Este trabajo fue apoyado por becas de SHO de los Institutos Nacionales de Salud (R01 GM092993), la Fundación Nacional de Ciencia (NSF premio CARRERA y DBI 0964216), la Oficina de Investigación Naval (ONR) Programa de Jóvenes Investigadores y el Premio de la Asociación de Minnesota de Biotecnología y Genómica Médicos. La fabricación del dispositivo se realizó en la Universidad de Minnesota Nanofabrication Center (NFC), que recibe el apoyo de la NSF a través de la Red de Infraestructura Nacional de Nanotecnología. Este trabajo también fue apoyado por becas a MR de los Institutos Nacionales de Salud (NS048357, R21 NS073684), la Sociedad Nacional de Esclerosis Múltiple (CA1060A11), el Applebaum, Hilton, Peterson y Sanford Fundaciones y la familia McNeilus. Los autores desean agradecer a Hyungsoon Im para obtener ayuda con las ilustraciones y Shailabh Kumar de asistencia con microscopía electrónica de barrido.

1. Microfabricación de micropocillos matriz de sustrato <ol><li> Comience con una oblea de silicio de 4 pulgadas con 100 nm de óxido térmico crecido. </li><li> Girar SPR-955 0,7 fotorresistente sobre la oblea a 4.000 rpm durante 30 seg. </li><li> Hornear en un placa caliente a 115 ° C durante 90 seg. </li><li> La exposicion fotoprotector. <ol><li> Utilice una máscara que creará 1 m agujeros dispuestos en una matriz hexagonal, con un período de 3 micras, donde la matriz cubre un área de 2 mm x 2 mm. </li><li> La exposicion de obleas en un paso a paso i-line utilizando un tamaño de paso de 6 mm y una exposición de 200 mJ / cm 2. </li></ol></li><li> Hornear en un placa caliente a 115 ° C durante 90 seg. </li><li> Desarrollar el uso de un desarrollador de centrifugado para depositar 2 charcos de CD-26 sobre la oblea para un tiempo de desarrollo total de 90 seg. </li><li> Enjuague bien con agua ultrapura DI y seque con N 2. </li><li> Grabe el óxido en un grabador con iones reactivos durante 6 minutos con 50 sccm de Ar, 25 sccm de CF 4 y 50 sccm de CHF 3 floala a una presión de 75 mTorr. </li><li> Silicio grabado en un ICP-zanja profunda grabador con iones reactivos durante 2 min con 63 sccm de C 4 F 8, 27 sccm de SF 6, 40 sccm de Ar, y 10 sccm de O 2 que fluye a una presión de 14 mTorr. </li><li> Enjuague en acetona, metanol, e isopropanol para eliminar resistir. </li><li> Colocar en un baño de H 2 SO 4 H 2 O 2 01:01 durante 10 minutos para limpiar la superficie. </li><li> Enjuague bien con agua ultrapura DI y seque con N 2. </li><li> Deposita 1.080 Å de Al 2 O 3 mediante deposición de capa atómica a 250 ° C. </li></ol> 2. Preparación de poli (dimetilsiloxano) escobilla de goma <ol><li> Si es posible, trabajar en una sala limpia. Trabajar de otro modo en el medio ambiente más limpio posible. </li><li> Obtener un plato de plástico Petri (u otro recipiente desechable limpio) con ≥ lados de 13 mm. </li><li> En ese plato, verter 5 g de agente de curado PDMS, seguido por 50 g de PDMS basagente de correo (o cualquier cosa con una relación 1:10 que en su mayoría va a llenar el plato de Petri). Mezclar bien con una pipeta de plástico desechable o varilla. </li><li> Eliminar las burbujas mediante la colocación de los PDMS mixtos en una cámara con una línea de vacío, aplicando vacío hasta que las burbujas salen de la mezcla (aproximadamente 30 min). </li><li> Si no está ya en la placa de Petri, vierta PDMS en placa de Petri, evitando la creación de burbujas de aire. </li><li> Curar durante la noche en una placa caliente a&gt; 50 ° C (más alto de calor durante menos tiempo también trabaja). </li><li> Con la nueva hoja de afeitar / limpio, corte cuidadosamente una pieza rectangular de aproximadamente 2,5 x 2,5 cm, dejando la superficie superior lo más limpio posible. Pelar con unas pinzas limpias. Se quita uno de los bordes (hasta 1,5 cm) presionando hacia abajo en la hoja de afeitar en un solo movimiento, para que el borde superior tan nítidas como sea posible (esto será el borde utilizado en el proceso de la escobilla de goma). </li><li> Limpiar con acetona, metanol, alcohol isopropílico y agua desionizada ultrapura, y después seque al aire con gas nitrógeno limpio. Almacene en un plato de Petri limpia. </li></ol> 3. Preparación de vesículas <ol><li> Recoger las soluciones madre de lípidos fosfatidilcolina de 25 mg / ml de huevo (PC) y 1 mg / ml de 1,2-dipalmitoil-sn-glicero-3-fosfoetanolamina-N - (lisamina rodamina B sulfonil) sal de amonio (Rho-DPPE) en cloroformo y una solución de 0,5 mg / ml de stock de monosialogangliósido GM1 en cloroformo / metanol (2:1 v / v). </li><li> En un vial de vidrio pequeño hacer una mezcla que resulta en 97% en moles de PC, 2% en moles GM1 y 1% en moles de Rho-DPPE mediante la adición de 18,9 l de 25 mg / ml de PC, 39,6 l de 0,5 mg / ml GM1 y 7,9 l de 1 mg / ml de Rho-DPPE utilizando jeringas de vidrio. </li><li> Nota:. El material suplementario para Nair et al 25 contiene una excelente hoja de cálculo Excel personalizable para calcular las cantidades de lípidos necesarios para crear las vesículas de cualquier composición deseada. </li><li> Colocar el vial en un desecador de vacío y eliminar el disolvente, dejando el vial al vacío durante 6 h. </li><li> Hacer una solución acuosa de 0,1M NaCl. Añadir 0,5 ml de la solución de NaCl 0,1 M al vial que contiene la película de lípido seca. </li><li> Dejar que la mezcla acuosa de lípidos para descansar durante la noche. </li><li> Mezcla, agitar brevemente para crear una suspensión de vesículas, a continuación, se somete a ultrasonidos durante 20 minutos en un sonicador de baño a temperatura ambiente. </li><li> Extruir la suspensión de vesículas a través de una membrana de policarbonato con 100 nm de tamaño de poro. Pase la suspensión de vesículas a través del filtro de 17x. </li><li> Guarde las vesículas extruidos en un vial de vidrio a 4 ° C. </li></ol> 4. Formación de rótula compatibles bicapas lipídicas <ol><li> Recoge 700 nm de diámetro de SiO2 cuentas. Las perlas se suministran en agua destilada con una concentración de reserva de 1,4 x 10 11 cuentas / ml. </li><li> En un tubo Eppendorf de 1,5 ml preparar una suspensión de perlas de 1 ml con una concentración de 1,5 x 10 10 perlas / ml mediante la adición de 107 l de solución de bolas de stock a 893 l de 0,1 M de NaCl. </li><li> Agite la suspensión para asegurar que es así mijo. </li><li> Centrifugar la suspensión a 1.700 xg durante 20 min y después se descartó el sobrenadante. Resuspender las perlas en 1 ml de 0,1 M de NaCl. Repita este paso dos veces más para lavar bien las cuentas. </li><li> Para formar los SSLBs, en un tubo Eppendorf de 1,5 ml añadir 25 l de la suspensión de perlas de SiO 2 a 200 l de 1 mg / ml de suspensión de vesículas. Vortex la mezcla y dejar reposar durante 1 hora a temperatura ambiente. En esta etapa la concentración SSLB debe ser 1,7 x 10 9 SSLBs / ml. </li><li> Se centrifuga a 1700 × g durante 20 min para sedimentar las SSLBs. El sedimento debería aparecer de color rosa debido a la ruptura de las vesículas con la Rho-DPPE sobre las perlas. </li><li> Desechar el sobrenadante y resuspender en 225 l de tampón fosfato salino (PBS), pH = 7,4. Repita los pasos 4.6 a 4.7 dos veces más para eliminar las vesículas intactas de la suspensión SSLB. </li></ol> 5. Asamblea de SSLB matriz <ol><li> Oblea Cleave de arrays de micropocillos que resulta en piezas rectangulares con 4-6 microwell arrays por pieza. </li><li> Vortex mezclar la suspensión SSLB, a continuación, colocar 10 l de SSLB suspensión en cada matriz de micropocillos. Lest resto durante 1 hora para permitir SSLBs se depositen en la superficie. </li><li> Lave suavemente el chip de gama micropocillos con PBS a partir de una botella de lavado, a continuación, sumergirse en un baño de PBS preparado en un plato llano. </li><li> Mientras sumergida, coloque suavemente la escobilla de goma PDMS ras en el chip de matriz de micropocillos y deslice suavemente por la superficie de 5x para eliminar SSLBs que no están inmovilizados en los pocillos. </li><li> Agarre el chip de gama micropocillos con pinzas y suavemente agite en el baño de PBS para eliminar cualquier exceso SSLBs. </li><li> Quite rápidamente el chip del baño de la escobilla de goma y colocar en un baño de PBS fresco hasta su uso posterior. Asegúrese de que la superficie superior del chip se mantiene húmedo para preservar los SSLBs. </li></ol> Nota: El método de montaje descrito anteriormente se puede utilizar para crear matrices de partículas de membrana naturales, como las partículas de mielina aisladas de cerebro de ratón, o phovesículas spholipid sin los SiO 2 soportes de talón. Este trabajo se describe en detalle en Wittenberg et al. 21 y resultados representativos se muestran abajo. 6. Toxina del Cólera Ensayo de unión <ol><li> Preparar una solución de 2 mg / ml de albúmina de suero bovino (BSA) en PBS. </li><li> Preparar una solución con la concentración deseada de Alexa 488-conjugado toxina del cólera subunidad B en PBS con 2 mg / ml de BSA. </li><li> Retire el chip de gama SSLB del baño de PBS y absorber la mayor parte de la solución de PBS usando un laboratorio de limpiar. Deja suficiente PBS en el chip para mantener la matriz SSLB hidratado. </li><li> Para evitar la unión no específica, añadir 200 l de 2 mg / ml de solución de BSA al chip de matriz SSLB. Deje reposar durante 1 hora en una caja húmeda. </li><li> Con una micropipeta, retirar 200 l de solución de la parte superior del chip de matriz SSLB. </li><li> Añadir 200 l de solución de la toxina del cólera en el chip de gama SSLB y dejar reposar durante 1 hora en una caja húmeda. </li><li> Lave suavemente el chip de gama SSLB con PBS a partir de una botella de lavado para eliminar cualquier toxina del cólera desatado. </li><li> Wick el exceso de PBS usando un laboratorio de limpiar. Antes de obtener imágenes de chip cubierta con una hoja de 24 mm x 40 mm cubierta. </li><li> Arrays de imagen con un microscopio vertical utilizando conjuntos de filtros adecuados para los fluoróforos de interés. </li><li> Analizar la intensidad de fluorescencia de SSLBs individuales de un conjunto con la función de análisis de partículas automatizado de software ImageJ. </li><li> Compilar intensidades medias de fluorescencia de SSLBs individuales en los histogramas que resumen las intensidades de SSLBs se encuentran en una matriz determinada. </li></ol>

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Spectroscopic+Detections.">Multiplexed Spectroscopic Detections.</a> Annu. Rev. Anal. Chem. 1 (1), 515-547 (2008).</li><li>Kundu, J., Levin, C. S., Halas, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+Monitoring+of+Lipid+Transfer+Between+Vesicles+and+Hybrid+Bilayers+on+Au+Nanoshells+Using+Surface+Enhanced+Raman+Scattering+(SERS).">Real-Time Monitoring of Lipid Transfer Between Vesicles and Hybrid Bilayers on Au Nanoshells Using Surface Enhanced Raman Scattering (SERS).</a> Nanoscale. 1 (1), 114-117 (2009).</li><li>Junesch, J., Sannomiya, T., Dahlin, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Properties+of+Nanohole+Arrays+in+Metal-Dielectric+Double+Films+Prepared+by+Mask-on-Metal+Colloidal+Lithography.">Optical Properties of Nanohole Arrays in Metal-Dielectric Double Films Prepared by Mask-on-Metal Colloidal Lithography.</a> ACS Nano. 6 (11), 10405-10415 (2012).</li><li>Wu, M., Holowka, D., Craighead, H. G., Baird, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Plasma+Membrane+Compartmentalization+with+Patterned+Lipid+Bilayers.">Visualization of Plasma Membrane Compartmentalization with Patterned Lipid Bilayers.</a> Proc. Natl. Acad. Sci. U.S.A. 101 (38), 13798-13803 (2004).</li><li>Hatzakis, N. S., Bhatia, V. K., Larsen, J., Madsen, K. L., Bolinger, P. Y., Kunding, A. H., Castillo, J., Gether, U., Hedegard, P., Stamou, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Curved+Membranes+Recruit+Amphipathic+Helices+and+Protein+Anchoring+Motifs.">How Curved Membranes Recruit Amphipathic Helices and Protein Anchoring Motifs.</a> Nat. Chem. Biol. 5 (11), 835-841 (2009).</li><li>Roizard, S., Danelon, C., Hassaine, G., Piguett, J., Schulze, K., Hovius, R., Tampe, R., Vogel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+G-Protein-Coupled+Receptors+in+Cell-Derived+Plasma+Membranes+Supported+on+Porous+Beads.">Activation of G-Protein-Coupled Receptors in Cell-Derived Plasma Membranes Supported on Porous Beads.</a> J. Am. Chem. Soc. 133 (42), 16868-16874 (2011).</li></ol>

Los autores no tienen intereses financieros en competencia a revelar.

En este trabajo se muestra que monodispersas de SiO 2 perlas recubiertas con bicapas lipídicas soportadas pueden ser dispuestos en matrices de micropocillos sin la necesidad de ligandos en las bicapas lipídicas o la superficie del sustrato, y las matrices se pueden utilizar para la caracterización de interacciones lípido-toxina. La constante de disociación hemos calculado para CTx/GM1 vinculante se compara favorablemente, dada la gran disparidad de los valores en la literatura, con un informe anterior po...

Membranas de bicapa lipídica son estructuras esenciales en la naturaleza. Membranas plasmáticas celulares y membranas de orgánulos están compuestas de bicapas de lípidos que incorporan una serie de moléculas que son necesarias para la vida. Muchos de los procesos que mantienen la vida ocurren en la superficie de las células o están mediadas por moléculas asociadas con membranas bicapa lipídica. De hecho, muchos productos farmacéuticos procesos diana o moléculas se encuentran sobre o en las membranas de 1,2. Por lo tanto, es necesario investigar analíticamente procesos, tales como reacciones químicas o acontecimientos de unión no covalentes que se producen en las superficies de membrana. Debido a que las membranas naturales pueden ser difíciles de aislar y / o de la interfaz con sensores, muchos investigadores emplean membranas modelo simplificado para llevar a cabo estudios analíticos. Un número de sistemas de membrana modelo se describen en la literatura, que van desde las vesículas gigantes que pueden ser decenas a cientos de micrómetros de diámetro a liposomas con dimensiones nanométricas 3,4. Alterntivamente, bicapas lipídicas planas depositados sobre soportes sólidos, es decir, soportadas bicapas de lípidos (SLBS), se pueden formar en un número de diferentes superficies y han sido ampliamente utilizados en aplicaciones biofísicas, bioquímicas, y de análisis 5. Acoplamiento SLBS con materiales eléctricos u ópticos permite la investigación de la bioquímica y biofísica de la membrana a través de la utilización de diferentes técnicas analíticas. La microscopía de fluorescencia 6, la electroquímica 7, 8 espectroscopia óptica, microscopía de sonda 9, resonancia de plasmón superficial 10, y espectrometría de masas 11 han sido empleados para estudiar la estructura y propiedades de SLBS. Matrices SLB ofrecen versatilidad adicional en el diseño de sensores para ensayos multiplex 12,13. Otras aplicaciones utilizan arrays SLB para imitar la unión que se forma entre las células inmunes 14. Métodos de preparación de matrices SLB han variado de microfluidic se acerca 15 de las que emplean las barreras físicas entre parches SLB adyacentes. 16 Otros grupos han utilizado métodos de impresión 17, 18 patrones fotoquímica y diversos enfoques nanoingeniería 19 para crear matrices SLB. En este artículo y el video adjunto se demuestra un método para formar matrices SLB depositando recubiertos-SLB SiO 2 cuentas en conjuntos ordenados de micropocillos 20. Nos referimos a los recubiertos de SLB SiO 2 cuentas como bicapas lipídicas soportadas esféricas (SSLBs). Esta técnica es una extensión del trabajo anterior que crea matrices de vesículas de fosfolípidos y biomembranas derivados de fuentes naturales 21, de donde también muestran ejemplos de resultados. Otros métodos para formar clusters de partículas de biomembrana o vesículas se han basado en los patrones de ligandos de direccionamiento específicos en las superficies que se asocian con ligandos complementarios contenidos en la superficie de la vesícula. Ejemplos incluyen biotina-asociación avidina 22,23 y esquemas de hibridación de ADN 24. Nuestro enfoque sólo requiere una matriz de micropocillos sin restos necesarias de orientación o de reconocimiento. El tamaño de SSLBs se define por el diámetro de los soportes de SiO 2 de talón, que tienen baja polidispersidad. Al ajustar el diámetro de micropocillos a sólo mayor que el diámetro SSLB, sólo un único SSLB se asienta en cada micropocillo. Un poli (dimetilsiloxano) (PDMS) de rasqueta retira entonces de la superficie de todos los SSLBs que no están inmovilizados en micropocillos. Los micropocillos y matrices SSLB resultantes tienen alta densidad (~ 10 5 SSLBs / mm 2) con 3 micras separación de centro a centro y periodicidad hexagonal. Al depositar en serie SSLBs con diferentes composiciones de lípidos, es posible crear matrices de múltiples componentes con SSLBs posicionado al azar. Para demostrar la capacidad de detección de matrices SSLB, hemos utilizado la interacción de la toxina del cólera (CTx) con un gangliósido (GM1) incorporado en los SSLBs. Conpartículas de membrana naturales, hemos sido capaces de detectar los lípidos específicos de células en matrices de múltiples componentes que contienen material de la membrana a partir de dos tipos de células diferentes.

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			<td height="42" style="height:42px;width:239px;">4-inch silicon wafers</td>
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			<td height="42" style="height:42px;width:239px;">Shipley MICROPOSIT CD-26 developer</td>
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			<td height="21" style="height:21px;width:239px;">i-line stepper</td>
			<td style="width:91px;">Canon</td>
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			<td height="42" style="height:42px;width:239px;">Vision 320 reactive ion etcher</td>
			<td style="width:91px;">Advanced Vacuum</td>
			<td style="width:119px;">Vision 320 RIE</td>
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			<td height="42" style="height:42px;width:239px;">Deep trench reactive ion etcher</td>
			<td style="width:91px;">Plasma Therm</td>
			<td style="width:119px;">SLR-770</td>
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			<td height="42" style="height:42px;width:239px;">Dow Corning Sylgard 184 poly(dimethylsiloxane) kit</td>
			<td style="width:91px;">Ellsworth Adhesives</td>
			<td style="width:119px;">184 SIL ELAST KIT 0.5KG</td>
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			<td height="42" style="height:42px;width:239px;">egg phosphatidylcholine</td>
			<td style="width:91px;">Avanti Polar Lipids</td>
			<td>840051C</td>
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			<td height="84" style="height:84px;width:239px;">1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) ammonium salt</td>
			<td style="width:91px;">Avanti Polar Lipids</td>
			<td>810158C</td>
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			<td height="42" style="height:42px;width:239px;">monosialoganglioside GM1&#160;</td>
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			<td height="42" style="height:42px;width:239px;">Silica beads</td>
			<td style="width:91px;">Bangs Laboratories</td>
			<td style="width:119px;">SS03N/4666</td>
			<td>Packaging on the bead container states the beads are 900 nm in diameter. However, after light-scattering and electron microscopy we determined the beads are roughly 700 nm in diameter.</td>
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			<td height="42" style="height:42px;width:239px;">Cholera toxin B-subunit, Alexa 488 conjugate</td>
			<td style="width:91px;">Molecular Probes</td>
			<td style="width:119px;">C-34775</td>
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			<td height="42" style="height:42px;width:239px;">Anti-oligodentrocyte antibody IgM O4, NorthernLights 557 conjugate</td>
			<td style="width:91px;">R&#38;D Systems</td>
			<td style="width:119px;">NL1326R</td>
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			<td height="42" style="height:42px;width:239px;">FM1-43</td>
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			<td style="width:119px;">T-3136</td>
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			<td height="42" style="height:42px;width:239px;">Eppendorf MiniSpin centrifuge</td>
			<td style="width:91px;">Fisher Scientific</td>
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formation of biomembrane microarrays with a squeegee-based assembly method

Membranas de bicapa lipídica forman las membranas plasmáticas de las células y definen los límites de orgánulos subcelulares. En la naturaleza, estas membranas son mezclas heterogéneas de muchos tipos de lípidos, contienen proteínas unidas a membrana y están decoradas con hidratos de carbono. En algunos experimentos, es deseable desacoplar las propiedades biofísicas o bioquímicas de la bicapa lipídica de las de la membrana natural. Tales casos requieren el uso de sistemas modelo tales como vesículas gigantes, liposomas o bicapas lipídicas soportadas (SLBS). Las matrices de SLBS son particularmente atractivos para aplicaciones de detección y imitando las interacciones célula-célula. Aquí se describe un nuevo método para la formación de matrices de SLB. Submicron de diámetro SiO 2 perlas se recubrieron primero con bicapas de lípidos para formar SLBS esféricas (SSLBs). Las perlas se depositan en una matriz de pocillos de micro-fabricada submicrónica de diámetro. La técnica de preparación utiliza un &quot;escobilla de goma&quot; para limpiar la superficie del sustrato, mientras que Leaving detrás SSLBs que se han asentado en los micropocillos. Este método no requiere ninguna modificación química del sustrato de micropocillos, ni ningún ligandos de dirección particulares sobre el SSLB. Los micropocillos están ocupados por perlas individuales debido a que el diámetro del pozo está sintonizado para ser justo más grande que el diámetro de la perla. Por lo general, más del 75% de los pozos están ocupadas, mientras que el resto permanece vacía. En búfer arrays SSLB muestran estabilidad a largo plazo de más de una semana. Múltiples tipos de SSLBs se pueden colocar en una sola matriz por deposición de serie, y las matrices se pueden utilizar para la detección, lo que se demuestra mediante la caracterización de la interacción de la toxina del cólera con el gangliósido GM1. También ponen de manifiesto que las vesículas de fosfolípidos sin los soportes de cuentas y biomembranas de fuentes celulares pueden ser dispuestos con el mismo método y los lípidos de membrana específicos de células se pueden identificar.

Cuando SiO 2 perlas se mezclan con una solución de vesículas compuestas de fosfolípidos, lípidos fluorescentes y otros lípidos, tales como gangliósidos, las vesículas se rompen en las superficies de las perlas de SiO 2 para formar SSLBs, como se muestra esquemáticamente en la Figura 1a. Después de lavar las SSLBs, una gota de solución SSLB se coloca en una matriz de micropocillos, y las perlas se dejó sedimentar a la superficie. (Figura 1b1) Esto tambié...

Watch this Scientific Journal Video about Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method at JoVE.com

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Bicapas lipídicas soportadas y partículas de membrana naturales son sistemas convenientes que pueden aproximarse a las propiedades de las membranas celulares y ser incorporadas en una gran variedad de estrategias de análisis. Aquí se demuestra un método para preparar compuestos de microarrays de soportados de lípidos de la bicapa recubiertos de SiO 2 perlas, vesículas de fosfolípidos o partículas de membrana naturales.

Formación de biomembrana Microarrays con un método basado en la Asamblea Rasero

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

formation-biomembrane-microarrays-with-squeegee-based-assembly

Research

JoVE Journal

Bioengineering

13.6K vistas.  University of Minnesota.  Bicapas lipídicas soportadas y partículas de membrana naturales son sistemas convenientes que pueden aproximarse a las propiedades de las membranas celulares y ser incorporadas en una gran variedad de estrategias de análisis. Aquí se demuestra un método para preparar compuestos de microarrays de soportados de lípidos de la bicapa recubiertos de SiO 2 perlas, vesículas de fosfolípidos o partículas de membrana naturales. 

Video: Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

Síntesis de oro Nanostar con un método de la Simiente de Plata crecimiento mediada por

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Investigación

Bioingeniería

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Microarrays de polímeros de alto rendimiento para el descubrimiento de Biomateriales

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Protocolo para Biofilm Streamer Formación en un dispositivo de microfluidos con Micro-pilares

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Fluorescencia Biomembranas Fuerza Sonda: La cuantificación simultánea de Cinética-Receptor ligando y encuadernación inducida Señalización Intracelular en una sola célula

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

Biomembrana Fabrication por la bicapa lipídica (SALB) Método-Solvente asistida

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning y Montaje de Microvasos 3D

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formación luz mediada y el patrón de hidrogeles para aplicaciones de cultivo celular

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Fabricación de pozos de agarosa personalizado para la siembra de células y tejido anillo autoensamblaje con moldes impresa en 3D

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

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.

Cuantificación simultánea de kynureninas seleccionadas analizadas por cromatografía líquida-espectrometría de masas en medio recogido de cultivos celulares oncológicos

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

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.