AFM Studies of the Effect of Temperature and Electric Field on the

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Langmuir 2009, 25, 1028-1037

AFM Studies of the Effect of Temperature and Electric Field on the Structure of a DMPC-Cholesterol Bilayer Supported on a Au(111) Electrode Surface Maohui Chen, Ming Li, Christa L. Brosseau, and Jacek Lipkowski* Department of Chemistry, UniVersity of Guelph, Guelph, Ontario, Canada N1G 2W1 ReceiVed August 29, 2008. ReVised Manuscript ReceiVed NoVember 5, 2008 Atomic force microscopy (AFM) was used to characterize a phospholipid bilayer composed of 70 mol % 1,2dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 30 mol % cholesterol, at a Au(111) electrode surface. Results indicate that addition of cholesterol relaxes membrane elastic stress, increases membrane thickness, and reduces defect density. The thickness and thermotropic properties of the mixed DMPC-cholesterol bilayer supported at the gold electrode surface are quite similar to the properties of the mixed membrane in unilamellar vesicles. The stability of the supported membrane at potentials negative to the potential of zero charge Epzc was investigated. This study demonstrates that the bilayer supported at the gold electrode surface is stable provided the applied potential (E - Epzc) is less than -0.3 V. At larger polarizations, swelling of the membrane is observed. Polarizations larger than -1 V cause electrodewetting of the bilayer from the gold surface. At these negative potentials, the bilayer remains in close proximity to the metal surface, separated from it by a ∼2 nm thick layer of electrolyte.

Introduction Phospholipid bilayers supported on planar solid surfaces serve as popular models of biomimetic membranes to study protein adsorption, peptide-membrane interactions, ion transport, lateral membrane inhomogeneities, phase transitions, and mechanical membrane properties.1-5 Such supported bilayer membranes (sBLMs) can be formed at surfaces of a variety of materials including glass, silicon, silicon nitride, quartz, and mica, using chiefly either a combination of Langmuir-Blodgett and Langmuir-Schaefer (LB-LS) deposition1,6 or vesicle fusion.1,7-16 * Corresponding author. (1) (a) Sackmann, E. Science 1996, 271, 43. (b) Tanaka, M.; Sackmann, E. Nature 2005, 437, 656. (c) Tanaka, M.; Sackmann, E. Phys. Status Solidi 2006, 203, 3452. (2) Williams, L. M.; Evans, S. D.; Flynn, T. M.; Marsh, A.; Knowles, P. F. R.; Bushby, J.; Boden, N. Langmuir 1997, 13, 751. (3) Johnston, L. J. Langmuir 2007, 23, 5886. (4) Schmidt, A.; Spinke, J.; Bayerl, T.; Sackmann, E.; Knoll, W. Biophys. J. 1992, 63, 1385. (5) Schuy, S.; Janshoff, A. Chem. Phys. Chem. 2006, 7, 1207. (6) (a) Tamm, L. K.; McConnel, H. M. Biophys. J. 1985, 47, 105. (b) Crane, J. M.; Tamm, L. K. Biophys. J. 2004, 86, 2965. (c) Stottrup, B. J.; Veach, S. L.; Keller, S. L. Biophys. J. 2004, 86, 2942. (d) Nikolov, V.; Lin, J.; Merzlyakov, M.; Hristova, K.; Searson, P. C. Langmuir 2007, 23, 13040. (7) Radler, J.; Strey, H.; Sackmann, E. Langmuir 1995, 11, 4539. (8) (a) Reviakine, I.; Brisson, A. Langmuir 2000, 16, 1806. (b) Richter, R. P.; Berat, R.; Brisson, A. R. Langmuir 2006, 22, 3497. (9) (a) Keller, C. A.; Kasemo, B. Biophys. J. 1998, 75, 1397. (b) Zhdanov, V. P.; Kasemo, B. Langmuir 2001, 17, 3518. (c) Ekeroth, J.; Konradsson, P.; Hook, F Langmuir 2002, 18, 7923. (d) Dimitrievski, K.; Kasemo, B. Langmuir 2008, 24, 4077. (10) (a) Leonenko, Z. V.; Carnini, A.; Cramb, D. T. Biochim. Biophys. Acta 2000, 1509, 134. (b) Leonenko, Z. V.; Finot, E.; Ma, H.; Dahms, T. E. S.; Cramb, D. T. Biophys. J. 2004, 86, 3783. (c) Leonenko, Z. V.; Merkle, D.; Shamrakov, L. G.; Lees-Miller, S. P.; Cramb, D. T. Biosen. Bioelectron. 2004, 20, 918. (d) Shamrakov, L. G.; Cramb, D. T. Can. J. Chem. 2005, 83, 1190. (11) (a) Oncins, G.; Picas, L.; Hernandez-Borrell, J.; Garcia-Manyes, S.; Sanz, F. Biophys. J. 2007, 93, 2713. (b) Garcia-Manyes, S.; Oncins, G.; Sanz, F. Biophys. J. 2005, 89, 1812. (c) Garcia-Manyes, S.; Oncins, G.; Sanz, F. Biophys. J. 2005, 89, 4261. (12) (a) Xie, F. A.; Yamada, R.; Gewirth, A. A.; Granick, S. Phys. ReV. Lett. 2002, 89, 246103. (b) Feng, Z. V.; Spurlin, T. A.; Gewirth, A. A. Biophys. J. 2005, 88, 2154. (c) Spurlin, T. A.; Gewirth, A. A. Biophys. J. 2006, 91, 2919. (d) Spurlin, T. A.; Gewirth, A. A. J. Am. Chem. Soc. 2007, 129, 11906. (e) Mou, J.; Yang, J.; Shao, Z. Biochemistry 1994, 33, 4439. (f) Mou, J.; Yang, J.; Huang, C.; Shao, Z. Biochemistry 1994, 33, 9981. (13) Kaasgaard, T.; Leidy, C.; Ipsen, J. H.; Mouritsen, O. G.; Jorgensen, K. Single Mol. 2001, 2, 105.

Lipid bilayers can also be tethered via functionalization to a gold surface to serve as platforms for biosensors.17 s-BLMs may also be directly deposited (formed by either the vesicle fusion18 or LB-LS19 methods) onto an electrode to study the effect of the static electric field on the membrane structure and stability. Natural biological membranes are frequently exposed to static electric fields on the order of 107-108 V m-1.20 The effect of the electric field on the membrane structure can be conveniently investigated using bilayers supported at a gold electrode surface, a metal which is chemically inert and behaves as an ideal capacitor over a large potential range. The electric field-driven transformations of bilayers formed from 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC) and from a mixture of DMPC and cholesterol have been investigated recently in this laboratory using photon polarization modulation infrared reflection absorption spectroscopy (PMIRRAS),18a-e,19 atomic force microscopy (AFM),18j scanning tunneling microscopy (STM),18f,i and neutron reflectivity (NR).18g,h Significant differences between the structure of a membrane containing only DMPC and a mixed DMPC–cholesterol membrane containing 30 mol% of cholesterol were observed in these studies.18d,e,i,19c (14) Seantier, B.; Breffa, C.; Felix, O.; Decher, G. Nano Lett. 2004, 4, 5. (15) Slade, A.; Luh, J.; Ho, S; Yip, C. M. J. Struct. Biol. 2002, 137, 283. (16) Malmsten, M. J. Colloid Interface Sci. 1995, 17, 106. (17) (a) Ko¨per, I. Mol. Biosyst. 2007, 3, 651. (b) Knoll, W.; Ko¨per, I.; Naumann, R.; Sinner, E.-K. Electrochim. Acta 2008, 53, 6680. (18) (a) Horswell, S. L.; Zamlynny, V.; Li, H.-Q.; Merrill, R. A.; Lipkowski, J. Faraday Discuss. 2002, 121, 405. (b) Zawisza, I.; Lachenwitzer, A.; Zamlynny, V.; Horswell, S. L.; Goddard, J. D.; Lipkowski, J. Biophys. J. 2003, 85, 4055. (c) Bin, X.; Zawisza, I.; Goddard, J. D.; Lipkowski, J. Langmuir 2005, 21, 330. (d) Bin, X.; Horswell, S. L.; Lipkowski, J. Biophys. J. 200589, 592. (e) Bin, X.; Lipkowski, J. J. Phys. Chem. B 2006, 110, 26430. (f) Xu, S.; Szymanski, G.; Lipkowski, J. J. Am. Chem. Soc. 2004, 126, 12276. (g) Burgess, I.; Szymanski, G.; Li, M.; Horswell, S. L.; Lipkowski, J.; Majewski, J.; Satija, S. Colloids Surf. B. Biointerfaces 2005, 40, 117. (h) Burgess, I.; Szymanski, G.; Li, M.; Horswell, S. L.; Lipkowski, J.; Majewski, J.; Satija, S. Biophys. J. 2004, 86, 1763. (i) Sek, S.; Xu, S.; Chen, M.; Szymanski, G.; Lipkowski, J. J. Am. Chem. Soc. 2008, 130, 5736. (j) Li, M.; Chen, M.; Sheepwash, E.; Brosseau, C. L.; Li, H.-Q.; Pettinger, B.; Gruler, H.; Lipkowski, J. Langmuir 2008, 24, 10313. (19) (a) Zawisza, I.; Bin, X. M.; Lipkowski, J. Langmuir 2007, 23, 5180. (b) Garcia-Araez, N.; Brosseau, C. L.; Rodriguez, P.; Lipkowski, J. Langmuir 2006, 22, 10365. (c) Brosseau, C. L.; Bin, X.; Roscoe, S. G.; Lipkowski, J. J. Electroanal. Chem. 2008, 621, 222. (20) Tsong, T. Y.; Astumian, R. D. Annu. ReV. Physiol. 1998, 50, 273.

10.1021/la802839f CCC: $40.75  2009 American Chemical Society Published on Web 12/29/2008

DMPC-Cholesterol Bilayer

Cholesterol is universally present in the plasma membranes of eukaryotic organisms.21 Cholesterol functions as a modulator of membrane fluidity, providing enhanced stiffness and rigidity to lipids in the liquid-crystalline phase. This property of cholesterol makes the proper functioning of many membrane-associated proteins possible. It is generally accepted that the high levels of cholesterol in the plasma membrane are important for mechanical coherence, resistance to mechanical fatigue, and maintenance of a high permeability barrier.21b,c Consequently, a wide variety of physical techniques have been employed to investigate mixed phospholipid and cholesterol monolayer or bilayer systems, as illustrated by several reviews.21c–e ThestudyofthemembranephasebehaviorofDMPC-cholesterol mixed bilayers, as a function of temperature and concentration of cholesterol, have been the focus of numerous studies (Mabrey et al.;22a Mortensen et al.;22b Needham et al.;22cAlmeida et al.22d). The gel to liquid crystalline state phase transition is observed at 23.9 °C for a pure DMPC bilayer.21a The phase transition becomes broad and progressively disappears with increasing cholesterol content. When cholesterol content exceeds 30%, only one liquid ordered phase exists in a broad range of temperatures from 5 to 40 °C.22d In this work, AFM was employed to study the structure of a mixed DMPC:cholesterol (abbreviated as DMPC-Chol, 70:30 mol%, respectively) bilayer deposited at a Au (111) electrode surface using either a combination of Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) techniques or by the fusion of small unilamellar vesicles (SUVs). This mol% of cholesterol represents the composition at which cholesterol forms either complexes23 with DMPC or is dispersed between the lipids in a hexagonal superlattice.24 At this composition and at a temperature of 18 °C, the DMPC-Chol bilayer exists predominantly in the ordered liquid crystalline state.22d AFM provides a means of both visualizing the lateral organization of lipids and measuring the thickness of the bilayer. These measurements were performed as a function of temperature and the potential applied to the electrode surface. The objectives of this study were 2-fold: (i) to integrate the present AFM data with results of previous PMIRRAS,18d,e,19c STM,18i and NR18g,h studies of this system into a comprehensive description of the mixed DMPC-Cholesterol bilayer supported at the gold electrode surface, (ii) to compare the behavior of the mixed bilayer to the properties of the pure DMPC bilayer (described in a companion paper18j) in order to assess the effect of cholesterol on the structure and stability of the supported bilayer.

Experimental Section Chemicals and Solutions. DMPC (Avanti Polar Lipids, Alabaster, AL, 99%) and cholesterol (Aldrich, 99%) were dissolved in chloroform (ACS HPLC grade, Milwaukee, WI) to make a stock solution (DMPC:Chol with 7:3 molar ratio). A 100 mM sodium (21) (a) Lipowski, R.; Sackmann, E., Eds. Structure and Dynamics of Membranes; Elsevier, Amsterdam, 1995. (b) Lemmich, J.; Mortensen, K.; Ipsen, J. H.; Honger, T.; Bauer, R.; Mouritsen, O. G. Eur. Biophys. J. 1997, 25, 293. (c) Yeagle, P. L. Biochim. Biophys. Acta 1985, 822, 267. (d) YeagleP. L. Biology of Cholesterol; CRC Press: Boca Raton, FL, 1988. (e) Finegold, L. Cholesterol and Membrane Models; CRC Press: Boca Raton, FL, 1993. (22) (a) Mabrey, S.; Mateo, P. I.; Sturtevant, J. M. Biochemistry 1978, 17, 2464. (b) Mortensen, K.; Pfeiffer, W.; Sackmann, E.; Knoll, W. Biochim. Biophys. Acta 1988, 945, 231. (c) Needham, D.; McIntosh, T. J.; Evans, E. Biochemistry 1988, 27, 4668. (d) Almeida, P. F. F.; Vaz, W. L.; Thompson, T. E. Biochemistry 1992, 31, 6739. (e) Sankaram, M. B.; Thompson, T. E. Biochemistry 1990, 29, 10676. (23) McConnell, H. M.; Vrljic, M. Annu. ReV. Biophys. Biomol. Struct. 2003, 32, 469. (24) Cannon, B.; Lewis, A.; Metze, J.; Thiagarajan, V.; Vaughn, M. W.; Somerharju, P.; Virtanen, J.; Huang, J.; Cheng, K. H. Phys. Chem. B. 2006, 110, 6339.

Langmuir, Vol. 25, No. 2, 2009 1029 fluoride (NaF, Sigma-Aldrich, 99.9%) solution was used as the supporting electrolyte for electrochemical measurements. Either a 50 or 1 mM NaF solution was used for AFM studies. This supporting electrolyte was used in earlier PMIRRAS studies done in this laboratory18d,e,19c in order to suppress the solubility of the BaF2 window. This electrolyte was also employed in the present work to facilitate comparison of data obtained from AFM and PMIRRAS. The NaF powder (Sigma-Aldrich, 99.9%) was cleaned in a UV ozone chamber (Jelight, Thief River Falls, MN) for 30-60 min to oxidize organic impurities. All solutions were prepared from water purified by a Milli-Q system (>18.2 MΩ cm). Electrochemical Measurements. Electrochemical measurements were carried out in an all-glass three-electrode electrochemical cell using the hanging meniscus configuration.18d,e,19c All glassware was cleaned in hot mixed acid (HNO3:H2SO4 1:3 v/v%) for approximately 45 min and rinsed thoroughly with Milli-Q water. The electrochemical cell and electrolyte were purged for ∼30 min with pure argon (BOC gas) before the measurement. Argon was continuously passed over the top of the electrolyte to prevent influx of oxygen during the measurement. The single crystal Au(111) working electrode (WE) and gold wire counter electrode (CE) were flame annealed using a Bunsen burner and quenched with Milli-Q water. The reference electrode (RE) was a saturated calomel electrode (SCE), connected to the cell via a salt bridge. For consistency with AFM measurements, all results will be presented versus Ag/AgCl in saturated KCl solution (SSCE) electrode (-50 mV vs SCE). The WE, CE, and RE were connected to a potentiostat (HEKA PG590). The analog signals from the WE were transmitted to a data acquisition interface (NI-DAQ 6052E, National Instruments). The DAQ board digitized the analog signals from the potentiostat and transmitted the digital signals to a computer for analysis. Custom software (generously supplied by Prof. Dan Bizzotto, University of British Columbia) was used to collect differential capacitance data. The differential capacitance curves were determined using a scan rate of 5 mV s-1 and an ac perturbation with a 25 Hz frequency and 5 mV rms amplitude. The differential capacitances were calculated from the in-phase and out-of-phase components of the ac signal, assuming a simple series resistor-capacitor equivalent circuit. Preparation of Gold Electrodes. The electrodes used in the AFM studies consisted of a vapor-deposited gold film. Figure S1a of the Supporting Information shows an AFM image of a ∼1 µm thick film of gold sputtered onto a standard microscope glass slide coated with ∼2 nm thick layer of Cr to ensure a good adhesion of the gold. The gold slide was prepared at the Fritz Haber Institute of the Max Planck Society in Berlin, Germany. The film is rough and consists of gold nanoparticles with an average diameter of ∼40 nm. The gold-coated glass slide was annealed either in a hydrogen-oxygen flame or in a muffle furnace at ∼650 °C to produce large Au crystallites. The AFM studies reported in this work were performed at atomically flat Au(111) terraces of a single grain shown in Figure S1b of the Supporting Information. Flame annealing is an established procedure to produce clean and well ordered surfaces of noble metal electrodes.25 Figure S1c of the Supporting Information shows that the limiting roughness does not change after annealing; however, lateral correlation length (grain size) increases by about 1 order of magnitude. To prevent chromium diffusion to the gold surface, all experiments described in this work were performed at electrodes which were flame annealed only once. Vesicle Fusion and LB-LS Deposition. Small unilamellar vesicles were prepared using the Barenholz procedure.26 A solution of DMPC and cholesterol in chloroform was placed in a test tube. The solvent was evaporated to dryness by vortexing the mixture under a stream of argon. Complete removal of the chloroform was achieved by placing the test tube in a vacuum desiccator for a minimum of 2 h. A sufficient volume of 50 mM NaF electrolyte was added to the dry lipid film to give a ∼1 mg mL-1 solution. The mixture was sonicated at 40 °C for ∼2 h in a bath sonicator (Aquasonic 50D) at a power (25) Clavilier, J. Flame-annealing and cleaning technique. In Interfacial Electrochemistry; Wieckowski, A., Ed.; Marcel Dekker: New York, 1999; p 231. (26) Barenholz, Y.; Gibbes, D.; Litman, B. J.; Goll, J.; Thompson, T. E.; Carlson, R. D. Biochemistry 1977, 16, 2806.

1030 Langmuir, Vol. 25, No. 2, 2009 of 45 W to form vesicles. The solution of vesicles was added to a glass cell using a 1 mL syringe. The final concentration of DMPC was about 1 × 10-4 M. Dynamic laser light scattering measurements performed on these solutions showed that the diameter of 99.75% of vesicles ranged from 38 to 69 nm, 0.24% of the vesicles were in the range 275 to 606 nm, and 0.01% of the vesicles were in the range 1989-5350 nm. A 100 nm syringe filter was used to remove the large vesicles. Unless otherwise stated, the fusion of vesicles at the Au(111) surface was performed at a temperature of ∼20 °C by injecting the stock solution of vesicles into the electrochemical cell containing 50 mM NaF solution (pH ∼ 9.0). Addition of divalent cations such as Ca2+ that facilitate vesicle fusion on mica8 was not employed since Ca2+ precipitates in the presence of fluoride ions. The second method used to form the bilayer was the LB-LS technique which was introduced by Tamm and McConnel6a and was used recently by several authors.6b-d Details of the procedure used in our laboratory have been described previously.19 Briefly, a few microliters of a stock solution of DMPC-cholesterol in chloroform was deposited at the air/water interface of the Langmuir trough (KSV5000,Finland).ThesolventwasevaporatedandDMPC-cholesterol molecules spread to form a monolayer that was compressed with the help of a movable barrier to a desired film pressure. The first monolayer was transferred at a film pressure of 40 mN m-1 using the LB method by vertical withdrawal of the electrode at a speed of 25 mm min-1. The transfer ratio was 1.0 ( 0.1. In the monolayer transferred onto the gold surface by the LB method, the head groups of DMPC face the metal surface and the hydrophobic hydrocarbon chains are directed towards the air. After emersion, the monolayer-covered gold electrode was allowed to dry for ∼1.5 h. The second leaflet was then transferred using the LS technique. The electrode covered by the first layer was horizontally brought into contact with the monolayer which was spread at the air/water interface of the Langmuir trough and compressed to a surface pressure of 40 mN m-1. In the monolayer transferred using the LS method, the acyl chains of the phospholipid are directed toward the metal surface and the polar heads are directed toward the air. Preliminary experiments were performed with bilayers transferred at a subphase temperature of ∼20 °C. However, these films were not homogeneous and contained many blisters with diameters between ∼50 and ∼100 nm and height of ∼10 nm. An AFM image of such a film is shown in Figure S2a of the Supporting Information. However, this problem was alleviated when the LB-LS transfer was carried out at an elevated subphase temperature (∼33 °C). Figure S2b of the Supporting Information shows an image of such a bilayer. The surface of the film transferred at ∼33 °C and then imaged at ∼20 °C is quite smooth and blister-free. AFM Measurements. Soft contact mode AFM and force distance curve measurements were carried out using V-shaped silicon nitride cantilevers with a nominal spring constant of 0.06 N/m (Digital Instruments) mounted to a scanner (AFMS1182, Molecular Imaging) and microscope (Molecular Imaging PicoSPM). The thermal tune method27 employed to calibrate three tips from the same batch gave the value of the force constant 0.07 ( 0.02 N/m that within the uncertainty limit was equal to the nominal value. The tips were exposed to ozone in a UV laminar flow cabinet for 30 min prior to use. The scan rate of imaging varied from 3.0 to 4.0 lines per second. A custom-designed cell equipped with a gold ring counter electrode and a SSCE reference electrode were used to perform electrochemical AFM experiments. The temperature dependence studies were carried out by placing the microscope with the scanner in a homemade environmental chamber. The temperature inside the chamber was precisely controlled ((0.1 °C) using a thermocouple and a temperature controller (Lakeshore). Cooling of the chamber was achieved by circulating cold water in a copper coil, placed inside the chamber. A second thermocouple (OMEGA, HH509R) was used to monitor the temperature inside the solution of the AFM cell. For each desired temperature, the measurement (imaging or force distance (27) Ohler, B. Practical advice on the determination of cantilever spring constant, Veeco Instruments Inc., www.veeco.com (accessed Nov 5, 2008).

Chen et al.

Figure 1. (a) Typical force curve measured on the DMPC-Chol bilayer supported at the Au(111) surface at open circuit potential (EOCP∼0.27V versus SSCE). (b) Histogram showing the distribution of the bilayer thicknesses determined from the force curves measured in 1 mM NaF at ∼20.0 °C.

curve measurements) was performed at least 0.5 h (equilibrium time) after the chamber reached the desired temperature. The force-distance curves were recorded by measuring the deflection of the cantilever versus the position of the sample mounted onto the piezoelectric translator. The original cantilever deflection versus piezo position curves were converted into force curves using software written in-house and the nominal value of the spring constant (∼0.06 N/m) provided by the supplier. The time needed to record one force curve was ∼1.5 s. The force curve measurements were repeated ∼100 times for the purpose of statistical data analysis. Soft contact mode imaging employs a repulsive force between the tip and the imaged sample. Preliminary experiments performed in 100 mM NaF solution demonstrated that at moderate separations between the tip and the DMPC-cholesterol bilayer covered gold surface, stronger attractive forces were observed than in the previously studied case of a pure DMPC bilayer.18j At smaller separations, the gradient of the force becomes comparable to the spring constant of the cantilever (∼0.06 N/m) causing mechanical instability of the tip and a tendency of the tip to “jump-to-contact” with the metal surface, through the film. This point is explained in Figure S3 of the Supporting Information. However, this problem could be alleviated by decreasing the electrolyte concentration and increasing the repulsive electrostatic force. Therefore, AFM studies reported in this work were performed using a 1 mM NaF solution. Figure 1a shows a typical force distance curve recorded on the DMPC-Chol bilayer in 1 mM NaF. The force-distance curve displays a weak repulsive interaction between the tip and the bilayercovered surface at large separations (∼7-80 nm), which changes to a largely increased repulsive interaction at separations less than ∼7 nm. At separations less than ∼5 nm, a characteristic discontinuity is observed followed by a vertical section corresponding to the direct interaction of the tip with the gold surface. The zero for the separation distance is determined by extrapolation of this section of the curve to the zero value of the force.28a The discontinuity corresponds to the penetration of the tip through the bilayer. In the soft contact mode, the images are acquired by applying a constant force just below the bilayer penetration force. The force distance curve is

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Figure 2. AFM deflection images of a DMPC-Chol bilayer formed by fusion of 1.2 mg/mL vesicles at a temperature of ∼22 °C and at the OCP. The images were acquired at (a) 15 min, (b) 25 min, (c) 45 min, and (d) 70 min after injection of vesicles into the AFM cell.

Figure 3. Comparison of AFM images of a Au(111) electrode surface covered by (a) a DMPC-Chol bilayer formed by vesicle fusion, (b) a DMPC-Chol bilayer deposited by the LB-LS technique, (c) a pure DMPC bilayer formed by vesicle fusion, and18j (d) a pure DMPC bilayer deposited by the LB-LS method.18j Parts a and b are deflection images, and parts c and d are MAC mode amplitude images. All images were acquired at a temperature of ∼20 °C.

sufficiently steep at that point to ensure good contrast without altering the structure of the bilayer. The “jump-in” distance corresponding to the discontinuity is usually taken as the film thickness.28 Figure 1b plots the distribution of the measured film thickness from 100 force distance curves. The mean value of the Gaussian fitting of the histogram plot, equal to 4.6 ( 0.2 nm, is taken as the film thickness. The “jump-in” distance measured from the force-distance curves gives the thickness of the compressed layer, and hence it is somewhat (28) (a) Butt, H.-J.; Cappella, B.; Kappl, M. Surf. Sci. Rep. 2005, 59, 1. (b) Butt, H.-J.; Stark, R. Colloid. Surf. A 2005, 252, 165.

smaller than the thickness of the film at equilibrium.28b Following Dufrene et al.,29 the elastic deformation δ produced by the AFM tip under a known load can be determined from the formula derived from a Hertzian model:

δ ) (9F2/16RE*2)1⁄3

(1)

where R is the radius of the tip curvature (∼20 nm) and E* is the effective compression modulus of the DMPC-Chol bilayer (∼7 ×

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Figure 4. AFM deflection images of the DMPC-Chol bilayer deposited at the Au(111) surface using the LB-LS method at the OCP and different temperatures as indicated in the images.

108 N m-2).30-32 By applying the Hertzian model, one assumes that the deformation has a pure elastic nature and that no change of the film structure (for example from liquid ordered to liquid disordered state) takes place during compression. The average load force F is determined from the distribution of the penetration force measured on the DMPC-Chol bilayer formed by LB-LS. The values of the penetration force and the elastic deformation estimated with the help of eq 1 are plotted as a function of the electrode potential in Figure S4 of the Supporting Information. Such deformation is ∼10% of the estimated thickness. Henceforth, all thicknesses reported are the corrected thickness values which are equal to the penetration distance plus the elastic deformation calculated with the help of eq 1. After this correction, the thickness determined from the distribution in Figure 1b amounts to 5.1 ( 0.2 nm. The uncertainty of the measured thickness will always be reported as the standard error σ/N, where σ is the standard deviation and N is the number of measurements. (29) (a) Dufrene, Y. F.; Boland, T.; Schneider, J. W.; Barger, W. R.; Lee, G. U. Faraday Discuss. 1998, 111, 79. (b) Dufrene, Y. F.; Lee, G. U. Biochim. Biophys. Acta 2000, 1509, 14. (c) Ebling, E.; Holscher, H.; Fuchs, H.; Anczykowski, B.; Schwarz, U. D. Nanotechnology 2006, 17, S221. (30) Brown, M. F.; Thurmond, R. L.; Dodd, S. W.; Otten, D.; Beyer, K. J. Am. Chem. Soc. 2002, 124, 8471. (31) McWhirter, J. L.; Voth, G. A. Biophys. J. 2004, 87, 3242. (32) Duwe, H. P.de; Sackmann, E. Physica A 1990, 163, 410.

Results and Discussion Effect of Cholesterol on the Structure of the DMPC-Chol Bilayer. The initial studies of DMPC-Chol bilayer formation were carried out at the open circuit potential (OCP) which has a value of ∼+270 mV versus SSCE and which is equal to the potential of zero charge (pzc) (Epzc ) 220 mV versus SCE or 270 mV versus SSCE).19c At this potential, the vesicles were observed to fuse quickly at the Au(111) surface. However, the morphology of the film formed by vesicle fusion underwent a slow transformation from initially rough to the final smooth state. Figures 2a-d show deflection images of the gold surface taken at different intervals of time after injection of vesicles into the solution. The film formed after 15 min (Figure 2a) shows no unfused vesicles; however, it is quite rough and stressed. The stress apparently relaxes with time, and after ∼70 min (Figure 2d) a smooth homogeneous film is observed. The thickness of this film, determined from the force distance curves, is equal to 3.8 ( 0.3 nm. Figure S5 in the Supporting Information shows images of a larger section of the electrode area covering several grains. It illustrates that similar film transformations are observed at surfaces of all grains of gold and that no phase segregation could be seen in these images. However, in contrast to mica or highly ordered pyrolytic graphite (HOPG), this gold sample allows one to obtain images of an atomically flat surface at one grain

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stack of multiple bilayers by Janiak et al.34b However, this value is in agreement with the “steric bilayer thickness” of 4.82 nm reported by Tristam-Nagle et al.34c For the DMPC-Chol bilayer with 30% cholesterol, the thickness of 5.1 ( 0.2 nm at 20 °C found in this study is somewhat higher than the “apparent thickness” of 4.6 nm by reported by Pencer et al.34a However, neutron diffraction studies on a stack of multiple DMPC-Chol bilayers with 30% Chol by Leonard et al.33a showed that the mixed bilayer is thicker by about 0.35 nm than the pure DMPC bilayer. When this number is added to the “steric bilayer thickness” of a pure DMPC bilayer34c one gets a thickness which is in agreement with the present study. Indeed, AFM studies shown here demonstrate that insertion of cholesterol increases the thickness of the DMPC bilayer produced by the LB-LS method by ∼0.4 nm, in good agreement with literature.33a,34a The lower values of the thickness for the bilayers formed by vesicle fusion indicate that molecules in these bilayers are either more tilted or less ordered. The fact that addition of cholesterol has a small effect on the film thickness is a further indication that films produced by this method are poorly ordered. The thickness of the bilayers determined by AFM are in agreement with tilt angles of the acyl chains reported in our earlier PMIRRAS studies.18c,d,19b,c The thickness d and the tilt angle R are related by the formula:

cos R ) d/5.4 Figure 5. (a) Surface roughness plotted as amplitude equal to 4 times standard deviation of surface heights and (b) periodicity (equal to the critical scaling length) plotted versus temperature. The roughness and the critical scaling length were calculated from the scaling analysis of the AFM images shown in Figure 4.

only. Consequently, the imaged area has to be restricted to dimensions less than 1 × 1 µm. This could also be seen in Figure S1b of the Supporting Information which shows an image of the native surface before addition of vesicles. Figures 3a and 3b compare the topography of a gold electrode covered by a film formed by the fusion of vesicles to that obtained by the LB-LS method. The two methods give smooth, homogeneous films which are similar in appearance. However, the thickness of the film formed by the LB-LS method (equal to 5.1 ( 0.2 nm) is significantly larger than the thickness of the bilayer formed by vesicle fusion. Figures 3c and 3d show images of pure DMPC bilayers, taken from a recent publication.18j Differences between the mixed DMPC-Chol and pure DMPC bilayers are striking. The mixed DMPC-Chol bilayers are smoother and thicker than bilayers composed of DMPC only. The thickness of bilayers produced by the LB-LS method is in good agreement with the thickness determined by neutron or X-ray diffraction methods33,34 for a stack of multiple bilayers. The values of the bilayer thickness measured by the diffraction methods depend on the interpretation of the electron density or scattering length density profiles. Thus, for pure DMPC, the thickness of 4.7 ( 0.2 nm at 20 °C is higher than the “apparent thickness” of 4.35 nm determined by Pencer et al.34a for unilamellar vesicles by small angle neutron scattering or to a value of 4.25 nm determined by X-ray diffraction studies of a (33) (a) Leonard, A.; Escrive, C.; Laguerre, M.; Pebay-Peyroula, E.; Neri, W.; Pott, T.; Katsaras, J.; Dufourc, E. J. Langmuir 2001, 17, 2019. (b) Marsan, M. P.; Muller, I.; Ramos, C.; Rodriguez, F.; Dufourc, E. J.; Czaplicki, J.; Milon, A. Biophys. J. 1999, 76, 351. (c) Dufourc, E. J.; Parish, E. J.; Chitrakorn, S.; Smith, I. C. P. Biochemistry 1984, 23, 6062. (34) (a) Pencer, J.; Nieh, M.-P.; Harroun, T. A.; Krueger, S.; Adams, C.; Katsaras, J. Biochim. Biophys. Acta 2005, 1720, 84. (b) Janiak, M. J.; Small, D. M.; Shipley, G. G. Biochemistry 1976, 15, 4575. (c) Tristam-Nagle, S.; Liu, Y.; Legleiter, J.; Nagle, J.-F. Biophys. J. 2002, 83, 3324.

(2)

where 5.4 nm is the thickness of the bilayer corresponding to a tilt angle of zero degrees. When the LB-LS method is used, at the OCP, the tilt angles correspond to 25 ( 3° (ref 19c) and 30 ( 3° (ref 19b) for DMPC-Chol and pure DMPC bilayers, respectively. These values correspond to thicknesses of 4.9 ( 0.2 and 4.7 ( 0.2 nm, respectively. Within the error bars, these thickness values are equal to the thicknesses measured by AFM. When the mixed DMPC-Chol bilayer is formed by vesicle fusion, the tilt angle is equal to 49 ( 3° (ref 18c) corresponding to a thickness of 3.5 ( 0.3nm. Within the experimental uncertainty limits, this number agrees with the thickness of 3.8 ( 0.3 nm determined by AFM in this work. The average tilt angle in the pure DMPC bilayer is 54 ( 3° (ref 18d) which corresponds to the bilayer thickness of 3.2 ( 0.3nm. This number is lower than the thicknesses of 4.2 ( 0.2 nm measured by AFM. We have already shown this difference in our previous publication.18 For randomly oriented chains the tilt angle is 54.4°. In bilayers produced by vesicle fusion the tilt angles of acyl chains are close to this angle, indicating that the bilayers are disordered. The lower values of the bilayer thickness estimated from the PMIRRAS data compared to the values obtained by AFM suggest that bilayers formed at the large surface electrode used in IR experiments are less ordered than bilayers formed at atomically smooth (111) surfaces of a single grain of gold. In summary, the present data confirm once more that the LB-LS method offers a better control of the film structure and allows for the formation of more ordered bilayers. The presence of cholesterol relaxes the undulation seen in the images of pure DMPC bilayers. We have recently demonstrated18j that undulations are caused by the elastic stress in the film induced by spontaneous curvature, which results from the packing of polar head groups in direct contact with the metal surface. The area occupied by the headgroup of the DMPC molecule is larger than the cross-section of two hydrocarbon chains. To compensate for the size mismatch, DMPC molecules (in a hydrated crystalline state) are packed with their polar heads alternatively displaced

1034 Langmuir, Vol. 25, No. 2, 2009

Chen et al.

Figure 6. (a) Corrected thickness of the DMPC-Chol bilayer (red line) and pure DMPC bilayer (blue line) plotted as a function of temperature. (b) Plot of the bilayer thickness in unilamellar vesicles determined by small-angle neutron scattering by Pencer et al.;34a (blue line) pure DMPC vesicles, and mixed DMPC-cholesterol vesicles with (pink line) 20% cholesterol and (green line) 47% cholesterol.

in a sawtooth manner.35 When polar head groups are located in one plane, the acyl chains must tilt or form a splay deformation which introduces spontaneous curvature and an elastic stress in a planar bilayer. The cholesterol molecule is buried in the chain region of the bilayer.33a In the presence of cholesterol the surface density of the head groups of DMPC molecules decreases and the separation between the head groups increases. These effects reduce the curvature and relax the stress induced by packing polar heads in the plane of the metal, and the undulation is lifted. Thermotropic Properties of the DMPC-Chol Bilayer. The thermotropic properties of the DMPC-Chol bilayer prepared by the LB-LS technique were investigated over the temperature range 14.0 °C to 31.8 °C at the OCP. This approach targeted detection and quantification of the main phase transition of the bilayer supported at the gold surface. AFM images were taken at an atomically smooth Au(111) terrace so that the features seen in the images represent the topography of the bilayer. Figure 4 shows representative AFM images of the DMPC-Chol bilayer at six selected temperatures. Only small changes of the topography could be seen in these images. Scaling analysis was employed to quantify the topographic information. The scaling analysis allows one to calculate the root-mean-square of the surface heights (or the standard deviation of surface heights) of the imaged surface ξ and the so-called critical scaling length Lc, which is a measure of periodicity of surface features in the direction parallel to the surface.36 For example, if scaling analysis is performed for an image of gratings consisting of parallel grooves, then ξ is equal to the peak to peak amplitude of the grooves divided by four and Lc is equal to the period of the grooves. The principle of the scaling analysis is explained in detail in Figure S6 of the Supporting Information. The amplitude and the critical scaling length of the surface features determined from the scaling analysis are plotted against temperature in Figures 5a and 5b, respectively. The results show that film becomes smoother with increasing temperature. However, since the bilayer is in the liquid ordered state at these temperatures,22d the film smoothing cannot be associated with a phase transition. Most likely heating releases stress induced in the bilayer during the LB-LS deposition. Figure 6a plots the thickness of the bilayer as a function of temperature, determined from the force curves. For the benefit of further discussion the thickness of the pure DMPC bilayer taken from our previous study18j is also plotted in this figure. In (35) Hauser, H.; Pascher, I.; Pearson, R. H.; Sundell, S. Biochim. Biophys. Acta 1981, 650, 21. (36) (a) Barabasi, A.-L.; Stanley, H. E. Fractal Concepts in Surface Growth; Cambridge University Press: New York, 1995. (b) Zhao, T.; Zagidulin, D.; Szymanski, G.; Lipkowski, J. Electrochim. Acta 2006, 51, 2255. (c) Malevich, D.; Baron, J.; Szymanski, G.; Lipkowski, J. J. Solid. State Electrochem. 2008, 12, 455.

addition, Figure 6b plots similar thickness data obtained from small angle neutron scattering (SANS) measurements on unilamellar vesicles of pure DMPC and two DMPC-Chol mixtures containing 20% and 47% cholesterol, compiled from the work of Pencer et al.34a The AFM data have large error bars which may reflect lower precision of the measurements or a less uniform distribution of the thickness in the membrane supported at the gold surface. However, the two sets of data show very similar dependence of the bilayer thickness on temperature, indicating a similarity between the properties of the bilayer supported at the gold electrode surface and the bilayer in unilamellar vesicles. The thickness of the pure DMPC bilayer changes abruptly by ∼0.5 nm across the phase transition, in agreement with X-ray diffraction studies performed on a stack of hydrated multiple bilayers.34b,c This change is chiefly due to chains melting upon the transition from the gel to the liquid crystalline state. The SANS data in Figure 6b show that the main phase transition temperature of the DMPC bilayer in vesicles is ∼24 °C, in agreement with literature.22a The phase transition temperature in the bilayer supported at the Au(111) surface is observed at ∼21-22 °C, which is ∼2 °C lower than in vesicles. Our previous study18j has shown that the bilayer at the metal surface is stressed and the two leaflets are poorly coupled. Therefore, it is easier to melt chains in such a bilayer. In contrast to pure DMPC, the thickness of the mixed DMPC-Chol bilayer changes gradually with temperature. The mixed bilayer is about 0.4 nm thicker than the pure DMPC bilayer. NMR studies demonstrated that addition of cholesterol reduces the acyl chain order in the gel state of DMPC bilayer.22e,37 This effect should cause a decrease in the bilayer thickness. At temperatures below the phase transition temperature of a pure DMPC bilayer, the increase of the bilayer thickness must be chiefly due to a decrease of the chain tilt angle, as demonstrated in our recent PMIRRAS study.19c In the liquid crystalline state of pure DMPC an addition of cholesterol increases the acyl chain order.22e In this case, higher values of the bilayer thickness are a result of the combined effects of both a reduction of the chain tilt angle and increased chain ordering. Potential Controlled Changes of the Bilayer Structure. The metal-solution interface behaves as a capacitor. According to the Helmholtz model, the capacitance of the interface is equal to C ) ε0ε/d, where ε0 and ε are the permittivity of vacuum and the relative permittivity of the bilayer, respectively, and d is the film thickness. A perfect defect-free bilayer is characterized by a low permittivity. In the presence of defects, water molecules penetrate into the bilayer, and as a result its permittivity increases. (37) (a) Vist, M. R.; Davis, J. Biochemistry 1990, 29, 451. (b) Miao, L.; Nielsen, M.; Thewalt, J.; Ipsen, J. H.; Bloom, M; Zuckermann, M. J.; Mouritsen, O. G. Biophys. J. 2002, 82, 1429.

DMPC-Cholesterol Bilayer

Figure 7. For the Au(111) electrode, dependence on the electrode potential of (a) differential capacitance: (dashed line) film-free surface in 0.1 M NaF solution; (red line) surface covered by the mixed DMPC-Chol bilayer; (blue line) surface covered by a pure DMPC bilayer. The bilayers were deposited on the Au(111) surface using the LB-LS technique; (b) the film roughness plotted as amplitude equal to 4 times standard deviation of surface heights and periodicity plotted as the critical scaling length determined from scaling analysis of AFM images in Figure 8; (c) the thickness of the mixed DMPC-Chol bilayer determined from the force distance curves.; (d) tilt angle of the acyl chains taken from recent PMIRRAS studies.19c Potentials measured versus the SSCE reference electrode, temperature was ∼20 °C.

This leads to an increase of the capacitance. Changes in both the bilayer thickness and the density of defects cause a change of the capacitance. Therefore, the measurement of the differential capacitance is a convenient means to investigate the effect of the electrode potential on the bilayer structure. Figure 7a plots the differential capacitance of the film-free Au(111) electrode and the electrode covered by the DMPC-Chol bilayer deposited using the LB-LS method. For comparison, a curve representing

Langmuir, Vol. 25, No. 2, 2009 1035

the electrode covered by a pure LB-LS DMPC bilayer is included in this figure. In the presence of the mixed DMPC-Chol bilayer the capacitance is much lower than in the presence of the pure DMPC bilayer. The minimum value of the capacitance for the DMPC-Chol bilayer is ∼1.7 µF cm-2 at E˜ 0.1V. Although this value is much lower than the minimum capacitance of the electrode covered by the pure DMPC bilayer (∼7 µF cm-2), it is still higher than the capacitance of a defect-free biological bilayer which is ∼0.8 µF cm-2 (ref 38). Therefore, the mixed DMPC-Chol bilayer contains far fewer defects than the pure DMPC bilayer, but is not defect-free. A compact bilayer is formed on the surface between ∼-0.1 and ∼+0.3 V vs SSCE, corresponding to (E - Epzc) from -0.37 to 0.03 V, characterized by a region of low capacitance. At E < -0.1V vs SSCE or (E - Epzc) < -0.37 V, the capacity increases, indicating onset of detachment of the film from the surface. Complete desorption of the film is reached at E