Electron-Transfer Processes at Electrodes Covered by Lipid Layers

Langmuir−Blodgett lipid films deposited onto glassy carbon electrodes were analyzed by comparing the voltammetric profiles of Fe(CN)63-/Fe(CN)64-, ...
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Electron-Transfer Processes at Electrodes Covered by Lipid Layers. Correlation with the Lipid Behavior at the Air-Water Interface M. Fernanda Mora, Natalia Wilke, and Ana M. Baruzzi* Instituto de Investigaciones en Fisicoquı´mica de Co´ rdoba (INFIQC), Departamento de Fisicoquı´mica, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´ rdoba, 5000 Co´ rdoba, Argentina Received October 22, 2002. In Final Form: April 15, 2003

Langmuir-Blodgett lipid films deposited onto glassy carbon electrodes were analyzed by comparing the voltammetric profiles of Fe(CN)63-/Fe(CN)64-, FeEDTA-/FeEDTA2-, Cu(NH3)42+/Cu(NH3)2+, and Ru(NH3)63+/Ru(NH3)62+ redox couples at these electrodes. A series of sphingolipids was analyzed, including a glicerocholine. A decrease in current depending on the lipid nature and on the couple was observed. The dependence on the lipid nature was analyzed by considering the behavior of the lipid monolayers at the air-aqueous-phase interface. The molecular area of the lipids, the dipole moment perpendicular to the interface, and the compressibility at the transfer pressure were obtained from the compression isotherms when each redox couple was present in the subphase. The results indicate that the electrochemical blocking depends mainly on the compressibility factor. Concerning the dependence on the redox reaction nature, it was found that, under these experimental conditions and for neutral lipids, as the electron-transferprocess rate increases, the electrochemical response is less sensitive to the presence of the lipid layers.

1. Introduction Monolayers formed at air-water interfaces (Langmuir films) and transferred to solid substrates (LangmuirBlodgett or self-assembled films) are used to study the properties and supramolecular structure of layers of lipids constituent of membranes.1 When the substrate of a Langmuir-Blodgett (L-B) film is an electrode, electrochemistry offers a good possibility for obtaining information concerning the interaction among the components, the electrical properties, and the permeability to different species of interest. The analysis of redox reactions at these modified interfaces gives additional information about the efficiency of the transfer process onto the solid substrate. In general, it is observed that the redox reaction occurs through pits and holes present in the lipid layer. When cyclic voltammetry is used as the electrochemical technique, the shape of the voltammograms depends on the coverage, arrangement, and size of the pits and holes and possible interactions between the different components of the systems.2,3 In other words, the degree of heterogeneity and the characteristics of the lipid layer on the substrate determine the response of the electrode and in turn depend on the forces that lead to a particular supramolecular arrangement. The aim of this paper is to find a relation between the behavior of a lipid monolayer at the air-aqueousphase interface and its blocking effect on an electrontransfer process when layers of this lipid cover the electrode. The characterization of this behavior is done by lateral pressure-molecular area and superficial potential-molecular area compression isotherms. With this purpose two couples are used: Cu(NH3)42+/Cu(NH3)2+ and * To whom correspondence should be addressed. Fax: +54-3514334188. E-mail: [email protected]. (1) Gaines, G. L. In Insoluble Monolayers at Liquid-Gas Interfaces; Prigogine, I., Ed.; Wiley-Interscience: New York, 1966. (2) Wilke, N.; Baruzzi, A. M. J. Electroanal. Chem. 2002, 67, 537. (3) Sabatani, E.; Rubinstein, I. J. Phys. Chem. 1987, 91, 6663.

FeEDTA-/FeEDTA2-. Previous results2 obtained for Ru(NH3)63+/Ru(NH3)62+ and Fe(CN)63-/Fe(CN)64- were also used for the analysis. Changes induced at the airaqueous-phase interface by the presence of these ions in the subphase were also analyzed. Cyclic voltammetry was used as the electrochemical technique. The series of lipid employed was dimirystoylphosphatidylcholine (dmpc), bovine brain sphingomyelin (sphm), galactocerebroside (galcer), and sulfatide (sul). 2. Experimental Section 2.1. Materials. The electrochemical measurements were carried out in a three-electrode conventional cell. Glassy carbon, used as working electrode, was commercially available (0.033 cm2 radius), polished with alumina of 1, 0.5, and 0.03 µm, and rinsed with ultrapure water. The counter electrode was a platinum mesh and the reference electrode, Ag|AgCl|Cl- (3 M). These experiments were performed in a solution containing 1 mM of the different redox species and 20 mM NaNO3 as the supporting electrolyte (analytical reagent, Mallinckrodt). The redox species used were K3Fe(CN)6 (proanalysis, Merck), Ru(NH3)6Cl3 (Stream Chemicals), Cu(NH3)42+ prepared from Cu(NO3)2 (Merck) and NH4OH (Carlo Erbo), and Fe(EDTA)prepared from Fe(NO3)3 (Merck) and disodium ethylendiamintetraacetate (EDTA,Merck). Ultrapure water produced by a MilliQ system (18 MΩ) was used for the subphase and for the electrochemical measurements. Dmpc, sphm, and galcer were from Avanti Polar Lipids, and sul was purified from bovine brain as previously described.4,5 2.2. Methods. 2.2.1. Lipid Monolayers. Lipids were spread on different subphases from solutions of approximately 1 nmol µL-1 prepared in chloroform:methanol (2:1). Details of the equipment used have been given in previous publications.6,7 The subphase temperature was kept at 25 ( 1 °C with a refrigerated Haake F3C thermocirculator. Surface pressure was measured with a platinized (4) Maggio, B.; Cumar, F. A. Brain Res. 1974, 77, 297. (5) Radin, N. S.; Brown, J. R Biochem. Prep. 1960, 7, 31. (6) Maggio, B.; Bianco, I. D.; Montich, G. G.; Fidelio, G. D.; Yu, R. K. Biochim. Biophys. Acta 1994, 1190, 137. (7) Carrer, D. C.; Maggio, B. J. Lipid Res. 1999, 40, 1978.

10.1021/la026732h CCC: $25.00 © 2003 American Chemical Society Published on Web 06/28/2003

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platinum plate. The surface potential was measured with an 241 95 Am plate as the surface electrode and a calomel reference electrode. At least triplicate monolayer isotherms were obtained and averaged. The molecular dipole moment perpendicular to the interface was calculated as in ref 8. The reciprocal of the compressibility, denominated the surface compressional modulus κ (mN m-1), was also used to describe monolayer properties. This is defined as1

( )

κ ) -Am

∂π ∂Am

T

where Am is the molecular area and is given in Å2 and π, the lateral surface pressure, is in mN m-1. 2.2.2. LB Film Transfer. Monolayers were spread over water and taken to the transfer pressure (40 mN m-1). Film transfer was accomplished as Langmuir and Schaefer proposed,1,9 that is, by gently allowing horizontal contact of the electrode surface with the lipid monolayer with a precision step motor; the electrode was maintained in that position for 30 s before lifting. This procedure was repeated four times. Every film was allowed to dry out for 30 min. The decrease in the total monolayer area as the electrode was lifted was measured as a control. This cannot be used for calculating a transfer rate because the electrode is embedded in Teflon onto whose surface the lipid is also adsorbed. 2.2.3. Electrochemical Measurements. Voltammetric experiments were performed using an Autolab electrochemical analyzer GPSTAT30. After the film transfer, the electrode was introduced in the solution and a few cycles were applied to attain a stationary condition of the surface. Then, the first subsequent cycle was recorded; this process was followed by a short time interval to recover the diffusion layer. At least triplicate voltammograms were performed; all the profiles shown are representative of the series of experiments. It is important to remark that the electrodes were only polished and not electrochemically activated to avoid an increase in hydrophilicity, improving in this way the adherence of the lipid layer. Unfortunately, with this pretreatment the rate constants of the redox couples are lower and less reproducible than on activated electrodes. This means that even with the polished layer-free electrode, there may be differences between duplicate experiments. As a consequence, it was very important to repeat the measurements several times to ensure reproducibility of the response with each lipid layer. For the same reason, the voltammograms obtained with the layer were always compared with the corresponding measurements on the bare electrode just before the coating was done. It is worth emphasizing that although the voltammograms at the bare electrode are different, the effect of the lipid layers is reproducible, and representative profiles of a series of experiments are shown.

Figure 1. Lateral pressure-molecular area (π-Am) compression isotherms of the different lipids over water (open symbols, left scale) and dipolar moment perpendicular to the interface (µp; solid symbols, right scale). Dmpc (square), galcer (circle), sphm (up triangle), and sul (down triangle).

3.1. Monolayer Isotherms. Information about the interaction between the lipid molecules at the airaqueous-phase interface was obtained in the presence and absence of the ions under study, using lateral pressuremolecular area and surface potential-molecular area isotherms. Figure 1 shows the lateral pressure-molecular area (π-Am) and the dipolar moment perpendicular to the interface-molecular area (µp-Am) compression isotherms at the air-aqueous-phase interface at 25 °C for monolayers of the different lipids studied in this work. They can be briefly described as follows: Dmpc. It is in a liquid-expanded state, with a collapse pressure of 49 ( 4 mN m-1 at a limiting mean molecular area of 40.4 Å2, in agreement with previous measurements.10 As the monolayer is compressed, the molecular

dipole moment increases up to a mean molecular area of about 95 Å2. Thereafter, it decreases gradually as the film is brought to a more closely packed state, revealing a contribution opposite to that of the hydrocarbon chains.11-13 Sphm. This lipid shows a two-dimensional phase transition at 13.0 ( 0.8 mN m-1 at a molecular area of 70 ( 2 Å2, and it collapses at 51 ( 2 mN m-1 at a limiting molecular area of 46.9 Å2. The resultant dipole moment µP of sphm is positive at all values of molecular area, but it is smaller in magnitude than that of dmpc and behaves in a different manner under compression. An initial increase of µP between 150 and 200 mD is observed, after which it remains insensitive to compression. The twodimensional phase transition is reflected as an upward inflection at molecular areas between 70 and 80 Å2. Sul. It forms a liquid-expanded film and collapses at 52 ( 2 mN m-1 at a limiting molecular area of 37.5 Å2. The dipole moment µP is near zero at the lift-off molecular area and acquires positive values reaching about 150 mD at the closest packing. Galcer. This lipid is liquid-condensed14 with a collapse pressure over water at about 44 ( 2 mN m-1, at a limiting molecular area of 42 Å2. In these monolayers µP is always positive, similar to dmpc and sphm, but completely different from sul. The only difference between galcer and sul is the presence of a -SO3 group in C3 of the galactose moiety in the polar headgroup of the anionic glycosphingolipid. The large decrease in µP of sul is due to the contribution of the negatively charged -SO3 group.15 For a more detailed discussion of the compression isotherms, see ref 8. When these π-Am compression isotherms are performed over the aqueous solutions used in the electrochemical measurements, negligible changes in the shape are observed. On the contrary, the dipole moments change for all lipid layers when these ions are present, this being evidence of interactions between the ions and the lipid

(8) Wilke, N.; Baruzzi, A. M.; Maggio, B. Langmuir 2001, 17, 3980. (9) Ulman, A. An Introduction to Ultrathin Organic Films. From Langmuir-Blodgget to Self-Assembly; Academic Press: San Diego, 1991. (10) Cevc, G.; Marsh, D. Phospholipid Bilayers. Physical Principles and Models. In Cell Physiology: A Series of Monographs; Bittar, E. E., Ed.; Wiley-Interscience: New York, 1987; Vol. 5.

(11) Carrer, D. C.; Maggio, B. J. Lipid Res. 1999, 40, 1978. (12) Maggio, B. J. Lipid Res. 1999, 40, 930.c¸ (13) Vogel, V.; Mo¨bius, D. Thin Solid Films 1987, 159, 73. (14) Maggio, B.; Cumar, F. A.; Caputto, R. Biochem. J. 1978, 171, 559. (15) Maggio, B. Prog. Biophys. Mol. Biol. 1994, 62, 55.

3. Results and Discussion

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Figure 2. Voltammograms of Cu(NH3)4+/Cu(NH3)22+ over bare (solid lines) and covered (dotted lines) electrodes. Composition of the electrochemical cell: 1 mM Cu(NO3)2 in NH4+/NH3 buffer and 20 mM NaNO3, pH ) 9. Electrode covered by dmpc (A), sphm (B), sul (C), and galcer (D). v ) 0.100 V s-1.

Figure 3. Voltammograms of Fe(EDTA)-/Fe(EDTA)2- over bare (solid lines) and covered electrodes (dotted lines). Composition of the electrochemical cell: 1 mM Fe(NO3)3, 10 mM EDTA, and 20 mM NaNO3. The electrode is covered by dmpc (A), sphm (B), sul (C), and galcer (D). v ) 0.100 V s-1.

monolayer, despite the small changes observed in the π-Am isotherms2,8 (data not shown). These isotherms were performed considering that although the transfer of the monolayer was done using pure water as subphase, the electrochemical experiments proceed in solutions containing 1 mM redox couple and 20 mM NaNO3. 3.2. Electrochemistry. At 0.100 V s-1, Cu(NH3)42+/ Cu(NH3)2+ and Ru(NH3)63+/Ru(NH3)62+ behave as reversible couples, while FeEDTA-/FeEDTA2- and Fe(CN)63-/ Fe(CN)64- as quasi-reversible, this last one being slower. Figure 2 shows representative current-potential profiles for the Cu(NH3)42+/Cu(NH3)2+ redox couple. The electrode was prepared by transferring four layers of dmpc (A), sphm (B), sul (C), or galcer (D). In all cases, a blocking of the surface with a corresponding decrease in current with respect to the bare electrode is observed. The effects promoted by dmpc, sphm, and sul (Figure 2A-C) are similar and lower than in the case of galcer (Figure 2D). For the FeEDTA-/FeEDTA2- redox couple, a decrease in current is also observed when four lipid layers are transferred (Figure 3). For this redox reaction, the effect is greater and more dependent on the lipid nature. The decrease in current follows the order galcer > sul > sphm

Mora et al.

> dmpc. In the case of galcer (Figure 3D), almost all the current is suppressed. For Ru(NH3)63+/Ru(NH3)62+, a blocking effect was only produced by sphm and galcer (see ref 2). On the contrary, for Fe(CN)63-/Fe(CN)64-, as well as for FeEDTA-/ FeEDTA2-, there is always a decrease in current in the order galcer > sphm > dmpc = sul (see ref 2). To have a better insight into the different parameters that characterize the lipid layers and their effect on the electrochemical response, the current, the molecular area, the dipolar moment, and the compressibility factor are plotted in Figure 4 for the different redox couples and lipids. Figure 4A shows the average cathodic current (peak current or maximum current in the cases where no peak is observed) normalized by the peak current of the voltammogram on the bare electrode (Inorm). The error bars were calculated by the average of at least three independent measurements. The blocking effect increases according to the order dmpc, sphm, and galcer for each redox couple, being more evident for the negatively charged couples, which in turn are the slowest. The effect promoted by sul, the charged lipid, is similar to that of sphm or dmpc, depending on the redox reaction. Figure 4B shows that at the transfer pressure, 40 mN m-1, the molecular area of the different lipids depends neither on the ions present in the subphase nor on the lipid nature, except in the case of sphm which occupies a slightly larger area. The dipole moment perpendicular to the interface, shown in Figure 4C, follows the order dmpc > sphm = galcer . sul. Sul presents the lowest µp over all subphases. In the case of galcer, µp is independent of the ion nature, indicating a low interaction with the ions in the subphase. The compressibility factor (κ) calculated from the π-Am isotherms is a measure of the compactness of the layers. Opposite to the molecular area and the dipole moment, Figure 4D shows that at π ) 40 mN m-1 the values of κ are different for each analyzed lipid and the order it follows (galcer > sphm = sul > dmpc) is the same for all couples, with galcer forming a quite packed layer and dmpc a much more expanded film. Figure 5 shows the normalized current as a function of the compressibility factor. This plot clearly shows that, for neutral lipids, as κ increases Inorm decreases. Galcer forms a condensed monolayer, promoting a great blocking effect; dmpc forms expanded monolayers with a little influence on the voltammetric profile. Sphm follows an intermediate behavior, which is not the case for sul, although both lipids have similar properties at the airaqueous-phase interface. The great differences in the electrochemical response of these two lipids indicate that the negative charge of sul plays an important role. It is possible that the charge introduces disorder to the layers or electrostatic interactions with the electroactive ion, explaining that the deviation of sul from the general tendency is bigger for the highly charged couples (Ru(NH3)63+/Ru(NH3)62+ and Fe(CN)63-/Fe(CN)64-). The electrochemical response also depends on the nature of the redox reaction. In this sense, Figure 4A shows that for the cationic couples, which in turn are the faster, the decrease in current promoted by the lipid layers is lower than for the anionic couples. Moreover, for the anionic couples, the dependence of the electrochemical blocking effect on κ is more evident. To analyze this dependence, Inorm was plotted vs the electron-transfer rate constant (k0) of each redox reaction (Figure 6), obtained by fitting the voltammograms on the bare electrode using the

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Figure 4. Effect of the lipid and redox couple nature on the electrochemical and isotherms parameters. Subphase or electrochemical solution composition: 1 mM K3Fe(CN)6 + 20 mM NaNO3 (white), 1 mM Ru(NH3)6Cl3 + 20 mM NaNO3 (gray), 1 mM Cu(NO3)2 + NH4+/NH3 buffer + 20 mM NaNO3 (oblique lines), and 1 mM Fe(NO3)3 + 10 mM EDTA + 20 mM NaNO3 (black). Parameters: (A) Average peak current (or maximum current when there is no peak) normalized by the peak current of the voltammogram in the absence of lipid layers for each lipid; (B) average molecular area at the transfer pressure (40 mN m-1); (C) average dipolar moment perpendicular to the interface at the transfer pressure (40 mN m-1); (D) compressibility factor at the transfer pressure (40 mN m-1).

. FeEDTA-/FeEDTA2- > Fe(CN)63-/Fe(CN)64-. Figure 6 shows that, for all neutral lipids, Inorm increases as k0 increases, which means that, under these electrochemical conditions (cyclic voltammetry with 0.100 V s-1 as sweep rate), the faster electron-transfer processes are less affected by the presence of the lipid than the slower. Sul, the charged lipid, has a different behavior with respect to the redox couple nature, indicating that the effect promoted by the presence of the lipid layers on the redox reaction is not so simple to understand. The reasons described above could explain why sul does not follow the general behavior. 4. Conclusions

Figure 5. Inorm (see text) as a function of the compressibility factor taken of the corresponding isotherm. Composition of the electrochemical cell: 1 mM K3Fe(CN)6 + 20 mM NaNO3 (squares), 1 mM Ru(NH3)6Cl3 + 20 mM NaNO3 (circles), 1 mM Cu(NO3)2 + NH4+/NH3 buffer + 20 mM NaNO3 (up triangles), and 1 mM Fe(NO3) + 10 mM EDTA + 20 mM NaNO3 (down triangles). Solid symbols: galcer, dmpc, and sphm. Open symbols: sul.

Nicholson method.16 The transfer rate constant k0 follows the order Ru(NH3)63+/Ru(NH3)62+ > Cu(NH3)42+/Cu(NH3)2+

For the series of lipids analyzed, the compressibility of each lipid at the air-aqueous-phase interface is an important factor determining the blocking effect promoted by the lipid layers on the electrode. At what level the compressibility factor of the lipid layer affects the electrochemical behavior of the redox couple cannot be answered at this stage. Probably it influences the electrochemical response in different ways. On one side, it is known that the transfer process of the lipid is improved (16) Nicholson, R. S. Anal. Chem. 1965, 37, 1351.

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potential, can occur on the electrode. Certainly, the response depends on all these factors. It has to be noted that this dependence of the normalized current with the compressibility factor can only be observed if the coverage of the electrode is enough, which in this case was attained by transferring four layers. If only one layer is transferred, the coverage is not enough.8 Concerning the dependence of the voltammetric behavior on the redox reaction nature, under these experimental conditions it is more sensitive to the presence of the lipid layer as the redox process rate decreases, except in the case of sul where factors such as the charge are influencing the response. Both the charge-transfer process itself and the mass transport regime of the electroactive ions to the surface can be influenced by the lipid layers. The electrochemical behavior of a redox reaction on electrodes modified by layers of these lipids is not greatly affected by the molecular area or by the dipole moment. Figure 6. Inorm (see text) as a function of the electron-transfer constant rate of the corresponding redox reaction. Electrode covered by dmpc (squares), sphm (circles), galcer (up triangles), and sul (down triangles).

as the layer is more rigid.9 On the other, a possible rearrangement and the appearance of pits in the lipid layers, which in turn depend on κ and on the applied

Acknowledgment. Financial support from the Consejo Nacional de Investigaciones Cientı´ficas y Tecnolo´gicas (CONICET), Secretarı´a de Ciencia y Tecnologı´a (SECyT) and Agencia Co´rdoba Ciencia is gratefully acknowledged. N. Wilke thanks CONICET for the fellowship granted. LA026732H