Development of Separation-Based Microfluidic Platforms to Study Intracellular Nitrosative and Oxidative Stress

Abstract

Nitrosative and oxidative stress are conditions caused by the overproduction of reactive nitrogen and oxygen species (RNOS), respectively. Nitric oxide (NO) and superoxide (O2•−) are the main sources of all RNOS. Macrophages are immunes cells that are known to produce large amounts of RNOS as part of the immune response. RNOS are capable of nitration, nitrosylation, oxidation, and peroxidation of crucial biomolecules, thereby inhibiting cellular function and causing cytotoxicity. Nitrosative and oxidative stress have been linked to neurodegenerative diseases, cardiovascular disease, and cancers. Therefore, a method to detect and monitor RNOS in macrophages is of the utmost importance. This dissertation describes the progress in developing separation-based microfluidic platforms to directly and indirectly measure RNOS in vitro. Most RNOS are highly reactive and unstable under physiological conditions and therefore extremely difficult to accurately quantitate. Microchip electrophoresis (ME) provides sub-minute separation times, making it possible to separate multiple RNOS in a single analysis before significant degradation occurs. First, methods were developed that utilized ME with electrochemical detection (EC) since many RNOS are electrochemically active. NO was detected indirectly through its degradation product nitrite (NO2–). NO2– and glutathione (common antioxidant) were separated and detected in macrophage cell lysates after stimulation with lipopolysaccharides (LPS) using ME-EC. Then this method was improved with the combination of transient isotachophoresis to stack NO2– and a platinum working electrode modified with platinum black to increase sensitivity. A cellulose acetate decoupler for ME-EC was also developed to reduce noise from the electric field. This approach should improve future separations through the use of higher electric field strengths and also allow coupling of ME-EC with single cell analysis devices. RNOS were also separated and detected with ME coupled to laser-induced fluorescence (LIF). LIF provides lower limits of detection compared with EC, but it requires derivatization of analytes prior to analysis. A method utilizing MitoSOX Red, a fluorescent probe specific for O2•−, was developed. O2•− production was monitored in macrophage cells stimulated with phorbol 12-myristate 13-acetate (PMA) while inhibiting superoxide dismutase, the primary cellular defense against O2•− production. Then DAF-FM DA, a NO-specific fluorescent probe, was added to this method. This allowed for the simultaneous detection of NO and O2•− in macrophages stimulated with both nitrosative and oxidative stress agents. In the future, both the ME-EC and ME-LIF developed in this dissertation will be applied to a single cell analysis (SCA) microfluidic system. SCA will provide information about the effects of cellular heterogeneity on RNOS production. Additionally, RNOS production due to disease-specific stimulants, such as amyloid-β for Alzheimer's disease, will be monitored.