Browsing by Subject "optical biosensor"
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Item Development and Characterization of Stable Glycoenzyme Conjugates(2014-11-07) Ritter, Dustin W.Optical glucose biosensors are being developed for long-term monitoring in diabetic individuals. These sensors rely upon the enzyme glucose oxidase, and loss of enzymatic activity leads to a need for frequent recalibration and eventually sensor replacement. Current enzyme stabilization strategies are effective, but generally result in a large increase in size and exclusion from the solution-phase. This sacrifice of native properties precludes the stabilized enzyme from incorporation into the aforementioned sensing platform, which requires that the enzyme be homogenously distributed and entrapped within a hydrogel. It is this incompatibility which provides the motivation for the development of new non-traditional enzyme stabilization strategies. Toward that end, this work focuses on the development and characterization of three enzyme modification strategies, all of which are intended to stabilize enzyme activity while permitting incorporation into an optical biosensing hydrogel. The first approach involves glycosylation site-targeted covalent attachment of poly(ethylene glycol) to glucose oxidase, which improves storage stability by 60%. The second approach builds upon the first, but subsequent modification of the poly(ethylene glycol)-modified glucose oxidase is performed to further stabilize the enzyme. This approach improves long-term storage stability by an order of magnitude. The final approach involves encasement of the glycoenzyme within a shell of albumin, wherein the inert protein is attached at the glycosylation sites in an orthogonal manner. This technique result in highly thermostable enzyme, retaining greater than 25 times more activity than native glucose oxidase following exposure to buffer at 60 ?C. In summary, enzyme deactivation is expected to be a major barrier in the realization of long-term glucose sensing with fully implantable optical glucose biosensors, and this work represents a step towards overcoming that hurdle. Each enzyme modification strategy yields a stabilized enzyme under certain conditions, whether it be long-term storage, elevated temperature, or exposure to various solvents/additives. This work enables the stabilized enzymes to be incorporated into hydrogels for evaluation under simulated in vivo conditions, followed by in vivo evaluation. Finally, it is expected that these enzyme stabilization approaches will be advantageous in other applications as well, including in vitro diagnostics, tissue engineering, and therapeutic biologicals.Item Dissolved oxygen and pH monitoring within cell culture media using a hydrogel microarray sensor(2009-05-15) Lee, Seung JoonProlonged exposure of humans and experimental animals to microgravity is known to be associated with a variety of physiological and cellular disturbances. With advancements in aerospace technology and prolonged space flights, both organism and cellular level understanding of the effects of microgravity on cells will become increasingly important in order to ensure the safety of prolonged space travel. To understand these effects at the cellular level, on-line sensor technology for the measurement and control of cell culture processes is required. To do this measurement, multiple sensors must be implemented to monitor various parameters of the cell culture medium. The model analytes used in this study were pH and dissolved oxygen which have physiological importance in a bioreactor environment. In most bioprocesses, pH and dissolved oxygen need to be monitored and controlled to maintain ionic strength and avoid hypoxia or hyperoxia. Current techniques used to monitor the value of these parameters within cell culture media are invasive and cannot be used to make on-line measurements in a closed-loop system. In this research, a microfabricated hydrogel microarray sensor was developed to monitor each anlyte. Either a pH or an oxygen sensitive fluorescent agent was immobilized into a hydrogel structure via a soft lithography technique and the intensity image of the sensor varied from the target analyte concentration. A compact detection system was developed to quantify concentration of each analyte based on the fluorescence image of the sensor. The system included a blue LED as an illumination source, coupling optics, interference filters and a compact moisture resistant CCD camera. Various tests were performed for the sensor (sensitivity, reversibility, and temporal/spatial uniformity) and the detection system (temporal/spatial stability for the light source and the detector). The detection system and the sensor were tested with a buffer solution and cell culture media off-line. The standard error of prediction for oxygen and pH detection was 0.7% and 0.1, respectively, and comparable to that of commercial probes, well within the range necessary for cell culture monitoring. Lastly, the system was coupled to a bioreactor and tested over 2 weeks. The sensitivity and stability of the system was affordable to monitor pH and dissolved oxygen and shows potential to be used for monitoring those analytes in cell culture media noninvasively.