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Direct in Situ Observation of a Lipid Monolayer-DNA Complex at the Air-Water Interface by X-ray Reflectometry Keitaro Kago,† Hideki Matsuoka,† Ryuji Yoshitome,† Hitoshi Yamaoka,*,† Kuniharu Ijiro,‡,§ and Masatsugu Shimomura‡ Department of Polymer Chemistry, Kyoto University, Kyoto 606-8501, Japan, Research Institute of Electronic Science, Hokkaido University, Sapporo 060-0812, Japan, and PRESTO, Japan Science and Technology Corporation (JST), Kawaguchi 332-0012, Japan Received September 25, 1998. In Final Form: May 17, 1999 Direct in situ observation of the fine structure of the cationic lipid monolayer-DNA complex on a water surface has been carried out by using an air-water interface X-ray reflectivity (XR) instrument. Interestingly, the thickness of the DNA layer was markedly thinner than the geometry of the cylindrical DNA molecule when the complex was deposited on the solid substrates; the thickness was determined to be 11 Å by XR measurement in a dried state, while the diameter of the DNA molecule is about 24 Å. However, the thickness in the complex on a water surface was estimated by in situ XR measurement to be about 25-28 Å, which is comparable to the geometry of the DNA molecule. Thus, the anomalously thin thickness was due to some experimental treatments, such as deposition on a solid substrate and/or drying. The structure of the monolayer and monolayer-polymer complex on the solid substrates in a dried state is not the same as that on a water surface. The possibility of some dynamic fluctuation of its structure was also suggested. These results strongly indicate the importance of a direct in situ study such as by the XR technique for the structural study of the monolayer and monolayer complex on a water surface.
Introduction Two-dimensional molecular self-assemblies such as monolayers and bilayers of amphiphilic molecules have been extensively studied. This is because they can be used as an “elementary structure” to construct a molecular superstructure. Monolayers of various kinds of functionalities have been constructed on a water surface and deposited on to substrates such as mica, glass, and silicon wafers. In this procedure, the control of the structure of the monolayer on a water surface is important. One trial for this purpose is the utilization of the complex of the water-soluble molecules (including polymers) with the (amphiphilic) monolayers on a water surface. Hydrogen bonding1-4 and electrostatic forces have been used as a driving force for the complex formation. Shimomura et al. reported the formation of a “polyion complex” between the monolayer consisting of charged molecules and the oppositely charged polymers at the air-water interface by an electrostatic force.5,6 One example is the complex of the cationic lipid and the DNA molecule.7-9 The merit of using polyion complex formation is that, by suitable combination of lipid and polymer, the cluster size can be * To whom correspondence should be addressed. † Kyoto University. ‡ Hokkaido University. § PRESTO. (1) Krauch, T.; Zaitsev, S. Yu.; Zubov, V. P. Colloids Surf. 1991, 57, 383. (2) Ahuja, R.; Caruso, P.; Mo¨bius, D.; Paulus, W.; Ringsdorf, H.; Wildburg, G. Angew. Chem., Int. Ed. Engl. 1993, 32, 1033. (3) Shimomura, M.; Nakamura, F.; Ijiro, K.; Taketsuna, H.; Tanaka, M.; Nakamura, H.; Hasebe, K. Thin Solid Films 1996, 284/285, 691. (4) Shimomura, M.; Nakamura, F.; Ijiro, K.; Taketsuna, H.; Tanaka, M.; Nakamura, H.; Hasebe, K. J. Am. Chem. Soc. 1997, 119, 2341. (5) Shimomura, M. Prog. Polym. Sci. 1993, 18, 295. (6) Miyano, K.; Asano, K.; Shimomura, M. Langmuir 1991, 7, 444. (7) Okahata, Y.; Kobayashi, T.; Tanaka, K. Langmuir 1996, 12, 1326. (8) Ijiro, K.; Shimomura, M.; Tanaka, M.; Nakamura, H.; Hasebe, K. Thin Solid Films 1996, 284/285, 780. (9) Ijiro, K.; Ikeda, T.; Shimomura, M.; Kago, K.; Matsuoka, H.; Yamaoka, H. Polym. Prepr. Jpn. 1998, 47, 767.
easily controlled and is fairly monodisperse, since it corresponds to the size of the polymer molecule. The detailed quantitative information of the exact structure of this kind of polyion complex on a water surface is obviously necessary to design and control the structure and functionality of the superstructure finally formed. However, such a study has been carried out after the deposition of the monolayer onto the solid substrates, not on a water surface. The structure in a “dried” state should not always be the same as that in a “wet” state. For example, by the surface plasmon technique, the thickness of the DNA molecule under the lipid monolayer was found to be about 9 Å,9 which is too small compared to the geometry of the DNA molecule, that is, about 24 Å.10 X-ray and neutron reflectometry (XR, NR) techniques are attractive to surface and interface scientists, since they provide information on the fine structure of the surface, the interface, and thin films.11-14 The thickness and the surface and interface roughness can be evaluated on the order of angstrom by these techniques. XR and NR techniques are recently being applied to the liquid systems. However, most of them use a strong X-ray source from synchrotron radiation (SR)15-20 and neutrons,15,21,22 which (10) Dickerson, R. E. Adv. Enzymol. 1992, 211, 67. (11) Russell, T. P. Mater. Sci. Rep. 1990, 5, 171. (12) Stamm, M. Adv. Polym. Sci. 1992, 100, 357. (13) Penfold, J.; Richardson, R. M.; Azrbakhsh, A.; Webster, J. R. P.; Bucknall, D. G.; Rennie, A. R.; Jones, R. A. L.; Cosgrove, T.; Thomas, R. K.; Higgins, J. S.; Fletcher, P. D. I.; Dickinson E.; Roser, S. J.; McLure, I. A.; Hillman, A. R.; Richards, R. W.; Staples, E. J.; Burgess, A. N.; Simister, E. A.; White, J. W. J. Chem. Soc., Faraday Trans. 1997, 93, 3899. (14) Bucknall, D. G.; Higgins, J. S. Rec. Res. Dev. Polym. Sci., in press. (15) Vaknin, D.; Kjær, K.; Als-Nielsen, J.; Lo¨sche, M. Biophys. J. 1991, 59, 1325. (16) Vaknin, D.; Kjær, K.; Ringsdorf, H.; Blankenburg, R.; Piepenstock, M.; Diederich, A.; Loesche, M. Langmuir 1993, 6, 1171. (17) Gregory, B. W.; Vaknin, D.; Gray, J. C.; Ocko, B. M.; Stroeve, P.; Cotton, T. M.; Struve, W. S. J. Phys. Chem. B 1997, 101, 2006. (18) Majewski, J.; Kuhl, T. L.; Gerstenberg, M. C.; Israelachvilli, J. N.; Smith G. S. J. Phys. Chem. B 1997, 101, 3122.
10.1021/la981352a CCC: $18.00 © 1999 American Chemical Society Published on Web 07/16/1999
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are very limited in use for experimentalists in related fields. Some investigators have developed the XR apparatus for the study of a monolayer on a water surface with a rotating anode X-ray generator.23-27 In 1996, Yamaoka et al. succeeded in constructing a specially designed XR apparatus for the study of a monolayer on a water surface for laboratory use.28-32 This apparatus has a Langmuir-Blodgett (LB) trough at the sample position; hence, the structure of the monolayer on a water surface can be studied as a function of the surface pressure, directly. Although this instrument has a conventional X-ray source (not synchrotron), it covers 8 decades of reflectivity, which is almost comparable to the SR apparatus.33 In fact, the structure of the phospholipid monolayer on a water surface could be studied in detail as a function of the surface pressure. A very clear Kiessig fringe was observed for the distearoylphosphatidylcholine monolayer on a water surface.28,29 The amphiphilic diblock copolymer monolayer on a water surface could be clearly detected, and the thickness of the hydrophobic part over the water surface and the hydrophilic part under it could be precisely determined.30 In this study, the fine structure of the lipid-DNA complex has been directly studied by the XR instrument in Kyoto. The measurements were made in situ, that is, for the monolayer complex on a water surface, and compared to the result in a dried state. Experimental Section Material. A cationic lipid, dimethyldioctadecylammonium (bromide salt, 2C18N+2C1, Sogo Pharmaceutical Co., Tokyo, Japan), was used as purchased. DNA from calf thymus (Sigma) was put into the LB trough (10 mg/L of DNA solution), and then a suitable amount of chloroform solution of 2C18N+2C1 was spread on its surface to form a lipid-DNA complex. X-ray Reflectivity. The XR was performed with an Air-Water Interface X-ray reflectometer in Kyoto. This apparatus was constructed by modification of RINT-TTR θ-θ rotating-anode X-ray system (Rigaku Corporation, Tokyo, Japan). The X-ray generator is a rotating anode type with a Cu target, and its maximum power is 60 kV-300 mA. The wavelength of the incident X-ray is 1.5406 Å (Cu KR1). On the sample stage, the Langmuir film balance and LB trough (USI System, Fukuoka, Japan) are mounted. The Langmuir film balance has a trough (19) Wu, X. Z.; Ocko, B. M.; Deutsch, M.; Sirota, E. B.; Sinha, S. K. Physica B 1996, 221, 261. (20) Chou, C. H.; Regan, M. J.; Pershan, P. S.; Zhou, X. L. Phys. Rev. E 1997, 55, 7212. (21) Hodge, P.; Towns, C. R.; Thomas, R. K.; Shackleton, C. Langmuir 1992, 8, 585. (22) Lu, J. R.; Li, Z. X.; Thomas, R. K.; Penfold, J.; J. Chem. Soc., Faraday Trans. 1996, 92, 403. (23) Lu, B. C.; Rice, S. A. J. Chem. Phys. 1978, 68, 5558. (24) Grundy, M. J.; Richardson, R. M.; Roser, S. J.; Penfold, J.; Ward, R. C. Thin Solid Films 1988, 159, 43. (25) Su, T. J.; Styrkas, D. A.; Thomas, R. K.; Baines, F. L.; Billingham, N. C.; Armes S. P. Macromolecules 1996, 29, 6892. (26) Hazallah, B.; Bosio, L.; Cortes, R.; Errafii, N. J. Chem. Phys. 1996, 93, 1202. (27) Vierl, U.; Cevc, G. Biochim. Biophys. Acta 1997, 1325, 165. (28) Matsuoka, H.; Yamaoka, H. Proc. Risø Int. Symp. Mater. Sci. 1997, 18, 437. (29) Yamaoka, H.; Matsuoka, H.; Kago, K.; Endo, H.; Eckelt, J.; Yoshitome, R. Chem. Phys. Lett. 1998, 295, 245. (30) Kago, K.; Matsuoka, H.; Endo, H.; Eckelt, J.; Yamaoka, H. Supramol. Sci. 1998, 5, 349. (31) Kago, K.; Fu¨rst, M.; Matsuoka, H.; Yamaoka, H.; Seki, T. Langmuir 1999, 15, 2237. (32) Kago, K.; Matsuoka, H.; Yoshitome, R.; Mouri, E.; Yamaoka, H. Langmuir 1999, 15, 4295. (33) Kago, K.; Endo, H.; Matsuoka, H.; Yamaoka, H.; Hamaya, N.; Tanaka, M.; Mori, T. J. Synchrotron Radiat. 1998, 5, 1304.
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Figure 1. XR profiles of the 2C18N+2C1 lipid monolayer-DNA complex on a water surface at different surface pressures. The curve for 9 mN/m is on scale, and others are shifted downward by every decade to avoid superposition.
barrier to change the surface pressure and a Wilhelmy type surface pressure sensor. The details of the apparatus have already been described elsewhere.28-30 X-ray reflectivity is a function of the refractive indices of the substances in the system. The complex refractive index n of a substance for X-rays is written as
n ) 1 - δ - iβ
(1)
where
δ)
F λ2 r (f + ∆f′) 2π e M
(2)
F λ2 r (∆f′′) 2π e M
(3)
β)
re is the classical electron radius, λ is the wavelength of the X-ray, (f + ∆f ′ + i∆f′′) is the complex atomic scattering factor, F is the density, and M is the atomic mass. The refractive index is a unique parameter representing the character of the substance; the layers are distinguished from each other only by the difference of their refractive indices. To obtain the film thickness, the surface and interface roughness, and the density, the XR profile was subjected to curve fitting. Calculation and data analysis for XR were done using a scientific software program invented by Rigaku Corporation and the scientific program MUREX118 (Multiple Reflection of X-rays).34 The procedure of data fitting and simulation is based on the theory of Parratt35 and Sinha et al.36 Results and Discussion Figure 1 shows the XR profiles for the 2C18N+2C1-DNA complex on a water surface at various surface pressures. The accumulation time for each angle was 6 s for the reflection angle (θ) range from 0 to 1° and 10 s for 1 to 3°. (34) Program for calculation/analysis of X-ray reflectivity and fluorescence intensity from multilayered thin films in grazing incidence/ exit X-ray experiments invented by Sakurai, K., National Research Institute for Metals, Tsukuba, Japan. (35) Parratt, L. G. Phys. Rev. 1954, 95, 359. (36) Sinha, S. K.; Sirota, E. B.; Garoff, S.; Stanley, H. B. Phys. Rev. B 1988, 38, 2297.
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Table 1. Structural Parameters for the 2C18N+2C1-DNA Complex on a Water Surface at Different Surface Pressures Estimated by X-ray Reflectivity Measurementsa surface pressure (mN/m) 9
19
29
39
δ β thickness roughness (×10-6) (×10-6) (Å) (Å) 2C18N+2C1 DNA(1) DNA(2) subphase 2C18N+2C1 DNA(1) DNA(2) subphase 2C18N+2C1 DNA(1) DNA(2) subphase 2C18N+2C1 DNA(1) DNA(2) subphase
2.00 3.44 3.53 3.57 2.32 3.31 3.45 3.57 2.36 3.31 3.45 3.57 2.42 3.30 3.47 3.57
0.0027 0.0124 0.0117 0.0114 0.0027 0.0124 0.0117 0.0114 0.0027 0.0124 0.0117 0.0114 0.0027 0.0124 0.0117 0.0114
7.6 28.2 13.6 12.1 26.1 12.4 13.0 27.9 13.2 14.6 25.4 11.3
0.6 0.2 1.3 0.7 1.9 0.2 1.4 0.2 0.2 0.2 1.3 0.7 3.3 1.4 0.1 1.0
a δ and β are the difference from unity of the real part and the coefficient of the imaginary part of the refractive index n, that is, n ) 1 - δ - iβ. The δ value is almost proportional to the electron density.
The dots are experimental points. Theoretically, the reflectivity has to be almost unity below the critical angle, since total reflection should occur, but the experimental data show lower reflectivity than unity. This may be due to the fact that the water surface is not perfectly flat; because the LB trough is small, the X-ray beam will hit the curved surface which is produced by the surface tension. At reflection angles θ of about 1 and 2-2.5°, Kiessig fringes are observed, although broad, for higher surface pressure conditions. The position of the Kiessig fringe shifted toward lower reflection angles, indicating an increase of the thickness with increasing surface pressure. The data were subjected to curve fitting. The experimental data were subjected to least-squares fitting using software with various initial parameters to find the best fit. Their consistency was confirmed so that the densities obtained from δ values (see eq 2) and by dividing the molecular mass by the occupied area and thickness agreed well. The solid lines in the figure are the best fit to the experimental data by model calculation. The parameters used for the fittings are summarized in Table 1. From these fitting parameters, we can draw a density map for the 2C18N+2C1-DNA complex on a water surface, which is shown in Figure 2. All the XR data obtained in this study could be fitted to some extent by a simple two-layer model consisting of air-lipid-DNA-water, but the agreement was not satisfactory. The head group cannot be distinguished from the lipid tail due to the low electron density of the head group (quaternized ammonium), although it was possible for phosphatidylcholine derivatives which have a big and high electron density head group.28,29 Instead, as shown in the figures, a three-layer model, air-lipid-DNA(1)-DNA(2)-water, showed better agreement with the XR data obtained. DNA(1) and DNA(2) layers have different electron densities. The electron density of the DNA(2) layer is higher than that of the DNA(1) layer. This means that there is less DNA and more water in the DNA(2) layer than in the DNA(1) layer. The thickness of the lipid part is less than the full-stretched length of the molecule (23 Å), indicating the tilt conformation of the chain, and it increased with increasing surface pressure. This can be attributed to the change of
Figure 2. Density map of the 2C18N+2C1 lipid monolayerDNA complex on a water surface for the direction perpendicular to the water surface at various surface pressures determined by the in situ XR measurements.
the packing of the lipid molecules (vertically aligned with increasing surface pressure) as was the case for our previous study for phospholipids.28,29 Under the lipid, there apparently exist two DNA layers. The thickness of the DNA(1) layer is about 25-28 Å, which is very close to the diameter of the cylindrical DNA molecule. The thickness of the DNA(2) layer is about 11-13 Å, which is thinner than the DNA(1) layer. Even though the better fit was obtained by using the three-layered model than by using the two-layered one, perfect agreement (like that obtained for the sample on a solid substrate) was not achieved. This may be due to complication of the structure of the sample on a liquid surface and due to the simplicity of the fitting model. The layer may form a more complex structure than a simple three-layered structure; the real structure may contain the lateral inhomogeneity and/or a non-Gaussian interface profile. One may think that the curve fitting, the profile itself, and the critical edge in Figure 1 are not satisfactory. We also do not think that the agreement in this figure is highly satisfactory. In fact, we can get better agreement if we do not consider the density of DNA layer, that is, if we use the larger δ value, which is proportional to the density. However, the density estimated by this fitting procedure was too large, more than two times larger than that which is physically reasonable for the DNA layer. Moreover, the estimated density is even higher than the one which was calculated by dividing the molecular mass by the summation of the van der Waals volume of all atoms in DNA, which is completely impossible physically. Hence, we have judged that the fitting results shown in Figure 1 are the most reasonable and probable ones, at least at this moment. The problem of this system which could not be clarified until now has been the extraordinary “thin” thickness of the DNA layer for the complex on the substrates in a dried state.9 It is due to the situation that no experimental technique could be applied to the structural study for the monolayer on a water surface. For comparison, the XR data in a dried state, which was for the sample deposited on a glass plate at 20 mN/m, are shown in Figure 3. The dots are the experimental data, and the solid line is the best results by model fitting. The thickness of the DNA layer under the lipid layer (thickness 7 Å) was estimated
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Figure 3. XR profile for the 2C18N+2C1 lipid monolayer-DNA complex on a glass plate in a dried state.
Letters
water surface predicted by the in situ XR measurements. The lipid molecules arranged on a water surface with a tilting angle which depends on the surface pressure. Under water, DNA molecules are adsorbed to the lipid monolayer to form a polyion complex by electrostatic force. Since we found two apparent DNA layers by an in situ XR experiment, there are at least two possibilities for the lipid-DNA complex, as shown in Figure 4. The thickness of the first layer of DNA is about 25-28 Å, which is comparable to the diameter of the cylindrical DNA molecule, as mentioned above. Hence, the first layer may be the DNA layer adsorbed to the lipid monolayer in a side-on manner. Under the first DNA layer there is the second DNA layer. It is obviously hard to expect that the negatively charged DNA is adsorbed on the first DNA layer, which is also negatively charged. Hence, it is appropriate to consider that the DNA molecules are not adsorbed to the lipid monolayer in a fully stretched conformation in a side-on manner. They do not form a simple layered structure (Figure 4b) but a flexible (wavy) conformation (Figure 4a), in which the trains correspond to the DNA(1) layer and the loops and tails to the DNA(2) layer. By the calculation of the density of each DNA layer obtained by the curve-fitting procedure, the volume fraction of DNA in the “first layer” was estimated to be approximately 70 vol % and that for the “second layer” was determined to be approximately 30 vol %. This situation is not in contradiction to the wavy model in Figure 4a. Such a structure may not be static, but dynamic and fluctuating. Although the XR data give us the timeaveraged structure, the real situation might be that some part of the DNA chain repeats adsorption and desorption to the lipid monolayer and that such a fluctuating part of the DNA chain also changes its location with time. Conclusion
Figure 4. Possible structure of the 2C18N+2C1 lipid monolayerDNA complex on a water surface estimated by detailed analysis of the in situ XR data: (a) wavy conformation; (b) two-layered structure.
to be 11 Å, which is markedly smaller than the geometrical size (diameter of cylindrical DNA). This thickness for a dried state, that is, 11 Å, is comparable to that obtained by the surface plasmon technique, 9 Å.9 By atomic force microscopy, no laterally heterogeneous structure was observed for the complex in the dried state; DNA molecules were bound to the lipid monolayer uniformly on the substrates. From these experimental results, the structures of the monolayer and the monolayer-polymer complex on the solid substrates in a dried state are not the same as those on a water surface. The DNA molecule might be destroyed in the drying process and/or by the interaction with the solid substrate. Thus, to know the exact structure of the monolayer systems on a water surface for the design and molecular construction of the superstructure for molecular devices, it is necessary to study it by the air-water XR technique. Figure 4 shows the schematic representation for the possible structure of the 2C18N+2C1-DNA complex on a
Our XR technique has a very high performance, which is enough for the study of a monolayer and a monolayerpolymer complex on a water surface. The fine structure of the complex and its change with surface pressure can be determined quantitatively by the in situ XR measurements. We confirmed that the structure and conformation of the DNA molecule in the lipid-DNA complex in a dried state are largely different from its real structure on a water surface. Apparently two DNA layers were found by the in situ XR measurements in the complex with the lipid monolayer on a water surface formed by electrostatic interaction. Hence, it is suitable to think that the conformation of the DNA molecule in the complex on a water surface is a rather wavy form. These observations reported here indicate the importance and necessity of the in situ investigations for the water surface systems such as a monolayer-polymer complex. Further experiments for the systems with different kinds of lipid and with DNA of different molecular weight are now in progress. Acknowledgment. This work was financially supported by the New Energy Development Organization (NEDO) project of the Ministry of International Trade and Industry of Japan and also by a Grant-in-Aid for Scientific Research on Priority Areas (Molecular Superstructure-Design and Creation) by the Ministry of Education, Science, Sports and Culture of Japan (08231237, 09217230, 08231201), to whom our sincere gratitude is due. LA981352A
