Time-resolved Fluorescence Spectroscopy Technique And Its Application In Biological Molecular Characterization

Compared with steady-state fluorescence spectrum analysis, time-resolved fluorescence spectrum of fluorescent substance can reflect more bioinformation, especially dynamic information about the microenvironment changes which happens on the fluorophores or the residues. On the basis of fluorescence principles and equipped with picosecond and femtosecond time-resolved fluorescence spectroscopy detection systems, the time-resolved fluorescence of several functional fluorescent probes has been studied. In this way, the properties of the macromolecules and the microenvironment changes can be characterized. In this dissertation for a Master’s degree, the study contents are demonstrated as follows:1. Fluorescein, which can be quenched by KI and has a quenched lifetime about23picoseconds, has been proved to be a better IRF reference dye at the green wavelength band than using scattering samples in our experiment. When using scattering samples, the detection wavelengths are supposed to be changed one by one which may bring in deviations due to the color effect of the system.2. Based on long lifetime phosphorescence characteristics of iridium complexes, measurement has been made to a series of iridium complexes under the anaerobic condition. The results showed that phosphorescence lifetime of the series of iridium complexes is about1millisecond, which makes it a relatively ideal oxygen probe. Confirmed by the experiment, the iridium complexes in methylene chloride solution are sensitive to the changes of dissolved oxygen.3. Due to good optical properties and sensitivity to the pH of the environment, fluorescein can be used as a good pH fluorescent probe by using the steady-state fluorescence intensity analysis and time-domain analysis method. In our experiment, the results showed that both of the methods can monitor the pH of the solution. But when pH is greater than8, the fluorescein is out of action.4. The measurement to the extent of NADH oxidation reduction state. Yellow fluorescent protein probes are more sensitive with the method of time-resolved fluorescence spectrum measurement than that of Steady-state fluorescence measurement.