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<p>Cytochrome c (Cyt c) is a heme-containing protein that is a component of the electron transport chain as well as the mitochondrial apoptotic pathway. It is released from the mitochondrial intermembrane space to the cytosol during apoptosis and is also thought to be a biomarker for cancer and liver disease. Therefore, an efficient Cyt c biosensor would be a very useful tool for studying apoptosis. Here we show the process of development of Cyt c-dependent aptazymes, derived by <em>in vitro </em>selection. These aptazymes consist of 3 components: 1) a substrate with a cleavage site that consists of a single ribonucleotide flanked by a fluorophore and quencher; 2) a DNAzyme (catalytic DNA) motif capable of cleaving the substrate; 3) an aptamer, a short piece of single- stranded DNA that can specifically bind Cyt c. When Cyt c is absent, the aptamer occludes the catalytic core of the DNAzyme and the fluorophore of the intact substrate is quenched. However, when Cyt c is present, the aptamer binds Cyt c, allowing the DNAzyme to cleave the embedded ribonucleotide, separating the fluorophore and quencher, resulting in a fluorescent signal. Simulations of <em>in vitro </em>selection of Cyt c- dependent aptazymes were also performed. The simulations revealed several methods that can improve the success rate of future <em>in vitro </em>selections of aptazymes.</p> <p>Further analysis of the previously derived DNAzyme DEC22-18 was also performed. A detailed understanding of this DNAzyme will allow it to be developed into a biosensor.</p> / Master of Science (MSc)

<p>The process of in vitro selection has led to the isolation of many catalytic DNA molecules, called deoxyribozymes, which can catalyze a range of biologically-relevant reactions. Despite these advances, questions still remain as to why DNA, which seems more suited to information storage than catalysis can efficiently catalyze chemical reactions. In this thesis, a group of deoxyribozymes that can catalyze their own phosphorylation using NTP substrates are used a model system to study how DNA is able to fold into complex structures necessary for catalysis. Using a variety of structural probing techniques, these studies elucidated a common secondary structure shared by three deoxyribozymes, which do not appear to share a common ancestor sequence. This suggests that this motif may be most efficient motif to catalyze self-phosphorylation by DNA. It also more generally demonstrates that DNA can undergo convergent evolution to reach the same complex folding arrangement. A fourth deoxyribozyme was found to fold into a complex tertiary structure containing a novel quadruplex-helix pseudoknot motif. The finding of this pseudoknot and comparison with other quadruplexes found in other functional nucleic acids led us to investigate whether these stable motifs could be incorporated into nucleic acid libraries to improve the process of in vitro selection and give researchers a better chance of isolating functional nucleic acids. Design and characterization of structured libraries revealed that DNA libraries could be made in which the majority of sequences are folded into quadruplex arrangements. The incorporation of this quadruplex scaffold into DNA sequence libraries may ease the isolation of functional nucleic acids that contain this useful structural motif. In the final part of this thesis, a self-phosphorylating deoxyribozyme was converted from a tool for study of DNA structure to a sensor for GTP and Mn²⁺, demonstrating that deoxyribozyme substrates can be converted into targets for biosensors.</p> / Doctor of Philosophy (PhD)

<p>Single-walled carbon nanotubes (SWNTs) are of special interest in current research due to their extraordinary mechanical, electronic and optical properties. Their unique structure, remarkable thermal and electrical conductivity, and high mechanical strength make SWNTs viable candidates for a wide range of device applications. However, pristine CNTs are not dispersible in most solvents, the main difficulties in CNT applications are related to their purification and solution-phase processing. In recent years, the supramolecular functionalization of SWNTs with conjugated polymers has received significant attention. Research within this field has been driven by the desire to find polymer structures that can selectively disperse certain nanotubes species with high efficiency.</p> <p>After a brief overview of the studies that are related to the investigation of the supramolecular interaction between various conjugated polymers and SWNTs (chapter 1), the synthesis of fluorene and thiophene-based conjugated polymers and their supramolecular complex formation properties with SWNTs are described (chapter 2, 3, 4, 5 and 6). In order to understand the effect that conjugated polymer structure has on formation of supramolecular complexes with SWNTs, various factors were investigated by: (1) altering the polymer backbone composition; (2) varying the polymer molecular weight; (3) introducing different solubilizing groups while the polymer backbone remained the same; (4) changing the polymer conformation. All of the resulting polymer-nanotube assemblies exhibit excellent solution stability in THF in the absence of excess unbound free polymer. The spectroscopic characterization of the polymer-SWNT complex materials indicated that the interaction between the conjugated polymers and SWNTs is strongly influenced by polymer structure.</p> <p>The interaction between a water soluble polythiophene derivative, poly[3-(3-N,N-diethylaminopropoxy)-thiophene] (PDAOT), and SWNTs is discussed in chapter 7. It is also demonstrated that the PDAOT-SWNT complexes form stable aqueous solutions that can be used for the fabrication of highly sensitive amperometric glucose biosensors.</p> / Doctor of Philosophy (PhD)

<p>The food-borne pathogen, <em>Listeria monocytogenes</em>, is a global health concern as it has been responsible for multiple food contamination outbreaks over the past century. Current detection methods like the enzyme-linked immunoassays (ELISA), and polymerase chain reaction (PCR) take over 24 h to attain results, are costly, require specialized equipment and trained personnel. In this study we investigated the use of functional nucleic acid (FNA) to develop a rapid and cost-effective detection method for <em>L. monocytogenes</em>. We carried out in<em> vitro</em> selection in order to isolate a fluorescently labeled DNA-RNA hybrid strand that can be bound and cleaved by specific endoribonucleases (RNase) from <em>L. monocytogenes</em>. We termed these DNA-RNA hybrid strands RNase-cleaved fluorescent substrate (RFS). Since no past studies have isolated RNases from <em>L. monocytogenes</em>, we first identified the genes based on sequence similarities with well characterized RNases. We purified and characterized RNase HII, RNase III and RNase G. Since this study focused primarily on developing RFS for RNase HII, we performed an in depth <em>in vitro</em> biochemical analysis to characterize this enzyme. We found that RNase HII from <em>L. monocytogenes</em> plays an important role in DNA replication and repair. Furthermore, we obtained six sequence classes by <em>in vitro</em> selection which could interact with RNase HII. The key nucleotide regions involved with RNase HII interactions were identified. In the final study, we showed the sequences isolated by <em>in vitro</em> selection could also be used as a tool to study ribonuclease function and identify new interaction between enzyme and substrate.</p> / Master of Science (MSc)

Microbial biosensor systems (MBS) are useful for analyte detection owing to their low cost, sensitivity, and selectivity for bioavailable analytes. Due to typically poor shelf-life and sensitivity to external conditions, there are few reports of MBS technology applied to simple analytical devices. The effectiveness of air-drying MBS in natural polymers was investigated as a novel preservation technique. Two colorimetric Escherichia coli MBS, a tetracycline-inducible reporter and an arsenate-inducible reporter, were dried on various substrates yielding novel MBS platforms. In proof-of-concept experiments performed in 96-well microplates, both systems demonstrated responsivity after air-drying in low concentrations of pullulan. However, the MBS were unresponsive following brief storage of 1 week. To improve the preservation of MBS, sensing strips were created by air-drying concentrated acacia gum-based MBS suspensions onto paper. Cells dried on these strips demonstrated responsivity upon solubilization in various tube-based assays. MBS sensing strip responsivity was demonstrated following storage for 6 weeks at 4 °C. Tetracycline-responsive sensing strips also performed well in assays using spiked lake water samples. Air-drying in natural polymers was an effective MBS preservation technique, and allowed for the creation of “mix and read” style assays which were simple, equipment-free and ready-to-use. / Thesis / Master of Science (MSc)

Point-of-care testing brings diagnosis and treatment monitoring to the site of the patient. It heavily relies on biosensors, which leverage the interactions between a target biomarker and a bioreceptor, to deliver fast and accurate results. However, non-specific binding of molecules and microorganisms on the biointerface can interfere with biomarker-bioreceptor interactions and diminish a biosensor’s sensitivity, specificity, and stability. In turn, this can lead to false diagnoses and ineffective treatments. Omniphobic-lubricant infused (OLI) coatings exhibit slippery, self-cleaning characteristics that repel untargeted molecules and microorganisms to augment the biosensor’s performance. In this work, we investigate the proficiency of OLI coatings in two specific applications: dissolved oxygen sensing and DNA biosensing. First, in water quality monitoring, an OLI coating is applied to the selectively permeable membranes of a dissolved oxygen sensor. Over a three-week incubation period in an environment with accelerated bacterial growth, the coated membranes exhibit a 160% higher reproducibility (10% deviation in sensitivity) and lower biofilm formation (96° static contact angle) in comparison to unmodified membranes (26%, 32°). The second application is in DNA biosensing, where a novel OLI coating uses carbon dioxide plasma activation to embed oligonucleotide probes. It demonstrates an optimized balance of slippery repellency (76° static contact angle, 10° sliding angle) and biosensing functionality, 19% longer clotting times than conventional blocking conditions, and equal sensitivity to PLL-PEG when capturing target DNA in whole blood. Going forward, our research will continue to expand the use of OLI coatings in biosensing applications, particularly exploiting its antibiofouling and anticoagulative capabilities. / Thesis / Master of Applied Science (MASc) / Biosensors are an integral tool in delivering quick and accurate point-of-care diagnosis and treatment monitoring. However, their performance can be impeded by the non-specific binding of undesirable molecules and microorganisms on the sensing surface. Omniphobic lubricant-infused (OLI) coatings have been shown to suppress biofouling and blood clotting on surfaces through exceptional repellency. This thesis focuses on the implementation of OLI coatings in biosensing applications. It investigates the antibiofouling capacity of an OLI coating on a membrane for dissolved oxygen detection. Then, it discusses a novel coating with integrated DNA biosensing functionality for working directly with blood samples. The enclosed work demonstrates that the OLI coating empowers biosensors to deliver more effective point-of-care testing.

Development of repellent surfaces which can supress bacteria adhesion, blood contamination and thrombosis, and non-specific adhesion on diagnostic devices has been a topic of intense research as these characteristics are in high demand. This thesis focused on design and development of omniphobic surfaces based on hierarchical structures and their application for preventing pathogenic contamination and biosensing. First, a flexible hierarchical heat-shrinkable wrap featuring micro and nanostructures, was developed with straightforward scalable methods which can be applied to existing surfaces. These surfaces reduced biofilm formation of World Health Organization-designated priority pathogens as well as minimized risk of spreading contamination from intermediate surfaces. This is due to the broad liquid repellency and the presence of reduced anchor points for bacterial adhesion on the hierarchical surfaces. Next, the developed surfaces were applied to minimize blood contamination and clot formation as well as facile integration of hydrophilic patterns. This led to droplet compartmentalization and was utilized for detection of Interleukin 6 in a rapid dip-based assay. Furthermore, in a review article the need for anti-viral or virus repellent surfaces and future perspectives were discussed as the global COVID-19 pandemic surged and attracted interest toward innovative technologies for suppressing the spread of pathogens. To address the pressing issue of non-specific adhesion in diagnostics devices, an omniphobic liquid infused electrochemical biosensor was developed. This was achieved by electroplating gold nanostructures on fluorosilanized gold electrodes. These electrodes demonstrated rapid and specific detection of Escherichia coli within an hour in complex biological liquids (blood, urine, etc.) without dilutions or amplification steps from clinical patient samples which are major bottle necks when rapid detection systems are sought for at the point of care. / Thesis / Doctor of Philosophy (PhD) / Repellent surfaces have a variety of applications in healthcare, for coating medical devices (e.g. indwelling implants, stethoscopes, and other external devices.), coating hospital surfaces for blood and pathogen repellency, and for developing anti-fouling diagnostic devices. Furthermore, they can be applied in the food sector for limiting contaminations, and in public areas on high-touch surfaces to eliminate the spread of infection. Therefore, there is a need for repellent surface which can be easily applied to surfaces with various form factors while having an easy fabrication method. Featuring hierarchical structures on a heat-shrinkable material, a repellent wrap was designed to be integrated on existing surfaces and repel pathogens and suppress the spread of infection as an intermediate surface. Similar concept was used for designing blood repellent surfaces which were patterned with hydrophilic regions for a rapid dip-based biosensing platform. Finally, surface textures on conductive materials with liquid infused repellent coatings were investigated for electrochemical biosensing in complex biological liquids.

Fiber-optic Fabry-Perot interferometric (FFPI) sensors have been widely used due to their high sensitivity, ease of fabrication, miniature size, and capability for multiplexing. However, direct measurement of self-assembled thin films, receptor immobilization process or biological reaction is limited in the FFPI technique due to the difficulty of forming Fabry-Perot cavities by the thin film itself. Novel methods are needed to provide an accurate and reliable measurement for monitoring the thin-film growth in the nanometer range and under various conditions. In this work, two types of fiber-optic multicavity Fabry-Perot interferometric (MFPI) sensors with built-in temperature compensation were designed and fabricated for thin-film measurement, with applications in chemical and biological sensing. Both the tubing-based MFPI sensor and microgap MFPI sensor provide simple, yet high performance solutions for thin-film sensing. The temperature dependence of the sensing cavity is compensated by extracting the temperature information from a second multiplexed cavity. This provides the opportunity to examine the thin-film characteristics under different environment temperatures. To demonstrate the potential of this structure for practical applications, immunosensors were fabricated and tested using these structures. Self-assembled polyelectrolytes served as a precursor film for immobilization of antibodies to ensure they retain their biological activity. This not only provides a convenient method for protein immobilization but also presents the possibility of increasing the binding capacity and sensitivity by incorporating multilayers of antibodies into polyelectrolyte layers. The steady-state measurement demonstrated the surface concentration and binding ratio of the immunoreaction. Analysis of the kinetic binding profile provided a fast and effective way to measure antigen concentration. Monitoring the immunoreaction between commercially available immunoglobulin G (IgG) and anti-IgG demonstrated the feasibility of using the MFPI sensing system for immunosensing applications. / Ph. D.

The electrostatic self-assembly (ESA) method presents an effective application in the field of biosensing due to the uniform nanoscale structure. In previous research, a single mode fiber (SMF) sensor system had been investigated for the thin-film measurement due to the high fringe visibility. However, compared with a SMF sensor system, a multimode fiber (MMF) sensor system is lower-cost and has larger sensing area (the fiber core), providing the potential for higher sensing efficiency. In this thesis, a multimode fiber-optic sensor has been developed based on extrinsic Fabry-Perot interferometry (EFPI) for the measurement of optical thickness in self-assembled thin film layers as well as for the immunosensing test. The sensor was fabricated by connecting a multimode fiber (MMF) and a silica wafer. A Fabry-Perot cavity was formed by the reflections from the two interfaces of the wafer. The negatively charged silica wafer could be used as the substrate for the thin film immobilization scheme. The sensor is incorporated into the white-light interferometric system. By monitoring the optical cavity length increment, the self-assembled thin film thickness was measured; the immunoreaction between immunoglobulin G (IgG) and anti-IgG was investigated. / Master of Science