Application Study Of Ultramicroelectrode And Capillary Electrophoresis With Electrochemical Detection In Bioanalysis

The appearance of the ultramicroelectrode in the 80s opened a new study approach under conditions where it was impossible to use electrochemistry ever before. In general an ultramicroelectrode means an electrode whose surface aera is smaller than the scale of the stationary layer of diffusion which is quickly developed after a disturbance of the electrochemical system. Micrometric size of these objects allows to very closely detect infinitesimal quantities of electroactives molecules with a kinetic solution often lower than the millisecond like catecholamine, glucose, uric acid, insulin, and nitric oxide.Capillary electrophoresis (CE), an important separation technique in modern science, appeared in 1980s and well developed since then. Due to its high separation efficiency and demand of ultra-small sample volume, capillary electrophoresis has important application in analytical chemistry, biological chemistry, environmental chemistry and so on. In combination with electrochemical detection (ED), CE offers high sensitivity and good selectivity for electroactive species, which has been demonstrated in our lab in the analyses of foods, traditional Chinese medicines and biological samples.The first chapter described the fabrication of platinized carbon fiber ultramicroelectrode and its application in the detection of species produced by Angeli's Salt. Angeli's Salt appears to be very good candidates to treat failing hearts that are characterized by pressure overload, poor contractile function and delayed relaxation. Moreover, it can be successfully combined with other drugs used in heart failure patients. Under physiological conditions, it is usually accepted that the aerobic decomposition of Angeli's salt produces nitrite (NO₂-) and nitroxyl (HNO), which dimerizes and leads to N2O. No consensus has yet been established on the formation of nitric oxide (NO) and/or peroxynitrite (ONOO-) by Angeli's salt. Because this salt has been shown to have pharmacological properties for the treatment of cardiovascular diseases, identification of its follow-up reactive intermediates is of increasing importance. In this work, we investigated the decomposition mechanism of Angeli's salt by voltammetry performed at platinized carbon fiber microelectrodes. By following the decomposition process of Angeli's salt, we showed that the mechanism depends on the experimental conditions. Under aerobic neutral and slightly alkaline conditions, the formation of HNO, NO₂-, but also of nitric oxide NO was demonstrated. In strongly alkaline buffer (pH>10), we observed the formation of peroxynitrite ONOO in the presence of oxygen. These electrochemical results are supported by comparison with UV spectrophotometry data.In the second chapter, ultramicroelectrode was used to study the catalysis kinetic of nNOS in different condition in vitro and to detect its catalysis products in such conditions. NOS, nitric oxide synthase, catalyzes the conversion of arginine to citrulline and nitric oxide (NO), which has especially important function in the brain, in the vascular system and in the immune system. But under some conditions, NOS can produce other reactive nitrogen species(RNS) and reactive oxygen species(ROS). There are many research work have been done for detecting these species. But most of them use indirect methods with chemiluminescence or fluorescence derivates(using fluorescent probe 4,5-diaminofluorescein (DAF-2)). The specificity of trapping compounds for NO is questioned by some researchers. Another disadvantage with these indirect methods is that they limit the feedback regulation by NO. In this chapter, we use ultramicroelectrode for direct detecting NO and some other RNS/ ROS. Because of the tiny size, the ultramicroelectrode would not change the environment around nNOS and realize the "real" detection.The third chapter presented the detection of urea, which has no UV absorption and no electroactivity under normal conditions in saliva sample by capillary electrophoresis-electrochemical detection. Urea essentially is the waste produced when the body metabolizes protein manufactured in the liver, by broken down protein or amino acids and ammonia, the kidneys transfer urea from the blood to the urine. Urea could be used for diagnosing diseases and disorders that affect the kidney, such as acute kidney failure or end-stage renal disease (ESRD). Comparing with the methods using urease, we have found urea can be oxidized at copper electrode in alkali solution. Combine with CE, we measured the quantity of urea in human saliva samples. By using this system, the demand of sample volume diminished to 60μL, that means no stimulation needed when collect the samples, which often happened in the other methods that could affect the concentration of urea.