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Immobilized lactate oxidase for development of a long-term implantable lactate sensor


Development of a continuous, long-term implantable lactate sensor has long been a goal in the field of biosensors. The current standard of care in treatment of diabetes mellitus involves measurement of only one metabolite, glucose, on an infrequent, discrete basis. Continuous monitoring would capture of all metabolic excursions so that treatment decisions could be made on complete time- series information. In addition, the measurement of the metabolite lactic acid would lead to an even better understanding of the patient's metabolic state. Lactate has also been shown to be of importance in many diseases involving compromised circulatory or pulmonary function, and athletes would benefit from continuous measurement of this variable. Lactate oxidase (LOx), the enzyme used in the construction of our lactate sensor, is known to be quite unstable with respect to enzymatic activity over time. This leads to reduced sensor lifetimes, which can be problematic for an implantable sensor due to the necessity for frequent replacement. This dissertation focuses on the characterization of LOx as it pertains to sensor design. LOx was immobilized in different constructs and the activity as a function of in vitro incubation time was measured. Two unique systems of immobilization were used here: one based on ionic forces and the other based on chemical cross-linking. It was found that good immobilization yield (the amount of active enzyme remaining after immobilization divided by the amount formulated) could be achieved in the ionic immobilization scheme; however, stability was lower than free enzyme in PBS in all cases. Protection of LOx via complexing with high concentrations of the oppositely charged polymer (a polycation) was essential for maximizing stability. Immobilization via chemical cross-linking in a bovine serum albumin matrix also had good process yields when minimum amounts of cross-linking agent were used. In addition, it was found that stability could be enhanced when compared to free enzyme in PBS via immobilizing the enzyme at a pH of approximately 5.5. The parameters found by experimentation were used to determine the linear range as a function of time for a sensor design model

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