Nous remercions le Dr Aaron P. Fox à l&#39;Université de Chicago, dans le laboratoire duquel ce protocole a été conçu à l&#39;origine, et William Roden (Université de Saint Louis) pour le développement ultérieur du protocole. Nous remercions la Fondation Whitehall et la National Science Foundation pour le soutien.

Partie 1: Préparation de la fibre de carbone <ol><li> Couper un faisceau de fibres de carbone (nous utilisons 7μm de diamètre) à une longueur qui est ~ 1,25 X la longueur de la pipette capillaire. </li><li> Retirez le dimensionnement par l&#39;ébullition ~ 100 ml d&#39;acétone dans un récipient et ajouter des fibres à la chaleur, de l&#39;acétone bouillante pendant 30 minutes ou plus. </li><li> Enlever les fibres de l&#39;acétone chaude, et le transfert d&#39;un bécher propre avec 50-100 ml d&#39;acétone fraîche. </li><li> Fibres sec en plaçant le paquet sur un plat ouvert 150 mm de Petri et Airdry nuit. </li></ol> Partie 2: tubes en verre au feu de polissage capillaire <ol><li> Incendie-polonaise chaque tube capillaire (nous utilisons des micro-hématocrite tubes capillaires, de 1.5mm de diamètre extérieur, diamètre intérieur 1,3 mm, 7,5 cm de long) en tenant le tube dans le centre avec une pince et en exposant la fin de la flame.Rotate du tube sur 3 fois alors que dans la flamme. </li><li> Inverser le tube et placez l&#39;extrémité opposée dans la flamme, encore une fois tourner 3 fois. </li><li> Placer les tubes polie au feu dans un plat en verre et le transfert au four jusqu&#39;à ce qu&#39;ils soient prêts à être remplis de fibres de carbone. </li></ol> Partie 3: Threading une fibre de carbone dans un tube capillaire <ol><li> Semblable à la description de Machado et al. (2008) et Mundroff et Wightman (2002), sucer la fibre dans le tube de verre en utilisant des tubes en plastique relié à un aspirateur avec un filtre. </li><li> Couper la fibre avec un ¼ de pouce d&#39;scalpel.Leave sur la fibre du tube. </li></ol> Partie 4: Tirer pipette-fibre dans un extracteur d&#39;électrode <ol><li> Placer le tube capillaire dans l&#39;extracteur d&#39;électrode (nous utilisons le Japon Narishige Modèle PP-830) et serrez les boutons pour tenir le tube capillaire en place. </li><li> Manipuler les commutateurs pour exécuter deux tractions, fixé à l&#39;extracteur réglages optimaux. </li><li> Une fois la pipette est tiré en deux, séparer les deux moitiés en coupant la fibre nue au centre avec des ciseaux. </li><li> Retirez chaque moitié d&#39;électrode et de tirer sur la fibre en saillie de briser le verre de la fibre seal.Trim avec des ciseaux afin que seul 1 / 8 pouce de fibre est laissé à l&#39;extérieur du tube. </li><li> Répétez les étapes 1-4 jusqu&#39;à environ 16 fibres électrodes sont retirées. </li></ol> Partie 5: Faire verre-époxy Seal <ol><li> Chauffer une aliquote de pré-faites de l&#39;époxy sur une plaque chauffante réglée sur 55 ° C pendant 6 minutes dans une hotte. </li><li> Alors que l&#39;époxy est le chauffage, enveloppez ~ 16 des électrodes tiré dans un paquet en utilisant du ruban. </li><li> Après la résine Epon est chauffé pendant 6 minutes, ajouter 0,7 grammes XOF MPDA de la résine chaude Epon (14%, p / v). Mélanger en remuant le flacon. </li><li> Trempez le faisceau d&#39;électrodes dans l&#39;époxy à chaud, plus solution.After durcisseur environ 20 secondes, une quantité suffisante d&#39;époxy entrera les astuces de verre par capillarité. </li><li> Défaire les électrodes bundle place, sur un carton avec des fentes et le lieu de ce bac dans un four réglé à 100 ° F. </li><li> Après au moins 48 heures, examiner chaque électrode sous un électrodes microscope.Discard dissection qui ne remplit pas à l&#39;époxy, ont une pointe brisées, ont plus d&#39;une fibre, etc.After inspectant chaque électrode, les replacer sur le support de carton et de retour dans le oven.Electrodes sont généralement bonnes pour environ un mois après la fabrication. </li></ol> Partie 6: coupe la fibre de carbone <ol><li> Placer l&#39;électrode de fibre de carbone avec soin sur une lame de verre avec du ruban téflon. </li><li> Visualisez la pointe d&#39;un microscope à dissection. </li><li> Utilisez une lame scalpel propre pour couper perpendiculairement à la fibre de carbone pour laisser une surface plate et propre pour le placement de la fibre de carbone contre une cellule. </li></ol>

<ol>
	<li>Mundorf, M. L., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amperometry+and+cyclic+voltammetry+with+carbon+fiber+microelectrodes+at+single+cells.">Amperometry and cyclic voltammetry with carbon fiber microelectrodes at single cells.</a> Curr Protocols in Neurosci. 6 (14), 1-6 (2002).</li><li>Machado, D. J., Montesinos, M. S., Borges, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Good+practices+in+single-cell+amperometry.">Good practices in single-cell amperometry.</a> Methods in Molec. Biol. 440, 297-313 (2008).</li></ol>

Ce protocole décrit comment générer et couper des électrodes ampérométriques. Pendant les enregistrements ampérométrique, une électrode en fibre de carbone est placée contre la surface d&#39;une cellule sécrétrice. L&#39;activité est observée comme exocytotique pics ampérométriques, qui indique le courant électrochimique provoquée par le transfert d&#39;électrons après oxydation des catécholamines. Avec la pratique et la découpe des électrodes ampérométriques, les niveaux de bruit peut être gra...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Epoxy Hardener 1,3-phenylene-diamene flakes</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>P23954-100G04 716AJ</td>
<td>Aliquots are stored in a dessicator and should not be exposed to light.</td>
</tr>
<tr>
<td>Carbon Fibers</td>
<td>Material</td>
<td>Goodfellow</td>
<td>C005725</td>
<td>&nbsp;</td>
<tr>
<td>Micro-Hematocrit Capillary Tubes</td>
<td>Material</td>
<td>Fisher</td>
<td>NC9836768</td>
<td>&nbsp;</td>
<tr>
<td>Epoxy Resin</td>
<td>Reagent</td>
<td>Miller-Stephenson</td>
<td>Epon Resin 828</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>Instrument</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Electrode Puller</td>
<td>Instrument</td>
<td>Narishige Japan</td>
<td>PP-830</td>
<td>1st pull = 24.8 degrees C; 2nd pull = 62.7 degrees C</td>
</tr>
<tr>
<td>Oven</td>
<td>Instrument</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>set to 100 degrees F.</td>
</tr>
<tr>
<td>Hot plate</td>
<td>Instrument</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>set to 55 degrees C</td>
</tr>		</tbody>
		</table>
		</div>

fabrication of amperometric electrodes

Électrodes en fibre de carbone sont cruciaux pour la détection de la libération de catécholamines à partir de vésicules dans des cellules individuelles pour des mesures ampérométrie. Ici, nous décrivons les techniques nécessaires pour produire un bruit faible (&lt;0,5 Pa) électrodes. Les techniques ont été modifiés à partir des descriptions publiées par les chercheurs précédents (1,2). Les électrodes sont faites par la préparation de fibres de carbone et de les enfiler individuellement dans chaque tube capillaire en utilisant un aspirateur avec un filtre pour aspirer la fibre. Ensuite, le tube capillaire avec la fibre est tirée par un extracteur d&#39;électrode, la création de deux moitiés, chacune avec une pointe à pointe fine. Les électrodes sont plongées dans chaude, époxy liquide mélangée avec le durcisseur pour créer un joint verre-époxy. Enfin, les électrodes sont placées dans un four pour durcir l&#39;époxy. Une manipulation soigneuse des électrodes est essentielle afin de s&#39;assurer qu&#39;elles sont faites de manière cohérente et sans dommage. Ce protocole montre comment fabriquer des électrodes ampérométriques et coupé pour l&#39;enregistrement à partir des cellules individuelles.

Watch this Scientific Journal Video about Fabrication of Amperometric Electrodes at JoVE.com

Fabrication of Amperometric Electrodes

Ce protocole décrit comment générer des électrodes en fibre de carbone. Les électrodes sont ensuite utilisés pour détecter la libération de catécholamines à partir des vésicules avec ampérométrie en fibre de carbone.

Fabrication d&#39;électrodes ampérométriques

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

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Electrochemical Analyses and Redox Titration

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14.3K vues.  Saint Louis University School of Medicine. Ce protocole décrit comment générer des électrodes en fibre de carbone. Les électrodes sont ensuite utilisés pour détecter la libération de catécholamines à partir des vésicules avec ampérométrie en fibre de carbone.

Vidéo: Fabrication of Amperometric Electrodes

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

Application de résine photopolymérisable adhésif dentaire pour les électrodes de montage ou des sondes de microdialyse dans les expériences chroniques

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

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Biologie

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues.  In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates.  The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive.  Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule.  Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks.    Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.  Micropatterning of several cell types was demonstrated on these substrates.   In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

Cellules Patterning sur des électrodes d&#39;indium optiquement transparents Tin Oxide

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

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.

Fabrication d&#39;un dispositif microfluidique pour la compartimentation du Neuron Soma et des axones

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

PDMS Device Fabrication and Surface Modification

Fabrication de dispositifs en PDMS et modification de surface

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.

Non du plasma Collage de PDMS pour fabrication bon marché de dispositifs microfluidiques

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 ...

Fabrication of the Thermoplastic Microfluidic Channels

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using polymer/nanoparticle composite microscale lysis and solid phase extraction columns. The recovered samples are concentrated, small volume samples that are PCRable, without any additional cleanup. Here, we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then, we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns. We demonstrate grafting and polymerization of a carbon nanotube/polymer composite column for bacterial cell lysis. We then show the lysis process followed by solid phase extraction of nucleic acids from the sample on chip using a silica/polymer composite column. The attached protocols contain detailed instructions on how to make both lysis and solid phase extraction columns.

Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns.

La fabrication des canaux microfluidiques thermoplastique

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

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass &lt;1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

Faire patch pipettes et électrodes Sharp avec un extracteur programmable

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. Lightweight recording devices carrying a high density array of tetrodes allow for the simultaneous monitoring of the activity of tens to hundreds of individual neurons. Here we describe a protocol for the fabrication of a micro-drive array with twenty one independently movable micro-drives. This device has been used successfully to record from hippocampal and cortical neurons in our lab. We show how to prepare a custom designed, 3-D printed plastic base that will hold the micro-drives. We demonstrate how to construct the individual micro-drives and how to assemble the complete micro-drive array. Further preparation of the drive array for surgical implantation, such as the fabrication of tetrodes, loading of tetrodes into the drive array and gold-plating, is covered in a subsequent video article.

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.

Micro-drive tableau pour les maladies chroniques In vivo Enregistrement: Disque de fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. ...

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].

Fabrication de constructions myogéniques de l&#39;ingénierie tissulaire

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

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

Fabrication et fonctionnement d&#39;un insert d&#39;oxygène pour les adhérents des cultures cellulaires

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...