Abstract
Exosomes are nano-sized (30-150 nm) extracellular vesicles (EVs) secreted by various cell types. They are easily accessible in biological fluids and contain specific disease biomarkers such as various proteins, lipids and genetic materials (e.g., miRNA, mRNA, DNA molecules as well as long-noncoding RNAs), which making them attractive for diagnosis and prognosis applications. In addition to disease diagnosis, utilization of exosomes as drug delivery carriers, vaccine agents and regenerative medicine have gained tremendous attention in recent years. Nevertheless, their clinical values have been hindered due to the lack of standardization of the established methods which created a pessimistic general atmosphere around exosome-based diagnosis and therapeutics. Improving the isolation purity, efficiency and detection accuracy in current exosome characterization and isolation techniques can benefit advancing the clinical translation of exosomes. To do this, substantial capital investment and efforts have been devoted to overcoming the key technical hindrances which made the exosome study an active research area and motivated the research direction in this thesis to open up new avenues towards the development innovative concepts and instruments for exosome research and applications. The challenges we intend to address in this thesis are related to both quantification and isolation of exosomes; we propose a novel exosome detection method based on fluorescence resonant energy transfer (FRET) principle which improved the accuracy and sensitivity of the exosome detection without imposing complicated and lengthy multistep processes. In addition, we further make use of a microfluidic based trapping device to capture exosomes enriched on the surfaces of the functionalized microbeads which facilitates on-chip elusion and lysis of the protein and RNA content for further molecular analysis including western blot and quantitative polymerase chain reaction (qPCR). This design also benefits the fluorescence-based exosome quantification by preventing the optical interference of the background noise. Moreover, in a novel acoustofluidic approach, implementing an elasticivnanocavity layer and application of surface acoustic waves allow capturing of individual nanoparticles. On-demand generation of nanoscale acoustic force gradients is a scalable method for massively multiplexed and organizing of individual submicron particles into discrete, single-particle traps, with the potential for widespread application in sorting, patterning, and size-selective capture exosomes. In addition, we demonstrated for the first time to integrate acoustic and dielectrophoretic force fields for an efficient sorting of the sub-micron particles and extracellular vesicles. Tuning the mechanical and electrical properties of the particles and medium benefits manipulation of a wide variety of particles by harnessing the acoustic and DEP forces simultaneously.Employing novel techniques such as integrated microfluidic systems will benefit exosomes as the next- generation biomarkers and therapeutic vehicles. We envision that exosome research will continue to grow with evolving our knowledge and technology regarding the isolation and characterization of exosomes.