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Contribution of Lateral Force and “Tapping Mode” Microscopies to the Study of Mixed Protein Langmuir-Blodgett Films F. Sommer,† S. Alexandre,‡ N. Dubreuil,‡ D. Lair,‡ Tran-minh Duc,§ and J. M. Valleton*,‡ URA 500 CNRS-UFR des Sciences, Universite´ de Rouen, 76821 Mont-Saint-Aignan, France, BIOPHY Research, 43 Boulevard du 11 Novembre 1918, 69603 Villeurbanne, France, and CENATS Universite´ Claude Bernard, Lyon I 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne, France Received June 13, 1996. In Final Form: October 31, 1996X Mixed Langmuir-Blodgett films of behenic acid and glucose oxidase have been elaborated. The resulting films have been studied by scanning force microscopy with the “tapping” mode, from the micrometer scale to the nanometer scale. The general organization of the mixed film and the enzymatic quasi 2D crystals have been observed. Lateral force microscopy, coupled with topographic data obtained with the contact mode, has made possible the distinction between enzyme and behenic acid domains.
Introduction Biosensors have been extensively developed in the three past decades.1,2 They are based on the coupling of a biomolecule able to recognize specifically a chemical or a biochemical species to a transducer able to convert the physical-chemical signal produced by the host-molecule interactions. Most of the biosensors are based on the use of enzymes. A lot of work has been devoted to the immobilization of enzymes in membranes of micro- to macroscopic thickness or on mineral interfaces. While the first type of immobilization leads to long response times, the latter one may lead to the enzyme denaturation. The Langmuir-Blodgett (LB) technique is used for transferring layers of amphiphilic molecules on solid substrates.3,4 This technique is very useful for elaborating very thin films in which enzymes may be incorporated. The biosensors based on LB technology5-11 utilize an active layer constituted of one or several layers of the amphiphilic molecule in which the enzyme is incorporated. The great advantage of LB technology over other techniques is to create an organization of very thin mixed layers which should lead to biosensors with very short response times. The basic technique consists in elaborating a monolayer of the amphiphilic molecule on a subphase containing the enzyme and then in transferring this monolayer on a solid substrate in order to form one, two, or more mixed layers. The most commonly used procedure of transfer is the Langmuir-Blodgett procedure, which consists in dipping * To whom correspondence should be addressed. † BIOPHY Research. ‡ Universite ´ de Rouen. § CENATS Universite ´ Claude Bernard. X Abstract published in Advance ACS Abstracts, January 1, 1997. (1) Lowe, C. R. Philos. Trans. R. Soc. London, Ser. B 1989, 324, 487. (2) Janata, J. Anal. Chem. 1992, 64, R196. (3) Roberts, G. Langmuir-Blodgett Films; Plenum: New York, 1990. (4) Barraud, A. J. Chim. Phys. 1985, 82, 683. (5) Fiol, C.; Valleton, J. M.; Delpire, N.; Barbey, G.; Barraud, A.; Ruaudel-Teixier, A. Thin Solid Films 1992, 210/211, 489. (6) Sriyudthsak, M.; Yamagishi, H.; Moriizumi, T. Thin Solid Films 1988, 160, 463. (7) Owaku, K.; Shinohara, H.; Ikariyama, Y.; Aizawa, M. Thin Solid Films 1989, 180, 61. (8) Anzai, J.; Osa, T. Sel. Electrode Rev. 1990, 12, 3. (9) Schumann, W.; Heyn, S. P.; Gaub, H. E. Adv. Mater. 1991, 3, 388. (10) Arisawa, S. E.; Arise, T.; Yamamoto, R. Thin Solid Films 1992, 209, 259. (11) Turko, I. V.; Lepesheva, G. I.; Chashchin, V. D. Thin Solid Films 1993, 230, 70.
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the substrate in a vertical position through the interface: a layer is transferred simultaneously on both sides of the substrate. For the characterization of LB films, scanning force microscopy is a unique tool12-18 with its different possibilities; few papers, however, dealing with artificial systems in which proteins are involved have been published.19-27 In a series of papers,25-27 we have reported our results obtained by scanning force microscopy for the study of mixed Langmuir-Blodgett films constituted of behenic acid and glucose oxidase transferred on HOPG by horizontal lifting or vertical methods. The images were essentially obtained with the contact mode. On the microscopic scale, the structure appears to be heterogeneous, enzyme aggregates being dispersed at random on the sample; these aggregates are covered by a behenic acid monolayer in which defects have been observed. At the molecular level, it was possible to observe the (12) Weisenhorn, A. L.; Egger, M.; Ohnesorge, F.; Gould, S. A. C.; Heyn, S. P.; Hansma, H. G.; Sinsheimer, R. L.; Gaub, H. E.; Hansma, P. K. Langmuir 1991, 7, 8. (13) Alves, C. A.; Smith, E. L.; Porter, M. D. J. Am. Chem. Soc. 1992, 114, 1222. (14) Meyer, E.; Overney, R.; Brodbeck, D.; Howald, L.; Luthi, R.; Frommer, J.; Guntherodt, H. J. Phys. Rev. Lett. 1992, 69, 1777. (15) Zasadzinski, J. A.; Viswanathan, R.; Madsen, L.; Garanes, J.; Schwartz, D. K. Science 1994, 263, 1726. (16) Bourdieu, L.; Ronsin, O.; Chatenay, D. Science 1993, 259, 798. (17) Peltonen, J. P. K.; He, P.; Rosenholm, J. B. Langmuir 1993, 9, 2363. (18) Chi, L. F.; Eng, L. M.; Graf, K.; Fuchs, H. Langmuir 1992, 8, 2255. (19) Egger, M.; Ohnesorge, F.; Weisenhorn, A. L.; Heyn, S. P.; Drake, B.; Prater, C. B.; Gould, S. A. C.; Hansma, P. K.; Gaub, H. E. J. Struct. Biol. 1990, 1003, 89. (20) Weisenhorn, A. L.; Drake, B.; Prater, C. B.; Gould, S. A. C.; Hansma, P. K.; Ohnesorge, F.; Egger, M.; Heyn, S. P.; Gaub, H. E. Biophys. J. 1990, 58, 1251. (21) Fiol, C.; Alexandre, S.; Delpire, N.; Valleton, J. M.; Paris, E. Thin Solid Films 1992, 215, 88. (22) Fujiwara, I.; Ohnishi, M.; Seto, J. Langmuir 1992, 8, 2219. (23) Ohnishi, S.; Hara, M.; Furuno, T.; Sasabe, H. Biophys. J. 1992, 63, 1425. (24) Liu, Z. F.; Manivannan, A.; Inokuchi, H.; Yanagi, H. J. Vac. Sci. Technol., B 1993, 11, 1766. (25) Alexandre, S.; Dubreuil, N.; Fiol, C.; Valleton, J. M. Microsc. Microanal. Microstruct. 1994, 5, 61. (26) Alexandre, S.; Dubreuil, N.; Fiol, C.; Malandain, J. J.; Sommer, F.; Valleton, J. M. Microsc. Microanal. Microstruct. 1994, 5, 359. (27) Dubreuil, N.; Alexandre, S.; Fiol, C.; Sommer, F.; Valleton, J. M. Langmuir 1995, 11, 2098.
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arrangement of enzyme molecules in quasi 2D crystal structures; it was also possible to distinguish the individual structure of the protein, revealing the two subunits which were previously described.28 On top of these molecules, behenic acid molecules were observed. In addition, we have presented a model of the structure consistent with the images and quantitative data (height profiles) obtained by this technique. The tapping mode and the friction (or lateral force) mode are also of great interest for studying such bicomponent films. The tapping mode advantages have been discussed by several authors.29-32 It begins to be used in liquid media32 with similar potential advantages as for the contact mode (liquid versus air). Friction mode has been used successfully for differentiating two spatially separated phases: mixtures of different fatty acids33-36 or polymers37,38 for example. The aim of this paper is two sided: on one hand, we wanted to test the limit resolution of the tapping mode with our mixed LB systems and in particular determine if it is possible to obtain molecular resolution for the enzyme; on the other hand, we wanted to find out if the friction mode is adequate for discriminating domains in which enzyme or behenic acid molecules are on top of the mixed system. Materials and Methods A Langmuir-Blodgett trough from ATEMETA (Paris) was utilized for the preparation of the samples; the dimensions of the trough are 50 cm × 6.5 cm; the volume of the liquid phase is 250 cm3. This system uses a plunging mobile barrier for compressing the amphiphilic molecules and a Wilhelmy balance for measuring the interfacial pressure. The temperature of the subphase was maintained at 22 °C. The elaboration of mixed layers of behenic acid and glucose oxidase was achieved using an aqueous solution of glucose oxidase as the subphase and a behenic acid solution in chloroform spread on its surface. Aqueous solutions were prepared with water obtained by a Millipore system involving deionization, reverse osmosis, and filtration (Milli-RO + Milli-Q). Glucose oxidase was obtained from Sigma (type VII; Aspergillus niger) and used without further purification. Its concentration in the subphase was 3.2 mg/L. Behenic acid, obtained from Sigma (purity: 99%), was prepared as a 10-3 M solution in chloroform (RP Normapur, purity > 99.2%), and an amount of 0.1 mL was spread with a capillary micropipet (Nichiryo). In order to obtain reproducible injections of behenic acid samples, a system was designed for positioning the pipet vertically, at a fixed height above the interface. After the behenic acid solution was spread, the enzyme molecules were allowed to adsorb onto the polar heads of behenic acid molecules or at the air/water interface for 45 min. A study of the behavior of the mixed system BA/GOx at the air/water (28) Hecht, H. J.; Kalisz, H. M.; Hendle, J.; Schmid, R. D.; Schomburg, D. J. Biol. Mol. 1993, 229, 153. (29) Spatz, J. P.; Sheiko, S.; Mo¨ller, M.; Winkler, R. G.; Reineker, P.; Marti, O. Nanotechnology 1995, 6, 40. (30) Parrat, D.; Sommer, F.; Solletti, J. M.; Duc, T. M. J. Trace Microprobe Tech. 1995, 13, 3, 343. (31) Wa¨livaara, B.; Warkentin, P.; Lundstro¨m, I.; Tengvall, P. J. Colloid Interface Sci. 1995, 174, 53. (32) Hansma, P. K. Appl. Phys. Lett. 1995, 64, 13, 1738. (33) Overney, R. M. Phys. Rev. Lett. 1992, 69, 12, 1777. (34) Overney, R. M.; Meyer, E.; Frommer, J.; Brodbeck, D.; Lu¨thi, R.; Howald, L.; Gu¨ntherodt, H. J.; Fujihira, M.; Takano, H.; Gotoh, Y.; et al. Nature 1992, 359, 133. (35) Overney, R. M.; Meyer, E.; Gu¨ntherodt, H. J.; Fujihira, M.; Takano, H. Thin Solid Films 1994, 240, 105. (36) Overney, R. M.; Meyer, E.; Frommer, J.; Brodbeck, D.; Lu¨thi, R.; Howald, L.; Gu¨ntherodt, H. J.; Fujihira, M.; Takano, H.; Gotoh, Y.; Wolter, O. Thin Solid Films 1992, 220, 132. (37) O’Shea, S. J.; Welland, M. E.; Rayment, T. Langmuir 1993, 9, 1826. (38) Andoh, Y.; Oguchi, S.; Kaneko, R.; Miyamoto, T. J. Phys. D: Appl. Phys. 1992, 25, A71.
Sommer et al. interface has been published recently.39 Then the mixed film was compressed upto 30 mN/m. After the compression, the film was transferred by the vertical method at this pressure. The transfers were performed on HOPG samples (highly oriented pyrolytic graphite) obtained from Le Carbone Lorraine (Paris); HOPG samples are square pieces 10 × 10 mm2, 1 mm thick. HOPG was freshly cleaved before any transfer and used without any particular processing. HOPG samples, initially in the air, were covered by two mixed layers by a down and up displacement through the interface. The rate of transfer was 1 cm/min. The feedback loop (control of the mobile barrier by the signal produced by the Wilhelmy balance) was kept closed in order to operate at a quasi constant interfacial pressure (30 mN/m). The transfer ratio may be estimated to be 0.9 ( 0.1; in addition, the regular shape of the transfer records (displacement of the dipping arm versus displacement of the mobile barrier) is a good indication that a Y-type double layer is formed. The scanning force microscopy image presented in this paper in the contact mode at high resolution was performed with a Nanoscope II, with a 1 µm scanner. Molecular resolution imaging was achieved in water by using the “liquid cell”. The cantilever used was characterized by a low spring constant (0.06 N/m). A standard tip of silicon nitride was used. The measurement was performed with the feedback loop on (constant force: 10-9 to 10-8 N). Lateral force microscopy and “tapping” mode (Digital Inc. trademark) measurements were performed with a Nanoscope III model from Digital Instruments (Santa Barbara, CA) with a 14 µm scanner. For lateral force measurements, soft cantilevers (0.06 N/m) were used. For the “tapping” mode, the cantilevers used were made of silicon; their spring constant was about 50 N/m; they operated in the air, at an oscillation frequency of 370 ( 20 kHz; and the amplitude of oscillations was determined by voltages between 80 and 120 mV. No processing was used on images obtained either with the “tapping” mode or with the friction measurements. The images are presented in height mode (palette of color for height: dark colors for low zones, light colors for high zones) and are top-view images, except the friction image (palette of color for friction levels: dark colors for low friction zones, light colors for high friction zones) and the second image obtained with the tapping mode, which corresponds to an image of the feedback signal.
Results Tapping Mode. Tapping mode allows us to obtain sharp images. Figure 1a corresponds to an image of the topography of the mixed bilayer constituted of behenic acid and glucose oxidase transferred on HOPG (image size, 1 µm × 1 µm; height scale, 25 nm). This image is characterized by randomly organized aggregates of enzyme molecules; the height of these aggregates already observed in our previous experiments25 corresponds to 1 or 2 layers of enzyme molecules. The lateral dimensions of these aggregates are between 200 and 400 nm; the areas corresponding to these dimensions represent approximately from 1000 to 5000 enzyme molecules. These enzyme aggregates are partly covered by a behenic acid layer presenting many defects. This layer corresponds to a monolayer. In different parts of Figure 1a, the behenic acid monolayer covering these aggregates is partly removed by the tip (see the upper part of the bigger aggregate in the upper left part of the image) in spite of the use of the tapping mode. In addition, small structures 10-20 nm wide also appear; they may be due either to behenic acid film collapse or to a very small number of glucose oxidase molecules trapped under the behenic acid monolayer. So far we have no element to decide what hypothesis is the more probable. (39) Dubreuil, N.; Alexandre, S.; Fiol, C.; Valleton, J. M. J. Colloid Interface Sci. 1996, 181, 393.
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Figure 2. Tapping mode image (amplitude signal) of the mixed behenic acid/glucose oxidase bilayer transferred on HOPG: image size, 175 nm × 175 nm. On this image, the arrangement of individualized enzyme molecules is clearly visible (domain A for example). In the left upper part of the image (domain B), the distances measured may correspond to the two subunits.
Figure 1. Tapping mode images of the mixed bilayer constituted of behenic acid and glucose oxidase transferred on HOPG: image size, 1 µm × 1 µm. (a) Topography image: The image is characterized by “high” aggregates of enzyme molecules. A behenic acid layer with many defects is partly covering these aggregates. (b) Feedback signal representation: This image, simultaneouly acquired with image 1a, reveals the characteristic parallel alignments of enzyme molecules.
The image obtained from the amplitude signal (the amplitude of the oscillation of the cantilever used as the feedback signal in the tapping mode) (Figure 1b) and simultaneously acquired with image 1a reveals the characteristic parallel alignments of enzyme molecules separated by 6 nm, already observed in our studies with the contact mode.25,27 These alignments are observed on the aggregates. Figure 1 is very useful for giving a global understanding of the structure of the BA/GOx mixed film transferred on HOPG and for identifying without any ambiguity the chemical nature of the different structures observed. Note that, in these first images (Figure 1b in particular), the alignments correspond at least in one direction to the molecular resolution. This is the reason why we tried to obtain a higher resolution, in order to observe individual
GOx molecules. This result was obtained by zooming in on a more reduced zone of an enzymatic aggregate partly covered by a behenic acid monolayer. The image obtained (Figure 2) (image size, 175 nm × 175 nm; amplitude signal) shows clearly the individual GOx molecules ordered in alignments. The orientation of the GOx molecules seems different in the different parts of this image: In the central part (zone A), the objects observed are 7 nm long and 5 nm wide, each of them corresponding to one GOx molecule. In the left upper part of the image (zone B), a zone quite different is visible: the objects observed in this zone are clearly smaller, and two of these objects have a size corresponding to the size of a single GOx molecule; this indicates that a single object corresponds to a unit of the GOx molecule which is constituted of two identical subunits. In order to compare the potential of the tapping mode with the contact mode, we compared these results with those obtained with the contact mode: the results obtained with the tapping mode are more reproducibly obtained than those obtained with the contact mode; moreover, no processing is necessary with the tapping mode except a flatten operation. However, with the contact mode it is possible to obtain higher resolution. An example is given here (Figure 3) of a result which could not be obtained with the tapping mode; this image obtained with the same system (mixed film of behenic acid and glucose oxidase) shows a film of behenic acid molecules covering the glucose oxidase molecules (alignments visible under the behenic acid film, separated by approximately 6 nm). The behenic acid molecules can be clearly individualized Lateral Force Microscopy. Lateral force microscopy has been used here to illustrate the possibility of identifying the chemical nature of the different structures observed in this binary system constituted only of behenic acid and glucose oxidase. Images of topography and friction mode have been acquired simultaneously; each of them is necessary for interpreting the other. The topographic image (Figure 4a) shows high structures: the enzyme aggregates observed in Figure 1. A large part of the area observed is covered by a behenic
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Figure 3. Contact mode image of the mixed behenic acid/ glucose oxidase bilayer transferred on HOPG: image size, 20 nm × 20 nm. The behenic acid molecules arranged on top of GOx molecules appear clearly. The alignments of the proteins are still visible under the behenic acid layer.
acid monolayer: the inset profile indicates a height of 3 nm, which can only correspond to a behenic acid monolayer. However, this monolayer, exhibiting many defects, is probably the second monolayer obtained during the second transfer step, and the hydrophobic tails of this monolayer are oriented upward. The observation of the lateral force image (acquired when scanning from left to right) (Figure 4b) reveals that this behenic acid layer is characterized by a high friction with the tip used (Si3N4). This indication helps to determine if enzymatic aggregates are covered by a behenic acid layer or not: in the left upper part, enzymatic aggregates observed on the topographic image almost disappear on the friction image; this indicates that these aggregates are not covered by the behenic acid film. On the contrary, the aggregates localized in the central part of the image appear covered by the behenic acid film characterized by an important friction. In a general way, the enzymatic structures are almost invisible in the friction image, and the coverage by the behenic acid film appears more clearly. Note that this type of image in the friction mode is not always obtained with such quality; we obtained in other cases the same type of result, but the friction itself may induce problems of sample scratching because of the interactions between the tip and the mixed film. These results indicate that the behenic acid film is characterized by a high friction with the tip. The part of the behenic acid which is in interaction with the hydrophobic tip is its hydrophobic tail; the strong interactions between the tip and the behenic acid domains with hydrophobic tails in an external position explain the important friction observed in these domains. The enzyme aggregates for which the hydrophilic/hydrophobic distribution is more balanced have lower interactions with the tip; this leads to a lower friction on the enzymatic aggregates. Conclusions-Discussion The results presented here concern “tapping” mode and lateral force measurements concerning mixed LangmuirBlodgett films of behenic acid and glucose oxidase. The two techniques reveal interesting potentialities competing with the contact mode or complementing it.
Figure 4. Topography and lateral force images of the same zone of the mixed behenic acid/glucose oxidase bilayer: image size, 2.5 µm × 2.5 µm. (a) Topography image: Enzyme aggregates appear surrounded by a behenic acid monolayer (the inset profile corresponds to the thickness of a single monolayer) covering only a part of the zone. (b) Lateral force image: The behenic acid monolayer is characterized by a high friction. It is possible to conclude that most of the enzyme aggregates are covered by a behenic acid monolayer (central part of the image in particular); some others are not covered (upper left part of the image).
The tapping mode allowed us to obtain a global perception of the organization of the mixed system with a higher quality than that of the contact mode (no scan scratches), revealing the repartition of the enzyme aggregates; in addition the molecular resolution has been obtained for the glucose oxidase. The resolution may be estimated to 1 nm; other measurements achieved with another tip gave a lower resolution (between 2 and 3 nm). This resolution seems to be an optimum; however, it is not sufficient for observing behenic acid molecules. A strong advantage of the tapping mode in the analysis of
Tapping Mode and LFM To Study Protein LB Films
such soft systems is the fact that, because of its nondestructive operating mode, minimum image processing is required. In order to illustrate the advantage of the contact mode on the angstrom scale, we have presented a higher resolution image showing clearly the behenic acid molecules on top of glucose oxidase molecules aligned in parallel structures. Lateral force or friction mode in conjunction with the contact or the tapping mode allowed us to discriminate between enzyme and BA domains. The hydrophobic part of the behenic acid molecules, which is the part in interaction with the hydrophobic probe, gives important
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friction characteristics. The friction mode gives some information on the nature of different components; this is especially interesting for a two-component system (plus the substrate) in which problems of attribution are minimized by the simultaneous utilization of topographic and friction results. These results reveal high heterogeneities of our mixed system on graphite; important rearrangements probably occur during and/or after the transfer. A similar work with muscovite mica, an hydrophilic substrate for which the interactions with the mixed fatty acid/protein will be quite different, is under way. LA960584B
