Immobilization of enzymes on inorganic nanoparticles
Immobilization of avidinylated enzymes (Glucose Oxidase or GOx and Horseradish Peroxidase or HRP) on inorganic particles was accomplished utilizing the affinity of avidin for biotin. We have synthesized biotinylated oxides (layered silicates and iron oxides) via a condensation reaction, and through a simple one step process, we have immobilized enzymes improving their thermal behavior, storage stability and behavior in different pH environments. Furthermore, a profound catalytic activity increase per mass (30-fold) was observed for HRP when immobilized on magnetic iron oxide particles (magnetite particles). This phenomenon proved to be independent of the immobilization steps and was observed even when particles and HRP were simply suspended together in a buffer solution. The activity increase was reversible and could be turned on and off with the addition and subtraction of the magnetic particles (with a Nd magnet). The results were reproduced using different activity assays and different batches of enzyme. Activity assays using particles with increasing magnetic properties showed a proportional increase on the enzymatic activity. The results suggest that the randomly distributed magnetic particles affect the paramagnetic species found in the catalytic cycle of HRP, changing the overall reaction rate. On a different approach modified silicates with immobilized gramicidin were evaluated as delivery vehicles for gramicidin to E. coli bacteria. Also a fluorescent protein immobilized on a biotinylated layered silicate was used to track the uptake of modified silicates to mammalian 9L Glioma cells. Finally, layered silicates and amphiphilic molecules were combined to develop a synthetic biomimetic membrane. The biomimetic membrane has the characteristics of a lipid bilayer membrane with similar thermotropic transitions. To evaluate the membrane's sensing capability, a sensing platform was developed that utilized the biomimetic membrane as the recognition element. The sensing capability was evaluated using saccharin as the analyte, a suspected carcinogen molecule already proven to interact with lipid bilayer membranes in a sensor setup.
enzyme immobilization; magnetite particles; Horseradish peroxidase; activity increase; glucose oxidase; layered silicates; biomimetic membrane; saccharin; sensor; avidinylated enzymes; biotinylated oxides; magnetic field effect
dissertation or thesis