Effect of a Polymer Cushion on the Electrical Properties and Stability of

Oct 30, 2009 - Janice Lin, John Szymanski, Peter C. Searson,* and Kalina Hristova*. Department of Materials Science and Engineering, Johns Hopkins ...
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Effect of a Polymer Cushion on the Electrical Properties and Stability of Surface-Supported Lipid Bilayers Janice Lin, John Szymanski, Peter C. Searson,* and Kalina Hristova* Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland 21218 Received August 28, 2009. Revised Manuscript Received October 9, 2009 A robust biomimetic cell membrane platform is critical for mechanistic studies of membrane protein channels. While polymer cushions are believed to facilitate the incorporation of membrane proteins in such a platform, a systematic characterization and optimization of such cushions is rarely performed. Here, we examine the influence of a polymer cushion on the electrical properties of supported 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayers produced via a Langmuir-Blodgett deposition/vesicle fusion assembly process on single-crystal silicon. We show that the resistance of DPhPC bilayers is maximized at the calculated crossover concentration of the polymer (5.9 mol % PEG-lipids). Additionally, these bilayers are sufficiently stable to allow impedance analyses to be performed for nearly 3 weeks. These studies reveal the optimal PEG concentration that yields electrically robust bilayers and demonstrate the utility of the platform for future studies of membrane protein channels and membrane active peptides.

Introduction Biomimetic membranes on planar solid supports are being developed to study the structure, function, and stability of membrane proteins in a controlled manner.1-6 The assembly of such membranes on conductive substrates allows electrical detection of ion permeation by electrochemical impedance spectroscopy (EIS).7-12 The architecture of supported membranes often includes a polymeric cushion, usually poly(ethylene glycol) (PEG), to raise the bilayer above the substrate.13,14 It is generally believed that this polymeric cushion facilitates the incorporation of membrane proteins with cytoplasmic domains by preventing denaturing contacts between the protein and the substrate.15,16 However, a systematic characterization and optimization of the PEG cushions in such studies is rarely performed. In this Article, we investigate the influence of the PEG cushion on the electrical properties of planar supported bilayers, with the goal of identifying PEG concentrations for which the electrical properties of the supported bilayer are optimal. 1,2-Diphytanoylsn-glycero-3-phosphocholine (DPhPC) bilayers are formed by Langmuir-Blodgett (LB) deposition and vesicle fusion on *To whom correspondence should be addressed. E-mail: [email protected] (P.C.S.); [email protected] (K.H.). (1) Sackmann, E. Science 1996, 271, 43–48. (2) Sackmann, E.; Tanaka, M. Trends Biotechnol. 2000, 18, 58–64. (3) Merzlyakov, M.; Li, E.; Hristova, K. Biointerphases 2008, 3, FA80–FA84. (4) Bayley, H.; Cremer, P. S. Nature 2001, 413, 226–230. (5) Plant, A. L. Langmuir 1999, 15, 5128–5135. (6) Richter, R. P.; Berat, R.; Brisson, A. R. Langmuir 2006, 22, 3497–3505. (7) Koper, I. Mol. BioSyst. 2007, 3, 651–657. (8) Vockenroth, I. K.; Fine, D.; Dodobalapur, A.; Jenkins, A. T. A.; Koper, I. Electrochem. Commun. 2008, 10, 323–328. (9) Raguse, B.; Braach-Maksvytis, V.; Cornell, B. A.; King, L. G.; Osman, P. D. J.; Pace, R. J.; Wieczorek, L. Langmuir 1998, 14, 648–659. (10) Zawisza, I.; Bin, X. M.; Lipkowski, J. Langmuir 2007, 23, 5180–5194. (11) Lin, J.; Merzlyakov, M.; Hristova, K.; Searson, P. C. Biointerphases 2008, 3, 33–40. (12) Nikolov, V.; Lin, J.; Merzlyakov, M.; Hristova, K.; Searson, P. C. Langmuir 2007, 23, 13040–13045. (13) Wagner, M. L.; Tamm, L. K. Biophys. J. 2000, 79, 1400–1414. (14) Naumann, C. A.; Prucker, O.; Lehmann, T.; Ruhe, J.; Knoll, W.; Frank, C. W. Biomacromolecules 2002, 3, 27–35. (15) Knoll, W.; Koper, I.; Naumann, R.; Sinner, E. K. Electrochim. Acta 2008, 53, 6680–6689. (16) Deverall, M. A.; Gindl, E.; Sinner, E. K.; Besir, H.; Ruehe, J.; Saxton, M. J.; Naumann, C. A. Biophys. J. 2005, 88, 1875–1886.

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single-crystal silicon. EIS is used to determine the bilayer resistance and capacitance as a function of the applied potential and PEG concentration. The bilayers are further characterized in terms of lipid mobility, platform homogeneity, and stability. These studies reveal the optimal PEG concentration that yields electrically robust bilayers and demonstrate the utility of the platform for future membrane protein studies.

Materials and Methods Silicon Substrate Preparation. Single side polished, n-type silicon wafers (Æ111æ, F = 0.001-0.005 Ω cm, Silicon Quest International) were cleaned by sonication for 15 min first in isopropyl alcohol, followed by acetone, and then again in isopropyl alcohol. All organic solvents were rinsed off thoroughly in deionized water before surface treatment in a 30% (v/v) hydrogen peroxide, 70% (v/v) sulfuric acid solution for 1 h. The silicon wafers were then rinsed thoroughly in deionized water and used within 1 h. The silicon wafers were submerged vertically into a Langmuir trough (Nima Technologies) for Langmuir-Blodgett deposition. Langmuir-Blodgett Deposition. A 1 mg/mL solution of DPhPC (Avanti Polar Lipids) and PEG-lipids (1,2-dipalmitoylsn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], Avanti Polar Lipids) was prepared in chloroform. The concentration of the PEG-lipids was varied from 0 to 24 mol %. An amount of 20 μL of the lipid solution was deposited at the air-water interface of the Langmuir trough (Figure S1a in the Supporting Information). After allowing a minimum of 30 min for the chloroform to evaporate, the lipids were compressed to a surface pressure of 32 mN/m. The silicon wafer was then raised vertically out of the trough at a rate of 15 mm/min, allowing the deposition of a PEG-supported lipid monolayer on the polished side of the silicon wafer (Figure S1b in the Supporting Information). A custom Teflon electrochemical cell was assembled on top of the monolayer with the working electrode area (0.814 cm2) defined by an O-ring. In some experiments, the working electrode area was decreased using smaller O-rings and/or a silicone mask (Invitrogen). An ohmic contact was formed by etching a small region at the edge of the wafer with 25% (v/v) hydrofluoric acid for 1 min to remove the native oxide and then applying indium gallium eutectic.

Published on Web 10/30/2009

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Vesicle Fusion. DPhPC vesicles of 100 nm diameter were prepared from a 1 mg/mL lipid solution by first evaporating the chloroform under a stream of nitrogen. The lipids were then further dried under vacuum for a minimum of 1 h. A buffer solution of 10 mM sodium phosphate and 100 mM potassium chloride was added to yield a lipid vesicle solution with a final concentration of 1 mg/mL. The solution was vortexed to ensure that all the lipids were suspended in buffer. The vesicles were extruded through an Avanti Mini-Extruder with a 100 nm polycarbonate membrane (Whatman); the solution was passed through the membrane a minimum of 10 times. A total of 400 μL of extruded vesicles was deposited over the PEG-supported lipid monolayer (Figure S1c in the Supporting Information). One hour was allowed for vesicle rupture and fusion to occur to create the lipid bilayer (Figure S1d in the Supporting Information). An additional 10 mL of buffer was added to the electrochemical cell prior to impedance analysis. PEG-Lipid Concentration Range. The concentration of PEG-lipids in the LB monolayers (lower bilayer leaflet) was varied, and the impedance of the bilayers was measured as described below. The concentration of PEG-lipids was varied between 0 and 24 mol %, 4 times the calculated crossover concentration for PEG2K, 5.9 mol %.17 The crossover concentration is the point at which the PEG chains are just touching each other. Below the crossover concentration is the “mushroom” regime where the PEG chains are widely spaced; above the crossover concentration is the “brush” regime where the PEG molecules are closely spaced and extended from the surface, forming the so-called “polymer brush”.17-21 The three PEG concentration regimes are depicted in Figure 1. Electrochemical Impedance Spectroscopy (EIS). Impedance spectroscopy was performed on a Solartron 1286/1255 electrochemical interface/frequency response analyzer. The electrochemical cell was configured with a platinum counter electrode and a Ag/AgCl (3 M NaCl) reference electrode22,23 (Figure S2a in the Supporting Information). All potentials were reported with respect to the Ag/AgCl reference (Ueq = 0.230 V SHE). Impedance spectra were recorded using a 20 mV perturbation at applied potentials from 1.0 to -1.0 V. All experiments were performed at room temperature and in the dark to prevent photoeffects. Impedance spectra were analyzed using a leastsquares fit (ZPlot software) to a circuit consisting of two RC loops in series with a single resistor (Figure S2b in the Supporting Information) to extract values for the membrane resistance, Rm, and capacitance, Cm. The series resistance Rs was on the order of 50 Ω cm2, the charge transfer resistance Rct was on the order of 106 Ω cm2, and the double layer capacitance Cp was on the order of 2 μF cm-2.11 Therefore, the dynamic range for electrical characterization of the supported lipid bilayer on silicon is about 102106 Ω cm2, corresponding to a frequency range of about 10104 Hz (Figure S3 in the Supporting Information). Impedance spectra were recorded for three bilayers at each PEG concentration studied, and the reported membrane resistance and capacitance values were the averages over the applied potential range. Fluorescence Recovery after Photobleaching (FRAP). FRAP was performed on bilayers supported on glass coverslips to assess lipid mobility at the optimal PEG concentration identified by electrical studies. The glass coverslips (Fisher Scientific) were cleaned and surface treated in the same manner as the silicon wafers, and the bilayers were prepared using LB deposition and (17) Kenworthy, A. K.; Hristova, K.; Needham, D.; McIntosh, T. J. Biophys. J. 1995, 68, 1921–1936. (18) deGennes, P. G. Macromolecules 1980, 13, 1069–1075. (19) Alexander, S. J. Phys. (Paris) 1977, 38, 983–987. (20) Milner, S. T.; Witten, T. A.; Cates, M. E. Macromolecules 1988, 21, 2610– 2619. (21) de Gennes, P. G. Adv. Colloid Interface Sci. 1987, 27, 189–209. (22) Oskam, G.; Schmidt, J. C.; Hoffmann, P. M.; Searson, P. C. J. Electrochem. Soc. 1996, 143, 2531–2537. (23) Oskam, G.; Schmidt, J. C.; Searson, P. C. J. Electrochem. Soc. 1996, 143, 2538–2543.

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Figure 1. Schematic depiction of the three concentration regimes of the PEG cushion. Below the crossover concentration is the “mushroom” regime (a). At the crossover concentration (b), the PEG chains are just touching each other. Above the crossover concentration is the “brush” regime (c). vesicle fusion as described above. The LB monolayer consisted of DPhPC and 5.9 mol % PEG-lipids. The vesicle solution consisted of DPhPC and 1 mol % N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)1,2-dihexadecanol-sn-glycerol-3-phosphoethanoamine, triethylammonium salt (NBD-PE) at a concentration of 1 mg/mL in buffer. Two glass coverslips placed back-to-back were submerged vertically into the Langmuir trough to ensure single sided LB deposition on each coverslip. After deposition, the coverslips were separated, cut to size, and positioned with the monolayer facing downward on a dry, cleaned glass slide supported by vacuum grease (Dow Chemical Co.) on the edges to avoid disruption of the monolayer. The vesicle solution was then added to the space between the coverslip and the slide, and incubated for 1 h. After incubation, the remaining free-floating vesicles were thoroughly washed away with deionized water. Photobleaching images were recorded using an Eclipse 600 flourescence microscope (Nikon) equipped with a mercury lamp and a SPOT RT camera (Diagnostic Instruments). Diffusion coefficients were determined using the boundary profile evolution (BPE) method.24 The presence of immobile fluorescent lipids was assessed by comparison of the fluorescence intensity of the bleached spot prior to bleaching and after complete recovery. Bilayer Stability. The stability of electrically optimal PEGsupported DPhPC bilayers assembled via Langmuir-Blodgett deposition/vesicle fusion was determined by measuring impedance spectra in the potential range from 1.0 to -1.0 V every day until the bilayer ruptured. Experiments were conducted at room temperature. The electrochemical cell was covered with parafilm to minimize solvent evaporation.

Results and Discussion Electrochemical Characterization of Bilayers with Different PEG-Lipid Concentrations. In this study, planar surface-supported bilayers were prepared using LB deposition of a monolayer containing different PEG-lipid concentrations, followed by vesicle fusion. A detailed description of the assembly process is given in Materials and Methods and is shown in Figure S1 in the Supporting Information. Prior to LB transfer, pressure-area isotherms were recorded for DPhPC monolayers (24) Merzlyakov, M.; Li, E.; Hristova, K. Langmuir 2006, 22, 1247–1253.

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Figure 2. Pressure-area isotherms of a DPhPC monolayer with different PEG-lipid concentrations.

at each PEG concentration at the air-water interface on the open trough (Figure 2). At this intermediate point in supported bilayer assembly, isotherms provide structural information about the monolayers. At PEG-lipid concentrations above the crossover concentration, a pronounced shoulder was observed in the pressure-area isotherms, which can be attributed to steric interactions (repulsion) between the PEG chains. This shoulder occurs at approximately 15 mN/m, which is consistent with previous results for 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/ PEG-lipid monolayers.13 After completing the supported bilayer via vesicle fusion, measurements were performed to determine its impedance at each PEG-lipid concentration studied. Figure 3 shows the resistance and capacitance versus applied potential for 5.9 mol % PEG-lipid in DPhPC bilayers (triangles). The resistance of DPhPC bilayers with a polymer cushion was independent of the applied potential from 1.0 to -0.8 V. At potentials negative to -0.8 V, the resistance decreased by 1 order of magnitude, suggesting field-induced breakdown of the bilayer. Similar results were obtained for all PEG-lipid concentrations studied. The average capacitance of the bilayer with 5.9 mol % PEG-lipids was 0.9 ( 0.1 μF cm-2 (Figure 3b). The bilayer can be modeled as a parallel plate capacitor with capacitance C = εε0/d, where ε0 is the permittivity of free space, ε is the relative permittivity of a lipid bilayer, and d is the bilayer thickness. Assuming ε=4, we calculated an effective membrane thickness of 4.0 nm, consistent with values reported in the literature.11 Figure 4 shows the average resistance and average capacitance in the potential-independent regime as a function of PEGlipid concentration. The average bilayer resistance (Figure 4a) was about 104 Ω cm2 for PEG-lipid concentrations ranging from 0.5 to 4 times the crossover concentration. We found that the standard deviation in bilayer resistance, obtained from three independently constructed bilayers, was lowest at 5.9 mol %, indicating that stable, reproducible, high resistance bilayers were formed at the crossover concentration. This finding is not surprising: the polymer provides uniform bilayer support near the crossover concentration, as depicted in Figure 1b. At high PEG-lipid concentrations, the average bilayer resistance remained high; however, the reproducibility decreased. For these PEG-lipid concentrations above the crossover, the pressurearea isotherms in Figure 2 indicated strong interactions between the PEG chains in the water subphase, below the DPhPC lipids located at the air-water interface. The presence of the polymer at high concentrations in the so-called “brush regime” (Figure 1c) is expected to induce lateral tension in the bilayer.25 We speculate that these interactions limit the ability of the DPhPC (25) Hristova, K.; Kenworthy, A. K.; McIntosh, T. J. Macromolecules 1995, 28, 7693–7699.

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Figure 3. Resistance (a) and capacitance (b) of DPhPC bilayers constructed with and without a 5.9 mol % PEG cushion. Results presented are experimental means (n = 3).

Figure 4. Resistance (a) and capacitance (b) of PEG-supported DPhPC bilayers with different PEG-lipid concentrations at 0 V. Average values for R and C were obtained from the potentialindependent regime. Each point represents the mean and standard deviation from three independent experiments.

lipids to pack reproducibly into high quality bilayers, which in turn leads to variations in bilayer resistance. In contrast, the average resistance was lower when the PEGlipid concentration was 0.25 times the crossover concentration (Figure 4a). This low resistance suggests the presence of defects in the bilayer. In this case, the average spacing between the PEG lipids (∼7 nm) is larger than the size of the PEG random Langmuir 2010, 26(5), 3544–3548

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coil (∼3.5 nm), such that the bilayer may be nonplanar, as depicted in Figure 1a, and prone to defects. As shown in Figure 4b, the bilayer capacitance was not significantly influenced by the variation in the PEG-lipid concentration. The lowest capacitance was observed at 0.5 and 1 times the crossover concentration. The average capacitance for these two concentrations was 0.87 ( 0.07 μF cm-2, giving an effective membrane thickness of 4.1 nm. At lower and higher PEG concentrations, the bilayer capacitance was slightly higher. This increase may be attributed to an increase in dielectric permittivity due to increased water penetration through the bilayer. In addition, the increase at 0.25 times the crossover concentration may be associated with the larger area of the nonplanar bilayer, as compared to that in the crossover case. Small defects are expected to significantly affect the resistance, but not so much the capacitance. Low values for the capacitance indicate relatively thick bilayers with low dielectric constant. Thus, we identify the crossover concentration as optimal for electrical measurements of the bilayers. This concentration yields bilayers with high reproducible resistance and low capacitance. Interestingly, this PEG-lipid concentration (∼6 mol %) is identical to the concentration that yields the most stable liposomes containing PEG-lipids (5-7 mol %).26 We next determined the impedance response of bilayers without a PEG cushion. Figure 3 compares the bilayer resistance and capacitance without a PEG cushion to the resistance and capacitance in the presence of 5.9 mol % PEG versus the applied potential. Figure 3a shows that the average resistance for bilayers without a PEG cushion was potential-dependent, increasing from about 104 Ω cm2 at -0.4 V, similar to bilayers with a PEG cushion, up to about 105 Ω cm2 at þ0.5 V. The capacitance of the bilayer without a PEG cushion was dependent on potential and was generally higher than the capacitance of bilayers with the PEG cushion (Figure 3b). Since the bilayer without a PEG cushion is in contact with the silicon support, the differences in impedance response of bilayers with and without a PEG cushion can be attributed to the influence of the reservoir between the bilayer and the support. Figure 3 also demonstrates that the reproducibility of the bilayers without a PEG cushion was much lower. Thus, the presence of the PEG cushion ensures the assembly of reproducible bilayers with parameters that do not depend on the applied transmembrane potential. Bilayer Homogeneity. To confirm that the measured bilayer resistances were determined by the bilayer itself and not by edge defects at the perimeter of the electrochemical cell, we varied the working electrode area and measured the impedance as a function of electrode area. Figure 5 shows that the average membrane resistance was independent of electrode area and hence independent of perimeter. This result demonstrates that the bilayer impedance is dominated by the area averaged properties and not by edge defects. Lipid Mobility. To further characterize the electrically optimal bilayer platform, we measured the mobility of fluorescently labeled lipids in the electrically optimized supported DPhPC bilayers using FRAP. Measurements were carried out at the PEG crossover concentration, where the bilayer exhibited the most reproducible resistance (lowest standard deviation) and lowest capacitance. Figure 6a shows fluorescence images after photobleaching a supported DPhPC bilayer containing 1 mol % NBD-PE as the fluorescent probe. From analysis of the intensity profiles (Figure 6b and c),24 we determined a diffusion coefficient (26) Tirosh, O.; Barenholz, Y.; Katzhendler, J.; Priev, A. Biophys. J. 1998, 74, 1371–1379.

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Figure 5. Bilayer resistance at three different working electrode areas. Results presented are experimental means (n = 3).

Figure 6. (a) Sequence of fluorescence images taken at t0 = 0 min, t1 = 2 min, and t2 = 4 min after photobleaching 5.9 mol % PEG2K-supported DPhPC bilayers labeled with 1 mol % NBDPE. Scale bars are 50 μm. (b) Intensity profile from the bleached to unbleached regions plotted from the outlined region and fitted to a Gaussian error function to determine the diffusion depth, w (w2= Dt; see ref 24 for details). (c) Diffusion coefficient D determined from a plot of w2 versus t as 1.5 ( 0.2 μm2/s (R = 0.99 ( 0.01).

of 1.5 ( 0.2 μm2/s (an average of three measurements). This is about a factor of 2 lower than values reported for POPC bilayers (2.3-2.7 μm2/s)24,27 and can be attributed to the interpenetration of the methyl groups of the DPhPC hydrocarbon chains. Similar mobilities were observed in DPhPC bilayers without a PEG cushion (results not shown). Furthermore, the presence of immobile fluorescent lipid fractions was assessed by comparing the fluorescence intensity of the bleached spot prior to bleaching and after complete recovery. No immobile fractions were observed in any of the FRAP experiments. (27) Merzlyakov, M.; Li, E.; Gitsov, I.; Hristova, K. Langmuir 2006, 22, 10145– 10151.

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Figure 7. Resistance (a) and capacitance (b) of two 5.9 mol % PEG-supported DPhPC bilayers at 0.2 V monitored as a function of time, until the bilayers ruptured and no bilayer impedance could be detected. Also shown is one PEG-supported DPhPC bilayer with excess vesicles removed from the electrochemical cell (3).

It has been hypothesized that a polymer cushion will “lift” the bilayer away from the substrate, decrease the lipid-substrate interactions, and, ultimately, increase mobility.2,13,28-30 To directly address this hypothesis, we previously measured and compared the mobility of lipids and incorporated proteins in 160 different POPC bilayers, with various PEG lengths and PEG-lipid concentrations, with PEG either chemically tethered to the surface or physically adsorbed. Surprisingly, we observed that the measured diffusion coefficients do not depend on the PEG molecular weight or the PEG-lipid concentration and are very similar to the values measured in the absence of PEG.27 The results of the current study, which utilized DPhPC instead of POPC, further confirm that the PEG cushion does not affect lipid mobility in supported bilayers assembled via LB deposition/vesicle fusion.27 Time-Dependence of the Electrical Properties of PEGSupported DPhPC Bilayers. We characterized the time-dependence of the electrical properties of the optimized PEG-supported DPhPC bilayers by measuring impedance spectra over an extended period of time. As shown in Figure 7a, in the presence of DPhPC vesicles, the bilayer resistance increased from about 104 Ω cm2 after formation to about 105 Ω cm2 after 1-2 days. The observed increase in bilayer resistance may be attributed to bilayer healing that results from the presence of excess vesicles in the buffer solution. We interpret these findings as an indication of the presence of imperfections in the supported DPhPC bilayer when it is first formed. With excess vesicles available in the buffer solution, additional lipids may have gradually incorporated into the bilayer, thus increasing the overall resistance. In the absence of (28) Munro, J. C.; Frank, C. W. Langmuir 2004, 20, 10567–10575. (29) Heibel, C.; Maus, S.; Knoll, W.; Ruhe, J. Org. Thin Films 1998, 695, 104– 118. (30) Shen, W. W.; Boxer, S. G.; Knoll, W.; Frank, C. W. Biomacromolecules 2001, 2, 70–79.

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excess DPhPC vesicles, we observed no change in the bilayer resistance over the first few days. The capacitance of DPhPC bilayers during the study did not change significantly (Figure 7b); the average capacitance of DPhPC bilayers in the presence of vesicles throughout the study was 0.92 ( 0.12 μF cm-2. In the absence of excess DPhPC vesicles, the average capacitance was 0.94 ( 0.18 μF cm-2. The two values are identical, implying that the presence of the vesicles does not influence the bilayer thickness. For bilayers in the presence of excess vesicles, the resistance remained high for about 20 days. After 20 days, the impedance of the bilayer could not be resolved: there was no inflection point in the Bode plots and only one peak in the phase angle. Both membrane resistance and capacitance were utilized to determine the time of bilayer rupture. First, the decrease in DPhPC bilayer resistance suggested the onset of bilayer rupture. Second, the inability to measure a membrane capacitance confirmed the bilayer’s disappearance. Our results indicated that after 20 days either the bilayer capacitance had increased above the value of the double layer capacitance or the bilayer resistance had decreased to values less than the series resistance. In either case, these results implied that the bilayer had ruptured. Without excess vesicles, bilayers remained stable for about 7 days. Thus, the results shown in Figure 7 provide evidence for “annealing” of PEG-supported DPhPC bilayers in buffer containing DPhPC vesicles. This annealing process leads to a 1 order of magnitude increase in resistance, up to about 105 Ω cm2. Furthermore, these bilayers exhibited high resistance for extended periods, nearly 3 weeks. This stability is exceptional when compared with free-standing membranes. Longer stabilities have been reported for bilayers that are chemically tethered to the substrate,31 but the chemical tethering may impair mobility of lipids and incorporated proteins. Thus, the bilayer platform characterized here combines stability, high lateral mobility, and excellent electric response.

Conclusions In this Article, we have investigated how the PEG cushion affects the electrical properties of a supported DPhPC bilayer platform that is being developed for studies of membrane proteins and ion channels in particular. We have identified the optimal concentration for PEG-lipids that maximizes the bilayer resistance while maintaining a low, physiologically relevant capacitance. Furthermore, we have demonstrated high lipid mobility as well as bilayer homogeneity and long-term stability for this platform. The described optimization of this biomimetic membrane platform will aid future studies of ion channels and other pore forming membrane proteins. Acknowledgment. Supported in part by NSF MCB 0718841. J.L. acknowledges support from the NSF IGERT NanoBio training grant. Supporting Information Available: Schematic depictions of the bilayer assembly process and the electrochemical setup, as well as examples of impedance and phase angle data. This material is available free of charge via the Internet at http://pubs.acs.org. (31) Vockenroth, I. K.; Ohm, C.; Robertson, J. W. F.; McGillivray, D. J.; Losche, M.; Koper, I. Biointerphases 2008, 3, FA68–FA73.

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