Development of a bienzymatic diagnostic system based on separately encapsulated urease and glucose oxidase for the simultaneous determination of urea and glucose in a single analytical sample

Enzymatic analysis methods are widely used for the determination of low-molecular-weight substances in various fields; however, they have several limitations. One promising approach to addressing these limitations is the encapsulation of enzymes in polyelectrolyte microcapsules (PMCs). In this study, the development of a bienzymatic diagnostic system based on two separately encapsulated enzymes—urease and glucose oxidase (GO)—is proposed for the first time, enabling the simultaneous determination of urea and glucose concentrations in a single sample. To achieve this, the influence of reaction products and substrates of urease on the activity of GO, and vice versa, was investigated. It was demonstrated that ammonia reduces the activity of glucose oxidase, while gluconic acid and hydrogen peroxide decrease the activity of urease, with the PMC shell exhibiting protective properties for the encapsulated form of urease. Other reaction components did not affect the enzyme activities. The linear range, coefficient of variation, and reusability of this diagnostic system were determined: the linear range for urea concentration detection was found to be between 10 and 50 mM (coefficient of variation 2 %) with 7 reuses of same PMC-urease; the linear range for glucose concentration detection was between 30 and 100 mM (coefficient of variation 8 %) with 13 reuses of same PMC-GO. This diagnostic system is suitable for determining urea and glucose concentrations in industrial biofuel production and in industrial fermenters using urea as a nitrogen source.