A FRET based assay for the quantification of synthetic and native lipid vesicles
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Typ
Examensarbete för masterexamen
Master Thesis
Master Thesis
Program
Applied physics (MPAPP), MSc
Publicerad
2018
Författare
Thorsteinsson, Konrad
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
Lipid nanoparticles, both of artificial and biological origin, have attracted significant attention in recent years. Biological lipid nanoparticles in the form of extracellular vesicles are involved in intercellular communication and biological material transport. Synthetic liposomes have also been proposed as promising drug delivery systems. In view of this broad interest, methods capable of accurately quantifying the content of lipid nanoparticles in a sample are urgently needed. To date, quantification is most commonly achieved by counting the particles after visualization, or by quantifying the total protein content in the case of particles of biological origin. In this thesis we present an alternative method allowing for the quantification of the total lipid surface area of an unknown sample. Our approach is based on Förster Resonance Energy Transfer (FRET), where the unknown lipid nanoparticle sample is sonicated with vesicles containing a FRET-fluorophore pair, leading to membrane fusion. The change in FRET fluorescence can then be correlated to the total surface area of the unknown sample. We first calibrated the method using synthetic vesicles of known surface area. We then tested the method on synthetic vesicles containing cholesterol, herpes simplex virus type 2, and two species of outer membrane vesicles secreted from E. coli bacteria. Finally, we benchmarked our results against alternative established methods and discussed potential and limitation of each. Our results indicate that the FRET assay is suitable to quantify all the lipid nanoparticle samples tested here and serves as a viable measurement technique to quantify lipid surface areas.
Beskrivning
Ämne/nyckelord
Fysik , Physical Sciences