Investgation Of Thiol Compounds Based On BODIPY Fluorescent Labeling Reagents

Sulfhydryl group(-SH) is the highest chemical activity group in cells. Thiol compounds play the essential role in regulating cell oxidation-reduction equilibrium, protecting the biofilm system, resisting to the oxidative stress and oxidation damage and so on. Numerous studies have shown that the abnormol levels of the thiols in physiological liquids closely associated with several diseases. Thiol compounds with low-molecular-weight are also important industrial raw materials that might cause environmental pollution problems. More and more attentions have been paid to identification and quantitative determination of thiol compounds because the important roles of thiol compounds in biological system and environment is came to be realized.Therefore, to develop the high sensitivity, easy and fast analysis method of thiol compounds is of great significance.Because of the thiol compounds with low-molecular-weight have no intrinsic fluorescence or chromophore, therefore, chemical derivative technique is adopted to attain high detection sensitivity. In this thesis, after derivatization between the thiol compounds and fluorescent probes, high performance liquid chromatography with fluorescence detection(HPLC-FL) and capillary electrophoresis with laser-induced fluorescence detection(CE-LIF) were applied to assay the thiol compounds in biological samples and environment water samples respectively.Fluorescent probe applied to the imaging of thiol compounds in the cell by fluorescent microscope. The research carried out the three chapters as follows:(1) CE-LIF and HPLC-FL were developed for simultaneous measurement of cysteine,homocysteine and glutathione. In these methods, N-( 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-yl) Iodoacetamide(BODIPY®507/545 IA) was applied for the precolumn derivatization. As is shown in this study, cysteine, homocysteine and glutathione could be separated in 20 min with HPLC, while in 9 min with CE, and the limits of detection(S/N=3)were 5 、 10 and 5 nmol/L respectively for fluorescence detection while 0.5 nmol/L for laser-induced fluorescence detection. The excellent linearity was obtained(R2=0.9967-0.9991).We have successfully separated and determined cysteine, homocysteine and glutathione in human urine and serum samples with recovery of 93.8-108.1% and 93.5-107.2%.(2) A HPLC-FL for the simultaneous determination of mercaptoethanol, propanethiol and butanethiol is established. Another BODIPY fluorescent dye,(1, 3, 5, 7-tetramethyl-8-bromomethyl-difluoroboradiaza-s-indacene, TMMB-Br) was used as precolumn fluorescent derivatizing reagent. The separation and the derivatization conditions were studied in detail. The derivatization of mercaptoethanol, propanethiol and butanethiol with TMMB-Br could be accomplished in only 15 min at 45℃. The limits of detection(S/N=3) were 5、10 and 10 nmol/L respectively and relative standard deviation of peak area and migrate time were from 0.39-2.31%and 0.19-0.83% respectively. This method was successfully applied to determine mercaptoethanol, propanethiol and butanethiol in the water samples with a recovery of95.9-105.7%, providing good results in terms of accuracy, precision and stable.(3) Cell disruption liquid was choosed to simulate the physiological conditions. In this study, the fluorescent probe(BODIPY®507/545 IA) responds to cysteine rapidly with the mild reaction conditions in simulated physiological condition. And a good linearity was obtained(R2=0.9948)of fluorescence intensity dependent on the concentration. The bioimaging was carried out in vitro,and biothiol compounds after derivatization could emit bright green fluorescence under fluorescent microscope. The result shows this fluorescent probe can rapidly respond to the endogenous biothiol compounds and shows stable, high selectivity, low biological toxicity, good biocompatibility and membrane permeability. HPLC-FL is developed for measurement of thiol compounds in cells, and a method of in-situ cell imaging and quantitative analysis of thiol compounds in cells is established...