Preparation of reusable enzyme strips using alkylamine and arylamine glass beads affixed on plastic strips for urea determination

Reusable strips of urease were prepared by covalently immobilizing it onto alkyl and arylamine glass beads, affixed on a plastic strip with a non-reactive adhesive. The immobilized urease retained its initial activity, prior to immobilization about 56% on alkylamine and 33.3% on arylamine. Maximum activity of alkylamine conjugated urease was attained at pH 6.5, 25 degrees C for 1 min, while arylamine conjugated urease showed maximum activity at pH 7.0, 40 degrees C, 15 min. The Km for urea was 4.65 and 16.66 mM, for alkyl and arylamine conjugated urease respectively. A method for determination of serum urea developed using these strips is based on measurement of NH3 generated with Nessler's reagent. The minimum detection limit permitted by the method was 10 and 5 mg/dL using alkyl and arylamine conjugated urease, respectively. Urea value in serum of healthy persons determined by this method, was in the range of 5-55 mg/dL with a mean of 26 mg/dL. The recovery of added urea was 82% for alkylamine and 85% for arylamine glass beads. A fair correlation (r=0.821) was found between urea values obtained by the standard urease kit and the present method. The alkylamine and arylamine conjugated urease stored at 4 degrees C in 0.02 M potassium phosphate buffer pH 7.0 were reused 100 and 90 times respectively, during the span of one month. The method offers advantage of enormous case in handling of immobilized urease.