Diese Arbeit wird teilweise durch das USDA National-Institut für Ernährung und Landwirtschaft, National Competitive Grants Program, National Research Initiative Programm 71,1 GJ 2007 finanziert als Grant No 2007-35503-18392 und National Institutes of Health, Institut für Allergie und Infektionskrankheiten , R33AI94514-03.

1. Spinnlösung Vorbereitung <ol><li> Bereiten Sie eine Reihe von Biopolymer-Konzentrationen (bis 30% 0,1%, w / v) untersucht werden und sicher sein, um den Feuchtigkeitsgehalt der Biopolymer-Pulver in diesen Berechnungen zu berücksichtigen. Für jede Konzentration, wiegen das Biopolymer (Stärke oder Pullulan) Pulver in einem 50 ml-Reagenzglas. Fügen wässrigen Dimethylsulfoxid (DMSO)-Lösung und einen Rührstab. </li><li> Das Röhrchen wird in kochendes Wasser unter ständigem Rühren auf einem Magnetrührer Heizplatte. </li><li> Nach ca. 1 Stunde, schalten Sie die Hitze und lassen Dispersion auf Raumtemperatur abkühlen. Die Dispersion ist dann bereit für rheologische Tests und Elektro. </li></ol> 2. Stationäre scherrheologie <ol><li> Aufwärmen der Rheometer und die Bühne Temperatur bei 20 ° C. Kalibrieren Sie den Abstand zwischen der Sonde (25 mm Konus verwendet werden) und die Bühne (Platte). </li><li> Last 0,41 ml der Biopolymer-Dispersion auf die Mitte der Bühne und senken Sie die Sonde, um die positi eingestelltauf (0,053 mm Abstand für 25 mm Konus). Stellen Sie sicher, dass die Dispersion gleichmäßig innerhalb der Lücke verbreitet. </li><li> Führen Sie die rheologischen Test mit den folgenden experimentellen Parameter: Sweep-Modus: log, Anfangsgeschwindigkeit: 100 s -1, Schlussrate: 0,1 s -1, Punkte pro Jahrzehnt: 10, Verzögerung, bevor Maßnahme: 5 sec, Messung Zeit: 10 sec, und Richtungen pro Takt: zwei (beide im Uhrzeigersinn und gegen den Uhrzeigersinn). </li><li> Rheologischen Daten analysieren, um geeignete Dispersions Konzentrationen für das Elektro Studien schätzen. <ol><li> Plotten scheinbaren Scherviskositäten gegen Schergeschwindigkeiten als Funktion der Polymerkonzentration. Für jede Stromkurve, ungefähre Null-Scherviskositäten η 0, durch die tatsächliche oder extrapolierten Werte (beispielsweise bei niedrigen Konzentrationen, wo die niedrige Scherratendaten unzuverlässig sind) für die scheinbare Viskosität bei 0,1 sec - 1. </li><li> Berechnen Sie die spezifische Viskosität: η sp = (η 0 - η s)/ Η s, wobei s η ist die Viskosität des Lösungsmittels. </li><li> Plotten spezifischen Viskositäten als Funktion der Konzentration. Identifizieren halbverdünnten unverflochtene und verstrickt Regime. Die halbverdünnten unverflochtene Regime beginnt mit dem niedrigen Konzentration Ende mit einem kleinen Hang, und die halbverdünnten Regime verstrickt hat eine größere Steigung nach der unverflochtene Regimes. Fit Potenzgesetz Regressionsmodelle in beiden Regimen. Die Leistungswerte sind die Pisten (Konzentrationsabhängigkeit) in halbverdünnten unverflochtene und verstrickt Regime auf einer log-log-Plot. Der Schnittpunkt der beiden Linien ist die Einbau Verstrickung Konzentration, c e. </li></ol></li></ol> 3. Das Elektroparametervariation <ol><li> Montieren Sie den Elektro Aufbau wie in Abbildung 1 dargestellt. Legen Sie die Spritze mit Dispersions entsprechender Zusammensetzung, zB 15% (w / v) Stärke oder Pullulan in 100% DMSO, auf die Spritzenpumpe. Clip der Hochspannungsleitung (postulierenIVE) an der Nadel. Schließen Sie den Gerinnungs Bad mit reinem Ethanol auf Masse durch Eintauchen der Masseleitung (negativ) in die Badewanne. Verwenden Sie eine Hebebühne, um den Abstand zwischen der Spritze und Nadel Koagulierbad einzustellen. Tauchen Sie ein Metallnetz in der Badewanne, um die Fasermatte nach Elektrospinning zu sammeln. </li><li> Drehen Sie das Biopolymer in der folgenden Parameterbereiche: Förderrate von 0,1 bis 0,4 ml / h, Entfernung von Spinn 5 bis 10 cm, und die Spannung von 0 bis 15 kV. <ol><li> Beginnen mit einer Spinn Abstand von 5 cm. Zum ersten Förderrate (0,1 ml / h), die Spannung bis Rampe langsam von 0 V Achten Sie auf die Form der Dispersion an der Nadelspitze extrudiert und beachten, wenn die Tropf Dispersion wird beschleunigt und dann länglich. </li><li> Beachten Sie die Spannung, bei der ein kleiner Strahl aus der Tropfenoberfläche eingeleitet, was electrospinnability der Lösung. Aufzeichnen der Spannung an dem ein kontinuierlicher Strahl einleitet, falls vorhanden. </li><li> Untersuchen Sie das komplette Programm für jede der drei parameters und Mitteilungs erfolgreichen Spinnbedingungen. Sammeln der Fasern nur, wenn es einen kontinuierlichen Strahl von der Spitze. </li></ol></li><li> Nach ein paar Minuten sammeln, spülen Sie die Fasermatte mit reinem Ethanol. Legen Sie die Fasermatte in einem Exsikkator mit Trockenmittel im Vakuum. </li><li> Wiederholen Sie für jede Biopolymer-Konzentration für eine vollständige Charakterisierung. </li></ol><img alt="Figur 1" fo:content-width="5in" src="/files/ftp_upload/51933/51933fig1highres.jpg" width="500" /> Abbildung 1 Schematische Darstellung des elektro Naßspinnen Einrichtung. Das Biopolymer Dispersion aus einer Spritzenpumpe extrudiert. Ein Hochspannungsgleichstromnetzteil bietet eine hohe Spannung an den stumpfen Nadel und Gelände die Gerinnungs Badewanne. Das Polymer-Jet von der Nadelspitze bewegt sich durch einen geraden Weg und entwickelt eine schnelle Schlag Weg (auch bekannt als Prügel Instabilität) dann. 4. Morphologische Charakterisierung<ol><li> Schneiden Sie ein Stück getrockneten Fasermatte und immobilisieren sie auf eine SEM-Stub mit Kohlenstoffband. </li><li> Legen Sie die Probe in die Stub SEM Instrument und Bilder zu erhalten für die Analyse. </li></ol>

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

Die Autoren erklären, dass sie nichts zu offenbaren haben.

Rheologie ist ein wesentliches Instrument, um die Verarbeitung von Polymeren, einschließlich konventioneller Faserspinn und Elektrospinning 13 studieren. Von dem stationären Scher rheologischen Untersuchungen, Polymer-Konformation und ihre Wechselwirkungen in verschiedenen Lösungsmitteln gelöst werden kann (Figuren 2 und 3). Bei Konzentrationen nicht hoch genug für Biopolymermolekülen miteinander überlappen, war ihre Konzentrationsabhängigkeit rund 1,4 (Abbild...

Elektrospinnen ist eine Technik, in der Lage, kontinuierliche Mikro um nanoskalige Fasern aus einer Vielzahl von Materialien ist. Es wird zunehmend akademischen und industriellen Interesse 1. Obwohl das Setup und die Praxis der Elektro scheinen einfach, bleibt die Fähigkeit, vorherzusagen und zu kontrollieren electrospinnability Fasereigenschaften eine Herausforderung. Der Grund mag in der Tatsache, dass es viele Faktoren, die das Elektrospinnverfahren 2 und den Prozess, vor allem den Weg von der Faser reiste, ist chaotisch 1 Einfluss liegen. Oft eine empirische &quot;Koch-und-aussehen&quot;-Ansatz wird für das Screening von potenziellen elektroverspinnbaren Materialien verwendet. Um jedoch eine bessere Kontrolle über das Elektrospinnverfahren und der resultierenden Fasereigenschaften, ein besseres Verständnis der Mechanismen, die electrospinnability regeln erforderlich gewinnen. Einige Forscher haben herausgefunden, dass molekulare Verhakung von Polymeren in der Spinnlösung ist eine essential Voraussetzung für eine erfolgreiche Elektro 3-5. Die Rheologie ist ein leistungsfähiges Werkzeug, um molekulare Konformation und Interaktion in Polymerdispersionen zu untersuchen. Zum Beispiel McKee et al. untersuchten die molekularen Konformation des linearen und verzweigten Poly (ethylenterephthalat-co-Ethylenisophthalat-Copolymer) in einem Lösungsmittel Chloroform / Dimethylterephthalat (3.7, v / v) enthält, und bestimmt, dass die Polymerkonzentration auf 2-2.5x sein die Verstrickung Konzentration für eine erfolgreiche Elektro 4. Es ist derzeit das Interesse an Fasern aus Biopolymeren wegen ihrer Vorteile in der biologischen Abbaubarkeit, Biokompatibilität und Erneuerbarkeit gegenüber den synthetischen Gegenstücke. Doch Praktiker stellen viele Herausforderungen, die sich in der Regel aus ihrer strukturellen Komplexität, Schwierigkeiten bei der thermischen Behandlung und schlechtere mechanische Eigenschaften. Stärke, in pflanzlichen Geweben gefunden wird, ist Among die am häufigsten vorkommenden Biopolymeren und preiswert auf der Erde. Reine Stärkefasern hergestellt unter Verwendung eines elektroNassSpinnApparatur wurden kürzlich beschriebenen 6. Pullulan ist ein extrazellulär durch bestimmte Bakterien lineares Polysaccharid. Der regelmäßige Wechsel von (1 → 4) und (1 → 6) Glucosidbindungen sind vermutlich für mehrere markante Eigenschaften Pullulan, einschließlich ausgezeichneter Faser / Filmbildungsfähigkeit 7,8 verantwortlich zu sein. Elektrospinnen von Pullulan Fasern aus wässriger Dispersion wurde durch eine Reihe von Forschern 9,10 berichtet. In unseren früheren Veröffentlichungen, die von zwei electrospinnability Biopolymere, Stärke 11 und 12 Pullulans, wurde diskutiert. Dieser Bericht konzentriert sich auf die Vorführung des Protokolls für die Verwendung rheologischen Prinzipien bei der Untersuchung des electrospinnability dieser beiden Biopolymere.

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

molecular entanglement and electrospinnability of biopolymers

Elektro ist eine faszinierende Technik, um aus einer Vielzahl von Materialien zu fertigen Mikro zu nanoskaligen Fasern. Für Biopolymere, wurde molekularen Verschlingung der Polymerbestandteile in der Spinnlösung gefunden, eine wesentliche Voraussetzung für eine erfolgreiche Elektro sein. Die Rheologie ist ein leistungsfähiges Werkzeug, um die molekulare Konformation und Interaktion von Biopolymeren zu untersuchen. In diesem Bericht zeigen wir das Protokoll für die Verwendung, um die Rheologie electrospinnability von zwei Biopolymeren, Stärke und Pullulan zu bewerten, aus ihren Dimethylsulfoxid (DMSO) / Wasser-Dispersionen. Wohlgeformte Stärke und Pullulan Fasern mit mittleren Durchmessern im Submikrometerbereich zu Mikrometer-Bereich erhalten. Electrospinnability wurde durch visuelle und mikroskopische Untersuchung des gebildeten Fasern ausgewertet. Durch Korrelieren der rheologischen Eigenschaften der Dispersionen, ihre electrospinnability zeigen wir, dass die molekulare Konformation, molekulare Verhakung und Scherviskosität beeinflussen Electrospinning. Rheologie ist nicht nur nützlich bei der Lösungsmittelsystem Auswahl und Prozessoptimierung, sondern auch für das Verständnis des Mechanismus der Faserbildung auf molekularer Ebene.

Fließkurven der Biopolymer-Dispersionen als Funktion der Konzentration Biopolymer und DMSO-Konzentration in dem Lösungsmittel erhalten. Zwei repräsentative Zahlen zeigen die Fließkurven der Stärke (2A) und Pullulans (2B) in Abhängigkeit von ihrer Konzentration in reinem DMSO Lösungsmittel. Die spezifischen Viskositäten wurden gegen Biopolymer-Konzentration (3A für Stärke und 3B für Pullulans) aufgetragen. Aus diesen Grundstücken wurden Verst...

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

Molecular Entanglement and Electrospinnability of Biopolymers

Elektro ist eine faszinierende Technik, die zur Mikro zu nanoskaligen Fasern aus einer Vielzahl von Materialien zu fertigen. Molekulare Verschlingung der Polymerbestandteile in der Spinnlösung ist wichtig für eine erfolgreiche Elektro. Wir präsentieren ein Protokoll für die Verwendung Rheologie, die electrospinnability von zwei Biopolymere, Stärke und Pullulan bewerten.

Molekulare Verschränkung und Electrospinnability von Biopolymeren

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

molecular-entanglement-and-electrospinnability-of-biopolymers

Research

JoVE Journal

Bioengineering

13.6K Aufrufe.  Pennsylvania State University.  Elektro ist eine faszinierende Technik, die zur Mikro zu nanoskaligen Fasern aus einer Vielzahl von Materialien zu fertigen. Molekulare Verschlingung der Polymerbestandteile in der Spinnlösung ist wichtig für eine erfolgreiche Elektro. Wir präsentieren ein Protokoll für die Verwendung Rheologie, die electrospinnability von zwei Biopolymere, Stärke und Pullulan bewerten. 

Serie: Molecular Entanglement and Electrospinnability of Biopolymers

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

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

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

Fluorescence Lifetime Imaging für molekulare Rotoren in lebenden Zellen

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

Forschung

Biotechnik

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

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

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

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

In-vitro-Assembly von Semi-künstliche molekulare Maschine und ihre Verwendung zum Nachweis von DNA-Schäden

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

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

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

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

Niedermolekulare Protein Enrichment auf mesoporösem Siliciumdioxid Dünnschichten für Biomarker Discovery

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

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

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

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

Biegesteifigkeit Maße von Biopolymeren mit Gliding Assays

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

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

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

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

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

Aplysia Ganglien-Vorbereitung für elektrophysiologische und molekulare Analysen einzelner Neuronen

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

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

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

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

Microscale Vortex-unterstützte Electroporator für Sequential Molekulare Liefer

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

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

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

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

Biofunktionalisierten Prussian Blue Nanopartikel für Molekulare Bildgebung Multimodale Anwendungen

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

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

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

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

Technik Molekulare Erkennung mit Bio-Mimetikum Polymers auf einwandigen Kohlenstoff-Nanoröhrchen

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

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

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

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

Biopartikel-Mikroarrays für chemotaktische und molekulare Analysen des menschlichen Neutrophilen-Schwärmens in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...