1 Nanostructural Control of Biological Molecules Arranged by Using Langmuir-Blodgett Films of Organo-modified Alminosilicate as a Template Shuntaro Arai1, Jun-Ichi Kusaka1,2, Munehiro Kubota3, Kei-ichi Kurosaka3,and Atsuhiro Fujimori1,* Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama, 338-8570, 2Graduate School of Science and Engineering, Yamagata University, Yonazawa, Yamagata, 992-8510, 3Kunimine Industries Co., Ltd. Iwaki, Fukushima, 972-8312, Japan. 1 (Received <Month> <Date>, <Year>; CL-<No>; E-mail: [email protected]) Clay Langmuir-Blodgett (LB) films play a template role in the formation of the lysozyme thin layer. Confirmation of chemisorption of biological molecules to the anionic montmorillonite surface is performed by comparison between IR spectra of multilayers of organo-clay and one chemisorbed lysozyme. The difference between height information of monolayers for only organo-modified alminosilicates and their adsorbed lysozyme indicates about 9 nm. This value is almost corresponded to twice for length of lysozyme along the long axis. Layered silicates, clay minerals, have played important roles in the modern technology.1 These materials have received considerable attention because of their potential use in numerous technical applications, ranging from nanocomposite materials to biomedical and personal care products.2 Clays are unique in the sense that they consist of negatively charged aluminosilicate layers kept together with exchangeable interlayer cations. Hence a large number of alkylammonium-modified layered silicates (organo-modified clays) have been developed, which are now widely used to form a large variety of nanocomposite materials with enhanced material properties.3 The Langmuir–Blodgett (LB) method is one of the most useful techniques to prepare ultra-thin films. In this method, floating molecular monolayers formed at an air/water interface are deposited on a solid substrate in a layer-by-layer way. Ultra-thin films of clay minerals were prepared by the LB method. Recently, the clay LB films have been hybridized with alkylammonium cations. When a solution of an amphiphilic alkylammonium salt is spread onto the clay suspension, the negatively charged clay platelets in the suspension are adsorbed onto a floating monolayer of the alkylammonium cation at an air–clay suspension interface. Previously, authors have reported formation of organomodified montmorillonite with extremely high surface coverage and localized negative charges in the bottom by modification method in oil/water interface.4-8 In this work, floating monolayers and multilayered films on solid of pre-formed organo-modified montmorillonites (MMTs) with high coverage and localized nagative carges in the bottom were constructed by Langmuir and LB method. In this case, buffer solution conatining lysozyme as cationic biomolecule have been used as subphase. The organomodifed alminosilicate LB films play a template role in the formation of the adsorbed lysozyme array. These LB multilayers of organo-modified MMTs adsorbed an enzyme were characterized by the π–A isotherm, infrared (IR) spectroscopy, and atomic force microscopic (AFM) observation. Natural Na+-montmorillonite was kindly supplied by Kunimine Co. with the cation-exchange capacity of 108.6 meq/100 g. The organophilic clay was prepared by cation exchange reactions of natural clay (aqueous dispersion) with 0.1 % aqueous solution of dimethyl dioctadecyl (DMDO) ammonium chloride. Four kinds of quaternary ammonium cations based on long-alkyl chain are used to hydrophobic parts of clays. In order to estimate hydrophobicity and ability of formation of ordered organized film, organo-modification agent having two alkyl-chains (DMDO) are applied. In this study, lysozyme were used as water-solved biological cationic enzyme (isoelectric point: pH11). The organo-modified clays were spread from toluene solution (approximately 10–4 M) onto buffer solution composed distilled water (approximately 18 MΩ·cm) containing lysozyme molecules. These monolayers were transferred onto CaF2 (IR samples) or mica (AFM samples) substrates at 15 °C using the LB and horizontal lifting method. IR spectra measured by system 2000 spectrometer (Parkin-Elmer Co. Ltd). The surface morphologies of the transferred monolayers were observed using a scanning probe microscope (Seiko Instrument, SPA300 with SPI-3800 probe station) at tapping mode utilizing microfabricated rectangular Si single crystal cantilevers with integrated pyramidal tips with a constant force of 1.7 Nm-1. Figure 1 shows IR spectra of LB multilayers of organomodified clay and they adsorbed lysozyme. In the case of monolayer on the water surface, obviously expanded tendency of π-A isotherm were confirmed in that adsorbed lysozyme (data not shown). The characteristics of spectrum in fig. 1(a) (a) vSi-O (b) vc=o vN-H Wavenumber / cm-1 Figure 1 IR spectra of multilayers for (a) organomodified alminosilicates (20 layers), and (b) their adsorbed lysozyme. (b) (a) 0 [μm] 1 0 3.6 [nm] 0 [μm] 1 13.0 [nm] 0 Figure 2 AFM images of X-type monolayers of (a) organo-modified alminosilicates and (b) their adsorbed lysozyme (pH 7, 30 mNm-1, and 15 °C). is existence of clear C-H and Si-O stretching vibrations. These bands based on the long alkyl chain on clay surface and silicate layers. In fig. 1(b), bands of vC=O and vN-H are clearly confirmed. These bans come from carboxyl and amino groups in lysozyme. Therefore, it seems that this result supports adsorption of biological enzyme to monolayer of organomodified MMTs. Figure 2 shows morphological estimation of these organized molecular films by AFM observation. The films shown in figs. 2(a) and (b) are corresponded to the X-type monolayers transferred by horizontal lifting method. Namely, both films expose hydrophilic clay surface or that adsorbed enzyme. In comparison of these images, the most important characteristic is difference between height information. Neat organo-clay monolayer have alkylated part with 2.5 nm and MMT part with 1.0 nm thickness. The height information in fig. 2(a) is well-corresponding to these values. This experimental fact indicates normal orientation of monolayer of organo-modified MMT on solid. From the result of fig. 2(b), the height information of adsorbed film indicates to 13.0 nm. The height of lysozyme along the long axis are corresponding to 4.5 nm9. Therefore, it seems that bimolecular layer adsorbed to MMT surface of normal oriented monolayer to the substrate. This speculation is supported by result of polarized IR spectroscopy and out-of plane X-ray diffraction at low angle side to their multilayers on glass substrate. Figure 3 shows schematic illustration of model of adsorbed double layered lysozyme / organo-modified alminosilicate monolayer on solid. It seems that interaction of Figure 4 AFM images of X-type monolayers of organomodified alminosilicates adsorbed lysozyme (pH 4 and 7, 10 mNm-1, and 15 °C) adsorption of cationic enzyme to the MMT correspond to electrostatic force. In addition, origin of formation of double layer structure for lysozyme assembly may correspond to COO–∙∙∙+H3N- interaction. Indeed, the adsorption condition of organo-MMT monolayer to cationic enzyme on air/water interface are pH7 and 30 mNm–1 in isotherm, and 15 °C in this case. Especially, high surface pressure condition of film transferring is easy to occur formation of piled up molecular film. Figure 4 shows adsorbed surface morphogical changes in X-type monolayer of organo-modified MMT depended on the transferring surface pressure and pH. In the case of low transferring surface pressure, low density single layered adsorption structure of lysozyme on organo-MMT monolayer are observed. Further, the amount of adsorbed enzyme increase with acidic condition10. Previously, dense packed lysozyme molecule observed by molecular resolution STM image. In the case of high surface pressure condition, formation of same type assembling structure are predicted. However, these like supramolecular structure generally lose an inherent activity of hydrolysis to saccharide. As mentioned above, organized molecular films of adsorbed enzyme have been constructed by means of the modified LB method using an organo-modified alminosilicate. In addition control of an amount of adsorbed molecules, adsorbed density, and adsorbed style is important to aiming at construction of new nanobio-materials. References 1 2 3 4 5 6 7 8 9 Figure 3 Schematic illustration of model of adsorbed double layered lysozyme / organo-modified alminosilicate monolayer on solid. 10 P. C. LaBarton, Z. Wang and T. Pinnavaia, J. Appl. Caly Sci., 15, 11 (1999). M. Alexandre and P. Dubois, Mater. Sci. Eng., 28, 1 (2000) R. A. Vaia, H. Ishii and E. P. Giannelis, Chem. Mater., 5, 1694 (1993). K. Kurosaka, M. Kubota, A. Fujimori, Japan Patent (2009), and (2011) A. Fujimori, J. Kusaka, R. Nomura, Polym. Eng. Sci., 51, 1099 (2011). A. Fujimori, SPE Plastic Res. Online, No.7, 1, (2011). A. Fujimori, J. Kusaka, M. Kubota, K. Kurosaka, RFP International, 6, 216 (2011). A. Fujimori, Y. Sugita, H. Nakahara, E. Ito, M. Hara, N. Matsuie, K. Kanai, Y. Ouchi, K. Seki, Chem. Phys. Lett., 387, 345 (2004). H. Shirahama, J. Lyklema, W. Norde, J. Colloid Interf. Sci., 139, 177, (1990) L. Haggerty, B.A. Watson, M.A. Barteau, A. M. Lenhoff, J. Vac. Sei. Technol. B, 9, 1219 (1991). 3 Graphical Abstract Textual Information Description (if any) Title Nanostructural Control of Biological Molecules Arranged by Using Langmuir-Blodgett Films of Organo-modified Alminosilicate as a Template Authors’ Names Shuntaro Arai, Jun-Ichi Kusaka, Munehiro Kubota, Kei-ichi Kurosaka, Atsuhiro Fujimori Graphical Information Ultrathin films of regularly adsorbed biological molecules were fabricated by means of the modified Langmuir-Blodgett (LB) method using an organo-modified alminosilicate. 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