A stable three-enzyme creatinine biosensor. 3. Immobilization of creatinine amidohydrolase and sensor development.

We describe the development of an extended use amperometric three-enzyme creatinine biosensor and the successful chemical modification and immobilization of the enzyme creatinine amidohydrolase using polyurethane prepolymers. Creatinine amidohydrolase is significantly stabilized by immobilization in polyurethane polymers. The half-life increases from six to more than 80 days in buffer at 37 degrees C. The effect of silver ions leached from amperometric reference electrodes on enzyme and sensor performance is discussed. The use of cellulose acetate cover membranes to prevent silver from reaching the enzyme is investigated. Sensors prepared with cover membranes have half-lives almost an order of magnitude greater than those prepared with no cover membrane over the silver electrode. The complete biosensor has been constructed on a clinical blood analyzer platform and is stable for many days.