Russ Algar

Fluorescence is a powerful tool for probing changes in structure on the nanometer scale. Förster resonance energy transfer (FRET) between fluorophores has a strong distance dependence in the 1-10 nm range, enabling indirect observation of minute positional changes. FRET has been used extensively for investigation of biomolecular systems and for the design of nanostructures for sensing biomolecules. This thesis describes the design of several FRET based nanostructures for fluorescence-based sensing of biomolecules. Molecular logic devices (MLDs) have the potential to make efficient biosensors which deliver complex information with a single output signal. These devices take multiple inputs and apply Boolean logic operations to produce a single binary (i.e. TRUE/FALSE) output. The unique molecular recognition abilities of DNA make it an ideal material for the construction of MLDs. Several DNA-based MLDs using FRET sensitized fluorescent emissions as output signals were devised. One of these devices was a protease biosensor designed to detect the presence and activity of a target protease, providing useful information about the target while reducing the rate of false-positive results. Though a working sensor was not ultimately achieved, important limitations on the design of this type of device were revealed. Two other devices were designed for the amplification of output signals from MLDs, an important aim to allow for more sensitive devices and more efficient logic circuits. Both devices were designed and tested using cascading DNA hybridization reactions to produce amplification of fluorescent output signals.Chemical ‘nose’ arrays utilize a panel of sensor elements which have non-specific but differing responses to targets to generate a unique ‘fingerprint’ pattern for each analyte. FRET between quantum dots (QDs) and dyes on peptides conjugated to QD surfaces can be used to determine rates of proteolysis. QDs with different surface chemistries can be used as individual sensor elements for the construction of a chemical ‘nose’ array to differentiate proteases, where the pattern of initial rates for the different surface chemistries represents the unique ‘fingerprint’ for a given protease. Three ionic QD surface ligands were synthesized, characterized, and tested for their effects on proteolysis and ability to differentiate between a panel of proteases.

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Quantum dots (QDs) are promising bioanalytical tools because of their outstanding optical properties, especially their bright and narrow photoluminescence, which offers capacity for multiplexed imaging. Along with flow cytometry, QDs of various colours allow for the detection and enumeration of multiple cell types simultaneously, which is critical for early and efficient screening for diseases, including cancers. Flow cytometry is widely recognized as the “gold-standard” technique for cell classification; however, the cost, size, and sophistication of the method make it unsuitable for use outside of specialized laboratories. A flow cytometry format amenable to point-of-care (POC) application thus has the potential to greatly increase its utility for diagnostics and health care. This thesis developed a smartphone-based flow cytometer by integrating a laser diode, a PDMS microfluidic chip, and a magnification system into a compact 3D-printed box that interfaced with a smartphone and its camera. Silica-quantum dot (SiO₂@QD) supra-nanoparticles were coated with either dextran or carboxymethyl dextran (CM-Dex), then conjugated to antibodies targeting different cell-surface antigens via tetrameric antibody complexes (TACs) or carbodiimide chemistry. Selective enumeration of multiple cancer cell lines was achieved by immunolabeling with a combination of red, orange, yellow, or green SiO₂@(QD-CM-Dex)-antibody conjugates. Smartphone videos of labeled cells under flow were analyzed with an algorithm in MATLAB that extracted PL colour features, classified the cells with a support vector machine model, and counted the cells. Future work with this prototype smartphone-based flow cytometer will aim to improve sensitivity, expand the classification and enumeration capabilities, and test its application with clinical samples.

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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.

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Concentric Förster resonance energy transfer (cFRET) based on fluorescent quantum dots as nanoscaffolds is a promising strategy for multiplexed bioanalysis and bioimaging. To expand the scope of prototypical cFRET strategy, which was limited to a particular combination of quantum dot (QD), peptides and fluorescent dyes, work in this thesis adopted a combination of an orange-emitting QD605 and red/deep-red fluorescent dyes Alexa Fluor 633 and Alexa Fluor 680 for the design of a long-wavelength cFRET configuration. This new configuration has shown certain superior properties compared to the prototypical one. Although more susceptible to photobleaching, the long-wavelength cFRET configuration offers much higher signal-to-background ratios in biological samples due to both the excellent brightness of the orange-emitting QD605 and long-wavelength excitation and emission. A rate analysis of both of the competitive and sequential energy transfer pathways revealed the dominant competitive pathway in the long-wavelength cFRET configuration, contrary to a dominant sequential pathway in the prototypical configuration. Moreover, to expand cFRET beyond peptide-linked configurations, an oligonucleotide-linked cFRET configuration was constructed and used to demonstrate the multiplexed detection of unlabeled target oligonucleotides through efficient toehold-mediated strand displacement. Overall, work in this thesis has contributed to evidence of cFRET as a general strategy and expanded it to a wider range of applications.

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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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