Continuous-flow analysis for glucose, triglycerides, and ATP with immobilized enzymes in tubular form.
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.
D000255:Adenosine Triphosphate / Q000032:analysis*; D001322:Autoanalysis; D004800:Enzymes, Immobilized; D005947:Glucose / Q000032:analysis*; D005949:Glucose Oxidase; D005954:Glucosephosphate Dehydrogenase; D005991:Glycerol Kinase; D006593:Hexokinase; D006735:Horseradish Peroxidase; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase; D008722:Methods; D014280:Triglycerides / Q000032:analysis*
D004800:Enzymes, Immobilized; D014280:Triglycerides; D000255:Adenosine Triphosphate; D005954:Glucosephosphate Dehydrogenase; D005949:Glucose Oxidase; D006735:Horseradish Peroxidase; D006593:Hexokinase; D005991:Glycerol Kinase; D008049:Lipase; D005947:Glucose