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. Planung der Experimente <ol><li> Bestimmen Sie das Volumen von Kupfer-Nanocomposites zur Synthese benötigt. Auf dieser Basis wählt eine Anzahl kleinvolumigen Flaschen (25 cm 2) oder größer Kolben, wie unten bei der Herstellung der Materialien angegeben. </li><li> Für diese Synthese, mit einem 37 ° C-Inkubator mit 5% CO 2 und mindestens 40% Luftfeuchtigkeit. Sicherzustellen, dass diese ein Inkubator ist und dass sie nicht so oft über den Zeitraum der Synthese (ca. 24 h) gestört werden. ACHTUNG: wiederholtem Öffnen und Schließen des Inkubators wird sicherlich dazu führen Temperaturschwankungen, die zu einer veränderten Synthese der Nanokomposit-Strukturen führen kann. </li></ol> 2. Herstellung von Materialien <ol><li> Bereiten alle Materialien frisch vor dem Beginn eines Versuchs, durch Zugabe von festem Material, Lösungsmittel direkt vor der Synthese beginnen soll. Keeping Stammlösungen von Cystin und Kupferausgangsstoffe in Flüssigkeit für lange Zeiten bevor das Experiment wird nicht empfohlen und kann zu unterschiedlichen Ergebnissen führen. Sobald Sie vom Anbieter geöffnet halten Ausgangsstoffe trocken durch Umwickeln der Oberseite des Behälters mit Parafilm. Anmerkung: Das folgende Protokoll ist als ein Beispiel für eine Reaktion in einer 25 cm2 Zellkulturflasche unter Verwendung von 7 &amp; mgr; l Cystin, 6,643 &amp; mgr; l steriles Wasser, und 350 ul CNPs verwendet. </li><li> Vorbereitung einer 2 mg / ml Lösung von Kupfer-Nanopartikeln durch Einwiegen mindestens 2 mg CNPs. Einweghandschuhe tragen bei diesem Schritt, um mögliche Kontakt des CNPs mit der Haut zu vermeiden. Legen Sie die Nanopartikel in einer leeren sterilen 16 ml Glasfläschchen. <ol><li> In die Durchstechflasche mit CNPs, fügen steril VE-Wasser in dem entsprechenden Volumen, um eine 2 mg / ml-Lösung zu machen und Vortex die Lösung für 20 Sekunden, um die Dispersion der Nanopartikel vor der Synthese beginnt bereitzustellen (mindestens 1 ml Gesamtvolumen wird empfohlen). Stellen Sie das Fläschchen mehr als die Hälfte Weg zu füllen nicht mit Wasser, da dies hemmt Mischen durch Vortexen. CNPs will schnell auf den Boden der Ampulle absetzen und wird dunkel in der Farbe (grau bis schwarz) angezeigt. </li><li> Beschallen die CNP-Lösung für 17 Minuten bei Raumtemperatur, um eine maximale Dispersion CNPs vor Beginn der Synthese bereitzustellen. In regelmäßigen Abständen überprüfen, um sicherzustellen, dass CNPs aufgrund Mischen einer Beschallung. Nach einer erfolgreichen Beschallung bleiben CNPs in Lösung für mindestens 30 min suspendiert, und die Lösung ist dunkel in der Farbe sein. </li></ol></li><li> Abwiegen ausreichende Masse von Cystin, um eine 72,9 mg / ml Lösung für die Synthese zu machen. Seit Cystin ist nicht in Wasser löslich ist direkt, legen Sie die gewogen Cystin in einer antistatischen Wägegefäß. <ol><li> In den Wiegebehälter, Cystin, setze ausreichend Volumen sterilen, 1 M NaOH, so daß der Cystin vollständig auflöst. Beispielsweise lösen sich 7,29 mg Cystin vollständig in 100 ul 1 M NaOH, um eine 72,9 mg / ml Lösung herzustellen. </li><li> Um sterile Bedingungen aufrechtzuerhalten, führen Sie diesen Schritt in einem sterilen Flussgewebekultur Kapuze. ACHTUNG: NaOHbei 1 M Konzentration ist ätzend, bei diesem Schritt, um den Kontakt von konzentrierter NaOH mit der Haut zu verhindern, so Einmalhandschuhe tragen </li></ol></li><li> Die Arbeit in einem sterilen Gewebekultur Kapuze, hinzufügen, 7 &amp; mgr; l von Cystin mit 6643 ul sterilem Wasser auf die sterile Synthese Kolben erste und ließ Inkubation 30 Min im Brutschrank bei 37 ° C mit dem Kolben entlüftet Kappe (lose), um eine wirksame Mischen. Resuspendieren 2 mg / ml Lösung CNP durch Vortexen für 30 Sekunden, da CNPs wird nach der Beschallungsschritt angesiedelt haben. <ol><li> Ausreichend CNP-Lösung auf die Synthese Kolben (unter Verwendung einer sterilen Technik), um die folgenden Komponentenverhältnisse beizubehalten: 1 Teile kombinieren Cystin, 50 Teile CNPs, und 949 Teilen sterilem Wasser in einem 25 cm 2 Zellkulturflasche, die Synthese zu starten. Zum Beispiel für eine 7 ml Volumen Synthese kombinieren 7 ul von Cystin-Stammlösung, 350 ul CNPs und 6.643 &amp; mgr; l sterilem Wasser. Setzen Sie die Kappe auf der Flasche und ziehen, so dass es secure. </li><li> Nach dem Kombinieren aller Komponenten für die Synthese, vorsichtig mischen in dem Kolben durch Verwirbelung 4-5 mal. Zeigen Kolben im CO 2 Inkubator und der Kolben durch Lösen der Entlüftungskappe, so dass es den Gasaustausch in und aus dem Kolben während der Synthese sein. </li></ol></li><li> Erlauben Synthese in den Inkubator für ungefähr 24 Stunden laufen. Während der Synthese kann man beobachten, wobei die Mikroskopie und durch das Auge, die Bildung von hochlinearen Composites. Hinweis: Der Prozess der Bildung der Strukturen kann sich plötzlich in dem Sinne, dass die Strukturen anfänglich schwer zu erkennen geschieht, dann schreitet Aussehen schnell zu einer zunehmender Dichte. Bildung kann daher vor 24 Stunden auftreten. Das Verfahren kann auch mit dem Auge beobachtet werden, wenn Strukturen größer und ihre Dichte zunimmt. Während die Erzeugung der Strukturen kann im Laufe der Zeit unter dem Mikroskop und mit dem Auge zu späteren Zeitpunkten beobachtet werden, kontinuierlich Unterbrechung der Synthesebedingungen und der Temperatur kann zu einer schlechten führenSynthese führt. </li><li> Beenden Synthese Biokomposite durch dichtes Verschließen des Synthesekolben und Speichern der Behälter in einem Kühlschrank (4 ° C). Strukturen, einmal erzeugt, bleiben stabil in dieser Form für mindestens ein Jahr. Etikett der Kolben mit Synthesebedingungen, einschließlich verwendeten Komponenten, Datum der Synthese sowie der Inkubationszeit der Synthese vor Beendigung. </li></ol> 3. Synthese Mit Kupfersulfat <ol><li> Führen Sie die Selbstorganisation Synthese durch Ersetzen CNPs mit Kupfersulfatsalz. Verwendung einer sterilen Technik lösen sich bei mindestens 2 mg an Kupfersulfat in einem ausreichenden Volumen von sterilem entionisiertem Wasser auf eine 2 mg / ml Lösung herzustellen. Die Kupfersulfat-Kristalle leicht in Lösung gehen bei dieser Konzentration, aber vortexen das Fläschchen, wenn nötig, und prüfen Sie nach Augenmaß, um sicherzustellen, dass alle Kristalle gelöst sind. </li><li> Nach der Herstellung der Kupfersulfat, durchzuführen Synthese, wie zuvor beschrieben, aber unter Ersatz CNPs mit dem Kupfersulfat. Hinweis: Selbstorganisierte Nanocomposites mit Kupfersulfat als Ausgangsmaterial erwiesen sich als viel konsequenter in endgültige Form als für Strukturen aus CNPs synthetisiert. </li><li> Beenden Sie die Synthese von Kupfersulfat Biokomposite wie für CNP-Composites (Schritt 2.6) und speichern sie langfristig bei 4 ° C. </li></ol> 4. Charakterisierung und Handhabung Biocomposites Post-Synthese <ol><li> Charakterisieren Biokomposite von CNPs und von Kupfersulfat mit weißen Lichtmikroskopie 9 und durch Elektronenmikroskopie 9 abgeleitet. <ol><li> Zur Charakterisierung und Prüfung von Biokomposite nach der Synthese von weißen Lichtmikroskopie, verwenden Sie ein inverses Mikroskop als Verbundwerkstoffe werden an der Bodenfläche des Kolbens innerhalb weniger Minuten der Verlegung der Kolben flach niederzulassen, und kann dann in den Mittelpunkt gerückt werden. Verwenden Sie das Hellfeld-Einstellung auf dem Mikroskop, um den Kontrast zwischen Biokomposite und dem flüssigen Medium zu maximieren. Composites aus CNPs und cop abgeleitetpro Sulfat beide scheinen klare bis opake in Farbe, aber nicht umgesetzten CNP Aggregate wird sehr dunkel in der Farbe angezeigt. <ol><li> Verwenden Sie eine Digitalkamera mit dem Mikroskop verbunden, um Bilder der Verbundwerkstoffe zu erfassen. Ein Bereich von Längen für die einzelnen Strukturen beobachtet werden. </li></ol></li><li> Zur Charakterisierung und Prüfung von Biokomposite nach der Synthese und nach Lagerung bei 4 ° C, ermöglichen Kolben auf Raumtemperatur für mindestens 15 Minuten kommen, wie Kolben wird Kondensation zunächst nach der Entnahme aus Kühlschrank, der verschleiern wirksam Schwerpunkt bei der Durchführung Mikroskopie zu bilden. Nachdem man Gleichgewichtseinstellung auf RT, wischen Sie die obere und untere Fläche des Kolbens mit einem sauberen Papiertuch, um die Mikroskopie Bildqualität zu maximieren. </li><li> Bei der Arbeit mit oder bildgebenden Verbundwerkstoffe, die langfristig gespeichert haben, Wirbel der Kolben für 30 Sekunden, um Klumpen von Verbundwerkstoffen, die während der in den Kühlschrank zu bilden distanzieren. Nach Vortex untersuchen die Strukturen mit einem umgekehrten micROSCOPE um sicherzustellen, dass Aggregate dissoziiert und wiederholen Vortexen wie nötig. </li><li> Verwenden invertierten Weißlichtmikroskopie, um die Wirksamkeit der Synthese für eine gegebene Experiment mit CNPs beurteilen. Beispielsweise belegen die Anwesenheit oder Abwesenheit von nicht umgesetztem Synthese CNPs in Kolben für CNP-abgeleiteten Biokomposite von Fläschchen mit verschiedenen Parametern wie Zeitpunkt der Synthese verwendet. Hinweis: Einzelne CNPs sind zu klein, um mit einem Lichtmikroskop beobachten, aber nicht umgesetzten CNP-Aggregate werden als Rundform und dunkle Objekte angezeigt werden, im Gegensatz zu den erfolgreich synthetisiert CNP-Verbundwerkstoffe, die eine High-Seitenverhältnis, lineare Form haben wird, und haben eine Reihe von verschiedenen Längen. Vermeiden Durchführung Synthese zu lange einer Zeit vor der Beendigung, da dies in stark verzweigten &quot;urchin&quot; artigen Strukturen, die schwer in die einmal gebildeten einzelnen Strukturen zu verteilen sind führen. </li><li> Verwenden invertiert weißen Lichtmikroskopie, um die Wirksamkeit zu beurteilender Synthese für ein gegebenes Experiment unter Verwendung von Kupfersulfat. Da Kupfersulfat geht vollständig in Lösung unter Verwendung dieses Protokolls wird die Lösung weniger dunkel als die Lösung aus der Synthese mit Hilfe CNPs angezeigt. Dokumentieren Sie die Größe und das Ausmaß der Kupfersulfat-Verbundwerkstoffen durch den Vergleich Fläschchen mit verschiedenen Synthesebedingungen wie Zeit der Synthese vor der Beendigung. Hinweis: Erfolgreich synthetisierten Verbundwerkstoffe wird eine Reihe von unterschiedlichen Längen zu zeigen. Vermeiden Durchführung Synthese zu lange einer Zeit vor der Beendigung, da dies in stark verzweigten Aggregaten von Verbundwerkstoffen, von denen einige &quot;Seeigel-like&quot; in Struktur ergeben, und die schwierig zu einmal gebildet einzelnen Strukturen zu verteilen sind . </li></ol></li><li> Konzentrations Biokomposite nach der Synthese, zentrifugieren Lösungen von Verbundwerkstoffen in einem Zentrifugenröhrchen. 6 ml entweder CNP-abgeleiteten Strukturen oder Kupfersulfat-abgeleiteten Strukturen in ein 15 ml Zentrifugenröhrchen. Zentrifuge für 10 minbei 500 · g bei RT unter Bildung eines Pellets. Für kleinere Volumina, fügen Sie 500 ul von Strukturen in Lösung zu 0,6 ml großen Tuben. Zentrifugation bei 2.000 × g bei Raumtemperatur für mindestens 10 min, um ein Pellet zu bilden. <ol><li> Nach Zentrifugieren für eine ausreichende Zeit (mindestens 10 min für microfuges), speichern die beobachtbare Pellet am Boden des Röhrchens, wo die Strukturen durch sorgfältiges Entfernen der überstehenden Flüssigkeit über dem Pellet konzentriert. Von Kupfersulfat abgeleitet Biocomposite Strukturen erscheinen in der Farbe blau und Strukturen aus CNPs abgeleitet sind dunkler (grau bis schwarz). </li><li> Noch ein Verbundstoffe an dieser Stange und den Vorgang im gleichen Röhrchen, um Strukturen zu konzentrieren, falls gewünscht. Zu der konzentrierten Pellets zu zerstreuen, fügen Sie das gewünschte Volumen der Lösung in das Röhrchen und Vortex für 10-30 sec. </li></ol></li><li> Beschallen Strukturen einmal gebildet, um die durchschnittliche Populationsgröße (Länge) der Strukturen zu bewegen, um Werte zu senken. Platz Strukturen in sterilem deionisiertem Wasser und Ultraschall behandeln zu least 10 min. Unter Verwendung dieses Verfahrens mit der Zeit geworden Strukturen fragmentiert und kleiner im mittleren Länge (siehe 6 des Textes). Dokumentänderungen in Verbund Größen mit unterschiedlichen Beschallungszeiten unter Verwendung eines invertierten Weißlicht-Mikroskop und Digitalkamera. </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.

Beim Auswerten potentiellen toxischen Wirkung von Nanomaterialien einschließlich CNPs, wurde beobachtet, daß über die langfristige, CNPs wurden aus einem anfänglich mehrere dispergierte Partikelverteilung auf eine größere, aggregierten Form (Figur 2) umgewandelt. In einigen Fällen sind diese hochaggregierten Formationen, die in der Zellkulturschale unter biologischen Bedingungen hergestellt wurden, gebildet hochlinearen Projektionen vom zentralen Aggregat, erinnert an die zuvor beschriebenen Kupf...

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

Das Ziel des Protokolls ist es, die Synthese von zwei neuartigen Biokomposite mit hohem Aspektverhältnis Strukturen zu beschreiben. Die Biokomposite bestehen aus Kupfer und Cystin, entweder mit Kupfer-Nanopartikel (CNP) oder Kupfersulfat trägt die metallische Komponente. Synthese in flüssiger unter biologischen Bedingungen (37 ° C) und der selbstorganisierten Verbundstoffen nach 24 h durchgeführt. Einmal gebildet, sind diese Verbundwerkstoffe in beiden flüssigen Medien in einer getrockneten Form sehr stabil. Die Verbundwerkstoffe skalieren von der Nano- bis Bereich in der Länge Mikro- und von einigen Mikrometern bis 25 nm im Durchmesser. Feldemissionsrasterelektronenmikroskopie mit energiedispersiver Röntgenspektroskopie (EDX) gezeigt, daß Schwefel in der NP-abgeleitete lineare Strukturen vorhanden, während sie nicht an dem Ausgangsmaterial CNP, wodurch bestätigt Cystin als Quelle für Schwefel in den endgültigen Nanokomposite . Während der Synthese dieser linearen Nano- und Mikroverbundwerkstoffe, ein breites Spektrum von Längen von structures in dem Synthesebehälter ausgebildet ist. Ultraschallbehandlung der flüssigen Mischung nach der Synthese wurde gezeigt, dass bei der Kontrolle der durchschnittlichen Größe der Strukturen durch Verminderung der Durchschnittslänge mit der erhöhten Zeit der Ultraschallbehandlung zu helfen. Da die gebildeten Strukturen sind sehr stabil, nicht agglomerieren und sich in der flüssigen Phase gebildet wird, kann auch zur Zentrifugation in Konzentrierung und Trennung gebildeten Verbundstoffe zu unterstützen.

1 zeigt ein Flussdiagramm schema der Syntheseschritte, die in dieser Arbeit beschriebenen linearen Biokomposite bilden. CNPs oder Kupfersulfat als Ausgangsmaterialien werden mit sterilem Wasser kombiniert, um eine 2 mg / ml-Lösung zu bilden, ist diese Lösung vermischt und beschallt, um eine gleichmäßige Mischung zu erhalten, und das Kupferlösung wird dann in dem folgenden Verhältnis zur Synthese vermischt: 949 Teile sterile Wasser: 50 Teile Kupfer-Mischung: 1 Teil Cystin-Stammlösung. Die tatsäch...

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

Hier präsentieren wir ein Protokoll, um neue, hochSeitenVerhältnis Biokomposite unter biologischen Bedingungen und in flüssigen Medien zu synthetisieren. Die Biokomposite skalieren von Nanometern bis Mikrometern im Durchmesser und Länge auf. Kupfer-Nanopartikel (CNP) und Kupfersulfat in Kombination mit Cystin sind die Schlüsselkomponenten für die Synthese.

Generation skalierbar, Metallic Hochseitenverhältnis Nanokomposite in einer biologischen Flüssigmedium

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 Aufrufe.  Centenary College of Louisiana.  Hier präsentieren wir ein Protokoll, um neue, hochSeitenVerhältnis Biokomposite unter biologischen Bedingungen und in flüssigen Medien zu synthetisieren. Die Biokomposite skalieren von Nanometern bis Mikrometern im Durchmesser und Länge auf. Kupfer-Nanopartikel (CNP) und Kupfersulfat in Kombination mit Cystin sind die Schlüsselkomponenten für die Synthese. 

Serie: 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).

Cellular Lipidextraktion für Targeted Stable Isotope Dilution Flüssigchromatographie-Massenspektrometrie-Analyse

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

Forschung

Biotechnik

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 der biologischen Baugruppen in Flüssig

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.

Synthese von Keratin-basierten Nanofaser für Biomedizinische Technik

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.

Magnetisches Wechselfeld-Responsive Hybrid Gelatine Mikrogele der gezielten Medikamentenapplikation

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.

Herstellung von Monodomäne Liquid Crystal Elastomere und Liquid Crystal Elastomer Nanokomposite

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.

Eine neue Technik zur Erzeugung und Observing Chemilumineszenz in einem biologischen Rahmen

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.

Automatisierte Roboter Liquid Handling Versammlung Modular DNA-Geräte

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.

Synthese von biokompatiblen Liquid Crystal Elastomerschäume als Zellgerüste für 3D Spatial Zellkulturen

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.

Schnelle, skalierbare Montage und Beladung von bioaktiven Proteinen und Immunstimulantien in diversen synthetischen darin über Flash-Nanoprecipitation

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.

Gleichzeitige Quantifizierung ausgewählter Kynurenine, analysiert durch Flüssigchromatographie-Massenspektrometrie 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 ...