Ultrafast Spectroscopic Study Of Fluorescence Dynamics Of Dye Molecules In Liquid Phase

Most biochemical reactions need to occur in liquid-phase environment.In life science research such as fluorescence cell imaging,the use and observation of the fluorescence emission behavior of specific dye molecules and their change patterns allow real-time probing of the p H,ion levels,and distribution of biomolecules such as target proteins in the intracellular liquid-phase microenvironment,playing an important role in cutting-edge life science research such as cell kinetics.In the liquid-phase environment,solvent effects such as intermolecular hydrogen bonding interactions and fluorescence quenching are prevalent and have important effects on the fluorescence emission and fluorescence kinetic processes of dye molecules.The study of the mechanism of these effects by means of ultrafast spectroscopy will contribute to the design of novel fluorescent probes,fluorescence imaging and other life science research fields.In this thesis,two common dye molecules,coumarin 153 and rhodamine 6G,were selected as objects of study,and the fluorescence spectra and fluorescence kinetic processes of these two dye molecules in a liquid-phase environment were investigated experimentally.Since the dye molecule coumarin 153 can form intermolecular hydrogen bonds with phenol,the fluorescence kinetics of the dye molecule coumarin 153 in liquid phase can be investigated by changing the content of phenol in the phenol-cyclohexane solvent mixture with the help of steady-state and ultrafast time-resolved fluorescence spectroscopy systems.On the other hand,since graphene oxide can lead to fluorescence quenching of dye molecule rhodamine 6G,by varying the concentration of graphene oxide and combining the steady-state spectroscopy with ultrafast time-resolved spectroscopy system,the excited state kinetics of rhodamine 6G can be probed under fluorescence quenching with different levels.The specific researches of this thesis are as follows.(1)The fluorescence emission and fluorescence kinetics of the dye molecule coumarin 153 in phenol-cyclohexane mixed solvents with molar ratios of 0,0.013,0.08,and 0.3,respectively,were investigated systematically using home-made steady-state fluorescence spectroscopy and time-resolved optical Kerr fluorescence spectroscopy system.It was found that the steady-state fluorescence spectrum of the dye molecule coumarin 153 was red-shifted with the increase of the molar ratio of the phenol-cyclohexane solvent mixture,and the degree of red-shift increased with the rise of the molar ratio.At the same time,the fluorescence emission frequency of coumarin 153 was found to be continuously red-shifted with increasing delay time by time-resolved optical Kerr fluorescence spectroscopy in the solvent mixture containing phenol,and the fluorescence peak shifting became more and more obvious with increasing the proportion of phenol in the solvent mixture.Since phenol is a polar molecule and has the ability of hydrogen donating,it is inferred that the above time-dependent fluorescence red-shift phenomenon may be related to solvent polarity or intermolecular hydrogen bonding interactions.To screen the two possible effects,we prepared a mixture of anisolecyclohexane with the same molar ratio of 0.3 and repeated the timeresolved optical Kerr fluorescence spectra measurements of the dye molecule coumarin 153.It was found that the fluorescence spectrum of the dye molecule coumarin 153 in the molar ratio of 0.3 of anisolecyclohexane mixture showed an overall red shift compared to that of cyclohexane alone,but no time-dependent red shift of the fluorescence spectrum was observed.It can be inferred that the increase of solvent polarity can only lead to the overall red shift of the fluorescence spectrum of the dye molecule coumarin 153,while the continuous red shift of the time-dependent fluorescence spectrum found in the phenol-cyclohexane solvent mixture is caused by the intermolecular excited state hydrogen bonds formed between the dye molecule coumarin 153 and phenol.As the proportion of phenol in the phenol-cyclohexane solvent mixture increases,the excited state hydrogen bonding is enhanced,the kinetic process of solvation is accelerated,and the rate of relaxation of the excited state hydrogen bonding complex to a lower free energy state increases accordingly,resulting in an accelerated red shift of the time-dependent fluorescence spectrum of the dye molecule coumarin 153.(2)The fluorescence quenching of the dye molecule rhodamine 6G induced by different concentrations of graphene oxide and its kinetic process were experimentally investigated using steady-state fluorescence spectroscopy,time-resolved fluorescence spectroscopy and ultrafast time-resolved transient absorption spectroscopy.During the experiments,we first measured the steady-state fluorescence spectra of the dye molecule rhodamine 6G.It was found that with the increase of graphene oxide concentration in the dye molecule rhodamine 6G-graphene oxide mixture solution,the fluorescence spectrum intensity of rhodamine 6G gradually diminished and the peak position of fluorescence spectrum was continuously red-shifted.On this basis,it was found that the fluorescence lifetime of the dye molecule rhodamine 6G gradually decreased with the increase of the proportion of graphene oxide in the mixed solution by timeresolved fluorescence spectroscopy measurements,and the decrease of fluorescence lifetime started to slow down after the concentration of graphene oxide reached 60 μg/m L.The excited state kinetics of the dye molecule rhodamine 6G was then experimentally measured using a femtosecond time-resolved transient absorption spectroscopy system.It was found that the intensity of the stimulated emission signal of the dye molecule rhodamine 6G gradually decreased with the increase of graphene oxide concentration in the mixed solution.At the same time,when the graphene oxide concentration reached 60 μg/m L,a new fast relaxation process appeared in the initial stage of the stimulated emission signal.This process became more and more obvious as the graphene oxide concentration continued to increase,and the relaxation process changed from the original double-exponential decay process to a triple-exponential decay process.It is shown that the electron transfer process between the excited dye molecule rhodamine 6G and graphene oxide can occur,which leads to the weakening of fluorescence intensity and the red shift of fluorescence wavelength.When the concentration of graphene oxide exceeds 60 μg/m L,the increase in the concentration of the R6G-GO complex will lead to an intensification of the photo-induced electrontransfer process,whereby the new relaxation process in the stimulated emission of R6 G is judged to be caused by the photo-induced electrontransfer process occurring in the R6G-GO complex.