Application of time-resolved laser-excited fluorimetry to bacteriology and virology
Abstract
Aminopeptidase profiling is an established bioanalytical technique used for the identification of bacterial pathogens. One of the undesirable features of this technique is the large number of solutions and corresponding measurements required to create these profiles. These vary from 30-40 and can take between 2-3 hours of a technician's time. The overall objective of this work has been a decrease in number of measurements by employing a multicomponent analysis. This has been accomplished through a mixture analysis of two sets of substrates one tagged with $\beta$-naphthylamine and the other with 6-aminoquinoline, incubated with the bacteria of interest to generate the corresponding aminopeptidase profile. The two established techniques of time and spectral resolution are combined to separate effectively the components of a fluorescent mixture. A simple matrix least squares method is found to be suitable for analysis of such data. The data is composed of the concatenation of two vectors, one consisting of the spectral information (wavelength) and the other time information (fluorescence decays). In this study it has been demonstrated that time and spectrally resolved fluorimetry can be employed to facilitate aminopeptidase profiling, without affecting the sensitivity of the technique. This study will be covered in Part I of this dissertation (Chapters 1-4). Substances of analytical, biological, and toxicological interest are often present only in ultratrace amounts in biological fluids. Chemical measurements, combining immunological processes and fluorescence detection, can provide powerful tools to detect very small quantities of analyte in complex biological systems. Fluorescence spectrometry is an analytical technique well known for its ultratrace limits of detection. Enzyme immunoassays can further increase the sensitivity by the chemical amplification process whereby one measures the accumulated products after the enzyme has been allowed to react with excess substrate for a fixed time period. In this work, the very low limits of detection of fluorescence immunoassays techniques and the enzyme amplification advantage have been brought together, through the employment of a laser-excited nanosecond time-resolved fluorimeter as a detection system. Also, in order to fully exploit the temporal and spectral resolution advantage for a high sensitivity ELISA technique, new ELISA reagent systems have been designed to tailor fit the ultratrace level measurement requirements. These reagents consist of $\beta$-naphthyl-phosphate and 8-hydroxyquinoline-phosphate along with low background buffer systems. This study will be covered in Part II of this dissertation (Chapters 5-6).
Degree
Ph.D.
Advisors
Lytle, Purdue University.
Subject Area
Analytical chemistry
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