Determination of cholesterol and cholesterol ester with novel enzyme microsensors

Enzyme microsensors using cholesterol oxidase (EC 1.1.3.6) and cholesterol esterase (EC 3.1.1.13) were developed for measuring cholesterol and cholesterol ester. The platinum microsensors (platinum diameter, 50 microns) were etched in hot aqua regia to create a cavity at their tip. A porous composite material prepared from acetylene black and Teflon emulsion was packed into this cavity and the redox mediator [Os(bpy)3](PF6)2 was monitored by cyclic voltammetry in the potential range of 200-900 mV. The microsensors were dipped overnight in buffer solution containing the desired enzyme to immobilize it on the tip by adsorption. Calibration curves for measurements of cholesterol and cholesterol ester, the effects of pH, temperature, and concomitant compounds, the lifetime of the microsensors, and their availability for measuring cholesterol and cholesterol ester in urine were examined. Under optimal conditions, the response of the sensors was linear in concentration ranges of 5 microM-0.47 mM cholesterol and 2 microM-1.00 mM cholesterol ester.