The development of rapid detection methods for bacterial contamination in the industrial agriculture sector is vital for improving food safety and public health. Furthermore, it is important to tailor these rapid methods for low-resource settings, because the majority of foodborne illness outbreaks occur in developing nations. Currently, the most widely used methods rely on nucleic acid testing using the polymerase chain reaction (PCR). This detection reaction provides repeatable results, is highly sensitive, and is highly specific, as it can detect a single strain within a species. However, PCR is reliant on proper sample pre-treatment to remove inhibitory contaminants which can affect downstream results, which leads to a trade-off between detection time and sensitivity of results. Rolling circle amplification (RCA) is another potential detection reaction which has the same advantages and is also better suited to low-resource settings, as it works at room temperature. This thesis reports on the development of a rapid sample preparation method that can be seamlessly integrated into simple PCR and is also well suited to low-resource settings due to the low cost and high availability of the required reagents. A modification of the hot sodium hydroxide plus tris (HotSHOT) lysis reaction was implemented to extract genomic DNA (gDNA), which was then captured onto cellulose filter paper, allowing for multiple samples to be simultaneously processed in under 30 minutes. This pre-treatment can even recover gDNA for detection from samples that would have caused complete inhibition of PCR. The calculated limit of detection (LoD) of extraction followed by simple PCR was similar to that of government-approved commercial kits, without needing a lengthy bacterial enrichment step. Improvements are needed to make this a truly quantitative detection system. Finally, our paper-based pre-treatment was integrated into an RCA reaction to detect at least 105 cells, which provides proof-of-concept for combining paper-based sample preparation with isothermal amplification of target nucleic acids. / Thesis / Master of Applied Science (MASc)

Semiconductor nanocrystals, otherwise known as Quantum dots (Q dots), are a new type of fluorophore that demonstrates many advantages over conventional organic fluorophores. These advantages offer the opportunity to improve known immunofluorescent methods and immunofluorescent biosensors for rapid and portable bacterial detection in water. The detection of the micro organism Escherichia coli O157:H7 by attenuation of a fluorophore’s signal in water was evaluated alone and in the presence of another bacterial species. A sandwich immunoassay with antibodies adhered to SU-8 as a conventional comparison to our novel attenuation detection was also conducted. The assays were tested for concentration determination as well as investigation for cross reactivity and interference from other bacteria and from partial target cells. In order to immobilize the capture antibodies on SU-8, an existing immobilization self-assembly monolayer (SAM) for glass was modified. The SAM was composed of a silane ((3-Mercaptopropyl) trimethoxysilane (MTS)) and hetero-bifunctional cross linker (N-γ-maleimidobutyryloxy succinimide ester (GMBS)) was utilized in this procedure. The SU-8 surface was activated using various acids baths and oxygenated plasma, and it was determined that the oxygenated plasma yielded the best surface attachment of antibodies. The use of direct fluorophore signal attenuation for detection of the target E. coli resulted in the lowest detectable population of 1x10¹ cfu/mL. It was not specific enough for quantitative assessment of target concentration, but could accurately differentiate between targeted and non-targeted species. The sandwich immunofluorescent detection on SU-8 attained the lowest detectable population of 1x10⁴ cfu/ml. The presence of Klebsiella pneumoniae in solution caused some interference with detection either from cross reactivity or binding site blocking. Partial target cells also caused interference with the detection of the target species, mainly by blocking binding sites so that differences in concentration were not discernable. The signal attenuation not only had a better lowest detectable population but also had less measurement error than the sandwich immunoassay on SU-8 which suffered from non-uniformed surface coverage by the antibodies.

The functionality of nucleic acids beyond genetics has attracted more attention over the past decades. Functional nucleic acids (FNA), including aptamers and nucleic acid-based enzymes, are well-known for their target binding and reaction catalysis abilities. FNA can be obtained through a technology called in vitro selection, which allows the isolation of customized FNA for various applications. In particular, FNA have received much interest in biosensing application. Their wide range of sensing targets, intrinsic stability, and high specificity have qualified them as the molecular recognition element in biosensors. This thesis explored the utilization of FNA to tackle real-world biosensing challenges, especially for pathogenic bacteria detection. The first project aimed to make the most use of in vitro selection to derive FNA that can meet the requirements of terminal applications. A few feasible approaches were proposed based on lessons from Mother Nature and validated by innovative scientist pioneers. In the second research project, I characterized an RNA-cleaving DNAzyme for Clostridium difficile infection diagnosis. This DNAzyme displayed high sensitivity and specificity for clinical C. difficile strains, making it a competitive candidate for a potential point-of-care diagnostic tool. In the next chapter, I incorporated a Legionella pneumophila-responsive RNA-cleaving DNAzyme into a bead-based assay for practical on-site detection. This assay exhibited a high stability and functionality in the cooling tower water samples, the real-world application environment. The following chapter was to optimize this assay further with a coupled rolling circle amplification strategy. This additional amplification speeded up the detection process, improved the limit of detection, and enabled the colorimetric results that are observable to the naked eye. These research aimed to advance the practical applications of FNA as key components in biosensors. I hope readers find this thesis insightful and inspirational for the development of the FNA field. / Thesis / Doctor of Philosophy (PhD)

Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide, accounting for over 600,000 deaths annually. The mortality rate of CRC can be significantly reduced if it is detected early, suggesting the importance of cancer screening in CRC management. Currently, colonoscopy is the gold standard for CRC diagnosis as it is accurate and reliable. However, it is an invasive procedure that is associated with risks of complications, which contributes to the lack of patient compliance in colonoscopy screening. Other noninvasive detection methods suffer from poor sensitivity and specificity. Thus, there remains a great demand for the development of a noninvasive and accurate test for CRC diagnosis. Recently, studies using next-generation sequencing techniques have revealed compositional changes in the intestinal microbiome associated with CRC, implicating the possibility of using fecal microbiome as potential diagnostic markers. Specifically, the level of the gram-negative bacterium, Fusobacterium nucleatum, has been shown to be elevated in CRC patients compared to healthy controls. The work described in this thesis aims to develop unique RNA-cleaving DNAzymes that can distinguish between healthy and CRC stool microbiomes. RNA-cleaving DNAzymes are single stranded DNA molecules that are extensively used as analytical tools for metal ion sensing and bacterial detection. We conducted an in vitro selection experiment and isolated a F. nucleatum-responsive RNA-cleaving DNAzyme sensor, named RFD-FN1, that is activated by a heat stable protein marker by this bacterium. RFD-FN1 is highly specific for F. nucleatum and it has a limit of detection of 107 CFU/mL without culture and a single cell when cultured for 36 hours. The discovery of this novel molecular probe for F. nucleatum presents an important step forward towards the development of a novel DNAzyme-based detection method for colorectal cancer. / Thesis / Master of Science (MSc)

With approximately 24,500 preterm children born annually in Canada and an estimated shortage of 6 million ounces of breast milk, the distribution of donor milk must be time-sensitive yet safe to efficiently meet this demand. Donor human milk banks take the greatest precautions to protect their users, but some of these microorganisms manage to circumvent the employed methods. The consumption of contaminated donor milk has the potential to be fatal particularly to the vulnerable, immunocompromised premature infants. The tools used by milk banks to ensure safe distribution rely heavily on the culture plate. It has been the gold standard in screening for microbiological specimens due to its wide availability, low cost, and simplicity. However, the procedural times for bacterial culture plates are tedious and long, lasting a minimum of 48 hours. Advances in microfluidics, particularly in combination with the concept of monitoring metabolites to indicate bacterial viability, hold much promise to significantly reducing the long processing times of culture plates. Combining the concept of compartmentalized culture and a chromogenic optical dye for the detection of metabolic changes as a diagnostic sensor would simplify the identification and quantification of microbial presence. The updated Rapid Culture Detection system is a nanowell device fabricated using polydimethylsiloxane (PDMS) that uses the oxygen-sensitive redox indicator Methylene Blue to determine the presence of bacteria. Preliminary studies have shown to detect bacteria in as little as 3.33 hours using these nanowells compared to the 24 hours required for microwell liquid culture (620%). Initial studies have also been conducted with human milk, indicating a slower detection than in LB media. The novel easy-to-use and low-cost Rapid Culture Detection system is a promising alternative detection tool for protecting infants from pathogenic illnesses caused by contaminated human milk and shortening the time required to access lifesaving nutrition. / Thesis / Master of Applied Science (MASc)

La contamination bactérienne des concentrés plaquettaires (CPs) représente le risque le plus fréquent d'infection transmise par transfusion. Malgré les progrès techniques accomplis dans le dépistage des agents infectieux, le risque de transmission de ces agents par transfusion ne peut encore être considéré comme totalement maîtrisé. Nous avons pu développer une méthode de détection par la technique de MALDI-TOF. Notre protocole permet une détection dans un délai de 8 heures à de faibles concentrations bactériennes.Nous avons évalué les performances d’un automate d’hémoculture, le VersaTREK, par rapport au Bactec, qui est fréquemment utilisé par les centres de transfusion. Nos résultats démontrent son intérêt dans la détection de la contamination bactérienne des CPs. Afin de caractériser l’origine de la contamination bactérienne, nous avons comparé, dans une étude rétrospective, les espèces bactériennes identifiées dans les hémocultures des patients de l’hôpital de la Timone, à celles identifiées dans des poches de CPs contaminés. Les espèces responsables de la contamination des CPs sont essentiellement issues de la flore cutanée. Elles sont donc différentes des espèces responsables d’épisodes de bactériémies.Ainsi, ce travail propose de nouvelles méthodes de détection de la contamination bactérienne. La mise en œuvre de ces nouvelles stratégies de diagnostic couplées à une meilleure connaissance de la prévalence et de l’origine de la contamination bactérienne des CPs pourrait permettre d’améliorer davantage la prévention et la réduction de la morbi-mortalité induite par une transfusion de CPs. / Bacterial contamination of platelet concentrates (PCs) is the most common risk of transfusion-transmitted infection. Despite better control of the elements of the technical progress achieved in the detection of infectious agents, the risk of transmission of these agents by transfusion cannot yet be considered as totally mastered. We have developed a method for detecting PCs contamination by MALDI-TOF. To the best of our knowledge, this is the first time that MALDI-TOF is used in the detection of microbial contamination in a complex medium such as PCs. Our protocol allows detection within 8 hours at low bacterial concentrations. In a second step, we evaluated the performance of a blood culture machine, VersaTREK®, compared to Bactec®, frequently used by transfusion centers. Our results demonstrate its interest in the detection of bacterial contamination of PCs. In order to characterize the origin of bacterial contamination of PCs, we compared in a retrospective study, bacterial species identified in patients' blood cultures treated at the Timone hospital, with those identified in infected PC units. The species responsible for the contamination of the PCs are mainly resulting from the cutaneous flora and are therefore different from the species responsible for episodes of bacteremia.Thus, this work proposes new methods for detecting bacterial contamination. The implementation of these new diagnostic strategies coupled with a better knowledge of the prevalence and origin of bacterial contamination of PCs could further improve the prevention and reduction of both morbidity and mortality induced by blood transfusion of PCs.

The food safety and human diagnostics markets are in need of faster working, reliable, sensitive, specific, low cost bioassays and biosensors for bacterial detection. This thesis reports the use of P22 bacteriophage tailspike proteins (TSP) immobilized on silanized silicon surfaces, roughened at a nano-scale, for specific capture and detection of Salmonella. Towards developing TSP biosensors, TSP immobilization characteristics were studied, and methods to improve bacterial capture were explored. Atomic force microscopy was used to count TSP immobilized on gold thin-films. Surface density counts are dependent on the immobilization scheme used. TSP immobilized on flat silicon (Si), silanized with 3-aminopropyltriethoxysilane and activated with glutaraldehyde, showed half the bacterial capture of gold thin-films. To improve bacterial capture, roughened mountain-shaped ridge-covered silicon (MSRCS) surfaces were coated with TSP and tested. Measurements of their bacterial surface density show that such MSRCS surfaces can produce bacterial capture close to or better than TSP-coated gold thin-films. / Biomedical Engineering

Genome-based detection methods provide the most conclusive means for establishing the presence of microbial species. A prime example of their use is in the detection of bacterial species, many of which are naturally vital or dangerous to human health, or can be genetically engineered to be so. However, current genomic detection methods are cost-prohibitive and inevitably use unique sensors that are specific to each species to be detected. In this thesis we advocate the use of combinatorial and non-specific identifiers for detection, made possible by exploiting the sparsity inherent in the species detection problem in a clinical or environmental sample. By modifying the sensor design process, we have developed new molecular biology tools with advantages that were not possible in their previous incarnations. Chief among these advantages are a universal species detection platform, the ability to discover unknown species, and the elimination of PCR, an expensive and laborious amplification step prerequisite in every molecular biology detection technique. Finally, we introduce a sparsity-based model for analyzing the millions of raw sequencing reads generated during whole genome sequencing for species detection, and achieve significant reductions in computational speed and high accuracy.

No description available.

Urine culture, the current gold standard for urinary tract infection (UTI) diagnosis, does not produce results in an acceptable length of time. An ultra-sensitive, cost-effective electrochemical biosensing platform with nanostructured microelectrodes was designed to address the need for a rapid, point-of-care (PoC) test that could achieve a sample-to-answer time in less than an hour. Printed circuit boards and metallized glass slides were processed using various techniques and then tested for their ability to form nanostructured microelectrodes. Peptide nucleic acid probes for the bacteria and yeast as well as ten probes for antibiotic resistance genes were designed and synthesized for use with the new platform. Validation of the sensor's specificity was performed using high concentrations (100nM) of synthetic DNA oligomers. Furthermore, a clinically relevant sensitivity of 103 cfu/mL was demonstrated by detecting 4 pathogen lysates (Staphylococcus saprophyticus, Pseudomonas aeruginosa, Enterococcus faecalis and Klebsiella pneumoniae) in a buffered solution.