Details

Autor(en) / Beteiligte

• Shear, Jason Ben

Titel

Detection advances for analysis of biological microenvironments using capillary electrophoresis

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

1995

Beschreibungen/Notizen

• To provide chemical insights into the mechanisms that underlie cellular function, approaches for improving the sensitivity, selectivity, and quantitative abilities of detection systems for capillary electrophoresis (CE) are investigated. A laser-induced fluorescence detection system is developed for CE that employs a charge-coupled device (CCD) detector to electronically track moving analyte bands and digitally filter fluorescence from background. Detection limits for rhodamine-labeled analytes are in the 10$\sp{-13}$ M range. To improve the utility of CE/LIF measurements for cellular samples, on-column fluorescence tagging procedures are developed. The ability of this CE/LIF system to analyze neurotransmitters present in biological matrices is demonstrated. To increase measurement periods in shot-noise-limited fluorescence detection with low-intensity excitation, an algorithm is developed that directs a reduction in the separation field when an analyte band is in the detection zone. Residence times of analytes in the detection zone can be increased nearly tenfold without significant increase in analysis time, and the limit of quantitation can be extended nearly to the limit of detection. When using optimized, shot-noise-limited LIF, modification of the detection system or separation field must be performed during a separation to achieve a detection sensitivity that is independent of analyte electrophoretic mobility. The highest possible signal-to-noise ratio for all analytes can be achieved by (1) reducing the data-acquisition rate and the excitation intensity during a separation by ${{1}\over {\tau}}$, where $\tau$ is the separation time; or (2) increasing the electric field and scanning the position of the detection zone so that all species migrate through the detection zone at the same velocity. Emphasis has been placed on the development of living-cell biosensor devices for improving the selectivity of detection with microcolumn separations. Detection of analyte bands separated by CE is accomplished using single-cell biosensors (SCB) that transduce analyte-receptor interactions into an observable chemical or electrophysiologic change. Analyses of complex biological mixtures can be performed with sensitivities that rival the best reported detection approaches, and CE/SCB systems can be engineered to respond to specific components of interest.