Continuous-flow analysis for glucose, triglycerides, and ATP with immobilized enzymes in tubular form

Abstract

Small-bore ("Autozyme") tubes with immobilized enzymes at the inner wall have been developed and studied for application in the Technicon "SMAC" high-speed continuous-flow biochemical analyzer. Tubes coated with glucose oxidase (D-glucose:oxygen oxidoreductase, EC 1.1.3.4) have been prepared for the assay of glucose, with colorimetric assay of the hydrogen peroxide produced; tubes coated with glycerol kinase (ATP:glycerol phosphotransferase, EC 2.7.1.30) for the enzymatic assay of triglycerides; tubes coated with hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) and glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NAD+ oxidoreductase EC 1.1.1.49) for the measurement of ATP, an intermediate product in assays for creatine kinase. With use of 10-15 cm lengths of Autozyme tube and SMAC hydraulics (150 samples per hour), assay sensitivity and carryover were similar to values for the corresponding free-enzyme methods. These immobilized enzymes were sufficiently stable for one to eight weeks of continuous use before replacemnt. We conclude that suitable bound-enzyme tubes can replace either single or multiple free-enzyme reagents in many continuous-flow assays at high sampling rates.