Study Of Mechanism And Application On Energy Transfer Between Fluorescein And Neutral Red

Fluorescence resonance energy transfer is an analytical method that is based on energy transfer between fluorescent substances.In comparison with a single dye, the method has higher sensitivity than the luminosity method. Compared with fluorescence method and resonance light scattering method, the method is characterizesd by few inferring substances, good reproduction. It is applied to the chemical analysis, the biological structure researches. This paper has carried on the study about the mechanism and related application of this method. This paper consists of five chapters:Chapter one:It reviewed the application and mechanism of non-radiation energy transfer of acidity dyestuff fluorescein and basic dyestuff. We also summarized its application of the method in analytical chemistry, with 62 literatures cited.Chapter two:The reactions between basic dyestuff neutral red(NR) and various acidity dyestuff including tetrabromofluorescein(TBF),tetraiodofluorescein(TIF),tetrachlorotetra-iodofluorescien(TCTIF), and tetrachlorotetrabromofluorescien(TCTBF) have been investiga-ted by the means of fluorescence spectra. The experimental results showed that NR strongly quenched the fluorescence produced by various studied acidity dyestuff, and this fluorescence quenching could be interpreted in terms of static quenching. From the calculation results of binding constants K for these dyes at various temperatures,it could be found that the increase in the electronegativity of substituting group of dyes molecules resulted in an increase in K,while the increase in reaction temperature led to a decrease in K. The interactions between Neutral red and various acidity dyestuffs were attributed to static-electricity gravitation which was confirmed by the calculation results of enthalpy and entropy for these reactions. According to the non-radiation energy transfer theory,the acceptor-donor distances(r) and energy transfer efficiencies between NR and various acidity dyestuff at various temperatures were obtained. These results further supported the conclusion that if r was within 7 nm, these reactions belonged to the single static quenching which was caused by the non-radiation energy transfer. It could be found some regularity among the parameters of energy transfer along with the increase in the electronegativity of substituting group of dyes. Chapter three:Tetrabromofluorescein-neutral red fluorescence energy transfer inhibition method for the determination of heparin. In the Britton-Robinson(B-R) buffer solutions of pH 6.65,the effective energy transfer could occur between TBF and NR. The fluorescence intensity of TBF was reduced and that of NR was strengthened at the same time. But if the heparin was added to this system,remarkable inhibition of the fluorescence energy transfer of TBF-NR pair was detected and the fluorescence intensity of TBF was strengthened. A new method for the determination of trace heparin was developed by using energy transfer from TBF to NR. The detection limit of this method was 0.071 mg/L. The linear range of determination for heparin was 0.1～2.0 mg/L. Among six times determination, the relative standard deviation was 1.39%～3.59% and the recoveries were 95.2%～105.3%. The method has been applied to the determination of heparin sodium in injection with satisfactory results.Chapter four:Determination of protein by method of tetrabromofluorescein-neutral red fluorescence reverse energy transfer. In the Britton-Robinson buffer solutions of pH 2.35, effective energy transfer could occur between tetrabromofluorescein (TBF) and neutral red(NR) due to static attraction. The fluorescence intensity of TBF was reduced and that of NR was increased at the same time. When human serum albumin(HSA) was added to this system, the fluorescence energy transfer between TBF-NR pair is disturbed, causing reverse reaction occur with resulted in the increase of fluorescence intensity of TBF and formation of HSA-TBF coordinate compound. A new method for the determination of trace HSA was established by the linear relationship between the fluorescence intensity of the coordinate compound and concentration of HSA. The detection limit of this method was 0.25 mg/L. The linear range of determination for HSA was 0.6～12.0 mg/L. Among six parallel experiments, the relative standard deviation was 1.94%～4.58% and the recoveries were 96.3%～104.9 %. The method was characterized by good stability and high selectivity. The results of the proteins in human serum samples were very close to those obtained using biuret spectrophotometric method.Chapter five:The study on energy transfer mechanism between fluorescein sodium and tetrabromofluorescein sodium in micelles of cationic surface active agent.In this paper, the fluorescence reaction of cationic surfaceactive agents (CSAA):cetyl trimethyl ammonium bromide(CTMAB) or hexadecyl pyridinium bromide(CPB) with tetrabromofluorescein sodium (TBF) in aqueous solution was investigated. It was found that fluorescence quenching of TBF appeared when it reacted with the cation monomer of a CSAA and a new stronger fluorescence was obtained when the ion-associates react with the micellate of CSAA. We investigated the condition of energy transfer between acidic fluorescent dyes in micelles of cetyl trimethyl ammonium bromide or Hexadecyl pyridinium bromide. It was indicated that in the micelles which were formed by cationic surface active agent with dyes embedded (cationic surface active agent and dyes were of opposite charge), when the concentration of cationic surface active agent reached to a certain value, the energy transfer could occur. In the value of two thirds of critical micelle concentration (CMC), the efficiency of energy transfer reached the maximum; when the concentration of cationic surface active agent went on increasing, the efficiency of energy transfer would be decreased. We also deduced the model of energy transfer between dyes in micelles and laws of it.