Date of Award

1997

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Steven A. Soper

Abstract

The research presented in this dissertation involves the construction of a near-infrared time-correlated single photon counting system which utilizes a mode-locked Ti:sapphire or pulsed diode laser as the excitation source and single photon avalanche diode as the photodetector and the subsequent study of two simple algorithms, the Maximum Likelihood Estimator (MLE) and the Rapid Lifetime Determination (RLD) methods, for fluorescence lifetime determination. The purpose of this research is to aid in the development of a low cost, highly efficient, and high throughput device for DNA sequencing which utilizes near-IR fluorophores which have similar absorption and emission properties but possess unique fluorescence lifetimes, which will allow the identity of the terminal base to be accomplished by lifetime discrimination. The performance of the MLE and RLD for measuring nanosecond and subnanosecond fluorescence lifetimes were evaluated in static solutions in the limits of low concentrations and high backgrounds from scattered photons generated from the solvent using experimental and Monte Carlo simulation results. The conclusions from these results were used in the on-line determinations of fluorescence lifetimes of components separated via capillary electrophoresis. For example, a capillary gel electrophoretic separation of near-IR dye-labeled C-terminated fragments was done with decay profiles of the separated fragments collected. The MLE lifetime value for decay profiles collected from 35 individual peaks was 581 $\pm$ 9 ps, which agreed with the high concentration value $(\tau\sb{f}=581$ ps). Also, a pulsed-diode laser based instrument with the counting electronics situated on a PC board was constructed and its performance evaluated by a CGE separation of A- and C-terminated DNA fragments. The results indicated lifetime values of 669 $\pm$ 42 ps and 528 + 68 ps for the A-terminated and C-terminated fragments, respectively, for the MLE. In order to study the effects of peak resolution on the fluorescence lifetime determination, Monte Carlo simulations were used to study the effect of electrophoretic resolution between peaks on the calculated lifetime using both the MLE and RLD algorithms. In addition, accuracy and precision were analyzed for the calculated lifetimes during the electrophoresis as a function of integration time.

ISBN

9780591766677

Pages

213

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