Glutamate and aspartate measurements in individual planaria by rapid capillary electrophoresis.

INTRODUCTION: Planaria present a unique model organism for studying primitive central nervous systems. The major mammalian excitatory neurotransmitters, glutamate and aspartate, have previously been measured in planaria via high pressure liquid chromatography (HPLC). A faster extraction and analysis procedure using capillary electrophoresis (CE) was developed which confirms the presence of these amino acids in single planaria homogenates. METHOD: Following homogenization and centrifugation of individual planaria in hydrochloric acid/acetonitrile, glutamate and aspartate were derivatized with naphthalene-2, 3-dicarboxaldehyde (NDA). The labeled amino acids were measured using capillary electrophoresis with laser-induced fluorescence (CE-LIF). RESULTS: CE-LIF electropherograms were generated in less than 1 min. The mean ± S.D. amounts of glutamate and aspartate were 1200 ± 500 and 1900 ± 700 pmol/mg-planarian (n=22), respectively. Spiked average recoveries of glutamate and aspartate were 96% and 91%, respectively. DISCUSSION: The high-throughput method provides the ability to quantitate changes in excitatory neurotransmitters under developmental or stimulatory conditions. The capability to monitor multiple neurotransmitter levels offers the opportunity to correlate behavioral responses with biochemical changes in planaria.

Gradient counterflow electrophoresis methods for bioanalysis.

CE has evolved as one of the most efficient separation techniques for a wide range of analytes, from small molecules to large proteins. Modern microdevices facilitate integration of multiple sample-handling steps, from preparation to separation and detection, and often rely on CE for separations. However, CE frequently requires complex geometries for performing sample injections and maintaining zone profiles across long separation lengths in microdevices. Two novel methods for performing electrophoretic separations, gradient elution moving boundary electrophoresis (GEMBE) and gradient elution isotachophoresis (GEITP), have been developed to simplify microcolumn operations. Both techniques use variable hydrodynamic counterflow and continuous sample injection to perform analyses in short, simple microcolumns. These properties result in instruments and microdevices that have minimal 'real-world' interfaces and reduced footprints. Additionally, GEITP is a rapid enrichment technique that addresses sensitivity issues in CE and microchips.

Capillary and microfluidic gradient elution isotachophoresis coupled to capillary zone electrophoresis for femtomolar amino acid detection limits.

In this work, gradient elution isotachophoresis was combined with capillary zone electrophoresis (GEITP-CZE) in a single microcolumn. The multistage approach addresses the issues of analyte resolution difficulties in GEITP, as well as poor concentration sensitivity in CZE. GEITP employs rapid electrophoretic focusing at a discontinuous ionic interface within a sample well generated through combined electroosmotic and hydrodynamic flows. The interface and enriched analytes are then pulled into a capillary or microchannel as the counter-flow is reduced for on-column detection. To transform GEITP-focused samples to CZE-based separation, the sample solution is replaced with CZE buffer solution while maintaining hydrodynamic flow to ensure migration toward the detector. The single solution switch and lack of polarity inversion allows for reproducible separations (typically <6% relative standard deviation in peak heights and <0.5% in migration times). Low-pressure hydrodynamic flow during CZE allowed for flexible resolution adjustment, with a linear increase versus the square root of migration time, without altering the separation column, field strength, or electrolyte system. As a first demonstration of the applicability of GEITP-CZE, a series of amino acids to be assayed for in future Mars exploration missions as indicators of biological life were studied. Separation of six amino acids, with limits of detection as low as 200 fM, were achieved using a capillary format with a total analysis time of 11 min. A glass-based microfluidic implementation is also demonstrated that can perform GEITP-CZE in 1 cm effective lengths.