Probe and bioconjugation strategies for laser-based time-resolved fluorometry in microbiological systems
Abstract
Laser-based pulse time-resolved fluorometry has been undergoing genesis the last twenty five years. Instrument technology has matured to the point that it is no longer the limiting factor to performing an analysis. What has, is the chemical systems that are being investigated. New fluorescent probe and bioconjugation strategies have been used to redesign these systems to take advantage of temporal resolution. For example, in the past, commercially available (9-anthroyloxy)steric acid probes could only be utilized to measure the bulk or average properties of lipid membranes. By employing time-resolved fluorometry, these probes can now be used as "molecular dipsticks" to directly investigate the viscosity of a lipid bilayer as a function of depth. On the other hand, for molecules that could not be purchased, they were synthesized to solve problems involved in finding long-lived fluorophores with functional groups that could be easily bioconjugated. Typical systems involved pyrene or naphthalene based emitters with primary amine or hydroxyl functionalities. These probes were targeted for such uses as DNA sequencing, DNA sequence detection, and monitoring genetically engineered organisms. Another strategy focussed on the use of a photo-induced reaction to generate a fluorophore with unique spectral properties. Based on excimer dynamics, the resultant excited state dimer will emit at a signature wavelength and have an unusual temporal profile. Consequently, these two characteristics can be used to our advantage. In an application described here, excimers were tried for reducing false positives in a DNA-based hybridization assay. On another note, an innovative algorithm was evaluated to extract continuous lifetime distributions from pulse time-resolved fluorescence decays. With the increasing complexity of microbiological systems, information once derived from discrete lifetime analysis became inadequate for describing lifetime kinetics in heterogenous environments. To help alleviate this problem, the Maximum Entropy Method (MEM) was explored by using both computer generated and real data sets.
Degree
Ph.D.
Advisors
Lytle, Purdue University.
Subject Area
Analytical chemistry
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.