© Copyright 1998 American Chemical Society
AUGUST 18, 1998 VOLUME 14, NUMBER 17
Letters Ion-Selective Lipid Bilayers Tethered to Microcontact Printed Self-Assembled Monolayers Containing Cholesterol Derivatives A. Toby A. Jenkins, Richard J. Bushby, Neville Boden, Stephen D. Evans,* Peter F. Knowles, Quanying Liu, Robert E. Miles, and Simon D. Ogier Centre for Self-Organising Molecular Systems, University of Leeds, Leeds, LS2 9JT, U.K. Received May 15, 1998 Microcontact printing has been used to prepare patterned self-assembled monolayers (SAMs) of cholesterylpolyethylenoxy thiol. These patterned SAMs were used as supports for the formation of integral supported lipid bilayers. Biofunctionality was confirmed by addition of valinomycin ionophores and gramicidin ion channels. Impedance spectroscopy of the resulting bilayer structures, incorporating valinomycin and the gramicidin, showed the expected ion selectivity.
The attachment of a lipid bilayer to a solid substrate, in such a way that it retains its biomimetic properties, has generated an increasing interest over recent years.1-17 * To whom correspondence should be addressed. E-mail:
[email protected]. (1) Stelzle, M.; Sackmann, E. Biochim. Biophys. Acta 1989, 981, 35142. (2) Brink, G.; Schmitt, L.; Tampe´, R.; Sackmann, E. Biochim. Biophys. Acta 1994, 1196, 227-230. (3) Ku¨hner, M.; Tampe´, R.; Sackmann, E. Biophys. J. 1994, 67, 217226. (4) Stelzle, M.; Weismu¨ller, G.; Sackmann, E. J. Phys. Chem. 1993, 97, 2974-2981. (5) Plant, A.; Gueguetchkeri, M.; Yap, W. Biophys. J. 1994, 67, 11261133. (6) Plant, A.; Brigham-Burke, M.; Petrella, E.; O’Shannessy, D. Anal. Biochem. 1995, 226, 342-348. (7) Terrettaz, S.; Stora, T.; Duschl, C.; Vogel, H. Langmuir 1993, 9, 1361-1369. (8) Duschl, C.; Liley, M.; Corradin, G.; Vogel, H. Biophys. J. 1994, 67, 1229-1237. (9) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197-210. (10) Lindholm-Sethson, B. Langmuir 1996, 12, 3305-3314. (11) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307-316. (12) Nakashima, N.; Yamaguchi, Y.; Eda, H.; Kunitake, M.; Manabe, O. J. Phys. Chem. 1997, 101, 215-220. (13) Nelson, A. Langmuir 1996, 12, 2058-2067. (14) Sackmann, E. Science 1996, 271, 43-48. (15) Williams, L. M.; Evans, S. D.; Flynn, T. M.; Marsh, A.; Knowles, P. F.; Bushby, R. J.; Boden, N. Langmuir 1997, 13, 751-757. (16) Williams, L. M.; Evans, S. D.; Flynn, T. M.; Marsh, A.; Knowles, P. F.; Bushby, R. J.; Boden, N. Supramol. Sci. 1997, 4, 513-517. (17) Cheng, Y.; Boden, N.; Bushby, R. J.; Clarkson, S.; Evans, S. D.; Knowles, P. F.; Marsh A. Langmuir 1998, 14, 839-844.
In principle, it should be possible to construct biosensors that are highly analyte specific, utilizing the specificity found naturally in cellular biology,18 in effect utilizing Nature’s evolutionary designed biosensing systems. There are several problems in achieving this. First, one needs to create a lipid bilayer, on a solid surface, that is sufficiently flexible and defect free such that ion channel activity can actually be measured. Second, the lipid membrane must be in a physical state in which incorporated biological molecules function as if in a natural system. Third, one needs to entrap water on the inner surface of the membrane to facilitate the incorporation of transmembrane proteins. Experiments on randomly selfassembled mixtures of cholesterylpolyethylenoxy thiol (CPEO3) and mercaptoethanol on gold have shown that although supported bilayers were formed, they were not sufficiently blocking to be characterized by electrochemistry. The move to using microcontact printed SAMs as described here has two perceived benefits. First, the area over which an integral bilayer has to span is reduced significantly. Second, as suggested by Groves et al.,19 (18) Darszon, A.; Vandenberg, C. A.; Sco¨nfeld, M.; Ellisman, M. H.; Spitzer, N. C.; Montal, M. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 239243. (19) Groves, J. T.; Wulfing, C.; Boxer, S. G. Biophys. J. 1996, 71, 2716-2723. (20) Boden, N.; Bushby, R. J.; Clarkson, S.; Evans, S. D.; Knowles, P. F.; Marsh A. Tetrahedron 1997, 53, 10939-10952.
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4676 Langmuir, Vol. 14, No. 17, 1998
transmembrane proteins will self-locate in supported bilayer structures on micropatterned substrates. In this communication, we demonstrate that it is possible to form phospholipid bilayers supported on a patterned, lipophilic SAM.8,15-17 As in our previous work, the tethered bilayers have been characterized by surface plasmon resonance and atomic force microscopy. We now describe how bilayer formation can be monitored using ac impedance spectroscopy, which was also used to demonstrate the ion transport selectivity of the ionophore valinomycin and the ion channel forming peptide, gramicidin. A lipophilic tether molecule consisting of cholesterol derivatized with a thiol-terminated trisethylenoxy chain (Figure 1), was synthesized to anchor the lipid to a gold substrate.20 A patterned CPEO3 SAM, with 15 µm × 15 µm square holes, was produced on the gold21 using microcontact printing (µCP).22 The “bare” wells thus created were subsequently filled with a mercaptoethanol SAM (Figure 1). All SAM solutions were 1 mM in HPLC grade dichloromethane. Supported bilayers of egg-phosphatidyl (egg-pc) choline were formed by the incubation of large unilamellar vesicles, produced by extrusion, with the SAM patterned substrate.11 Immediately before addition of the vesicles, the micropatterned SAM was measured by impedance spectroscopy. On addition of vesicles, during the incubation, impedance measurements were made every minute for the first 10 measurements (when the impedance changes most quickly) and then at longer intervals for a period of up to 18 h. All impedance measurements were made at room temperature, 19 °C, well above the egg-pc phase transition temperature of around 1 °C. Each (21) The gold substrate was formed by evaporation of 5 nm of chromium followed by 150 nm of gold onto high quality glass microscope slides at a pressure of 2 × 10-6 mb. The gold substrate (working electrode) is connected by a 1 mm wide gold track to the instruments leads. (22) Wilbur, J. L.; Kumar, A.; Biebuyck, H. A.; Kim, E.; Whitesides, G. M. Nanotechnology 1996, 7, 452-457. (23) The impedance of the bare gold, in the absence of a redox couple, is dominated by the double layer capacitance of the substrate, with an experimentally determined value of 40 µF cm-2. This result is higher than that typically quoted for smooth bare gold and is probably due to the roughness of the surface.24 The adsorption of a single component CPEO3 from solution (nonstamped) onto a bare gold substrate presents a blocking SAM. The capacitance of such a monolayer derives from the dielectric properties of the SAM and has a value of 0.85 µF cm-2 in 0.1 M KCl. Measurements on single-component stamped (nonpatterned) CPEO3 SAMs gives a capacitance of 2.5 µF cm-2, significantly higher than the nonstamped CPEO3 SAMs. This higher capacitance can be readily ascribed to the presence of defects in the stamped films. Assuming the defect regions exhibit the same capacitance as bare gold would equate to a 4% defect concentration (i.e., R ) 0.96). The adsorption of a pure mercaptoethanol SAM on gold gave capacitance values of around 9.2 µF cm-2 in 0.1 M KCl. Theoretically, a perfect blocking SAM of mercaptoethanol would have a capacitance of 4 µF cm-2 (assuming a thickness of 5 Å and r to be 2.25). Hence, an approximate calculation would suggest that 70% of the available surface sites must be blocked (θ ) 0.7). The micropatterned SAM consists of two components: the CPEO3 system and mercaptoethanol. The capacitance of these SAMs derives from three separate contributions: the areas of CPEO3 SAM, areas of mercaptoethanol SAM, and areas of gold at the bottom of defects. Typical capacitance values of such patterned SAMs are close to 6.0 µF cm-2 in 0.1 M KCl. These experimentally determined values are a factor of 2.4 greater than those predicted theoretically (based on the values obtained for the stamped single component surfaces). Note, this calculation assumes that all defects within the CPEO3 layer are “backfilled” with mercaptoethanol. However if only 90% of these defects were back filled, then we would expect to obtain a value close to that observed. Unilamellar egg-pc lipid vesicles made by extrusion were added to single component unstamped (solution adsorbed) CPEO3 SAMs, and the impedance of the system was measured over a period of 3 h. The final capacitance of the SAM and lipid monolayer was 0.58 µF cm-2. This is close to the value predicted theoretically for an egg-pc monolayer-CPEO3 of around 0.44 µF cm-2, suggesting that the SAMlipid system is blocking. Measurements of egg-pc vesicles unrolling on the single component stamped CPEO3 SAMs give a final capacitance of 0.95 µF cm-2 suggesting a higher defect concentration in this system.
Letters
impedance measurement was made over the frequency range 50 kHz to 300 mHz, with a 20 mV peak-peak ac signal at the open circuit potential of the cell. The lipid was dispersed in 0.1 M BaCl2, which was used as the electrolyte to follow the formation of the bilayer. For ion selectivity measurements, the electrolyte was exchanged with four times the cell volume of the new electrolyte. The molar concentration of the electrolyte was maintained at 0.1 M in each case. To understand the impedance spectra of the lipidpatterned SAM system, the impedance of the “individual” components making up the system were independently measured, where possible. These results are presented in footnote 23. The capacitance values were determined by fitting the high-frequency (50 kHz to 1 kHz) region of the impedance spectra. A semicircle (modeling a series RC circuit) was fitted to the data points obtained by plotting real admittance, divided by frequency (Y′/ω), against imaginary admittance, divided by frequency (Y′′/ ω) (Figure 2b), as described by Lingler et al.25 Using the Solartron Z plot software to fit data gave estimated capacitance value errors of 4% or less. The adsorption kinetics of egg-pc lipid on the patterned SAM, as followed by the change in capacitance, was similar to those reported by Lingler et al.25 The initial rapid decrease in capacitance is attributed to the adsorption and spontaneous rupture of the vesicles on the surface. The slower decay of the capacitance for times greater than 20 min is attributed to bilayer spreading and selforganization of the lipids on the surface. Such a two-step adsorption process is in agreement with SPR results on vesicle interactions with nonpatterned SAMs, carried out by ourselves and other workers.15,25 The final capacitance of the lipid-SAM system was between 0.9 and 1.0 µF cm-2. The lipid-coated CPEO3 functionalized regions (region 1, Figure 1b) may be represented as a capacitance, C1 (equation 2a), which can be considered as having contributions from an ideal film plus defects (C1 has a value of 0.95 µF cm-2).23 In the “well” regions (region 2, Figure 1b) the capacitance C2, given by eq 2b, can be considered to have two main contributions, one from the bilayercovered portions of the surface, Cb, and the second originating from the mercaptoethanol-coated substrate, Cs. If φ represents the surface coverage of CPEO3 regions, then (1 - φ) will represent the area of the “wells” and the net capacitance is given by eq 1.
Ctotal ) φC1 + (1 - φ)C2
(1)
In order minimize the number of parameters introduced in our model, we assume that the fractional lipid bilayer coverage in the mercaptoethanol regions and the fractional lipid monolayer coverage in the CPEO3 regions are the same, and we denote this as β. Thus the capacitance, C1, has three contributions: the first is from the lipid monolayer (Clip) in series with the CPEO3 SAM (CSAM), the second the CPEO3 SAM without adsorbed lipid (CSAM), and the third from defects in the CPEO3 SAM, the gold double layer capacitance, (1 - R)Cau, where R is the coverage of CPEO3 SAM. Equation 2b describes the capacitance of the lipid bilayer adsorbed on the mercaptoethanol SAM, region 2. In this case the capacitance has contributions from the bilayer (Cb) in series with a defect containing mercaptoethanol SAM (Cmer), where θ repre(24) Piela, B.; Wrona, P. K. J. Electroanal. Chem. 1995, 388, 69-79. (25) Lingler, S.; Rubinstein, I.; Knoll, W.; Offenha¨usser, A. Langmuir 1997, 13, 7085-7091.
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Langmuir, Vol. 14, No. 17, 1998 4677
Figure 1. (a) Construction of patterned SAM, formation of egg-pc lipid bilayer on a cholesterylpolyethylenoxy thiol stamped SAM on gold. (b) The graphical and electrical models of the CPEO3-lipid system.
sents the surface coverage of the mercaptoethanol and the capacitance contribution from the defects is given by (1 - θ)Cau.
C1 ) β(Cchol-1 + Clip-1)-1 + (1 - β)Cchol where:
Cchol ) RCsam + (1 - R)CAu C2 ) β(Cb-1 + Cs-1)-1 + (1 - β)Cs
where:
(2a)
(2b)
Cs ) θCmer + (1 - θ)CAu
The total capacitance Ctotal can be represented by the equivalent circuit shown in Figure 1b. This decreases linearly with increasing lipid coverage β, eq 3.
Ctotal ) φC1 + (1 - φ)(β(Cb-1 + Cs-1)-1 + (1 - β)Cs) (3) Using “ideal” values we estimate Ctotal to be 0.47 µF cm-2.
Experimentally, for lipids adsorbed on microcontact printed SAMs, we find values between 0.9 and 1.0 µF cm-2, which would correspond to a β (lipid coverage) variation between 0.84 and 0.88. Using the Boukamp impedance analysis software,26 the expected impedance response for the theoretical circuit shown in Figure 1 has been simulated. It was found that for sufficiently high values of Rlip, Rd, and Rval (>200 kΩ cm-2) only one time constant on the Nyquist (Z′, -jZ′′) plot was observed, this is in agreement with our experimental data, Figure 2b. The resistance of the system was determined by fitting the impedance spectrum, obtained between 50 kHz and 300 mHz, to a parallel RC circuit. The fitting procedure extrapolated the data plotted in the complex plane (Z′ vs -jZ′′) to the real axis (Z′), Figure 2a. This provides an estimate of the resistance of the system at very low frequencies ( Cs+ > Li+ > Na+ > Ba2+; thus we would expect the conductance of a bilayer containing functional valinomycin to decrease in the same order.27 Figure 3 shows the experimentally determined conductances for bilayers in the presence (circles) and absence (triangles) of valinomycin under 0.1 M KCl and 0.1 M BaCl2 solutions. The control experiment shows no selectivity for potassium or barium while in the presence of valinomycin we find that the conductance varies by a factor of 2. Experiments with the ion channel forming peptide gramicidin also show the expected selectivity, Cs+ > K+ > Na+ . Ba2+ (all with chloride anions at 0.1 M).28,29 Membranes were found to be stable for a minimum of 24 h. Figures 2 and 3 both show the successful formation of a lipid bilayer on microcontact printed SAMs and importantly, for future biosensor applications, the selective functionality of valinomycin within the bilayer. The latter implies that conductance is dominated by transport through the ionophore rather than defects induced in the bilayer. The combination of the CPEO3 molecules and microcontact printing has allowed us to develop a unique new method for supporting lipid bilayers (on solid supports), while retaining the biological functionality of incorporated ionophores. Acknowledgment. This work was supported by funding from the BBSRC and the Defence and Evaluation Research Agency (DERA). LA980581U
Figure 3. Ion selectivity for Ba2+ and K+ of a lipid bilayer (1) without valinomycin and (2) with 0.5 mol % valinomycin.
contribution to the impedance can be effectively ignored. As a result, we can write
(27) Steinem, C.; Janshoff, A.; Ulrich, W. P.; Sieber, M.; Galla, H. J. Biochim. Biophys. Acta 1996, 1279, 169-180. (28) Hill, B. Ionic Channels of Excitable Membranes, 2nd ed.; Sinauer Associates, 1992; pp 305-312. (29) Woolley, G. A.; Wallace, B. A. J. Membr. Biol. 1992, 129, 109136.
