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Langmuir 1998, 14, 1674-1679
Oriented Bacteriorhodopsin/Polycation Multilayers by Electrostatic Layer-by-Layer Assembly Jin-An He,† Lynne Samuelson,‡ Lian Li,§ Jayant Kumar,† and Sukant K. Tripathy*,† Center for Advanced Materials, Departments of Chemistry and Physics, University of Massachusetts Lowell, Lowell, Massachusetts 01854, Science and Technology Directorate, U.S. Army Natick Research, Development and Engineering Center, Natick, Massachusetts 01760, and Molecular Technologies Inc., Westford, Massachusetts 01886 Received December 4, 1997 The purple membrane (PM) containing bacteriorhodopsin (BR) is a promising biomaterial for many potential optical and optoelectronic device applications. Organized, heterogeneous assemblies using polycationic poly(dimethyldiallylammonium chloride) (PDAC) and PM fragments have been successfully constructed by the spontaneous alternating adsorption of PDAC and PM. The fabrication process of the multilayers was followed by UV-vis absorption spectroscopy and ellipsometry. The results indicate that the deposition process is linear and highly reproducible from layer to layer and that a monomolecular film of PM may be obtained in each PDAC/PM bilayer by controlling the adsorption time. Second harmonic generation measurements from the composite films gave a second-order susceptibility χ(2) of 8.1 × 10-9 esu and confirm that the PM fragments are arrayed with a high degree of orientation and acentric polar order in the films. Atomic force microscopy images provided the surface morphology of sequential layers of PDAC and PM fragments. Relative PDAC/PM bilayer thicknesses of 55 Å were observed, and the homogeneity of the layers was found to improve as the number of layers increased.
Introduction In the past decade there has been a tremendous surge toward the characterization, modification, and processing of biomaterials for device applications. These applications are envisaged in such areas as bioelectronics, biosensors, and biooptics and in the emerging area of biocomputing.1,2 All these applications however, require using easily available, stable, and multifunctional biomaterials that may be organized into architectures with optimal spatial arrangements and design to elicit the desired functional properties. Bacteriorhodopsin (BR) is found in the cell membrane of Halobacterium salinarium where it forms natural two-dimensional crystalline arrays in the purple membrane (PM).3 Long-term stability against thermal, chemical, and photochemical degradation, together with desirable photoelectric and photochromic properties, has made BR one of the most promising biological candidates for device applications. As a result, researchers have prepared molecularly oriented BR solid thin film devices using a variety of processing techniques.4 For example, a molecular electronic device which is based on the photoelectricity of BR has been designed for motion and direction detection.5,6 The photochromic properties of BR on the other hand have been shown to provide the technological basis for recording media for optical and holographic information storage and processing.7,8 The * Corresponding author. † University of Massachusetts Lowell. ‡ U.S. Army Natick Research, Development and Engineering Center. § Molecular Technologies. (1) Birge, R. R. Annu. Rev. Phys. Chem. 1990, 41, 683. (2) Takei, H.; Lewis, A.; Chen, Z. P.; Nebenzahl, I. Appl. Opt. 1991, 30, 500. (3) Oesterhelt, D.; Stoeckenius, W. Nature New Biol. 1971, 233, 149. (4) Chen, Z. P.; Birge, R. R. TIBTECH 1993, 11, 292. (5) Miyasaka, T.; Koyama, K. Appl. Opt. 1993, 32, 6371. (6) Miyasaka, T.; Koyama, K.; Otoh, I. Science 1992, 255, 342. (7) Birge, R. R. Am. Sci. 1994, 82, 348.
attempts for immobilizing and processing PM fragments onto solid supports have included the conventional Langmuir-Blodgett (LB) method,9 electrical sedimentation,10 chemiadsorption,11 and an unprecedented molecular recognition technique using antigen-antibody interaction.12 Physical characterization and photoelectric measurements have indicated that PM films formed by many of the above methods are ordered to some degree, though it is difficult to determine and optimize quantitatively the extent of orientation achieved using these conventional techniques. However, PM films that were formed using the antigenantibody recognition technique have demonstrated PM fragments to be highly ordered and the highest efficiency of photoelectric conversion reported to date have been obtained from these films.13 It is apparent that the resulting photoelectric signal is greatly dependent on the orientation of the PM fragments in the film, and it is well worthwhile to develop simpler and convenient techniques to prepare such highly ordered PM films. Recently, a novel and simple technique for ultrathin film assembly has been developed using the alternate layer-by-layer electrostatic deposition of oppositely charged polyelectrolytes.14,15 Furthermore, this approach has been extended and successfully applied to a wide range of charged molecules such as globular proteins,16,17 en(8) Wolperdinger, M.; Hampp, N. Biophys. Chem. 1995, 56, 189. (9) Miyasaka, T.; Koyama, K. Thin Solid Films 1992, 210/211, 146. (10) Keszthelyi, L. Biochim. Biophys. Acta 1980, 598, 429. (11) Brizzolara, R. A. BioSystems 1995, 35, 137. (12) Koyama, K.; Yamaguchi, N.; Miyasaka, T. Science 1994, 265, 762. (13) Koyama, K.; Yamaguchi, N.; Miyasaka, T. Adv. Mater. 1995, 7, 590. (14) Decher, G. Science 1997, 277, 1232. (15) Ferreira, M.; Cheung, J. H.; Rubner, M. F. Thin Solid Films 1994, 244, 806. (16) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (17) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3427.
S0743-7463(97)01336-X CCC: $15.00 © 1998 American Chemical Society Published on Web 02/13/1998
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zymes,18 nanoparticles,19 organic and polymeric microcrystals,20 ceramics and clay,21 organic dyes,22 DNA,23 and viruses24 by choosing suitable polyions as the counterions. In all these assemblies, the limiting factor in multilayer growth was to obtain sufficient adsorption of each polyion such that a complete charge reversal occurred (not merely neutralization) at the surface after each step. This concept of complete charge reversal was effectively utilized recently by us to build acentric multilayer films of polyelectrolytes containing nonlinear optical chromophores.25 Significant second-order nonlinearity was achieved in these systems. For biomaterials, this electrostatic adsorption technique offers several advantages over conventional approaches such as the LB method. These include, applicability to a large variety of watersoluble proteins, mild assembly conditions, and avoidance of denaturation of the biomaterial, among others. Layer thickness and organization can be further controlled without the need for specialized instrumentation and timeconsuming procedures. In the present work, we demonstrate that electrostatic deposition is an effective and facile method for preparing highly ordered acentric ultrathin assemblies of PM fragments. Several characterization methods, including UV-vis absorption, ellipsometry, second harmonic generation (SHG), and atomic force microscopy (AFM), verified that the PM fragments have been electrostatically layered into a preferred hierarchical structure. Experimental Section Materials. BR in the form of the purple membrane (PM) was isolated from Halobacterial halobium R1M1 strain as described by Oesterhelt and Stoeckenius.26 Its purity was confirmed using the absorption ratio of A280 nm/A570 nm. This ratio was 2.0 for our sample, which indicated a good quality sample.27 The PM fragments were stored at 4 °C in Milli-Q water. In the adsorption experiments, the PM suspension was used at a concentration of 0.5 mg/mL of BR (1.2 O. D. at 570 nm calculated according to ) 63 000 M-1 cm-1 and MW ) 26 000) and the pH was adjusted to 9.4 using 0.1 M NaOH aqueous solutions before each experiment. Figure 1 is an absorption spectrum of PM dissolved in Milli-Q water (pH 9.4). The absorption maximum is still found at around 568 nm and is similar to that which is observed for PM under neutral physiological conditions. This indicates that the spectral properties of PM patches are not changed in alkaline medium. Commercially available poly(dimethyldiallylammonium chloride) (PDAC, medium molecular weight, 20 wt % in water) was purchased from Aldrich Chemical Co. and used as the polycation without further purification. The polyelectrolyte was dissolved in Milli-Q water at a concentration of 2.0 mg/mL containing 0.5 M NaCl and a pH of 6.8 for the multilayer adsorption. Milli-Q water was also used for all rinsing steps during the adsorption process. The polycation/PM composite films were deposited onto the following solid supports for detailed characterization: quartz slides for UV-vis absorption measurements, glass slides for (18) Onda, M.; Lvov, Y.; Ariga, K.; Kunitake, T. Biotech. Bioeng. 1996, 51, 163. (19) Sun, Y. P.; Hao, E. C.; Zhang, X.; et al. Langmuir 1997, 13, 5168. (20) Tripathy, S. K.; Katagi, H.; Kasai, H.; Balasubramanian, S.; Oshikiri, H.; Kumar, J.; Oikawa, H.; Okada, S.; Nakanishi, H. To be published in Jpn. J. Appl. Phys. (21) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. Langmuir 1996, 12, 3038. (22) Ariga, K.; Lvov, Y.; Kunitake, T. J. Am. Chem. Soc. 1997, 119, 2224. (23) Lvov, Y.; Decher, G.; Sukhorukov, G. Macromolecules 1993, 26, 5396. (24) Lvov, Y.; Flaas, H.; Decher, G.; et al. Langmuir 1994, 10, 4232. (25) Wang, X.; Balasubramanian, S.; Li, L.; Jiang, X.; Sandman, D. J.; Rubner, M. F.; Kumar, J.; Tripathy, S. K. Macromol. Chem. Rapid Commun. 1997, 18, 451. (26) Oesterhelt, D.; Stoeckenius, W. Methods Enzymol. 1974, 31, 667. (27) Oesterhelt, D. Nature 1989, 338, 16.
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Figure 1. UV-vis absorption spectrum of PM fragments dissolved in Milli-Q water (pH 9.4, adjusted by 0.1 M NaOH aqueous solution).
Figure 2. Schematic of PDAC/PM alternate assembly using a negatively charged solid support. ellipsometry (one side frosted) and SHG, and silicon wafers for AFM. All solid supports were pretreated for 30 min in an ultrasonic bath at 25 °C in a mixture of ethanol/acetone/ chloroform (2/1/1, v/v/v) and then again in 2% KOH aqueous solution under the same conditions. After treatment, the slides were thoroughly rinsed with Milli-Q water and stored in Milli-Q water prior to use. This pretreatment procedure was used to partially hydrolyze the surface of the support substrate and render a net negative surface charge for subsequent polycation (PDAC) adsorption. Alternate Sequential Adsorption. The fabrication of PDAC/PM ultrathin composite assemblies is schematically illustrated in Figure 2 and is described as follows. A solid support with a negatively charged planar surface is immersed in the solution of PDAC for 5 min, and a monolayer of the polycation is adsorbed. After being rinsed in Milli-Q water for 2 min and then dried with nitrogen, the modified substrate is transferred into a 0.5 mg/mL PM suspension (pH 9.4) for 5 min, rinsed with water (pH 9.4) for 2 min and again dried by nitrogen. This process is repeated until the desired number of bilayers of PDAC/PM is obtained. All adsorption procedures were carried out at room temperature (approximately 18 °C). The process was periodically interrupted (as necessary) to carry out absorption and ellipsometric measurements to monitor each layer’s adsorption. Characterization Methods. UV-vis absorption spectra were obtained using a Perkin-Elmer Lambda-9 UV/vis/nearinfrared spectrophotometer. AFM studies were performed with a Nanoprobe atomic force microscope (Park Scientific, CA). The AFM was operated in the contact mode using a standard silicon nitride cantilever. The 5 µm × 5 µm images were recorded with a scan rate of 2 s-1 in ambient air. All images were stable with time, reproducible, and consistent throughout the sample. The ellipsometric measurements were carried out with a Rudolph Research/Auto EL III ellipsometer. A helium-neon laser served as the light source (wavelength 632.8 nm), and an angle of incidence of 70° was used. For these measurements, a series of stepped multilayers was deposited onto a single glass slide to eliminate any discrepancy from slide to slide. The area of the incident laser beam on the surface was approximately 1.5 mm2 so the measured data actually reflects an average value of film thickness from this area. All data reported are an average values obtained from three separate measurements from different regions of the sample. The second-order nonlinear optical susceptibility χ(2) of composite PDAC/PM films was measured using the SHG technique
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Figure 3. Dependence of the adsorbed relative amount of PM on pH of the PM suspensions. The solid support was sequentially submerged into PDAC and PM solution through four cycles. with a p-polarized fundamental laser beam at 1064 nm from a Q switched Nd:YAG laser (Quantel 660A). The p-polarized SHG signal, selected with an interference filter at 532 nm, was detected by a photomultiplier tube and averaged with a boxcar integrator (Stanford Research System). A Y-cut quartz crystal was used as the reference. By comparison of the SHG intensity from the multilayers with that from the quartz crystal, the second-order nonlinear optical susceptibility χ(2) of the sample was determined.25
Results and Discussion Effect of pH. The PM fragments have two faces, which are named the cytoplasmic and extracellular sides. It is well-known that in neutral aqueous solution, the two faces of PM are both negatively charged so the PM fragments can be sedimented in an orderly fashion onto the surface of an electrode using an electric field.10 Therefore, on the basis of surface charge of PM, it was expected that the negatively charged PM fragments could also be attracted onto a polycation surface using simple electrostatic layerby-layer adsorption. However, when this adsorption process was initially investigated using an alternate dipping of the solid support first into PDAC solution and then the PM suspension (0.5 mg/mL, pH 7.0), only a very small PM absorbance increase at around 570 nm was observed for the first four bilayers. Further adsorption was not possible as deposition of subsequent layers was attempted. This behavior suggested that the charge density of PM in neutral medium was too low to afford enough Coulombic force to sufficiently interact with the cationic charges of the PDAC layer. For this technique to work, not only it is necessary that the PM electrostatically bind to the PDAC but also it must provide sufficient coverage such that the surface is eventually rendered negatively charged for the layering process to continue. Thus, the extent of the charge surface density of the PM fragments becomes a key point in the fabrication of polyion/ PM assemblies by electrostatic attraction. One convenient way to adjust the charge density of a polyelectrolyte fragment or cluster is by changing pH or ionic strength of the solution. In these studies, the negative charge density and adsorption of the PM fragments was enhanced by increasing the pH of the PM solution. Figure 3 shows a plot of the relative adsorption amounts of PM in a four bilayer assembly ((PDAC/PM)4) characterized by the absorbance at 563 nm vs the pH of the PM solution. As shown, the adsorbed amount of (PDAC/PM)4 increases with an increase in the pH of the PM solution. This result confirms that PM patches are more readily adsorbed onto the polycation layer as a negatively charged cluster multicounterion under more
He et al.
Figure 4. Film thickness of one bilayer of PDAC/PM plotted against the adsorption time of the solid support in PM solution.
alkaline conditions. A possible explanation for this behavior is that there are several competitive interactions between molecules which can cause aggregation and spontaneous assembly. These molecular interactions include hydrophobic and hydrophilic interactions, hydrogen and covalent bonding, van der Waals forces, and Coulombic interaction.28 It is reasonable to believe that these interactions are taking part to different extents in the adsorption process between PM and PDAC under the given conditions. However, at high pH, when the negative charge density of the PM is large enough, the electrostatic interaction becomes the dominant driving force and the role of the other molecular forces are reduced in the adsorption process. In acidic and neutral media, a smaller amount of PM was adsorbed with each layer, and adsorption eventually stopped after several layers. This is due to the other molecular interactions hindering and interfering with the Coulombic forces between charged species which are needed for electrostatic adsorption.29 Adsorption Time. In previous studies of the electrostatic layering of proteins, an aggregation behavior within the protein layer was observed.16,30 Many factors are believed to lead to the aggregation of protein, such as solution concentration, pH, ionic strength, and adsorption time. Moreover, it is difficult to control the degree of aggregation of the proteins once the aggregation process has been initiated. Consequently, some domains are formed in the protein layer, which makes the subsequent film growth random, inhomogeneous, and increasingly difficult. To understand the aggregation behavior and ultimately the film formation of BR during the adsorption process, the change in thickness of the films with adsorption time in the PM solution was measured. The results are given in Figure 4. When the glass slide with one layer of PDAC deposited was immersed into a 0.5 mg/mL PM solution (pH 9.4) for 5 min, the thickness of the film was measured to be 55 Å. This value is consistent with the sum of the thicknesses of one layer of PM and one layer of PDAC, where the known thickness of PM fragments varies from 45 to 50 Å according to different measurement methods,31,32 and the thickness of a PDAC monolayer is (28) Muthukumar, M.; Ober, C. K.; Thomas, E. L. Science 1997, 277, 1225. (29) Balasubramanian, S.; Wang, X.; Wang, H. C.; Yang, K.; Li, L.; Kumar, J.; Tripathy, S. K. Azo chromophore functionalized polyelectrolytes: 2. Acentric self-assembly through a layer-by-layer deposition process. Submitted to Chem. Mater. (30) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. Thin Solid Films 1996, 284/285, 797. (31) Niemi, H. E.-M.; Ikonen, M.; Levlin, J. M.; Lemmetyinen, H. Langmuir 1993, 9, 2436. (32) Shen, Y.; Safinya, C. R.; Liang, K. S.; et al. Nature 1993, 366, 48.
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Figure 5. Linear increase of multilayer thickness with the number of cycles of alternate PDAC/PM adsorption.
approximately 10 Å depending on the solution concentration, pH and ionic strength.16,33 Therefore, a single layer of PM fragments had been deposited onto the PDAC layer after a 5 min adsorption time. When adsorption time was increased to 10 min, the final thickness of the film increased to 100 Å which indicated that some form of aggregation had occurred and possibly that a second layer of PM had adsorbed along with the first PM layer. The glass slide was then kept in the PM solution for another 20 min, and no further increase of the film thickness was observed. Unlike some globular proteins, such as glucose oxidase and hemoglobin, their aggregation in electrostatic layering can be as high as several molecular layers.30 For PM on the average, only one additional layer of PM fragments could be aggregated with the original PM monolayer. This is perhaps related to the larger size of the PM fragments (usually 0.5-1.0 µm in diameter, and about 5.0 nm in thickness), thus making it sterically more difficult to aggregate excessive amounts of PM. Multilayer Growth. On the basis of the results presented to this point, it is clear that PDAC/PM composite monolayer films may be prepared using electrostatic adsorption. The next question to address was whether well-defined multilayer deposition was feasible using this process. To build multilayers, the negatively charged solid support was alternately immersed into a 2.0 mg/mL PDAC solution for 5 min and then immersed into a 0.5 mg/mL PM suspension (pH 9.4) for another 5 min, and this was repeated until the desired number of layers was obtained. It was necessary to rinse the films with water of the same pH and then dry with nitrogen before proceeding to the next step each time. With the above multilayer fabrication procedure, highly reproducible films with controlled thickness were obtained. The determination of film thickness with the number of bilayers was carried out using ellipsometry, and the results are given in Figure 5. As shown, the bilayer thickness increases linearly with the number of PDAC/PM bilayers, and the slope of the line (55 Å/bilayer), is equivalent to the bilayer thickness. These results confirm that the multilayer growth of the PDAC/PM assembly is regular and reproducible and that the orientation of PM on the solid support is such that the plasma membrane plane of PM is parallel to the solid support plane. Further evidence for uniform multilayer growth of PDAC/PM assemblies is obtained from UV-vis absorption spectroscopy. Figure 6 shows the UV-vis spectra for a sequence of PDAC/PM bilayers at each consecutive step of the multilayer assembly process. The signature (33) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210/ 211, 831.
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Figure 6. UV-vis absorption spectra of PDAC/PM multilayers on a quartz slide. The curves, from bottom to top, correspond to absorption of 2, 4, 6, 8, 10, 12, and 14 alternate PDAC/PM adsorbed bilayers, respectively. The inset shows the increases of absorbances at 278 and 563 nm with the number of bilayers. The baseline increase is due to light scatting of the PDAC/PM multilayers, its contribution had been deducted from the total absorbance.
absorption bands for BR at 278 and 563 nm are observed to increase proportionately with each additional bilayer deposition as shown in the inset of the figure. This again supports the thesis that the electrostatic deposition is a linear process and that each transfer contributes an equal amount of PM fragments to the film assembly. The UV-vis data also show that the deposited PM fragments in the PDAC/PM multilayers are not denatured as the characteristic absorption bands of PM are still observed. There is however a slight blue shift of about 5 nm for the peak at 563 nm when compared to the absorption of the PM in solution. A similar blue shift in a dried PM film was previously observed with a BRsoya-PC LB film. This behavior was explained by the dehydration of the Schiff base of the retinal chromophore in BR in the absence of water;34 while in the presence of water, the proton of the Schiff base is localized and a red shift is observed.35,36 The absorbance for each deposited bilayer of PDAC/PM was calculated to be 1.5 × 10-3 at 563 nm from the slope of the line shown in the inset of Figure 6. This absorbance is due to PM only as PDAC contributes no absorption in this wavelength range. It is interesting to compare this value with that of BR-soya-PC LB films. In the LB films, the absorbance of a BR monolayer is in the range of (0.320.45) × 10-3 at 570 nm depending on the weight ratio of BR/soya-PC and the deposition pressure of the film.37,38 Our value is 3-5 times higher than that observed with the LB system. This result suggests that the PM fragment assemblies prepared using this spontaneous layer-by-layer assembly are organized into a more compact, dense monolayer formation. In contrast, soya-PC must occupy part of the area in the BR-soya-PC LB film, and this dilution effect leads to the decrease of the absorbance of the BR monolayer. This improved packing and organization of the PM in these electrostatically adsorbed films is expected to offer a number of advantages over previous (34) Ikonen, M.; Peltonen, J.; Vuorimaa, E.; Lemmetyinen, H. Thin Solid Films 1992, 213, 277. (35) Hwang, S.-B.; Korenbrot, J. I.; Stoeckenius, W. Biochim. Biophys. Acta 1978, 509, 300. (36) Hildebrant, P.; Stockburger, M. Biochemistry 1984, 23, 5539. (37) Korenbrot, J. I. Methods Enzymol. 1982, 88, 45. (38) Hwang, S.-B.; Korenbrot, J. I.; Stoeckenius, W. J. Membr. Biol. 1977, 36, 115.
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Figure 7. SH intensity as a function of incident angle of the fundamental laser beam from a double-sided self-assembled PDAC/PM multilayer (12 layers).
BR films for device fabrication, because the sensitivity and performance of the devices is greatly dependent on the amount and orientation of the BR in the twodimensional configurations. Second Harmonic Generation (SHG). The nonlinear optical (NLO) properties of PM fragments has been previously investigated, and it was determined that the retinal chromophore of BR exhibits a very large secondorder hyperpolarizability.39 Moreover, PM patches can be easily oriented by sedimentation from solution onto the surface of an electrode and this further enhances the second-order susceptibility of PM films.40 The better the orientation and acentric order of PM in the films, the larger the NLO coefficient. The second-order susceptibility χ(2) of the PDAC/PM films (12 bilayers) was measured and determined to be 8.1 × 10-9 esu. This value is larger than that of the electrophoretically sedimented film of BR (5.4 × 10-9 esu),41 which indicates that the degree of order of PM fragments in PDAC/PM multilayers is better than that obtained in electric-field-oriented PM films. The relationship between the transmitted second harmonic(SH) intensity of the double-sided multilayers (PDAC/ PM films deposited on both sides of the glass slide), and the incident angle is shown in Figure 7. The interference pattern arises from the phase difference between the SH waves generated at either side of the glass substrate during propagation of the fundamental wave. Complete extinction appeared for destructive interference over the whole film, and this indicated that the PDAC/PM multilayers deposited on both sides of the glass slide are uniform.42 AFM Studies. Ellipsometry, UV-vis absorption, and SHG all confirmed the feasibility of preparing oriented PDAC/PM multilayer assemblies by electrostatic layerby-layer deposition. These techniques, however, given the large size of the sampling area, provide only an average value over that particular area and can only characterize the film homogeneity down to the micrometer range. Atomic force microscopy (AFM) provided further detailed information involving the surface morphology and the homogeneity of the deposited films down to the nanometer scale. Figure 8 shows AFM images of the polycation layer (Figure 8a), one bilayer of PDAC/PM with PM as the outer layer (Figure 8b), and two bilayers of PDAC/PM (Figure 8c). In Figure 8a, before PM adsorption, the polymer surface is observed to be extremely smooth with almost no surface features and is consistent with other reported (39) Huang, J.; Chen, Z. P.; Lewis, A. J. Phys. Chem. 1989, 93, 3314. (40) Huang, J.; Lewis, A. Biophys. J. 1989, 55, 835. (41) Bouevitch, O.; Lewis, A. Opt. Commun. 1995, 116, 170. (42) Li, D.; Ratner, M. A.; Marks, T. J.; et al. J. Am. Chem. Soc. 1990, 112, 7389.
Figure 8. AFM images of (a) PDAC layer on a silicon wafer before PM adsorption, (b) one layer of PM adsorbed to the PDAC layer, and(c) two bilayers of PDAC/PM film.
polymer surfaces.43,44 Adsorption of PM to the PDAC surface results in the formation of a monolayer of PM as shown by the large patches dispersed across the surface. The cross-sectional analysis indicated that the thickness of a large patch is about 55 Å, and this value was found to increase to 110 Å at the boundary of two patches of PM and is most likely due to their partial overlap. This may happen as a large PM fragment tries to fit in a space smaller than its size defined by already adsorbed membrane fragments. When two bilayers of PDAC/PM were adsorbed on the silicon, the adsorption pattern of the top PM layer appears to be the same, indicating the same absorption kinetics. However, the uniformity is somewhat improved, and there is a slight lack of contrast that may be the result of imaging a thicker soft film. The orientation of the PM, that is whether the cytoplasmic or extracelluar side faces the solid support or electrode, is critical in determining the magnitude of the photoelectric behavior of the assembly. The study of Koyama and co-workers showed that the most intense photocurrents are observed when the cytoplasmic side of the PM is directed toward the electrode, whereas only barely measurable responses are observed when the PM is oriented in the opposite direction.12,13 At this point, we know that we have obtained PDAC/PM multilayers, with a single molecular layer of PM in each bilayer, and the order of the stacking is the same in the multibilayers given the second harmonic response. However, we have not yet determined which side of the PM is adsorbed toward the solid support. Fisher and co-workers showed that PM fragments adsorb preferentially, most likely due to the difference of surface charge density between the two PM sides, to a cationic glass surface with the extracellular (43) Chen, X.; Davies, M. C.; Roberts, C. J.; et al. Langmuir 1997, 13, 4106. (44) Baty, A. M.; Suci, P. A.; Tyler, B. J.; et al. J. Colloid Interface Sci. 1996, 177, 307.
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side facing the glass.45 Future studies of our electrostatically prepared PM assemblies will include photocurrent and proton pumping to determine the orientation of the BR with respect to the solid support. Conclusion In this work, it has been shown that highly ordered PDAC/PM multilayers can be successfully fabricated using consecutive layer-by-layer electrostatic adsorption. Through judicious control of pH and adsorption time, welldefined PDAC/PM multilayer assemblies may be prepared with controlled thickness and organization. UV-vis absorption spectra and ellipsometry confirmed that the spontaneous growth of PDAC/PM bilayers is a successive and highly reproducible ordered process where a monomolecular layer of PM is deposited each time. By (45) Fisher, K. A.; Yanagimoto, K.; Stoeckenius, W. J. Cell Biol. 1978, 77, 611.
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comparing the absorbance of BR in BR-soya-PC LB film with that of our PDAC/PM composite films, it can be concluded that the PM patches are more densely packed in these films than in the LB assemblies. Moreover, the value of the second-order susceptibility χ(2) of the PDAC/ PM films is larger than that observed with electric-fieldsedimented BR films, which suggests that the array of PM fragments in these PDAC/PM multilayers is highly ordered. Electrostatic deposition of PM may be an effective and facile method for the fabrication of a host of bioelectronic and biooptical ultrathin devices. Acknowledgment. Financial support from US Army NRDEC through a grant from Batelle is gratefully acknowledged. Discussions with Dr. Joseph Akarra at the US Army NRDEC, Prof. Michael Rubner at MIT and technical support in the AFM studies by Mr. Ram Nagarajan at U. Mass. Lowell are gratefully acknowledged. LA971336Y
