Amperometric assay of creatinine in urine by flow injection analysis based on conjugated reactions of immobilized enzymes. Simultaneous compensation of endogenous ammonia.

A flow-injection analysis biosensor system was developed for the amperometric assay of creatinine based on coupled reactions of three immobilized enzymes, using an oxygen electrode as the detection device. The ammonia produced by creatinine deiminase-catalyzed hydrolysis of creatinine was further converted into L-glutamate with two sequentially aligned enzyme reactors: glutamate dehydrogenase and glutamate oxidase. Endogenous ammonia was simultaneously compensated with a double peak recording system, where the flow was split after sample injection and rejoined before the glutamate dehydrogenase reactor. The system gave linear calibration in a range of 0.1-2.0 mM for creatinine and the first peak of ammonia, and 0.1-3.0 mM for the second peak of ammonia. One run was completed within two minutes. The system can be readily applied to the assay of creatinine in urine and showed good correlation with that from the currently used Jaffe method.