Capillary and microchip electrophoresis for the analysis of biological samples.

摘要

Electrophoretic methodologies have been developed and employed in the chapters of this thesis for the analysis of various biological samples from Drosophila head homogenates to single mammalian cells and autocatalytic ribonucleic acids. Chapter 1 discusses resolution of fourteen biogenic amines and metabolites with similar mobilities by employing micellar electrokinetic capillary chromatography (MEKC) coupled to amperometric electrochemical detection (ECD). The optimization of separation conditions to achieve resolution of analytes of biological significance within 20 minutes in a single separation is presented. These molecules include dopamine, epinephrine, norepinephrine, octopamine, L-3, 4-dihydroxyphenylalanine, tyramine, and serotonin as well as metabolites 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 3-methoxytyramine in addition to metabolites specific to invertebrates including N-acetyl dopamine, N-acetyl octopamine, and N-acetyl serotonin. The separation conditions have been optimized and result in excellent reproducibility and predictable peak shifting, thus enabling identification of several metabolites along with their biogenic amine precursors in biological samples, specifically from the fruit fly Drosophila melanogaster. The separation method is sensitive, selective and quantitative as demonstrated by its capacity to detect changes in tyramine, octopamine, and N-acetyl octopamine present in the head homogenates of the Canton-S and mutant inactive1 Drosophila lines. Quantitative analysis of metabolites in conjunction with their biogenic amine precursors in a single separation offers tremendous potential to understand the physiological processes and underlying mechanisms mediated by various biogenic amines in Drosophila and other animals.;The separation method developed has been adapted for use in a small-volume separation of single Drosophila heads and has been employed in Chapter 3 to demonstrate the measurement of neurochemical variability between individual Drosophila heads. Reproducible homogenization of single Drosophila heads in 250-nL volumes provides a four-fold increase in sensitivity without overloading the electrochemical detector. Importantly, this increase in sensitivity has led to the unique detection of analytes, such as N-acetyl tyramine and octopamine in Drosophila under these conditions. Notably, analytes including L-3, 4-dihydroxyphenyl alanine, N-acetyl octopamine, N-acetyl dopamine, N-acetyl tyramine, N-acetyl serotonin, octopamine, dopamine, tyramine and serotonin have been consistently identified in single head homogenates corresponding well with previous MEKC population studies on Drosophila. (Abstract shortened by UMI.).