Advancing Intracellular HNO Delivery Quantification and Integrated Strategies for Enhancing Sensitivity Through Monolith-Based Online Preconcentration Using Capillary Electrophoresis-Laser Induced Fluorescence Detection

Abstract

Capillary electrophoresis (CE) is a widely employed technique in life sciences, with applications in environmental, clinical, pharmaceutical, and food analysis. Among its various modes, capillary zone electrophoresis (CZE) stands out as the most commonly used due to its efficiency in achieving rapid separation. The flat flow profile within small-diameter capillaries further enhances separation efficiency, making it particularly effective for analyzing large biomarkers, as well as charged biomolecules such as proteins and signaling molecules. While UV detection is frequently utilized, techniques like laser-induced fluorescence (LIF) offer increased sensitivity. However, limitations arise from the ultra-small sample injection volume and the short optical path length, which can be mitigated by employing on-capillary preconcentration techniques, such as transient capillary isotachophoresis (tITP). Furthermore, CE-based flow-gated online injections prove indispensable for achieving high temporal resolution in monitoring time-sensitive reactions, enabling the study of dynamic processes and kinetics in a range of systems.The dissertation focuses on advancing the analysis of trace compounds by utilizing CZE, tITP-CZE, and flow-gated methods. It explores the quantitative analysis of intracellular nitroxyl (HNO) delivery, employing a fluorescent thiol-based trapping agent that eliminates the need for additional labeling steps. Through the use of CZE-LIF, HNO can be detected with good sensitivity, allowing for precise differentiation from other signals. It further introduces an analytical process that combines preconcentration through counterflow tITP within a monolith with CZE separation. Counterflow tITP-CZE addresses challenges such as flow dispersion and provides a stable porous medium for effective analyte preconcentration. Moreover, the addition of tITP-CZE in a flow-gated online injection system holds promise for the analysis of trace compounds and immunoassays, with flow-gated injections proving particularly valuable when combined with on-capillary preconcentration techniques. In conclusion, this study lays the foundation for three distinct capillary electrophoresis-based investigations: quantifying intracellular HNO delivery using a label-free thiol-based fluorescent trapping molecule, integrating a transient monolith within the capillary alongside counterflow isotachophoresis for the preconcentration and separation of trace analytes with distinguished structural modifications, and exploring the potential utilization of flow-gated online injections coupled with tITP-CZE for the analysis of dynamic processes.