<p>Research in paper-based analytical devices has been increasing in recent years. Before technology transfer and market acceptance, these paper-based sensors have to be validated with field samples. In this study, we have made an attempt to evaluate the effectiveness of paper-based sensors to detect pesticides in real world samples. Generation 1 biosensor has been modified to be user friendly. There is no difference in the performance of generation 2 sensors; they detect pesticides based on colorimetric assay. The assay protocol involves first introducing the sample to the sensing zone by pipetting the water sample. Following an incubation period of 15min, the substrate end of the sensor is dipped into the sample to move paper bound indoxyl acetate (IDA) to the sensing region to initiate the enzyme-catalyzed hydrolysis of the substrate, resulting in the development of blue color. The presence of pesticide is indicated by either a decrease in color intensity or with no color development at all.</p> <p>To evaluate the effectiveness of biosensor in detecting pesticides in real world samples, a field study was conducted in four villages of southern India. Water samples from different aquatic environment including both surface water and ground water, were tested using generation 2 paper-based sensors. The paper-based sensors were capable of detecting organophosphorus pesticides in real world samples. The results were confirmed using GC-MS.</p> <p>The presence of higher concentration of dibutyl phthalate (in the range of 100uM to 10mM) in water can be a potential interference for the paper-based assay for the detection of pesticides in water. The paper-based biosensor assay platform can detect pesticides in the environmental samples and results have been validated by GC-MS. But for transfer of technology to the industry, further optimization is required to improve the stability of substrate to withstand atmospheric temperature fluctuations thus allowing the storage and shipment of the biosensor strips. Additionally to conduct reliable assays and obtain consistent results, the fabrication of biosensor strips needs to be improved to maintain the consistent volumes of bioinks impregnated on paper support.</p> / Master of Applied Science (MASc)

Lateral flow biosensors are analytical devices that detect biomaterials with physicochemical signals, such as optical signals. Unlike other biosensors, lateral flow biosensors are based on porous membranes, which use capillary force to transport biomaterials spontaneously. However, lateral flow biosensors are fabricated in batch mode, which means that membranes need to be cut from the rolls, pretreated, and assembled using a step-by-step process. Thus, there is a need for a more efficient manufacturing process. This thesis aims to accelerate the fabrication process by developing a method wherein the whole device is printed directly, including the printable substrates, as well as by developing a clog-free process for depositing expensive reagents. These novel printable porous media were developed using printing inks that contained various pigments and polymer binders. To this end, candidate formulations were screened from nine hundred inks formulations via wicking experiments. The results of these tests showed that the most promising formulations were based on calcium carbonates and latex polymers. This formulation was then used to develop printable porous media that can easily be printed into complex patterns, with changeable wicking speeds within each pattern. In addition, a bio- colorimetric assay of alkaline phosphates conducted on these porous media showed strong color signals that were comparable to the traditional membrane-based lateral flow strips. Clog-free printing processes were investigated by using a piezoelectric inkjet printer to print silica sols and six nanoparticle inks. The results of these tests showed that the vibration of the piezoelectric layer and the deposition of particles on the printhead surfaces induced clogging issues. Over time, the silica sols formed multilayer deposits on the print head surface, which subsequently detached due to the vibration of the piezoelectric layer. Consequently, these large sheets of silica clogged the nozzles during printing. This clogging issue was eliminated by adjusting the pH value of the silica sol inks to 3.1. The hydrophobic cationic polystyrene nanoparticles form a sub-monolayer on the printhead surface, which causes air entrainment and promotes air bubble adhesion into the interior of the print head surface when the piezoelectric layer deforms. Thus, alternate surface chemistries for the print head and ink particle surfaces may be required in order to print hydrophobic ink materials. Overall, this enhanced understanding of these clogging mechanisms helps to explain why printer performance varies when different particles are used. / Thesis / Doctor of Philosophy (PhD) / Many devices in our day-to-day lives incorporate lateral flow biosensors, for example, home pregnancy test kits. These tests allow users to obtain results within 30 minutes by simply applying a few droplets of urine onto a test strip. However, these biosensors are largely manufactured using manual processes: workers cut strips (also called substrates) from sheets, deposit reagents onto the strips, and then assemble the pretreated strips into devices. As such, these processes are time consuming and less productive. To accelerate the manufacturing process, we developed printable porous substrates and a clog-free printing process for depositing expensive reagents onto the substrates. Novel porous media can be flexibly printed into complex patterns using pigment- based inks. Moreover, the use of different pigments within the designed patterns enables these porous media to control wicking velocity. In addition to printable porous substrates, the research in this thesis shows that the manufacturing process can be improved by using piezoelectric inkjet printers. The use of these printers not only allows the expensive reagents to be precisely deposited onto the substrates, but it also offers a more cost-effective method of doing so. Finally, in order to ensure the printing process remained clog-free, we systematically investigated clogging mechanisms by printing with different polymers and nanoparticles.

Advances in our ability to detect biological targets relevant to human health have come from the engineering of biological molecules into assemblies capable of performing target-induced signal generation. Such assemblies, known as biosensors, are composed of a molecular recognition element (MRE) and a signal generating transduction element. One MRE class that has received great attention in recent years is functional nucleic acids, which include DNA aptamers and DNAzymes. Since 1990, a large number of functional nucleic acids have been reported. However, broad commercial use of functional nucleic acids in applications that benefit human health is sparse. The goal of this thesis is to expand the usefulness of functional nucleic acids. The thesis is made of four projects. In the first project I developed a simple colorimetric biosensor for the detection of a toxic metal ion using a reported RNA-cleaving DNAzyme coupled with urease as the signal reporter. This is followed by a project where I developed a highly effective method for the synthesis and purification of the DNA-urease conjugate needed for the biosensor. I then turned my attention to the search for high-affinity DNA aptamers that bind VEGF-165, an important human protein found to be relevant in the progression of cancers. Given that VEGF-165 is a homodimeric protein, in my third project I looked into the suitability of reported DNA aptamers for this protein for the creation of dimeric aptamers with higher binding affinity. I examined multiple factors that may affect the successful engineering of dimeric aptamers and determined that none of the existing aptamers are compatible for creating a productive dimeric aptamer. With this finding, I made an effort to create our own aptamers for this protein target. I was able to isolate a new aptamer that appears to be an excellent candidate for creating a higher affinity DNA aptamer. Overall, my work adds to our increasing appreciation of the functional capability demonstrated by single-stranded DNA molecules. More importantly, I hope the methods I have developed and new functional DNA molecules I have generated in this thesis will continue to drive the development of the functional nucleic acid field and contribute to the health research community’s efforts to increase human longevity. / Thesis / Doctor of Philosophy (PhD)

The need for a non-invasive, accurate, easy-to-use, and cost-effective colorectal cancer (CRC) detection device is apparent in the low survival rates seen in late-stage diagnoses. Once CRC has progressed past stage I, the 5-year survival rate drops significantly, and treatment options become less favourable. The best way to treat CRC is to catch it early. The development of an RNA-cleaving fluorogenic DNAzyme (RFD) holds the potential to remediate this deficiency. A DNAzyme, called RFD-FN1, was identified from a synthetic random-sequence DNA library to selectively bind to an unknown target associated with Fusobacterium nucleatum, which has been found to be overabundant in pre- and cancerous colorectal tissue and stool. Target recognition by the DNAzyme induces the cleavage of a fluorogenic substrate and generates a fluorescent signal to indicate the presence of the bacterium. This thesis outlines the efforts made towards functionalizing the F. nucleatum-responsive probe in stool samples to create a non-invasive screening test. RFD-FN1 is selective towards a heat-stable F. nucleatum protein, but its limit of detection is only 10^7 CFU/mL. Although able to detect spiked concentrations of F. nucleatum cells in processed stool samples, the use of heat, filtering, centrifugation, antibiotics, culturing or serial dilutions are not sufficient to detect the F. nucleatum that is naturally present in the diseased samples. Experiments designed to enrich the target concentration revealed that the target is not produced consistently in any growing condition tested. Size exclusion chromatography and mass spectrometry analysis identified five potential targets that RFD-FN1 may be responding to. Three candidate targets were cloned and purified, but they failed to induce RFD-FN1’s activity. Due to the COVID-19 pandemic, the purification of the final two proteins was not completed. Purifying the two candidate targets and testing their ability to induce RFD-FN1 represents future research efforts. If the target for the DNAzyme is confirmed, a reselection for a more sensitive DNAzyme, that can function in human stool, can be attempted. / Thesis / Master of Health Sciences (MSc)

Oxidative stress-inducing chemical contamination in the environment is a significant concern for public health. The depletion of antioxidants by these chemicals results in oxidative stress which may cause detrimental effects in many cell types. For example, multiple stress responses may be activated in bacteria and several disorders including neurodegenerative disorders may occur in mammalian organisms. Oxidative chemicals also have negative effects on engineered water systems as an oxidative stress response in bacteria has been implicated to cause process failure in wastewater treatment facilities. Therefore, it is essential to monitor oxidative chemical contamination in water environments to provide early warning of potential negative effects. Whole-cell biosensors that indicate bacterial stress responses to oxidative toxic agents can be powerful tools in environmental monitoring. An oxidative stress response found in many Gram-negative heterotrophic bacteria called the glutathione-gated potassium efflux (GGKE) mechanism is a good biological indicator to be used in a biosensor designed to detect the presence of oxidative chemicals in water. The authors of this study propose the development of a GGKE biosensor using an environmental strain of Pseudomonas aeruginosa. The abundance of the global antioxidant glutathione, the gating compound in GGKE, in various cell types suggests that there may be connections between the responses of the different cell types to oxidative stress. In this study, specific oxidative stress responses in two distantly related cell types were studied: the GGKE mechanism in Gram-negative heterotrophic bacteria, and mitochondrial dysfunction in rat brain cells. Furthermore, the use of an octanol-based emulsification method for the immobilization of P. aeruginosa in calcium alginate microbeads was evaluated for long-term mechanical stability, viability, and GGKE response of the immobilized cells. The immobilization of cells is an important factor in the design of a whole-cell biosensor, and must yield viable and active cells over time. This study showed that the dose-dependent responses of GGKE in Pseudomonas aeruginosa cells and of mitochondrial dysfunction in a mixed culture of rat brain cells to a model oxidative electrophilic chemical, N-ethylmaleimide, correspond well to each other. We also showed that both responses are accompanied by the depletion of intracellular glutathione, which precedes the GGKE response in P. aeruginosa as well as mitochondrial damage in rat brain cells. Thus, this study suggests that bacterial responses to oxidative stress involving glutathione, such as GGKE, could potentially be used as an early warning to predict the presence of bioavailable oxidative chemicals that can induce oxidative stress in eukaryotic systems. Although further research is needed, this suggests that bacterial stress response biosensors may be used to predict oxidative stress responses in mammalian brain cells. The octanol-based emulsification method produced P. aeruginosa encapsulated alginate microbeads with an average diameter of 200 μm. The microbeads were mechanically stable in solutions containing up to 20 mg/L K+ for 15 days. LIVE/DEAD® and specific oxygen uptake rate (SOUR) analyses showed that the microbead-immobilized cells recovered their membrane integrity within 5 days but not their net respiration potential. The microbead immobilized cells had no net GGKE potential in response to 50 mg/L N-ethylmaleimide after 14 days whereas water-based alginate bead (2mm) immobilized cells did, albeit at a reduced level to planktonic cells. Confirmation experiments revealed that octanol impeded cellular activities of the immobilized cells. Overall, this study showed that the octanol-based emulsification method is not suitable for the immobilization of P. aeruginosa for use in the GGKE biosensor and other microscale immobilization methods should be evaluated. / Master of Science

Additive manufacturing in the form of 3D printing is a revolutionary technology that has developed within the last two decades. Its ability to print an object with accurate features down to the micro scale have made its use in medical devices and research feasible. A range of life-saving technologies can now go from the laboratory and into field with the application of 3D-printing. This technology can be applied to medical diagnosis of patients in at-risk populations. Living biosensors are limited by being Genetically Modified Organisms (GMOs) from being employed for medical diagnosis. However, by containing them within a 3D-printed enclosure, these technologies can serve as a vehicle to translate life-saving diagnosis technologies from the laboratory and into the field where the lower cost would allow more people to benefit from inexpensive diagnosis. Also, the GMO biosensors would be contained with a press-fit, ensuring that the living biosensors are unable to escape into the environment without user input. In addition, 3D-printing can also be applied to reduce the cost of lab-based technologies. Cell patterning technology is a target of interest for applying more cost-effective technologies, as elucidation of the variables defining cell patterning and motility may help explain the mechanics of cancer and other diseases. Through the use of a 3D-printed stamp, bacterial cells can be patterning without the use of a clean room, thus lowering the entry-barrier for researchers to explore cell patterning. With the commercialization of 3D-printing an opportunity has arisen to transition life-saving technologies into more cost-effective versions of existing technologies. This would not only allow more research into existing fields, but also to ensure that potentially life-saving technologies reach the people that need them. / Ph. D.

Biosensors are devices that use biological components to detect important analytes. Biosensing systems have various applications in areas such as medicine, environmental monitoring, and process control. Classical biosensors are often based on bacteria or purified enzymes that have limitations on efficiency or stability. I have developed several new biosensors to overcome these disadvantages. Two preliminary biosensors were first created based on the extremely strong and specific interaction between biotin and (strept)avidin. Both biosensors showed high sensitivity and reliability for measuring biotin with detection limits of 50-1000 pg/ml and 20-100 ng/ml, respectively. Following these, a new biosensor was developed by coupling a mobile, functionalized microsurface with cell-free expression approaches. This biosensor demonstrated a dynamic range of 1- 100 ng/ml. In addition, I also explored the possibility of combining these biosensing systems with engineered living cells. By leveraging the tools of synthetic biology, a genetic circuit was designed, constructed, and inserted into bacteria for enhanced biotin biosynthesis in vivo. Upon induction, a 17-fold increase in biotin production was measured in the engineered cells in comparison to wild type cells using the biosensors created herein. These new biosensors, particularly the mobile biosensing modality, form a building block for advanced biosensing and drug delivery systems due to enhancements in mobility and specificity. In the future, these biosensing and cellular production systems could impact a range of fields ranging from biomedicine to environmental monitoring. / Master of Science

To improve the process stability of wastewater treatment plants, the construction of a whole-cell bacterial biosensor is explored to harness the natural stress response of the bacterial cells. The stress response selected in this work is the glutathione-gated potassium efflux (GGKE) system, which responds to electrophilic stress by effluxing potassium from the interior to the exterior of the cell. Thus, the bulk potassium in solution can be monitored as an indicator of bacterial stress. By utilizing this stress response in a biosensor, the efflux of potassium can be correlated to the stress response of the immobilized culture, providing an early warning system for electrophilic shock. This type of shock is a causative factor in many process upset events in wastewater treatment plants, so the application of the sensor would be an early warning device for such plants. The research conducted here focused on the biological element of the biosensor under development. Three immobilization matrices were explored to determine the cell viability and potassium efflux potential from immobilized cells: a calcium alginate, a photopolymer, and a thermally reversible gel. The calcium alginate was unstable, and dissolved after five days, such that the long-term impact of immobilization on the cells could not be determined in the matrix. The photopolymer resulted in very low actvity and viability of immobilized cellsOf the three matrices tested, indicating that the composition of the polymer was toxic to the cells. Of the matrices tested, the thermally-reversible gel showed the best response for further study, in that the matrix did not inhibit cell activity or potassium efflux. / Master of Science

Ionic electroactive polymers have been developed as mechanical sensors or actuators, taking advantage of the electromechanical coupling of the materials. This research attempts to take advantage of the chemomechanical and chemoelectrical coupling by characterizing the transient response as the polymer undergoes an ion exchange, thus using the polymer for ionic sensing. Nafion™ is a biocompatible material, and an implantable polymeric ion sensor which has applications in the biomedical field for bone healing research. An ion sensor and a strain gauge could determine the effects of motion allowed at the fracture site, thus improving rehabilitation procedures for bone fractures. The charge sensitivity of the material and the capacitance of the material were analyzed to determine the transient response. Both measures indicate a change when immersed in ionic salt solutions. It is demonstrated that measuring the capacitance is the best indicator of an ion exchange. Relative to a flat response in deionized water (±2%), the capacitance of the polymer exhibits an exponential decay of ~25% of its peak when placed in a salt solution. A linear correlation between the time constant of the decay and the ionic size of the exchanging ion was developed that could reasonably predict a diffusing ion. Tests using an energy dispersive spectrometer (EDS) indicate that 90% of the exchange occurs in the first 20 minutes, shown by both capacitance decay and an atomic level scan. The diffusion rate time constant was found to within 0.3% of the capacitance time constant, confirming the ability of capacitance to measure ion exchange. / Master of Science

DNA detection technology has developed rapidly due to its extensive application in clinical diagnostics, bioengineering, environmental monitoring, and food science areas. Currently developed methods such as surface Plasmon resonance (SPR) methods, fluorescent dye labeled methods and electrochemical methods, usually have the problems of bulky size, high equipment cost and time-consuming algorithms, so limiting their application for in vivo detection. In this work, an intrinsic Fabry-Perot interferometric (IFPI) based DNA sensor is presented with the intrinsic advantages of small size, low cost and corrosion-tolerance. This sensor has experimentally demonstrated its high sensitivity and selectivity. In theory, DNA detection is realized by interrogating the sensor's optical cavity length variation resulting from hybridization event. First, a microgap structure based IFPI sensor is fabricated with simple etching and splicing technology. Subsequently, considering the sugar phosphate backbone of DNA, layer-by-layer electrostatic self-assembly technique is adopted to attach the single strand capture DNA to the sensor endface. When the target DNA strand binds to the single-stranded DNA successfully, the optical cavity length of sensor will be increased. Finally, by demodulating the sensor spectrum, DNA hybridization event can be judged qualitatively. This sensor can realize DNA detection without attached label, which save the experiment expense and time. Also the hybridization detection is finished within a few minutes. This quick response feature makes it more attractive in diagnose application. Since the sensitivity and specificity are the most widely used statistics to describe a diagnostic test, so these characteristics are used to evaluate this biosensor. Experimental results demonstrate that this sensor has a sensitivity of 6nmol/ml and can identify a 2 bp mismatch. Since this sensor is optical fiber based, it has robust structure and small size ( 125μm ). If extra etching process is applied to the sensor, the size can be further reduced. This promises the sensor potential application of in-cell detection. Further investigation can be focused on the nanofabrication of this DNA sensor, and this is very meaningful topic not only for diagnostic test but also in many other applications such as food industry, environment monitoring. / Master of Science