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Langmuir 1998, 14, 648-659
Tethered Lipid Bilayer Membranes: Formation and Ionic Reservoir Characterization Burkhard Raguse,* Vijoleta Braach-Maksvytis, Bruce A. Cornell, Lionel G. King, Peter D. J. Osman, Ron J. Pace,† and Lech Wieczorek Co-operative Research Centre for Molecular Engineering & Technology, 126 Greville Street, Chatswood, NSW 2067 Australia Received October 15, 1997X Using novel synthetic lipids, a tethered bilayer membrane (tBLM) was formed onto a gold electrode such that a well-defined ionic reservoir exists between the gold surface and the bilayer membrane. Self-assembled monolayers of reservoir-forming lipids were first adsorbed onto the gold surface using gold-sulfur interactions, followed by the formation of the tBLM using the self-assembly properties of phosphatidylcholinebased lipids in aqueous solution. The properties of the tBLM were investigated by impedance spectroscopy. The capacitance of the tBLM indicated the formation of bilayer membranes of comparable thickness to solvent-free black (or bilayer) lipid membranes (BLM). The ionic sealing ability was comparable to those of classical BLMs. The function of the ionic reservoir was investigated using the potassium-specific ionophore valinomycin. Increasing the size of the reservoir by increasing the length of the hydrophilic region of the reservoir lipid or laterally spacing the reservoir lipid results in an improved ionic reservoir. Imposition of a dc bias voltage during the measurement of the impedance spectrum affected the conductivity of the tBLM. The conductivity and specificity of the valinomycin were comparable to those seen in a classical BLM.
The ability to design and build nanostructured architectures through the self-assembly of monolayer,1-7 bilayer,8-19 and multilayer,20-24 membranes is one of the current challenges in chemistry and physics. Bilayer * To whom correspondence should be addressed. E-mail:
[email protected]. † Permanent Address: Chemistry Department, Faculty of Science, Australian National University, Canberra, ACT Australia. X Abstract published in Advance ACS Abstracts, December 15, 1997. (1) Ulman, A. Introduction to Ultrathin Organic Films from Langmuir-Blodgett to Self-Assembly; Academic Press: New York, 1991. (2) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321-335. (3) Bain, D. C.; Evall, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7155-7164. (4) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 71647175. (5) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12-20. (6) Schessler, H. M.; Karpovich, D. S.; Blanchard, G. J. J. Am. Chem. Soc. 1996, 118, 9645-9651. (7) Kim, T.; Chan, K. C.; Crooks, R. M. J. Am. Chem. Soc. 1997, 119, 189-193. (8) Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580583. (9) Sackmann, E. Science 1996, 271, 43-48. (10) Stelzle, M.; Weissmuller, G.; Sackmann, E. J. Phys. Chem. 1993, 97, 2974-2981. (11) Fare, T. L. Langmuir 1990, 6, 1172-1179. (12) Ti Tien, H.; Salamon, Z. Bioelectrochem. Bioenerg. 1989, 22, 211-218. (13) Gu, L.; Wang, L.; Xun, J.; Ottova-Leitmannova, A.; Ti Tien, H. Bioelectrochem. Bioenerg. 1996, 39, 275-283. (14) Florin, E. L.; Gaub, H. E. Biophys. J. 1993, 64, 375-383. (15) Spinke, J.; Yang, J.; Wolf, H.; Liley, M.; Ringsdorf, H.; Knoll, W. Biophys. J. 1992, 63, 1667-1671. (16) Miller, C.; Cuendet, P.; Gratzel, M. J. Electroanal. Chem. 1990, 278, 175-192. (17) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197-210. (18) Plant, A. L. Langmuir 1993, 9, 2764-2767. (19) Plant, A. L.; Gueguetchkeri, M.; Yap, W. Biophys. J. 1994, 67, 1126-1133. (20) Decher, G. In Comprehensive Supramolecular Chemistry; Pergamon Press: New York, 1996; Vol. 9, Chapter 14. (21) Netzer, L.; Iscovici, R.; Sagiv, J. Thin Solid Films 1983, 99, 235.
membranes formed onto solid supports are becoming particularly attractive as model systems that can be used to mimic the structural, sensing, and transport roles of biological membranes.8,25-36 Supported bilayer membranes (sBLMs) were developed in order to overcome the extreme fragility of the classical black (or bilayer) lipid membrane (BLM).37 BLMs, once formed, typically survive for minutes to hours and are very sensitive toward vibration and mechanical shocks. In contrast, the sBLMs have improved stability, surviving for hours to days with little sensitivity toward mechanical (22) Yang, H. C.; Aoki, K.; Hong, H.-G.; Sackett, D. D.; Arendt, M. F.; Yau, S.-L.; Bell, C. M.; Mallouk, T. E. J. Am. Chem. Soc. 1993, 115, 11855. (23) Horne, J. C.; Blanchard, G. J. J. Am. Chem. Soc. 1996, 118, 12788-12795. (24) Ariga, K.; Lvov, Y.; Kunitake, T. J. Am. Chem. Soc. 1997, 119, 2224-2231. (25) McConnell, H. M.; Watts, T. H.; Weis, R. M.; Brian, A. A. Biochim. Biophys. Acta 1986, 864, 95-106. (26) Stenger, D. A.; Fare, T. L.; Cribbs, D. H.; Rusin, K. M. Biosens. Bioelectron. 1992, 7, 11-20. (27) Gilardoni, A.; Margheri, E.; Gabrielli, G. Colloids Surf. 1992, 68, 235-242. (28) Seifert, K.; Fendler, K.; Bamberg, E. Biophys. J. 1993, 64, 384391. (29) Stelzle, M.; Sackmann, E. Biochim. Biophys. Acta 1989, 981, 135-142. (30) Elender, G.; Kuhner, M.; Sackmann, E. Biosens. Bioelectron. 1996, 11, 565-577. (31) Steinem, C.; Janshoff, A.; Ulrich, W.-P.; Sieber, M.; Galla, H.-J. Biochim. Biophys. Acta 1996, 1279, 169-180. (32) Heysel, S.; Vogel, H.; Sanger, M.; Sigrist, H. Protein Sci. 1995, 4, 2532-2544. (33) Nikolelis, D. P.; Siontorou, C. G.; Krull, U. J.; Katrivanos, P. L. Anal. Chem. 1996, 68, 1735-1741. (34) Krull, U. J.; Heimlich, M. S.; Kallury, K. M. R.; Piunno, P. A. E.; Brennan, J. D.; Brown, R. S.; Nikolelis, D. P. Can. J. Chem. 1995, 73, 1239-1250. (35) Rothe, U.; Aurich, H. Biotechnol. Appl. Biochem. 1989, 11, 1830. (36) Naumann, R.; Jonczyk, A.; Kopp, R.; van Esch, J.; Ringsdorf, H.; Knoll, W.; Graber, P. Angew. Chem., Int. Ed. Engl. 1995, 34, 20562058. (37) Ti Tien, H. Bilayer Lipid Membranes (BLM): Theory and Practice; Marcel Dekker; New York, 1974.
S0743-7463(97)01123-2 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/14/1998
Tethered Lipid Bilayer Membranes
perturbation. However, the classical BLM has the advantage of possessing an essentially unlimited ionic reservoir on each side of the bilayer membrane and has therefore been used extensively as a model system for studying the electrical properties of lipid membranes and incorporated membrane ionophores. Supported BLMs have been formed onto glass or silica onto unfunctionalized metal surfaces,16,25,30,38 surfaces,11-13,26,29,33 or onto self-assembled alkanethiol monolayers.10,14,18,19,28,31 Methods for the bilayer formation have included the Langmuir-Blodgett technique,11,25-27,29,31 vesicle fusion onto the substrate,15,18,19,25,31,32 spontaneous thinning of lipid/decane mixtures,12-14,28,31,33 and adsorption of charged lipids onto oppositely charged surfaces.10,31 The main drawback of the sBLMs produced to date is that they lack a well-defined ionic reservoir on both sides of the membrane. This limits the utility of these structures in studying membrane transport functions of natural or synthetic ion carriers and channels. Examples of sBLMs with hydrophilic groups between the substrate and the membrane have been reported.15,17,35,36 However these reports deal largely with producing a space between the solid substrate and the membrane in order to accommodate the extramembrane domains of large membrane proteins, not with providing or characterizing an ionic reservoir.35,36 Lang17 does report the formation of an ionic reservoir if imperfect, chemisorbed monolayers are used to form the initial self-assembled monolayer. However, the membranes appear to be noninsulating (specific resistances of the membranes are ∼104 Ω cm2 ), and the process of producing the reservoir-forming defects appears uncontrollable. In order to further develop the scientific and practical potential of bilayer membranes in biomimetic chemistry and biophysics, we have set out to produce a tethered lipid bilayer that possesses the following characteristics: (a) a fluid yet highly insulating lipidic core, (b) a functional, well-defined ionic reservoir on each side of the membrane, (c) a high degree of mechanical, chemical, and biochemical stability, (d) accessibility to electrical measurements, and (e) ease and reliability of production. We wish to report a tBLM system which closely resembles the classical BLM in terms of thickness, ionophore incorporation, and conduction characteristics and yet is more stable and easier to produce than current sBLMs.
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Figure 1. Schematic representation of the tethered bilayer membrane where (A) is the mobile lipid that makes up the bulk of the membrane, (B) is the reservoir lipid that defines the ionic reservoir and tethers the membrane to the gold surface, (C) is the spacer molecules used to laterally space the reservoir lipids, and (D) is the valinomycin ionophore used to modulate the membrane conductivity. Scheme 1a
Results and Discussion Structure and Synthesis of Membrane Components. The basic tBLM structure is shown schematically in Figure 1. The mobile lipid (A) forms the bulk of the bilayer membrane. The hydrophilic portion of the reservoir lipid (B) defines the ionic reservoir between the gold electrode and the bilayer membrane. The hydrophobic portion of the reservoir lipid incorporates into the bilayer membrane and thus tethers the membrane to the electrode surface. Reservoirs of approximately 0, 20, 40, and 60 Å length were investigated. An unsymmetrical (benzyl alkyl) disulfide was used to attach the reservoir lipid to a gold surface. The thiobenzyl group was essential in laterally spacing the hydrophilic reservoir and providing an ionic reservoir. Spacer molecules (C) were used to further control the lateral spacing between the reservoir lipids. The spacer molecules are small, hydrophilic disulfide-containing molecules such as dithiodiglycolic acid 16. The potassium(38) Ariga, K.; Okahata, Y. J. Am. Chem. Soc. 1989, 111, 56185622.
a (a) (i) thiourea, ethanol, reflux, (ii) OH- ; (b) BnSCl, DCM, room temperature; (c) succinic anhydride, pyridine, room temperature, 24 h; (d) tetraethylene glycol, DCC, DMAP, DCM, room tempterature, 48 h; (e) 4, 6, or 8, DCC, DMAP, DCM, room temperature, 24 hr; (f) triphenyl phosphine, ethanol, room temperature, 48 h.
specific valinomycin ion carrier (D) was used to modulate the conductivity of the membrane and investigate the function of the ionic reservoir. The synthesis and structure of the reservoir lipids is shown in Scheme 1. The utility of using gold-sulfur interactions to produce self-assembled monolayers (SAMs)
650 Langmuir, Vol. 14, No. 3, 1998
of alkanedisulfides on gold surfaces is well established.1-7 In order to use the same methodology to form SAM’s of the reservoir lipids on the gold electrode, a disulfide group was incorporated into the reservoir lipid structure. The hydrophilic ion reservoir is formed from tetraethylene glycol units joined via succinate esters. Through joining one, two, or three of these units together, ionic reservoirs of varying lengths could be readily produced. Finally, the phytanyl groups of the reservoir lipid form part of the lipid bilayer membrane and tether the lipid membrane system onto the solid substrate. Thus, the monotosylate of tetraethylene glycol39 1 was reacted with thiourea in ethanol to give the thiouronium salt, which was hydrolyzed with sodium hydroxide to give the thiotetraethylene glycol 2. Thiotetraethylene glycol 2 was reacted with benzylsulfenyl chloride (generated from benzylthiol and N-chlorosuccinimide) to give benzyl tetraethylene glycol disulfide 3. Reaction with succinic anhydride gives the hemisuccinate ester 4. Further elaboration of 4 involves the successive ester formation with tetraethylene glycol groups in the presence of DCC and a catalytic amount of DMAP to yield compound 5, followed by reaction with succinic anhydride to give compound 6. The tetraethylene glycol adduct 7 and the hemisuccinate adduct 8 were then produced in analogous fashion. The final step in the synthesis of the reservoir lipids was coupling of phytanol to the reservoir component using DCC/DMAP. Phytanol40 9 was produced by hydrogenation of commercially available phytol over Raney Nickel at 1 atm H2 as a 3R/S mixture of diastereoisomers. Coupling of phytanol 9 to the “single-length” reservoir (i.e. made up of one tetraethylene glycol unit plus one hemisuccinate ester) 4 gives the single-length reservoir phytanyl lipid 10 (SLP); coupling to reservoir 6 gives the “double-length” reservoir phytanyl lipid 11 (DLP); and coupling to reservoir 8 gives the “triple-length” phytanyl reservoir lipid 12 (TLP). The thiobenzyl group of the reservoir lipids was incorporated into the molecules as a means of spacing the tetraethylene glycol chains apart during SAM formation. It was thought that close packing of the tetraethylene glycol chains would restrict the ion reservoir capacity. In order to confirm this, the thiobenzyl group was removed by reduction of the DLP reservoir lipid 11 with triphenylphosphine to give the reservoir lipid thiol 13. The mobile lipids that are generally incorporated into the tBLM are two types of glycero-ether lipid, namely the 2,3-di-O-phytanyl-sn-glycerol 14 (DPG)41 and the corresponding 2,3-di-O-phytanyl-sn-phosphatidylcholine lipid 15 (DPEPC).41 The use of phytanyl groups improves both the chemical stability and the fluidity over those of conventional unsaturated hydrocarbons. Similarly, ether groups, used in place of the more labile ester groups, are resistant to hydrolysis and enzyme attack. Membrane Formation. Formation of the bilayer membrane was achieved in a two-step process. In the first step, the self-assembly properties of the sulfur-gold interaction were used to produce SAM’s of the reservoirforming components. This involved immersing freshly evaporated gold-coated glass microscope slides in an ethanolic solution of SLP, DLP, or TLP reservoir lipid 10, 11, 12, and spacer molecule 16 for 24 h. Subsequently, (39) Markovskii, L. N.; Rudkevich, D. M.; Kal’chenko, V. I.; Tsymbal, I. F. Zh. Org. Khim. 1990, 26, 2425-2433. (40) Jellum, E.; Eldjarn, L.; Try, K. Acta Chem. Scand. 1966, 20, 2535-2538. (41) Yamauchi, K.; Doi, K.; Kinoshita, M.; Kii, F.; Fukuda, H. Biochim. Biophys. Acta 1992, 1110, 171-177.
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Figure 2. Schematic of the experimental setup used for the preparation and impedance measurement of the tBLM. A machined Teflon well is clamped tightly onto the gold-covered microscope slide by means of a metal surround that sits on the lower lip of the Teflon well. The internal diameter of the well defines an electrode area of 16 mm2. The impedance spectrometer is connected to the reference and counter electrodes by immersion in the aqueous buffer and to the gold surface in order to complete the circuit.
the gold slides were rinsed with ethanol and stored in ethanol at 4 °C. Immediately prior to use, the electrodes were dried in a stream of air and assembled into a Teflon cell, as shown in Figure 2. The second step in the formation of the membrane uses an in-situ solvent-dilution method of bilayer formation onto hydrophobic SAMs similar to that used by Miller.16 The method involves placing a small quantity of the bilayer-forming lipid, dissolved in a water miscible solvent such as ethanol, onto the SAM, followed by rapid and vigorous rinsing with excess aqueous solution. During the addition and rinsing of the surface with the aqueous solution the lipid/water phase of certain lipids selfassembles to form the tBLM and excess lipid is rinsed away. The prime advantage of this method is the simplicity and speed with which a functional bilayer membrane can be formed. Individual membranes can be produced in less than a minute and are immediately available for use. Impedance Characterization of the tBLM. Impedance spectroscopy was used to compare the solvent-dilution method of membrane formation discussed above with the more commonly employed methods of sBLM formation such as detergent dilution31 or vesicle adsorption onto SAMs.15,18,19,25,31,32 A number of reports10,11,16-19,31 have shown that impedance spectroscopy is a useful tool for the characterization of sBLMs. Thus the capacitances of SAMs and lipid bilayers can be obtained from the impedance at high frequency (eq 1).
|Z| ) 1/(2πfC)
(1)
Where |Z| is the modulus of the impedance in ohms, f is the frequency (in Hz) at which |Z| is measured, and C is the capacitance (in F). From the value of the capacitance the thickness of the hydrocarbon region can be estimated using eq 2
d)
0A C
(2)
where d is the thickness of the hydrocarbon region, 0 is the permittivity of free space, is the dielectric constant of the hydrocarbon membrane, A is the area of the membrane, and C is the capacitance. Although there is some uncertainty as to the actual value of for the
Tethered Lipid Bilayer Membranes
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Table 1. Comparison of Capacitance and Thickness Parameters of the tBLM and Solvent-Free BLMs capacitancea lipid
literature
C16SH only C18:1PC C20:1PC C22:1PC C24:1PC DPEPC DPEPC/DPG
1.00b 0.72 ( 0.02c N/A 0.57 ( 0.02c 0.48 ( 0.03c N/A N/A
C16SH +
lipidd
0.94 ( 0.05f 0.62 ( 0.02 0.57 ( 0.02 0.53 ( 0.02 0.48 ( 0.02 0.58 ( 0.03 0.50 ( 0.02
thicknessg (Å) DLP +
lipide
0.68 ( 0.03 0.62 ( 0.02 0.60 ( 0.03 0.56 ( 0.02 0.62 ( 0.02 0.52 ( 0.02
literature
C16SH + lipid
DLP + lipid
18.6b 25.8 ( 0.8c N/A 32.7 ( 1.3c 38.6 ( 2.2c N/A N/A
19.7 ( 0.5f 30.1 ( 1.0 32.6 ( 1.1 35.2 ( 1.3 38.6 ( 1.6 32.1 ( 1.6 37.1 ( 1.6
27.4 ( 1.2 29.4 ( 1.0 31.0 ( 1.4 33.6 ( 1.2 30.0 ( 1.0 35.8 ( 1.4
a Membrane capacitance (C , µF/cm2) calculated from the impedance at 1000 Hz. b Value for the hexadecanethiol monolayer from ref m 14. c Value for solvent-free BLMs from ref 43. dCapacitance of the bilayer formed on a hexadecanethiol SAM. e Capacitance of the bilayer formed on a DLP reservoir lipid 11 SAM. f Value of the hexadecanethiol monolayer. g Calculated from capacitance using ) 2.1, error ) (standard deviation.
hydrocarbon region of a bilayer, a reasonable assumption10,14,27,36,37 for the value for the dielectric constant of 2.1 is generally used in calculations. As a reference for subsequent measurements, the capacitance of monolayers of hexadecanethiol (HD) was determined from the impedance at 1000 Hz. The value of 0.94 ( 0.05 µF/cm2 obtained compares favorably with the literature value 1.0 µF/cm2.14,42 Calculations of the thickness of the monolayer therefore give values of 19.7 ( 0.5 Å for our system compared to values of 18.6 Å for the literature value derived from electrical measurements. Theoretical values, obtained from a structural computer model,44 for an all-trans hexadecanethiol SAM tilted at 20-30° from the normal give length values of 18.6-20.3 Å. As stated above, in order to validate the methodology of tBLM formation, monolayers of various mono-unsaturated diacylphosphatidylcholine lipids (i.e. C18:1PC, C20: 1PC, C22:1PC, and C24:1PC lipids) were formed onto SAMs of hexadecanethiol. The impedance at 1000 Hz was measured, and from this data the capacitance and the bilayer thickness were calculated using eqs 1 and 2 (Table 1). By subtracting the constant value of 19.7 Å of the hexadecanethiol monolayer from the bilayer value, the thickness of the monolayer of the phosphatidylcholine lipid was calculated. As a valid comparison, the thickness value obtained from capacitance measurements for a corresponding half bilayer produced by the solvent-free BLMs as determined by Benz43 was used. Figure 3 shows the results from the two different methods. As can be seen, there is excellent agreement between the data. The value of 12.2 Å obtained by Fettiplace45 for a half bilayer from egg lecithin (a mixture of C16 and C18 unsaturated PC lipids) in a solvent-free BLM is also in good agreement with the values obtained. From the slope of the data of the thickness of the unsaturated PC lipids against the number of methylene units in Figure 3 an increase in thickness of 1.41 ( 0.08 Å per CH2 is obtained. This may be compared with a value of 1.4 Å per CH2 obtained by Benz43 for solvent-free BLMs and 1.3-1.5 Å per CH2 obtained by Porter42 for alkanethiol SAMs. Benz and others43 have noted that there is a significant difference between bilayer thickness values obtained in the presence or absence of a hydrocarbon solvent such as decane. The presence of decane leads to much thicker membranes by electrical measurements; for example, half(42) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559-3568. (43) Benz, R.; Frohlich, O.; Lauger, P.; Montal, M. Biochim. Biophys. Acta 1975, 394, 323-334. (44) Chem 3D; CambridgeSoft Corporation: Cambridge, MA. (45) Fettiplace, R.; Andrews, D. M.; Haydon, D. A. J. Membr. Biol. 1971, 5, 277-296.
Figure 3. Lipid thickness as determined from capacitance measurements. Values for the monolayer of diacyl PC lipid membranes formed on HD SAM’s (O) were calculated by subtracting the measured thickness of the HD SAM (19.7 Å) from the total bilayer thickness. This is compared with the values obtained for the half-bilayer thickness of solvent-free BLMs of diacyl PC lipids (0).43
bilayer thicknesses of 23.9 to 25.8 Å have been obtained for C18:1PC lipids in the presence of decane46,47 compared to 12.9 ( 0.4 Å obtained in solvent-free BLMs by Benz.43 Other authors have also found that larger specific capacitances are obtained in the absence of alkanes on sBLM systems.10,14,18,19,31,48 For instance, Plant18,19 determined specific capacitance values for 1-palmitoyl-2oleoylphosphatidylcholine assembled onto an octadecanethiol SAM by vesicle adsorption and obtained values of 0.64-0.68 µF/cm2 in the absence of decane solvent. In the presence of decane the initial capacitance was found to be 0.42 µF/cm2, which over a period of 16 h increased to a value of 0.60 µF/cm2. The question as to whether these larger capacitances (smaller calculated membrane thickness values) for solvent free systems reflect the real thickness of the hydrocarbon region of the bilayer membranes or whether they are due to, for instance, an increased value of due to water penetration into the hydrocarbon region has not been totally resolved. Formation of the bilayer membrane on a SAM of DLP reservoir lipid 11 gave similar results to those obtained on the hexadecanethiol SAM’s (Table 1). The thickness of the bottom half of the bilayer is expected to be dominated (46) Requena, J.; Haydon, D. A. Fed. Proc. 1974, 33, No. 5 Part II. (47) Cherry, R. J.; Chapman, D. J. Mol. Biol. 1969, 40, 19-32. (48) Montal, M.; Mueller, P. Proc. Natl. Acad. Sci. U.S.A. 1972, 69, 3561-3566.
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by the phytanol chains of the DLP reservoir lipid 11, although we assume that incorporation of the mobile lipid into the bottom layer also occurs. The exact thickness of the bottom half of the bilayer formed on a DLP reservoir lipid 11 SAM in the presence of the mobile lipid is therefore not readily obtainable from these measurements. A least squares fit to the data shown in Table 1, for the PC lipid bilayer membrane formed on DLP reservoir lipid 11, gives a value of 1.02 ( 0.07 Å per CH2. The smaller value compared with results obtained on a hexadecanethiol SAM (1.41 ( 0.08 Å per CH2) presumably reflects a more disordered, fluid system due to the phytanol reservoir lipid producing slightly thinner membranes. Measurements of two phytanyl-based lipids showed that the apparent thickness of DPEPC 15 was approximately the same as that of the C20:1PC lipid, whereas a 7:3 mixture of DPEPC/DPG was equivalent to a C22:1PC to C24:1PC lipid (Table 1). The DPEPC/DPG lipid mixture was developed in order to improve the fluidity, the seal, and the stability of the membrane.8 For instance, the current stability of a tBLM formed with DPEPC/DPG is >8 weeks at room temperature. Membrane Conduction Characteristics. Impedance spectroscopy was also used to characterize the conductance properties of the system. A simple but extremely useful tool for the characterization of the system was the use of valinomycin49 ionophore incorporated into the tBLM. This potassium-selective ion carrier enables observation of potassium-mediated ion flux through the membrane. The conductance level can be used to characterize the ion reservoir capacity of the tBLM. Furthermore, simply by changing the cation from potassium to sodium in the buffer, it is possible to check the membrane integrity during the course of an experiment. Other ion carriers such as nonactin and lasolocid as well as ion channels such as gramicidin can also be incorporated into the tBLM system and will be reported in a separate publication. Figure 4a shows a typical Bode plot of a conducting (i.e. in the presence of valinomycin) and a nonconducting (i.e. in the absence of valinomycin) tBLM in an aqueous bathing solution containing 10 mM KCl/90 mM NaCl. The tBLM was produced from a mobile lipid consisting of a 7:3 ratio of DPEPC/DPG formed on a SAM of DLP reservoir lipid 11. For the conducting tBLM, the DPEPC/DPG lipid solution contained valinomycin in a ratio of 200:1 total mobile lipid to valinomycin. Unless otherwise specified, all further experiments are carried out on this system. The essential nature of the hydrophilic reservoir lipid in providing an ionic reservoir next to the gold electrode can be most readily seen by comparison of the tBLM with the conductivity of a supported bilayer membrane formed onto a hexadecanethiol first layer (Figure 4c). In the case of the supported bilayer there is of course no ionic reservoir except through adventitious defect sites in the SAM;10,17 hence, there is minimal change in conductivity on addition of KCl. Three experimentally derived parameters are used to characterize the tBLM. These are (i) the membrane capacitance parameter (CM) of a valinomycin-free membrane as measured from the impedance at 1000 Hz and calculated using eq 1, (ii) the Helmholtz capacitance parameter (CH) of a conductive membrane (containing valinomycin) as measured from the impedance at 0.1 Hz and calculated using eq 1, and (iii) the admittance (Yφ min) at which the phase relationship (φ) between the excitation (49) Hilgenfeld, R.; Saenger, W. Top. Curr. Chem. 1982, 101, 1-82.
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and the resultant current is minimum (see arrow a in Figure 4a and b). The values for CM derived from the impedance at 1000 Hz are a reasonable approximation of the total capacitance as long as the background leakage conductivity of the tBLM is low and the slope of the modulus of impedance versus frequency is -1 on a log-log plot. As can be seen in Figure 4a (in the absence of valinomycin ionophore), the tBLM system satisfies these criteria. Similarly, the value for CH derived from the admittance at 0.1Hz is a reasonable approximation of the capacitance due to the ionic double layer next to the gold surface for a conductive tBLM system (e.g. Figure 4a in the presence of valinomycin). A simplified electrical analogue model for the tBLM system is shown in Figure 4d. The analogue model shown in Figure 4d is used as a simple conceptual model of the tBLM system for the experimentally derived values of CM, CH, and Yφ min. In this model the membrane capacitance (CM) is bypassed by a variable ohmic resistance (RM: representing the ionophore-mediated ion flux as well as any background ion leakage). The membrane capacitance is in series with an effective Helmholtz capacitance (CH) due to the ionic double layer next to the gold surface. The total capacitance of a nonconductive tBLM is therefore C ) CMCH/(CM + CH). Using a least squares fit of the model shown in Figure 4d, values of CH ) 5.1 µF/cm2, CM ) 0.56 µF/cm2, and RM > 5 × 106 Ω/cm2 are calculated. This may be compared with CH ) 5.0 µF/cm2 derived directly from the impedance at 0.1 Hz for the conducting membrane and CM ) 0.52 µF/cm2 derived directly from the impedance at 1000 Hz for a valinomycin-free (nonconducting) membrane. As the CH (∼5 µF/cm2) is relatively large compared to the CM (∼0.5 µF/cm2), its contribution to the overall capacitance is Li+. This reflects the expected selectivity for valinomycin.49 In the method of assembly of the tBLM described here, the ionophore is included in the lipid matrix during the formation of the tBLM, whereas most classical BLM measurements are made by introducing the ionophore into the aqueous bathing solution. BLM measurements have shown that a concentration of 10-7 M valinomycin in the aqueous bathing solution yields final lipid/valinomycin ratios in the range 100:1 to 600:1.50,52 Although (52) Boheim, G.; Hanke, W.; Eibl, H. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 3403-3407.
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caution is required when comparing different systems it was interesting to note that the two methods of preparation (i.e. premixing the valinomycin with the mobile lipid mixture at a 200:1 ratio or incorporation of valinomycin into the lipid membrane from an aqueous solution at 10-7 M valinomycin) gave approximately equivalent results for the tBLM system. Thus tBLMs were prepared without valinomycin, followed by exchange of the aqueous bathing solution with a 10-7 M valinomycin in 10 mM KCl/90 mM NaCl solution. After equilibration (5-60 min) the conduction ((1.1 ( 0.2) × 10-4 S/cm2) was very similar to the conduction of tBLMs ((0.7 ( 0.1) × 10-4 S/cm2) made from the valinomycin premixed with DPEPC/DPG, using a lipid to valinomycin ratio of 200:1. Effect of dc Bias Voltage on the Conductance. An intriguing effect of having a restricted reservoir was found when applying a dc bias voltage of between (500 mV superimposed on the 50 mV ac excitation. This dc bias influenced both the conductance and the Helmholtz capacitance (as defined above) of the system. Figure 7a shows the effect of the dc bias voltage on the conductance of SLP, DLP and TLP reservoir lipids 10, 11, and 12 at 10 mM KCl/90 mM NaCl using the standard DPEPC/DPG/valinomycin fluid lipid layer. A positive voltage denotes that the gold electrode is positive relative to the reference electrode. As can be seen, at positive voltages the conductance of the system is reduced dramatically, falling to the level of detection. Thus for the SLP 10, reduction in conductance occurs between -100 and +100 mV, for DLP 11, it occurs between 0 and +200 mV, and for TLP 12, it occurs between +100 and +300 mV. That is, larger reservoirs are less sensitive toward the dc bias voltage than shorter reservoirs. At dc bias voltages more negative than the respective threshold values, the conductance is constant. The effect of switching the conductivity of the tBLM system by reversing the dc bias voltage is fast (>90% change in 99.99% pure. Immediately after gold deposition the electrodes were removed from the evaporator and immersed into an ethanolic solution containing the reservoir lipids. Unless otherwise stated the concentration of the reservoir lipid 10, 11, or 12 was 0.25 mM and immersion time was 24 h. When the spacer molecule 16 was added to the solution, the concentration of the reservoir lipid was kept constant at 0.25 mM with 0-0.25 mM spacer 16 added. After adsorption of the SAM, the electrodes were rinsed with ethanol and stored at 4° in ethanol until ready for use, generally within 3 days. Prior to use, the electrodes were briefly dried in air and a Teflon well was clamped onto the gold-coated slide, as shown in Figure 2. The diameter of the working electrode as defined by the teflon well was approximately 16 mm2. The volume of the well was approximately 0.2 mL. The tBLM formation was completed by adding an ethanolic solution of lipid (5 µL, 10 mM) to the electrode surface, followed by vigorous rinsing with two times 0.5 mL of 0.1 M NaCl using a syringe. The tBLMs were left covered with 0.1 M NaCl solution and were ready for immediate use. The lipids used were C18: 1PC, C20:1PC, C22:1PC, C24:1PC, DPEPC, or DPEPC/DPG (7:3 ratio). For experiments with valinomycin ionophore, the valinomycin was generally premixed into the ethanolic lipid solution at ratios of between 20:1 and 20 000:1 total lipid/valinomycin. Buffer changes were carried out by exchanging the total well volume several times with the new buffer using a syringe. Potassium chloride solutions at
