Langmuir 1992,8, 2219-2222
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Atomic Force Microscopy Study of Protein-Incorporating Langmuir-Blodget t Films Ichiro Fujiwara,; Michihiro Ohnishi, and Jun’etsu Set0 SONY Research Center, 174 Fujitsuka-cho, Hodogaya-ku, Yokohama, 240, Japan Received February 28,1992. In Final Form: May 7, 1992
The microscopic surfacemorphology of glucose oxidase protein-incorporatingarachidicacid LangmuirBlodgett (LB) films were investigated using atomic force microscopy (AFM)and replica transmission electron microscopy (TEM). AFM images showing clear morphology of protein molecules were observed over a 100-600 nm area. It is indicated that the glucose oxidase molecules make their aggregates, and glucose oxidase aggregates are randomly and sparsely distributed with an average size estimated to be 20-50 nm in diameter. The size and distribution are in fair agreement with those observed by replica TEM. Furthermore, high-resolution images of arachidic acid molecules on top of the protein molecules were successfully observed for the first time. The images show two-dimensional regular structures and the periods of regularity of the arachidic acid molecules are 0.54 and 0.48 nm, respectively. The area per molecule is estimated to be 0.26 nm2. This value is consistent with that obtained from the isotherm of a monolayer of arachidic acid. Our observations provide rough confirmation of our model of the structure of glucose oxidase-incorporating arachidic acid LB film. 1. Introduction Langmuir-Blodgett (LB) film technology is one of the important technologiesfor future molecular electronic and bioelectronic device5.l We have demonstrated various kinds of device applications of LB film technology, such as optical memory,2bioreactor? and liquid crystal aligning layers for liquid crystal d i ~ p l a y . ~ Protein-incorporatingLB filmswere extensivelystudied from the standpoint of application for bioelectronic devices. We have already reported a multistage bioreactor using protein-incorporating LB films as a prototype of bioelectronic device^.^ Concerning the structure of protein-incorporating LB films, we have also obtained preliminary experimental data using X-ray photoelectron spectroscopy (XPS) and replica transmission electron microscopy (TEMh5 It is necessary to investigate the microscopicmorphology of the protein-incorporating LB films in order to improve the quality of the LB films. However, it is not enough to characterize the fine structure of the LB films using conventional analytical methods such as optical microscopy and scanning electron microscopy because of the limited resolution. TEM and low energy electron diffraction (LEED) have been used to observe high-resolution images of thin films, but these electron beam techniques have the disadvantage of damaging organic thin films. Recently, scanning tunneling microscopy (STM) has gained much interest because of the high resolution achievable in principle. Since the first STM image of a fatty acid LB film was demonstrated! several workers have reported STM images of various kinds of LB f i i ~ . ~ - l O
Since most of the LB films are insulating, classical tunneling theory cannot explain the contrast mechaniem. Several interpretations of the STM images, such as a resonant tunneling mechanism, have been proposed.l1 However, there has been some controversy over the interpretations of the STM images of insulating LB films. This controversy over the contrast mechanism of STM images is avoided by use of atomic force microscopy (AFM).12 Since AFM uses atomic or molecular interaction forces between the cantilever and the sample as a probe, AFM offers the possibility of imaging the surface on a molecular scale without the requirement of conducting samples, such as polymers and proteins.13J4 Molecularresolution AFM images of fatty acid LB f i iwere reported very recently.15J6 Nanometer scale defects in fatty acid LB films were also reported using AFM.17 The interpretations of the AFM images are important and it is desirable to use other analytical method or computer simulation for the confirmation of the interpretation. In this paper, the microscopic structure of the proteinincorporating LB films (glucose oxidase-incorporating arachidic acid LB films) was investigated by AFM and replica TEM. The AFM image was compared with the replica TEM image. The microscopic morphology of the protein-incorporating LB films is discussed. 2. Experimental Section Glucose oxidase protein (Sigma Chemical) was dissolved in ultrapure water (Millipore) at a concentration of 500 mg/L. Arachidicacid was used to adsorb glucoseoxidase because glucose
(IO)Fujiwara, 1.; Ishimoto, C.; Seto, J. J. Vac. Sci. Technol. 1991, B9 (2), 1148. (11) Mizutani,M.; Sigeno, M.; Kajimura, K. Appl. Phys. Lett. 1990, (1) Roberts, G., Ed.Langmuir-Blodgett Films; Plenum Press: New 56,1974. York, 1990. (12) Binnig, G.;Quate,C. F.; Gerber, G. Phys.Reu.Lett. 1986,12,930. (2) Ishimoto, C.; Tomimuro, H.; Seto, J. Appl. Phys. Lett. 1986, 49, (13) Marti,O.;Ribi,H.;Drake,B.;Albrecht,T.R.;Quate,C.F.;Hanema, 1677. P. K. Science 1988,239, 50. (3) Ohniahi, M.; Ishimoto, C.; Seto, J. J. Chem. SOC.Jpn., in press. (14) Drake, B.;Prater, C. B.; Weisenhom, A. L.; Goulg, S. A. C.; Al(4) Baker, S.;Seki, A.; Seto, J. Thin Solid Films 1989, 180, 263. brecht, T. R.; Quate, C. F.; Cannell, D. S.; Hansma, H. G.; Hansma, P. (5)Ohnishi, M.; Aoki, M.; Ishimoto, C.; Seto, J. to be submitted for K. Science 1989,243,1586. publication. (15) Meyer, E.; Howald, L.; Ovemy, R. M.; Heinzelmann, H.; From(6) Smith, D. P. E.; Bryant, A.; Quate, C. P.; Rabe, J. P.; Gerber, C.; mer, J.; Guntherode, H. J.; Wager, T.; Schier, H.; Roth, S. Nature 1991, Swalen, J. D. Natl. Acad. Sci. U.S.A. 1987,84,969. (7) Mizutani,W.;Shigeno,M.;Saito,K.;Watanabe,K.;Sugi,M.;Ono, 349, 398. (16) Weisenhom,A.L.;Egger,M.;Ohnesorge,F.;Gould,S.A.C.;Heyn, M.; Kajimura, K. Jpn. J. Appl. Phys. 1988,27, 1803. S.P.; Hansma, H. G.; Sinsheimer, R. L.; Caub, H. E.; Hansma, P. K. (8) Fuchs, H.; Akari, S.; Dransfeld, K. 2.Phys., in press. Langmuir 1991, 7, 8. (9) Dovek, M. M.; Albrecht, T. R.; Kuan, S. A. J.; Lang, C. A.; Emch, (17) Hansma, H. G.; Could, S. A. C.; Hansma, P. K.; Gaub, H. E.; R.; Grutter, P.; Frank, C. W.; Pease, R. F. W.; Quate, C. F. J. Microsc. Longo, M. L.; Zasadzinski, J. A. N. Langmuir 1991, 7,1051. 1988,152, 229. 0743-7463f9212408-2219$03.00/0
0 1992 American Chemical Society
2220 Langmuir, Vol. 8, No. 9,1992 oxidase by itself does not form a monolayer a t the air-water interface. The spreading solvent of the amphiphilic materials was prepared in chloroform a t a concentration of 1 mg/ml. The glucose oxidase-incorporatingarachidic acid LB film was prepared by the Fromherz method.18 A Fromherztrough (MayerFeintechnik) which has eight compartment troughs with two barriers was used for LB film deposition. The subphase solution is placed in several trough compartments. Then, a monolayer of arachidic acid molecules is organized on the surface of the subphase. A protein solution ins placed in the adjoining trough compartment. The monolayer is then moved over the surface of the protein solution to allow the protein to adsorb to the monolayer. This composite monolayer with adsorbed protein is returned onto the pure subphase. The surface pressure of the monolayer of arachidic acid was 30 mN/m through the adsorption step and that of the mixed monolayer was 20 mN/m. The time allowed for adsorption was about 1 h. Mica with a surface which was freshly cleaved was used for the substrate. The composite monolayer was then deposited onto the mica substrate. Since the surface of the mica substrate was hydrophilic,the LB film deposition started on the upward stroke. The surface pressure of the composite monolayer on the subphase was 30-50 mN/m for deposition of the glucose oxidaseincorporating arachidic acid LB film monolayer. An AFM instrumentwith an opticaldeflectiondetection system (Digital Instrument; Nanoscope 11)was used in this experiment. In this instrument, a red light from a visible semiconductor laser is focused onto the edge of the cantilever in order to measure its deflection. The reflection from the back of the cantilever is detected using two segmented photodetectors and is amplified. All of the AFM measurements were carried out a t room temperature operating in air. In order to obtain high-resolution images, the AFM was operated with repulsive forceson the order of several nanonewtons. A microfabricated silicon nitride cantilever with a spring constant of 0.1-0.5 N/m was used with a 700 nm scanner. The sample was glued to a steel disk and placed on an electrically grounded magnetic disk on top of a piezoelectric translator which moves in three orthogonal directions. The constant force mode with scan rates of 4-16 Hz was mainly used for this experiment. Low-pass filter treatment was carried out in order to reduce the noise component of the signal. A plasma polymerization instrument (Ushio, PNR-110) was used for replica TEM experiments.lg The plasma-polymerized ethylenefilm was prepared on the protein-incorporatingarachidic acid LB film on a covered glass plate. The sample was treated with hydrofluoric acid in order to solve organic film coated glass for the TEM experiment of the resultant replica film. The UV absorption spectrum of the LB film was measured using a spectrophotometer (Beckman, DU-50).
3. Results and Discussion Before characterization of the glucose oxidase-incorporating LB film with the AFM, the UV absorption spectrum of the LB film was measured. The absorption band at 280 nm which was assigned to the absorption of the glucose oxidase was observed. This suggests that glucose oxidase proteins were really incorporated in the LB film. AFM images of glucose oxidase-incorporatingarachidic acid LB films showing clear surface morphology were observed over a 100-600 nm area. Figure 1 shows the typical AFM image of the LB film monolayer at 600 nm of scan area. This image is stable and is unchanged after severalminutes of continuous observation. We measured the force curve and checked its reproducibility. The constant force between the cantilever and the surface of the sample estimated from the force curve is on the order of several nanonewtons. During the measurement we (18)Fromherz, P. FEBS Lett. 1970,II,205. (19)Tanaka, A.; Yamaguchi, M.; Iwashaki, T.; Iriyama, K. Chem.Lett. 1989,1219.
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Figure I. AFM image of glucose oxidase-incorporatingarachidic acid LB film monolayer on the mica substrate, scan area 600 nm X 600 nm. Low-pass filter treatment was carried out in order to reduce the noise component of the signal.
observed no damage to the LB films caused by the cantilever. But when the constant force between the cantilever and the surface of the sample is high, it was found that the cantilever damaged the LB films. In this case, a hole could be seen near the center of the scan area by reducing the constant force and expanding the scan area. In Figure 1,the lighter part of the image indicates the higher part of the surface and the darker part of the image indicates the lower part of the surface. Surface corrugation can be observed in Figure 1. The higher parta are randomly and sparsely distributed in the scan area. In the procedure of the Fromherz method, the area of the monolayer of arachidic acid does not change during the adsorption of the glucose oxidase protein. Therefore, the arachidic acid molecules must remain homogeneously distributed at the air-water interface. We expect the structure of the deposited LB films to be similar to the structure of the monolayer at the air-water interface. The adsorption of the protein can be random and sparse, and the arachidic acid molecules are too small to be observed in the scan area in Figure 1. Therefore, the higher parts of the corrugation in the AFM image correspond to glucose oxidase protein molecules. The protein molecules are randomly and sparsely distributed in the LB film and the average size is 20-50 nm in diameter. Figure 2 shows the replica TEM image of glucose oxidase incorporating arachidic acid LB film. The replica TEM technique has the advantage of giving little damage to the LB film structure.lg In this technique, a dc plasma polymerized replica is used to obtain the image shown in Figure 2. A fine mosaic structure can be observed. The black parts of the mosaic structure indicate higher parts of the corrugation and the white parts indicate lower parta of the corrugation. The black parts which correspond to the higher parts in Figure 1are protein molecules and the size is about 50 nm in diameter. The protein molecules are randomly and sparsely distributed in the LB film. This size and distribution are almost the same as in Figure 1. Therefore, the interpretation of the surface morphology of the AFM image shown in Figure 1is confirmed by the replica TEM technique. The glucose oxidase molecules are approximately spher-
AFM Study of Protein-Incorporating LB Films
Langmuir, Vol. 8,No.9,1992 2221
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Figure 2. "EM image of the plasma polymerized replica of glucoseoxidase-incorporating arachidic acid LB film. Dark parts of the mosaic structure correspond to the lighter parts in Figure 1.
II1111I1II
Figure 4. Enlarged AFM image of glucose oxidase-incorporating arachidic acid LB film monolayer on the mica substrate, scan area 100 nm X 100 nm. Low-pass filter treatment was carried out in order to reduce the noise component of the signal.
Figure 3. Model of the microscopic structure of glucose oxidase incorporating arachidic acid LB film. Spheres indicate glucose oxidase protein molecules and rodlike molecules indicate arachidic acid molecules. Glucoseoxidase proteins make their aggregates and arachidic acid molecules were located on top of the protein aggregates and on the mica substrate.
ical with an average size of 8 nm in diameter. This size is smaller than that of the corrugation shown in Figure 1. This indicates that each corrugation in the AFM image is composed of several glucose oxidase protein molecules. This may be caused by the aggregation of the protein molecules at the air-water interface when they adsorbed to the arachidic acid molecules. Figure 3 shows the model of the microscopic structure of the glucose oxidase-incorporating arachidic acid LB film expected from the deposition process of the Fromherz method. Spheres indicate glucose oxidase protein molecules and rodlike molecules indicate arachidic acid molecules. Glucose oxidase proteins make their aggregates, and the arachidic acid molecules are homogeneously distributed and are densely packed on the surface of the substrate, which is either mica or protein molecules. The glucose oxidase protein molecules are randomly and sparsely distributed on the surface. The model shown in Figure 3 is supported by angle-resolved XPS measurement? From this model, the lower parts of the corrugation correspond to arachidic acid molecules. Figure 4 shows the enlarged image of Figure 1at 100nm of scan area. It is seen that the height of the surface corrugation is not homogeneous and depends on the location in the substrate. As described above, it is assumed that arachidic acid molecules are located both at the higher parts of the corrugation and at the lower parts of the corrugation. Again, glucose oxidase protein moleculesare randomly and sparsely distributed on the surface of the mica substrate. In order to confirm the model structure shown in Figure 3, we tried to observe high-resolution images of the corrugation. Figure 5 showsthe AFM image of an arachidic acid LB film on top of the protein molecules. This image
Figure 5. AFM image of arachidic acid molecules on top of glucose oxidase protein molecules, scan area 4 nm X 4nm. Lowpass filter treatment was carried out in order to reduce the noise component of the signal.
is stable and is unchanged after several minutes of continuous observation. We measured the force curve and found that the image was observed when the constant force was as low as several nanonewtons. We also checked reproducibility of the image by changingthe magnification of the scan area and found that this imagewas not a thermal drift and the mica substrate. But this image was observed at limited scan area. Because the surface of the protein is not very flat, the AFM images of the arachidic acid molecules on top of it became a little bit noisy for larger scan areas. The image in Figure 4 shows that a twodimensional regular structure was observed in molecular resolution. In order to measure the regularity of the image, two-dimensional fast Fourier transform treatment was carried out. Figure 6 shows the two-dimensional fast Fourier trans-
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Figure 6. Two-dimensional fast Fourier transform of Figure 4. Two bright spots which indicate the regularity of the image can be seen. Each spot corresponds to the regularity of the 0.48 and the 0.52 nm periods. The area per molecule is estimated to be about 0.26 nm2. Table I. List of the Size of the Glucose Oxidase Protein-IncorporatingArachidic Acid LB Film regularity protein (glucoseoxidase) arachidicacid
20-50 nm
in diameter 0.48 nm 0.54 nm
commenta glucose oxidase aggregation aredmolecule 0.26 nm2
form image of Figure 5. Two bright spots which indicate the regularity of the image can be seen. Each spot corresponds to the regularity of the 0.48 and the 0.52 nm periods. The area per molecule is estimated to be 0.26 nm2. The area per molecule corresponds to the limiting area per molecule of the monolayer of arachidic acid. This value can be estimated from the isotherm of the arachidic acid and is known to be about 0.22 nm2.1 The limiting area is close to the value occupied by an arachidic acid molecule in a single crystal, thus confirming the close packed layer as a two-dimensional solid. The area per molecule calculated from the AFM image is consistent with that obtained from the isotherm of a monolayer of arachidicacid. This confirmedthat the AFM image really shows arachidic acid molecules on top of the proteins. XPS data also suggested that the arachidic acid molecules are located on top of the protein molecule^.^ Therefore, molecular-resolution images of arachidic acid moleculeson top of the protein moleculeswere successfully observed for the first time. Table I shows the summary of the size of the molecules observed by AFM. Glucose oxidase molecules make their
aggregates and glucose oxidase aggregates were randomly and sparsely distributed with an average diameter of 2050 nm. In the case of the limiting area, the arachidic acid molecules are arranged almost perpendicular to the airwater interface. Thus, the arachidic acid molecules are arranged regularly on top of the protein molecules and their long molecular axes oriented almost parallel to the substrate normal. The periods of regularity of the arachidic acid molecules are 0.48 and 0.54 nm, respectively. This picture is consistent with our model of the LB film. Therefore, our observation provides a rough confirmation of the model of the structure of the glucose oxidaseincorporating arachidic acid LB film. We tried to observe the AFM image of the lower parts of the corrugation. But, clear AFM images could not be obtained. At the moment, arachidic acid molecules can be observed on the protein molecules but not directly on the mica substrate. This may be because the intermolecular interactions between arachidic acid molecules and the substrate are different. The intermolecular interactions between arachidic acid and protein molecules appear to be stonger than that between arachidic acid molecules and the mica substrate and that protein molecules appear to be a good substrate for observation of LB films with the AFM. 4. Conclusions The microscopic surface morphology of the glucose oxidase-incorporatingarachidic acid LB film was clarified using atomic force microscopy (AFM) and replica TEM. AFM images showing clear morphology of protein molecules were observed over a 100-600 nm area. It is indicated that the glucoseoxidase protein moleculesmade their aggregatesand the protein aggregateswere randomly and sparsely distributed with an average diameter of 2050 nm. The size and distribution are in fair agreement with observation by replica TEM. Furthermore, molecular resolution images of arachidic acid molecules on top of the protein molecules were successfullyobserved for the first time. The image shows two-dimensional structure and the periods of regularity of the arachidic acid molecules are 0.48 nm and 0.54 nm, respectively. The area per molecule is about 0.26 nm2and this value is consistent with that obtained from the isotherm of a monolayer of arachidic acid. Our observations provide rough confirmation of our model of the structure of glucose oxidase-incorporating arachidic acid LB film.
Acknowledgment. We gratefully acknowledge the helpful discussions with Professor Quate of the Stanford University and Dr. Yamada of the National Research Laboratory of Metrology and contributions of M. Aoki, Dr. Ishimoto, R. Minatoya, and T. Ishibashi of the SONY Research Center. Registry No. Glucoseoxidase,9001-37-0; arachidicacid, W630-9.
