Langmuir 1989,5, 35-41
35
Spontaneous Assembly of Phosphatidylcholine Monolayers via Chemisorption onto Gold' Wojciech Fabianowski,2apbLouis C. Coyle,2aBruce A. Weber,2a Richard D. Granata,2aDavid G. Castner,2cAndrzej Sadownik,2aydand Steven L. Regen*p2a Department of Chemistry and Zettlemoyer Center for Surface Studies, Lehigh University, Bethlehem, Pennsylvania 18015, and the National ESCA Surface Analysis Center for Biomedical Problems, Department of Chemical Engineering, BF-10, University of Washington, Seattle, Washington 98195 Received June 22, 1988. I n Final Form: August 5, 1988 Phosphatidylcholine molecules bearing thiol and disulfide moieties at the terminus of both the sn-1 and sn-2 chains have been synthesized and chemisorbed onto freshly evaporated gold film. A combination of ellipsometry, contact angle measurements, heterogeneous electron-transfer properties, and X-ray photoelectron spectroscopy has shown that 1,2-bis(16-mercaptohexadecanoyl)-sn-glycero-3-phosphocholine and l,Bbis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine yield chemisorbed monolayers which are (i) densely packed, (ii) macroscopically uniform, (iii) strongly hydrophilic, and (iv) oriented such that the polar head groups extend outward from the surface and the sulfur moieties are in intimate contact with the gold. Ellipsometry and contact angle measurements indicate that analogous surfaces can be produced from l,2-bis(ll-mercaptoundecanoyl)-sn-glycero-3-phosphocholineand 1,2-bis[l2-(isolipoy1oxy)dodecanolyl]-sn-glycero-3-phosphocholine.The potential for adjusting and utilizing this new class of "biomembrane-like" surfaces for biochemistry, biology, and medicine is briefly discussed.
Introduction Understanding the relationships that exist between the chemical composition, structure, and function of cell membranes represents one of the most difficult challenges presently facing chemistry and biology. Because biological membranes are highly complex at the molecular level,3a variety of model systems have been developed for mechanistic studies; the most popular of these include (i) lipid monolayers assembled at the gas-water interface: (ii) bilayer lipid membranes (BLMs),~(iii) multilamellar and unilamellar vesicles: (iv) planar phospholipid monolayers and bilayers supported on solid surfaces, i.e., LangmuirBlodgett (LB) and (v) polymerized analogues of i-iv.'O The investigation of monolayers at the gas-water in(1) This research was supported in part by the National Science Foundation (CHE 87-00833) and by the 3M Co., St. Paul, MN. The XPS studies carried out in this work were made possible through a grant from the National Institutes of Health, Division of Research Resources (RR 01296). (2) (a) Lehigh University. (b) On leave from the Warsaw Technical University. (c) University of Washington. (d) On leave from the Warsaw Academy of Agriculture. (3) Singer, S. J.; Nicolson, G. L. Science (Washinton,D C ) 1972,175, 720. (4) Cadenhead, D. A. In Structure and Properties of Cell Membranes; Press, G., Benga, Eds.; CRC: Boca Raton, FL, 1985; Vol. 111. (5) Tien, H. T. Bilayer Lipid Membranes (BLM) Theory and Practice; Marcel Dekker: New York, 1974. (6) Bangham, A. D. Prog. Biophys. Mol. Biol. 1968, 18, 29. Tyrrell, D. A.; Heath, T. D.; Colley, C. M.; Ryman, B. E. Biochim. Biophys. Acta 1976,457, 259. Chapman, D. In Membrane Structure and Function; Bittar, E. E., Ed.; Wiley: New York, 1980; p 103. Jain, M. H.; Wagner, R. C. Introduction to Biological Membranes; Wiley-Interscience: New York, 1980. Liposomes: From Physical Structure to Therapeutic Applications; Knight, C. G., Ed.; Elsevier/North Holland Biomedical: Cambridge, 1981. Fendler, J. H. Membrane Mimetic Chemistry;Wiley-Interscience: New York, 1982. (7) Hafeman, D. G.; von Tscharner, V.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1981,78,4552. Nakanishi, M.; Brian, A. A,; McConnell, H. M. Mol. Immunol. 1983, 20, 1227. Watts, T. H.; Gaub, H. E.; McConnell, H. M. Nature (London) 1986,320, 179. (8)Johnston. D. S.; Sanahera, S.: Manion-Rubio,A. Biochim. Biophys. - . Acta 1980,602, 213. (9) McConnell. H. M.: Watts. T. H.: Weis. R. M.: Brian, A. A. Biochim. Bioshys. Acta 1986,864, 95. (10) Review: Bader, H.; Dorn, K.; Hupfer, B.; Ringsdorf, H. In Polymer Membranes; Gordon, M., Ed.; Springer-Verlag: New York, 1985; p '
1.
terface provides valuable insight into the compressibility, packing behavior, and miscibility of phospholipids. Vesicles and BLMs are useful for studying the permeability behavior of lipid bilayers; vesicles have also been extensively used to probe lipid conformation, fluidity, phase transition behavior, miscibility, and diffusion." Recently, LB films derived from phospholipids have been shown to serve as powerful tools for studying the molecular basis of cellular recognition? Principal advantages that have been cited for planar lipid membranes over vesicles are (1) their greater efficacy in evoking specific responses involving the recognition of gene products of the major histocompatibility complex, (2) their suitability for examination of single cell-target membrane interactions by optical microscopy, and (3) their usefulness for measuring specific cell binding to a target membrane and for studying biochemical events in the region of membrane-membrane contact. In this paper we describe a new class of biomembrane models which are intended to serve as complements to phospholipid-based LB films. Specifically, we report the preparation of phosphatidylcholine monolayers chemisorbed onto supported evaporated gold film.12 Our rationale for constructing such surfaces was based on the following considerations: First, assembling phospholipid monolayers via chemisorption should be experimentally simpler than LB film preparation and should yield more reproducible surfaces. While the formation of LB films is relatively straightforward in principle, monolayer manipulations using a film balance are tedious and require extreme care in order to obtain high reprod~cibi1ity.l~ Second, chemisorbed monolayers should be amenable to direct analysis of surface hydrophilicity via standard contact angle measurement^.'^^'^ With conventional (11)Small, D. M. Physical Chemistry of Lipids; Plenum: New York, 1986; Chapter 12. (12) A preliminary account of this work has previously been published: Diem. T.: Czaika, B.; Weber, B.; Reaen, S. L. J. Am. Chem. SOC.1986, 108, 6094. (13) Arnett, E. M.; Chao, J.; Kinzig, B. J.; Stewart, M. V.; Thompson, 0.;Verbiar, R. J. J.Am. Chem. SOC.1982, 104, 389. (14)Andrade, J. D. In Surfc 'e and Interfacial Aspects of Biomedical Polymers; Andrade, J. D., Ed.; Plenum: New York, 1985; Vol. 1,p 249.
0 1989 American Chemical Society
36 Langmuir, Vol. 5, No. 1, 1989 monomeric LB films, such measurements are normally precluded because of the strong tendency toward redeposition of the lipids at the gas-water interface when the support is removed from water into air.16 Because chemisorbed monolayers should be strongly bound to the solid support, it should be possible to analyze their hydrophilicity directly. Such data would not only be of immediate value for the characterization of film structure but should also help to define surface structure-bioactivity relationships. For example, the adhesion properties of these surfaces toward proteins and whole cells are likely to depend (in part) on their surface hydrophilicity. Third, the structure and biological characteristics of chemisorbed phospholipid monolayers should, in principle, be adjustable through manipulation of the lipid chains. Specifically, the ability to systematically vary the lengths of the sn-1 and sn-2 fatty acid chains should allow one to “pin down” each lipid component of the monolayer in a controlled manner. It should be possible, therefore, to modulate the extent of hydrocarbon exposure at the film surface. This feature could have important implications in terms of adjusting the biocompatibility of such surface~.~’ Fourth, phospholipid monolayers chemisorbed onto an electroactive support could provide the basis for unique electron-transfer experiments across “biomembrane-like” surfaces. One could envision, for example, the inclusion of selected membrane-bound redox agents such as cytochrome oxidase into chemisorbed phospholipid monolayers on gold for mechanistic studies.18 Fifth, spontaneous assembly methods are applicable to a variety of surface geometries (LB methods are limited to planar surfaces). This fact, together with the possibility of controlling the biocompatibility of these films, could make them particularly attractive as novel biomaterials for device applications, e.g., artificial organs, catheters, etc. Motivated by all of these considerations, we have begun a program aimed at synthesizing and characterizing a variety of phospholipid monolayers chemisorbed onto solid supports. In the present work, we describe the results that we have obtained from studies which take advantage of spontaneous assembly methods previously introduced by Nuzzo and Allara.12J”21 In particular, these workers have shown that a variety of substituted organic disulfides can be chemisorbed onto gold film, yielding densely packed, stable, and oriented monolayers. Analogous monolayer films have also been reported when thiol groups are used as the chemisorptive (15)Adamson, A. W. Physical Chemistry of Surfaces; Wiley-Interscience: New York, 1976;p 333. (16)Albrecht, 0.;Johnston, D. S.; Villaverde, C.; Chapman, D. Biochim. Biophys. Acta 1982,687,165. (17)Ratner, B. D.In Biomaterials: Interfacial Phenomena and Applications;Cooper, S. L., Peppas, N. A., EMS.; Advances in Chemistry 199; American Chemical Society: Washington, D.C., 1982;Chapter 2, p 9. Chapman, D. U.S. Patent 4 348329,1982. (18)Di Gleria, K.;Hill, H.A. 0.; Lowe, V. J.; Page, D. J. J. Electroanal. Chem. 1986,213,833. (19)Nuzzo, R. G.; Allara, D. L. J . Am. Chem. SOC.1983,105,4481. (20) Nuzzo, R.G.; Fusco, F. A.; Allara, D. L. J.Am. Chem. Soc. 1987, in9 2.158
- - - 7
(21)Dubois, L. H.; Zegarski, B. R.; Nuzzo, R. G. Proc. Natl. Acad. Sci. U.S.A. 1987,84,4739. (22)Finklea, H. 0.; Nelendez, J. A. Spectroscopy (Springfield,Oreg.)
-..
19116 -, I 47 ---”)
(23)Finklea, H.0.; Avery, S.; Lynch, M.; Furtach, T.Langmuir 1987, 3,409. (24) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J.Am. Chem. SOC.1987,109,3559. (25)Sabatani, E.;Maoz, R.; Sagiv, J.; Rubinstein, I. J . Electroanal. Chem. 1987,219,365.Rubinstein, I.; Sabatani, E. J . Phys. Chem. 1987, 91,6663. Rubinstein, I.; Steinberg, S.; Tor, Y . ; Shanzer, A.; Sagiv, J. Nature (London) 1988,332,426.
Fabianowski et al. Specific phosphatidylcholine molecules that we have chosen to study are shown below. Lipids 1 and 2 (i.e.,
1, n = 10 2, n = 15
3
I
OrC
CZO
I
I
4,
1,2-bis(ll-mercaptoundecanoyl)-sn-glycero-3-phosphocholine and 1,2-bis(l6-mercaptohexadecanoyl)-snglycero-3-phosphocholine,respectively), having thiol groups at the terminus of each of the sn-1 and sn-2 chains, were expected to form stable phospholipid monolayers on gold, having the choline head groups extended outward from the surface. Two disulfide-bearing phosphatidylcholine molecules which were also chosen for this study are 1,2-bis[ 12-(lipoyloxy)-dodecanoyl] -sn-glycero-3phosphocholine (3) and its structural isomer 1,2-bis[12(isolipoyloxy)dodecanolyl]-sn-glycero-3-phosphocholine (4). Each of these lipids, having one heterocyclic disulfide group per chain terminus, was also expected to form a Chemisorbed film. Depending on their packing density, such monolayers could exist either in a solid-analogousor a liquid-analogous state. Examination of space-filling models (CPK) indicates that a densely packed monolayer of 3, having an all-anti conformation, and having each sulfur atom chemisorbed onto a gold surface, should exhibit significant tilt relative to the surface normal due to the placement of the disulfide moieties. In contrast, an analogous all-anti, tightly packed and chemisorbed monolayer of 4 would be expected to “sit” perpendicular to the surface. If chemisorbed film of 3 and 4 were, in fact, “solid-like” in nature, significant differences in film thickness might exist between these monolayers, and such differences might be observable via ellipsometry measurements. Similarly, lipid 2 would be expected to show a greater film thickness relative to 1, due to ita longer fatty acid chains. Thus, comparison of film thicknesses between 1 and 2 (and between 3 and 4) should give an indication (26)Troughton, E.B.; Bain, C. D.; Whitesides, G. M.; Nuzzo, R. G.; Allara, D. L.; Porter, M. D. Langmuir 1988,4,365. (27)Nuzzo, R. G.;Zegarski, B. R.; Dubois, L. H. J. Am. Chem. SOC. 1987,109, 733.
Langmuir, Vol. 5, No. 1, 1989 37
Assembly of Phosphatidylcholine Monolayers of the packing density and orientation of the chemisorbed monolayers.
Results and Discussion Phospholipids. We have previously reported the synthesis of lipids 1-3 as part of our ongoing efforts in the polymerized vesicle area.28,29Lipid 4 was prepared in a manner which was exactly analogous to that used for the synthesis of 3, except that isolipoic acid was employed.30 Specifically, esterification of the cadmium chloride complex of sn-glycero-3-phosphocholine(GPCCdCl,) with 12-(tetrahydropyrany1oxy)dodecanoicacid, using dicyclohexylcarbodiimide (DCC) as the condensing agent and 4-(dimethy1amino)pyridine (DMAP) as a catalyst, produced the corresponding protected phosphatidylcholine; subsequent deprotection, via ion-exchange-catalyzed methanolysis, afforded 1,2-bis(l2-hydroxydodecanoyl)sn-glycero-3-phosphocholine.29 Esterification of this dihydroxylecithin with isolipoic acid anhydride afforded an 81% isolated yield of 4. Chemisorption of Thiol- and Disulfide-Bearing Phospholipids onto Gold. Immersion of a gold-coated slide into a M methanolic solution of 1 and subsequent solvent rinsing afforded a very hydrophilic surface having advancing contact angles, 8, ranging between 10’ and 15” and film thicknesses between 17 and 19 A (Table I). Phospholipid 2 produced an even more hydrophilic surface (0 < lo’), with film thicknesses between 21 and 24 A. Chemisorption of 3 and 4 on gold resulted in the formation of surfaces with film thicknesses in the range 19-24 and 24-32 A, respectively; surface hydrophilicities were characterized by advancing contact angles of 12-19’ and 13-20’, respectively. We believe that at least part of the observed variation in film thicknesses is derived from optical measurements made on “bare” gold. Specifically, adventitious surface contamination is likely to have some influence on the optical constants measured for gold. Variations in the hydrophilicity of the lipid-modified surfaces are presumed to arise from surface contamination and/or a variation in surface structure. All of the spontaneously assembled monolayers discussed above remained chemisorbed to the gold surface after immersion in the depositing solvent (pure ethanol or methanol) for 24 h at room temperature; i.e., no significant change in contact angles and film thickness was detected. By use of similar experimental procedures, a non-sulfur-bearing phosphatidylcholine (1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC)) produced surfaces on gold, with hydrophilicities and apparent film thicknesses that were similar to those obtained with solvent alone (Table I). Barrier Properties of Spontaneously Assembled Monolayers toward Heterogeneous Electron Transfer. In order to judge, qualitatively, the density and macroscopic uniformity of coverage by the monolayers formed from 2 and 3 on gold, we have examined their barrier properties toward heterogeneous electron transfer. Specifically, we have used Fe(CN)63-as an electrochemical probe t o test for structural defects within each of the chemisorbed monolayer^.^^ Figure 1 shows the cyclic voltammetric potential-current (E-i) curves for unmodified gold with 1mM Fe(CN),% as (28) Samuel, N. K. P.; Singh, M.; Yamaguchi, K.; Regen, S. L. J.Am. Chem. SOC.1985,107,42. (29) Sadownik, A.; Stefely, J.; Regen, S. L. J. Am. Chem. SOC.1986, 108. 7789. - I
(30) Bergson, G.; Frisel, C. Acta Chem. Scand. 1964, 18, 2000.
Table I. Surface Modification of Gold modifier”
1 2 3 4
DPPC none
depositing solvent methanol methanol methanol methanol methanol methanol
deg 10-15 104.24In our hands, monolayers of octadecyl mercaptan on gold resulted in a greater than 300-fold reduction in redox currents relative to "bare" gold (Figure 2, Table 11). With monolayers of 2 and 3 chemisorbed onto gold, we have observed the presence of substantial electrochemical barriers (Figures 3 and 4 and Table 11). While an apparent oxidation current could be detected for 2 (as a shoulder), monolayers of 3 on gold did not exhibit unambiguous redox currents. Both in the absence and in the presence of Fe(CNIG3-,only strong capacitive currents could be detected. For purposes of comparison, therefore, we have determined currents for 3 on gold at a switching potential of +0.5 V. Nonetheless, it is clear from Figure 3 that 3 provides a substantial barrier toward Fe(CN):and that the monolayer is densely packed. X-Ray Photoelectron Spectroscopy (XPS). Survey scans for 2 and 3 chemisorbed onto gold over a binding
0 N
P S
15.6 1.7 2.4 1.5
~
19.1 2.3 3.1 1.1
Takeoff angle.
energy (BE) range 0-1000 eV (55O takeoff angle)33confirmed the presence of all of the expected elements. High-resolution spectra recorded for C 1s (55O takeoff angle) of both 2 and 3 revealed peaks at 285,287, and 289.5 eV. The 285-eV peak contains about 60% of the total C 1s area and is assigned to the C-C and C-H atoms of the molecule. Peaks appearing at 287 eV (ca. 25-30%) are assigned to the C-0, C-N, and C-S atoms; those at 289.5 eV (ca. 5-10%) are assigned to the C 0 2 atom. The sulfur 2p peak found for 2 chemisorbed on gold appeared at 162.4 eV. This BE is similar to that observed for the S 2p of chemisorbed dimethyl disulfide on gold (162.7 eVh2' In contrast, 3 on gold exhibited two distinct S 2p peaks of similar intensity: one appeared at 162 and the other at 164 eV. The presence of this second peak strongly suggests that not all of the sulfur atoms of the heterocyclic disulfide groups are strongly chemisorbed to the Au surface. Specifically, this value is very close to the 164.8-eV BE which has previously been assigned to physisorbed multilayers of dimethyl disulfide on gold.n Single N 1s and P 2p peaks were observed in all cases, with BEs of 403.2 and 134.2 eV, respectively. In an effort to further substantiate the presence of 2 and 3 on gold, and also to obtain evidence for orientation, X P S studies were carried out with two different takeoff angles. Specifically, elemental compositions were calculated from detailed scans of the Au 4f, C Is, 0 Is, N Is, P 2p, and S 2p photoemission lines, using takeoff angles of Oo (deepest sampling depth) and 80° (most surface sensitive). Tables 111 and IV summarize the surface compositions detected, under the experimental conditions used. In addition, a comparison of "apparent" elemental compositions for 2 and 3, (80' takeoff angle), with the corresponding theoretical compositions is presented in Table V. (33) The takeoff angle is defined as the angle between the surface normal and the axis of the analyzer lens.
Assembly of Phosphatidylcholine Monolayers
Langmuir, Vol. 5, No. 1, 1989 39
Table IV. XPS-Determined Atomic Composition of 3 on Gold XPS atomic % element 00 a 80" a ~
C 0
N P
S a
14.8 60.6 19.6 0.9 2.2 2.0
Takeoff angle. 0
Table V. Comparison of Experimental and Theoretical Phosphatidylcholine Atomic Compositions (80" Takeoff Angle) atomic % 3
2
element
theory
XPS
theory
XPS
C
76.6 15.4 1.9 1.9 3.9
71.1
72.7 18.2 1.5 1.5 6.1
71.1 23.0 1.0 2.6 2.3
~~
0 N P
S
I
~~
23.9 54.2 17.7 0.5 1.4 2.3
AU
36,
21.6 2.6 3.5 1.2
Qualitatively, Tables I11 and IV show that the N, P, and 0 concentrations increase and the S concentration decreases when a higher takeoff angle is used. Since the N, P, and 0 atoms are located in the head group region of the molecule, and the S atoms are positioned at the ends of the hydrocarbon chains, these results infer an orientation in which the choline head groups are present at the vacuum-lipid interface and the sulfur moieties are at the lipid-gold interface. The fact that the C concentration increases and the Au concentration decreases as the takeoff angle is increased is also consistent with a lipid monolayer "sitting" on top of the Au surface; i.e., a larger amount of C and a lower amount of Au are predicted as the sampling depth is decreased. XPS compositional data obtained for 2 and 3 on gold, with a takeoff angle of 80°,are in good agreement with the theoretical values (Table V). For this comparison, we have specifically chosen the "high takeoff angle data", since these XPS signals should originate primarily from the lipid layer itself. The fact that the XPS concentrations for the atoms located in the head group region (N, P, and 0)are generally higher than the theoretical values, while the C and S are lower, is also consistent with a model in which the lipids are oriented such that the phosphocholine groups are at the vacuum-lipid interface. Film thicknesses for 2 and 3 on gold were estimated by measuring the XPS signal intensity of Au 4f7/2(takeoff angle of Oo) before and after 2 min of etching with Xe+. This 2-min Xet etch was sufficient to completely remove both lipids from gold; Le., only Au could be detected by XPS. Thickness values were estimated by use of eq 1
IAu = IAute-d/X
(1)
where IAu is the Au 4f7 intensity of the Au substrate with the lipid monolayer, jAutis the Au 4f7 intensity of the clean Au surface, d is the monolayer tiickness, and X is the inelastic mean free path (IMFP) of the Au 4fTI2photoelectrons in the phospholipid monolayer. The specific IMFP value that we have used in this equation, calculated from equations of Seah and Dench,%was 34 A. This IMFP predicts that the sampling depth (3 times the IMFP) should decrease from ca. 100 to 20 A as the takeoff angle (34) Seah, M.P.;Dench, W. A. SIA Surf. Interface Anal. 1979,1, 2.
I 20
I
I 40
I
I 60 AREA
I
I
80
I
I
100
,x 120
I
140
,
I
,
160
180
molecule)
Figure 5. Surface pressure-area isotherm for 2 at 25
OC.
increases from Oo to 80°. With eq 1, both of the monolayers formed from 2 and 3 on gold were found to be ca. 22 A,which is in excellent agreement with film thicknesses estimated by ellipsometry. Surface Site Density. In order to estimate the number of molecules of 2 present per square centimeter of geometric surface area on gold, we have subjected this modified film to hydrolysis using 5.4 M HC1. Quantitative analysis of the liberated phosphate using a colorimetric assay (see Experimental Section) for three independent samples revealed lipid loadings of 4.8,4.3, and 5.4 X 1014 lipids/cm2. On the basis of surface pressure-area isotherms measured for 2 at the air-water interface (Figure 5), it is clear that the minimal area that this lipid can occupy in a highly compressed state is ca. 40 A2. Theoretically, therefore, a maximum of ca. 2.5 X 1014molecules of 2 may be chemisorbed onto 1cm2 of gold. The higher "apparent" loading that we have observed is a likely consequence of the true surface area being greater than the geometrical area; i.e., the gold surface is not perfectly flat at the molecular level.20@~ssInterestingly, the loading that we have determined for 2 on gold is very similar to that which has previously been estimated for a tritiated form that was chemisorbed of trans-4,5-diacetyl-l,2-dithiane onto similar gold film (Le., 4.4 X 1014).20
Conclusions Taken as a whole, all of the above results obtained for 1-4 chemisorbed onto gold infer film structures in which the lipids are present as densely packed, macroscopically uniform monolayers having the polar phosphocholine groups extending outward from the surface and the sulfur moieties in intimate contact with the gold. While our ellipsometry data must be viewed with caution, for reasons discussed above, they clearly imply the presence of densely packed and oriented monolayers. Absolute thickness values are in approximate agreement with thicknesses estimated from CPK space-filling models, if it is assumed that densely packed and fully extended monolayers are oriented perpendicular to the support. The larger thickness observed for films derived from 2, as compared with those produced from 1 (and for 4 as compared with 3), is consistent with such an orientation and with a dense packing of the chains. In addition, the excellent agreement between the monolayer thickness of 2 on gold determined from ellipsometry, with that estimated by XPS, further strengthens the case for dense packing. The fact that a substantial reduction in peak currents is (35) Binnig, G.;Rohrer, H. Angew. Chem.,Znt. Ed. Engl. 1987,26,606. (36) Giambattista, B.; McNairy, W. W.; Slough, C. G.; Johnson, A,; Bell, L. D.; Coleman, R. V.; Schneir, J.; Sonnenfeld, R.; Drake, B.; Hansma, P. K. h o c . Natl. Acad. Sci. U.S.A. 1987,84, 4671.
40 Langmuir, Vol. 5, No. 1, 1989 observed for Fe(CN)63-,in and of itself, provides compelling evidence for dense and uniform packing of monolayers of 2 and 3 on gold. Finally, the high wettability of monolayers of 1-4 chemisorbed on gold, together with their dense packing and strong affinity to the support (as judged by their stability in the presence of ethanol or methanol), infer outer surface compositions rich in the polar zwitterionic phosphocholine group and lipid-gold interfaces that are rich in sulfur. XPS results that have been obtained with 2 and 3 on gold establish that both lipids are deposited intact on the gold surface and provide further support for oriented monolayers. The S 2p signal for 2 on gold makes a compelling argument for all of the sulfur being strongly chemisorbed onto the metal. In the case of 3, two types of sulfur exist; one is chemisorbed to the gold, and the other appears to be either not adsorbed or physisorbed to the gold. This result, together with the fact that the monolayer is stable in the presence of ethanol, suggests that a significant percentage of 3 is bound via a single heterocyclic disulfide group and/or one of the two sulfur atoms of eac5 disulfide moiety. Although we cannot rule out the possibility that the monolayer is comprised of two separate populations of 3 (Le., one having both of the heterocycles chemisorbed onto gold and the other having both heterocycles physisorbed), we believe this to be unlikely, given the monolayer's stability in ethanol. Finally, we note that phospholipids in natural biomembranes exist predominantly in the liquid-crystalline phase.37 On the basis of the experimental methods that we have used for film characterization, we are unable to judge the "phase" properties of these chemisorbed monolayers. The fact that 2 affords the most hydrophilic film implies that it has the highest surface density of phosphocholine groups. At the present time, however, it is not clear whether or not this strong hydrophilicity also reflects an increase in the packing density of the film; e.g., a more "solid-like'' film would be expected to minimize exposure of the lipophilic hydrocarbon chain at the lipid-water interface and maximize surface hydrophilicity. Studies that are now in progress are aimed at (1) defining the conformation and orientation of these chemisorbed lipids via a detailed reflection infrared analysis, (2) synthesizing analogous film structures in which the lengths of the sn-1 and sn-2 chains are systematically varied, (3) evaluating the biological properties of these supported films (e.g., adhesion properties toward proteins and cells), and (4) using chemisorbed phospholipid monolayers as a framework from which to construct more sophisticated "biological-like" surfaces.
Experimental Section General Methods. Unless stated otherwise, all reagents and chemicals were obtained from commercial sources and used without further purification. House-deionized water was further purified by using a Millipore Milli-Q filtering system containing one carbon and two ion-exchange stages. sn-Glycero-3phosphorylcholine was purchased from Sigma Chem. Co. as the CdCl, complex. Isolipoic acid30 and 1,2-bis(l2-hydroxydodecanoyl)-sn-glycero-3-phosphocholineBwere prepared by using procedures similar to those described in the literature. Chloroform and methanol used for chromatography were reagent grade (Fisher). Dichloromethane (Aldrich, Gold Label) was used as obtained. Methanol (Fisher) and absolute ethanol (Midwest Solvent Co.) were purified by distillation over 4A molecular sieves (8-12 mesh). 4-(Dimethy1amino)pyridine (DMAP, Aldrich) was (37) Wilkinson, D. A.; Nagle, J. F. In Liposomes: From Physical Structure to Therapeutic Applications; Knight, C. G., Ed.; Elsevier: New York, 1981; p 273.
Fabianowski et al. recrystallized once from toluene prior to use. AG-501-X8D was obtained from Bio-Rad Laboratories and was washed with eluting solvent prior to use. Octadecyl mercaptan (Aldrich Chem. Co.) was purified by column chromatography (silica, hexane) prior to use. 'H NMR, IR, and UV spectra were recorded on JEOL FX 9OQ, Beckman Acculab 7, and Bausch & Lomb Spectronic 2000 spectrometers, respectively. Chemical shifts are reported relative to tetramethylsilane. Elemental analyses were performed by Robertson Laboratory, Inc. (Florham Park, NJ). Chromatographic procedures were similar to those previously described.2g Glass vials that were used for spontaneous assembly preparations were cleaned by immersion in concentrated sulfuric acid (24 h), followed by rinsing with Milli-Q water and by freshly distilled solvent methanol or ethanol. 1 ,%-Bis[12-( isolipoyloxy )dodecanolyl]-sn -glycero-3phosphocholine (4). Procedures used for the preparation of 4 were exactly analogous to those previously described for the preparation of 3, except that isolipoic acid was used in place of lipoic acid.29,30 Esterification of 0.19 mmol of 1,2-bis(12hydroxydodecanoyl)-sn-glycero-3-phosphocholine with 0.5 mmol of isolipoic acid anhydride in dichloromethane, using 0.4 mmol of DMAP as a catalyst, afforded after workupz90.159 g (81%) of 4, as a yellow solid. Similar to 3, compound 4 should be stored as a dilute (ca. 1%) dichloromethane solution at 0 "C in the dark IR (KJ3r) v m 1738, V N ( 962,1045,1090 ~ ~ cm-'; 'H NMR (CDC1,) 6 1.28 (s, 36 H CH,), 1.35-1.85 (m, 12 H, isolipoic chain CH2),2.30 (t, 8 H, CH,COZ), 2.45-3.35 (m, 10 H, isolipoic ring), 3.40 (s, 9 H, N(CH,),), 4.05 (t, 4 H, CH20C=O), 3.78-4.58 (m, 8 H, CH2, NCHz), 5.20 (m, 1 H, CHO). Anal. Calcd for CaHMOl2NPS4: C, 55.95; H, 8.61; N, 1.36; P, 3.01; S, 12.44. Found C, 55.72; H, 8.41; N, 1.38; P , 2.54; S, 11.81. Gold Slides. Freshly evaporated gold slides were prepared by vapor deposition of ca. 1000 A of gold (Materials Research Corporation, 99.999% purity) onto 1cm X 3 cm glass slides that were primed with 150 A of chromium (Alpha, Inc.). A VEECO Model VE 776 thermal evaporator (available to us at the Sherman Fairchild Center for Solid-state Studies, Lehigh University) was used for all film preparations. This evaporator employed a resistively heated tungsten basket, operating with a base pressure of 1 X lo4 Torr. While no special precautions were taken after removal of gold slides from the evaporator, the slides were immersed in the appropriate depositing solution with minimal delay; typically, they were immersed within 1h after removal from the vacuum chamber. A 1-h time period was sufficient for measurements of optical constants required for ellipsometry. Spontaneous Monolayer Assembly. Typically, monolayer films were assembled by immersing the 1 x 3 cm gold-coated substrates into precleaned 5-mL vials that contained ca. 3 mL of an unstirred solution M) of lipid a t ambient temperature (usually 20-25 OC) for 24 h. The slides were then washed by immersing them into 3 mL of pure solvent and gently agitating them (each slide was moved in and out of the solvent 20 times). Finally, the slides were allowed to air-dry for ca. 10 min prior to ellipsometry and contact angle measurements. All manipulations of the gold slides were performed with Teflon forceps to prevent contamination and avoid mechanical damage of the surface. Contact Angle Measurements. Contact angles were measured by using a Rame-Hart Model 100 contact angle goniometer under ambient conditions (in all cases, measurements were made with relative humidities between 50% and 70%). All angles were measured within 15 s after placing the water droplet on the modified surface. Advancing angles were measured just prior to the advance of the drop when its volume is increased. The volume of the drop used was 5 pL. Increasing the drop size to 10 pL had a negligible effect on the observed contact angles. All reported values are the average of at least six water drops on each sample, a t different locations on the surface of the gold slide. Ellipsometry Measurements. Thicknesses of chemisorbed monolayers were determined by using a Rudolph Model AutoEL-I1 computerized ellipsometer operating with a laser beam having a wave length of 6328 A. The incidence angle used in all cases was 70'. Film thicknesses were calculated from the differences in the measurements between "bare" and lipid-modified gold. Optical constants ( N , and K,) were measured prior to immersion in the lipid solution, and a refractive index value of 1.50 was assumed in order to calculate the apparent film thick-
Assembly of Phosphatidylcholine Monolayers nesses. Measurements were made at no less than eight different locations on the surface of the gold slide. Thickness values were computed by using a Rudolph Film 85 Transparent Single Film Program (no. 13). Monolayer Formation of 2 at the Gas-Water Interface. Surface pressure-area isotherms were recorded with a computerized MGW Lauda film balance equipped with a thermal controller. Lipid 2 was spread from a hexane/ethanol solution (9/1, v/v) a t the gas-water interface (25 "C). The aqueous subphase consisted of house-deionized water that was purified by passage through a Milli-Q deionizer and purged with nitrogen. All monolayer experiments were conducted under a nitrogen atmosphere. Electrochemical Measurements. Cyclic voltammetry was performed by using a corrosion voltamograph BAS potentiostat and a 7046 Hewiett-Packard X-Y recorder, which used a conventional three-electrode cell. The electrode was mounted in a gold-plated clamp with a spacer, so that a constant surface area (2 cm2)was exposed to the electrolyte. All electrolyte solutions were freshly prepared by using deionized water from a Milli-Q purification system and reagent grade KC1 and K3Fe(CN)e (Fisher). Before electrochemical measurements, the electrolyte solution was purged with argon for 15 min. Typically, the cyclic voltamogram was run in the potential range between -0.2 and +0.5 V, using Ag/AgC1(3 N NaCI; equivalent potential to SCE) as the reference electrode and employing a scan rate of 100 mV/s. The first scan was started from the open circuit potential in the positive direction. Phosphorus Analysis. Typically, a 1 X 3 cm lipid-modified gold-coated slide was placed in a 9-cm-diameter Petri dish with the gold surface facing down. A 2.5-mL volume of 5.4 M HC1 was then carefully pipetted into the dish. Care was taken so that the bare glass side of the slide was not exposed to the acidic solution or to the "Milli-Q" water, which was used in subsequent washings. The dish was covered with a second (inverted) Petri dish, placed on a hot plate (ca. 40 "C) for 1h, and then allowed to stand for 6 h at ambient temperature. The acidic solution was then transferred from the dish to a test tube (the slide was carefully lifted on one side by using a clean glass rod in order to remove a portion of the solution which was trapped under the slide) and slowly concentrated to dryness by heating at ca. 40 "C. The gold-coated slide was washed 3 times by gentle warming in the presence of 5 mL of "Milli-Q water" (40 "C, 1 h), the washings were added to the test tube, and the contents was again concentrated to dryness. Residual phosphorus was analyzed by using colorimetric methods which were similar to those previously d e s ~ r i b e d . ~Phosphorus ~ analysis determined from blank experiments indicated the equivalent of ca. 4 x 10" phosphorus atoms/cm2. Samples, containing chemisorbed 2, were corrected by subtraction of this blank value. X-ray Photoelectron Spectroscopy. The monochromatic A1 K a X-ray source, hemispherical analyzer, multichannel de(38) Regen, S. L.; Kirszensztejn, P.; Singh, A. Macromolecules 1983, 16, 335.
Langmuir, Vol. 5,No. l, 1989 41 tector, differentially pumped Leybold-Heraeus ion gun, and variable-angle sample stage features of a Surface Science Instrument (SSI) SSX-100 spectrometer system were utilized for all XPS experiments. Samples that were shipped from Lehigh to the University of Washington for XPS analysis were stored in sealed vials and were covered with methanol. Each sample was removed from its storage vial and mounted directly on the variable-angle sample stage. The sample was then immediately inserted into the spectrometer and analyzed. Elemental compositions were calculated from detailed scans (20-eV regions a t an analyzer pass energy of 150 eV and spot size of 1000 pm) of the Au 4f, C le, 0 Is, N ls, P 2p, and S 2p photoemission lines at takeoff angles of 0" and 80". High-resolution C 1s spectra (at an analyzer pass energy of 25 eV and an X-ray spot size of lo00 pm) were acquired a t a takeoff angle of 55". Survey scans over a binding energy range 0-lo00 eV with an analyzer pass energy of 150 eV and an X-ray spot size of 1000 pm were acquired a t a takeoff angle of 55" to determine which elements were present on the outer surface of each sample. In order to minimize X-ray damage of the sample, a new spot on the sample was analyzed when the takeoff angle was changed. All samples appeared laterally homogeneous. The experimental peak areas were numerically integrated and normalized with the SSI software package to account for the number of scans, the number of channels per electronvolt, the Scofield photoionization cross section,39and the sampling depth. The normalized peak areas were then used to calculate the surface elemental composition. The SSX-100 transmission function for a pass energy of 150 eV was assumed to be constant over the binding energy range spanned by the Au, P, S, C, N, and 0 lines.@ The sampling depth was assumed to vary as KEO.', where KE is the kinetic energy of the photoelectron^.^^ The instrument BE scale was calibrated by setting the Au 4f7/2 BE to 83.93 eV and the metallic Cu 2p3/2 BE to 932.47 eV. The C 1s high-resolution spectra were resolved into individual Gaussian peaks by using a least-squares curve-fitting program provided by SSI.
Acknowledgment. We are grateful to Ralph Nuzzo (AT&T Bell Laboratories), Dave Allara (Pennsylvania State University), and George Whitesides (Harvard University) for many valuable discussions regarding spontaneous assembly methods. We are also grateful to our colleagues, Tadeusz Diem, Michael Markowitz, and Nancy Dodrer (Lehigh) and to Robert D. McElhaney, Jr. (University of Washington), for valuable technical assistance. Registry No. 1, 87050-11-1; 2,93404-44-5; 3, 116405-86-8; 4, 117465-69-7;Au, 7440-57-5; isolipoic acid anhydride, 117342-07-1; 1,2-bis(12-hydroxydodecanoyl)-sn-glycero-3-phosphocholine, 117465-70-0. (39) Scofield, J. H. J. Electron. Spectrosc. Relat. Phenom. 1976,8,1!29. (40) Application Note, Surface Science Instruments (Mountain View, CA), March, 1987.
