YG es apoyado por una beca del BID. IN es un tipo Alon y miembro de la facultad ‭ Edmond J. ‬ Safra Centro de Bioinformática de la Universidad de Tel Aviv ‭. ‬ Esta investigación fue apoyada por la ISF beca 1499/10.

1. Creación de un Master cinta Scotch-5 <ol><li> Dibujar o imprimir diseño deseado microcanal, a escala, en el papel. En nuestro caso, el diseño se compone de dos canales en forma de Y, cada uno de 3 mm de ancho (Figura 2). </li><li> Cubra un portaobjetos de vidrio con capas de cinta adhesiva. El número de capas determinará la altura del canal (alrededor de 60 micras por capa). Utilizamos tres capas de cinta scotch. </li><li> Coloque el esquema de trazado sobre una superficie plana. Alinear a la diapositiva en el patrón de diseño. Cortar cuidadosamente la cinta adhesiva en el portaobjetos de vidrio con un bisturí de acuerdo con el diseño. </li><li> Retire la cinta Scotch de todas las regiones de la lámina de vidrio, excepto aquellos en el diseño del microcanal. </li><li> Colocar el portaobjetos en un horno de calentamiento a 65 ° C durante 2-3 min. Limpie suavemente con etanol. </li></ol> 2. Fabricar un dispositivo microfluídico de PDMS <ol><li> Mezclar la base y componentes de curado de PDMS como se recomienda por el fabricante. Utilizamos una mezcla 10:1relación de base a agente de curado. Verter aproximadamente 30 ml de mezcla de PDMS en una placa Petri a una altura de ~ 0,5 cm. Desgasificar el PDMS en el vacío si es necesario. Sumerja el portaobjetos en el PDMS con la cinta Scotch estampado mirando hacia arriba. La colocación del patrón después de que el PDMS evita la formación de burbujas de aire entre la corredera y fondo del plato. Curar durante 48 horas a 65 ° C, lo que hace que la antena es horizontal utilizando un nivel de burbuja. </li><li> En una nueva placa de Petri 90 mm vierta 3 ml de mezcla de PDMS, resultando en una capa de aproximadamente 0,5 mm de profundidad. (Si una recubridora de rotación está disponible, se puede utilizar para esta etapa). </li><li> Degas y curar como en 2.1. </li><li> Suavemente cortar el dispositivo de microfluidos al tamaño deseado con un escalpelo. Esta capa se denomina la &quot;capa de flujo&quot;. </li><li> Cortar un trozo de tamaño similar de capa delgada PDMS de la segunda placa de Petri. Esto se denomina &quot;capa de pozos&quot;. </li><li> Utilice un golpeador biopsia de tamaño apropiado (Usamos 1,2 mm ID) para perforar agujeros para las entradas y salidas en la capa de flujo. </li&gt; <li> Coloque la capa de pozos en un portaobjetos de vidrio de espesor sobre la disposición de diseño y rompa los pozos, utilizando 2 mm ID golpeador biopsia, en alineación con los microcanales de la presentación. </li><li> Limpiar ambas capas de PDMS, así como un 24 mm x 60 mm cubreobjetos de vidrio con etanol. Seque y mantener limpio. </li><li> Plasma tratar tanto el cubreobjetos de vidrio y la capa de PDMS pozos utilizando estándar grabador de plasma (por no reversible bonding) o un tratador corona de mano 6 para la unión reversible. Colocar con cuidado la capa de pozos en la parte superior del cubreobjetos para causar la adhesión (Figura 3). Frote suavemente las burbujas de aire (si es necesario). </li></ol> 3. Celular Experimento de imágenes <ol><li> Asegúrese de tener listo el soporte deseado en jeringas apropiadas conectados a tubos de plástico flexible con un diámetro interno de 0,02 &quot;(Tygon) en la bomba de jeringa. </li><li> Crecer S. cerevisiae a la fase deseada en YPD líquido. Vortex a fondo y el lugar 300 μl de cada cepa en un tubo de microcentrífuga. </li><li> Coloque suavemente 1 l de Concanavalina A (Sigma) 2 mg / ml en cada pocillo. Lavar el exceso de Concanavalina A partir de cada pocillo dos veces con cuidado pipeteando agua. </li><li> Mientras que la Concanavalina A se está secando, lavar dos veces con 300 cepas l de medio SC falta de glucosa. Lavadora glucosa residual de las paredes celulares ayuda a la adherencia adecuada a la concanavalina A. </li><li> Células Vortex a fondo. Pipetear 0,75 l o menos de cada suspensión de células en su propio pozo (Figura 3). Lave suavemente a las células residuales con medio rico. Los siguientes dos pasos deben realizarse rápidamente a fin de evitar que las células se seque. </li><li> Plasma tratar el chip y los pozos de fondo con el tratador corona de mano, teniendo cuidado de no golpear a los pozos directamente. Colocar con cuidado el chip en los pocillos, prestando mucha atención a la alineación entre los dos (este paso se puede realizar en un estereoscopio). Presione suavemente para adherir las dos capas de la EPMS. Lentamente llene completamente el dispositivo con aproximadamente 50 l medio rico, asegurándose de que no hay burbujas de aire se dejan dentro de la canal. </li><li> Conecte el dispositivo, la inserción de los tubos en las entradas apropiadas e iniciar el flujo de los medios de comunicación. Tener cuidado de que no hay burbujas de aire se introduce en el dispositivo, ya que pueden ser difíciles de eliminar, esto se puede lograr mediante la desconexión de una entrada o salida y golpeando suavemente sobre el dispositivo en el que las burbujas son, con cuidado de no romper el cubreobjetos de vidrio. </li><li> Colocar bajo microscopio y buscar los puntos apropiados de formación de imágenes en cada pocillo. </li><li> Mantener las células bajo un flujo de medio rico durante 1 hora o más para permitir la recuperación, si es necesario. </li><li> Iniciar el experimento: se utiliza el flujo constante de cada una de las dos bombas de jeringa para evitar el reflujo de los medios de comunicación, el cambio de velocidades relativas de flujo como se desee. Ajuste de la bomba A a 0,8 ml / hr y B de la bomba a 0,2 ml / hr resultados en medio A que fluye sobre 80% de la anchura del canal (Figura 4). Esto puede ser dinámicamente ccolgado mediante el cambio de los dos caudales. Las nuevas condiciones estabilizar dentro de segundos a lo largo de toda la longitud de la canal 2. </li></ol>

<ol>
	<li>Dertinger, S. K., Jiang, X., Li, Z., Murthy, V. N., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradients+of+substrate-bound+laminin+orient+axonal+specification+of+neurons.">Gradients of substrate-bound laminin orient axonal specification of neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 12542-12547 (2002).</li><li>Hersen, P., McClean, M. N., Mahadevan, L., Ramanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+processing+by+the+HOG+MAP+kinase+pathway.">Signal processing by the HOG MAP kinase pathway.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 7165-7170 (2008).</li><li>Paliwal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAPK-mediated+bimodal+gene+expression+and+adaptive+gradient+sensing+in+yeast.">MAPK-mediated bimodal gene expression and adaptive gradient sensing in yeast.</a> Nature. 446, 46-51 (2007).</li><li>Bennett, M. R., Hasty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+measuring+gene+network+dynamics+in+single+cells.">Microfluidic devices for measuring gene network dynamics in single cells.</a> Nat. Rev. Genet. 10, 628-638 (2009).</li><li>Shrirao, A. B., Perez-Castillejos, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+Fabrication+of+Microfluidic+Devices+by+Replicating+Scotch-tape+Masters.">Simple Fabrication of Microfluidic Devices by Replicating Scotch-tape Masters.</a> Chips &#38; Tips. , (2010).</li><li>Haubert, K., Drier, T., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS+bonding+by+means+of+a+portable,+low-cost+corona+system.">PDMS bonding by means of a portable, low-cost corona system.</a> Lab Chip. 6, 1548-1549 (2006).</li><li>Gerber, D., Maerkl, S. J., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+microfluidic+approach+to+generating+protein-interaction+networks.">An in vitro microfluidic approach to generating protein-interaction networks.</a> Nat. Methods. 6, 71-74 (2009).</li></ol>

No hay conflictos de interés declarado.

En este trabajo se presenta un método de sobremesa sencillo para crear un dispositivo de microfluidos que permite las siguientes cepas de levadura de manera simultánea en condiciones dinámicas. Después de varias cepas en un canal proporciona una herramienta fiable para la comparación de la dinámica de las respuestas de células individuales en múltiples cepas. Una ventaja de nuestro enfoque es la capacidad para fabricar el dispositivo con técnicas sencillas sin la necesidad de una sala limpia. De hecho, también...

Las células constantemente reaccionar a un entorno que cambia dinámicamente, cambiando su metabolismo, el perfil de la transcripción y de las funciones celulares. Para estudiar estos fenómenos, los métodos que puedan cuantificar dichos cambios son necesarios. Un tipo de lectura para tales respuestas es el cambio en la localización de los factores de transcripción relacionados con el estrés en respuesta a estrés nutricional. Los dispositivos microfluídicos 1 se han utilizado para manipular dinámicamente condiciones ambientales a las células 2-4. Se presentan varias ventajas para imágenes de células vivas: el medio ambiente de las células puede ser controlada con precisión y cambiar dinámicamente; células pueden ser vivo-imagen formada en todo momento, incluyendo durante la manipulación de las condiciones externas, y volúmenes mínimos de reactivos se requieren. Un diseño muy simple para un dispositivo de microfluidos, permitiendo alternancia entre dos condiciones, es un dispositivo en forma de Y 2. Nos utilizan habitualmente en forma de Y que permiten que los dispositivos de microfluidosaplicación de cambios dinámicos en las células en el canal. Usamos este dispositivo para seguir los cambios de localización de marcado con fluorescencia factores de transcripción relacionados con el estrés en las células de levadura. Seguimos estos cambios en las nuevas condiciones nutricionales. Por ejemplo, se puede proporcionar un impulso (o una serie de pulsos) de la glucosa que contienen medio dentro de un período de no glucosa-medio, con una resolución temporal muy fina. A menudo, en estos experimentos, se está interesado en comparar las respuestas de las cepas celulares diferentes (por ejemplo, mutantes diferentes, o diferentes cepas salvajes). El canal en forma de Y está limitado a una cepa en cada canal - si más de una cepa se utiliza, no hay ninguna manera sencilla de distinguir entre las cepas. Para superar esto, los canales se pueden utilizar diferentes. Si queremos aplicar las mismas condiciones dinámicas a múltiples cepas, nos gustaría combinar el concepto Y-canal con múltiples canales. Hay dos desafíos en la implementación de esta solución: Puede ser difícil de aplicar tél mismas condiciones al mismo tiempo a todos los canales, y no hay una limitación geométrica con el número de Y-canales que se pueden montar en un solo dispositivo. Estábamos por lo tanto buscando una manera para ver varias cepas en un canal en condiciones dinámicas. Aquí se describe un dispositivo de microfluidos de dos capas destinado a cepas de imágenes múltiples en un canal bajo las mismas condiciones dinámicas. La capa inferior consiste en PDMS delgada con una fila de agujeros. Esta capa está unida al cubreobjetos de vidrio en la creación de los pozos que vamos a colocar las diferentes cepas, adhiriéndose con concanavalina A para el vidrio. La segunda capa es un dispositivo microfluídico en forma de Y creado usando el método de la cinta adhesiva 5. Después de colocar las cepas en los pocillos de la segunda capa es rápidamente alineado y conectado a la primera, la creación de un canal con varios pozos que contienen las diferentes cepas. Este último dispositivo permite tras varias cepas en un canal, donde todoscepas se someten a las mismas condiciones que en el mismo tiempo exacto. Todo el diseño y el proceso de producción se puede hacer en el laboratorio, utilizando medios sencillos, sin litografía blanda.

<table border="1"><tbody><tr><td> Reactivo / Equipo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> PDMS-SYLGARD 184 </td><td> Dow Corning EE.UU. </td><td></td></tr><tr><td> Desecador de vacío </td><td> Nalgene </td><td> 5310-0250 </td></tr><tr><td> Biopsia pegadores </td><td> Ted Pella Inc. </td><td> Harris Uni-Core 15.076 (2 mm), 15.074 (1,2 mm) </td></tr><tr><td> Las bombas de jeringa </td><td> Chemyx </td><td> Fusion 200 </td></tr><tr><td> Corona tratador </td><td> Technic Electro-productos </td><td> BD-20 </td></tr><tr><td> Tygon tubería </td><td> Tygon </td><td> S-54-HL </td></tr><tr><td> Concanavalina-A </td><td> Sigma </td><td> C7275 </td></tr><tr><td> Cinta adhesiva </td><td> 3M Scotch </td><td> Cinta transparente 1/2 &quot; </td></tr></tbody></table>

a microfluidic device for studying multiple distinct strains

El estudio de las respuestas celulares a los cambios ambientales plantea muchos retos experimentales: las células necesitan para formar imágenes en condiciones cambiantes, a menudo de una manera comparativa. Placas de pocillos múltiples, se utilizan habitualmente para comparar diferentes cepas o líneas celulares, pero permiten un control limitado sobre la dinámica del medio ambiente. Los dispositivos microfluídicos, por otra parte, permite el control exquisito dinámico sobre las condiciones del entorno, pero es difícil de imagen y distinguir más de unas pocas cepas de ellos. Aquí se describe un método para fabricar fácil y rápidamente un dispositivo de microfluidos capaz de aplicar las condiciones de cambio dinámico a múltiples cepas de levadura distintas en un canal. El dispositivo está diseñado y fabricado por medios sencillos sin necesidad de litografía blanda. Se compone de un canal de flujo en forma de Y unido a una segunda capa albergar micropocillos. Las cepas se colocan en pocillos separados, y fotografiado bajo las exactas condiciones dinámicas mismos. Nos demo denstrate el uso del dispositivo para la medición de las respuestas de proteína de localización a los pulsos de cambios de nutrientes en diferentes cepas de levadura.

Para demostrar la separación entre las diferentes cepas que fotografiado dos cepas de levadura distinguibles en pocillos alternos. La formación de imágenes completo pozos muestra ninguna fuga entre los pocillos de células (Figura 5a, b). Ambas cepas tienen la MSN2 factor de transcripción etiquetados con YFP. Para probar el efecto simultáneo de las condiciones cambiantes dinámicamente, cambiamos las velocidades de flujo en los dos canales de entrada, creando un paso de medio sin glucosa, lo que re...

Watch this Scientific Journal Video about A Microfluidic Device for Studying Multiple Distinct Strains at JoVE.com

A Microfluidic Device for Studying Multiple Distinct Strains

Se presenta un método simple para producir los dispositivos de microfluidos capaces de aplicar similares condiciones dinámicas a múltiples cepas distintas, sin la necesidad de una sala limpia o litografía blanda.

Un dispositivo de microfluidos para el estudio de varias cepas distintas

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

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Research

JoVE Journal

Bioengineering

8.7K vistas.  Tel Aviv University. Se presenta un método simple para producir los dispositivos de microfluidos capaces de aplicar similares condiciones dinámicas a múltiples cepas distintas, sin la necesidad de una sala limpia o litografía blanda.

Video: A Microfluidic Device for Studying Multiple Distinct Strains

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

Dispositivo de microfluidos para volver a crear un microambiente tumoral<em&gt; In Vitro</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Investigación

Bioingeniería

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

Mezcladores de microfluidos para el estudio plegamiento de proteínas

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

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 ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

Analizando Mezcla falta de homogeneidad en un dispositivo de microfluidos por Microscale Schlieren Técnica

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Totalmente automatizada de dispositivos de microfluidos para centrífuga Ultrasensible detección de proteínas a partir de sangre entera

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Co-cultivo de estar con microbioma intestinal humana microconstruidos Villi en una Gut-on-a-Chip dispositivo de microfluidos

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

Seca de la película basada en la fotoresistencia el Biosensor electroquímico de microfluidos plataforma: Fabricación de dispositivo, preparación del análisis de la en-viruta y el funcionamiento del sistema

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

Perfusable Red Vascular con un modelo de tejido en un dispositivo de microfluidos

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Generación de condiciones ambientales dinámicas utilizando un dispositivo microfluídico de alto rendimiento

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Configuración de un dispositivo microfluídico para la producción de tapones combinatorios

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

Autor destacado: Integración de enfoques computacionales y experimentales en oncología de precisión

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

Dispositivo microfluídico bicapa para la producción de tapones combinatorios

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...