An Electrochemical Study of Antibiotic Ionophores in Self-Assembled

antibiotic ionophores, nystatin, a polyene which transports primarily anions across ... azide ions per nystatin pore and 57.2 ( 1.1 water molecules or...
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Langmuir 1997, 13, 5962-5968

An Electrochemical Study of Antibiotic Ionophores in Self-Assembled Lipid Layers at Platinum Hamilton J. Maguire and Sharon G. Roscoe* Chemistry Department, Acadia University, Wolfville, Nova Scotia, Canada B0P 1X0 Received June 4, 1996. In Final Form: July 7, 1997X Self-assembled lipid layers (SALL) have been formed on a platinum electrode surface from aqueous solutions of potassium oleate and lithium γ-linolenate at 299 K in a pH 7.0 phosphate buffer. Cyclic voltammetric measurements showed a significant decrease in the surface charge density due to oxide formation on the electrode surface in an aqueous solution in the presence of the lipids. A lipid barrier was formed that was impermeable to the transport of water and azide ions to the electrode surface. The antibiotic ionophores, nystatin, a polyene which transports primarily anions across membranes, and alamethicin, an antibiotic peptide which transports anions and excludes cations, were introduced into the SALL in separate experiments. The surface charge density increased in the presence of these ionophores as a result of diffusion of water to the electrode surface through the apparent ionophore pores. Azide ions diffused through the ionophore-incorporated SALL and were detected and measured electrochemically by a decrease in surface charge density from oxide due to the blocking of the electrode surface by the adsorbed azide ions. The electrode surface coverage was determined to be 16.4 ( 0.4 water molecules or 15.6 ( 1.8 azide ions per nystatin pore and 57.2 ( 1.1 water molecules or 56.6 ( 3.8 azide ions per alamethicin pore. These results agree with calculations for monolayer coverage based on geometrical dimensions of the molecules.

Introduction There has been a great deal of interest in the transport of small molecules across biological membranes in order to understand the mechanisms of drug delivery systems1-3 and in the development of biosensors.4-7 Lipid bilayers can be used as model systems to simulate natural membranes in experimental situations. In the past, membranes were formed by pulling a lipid solution across a small orifice in a thin plastic film.8 These experimental bilayers were used to investigate electrical conductance,8-10 ion permeability,8,9,11 and diffusion through membranes.12 More recent work has involved the incorporation of antibiotics,9,13,14 toxins,14,15 and viruses14 into membranes. Electrochemical studies have used mercury,16-22 gold,23-28 * Author to whom correspondence should be addressed. X Abstract published in Advance ACS Abstracts, October 1, 1997. (1) Venema, F. R.; Weringa, W. D. J. Colloid Interface Sci. 1988, 125, 484. (2) Guerra, F. J.; Neumann, J. M.; Huynh-Dinh, T. Tetrahedron Lett. 1987, 28, 3581. (3) Michaelis, L.; Moore, M. J. Top. Lipid Res. 1986, 264. (4) Nikolelis, D. P.; Brennan, J. D.; Brown, R. S.; McGibbon, G.; Krull, U. J. Analyst 1991, 116, 1221. (5) Nikolelis, D. P.; Brennan, J. D.; Brown, R. S.; Krull, U. J. Anal. Chim. Acta 1992, 257, 49. (6) Nikolelis, D. P.; Krull, U. J. Talanta 1992, 39, 1045. (7) Nikolelis, D. P.; Krull, U. J. Electroanalysis 1993, 5, 539. (8) Finkelstein, A.; Holz, R. In Membranes: Lipid Bilayers and Antibiotics; Eisenman, G., Ed.; Marcel Decker: New York, 1973; Vol. 2, pp 377-408. (9) Kleinburg, M. E.; Finkelstein, A. J. Membr. Biol. 1984, 80, 257. (10) Yamanaka, T.; Tano, T.; Tozaki, K.; Hayashi, H. Chem. Lett. 1994, 1143. (11) Medoff, G.; Kobayashi, G. A. In Antifungal Chemotherapy; Speller, D. C. E., Ed.; John Wiley & Sons Ltd.: New York, 1980; pp 3-33. (12) O’Neill, L. J.; Miller, J. G.; Petersen, N. O. Biochemistry 1986, 25, 177. (13) Rizzo, V.; Stankowski, S.; Schwarz, G. Biochemistry 1987, 26, 2751. (14) Marshall, G. R.; Beusen, D. D. In Biomembrane Electrochemistry; Blank, M., Vodyanoy, I., Eds.; American Chemical Society: Washington, DC, 1994; pp 259-314. (15) Rex, S. Biophys. Chem. 1996, 58, 75. (16) Becucci, L.; Moncelli, M. R.; Guidelli, R. J. Electroanal. Chem. 1996, 413, 187. (17) Nelson, A. Langmuir 1996, 12, 2058. (18) Nelson, A. J. Chem. Soc., Faraday Trans. 1993, 89, 2799. (19) Nelson, A. J. Chem. Soc., Faraday Trans. 1993, 89, 3081.

S0743-7463(96)00545-8 CCC: $14.00

and carbon29-31 electrodes as the substrate for the formation of lipid monolayers and bilayers. Some of these studies have used the formation of covalent bonds between the sulfur of the thiol functional group and the metal surface to form a stable monolayer24,25 from ethanolic solutions. Modified electrodes have also been made by transferring insoluble surfactant layers from a LangmuirBlodgett trough to a gold electrode by touching the surface containing the monolayer.26-28 Antibiotic ionophores such as nystatin and alamethicin, which are both anion and cation ionophores, are used to facilitate the transport of ions through the hydrophobic regions of bilayers. Nystatin (926 g mol-1) is an effective antifungal, antiprotozoal, antitumor, and antiviral agent32 with the structure33 shown in Figure 1a. When it is incorporated into a membrane, nystatin forms a “barrelstave” type of structure where the polyene backbone interacts with the fatty acid tails, the sugar interacts with the polar fatty acid heads and the hydroxyl and carbonyl groups line the pore.8,33 Two barrel-staves form a dimer end-to-end to span the membrane, and these are in equilibrium with the monomers. The pore diameter is 0.8-1 nm depending on whether the number of molecules associated to form the pore is 8-10. Alamethicin (2 200 (20) Miller, I. R.; Doll, L.; Lester, D. S. Bioelectrochem. Bioenerg. 1992, 28, 85. (21) Pospisil, L.; Muller, E.; Emons, H.; Dorfler, H. D. J. Electroanal. Chem. Interfacial Electrochem. 1984, 170, 319. (22) Kozarac, Z.; Klaric, R.; Dragcevic, D.; Cosovic, B. Colloids Surf. 1991, 56, 279. (23) Nakashima, N.; Taguchi, T. Colloids Surf. A 1995, 103, 159. (24) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197. (25) Lang, H.; Duschi, C.; Gratzel, M.; Vogel, H. Thin Solid Films 1992, 210, 818. (26) Bizzotto, D.; Noel, J. J.; Lipkowski, J. J. Electroanal. Chem. 1994, 369, 259. (27) Bizzotto, D.; Lipkowski, J. Prog. Surf. Sci. 1995, 50, 237. (28) Bizzotto, D.; Lipkowski, J. J. Electroanal. Chem. 1996, 409, 33. (29) Blaha, C. D.; Liu, D.; Phillips, A. G. Biosens. Bioelectron. 1996, 11, 63. (30) Bianco, P.; Haladjian, J. Electroanalysis 1995, 7, 442. (31) Kaufmann, J. M.; Chastel, O.; Quarin, G.; Patriarche, G. J.; Khodari, M. Bioelectrochem. Bioenerg. 1990, 23, 167. (32) Witzke, N. M.; Bittman, R. Biochemistry 1984, 23, 1668. (33) Gennis, R. B. In Biomembranes: Molecular Structure and Theory; Cantor, C. R., Ed.; Springer-Verlag: New York, 1989; pp 127-130, 286-291.

© 1997 American Chemical Society

Electrochemical Study of Antibiotic Ionophores

Langmuir, Vol. 13, No. 22, 1997 5963 Table 1. Capillary Gas Chromatography Analysis of Potassium Oleate and Lithium γ-Linolenatea potassium oleate

Figure 1. Structures of the ionophores used in this study: (a) nystatin, (b) alamethicin.

g mol-1), on the other hand, is composed of 20 amino acids arranged in an R-helix33 (Figure 1b). The average number of alamethicin molecules incorporated into a barrel-stave structure in membranes is between 8 and 1013 resulting in a pore diameter as large as 1.5 nm.34 The alamethicin pore is voltage-gated, in which the opening of the pore requires a potential difference of 100 mV between the sides of the membrane.13,33,34 The present study reports (i) the SALL of soluble salts of fatty acids at the platinum electrode surface directly from an aqueous solution, (ii) the incorporation of ionophores into the SALL, and (iii) the transport of ions through ionophore-incorporated SALL to the electrode surface. The platinum electrode which supports the SALL is used as a sensitive detector for the transport of ions such as azide through ionophores to the electrode surface. The SALLs were formed from γ-linolenate and oleate on a platinum electrode surface in a pH 7.0 phosphate buffer solution at 299 K. Incorporation of the ionophores, nystatin and alamethicin, separately into the SALLs allowed the transport of water molecules through the lipid barrier to the electrode surface which was detected and measured by oxide deposition on the electrode surface. The azide ion is known to adsorb very strongly to a platinum electrode surface, resulting in the blocking of the oxide deposition normally seen in aqueous solutions.35,36 The transportation of azide ion through these ionophores was also measured but in this case by the decrease in oxide formation due to the competitive adsorption of azide ion which blocks oxide deposition. These studies provide a model of self-assembling lipid layers for investigations into the transportation of species such as ions and organic molecules through lipid barriers from aqueous solutions and may provide a useful medium in the development of biosensors. Experimental Section (i) Chemicals and Solutions. Solutions of nystatin, alamethicin, sodium azide (Sigma Chemical Co.), potassium oleate (Fluka Chemical Co.), and lithium γ-linolenate (Efamol Research Institute) were all prepared by dissolving the reagents in 0.05 M phosphate buffer at pH 7.0. Capillary gas chromatographic analyses (Efamol Research Institute) of the potassium oleate and lithium γ-linolenate are found in Table 1. The buffer was made with monobasic anhydrous KH2PO4 (Sigma Chemical Co.) in conductivity water (Nanopure, resistivity 18.0 MΩ cm) and 0.10 mol L-1 sodium hydroxide (concentrated volumetric solution, BDH Chemical Co.). Sulfuric acid solutions of 0.5 M were prepared using Aristar grade, BDH Chemical Co., for calibrating the platinum electrode surface area. Nitrogen gas (Linde, (34) Esposito, G.; Carver, J. A.; Boyd, J.; Campbell, I. D. Biochemistry 1987, 26, 1043. (35) Roscoe, S. G.; Conway, B. E. J. Chem. Soc., Chem. Commun. 1988, 900. (36) Roscoe, S. G.; Conway, B. E. J. Electroanal. Chem. 1988, 249, 217.

lithium γ-linolenate

identification

mass percent

identification

mass percent

air 14:0 16:0 16:1 ω7 18:0 18:1 ω9 18:2 ω6 20:1 ω9

0.40 1.92 12.38 3.59 6.89 55.59 9.40 1.48

16:0 16:1 ω7 18:0 18:1 ω9 18:2 ω6 18:3 ω6 18:3 ω3 other

0.51 0.77 0.19 3.66 16.02 76.68 0.58 1.61

a CGC analyses performed by the Efamol Research Institute, Kentville, NS, Canada.

commercial purity 99.7%) was deoxygenated by passage through a copper furnace at 573 K and dried by passage through a column of molecular sieves. The nitrogen gas was then used to deoxygenate the solution in the electrochemical cell, which also served to mix the bulk solution. (ii) Electrochemical Equipment and Techniques. Experiments were made in an all-glass, three-compartment electrochemical cell, with compartments for each of the Pt wire working electrode, Pt wire counter electrode, and saturated calomel reference electrode. All of the compartments were fitted with a ground-glass top which allowed the insertion of an electrode and the venting of gases through miniature glass bubblers. A nitrogen bubbler was inserted into the working electrode compartment which ensured a well-mixed solution and removal of oxygen. The working and counter electrodes were constructed of high-purity platinum wire (99.99%, Johnson, Matthey and Mallory Ltd.) which was sealed through soft glass tubing. The saturated calomel reference electrode was prepared using a standard procedure.37 Cyclic voltammograms were obtained using a potentiostat (Hokuto Denko Ltd., Model HD-301) and function generator (Hokuto Denko Ltd., model HD-111) to produce a repeating triangular potential sweep between -0.70 and +1.00 V at a sweep rate of 500 mV s-1. Both a well-mixed solution from bubbling nitrogen and a fast sweep rate were used to eliminate the effects of diffusion. The current and potential response from the potentiostat was recorded on an X-Y recorder (Allen Datagraph, Inc., Model 720M). (iii) Experimental Methodology. Before the cyclic voltammograms were recorded, the electrochemical cell containing the phosphate buffer, pH 7.0, and the other solutions in separate containers were placed in a constant-temperature bath, fitted with a Julabo temperature regulator, maintained at 299 K for 1/ h prior to the start of each experiment. During this time, the 2 phosphate buffer in the cell was continuously purged with nitrogen gas to ensure the removal of dissolved oxygen. This was verified by the profile of the cyclic voltammograms recorded at intervals before the start of the experiment. Cyclic voltammograms were recorded when steady-state conditions were attained as shown by continuously identical tracings. The surface charge from the underpotential deposition on the platinum electrode surface38 was determined by integration of the oxide reduction region (OR) of the cyclic voltammogram below the double-layer region shown by the dotted line (Figure 2). The entire cyclic voltammogram was scanned with a Hewlett Packard Scan Jet IIcx scanner, and then the figure was digitized using Unscanit software (Silk Scientific Software). The area of the oxide reduction region was determined by entering the digitized image into a computer integration program. The real surface area of the electrode was calculated by measuring the area of the hydrogen reduction region, HR, of a cyclic voltammogram of a solution of 0.5 M H2SO4.38 The surface charge density, QOR, was obtained by dividing the surface charge by the real surface area of the working electrode. The surface charge density due to azide, QN3-, was determined from the difference between the surface charge density, QOR, due to oxide (37) Kennedy, J. H. Analytical Chemistry Principles; Harcourt Brace Jovanovich Publishers: San Diego, CA, 1984; p 482. (38) Conway, B. E.; Angerstein-Kozlowska, H. Acc. Chem. Res. 1981, 41, 49.

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Figure 2. Cyclic voltammogram of phosphate buffer alone, pH 7.0, at 299 K, recorded with sweep rates of 500 mV s-1: HR, hydrogen reduction region; HO, hydrogen oxidation region; OO, oxide deposition region; OR, oxide reduction region, showing the region for integration below the dotted line. formation for the ionophore-incorporated SALL in the absence and presence of azide. The results of the experiments are described in the following manner: (i) In order to characterize the behavior of each of these species at the electrode surface, measurements were made with the phosphate buffer alone, followed by measurements of each of the compounds (sodium azide, nystatin, and alamethicin) individually in the phosphate buffer. (ii) Lipids (γ-linolenate and oleate) were individually added to a phosphate buffer solution to determine the optimum bulk concentration for SALL formation. (iii) Once the SALL was formed, sodium azide was added to the bulk solution in increasing concentrations to determine whether azide ions were able to penetrate through the SALL to the electrode surface in the absence of an incorporated ionophore. (iv) Using the optimum concentration of lipid in the phosphate buffer, the SALL was then formed on the electrode surface. Studies were made with increasing concentrations of the ionophores in the bulk solution to measure the incorporation of each ionophore (nystatin and alamethicin) in each SALL (γ-linolenate and oleate). Cyclic voltammetric measurements were used to determine quantitatively the deposition of oxide on the electrode surface due to the transport of water through apparent pores formed by the ionophores in the SALL. Aliquots of sodium azide were then added to the cell with cyclic voltammograms recorded after each addition of azide. (v, vi) These measurements allowed calculations of the total area exposed by the apparent pores through the SALL, the number of pores formed in the SALL, and the number of molecules adsorbed per pore.

Results and Discussion (i) Electrochemical Effects of Azide and Ionophores Individually in the Phosphate Buffer Electrolyte Solution. Cyclic voltammetric measurements were first made with the phosphate buffer electrolyte alone (Figure 2). The surface charge density due to oxide formation, QOR, determined from the area of the oxide reduction region of the cyclic voltammogram of the phosphate buffer was calculated to be 525 µC cm-2. Aliquots of a sodium azide solution were then added to the phosphate buffer to give a bulk solution concentration ranging from 3.30 × 10-5 to 5.80 × 10-3 M, with cyclic voltammograms recorded after each addition of azide in order to measure the electrochemical response to the azide ions in the absence of lipids and ionophores. Figure 3 shows the dramatic decrease in the oxide reduction region of the cyclic voltammogram due to the blocking of the surface of the electrode by the azide ions, similar to the results obtained by Roscoe and Conway35,36 when azide was added to a basic solution. The peaks in the cathodic region of the cyclic voltammogram between -0.4 and -0.6 V are due to nitrogen evolution from the two-electrontransfer reduction of azide ions.35,36 As the azide concentration in the bulk solution increased, QOR decreased.

Maguire and Roscoe

Figure 3. Cyclic voltammogram of sodium azide (5.80 × 10-3 M) in phosphate buffer, pH 7.0, at 299 K, recorded with sweep rates of 500 mV s-1.

Figure 4. Cyclic voltammograms in phosphate buffer, pH 7.0, at 299 K, recorded with sweep rates of 500 mV s-1: solid line, potassium oleate (6.90 × 10-6 M); dotted line, potassium oleate (6.90 × 10-6 M) + nystatin (1.46 × 10-5 M).

QOR was 24 µC cm-2 for an azide bulk solution concentration of 5.80 × 10-6 M which is only 5% of QOR for the phosphate buffer alone. Finally, each of the ionophores, nystatin and alamethicin, was added to a phosphate buffer electrolyte individually, in order to examine the surface adsorption behavior of each ionophore at the platinum electrode surface in the absence of a SALL. The surface adsorption was measured by an increase in the charge in the anodic oxidation region and a corresponding decrease in the oxide reduction region, due to a partially blocked deposition of surface oxide. Nystatin adsorbed slightly onto the electrode surface with concentrations up to 1.16 × 10-4 M, as the QOR decreased only to 486 µC cm-2 which is 93% of QOR in the phosphate buffer alone. Similarly, slight adsorption of alamethicin occurred with concentrations up to 2.76 × 10-6 M. (ii) Electrochemical Effects of Lipids in the Phosphate Buffer Solution and the Formation of SALL. Lipids were individually added in small aliquots to a phosphate buffer solution to determine the optimum bulk concentration for SALL formation. Potassium oleate was added to give a bulk solution concentration of 6.90 × 10-6 M, and similarly lithium γ-linolenate was added to give a bulk solution concentration of 1.92 × 10-5 M. These separate additions caused the profile of the cyclic voltammogram to collapse gradually during 20-30 min of potential cycling (Figures 4 and 5). QOR obtained once steady-state conditions were attained was 3 µC cm-2 for the oleate SALL and 69 µC cm-2 for the γ-linolenate SALL. These values are 0.6% and 13%, respectively, of QOR of 525 µC cm-2 for the phosphate buffer alone. Thus a hydrophobic barrier is formed with the oleate SALL which is impermeable to the transport of water molecules and

Electrochemical Study of Antibiotic Ionophores

Figure 5. Cyclic voltammogram of lithium γ-linolenate (1.92 × 10-5 M) in phosphate buffer, pH 7.0, at 299 K, recorded with sweep rates of 500 mV s-1.

thereby prevents them from reaching the electrode surface as indicated by the removal of the hydrogen underpotential deposition regions (shown in Figure 2 as HR and HO). Although a barrier was formed with the γ-linolenate SALL, it was less hydrophobic than the oleate SALL. It is important to note that, in the absence of potential cycling, no self-assembling of the lipids occurs at the electrode surface. Because the number of layers formed by these lipids is not known, the term “self-assembled lipid layers” or SALL is used to describe these lipid barriers. (iii) Electrochemical Effects of the SALL Barrier on the Transport of Azide Anions to the Electrode Surface. Once the SALL was formed, sodium azide in phosphate buffer was added to the bulk solution in increasing concentrations to give a bulk solution concentration of azide ranging from 3.30 × 10-5 to 5.80 × 10-3 M. Cyclic voltammograms were recorded after each addition to determine the permeability of azide ion through the SALL in the absence of ionophores. With either the oleate or γ-linolenate SALL, there was no change in the QOR value and no evidence of cathodic nitrogen evolution due to the reduction of azide ions at the electrode surface.35,36 This indicated that the azide ions were unable to penetrate the SALL and contact the electrode surface. (iv) Electrochemical Effects of the SALL Barrier on the Transport of Water and Azide Anions to the Electrode Surface in the Presence of Ionophore. The optimum concentration of lipid in the phosphate buffer (i.e., an oleate concentration of 6.90 × 10-6 M or a γ-linolenate concentration of 1.92 × 10-5 M) was then used to form the SALL on the electrode surface. Studies were made with increasing concentrations of the ionophores in the bulk solution to measure the incorporation of each ionophore (nystatin and alamethicin) in each SALL (γ-linolenate and oleate). Cyclic voltammetric measurements were used to determine quantitatively the deposition of oxide on the electrode surface due to the transport of water through apparent pores formed by the ionophores in the SALL. An example of a cyclic voltammogram of a SALL in the presence of an ionophore is shown by the dotted line in Figure 4 for 1.46 × 10-5 M nystatin in the bulk solution, added after the oleate SALL was formed, as shown by the solid line. Figure 6 shows the relationship of QOR versus concentration of nystatin in the bulk solution for (i) the phosphate buffer alone (in the absence of nystatin and SALL), (ii) the phosphate buffer with an oleate SALL (in the absence of nystatin), (iii) the phosphate buffer with increasing concentrations of nystatin (in the absence of an oleate SALL), and (iv) the phosphate buffer with an oleate SALL and with increasing concentrations of nystatin. The QOR value for the phosphate buffer alone of 525 µC cm-2 is

Langmuir, Vol. 13, No. 22, 1997 5965

Figure 6. Surface charge density due to oxide versus nystatin bulk concentration for (i) b, gradual addition of nystatin to a phosphate buffer in the absence of an oleate SALL; (ii) O, gradual addition of nystatin to a phosphate buffer with an oleate SALL; (iii) 2, specific aliquot additions of nystatin to separate oleate SALLs. The dotted and dashed lines give the surface charge densities for the phosphate buffer alone and the phosphate buffer with an oleate SALL, respectively.

shown by a dotted line, and that for the phosphate buffer with an oleate SALL of 3 µC cm-2, by the dashed line. However, in the presence of increasing concentrations of nystatin in the phosphate buffer solution in the absence of an oleate SALL, the QOR decreased due to a small adsorption and blocking effect by the nystatin. When nystatin was added in increments to give a concentration range of 6.37 × 10-6-1.01 × 10-4 M to the bulk solution in which the SALL had already formed, the QOR gradually increased to a plateau value of 370 µC cm-2 due to the transport of water through the apparent pores formed by the ionophores. However, this value was significantly smaller than the QOR of 488 µC cm-2 observed for nystatin in the phosphate buffer in the absence of the SALL. This indicates that the SALL remained contiguous on the electrode surface in the presence of the ionophore. The maximum concentration of ionophores on the surface may be represented by a hexagonal close packing of the pores. In this case, the minimum area remaining in a square containing a circle (representing a pore) with sides touching those of the square is 23% of the total area. The surface charge density of the oxide with the highest concentration of nystatin was about 70% of the oxide formed in the presence of the phosphate buffer alone. Therefore, a minimum of 30% of the available area was covered by lipids, which also suggested that the SALL remained contiguous even in the presence of the highest concentration of nystatin. Five separate experiments were then made with a specific amount of nystatin added to the bulk solution, which allowed incorporation of different amounts of nystatin into the SALL (Figure 6). Aliquots of a sodium azide solution in phosphate buffer were added to the cell in each experiment to give a bulk solution concentration ranging from 3.30 × 10-5 to 5.65 × 10-3 M. Cyclic voltammograms were recorded after each addition of azide to measure the transport of azide ion through the pores by a decrease in oxide deposition due to the blocking effect by competitively adsorbed azide ions. In each experiment, QOR for oxide formation decreased with increasing azide concentration in the bulk solution until essentially all of the oxide formation was inhibited by the transported azide ion, independent of the nystatin bulk solution concentration (Figure 7a). Conversely, the surface charge density due to azide, QN3-, increased as the azide concentration in the bulk solution increased due to the blocking of the oxide formation (Figure 7b). As the nystatin concentration was increased, the plateau values for QN3- also increased,

5966 Langmuir, Vol. 13, No. 22, 1997

Maguire and Roscoe

Figure 8. Effect of surface concentration of oxide versus surface concentration of azide with different concentrations of nystatin ionophore with an oleate SALL. Concentration of nystatin in the bulk solution: O, 6.53 × 10-6 M; 4, 1.33 × 10-5 M; 0, 2.65 × 10-5 M; 3, 4.91 × 10-5 M; ], 1.01 × 10-4 M.

Figure 7. (a) Surface charge density due to oxide versus azide concentration for a nystatin ionophore with an oleate SALL. (b) Surface charge density due to azide versus azide concentration for a nystatin ionophore with an oleate SALL. Concentration of nystatin in the bulk solution: O, 6.53 × 10-6 M; 4, 1.33 × 10-5 M; 0, 2.65 × 10-5 M; 3, 4.91 × 10-5 M; ], 1.01 × 10-4 M.

which suggested that a greater number of apparent pores had been formed, and hence there was greater electrode surface area available for adsorption. Similar measurements were made with a γ-linolenate SALL. Aliquots of nystatin solution were added to the bulk solution to give a concentration range of 5.02 × 10-77.38 × 10-5 M. At the highest nystatin concentration, QOR had increased to a plateau value of 259 µC cm-2, which was lower than that for the nystatin-incorporated oleate SALL. Similarly, five separate experiments were made with the incorporation of specific amounts of nystatin in the γ-linolenate SALL, and aliquots of a sodium azide solution were added to the bulk solution in the cell to measure transport of the azide ion. Only the final results of these experiments are shown and described later. The experiments which were made with the nystatin ionophore with both oleate and γ-linolenate SALLs were repeated systematically for an alamethicin ionophore in each of the SALLs. After an oleate SALL was formed on the platinum electrode, aliquots of an alamethicin solution were added to the bulk solution of the cell to give concentrations that ranged from 2.31 × 10-8 to 4.60 × 10-6 M. At the highest alamethicin concentration QOR was 69 µC cm-2, which was much lower than QOR for either the nystatin-incorporated oleate or γ-linolenate SALL. In order to increase the incorporation of alamethicin, the working electrode was removed from the cell, cleaned in mixed acid (50/50 18.0 M H2SO4/15.9 M HNO3 (v/v)), rinsed with Nanopure water, and replaced in the cell. The lipids and alamethicin were able to self-assemble in cooperation with one another onto the clean electrode surface. This resulted in a more open structure as a result of the greater incorporation of alamethicin, since the QOR value of 404 µC cm-2 after the new SALL was formed was much higher than that determined for the previous SALL. However,

the QOR value was not as high as the QOR value in the presence of phosphate buffer alone, which indicated that the SALL had re-formed on the electrode surface. These experiments were repeated with a γ-linolenate SALL. The QOR value of 223 µC cm-2 after the alamethicin-incorporated SALL formed was higher than the previous SALL but smaller than the values for any of the previous ionophore-incorporated SALLs. The results of these measurements are shown with the final results. (v) Determination of Surface Concentrations for Oxide and Azide within the Ionophore Pores. The surface concentration, Γ (molecules cm-2), for both the oxide and the azide ion was calculated by the following formula:

Γ)

QNA nF

where Q is the surface charge density (C cm-2), NA Avogadro’s number, n the number of electrons transferred per molecule, and F the Faraday constant (C mol-1). Γ was calculated using n equal to 1, for a one-electrontransfer reaction for adsorption of hydroxide and azide at the electrode surface. The surface concentration due to oxide formation, ΓOR, was calculated using the value of QOR to replace that of Q in the above equation, assuming the reduction process to proceed as follows:39

PtOH + e- f Pt + OHAlthough a number of forms of oxide can occur at high anodic potentials, reduction of oxide was treated as a oneelectron-transfer process for the adsorption and removal of OH species. The surface concentration due to azide, ΓN3-, was calculated in a similar manner, except that QN3was used to replace Q in the equation. Plots of ΓOR versus ΓN3- show the effects of bulk concentration of the ionophores on the competitive behavior of the transported molecules at the electrode surface for the nystatin-incorporated oleate SALL (Figure 8). The surface concentration of adsorbed species increased with increasing nystatin bulk concentration due to the greater number of apparent pores formed in the SALL, resulting in a larger exposed surface area. Similar results were obtained for the nystatin-incorporated γ-linolenate SALL, the alamethicin-incorporated oleate SALL, and the alamethicin-incorporated γ-linolenate SALL. (39) Vassiliev, Yu.B.; Bagotzky, V. S.; Gromyko, V. A. J. Electroanal. Chem. 1984, 178, 247.

Electrochemical Study of Antibiotic Ionophores

Figure 9. Number of nystatin pores formed in a SALL versus concentration of nystatin for the oleate and γ-linolenate SALLs: b, oleate SALL; 2, γ-linolenate SALL.

Langmuir, Vol. 13, No. 22, 1997 5967

Figure 11. Effect of surface concentration of oxide versus surface concentration of azide per nystatin pore with different concentrations of nystatin ionophore in an oleate SALL. Concentration of nystatin in the bulk solution: O, 6.53 × 10-6 M; 4, 1.33 × 10-5 M; 0, 2.65 × 10-5 M; 3, 4.91 × 10-5 M; ], 1.01 × 10-4 M. Table 2. Surface Concentrations in Molecules per Pore for the Nystatin-Incorporated Oleate SALL

Figure 10. Number of alamethicin pores formed in a SALL versus concentration of alamethicin for the oleate and γ-linolenate SALLs: 9, oleate SALL; 1, γ-linolenate SALL.

(vi) Determination of the Number of Ionophore Pores and the Surface Concentration per Pore. The number of ionophore pores in a SALL, P, was calculated using the equation

P)

QORPAe QORAp

where QORP is the oxide reduction surface charge density in the presence of lipid and ionophore (C cm-2), Ae the surface area of the platinum working electrode (cm2), QOR the oxide surface charge density in phosphate buffer (C cm-2), and Ap the area of the ionophore pore (cm2). Ap was calculated using a circular model, Ap ) πr2, with r equal to 0.40 nm for nystatin33 and 0.75 nm for alamethicin.33,34 As the nystatin concentration in the bulk solution increased, the number of apparent ionophore pores increased to a plateau value of ∼5.5 × 1013 pores in the oleate SALL and ∼4.5 × 1013 pores in the γ-linolenate SALL (Figure 9). The number of apparent alamethicin pores was less, with a maximum near 2 × 1013 pores in the oleate SALL and 1 × 1013 pores in the γ-linolenate SALL for the highest concentration used (Figure 10). Rizzo et al.13 also reported that alamethicin incorporation into a SALL reached a limiting value with higher concentrations of alamethicin. The surface concentration per ionophore pore, Γp, was then determined using

Γp )

ΓAe P

where Γ is the surface concentration (molecules cm-2), Ae the surface area of the electrode (cm2), and P the number

[nystatin] (M)

no. of H20 molecules/pore

no. of N3ions/pore

6.53 × 10-6 1.33 × 10-5 2.65 × 10-5 4.91 × 10-5 1.01 × 10-4

16.1 16.7 16.5 16.6 16.4

13.8 16.5 14.4 15.2 14.0

Table 3. Surface Concentrations in Molecules per Pore for the Nystatin-Incorporated γ-Linolenate SALL [nystatin] (M)

no. of H20 molecules/pore

no. of N3ions/pore

6.37 × 10-6 1.37 × 10-5 2.58 × 10-5 5.23 × 10-5 9.99 × 10-5

16.6 16.0 16.5 16.3 16.3

16.6 16.0 16.5 16.3 16.3

of apparent ionophore pores in the SALL. A comparison of the oxide surface concentration versus the azide surface concentration in molecules per pore for each of the different concentrations of nystatin incorporated into the oleate SALL (Figure 11) showed very consistent results for all the experiments. Similar results were found with the γ-linolenate SALL. These results suggest that nystatin formed the same sized pore in each SALL. The surface concentration of azide was calculated from the experiments using the highest concentration of azide in the bulk solution of the cell. The numerical values for the number of molecules of water adsorbed per pore based on oxide formation and the number of azide ions adsorbed per pore are shown in Tables 2 and 3 for the nystatin-incorporated oleate and γ-linolenate SALL, respectively. The results for the nystatin-incorporated SALL were in good agreement with 16.4 ( 0.4 water molecules and 14.8 ( 1.7 azide ions adsorbed per pore with the oleate SALL and 16.3 ( 0.3 water molecules and 16.3 ( 0.3 azide ions per pore with the γ-linolenate SALL. Similarly, good agreement between the results was obtained for the experiments with the alamethicinincorporated SALL in an oleate SALL and the γ-linolenate SALL. The numerical values for the number of molecules and ions adsorbed per pore for the alamethicin-incorporated SALL are shown in Table 4 for the oleate SALL and Table 5 for the γ-linolenate SALL. Similar values were obtained for SALL with both 57.5 ( 0.9 water molecules and 56.3 ( 3.5 azide ions per pore in the oleate SALL and 56.9 ( 1.0 water molecules and 56.9 ( 1.0 azide ions per pore in the γ-linolenate SALL. The surface concentration

5968 Langmuir, Vol. 13, No. 22, 1997

Maguire and Roscoe

Conclusions

Table 4. Surface Concentrations in Molecules per Pore for the Alamethicin-Incorporated Oleate SALL [alamethicin] (M)

no. of H20 molecules/pore

no. of N3ions/pore

5.65 × 10-7 1.05 × 10-6 1.76 × 10-6 2.21 × 10-6 4.44 × 10-6

57.4 56.6 57.2 58.3 58.0

57.4 56.6 57.0 57.8 52.8

Table 5. Surface Concentrations in Molecules per Pore for the Alamethicin-Incorporated γ-Linolenate SALL [alamethicin] (M)

no. of H20 molecules/pore

no. of N3ions/pore

5.72 × 10-7 1.15 × 10-6 2.33 × 10-6 4.55 × 10-6

57.1 57.9 56.2 56.3

57.1 57.9 56.2 56.3

Table 6. Number of Water Molecules and Azide Ions Transported per Nystatin and Alamethicin Pores Based on Geometrical Considerations moiety

radius (nm)

area (nm2)

no. of water molecules/pore

no. of azide ions/pore

nystatin alamethicin water azide

0.40 0.75 0.097 0.10

0.50 1.8 0.030 0.031

17 60

16 56

per alamethicin pore was 3.5 times larger than that for the nystatin pore. Geometrical calculations were made to compare the experimental number of water molecules or azide ions adsorbed per apparent pore with the theoretical number that would fit into the surface area exposed by the pores (Table 6). The number of molecules per pore was determined by dividing the area of the pore by the area of the adsorbing species. Under anodic potentials the water molecule adsorbs onto the electrode surface through the oxygen atom. The area occupied by a water molecule was calculated using a circular model of radius 0.097 nm using the length of the O-H bond in water.40 Similarly under anodic potentials, the azide ion adsorbs through an end-on adsorption.36 The radius of the nitrogen atom in hydrazoic acid of 0.10 nm41 was used as the basis for determining the area of the azide ion. Thus, the experimentally determined values of 16.4 ( 0.4 water molecules or 15.6 ( 1.8 azide ions per apparent nystatin pore compare very well with the theoretical values of 17 water molecules or 16 azide ions per nystatin pore. Similarly for alamethicin, the experimentally determined values of 57.2 ( 1.1 water molecules or 56.6 ( 3.8 azide ions per apparent pore compare very well with the theoretical values of 60 water molecules or 56 azide ions per alamethicin pore. These consistent results therefore give support to the interpretation of the formation of pores in the SALLs by these ionophores. (40) Bowser, J. R. Inorganic Chemistry; Brooks/Cole Publishing Company: Pacific Grove, CA, 1993; pp 132-133. (41) Pauling, L. The Nature of the Chemical Bond; Cornell University Press: Ithaca, NY, 1960; pp 270-273.

The present study reports the self-assembling of soluble salts of fatty acids at the platinum electrode surface directly from an aqueous solution and examines the incorporation of ionophores into the SALL. The platinum electrode which supports the SALL is used as a sensitive surface detector for the transport of ions such as azide through ionophores to the electrode surface. Model SALLs that were impermeable to water and azide ions in the absence of ionophores were formed from oleate and less efficiently from γ-linolenate on a platinum electrode surface in a pH 7.0 phosphate buffer solution at 299 K. The number of apparent pores developed in an oleate or a γ-linolenate SALL as a result of the incorporation of nystatin in the SALL was dependent on the nystatin concentration in the bulk solution up to a limiting value. A similar behavior was observed with alamethicin. The azide ion proved to be an excellent species to examine transport through the ionophores due to the high sensitivity of the platinum electrode for this ion. A good correlation was obtained for surface monolayer coverage per pore between the calculations from electrochemical measurements and calculations based on geometrical considerations which was consistent with the formation of pores in the SALLs by the ionophores. Thus, a model SALL for use in aqueous solutions is reported which in many ways reflects the behavior of natural membranes in that the SALL is fluid, is self repairing, and is able to incorporate certain ionophores. Thus, it provides a means for examining the effects of hydrophobicity and fluidity of self-assembled lipid layers with other molecules in an aqueous environment. For example, the SALL provides another medium for the study of immobilization of proteins from aqueous solutions, such as hormone receptors and antibodies, under nondenaturing conditions and in welldefined orientations. The SALL formed in this study does not require nanometer thick water layers or ultrathin soft polymer cushions to maintain the thermodynamic and structural properties of the free SALL.42 Because the SALL is formed directly onto the platinum electrode surface, detection and quantitative analyses may be made of electroactive species which are able to penetrate the film. Therefore, it may be suitable as a biosensor for studies of toxins and antibiotics. Acknowledgment. The authors thank Efamol Research Institute, Kentville, NS, Canada, for providing the lithium γ-linolenate and performing the CGC analyses on the potassium oleate and the lithium γ-linolenate used in the study and James R. Roscoe for writing the computer integration program used in determining cyclic voltametric areas. Grateful acknowledgement is also made to the Natural Science and Engineering Research Council of Canada for support of this research. LA9605450 (42) Sackmann, E. Science 1996, 271, 43.