Anal. Chem. 2006, 78, 6535-6541
Alternating Current Impedance Imaging of High-Resistance Membrane Pores Using a Scanning Electrochemical Microscope. Application of Membrane Electrical Shunts To Increase Measurement Sensitivity and Image Contrast Eric Nathan Ervin and Henry S. White*
Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112 Lane A. Baker and Charles R. Martin*
Department of Chemistry, Center for Research at the Bio/Nano Interface, University of Florida, Gainesville, Florida 32611-7200
Whether an individual pore in a porous membrane can be imaged using scanning electrochemical microscopy (SECM), operated in ac impedance mode, is determined by the magnitude of the change in the total impedance of the imaging system as the SECM tip is scanned over the pore. In instances when the SECM tip resistance is small relative to the internal pore resistance, the total impedance changes by a negligible amount, rendering the pore invisible during impedance imaging. A simple solution to this problem is to introduce a low-impedance electrical shunt (i.e., a salt bridge) across the membrane. This principle is demonstrated by imaging polycarbonate membranes (6-12-µm thickness) containing between 1 and 2000 conical-shaped pores (60-nm- and 2.5-µm-diameter openings) using an ∼1-µm-radius Pt tip. Theory and experiments show that image contrast (the change in ac current measured as the probe is scanned over the pore) is inversely proportional to the total resistance of the membrane and can be increased by a factor of ∼50× by introducing a low-resistance electrical shunt across the membrane. Remarkably, SECM images of membranes containing a single high-resistance (∼1GΩ) pore can only be imaged by short-circuiting the membrane. Image contrast also becomes independent of membrane resistance when an electrical shunt is used, allowing for more quantitative comparisons of the features in ac impedance images of different membranes. Alternating current (ac) impedance imaging of surfaces using a scanning electrochemical microscope (SECM) is a relatively new scanning probe method that is based on measuring the variation in electrical impedance as a small metal SECM tip is scanned over a surface.1-5 An advantage of ac impedance imaging is that it does not require the presence of an electroactive species in the solution, * To whom correspondence should chem.utah.edu;
[email protected]. 10.1021/ac060577k CCC: $33.50 Published on Web 08/19/2006
be
addressed.
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© 2006 American Chemical Society
as is necessary in conventional SECM imaging modes.6 Relaxation of the requirement of having a redox couple present will provide additional opportunities for SECM, especially in imaging and measuring transport across biological membranes where the introduction of oxidants and reductants (i.e., the redox mediator) may interfere with the function of the sample.7-10 We recently reported ac impedance imaging of a porous polycarbonate membrane.3 The measurement of membrane pore structure is based on measuring the impedance, using phase-sensitive detection, between a scanning SECM tip, in close contact to the membrane, and a large auxiliary electrode positioned in the solution on the opposite side of the membrane. In ac imaging, a pore opening is observed only if the total impedance between the SECM tip and the auxiliary electrode changes significantly as the tip passes over the pore.3 Consider the simple cylindrical pore depicted in Scheme 1, which has a (1) Gabrielli, C.; Huet, F.; Keddam, M.; Rousseau, P.; Vivier, V. J. Phys. Chem. B 2004, 108, 11620-11626. (2) (a) Katemann, B. B.; Schulte, A.; Calvo, E. J.; Koudelka-Hep, M.; Schuhmann, W. Electrochem. Commun. 2002, 4, 134-138. (b) Katemann, B. B.; Inchauspe, C. G.; Castro, P. A.; Schulte, A.; Calvo, E. J.; Schuhmann, W. Electrochim. Acta 2003, 48, 1115-1121. (c) Etienne, M.; Schulte, A.; Schumann, W. Electrochem. Commun. 2004, 6, 288. (3) Ervin, E. N.; White, H. S.; Baker, L. A. Anal. Chem. 2005, 77, 5564-5569. (4) (a) Alpuche-Aviles, M. A.; Wipf, D. O. Anal. Chem. 2001, 73, 4873-4881. (b) Horrocks, B. R.; Schmidtke, D.; Heller, A.; Bard, A. J. Anal. Chem. 1993, 65, 3605-3614. (5) Baranski, A. S.; Diakowski, P. M. J. Solid State Electrochem. 2004, 8, 683692. (6) Bard, A. J.. Mirkin, M. V., Eds. Scanning Electrochemical Microscopy; Marcel Dekker: New York, 2001. (7) Macpherson, J. V.; Jones, C. E.; Barker, A. L.; Unwin, P. R. Anal. Chem. 2002, 74, 1841-1848. (8) Tsionsky, M.; Cardon, Z. G.; Bard, A. J.; Jackson, R. B. Plant Physiol. 1997, 113, 895-901. (9) (a) Cai, C.; Liu, B.; Mirkin, M. V. Anal. Chem. 2002, 74, 114-119. (b) Liu, B.; Rotenberg, S. A.; Mirkin, M.V. Anal. Chem. 2002, 74, 6340-6348. (c) Liu, B.; Rotenberg, S. A.; Mirkin, M. V. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 9855-9860. (10) (a) Uitto, O. D.; White, H. S. Pharm. Res. 2003, 20, 646-652. (b) Uitto, O. D.; White, H. S.; Aoki, K. Anal. Chem. 2002, 74, 4577-4582, and references therein.
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Scheme 1. Resistances That Contribute to the Total Impedance as the Tip Is Scanned Across a Pore
Figure 1. Schematic of cell and instrumentation for impedance imaging, showing the bypass channel used to provide an electrical short between the upper and lower compartments.
large length-to-diameter ratio. The resistances that contribute to the overall impedance between the tip and auxiliary electrode include the tip resistance (Rtip), the pore entrance resistance (Rentry), the internal pore resistance (Rinternal), and the pore exit resistance (Rexit). For a membrane containing a single pore, the total resistance is given by
Rtot ) Rtip + (Rentry + Rinternal + Rexit)
(1)
where the parentheses contain the resistances associated with the membrane. An impedance image of the pore can be obtained if the combined change in Rtip and Rexit , i.e., (∆Rtip + ∆Rexit), as the tip passes over the pore, is significant in comparison to the total resistance given by eq 1. Thus, the ability to resolve an individual pore opening by SECM impedance imaging depends on the ratio (∆Rtip + ∆Rexit)/Rtot. For the long narrow pore depicted in Scheme 1, Rinternal > Rtip and Rinternal . Rexit. Thus, regardless of whether (∆Rtip + ∆Rexit) is significant, the ratio (∆Rtip + ∆Rexit)/Rtot will always be small, thereby reducing the image contrast. In this report, we have systematically imaged membranes containing between 1 and 2000 pores as a means to investigate the dependence of image contrast on Rtot. Because the pores represent parallel ionic pathways, Rtot decreases as the number of pores, N, increases, eq 2. Thus, as N increases, the quantity
Rtot ) Rtip + (Rentry + Rinternal + Rexit)/N
(2)
(∆Rtip + ∆Rexit)/Rtot increases, yielding higher contrast in the SECM images. This prediction is quantitatively demonstrated in this report. Equation 2 suggests that the maximum contrast in impedance imaging, (∆Rtip + ∆Rexit)/Rtip., occurs as N f ∞ and Rtot f Rtip. We demonstrate in this report that the optimal contrast can also be obtained for a membrane with few pores (small N) by introducing an artificial electrical shunt across the membrane, effectively short-circuiting the two resistances that do not depend on the position of the tip, i.e., Rentry and Rinternal. To provide a low resistant path, a standard salt bridge, referred to here as a “bypass 6536 Analytical Chemistry, Vol. 78, No. 18, September 15, 2006
channel”, is used to connect the upper and lower solutions of the diffusion cell, allowing current to flow around the membrane, Figure 1. In addition to providing the maximum possible image contrast, the image contrast is also independent of the membrane properties when an electrical shunt is used, since Rtot ) Rtip regardless of the total membrane resistance. EXPERIMENTAL SECTION Chemicals and Materials. KCl, NaOH, and NaCN (all from Mallinckrodt) where used as received. Ferrocenylmethyltrimethylammonium hexaflourophosphate (FcTMA+) was prepared by metathesis of the iodide salt (Strem, 99%) with ammonium hexaflourophosphate (Strem, 99%). The crystals were collected by vacuum filtration and recrystallized from water. All solutions were prepared using 18 M Ω‚cm H2O from a Barnstead E-pure water purification system. Two types of track-etched polycarbonate membranes11 were used in these studies. The first was an ∼6-µm-thick membrane with a nominal pore density of ∼8 × 104 pores/cm2. The second membrane was ∼12 µm thick with a pore density of either ∼1.0 × 104 or ∼50 pores/cm2. In all cases, the membranes contained conically shaped pores having small and large opening diameters of ∼60 nm and ∼2.5 µm, respectively, as determined by scanning electron microscopy. Preparation and characterization of the membranes have been described elsewhere.12 Scanning Electrochemical Microscopy. The SECM cell and instrumentation for imaging membranes, Figure 1, have previously been described13 and are identical to the system recently described for ac imaging.3 Very briefly, the two-piece horizontal diffusion cell is constructed from Teflon, allowing for the positioning of a membrane between the two compartments containing aqueous solutions. The membrane is sandwiched between two glass slides, each containing a 0.9-mm-radius hole to allow solution contact. The number of membrane pores separating the upper and lower compartments of the SECM cell was varied by using membranes of different pore densities and by offsetting the holes in the glass slides apart from one another in order to control the area of the (11) Fleischer, R. L.; Price, P. B.; Walker, R. M. Nuclear Tracks in Solids, Principles and Applications; University of California Press: Berkeley, CA, 1975. (12) Li, N.; Yu, S.; Harrell, C. C.; Martin, C. R. Anal. Chem. 2004, 76, 20252030. (13) Lee, S.; Zhang, Y.; White, H. S.; Harrell, C. C.; Martin, C. R. Anal. Chem. 2004, 76, 6108-6115.
membrane exposed to both solutions. The membrane pore density was multiplied by the area of the exposed membrane to obtain an estimate of the number of pores. High-vacuum grease (Dow Corning) was used to seal the membrane between the slides, and the glass slides were clamped between the two halves of the diffusion cell. Images were obtained by measuring the impedance between the SECM tip and a 1.5-mm-radius Pt disk electrode positioned on the opposite side of the membrane. A 10-mV rms ac signal from a lock-in amplifier (Stanford Research Systems R810) was applied between the tip and Pt electrode and the ac current (iac) response measured by phase-sensitive detection. Two different potentiostats were used based on the required sensitivity. A Pine AFRDE5 bipotentiostat was used for high current measurements (>100 nA), while a Dagan Chem-Clamp voltammeter/amperometer was used for lower current measurements. The position of the SECM tip is controlled with ∼20-nm precision using piezoelectric inchworm microtranslation stages with optical encoding (8200, EXFO) interfaced via a controller box to LabView. Positioning of the SECM tip in proximity to the membrane surface was accomplished by monitoring the decrease in the ac current as the tip is moved toward the membrane. The absolute distance between the tip and surface was measured after imaging by recording the tip travel distance needed to bring the tip in contact with the membrane surface (resulting in a sudden leveling off the ac current). With practice, it is possible to reproducibly position the tip at a separation distance of 1 ( 0.5 µm, prior to measuring the absolute distance by making contact with the surface. SECM Tip Preparation. Preparation and characterization of the conical-shaped, glass-sealed Pt SECM tips used in this study have been detailed in a previous report3 and closely follow literature methods.14,15 A slight modification of our previously reported procedure used here is that the Pt wire was electrochemically etched in a 6 M NaCN, 0.1 M NaOH solution by applying a 100-Hz, 4-V peak-to-peak signal until a sharp tip was obtained, as observed by optical microscopy (Caution: NaCN solutions should be handled with extreme care.). The exposed Pt tip is conical shaped. Tip radii were determined by voltammetric measurements as previously described, using the expression for the diffusion-limited current at a conical electrode reported by Mirkin and Zoski,16 and by scanning electron microscopy, as described previously. Approximately equal size tips (1 ( 0.2 µm radii) were employed to record all data in order to allow for meaningful comparison of image contrast in different images. RESULTS AND DISCUSSION As discussed in the introduction, the ability to observe a pore in a SECM impedance image depends on whether there is a significant variation in the total impedance as the tip scans over the pore opening. We quantify this concept by measuring the image contrast as a function of the membrane resistance. The latter is determined by the number of pores in the membrane, N, eq 2, which can be readily adjusted by the methods described in the Experimental Section. (14) Zoski, C. G.; Liu, B.; Bard, A. J. Anal. Chem. 2004, 76, 3646-3654. (15) (a) Penner, R. M.; Heben, M. J.; Lewis, N. S. Anal. Chem. 1989, 61, 16301636. (b) Heben, M. J.; Dovek, M. M.; Lewis, N. S.; Penner, R. M.; Quate, C. F. J. Microsc. (Oxford, UK) 1988, 152, 651-661. (16) Zoski, C. G.; Mirkin, M. V. Anal. Chem. 2002, 74, 1986-1992.
Membrane Resistance as a Function of N. The resistance of a membrane containing N identical pores can be evaluated from the expression Rm ) (Rentry + Rinternal + Rexit) /N, where Rentry ) 1/4κrentry, Rinternal ) lp/(πκrentryrexit) and Rexit ) 1/4κrexit. In these expressions, κ is the solution conductivity, rentry and rexit are the radii of the small and large pore openings, respectively, and lp is the length of the pore (i.e., the membrane thickness). In the imaging experiments reported below, the upper compartment of the cell contains 1 mM KCl, while the lower compartment contains 10 mM KCl. The different KCl concentrations are chosen to reduce the SECM tip capacitance while simultaneously decreasing Rentry and Rinternal. As previously described, this strategy, while not absolutely necessary for imaging, improves the image contrast and simplifies both the measurement and analysis.3 Using the above equations and appropriate KCl concentrations, Rexit in the upper solution of the diffusion cell (1 mM KCl) is computed to be ∼16.7 MΩ, while Rentry in the lower solution (10 mM KCl) is ∼56 MΩ. In computing Rinternal, we assume that the pore is filled with 5 mM KCl (the approximate average value of the KCl concentrations in the upper and lower compartments) yielding Rinternal ∼ 680 MΩ for a 6-µm-thick membrane and Rinternal ∼ 1.4 GΩ for a 12-µm-thick membrane. Experimental measurements of the membrane resistance (vide infra) indicate that this approximation is quite reasonable. Finally, the SECM tip resistance is given by Rtip ) 1/2πκrt , which, for a 1-µm-radius tip in a 1 mM KCl solution, is equal is ∼10.6 MΩ. Comparing the above values of Rexit, Rentry, Rinternal, and Rtip indicates that the internal resistance of the pore dominates both the membrane and overall system resistances when N is small. For instance, for a 12-µm-thick membrane, Rinternal is ∼100 times larger than either of the two resistances that depend on the tip position, Rexit and Rtip. Thus, the image contrast in an SECM impedance image of a single pore 12 µm-thick membrane is anticipated to be very low. Below, we show by experiment that the image contrast is negligibly small in this case; i.e., the pore cannot be observed by ac SECM. Steady-state dc i-V curves were recorded to verify the value of N based on the membrane pore density and the area of the membrane in contact with the solutions (see Experimental Section). A potentiostat was used to slowly scan the potential between two Ag/AgCl electrodes, placed ∼3 cm from each side of the membrane, between -400 and 400 mV. Figure 2A shows the i-V curve for a 6-µm-thick membranes containing ∼2000 and ∼1000 pores, while Figure 2B shows the i-V curve for a 12-µmthick membrane containing 1 pore. The straight lines plotted in Figure 2 represent the predicted i-V curves based on the membrane resistance, Rm, computed as described above. Agreement to within a factor of 50% between the slopes of the experimental and predicted i-V is obtained in each case, demonstrating the ability to compute approximate values of Rm based on N and the pore geometry. As a side note to this section, we frequently observe nonlinear i-V curves immediately after immersing the membranes in solution. Linear i-V curves, such as the examples shown in Figure 2, are obtained after the membrane has been in contact with the solution for extended periods (>3 h). A number of examples of nonlinear and linear i-V curves are presented in the Supporting Information file. We did not pursue investigations of this pheAnalytical Chemistry, Vol. 78, No. 18, September 15, 2006
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Figure 2. (A) i-V curves for ∼6-µm-thick membranes containing ∼2000 (triangles) and ∼1000 (squares) pores. The number of pores was estimated from the exposed area of the membrane and the membrane pore density. The solid lines represent the predicted i-V curves for membranes containing 2000 and 1000 pores, respectively, based on the resistance estimated in the text. (B) i-V curve for a ∼12-µm-thick membrane containing a single pore. The solid line represents the predicted i-V curve. All i-V curves were obtained using two Ag/AgCl electrodes located on opposite sides of the membrane. The upper and lower compartments of the SECM cell contained 1 and 10 mM KCl, respectively.
nomenon, whose origin appears to be associated with slow equilibration of the pore interior after contact with the ionic solutions. Dependence of Image Quality on Membrane Resistance. Impedance images of individual pores in 6-µm-thick membranes containing ∼2000, ∼1000, and ∼10 pores are shown in Figure 3. The images were obtained using a 1-kHz, 10-mV rms ac input voltage. The images were recorded using different SECM tips (1 ( 0.2 µm radius), and at a tip-to-membrane distance of ∼1 µm; the influence of variable tip-to-membrane distance is discussed below. The images correspond to the spatial variation in the ac current as the tip is rastered across the membrane surface. The lighter peak-shaped feature in each image corresponds to a higher ac current above a pore opening. In a recent report, we demonstrated that the magnitude of the ac current at f ) 1 kHz is inversely proportional to the total resistance to the SECM imaging/membrane system. Thus, the magnitude of the ac current plotted in Figure 3 is directly proportional to the inverse of the total system resistance. Reference 3 describes in detail the equivalent circuit and the frequency response of the ac impedance SECM. Each image in Figure 3 is normalized to its own background ac current measured away from the pore opening; all three images are plotted on the same scale (absolute values of the background current are given in the caption). This normalization allows for a 6538
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Figure 3. SECM impedance images (35 × 35 µm) of the large opening (2.5-µm diameter) of an individual conical-shaped pore in an ∼6-µm-thick membrane containing (A) ∼2000, (B) ∼1000, and (C) ∼10 pores. The images were obtained using a ∼1-µm-radius Pt SECM tip, with 1 and 10 mM KCl in the upper and lower solutions, respectively. Ac input signal: f ) 1.0 kHz and Vin ) 10 mV rms. All images are normalized to their respective current measured far from the pore opening. Absolute values of the background current used for normalization: (A) 4.3, (B) 3.7, and (C) 0.1 nA.
simpler comparison of the image contrast in the images of membranes containing different numbers of pores. Inspection of Figure 3 clearly shows that image contrast decreases dramatically as N decreases. To quantify this relationship, we define the SECM image contrast as the percent relative increase in the ac current, eq 3, where ipeak is the ac current when the SECM tip is above
image contrast ) [(ipeak - ibackground)/ibackground] × 100 (3)
the pore opening and ibackground is the current measured away from the pore opening. In Figure 3, the image contrast decreases in the order 30, 14, and 3% for N ∼ 2000, 1000, and 10, respectively. Images of pores in membranes with N < 10 could not be obtained, a consequence of a vanishingly small image contrast, as will be demonstrated below. Figure 4 shows image contrast plotted as a function of the inverse value of the membrane resistance, Rm-1. Values of Rm were determined from the dc i-V curves as previously described, and image contrast was computed from ac images using eq 3. Image contrast was plotted as a function of Rm-1 (rather than Rm) because Rm-1 increases linearly with increasing N. In order to minimize the error that results from the dependence of the peak ac current on the tip-to-membrane distance, the values of image contrast plotted in Figure 4 were determined from repetitive imaging of pores in the same membrane. In these
over (R over tip and R pore),
away Rbackground ) R away tip + R pore /N
(6)
and away -1 -1 -1 + (R over (7) Rpeak ) R over tip + [(R pore /(N - 1)) pore) ] away over over away ) R over tip + (R pore R pore)/((N - 1)R pore + R pore )
Figure 4. Image contrast (eq 3) of SECM impedance images plotted as a function of the inverse of the membrane resistance (determined from steady-state i-V curves). Data were obtained from impedance images of the large opening (2.5-µm diameter) of conical-shaped pores. All data where measured with a ∼1-µm-radius Pt SECM tip, and with 1 and 10 mM KCl in the upper and lower solutions, respectively. Ac input signal: f ) 1.0 kHz and Vin ) 10 mV rms. The error bars represent the reproducibility of i-V slopes and image contrast values recorded using the same membrane. The data point located at the origin corresponds to a 0.96 GΩ membrane containing a single pore.
In writing eqs 6-8, we note that when the tip is positioned away from a pore opening at the membrane surface, the tip resistance is in series with N identical parallel pore resistances (eq 6). When the tip is over a pore opening, there are N - 1 pore resistances in parallel with 1 pore resistance that is directly beneath the tip. The parallel resistances are again in series with the tip resistance (eqs 7 and 8). Substituting eqs 6 and 8 into eq 5 yields
(ipeak - ibackground)/ibackground )
[(
R away tip +
experiments, the tip was retracted far from the membrane and then returned to an estimated separation distance of 1 µm. The averaged values of image contrast from a minimum of three measurements are plotted, along with the corresponding estimate of the error. Figure 4 shows that the image contrast increases from an immeasurably small value (100 MΩ), it is very difficult to locate and image a pore due to the low image contrast, as described above. However, the theory and experiments presented above suggest that image contrast can be greatly improved in this situation by providing a low-resistance pathway in parallel with membrane. Experimentally, this is readily accomplished by connecting the aqueous solutions on opposite sides of the membrane with a salt bridge containing a high concentration of electrolyte, as shown in Figure 1. We refer to the salt bridge in this application as a “bypass channel” because its purpose is to provide an electrical bypass around the resistive membrane. The bypass channel is equivalent to introducing a large number of virtual pores in the membrane (N f ∞). The analysis presented above indicates that this simple strategy should increase the SECM image contrast by a factor of ∼60 when imaging a pore in a 12-µm-thick membrane. To demonstrate the principle of using a bypass channel, a 12µm-thick membrane containing a single conical-shaped pore was mounted in the SECM cell with 1 and 10 mM KCl solutions in the upper and lower compartments. The membrane resistance was measured to be ∼1.0 GΩ from the dc i-V curve (in agreement with the estimated 1.4 GΩ based on pore geometry, 6540 Analytical Chemistry, Vol. 78, No. 18, September 15, 2006
Figure 5. SECM impedance images (150 × 150 µm) of a ∼12µm-thick membrane containing a single conical-shaped pore (2.5µm-diameter pore opening) obtained with the bypass channel opened, closed, reopened, and reclosed. The images where obtained using a 1-µm-radius Pt SECM tip, with 1 and 10 mM KCl in the upper and lower solutions, respectively. Ac input signal: f ) 1.0 kHz and Vin ) 10 mV rms. The images are normalized to their respective current measured far from the pore opening. The absolute values of the background current used for normalization are 5 (“open”) and 0.08 nA (“closed”).
vide supra). In the absence of the bypass channel, the pore could not be located or imaged. Thus, to start the experiment, the ends of a 10-cm-long, 0.5-cm-i.d. Tygon tube, filled with 1 mM KCl, were immersed into the solutions of the upper and lower compartments. The SECM tip was brought down into close contact with the membrane (∼5 µm) and the single pore was locate and imaged, Figure 5. The bypass channel was then carefully disconnected from the upper compartment, and the same region of the membrane was imaged without retracting the SECM tip. As demonstrated in Figure 5, the image of the pore vanishes when the bypass channel is disconnected. When the bypass channel was reimmersed in the upper compartment, the pore reappeared in the SECM image. The bypass channel was repeatedly removed and replaced in the upper solution, with excellent image reproducibility (Figure 5). In addition, this method was repeated with different membranes and tips, yielding similar results (see Supporting Information for an additional example of this experiment). The contrast in the images shown in Figure 5 when employing the bypass channel is ∼25%. This value is similar to the image
contrast observed for polycarbonate membranes containing ∼2000 pores (Figures 3 and 4). An additional consequence of using the bypass channel is that the contrast observed in the SECM images is no longer a function of the membrane resistance. This can be readily deduced from eq 16, where the image contrast is only a function of Rtip and ∆Rtip. This result should simplify quantitative comparisons of ac impedance images of pores in different membranes. Employing a bypass channel is a remarkably simple means to greatly enhance the contrast in ac impedance SECM images of high-resistance membranes and should be generally useful in other applications where the dominant pore resistance is independent of the SECM tip position. For instance, the ionic resistance of protein ion channels that transport small ions, e.g., R-hemolysin or OmpF porin, is dominated by an internal channel resistance of ∼1 GΩ.17 Imaging a protein ion channel in a membrane that contains a small number of proteins would thus be impossible without the use of a bypass channel. (17) (a) Rostovtseva, T. K.; Nestorovich, E. M.; Bezrukov, S. M. Biophys. J. 2002, 82, 160. (b) Gu, L.-Q; Braha, O.; Conlan, S.; Cheley, S.; Bayley, H. Nature 1999, 398, 686.
CONCLUSION Image contrast in ac impedance SECM of membranes increases dramatically as the membrane resistance decreases. Experiments and theory presented in this report demonstrate that, in instances where the SECM tip resistance is small relative to the internal pore resistance, the total impedance changes by a negligible amount during imaging, resulting in insufficient image contrast to obtain images. A simple and highly effective solution to this problem is to shunt the ion current around the membrane using a low-impedance bypass channel. ACKNOWLEDGMENT This research was supported by the Defense Advanced Research Project Agency. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review March 29, 2006. Accepted July 25, 2006. AC060577K
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