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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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