Development of an amperometric sensor system for use in real-time monitoring of endocrine events.

摘要

An understanding of dynamics of hormone secretion has been hampered by shortcomings in the techniques available for studying secretory events. Blood sampling measures hormonal signals only after dilution, dispersion, and clearance of hormones in systemic blood. Further, blood withdrawal can alter the hormonal balance one is trying to measure, and therefore the number and frequency of samples taken are limited.; Characterization of the rapid dynamics of hormone secretion and control necessitates the development of a new methodology able to monitor endocrine metabolism rapidly, and without affecting the intricate endocrine balance. This thesis describes the design, characterization, and implementation of an amperometric biosensor system which performs cyclic voltammetry, and is capable of monitoring endocrine metabolism on-line and in real-time. Temporal resolution on the order of seconds was achieved through the use of a high speed computer interface. These time scales are an order of magnitude greater than can be obtained realistically and cost effectively using prior methodologies.; This sensor system was used to monitor the depletion of ascorbic acid from the luteinized ovary in vivo. It was also used to monitor adrenal gland metabolism and response to external environmental stimuli in vivo. In addition, the sensor was able to monitor the prerelease of ascorbate prior to catecholamine secretion from the adrenals which has been described in vitro but heretofore never observed in vivo. Finally, the sensor system was integrated into a microperifusion chamber to monitor metabolism of the anterior pituitary gonadotropes and corticotropes. The response of the sensor from these cells demonstrated that multiple intracellular second messenger systems are responsible for the release of hormones from the gonadotropes in response to GnRH stimulation.; It also showed that CRF and AVP elicit hormone release from the corticotropes through two different intracellular mechanisms resulting in very different dynamics of secretion of the same hormone, ACTH. These results point out many of the advantages this sensor system has over conventional methods of study, which utilize blood withdrawal followed by immunoassay.