Study On The Determination Of Natural Exogenous Cytokinins In Plants By Resonance Rayleigh Scattering And Fluorescence Spectra Technology And Their Analytical Application

Natural exogenous cytokinins play a key role in agriculture, fruits, tissue cultures in gardening plants and the development of potential anticancer drugs. Therefore, its analysis is very important. At present, many quantitative-analysis methods used to determine6-BA and Kinetin have already been reported, such as spectrophotometric, Voltammetric, capillary-electrophoresis and high performance liquid chromatography (HPLC), etc. These methods have their own advantages and disadvantages, but most of which need to degrade by heating, have complex operations and low sensitivity. At the mean time, development of RRS method with a lower price apparatus and simply operation to gain fuller and more generous information on natural exogenous cytokinins in plants react with some chemicals is considerable and significant.This project was supported by the National Natural Science Foundation of China (No.21175015) and Imbursement Help of Education Committee of Chongqing, China (No.KJ101308). And the main work of this thesis is shown as follows:1. The absorption and resonance rayleigh scattering spectra of6-Benzylaminopurine—Cu(Ⅱ)—Triphenylmthane Dyes systems and their analytical applicationIn HAc-NaAc buffer medium with pH5.37-5.58,6-Benzylaminopurine (6-BA) can react with Cu(II) and Triphenylmthane dyes to form2:2:1(Lissamine green system),3:3:1(Fast green system) and2.5:2.5:1(water-soluble Aniline blue system) ternary ion-associations, respectively, which resulted in the change of the absorption spectra, the great enhancement of RRS and Frequency Doubling Scattering (FDS) intensities. The maximum RRS wavelengths are all located at372nm. And the increasement amount of absorption, the enhancement degree of RRS (ΔIRRS), FDS (ΔIFDS) were all proportional to the concentration of6-BA during a certain range. The detection limits of methods for Lissamine green system were5.48ng-mL"1,119.70ng-mL-1and64.20ng-mL-1, respectively. Therefore, the sensitive method for determination of trace6-BA with RRS spectrum in6-BA—Cu(Ⅱ)—LG system has been developed, RRS method could be applied to the determination of6-BA in bean sprouts, and with satisfaction results. In this paper, we used Gaussview3.07and Gaussian03W softwares which were at a B3LYP/6-31G (base group) method based on the density functional theory (DFT) to calculate the charge distribution of6-BA. Furthermore, the reaction mechanism and the main reasons for the enhancement of RRS of the Lissamine green system were preliminarily discussed.2. Effects of the tnteraction between Na2W04-6-Benzylaminopurine Anionic Chelate with Rhodamine6G on the resonance rayleigh scattering and fluorescence spectra and their analytical applicationsIn pH=4.99-6.06Britton-Robinson buffer medium,6-Benzylaminopurine (6-BA) can react with Na2WO4to form1:1anionic chelate [6-BA-WO4]2-, which can further react with Rhodamine6G to form ternary ion-associations at room temperature. It resulted in the remarkable enhancement of Resonance Rayleigh Scattering (RRS), and the new RRS spectra were emerged. In the meantime the fluorescence of solution was quenching. The maximum excitation (λex) and emission (λem) wavelengths of the fluorescence of the system were at290nm and559nm, respectively. The maximum RRS wavelength was near by316nm. The intensities of RRS enhancing (ΔIRRS) and Fluorescence quenching (ΔIF) were directly proportional to the concentration of6-BA in a certain range. So, the RRS method and fluorescence quenching method for the determination of trace amounts of6-BA has been developed, under optimum conditions, the linear ranges and the detection limits of two methods were0.05-15.00μg-mL-1and8.2ng-mL-1(RRS),0.50-15.00μg-mL"1and17.0ng-mL-1(Fluorescence quenching), respectively. It was found that the RRS method was obviously superior to fluorescence quenching method. The optimum conditions and the influencing factors of two methods were investigated in the study. The influences of coexist substances were tested by RRS, which showed the method has a good selectivity. This method has been applied to determinate6-BA in cucumber and green pepper samples with satisfactory results. In the paper, the charge distribution of6-BA was calculated using the B3LYP/6-31G (base group) method based on the density functional theory (DFT), and the reaction mechanism of the ternary ion-associations system was preliminarily discussed. Basing on the signal of polarized synchronous light scattering (PSLS), Polarization (P) in reaction and the form of resonance light scattering were analyzed, and the main reasons for the enhancement of RRS were discussed.3. Studies on the Interaction of Kinetin-induced Fluorescence Spectroscopic changes of Aromatic Amino AcidsA highly sensitive method was developed for determination of trace Kinetin, which reacted with aromatic amino acids, including tyrosine, tryptophan and phenylalanine by fluorescence quenching method in room temperature (12℃-30℃) and Britton-Robinson buffer medium of pH2.02-3.79. The calibration curves of fluorescence quenching method on three systems were linear over Kinetin concentration range0.025μg/mL-2.8μg/mL, correlation coefficient (r) was0.9959(KT—D-Trp system);0.050μg/mL-2.5μg/mL, r was0.9967(KT—D-Tyr system);0.25μg/mL-10.0μg/mL, r was0.9955(KT—L-Phe system), respectively. In KT—D-Trp system, this method has been applied to determine trace Kinetin in real samples, such as persimmon skin and plum-skin, and with satisfaction results:the recoveries were within the range of92%-110%(persimmon skin) and90%-102%(plum-skin), The relative standard deviations (RSD) was in the range of0.58%-4.64%(persimmon skin) and0.39%-2.77%(plum-skin).4. Effects of the Interaction between Kinetin with BSA and HSA on the Fluorescence Spectra and Their Analytical ApplicationsIn the room temperature, the interaction of Kinetin in Britton-Robinson buffer medium at approximately pH7.00with bovine serum albumin (BSA) and human serum albumin (HSA) have been studied by fluorescence spectroscopic methods. Fluorescence data revealed the attendance of Kinetin could quench the intrinsic fluorescence of proteins and the presence of only one binding site on BSA and HSA within the concentration ranges of3.79×10-7-7.58×10-7mol/L and3.76×10-7-7.52×10-7mol/L respectively. The quenched fluorescence intensities (ΔF) in a certain range were linearly related to the concentration of Kinetin, the parameters of the calibration curves for KT-BSA system and HSA-KT system were:the detection limit (3a) were1.63×10-7mol/L and1.79×10-7mol/L; the linear ranges were9.28×10-6mol/L-8.12×10-5mol/L and9.28×10-6mol/L-1.04×10-4mol/L; Correlation coefficient(r) were0.9941and0.9991, respectively. The molar ratio method was used to analyze influence on conformational transition of two proteins with the interaction with KT by UV absorption spectra. Some influencing factors and effect of coexisting substances were investigated which suggested a good selectivity of this established method. The method has been applied to the determination of trace Kinetin in real samples, including tomato skin, balsam pear skin and grape skins. Furthermore, the influence of temperature on systems’fluorescence spectra was observed, with experimental data and Stern-Volmer graphs revealed that the interaction between KT and proteins were mainly static quenching reaction.