Development of microfluidic methods for the determination of products of nitrosative and oxidative stress

Abstract

When the pro-inflammatory and anti-inflammatory immune responses becomeimbalanced, an excess of pro-inflammatory reactive nitrogen and oxygen species (RNOS) may be produced, which in turn can react with biomolecules such as proteins, lipids, and DNA. These reactions result in structural modifications that can ultimately lead to functional alterations, cell damage, or even cell death. As RNOS are highly reactive and short-lived at physiological pH (t1/2 < 1 s), rapid microfluidic methods allow for near real-time measurement of these species in biological samples. Additionally, electrophoretic separation techniques such as capillary electrophoresis (CE) and microchip electrophoresis (ME) are useful for the efficient separation of small, ionic species such as RNOS. In this dissertation, several novel microfluidic and electrophoretic approaches for studying cellular nitrosative and oxidative stress via monitoring of RNOS (e.g., nitric oxide, peroxynitrite), their metabolites (e.g., nitrite), and their reactive products (e.g., 3-nitrotyrosine) are described. First, a method using ME with electrochemical detection (EC) was developed to detect nitrite in cell lysates isolated from stimulated macrophages as an indicator of nitric oxide production. This method features transient isotachophoresis stacking and a platinum black working electrode for enhanced signal and sensitivity for nitrite. Additionally, progress towards a second ME-EC method with dual electrochemical detection is reported that is selective for nitrotyrosine and nitrotyrosine-containing peptides. A complementary CE-UV method for the separation of several nitrated and non-nitrated peptides was also developed and has been applied to the identification of reaction products of tyrosine under acidic and oxidizing conditions. Finally, stereolithographic 3D printing was used to develop a low-volume module for boronate affinity enrichment that will ultimately be integrated with other modules and used in a plug-and-play system for capture and detection of derivatized, nitrated peptides. Together, these methods provide a suite of approaches for investigating nitrosative and oxidative stress in cellular systems.