D.C.H., M.B. and F.K. were supported by an EPSRC Follow on Fund. B.L-V and D.S. were each supported by MRC PhD studentships. K.C. was supported by funding from the UK Regenerative Medicine Platform.

1. Synthèse de PHNGAD (poly [1,6-hexanodiol / néopentylglycol / di (éthylène glycol), de l&#39;acide adipique--ALt] diol) <img alt="Schéma 1" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme1.jpg" width="500" /> Schéma 1: Synthèse de PHNAGD représentation schématique de la synthèse de PHNAGD.. PHNAGD a été préparé par la réaction de 1,6-Hexanodiol, le diéthylène glycol, neoppentyl glycol et d&#39;acide adipique. PHNAGD, Poly [1,6-hexanodiol / néopentylglycol / di (éthylène glycol), l&#39;acide adipique--ALt] diol. <ol><li> Appliquer un traitement thermique à des monomères de 1,6-hexanediol, le di (éthylène glycol) et du néopentylglycol à 40 ° C pendant 48 heures dans une étuve à vide pour éliminer l&#39;eau résiduelle. Laisser froid jusqu&#39;à température ambiante sous vide. </li><li> Ajouter 22 mmol de chaque monomère et d&#39;acide adipique (55 mmol) dans un ballon à fond rond à deux cols équipé d&#39;un barreau agité et raccordé à un appareil de Dean-Stark. </li><li> Placez tout l&#39;ensemble sous vide et chauffer doucement la glassware à 40 ° C pendant 6 heures, afin d&#39;éviter toute absorption d&#39;humidité au cours de l&#39;addition de la substance chimique dans le ballon. </li><li> Ajouter à la seringue, goutte à goutte, 0,0055 mole de titane de catalyseur (IV) de butoxyde. </li><li> On agite le mélange réactionnel à 180 ° C, sous une atmosphère de N2 pendant 24 heures, et recueillir l&#39;eau résiduelle dans le piège de Dean-Stark. Laissez le produit refroidir à température ambiante. </li></ol> Synthèse de 2 PU134 <img alt="Schéma 2" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme2.jpg" width="500" /> Schéma 2: Synthèse de PU134 représentation schématique de la synthèse de polyuréthanne PU134 134 a été préparé par la réaction de 1,0 équivalent d&#39;un PHNGAD avec 2,0 équivalents d&#39;un 4,4 &#39;-méthylène bis (isocyanate de phényle), suivi par l&#39;addition de 1,0. équivalents d&#39;un agent d&#39;allongement de chaîne 1,4-butanediol. <ol><li> Mélanger un équivalent de la PHNGAD polyol (Mn ~ 1800 Da, 3,2 mmol) avec deux équivalents de 4»4-méthylène-bis (isocyanate de phényle) (6,4 mmol) dans anhydre N, N-diméthylformamide (12 ml). </li><li> On agite le mélange réactionnel à 70 ° C, sous une atmosphère de N2. </li><li> Ajouter à la seringue, goutte à goutte le titane de catalyseur (IV) butoxyde (0,8% en poids). </li><li> Après 1 heure, ajouter un équivalent de l&#39;allongeur de chaîne 1,4-butanediol (3,2 mmole). Augmenter la température à 90 ° C et agiter pendant 24 heures sous une atmosphère de N 2. </li><li> Après la réaction, collecter le polyuréthanne par précipitation par addition d&#39;éther diéthylique (hexane ou de l&#39;eau peut aussi être utilisé) goutte à goutte dans la solution de réaction jusqu&#39;à ce que la précipitation se produit. </li><li> Centrifuger la solution à 5300 g pendant 5 min. </li><li> Décanter le surnageant et sécher à 40 ° C dans une étuve à vide jusqu&#39;à ce que le solvant s&#39;évapore. Remarque: Afin de garantir que le produit final de la réaction possède les bons paramètres en ce qui concerne la distribution de la masse moléculaire, le grou fonctionnelps du polymère, et les températures de fusion et de transition vitreuse, diverses techniques analytiques et les méthodes peuvent être utilisées, telles que la Chromatographie de permeation sur gel ou par spectroscopie FITR. </li></ol> 3 Préparation des solutions de PU134 <ol><li> Peser 200 mg de PU134 dans une bouteille en verre. </li><li> Diluer PU134 à une concentration finale de 2% dans un certain nombre de solvants: chloroforme, un mélange de chloroforme et de toluène à 1: 1, le tétrahydrofuranne, et la combinaison de tétrahydrofurane et de dichlorométhane dans le rapport 1: 1. Remarque: L&#39;élection du droit solvant varie en fonction du polymère à utiliser. Différents solvants possèdent des points d&#39;ébullition différents qui peuvent influer sur la solubilisation du polymère. </li><li> Agiter la solution vigoureusement pendant 20 min à température ambiante en utilisant un agitateur à une vitesse de 200 MOT / min, jusqu&#39;à ce que la solution devienne homogène et aucun précipité n&#39;est observé. </li></ol> 4. revêtement de lames de verre avec PU134 <ol><li> Placez un roue 15 mm 2 lamelle sur la tournette. </li><li> Appliquer 50 ul de la solution de chaque PU134 à l&#39;aide d&#39;une pipette. Ajuster le volume de la solution de PU134 en conséquence de la taille de la lamelle couvre-objet en maintenant le volume nécessaire à la surface rapport proportionnel. </li><li> Faites tourner chaque lamelle pendant 7 secondes à 23 x g. </li><li> Lamelle air sec à température ambiante pendant au moins 24 heures avant la stérilisation. </li></ol> 5. irradiation de Coverslip <ol><li> Gamma-irradier polymère lamelle revêtue par application d&#39;une dose de 10 Grays l&#39;aide d&#39;un irradiateur de laboratoire pendant 12 min. </li><li> UV irradier revêtu de polymère en utilisant une lamelle 30 W, lampe UV pour 16 min de chaque côté. </li><li> Placer la lamelle revêtue de polymère dans une plaque de culture de tissu approprié en fonction de la taille de la diapositive. </li></ol> 6. microscopie électronique à balayage <ol><li> Or polymère manteau lamelle par pulvérisation pour 200 secondes dans une atmosphère de 5 x 10 -1 millibars de pression. </li><li> Capturez le micrographies de la lamelle couvre-objet revêtu de polymère en utilisant un microscope électronique à balayage à une tension d&#39;accélération de 20 kV en mode de formation d&#39;image d&#39;électrons secondaires. </li></ol> 7. microscopie à force atomique Observations <ol><li> Balayer une zone de 20 x 20 um de la surface du polymère. </li><li> Mettre en place une vitesse de balayage de 1,32 Hz à 1,60 Hz. </li><li> Mettre en place une résolution de 512 x 512 pixels dans la région balayée. </li><li> Calculer la moyenne quadratique (RMS ou Rq) du revêtement à l&#39;aide de la moyenne des écarts de hauteur à partir de prises au plan moyen de données d&#39;image exprimée en tant que: <img fo:content-width="1in" src="/files/ftp_upload/51723/51723eq1.jpg" width="123" /> Où Zi est la valeur courante de Z, et N est le nombre de points à l&#39;intérieur de la zone donnée. </li><li> 7.5 Calculer l&#39;écart ou rugosité de surface moyenne (Ra) de l&#39;image en utilisant, <img fo:content-width="1.5in" src="/files/ftp_upload/51723/51723eq2.jpg" width="154" /> Où Z (x) est la fonction qui décrit til analyse le profil de surface en termes de hauteur (Z) et la position (x) de l&#39;échantillon sur la longueur d&#39;évaluation de &quot;L&quot;. Ra représente la valeur moyenne de la surface par rapport au plan médian. </li></ol> 8. culture cellulaire et de la différenciation <ol><li> Culture et différencier la lignée humaine de cellules souches embryonnaires (CSEh) H9 comme décrit dans Hay 17. </li><li> Détacher les cellules en utilisant un réactif de dissociation et les Réensemencement sur ​​des lames revêtues PU134 à 9 jours du processus de différenciation, en présence d&#39;un milieu sans sérum comme décrit dans Szkolnicka 18,19. Remarque: L&#39;utilisation d&#39;une dissociation enzymatique des cellules est préférable de détachement de cellule physique. </li></ol> 9. cytochrome P450 de dosage fonctionnel <ol><li> Mesurer l&#39;activité du CYP3A suivant les instructions du fabricant. </li><li> Incuber CSEh hépatocytes dérivés au jour 13 ou 19 jours, et les médias sans cellules - comme contrôle négatif, avec le substr appropriémangé pendant 5 heures à 37 ° C. </li><li> Recueillir les surnageants de cellules et les médias, effectuer l&#39;essai selon les instructions du fabricant. </li><li> Mesurer le niveau de l&#39;activité de CYP et normalisée par rapport à la zone de surface (cm 2). </li></ol> 10 Immunocoloration <ol><li> Laver CSEh hépatocytes dérivé avec du PBS, pendant 1 min, répéter deux fois. </li><li> Ajouter glacée 100% de méthanol pour fixer les cellules, dans -20 ° C pendant 10 min. </li><li> Laver les cellules avec du PBS pendant 5 min et répéter deux fois. </li><li> Incuber les cellules avec du PBS / T (0,1% de Tween) / 10% de BSA pendant 1 heure à température ambiante. </li><li> Aspirer la solution de PBST et ajouter l&#39;anticorps primaire approprié dilué dans PBS / T (0,1% de Tween) / BSA à 1%, incubation à 4 ° C avec agitation douce O / N. </li><li> Laver les cellules avec du PBS / T (0,1% de Tween) / BSA à 1% pendant 5 minutes et répéter trois fois. </li><li> Diluer l&#39;anticorps approprié dans du PBS / T (0,1% de Tween) / 1% de BSA, ajouter aux cellules et incuber dans l&#39;obscurité à température ambiante pendant 1 h sous agitation douce. </li><li> Laver les cellules avec du PBS pendant 5 min et répéter trois fois. </li><li> Ajouter MOWIOL 488 (contenant DAPI 1: 1,000) à chaque puits, ajouter une lamelle doucement pour réduire le nombre de bulles d&#39;air. Magasin cellules fixé à 4 ° C dans l&#39;obscurité. Observer la coloration à l&#39;aide d&#39;un microscope avec filtre approprié et d&#39;une lampe fluorescente. Les anticorps primaires et secondaires optimisés sont répertoriés dans le tableau 1. </li></ol>

<ol>
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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV+and+gamma+irradiation+effects+on+surface+properties+of+polyurethane+derivate+from+castor+oil.">UV and gamma irradiation effects on surface properties of polyurethane derivate from castor oil.</a> Pol&#237;meros. 23 (3), 305-311 (2013).</li><li>Rosu, L., Cascaval, C. N., Ciobanu, C., Rosu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+UV+radiation+on+the+semi-interpenetrating+polymer+networks+based+on+polyurethane+and+epoxy+maleate+of+bisphenol+A.">Effect of UV radiation on the semi-interpenetrating polymer networks based on polyurethane and epoxy maleate of bisphenol A.</a> Journal of Photochemistry and Photobilogy A: Chemistry. 169 (2), 177-185 (2005).</li><li>Yang, X. F., Tallman, D. E., Bierwagen, G. P., Croll, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blistering+and+degradation+of+polyurethane+coatings+under+different+accelerated+weathering+tests.">Blistering and degradation of polyurethane coatings under different accelerated weathering tests.</a> Polymer Degradation and Stability. 77 (1), 103-109 (2002).</li></ol>

D.C.H. is CSO, Director, founder and a shareholder in FibromEd Products Ltd. M.B. and J.P.I. are founder shareholders in FibromEd Products Ltd.

Many of the current methods used to generate hepatocytes from stem cells rely on undefined matrices of animal origin. These substrates can be costly and highly variable, affecting cell function and stability, representing a significant barrier to application. Therefore, we performed a screen for synthetic materials which support the culture of stem cell derived hepatocytes. We have identified, a simple polyurethane (PU134), formed by polymerizing PHNGAD, MDI and an extender, that in combination with a robust hepatocyte d...

Les matières biologiques ont été largement utilisés dans l&#39;entretien et la différenciation des cellules souches pluripotentes 1. Tout en permettant, ces substrats biologiques contiennent souvent une multitude de composants définis. Matrigel est un substrat communément utilisé pour la culture de cellules souches et de la différenciation. Malheureusement, sa composition variable influe sur la fonction des cellules et le phénotype. Bien qu&#39;une grande variété de matrices biologiques alternatives, mieux définis ont été utilisés 2-7, leur origine animale ou pauvres évolutivité en fait des candidats inappropriés pour la production industrielle. Par conséquent, l&#39;identification des alternatives synthétiques, à composition définie et des performances fiables, sont des objectifs clés de la recherche sur les cellules souches. Dans une tentative pour surmonter les limitations des indéfinis substrats de culture de cellules, des collaborations interdisciplinaires entre la chimie et de la biologie ont identifié des matériaux synthétiques ayant la capacité de supporter le phénotype cellulaire. Synthsubstrats étiques sont évolutives, rentables, et peuvent être fabriqués dans des structures 3D complexes, imitant l&#39;environnement in vivo. En raison de ces propriétés des substrats synthétiques ont été largement utilisés pour supporter et entraîner la différenciation de nombreux types de cellules 8-10. Essais de débit de pointe et haute ont facilité la sélection rapide de matériaux synthétiques, de grandes bibliothèques, et livré de nouveaux matériaux aux propriétés flexibles avec de larges applications dans la recherche et le développement 11-13 biomédicale. Utilisant à haut débit, la technologie de dépistage micro-réseau polymère, nous avons identifié rapidement d&#39;une simple polyuréthane (PU134), adapté pour l&#39;entretien des hépatocytes dérivés de cellules souches humaines. Ce polymère a été jugée supérieure à des substrats provenant des animaux en ce qui concerne la différenciation et la fonction des hépatocytes 14-16. Nous avons par la suite optimisé le processus conditions de revêtement, de la topographie et de stérilisation pour accéder à effetssur le rendement en polymère stabilisant fonction des hépatocytes et la durée de vie. Ceci a des implications importantes en ce qui concerne la compréhension des fondamentaux de la biologie des hépatocytes pour la modélisation à base de cellules et des applications en médecine régénérative. La technologie décrite ici est un exemple de la façon dont la surface d&#39;un polymère synthétique peut être optimisée pour conserver le phénotype cellulaire. Nous croyons que la combinaison de cette technologie avec un hépatocyte libre protocole de différenciation de sérum efficace a le potentiel de fournir une production évolutive des hépatocytes pour l&#39;utilisation de la modélisation in vitro et la médecine régénérative.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Synthesis, preparation, coating and characterization of polymer PU134 coated coverslips</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Edmun B&#252;hler</td>
			<td>KS-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Irradiator</td>
			<td>CIS Biointernational</td>
			<td>IBL 637&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Specialty Coating System</td>
			<td>&#160;P-6708</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope&#160;</td>
			<td>Philips</td>
			<td>XL30CPSEM</td>
			<td></td>
		</tr>
		<tr>
			<td>Atomic Force Microscope</td>
			<td>DimensionV Nanoscope, VEECO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p4-GLO CYP3A4</td>
			<td>Promega</td>
			<td>V8902</td>
			<td></td>
		</tr>
		<tr>
			<td>UV bulb</td>
			<td>ESCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoScope analysis software</td>
			<td>VEECO</td>
			<td></td>
			<td>version 1.20</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Olympus</td>
			<td>TH45200</td>
			<td>Use Volocity 4 Software</td>
		</tr>
		<tr>
			<td>Tissue culture plates</td>
			<td>Corning, UK&#160;</td>
			<td>3527</td>
			<td></td>
		</tr>
		<tr>
			<td>glass slides</td>
			<td>Scientific Laboratory Supplies</td>
			<td>MIC3308</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethylene glycol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>93171</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;1,6-hexanediol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>240117</td>
			<td></td>
		</tr>
		<tr>
			<td>Neopentyl glycol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>408255</td>
			<td></td>
		</tr>
		<tr>
			<td>Adipic acid</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>9582</td>
			<td></td>
		</tr>
		<tr>
			<td>anhydrous N,N-Dimethylformamide</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>227056</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>676845</td>
			<td></td>
		</tr>
		<tr>
			<td>titanium (IV) butoxide&#160;</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>244112</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-butanediol&#160;</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>493732</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum oven</td>
			<td>Thermoscientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4,4&#8217;-Methylenebis(phenyl isocyanate)</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>101688</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrahydrofurane</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>401757</td>
			<td></td>
		</tr>
		<tr>
			<td>Sputter coater</td>
			<td>Bal-Tec SCD 050</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Inmunostaining</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline (-MgCl2, -CaCl2)</td>
			<td>Gibco</td>
			<td>10010031</td>
			<td>&#160;Store at room temperature</td>
		</tr>
		<tr>
			<td>PBST, PBS made up with 0.1% TWEEN 20&#160;</td>
			<td>&#160; Scientific Laboratory Supplies Ltd</td>
			<td>EC607&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>&#160; Scientific Laboratory Supplies Ltd</td>
			<td>CHE5010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich, UK</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>MOWIOL 488 DAPI</td>
			<td>Calbiochem</td>
			<td>475904</td>
			<td>Made up in Tris HCL and glycerol as per manufacturers instructions</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cell culture and Functional assay</td>
		</tr>
		<tr>
			<td>CYP3A activity pGLO kit</td>
			<td>Promega</td>
			<td>V8902</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepatozyme</td>
			<td>Gibco</td>
			<td>17705021</td>
			<td></td>
		</tr>
		<tr>
			<td>TryLE express</td>
			<td>Life Technologies</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/12604013">12604013</a></td>
			<td></td>
		</tr>
	</tbody>
</table>

stabilizing hepatocellular phenotype using optimized synthetic surfaces

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.

Polymer solvent influences the topography of the polymer coated surface
Polyurethane 134 was solubilized in chloroform, either alone or in combination with toluene or tetrahydrofuran or dichloromethane and the glass slides spin-coated with the different formulations. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to characterize the physical properties of the polymer coatings (Figure 1). The coating obtained using toluene or chl...

Watch this Scientific Journal Video about Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces at JoVE.com

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

This article will focus on developing polymer coated surfaces for long-term, stable culture of stem cell derived human hepatocytes.

Stabiliser hépatocellulaire phénotype en utilisant des surfaces synthétiques optimisés

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing ...

stabilizing-hepatocellular-phenotype-using-optimized-synthetic

Research

JoVE Journal

Chemistry

10.2K vues.  University of Edinburgh. This article will focus on developing polymer coated surfaces for long-term, stable culture of stem cell derived human hepatocytes.

Vidéo: Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

Liaison covalente de BMP-2 sur les surfaces en utilisant une approche monocouche auto-assemblée

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Recherche

Chimie

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.

The objective of this research was to form synthetic plant cell wall tissue using layer-by-layer assembly of nanocellulose fibrils and isolated lignin assembled from dilute aqueous suspensions.&#160; Surface measurement techniques of quartz crystal microbalance and atomic force microscopy were used to monitor the formation of the polymer-polymer nanocomposite material.

Vers Biomimicking Bois: Films Fabrication libre-debout de nanocellulose, lignine, et un polycation synthétique

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and ...

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3]2+ (bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of novel materials. Coupled with analysis by in situ secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS), soft landing provides unprecedented insights into the interactions of well-defined species with surfaces.

In Situ SIMS et spectroscopie IR des surfaces bien définies Préparé par Soft Landing de Ions Mass-sélectionnés

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Méthodologie synthétique pour asymétrique Ferrocene dérivés Systems Bio-conjugués via solide méthodologie à base de résine de phase

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional units (e.g., domains). Traditional consensus-building models fail to account for interpositional dependencies &#8211; functionally required covariation of residues that tend to appear simultaneously throughout evolution and across the phylogentic tree. These relationships can reveal important clues about the processes of protein folding, thermostability, and the formation of functional sites, which in turn can be used to inform the engineering of synthetic proteins. Unfortunately, these relationships essentially form sub-motifs which cannot be predicted by simple &#8220;majority rule&#8221; or even HMM-based consensus models, and the result can be a biologically invalid &#8220;consensus&#8221; which is not only never seen in nature but is less viable than any extant protein. We have developed a visual analytics tool, StickWRLD, which creates an interactive 3D representation of a protein alignment and clearly displays covarying residues. The user has the ability to pan and zoom, as well as dynamically change the statistical threshold underlying the identification of covariants. StickWRLD has previously been successfully used to identify functionally-required covarying residues in proteins such as Adenylate Kinase and in DNA sequences such as endonuclease target sites.

Synthetic protein sequences based on consensus motifs typically ignore co-evolving residues, that imply interpositional dependencies (IPDs). IPDs can be essential to activity, and designs that disregard them may result in suboptimal results. This protocol uses StickWRLD to identify IPDs and help inform rational protein design, resulting in more efficient results.

Optimisation des protéines synthétiques: Identification des dépendances interposition indicatrice constructivement et / ou les résidus fonctionnellement liées

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

Un protocole optimisé pour le radiomarquage efficace de nanoparticules d&#39;or à l&#39;aide d&#39;un<sup&gt; 125</sup&gt; I-étiqueté Azide prothétique Groupe

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Color Spot Test As a Presumptive Tool for the Rapid Detection of Synthetic Cathinones

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and other border protection agencies globally. Color testing is a presumptive identification technique indicating the presence or absence of a particular drug class using rapid and uncomplicated chemical methods. Owing to their relatively recent emergence, a color test for the specific identification of synthetic cathinones is not currently available. In this study, we introduce a protocol for the presumptive identification of synthetic cathinones, employing three aqueous reagent solutions: copper(II) nitrate, 2,9-dimethyl-1,10-phenanthroline (neocuproine) and sodium acetate. Small pin-head sized amounts (approximately 0.1-0.2 mg) of the suspected drugs are added to the wells of a porcelain spot plate, and each reagent is then added dropwise sequentially before heating on a hotplate. A color change from very light blue to yellow-orange after 10 min indicates the likely presence of synthetic cathinones. The highly stable and specific test reagent has the potential for use in the presumptive screening of unknown samples for synthetic cathinones in a forensic laboratory. However, the nuisance of an added heating step for the color change result limits the test to laboratory application and decreases the likelihood of an easy translation to field testing.

Here we present a simple, inexpensive, and selective chemical spot test protocol for the detection of synthetic cathinones, a class of new psychoactive substances. The protocol is suitable for use in various areas of law enforcement that encounter illicit material.

Couleur Test ponctuel comme un outil présomptif pour la détection rapide des Cathinones de synthèse

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

Caractérisation des polymères synthétiques via Matrix Assisted Laser Desorption ionisation temps de spectrométrie de masse (MALDI-TOF) de vol

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

Light-driven Molecular Motors on Surfaces for Single Molecular Imaging

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces are demonstrated. This work requires careful design, considerable synthetic effort, and proper analysis. The rotary motion of the molecular motor in solution is shown by 1H NMR and UV-vis absorption spectroscopy techniques. In addition, the method to graft the motor onto an amine-coated quartz is described. This method helps to gain more insight into molecular machines.

The manuscript describes how to synthesize and graft a molecular motor on surfaces for single molecular imaging.

Moteurs moléculaires axée sur la lumière sur les Surfaces pour l’imagerie moléculaire unique

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces ...

A Facile Synthetic Method to Obtain Bismuth Oxyiodide Microspheres Highly Functional for the Photocatalytic Processes of Water Depuration

Bismuth oxyhalide (BiOI) is a promising material for sunlight-driven-environmental photocatalysis. Given that the physical structure of this kind of materials is highly related to its photocatalytic performance, it is necessary to standardize the synthetic methods in order to obtain the most functional architectures and, thus, the highest photocatalytic efficiency. Here, we report a reliable route to obtain BiOI microspheres via the solvothermal process, using Bi(NO3)3 and potassium iodide (KI) as precursors, and ethylene glycol as a template. The synthesis is standardized in a 150 mL autoclave, at 126 &#176;C for 18 h. This results in 2-3 &#181;m-sized mesoporous microspheres, with a relevant specific surface area (61.3 m2/g). Shortening the reaction times in the synthesis results in amorphous structures, while higher temperatures lead to a slight increase in the porosity of the microspheres, with no effect in the photocatalytic performance. The materials are photo-active under UV-A/visible light irradiation for the degradation of the antibiotic ciprofloxacin in water. This method has demonstrated to be effective in interlaboratory tests, obtaining similar BiOI microspheres in Mexican and Chilean research groups.

This article describes a synthetic method to obtain bismuth oxyiodide microspheres, which are highly functional to perform the photocatalytic removal of organic pollutants, such as ciprofloxacin, in water under UV-A/visible light irradiation.

Une méthode de synthèse Facile d’obtenir Bismuth Oxyiodide microsphères très fonctionnel pour les procédés photocatalytiques de dépuration de l’eau

Bismuth oxyhalide (BiOI) is a promising material for sunlight-driven-environmental photocatalysis. Given that the physical structure of this kind of ...