Doctor of Philosophy (PhD)
Efforts to improve the technology for DNA analyses stimulated the work presented here to develop a series of fluorescent probes that would aid in the increased multiplexing capabilities allowing the ability to detect multiple targets in a single measurement. Typically, spectral discrimination has been used to identify each DNA fragment in sequencing or diagnostic applications by means of unique emission properties from several labeling dyes. This has been accomplished with labels that fluoresce in the visible region. The limitations with these dyes is the broad emission profile making discrimination difficult, high intrinsic biological fluorescence and structural differences of the dyes leading to differences in the electrophoretic mobility of labeled oligonucleotides, complicating sorting using electrophoresis. The use of labeling dyes that fluoresce in the near-IR has several advantages including reduced background interference, increased sensitivity due to decreases in Raman scattering, and can be excited using inexpensive diode lasers allowing time-resolved measurements to be carried out. Thus, lifetime discrimination in parallel with color discrimination can potentially offer increased multiplexing capabilities for a variety of DNA analysis applications. Strategies are reported that produce symmetrical metal-free and metallo-phthalocyanine dyes, Pc and MPc, respectively, that contain various numbers of water solubilizing carboxylic acid groups on their periphery that provide a dual role by also serving as functional groups to covalently link primary amine-containing targets to these dyes. The influence of the number of solubilizing groups and metal center on the spectral and photophysical properties were evaluated. MPc dyes containing 4, 8, or 16 carboxylic acid groups exhibited similar absorption and emission maxima (677 nm and 686 nm, respectively) with the molar absorptivity of the Q-band ~105 M-1cm-1. Results indicated that the fluorescence lifetimes and quantum yields varied as a function of the metal center; the degree of carboxylation did not significantly alter these properties in DMSO, but did mediate the solubility and aggregation states when placed in aqueous solvents. Results also showed that the conjugate, produced by covalently linking an MPc to biological entities, generated a red-shift in the emission maximum with a fluorescence lifetime shorter than that of the native MPc dye.
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Verdree, Vera Tamisha, "The spectral and photophysical characterization of water solubilizing metal phthalocyanines for highly sensitive detection in biological studies" (2007). LSU Doctoral Dissertations. 1370.
Steven A. Soper