Ce travail est financé en partie par l&#39;Institut national de l&#39;USDA pour l&#39;alimentation et l&#39;agriculture, Programme national de l&#39;octroi des subventions, Programme national de l&#39;Initiative de recherche 71,1 exercice 2007, Grant n ° 2007-35503-18392, et les instituts nationaux de la santé, Institut pour l&#39;allergie et des maladies infectieuses , R33AI94514-03.

1. Dope Spinning Préparation <ol><li> Préparez une gamme de concentrations de biopolymères à étudier (0,1% à 30%, p / v) et assurez-vous de considérer la teneur en humidité de la poudre de biopolymère dans ces calculs. Pour chaque concentration, peser le biopolymère (amidon ou le pullulane) en poudre dans un tube à essai de 50 ml. Ajouter aqueuse de diméthylsulfoxyde (DMSO) solution et une barre d&#39;agitation. </li><li> Placer le tube dans l&#39;eau bouillante avec une agitation constante sur un agitateur magnétique chauffant. </li><li> Après environ 1 h, éteindre le feu et laisser refroidir la dispersion à la température ambiante. La dispersion est alors prêt pour les essais rhéologiques et électrofilage. </li></ol> 2. Steady cisaillement Rhéologie <ol><li> Faire chauffer le rhéomètre et régler la température de l&#39;étape à 20 ° C. Calibrer l&#39;écart entre le (mm cône 25 utilisé) sonde et la scène (plaque). </li><li> Charge 0,41 ml de la dispersion de biopolymère sur le centre de la scène et descendre la sonde pour régler la positisur (écart de 0,053 mm pour 25 mm cône). Assurez-vous que la dispersion s&#39;étend uniformément dans l&#39;intervalle. </li><li> Effectuer le test rhéologique avec les paramètres expérimentaux suivants: mode de balayage: log, le taux initial: 100 sec -1, le taux final: 0,1 sec -1, les points par décennie: 10, délai avant mesure: 5 secondes, mesurer le temps: 10 sec, et les directions par mesure: deux (deux dans le sens horaire et anti-horaire). </li><li> Analyser les données rhéologiques pour estimer les concentrations de dispersion appropriées pour électrofilature essais. <ol><li> Tracer les viscosités de cisaillement apparente par rapport aux taux de cisaillement en fonction de la concentration de polymère. Pour chaque courbe de débit, approximatives viscosités de cisaillement zéro, η 0, par les valeurs réelles ou extrapolées (par exemple, à de faibles concentrations où les données à faible taux de cisaillement ne sont pas fiables) pour la viscosité apparente à 0,1 sec - 1. </li><li> Calculer viscosité spécifique: η sp = (η 0 - η s)/ η s, où η s est la viscosité du solvant. </li><li> Tracer les viscosités spécifiques en fonction de la concentration. Identifier les régimes non enchevêtrées et entremêlées de semi-dilution. Le régime de semi-dilution non enchevêtrées commence à partir de l&#39;extrémité inférieure de concentration avec une petite pente, et le régime de semi-dilution empêtré a une plus grande pente sur le régime non enchevêtrées. Modèles en loi de puissance régression Fit dans les deux régimes. Les valeurs de puissance sont les pistes (de dépendance de concentration) dans les régimes non enchevêtrées et entremêlées de semi-dilution sur un terrain log-log. L&#39;intersection des deux lignes ajustées est la concentration d&#39;enchevêtrement, c e. </li></ol></li></ol> 3. Électrofilage paramètre Variation <ol><li> Assembler l&#39;installation d&#39;électrofilage, comme illustré sur la figure 1. Charger la seringue avec la composition de dispersion approprié, par exemple, 15% (p / v) de l&#39;amidon ou du pullulane dans 100% de DMSO, sur la pompe à seringue. Fixez le fil à haute tension (positive) à l&#39;aiguille. Connecter le bain de coagulation contenant de l&#39;éthanol pur à la masse en immergeant le fil de terre (négative) dans le bain. Utilisation d&#39;un vérin de laboratoire pour régler la distance entre l&#39;aiguille de seringue et le bain de coagulation. Immerger un treillis métallique dans le bain pour recueillir le mat de fibres après l&#39;électrofilage. </li><li> Faites tourner la biopolymère dans le paramètre suivant varie: débit d&#39;alimentation de 0,1 à 0,4 ml / h, la distance filature de 5 à 10 cm, et la tension de 0 à 15 kV. <ol><li> Commencer avec une distance de filage de 5 cm. Pour la première vitesse d&#39;alimentation (0,1 ml / h), la rampe de la tension lentement de 0 V. Faites attention à la forme de la dispersion extrudé à la pointe de l&#39;aiguille et noter lorsque la dispersion de gouttes est accéléré et allongée. </li><li> Notez la tension à laquelle un petit jet lancé à partir de la surface de la goutte, ce qui indique electrospinnability de la solution. Enregistrer la tension à laquelle un jet continu entame, le cas échéant. </li><li> Examinez la gamme complète pour chacun des trois parameters et notes conditions de filage succès. Recueillir les fibres seulement quand il ya un jet continu de la pointe. </li></ol></li><li> Après quelques minutes de collecte, de rincer le matelas de fibres avec de l&#39;éthanol pur. Placer le matelas de fibres dans un dessiccateur contenant un agent déshydratant sous vide. </li><li> Répétez l&#39;opération pour chaque concentration de biopolymère pour la caractérisation complète. </li></ol><img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51933/51933fig1highres.jpg" width="500" /> Figure 1: Représentation schématique de la configuration de l&#39;électro-filage au mouillé. Dispersion de biopolymère est extrudé à partir d&#39;une pompe à seringue. Un courant continu haute tension alimentation fournit la haute tension à l&#39;aiguille émoussée et motifs du bain de coagulation. Le jet de la pointe de l&#39;aiguille de polymère se déplace dans le droit chemin et se développe ensuite un chemin à fouetter rapide (aka fouetter instabilité). 4 Caractérisation morphologique<ol><li> Coupez un morceau de tapis de fibre séchée et l&#39;immobiliser sur un MEB à l&#39;aide de ruban de carbone. </li><li> Chargez l&#39;échantillon talon dans l&#39;instrument SEM et obtenir des images pour l&#39;analyse. </li></ol>

<ol>
	<li>Greiner, A., Wendorff, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+self-assembled+nanofibers+by+electrospinning.">Functional self-assembled nanofibers by electrospinning.</a> Self-aseembled nanomaterials. 1, 107-171 (2008).</li><li>Ramakrishna, S., Fujihara, K., Teo, W. E., Lim, T. C., Ma, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An Introduction to Electrospinning and Nanofibers. , (2005).</li><li>Klossner, R. R., Queen, H. A., Coughlin, A. J., Krause, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+Chitosan&#8217;s+Rheological+Properties+and+Its+Ability+to+Electrospin.">Correlation of Chitosan&#8217;s Rheological Properties and Its Ability to Electrospin.</a> Biomacromolecules. 9 (10), 2947-2953 (2008).</li><li>McKee, M. G., Wilkes, G. L., Colby, R. H., Long, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlations+of+Solution+Rheology+with+Electrospun+Fiber+Formation+of+Linear+and+Branched+Polyesters.">Correlations of Solution Rheology with Electrospun Fiber Formation of Linear and Branched Polyesters.</a> Macromolecules. 37 (5), 1760-1767 (2004).</li><li>McKee, M. G., Hunley, M. T., Layman, J. M., Long, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+rheological+behavior+and+electrospinning+of+cationic+polyelectrolytes.">Solution rheological behavior and electrospinning of cationic polyelectrolytes.</a> Macromolecules. 39 (2), 575-583 (2006).</li><li>Kong, L., Ziegler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+pure+starch+fibers+by+electrospinning.">Fabrication of pure starch fibers by electrospinning.</a> Food Hydrocolloids. 36, 20-25 (2014).</li><li>Singh, R. S., Saini, G. K., Kennedy, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pullulan:+microbial+sources,+production+and+applications.">Pullulan: microbial sources, production and applications.</a> Carbohydrate Polymers. 73 (4), 515-531 (2008).</li><li>Leathers, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotechnological+production+and+applications+of+pullulan.">Biotechnological production and applications of pullulan.</a> Applied Microbiology and Biotechnology. 62 (5), 468-473 (2003).</li><li>Karim, M. R., Lee, H. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+electrospun+pullulan/montmorillonite+nanofiber+mats+in+aqueous+solution.">Preparation and characterization of electrospun pullulan/montmorillonite nanofiber mats in aqueous solution.</a> Carbohydrate Polymers. 78 (2), 336-342 (2009).</li><li>Stijnman, A. C., Bodnar, I., Hans Tromp, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+of+food-grade+polysaccharides.">Electrospinning of food-grade polysaccharides.</a> Food Hydrocolloids. 25 (5), 1393-1398 (2011).</li><li>Kong, L., Ziegler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+molecular+entanglements+in+starch+fiber+formation+by+electrospinning.">Role of molecular entanglements in starch fiber formation by electrospinning.</a> Biomacromolecules. 13 (8), 2247-2253 (2012).</li><li>Kong, L., Ziegler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheological+aspects+in+fabricating+pullulan+fibers+by+electro-wet-spinning.">Rheological aspects in fabricating pullulan fibers by electro-wet-spinning.</a> Food Hydrocolloids. 38, 220-226 (2014).</li><li>Han, C. 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> Fiber Spinning Rheology and Processing of Polymeric Materials: Volume 2: Polymer Processing. , 257-304 (2007).</li><li>Morris, E. R., Cutler, A. N., Ross-Murphy, S. B., Rees, D. A., Price, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+and+shear+rate+dependence+of+viscosity+in+random+coil+polysaccharide+solutions.">Concentration and shear rate dependence of viscosity in random coil polysaccharide solutions.</a> Carbohydrate Polymers. 1 (1), 5-21 (1981).</li><li>Thompson, C. J., Chase, G. G., Yarin, A. L., Reneker, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+parameters+on+nanofiber+diameter+determined+from+electrospinning+model.">Effects of parameters on nanofiber diameter determined from electrospinning model.</a> Polymer. 48 (23), 6913-6922 (2007).</li></ol>

Les auteurs déclarent qu&#39;ils n&#39;ont rien à divulguer.

La rhéologie est un outil essentiel pour étudier le traitement des polymères, y compris le filage de fibres classiques et électrofilage 13. D&#39;après les études régulières cisaillement rhéologiques, la conformation des polymères et leurs interactions dans différents solvants peuvent être résolus (figures 2 et 3). A des concentrations suffisamment élevées pour ne pas les molécules de biopolymères se chevauchent les uns aux autres, leur dépendance à la conc...

Électrofilage est une technique qui est capable de produire en continu de micro-fibres à l&#39;échelle nanométrique dans une grande variété de matériaux. Il a gagné en intérêt académique et industrielle 1. Bien que la configuration et la pratique de électrofilature semblent simples, la capacité de prévoir et de contrôler electrospinnability propriétés de la fibre reste un défi. La raison peut résider dans le fait qu&#39;il ya beaucoup de facteurs qui influent sur ​​le processus de électrofilature 2 et le processus, en particulier le chemin parcouru par la fibre, est chaotique 1. Souvent, une approche empirique &quot;cook-and-look&quot; est utilisé pour le dépistage des matériaux electrospinnable potentiels. Cependant, pour obtenir un meilleur contrôle sur le processus de électrofilature et les propriétés des fibres obtenues, une compréhension plus complète des mécanismes qui régissent electrospinnability est nécessaire. Plusieurs chercheurs ont constaté que l&#39;enchevêtrement moléculaire de polymères dans la solution de filage est une essential condition préalable pour l&#39;électrofilage réussie de 3 à 5. La rhéologie est un outil puissant pour sonder la conformation moléculaire et d&#39;interaction dans des dispersions de polymères. Par exemple, McKee et al. étudié la conformation moléculaire linéaire et ramifié poly (éthylène téréphtalate-co-isophtalate d&#39;éthylène), les copolymères dans un solvant contenant un mélange chloroforme / téréphtalate de diméthyle (3.7, v / v), et a déterminé que la concentration en polymère doit être 2-2.5x la concentration d&#39;enchevêtrement pour électrofilature succès 4. Il est actuellement un regain d&#39;intérêt dans les fibres de biopolymères en raison de leurs avantages en biodégradabilité, la biocompatibilité et de renouvellement vis-à-vis de leurs homologues synthétiques. Pourtant, les praticiens sont confrontés à de nombreux défis découlant généralement de leur complexité structurelle, de la difficulté dans le traitement thermique et les propriétés mécaniques inférieures. Amidon, qui se trouve dans les tissus végétaux, est among biopolymères les plus abondants et bon marché sur la terre. Fibres d&#39;amidon pur fabriqués en utilisant un appareil d&#39;électro-filage au mouillé ont été récemment décrit 6. Le pullulane est un polysaccharide linéaire de façon extracellulaire produite par certaines bactéries. L&#39;alternance régulière de (1 → 4) et (1 → 6) glucosidiques sont soupçonnés d&#39;être responsables de plusieurs propriétés distinctives de pullulane, y compris une excellente fibre / filmogène capacité 7,8. Électrofilage de fibres de dispersion aqueuse de pullulane a été rapportée par plusieurs chercheurs 9,10. Dans nos publications antérieures, le electrospinnability de deux biopolymères, amidon 11 et 12 pullulane, a été discutée. Ce rapport met l&#39;accent sur la démonstration du protocole pour l&#39;utilisation de principes rhéologiques à l&#39;enquête de la electrospinnability de ces deux biopolymères.

<table border="0" cellpadding="0" cellspacing="0" width="762">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:253px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:175px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gelose 80 starch</td>
			<td style="width:253px;">Ingredion</td>
			<td style="width:119px;"></td>
			<td>Used as it is</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pullulan</td>
			<td style="width:253px;">Hayashibara Co. Ltd</td>
			<td style="width:119px;"></td>
			<td>Used as it is</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethyl Sulfoxide</td>
			<td style="width:253px;">BDH Chemicals</td>
			<td style="width:119px;">BDH1115-4LP</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Ethanol</td>
			<td style="width:253px;">VWR International</td>
			<td style="width:119px;">89125-172</td>
			<td>200 proof</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:253px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rheometer</td>
			<td style="width:253px;">TA Instruments</td>
			<td style="width:119px;">ARES&#160;</td>
			<td>50 mm cone and plate geometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe (10 mL)</td>
			<td style="width:253px;">Becton, Dickinson and Company</td>
			<td style="width:119px;">309604</td>
			<td>Syringe with Luer-Lok&#174; Tip</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">High voltage generator</td>
			<td style="width:253px;">Gamma High Voltage Research, Inc.</td>
			<td style="width:119px;">ES40P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe pump</td>
			<td style="width:253px;">Hamilton Company</td>
			<td style="width:119px;">81620</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Environmental scanning electron microscope</td>
			<td style="width:253px;">FEI Company</td>
			<td style="width:119px;">Quanta 200</td>
			<td>for starch fibers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Environmental scanning electron microscope</td>
			<td style="width:253px;">Phenom-World</td>
			<td style="width:119px;">Phenom G2 Pro</td>
			<td>for pullulan fibers</td>
		</tr>
	</tbody>
</table>

molecular entanglement and electrospinnability of biopolymers

Électrofilage est une technique pour fabriquer des micro fascinant de fibres à l&#39;échelle nanométrique dans une grande variété de matériaux. Pour biopolymères, enchevêtrement moléculaire des polymères constitutifs de la solution de filage a été trouvé à être une condition préalable essentielle pour électrofilature succès. La rhéologie est un outil puissant pour sonder la conformation moléculaire et l&#39;interaction des biopolymères. Dans ce rapport, nous démontrons le protocole à utiliser pour évaluer la rhéologie electrospinnability de deux biopolymères, de l&#39;amidon et le pullulane, de leur sulfoxyde de diméthyle (DMSO) / dispersion d&#39;eau. Bien formés amidon et fibres de pullulane avec des diamètres moyens dans la submicronique pour ordre du micron ont été obtenus. Electrospinnability a été évaluée par observation visuelle et microscopique des fibres formées. En corrélant les propriétés rhéologiques des dispersions à leur electrospinnability, nous démontrons que la conformation moléculaire, enchevêtrement moléculaire et la viscosité de cisaillement affectent tous élusrospinning. La rhéologie est non seulement utile dans le choix du système de solvant et d&#39;optimisation de processus, mais également à la compréhension du mécanisme de formation de la fibre à un niveau moléculaire.

les courbes d&#39;écoulement des dispersions de biopolymères, en fonction de la concentration du biopolymère et la concentration en solvant DMSO ont été obtenus. Deux figures représentatives montrent les courbes d&#39;écoulement de l&#39;amidon (figure 2A) et le pullulane (figure 2B) en fonction de leur concentration dans le solvant de DMSO pur. Les viscosités spécifiques ont été tracées par rapport à la concentration de biopolymère (figure 3A pour l&#39;a...

Watch this Scientific Journal Video about Molecular Entanglement and Electrospinnability of Biopolymers at JoVE.com

Molecular Entanglement and Electrospinnability of Biopolymers

Électrofilage est une technique utilisée pour fabriquer fascinant de micro-fibres à l&#39;échelle nanométrique dans une grande variété de matériaux. Enchevêtrement moléculaire des polymères constitutifs de la solution de filage est essentiel pour la réussite électrofilage. Nous présentons un protocole pour l&#39;utilisation de la rhéologie d&#39;évaluer la electrospinnability de deux biopolymères, amidon et pullulane.

Enchevêtrement moléculaire et Electrospinnability de biopolymères

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular ...

molecular-entanglement-and-electrospinnability-of-biopolymers

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Bioengineering

13.6K vues.  Pennsylvania State University.  Électrofilage est une technique utilisée pour fabriquer fascinant de micro-fibres à l'échelle nanométrique dans une grande variété de matériaux. Enchevêtrement moléculaire des polymères constitutifs de la solution de filage est essentiel pour la réussite électrofilage. Nous présentons un protocole pour l'utilisation de la rhéologie d'évaluer la electrospinnability de deux biopolymères, amidon et pullulane. 

Vidéo: Molecular Entanglement and Electrospinnability of Biopolymers

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

Imagerie de vie de fluorescence des rotors moléculaires dans les cellules vivantes

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

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Bio-ingénierie

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

Assemblage in vitro de la semi-artificielle machine moléculaire et son utilisation pour la détection de lésions de l&#39;ADN

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

Faible enrichissement moléculaire des protéines de poids sur les films minces de silice mésoporeuse de recherche de biomarqueurs

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9.
	Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
	This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

Mesures rigidité en flexion des biopolymères en utilisant des essais de vol à voile

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e.&#160;low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Increasing the PEG molecular weight increased microgel diameter and swelling ratio. Simple adaptations to the PEG microgel precipitation reaction are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

Caractéristiques des précipitations formées polyéthylène Microgels glycol sont commandés par le poids moléculaire des réactifs

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

Aplysie Préparation des ganglions pour les analyses électrophysiologiques et moléculaires de neurones simples

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Electroporateur pour livraison séquentielle moléculaire microscopique assistée Vortex-

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Biofunctionalized bleu de Prusse nanoparticules pour l&#39;imagerie moléculaire multimodale Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

Reconnaissance moléculaire Ingénierie avec Polymères Bio-mimétiques sur simple Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

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.

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.