The authors would like to acknowledge the technical assistance of Alfred Gunasekaran in electron microscopy studies at the Institute of Micromanufacturing at Louisiana Tech University, and Dr. Jim McNamara for assistance with additional microscopy studies. The work described was supported in part by Louisiana board of Regents PKSFI Contract No. LEQSF (2007-12)-ENH-PKSFI-PRS-04 and the James E. Wyche III Endowed Professorship from Louisiana Tech University (to M.D.).

1. Planification d&#39;expériences <ol><li> Déterminer le volume de nanocomposites de cuivre nécessaires pour la synthèse. Sur cette base, choisir un certain nombre de petits flacons de volume (25 cm 2), ou les grands flacons, comme indiqué ci-dessous dans la préparation des matériaux. </li><li> Pour cette synthèse, en utilisant un incubateur à 37 ° C avec 5% de CO 2 et au moins 40% d&#39;humidité. Assurez-vous que un tel incubateur est disponible et qu&#39;il ne sera pas perturbé à plusieurs reprises au cours de la période de synthèse (environ 24 h). ATTENTION: l&#39;ouverture et la fermeture répétées de l&#39;incubateur va certainement causer des fluctuations de température qui peuvent résulter de la synthèse modifiée des structures nanocomposites. </li></ol> 2. Préparation des matériaux <ol><li> Préparer tous les nouveaux matériaux avant le début d&#39;une expérience, en ajoutant des matériaux solides aux solvants juste avant la synthèse est de commencer. Garder solutions de stockage de matériaux de cystine et de départ de cuivre dans le liquide pour de longues durées bvant l&#39;expérience est déconseillé et peut conduire à des résultats variables. Une fois ouvert auprès du fournisseur, garder les matières sèches en enveloppant le haut du conteneur avec du Parafilm départ. Remarque: Le protocole suivant est utilisé comme un exemple de réaction dans un 25 flacon de culture cm 2 de cellules en utilisant 7 ul de cystine, 6,643 pi d&#39;eau stérile, et 350 ul de CNPs. </li><li> Préparer une solution à 2 mg / ml de nanoparticules de cuivre en pesant au moins 2 mg de CNPs. Porter des gants jetables lors de cette étape pour éviter tout risque de contact avec la peau du CNPS. Placez les nanoparticules dans un vide stérile 16 ml flacon en verre. <ol><li> Pour le flacon contenant CNPS, ajouter de l&#39;eau déminéralisée stérile dans le volume approprié de faire une solution à 2 mg / ml et vortex la solution pendant 20 secondes afin de fournir dispersion des nanoparticules de synthèse commence avant (au moins 1 ml volume total est recommandé). Ne pas remplir le plus de la moitié du flacon avec de l&#39;eau car cela va inhiber le mélange au vortex. CNPs will régler rapidement au fond du flacon et apparaîtra de couleur foncée (gris au noir). </li><li> Soniquer la solution CNP pour 17 min à température ambiante pour assurer une dispersion maximale de CNPs avant le début de la synthèse. Vérifiez régulièrement pour vous assurer que CNPs confondez due à ultrasons. Après un traitement par ultrasons succès, CNPs restent en suspension dans la solution pendant au moins 30 min et la solution sera de couleur foncée. </li></ol></li><li> Peser une masse suffisante de la cystine pour former une solution / ml de 72,9 mg pour la synthèse. Depuis cystine est pas directement soluble dans l&#39;eau, placez la cystine pesé dans un récipient de pesée antistatique. <ol><li> Dans le récipient contenant la cystine pesage, ajouter un volume suffisant de, 1 M de NaOH stérile, de sorte que la cystine se dissout complètement. Par exemple, dissoudre 7,29 mg de cystine complètement dans 100 pi de NaOH 1 M, de faire un / ml solution 72,9 mg. </li><li> Pour maintenir des conditions stériles, réaliser cette étape dans un tissu d&#39;écoulement culture hotte stérile. ATTENTION: NaOHà 1 M concentration est caustique, donc porter des gants jetables lors de cette étape pour éviter le contact de NaOH concentré avec la peau </li></ol></li><li> Travailler dans une culture tissulaire hotte stérile, ajouter 7 pi de cystine avec 6643 ul d&#39;eau stérile dans le flacon de synthèse stérile premier, et laisser incuber pendant 30 minutes dans l&#39;incubateur à 37 ° C avec le bouchon de flacon ventilé (en vrac) pour fournir efficace mélange. Reprendre le / ml solution CNP 2 mg par vortex pendant 30 secondes, depuis CNPs aura réglé après l&#39;étape de traitement par ultrasons. <ol><li> Ajouter CNP solution dans le ballon de synthèse (en utilisant une technique stérile) afin de maintenir les rapports de composants suivants: combiner une cystine parties, 50 parties CNPS, et 949 parties d&#39;eau stérile dans un 25 cm2 ballon de culture de cellule pour commencer la synthèse. Par exemple, pour un volume de synthèse 7 ml, mélanger 7 pi de solution cystine boursier, 350 pi du CNPS, et 6643 pi d&#39;eau stérile. Replacez le bouchon sur le flacon et serrer de sorte qu&#39;il est sécure. </li><li> Après combinant tous les composants pour la synthèse, mélanger doucement dans le ballon en secouant 4-5 fois. Placer la fiole dans incubateur à CO 2 et évacuer la fiole en dévissant le bouchon de sorte qu&#39;il y ait un échange de gaz dans et hors de la fiole pendant la synthèse. </li></ol></li><li> Laisser la synthèse afin de fonctionner dans l&#39;incubateur pendant environ 24 h. Lors de la synthèse, on peut observer, à la microscopie et à l&#39;oeil, la formation de composites très linéaires. Remarque: Le processus de formation des structures peut arriver tout à coup dans le sens que les structures sont d&#39;abord difficile à détecter, puis apparition procède rapidement à une augmentation de la densité. Formation peut donc se produire avant 24 h. Le processus peut également être observée à l&#39;oeil fois que les structures deviennent plus grandes et leur densité augmente. Alors que la génération des structures peut être observée au fil du temps sous le microscope et à l&#39;œil à des moments plus tard, interrompant continuellement les conditions de synthèse et la température va conduire à une mauvaiseles résultats de la synthèse. </li><li> Mettre fin à la synthèse de biocomposites en coiffant hermétiquement la fiole de synthèse et le stockage du récipient dans un réfrigérateur (4 ° C). Structures, une fois générés, restent stables sous cette forme pendant au moins un an. Etiqueter le flacon avec des conditions de synthèse, y compris les composants utilisés, la date de la synthèse, et le temps d&#39;incubation de la synthèse avant la fin. </li></ol> 3. Synthèse Utilisation de sulfate de cuivre <ol><li> Réaliser la synthèse auto-assemblage en remplaçant CNPs avec du sel de sulfate de cuivre. En utilisant une technique stérile, dissoudre au moins 2 mg de sulfate de cuivre dans un volume suffisant d&#39;eau déminéralisée stérile de faire un / ml, solution à 2 mg. Les cristaux de sulfate de cuivre vont facilement dans la solution à cette concentration, mais Vortex le flacon si nécessaire, et inspectent visuellement à assurer que tous les cristaux sont dissous. </li><li> Après la préparation du sulfate de cuivre, de réaliser la synthèse comme décrit précédemment, mais en remplaçant CNPs avec le sulfate de cuivre. Remarque: nanocomposites auto-assemblées à l&#39;aide de sulfate de cuivre comme matériau de départ ont été trouvés à être beaucoup plus cohérent dans la forme finale que pour des structures synthétisées à partir de PNC. </li><li> Terminer la synthèse de biocomposites de sulfate de cuivre pour les composites CNP (étape 2.6) et les stocker à long terme à 4 ° C. </li></ol> 4. Caractérisation et la manipulation des Biocomposites post-synthèse <ol><li> Caractériser biocomposites dérivés de CNPs et de sulfate de cuivre par microscopie à lumière blanche 9 et par microscopie électronique 9. <ol><li> Pour la caractérisation et l&#39;inspection des biocomposites post-synthèse par microscopie de lumière blanche, utiliser un microscope inversé comme composites se déposent à la surface inférieure de la fiole à quelques minutes de la pose du flacon plat, et peuvent ensuite être mis en évidence. Utilisez le réglage de champ clair sur le microscope pour optimiser le contraste entre les biocomposites et le milieu liquide. Composites dérivés de PNC et flicpar sulfate seront tous deux être clair à opaque en couleur, mais agrégats CNP pas réagi apparaît une couleur très sombre. <ol><li> Utilisation d&#39;un appareil photo numérique connecté au microscope pour capturer des images des composites. Une gamme de longueurs pour les structures individuelles sera observée. </li></ol></li><li> Pour la caractérisation et l&#39;inspection des biocomposites post-synthèse et après stockage à 4 ° C, permettent flacons à venir à la température ambiante pendant au moins 15 min en flacons formeront condensation initialement lors de l&#39;enlèvement du réfrigérateur, qui obscurcir efficace en se concentrant tout en effectuant l&#39;imagerie microscopique. Après avoir laissé l&#39;équilibre à la température ambiante, essuyer les surfaces supérieure et inférieure de la fiole avec une serviette en papier propre pour maximiser la qualité de l&#39;imagerie microscopique. </li><li> Lorsque vous travaillez avec ou imagerie composites qui ont été stockées à long terme, vortex le flacon pendant 30 secondes à se dissocier des touffes de composites qui forment alors dans le réfrigérateur. Après vortex, inspecter les structures avec un micro inverséroscope pour assurer que les agrégats ont dissocié, et répéter vortex si nécessaire. </li><li> Utilisez microscopie inversée de lumière blanche pour évaluer l&#39;efficacité de la synthèse d&#39;une expérience donnée en utilisant CNPs. Par exemple, le document de la présence ou de l&#39;absence de CNPs qui n&#39;a pas réagi dans des flacons de synthèse utilisés pour biocomposites CNP-dérivés de flacons avec différents paramètres tels que le temps de synthèse. Remarque: individuel CNPs sont trop petits pour observer avec un microscope optique, mais qui n&#39;a pas réagi CNP regroupe apparaîtra comme forme arrondie et objets sombres, contrairement aux CNP-composites synthétisés avec succès ce qui aura un rapport de haute aspect, forme linéaire, et aura une gamme de longueurs différentes. Éviter d&#39;effectuer la synthèse pendant trop longtemps d&#39;une période de temps avant la fin, car cela va entraîner très ramifiées structures de type «oursins», qui sont difficiles à disperser dans des structures individuelles une fois formés. </li><li> Utilisez la microscopie de lumière blanche inversée pour évaluer l&#39;efficacitéde la synthèse pour une expérience donnée en utilisant du sulfate de cuivre. Depuis le sulfate de cuivre va pleinement dans la solution en utilisant ce protocole, la solution apparaît moins sombre que la solution de synthèse en utilisant CNPs. Document, la taille et l&#39;étendue des composites de sulfate de cuivre en comparant les flacons avec différentes conditions de synthèse tel que le temps de synthèse avant la fin. Remarque: composites synthétisé avec succès montreront une gamme de longueurs différentes. Éviter d&#39;effectuer la synthèse pendant trop longtemps d&#39;une période de temps avant la fin, comme cela se traduira par des agrégats hautement branchés de composites, dont certaines seront &quot;oursin-like&quot; dans la structure, et qui sont difficiles à disperser dans des structures individuelles une fois formés . </li></ol></li><li> Pour concentrer biocomposites post-synthèse, solutions centrifugeuses de composites dans un tube de centrifugeuse. Ajouter 6 ml de chacune des structures CNP-structures dérivées ou dérivées de sulfate de cuivre dans un tube de centrifugeuse de 15 ml. Centrifuger pendant 10 minà 500 x g à la température ambiante pour former une pastille. Pour les plus petits volumes, ajouter 500 pi de structures dans une solution à 0,6 ml tubes de taille. Centrifuger à 2000 xg à température ambiante pendant au moins 10 min pour former une pastille. <ol><li> Après centrifugation pendant une durée suffisante (au moins 10 min pour microfuges), à l&#39;exception du culot observable au fond du tube, où les structures sont concentrés en éliminant soigneusement le liquide surnageant au-dessus de la pastille. structures biocomposites issus de sulfate de cuivre apparaissent en bleu dans la couleur et des structures dérivées de CNPs sont plus sombres (gris au noir). </li><li> Ajouter plus de composites à ce tube et répéter le processus dans le même tube de se concentrer structures si désiré. Pour disperser les pastilles concentrées, ajouter le volume désiré de solution dans le tube, et pour 10 à 30 vortex sec. </li></ol></li><li> Structures Soniquer fois formés, pour déplacer la taille moyenne de la population (de longueurs) des structures à des valeurs inférieures. La place des structures dans l&#39;eau déminéralisée stérile et soniquer au least 10 min. Grâce à ce procédé, au fil du temps, les structures deviennent fragmentées et de plus petite longueur moyenne (voir Figure 6 du texte). Les modifications des documents dans les tailles composites avec différents temps de sonication en utilisant un microscope à lumière blanche inversé et appareil photo numérique. </li></ol>

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II. Copper (II) with Cystine and Related Ligands.</a> Inorganic Chemistry. 2, 843-849 (1963).</li><li>Hallman, P., Perrin, D., Watt, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+computed+distribution+of+copper+(II)+and+zinc+(II)+ions+among+seventeen+amino+acids+present+in+human+blood+plasma.">The computed distribution of copper (II) and zinc (II) ions among seventeen amino acids present in human blood plasma.</a> Biochem. J. 121, 549-555 (1971).</li><li>Jensen, L. S., Maurice, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+sulfur+amino+acids+on+copper+toxicity+in+chicks.">Influence of sulfur amino acids on copper toxicity in chicks.</a> The Journal of nutrition. 109, 91-97 (1979).</li><li>Lee, Y., Choi, J. R., Lee, K. J., Stott, N. 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Authors have nothing to disclose.

Alors que l&#39;évaluation des effets toxiques potentiels des nanomatériaux dont CNPS, il a été observé que sur le long terme, PNC ont été transformés à partir d&#39;une distribution de particules initialement plus dispersée à une forme agrégée plus grande (Figure 2). Dans certains cas, ces formations très agrégés qui ont été produites dans le plat de culture cellulaire, dans des conditions biologiques, formés projections très linéaires de l&#39;agrégat central, rappelant cuivre d?...

Copper is a highly reactive metal that in the biological world is essential in some enzyme functions 1,2, but in higher concentrations is potently toxic including in the nanoparticulate form 3,4. Concern over copper toxicity has become more relevant as CNPs and other copper-based nanomaterials are utilized, due to the increased surface area/mass for nanostructures. Thus, even a small mass of copper, in nanoparticle form, could cause local toxicity due to its ability to penetrate the cell and be broken down into reactive forms. Some biological species can complex with and chelate metal ions, and even incorporate them into biological structures as has been described in marine mussels 5. In studying the potential toxic effects of nanomaterials 4, it was discovered that over time, and under biological conditions used for typical cell culturing (37 &#176;C and 5% CO2), stable copper biocomposites could be formed with a high-aspect ratio (linear) structure.
By a process of elimination, the initial discovery of these linear biocomposites, which occurred in complete cell culture media, was simplified to a defined protocol of essential elements needed for the biocomposites to self-assemble. Self-assembly of two types of highly linear biocomposites was discovered to be possible with two starting metal components: 1) CNPs and 2) copper sulfate, with the common biological component being cystine. Although more complex, so called &#8220;urchin&#8221; and &#8220;nanoflower&#8221; type copper-containing structures with nanoscale and microscale features have been previously reported, these were produced under non-biological conditions, such as temperatures of 100 &#176;C or greater 6-8. To our knowledge, synthesis of individual, linear copper-containing nanostructures that are scalable in liquid phase under biological conditions has not been previously described. 
One of the starting materials utilized for synthesis of nanocomposites, namely CNPs, has been reported previously to be very toxic to cells 4. It has recently been reported that after the nanocomposites are formed, these structures are less toxic on a per mass basis than the starting NPs 9. Thus, the synthesis described here may be derived from a biological and biochemical reaction that has utility in stabilizing reactive copper species, both in the sense of transforming the NP form into larger structures and in producing composites less toxic to cells. 
In contrast to many other nanomaterial forms which are known to aggregate or clump upon interaction with biological liquid media 10,11, once formed, the highly linear composites described here avoid aggregation, possibly due to a redistribution of charge which has been previously reported 9. As detailed in the current work, this avoidance of aggregation is convenient for the purposes of working with the structures once formed for at least 3 reasons: 1) composite structures once formed may be concentrated using centrifugation and then easily dispersed again using vortex mixing; 2) formed structures can be decreased in average size by sonication for different periods of time; and 3) the formed linear structures may provide an additional tool for avoiding the recently described &#8220;coffee ring effect&#8221; 12 and thus provide a dopant for creating more evenly distributed coatings of materials, especially those containing spherical particulates.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mini Vortexer</td>
			<td>VWR (https://us.vwr.com)</td>
			<td>58816-121</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator Model # 2425-2</td>
			<td>VWR (https://us.vwr.com)</td>
			<td>Contact vendor</td>
			<td>Current model calalog # 98000-360</td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge (Refrigerated Microcentrifuge)</td>
			<td>Labnet (http://labnetinternational.com/)</td>
			<td>C2500-R</td>
			<td>Model Prism R</td>
		</tr>
		<tr>
			<td>Cell Culture Centrifuge Model Z323K</td>
			<td>Labnet (http://labnetinternational.com/)</td>
			<td>Contact vendor</td>
			<td>Current model Z206A catalog # C0206-A</td>
		</tr>
		<tr>
			<td>Sonicator (Ultrasonic Cleaner)</td>
			<td>Branson Ultrasonics Corporation (http://www.bransonic.com/)</td>
			<td>1510R-MTH</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>Sartorius (http://dataweigh.com)</td>
			<td></td>
			<td>Model CP225D similar model CPA225D</td>
		</tr>
		<tr>
			<td>Olympus IX51 Inverted Light Microscope</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus DP71 microscope digital camera</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>external power supply unit- white light for Olympus microscope</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>TH4-100</td>
			<td></td>
		</tr>
		<tr>
			<td>10x, 20, and 40x microscope objectives</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>Hitachi (http://hitachi-hitec.com/global/em/sem/sem_index.html)</td>
			<td>model S-4800</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>Zeiss (http://zeiss.com/microscopy/en_de/products.html)</td>
			<td>model Libra 120</td>
			<td></td>
		</tr>
		<tr>
			<td>Table Top Work Station Unidirectional Flow Clean Bench</td>
			<td>Envirco (http://envirco-hvac.com)</td>
			<td>model PNG62675</td>
			<td>Used for sterile cell culture technique</td>
		</tr>
	</tbody>
</table>

generation of scalable, metallic high-aspect ratio nanocomposites in a biological liquid medium

L&#39;objectif de ce protocole est de décrire la synthèse de deux nouveaux biocomposites avec des structures de ratio d&#39;aspect élevé-. Les biocomposites sont en cuivre et la cystine, soit avec des nanoparticules de cuivre (CNPS) ou du sulfate de cuivre qui contribue le composant métallique. La synthèse est effectuée dans un liquide dans des conditions biologiques (37 ° C) et la forme des composites auto-assemblées après 24 h. Une fois formés, ces composites sont très stables dans les deux milieux liquides et sous une forme séchée. Les composites échelle du nano aux micro gamme de longueur, et de quelques microns à 25 nm de diamètre. La microscopie électronique à balayage à émission de champ avec une spectroscopie à dispersion d&#39;énergie aux rayons X (EDX) a montré que le soufre était présent dans les structures linéaires NP-dérivés, tandis qu&#39;il est absent de la matière CNP départ, ce qui confirme la cystine en tant que source de soufre dans les nanocomposites finaux . Lors de la synthèse de ces nano et micro-composites linéaires, un large éventail de longueurs de structures est formée dans le récipient de synthèse. La sonication du mélange liquide après la synthèse a été démontrée pour aider à contrôler la taille moyenne des structures en diminuant la longueur moyenne avec l&#39;augmentation du temps de sonication. Étant donné que les structures formées sont très stables, ne pas agglomérer, et sont formées dans la phase liquide, la centrifugation peut également être utilisée pour aider à concentrer et séparer les composites formés.

La figure 1 montre un organigramme schématique des étapes de synthèse pour former les biocomposites linéaires décrits dans cet ouvrage. CNPs ou sulfate de cuivre comme matières de départ sont combinés avec de l&#39;eau stérile pour former une / ml, solution 2 mg, cette solution est mélangé et traité par ultrasons pour fournir un mélange homogène, et cette solution de cuivre est ensuite mélangé dans le rapport suivant de synthèse: 949 parties stérile l&#39;eau: 50 parties de mélange de...

Watch this Scientific Journal Video about Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium at JoVE.com

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

Ici, nous présentons un protocole à synthétiser de nouveaux, de haute rapport d&#39;aspect biocomposites dans des conditions biologiques et dans des milieux liquides. Les biocomposites échelle du nanomètre au micromètre de diamètre et de la longueur, respectivement. nanoparticules de cuivre (CNPS) et du sulfate de cuivre combinés avec la cystine sont les éléments clés pour la synthèse.

Génération des solutions évolutives de métal de très haute-Aspect Ratio nanocomposites dans un milieu liquide biologique

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

generation-scalable-metallic-high-aspect-ratio-nanocomposites

Research

JoVE Journal

Bioengineering

9.0K vues.  Centenary College of Louisiana.  Ici, nous présentons un protocole à synthétiser de nouveaux, de haute rapport d'aspect biocomposites dans des conditions biologiques et dans des milieux liquides. Les biocomposites échelle du nanomètre au micromètre de diamètre et de la longueur, respectivement. nanoparticules de cuivre (CNPS) et du sulfate de cuivre combinés avec la cystine sont les éléments clés pour la synthèse. 

Vidéo: Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

L&#39;extraction des lipides cellulaires pour ciblées dilution isotopique stable analyse de spectrométrie de masse chromatographie liquide-

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Recherche

Bio-ingénierie

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

In situ TEM d’assemblages biologiques dans un liquide

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Synthèse de nanofibres à base de kératine de génie biomédical

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Magnétique alternatif microgels de gélatine hybride sensible au champ de libération contrôlée de médicaments

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

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

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

Préparation de monodomaine à cristaux liquides élastomères et élastomères cristaux liquides nanocomposites

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

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

Une nouvelle technique de production et d&#39;observation de chimiluminescence en milieu biologique

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Automatisé de robotique Liquid Handling assemblage de dispositifs modulaires ADN

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Synthèse de cristaux liquides biocompatibles mousses élastomères comme échafaudages cellulaires pour les cultures de cellules spatiales 3D

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

Assemblage rapide, évolutive et dans divers Nanocarriers synthétique Via Flash nanoprécipitation apport de protéines bioactives et Immunostimulants

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

Quantification simultanée de kynurenines sélectionnées analysées par chromatographie liquide-spectrométrie de masse dans le milieu recueilli à partir des cultures de cellules cancéreuses

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...