue arrows, respectively, Figure 3). Figure 3 illustrates all these three components, for both cations (green sphere) and anions (purple sphere), at four equipotential lines to highlight the marked difference in ion transport between the inner and outer solutions for V = ±500 mV. The total ion transport rate (black arrows) of each ion species for each applied potential remains constant across the designed dashed lines, verifying that mass and charge are conserved in the system and that the sum of the electrophoretic and diffusive components will always be the same. Based on the polarity of the applied voltage, cations/anions will get attracted/repelled resulting in electrophoretic ion transport either in or out of the nanopipette tip (dotted black line, Figure 3). Additionally, any gradients in the ion concentration (color map in the background of Figure 3) give rise to diffusive ion transport with both species moving toward (with V = −500 mV) or away from the tip interface (with V = 500 mV).
Figure 3 shows that the total ion transport rate at −500 mV is lower than the rate at 500 mV by 75%, which is in agreement with the experimental and simulated i–V responses presented in Figure 1b. It is important to note that the electrophoretic transport dominates diffusion in all cases. To summarize, when V = 500 mV, there are a larger number of ions flowing across the nanopipette tip aperture over time, which results in a higher current magnitude (Table ST6.2, Supporting Information), demonstrating that an asymmetric ion mobility is responsible for the observed above-bulk conductivity. In contrast, when V = −500 mV, there is a low number of ions flowing across the nanopipette tip aperture over time, resulting in a much lower current magnitude (Table ST6.1, Supporting Information), which again is consistent with the experimental data. Figure 3 shows that PEG acts as an anion-selective membrane in the external solution. When V = −500 mV, the cations in the nanopipette flow away from the sensing region toward the inner electrode, while cations in the external solution flow with a lower transport rate toward the nanopipette aperture. This creates a depletion region. On the contrary, when V = + 500 mV, the cations in the nanopipette flow from the inner electrode to the nanopipette tip, while cations at the nanopipette tip flow to the bath solution with hindered ion transport, creating an ion-enriched region.

Mechanism of Current Enhancement upon dsDNA Translocation

DNA molecules carry a negative surface charge and form counterion clouds when immersed in electrolyte solutions (0.1 M KCl). (7,46) In standard conditions (no PEG) and under negative potentials (−500 mV), the temporary increase in the current magnitude recorded during dsDNA translocation is due to a combination of the additional ions carried by the molecule to the sensing region of the nanopipette and the temporary change in the concentration and transport properties of ions in solution, which results in a temporary higher conductivity. (7)
In the presence of PEG, the physics related to the generated current upon dsDNA translocation through the nanopipette aperture is considerably more complex. Our previous work on the polymer–electrolyte nanopore showed that the single-molecule capture physics is remarkably different. In PEG, the capture of DNA follows a linear relationship between the molecule count and the applied voltage, suggesting a diffusion-limited regime in contrast to a barrier-limited regime in the absence of PEG. (26) As previously explained, the nanopipette shows a remarkable ion depletion at the tip region with very few ions transporting through the interface when −500 mV is applied (see the ion concentrations in Figure 2a and transport in Figure 3a), while the external solution is mainly populated by anions, with cation transport hindered by intercalation in the PEG molecules. (27) In these conditions, the counterion cloud carried by the dsDNA molecule certainly contributes to the temporal increase of the ion concentration, and thus the conductivity, of the system. However, this is not sufficient to explain the drastic current enhancement recorded experimentally. In fact, the charge carried by the translocating dsDNA molecule is the same regardless of the presence or absence of PEG in the external solution; thus, the increased conductivity should be approximately equal in both cases (see Section S7.1, Supporting Information).
We explored whether the mechanical interactions between dsDNA and PEG molecules at the interface of the internal and external solutions could temporarily alter the ion concentrations at the tip region. Briefly, we considered a rectangular protrusion of the domain inside the nanopipette (inner solution) toward the bath domain (external solution) to get a simplistic model of the interface shift caused by the translocation of dsDNA, as shown in Figure 4a. We performed a parametric study by varying the size of this protrusion (Δz) from z = 0 to z = –30 nm with 2 nm steps. Figure 4b presents the simulated average ion concentration along the symmetry axis for three different interface displacements (0, 2, and 30 nm). As the interface moves further away from the nanopipette tip opening (z = 0 nm), the number of ions in the nanopipette’s sensing region increases, resulting in an enhanced current value. We found that an interface displacement of 16 nm toward the external solution is sufficient to cause an increase in the ion current to match the current peak maxima measured experimentally for the translocation of a single 4.8 kbp dsDNA molecule (Sections S7.2 and S7.3, Supporting Information). This current enhancement is due to a 33% increase in the ion concentration in the nanopipette sensing region (0 < z < 20 nm) caused by this shift in the interface.

Figure 4

Figure 4. Proposed mechanism of current enhancement upon translocation of a dsDNA molecule. (a) Translocation of a dsDNA molecule through the nanopipette causes a temporary displacement of the interface (Δz) between the pore and external solution (blue dashed line), which results in a temporary ion enrichment in the nanopipette tip region (note that the illustrations are not in scale, and geometries were chosen for illustration purposes only). (b) Simulated average ion concentration along the axis of symmetry (r = 0 nm) for 0 nm (black), 2 nm (cyan), and 30 nm (magenta) interface displacements. (c) Simulated (black curve) and experimental (colored points) current peak maxima (Δi) for different interface displacements toward the external solution and sizes of dsDNA molecules translocating through the nanopipette tip aperture toward the bath, respectively. The error bars represent the standard deviation of the experimental current peak maxima values. The horizontal coordinate of experimental data points was chosen according to the expected Δz (Table ST7.1, Supporting Information). Further details on the interface displacement simulations and the experimental translocation data are included in the Supporting Information (Section S7).

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To summarize, we found that the translocation of dsDNA molecules through the pore causes a temporary displacement of the interface, which results in a shift of the ion depleted region toward the bath. The consequence is ion enrichment in the sensing region inside the nanopipette, which results in higher conductivity and thus higher measured currents (Figure 4a). We speculate that the time required for the ion concentrations in the nanopipette sensing region to return to the pre-translocation values is at the origin of the longer time required to restore the initial baseline current (Figures 1c and SF4.8, Supporting Information). Note that in our simulations, we simplistically assume that the interface between the pore and the external solution without DNA is a straight line at z = 0 nm (no mixing, blue dashed line in Figure 4). The interface is likely to be more diffuse than represented in the model. However, the balancing of the fluxes shown in Figure 3 must still ultimately occur beyond the transition region. Thus, while the model does not capture the detail at the interface exactly, it does capture the reason. Using a more sophisticated model for the interface would certainly improve the accuracy of our calculations but not the level of our understanding of the system.
Based on this mechanism, we expect various dsDNA molecule sizes to have different effects on the translocation current, as recently reported by Confederat et al. (47) for DNA origami. For instance, longer dsDNA molecules would displace the interface further toward the external solution. To test this hypothesis, we repeated the same experiment as the one illustrated in Figure 1 using a range of sizes of dsDNA as the analyte (0.7–7 kbp) with and without PEG in the outside bath (Figure 4c and Sections S7.3 and S7.4, Supporting Information). In PEG, experimental current peak maxima for the translocation of dsDNA molecules with sizes from 0.7 up to 7 kbp are in close agreement with the trend obtained from the simulated current values due to interface displacements, as shown in Figure 4c and Table ST7.1 in the Supporting Information. In the no PEG case, not only there is no evident correlation, but the detection is limited to molecules with a minimum size of 4.8 kbp (Figure SF7.2c, Supporting Information). These findings confirm our initial hypothesis that the current enhancement in the presence of PEG 35K upon dsDNA translocation cannot be explained only in terms of additional ions carried by the analyte, supporting the new pore-centric theory recently reported by Lastra et al. (22) for a system based on a pore’s flux imbalance, but a mechanical interaction between the analyte and PEG molecules at the nanopipette tip opening must be taken into account.
To further support this, we experimentally verified that the voltammetric responses and current enhancement caused by PEG disappear when a positive pressure is applied at the back of the nanopipette to force PEG molecules away from the tip opening (Section S7.5, Supporting Information). This result shows that the PEG effect is completely canceled by disrupting the interface, underpinning the importance of the latter to the observed current enhancement.

Conclusions

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To summarize, we developed a finite-element model to improve our understanding of the dramatic current enhancement observed upon dsDNA molecule translocation through a nanopipette to an external solution containing 50% (w/v) PEG 35K. This system was successfully simulated by assuming a decrease/increase in the diffusion coefficients of cations/anions, respectively, due to the cation-binding properties of PEG. We observed that the characteristic i–V response in the presence of PEG is due to voltage-dependent ion concentrations at the tip region with ion enrichment at positive and ion depletion at negative potentials. A similar behavior was noticed in the asymmetric transport rates for each ion species across the tip orifice, resulting in higher currents at a positive applied bias compared to negative. Furthermore, we demonstrated that conventional mechanisms of current enhancement based on additional ions carried by the analyte are not sufficient to fully explain our system. Hence, we proposed a novel mechanism supported by experimental evidence, which relies on mechanical interactions between the translocating analyte and the interface between the solutions. We proved that such interactions could lead to alteration of the ion distribution at the tip orifice, which can result in temporary current increases. We expect that this work can provide a new paradigm in nanopore sensing, where the alteration of the ion transport properties of the external solution can be harnessed to provide enhanced SNRs allowing for the biochemical and structural analysis of proteins and other biomolecules.

Materials and Methods

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Nanopipette Fabrication

Quartz capillaries of 1.0 mm outer diameter and 0.5 mm inner diameter (QF100-50-7.5; Sutter Instrument) were used to fabricate the nanopipette using the SU-P2000 laser puller (World Precision Instruments). A two-line protocol was used, line 1: HEAT 750/FIL 4/VEL 30/DEL 150/PUL 80, followed by line 2: HEAT 625/FIL 3/VEL 40/DEL 135/PUL 150. The pulling protocol is instrument-specific, and there is variation between different SU-P2000 pullers.

External Bath Preparation

To generate 10 mL of the 50% (w/v) PEG 35K (Sigma Aldrich; 94646) 0.1 M KCl solution, 1 mL of a 1 M KCl solution, 4 mL of ddH2O, and 5 g of PEG 35K were mixed inside a tube. The tube was then left inside a 70 °C incubator for 2 h, followed by overnight incubation at 37 °C. The tubes were then left on a bench for 4 h to reach room temperature prior to use. All electrolytes were stored at room temperature.

Double-Stranded DNA Preparation

To prepare the individual dsDNA samples, the GeneRuler 1 kbp plus DNA Ladder (SM1331: Thermo Fisher) was separated via a 0.8% agarose gel. The individual bands (0.7, 1.5, 2, 3, 4, 4.8, and 7 kbp) were physically isolated with a blade, and the dsDNA was extracted using the Monarch DNA Gel Extraction Kit according to the manufacturer specifications (T1020; New England Biolabs Inc.). The extracted dsDNA was further purified using the Genomic Clean and Concentrator Kit (D4010; Zymo Research). All dsDNA was eluted in the Monarch DNA Elution Buffer (T1016L; New England BioLabs Inc.) and stored at −20 °C. All the dsDNA was then diluted from stock to 0.3 nM with 0.1 M KCl.

Ion Current Trace Recording

The nanopipettes were all filled with 0.3 nM dsDNA diluted in 0.1 M KCl (P/4240/60; Fisher Scientific) and fitted with a Ag/AgCl working electrode. The nanopipettes were immersed into the electrolyte bath containing or not containing PEG 35K with a Ag/AgCl reference electrode. The ionic current trace was recorded using a MultiClamp 700B patch-clamp amplifier (Molecular Devices) in voltage-clamp mode. The signal was filtered using a low-pass filter at 20 kHz and digitized with Digidata 1550B at a 100 kHz sampling rate and recorded using the software pClamp 10 (Molecular Devices).

Finite-Element Modeling

Finite-element simulations were performed with COMSOL Multiphysics 5.6 (COMSOL Inc.).

Data Availability

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The IV curves, ion current traces, and simulation COMSOL report, input parameters and definition, geometry, physics, boundary conditions, and mesh modeling files associated with this paper are openly available from the University of Leeds data repository at https://doi.org/10.5518/1274.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnanoscienceau.2c00050.

  • Supporting Information contents: simulation overview and verification; definition of simulation input parameters; influence of external solution viscosity; reproducibility of experimental data; ion concentrations at the nanopipette tip region; ion transport and the definition of the sensing region; interface displacement model; and effect of an externally applied pressure on the voltammogram and dsDNA translocation (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Authors
  • Authors
    • Fabio Marcuccio - School of Electronic and Electrical Engineering, University of Leeds, LeedsLS2 9JT, U.K.;  Bragg Centre for Materials Research, University of Leeds, LeedsLS2 9JT, U.K.;  Orcidhttps://orcid.org/0000-0003-4816-2896
    • Dimitrios Soulias - School of Electronic and Electrical Engineering, University of Leeds, LeedsLS2 9JT, U.K.;  Bragg Centre for Materials Research, University of Leeds, LeedsLS2 9JT, U.K.;  Orcidhttps://orcid.org/0000-0003-4700-6744
    • Chalmers C. C. Chau - School of Electronic and Electrical Engineering, University of Leeds, LeedsLS2 9JT, U.K.;  Bragg Centre for Materials Research, University of Leeds, LeedsLS2 9JT, U.K.;  School of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, LeedsLS2 9JT, U.K.;  Orcidhttps://orcid.org/0000-0002-3134-6798
    • Sheena E. Radford - School of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, LeedsLS2 9JT, U.K.;  Orcidhttps://orcid.org/0000-0002-3079-8039
    • Eric Hewitt - School of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, LeedsLS2 9JT, U.K.
  • Author Contributions

    F.M. and D.S. contributed equally to this work. CRediT: Fabio Marcuccio conceptualization (equal), investigation (equal), methodology (equal), visualization (equal), writing-original draft (equal); Dimitrios Soulias conceptualization (equal), investigation (equal), methodology (equal), visualization (equal), writing-original draft (equal); Chalmers C. C. Chau conceptualization (supporting), data curation (supporting), investigation (supporting), methodology (supporting), visualization (supporting), writing-review & editing (supporting); Sheena E Radford methodology (supporting), project administration (supporting), resources (supporting), supervision (supporting), writing-review & editing (supporting); Eric Hewitt methodology (supporting), project administration (equal), resources (supporting), supervision (equal), writing-review & editing (equal); Paolo Actis funding acquisition (lead), project administration (lead), supervision (lead), writing-review & editing (supporting); Martin Andrew Edwards conceptualization (lead), formal analysis (supporting), investigation (supporting), project administration (supporting), resources (supporting), supervision (supporting), validation (supporting), writing-original draft (supporting).

  • Funding

    F.M. and P.A. acknowledge funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie MSCA-ITN grant agreement no. 812398, through the single entity nanoelectrochemistry, SENTINEL, project. D.S. acknowledges funding from the University of Leeds. S.E.R. holds a Royal Society Professorial Fellowship (RSRP\R1\211057). P.A. and C.C. acknowledge funding from the Engineering and Physical Science Research Council UK (EPSRC) Healthcare Technologies for the grant no. EP/W004933/1. For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We thank Prof Joshua B. Edel (Imperial College London) for generously providing the MATLAB script used for event analysis in this study. We also thank Prof Aleksei Aksimentiev (University of Illinois, Urbana Champaign) for illuminating discussions. We thank Dr. Nataricha Phisarnchananan (University of Leeds) for performing the viscosity measurement of the electrolyte. We thank Fabio Crameri for providing the color maps used for the simulation plots (Scientific Colour Maps, Zenodo, http://doi.org/10.5281/zenodo.1243862).

Abbreviation

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dsDNA

double-stranded DNA

PEG

poly(ethylene) glycol

SF

supporting figure

SE

supporting equation

ST

supporting table

References

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    Reducing a DNA mol.'s translocation speed in a solid-state nanopore is a key step toward rapid single mol. identification. Here we demonstrate that DNA translocation speeds can be reduced by an order of magnitude over previous results. By controlling the electrolyte temp., salt concn., viscosity, and the elec. bias voltage across the nanopore, we obtain a 3 base/μs translocation speed for 3 kbp double-stranded DNA in a 4-8 nm diam. silicon nitride pore. Our results also indicate that the ionic cond. inside such a nanopore is smaller than it is in bulk.
  18. 18
    Rabinowitz, J.; Edwards, M. A.; Whittier, E.; Jayant, K.; Shepard, K. L. Nanoscale Fluid Vortices and Nonlinear Electroosmotic Flow Drive Ion Current Rectification in the Presence of Concentration Gradients. J. Phys. Chem. A 2019, 123, 8285– 8293,  DOI: 10.1021/acs.jpca.9b04075
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    18
    Nanoscale Fluid Vortices and Nonlinear Electroosmotic Flow Drive Ion Current Rectification in the Presence of Concentration Gradients
    Rabinowitz Jake; Whittier Elizabeth; Jayant Krishna; Shepard Kenneth L; Edwards Martin A
    The journal of physical chemistry. A (2019), 123 (38), 8285-8293 ISSN:.
    Ion current rectification (ICR) is a transport phenomenon in which an electrolyte conducts unequal currents at equal and opposite voltages. Here, we show that nanoscale fluid vortices and nonlinear electroosmotic flow (EOF) drive ICR in the presence of concentration gradients. The same EOF can yield negative differential resistance (NDR), in which current decreases with increasing voltage. A finite element model quantitatively reproduces experimental ICR and NDR recorded across glass nanopipettes under concentration gradients. The model demonstrates that spatial variations of electrical double layer properties induce the nanoscale vortices and nonlinear EOF. Experiments are performed in conditions directly related to scanning probe imaging and show that quantitative understanding of nanoscale transport under concentration gradients requires accounting for EOF. This characterization of nanopipette transport physics will benefit diverse experimentation, pushing the resolution limits of chemical and biophysical recordings.
  19. 19
    Lan, W. J.; Edwards, M. A.; Luo, L.; Perera, R. T.; Wu, X.; Martin, C. R.; White, H. S. Voltage-Rectified Current and Fluid Flow in Conical Nanopores. Acc. Chem. Res. 2016, 49, 2605– 2613,  DOI: 10.1021/acs.accounts.6b00395
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    19
    Voltage-Rectified Current and Fluid Flow in Conical Nanopores
    Lan, Wen-Jie; Edwards, Martin A.; Luo, Long; Perera, Rukshan T.; Wu, Xiaojian; Martin, Charles R.; White, Henry S.
    Accounts of Chemical Research (2016), 49 (11), 2605-2613CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    A review. Ion current rectification (ICR) refers to the asym. potential-dependent rate of the passage of soln. ions through a nanopore, giving rise to elec. current-voltage characteristics that mimic those of a solid-state elec. diode. Since the discovery of ICR in quartz nanopipettes two decades ago, synthetic nanopores and nanochannels of various geometries, fabricated in membranes and on wafers, have been extensively investigated to understand fundamental aspects of ion transport in highly confined geometries. It is now generally accepted that ICR requires an asym. elec. double layer within the nanopore, producing an accumulation or depletion of charge-carrying ions at opposite voltage polarities. Our research groups have recently explored how the voltage-dependent ion distributions and ICR within nanopores can induce novel nanoscale flow phenomena that have applications in understanding ionics in porous materials used in energy storage devices, chem. sensing, and low-cost elec. pumping of fluids. In this Account, we review our most recent investigations on this topic, based on expts. using conical nanopores (10-300 nm tip opening) fabricated in thin glass, mica, and polymer membranes. Measurable fluid flow in nanopores can be induced either using external pressure forces, elec. via electroosmotic forces, or by a combination of these two forces. We demonstrate that pressure-driven flow can greatly alter the elec. properties of nanopores and, vice versa, that the nonlinear elec. properties of conical nanopores can impart novel and useful flow phenomena.Electroosmotic flow (EOF), which depends on the magnitude of the ion fluxes within the double layer of the nanopore, is strongly coupled to the accumulation/depletion of ions. Thus, the same underlying cause of ICR also leads to EOF rectification, i.e., unequal flows occurring for the same voltage but opposite polarities. EOF rectification can be used to elec. pump fluids with very precise control across membranes contg. conical pores via the application of a sym. sinusoidal voltage. The combination of pressure and asym. EOF can also provide a means to generate new nanopore elec. behaviors, including neg. differential resistance (NDR), in which the current through a conical pore decreases with increasing driving force (applied voltage), similar to solid-state tunnel diodes. NDR results from a pos. feedback mechanism between the ion distributions and EOF, yielding a true bistability in both fluid flow and elec. current at a crit. applied voltage. Nanopore-based NDR is extremely sensitive to the surface charge near the nanopore opening, suggesting possible applications in chem. sensing.
  20. 20
    Perera, R. T.; Johnson, R. P.; Edwards, M. A.; White, H. S. Effect of the Electric Double Layer on the Activation Energy of Ion Transport in Conical Nanopores. J. Phys. Chem. C 2015, 119, 24299– 24306,  DOI: 10.1021/acs.jpcc.5b08194
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    Effect of the electric double layer on the activation energy of ion transport in conical nanopores
    Perera, Rukshan T.; Johnson, Robert P.; Edwards, Martin A.; White, Henry S.
    Journal of Physical Chemistry C (2015), 119 (43), 24299-24306CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
    Measured apparent activation energies, EA, of ion transport (K+ and Cl-) in conical glass nanopores are reported as a function of applied voltage (-0.5 to 0.5 V), pore size (20-2000 nm), and electrolyte concn. (0.1-50 mM). EA values for transport within an elec. charged conical glass nanopore differ from the bulk values due to the voltage and temp.-dependent distribution of the ions within the double layer. Remarkably, nanopores that display ion current rectification also display a large decrease in EA under accumulation mode conditions (at applied neg. voltages vs. an external ground) and a large increase in EA under depletion mode conditions (at pos. voltages). Finite element simulations based on the Poisson-Nernst-Planck model semiquant. predict the measured temp.-dependent cond. and dependence of EA on applied voltage. The results highlight the relationships between the distribution of ions with the nanopore, ionic current, and EA and their dependencies on pore size, temp., ion concn., and applied voltage.
  21. 21
    Luo, L.; Holden, D. A.; Lan, W.-J.; White, H. S. Tunable Negative Differential Electrolyte Resistance in a Conical Nanopore in Glass. ACS Nano 2012, 6, 6507– 6514,  DOI: 10.1021/nn3023409
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    Tunable Negative Differential Electrolyte Resistance in a Conical Nanopore in Glass
    Luo, Long; Holden, Deric A.; Lan, Wen-Jie; White, Henry S.
    ACS Nano (2012), 6 (7), 6507-6514CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    Liq.-phase neg. differential resistance (NDR) is obsd. in the i-V behavior of a conical nanopore (∼300 nm orifice radius) in a glass membrane that separates an external low-cond. 5 mM KCl soln. of DMSO/H2O (vol./vol. 3:1) from an internal high-cond. 5 mM KCl aq. soln. NDR appears in the i-V curve of the neg. charged nanopore as the voltage-dependent electroosmotic force opposes an externally applied pressure force, continuously moving the location of the interfacial zone between the two miscible solns. to a position just inside the nanopore orifice. An ∼80% decrease in the ionic current occurs over less that a ∼ 10 mV increase in applied voltage. The NDR turn-on voltage is tunable over a ∼ 1 V window by adjusting the applied external pressure from 0 to 50 mmHg. Finite-element simulations based on soln. of Navier-Stokes, Poisson, and convective Nernst-Planck equations for mixed solvent electrolytes within a neg. charged nanopore yield predictions of the NDR behavior that are in qual. agreement with the exptl. observations. Applications in chem. sensing of a tunable, soln.-based elec. switch based on the NDR effect are discussed.
  22. 22
    Lastra, L. S.; Bandara, Y. M. N. D. Y.; Nguyen, M.; Farajpour, N.; Freedman, K. J. On the Origins of Conductive Pulse Sensing inside a Nanopore. Nat. Commun. 2022, 13, 2186,  DOI: 10.1038/s41467-022-29758-8
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    On the origins of conductive pulse sensing inside a nanopore
    Lastra, Lauren S.; Bandara, Y. M. Nuwan D. Y.; Nguyen, Michelle; Farajpour, Nasim; Freedman, Kevin J.
    Nature Communications (2022), 13 (1), 2186CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)
    Nanopore sensing is nearly synonymous with resistive pulse sensing due to the characteristic occlusion of ions during pore occupancy, particularly at high salt concns. Contrarily, conductive pulses are obsd. under low salt conditions wherein electroosmotic flow is significant. Most literature reports counterions as the dominant mechanism of conductive events (a mol.-centric theory). However, the counterion theory does not fit well with conductive events occurring via net neutral-charged protein translocation, prompting further investigation into translocation mechanics. Herein, we demonstrate theory and expts. underpinning the translocation mechanism (i.e., electroosmosis or electrophoresis), pulse direction (i.e., conductive or resistive) and shape (e.g., monophasic or biphasic) through fine control of chem., phys., and electronic parameters. Results from these studies predict strong electroosmosis plays a role in driving DNA events and generating conductive events due to polarization effects (i.e., a pore-centric theory).
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    White, H. S.; Bund, A. Ion Current Rectification at Nanopores in Glass Membranes. Langmuir 2008, 24, 2212– 2218,  DOI: 10.1021/la702955k
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    Ion Current Rectification at Nanopores in Glass Membranes
    White, Henry S.; Bund, Andreas
    Langmuir (2008), 24 (5), 2212-2218CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    The origin of ion current rectification obsd. at conical-shaped nanopores in glass membranes immersed in KCl solns. has been investigated using finite-element simulations. The ion concns. and fluxes (due to diffusion, migration, and electroosmotic convection) were detd. by the simultaneous soln. of the Nernst-Planck, Poisson, and Navier-Stokes equations for the two-ion (K+ and Cl-) system. Fixed surface charge on both the internal and external glass surfaces that define the pore structure was included to account for elec. fields and nonuniform ion cond. within the nanopores and elec. fields in the external soln. near the pore mouth. We demonstrate that previous observations of ion current rectification in conical-shaped glass nanopores are a consequence of the voltage-dependent soln. cond. in the vicinity of the pore mouth, both inside and outside of the pore. The simulations also demonstrate that current rectification is maximized at intermediate bulk ion concns., a combination of (i) the elec. screening of surface charge at high concns. and (ii) a fixed no. of charge-carrying ions in the pore at lower concn., which are phys. conditions where the voltage dependence of the cond. disappears. In addn., we have quant. shown that electroosmotic flow gives rise to a significant but small contribution to current rectification.
  24. 24
    Wei, C.; Bard, A. J.; Feldberg, S. W. Current Rectification at Quartz Nanopipet Electrodes. Anal. Chem. 1997, 69, 4627– 4633,  DOI: 10.1021/ac970551g
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    Current rectification at quartz nanopipet electrodes
    Wei, Chang; Bard, Allen J.; Feldberg, Stephen W.
    Analytical Chemistry (1997), 69 (22), 4627-4633CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Ag/AgCl ref. electrodes fabricated from pulled quartz tubes with orifice radii of 20 nm to 20 μm were characterized in KCl solns. of different concns. by cyclic voltammetry. Linear current-voltage (i-V) dependence was obsd. with micropipet electrodes (with micrometer-sized tips) with the same concn. (0.01-1 M) of KCl inside and outside the pipet, but current rectification was found at nanopipet electrodes at KCl concns. of ≤0.1 M (with nanometer-sized tips). This is attributed to formation of a diffuse elec. double layer within the tip orifice. The effects of electrode size, electrolyte concn., and soln. pH on the nonlinear i-V behavior of these electrodes were studied. A model for the obsd. behavior shows the rectification to be caused by the permselectivity in the tip region and the geometric asymmetry of the tip orifice. This phenomenon may be important in studies of ion transport in charged channels and porous membranes.
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    Chau, C. C.; Radford, S. E.; Hewitt, E. W.; Actis, P. Macromolecular Crowding Enhances the Detection of DNA and Proteins by a Solid-State Nanopore. Nano Lett. 2020, 20, 5553– 5561,  DOI: 10.1021/acs.nanolett.0c02246
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    Macromolecular Crowding Enhances the Detection of DNA and Proteins by a Solid-State Nanopore
    Chau, Chalmers C.; Radford, Sheena E.; Hewitt, Eric W.; Actis, Paolo
    Nano Letters (2020), 20 (7), 5553-5561CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
    Nanopore anal. of nucleic acid is now routine, but detection of proteins remains challenging. Here, the authors report the systematic characterization of the effect of macromol. crowding on the detection sensitivity of a solid-state nanopore for circular and linearized DNA plasmids, globular proteins (β-galactosidase), and filamentous proteins (α-synuclein amyloid fibrils). A remarkable ∼1000-fold increase in the mol. count for the globular protein β-galactosidase and a 6-fold increase in peak amplitude for plasmid DNA under crowded conditions. were obsd. Also macromol. crowding facilitates the study of the topol. of DNA plasmids and the characterization of amyloid fibril prepns. with different length distributions. A remarkable feature of this method is its ease of use; it simply requires the addn. of a macromol. crowding agent to the electrolyte. The authors therefore envision that macromol. crowding can be applied to many applications in the anal. of biomols. by solid-state nanopores.
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    Chau, C.; Marcuccio, F.; Soulias, D.; Edwards, M. A.; Tuplin, A.; Radford, S. E.; Hewitt, E.; Actis, P. Probing RNA Conformations Using a Polymer–Electrolyte Solid-State Nanopore. ACS Nano 2022, 16, 20075– 20085,  DOI: 10.1021/acsnano.2c08312
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    26
    Probing RNA Conformations Using a Polymer-Electrolyte Solid-State Nanopore
    Chau, Chalmers; Marcuccio, Fabio; Soulias, Dimitrios; Edwards, Martin Andrew; Tuplin, Andrew; Radford, Sheena E.; Hewitt, Eric; Actis, Paolo
    ACS Nano (2022), 16 (12), 20075-20085CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    Nanopore systems have emerged as a leading platform for the anal. of biomol. complexes with single-mol. resoln. The conformation of biomols., such as RNA, is highly dependent on the electrolyte compn., but solid-state nanopore systems often require high salt concn. to operate, precluding anal. of macromol. conformations under physiol. relevant conditions. Here, we report the implementation of a polymer-electrolyte solid-state nanopore system based on alkali metal halide salts dissolved in 50% w/v poly(ethylene) glycol (PEG) to augment the performance of our system. We show that polymer-electrolyte bath governs the translocation dynamics of the analyte which correlates with the phys. properties of the salt used in the bath. This allowed us to identify CsBr as the optimal salt to complement PEG to generate the largest signal enhancement. Harnessing the effects of the polymer-electrolyte, we probed the conformations of the Chikungunya virus (CHIKV) RNA genome fragments under physiol. relevant conditions. Our system was able to fingerprint CHIKV RNA fragments ranging from ∼300 to ∼2000 nt length and subsequently distinguish conformations between the co-transcriptionally folded and the natively refolded ∼2000 nt CHIKV RNA. We envision that the polymer-electrolyte solid-state nanopore system will further enable structural and conformational analyses of individual biomols. under physiol. relevant conditions.
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    Zhang, Z.; Ohl, M.; Diallo, S. O.; Jalarvo, N. H.; Hong, K.; Han, Y.; Smith, G. S.; Do, C. Dynamics of Water Associated with Lithium Ions Distributed in Polyethylene Oxide. Phys. Rev. Lett. 2015, 115, 198301  DOI: 10.1103/PhysRevLett.115.198301
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    Dynamics of water associated with lithium ions distributed in polyethylene oxide
    Zhang, Zhe; Ohl, Michael; Diallo, Souleymane O.; Jalarvo, Niina H.; Hong, Kunlun; Han, Youngkyu; Smith, Gregory S.; Do, Changwoo
    Physical Review Letters (2015), 115 (19), 198301/1-198301/6CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
    The dynamics of water in polyethylene oxide (PEO)/LiCl soln. has been studied with quasielastic neutron scattering expts. and mol. dynamics (MD) simulations. Two different time scales of water diffusion representing interfacial water and bulk water dynamics have been identified. The measured diffusion coeff. of interfacial water remained 5-10 times smaller than that of bulk water, but both were slowed by approx. 50% in the presence of Li+. Detailed anal. of MD trajectories suggests that Li+ is favorably found at the surface of the hydration layer, and the probability to find the caged Li+ configuration formed by the PEO is lower than for the noncaged Li+- PEO configuration. In both configurations, however, the slowing down of water mols. is driven by reorienting water mols. and creating water-Li+ hydration complexes. Performing the MD simulation with different ions (Na+ and K+) revealed that smaller ionic radius of the ions is a key factor in disrupting the formation of PEO cages by allowing spaces for water mols. to come in between the ion and PEO.
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    Ren, C.; Tian, W.; Szleifer, I.; Ma, Y. Specific Salt Effects on Poly(Ethylene Oxide) Electrolyte Solutions. Macromolecules 2011, 44, 1719– 1727,  DOI: 10.1021/ma1027752
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    Specific Salt Effects on Poly(ethylene oxide) Electrolyte Solutions
    Ren, Chun-lai; Tian, Wen-de; Szleifer, Igal; Ma, Yu-qiang
    Macromolecules (Washington, DC, United States) (2011), 44 (6), 1719-1727CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
    Exploring the mechanism of specific salt effects in electrolyte solns. is an old and attractive subject. It has been gradually realized that the competition at the mol. level plays an important role. Aiming to include mol. details as many as possible, we combine mol. dynamics (MD) simulations with a mol. theory to study specific salt effects on poly(ethylene oxide) (PEO) solns. with the addn. of monovalent salt. Radial distribution functions obtained from MD simulations provide microscopic structures of different components as well as interactions between various species. On the basis of these interactions, we construct the mol. theory with four assumptions: (1) an ion along with bound water in the first shell works as a single entity; (2) short-ranged interactions among various species are modeled as hydrogen-bonding interactions; (3) the ability of a hydrated ion to provide donors/acceptors for hydrogen bonding is governed by the charge d.; (4) contact ion pairs are included, esp. in the cases of small cations. The mol. theory is generalized with the explicit inclusion of ion-PEO, ion-water, ion-ion, water-water, and water-PEO hydrogen bonds. This means the mol.-scale structure and interaction are included within the frame of the theory. Theor. calcd. cloud points verify that the salting-out ability for alkali metal ions follows the series of K+ > Rb+ > Cs+ > Na+ > Li+, which is in agreement with the exptl. observations. Here, the competition among ion-PEO, ion-water, and water-PEO interactions and the impact of steric repulsions induced by the introduction of ions are two essential factors detg. the phase behavior of PEO solns. The combined methods bridge the microscopic interactions and structures to the macroscopic behavior.
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    Poudel, L.; Podgornik, R.; Ching, W.-Y. The Hydration Effect and Selectivity of Alkali Metal Ions on Poly(Ethylene Glycol) Models in Cyclic and Linear Topology. J. Phys. Chem. A 2017, 121, 4721– 4731,  DOI: 10.1021/acs.jpca.7b04061
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    29
    The Hydration Effect and Selectivity of Alkali Metal Ions on Poly(ethylene glycol) Models in Cyclic and Linear Topology
    Poudel, Lokendra; Podgornik, Rudolf; Ching, Wai-Yim
    Journal of Physical Chemistry A (2017), 121 (24), 4721-4731CODEN: JPCAFH; ISSN:1089-5639. (American Chemical Society)
    The effects of hydration and alkali metal (K, Na, and Li) bonding on two structural variants of polyethylene glycol (PEG), a cyclic (18-crown-6) configuration and a linear chain model with two different lengths, are studied by ab initio d. functional calcns. A total of 24 structural models are constructed, with different conformations of the PEG chain and its mol. environment. Detailed comparisons of the results enable us to obtain conclusive evidence on the effect of the different components of the soln. environment on the PEG structural variants. The results include the binding energy, the partial charge distribution, the solvation effect, interfacial hydrogen bonding and cohesion between different structural units in the system composed of PEG, alkali metal ions and water. Based on these comprehensive and precise comparisons, we conclude that the ion-PEG interaction is strongly influenced by the presence of solvent and the charge transfer in PEG complex depends strongly on the topol., the type of alkali metal ion, and the solvent. The interaction between alkali metal ions in the two PEG models does not always scale with the ion size but depends on their local environment.
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    Tao, Z.; Cummings, P. T. Molecular Dynamics Simulation of Inorganic Ions in PEO Aqueous Solution. Mol. Simul. 2007, 33, 1255– 1260,  DOI: 10.1080/08927020701697691
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    Molecular dynamics simulation of inorganic ions in PEO aqueous solution
    Tao, Z.; Cummings, P. T.
    Molecular Simulation (2007), 33 (14-15), 1255-1260CODEN: MOSIEA; ISSN:0892-7022. (Taylor & Francis Ltd.)
    Solid polymer electrolytes (SPEs), esp. the ones dissolving lithium ions in poly ethylene oxide (PEO) polymer by the bonds between ether oxygen and cations, have long been investigated with the goals of developing batteries with high energy d. It has been accepted that most ions move through the amorphous polymer phase and their mobility depends crucially on the soln. environment, though the detailed transport mechanism is not fully developed. Recently, ternary mixts. composed of PEO/salts in aq. soln. have been shown to display more attractive properties than binary SPE mixts. Numerous expts. have found a dramatically changed environment for the cations and increased ionic cond. of polymer/salts electrolytes for increased relative humidity, suggesting that the coupling between polymer chains and cations may be weakened due to the existence of water mols. In this paper we report mol. dynamics (MD) simulation, using an optimized force field that includes polarizabilities via the dynamic shell model, to study the structural properties of inorg. ions in PEO aq. soln. and the competitive solvation of ions between water and polymer oxygen. Our simulation results show that ions are solvated more favorably by water than by polymer. This conclusion is in a good agreement with neutron diffraction by isotropic substitution (NDIS) expts.
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    Giesecke, M.; Hallberg, F.; Fang, Y.; Stilbs, P.; Furó, I. Binding of Monovalent and Multivalent Metal Cations to Polyethylene Oxide in Methanol Probed by Electrophoretic and Diffusion NMR. J. Phys. Chem. B 2016, 120, 10358– 10366,  DOI: 10.1021/acs.jpcb.6b08923
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    Binding of Monovalent and Multivalent Metal Cations to Polyethylene Oxide in Methanol Probed by Electrophoretic and Diffusion NMR
    Giesecke, Marianne; Hallberg, Fredrik; Fang, Yuan; Stilbs, Peter; Furo, Istvan
    Journal of Physical Chemistry B (2016), 120 (39), 10358-10366CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
    Complex formation in methanol between monodisperse polyethylene oxide (PEO) and a large set of cations was studied by measuring the effective charge acquired by the PEO upon complexation. Quant. data were obtained at the low ionic strength of 2 mM (for some salts, also between 0.5 mM and 6 mM) by a combination of diffusion NMR and electrophoretic NMR expts. For strongly complexing cations, the magnitude of the acquired effective charge was in the order of 1 cation per 100 monomer units. For monovalent cations, the relative strength of binding varies as Na+ < K+ ≈ Rb+ ≈ Cs+ while Li+ exhibited no significant binding. All polyvalent cations bind weakly, except for Ba2+ that exhibited strong binding. Anions do not bind as is shown by the lack of response to the chem. nature of anionic species (perchlorate, iodide or acetate). Diffusion expts. show directly that the acetate anion with monovalent cations does not assoc. to PEO. Considering all cations, we find that the obsd. binding does not follow any Hofmeister order. Instead, binding occurs below a crit. surface charge d. which indicates that the degree of complexation is defined by the solvation shell. A large solvation shell prevents the binding of most multivalent ions.
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    Siwy, Z. S. Ion-Current Rectification in Nanopores and Nanotubes with Broken Symmetry. Adv. Funct. Mater. 2006, 16, 735– 746,  DOI: 10.1002/adfm.200500471
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    Ion-current rectification in nanopores and nanotubes with broken symmetry
    Siwy, Zuzanna S.
    Advanced Functional Materials (2006), 16 (6), 735-746CODEN: AFMDC6; ISSN:1616-301X. (Wiley-VCH Verlag GmbH & Co. KGaA)
    This article focuses on ion transport through nanoporous systems with special emphasis on rectification phenomena. The effect of ion-current rectification is obsd. as asym. current-voltage (I-V) curves, with the current recorded for one voltage polarity higher than the current recorded for the same abs. value of voltage of opposite polarity. This diode-like I-V curve indicates that there is a preferential direction for ion flow. Exptl. evidence that ion-current rectification is inherent to asym., e.g., tapered, nanoporous systems with excess surface charge is provided and discussed. The fabrication and operation of asym. polymer nanopores, gold nanotubes, glass nanocapillaries, and silicon nanopores are presented. The possibility of tuning the direction and extent of rectification is discussed in detail. The theor. models that have been developed to explain the ion-current rectification effect are also presented.
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    Momotenko, D.; Cortés-Salazar, F.; Josserand, J.; Liu, S.; Shao, Y.; Girault, H. H. Ion Current Rectification and Rectification Inversion in Conical Nanopores: A Perm-Selective View. Phys. Chem. Chem. Phys. 2011, 13, 5430– 5440,  DOI: 10.1039/C0CP02595J
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    Ion current rectification and rectification inversion in conical nanopores. A perm-selective view
    Momotenko, Dmitry; Cortes-Salazar, Fernando; Josserand, Jacques; Liu, Shujuan; Shao, Yuanhua; Girault, Hubert H.
    Physical Chemistry Chemical Physics (2011), 13 (12), 5430-5440CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)
    Ionic transport in charged conical nanopores is known to give rise to ion current rectification. The rectification direction can be inverted when using electrolyte solns. at very low ionic strengths. To elucidate these phenomena, electroneutral conical nanopores contg. a perm-selective region at the tip were investigated and shown to behave like classical charged nanopores. An anal. model is proposed to account for these rectification processes.
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    Wen, C.; Zeng, S.; Li, S.; Zhang, Z.; Zhang, S.-L. On Rectification of Ionic Current in Nanopores. Anal. Chem. 2019, 91, 14597– 14604,  DOI: 10.1021/acs.analchem.9b03685
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    On Rectification of Ionic Current in Nanopores
    Wen, Chenyu; Zeng, Shuangshuang; Li, Shiyu; Zhang, Zhen; Zhang, Shi-Li
    Analytical Chemistry (Washington, DC, United States) (2019), 91 (22), 14597-14604CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Rectification of ionic current, a frequently obsd. phenomenon with asym. nanopores varying in geometry and/or surface charge, has been utilized for studies of microfluidic circuits, nanopore sensors, and energy conversion devices. However, the physics behind the rectification phenomenon deserves further anal., and the involved processes need renewed organization; however, the origin is known, and numerous simulations based on the Poisson-Nernst-Planck formalism provide details of the observation. Here, we present an anal. model by identifying the causal chain connecting the key phys. factors and processes leading to rectification: the charge present on the pore sidewalls causing the selectivity of ion fluxes through the pore, the selectivity inducing enrichment-depletion of ions around the pore, and the established ion concn. gradient rendering the elec. field redistribution in the pore. Our anal. model that considers nanopore geometry, surface charge d., and electrolyte concn. calcs. the ionic current and corresponding rectification factor at given bias voltages. The model is validated by numerical simulations, and the model results agree well with exptl. data. It is, therefore, a useful tool not only for gaining phys. insights into ionic current rectification but also for providing practical guidelines in designing nanopore- and nanopipette-based ion sensors for a range of applications.
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    Charron, M.; Briggs, K.; King, S.; Waugh, M.; Tabard-Cossa, V. Precise DNA Concentration Measurements with Nanopores by Controlled Counting. Anal. Chem. 2019, 91, 12228– 12237,  DOI: 10.1021/acs.analchem.9b01900
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    Precise DNA Concentration Measurements with Nanopores by Controlled Counting
    Charron, Martin; Briggs, Kyle; King, Simon; Waugh, Matthew; Tabard-Cossa, Vincent
    Analytical Chemistry (Washington, DC, United States) (2019), 91 (19), 12228-12237CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Using a solid-state nanopore to measure the concn. of clin. relevant target analytes, such as proteins or specific DNA sequences, is a major goal of nanopore research. This is usually achieved by measuring the capture rate of the target analyte through the pore. However, progress is hindered by sources of systematic error that are beyond the level of control currently achievable with state-of-the-art nanofabrication techniques. In this work, we show that the capture rate process of solid-state nanopores is subject to significant sources of variability, both within individual nanopores over time and between different nanopores of nominally identical size, which are absent from theor. electrophoretic capture models. We exptl. reveal that these fluctuations are inherent to the nanopore itself and make nanopore-based mol. concn. detn. insufficiently precise to meet the stds. of most applications. In this work, we present a simple method by which to reduce this variability, increasing the reliability, accuracy, and precision of single-mol. nanopore-based concn. measurements. We demonstrate controlled counting, a concn. measurement technique, which involves measuring the simultaneous capture rates of a mixt. of both the target mol. and an internal calibrator of precisely known concn. Using this method on linear DNA fragments, we show empirically that the requirements for precisely controlling the nanopore properties, including its size, height, geometry, and surface charge d. or distribution, are removed while allowing for higher-precision measurements. The quant. tools presented herein will greatly improve the utility of solid-state nanopores as sensors of target biomol. concn.
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    Nanometer-scale pores have demonstrated potential for the elec. detection, quantification, and characterization of mols. for biomedical applications and the chem. anal. of polymers. Despite extensive research in the nanopore sensing field, there is a paucity of theor. models that incorporate the interactions between chems. (i.e., solute, solvent, analyte, and nanopore). Here, we develop a model that simultaneously describes both the current blockade depth and residence times caused by individual poly(ethylene glycol) (PEG) mols. in a single α-hemolysin ion channel. Modeling polymer-cation binding leads to a description of two significant effects: a redn. in the mobile cation concn. inside the pore and an increase in the affinity between the polymer and the pore. The model was used to est. the free energy of formation for K+-PEG inside the nanopore (≈ -49.7 meV) and the free energy of PEG partitioning into the nanopore (≈ 0.76 meV per ethylene glycol monomer). The results suggest that rational, phys. models for the anal. of analyte-nanopore interactions will develop the full potential of nanopore-based sensing for chem. and biol. applications.
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    The authors demonstrate rectification of ionic transport in a nanofluidic diode fabricated by introducing a surface charge discontinuity in a nanofluidic channel. Device current-voltage (I-V) characteristics agree qual. with a 1-dimensional model at moderate to high ionic concns. This study illustrates ionic flow control using surface charge patterning in nanofluidic channels under high bias voltages.
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    Recently reported exptl. and theor. studies of nanofluidic nonlinear devices, such as bipolar and unipolar ionic diodes, have yet to answer the question about the possibility of their further miniaturization. We theor. investigate the effects of size redn., applied bias, and soln. ionic strength in such devices. We compare the numerical solns. of the Poisson, Nernst-Planck (PNP), and Navier-Stokes (NS) equations with their one-dimensional, anal. approxns. We demonstrate that the contribution of electroosmosis is insignificant and find anal. approxns. to PNP for bipolar and unipolar diodes that are in good agreement with numerical 3D solns. We identify the minimal dimensions for such diodes that demonstrate ion current rectification behavior and demonstrate the importance of the edge effect in very short diodes.
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    Ionic transport at the nanoscale features phenomena that are not obsd. in larger systems. Nonlinear current-voltage curves characteristic of ionic diodes as well as ion selectivity are examples of effects obsd. at the nanoscale. Many man-made nanopore systems are inspired by biol. channels in a cell membrane, thus measurements are often performed in aq. solns. Consequently, much less is known about ionic transport in nonaq. systems, esp. in solid-state electrolytes. Here we show ionic transport through single pores filled with gel electrolyte of poly(Me methacrylate) (PMMA) doped with LiClO4 in propylene carbonate. The system has no liq. interface and the ionic transport occurs through the porous gel structure. We demonstrate that a conically shaped nanopore filled with the gel rectifies the current and works as a solid-state ionic diode.
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    Nanopores that exhibit ionic current rectification (ICR) behave like diodes such that they transport ions more efficiently in one direction than in the other. Conical nanopores have been shown to rectify ionic current, but only those with at least 500 nm in length exhibit significant ICR. Here, through the finite element method, we show how ICR of conical nanopores with lengths below 200 nm can be tuned by controlling individual charged surfaces, i.e., the inner pore surface (surfaceinner) and exterior pore surfaces on the tip and base side (surfacetip and surfacebase). The charged surfaceinner and surfacetip can induce obvious ICR individually, while the effects of the charged surfacebase on ICR can be ignored. The fully charged surfaceinner alone could render the nanopore counterion-selective and induces significant ion concn. polarization in the tip region, which causes reverse ICR compared to nanopores with all surfaces charged. In addn., the direction and degree of rectification can be further tuned by the depth of the charged surfaceinner. When considering the exterior membrane surface only, the charged surfacetip causes intrapore ionic enrichment and depletion under opposite biases, which results in significant ICR. Its effective region is within ∼40 nm beyond the tip orifice. We also found that individual charged parts of the pore system contributed to ICR in an additive way because of the additive effect on the ion concn. regulation along the pore axis. With various combinations of fully/partially charged surfaceinner and surfacetip, diverse ICR ratios from ∼2 to ∼170 can be achieved. Our findings shed light on the mechanism of ICR in ultrashort conical nanopores and provide a useful guide to the design and modification of ultrashort conical nanopores in ionic circuits and nanofluidic sensors.
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  • Abstract

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    Figure 1

    Figure 1. Schematic and representative data for current-voltage and conductive-pulse measurements of dsDNA translocation through a nanopipette. (a) Nanopipette (25 nm pore diameter), filled with a 0.3 nM solution of 4.8 kbp dsDNA in 0.1 M KCl, is immersed in a solution of the same electrolyte with and without the presence of 50% (w/v) PEG 35K. The application of a negative potential to a Ag/AgCl quasi-reference electrode inside the nanopipette with respect to a ground electrode in the external solution causes outbound migration of DNA molecules. (b) Experimental (curves) and simulated (points) voltammograms of the nanopipette in the presence (orange) and absence (gray) of PEG in the outside solution. (c) Representative current trace recorded upon translocation of a dsDNA molecule through the nanopipette aperture with (orange trace) and without (gray trace) the presence of PEG in the external solution. Further examples of voltammetry and current traces from this and other nanopipettes are included in the Supporting Information (Section S4).

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    Figure 2

    Figure 2. Simulated ion distributions close to the nanopipette tip at ±500 mV in the presence and absence of PEG in the external solution. Average concentration (Cavg = 1/2([K+] + [Cl–])) with (a, b) and without (c, d) PEG in the external solution for an applied voltage of (a, c) −500 mV and (b, d) 500 mV. (e) Average ion concentrations along the nanopipette axis of symmetry (red dashed line in panel a) in the presence (orange) and absence (gray) of PEG for negative (dashed curves) and positive (solid curves) bias applied. Note that average ion concentrations under different applied potentials and individual cation and anion concentration distributions are included in the Supporting Information (Section S5).

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    Figure 3

    Figure 3. Visualization of the relative contributions of different physical processes to the transport rates of K+ and Cl– at −500 mV (a) and +500 mV (b) with PEG in the outer solution. The lengths of the arrows represent the magnitude of the total transport rate (black) across the respective equipotential line (dashed gray: I, II, III, and IV), which is the sum of electrophoretic (red) and diffusive (blue) contributions. In addition, the arrows being parallel to the z axis and the ion positions were selected for illustration purposes only. Arrows for negligible diffusive contributions are not shown in the plot for ease of representation. The color map in the background represents the average ion concentration, and the dotted line at the nanopipette aperture represents the interface between the inner and the external solution. Further details on the transport calculations and the individual values for the transport rate of each ion for each boundary are included in the Supporting Information (Section S6).

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    Figure 4

    Figure 4. Proposed mechanism of current enhancement upon translocation of a dsDNA molecule. (a) Translocation of a dsDNA molecule through the nanopipette causes a temporary displacement of the interface (Δz) between the pore and external solution (blue dashed line), which results in a temporary ion enrichment in the nanopipette tip region (note that the illustrations are not in scale, and geometries were chosen for illustration purposes only). (b) Simulated average ion concentration along the axis of symmetry (r = 0 nm) for 0 nm (black), 2 nm (cyan), and 30 nm (magenta) interface displacements. (c) Simulated (black curve) and experimental (colored points) current peak maxima (Δi) for different interface displacements toward the external solution and sizes of dsDNA molecules translocating through the nanopipette tip aperture toward the bath, respectively. The error bars represent the standard deviation of the experimental current peak maxima values. The horizontal coordinate of experimental data points was chosen according to the expected Δz (Table ST7.1, Supporting Information). Further details on the interface displacement simulations and the experimental translocation data are included in the Supporting Information (Section S7).

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  • References

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      Abstr.: Rapid advances in nanopore technologies for sequencing single long DNA and RNA mols. have led to substantial improvements in accuracy, read length and throughput. These breakthroughs have required extensive development of exptl. and bioinformatics methods to fully exploit nanopore long reads for investigations of genomes, transcriptomes, epigenomes and epitranscriptomes. Nanopore sequencing is being applied in genome assembly, full-length transcript detection and base modification detection and in more specialized areas, such as rapid clin. diagnoses and outbreak surveillance. Many opportunities remain for improving data quality and anal. approaches through the development of new nanopores, base-calling methods and exptl. protocols tailored to particular applications.
    4. 4
      Xu, X.; Valavanis, D.; Ciocci, P.; Confederat, S.; Marcuccio, F.; Lemineur, J.-F.; Actis, P.; Kanoufi, F.; Unwin, P. The New Era of High Throughput Nanoelectrochemistry. Anal. Chem. 2023,  DOI: 10.1021/acs.analchem.2c05105
      There is no corresponding record for this reference.
    5. 5
      Houghtaling, J.; Ying, C.; Eggenberger, O. M.; Fennouri, A.; Nandivada, S.; Acharjee, M.; Li, J.; Hall, A. R.; Mayer, M. Estimation of Shape, Volume, and Dipole Moment of Individual Proteins Freely Transiting a Synthetic Nanopore. ACS Nano 2019, 13, 5231– 5242,  DOI: 10.1021/acsnano.8b09555
      5
      Estimation of Shape, Volume, and Dipole Moment of Individual Proteins Freely Transiting a Synthetic Nanopore
      Houghtaling, Jared; Ying, Cuifeng; Eggenberger, Olivia M.; Fennouri, Aziz; Nandivada, Santoshi; Acharjee, Mitu; Li, Jiali; Hall, Adam R.; Mayer, Michael
      ACS Nano (2019), 13 (5), 5231-5242CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      This paper demonstrates that high-bandwidth current recordings in combination with low-noise silicon nitride nanopores make it possible to det. the mol. vol., approx. shape, and dipole moment of single native proteins in soln. without the need for labeling, tethering, or other chem. modifications of these proteins. The anal. is based on current modulations caused by the translation and rotation of single proteins through a uniform elec. field inside of a nanopore. We applied this technique to nine proteins and show that the measured protein parameters agree well with ref. values but only if the nanopore walls were coated with a nonstick fluid lipid bilayer. One potential challenge with this approach is that an untethered protein is able to diffuse laterally while transiting a nanopore, which generates increasingly asym. disruptions in the elec. field as it approaches the nanopore walls. These "off-axis" effects add an addnl. noise-like element to the elec. recordings, which can be exacerbated by nonspecific interactions with pore walls that are not coated by a fluid lipid bilayer. We performed finite element simulations to quantify the influence of these effects on subsequent analyses. Examg. the size, approx. shape, and dipole moment of unperturbed, native proteins in aq. soln. on a single-mol. level in real time while they translocate through a nanopore may enable applications such as identifying or characterizing proteins in a mixt., or monitoring the assembly or disassembly of transient protein complexes based on their shape, vol., or dipole moment.
    6. 6
      Yusko, E. C.; Bruhn, B. R.; Eggenberger, O. M.; Houghtaling, J.; Rollings, R. C.; Walsh, N. C.; Nandivada, S.; Pindrus, M.; Hall, A. R.; Sept, D.; Li, J.; Kalonia, D. S.; Mayer, M. Real-Time Shape Approximation and Fingerprinting of Single Proteins Using a Nanopore. Nat. Nanotechnol. 2017, 12, 360– 367,  DOI: 10.1038/nnano.2016.267
      6
      Real-time shape approximation and fingerprinting of single proteins using a nanopore
      Yusko, Erik C.; Bruhn, Brandon R.; Eggenberger, Olivia M.; Houghtaling, Jared; Rollings, Ryan C.; Walsh, Nathan C.; Nandivada, Santoshi; Pindrus, Mariya; Hall, Adam R.; Sept, David; Li, Jiali; Kalonia, Devendra S.; Mayer, Michael
      Nature Nanotechnology (2017), 12 (4), 360-367CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      Established methods for characterizing proteins typically require phys. or chem. modification steps or cannot be used to examine individual mols. in soln. Ionic current measurements through electrolyte-filled nanopores can characterize single native proteins in an aq. environment, but currently offer only limited capabilities. The zeptoliter sensing vol. of bilayer-coated solid-state nanopores can be used to det. the approx. shape, vol., charge, rotational diffusion coeff. and dipole moment of individual proteins. To do this, the authors developed a theory for the quant. understanding of modulations in ionic current that arise from the rotational dynamics of single proteins as they move through the elec. field inside the nanopore. The approach allows the authors to measure the five parameters simultaneously, and they can be used to identify, characterize and quantify proteins and protein complexes with potential implications for structural biol., proteomics, biomarker detection and routine protein anal.
    7. 7
      Wang, V.; Ermann, N.; Keyser, U. F. Current Enhancement in Solid-State Nanopores Depends on Three-Dimensional DNA Structure. Nano Lett. 2019, 19, 5661– 5666,  DOI: 10.1021/acs.nanolett.9b02219
      7
      Current Enhancement in Solid-State Nanopores Depends on Three-Dimensional DNA Structure
      Wang, Vivian; Ermann, Niklas; Keyser, Ulrich F.
      Nano Letters (2019), 19 (8), 5661-5666CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      The translocation of double-stranded DNA through a solid-state nanopore may either decrease or increase the ionic current depending on the ionic concn. of the surrounding soln. Below a certain crossover ionic concn., the current change inverts from a current blockade to current enhancement. In this paper, we show that the crossover concn. for bundled DNA nanostructures composed of multiple connected DNA double-helixes is lower than that of double-stranded DNA. Our measurements suggest that counterion mobility in the vicinity of DNA is reduced depending on the three-dimensional structure of the mol. We further demonstrate that introducing neutral polymers such as polyethylene glycol into the measurement soln. reduces electroosmotic outflow from the nanopore, allowing translocation of large DNA structures at low salt concns. Our expts. contribute to an improved understanding of ion transport in confined DNA environments, which is crit. for the development of nanopore sensing techniques as well as synthetic membrane channels. Our salt-dependent measurements of model DNA nanostructures will guide the development of computational models of DNA translocation through nanopores.
    8. 8
      Chang, H.; Kosari, F.; Andreadakis, G.; Alam, M. A.; Vasmatzis, G.; Bashir, R. DNA-Mediated Fluctuations in Ionic Current through Silicon Oxide Nanopore Channels. Nano Lett. 2004, 4, 1551– 1556,  DOI: 10.1021/nl049267c
      8
      DNA-Mediated Fluctuations in Ionic Current through Silicon Oxide Nanopore Channels
      Chang, H.; Kosari, F.; Andreadakis, G.; Alam, M. A.; Vasmatzis, G.; Bashir, R.
      Nano Letters (2004), 4 (8), 1551-1556CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      Single mol. sensors in which nanoscale pores within biol. or artificial membranes act as mech. gating elements are very promising devices for the rapid characterization and sequencing of nucleic acid mols. The two terminal elec. measurements of translocation of polymers through single ion channels and that of ssDNA mols. through protein channels have been demonstrated, and have sparked tremendous interest in such single mol. sensors. The prevailing view regarding the nanopore sensors is that there exists no elec. interaction between the nanopore and the translocating mol., and that all nanopore sensors reported to-date, whether biol. or artificial, operate as a coulter-counter, i.e., the ionic current measured across the pore decreases (is mech. blocked) when the DNA mol. transverses through the pore. We have fabricated nanopore channel sensors with a silicon oxide inner surface, and our results challenge the prevailing view of exclusive mech. interaction during the translocation of dsDNA mols. through these channels. We demonstrate that the ionic current can actually increase due to elec. gating of surface current in the channel due to the charge on the DNA itself.
    9. 9
      Smeets, R. M. M.; Keyser, U. F.; Krapf, D.; Wu, M.-Y.; Dekker, N. H.; Dekker, C. Salt Dependence of Ion Transport and DNA Translocation through Solid-State Nanopores. Nano Lett. 2006, 6, 89– 95,  DOI: 10.1021/nl052107w
      9
      Salt Dependence of Ion Transport and DNA Translocation through Solid-State Nanopores
      Smeets, Ralph M. M.; Keyser, Ulrich F.; Krapf, Diego; Wu, Meng-Yue; Dekker, Nynke H.; Dekker, Cees
      Nano Letters (2006), 6 (1), 89-95CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      We report exptl. measurements of the salt dependence of ion transport and DNA translocation through solid-state nanopores. The ionic conductance shows a three-order-of-magnitude decrease with decreasing salt concns. from 1 M to 1 μM, strongly deviating from bulk linear behavior. The data are described by a model that accounts for a salt-dependent surface charge of the pore. Subsequently, we measure translocation of 16.5-μm-long dsDNA for 50 mM to 1 M salt concns. DNA translocation is shown to result in either a decrease ([KCl] > 0.4 M) or increase of the ionic current ([KCl] < 0.4 M). The data are described by a model where current decreases result from the partial blocking of the pore and current increases are attributed to motion of the counterions that screen the charge of the DNA backbone. We demonstrate that the two competing effects cancel at a KCl concn. of 370 ± 40 mM.
    10. 10
      Zhang, Y.; Wu, G.; Si, W.; Ma, J.; Yuan, Z.; Xie, X.; Liu, L.; Sha, J.; Li, D.; Chen, Y. Ionic Current Modulation from DNA Translocation through Nanopores under High Ionic Strength and Concentration Gradients. Nanoscale 2017, 9, 930– 939,  DOI: 10.1039/C6NR08123A
      10
      Ionic current modulation from DNA translocation through nanopores under high ionic strength and concentration gradients
      Zhang, Yin; Wu, Gensheng; Si, Wei; Ma, Jian; Yuan, Zhishan; Xie, Xiao; Liu, Lei; Sha, Jingjie; Li, Deyu; Chen, Yunfei
      Nanoscale (2017), 9 (2), 930-939CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
      Ion transport through nanopores is an important process in nature and has important engineering applications. To date, most studies of nanopore ion transport have been carried out with electrolytes of relatively low concns. In this paper, we report on ionic current modulation from the translocation of dsDNA through a nanopore under high ionic strength and with an electrolyte concn. gradient across the nanopore. Results show that in this case, DNA translocation can induce either neg. or pos. ionic current modulation, even though usually only downward peaks are expected under this high ion concn. Through a series of expts. and numerical simulations with nanopores of different diams. and concn. gradients, it is found that the pos. pulse is due to extra ions outside the elec. double layer of the DNA that are brought into the nanopore by the enhanced electroosmotic flow (EOF) with the neg. charged DNA inside the nanopore.
    11. 11
      Kesselheim, S.; Müller, W.; Holm, C. Origin of Current Blockades in Nanopore Translocation Experiments. Phys. Rev. Lett. 2014, 112, 018101  DOI: 10.1103/PhysRevLett.112.018101
      11
      Origin of current blockades in nanopore translocation experiments
      Kesselheim, Stefan; Mueller, Wojciech; Holm, Christian
      Physical Review Letters (2014), 112 (1), 018101/1-018101/5CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
      We present a detailed investigation of the ionic current in a cylindrical model nanopore in the absence and the presence of a double stranded DNA homopolymer. Our atomistic simulations are capable of reproducing almost exactly the exptl. data obtained by Smeets et al., including notably the crossover salt concn. that yields equal current measurements in both situations. We can rule out that the obsd. current blockade is due to the steric exclusion of charge carriers from the DNA, since for all investigated salt concns. the charge carrier d. is higher when the DNA is present. Calcns. using a mean-field electrokinetic model proposed by van Dorp et al. fail quant. in predicting this effect. We can relate the shortcomings of the mean-field model to a surface related mol. drag that the ions feel in the presence of the DNA. This drag is independent of the salt concn. and originates from electrostatic, hydrodynamic, and excluded vol. interactions.
    12. 12
      Lastra, L. S.; Bandara, Y. M. N. D. Y.; Sharma, V.; Freedman, K. J. Protein and DNA Yield Current Enhancements, Slow Translocations, and an Enhanced Signal-to-Noise Ratio under a Salt Imbalance. ACS Sens. 2022, 7, 1883– 1893,  DOI: 10.1021/acssensors.2c00479
      12
      Protein and DNA Yield Current Enhancements, Slow Translocations, and an Enhanced Signal-to-Noise Ratio under a Salt Imbalance
      Lastra, Lauren S.; Bandara, Y. M. Nuwan D. Y.; Sharma, Vinay; Freedman, Kevin J.
      ACS Sensors (2022), 7 (7), 1883-1893CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
      Nanopores are a promising single-mol. sensing device class that captures mol.-level information through resistive or conductive pulse sensing (RPS and CPS). The latter has not been routinely utilized in the nanopore field despite the benefits it could provide, specifically in detecting subpopulations of a mol. A systematic study was conducted here to study the CPS-based mol. discrimination and its voltage-dependent characteristics. CPS was obsd. when the cation movement along both elec. and chem. gradients was favored, which led to an ∼3x improvement in SNR (i.e., signal-to-noise ratio) and an ∼8x increase in translocation time. Interestingly, a reversal of the salt gradient reinstates the more conventional resistive pulses and may help elucidate RPS-CPS transitions. The asym. salt conditions greatly enhanced the discrimination of DNA configurations including linear, partially folded, and completely folded DNA states, which could help detect subpopulations in other mol. systems. These findings were then utilized for the detection of a Cas9 mutant, Cas9d10a-a protein with broad utilities in genetic engineering and immunol.-bound to DNA target strands and the unbound Cas9d10a + sgRNA complexes, also showing significantly longer event durations (>1 ms) than typically obsd. for proteins.
    13. 13
      Restrepo-Pérez, L.; Joo, C.; Dekker, C. Paving the Way to Single-Molecule Protein Sequencing. Nat. Nanotechnol. 2018, 13, 786– 796,  DOI: 10.1038/s41565-018-0236-6
      13
      Paving the way to single-molecule protein sequencing
      Restrepo-Perez, Laura; Joo, Chirlmin; Dekker, Cees
      Nature Nanotechnology (2018), 13 (9), 786-796CODEN: NNAABX; ISSN:1748-3387. (Nature Research)
      A review. Proteins are major building blocks of life. The protein content of a cell and an organism provides key information for the understanding of biol. processes and disease. Despite the importance of protein anal., only a handful of techniques are available to det. protein sequences, and these methods face limitations, for example, requiring a sizable amt. of sample. Single-mol. techniques would revolutionize proteomics research, providing ultimate sensitivity for the detection of low-abundance proteins and the realization of single-cell proteomics. In recent years, novel single-mol. protein sequencing schemes that use fluorescence, tunnelling currents and nanopores have been proposed. Here, we present a review of these approaches, together with the first exptl. efforts towards their realization. We discuss their advantages and drawbacks, and present our perspective on the development of single-mol. protein sequencing techniques.
    14. 14
      Thiruraman, J. P.; Masih Das, P.; Drndić, M. Stochastic Ionic Transport in Single Atomic Zero-Dimensional Pores. ACS Nano 2020, 14, 11831– 11845,  DOI: 10.1021/acsnano.0c04716
      14
      Stochastic Ionic Transport in Single Atomic Zero-Dimensional Pores
      Thiruraman, Jothi Priyanka; Masih Das, Paul; Drndic, Marija
      ACS Nano (2020), 14 (9), 11831-11845CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      We report on single at. zero-dimensional (0D) pores fabricated using aberration-cor. scanning transmission electron microscopy (AC-STEM) in monolayer MoS2. Pores are comprised of a few atoms missing in the two-dimensional (2D) lattice (1-5 Mo atoms) of characteristic sizes from ~ 0.5 to 1.2 nm, and pore edges directly probed by AC-STEM to map the at. structure. We categorize them into ~ 30 geometrically possible zigzag, armchair, and mixed configurations. While theor. studies predict that transport properties of 2D pores in this size range depend strongly on pore size and their at. configuration, 0D pores show an av. conductance in the range from ~ 0.6-1 nS (bias up to 0.1 V), similar to biol. pores. In some devices, the current was immeasurably small and/or pores could not be wet. Furthermore, current-voltage (I-V) characteristics are largely independent of bulk molarity (10 mM to 3 M KCl) and the type of cation (K+, Li+, Mg2+). This work lays the exptl. foundation for understanding of the confinement effects possible in at.-scale 2D material pores and the realization of solid-state analogs of ion channels in biol.
    15. 15
      Fragasso, A.; Schmid, S.; Dekker, C. Comparing Current Noise in Biological and Solid-State Nanopores. ACS Nano 2020, 14, 1338– 1349,  DOI: 10.1021/acsnano.9b09353
      15
      Comparing Current Noise in Biological and Solid-State Nanopores
      Fragasso, Alessio; Schmid, Sonja; Dekker, Cees
      ACS Nano (2020), 14 (2), 1338-1349CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      A review. Nanopores bear great potential as single-mol. tools for bioanal. sensing and sequencing, due to their exceptional sensing capabilities, high-throughput, and low cost. The detection principle relies on detecting small differences in the ionic current as biomols. traverse the nanopore. A major bottleneck for the further progress of this technol. is the noise that is present in the ionic current recordings, because it limits the signal-to-noise ratio (SNR) and thereby the effective time resoln. of the expt. Here, the authors review the main types of noise at low and high frequencies and discuss the underlying physics. Moreover, the authors compare biol. and solid-state nanopores in terms of the SNR, the important figure of merit, by measuring translocations of a short ssDNA through a selected set of nanopores under typical exptl. conditions. The authors find that SiNx solid-state nanopores provide the highest SNR, due to the large currents at which they can be operated and the relatively low noise at high frequencies. However, the real game-changer for many applications is a controlled slowdown of the translocation speed, which for MspA was shown to increase the SNR > 160-fold. Finally, the authors discuss practical approaches for lowering the noise for optimal exptl. performance and further development of the nanopore technol.
    16. 16
      Rosenstein, J. K.; Wanunu, M.; Merchant, C. A.; Drndic, M.; Shepard, K. L. Integrated Nanopore Sensing Platform with Sub-Microsecond Temporal Resolution. Nat. Methods 2012, 9, 487– 492,  DOI: 10.1038/nmeth.1932
      16
      Integrated nanopore sensing platform with sub-microsecond temporal resolution
      Rosenstein, Jacob K.; Wanunu, Meni; Merchant, Christopher A.; Drndic, Marija; Shepard, Kenneth L.
      Nature Methods (2012), 9 (5), 487-492CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      Nanopore sensors have attracted considerable interest for high-throughput sensing of individual nucleic acids and proteins without the need for chem. labels or complex optics. A prevailing problem in nanopore applications is that the transport kinetics of single biomols. are often faster than the measurement time resoln. Methods to slow down biomol. 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 mols. as well as current signatures during mol. passage events that shed light on submol. DNA configurations in small nanopores.
    17. 17
      Fologea, D.; Uplinger, J.; Thomas, B.; McNabb, D. S.; Li, J. Slowing DNA Translocation in a Solid-State Nanopore. Nano Lett. 2005, 5, 1734– 1737,  DOI: 10.1021/nl051063o
      17
      Slowing DNA Translocation in a Solid-State Nanopore
      Fologea, Daniel; Uplinger, James; Thomas, Brian; McNabb, David S.; Li, Jiali
      Nano Letters (2005), 5 (9), 1734-1737CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      Reducing a DNA mol.'s translocation speed in a solid-state nanopore is a key step toward rapid single mol. identification. Here we demonstrate that DNA translocation speeds can be reduced by an order of magnitude over previous results. By controlling the electrolyte temp., salt concn., viscosity, and the elec. bias voltage across the nanopore, we obtain a 3 base/μs translocation speed for 3 kbp double-stranded DNA in a 4-8 nm diam. silicon nitride pore. Our results also indicate that the ionic cond. inside such a nanopore is smaller than it is in bulk.
    18. 18
      Rabinowitz, J.; Edwards, M. A.; Whittier, E.; Jayant, K.; Shepard, K. L. Nanoscale Fluid Vortices and Nonlinear Electroosmotic Flow Drive Ion Current Rectification in the Presence of Concentration Gradients. J. Phys. Chem. A 2019, 123, 8285– 8293,  DOI: 10.1021/acs.jpca.9b04075
      18
      Nanoscale Fluid Vortices and Nonlinear Electroosmotic Flow Drive Ion Current Rectification in the Presence of Concentration Gradients
      Rabinowitz Jake; Whittier Elizabeth; Jayant Krishna; Shepard Kenneth L; Edwards Martin A
      The journal of physical chemistry. A (2019), 123 (38), 8285-8293 ISSN:.
      Ion current rectification (ICR) is a transport phenomenon in which an electrolyte conducts unequal currents at equal and opposite voltages. Here, we show that nanoscale fluid vortices and nonlinear electroosmotic flow (EOF) drive ICR in the presence of concentration gradients. The same EOF can yield negative differential resistance (NDR), in which current decreases with increasing voltage. A finite element model quantitatively reproduces experimental ICR and NDR recorded across glass nanopipettes under concentration gradients. The model demonstrates that spatial variations of electrical double layer properties induce the nanoscale vortices and nonlinear EOF. Experiments are performed in conditions directly related to scanning probe imaging and show that quantitative understanding of nanoscale transport under concentration gradients requires accounting for EOF. This characterization of nanopipette transport physics will benefit diverse experimentation, pushing the resolution limits of chemical and biophysical recordings.
    19. 19
      Lan, W. J.; Edwards, M. A.; Luo, L.; Perera, R. T.; Wu, X.; Martin, C. R.; White, H. S. Voltage-Rectified Current and Fluid Flow in Conical Nanopores. Acc. Chem. Res. 2016, 49, 2605– 2613,  DOI: 10.1021/acs.accounts.6b00395
      19
      Voltage-Rectified Current and Fluid Flow in Conical Nanopores
      Lan, Wen-Jie; Edwards, Martin A.; Luo, Long; Perera, Rukshan T.; Wu, Xiaojian; Martin, Charles R.; White, Henry S.
      Accounts of Chemical Research (2016), 49 (11), 2605-2613CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review. Ion current rectification (ICR) refers to the asym. potential-dependent rate of the passage of soln. ions through a nanopore, giving rise to elec. current-voltage characteristics that mimic those of a solid-state elec. diode. Since the discovery of ICR in quartz nanopipettes two decades ago, synthetic nanopores and nanochannels of various geometries, fabricated in membranes and on wafers, have been extensively investigated to understand fundamental aspects of ion transport in highly confined geometries. It is now generally accepted that ICR requires an asym. elec. double layer within the nanopore, producing an accumulation or depletion of charge-carrying ions at opposite voltage polarities. Our research groups have recently explored how the voltage-dependent ion distributions and ICR within nanopores can induce novel nanoscale flow phenomena that have applications in understanding ionics in porous materials used in energy storage devices, chem. sensing, and low-cost elec. pumping of fluids. In this Account, we review our most recent investigations on this topic, based on expts. using conical nanopores (10-300 nm tip opening) fabricated in thin glass, mica, and polymer membranes. Measurable fluid flow in nanopores can be induced either using external pressure forces, elec. via electroosmotic forces, or by a combination of these two forces. We demonstrate that pressure-driven flow can greatly alter the elec. properties of nanopores and, vice versa, that the nonlinear elec. properties of conical nanopores can impart novel and useful flow phenomena.Electroosmotic flow (EOF), which depends on the magnitude of the ion fluxes within the double layer of the nanopore, is strongly coupled to the accumulation/depletion of ions. Thus, the same underlying cause of ICR also leads to EOF rectification, i.e., unequal flows occurring for the same voltage but opposite polarities. EOF rectification can be used to elec. pump fluids with very precise control across membranes contg. conical pores via the application of a sym. sinusoidal voltage. The combination of pressure and asym. EOF can also provide a means to generate new nanopore elec. behaviors, including neg. differential resistance (NDR), in which the current through a conical pore decreases with increasing driving force (applied voltage), similar to solid-state tunnel diodes. NDR results from a pos. feedback mechanism between the ion distributions and EOF, yielding a true bistability in both fluid flow and elec. current at a crit. applied voltage. Nanopore-based NDR is extremely sensitive to the surface charge near the nanopore opening, suggesting possible applications in chem. sensing.
    20. 20
      Perera, R. T.; Johnson, R. P.; Edwards, M. A.; White, H. S. Effect of the Electric Double Layer on the Activation Energy of Ion Transport in Conical Nanopores. J. Phys. Chem. C 2015, 119, 24299– 24306,  DOI: 10.1021/acs.jpcc.5b08194
      20
      Effect of the electric double layer on the activation energy of ion transport in conical nanopores
      Perera, Rukshan T.; Johnson, Robert P.; Edwards, Martin A.; White, Henry S.
      Journal of Physical Chemistry C (2015), 119 (43), 24299-24306CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
      Measured apparent activation energies, EA, of ion transport (K+ and Cl-) in conical glass nanopores are reported as a function of applied voltage (-0.5 to 0.5 V), pore size (20-2000 nm), and electrolyte concn. (0.1-50 mM). EA values for transport within an elec. charged conical glass nanopore differ from the bulk values due to the voltage and temp.-dependent distribution of the ions within the double layer. Remarkably, nanopores that display ion current rectification also display a large decrease in EA under accumulation mode conditions (at applied neg. voltages vs. an external ground) and a large increase in EA under depletion mode conditions (at pos. voltages). Finite element simulations based on the Poisson-Nernst-Planck model semiquant. predict the measured temp.-dependent cond. and dependence of EA on applied voltage. The results highlight the relationships between the distribution of ions with the nanopore, ionic current, and EA and their dependencies on pore size, temp., ion concn., and applied voltage.
    21. 21
      Luo, L.; Holden, D. A.; Lan, W.-J.; White, H. S. Tunable Negative Differential Electrolyte Resistance in a Conical Nanopore in Glass. ACS Nano 2012, 6, 6507– 6514,  DOI: 10.1021/nn3023409
      21
      Tunable Negative Differential Electrolyte Resistance in a Conical Nanopore in Glass
      Luo, Long; Holden, Deric A.; Lan, Wen-Jie; White, Henry S.
      ACS Nano (2012), 6 (7), 6507-6514CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      Liq.-phase neg. differential resistance (NDR) is obsd. in the i-V behavior of a conical nanopore (∼300 nm orifice radius) in a glass membrane that separates an external low-cond. 5 mM KCl soln. of DMSO/H2O (vol./vol. 3:1) from an internal high-cond. 5 mM KCl aq. soln. NDR appears in the i-V curve of the neg. charged nanopore as the voltage-dependent electroosmotic force opposes an externally applied pressure force, continuously moving the location of the interfacial zone between the two miscible solns. to a position just inside the nanopore orifice. An ∼80% decrease in the ionic current occurs over less that a ∼ 10 mV increase in applied voltage. The NDR turn-on voltage is tunable over a ∼ 1 V window by adjusting the applied external pressure from 0 to 50 mmHg. Finite-element simulations based on soln. of Navier-Stokes, Poisson, and convective Nernst-Planck equations for mixed solvent electrolytes within a neg. charged nanopore yield predictions of the NDR behavior that are in qual. agreement with the exptl. observations. Applications in chem. sensing of a tunable, soln.-based elec. switch based on the NDR effect are discussed.
    22. 22
      Lastra, L. S.; Bandara, Y. M. N. D. Y.; Nguyen, M.; Farajpour, N.; Freedman, K. J. On the Origins of Conductive Pulse Sensing inside a Nanopore. Nat. Commun. 2022, 13, 2186,  DOI: 10.1038/s41467-022-29758-8
      22
      On the origins of conductive pulse sensing inside a nanopore
      Lastra, Lauren S.; Bandara, Y. M. Nuwan D. Y.; Nguyen, Michelle; Farajpour, Nasim; Freedman, Kevin J.
      Nature Communications (2022), 13 (1), 2186CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)
      Nanopore sensing is nearly synonymous with resistive pulse sensing due to the characteristic occlusion of ions during pore occupancy, particularly at high salt concns. Contrarily, conductive pulses are obsd. under low salt conditions wherein electroosmotic flow is significant. Most literature reports counterions as the dominant mechanism of conductive events (a mol.-centric theory). However, the counterion theory does not fit well with conductive events occurring via net neutral-charged protein translocation, prompting further investigation into translocation mechanics. Herein, we demonstrate theory and expts. underpinning the translocation mechanism (i.e., electroosmosis or electrophoresis), pulse direction (i.e., conductive or resistive) and shape (e.g., monophasic or biphasic) through fine control of chem., phys., and electronic parameters. Results from these studies predict strong electroosmosis plays a role in driving DNA events and generating conductive events due to polarization effects (i.e., a pore-centric theory).
    23. 23
      White, H. S.; Bund, A. Ion Current Rectification at Nanopores in Glass Membranes. Langmuir 2008, 24, 2212– 2218,  DOI: 10.1021/la702955k
      23
      Ion Current Rectification at Nanopores in Glass Membranes
      White, Henry S.; Bund, Andreas
      Langmuir (2008), 24 (5), 2212-2218CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      The origin of ion current rectification obsd. at conical-shaped nanopores in glass membranes immersed in KCl solns. has been investigated using finite-element simulations. The ion concns. and fluxes (due to diffusion, migration, and electroosmotic convection) were detd. by the simultaneous soln. of the Nernst-Planck, Poisson, and Navier-Stokes equations for the two-ion (K+ and Cl-) system. Fixed surface charge on both the internal and external glass surfaces that define the pore structure was included to account for elec. fields and nonuniform ion cond. within the nanopores and elec. fields in the external soln. near the pore mouth. We demonstrate that previous observations of ion current rectification in conical-shaped glass nanopores are a consequence of the voltage-dependent soln. cond. in the vicinity of the pore mouth, both inside and outside of the pore. The simulations also demonstrate that current rectification is maximized at intermediate bulk ion concns., a combination of (i) the elec. screening of surface charge at high concns. and (ii) a fixed no. of charge-carrying ions in the pore at lower concn., which are phys. conditions where the voltage dependence of the cond. disappears. In addn., we have quant. shown that electroosmotic flow gives rise to a significant but small contribution to current rectification.
    24. 24
      Wei, C.; Bard, A. J.; Feldberg, S. W. Current Rectification at Quartz Nanopipet Electrodes. Anal. Chem. 1997, 69, 4627– 4633,  DOI: 10.1021/ac970551g
      24
      Current rectification at quartz nanopipet electrodes
      Wei, Chang; Bard, Allen J.; Feldberg, Stephen W.
      Analytical Chemistry (1997), 69 (22), 4627-4633CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Ag/AgCl ref. electrodes fabricated from pulled quartz tubes with orifice radii of 20 nm to 20 μm were characterized in KCl solns. of different concns. by cyclic voltammetry. Linear current-voltage (i-V) dependence was obsd. with micropipet electrodes (with micrometer-sized tips) with the same concn. (0.01-1 M) of KCl inside and outside the pipet, but current rectification was found at nanopipet electrodes at KCl concns. of ≤0.1 M (with nanometer-sized tips). This is attributed to formation of a diffuse elec. double layer within the tip orifice. The effects of electrode size, electrolyte concn., and soln. pH on the nonlinear i-V behavior of these electrodes were studied. A model for the obsd. behavior shows the rectification to be caused by the permselectivity in the tip region and the geometric asymmetry of the tip orifice. This phenomenon may be important in studies of ion transport in charged channels and porous membranes.
    25. 25
      Chau, C. C.; Radford, S. E.; Hewitt, E. W.; Actis, P. Macromolecular Crowding Enhances the Detection of DNA and Proteins by a Solid-State Nanopore. Nano Lett. 2020, 20, 5553– 5561,  DOI: 10.1021/acs.nanolett.0c02246
      25
      Macromolecular Crowding Enhances the Detection of DNA and Proteins by a Solid-State Nanopore
      Chau, Chalmers C.; Radford, Sheena E.; Hewitt, Eric W.; Actis, Paolo
      Nano Letters (2020), 20 (7), 5553-5561CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      Nanopore anal. of nucleic acid is now routine, but detection of proteins remains challenging. Here, the authors report the systematic characterization of the effect of macromol. crowding on the detection sensitivity of a solid-state nanopore for circular and linearized DNA plasmids, globular proteins (β-galactosidase), and filamentous proteins (α-synuclein amyloid fibrils). A remarkable ∼1000-fold increase in the mol. count for the globular protein β-galactosidase and a 6-fold increase in peak amplitude for plasmid DNA under crowded conditions. were obsd. Also macromol. crowding facilitates the study of the topol. of DNA plasmids and the characterization of amyloid fibril prepns. with different length distributions. A remarkable feature of this method is its ease of use; it simply requires the addn. of a macromol. crowding agent to the electrolyte. The authors therefore envision that macromol. crowding can be applied to many applications in the anal. of biomols. by solid-state nanopores.
    26. 26
      Chau, C.; Marcuccio, F.; Soulias, D.; Edwards, M. A.; Tuplin, A.; Radford, S. E.; Hewitt, E.; Actis, P. Probing RNA Conformations Using a Polymer–Electrolyte Solid-State Nanopore. ACS Nano 2022, 16, 20075– 20085,  DOI: 10.1021/acsnano.2c08312
      26
      Probing RNA Conformations Using a Polymer-Electrolyte Solid-State Nanopore
      Chau, Chalmers; Marcuccio, Fabio; Soulias, Dimitrios; Edwards, Martin Andrew; Tuplin, Andrew; Radford, Sheena E.; Hewitt, Eric; Actis, Paolo
      ACS Nano (2022), 16 (12), 20075-20085CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      Nanopore systems have emerged as a leading platform for the anal. of biomol. complexes with single-mol. resoln. The conformation of biomols., such as RNA, is highly dependent on the electrolyte compn., but solid-state nanopore systems often require high salt concn. to operate, precluding anal. of macromol. conformations under physiol. relevant conditions. Here, we report the implementation of a polymer-electrolyte solid-state nanopore system based on alkali metal halide salts dissolved in 50% w/v poly(ethylene) glycol (PEG) to augment the performance of our system. We show that polymer-electrolyte bath governs the translocation dynamics of the analyte which correlates with the phys. properties of the salt used in the bath. This allowed us to identify CsBr as the optimal salt to complement PEG to generate the largest signal enhancement. Harnessing the effects of the polymer-electrolyte, we probed the conformations of the Chikungunya virus (CHIKV) RNA genome fragments under physiol. relevant conditions. Our system was able to fingerprint CHIKV RNA fragments ranging from ∼300 to ∼2000 nt length and subsequently distinguish conformations between the co-transcriptionally folded and the natively refolded ∼2000 nt CHIKV RNA. We envision that the polymer-electrolyte solid-state nanopore system will further enable structural and conformational analyses of individual biomols. under physiol. relevant conditions.
    27. 27
      Zhang, Z.; Ohl, M.; Diallo, S. O.; Jalarvo, N. H.; Hong, K.; Han, Y.; Smith, G. S.; Do, C. Dynamics of Water Associated with Lithium Ions Distributed in Polyethylene Oxide. Phys. Rev. Lett. 2015, 115, 198301  DOI: 10.1103/PhysRevLett.115.198301
      27
      Dynamics of water associated with lithium ions distributed in polyethylene oxide
      Zhang, Zhe; Ohl, Michael; Diallo, Souleymane O.; Jalarvo, Niina H.; Hong, Kunlun; Han, Youngkyu; Smith, Gregory S.; Do, Changwoo
      Physical Review Letters (2015), 115 (19), 198301/1-198301/6CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
      The dynamics of water in polyethylene oxide (PEO)/LiCl soln. has been studied with quasielastic neutron scattering expts. and mol. dynamics (MD) simulations. Two different time scales of water diffusion representing interfacial water and bulk water dynamics have been identified. The measured diffusion coeff. of interfacial water remained 5-10 times smaller than that of bulk water, but both were slowed by approx. 50% in the presence of Li+. Detailed anal. of MD trajectories suggests that Li+ is favorably found at the surface of the hydration layer, and the probability to find the caged Li+ configuration formed by the PEO is lower than for the noncaged Li+- PEO configuration. In both configurations, however, the slowing down of water mols. is driven by reorienting water mols. and creating water-Li+ hydration complexes. Performing the MD simulation with different ions (Na+ and K+) revealed that smaller ionic radius of the ions is a key factor in disrupting the formation of PEO cages by allowing spaces for water mols. to come in between the ion and PEO.
    28. 28
      Ren, C.; Tian, W.; Szleifer, I.; Ma, Y. Specific Salt Effects on Poly(Ethylene Oxide) Electrolyte Solutions. Macromolecules 2011, 44, 1719– 1727,  DOI: 10.1021/ma1027752
      28
      Specific Salt Effects on Poly(ethylene oxide) Electrolyte Solutions
      Ren, Chun-lai; Tian, Wen-de; Szleifer, Igal; Ma, Yu-qiang
      Macromolecules (Washington, DC, United States) (2011), 44 (6), 1719-1727CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
      Exploring the mechanism of specific salt effects in electrolyte solns. is an old and attractive subject. It has been gradually realized that the competition at the mol. level plays an important role. Aiming to include mol. details as many as possible, we combine mol. dynamics (MD) simulations with a mol. theory to study specific salt effects on poly(ethylene oxide) (PEO) solns. with the addn. of monovalent salt. Radial distribution functions obtained from MD simulations provide microscopic structures of different components as well as interactions between various species. On the basis of these interactions, we construct the mol. theory with four assumptions: (1) an ion along with bound water in the first shell works as a single entity; (2) short-ranged interactions among various species are modeled as hydrogen-bonding interactions; (3) the ability of a hydrated ion to provide donors/acceptors for hydrogen bonding is governed by the charge d.; (4) contact ion pairs are included, esp. in the cases of small cations. The mol. theory is generalized with the explicit inclusion of ion-PEO, ion-water, ion-ion, water-water, and water-PEO hydrogen bonds. This means the mol.-scale structure and interaction are included within the frame of the theory. Theor. calcd. cloud points verify that the salting-out ability for alkali metal ions follows the series of K+ > Rb+ > Cs+ > Na+ > Li+, which is in agreement with the exptl. observations. Here, the competition among ion-PEO, ion-water, and water-PEO interactions and the impact of steric repulsions induced by the introduction of ions are two essential factors detg. the phase behavior of PEO solns. The combined methods bridge the microscopic interactions and structures to the macroscopic behavior.
    29. 29
      Poudel, L.; Podgornik, R.; Ching, W.-Y. The Hydration Effect and Selectivity of Alkali Metal Ions on Poly(Ethylene Glycol) Models in Cyclic and Linear Topology. J. Phys. Chem. A 2017, 121, 4721– 4731,  DOI: 10.1021/acs.jpca.7b04061
      29
      The Hydration Effect and Selectivity of Alkali Metal Ions on Poly(ethylene glycol) Models in Cyclic and Linear Topology
      Poudel, Lokendra; Podgornik, Rudolf; Ching, Wai-Yim
      Journal of Physical Chemistry A (2017), 121 (24), 4721-4731CODEN: JPCAFH; ISSN:1089-5639. (American Chemical Society)
      The effects of hydration and alkali metal (K, Na, and Li) bonding on two structural variants of polyethylene glycol (PEG), a cyclic (18-crown-6) configuration and a linear chain model with two different lengths, are studied by ab initio d. functional calcns. A total of 24 structural models are constructed, with different conformations of the PEG chain and its mol. environment. Detailed comparisons of the results enable us to obtain conclusive evidence on the effect of the different components of the soln. environment on the PEG structural variants. The results include the binding energy, the partial charge distribution, the solvation effect, interfacial hydrogen bonding and cohesion between different structural units in the system composed of PEG, alkali metal ions and water. Based on these comprehensive and precise comparisons, we conclude that the ion-PEG interaction is strongly influenced by the presence of solvent and the charge transfer in PEG complex depends strongly on the topol., the type of alkali metal ion, and the solvent. The interaction between alkali metal ions in the two PEG models does not always scale with the ion size but depends on their local environment.
    30. 30
      Tao, Z.; Cummings, P. T. Molecular Dynamics Simulation of Inorganic Ions in PEO Aqueous Solution. Mol. Simul. 2007, 33, 1255– 1260,  DOI: 10.1080/08927020701697691
      30
      Molecular dynamics simulation of inorganic ions in PEO aqueous solution
      Tao, Z.; Cummings, P. T.
      Molecular Simulation (2007), 33 (14-15), 1255-1260CODEN: MOSIEA; ISSN:0892-7022. (Taylor & Francis Ltd.)
      Solid polymer electrolytes (SPEs), esp. the ones dissolving lithium ions in poly ethylene oxide (PEO) polymer by the bonds between ether oxygen and cations, have long been investigated with the goals of developing batteries with high energy d. It has been accepted that most ions move through the amorphous polymer phase and their mobility depends crucially on the soln. environment, though the detailed transport mechanism is not fully developed. Recently, ternary mixts. composed of PEO/salts in aq. soln. have been shown to display more attractive properties than binary SPE mixts. Numerous expts. have found a dramatically changed environment for the cations and increased ionic cond. of polymer/salts electrolytes for increased relative humidity, suggesting that the coupling between polymer chains and cations may be weakened due to the existence of water mols. In this paper we report mol. dynamics (MD) simulation, using an optimized force field that includes polarizabilities via the dynamic shell model, to study the structural properties of inorg. ions in PEO aq. soln. and the competitive solvation of ions between water and polymer oxygen. Our simulation results show that ions are solvated more favorably by water than by polymer. This conclusion is in a good agreement with neutron diffraction by isotropic substitution (NDIS) expts.
    31. 31
      Giesecke, M.; Hallberg, F.; Fang, Y.; Stilbs, P.; Furó, I. Binding of Monovalent and Multivalent Metal Cations to Polyethylene Oxide in Methanol Probed by Electrophoretic and Diffusion NMR. J. Phys. Chem. B 2016, 120, 10358– 10366,  DOI: 10.1021/acs.jpcb.6b08923
      31
      Binding of Monovalent and Multivalent Metal Cations to Polyethylene Oxide in Methanol Probed by Electrophoretic and Diffusion NMR
      Giesecke, Marianne; Hallberg, Fredrik; Fang, Yuan; Stilbs, Peter; Furo, Istvan
      Journal of Physical Chemistry B (2016), 120 (39), 10358-10366CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      Complex formation in methanol between monodisperse polyethylene oxide (PEO) and a large set of cations was studied by measuring the effective charge acquired by the PEO upon complexation. Quant. data were obtained at the low ionic strength of 2 mM (for some salts, also between 0.5 mM and 6 mM) by a combination of diffusion NMR and electrophoretic NMR expts. For strongly complexing cations, the magnitude of the acquired effective charge was in the order of 1 cation per 100 monomer units. For monovalent cations, the relative strength of binding varies as Na+ < K+ ≈ Rb+ ≈ Cs+ while Li+ exhibited no significant binding. All polyvalent cations bind weakly, except for Ba2+ that exhibited strong binding. Anions do not bind as is shown by the lack of response to the chem. nature of anionic species (perchlorate, iodide or acetate). Diffusion expts. show directly that the acetate anion with monovalent cations does not assoc. to PEO. Considering all cations, we find that the obsd. binding does not follow any Hofmeister order. Instead, binding occurs below a crit. surface charge d. which indicates that the degree of complexation is defined by the solvation shell. A large solvation shell prevents the binding of most multivalent ions.
    32. 32
      Siwy, Z. S. Ion-Current Rectification in Nanopores and Nanotubes with Broken Symmetry. Adv. Funct. Mater. 2006, 16, 735– 746,  DOI: 10.1002/adfm.200500471
      32
      Ion-current rectification in nanopores and nanotubes with broken symmetry
      Siwy, Zuzanna S.
      Advanced Functional Materials (2006), 16 (6), 735-746CODEN: AFMDC6; ISSN:1616-301X. (Wiley-VCH Verlag GmbH & Co. KGaA)
      This article focuses on ion transport through nanoporous systems with special emphasis on rectification phenomena. The effect of ion-current rectification is obsd. as asym. current-voltage (I-V) curves, with the current recorded for one voltage polarity higher than the current recorded for the same abs. value of voltage of opposite polarity. This diode-like I-V curve indicates that there is a preferential direction for ion flow. Exptl. evidence that ion-current rectification is inherent to asym., e.g., tapered, nanoporous systems with excess surface charge is provided and discussed. The fabrication and operation of asym. polymer nanopores, gold nanotubes, glass nanocapillaries, and silicon nanopores are presented. The possibility of tuning the direction and extent of rectification is discussed in detail. The theor. models that have been developed to explain the ion-current rectification effect are also presented.
    33. 33
      Momotenko, D.; Cortés-Salazar, F.; Josserand, J.; Liu, S.; Shao, Y.; Girault, H. H. Ion Current Rectification and Rectification Inversion in Conical Nanopores: A Perm-Selective View. Phys. Chem. Chem. Phys. 2011, 13, 5430– 5440,  DOI: 10.1039/C0CP02595J
      33
      Ion current rectification and rectification inversion in conical nanopores. A perm-selective view
      Momotenko, Dmitry; Cortes-Salazar, Fernando; Josserand, Jacques; Liu, Shujuan; Shao, Yuanhua; Girault, Hubert H.
      Physical Chemistry Chemical Physics (2011), 13 (12), 5430-5440CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)
      Ionic transport in charged conical nanopores is known to give rise to ion current rectification. The rectification direction can be inverted when using electrolyte solns. at very low ionic strengths. To elucidate these phenomena, electroneutral conical nanopores contg. a perm-selective region at the tip were investigated and shown to behave like classical charged nanopores. An anal. model is proposed to account for these rectification processes.
    34. 34
      Wen, C.; Zeng, S.; Li, S.; Zhang, Z.; Zhang, S.-L. On Rectification of Ionic Current in Nanopores. Anal. Chem. 2019, 91, 14597– 14604,  DOI: 10.1021/acs.analchem.9b03685
      34
      On Rectification of Ionic Current in Nanopores
      Wen, Chenyu; Zeng, Shuangshuang; Li, Shiyu; Zhang, Zhen; Zhang, Shi-Li
      Analytical Chemistry (Washington, DC, United States) (2019), 91 (22), 14597-14604CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Rectification of ionic current, a frequently obsd. phenomenon with asym. nanopores varying in geometry and/or surface charge, has been utilized for studies of microfluidic circuits, nanopore sensors, and energy conversion devices. However, the physics behind the rectification phenomenon deserves further anal., and the involved processes need renewed organization; however, the origin is known, and numerous simulations based on the Poisson-Nernst-Planck formalism provide details of the observation. Here, we present an anal. model by identifying the causal chain connecting the key phys. factors and processes leading to rectification: the charge present on the pore sidewalls causing the selectivity of ion fluxes through the pore, the selectivity inducing enrichment-depletion of ions around the pore, and the established ion concn. gradient rendering the elec. field redistribution in the pore. Our anal. model that considers nanopore geometry, surface charge d., and electrolyte concn. calcs. the ionic current and corresponding rectification factor at given bias voltages. The model is validated by numerical simulations, and the model results agree well with exptl. data. It is, therefore, a useful tool not only for gaining phys. insights into ionic current rectification but also for providing practical guidelines in designing nanopore- and nanopipette-based ion sensors for a range of applications.
    35. 35
      Charron, M.; Briggs, K.; King, S.; Waugh, M.; Tabard-Cossa, V. Precise DNA Concentration Measurements with Nanopores by Controlled Counting. Anal. Chem. 2019, 91, 12228– 12237,  DOI: 10.1021/acs.analchem.9b01900
      35
      Precise DNA Concentration Measurements with Nanopores by Controlled Counting
      Charron, Martin; Briggs, Kyle; King, Simon; Waugh, Matthew; Tabard-Cossa, Vincent
      Analytical Chemistry (Washington, DC, United States) (2019), 91 (19), 12228-12237CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Using a solid-state nanopore to measure the concn. of clin. relevant target analytes, such as proteins or specific DNA sequences, is a major goal of nanopore research. This is usually achieved by measuring the capture rate of the target analyte through the pore. However, progress is hindered by sources of systematic error that are beyond the level of control currently achievable with state-of-the-art nanofabrication techniques. In this work, we show that the capture rate process of solid-state nanopores is subject to significant sources of variability, both within individual nanopores over time and between different nanopores of nominally identical size, which are absent from theor. electrophoretic capture models. We exptl. reveal that these fluctuations are inherent to the nanopore itself and make nanopore-based mol. concn. detn. insufficiently precise to meet the stds. of most applications. In this work, we present a simple method by which to reduce this variability, increasing the reliability, accuracy, and precision of single-mol. nanopore-based concn. measurements. We demonstrate controlled counting, a concn. measurement technique, which involves measuring the simultaneous capture rates of a mixt. of both the target mol. and an internal calibrator of precisely known concn. Using this method on linear DNA fragments, we show empirically that the requirements for precisely controlling the nanopore properties, including its size, height, geometry, and surface charge d. or distribution, are removed while allowing for higher-precision measurements. The quant. tools presented herein will greatly improve the utility of solid-state nanopores as sensors of target biomol. concn.
    36. 36
      Ivanov, A. P.; Actis, P.; Jönsson, P.; Klenerman, D.; Korchev, Y.; Edel, J. B. On-Demand Delivery of Single DNA Molecules Using Nanopipets. ACS Nano 2015, 9, 3587– 3595,  DOI: 10.1021/acsnano.5b00911
      36
      On-Demand Delivery of Single DNA Molecules Using Nanopipets
      Ivanov, Aleksandar P.; Actis, Paolo; Jonsson, Peter; Klenerman, David; Korchev, Yuri; Edel, Joshua B.
      ACS Nano (2015), 9 (4), 3587-3595CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      Understanding the behavioral properties of single mols. or larger scale populations interacting with single mols. is currently a hotly pursued topic in nanotechnol. This arises from the potential such techniques have in relation to applications such as targeted drug delivery, early stage detection of disease, and drug screening. Although label and label-free single mol. detection strategies have existed for a no. of years, currently lacking are efficient methods for the controllable delivery of single mols. in aq. environments. In this article we show both exptl. and from simulations that nanopipets in conjunction with asym. voltage pulses can be used for label-free detection and delivery of single mols. through the tip of a nanopipet with "on-demand" timing resoln. This was demonstrated by controllable delivery of 5 kbp and 10 kbp DNA mols. from solns. with concns. as low as 3 pM.
    37. 37
      Steinbock, L. J.; Lucas, A.; Otto, O.; Keyser, U. F. Voltage-Driven Transport of Ions and DNA through Nanocapillaries. Electrophoresis 2012, 33, 3480– 3487,  DOI: 10.1002/elps.201100663
      37
      Voltage-driven transport of ions and DNA through nanocapillaries
      Steinbock, Lorenz J.; Lucas, Alex; Otto, Oliver; Keyser, Ulrich F.
      Electrophoresis (2012), 33 (23), 3480-3487CODEN: ELCTDN; ISSN:0173-0835. (Wiley-VCH Verlag GmbH & Co. KGaA)
      We study the effect of salt concn. on the ionic conductance and translocation of single DNA mols. through nanocapillaries made out of quartz glass. DNA translocation expts. were performed in aq. soln. for concns. of KCl between 10 mM and 2 M while ion conductance was characterized from 1 mM to 2 M KCl concn. Here, we develop a model for the conductance of conical nanocapillaries taking into consideration the surface charge of the quartz glass. We demonstrate that the conductance of our nanocapillaries shows similar behavior to silicon oxide nanopores at low and high KCl concns. Finally, we show that DNA translocations in high KCl concns. (400 mM-2 M) cause a redn. in the ionic current. In contrast, DNA translocations at low KCl concns. (10-300 mM) lead to increases in the ionic current. Our new results, which until now have not been shown for nanocapillaries, can be well understood with an adapted model.
    38. 38
      Reiner, J. E.; Kasianowicz, J. J.; Nablo, B. J.; Robertson, J. W. F. Theory for Polymer Analysis Using Nanopore-Based Single-Molecule Mass Spectrometry. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 12080– 12085,  DOI: 10.1073/pnas.1002194107
      38
      Theory for polymer analysis using nanopore-based single-molecule mass spectrometry
      Reiner, Joseph E.; Kasianowicz, John J.; Nablo, Brian J.; Robertson, Joseph W. F.
      Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (27), 12080-12085, S12080/1-S12080/4CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Nanometer-scale pores have demonstrated potential for the elec. detection, quantification, and characterization of mols. for biomedical applications and the chem. anal. of polymers. Despite extensive research in the nanopore sensing field, there is a paucity of theor. models that incorporate the interactions between chems. (i.e., solute, solvent, analyte, and nanopore). Here, we develop a model that simultaneously describes both the current blockade depth and residence times caused by individual poly(ethylene glycol) (PEG) mols. in a single α-hemolysin ion channel. Modeling polymer-cation binding leads to a description of two significant effects: a redn. in the mobile cation concn. inside the pore and an increase in the affinity between the polymer and the pore. The model was used to est. the free energy of formation for K+-PEG inside the nanopore (≈ -49.7 meV) and the free energy of PEG partitioning into the nanopore (≈ 0.76 meV per ethylene glycol monomer). The results suggest that rational, phys. models for the anal. of analyte-nanopore interactions will develop the full potential of nanopore-based sensing for chem. and biol. applications.
    39. 39
      Karnik, R.; Duan, C.; Castelino, K.; Daiguji, H.; Majumdar, A. Rectification of Ionic Current in a Nanofluidic Diode. Nano Lett. 2007, 7, 547– 551,  DOI: 10.1021/nl062806o
      39
      Rectification of Ionic Current in a Nanofluidic Diode
      Karnik, Rohit; Duan, Chuanhua; Castelino, Kenneth; Daiguji, Hirofumi; Majumdar, Arun
      Nano Letters (2007), 7 (3), 547-551CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      The authors demonstrate rectification of ionic transport in a nanofluidic diode fabricated by introducing a surface charge discontinuity in a nanofluidic channel. Device current-voltage (I-V) characteristics agree qual. with a 1-dimensional model at moderate to high ionic concns. This study illustrates ionic flow control using surface charge patterning in nanofluidic channels under high bias voltages.
    40. 40
      Vlassiouk, I.; Smimov, S.; Siwy, Z. Nanofluidic Ionic Diodes. Comparison of Analytical and Numerical Solutions. ACS Nano 2008, 2, 1589– 1602,  DOI: 10.1021/nn800306u
      40
      Nanofluidic Ionic Diodes. Comparison of Analytical and Numerical Solutions
      Vlassiouk, Ivan; Smirnov, Sergei; Siwy, Zuzanna
      ACS Nano (2008), 2 (8), 1589-1602CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      Recently reported exptl. and theor. studies of nanofluidic nonlinear devices, such as bipolar and unipolar ionic diodes, have yet to answer the question about the possibility of their further miniaturization. We theor. investigate the effects of size redn., applied bias, and soln. ionic strength in such devices. We compare the numerical solns. of the Poisson, Nernst-Planck (PNP), and Navier-Stokes (NS) equations with their one-dimensional, anal. approxns. We demonstrate that the contribution of electroosmosis is insignificant and find anal. approxns. to PNP for bipolar and unipolar diodes that are in good agreement with numerical 3D solns. We identify the minimal dimensions for such diodes that demonstrate ion current rectification behavior and demonstrate the importance of the edge effect in very short diodes.
    41. 41
      Vilozny, B.; Wollenberg, A. L.; Actis, P.; Hwang, D.; Singaram, B.; Pourmand, N. Carbohydrate-Actuated Nanofluidic Diode: Switchable Current Rectification in a Nanopipette. Nanoscale 2013, 5, 9214– 9221,  DOI: 10.1039/C3NR02105J
      41
      Carbohydrate-actuated nanofluidic diode: switchable current rectification in a nanopipette
      Vilozny, Boaz; Wollenberg, Alexander L.; Actis, Paolo; Hwang, Daniel; Singaram, Bakthan; Pourmand, Nader
      Nanoscale (2013), 5 (19), 9214-9221CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
      Nanofluidic structures share many properties with ligand-gated ion channels. However, actuating ion conductance in artificial systems is a challenge. We have designed a system that uses a carbohydrate-responsive polymer to modulate ion conductance in a quartz nanopipette. The cationic polymer, a poly(vinylpyridine) quaternized with benzylboronic acid groups, undergoes a transition from swollen to collapsed upon binding to monosaccharides. As a result, the current rectification in nanopipettes can be reversibly switched depending on the concn. of monosaccharides. Such mol. actuation of nanofluidic conductance may be used in novel sensors and drug delivery systems.
    42. 42
      Plett, T. S.; Cai, W.; Le Thai, M.; Vlassiouk, I. V.; Penner, R. M.; Siwy, Z. S. Solid-State Ionic Diodes Demonstrated in Conical Nanopores. J. Phys. Chem. C 2017, 121, 6170– 6176,  DOI: 10.1021/acs.jpcc.7b00258
      42
      Solid-State Ionic Diodes Demonstrated in Conical Nanopores
      Plett, Timothy S.; Cai, Wenjia; Le Thai, Mya; Vlassiouk, Ivan V.; Penner, Reginald M.; Siwy, Zuzanna S.
      Journal of Physical Chemistry C (2017), 121 (11), 6170-6176CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
      Ionic transport at the nanoscale features phenomena that are not obsd. in larger systems. Nonlinear current-voltage curves characteristic of ionic diodes as well as ion selectivity are examples of effects obsd. at the nanoscale. Many man-made nanopore systems are inspired by biol. channels in a cell membrane, thus measurements are often performed in aq. solns. Consequently, much less is known about ionic transport in nonaq. systems, esp. in solid-state electrolytes. Here we show ionic transport through single pores filled with gel electrolyte of poly(Me methacrylate) (PMMA) doped with LiClO4 in propylene carbonate. The system has no liq. interface and the ionic transport occurs through the porous gel structure. We demonstrate that a conically shaped nanopore filled with the gel rectifies the current and works as a solid-state ionic diode.
    43. 43
      Ma, L.; Li, Z.; Yuan, Z.; Huang, C.; Siwy, Z. S.; Qiu, Y. Modulation of Ionic Current Rectification in Ultrashort Conical Nanopores. Anal. Chem. 2020, 92, 16188– 16196,  DOI: 10.1021/acs.analchem.0c03989
      43
      Modulation of ionic current rectification in ultrashort conical nanopores
      Ma, Long; Li, Zhongwu; Yuan, Zhishan; Huang, Chuanzhen; Siwy, Zuzanna S.; Qiu, Yinghua
      Analytical Chemistry (Washington, DC, United States) (2020), 92 (24), 16188-16196CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Nanopores that exhibit ionic current rectification (ICR) behave like diodes such that they transport ions more efficiently in one direction than in the other. Conical nanopores have been shown to rectify ionic current, but only those with at least 500 nm in length exhibit significant ICR. Here, through the finite element method, we show how ICR of conical nanopores with lengths below 200 nm can be tuned by controlling individual charged surfaces, i.e., the inner pore surface (surfaceinner) and exterior pore surfaces on the tip and base side (surfacetip and surfacebase). The charged surfaceinner and surfacetip can induce obvious ICR individually, while the effects of the charged surfacebase on ICR can be ignored. The fully charged surfaceinner alone could render the nanopore counterion-selective and induces significant ion concn. polarization in the tip region, which causes reverse ICR compared to nanopores with all surfaces charged. In addn., the direction and degree of rectification can be further tuned by the depth of the charged surfaceinner. When considering the exterior membrane surface only, the charged surfacetip causes intrapore ionic enrichment and depletion under opposite biases, which results in significant ICR. Its effective region is within ∼40 nm beyond the tip orifice. We also found that individual charged parts of the pore system contributed to ICR in an additive way because of the additive effect on the ion concn. regulation along the pore axis. With various combinations of fully/partially charged surfaceinner and surfacetip, diverse ICR ratios from ∼2 to ∼170 can be achieved. Our findings shed light on the mechanism of ICR in ultrashort conical nanopores and provide a useful guide to the design and modification of ultrashort conical nanopores in ionic circuits and nanofluidic sensors.
    44. 44
      Scott, E. R.; White, H. S.; Bradley, P. J. Iontophoretic Transport through Porous Membranes Using Scanning Electrochemical Microscopy: Application to in Vitro Studies of Ion Fluxes through Skin. Anal. Chem. 1993, 65, 1537– 1545,  DOI: 10.1021/ac00059a010
      44
      Iontophoretic transport through porous membranes using scanning electrochemical microscopy: application to in vitro studies of ion fluxes through skin
      Scott, Erik R.; White, Henry S.; Phipps, J. Bradley
      Analytical Chemistry (1993), 65 (11), 1537-45CODEN: ANCHAM; ISSN:0003-2700.
      Scanning electrochem. microscopy (SECM) is used to map localized iontophoretic fluxes of electroactive species through porous membranes. A method is described that allows both the rate of transport of species from a microscopic pore and the pore's diam. to be measured. SECM images and analyses of synthetic porous membranes (track-etched polycarbonate and mica membranes) and hairless mouse skin are reported. Preliminary anal. of SECM images of the mouse skin indicates that a significant percentage of the iontophoretic flux occurs through pores assocd. with hair follicles.
    45. 45
      Morgan, H.; Green, N. G. AC Electrokinetics: Colloids and Nanoparticles; Research Studies Press, 2003.
      There is no corresponding record for this reference.
    46. 46
      Kowalczyk, S. W.; Wells, D. B.; Aksimentiev, A.; Dekker, C. Slowing down DNA Translocation through a Nanopore in Lithium Chloride. Nano Lett. 2012, 12, 1038– 1044,  DOI: 10.1021/nl204273h
      46
      Slowing down DNA Translocation through a Nanopore in Lithium Chloride
      Kowalczyk, Stefan W.; Wells, David B.; Aksimentiev, Aleksei; Dekker, Cees
      Nano Letters (2012), 12 (2), 1038-1044CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      The charge of a DNA mol. is a crucial parameter in many DNA detection and manipulation schemes such as gel electrophoresis and lab-on-a-chip applications. Here, we study the partial redn. of the DNA charge due to counterion binding by means of nanopore translocation expts. and all-atom mol. dynamics (MD) simulations. Surprisingly, we find that the translocation time of a DNA mol. through a solid-state nanopore strongly increases as the counterions decrease in size from K+ to Na+ to Li+, both for double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA). MD simulations elucidate the microscopic origin of this effect: Li+ and Na+ bind DNA stronger than K+. These fundamental insights into the counterion binding to DNA also provide a practical method for achieving at least 10-fold enhanced resoln. in nanopore applications.
    47. 47
      Confederat, S.; Sandei, I.; Mohanan, G.; Wälti, C.; Actis, P. Nanopore Fingerprinting of Supramolecular DNA Nanostructures. Biophys. J. 2022, 121, 4882– 4891,  DOI: 10.1016/j.bpj.2022.08.020
      47
      Nanopore fingerprinting of supramolecular DNA nanostructures
      Confederat, Samuel; Sandei, Ilaria; Mohanan, Gayathri; Walti, Christoph; Actis, Paolo
      Biophysical Journal (2022), 121 (24), 4882-4891CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      DNA nanotechnol. has paved the way for new generations of programmable nanomaterials. Utilizing the DNA origami technique, various DNA constructs can be designed, ranging from single tiles to the self-assembly of large-scale, complex, multi-tile arrays. This technique relies on the binding of hundreds of short DNA staple strands to a long single-stranded DNA scaffold that drives the folding of well-defined nanostructures. Such DNA nanostructures have enabled new applications in biosensing, drug delivery, and other multifunctional materials. In this study, we take advantage of the enhanced sensitivity of a solid-state nanopore that employs a poly-ethylene glycol enriched electrolyte to deliver real-time, non-destructive, and label-free fingerprinting of higher-order assemblies of DNA origami nanostructures with single-entity resoln. This approach enables the quantification of the assembly yields for complex DNA origami nanostructures using the nanostructure-induced equiv. charge surplus as a discriminant. We compare the assembly yield of four supramol. DNA nanostructures obtained with the nanopore with agarose gel electrophoresis and at. force microscopy imaging. We demonstrate that the nanopore system can provide anal. quantification of the complex supramol. nanostructures within minutes, without any need for labeling and with single-mol. resoln. We envision that the nanopore detection platform can be applied to a range of nanomaterial designs and enable the anal. and manipulation of large DNA assemblies in real time.

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    • Supporting Information contents: simulation overview and verification; definition of simulation input parameters; influence of external solution viscosity; reproducibility of experimental data; ion concentrations at the nanopipette tip region; ion transport and the definition of the sensing region; interface displacement model; and effect of an externally applied pressure on the voltammogram and dsDNA translocation (PDF)


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