Coupled reactions of immobilized enzymes and immobilized substrates: clinical application as exemplified by amylase assay.
We described a partitioned enzyme-sensor system, which incorporates an immoblized substrate and three or more discrete immobilized enzymes. This instrument measures alpha-amylase activity by passing the solution containing alpha-amylase over a column packed with immobilized starch. The resulting oligosaccharides are successively exposed to a column or columns containing immobolized glucose oxidase, catalase, glucoamylase or maltase, and glucose oxidase. The resulting hydrogen peroxide is detected by a three-electrode amperometric cell. All immobilized reagents were immobilized on a particulate, porous alumina to allow rapid and constant flow rate. With use of less than optimum immobilized reagents, alpha-amylase activity has been measured from about 5 to 200 kU/liter with a 50 microliter sample size. Lack of sensitivity is predominantly attributable to the low activity and low stability of immobilized maltase and glucoamylase. We believe that a clinical test using this system is feasible and desirable because the immobilized reagent system should allow for testing of alpha-amylase with excellent precision, convenience to the operator, and low cost.
D000681:Amylases / Q000032:analysis*; D000818:Animals; D002374:Catalase; D004800:Enzymes, Immobilized; D005087:Glucan 1,4-alpha-Glucosidase; D005949:Glucose Oxidase; D008722:Methods; D010179:Pancreas / Q000201:enzymology; D013213:Starch; D013552:Swine; D000516:alpha-Amylases / Q000032:analysis*
D004800:Enzymes, Immobilized; D013213:Starch; D005949:Glucose Oxidase; D002374:Catalase; D000681:Amylases; D000516:alpha-Amylases; D005087:Glucan 1,4-alpha-Glucosidase