Langmuir 1992,8, 1627-1632
1627
Electrodes Modified with Cast Lipid Films: The Effects of Lipid Composition on the Electrochemistry of Incorporated Amphiphilic Viologens Maria T. Rojas, Mei Han,and Angel E. Kaifer* Chemistry Department, University of Miami, Coral Gables, Florida 33124 Received January 29, 1992. In Final Form: March 16, 1992
The cyclic voltammetric behavior of N-ethyl-N’-octadecylviologen, 12+, and N-(cholesteryl acetate)N’-ethylviologen, 22+,was studied in phosphatidylcholinefiis cast on the surfaceof glassy carbonelectrodes. Both viologens exhibited diffusion-controlled voltammetric behavior under these conditions. Visible spectroscopy after exhaustive one-electron reduction of the viologen-containinglipid films revealed a low level of dimerization in spite of the large local concentration of reduced viologen, l+. Apparent diffusion coefficients for 12+ were found to be essentially independent from its film concentration, suggesting that charge propagation takes place via actual diffusion of the amphiphilic viologen in the lipid film. Voltammetric currents for both 12+ and 22+were observed to be highly dependent on the degree of saturation of the lipid’s fatty acyl residues, generally increasing with the degree of unsaturation. Currents also increased with temperature, but no clear break points, indicative of phase transitions, were detected. Cholesterol addition to fully unsaturated lipid films did not affect the voltammetric currents while it increased the currents observed in fully saturated lipid films.
Introduction Substantial research effort has been devoted recently to the investigation of electrodes modified with films of amphiphilic molecules. Although most of the work currently available in the literature focuses on films of monolayer thickness formed by the self-assembly of alkylorganosulfur compounds’ or alkylsilanes,2multilayer films have also received some attention3 In some instances, these interfacial structures have been utilized to measure rate constants for rapid heterogeneous electron transfer reaction^.^ Chidsey has studied long range electron transfer using a self-assembled monolayer containing covalently-attached ferrocene group^.^ Interesting chemical (1) (a) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. SOC.1987,109,3559. (b) Finklea, H. 0.; Avery, S.; Lynch, M.; Furtsch, T. Langmuir 1987,3,409. (c) Troughton, E. B.; Bain, C. D.; Whitesides, G. M.; Nuzzo, R. G.; Allara, D. L.; Porter, M. D. Langmuir 1988,4,365. (d) Strong, L.; Whitesides, G . M. Langmuir 1988,4, 546. (e) Bain, C. D.; Troughton, E. B.; Tao, Y. T.; Evall, J.;Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321. (f) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1989,111,7155. (g) Bain, C. D.; Whitesides, G . M. J. Am. Chem. Soc. 1989, 111, 7164. (h) Bain, C. D.; Biebuyck, H. A.; Whitesides, G . M. Langmuir 1989,5,723. (i) Bain, C. D.; Whitesides, G. M. Langmuir 1989,5,1370. Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. SOC.1990,112,558. (k) Chidsey, C. E. D.; Bertozzi, C. R.; Putvinski, T. M.; Mujsce, A. J. J . Am. Chem. Soc. 1990,112,4301. (1) Whitesides, G. M.; Laibinis, P. E. Langmuir 1990, 6,87. (m) Chidsey, C. E. D.; Loiacono, D. N. Langmuir 1990,6,682. (n) Finklea, H. 0.;Snider, D. A.; Fedyk, J. Langmuir 1990,6,682. ( 0 ) Walczak, M. M.; Chung, C.; Stole, S. M.; Widrig, C. A.; Porter, M. D. J.Am. Chem. Soc. 1991,113,2370. (p) Widrig, C. A,; Alves, C. A.; Porter, M. D. J. Am. Chem. SOC.1991, 113, 2805. (q) Widrig, C. A.; Chung, C.; Porter, M. D. J. Electroanal. Chem. 1991,310,335. (r) Walczak, M. M.; Popenoe, D. D.; Deinhammer, R. S.; Lamp, B. D.; Chung, C.; Porter, M. D. Langmuir 1991, 7, 2687. (s) Miller, C.; Cuendet, P.; Gratzel, M. J. Phys. Chem. 1991,95,877. (2) (a) Sagiv, J. J. Am. Chem. SOC.1980,102,92. (b) Sagiv, J. Isr. J. Chem. 1979,18, 346. ( c ) Netzer, L.; Sagiv, J. J. Am. Chem. Soc. 1983, 105,647. (d) Netzer, L.; Iscovici, R.; Sagiv, J. Thin Solid Films 1983,99, 235; 1983,100,67. (e) Maoz, R.; Sagiv, J. J. Colloid Znterface Sci. 1984, 100,465. (f) Gun, J.; Iscoici, R.; Sagiv, J. J . Colloid Interface Sci. 1984, 101, 201. (g) Maoz, R.; Sagiv, J. Thin Solid Films 1985, 132, 135. (h) Pomerantz, M.; Segmuller, A.; Netzer, L.; Sagiv, J. Thin Solid Films 1985,132, 153. (i) Gun, J.; Sagiv, J. J. Colloid Interface Sci. 1986,112, 457. ti) Cohen, S. R.; Naaman, R.; Sagiv, J. J.Phys. Chem. 1986,90,3045. (3) (a) Lee, H.; Kepley, J. K.; Hong, H. G.; Akhter, S.; Mallouk, T. E. J.Phys. Chem. 1988,92,2597. (b) Hong, H.-G.; Mallouk, T. E. Langmuir 1991, 7, 2362. (4) (a) Sabatini, E.; Rubinstein, I.; Maoz, R.; Sagiv, J. J. Electroanal. Chem. 1987, 219, 365. (b) Sabatini, E.; Rubinstein, I. J. Phys. Chen. 1987, 91, 6663.
properties can be engineered by introducing bindine or redox-active’ groups inside the monolayer structures. Although these interfacial structures offer much hope of providing substantial control on the reactivity of electrode surfaces, it would be desirable to develop other methods for the easy modification of electrodes with amphiphilic molecules. In particular, methods that do not involve the synthesis of organosulfur or silane derivatives would be quite convenient. Several years ago, Tanakaet al. reported that the electrochemistry of hydrophilic species is essentially blocked on electrodes covered by palmitic acid layers? Inspired by this finding, we introduced and studied a new type of electrode which is modified with cast lipid films.g We demonstrated that the lipid films exhibit permselectivity, blocking the access of hydrophilic species to the electrode surface while permitting the incorporation of amphiphilic species. Therefore, when redox-active amphiphiles are used, their electrochemistry inside the lipid film can be easily recorded. The permselective properties of these electrodes have attracted some attention due to their potential application to pharmaceutical analysis.l0 Recently, we have reported that the addition of charged surfactants to zwitterionic phosphatidylcholine (PC) films allows the fine-tuning of the redox potentials of an amphiphilic viologen incorporated inside (5) Chidsey, C. E. D. Science 1991,251, 919. (6) (a) Rubinstein, I.;Steinberg,S.;Tor, Y.;Shanzer,A.;Sagiv, J.Nature 1988,332,426. (b) Steinberg, S.; Tor, Y.; Sabatani, E.: Rubinstein, I. J. Am. Chem. SOC.1991,113,5176. (7) (a) Facci, J. S. Langmuir 1987,3, 525. (b) Diaz, A,; Kaifer, A. E. J. Electroanul. Chem. 1988,249, 333. (c) Donohue, J. J.; Buttry, D. A. Langmuir 1989,5,671. (d) Van Galen, D. A.; Majda, M. Anal. Chem. 1988,60, 1549. (e) Widrig, C. A.; Majda, M. Langmuir 1989,5,689. (f) Lee, K. A. Bunding Langmuir 1990,6, 709. (g) DeLong, H. C.; Buttry, D. A. Langmuir 1990,6,1319. (h) Creager, S. E.; Collard, D. M.; Fox, M. A. Langmuir 1990,6, 1617. (i) Kunitake, M.; Akiyoehi, K.; Kawatana, K.; Nakaahima, N.; Manabe, 0.J. Electroanal. Chem. 1990,292,277. ti) Gomez, M.; Li, J.; Kaifer, A. E. Langmuir 1991, 7, 1571. (k) Gomez, M.; Li, J.; Kaifer, A. E. Langmuir 1991, 7,1797. (1) Rowe, G. K.; Creager, S . E. Langmuir 1991,7,2307. (m)De Long, H. C.; Donohue, J. J.; Buttry, D. A. Langmuir 1991, 7, 2196. (n) Bilewicz, R.; Majda, M. Langmuir 1991, 7, 2794. (8)Tanaka, K.; Tamamushi, R. J . Electroanal. Chem. 1987,236,305. (9) Garcia, 0. J.; Quintela, P. A.; Kaifer, A. E. Anal. Chem. 1989,61, 979. (10) (a) Chastel, 0.; Kauffman, J. M.; Patriarche, G. J.; Christian, G. D. Anal. Chem. 1989,61,170. (b) Wang,J.;Lu, 2.Anal. Chem. 1990,62, 826.
1992 American Chemical Society
Rojas et al.
1628 Langmuir, Vol. 8, No. 6, 1992
the PC film.” Rusling and co-workershave also described the properties of electrodes modified with cast films of cationic surfactants, such as dioctadecyldimethylammonium bromide and didodecyldimethylammonium bromide.12 Modification of the electrode surface via casting of lipid films is attractive as it offers the possibility of using these electrodes for the study of electron transfer reactions in an environment that resembles the structural features of biological membranes. It thus becomes imperative to prove that the cast lipid films show a substantial degree of multilayer organization in order to use them as reasonable models for biomembranes. For instance, it is well-known that the fluidity of actual biomembranes is controlled by the degree of unsaturation of the fatty acid chains in the constituent lipids, as well as by the membrane’s cholesterol content.13 Saturated fatty acyl residues promote the rigid or cystalline state due to the favorable packing characteristics of all-trans alkyl chains. Conversely, the presence of unsaturations introduces bends in the hydrocarbon chains that foster a more disorganized packing arrangement, i.e., a greater extent of membrane fluidity. Membrane fluidity can be quantitated by the so-called transition or melting temperature which expresses the temperature at which the membrane undergoes a phase transition from the gel or crystalline state to the fluid state. Generally speaking, membranes formed by lipids having longer fatty acyl chains tend to have higher transition temperat~re5.l~Similarly, the greater the degree of saturation of the chains, the higher the transition temperature. Lipid composition is the preferred mechanism by which procaryotic organisms regulate the fluidity of their cell membranes. Many eucaryotic organisms also use cholesterol as a key regulator of membrane fluidity. Its usual role is to decrease the fluidity of the membrane by sterically blocking empty volumes created by the presence of unsaturations in the lipids. However, at high concentrations it may exert the opposite effect. In fact, contradictory evidencehas been found regarding the effects of cholesterol on the fluidity of simple membrane m0de1s.l~ We decided to investigate some of these phenomena using electrodes modified with cast lipid layers made with natural (egg) PC, a fully saturated synthetic lipid, ~ - a distearoylphosphatidylcholine (DSPC), and a fully unsaturated synthetic lipid, L-a-dioleoylphosphatidylcholine (DOPC). We have also performed experiments with lipid layers containing cholesterol and compared the results to those obtained with the lipids in the absence of cholesterol. Throughout these experiments we used two amphiphilic viologens as redox probes. The viologens appropriately possess either an alkyl chain (12+) or a cholesteric residue ( 2 9 as the lipophilic groups. Detailed structures are given below. Experimental Section Materials. N-Ethyl-N’-octadecyl-4,4’-bipyridiniumdibro-
mide (1Brz) was synthesized as reported elsewhere.7k The bis(hexafluorophosphate) salt was obtained by simple counterion exchange with NHSFe. N-(Cholesterylacetate)-N’-ethyl-4,4’bipyridinium bis(hexafluorophosphate) was prepared by the reaction of cholesteryl iodoacetate with l-ethyl-4-(4’-pyridyl)pyridinium bromide in refluxing acetonitrile, followed by counterion exchange using NHdPFs. Full details of the synthesis and (11) Han, M.; Kaifer, A. E. J.Chem. SOC.,Chem. Commun. 1990,1698. (12) Rusling, J. F.;Zhang, H. Langmuir 1991, 7, 1791. (13)Stryer, L. Biochemistry, 3rd ed.; Freeman: New York, 1988; Chapter 12. (14) Chapman, D.In Membrane Fluidity in Biology; Aloia, R. C., Ed.; Academic Press: New York, 1983; Vol. 2, pp 55-61. (15) Matubayasi, N.; Mataunaga, R.; Motomura, K. Langmuir 1989, 5, 1048.
22+ purification of this compound will be given in a forthcoming paper:16 All lipids were purchased either from Sigma or Princeton Lipids. No measurable difference was found in our experiments between the materials from these two suppliers. All aqueous solutions were freshly prepared with distilled water further purified with a Barnstead Nanopure four-cartridge system. Allother chemicalswere analytical grade. Indium-doped tin oxide electrodes were purchased from Delta Technologies. Equipment. Electrochemical experiments were performed with Princeton Applied Research instrumentation which has been already described in detail.17 Absorption spectra were recorded with a Hewlett-Packard 8452A spectrophotometer. Procedures. Electrodes modified with cast lipid films were prepared by a simple method. A measured microliter volume of a freshly prepared chloroform solution of the lipid was deposited at the tip of a glassy carbon (GC) electrode which had been previously conditioned by polishing with 0.05-pm alumina on a felt surface. The electrode was rotated (200 rpm for 5-10 min) to allow the evaporation of the chloroform in air. When the electrode was cast with pure lipid, the viologen probe was incorporated by immersing the lipid-covered electrode in a millimolar solution of the redox-active probe. More commonly, the viologen was preincorporated in the lipid film by dissolving it in the lipid/chloroform solution used for casting. In this case the electrode was equilibrated prior to any voltammetric measurementa in 0.100 M phosphate buffer solution (pH = 7.0) for 5-10 min. Immediately afterward, the electrode potential was cycled at 100 mV/s between 0.0 and -1.2 V vs SSCE during 10 min to allow the equilibration of the lipid f i b . Current-potential curves usually reached an approximate steady state after this cycling time. Therefore, voltammetric measurements were always recorded immediately after this pretreatment protocol was completed. For variable temperature voltammetric experiments, a nonisothermal cell design was utilized in which the reference electrode compartment was separated from the main compartment and kept at constant laboratory temperature. The main compartment was immersed in a temperature-controlled bath that was set to the chosen temperature value. Spectrophotometric measurements were recorded using optically transparent tin oxide electrodes, cut to size in order to fit inside a regular 1-cm cuvette. These were covered with the cast lipid f i i and placed inside the cuvette which had been previously filled with deoxygenated phosphate buffer aqueous solution.
Results and Discussion Figure 1 shows the cyclic voltammetric response of a GC electrode covered with 4.1 X lo-* mol.cm-2 of leBr2 and 4.1 X lo-’ mol.cm-2 of PC and immersed in aqueous phosphate buffer solution. This level of coverage results in a lipid film having a thickness of approximately 3.7 pm, as estimated from a volume of approximately 1.5 nm3per PC molecule in multilayer assemblies.ls Two sets of waves are clearly visible corresponding to the two consecutive monoelectronic reductions of the viologen subunit (12+/ 1+ and l + / l ) , Both redox processes exhibit behavior (16) Li, J.; Kaifer, A. E. To be submitted. (17) Quintela, P. A.; Kaifer, A. E. Langmuir 1987, 3, 769. (18) Tanaka, K.; Masanaga, M.; Tanaka, T. J.Am. Chem. SOC.1986, 108,5448.
Langmuir, Vol. 8, No. 6,1992 1629
Electrodes Modified with Cast Lipid Films
1
0.0
.l.2
POTENTIAL, V vs SSCE
Figure 1. Cyclicvoltammogram(50mV/s) in0.100M phosphate buffer (pH = 7.0) of A GC electrode (0.08cm2)covered with 4.1 X mol/cm2 lBr2 and 4.1 x mol/cm2 egg PC. characteristic of reversible redox couples. This is significant because the aqueous voltammetric behavior of amphiphilic viologens, such as 12+,is usually complicated by precipitation of the reduced forms, l+ and 1, on the electrode ~ u r f a c e . ' ~Amphiphilic .~~ viologens yield reversible voltammetric waves in nonaqueous solvents, like acetonitrile or dimethylformamide, that are similar to those in Figure 1.20The measured half-wave potentials for the 12+/1+ and 1+/1 couples are also more positive than expected for these processes in aqueous solution. Therefore, the voltammetric behavior of 12+inside the cast lipid film clearly suggests that the viologen subunits reside in an essentially nonaqueous or water-depleted environment. This, in turn, suggests that a certain level of molecular organization exists within the lipid film. Previous work on cast lipid films contacting aqueous media indicates that the lipid films adopt the expected lamellar multilayer structure.21 Our voltammetric results are consistent with this lamellar arrangement, suggesting that the presence of 12+at the 9.1 mol ?& level in the lipid film does not affect its degree of molecular organization. This is indeed expected because the overall molecular shape of the amphiphilic viologen is similar to that of phosphatidylcholine. The first cathodic peak current for the reduction of 12+ increases linearly with the square root of the scan rate. This fact, along with the shape of the voltammetric waves, indicates that the electrochemistry of 12+inside the lipid film is diffusion controlled. This is an interesting result in itself, since lipid bilayer membranes are rather impermeable to ions, a t least in the absence of ionophores, carriers, or channel-forming species. However, the observation of sizable faradaic currents in our voltammetric experiments with GC/PC(12+) electrodes indicates that substantial ionic currents pass through the lipid film in order to maintain electroneutrality conditions, in response to the electrochemical conversions. Large ionic currents across a rather thick (3.7 bm) lipid layer would be difficult to explain if the lipid film is viewed as a rigidly organized multilayer assembly. However, our experiments with PC lipid films were done at 25 "C, clearly above the phase transition temperature observed for PC in bilayer and multilayer assemblies.22 Thus, the lipid film is in the fluid (19) Diu, A.; Quintela, P. A.; Schuette, J. M.; Kaifer, A. E. J. Phys. Chem. 1988,92, 3537. (20) Lu, T.; Cotton, T. M.; Hurst, J. K.; Thompson, D. H. P. J.Electroanal. Chem. 1988, 246, 337. (21) (a) Okahata, Y.; Taguchi, K.; Seki, T. J. Chem. SOC.,Chem. Commun. 1985,1122. (b) Okahata, Y.; Ebato, H.; Taguchi, K. J. Chem. Soc., Chem. Commun. 1987, 1363. (c) Okahata, Y.; Shimizu, 0. Langmuir 1987,3, 1171. (d) Okahata, Y.; Ye,X. J. Chem. SOC.,Chem. Commun. 1989, 1147. (22) Bretscher, M. S. Science 1973, 181,622.
I
I
1
I
300
I
I
I
1
1
I
800
Figure 2. Visible absorption spectra of 1+ inside PC films: (A) 4.8 mol % of lBr2 in egg PC film, absorbance scale 0-0.16; (B) 9.1 mol % of lBr2 in egg PC film, absorbance scale 0-0.42.
state which, by itself, relaxes the degree of molecular organization. Furthermore, the lipid film may contain defective regions in which the molecular organization is looser. Both of these factors probably contribute to facilitate the passage of ionic currents driven by the electrochemical conversions in the film. Therefore, the voltammetric behavior of 12+ in the lipid film suggesta that the PC molecules aggregate to form an organized assembly which prevents the precipitation of the reduced viologen forms 1+ and 1. However, the passage of sizable ionic currents across a thick lipid film is not compatible with a rigidly organized, hydrophobic structure. These two seemingly opposing findings can be understood by assuming that the lipid film consists of a large number of well-organized multilayer microdomains that are interconnected by regions exhibiting poor molecular organization. Viologen cation radicals are known to dimerize reversibly in aqueous media.23 Dimerization appears to be at least partially driven by the increased lipophilicity of the cation radical as compared to the parent dication. The optical absorption differences between monomeric and dimeric cation radicals are well e~tablished.~~" Therefore, the extent of 1+ dimerization can also be used to qualitatively assess the degree of molecular organization and water exclusion prevalent inside the lipid film. Figure 2B shows the visible spectrum of a PC film containing 9.1 mol 9% of 12+exhaustively reduced at -0.7 V vs SSCE to ensure the complete conversion to l+. The ratio of absorbances at 364 and 394 nm can be utilized to estimate the dimer to monomer (D/M) ratio using an approximate method previously developed by our group and applied to the dimerization of the methylviologen cation radical in mi(23) (a) Kosower, E. M.; Cotter, J. L. J.Am. Chem. SOC. 1964,86,5524. (b) Bird, C. L.; Kuhn, A. T. Chem. SOC.Rev. 1981, 10,49.
1630 Langmuir, Vol. 8,No. 6,1992 cellar solutions.24 By use of this method, the D/Mratio was found to be approximately 0.5 in this case (A364IA304 = 0.75). A similar experiment performed with a lipid film initially containing 4.0 mol 5% of 12+ yielded the visible spectrum shown in Figure 2A which shows aA36$Amvalue of 0.6 which corresponds to a D/Mratio of 0.25. Under the last set of conditions, the effective concentration of cation radical inside the lipid layer is about 0.11 M! The low concentration of dimer in the lipid film implies that the level of molecular organization is high enough to overcome the large tendency of these cation radicals to dimerize. Alternatively, the lipid film may exclude water well enough so that the tendency of the cation radicals to dimerize is greatly decreased inside the film. In any instance, these spectral results are consistent with the molecular organization of the lipid film into a multilayer assembly. Charge Propagation inside the Lipid Film. Electrodes covered or modified with cast lipid films incorporating the viologen derivative 12+ exhibit voltammetric behavior that can be characterized as diffusion controlled. This is not unusual in modified electrodes. Electroactive species confined to modified layers on electrode surfaces usually exhibit diffusion controlled behavior, especially if the thickness of the modified layer is larger than a few monolayers. In these situations, questions about the mechanism of propagation of the electrochemical conversion (the so-called charge propagation) across the modified layer invariably arise. Two mechanisms have been invoked to explain charge propagation: (i) actual diffusion of the electroactive probe through the modified film and (ii) electron hopping among neighboringprobes in different oxidation states. The balance between these contributions can be quantitatively expressed by the well-known Rahms-Duff equation25 Dapp= D + ( ~ / 4 ) 6 ~ k , , C
(1)
where Dappis the measured (apparent)diffusion coefficient, D is the actual diffusion coefficient of the electroactive probe, 6 is the average distance between neighboring probes in the modified film, k,, is the rate constant for the probe's electron self-exchangeprocess, and C is the effective probe concentration in the modified film. Typically, a linear increase of Dapp with C is taken as evidence for the predominance of the second term (electron hopping) in eq 1. Conversely, independence of Dappfrom C reveals the predominance of the first term (physical diffusion) on the charge propagation b e h a v i ~ r . ~ ~ ! ~ ' In order to address the mechanism of charge propagation across the cast lipid films, we measured the apparent diffusion coefficient using cyclic voltammetry with lipidcovered electrodes containing varying concentrations of 12+. In order to ensure that the diffusion layer in these experiments would be confined within the thickness of the lipid films, we cast thicker lipid layers than usual, with a nominal coverage of 2.1 X 10-6 mol/cm2of lipid and varying coverages of 12+. This coverage level resulted in lipid films having an estimated thickness of 17 pm. The results are plotted in Figure 3. The Dappvalues tend to decrease as the film concentration of 12+ increases. This clearly demonstrates that electron hopping is not the predominant mechanism of charge propagation. In fact, (24) Quintela, P. A.; Diaz, A.; Kaifer, A. E. Langmuir 1988,4, 663. (25) (a) Dahms, H. J . Phys. Chem. 1968,72,362. (b)Ruff, I.; Friedrich, V. J. J . Phys. Chem. 1971, 75,3297. (26) For instance, see (a) Facci,J.; Murray, R. W. J. Electroanal. Chem. 1981,124,339. (b) Facci, J.; Murray, R. W. J. Phys. Chem. 1981,85,2870. (c) Buttry, D.; Anson, F. C. J. Electroanal. Chem. 1981, 130, 333. (d) Oyama, N. J . Electroanal. Chem. 1982,139, 371. (27) Reference 14, pp 21-22.
a
;5
s,
U
0.00
r
0.02
0.04
0.06
0.08
0.10
0.12
Film concentration of viologen, mol/L
Figure 3. Dependence of the diffusion coefficient of 12+ on ita concentration inside lipid f i i made by casting 1.7 X 10-8mol/ cmz of PC and 4.7 x lO-' mol/cmzof DSPC. Temperature = 22 OC.
-.
(A) Transversal movement orflip-flop.
I
=--
k-1
a==-
o==r----(\
(B) Lateral movement.
Figure 4. Schematic representation of the two fundamental diffusional events in bilayer lipid membranes. decreasing Dapp values with increasing concentration of redox probe have been observed before in other reports on modified electrodes. The reasons for this behavior may vary from system to system but are usually related to the complexity of the charge propagation process which is not entirely well represented by eq 1. In our case, the decrease of DaPqvaluesin the concentration range surveyed is not quite significant and we interpret the data in Figure 1 as a strong indication that charge propagation in the cast lipid films is accomplished essentially via actual diffusion of the amphiphilic viologen species through the lipid multilayer assembly. At this point it is tempting to address the several types of viologen motions that can take place in the lipid film. For instance, one can consider transversal (flip-flop motion, see Figure 4A) and lateral diffusion (see Figure 4B) of the viologen in the lipid multilayer. The fist one is typically a very slow process in lipid bilayer membranes while the latter one is much faster. However, it is indeed an oversimplificationto assume that transversal and lateral diffusion can be distinguished throughout a very thick lipid film. It is likely that the lipid film is composed of many ordered microdomains randomly oriented in relation to one another. Thus, both transversal and lateral diffusional movements can contribute to the propagation of the electrochemical conversions driven by the electrode surface. Because of the relative rates of these two diffusional processes, one must conclude that charge prop-
Electrodes Modified with Cast Lipid Films
Langmuir, Vol. 8,No. 6,1992 1631
e t-
4 U
c
1
i
I
I
I
B
-1.2
3-
0.0
POTENTIAL, V vs SSCE
Figure 5. Cyclic voltammetric response (50mV/s) in 0.100 M phosphats buffer (pH = 7.0) of a GC electrode (0.08 cm2)covered with 4.1 X 10-8 mol/cm2 of lBr2 and 4.1 X lo-' mol/cm2 of lipid (A) the lipid is pure DOPC; (B) the lipid is pure DSPC.
agation takes place via lateral diffusion of the viologen in the organized microdomains within the lipid assembly. Furthermore, the diffusion coefficients of 12+measured in our experiments fall within the range of lateral diffusion coefficients that have been determined for a variety of molecules in biomembranes. Lipid Composition Studies. The prevalent nature of viologen diffusion as the main mechanism of charge propagation gives rise to the distinct possibility of controllingthe extent of the electrochemical conversionsinside the lipid films. This would be feasible if one could manipulate the fluidity of the lipid film which, in turn, should govern the rate of diffusion of the amphiphilic viologen through the lipid assembly. As mentioned above Nature uses two approaches to control membrane fluidity: the first one relies on the degree of unsaturation of the lipid's fatty acyl residues while the second one makes use of cholesterol. We have explored both methods. In this section, we describe the results obtained with electrodes modified with cast lipid layers which were prepared with mixtures of pure synthetic lipids, such as DSPC and DOPC. This allowed us to adjust the degree of unsaturation of the lipid assembly and assess its effect on the electrochemicalbehavior of 12+.The results obtained using cholesterol as a fluidity modifier of the lipid assembly are described in a later section. The effect of the degree of unsaturation on the electrochemistry of 12+inside films of pure synthetic lipids is dramatically illustrated by the voltammograms of Figure 5. The cyclic voltammetric response of a GC/DOPC(12+) electrode is shown in Figure 5A and is similar to the behavior of a GC/PC(12+)electrode (see Figure 1). In clear contrast, Figure 5B shows the cyclic voltammetric response of a GC/DSPC(12+) electrode. The lipid and viologen
coverages in the electrodes whose voltammetric behavior is shown in Figure 5 were identical, as were the voltammetric conditions. Therefore, the striking difference in voltammetric currents observed for the viologen reductions is related solely to the different nature of the fatty acyl residues of DSPC and DOPC, with the low currents observed inside the saturated lipid DSPC contrasting with the sizable currents recorded inside unsaturated DOPC. This result can easily be understood in terms of fluidity. The phase transition temperature of pure DSPC bilayers has been reported as 61 0C.22 Therefore, our room temperature experiments were done with the cast lipid assembly in the gel or crystalline state. The rigidity of the lipid film explains then the extremely low voltammetric currents observed under these conditions. The much larger currents observed with the DOPC film are consistent with the more fluid state of the multilayer assembly which is predicted from the lower phase transition temperature of this unsaturated lipid (-10 "C). Since DSPC and DOPC films represent extremes in the degree of saturation, it is interesting to explore the properties of lipid layers of intermediate Composition (varyingDSPC/DOPC ratios). Figure 6shows the cathodic redox couple as a function of peak current for the 12+/1+ the molar fraction of saturated lipid DSPC. As expected from the results with pure lipid films,the current decreases monotonically with an increasing degree of saturation of the lipid's acyl residues. AU these data are quite compelling because they clearly indicate that the electrochemistry of 12+confined within cast lipid films follows trends that can be easily predicted from well-known lipid membrane properties. These data also argue strongly in favor of a substantial level of molecular organization in the cast lipid films. The fact that lipid membranes exhibit temperaturedependent fluidity prompted us to explore the voltammetric behavior at several temperatures of 12+in lipid films made with varying DSPC/DOPC ratios. Figure 7 showsthe cathodic peak current for the 12+ 1+conversion as a function of temperature for several lipid compositions. The data clearly demonstrate two trends: (i) at constant lipid composition, currents increase with increasing temperature; (ii) at constant temperature, currents increase with increasing DOPC/DSPC ratio. The second trend, of course, confirms at several temperatures what we had already observed at room temperature (see data in Figure 6). The first trend is also consistent with a correlation between voltammetric currents and fluidity. As the temperature increases, so does the fluidity of the lipid film
-
Rojas et al.
1632 Langmuir,Vol. 8, No. 6,1992
Table I. Electrochemical Parameters of l ( P F & a n d 2(PF& inside Lipid Films of Varying Composition
=
E $?
i
8
12
.
O
I
I
A 0
'{AA
o L : 270
A
: 282
t
0
;
:
294
I
:
;
~
306
318
" 330
TEMPERATURE. K
Figure 7. Temperature effects on the first cathodic peak current for 12+ inside mixed DSPC/DOPCfilms. The molar ratios of DOPC are ( 0 )0, (A)0.2,( 0 )0.4,( 0 )0.6,and (m) 0.8.
and this translates into large current levels. However, one would expect to detect a sharp break point in the current-temperature behavior reflecting the melting of the multilayer assembly, that is, the gel-to-fluid phase transition. However, no clear break points are observed in the current-temperature behavior at any of the surveyed lipid compositions. The lack of sharply defined transition points in the current-temperature behavior of these lipid films is somewhat surprising. Rusling and co-workers have detected clear transition temperatures in similar experiments using cast dioctadecyldimethylammonium bromide films incorporating ferrocyanide ions as electroactive probes.12 The lack of transition points in our experimental results might be related to several factors. We have already postulated that the lipid film can be seen as a collection of randomly oriented, multilayer microdomains. These microdomains have lamellar (multilayer) organization. However, the interfaces between neighboring microdomainsmight be composed of 'defective regions" in which the molecular organization is looser. As was pointed out before, the defective regions can have a substantial influence on the overall electrochemical behavior of the film. This influence might be significant enough to "smooth out" the effects of the phase transition in the organized microdomains. Effects of Cholesterol. Cholesterol is frequently utilized as a fluidity modifier in lipid membranes. This factor led us to perform a series of experiments incorporating cholesterol into the composition of the cast lipid films. In fact, cholesterol was used in two different ways: (i) as the lipophilic tail of the amphiphilic viologen probe; (ii) as a mere additive to the lipid film. We have synthesized an amphiphilic viologen that contains a cholesteric moiety as the lipophilic tail (see Experimental Section). Because this compound was obtained as the bis(hexafluorophosphate) salt, 2(PF&, we decided to compare ita voltammetric behavior inside a variety of lipid films with that of the octadecyl analog, l(PF&. The results are shown in Table I. Several general trends emerge from the data. For instance, in a lipid film of similar composition, apparent Do values are always higher for 1 2 + than for 22+. Thus, the diffusional movement of the cholesteric viologen derivative through the multilayer assembly appears to be more difficult than that of the octadecyl analog. This is true for all lipid compositions surveyed and probably reflects the bulkier nature of the
viologen/lipid
% chol
El? V
1(PFs)dEggPc l(PF&/Egg Pc l(PF6)dDOPC l(PFe)dDOPC l(PF6)dDSPC l(PF6)dDSPC 2(PFs)dEgg Pc 2(PF&/Egg PC 2(PF6)dDOPC 2(PFs)dDOPC 2(PF6)dDSPC 2(PFs)dDSPC
0
25 0 25 o 25 0 25 0 25 o 25
-0.50 -0.50 4.50 -0.49 c -0.51 -0.43 -0.41 -0.44 -0.42 c -0.40
Ez? V -0.85 -0.84 -0.80
Do,b cm2/s (1k 1) X 1o-B (2k 2) X 10-B (2h 1)X 1o-B (2k 1) X le
C
C
-0.81 -0.85 -0.83 -0.85 -0.84
(6i 2) X (6k 3) X (2i 2) X (6i4)X (5t2)X
C
C
-0.79
(4i 4) X
-0.80
10-l' 10-1' lWIO 10-l'
E1 and E2 are the half-wavepotentials (Vvs SSCE)for the 12+/1+ and 1+/1redox couples, respectively. Error margins are standard deviations obtained from at leaat three independent determinations. c Currenta were too small to allow the determination of accurate values.
*
cholesteryl residue as compared to a simple octadecyl chain. DOPC and egg PC films exhibit similar properties with both redox probes. This is expected owing to egg PC's high degree of unsaturation. In contrast, both 12+and 22+ give rise to almost undetectable peak currents in pure DSPC films. Again, this is ascribed to the gel (rigid)nature of DSPC films at room temperature. Another general trend that is clear from the data is the lack of a defined effect from the addition of 25 mol % cholesterol to either DOPC or egg PC lipid fiis. However, cholesterol addition to DSPC films increases the voltammetric response of these lipid films to the point where peak potentials and currents become measurable. In effect, then, cholesterol addition expands the rigid crystalline nature of DSPC films at room temperature, favoring the diffusion of the viologen probes through the multilayer assembly. Thus the addition of cholesterolto DSPC films has an effect similar to an increase in the degree of unsaturation. In summary, our results indicate that cholesterol addition (at the 25 mol % level) does not significantly alter the rate of diffusion of either viologen probe inside lipid films containing a large fraction of unsaturated acyl residues. However, a similar level of cholesterol has a measurable effect on fully saturated lipid films increasing their fluidity as assessed from the voltammetric currents detected with both viologen probes.
Conclusions
Thiiwork has shown that cast phosphatidylcholine f i i provide a very useful matrix to conduct voltammetric experiments with amphiphilic electroactive species, such as viologens 12+ and 22+. The cyclic voltammetric results suggest that the lipid film is composed of organized multilayer microdomains connected by regions of loose molecular order. Charge propagation across the lipid films seems to be diffusional. As a result of this, the extent of the electrochemical conversions within the film can be controlled by adjusting the fluidity of the lipid film. We have shown that this is the case by measuring the voltammetric currents as a function of (i) degree of unsaturation of the lipid's fatty acyl residues, (ii) temperature, and (iii) cholesterol content.
Acknowledgment. This research was supported by the National Science Foundation (Grant CHE-9000531). We are grateful to Jing Li for the sample of 2(PF& used in this work.
