Spectrophotometric flow-injection determination of urea in body fluids by using an immobilized urease reactor

A flow system involving a packed-bed enzyme reactor (volume 180 μl) with urease immobilized covalently on poly(glycidyl methacrylate)-coated porous glass is used for determining urea in blood serum and urine. Enzymatically produced ammonia is converted to an indophenolate dye (by oxidative coupling with hypochlorite and sodium salicylate), which is detected spectrophotometrically at 700 nm. The calibration graph is rectilinear for 25–500 μM urea when injecting samples (75 μl) diluted 1:50 for serum or 1:1000 for urine at a frequency of 60 h^?1; the relative standard deviation is 1.1% for ten injections of 300 μM urea. The immobilized urease is stabilized by the addition of disodium EDTA, sodium azide and 2-mercaptoethanol to a 0.2 M phosphate buffer (pH 6.9) used as the carrier stream, which serves also as a preservative for longterm storage of the urease reactor packing at 4°C.