Langmuir 2004, 20, 701-707
701
Scanning Electrochemical Microscopy (SECM) Studies of Oxygen Transfer across Phospholipid Monolayers under Surface Pressure Control: Comparison of Monolayers at Air/Water and Oil/Water Interfaces Susan Cannan,† Jie Zhang,†,§ Frank Grunfeld,‡ and Patrick R. Unwin*,† Department of Chemistry, University of Warwick, Coventry, CV4 7AL, United Kingdom, and Nima Technology Limited, The Science Park, Coventry, CV4 7EZ, United Kingdom Received May 29, 2003. In Final Form: August 30, 2003 Scanning electrochemical microscopy has been used in combination with a specially designed Langmuir trough to compare the kinetics of oxygen transfer across an L-R-phosphatidylethanolamine, distearoyl monolayer spread at three different interfaces: air/water, air/water in contact with an oil lens, and oil/ water. The monolayer is shown to reduce the kinetics of interfacial transport, and rate constants for the transport of oxygen across each interface, at different surface pressures, have been evaluated. The results obtained for each interface are compared, and the implications for studies of passive diffusion across cell membranes are discussed.
Introduction Diffusion across membranes is fundamental to a myriad of biological processes and has thus been widely studied in both model systems and living cells.1 Greater understanding of these processes, especially in a quantitative way, has practical applications in areas such as the development of drug delivery systems, artificial organs, and tissue engineering.2 The cellular membrane plays a key role in controlling the transport of neutral molecules and ions into and out of the cell, thus maintaining both concentration and electrochemical gradients. Investigations into the complex structure-function relationships that exist in biomembranes have led to the development of a number of model systems, which have various merits.3 The model that most closely approaches the structural and electrical properties of a cell membrane is a bilayer lipid membrane (BLM),4 which has been used extensively to further our understanding of charge and molecular transfer across biological interfaces.5-7 Experimentally, BLMs can readily be formed by several methods.8 However, the use of BLMs presents some * To whom correspondence should be addressed. Tel: +44-247652-3264. Fax: +44-24-7652-4112. E-mail: p.r.unwin@ warwick.ac.uk. † University of Warwick. ‡ Nima Technology Ltd. § Present address: School of Chemistry, Monash University, Victoria 3800, Australia. (1) Stein, W. D. Transport and Diffusion across Cell Membranes; Academic Press: Orlando, FL, 1986. (2) Fournier, R. L. Basic Transport Phenomena in Biomedical Engineering; Taylor and Francis: Philadelphia, 1999. (3) Gennis, R. B. Biomembranes: Molecular structure and Function; Springer-Verlag: New York, 1989. (4) Volkov, A. G.; Deamer, D. W.; Tanelian, D. L.; Markin, V. S. Liquid Interfaces in Chemistry and Biology; John Wiley & Sons: New York, 1998. (5) Tien, H. T. Bilayer Lipid Membranes (BLM): Theory and Practice; Marcel Dekker: New York, 1974. (6) Tsionsky, M.; Zhou, J. F.; Amemiya, S.; Fan, F.-R. F.; Bard, A. J.; Dryfe, R. A. W. Anal. Chem. 1999, 71, 4300. (7) Amemiya, S.; Ding, Z.; Zhou, J. F.; Bard, A. J. J. Electroanal. Chem. 2000, 483, 7. (8) Tien, H. T.; Ottova-Leitmannova, A. Membrane Biophysics as Viewed from Experimental Bilayer Lipid Membranes; Elsevier: Amsterdam, 2000.
practical difficulties, not least because they are physically unstable9 and their interfacial potential cannot be accurately controlled or measured.10 A more practical alternative is the formation of a phospholipid monolayer at an oil/water interface. This system is characterized by high physical stability and the facility to control the potential drop across the monolayer.11 The kinetics of ion transfer (IT)10,12-15 and electron transfer (ET)16,17 has been studied at phospholipid monolayers formed at the interface between two immiscible electrolyte solutions (ITIES). Some research has shown that the presence of a phospholipid monolayer can inhibit the rate of ET16 and IT,10,15 while others have reported enhanced transfer rates.18,19 Kakiuchi and co-workers demonstrated that the state of the monolayer was an important factor in determining the overall transfer kinetics of IT; in the liquid expanded (LE) state, the monolayer had no effect on IT, but the rate was lowered when the monolayer was in the liquid condensed (LC) state.12 Scanning electrochemical microscopy (SECM) has proved to be a powerful technique for probing the kinetics of both IT7,15 and ET20-22 across monolayers at ITIES and also for (9) Grandell, D.; Murtoma¨ki, L. Langmuir 1998, 14, 556. (10) Cunnane, V. J.; Schiffrin, D. J.; Fleischmann, M.; Geblewicz, G.; Williams, D. J. Electroanal. Chem. 1988, 243, 455. (11) Kakiuchi, T. In Liquid-Liquid Interfaces. Theory and Application; Volkov, A. G., Deamer, D. W., Eds.; CRC Press: Boca Raton, FL, 1996. (12) Kakiuchi, T.; Kotani, M.; Noguchi, J.; Nakanishi, M.; Senda, M. J. Colloid Interface Sci. 1992, 149, 279. (13) Grandell, D.; Murtoma¨ki, L.; Sundholm, G. J. Electroanal. Chem. 1999, 469, 72. (14) Liljeroth, P.; Ma¨lkia¨, A.; Cunnane, V. J.; Kontturi, A.-K.; Kontturi, K. Langmuir 2000, 16, 6667. (15) Zhang, J.; Unwin, P. R. Langmuir 2002, 18, 2313. (16) Cheng, Y.; Schiffrin, D. J. J. Chem. Soc., Faraday Trans. 1994, 90, 2517. (17) Tsionsky, M.; Bard, A. J.; Mirkin, M. V. J. Am. Chem. Soc. 1997, 119, 10785. (18) Kontturi, A.-K.; Kontturi, K.; Murtoma¨ki, L.; Quinn, B.; Cunnane, V. J. J. Electroanal. Chem. 1997, 424, 69. (19) Manzanares, J. A.; Allen, R. M.; Kontturi, K. J. Electroanal. Chem. 2000, 483, 188. (20) Wei, C.; Bard, A. J.; Mirkin, M. V. J. Phys. Chem. B 1995, 99, 16042.
10.1021/la034943w CCC: $27.50 © 2004 American Chemical Society Published on Web 01/08/2004
702
Langmuir, Vol. 20, No. 3, 2004
studying IT across BLMs.6,23,24 Particular attributes of the SECM approach are the high mass transfer rates available, allowing the study of rapid interfacial processes, and the ability to make spatially resolved measurements. The latter proved particularly powerful in visualizing differences in ET rates across mixed phospholipid monolayers.21 The effect of monolayers on the transfer of neutral molecules has been studied to a lesser extent, at both the ITIES25 and an air/water interface.26,27 The advantage of the air/water system was that measurements could be made directly in a Langmuir trough, so allowing the surface pressure and molecular area of the monolayer to be adjusted and controlled. A model monolayer system intermediate between the air/water and liquid/liquid interface is that proposed by Mo¨hwald and co-workers:28,29 an air/water interface, with an adsorbed monolayer, in contact with a thin oil layer (nanometer dimensions). In this case, the thin oil film, resembling a lens, is formed by contacting a phospholipid monolayer at an air/water interface with a reservoir of oil.30 Unlike the thicker oil/water interface, the thin oil film allows techniques such as X-ray diffraction,29 X-ray reflectivity,31 and ellipsometry31,32 to be used to study the structure of the monolayer at the interface. Pressurearea isotherms of phospholipid monolayers spread under these conditions have been thoroughly investigated and compared to similar measurements made at the air/water and oil/water interfaces;28,32 however, there have been no reported studies of the kinetics of molecular transfer across a monolayer formed at the air/thin oil interface. In this paper, we compare the kinetics of oxygen transfer across an L-R-phosphatidylethanolamine, distearoyl (DSPE) monolayer spread at three different interfaces: air/water, air/water in contact with an oil lens, and oil/ water. For these studies, we use a combination of SECM with a specially designed Langmuir trough, which is applicable to each of these different interfaces.33 As already mentioned, the Langmuir trough apparatus allows precise control of both the surface pressure and surface coverage of the monolayer at the interface.9,13,14,34 In particular, pressure-area isotherms enable the state of the monolayer at a specific surface pressure to be ascertained. The study of these various interfaces is of interest because it has been suggested that a monolayer at the n-alkane/water interface is a closer analogue for a leaflet of bilayer vesicles than a monolayer at the air/water interface.35 The intracellular transfer of oxygen is of paramount importance in bioenergetics and is an active area of (21) Delville, M.-H.; Tsionsky, M.; Bard, A. J. Langmuir 1998, 14, 2774. (22) Tsionsky, M.; Bard, A. J.; Mirkin, M. V. J. Am. Chem. Soc. 1997, 119, 10785. (23) Yamada, H.; Matsue, T.; Uchida, I. Biochem. Biophys. Res. Commun. 1991, 180, 1330. (24) Matsue, T.; Shiku, H.; Yamada, H.; Uchida, I. J. Phys. Chem. 1994, 98, 11001. (25) Strutwolf, J.; Zhang, J.; Barker, A. L.; Unwin, P. R. Phys. Chem. Chem. Phys. 2001, 3, 5553. (26) Slevin, C. J.; Ryley, S.; Walton, D. J.; Unwin, P. R. Langmuir 1998, 14, 5331. (27) Zhang, J.; Unwin, P. R. Langmuir 2002, 18, 1218. (28) Thoma, M.; Pfohl, T.; Mo¨hwald, H. Langmuir 1995, 11, 2881. (29) Brezesinski, G.; Thoma, M.; Struth, B.; Mo¨hwald, H. J. Phys. Chem. 1996, 100, 3126. (30) Aveyard, R.; Binks, B. P.; Cooper, P.; Fletcher, P. D. I. J. Prog. Colloid Polym. Sci. 1990, 81, 36. (31) Thoma, M.; Schwendler, M.; Baltes, H.; Helm, C. A.; Pfohl, T.; Riegler, H.; Mo¨hwald, H. Langmuir 1996, 12, 1722. (32) Harke, M.; Motschmann, H. Langmuir 1998, 14, 313. (33) Zhang, J.; Strutwolf, J.; Cannan, S.; Unwin, P. R. Electrochem. Comm. 2003, 5, 105. (34) Grandell, D.; Murtoma¨ki, L.; Sundholm, G. J. Electroanal. Chem. 1999, 463, 242. (35) Gruen, D. W. R.; Wolfe, J. Biochim. Biophys. 1982, 688, 572.
Cannan et al.
research.36-38 The adequate supply of oxygen to cells is necessary for the growth of multicellular systems; lack of oxygen can cause cell death or necrosis. Given the importance of oxygen transport in living organisms and our previous studies of model systems,25,26 we have focused on oxygen transfer kinetics to compare the behavior of the different interfaces. The results obtained are used to draw conclusions on the factors controlling passive diffusion across model membranes. Experimental Section Chemicals. The aqueous subphase was 25 mM phosphate buffer, pH 7, prepared from potassium dihydrogen phosphate and sodium hydroxide (both BDH AnalaR), using Milli-Q reagent water (Millipore Corp.). Sodium chloride (BDH AnalaR) was added to give an ionic strength of 0.1 M. The solution used to spread the monolayer typically contained 0.3 mg/mL of DSPE (99%, Sigma-Aldrich) and was deposited onto the aqueous phase from a solution of chloroform/methanol (both BDH HPLC grade solvents) in a 5:1 v/v ratio. Decane was used as supplied (99%, Lancaster). Apparatus. The Langmuir trough (Nima Technology, Coventry, U.K.) was designed specifically to compress monolayers at the liquid/liquid interface. It was based on a design by Brooks and Pethica,39 who used an all-glass construction. We have opted for poly(tetrafluoroethylene) (PTFE), as this contaminates the subphase less and can be machined more precisely. The liquidliquid trough has a special PTFE barrier system, which has its surfaces matched to the subphase that it is confining; the aqueous subphase is confined by hydrophilic surfaces, and the oil phase by hydrophobic surfaces. In this way, leakage and crosscontamination are eliminated and the monolayer is trapped at the liquid/liquid interface. The trough was machined from a single piece of pure PTFE, deep enough to accommodate two subphases; 4.5 mm was required for the aqueous subphase, and 2 mm for the oil subphase. An insert barrier with a ledge 10 mm wide, upon which the barriers ran, was then made to fit snugly inside the periphery of the trough, as illustrated in Figure 1. For the experiments described herein, where the aqueous phase was more dense than the oil phase, the inside edges of the insert barrier and the lower face of the barrier were etched with TetraEtch (W. L. Gore & Associates) to render the PTFE hydrophilic. Hence, all adjoining interfacial surfaces that contained the aqueous phase were hydrophilic and all adjoining interfacial surfaces containing the oil phase were hydrophobic. This ensured that the amphiphile was trapped at the liquid/liquid interface. The Langmuir trough was positioned inside a home-built glovebox. A 25 µm diameter Pt disk submarine working electrode,40 characterized by an RG value of 10 (ratio of glass insulator radius to electrode radius), was mounted vertically with respect to the interface, using a small PTFE block and a glass capillary (2 mm o.d., 8 cm length) as shown schematically in Figure 2. The Pt ultramicroelectrode (UME) was manipulated using a set of x,y,z stages (M-433, Newport Corp., CA). A piezoelectric positioner and controller (models P843.30 and E662, Physik Instrumente, Waldbronn, Germany), to which the glass capillary was attached, was used for fine positioning in the z-direction (normal to the interface). The potential of the Pt working electrode was controlled by a triangular wave/pulse generator (Colburn Electronics, Coventry, U.K.), and the current was measured with a home-built current follower. All electrochemical measurements were made in a twoelectrode arrangement with a piece of silver wire acting as a quasi-reference electrode (AgQRE). All potentials are reported with respect to this electrode. Data were recorded using a Lab (36) Subczynski, W. K.; Hyde, J. S. Adv. Exp. Med. Biol. 1998, 454, 399. (37) Subczynski, W. K.; Hyde, J. S.; Kusumi, A. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 4474. (38) Dutta, A.; Popel, A. S. J. Theor. Biol. 1995, 176, 433. (39) Brooks, J. H.; Pethica, B. A. Trans. Faraday Soc. 1964, 60, 208. (40) Slevin, C. J.; Umbers, J. A.; Atherton, J. H.; Unwin, P. R. J. Chem. Soc., Faraday Trans. 1996, 92, 5177.
Oxygen Transfer across Phospholipid Monolayers
Langmuir, Vol. 20, No. 3, 2004 703
Figure 1. A drawing of the Langmuir trough designed to study liquid/liquid interfaces (top). Cross section through the trough (bottom). by adding a known volume (40-60 µL) of the DSPE spreading solution from a microliter syringe (100 µL, Hamilton, Reno, NV). The solvent was left to evaporate for approximately 5-10 min. For studies of the monolayer at the air/water interface, the system was then ready for SECM approach curve measurements. The formation of the thin oil interface was achieved by carefully adding 8-10 mL of decane to the reservoir around the subphase and allowing it to spread over the monolayer (1 h). The thick oil layer (ca. 3 mm) was formed in a similar way but using up to 100 mL of decane. For all studies, pressure-area isotherms were run and then pressures were selected at which SECM measurements were subsequently made. The potential required to effect the diffusion-controlled reduction of oxygen at the Pt UME was determined by running a voltammetric wave for oxygen reduction in the bulk subphase; a potential between -0.6 and -0.65 V was typically used. Before any electrochemical measurements were performed, the Pt electrode was pretreated in the bulk solution by pulsing the potential for 1 s to a value of 1 V and then holding at -0.6 V for 15 s. Approach curves presented in this paper have been normalized by the steady-state bulk current for oxygen reduction (I∞). All measurements were made at a temperature of 295 ( 1 K in an air-conditioned room.
Figure 2. Schematic (not to scale) showing the experimental arrangement for the studies of oxygen transfer across a phospholipid monolayer spread at an oil/water interface. PC-1200 card (National Instruments, Austin, TX). Surface tension measurements were made using a Wilhelmy paper plate attached to an electrobalance (model PS4, Nima Technology). Procedures. The surface of the subphase was cleaned by aspiration, and an SECM approach curve for the reduction of oxygen was run to the clean interface. This involved holding the UME at a potential to promote the diffusion-controlled reduction of oxygen, while slowly (1 µm s-1) translating the UME toward the interface in the normal direction using the piezoelectric positioner and controller.26,33 The monolayer was then formed
Results and Discussion Pressure-Area Isotherms. Pressure (Π)-area (A) isotherms were run for each type of interface, typically at a compression rate of 10 cm2 min-1, from a surface area of 180 cm2. The surface pressure (Π) of the monolayer was taken as the difference between the surface tension of a clean air/water interface (γ0) and the measured surface tension in the presence of the monolayer (γ) (and oil for the oil/water systems). Figure 3 shows typical isotherms measured at each interface. The isotherm for DSPE at an air/water interface is typical for lipids with a PE headgroup.29 The collapse point at an area of approximately 40 Å2/molecule corresponds to the cross-sectional area of
704
Langmuir, Vol. 20, No. 3, 2004
Figure 3. Pressure-area isotherms for the compression of a DSPE monolayer spread at the interface between (a) air/water, (b) thin decane film/water, and (c) thick decane/water.
the two saturated alkyl chain tailgroups.3 At both types of oil/water interface, the shape of the isotherm is very different. This is due to the decane penetrating the monolayer to some extent, stabilizing the fluid phase42 and causing a larger molecular area in this state, compared to that at the air/water interface. On compression, the pressure steadily increases as the decane is squeezed out of the interface, eventually reaching the situation where the LC state of the DSPE monolayer is similar to that at an air/water interface (Π > 32 mN m-1 for the thin oil, Π > 35 mN m-1 for the thick oil). In the case of the thick decane layer, the lower slope in this region of the Π-A isotherm indicates that some decane remains at the interface, until it is eventually squeezed out at the collapse point (54 mN m-1). This is not as obvious in the thin decane layer, which has a slope in the latter stages of the isotherm (small values of A) that almost matches that at the air/ water interface and a collapse point at 40 Å2 per molecule. This suggests that, in this case, most of the decane has been squeezed out during the compression of the monolayer at the interface. SECM Approach Curves at the Air/Water Interface. The Pt UME was positioned manually approximately 30 µm from the interface and then scanned toward the surface at a rate of 1 µm s-1 using the piezoelectric positioner, with the diffusion-limited oxygen reduction current measured as a function of position. The approach process was stopped as soon as a maximum current was observed to prevent the electrode pushing through the interface.26,33 Two typical approach curves to a clean interface, run on separate days, are shown in Figure 4. The current increases as the probe approaches the interface because of induced oxygen transfer from air to water,26 as shown in Figure 2. This response was used to determine the distance of closest approach of the electrode to the interface by fitting the curve to the theoretical characteristic for diffusion-controlled oxygen transfer across the air/water interface, as previously established.26 Figure 4 illustrates that the experimental data match well to the theoretical behavior and that there is a high degree of reproducibility. The experimental data only deviate from theory at distances close to the interface where a flattening out of the curve occurs. This is indicative of a thin layer of water becoming trapped by surface tension as the UME approaches the interface. The maximum normalized current recorded of 9.5 ( 0.1 corresponded to a distance, d, of 1.1 ( 0.1 µm. (41) Thoma, M.; Mo¨hwald, H. J. Colloid Interface Sci. 1994, 162, 340. (42) Kwak, J.; Bard, A. J. Anal. Chem. 1989, 61, 1221.
Cannan et al.
Figure 4. Approach curves for the reduction of oxygen at a Pt UME as it was translated toward a clean air/water interface (solid lines) matched to the theory for the diffusion-controlled transfer of oxygen ()).
Figure 5. Schematic for the transfer of oxygen across a monolayer spread at an air/water or oil/water interface.
Since the kinetics of oxygen transport is fast and SECM measurements are most accurate at the closest distance from the interface,42 kinetic information, with the monolayer present, was obtained by measuring the maximum current in an approach curve at this distance of closest approach. This is equivalent to using the SECM as an ultrathin layer cell and making measurements under the optimal conditions of high mass transport rates, needed to compete with the fast interfacial kinetics. The presence of the monolayer was confirmed to have no effect on the distance of closest approach, by running approach curves for the oxidation of ferrocyanide over the range of surface pressures used. These were in complete agreement with theory for hindered diffusion43 and yielded the same distance of closest approach. Approach curves were run at different surface pressures for DSPE spread at an air/water interface. As the surface pressure of the monolayer was increased, it was found that the magnitude of the normalized current decreased, indicating that the state of compression of the DSPE monolayer had an effect on the transfer of oxygen across the interface. This is consistent with our findings on the transfer of both oxygen and bromine across fatty alcohol monolayers26,27 at the air/water interface. A schematic for oxygen transfer across a monolayer at a water surface in contact with either an oil or air phase is shown in Figure 5. We consider the case where passive diffusion across the interface, rate constant k, limits the rate, and the entry and exit to the monolayer are rapid, so that equilibrium is established by the local oxygen concentrations. The net flux of oxygen across the monolayer, j, is then given by
j ) k(cmon - cmon,aq)
(1)
where cmon is the interfacial concentration of oxygen in the monolayer at the air or oil boundary and cmon,aq is the (43) Barker, A. L.; Gonsalves, M.; Macpherson, J. V.; Slevin, C. J.; Unwin, P. R. Anal. Chim. Acta 1999, 385, 223.
Oxygen Transfer across Phospholipid Monolayers
Langmuir, Vol. 20, No. 3, 2004 705
Figure 6. Working curve of normalized current vs log K modeled at a distance of closest approach of 1.1 µm.
interfacial concentration of oxygen in the monolayer at the aqueous boundary. Since the partition coefficient of oxygen between the monolayer and aqueous phase is given by
cmon,aq Kp ) caq,i
(2)
j ) k(cmon - Kpcaq,i)
(3)
When there is no flux across the monolayer and the aqueous phase has a uniform (bulk) concentration, c/aq, then
cmon c/aq
(4)
Hence
j ) kKpc/aq(1 - C)
(5)
where C ) caq,i/c/aq. Defining a permeability coefficient for transfer,
k' ) kKpc/aq
(6)
equation 5 simplifies further to
j ) k′(1 - C)
the normalized current ratio, I(d)/I∞, to K, the normalized rate constant,
K)
then
Kp )
Figure 7. Approach curves for the reduction of oxygen at a Pt UME as it was translated toward a DSPE monolayer spread at an air/water interface at different surface pressures (solid lines). From top to bottom, the solid lines correspond to 10, 24, 33.5, and 44 mN m-1. The dashed lines represent theoretical approach curves for diffusion-controlled oxygen transfer (top) and then log K values of 1.08, 0.87, 0.60, and 0.47 (bottom).
(7)
Equation 7 turns out to be identical to the flux expression used in an earlier SECM model to describe dissolution,44 and so the results from that model, relating the steadystate normalized tip current to the kinetics of the interfacial process, may be used directly. Note that this analysis assumes that the diffusivity and concentration of O2 in the second phase are sufficiently high to maintain cmon at the equilibrium value. We have shown previously25,26 that this is an excellent assumption given the high relative solubility and diffusion coefficient of O2 in these phases, compared to water. A comprehensive treatment outlining the conditions under which depletion in the second phase is negligible has been given elsewhere.45 A working curve (Figure 6) was obtained at the distance of closest approach, defined earlier as 1.1 ( 0.1 µm, relating (44) Macpherson, J. V.; Unwin, P. R. J. Phys. Chem. 1996, 100, 19475. (45) Barker, A. L.; Macpherson, J. V.; Slevin, C. J.; Unwin, P. R. J. Phys. Chem. B 1998, 102, 1586.
k′a Dc/aq
(8)
Here, I(d) is the distance-dependent steady-state current, a is the radius of the UME, and D is the diffusion coefficient for oxygen in water. From this curve, K values were assigned to the measured normalized currents, taken at the distance of closest approach, for each surface pressure. The sigmoidal shape of the working curve makes it clear that at high K, where the current values lie on the plateau, the rate of oxygen transfer approaches the diffusioncontrolled limit and values for K cannot be apportioned with accuracy from corresponding current measurements. However, in the rising section of the working curve, -0.2 < log K > 1.8, accurate determination of K is possible, because of the strong dependence of the current on K. Based on the values of K obtained from the current measurements at the distance of closest approach, full approach curves were simulated for the entire data set and found to match well to the experimental data (Figure 7). Table 1 lists the normalized currents, measured at the distance of closest approach, and the corresponding rate constants for the transfer of oxygen at each surface pressure investigated. SECM Approach Curves at the Decane/Water Interface. These were run at both types of decane/water interfaces (thin and thick oil), and the data were analyzed as already described. The approach to a thick decane/ water interface, in the absence of a monolayer, indicated that oxygen transfer was diffusion-controlled. During approach curve measurements, with the monolayer present at fixed surface pressures, the surface pressure was independently maintained via a feedback control between the pressure sensor and the trough barriers. A barrier speed of 2.5 cm2 min-1 or less was required to keep the pressure constant in the high-pressure region, which was found to have no effect on the rate of transport to the UME. This was confirmed by control measurements in which comparable induced oxygen transfer approach curves were recorded at a clean water/decane interface with the barrier position either fixed or translated at a speed of 3 cm2 min-1 during the measurement.33 The distance of closest approach of the probe UME to both types of decane/water interface differed from the 1.1 ( 0.1 µm measured for the air/water interface. For the thin and thick decane layers, these distances were 2.5 ( 0.1
706
Langmuir, Vol. 20, No. 3, 2004
Cannan et al.
Table 1. Normalized Currents, I/I∞, at the Distances of Closest Approach and Corresponding Rate Constants, k′, for the Transfer of Oxygen across a DSPE Monolayer Spread at the Interface between Air/Water, Thin Decane Film/Water, and Thick Decane/Water at Various Surface Pressures (Π)a air/water
thin decane/water
Π/mN m-1
I/I∞
k′/10-8 mol cm-2 s-1
5 10 14 24 33.5 38 44
5.55 ( 0.10 5.11(0.10 4.84 ( 0.08 4.05 ( 0.08 2.84 ( 0.06 2.59 ( 0.06 2.34 ( 0.05
5.9 ( 0.2 5.1 ( 0.2 4.5 ( 0.15 3.1 ( 0.1 1.7 ( 0.06 1.5 ( 0.06 1.2 ( 0.05
a
thick decane/water
Π/mN m-1
I/I∞
k′/10-8 mol cm-2 s-1
21 28 37 40 43 45
3.90 ( 0.08 3.66 ( 0.07 2.91 ( 0.04 2.70 ( 0.04 2.71 ( 0.04 2.43 ( 0.04
10.5 ( 0.25 7.0 ( 0.2 3.0 ( 0.1 2.4 ( 0.08 2.4 ( 0.08 1.8 ( 0.08
Π/mN m-1
I/I∞
k′/10-8 mol cm-2 s-1
41 44 46 48 50 52 54
5.94 ( 0.10 5.41 ( 0.10 4.96 ( 0.08 4.61 ( 0.08 4.23 ( 0.08 3.79 ( 0.06 3.30 ( 0.06
21 ( 0.5 11 ( 0.25 7.8 ( 0.2 6.1 ( 0.2 4.5 ( 0.15 3.5 ( 0.1 2.4 ( 0.08
Note that the distances of closest approach are different for each system (see the text).
Figure 8. SECM approach curves to a DSPE monolayer spread at a thick decane/water interface. From top to bottom: Π ) 41, 48, and 52 mN m-1.
Figure 9. Plot of ln k′ vs Π for the transfer of oxygen across a DSPE monolayer spread at the interface between (a) air/ water, (b) thin decane film/water, and (c) thick decane/water.
µm and 1.6 ( 0.1 µm, respectively. Typical SECM approach curves to DSPE adsorbed at the thick decane/water interface at specific surface pressures are shown in Figure 8. The rate constants for the transfer of oxygen across both types of decane/water interface, deduced by measuring the tip current at the distance of closest approach over the range of surface pressures studied, are given in Table 1. Working curves, similar to that shown in Figure 6, were calculated to allow measured tip currents to be converted to corresponding rate constants. Comparison of Oxygen Transfer across Monolayers at Air/Water and Oil/Water Interfaces. The relationship between k′ and Π is compared in Figure 9 for the three different interfaces. These are represented as semilogarithmic plots of ln k′ versus Π to highlight the range of k′ measured. For the decane/water interfaces, oxygen transfer was diffusion-controlled up to surface pressures of approximately 20 mN m-1 for the thin layer
and 40 mN m-1 for the thicker layer. At higher pressures, the rate of oxygen transfer decreased rapidly, more so at the thick decane interface, as indicated by the steep gradients of the trendlines in Figure 9. The data show that in the LE state, the DSPE monolayer presents no barrier to the flux of oxygen, on the time scale of SECM measurements, at any of the interfaces used herein. However, as the monolayer becomes more compact and its state more ordered, the rate of oxygen transfer decreases. This occurs at values of Π > 5 mN m-1 at the air/water interface, where the LC state is observed, but at significantly higher pressures for both of the decane/ water interfaces. Fluorescence microscopy, ellipsometry, and X-ray diffraction techniques have been used to study the structural changes of the closely related phospholipid L-R-phosphatidylethanolamine, dipalmitoyl (DPPE) at thin and thick oil/water interfaces, in different states.29,31,46 These investigations have concluded that at both types of oil/ water interface the fluid, LE state of the lipid is stabilized as the oil molecules become incorporated into the monolayer, reducing the tilt angle of the tails by decreasing the repulsive forces between the headgroups.29 If the oil molecules and lipid alkyl chain tailgroups are the same length, they will also mix in the LC state, and the Π-A isotherm exhibits a plateau between the LE and LC transition.41 DSPE and decane do not have similar length chains, yet the increased molecular area and the shape of the isotherms are clear indications that some decane is present within the monolayer until high pressures are reached, at which point decane is eventually squeezed out. At any given value of Π, the flux of oxygen across the DSPE monolayer is greatest at the thick decane/water interface, then diminishes at the interface between thin decane/water, and finally is slowest at the air/water interface. This sequence reflects the change in the organization of the monolayer at each interface, which becomes more ordered as the interface changes from thick oil/water to thin oil/water and then to air/water. At the air/water interface, the molecules of DSPE are orientated with their hydrophilic headgroups dissolved in the aqueous phase and the hydrophobic tails out of the water. Due to the relative sizes of the head- and tailgroups in DSPE molecules (the cross-sectional area of the alkyl chains is larger than that of the phosphatidylethanolamine headgroup), the packing of the monolayer is determined by the hydrocarbon tails.32 Cohesive forces between the hydrocarbon chains, which increase with decreasing A, and the hydrophobic nature of the tailgroup maintain a high degree of order in the monolayer. However, for the oil/water interfaces, the presence of decane at the interface, which (46) Thoma, M.; Mo¨hwald, H. Colloids Surf. 1995, 95, 193.
Oxygen Transfer across Phospholipid Monolayers
can penetrate between the alkyl tailgroups of the lipid, reduces these attractive forces and allows greater lateral freedom of the molecules at the interface.47 In this more disorganized state, the studies in this paper have clearly shown that the barrier to oxygen transfer is reduced. Only at high pressures, when most of the decane has been excluded from the interface, does the flux of oxygen across the adsorbed monolayer diminish. This is most apparent at the interface between thick decane/water since more decane is available to permeate the monolayer and, based on the isotherm data, this solvent is only removed at high pressures (Π > 40 mN m-1). At the highest pressures accessed in these studies, the rate constants for all three systems converge in the limit where ln k′-Π plots are extrapolated to high pressure. This indicates that the organization of the monolayer is similar at this limit and largely independent of whether air or oil is the second phase in contact with the aqueous phase. Conclusions SECM has been combined with a Langmuir trough to enable the study of oxygen transfer kinetics across liquid/ liquid interfaces under surface pressure control. The rate of oxygen transfer across a DSPE monolayer has been measured at an air/water interface and two types of oil/ water interface as a function of interfacial surface pressure. At all three interfaces, oxygen transfer is diffusioncontrolled when the monolayer is in the LE state, but the rate of transfer diminishes with increasing surface pressure in the LC state. For any given surface pressure, the rate constant for oxygen transfer decreases as the interface changes from thick oil/water to thin oil/water to air/water. The monolayer is therefore most permeable to oxygen at (47) Davies, J. T.; Rideal, E. K. Interfacial Phenomena; Academic Press: London, 1963.
Langmuir, Vol. 20, No. 3, 2004 707
a thick oil/water interface where the hydrocarbon tail region of the phospholipid is less ordered. However, at the very highest limiting surface pressures, the permeability coefficients for all three systems are similar, which is consistent with the idea that the interface is primarily occupied by the monolayer and that at the highest pressures the organization of this layer is similar for all three interfaces. It is interesting to note that the amount of decane present at the interface influences the permeability of the monolayer to oxygen, since BLMs, widely used as model membrane systems and usually prepared as black lipid membranes, often contain residual organic solvents.5 Our studies suggest that the quantity of organic solvent, frequently decane, which remains incorporated in the BLM system could be an important factor in determining transport across such membranes and that this needs careful consideration when such BLMs are used in permeability studies. Having established that quantitative measurements can be made on solute transfer across monolayers at liquid/ liquid interfaces under surface pressure control, there is now scope for using this technique further for other systems. The investigation of charge transfer (electron and ion transfer) across such layers would be of particular interest, although this would most likely require the use of more polar organic solvents. Work in this direction is underway in our laboratory. Acknowledgment. The support of this work by the EPSRC (GR/M 90948) is acknowledged. S.C. thanks EPSRC for a studentship, and J.Z. gratefully acknowledges scholarships from the ORS scheme, Avecia, and the University of Warwick. LA034943W
