Langmuir 2003, 19, 5567-5569
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Highly Electrically Insulating Tethered Lipid Bilayers for Probing the Function of Ion Channel Proteins Samuel Terrettaz, Michael Mayer,† and Horst Vogel* Institute of Biomolecular Sciences, Swiss Federal Institute of Technology Lausanne, CH-1015 Lausanne, Switzerland Received February 5, 2003. In Final Form: May 9, 2003 A method is presented to form gold-electrode-tethered lipid bilayers with exceptionally high electrical resistances. Electrical impedance spectroscopy is used to monitor the bilayer incorporation of a ligandgated ion channel protein and the modulation of its channel activity by the selective binding of an antibody. Due to the low defect density of the tethered membrane, the effect of a few channels can be resolved thus opening the way to single-channel experiments on this highly stable and versatile platform. In turn, a minute quantity of analyte, here antibodies, can be measured, which is of great interest for bioanalytics.
Ion-channel-forming proteins are indispensable in cellular signaling. The direct electrical measurement of ion channel activity by using planar (black) lipid membrane (BLM) and patch clamp technologies is of utmost importance to elucidate the function of ion channels in signal transduction processes and their role as central targets for medicaments. The classical technologies1 are timeconsuming and therefore not suited for an efficient investigation of a large number of either channel proteins or potential channel function modifying ligand molecules. Recent proposals to use array and chip technologies improve the efficiency of probing electrical ion channel activities.2 Electrical impedance measurements on artificial or native membranes tethered to electrodes offer in this context one of the most promising approaches. Tethered membranes show an exceptional mechanical and functional stability of weeks3 and were already used for detecting electrically a number of interesting ligandreceptor interactions.4 The sensitivity of such impedance measurements has however been severely limited by the relatively low electrical resistance of the tethered membranes. Here we report on the formation of tethered lipid bilayers with an unprecedented high electrical resistance of 7 MΩ * To whom correspondence should be addressed. Horst Vogel, EPFL-LCPPM, CH 1015 Lausanne, Switzerland. E-mail:
[email protected]. † Present address: Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St. (Box 53), Cambridge, MA 02138. (1) (a) Sakmann, B.; Neher, E. Single-Channel Recording; Plenum Press: New York, 1983. (b) Schlue, W. R.; Hanke, W. Planar Lipid Bilayers; Academic Press: London 1993. (2) (a) Bayley, H.; Cremer, P. S. Nature 2001, 413, 227-230. (b) Heyse, S.; Stora, T.; Schmid, E.; Lakey, J. H.; Vogel, H. Biochim. Biophys. Acta 1998, 1376, 319-338. (c) Fromherz, P.; Kiessling, V.; Kottig, K.; Zeck, G. Appl. Phys. A 1999, 69, 571-576. (d) Schmidt, C.; Mayer, M.; Vogel, H. Angew. Chem., Int. Ed. 2000, 39, 3137-3140. (e) Hennesthal, C.; Steinem, C. J. Am. Chem. Soc. 2000, 122, 8085-8086. (f) Jenkin, A. T. A.; Boden, N.; Bushby, R. J.; Evans, S. D.; Knowles, P. F.; Miles, R. E.; Ogier, S. D.; Scho¨nherr, H.; Vancso, G. J. J. Am. Chem. Soc. 1999, 121, 5274-5280. (3) (a) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197-210. (b) Plant, A. L. Langmuir 1999, 15, 5128-5135. (4) (a) Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. J.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580583. (b) Steinem, C.; Janshoff, A.; Von dem Bruch, K.; Reihs, K.; Goossens, J.; Galla, H.-J. Bioelectrochem. Bioenerg. 1998, 45, 17-26. (c) Stora, T.; Lakey, J.; Vogel, H. Angew. Chem., Int. Ed. 1999, 38, 389-392. (d) Terrettaz, S.; Ulrich, W.-P.; Vogel, H.; Hong, Q.; Dover, L. G.; Lakey, J. H. Protein Sci. 2002, 11, 1917-1925. (e) Naumann, R.; Baumgart, T.; Gra¨ber, P.; Jonczyk, A.; Offenha¨usser; A.; Knoll, W. Biosens. Bioelectron. 2002, 17, 25-34.
cm2, the subsequent detection of only a few synthetic ligand-gated ion channels (SLIC) incorporated in the tethered lipid bilayer, and finally the modulation of the channel activity by the selective binding of an antibody.5 These results are a major milestone toward measuring single ion channel gating kinetics on multiarray electrodes. Experimental Section Chemicals. Synthesis of SLIC 1 and thiolipid 2 (Figure 1) is described elsewhere5,6 and in the Supporting Information. For the sources of other chemicals, see the Supporting Information. The buffer comprised 5 mM sodium phosphate (pH 7.4) and 0.1 M KCl in deionized water. Layer Formation. Gold electrodes were prepared immediately before use by evaporating (5 × 10-6 mbar) a 45 nm thick gold film through a metallic mask onto a glass slide silanized with 3-mercaptopropyltrimethoxysilane. First a self-assembled monolayer (SAM) of 2 was formed by incubating a gold electrode in a detergent solution of 2 (1 mg/mL, 48 mM octylglucoside in buffer) for 12 h followed by washing with a 48 mM octylglucoside solution in buffer. The lipid bilayer was then completed by covering the SAM of 2 with a mixed monolayer of DphytPC and cholesterol formed by dilution of a detergent solution of DphytPC/ cholesterol (9/1) below the critical micellar concentration of octylglucoside.7 Impedance spectroscopy (IS) was done as described elsewhere.4c Special care had to be taken to measure the lowfrequency range from 0.1 Hz to 1 mHz using an amplification of 108 V/A and long time constants between 1 and 100 s.
Results and Discussion Electrical Properties of the Tethered Lipid Bilayers. A new synthetic thiolipid 2, similar to the one reported by Cornell et al.,4a was used throughout this work. Most notably, this lipid consists of a single phytanoic acid coupled to a hydrophilic spacer which is terminated by a thiol group (Figure 1). This thiolipid has a smaller cross section than phospholipids and therefore is expected to form a first monolayer with a higher lipid density on a solid support. The lower electrical resistance of supported lipid bilayers has been related to their reduced lipid density as compared to unsupported membranes such as BLM.8 (5) Terrettaz, S.; Ulrich, W.-P.; Guerrini, R.; Verdini, A.; Vogel, H. Angew. Chem., Int. Ed. 2001, 40, 1740-1743. (6) Heyse, S.; Ernst, O. P.; Dienes, Z.; Hofmann, K. P.; Vogel, H. Biochemistry 1998, 37, 507-522. (7) Terrettaz, S.; Stora, T.; Duschl, C.; Vogel, H. Langmuir 1993, 9, 1361-1369. (8) Wiegand, G.; Arribas-Layton, N.; Hillebrandt, H.; Sackmann, E.; Wagner, P. J. Phys. Chem. B 2002, 106, 4245-4254.
10.1021/la034197v CCC: $25.00 © 2003 American Chemical Society Published on Web 06/10/2003
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Langmuir, Vol. 19, No. 14, 2003
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Figure 1. Chemical structure of SLIC 1 (one-letter amino acid code) and thiolipid 2. X (ornithine) allows the synthesis of branched polypeptides. 1 and 2 attach via terminal SH-groups to gold surfaces. Length scales of 1 and 2 are schematic and not identical.
Figure 2. (A) Real part of the impedance spectrum of a 3.34 mm2 disk gold electrode covered by a lipid bilayer comprising a tethered monolayer of the thiolipid 2 and a mixed monolayer of DphytPC and cholesterol (molar ratio 9/1). 199 data points equally spaced on the log scale between 0.1 and 20 000 Hz and 5 additional data points (b) between 1 mHz and 0.1 Hz were measured. The impedance spectrum of the lipid bilayer is represented by a parallel combination of the resistance Rm and the capacitance Cm, while Cdl and Rs represent the double-layer capacitance and the resistance of the solution, respectively. Impedance spectra of electrically less dense tethered lipid bilayers with the molecular composition previously used for antibody binding to SLIC in a tethered lipid bilayer (ref 5) prepared by 1 h (dashed line) and 12 h (dotted line) thiophospholipid adsorption are shown to highlight the considerable increase of layer resistance in our present case. (B) Corresponding imaginary parts of the impedance spectra.
According to Nagle and Scott’s theory,9 ion permeability of the lipid bilayer is related to lateral fluctuations that depend on the layer compressibility (and lipid density). In this respect, an additional increase of the electrical insulation of supported phospholipid layers by cholesterol could also be explained. The real and imaginary parts of the impedance spectrum of a tethered lipid bilayer on an electrode are shown in (9) Nagle, J. F.; Scott, H. L. Biochim. Biophys. Acta 1978, 513, 236243.
Figure 2 together with the impedance spectra of two supported lipid bilayers of lower resistances made with a previously reported sulfur-bearing phospholipid.5 The impedance spectrum of each of the three curves can be fitted by the electrical equivalent circuit shown in the insert of Figure 2 differing in the particular value of the membrane resistance. The capacitance values of the lipid bilayer Cm and of the electrode double-layer Cdl were identical to those determined previously;4e,5 this finding confirms the model of a lipid bilayer decoupled from the electrode by a hydrated cushion.4a-d,5 The electrical characteristics of the lipid bilayer dominated the impedance spectrum in the whole available frequency range and could be determined very accurately, while Cdl (approximately 10 µF/cm2) was obtained only from the lowest frequency range. Cm was measured to be 0.67 ( 0.06 µF/cm2 in excellent agreement with previous work.4a-d,5 Interestingly, the nonideal frequency dependence of the membrane capacitance (CPE effect) reported earlier was not present for layers with a resistance Rm > 105 Ω.5 This could be either due to a low concentration of membrane defects or to a better estimation of the dielectric properties of the lipid bilayer in a wider frequency range. The most striking result of our measurements is the substantial increase of the membrane resistance, compared to previously reported cases.4,5 The membrane resistance is visible as a plateau in the real part of the impedance spectra at low frequencies (Figure 2A). Values above 2 × 108 Ω have been reached for the newly reported membranes, which translate into an unprecedented membrane resistance of 7 MΩ cm2. For comparison, the electrical resistances of unsupported planar lipid bilayers (BLM) are around 10 MΩ cm2.1b SLIC Addition. A synthetic ligand-gated ion channel SLIC 1 (Figure 1) comprising four channel-forming amphipathic membrane-spanning R-helices each connected to a (NANP)3 sequence has been shown to bind the monoclonal antibody Sp3E9 with high affinity and selectivity in a former study.5 SLIC was directly attached to the gold electrode prior to the formation of the lipid bilayer; but due to the lower electrical resistance of our former tethered membranes, we could detect antibody binding only in the presence of relatively high SLIC concentrations in the lipid bilayer.5 In the experiments reported here, SLIC inserted into a preformed highly insulating tethered lipid bilayer at low concentration from the aqueous phase, in analogy to protocols used in classical BLM experiments. SLIC has such a high affinity to the tethered lipid bilayer that no dc voltage was required for its bilayer insertion. In Figure 3A, the incorporation of
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Langmuir, Vol. 19, No. 14, 2003 5569
Antibody Binding to SLIC. Selective antibody binding to SLIC in the lipid bilayer increased the membrane resistance as a function of the concentration of the antibody Sp3E9 in the aqueous phase (Figure 3B). The observed response corresponds to a total closure of 95 SLIC channels of 90 pS conductance. With the present signal-to-noise ratio, the closure of a few individual channels could be detected. As seen in Figure 2A, the resistance of an electrically blocking tethered bilayer is shifted to the very low frequencies of the impedance spectrum as the time constant RmCm of the membrane increases. The modulation of the membrane resistance by the gating of channel proteins should therefore be measured in the same frequency range. The requirement of a detection at a very low frequency precludes the measurement of any kinetics other than the slow diffusion of the ligand to the membrane-bound receptor.
Figure 3. (A) Real part of the electrical impedance (measured at 0.01 Hz) of a tethered lipid bilayer after the addition of 7 × 10-7 M (a) of SLIC to the aqueous phase. Single-channel recordings on BLM were done at -30 mV in 1 M NaCl, 10 mM Tris-HCl, pH 7.4, as described elsewhere (ref 12). The singlechannel events shown correspond to a conductance level of up to 90 pS at the buffer conditions used for IS. (B) Real part of the electrical impedance (measured at 0.01 Hz) of the tethered lipid bilayer containing SLIC after the addition (a) of 10-6 M of the antibody Sp3E9.
SLIC in a tethered lipid bilayer is reflected as a drop in membrane resistance measured on the real part of the impedance at 0.01 Hz. Upon addition of 0.7 µM SLIC in the aqueous phase, the layer resistance was seen to decrease immediately and saturate in about 30 min. The slow kinetics allowed interruption of the bilayer insertion process by washing with buffer and thus keeping the number of SLIC molecules in the membrane as low as possible in a controlled way. Single conductance states of up to 90 pS were measured for SLIC in BLM (Figure 3A insert). The decrease of the membrane resistance after incorporation of SLIC in tethered lipid membranes shown in Figure 3A can be explained by the presence of only about 200 open SLIC channels of 90 pS conductance. No capacitance change could be detected during the bilayer incorporation of SLIC, which confirms the membrane’s integrity. In the presence of higher concentrations of SLIC, a large increase of the capacitance could be observed indicating disruption of the membrane. Tethered bilayers however sustained a considerably higher SLIC concentration than BLM in agreement with similar observations made with melittin.10 (10) Becucci, L.; Guidelli, R.; Liu, Q.; Bushby, R. J.; Evans, S. D. J. Phys. Chem. B 2002, 106, 10410-10416.
Conclusions Tethered lipid bilayers with an extremely high resistance can be formed on a gold electrode. With a further optimization of membrane composition and electrode miniaturization, gigaOhmic resistances should be obtained. Especially, as the surface area of the electrode decreases, the membrane leakage through defects might be reduced. Therefore single-channel experiments are likely to be done on microelectrodes. The resolution in our present impedance measurements is so high (Figure 2B) that the blocking of only a few large pore molecules can already be detected. However, the kinetics of opening and closing of single channels cannot be measured with an ac voltage at low frequency. Alternative techniques with a time resolution in the millisecond range such as the time domain impedance spectroscopy proposed by Wiegand et al.11 would be required to record channel dynamics in tethered lipid bilayers. Measuring channel modulation of a few SLICs offers in turn the possibility to detect very small quantities of analytes such as antibodies. The ability to modify the detector element in SLIC could thus deliver a generic tool for ultrasensitive biosensing. Acknowledgment. This work was financially supported by the TopNano 21 program. The authors are grateful to Andreas Heusler for synthesizing the thiolipid. Supporting Information Available: Sources of chemicals and details on the synthesis of the thiolipids. This material is available free of charge via the Internet at http://pubs.acs.org. LA034197V (11) Wiegand, G.; Neumaier, E.; Sackmann, E. Rev. Sci. Instrum. 2000, 71, 2309. (12) Pawlak, M.; Meseth, U.; Dhanapal, B.; Mutter, M.; Vogel, H. Protein Sci. 1994, 3, 1788-1805.
