No description available.

Antibiotic-resistant bacterial infections account for millions of human fatalities each year, with drug susceptibility testing (DST) affordability being limited by instrumentational constraints, monetary and/or time expenses. Molecular diagnostics performed at the point of care can provide a solution. Split probes coupled with label-free reporters like Fluorescent Light-up APtamers (FLAPs) are promising for point-of-care DST as they offer the needed selectivity towards point mutations inducive of drug resistance. This project aims at bridging the gap in FLAP applications for molecular diagnostics with a focus on multiplexing the analysis. Due to the limited number of DNA FLAPs available, we explored the ability of one of the most efficient DNA FLAPs - dapoxyl binding aptamer (DAP) - to bind fluorogens with different spectral properties. We performed the rational mutagenesis of the DAP dye-binding core to reveal any sequence-function correlations and to identify prospective orthogonal FLAP-dye pairs. Orthogonal FLAPs were used as scaffolds to design split dapoxyl aptameric (SDA) probes, which targeted a fragment of the katG gene from Mycobacterium tuberculosis complex associated with bacterial resistance to a first-line antituberculous drug isoniazid (INH). The probes were tested to differentiate the nucleic acid targets with single-nucleotide variations corresponding to isoniazid-susceptible (INHS) and isoniazid-resistant (INHR) bacterial phenotype in a multiplex fashion. With proper optimization of the probes, they can find an application in ratiometric analysis of heterogeneous bacterial populations composed of both drug-susceptible and drug-resistant strains and thus help in initial diagnosing of infectious diseases and in monitoring the therapy outcomes.

Peptide hormones are important biomolecules that transduce downstream effects such as cell proliferation, regulation, and gene expression. Their levels have been upregulated in various disorders such as cancer, yet detection methods are lacking. We designed two split aptamer-based assays for the detection of a peptide hormone – Somatostatin (SST) – with different signal readouts: fluorescent readout based on light-up aptamers and the colorimetric readout of ABTS peroxidation from a G-quadruplex. We used an already selected split-aptamer –SSTA5–for SST for our designs and we had expected the developed detection systems to exhibit detection and quantification capabilities that would hopefully allow their use for SST monitoring in clinical samples. However, our experiments did not support the hypothesis of this project and SST was not able to be detected using either of our fluorescent or colorimetric methods. To determine if the SSTA5 aptamer could bind SST appropriately, Förster resonance energy transfer (FRET) was used. We verified that there was no energy transfer between two covalently-attached light-sensitive molecules (one attached to each part of the split SSTA5 aptamer); thus, we theorize that the aptamer does not hybridize in the presence of the tetra decapeptide SST. Therefore selection of another, more appropriate, aptamer for SST will be needed for further aptameric-based detection methods. Once this is accomplished, our methodologies could be re-applied for detection of SST which could lead to real-time detection of essential hormonal levels in patients.

Due to the misuse of antibiotics, multi-drug resistant (MDR) bacteria have become more rampant in our society; these MDR have given rise to diseases that are not readily curable. One such agent is the Mycobacterium tuberculosis complex, which is a causative agent of tuberculosis (TB). Timely diagnostics of the bacterial infection and detection of bacterial drug-susceptibility profiles helps to initiate the necessary treatment in a timely fashion and to limit transmission of the disease. For more affordable detection of bacterial diseases, such as TN, tag-free split aptamer probes are advantageous. This proposal aims at designing split aptamer probes for detection of point mutations in the rpoB and katG genes of M. tuberculosis that are associated with resistance to two front-line antibiotics – rifampin and isoniazid, respectively, which causes MDR-TB. The probes will be designed and tested with synthetic oligonucleotide mimics of the bacterial genes in terms of their limit of detection and selectivity in discriminating the targets with single-nucleotide substitutions.

No description available.

<p>Adaptation of nucleic acid <em>in vitro</em> selection for whole cell targets has been demonstrated to be an effective means of isolating useful sequences with applications in biomarker detection and therapeutics. The problem of efficient delivery of materials across cell membranes is common to a variety of research and medical fields. Existing aptamers isolated in surfacing binding selections have been successfully adapted for cell targeted therapies through complex modifications. However, better aptamers may be derived from a selection optimized to isolate internalized sequences directly. A cell selection experiment with the goal of identifying circular random-sequence DNA aptamers with the ability to facilitate their own internalization into MCF7 cells was conducted. Several classes of sequences isolated from this selection were shown to target cell nuclei at a rate significantly greater than control sequences as determined by qPCR relative recovery assays supported by <em>in situ</em> RCA fluorescence microscopy data. The localization of functional DNA sequences at the subcellular and intercellular levels suggests a receptor mediated mechanism. Techniques for the selection, purification and fluorescent detection of small circular DNAs were also developed for this study. Further work to characterize and identify targets should be pursued to better understand the mechanism of internalization and judge the suitability of G18d sequences as a delivery platform.</p> / Master of Science (MSc)

Clostridium difficile, the causative agent of C. difficile infection (CDI), causes hundreds of thousands of hospital-acquired infections in the United States and Canada annually. Furthermore, the prevalence and severity of CDI has been on the rise in developed countries, especially with the appearance of “hypervirulent” strains. Detection of CDI is thus of great importance. Traditional detection methods can be time consuming or lack the desired sensitivity. On the other hand, aptamers pose great prospects as diagnostic and therapeutic agents. Aptamers are nucleic acid ligands with molecular recognition capabilities rivaling those of antibodies. They are obtained by a process of in vitro selection known as systematic evolution of ligands by exponential enrichment (SELEX). However, the current approach may result in aptamers that experience non-specific binding in complex or biological samples. Here, we propose a multiple domain selection (MDS) method for aptamer isolation. This method utilizes independent selections on separate components of a single target in order to obtain uniquely specific aptamers. The aptamers can then be unified into a heterobivalent construct able to recognize two sites on one target. We hypothesize the combined aptamer would result in greater affinity and specificity for the target, resulting in greatly increased aptamer utility in current and future applications. In the current study, we have cloned and purified full length C. difficile DnaK as well as the N-terminal domain (NTD) and C-terminal domain (CTD) of the protein. MDS was performed on each target and the resulting aptamers were combined into a heterobivalent construct. The construct resulted in an approximately 100-fold affinity increase relative to the single aptamer for DnaK, and could detect much smaller quantities of target. Although it experienced low level recognition of high concentrations of purified E. coli DnaK, there was no detectable non-specific binding in several biological samples. / Thesis / Master of Science (MSc)

<p> The purpose of this research is to develop a general method for capturing and separating specific targets. Nucleic acid aptamers are short sequences of single-stranded DNA or RNA which have the ability to bind to the small organic or inorganic molecules such as protein and metal ions with high binding affinity. In this study, a bioconjugate re-usable system was developed. It can reversibly load or unload DNA aptamers.</p> <p> To allow separation, a thermally responsive polymer (N-isopropylacrylamide, PNIPAM) is used. This polymer can undergo a reversible phase transition upon adding NaCl and/or increasing temperature. A short sequence of single stranded DNA (ssDNA) was coupled to PNIPAM. The ssDNA will experience a reversible phase transition because of the PNIPAM.</p> <p> The DNA sequence for an aptamer can be extended to contain a sequence that is complementary to that of the ssDNA coupled to PNIPAM. Adding this extended aptamer to the conjugate will result in spontaneous hybridization of the two strands of DNA. These strands can be separated using an agent (e.g. urea) that destroys hydrogen bonding. The conjugate can be recovered using a reversible inverse phase transition.</p> <p> The same PNIPAM-ssDNA conjugate can be used reversibly for coupling different aptamers. The aptamers did not lose their binding ability when coupled with PNIPAM-ssDNA conjugates. In the process of precipitation separation targets, the PNIPAM-ssDNA conjugate showed little loss with applied phase transition. Moreover, the coupling efficiency of the ssDNA to PNIPAM conjugates was determined. The binding ability of the ATP aptamers to ATP was also investigated.</p> / Thesis / Master of Applied Science (MASc)

Asthma is a chronic disease affecting over 300 million people worldwide. Airway inflammation is a central feature of asthma. Quantitative sputum cytometry is the most validated method to assess this and to adjust anti-inflammatory therapy, yet it is underutilized due to rigorous processing that requires expensive specialized technicians. To address these limitations, this thesis focuses on the development of several point of need biosensors that rapidly quantify respiratory biomarkers as alternatives to traditional laboratory tests. The project began by developing a paper based sensor for detection of myeloperoxidase (MPO), a neutrophil biomarker. A test was developed using commercially available antibodies, showing direct correlation between the test line colour intensity and total neutrophils. This work was expanded to include a second protein target, eosinophil peroxidase (EPX), for identification of eosinophils. Although the test performed well using neat samples, it failed to identify EPX in clinical sputum samples. Analyzing pre-treatment methods identified that a quick immunoprecipitation technique using protein A/G beads followed by syringe filtration allowed for the device to successfully quantify EPX, eliminating the need for a centrifuge. However, the limited supply of commercial anti-EPX antibodies combined with the need for sample pre-treatment prompted investigation into alternative detection avenues. Nucleic acid aptamers were explored, with aptamer selection for EPX producing several aptamer candidates. Binding affinity and specificity tests were performed, with the T1-5 aptamer emerging. T1-5 was capable of selectively binding EPX over MPO with high affinity. This aptamer was integrated into a simple pull-down assay, capable of detecting EPX with an order of magnitude lower limit of detection than the antibody test. Overall this work has developed multiple sensors with the potential to overcome the limitations of accessibility to sputum cytometry, rapidly identify the presence and type of airway inflammation, and deliver personalized treatment strategies that not only reduce the global healthcare burden, but also greatly improve a patient’s quality of life. / Thesis / Doctor of Philosophy (PhD)

Gold nanoclusters possess both theoretical and practical importance in the development of ultrasensitive biosensors based on surface-enhanced Raman spectroscopy (SERS). Manipulation of gold nanoclusters in a predictable and reproducible manner for the application of refined biochemical analysis still remains challenging. In this study, high-purity gold nanoclusters are isolated via a simple method based on density gradient centrifugation. Three distinct bands including monomers, small aggregates (2-4 nanospheres), and large aggregates (>5 nanospheres) can be separated via density gradient centrifugation. The isolated gold nanoclusters greatly enhance the Raman intensity of the trapped dye molecules such that single nanocluster detection is feasible. To develop a gold nanoparticle-based biosensor for influenza virus, effort was also made to modify recognition moieties such as aptamers to gold nanoparticles via distinct approaches. The increase of hydraulic diameter and the shift of optical absorbance spectrum indicate the success of surface modification to gold nanoparticles. / Master of Science