Continuous-flow/stopped-flow system incorporating two rotating bioreactors in tandem: application to the determination of alkaline phosphatase activity in serum.
Two rotating bioreactors in tandem have been incorporated into a continuous-flow/stopped-flow sample/reagent processing setup for the determination of alkaline phosphatase (EC3.1.3.1) activity in serum samples. The strategy circumvents incompatibility of buffer systems as well as that of the immobilized enzymes utilized in the bioreactors (alkaline phosphatase and alcohol oxidase, EC 1.1.3.13). The determination is indirect in nature although recorded responses are directly related to the enzyme activity in the sample. It couples the following enzyme-catalyzed reactions: (1) hydrolysis of p-nitrophenyl dihydrogen phosphate catalyzed by alkaline phosphatase, (2) enzymatic reaction between unreacted p-nitrophenyl dihydrogen phosphate with methanol, and (3) conversion of the residual methanol to the corresponding aldehyde and H2O2, catalyzed by alcohol oxidase. The H2O2 is amperometrically determined at a stationary Pt-ring electrode (applied potential + 0.600 V vs a Ag/AgCl, 3.0 M NaCl reference).
Department of Chemistry, Oklahoma State University, Stillwater 74078-0447.
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D000429:Alcohol Oxidoreductases / Q000097:blood; D000469:Alkaline Phosphatase / Q000097:blood*; D002623:Chemistry Techniques, Analytical / Q000379:methods; D004800:Enzymes, Immobilized / Q000097:blood*; D017022:Flow Injection Analysis / Q000379:methods*
D004800:Enzymes, Immobilized; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D000469:Alkaline Phosphatase