| Description |
This work develops a Flow- Electrial- Split-Flow Lateral Transport Thin (Fl-El- SPLITT) enhanced separation system to separate exosomes from a minimally processed biological fluid. New simulation, electroimpedance spectroscopy (EIS) and hydrolytic products analysis developments improve understanding in thin electrode flow channels. Exosomes are tiny biological vesicles involved in cancer metastasis, paracrine level cell signaling, and other biological processes. SPLITT and related techniques are implemented as a means of processing high volumes of sample with the overall goal of better therapeutics for cancer and other diseases. The newly developed Fl-El-SPLITT is a general particle separation device and this dissertation also forms the theoretical, and practical groundwork for its future development. This work achieves the first separation of B16-F10 mouse melanoma exosomes into subtypes using asymmetrical flow field flow fractionation (FFF), and also the first separation of exosomes using electrical field flow fractionation. Some effects of exosome buffer substitution are explored for yield, mobility, and separation differences. The best achieved separation efficiencies were ~60% and ~75% for two SPLITT designs, but actual U87 exosomes and oncosomes were ~26%. The methods, design, and validation for the variations of the Fl-El-SPLITT are detailed. Additional continuous flow separation techniques are applied to particle separations. Existing theory for El-SPLITT is limited to DC fields. This work expands applicability to DC, AC, and cross flows through a new simulation and comparison with theory and iv experiment. Voltage effects on total particle recovery are shown. This work details the first use and analysis of EIS on an El-FFF/El-SPLITT system. EIS abilities are shown through analysis of AC and DC voltage, frequency, salt concentration, and device geometry. Advantages of EIS for El-FFF/Fl-El-SPLITT characterization include: speed, simplicity, curvefitting to multiple electrical models, evaluation of phase and current information, and visualization across multiple frequencies at once. This work also contains a theoretical development of equations for the current that is required for hydrolysis based upon either the volume of gas produced or the change in fluid conductivity. Calculated currents are less than measured currents and show consistency, and relaxation of byproducts. A new effective current is proposed for El-FFF/Fl-El-SPLITT based upon hydrolysis. |
