Modern enzyme-linked bioanalytical methods.

Abstract

Research concerning the development of various enzyme-linked bioanalytical methods is described. Riboflavin binding protein (RBP) is used as the specific binder in new enzyme-linked competitive binding assays for riboflavin. Analytical studies dealing with the development of biosensors based on novel asymmetric ion-selective cellulose acetate membranes are also presented. Both heterogeneous and homogeneous competitive binding assay protocols for the determination of riboflavin are detailed. These assays are based on the competition between analyte vitamin molecules and enzyme-riboflavin conjugates for a limited number of RBP sites. For the heterogeneous assay, a glucose-6-phosphate dehydrogenase-3-carboxymethylriboflavin conjugate is used in conjunction with immobilized RBP. As the concentration of riboflavin in the sample increases, less enzyme-riboflavin conjugate associates with the immobilized binder, resulting in lower enzymatic activity bound to the solid phase. For the much faster homogeneous protocol, a malate dehydrogenase-3-carboxymethylriboflavin conjugate is utilized in conjunction with soluble RBP. In the absence of the vitamin, the catalytic activity of the enzyme-riboflavin conjugate is inhibited up to 71%. In the presence of riboflavin, activity is regained in proportion to the concentration of vitamin present. Under optimized conditions, both heterogeneous and homogeneous assays are shown to offer adequate detection limits and selectivities for direct measurement of riboflavin in human urine and multivitamin preparations without sample pretreatment. The potentiometric response properties of ammonium, carbonate and proton selective electrodes prepared by incorporating appropriate neutral carriers within novel asymmetric cellulose acetate membranes are reported. The hydrolyzed surface of the asymmetric membranes can be activated in aqueous solution with carbonyldiimidazole for the direct immobilization of proteins to the surface of the membranes, without loss in potentiometric ion response. A novel approach for enhancing the substrate sensitivity of ion-selective electrode-based enzyme probes is also introduced. The concept involves the use of two working enzyme electrodes in a differential potentiometric cell arrangement. Since each sensor responds selectively to the same substrate but in opposite directions, the potential difference between the two enzyme electrodes is greater than that obtained when each enzyme probe is measured alone versus a standard reference electrode.