Los canales de microfluidos son fabricados en una poliolefina cíclica (Zeonex ® 690R, Zeon Chemical Inc., Louisville, KY). Zeonex tiene una temperatura de transición vítrea (Tg) de 136 ° C y UV es transparente, lo cual es esencial para la química de la luz dirigida utilizado en la formación monolito porosa. Los microcanales son fabricados por micro-estampado en caliente con una NiCo electroformada maestro de molde, que tiene la negativa (inversa) características de la plataforma de microfluidos. El estampado en caliente se realiza de la siguiente manera: <ol><li> El molde y el sustrato de polímero se colocan entre dos placas metálicas paralelas. </li><li> Los platos se calientan hasta la temperatura de estampación (166 ° C), que es de 30 º C por encima de la Tg de Zeonex. </li><li> Los platos se prensan junto durante unos 5 min y la presión se mantiene a 250 psi. </li><li> El molde y el sustrato se retiran de las placas se calienta, se deja enfriar durante 1-2 minutos, y luego manualmente separados unos de otros. </li><li> Pozos de diámetro de 1,5 mm son perforados en los extremos de los canales para la introducción de la muestra y recolección. </li><li> El sustrato de plástico en relieve es entonces adherido térmicamente con otro pedazo de Zeonex formulario adjunto a los canales de microfluidos. El sustrato en relieve y la pieza de la cubierta se puede UV-ozono o plasma tratado antes de la unión térmica para mejorar la eficiencia de sellado y garantizar la fidelidad de la estructura del canal cerrado (Bhattacharyya y Klapperich, 2007). </li></ol> Fabricación de extracción en fase sólida (SPE) de columna dentro de los canales de microfluidos de plástico La fabricación de la fase sólida es un proceso de dos pasos. Al principio, los microcanales son fabricados superficie modificada con el fin de mejorar la adherencia de la columna de la SPE en el dispositivo de plástico. Injerto a través de fotopolimerización superficie se utiliza para funcionalizar las paredes de los microcanales poliméricos. El procedimiento de injerto es el siguiente: <ol><li> Los microcanales se llena con una mezcla de 1:1 de diacrilato de etileno (EDA) y metacrilato de metilo (MMA) con 3% de benzofenona, que es un fotoiniciador de hidrógeno abstracción. La EDA, MMA y benzofenona son adquiridos de Sigma-Aldrich, St. Louis, MO. </li><li> El chip es entonces irradiados con UV durante 10 minutos a 254 nm de longitud de onda UV y 200 mJ / cm 2 de la energía en un instrumento de la exposición ultravioleta (CL-1000 Crosslinker UV, UPV Inc., Upland, CA). El injerto se produce a través de la polimerización abstracción de H y los resultados de las cadenas de polímero anclada en la superficie, que se incorporan en la mezcla de polimerización utilizada para la preparación del monolito en el segundo paso. </li><li> El monómero se elimina el exceso de los canales de lavado con 15 l de metanol con aspiración al vacío. </li></ol> Después del proceso de photografting, un polímero microporoso monolito se forma dentro de los microcanales que atrapan las partículas de sílice para la extracción de ADN. El monolito porosa se ​​prepara in situ de la siguiente manera: <ol><li> Los canales de superficie modificada se llena con una mezcla de precursores monolito compuesto de Buma (15% en peso), EDMA (10% en peso), 1-dodecanol (52,5% en peso), ciclohexanol (22,5% en peso), DMPAP (en peso del 1% con respecto a los monómeros) y 0,7 micras partículas de sílice (15% con respecto al volumen total de la mezcla de pre-polímero). El microesferas de sílice fueron adquiridos de Polysciences, Inc. (Warrington, PA) y los monómeros y disolventes porogenic son adquiridos de Sigma-Aldrich, St. Louis, MO. </li><li> El chip se irradia con luz UV en el reticulador UV a 200 mJ / cm 2 de 1.1 minutos para iniciar la polimerización, el monómero y se elimina el exceso de los canales de lavado con 15 l de metanol con aspiración al vacío. </li><li> Las conexiones de fluidos (NanoPorts conexiones TM, Upchurch Scientific, Oak Harbor, WA) se unen después de la introducción y pozos de captación de los canales de microfluidos. </li><li> El monolito porosa se lava de nuevo con metanol durante un mínimo de 30 minutos utilizando la bomba de jeringa con un caudal de 100mL/hour. </li></ol> Fabricación de CNT lisis monolito dentro de los canales de microfluidos de plástico La fabricación de la lisis CNT monolito (nanotubos de carbono) es un proceso de dos pasos. En primer lugar, los microcanales son fabricados superficie modificada (injertados) en la misma forma que para las columnas de SPE en el dispositivo de plástico. Después del proceso de photografting, un polímero microporoso monolito se forma dentro del microcanal que está impregnado con nanotubos de carbono de pared múltiple. El monolito porosa se ​​prepara in situ de la siguiente manera: <ol><li> Multi-nanotubos se resuspenden en ciclohexanol a una concentración de 2.27m. Esta es la resuspensión ultrasonicated durante 30 minutos a 50% de la amplitud y el 50% ciclo de trabajo con una célula Sonifier disruptor (Sonifier S-250D, fabricado por ultrasonidos Branson, Danbury, CT). Los ultrasonidos proporcionan una suspensión de 0,5 eficaz y estable de la CNT. Los nanotubos de carbono fueron adquiridos de Nanolabs (Brighton, MA). </li><li> Los canales de superficie modificada se llena con una mezcla de precursores similares monolito sólo que ahora la solución de ciclohexanol + CNT se utiliza. La mezcla consiste monolito de Buma (15% en peso), EDMA (10% en peso), 1-dodecanol (52,5% en peso), y ciclohexanol + CNT (22,5% en peso). A esta solución la DMPAP fotoiniciador se añade (1,13% en peso con respecto a los monómeros). Los monómeros, disolventes y porogenic DMPAPA son adquiridos de Sigma-Aldrich, St. Louis, MO. </li><li> El chip se irradia con luz UV en el reticulador UV a 120 mJ / cm 2 para 0,9 minutos para iniciar la polimerización. El dispositivo se voltea 180 grados en la crossliker e irradiado por otros 0,9 minutos a 120 mJ / cm 2. El monómero se elimina el exceso de los canales de lavado con 50 l de metanol con aspiración al vacío. </li><li> Las conexiones de fluidos (NanoPorts conexiones TM, Upchurch Scientific, Oak Harbor, WA) se unen después de la introducción y pozos de captación de los canales de microfluidos. </li></ol> Preparación de muestras de bacterias para su uso con CNT lisis monolito La preparación de la muestra para una muestra bacteriana consiste en tomar una medida de la densidad óptica a 600 nm para asegurar que la muestra no es muy concentrada con el fin de evitar la obstrucción del canal. <ol><li> Tome una lectura de DO a 600 nm con la bacteria en los medios de comunicación, esto se hace con un BioPhotometer (Eppendorf BioPhotometer, Eppendorf Scientific, Inc., Westbury, NY). Diluir la muestra hasta que las lecturas de OD dentro del rango 0.18-0.25 A. </li><li> Centrifugar la muestra a 6500 rpm durante 5 minutos y decantar el sobrenadante. </li></ol> Lisis <ol><li> Resuspender las bacterias en GuSCN * + 0.01% SDS + 4% proteinasa K (0.8mg/ml). Agitar brevemente (1 ml de bacterias en los medios de comunicación deben ser resuspendido en 1 ml GuSCN). Proteinasa K fue agregado a la solución para ayudar con la desnaturalización de las proteínas que se unen al ADN bacteriano y para inhibir la DNAases y RNAases. Un beneficio adicional de la proteinasa K es que también ayuda a romper las proteínas de la pared celular, lo que es deseable, ya que tanto bacterias gram-positivas y gram-negativos paredes celulares de las bacterias contienen grandes cantidades de proteínas (aunque bacterias gram-negativas suelen tener más proteínas ). El buffer caotrópico además ayuda a la proteinasa K con desnaturalización proteínas, mientras que el detergente rompe la pared celular. Proteinasa K y tiocianato de guanidinio (GuSCN) fueron adquiridos de Qiagen Inc. (Valencia, CA). SDS (dodecil sulfato de sodio) fue adquirido de Pierce (Rockford, IL). </li><li> Inmediatamente la carga de la muestra en una jeringa conectada a un tubo PEEK. Aspirar la jeringa para que no haya aire en el sistema. Conecte el tubo a la NanoPort y el canal. </li></ol> Una bomba de jeringa KDS100 (fabricado por KD Scientific, Holliston, MA) fue utilizado para los experimentos y el caudal utilizado es de 450 l / h para todos los pasos. El flujo del canal de la lisis de la muestra en 450ul/hr. <ol><li> Recoger la muestra y proceder a la extracción de ADN a través de la SPE (véase el protocolo de extracción de ADN). NOTA: Esta muestra bacteriana no tendrá que seguir el paso de carga 2, ya que la suspensión es GuSCN *. </li></ol> Protocolo de extracción de ADN El procedimiento de extracción consiste en tres pasos: <ol><li> De carga. </li><li> Washington </li><li> Eluir. </li></ol> Bomba de jeringa KDS100 (fabricado por KD Scientific, Holliston, MA) fue utilizado para los experimentos y la velocidad de flujo usada fue de 300 l / h para todos los pasos. Carga <ol><li> La condición del canal con buffer caotrópico (GuSCN *, tiocianato de guanidinio) durante 1-2 minutos antes de comenzar el experimento. </li><li> Mezclar la muestra con tampón caotrópico en proporción de 1:1. </li><li> El flujo de mezcla de la muestra a través del canal durante 3 min. </li></ol> Lavar <ol><li> Lavar el canal con el 70% de etanol durante 2 min. </li><li> Lavar el canal con etanol al 100% durante 2 min. </li></ol> Eluir <ol><li> Eluir el ADN extraído en agua Millipore durante 3 min. Recoge 15 l de agua purificada, lista para PCR de ADN. </li></ol> * El GuSCN del kit de Qiagen (Buffer RLT) se utilizó.

<ol>
	<li>Bhattacharyya, A., Klapperich, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+chemical+analysis+of+plasma+and+ultraviolet-ozone+surface+treatments+for+thermal+bonding+of+polymeric+microfluidic+devices.">Mechanical and chemical analysis of plasma and ultraviolet-ozone surface treatments for thermal bonding of polymeric microfluidic devices.</a> Lab Chip. 7, 876-882 (2007).</li></ol>

The authors have nothing to disclose.

fabrication of the thermoplastic microfluidic channels

En nuestro laboratorio, hemos logrado ácidos nucleicos aislados directamente de los volúmenes de microlitro y submicroliter de la sangre humana orina y las heces con polímero / nanopartículas compuestas lisis micro y sólidas columnas de extracción en fase. Las muestras recuperadas son muestras de concentrado, pequeño volumen que se PCRable, sin ningún tipo de limpieza adicional. En este sentido, se muestra cómo fabricar chips de microfluidos con termoplástico estampado en caliente y sellado por calor. Luego, se demuestra cómo utilizar la luz en la superficie in situ dirigidos injerto y la polimerización a través del chip sellado para formar las columnas compuestas en fase sólida. Demostramos injerto y la polimerización de un nanotubo de carbono / polímero columna compuesta para la lisis celular de las bacterias. A continuación, muestran el proceso de lisis seguido de la extracción en fase sólida de los ácidos nucleicos de la muestra en el chip con un contenido de sílice / columna de compuesto de polímero. Los protocolos adjuntos contienen instrucciones detalladas sobre cómo hacer que tanto la lisis y la extracción en fase sólida columnas.

Watch this Scientific Journal Video about Fabrication of the Thermoplastic Microfluidic Channels at JoVE.com

Fabrication of the Thermoplastic Microfluidic Channels

Aquí se demuestra cómo fabricar chips de microfluidos con termoplástico estampado en caliente y sellado por calor. Luego se muestra cómo utilizar la luz en la superficie in situ dirigidos injerto y la polimerización a través del chip sellado para formar las columnas compuestas en fase sólida.

Fabricación de los canales de microfluidos termoplástico

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using ...

fabrication-of-the-thermoplastic-microfluidic-channels

Research

JoVE Journal

Biology

13.3K vistas.  Boston University. Aquí se demuestra cómo fabricar chips de microfluidos con termoplástico estampado en caliente y sellado por calor. Luego se muestra cómo utilizar la luz en la superficie in situ dirigidos injerto y la polimerización a través del chip sellado para formar las columnas compuestas en fase sólida.

Video: Fabrication of the Thermoplastic Microfluidic Channels

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Fabricación de un dispositivo de microfluidos para la compartimentación de las neuronas y los axones Soma

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Investigación

Biología

PDMS Device Fabrication and Surface Modification

PDMS fabricación de dispositivos y modificación de la superficie

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

No plasmáticos de unión de PDMS para la Fabricación de bajo costo de los dispositivos de microfluidos

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

Microfluidic Applications for Disposable Diagnostics

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

Las solicitudes de microfluidos para el diagnóstico desechables

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new ...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Parche de grabación Clamp de canales iónicos expresados ​​en ovocitos de Xenopus

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

Fabricación de electrodos amperométricos

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

Fabricación de construcciones de ingeniería tisular miogénica

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

La sonda de microfluidos: Operación y Uso de tratamiento de superficies localizadas

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagénesis y análisis funcional de los canales iónicos heteróloga se expresa en células de mamífero

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

Un ensayo de cribado para la identificación de moduladores fluorescente de canales GIRK

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...