The metabolism of amino acids produces important chemical signaling molecules called neurotransmitters, which are responsible for carrying out important actions within the human body. There are approximately one hundred identified neurotransmitters. Neurotransmitter study is important due to their involvement in biological, physiological, pharmacological, and pathological functions. Commonly employed methods for neurotransmitter detection are mainly based upon microdialysis. However, the methods suffer from disadvantages. Microdialysis fails to determine the absolute concentration of analytes and therefore requires it to be tied in with an analytical technique such as high performance liquid chromatography or capillary electrophoresis. Although high performance liquid chromatography is the most powerful analytical technique to date, it necessitates high maintenance and suffers from poor temporal resolution. While capillary electrophoresis affords more rapid separations than high performance liquid chromatography, it suffers from poor concentration limits of detection and requires large sample dilutions of highly conductive samples, such as biological fluids. Consequently, research is focused on detection of various amino acids and neurotransmitters employing novel analytical techniques along with traditional capillary electrophoresis.
First, a method was developed using traditional capillary electrophoresis with laser induced fluorescence detection to detect two major excitatory neurotransmitters, glutamate and aspartate in planaria. The method was later applied to detect several biogenic amines using micellar electrokinetic chromatography with laser induced fluorescence detection in planaria to study the effect of feeding on the levels of biogenic amines within individual planaria homogenates.
The concentration sensitivity issue of capillary electrophoresis led to the use of a new method for sensitive neurotransmitter measurements, gradient elution isotachophoresis. Gradient elution isotachophoresis is an efficient capillary-based enrichment and separation technique based on balancing hydrodynamic counter-flow against electrophoresis. Enrichment is achieved with the aid of high concentrations of leading electrolyte in the counter-flow solution that creates an ionic interface near the capillary inlet. Discrete electrolyte spacers or carrier ampholyte mixtures are used to separate analyte zones. The method was applied to the enrichment and separation of physiologically relevant concentrations of aspartate and glutamate labeled with dansyl chloride, phenyl isothiocyanate, or carboxyfluorescein, succinimidyl ester in artificial cerebrospinal fluid using ultraviolet absorbance detection. Finally, gradient elution isotachophoresis was combined with capillary zone electrophoresis to eliminate the use of spacers and provide rapid separations and enrichment. The technique was applied for the detection of biogenic amines in a glass microfluidic device.
|Advisor:||Shackman, Jonathan G.|
|Commitee:||Nicholson, Allen W., Spano, Francis C., Strongin, Daniel R.|
|School Location:||United States -- Pennsylvania|
|Source:||DAI-B 72/04, Dissertation Abstracts International|
|Subjects:||Neurosciences, Analytical chemistry, Biochemistry|
|Keywords:||Amino acids, Biogenic amines, Capillary electrophoresis, Gradient elution isotachophoresis, Microfluidics, Neurotransmitters, Preconcentration techniques|
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