UBC Theses and Dissertations
FRET–based proteolytic activity assays on quantum dots Wu, Miao
Proteases play crucial roles in a multitude of biological processes. However, the behavior of proteases is different when the hydrolysis process occurs at the surface of nanoparticles when compared with that in bulk solution. Preliminary studies have reported an enhancement of hydrolase activity when multiple substrates are conjugated on a nanoparticle surface. The differences in activity and kinetic profiles were partly attributed to interactions between the hydrolase and the nanoparticle surface. Such phenomena have revealed the importance of studying the effect of nanoparticle surface properties on proteolysis. One of the most widely used nanoparticles in bioanalytical applications are quantum dots (QDs). In this work, QDs were used as a scaffold for the study of proteolytic activity on the surface of a nanoparticle where multiple copies of peptide substrate were co-localized, surface chemistry could be varied, and the progress of proteolysis tracked by Förster resonance energy transfer (FRET). The surface was modified with four different types of anionic, small-molecule ligand coatings that are commonly used in the literature: CYS (Cysteine), DHLA (Dihydrolipoic acid), GSH (Glutathione) and MPA (Mercaptopropionic acid). Difference in properties, such as the relative charge on the QDs, appeared to have a large effect on the rate of proteolysis, enhancing or inhibiting protease activity relative to bulk solution. Kinetic profiles were compared for two model proteases, trypsin and thrombin, that hydrolyze the same substrate. Of these two model proteases, thrombin was more sensitive to the QD coating and had a more varied response to different coatings. These results may provide a new way to adjust sensitivity and selectivity in proteolytic assays in vitro. Further, as a first step toward studying proteolysis in biological systems, the QD-FRET method used to track proteolysis in vitro has been adapted to fluorescence microscopy, which enables measurement of spatially heterogeneous protease activity, such as would be encountered with cultured cells. Optical parameters, such as exposure time and excitation intensity are optimized, and calibration samples and homogeneous proteolytic assays were compared between measurements with an epifluorescence microscope and a fluorescence plate reader. Proof-of-concept for heterogeneous proteolytic assays was also demonstrated.
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Attribution 2.5 Canada