Alternating Current Potentiometric Scanning Ion Conductance

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Alternating Current Potentiometric Scanning Ion Conductance Microscopy (AC-PSICM) Lushan Zhou, Yi Zhou, Wenqing Shi, and Lane A Baker J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.5b03120 • Publication Date (Web): 27 May 2015 Downloaded from http://pubs.acs.org on June 2, 2015

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The Journal of Physical Chemistry

[Submitted to The Journal of Physical Chemistry C as an article]

Alternating

Current

Potentiometric

Scanning

Ion

Conductance

Microscopy (AC-PSICM) Lushan Zhou, Yi Zhou, Wenqing Shi, and Lane A. Baker*

Department of Chemistry Indiana University 800 E. Kirkwood Avenue Bloomington, Indiana 47405

*Author to whom correspondence should be addressed.

E-mail: [email protected]; Phone: (812) 856-1873; Fax: (812) 856-8300 Submitted: 04-01-2015

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ABSTRACT

Studies of ion transport at small length scales inform the fundamental understanding of various biophysical processes. Here, we describe a new method, alternating current potentiometric scanning ion conductance microscopy (AC-PSICM), which measures ion transport through nanopores as a function of AC perturbations over a range of frequencies (5 Hz – 50 kHz). Phase and amplitude of local potential in the vicinity of nanopores in polymer membranes were captured with a nanopipet. Phase was found to be sensitive to local conductive pathways (nanopores in this case) and can be used to quantify single nanopore resistance. Investigation of phase approach curves and lateral phase distributions with single nanopore samples predicted four distinct frequency ranges for resolving heterogenous conductive pathways within a sample, which were confirmed with line profile measurements of the phase response in samples with different sized nanopores. AC-PSICM is suitable for ion transport studies at the nanometer scale, and can be used to access wide ranges of time scales. Phase mapping shows promise for visualization of heterogeneous transport pathways and could be used in future studies to examine conductance at cell and tissue interfaces.

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INTRODUCTION

Scanning ion conductance microscopy (SICM) has proven well suited for studies of nanometer scale ion transport for both synthetic membranes1-4 and biological samples.5,6 When combined with noncontact imaging of surface topography, SICM presents a powerful tool for the study of a wide range of ion transport related physiological processes at subcellular resolution.7-9 SICM uses an electrolyte filled nanopipet to scan over a sample surface in electrolyte solution,10 where probe-sample distance is controlled by monitoring ion current generated between an electrode inside the pipet and an electrode in the bath solution.11 Additional driving forces, such as concentration or potential gradients, can be used to drive ion flux for studies of local ion transport via SICM.3 To increase sensitivity, a potential measurement technique, potentiometric scanning ion conductance microscopy (P-SICM), was developed.5 In PSICM a dual-barrel nanopipet is used with one barrel providing conventional ion current feedback control and the second barrel measuring variations in potential. In this work, we extended the previously described P-SICM technique to AC fields by perturbing the sample of interest with a sinusoidal transmembrane potential for a range of frequencies (5 – 50,000 Hz) and recording localized electrochemical impedance measurements. AC impedance (also known as electrochemical impedance spectroscopy (EIS)) is a powerful nondestructive technique widely used to understand electrochemical processes,12,13 electrophysiological properties,14-16 and to design biosensors.17 The EIS technique studies the impedance of a system of interest as a function of frequency18 and has been used as a routine bulk characterization method for

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various systems. The incorporation of AC impedance measurements to scanning electrochemical microscopy (SECM)19 has successfully demonstrated the utility of impedance measurements in studying local properties of interfaces such as visualization of interfacial corrosion,20-22 surface reactivity,23,24 impedance distribution at surfaces25 and surface topography.26-30 The use of an AC signal in SICM has also been reported recently as new SICM imaging modes, where an AC perturbation is applied to the pipet electrode to generate a modulated ion current and the resultant AC ion current is used to control probe-sample distance during scan.31,32 These approaches that use an AC response as feedback control are promising in increasing the imaging speed and in avoiding disruption of solution due to the physical movement of probe as in the commonly used SICM distance modulation imaging mode. Here, we take a different approach, and apply an AC voltage across the sample (as opposed to the probe) to induce ion transport through the conductive pathways within the sample and the resultant local alternating signal is measured and analyzed to resolve and quantify the properties of the sample conductive pathways. We demonstrate measurement of AC phase provides a route to distinguish differences in local conductivity at sample features and can be used in conjunction with equivalent circuit models to provide a more sensitive signal to quantify feature resistance. Compared to DC measurements, AC measurements provide additional advantages. First, more stable potential control can be achieved because DC drift in electrode potentials can be minimized. Second, AC measurements are better suited for transport studies within complex biological samples. Resistances from different parts of the sample can be separated based on distinct frequency responses of the sample. Third, use of an AC transmembrane potential can

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minimize possible sample damage caused by a constant applied bias. Fourth, as applied here, the AC-PSICM technique provides opportunities to study ion transport interactions at a much smaller time scale when measurements are done at higher frequencies. Work here describes the basis for evaluation of the AC response in PSICM measurements for nanoporous membranes that can be used to guide future development of AC methods for measurements at biological interfaces.

EXPERIMENTAL SECTION Chemicals and Materials. Solutions were prepared with Milli-Q water (18.2 MΩ·cm at 25 °C, Millipore Corp., Danvers, MA). Potassium chloride (Mallinckrodt, Philipsburg, NJ) solution with concentration 0.1 M was used as electrolyte for SICM measurements. Sodium hypochlorite (13% active chlorine, Acros, Morris Plains, NJ) and potassium iodide (Mallinckrodt, Philipsburg, NJ) were used to prepare nanoporous membranes. Clear, water-resistive epoxy (Devcon, Riviera Beach, FL) was utilized to isolate a single nanopore in a membrane.

Instrumentation for AC-PSICM. A ScanIC scanning ion conductance microscope (ionscope, London, U.K.) was modified as shown in Figure 1 to realize AC-PSICM. Samples analyzed consisted of a membrane mounted between two chambers of a conductivity cell with 0.1 M KCl on both sides. A single barrel quartz nanopipet filled with 0.1 M KCl was used to measure the local potential changes (vs RE) via the potential electrode (UE) connected to a

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differential amplifier. Local potential variations were induced by transmembrane potentials (VTM) applied to a Ag/AgCl working electrode (WE) located in the lower chamber. An AC sinusoidal VTM (2 Vpp, frequency = 5 – 50,000 Hz) was utilized and local potential deflections (amplitude and phase) were extracted with a lock-in amplifier (LIA, SR530, Stanford Research Systems, Sunnyvale, CA), with the synchronized output of the function generator used as external reference signal. The LIA time constant was set at 10-30 ms for high frequencies and 30-300 ms for frequencies below 80 Hz (1 ms was used for 10 Hz and below in some cases). A platinum counter electrode (CE) placed in the top chamber connected to a custom-built CE driver served to prevent potential fluctuations at the reference electrode (RE). The response of two 100 kΩ calibration resistors were recorded to remove these artifacts from the measurement electronics (primarily from CE driver and differential amplifier), with the assumption that the resistors displayed a flat response over the frequency range of interest (5 – 50,000 Hz). Additionally, smaller transmembrane potentials (e.g. 0.01 Vpp) were found to display similar phase response, although diminished amplitudes.

Membrane preparation and characterization. Nanoporous membranes were prepared from ion-tracked polyimide films (track density 104 tracks/cm2, thickness 25 µm, it4ip, Belgium) through the track-etch process, as previously described.33 Briefly, films were first etched in 13% hypochlorite solution at 70 °C and then immersed in 1 M potassium iodide solution for 30 min to neutralize residual etchant and rinsed with Milli-Q water (Millipore Corp., Danvers, MA). Etching

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time was controlled to yield cylindrical nanopores with various diameters. For example, 12 min etching gave pores with ~680 nm diameter (Figure 2a). Membranes with different sized nanopores were fabricated by selectively enlarging pores with a focused ion beam (FIB, Zeiss Auriga, Oberkochen, Germany). An epoxy painting method was used to isolate one single pore.3 In this method, an etched nanopore membrane was mounted on a glass slide, which was then placed on an inverted optical microscope (Nikon TE200, Melvile, NY, USA). Water-resistive epoxy was then applied with a fine brush to isolate a single pore, as indicated by a dashed circle in Figure 2a. The membrane was then mounted onto a conductivity cell filled with 0.1 M KCl. To characterize ion transport across the isolated single pore, a previously reported four-electrode SICM was first utilized.3 Briefly, a nanopipet placed in the top chamber (Figure 2b) was used as the probe to scan over the membrane. A DC transmembrane potential was applied to WE (vs RE) and ion current was recorded by the scanning pipet. Ion current across the membrane generated by the VTM induced significant changes in pipet current (-∆I) when the nanopipet scanned over the nanopore, Figure 2c (VTM = ±0.9 V). Nanopore position was determined in this manner for subsequent AC-PSICM measurements.

RESULTS/DISCUSSION Local impedance measurements Local impedance measurements were first performed on single nanopore membranes. Ion transport through the nanopore was characterized with the four-

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electrode SICM as described in the experimental section. The pipet was then held over the center of the nanopore at a probe-sample distance (Dps) of ~300 nm, and both amplitude and phase of local potential deflections were recorded over the frequency range of 5 – 50,000 Hz. The same measurements were repeated when pipet was held over the surface, but away from nanopore center at the same Dps. For experiments here, the measured local potential amplitudes represent relative values,5 and as such, further discussion is primarily focused on phase measurements. Response measured at the aforementioned two positions as analyzed by Bode plots (phase angle vs frequency) are shown in Figure 3a (black square: over nanopore center; red circle: over membrane area). Both plots share the same trend, phase angle initially increases with frequency and then decreases after reaching a maximum value. Comparison of the two plots indicates that phase response can be clearly differentiated for the nanopore vs the membrane area, i.e. phase can resolve conductive pathways within the sample. This differentiation is most pronounced at low frequencies. To interpret local impedance measurements, an equivalent circuit model was proposed (Figure 3b), where Rpore represents the resistance of the nanopore and Cmem refers to membrane capacitance. Access resistance near the entrance of the nanopore due to field constriction34 and access resistance from the nanopipet tip are combined and represented as Racc. Solution resistance (Rsol) exists between the sample and WE or RE on each side of the membrane. Capacitance of solution and electrodes are not addressed in this circuit model. Solution capacitance is neglected because 0.1 M KCl used in experiments is approximated as purely conductive. For electrode capacitance, Ag/AgCl is very close to an “ideal” nonpolarizable electrode, which, by definition, has

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negligible capacitive charging at the electrode/solution interface and also low charge transfer resistance. The nanopipet serves as a potential probe for sensing local solution potential measured with a high impedance differential amplifier, where minimal current is passed. Therefore, resistance or capacitance associated with the nanopipet are not included in the equivalent circuit. In the equivalent circuit, Rpore and Cmem respond as a parallel RC circuit, while other resistances (Rsol and Racc) form a series RC circuit with Cmem. Combination of these two basic RC circuits results in “peak-shaped” Bode plots. Experimental data shown in Figure 3a support this model, where the low frequency range behaves as an RC in parallel and the high frequency range behaves as an RC in series. Estimations of the RC time constant for the single pore system studied here also agree well with this model. Bulk impedance (Supporting Information, Figure S1a,b) and current-voltage characterization (Figure S1c) was performed with the single nanopore membrane used (Figure 2a). The nanopore resistance was found to be 52.2 MΩ, and membrane capacitance was 33.9 pF. Access resistance encountered (Racc) is normally on the order of 1 MΩ. For instance, the nanopore access resistance (Racc, pore) can be estimated by the following equation.35

1

, = 4

(Equation 1)

Here, = 1.25 ∙ , is the specific conductivity of 0.1 M KCl at room temperature and refers to the pore inner radius. The pore access resistance (, ) was found to be 0.582 MΩ for the 687 nm i.d. nanopore used here. The nanopipet access resistance 9



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( , ), generated from the current squeezing effect36 when the nanopipet is brought close to a surface, is also small (~0.25 MΩ for pipets used in this study (Figure S2)). Rsol for 0.1 M KCl is even smaller than Racc and hence can be neglected. These values together yield a Racc value on the order of 1 MΩ. This estimation suggests the parallel RC part of the circuit has a larger time constant due to the significantly larger R value, and thus the parallel RC dominates the response over lower frequencies, while the response at higher frequencies is due to the series RC part of the equivalent circuit. Figure 3a shows two Bode plots measured over the nanopore center and over the membrane area (at close Dps) which overlap at high frequencies, which indicates little or no difference for Racc and Cmem. Differences are observed in the low frequency regime, which suggests a change in Rpore value when measured at both positions. However, the value of Rpore itself should not physically change. To be specific, the applied transmembrane potential generates a potential profile around the nanopore (Figure 3a, inset). A smaller AC potential is measured when the probe is farther away from the field center (the pore).35 For our model, this can be reflected in the measured resistance of the pore, which we term the apparent pore resistance, R’pore. Therefore, a larger R’pore is obtained when the pipet is held farther away from the nanopore center, which explains the difference observed for the aforementioned Bode plots. To support this model, impedance measurements were also performed over the pore center and over the membrane area at Dps =12.5 µm. As shown in Figure 3a, the probe was first held at the field/nanopore center (position 1), and was then moved laterally (position 3), vertically (position 2) and both laterally and vertically (position 4) away from the field/nanopore center. Bode plots obtained for positions 2-4 show a

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similar phase response, in contrast to the response at position 1.

Together, these

results indicate local conductive pathways can be resolved with phase and the proposed circuit model for a single nanopore membrane is suitable to qualitatively explain the measured phase response.

Quantification of nanopore resistance To quantitatively understand local impedance data with the circuit model, systematic measurements were carried out over the center of a single nanopore at various Dps, and Bode plots recorded were fit to the proposed equivalent circuit model. The equivalent circuit is simplified as shown in Figure 4a, inset. Rs is the sum of both Rsol and Racc in Figure 3b, and Rpore is labeled as R’pore here to represent the apparent Rpore value measured. For this circuit, the phase angle can be written as a function of frequency expressed by Equation 2.

= tan

(

!"'#$%& )*&*

(

"'#$%& + ", + !( )*&* ( "'#$%& ",

-

(Equation 2)

Here, . = 2πf, and values for constants R’pore, Rs and Cmem can be found from fitting of vs f plots for each Dps. For all fittings, Cmem was fixed at 33.9 pF, which is the single pore membrane capacitance obtained from bulk impedance measurements (Figure S1a,b). This was done because Cmem represents the whole membrane area between WE and RE in the proposed equivalent circuit, and Cmem must not change with probe position.

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Table 1 displays values for R’pore and Rs from fitting with coefficient of determination (R2) for each different Dps. Values for coefficient of determination is close to 1, which suggests a good fit with the equivalent circuit model. From values of resistances from fitting and the capacitance value, Bode plots (phase and amplitude) and Nyquist plots (impedance value plotted on complex plane) are found to be in good agreement with plots constructed from experimental data (Figure S3). The R’pore values, as expected, are all larger than the actual nanopore resistance and increase with Dps, while Rs remains constant except for the one value measured when the pipet was close to the sample surface (Dps = 0.3 µm). Further, both resistances were plotted against Dps. The apparent nanopore resistance increases linearly with Dps as shown in Figure 4a. This trend can be rationalized from a simple picture of the resistance to ion flow due to the gap between pore center and probe tip opening. Ions need to travel from the field center through the solution to the pipet tip in order to be sensed and this path causes the attenuated potential or increased resistance measured with the pipet tip. This also predicts that the actual nanopore resistance can be measured at Dps = 0. Extrapolation of the trend line to zero distance yields 48.0 MΩ (Figure 4b), which agrees well with the nanopore resistance determined from bulk measurements (52.2 MΩ). Therefore, the resistance of local conductive features can be determined from phase measurements. Fitted values for Rs remain essentially constant when the probe is micrometers away from the pore center, while a significant increase in Rs is seen when the probe is close to pore center. This could be due to the increase in pipet access resistance when close to the surface, where this value is insignificant when the probe is retracted from the surface. Upon further examination of the circuit models (Figure 3b and Figure 4a

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inset), local potential measured with the nanopipet is the potential difference between RE and the pipet tip, which is the potential drop across the upper Rsol and Racc (or more accurately, somewhere within Racc in Figure 3b). At a certain frequency, current passing through these resistors is the same. Therefore, the measured voltage magnitude scales with the sum of the upper Rsol and Racc and hence scales with Rs in the simplified circuit (Figure 4a inset). Peak voltage values measured with the nanopipet at 5 Hz were plotted against Dps and were overlaid with the Rs vs Dps curve in Figure 4c. Because Rs values are obtained solely from phase measurement, the fact that Rs follows the same trend with the measured magnitude values further supports the equivalent circuit model used for our single nanopore system.

Phase approach curves and lateral phase distribution In the AC-PSICM described here, phase measurement has shown utility in differentiation of local conductive pathways and in quantification of the resistance of a single nanopore with a fixed-position measurement mode. Use of phase to resolve heterogeneous conductive pathways within a sample in an imaging mode was also investigated. Phase measurements have several advantages compared to amplitude measurements. First, amplitude is a relative measurement for both current2,3 and potentiometric measurements.5 For current measurements with a nanopipet, only a portion of the total current from the feature under study is collected with the pipet electrode. In potentiometric measurements, potential deflections at two different Dps must be recorded for each position and the difference in potential deflections is then used to calculate the local conductance value as described previously for potentiometric

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scanning ion conductance microscope (P-SICM).5 Phase, however, is not affected by the signal intensity measured by the nanopipet. Second, while in this validation of the method modest VTM (2 Vpp) was used, smaller VTM can be used for future biological studies, we expect with favorable signal to noise ratios. Third, phase measurement can be more selective than amplitude measurement. Phase is sensitive to frequency and different conductive features (with different R and C values) within a sample can show unique frequency dependent phase responses. In other words, the optimal frequencies for resolving different features can vary and hence phase provides an opportunity to selectively resolve heterogeneous conductive pathways within one sample by adjusting the perturbation frequency. Therefore, phase approach curves and lateral phase distributions were examined to investigate the use of phase to resolve different conductive pathways within a sample. Again, the simplest case of a single nanopore membrane was used initially. To perform an “approach curve”, a pipet was held 300 nm above the nanopore center and was then retracted with a step size of 2.5 µm. Measurements were taken at each step. At each step, phase angle measured by the nanopipet was recorded for frequencies over the range of 5 to 50 kHz. By plotting phase angle of the signal against Dps, a “phase approach curve” was obtained. Figure 5a shows phase approach curves for 4 selected frequencies measured with the same sample discussed previously. As the probe approached the nanopore center, a decrease in phase angle was seen for all curves. However, a noticeable difference in shape was found among the four curves. Approach curves at 30 or 200 Hz have a plateau region at larger Dps and a steep region when the probe is close to the sample surface. For approach curves measured at 5 Hz,

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changes were much more gradual. A sharper approach curve is ideal for resolving different features within a sample in that the “field” of each feature is more confined and the probe only detects a change at a closer distance. In other words, the interference of responses from features that are close to each other is minimized. On the other hand, the absolute change in phase angle determines the sensitivity in resolving a feature. For 5 or 30 Hz curves, a much larger phase angle difference is seen when the probe approaches the surface from far away compared to the 200 Hz approach curve. As for the approach curve obtained at a much higher frequency, 1000 Hz in Figure 5a, little change in phase angle with Dps in the measured distance range is observed. In sum, the optimal frequency that gives the best spatial resolution (sharp approach curve) and the best sensitivity among the four selected frequencies shown here is 30 Hz. The same conclusion can be drawn with the lateral phase distribution (Figure 5b), where phase angle is plotted against the probe’s lateral distance from the nanopore center for various frequencies. In this case, all measurements were taken at a constant Dps of 300 nm. In Bode plots shown in Figure 3a, the optimal frequency, 30 Hz, falls into the “peak frequency” regime at which the phase angle reaches a maximum value. A larger selection of phase approach curves as well as the lateral phase distribution is shown in Figure S4 and suggests characteristic frequency regimes in terms of spatial resolution and sensitivity of resolving sample heterogeneity as shown in Table 2. Together, these data indicate that phase can be used for resolution of heterogeneous conductive pathways within a sample and optimal frequencies can be found from “peak frequencies” in the Bode plot.

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AC-PSICM for resolving heterogeneous nanoporous membrane To demonstrate frequency - resolved measurements for different conductive pathways within a sample, a polyimide membrane with two different sized nanopores was tested. The membrane was fabricated with the same track-etch process described in the experimental section and yielded nanopores with uniform size within the membrane. A focused ion beam was then utilized to enlarge one of the two pores close to each other (Figure S5a). The membrane area with two pores was then isolated with epoxy and mounted onto a conductivity cell in the same manner as for the single pore sample. The resistance of the FIB enlarged pore became significantly smaller as can be seen from the SICM current images (Figure S5b) or the current-voltage plots for both nanopores measured with a nanopipet held above the pore center when VTM was swept from -0.5 V to 0.5 V (Figure S5c). Figure 6a shows Bode plots measured at the center of each nanopore. Phase angles recorded with pore 2, which is the enlarged nanopore (i.d. 1.16 µm), are smaller than those for pore 1 (i.d. 657 nm) at all frequencies. This observation agrees with the equivalent circuit model where smaller Rpore value leads to smaller phase angle. Phase line profiles across two pores were collected in fixed-position mode (Dps = ~280 nm). Figure 6b is an ion current image of the two pore area of the membrane and Figure 6c displays the phase line profiles obtained for four selected frequencies representative of the four regimes discussed in Table 2 (see individual plot at each frequency in Figure S6 in supporting information). The measured phase angle decreases over pores and hence pores appear as negative peaks in line profiles. Two pores are clearly resolved for all frequencies except for 2000 Hz, where only a small peak is seen for Pore 2. At 70

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Hz, which is in the “peak frequency” regime, two narrow, symmetric, well separated peaks associated with the two nanopores are observed. At the 500 Hz trace, separated response for two nanopores similar to the 70 Hz signal is observed. However, the peak intensity is significantly reduced at 500 Hz, which makes the features harder to identify, especially for Pore 1. At 5 Hz, peaks observed are rather deep and broad. Although sufficient contrast for feature identification exists, the two peaks already start to overlap in the middle and the difference in peak intensity for the two pores is less significant than at 70 Hz. These observations further support the findings from the phase approach curve measurements.

CONCLUSIONS We have established a method for measurement of ion transport under alternating current conditions at the nanometer scale. Phase was found to be sensitive to local conductive pathways within the sample and quantification of the nanopore resistance with phase was achieved by fitting to a proposed equivalent circuit model. Examination of phase approach curves and the lateral phase distribution on a single nanopore sample showed that phase can be used to differentiate heterogeneous conductive pathways within a sample. Further, the optimal frequency for resolving different features within a sample was found to be the “peak frequency” in the phase Bode plot for a given system. A phase line profile across two different sized nanopores within one sample confirmed the frequency selection model and preliminarily demonstrated the possibility of studying sample conductance heterogeneity with ACPSICM in an imaging fashion. This “phase imaging” method for transport studies is

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promising if a proper way to decouple ion current and probe-sample distance control is developed. Further, for routine imaging applications, time considerations of impedance techniques should be considered carefully (see supporting information).

And such

“phase imaging” method can be applied to various studies, including ion transport through biological membranes and ion-selective synthetic membranes. In future measurements of biological systems, careful consideration of the RC values for the sample under study (and in relation to the probe properties) must be considered. In particular, to determine the maximum response in signal (phase response), as demonstrated here for pores of known geometry.

For ion channels of constant

dimensions, these values could be estimated from known crystal structures, however the resolution of SICM likely precludes measurement of the contribution of individual channels. We expect in the short term that techniques like this will find application in measurement of heterogeneous transport at tissue interfaces, where micron scale (cellto-cell) interactions present problems that are presently more tractable.

ACKNOWLEDGEMENTS Support from the National Institutes of Health (R01DK084059) and Indiana University is acknowledged. The authors thank the Nanoscale Characterization Facility and Mass Spectrometry Facility at Indiana University for scanning electron microscope, FIB (acquired through the National Science Foundation MRI program (0923064)) and MALDI mass spectrometer use. IU Electronic and Mechanical Instruments Services are acknowledged for assistance in building tools necessary for this study.

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Supporting Information Available: Bulk membrane characterization, calculations and estimations of pipet conductance, fitting results from equivalent circuit models, and considerations relevant to frequency selection and image acquisition. This material is available free of charge via the Internet at http://pubs.acs.org. REFERENCES (1)

Chen, C.-C.; Baker, L. A. Effects of Pipette Modulation and Imaging Distances on Ion Currents Measured with Scanning Ion Conductance Microscopy (SICM). Analyst 2011, 136, 90-97.

(2)

Chen, C.-C.; Zhou, Y.; Baker, L. A. Single-Nanopore Investigations with Ion Conductance Microscopy. ACS Nano 2011, 5, 8404-8411.

(3)

Zhou, Y.; Chen, C.-C.; Baker, L. A. Heterogeneity of Multiple-Pore Membranes Investigated with Ion Conductance Microscopy. Anal. Chem. 2012, 84, 3003-3009.

(4)

Chen, C.-C.; Derylo, M. A.; Baker, L. A. Measurement of Ion Currents through Porous Membranes with Scanning Ion Conductance Microscopy. Anal. Chem. 2009, 81, 4742-4751.

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Chen, C.-C.; Zhou, Y.; Morris, C. A.; Hou, J.; Baker, L. A. Scanning Ion Conductance Microscopy Measurement of Paracellular Channel Conductance in Tight Junctions. Anal. Chem. 2013, 85, 3621-3628.

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Korchev, Y. E.; Negulyaev, Y. A.; Edwards, C. R. W.; Vodyanoy, I.; Lab, M. J. Functional Localization of Single Active Ion Channels on the Surface of a Living Cell. Nat. Cell Biol. 2000, 2, 616-619.

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Shevchuk, A. I.; Gorelik, J.; Harding, S. E.; Lab, M. J.; Klenerman, D.; Korchev, Y. E. Simultaneous Measurement of Ca2+ and Cellular Dynamics: Combined Scanning Ion Conductance and Optical Microscopy to Study Contracting Cardiac Myocytes. Biophys. J. 2001, 81, 1759-1764.

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Shin, W.; Gillis, K. D. Measurement of Changes in Membrane Surface Morphology Associated with Exocytosis Using Scanning Ion Conductance Microscopy. Biophys. J. 2006, 91, L63-L65.

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Gorelik, J.; Ali, N. N.; Sheikh Abdul Kadir, S. H.; Stojkovic, P.; Armstrong, L.; Sviderskaya, E. V.; Negulyaev, Y. A.; Klenerman, D.; Bennett, D. C.; Lako, M.; et al. Non-Invasive Imaging of Stem Cells by Scanning Ion Conductance Microscopy: Future Perspective. Tissue Eng., Part C 2008, 14, 311 -318.

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FIGURE CAPTIONS

FIGURE 1. Schematic of AC-PSICM. The nanopore membrane is mounted on a conductivity cell and is immersed in 0.1 M KCl electrolyte. The working electrode (WE) applies a 2 Vpp transmembrane AC potential (5 Hz - 50 kHz) with respect to a reference electrode (RE), which is held at ground potential. Counter electrode (CE) is connected to a counter electrode driver. A differential amplifier is used to measure local potential (via a potential electrode, UE) vs RE, which is then fed to a lock-in amplifier for phase and amplitude measurements.

FIGURE 2. (a) Optical image of a single nanopore membrane. Boundary of painted epoxy for single pore isolation can be seen and the isolated nanopore is indicated with the dashed circle. Inset: scanning electron microscopy (SEM) image of the isolated single pore in (a), nanopore diameter is 689 nm. (b) Illustration of the four-electrode SICM setup: pipet electrode (PE) inside the nanopipet is used to control pipet position as well as to record ion current images. (c) SICM ion current images of the single pore shown in (a) obtained under -0.9 V and +0.9 VTM.

FIGURE 3. (a) Bode plots measured over the nanopore center and over a flat membrane area at both Dps = 300 nm and Dps = 12.5 µm. Inset shows a side view of the 22


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nanopore. Numbers indicate the positions in the local electric field where each plot in (a) was recorded. (b) Proposed equivalent circuit for single pore system: Rsol represents solution resistance, Racc is combined probe and nanopore access resistance, Rpore is nanopore

resistance,

Cmem

is

membrane

capacitance

and

VTM

is

applied

transmembrane potential.

FIGURE 4. (a) Fitted values for apparent pore resistance plotted against Dps yield a linear relationship (coefficient of determination R2 = 0.9955). Inset: simplified equivalent circuit for single pore system. Rs is the combination of solution resistance and access resistances, R’pore is the apparent pore resistance and Cmem is membrane capacitance. Blue and green dashed boxes indicate the series RC and parallel RC components in the circuit, respectively. (b) The extrapolated line in (a). At Dps = 0 µm, R’pore was found to be 48 MΩ. (c) Overlaid graphs of fitted values for Rs and measured local potential peak amplitude vs Dps share the same trend.

FIGURE 5. A selection of phase approach curves (a) and lateral phase distributions (b) at different frequencies. The four frequencies shown here represent the four representative regimes of phase response summarized in Table 2.

FIGURE 6. (a) Bode plots (phase) for two different sized pores shown in ion current image in (b) within one sample. The inner diameters are 657 nm and 1.16 µm for pore 1 and 2, respectively. (b-c) SICM ion current image (b) and corresponding phase line profiles measured along a line (indicated with the white dashed line in ion current image)

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across two different sized nanopores. Peaks at ~14 µm and ~52 µm in phase line profiles correspond to pore 1 and pore 2 in the ion current image respectively.

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TABLE

Table 1. Values for each parameter in the simplified equivalent circuit in Figure 4a as determined from fitting.*

345 (78)

:>4;

Alternating Current Potentiometric Scanning Ion Conductance

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