A New Class of Thiolipids for the Attachment of Lipid Bilayers on Gold

Langmuir 1994,10, 197-210

197

A New Class of Thiolipids for the Attachment of Lipid Bilayers on Gold Surfaces Holger Lang, Claus Duschl, and Horst Vogel’ Institute of Physical Chemistry, Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland Received October 13, 1 9 9 P

A new class of lipid molecules is synthesized, based on two dipalmitoylphosphatidic molecules, each extended at the lipid phosphate by a hydrophilic spacer chain of ethoxy groups of variable length, which are then coupled as a bilipid via a terminal disulfide group at the hydrophilic spacer. These anchorbearing “thiolipids” can attach to gold substrates by forming stable gold-sulfur bonds. In this way we can couple lipid bilayers to gold surfaces, with the possibility of preserving a water layer between the support and the first monolayer. The thiolipid molecules are characterized on a Langmuir film balance using fluorescencemicroscopy. The molecular areas of the thiolipids on the water surface are determined to be 80-90 A 2 at a fully compressed state. The thiolipid monolayers show a typical first-order phase transition on the water surface with regular, starlike domains. The formation of thiolipid-attached monoand bilayerson gold surfacesis investigatedby surfaceplasmon resonance (SPR),impedancemeasurements, and cyclic voltammetry. Four different supported membrane systems are studied in detail: (i) pure thiolipid layers; (ii) mixed lipid bilayers containing a first pure thiolipid monolayer and a second one of conventionalphospholipids;(iii)bilayers, where the first gold-attachedmonolayer is composedof a mixture of thio- and conventional phospholipids with another second phospholipid layer on top; (iv) monolayera of pure 1-hexadecanethioland layers with a second phospholipid film on top of the 1-hexadecanethiol. The electrochemicalexperiments reveal electrically blocking layers for all lipid systems investigated with specific resistances of lO4-lOs Q cm2. The capacitance values for pure thiolipid bilayers are in the range of 0.6-0.7 pF/cm2 for the pure thiolipid bilayers and 0.7-0.8 pF/cm2 for the mixed thiolipid/phospholipid bilayers, which is comparable to the values found for unsupported, so-called black lipid membranes. SPR measurements confirm qualitatively the results of the electrochemical experiments.

I. Introduction Artificial lipid mono- and bilayers have been used extensively to study the basic principles of self-organized molecular layers especially in the context as models of biological membranes. In particular, the incorporation of proteins in a functionally active state in lipid bilayers, has enabled the elucidation of structure-function relationships of many biologically important membrane proteins under defined conditions.14 There is a new, fast growing interest in the realization of long-lasting, stable lipid layers on solid supports, for application in molecular (bio)electronics and (bio)sensors, as well as for fundamental structural studies using scanning probe techniques.kl1 One of the most attractive goals would be the incorporation of intrinsic membrane proteins into such supported lipid layers, either for the development of highly selective sensor devices based on natural signal recognition and amplification principles or for the immobilization of macromolecules for structural studies. Abstract Dublished in Aduance ACS Abstracts. December 1. 1993. (1) Fendler, J.H. MembraneMimetic Chemistry;Wiley-Intarscience: New York, 1982. (2) Miller, C. Ion Channel Reconstitution; Plenum: New York, 1986. (3) Cecv, G.;Marsh, D. Phospholipid Bilayers; Wiley-Interscience: New York, 1987. (4) Gennis, R. B. Biomembranes; Springer: Heidelberg, 1989. (5) Kuhn,H. ThinSoZidFilms 1989,178,l. Hong,F.T.,Ed.MolecuZur Electronics: Bioeensors and Biocomputers; Plenum: New York, 1989. (6) Roberts. G., Ed. Lannmuir-Blodaett - Film:Plenum: New York, 1990. (7) Ulman,A. An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self-Assembly; Academic: San Diego, CA, 1991. (8)Swalen,J.D.;Allara, D. L.;Andrade,J. D.; Chandroes, E. A.;Garoff, S.; Israelachvili, J.; McCarthy,T. J.; Murray, R.; Pease, R. F.; Rabolt, J. F.; Wynne, K. J.; Yu, H. Langmuir 1987,3,932. (9) Frommer, J. Angew. Chem. 1992,104,1325. (10) Engel, A. Annu. Rev. Biophys. Biophys. Chem. 1991,20, 79. (11) Rademacher,M.; Tillmann, R. W.;Fritz, M.; Gaub, H. E. Science 1992,257, 1900. ~~

For the above mentioned application, usually, the lipid compounds are adsorbed on the surface of metals and glasses, but also semiconductors, and natural products such as muscovite mica, either by physisorption or by covalent cross-linkage. Lipid layers attached by noncovalent bonds to the solid support are less stable than those fixed covalently. Classical examples of the first case are the transfer of Cd or Ca salts of long-chain fatty acids to solid supports by Langmuir-Blodgett-Kuhn (LBK) techniques.&’ In the second case one of the well-known examples is silanization, especially of glass, e.g. with alkylchlorosilanes which, under elimination of HC1, form stable, covalent Si-0-Si bonds with the -OH groups on the glass surface.12 In general the silanization method is applicable for the controlled formation of hydrophobic surfaces. Furthermore, this method has been extended by using alkoxyailanes instead of chlorosilanes to covalently attach molecular surface layers which contain exposed, terminal -OH, -SH, -COOH, or -NH groups, to mention a few.18 Another strategy is the covalent attachment of sulfurbearing compounds on zero-valent metals such as Au, Ag, Pt, and Cu. The sulfur-bearing compounds are applied mainly in their low-oxidation states as thiols, thioethers, and disulfides. Most of the work has been performed using linear alkanethiols and their substituted analogues, dialkyl ~

(12) Sagiv, J. J. Am. Chem. SOC.1980,102,92. Netzer, L.;kovici, J.; Sagiv, J. Thin Solid Film 1989,100,67. Maoz, R.; Sagiv, J. J. Colloid Interface Sci. 1984, 100, 465. Gun,J.; Iecovici, R.; Sagiv, J. J. Colloid Interface Sci. 1984,101,201. Till”, N.; Ulman,A.; Schddkraut, J. S.; Penner, T. L.J . Am. Chem. SOC.1988,110,6136. Waaserman,5.R.;Tao, Y. T.; Whitesides, G. M. Langmuir 1989, 5, 1074. Waaserman, 5,R.; Whitesides, G. M. Tidswell, I. M.; Ocko, B. M.; Pershan, P. S.; Axe, J. D. J. Am. Chem. SOC.1989,111, 5852. (13) Miller, J. D.;Lhida, H. Langmuir 1986,2,127. Haller, I. J. Am. Chem. SOC.1987,100,8050. Yee, J. K.; Parry, D. B.; Caldwell, K. D.; Harris,J. M.Langmuir 1991,7,307. Kessel, C. R.; Granick,S. Langmuir 1991, 7, 532.

0743-7463/94/2410-0197$04.50/00 1994 American Chemical Society

Lang et al.

198 Langmuir, Vol. 10,No. 1, 1994

thioethers and dialkyl s~lfides,~J* but also more sterically complicated structures such as phospholipids with the sulfur at the termini of the hydrophobic alkyl chains have been used.ls Several fundamental studies have been undertaken in order to investigate the mechanism involved in the adsorption process of the thio compounds, as well as the structures of the resulting molecular layers on gold surfaces.1'3-29 Unfortunately, the supported lipid layers described so far are, in general, not suitable for reconstituting intrinsic membrane proteins in a functionally active form. This is mainly due to the particular structure of these layers, which are too rigid, are very often crystalline, contain an unsuitably high proportion of hydrophobic to polar parts in the molecules, and, furthermore, are directly attached to the solid support excludingthe space and water required for the proper folding of the extramembraneous parts of a membrane protein. I t is the topic of this paper to present a new concept for the covalent attachment of lipid bilayers to a solid support (here gold) combining the required properties of an artificial membrane for protein insertion, with the wellestablished ways of lipid fixation by sulfur groups to gold surfaces.30 For a membrane protein to function correctly, it is of fundamental importance to have water on both sides of the lipid bilayer into which the protein is incorporated. In order to fulfill this requirement, we attached the lipid bilayer via a hydrophilic spacer chain to the solid support, using specially synthesized lipids bearing oligoethylene glycols of variable lengths. These chains are resistant to acidsand bases and have been shown to preserve the biological activity of many membrane proteins when present in such detergents as,e.g., TWEEN or Triton (see ref 4, p 90). The synthesis was performed using a commerciallyavailable dialkylglycerophosphatidic acid to which the ethylene glycol spacers were coupled directly. In order to prevent competition reactions between thiol and alcohol groups in the coupling reaction of the spacers to the phosphatidic acids, a disulfide group was selected as the functional terminus at the hydrophilic spacers. Recent studies indicate that disulfides adsorb on gold similarly to thiols without any loss of stability of the resulting 1ayer.ls (14) Bain, C. D.; Whitesides, G. M. Angew. Chem. 1989,101,522 (and referencea therein). Walczak, M. M.; Chug, C.; Stole, S. C.; Widrig, C. A.;Porter,M.D.J.Am.Chem.Soc. 1991,113,2370. Evans,S.D.;Urankar, E.; Ulman, A.; Ferris, N.J. Am. Chem. SOC.1991,113,4121. (15) Diem, T.; Czajka, B.; Weber, B.; Regen, S. L. J. Am. Chem. SOC. 1986,108,6094. Fabianowski, W.; Coyle, L. C.; Weber, B. A.; Granata, R. D.; Castner, D. G.; Sadownik, A.; Regen, S. L. Langmuir 1989,5,35. (16) Porter, M. D.; Bright,T. B.; Allara, D. L.; Chidsey, C. E. D. J . Am.

Chem. SOC.1987,109,3559. (17) Finklea, H. 0.;Avery, S.; Lynch, M.; Furtsch, T. Langmuir 1987,

3,409. (18) Troughton, E. B.; Bain, C. D.;

Whitesides, G. M.; Nuzzo, R. G.; Allara, D. L;Porter, M. D. Langmuir 1988,4,365. (19) Strong, L.; Whitesides, G. M. Langmuir 1988,4,546. (20) Ulman, A.;Eilers, J. E.; Tillman, N.Langmuir 1989,5, 1147. (21) Bain,C. D.;Troughton,E.B.;Tao,Y.T. T.;Evall,J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989,111, 321. (22) Bain, C. D.; Evall, J.; Whitesides,G. M. J. Am. Chem. SOC.1989,

111, 7155. (23) Whitesides, G. M.; Laibinis, P. E. Langmuir 1990, 6, 87. (24) Stole, S. M.; Porter, M. D. Langmuir 1990,6, 1199. (25) Widrig, C. A.; Alves, C. A,; Porter, M. D. J . Am. Chem. SOC.1991, 113, 2806. (26) Chaudhury, M. K.; Whitesides, G. M.Science 1992,255, 1230. (27) Folkers,J. P.; Laibinis, P. E.; Whitesides, G. M. Langmuir 1992, 8, 1330. (28) Edinger, E.; GBlzhiuser, A.; Demota, K.; Well, Ch.; Grunze, M. Langmuir 1993,9,4. (29) Bertileeon, L.; Liedberg, B.Langmuir 1993,9, 141. (30) A preliminary account of this work has previously been pub-

lished Lang, H.; Duechl, C.; Griitzel, M.; Vogel, H. Thin Solid Films 1992,210/211,818.

This new class of lipids, named "thiolipids" in the following, was first characterized on a water-air interface by means of a Langmuir film balance combined with epifluorescence microscopy, in order to investigate the phase behavior of the lipid monolayers and compare their properties with those of well-characterized conventional phospholipids. Subsequently, different protocols were developed to couple mono- and bilayers to gold surfaces via the thiolipids, using self-assemblyand LBK techniques. In particular we investigated the formation of lipid bilayers composed of (i) pure thiolipids, (ii) a gold-attached thiolipid monolayer with a second conventional phospholipid monolayer on top, and (iii) finally a first mixed monolayer of thiolipids and phospholipids on gold, with a second phospholipid monolayer on top. The layer formation was detected in situ by electrical impedance measurements, cyclic voltammetry, and surface plasmon resonance. 11. Experimental Section Materials and General Methods. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC), l-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC), and 1,2-dipalmitoyl-sn-glycero-3phosphatidic acid disodium salt (DPPA) 8 were purchased either from Fluka or Avanti; n-octyl-8-D-glucopyranoside (OG) was purchased either from Bachem or Sigma. These compounds, as well as the synthetic lipids 10, 11,12,13, and 14 (Figure 4), were stored at -18 "C in a desiccator. Ethanol (puriss.), pyridine (puriss.), dichloromethane (puriss.), diethyl ether (puriss.), potassium chloride (puriss.), 1-hexadecanethiol (pract.), 2,4,6triisopropylbenzenesulfonylchloride (TPS) (9), bis(2-hydroxyethyl) disulfide (5), diethylene glycol monochlorohydrine (l),and triethylene glycol monochlorohydrine (3) were obtained from Fluka (Buchs, Switzerland). Pyridine was f i s t distilled over potassium hydroxide followed by chlorosulfonic acid and finally over calcium hydride. Dichloromethane and diethyl ether were distilled over calcium chloride and sodium, reapectively. All other products were used without further purification. The water used was doubly deionized and treated on activated charcoal using a Millipore-Q system. Thin-layer chromatography (TLC) was carried out on precoated plates (0.2 mm silica gel on aluminum support; E. Merck silica gel 60 F 254). The products were visualized by dipping the plate briefly into a solution of phosphomolybdic acid (4 g in 100 mL of ethanol), followed by heating of the plate. Column chromatography was performed with silica gel 60 (E. Merck, particle size 70-230 mesh) at ambient pressure. 'H-NMR spectra were measured with samples dissolved in CDCb, or a mixture of CDCla/CDsOD(2/1),at room temperature, using a Bruker AC-250 spectrometer operating at 250 MHz unless otherwise stated. Chemical shifts 6 are given in ppm, usingTMS as internal standard. Elemental analyses were carried out in the Microanalytical Laboratory of Ciba Geigy, Basel, Switzerland. 1-Mercaptodiethylene Glycol (2). The compound was synthesized by modifying published reaction schemes for the preparation of aliphatic mercaptanes.s* A 10-g(80mmol) portion of diethylene glycol monochlorohydrin 1 and 30 g (400 mmol) of sodium hydrogen sulfide monohydrate were dissolved in 200 mL of ethanol and heated to 60 "C. A mixture of 20 mL of concentrated hydrochloric acid (d = 1.19) and 100mL of ethanol was then added dropwise over a period of 6 h. To destroy the remaining inorganic sulfide,the addition of ethanolic hydrochloric acid was continued at room temperature until the suspension grew pale. The precipitated solid was filtered off and the solvent evaporated. The residue was dissolved in 50 mL of cold ethanol and filtered again, to obtain a yellow oil after evaporation of the solvent. The crude product was purified by bulb-to-bulb distillation (2 X 10-1 mbar, 160 "C), yielding 6.4 g (66%) of a nearly colorless oil. 1H-NMR (CDCb): 6 = 1.54 (t,J = 8.2 Hz, (31) Ellis, L. M., Jr.; Reid, E. E. J. Am. Chem. SOC.1932,54, 1674. Gilman, H.; Plunkett, M. A.; Tolman, L.; Fullhart, L.; Broadbant, H. S.

J . Am. Chem. SOC.1945,67, 1845.

Lipid Bilayers on Gold

Langmuir, Vol. 10, No. 1, 1994 199

lH, -SH); 2.2 (b, lH, -OH); 2.64-2.73 (2t, J1 = 6.3 Hz, J 2 = 8.2 Hz, 2H, -CHrSH); 3.53-3.63 (m, 4H, -CH,-O-CHz-); 3.69-3.74 (m, 2H, -CH2-OH). 1-MercaptotriethyleneGlycol (4). 4 was analogously prepared using 10g (60mmol) of triethylene glycol monochlorohydrin 3,22.2 g (300 mmol) of sodium hydrogen sulfide monohydrate, and 15 mL of hydrochloric acid (d = 1.19). Bulb-to-bulb distillation was carried out at 5 X le2mbar and about 200 "C, yielding 5.4 g (55%) of a colorless oil. 1H-NMR (CDCls): 6 = 1.57 (t, J = 8.2 Hz, lH, -SH); 2.38 (b, lH, -OH); 2.64-2.72 (2t, J1=6.4 Hz, J 2 = 8.2 Hz, 2H, -CHz-SH); 3.57-3.66 (m, 8H, -CH2O-CH2-); 3.67-3.75 (m, 2H, -CH2-OH). Bis[5-hydroxy-3-oxapntyl] Disulfide (6). The compound was synthesized by modifying published reaction schemes for the preparation of dialkyl A3-g (24.6mmol) portion of 1-mercaptodiethylene glycol (2) was dissolved in 100 mL of methanol and mixed with a solution of potassium carbonate (1.7 g, 12.3 mmol in 50 mL of water). A 3.1-g (12.3 mmol) portion of iodine in 100 mL of methanol was added dropwise at room temperature, thus precipitating potassium iodide. If a remaining yellow color was observed, a small amount of sodium sulfite was added until decoloration. After evaporating to dryness, the residue was suspended in 30 mL of ethanol and the potassium iodide removed by filtration after cooling. The resulting solution was evaporated once again. Purification followed by quick bulbto-bulb distillation (10-2 mbar, 200 "C), yielding 2.6 g (87%) of a barely colored oil. 1H-NMR (CDCls) 6 = 2.66 (b, 2H, -OH); 2.91 (t, J = 6.3 Hz, 4H, -CH2-S); 3.55-3.58 (m, 4H, -CH2-CH2OH); 3.7-3.76 (m, 8H, -CHrOH, -CHZ-CH~-S). Bis[S-hydroxy-3,6-dioxaoctyl]Disulfide (7). 7 was prepared analogously to 6 using 3 g (18 mmol) of l-mercaptotriethylene glycol (4), 2.3 g (9 mmol) of iodine, and 1.3 g (9.5 "01) of potassium carbonate. To eliminate the inorganic salt, the product was dissolved in methylene chloride and purified over a 5 cm X 1cm silica gel column, using CH2C12:MeOH (100:5) as eluent. Afterward, it was dried under vacuum for some hours yielding 2.2 g (74%) of a nearly colorless oil. 'H-NMR (CDCls) 6 = 2.88 (t, J = 6.6 Hz, 6H, -OH, -CHz-S); 3.55-3.58 (m, 4H, -CH&H2-OH); 3.6-3.75 [m, 16H; U C H A H 2 - O - (6 = 3.63, 8); CH2-CH2-S (6 = 3.72, t, J = 6.6 Hz); CHz-OH]. Bis[2-(1,2-dipalmitoyl-s~-glycero-3-phosphoryl)ethyl] Disulfide Monohydrate (10). TPS has been originally used m a coupling agent for the synthesis of internucleotide bondss and has subsequently found application for the coupling of alcohols to phosphatidic acids.% Here the following procedure was applied. A 100-mg (144 pmol) portion of well-dried 1,2dipalmitoyl-sn-glycero-3-phosphatidic acid disodium salt (8) and 87 mg (288 pmol) of TPS 9 were suspended in 5 mL of anhydrous pyridine, with ultrasonication. The mixture was warmed briefly until a clear solution wm obtained and then stirred for 15 min. A 9-mg (58 mmol) portion of bis(2-hydroxyethyl) disulfide (5) (previouslyevaporated several times from a solution of anhydrous pyridine) was dissolved in 1mL of anhydrous pyridine and added to the solution prepared above. The mixture was stirred overnight at room temperature, with exclusion of moisture. Next, 1mL of anhydrous methylene chloride was added. The mixture was warmed briefly and stirring continued for another hour. One milliliter of water was added and, after 15 min, the mixture was again warmed briefly and finally evaporated to dryness under reduced pressure (to prevent foaming during the evaporation, some toluene was added). The obtained yellowish residue was suspended in 20 mL of anhydrous diethyl ether by ultrasonication and filtered. The filtrate was evaporated and purified on a silica gel column (30 cm X 1cm). The column was first washed with 100 mL of chloroform, the product then being eluted using a gradient of CHC13/MeOH(1W3 100:5). To eliminate traces of silica gel, the product was dissolved once more in 20 mL of diethyl ether and filtered. Recrystallization was performed by dissolution of 1 mL of chloroform and the subsequent addition

-

(32) McAllan, D. T.; Cullum, T. V.; Dean, R. A.; Fidler, F. A. J. Am. Chem. SOC. 1951, 73, 3627. (33) Lohrmann, R.;Khorana, H. G. J.Am. Chem. SOC.1966,88,829. (34) Aneja, R.;Chada,J. S.;Davies, A. P. Tetrahedron Lett. 1969,48, 4183. Aneja, R.;Chada,J. S.;Davies, A. P.Biochim.Biophys.Acta 1970, 48, 4183. Aneja, R.;Davies, A. P. Chem. Phys. Lipids 1970, 4 , 60.

of 10 mL of acetone. After 5 h at -15 "C, the white precipitate was isolated by filtration and dried under vacuum, yielding 47 mg (57% , based on the disulfide) of a colorless solid. Rf0.56 (CHCWMeOH/HzO(65254)). 'H-NMR (CDCldCDsOD (2:l)) 6 = 0.88 (t, J = 6.3 Hz, 12H, -CHs); 1.27-1.4 [m, 96H, -CH2 (c4-c16)1;1.61 [b, 8H, -CH2 (C3)l;2.28-2.36 [m, 8H, -CH2 ((2211; 3.02 (t,J 6.3 Hz, 4H, 4H2-S); 4.00 (t, J = 6.3 Hz, 4H, S-CH2CH2-); 4.17-4.45 (m, 12H, PO-CHz-CHCHI. Anal. Calcd for C,~HI~~OI&PZ-H~O: C, 61.98; H, 10.26; S, 4.47; P, 4.32. Found: C, 60.65; H, 9.50; S, 4.07; P, 4.23. Bis[ 5 4 1,2-dipalmitoyl-sn-glycero-3-phosphoryl)-3-oxapentyl] Disulfide Monohydrate (11). 11 was analogously prepared using 78 mg (288 pmol) of TPS 9,14 mg (58 pmol) of bis[5-hydroxy-3-oxapentylldisulfide (6), and 100mg (144 pmol) of 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid disodium salt (8). Purification and recrystallization were carried out as described before, with the exception that the gradient used in this case was CHCldMeOH (100:3 100:4). The yield was 64 mg (72%, based on the disulfide) of a colorless solid. Rf0.59 (CHCldMeOH/H20 (65:25:4). 'H-NMR (CDCldCDsOD (2:l)): 6 = 0.88 (t, J = 6.5 Hz, 12H, -CH3); 1.27-1.35 [m, 96H, -CH2 (C,-CldI; 1.61 [b, 8H, -CHa(Ca)l; 2.28-2.36 [m, 8H, -CH2(C2)1; 2.94 (t,J = 6.3 Hz, 4H, -CHrS); 3.68 (b, 4H, PO-CHz-CHz-); 3.77 (t,J = 6.3 Hz, 4H, S-CH~-CHZ-);3.97-4.03 (m, 8H, POCH2-); 4.14-4.44 (m, 4H,-CH2-O-CO-); 5.23 (b, 2H, >CH). Anal. Calcdfor C,eH16~01&P2.H20: C, 61.55; H, 10.07; S, 4.21; P, 4.07. Found C, 60.18; H, 9.44; S, 4.07; P, 4.05. Bis[ 8 4 1,2-dipalmitoyl-sm-glycero-3-phosp horyl)3,6-dioxaoctyl] Disulfide Monohydrate (12). 12 was analogously prepared using 78 mg (288 pmol) of TPS 19,19 mg (57 pmol) of bis[8-hydroxy-3,6-dioxaoctyl]disulfide (7), and 100mg (144 pmol) acid disodium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphatidic (8). Purification and recrystallization were carried out as described for 11, yielding 63 mg (69%,based on the disulfide) of a colorless solid. Rf 0.63 (CHCUMeOH/H20 (65:254)). 'HNMR (CDCldCDsOD(2:l)): 6 = 0.88 (t,J = 6.5 Hz, 12H,-CH3); 1.27-1.35 [m, 96H, -CHz(Cd-C16)1;1.61 [b, 8H, -CH2(Cs)l; 2.282.36 [m, 8H, -CH2 (C2)l; 2.94 (t, J = 6.3 Hz, 4H, -CHz-S); 3.67 (b, 12H, C-O-CH2-CH2-04); 3.76 (t,J = 6.3 Hz, 4H, S-CH2CHz-); 3.99-4.03 (m, 8H, PO-CHz-); 4.14-4.44 (m, 4H, -CH2O-CO-); 5.23 (b, 2H, >CHI. Anal. Calcd for C E ~ H ~ W O ~ ~ S ~ PyH20: C, 61.16; H, 10.14; S, 3.98; P, 3.85. Found: C, 61.12; H, 10.03; S, 3.88; P, 3.75. 1-Chloro-5-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)-3oxapentane (13). A 262-mg (866 @mol)portion of TPS 9,135 mg (1.08 mmol) of diethylene glycol monochlorohydrin (l),and 7 mL of anhydrous pyridine were stirred for 30 min, with exclusion of moisture. Then, 150 mg (217 pmol) of well-dried 1,2dipalmitoyl-sn-glycero-3-phosphatidic acid disodium salt was added and the suspension was first ultrasonicated and then warmed briefly. The resulting clear solution was stirred for 3 h at room temperature before 1 mL of anhydrous methylene chloride was added. The further steps were carried out analogously to 10. Column chromatography was performed on silica gel (30 cm X 1cm), using a gradient of CHCla CHCUMeOH (100:4). The yield was 92 mg (80%,based on the phosphatidic acid) of a colorless solid. Rf0.67 (CHCldMeOH/H20 (65:254)). 1H-NMR (CDCl3) (Bruker AC-P 200 operating at 200 MHz): 6 = 0.88 (t,J = 6.4 Hz, 6H, -CHs); 1.26-1.33 [m, 48H, -CH2 (C4ClS)]; 1.59 [b, 4H, -CH2 (C3)l;2.25-2.35 [m, 4H, -CH2 (C2)l;2.61 (b, lH, P-OH); 3.65-3.87 (m, 6H,-CH2UCH2-,-CHAl); 3.984.10 (b, 4H, PO-CH2-1; 4.14-4.49 (m, 2H, -CH2-O-CO-); 5.3 (b, lH, >CH). Anal. Calcd for C&,&PCl.H20: C, 60.06; H, 9.91; P, 4.00; C1, 4.58. Found: C, 59.94; H, 9.99; P, 3.87; C1, 4.49. 1-Chloro-5-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)3oxaoctane (14). 14 was analogously synthesized as 13 but using 1mmol of triethylene glycol monochlorhydrin. Rf0.69 (CHCld MeOH/H20 (65254)). 'H-NMR (CDCls) (Bruker AC-P 200 operating at 200 MHz): 6 = 0.88 (t, J = 6.4 Hz, 6H, -CH3); 1.261.33 [m, 48H, -CH2 (C4-Cla)l; 1.60 [b, 4H, -CH2 (C3)l;2.26-2.36 [m, 4H,-CH2 (Cz)];2.61 (b, 1H,P-OH); 3.65-3.87 (m, 10H,-CHr O-CH2-, -CHrCl); 3.99-4.06 (b, 4H, PO-CHs-); 4.14-4.49 (m, 2H, -CH2-0-CO-); 5.3 (b, l H , >CH). Anal. Calcd for C41H~01&%HzO:C, 61.70; H, 10.13; P, 4.03; C1, 3.89. Found: C, 60.01; H, 10.10; P, 3.92; C1, 4.20.

-

-

Lang et al.

200 Langmuir, Vol. 10, No. 1, 1994 view from above

cross-section from the side

I

Ag'AgC'

Cr (5-10 nm)

0

p-Si (about0.3mm) AU (200nm)

Figure 1. Configuration of the gold electrodes used for the electrochemical experiments. Monolayer Experiments. The monolayer experimentswere performed on a commercial Langmuir trough (Riegler & Kirstein),a which was mounted on the three-directional, motorstepper driven translation stage of a Zeiss Axiotron microscope. The epifluorescence micrographs (excitation by a 50-W Hg-Xe lamp with an interference filter at 450-490 nm combined with a dichroitic mirror >510 nm, cut off filter >520 nm) were recorded with a SIT-video camera (Hamamatsu),directly attached to the microscope, and were stored on a video recorder. The images shown were taken from the video screen and correspond to 80 X 120 pm2 on the water surface. The thiolipids were dissolved in chloroform (1mg/mL) together with 1mol % fluorescent labeled 1,2-dimyristoyl-sn-glycero-3phosphoethanolamine(NBD-PE, Molecular Probes, Eugene, OR) and spread on the surface of deionized (Millipore) water. Gold Electrodes. The samples were purchased from the "Centre Suisse d'Electronique et de Microtechnique, Neuchfitel, Switzerland". The texture of the sample is shown in Figure 1. In order to clean the gold surface, the whole sample was immersed for 1min in a mixture of 1g of potassium bichromate in 100 mL of 98% sulfuric acid at 100 "C. Next, the sample was washed abundantlywith deionized water. This procedure was performed twice before placing the sample in the aqueous lipid dispersions. If an ethanolic alkanethiol solution was used, the sample was finally rinsed briefly with ethanol before immersing. The slides were transferred into the appropriatelipid solution withoutdelay. Preparation oft he Lipid Dispersions. The corresponding amounts of OG and of the lipids were ultrasonicated in 1mL of aqueous 0.1 M KCl, until clear dispersions were obtained. All dispersions were prepared freshly and used within 1day. The thiolipid dispersions of lower concentration were prepared by diluting the stock sample with OG (48 mM, in 0.1 M KC1). The l-hexadecanethiol solution (3.9 mM) was prepared in deoxygenized ethanol, with ultrasonicationunder exclusion of air. The solution was used immediately. Performance of the Self-Assemblyand Formation of the Mono- and Bilayers. Gold electrodes (1mm2)were connected to the measurement cell shown in Figure 2. Initially, 50 pL of 0.1 M KCl was added to the cell to determine the electrical propertiesof the bare gold surface. Then, 0.5 mL of the thiolipid solution was added. The adsorption of the lipid was followed by measuring continuously the two current components. For the formation of the thiolipid monolayers, the self-assembly was stopped after appropriate incubation times and the resulting layer washed abundantly with a solution of OG (48 mM, in 0.1 M KC1) until a stable signal was reached. For the succeeding bilayer formation on the previously-existing monolayer, 0.2 mL of a dispersion of the conventional lipid DMPC, DPPC, or POPC (1mg/mL, 48 mM OG) was placed to the cell and diluted slowly with small portions of 0.1 M KC1 until no further decrease of the current response could be evoked by dilution. In the case of mixed bilayers, the self-assemblywas interrupted after a specific time (in general 2-3 min) and the resulting layer (35) Riegler, J. E. Rev. Sci. Instrum. 1988,59, 2220.

sample Figure 2. Diagram of the measurement cell for the electrochemical experiments.

was washed abundantly with a solution of OG (48 mM, in 0.1 M KC1). The thus-formed, imperfect monolayer was treated with the dispersion of a conventionallipid as described for the thiolipid monolayer. When a l-hexadecanethiol monolayer was used, the selfassembly was stopped after 8 h and the sample was washed with ethanol and dried under air for 30 min. The formation of a second layer on this monolayer using one of the conventional lipid was performed as described for the thiolipids. Surface Plasmon Resonance (SPR). In an attenuated total reflection (ATR) scan, the excitation of the surface plasmon waves is seen by a pronounced dip in the reflectivity. The optical properties of the thin organic film were derived by fitting the experimental reflectivity-vs-angle (8-28) scans with curves calculated according to the Fresnel equations. The time-dependent measurements were taken at a fixed angle at the steepest part of the resonance curve at an angle slightly smaller than the resonance angle. A shift of the reflection minimum to greater angles due to the adsorptionof an organic layer caused an increase in the measured reflectivity. The surface plasmon resonance measurements were performed on a home-made computerized reflection apparatus as described in detail elsewhere.% At the resonance angle, the incident laser beam ( H e N e laser, 632.8 nm, p-polarized) couples via an unilateral, high index prism (SF 10, n = 1.723) to the surface plasmon mode in a thingold film, using the Kretschmann coupling scheme.37 Impedance Measurements. A sinusoidal single frequency voltage (120 Hz, ac 117.5 mV, dc 0 mV vs Ag/AgCl in 0.1 M KCl) was applied to the cell and the resulting potential drop at a resistor Ro of 1kQ was measured and analyzed by a lock-in amplifier as the sum of two harmonics, cp and cp + 90". The amplitude of one of the harmonics was taken as a Faradaic current, while the other was considered to correspond to capacitive current. The instrument used was a two-phase lock-in amplifier, Model 5206 from EG&G Brookdeal Electronics, Princeton Applied Research, equipped with an audio frequency plug-in card (operatingin the frequency range of 20 Hz to 20 kHz). Cyclic Voltammetry. Cyclic voltammetry was performed using an electrochemical analyzer "BAS loo", Bioanalytical System, Inc., which used a conventionalthree-electrode cell. With this method, a sawtooth voltage profile is applied to a circuit, with the resulting current response being recorded. Typically the cyclicvoltammograms were run in the potential range between -0.2 and +0.1 V using Ag/AgCl (0.1 M KC1) as the reference electrodeand Pt as the working electrode, and employing a sweep rate of 51.2 mV/s. The first scan was started from the open circuit potential in the negative direction. Evaluation of the Electrical Properties of the Supported Layers. ( i ) From Impedance Measurements. In order to determine the capacitance and the resistance of lipid layers covering the gold electrode, we modeled the electrochemical (36) Terrettaz, S.; Stora, T.; Duschl, C.; Vogel, H. Langmuir 1993,9, 1361. (37) Kretschmann, E. Opt. Commun. 1972,6,185.

Langmuir, Vol. 10, No. 1, 1994 201 H3C H3C UO I

I

I I

I

I

-

I

I uc

:

electrolyte

I I

electrode

+

: I I

CH3 CH3

\lo il ::: ;;

0, 0,

H2C-CH

layer

n=3

\

CH2

I

I

HC-CH2 / o /o

12

I

9 HO-Y=O

Figure 3. Simplified equivalent circuit of the electrochemical measurement cell composed of the lipid layer of resistance R1 and capacitance C and the electrolyte of resistance R,. U,is the applied sinusoidal voltage of 17.5 mV and response across a resistor R,.

U,the measured

[o.CH~cH2]n\

measuring cell by the electricalequivalent circuit shown in Figure 3 which is based on several simplifying assumptions. Firstly, the capacitance of the measurement cell with a bare gold electrode in 0.1 M KC1solution is supposedly dominated by the Helmholtz layer of the ionic components of the buffer, typically in the range of 30 kF/cm2. This value is an order of magnitude greater than that of the capacitance of a lipid layer and can therefore be neglected, to a good approximation. Furthermore, because the resistances of the electrolyte and the bare electrode are in series, they can be combined as Re. UOis the applied sinusoidal voltage and U,the measured response across the internal resistor Ro of the instrument. Assigning Z as the impedance of the measurement cell, composedof the electrolyte,the layer, and the electrode, U,for the described circuit can be derived as follows

uo=zz+u,

s-s

/[CH2\CH;o]n

9

HO-y-0

(1)

with

9

O=Y-OH

0 . CH2/CH2\ 0/CH2\

CI CH;

Figure 4. Chemical structure of the synthesized lipids. Z,d=Ro+Z

and

I=UdZ,d

(2)

one obtains

U,= U&d (2+ Ro)

(3)

Z is defined as follows: (4)

This leads to the following expression for U,

uc= u," + iuc'M

u,"

UCm= U&dRl With

Z = Re + ( l / R l+ id)-'

u,"

Applying the same simplification to the denominator of the real term of U,and, furthermore, neglecting the second part of the numerator, the real component of the voltage response is

one obtains

(5)

oCRJt? = uo (6) (R, + Re + R1)*+ (Ro + Re12( W C R ~ ) ~ Ro(Ro+ Re + Rl) + Ro(Ro+ Re) (oCR1l2 (R, + Re + R112+ (Ro+ Re)' (wCRl)2

= uo

(7)

where Ucmand Ucmrepresent the real and imaginary components, respectively. Considering once again the imaginary term of U,,one notes that for Rl >> Ro, Re and a frequency of 120 Hz, the denominator is determined by R12. This leads to the following simplification

UCM= UooCRo

Rl= UdZf

(9)

The resistance of the layer can thus be evaluated by measuring the faradaic current Zf of the system. Although this approximation leads to a greater error than for the imaginary term, it yields reliable data for qualitative comparison between the different layers. (ii)From Cyclic Voltammetry. The capacitance of electrically blocking supported lipid layers can also be determined by cyclic voltammetry, applying the following relations Q=CU dQ/dt = C(dU/dt)

(10)

C = I(dU/dt)-'

(12)

(11)

where I is half of the current difference between the forward and the reverse scans in the voltammogram and dU/dt is the sweep rate (51.2 mV/s).

with

u p = IJl0 one obtains

c = IJ(uU0)

u,m = I$o

(8)

The capacitance of the organic layer can thus be determined by measuring the capacitive current Z, of the system.

111. Results Lipid Synthesis. The disulfide-bearing and the chlorine-bearing lipids, with the structure shown in Figure 4, were prepared by direct esterification of the phosphate group of DPPA with the appropriate alcohol using TPS as condensing agent (Scheme 1,iii). The synthesis of the

Lang et al.

202 Langmuir, Vol. 10, No. 1, 1994

-

Scheme 1

H (OCH2CH2 ),CI

I CI

IE

(9

H (0CH2CH2 ),SH

Y

Y

e!

I e!

a.

8 a! v)

hydroxy-bearing chains followed the same procedure for all compounds used. We started with the monochlorohydrine of a glycol substituting the chloride for a thiolate anion (Scheme 1,i). The disulfides were formed from the thiols by oxidation with iodine (Scheme 1,ii). The yield of chlorine-bearing lipid was 80%. Due to the double coupling reaction the yield was slightly lower, around 70%, for the three thiolipids. Lipid Monolayers at the Air-Water Interface. The compression of monomolecular films of amphiphilic molecules on a water surface of a Langmuir trough with simultaneous pressure reading via a Wilhelmy plate (pressure-vs-area isotherms) gives detailed information on the molecular dimensions, packing properties, and phase transitions of the films.38 Fluorescence microscopy allows the observation of lateral structure formation in the monolayer during phase tran~ition~9-4~ due to different solubilities of the introduced dye dopant in different phases. The combination of these techniques represents one of the standard characterizations of amphiphilic molecules. The pressure-vs-area/molecule isotherms of the three thiolipids are depicted in Figure 5. While the isotherms of the thiolipids 11 and 12 show a clear break, indicating a first-order phase transition from a liquidexpanded to a solid-condensed state at approximately 14 and 25 mN/m, respectively, the isotherm of thiolipid 10 lacks this feature. There, from a flat region at .rr = 0 mN/m the curve rises steeply without an intermediate transition region. This establishes a clear monotonic dependence of the transition pressure of the film on the spacer length of the thiolipids, with the longest spacer causing the highest pressure break. The areas per molecule in the films at 35 mN/cm2 of thiolipid 10, 11, and 12 are 75,80, and 81 A2, respectively, and compare well with the areas occupied by four extended hydrocarbon chains aligned along the normal to the water surface. The fluorescent micrographs of thiolipids 11 and 12 (Figure 6) show star-shaped domains which contain no fluorescent dopant and which emerge at the onset of the coexistence plateau of the phase transition, as for many conventional lipid sy~tems.3~"'~ The superstructures in which the domains arrange themselves are indicative of a repulsive interaction between the domains. While the domains are very regular for the lipid 12, the patterns of lipid 11 are much more irregular in shape and contain filamentous and dendrite-like elongations. Interestingly, the chlorine-bearing lipids 13 and 14 show similarities to their thiolipid partners of the same spacer length. Ap~

~~

(38) Adamson, A. W. Physical Chemistry of Surfaces; Wiley: New York, 1976. (39) Peters, R.; Beck, K. Proc. Nutl. Acad. Sci. U.S.A. 1983,80,7183. (40) LBsche, M.; Sackmann, E.; MBhwald,H. Ber. Bunsen-Ges. Phys. Chem. 1983,87,848. (41) McConnell, H. M.; Tamm,L. K.; Weis, R. M. Proc. Natl. Acad. Sci. U.S.A. 1984,81,3249. (42) Knobler, C. M. Science 1990, 249, 870. (43) MBhwald,H. Annu. Rev. Phys. Chem. 1990,41,441. McConnell, H. M. Annu. Rev.Phys. Chem. 1991,42,171.

BO

100

120

140

160

180

200

220

AredMolecule [A 21

Figure 5. Pressuremolecular area isotherms of thiolipids 10, 11, and 12 (compression rate, 3 Az/min; subphase, water; temperature, 22 "C).

parently, the length of the hydrophilic spacer group influences the pattern formation between the solid and fluid coexisting phases. During expansion, the patterns melt again and disappear completely at the discontinuity in the isotherms. This, together with the fact that there is almost no hysteresis between the compression and the expansion isotherms, is a clear sign that the system is reversible. The behavior of the monolayers formed from thiolipid 10 when observed by the fluorescencemicroscope is quite different. Immediately on spreading the lipid solution at T = 0 mN/m irregular structures appear. During compression, only the ratio between dark and bright areas increases, an effect which can be understood as a simple increase in the degree of crystallization. Similar behavior was found for the lipids in which, compared to the thiolipids, the sulfur is substituted by chloride. The break pressure indicating the phase transition showsthe same qualitative dependence on the spacer length as for the thiolipids. However, compared to the respective thiolipids with the same spacer, the onset of phase transition occurs at even higher pressures: 24 mN/m for lipid 13 and at 31 mN/m for lipid 14. The shapes and sizes of the domains formed from the chloride lipids resemble those of the thiolipids very strongly (see Figure 6). Electrochemical Measurements. Formation of the Self-Assembled Mono- a n d Bilayers by Adsorption of Disulfide-Bearing Lipids from Micelles onto Gold. The adsorption of the thiolipids onto gold was carried out in an aqueous medium. It is necessary to solubilize the lipids in the form of small micelles with the disulfide groups accessible from the aqueous phase in order to make the reaction with the gold surface possible. For this purpose, the detergent OG was used which has a high critical micellar concentration (cmc) of about 20 mM at 20 "C and therefore can be eliminated easily from the resulting thiolipid layer by washing with water. After 3 days of incubation of the gold substrates in a solution of 0.7 mM thiolipid and 48 mM detergent in aqueous 0.1 M KCl, the self-assembly appeared to be complete according to impedance measurements. The layers thus formed do not change their electrical characteristics after washing with water. Thiolipid monolayers were obtained by washing the samples thoroughly with 48 mM detergent solution directly after the SA, removing the second, only physisorbed layer. The hydrophobic surface of the monolayer was then used as a substrate for the formation of lipid bilayers. For this application, the performed thiolipid monolayers were treated with an aqueous solution of 0.1 M KC1 containing

Lipid Bilayers on Gold

Langmuir, Vol. 10, No. 1, 1994 203

-

Figure 6. Fluorescence microscopic pictures taken from various thiolipid and corresponding chlorine lipid monolayers on the water surface at a surface pressure of 30 mN/m (well above the phase transition) a t 22 "C: (A) thiolipid 11; (B) thiolipid 12; (C) chlorinebearing lipid with two spacer units (lipid 13); (D) chlorine-bearing lipid with three spacer units (lipid 14). All photographs show an area of approximately SOx EO pm2.

t-

" I

I

'

capacitive faradaic

Time Figure 7. Self-assembly of a DMPC lipid monolayer on a gold electrode covered by a covalently attached monolayer of thiolipid 12. Conditions: The thiolipid covered gold electrode was placed in the electrochemical cell and covered with 0.2 mL of a lipid dispersion of 48 mM OG and 1mg/mL DMPC. The capacitive and Faradaic currents were measured during stepwise addition of 0.1 M KCl solution. During this process, the concentration of OG was diluted below its cmc thus forming a second lipid monolayer of DMPC.

41 mM OG and 4 mM of the conventional lipids DPPC, DMPC, or POPC, respectively. The physisorption of these lipids was achievedby diluting the solutions stepwise with electrolyte below the cmc of the detergent. This process was recorded by following simultaneously the capacitive and the faradaic current response of the system. An exampleof the simultaneous recording of the faradaic and capacitive currents during the formation of such a bilayer is shown in Figure 7. The chemisorbed thiolipid layers were stable enough to be reused several times for the formation of different bilayers simply by removing the second layers with detergent solution. Another type of a mixed supported bilayer was formed

on an imperfect thiolipid monolayer. In this case the SA process of thiolipid 12 on gold was stopped after 2-3 minutes and the sample was treated with detergent solution, removing nonchemisorbed material. The formation of the bilayer was accomplished as described before, resulting in similar curves for the current responses. Kinetics of the Layer Formation. Here, we investigated the adsorption behavior of the individual disulfidebearing lipids on gold surfaces. The electrodes were immersed in the corresponding detergent-lipid solutions, and the process of layer formation was observed by impedance measurements following the formation of an insulating barrier. This method allows the continuous and simultaneous observation of the capacitive and the faradaic current response of the electrochemical cell. Because the experiments were all performed at detergent concentrations well above the cmc, we expect to preferentially observe the formation of the first thiolipid monolayer on the gold surface under these conditions. Firstly, we determined the influence of the ratio of thiolipid and detergent in the adsorption solution on the electrochemical events. Therefore, the concentration of the lipid was varied between 0 and 0.7 mM, keeping the detergent concentration constant at 48 mM, which is approximately twice its cmc. The measured capacitive current-vs-time curves using thiolipid 11 are shown in Figure 8. For all experiments, the curves start at about 3.4 PA. According to eq 8 one calculates a capacitance of 27 pF/cm2, a value which is dominated by the Helmholtz layer in 0.1 M KCl on bare gold. An interesting, but not unexpected, result is the fact that the pure detergent alone

204 Langmuir, Vol. 10, No. 1, 1994

7xIOE-4M 7XlOE-5 M

A

"

I

0

.~

,

.

200

,

Lung et al.

.

,

400

.

Time

-

I

800

600 [E]

Figure 8. Formation of a monolayer of thiolipid 11 on a gold electrode. 11 was dissolved at the particular concentrations indicated in the figure in 48 mM OG, 0.1 M KCl at room temperature.

0

100

U [mV]

so,

40 30

0

200

V.I

-100 -200 Ag I AgCl I KCI

Figure 10. Cyclic voltammograms (A) of a freshly cleaned bare gold electrode, and (B)of thiolipid 12 bilayera formed on this particular gold electrode. Conditione: 0.1 M KCl, sweep rate 51.2 mV/s. For each caw several consecutive scans were superimposed.

I

I Table 1. Molecular Propertier of Thiolipid Bilayen and 40

20

400

Time

[SI

600

800

Figure 9. Time course of the phase angle during the adsorption of thiolipid 11 on a gold electrode. The insert is an expanded region of the graph. Conditions: 0.7 mM 11,48mM OG, 0.1 M KCl. modified the interface between the gold and the electrolyte by decreasing the capacitive current, albeit only weakly. It is not clear whether this was due to an attachment of the polar OH groups of the glucosidic part to the gold or a modification of the Helmholtz layer or both. This process, however, could be reversed by extensive washing of the electrode with buffer. In the case of the thiolipid, the rate of layer formation depends strongly on the lipid concentration. Similar results were obtained also with thiolipids 10 and 12. Taking together the results obtained so far for layer formation of thedifferent thiolipids on gold, the adsorption kinetics are all composed of at least two different processes. The first process has a characteristic time of seconds or minutes while the slower one takes hours and is apparently completed only after days. Although small changes in the capacitance of the supported thiolipid layers were observed even after several hours, the gold electrode is nearly totally covered by the lipid layer after only a few minutes. This can be nicely demonstrated by measuring the phase angle of the impedance @ = arctan(lJlf), which is an indication of the completeness of the lipid layer on the gold surface. As an example, the time-course of the phase angle during the adsorption process of thiolipid 11 is shown in Figure 9. @ adopts a value of nearly 90° after only a few minutes of incubation time, indicating the presence of an almost totally blocking lipid layer (no membrane translocation of charged species). Accordingto this finding we can regard the layers as purely capacitive to a good approximation. Under these conditions, the capacitance of the supported lipid layers can also be determined by cyclic voltammetry. For this particular investigation, gold electrodes were

Correrponding Subatrate-AttachedMonolayers on Gold Surfacer after Different Timer of Incubating the Eleotrode. capacitance (fiF/cm2)ofbilayers incubation (gold-attachedmonolayers) ~

time (h) lipid 10 lipid 11 livid 12 1 1.51 kO.19 0.81 0.11 0.75 0.06 2 1.14* 0.10 0.72 0.06 0.65 0.06 8 0.80* 0.10 0.62. 0.07 0.59 0.09 72 0.71 0.14 (1.41)0.59 0.06 (1.20) 0.51 & 0.03 (1.14)

*** *

tr

2.25

* *

thickness (A) of bilayera (gold-attached monolayers)* after 72 h of adsorption lipid 10 lipid 11 lipid 12 28.0 (14.1) 33.8 (16.6) 39.0(17.5)

a A gold electrode was immersed in a dispersion of the particular thiolipid in 48 mM OG for the time indicated and subsequently stepwiee diluted and washed with 0.1 M KCl solution. The capacitance values of the thus formed lipid bilayers were measured for each sample by both the impedance method and cyclic voltammetry in the same electrochemical cell. Corresponding capacitance valuea for the gold-attachedmonolayerawere subsequently memured by the same two methods after washing the bilayer containing gold electrode fiist by 48 mM OG solution in 0.1 M KCl followed by washing with 0.1M KCl. The two measuring methods gave identical results within experimental uncertainty. T h e values listed represent the average of five different layer formations. Calculated from the corresponding capacitance values according to eq 13 using t, = 2.25 and e, = 8.85 X 10-l2 A s V-l m-1.

*

incubated with asolution of48 mM detergent and 0.7 mM thiolipid for 1 h, 2 h, 8 h, and 3 days and then washed thoroughly with buffer solution. Figure 10showsthe cyclic voltammograms of a bare gold electrode and of a corresponding electrode aftar deposition of a thiolipid 12 bilayer. Table 1 shows the results of the different thiolipids obtained after different incubation times. These results support the existence of a bilayer. Moat importantly, the values of the capacitance of the lipid layers also determined by the impedance method are in full agreement with those obtained by the cyclic voltammograms. From the known capacitance values, the thickness, d , of the hydrocarbon chain part of the finally completed,

Lipid Bilayers on Gold

Langmuir, Vol. 10, No. 1, 1994 205

supported lipid bilayers can be calculated using the formula: d = e,ao(C/A)-' (13) where CIA is the capacitance per unit area, e, the dielectric constant of the hydrocarbon region, and eo the permittivity of vacuum. It has been shown for the case of planar, freestanding phospholipid bilayers that eq 13 is a reasonably good approximation, since the capacitance of the phospholipid polar headgroup region is considerably higher than that of the lipid hydrocarbon region.Mt45 For the hydrated polar headgroup region, including the spacer chains, of a thiolipid layer the same arguments should be valid. Taking a value of cr = 2.25 for the dielectric constant of the thiolipid hydrocarbon chains, in analogy to corresponding data of saturated crystalline hydrocarbons or polyethylene,* the thickness calculated for the lipid bilayers which were incubated for 72 h are 2434, and 39 A for thiolipids 10, 11,and 12, respectively. For a perfectly ordered lipid bilayer with the alkane chains in an all-trans conformation and not tilted to the membrane plane, one would expect a thickness of 38 A for the hydrocarbon chains. This fits perfectly with the calculated thickness of the thiolipid 12. If the chains of thiolipids 10 and 11 in the gold-supported bilayers adopt an all-trans conformation, they would be tilted from the membrane normal by 42O and 27O, respectively. However, a certain disorder in the thiolipid hydrocarbon chains would also explain the reduced chain thickness for the latter cases. Capacitance data cannot distinguish between these possibilities. Formation of Bilayers on Preformed, Chemisorbed Monolayers. Monolayers of either thiolipid or l-hexadecanethiol on gold were used as a base for the formation of bilayers, with conventional lipids as a second layer. Consequently, a chemisorbed thiolipid or hexadecane monolayer was treated with solutions of either DPPC, DMPC, or POPC and the capacitances of the finally formed bilayers were measured. To test whether or not the proposed model circuit in Figure 3 and the corresponding mathematical descriptions of eqs 6 and 7 fit our system acceptably, the two voltages U,RE and U:M were measured at different frequencies. To this end, a gold-supported bilayer was used, composed of a chemisorbed monolayer of 1-hexadecanethiol and a second monolayer of DMPC. The calculated curves and the measured values for the imaginary and the realvoltage terms are shown in Figure 11. In both cases, the calculated curves fit the measured data quite well in the frequency range of 20-500 Hz. The parameters obtained by this fitting give a layer capacitance, C, of 7 nF (which corresponds to 0.7 pF/cm2) and a layer resistance, R1,of 5.5 MQ which corresponds to a specific resistance of 5.5 104 M Q cm2. The resistance Re, which represents the electrolyte and the electrode, is about 1 kQ, in good agreement with the value expected for the sum of the electrode and that of the electrolyte. The capacitance values measured by the impedance method for the different combinations of l-hexadecane(44)Hanai, To;Haydon, D. A.; Taylor, J . Proc. R. SOC.London 1964, A281,377. Hanai, T.; Haydon, D. A,; Taylor, J. J. Theoret. Biol. 1965, 9,278. Fettiplace, R.; Gordon, L. G. M.; Hladky, S. B.; Requena, 3.; Zingeheim,H. P.; Haydon, D. A. Methods in Membrane Biology. Kom, E. D., Ed.; Plenum: New York, 1975; Vol. 4,p 1. Dilger, J. P.; Fisher, L. R.; Haydon, D. A. Chem. Phys. Lipids 1982,30, 159. (45) Lliuger,P.; Leealauer, W.; Marti,E.; Richter, J.Baochim.Biophys. Acta 1967,135,20. Benz,R.;Fr(ihlich, O.;Liiuger,P.;Montal, M.Biochim. Biophys. Acta 1975, 394, 323. (46)Lanza, V. L.;Herrman, D. B. J. Polym. Sei. 1958, 28, 622. Handbook of Chemistry and Physics;Weat, R. C., Ed.; CRC: Cleveland, OH, 1973.

A

0

REAL

1

100 200 300 400 500 600

frequency [Hz]

400

IMAQINARY

0

100 200 300 400 500 600

frequency [Hz]

Figure 11. Electrical characteristics of a supported lipid bilayer on a gold electrode. The bilayer is composed of a l-hexadecanethiol monolayer, chemically attached to the gold surface, and a second monolayer of DMPC on top. A sinusoidal voltage of 17.5 mV in the frequency range between 20 and 500 Hz was applied to the gold electrode and the voltage response was measured by the impedance technique. Measured pointa and calculated curves are as follows: (A) for the voltage response in phase with the stimulus, i.e. the real part Ucm of eq 7; (B)for the corresponding case with the voltage response 90° out of phase with the simulus, Le. Ucmof eq 6.

thiol or thiolipid/phospholipid mixed, supported bilayers are summarized in Table 2. The most striking result is that the capacitance values of the bilayers are practically identical for a particular class of phospholipidsas a second layer, irrespective whether we used one of the thiolipids (11,12) or 1-hexadecanethiolasa first layer. The exception is thiolipid 10 as a base, which always yields bilayers of higher capacitance values. Under the assumption that the capacitance of the whole bilayer, Chi, is composed of the capacitance Cmonol, the measured capacitance of the chemisorbed thiolipid or hexadecane monolayer, and in series the capacitances Cmono2 of the second monolayers consisting of the conventional lipids, the following formula is valid The calculated values of Cmono2 obtained for the second phospholipid monolayers formed on the first layer of thiolipids 10, 11, and 12 are in the range of 1.9-2.7 pF/cm2. By comparing the values which are found on the thiol with the values on the different thiolipids 10,11, and 12, no clear difference in the degree of perfection of the second layers with respect to the first layer can be observed, except that the standard deviations are smaller for the thiol system than for the thiolipids. This means that in addition to alkanethiols, the thiolipids are useful as a base for the formation of bilayers. The values of the specific resistance of the bilayers, composed of one monolayer of thiolipid and another monolayer of a conventionallipid, were found to be in the range of lo4 Q cm2. These values are by a factor of 1000 smaller than those for unsupported bilayer lipid membranesul45 but in the same range as the bilayers with 1-hexadecanethiol as first layer. Formation of Mixed Bilayers on Imperfect, Chemisorbed Monolayers. Finally, we tried to build lipid bilayers, the first monolayer of which is composed of two different lipids. In contrast to the bilayers described before, where the first layer was assumed to be composed of pure thiolipids and the second one of a conventional lipid, a mixed first layer was now required. This was performed by interrupting the adsorption process after a short period of time, resulting in an imperfect monolayer. The values for the capacitance equivalents of the thus formed layers are between 2.5 and 7.5 pF/cm2. In the following step, this imperfect monolayer was completed by a conventional lipid, to form a bilayer. In Table 2 the values are given for three conventional lipids, using

Lung et al.

206 Langmuir, Vol. 10, No. 1, 1994 Table 2. Electrical and Optical Properties of Supported Lipid Mono- and Bilayers on Gold Surfaces second layerb

thiolipid 10 thiolipid 11 thiolipid 12 1-hexadecanethiol mixed layer of 12/phospholipide

1.41 1.20 1.14 1.13

0.21-0.26 0.35-0.42 0.26

0.71 0.59 0.51

0.32-0.50 0.55-0.80

0.83 0.72 0.72 0.71 1.00

0.42 0.45

0.92 0.80 0.80 0.76 0.82

0.30 0.30

0.93 0.77 0.78 0.78 0.78

0.35 0.38(0.40) 0.30 (0.35)

0 First monolayer on gold substrate of the lipids indicated. Bilayers with the second layer formed on top of the first by SA of the corresponding lipids from an OG dispersion, or in some cases of the SPR measurements, in addition from vesicle dispersions for the values indicated in parentheses. Bilayers of the particular thiolipid used for the first layer. The values of the capacitance of the gold-attached monolayers, Cmonol,and the completed bilayer, Chi, given as pF/cm2, were measured by the impedance method; the corresponding values of the shift of the resonance angle in the SPR for the first monolayer, ABmonol, and of the second monolayer (not bilayer!), ABmm2, are given in degree8.a An imperfect monolayer of thiolipid 12 was first formed on gold by interrupting the adsorption process of thiolipid 12 after a few minutes and then completing the first and-the second layer by the phospholipids indicated.

thiolipid 12 in the adsorption step. Each value contains at least six bilayer formations on three different thiolipid samples. No systematic influence on the final bilayer capacitance due to the imperfect thiolipid monolayer was observed if samples in the capacitance range given above were used. The bilayer formation failed if samples having a capacitance higher than 10 pF/cm2for the thiolipid layer were used. The resistance values for this form of bilayer are in the range 0.5-1.2 MO per 1 mm2 sample (which corresponds again to approximately lo4 Q cm2) and are therefore comparable to the bilayers composed of two pure lipid monolayers. The degree of coverage of the imperfect thiolipid monolayer used for the formation of the mixed lipid layer can be estimated by describing the electrode as an electrical equivalent circuit composed of two parallel capacitors. The measured capacitance Csample of the sample electrode is then given by

One term, C,,,, corresponds to that part of the electrode which is covered by the thiolipid layer and the other,,,C to the uncovered part of the gold surface. A similar relation holds for the total area Asample for the sample electrode Asample

=4

0 ,

+AUCOV

(16)

where A,, is that part of the electrode which is covered by a thiolipid layer and A,,,, corresponds to that part which remains uncovered. Combining eqs 15 and 16 leads of the to a simple relation for the fraction x = Acov/Asample electrode covered by a lipid bilayer

The capacitance of the Helmholtz layer on bare gold is 27 pF/cm2 as described before; for a monolayer of thiolipid 12 it is 1.14 pF/cm2. Supposing that the structure of the Helmholz layer on the uncovered parts of the sample is the same as that on bare gold allows one to calculate the covered fraction x of the electrode area. Applying eq 17 to the values of the capacitance of the imperfect thiolipid layer, avalue of 75-95% coverage is obtained. This means that 5 2 5 % of the total sample surface is still free and therefore accessible for possible incorporation of proteins. It should, however, be noted that this is only an approximation, using a very simplified model. The uncertainty is the capacitance of the Helmholtz layer, which in this

model was assumed to be undisturbed by the hydrophobic environment of the adsorbed thiolipids. Surface Plasmon Resonance. Plasmon surface polaritons are mixed excitations of the oscillation of a nearlyfree electron gas and an evanescent electromagnetic field at a metal-dielectric interface. They can only propagate along this interface and their physical characteristics are described by the dispersion relation, which correlates their momentum with their energy and which is determined by the optical architecture in the vicinity of the interface. Both energy and momentum of the surface plasmon waves have to be matched when excited by normal plane light waves. Thus, via the coupling conditions the optical properties of thin organic films at a metal interface can be deduced to a very high accuracy.47 After characterization of the thin gold layer, the formation of the SA films was monitored on-line by recording the intensity increase of the reflected light at an angle slightly smaller than the resonance angle of the bare gold. The adsorption process was judged to be finished after approximately 4 h (visually there was no further change of the films over longer periods). The angle shifts of the resonance minima which are proportional to the optical thicknesses An d (An is the refractive index difference between the organic film and the surrounding medium, d is the geometrical thickness of the film) of the different thiolipid SA films are summarized in Table 2. They were taken after the final rinsing procedure. As already outlined, the films contain a bilayer in which one monolayer is covalently bound to the gold substrate. Table 2 also includes the resonance angle shifts after removing the unbound monolayers by flushing the surface with an OG solution leaving only the directly attached thiolipid monolayers. Surprisingly, when comparing the optical data with the electrochemical ones, the relative differences in the optical thicknesses between the various thiolipid films are more pronounced than for the electrochemical measurements. Especially, the optical data of the layers formed from thiolipid 10 show a low optical thickness which may indicate a low monolayer coverage. We will discuss this below. To explore the potential of building up mixed bilayers, we investigated and compared the spontaneous formation of a second monolayer on a preformed thiolipid monolayer by SA of phospholipids out of (i) detergent and (ii) vesicle dispersions. On thiolipid 10, no reproducible results were obtained. There, the assembled phospholipid layers could not be completely washed away with OG, pointing to a (47) Raether, H. In Physics of Thin Film;Hass, G., Francombe, M. H., Hoffmann, R. W., Eds.;Wiley: New York, 1977;Vol. 9, p 145. Knoll, W. MRS Bull. 1991,16, 29.

Lipid Bilayers on Gold

Langmuir, Vol. 10,No. 1, 1994 207

I"

-50

50

150 250

350

450

550

Time [SI

Figure 12. Reflected intensity versus time (SPR experiment) of a gold surface covered with a monolayer of thiolipid 12 after incubation (startingat time 0) of POPC from a detergent solution (above)and a vesicle dispersion (below),respectively. Arrows at the upper curve indicate washing with 0.1 M KC1 (see Materials

and Methods). The reflected light was recorded at an angle slightly smaller than the resonance angle. The increase of the reflected intensity is due to a shift of the resonance curve.

possible intercalation of the phospholipids into the thiolipid film. Figure 12 compares the time courses of the formation of POPC monolayers on a covalently bound thiolipid 12 monolayer performed by the two methods. The changes in reflectivity coincide with an angle shift A8 = 0.38O and A8 = 0.4' in pure water. When the same experiments were performed in 0.1 M KC1 solution only the OG yielded similar results to those obtained in pure water, but considerably higher angle shifts were observed using the vesicle assembly procedure, very likely due to multilayer formation. The completion of a supported bilayer with POPC as a second layer resulted for all cases investigated, including l-hexadecanethiol, in angular shifts between 0.3O and 0 . 4 O , i.e. on the average A8 = 0.35O.These results, together with the angle shifts of other combinations of phospholipids on thiolipid layers are summarized in Table 2 (see ref 30 for some representative reflectivityvs-angle scans). In the case of DPPC and DMPC, bilayers on the thiolipid monolayers were only formed using lipid detergent dispersions as the adsorption process, since lipid vesicle dispersions always resulted in multilayer formation. The measured angular shifts were A8 = 0.30°for the DMPC monolayer and A8 = 0.42-0.45O for DPPC monolayers on thiolipids 11 and 12. Applying two refractive indices, n = 1.45 and n = 1.5, as lower and upper limits and using the appropriate SPR angle shifts, one can estimate a range of thicknesses for the phospholipid monolayers of 14-19 A for DMPC, 19-29 A for DPPC, and 14-25 A for POPC. Although there is some uncertainty in the accurate value of the thickness of the layers, the results are consistent with the formation of a single monolayer of phospholipids on the thiolipid-covered gold substrate.

Discussion The experimental results of the monolayer, the electrochemical capacitance, and the surface plasmon resonance measurements show that the three thiolipids synthesized exhibit an amphiphilic character and form well-defined mono- and bilayers on gold substrates by selfassembly. The length of the hydrophilic spacer has an influence on the structure of the films, both on the water and on the gold surface. Thiolipid Monolayers on the Water Surface. The monotonic increase of the transition pressure with longer spacers in the Langmuir experiments suggests an additional, spacer-dependent interaction between the thiolipid molecules. As the same tendency in the discontinuities of the isotherms was also observed with the chlorinecontaining lipids 13 and 14, incorporating hydrophilic

spacers (CH~CHZO),with n = 2 and 3, respectively, we attribute this behavior to the spacer rather than to the twin character of the thiolipid. On comparison of these results with the phase behavior of DPPA43the additional interaction in thiolipids is repulsive. It might be induced by the increased hydration of the spacer headgroups. The spacer groups also influence the fluorescence microscopypatterns (Figure 6) of coexisting fluid and solid domains of the thiolipids on the water surface. In particular, the regular starlike pattern observed for thiolipid 12 is unusual for a lipid film. Similar patterns have previously only been observed for J-aggregates.& Thiolipid Mono- and Bilayers on Gold Surfaces. The trends in the transition pressures seem to contradict our findings that the self-assembled layers, formed by the moleculeswith the longer spacers, show a higher molecular integrity and also a higher optical density. An explanation of this behavior might be that the increased rigidity of the thiolipids with the shorter spacer observed in the monolayers on the water surface is also present in the micelles. This would lead to problems in filling the holes or defects in the SA films after the initial adsorption process, as single molecules could not easily exchange from the micelles to the gold surface. Furthermore, the longer spacer gives the thiolipids more flexibility to arrange themselves according to the packing requirements of their hydrocarbon chains. The spacer should also ensure an increased decoupling from the rigid, and on a molecular scale, relatively rough gold surface. It is quite interesting to compare the kinetics of bilayer formation as observed by the two different type of methods, electrochemical and SPR measurements, respectively. Both techniques observe at least two different processes: A fast one in the second to minute range and a slower one occurring over hours. Although we have not performed a detailed investigation of the kinetics of the thiolipid adsorption, we interpret our present data qualitatively as follows. The fast process reflects the coadsorption of thiolipids and detergents on the gold surface, corresponding to an incomplete, first thiolipid monolayer. However, in order to prevent contact between the lipid hydrocarbon chains and the water phase, the formation of a thiolipid monolayer on gold must be accompanied by formation of second layer on top of the first, creating a typical bilayer configuration with the polar groups of the second layer directed toward the aqueous environment. As long as the detergent concentration is above the cmc, this second layer will certainly be composed either of pure detergent or, more realistically, of a mixture of detergent and thiolipid molecules. The second, slow process observed then involves a further completion of the first layer, by closing still-existing holes. Already adsorbed lipid molecules may sterically hinder the docking of further thiolipid molecules to the gold surface. At a certain coverage, new thiolipid molecules may only adsorb to the gold if the already adsorbed thiolipid molecules rearrange, including the breaking and re-formation of certain Au-S bonds. This slows down the adsorption rate considerably, as the first monolayer approaches completion. According to SPR measurements, the slow process of mass adsorption is completed after 2-4 h. On the other hand, small changes in the capacitance of the formed bilayers are still observed after 2-3 days. We interpret these slow capacitance changes as an internal structural reorganization of the supported lipid bilayer, without significant, additional mass adsorption. (48) Duschl,C.;Kemper,D.;Frey,W.;Meller,P.;Ringsdorf,H.;Knoll, W.J. Chem. Phys. 1989,93,4587.

208 Langmuir, Vol. 10, No. 1, 1994

Lang et al.

According to the data in Tables 1 and 2, the electrochemical and the optical properties of the gold-supported thiolipid mono- and bilayers show a dependence on the length of the hydrophilic spacer groups although the main lipid backbone remains identical in the three different thiolipids. The understanding of these effects is important for designing lipid-based functionalized surfaces. The largest difference was observed between thiolipid 10 and 11. Interestingly, the electrical and optical properties of the thiolipid 12 monolayer resemble those of a hexadecanethiol monolayer on gold. In principle, the electrochemical and the optical data both contain information about the molecular properties of the self-assembled lipid films. For example, the capacitance datacan be evaluated to deliver the thickness of the lipid hydrocarbon chains in the framework of the model used for eq 13. This procedure has been applied quite successfully to planar unsupported lipid bilayers in aqueous medium. However, one has to be aware that it is based on a simplified description of a planar lipid bilayer, similar to our equivalent circuit in Figure 3. Equation 13 describes the capacitance of the hydrocarbon part properly only if the capacitance of the polar headgroup region is higher than the capacitance of the lipid hydrocarbon part by a factor of about 100 (see ref 44 for a detailed discussion of this subject). This is the case for planar unsupported lipid bilayers, but here the question arises, whether also this holds for supported thiolipid layers on gold substrates. It is difficult to find a definite answer since there is no detailed structural data on the thiolipid spacer groups available. Intuitively one would argue that the spacer groups are fully hydrated, similar to the (CHzCHzO-) chains in detergents such as Tween and Tritons4 Under these circumstances eq 13 would be valid. The next question then arises of how to calculate the layer thickness of the lipid hydrocarbon region and what the values obtained imply in terms of a structural model. In addition to the experimental error, another uncertainty arises in the choice of the value of er to be used, or even whether the same value can be used for all different layers. Literature values for fluid planar lipid bilayers range from e, = 2.0 to 2.244145 The thiolipids used here consist of saturated C16 fatty acid (palmitic acid) chains. At room temperature the thiolipid bilayers are therefore in the socalled ordered or crystalline state. A value of er = 2.25, typical for crystalline hydrocarbons, would seem to be appropriate.*G Taking this value, one calculates from the capacitance of a 1-hexadecanethiol monolayer on gold (C = 1.13 pF/cm2, Table 2) a hydrocarbon thickness of 18A which means that a fully stretched, 20 A long hexadecane chain of the thiol would be tilted by 28O to the normal of the gold support. This is in excellent agreement with recent FTIR measurements on long chain thiolates on gold surfaces.49It should be noted in this respect that the model used for the evaluation of the capacitance of hexadecanethiol is an appropriate description for evaluating the layer capacitance, as has been proven elsewhere by a detailed impedance spectroscopicinvestigation over a wide frequency range.36 For the gold-supported bilayers of thiolipids 10, 11, and 12, the corresponding thicknesses of the hydrocarbon chain layers are calculated to be 28,34, and 39 A, respectively. For thiolipid 12, this value fits nicely with a perfectly ordered lipid bilayer, with the hydrocarbon chains oriented parallel to the membrane

normal. For the other two thiolipids, two structural possibilities remain, either a considerable tilt or disorder of the lipid chains or a combination of both. From capacitance data alone it is impossible to decide which is the correct answer. The results of the SPR experiments might help in this respect. Interestingly, there are small, but defined differences between the capacitance values of the corresponding thiolipid bilayers and monolayers. The thickness of the gold-attached monolayer is always slightly smaller than the corresponding half of a bilayer (see Table 1). This indicates that the second monolayer in the lipid bilayer structurally interacts and influences the first one. In the present case of crystalline and identical lipid layers, the mutual interaction increases the chain order. However, a contrary effect might also be possible as discussed for the mixed layers below. The optical properties of the thiolipid films are in qualitative agreement with the electrochemical characteristics. As for the capacitance data, the problem arises of how to analyze the SPR-measured angle shifts quantitatively in terms of a mass coverage. The central difficulty is to chose the right refractive index values. For supported layers in the condensed state, one finds refractive index values in the range of n = 1.45-1.55.6*mThe according values for the fluid state are not found in the literature, but as a lower limit a refractive index of 1.4 seems to be justified. Taking the values for crystalline films, this results in an uncertainty of a factor of about 2 in the calculation of the optical layer thickness. Within this range, the optical data of the layer thickness of thiolipid 12 and hexadecanethiol fit reasonably well with the picture obtained from the electrochemical measurements. Furthermore, the measured decrease of the SPR angle shifts for thiolipids 10 would indicate a lower mass density of adsorbed lipids which in turn would favor a model where the lipid chains in these particular layers are less densely packed or more disordered than in the case of thiolipid 12 or hexadecane layers. We will discuss this problem from another point of view a t the end. Mixed lipid bilayers on gold surfacesare particularly interesting for biological applications. The formation of a second phospholipid monolayer on a first thiolipid monolayer allows the defined completion of a bilayer arrangement which is able to accommodate functional biological macromolecules such as glycolipids%and lipid anchored proteins51and finally to serve as a matrix for the reconstitution of intrinsic membrane proteins in the case where the first monolayer is composed of a mixture of thiolipids and fluid phospholipid molecules. The following points are remarkable in the electrochemical characterization of the mixed supported layers. (i) The formation of a second phospholipid monolayer on top of an already existing chemisorbed thiolipid yields highly reproducible capacitance values for the supported lipid bilayers. The variation between different experiments is in the range of h0.03 pF/cm2. (ii) The capacitance values are practically identical for any one sort of phospholipid irrespective of whether the first layer is thiolipid 11,12, or 1-hexadecanethiol. Again, the exceptions are the layers on thiolipid 10, which result, for each lipid class, in higher values of cbj. These results are of importance for the structural interpretation of the capacitance data. They suggest that the second phos-

(49) Nuzzo,R. G.; Dubois, L. H.; A h a , D. L. J.Am. Chem. Soc. 1990, 112,558. Stole, S. M.; Porter, M. D. Langmuir 1990,6,1199. Laibinis,

(50) Pockrand, I.; Swalen, J. D.; Gordon, J. D., II; Philpott, M . R.Surf. Sci. 1977, 74, 213. (51) Bouvier,J.; Etges, R.; Bordier, C. J.Bioi. Chem. 1981,260,15504. Ferguson, M. A.; Williams,A. F.Annu. Rev. Biochem. 1988,57,285. Low, G. M.; Saltiel, A. R. Science 1988, 239, 268.

P. E.; Whitesides, G. M.; Allara, D. L.; Tao, Y. T.; Parikh, A. N.; Nuzzo, R. G.J. Am. Chem. SOC.1991, 113, 7152. Bertihson, L.; Liedberg, B. Langmuir 1993, 9, 141.

Lipid Bilayers on Cold

pholipid layer also structurally influences the first one: because the clear differences seen in pure mono- and bilayers between thiolipid 11 and 12 (Table 2) are not present in the mixed bilayers. It should be noted, that in the case of phospholipid monolayers formed on top of silanized glass plates, the first silane hydrocarbon layer has an influence on the lateral mobility of the second phospholipid layer.52 In turn, the formal decoupling of the capacitance of a lipid bilayer into the individual contributions of the two monolayers according to eq 14 might result in an unrealistic description of the real layer properties. In this context it is useful to remember the differences between the capacitance measurements and the SPR experiments. In an electrochemical experiment, we measure either the first thiolipid monolayer or the completed bilayer. There is no way to obtain the capacitance of the second lipid layer independently. On the other hand, in an SPR angle scan we can observe the adsorption of the first thiolipid layer and, in turn, that of the second. (iii) The capacitance values of the mixed bilayers are clearly higher than those of the pure thiolipid films. The Cbivaluesincrease in the order 0.72 (DPPC), 0.78 (POPC), 0.80 pF/cm2 (DMPC), in agreement with the decrease of the bilayer thicknesses determined in the corresponding pure phospholipid bilayers by X-ray diffra~tion.~ These relative differences are also reflected by the SPR angle shifts measured for the formation of the second phospholipid layers. (iv) In the case of DMPC and POPC, mixed bilayers on imperfect thiolipid monolayers show capacitance values similar to those found for bilayers formed on an already completed thiolipid monolayer. Only in the case of DPPC were much higher capacitance values found, indicating that the phospholipids have to be above (POPC) or around (DMPC)the ordered *-, fluid phase transition temperature to complete the holes in the first layer. Taking together the results of our supported lipid layers in the context of already published optical and electrical properties of other lipid films, one can distinguish three cases: (i) Apparently solvent-free planar, unsupported lipid bilayers in a fluid state show capacitance values of 0.7-0.8 pF/cm2,53verysimilar to those of our gold-supported mixed membranes. (ii) This is in contrast to the supported crystalline thiolipid bilayers with capacitance values of 0.5-0.6 pF/ cm2, which are comparable to the equivalent thioalkane monolayers of equivalent hydrocarbon chain length, with 1.15 pFlcm2. This latter value is in agreement with previously published data on alkanethiols.” (iii) Finally, there are the Langmuir-Blodgett films of long chain fatty acid salts such as Cd arachidate on metal electrodes. Capacitance values of 1.2 pF/cm2 were found for bilayers of such systems.55 The question remains of how to correlate the electrochemical results with those from the SPR measurements. One point which has not been taken into account up to now is the optical contrast. In general, the angle shift of the SPR is proportional to the optical thickness, which is a good measure for the mass or molecular coverage. (52) Merkel, R.; Sackmann, E.; Evans, E. J.Phys. (Paris) 1989,50, 1535. (53) Benz, R.; Gisin, B. F. J. Membr. Biol. 1978,40, 293. Schindler, H. G.; Qua&, U.h o c . Natl. Acad. Sci. U.S.A. 1980, 77, 3052. (54) Porter,M. D.;Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. SOC.1987,109,3559. Chidsey, C. E. D.; Loiacono, I).N. Langmuir 1990,6,682. (55) Stelzle, M.; Sackmann,E. Biochim.Biophys. Acta 1989,981,135.

Langmuir, Vol. 10, No. 1, 1994 209

However, if the contrast of the refractive indices between the layer and the surrounding medium decreases, due to disorder in the hydrocarbon chains, this translation into mass coverage becomes more and more arbitrary. Especially for less densely packed, disordered films, resembling layers in a fluid state, the refractive index is not much higher than that of pure water. This leads us to the conclusion that, although all the thiolipid layers possess very good electrical blocking properties, this does not necessarily mean that the chain packing is the same for all the different thiolipid films. In this spirit we calculate a thickness for the thiolipid monolayers from the capacitance values using the “optial” dielectric constant of bulk polyethylene of 2.25 (see Table 1, last line) and deriving then the refractive indices for these films. This gives for the thiolipid 10 film a value of 1.4, for the thiolipid 11film a value of 1.44, and for the film of thiolipid 12, with the longest spacer, a refractive index n = 1.48. The former value coincides with values obtained from fluid layers, whereas the latter is typical for a condensed film. The properties of the phospholipid layers resemble more closely those of LB films. This could mean that in this case, in spite of forming optically compact films, the defect density is higher and, therefore, the apparent dielectric constant, as well. Finally we want to compare the SA from a lipid/ detergent solution to that from vesicle dispersion. The SA technique from detergent solution gives reproducible results for the SPR angle shift for DMPC and POPC, independent of the salt content in the solution. The method which works from vesicle dispersion requires more defined conditions. Only vesicles prepared from POPC in pure (Millipore)water caused a thickness increase which could be unambiguously assigned to the formation of a monolayer. As a POPC membrane at room temperature is in a fluid state, our results confirm the findings of Spinke et al.= They used DMPC vesicles (phase transition temperature of DMPC T, = 23 “C) and obtained only reproducible results, consistent with the formation of a monolayer when maintaining their solution above phase transition temperature. But in contrast to their results, our experiments with a 0.1 M KC1 solution produced considerably thicker layers. Probably, depending on the vesicle size, subtle changes in the mobility of the molecules in the membrane influence the surface tension of the vesicleswhich then plays an important role in the spreading properties of the vesicles on the hydrophobic support. Finally, we would like to point out that the phospholipid layer formation on the thiolipid films gives identical results to those obtained on long chain alkanethiols which shows again the quality of the thiolipid films as far as the arrangement of the hydrocarbon chains is concerned.

Conclusion The present work demonstrates a new way to attach lipid bilayers to gold substrates. Of particular importance for biological applications are the phospholipid bilayers which are covalently fixed to the gold surface by thiolipids in the lipid monolayer facing the substrate. These supported bilayers are distinguished by their extreme simplicity of preparation. They form by SA in a dilution process starting from mixed lipid detergent solutions. The resulting gold-attached bilayers are mechanically and chemically stable for several days to weeks. This opens the door for a number of applications. The most obvious (56) Spinke, J.; Yang, J.; Wolf, H.; Liley, M.; Ringsdorf, H.; Knoll, W. Biophys. J. 1992, 63, 1667.

210 Langmuir, Vol. 10, No. 1, 1994 is the incorporation of membrane-spanning proteins in a functionally active form into the gold-supported bilayers. Using, for instance, membrane protein receptors, it might be possible to detect the ligand binding to these receptors by a number of different surface sensitive techniques where gold surfaces may be applied, such as impedance spectr0scopy,5~surface plasmon resonance,s8and piezoelectric measurementa.59 In general, the immobilized membranes are certainly useful for the development of new biosensorsm adopting biological signal transduction and amplification processes. Nanotechnology is another interesting field of application, for instance, by producing defined structures on the gold substrate surface by phase separations in mixed lipid layers of conventional lipids and thiolipids. (57) Macdonald,J.R.,Ed.ZmpedanceSpectroscopy;Wiley: New York, 1987. (58) JBnsson, U.; Malmquist, M. Adu. Biosens. 1992, 2, 291. (59)Deakii, M. R.; Buttry, D. A. Anal. Chem. 1989, 61, 1147A. McCallum,J. J. Analyst 1989,114,1173. Ngeh-Ngwainbi,J.; Suleiman, A. A.; Guilbault, G. G. Biosens. Bioelectron. 1990,5, 13. (60) Reviews on biosensors: Turner, A. P. F.; Karube, I.; Wilson, G. S. Biosensors, Fundamentals and Applications; Oxford University Press: Oxford, 1987. Mosbach, K., Ed. Methods Enzymol. 1988, 137. Janata, J.; Bezegh, A. Anal. Chem. 1988,60,62R. Scheller, F.; Schubert, F.; Pfeiffer, D.; Hintache, R.; Dransfeld, I.; Renneberg, R.; Wollenberger, U.; Riedel, K.; Pavlova, M.; Kiihn, M.; MUer, H. G.; Tan, P.; Hoffmann, W.; Moritz, W. AnaZyst 1989,114,653. Lowe, C. R. Philos. Trans. R. SOC. London 1989, B324,487. Camman, K.; Lemke, U.; Rohen, A.; Sander, J.; Wilken, H.; Winter, B. Angew. Chem. 1991, 103, 519. Wise, D. L., Wingard, L. B., Jr.; Eds.; Biosensors with Fiberoptics; Humana: Clifton, NJ, 1991.

Lang et al.

ATR cmc DMPC DPPA DPPC

LBK NBD-PE NMR OG POPC SA

SPR TLC TPS

Glossary attenuated total reflection critical micellar concentration 1,2-dimyristoyl-sn-glycero-3-phosphocholine 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid, disodium salt 1,2-dipalmitoyl-sn-glycero-3-phosphocholine Langmuir-Blodgett-Kuhn N-(7-nitrobenz-2-oxa-l,3-diazol-4yl)-l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine nuclear magnetic resonance n-octyl-8-D-ghcopyranoside l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine self-assembly surface plasmon resonance thin-layer chromatography 2,4,6-triisopropylbenzenesulfonylchloride

Acknowledgment. We thank Dr. M. Liley and Dr. M. Pawlak for critically reading the manuscript. The work was supported by grants from the Swiss NationalScience Foundation (PN 24 and SPP Biotechnologie 5002-35180), the Roche Research Foundation, and the European Institute of Technologie (project no. B173).