El apoyo financiero proporcionado por el NIH subvención HL89764, subvenciones NSF 0546012, 0730503 y 0725528.

A. biotinilación de ADN ADN plásmido se biotinilado covalentemente con las etiquetas de biotina guanina específicas como se describe por el fabricante (Mirus Bio, Madison, WI), pero a escala para que las etiquetas de ~ 1-2 biotina por ADN. ADN plásmido (pEGFP-C1, 4,9 kb, Clontech, Mountain View, CA) fue etiquetado en este protocolo. <ol><li> Disolver la cantidad deseada de pDNA en buffer TE de DNasa y RNasa libre (biología molecular, grado de calidad) de agua para hacer una solución de ADN 1μg/μL. </li><li> Llevar a cabo la reacción de marcaje con las mezclas de reacción siguiente. Añadir la etiqueta lo último reactivo. </li></ol> Para la reacción de ADN 100 microgramos: <table border="1"><tbody><tr><td> Libre de DNasa y RNasa libre de agua </td><td> 75 l </td></tr><tr><td> Buffer 10X Un etiquetado </td><td> 20 l </td></tr><tr><td> 1μg/μL ADN </td><td> 100 L </td></tr><tr><td> Etiqueta de TI reactivo </td><td> 5 l </td></tr><tr><td> Volumen Total </td><td> 200 l </td></tr></tbody></table><ol start="3"><li> Incubar la reacción a 37 ° C durante 1 hora. </li><li> Purificar la muestra marcada por el etanol o isopropanol precipitación siguiendo protocolos estándar. </li></ol> Nota: Las columnas de filtración en gel base puede conducir a la absorción UV de alta o de fluorescencia de fondo, que puede afectar a la cuantificación del ADN o la caracterización de fluorescencia. Nota: El nivel de biotinilación puede ser determinado por las pruebas basadas en HABA. B. Etiquetado de los polímeros catiónicos Cy5- Quitosano (390 kDa, 83,5% desacetilado, Vanson, Redmond, WA) fue utilizado como un polímero catiónico modelo en este estudio. Las aminas primaria gratuita en la red troncal de polímero quitosano se marcaron con Cy5-NHS (Amersham Biosciences, Piscataway, NJ). <ol><li> Para facilitar la conjugación completa de tinte Cy5, calcular la cantidad necesaria de Cy5-NHS de tal manera que la proporción molar de Cy5: aminas primarias es de 1: 200. </li><li> Ajustar el pH de la solución de quitosano (en tampón acetato 25 mM) a ~ 6,5 por adición de NaOH. Tenga en cuenta que la reacción del NHS es más eficiente a pH básico, pero la solubilidad del quitosano aquí limita el rango de pH de trabajo. </li><li> Mientras se agita, se añade lentamente la cantidad calculada de Cy5-NHS (1 mg / ml DMSO) a la solución de quitosano en forma gota a gota. </li><li> Agite la mezcla en la oscuridad a temperatura ambiente durante la noche. </li><li> Para purificar, diálisis con 10k MWCO Slide-A-Lyzer (Pierce) durante 2 horas contra tampón acetato 1% a temperatura ambiente en la oscuridad. </li><li> Vuelva a colocar buffer y otra diálisis de 2 horas a temperatura ambiente en la oscuridad. </li><li> Vuelva a colocar buffer y diálisis durante la noche a 4 ° C en la oscuridad. </li><li> Tienda purificada polímero marcado a -20 ° C. </li></ol> Nota: En este estudio, una curva estándar se construye mediante la medición de la intensidad de emisión de Cy5-NHS éster a 670 nm. Caracterizar la densidad de etiquetado a través de la medición de la emisiones que se obtengan a 670 nm de Cy5 marcado con quitosano en la curva estándar. Absorbancia también se puede utilizar para determinar el etiquetado de eficiencia, pero no se realizó aquí. C. Preparación de QD-ADN marcado y Cy5 polímeros La relación molar de pDNA a QD se mantuvo en exceso (pDNA: QD ≈ 1: 2) para asegurar la conjugación completa de los puntos cuánticos de pDNA. El número de puntos cuánticos etiquetados en cada pDNA se puede estimar a través de TEM imágenes u otros recursos equivalentes. En nuestro estudio, el número de puntos cuánticos por pDNA se estima en ~ 3.1 por TEM y espectroscopia de moléculas individuales. Use un Millipore Milli-Q agua gradiente (&gt; 18,0 MW, 0.2um filtrados) durante la preparación. <ol><li> Calcular la cantidad necesaria de quitosano para 10μg pDNA según deseada N / P, la relación teórica de aminas protonadas en la solución de quitosano a los fosfatos negativos en la solución de ADN. </li><li> Añadir estreptavidina-funcionalizados 605QDs (Qdot 605 ITK, Invitrogen, Carlsbad, CA) en la solución de biotina pDNA. </li><li> Incubar la solución a temperatura ambiente en la oscuridad durante 15 min. </li><li> Agregue el QD-ADN marcado en 50 mM solución de sulfato de sodio para hacer el volumen final de 200μL. </li><li> Diluir Cy5-quitosano, según deseada N / P, con agua Milli-Q para hacer que el volumen final de 200μL. </li></ol> Nota: Mantenga la reacción en la oscuridad para evitar photobleaching posible. Importante: Tenga cuidado al utilizar el Qdot 605 ITK ™ estreptavidina conjugada (la serie CTI), como puntos cuánticos en este catálogo están diseñados con el propósito de FRET. La serie Qdot regulares se conjugan con una capa de PEG para evitar la unión no específica, especialmente para el etiquetado de celular. Sin embargo, esta capa adicional amplía la distancia donador-aceptor, lo que resulta en rrelucida la eficiencia de transferencia de energía. D. La fabricación de la SU-8 Masters usando fotolitografía estándar <ol><li> Oblea de silicio se pirañas limpia y al horno a 200 ° C durante 5 min. </li><li> Por el espesor de 25μm maestro designado, capa fotosensible girar la negativa (SU-8 2025, Microchem, Newton, MA) en la oblea de Si a 2000 rpm durante 30 segundos. </li><li> Soft hornear la oblea en una placa con una rampa de 65 ° C / hr a 95 ° C. </li><li> Exponga a la luz UV (365 nm) para 250mJ/cm 2 a través de una máscara de la película (CAD / Servicios de arte, Bandon, Oregón) que contiene el diseño de microcanales. </li><li> Después de la exposición hornear la oblea en una placa con una rampa de 65 ° C / hr a 95 ° C. </li><li> Desarrollar la oblea con SU-8 desarrollador fotosensible. </li><li> La oblea patrón es difícil al horno en una placa con una rampa de 65 ° C / hr a 200 ° C. Mantener la oblea a 200 ° C durante al menos 5 horas, luego se enfrían gradualmente la oblea a temperatura ambiente. </li></ol> Importante: rampa gradual durante el SU-8 proceso de cocción maestro es necesario, de lo contrario el SU-8 puede separarse de la estructura de la oblea de silicio o grietas en la estructura de SU-8 puede ser inducida por el estrés de liberación. E. Replica Moldeo por inyección de PDMS de los maestros y la vinculación a la tapa de vidrio <ol><li> El maestro de SU-8 se coloca en un recipiente pesado. </li><li> Mezcla de silicona y elastómeros agente de curado (poli (dimetilsiloxano), PDMS, Sylgard 184, Dow Corning, Midland, MI) en un 10: 1 relación. </li><li> Vierta la mezcla de PDMS sobre el SU-8 maestros y abandonar el barco con un peso de un secador de vacío para eliminar las burbujas. </li><li> Curar el PDMS a 65 ° C durante 1-2 horas. </li><li> Pelar la franja de PDMS del molde maestro Si. </li><li> Golpe canal entradas y salidas del dispositivo de simulación. </li><li> Limpie la tira de PDMS y cubierta de vidrio con etanol y secar al aire. </li><li> El tratamiento de la limpieza PDMS tira y cubierta de vidrio con plasma de oxígeno (20 W para 1 minuto). </li><li> Inmediatamente de bonos de la franja de PDMS con cubierta de vidrio. </li><li> Deje el chip de microfluidos en condiciones de servidumbre en el horno a 95 ° C durante la noche. </li></ol> Importante: El tratamiento de plasma y hornear durante la noche son esenciales para la resistencia de unión mejorada. F. Monitor de la formación de ADN Nanocomplexes En el dispositivo de microfluidos <ol><li> Llene el canal de microfluidos con agua (para asegurar que no haya burbujas en el canal de microfluidos), antes de cargar los reactivos para asegurar el flujo suave durante el experimento. </li><li> Cargar el ADN QD-etiquetado y marcado con Cy5 soluciones de quitosano en dos jeringas de vidrio individuales, a través de la tubería se describe en el vídeo. </li><li> Conecte el tubo con las dos entradas de dispositivos de microfluídica. Tenga cuidado de no introducir aire durante el proceso. Establecer la velocidad de flujo en 20nL/min (PHD-2000 bomba de jeringa, Holliston, MA), bajo condiciones de flujo laminar. </li><li> Compruebe los microcanales bajo el microscopio. </li><li> Cuando el flujo es estable (~ 15 a 20 minutos), QD mediada por FRET deben ser observadas en el centro del canal. </li><li> Tome fotografías de fluorescencia (CCD enfriado, Qimaging, BC, Canadá) en diferentes lugares a lo largo del canal. </li><li> Analizar las imágenes de fluorescencia con ImageJ y OriginLab. </li></ol><img src="/files/ftp_upload/1432/1432fig1.jpg" alt="Figura 1" /> Figura 1. QD-FRET proporciona una indicación sensible de la aparición de Nanocomplexes ADN de auto-ensamblaje <ol style="list-style-type:lower-alpha;"><li> Puntos cuánticos mediada por la transferencia de energía de resonancia fluorescente (FRET-QD) puede proporcionar una indicación cuantitativa y muy sensible de la estabilidad Polyplex en entornos de extra-o intra-celular, que permite la detección inequívoca de la aparición de las interacciones entre el ADN y el portador del gen. La pareja FRET, 605QD y Cy5, fue elegido basado en la maximización de solapamiento espectral entre el donante y el receptor y minimizar los posibles cross-talk. Para esta pareja, la distancia de Förster es 69.4Å 3. </li><li> Auto-ensamblaje de la nanocomplexes ADN QD-FRET. ADN plásmido aniónicos (pDNA) y el portador del gen catiónico se marcaron con QD (donante de energía) y Cy5 (aceptor de energía), respectivamente. QD-FRET nanocomplexes se formaron a través de coacervación compleja electrostática. A la excitación a 488 nm, QD-FRET mediada Cy5 emisiones indicó la formación de un nanocomplex compacto e intacta. El tiempo de residencia (R t) se puede calcular de acuerdo a la distancia (x), que mide desde donde las dos corrientes se encuentran en la posición de la reacción que se investiga, y la velocidad media del flujo (v). Debido a la naturaleza del flujo laminar, la mezcla sólo se lleva a cabo en la interfaz (el centro de cada imagen), lo que permite calcular de forma precisa el transporte de masa en función de t R. Resolución temporal puede ser ajustado por la variación de la applied caudales. (Recuadro) FRET mediada por la señal se observó inmediatamente en la interfaz cuando las dos corrientes se reunieron, lo que indica que la unión fue rápida, que ocurre dentro de unos pocos milisegundos de acuerdo con los caudales aplicados. Barra de escala: 100μm. </li></ol>

<ol>
	<li>Ho, Y. P., Chen, H. H., Leong, K. W., Wang, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+intracellular+stability+and+unpacking+of+DNA+nanocomplexes+by+quantum+dots-FRET.">Evaluating the intracellular stability and unpacking of DNA nanocomplexes by quantum dots-FRET.</a> J Control Release. 116, 83-89 (2006).</li><li>Chen, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+comparison+of+intracellular+unpacking+kinetics+of+polyplexes+by+a+model+constructed+from+quantum+dot-FRET.">Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum dot-FRET.</a> Mol. Ther. 16, 324-332 (2008).</li><li>Zhang, C. Y., Yeh, H. C., Kuroki, M., Wang, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Quantum-Dot-Based+DNA+Nanosensor.">Single-Quantum-Dot-Based DNA Nanosensor.</a> Nat Mat. 4, 826-831 (2005).</li></ol>

The authors have nothing to disclose.

<ul><li> Importancia de nuestro trabajo: <ol><li> Este es el primer intento de controlar poliméricos ADN nanocomplex de auto-ensamblaje cinética en tiempo real (resolución de milisegundos) a través de QD-FRET respuestas dentro de un chip de microfluidos sencillos. </li><li> QD mediada por FRET proporciona una indicación muy sensible y cuantitativo de la aparición de las interacciones moleculares y en todo el proceso de auto-ensamblaje, mientras que microfluidos ofrece un microambiente bien controlados para analizar espacialmente el proceso durante la síntesis de ADN nanocomplex. </li><li> La integración de la microfluídica y nanofotónica sugiere un enfoque nuevo e interesante para investigar cualquier tipo de reacciones de complejos. </li><li> El resultado de QD-FRET nanocomplexes ADN poliméricos podría ser fácilmente aplicada para el establecimiento de relaciones estructura-función. 1,2 </li></ol></li></ul>

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		<div>
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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>SU-8</td>
<td>&nbsp;</td>
<td>MicroChem</td>
<td>SU-8 2025</td>
<td>&nbsp;</td>
<tr>
<td>PDMS</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<tr>
<td>Plasmid DNA</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>pEGFP-C1</td>
<td>4.9 kb, MW = 3.3 x 106</td>
</tr>
<tr>
<td>LabelIT Biotin Labeling Kit</td>
<td>&nbsp;</td>
<td>Mirus Bio LLC</td>
<td>MIR 3400</td>
<td>Standard protocol yields labeling efficiency of approximately one label every 20-60 bp of double-stranded DNA, The density of labeling was adjusted in this work.</td>
</tr>
<tr>
<td>Streptavidin 605QD</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>Qdot&reg; 605 ITK&trade; Streptavidin Conjugate</td>
<td>&nbsp;</td>
<tr>
<td>Cy5-NHS Ester</td>
<td>&nbsp;</td>
<td>Amersham Biosciences</td>
<td>PA15101</td>
<td>&nbsp;</td>
<tr>
<td>Chitosan</td>
<td>&nbsp;</td>
<td>Vanson</td>
<td>390 kDa </td>
<td>83.5% deacetylated</td>
</tr>
<tr>
<td>Cover Glass</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-545C</td>
<td>No. 1; Size: 40 x 22mm</td>
</tr>
<tr>
<td>Gastight Glass Syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>TLL series</td>
<td>50&mu;L to 500&mu;L depending on sample volume</td>
</tr>
<tr>
<td>Tygon Tubing</td>
<td>&nbsp;</td>
<td>SmallParts</td>
<td>0.02 ID, 100ft</td>
<td>Tygon Tubes Microbore, 0.02 ID, 100ft</td>
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<tr>
<td>Connector</td>
<td>&nbsp;</td>
<td>SmallParts</td>
<td>HTX-23R</td>
<td>Customized in length of 0.750"</td>
</tr>
<tr>
<td>Syringe Pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>PHD-2000</td>
<td>&nbsp;</td>
<tr>
<td>CCD</td>
<td>&nbsp;</td>
<td>Qimaging</td>
<td>Intensified Retiga Cooled</td>
<td>&nbsp;</td>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>BX-51</td>
<td>100W mercury arc lamp</td>
</tr>
<tr>
<td>ImageJ</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>v1.36b</td>
<td>http://rsb.info.nih.gov/ij</td>
</tr>
<tr>
<td>Origin Pro8</td>
<td>&nbsp;</td>
<td>OriginLab</td>
<td>Student Version</td>
<td>&nbsp;</td>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>475AF40</td>
<td>Excitation filter in both channels</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>595AF60</td>
<td>Emission filter in 605QD channel</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>670DF40</td>
<td>Emission filter in QD-FRET channel</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>500 DRLP</td>
<td>Long pass dichroic in 605QD channel</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>595DRLP</td>
<td>Long pass dichroic in QD-FRET channel</td>
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combining qd-fret and microfluidics to monitor dna nanocomplex self-assembly in real-time

Los avances en genómica continuará impulsando el desarrollo de terapias que pueden dirigirse a la patogénesis a nivel celular y molecular. Por lo general funcional dentro de la célula, basadas en ácidos nucleicos terapéuticos requieren un eficiente sistema de suministro intracelular. Un enfoque ampliamente adoptado es el ADN complejo con un portador del gen para formar nanocomplexes electrostática a través de auto-ensamblaje, facilitando la captación celular de ADN, mientras que lo protege contra la degradación. El desafío radica en el diseño racional de los portadores del gen eficiente, ya que la disociación prematura o excesivamente unión estable sería perjudicial para la captación celular y la eficacia terapéutica. Nanocomplexes sintetizado por el volumen de mezcla mostró una amplia gama de comportamiento intracelular desembalaje y el tráfico, que se atribuyó a la heterogeneidad en el tamaño y la estabilidad de nanocomplexes. Esta heterogeneidad dificulta la evaluación precisa de la cinética de auto-ensamblaje y se suma a la dificultad en la correlación de sus propiedades físicas a las eficiencias de transfección o bioactividad. Se presenta una nueva convergencia de la nanofotónica (es decir, QD-FRET) y microfluídica para caracterizar la cinética en tiempo real de la nanocomplex auto-ensamblaje bajo flujo laminar. QD-FRET proporciona una indicación muy sensible a la aparición de las interacciones moleculares y medida cuantitativa de todo el proceso de síntesis, mientras que microfluidos ofrece un microambiente bien controlados para analizar espacialmente el proceso con una alta resolución temporal (~ milisegundos). Para el sistema de modelo de nanocomplexes poliméricos, dos etapas distintas en el proceso de auto-ensamblaje fueron capturados por esta plataforma analítica. El aspecto cinética del proceso de auto-ensamblaje obtenido en la microescala sería especialmente valioso para microreactor basada en las reacciones que son relevantes para muchas aplicaciones de micro-y nano-escala. Además, nanocomplexes puede ser personalizado mediante un diseño adecuado de los dispositivos de microfludic, y el resultado QD-FRET nanocomplexes polímeros de ADN pueden ser fácilmente aplicada para el establecimiento de relaciones estructura-función.

Watch this Scientific Journal Video about Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time at JoVE.com

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Se presenta un nuevo y poderoso de integración de la nanofotónica (QD-FRET) y microfluídica para investigar la formación de polyplexes polielectrolito, que se espera que proporcione un mejor control y síntesis de polyplexes uniforme y personalizable para a base de ácido nucleico futuras terapias.

La combinación de QD-FRET y microfluídica para monitorear Nanocomplex ADN auto-ensamblado en tiempo real

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

combining-qd-fret-microfluidics-to-monitor-dna-nanocomplex-self

Research

JoVE Journal

Biology

11.0K vistas.  Johns Hopkins University. Se presenta un nuevo y poderoso de integración de la nanofotónica (QD-FRET) y microfluídica para investigar la formación de polyplexes polielectrolito, que se espera que proporcione un mejor control y síntesis de polyplexes uniforme y personalizable para a base de ácido nucleico futuras terapias.

Video: Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and biogeochemical flux. However, to take advantage of nutrient patches in the ocean, swimming microbes must overcome the influences of physical forces including molecular diffusion and turbulent shear, which will limit the availability of patches and the ability of bacteria to locate them. Until recently, methodological limitations have precluded direct examinations of microbial behaviour within patchy habitats and realistic small-scale flow conditions. Hence, much of our current knowledge regarding microbial behaviour in the ocean has been procured from theoretical predictions. To obtain new information on microbial foraging behaviour in the ocean we have applied soft lithographic fabrication techniques to develop 2 microfluidic devices, which we have used to create (i) microscale nutrient patches with dimensions and diffusive characteristics relevant to oceanic processes and (ii) microscale vortices, with shear rates corresponding to those expected in the ocean. These microfluidic devices have permitted a first direct examination of microbial swimming and chemotactic behaviour within a heterogeneous and dynamic seascape. The combined use of epifluorescence and phase contrast microscopy allow direct examinations of the physical dimensions and diffusive characteristics of nutrient patches, while observing the population-level aggregative response, in addition to the swimming behaviour of individual microbes. These experiments have revealed that some species of phytoplankton, heterotrophic bacteria and phagotrophic protists are adept at locating and exploiting diffusing microscale resource patches within very short time frames. We have also shown that up to moderate shear rates, marine bacteria are able to  fight the flow  and swim through their environment at their own accord. However, beyond a threshold high shear level, bacteria are aligned in the shear flow and are less capable of swimming without disturbance from the flow. Microfluidics represents a novel and inexpensive approach for studying aquatic microbial ecology, and due to its suitability for accurately creating realistic flow fields and substrate gradients at the microscale, is ideally applicable to examinations of microbial behaviour at the smallest scales of interaction.  We therefore suggest that microfluidics represents a valuable tool for obtaining a better understanding of the ecology of microorganisms in the ocean.

The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described.

Respuesta quimiotáctica de los microorganismos marinos a escala micro nutrientes Capas

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and ...

Investigación

Biología

Studies of Bacterial Chemotaxis Using Microfluidics - Interview

Los estudios de quimiotaxis bacteriana utilizando microfluídica - Entrevista

Applying Microfluidics to Electrophysiology

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities.  Specifically, the motivation for the microfluidic brain slice device is discussed including how the device docks to standard perfusion chambers and the technique of passive pumping which is used to deliver boluses of neuromodulators to the brain slice.  By simplifying the device design, we are able to achieve a practical solution to the current unmet electrophysiology need of applying multiple neuromodulators across multiple regions of the brain slice.  This is achieved by substituting the standard coverglass substrate of the perfusion chamber with a thin microfluidic device bonded to the coverglass substrate.  This was then attached to the perfusion chamber and small holes connect the open-well of the perfusion chamber to the microfluidic channels buried within the microfluidic substrate.  These microfluidic channels are interfaced with ports drilled into the edge of the perfusion chamber to access and deliver stimulants.  This project represents how the field of microfluidics is transitioning away from proof-of concept device demonstrations and into practical solutions for unmet experimental and clinical needs.

La aplicación de microfluídica para Electrofisiología

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities.  Specifically, the motivation for the ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Microfluídica digitales para el procesamiento automatizado de Proteómica

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

La determinación de perfiles genéticos de expresión en C. elegans Utilización de microarrays y PCR en tiempo real

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

Combinando Manipulación sola molécula y de imagen para el estudio de las interacciones proteína-ADN

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Imágenes en tiempo real de Soltero Engineered ARN transcritos en células vivas utilizando Ratiometric bimoleculares Balizas

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Alta resolución Cuantificación de celulosa cristalina acumulación en<em&gt; Arabidopsis</em&gt; Las raíces para monitorear la célula específica de tejido de pared Modificaciones

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Detección y caracterización de autoensamblaje de proteínas en Vivo por citometría de flujo

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. Upon wounding of a leaf, an increase in cytosolic calcium ion concentration (Ca2+ signal) occurs at the wound site. This signal is rapidly transmitted to undamaged leaves, where defense responses are activated. Our recent research revealed that glutamate leaking from the wounded cells of the leaf into the apoplast around them serves as a wound signal. This glutamate activates glutamate receptor-like Ca2+ permeable channels, which then leads to long-distance Ca2+ signal propagation throughout the plant. The spatial and temporal characteristics of these events can be captured with real-time imaging of living plants expressing genetically encoded fluorescent biosensors. Here we introduce a plant-wide, real-time imaging method to monitor the dynamics of both the Ca2+ signals and changes in apoplastic glutamate that occur in response to wounding. This approach uses a wide-field fluorescence microscope and transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-based Ca2+ and glutamate biosensors. In addition, we present methodology to easily elicit wound-induced, glutamate-triggered rapid and long-distance Ca2+ signal propagation. This protocol can also be applied to studies on other plant stresses to help investigate how plant systemic signaling might be involved in their signaling and response networks.

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

Wide-Field, Imágenes en tiempo real de señales de heridas locales y sistémicas en Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. ...