12054 DOI: 10.1021/la101084b Langmuir 2010, 26(14), 12054–12059Published on Web 05/06/2010

pubs.acs.org/Langmuir

© 2010 American Chemical Society

Electrically Addressable, Biologically Relevant Surface-Supported Bilayers

Janice Lin, John Szymanski, Peter C. Searson,* and Kalina Hristova*

Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland 21218

Received March 17, 2010. Revised Manuscript Received April 22, 2010

The assembly of electrically addressable, planar supported bilayers composed of biologically relevant lipids, such asthose used in vesicular systems, will greatly enhance the experimental capabilities in membrane and membrane proteinresearch. Here we assess the electrical properties of bilayers composed of a wide range of physiologically relevant lipidsand lipid combinations. We demonstrate that robust, biologically relevant, planar supported bilayers with highresistance composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 25 mol % cholesterol can beconstructed with high reproducibility. Furthermore, to enable studies of pore-forming peptides, which are commonlycationic, we demonstrate the construction of bilayers with biologically relevant outer leaflets incorporating up to 10 mol %negatively charged lipids. Unique features of the platform are that (1) the substrate is commercially available, atomicallysmooth, single-crystal silicon, (2) the polymer cushion allows for the natural incorporation of membrane proteins, and(3) the platform is highly reproducible.

Introduction

Over the past 25 years, researchers have developed and utilizedsurface-supported bilayers as model systems for studying thestructure, assembly, and function of biological membranes.1,2

These model structures are especially well suited for studies oftransport properties through incorporated pores or channels3

because they can be constructed on planar conducting substratesand the transmembrane potential can be easily controlled. Vari-ous approaches have been utilized to create bilayers for electricalcharacterization using self-assembly,4,5 Langmuir-Blodgett(LB),6 and Langmuir-Schaffer (LS) techniques.7 Further modi-fications include the incorporation of a polymer cushion ofpoly(ethylene glycol) (PEG) to raise the bilayer from the substrateand allow room for the natural conformation of the extramem-branous regions of membrane proteins.7,8

Electrochemical impedance spectroscopy (EIS) is widely usedto characterize ion channels and pores in supported bilayers. Tomaximize the dynamic range for the characterization of transportthrough open ion channels, the surface-supported bilayers musthave a highmembrane resistance. This requirement often dictatesthat particular types of lipids suitable for electrochemical characte-rization are used, such as 1,2-diphytanoyl-sn-glycero-3-phosphocho-line (DPhPC)4,5 or 1,2-dimyristoyl-sn-glycero-3-phospho-choline (DMPC).7 DPhPC and DMPC, however, are not foundin mammalian cells and are thus not physiologically relevant.Furthermore, the use of these lipids precludes comparison tocomplementary biophysical studies of vesicular systems composed

of biologically relevant lipids. Although vesicular systems are notsuitable for electrical measurements, they are widely used for thestructural and thermodynamic characterization of membrane-associated proteins. Therefore, the assembly of electrically addres-sable bilayers composed of biologically relevant lipids, such asthose used in vesicular systems, will greatly enhance the experi-mental capabilities in membrane and membrane protein research.

Here we assess the electrical properties of bilayers composed ofa wide range of physiologically relevant lipids and lipid combina-tions (Table 1).We demonstrate that biologically relevant, planarsupported bilayers with high resistance and high reproducibilitycomposed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC) and 25 mol % cholesterol can be constructed. Moreimportantly, to enable studies of pore-forming peptides, whichare commonly cationic, we demonstrate the construction ofbilayers with biologically relevant outer leaflets incorporatingup to 10 mol % negatively charged lipids. The unique features ofthis platform are that (1) the substrate is commercially available,atomically smooth, single-crystal silicon, (2) the polymer cushionallows for the natural incorporation of membrane proteins, and(3) the platform is highly reproducible.

Materials and Methods

Silicon Substrate Preparation. Single-side-polished, n-typesilicon wafers (Æ111æ, F = 0.001-0.005 Ω cm, Silicon QuestInternational) were cleaned by sonication for 15 min first inisopropyl alcohol, then in acetone, and then again in isopropylalcohol. Each silicon wafer was rinsed thoroughly in deionizedwater before surface treatment in a 30% (v/v) hydrogen peroxide,70% (v/v) sulfuric acid solution for 1 h.The siliconwaferwas thenrinsed thoroughly in deionized water and used within 1 h. Thesilicon wafer was submerged vertically into a Langmuir trough(Nima Technologies) for LB deposition.

Langmuir-Blodgett Deposition. A 1 mg mL-1 solution oflipids (Avanti Polar Lipids) and 5.9 mol % PEG2K lipids (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy(poly-(ethylene glycol))-2000], Avanti Polar Lipids) was prepared inchloroform. Twenty-five microliters of the lipid solution wasdeposited at the air-water interface on theLangmuir trough.Afterallowing aminimumof 30min for the chloroform to evaporate, thelipids were compressed to a surface pressure of 32 mN m-1. The

*To whom correspondence should be addressed: searson@jhu.edu andkh@jhu.edu(1) Sackmann, E. Science 1996, 271, 43–48.(2) Sackmann, E.; Tanaka, M. Trends Biotechnol. 2000, 18, 58–64.(3) Vockenroth, I. K.; Atanasova, P. P.; Long, J. R.; Jenkins, A. T.; Knoll, W.;

Koper, I. Biochim. Biophys. Acta 2007, 1768, 1114–1120.(4) Atanasov, V.; Knorr, N.; Duran, R. S.; Ingebrandt, S.; Offenhausser, A.;

Knoll, W.; Koper, I. Biophys. J. 2005, 89, 1780–1788.(5) 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.(6) Nikolov, V.; Lin, J.; Merzlyakov, M.; Hristova, K.; Searson, P. C. Langmuir

2007, 23, 13040–13045.(7) Chen, M. H.; Li, M.; Brosseau, C. L.; Lipkowski, J. Langmuir 2009, 25,

1028–1037.(8) Wagner, M. L.; Tamm, L. K. Biophys. J. 2000, 79, 1400–1414.

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siliconwafer was then raised vertically out of the trough at a rate of15 mm min-1, allowing the deposition of a PEG-supported lipidmonolayer on the polished side of the silicon wafer.

A custom Teflon electrochemical cell was assembled on top ofthe monolayer with the working electrode area (0.814 cm2)defined by an O-ring. An ohmic contact was formed by etchinga small regionat the edgeof thewaferwith 25%(v/v) hydrofluoricacid for 1 min to remove the native oxide and then applying anindium gallium eutectic.

Vesicle Fusion. Vesicles (100 nm diameter) were preparedfrom a 1 mg mL-1 lipid solution by first evaporating the chloro-form under a stream of nitrogen. The lipids were then furtherdried under vacuum for a minimum of 1 h. A buffer solution of10 mM sodium phosphate and 100 mM potassium chloride wasadded to yield a lipid vesicle solutionwith a final concentration of1mgmL-1. The solutionwas vortexmixed to ensure that all of thelipids were suspended in buffer. The vesicles were passed throughan extruder (Avanti Mini-Extruder) with a 100 nm polycarbo-nate membrane (Whatman) a minimum of 10 times. The PEG-supported lipidmonolayerwas incubatedwith 400 μLof extrudedvesicles for at least 1 h to allow for vesicle rupture and fusion tooccur and complete the lipid bilayer. An additional 10 mL ofbuffer was added to the electrochemical cell prior to impedanceanalysis. Experiments were performed on supported bilayersstored in buffer at room temperature in the dark without remov-ing the excess vesicles.

Incorporation of Negatively Charged Lipids. Lipid vesicleswere composed primarily of either DPhPC or 25 mol % choles-terol and POPC. Two negatively charged lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(10-rac-glycerol) (POPG), were eachseparately incorporated into lipid vesicles in increments of 5mol %. POPS has a phosphate and a terminal serine moiety withcharged carboxylic acid and amine groups; POPG has a terminalglycerol moiety and hence has only a charged phosphate in theheadgroup.

Electrochemical Impedance Spectroscopy (EIS). EIS wasperformed using a Solartron 1286/1255 electrochemical interface/frequency response analyzer. The electrochemical cell was con-figured with a platinum counter electrode and a Ag/AgCl (3 MNaCl) reference electrode. All potentials were reported withrespect to the Ag/AgCl reference (Ueq=0.230 V SHE). Impe-dance spectra were recorded using a 20 mV perturbation atapplied potentials from 0.5 to -0.5 V. All experiments wereperformed in 10 mM sodium phosphate and 100 mM potassium

chloride buffer solution at room temperature in the dark toprevent photoeffects. Using a nonlinear least-squares fit (ZPlot,Scribner Associates), impedance spectra were fit to a circuitconsisting of two RC loops in series with a single resistor(Figure 1) to extract values for the membrane resistance, Rm,and capacitance, Cm.

9,10 A key requirement for detecting themembrane impedance is that the membrane capacitance Cm isless than the capacitance of the electrode interface Cp. This isachieved by using highly doped silicon.9 For each unique lipidbilayer composition, impedance spectra were recorded for threeindependently constructed bilayers after 1 h of vesicle incubation.Student t tests were performed to determine the statistical sig-nificance (R=0.05). Finally,we characterized the bilayer stabilityby measuring the bilayer’s impedance until it ruptured.

Results and Discussion

All surface-supported bilayers were formed by LB depositionfollowedby vesicle fusion (VF).DPhPCbilayers are considered to

Table 1. Summary of Mean Capacitances and Resistances of DPhPC and POPC Bilayer Compositions with Cholesterol and Negatively Charged

Lipids (POPS and POPG) Incorporated in Varying Molar Concentrations

bilayer composition

lower leafleta upper leaflet capacitance (F cm-2) resistance (Ω cm2) bilayerb

DPhPC DPhPC 8.8� 10-7( 1.1� 10-7 7421( 1587√

DPhPC, 5% POPG 8.9� 10-7( 3.8� 10-8 10 600 ( 3411√

DPhPC, 10% POPGDPhPC, 5% POPS 9.7� 10-7( 1.2� 10-8 2240( 1033DPhPC, 10% POPSDPhPC, 5% POPC 1.0� 10-6( 5.6� 10-8 27 400( 8036

√

POPC, 25% CH 1.0� 10-6( 1.8� 10-8 2641( 1232√

c

POPC, 25% CH, 5% POPS 1.3� 10-6( 2.2� 10-7 19 200 ( 16817POPC, 25% CH, 10% POPS 1.2� 10-6( 1.0� 10-7 13 800 ( 665

√

POPC, 25% CH, 15% POPSPOPC, 25% CH, 5% POPG 9.1� 10-7( 3.7� 10-8 23 800( 8393

√

POPC, 25% CH, 10% POPGPOPC POPC 1.2� 10-6( 2.1� 10-8 855( 490POPC, 25% CH POPC, 25% CH 1.1� 10-6( 2.5� 10-7 8550( 2603

√

POPC, 25% CH, 5% POPSPOPC, 25% CH, 10% POPS

Data reported here are from impedance measurements taken after 1 h of vesicle incubation. aAll lower leaflet compositions included5.9 mol% PEG2K-lipids to serve as the polymer cushion. bA qualitative assessment is made of whether an electrically addressable bilayerwith experimental utility could be formed from each composition. cDPhPC lower leaflet with POPC, 25 mol % cholesterol upper leafletbilayers attained high membrane resistance, approximately 7� 104 Ω cm2, after a few days.

Figure 1. Schematic of the Langmuir-Blodgett/vesicle fusionPEG-supported bilayer construct and the EIS three-electrode(counter electrode, reference electrode, and working electrode)experimental setup. To the right is the electrical circuit model ofa surface-supported bilayer to which the impedance and phase-angle data was fit. It consists of an RC loop associated with thebilayer in series with an RC loop associated with the siliconelectrode interface. The solution resistance Rs is on the order of50 Ω cm2, the semiconductor charge-transfer resistance Rct is onthe order of 106 Ω cm2, and the double-layer parallel capacitanceCp is approximately 2-4 μF cm-2. Rm and Cm are the membraneresistance and capacitance, respectively.

(9) Lin, J.; Merzlyakov, M.; Hristova, K.; Searson, P. C. Biointerphases 2008, 3,33–40.

(10) Orazem, M. E.; Tribollet, B. Electrochemical Impedance Spectroscopy; JohnWiley & Sons: Hoboken, NJ, 2008.

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be ideal for electrical measurements because the intercalation ofmethyl groups on the acyl chains results in high electricalresistance, thereby maximizing the dynamic range for studies ofmembrane protein channels and pores. We thus started withDPhPC and took a sequential approach to developing a biologi-cally relevant bilayer platform. We first incorporated a neutral,zwitterionic lipid (POPC) and negatively charged lipids (POPGand POPS) into the upper leaflet of DPhPC bilayers. This greatlyenhances the versatility of the platform because POPG lipidscomprise approximately 25% of bacterial membranes whereasPOPS is the predominant negatively charged lipid in mammalianmembranes.11-13 Negatively charged lipids in concentrations ofup to about 10 mol % are commonly found in cell membranes,and they play a key role in the specific electrostatic targeting ofantibiotics and pore-forming peptides to bacterial membranes.POPC is a ubiquitous lipid found in mammalian cell membranesthroughout the body. Second, we considered a bilayer with aDPhPC lower leaflet and a POPC upper leaflet with the additionof cholesterol, POPG, or POPS. Third, we investigated POPC-based bilayers. Finally, we summarized the stability of thesebilayers and identified the optimal, biologically relevant bilayerplatforms for specific applications.DPhPCBilayers IncorporatingPOPG,POPS, orPOPC

in the Upper Leaflet. Figure 2 shows representative impedanceand phase-angle spectra for DPhPC bilayers containing 5 mol %POPC, POPG, or POPS in the upper leaflet and supported by a

5.9 mol % PEG-lipid cushion. The corresponding spectra forDPhPC bilayers are provided for comparison. The characteristicsignature of a robust bilayer is an inflection point in the Bode plot(Figure 2a) separating two capacitive relaxations. The higher-frequency component is due to the bilayer, and the lower-frequency component is derived from the silicon interface.9 Thetime constants for the two relaxations are clearly seen in the phaseangle plot (Figure 2b). The addition of 5mol%POPC, POPG, orPOPS to the DPhPC upper leaflet results in spectra with clearlydefined membrane impedance at high frequencies (∼10-102 Hz),indicative of the formation of a high-resistance bilayer. At theseconcentrations, the construction of these bilayers using LB/VFwas very reproducible. The incorporation of greater than 5mol%POPG or POPS did not yield observable membrane impedance,suggesting the formation of defective bilayers. However, theincorporation of up to 10 mol % POPC in the upper leaflet stillyielded very robust, reproducible bilayers.

Figure 3 shows the resistance and capacitance versus appliedpotential for these bilayers, extracted from the impedance spectrameasured after 1 h of vesicle incubation. The average resistance ofstandard DPhPC bilayers at 0 V was 9.0( 1.4 � 103 Ω cm2. Themembrane resistance is very reproducible and is very close tovalues reported previously.14We first investigated the influence ofpalmitoyl acyl chains on DPhPC lipid packing by incorporating5 mol % POPC, a zwitterionic lipid with the same acyl chains asPOPG and POPS, into the upper leaflet. The result was a signi-ficant increase in bilayer resistance up to 2.7 ( 0.8 � 104 Ω cm2

(p = 0.03).

Figure 2. Impedance Z (a) and phase-angle θ (b) data and fits of5.9 mol % PEG-lipid supported bilayers of DPhPC incorporating5 mol% POPC, POPG, or POPS in the upper leaflet as a functionof frequency at 0 V. The impedance was measured after 1 h ofvesicle incubation. The dynamic range for electrical characteriza-tion of the supported lipid bilayer on silicon is about 102-106

Ω cm2, corresponding to a frequency range of about 10-104 Hz.The impedance spectra show two capacitive regimes, one at higherfrequencies associatedwith the bilayer andone at lower frequenciesassociated with the electrode interface. The phase-angle minimaoccur at the same frequency at which the membrane resistance ismeasured.

Figure 3. Resistance (a) and capacitance (b) of DPhPC bilayersincorporated with 5 mol % POPS, 5 mol % POPG negativelycharged lipids, or 5 mol% POPC in the upper leaflet as a functionof potential. The impedance was measured after 1 h of vesicleincubation. Values shown are experimental means (n= 3).

(11) Dowhan, W. Annu. Rev. Biochem. 1996, 66, 199–232.(12) Nomura, K.; Inaba, T.; Morigaki, K.; Brandenburg, K.; Seydel, U.;

Kusumoto, S. Biophys. J. 2008, 95, 1226–1238.(13) Spector, A. A.; Yorek, M. A. J. Lipid Res. 1985, 26, 1015–1035.

(14) Lin, J.; Szymanski, J.; Searson, P. C.; Hristova, K. Langmuir 2010, 26,3544–3548.

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This result was unexpected because the steric mismatch be-tween the palmitoyl and diphytanoyl acyl chains is expected todisrupt the lipid packing, thus decreasing membrane resistancerelative to that of DPhPC bilayers. However, after a few days theDPhPC membrane resistance increased to nearly 105 Ω cm2 andthe DPhPC membrane with 5 mol % POPC showed a very smallincrease (Figure 4a). In previous work, we attributed the increasein resistance with time to a bilayer annealing process where lipidsfrom excess vesicles sealed up imperfections in the bilayer.14

Bilayers in buffer without vesicles did not show any increase inresistance with time. We thus propose that the addition of POPCto DPhPC vesicles increased the rate of bilayer formation andannealing by facilitating vesicle fusion.

Next, we incorporated a negatively charged lipid, POPG orPOPS, into the upper leaflet of DPhPC bilayers. As seen inFigure 3a, the incorporation of either 5 mol % POPG or POPSinto the upper leaflet resulted in significant decreases in mem-brane resistance compared to thatmeasured in the 5mol%POPCsystem (p=0.01 and 0.02). We propose that the decrease inmembrane resistance upon addition of POPG or POPS is likelydue to charge repulsion between the headgroups: POPC is azwitterionic lipid whereas POPG and POPS are negativelycharged. This hypothesis is supported by molecular dynamicssimulations that demonstrate a decrease in the electrostaticpotential and hence an increase in the permeability of POPGbilayers as compared to those of POPC bilayers.15

Furthermore, the membrane resistances of both 5 mol %POPG, DPhPC and 10 mol % POPS, DPhPC bilayers changedonly minimally with time, suggesting that their maximum resis-tances had been attained by the first day (Figure 4a). These resultssuggest that the kinetics of membrane resistance developmentdepends on the lipid composition of the vesicles that fuse to createthe upper leaflet. Vesicles composed of onlyDPhPC require more

time for the vesicle fusion and bilayer annealing process to go tocompletion whereas doping these vesicles with (1) a lipid with adifferent acyl chain or (2) a negatively charged lipid increased therate of these processes such that the bilayer’s maximum resistancewas attained in the first 24 h.

The average capacitance of standardDPhPCbilayerswas 0.9(0.1 μF cm-2 (Figure 3b). Electrically, the bilayer can be modeledas a parallel plate capacitor with capacitanceC= εεo/d, where ε isthe relative permittivity of the lipid bilayer, εo is the permittivityof free space, and d is the bilayer thickness. Taking ε = 4,16 wecalculate an effective membrane thickness of 4.0 nm, consistentwith values reported in the literature.4,16,17 The addition of 5mol%POPC, POPG, or POPS to the outer leaflet minimally increasedthe capacitance up to about 1.0 μF cm-2. The slight increase incapacitance may be attributed to a small increase in relativepermittivity due to increased water penetration associated withthe steric mismatch between the palmitoyl and diphytanoyl acylchains and/or the introduction of charge into the bilayer, in thecase of POPG and POPS.

The capacitance of bilayers with negatively charged lipidschanged only minimally with time (Figure 4b). We observed aslight decrease in capacitance after 1 or 2 days, which suggests aslight increase in bilayer thickness or a decrease in permittivityassociated with bilayer annealing.Bilayers with DPhPC Lower Leaflets and POPC/Cho-

lesterol-Based Upper Leaflets. The second step in our devel-opment of a physiologically relevant platform was to form ahybrid bilayer with an upper leaflet composed of POPC and25 mol % cholesterol and a lower leaflet composed of DPhPC.This asymmetric bilayer presents a physiologically relevant outerleaflet suitable for peptide binding studies that also maintains afoundation for high membrane resistance in the lower leaflet.Cholesterol was included because it is a main component ofmembranes and it is known to impart both structural rigidity andcohesive flexibility to cellular membranes and is integral tosignaling pathways.18,19 Cholesterol is structurally smaller thanphospholipids and can potentially increase the packing density ofPOPC bilayers;20 thus it is expected to create a more cohesivebilayer with higher resistance. However, the resistance of thisasymmetrical bilayer decreased to 2.6( 1.2� 103Ω cm2, which isabout 3 times lower than that measured for DPhPC-only bilayersafter 1 h of vesicle incubation (Figure 5a).

After 3 days, the membrane resistance increased from approxi-mately 2.6� 103 to approximately 7.1� 104 Ω cm2 in POPC and25 mol % cholesterol upper leaflet, DPhPC lower leaflet bilayers(Figure 6a). In this case, we propose that the addition ofcholesterol to POPC vesicles retards vesicle fusion kinetics, thusslowing down the rate of bilayer formation and annealing. Notethat the high resistance observed after 3 days also suggests thatcholesterol created a highly cohesive, high-resistance bilayer withPOPC lipids, resulting in a physiologically relevant and electri-cally comparable platform to DPhPC-only bilayers.

The additional integration of negatively charged lipids into thezwitterionic asymmetrical bilayer increased the membrane resis-tance measured after 1 h of vesicle incubation (Figure 5a). POPS(10mol%) in the upper leaflet increased themembrane resistanceto 1.4( 0.1� 104Ω cm2, and the incorporation of 5mol%POPG

Figure 4. Development of resistance (a) and capacitance (b) ofDPhPC bilayers incorporated with 5 mol % POPS, POPG, orPOPC in the upper leaflet with time. The time evolution fromDPhPC-only bilayers is provided as a reference.14

(15) Zhao, W.; Rog, T.; Gurtovenko, A. A.; Vattulainen, I.; Karttunen, M.Biophys. J. 2007, 92, 1114–1124.

(16) Purrucker, O.; Hillebrandt, H.; Adlkofer, K.; Tanaka, M. Electrochim.Acta 2001, 47, 791.

(17) Gritsch, S.; Nollert, P.; Jahnig, F.; Sackmann, E. Langmuir 1998, 14, 3118.(18) Needham, D.; Nunn, R. S. Biophys. J. 1990, 58, 997–1009.(19) Rawicz, W; Smith, B. A.; McIntosh, T. J.; Simon, S. A.; Evans, E. Biophys.

J. 2008, 94, 4725–4736.(20) Pandit, S. A.; Chiu, S.W.; Jakobsson, E.; Grama, A.; Scott, H. L.Langmuir

2008, 24, 6858–6865.

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further increased themembrane resistance to 2.6( 0.8� 104Ω cm2.Larger concentrations of POPS or POPG destabilized the mem-brane such that no bilayer could be detected by impedancespectroscopy. This supports the hypothesis that doping vesicleswith negatively charged lipids enhanced vesicle fusion kinetics,regardless of the presence of cholesterol. Furthermore, we againobserved only a minimal increase in membrane resistance withtime for negatively charged 10 mol % POPS, 25 mol % choles-terol, and the POPC upper leaflet, DPhPC lower leaflet bilayer(Figure 6a). Unlike in the previous case of POPG or POPS inDPhPC bilayers, the membrane resistance instead increased intheir presence in the POPC and 25 mol % cholesterol upperleaflet; this suggets that cholesterol in the membrane was able tocounteract some of the effects of charge repulsion.

Theaverage capacitanceof the asymmetrical POPCand25mol%cholesterol upper leaflet, DPhPC lower leaflet bilayer was 1.0(0.02 μF cm-2, which is only slightly higher than that of aDPhPC-only bilayer (Figure 5b). This may be attributed to aslight increase in relative permittivity due to a slight decreasein packing efficiency and increased water penetration in the25 mol % cholesterol and POPC upper leaflet as compared tothose in the DPhPC leaflet. With the addition of 5 mol % POPG,the membrane capacitance was comparable to DPhPC-only bi-layers. However, with the addition of 10 mol % POPS, thecapacitance increased significantly to 1.2 ( 0.1 μF cm-2 (p =0.03), suggesting either bilayer thinning or increased relativepermittivity that was potentially due to the increased permeabilityand local defects arising from the aforementioned electrostaticrepulsion in the headgroups. The capacitance of negatively charged

bilayers changed only minimally with time (Figure 6b). We againobserved a slight decrease in capacitance after 1 or 2 days, whichsuggests a slight increase in overall bilayer thickness or a change inpermittivity.POPC-BasedBilayers.The third step in our explorationwas

to characterize symmetrical POPC-based bilayers in order toobtain baseline characteristics before the addition of negativecharge to a biologically relevant platform. The resistance ofPOPC-only bilayerswas about 103Ω cm2, which is about anorderof magnitude lower than the resistance of the DPhPC-onlybilayers (Figure 7). The difference in resistance between DPhPCand POPC bilayers is likely due to the different structure of theacyl chains. As discussed above, themethyl groups on theDPhPCchains have a propensity for entanglement with adjacent chains,resulting in high membrane resistance due to tighter packingin the hydrophobic core.4 In contrast, POPC lipids have oneunsaturated, and therefore kinked, chain that precludes cohesivepacking with each other.

The addition of 25mol% cholesterol to POPCbilayers resultedin membrane resistances comparable to those of DPhPC-onlybilayers (Figure 7). Furthermore, the electrical characteristics ofthese membranes were very reproducible, as can be seen from thesmall standard deviation inmembrane resistance and capacitance.This demonstrates an uncharged, biologically relevant alternativeto DPhPC bilayers. The capacitances of the DPhPC and POPCbilayers were ∼0.9 and ∼1.2 μF cm-2, respectively. Both bilayersare about 5 nm thick and have a hydrophobic core that is 2.6 to2.7 nm thick.21,22 The slightly larger capacitance of the POPCbilayers compared to that of the DPhPC bilayers is likely due to asmall increase in relative permittivity arising from the increasein area per molecule, and hence a larger water content, in POPC

Figure 5. Resistance (a) and capacitance (b) of asymmetricalbilayers composed of POPC and 25 mol % cholesterol incorpo-rated with 10 mol % POPS or 5 mol % POPG negatively chargedlipids in the upper leaflet as a function of potential. The lowerleaflets were composed of DPhPC lipids with 5.9 mol % PEG2Klipids. Impedancewasmeasuredafter 1 h of vesicle incubation.Thevalues shown are experimental means (n= 3).

Figure 6. Development of resistance (a) and capacitance (b) ofasymmetric bilayers composed of POPC and 25mol% cholesterolincorporated with 10 mol % POPS in the upper leaflet with time.The time evolution from DPhPC-only bilayers is provided as areference.14

(21) Hung, W. C.; Chen, F. Y.; Huang, H. W. Biochim. Biophys. Acta 2000,1467, 198–206.

(22) Kucerka, N.; Tristram-Nagle, S.; Nagle, J. F. J. Membr. Biol. 2005, 208,193–202.

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bilayers. Similarly, the decrease in capacitance from 1.2 to1.1 μF cm-2 upon addition of cholesterol to POPC bilayers islikely due to a decrease in relative permittivity.

POPC bilayers with 5 and 10 mol % cholesterol also exhibitedhigh membrane resistance (data not shown) but were susceptibleto electroporation in the potential range from 0.5 to -0.5 V (vsAg/AgCl). Cholesterol aggregation in cellular membranes, theformation of lipid rafts,23 and other hetereogeneities can poten-tially counteract the effects of increased cohesiveness and lead todifficulties in reproducibility. Therefore, there appeared to be aminimum concentration of cholesterol between 10 and 25 mol %required to achieve POPC bilayers with high resistance that maybe utilized over the same operating regime as DPhPC bilayers.

The final step inour development of an electrically addressable,biologically relevant supported bilayer platform was to integratenegatively charged lipids into bilayers comprising POPC and25 mol % cholesterol. However, our attempts to create such aplatform were unsuccessful because even low concentrations ofeither POPS or POPG resulted in impedance spectra where themembrane impedance could not be resolved.Summary of Bilayer Performance and Stability. A sum-

mary of membrane resistances and capacitances measured for allbilayer compositions is presented in Table 1. As discussedpreviously, for transport studies through membrane proteins, asurface-supported bilayer platform should have a high resistancetomaximize the dynamic range and should have a physiologically

relevant capacitance in the range of about 0.8-1.0 μF cm-2. Onthe basis of the results presented here, a symmetric POPC and25 mol % cholesterol bilayer provides the optimal, most physio-logically relevant platform with only zwitterionic lipids. Forstudies where the incorporation of charged lipids is desirable,the optimal platform is an asymmetrical bilayer with POPC,25 mol % cholesterol, a 5 mol % POPG upper leaflet, and aDPhPC lower leaflet.

One question that may arise for asymmetrical bilayers iswhether lipid equilibration between the two leaflets may be theunderlying cause of the observed kinetics. In vivo lipid flip-flopacross leaflets is controlled andminimized by lipid translocases;24

however, in biomimetic-supported bilayer systems, this mechan-ism cannot be prevented. It has been shown that using the LB/VFtechnique to create PEG-supported lipid bilayers, as opposed tousing LB/LS to create bilayers without a PEG cushion, retainedthe lipid asymmetry of a bilayer for about 2 h after its construction.25

With time, this phenomenon of lipid equilibration is likely occurringin thebilayerspresentedhere such that lipidsother thanDPhPCmayintegrate into the lower leaflet. If this is functionally significant, thenwe should observe a decrease in membrane resistance due to thecreation of short-circuit paths across the bilayer. However, withinthe first five days we observed only stable or increasing membraneresistances, suggesting that this phenomenon does not negativelyimpact the electrical properties of these bilayers.

Finally, note that all bilayers with lipid compositions that wereelectrically addressable were stable for a minimum of 5 days.Symmetric DPhPC bilayers were previously observed to be stablefor more than 3 weeks,14 so it appears that the incorporation ofnegative charge decreased the long-term stability of supportedbilayers. Nonetheless, the observed stability is exceptionally longwhen compared to that of black lipid membranes (BLMs), whichtypically rupture within several hours.26

Conclusions

Although DPhPC bilayers are commonly used as a platformfor electrochemical studies, it is uncommon to find them beingutilized in many biochemical and biophysical studies. Here wedescribed electrically addressable, biologically relevant bilayersthat incorporate (1) POPCand 25mol%cholesterol and (2) up to5 mol % POPG and 10 mol % POPS negatively charged lipids.We demonstrated that bilayers of POPC and 25 mol % choles-terol, a composition thatmimics themammalianmembrane, havethe same electrical characteristics as DPhPC bilayers. For studiesof soluble, cationic, pore-forming peptides such as antibiotics andother cell-penetrating peptides, we demonstrated electricallyaddressable negatively charged bilayers with biologically relevantupper leaflets that can be utilized to attract these peptideselectrostatically to the membrane interface. Thus, POPC andcholesterol bilayers produced via LB/VF deposition on single-crystal silicon are a suitable model system for studies of processesinvolving ion channels and pore-forming peptides in an environ-ment that closely mimics biological membranes.

Acknowledgment. We gratefully acknowledge support fromNSF MCB 071881 and NSF IGERT 0549350.

Figure 7. Resistance (a) and capacitance (b) of symmetricalDPhPC and POPC as well as POPC with 25 mol % cholesterolbilayers as a functionof potential. Impedancewasmeasured after 1h of vesicle incubation. The values shown are experimental means(n= 3).

(23) Lingwood, D.; Simons, K. Science 2010, 327, 46–50.

(24) Daleke, D. L. J. Lipid Res. 2003, 44, 233–242.(25) Kiessling, V.; Wan, C.; Tamm, L. K. Biochim. Biophys. Acta 2009, 1788,

64–71.(26) Anrather, D.; Smetazko, M.; Saba, M.; Alguel, Y.; Schalkhammer, T.

J. Nanosci. Nanotechnology 2004, 4, 1–22.