Isolation and Detection of Extracellular Vesicles

Show simple item record

dc.contributor Helsingin yliopisto, Matemaattis-luonnontieteellinen tiedekunta, Kemian laitos fi
dc.contributor University of Helsinki, Faculty of Science, Department of Chemistry en
dc.contributor Helsingfors universitet, Matematisk-naturvetenskapliga fakulteten, Kemiska institutionen sv Tear, Jing Ying Crystal 2017
dc.identifier.uri URN:NBN:fi-fe2017121155628
dc.description.abstract Extracellular vesicles (EVs) are bilayer nanoparticles ranging from 40 nm to 5 μm in size and are mainly categorized as exosomes, microparticles, and apoptotic bodies. Recent studies reveal their involvement in various metabolic mechanisms in the human body. In particular, certain types of platelet-derived EVs found in plasma and serum are related to blood coagulation diseases and strokes. Merely, the diversity and heterogeneity of EVs in cellular systems and the lack of their isolation and detection methods are known about EVs. The fact is that a wide range of nomenclatures and basic questions regarding their morphology, size, and phenotype has remained debatable. As a result, there is a need to discover and standardize a simple and robust protocol for the isolation and detection of EVs. The literature part of the thesis covers an overview of what is defined as EVs and their functions undertaken, followed by a summary of the conventional methods for isolation and detection of EVs. The literature part finally concludes with an insight into upcoming and novel isolation and detection methods. Ultracentrifugation is the “golden” method used in the isolation of EVs. The long extraction times and varying reproducibility makes ultracentrifugation less viable, resulting in the exploration of other methods including size exclusion chromatography and immunoaffinity methods. Currently, none of these techniques are able to effectively distinguish between subtypes of EVs and matrix contaminants such as proteins remaining in the isolates. Each isolation method has its own edging advantages and disadvantages. Nevertheless, immunoaffinity methods showed greater potential in EVs extraction due to their high specificity and selectivity to process purified EVs samples. Detection methods are hindered by the minute sizes of EVs and the presence of contaminants in the isolates. Most detection methods involve the use of antibodies to detect or microscopic imaging to verify their presence based on their morphology. The trend is, however, skewing towards more reliable detection methods such as the use of mass spectrometry and microfluidic devices. The aim of the experimental portion is the ability to isolate purified platelet-derived EVs from complex human plasma samples and enable fast isolation of EVs. The use of immunoaffinity chromatography using antibody immobilized carbonyldiimidazole (CDI) monolithic disk was explored. Studies using specific platelet EV biomarker, anti-human CD61 antibody had successfully isolated platelet-derived EVs from plasma samples. In these studies, diluted plasma in phosphate buffer saline (PBS) solution (1:20 v/v) was injected through the housing setup containing the anti-human CD61 antibody immobilized monolithic disk. The eluate was obtained by injection of carbonate-bicarbonate solution and neutralized with hydrochloric acid. Detection analyses were then performed with nanoparticle tracking analysis (NTA), protein assays, Western blot and transmission electron microscopy (TEM). The average particle counts obtained in plasma isolates were found to achieve 10,000 particles more than isolates obtained after ultra-centrifugation reported in the literature. Positive presence of cytosolic protein TSG101 and transmembrane protein CD9 in Western blots, and transmission electron microscopic images of circular bilayer particles confirmed the isolation of platelet-derived EVs. Optimization studies also showed the inverse relationship between the flow rate in isolation step and the yield of platelet-derived EVs obtained. Affinity chromatography using antibody immobilized monolithic disk proved its success in the quick isolation (≤45 minutes) of platelet-derived EVs from plasma samples. The fast, miniaturized, compact setup, and simple methodology make the method viable for automation such as high throughput screening and suitable for applications of microfluidic microchips and censoring techniques on-line coupled for non-invasive and portable diagnostics. en
dc.language.iso eng
dc.publisher Helsingin yliopisto fi
dc.publisher University of Helsinki en
dc.publisher Helsingfors universitet sv
dc.title Isolation and Detection of Extracellular Vesicles en
dc.type.ontasot pro gradu -tutkielmat fi
dc.type.ontasot master's thesis en
dc.type.ontasot pro gradu-avhandlingar sv
dc.subject.discipline Analytical Chemistry en
dc.subject.discipline Analyyttinen kemia fi
dc.subject.discipline Analytisk kemi sv
dct.identifier.urn URN:NBN:fi-fe2017121155628

Files in this item

Files Size Format View
thesis-crystal-final1.pdf 3.507Mb application/pdf View/Open

This item appears in the following Collection(s)

Show simple item record