Coupled enzyme reactions in multicompartment microparticles

Spherical biopolymer particles have been fabricated, applying coprecipitation with calcium carbonate, followed by cross-linking of the macromolecules and dissolution of the inorganic support. Particles made of roughly 80% horseradish peroxidase (HRP) as well as glucose oxidase (GOX) were prepared and enzyme activities were confirmed, applying the Amplex Red assay. The enzyme particles were reusable for at least six times, with a remaining activity of 30-50% from the initial one. When multiple coprecipitation steps and one or several cross-linking procedures were applied, multicompartment particles were obtained. Each of the resulting concentric compartments could be independently loaded with biomolecules. Three coupled enzymes, beta-glucosidase (beta-Glu), GOX, and HRP have been incorporated stepwise in such particles. Each of these enzymes was located in a separate compartment, in a desired sequence, and at a defined position. The distance between the enzyme containing compartments was also varied, including spacing compartments consisting of bovine serum albumin (BSA). When fluorogenic substrates for beta-Glu and HPR were used, the start and the end of the coupled enzyme reaction were visualized and recorded inside of individual particles, applying confocal laser scanning microscopy. A strong influence of the spacing on the reaction kinetics of the last enzyme was observed, suggesting an impaired diffusion of the intermediate products of the chain reaction through the spacing compartments made of BSA. The influence of the spacing between compartments containing different enzymes on the reaction kinetics was demonstrated on the microscopic scale within one microparticle, which to the best of our knowledge was not achieved until now.