Langmuir 1998, 14, 2573-2576
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Notes Determination of the Surface Concentration of Crown Ethers in Supported Lipid Membranes by Capacitance Measurements Samuel Terrettaz, Horst Vogel,* and Michael Gra¨tzel Institute of Physical Chemistry, Federal Institute of Technology, CH-1015 Lausanne, Switzerland Received July 28, 1997. In Final Form: February 4, 1998
Introduction Physical and chemical properties of an interface can be controlled by a molecular layer of organic molecules. Both fundamental and technology-oriented studies in this fastgrowing field have been reviewed.1 Supported lipid layers represent a simplified version of a biological membrane at which some interfacial processes can be investigated by surface-sensitive techniques.2 After incorporation of selective molecules in the layers, molecular recognition events at a lipid layer surface can be used to detect the presence of a given analyte in solution. To develop this field further, it is important to establish different techniques for preparing molecular layers with desired properties, reproducibly. It is also very useful to test the sensitivity of different measurement techniques. We have already reported the specific detection of K+ and Ca2+ using supported lipid monolayers containing ionophores by impedance techniques.3,4 Through ion complexation by the neutral ligand, electrical charges are incorporated in the low-dielectric-constant environment of the lipid layer, leading to an increase of the capacitance. Self-assembly (SA) is an efficient method for forming the lipid/ionophore molecular layer on a solid support. Starting from a mixed lipid/ionophore solution in organic solvents, one finds that a simple dilution step by water or electrolyte leads to the spontaneous formation of a single-lipid/ionophore monolayer on a hydrophobic support. Unfortunately, the resulting mixed layer on the support is not necessarily of the same molecular composition as the starting solution.3 However, for quantitative ion-binding studies the molecular lipid/ionophore composition is necessary. Here, we show another way of producing an ionophore containing a lipid monolayer from a micellar solution and a simple procedure to determine its molecular composition by capacitance measurements.
(DMB) was a gift from Prof. E. sz Kova´ts. All other chemicals were reagent grade and used without further purification. The electrolyte comprised 0.1 M KCl or CaCl2 and 10 mM Tris-HCl pH 7.5. The aqueous ionophore solutions of C14-18-crown-6 were sonicated in a bath-type sonicator until clear solutions were obtained. The water used was purified via an ion exchanger (Milli Q system, Millipore). Circular areas of bare silicon (0.01 cm2) in insulated n-type silicon wafers were covered by a 35-Å SiO2 layer and then silanized by DMB in order to yield a hydrophobic surface. The silanization was performed in a sealed ampule at 240 °C for 72 h according to a method known for producing high-coverage densities.5 The lipid layers were formed by the following procedure directly in the electrochemical cell. After an extensive washing, the silanized electrodes were left in about 5 mL ethanol and then 50-100 mL of a 1 mM lipid solution in ethanol were added and mixed vigorously with a syringe. After about 5 min, the lipid solution was diluted rapidly with several milliliters of electrolyte. Then the electrolyte was replaced several times to get rid of the excess lipid. K+ sensitive layers were formed by introducing aqueous solutions of C14-18-crown-6 in the electrolyte. The incorporation of ionophores in the POPC layer could be stopped at any time by rinsing with the electrolyte. For the estimation of the membrane composition presented in Figure 2, the incorporation of ionophores in the membrane was pursued in 0.1 M CaCl2 until a constant admittance value was obtained. The time required for reaching the steady state is dependent on the ionophore concentration and could extend to several hours for the smallest concentration used. The admittance measurements were performed using a twochannel lock-in amplifier (EG&G model 5206) at 120 Hz, applying 20 mV RMS to the electrochemical cell and a dc bias of -1.3 V (vs Ag/AgCl/0.1 M KCl). A large AgCl-coated Ag wire served as the counter/reference electrode. A high electrolyte concentration (0.1 M KCl or 0.1 M CaCl2) was chosen to minimize the electrolyte resistance and double-layer effects. All the experiments were done at room temperature. Under these conditions the impedance of the whole electrochemical cell is almost completely capacitive. After deposition of a POPC layer, the imaginary and the real part of the admittance of the electrochemical cell are (4.5 ( 0.4) × 10-6 Ω-1 and (1.8 ( 0.7) × 10-7 Ω-1, respectively. The capacitance is the ratio of the imaginary part of the admittance on the radial frequency. In the model of two capacitors in series, a capacitance can be attributed to a lipid layer from measurements with and without the additional layer. The changes of the real part of the admittance are given as additional information but are not analyzed quantitatively. A detailed description of the applied procedure (electrode derivatization, electrochemical setup, and evaluation of the data) can be found in references.3,4
Results and Discussion Experimental Section 1-Palmitoyl-2-oleoyl-sn-3-glycerophosphocholine (POPC) was from Avanti Polar Lipids and 1,4,7,10,13,16-hexaoxacyclooctadecane (abbreviated as 18-crown-6) was purchased from Fluka. Tetradecyl-18-crown-6 (C14-18-crown-6) was a gift from Prof. P. Tundo. (3,3′-dimethylbutyl)dimethyl(dimethylamino)silane * To whom correspondence should be addressed. (1) Ulman, A. An Introduction to Ultrathin Organic Films from Langmuir-Blodgett to Self- Assembly; Academic Press: San Diego, 1991. (2) Sackmann, E. Science 1996, 271, 43. (3) Miller, C.; Cuendet, P.; Gra¨tzel, M. J. Electroanal. Chem. 1990, 278, 175. (4) Terrettaz, S.; Vogel, H.; Gra¨tzel, M. J. Electroanal. Chem. 1992, 326, 161.
With the reported procedure, monolayers of lipids can be assembled very easily and reproducibly at the interface between an aqueous solution and a hydrophobic electrode. POPC layers are very stable and no modification of their electrical properties can be detected over a period of several days. The obtained capacitance of 1.4 ( 0.1 µF/cm2 is among the lowest reported for a supported monolayer of phospholipids.3,4 These monolayers are thus associated with a low density of defects and a low content of polar solvent (ethanol or water). Impedance is a very sensitive (5) Szabo´, K.; Le Ha, N.; Schneider, P.; Zeltner, P.; Kova´tz, E. sz. Helv. Chim. Acta 1984, 67, 2128.
S0743-7463(97)00838-X CCC: $15.00 © 1998 American Chemical Society Published on Web 04/11/1998
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technique to describe the properties of an insulating layer because a small number of defects will drastically affect the measurement. Resistivities of lipid layers, especially, are seen to vary significantly with the procedure used for the layer formation.6 In our system, thanks mainly to the insulation provided by the silica, the electrical resistance of the molecular layer does not affect the measured impedance practically. The capacitance of our layer is compared hereafter with values obtained in similar systems. A more detailed discussion can be found elsewhere.6 A capacitance of 2 and 4 µF/cm2 has been obtained by SA from detergent solution or from unilamellar vesicle solution, respectively.7 Using the LangmuirBlodgett (LB) technique, layers with slightly lower capacitances (1.1 µF/cm2) have been obtained under certain conditions.4,6 Transferring a lipid monolayer from the water surface to a solid support is probably accompanied by some defect formation, as indicated by the higher capacitance values usually found in the LB layers.7-9 In our configuration, the capacitance of a POPC monolayer is the same in 0.1 M KCl and 0.1 M CaCl2.10 To display some cation selectivities, ionophores have to be introduced in the system. The cavity of 18-crown-6 is especially designed for the selective complexation of K+. In the aqueous phase, 18-crown-6 has an association constant of approximately 100 M-1 with K+ 11 and shows a negligible affinity to Ca2+.12,13 The addition of 18-crown-6 and its more hydrophobic derivative C14-18-crown-6 in the aqueous solution is discussed below. The admittance changes of a supported POPC monolayer induced by both ionophores in 0.1 M KCl and CaCl2 are illustrated in Figure 1. When 10-1 M 18-crown-6 is added to a 0.1 M KCl solution in the electrolyte, the capacitance of the supported POPC monolayer changes abruptely to a higher value. After thermolayer is washed with 0.1 M KCl, the monolayer capacitance returns to its initial value. In the presence of only Ca2+ cations, the capacitance of the POPC layer is hardly affected by 18-crown-6. This quick and reversible capacitance change obtained in the presence of K+ resembles the one already observed for a membrane composed of C14-18-crown-6 and POPC.3 However, the increase of the capacitance of the POPC monolayer in the presence of both K+ and 18-crown-6 is much smaller than the response to K+ of a monolayer containing C14-18crown-6 and POPC.14 In addition to modest amplitude, the perfect reversibility of the electrical response indicates that the interaction between 18-crown-6 and POPC is confined near the monolayer surface. If, instead of 18-crown-6, the more hydrophobic C1418-crown-6 is added to the electrolyte, the capacitance changes are drastically enhanced. Even at a concentration of 10-4 M C14-18-crown-6, the capacitance increase is much larger than the one induced by 10-1 M 18-crown-6 (6) Lindholm-Sethson, B. Langmuir 1996, 12, 3305. (7) Terrettaz, S.; Stora, T.; Duschl, C.; Vogel, H. Langmuir 1993, 9, 1361. (8) Stelzle, M.; Sackmann, E. Biochim. Biophys. Acta 1989, 981, 135. (9) Fare, T. L. Langmuir 1990, 6, 1172. (10) No significant capacitance change is induced at a silanized electrode with and without a POPC layer by the replacement of 0.1 M CaCl2 by 0.1 M KCl. Variations of the POPC/electrolyte interface due to ionic strength or to selective ion binding have a negligible effect in our system. (11) Frensdorff, H. J. Am. Chem. Soc. 1971, 93, 600. (12) Hirooka, M. Crown Compounds; Elsevier: Amsterdam, 1982. (13) Matsumura, H.; Furusawa, K. J. Colloid Interface Sci. 1988, 124, 385. (14) For instance, the layer capacitance is increased by 7%, 3%, and 0% when [18-crown-6] ) 10-1, 10-2, and 10-3 M are respectively added to 0.1 M KCl. In comparison, the capacitance of a lipid layer containing C14-18-crown-6 can be increased to more than 50% in the presence of only 10-3 M KCl.
Notes
Figure 1. (A) Traces of the imaginary, Yi, and the real, Yr, admittance of a Si/SiO2/DMB electrode coated with a pure POPC layer in 0.1 M KCl electrolyte and their variations (1) after addition of 0.1 M 18-crown-6 to the aqueous phase, (2) after washing with excess of electrolyte, (3) after addition of 10-4 M C14-18-crown-6 to the aqueous phase, and (4) after washing with the electrolyte. Note the quick, reversible, and relatively small change of the admittance induced by 18-crown-6, compared to the slower, under our rinsing conditions, irreversible, and much larger effect of the C14-18-crown-6 at a 1000fold lower concentration. In addition, the molecular formula of the corresponding ionophores are shown. (B) Same experiment as (A) but in 0.1 M CaCl2 with similar qualitative effects. The increase of the admittance is, however, much weaker because of the very low affinity of Ca2+ ions to the ionophores; practically no ions can be introduced in the lipid layer (notice the change of scale compared to (A)). The interaction of 18-crown-6 with POPC in 0.1 M CaCl2 is negligible at the present resolution.
(Figure 1). However, the kinetics is much slower and a stable value is only obtained after several hours. The capacitance decreases clearly after washing with pure electrolyte, but the initial value cannot be reached under these experimental conditions. The interaction with C1418-crown-6 results in a net capacitance increase which
Notes
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should be related to some structural changes in the lipid layer. Furthermore, the modified layer displays now exactly the same very large K+ sensitivity as the membranes consisting of lipids and ionophores. Depending on the ionophore ratio, an increase of more than 50% of the layer capacitance can be obtained in the presence of 10-3 M KCl. Other characteristics such as the stability of the response are also the same as reported in the previous work.3 It is therefore reasonable to assume that similar layers containing both C14-18-crown-6 and POPC are also obtained with this method. Because of the very low affinity between Ca2+ and the ionophore, the increase of the capacitance in 0.1 M CaCl2 is interpreted solely as the incorporation of the C14-18-crown-6 molecules in the POPC layer. In 0.1 M KCl, the very large response is the result of both the ionophore incorporation and the binding to K+ (Figure 1A). This process can be used to prepare layers with a given number of ionophores and therefore with a given sensitivity. The slow incorporation of C14-18-crown-6 can be followed in real time and interrupted simply by removing the ionophores from the bathing solution. The ionophore content of any membrane can be estimated from its capacitance value. The procedure is applied in the following to determine the molar composition of layers obtained in 0.1 M CaCl2 and at the steady state.15 The extent of ionophore incorporation at the steady state depends on the concentration of C14-18-crown-6 in 0.1 M CaCl2 and can be calculated from the measured capacitance. The K+ response in the presence of 10-3 M KCl is then used to confirm the ionophore ratio of those membranes and to get the number of K+ bound to the membrane. The corresponding values of the capacitance and the relation between them are reported in Figure 2. To relate the measured capacitance in 0.1 M CaCl2 to the composition of the layer, the total capacitance of the supported membrane, Clayer, is modeled as the sum of parallel capacitors of the crown ether and the POPC, respectively.
Clayer ) XCionophore + (1 - X)Clipid
(1)
where X is the surface fraction occupied by C14-18-crown6. Clipid and Cionophore are the capacitance of the pure POPC and C14-18-crown-6 monolayers (measured in 0.1 M CaCl2) and their value is 1.3 and 2.6 µF/cm2, respectively. X values of 0.14, 0.60, and 1 are obtained for membranes formed with C14-18-crown-6 concentrations of 10-5, 5 × 10-4, and 10-4 M, respectively (Figure 2B). This treatment supposes that the influence of an ionophore molecule on the insulating properties of the neighbor lipid molecules can be neglected. The interaction of C14-18-crown-6 with POPC is quite strong, and a partition constant between the organic layer and the aqueous phase can be calculated to be approximately 104 for the concentrations investigated.16 Interestingly, layers formed from a solution of C14-18-crown-6 at steady state display the same capacitance and the same sensitivity to K+ as pure ionophore (15) We want to point out that the critical micellar concentration and the partition constant between the POPC layer and the aqueous solution are different when C14-18-crown-6 is bound or not to a cation. Therefore, for the quantitative description, all the sensitive layers are formed in 0.1 M CaCl2. Only after the rinsing step with 0.1 M CaCl2 is KCl added to the electrolyte. (16) The partition constant Kp is defined as the ratio of the surface concentration of bound ionophores to the free concentration of C1418-crown-6 in the aqueous solution divided by the thickness of the organic layer. An area per molecule of 100 Å2 for C14-18-crown-616 and 70 Å2 for POPC has been used to get the surface concentration of ionophores from the surface fraction, X.
Figure 2. (A) Steady-state capacitance of the sensitive monolayer in 0.1 M CaCl2 (b) and in 10-3 M KCl and 0.1 M CaCl2 (O) versus the concentration of C14-18-crown-6 in the electrolyte. (B) Capacitance of the sensitive monolayer in 0.1 M CaCl2 versus the surface fraction of the electrode covered by the ionophore, calculated with eq 1. (C) Capacitance of the sensitive monolayer in the presence of 10-3 M KCl versus the surface fraction of the electrode covered by the ionophore.
layers prepared by SA from ethanol. Furthermore, if a supported POPC layer is contacted with an electrolyte containing more than 10-4 M C14-18-crown-6, the same limiting capacitances are obtained, but now faster. One possible explanation is thus to consider this layer as to contain a high concentration of ionophores; in the extreme case it might be composed of ionophores only. Knowing the fraction of C14-18-crown-6 in a membrane, we would like to get the fraction, θ, of ionophores bound to K+ for a given KCl concentration (10-3 M) in the electrolyte. So we can write
Clayer ) θXCK+ + (1 - θ)XCionophore + (1 - X)Clipid (2) where X, Cionophore, and Clipid are known from measurements in 0.1 M CaCl2 and Clayer is measured. CK+, the capacitance of a monolayer of C14-18-crown-6 where all the ionophores molecules have bound K+, is not easily reached experimentally. Additional information is thus needed to get the value of θ. The association constant is supposed to be similar to the association constant of K+ and 18-crown-6 in water as shown by Matsumara et al. for dicyclohexano18-crown-613 and octadecyloxymethyl-18-crown-617 at the air/water interface. Using an adsorption isotherm from Langmuir type and an association constant of 100 M-1, we calculate that 9% of the ionophores are liganded with (17) Matsumura, H.; Watanabe, T.; Furusawa, K.; Inokuma, S.; Kuwamura, T. Bull. Chem. Soc. Jpn. 1987, 60, 2747.
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K+ for [K+] ) 10-3 M. This treatment neglects for simplicity the influence of the electrical double-layer potential at these high electrolyte concentrations. The local concentration of K+ at the interface is thus supposed to be the same as its value in the bulk. If we admit that the area per C14-18-crown-6 molecule is unchanged during K+ complexation, we can introduce this value of θ in eq 2. The expected linear relationship between Cmembrane and θ is experimentally observed and reported in Figure 2C. Thus, we have shown that the increase of the capacitance is related quantitatively to the incorporation of ionophores and of ionic charges in the lipid matrix. Conclusion In summary, the following results have been obtained in the present communication. The higher hydrophobicity of the C14-18-crown-6 ionophore results in a different interaction with a POPC monolayer than that of the 18-
Notes
crown-6. While 18-crown-6 interacts weakly and reversibly to a lipid monolayer, C14-18-crown-6 molecules replace some lipid molecules irreversibly and give the layer a long-lasting K+ sensitivity. The capacitance increase of the monolayer due to C14-18-crown-6 incorporation and to selective K+ complexation allows one to calculate the composition of any layer. This new way of producing ionsensitive monolayers allows a good control on the layer composition because the slow ionophore insertion can be interrupted when a certain capacitance value (a certain ionophore content) is obtained. A fine-tuning of the membrane sensitivity is thus possible. Acknowledgment. This work was supported by the Swiss National Science Foundation, Priority Programme on Biotechnology, Grant 5002-35180. LA9708389
