5850
Langmuir 1998, 14, 5850-5857
Formation and Characterization of Supported Hexadecanethiol/Dimyristoyl Phosphatidylcholine Hybrid Bilayers Containing Gramicidin D Joomi Ha, Charles S. Henry, and Ingrid Fritsch* Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701 Received December 18, 1997. In Final Form: August 10, 1998 Polarization-modulation Fourier transform infrared reflection spectroscopy (PM-FTIR), ellipsometry, and a comparison of capacitance determinations by cyclic voltammetry and ac impedance have been performed on supported hybrid bilayers. The hybrid bilayers were constructed by fusion of vesicles of dimyristoyl phosphatidyl choline (DMPC), which form the top layer, with ethanol-rinsed, self-assembled monolayers (SAMs) of hexadecanethiol on gold, which form the bottom layer. A strategy for incorporating Gramicidin D (gD) which is 85% Gramicidin A, an ion channel forming polypeptide, is described. PMFTIR reproducibly demonstrates that bilayers are, indeed, formed and that gD can be incorporated into the assembly from the phospholipid vesicles. X-ray photoelectron spectroscopy of the bilayers showed evidence of the presence of DMPC and gD, although results were not reproducible, presumably due to sample damage during analysis. The capacitance in KNO3 solution for the gD-containing bilayer is higher than that for bilayers without gD. The opposite trend occurs for solutions containing Mg(NO3)2. Experimental evidence appears to indicate that this effect is more likely due to compositional changes that gD induces in the bilayer rather than discrimination of ion permeation due to channel formation. Cyclic voltammetry (0.100 V/s scan rate) of SAM and SAM/phospholipid hybrid layers (measured at either 0.4 or 0.0 V vs Ag/AgCl saturated KCl) consistently gives higher capacitance values than ac impedance (at 0.0 V dc).
Introduction The incorporation of membrane proteins and enzymes into modifying layers on surfaces is of interest for model systems of biomembranes and for the development of chemical sensors. Membrane protein structure and function are highly dependent on the surrounding environment, and thus, it is essential to design materials on surfaces that provide the necessary characteristics to host such proteins. Important and challenging goals are finding ways to incorporate proteins into the biomembranes and to characterize the resulting assemblies. This report describes the formation of hybrid bilayers of phospholipids and self-assembled monolayers on gold, a method to incorporate a polypeptide, Gramicidin D, into the bilayer, and characterization primarily by polarization modulation Fourier transform infrared reflection absorption spectroscopy (PM-FTIR), electrochemistry, and ellipsometry. Because phospholipids constitute a major component of biological membranes, they serve as natural candidates for model systems. Two main methods have been used to deposit ordered layers of phospholipids onto surfaces. Langmuir-Blodgett (LB) techniques have been used to assemble phospholipids onto a variety of surfaces, including freshly cleaved mica,1 highly ordered pyrolytic graphite,1 germanium,2-5 ZnSe,6 metals,7,8 quartz,9 and Si/SiO2 * To whom correspondence should be addressed. Telephone: (501) 575-3289. Fax: (501) 575-4049. E-mail:
[email protected]. (1) Solletti, J. M.; Botreau, M.; Sommer, F.; Brunat, W. L.; Kasas, S.; Duc, T. M.; Celio, M. R. Langmuir 1996, 12, 5379. (2) Fringeli, U. P.; Apell, H.-J.; Fringeli, M.; La¨uger, P. Biochim. Biophys. Acta 1989, 984, 301. (3) Tantulian, S. A.; Jones, R. R.; Reddy, L. G.; Stokes, D. L.; Tamm, L. K. Biochemistry 1995, 34, 4448. (4) Wenzl, P.; Fringeli, M.; Goette, J.; Fringeli, U. P. Langmuir 1994, 10, 4253. (5) Gregory, B. W.; Dluhy, R. A.; Bottomley, L. A. J. Phys. Chem. 1994, 98, 1010.
electrodes modified with organosilanes.10 Vesicle fusion techniques involve hydrophobic, hydrophilic, or electrostaticcouplingofphospholipidvesiclestoasubstrate.3,4,9,11-19 An advantage over LB methods is the simplicity and natural arrangement inherent in self-assembly of the phospholipids. To afford the desired interaction with phosphohlipids, the surface of the substrate may be premodified with molecules, such as a self-assembled monolayer (SAM)20-24 of organothiols or organodisulfides on gold. SAMs have the benefit that the molecules (6) Naydenova, S.; Petrov, A. G.; Yarwood, J. Langmuir 1995, 11, 3435. (7) Fare, T. L.; Rusin, K. M.; Bey, P. P., Jr. Sens. Actuators, B 1991, B3, 51. Powder Technol. 1991, 51. (8) Duevel, R. V.; Corn, R. M.; Liu, M. D.; Leidner, C. R. J. Phys. Chem. 1992, 96, 468. (9) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307. (10) Miller, C.; Cuendet, P.; Gratzel, M. J. Electroanal. Chem. 1990, 278, 175. (11) Plant, A. Langmuir 1993, 9, 2764. (12) Plant, A.; Gueguetchkeri, M.; Yap, W. Biophys. J. 1994, 67, 1126. (13) Plant, A. L.; Brigham-Burke, M.; Petrella, E. C.; O’Shannessy, D. J. Anal. Biochem. 1995, 226, 342. (14) Meuse, C. W.; Niaura, G.; Lewis, M. L.; Plant, A. L. Langmuir 1998, 14, 1604. (15) Steinem, C., Janshoff, A., Ulrich, W.-P., Sieber, M., Galla, H.-J. Biochim. Biophys. Acta 1996, 1279, 169. (16) Ha, J.; Henry, C. S.; Fritsch, I. ECS Proceedings of the 189th National Meeting Symposium on New Directions in Electroanalytical Chemistry; The Electrochemical Society: Pennington, NJ, 1996; Vol. 96-9, p 197. (17) Stelzle, M.; Weissmuller, G.; Sackmann, E. J. Phys. Chem. 1993, 96, 2974. (18) Hawkridge, F. M.; Cullison, J. K.; Hawkridge, F. M.; Nakashima N.; Yoshikawa, S. Langmuir 1994, 10, 877. (19) Pandey, P. C.; Aston, R. W.; Weetall, H. H. Biosens. Bioelectron. 1995, 10, 669. (20) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 4481. (21) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, D. E. D. J. Am. Chem. Soc. 1987, 109, 3559. (22) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321.
S0743-7463(97)01392-9 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/12/1998
Hybrid Bilayers Containing Gramicidin D
chemisorb rather than physisorb and provide an ordered foundation with designed chemical properties. Several kinds of molecules have been incorporated into supported hybrid bilayers formed from SAMs and phospholipid vesicles to impart different effects. Plant11 reports changes in permeability of hybrid bilayers caused by incubating bilayers with the pore-forming peptide mellitin. A glucose sensor has been designed19 where tetracyanoquinodimethane (TCNQ) resides within hybrid bilayers and serves as a mediator between the underlying electrode and the overlying, cross-linked glucose oxidase. Hawkridge and co-workers18 used enzyme reconstitution procedures to immobilize bovine cytochrome c into hybrid bilayers to shuttle electrons between electrodes and cytochrome c in solution. Others have formed hybrid lipid trilayers15,17 on solid supports by associating charged vesicles to charged monolayers via electrostatic interactions. Such films have been used to induce specific adsorption of streptavidin with membrane-incorporated biotinylated lipids.17 More recently, Cornell and co-workers25 reported an elegant way (although not involving vesicle fusion methods) to construct a universal chemical sensor, by creating on a gold substrate a lipid bilayer assembly containing free-moving lipids, surface-attached lipids, membranespanning lipids that are surface-attached and contain antigen-binding fragments, and gramicidin A (gA) either derivatized with thiol groups or antigen-binding fragments. The bilayer is formed so that there is a fluid reservoir between the first lipid layer and the surface. In the presence of large analytes or in the absence of small analytes, the derivatized gA links with the tethered membrane-spanning lipid and is inhibited from aligning with a surface-attached gA in the bottom layer, resulting in a change in the electrochemical response. Gramicidin A is an ion-channel-forming peptide and is one of the best characterized and most extensively studied membrane polypeptides.26-28 It is an antibiotic that is isolated from Bacillus brevis29 and is active against Grampositive bacteria.30 It consists of an alternating L,Dpentadecapeptide with the primary sequence HCO-L-ValGly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-(L-Trp-D-Trp)3L-Trp-N-HCH2CH2OH.31 The 3-dimensional conformation of gA is complex and dependent upon its environment.28,32 In biological or model membrane systems, gA adopts an ion channel conformation which allows the passage of water and small, monovalent cations. The channel is in the form of two β6.3-helical monomers that dimerize end to end with the formyl-NH ends associated in the center of a lipid bilayer.28 The length of the dimer is ∼26 Å. The peptide backbone forms a hydrophilic pore that has a diameter of about 4 Å.33 Because of its simple composition and characteristic functional dependence on structure, the native (underivatized) form of Gramicidin A can be used as a probe to evaluate modifying layers on electrodes. The value of (23) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112, 5258. (24) Li, T. T. T.; Weaver, M. J. J. Am. Chem. Soc. 1984, 106, 6107. (25) Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580. (26) Andersen, O. S.; Koeppe, R. E. Phys. Rev. 1992, 72, S117. (27) Koeppe, R. E., II; Hodgson, K. O.; Stryer, L. J. J. Mol. Biol. 1978, 121, 41. (28) Killian, J. A. Biochim. Biophys. 1992, 1113, 391. (29) Dubos, R. J. J. Exp. Med. 1939, 70, 1. (30) Hotchkiss, R. D. Adv. Enzymol. 1944, 4, 153. (31) Sarges, R.; Witkop, B. J. Am. Chem. Soc. 1965, 87, 2011. (32) Killian, J. A., Nicholson, L. K., Cross, T. A. Biochim. Biophys. Acta 1988, 943, 535. (33) Urry, D. W.; Goodall, M. C.; Glickson, J. D.; Mayers, D. F. Proc. Natl. Acad. Sci. U.S.A. 1971, 68, 1907.
Langmuir, Vol. 14, No. 20, 1998 5851
monitoring changes in vibrational bands of gramicidin incorporated into lipid films deposited by LB and casting methods has been demonstrated.34,35 However, few examples of native gramicidin in lipid layers on electrodes have been reported. Nelson36,37 has presented cyclic voltammetric evidence of the selective permeability of gramicidin D, gD (a mixture containing 70-85% gA and gramicidins B and C), toward Tl+ over Cd2+ at mercury drop electrodes. The surface of the mercury was modified with dioleoyl phosphatidylcholine and bovine brain phosphatidylserine incorporating gD with the LangmuirBlodgett technique. These results are consistent with gA in the ion channel conformation. This system is not easily conducive to further evaluation by spectroscopy due to the nature of mercury. Steinhem et al.15 have evaluated by impedance analysis different preparation techniques for supported lipid layers. They incorporated gD into one type of supported hybrid trilayer, consisting of a SAM of 3-mercaptopropionic acid, covered with two layers of dimethyldioctadecylammoniumbromide (DODAB), formed from fusion of DODAB vesicles. Again, spectroscopic characterization was not performed. However, electrochemical behavior was observed in the presence of Cs+ and Sr2+ that could be interpreted as gramicidin channels controlling ion permeation through the film. These results are consistent with those of Nelson. Electrochemical measurements alone are not sufficient to characterize phospholipid-modified electrodes, although electrochemical evaluation of faradaic and nonfaradaic processes is carried out widely.7,10-12,15-19,25,38,39 Although many spectroscopic studies of phospholipid films on surfaces have been reported,1-6,8,9,13,14,16-18,34,35,38 only a few are of supported hybrid bilayers formed from SAMs and phospholipid vesicles. Those include ellipsometry16,19 and surface plasmon resonance analysis.13,16,17 Demonstrating the importance of additional spectroscopic studies, Meuse et al.14 recently reported surface-enhanced Raman spectroscopy (SERS) and reflection absorption infrared spectroscopy (RAIRS) of supported hybrid bilayers that reveal structural changes in hexadecanethiol and octadecanethiol SAMs after the phospholipid layer of either dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC) or their deuterated analogues is deposited. Here, we report information complementary to the existing literature. First, the importance of the presence of an underlying SAM (formed in the presence and absence of gD) on the assembly of the phospholipid layer (with and without gD) is demonstrated by ellipsometry. Second, we present PM-FTIR spectra of gold surfaces modified by hybrid bilayers formed from hexadecanethiol SAMs and dimyristoyl phosphatidylcholine vesicles. Third, for the first time, vibrational spectroscopic data are presented for supported hybrid bilayers containing native (underivatized) gD. Fourth, the capacitance of monolayers and hybrid bilayers was determined to further elucidate the structure, and we report capacitance values that seem to be technique dependent. Experimental Section Materials. Hexadecanethiol (C16SH, Aldrich) solutions were prepared as described previously.16 The C16SH was filtered through alumina (Brockman, neutral, activity I) prior to the preparation of fresh derivatization solutions. Absolute ethanol (34) Okamura, E.; Umemura, J.; Kakenaka, T. Biochim. Biophys. Acta 1986, 886, 68. (35) Lukes, P. J.; Petty, M. C.; Yarwood, J. Langmuir 1992, 8, 3435 (36) Nelson, A. J. Electroanal. Chem. 1991, 303, 221. (37) Nelson, A. J. Chem. Soc., Faraday Trans. 1993, 89, 2799.
5852 Langmuir, Vol. 14, No. 20, 1998 (100%, Millenium Petrochemical), magnesium nitrate hexahydrate (99.995%, Aldrich), potassium nitrate (99.999%, Acros, FairLawn, NJ), sulfuric acid (Fisher), methanol (HPLC grade, Fisher), CHCl3 (HPLC grade, Fisher), and hydrogen peroxide (30%, Fisher) were used as received. Zero-grade argon and nitrogen (Air Products) were used to purge the solutions and maintain an inert atmosphere in a glovebag (Instruments for Research and Industry, Cheltenham, PA) during surface derivatization. Milli-Q RG (Millipore) DI water was used for all aqueous solutions and rinsing. Dimyristoyl phosphatidylcholine (DMPC), obtained from Avanti Polar Lipids, and gramicidin D (gD), obtained from Sigma Chemical Co., were used without further purification. Gramicidin D is a mixture of gramicidin A, B, and C, of which gA is the major component (∼85%). Substrate Preparation. Gold substrates were prepared as described previously.16 In an Edwards E306A thermal evaporator, approximately 50 Å of chromium from chromium-plated tungsten rods (R. D. Mathis) was deposited as an adhesion layer, followed by 2000-2500 Å of gold (Canadian Maple Leaf, 99.9% or Credit Suisse, 99.99%) onto clean silicon wafers (Silicon Quest International).22 Immediately prior to modification by SAMs, gold substrates were cleaned in piranha solution, which is a 3:7 solution of 30% H2O2 and concentrated H2SO4. Caution! This solution is very corrosive and must be handled with extreme care. Substrates were then rinsed thoroughly in DI water. Monolayer and Bilayer Preparations. Monolayers and bilayers were prepared in a similar manner as described previously,16 except with rigorous exclusion of air. There is growing support that air (or more likely the ozone in the air) oxidizes the thiolates in SAMs.40-45 Therefore, to ensure that the SAMs were free of the more weakly bound oxidized products, air-exclusion during sample preparation was enforced. Solution preparation and monolayer formation were performed in an Arpurged glovebag to minimize air-oxidation of thiolates. SAMs were formed by soaking Au substrates in derivatizing solutions of 1 mM C16SH or 0.1 mM gD in 1 mM C16SH (in ethanol) for ∼12 h. Electrodes were removed from solution and rinsed with ethanol prior to performing experiments. Samples were only exposed to air when placed in the dry, CO2-free, air-purged (Balston) sample chamber of the PM-FTIR. Vesicle suspensions of DMPC and DMPC + gD (mole ratio: 28 DMPC to 1 gD) used for bilayer formation were prepared according to published methods.46 To 100 µg of dried gD (from 50 µM gD/CH3OH stock solution) 1.5 µmol of DMPC was added (from 1 mg/10 µL DMPC/CH3OH stock) along with 90 µL of CH3OH and 100 µL of CHCl3. The solution was mixed, and the resulting suspension was dried under vacuum overnight to remove the organic solvent. The dried gD/lipid mixtures were resuspended in 500 µL of Ar-purged DI water and sonicated for 2 h in a 55 °C bath. The gramicidin concentrations in vesicle solutions were determined by measuring the absorbance at 280 nm ( ) 20 840 M-1 cm-1)47 using an HP 8452A diode array spectrophotometer. Typical gD concentrations are from 0.9 to 1.4 mM. The conformation of gD in vesicles was determined by circular dichroism (CD) measurements that were obtained at room temperature using a JASCO 710A spectrometer.16 The characteristic positive peaks at 218-220 nm and 235-236 nm, a positive minimum at 229-230 nm and a negative ellipticity below 208 nm, indicate that gA exists in a β6.3 channel conformation in the DMPC vesicles.48 (38) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197. (39) Ding, L.; Li, J.; Dong, S.; Wang, E. J. Electroanal. Chem. 1996, 416, 105. (40) Li, Y.; Huang, J.; Melver, R. T., Jr.; Hemminger, J. C. J. Am. Chem. Soc. 1992, 114, 2428. (41) Tarlov, M. J.; Newman, J. G. Langmuir 1992, 8, 1398. (42) Scott, J. R.; Baker, L.; Everett, W. R.; Wilkins, C. L.; Fritsch, I. Anal. Chem. 1997, 69, 2336. (43) Norrod, K. L.; Rowlen, K. L. J. Am. Chem. Soc. 1998, 120, 2030. (44) Zhang, Y.; Terrill, R. H.; Bohn, P. W. J. Am. Chem. Soc. 1998, 120, 2034. (45) Schoenfisch, M. H.; Pemberton, J. E. J. Am. Chem. Soc. 1998, 120, 4502. (46) Greathouse, D.; Hinton, J.; Kyung, K.; Koeppe, R. E. Biochemistry 1994, 33, 4291. (47) Turner, G. L.; Hinton, J. F.; Koeppe, R. E.; Parli, J. A.; Millett, F. F. Biochim. Biophys. Acta 1983, 756, 133.
Ha et al. Hybrid bilayers were prepared in an Ar-purged glovebag immediately prior to use. They were formed by soaking the SAMcoated substrates in aqueous suspensions of vesicles of either DMPC or DMPC + gD for 24 h, followed by rinsing with water. Samples were then allowed to dry in the Ar-atmosphere. Electrochemical Measurements. The electrochemical cell consisted of a standard three-electrode system. The reference was a Ag/AgCl (saturated KCl) electrode. A platinum flag electrode served as the auxiliary electrode. Immediately prior to electrochemical experiments, solutions were purged thoroughly in a closed cell to minimize the presence of oxygen. Capacitance, C, of alkanethiol monolayers and hybrid bilayers was determined by two different methods, cyclic voltammetry and ac impedance. Cyclic voltammetry was performed using a computerinterfaced potentiostat (BioAnalytical Systems, 100B with 100W software). Cyclic voltammetry was performed over two different potential ranges: between 0.450 and 0.300 V and between 0.100 V and -0.100 V, in 0.1 M KNO3 (reagent grade, 99.999%) and in 0.1 M Mg(NO3)2 (reagent grade, 99.995%). The equation C ) ic/νA was used to calculate the area-normalized capacitance. The ic is the charging current measured at a given potential (0.400 or 0.0 V) on the first scan, ν is the scan rate, and A is the electrode area. The ac impedance measurements were made using an EG&G PAR M273A potentiostat, M388 Electrochemical Impedance Systems software, and EG&G PAR M5210 lock-in amplifier. A sinusoidal ac signal was applied at frequencies between 10 and 64 000 Hz. Measurements were made in 0.1 M KNO3 and 0.1 M Mg(NO3)2 with a 0.010 V amplitude at 0.0 V vs Ag/AgCl (saturated KCl) reference. To determine the area-normalized capacitance, an equivalent circuit was assumed, like that used by Plant et al.,12 Lang et al.,38 and Steinem et al.15 Extrapolation of the sloped lines in bode plots (absolute impedance vs log of angular frequency, ω) to the y-axis (where log(ω) ) 0) gives a value |Z|. Then, C was calculated from C ) 1/(|Z|A).49 Errors that are reported in tables represent 1 standard deviation. Ellipsometry. The procedure for measuring film thicknesses in air was described previously.16 A Rudolph Research Model 43603 ellipsometer equipped with a 5 mW helium-neon laser light source (632.8 nm) and with a 70°angle of incidence was used to measure the monolayer and bilayer thicknesses. Six measurements were obtained at various sites on each freshly cleaned Au substrate and subsequently modified surfaces. The change in polarization state and the phase change of the electric field associated with the light beam were determined. The averages of these measurements were used to calculate the film thickness on each modified substrate. A refractive index of 1.45 for hydrocarbon layers on gold was assumed.22 Errors that are reported in tables represent 1 standard deviation. Infrared Spectroscopy. IR spectra of powder samples in KBr pellets were obtained in transmission mode with a Mattson Research Series Fourier transform infrared spectrometer, using a DTGS detector. The resolution was 2 cm-1. Infrared spectra of the modified electrodes were obtained with the same instrument, but using an external sample chamber with a polarization modulation accessory. The IR beam was focused onto the sample at an incident angle of 77°. The beam was p-polarized and passed through a ZnSe Series II photoelastic modulator (Hinds) operating at 37 kHz before reaching the sample. The reflected beam was detected using a liquid nitrogen cooled HgCdTe detector. Spectra were taken with 2 cm-1 resolution and a half wavenumber of 2400 cm-1. PM-FTIR spectra were normalized by fitting the entire differential reflectance spectra to ninth order polynomial backgrounds using FitIT curve fitting software (Mattson). After curve fitting, spectra were truncated and converted to absorbance using a WinFirst macro, written in-house under the specifications of Mattson.50 (48) Urry, E. W.; Spisni, A.; Khaled, M. A. Biochem. Biophys. Res. Commun. 1979, 88, 940. (49) Electrochemical Impedance Plots. Appendix A, In EG & G PAR Models 378 & 388 Electrochemical Impedance Systems Instructions Manual; EG & G Instruments Corp.: Princeton, NJ, 1983-1989; p 45. (50) Everett, W. R. Ph.D. Dissertation, University of Arkansas, 1996.
Hybrid Bilayers Containing Gramicidin D
Langmuir, Vol. 14, No. 20, 1998 5853
Table 1. Monolayer and Bilayer Thickness As Determined by Ellipsometry thickness (Å)
modifying layers C16SH C16SH/DMPC C16SH/DMPC + gD DMPC a
modifying layers
thickness (Å)
22.2 ( 1.5a C16SH + gD 19.6 ( 2.0a 43.6 ( 1.0a C16SH + gD/DMPC 40.6 ( 1.8 44.0 ( 0.8 C16SH + gD/DMPC + gD 44.5 ( 0.6a 19.5 ( 11.2
From ref 16.
Results and Discussion Characterization with Ellipsometry. We have previously used ellipsometry to characterize gold surfaces that have been modified by fusion of phospholipid vesicles of DMPC with self-assembled monolayers of hexadecanethiol.16 Those results, which are duplicated in Table 1, demonstrate that this procedure forms bilayers reproducibly. The first layer consists of a monolayer of 22.2 ( 1.5 Å of hexadecanethiol (or 19.6 ( 2.0 Å if formed in the presence of gD), and the second layer consists of phospholipids, which, depending on the first layer and whether gA is present, increases the thickness by an additional 21 to 25 Å. Here, we report a more thorough analysis of these layers using ellipsometry, including exploration of the extent of vesicle physisorption and contribution of gD to total thickness of each layer. These results are also reported in Table 1. The results for the film formed on a bare gold sample that has been placed in a suspension of DMPC vesicles is 19.5 ( 11.2 Å. The average thickness of physisorbed DMPC on gold is lower than that of a DMPC layer formed at a C16SH SAM. This could be due to lower coverage and less order. In addition, the large standard deviation implies that the DMPC physisorbed to gold is not uniformly distributed. Thus, it appears that the SAM is necessary to initiate reproducible and specific fusion of DMPC vesicles with the surface. On the basis of surface coverage studies,16 the presence of gD in the thiol solution during assembly of C16SH onto gold appears to cause a decrease in the total C16SH that attaches to the surface. However, the average thickness for a SAM, formed with gD in solution, is lower, but within error at 95% confidence of that of a pure C16SH SAM. To further investigate the effect of gD on the modified surfaces, we obtained additional ellipsometry results for hybrid bilayers in which gD was present during formation of only one of the two layers, either in the first layer, gD + C16SH, or in the second layer, DMPC + gD. The thicknesses of these two types of hybrid bilayers are significantly different at the 95% confidence level. The presence of gD during formation of the first layer has a significant impact on the structure of the bilayer. This is consistent with our previous surface coverage results and ellipsometry of the monolayers. However, when gD is present, with the lipid used for forming both the first and second layers (C16SH + gD/DMPC + gD), the total film thickness is the greatest of all of these combinations. This seems to indicate that DMPC + gD vesicles can fill in gaps or defects in the underlying layer better than DMPC alone. Although ellipsometry serves as a sensitive measure of thickness, there remain unanswered questions about the composition and structure of the layers on the surface, especially in the presence of gD. In addition, the calculation of thickness from the ellipsometry measurements involves the assumption that the refractive index is the same for C16SH, phospholipids, and gA. Thus, it is
Table 2. IR Peak Assignments for Monolayers and Bilayers peak
C16SH
CH3 νas(ip) CH3 νas(op) CH3 νs(FR) CH3 νs(FR) CH2 νas CH2 νs CH3 δ amide A amide I amide II CdO PO2- νas PO2- νs
2964
C16SH + C16SH/ C16SH + gD/ DMPC/ gD DMPC DMPC + gD KBr 2964
2962
gD/ KBr 2960a
2962 2956
2877
2877
2877
2878
2918 2850
2918 2850
2917 2850 1457
2918 2850 1458 3280 1660 1546 1739 1261 1099
1739 1260 1100
2872a 2932a 2919 2850 1468
1457 3278 1637 1536
1736 1252 1092
a Assignment from refs 55 and 57: “CH and CH group 2 3 symmetric and asymmetric stretching”.
essential that other techniques be used to further elucidate the structure and verify the validity of the ellipsometry results. Characterization with PM-FTIR. There has been a substantial number of structural analyses on phospholipid films performed in air by infrared techniques. Some studies involve external reflectance,5,8,14,35 but most use attenuated total reflectance (ATR) IR.2-6,34,35,51 The phospholipid films of those investigations were deposited onto substrates using LB deposition, casting, or a combination of LB- and vesicle-deposition methods. Until recently, infrared spectra had not been reported for supported hybrid bilayers formed from SAMs and phospholipid vesicles. Meuse et al.14 studied the effect of phospholipids on the underlying structure of the SAMs with RAIRS and SERS. Here, PM-FTIR was used to evaluate structure and composition of surfaces modified with C16SH SAMs and DMPC with and without gD. Polarization modulation offers an advantage over standard external reflectance IR techniques in that a background sample is not required.52 Several IR bands of gA34,35,53-57 have different frequencies from DMPC34 and from alkanethiols,21,23 so that compositional variation in the hybrid bilayers can be monitored. Table 2 summarizes the peak positions and assignments for PM-FTIR spectra for films containing different combinations of C16SH, gD, and DMPC and for transmission FTIR spectra of gD and DMPC in a KBr pellet. Figure 1 shows representative IR spectra for C16SH SAMs, C16SH/DMPC bilayers, and DMPC dispersed in KBr. In the PM-FTIR spectrum of a C16SH SAM (Figure 1a), the ∼ 0.0012 absorbance for the νas(CH2) band falls within the range of absorbance values reported for organothiols having monolayer coverage. The reported values extend from as low as 0.0008 AU for the longerchain C18SH SAMs on Au14 to as high as 0.0035 AU for C16SH SAMs on Au.23 Other points to note in Figure 1a are the hydrocarbon peak positions. The bands at 2918 and 2850 cm-1 are assigned to the asymmetric, νas, and symmetric, νs, CH2 modes, respectively.21 That at 2964 cm-1 is assigned to the in-plane, CH3 asymmetric stretching mode (CH3 νas(ip)). An out-of-plane CH3 asymmetric (51) Frey, S.; Tamm, L. Biophys. J. 1991, 60, 922. (52) Duevel, R. V., Corn, R. M. Anal. Chem. 1992, 64, 337. (53) Urry, D. W.; Shaw, R. G.; Trapane, T. L.; Prasad, K. U. Biochem. Biophys. Res. Commun. 1983, 114, 373. (54) Naik, V. M.; Krimm, S. Biophys. J. 1986, 49, 1131. (55) Naik, V. M.; Krimm, S. Biophys. J. 1986, 49, 1147. (56) Veatch. W. R.; Fossel, E. T.; Blout, E. R. Biochemistry 1974, 13, 5249. (57) Iqbal, Z.; Weidekamm, E. Arch. Biochem. Biophys. 1980, 202, 639.
5854 Langmuir, Vol. 14, No. 20, 1998
Figure 1. PM-FTIR spectra of (a) ethanol-rinsed SAM of C16SH and (b) C16SH/DMPC bilayer. Transmission IR (c) of dried DMPC in a KBr pellet.
stretching band (CH3 νas(op)) for SAMs is not easily distinguished in the spectra from noise, consistent with previous reports.21 The CH3 symmetric stretch fermi resonance mode (CH3 νs(FR)) is split, having one peak at 2877 cm-1 and the other as a shoulder (at approximately 2937 cm-1) on the CH2 asymmetric band.58 The CH stretching frequencies are in good agreement, within (1 cm-1, of values reported in the literature for long-chain alkanethiol monolayers on Au21,23 and are within the resolution (2 cm-1) of our measurements. The spectrum for a C16SH/DMPC hybrid bilayer is shown in Figure 1b. The absorbance values of the CHstretching bands are essentially doubled from that of the SAM. We interpret this to mean that a bilayer has formed. These data are consistent with the ellipsometry measurements. The DMPC has 12 methylene carbons in the alkyl chains, not 15, and thus we would also expect to see an 80% increase in the absorbance of the CH2 bands, if no significant changes in orientation occur. The actual increase is 78.7 ( 3.2% AU for the CH2 νas band. We would expect a doubling of the CH3 absorbances. The actual increase is 95.4 ( 16.8% AU for the νas(ip) band. Other evidence for the presence of phospholipids includes ester carbonyls at 1739 cm-1 and the asymmetric PO2and symmetric PO2- stretches at 1260 and 1100 cm-1, respectively.5,14,35 The relative magnitude of the carbonyl stretch varies from sample to sample by (0.0006 AU, which might be an indicator of small variations in the orientation of the headgroup or in coverage. The only frequencies of the CH-stretching modes for the C16SH/ DMPC bilayers that differ from those for the C16SH SAM are the CH3 νas(ip) (2962 cm-1) and the CH2 νas (2917 cm-1) modes. However, these are within the resolution of our measurements and might not be significant. In addition, (58) Dubois, L. H.; Zegarski, B. R.; Nuzzo, R. G. J. Chem. Phys. 1993, 98, 678.
Ha et al.
Figure 2. PM-FTIR spectra of (a) ethanol-rinsed SAM of C16SH + gD and (b) C16SH + gD/DMPC + gD bilayer. Transmission IR (c) of dried gD in a KBr pellet.
if a monolayer consists of alkyl chains in all-trans configuration, the CH2 νas band will occur at 2918 cm-1, as compared to 2924 cm-1 for films with a more disordered, liquidlike structure.21 Overall, our results indicate that the structures of the hydrocarbon chains in the DMPC layer are similar to those in the SAM. Okamura et al.34 observed similar frequencies for CH2 νas (2918.1 ( 0.1 cm-1) and CH2 νs (2850.4 ( 0.1 cm-1) modes of DMPC multilayers when cast on germanium ATR plates from a chloroform solution. Figure 1c shows the transmission IR spectra for DMPC in a KBr pellet for comparison. The CH2 νas (2919 cm-1) and νs (2850 cm-1) modes and the CH3 νas(op) mode (2956 cm-1) are at frequencies consistent with crystalline hydrocarbon substances.21 The ester carbonyl peak for DMPC in the KBr pellet (1736 cm-1) is lower by 3 cm-1 than that in the hybrid bilayer. Those bands for the PO2asymmetric stretching mode at 1252 cm-1, and the PO2symmetric stretching mode, observed at 1092 cm-1, are lower for DMPC in the KBr pellet than that in the hybrid bilayer by 8 cm-1. The structure of DMPC in the hybrid bilayers is clearly different from that in the powder form. Figure 2 shows the PM-FTIR spectra for the C16SH + gD monolayer and the C16SH + gD/DMPC + gD bilayer and the transmission spectrum of gD in a KBr pellet. In the spectrum for ethanol-rinsed C16SH + gD (Figure 2a), there are no characteristic IR bands for gA. Thus, it appears that gD is not present in sufficient concentrations in the SAMs formed from C16SH + gD to be detected. In addition, the absorbance for the CH-stretching modes is only approximately 90% of that for a SAM formed from C16SH alone. Interestingly, the frequencies of the CHstretching modes are identical to those for the pure C16SH SAM. This indicates that changes in the monolayer structure due to assembly in the presence of gD are not detectable with PM-FTIR. X-ray photoelectron spectroscopy (XPS) measurements were also used to extract information regarding surface composition (see Supporting Information). This method has a sensitivity of 0.01-0.3%
Hybrid Bilayers Containing Gramicidin D
Langmuir, Vol. 14, No. 20, 1998 5855
Table 3. Capacitance Values for Monolayers and Bilayers Obtained from CV at 0.4 and 0.0 V and AC Impedance at 0.0 V C (µF/cm2)
C (µF/cm2)
C (µF/cm2)
modifying layers
0.1 M KNO3a
0.1 M Mg(NO3)2a
0.1 M KNO3a,e
0.1 M Mg(NO3)2a,e
0.1 M KNO3b,e
0.1 M Mg(NO3)2b,e
0.1 M KNO3c,e
0.1 M Mg(NO3)2c,e
C16SH C16SH + gD C16SH/DMPC C16SH + gD/DMPC + gD
3.05 ( 0.07a 4.80 ( 0.46a 2.75 ( 0.08a 3.69 ( 0.17a
7.40 ( 0.26a 5.79 ( 0.19a 4.60 ( 0.14a 3.39 ( 0.07a
2.98 ( 0.04 4.50 ( 0.03 1.78 ( 0.03 2.08 ( 0.03
4.80 ( 0.29 3.85 ( 0.31 2.51 ( 0.07 2.08 ( 0.04
1.53 ( 0.06 2.08 ( 0.02
2.51 ( 0.07 2.08 ( 0.04
0.90 ( 0.08 1.31 ( 0.55
1.40 ( 0.43 1.21 ( 0.17
a Data obtained from CV measurements at 0.4 V vs Ag/AgCl (saturated KCl). b Data obtained from CV measurements at 0.0 V vs Ag/AgCl (saturated KCl). c Data obtained from ac impedance measurements at 0.0 V vs Ag/AgCl (saturated KCl). d From ref 16. e Purified electrolytes, KNO3 (99.999%) and Mg(NO3)2 (99.995%) and air-free preparation and analysis of sample.
of the elemental composition of the surface59 and thus provides an analysis method complementary to IR, which can be a factor of 10 less sensitive.60 Peaks for C(1s) and S(2p) that are similar to those for the C16SH SAM alone were observed and there are no new peaks for N(1s), O(1s), or C(1s) that correspond to gD. Thus, XPS is consistent with PM-FTIR of C16SH + gD monolayers. Figure 2b shows that in a bilayer of C16SH + gD/DMPC + gD, the absorbance bands in the CH-stretching region is approximately two times the absorbance of monolayers. Gramicidin is present in the film this time, as demonstrated by the prominent amide I peak (CdO stretch) at 1660 cm-1, the amide II peak (coupled CN stretch and NH bending) at 1546 cm-1, and the weak, broad Amide A band (NH stretch) at 3280 cm-1.34,35 Also present is the sharp band at 1739 cm-1 for the ester carbonyl of DMPC, as well as the peaks assigned to asymmetric and symmetric PO2stretching modes. The absorbance of the ester carbonyl band is higher than that for the C16SH/DMPC bilayers. This may be an indication that there is an orientational change in the DMPC headgroup relative to the surface in the presence of gD or a compositional change. The magnitude of this band does vary, however, from bilayer to bilayer by as much as 15% AU relative standard deviation. Figure 2c shows the transmission IR spectrum obtained from gD in a KBr pellet. The characteristic bands are the amide A at 3278 cm-1, the amide I at 1637 cm-1, and the amide II at 1536 cm-1.55,57 These are lower in frequency by 2, 23, and 10 cm-1, respectively, than the corresponding bands in the hybrid bilayers containing gD. It has been reported that the frequency shift of the amide I band suggests a change in environment around the gramicidin molecules and/or a conformational change caused by the introduction of gramicidin into the lipid layer.34,35 XPS spectra of hybrid bilayers with and without gD was not as informative as PM-FTIR. The presence of N(1s) and P(2p) peaks was highly sporadic from sample to sample. However, PM-FTIR spectra before and after XPS analysis of the same sample was identical. Perhaps sample damage to the phospholipid layer in the small X-ray irradiated area occurs, but is not sufficient to perturb enough of the sample to affect IR analysis, which covers an area ∼100 times larger. Capacitance in KNO3 and Mg(NO3)2 Electrolyte. Electrochemical analysis can provide information about (59) Brundle, C. R. X-ray Photoelectron Spectroscopy. Chapter 5.1 In Encyclopedia of Materials Characterization; Brundle, R. C., Evans, C. A., Jr., Wilson, S., Eds.; Butterworth-Heinemann, Boston, MA, 1992; p 282. (60) Cox, J. N. Fourier Transform Infrared Spectroscopy. Chapter 8.1 In Encyclopedia of Materials Characterization; Brundle, R. C., Evans, Jr., C. A., Wilson, S., Eds., Butterworth-Heinemann, Boston,MA, 1992; p 416. (61) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications; John Wiley & Sons: New York, 1980; pp 500-511.
the barrier properties of the modified surfaces to solution species. The capacitance at an electrode surface depends on the composition of the electrolyte, solvent, the nature of the electrode surface, and the applied potential.61 There are numerous reports that draw general conclusions about SAMs and phospholipid layers on electrodes based on capacitance measurements using ac impedance7,10,11,12,15,17,19,38,39 and cyclic voltammetry.16,18,19,21,36-39 Here, several points are addressed. (1) We report capacitance values for SAMs with and without gD (Table 3) obtained by CV at 0.4 and 0.0 V and by ac impedance at 0.0 V and relate the results to capacitance values in the literature. (2) Once we demonstrate that our SAMs are comparable to those of others, the change in capacitance from monolayers to bilayers is contrasted and correlated to results from spectroscopy. (3) The effect of the presence and absence of gD in monolayers and bilayers on capacitance in potassium- and magnesium-containing nitrate salts is presented. These two salts were chosen to evaluate the relative permeation of mono- and dications through the films, respectively. The capacitance values in Table 3 obtained by CV at 0.4 V are generally quite higher than those reported by others for SAM and phospholipid modified electrodes. The capacitance for the C16SH/DMPC bilayer in purified 0.1 M KNO3 is ∼2.6 times greater than those reported by Plant11 for C18SH/POPC bilayers on Au in 0.010 M KCl and ∼1.6 times greater than that for the C10SH/POPC bilayer in Tris-buffered saline.12 These differences may be due to the manner in which the capacitance was determined and not necessarily due to large differences in sample preparation or composition. We compared the results from different electrochemical techniques for the monolayer-modified surfaces (C16SH and C16SH + gD). The capacitances for C16SH and C16SH + gD SAMs using CV at 0.0 V were 49-53% lower in 0.1 M KNO3 and 46-47% lower in 0.1 M Mg(NO3)2 than results obtained by CV at 0.4 V. For example, the capacitance of C16SH SAM from CV at 0.0 V in 0.1 M KNO3 (99.999%) electrolyte was 1.53 ( 0.06 µF/cm2, but is 2.87 ( 0.04 µF/cm2 at 0.4 V. However, our value for CV at 0.0 V is still quite higher than that for Plant11 for C18SH SAMs (0.96 ( 0.13 µF/cm2) using ac impedance at 0.0 V or for Wang and co-workers39 for C16SH SAMs (0.96 ( 0.03 µF/ cm2) using ac impedance at 0.22 V. We also performed ac impedance at 0.0 V, which gave a capacitance for C16SH SAMs of 0.90 ( 0.08 µF/cm2. This value is in agreement with those previously reported. Thus, we conclude that our sample preparation procedure must be valid. Previously, Wang and co-workers39 compared several electrochemical determinations of capacitance (CV, ac impedance, and chronoamperometry/chronocoulometry) of various preparations of supported phospholipid membranes (however, none involved vesicles). Although they assume the same equivalent-circuit models, their capaci-
5856 Langmuir, Vol. 14, No. 20, 1998
tances are essentially independent of technique. To dismiss concerns that our apparent “method-dependent capacitances” are not instead due to variations in sample composition, we tested the same sample with different electrochemical methods in different sequences and obtained results that are highly reproducible and similar to those shown in Table 3. Table 3 also shows that SAMs prepared from solutions of C16SH + gD exhibit the same trends in capacitance when compared to pure C16SH SAMs, regardless of the technique. Although the absolute values are higher using CV at 0.4 V than the other electrochemical methods, a comparison of the relative capacitances of differently modified surfaces with the same technique should be satisfactory. There are two sets of capacitance values determined from CV at 0.4 V in Table 3 for monolayers and bilayers. The first set of values was obtained in reagent grade 0.1 M KNO3 and 0.1 M Mg(NO3)2 (from Mg(NO3)2‚6 H2O) and for samples which were not protected from air during transfers to and from the electrochemical cell. These were reported previously.16 It has been suggested that SAMs air oxidize upon exposure to air (and especially ozone),40-45 which may change the capacitance of the layers. Also, the impurities in the electrolytes used previously, and in particular those of the Mg-containing salt, may have led to capacitance values that could not be accurately predicted. Thus, a second set of capacitance values (Table 3) were obtained in solutions prepared from higher purity salts, KNO3 (99.999%) and Mg(NO3)2‚6 H2O (99.995%), and for which samples had not been exposed to air at any point in the preparation or during electrochemical analysis. Most of the values for both electrolytes in the second set are lower than those in the first, which is consistent with fewer defects in the ensembles of molecules. Overall, the trend in capacitance of monolayers when compared to bilayers is consistent with the corresponding spectroscopic data. Specifically, the decrease in capacitance from monolayers when compared to bilayers is close to 50%, which is expected if the bilayer is double the thickness of the monolayer and if the molecules in the two layers are similar. There is a more dramatic decrease in capacitance from monolayers to bilayers for the gDcontaining films (46% for KNO3 and 54% for Mg(NO3)2) than for those without gD (40% for KNO3 and 48% for Mg(NO3)2). This agrees with ellipsometry which shows a 127% and 96% increase in thickness for bilayers with and without gD, respectively. PM-FTIR is also consistent, which shows lower intensity C-H vibrational modes when capacitance in KNO3 increases. Examples include comparisons of SAMs of C16SH to SAMs of C16SH + gD and bilayers of C16SH/DMPC to bilayers of C16SH + gD/ DMPC + gD. Determination of capacitance in different electrolytes can provide valuable insights into film structure and composition that may not be readily discernible from spectroscopic data. For example, the relative capacitance trends in Mg(NO3)2, for films with and without gD, are opposite those of KNO3. In both purities of electrolyte, higher capacitance is obtained in KNO3 when gD is present in the derivatizing solutions to form the monolayers (3438%) and bilayers (14-24%). In Mg(NO3)2 there is a decrease in capacitance from C16SH SAMs to C16SH + gD SAMs of 22% and 20% for the first and second sets in Table 3, respectively. For bilayers, the decrease in capacitance in Mg(NO3)2 when gD is present is 26% and 17%, respectively. Although the absolute capacitance values for the Mg(NO3)2 electrolyte in the second set are lower than for the first set, the trends observed in the
Ha et al.
absence and presence of gD are similar and reproducible. These results seem to indicate that the ion channel conformation of the native gA may serve to discriminate between K+ and Mg2+. This conclusion is also consistent with results from Nelson36,37 and Galla and co-workers.15 However, based on the new spectroscopic evidence described above, the C16SH + gD monolayer does not contain detectable amounts of gD. Thus, for that modified surface, one cannot propose that selective permeation is based on ion channel formation but rather that the presence of gD in the derivatizing solution somehow changes the SAM film structure and perhaps its composition. Structural and compositional changes may also contribute to the selective-ion effect for bilayers with and without gD. Conclusions Hybrid biomembrane-like materials hold great potential for many applications. As they become more representative of real biomembranes, they will contain increasing numbers of components. Thus, investigations into their construction, characterization, and limitations are imperative. We presented here aspects that are important to both the construction and characterization of hybrid bilayers formed from hexadecanethiol and DMPC, both with and without native gD. We demonstrated that the SAM is essential for uniform and reproducible deposition of phopholipids onto a surface from vesicles and that a bilayer is indeed formed and showed the effect gD has on the thickness of bilayers. Also, the incorporation of native gD into these hybrid bilayers from vesicles enhances permeability to KNO3, having the opposite effect to Mg(NO3)2. This may be due to structural or compositional changes that the gD induces or due to channel formation. However, because the C16SH monolayers formed in the presence of gD also exhibit this behavior, we suspect that structural and compositional changes are most likely responsible for this effect. This work is important to future research on the incorporation of species into supported hybrid biomembranes. It may be necessary in some cases to tether the species directly to the surface, as was done with gA by Cornell and co-workers.25 Crucial to understanding supported hybrid biomembranes is the characterization of the films with multiple techniques. We showed that the determination of capacitance, which is a common evaluation parameter for these systems, sometimes depends on the electrochemical technique used. When ac impedance at 0.0 V vs Ag/AgCl (saturated KCl) was used to measure capacitance of C16SH SAMs, we observed values similar to those in the literature. Measurements at 0.0 V using the first scan in CV gave higher values. Those at 0.4 V using CV are the highest for the methods evaluated here. However, the relative trends in capacitance from one modified surface to another are consistent within a given technique. Vibrational analysis using RAIRS and SERS of supported hybrid bilayers formed from SAMs and phospholipid vesicles provides a valuable addition to structural and compositional analysis, and its importance has been recently demonstrated.14 Evaluation by PM-FTIR, which has been performed on these systems for the first time here, yields spectra that are consistent with monolayer and bilayer formation, supports the notion that the bilayers are highly ordered, and confirms the incorporation of gD from phospholipid vesicles in solution. Ellipsometry data support these conclusions. Numerous other analytical methods could be used, such as XPS for elemental analysis. XPS corroborates the other data that suggest gD is not present in detectable amounts in ethanol-rinsed SAMs of C16SH + gD. However, XPS analysis of bilayers
Hybrid Bilayers Containing Gramicidin D
Langmuir, Vol. 14, No. 20, 1998 5857
gave irreproducible results, presumably because of sample damage or other changes that occur in the phospholipid layer.
and Dr. Neal Watkins are also thanked for use of the ellipsometer and helpful discussions.
Acknowledgment. Financial support was provided by the National Science Foundation, Grant EHR-9180768 and NSF Career Award CHE-96-24114. We acknowledge Professor Roger Koeppe, II, and Dr. Denise Greathouse for helpful discussions on gA and for the use of equipment to make and characterize vesicles. Professor David Paul
Supporting Information Available: Figures showing XPS spectra of SAM-modified electrodes and text containing a discussion of those and hybrid bilayer analyses (6 pages). Ordering and Internet access information is given on any current masthead page. LA971392Z
