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 JoVE Biology

Fabrication of Amperometric Electrodes

1, 2, 1

1Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, 2Yale University School of Medicine

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    Summary

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

    Date Published: 5/04/2009, Issue 27; doi: 10.3791/1040

    Cite this Article

    Pike, C. M., Grabner, C. P., Harkins, A. B. Fabrication of Amperometric Electrodes. J. Vis. Exp. (27), e1040, doi:10.3791/1040 (2009).

    Abstract

    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 (<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.

    Protocol

    Part 1: Carbon Fiber Preparation

    1. Cut a bundle of carbon fibers (we use 7μm diameter) at a length that is ~1.25 X the length of the capillary pipette.
    2. 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.
    3. Remove fibers from the warm acetone, and transfer to a clean beaker with 50-100 mL of fresh acetone.
    4. Dry fibers by placing the bundle on an open 150 mm Petri dish and airdry overnight.

    Part 2: Fire-polishing Glass Capillary Tubes

    1. Fire-polish each capillary tube (we use micro-hematocrit capillary tubes,1.5mm outer diameter, 1.3mm inner diameter, 7.5cm long) by holding the tube in the center with forceps and exposing the end to the flame.Rotate the tube about 3 times while in the flame.
    2. Invert the tube and place the opposite end into the flame, again rotating 3 times.
    3. Place the fire-polished tubes in a glass dish and transfer to the oven until they are ready to be filled with carbon fibers.

    Part 3: Threading a Carbon Fiber into a Capillary Tube

    1. Similar to the descriptions by Machado et al. (2008) and Mundroff and Wightman (2002), suck the fiber into the glass tube using plastic tubing connected to a vacuum with a filter.
    2. Cut the fiber using a scalpel.Leave ¼ inch of fiber out of tube.

    Part 4: Pulling Pipette-Fiber in an Electrode Puller

    1. Place capillary tube into the electrode puller (we use Narishige Japan Model PP-830) and tighten the knobs to hold the capillary tube in place.
    2. Manipulate the switches to execute two pulls, set at the optimal puller settings.
    3. Once the pipette is pulled in half, separate the two halves by cutting the bare fiber at the center using scissors.
    4. Remove each electrode half and pull on the protruding fiber to break the glass seal.Trim the fiber with scissors so that only 1/8 inch of fiber is left outside of the tube.
    5. Repeat steps 1-4 until about 16 fiber-electrodes are pulled.

    Part 5: Making Epoxy-Glass Seal

    1. Heat a pre-made aliquot of epoxy on a hot plate set to 55°C for 6 minutes in a fume hood.
    2. While epoxy is heating, wrap ~16 of the pulled electrodes into a bundle by using tape.
    3. After the Epon Resin is heated for 6 minutes, add 0.7 grams xof MPDA to the hot Epon Resin (14%, w/v).Mix by swirling the vial.
    4. Dip the bundle of electrodes into the hot epoxy plus hardener solution.After about 20 sec, a sufficient amount of epoxy will enter the glass tips via capillary action.
    5. Undo the bundle, place electrodes onto cardboard with slots and place this tray in an oven set to 100°F.
    6. After at least 48 hours, examine each electrode under a dissecting microscope.Discard electrodes that did not fill with epoxy, have a smashed tip, have more than one fiber, etc.After inspecting each electrode, place them back on the cardboard holder and back into the oven.Electrodes are usually good for about 1 month after fabrication.

    Part 6:Cutting the Carbon Fiber

    1. Place carbon fiber electrode carefully onto a glass slide with Teflon tape.
    2. Visualize the tip under a dissecting microscope.
    3. Use a clean scalpel blade to cut perpendicularly across the carbon fiber to leave a flat clean surface for placement of the carbon fiber against a cell.

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    Discussion

    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.

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    Disclosures

    Acknowledgements

    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.

    We thank the Whitehall Foundation and National Science Foundation for support.

    Materials

    Name Type Company Catalog Number Comments
    Epoxy Hardener 1,3-phenylene-diamene flakes Reagent Sigma-Aldrich P23954-100G04 716AJ Aliquots are stored in a dessicator and should not be exposed to light.
    Carbon Fibers Material Goodfellow C005725
    Micro-Hematocrit Capillary Tubes Material Fisher Scientific NC9836768
    Epoxy Resin Reagent Miller-Stephenson Epon Resin 828
    Dissecting Microscope Instrument
    Electrode Puller Instrument Narishige International PP-830 1st pull = 24.8 degrees C; 2nd pull = 62.7 degrees C
    Oven Instrument set to 100 degrees F.
    Hot plate Instrument set to 55 degrees C

    References

    1. Mundorf, M.L. & Wightman, R.M. Amperometry and cyclic voltammetry with carbon fiber microelectrodes at single cells. Curr Protocols in Neurosci. 6.14.1-6.14.22 (2002).
    2. Machado, D.J., Montesinos, M.S. & Borges, R. Good practices in single-cell amperometry. Methods in Molec. Biol. 440, 297-313 (2008).

    Comments

    7 Comments

    The parameters of the electrode puller are correct or not. 1st pull=²4.8 degrees C; ²nd pull=6².7 degrees C.
    ²4.8 degrees C is a room temperature. I guess 1st pull=6².7 degrees C. But the ²nd pull=? Thanks
    Reply

    Posted by: AnonymousMarch 1, 2010, 11:05 AM

    Yes, for this Narishige Model PP-830 puller, we have our puller set to 6².7 degrees and the second pull to 41 degrees. Sorry for the confusion. Amy Harkins
    Reply

    Posted by: AnonymousMarch 1, 2010, 4:02 PM

    Hi Amy, Thank you so much for your response. Now I have three questions again.
    One is the temperature of epoxy. Actually, we purchased EPON Resin 8²8 from HEXION, which is a sticky fluid. In terms of your video, it is heated on a hot plate set to 55°C for 6 minutes in a fume hood, then add MPDA. But we found it is still a little sticky after 6 mins at 55°C and MPDA is not dissolved completely. Do you think 55°C is high enough to dissolve MPDA or not?
    Another questiton that I have is regarding the oven temperature, Is 100°F for 48 hours sufficient for the epoxy to dry?
    The third question is about the making the epoxy-glass seal. According to the video, it is twice for 5 s. But based on the text, it is ²0 seconds, which one is correct?
    Thank you very much!
    Reply

    Posted by: AnonymousMarch 5, 2010, 2:57 PM

    We found the temperatures empirically as reported for the heating of the epon. You may need to raise the temperature. The idea is to add MPDA, swirl and have it dissolve within 3-5 sec with vigorous swirling.

    Yes, 100 degree oven is fine for epoxy to dry, 48 hrs. If it dŒsn't dry in this time, it isn't the temp of the oven, but the mix of the epoxy and relative ratios of epon to MPDA.

    For the dips of the glass electrodes into the heated, mixed epoxy, we typically use two dips each for about 5 sec. This again will depend on the ratio and mix/viscosity of the epoxy on the heat plate. If you raise the temp on the epoxy mix, it should work better. If the epoxy cures too quickly, the electrodes will be captured in the glass vial of hardened epoxy. We vary the hardener amount and don't change the epon when we find the optimal conditions. We don't know for certain, but it seems that the hardener is the variable in this equation, and might need to have more hardener added. Although we have no proof, we have thought that the hardener may be affected by humidity and so we store it in a dessicator.
    Reply

    Posted by: AnonymousMarch 5, 2010, 3:24 PM

    Hi Amy, 100 degree oven temperature for drying the electrode is F or C? But the paper you cited (1) reported that the temperature is 100 to 150 C degrees.
    Another question is when you say "Remove each electrode half and pull on the protruding fiber to break the glass seal.Trim the fiber with scissors so that only 1/8 inch of fiber is left outside of the tube. " After pulling should the tip of the glass electrode still be intact or broken. When we tried to pull the carbon fiber out a little the glass tip is not broken, would this still allow the epoxy to enter into the electrode or not? Is the time 5 s for two times enough to allow the epoxy to enter?
    I really appreciate your kindness. Thank you.
    Reply

    Posted by: AnonymousMarch 5, 2010, 5:21 PM

    Yes, 100 C for oven temp.

    When pulling the fiber with fingers away from the glass before cutting the end off, you will allow the fiber to be "free" and not stuck to the glass that heated while pulling the sharp electrode. It dŒsn't "break" the tip of the glass electrode, that is still very much intact.

    Yes, it is plenty of space and time to fill the electrodes with ² dips of 5 sec each. When the epoxy is liquid enough you will see that it has filled the tips of each of the electrodes, easily seen by eye without a microscope. Twice dipping, 5 sec each dip or so, should be plenty to fill and well-seal the electrode around the fiber as the epoxy cures and hardens in the oven.
    Reply

    Posted by: AnonymousMarch 5, 2010, 5:45 PM

    Hello Amy, just came aross this. Great! We're having quite some trouble with the commercial ones; will ask my student to try out your protocol soon.
    Best, Jan
    Reply

    Posted by: AnonymousJuly 30, 2010, 9:01 AM

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