Use of immobilized enzymes in automated clinical analysis: determination of uric acid and glucose using immobilized enzymes in column form.

We studied the use of immobilized enzymes, covalently bound to alkylaminosilane derivative of porous glass, to automated clinical analysis on uric acid and glucose in blood, serum and urine. A microcolumn with an immobilized enzyme was prepared and used in an AutoAnalyzer I continuous flow system. Uricase (EC 1.7.3.3) from Candida utilis and glucose oxidase (EC 1.1.3.4) from Aspergillus niger were immobilized for the determination of uric acid and glucose, respectively. Hydrogen peroxide produced by these oxidases was colorimetrically determined using horse-radish peroxidase (EC 1.11.1.7) and a hydrogen acceptor in solution. Sensitivity and wash charactertistics of a column with immobilized enzyme, 1.5 mm of inner diameter and up to 40 mm in length, were satisfactory at an assay speed of 50 samples per hour. The results correlated well with those obtained by other well established methods utilizing the AutoAnalyzer system. The immobilized enzymes were sufficiently stable for at least two months of 2000 tests when used repeatedly. Clinical trials proved that this method is capable of replacing the soluble enzyme method, giving reliable and reproducible results at lower cost.