Modulation of Charge Density and Charge Polarity of Nanopore Wall

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Modulation of Charge Density and Charge Polarity of Nanopore Wall by Salt Gradient and Voltage Chih-Yuan Lin, Elif Turker Acar, Jake W. Polster, Kabin Lin, Jyh-Ping Hsu, and Zuzanna S. Siwy ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b01357 • Publication Date (Web): 26 Jul 2019 Downloaded from pubs.acs.org on July 27, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Modulation of Charge Density and Charge Polarity of Nanopore Wall by Salt Gradient and Voltage

Chih-Yuan Lin,1,2,+ Elif Turker Acar,1,3,+ Jake W. Polster,4 Kabin Lin,1,5 Jyh-Ping Hsu,2,6,* Zuzanna S. Siwy1,4,7,* 1Department

of Physics and Astronomy, University of California, Irvine, California 92697, United States

2Department 3Department

of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan of Chemistry, Faculty of Engineering, Istanbul University - Cerrahpasa, 34320 Avcılar-Istanbul, Turkey

4Department 5School

of Chemistry, University of California, Irvine, California 92697, United States

of Mechanical Engineering and Jiangsu Key Laboratory for Design and Manufacture

of Micro-Nano Biomedical Instruments, Southeast University, Nanjing 211189, China 6Department

of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 10617, Taiwan

7Department

of Biomedical Engineering, University of California, Irvine, California 92697, United States

+ These authors contributed equally.

* Corresponding authors: E-mail: [email protected] (Zuzanna S. Siwy); [email protected] (Jyh-Ping Hsu)

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Abstract Surface charge plays a very important role in biological processes including ionic and molecular transport across a cell membrane. Placement of charges and charge patterns on walls of polymer and solid-state nanopores allowed preparation of ion selective systems as well as ionic diodes and transistors to be applied in building biological sensors and ionic circuits. In this manuscript we show that surface charge of a 10 nm in diameter silicon nitride nanopore placed in contact with a salt gradient is not a constant value but rather it depends on applied voltage, and magnitude of the salt gradient. We found that even when a nanopore was in contact with solutions of pH equivalent to the isoelectric point of the pore surface, the pore walls became charged with voltage-dependent charge density. Implications of the charge gating for detection of proteins passing through a nanopore were considered as well. Experiments performed with single 30 nm in length silicon nitride nanopores were described by continuum modeling, which took into account surface reactions on the nanopore walls and local modulation of the solution pH in the pore and at the pore entrances. The results revealed that manipulation of surface charge can occur without changing pH of the background electrolyte, which is especially important for applications where maintaining pH at a constant and physiological level is necessary. The system presented also offers a possibility to modulate polarity and magnitude of surface charges in a two-electrode set-up, which previously was accomplished in more complex multi-electrode systems. Keywords: ion transport, nanopore, fluidic diode, debye length, charge regulation


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Biological channels in a cell membrane function in highly asymmetric ionic conditions, such that the medium inside the cell contains a higher concentration of potassium ions than the extracellular medium.1 In many cells, the sodium and calcium gradients have the opposite direction, so that concentration of the ions on the extracellular side is higher. Mimicking the conditions of biological ion channels, solid state and polymer nanopores were also subjected to salt concentration gradients with applications in ion gating and pumping,2-6 detection of particles and molecules in the resistive-pulse technique,7-13 energy conversion,14-17 and desalination.18, 19 As an example, introducing a salt gradient across a silicon nitride nanopore led to a significant enhancement of the DNA capture rate, which sped up the analysis.7 A similar concept to control transport of biomolecules and nanoparticles was adopted in other nanopore systems,11,

20

and explained via the effects of locally enhanced electric field and

osmotic flow.13, 21, 22 Salinity power generation, i.e. converting Gibbs free energy into electricity, has also become a promising application of ion selective nanopores and porous membranes placed in contact with a salt concentration gradient. Driven by the gradient, the counter-ions preferentially diffuse from the high concentration side to the low concentration side of a nanoporous membrane, producing clean and sustainable energy by means of osmotic current and diffusion potential.14-16 Another technological application of porous membranes in contact with salt gradient is desalination.23, 24 Recently developed nanopore systems provide solutions to lower energy consumption and improved throughput of desalination and nanofiltration technologies.25, 26 Single nanopores with well-defined geometry and surface characteristics provide an excellent model system to understand ionic and molecular transport at the nanoscale in a wide variety of experimental conditions.27 Several previous reports discussed the mechanisms of


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ion transport under an asymmetric salt concentration in conical28-30 and cylindrical5,

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nanopores. For example, conically shaped nanopores with negative surface charges exhibited diffusion current whose magnitude was dependent on the direction of the concentration gradient.28 The findings were explained via concentration dependent thickness of the electric double layer (EDL) at the narrowest part of the pore, which governs the system behavior. In addition, cylindrical nanopores were reported to function as ionic diodes when placed in a salt concentration gradient.31 The application of salt gradient to tune ionic selectivity was reported as well.32 All the above mentioned transport effects were described by continuum models based on the Poisson-Nernst-Planck and Navier-Stokes equations, which revealed local ion accumulation and depletion in response to both voltage bias and salt gradient. The models treated surface charge density as a parameter determined only by the type of material or chemical modification as well as pH. Even though dependence of surface charge density on salt concentration was postulated before,34, 35 this effect was not taken into account when describing transport through nanopores in contact with a salt gradient. In this manuscript we reveal that a combination of salt gradient and voltage can modulate and induce surface charge in nanopores even when they are in contact with a solution of pH equivalent to the isoelectric point of the nanopore material. Consequently, nanopores that are not charged at 0 V produce ion current rectification when electric field is applied. We also identified experimental conditions where the nanopore surface is positively charged for one voltage polarity and negatively charged for the opposite voltage polarity. The manuscript provides a complete numerical treatment of ion current through silicon nitride (SiNx) nanopores at different pH values, salt gradients and voltages. The model incorporates three protonation/deprotonation reactions that can take place on SiNx surfaces, and the simulated transport properties are in a very good agreement with experimental data. This study highlights the influence of the salt gradient on the local surface charge density, which


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in turn modulates ion current and ion current rectification.

Results and Discussion All experiments were performed with single nanopores prepared in 30 nm thick silicon nitride membranes by the process of dielectric breakdown.36-38 As illustrated in Figure 1, one reservoir was filled with an aqueous KCl solution of a higher salt concentration (e.g., 1000, 100 or 10 mM) while the other reservoir contained solution of a lower salt concentration, 1 mM KCl, with the same pH value. Three pH values were considered, pH 3, pH 6 and pH 8. Due to different salt concentrations on each side of the membrane, both cations and anions are driven by diffusion from the high concentration side toward the low concentration side. Figure 2 shows current-voltage characteristics of a 10 nm in diameter and 30 nm in length nanopore recorded at salt gradients of 100 mM/1 mM and 1000 mM/1 mM KCl. The measurements indicate that the nanopore rectifies current in a pH dependent fashion. At pH 8, currents at positive voltages are larger than those at negative voltages, showing a diode-like current-voltage curve, referred to as ion current rectification (ICR). Acidic conditions of pH 3 also lead to ion current rectification but in the opposite direction, i.e. positive currents are smaller in magnitude than negative currents. These experiments are in agreement with the previously reported isoelectric point of silicon nitride (~pH 6);39, 40 consequently, the polarity of surface charges switches from negative at pH 8 to positive at pH 3. We were, however, surprised to see that under the salt gradient the nanopore exhibited ion current rectification even at pH 6, a condition where the silicon nitride surface is predicted to be neutral (Figure 2a,c). This finding conflicts with the conventional thought that a cylindrical nanopore in contact with a salt concentration gradient can rectify ion current only if the pore walls contain excess surface charge.27, 41 Our experiments suggested that the imposed concentration gradient might modulate local surface charges of the nanopore in a


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voltage-dependent manner. Figures S1 contains recordings for the same nanopore in symmetric electrolyte concentrations, and indicates that without the salt gradient the nanopore’s current-voltage curves were mostly linear (with exception of 100 mM, 1 M at pH 8 and pH 3). Figures S2-S4 show recordings for three additional nanopores that were independently prepared by the dielectric breakdown process. A 5 nm nanopore in Figure S2 exhibited linear I-V curves in nearly all cases with symmetric ionic concentrations on both sides of the membrane (with exception of weak rectification in 100 mM, pH 6); the nanopore rectified current at pH 6 when salt gradients were introduced (this nanopore was not studied at pH 3 or pH 8). In Figure S3, a nanopore with a diameter of 7 nm was examined, and did not rectify at any of the symmetric cases considered. Under salt gradients, the nanopore in Figure S3 exhibited qualitatively similar behavior to the nanopore shown in the main manuscript. We also show findings for a 20 nm nanopore in symmetric and asymmetric salt conditions in Figure S4. All these recordings indicate that the effect of ion current rectification at pH 6 under salt gradient is robust and independent of whether in symmetric conditions the pores showed linear I-V curves in all pH values/concentrations or they rectified in few cases. As an example, the pore shown in the main manuscript and the pore in Figure S4 displayed rectification in symmetric conditions at pH 3, and I-V curves of the two nanopores were inverted with respect to one another. The rectification without salt gradient can stem from possible differences in the structure between independently prepared nanopores;42 these variations in local pore geometry do not however affect the main findings of our work.

In order to provide evidence that the effect of ion current rectification at SiNx surface isoelectric point conditions is independent to the method of nanopore preparation, we also performed experiments with a nanopore drilled by electron beam in a transmission electron


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microscope.43 A series of experiments in symmetric and asymmetric salt conditions is shown in Figure S5. In addition, this nanopore was probed at pH 4 and pH 5, because, as we explain below, it was characterized by an isoelectric point between 4 and 5.44, 45 This observation is consistent with earlier reports that showed surface properties of silicon nitride nanopores depended on the method of preparation.39,

44-51

Similar to the nanopores prepared by the

dielectric breakdown process, the TEM nanopore also exhibited rectification at pH values where the surface was predicted to be neutral.

Current-voltage curves recorded in salt gradients at pH 3 and pH 8 were also analyzed in terms of current rectification magnitude. We realized that the nanopores shown in Figures 2 and S4 rectified to a larger extend in the acidic conditions (Figure 2e,f), suggesting the charge density at pH 3 was higher than at pH 8. In order to capture the dependence of surface charge on pH we considered the following three protonation reactions:46, 47

SiOH  SiO   H 

(1)

SiOH 2   SiOH  H 

(2)

SiNH 3  SiNH 2  H 

(3)

Surface charge density can then be calculated as:

 [H  ]s 2 [H  ]s  K a1   a Ka 2 Ka 3 a  s  1018 eN t    2 [H  ]s a  b   a  b [H  ]s  K a1  [H ]s 1 Ka 2 Ka 3 

    

where e is the elementary charge, and N t   a  b is the total site density of the functional groups.  a and b are the site densities of silanol and amine groups, respectively. [H  ]s denotes the proton concentration on the nanopore surface.52 More detailed description of the modeling can be found in Supporting Information.


(4)

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Figure 1. Schematic illustration of the experiment setup and the pH-regulated surface. One reservoir was filled with an aqueous KCl solution of a higher salt concentration while the other reservoir contained solution of a lower salt concentration, 1 mM KCl, with the same pH value. The transmembrane potential was applied with two Ag/AgCl electrodes with the ground electrode placed in the 1 mM KCl solution. Due to the protonation/deprotonation of functional groups on the silicon nitride surface, the nanopore is negatively (positively) charged at the solution pH above (below) the isoelectric point of silicon nitride surface. The imposed salt gradient and applied voltage lead to voltage-dependent charge density, voltage-dependent ionic concentrations in the pore, and ion current rectification.

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Figure 2. Current-voltage characteristics of a 10 nm in diameter SiNx nanopore that is placed between two different electrolyte solutions with low (1 mM) and high (100 or 1000 mM) KCl concentrations of the same pH. Three pH conditions were considered, pH 3, pH 6, and pH 8. Panels in the left column show experimental data, while the panels on the right present modeling results. (a, b) Results for the salt gradient of 100 mM/1 mM; (c, d) Results for the salt gradient of 1000 mM/1 mM. (e, f) ICR as a function of pH and salt gradient calculated such that ICR is equal or larger than 1, which facilitates comparative analysis. ICR at pH 8 (pH 3 and pH 6) is calculated as a ratio of current magnitudes at +1 V and -1 V (ratio of current magnitudes at -1 V and at +1 V. Dissociation constant and density of charge groups used in the simulations are given in the Materials and Methods section.

In order to describe the experimental results quantitatively, we utilized a continuum model that takes into account local pH modulation of surface charges. To this end, concentration of multiple ionic species, including K+, Cl, H+, and OH ions, was introduced 9 ACS Paragon Plus Environment

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into the Poisson-Nernst-Planck and Navier-Stokes equations (see Materials and Method). This highly coupled model calculated local surface charge based on all ionic concentrations set by the conditions of our experiments, including salt concentration and voltage. As shown in Figure 2b,d, the simulated I-V curves are in a good agreement with the experimental data (Figure 2a,c), suggesting that the results from the numerical model can offer insight into the mechanism of the nanopore transport properties. The current-voltage curves recorded at a salt gradient of 10 mM/1 mM is provided in Figure S6 of the Supporting Information. We will begin the analysis with the two extreme pH values studied, pH 8 and pH 3. It is generally accepted that silicon nitride nanopores are negatively charged at pH 8, implying that cations are the majority of transported ions. If a positive voltage bias (e.g., +1 V) is applied, with the positive potential at the side with higher concentration (Figure 1), the driving force of the external electric field and the salt gradient move cations in the same direction, yielding accumulation of K+ within the nanopore (Figures 3a and S7) and a larger current. In contrast, if a negative voltage bias (e.g., 1 V) is applied, the direction of the external electric field is opposite to the salt gradient, resulting in a relatively low ionic concentrations in the nanopore, and accordingly, a smaller current (Figure 3a). At pH 3, the situation is reversed since with excess positive surface charge, anions are the majority carriers, and consequently the I-V curve is flipped. Figure S7 a-c confirms that for the salt gradient of 100 mM/1 mM, the total ionic concentration within the nanopore at +1 V (1 V) is significantly higher than that at 1 V (+1 V) for pH 8 (pH 3), which is in accordance with the observed rectification phenomenon in Figure 2. For the largest salt gradient considered, 1000 mM/1 mM (Figure S7 d-f), ionic distributions are similar to those shown in Figure S7 a-c, however the voltage-induced accumulation and depletion of ions is less pronounced due to thinner EDL, as reported previously.31, 32


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The profiles shown in Figure 3 also revealed that concentrations of protons and hydroxide ions in the pore were voltage dependent as well. Note that this figure is focused on the pore entrances and interior region, and complementary Figures S8, S9 show a larger portion of the reservoirs on both side of the membrane where the concentrations of individual ions reach their bulk values. At pH 8 and positive voltages, accumulation of potassium ions in the nanopore is accompanied by enhanced OH concentration, which we believe is dictated by the electroneutrality requirement. At pH 3 and negative voltages, accumulation of chloride ions and protons was observed. Voltage and salt gradient enhancement of protons and hydroxides participates in the modulation of the pore wall’s surface charge.

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Figure 3. Axial variations of cross-section-averaged concentrations for individual ions at +1 V and 1 V (red) for a 10 nm nanopore placed in contact with a salt gradient of 100 mM/1 mM KCl, at three levels of pH. (a, c, e) K+ and Cl ions; (b, d, f) H+ and OH ions. Yellow regions highlight the nanopore interior. The reservoir on the right contains 1 mM KCl and the ground electrode. Figure S8 shows larger portions of the reservoirs where the ionic concentrations reach their bulk values.

We further plotted the axial variations of surface charge densities along the pore surface for various combinations of applied voltage and pH, as shown in Figures 4 and S10. Due to the presence of the salt gradient, the surface charge density,  s , was not a constant parameter but rather varied considerably along the axis for both pH 8 and pH 3 even if no voltage is applied (i.e., 0 V). The surface-averaged charge density calculated from the numerical model,  s , is given in Table 1. In general, the values obtained numerically are smaller than those estimated by eqn. (4). In addition,  s at pH 3 is larger than  s at pH 8, suggesting the nanopore has a higher rectification capability when immersed in pH 3 solution. Table S1 also shows the magnitude of  s increases with increasing salt concentration, which is related to the accumulation of counterions in the pore that, in turn, replace H+ or OH- ions; as the salt concentration increases, K+ ions at pH 8 ions replace H+ (Cl ions at pH 3 replace OH) at the surface, yielding a higher charge density.53 12 ACS Paragon Plus Environment

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Figures 4 and S10 reveal that the magnitude of  s was also modulated by the voltage polarity, compare  s for +1 V and 1 V. Note that due to the effect of concentration polarization (CP),54, 55 the two pore entrances can have significantly different magnitudes of

 s . Figure 4e,f shows that  s at pH 3 and +1 V varies considerably along the nanopore wall, and drops abruptly near the nanopore opening in contact with the lower salt concentration. The sudden drop in  s is consistent with the depletion zone created at this location, which lowers the local ionic concentration of K+ and H+ (see black solid curves of Figure 3e,f), and consequently diminishes the magnitude of  s . The effect of ionic concentrations on  s is especially pronounced at pH 3, because surface charge at this condition stems from the protonation reactions of the silanol (eq. (2)) and amine (eq. (3)) groups, and is highly sensitive to H+ concentration. At the opposite voltage polarity of – 1 V, the depletion zone is weaker, because it is created at the nanopore opening in contact with higher ionic concentrations.

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Figure 4. Axial variation of surface charge density of the pore wall  s at three levels of applied voltage. (a, b) pH 8; (c,d) pH 6; (e, f) pH 3. Left column: 100 mM/1 mM; right column: 1000 mM/1 mM. The silicon nitride nanopore we modeled was 10 nm in diameter and 30 nm in length. The reservoir on the right contains 1 mM KCl and the ground electrode.

Table 1. Surface-averaged charge density  s (mC/m2) of the pore wall calculated at three levels of bulk pH without applied voltage (0 V). Bulk pH

3

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Eqn (4)

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Table 2. Surface-averaged charge density  s (mC/m2) of the pore wall calculated at three levels of bulk pH, and +/1 V. The values were obtained numerically from the pH modulation model. Bulk pH 100 mM/1 mM

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Ion current rectification observed at pH 6 can also be explained by the model (see blue dotted lines of Figure 2b,d), which revealed that even when the pore is neutral at 0 V, it acquires effective charge when voltage is applied (Figure 4c,d). As an example, let’s consider the case of positive voltages when potassium ions are transported down the electric potential and concentration gradients, while chloride ions move against the concentration gradient. At +1 V, concentration of K+ ions in the pore stays on a level of ~60 mM, and is accompanied by a significant enhancement of OH and depletion of H+ (Figure 3c,d), similar to results seen at pH 8 (Figure 3a,b). Here also the enhanced OH concentration is believed to be dictated by the electroneutrality requirement; indeed, the K+ and OH axial distributions have a very similar character. As shown in Figure 5a, the OH ion concentration increases considerably with increasing positive potentials while the H+ concentration becomes depleted; consequently a negative surface charge is induced on the pore walls. For the opposite negative voltage polarity, the H+ (OH-) concentration is largely enhanced (depleted) (Figure 5b), because chloride ions are preferentially accumulated in the pore. In this case, the profiles of Cl and H+ follow the same axial and voltage dependence, and the effective charge of the 15 ACS Paragon Plus Environment

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pore wall becomes positive.

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Figure 5. Voltage-dependence of axial profiles of cross-section-averaged concentrations of (a) OH  and (b) H+ ions in a 10 nm in diameter nanopore placed in contact with 100 mM/1 mM KCl gradient, pH 6. Yellow regions highlight the nanopore interior. The reservoir on the right contains 1 mM KCl and the ground electrode. Figure S9 shows larger portions of the reservoirs where the ionic concentrations reach their bulk values.

Figure 4c,d shows the magnitude of surface charge at pH 6 induced by salt gradients and voltage. Note that, regardless of the degree of the salt gradient,  s approaches zero at 0 V. The figure also reveals that in order to obtain finite  s and ion current rectification, the imposed salt gradient has to be sufficiently large, e.g., 1000 mM/1 mM, 100 mM/1 mM, or 1000 mM/10 mM (Figures 4c,d and S11). When a nanopore is in contact with 10 mM/1 mM, pH 6, the induced charge is very low (a few mC/m2), and the experimental as well as simulated I-V curves are nearly linear (Figures S6 and S10b). We also realized that, at pH 6, the magnitude of the  s at 1 V was significantly larger than that at +1 V; the average magnitude of  s at 1 V is ~4 and 6 times larger than  s at +1 V for salt gradients of 100 mM/ 1mM and 1000 mM/1 mM, respectively (Table 2). Unequal magnitude of induced 16 ACS Paragon Plus Environment

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charge in positive and negative voltages was found crucial for ion current rectification to occur at pH 6. Figure S12 shows results of numerical modeling that assumed the magnitude of surface charge induced by voltage was equal in magnitude but opposite in polarity. In this case, the nanopore did not rectify the current independent of whether only K+ and Cl were considered or when all four species were included in the simulations. Finally, for the highest concentration gradient of 1000 mM/1 mM at pH 6 (Figures 2c and S7e) only weak rectification is seen for the 10 nm nanopore, even though the induced surface charge is voltage polarity asymmetric. We believe this result can be again explained by screening of the surface charges at high salt concentrations; this claim is also supported by lower rectification in the 1000 mM/10 mM gradient compared to rectification in the 100 mM/1 mM gradient (Figures 2 and S11). Note that the narrower (5 nm and 7 nm) nanopores shown in Figures S2, S3 rectify current at pH 6 for both salt gradients. As the next step, we calculated the magnitude of  s as a function of voltage in symmetric salt concentrations. The results in Table S2 indicate that, at pH 6,  s is indeed insensitive to the applied voltage and remains close to 0. Tables 1 and S2 confirm that, if the bulk pH is close to the isoelectric point of the pore wall, formation of excess surface charge requires both salt gradient and applied voltage. The same model was applied to describe the experimental data recorded for the TEM drilled nanopore shown in Figure S5. Simulated current-voltage curves for different parameters of the surface groups (pKa, charge density and composition) are included in Figure S13. Under salt gradients, at pH 3 and pH 8 the pore rectified in a similar manner as the nanopores prepared by the dielectric breakdown process. At pH 6 and pH 5, however, the nanopore still rectified in the same direction as at pH 8, suggesting the nanopore isoelectric point was lower than for the nanopores prepared by the dielectric breakdown (Figure S5). Finally, the recordings and modeling at pH 4 and pH 5 confirmed that the isoelectric point of 17 ACS Paragon Plus Environment

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this nanopore was between pH 4 and pH 5; at both pH values we identified conditions when the nanopore walls were negatively charged for positive voltages and positively charged at negative voltages such that ion current rectification could still be observed (Figure S13). The possibility of inducing surface charge with concentration gradient at pH 6 helped us understand the non-linear current increase that was observed at negative voltages above 0.5 V at pH 8 and 1000 mM/1 mM KCl (red symbols and curve in Figure 2c,d). We postulated this non-linear ion current behavior was related to a change in the surface charge, because as shown in Figure 4b, the surface charge density at +1 V is negative as expected at pH 8, but assumes a positive value for 1 V. Figure 6a shows that the change of surface charge with voltage is gradual. The magnitude of  s being negative at 0 V, decreases with the increase of negative voltages, and at ~ 0.3 V becomes positive. Increasing the voltage beyond 0.3 V leads to more positive surface charge, which is responsible for the increase of the current and reduced ICR. We believe that the switch of surface charge polarity at negative voltages occurs, because the imposed electric field and salt gradient act on K+ in the opposite directions. Consequently, in order to reach electroneutrality, protons accumulate in the pore in a voltage-dependent manner and induce formation of positive surface charge (Figure 6b). The effect of proton accumulation on local surface charge was found very robust, because changing protons mobility in our simulations did not lead to significant changes in the predicted I-V curves (Figure S14). A similar character of current-voltage curves at pH 8 was also observed for the nanopore shown in Figure S4.


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Axial Position (nm) Figure 6. Axial variation of (a) surface charge density  s , (b) H+ concentration, and (c) electroosmotic velocity for negative voltages applied across a nanopore in contact with a 1000 mM/1 mM salt gradient at pH 8. Yellow region highlights the nanopore interior. The nanopore was 10 nm in diameter and 30 nm in length.

Our experiments and modeling at pH 8 suggested that a negatively charged pore when exposed to sufficiently high salt gradient can become positively charged at voltages of one polarity. Similar considerations at pH 3 revealed that the system remained positively charged in all salt conditions examined. The small difference in the  s magnitude for the 1000 mM/1 mM and 100 mM/1 mM gradients (Table 2) implies that the reduction of ICR ratio for the former gradient can be attributed to thinner EDL, and weaker dependence of ionic


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concentrations on voltage (Figures S7 and S15). We also realized that the voltage-dependent switch of surface charge polarity at pH 8 and pH 6 could lead to modulation of electroosmotic flow (EOF)56 inside the nanopore (Figures 6c and 7). The notable conclusion from the simulations is that at pH 8 and pH 6 the direction of EOF is voltage polarity independent (Figure 7a,b). At +1 V and 1000 mM/1 mM KCl gradient, the pore wall is negatively charged, thus EOF’s direction is determined by K+ ions moving from the more concentrated solution towards 1 mM KCl. At 1 V however, the pore acquires positive surface charges, which lead to accumulation of Cl ions at the surface, and EOF is again directed towards 1 mM KCl. As discussed above, for all salt gradients at pH 3, the pore walls remained positively charged, thus the direction of EOF switched for opposite voltage polarities. Note also that at pH 3 and pH 8, EOF velocity is the highest for the voltage polarity where the currents were higher, thus at conditions at which the pore walls were more charged.


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Figure 7. Electroosmotic (EOF) velocity profiles (m/s) in a nanopore placed in contact with a salt gradient of 1000 mM/1 mM for +1 V and 1 V, and three levels of pH. EOF velocities for 100 mM/1 mM salt gradient are shown in Figure S16. The top reservoir contains 1 mM KCl and the ground electrode.


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Figure 8. Modeling for translocation of a globular protein through a nanopore placed in contact with a concentration gradient of 1000 mM/1 mM KCl (solid curves), and symmetric salt conditions (1M KCl) (dashed curves). The hydrodynamic radius and isoelectric point of the protein were assumed as 3 nm and 4.7, respectively.57-59 (a) Translocation velocity and (b) charge density of a protein at pH 6 and +0.5 V. (c) Translocation velocity and (d) charge density of a protein at pH 6 and 0.5 V. Note that at +0.5 V the protein passes from right to left in the scheme presented (upper left), and the translocation velocity is negative. For

0.5 V the protein moves from left to right (upper left scheme), and the translocation velocity is positive. EP and EO denote electrophoresis and electroosmosis, respectively. 22 ACS Paragon Plus Environment

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Inspired by the modulations of surface charge density and EOF in a nanopore, we asked a question whether the charge regulation could also occur for a molecule or particle passing through a pore placed in a salt gradient. If a molecule changed its charge state during translocation, one should be able to identify experimental conditions where the translocation velocity is tunable. To this end we considered a 6 nm in diameter globular protein characterized with an isoelectric point of 4.7,58, 59 passing through a pore subjected to a 1000 mM/1 mM KCl salt gradient at pH 6. Effective charge of the protein as a function of local pH is shown in Figure S17. When the protein is placed on the side of the membrane with 1 mM KCl, it will pass through the pore at positive voltages, and its electrokinetic transport will be opposed by EOF. The model predicted the protein’s effective charge would decrease during the translocation, further diminishing the electrophoretic force acting on the protein, and the passage velocity (Figure 8b). When the protein reaches the axial position of ~5 nm its velocity drops to 0 m/s, indicating a possibility of the molecule getting trapped in the pore. The process of protein trapping can be further influenced by the density of charge sites, N t , on the pore walls, as shown in Figure S18. When the protein is placed on the side of the nanopore in contact with 1 M KCl, its passage can be induced with negative voltages (Figure 8c). The modeling revealed that not only the pore walls become positively charged in this case (Figure 4), but the protein acquires positive charge as well (Figure 8d). The protein’s charge is lower than that of the pore walls, so that translocation still occurs for negative voltages. Note that the molecule slows down in the pore for these conditions as well, however no axial position with Up = 0 was identified. The situation in the absence of salt gradient at pH 6 was also considered (see dashed curves of Figure 8a,c). As expected, the trapping and large modulations of the translocation velocity are no longer observed. In this case, the protein always carries negative surface 23 ACS Paragon Plus Environment

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charge that is insensitive to voltage and direction of translocation. Finally, Figure S19 explores the case of pH 8 with the same salt gradient of 1000 mM/1 mM. Positive voltages were found to prevent the molecule from entering, while negative voltages caused the protein to speed up along the pore axis. These results suggest that performing detection of this protein at pH 6 offers a larger control over the translocation process and detection. Optimizing translocation velocity of proteins with different isoelectric points under salt gradient will require modeling of surface charge properties of the pore walls and the proteins at different voltages and pH values.

Conclusions This manuscript presents experimental and modeling results that suggest the surface charge of nanopores placed in contact with a salt concentration gradient is modulated by the applied voltage. A nanopore that is uncharged at 0 V at any salt conditions, when placed in a salt gradient can become positively charged at one voltage polarity and negatively charged at the opposite polarity. Even if a nanopore is charged in the absence of electric field, the pore’s walls can switch polarity when subjected to sufficiently high salt gradients and voltage. The experiments are accompanied by a numerical model, developed to capture local pH, salt concentrations, and dissociation level of surface groups as a function of salt gradient and voltage. The charge modulation model we described also applies for objects passing through a nanopore, such as proteins. We found that a salt gradient across a nanopore can tune the effective charge and translocation velocity of a protein. Detection of molecules and particles as they pass through a pore in the resistive-pulse technique is the basis of nanopore analytics, and controlling the passage velocity has been identified as one of the most pressing issues to improve the technology.60, 61 Our work suggests that a nanopore can tune the object velocity


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without any chemical modifications of the pore walls, but rather by performing the experiment in a salt gradient and placing the object to be detected on either side of the membrane.

Materials and Method Pore preparation. Four nanopores reported in this manuscript (diameters of 5 nm, 7nm, 10 nm, and 20 nm) were prepared in 30 nm thick silicon nitride films (50 x 50 µm2, Norcada Product) by the dielectric breakdown process, as reported before.37,

38

Briefly, a chip was

placed in a custom-made conductivity cell and exposed to 1 M KCl at pH 1.6. Voltage of 12 V was applied using two Ag/AgCl electrodes. An additional 8 nm in diameter nanopore was drilled in a homemade 15-nm-thick silicon nitride film by using 200 kV electron beam irradiation of the JEOL 2100F transmission electron microscope.43

Transmembrane current

was monitored during the process, and the process was stopped when the current increased ~100 nA from the background level.54 As prepared pore was subsequently characterized by recording an I-V curve, which quantified the pore resistance. The resistance was used to estimate the pore diameter assuming cylindrical shape of the pore.

Current measurement. Current−voltage curves were recorded for different salt concentrations (10 mM/1 mM, 100 mM/1 mM and 1000 mM/1 mM) at various pH values (pH 3, pH 6 and pH 8) with a Keithley 6487 picoammeter/voltage source (Keithley Instruments, Cleveland, OH) using two Ag/AgCl pellet electrodes. The voltage was swept between −1 V and +1 V with 0.1 V steps. At least three scans were recorded for every set of experimental conditions, and the data shown represent averages of the scans. The current was stable and showed only few % variability between scans (Figure S20).


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Theoretical modeling. Electrokinetic ion and protein transport in a cylindrical nanopore were modelled by the coupled Poisson-Nernst-Planck and Navier-Stokes equations, and solved using the commercial finite-element software COMSOL Multiphysics. To capture electrochemical properties of the silicon nitride nanopore, the pore wall surface had two types of functional groups with associated dissociation reactions as described by eq. (1-3).46, 47 The equilibrium dissociation constants of these reactions were, respectively, defined as

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3

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respectively; [H  ]s is the molar concentration of proton on the pore surface. Note that the silicon nitride surface might contain primary (SiNH2), secondary (Si2NH), and tertiary amine (Si3N) groups.39 Since all the amine groups are basic, to facilitate the modeling, one reaction was considered. Typically, amines are assumed to constitute one third of the reactive sites, while two-thirds are silanol groups.44 Surface charge density of the nanopore,  s , can be then expressed by eq. (4) given in the main text. It is known that the surface property of SiNx depends heavily on its preparation process,51,

62

thus we considered and tested a set of

reported parameters such as dissociation constants and site densities (see Figures S13, S21 and Table S3). Figures S22 and S23 show that the reported here behavior of nanopores under a salt gradient is insensitive to the presence of buffer ions and charged conditions on the membrane outer surfaces. Figure 2 in the main text was obtained using the following parameters: pK a1  6 , pK a 2  2 , pK b  10 , and N t  2 sites/nm2.


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Acknowledgements Research was supported as part of the Center for Enhanced Nanofluidic Transport, an Energy Frontier Research Center funded by the U.S. Department of Energy (DOE), Office of Science, Basic Energy Sciences (BES), under Award # DE-SC0019112 (development of the model and data analysis, preparation of nanopores by dielectric breakdown and transmission electron microscopy). C.Y.L., J.P., and Z.S were supported by the award. E.T.A. was supported by The Scientific and Technological Research Council of Turkey (TUBITAK), 2219-International Postdoctoral Research Fellowship Program (App. No: 1059B191600613), and the Istanbul University Cerrahpasa, Engineering Faculty Chemistry Department for additional financial support. Supporting Information The information is free of charge on the ACS Publication website: Additional experimental recordings and details of numerical modeling. Additional experimental data for dielectric breakdown nanopores (Figures S1-S4) and a TEM nanopore (Figure S5); experimental and simulated current-voltage curves and charge density for the salt gradient of 10 mM/1 mM (Figures S6 and S10); profiles of ionic concentrations (Figures S7-S9); modeling of current-voltage curves and surface charge density performed at the salt gradient of 1000 mM/10mM (Figure S11); modeling of current-voltage curves at pH 6 for various combinations of surface charge distribution (Figure S12); simulated current-voltage characteristics for a TEM nanopore (Figure S13); effect of diffusivity of proton ions on current-voltage curves (Figure S14); profiles of ionic concentrations and electroosmotic flow for the salt gradient of 1000 mM/1 mM (Figures S15-S16); calculated surface-averaged charge density for various experimental conditions (Tables S1-S2); modeling of protein translocation at pH 8 with salt and voltage modulated charge density on the pore walls and the protein (Figures S17-S19); few subsequent recordings of current-voltage curves (Figure S20); detailed description of the pH-regulation model; modeling of current-voltage curves for various dissociation properties of the surface groups (Figure S21 and Table S3); effects of 27 ACS Paragon Plus Environment

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buffer ions and charge conditions of the membrane on current-voltage curves(Figures S22-S23). References 1. Hille, B., Ion Channels of Excitable Membranes. 3rd ed.; Sinauer: Sunderland, MA, 2001. 2. Wang, J.; Fang, R. C.; Hou, J.; Zhang, H. C.; Tian, Y.; Wang, H. T.; Jiang, L., Oscillatory Reaction Induced Periodic C-Quadruplex DNA Gating of Artificial Ion Channels. ACS Nano 2017, 11, 3022-3029. 3. Siwy, Z.; Fulinski, A., Fabrication of a Synthetic Nanopore Ion Pump. Phys. Rev. Lett. 2002, 89, 198103. 4. He, Y.; Gillespie, D.; Boda, D.; Vlassiouk, I.; Eisenberg, R. S.; Siwy, Z. S., Tuning Transport Properties of Nanofluidic Devices with Local Charge Inversion. J. Am. Chem. Soc. 2009, 131, 5194-5202. 5. Qiu, Y. H.; Lucas, R. A.; Siwy, Z. S., Viscosity and Conductivity Tunable Diode-Like Behavior for Meso- and Micropores. J. Phys. Chem. Lett. 2017, 8, 3846-3852. 6. Lin, C. Y.; Yeh, L. H.; Hsu, J. P.; Tseng, S., Regulating Current Rectification and Nanoparticle Transport Through a Salt Gradient in Bipolar Nanopores. Small 2015, 11, 4594-4602. 7. Wanunu, M.; Morrison, W.; Rabin, Y.; Grosberg, A. Y.; Meller, A., Electrostatic Focusing of Unlabelled DNA into Nanoscale Pores Using a Salt Gradient. Nat. Nanotechnol. 2010, 5, 160-165. 8. Qiu, Y. H.; Lin, C. Y.; Hinkle, P.; Plett, T. S.; Yang, C.; Chacko, J. V.; Digman, M. A.; Yeh, L. H.; Hsu, J. P.; Siwy, Z. S., Highly Charged Particles Cause a Larger Current Blockage in Micropores Compared to Neutral Particles. ACS Nano 2016, 10, 8413-8422. 9. Nova, I. C.; Derrington, I. M.; Craig, J. M.; Noakes, M. T.; Tickman, B. I.; Doering, K.; Higinbotham, H.; Laszlo, A. H.; Gundlach, J. H., Investigating Asymmetric Salt Profiles for Nanopore DNA Sequencing with Biological Porin MspA. PLoS One 2017, 12, e0181599. 10. He, Y. H.; Tsutsui, M.; Scheicher, R. H.; Fan, C.; Taniguchi, M.; Kawai, T., Mechanism of How Salt-Gradient-Induced Charges Affect the Translocation of DNA Molecules through a Nanopore. Biophys. J. 2013, 105, 776-782. 11. Sha, J. J.; Shi, H. J.; Zhang, Y.; Chen, C.; Liu, L.; Chen, Y. F., Salt Gradient Improving Signal-to-Noise Ratio in Solid-State Nanopore. ACS Sens. 2017, 2, 506-512. 12. Ivica, J.; Williamson, P. T. F.; de Planque, M. R. R., Salt Gradient Modulation of MicroRNA Translocation through a Biological Nanopore. Anal. Chem. 2017, 89, 8822-8829. 13. Chou, T., Enhancement of Charged Macromolecule Capture by Nanopores in a Salt Gradient. J. Chem. Phys. 2009, 131, 034703. 14. Siria, A.; Poncharal, P.; Biance, A. L.; Fulcrand, R.; Blase, X.; Purcell, S. T.; Bocquet, L., Giant Osmotic Energy Conversion Measured in a Single Transmembrane Boron Nitride Nanotube. Nature 2013, 494, 455-458. 15. Feng, J. D.; Graf, M.; Liu, K.; Ovchinnikov, D.; Dumcenco, D.; Heiranian, M.; Nandigana, V.; Aluru, N. R.; Kis, A.; Radenovic, A., Single-Layer MoS2 Nanopores as Nanopower Generators. Nature 2016, 536, 197-200. 16. Tseng, S.; Li, Y.-M.; Lin, C.-Y.; Hsu, J.-P., Salinity Gradient Power: Influences of Temperature and Nanopore Size. Nanoscale 2016, 8, 2350-2357. 17. Gao, J.; Guo, W.; Feng, D.; Wang, H. T.; Zhao, D. Y.; Jiang, L., High-Performance Ionic Diode Membrane for Salinity Gradient Power Generation. J. Am. Chem. Soc. 2014, 136, 12265-12272. 18. Heiranian, M.; Farimani, A. B.; Aluru, N. R., Water Desalination with a Single-Layer 28 ACS Paragon Plus Environment

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Modulation of Charge Density and Charge Polarity of Nanopore Wall

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