Application Of Organic Dye-Copper Complex On Fluorimetric Analysis Of Cysteine

Thiol compounds are important biologically active materials in organism. They serve as antioxidant in cell and participate in a variety of important cellular functions, such as synthesis of protein and DNA, material delivery and metabolism process. As a very important thiol-containing compound, L-cysteine plays key roles in cellular functions, for instance antibacterial and detoxification, etc. Some diseases are induced by cysteine decompensation resulting in the change of cysteine concentration in organism. Thus, some diseases can be diagnosed via cysteine determination in biological systems. Development of simple, quick and exact method for qualitative and quantitative analysis for cysteine is of importance.Cysteine acts as reducing agent and copper(II) serves as oxidant. Redox reaction between them is easily occurred which could be used for recognizing cysteine. It is well known ortho-dihydroxy aromatic hydrocarbon or similar structure compounds can bind with copper(II) and form stable metal complex. Thus, some organic fluorescent compounds containing the structure as Tiron, Alizarin Red S and Morin and their copper complex were selected as host for sensing amino acid. The interaction between organic metal complex and amino acid was investigated. The effect of aromatic hydrocarbon structure on determining sensitivity for cysteine was discussed. Several highly selective and sensitive fluorescence assays for L-cysteine were developed. This thesis consists of five chapters.In chapter 1, the development of study on cysteine and glutathione was reviewed. Especially, various determination methods of cysteine were discussed in detail that included high performance liquid chromatography, flow injection, voltammetry, chemiluminescence method, capillary zone electrophoresis, spectrophotometry and spectroflourimetry.In chapter 2, the interaction between Tiron-Cu(II) and cysteine was investigated via fluorescence and absorption spectroscopy. The complex Tiron-Cu(II) formed at 80°C and it emitted weak fluorescence in pH 8.0 Britton-Robinson buffer which excitation wavelength and emission wavelength was 290 nm and 350 nm, respectively. The addition of L-cysteine into Tiron-Cu(II) system resulted in fluorescence enhancement at 350 nm which was promotional to the L-cysteine concentration. A novel spectrofluorimetric method for the determination of cysteine was developed with good linear calibration of cysteine concentration ranging from 7.46×10^-7 to 2.20×10^-5 mol·L^-1 and the detection limit as 7.46×10^-8mol·L^-1. The proposed method was applied to determine cysteine in synthetic amino acid samples with the recovery 94.0～100.7%.In chapter 3, the interaction between Alizarin Red S (ARS)-H₃BO₃ and Cu(II)was investigated via fluorescence and absorption spectroscopy. In order to optimize determining condition and make the determination easy operate, Alizarin Red S was selected as a ligand for copper. Obviously, Alizarin Red S contains anthracene group which promise longer excitation and emission wavelength than that of Tiron. In pH 6.55 B-R buffer, ARS-H₃BO₃ emitted strong fluorescence which excitation and emission wavelength was 435 nm and 587 nm respectively. The fluorescence intensity at 587nm was quenched by the addition of Cu(II) into ARS-H₃BO₃ system and the color changed from yellow to red. It was assumed that the addition of Cu(II)led ARS-H₃BO₃ to decompound and ARS-Cu(II) complex formed. A good linear response of fluorescence intensity as a function of Cu(II) concentration was obtained ranging from 1.0×10^-6 mol·L^-1 to 2.4×10^-5 mol·L^-1 ( r = 0.996) with the detection limit as 1.01×10^-7mol·L^-1. The proposed method was applied to determine Cu(II) in discharged water and the recovery was 95.5～101.0%.In chapter 4, the interaction between Alizarin Red S (ARS)-Cu(II) and cysteine was investigated via fluorescence and absorption spectroscopy. An ideal fluorescent probe posses high quantum yield, longer excitation wavelength and longer emission wavelength which promise to provide sensitive method and avoid burn of tissue and fluorescent bleaching. Accordingly ARS-H₃BO₃ system was a good fluorescent assay for cysteine. In pH 6.51 Britton- Robinson buffer, addition of L-cysteine into ARS-Cu(II) system resulted in a fluorescence enhancement because cysteine reduced Cu(II) to Cu(I) which led ARS-Cu(II) decompound and ARS was released. A good linear response of fluorescence intensity as a function of cysteine concentration was obtained ranging from 1.08×10^-6 to 4.0×10^-5 mol·L^-1 (r = 0.9994) with the limit of detection as 1.08×10^-7 mol·L^-1. The proposed method was applied to determine cysteine in protein hydrolysate of fresh porcine blood with recovery of 93.2-100.2%.In chapter 5, the interaction between Morin-Cu(II) complex and Cysteine was investigated via fluorescence and absorption spectroscopy. Morin possesses largeÏ€conjugated system and emits strong fluorescence. Although the compound doesn't contain ortho-dihydroxy group, oxygen of pyran and hydroxyl of phenyl cooperate to bind with copper and form stable metal complex. In pH 7.4 Britton-Robinson buffer, addition of Cysteine into Morin-Cu(II) system resulted in immediate fluorescence enhancement at 539 nm which was promotional to the Cysteine amount. A novel spectrofluorimetric method for the determination of Cysteine (Cys) has been developed with good linear calibration of Cysteine concentration ranging from 6.52×10^-7 to 2.20×10^-5 mol·L^-1 and the detection limit as 6.52×10^-8mol·L^-1. The proposed method was applied to determine cysteine in protein hydrolysate of fresh porcine blood and the recovery was 91.3-94.9%.