QUANTITATION OF CHOLINE IN THE EXTRACELLULAR FLUID OF BRAIN-TISSUE WITH AMPEROMETRIC MICROSENSORS

Amperometric microsensors for the detection of choline in the extracellular fluid of brain tissue have been prepared by immobilizing horseradish peroxidase and choline oxidase onto carbon fiber microcylinder electrodes with a cross-linkable redox polymer. The microcylinders have diameters of 7 or 10 mu m and lengths of 200-400 mu m. To detect choline, the microsensors are operated at an applied potential of -0.1 V vs SCE. At this potential, ascorbate and other easily oxidizable interferent molecules present in brain tissue are not detected by the electrode. Ascorbate, however, can interfere with the response to choline by acting as a reducing agent in the enzyme-containing polymer film. So, a Nafion overlayer is required in order to reliably detect choline in the presence of physiologically relevant concentrations of ascorbate (similar to 200 mu M). The Nafion-coated microsensors have a detection limit of similar to 5 mu M choline and give a linear response beyond 100 mu M when calibrated in vitro at 37 degrees C. Exposure of the microsensors to brain tissue for several hours causes less than a 10% loss in redox polymer surface coverage and less than a 25% loss in sensitivity to choline. To assess the ability of the microsensors to monitor choline levels in brain tissue, small volumes of a choline solution were injected into brain tissue at a site about I mm away from a microsensor. The current arising at the microsensor was converted to choline concentration by calibrating the sensor following the in vivo experiment. The resultant choline concentrations were in excellent agreement with those predicted by appropriate diffusion equations.