Langmuir 1991, 7, 152-155
152
Fibrous Structure in a Flavin Monolayer Observed by Dark-Field Electron Microscopy Osamu Wada,' Sonoko Suzuki, Satoshi Ueyama,? Hiroaki Kawakubo,? and Satoru Isodat Materials & Electronic Devices Laboratory, Central Research Laboratory, Mitsubishi Electric Corp., 8-1 - 1 Tsukaguchi-Honmachi, Amagasaki, Hyogo 661, Japan Received August 30, 1989. In Final Form: June 4, 1990
A microscopic study on a monolayer of flavin (7,8-dimethyl-3,10-dinonylisoalloxazine (DNI)),which is typical of functional groups of redox proteins, has been performed using dark-field electron microscopy. It was found that the flavin monolayer has a fibrous structure with good crystallinity and no holes over the region of a few micrometers squared. From the electron diffraction pattern, it was also found that the orientation of the fiber axis is virtually ~ / off 6 the lifting direction during preparation of the monolayer. For molecular orientation, it was clarified from the FT-IR measurement that the long axis of the isoalloxazine ring is oriented perpendicularly to the lifting direction of the substrate. Introduction The molecular orientation of proteins in the biomembrane plays an important role for high efficiency and selectivity of biological reactions such as photosynthesis and electron transfer. In the case of the electron transport system in the biomembrane, proteins take a specific intermolecular orientation in which prosthetic groups like heme and flavin can take a certain geometry; hence highly efficient electron transfer can take place between prosthetic groups.' Accordingly, it may be considered that the electron transfer in molecular organization formed in vitro can also be controlled if it is possible to assemble the molecules in the same way as in the biomembrane.2 From this standpoint, it is of great importance to evaluate and control the orientation of the functional group in the molecular organization. To control the orientation of the functional groups, the Langmuir-Blodgett (L-B) method is considered to be one of the most useful methods. Recently, many kinds of L-B films have come to be studied and are expected to be applied in many fields, especially molecular electronics. Structural analysis is very important for understanding the relationship between the function and the structure of L-B films in these studies. Structural analysis of the L-B films has been carried out by many methods. In particular, microscopic methods are useful for studying the surface structure of the L-B films. Fluoroescence microscopy has an advantage of direct observation of the monolayer spread on the s u b p h a ~ e . ~Scanning -~ tunneling microscopy (STM) is a recent method for atomic scale observation of L-B film^.^^^ Transmission electron microscopy (TEM) studies have been performed by using the shadowing technique*-1° and t
Central Research Laboratory.
(1) Chance, B. et al., Eds. Tunneling in Biological System; Academic Press: New York, 1980. (2) Isoda, S.; Maeda, M. Proceedings of the 6th Symposium on Future Electron Deuices; Osaka, 1987, p 285. (3) Lbshe, M.; Sachmann, E.; Mbhwald, H. Ber. Bunsen-Ges. Phys. Chem. 1983,87, 848. (4) Loshe, M.; Mbhwald, H. Reu. Sci. Instrum. 1984, 55, 1968. (5) Gobel, H. D.; Mbhwald, H. Thin Solid Films 1988,159,63. (6) Smith, D. P. E.; Bryant, A.; Quate, C. F.; Rabe, J. P.; Gerber, Ch.; Swalen, J. D. Proc. Natl. Acad. Sci. U.S.A. 1987,84, 969. (7) Mizutani, W.; Shigeno, M.; Saito, K.; Watanabe, K.; Sugi, M.; Ono, M.; Kajimura, K. J p n . J . Appl. Phys. 1988,27, 1803. (8)Ries, H. E.; Kimball, W. A. J . Phys. Chem. 1955,59, 94. (9) Fischer, A,; Sackmann, E. J. Phys. (Les Ulis, Fr.) 1984,45, 517. (10) Allain, M.; Benattar, J. J.; Rieutord, F.; Robin, P. Europhys. Lett. 1987, 3, 309.
phase contrast to clarify the structure and mechanism of formation of the L-B films. Uyeda et al. reported that there exist densely scattered small holes in the stearic acid monolayer by using the dark-field image technique of TEM.14 Since then, L-B films have been studied by this technique.15J6 Such holes and defects can also be observed by the shadowing t e c h n i q ~ e , but ~ J ~the dark-field image technique enables direct observation of the monolayer and a transmission electron diffraction (TED) study at the same area as that of the TEM observation. We adopted, therefore, the dark-field image technique to study the fine structure and the crystallinity of the L-B films composed of functional molecules such as flavin and heme which are typical of prosthetic groups for electron transport function in proteins. In this work, we have studied a monolayer of 7,8-dimethyl-3,10-dinonylisoalloxazine (DNI) which is one of the flavin derivatives. By the study of the polarized transmission spectra of the FTIR spectro~copy,'~ the authors already reported that DNI molecules in the L-B film on CaF2 take a specific biaxial orientation. This result means that the DNI molecules are arranged regularly on a macroscopic scale and suggests that the DNI monolayer may have some crystallinity. Therefore, fine structure, crystallinity, and molecular orientation of the flavin monolayer have been investigated by TEM, TED, XD, and FT-IR methods. Experimental Section 7,8-Dimethyl-3,10-dinonylisoalloxazine (DNI)moleculeswere used for the flavin monolayer films. DNI was purchased from Hamari Chemicals Co. Ltd. The molecular structure of DNI is shown in Figure la. The DNI molecule has two alkyl chains attached to the isoalloxazine ring. A DNI monolayer was deposited o n a substrate by the L-B method with a Langmuir trough equipped with a film balance. DNI solution in chloroform (1.0 mM) was spread o n BaClzsolution (0.1 mM, adjustedtopH 9.0 with NaOH solution, 17.2 O C ) . The DNI monolayer (11)Fischer, A,; Sackmann, E. Nature 1985,313, 299. (12) Fischer, A.; Sackmann, E. J. Colloid Interface Sci. 1986,112, 1. (13) Frey, W.; Schneider, J.; Ringsdorf, H.; Sackmann, E. Macromolecules 1987, 20, 1312. (14) Uyeda, N.; Takenaka, T.; Aoyama, K.; Matsumoto, M.; Fujiyoshi, Y. Nature 1987,327, 319. (15) Wright, A. C.; Luk, S.; Williams, J. 0. In Proceedings of the 9th
European Congress on Electron Microscopy; Goodhew, P. J., Dickinson, H. G., Eds.; IOP Publishing Ltd: Bristol, 1988; Vol. 2, p 301. (16) Inoue, T.; Yase, K.; Okada, S.; Matauda, H.; Nakanishi, H.; Kato, M. Jpn. J. Appl. Phys. 1988,27, L1635. (17) Suzuki, S.; Isoda, S.; Maeda, M. Jpn. J. Appl. Phys. 1989, 28, 1673.
0743-7463/91/2407-0152$02.50/00 1991 American Chemical Society
Fibrous Structure in a Flavin Monolayer
Langmuir, Vol. 7, No. 1, 1991 153
LH
c
'yr
1
""I
It \ E
0
50
100
150
200
A /g molecule' (b)
Figure 1. (a) Molecular structure of 7,8-dimethyl-3,10dinonylisoalloxazine (DNI). (b) Pressure (+area ( A ) isotherm of the DNI monolayer. A t the pressure of 25 "em-*, which is used as the constant pressure a t the deposition of the DNI monolayer, the molecular area is 49 A*. was deposited a t a constant surface pressure (r)of 25 mN.m-1 onto the substrate a t the lifting speed of 5 mmemin-*. A t this surface pressure, the molecular area ( A ) was estimated a t 49 A2 from the pressure-area ( P A ) isotherm shown in Figure lb. The depositing procedure is dipping the substrate into the subphase before spreading and compressing the molecules and lifting the substrate to deposit the monolayer on the substrate. The deposited film was considered to be a monolayer with the isoalloxazine ring in contact with the hydrophilic substrate. The initial spreading molecular area was 120 A*, and the transfer ratio was 1.18 on a carbon substrate. For TEM observation, a thin carbon film was used as the substrate. The thin carbon film was prepared by vacuum evaporation onto a mica (001) surface. The carbon film was separated from the mica on the water surface and then placed onto a semicircular microgrid. The surface of the carbon film was treated to be hydrophilic by a plasma-etching procedure. The semicircular microgrid was set to a certain direction in order to examine the directional dependence of the DNI monolayer on the lifting direction. TEM observation was carried out by using a JEOL JEM-2000FX type electron microscope a t an accelerating voltage of 200 kV with the minimum dose system in order to decrease the radiation damage of the L-R films." Polarized transmission FT-IR spectra of the DNI L-B film (Y-type, 11 monolayers) were measured by using the CaF2 substrate covered with the same carbon film as used for TEM observation. X-ray measurement was carried out by using the same specimen (11 monolayers) as that used for the FT-IR measurement for consistency of the experiments.
Results and Discussion Figure 2 shows a dark-field image of the DNI monolayer. As shown in the figure, the DNI monolayer appears as a bright image on a dark background which reflects the carbon substrate. This contrast of the monolayer to the carbon substrate is almost the same as that reported by Uyeda et al.,I4 whereas the image of the DNI monolayer is found to be completely different from that of the stearic acid monolayer. Several characteristics are observed in this dark-field electron micrograph. The most important characteristic is that the DNI monolayer has a fibrous structure which consists of many planar fibrils oriented in the same direction. The fibrils are a few micrometers long, and their minimum width is about 30 nm as observed
Figure 2. Dark-field image of DNI monolayer. The monolayer has a bright image on a dark background corresponding to the carbon substrate. The monolayer has a fibrous structure with planar fibrils. The direction of the fibrils is about r/6 off the lifting direction of the substrate. The minimum width of the fibril observed is about 30 nm a t the edge of the monolayer.
at the edge of the monolayer. Such as anisotropic structure has not previously been reported for L-B films of aliphatic acids. This fibrous structure may be attributed to the molecular interaction among the DNI molecules and to the interaction between the DNI molecule and the carbon surface during preparation of the DNI monolayer. To clarify the influence of the preparation procedure on the structure of the DNI monolayer, the relationship between the long axis of the fibrils and the lifting direction of the substrate was investigated by using the semicircular microgrid. It was found that the direction of the fibrils was about n/6 off the lifting direction, as shown in Figure 2. Another important characteristic is that there are no holes over the region of a few micrometers squared in the DNI monolayer, although cracks exist a t the edge of the monolayers. This result shows that defects in the DNI monolayer are much fewer than those previously observed in the monolayer of aliphatic acid, which has densely scattered holes. Accordingly, the DNI L-B film is considered to be a promising material for molecular electronic devices. Crystallinity of the DNI monolayer was examined by electron diffraction. Figure 3a shows a transmission electron diffraction (TED) pattern of the DNI monolayer. In this TED pattern, it should be noted that all diffracted spots, which have symmetrical arcs, are aligned in line. Such a TED pattern is typical of a fibrous polycrystalline pattern as observed in polymer materials with a specific crystal axis (called a fiber axis) perpendicular to the line of the diffracted spots (called a layer line). When the monolayer was tilted against the incident electron beam by rotating the semicircular microgrid around the fiber axis from 0 to n/6,the TED showed almost the same pattern with the slight and continuous change of the lattice parameters. This result shows t h a t the azimuthal crystalline orientation around the fiber axis has a slight distribution but not a random distribution as observed in the bulk fibrous polycrystals. This means that the DNI molecules have a specific orientation with the hydrophilic group attached to the surface of the carbon surface. I t is considered, as a whole, that the DNI monolayer takes the fibrous structure consisting of the planar singlelayer microcrystals with a preferential orientation along the fiber axis. The direction of the long axis of the fibrils observed in Figure 2 coincides with the fiber axis. Furthermore, the crystallinity of the DNI monolayer is found to be high due to the sharpness of the diffraction spots. This means that the DNI molecules are regularly packed in the microcrystal. This type of fibrous structure has not been observed so far for one-monolayer L-B films
Wada et al.
154 Langmuir, Vol. 7, No. 1, 1991
.-c C >r
E'
dimhon
,
polalued direction
0
P
v
:
o(jjjfzJA2
I
0
0
0
I
Figure 4. Molecular orientation of DNI L-B film (Y-type, 11 monolayers). (a) Dependence of absorbance of the two C=O stretching bands on rotational angel (4) of substrate to the electric vector of the polarized incident beam. The direction of the polarized incident beam is parallel to the lifting direction of the substrate a t 4 = 0. (b) Schematic diagram of relationship between DNI molecule and lifting direction. The short axis of the isoalloxazine ring stands obliquely a t the angle of 8 to the substrate.
0
0
G
c2-0
(b)
SH
4.3E
I
0
:
Ob4.78
Figure 3. (a) Electron diffraction pattern of DNI monolayer. Diffracted spots are aligned in line (called layer lines). The direction of the fiber axis, which is perpendicular to the layer line, is found to be parallel to the direction of the fibrils. The direction of the fiber axis is about n/6 off the lifting direction. (b) Diagram of 10 intensive diffracted spots of TED pattern. Lattice parameters are determined to be 5.0, 4.7, and 4.3 A, respectively. (c) Pseudohexagonal lattice structure of the DNI monolayer determined from TED pattern. The area occupied by a single lattice point (shaded part) is estimated to be 25 A*.
from either the crystallographical or the morphological standpoint, although fibrous morphology was reported for many kinds of L-B films of diacetylene derivatives by TEM.1%20 To determine the crystalline structure of the DNI monolayer, we analyzed 10 intensive diffraction spots shown in Figure 3b. Lattice parameters were estimated to be 5.0, 4.7, and 4.3 A, respectively. From this analysis, the crystalline structure of the DNI monolayer was found to be a pseudohexagonal packing structure as shown in Figure 3c. The crystallinity along the normal direction to the substrate was investigated by XD for the same sample as that used for the FT-IR measurement (11 monolayers). From the periodic reflection peaks, the spacing for the periodicity was found to be 29 A. If the L-B film is considered to be the Y-type, the thickness of the monolayer was estimated to be 14.5 A. This result shows that the DNI L-B film was deposited regularly. The long axis of the isoalloxazine ring takes in-plane orientation to the substrate plane." From these results, the alkyl chain of the DNI molecule, having a net length of about 12 A, is considered to stand almost perpendicular to the surface of the carbon substrate. We assumed, therefore, that the 10 intensive diffraction spots in Figure 3b are originated (18) Sarkar, M.;Lando, J. B. Thin Solid Films 1983,s. 119. (19) Tieke, B.; Weiss, K. J. Colloid Interface Sci. 1984, 202, 129. (20) Kajzar, F.; Rothberg, L.; Etemad, S.; Chollet, P. A.; Grec, D.; Boudet, A.; Jedju, T. Thin Solid Films 1988, 260,373.
from the alkyl chains of the DNI molecules. Under this assumption, a single lattice point in Figure 3c corresponds to a single alkyl chain. Since the area occupied by the single lattice point is estimated to be 25 A2 from the TED pattern, the area occupied by the single DNI molecule which has two alkyl chains is 50 A2. This obtained value for the molecular area almost corresponds to that obtained from the x-A isotherm (49 A2)in Figure l b a t the surface pressure of 25 mN0m-l. The molecular area of 49 A2 obtained from the x-A isotherm is considered to coincide with the area of the isoalloxazine ring which is packed with the short axis standing obliquely to the film plane.17 The geometrical plane area of the isoalloxazine ring was estimated to be about 65-70 A2 from both the extrapolation of the x-A isotherm to x = 0 and the molecular model. From these values, the tilt angle of the short axis to the substrate may be about 40-45". Therefore, the isoalloxazine ring is considered to be packed in the same pseudohexagonal structure in which the alkyl chains are regularly packed. Thus, the fine morphology and the crystallinity of the DNI monolayer were analyzed by TEM. The molecular orientation, however, is hardly clarified by TEM and TED. Therefore, we used the FT-IR method to estimate the molecular orientation of the isoalloxazine ring in the DNI monolayer. We measured polarized transmission FT-IR spectra of the DNI L-B film (11 monolayers) on the substrates of CaF2 covered with a thin carbon film to compare the results with the TEM observation. We used the absorption band a t 1653 cm-' which was assigned to the stretching mode of the carbonyl group C d . 2 1 Figure 4a shows the dependence of the absorbance for the C2=0 band on the rotational angle of the substrate around the polarized incident beam. As shown in the figure, the absorbance changes with the rotational angle. This shows that the isoalloxazine ring does not take a random orientation. The absorbance of the CFO band shows a maximum a t the angle of about 2x/3 and a minimum a t the angle of about x/6. This result suggests that the long axis of the isoalloxazine ring is oriented almost perpendicularly to the lifting direction of the substrate. The orientation of the isoalloxazine ring on the carbon substrate was found to be different from that on the CaF2 substrate, (21) A b , M.;Kyogoku, Y.;Kitagawa, T.; Kawano, K.; Ohishi, N.; TakaiSuzuki, A.; Yagi, K. Spectrochim. Acta 1986,42A, 1059.
Langmuir, Vol. 7, No. 1, 1991 155
Fibrous Structure in a Flavin Monolayer
in which the long axis is oriented almost parallel to the lifting d i r e ~ t i 0 n . I This ~ difference may be due to the surface properties of the substrates. Figure 4b shows a schematic diagram of the relationship between the isoalloxazine ring and the lifting direction of the substrate.
Conclusion TEM, FT-IR, and XD measurements show that (1)the DNI monolayer has a fibrous structure, (2) the DNI molecules are packed in a pseudohexagonal structure in the monolayer, (3) the isoalloxazine rings take a specific orientation, and (4) the DNI monolayer is regularly deposited with the alkyl chains almost standing normal to the surface of the substrate. Summarizingthe above conclusions,the structural model of the DNI monolayer is schematically shown in Figure 5. The microcrystals, which are the constitutional units of the fibril, are oriented to the direction about r/6 off the lifting direction. Figure 5b shows the cross-sectional diagram of the microcrystals with the fiber axis normal to this figure. The microcrystals take a slightly distributed orientation around the fiber axis (c axis). As a result, the direction of the alkyl chains (a axis) takes a slightly different orientation in each microcrystal. As for the molecular electronic function of the DNI monolayer, we reported that stable cyclic voltamograms of the DNI monolayer on the Au electrode were observed based on the redox reaction of the isoalloxazine ring, and the molecular interaction among the isoalloxazine rings was found to be rather strong.22 The latter result is considered to be closely related to the high crystallinity of the DNI monolayer. Furthermore, we are now studying the e l e c t r ~ c h e m i c a and l ~ ~ photoelectric properties of molecular heterojunction through flavin-porphyrin multilayers. We believe that these electronic properties are based on the structure of the flavin-porphyrin multilayers and can be applied to electronic devices. Acknowledgment. We thank Dr. H. Nakahara for his helpful discussion. This work was performed under the
carbon substrate /
I
I
(a)
(b)
Figure 5. Schematic diagram of structural model for DNI monolayer. Planar (a) and cross-sectional (b) diagram of microcrystals. In the cross-sectional diagram, the a axis of the microcrystal is parallel to the alkyl chains. The microcrystals take a slightly distributed orientation around the fiber axis (c axis). G.B. stands for grain boundary between the microcrystals.
management of FED (the R & D Association for Future Electron Devices) as a part of the R & D of Basic Technology for Future Industries sponsored by NED0 (New Energy and Industrial Technology Development Organization).
(22) Ueyama, S.;I d a , S.;Maeda, M. J.ElectroanaL Chem. 1989,264, 149.
( 2 3 )Ueyama, S.;Isoda, S.; Maeda, M. J . ElectroanaL Chem. In press.
Registry No. DNI, 122188-09-4.
