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Precipitation-Induced Voltage-Dependent Ion Current Fluctuations in Conical Nanopores Laura Innes,†,‡ Matthew R. Powell,†,‡ Ivan Vlassiouk,§ Craig Martens,| and Zuzanna S. Siwy*,† Department of Physics and Astronomy, UniVersity of California, IrVine, California 92697, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830, and Department of Chemistry, UniVersity of California, IrVine, California 92697 ReceiVed: NoVember 13, 2009; ReVised Manuscript ReceiVed: March 27, 2010
Single conically shaped nanopores produce stable ion current fluctuations when in contact with weakly soluble salts, such as calcium hydrogen phosphate (CaHPO4) and cobalt hydrogen phosphate (CoHPO4). The pore spontaneously switches between high and low conductance states, called open and closed states, respectively. Pore opening and closing are linked to the dynamic formation of the calcium and cobalt precipitates at the small opening of the pore. The probabilities of pore opening and closing are voltage-dependent, and this characteristic of ion current signal is known for biological voltage-gated channels. We show that new types of ion current fluctuations are obtained in conditions at which precipitates of CaHPO4 and CoHPO4 can form in the pore at the same time. 1. Introduction There has been a great deal of interest in the preparation of pores which mimic the behavior of biological channels in a cell membrane.1-3 Biological channels are responsive to external stimuli such as change in the transmembrane potential, the binding of a chemical or variations of mechanical pressure.4 Mimicking the voltage-responsive property in synthetic nanopores is especially attractive since voltage response provides the quickest feedback and such voltage-controlled pores can be the basis for ionic circuits in biosensing, lab-on-a-chip, and artificial cells systems. Voltage sensing is manifested as rectifying and diode-like current-voltage curves as well as voltage-dependent switching of a pore between its high and low conductance states.4,5 The latter property is usually called voltage gating in biology nomenclature.4 It is important to mention that when creating biomimetic voltage-responsive structures, researchers often focus on mimicking the transport properties of the biochannels, not the exact mechanisms that occur in these biosystems. Ion current-rectification for example can be obtained by several very different physicochemical mechanisms.5 In this article we analyze the effect of voltage gating in single conically shaped nanopores prepared in a polymer film. The pores are in contact with a solution of weakly soluble salts such as calcium hydrogen phosphate (CaHPO4) and cobalt hydrogen phosphate (CoHPO4) in 0.1 M KCl. We have shown recently that negative surface charges on the walls of conical nanopores together with the externally applied voltage lead to an increase of ionic concentrations in the pore so that precipitation can occur.6,7 The precipitation is localized in the pore and possibly at its entrance, but not in the bulk where the ionic concentrations do not exceed the solubility product. In the current-voltage curves, the nanoprecipitation is observed as negative incremental resistance; thus there is a voltage range for which at higher voltages lower average values of currents are recorded.6,7 The precipitates are very unstable, and their dynamic formation and * Corresponding author: e-mail,
[email protected]; phone (949) 824-8290. † Department of Physics and Astronomy, University of California. ‡ These authors contributed equally to this work. § Oak Ridge National Laboratory. | Department of Chemistry, University of California.
dissolution can be seen as a subsequent blocking and opening of the pores. In this article we present an analysis of ion current fluctuations induced by CaHPO4, CoHPO4, and mixtures of the salts. The data are analyzed using the approach which is typically applied for biological voltage-gating channels. We examine the probability of single pores to be in the open and closed states, which correspond to the high and low conductance states, respectively. We also analyze the so-called dwell times, which describe how long a pore stays in subsequent open or closed states. The analysis revealed a very strong dependence of the switching kinetics with the applied voltage. The shapes of the current instabilities are studied as well: the pore openings and closings observed with CaHPO4 and CoHPO4 precipitates are characterized by a rapid pore opening (burst of high current) followed by a gradual blocking of the pore (low current), which we attributed to the time needed to form a nanoprecipitate. We also show that exposing a nanopore to a mixture of cobalt and calcium salts leads to new patterns of ion current fluctuations, which are not observed when the salts are studied individually. Most notably, we identify conditions at which the ion current through single pores produces rectangular-shaped openings and closings, a behavior that resembles gating in biological channels. 2. Experimental Section 2.1. Preparation of Single Pores. Single conically shaped nanopores were prepared by the track-etching technique in 12 µm thick polyethylene terephthalate (PET) films, as described before.8 The technique entails irradiation of the films by single swift heavy ions, e.g., gold or uranium ions, which leads to the formation of a local damage in the foil, the so-called latent track. The irradiation was performed at the linear accelerator of the Institute for Heavy Ions Research (GSI), Darmstadt, Germany. Chemical etching of these single-ion irradiated foils resulted in the formation of single pores.9 In order to obtain conically shaped nanopores, the irradiated polymer foils were etched asymmetrically from one side in 9 M NaOH. The other side of the membrane was protected against the etching by an acidic stopping solution.10 The etching process was performed in a conductivity cell characterized by hundreds of GΩ seal resis-
10.1021/jp910815p 2010 American Chemical Society Published on Web 04/15/2010
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Figure 1. Fragment of fluctuating ion current recording with distinct states of high and low conductance. Base, event, and rejection lines were marked, which allowed us to divide the signal into open and closed states characterized with high and low current values, respectively. The pore openings and closings are caused by the dynamic formation of nanoprecipitates in the pore and the close vicinity to the pore opening. The state of pore opening characterized by the maximum current corresponds to residual precipitates still present in the pore. (See the scheme on the right.)
tance as measured by Keithley picoammeter/voltage source 6487 and an amplifier Axopatch 200B (Molecular Devices, Inc.). The pore fabrication was controlled by monitoring the etching current when 1 V was applied across the membrane using platinum electrodes. The current through the pore was measured with an amplifier Axopatch 200B (Molecular Devices Inc.) connected to the computer via the Digidata 1322A. In the beginning of the etching process the current was zero. We stopped the etching when the breakthrough current reached =100 pA. Replacing the 9 M NaOH solution with the acidic stopping medium halted the etching. 2.2. Ion Current Recordings. Ion current was recorded in the same cell in which the pores were etched. The compartments of the cell were thoroughly washed and filled with a given electrolyte. Two Ag/AgCl electrodes were used for applying the transmembrane potential and measuring the ion current. Since Ag/AgCl electrodes are to a large degree nonpolarizable and very stable, we could build a two-electrode setup in which one electrode placed at the tip (the narrow end) of a conical nanopore was grounded. The other electrode, placed at the base (the big opening) of a pore, was used to apply a given potential difference with respect to the ground electrode. Transport properties of single nanopores were studied in 1 M KCl, as well as in 0.1 M KCl with and without added calcium chloride and cobalt chloride in the presence of 2 mM phosphate buffer (PBS), pH 8. The recordings in 1 M KCl were used for estimating the pore size using the electrochemical method reported before.10 Recordings in 0.1 M KCl, pH 8, were treated as a control measurement. The time series of ion current were recorded with 10 kHz sampling frequency and filtered with the built-in Bessel filter of 2 kHz. The voltage was changed with 100 mV steps from +1000 to -1000 mV. Each recording was 2 min long. Current-voltage curves were found by calculating arithmetic average values of the current signals for a given voltage together with the standard deviation. The high seal resistance of the cell reduces the offset current to sub-picoamperes so that the measured current signals were not affected by the offset. 2.3. Analysis of Time Series. The recordings of ion current in time were analyzed using the commercial software Clampfit 9.2 (Molecular Devices, Inc.). The built-in tools allowed us to
find the events of pore opening and closing as well as the duration of the subsequent open and closed states, or the socalled dwell times. An opening event corresponded to a state with a high current level, comparable to the ion current value recorded in 0.1 M KCl (Figures 1 and 2). A closed state of the pore corresponded to low currents, typically ∼-50 pA. The events were found by defining the position of the baseline, the event line, and the rejection line (see Figure 1). The baseline was set at the closed state of the pore, while the detection line was positioned a little below the baseline in order to detect all events of pore opening (seen as downward peaks in Figure 1). Once these were determined, the lower limit of the amplitude of the peak could be set, which enabled us to remove very short or/and insufficiently deep peaks, and not count them as events. An event was then determined by any peak that reached beyond the half way point of the average maximum current. The peaks located above the rejection line were then deleted (Figure 1). The Clampfit software identified the beginning and the end of each open and closed state. These data were then transferred into Excel, which allowed for a convenient calculation of the dwell times. Duration of subsequent open and closed states, number of open and closed states, and the probability of the pores to be in the open and closed state were calculated for each data series. 3. Results and Discussions Transport properties of single conical nanopores were studied in 0.1 M KCl, pH 8 (2 mM phosphate buffer), and compared with the recordings in the presence of CaCl2 and CoCl2. We would change the ratio of the calcium and cobalt salts in the solution keeping their total concentration constant of 0.2 mM. The experiments were done in a basic pH to ensure the presence of negative charges on the pore walls due to the deprotonated carboxyl groups that are created in the process of heavy ion irradiation and chemical etching. The density of these carboxyl groups was estimated to be 1 per nm2.11 3.1. Current-Voltage Curves. Current-voltage curves of a single conical nanopore with the small opening ∼2 nm before and after adding of the divalent salts are shown in Figure 2. In
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Figure 3. (a) Current-voltage curve of a single conical nanopore with the narrow opening of 8 nm in diameter recorded in 0.1 M KCl, 0.2 mM CoCl2, pH 8 (2 mM PBS). (b) Probabilities of pore opening (red) and closing (black) in 0.1 M KCl, 0.2 mM CoCl2, pH 8.
Figure 2. Current-voltage curves recorded through a single conical nanopore with the narrow opening of 2 nm in diameter. The current values are arithmetic averages of 2 min long recordings: (a) control recordings in 0.1 M KCl, pH 8 (2 mM PBS); (b) current-voltage curve obtained in 0.1 M KCl and mixture of 0.05 mM CoCl2 and 0.15 mM CaCl2; (c) and (d) current-voltage curves recorded in 0.2 mM CaCl2 and 0.2 mM CoCl2, respectively; (e) peak current determined as the average maximum current in the open state of the pore in 0.05 mM CoCl2 and 0.15 mM CaCl2.
0.1 M KCl, pH 8, the conical nanopore rectifies the current due to breaking symmetry of the electric potential of interactions between cations and the negative charges on the pore walls (Figure 2a).12-14 The observed current is carried primarily by cations. This selectivity toward positively charged ions was shown previously by measuring the so-called reversal potential established across a membrane in contact with a KCl concentration gradient.12,15,16 The cationic selectivity of the pores was further confirmed by modeling ion current using the continuum approach of the Poisson-Nernst-Planck equations,15 Monte Carlo,17 and molecular dynamics.18 The preferential direction of the cation movement (currents for negative voltages) is from the narrow opening to the wide opening of the cone. Upon addition of divalent cations in the presence of the phosphate buffer, the current-voltage curve flipped, so that the average currents for positive voltages are higher than the average currents for the corresponding negative voltages.7 The change in current-voltage curves was explained by the formation of CaHPO4 and CoHPO4 precipitates. Figure 2 shows currentvoltage curves obtained when the pore was in contact with 0.05 mM CoCl2 and 0.15 mM CaCl2 (Figure 2b) in comparison with the recordings in 0.2 mM CaCl2 (Figure 2c) and 0.2 mM CoCl2 (Figure 2d). With divalent cations present in the solution, ion currents for negative voltages were very unstable showing transient closings and openings of a pore (see Figure 1), which resulted in a big standard deviation shown in the current-voltage curves (Figure 2b-d). These current instabilities were attributed to the formation and dissolution of CaHPO4 and CoHPO4 precipitates in the nanopore and possibly at its narrow entrance.7 Control experiments were performed to exclude the possibility of the precipitates formation in the bulk solution. We addition-
ally showed that the current instabilities are formed only at basic solutions and when the solution of divalent ions is present at the narrow opening of the pore (Figures S1-S3, Supporting Information). In the situation when only the big opening of the pore is in contact with CaCl2 or CoCl2, the ion current signals remained stable (Figure S1). Numerical solutions of the Poisson-Nernst-Planck equations also confirmed that inside the pore and in the close vicinity to the pore narrow opening, the ionic concentrations of any cation can increase to values that are even 2 orders of magnitude higher than these in the bulk solution, which promotes the precipitation.7,14 The modeling for a different precipitation system was reported by us before;7 similar calculations for CaHPO4 are shown in the Supporting Information (Figure S4). For negative applied voltages, the ionic concentrations in the pore and its narrow pore opening dramatically increase, which suggests the possibility of precipitate formation. In contrast, at positive voltages, concentrations of all ions are lower than those in the bulk solution so that precipitates cannot be formed.7,14 The asymmetric influence of the applied voltage on ionic concentrations in the pore explains why the current instabilities occur only for one voltage polarity. We also studied voltage dependence of the average peak current, thus maximum value of the current in the open state of a pore averaged over all pore events (Figure 2e). In the majority of the current recordings, the peak current was lower than the current in 0.1 M KCl measured before adding the divalent salts. The discrepancy between the peak and the control currents suggests that a residual precipitate is present in the pore or/and the vicinity of the narrow opening all the time, and the current fluctuations that we observe might be due to dissolution or disintegration of only a part of the precipitate. Possible mechanisms of the pore opening will be described below. Figure 2b shows the data recorded in the presence of 0.05 mM Co2+ and 0.15 mM Ca2+, but similar behavior of current-voltage curves has been observed for all studied ratios of cobalt and calcium salts concentrations. For pores with diameters larger than 5 nm, in the presence of 0.2 mM CoCl2 the CoHPO4 precipitation was observed only for higher negative voltages, as shown in Figure 3 for an 8 nm pore. The precipitates were not sufficiently large to totally obstruct the pore opening, and the currents would be blocked only partially. 3.2. Ion Current Series in Time. Current-voltage curves represent average transport properties and cannot reveal the subtleties in differences in ion current behavior observed in systems with various concentration ratios of cobalt and calcium salts (Figure 2b-d). We therefore studied the behavior of ion current in time. These recordings were analyzed by dividing the data series for each voltage into open and closed states, which allowed us to find their subsequent durations.19 Then, we calculated the probability of the pore to be in the open and
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Figure 4. Transport properties of a single conical nanopore with a diameter of 2 nm studied in 0.1 M KCl, 0.2 mM CoCl2, pH 8 (2 mM PBS): (a) probability of pore opening (red) and closing (black) as a function of applied voltage; (b) number of pore openings in 2 min long recordings as a function of voltage; (c-e) examples of ion current signals in time recorded at -200 mV (c), -600 mV (d), and -1000 mV (e), as indicated in the figure.
closed state, equal to the percentage of the time for which the current was above or below the threshold value, respectively (Figure 1). We also looked at the number of openings and closings in a 2-min time period and average values of dwell times in the open and closed states. 3.3. Probability of Opening and Closing. Figure 4 shows the probability of a single 2 nm conical pore to be in its closed and open states as a function of voltage for 0.1 M KCl, 0.2 mM CoCl2, and 2 mM PBS, pH 8. For positive voltages and low values of negative voltages the pore is open and does not show any instability. There is a voltage threshold for which we see a sudden emergence of current fluctuations. The onset of current instabilities is also defined by the beginning of the negative incremental resistance. A very dramatic change of the pore behavior is then observed: once a pore starts fluctuating (at -200 mV for the recordings shown in Figure 4c) it does not come back to the quiet open state unless the voltage is switched back to positive voltages or lower negative voltages. We found that the probabilities of pore opening and closing are a function of the transmembrane potential, which for voltages below ∼-400 mV reaches a plateau. For all studied pores with diameter
