Electrochemical and PM-IRRAS Studies of the ... - ACS Publications

330

Langmuir 2005, 21, 330-347

Electrochemical and PM-IRRAS Studies of the Effect of the Static Electric Field on the Structure of the DMPC Bilayer Supported at a Au(111) Electrode Surface Xiaomin Bin, Izabella Zawisza, John D. Goddard, and Jacek Lipkowski* Department of Chemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Received May 25, 2004. In Final Form: September 4, 2004 Differential capacity, charge density measurements, and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) were employed to study the fusion of small unilamellar vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) on a Au(111) electrode surface. The differential capacity and charge density data showed that the vesicles fuse onto the gold surface at charge densities between -10 µC/cm2 < σM < 10 µC/cm2 to form a bilayer. When σM < -10 µC/cm2, the film is detached from the surface but it remains in close proximity to the surface. PM-IRRAS experiments provided IR spectra for the bilayer in the adsorbed and the desorbed state. Ab initio normal coordinate calculations were performed to assist interpretation of the IR spectra. The IR bands were analyzed quantitatively, and this analysis provided information concerning the conformation and orientation of the acyl chains and the polar head region of the DMPC molecule. The orientation of the chains, hydration, and conformation of the headgroup of the DMPC molecule strongly depend on the electrode potential.

1. Introduction Bilayers of phospholipids supported at a solid surface constitute an attractive model of a biological membrane to study membrane processes.1-4 The supported phospholipid membranes are usually formed by spreading small unilamellar vesicles (SUVs) onto a hydrophilic surface of glass or freshly cleaved mica.1,5-7 The bilayers may also be deposited onto a metal electrode surface, where they can be exposed to static electric fields on the order of 107-108 V/m that are comparable in magnitude to the field acting on a biological membrane.8 This approach constitutes a very convenient means to mimic the static electric field acting on a biological membrane and to study field-driven membrane processes. Several strategies have been used to deposit a membrane onto an electrode surface.9 Investigations of transmembrane proteins require matrices with a water reservoir on the two sides of the membrane. Such models have been built by fusing SUVs onto an electrode modified with hydrophilic thiols.10-16 Monolayers of phospholipids (1) Sackmann, E. Science. 1996, 271, 43. (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) Bayerl, M. M.; Bloom, T. Biophys. J. 1990, 58, 357. (4) Schmidt, A.; Spinke, J.; Bayerl, T.; Sackmann, E.; Knoll, W. Biophys. J. 1992, 63, 1385. (5) Leonenko, Z. V.; Carnini, A.; Cramb, D. T. Biochim. Biophys. Acta 2000, 1509, 131. (6) Raedler, J.; Strey, H.; Sackmann, E. Langmuir 1995, 11, 4539. (7) Xie, A. F.; Yamada, R.; Gewirth, A. A.; Garnick, S. Phys. Rev. Lett. 2002, 89, 246103. (8) Tsong, T. Y.; Astumian, R. D. Annu. Rev. Physiol. 1998, 50, 62. (9) Guidelli, R.; Aloisi, G.; Becucci, L.; Dolfi, A.; Moncelli, M. R.; Buoninsegni, F. T. J. Electroanal. Chem. 2001, 504, 1. (10) Cornell, B. A.; Braach-Maksvytis, V.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. L. Nature 1997, 387, 580. (11) Williams, L. M.; Evans, S. D.; Flynn, T. M.; Marsh, A.; Knowles, P. M.; Bushby, R. J.; Boden, N. Langmuir 1997, 13, 751. (12) Raguse, B.; Braach-Maksvytis, V.; Cornell, B. A.; King. L. G.; Osman, P. D. J.; Pace, R. J.; Wieczorek, L. Langmuir 1998, 14, 648. (13) Stora, T.; Lakey, J. H.; Vogel, H. Angew. Chem., Int. Ed. 1999, 38, 389. (14) Becucci, L.; Guidelli, R.; Lin, Q. Y.; Bushby , R.J.; Evans, S. D. J. Phys. Chem B 2002, 106, 10410.

deposited at a mercury electrode surface have been used to study surface-attached or imbedded proteins or peptides.17-22 Bilayers also have been formed at an electrode surface either by direct fusion of SUVs23-27 or by depositing a monolayer of a phospholipid onto a monolayer of thiols tethered to a gold electrode surface.28-32 In the past, the model biomembranes at electrode surfaces have been investigated chiefly using electrochemical techniques. These methods measure average properties and do not provide insight into the structure of the membrane at the molecular level. Recently, we have developed in situ photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS)33 and have applied this technique to study the potential controlled (15) Naumann, R.; Schiller, M. S.; Giess, F.; Grohe, B,; Hartman, K. B.; Karcher, I.; Koper, I.; Luben, Jorn.; Vasilev, Krasimir.; Knoll, W. Langmuir 2003, 19, 5435. (16) Wright, J. E. I.; Fatih, Khalid.; Brosseau, C. L.; Omanovic, S.; Roscoe, S. G. J. Electroanal. Chem. 2003, 550, 41. (17) Bizzotto, D.; Nelson, A. Langmuir 1998, 14, 6269. (18) Miller, I. R. In Topics in bioelectrochemsitry and Bioenergetics; Millazzo, G., Ed.; Wiley: Chichester, 1981; p 194. (19) Prieto, F.; Navarro, I.; Rueda, M. J. Electroanal. Chem. 2003, 550, 253. (20) Moncelli, M. R.; Becucci, L.; Nelson, A.; Guidelli, R. Biophys. J. 1996, 70, 2716. (21) Nelson, A.; Benton, A. J. Electroanal. Chem. 1986, 202, 253. (22) Nelson, A.; Bizzotto, D. Langmuir 1999, 15, 7031. (23) Stauffer, V.; Stoodly, R.; Agak, J. O.; Bizzotto, D. J. Electroanal. Chem. 2001, 516, 73. (24) Hepel, M. J. Electroanal. Chem. 2001, 509, 90. (25) Horswell, S. L.; Zamlynny, V.; Li, H.-Q.; Merrill, A. R.; Lipkowski, J. Faraday Discuss. 2002, 121, 405. (26) Zawisza, I.; Lachenwitzer, A.; Zamlynny, V.; Horswell, S. L.; Goddard, J. D.; Lipkowski, J. Biophys. J. 2003, 85, 4055. (27) Burgess, I.; Li, M.; Horswell, S. L.; Szymanski G.; Lipkowski, J.; Majewski, J.; Satija, S. Biophys. J. 2004, 86, 1763. (28) Lingler, S.; Rubinstein, I.; Knoll, W.; Offenhausser, A. Langmuir 1997, 13, 7085. (29) Buoninsegni, F. T.; Herrero, R.; Moncelli, M. R. J. Electroanal. Chem. 1998, 452, 33. (30) (a) Plant, A. L. Langmuir 1999, 15, 5128. (b) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197. (31) Krysinski, P.; Zebrowska, A.; Michota, A.; Bukowska, J.; Becucci, L.; Moncelli, M. R. Langmuir 2001, 17, 3852. (32) Gregory, B. W.; Dluhy, R. A.; Bottomley, L. A. J. Phys. Chem. 1994, 98, 1010.

10.1021/la048710w CCC: $30.25 © 2005 American Chemical Society Published on Web 12/04/2004

DMPC Bilayer Supported at a Au(111) Electrode Surface

Langmuir, Vol. 21, No. 1, 2005 331

transformations of phospholipid bilayers at a Au(111) electrode surface.25,26,34 We have demonstrated that PMIRRAS is a powerful tool to monitor potential induced reorientation, conformation, and hydration of phospholipid molecules in bilayers supported at an electrode surface. The purpose of the present work was to apply PM-IRRAS to describe the electric field-driven changes in the structure of a bilayer formed by 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at a Au(111) electrode surface. The (111) plane of gold is energetically most stable and allows one to study thin organic film over a broad range of potentials and charge densities. This study was initiated by Horswell et al.25 Since this first publication, we have made significant methodological improvements that allowed us to investigate absorption bands of the phosphate and choline groups. Further, new protocols have been developed to determine the angle between the direction of the transition dipole of a specific IR band and the direction normal to the surface. As a result of these improvements, we are able to provide a detailed description of the influence of the static electric field on the packing, orientation, conformation, and hydration of the polar head region of DMPC molecules in the supported bilayer. These studies provide background for future work on the electric-field-assisted changes in the conformation of proteins and peptides embedded into a model biological membrane.

1 h. After cooling, it was rinsed with copious amounts of pure water. The working electrode was flame-annealed and introduced into the cell under the protection of a drop of water. It was then positioned in a hanging meniscus configuration.37 Cyclic voltammetry (CV) and differential capacitance (DC) measurements of pure electrolyte were used to check the cleanliness of the system.38 The electrochemical instruments consisted of a Heka potentiostat/ galvanostat 600 (HEKA, Lambrecht/Pfalz,Germany) and a 7265 DSP lock-in amplifier (EG&G Instruments, Cypress, CA). All data were acquired via a plug-in acquisition board (RC Electronics, Santa Barbara, CA) and in-house software. The differential capacity of the metal-solution interface was measured by superimposing a small sinusoidal perturbation of 5 mV r.m.s and 25 Hz on a 5 mV/s voltage ramp. The in-phase and out-ofphase components of the alternating current were measured using the lock-in amplifier. Chronocoulometry was applied to determine the charge density at the electrode surface. The gold electrode was held at a base potential Ebase ) -0.8 V for 30 s. Then, the potential was stepped to a variable potential Ec for t ) 120 s to obtain complete spreading of vesicles on the electrode surface. Next, the potential was stepped to the desorption potential Edes ) -1.2 V (SSCE) for 0.15 s. The integration of the current transients gives the difference between charge densities at potentials Ec and Edes. The same set of experiments was performed at the Au(111) electrode in the solution without DMPC vesicles. The absolute charge densities were calculated using potential of zero charge (pzc) Epzc ) 0.30 V vs (SSCE). The solutions were stirred to enhance mass transport to the electrode. Surface pressure was calculated by integration of charge density curves. 2.2. Spectra Collection and Processing. The PM-IRRAS setup consisted of a Nicolet Nexus 870 spectrometer, equipped with an external optical bench, MCT-A detector TRS50 MHz (Nicolet, Madison, WI), photoelastic modulator (PEM), (Hinds Instruments PM-90 with II/ZS50 ZnSe 50 kHz optical head, Hillsoboro, OR), and demodulator (GWC Instruments synchronous sampling demodulator, Madison, WI). The electrode potentials were controlled via a potentiostat (EG&G, PAR Model 362, Princeton, NJ) using in-house software, Omnic Macro, and a digital-to-analog converter (Omega, Stamford, CT). In addition, Omnic Macro was used to collect and to save spectra. The IR window was a BaF2 1 in. equilateral prism. It was washed in water and methanol and then cleaned in an ozone UV chamber (UVO-cleaner, Jelight, Irvine, CA) for 20 min before being assembled in the cell. DMPC vesicles prepared in D2O or H2O were injected to the cell, and then argon (BOC Gases, Mississauga, Ontario, Canada) was gently bubbled for 2 h to deaerate the solution. DMPC vesicles were fused to form a bilayer on the surface of the working electrode. The potential of the WE was set initially at -1.0 V vs Ag/AgCl, and then spectra were acquired at a series of potentials, which were programmed as a cyclic sequence of 0.1 or 0.2 V potential steps. In total, 20 cycles of 400 scans each were performed to give 8000 scans at every applied potential. The resolution of the instrument was 2 cm-1. At the end of the experiment, blocks of scans were individually checked for anomalies before averaging using in-house software. The angle of the incident infrared beam and the thickness of the electrolyte layer between the prism and the electrode were adjusted individually for different spectral regions. The measurements of IR spectra were carried out with the PEM set for half-wave retardation at 2900 cm-1 for the CH stretching region. The angle of incident light was set to 53°, and the electrolyte thickness was ca. 5.8 µm. These parameters gave comparable intensities of p- and s-polarized light inside the thin layer cavity and, hence, allowed for the cancellation of the IR absorption by vesicles that did not spread at the electrode surface. In the CdO stretching and the CH bending region, the maximum PEM efficiency was set to 1600 cm-1. The angle of incident light was 60°, and the electrolyte layer was 6.0 µm. In the phosphate group stretching region, the PEM maximum efficiency was set to 1200 cm-1 (νas(PO2-)) and 1000 cm-1 (νs(PO2-)). The angle of incident light was 57°, and the gap thickness was 6.4 and 5.0 µm for asymmetric and symmetric phosphate stretches, respectively. For CH stretching, CO stretching, and the symmetric phosphate

2. Experimental Section 2.1. Electrodes, Solutions, and Materials. Au(111) single crystals were used as working electrodes (WE). They were grown, oriented, and cut in our laboratory. The counter electrode (CE) used for electrochemical measurements was a Au coil, whereas a cylindrical Pt foil was used as the CE for spectroelectrochemical measurements. A saturated calomel electrode (SCE) was used as the reference electrode (RE) in electrochemical experiments and a Ag/AgCl (3M KCl) reference electrode (SSCE) (E ) -40mV versus the SCE) was used in the PM-IRRAS measurements. In this paper, all potentials are reported on the SSCE scale. A 0.1 M NaF (Merck, Suprapur, Darmstadt, Germany) solution was used as the supporting electrolyte in electrochemical and PMIRRAS experiments. All aqueous solutions were prepared from ultrapure water, purified by a Milli-Q UV plus (resistivity higher than 18.2 MΩ cm, Millipore, Molsheim, France) water system. The solvent for the IR experiments was D2O (Cambridge Isotope Laboratories, Inc., Cambridge, MA) or H2O. 1,2-Dimyristoylsn-glycero-3-phosphocholine (DMPC) was purchased from (Sigma, 99.0+%,St Louis, MO) and used without further purification. All measurements were carried out at room temperature (20 ( 2 °C), which is below the phase transition temperature of DMPC (24 °C).35 The procedure described by Barenholtz et al.36 was used to prepare the SUVs. A 10 mg/mL chloroform (Aldrich, ACS HPLC grade, Milwaukee, WI) solution of DMPC was used as stock solution. The stock solution (0.2 mL) was dried by vortexing in a test tube under a flow of argon. To remove the residue of the solvent, dried DMPC film was placed in a vacuum desiccator for at least 2 h. Next, 2 mL of 0.1M NaF electrolyte was added to the dry lipid and the mixture was sonicated at 35 °C for at least 1 h. (Usually the mixture became translucent after 20-30 min.) The solutions of vesicles were added to a glass cell using a 1 mL syringe. The final concentration of DMPC was 6 × 10-5 M in the electrochemical cell and 1 × 10-4 M in the IR cell, respectively. Prior to experiments, glassware was cleaned in a hot mixture of concentrated nitric and sulfuric acids (ratio ≈ 1:3) for at least (33) Zamlynny, V.; Zawisza, I.; Lipkowski, J. Langmuir 2003, 19, 132. (34) Zawisza, I.; Bin, X.; Lipkowski, J. Bioelectrochem. Bioenerg. 2004, 63, 137. (35) Israelachvili, J. Intermolecular and surface forces; Academic Press: New York, 1985; Chapter 17, p 366. (36) Barenholz, Y.; Gibbes, D.; Litman, B. J.; Goll, J., Thompson, T. E.; Carlson, R. D. Biochemistry 1977, 16, 2806.

(37) Dickertmann, D.; Schultze, J. W.; Koppitz, F. D. Electrochim. Acta 1976, 21, 967. (38) Richer, J.; Lipkowski, J. J. Electrochem. Soc. 1985, 133, 121.

332

Langmuir, Vol. 21, No. 1, 2005

Bin et al.

stretching regions, D2O was used as the solvent, and for the asymmetric phosphate stretching and the CH2 bending regions, the solvent was H2O. The thickness of the thin layer was determined by comparing the experimental reflectivity spectrum of the thin layer cell, attenuated due to the layer of solvent between the electrode and the IR window, to the reflectivity curve calculated from the optical constants of the cell components.39 The demodulation technique developed in Corn’s laboratory33,40-41 was applied in this work. A modified version of a method described by Buffeteau et al.42 was used to correct the average intensity IA(ω) and the intensity difference ID(ω) for the PEM response functions and for the difference in the optical throughputs for p- and s-polarized light.33 Finally, the measured spectra had to be corrected for background due to the absorption of IR photons in the thin layer cavity. The spline interpolation technique described by Zamlynny et al.33 was used for the background correction. When all these corrections are introduced, the background-corrected spectrum plots ∆S(ω), which is the absorbance of the film of adsorbed molecules:

∆S(ω) )

(Is - Ip) (Is + Ip)/2

) 2.3A ) 2.3Γ

(1)

where  is the decimal molar absorption coefficient and Γ is the surface concentration of the absorbing molecules.

3. Results and Discussion 3. 1 Electrochemical properties. Spreading of DMPC vesicles onto a Au(111) electrode surface can be conveniently studied with the help of differential capacity. Figure 1a shows the differential capacity curve recorded for the supporting electrolyte alone and in the presence of DMPC vesicles in the solution. The positive potentials are limited by surface oxidation of gold, whereas the onset of hydrogen evolution defines the negative potential limit. The curves recorded in the presence of vesicles merge with that of the pure electrolyte when E < -1.1 V, indicating that DMPC does not adsorb at the gold surface at these negative potentials. A pseudo-capacity peak is observed at E ) -0.89 V, which indicates the onset of DMPC adsorption. At E ) ∼-0.5 V, another phase transition is observed, which is manifested by a steep drop of the differential capacity to C ) ∼10 µF/cm2. At potentials more positive than -0.4 V, a film of DMPC is formed at the Au(111) surface and the differential capacity slowly decreases to 8.5 µF/cm2 at 0.1 V. Independent AFM experiments performed on this system indicated that the film is a bilayer in the ripple phase.43 The AFM images revealed also that the film contains many defects and may be seen as an assembly of rafts separated by cracks filled with the solvent. The capacity of a metal electrode surface covered by a defect-free phospholipid bilayer C1 should be equal to ∼0.8 µF/cm.29 We may use the following equation:44

θ)

C0 - C C0 - C1

(2)

to estimate the coverage (θ) of the gold electrode surface by the DMPC bilayer. In eq 2, C0 and C are the capacities (39) Li, N.; Zamlynny, V.; Lipkowski, J.; Henglein, F.; Pettinger, B. J. Electroanal. Chem. 2002, 524/525, 43. (40) Barner, B. J.; Green, M. J.; Saez, E. I.; Corn, R. M. Anal. Chem. 1991, 63, 55. (41) Green, M. J.; Barner, B. J.; Corn, R. M. Rev. Sci. Instrum. 1991, 62, 1426. (42) Buffeteau, T.; Desbat, B.; Blaudez, D.; Turlet, J. Appl. Spectrosc. 2000, 54, 1646. (43) Li, M.; Lipkowski, J. Langmuir, in preparation. (44) Damaskin, B. B.; Petrii, O. A.; Batrakov, V. V. Adsorption of organic compounds on electrodes; Nauka: Moscow, 1968.

Figure 1. (a) Differential capacity, C, vs potential, E, curves at the Au(111) electrode in 0.1 M NaF (- - -) and 0.1 M NaF + 6 × 10-5 M DMPC (s). (b) Charge density, σM, vs potential, E, curves at the Au(111) electrode in 0.1 M NaF (filled squares) and 0.1 M NaF + 6 × 10-5 M DMPC (open squares). Inset: Surface pressure, π, vs potential, E, plot calculated by integration of the charge density curves.

of the film-free and film-covered surface. At E ) ∼0.1 V, C is equal to ∼8.5 µC/cm2, giving the surface coverage θ ) 74%. Figure 1b plots charge density curves determined from chronocoulometric experiments. These data are consistent with the differential capacity measurements. They show that DMPC vesicles spread on the Au surface in the potential range -0.4 < E < 0.4 V. In this potential region, the charge density of gold covered by the DMPC bilayer changes from ∼-10 µC/cm2 to ∼10 µC/cm2. The capacity and the charge density data can be combined to estimate the potential drop between the metal and the electrolyte solution (∆φM-S), which is approximately equal to the potential drop across the membrane, using the formula:45

∆φM-S )

σM + χM C

(3)

where χM is the surface potential of the membrane. Our data show that the membrane is lifted from the metal surface when the first term is more negative than -0.75 V. Figure 1b shows that the adsorption of the bilayer at the electrode surface causes a small, ∼150 mV, shift of the potential of zero charge in the negative direction. The shift of the pzc (EN) is described by46 EN ) χM - χw w ) Γ(µorg ⊥ - nµ⊥ )/, where χw is the surface potential of (45) Becucci, L.; Moncelli, M. R.; Guidelli, R. Langmuir 2003, 19, 3386.

DMPC Bilayer Supported at a Au(111) Electrode Surface

Langmuir, Vol. 21, No. 1, 2005 333

water molecules at the film free electrode surface, Γ is the surface concentration of DMPC,  is the permittivity, µ⊥org and µ⊥w are the components of the permanent dipoles of the DMPC and water molecules in the direction normal to the surface, and n is the number of water molecules displaced from the surface by one molecule of DMPC. At the pzc of the Au(111)-solution interface, water molecules are expected to have a small preferential orientation with the oxygen atom directed toward the metal, and hence, 47 µw and its sign should be ⊥ should be rather small negative. Therefore, the absolute value of the dipole potential due to the asymmetry in the orientation of DMPC molecules in the two leaflets (µorg ⊥ ) should be somewhat higher than 150 mV. Incidentally, this estimate agrees quite well with the value of the surface potential in freestanding bilayers of phosphatidylcholines, estimated to be ∼280 mV.48 These estimates indicate that the bilayer is lifted from the metal when ∆φM-S is more negative than -0.5V. The area between the charge density curves for the supporting electrolyte and the electrolyte containing DMPC vesicles corresponds to the surface pressure of the bilayer (lowering of the surface energy due to the presence of the membrane), which can be calculated using the following equation:46

of the transition dipole moment, and 〈E2〉 is the mean square electric field strength of the photon. At a gold electrode surface, the direction of the electric field of a p-polarized photon is normal to the surface. For randomly oriented molecules, cos2 θ ) 1/3. Hence, the angle, θ, for a given vibration can be determined from the formula:49

cos2 θ )

∫Aexpdv 3∫Acaldv

(6)

where θ is the angle between directions of the electric field of the photon and the transition dipole of the vibrational mode in the molecule, |µ| is the absolute value

where Aexp is the experimental background-corrected integrated intensity of a given IR band and Acal is the integrated intensity of the band calculated from the optical constants for a film of randomly oriented DMPC molecules whose thickness corresponds to the thickness of the DMPC bilayer. The Acal has to be calculated using the optical matrix method for reflection from an interface consisting of four phases: Au/DMPC/D2O or H2O/BaF2.33,39,50 Once the direction of the transition dipole with respect to the surface normal is known, the orientation of the DMPC molecules in the film may be determined by relating the direction of the transition dipole to the geometry of the molecule. The optical constants for DMPC were determined from transmission spectra using the procedure described in refs 33, 39, and 49. The transmission spectra of DMPC vesicles in D2O and H2O and a solution of DMPC in CCl4 were measured in a flow cell, consisting of two BaF2 flat windows separated by a 25 µm thick Teflon spacer. The DMPC concentration in D2O was 0.6286%, in H2O was 0.7619%, and in CCl4 was 0.6190% (v/v). The optical constants for Au and BaF2 were taken from ref 51, while the optical constants for D2O and H2O were taken from ref 52. The attenuation coefficient, k, was determined from the transmission spectra, and the refractive index, n, was then calculated from k using the Kramers-Kroening transformation as described in refs 33, 39, and 49. The average refractive index at infinite frequency, n∞ ) 1.4, was used for the whole spectral region.53,54 The optical constants for a solution of DMPC vesicles in D2O (3000-2800, 1800-1520, and 1120-900 cm-1) or H2O (1520-1120 cm-1) are shown in Figure 2. In the CH stretching region (Figure 2a), four peaks are seen at 2956, 2922, 2873, and 2853 cm-1 originating from CH3 asymmetric, CH2 asymmetric, CH3 symmetric, and CH2 symmetric stretches, respectively. Panel 2b shows the CdO stretching and the CH bending region of the IR spectrum. The CdO stretching is located at ∼1735 cm-1. A busy spectral region between 1550 and 1300 cm-1 corresponds to CH bending bands. The methylene scissoring mode is located at 1468 cm-1. The low-frequency shoulder corresponds to the CH3 bending band located at ∼1460 cm-1. The corresponding symmetric band is located at 1378 cm-1. The asymmetric bending (δas) modes of the methyl groups attached to the nitrogen atom in the choline moiety appear at 1490 and 1480 cm-1. The symmetric bending modes (δs) of the methyl group in the choline moiety are seen at 1418 and 1378 cm-1. Panel 2c shows the PdO stretching region in the phosphate group of the DMPC molecule.

(46) Lipkowski, J.; Stolberg, L. In Adsorption of molecules at metal electrodes; Lipkowski, J., Ross, P. N., Eds.; VCH Publishers: New York. 1992; pp 171-238. (47) Lipkowski, J.; Nguyen, C.; Huong, Van.; Hinnen, C.; Parsons, R.; Chevalet, J. J. Electroanal. Chem. 1983, 143, 375. (48) (a) Brockman, H Chem. Phys. Lipids 1994, 73, 57. (b) Clarke, R. J. Biochim. Biophys. Acta. 1997, 1327, 269. (49) (a) Allara, D. L.; Swalen, J. D. J. Phys. Chem. 1982, 86, 2700. (b)Allara, D. L.; Nuzzo, R. G. Langmuir 1985, 1, 52.

(50) Popenoe, D. D.; Stole, S. M.; Porter, M. D. Appl. Spectrosc. 1992, 46, 79. (51) Palik, E. Handbook of Optical Constants of Solid II; Academic Press: San Diego, 1998. (52) Bertie, J. E.; Ahmed, M. K.; Eysel, H. H. J. Phys. Chem. 1989, 93, 2210. (53) Frey, S.; Tamm, L. K. Biophys. J. 1991, 60, 922. (54) Flasch, C. R.; Gericke, A.; Mendelsohn, R. J. Phys. Chem B 1997, 101, 58.

π ) γo - γ )

E (σM - σM )dE ∫E)-1.25V 0

(4)

where γ0 and γ are the surface energies and σM and σM0 are the charge densities in the absence and presence of vesicles in solution, respectively. The surface pressure curve is shown in the inset to Figure 1b. It quantitatively describes the energetics associated with the spreading of the vesicles into a membrane at the metal-solution interface. The surface pressure plot is bell shaped with a maximum of approximately 56 mN/m1 at E ) ∼0.5 V. At potentials close to the pzc, the surface pressure is large and, hence, the bilayer is very stable at the electrode surface. However, the surface pressure decreases in a quasi-parabolic fashion with potential. This behavior shows that charging the metal leads to a significant decrease in the film pressure, with the bilayer being progressively detached from the metal surface. However, independent neutron reflectivity experiments27 have shown that when the bilayer is detached from the electrode surface at negative potentials, it remains in close proximity to the electrode separated from the gold surface by an ∼1 nm thick layer of the electrolyte. 3.2. FTIR Studies. 3.2.1. Optical Constants and Band Assignments. When linearly polarized light is absorbed by molecules adsorbed on a reflecting metal surface, the integrated intensity of the absorption band is proportional to the square of the dot product of the transition dipole, µ, and the electric field of the photon, E:33,49

∫Adv ∝ |µ‚E|2 ∝ cos2 θ|µ|2〈E2〉

(5)

334

Langmuir, Vol. 21, No. 1, 2005

Figure 2. Isotropic optical constants of DMPC calculated from the transmittance of its 0.6286% (v/v) solution in (a-c) D2O and 0.7619% (v/v) in H2O (b, c). (a) CH stretching region, (b) CdO stretching region, (c) PO stretching region, n (- - -) refractive index, k (s) attenuation coefficient.

The asymmetric νas(PO2-) is located at ∼1230 cm-1 and is overlapped with the νas(C-O-C) stretching in the ester group at ∼1180 cm-1. The symmetric νs(PO2-) stretching band is placed at 1087 cm-1 and is overlapped with the νas(C-O-[P]) stretching at 1066 cm-1. The peak located at 970 cm-1 is the asymmetric stretch of the C-N bond in the choline group, νas(CN+(CH3)3). DMPC is a zwitterionic molecule, consisting of two 14carbon saturated acyl chains, a glycerol backbone, a negatively charged phosphate group, and a positively charged choline group. Figure 3 shows a model of the DMPC molecule and the directions of the transition dipoles of the major bands whose positions are shown in Figure 2.55-57 Below, we will discuss the PM-IRRAS spectra for a DMPC bilayer on the Au(111) surface. The behavior of

Bin et al.

the acyl chain region will be described first and the polar headgroup region next. 3.2.2. Acyl Chains. A DMPC molecule contains 24 methylene groups in the two acyl chains and two methylene groups in its glycerol part. The CH2 groups of the chains dominate the methylene bands. Consequently, these bands provide information concerning conformation, orientation, and physical state of the chains. Figure 4a shows the IR spectra in the CH stretching region. The top dashed and the second solid lines plot bands for the bilayer of randomly oriented molecules, calculated from the transmission spectra of a solution of DMPC in CCl4 (dashed line) and a dispersion of DMPC vesicles in D2O (thick line), respectively. The four curves in the bottom plot the PM-IRRAS spectra for the bilayer of DMPC at the electrode surface at selected electrode potentials. This spectral region consists of four overlapping bands corresponding to νas(CH3), νas(CH2), νs(CH3), and νs(CH2) and two Fermi resonances between the overtones of the symmetric bending mode and symmetric methyl and methylene stretches.58 To extract quantitative information concerning the methylene bands, this spectral region had to be deconvoluted. The band deconvolution is shown in Figure 4b. In the DMPC bilayer spread at the Au surface, the νas(CH2) and the νs(CH2) bands are located at ∼2920 and 2851 cm-1, respectively. In a CCl4 solution of DMPC, their positions are 2927 and 2856 cm-1, in a dispersion of DMPC vesicles in D2O these bands appear at 2923 and 2853 cm-1, respectively. The full-widths at half-maximum (fwhm) of νas(CH2) and νs(CH2) in the bilayer supported at the gold electrode are equal to ∼16 and ∼9 cm-1, respectively. In the aqueous dispersion of DMPC vesicles, the fwhm of νas(CH2) and νs(CH2) are equal to 18 and 12 cm-1, respectively, and in DMPC dissolved in CCl4, they are equal to 21 and 12 cm-1, respectively. In the bilayer supported at the Au(111) surface, the frequencies of CH2 stretching bands are red-shifted and the bands are narrower in comparison to the same bands in the dispersion of vesicles and in the CCl4 solution. The frequencies of the band center lower than 2920 cm-1 for νas(CH2) and lower than 2850 cm-1 for νs(CH2) are characteristic for the gel state of the bilayer in which the acyl chains are fully stretched and assume the all-trans conformation.59-62 Higher frequencies of methylene bands indicate a presence of gauche conformations and melting of the chains. In the bilayer at the Au(111) electrode surface, the frequencies of the νas(CH2) and the νs(CH2) bands are at the borderline between the two states. They indicate that the acyl chains of the DMPC molecules are predominantly in the all-trans conformation. However, a small fraction of the chains has melted. This property is characteristic of the ripple phase.62,63 The fact that the half-width of these bands is lower than that in the spectrum for the aqueous dispersion of vesicles indicates a lower mobility of DMPC molecules in the bilayer supported at the gold surface. Figure 4c shows that the position and the half-width of (55) Fringeli, U. P. Z. Naturforsch. 1997, 32C, 20. (56) Fringeli, U. P.; Guenthard, H. H. Mol. Biol. Biochem. Biophys. 1981, 31, 270. (57) Fringeli, U. P. Biophys. J. 1981, 34, 173. (58) MacPhail, R. A.; Strauss, H. L.; Snyder, R. G.; Elliger, C. A. J. Phys. Chem. 1984, 88, 334. (59) Snyder, R. G.; Hsu, S. L.; Krimm, S. Spectrochim. Acta 1977, 34, 395. (60) Cameron, D. G.; Casal, H. L.; Mantsch, H. H. Biochemistry 1980, 19, 3665. (61) Snyder, R. G.; Strauss, H. L.; Elliger, C. A. J. Phys. Chem. 1982, 86, 5145. (62) Brandenburg, K.; Snyder, R. G. Z. Naturforsch. 1986, 41C, 453. (63) Heimburg, T. Biophys. J. 2000, 78, 1154.

DMPC Bilayer Supported at a Au(111) Electrode Surface

Langmuir, Vol. 21, No. 1, 2005 335

Figure 3. Schematic diagram of the DMPC molecule and the directions of the transition dipole moments of the major IR bands.

the νas(CH2) band depends on the electrode potential. Lower frequencies and narrower bands are observed at E < -0.4 V, indicating that the bilayer is more ordered when it is detached from the gold surface (with the exception of the last point for E ) -1.0 V). Higher frequencies and band broadening observed at E > -0.4 V indicate that the bilayer is less ordered when it is adsorbed and is in direct contact with the gold surface. However, the increase of the band frequency and the band broadening that accompany the potential-controlled adsorption of the bilayer are much smaller than the corresponding changes observed during the temperature-induced gel-liquid crystalline phase transition.25 Apparently, the disorder introduced by adsorption of the bilayer at the metal surface is very small in comparison to the disorder induced by the thermal melting of the chains. The curve recorded by changing the electrode potential in the positive direction does not overlap with the curve corresponding to the change of E in the negative direction. Such hysteresis is typical for phospholipid bilayers at electrode surfaces and has been observed in previous studies.25-27 Similar changes in the band position and width were observed for the νs(CH2) band, but they were much smaller than for the νas(CH2) band. Figure 4a shows that the intensities of the methylene stretching bands change with potential. The intensity is low at negative potentials where the bilayer is detached from the metal and increases at more positive E when the bilayer adsorbs on the gold surface. This behavior indicates that adsorption/detachment of the film have an influence on the orientation of DMPC molecules and, consequently, on the angle, θ, between the direction of the transition dipole of a given vibration and the surface normal. The angle, θ, can be calculated with the help of eq 6. Figure 5a and b plot θs(CH2) and θas(CH2) as functions of the electrode potential. The angles, θs(CH2) and θas(CH2), have comparable magnitude, and this property indicates that

a rotational motion of DMPC molecule or a wobbling in a cone are not constrained.64,65 The integrated band intensities are affected by systematic errors of the background correction and the band deconvolution procedure. However, since θ is determined from the ratio of band intensities, these errors cancel out to a large extent if the same analysis is applied to the PM-IRRAS and transmission spectra from which the optical constants are calculated. Different background corrections and different deconvolution procedures used in the data analysis gave angles that differed by no more than 3°. This number is also a measure of the reproducibility of our measurements because the uncertainties of the background correction and the deconvolution procedures constituted the major source of the error of the tilt angle measurement. However, transition dipoles for several IR bands have the same direction. This property allowed us to measure the same tilt angles from bands in different spectral regions, having different backgrounds and band deconvolution conditions. The agreement among these tilt angles was also at the level of 3° or better. In principle, an additional error in the reported values of the tilt angle may arise from small differences between conformations of DMPC molecules in vesicles and in the bilayer film. However, the differences between integrated intensities of the CH2 stretch bands in the solution of DMPC in CCl4 and in the dispersion of DMPC vesicles are small (2% for the symmetric and 4% for the asymmetric stretches) despite large differences between the conformation state. Since the differences between the conformation state of DMPC in the bilayer and in the suspension of vesicles are much smaller, this error is most likely negligible. (64) Aussenac, F.; Laguerre, M.; Schmitter, J.-M.; Dufourc, E. J. Langmuir 2003, 19, 10468. (65) Pascher, I.; Lundmark, M.; Nyholm, P.-G.; Sundell, S. Biochim. Biophys. Acta 1992, 1113, 339.

336

Langmuir, Vol. 21, No. 1, 2005

Bin et al.

plotted as a function of the electrode potential in Figure 5c. The tilt angle, θ, of the hydrocarbon chains in the DMPC bilayer supported at the gold electrode surface changes from ∼35° at potential range -1.0 < E < -0.5 V to ∼55° at E > -0.4 V. For negative potentials, the tilt angle for the bilayer supported at the electrode surafce is equal to the tilt angle determined for hydrated multibilayers of DMPC in the liquid crystalline state at ∼35 °C67a and is somewhat higher than the angle of 28° determined for a dry film of DMPC deposited at a CaF2 surface at room temperature.67b It also agrees favorably with the value 29°-33° for thin hydrated films of DPPC reported in refs 55 and 56. However, eq 7 is strictly applicable for a film with all molecules in the trans conformation. In our case, the trans conformation is predominant. Nevertheless, the film contains a certain percentage of gauche conformers. Their presence introduces an error to reported values of the chain tilt angle whose magnitude is difficult to assess. To compare our results with the rich literature concerning X-ray diffraction data for a stack of DMPC bilayers, it is convenient to calculate the area per molecule (S) with the help of the formula:73

S cos θchain ) 2Σ

Figure 4. (a) PM-IRRAS spectra in the CH stretching region of a DMPC bilayer on a Au(111) electrode in 0.1 M NaF/D2O solution at potentials indicated on the figure. The top two traces plot spectra calculated for 5.5 nm thick film of randomly oriented DMPC molecules calculated using optical constants of DMPC for vesicles dispersed in D2O (s) and solution of DMPC in CCl4 (- - -). (b) Example of a deconvolution of the overlapping νas (CH3), νas (CH2), νs (CH3), and νs (CH2) bands and two Fermi resonances between the overtones of the symmetric bending mode and symmetric methyl FRvs(CH2) and methylene stretches FRvs(CH3) modes for the DMPC bilayer at E ) -0.5 V. (c) Peak position of νas (CH2) (9) positive, (0) negative potential step; and the full-widths at half-maximum (fwhm) of νas(CH2) (b) positive, (O) negative potential steps. For the fwhm, the line is a guide to the eye, not a fit to the data.

For a fully stretched all-trans conformation of acyl chains, θs(CH2), θas(CH2), and the chain tilt angle θchain are related by the formula:66

cos2 θas + cos θs + cos2 θchain ) 1

(7)

Therefore, eq 7 allows one to calculate the chain tilt angle

(8)

where 2Σ is the cross sectional area of the two acyl chains; for DMPC in the gel state, 2Σ ) 38 Å2, and in the liquid crystalline state, 2Σ ) 48.8 Å2.65 In the present case, the chains are predominantly in the all-trans conformation and 2Σ ) 38 Å2. At negative potentials, the area per molecule is equal to 46.4 Å2. This number is very close to the value of 47 Å2 determined for the gel phase of a stack of fully hydrated bilayers.68,69 The chain tilt angle and hence the area per molecule depend on the hydration state of the bilayer and are higher in the fully hydrated state.67,70,71 The magnitude of the area per molecule determined for the film of DMPC at negative potentials indicates that the bilayer is fully hydrated. The change of the tilt angle upon adsorption is dramatic. In the adsorbed state, the area per molecule increases to 66.2 Å2. This number is somewhat higher than the area per DMPC molecule in the liquid crystalline state of the bilayer, for which the literature reports values that range from 59.5 to 63.4 Å2.72 An expansion of the molecular area of phospholipids to a value in the range between 60-70 Å2 indicates a significant change in the packing of the polar head region and is possible only in the presence of water molecules that act as spacers forming bridges between the headgroups.73 Therefore, the increase of the tilt angle of the chains is expected to be accompanied by an increase in the hydration of the polar head region of the bilayer. However, in the adsorbed state, the tilt angle is very close to the value of 54.5° expected for randomly oriented (66) Umemura, J.; Kamata, T.; Kawai, T.; Takenaka, T. J. Chem. Phys. 1990, 94, 62. (67) (a) Ter-Minassian-Saraga, L.; Okamura, E.; Umemura, J.; Takenaka, T. Biochim. Biophys. Acta 1988, 946, 417. (b) Nabedryk, E.; Gingold, M. P.; Breton, J. Biophys. J. 1982, 38, 243. (68) Tristram-Nagle, S.; Zhang, R.; Suter, R. M.; Worthington, C. R.; Sun, W.-J.; Nagle, J. F. Biophys. J. 2002, 83, 3324. (69) Tristram-Nagle, S.; Liu, Y.; Legleiter, J.; Nagle, J. F. Biophys. J. 1993, 64, 1097. (70) Katsaras, J. D.; Yang, S.-C.; Epand, R. M. Biophys. J. 1992, 63, 1170. (71) Smith, G. S.; Sirota, E. B.; Safinya, C. R.; Clark, N. A. Phys. Rev. Lett. 1988, 60, 813. (72) Nagle, J. F.; Tristram-Nagle, S. Biochim. Biophys. Acta 2000, 1469, 159. (73) Hauser, H. Pascher, I.; Pearson, R. H.; Sundell, S. Biochim. Biophys. Acta 1981, 650, 21.

DMPC Bilayer Supported at a Au(111) Electrode Surface

Langmuir, Vol. 21, No. 1, 2005 337

Figure 5. Dependence of the angle (θ) between directions of the transition dipole moment and the electric field of the photon (normal to the surface) on potential for the DMPC bilayer on the Au(111) electrode in 0.1 M NaF/D2O solution for (a) νs(CH2), (b) νas(CH2), and (c) the direction of the acyl chain; (9) positive, (0) negative potential steps.

molecules. Therefore, the data in Figure 5 may also be interpreted in terms of a dramatic disorder introduced into the film by adsorption onto the electrode surface. In that case, however, a significant melting of the chains should be observed along with a significant blue shift of the CH2 stretch band positions. Further, in our previous work,25 we have demonstrated that in the adsorbed state the bilayer undergoes the gel-liquid crystalline phase transition at ∼24 °C, which is a similar temperature to that in the suspension of vesicles. With the help of this additional information, we may exclude the possibility that adsorption of the bilayer causes total disorder and a random orientation of DMPC molecules. The CH2 bending band (δ(CH2)) is very sensitive to the interchain interaction and, hence, provides useful information concerning packing of the acyl chains in the

bilayer.74-78 The chains are known to pack into triclinic, orthorhombic, monoclinic, and hexagonal subcells65 which define chain positions with respect to the lateral neighbors. A splitting of the CH2 band is observed when chains are packed into an orthorhombic or monoclinic subcell. In contrast, a single δ(CH2) band is observed if the chains are packed into a hexagonal or triclinic subcell.74 Figure 6a shows bands in the CH bending spectral region. The top and the second lines plot the spectra for (74) Snyder, R. G. J. Mol. Spectrosc. 1961, 7, 116. (75) Tasumi, M.; Shimanouchi, T. J. Chem. Phys. 1965, 43, 1245. (76) Snyder, R. G.; Liang, G. L.; Strauss, H. L.; Mendelsohn, R. Biophys. J. 1996, 71, 3186. (77) Snyder, R. G.; Goh, M. C.; Srivatsavoy, V. J. P.; Strauss, H. L.; Dorset, D. L. J. Phys. Chem. 1992, 96, 10008. (78) Pelletier, I.; Laurin, I.; Buffeteau, T.; Desbat, B.; Pezolet, M. Langmuir 2003, 19, 1189.

338

Langmuir, Vol. 21, No. 1, 2005

Figure 6. (a) PM-IRRAS spectra in the CH bending modes region of a DMPC bilayer on a Au(111) electrode in 0.1 M NaF/ D2O solution at different potentials marked on the figure. The two top traces plot the calculated spectra for 5.5 nm thick film of randomly oriented molecules using optical constants of DMPC for vesicles dispersed in D2O (s) and solution of DMPC in CCl4 (- - -). (b) Example of a deconvoluted spectrum of the overlapping δas(N+(CH3)3), δ(CH2), and δas(C(CH3)) modes at E ) -0.5 V. (c) The dependence of the angle (θ) between the direction of the transition dipole moment and the surface normal on potential in the DMPC bilayer on the Au(111) electrode in 0.1 M NaF/ D2O solution for δs(CH2). (9) positive, (0) negative potential steps.

the CCl4 solution of DMPC and the aqueous dispersion of vesicles, respectively. The bottom curves plot the spectra for the bilayer at the Au(111) electrode surface at selected electrode potentials. This busy region consists of several overlapping CH bending modes whose deconvolution is shown in Figure 6b. Significantly, only one methylene bending mode centered at 1468 cm-1 is present in these spectra. This indicates that chains are laterally packed either in a tilted hexagonal or triclinic subcell.

Bin et al.

Figure 6a shows that the intensity of the δ(CH2) depends on the electrode potential, indicating that the direction of the transition dipole of this mode changes with E. Figure 6c plots the angle between the transition dipole and surface normal for the δ(CH2) mode, calculated with the help of eq 6. The angle changes from 69° at negative potentials to 56° at positive potentials. The transition dipole of δ(CH2) is parallel to the bisector of the H-C-H angle and has the same direction as the transition dipole of the νs(CH2) vibration.55 Indeed, the numerical values of the angles between the directions of the transition dipoles of the δ(CH2) and νs(CH2) and the surface normal agree within 1°. Different spectral regions provide consistent structural information. 3.2.3. The Headgroup Region. 3.2.3.1. The Glycerol Ester Group. Two bands correspond to the vibrations of the glycerol ester group: the CdO stretch at ∼1740 cm-1 (ν(CdO)) and the asymmetric C-O-C stretch at ∼1180 cm-1. Figure 7a shows the CdO ester group stretching bands. The dashed top line plots the band for the CCl4 solution of DMPC, and the second solid line marks the band in the spectrum of DMPC vesicles dispersed in D2O. The four traces in the lower portion of the figure plot bands of the bilayer at the gold electrode surface for selected electrode potentials. The transmission spectra show that the shape and the position of the ν(CdO) band depend on the nature of the solvent. In the dispersion of DMPC, the ν(CdO) band is broad because it is composed of two overlapping bands with maxima at 1744 and 1734 cm-1, which correspond to the presence of non-hydrogen-bonded and hydrogen-bonded ester groups, respectively.79-81 In the CCl4 solution, the ν(CdO) band has a maximum at 1740 cm-1 and is narrower because absorption by the nonhydrogen-bonded ester groups is predominant in this solvent. However, this band has a shoulder at ∼1730 cm-1 due to the presence of residual water of hydration. These examples and rich literature data82-90 indicate that the ν(CdO) bands can provide useful information concerning the potential-induced changes in the local environment of the carbonyl ester group and particularly changes in the hydration of the ester group. For the bilayer on the Au(111) surface, Figure 7b shows changes of the ν(CdO) band position with potential. In the desorbed film, the maximum of the absorption of ν(CdO) band is located at 1740 cm-1, indicating that the non-hydrogen-bonded state dominates the spectrum. At E > -0.4 V, the band at 1730 cm-1 grows and causes a red shift of the ν(CdO) overall band position to 1738 cm-1. At potentials higher than 0.2 V, the frequency of the ν(CdO) band increases again to 1739 cm-1. The inset in Figure 7b shows that the plot of the ν(CdO) band maximum and the differential capacity curve have similar profiles. The spectroscopic and the electrochemical data (79) Blume, A.; Hu¨bner, W.; Messner, G. Biochemistry 1988, 27, 8239. (80) Hu¨bner, W.; Mantsch, H. H. Biophys. J. 1991, 59, 1261. (81) Lewis, R. N. A. H.; McElhaney, R. N.; Pohle, W.; Mantsch, H. H. Biophys. J. 1994, 67, 2367. (82) Lewis, R. N. A. H.; McElhaney, R. N. Chem. Phys. Lipids 1998, 96, 9. (83) Fringeli, U. P.; Gu¨nthard, H. H. Biochim. Biophys. Acta 1976, 450, 101. (84) Wong, P. T. T.; Mantsch, H. H. Chem. Phys. Lipids 1988, 46, 213. (85) Mantsch, H. H.; McElhaney, R. N. Chem. Phys. Lipids 1991, 57, 213. (86) Blume, A.; Hu¨bner, W.; Messner, G. Biochemistry 1988, 27, 8239. (87) Hu¨bner, W.; Mantsch, H. H. Biophys. J. 1991, 59, 1261. (88) Lewis, R. N. A. H.; McElhaney, R. N.; Pohle, W.; Mantsch, H. H. Biophys. J. 1994, 67, 2367. (89) Lewis, R. N. A. H.; McElhaney, R. N. Biochemistry 1990, 28, 7946. (90) Lewis, R. N. A. H.; McElhaney, R. N. Biophys. J. 1993, 65, 1866.

DMPC Bilayer Supported at a Au(111) Electrode Surface

Figure 7. (a) PM-IRRAS spectra in the ν(CdO) stretching region of a DMPC bilayer on a Au(111) electrode in 0.1 M NaF/ D2O solution at potentials indicated on the figure. The top two traces plot spectra calculated for 5.5 nm thick DMPC bilayer using optical constants of DMPC for vesicles dispersed in D2O (s) and solution of DMPC in CCl4 (- - -). (b) The dependence of the position of the maximum absorption frequency of the ν(CdO) stretch on the electrode potential in a DMPC bilayer at a Au (111) surface; (9) positive and (0) negative potential steps. Inset: Differential capacity, C, of the Au(111) electrode DMPC covered by the DMPC bilayer. (c) Angle (θ) between directions of transition dipole moment of ν(CdO) and the surface normal on the electrode potential for a DMPC bilayer at a Au(111) electrode in 0.1 M NaF/D2O solution; (9) positive, (0) negative potential step.

correlate very well. The IR data demonstrate that the adsorption/detachment of the bilayer leads to significant changes in the hydration of the ester group. The ester carbonyl group is less hydrated at negative potentials, where the bilayer is detached from the gold surface. It is much better hydrated when the bilayer is adsorbed at the gold surface at E > -0.4 V. When the bilayer is in direct

Langmuir, Vol. 21, No. 1, 2005 339

contact with the metal surface, more water penetrates into its polar head region. This behavior is consistent with the change of the area per molecule discussed in the previous section. Figure 7a shows that the intensity of the ν(CdO) band increases by a factor of 2 on moving from negative to positive potentials. Such a large increase cannot be explained by hydration effects and indicates that the orientation of the CdO groups changes with the electrode potential. Equation 6 may be used again to calculate the angle between the direction of the transition dipole of the ν(CdO) band and the normal to the surface. The calculated angle is a measure of the average orientation of the two ester groups. Figure 7c plots the mean θCdO as a function of the electrode potential. The tilt angle is about 70° at negative potential, when the DMPC bilayer is detached from the gold surface. This number compares well with the literature that reports an angle of 67° for a dry film of DMPC at room temperature67b and angles of 65° and 64° for dry and hydrated multibilayers of DMPC in the gel state (at ∼28 °C). When the DMPC bilayer absorbs onto the Au(111) surface, θCdO decreases to ∼62°. Fringeli reported that the direction of the transition dipole moment of the carbonyl stretch is parallel to the CdO bond.55 However, crystallographic data73 show that the CdO bonds of a DMPC molecule of the β and γ acyl chains are located in different planes and that the angle between these bonds is approximately 90°. Further, the DMPC molecule may assume two different conformations (molecules A and B) in which the CdO bonds in the β chain are oriented in different directions.73 Recent NMR and molecular dynamics simulations indicate that a bilayer consists of 80% DMPC-A and 20% DMPC-B.64 We have performed ab initio normal coordinate quantum chemical calculations described in the Appendix to determine the direction of the transition dipole of the Cd O stretch band. In addition, the Supporting Information shows animated vibrations corresponding to the ∼1740 cm-1 band. The results of the normal coordinate calculations show that the vibrations of the CdO groups in the β and γ acyl chains are coupled and as a result the CdO bands split into two bands corresponding to the in-phase and the outof-phase motions of the oxygen atoms. In the case of the DMPC-A conformation, the in-phase and the out-of-phase bands are separated by ∼5 cm-1 and the coupling is weak. The transition dipoles of the two bands are almost perpendicular to the chains; however, their direction rotates in the plane normal to the chains. In the DMPC-B molecule, the two bands are separated by only 1 cm-1 and the vibrations are strongly coupled. The transition dipole of the in-phase mode is nearly perpendicular to the direction of the chains. However, the transition dipole of the out-of-phase mode is nearly parallel to the chains. The normal coordinate calculations demonstrate that the ν(CdO) band has a very complex structure. Nevertheless, a comparison of Figures 5c and 7c shows that the chain tilt angle and the angle of the transition dipole of the ν(CdO) band change with potential in the opposite direction. This behavior indicates that the transition dipole of the ν(CdO) band is oriented at approximately 90° with respect to the acyl chains and that the conformation A is the dominant structure of the DMPC molecules. The asymmetric C-O-C stretch of the ester groups in the β and γ acyl chains is observed at a frequency of ∼1190 cm-1 and overlaps with the asymmetric PO2- stretch at ∼1240 cm-1. Figure 8 shows IR bands in this spectral region. Figure 8a plots spectra calculated from the transmission measurements for a solution of DMPC in

340

Langmuir, Vol. 21, No. 1, 2005

Figure 8. (a) PM-IRRAS spectra for the νas(PO2-) region of a DMPC bilayer on a Au(111) electrode in 0.1 M NaF/H2O solution at potentials indicated on the figure. The top two traces plot spectra calculated for 5.5 nm thick DMPC bilayer using optical constants of DMPC for vesicles dispersed in H2O (s) and solution of DMPC in CCl4 (- - -). (b) Example of the deconvolution of a spectrum from part (a). (c) Angle (θ) between directions of the transition dipole moment of νas(C-O-C) and the surface normal plotted against the electrode potential for a DMPC bilayer on a Au(111) electrode in 0.1 M NaF/D2O solution; (9) positive, (0) negative potential steps. The lines in the panel (c) are guides to the eye, not fits to the data.

CCl4, the aqueous solution of vesicles, and the PM-IRRAS spectra of the bilayer at the Au(111) electrode surface. The band deconvolution is shown in Figure 8b. The bands are weak, and hence, the signal-to-noise ratio (S/N) is relatively poor in this spectral region. Nevertheless, the bands can be deconvoluted and analyzed. Figure 8c plots the angle between the direction of the transition dipole of the ∼1190 cm-1 band and the surface normal as a function of the electrode potential.

Bin et al.

The C-O-C vibrations of the ester group are strongly coupled with wagging modes.55 Indeed, the ab initio calculations described in the Appendix show that estergroup vibration is strongly coupled with the wagging motions of the tails. This coupling could be seen in the movie shown in the Supporting Information of this paper. In contrast, the calculations also show that the coupling of the vibrations of the -C(O)-O-C- groups in the β and γ chains is rather weak. The ester group in the γ chain is coplanar with the chain, while the ester group of the β chain is out of the plane of the chain.73 According to Fringeli,55 the C-O-C band of the γ chain is observed at ∼1180 cm-1 and the direction of the transition dipole of this band is parallel to the chain. The frequency of the C-O-C band in the β chain is shifted to lower frequencies, and the direction of its transition dipole is no longer parallel to the chain. The C-O-C band in Figure 8a is observed at ∼1190 cm-1, and hence, it may be considered as being dominated by the vibrations of the C-O-C group in the γ chain. The solid and the dashed lines in Figure 8c plot the chain tilt angles previously shown in Figure 5c. While the scatter of the experimental points for the C-O-C band is large due to the poor S/N and uncertainties in the background correction and the band deconvolution procedure, we cannot claim that they fit the lines. However, the angles for the transition dipole of the C-O-C band are certainly in the same range as the tilt angles of the chains consistent with Fringeli’s interpretation. 3.2.3.2. Phosphate Group. The bands of the phosphate group include asymmetric νas(PO2-) and symmetric νs(PO2-) stretches of the PO2- moiety and two bands corresponding to complex asymmetric vibrations of the phosphate ester group (νas(C-O[P])). The asymmetric stretch of the phosphate group overlaps with the C-O-C stretch of the ester group, and these bands are shown in Figure 8a. The νas(PO2-) band has a maximum at 1255 cm-1 in the CCl4 solution. In the aqueous dispersion of vesicles, the band is broader and its maximum is redshifted to 1237 cm-1. In fact, this is a composite band that has two components centered at 1228 and 1246 cm-1. The lower-frequency component is assigned to the hydrated phosphate group, while the higher-frequency band is assigned to the non-hydrogen-bonded phosphate group.91,92 In the PM-IRRAS spectra, the noise level in the region of the νas(PO2-) band was too high to allow for band deconvolution and a detailed analysis of the band shape. Nevertheless, the data show that the band center shifts toward lower frequencies on moving from negative to positive potentials. This behavior indicates a stronger hydration of the phosphate group when the bilayer is adsorbed at the electrode surface, consistent with the behavior of the CdO group discussed earlier. Figure 9a shows the 1125-1000 cm-1 spectral region that contains the symmetric phosphate group stretch νs(PO2-) at ∼1090 cm-1 overlapping with two asymmetric νas(C-O[P]) stretching bands at ∼1070 and 1055 cm-1.26,55,56,92 The two top spectra correspond to randomly oriented molecules. They were calculated from the data obtained from the transmission measurements for a solution of DMPC in CCl4 (dashed line) and a suspension of DMPC vesicles in D2O (thick solid line). The four traces in the lower portion of the figure plot the PM-IRRAS spectra acquired for selected electrode potentials, indicated in the figure. The band deconvolution is shown in Figure 9b. The νs(PO2-) band has maximum at 1091 cm-1 in the (91) Hubner, W.; Blume, A. Chem. Phys. Lipids 1998, 96, 99. (92) Casal, H. L. Mantsch, H. H. Biochim. Biophys. Acta 1984, 779, 381.

DMPC Bilayer Supported at a Au(111) Electrode Surface

Langmuir, Vol. 21, No. 1, 2005 341

Figure 9. (a) PM-IRRAS spectra for νs(PO2-) region of a DMPC bilayer on a Au(111) electrode in 0.1 M NaF/D2O solution at potentials indicated on the figure. The top two traces plot spectra calculated for 5.5 nm thick DMPC bilayer using optical constants of DMPC for vesicles dispersed in D2O (s) and solution of DMPC in CCl4 (- - -). (b) Example of the deconvolution of a spectrum in panel (a).

They are also perpendicular to the line connecting the two esterified oxygen atoms of the phosphate group. Hence, eq 7 can be used to calculate the angle between the -O-P-O- line and the surface normal. The changes of this tilt angle with potential are shown in Figure 10c. The angle changes from ∼32° at negative potentials where the bilayer is detached from the metal surface to ∼38° at E > -0.4 V where it is absorbed at the gold electrode surface. Figure 9b shows that the νas(C-O[P]) band of the ester phosphate group may be deconvoluted into two components. The lower-frequency component corresponds to the C-O stretch of the phosphate ester group bonded to the choline moiety. The higher-frequency component corresponds to the ester phosphate group attached to the glycerol moiety.26 For the suspension of DMPC vesicles in D2O, the deconvoluted bands have absorption maxima at 1055 and 1068 cm-1. In the CCl4 solution of DMPC, these peaks appear at 1065 and 1074 cm-1. For the DMPC bilayer on the electrode surface, the lower-frequency νas(C-O[P]) band does not change with potential and is centered at ∼1056 cm-1. However, the position of the higher-frequency band changes from 1073 cm-1 at E < -0.4 V to 1070 cm-1 at E >-0.4 V. This red shift can be explained by an increased hydration of the polar group region in the bilayer adsorbed on the electrode surface. The peak around 1070 cm-1 originates from the νas(CO[P]) stretch of the ester group attached to glycerol moiety. This vibration is coupled to the P-O stretch and C-O stretch in the glycerol part of the lipid molecule. This part of the DMPC molecule becomes more hydrated when the film is adsorbed on the electrode surface. On the other hand, the lower-frequency band involves vibrations of the ester group bonded to the choline group, which is less affected by hydration.26 Figure 11a and b plot the angles between directions of the transition dipoles of the two νas(C-O[P]) stretches and the surface dipoles. The normal coordinate analysis described in ref 26 showed that the direction of the transition dipole of the 1056 cm-1 band is located in the plane of the PO2- group parallel to the line joining the two nonesterified oxygen atoms of the O-P-O moiety. In fact, it has the same direction as the transition dipole of the νas(PO2-) band. Indeed, a comparison of Figures 10a and 11a shows that the angles between the direction of the transition dipoles of the νas(PO2-) and νas(C-O[P]) bands have comparable magnitude. Clearly, consistent information is obtained from the analysis of the IR bands in different spectral regions. The direction of the transition dipole of the ∼1070 cm-1 band is parallel to the C-O bond of the P-O-C line. The tilt angle of this transition dipole changes from ∼40° at negative potentials to ∼65° at positive potentials. This is a larger change than for the -O-P-O- line observed in Figure 10c. This behavior suggests that not only the tilt of the phosphatidyl-choline group changes with potential but also the group rotates when E is changed from negative to positive values. 3.2.3.3. Choline Moiety. Information concerning the choline group can be extracted from the 1500-1350 cm-1 region where C-H bending modes of the methyl groups in the N+(CH3)3 moiety are located and in the 850 and 1000 cm-1 regions where C-N stretching bands can be found. The C-H bending bands of the choline group overlap with the bending modes of the terminal methyl groups of the acyl chains. The deconvolution of this spectral region is shown in Figure 6b. The asymmetric bending modes of the methyl groups attached to the nitrogen atom

aqueous dispersion of vesicles. The band position is blueshifted to 1098 cm-1 in the CCl4 solution. In the bilayer supported on the Au electrode surface, the maximum of the νs(PO2-) band shifts from ∼1094 to ∼1089 cm-1 when the potential changes from -1.0 V to 0.4 V. Simultaneously, the fwhm of the band increases from ∼17 to ∼20 cm-1. Consistent with the results for other spectral regions, these changes indicate that the phosphate group is more hydrated when the bilayer is absorbed on the electrode surface. Assuming that the absolute values of the transition dipoles of the phosphate group are approximately equal for the supported bilayer and for the unilamellar vesicles, the angle between the direction of the transition dipole and the surface normal can be determined with the help of eq 6. The direction of the transition dipole of νas(PO2-) band is parallel to the line connecting the two nonesterified oxygen atoms of the phosphate group. The transition dipole of νs(PO2-) stretch is pointed along the bisector of the PO2group.26,55,56 Figure 10a and b plot the angle between the directions of the transition dipoles of the asymmetric and the symmetric stretches and the surface normal (θas(PO2-) and θs(PO2-) as a function of the electrode potential. The angles θas(PO2-) and θs(PO2-) change when the bilayer is adsorbed/desorbed, indicating that the potential controlled adsorption/desorption process leads to a reorientation of the phosphate group. The transition dipoles of the asymmetric and symmetric stretches are located in the plane of the nonesterified group and are perpendicular to each other.26,55,56

342

Langmuir, Vol. 21, No. 1, 2005

Bin et al.

Figure 10. Dependence of the angle (θ) between directions of the transition dipole moment and the normal to the surface on the electrode potential for the DMPC bilayer on the Au(111) electrode (a) νas(PO2-) in 0.1 M NaF/H2O solution, (b) νs(PO2-) 0.1 M NaF/D2O, and (c) tilt angle between the line connecting two esterified oxygen atoms and the surface normal; (9) positive, (0) negative potential steps. The lines are guides to the eye. Cartoons show the orientation of the phosphate group and the directions of the as and s phosphate stretching transition dipole moments.

in the choline group appear at ∼1490 and ∼1480 cm-1 and the symmetric modes are at ∼1420 and ∼1380 cm-1. The symmetric bands are much weaker and more difficult to discern from the background in the PM-IRRAS spectrum. Therefore, we will restrict further discussion to the asymmetric bands. In the transmission spectrum of DMPC dissolved in CCl4, the two δas(N+(CH3)3) bands appear at 1481 and 1493 cm-1. In the spectrum of DMPC vesicles in D2O, the two δas(N+(CH3)3 appear at 1478 and 1491 cm-1. In the spectrum of the DMPC bilayer supported at the Au electrode surface, the center of the higher-frequency band

is observed at 1491 cm-1 independently of the electrode potential. The center of the lower-frequency band changes from a value of 1480 cm-1 at E < -0.4 V to 1478 cm-1 when E > -0.4 V. Such a red shift may indicate that the overall volume of the choline group decreases on moving from negative to positive potentials.84 Overall, changes of the band shape and the band position for the choline group are much smaller than for the carbonyl and phosphate groups discussed earlier. This behavior indicates that the choline group is not hydrogen bonded to water. Only those C-N stretching bands with frequencies higher than ∼900 cm-1 were measured in our experiment

DMPC Bilayer Supported at a Au(111) Electrode Surface

Langmuir, Vol. 21, No. 1, 2005 343

Figure 11. Dependence of the angle (θ) between the transition dipole moment and the surface normal for the DMPC bilayer at Au(111) electrode in 0.1 M NaF/D2O for the (C-O[P]) stretching bands at (a) 1050 cm-1 and (b) 1070 cm-1; (9) positive, (0) negative potential steps. The lines are guides to the eye, not fits to the data. Cartoons show the orientation of the phosphate group and the directions of the as and s phosphate stretching dipole moments.

due to proximity of the cutoff frequency of the BaF2 prism (ca. 800 cm-1) and low performance of photoelastic modulator at these low frequencies. The transmission spectra in Figure 12a show three bands at ∼970, ∼950, and ∼925 cm-1 in the 1000-900 cm-1 region. In the PMIRRAS spectra, the band at ∼925 cm-1 is not present. The strongest (νasC-N+(CH3)3) is located at 973 cm-1 in dispersion of vesicles in D2O and at 971 cm-1 in the CCl4 solution. In the PM-IRRAS spectra, the C-N asymmetric stretch is localized at 972 cm-1, and its position is not dependent on the electrode potential position. Consistent with the behavior of the C-H bending modes of the choline group, the position and the shape of the C-N stretch bands depend weakly on the solvent and do not change with the electrode potential. The directions of the transition dipoles of the choline group vibrations were determined by Fringeli.55,56 Considering the -N(CH3)3 fragment as a subsystem with C3v symmetry, the symmetry of the δas(N+(CH3)3) at ∼1490 cm-1 was classified as A1 and the symmetry of the δas(N+(CH3)3) at ∼1480 cm-1 as E. This gives the direction of the transition dipole of the higher-frequency band to be

parallel to the C3 axis of the choline group, while the direction of the transition dipole of the lower-frequency mode is located in the plane of the three methyl groups and perpendicular to the C3 axis. The cartoons in Figure 12 show the directions of the transition dipoles of the ∼1480 and ∼1490 cm-1 bands. The angles between the directions of these transition dipoles and the surface normal are plotted against the electrode potential in Figure 12b and c. The values of these angles are quite large and display a weak dependence on the electrode potential. Cs symmetry of the R- N(CH3)3 group was assumed for the C-N stretching band.55 When the site symmetry is lowered to Cs, the (νasC-N+(CH3)3) band splits into three components. The doublet at ∼970 cm-1 is anti-symmetric, and its transition dipole is located in the plane of the three methyl groups, while the band at ∼920 cm-1 is symmetric and its dipole is parallel to the C3 axis. The direction of the (νasC-N+(CH3)3) band at ∼970 cm-1 is shown in the cartoon. The angle between the direction of the transition dipole of the ∼970 cm-1 band and the surface normal is plotted against potential in Figure 12d. Indeed, it has similar magnitude and displays a similar change

344

Langmuir, Vol. 21, No. 1, 2005

Bin et al.

Figure 12. (a) PM-IRRAS spectra in the CN stretch of choline group νas(CN+(CH3)3) region for a DMPC bilayer at a Au(111) electrode in 0.1 M NaF/D2O solution at different potentials marked on the figure. The top two traces plot spectra calculated for 5.5 nm thick DMPC bilayer using optical constants of DMPC for vesicles dispersed in D2O (s) and solution of DMPC in CCl4 (- - -). Panels (b), (c), and (d): the dependence of the angle (θ) between the transition dipole moment and the surface normal for the DMPC bilayer at the Au(111) electrode in 0.1 M NaF/D2O for νas(CN+(CH3)3), δas(CN+(CH3)3) (∼1490 cm-1) and δas(CN+(CH3)3) (∼1480 cm-1) respectively; (9) positive, (0) negative potential steps. The lines in panels (b-d) are guides to the eye, not fits to the data. Cartoons show the orientation of the phosphate group and the directions of the as and s phosphate stretching transition dipole moments.

with potential as the δas(N+(CH3)3) at ∼1480 cm-1. This behavior indicates that the choline group may rotate freely with respect to the C3 axis both in the desorbed and adsorbed bilayer. The data in Figure 12c show that in the desorbed state at E < -0.4 V, the C-N bond of the choline moiety (C3 axis of the choline group) forms an angle of ∼75° with the normal to the surface. The angle increases to ∼79° when the bilayer adsorbs on the gold surface at E > -0.4 V. The tilt angle of the C-N bond is higher than the values

reported in the literature.39,47,52 The C3 axis was found to be tilted by 64°-61° in dry films and by 56°-51° in hydrated films. This behavior indicates that the charge density on the metal surface has significantly influenced the conformation of the positively charged choline group. 4. Summary and Conclusions We have employed charge density measurements and PM-IRRAS spectroscopy to study field-driven transformations of a DMPC bilayer formed by fusion of unilamellar

DMPC Bilayer Supported at a Au(111) Electrode Surface

Langmuir, Vol. 21, No. 1, 2005 345

Table 1. Comparison of the Angles between the Directions of Transition Dipoles and the Surface normal for the Adsorbed and Desorbed States of DMPC at the Au(111) Electrode Surface and the Angles for a DMPC Bilayer Supported Either at CaF267bor Ge67a

vibrational mode

Au(111) at F > 6 × 108 V/m (desorbed state) at ∼20°C

Au(111) at F < 6 × 108 V/m (adsorbed state) at ∼20°C

hydrated bilayer at Ge67a at ∼29°C

dry bilayer at Ge67a at ∼29°C

air-dried bilayer at CaF267b

vas(CH2) vs(CH2) θchain δ(CH2) v(CdO) v(C-O-C) vas(PO2-) va(PO2-) δas(N+(CH3)3) vas(N+(CH3)3)

64° 68° 35° 69° 70° 40° 62° 77° 75° 66°

52° 57° 55° 56° 63° 58° 57° 75° 80° 66°

66° 66° 35° 66° 65° 52° 64° 57° 56°

71° 71° 26° 71° 64° 48° 66° 56° 64°

70° 72° 17° 69° 67°

vesicles at the gold electrode surface. The electrochemical data indicate that the vesicles fuse to form a bilayer in direct contact with the metal surface when the charge density at the metal is less than 10 µC/cm2. At higher charge densities at the metal surface, the bilayer is lifted (desorbed from the metal). Independent neutron reflectivity experiments demonstrated that, in the desorbed state, the lifted bilayer remains in close proximity to the electrode separated from the gold surface by a thin (∼1 nm thick) cushion of the solvent (water).27 The spectroscopic data provided the molecular-level information concerning the effect of the electric field on the tilt of the acyl chains and the conformational changes in the polar head region of the DMPC molecule. Table 1 lists angles between directions of transition dipoles and the surface normal for the major IR bands of the DMPC molecule in the desorbed and the adsorbed states of the bilayer. For comparison, literature values of the tilt angles for the DMPC bilayer supported either at a Ge or CaF2 window are also reported in this table. The results show that in the desorbed state the tilt of the chains assumes a similar tilt angle to that observed in the hydrated bilayer supported at a Ge crystal surface. However, the frequencies of the CH2 stretch bands indicate that the chains are predominantly all trans stretched in the bilayer supported at gold at 20 °C, while the chains are melted and contain gauche conformations in the bilayer supported at a Ge crystal at 29 °C. One also can observe significant differences between the angles of the transition dipoles with the surface normal for the headgroup region of the bilayers deposited at gold and Ge crystals. Clearly, the head assumes different conformations at these two solid supports. The literature data for the solid and hydrated films included in Table 1 show that hydration of the film has also a profound influence on the chains tilt and the polar head conformation. For the bilayer deposited at a gold electrode surface, the differences between the chain tilt angles and conformations of the polar head region in the adsorbed and desorbed states are dramatic. The molecular models in Figure 13 show the chain orientation and the polar head conformation for the two states of the bilayer. The models were constructed using crystallographic data and conformation angles for the DMPC molecule reported by Hauser et al.73 The chains were then tilted, and the bonds in the polar head region were rotated to give the tilt angles reported in Table 1. The result shows that structural changes between the desorbed and adsorbed state of the film allow the maximum contact of the polar head and specifically the phosphate group with the metal. This transformation causes the chains to tilt and the heads to be less densely packed. It opens a space for water to

63°

penetrate the polar head region. Indeed, our PM-IRRAS data show that the transition from the desorbed to the adsorbed state of the bilayer involves a significant increase in the hydration of the ester and phosphate groups. We would like to emphasize that this change in hydration is caused by a change in the packing of the polar heads. When the bilayer is separated from the solid surface by a cushion of the solvent, the polar heads are packed in a zigzag fashion that gives a minimum area per molecules, as shown by Hauser et al.73 When the bilayer is adsorbed at the metal surface, all polar heads are located in the plane of the metal surface. This causes the area per molecule to increase and opens space that is filled by water molecules. The electrochemical data indicate that the bilayer undergoes a further transformation when the charge density at the metal changes sign at ∼0.2 V(Ag/AgCl). The IR data indicate that further changes in the conformation of the polar head take place in this region. However, the angles between the directions of the transition dipoles of the major bands and the surface normal are large and, hence, the band intensity is low. The scatter of the experimental points prevents more-detailed analysis of these changes. In conclusion, we have demonstrated that a combination of electrochemistry and PM-IRRAS spectroscopy allows one to describe the potential-driven transformation of a phospholipid bilayer supported at a Au(111) surface at the molecular level. Our next goal is to incorporate ion channels into the model membrane supported at the electrode surface and to use the same methodology to study conformational and structural changes that are responsible for voltage-gated processes that occur in biological membranes.

Figure 13. Scheme of orientation of the DMPC molecule in the bilayer at (a) E ) -0.8 V (desorbed film) and (b) E ) 0.2 V (adsorbed film).

346

Langmuir, Vol. 21, No. 1, 2005

Bin et al.

Table 2. Predicted Vibrational Frequencies of the A and B Forms of the DMPC Moleculea predicted harmonic vibrational frequency cm-1

scaled vibrational frequency cm-1

description of the normal mode A

1990 1985 1368

1782 1777 1225

1345

1204

1992 1991 1394

1783 1782 1248

1381

1236

CdO stretches coupled out of phase CdO stretches coupled in phase Asymmetric C(O)-O-C stretches coupled out of phase. Wagging motions of the methylenes also are involved in this mode. Asymmetric C(O)-O-C stretches coupled in phase. Wagging motions of the methylenes also are involved in this mode. B

a

CdO stretches coupled out of phase CdO stretches coupled in phase Asymmetric C(O)-O-C stretches coupled in phase. Wagging motions of the methylenes also are involved in this mode. Asymmetric C(O)-O-C stretches coupled in phase. Wagging motions of the methylenes also are involved in this mode.

Carbonyl stretch and C(O)-O-C regions.

5. Appendix Ab Initio Quantum Mechanical Calculations. Ab initio predictions of the geometries and harmonic vibrational frequencies of isolated A and B forms of DMPC were made using the Gaussian98 program suite.93 Due to the relatively large size of the DMPC molecule, C36H72NO8P, the ab initio Hartree-Fock method with the splitvalence plus polarization 6-31G(d) basis set was adopted. The initial starting geometries of the A and B forms were modeled after the crystal structure.94 Hydrogens were added with the Gaussview modeling program. The two waters of hydration for each form were removed, and preliminary geometry optimizations were performed with molecular mechanics and the semi-empirical PM3 method. Hartree-Fock geometries were then determined with a smaller 3-21G* basis set, and then finally the larger basis set 6-31G(d)-optimized structures and harmonic vibrational frequencies were computed. The predicted harmonic vibrational frequencies were scaled by 0.8953 (see ref 95) prior to comparison with experimental values. Table 2 lists the predicted and scaled vibrational frequencies. The scaled frequencies are still about 40-20 cm-1 higher than the experimentally measured frequencies of the v(CdO) and v(C(O)-O-C) bands. However, most of this difference may be attributed to the hydration of the ester group that was neglected in the calculations. Normal modes were inspected visually using the Gaussview program. The modes were assigned to atomic motions, and the directions of the dipole moment transition vector noted for intense vibrations. These assignments were used in the interpretations of the experimental spectroscopic measurements, particularly for the carbonyl stretches and the C(O)-O-C asymmetric stretches. The (93) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.; Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B. G.; Chen, W.; Wong, M. W.; Andres, J. L.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian 98, revision A.11; Gaussian, Inc.: Pittsburgh, PA, 1998. (94) Pearson, R. H.; Pascher, I. Nature 1979, 281, 499. (95) Scott, A. P.; Radom, L. J. Phys. Chem. 1996, 100, 16502.

calculations show unambiguously that the v(CdO) vibrations of the carbonyl group in the β and γ chains are coupled and that the CdO band consists of two overlapping bands corresponding to the out-of-phase and in-phase movement of the oxygen atoms of the carbonyl group. The displacement of the oxygen atoms during the CdO groups vibrations is shown Figure 14. In addition, the movement of the carbonyl group atoms which can be seen in the animated version of this figure given in the Supporting Information of this paper. The coupling is stronger for the DMPC-B than for the DMPC-A molecule. The yellow arrow in Figure 14 shows the direction of the transition dipole of the CdO group vibration. The transition dipoles of the out-of-phase and in-phase bands have different directions. The transition dipoles of the CdO stretches point in different directions in the DMPC-A and DMPC-B forms. For the C(O)-O-C stretch, the normal coordinate calculations demonstrate that the vibrations of the ester groups in the β and γ chains are very weakly coupled.

Figure 14. Molecular models showing the out-of-phase and in-phase vibrations of the CdO groups in DMPC A and DMPC B. The red arrows show the directions of motion of the oxygen atoms. The yellow arrows are the directions of the transition dipoles.

DMPC Bilayer Supported at a Au(111) Electrode Surface

Langmuir, Vol. 21, No. 1, 2005 347

However, the vibration of the C(O)-O-C group is strongly coupled to the wagging motion of the methylenes of the acyl chain. The movements of atoms corresponding to the vibration of the two C(O)-O-C groups and the coupling of these vibrations to the wagging movement of the chains can be viewed in a movie in the Supporting Information of this paper. When the energy optimization is performed for an isolated DMPC molecule, the acyl chain positions change and no longer correspond to the positions in the original crystallographic data. Since the chain-wagging movement is coupled to the C(O)-O-C group vibration, the directions of the transition dipoles determined from

the calculations are not directly comparable with experiment. These transition dipoles are not shown. Acknowledgment. This work was funded by NSERC Discovery Grants. J.L. acknowledges the Canada Foundation of Innovation (CFI) for a Canada Research Chair. Supporting Information Available: Animated vibrations corresponding to the ∼1740 cm-1 band and the ester-group vibration strongly coupled with the wagging motions of the tails. This material is available free of charge via the Internet at http://pubs.acs.org. LA048710W