Tethered Mono- and Bilayer Lipid Membranes on Au and Hg

A simple thiolipid molecule was synthesized by covalent linking of mercaptopropionic acid with phosphatidylethanolamine. The integrity of thiolipid ...
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Tethered Mono- and Bilayer Lipid Membranes on Au and Hg P. Krysin´ski,*,† A. Z˙ ebrowska,† A. Michota,‡ J. Bukowska,‡ L. Becucci,§ and M. R. Moncelli§ Laboratory of Electrochemistry and Laboratory of Molecular Interactions, Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland, and Laboratory of Electrochemistry, Department of Chemistry, University of Florence, via G. Capponi 9, 50-121 Florence, Italy Received November 29, 2000. In Final Form: March 29, 2001 A simple thiolipid molecule was synthesized by covalent linking of mercaptopropionic acid with phosphatidylethanolamine. The integrity of thiolipid self-assembled monolayers supported on gold and mercury electrodes was tested by electrochemical techniques both in the absence and in the presence of hydrophilic redox couples. The organization of the thiolipid monolayer supported on gold was also assessed by surface-enhanced Raman spectroscopy both in air and in aqueous solution. The thiolipid monolayer was then covered with a phospholipid monolayer to form a bilayer lipid membrane tethered to the electrode surface (t-BLM), and the electrochemical tests were repeated using cyclic voltammetry, differential capacitance, impedance spectroscopy (IS), and chronocoulometry. These bilayers possess an exceptionally good mechanical stability while exhibiting electrochemical properties similar to those of conventional BLMs. However, no substantial “ionic reservoir” separating the t-BLM from the electrode surface was observed by IS. Most probably, this is due to the insufficient length of the mercaptopropionic spacer and its gauche conformation related to the electrode surface.

Introduction So far, the experimental models of biomembranes that have been more extensively investigated are the bilayer lipid membranes (BLMs). They consist of two adjacent lipid monolayers (the lipid bilayer), with the hydrocarbon tails in contact to form the hydrophobic interior of the bilayer, and the polar heads turned toward the aqueous solutions that bath the two sides of the bilayer and mimic the intra- and extracellular fluids of biomembranes. BLMs are not suitable for biosensor technology since they are extremely fragile, metastable systems.1 In addition, these systems do not lend themselves to investigation with surface-sensitive techniques. In an effort to overcome this problem, solid-supported membranes (SSMs) have been devised.2-5 Great interest has been focused on self-assembled films that are attached to the substrate via a covalent linkage between the selfassembled molecules and the solid support, yielding structures of long-term stability. Monolayers of alkanethiols on gold are probably the most widely used and best characterized of all self-assembled films to date. The deposition of a second phospholipid monolayer on top of the first chemisorbed alkanethiol monolayer, yielding the so-called supported hybrid bilayer membranes (HBMs), provides a bimolecular film on electrodes which has gained increasing importance as a biomimetic system.6-13 How†

Laboratory of Electrochemistry, University of Warsaw. Laboratory of Molecular Interactions, University of Warsaw. § Laboratory of Electrochemistry, University of Florence. ‡

(1) Krysin´ski, P., Tien, H. T.; Ottova, A. Biotechnol. Prog. 1999, 15, 974. (2) Sackmann, E. Science 1996, 271, 43. (3) Ottova-Leitmannova, A.; Tien, H. T. Prog. Surf. Sci. 1992, 41, 337. (4) Tien, H. T.; Wurster, S. H.; Ottova, A. L. Bioelectrochem. Bioenerg. 1997, 42, 77. (5) Bilayer lipid membranes and other lipid-based methods. Handbook of Chemical and Biological Sensors; Taylor R. F., Schultz J. S., Eds.; Institute of Physics Publishing: Philadelphia, PA, 1996.

ever, the major drawback of HBMs is the presence of a hydrophobic, solidlike alkanethiol monolayer under the outer phospholipid monolayer. Having been recognized that, for the intrinsic membrane proteins to retain their functional activity, the bilayer must be provided with aqueous “ionic reservoirs” on both sides, a novel class of lipids was synthesized. These lipids, called “thiolipids”, consist of a hydrophilic chain (a “spacer”) covalently linked on one side to the polar head of a phospholipid molecule and terminated on the other side with a functional thiol group for anchoring to the surface of a metal, e.g., a gold electrode.14-20 With these thiolipid molecules replacing the alkanethiol monolayer of HBMs, a biomimetic membrane is formed, referred to as a tethered bilayer lipid membrane (t-BLM), which closely resembles the phos(6) Seifert, K.; Fendler, K.; Bamberg, E. Biophys. J. 1993, 64, 384. (7) Steinem, C.; Janshoff, A.; Ulrich, W. P.; Sieber, M.; Galla, H.-J. Biochim. Biophys. Acta 1996, 1279, 169. (8) Plant, A. L. Langmuir 1999, 15, 5128. (9) Plant, A. L.; Gueguetchkeri, M.; Yap, W. Biophys. J. 1994, 67, 1126. (10) Lingler, S.; Rubinstein, I.; Knoll, W.; Offenha¨usser, A. Langmuir 1997, 13, 7085. (11) Meuse, C. W.; Niaura, G.; Lewis, M. M. L.; Plant, A. L. Langmuir 1998, 14, 1604. (12) Meuse, C. W.; Krueger, S.; Majkrzak, C. F.; Dura, J. A.; Fu, J.; Connor, J. T.; Plant, A. L. Biophys. J. 1998, 74, 1388. (13) Tadini Buoninsegni, F.; Herrero, R.; Moncelli, M. R. J. Electroanal. Chem. 1998, 452, 33. (14) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197. (15) Duschl, C.; Liley, M.; Corradin, G.; Vogel, H. Biophys. J. 1994, 67, 1229. (16) Bunjes, N.; Schmidt, E. K.; Jonczyk, A.; Rippmann, F.; Beyer, D.; Ringsdorf, H.; Gra¨ber, P.; Knoll, W.; Naumann, R. Langmuir 1997, 13, 6188. (17) Steinem, C.; Janshoff, A.; von dem Bruch, K.; Reihs, K.; Goossens, J.; Galla, H-J. Bioelectrochem. Bioenerg. 1998, 45, 17. (18) Williams, L. M.; Evans, S. D.; Flynn, T. M.; Marsh, A.; Knowles, P. F.; Bushby, R. J.; Boden, N. Langmuir 1997, 13, 751; Supramol. Sci. 1997, 4, 513. (19) Raguse, B.; Braach-Maksvytis, V.; Cornell, B. A.; King, L. G.; Osman, P. D. J.; Pace, R. J.; Wieczorek, L. Langmuir 1998, 14, 648. (20) 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, 580.

10.1021/la0016625 CCC: $20.00 © 2001 American Chemical Society Published on Web 05/23/2001

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pholipid matrix of biomembranes.9-12 The rationale behind such an approach is 2-fold: (i) realizing a robust bilayer system compatible with the technological requirements of nanoelectronics, while retaining its biocompatibility for biosensor applications; (ii) providing a stable model system for investigating the functions of integral proteins incorporated in the bilayer and the influence of agonists and antagonists upon these functions. Following these lines, the development of biomimetic membranes consisting of tethered lipid bilayers should meet a number of requirements: (i) they should be robust enough for long-term stability and be easily and reproducibly prepared; (ii) they should be as flexible as the lipid films above the transition temperature from the gel to the liquid crystalline state; (iii) they should have water on both sides of the lipid bilayer; (iv) they should be sufficiently free from pinholes and other defects that might provide preferential pathways for electron and ionic transport across the lipid bilayer. The latter requirement is crucial for the realization of biomimetic membranes sufficiently blocking as to characterize ion channel activity by electrochemical means without the disturbing presence of stray currents due to defects. In the present work a simple thiolipid molecule was synthesized and the integrity of thiolipid self-assembled monolayers supported on gold and mercury electrodes was tested by electrochemical techniques both in the absence and in the presence of hydrophilic redox couples. Next, the thiolipid monolayer was covered with a phospholipid monolayer as in refs 13 and 21, and the electrochemical tests were repeated using cyclic voltammetry, differential capacitance, impedance spectroscopy (IS), and chronocoulometry. The organization of the thiolipid monolayer supported on gold was also assessed by means of surfaceenhanced Raman spectroscopy both in air and in aqueous solution. Experimental Section Chemicals. All chemicals were of the highest purity commercially available: L-cephalin (dipalmitoylphosphatidylethanolamine, DPPE), res. grade (Serva, synthetic, 97-99%), dioleoylphosphatidylcholine (DOPC) (Lipid Products, England), 3mercaptopropionic acid (MPA) (Merck, >98%), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (Aldrich, 98+%), LiClO4 (Aldrich, ACS grade), CHCl3 (Lachema, reagent grade), K3Fe(CN)6‚3H2O (Sigma, 99%), KCl (POCh, reagent grade), gramicidin D (Sigma). Aqueous solutions were prepared from water of high purity (Milli-Q). Synthesis and Characterization. The synthesis of DPPEmercaptopropionamide was carried out following the EDC coupling procedure described by Katz et al.22 and Uragami et al.23 This procedure can be summarized as follows. To a solution containing DPPE (0.03 mmol, 20 mg), triethylamine (2 µL), and CHCl3 (2 mL), a second solution composed of EDC (0.03 mmol, 5.7 mg), mercaptopropionic acid (0.03 mmol, 2.61 mL), and CHCl3 (2 mL) was added dropwise with continuous stirring. The total addition time was ca. 5 min. The reaction mixture was stirred for 24 h at room temperature under an argon atmosphere. The reaction progress was monitored by thin-layer chromatography. Next, the solvent was removed under reduced pressure. The residue so obtained was purified chromatographically (silica, CHCl3/MeOH/H2O, 20:5:1 v/v/v eluent), yielding 19.78 mg of crude product in the form of a pale yellow solid, which was used without further purification. This product was identified as DPPEmercaptopropionamide (DPPE-MPA) by 1H NMR (Varian Unity (21) Krysinski, P.; Moncelli, M. R.; Tadini-Buoninsegni, F. Electrochim. Acta 2000, 45, 1885. (22) Katz, E.; Itzhak, N.; Willner, I. Langmuir 1993, 9, 1392. (23) Uragami, M.; Dewa, T.; Inagaki, M.; Hendel, R. A.; Regen, S. L. J. Am. Chem. Soc. 1997, 119, 3797.

Langmuir, Vol. 17, No. 13, 2001 3853 Plus, 500 Hz, CDCl3) and by IR (FTIR-8201PC/8601PC Shimadzu spectrophotometer). Instrumentation. Pressure vs area and surface potential vs area isotherms of the DPPE-MPA thiolipid thus synthesized were recorded by the use of Langmuir troughs (KSV Mini LB 5000 and KSV SPM LB 5001) supplied with a surface potential sensor (KSV Ltd., Finland). Monolayers were spread by using thiolipid solutions of concentration 1.98 mg/mL in CHCl3. Electrochemical measurements on gold-supported films were conducted with a PC-controlled, custom-built potentiostat/ galvanostat (KSP Electronics, Poland) as well as an Autolab instrument (Eco Chemie) with FRA-DSG module, using a conventional small three-electrode cell with a Pt wire as counter electrode. All potentials are referred to an Ag|AgCl|1 M KClaq reference electrode. Potassium hexacyanoferrate(II) solutions used for integrity tests on mono- and bilayers were deaerated by bubbling with argon for 10 min before measurements. The conformation of the thiolipid molecule in CHCl3 was monitored by Raman spectroscopy, while the organization of the gold-supported thiolipid was monitored by surface-enhanced Raman spectroscopy (SERS). Raman and SERS spectra were recorded with a Jobin-Yvon Spex T64000 Raman microscope equipped with a Kaiser holographic notch filter, 1800 grooves/ mm holographic grating and 1024 × 256 pixel nitrogen-cooled CCD detector. The microscope attachment was based on an Olympus BX40 system with a 50× long-distance objective (LMPLFL50x/0.50). A Laser-Tech model LJ-800 mixed argon/ krypton laser provided excitation radiation of 647.1 nm. Electrochemical measurements on mercury-supported films were carried out with an Autolab instrument (Eco Chemie) supplied with a FRA2 module for impedance measurements, SCAN-GEN scan generator, and GPES3 software. Chronocoulometric measurements of Tl+ ion reduction were carried out with a wholly computerized apparatus24 following a procedure described elsewhere.25 The microprocessor used to control all the operations was a model NOVA 4X from Data General (Westboro, MA), whereas an Amel model 551 (Milano, Italy) fastrise potentiostat with a rise time of 0.1ms was employed for the potentiostatic control of the three-electrode system. The homemade hanging mercury drop electrode (HMDE), the cell, and the experimental setup for coating the mercury drop are described elsewhere.26,27 Electrode Functionalization. Polycrystalline gold-ball electrodes (Au, 99.99%) for electrochemical measurements were first cleaned in the reductive flame of a Bunsen burner and then cyclically polarized in aqueous 1 M HClO4 over the potential range from -300 to +1500 mV, as described elsewhere.28 A DPPE-MPA monolayer was covalently self-assembled on gold by keeping a clean gold-ball electrode immersed in a 1.2 × 10-3 M solution of DPPE-MPA in CHCl3 for about 3 days. The thiolipid-coated gold (henceforth briefly referred to as t-DPPE, where t stands for tethered) was then removed from the solution, rinsed with chloroform, and dried. After these operations, the quality of the t-DPPE was checked by differential capacitance measurements in aqueous 0.5 M KCl or 0.1 M LiClO4 and by cyclic voltammetry in the above supporting electrolytes containing 1 × 10-3 M K4Fe(CN)6. In SERS experiments the thiolipid monolayer was self-assembled on a roughened gold surface. Briefly, the roughening procedure consisted of 20 oxidationreduction cycles between -0.6 and +1.2 V vs SCE at a rate of 0.5 V/s in 0.1 M KCl, pausing at -0.6 V for 8 s and at 1.2 V for 3 s. Finally, the roughened gold electrode was held at -0.6 V for 5 min. The electrode was then thoroughly rinsed with deionized water, dried, and immersed in the monolayer-forming solution in benzene. After the t-DPPE was characterized, a second DOPC monolayer was transferred on top of it. For this purpose, a procedure (24) Carla`, M.; Sastre de Vicente, M.; Moncelli, M. R.; Foresti, M. L.; Guidelli, R. J. Electroanal. Chem. 1988, 246, 283. (25) Ruiz, J.; Foresti, M. L. J. Chem. Soc., Faraday Trans. 1988, 84, 4299. (26) Moncelli, M. R.; Becucci, L. J. Electroanal. Chem. 1997, 433, 91. (27) Moncelli, M. R., Becucci, L.; Nelson, A.; Guidelli, R. Biophys. J. 1996, 70, 271628. (28) Krysinski, P.; Brzostowska-Smolska, M. J. Electroanal. Chem. 1997, 424, 61.

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Figure 1. Pressure vs area isotherms of Langmuir-Blodgett films of DPPE and DPPE-MPA. The dashed line shows the change in surface potential following DPPE-MPA compression. described in refs 13 and 21 was adopted. Briefly, the t-DPPE was immersed into an aqueous electrolyte on whose surface a DOPC film had been previously spread. In the resulting bilayer the alkyl chains of the thiolipid monolayer are in contact with those of DOPC thanks to hydrophobic interactions, while the polar heads of DOPC face the solution. This bilayer system was then characterized electrochemically in situ in the same cell used for the transfer of the DOPC monolayer. To prepare a t-DPPE on a mercury support, a thermostated mercury drop formed at the tip of a HMDE was kept in a 1.2 × 10-3 M solution of DPPE-MPA in CHCl3 for about 1 h. The drop was then immersed in an electrolysis cell containing aqueous 0.1M KCl. After the applied potential was scanned from -0.30 to -1.50 V and vice versa three or four times, a stable capacitance vs potential curve was attained. The transfer of a second DOPC monolayer on top of the mercury-supported t-DPPE was carried out as on the gold-supported one.

Results and Discussion DPPE-MPA Monolayers at the Air/Water Interface. The surface behavior of DPPE-MPA is shown in Figure 1. The pressure vs area isotherms shown in this figure indicate that the surface area per molecule is ca. 90 Å2, which is larger that that observed for the DPPE molecule alone (ca. 65 Å2). Moreover, the collapse pressure of DPPE-MPA, which is accompanied by a plateau on the corresponding surface potential vs area curve, amounts to 16 mN/m and is therefore appreciably lower than that, ca. 63 mN/m, of DPPE alone; no observable phase transition precedes the collapse of this film. A further feature, not shown in this figure to avoid confusion, is represented by the invariance of the isotherm following consecutive compressions and decompressions of the surface film, a behavior typically associated with the fluidity of the surface film. All these observations lead to the conclusion that even the addition of a relatively short spacer chain to the lipid head drastically changes the hydrophilicity of this group, affecting the overall hydrophilic/hydrophobic balance of the thiolipid molecule. Apparently, the area per molecule appears to be comparable with that estimated for the spacer chain lying on the interface, with the hydrophilic oxygen and nitrogen

Figure 2. Raman (a) and SERS (b, c) spectra of DPPE-MPA.

atoms of the amide group directed toward the aqueous solution. These conclusions are strongly supported by the Raman spectra of DPPE-MPA in methanol. In the diagnostic region for the C-S stretching vibration of the molecule under investigation (600-800 cm-1, Figure 2a), one can observe essentially one band at 668 cm-1. This strong band is characteristic for the gauche conformer of the S-C-C chain.29,30 Thus, even in the bulk of a solvent of relatively low dielectric constant such as CH3OH, the gauche conformer is the most stable one. Under these conditions the Langmuir-Blodgett transfer of the thiolipid on gold, carried out near the collapse pressure, yields a t-DPPE poorly insulating toward the hydrophilic redox probe K4Fe(CN)6 in aqueous 0.1 M LiClO4. Thus, the oxidation and reduction voltammetric peaks of this redox couple, typical of a diffusion-limited process, are still observable, albeit with a peak-to-peak separation greater than 200 mV. Cyclic Voltammetry and Capacitance Measurements of DPPE-MPA Monolayers and DPPE-MPA/ DOPC Bilayers on Gold and Mercury. To assess the organization of t-DPPE on a roughened polycrystalline gold support, the SERS spectra of this tethered monolayer were recorded both in air and in aqueous 0.1 M LiClO4. The results are shown in parts b and c of Figure 2, respectively. Again, these spectra show that even though the trans conformation (720 cm-1) is the main structure in air, the same monolayer in contact with 0.1 M LiClO4 adopts mainly the gauche conformation of the S-C-C chain of the spacer (643.5 cm-1). In view of all these results, no perfect passivating behavior of the thiolipid monolayer on gold is to be (29) Bryant, M. A.; Pemberton, J. E. J. Am. Chem. Soc. 1991, 113, 3629, 8284. (30) Kudelski, A.; Hill, W. Langmuir 1999, 15, 3162.

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Figure 5. Capacitance vs potential curves of a DPPE-MPA monolayer (a, a′) and of a DPPE-MPA/DOPC bilayer (b, b′) supported on gold (a, b) and on mercury (a′, b′). The upper scale refers to gold and the lower one to mercury.

Figure 3. Cyclic voltammograms of 1 × M K4Fe(CN)6 in aqueous 0.1 M LiClO4 on a DPPE-MPA monolayer (a) and on a DPPE-MPA/DOPC bilayer (b) supported on gold. The inset shows the same cyclic voltammogram on bare gold. 10-3

Figure 4. Cyclic voltammogram of 1 × 10-3 M Fe(III) in aqueous 0.1 M lithium citrate on a DPPE-MPA/DOPC bilayer supported on mercury. The inset shows the same cyclic voltammogram on bare mercury.

expected. Indeed, the cyclic voltammogram of 1 × 10-3 M Fe(CN)64- in aqueous 0.1 M LiClO4 on gold-supported t-DPPE (Figure 3, curve a) shows relatively large currents with an exponential behavior. The lack of a sigmoidal shape of the cyclic voltammograms, typical of the presence of an appreciable number of pinholes, seems to indicate that the DPPE-MPA monolayer is substantially pinhole free; however, its molecules do not form a well-ordered monomolecular film totally impermeable to the hydrophilic Fe(CN)64- redox probe. The addition of a second DOPC monolayer on top of the tethered thiolipid improves the blocking properties of the resulting DPPE-MPA/DOPC bilayer, as shown by the voltammogram of 1 × 10-3 M Fe(CN)64- in 0.1 M LiClO4 (Figure 3, curve b) on such a bilayer; this denotes a well-ordered, defect-free structure. Similar experiments carried out for the DPPE-MPA/ DOPC bilayer tethered to a mercury electrode in the presence of the citrate complex of Fe(III) show the blocking properties of this bilayer toward this inorganic redox couple. Figure 4 shows the cyclic voltammogram of Fe(III) citrate complex both on bare and on bilayer-coated

mercury. It is apparent that the electron transfer process of the Fe(III)/Fe(II) couple is almost completely suppressed. The capacitance vs potential curves of the gold supported DPPE-MPA monolayer and DPPE-MPA/DOPC bilayer were recorded with a two-electrode system making use of the capacitance-to-period conversion method31 to test the good quality and stability of these films. Over the potential range from -0.2 to +0.5 V the C vs E plot for the DPPEMPA monolayer (Figure 5, curve a) shows a curvature, with a flat minimum of about 1.8 ( 0.1 µF cm-2. This value is practically coincident with the differential capacitance of DOPE and DOPC monolayers noncovalently self-assembled on mercury.27,32 This indicates that the contribution to the capacitance from the short and relatively hydrophilic mercaptopropionic chain is negligible with respect to that of the DPPE molecule. The C vs E curve for the DPPE-MPA/DOPC bilayer in Figure 5, curve b, is much flatter, with a minimum of 0.9 ( 0.08 µF cm-2. This value coincides with the differential capacitance expected for two phospholipid monolayers in series, each with a typical differential capacitance of 1.8 µF cm-2. This excludes a partial interdigitation of the contacting alkyl chains of the two lipid monolayers composing the bilayer. Curves a′ and b′ in Figure 5 show the C vs E plots for a mercury-supported DPPE-MPA monolayer and a DPPE-MPA/DOPC bilayer over the potential range from -0.3 and -0.9 V. While the differential capacitance for the monolayer is somewhat higher on Hg than on Au, that for the bilayer is practically identical on the two metals. ac-Impedance Spectroscopy of DPPE-MPA Monolayers and DPPE-MPA/DOPC Bilayers on Gold and Mercury. To assess the functional quality and usefulness of our system to provide the biomimetic system for possible incorporation of membrane proteins, the ac-impedance analysis of DPPE-MPA/DOPC bilayers was performed on both gold and mercury electrodes. Figure 6 shows the Bode plot for the gold-supported bilayer in 0.5 M KCl at +500 mV. As can be seen from this figure, this system reveals its complex behavior, as the simplest equivalent circuit describing our results obtained from the fitting procedure contains a constant phase element. In general, the equivalent circuit consists of the resistance, RΩ ) 69 Ω, of the supporting electrolyte, in series with a parallel (31) Kalinowski, S.; Figaszewski, Z. Meas. Sci. Technol. 1995, 6, 1043. (32) Nelson, A.; Benton, A. J. Electroanal. Chem. 1986, 202, 253.

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Figure 6. Impedance spectrum |Z(f)| and phase angle for the DPPE-MPA/DOPC bilayer supported on polycrystalline gold in aqueous 0.5 M KCl at +0.5 V. The solid curves are results of the fitting procedure based on the equivalent circuit shown in the inset.

Figure 7. Impedance spectrum |Z(f)| and phase angle for the DPPE-MPA/DOPC bilayer supported on mercury in aqueous 0.1 M KCl at -0.5 V. The solid curves are results of the fitting procedure based on the equivalent circuit shown in the inset.

arrangement of the resistance, Rl ) 0.25 MΩ cm2, and capacitance, Cl ) 1.88 µF cm-2, of the outer phospholipid monolayer; this is in series with a further parallel arrangement of a resistance, Rm ) 0.54 MΩ cm2, and a constant phase element, Q, ascribable to the tethered thiolipid monolayer underlying the outer phospholipid monolayer. The presence of this element reflects an imperfect organization of the thiolipid monolayer on a polycrystalline gold electrode, as expected when taking into account the present SERS studies (Figure 2c). The fitting procedure also provides the value of parameter n ) 0.85, which reflects the hybrid nature of Q in the equivalent circuit: for n ) 1 Q represents a pure capacitance, while for n ) 0.5, it can be approximated by a Warburg impedance. Even though the resistance of the present bilayer extracted from the fitting procedure is much higher than those of a number of thiolipid|lipid bilayers anchored on gold,14-17 the imperfect behavior of the thiolipid monolayer directed us toward the defectfree, model surface of liquid mercury. Figure 7 shows the Bode plot for the mercury-supported bilayer in 0.1 M KCl. This can be interpreted on the basis of the simple equivalent circuit shown in the same figure. It consists of the resistance RΩ of the aqueous solution, in series with a parallel arrangement of the resistance Rb and capacitance Cb of the lipid bilayer. At frequencies higher than 104 Hz the overall impedance is entirely determined by the frequency-independent resistance RΩ ) 320 Ω cm2. Over the intermediate frequency range from

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104 to 3 Hz the impedance is mainly determined by the capacitance Cb, as appears from the almost unit slope of the Bode plot and from a phase angle close to 90°. At still lower frequencies the resistance Rb starts to contribute to the current, and hence the slope of the Bode plot tends to decrease. Fitting of the Bode plot to the equivalent circuit yields a capacitance Cb ) 0.9 µF cm-2 and a resistance Rb ) 0.25 MΩ cm2. It should be noted that the resistance of the present tethered lipid bilayer is again about 1 order of magnitude greater than those of a number of thiolipid|lipid bilayers anchored on gold.14-17 This indicates that the defect-free surface of liquid mercury favors the formation of lipid bilayers with a minimum number of pinholes as was confirmed earlier (see Figure 4). The monomer of gramicidin D incorporated in a mercury-supported phospholipid monolayer has often been reported to act as an ion channel toward Tl+ ion, thus allowing its penetration across the monolayer and its electroreduction to thallium amalgam.33-37 The length of this pentadecapeptide matches that of a phospholipid monolayer, thus permitting it to span the whole monolayer. Gramicidin is also capable of inducing monocation transport in lipid bilayers. In this case the membrane conductance increases with the square of the gramicidin concentration in the aqueous phase, indicating that the conductance channel is a gramicidin dimer.38-41 It was therefore interesting to test the ability of gramicidin to allow thallous ion electroreduction on mercury across the DPPE-MPA/DOPC bilayer. To this end, the potentialstep chronocoulometric technique was adopted, by carrying out a series of potentials steps from a fixed initial potential Ei ) -0.30 V, at which Tl+ ion is still electroinactive and the lipid bilayer is well organized, to progressively more negative potentials E. The charge Q(t,E) following these potential steps was recorded as a function of time. Figure 8 shows curves of the charge Q (100 ms, E) measured 100 ms from the instant of each potential step against the applied potential E for 4 × 10-4 M Tl+ electroreduction from aqueous 0.1 M KCl on bilayer-coated mercury, in the presence of different concentrations of gramicidin in the aqueous phase. The ill-defined limiting charge of these Q vs E curves is controlled both by diffusion of Tl+ ions toward the bilayer and by a purely chemical binding step at the mouth of the channel.35,37,42 An approximate expression for the rate constant k of this chemical step, based on the diffusion-layer approximation, is the following:43

Ql δk ) D Qd - Ql

(1)

Here Qd is the diffusion limiting charge, as attained at a bare mercury electrode (curve e in Figure 8), Ql is the limiting charge at the bilayer-coated electrode, which depends on the bulk gramicidin concentration cG, D is the diffusion coefficient of the Tl+ ions, and δ is the diffusion(33) Nelson, A. J. Electroanal. Chem. 1991, 303, 221. (34) Nelson A. J. Chem. Soc., Faraday Trans. 1993, 89, 2799. (35) Nelson A. Langmuir 1996, 12, 2058. (36) Nelson, A. Langmuir 1997, 13, 5644. (37) Rueda, M.; Navarro, I.; Ramirez, G.; Prieto, F.; Prado, C.; Nelson, A. Langmuir 1999, 15, 3672. (38) Tosteson, D. C.;Andreoli, T. E.; Tieffenberg, M.; Cook P. J. Gen. Physiol. 1968, 51, 373S. (39) Gondola, M. C. Biochim. Biophys. Acta 1970, 219, 471. (40) Hladky, S. B.; Haydon, D. A. Biochim. Biophys. Acta 1972, 274, 294. (41) Bamberg, E., Lau¨ger P. J. Membr. Biol. 1973, 11, 177. (42) Andersen, O. S. Biophys. J. 1983, 41, 147. (43) Becucci, L.; Moncelli, M. R.; Guidelli, R. Unpublished results.

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ion channel; on the other hand, no appreciable decrease in Rb is to be expected, due to the lack of a substantial ionic reservoir on the metal side of the bilayer that might accommodate the K+ ions, most probably because of the insufficient length of the spacer and its gauche conformation. The presence of such a hydrophilic reservoir can be realized with a hydrophilic spacer interposed between the lipid bilayer and the metal surface.19 In this case, the decrease in Rb due to the permeating K+ ions is revealed by a typical inflection in the Bode plot, which marks a shift in the control of the impedance from Cb to Rb and then to the capacitance of the hydrophilic spacer, as the frequency is progressively decreased. The high conductance of the gramicidin-containing bilayer in the presence of Tl+ ion is simply due to the practically unlimited reservoir of Tl+ ions represented by the mercury drop at potentials of thallium amalgam formation. Conclusions Figure 8. Plots of the chronocoulometric charge Q (100 ms, E) vs E for the electroreduction of 4 × 10-4 M Tl+ ion on a DPPE-MPA/DOPC bilayer supported on mercury in the presence of 0 (a), 1 × 10-7 (b), 2 × 10-7 (c), and 3 × 10-7 M (d) gramicidin D. Curve e is the same plot on bare mercury. The inset shows a plot of ln(δk/D) vs ln cG as obtained from curves a to e.

layer thickness at the electrolysis time at which Ql and Qd are measured (100 ms in the present case). The inset of Figure 8 shows a plot of ln(δk/D), as estimated from eq 1, against ln cG. The slope of this plot equals 2.1. This is explained by considering that the rate of Tl+ binding at the mouth of the channel is proportional to the concentration Γd of the gramicidin dimers spanning the bilayer. In view of the dimerization equilibrium between monomers and dimers in the bilayer,41 Γd is proportional to the square, Γm2, of the concentration of gramicidin monomers incorporated in the bilayer. If Γm is .Γd and we are far from saturation, then Γm is practically proportional to the gramicidin concentration cG in the bulk solution, and k is second order with respect to cG, in agreement with the slope of the plot in the inset of Figure 8. Unfortunately, a similar analysis on a gold electrode covered with DPPE-MPA/DOPC bilayer could not be performed, since the observable reduction of Tl+ ions and subsequent deposition of metallic thallium immediately resulted in the destruction of the bilayer. The Bode plot of a mercury-supported DPPE-MPA/ DOPC bilayer incorporating gramicidin from a 10-7 M solution containing 0.1 M KCl but no Tl+ ions has a shape similar to that in Figure 7 and can be interpreted on the basis of the same equivalent circuit; in particular, the incorporation of gramicidin leaves the resistance Rb of the bilayer substantially unaltered, while it increases its capacitance Cb from 0.9 to 1.6 µF cm-2. The increase in Cb is due to the movement of K+ ions to and fro along the

Mono- and bimolecular phospholipid films tethered to the surface of gold and mercury electrodes via a short mercaptopropionamide side chain were investigated. The organization of the first monolayer directly coupled to the metallic surface is strongly affected by the conformation adopted by this chain at the interface. It should be noted, however, that the electrical properties of the present tethered lipid bilayer are greatly improved with respect to a number of thiolipid|lipid bilayers anchored to gold.14-17 Experiments with gramicidin D incorporated in the tethered bilayers show that the present system retains its fluidlike structure, allowing the gramicidin monomers to form dimers acting as ion channels for Tl+ ions. However, the impedance analysis of the bilayer in the presence of only K+ ions (no Tl+) shows the absence of a substantial ionic reservoir interposed between the bilayer and the electrode surface. This is quite probably due to the insufficient spacer length and to the gauche conformation adopted by the side chain at the surface. These preliminary results indicate that the proper tailoring of a spacer chain including more hydrophilic groups and allowing for hydrogen bond formation should improve the capability of trapping water molecules as well as ions between the lipid bilayer and the electrode surface. This, in turn, should lead to the realization of a biomimetic membrane anchored to the electrode surface, acting as a matrix for the immobilization of specific biocatalysts, e.g., membrane integral proteins in a functionally active form. Acknowledgment. We thank Ms. Anna Kucharska (M.Sc.), from the Laboratory of Stereocontrolled Organic Synthesis, for her help in the synthesis of MPA-DPPE. Funds were provided by the Department of Chemistry Grant BW-1483/9/2000 and the State Committee for Scientific Research Grant 3T09A11719. NATO HTECH Linkage Grant 970527 is also gratefully acknowledged. LA0016625