Russ Algar

Associate Professor

Relevant Degree Programs

 
 

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2021)
Development and evaluation of luminescent nanoparticles for immunolabeling and imaging via microscopy and smartphone-based devices (2021)

Fluorescent nanomaterials are of interest for applications in bioanalysis and imaging due to their potential for high brightness, robust photostability, diverse surface functionalization, and special size-dependent properties. Cellular immunolabeling with antigen-targeting biomolecules (e.g. antibodies, aptamers) is one such application, and has numerous applications in biomedical research and for laboratory-based or point-of-care molecular diagnostics for health care. For example, immunolabeling is a commonly used technique for the diagnostic screening of cancerous cells and relies on the detection of antigens that are specific to malignant cells. However, integrating fluorescent nanomaterials within molecular diagnostic assays can be challenging— optimization to the physicochemical properties of a nanomaterial and its surface chemistry is necessary. When optimized, fluorescent nanoparticles have the ability to address key challenges in bioanalysis and imaging, including enhanced sensitivity and enabling the use of mass-produced consumer technologies, such as smartphones, as a platform for point-of-care diagnostics. This thesis presents key contributions towards the development of fluorescent nanoparticles for applications in bioanalysis and imaging, and particularly immunofluorescent labeling of cells. These materials include quantum dots (QDs), single-chain polymer nanoparticles (SCPNs), polymer dots (Pdots), and composite supra-nanoparticles that comprise iron oxide nanoparticles/QDs and silica nanoparticle/QDs. Breast cancer cells were labeled and imaged using various antibody conjugates of these nanoparticles. With several of the materials, dextran is demonstrated to be an ideal surface coating. Benefits include improved colloidal stability, reduced non-specific binding, and utility as a biochemical handle for the assembly of tetrameric antibody complexes (TACs) for specific cellular immunolabeling. Despite several advantages, TACs had not been used with fluorescent nanoparticles prior to this thesis. In addition, the exceptional brightness of the supra-nanoparticles enabled their utilization for smartphone-based imaging of immunolabeled cells. This imaging is done with a device manufactured by 3-D printing, which, in conjugation with the brightness of the materials, avoids the typical trade-off between optimal sensitivity and portability, simplicity, and low cost. Overall, this thesis enriches the science of cellular immunolabeling and imaging via novel materials, novel immunoconjugation methods, and novel devices.

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Development and characterization of semiconducting polymer dots for applications in bioanalysis (2020)

Semiconducting polymer dots (Pdots) are rapidly gaining popularity as fluorescent probes in bioanalysis and imaging. These materials have several remarkable and highly advantageous properties, including their extremely high per particle brightness, large one- and two-photon absorption cross sections, biocompatibility, and ease of preparation. However, being a new material, Pdots suffer from several key limitations that must be addressed before they may find widespread use in bioanalysis. Pdots are typically synthesized by the manual nanoprecipitation method which suffers from poor control and reproducibility. Although energy transfer-based chemical and biological sensors have been developed using Pdots, their photophysics and mechanisms of energy transfer are poorly understood, limiting the rational design of such sensors. Being held together by relatively weak entropic forces, Pdots are only moderately stable and are often prone to non-specific binding due to their surface chemistries. To date, relatively few surface and bioconjugate chemistries have been reported. This thesis presents the development of a novel, flow-based synthetic method for Pdots yielding improved reproducibility, tuneable particle sizes, and the ability to synthesize Pdots on small (1 mL) and large (100 mL) scales. A comprehensive study of energy transfer between Pdot donors and organic dye acceptors is also presented, and considers the frameworks of Förster Resonance Energy Transfer (FRET), Dexter energy transfer, and photoinduced electron transfer (PET). The results suggest that FRET alone is not sufficient to describe energy transfer in such systems, and that Dexter ET and PET likely also contribute. Thirdly, a surface coating material based on dextran, a biosynthetic glucan, was used to functionalize the Pdot surface and enabled preparation of immunoconjugates using tetrameric antibody complexes (TACs), resulting in improved performance in proof-of-concept immunoassay and cellular imaging applications. Overall, this thesis presents key contributions to the development of Pdots for applications in bioanalysis, including their synthesis, a deeper understanding of their photophysics, and enhanced performance in biosensing and imaging.

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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 - 2020)
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 Forster 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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