Name: fabrication of amperometric electrodes
Uploaded: 2009-05-04T00:00:00
Description: Watch this Scientific Journal Video about Fabrication of Amperometric Electrodes at JoVE.com

<p class="jove_content">We thank Dr. Aaron P. Fox at the University of Chicago, in whose laboratory this protocol was originally designed, and William Roden (Saint Louis University) for further development of protocol.</p>
<p class="jove_content">We thank the Whitehall Foundation and National Science Foundation for support.</p>

<p class="jove_title">Part 1: Carbon Fiber Preparation</p>
<ol>
<li>Cut a bundle of carbon fibers (we use 7&mu;m diameter) at a length that is ~1.25 X the length of the capillary pipette.</li>
<li>Remove the sizing by boiling ~100 mL of acetone in a container and adding the fibers to the hot, boiling acetone for 30 minutes or longer.</li>
<li>Remove fibers from the warm acetone, and transfer to a clean beaker with 50-100 mL of fresh acetone.</li>
<li>Dry fibers by placing the bundle on an open 150 mm Petri dish and airdry overnight. </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> <em style='font-style: italic'>Curr Protocols in Neurosci</em>. <b>6</b> (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> <em style='font-style: italic'>Methods in Molec. Biol</em>. <b>440</b>, 297-313 (2008).</li></ol>

<p class="jove_content">This protocol describes how to generate and cut amperometric electrodes.  During amperometric recordings, a carbon fiber electrode is placed against the surface of a secretory cell. Exocytotic activity is observed as amperometric spikes, which indicates the electrochemical current caused by the transfer of electrons after oxidation of the catecholamines.  With practice making and cutting amperometric electrodes, noise levels can be greatly improved over many of the commercially available electrodes.</p>

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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

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.

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

Fabrication of Amperometric Electrodes

<p class="jove_content">This protocol describes how to generate carbon fiber electrodes.  The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.</p>

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

Research

JoVE Journal

Biology

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 ...

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 ...

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 ...

PDMS Device Fabrication and Surface Modification

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 ...

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 ...

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 ...

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 ...

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 ...

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

Fabrication and Use of MicroEnvironment microArrays MEArrays

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...