Item Details

title

ANALYSIS BY CAPILLARY ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION: FROM SMALL MOLECULES TO WHOLE CELLS

author

Massie, Tara

abstract

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is a versatile and sensitive analytical tool with many potential applications. Its advantages over other chromatographic methods include minimal solvent and sample requirements, low waste volumes, simple instrumentation, and diversity of analytes studied. CE is one of the most efficient separation techniques available for the analysis of both large and small molecules, but because few analytes are natively fluorescent, it is necessary to bind them with fluorescent tags if LIF detection is to be employed. These tags increase sensitivity and provide additional separation from components of the sample that do not bind the tags. In this dissertation, four applications of CE-LIF for the determination of tagged analytes are documented. First, a rapid CE-LIF method was developed for the determination of gamma-aminobutyric acid (GABA) and other important brain biogenic amines and amino acids present in biological samples at low levels. The high efficiency and sensitivity necessary to identify toxicologically relevant changes in neurotransmitter levels in brain tissue and microdialysates following Mn exposure was achieved, and the method required minimum sample work-up and quantities of sample for analysis. A linear relationship between the concentration of GABA and the CE-LIF peak height was established, with correlation coefficients of 0.989 or better for four neurotransmitters. Second, the use of two squarylium-based fluorescent dyes (bis-SQHN-4d and SQHN-3c) as noncovalent protein labels was investigated. The fluorescence emissions of both dyes were enhanced upon complexation with model proteins, demonstrating promise as noncovalent fluorescent protein labels. Third, the nature of interactions between two boronic acid-based probes (SQTM-BA1 and ANQW-BA1) and two model proteins (bovine serum albumin (BSA) and hemagglutinin (HA)) have been explored. The complex between SQTM-BA1 and BSA was the only complex to show increased fluorescence emission, and the role of electrostatic interactions between SQTM-BA1 and BSA in complex formation was explored. Lastly, CE-LIF methods were developed to label and quantify turnip yellow mosaic virus (TYMV). Noncovalent labels Red-1c and NN127 effectively labeled the surface proteins of the virus, and the labeled viruses were then analyzed by CE-LIF. The CE-LIF methods developed herein for the analysis of neurotransmitters, free solution proteins, and viruses demonstrate the versatility of the technique, especially when coupled with fluorescent labeling.

subject

Capillary Electrophoresis

Laser-Induced Fluorescence

Neurotransmitters

Noncovalent Labeling

Squarylium

Virus

contributor

Colyer, Christa L (committee chair)

Jones, Bradley T (committee member)

Jones, Paul B (committee member)

Noftle, Ronald E (committee member)

date

2011-02-16T21:42:28Z (accessioned)

2011-04-07T08:30:06Z (available)

2010 (issued)

degree

Chemistry (discipline)

embargo

2011-04-07 (terms)

identifier

http://hdl.handle.net/10339/30413 (uri)

language

en (iso)

publisher

Wake Forest University

type

Dissertation