Diese Arbeit wurde durch Gewährung-in-Hilfe für junge Wissenschaftler (16K17956) vom Ministerium für Bildung, Kultur, Sport, Wissenschaft und Technologie von Japan, der Kyoto-Techno Center und The Mitani Stiftung für Forschung und Entwicklung unterstützt.

1. Instrumente <ol><li> Polarisationsvorrichtung <ol><li> Um eine Polarisationsvorrichtung zu konstruieren, liefert eine Lichtquelle mit einer Halogenlampe, eine Lichtführung, Polarisatoren, ein Probentisch, ein optischer Schiene, Stange steht und eine digitale Spiegelreflexkamera (siehe Abbildung 1C und Liste für die Polarisations Geräteteile). </li></ol></li></ol> 2. Herstellung der Biopolymer-Lösung <ol><li> Polysaccaridlösungen <ol><li> Löst sacran 7 (0,5 g) in reinem Wasser (100 ml) durch Rühren bei ~ 80 ° C für mehr als 12 h. Während der Auflösung, decken sie die Behälter mit Plastikfolie, um Verdunstung zu verhindern. Bereiten einer wäßrigen Lösung von Xanthangummi in der gleichen Weise. </li><li> Kühlen Sie die Lösung auf ~ 25 ° C 0,5 Gew% ige wässrige Lösungen zu erhalten. </li><li> Zentrifugiere die sacran Lösung zu entfernen Verunreinigungen (48.400 xg, 4 ° C, 1 h, 3 timn). </li></ol></li><li> MT - Lösung <ol><li> Vorbereiten eine 0,5 wt% Tubulin - Lösung (1 ml) in einem Britton-Robinson - Puffer (80 mM Piperazin- N, N &#39;-Bis (2-ethansulfonsäure) (PIPES), 1 mM Ethylenglycol-bis (β-aminoethylether) - N, N, N &#39;N&#39; -tetraessigsäure (EGTA) und 5 mM MgCl 2, pH 6,8) auf Eis 8. </li><li> Verwenden 0,5 wt% Tubulin-Lösung (50 ul) und Guanosin-5&#39; - [(α, β) -methyleno] triphosphat (GpCpp) (5 &amp; mgr; l), um eine GpCpp Tubulin enthaltenden Lösung hergestellt (50 ul). bei 37 ° C inkubieren 3 h einen stabilen Kern MT zu erhalten. HINWEIS: Die Rolle der GpCpp ist MT Bildung zu unterstützen und vollständig die Depolymerisation von MT an Tubulin zu unterdrücken. </li><li> Mischen Sie die 0,5 wt% Tubulin-Lösung (950 ul) und die GpCpp enthaltenden Tubulin-Lösung (50 ul) bei ~ 25 ° C für 1 Tag eine stabile 0,5 wt% MT-Lösung zu erhalten. </li></ol></li><li> DNA - Lösung<ol><li> Vorbereiten eine 0,5 wt% DNA-Lösung (1 ml) in Tris-EDTA-Pufferlösung (10 mM Tris, pH 8,0, mit 1 mM EDTA). </li></ol></li><li> Halten Sie die 0,5 wt% Biopolymer Probenlösungen bei 25 ° C für die Trocknung Experiment. </li></ol> 3. Trocknen Experimente und Beobachtung unter Kreuz-polarisiertes Licht <ol><li> Lösungen in einer einseitig offenen Zellen (1A) <ol><li> Geschnitten , um eine Silizium - Platte (siehe Materialliste) in eine geeignete Form mit einer Dicke von 1 mm (Innenmaß von 5-15 mm, 1 mm, und ~ 20 mm; 1A). <ol><li> Zubauen eine einseitig offene Zelle eines Siliziumabstandshalters zusammengesetzt mit Innenmaß von 5-15 mm, 1 mm, und ~ 20 mm und zwei nicht-modifizierten Glas-Objektträger (76 mm × 1 mm × 26 mm). Fix beide Seiten der Zelle mit Doppel Clips im Voraus, um die Probenlösung zu halten austritt. </li></ol></li><li> Langsam jede der 0,5 Gew% Biopolymer-Lösung (100-300 ul) eine ~ 1 mm Porengröße Pipettete Spitze jeder Zelle bei ~ 25 ° C. Entfernen von Luftblasen aus den Zellen unter Verwendung einer Spritzennadel. </li><li> Platzieren Sie die Zellen in einem Ofen mit einer Luft Zirkulator bei 60 ° C unter Atmosphärendruck für die Verdampfung; die Verdampfungsrichtung, die der Schwerkraft entgegengesetzt. </li></ol></li><li> Beobachtungen unter kreuzpolarisiertem Licht (1B-1C) <ol><li> Bietet gerade sichtbares Licht über eine 100 W Halogenlampe mit einer flachen Oberflächen-Lichtquelle über einen breiten Bereich (80 mm x 80 mm). Stellen Sie die Polarisatoren 45 ° und 135 ° , um den Halter (Abbildung 1C). </li><li> Fix die Positionen der Lichtquelle, Polarisatoren, Probentisch, und eine optische Kamera Schiene und Stange steht (1C) verwendet wird . Platzieren Sie den Probentisch zwischen den beiden Polarisatoren (der Abstand zwischen den Polarisatoren sollte ~ 5 cm). Stellen Sie die Kamera ~ 20 cm von dem Probentisch Fokussierung zu ermöglichen. </li><li> Zu bestimmten Zeiten, legen Sie die Proben aus Schritt 3.1.3 zwischen dem polarizers auf der Bühne parallel zu der XZ-Ebene und die Vorrichtung mit einem schwarzen Vorhang bedecken; die eigentliche Vorrichtung ist in 1C gezeigt. </li><li> Fotografieren Sie die Proben durch lineare gekreuzten Polarisatoren eine digitale Spiegelreflexkamera mit einem Standard-Zoomobjektiv (siehe Materialliste). Steuern Sie die Kamera-Einstellungen, wie Brennweite, Computer-Software (siehe Materialliste). </li></ol></li><li> Räumlich-zeitliche Analyse der durchgelassenen Lichtintensität (1C) <ol><li> Um die Änderung des Orientierungsordnungsparameters in dem Trocknungsprozess zu bewerten, sammeln Fotografien stündlich für 24 h. </li><li> Messen Sie die übertragene Lichtintensität entlang der Mittellinie in der Z-Richtung als Grauwert eines Bildverarbeitungsprogramms unter Verwendung von (zB ImageJ). </li><li> Eine Grafik mit dem Grauwert in Abhängigkeit von dem Abstand von der oberen offenen Seite. </li></ol></li><li> Mikroskopische Beobachtungen unter kreuzpolarisierten light (1D) <ol><li> Um die Methoden zu überprüfen, machen mikroskopische Beobachtungen mit einem Polarisationsmikroskop mit einer CCD - Kamera ausgestattet 9. Halten erster Ordnung Verzögerungsplatte im Strahlengang. Kontrollieren Sie die Bedingungen für die Fotos eine PC-Software. </li></ol></li></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>

Die Autoren haben nichts zu offenbaren.

Es war manchmal schwierig, für die Kamera auf der Probe zu konzentrieren, aufgrund der durchgelassenen Lichtintensität zu gering zu sein. In solchen Fällen auf der Bühne eine erweiterte transparenten Kunststoffilm Platzieren half, den Fokus zu arrangieren. Die Begrenzung der beobachtbaren Auflösung war abhängig von der Kameralinse, ~ 10 &amp; mgr; m in diesem Fall. Die beobachtbare Begrenzung der Probendicke, &amp; Delta; y, waren abhängig von der maximalen Lichtintensität der Lampe, ~ 10 mm in diesem Fall. ...

Durch die Fokussierung auf den starren, stabförmigen Strukturen von Biopolymeren, dynamisch weichen Materialien wurden für verschiedene Anwendungen, einschließlich Polysaccharids Biofilm Matrices 1 „aktiven Gele“ bestehend aus Zytoskelett - Proteinen 2 und „DNA origami“ gewünschten Formen 3 verwendet. Um zu klären, haben die strukturellen Eigenschaften, viele Strategien wurden untersucht, wie die Transmissionselektronenmikroskopie, Rasterelektronenmikroskopie, Rasterkraftmikroskopie und konfokale Fluoreszenzmikroskopie. Da jedoch diese Methoden meist in einem ausgetrockneten oder statischen Zustand durchgeführt werden, ist es schwierig, die dynamischen Verhalten in der makroskopischen Ebene zu erklären, wie es in der tatsächlichen lebenden Systemen gesehen. Vor kurzem haben wir beobachtet , erfolgreich das dynamische Verhalten von Biopolymeren auf die wässrige Luft-LC - Schnittstelle durch polarisiertes Licht 4. Bei der Visualisierung der Struktur ausgerichtet, während das Trocknen des BiopolymersLösung zeigten die zeitlichen Veränderungen Selbst Integration von Biopolymeren auf der instabilen Luft-LC-Schnittstelle. Hier beschreiben wir ein Protokoll für die Trocknung von LC-Biopolymer-Lösungen an der Luft-LC-Schnittstelle unter Verwendung von polarisiertem Instrumenten. Durch Auswertung des Orientierungsordnungsparameters in der Seitenansicht der Fluidphase in einer offenen einseitiges-Zelle im Gegensatz Phase auf andere Analysen der LC , dass 5 nicht die Ansicht , Trocknen, 6 wurde die LC Dynamik während des Trocknungsvorganges hier untersucht . Die Kombination der Zelle der Verdampfung und die Verwendung von polarisiertem Instrumenten zur makroskopischen Überwachung mit einer kontrollierten Verdampfungsrichtung erlaubt. Darüber hinaus war es möglich , die Trocknungs Aufzeichnungen durch die Konzentration auf den kristallinen Strukturen der adsorbierten Mikrodomänen zu validieren, die durch das Molekulargewicht beeinflußt wurde, Konzentration, usw. Um die Wirksamkeit des Verfahrens wird die Trocknung Pro zu demonstrierenzesse basischer Biopolymeren mit starren Stabformen, wie Polysaccharide, Mikrotubuli (MTs), und DNA, wurden untersucht. Wir haben uns für diese Biopolymere, weil sie typische Beispiele für hierarchische Makromoleküle mit megamolecular Gewichte sind, und ihre intermolekularen Wechselwirkungen ermöglichen ihnen LC Staaten zu bilden.

<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

Lebende Organismen, die Wasser verwenden, sind immer anfällig in der Umwelt zu trocknen. Ihre Aktivitäten werden angetrieben von Biopolymer-basierten Mikro- und Makrostrukturen, wie in den Fällen gesehen Wasser in Leitbündel bewegten und feuchtigkeitsspendende Wasser in Hautschichten. In dieser Studie haben wir ein Verfahren zur Bewertung der Wirkung von wässrigem flüssigkristallinen (LC) Lösungen von Biopolymeren beim Trocknen zusammen. Als LC Biopolymere megamolecular Gewicht haben, wählten wir Polysaccharide, Cytoskelett-Proteine ​​und DNA zu untersuchen. Die Beobachtung von Biopolymer-Lösungen während zeigt aus der instabilen Luft-LC-Schnittstelle beginnend milliscale Selbstintegration unter polarisiertem Licht trocknet. Die Dynamik der wässrigen LC-Biopolymer-Lösungen kann durch Verdampfen von Wasser aus einer einseitig offenen Zellen überwacht werden. Durch die Analyse der Bilder kreuzpolarisiertem Licht genommen, ist es möglich , die räumlich-zeitlichen Veränderungen des Orientierungsordnungsparameter zu erkennen. DiesVerfahren für die Charakterisierung von nicht nur künstlichen Materialien in verschiedenen Bereichen, aber auch natürliches Leben Geweben nützlich sein. Wir glauben, dass es ein Bewertungsverfahren für weiche Materialien in der biomedizinischen und Umweltbereich bieten.

<html> Verwendung der Vorrichtung wie in 1 gezeigt ist , um die Selbst Integration von Mikrodomäne macrodomain auf einer trocknenden Luftschnittstelle-LC bewertet wurde (2A). Da die erste Demonstration des Trocknungs Experiment wurden zwei Arten von megamolecular Polysacchariden, sacran (M w = 1,9 x 10 7 g mol -1) und Xanthangummi (4,7 x 10 6 g mol -1), wurden verglichen. 2B...

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

Ein Verfahren zur Trocknung induzierte Selbst Integration von megamolecular Biopolymeren auf der Luft-Flüssigkeit-kristalline Grenzfläche ist vorgesehen. Diese Methodik wird nützlich sein, nicht nur für das Verständnis der makroskopischen Potentiale von Biopolymeren, sondern auch als ein Bewertungsverfahren für weiche Materialien in der biomedizinischen und Umwelt.

Methoden zur Selbst Integration von Megamolecular Biopolymere auf der Trocknungsluft-LC-Schnittstelle

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 Aufrufe.  Japan Advanced Institute of Science and Technology.  Ein Verfahren zur Trocknung induzierte Selbst Integration von megamolecular Biopolymeren auf der Luft-Flüssigkeit-kristalline Grenzfläche ist vorgesehen. Diese Methodik wird nützlich sein, nicht nur für das Verständnis der makroskopischen Potentiale von Biopolymeren, sondern auch als ein Bewertungsverfahren für weiche Materialien in der biomedizinischen und Umwelt. 

Serie: 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.

Synthese von Poly (<em&gt; N</em&gt; Isopropylacrylamid) Janus Microhydrogels für anisotroper Thermo-Ansprechempfindlichkeit und organophiler / Hydrophilie Belastbarkeit

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

Forschung

Chemie

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.

Orientationaler Übergang in einem Flüssigkristall, der durch das thermodynamische Wachstum von Grenzflächenbetonplatten ausgelöst wird

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

Ingenieurwesen

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.

Grenzflächenphysikalische Strukturen von Polymeren und Biomakromolekülen, die über sum frequency generation Vibrational Spectroscopy aufgedeckt werden

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

Biochemie

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.

Vorbereitung der Flüssigkristall-Netzwerke für makroskopische oszillierende Bewegung, die durch Licht induzierten

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.

Mikrofluidische Vorbereitung der flüssigen kristallenen Elastomer Aktoren

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

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.

Automatisierte Lipid Bilayer Membrane Formation eines Polydimethylsiloxans Dünnschicht unter Verwendung

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

Biotechnik

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.

Selbstmontage von Hybrid-Lipidmembranen, dotiert mit hydrophoben organischen Molekülen an der Wasser-Luft-Schnittstelle

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.

Kristallisation von Proteinen auf Chip durch Mikrodialyse für In Situ-Röntgenbeugungsstudien

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

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Einzelmoleküldiffusion und Assemblierung auf polymerüberfüllten Lipidmembranen

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

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.

Die Kristallisation von Membranproteinen in Lipidische Mesophasen

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