FSCV for Neurotransmitter Study: Experimental Methods

SeePart 1 here.

To construct carbon fiber microelectrodes (CFMEs) takes practice and a steady hand. In the case of T-650 fiber electrodes, the fiber is aspirated into glass capillaries, while the larger diameter fibers are cut to length and manually inserted, often with the aid of a stiff metal wire. Each tube is pulled into two electrodes with a micropipette puller so that some fiber is exposed at the pulled end. The exposed fiber is then trimmed to about 50 µm for cylindrical electrodes, and all the way to the glass for disk electrodes. The electrode tip is then sealed with epoxy. Once the epoxy has set, the tips are ground smooth to the desired angle on a very fine, rotating grindstone. After soaking for 30 minutes in isopropanol, they are ready to be used as the working electrode in the cyclic voltammetry apparatus. An Ag/AgCl wire is used as the reference electrode.

Several different buffers are commonly used for these experiments, including a tris buffer at pH 7.4, and a phosphate buffer at ph 6.5. The primary buffer used was made to a pH of 6.2, and referred to as the fly buffer. It was adapted from Schneider’s insect medium, which is often used to culture insect nerve tissue. Manufactured octopamine hydrochloride and tyramine hydrochloride were made up as stock solutions in 0.1 M perchloric acid, and stored in a refrigerator. Before each experiment, the stock solutions were diluted with buffer to the appropriate concentration.

Carbon-Fiber Microelectrode (CFME).

Experiments are carried out inside a faraday cage as ambient electromagnetic waves can greatly distort the sensitive readings. Data is collected using cyclic voltammetry software and a potentiostat to apply a specified waveform. Phenolamines are tested using flow-injection analysis, with five second injections to imitate rapid biological changes. The working electrode tip is placed at the output of a flow cell. Buffer is continually pumped through the cell using a syringe pump. The CV software controls an air actuator which is mounted on a 6-port HPLC loop injector, allowing the sample to flow past the electrode. An average of ten pre-injection cyclic voltammograms are used to background subtract the data. The results are displayed as a current versus time trace, called a cyclic voltammogram (CV), and as a color plot, which displays current, time, and voltage.

Color plots and resulting Cyclic Voltammograms, with and without analyte present.

Nation coatings are applied to electrode surfaces using the same potentiostat/software arrangement as in previously described electrochemical experiments. The liquid Nation is placed in a dish, and the reference electrode is inserted. A piecemeal waveform is applied, and the working electrode tip is dipped for 30 seconds into the liquid. The electrode is then removed and heated for 10 minutes at 70°C. After one hour, the Nafion-coated electrodes are used to test for tyramine and octopamine in the same manner as before, with the results being compared to pre-Nafion data for each electrode.