Name: methods for the self-integration of megamolecular biopolymers on the drying air-lc interface
Uploaded: 2017-04-07T12:30:00
Description: Watch this Scientific Journal Video about Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface at JoVE.com

This work was supported by grant-in-aid for Young Scientists (16K17956) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, The Kyoto Technoscience Center, and The Mitani Foundation for Research and Development.

1. Instruments
<ol>
	<li>Polarization device
	<ol>
		<li>To construct a polarization device, provide a light source with a halogen lamp, a light guide, polarizers, a sample stage, an optical rail, rod stands, and a digital single-lens reflex camera (see Figure 1C and Materials List for the polarization device parts).</li>
	</ol>
	</li>
</ol>
2. Preparation of the Biopolymer Solution
<ol>
	<li>Polysaccharide solutions
	<ol>
		<...

<ol>
	<li>Flemming, H. -. C., Wingender, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biofilm+matrix.">The biofilm matrix.</a> Nat. Rev. Microbiology. 8 (9), 623-633 (2010).</li><li>Osada, Y., Kawamura, R., Sano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hydrogels of cytoskeletal proteins. , (2016).</li><li>Rothemund, P. W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+DNA+to+create+nanoscale+shapes+and+patterns.">Folding DNA to create nanoscale shapes and patterns.</a> Nature. 440, 297-302 (2006).</li><li>Okeyoshi, K., Okajima, M. K., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milliscale+self-integration+of+megamolecule+biopolymers+on+a+drying+gas-aqueous+liquid+crystalline+interface.">Milliscale self-integration of megamolecule biopolymers on a drying gas-aqueous liquid crystalline interface.</a> Biomacromolecules. 17 (6), 2096-2103 (2016).</li><li>De Gennes, P. G., Prost, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The physics of liquid crystals. , (1993).</li><li>De Gennes, P. G., Brochard-Wyart, F., Quere, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Capillarity and wetting phenomena: drops, bubbles, pearls, waves. , (2003).</li><li>Okajima, M. K., Kaneko, D., Mitsumata, T., Kaneko, T., Watanabe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyanobacteria+that+produce+megamolecules+with+efficient+self-orientations.">Cyanobacteria that produce megamolecules with efficient self-orientations.</a> Macromolecules. 42 (8), 3058-3062 (2009).</li><li>Okeyoshi, K., Kawamura, R., Yoshida, R., Osada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermo-+and+photo-enhanced+microtubule+formation+from+Ru(bpy)32+-conjugated+tubulin.">Thermo- and photo-enhanced microtubule formation from Ru(bpy)32+-conjugated tubulin.</a> J. Mater. Chem. B. 2, 41-45 (2014).</li><li>Matsuo, E. S., Tanaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+discontinuous+volume-phase+transition+of+gels.">Kinetics of discontinuous volume-phase transition of gels.</a> J. Chem. Phys. 89, 1695-1703 (1988).</li><li>Okajima, K., Mishima, R., Amornwachirabodee, K., Mitsumata, T., Okeyoshi, K., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+swelling+in+hydrogels+formed+by+cooperatively+aligned+megamolecules.">Anisotropic swelling in hydrogels formed by cooperatively aligned megamolecules.</a> RSC Adv. 5, 86723-86729 (2015).</li><li>Joshi, G., Okeyoshi, K., Okajima, M. K., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+control+of+diffusion+and+swelling+in+megamolecular+polysaccharide+hydrogels.">Directional control of diffusion and swelling in megamolecular polysaccharide hydrogels.</a> Soft Matter. 12, 5515-5518 (2016).</li></ol>

It was sometimes difficult for the camera to focus on the sample due to the transmitted light intensity being too low. In such cases, placing an extended transparent plastic film on the stage helped to arrange the focus. The limitation of the observable resolution was dependent on the camera lens, ~10 &#181;m in this case. The observable limitation of the sample thickness, &#916;y, was dependent on the maximum light intensity of the lamp, ~10 mm in this case.
The advantage of the device shown ...

By focusing on the rigid, rod-shaped structures of biopolymers, dynamic soft materials have been used for various applications, including polysaccharide biofilm matrices1, &#34;active gels&#34; composed of cytoskeletal proteins2, and &#34;DNA origami&#34; of desired shapes3. To clarify the structural properties, many strategies have been explored, such as transmission electron microscopy, scanning electron microscopy, atomic force microscopy, and confocal fluorescence microscopy. However, because these methods are mostly undertaken in a dried or static state, it is difficult to explain the dynamic behaviors in macroscopic scales, as seen in actual living systems. Recently, we successfully observed the dynamic behavior of biopolymers on the aqueous air-LC interface through polarized light4. During the visualization of the oriented structure while drying the biopolymer solution, the temporal changes indicated self-integration of biopolymers on the unstable air-LC interface.
Here, we describe a protocol for the drying of LC biopolymer solutions at the air-LC interface using polarized instruments. As opposed to other analyses of the LC phase that do not consider drying5,6, the LC dynamics during the drying process were investigated here by evaluating the orientational order parameter&#160;&#160;in the lateral view of the fluid phase in a one-side-open cell. The combination of the cell evaporation and the use of polarized instruments allowed for macroscopic monitoring with a controlled evaporation direction. In addition, it was possible to validate the drying records by focusing on the crystalline structures of the adsorbed microdomains, which were affected by molecular weight, concentration, etc. To demonstrate the effectiveness of the method, the drying processes of basic biopolymers with rigid rod shapes, such as polysaccharides, microtubules (MTs), and DNA, were investigated. We chose these biopolymers because they are typical examples of hierarchical macromolecules with megamolecular weights, and their intermolecular interactions enable them to form LC states.

<table border="0" cellpadding="0" cellspacing="0" width="617">
	<tbody>
		<tr height="69">
			<td height="69" style="height:69px;width:172px;">sacran</td>
			<td style="width:151px;">Green Science Materials Inc., Japan</td>
			<td style="width:131px;"></td>
			<td style="width:163px;">From Aphanothece sacrum. 
			Mw = 1.9 &#215; 107 g mol-1</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:172px;">xanthan gum</td>
			<td style="width:151px;">Taiyo Kagaku Co., Japan</td>
			<td style="width:131px;">Neosoft XC</td>
			<td style="width:163px;">From Xanthomonas campestris. 
			Mw = 4.7 &#215; 106 g mol-1</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">tubulin</td>
			<td style="width:151px;">Cytoskeleton, Inc., USA</td>
			<td style="width:131px;">T240</td>
			<td style="width:163px;">From porcine brain.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">GpCpp</td>
			<td style="width:151px;">Jena Bioscience, Germany</td>
			<td style="width:131px;">NU405L</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">piperazine-N,N&#8242;-bis(2-ethanesulfonic acid)</td>
			<td style="width:151px;">Sigma-Aldrichi, Co. LLC.</td>
			<td style="width:131px;">P6757-500G</td>
			<td style="width:163px;">PIPES</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:172px;">ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid</td>
			<td style="width:151px;">Dojindo Molecular Technologies, Inc.</td>
			<td style="width:131px;">342-01314</td>
			<td style="width:163px;">EGTA</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">MgCl2-6H2O</td>
			<td style="width:151px;">Wako Pure Chemical Industries, Ltd.</td>
			<td style="width:131px;">135-15055</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">KOH</td>
			<td style="width:151px;">Wako Pure Chemical Industries, Ltd.</td>
			<td style="width:131px;">162-21813</td>
			<td style="width:163px;">pellet</td>
		</tr>
		<tr height="21">
			<td height="58" style="height:58px;width:172px;">DNA</td>
			<td rowspan="2" style="width:151px;">Sigma-Aldrich, Co. LLC.</td>
			<td rowspan="2" style="width:131px;">D1626</td>
			<td rowspan="2" style="width:163px;">From salmon testes. 
			Mw = 1.3 &#215; 106 Da (~2000 bp)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">Tris-EDTA buffer solution</td>
			<td style="width:151px;">Sigma-Aldrich, Co. LLC.</td>
			<td style="width:131px;">T9285-100ML</td>
			<td style="width:163px;">10 mM Tris, pH 8.0, with 1 mM EDTA</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">slide glass</td>
			<td style="width:151px;">Matsunami Glass Ind., Ltd., Japan</td>
			<td style="width:131px;">S1111</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">silicon rubber sheet</td>
			<td style="width:151px;">Asone Co.</td>
			<td style="width:131px;">6-611-32</td>
			<td style="width:163px;">Thickness: 1 mm</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">centrifuge</td>
			<td style="width:151px;">Beckman-Coulter, Inc., USA</td>
			<td style="width:131px;">Avanti J-25</td>
			<td style="width:163px;">equipped with a JA-20 rotor</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">light source</td>
			<td style="width:151px;">Sumita Optical Glass, Inc., Japan</td>
			<td style="width:131px;">LS-LHA</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">light guide</td>
			<td style="width:151px;">Sumita Optical Glass, Inc., Japan</td>
			<td style="width:131px;">GF7.2-1-L1500R-M80 (AAAR-015M)</td>
			<td style="width:163px;">80 mm &#215; 80 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">halogen lamp</td>
			<td style="width:151px;">Ushio Inc., Japan</td>
			<td style="width:131px;">JCR 15V150WBN</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">polarizer</td>
			<td style="width:151px;">Luceo, Co.,Ltd.</td>
			<td style="width:131px;">POLAX-42S</td>
			<td style="width:163px;">40 x 80 x 2t (45&#730;)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">KMH-80</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">sample stage</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">TARW-25503L</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">sample holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">SHA-25RO</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">rod</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">ROU-12-40</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">posts holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">RS-6-40</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">posts holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">RS-12-60</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">posts holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">RS-12-80</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">posts holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">RS-12-130</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">carrier</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">CAA-25LS</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">camera holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">CMH-2</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">medium optical rail</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">OBA-500SH</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">lenstube</td>
			<td style="width:151px;">Tomytech, BORG</td>
			<td style="width:131px;">lenstube BK</td>
			<td style="width:163px;">80&#966;, L25 mm&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">lenstube</td>
			<td style="width:151px;">Tomytech, BORG</td>
			<td style="width:131px;">lenstube BK</td>
			<td style="width:163px;">80&#966;, L50 mm&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">multiband</td>
			<td style="width:151px;">Tomytech, BORG</td>
			<td style="width:131px;"></td>
			<td style="width:163px;">80&#966;&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">V plate</td>
			<td style="width:151px;">Tomytech, BORG</td>
			<td style="width:131px;">V plate 60S</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">plate holder</td>
			<td style="width:151px;">Viexen, Co.,Ltd.</td>
			<td style="width:131px;">plate holder SX</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">EOS Kiss X7i&#160;</td>
			<td style="width:151px;">Canon Inc., Japan</td>
			<td style="width:131px;">8594B001</td>
			<td style="width:163px;">with a standard zoom lens,&#160; EFP 18-55 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">photographic software</td>
			<td style="width:151px;">Canon Inc., Japan</td>
			<td style="width:131px;">EOS Utility</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">PC</td>
			<td style="width:151px;">Microsoft</td>
			<td style="width:131px;">Surface</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">polarization microscope</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:131px;">BX51</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">first order retardation plate</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:131px;">U-TP530</td>
			<td style="width:163px;">&#955; = 530 nm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">CCD camera</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:131px;">DP80</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">photographic software</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:131px;">cellSens Standard</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">Java-based image processing program</td>
			<td style="width:151px;">the National Institutes of Health</td>
			<td style="width:131px;">ImageJ</td>
			<td style="width:163px;"></td>
		</tr>
	</tbody>
</table>

methods for the self-integration of megamolecular biopolymers on the drying air-lc interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

Using the device as shown in Figure 1, the self-integration from microdomain to macrodomain on a drying air-LC interface was evaluated (Figure 2A). As the first demonstration of the drying experiment, two kinds of megamolecular polysaccharides, sacran (Mw = 1.9 x 107 g mol-1) and xanthan gum (4.7 x 106 g mol-1), were compared. Figure 2B shows photographs of the solutions in t...

Watch this Scientific Journal Video about Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface at JoVE.com

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and ...

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