UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Evaluating small molecule and macromolecular ligands for tuning protease activity on QD probes Jeen, Yow Ting

Abstract

Quantum dots (QDs) provide a promising platform for fluorescence-based assays. Their non-trivial surface area, bright photoluminescence (PL), and photostability can be coupled to Förster resonance energy transfer (FRET) to enable sensitive detection of proteolytic activities. To further develop QD probes and fine-tune the interactions between QDs and enzymes, the QD ligand library was expanded to include a zwitterionic carboxybetaine, anionic serine-appended lipoic acid, and a glucitol-functionalized lipoic acid. Ligands are a necessary part of QD probes as QDs are often synthesized with hydrophobic ligands and ligand exchange is needed to render QDs colloidally stable in aqueous solutions. Since the ligands are at the interface of the nanocrystal surface and the bulk solution, a variety of available QD surface chemistry is needed to optimize ligand selection for different analytes and matrices. The pH and ionic stability of the new ligand-coated QDs were evaluated and compared to the commonly used dihydrolipoic acid (DHLA) and glutathione-coated QDs. Three model proteases were studied to evaluate their activities on the different ligand-coated QDs. The variations in the proteolytic activities on the different QDs could be used to distinguish between the three enzymes. As the ligand library continues to expand, a combination of these QDs can be used to identify an unknown enzyme in a microarray format. Biomolecules conjugated to QDs provide yet another strategy to manipulate proteolytic activity on QDs. As a proof-of concept, a peptide sequence based on protease-activated-receptor 1 (PAR1) was displayed on QD-substrate conjugate to mimic the display of PAR1 on cellular surface. The PAR1-displaying zwitterionic QDs were associated with enhanced relative initial rate compared to the control by as much as 15-fold. These results highlight the importance of the QD surface chemistry and that different elements can have a synergistic effect when assembled together on the QD platform. Similar to what is seen in biology, the selectivity of QD probes can be tuned through additional allosteric interactions instead of substrate recognition sites for the protease active site alone.

Item Citations and Data

License

Attribution-NonCommercial-NoDerivatives 4.0 International

Usage Statistics