Item Details

abstract

The importance of biomolecules to many fields has driven the development of more efficient and selective analytical methods for the examination of such compounds. Part of this development includes organic dye molecules to act as noncovalent fluorescent labels to aid biomolecule detection. Noncovalent binding of dyes through hydrophobic, electrostatic, and other interactions with biological molecules can result in improved sensitivity by assays using capillary electrophoresis with laser induced fluorescence detection (CE-LIF). Results from such studies provide much needed information regarding the formation of noncovalent complexes as well as a means of quantifying biomolecules. Presented herein are results from spectroscopic and CE-LIF experiments focused on the characterization and application of five new asymmetric squarylium dyes containing variations in placement and number of carboxylic acid residues, as potential fluorescent probes for proteins and viruses. Bis-SQ-4d, which contains a double squaric acid center, was found to be a successful noncovalent label for human serium albumin (HSA), myoglobin, transferrin, trypsinogen, and bovine serium albumin (BSA) and was also used in the separation of a complex mixture of these proteins. Also examined were dyes SQHN-3c, SQHN-102, SQHN-103, and SQHN-105, which contain a single squaric acid center with various carboxylic acid functional groups. Despite structural similarities, these dyes possess very distinct pKa values along with spectral and binding characteristics, underscoring the importance of even subtle changes in dye structure towards the utility of a fluorescent probe in CE-LIF assays. In addition to the application of these dyes as noncovalent labels for proteins, SQHN-102 (λmax=634 nm) also showed affinity towards two plant viruses (turnip yellow mosaic virus, TYMV and cowpea mosaic virus, CPMV). Optimization of experimental conditions necessary for virus or protein binding sometimes comes at the expense of separation efficiency. However, such a compromise was accommodated by using CE-LIF for these studies, since this technique served both as a tool to study the binding (by way of frontal analysis, CE-FA, and nonequilibrium capillary electrophoresis of equilibrium mixtures, NECEEM) and to study the separation of labeled analytes, with the aim to guide future design of more selective and sensitive dyes for bioanalysis.

subject

binding constants

capillary electrophoresis

fluorescent labels

noncovalent labels

protein labeling

squarylium dyes

contributor

Rockett, Stephanie (author)

Colyer, Christa L (committee chair)

Hinze, Willie L (committee member)

Comstock, Lindsay R (committee member)

Dubey, Purnima (committee member)

Jones, Paul B (committee member)

date

2013-01-09T09:35:11Z (accessioned)

2015-01-09T09:30:08Z (available)

2012 (issued)

degree

Chemistry (discipline)

embargo

2015-01-09 (terms)

identifier

http://hdl.handle.net/10339/37651 (uri)

language

en (iso)

publisher

Wake Forest University

title

Characteristics and Performance of Novel Squarylium Dyes as Fluorescent Probes for the Analysis of Proteins and Viruses by CE-LIF

type

Dissertation