The overall objective is to develop, test, and confirm sensor and sensor- like techniques for the measurement of insulin with high temporal and spatial resolution. The work focuses on two approaches. One is the use of an amperometric electrode to measure insulin secreted from individual cells. The other is based on combining capillary zone electrophoresis (CZE) with microdialysis sampling for in vivo measurements. An insulin-sensitive microelectrode has been developed and used to make amperometric recordings at single pancreatic beta-cells. Application of glucose to a cell results in a series of current spikes when the electrode is near the cell. The current spikes may be due to oxidation of packets of insulin secreted by exocytosis.
The first aim i s to confirm that insulin is the detected substance using a variety of tests including independent chemical analysis and pharmacological evaluation. Secondly, the hypothesis that the spikes are due to exocytosis will be tested using multiple electrode measurements and evaluation of spike frequency, area, and shape. Improvements in time resolution and stimulation conditions will be required to perform these tests. Other improvements will also be pursued including: 1) combination with glucose and oxygen electrodes to make simultaneous measurements at a cell and 2) using smaller electrodes to observe spatial heterogeneity of release from a cell. The technique will be useful in studying many aspects of insulin secretion including exocytosis, stimulus-secretion coupling, and pharmacological actions. Given the role of insulin secretion in diabetes, this technique could have a significant health impact. For insulin measurements in more complex environments, the coupling of microdialysis with a CZE/laser induced fluorescence assay will be explored. Insulin will be """"""""tagged"""""""" by adding a fluorescently-labeled antibody fragment to the dialysate. The antibody and antibody/insulin complex will be rapidly separated by electrophoresis. Variations of this analysis will also be explored including a competitive assay and assays for other peptides of interest. It is expected that minute time resolution with quantitative recovery from the dialysis probe will be possible. This technique may prove useful in clinical settings and for in vivo measurements. The technique may also be useful as a general approach to developing sensors for other peptides.
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