Studies of physiological cations, triglycerides and lipoprotein-associated phospholipase A2 biosensors

• Main Authors: Wei-yin Liao, 廖偉茵
• Other Authors: Chung-Chiun Liu
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/66815163272406053449

博士 === 國立成功大學 === 化學工程學系碩博士班 === 96 === This dissertation is focused on two important types of biosensors, (1) potentiometric ion sensors and (2) amperometric biosensors for determining triglycerides (TG) and lipoprotein-associated phospholipase A2 (Lp-PLA2). Both of these kinds of sensors were fabricated by microfabrication technology to achieve the final goal of being useable tools for real-time monitoring of body chemistry. For example, the glucose meter was created for people to detect glucose levels in blood at anytime and anywhere. This kind of instrument ensures that people can spend less time to be concerned about their health than they used to be. There is no doubt that the sensors used in the glucose meter can provide a convenient way to assist people in measuring blood glucose. Hence, the development of the biosensors, which can detect the biomarkers of disease will help people predict the possibility of disease happing. For this reason, developing a chip having multiple sensors for the evaluation of significant physiological cations and biomarkers of diseases (Lp-PLA2 and TG) is the motivation of this research. The potentiometric ion sensors in this study were created in two different forms i.e. as sensor array chips and microfluidic devices. An ion sensor array chip is a chip which can measure the pH, K+, Na+, NH4+ and Ca2+ ion concentrations of biological samples. In creating this array, it is necessary to develop a chip type reference electrode to improve the practicability and preciseness. Here, the fabrication of a planar-form, chip type reference electrode using a screen-printing method was developed to replace a commercial reference electrode. The reference electrode chip uses agar gel as the inner electrolyte and chloroprene rubber for the liquid junction and insulator. It was shown that the reference electrode chip is insensitive to most of the physiologically important ionic species. Integration of the reference electrode chip with four different ion selective electrodes (potassium, sodium, ammonium and calcium) and a pH indicator, on a substrate, to form an ion sensor array chip was performed. There was no significant difference between the sensitivity obtained from ion selective electrodes employing our reference electrode chip and from ion selective electrodes formed with a commercial reference electrode. These results indicate that our fabricated reference electrode was at least equal to commercial reference electrodes, in terms of sensitivity, while being compatible with potentiometric ion sensors fabricated on the same substrate as in an ion sensor array. A microfluidic device with an all-solid-state potentiometric biosensor array was developed by using microfabrication technology. The sensor array included a pH indicator, potassium and calcium ion-selective microelectrodes. The detection system was integrated with a micro-pneumatic pump which can continuously drive fluids into the microchannel through sensors. The sensor array microfluidic device showed near Nerstian responses. Heart disease has become a major heath concern for entire population. Recently, not only TG but Lp-PLA2 was also an important marker of cardiovascular disease. In this study, detection and quantification of Lp-PLA2 and TG using an iridium modified carbon based biosensor were successfully carried out. The detection procedure was based on measuring the enzymatically produced hydrogen peroxide and NADH from the reactions of methyl ester hydrolysis by Lp-PLA2 and glycerol oxidation by glycerol dehydrogenase. From the results, methyl butyrate can be as the substrate for Lp-PLA2 assay. The detection of Lp-PLA2, in the concentration range 0 to 150 Uml-1, was established as following a linear relationship with a sensitivity of 1.45 nAU-1 in bovine serum. A linear response to glyceryl tributyrate in the concentration range of 0 to 10 mM and a sensitivity of 7.5 nA(mM)-1 in bovine serum. The potential interference of species such as uric acid (UA) and ascorbic acid (AA) was assessed. The incorporation of a selected surfactant and an increase in the incubation temperature appeared to enhance the performance of this biosensor. The conditions for the determination of Lp-PLA2 and TG levels in bovine serum using this biosensor were optimized.