Analysis of Protein Aggregates and Synthetic Nanoparticles Using Capillary Electrophoresis with Light Scattering and Fluorescence Detection

Abstract

The goal of the research presented in this dissertation is to develop capillary electrophoresis (CE) based analysis techniques for the characterization of individual particles including amyloid beta (Aâ) peptide aggregates and to apply these methods to study the aggregation of Aâ peptides. Protein self-assembly into ordered aggregates is a common and important process in normal biological function, but this process is also involved in several diseases. Amyloid beta peptide aggregation is linked to Alzheimer’s disease. Thioflavin T (ThT) has been used as a fluorescent probe for the detection of Aâ aggregation. Based on previous observations of false positive fluorescent signals due to the presence of non-amyloid particles in Aâ aggregation studies using CE with ThT fluorescence, a spectroscopic study of ThT in the presence of synthetic polystyrene (PS) beads was conducted (Chapter 2). The study demonstrated ThT fluorescence enhancement in the presence of PS beads, similar to that observed for Aâ fibrils. Further spectroscopic investigation of ThT showed an absence of any ThT micelle formation, in contrast to other published work. It is important to analyze individual particles in heterogeneous samples of protein aggregates such as solutions of aggregating Aâ peptide in order to understand aggregation mechanisms, and CE is a good technique for such analysis. Hence, a CE system with three channel laser light scattering (LLS) and laser-induced fluorescence (LIF) detection (CE-LLS2-LIF) was developed and optimized using fluorescent and nonfluorescent polystyrene (PS) beads (Chapter 3). The instrument was designed for simultaneous collection of LLS at 20° and 90° and LIF at 90° with respect to the source. Detection of individual PS beads as small as 80 nm diameter was demonstrated using LLS at 90°. The CE-LLS2-LIF system was then used for the characterization of individual Aâ aggregates and for the study of Aâ peptides aggregation (Chapter 3). The CE-LLS2-LIF system was also used to analyze individual Aâ aggregates, PS beads, quantum dot nanocrystals, and cyanine based organic functional particles. Different particle types were separated, detected individually and characterized based on their electrophoretic mobilities, fluorescence and light scattering at 20° and 90° (Chapter 4).