Surface immobilization of organophosphorus hydrolase for biosensor based spectroscopic detection of an organophosphorus derivative
Date of Award
Doctor of Philosophy (Ph.D.)
First Committee Member
Roger M. Leblanc - Committee Chair
This dissertation explores several immobilization techniques of organophosphorus hydrolase (OPH). OPH is a very important broad specificity enzyme which catalyzes the hydrolysis reaction of organophosphorus (OP) compounds. OP compounds are commercially utilized in several different forms as insecticides and pesticides. Due to their structural resemblance to nerve agents, they must be monitored environmentally. The significance of OPH has revolutionalized material science especially in the area of designing enzyme-based sensors.OPH was immobilized by both physical adsorption techniques and through chemical attachment via covalent reaction to a quartz slide. Four methods of immobilization were explored. The fabricated thin-films were characterized by surface chemistry isotherms and spectroscopic techniques, and the topographies were examined by imaging microscopy.Immobilization leads to increased stability of the enzyme compared to solution. Native OPH is a globular enzyme made up of several types of secondary structure. The secondary stricture was investigated by looking at the change in conformation of the enzyme upon immobilization. The bioactivity of the immobilized enzyme was tested, by exposing the sensing system to different concentrations of paraoxon (PXN) aqueous solution. The presence of PXN was indicated by spectroscopic detection of the hydrolysis product, para-nitrophenol and also by photonic detection due to changes in the fluorescence intensity of the film. Several of the immobilized OPH films showed a sensitivity of 10-9 M to PXN aqueous solution. The advantages of the developed sensing systems were a fast detection time, good reproducibility and recovery of enzyme activity. The experimental data produced have confirmed that OPH can be efficiently utilized as an enzymatic biosensor. All models for OPH biosensors herein can be used as prototypes for biosensor design.
Constantine, Celeste A., "Surface immobilization of organophosphorus hydrolase for biosensor based spectroscopic detection of an organophosphorus derivative" (2004). Dissertations from ProQuest. 2105.