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Title page for ETD etd-03272009-144927


Type of Document Dissertation
Author Wilburn, Jeremy
Author's Email Address jeremy.wilburn@gmail.com
URN etd-03272009-144927
Title Electrochemical Metabolic Profiling of Live Cells and Pancreatic Islets
Degree PhD
Department Chemistry
Advisory Committee
Advisor Name Title
Dr. David E. Cliffel Committee Chair
Keywords
  • multianalyte microphysiometry
  • scanning electrochemical microscopy
  • targeted metabolic analysis
Date of Defense 2009-02-23
Availability unrestricted
Abstract
The past decade has witnessed the birth of the field of Metabolomics, which aims to study the "chemical fingerprints" left behind by specific cellular processes. The chemical fingerprints themselves consist of the totality of metabolites within a biological entity. While the goal of Metabolomics is to take a snapshot of all metabolites at a given time, there are also subgenres that focus on small numbers of metabolites (e.g., metabolic profiling) or individual metabolite measurements (e.g., targeted metabolic analysis).

The object of the studies presented in this dissertation is to expand the capabilities of the instrumental platforms of Multianalyte Microphysiometry (MAMP) and Scanning Electrochemical Microscopy (SECM) toward metabolic profiling and targeted metabolic analyses, and thus expand the state-of-the-art in electrochemical metabolic studies. In that respect, these techniques were applied toward MAMP studies of organophosphate toxicity in a model neuronal cell line through the incorporation of new sensors for extracellular calcium and dopamine, in order to develop and refine a computational model of neuronal cellular responses to the toxins. The targeted analysis of insulin secretion was studied in the MAMP by introducing a new experimental protocol utilizing continuous flow, rather than the previous flow/stop-flow parameters. In order to enable targeted analysis of insulin production in SECM, a pre-existing insulin sensor was adapted for use with a 7 um carbon-fiber ultramicroelectrode. This new sensor successfully detected insulin exocytosis by single islets as well as differentiated levels of insulin production at physiologically low (2.8 mM) and high (16.7 mM) glucose levels. In order to perform similar targeted analysis of lactate production and glucose uptake by individual live cells, 25 um Pt electrodes were modified with films of lactate oxidase and glucose oxidase respectively, resulting in the first published measurements of live cells using enzyme-modified ultramicroelectrodes. Finally, SECM imaging was used for respiratory targeted analysis of single live cells and pancreatic islets as well as for the potential of the peptaibol antibiotic alamethicin to function as a microfluidic valve when incorporated in a lipid bilayer membrane.

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