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Experimental Investigation of Dynamic Deprotonation/ Protonation of Highly Charged Particles Yinghua Qiu, Anna Dawid, and Zuzanna S. Siwy J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b01919 • Publication Date (Web): 03 Mar 2017 Downloaded from http://pubs.acs.org on March 3, 2017

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The Journal of Physical Chemistry

Experimental Investigation of Dynamic Deprotonation/Protonation of Highly Charged Particles Yinghua Qiu,1 Anna Dawid,1,2 Zuzanna S. Siwy*1,3,4 1

Department of Physics and Astronomy, University of California, Irvine, California 92697, United States 2

College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences,

Centre of New Technologies, University of Warsaw, Stefana Banacha 2C, 02-097 Warsaw, Poland 3

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

4

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

*

Corresponding Author: [email protected], Tel. 949-824-8290

ACS Paragon Plus Environment


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Abstract:

Single pores have found application in detecting and characterizing individual objects such as cells, particles, and even individual molecules. The experimental approach, called resistive-pulse technique, is often performed at symmetric electrolyte conditions so that the properties of the passing object remain constant in the course of measurement and translocation. Here we report experiments with highly charged mesoparticles passing through pores placed in contact with a pH gradient, and demonstrate that this set-up allows probing protonation and deprotonation of the particles.

Based

on

fast

diffusion

of

protons,

and

sub-millisecond

deprotonation/protonation kinetics of carboxyl groups, we expected the particles would change their ionization state within few milliseconds. However, our results show that the kinetics of protonation and deprotonation of the highly charged particles is significantly slower, and exceeds 100 ms. We hypothesize that condensation of counterions that occurs on the particles at higher pH is responsible for the modified rates of protonation. The slowed down deprotonation is attributed to modified local pH of the solution next to a highly charged surface. In addition, we show how electroosmotic flow of neutral particles through a pore in contact with pH gradient can probe modulations of local surface charge properties of the pore by voltage polarity.


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Introduction. When solid surfaces are in contact with aqueous solutions, they can carry excess charges due to the deprotonation/protonation of surface chemical groups or adsorption of charged species.1 A charged surface introduces modulations in local ionic concentrations and the so-called electrical double-layer is formed.1,2 In a pore or channel, charged walls in combination with an applied voltage can lead to electroosmotic flow of the solution and all dissolved in it species. Thus, neutral colloids can pass through the pore by electroosmosis, with velocity that is proportional to the surface potential. Charged particles on the other hand translocate by a combination of electroosmosis and electrophoresis.2 Passage of particles through single pores is the basis of the resistive-pulse technique. Translocation of individual objects is observed as a transient change of the transmembrane current, called a pulse.3,4 Resistive-pulse technique is used to detect objects as well as characterize their physical properties including size,4,5 shape,6,7 surface charge,5, 8, 9 and even mechanical properties.10-12 During the experiment these properties are typically assumed not to change in the course of translocation, so that e.g. passage time of a charged particle can be related with the particle zeta potential and surface charge.2,

5, 13

This assumption is justified since the ionic strength and pH of

solution on both sides of the membrane are often the same. In this manuscript we describe resistive-pulse experiments of detecting neutral and charged colloids using single pore membranes placed in contact with a pH gradient. The charged colloids carried carboxyl groups whose deprotonation state was regulated by the solution pH.1 Electrokinetic transport of particles in both directions, from lower to higher pH and vice versa, was performed and compared to recordings under symmetric electrolyte conditions. We show that this experimental set-up allows probing kinetics of deprotonation/protonation of particles passing between solutions of different pH values. Experiments were performed with two types of charged colloids that differed in their surface charge density by a factor of three. The more charged colloids were reported before to undergo the effect of ion condensation,14-19 leading to lowering the particle zeta potential and significant slowing down of the translocation.20 Here, we present



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experimental data claiming that the kinetics of deprotonation/protonation of the colloids with condensed ions is significantly slower than what could be predicted based on earlier experiments,21,22 and can exceed 100 ms. Methods: Experiments and Modeling Preparation of pores: Experiments were performed with single pores prepared in polymer films by the track-etching technique.23 12 µm thick films of polyethylene terephthalate (PET), as well as 30 and 60 µm thick polycarbonate (PC) films were used in measurements. The pore preparation started with single-ion irradiation performed at the Institute for Heavy Ions Research in Darmstadt, Germany.24 Etching the irradiated films in diluted NaOH at elevated temperature led to cylindrical pores whose diameter increased with the etching time.25 The effective pore diameter was estimated through the pore resistance measured in 1 M KCl at pH 8.26 Pores used in the experiments had opening diameter of ~1 µm, and the etching process led to thinning of the membranes by a similar amount. PET pores usually have rough walls due to laminar and semicrystalline structure of the polymer material.27 PC is amorphous and PC pores feature smooth walls and cylindrical shape with one or two narrowed entrances.6 Detecting particles: The particles were purchased from Bangs Laboratories (Fisher, IN, USA). 410 and 400 nm carboxylated, as well as 400 nm unmodified polystyrene particles were used in the experiments. The density of carboxyl groups on the 400 nm carboxylated spheres was ~3 times higher as that of 410 nm ones. Based on the technical specifications from the manufacturer, the density of carboxyl groups on the particles are 10 and 3.2 per nm2 for the 400 and 410 nm charged spheres, respectively.28 The high densities of the groups suggest that multiple carboxyl groups reside on polymer chains exposed to the solution.14 Particle suspensions (~109 particles per mL) were prepared using 0.1 M KCl solution at pH 5.5, 8 and 10, containing 0.1% (v/v) Tween 80. A particle suspension was placed in one chamber of the conductivity cell, and the other chamber was filled with KCl solution without particles. Two Ag/AgCl electrodes were used to apply transmembrane potential and measure current. Ionic current measurements were conducted with Axopatch 200B


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and 1322A Digidata (Molecular Devices, Inc.) under a sampling frequency of 20 kHz. The recorded data were subjected to a low-pass Bessel filter of 1 kHz. In all experiments, the particles passed through a given pore in the same direction, so that changing pH gradient required changing solutions in both chambers of the conductivity

cell.

During

the

experiment,

neutral

particles

were

moved

by

electroosmosis towards a negatively biased electrode; carboxylated particles moved in the direction of electrophoresis towards a positively biased electrode, as shown in Figure S1. Numerical modeling: Coupled Poisson-Nernst-Planck and Navier-Stocks equations were solved with Comsol Multiphysics 4.4 package for single pores with opening diameters of 800 and 785 nm. The temperature was set as 298 K. The mesh size of 0.2 nm was used for the charged pore surface; for the charged boundary of the reservoirs the mesh of 0.5 nm was chosen. Diffusion coefficient for potassium and chloride ions was assumed equal to the bulk value of 2 × 10 9 m2/s. Figure 1 shows geometry of the −

modeled pore under a pH gradient; the pore length was 11 µm, the pore diameter 800 nm. All simulations were performed in 100 mM KCl as the bulk electrolyte assuming dielectric constant of water of 80. Surface charge was placed on the pore walls and on the membrane outside surfaces. Details of all boundary conditions are shown in Table S1. For lower pH, the surface charge density, σw, was set as -0.02 C/m2, for higher pH we set σw= -0.04 C/m2. On the pore walls, the boundary DE was divided into 11 equidistant parts with the surface charge density changing from -0.02 to -0.04 C/m2 or from -0.04 to -0.02 C/m2, representing the two directions of pH gradient. This model was used to calculate velocity profiles and ion concentrations. Numerical modeling of resistive-pulses caused by particles with different surface charge densities required using a denser mesh. In order to reach model convergence, the pore length was limited to 5.5 µm (Figure S2, Table S2).



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Figure 1. Scheme of the Comsol simulation model. The yellow part represents the membrane, and the blue part is the solution. The boundary conditions used are listed in Table S1. The dielectric constants of water and polymer were set as 80 and 4, respectively. The surface charge density of the pore walls was changed discontinuously in 11 equidistant steps.


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Results and Discussion

Figure 2. (a) Scheme of the experimental setup. Solutions with and without particles were placed in the left and right reservoirs, respectively. The pH of solutions on both sides of the membrane was 5.5, 8 or 10. Single micropores in polyethylene terephthalate (PET) and polycarbonate (PC) were used in all measurements. (b) An example ion current pulse corresponding to a passage of a single 400 nm in diameter neutral particle through a ~1530 nm in diameter PET pore under pH 8. This translocation event reflects topography of the pore.27 The purpose of our experiments was to probe protonation/deprotonation kinetics of charged particles as they pass through a pore in contact with a pH gradient. Based on earlier experiments in symmetric pH conditions,14, 20 we expected that changes of the effective surface charge densities of particles via protonation/deprotonation could be observed via changes in the shape and magnitude of resistive pulses. Pores used in the experiments also contain carboxyl groups thus the set pH gradient could affect not only the charged state of the particles but also the pore walls.1 Charged particles pass through a charged pore by a combination of electrophoresis and electroosmosis (Figure S1) whose properties are sensitive to pH and its gradient. Thus we wanted first to identify the optimal conditions to investigate protonation state of particles, with minimum effects of other phenomena on resistive pulses.



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Passage of particles through single pores was performed using an experimental set-up shown in Figure 2 with single pores prepared in PET and PC films; both types of pores contain carboxyl groups on the pore walls.26, 29 As reported recently, the range of pH which modulates surface charge density via protonation/deprotonation is dependent on the density of carboxyl groups.14, 20 When the groups are present at densities below 1 group per nm2, the maximum surface charge density is achieved at ~pH 7; increasing pH further does not deprotonate more groups.14 When the density of carboxyl groups is higher, maximum deprotonation state is reached only at pH 11.14 Density of carboxyl groups in PET and PC pores can be as high as ~1 group per nm2, and 0.5 group per nm2 (estimated from chemical structure of the polymer), respectively, thus according to our previous modeling,14 surface charge on PET pores might be sufficiently high to induce pH modulation over the wider pH range. Dynamic modulation of surface charge properties of a PET pore placed in contact with pH gradient was probed using neutral particles. Due to the negatively charged walls, application of electric field leads to electroosmotic flow of the whole solution, so that the local pH of the pore is affected by the voltage and its polarity. We investigated how electroosmosis in symmetric and asymmetric pH conditions influenced passage time and resistive-pulse amplitude of the particles. We first chose pH 5.5 and 8, which were expected to lead to different ionization states of the polymer walls, with pH 8 assuring a higher deprotonation degree. Experiments were performed with 400 nm in diameter neutral polystyrene particles in symmetric pH conditions as well as under pH gradients, so that the particles traveled from a solution of pH 5.5 to pH 8 and in the reversed direction (Figure 3). We found that the pulse amplitude is insensitive to pH on either side of the membrane and the recorded ∆I/I differed by less than 6% between all pH conditions (Figure S3). As expected, however, the translocation time showed a clear pH dependence. Since the electroosmotic velocity increases with the increase of the surface zeta potential,2 the passage time of the neutral particles through a PET pore at symmetric pH 8 conditions was indeed shorter than when the pore was in contact with pH 5.5 solutions. Less intuitive results were obtained when a pH gradient was established across the same pore. When the particles’ passage started at pH 5.5, their translocation time was nearly the same as in the case with symmetric pH 5.5 conditions


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(Figure 3). The same effect was observed in experiments in which the particles passed from pH 8 to pH 5.5 – again the passage time was comparable to the values obtained when the pore was in contact with pH 8 solutions on both sides. Note that the particles were always placed on the same side of the membrane, so that probing particles passage from pH 5.5 to pH 8 and in the opposite direction required changing solutions in both chambers of the conductivity cell. This protocol assured that the translocation times recorded were not affected by a possible asymmetry stemming from the pores’ rough geometry.30

Figure 3. Passage time of 400 nm neutral particles under four different pH conditions across a single PET pore with a diameter of 1530 nm. All particle suspensions were prepared in 0.1 M KCl. Our results can be understood qualitatively by analysis of Figure 2, which presents a scheme of a pore in contact with a pH gradient and applied bias. The key point to understand the results is the realization that electroosmosis moves the whole solution through the pore. Consequently, if a transport of particles occurs by electroosmosis, the pore becomes filled with the feed solution.2, 31 The pore entrance in contact with pH 5.5 is charged weaker, i.e. it features lower zeta potential compared to the case when the pore entrance is in contact with pH 8. When the particles start the translocation from the side kept at pH 5.5, the pore becomes filled with the slightly acidic solution resulting in



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lowering of zeta potential of the pore walls, and of electroosmotic velocity. In the case when the translocation starts on the side of the membrane with pH 8, electroosmotic flow will fill the pore with a solution of higher pH, and consequently higher electroosmotic velocities were observed.

Figure 4. (a) Electroosmotic flow velocity and (b) pressure distribution along the axis of a negatively charged pore with its surface charge density changing in a step-wise manner from -0.02 (pH 5.5) to -0.04 C/m2 (pH 8). The arrow on the top of each figure shows the flow direction. The voltage was set as 1 V and solution concentration of 0.1 M KCl was used. The pore modeled was 800 nm in diameter and 11 µm in length.

Numerical modeling based on solving coupled Poisson-Nernst-Planck and Navier Stokes equations was used to examine properties of solution velocity in an 11 µm long pore in contact with pH gradient.32,33 Dissimilar pH on both sides of the pore is expected to cause a surface charge density gradient along the pore axis. Thus, in the modeling, one entrance of the pore had surface charge density of -0.02 C/m2 (representing pH 5.5) the other one -0.04 C/m2 (pH 8) (Figure 1); the charge changed discontinuously with 11 equidistant steps by 0.002 C/m2. Figure 4a,b shows distribution of velocity and pressure profiles along the pore axis. Similar to results previously published by Herr et al.34 for a channel with a junction between two zones with different values of zeta potential,


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velocity distribution in our structure is also dependent on both axial and radial positions (Figure 5). The radial profile of the solution velocity changes with the change of surface charge density of the pore walls. In the less (more) charged zones, the velocity reaches maximum (minimum) at the pore axis. Position dependent velocity and the necessity to fulfill the continuity equation lead to formation of local pressure gradients shown in Figure 4b. Presence of the solution flow naturally fills the pore with the feed solution.

Figure 5. Comsol simulations of electroosmotic fluid flow in a pore with discontinuous surface charge density from -0.02 to -0.04 C/m2 (left) and from -0.04 to -0.02 C/m2 (right). The surface charge density changed along the axis in 11 equidistant steps. 0.1 M KCl and 1 V of transmembrane potential was used in the simulations. Boundary conditions are listed in Table S1.



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In order to visualize a possible shape of the distribution of protons along the pore axis for the two directions of electroosmotic flow, we modeled an artificial case of a pore in contact with a concentration gradient of positive ions. For demonstration purposes and feasibility of calculations, we considered a threefold concentration gradient of 0.1 and 0.3 M. Figure 6 shows that indeed, for 1 V potential difference, electroosmosis fills nearly the whole pore volume with the feed solution i.e. the concentration of positive ions along the pore axis is equal to the concentration in the solution which is pulled into the pore. We also tested electroosmosis through PET pores using pH 8 and pH 10 conditions.20 These experiments allowed us to probe whether the effective surface charge density on the pore walls is changing beyond pH 7. We found the passage time of neutral particles at pH 10 was ~20% shorter compared to the passage time at pH 8 suggesting the density of carboxyl groups on PET was sufficiently high to induce small modulation of surface charge densities at basic pH conditions14 (Figure S4). Similar to the results with the gradient of pH 5.5 and pH 8, the amplitude of resistive pulses was nearly independent of the pH conditions of solutions on either side of the membrane (not shown).


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Figure 6. Distribution of concentration of positive ions along the axis of a pore placed in contact with 0.1 M and 0.3 M 1:1 electrolyte at ±1 V. In (a-b) the walls surface charge density varied discontinuously (in 11 equidistant steps) from -0.02 C/m2 at one pore entrance to -0.04 C/m2 on the other entrance; in (c) the pore walls had a constant charge density of -0.04 C/m2. The yellow part shows the pore region. Boundary conditions are listed in Table S1.The arrows show the direction of the electroosmotic flow in the pore.



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Probing the effect of electroosmosis in PC pores was more challenging. Lower surface charge density35 and increased hydrophobicity36 of the polymer compared to PET prevents neutral polystyrene particles to pass through. However, since no passages of neutral polystyrene particles were observed either at pH 8 or pH 10 we concluded that electroosmotic flow in these pores was minimal. As reported by us before,14 we did observe extremely infrequent passages of another type of neutral particles composed of poly methyl methacrylate (PMMA). Passage time of these particles through PC pores exceeded 1 s at 0.8 V, which supports our conclusion on very low zeta potential of PC pores. Electroosmotic transport of neutral particles through PET pores revealed that surface charge densities of the pores are modulated even at pH values as high as pH 10. We realized that in order to probe the change of protonation state of particles we needed to work with PC pores and at pH gradient of pH 8 and pH 10,

14, 20

which will keep the

surface charge properties of the pore pH independent. In this case, we expected the shape and duration of resistive-pulses would be maximally sensitive to the change of protonation state of particles. We used two types of carboxylated particles, which differed in the carboxyl groups density by a factor of 3. The highly charged particles were 400 nm in diameter and were previously shown to change their protonation state for pH values up to pH 10;14, 20 the increasing surface charge density of the particles was observed as pH sensitive duration and amplitude of resistive-pulses, summarized below. The less charged particles, 410 nm in diameter, were only weakly responsive to pH changes above pH 8. Figure 7 shows passage examples of charged 400 and 410 nm particles through a single PC pore at symmetric electrolyte conditions of pH 8 and pH 10 as well as pH gradient of pH 8/pH 10. As previously reported, PC micropores are cylindrical along the majority of their length with exception of narrow entrances.6, 37 The 410 nm particles with lower density of carboxyl groups produce resistive-pulses, which trace the pore shape at both pH 8 and pH 10 conditions, and cause a relative current change that is


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consistent with their volume.4 The pulses contain larger current drops in the beginning and end (marked with symbols # and @ in Figure 7), reflecting the narrow entrances, and a flat medium section corresponding to the cylindrical interior. However, the shape and magnitude of pulses created by the highly charged 400 nm in diameter particles are different.

14

Let’s look first at the data at symmetric pH conditions. The particles when

entering the pore cause a current drop (marked as #, Figure 7) that is larger than the current drop observed with the less charged particles.

14

On the other hand, the exit of

the highly charged particles is accompanied by a current increase above the baseline level (marked as * in Figure 7). The effects were discussed in previous papers as resulting from concentration polarization and changes of local ionic concentrations at both ends of the pore.14, 38, 39 Since the negatively charged particles move in the same direction as chloride ions, they deplete chloride ions at the pore entrance so that a larger current blockage is observed in the beginning of the translocation. As the particle exits the pore, its counterions increase local concentration of potassium ions; since potassium ions are sourced from the pore exit, a current increase is observed. We therefore postulated that the enhanced current decrease when the particle enters the pore is informative on the particle charge i.e. protonation state in the feed solution. Consequently, the current increase at the end of the translocation is expected to carry information on the protonation state in the solution on the opposite side of the membrane.



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Figure 7. (a) Example passages of charged 410 nm (blue) and 400 nm (red) spheres through a 30 µm long PC pore with diameter of ~1150 nm in symmetric pH conditions of pH 8 and pH 10 as well as pH gradients of pH10/pH8, pH8/pH 10 at 1V. The background electrolyte was 0.1 M KCl. The density of carboxyl groups on the 400 nm particles was three times higher than the density of carboxyl groups on the 410 nm spheres. The # and * denote the current decrease and increase when the highly charged particles entered and exited the pore, respectively. The # and @ denote the first and second peak in pulses of the less charged particles. (b) Scheme showing analysis of ion current pulses of the highly charged particles.


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In order to support the hypothesis, we performed Comsol modeling of ion current pulses of particles characterized with a steadily increased surface charge density (Figure S2). A nearly linear dependence was found between the particle surface charge and the amplitude of current enhancement accompanying the particle exit (Figure 8), suggesting the current enhancement carries information on the degree of particle deprotonation.20

Figure 8. (a) Simulation results of resistive-pulses caused by particles with different surface charge densities in 0.1 M KCl at 0.5 V. (b) Relative current increase caused by the particles as they exit the pore as a function of particle surface charge density. The particle was placed at the position where the current increase appears in (a). The surface charge density of the pore was set as −0.04 C/m2. Boundary conditions used in the simulations are listed in Table S2.



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Figure 9. (a) Values of ion current decrease (marked as # in Figure 7) and (b) current increase (marked as * in Figure 7) in the resistive pulse events of highly charged 400 nm spheres obtained with a ~1150 nm in diameter PC pore. All particle suspensions were prepared in 0.1 M KCl pH 8 or 10. The legend indicates pH gradients and direction of the particle transport. (c) and (d) show values from (a) and (b) normalized by the baseline current shown in Figure S5. Quantitative analysis of the resistive pulses included the magnitude for current decrease and increase as well as the passage time in all pH conditions considered (Figure 7b). Figure 9a, b shows the absolute magnitude of the enhanced current decrease at the pore entrance and current increase for the highly charged 400 nm in diameter particles. For symmetric pH conditions, both current decrease and increase are higher at pH 10 than at pH 8. Note, that due to microscale of pores used in the experiments, the ionic current governed by surface charges contributed to the overall


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current nearly insignificantly;2, 40 consequently, the baseline current was not affected by the solution pH (Figure S5). Analysis of the peaks’ magnitudes allowed us to conclude that when the particles enter the pore from the same pH, they cause nearly the same current drop at the pore entrance, independent of the pH of the solution on the opposite side of the membrane (Figure 9 a, c). Thus, as expected, the first peak corresponding to the current decrease (‘#’ in Figure 7) depends only on the properties of the solution from which the particles are sourced. We further hypothesized that when a highly charged particle passes through a pore from pH 8 to pH 10, the current increase at the exit (‘*’ in Figure 7) would be comparable to that observed in symmetric pH 10 conditions, since at the pore exit the particle would be in contact with pH 10 solution. A similar effect was expected for particles passing in the opposite direction: particles passing from pH 10 to pH 8 should cause a current increase similar to that in symmetric pH 8 conditions. However, as shown in Figure 9b,d when a highly charged particle exits the pore, the current increase it causes does depend on the pH of the solution from which it was sourced. Moreover, the difference in the peak magnitude between the data recorded in symmetric pH 10 conditions, and when the particles moved from pH 8 to pH 10 increases with voltage (Figure 9b). The same conclusions can be drawn when comparing symmetric pH 8 data with recordings pH 10 to pH 8. The data suggest that the translocation time is insufficient to complete protonation/deprotonation of the passing particles. This finding was surprising, because the translocation times at lower voltages were as high as 100 ms (Figure S6). Moreover, based on mobility of protons of 36.2310−8 m2V−1s−1,41 and electric field in the pore of ~105 V/m, protons would need only ~0.3 ms to pass through the whole pore length. Thus, the particles could indeed be expected to complete their protonation/deprotonation during the process of translocation, especially in the light of earlier reports on sub-milliseconds deprotonation/protonation kinetics,21, 22 We used the data of ion current increase shown in Figure 9 to estimate the degree of particles deprotonation assuming that the particles are maximally deprotonated in symmetric pH 10 conditions. This assumption is in agreement with the maximum



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magnitude of current increase observed in the symmetric pH 10 case, ∆ . Lower magnitude of current increase is indicative of lower effective surface charge density thus incomplete deprotonation. Consequently, in order to obtain the degree of deprotonation in the remaining pH conditions (symmetric pH 8 and two directions of pH 8/pH 10

gradient) we divided the observed current increase by ∆ .

Figure 10. Deprotonation degrees of the carboxylated surfaces of charged 400 nm particles under different pH gradients. We set the deprotonation ratio as 1 in the symmetric pH 10 case, for which the maximum current enhancement upon particle exit

was recorded, marked as ∆ in the main text. The other numbers represent the

ratio of current increase obtained in the other three pH arrangements and ∆ .

This analysis (Figure 10) confirmed the particles starting at pH 10 did not undergo complete protonation when being transported to pH 8; similarly, particles which started at pH 8, did not undergo complete deprotonation at the end of the transit towards the reservoir kept at pH 10. In order to support the conclusion on the insufficient time for the particles to undergo complete protonation/deprotonation when in the pore, we performed experiments with a pore that was twice as long as the one used to collect data shown in Figures 7, 9 and 10. We expected the longer pore would allow observation of higher degrees of


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protonation/deprotonation compared to the results in the shorter pore. Figure S7 summarizes our results and confirms that in longer pores, most probably due to longer translocation times, larger changes of protonation can indeed be obtained.

Figure 11. Scheme of (a) protonation (b) and deprotonation processes on a charged surface with a high density of carboxyl groups.

The question however remains what is the physical basis for the extended protonation/deprotonation times of the highly charged particles. Figure 11 shows a scheme of a modified chemical and electrical environment a highly charged surface induces. We hypothesize that for particles traveling from pH 10 to pH 8, the process of protonation can be hindered by ion condensation17-19 that had prior occurred at pH 10 at the solid-liquid interface. In our previous work, the existence of ion condensation was supported by an anomalously lowered zeta potential of the particles at pH 10 leading to slowed down electrokinetic translocation, and saturation of electrokinetic mobility for pH values above 9.20



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Using models of highly charged colloids developed before16 we were able to predict the percentage of counterions, , which underwent ion condensation. The formula uses electrokinetic velocity, µ, measured from the translocation time, as the main experimental observable:

θ = 1−

η a (1 + aκ ) Ze

µ

where η, a, Z, κ, and e stand for water viscosity, particle diameter, the inverse of the Debye length, and the elementary charge, respectively. The formula predicts that more than 90% of counterions are present in the condensed form. We hypothesize the condensed ions hinder access of protons to the particle’s surface and the protonation process. We also analyzed a possible mechanism behind slowed down deprotonation observed when the particles passed from pH 8 to pH 10. We believe this effect could stem from locally decreased concentration of hydroxide ions, caused by the presence of the negative charges on the pore walls (Figure 11b). Decreased concentration of OHcould be responsible for hindered further deprotonation.

Finally, we analyzed the magnitude of the two peaks of current decrease observed in the passage of the less charged 410 nm in diameter particles (Figure 7). Since these particles did not modulate the magnitude of the resistive-pulses (Figure S8), we concluded the same approach, as applied to the highly charged particles, could not be used to probe kinetics of their protonation and deprotonation. In the future studies we will address this issue by considering position dependent translocation velocity, longer pores, and different pH gradients.

Conclusions. In this manuscript we report resistive-pulse experiments of neutral and charged particles with micropores placed in contact with a pH gradient. Our results indicate that the presence of dissimilar pH conditions on both sides of the membrane causes


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modified electroosmotic flow that becomes dependent on the direction of the imposed pH gradient. Our experimental set-up also allowed us to probe protonation and deprotonation kinetics of highly charged particles. The particles cause enhanced amplitudes of current decrease and increase in the beginning and end of their translocation, which can be correlated with their protonation state. Our analysis revealed that highly charged particles did not undergo complete protonation or deprotonation when in the pore even though the passage times reached 100 ms at lower voltages. In order to explain this effect, a hypothesis was presented pointing to the importance of the condensed ions and modulated pH close to the surface for the protonation/deprotonation process. Our future efforts will be focused on identifying an experimental system, which could be applicable to studying protonation of particles with different densities of carboxyl groups. Supporting Information. Details of experimental set-up and numerical modeling as well as additional recordings of particle passage through single pores.

Acknowledgments. This research was supported by the National Science Foundation (CHE 1306058). We acknowledge GSI Helmholtzzentrum für Schwerionenforschung in Darmstadt, Germany for providing irradiated membranes. References. (1)

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