The application of immobilized enzymes in analytical chemistry.

Abstract

The use of immobilized enzymes (and cells) in flow analyses and some determinants of the performance of the resulting analytical systems have been examined; the detector used in most cases was the LKB 2017-30 flow microcalorimeter. The detector time constant and sensitivity, reactor enzyme activity, flow rate and the mode of sample injection were primary determinants of system characteristics such as, sensitivity, minimum detectable concentration, linearity range and speed analysis. These relationships were later applied in the determination of the reaction enthalpy. Glucose, urea, fructose, mannose, glucoseamine, the cofactor ATP, vitamin C and hydrogen peroxide were also determined using the appropriate immobilized enzymes. The kinetics of the reactions catalysed by immobilized urease, glucose oxidase, acetylcholinesterase or ascorbate oxidase was studied via flow microcalorimetry. Apparent intrinsic kinetic parameters, external and internal substrate moduli, as well as the corresponding effectiveness factors were determined. In all cases,the Michaelis constant (Km) was increased and the observed activity severely limited as a consequence of the slow diffusion of substrate. Flow analysis using immobilized cells and microcalorimetric monitoring was also examined. This combination is well suited for broad-spectrum analysis. The effects of selected organic solvents (permeablizing agents) on the linearity range (approximately given by the Km) was also of some interest. The specificity (estimated as (dQ/dt)max/KmaPP) for a range of substrate was proposed as a means of differentiating between closely related strains of cells. Electrochemical monitoring of immobilized enzyme reactions may rival the thermochemical approach in its widespread applicability and simplicity. Both potentiometric and amperometric modes were used; the latter in connection with flow analyses. Two amperometric enzyme reactors and a flow-through enzyme electrode were constructed and successfully used in substrate determinations.