The Application Of Fluorescence Analysis To Bioscience

As one of the modern instrumental analytical methods,the fluorescence analysi s is developing fast in recent years and playing a important role in the study o n bioscience due to its high sensitivity and selectivity. The thesis is focus on the application of fluorescence analysis in enzyme immunoassay, pharmaceutical analysis and in the study on the interaction between medicine and deoxyribonucl eic acid and protein. The thesis includes four parts:Chapter 1 The applications of fluorescence analysis to the Immunoassay, pharmace utical analysis and to the study of the interaction between medicines and biomac romolecule The applications of fluorescence analysis in the three major fields, fluoresce nce immunoassay, pharmaceutical analysis and the interaction between the medicin e and biomacromolecule were reviewed in detail. Chapter 2 Study on the determination of HRP by Catalytic Spectrometry and the As say of (-fetoprotein Protein by micelle enhanced Fluorescence Enzyme-Linked Immu noassay 1. Study on the Determination of HRP by Micellar Enhanced Fluorimetry and the Determination of AFP by Micelle Enhanced Fluorescence Enzyme Linked Immunoassay. A micellar enhanced catalytic fluorimetry for determination of HRP was estab lished, in which the o- phenlenediamine is as hydrogen donor and Triton x-100 as a sensitized agent. The effect of various surfactants was studied and the sensi tized mechanism of surfactants on the fluorescence strength of products was disc ussed also. In the selected conditions, the fluorescence strength of the product s was proportional to the concentration of HRP between 10~200 pg/mL with a linea r regression coefficent 0.9956. Based on the study, a micellar enhanced fluoresc ence enzyme linked immunoassay for the determination of AFP was proposed and the samples were determined with a satisfactory result compared with conventional e nzyme linked immunoassay. 2. Study on the New Determining method for HRP A new catalytic spectrometry was developed, in which the reduced rhodamine B a s a donor of hydrogen. The mechanism was discussed and the enzymatic reaction co nditions were investigated. In the reaction system of HRP containing H2O2 and th e reduced rhodamine B reduced rhodamine B can be oxidized by H2O2 catalyzed by H RP, induced by o-phenylenediamine. The result shows that, the red oxidized produ cts has a absorption maximum peak at 566 nm same as that of rhodamine B. In the certain conditions, the absorbance of the products was proportional to the conce ntration of HRP in the range of 12.0~250pg/mL with the RSD of 3.2(, and the line ar regression coefficient of 0.9989.Chapter 3 The Spectroscopic Study of Comptothecin 1. The study of comptothecin by fluorescence and UV spectroscopy The spectroscopic properties of CPT in solutions were studied systematically.2.The distribution of different forms of CPT . The distribution of lacton camptothecin and ring-opened form (Carboxylate form ) was studied by first derivative ratio spectrum of UV-vis absorptive and fluore scence spectrometry. In different pH aqueous buffer solutions, it was shown that the CPT exists mainly in the opened-ring form (Carboxylate form) in physiologic al pH(pH7.4).3. The Interaction on CPT and BSA The interaction between camptothecin(CPT) and bovine serum albumin(BSA) in the physilogical pH(pH7.4) was studied by UV-visible absorption spectrometry and fl uorospectrometry. When CPT interacts with BSA, the forming constant of CPT-BSA c omplex was determined as 1.28(105L(mol-1 and the number of binding sites was 0.8 3. The experiment showed that the quenching process is a static interaction.4. Study on the inclusion of CPT and (-cyclodextrin The inclusive complex of camptothecin and (-cyclodextrin((-CD) was studied by fluorescence spectroscopy, and the forming mechanism of the inclusion complex w as discussed. The results show that camptothecin can form a inclusive complex wi th (-CD with a molar ratio of 1:1. Th...