Langmuir 1998, 14, 6269-6273
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Continuing Electrochemical Studies of Phospholipid Monolayers of Dioleoyl Phosphatidylcholine at the Mercury-Electrolyte Interface Dan Bizzotto† and Andrew Nelson* Plymouth Marine Laboratory, Plymouth, U.K. Received March 18, 1998. In Final Form: July 1, 1998 The electrochemical properties of phospholipid monolayers of dioleoyl phosphatidylcholine (DOPC) spread from the gas/solution interface on mercury surfaces are quantitatively investigated in this paper. These layers display multiple states which interconvert through phase transitions characterized by two sharp capacitive peaks. Potential pulse techniques (chronocoulometry) were used to quantitatively investigate the properties of the DOPC monolayer on a mercury electrode. Charge density and the resulting film pressure due to DOPC spreading at the Hg/solution interface were determined. Results indicate that the lipid layers are displaced from the mercury surface at negative potentials in excess of -1.8 V. The potential of maximum film pressure or stability of the lipid monolayer and the shift in the potential of zero charge due to lipid transfer to the mercury surface were estimated as -0.4 and +0.435 V versus Ag/AgCl (saturated KCl), respectively. The similarity of the DOPC monolayer properties on mercury to the insoluble surfactant monolayer properties on single-crystal gold electrodes is noted. The spread DOPC layer and specifically the first phase transition was further characterized utilizing Tl+ and Cd2+ reduction. From potentials of -0.65 V to the potential coincident with the first phase transition, the permeability of the layer to these metal ions increases with an increase in the applied negative potential. The second phase transition represents a process involving the growth and coalescence of defects.
1. Introduction Phospholipid monolayers spread at the mercury-water interface have been studied by several groups.1-5 The method of depositing phospholipid layers onto mercury surfaces was originally developed by Miller1 and subsequently applied by Nelson2. Guidelli6-9 has also made a substantial contribution to this area of work. The system represents exactly half a bilayer and has proven to be a powerful membrane model. By the same token the model has a general application for examining lipid interactions3 and also shows promise as a biosensor. In particular, the behavior of the lipid monolayer on the mercury surface in response to changes in potential is of interest. Dioleoyl phosphatidylcholine (DOPC) monolayers undergo two pronounced phase transitions characterized by two capacitance peaks respectively at potentials more negative than -0.5 V versus the potential of zero charge (PZC) of the mercury-water interface.2 These phase transitions have already been well-characterized experimentally2 and their form is dependent on the interaction of the lipid with biologically active species in solution.3 Predictive models of the lipid phase transitions4,5 show that they represent a rather complicated reorientation whereby the lipid monolayer reverted first to a two-phase system of a thick and thin monolayer and finally to a pored bilayer. * To whom correspondence should be addressed. Fax: 44-1752633102. Tel.: 44-1752-633290. E-mail:
[email protected]. † Present address: Department of Chemistry, University of British Columbia, British Columbia, Canada. E-mail: bizzotto@ chem.ubc.ca. (1) Miller, I. R. in Topics in Biochemistry and Bioenergetics; John Wiley and Sons: New York, 1981; pp 161-224. (2) Nelson, A.; Auffret, N. J. Electroanal. Chem. 1988, 244, 99. (3) Nelson, A.; Auffret, N.; Borlakoglu, J. Biochim. Biophys. Acta 1990, 1021, 205-216. (4) Leermakers, F. A. M.; Nelson, A. J. Electroanal. Chem. 1990, 278, 53-72. (5) Nelson, A.; Leermakers, F. A. M. J. Electroanal. Chem. 1990, 278, 73-83.
The work described in this paper had two aims. One objective was to characterize DOPC monolayer properties on mercury using a quantitative approach that was employed studying the properties of an insoluble monolayer spread on single-crystal Au electrodes.10 These techniques are directly applicable to this system yielding charge density measurements from which the film pressure as a function of the electrode potential at the Hg interface can be determined using thermodynamic arguments. Another objective was to study further the two phase transitions. First, Tl(I) and Cd(II) reduction was investigated using a microelectrode array model to quantify the barrier properties of the DOPC layer in a similar way to previous work11 with the aim of describing the changes in the monolayer coincident with the first phase transition. These permeability studies of the layer were carried out as a prerequisite to concurrent work12,13 where the transport properties of ion channels in the layer are investigated. Second, in the absence of electroactive species in solution, potential step techniques were performed in order to better understand the mechanism of the second phase transition. 2. Experimental Section Electrolytes (0.1 mol dm-3 KCl, BDH Chemicals Ltd., calcined 500 °C; 0.1 mol dm-3 NaF, Merck Suprapure) were prepared in 18.2 MΩ Milli Q water and deaerated with special grade argon (6) Moncelli, M. R.; Becucci, L.; Nelson, A.; Guidelli, R. Biophys. J. 1996, 70, 2716-2726. (7) Becucci, L.; Moncelli, M. R.; Guidelli, R. J. Electroanal. Chem. 1996, 413, 187-193. (8) Moncelli, M. R.; Becucci, L.; Guidelli, R. Biophys. J. 1994, 66, 1969-1980. (9) Moncelli, M. R.; Becucci, L.; Herrero, R.; Guidelli, R. J. Phys. Chem. 1995, 99, 9940-9951. (10) Bizzotto, D.; Noe¨l, J. J.; Lipkowski, J. J. Electroanal. Chem. 1994, 369, 259-265. (11) Nelson, A.; van Leeuwen, H. P. J. Electroanal. Chem. 1989, 273, 183-199. (12) Nelson, A. Langmuir 1997, 13, 5644-5651. (13) Nelson, A. Langmuir 1996, 12, 2058-2067.
S0743-7463(98)00314-X CCC: $15.00 © 1998 American Chemical Society Published on Web 09/19/1998
6270 Langmuir, Vol. 14, No. 21, 1998 before each experiment. A blanket of argon gas was maintained above the electrolyte during the experiment. Monolayers of DOPC (semi-synthetic grade, Lipid Products, U.K.) were prepared as before2 by spreading a pentane (Fisons, HPLC grade) solution of DOPC (1 mg/mL) at the argon-electrolyte interface in the electrochemical cell. A fresh mercury drop extruded in the Ar atmosphere (area, A ) 0.0088 cm-2) was coated with the monolayer from the argon-electrolyte interface prior to each experiment. A Metrohm potentiostat was used (E506 Polarecord) with a PAR 5110 lock-in-amplifier and data were recorded with a Maclab (16 bit, 100 kHz) data acquisition system. The Maclab system was also used to stimulate the cell potential. In this paper, all potentials are quoted versus the Ag/AgCl:saturated KCl reference electrode. For capacity measurements, the cell was stimulated with an AC waveform (75 Hz, 4.5 mV rms) superimposed on a voltage ramp (scan rate, 5 mV s-1). The capacity was calculated from the out-of-phase and in-phase components of the current assuming a RC circuit. Chronocoulometry was used to measure the charge density for bare and DOPC-covered Hg surfaces following the techniques used for gold surfaces.10 A series of potential steps were performed covering the potentials between -0.2 and -1.8 V. Prior to each potential step measurement, an initial potential of -0.4 V was used to ensure that the monolayer was in a reproducible state. The potential was stepped to predetermined values between -0.2 and -1.8 V (Esprd) for 10 ms, which is enough time for the establishment of equilibrium.14 The potential was then pulsed for 15 ms to a value at which the monolayer is displaced from the mercury surface (Edis) and returned to the initial potential (-0.4 V) for >5 s. The current transient resulting from the potential step between Esprd and Edis was integrated to give relative charge density values. These were converted to absolute values using the charge density determined with a nonspecifically adsorbed electrolyte (NaF) in the absence of an adsorbant. The absolute charge densities were plotted against the potential, Esprd. The validity of these charge density estimations depended on the complete displacement of the lipid layer from the mercury taking place at Edis within the measurement time. For this reason Edis was varied from -1.75 to -1.9 V in separate experiments to establish that complete monolayer displacement was indeed being attained. The DOPC monolayer was characterized by differential capacity measurements before and after the potential step experiments to ensure that the monolayer was not significantly altered during the pulsing procedure. A series of potential step experiments were done to characterize Tl(I) and Cd(II) reductions in the potential region leading to and covering the first phase transition. Both the Tl(I)/Tl(Hg) and Cd(II)/Cd(Hg) couples are kinetically fast on the bare electrode surface15 and the DOPC monolayer inhibits the reduction of these ions by blocking the electrode surface. As a result, alterations in the characteristics of the faradaic reaction can be used to investigate the permeability to ions of the DOPC monolayers. The potential (E) was held at -0.3 V for 1 ms and then stepped to successively more negative values (up to -1.0 V) for 100 ms for both the coated and uncoated electrode. The current (i) was recorded at a 10-kHz sampling frequency with a 5-kHz low-pass prefilter. The current transients were analyzed in the following manner. First, the current (I) at defined experimental times (t) during the transient was plotted against the potential (-E) to give a current-sampled voltammogram (i versus -E) plot. Second, the current transients were fit to the random microelectrode model of Scharifker.16 The principle of this analysis is that an impermeable monolayer with areas permeable to metal ions is represented as a surface with a random array of disk microelectrodes of equal size. The disk microelectrodes correspond to areas of the new evolving phase of the lipid which is permeable to metal ions. This model assumes that the number of permeable areas and their size do not evolve with time. Potential step characterization of the second phase transition of the DOPC monolayer was carried out in a solution with no (14) Noel, J.; Bizzotto, D.; Lipkowski, J. J. Electroanal. Chem. 1993, 344, 343-354. (15) Galus, Z. Fundamentals of Electrochemical Analysis; Ellis Horwood: Chichester, 1976. (16) Scharifker, B. R. J. Electroanal. Chem. 1988, 240, 61-76.
Bizzotto and Nelson
Figure 1. (a) Capacity of a Hg electrode in 0.1mol dm-3 KCl (- - -) and the electrode covered by DOPC (s, cathodic scan; - - -, anodic scan). (b) Charge density (σM) of a Hg electrode in 0.1 mol dm-3 NaF (b), 0.1 mol dm-3 KCl (O), and covered by DOPC in 0.1 mol dm-3 KCl (2). (c) The film pressure (πMS) of DOPC on Hg calculated using the charge density measured in (b) where chloride adsorption on Hg is absent. Capacity, σM and πMS plotted versus the potential (-E). electroactive species present. Initially, the potential was held at -0.6 V and then stepped to -0.975 V for 10 ms. Subsequently, the potential was stepped to successive values more negative than this one and the current transients measured. The work described in this paper was carried out on DOPC monolayers which were reproducible in structure and properties. The coverage and stability of the monolayer was easily checked from observation of the capacitance versus potential plots. When the DOPC coverage was lower than that required for a condensed monolayer, the height of the first capacitance peak relative to the second was decreased and the monolayer capacitance was increased2 from its minimum value (see later). When the DOPC coverage was higher than that characteristic of a condensed monolayer, the second capacitance peak was lowered relative to the first and the onset of the first capacitance peak was broadened.5 The successful deposition of stable condensed monolayers of reproducible coverage depended to a large extent on the pretreatment of the glass capillary supporting the mercury drop electrode. The inside of the capillary was silanized to facilitate the stability of the mercury drop. The silanization on the outside of the capillary was removed to ensure the stability of the DOPC monolayer on the mercury surface. This monolayer stability depended on the development of a seal between the DOPC monolayer and the unsalinized outer glass surface of the capillary tip. The lipid-glass seal was facilitated by dipping the capillary tip several times through the lipid-covered gas-water interface prior to depositing the monolayer on the mercury surface. Using these techniques, the DOPC monolayers were stable during the rather extensive series of potential step experiments.
3. Results and Discussion 3.1. Capacity and Charge Density Measurements. The differential capacity of a spread layer of DOPC onto Hg was measured for both anodic and cathodic scans (Figure 1a). The DOPC monolayer displays a low-capacity region (1.85 µF cm-2), two sharp pseudocapacitance peaks,
Phospholipid Monolayers of DOPC
(peaks 1 and 2 at -0.94 and -1.02 V, respectively), and a further broader pseudocapacitance peak (peak 3 at -1.275 V) at more negative potentials. Peaks 1, 2, and 3 correspond to structural reorientations of the monolayer and peaks 1 and 2 represent phase transitions 1 and 2, respectively, of the lipid monolayer.2,4,5 The cathodic and anodic scans are exactly the same when the negative switching potential is limited to -1.1 V. In contrast, a large difference is noted between the cathodic and anodic scans when the negative potential scan is taken beyond peak 3 to -1.8 V as shown in Figure 1a. In fact, peak 3 displays a large hysteresis with respect to the scan direction similar to the monolayer displacement peaks of insoluble amphiphile layers on single-crystal gold electrodes.10,17,18 At potentials of -1.8 V the capacitance measured in the anodic scan for the covered and uncovered mercury surface is equal. In addition, when the potential is returned to more positive values, the capacitance peaks and low-capacitance region of the capacitance-potential curves are indicative of a Hg electrode partially covered by lipid (see ref 2). From this evidence and by analogy to the spectroelectrochemical experiments performed on the amphiphile-covered gold electrodes,18 it is reasoned that at very negative potentials of about -1.8 V the DOPC layers are in fact displaced from the mercury surface to form aggregate structures near the electrode surface which respread at less negative potentials. This potentialdependent wetting and spreading phenomena has been elegantly visualized by Zutic in recent work.19 The charge density curves (σM vs -E) measured for the DOPC-coated and -uncoated electrode are shown in Figure 1b. Here, the negative displacement potential employed was -1.9 V. Other displacement potentials of -1.75, -1.80, and -1.85 V were also investigated. The requirement for the monolayer displacement potential is that the layer is displaced within the measurement time period of the step to Edis (in this case 15 ms) as well as not being so negative as to change the layer upon respreading as is seen in the slow sweep capacity measurements. This was checked by measuring the capacity (-0.4 to -1.2 V) of the layer before and after step experiments, revealing no loss in lipid coverage. The displacement potential can be checked by sequentially changing Edis in the experiments and watching for consistent σM versus -E plots for the adsorbed phospholipid. This was in fact observed for the two most negative displacement potential values. The charge density curve for the DOPC-coated Hg shows three inflections corresponding to peaks 1 and 2 and the monolayer displacement process (peak 3) in Figure 1a. The PZC measured for Hg in contact with a 0.01 mol dm-3 NaF electrolyte was found to be -0.365 V which is 0.035 V more positive than that measured by Grahame.20 This value obtained in the absence of specific adsorption on the Hg surface was used throughout this study to calculate absolute charge values. The capacitance-potential curve for the uncoated Hg in KCl was not used in this analysis due to the specific adsorption of Cl- on Hg. However, the curve for the DOPC-coated Hg in KCl can be used since the specific adsorption of Cl- is precluded by the presence of DOPC on the mercury. The PZC of DOPC-coated Hg can be shown by extrapolation of the charge density measurements in the potential range positive of -0.8 V to be +0.070 V. It may be estimated that this positive potential shift (∆E ) +0.435 V ( 1 mV 95% confidence (17) Bizzotto, D.; Noe¨l, J.; Lipkowski, J. Thin Solid Films 1993, 248, 69-77. (18) Bizzotto, D.; Lipkowski, J. J. Electroanal. Chem. 1996, 409, 33. (19) Ivosevic, N.; Zutic, V. Langmuir 1998, 14, 231-234. (20) Grahame, D. C. Chem. Rev. 1947, 41, 441-501.
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Figure 2. Normalized current-potential (i/i0 vs -E) plots for the reduction of Cd2+ (a) and Tl+ (b) in 0.1 KCl mol dm-3 on a Hg electrode (0) and on an electrode covered by DOPC at sampling times of 10 (3), 25 (open sideways triangle), 50 (4), 75 (]), and 100 ms (O).
level) of the PZC due to lipid transfer to mercury corresponds to a positive surface potential of the spread DOPC layer.21 This is in very good agreement with the positive surface potential of 0.450 V for monolayer films of phosphatidylcholine at the gas-water interface compressed to 48 mN m-1.22-24 Integration of the difference between the σM versus -E plots for the DOPC-coated electrode and the electrode in 0.1 mol dm-3 NaF yields the film surface pressure (πMS) due to the presence of the lipid at the mercury/electrolyte interface as shown in Figure 1c. The maximum value of the film pressure (52 mN m-1) closely corresponds to the equilibrium spreading pressure of DOPC at the air/water interface.23,25 The potential (-0.4 V) characteristic of this value is equivalent to the potential of maximum stability of the lipid monolayer relative to its displacement by water. This is also the potential where the charge density curve recorded for the DOPC-coated Hg surface crosses that measured for the uncoated Hg surface in 0.1 mol dm-3 NaF. The long tail of small film pressure values in the negative potential region (
