The method is significant because it enables neurochemical detection with high spatial and temporal resolution which can potentially enhance in vivo methods of neurochemical detection. The main advantage of this technique is that it is a quick, easy and reproducible method to enhance the sensitivity and temporal resolution of neurotransmitter detection. Demonstrating the procedure will be Sanuja Mohanaraj and Pauline Wonnenberg, graduate students from my laboratory.
To begin, separate carbon fiber material into individual strands and pull a single seven-micrometer diameter carbon fiber from a strand. Connect a vacuum line to a borosilicate glass capillary and aspirate the carbon fiber into the capillary. Then cut a 10-centimeter by 25-centimeter piece of cardboard to serve as an electrode holder.
Tape a paper towel around the cardboard as a support, then insert the capillary into the electrode holder and carefully secure it in a vertical capillary puller. Configure the capillary puller to pull the glass capillary to a fine taper for electrode materials and start it. Once pulling finishes and the heating coil has cooled, cut the carbon fiber connecting the tube-pulled electrodes.
Carefully remove the microelectrodes from the capillary puller. Guided by a stereoscope or microscope, use a sharp blade or surgical scissors to trim the protruding carbon fiber on each electrode to about 100 to 150 micrometers in length. Next, in a 25-milliliter vial, use a cotton swab to mix 10 grams of epoxy with 0.2 milliliters of hardener.
Fill another vial with acetone. For each electrode, immerse the carbon fiber tip in epoxy for 15 seconds and then dip it in acetone for three seconds to remove excess epoxy. After epoxying, the epoxied electrodes are then cured in the oven for three hours at 125 degrees Celsius.
Next, use a micromanipulator to place a carbon fiber microelectrode in a silver-silver-chloride reference electrode in a 0.5 millimolar solution of chloroauric acid in 0.1 molar aqueous potassium chloride. Connect the electrodes to a potentiostat with a carbon fiber microelectrode as the working electrode. Scan the electrode from 0.2 volts to minus one volt at 50 millivolts per second for 10 cycles to perform electrode deposition.
It is critical to optimize the parameters for depositing the gold coating. Too much coating will cause noise and signal overload while too little coating will not enhance neurochemical detection. Before the test, prepare a 10-millimolar stock solution of dopamine in perchloric acid and about a liter of pH 7.4 PBS-based buffer in deionized water.
Pipette one microliter of the dopamine stock solution into 10 milliliters of buffer to make an approximately one micromolar dopamine solution. Then connect a carbon fiber microelectrode and a silver-silver chloride reference electrode to a potentiostat. Fix the carbon fiber electrode and the reference electrode in the head stage of the flow cell apparatus and use the micromanipulator to lower them into the flow cell.
Draw 60 milliliters of the PBS buffer into a syringe. Fill the flow cell with buffer, and mount the syringe in a syringe pump. Start flowing buffer through the flow cell at a rate of one milliliter per minute.
Then configure the potentiostat to scan for minus 0.4 volts to 1.3 volts at 10 hertz and 400 volts per second. Briefly apply the waveform to the microelectrode, observe the oscilloscope, and adjust the gain to prevent overloading. Let the microelectrode equilibrate for 10 minutes in the buffer.
Then draw the diluted dopamine solution into a syringe and connect it to the injection port of the flow cell. Set the total run time on the potentiostat to 30 seconds. Start recording the measurements, wait 10 seconds, and then inject 0.2 milliliters of dopamine solution into the flow cell.
When the run finishes, process the data with high definition cyclic voltammetry analysis software. Let the microelectrode re-equilibrate for 10 minutes before performing another test. When the tests are finished, clean the flow cell by injecting three milliliters of water and three milliliters of air into the buffer and injection ports three times.
Coated carbon fibers were imaged with scanning electron microscopy. The thickness and particle size of the gold nanoparticle coatings can be controlled by the electrode deposition time. 20 minutes of electrode deposition yielded a thick gold coating with sharp ridges while five minutes yielded a thin uniform gold coating.
Gold nanoparticle coated carbon-fiber microelectrodes had significantly higher peak oxidative currents and faster electron transfer kinetics than unmodified electrodes. The gold nanoparticle coating had no significant effect on the stability of the electrode responses as demonstrated here in a solution of dopamine. Both bare and gold nanoparticle coated electrodes responded linearly to scan rate changes with a much greater magnitude of change in the gold-coated electrodes.
This indicated that dopamine absorption could be controlled via the scan rate. Both bare and gold nanoparticle-coated electrodes responded linearly between dopamine concentrations of 100 nanomolar to 10 micromolar. An asymptotic curve was observed at higher concentrations, indicating that dopamine is supersaturated at the electrode surface.
The ability to detect neurochemical changes on a faster time scale and at higher sensitivities will help answer complex questions in neuroscience. This method also has uses in analytical chemistry, metabolomics, and environmental science. While it is easy to learn, visual demonstration is critical for learning to make, modify, and test the microelectrodes.
Future directions for this method include adjusting the deposition of gold and other coatings to account for thickness, size, shape, and morphology to optimize the detection of specific neurotransmitters. Researchers should practice handling small fibers under a microscope before trying this technique. Also, neurotransmitter stock solution should be prepared in a fume hood because they use 1 molar perchloric acid.
