UC Riverside

There are multiple challenges and limitations in the current separation methods for large biological complexes, including insufficient resolution, time-consuming pre-treatment processes and downstream analysis, potential analyte-column interactions leading to denaturation or adsorption, and the need for improving efficiency in separation and characterization when dealing with large complexes with small molecules simultaneously. In this dissertation, I aim to develop improved solutions and strategies to overcome these challenges, focusing on two powerful analytical separation techniques: Asymmetrical Flow Field-Flow Fractionation (AF4) and Capillary Electrophoresis (CE). AF4, a separation technique without a stationary phase, has proven valuable in separating large analytes with sizes ranging from nanometers to micrometers over the past three decades, offering an effective alternative to Size Exclusion Chromatography (SEC). Meanwhile, CE has matured into a robust analytical separation technique, featuring advantages like minimal sample consumption, rapid analysis, high resolution, and efficiency.In Chapter 2, the offline coupling of AF4 and CE is detailed for extracellular vesicle (EV) analysis. EVs, as important mediators of cell-to-cell communication capturing great research interests to study their functions and explore their utility in biomedical practices, pose challenges in purification due to their heterogeneity and low abundance in biological samples. AF4 provides rapid size-based separation, while CE resolves EVs from contaminants with similar sizes eluted from AF4. The coupled AF4-CE system demonstrates efficiency in monitoring EV secretion from cells in the cell culture media, and detecting serum EVs, offering rapid quantification in biological samples. Chapter 3 introduces the coupling system of AF4 with Large Volume Sample Stacking/CE (LVSS/CE) to obtain high-purity EVs for downstream protein profiling. This two-dimensional separation method first separates EVs from smaller serum proteins by AF4 and then resolves EVs from non-vesicular matrix components by CE following LVSS. The approach enhances EV isolation and collection from LVSS/CE, enabling EV immuno-fluorescence labeling and permitting the identification of EV proteins through proteomic analysis. Chapter 4 presents an online separation and characterization platform for mRNA and mRNA-loaded lipid nanoparticles (LNPs) using AF4 with multi-detectors (MD-AF4). LNPs, promising in drug delivery, present challenges in characterization. The MD-AF4 method achieves exceptional resolution between mRNA-LNPs and mRNAs, offering comprehensive and multi-attribute particle characterizations within a single injection, including size distribution, batch-to-batch variability, shape factor and encapsulation efficiency. The platform also proves potential for monitoring LNP stability under various stress conditions. This dissertation highlights the significance of AF4 and CE in advancing the analysis of large biological complexes. The novel methodologies introduced herein hold promise for consolidating our understanding of complex biological matrices, and advancing biomedical applications.