Co-immobilization of enzymes with the help of a dendronized polymer and mesoporous silica nanoparticles

The two enzymes Aspergillus sp. glucose oxidase (GOD) and horseradish peroxidase (HRP) were co-immobilized on solid silica supports in a spatially controlled way by using mesoporous silica nanoparticles (Hiroshima Mesoporous Materials, HMM) and a polycationic dendronized polymer (denpol). The silica support was first coated with the denpol, followed by the deposition of the mesoporous silica nanoparticles into which - in a next step - GOD was adsorbed. Finally, the GOD-loaded silica nanoparticles were coated with a denpol-HRP conjugate constituting of several HRP molecules which were covalently bound to the denpol via bis-aryl hydrazone (BAH) bonds. The entire immobilization process was followed in real time with quartz crystal microbalance with dissipation monitoring (QCM-D). The activities and storage stabilities of the co-immobilized enzymes were determined by analyzing a two-step cascade reaction involving the two immobilized enzymes GOD and HRP. d-glucose and o-phenylenediamine (OPD) were used as substrates for GOD and HRP, respectively. The cascade reaction - in which intermediate hydrogen peroxide was formed from d-glucose and dissolved O₂ with GOD - was shown to take place. The immobilized enzymes remained fairly stable for at least 2 weeks if stored in contact with an aqueous solution of pH = 7 at 4 °C. If, however, denpol-BAH-GOD coated HRP-loaded mesoporous silica nanoparticles were used (the reversed situation), the cascade reaction was not effective. This was probably due to slow diffusion of hydrogen peroxide from the surface-exposed GOD to the particle-trapped HRP, and/or due to an inefficient loading of active HRP inside the particles. Overall, the combination of two enzyme immobilization methodologies - enzymes adsorbed within mesoporous silica nanoparticles and enzymes adsorbed as denpol-BAH-enzyme conjugates - allows the spatially controlled localization of different types of enzymes in a simple way. Possible applications of the concept are in the field of bioelectrode fabrication.