Formation and Characterization of Planar Phospholipid Bilayers

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Formation and Characterization of Planar Phospholipid Bilayers Supported on TiO2 and SrTiO3 Single Crystals Tammy E. Starr and Nancy L. Thompson* Department of Chemistry, Campus Box 3290, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290 Received July 13, 2000. In Final Form: September 5, 2000 Supported phospholipid bilayers have been prepared on rutile TiO2 and SrTiO3 single-crystal substrates. Fluidlike bilayers were successfully deposited onto each surface by Langmuir-Blodgett and LangmuirSchaeffer methods and the vesicle adsorption and fusion method. The characteristics of membranes supported on TiO2 and SrTiO3 single crystals were evaluated using fluorescence pattern photobleaching recovery, fluorescence imaging, and differential interference contrast imaging, and the results were compared to similar preparations on fused silica. Diffusion coefficients of a fluorescent lipid in membranes supported on all substrates were on the order of 10-8 cm2/s, and fractional mobilities were 74-99%. Measured diffusion coefficients were faster for fluorescent lipids in bilayers on TiO2 and SrTiO3 than in bilayers on fused silica, and some sample types exhibited two diffusing species rather than a single diffusing species. Both the intensities and bleachabilities of the fluorescent lipids were significantly reduced for bilayers on TiO2 and SrTiO3 as compared to fused silica. A new theoretical model for interpreting fluorescence pattern photobleaching recovery data for fluorescent lipid diffusion in planar membranes is described. The ability to form planar phospholipid bilayers on TiO2 and SrTiO3 is expected to facilitate new developments in total internal reflection fluorescence microscopy, surface-based assays of importance in biotechnology, and biosensors.

Introduction Planar supported phospholipid bilayers are widely used as model systems for the study of biological membranes and are of growing interest in the areas of biosensors and biofunctionalized surfaces.1-7 At least three methods for depositing bilayers onto planar surfaces have been reported. The classic method is the sequential transfer of two monolayers from the air-water interface via Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) techniques.8,9 An alternative method is the adsorption of phospholipid vesicles to the surface and their subsequent fusion to form a bilayer.10,11 The third method is a hybrid of the two previous methods, in which a LB monolayer is first deposited and the second leaflet is formed by vesicle fusion.12 Planar supported bilayers have been deposited onto numerous different substrate surfaces including glass,13 fused silica,14 oxidized silicon,8 mica,15,16 and thin * To whom correspondence should be addressed. Phone: (919) 962-0328. Fax: (919) 966-3675. E-mail: [email protected]. (1) Sackmann, E. Science 1996, 271, 43. (2) Nikolelis, D. P.; Hianik, T.; Krull, U. J. Electroanalysis 1999, 11 (1), 7. (3) Heyse, S.; Stora, T.; Schmid, E.; Lakey, J. H.; Vogel, H. Biochim. Biophys. Acta 1998, 1376 (3), 319. (4) Plant, A. L. Langmuir 1999, 15 (15), 5128. (5) Thompson, N. L.; Pearce, K. H.; Hsieh, H. V. Eur. Biophys. J. 1993, 22, 367. (6) Hollars, C. W.; Dunn, R. C. Biophys. J. 1998, 75, 342. (7) Yang, J.; Kleijn, J. M. Biophys. J. 1999, 76 (1), 323. (8) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105. (9) Wright, L. L.; Palmer, A. G.; Thompson, N. L. Biophys. J. 1988, 54, 463. (10) Brian, A. A.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 6159. (11) Pearce, K. H.; Hiskey, R. G.; Thompson, N. L. Biochemistry 1992, 31, 5983. (12) Kalb, E.; Frey, S.; Tamm L. K. Biochim. Biophys. Acta 1992, 1103, 307. (13) Cremer, P. S.; Boxer, S. G. J. Phys. Chem. B 1999, 103, 2554. (14) Lagerholm, B. C.; Starr, T. E.; Volovyk, Z. N.; Thompson, N. L. Biochemistry 2000, 39 (8), 2042. (15) Egawa, H.; Furusawa, K. Langmuir 1999, 15, 1660.

films of platinum,17 gold,18 silver,19 TiO2,20,21 indium tin oxide,7 and polymers.22-26 Two additional hydrophilic surfaces, single-crystal rutile TiO2 and single-crystal SrTiO3, are potentially useful substrates for supported planar bilayers. Both surfaces have been studied extensively by macroscopic techniques27 and by atomic force microscopy (AFM) and scanning tunneling microscopy (STM).28-33 AFM/STM studies confirm that both surfaces are flat and smooth28,34 with (16) Zasadzinski, J. A. N.; Helm, C. A.; Longo, M. L.; Weisenhorn, A. L.; Gould, S. A. C.; Hansma, P. K. Biophys. J. 1991, 59, 755. (17) Puu, G.; Gustafson, I. Biochim. Biophys. Acta 1997, 1327, 149. (18) Bunjes, N.; Schmidt, E. K.; Jonczyk, A.; Rippmann, F.; Beyer, D.; Ringsdorf, H.; Graber, P.; Knoll, W.; Naumann, R. Langmuir 1997, 13 (23), 6188. (19) Salamon, Z.; Wang, Y.; Tollin, G.; Macleod, H. A. Biochim. Biophys. Acta 1994, 1195, 267. (20) Csu´cs, G.; Ramsden, J. J. Biochim. Biophys. Acta 1998, 1369, 61. (21) Sinner, A.; Offenha¨usser, A. Thin Solid Films 1998, 327-329, 758. (22) Elender, G.; Ku¨hner, M.; Sackmann, E. Biosens. Bioelectron. 1996, 11 (6-7), 565. (23) Majewski, J.; Wong, J. Y.; Park, C. K.; Seitz, M.; Israelvachvili, J. N.; Smith, G. S. Biophys. J. 1998, 75 (5), 2363. (24) Wong, J. Y.; Park, C. K.; Seitz, M.; Israelvachvili, J. Biophys. J. 1999, 77 (3), 1458. (25) Wong, J. Y.; Majewski, J.; Seitz, M.; Park, C. K.; Israelvachvili, J.; Smith, G. S. Biophys. J. 1999, 77 (3), 1445. (26) Kuhner, M.; Tampe, R.; Sackmann, E. Biophys. J. 1994, 67 (1), 217. (27) Henrich, V. E.; Cox, P. A. The Surface Science of Metal Oxides; Cambridge University Press: Cambridge, U.K., 1994. (28) Polli, A. D.; Wagner, T.; Ru¨hle, M. Surf. Sci. 1999, 429, 237. (29) Sekiguchi, S.; Fujimoto, M.; Nomura, M.; Sung-Baik, C.; Tanaka, J.; Nishihara, T.; Min-Gu, K.; Hyoung-Ho, P. Solid State Ionics 1998, 108, 73. (30) Sta¨uble-Pu¨mpin, B.; Ilge, B.; Matijasevic, V. C.; Scholte, P. M. L. O.; Steinfort, A. J.; Tuinstra, F. Surf. Sci. 1996, 369, 313. (31) Fan, F.-R. F.; Bard, A. J. J. Phys. Chem. 1990, 94, 3761. (32) Rohrer, G. S.; Henrich, V. E.; Bonnell, D. A. Science 1990, 250, 1239. (33) Jarvis, S. P.; Tokumoto, H.; Yamada, H.; Kobayashi, K.; Toda, A. Appl. Phys. Lett. 1999, 75 (24), 3883. (34) Fujishima, A.; Chikuni, M.; Kojima, E.; Kitamura, A.; Watanabe, M. S. T. Nature 1997, 388, 431.

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microroughnesses on the order of e15 nm.31,35 TiO2 and SrTiO3 have both been used as photocatalytic electrodes in the photoelectrolysis of water and as substrates for adsorption studies of metals on transition-metal oxides.27 The TiO2 single-crystal surface has been examined for its antifogging, self-cleaning properties,34 and it has been used as a substrate for the photodecomposition and thermal degradation of organic monolayers.36-38 The surface of the SrTiO3 single crystal is most commonly used as a substrate for high-temperature superconducting oxide films.39-42 Although TiO2 and SrTiO3 single crystals have been used as substrates for many applications, they have not yet been used as substrates for planar supported bilayers. The ability to deposit supported phospholipid bilayers onto the TiO2 and SrTiO3 single-crystal surfaces in combination with their semiconducting properties and high dielectric constants21,32,43 may make these surfaces very applicable in the fields of biomembranes and biosensors. These substrates also have very high refractive indices (≈2.5) and are therefore of interest in total internal reflection fluorescence microscopy.44 Whereas the evanescent wave depth at the interface of water and fused silica is typically 800 Å, this depth at the interface of water and TiO2 or SrTiO3 is predicted to be as low as 180 Å.5 In the work described herein, the integrities of supported phospholipid bilayers composed of mixtures of palmitoyloleoyl-phosphatidylcholine and cholesterol containing the fluorescent lipid analogue nitrobenzoxadiazol-phosphocholine (NBD-PC) on TiO2 and SrTiO3 single crystals have been examined by fluorescence pattern photobleaching recovery (FPPR), fluorescence imaging, and differential interference contrast (DIC) imaging. Supported phospholipid bilayers formed by both LB/LS monolayer deposition and by vesicle adsorption and fusion were evaluated on each substrate. The diffusion coefficients, fractional mobilities, and visual properties have been compared with results from similar bilayer preparations on planar fused silica substrates. Materials and Methods Substrate Preparation. Epitaxially polished SrTiO3 (randomly oriented, 25 mm × 25 mm × 1 or 0.5 mm) and rutile TiO2 (randomly oriented, 20 mm × 20 mm × 1 or 0.5 mm) singlecrystal substrates (First Reaction, Hampton Falls, NH) and fused silica substrates (25 mm × 25 mm × 1 mm) (Quartz Scientific, Fairport Harbor, OH) were purchased commercially. According to the manufacturer’s specifications, the roughnesses of the bare TiO2 and SrTiO3 surfaces were 1-12 Å over a horizontal scan distance of 100 µm. These roughnesses were confirmed, and the roughness of the fused silica substrates was determined to be approximately 10-20 Å over 15 µm by AFM (Nanoscope 3A, Digital Instruments, Santa Barbara, CA). All substrates were cleaned by boiling in detergent (Lot 08778, ICN, Aurora, OH), bath sonicating, rinsing thoroughly with deionized water, and drying at 160 °C. Immediately before bilayer deposition, substrates were cleaned in an argon ion plasma (15 min, 25 °C) (35) Szot, K.; Speier, W. Phys. Rev. B 1999, 60 (8), 5909. (36) Sawunyama, P.; Fujishima, A.; Hashimoto, K. Langmuir 1999, 15, 3551. (37) Onishi, H.; Iwasawa, Y. Langmuir 1994, 10, 4414. (38) Onishi, H.; Yamaguchi, Y.; Fukuii, K.; Iwasawa, Y. J. Phys. Chem. 1996, 100, 9582. (39) Han, K.; Yu-Zhang, K. Philos. Mag. B 1999, 79 (6), 897. (40) Gupta, M. K.; Vyas, J. C.; Gandhi, D. P.; Muthe, K. P.; Aswal, D. K.; Gupta, S. K.; Kothiyal, G. P.; Sabharwal, S. C. J. Cryst. Growth 1995, 156 (1-2), 74. (41) Moller, P. J.; Komolov, S. A.; Lazneva, E. F. Surf. Sci. 1999, 425 (1), 15. (42) Matijasevic, V. C.; Ilge, B.; Sta¨uble-Pu¨mpin, B.; Rietveld, G.; Tuinstra, F.; Mooij, J. E. Phys. Rev. Lett. 1996, 76, 4765. (43) Yoon, J.-W.; Miyayama, M. Appl. Phys. Lett. 1999, 74 (5), 738. (44) Thompson, N. L.; Lagerholm, B. C. Curr. Opin. Biotechnol. 1997, 8 (1), 58.

Starr and Thompson (PDC-3XG, Harrick Scientific, Ossining, NY). Fused silica substrates were discarded after one use. TiO2 and SrTiO3 substrates were soaked in acetone, rinsed with deionized water, soaked in 2-propanol, rinsed again with deionized water, and then cleaned for reuse as described above. TiO2 and SrTiO3 substrates were repolished (First Reaction) as necessary. Phospholipid Vesicles. Small unilamellar vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol (Chol), and 1-acyl-2-[12-[7-nitro-2-1,3-benzoxadiazol-4yl]aminododecanoyl]-sn-glycero-3-phosphocholine (NBD-PC) (Avanti Polar Lipids, Birmingham, AL) were prepared. Suspensions containing 2 mM POPC/NBD-PC (98:2, mol:mol) or POPC/ Chol/NBD-PC (68:30:2, mol:mol:mol) in deionized water were tip sonicated to form vesicles as previously described.11,14 Vesicle suspensions were clarified immediately before use by sedimentation at 130 000g for 30 min. Planar Bilayers. Supported planar bilayers were deposited onto clean substrates either by vesicle adsorption and fusion or by LB/LS methods.11,14 Planar bilayers formed by vesicle adsorption and fusion were constructed by applying 65 µL of vesiclecontaining solution to the substrates (30 min, 25 °C) and reapplying another 65 µL of vesicle-containing solution (1 h, 25 °C), followed by rinsing with 3 mL of Tris buffer (0.05 M, pH 7.4). LB/LS bilayers were prepared according to standard techniques.8,9 The appropriate lipid mixture was dissolved in hexane/ ethanol (9:1) to produce 1 mM POPC/NBD-PC (98:2, mol:mol). A volume corresponding to 100 Å2/molecule was spread onto the air-water interface of a Langmuir trough (model 4, Joyce-Loebl, Gateshead, U.K.) filled with deionized water (18 MΩ cm). After a wait of 10 min for the solvent to evaporate, the monolayer at the interface was compressed slowly (1-2 Å2 molecule-1 min-1) to a surface pressure of 35 mN/m. While the surface pressure was maintained at 35 mN/m, the substrates were raised perpendicularly through the interface at a speed of 4 mm/min to apply a monolayer, and the second leaflet of the bilayer was applied by lowering the monolayer-covered substrates parallel to and through the interface. Bilayers were then rinsed with 3 mL of Tris buffer. Fluorescence and DIC Imaging. Epifluorescence and DIC images were obtained using an instrument consisting of an inverted microscope (Zeiss Axiovert 10 with 100× 1.30 NA PlanNeoFluar objective) and a charge-coupled device (CCD) camera (MicroMax, 782 × 582 chip, Princeton Instruments). All micrographs are 12-bit digitized images with 0.08 µm pixel resolution. Fluorescence Pattern Photobleaching Recovery. FPPR measurements were carried out on an instrument consisting of an argon ion laser (Innova 90-3, Coherent, Palo Alto, CA), an inverted optical microscope (Zeiss Axiovert 35), and a singlephoton counting photomultiplier (31034A, RCA, Lancaster, PA). The radii of the illuminated and observed circular areas in the image plane, formed by the beam and an image plane aperture, were 70 and 50 µm, respectively. Other experimental parameters were as follows: excitation wavelength, 488.0 nm; objective, 40 × 0.75 NA; observation and bleach powers, 0.3-5 µW and 0.5 W; bleach pulse duration, 25-350 ms; bleach depths, 20-75%. A series of parallel stripes was created by placing a Ronchi ruling with 50 lines/in. in a back image plane. The resulting ruling periodicity in the sample plane was 19.5 µm. Fluorescence recovery was monitored for 40-100 s after photobleaching, and data were curve-fit to theoretical forms by using the Mathematica (Wolfram Research Inc., Champaign, IL) software package. All fit parameters were independent of the bleaching depth.

Results Formation and Characterization of Supported Planar Phospholipid Bilayers. Planar phospholipid bilayers were successfully deposited on rutile TiO2 substrates, on SrTiO3 single-crystal substrates, and on fused silica substrates. Membranes were formed on all three substrate types by the adsorption and fusion of probesonicated vesicles composed of POPC/NBD-PC (98:2, mol: mol) and POPC/Chol/NBD-PC (68:30:2, mol:mol:mol). Bilayers composed of POPC/NBD-PC (98:2, mol:mol) were also formed on the three types of substrates with the LB/

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Figure 1. Epifluorescence images of supported planar bilayers. Bilayers were prepared by (a-c) the adsorption and fusion of POPC vesicles, (d-f) the adsorption and fusion of POPC/Chol vesicles, and (g-i) LB deposition of POPC. Bilayers were supported on (a, d, and g) fused silica, (b, e, and h) SrTiO3, and (c, f, and i) TiO2.

LS technique. The visual properties of supported membranes on TiO2 and SrTiO3 substrates were examined with fluorescence and DIC microscopy and compared to those of similar bilayer preparations on fused silica substrates. The diffusional properties of the fluorescent lipid probe NBD-PC in the supported bilayers were characterized with FPPR. Fluorescence Images. Figure 1 shows typical epifluorescence images of supported bilayers for each of the three preparation types on each of the three substrate types. Membranes prepared by the adsorption and fusion of POPC and POPC/Chol vesicles on fused silica substrates appeared uniformly fluorescent within optical resolution (Figure 1a,d). On the contrary, membranes prepared by the adsorption and fusion of POPC vesicles on SrTiO3 and TiO2 substrates appeared as a uniform background coverage with numerous small ( D2 and λ is the rate of transport of the rapidly diffusing species through the 1/e2 radius of the Gaussianshaped illuminated area. The model is simplified to a system consisting of only one mobile population and one immobile population by allowing f2 ) 0 (eq 4). When the illuminated area is large compared to the stripe periodicity (λ f 0), eq 4 reduces to eq 3 with n ) 3 and D3 ) 0. In the absence of the ruling, a f ∞, ki f 0, and eq 4 reduces to

F(t) ≈ 1 - β + βf1λt F(-)

(5)

The degree to which the observed fluorescence recoveries were due in part to the exchange of bleached molecules from inside with unbleached molecules from outside the large illuminated area as opposed to exchange between the striped illuminated regions was assessed by fitting data obtained in the absence of the ruling to eq 5. The data were always lines with small positive slopes. The value of the product f1λ was obtained from the intercepts and slopes of these data when fit to lines. This parameter ranged from 3.2 × 10-3 to 2.7 × 10-4 s-1, corresponding to 1-16% recovery after 50 s. The parameter f1λ was strongly correlated with the NBD-PC diffusion coefficient; larger f1λ values corresponded to samples with faster diffusion. Lateral Mobility of NBD-PC in Supported Bilayers. The diffusional characteristics of NBD-PC in supported bilayers were examined by using FPPR. With the ruling in place, the fluorescence recovered to a value approximately equal to the midpoint of the prebleach and immediate postbleach fluorescence after 1-2 min for all nine sample types. This result indicates qualitatively that most of the NBD-PC molecules were laterally mobile with diffusion coefficients on the order of 10-9 to 10-8 cm2 s-1. Fluorescence recovery curves were first fit to the two functional forms which describe a single mobile component and a single immobile component, in the absence and presence of contributions arising from diffusion from outside of the illuminated area (eq 3 with n ) 2 and D2 ) 0 and eq 4 with f2 ) 0). The parameter a and product f1λ were fixed at their previously determined values, and the free parameters were β, D1, and f1. The χ2 goodnessof-fit parameters were calculated for the best fits to both equations. For all sample types, the average χ2 values for fits to eq 4 were slightly less than those for fits to eq 3. T-test analysis taking into account the uncertainties and number of curves taken for each sample type indicated that the two χ2 values for all samples composed of POPC/ NBD (98:2, mol:mol) regardless of the substrate type and (47) Starr, T. E.; Thompson, N. L. Manuscript in preparation.

preparation were statistically different with 80-98% certainty.48 The two χ2 values for samples containing cholesterol were statistically different with only e50% certainty. Because the values of f1λ for cholesterolcontaining samples were ∼10-4, eq 3 ≈ eq 4. Thus, it was determined that eq 4 was a better (or at least an equivalent) theoretical model for all data, and further analysis was carried out using only eq 4. Fluorescence recovery curves were next fit to the more general functional form describing an immobile component and two mobile components (eq 4). The parameters a and f1λ were fixed at their previously determined values. The free parameters were β, D1, D2, f1, and f2. F-statistic analysis of the χ2 goodness-of-fit parameters9 and comparison of the reduced χ2 values indicated that the majority of fluorescence recovery curves for Langmuir-Blodgett bilayers and for cholesterol-containing bilayers on all three substrates was adequately fit by the one mobile component model (eq 4 with f2 ) 0). Diffusion coefficients of NBD-PC in LB bilayers and in cholesterol-containing bilayers were in the ranges of (1.4-2.1) × 10-8 and (0.4-1.6) × 10-8 cm2 s-1, respectively. Fractional mobilities ranged from 73 to 97%. For these bilayers, there was little evidence for a second mobile component. On the contrary, recovery curves for planar bilayers formed by the adsorption and fusion of POPC/NBD (98:2, mol:mol) vesicles were often better fit by the two mobile components model (eq 4). The two diffusion coefficients were on the order of 10-8 and e10-9 cm2 s-1, respectively, and the total mobilities ranged from 89 to 99% with the proportions of fast and slow components varying with the substrate. There was also more heterogeneity from area to area on these samples, as evidenced by the larger uncertainties in the best-fit values for the diffusion coefficients and fractional mobilities. Representative recovery curves and their corresponding fits to the one and two mobile components models are shown in Figure 3. The diffusion coefficients, mobile fractions, and reduced χ2 values49 obtained from the one mobile component model and the two mobile components model (bilayers formed from POPC vesicles) are shown in Table 2. Diffusion coefficients of the NBD-PC lipid analogue were faster in all bilayer types when supported on TiO2 or SrTiO3 than in similar bilayers supported on fused silica. The presence of cholesterol slowed the diffusion in bilayers supported on all three substrates. Mobile fractions were larger for all bilayer compositions and preparations for samples on fused silica surfaces than for corresponding bilayers supported on TiO2 or SrTiO3. Similarly, mobile fractions were larger for LB bilayers on all surfaces than for bilayers formed from vesicles on each surface. Discussion Phospholipid bilayers have been successfully deposited onto fused silica, TiO2 single-crystal, and SrTiO3 singlecrystal planar substrates by vesicle adsorption and fusion. Planar bilayers prepared by the fusion of POPC/NBD-PC vesicles appeared uniformly fluorescent when supported on fused silica, but numerous small bright defects were present in the bilayer when the support was TiO2 or SrTiO3. Features present in DIC images of these samples suggested that the defects were large unfused vesicles or (48) Johnson, R.; Bhattacharyya, G. Statistics Principles and Methods; John Wiley and Sons: New York, 1987. (49) Taylor, J. An Introduction to Error Analysis: The Study of Uncertainties in Physical Measurements; Oxford University Press: Mill Valley, CA, 1982.

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Figure 3. Representative best fits of FPPR recovery curves to theoretical forms. These plots show two typical fluorescence recovery curves and their best fits to eq 4 for both the one- and two-component models. The data are for (a and b) a POPC LB bilayer on TiO2 and (c and d) a bilayer formed by the adsorption and fusion of POPC vesicles on SrTiO3. The values of f1λ were (a and b) 8.8 × 10-4 and (c and d) 3.1 × 10-3. The best-fit parameters for the two curves were (a) D1 ) 1.6 × 10-8 cm2 s-1, f1 ) 0.94, β ) 0.77; (b) D1 ) 1.6 × 10-8 cm2 s-1, D2 ) 1.0 × 10-9 cm2 s-1, f1 ) 0.92, f2 ) 0.08, β ) 0.79; (c) D1 ) 2.2 × 10-8 cm2 s-1, f1 ) 0.74, β ) 0.77; (d) D1 ) 5.1 × 10-8 cm2 s-1, D2 ) 5.2 × 10-9 cm2 s-1, f1 ) 0.66, f2 ) 0.23, β ) 0.85. The reduced χ2 goodness-of-fit parameters were (a) 0.89, (b) 0.94, (c) 1.26, and (d) 0.93. The insets show the residuals. Table 2. Diffusion Coefficients and Mobile Fractions for NBD-PC in Supported Bilayers preparation and substrate

diffusion coefficient 1 (10-8 cm2 s-1)

fused silica TiO2 SrTiO3

1.4 ( 0.2 1.6 ( 0.5 2.1 ( 0.5

fused silica TiO2 SrTiO3

0.40 ( 0.03 1.6 ( 0.6 1.5 ( 0.5

fused silica TiO2 SrTiO3

0.62 ( 0.18 2.5 ( 0.8 3.1 ( 0.5

fused silica TiO2 SrTiO3

2.9 ( 0.9 3.8 ( 0.7 4.3 ( 0.9

diffusion coefficient 2 (10-8 cm2 s-1)

mobile fraction 1 (%)

Langmuir-Blodgett

mobile fraction 2b (%)

POPCd

Vesicle Fusion POPC/Chold

Vesicle Fusion POPCd

Vesicle Fusion POPCe,f 0.32 ( 0.12 53 ( 7 0.34 ( 0.18 64 ( 17 0.29 ( 0.13 71 ( 16

46 ( 6 26 ( 13 18 ( 13

a

total mobile fraction (%)

χ2redc

97 ( 3 85 ( 7 81 ( 7

0.97 ( 0.05 0.95 ( 0.04 0.96 ( 0.04

92 ( 6 73 ( 10 78 ( 9

0.99 ( 0.03 0.95 ( 0.07 0.95 ( 0.05

80 ( 9 75 ( 10 74 ( 9

1.1 ( 0.1 0.99 ( 0.06 1.1 ( 0.1

99 ( 2 90 ( 10 89 ( 10

0.99 ( 0.06 0.97 ( 0.05 0.99 ( 0.06

a All values are averages over 30-34 curves on 5 different samples, and uncertainties are standard deviations of the mean. b The characteristic times for the slow diffusion coefficient, k2-1 ≈ 70-170 s, are approximately equal to the acquisition times, so values reported for D2 should be regarded as upper limits. c A reduced χ2 goodness-of-fit parameter 3. Average F values for planar membranes prepared by POPC vesicle fusion supported on fused silica, TiO2, and SrTiO3 were 68, 10, and 18 respectively, and the proportions of curves better fit by the model for two mobile components were 84%, 72%, and 63%.

vesicle aggregates embedded in or adsorbed to the bilayer. The presence of unfused vesicles on TiO2 and SrTiO3 is consistent with the previous observation that supported membranes prepared similarly on an SiO2 waveguide sputtered with TiO2 are composed of 80% bilayer and 20% vesicles or vesicle aggregates.20 The addition of 30 mol % cholesterol to the POPC vesicles resulted in uniformly fluorescent bilayers supported on

each of the three substrates. It has been reported previously that the presence of cholesterol improves the quality of LB bilayers supported on polymer films by suppressing the formation of buds.22 In the current measurements, the addition of cholesterol improved the quality of bilayers formed by vesicle fusion by either enhancing the vesicle fusion or inhibiting vesicle adhesion. In both cases, the observations may be explained by the

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suppression of undulation forces in the presence of cholesterol as a result of the increase in membrane tension and membrane bending moduli.22 Phospholipid bilayers were also deposited onto each of the three substrates by LB/LS methods. These LB bilayers exhibited small heterogeneities in the fluorescence intensity on the order of optical resolution in size that were not detectable by DIC imaging. Similar submicron heterogeneities in LB monolayers and bilayers composed of DPPC have been reported previously.50-52 The relative intensities and bleaching properties of the fluorescent lipid NBD-PC varied with the substrate on which bilayers were supported. The reduced fluorescence intensities of planar bilayers supported on TiO2 and SrTiO3 are most likely due to fluorescence quenching by the metaloxide substrates. The increase in fluorescence quenching by TiO2 and SrTiO3 is predicted by and consistent with theoretical models for the fluorescence emission of fluorophores near dielectric and metal surfaces53,54 as well as previous experimental results concerning fluorescence near semiconductors.55 The experimental observation that an increase in quenching leads to a decrease in the rate of photobleaching is also consistent with theoretical models54,56 and previously reported measurements.54 FPPR experiments indicated that planar bilayers prepared by both methods, composed of both phospholipid mixtures, and supported on all three substrates were fluidlike. Diffusion coefficients and fractional mobilities of NBD-PC in the bilayers were on the order of 10-8 cm2 s-1 and 73-97%. The values of 1.4 × 10-8 cm2 s-1 and 97% are consistent with previous results for POPC LB bilayers supported on fused silica.57,58 Planar bilayers prepared by the adsorption and fusion of POPC/NBD-PC vesicles exhibited two mobile species when supported on all three substrates. This existence of two different mobile species in a planar bilayer supported on fused silica and composed of POPC has been observed previously by single-molecule microscopy.58 The observed diffusion coefficients and fractional mobilities are in the same range as those previously reported.10-12,58,59,60 Samples prepared by the adsorption and fusion of cholesterol-containing vesicles exhibited homogeneous diffusion regardless of the substrate type. The presence of cholesterol slowed the diffusion coefficients as expected.61,62 In addition to effects due to the preparation method and phospholipid composition of supported membranes, general trends in the diffusion coefficients and fractional mobilities were observed as a function of the substrate type. This observation might be explained by differences in the separations between the planar membranes and

the supports. In a comparison of bilayers supported on fused silica to bilayers supported on TiO2 or SrTiO3, the structure, surface charge density,63,64 and surface roughness of the substrate supports vary. Therefore, the van der Waals, electrostatic, and hydration forces differ for bilayers on different substrates.13,65 The undulation forces are different in the absence than in the presence of cholesterol. If the attractive van der Waals forces dominate the repulsive electrostatic and hydration forces less heavily on TiO2 and SrTiO3 than on fused silica, the bilayers would have greater distances (and therefore thicker water layers between the substrates and bilayers) from the TiO2 and SrTiO3 surfaces than from the fused silica surface. A larger separation between the membrane and the substrate could facilitate faster phospholipid diffusion because the frictional contact between the substrate with its associated water and the mobile phospholipids would be reduced.66 Fractional mobilities also varied with the substrate and method of bilayer preparation. Mobile fractions for all sample preparations and compositions were lower on TiO2 and SrTiO3. For samples prepared by POPC vesicle fusion, this decrease was due at least in part to unfused vesicles. Another possible source may be imperfections, such as scratches, in the substrates, caused by handling and reuse. Previous studies have shown that a mobile bilayer is not capable of healing or recovering over a scratch.67 The ability to form supported phospholipid bilayers on TiO2 and SrTiO3 single-crystal surfaces may facilitate a number of new applications. There has recently been much development using supported membranes in the area of biosensors.1 Two such developments are the micropatterning and compartmentalization of lipid bilayers on solid supports68-71 and the application of a potential across the supported membrane to produce a concentration gradient71,72 or to use as an electrode.7,21 TiO2 and SrTiO3 singlecrystal substrates may be candidates for substrates to further advance these fields. Another technology in which TiO2 and SrTiO3 supported bilayers will be useful is total internal reflection fluorescence microscopy.5,44,74-76 Traditionally, fused silica (n ) 1.47) is used as a solid support to create evanescent fields that penetrate approximately 700-1000 Å into the aqueous phase. TiO2 (n ) 2.60) and SrTiO3 (n ) 2.41) could be used to create evanescent waves as thin as 180 Å. Such a decrease in the evanescent depth may allow the measurement of weaker equilibrium constants and the associated kinetic rates by total internal reflection fluorescence microscopy. Given a constant binding site density, the lower limit of the equilibrium constant measurable is

(50) Hwang, J.; Tamm, L. K.; Bo¨hm, C.; Ramalingam, T. S.; Betzig, E.; Edidin, M.; Science 1995, 270, 610. (51) Tamm, L. K.; Bo¨hm, C.; Yang, J.; Zhifeng, S.; Hwang, J.; Edidin, M.; Betzig, E. Thin Solid Films 1996, 285, 813. (52) Hollars, C. W.; Dunn, R. C. Biophys. J. 1998, 75, 342. (53) Hellen, E. H.; Axelrod, D. J. Opt. Soc. Am. B 1987, 4 (3), 337. (54) Enderlein, J. Chem. Phys. 1999, 247, 1. (55) Nakache, M.; Schreiber, A. B.; Gaub, H.; McConnell, H. H. Science 1985, 317 (5), 75. (56) Song, L.; Hennink, E. J.; Young, T.; Tanke, H. J. Biophys. J. 1995, 68 (6), 2588. (57) Schmidt, T.; Schu¨tz, G. J.; Baumgartner, W.; Gruber, H. J.; Schindler, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 2926. (58) Schu¨tz, G. J.; Schindler, H.; Schmidt, T. Biophys. J. 1997, 73, 1073. (59) Lee, G. M.; Ishihara, A.; Jacobson, K. A. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 6274. (60) Tendian, S. W.; Lentz, B. R.; Thompson, N. L. Biochemistry 1991, 30, 10991. (61) Rubenstein, J. L. R.; Smith, B. A.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1979, 76 (1), 15. (62) Ladha, S.; Mackie, A. R.; Harvey, L. J.; Clark, D. C.; Lea, E. J. A.; Brullemans, M.; Duclohier, H. Biophys. J. 1996, 71, 1364.

(63) Yates, D. E.; Healy, T. W. J. Chem. Soc., Faraday Trans. 1 1980, 76, 9. (64) Attard, P.; Antelmi, D.; Larson, I. Langmuir 2000, 16, 1542. (65) Israelachvili, J. Intermolecular and Surface Forces; Academic Press: London, 1991. (66) Gyo¨rvary, E.; Wetzer, B.; Sleytr, U. B.; Sinner, A.; Offenha¨usser, A.; Knoll, W. Langmuir 1999, 15, 1337. (67) Cremer, P. S.; Groves, J. T.; Kung, L. A.; Boxer, S. G. Langmuir 1999, 15, 3893. (68) Groves, J. T.; Ulman, N.; Boxer, S. G. Science 1997, 275, 651. (69) Groves, J. T.; Ulman, N.; Cremer, P. S.; Boxer, S. G. Langmuir 1999, 14, 3347. (70) Hovis, J. S.; Boxer, S. G. Langmuir 2000, 16, 894. (71) Yang, T.; Simanek, E. E.; Cremer, P. Anal. Chem. 2000, 72, 2587. (72) van Oudenaarden, A.; Boxer, S. G. Science 1999, 285, 1046. (73) Groves, J. T.; Boxer, S. G.; McConnell, H. M. J. Phys. Chem. B 2000, 104, 119. (74) Phimphivong, S.; Kolchens, S.; Edmiston, P. L.; Saavedra, S. S. Anal. Chim. Acta 1995, 307 (2-3), 403. (75) Axelrod, D. Methods Cell Biol. 1993, 30, 245. (76) Tamm, L. K. In Molecular Luminescence Spectroscopy; Schulman, S. G., Ed.; John Wiley & Sons: New York, 1993; Part 3, Chapter 7.

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directly proportional to the evanescent depth.77 In addition, thinner evanescent fields would allow one to probe the environment and behavior of molecules very near phospholipid bilayers deposited on these high refractive index substrates. Acknowledgment. This work was supported by National Science Foundation Grant MCB9728116. We (77) Thompson, N. L.; Palmer, A. G.; Wright, L. L.; Scarborough, P. E. Comments Mol. Cell. Biophys. 1988, 5 (2), 109.

Starr and Thompson

thank Kenneth A. Jacobson for permission to use the optical imaging instrumentation and for his insightful conversations, Christian Dietrich for his help in acquiring the images and for valuable suggestions, and Randall C. Cush for assistance with assembling the FPPR instrumentation. AFM analysis of bare substrates was generously carried out by Glenn C. Ratcliff and Dorothy A. Erie.

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