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

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

In an effort to provide wastewater treatment operators with the capability to be proactive in assessing and solving deflocculation events, this study has tested the components of a biosensor to predict deflocculation and investigated the mechanistic cause of deflocculation relating to chlorination of activated sludge cultures. In order to effectively manage upset events, it is necessary to know the source of an upset and the causative mechanism that the source initiates. The Glutathione-gated potassium efflux (GGKE)induced activated sludge deflocculation biosensor incorporates novel microtechnology with a whole cell biological element to predict deflocculation from electrophilic sources. This sensor utilizes microfluidic channels to conduct influent wastewater across a biofilm of Eschericia coli K 12 and monitors the bacterial response to the influent. The bacterial response, which is efflux of K+ ion from the cytoplasm, is monitored with a fluorescence-based sensor called an optode. The components of the system satisfy the project requirements, which include minimal expense (both operation and manufacture), on-line capability and minimal maintenance. The research conducted to date demonstrates the ability of the components of the biosensor to fulfill the design requirements. The optode K+ detector successfully measured an increase in soluble K+ following the exposure of E. coli K-12 to the electrophile N ethyl malemide. The manufacture of the microfluidic device has been completed and the device has demonstrated the ability to conduct influent under negative pressure across an established biofilm with the optode in place. The establishment of a biofilm under expected hydrodynamic conditions has also been completed. Future research efforts will include integrating the components of the biosensor into a working prototype that will be capable monitoring the reaction of bacteria to the presence of electrophilic compounds in wastewater. Sensors of this nature will provide operators with the early warning necessary to be proactive against toxic upsets rather than reactive. The knowledge needed to create a biosensor resides in the identification of bacterial response mechanisms that cause upset events in wastewater treatment facilities. The biosensor that has been developed relies on the discovery of the link between electrophile-induced GGKE and activated sludge deflocculation. Research has been concluded, which expands the role of GGKE and activated sludge deflocculation to include chlorine-induced GGKE. Through a series of laboratory-scale reactors, a relationship has been established between chlorine addition to control filamentous bulking, increased soluble K+ levels and an increase in effluent suspended solids . The results demonstrate that the addition of chlorine to control filamentous bulking may elicit the GGKE mechanism, initiating activated sludge deflocculation, similar to observations of chlorination at full-scale activated sludge wastewater treatment facilities. Establishing a mechanistic cause of deflocculation related to chlorination will permit operators to apply chlorine in a manner that may avoid deflocculation, rather than reacting to deflocculation after it has occurred. / Master of Science

This research presents a novel biosensor which utilizes the refractive index sensitivity of a fiber optic long-period grating. The long period grating couples light from the forward propagating guided core mode of a single-mode fiber into discrete circularly symmetric cladding modes. Due to imperfections in the cladding surface, loss bands are seen in the transmission spectrum corresponding to the coupled wavelengths. Based on the phase-matching condition between the coupling and coupled modes, the loss bands shift with changes in the refractive index of the surrounding medium. The grating surface is chemically treated to covalently bond antibody to the cladding of the sensor. Treatment with the proper antigen increases the effective index seen by the cladding modes and affects the placement of the loss bands. This sensor demonstrates specific antigen binding capacity with loss band shifts of 10 nm or more. The device offers several advantages over the widely used Enzyme-Linked Immuno-Sorbent Assays. Diagnostic applications can be expanded beyond the tests presented here. / Master of Science

La microbalanza de cristal de cuarzo (QCM) se usa como técnica alternativa de análisis químico, donde las aplicaciones dependen directamente de la sensibilidad del cristal. Por tanto, es el parámetro más importante que determina el uso de los cristales de cuarzo frente a otras técnicas. La ecuación de Sauerbrey, teóricamente relaciona la variación de la densidad de masa en la superficie del cristal con el cambio de la frecuencia y al mismo tiempo predice que la sensibilidad aumenta en la misma proporción que el cuadrado de la frecuencia fundamental de resonancia serie del cristal. Aumentar la frecuencia fundamental de trabajo para aumentar la sensibilidad, es una necesidad; el objetivo principal de esta tesis doctoral es estudiar los sistemas de caracterización de resonadores de cuarzo, que se usan en la actualidad y proponer un sistema o una mejora a los ya existentes que permitan el uso de cristales de cuarzo de alta frecuencia de fundamental (HFF-QCM). De los sistemas que en la actualidad caracterizan a la microbalanza de cuarzo, se destacan los analizadores de impedancia y los osciladores, como métodos de caracterización de HFF-QCM. Los analizadores, por su gran tamaño y coste, quedan relegados a su uso como instrumento de referencia en el laboratorio. Los osciladores en cambio, por su bajo coste, su capacidad de integración, su sencillez del circuito y la monitorización continua de frecuencia de oscilación, lo hacen adecuados como interfaz para sistemas sensores basados en QCM. En esta tesis, se propone un sistema oscilador, basado en un oscilador diferencial equilibrado, como sistema de caracterización de cristales a 10 MHz, estudiándose el comportamiento del circuito, la capacidad de este sistema para compensar la capacitancia paralela del cristal, las aplicación de este tipo de interfaz para la caracterización de líquidos y finalmente se desarrollo una aplicación como inmunosensores piezoeléctricos para la detección del pesticida Carbaryl. / Montagut Ferizzola, YJ. (2011). Sistema Oscilador Mejorado para Aplicaciones de Microbalanza (QCM) en Medios Líquidos y Propuesta de un Nuevo Método de Caracterización para Biosensores Piezoeléctricos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/9688