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>
		<li>Dissolve sacran7 (0.5 g) in pure water (100 mL) by stirring at ~80 &#176;C for more than 12 h. During the dissolution, cover the container with plastic wrap to prevent evaporation. Prepare an aqueous solution of xanthan gum in the same manner.</li>
		<li>Cool the solutions at ~25 &#176;C to obtain 0.5 wt% aqueous solutions.</li>
		<li>Centrifuge the sacran solution to remove impurities (48,400 x g, 4 &#176;C, 1 h, 3 times).</li>
	</ol>
	</li>
	<li>MT solution
	<ol>
		<li>Prepare a 0.5 wt% tubulin solution (1 mL) in a Britton-Robinson buffer (80 mM piperazine-N,N&#8242;-bis(2-ethanesulfonic acid) (PIPES); 1 mM ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA); and 5 mM MgCl2, pH 6.8) on ice8.</li>
		<li>Use 0.5 wt% tubulin solution (50 &#181;L) and guanosine-5&#39;-[(&#945;,&#946;)-methyleno]triphosphate (GpCpp) (5 &#181;L) to prepare a GpCpp-containing tubulin solution (50 &#181;L). Incubate at 37 &#176;C for 3 h to obtain a stable MT nucleus. 
		NOTE: The role of GpCpp is to support MT formation and to completely suppress the depolymerization of MT to tubulin.</li>
		<li>Mix the 0.5 wt% tubulin solution (950 &#181;L) and the GpCpp-containing tubulin solution (50 &#181;L) at ~25 &#176;C for 1 day to obtain a stable 0.5 wt% MT solution.</li>
	</ol>
	</li>
	<li>DNA solution
	<ol>
		<li>Prepare a 0.5 wt% DNA solution (1 mL) in Tris-EDTA buffer solution (10 mM Tris, pH 8.0, with 1 mM EDTA).</li>
	</ol>
	</li>
	<li>Keep the 0.5 wt% biopolymer sample solutions at 25 &#176;C for the drying experiment.</li>
</ol>
3. Drying Experiments and Observation under Cross-polarized Light
<ol>
	<li>Solutions in a one-side-open cell (Figure 1A)
	<ol>
		<li>Cut a silicon sheet (see Materials List) into an appropriate shape with a thickness of 1 mm (inner dimension of 5-15 mm, 1 mm, and ~20 mm; Figure 1A).
		<ol>
			<li>Assemble a one-side-open cell composed of a silicon spacer with inner dimension of 5-15 mm, 1 mm, and ~20 mm and two non-modified glass slides (76 mm &#215; 1 mm &#215; 26 mm). Fix both sides of the cell with double clips in advance to keep the sample solution from leaking out.</li>
		</ol>
		</li>
		<li>Slowly add each of the 0.5 wt% biopolymer solution (100-300 &#181;L) using a ~1-mm pore-size pipette tip to each cell at ~25 &#176;C. Remove air bubbles from the cells using a syringe needle.</li>
		<li>Place the cells in an oven with an air circulator at 60 &#176;C under atmospheric pressure for evaporation; the evaporation direction is opposite to that of gravity.</li>
	</ol>
	</li>
	<li>Observations under cross-polarized light (Figure 1B-1C)
	<ol>
		<li>Provide straight visible light via a 100 W halogen lamp with a flat-surface light source over a wide area (80 mm x 80 mm). Adjust the polarizers to 45&#176; and 135&#176; using the holders (Figure 1C).</li>
		<li>Fix the positions of the light source, polarizers, sample stage, and camera using an optical rail and rod stands (Figure 1C). Place the sample stage between the two polarizers (the distance between the polarizers should be ~5 cm). Place the camera ~20 cm from the sample stage to allow focusing.</li>
		<li>At given times, place the samples from step 3.1.3 between the polarizers on the stage parallel to the XZ-plane and cover the device with a black curtain; the actual device is shown in Figure 1C.</li>
		<li>Photograph the samples through linear crossed polarizers using a digital single-lens reflex camera with a standard zoom lens (see Materials List). Control the camera settings, such as focal distance, using computer software (see Materials List).</li>
	</ol>
	</li>
	<li>Spatio-temporal analysis of the transmitted light intensity (Figure 1C)
	<ol>
		<li>To evaluate the change of the orientational order parameter&#160;in the drying process, collect photographs hourly for 24 h.</li>
		<li>Measure the transmitted light intensity along the centerline in the Z-direction as a gray value using an image processing program (e.g., ImageJ).</li>
		<li>Plot a graph of the gray value as a function of the distance from the upper open side.</li>
	</ol>
	</li>
	<li>Microscopic observations under cross-polarized light (Figure 1D)
	<ol>
		<li>To check the methods, make microscopic observations with a polarization microscope equipped with a CCD camera9. Keep a first-order retardation plate in the light path. Control the conditions for the photos using a PC software.</li>
	</ol>
	</li>
</ol>

<ol><li>Flemming, H. -C., Wingender, J. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/20676145">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aOsada%2C+Y+%22Hydrogels+of+cytoskeletal+proteins%22">Hydrogels of cytoskeletal proteins</a>. , Springer. (2016).</li>
<li>Rothemund, P. W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Folding+DNA+to+create+nanoscale+shapes+and+patterns%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Milliscale+self-integration+of+megamolecule+biopolymers+on+a+drying+gas-aqueous+liquid+crystalline+interface%5BTitle%5D)+AND+%22Biomacromolecules%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aDe+Gennes%2C+PG+%22The+physics+of+liquid+crystals%22">The physics of liquid crystals</a>. , Oxford University Press. Oxford, UK. (1993).</li>
<li>De Gennes, P. G., Brochard-Wyart, F., Quere, D. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aDe+Gennes%2C+PG+%22Capillarity+and+wetting+phenomena%3A+drops%2C+bubbles%2C+pearls%2C+waves%22">Capillarity and wetting phenomena: drops, bubbles, pearls, waves</a>. , Springer. (2003).</li>
<li>Okajima, M. K., Kaneko, D., Mitsumata, T., Kaneko, T., Watanabe, J. <a target="_blank" href="http://pubs.acs.org/doi/abs/10.1021/ma802829x">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://pubs.rsc.org/en/content/articlelanding/2014/%E2%80%8Btb/c3tb21242d">Thermo- and photo-enhanced microtubule formation from Ru(bpy)32+-conjugated tubulin.</a> J. Mater. Chem. B. 2, 41-45 (2014).</li>
<li>Olympus. Support BX51P. , Available from:
 <a target="_blank" href="http://www.olympus-ims.com/microscope/bx51p/">http://www.olympus-ims.com/microscope/bx51p/</a> (2016).</li>
<li>Matsuo, E. S., Tanaka, T. <a target="_blank" href="http://aip.scitation.org/doi/abs/10.1063/1.455115">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://pubs.rsc.org/en/Content/ArticleLanding/2015/RA/C5RA18585H">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/pubmed?term=((Directional+control+of+diffusion+and+swelling+in+megamolecular+polysaccharide+hydrogels%5BTitle%5D)+AND+%22Soft+Matter%22%5BJournal%5D)">Directional control of diffusion and swelling in megamolecular polysaccharide hydrogels.</a> Soft Matter. 12, 5515-5518 (2016).</li>
</ol>

The authors have nothing to disclose.

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><tbody><tr><td>sacran</td><td>Green Science Materials Inc., Japan</td><td></td><td>From Aphanothece sacrum.&lt;br/&gt; M&lt;sub&gt;w&lt;/sub&gt; = 1.9 &amp;times; 10&lt;sup&gt;7&lt;/sup&gt; g mol&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>xanthan gum</td><td>Taiyo Kagaku Co., Japan</td><td>Neosoft XC</td><td>From Xanthomonas campestris.&lt;br/&gt; M&lt;sub&gt;w&lt;/sub&gt; = 4.7 &amp;times; 10&lt;sup&gt;6&lt;/sup&gt; g mol&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>tubulin</td><td>Cytoskeleton, Inc., USA</td><td>T240</td><td>From porcine brain.</td></tr><tr><td>GpCpp</td><td>Jena Bioscience, Germany</td><td>NU405L</td><td></td></tr><tr><td>piperazine-&lt;em&gt;N,N&amp;prime;&lt;/em&gt;-bis(2-ethanesulfonic acid)</td><td>Sigma-Aldrichi, Co. LLC.</td><td>P6757-500G</td><td>PIPES</td></tr><tr><td>ethylene glycol-bis(&amp;beta;-aminoethyl ether)-&lt;em&gt;N,N,N&#039;,N&#039;&lt;/em&gt;-tetraacetic acid</td><td>Dojindo Molecular Technologies, Inc.</td><td>342-01314</td><td>EGTA</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;-6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Wako Pure Chemical Industries, Ltd.</td><td>135-15055</td><td></td></tr><tr><td>KOH</td><td>Wako Pure Chemical Industries, Ltd.</td><td>162-21813</td><td>pellet</td></tr><tr><td>DNA</td><td>Sigma-Aldrich, Co. LLC.</td><td>D1626</td><td>From salmon testes.&lt;br/&gt;Mw = 1.3 &amp;times; 10&lt;sup&gt;6&lt;/sup&gt; Da (~2,000 bp)</td></tr><tr><td>Tris-EDTA buffer solution</td><td>Sigma-Aldrich, Co. LLC.</td><td>T9285-100ML</td><td>10 mM Tris, pH 8.0, with 1 mM EDTA</td></tr><tr><td>slide glass</td><td>Matsunami Glass Ind., Ltd., Japan</td><td>S1111</td><td></td></tr><tr><td>silicon rubber sheet</td><td>Asone Co.</td><td>6-611-32</td><td>Thickness: 1 mm</td></tr><tr><td>centrifuge</td><td>Beckman-Coulter, Inc., USA</td><td>Avanti J-25</td><td>equipped with a JA-20 rotor</td></tr><tr><td>light source</td><td>Sumita Optical Glass, Inc., Japan</td><td>LS-LHA</td><td></td></tr><tr><td>light guide</td><td>Sumita Optical Glass, Inc., Japan</td><td>GF7.2-1-L1500R-M80 (AAAR-015M)</td><td>80 mm &amp;times; 80 mm</td></tr><tr><td>halogen lamp</td><td>Ushio Inc., Japan</td><td>JCR 15V150WBN</td><td></td></tr><tr><td>holder</td><td>Sigmakoki, Co.,Ltd.</td><td>KMH-80</td><td></td></tr><tr><td>sample stage</td><td>Sigmakoki, Co.,Ltd.</td><td>TARW-25503L</td><td></td></tr><tr><td>sample holder</td><td>Sigmakoki, Co.,Ltd.</td><td>SHA-25RO</td><td></td></tr><tr><td>rod</td><td>Sigmakoki, Co.,Ltd.</td><td>ROU-12-40</td><td></td></tr><tr><td>posts holder</td><td>Sigmakoki, Co.,Ltd.</td><td>RS-6-40</td><td></td></tr><tr><td>posts holder</td><td>Sigmakoki, Co.,Ltd.</td><td>RS-12-60</td><td></td></tr><tr><td>posts holder</td><td>Sigmakoki, Co.,Ltd.</td><td>RS-12-80</td><td></td></tr><tr><td>posts holder</td><td>Sigmakoki, Co.,Ltd.</td><td>RS-12-130</td><td></td></tr><tr><td>carrier</td><td>Sigmakoki, Co.,Ltd.</td><td>CAA-25LS</td><td></td></tr><tr><td>camera holder</td><td>Sigmakoki, Co.,Ltd.</td><td>CMH-2</td><td></td></tr><tr><td>medium optical rail</td><td>Sigmakoki, Co.,Ltd.</td><td>OBA-500SH</td><td></td></tr><tr><td>lenstube</td><td>Tomytech, BORG</td><td>lenstube BK</td><td>80&amp;phi;, L25 mm&amp;nbsp;</td></tr><tr><td>lenstube</td><td>Tomytech, BORG</td><td>lenstube BK</td><td>80&amp;phi;, L50 mm&amp;nbsp;</td></tr><tr><td>multiband</td><td>Tomytech, BORG</td><td></td><td>80&amp;phi;&amp;nbsp;</td></tr><tr><td>V plate</td><td>Tomytech, BORG</td><td>V plate 60S</td><td></td></tr><tr><td>plate holder</td><td>Viexen, Co.,Ltd.</td><td>plate holder SX</td><td></td></tr><tr><td>EOS Kiss X7i&amp;nbsp;</td><td>Canon Inc., Japan</td><td>8594B001</td><td>with a standard zoom lens,&amp;nbsp; EFP 18-55 mm</td></tr><tr><td>photographic software</td><td>Canon Inc., Japan</td><td>EOS Utility</td><td></td></tr><tr><td>PC</td><td>Microsoft</td><td>Surface</td><td></td></tr><tr><td>polarization microscope</td><td>Olympus</td><td>BX51</td><td></td></tr><tr><td>first order retardation plate</td><td>Olympus</td><td>U-TP530</td><td>&amp;lambda; = 530 nm</td></tr><tr><td>CCD camera</td><td>Olympus</td><td>DP80</td><td></td></tr><tr><td>photographic software</td><td>Olympus</td><td>cellSens Standard</td><td></td></tr><tr><td>Java-based image processing program</td><td>the National Institutes of Health</td><td>ImageJ</td><td></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 ...

methods-for-self-integration-megamolecular-biopolymers-on-drying-air

Research

JoVE Journal

Bioengineering

6.0K Views.  Japan Advanced Institute of Science and Technology.  Een werkwijze voor het drogen geïnduceerde zelfintegratie van megamolecular biopolymeren op de lucht-vloeistof grensvlak kristallijn is hier voorzien. Deze methodologie zal niet alleen nuttig zijn voor het begrijpen van de macroscopische mogelijkheden van biopolymeren, maar ook als een evaluatiemethode voor de zachte materialen in biomedische en milieugebied. 

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

Synthesis of Poly(N-isopropylacrylamide) Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this unique property, Janus microparticles have been recognized as a new class of materials, thereby attracting a great deal of attention from various research fields. The versatility of these microparticles has been exemplified through their uses as building blocks for self-assembly, electrically responsive actuators, emulsifiers for painting and cosmetics, and carriers for drug delivery. This study introduces a detailed protocol that explicitly describes a synthetic method for designing novel Janus microhydrogels composed of a single base material, poly(N-isopropylacrylamide) (PNIPAAm). Janus microdroplets are firstly generated via a hydrodynamic focusing microfluidic device (HFMD) based on the separation of a supersaturated aqueous NIPAAm monomer solution and subsequently polymerized through exposure to UV irradiation. The resulting Janus microhydrogels were found to be entirely composed of the same base material, featured an easily identifiable compartmentalized morphology, and exhibited anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability. We believe that the proposed method introduces a novel hydrogel platform with the potential for advanced synthesis of multi-functional Janus microhydrogels.

Synthesis of PolyN-isopropylacrylamide Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

We present a protocol to synthesize Janus microhydrogels composed entirely of the same base material, poly(N-isopropylacrylamide) (PNIPAAm), with a clearly compartmentalized structure base on the phase separation of a supersaturated NIPAAm monomer solution. The synthesized Janus microhydrogels show unique properties such as anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability.

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this ...

Chemistry

Orientational Transition in a Liquid Crystal Triggered by the Thermodynamic Growth of Interfacial Wetting Sheets

In liquid crystal (LC) physical chemistry, molecules near the surface play a great role in controlling bulk orientation. Thus far, mainly to achieve desired molecular orientation states in LC displays, the "static" surface property of LCs, so-called surface anchoring, has been intensively studied. As a rule of thumb, once the initial orientation of LCs is "locked" by specific surface treatments, such as rubbing or treatment with a specific alignment layer, it hardly changes with temperature. Here, we present a system exhibiting an orientational transition upon temperature variation, which conflicts with the consensus. Right on the transition, the bulk LC molecules experience the orientational rotation, with 90&#176; between the planar (P) orientation at high temperatures and the vertical (V) orientation at low temperatures in the first-order transitional manner. We have tracked thermodynamic surface anchoring behavior by means of polarizing optical microscopy (POM), dielectric spectroscopy (DS), high-resolution differential scanning calorimetry (HR-DSC), and grazing incidence X-ray diffraction (GI-XRD) and reached a plausible physical explanation: that the transition is triggered by a growth of surface wetting sheets, which impose the V orientation locally against the P orientation in the bulk. This landscape would provide a general link explaining how the equilibrium bulk orientation is affected by surface-localized orientation in many LC systems. In our characterization, POM and DS are advantageous by offering information on the spatial distribution of the orientation of LC molecules. HR-DSC gives information about the precise thermodynamic information on transitions, which cannot be addressed by conventional DSC instruments due to limited resolution. GI-XRD provides information on surface-specific molecular orientation and short-range orderings. The goal of this paper is to present a protocol for preparing a sample that exhibits the transition and to demonstrate how the thermodynamic structural variation, both in the bulk and on surfaces, can be analyzed through the abovementioned methods.

Here, we present a protocol to trigger an orientational transition of a liquid crystal in response to temperature. Methodologies are described for preparing a sample in order to observe the transition and the detailed transitional evolution.

In liquid crystal (LC) physical chemistry, molecules near the surface play a great role in controlling bulk orientation. Thus far, mainly to achieve ...

Engineering

Interfacial Molecular-level Structures of Polymers and Biomacromolecules Revealed via Sum Frequency Generation Vibrational Spectroscopy

As a second-order nonlinear optical spectroscopy, sum frequency generation (SFG) vibrational spectroscopy has widely been used in investigating various surfaces and interfaces. This non-invasive optical technique can provide the local molecular-level information with monolayer or submonolayer sensitivity. We here are providing experimental methodology on how to selectively detect the buried interface for both macromolecules and biomacromolecules. With this in mind, interfacial secondary structures of silk fibroin and water structures around model short-chain oligonucleotide duplex are discussed. The former shows a chain-chain overlap or spatial confinement effect and the latter shows a protection function against the Ca2+ ions resulting from the chiral spine superstructure of water.

Being comprehensively utilized, sum frequency generation (SFG) vibrational spectroscopy can help to reveal chain conformational order and secondary structural change happening at polymer and biomacromolecule interfaces.

As a second-order nonlinear optical spectroscopy, sum frequency generation (SFG) vibrational spectroscopy has widely been used in investigating various ...

Biochemistry

Preparation of Liquid Crystal Networks for Macroscopic Oscillatory Motion Induced by Light

A strategy based on doped liquid crystalline networks is described to create mechanical self-sustained oscillations of plastic films under continuous light irradiation. The photo-excitation of dopants that can quickly dissipate light into heat, coupled with anisotropic thermal expansion and self-shadowing of the film, gives rise to the self-sustained deformation. The oscillations observed are influenced by the dimensions and the modulus of the film, and by the directionality and intensity of the light. The system developed offers applications in energy conversion and harvesting for soft-robotics and automated systems.
The general method described here consists of creating free-standing liquid crystalline films and characterizing the mechanical and thermal effects observed. The molecular alignment is achieved using alignment layers (rubbed polyimide), commonly used in the display manufacturing industry. To obtain actuators with large deformation, the mesogens are aligned and polymerized in a splay/bend configuration, i.e., with the director of the liquid crystals (LCs) going gradually from planar to homeotropic through the film thickness. Upon irradiation, the mechanical and thermal oscillations obtained are monitored with a high-speed camera. The results are further quantified by image analysis using an image processing program.

The goal of the protocol is to create liquid crystalline polymer films that can mechanically oscillate under continuous light irradiation. We describe in great detail the conception of free-standing films, from the liquid crystal alignment method to the photo-actuation. The experimental protocol applied to prepare this material is broadly applicable.

A strategy based on doped liquid crystalline networks is described to create mechanical self-sustained oscillations of plastic films under continuous ...

Microfluidic Preparation of Liquid Crystalline Elastomer Actuators

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation usually consists in the formation of droplets containing low molar mass liquid crystals at elevated temperatures. Subsequently, these particle precursors are oriented in the flow field of the capillary and solidified by a crosslinking polymerization, which produces the final actuating particles. The optimization of the process is necessary to obtain the actuating particles and the proper variation of the process parameters (temperature and flow rate) and allows variations of size and shape (from oblate to strongly prolate morphologies) as well as the magnitude of actuation. In addition, it is possible to vary the type of actuation from elongation to contraction depending on the director profile induced to the droplets during the flow in the capillary, which again depends on the microfluidic process and its parameters. Furthermore, particles of more complex shapes, like core-shell structures or Janus particles, can be prepared by adjusting the setup. By the variation of the chemical structure and the mode of crosslinking (solidification) of the liquid crystalline elastomer, it is also possible to prepare actuating particles triggered by heat or UV-vis irradiation.

This article describes the microfluidic process and parameters to prepare actuating particles from liquid crystalline elastomers. This process allows the preparation of actuating particles and the variation of their size and shape (from oblate to strongly prolate, core-shell, and Janus morphologies) as well as the magnitude of actuation.

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs &#8212; polysiloxane-based and epoxy-based &#8212; are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 &#176;C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers can be readily functionalized and have been used in various applications such as bio-sensors and bio-devices. In this study, we introduced a planar organic molecule: copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) to dope lipid membranes. The CuPc/lipid hybrid membrane forms at the water/air interface by self-assembly. In this membrane, the hydrophobic CuPc molecules are located between the hydrophobic tails of lipid molecules, forming a lipid/CuPc/lipid sandwich structure. Interestingly, an air-stable hybrid lipid bilayer can be readily formed by transferring the hybrid membrane onto a Si substrate. We report a straightforward method for incorporating nanomaterials into a lipid bilayer system, which represents a new methodology for the fabrication of biosensors and biodevices.

We report a protocol for producing a hybrid lipid membrane at the water/air interface by doping the lipid bilayer with copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) molecules. The resulting hybrid lipid membrane has a lipid/CuPc/lipid sandwich structure. This protocol can also be applied to the formation of other functional nanomaterials.

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers ...

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (&#60;1 &#181;L) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This paper details the fabrication protocol of microfluidic chips developed for on-chip protein crystallization with the dialysis method and in situ X-ray diffraction experiments. The microfabrication process makes it possible to integrate a semipermeable regenerated cellulose dialysis membrane with any molecular weight cut-off, between two layers of the chip.

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

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

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