Este trabajo está financiado en parte por el Instituto Nacional de USDA para la Agricultura y la Alimentación, el Programa Nacional de Becas Competitivas, Programa Nacional de la Iniciativa de Investigación de 71.1 el año fiscal 2007 como subvención No. 2007-35503-18392, y los Institutos Nacionales de Salud, Instituto de Alergias y Enfermedades Infecciosas , R33AI94514-03.

1. Spinning Preparación Dope <ol><li> Prepare una serie de concentraciones de biopolímero que se investigue (0,1% a 30%, w / v) y asegúrese de considerar el contenido de humedad del polvo de biopolímero en estos cálculos. Para cada concentración, se pesa el biopolímero (almidón o pululano) polvo en un tubo de ensayo de 50 ml. Añadir dimetilsulfóxido acuoso solución (DMSO) y una barra de agitación. </li><li> Se coloca el tubo en agua hirviendo con agitación constante en una placa caliente de agitación magnética. </li><li> Después de aproximadamente 1 hora, apagar el fuego y deje que la dispersión se enfríe a temperatura ambiente. La dispersión es entonces listo para la prueba reológica y electrospinning. </li></ol> 2. Steady Shear Reología <ol><li> Caliente el reómetro y ajustar la temperatura fase a 20 ° C. Calibrar la brecha entre el (mm cono 25 se utiliza) de la sonda y la etapa (placa). </li><li> Cargar 0,41 ml de la dispersión de biopolímero en el centro del escenario y bajar la sonda para establecer el positien (brecha de 0,053 mm para 25 mm cono). Asegúrese de que la dispersión se extiende de manera uniforme dentro de la brecha. </li><li> Realice la prueba reológica con los siguientes parámetros experimentales: modo de barrido: registro, tasa inicial: 100 seg-1, tipo final: 0,1 seg-1, puntos por década: 10, retraso antes de medida: 5 seg, medir el tiempo: 10 seg, y direcciones por compás: dos (tanto en sentido horario y en sentido antihorario). </li><li> Analizar los datos reológicos para estimar las concentraciones de dispersión apropiados para electrospinning ensayos. <ol><li> Trazar viscosidades de cizallamiento aparente frente a velocidades de cizalladura como una función de concentraciones de polímero. Para cada curva de flujo, aproximados viscosidades de cizallamiento cero, η 0, por los valores reales o extrapolados (por ejemplo, a bajas concentraciones, donde los datos de baja velocidad de cizallamiento son poco fiables) para la viscosidad aparente a 0,1 seg - 1. </li><li> Calcular la viscosidad específica: η sp = (η 0 - η s)/ η s, donde s η es la viscosidad del disolvente. </li><li> Trazar viscosidades específicas como una función de la concentración. Identificar los regímenes sin entrelazar y enredadas semidilute. El régimen sin entrelazar semidilute comienza desde el extremo más bajo de concentración con poca pendiente, y el régimen semidilute enredado tiene una mayor pendiente siguiendo el régimen sin entrelazar. Modelos de regresión de ley de potencia en forma en ambos regímenes. Los valores de potencia son las laderas (dependencia de la concentración) en los regímenes sin entrelazar y enredadas semidilute en un gráfico log-log. La intersección de las dos líneas de armarios es la concentración enredo, c e. </li></ol></li></ol> 3. electrospinning variación de parámetros <ol><li> Montar la configuración de electrospinning como se muestra en la Figura 1. Cargar la jeringa con la dispersión de la composición apropiada, por ejemplo, 15% (w / v) de almidón o pululano en 100% de DMSO, a la bomba de jeringa. Sujete el cable de alta tensión (positIVE) a la aguja. Se conecta el baño de coagulación que contiene etanol puro a tierra sumergiendo el cable de tierra (negativo) en el baño. Use un gato de laboratorio para ajustar la distancia entre la aguja de la jeringa y baño de coagulación. Sumergir una malla metálica en el baño para recoger la estera de fibra después de electrospinning. </li><li> Haga girar el biopolímero en rangos de los siguientes parámetros: velocidad de alimentación de 0,1 a 0,4 ml / h, la distancia de giro de 5 a 10 cm, y el voltaje de 0 a 15 kV. <ol><li> Comience con una distancia de giro de 5 cm. Por primera velocidad de avance (0,1 ml / hr), aumento de la tensión lentamente desde 0 V. Preste atención a la forma de la dispersión extruido en la punta de la aguja y anote cuando la dispersión de goteo se acelera y luego alargada. </li><li> Tenga en cuenta la tensión a la que un pequeño chorro de inició de la superficie de la gota, lo que indica electrospinnability de la solución. Registre el voltaje en el que un chorro continuo inicia, si los hubiere. </li><li> Examine la gama completa para cada uno de los tres Parameters y fichas condiciones de hilatura éxito. Recoger las fibras sólo cuando hay un chorro continuo de la punta. </li></ol></li><li> Después de unos minutos de la recolección, enjuague la estera de fibra con etanol puro. Coloque la estera de fibra en un desecador que contiene desecante al vacío. </li><li> Repita el procedimiento para cada concentración biopolímero para la caracterización completa. </li></ol><img alt="Figura 1" fo:content-width="5in" src="/files/ftp_upload/51933/51933fig1highres.jpg" width="500" /> Figura 1. Dibujo esquemático de la configuración de electro-hilado en húmedo. La dispersión biopolímero se extruye a partir de una bomba de jeringa. Una fuente de alimentación de CC de alto voltaje proporciona alta tensión a la aguja de punta roma y el recinto del baño de coagulación. El chorro de polímero a partir de la punta de la aguja se desplaza a través de un camino recto y luego se desarrolla un camino rápido para batir (también conocido como azotes inestabilidad). 4. Caracterización Morfológica<ol><li> Corte un pedazo de manta de fibra seca e inmovilizarlo sobre un trozo SEM utilizando cinta de carbono. </li><li> Cargue el talón de la muestra en el instrumento SEM y obtener imágenes para el análisis. </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>

Los autores declaran que no tienen nada que revelar.

La reología es una herramienta esencial para estudiar el procesamiento de polímeros, incluyendo la hilatura de fibra convencional y electrospinning 13. De los estudios de cizalladura constantes reológicas, la conformación del polímero y sus interacciones en diferentes disolventes pueden ser resueltos (Figuras 2 y 3). A concentraciones no son suficientemente altos para las moléculas de biopolímero se solapen uno con el otro, su dependencia de la concentración fue de al...

Electrospinning es una técnica que es capaz de producir micro continua a las fibras de nano-escala a partir de una amplia variedad de materiales. Se ha ganado un creciente interés académico e industrial 1. Aunque la configuración y la práctica de electrospinning parecen sencillo, la capacidad de predecir y controlar electrospinnability propiedades de la fibra sigue siendo un reto. La razón puede estar en el hecho de que hay muchos factores que influyen en el proceso de electrospinning 2 y el proceso, especialmente la trayectoria recorrida por la fibra, es caótico 1. A menudo, un enfoque empírico &quot;cocinar-y-mirar&quot; se utiliza para la detección de materiales electrospinnable potenciales. Sin embargo, para tener un mejor control sobre el proceso de electrospinning y propiedades de la fibra resultantes, una comprensión más completa de los mecanismos que gobiernan electrospinnability se requiere. Varios investigadores han encontrado que el entrelazamiento molecular de polímeros en la disolución de hilatura es un infll requisito previo para electrospinning éxito 3 5. La reología es una poderosa herramienta para sondear conformación molecular y la interacción en dispersiones de polímeros. Por ejemplo, McKee et al. investigado la conformación molecular de lineal y ramificado de poli (tereftalato de etileno-co-isoftalato de etileno) copolímeros en un disolvente que contiene cloroformo / tereftalato de dimetilo (7/3, v / v), y ha determinado que la concentración de polímero tenía que ser 2-2,5 veces la concentración de entrelazamiento para electrospinning éxito 4. En la actualidad existe un interés renovado en las fibras de biopolímeros debido a sus ventajas en biodegradabilidad, biocompatibilidad, y renovabilidad vis-à-vis sus homólogos sintéticos. Sin embargo, los profesionales se enfrentan a muchos retos derivados generalmente de su complejidad estructural, la dificultad en el procesamiento térmico y las propiedades mecánicas inferiores. El almidón, que se encuentra en los tejidos vegetales, es among los biopolímeros más abundantes y de bajo costo en la tierra. Fibras de almidón puro fabricados utilizando un aparato electro-hilatura en húmedo se describió recientemente 6. El pululano es un polisacárido lineal producida extracelularmente por ciertas bacterias. La alternancia regular de (1 → 4) y (1 → 6) enlaces glucosídicos se cree que son responsables de varias propiedades distintivas de pululano, incluida la fibra excelente / formadores de 7,8 capacidad de película. Electrospinning de fibras de dispersión acuosa de pululano ha sido reportado por un número de investigadores 9,10. En nuestras publicaciones anteriores, el electrospinnability de dos biopolímeros, almidón 11 y 12 de pululano, se ha discutido. Este informe se centra en la demostración del protocolo para la utilización de principios reológicas en la investigación de la electrospinnability de estos dos biopolímeros.

<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

Electrospinning es una técnica fascinante para fabricar micro fibras a nano-escala a partir de una amplia variedad de materiales. Para biopolímeros, enredo molecular de los polímeros constituyentes en la solución de hilado se encontró que era un requisito previo esencial para electrospinning éxito. La reología es una herramienta poderosa para sondar la conformación molecular y la interacción de los biopolímeros. En este informe, demuestran el protocolo para la utilización de reología para evaluar la electrospinnability de dos biopolímeros, almidón y pullulan, desde su dimetilsulfóxido (DMSO) / dispersiones de agua. Se obtuvieron de almidón y fibras de pululano bien formados con diámetros medios en el rango submicrónico para micrones. Electrospinnability se evaluó por observación visual y microscópica de las fibras formadas. Al correlacionar las propiedades reológicas de las dispersiones a su electrospinnability, nos demuestran que la conformación molecular, enredo molecular y viscosidad de corte, afectan electorospinning. Reología no sólo es útil en la selección del sistema disolvente y la optimización de procesos, sino también en la comprensión del mecanismo de formación de la fibra en un nivel molecular.

Se obtuvieron curvas de flujo de las dispersiones de biopolímero como una función de la concentración de biopolímero y la concentración de DMSO en disolvente. Dos figuras representativas muestran las curvas de flujo de almidón (Figura 2A) y pululano (Figura 2B) como una función de su concentración en disolvente DMSO puro. Las viscosidades específicas se representaron frente a la concentración de biopolímero (Figura 3A para el almidón y la Figura 3B</...

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

Molecular Entanglement and Electrospinnability of Biopolymers

Electrospinning es una técnica fascinante utilizado para fabricar fibras micro a nano-escala a partir de una amplia variedad de materiales. Entrelazamiento molecular de los polímeros constituyentes en la disolución de hilatura es esencial para electrospinning éxito. Se presenta un protocolo para la utilización de reología para evaluar la electrospinnability de dos biopolímeros, almidón y pululano.

Molecular Enredo y Electrospinnability de Biopolímeros

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

13.7K vistas.  Pennsylvania State University.  Electrospinning es una técnica fascinante utilizado para fabricar fibras micro a nano-escala a partir de una amplia variedad de materiales. Entrelazamiento molecular de los polímeros constituyentes en la disolución de hilatura es esencial para electrospinning éxito. Se presenta un protocolo para la utilización de reología para evaluar la electrospinnability de dos biopolímeros, almidón y pululano. 

Video: 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.

Imágenes de fluorescencia vida de rotores moleculares en las células vivas

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

Investigación

Bioingeniería

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.

En la Asamblea in vitro de la semi-artificial de la máquina molecular y su utilización para la detección del daño del ADN

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

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

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

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

De bajo peso molecular de proteínas en el enriquecimiento de las películas delgadas de silicio mesoporosos para el descubrimiento de biomarcadores

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.

Medidas de flexión rigidez de biopolímeros utilizando ensayos de Vuelo a Vela

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 Preparación De Ganglios Para Análisis Electrofisiológicos Y Moleculares De Neuronas Individuales

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.

Vortex asistida Microscale Electroporador para entrega secuencial Molecular

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.

Biofuncionalizadas prusiana nanopartículas azul para Multimodal Molecular Aplicaciones de imágenes

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.

Ingeniería Molecular Recognition con polímeros biomiméticos en una sola pared nanotubos de carbono

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.

Métodos para la auto-integración de Megamolecular Biopolímeros en el Aire-LC Interfaz de secado

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.

Microarrays de biopartículas para el análisis quimiotáctico y molecular del enjambre de neutrófilos humanos in vitro

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