Real-time assessment of spatial and temporal coupled catalysis within polyelectrolyte microcapsules containing coimmobilized mucose oxiase and peroxidase

The encapsulation of biological enzymes within polyelectrolyte microcapsules is an important step toward microscale devices for processing and analytical applications, one which could be applied to the realization of minimally invasive sensing technology. In this work, the encapsulation and functional characterization of a bienzymatic coupled catalytic system within polyelectrolyte microcapsules is described. The two components, glucose oxidase (GOx) and horseradish peroxidase (HRP), were coprecipitated with calcium carbonate microspheres, followed by layer-by-layer assembly to form ultrathin polymer film coatings that act as capsule walls after removal of the sacrificial carbonate cores. Encapsulated concentrations of GOx and HRP were determined to be 19.7 +/- 1 1.0 and 29.4 +/- 3.6 mg/mL, respectively. An 85% decrease in the rate of glucose consumption relative to GOx and HRP in free solution was observed, which is attributed to substrate diffusion limitations. To further understand the temporal and spatial dynamics of the two-step reaction, a technique for monitoring microscale glucose consumption was developed using confocal imaging techniques. Time-based acquisition of capsule/Amplex Red suspensions was performed, from which it was observed that the high concentration of enzyme immobilized within the capsule walls resulted in a greater rate and quantity of glucose consumption at the capsule periphery when compared to glucose consumption within the capsule interior. These findings demonstrate the function of a bienzymatic catalytic system within the controlled environment of polyelectrolyte microspheres and a novel approach to analysis of the internal reactions using confocal imaging that will allow direct comparison with reaction -diffusion modeling and further explorations to optimize the distribution and activity of the encapsulated species.