Russ Algar

Associate Professor

Relevant Degree Programs

 

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2019)
Design of quantum dot and smartphone-based luminescent bioassay platforms for prospective point-of-care diagnostics (2017)

Smartphones are essential components of daily life. These devices feature built-in cameras and light sources, data storage, and wireless data transmission, making them emerging devices for optical imaging and diagnostic bioassays. To date, the majority of smartphone-based diagnostics have been developed for colourimetric assays, which often suffer from limited multiplexing capability and poor sensitivity. In general, fluorescence-based assays offer greater sensitivity and multiplexing capacity, and in combination with smartphone platform may help to overcome these limitations. This thesis describes research toward the development of smartphone platforms for fluorescence-based bioassays using quantum dots (QDs) and Förster resonance energy transfer (FRET), and addresses two critical challenges: multiplexing and analysis of biological sample matrices. Multiplexing was achieved by matching the built-in RGB (red-green-blue) channels of smartphone cameras with the narrow, bright, and tunable emission of QDs. The QDs provided superior brightness in comparison to traditional fluorescent dyes and proteins, and served as excellent FRET donors in assays that used proteases as model analytes. Up to threeplex assays were demonstrated for the detection of trypsin, chymotrypsin, and enterokinase. The analytical performance of the smartphone-based platform matched that of a bench-top spectrofluorimeter, where the smartphone was a fraction of the cost and size. A smartphone based platform was also developed for detection of analytes in serum and whole blood. Most clinical samples will take this form and necessitate careful assay design to overcome challenges associated with physical, optical and chemical properties of whole blood. Blood is strongly absorbing, scattering, autofluorescent, and contains high concentrations of proteins and small molecules. A well-thought-out combination of QDs, FRET, and a paper-in-PDMS chip enabled direct, single-step and quantitative fluorescence-based detection of thrombin activity in whole blood. The research in this thesis is a foundation for the development of novel point-of-care diagnostics assays with consumer electronics that could help enable personalized health care.

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Master's Student Supervision (2010 - 2018)
Evaluating small molecule and macromolecular ligands for tuning protease activity on QD probes (2018)

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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A long-wavelength quantum dot-concentric Förster resonance energy transfer configuration: characterization and application in a multiplexed hybridization assay (2017)

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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FRET–based proteolytic activity assays on quantum dots (2014)

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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