Rapid, High Sensitivity Capillary Separations for the Analysis of Biologically Active Species

Abstract

A series of rapid, high sensitivity capillary electrophoresis (CE) separation systems have been developed for the analysis of biological analytes and systems. A majority of the work has focused on development of novel instrumentation, in which new injection and detection strategies were investigated to improve the sensitivity of fast CE. A novel optical injection interface for capillary zone electrophoresis based upon the photophysical activation of caged dye attached to the target analyte was developed. The primary advantage of this approach is the lower background and background-associated noise resulting from reduced caged-fluorescein emission in conjunction with the high quantum yield of the resulting fluorescein. Improved detection limits were obtained compared to those observed in photobleaching-based optical gating. A primary drawback of photolytic optical gating CE is the lack of available caged-dye analogs with sufficiently fast reaction kinetics for online derivatization. To overcome this limitation, we have developed a chemical derivatization scheme for primary amines that couples the fast kinetic properties of o-phthaldialdehyde (OPA) with the photophysical properties of visible, high quantum yield, fluorescent dyes. The feasibility of this approach was evaluated by using an OPA/fluorescent thiol reaction, which was used to monitor neurotransmitter mixtures and proteins. The utilization of a high power ultraviolet light emitting diode for fluorescence detection in CE separations has been introduced to analyze a range of environmentally and biologically important compounds, including polyaromatic hydrocarbons and biogenic amines, such as neurotransmitters, amino acids and proteins, that have been derivatized with UV-excited fluorogenic labels. To understand cellular chemistry, it is imperative that single cells should be studied. This work was focused on developing CE based method to characterize the cellular uptake of TAT-EGFP. We demonstrated TAT mediated delivery of EGFP protein into HeLa cells and TAT-EGFP loaded single cell was analyzed by CE-LIF to determine the intracellular EGFP content. An application of CE-LIF for the determination of biogenic amine levels in the antennal lobes of the Manduca sexta is also explored and methods were developed to analyze a single antennal lobe dissected from moths. The lobe was digested and contents were labeled with the fluorogenic dye prior to CZE analysis.