Co-encapsulated urease and glucose oxidase in polyelectrolyte microcapsules for simultaneous glucose and urea determination

Background Clinical monitoring of metabolic disorders requires stable tools for simultaneous analyte detection. However, conventional free-enzyme assays suffer from rapid degradation, poor operational stability, and single-use limitations. Herein, we report a novel bi-enzymatic diagnostic system based on the rational co-encapsulation of glucose oxidase (GOx) and urease within polyelectrolyte microcapsules (PMCs) for the simultaneous determination of glucose and urea. Results Fabricated via layer-by-layer assembly, the PMCs achieved high enzyme loading (73 ± 2% for GOx and 93 ± 5% for urease). Co-encapsulation significantly reduced the GOx Michaelis constant from 10.1 to 2.5 mM. The system exhibited linear ranges of 0.5–3.0 mM (glucose) and 1–40 mM (urea), covering clinically relevant concentrations. It maintained performance for over 61 measurement cycles and a 27-day shelf-life at room temperature. Direct analysis of human saliva revealed that the PMCs effectively shield enzymes from matrix interferences, yielding significantly lower data dispersion and higher reproducibility than standard Clark-electrode and Berthelot reference methods. Significance By combining multi-cycle reusability, room-temperature stability, and enhanced precision in raw biological fluids, this co-encapsulation strategy overcomes the fundamental trade-offs of traditional immobilization. The developed PMCs provide a robust, cost-effective, and comprehensive platform for point-of-care metabolic diagnostics and non-invasive monitoring, establishing a generalizable framework for future multi-enzymatic biosensing applications.