The continuing rise of antibiotic resistance (AR) is an emerging public health problem. This problem is further compounded by a reduction in the development of novel antibiotics to combat these persistent pathogens. This is due to the cost to bring the drug to market and the low likelihood of developing a successful drug. However, one alternative and promising approach is the application of bacteriophage (phage) viruses to selectively infect and kill pathogenic bacteria. These viruses are ubiquitous and innumerable, making them a compelling therapeutic alternative. However, this innumerable nature has a downside for therapy development. Current methods are limited in their throughput, generation of quantitative data regarding phage-host interactions, and are not amenable to multiplexing or parallel screening (i.e. scalability). Because phage therapy is personalized and requires screening collections of phages to identify efficacious phages to formulate into a therapeutic cocktail, new methods are required to address these gaps. Traditional screening methods include plaque and kinetic growth assays, but these assays are not representative of native conditions and are limited in multiplexing and scalability. Phage-layer Interferometry (PLI), engineered multiplex compatible reporter phages (MultiPhlex), and an indirect phage susceptibility assay called Phage-Assisted Droplet Sorting (PhADS) are aimed at addressing these current challenges to the field of phage therapy. PLI was developed to provide a standardized method for generating quantitative data regarding phage-host interactions. The development of MultiPhlex is ongoing but seeks to address the current inability to multiplex phage screening by developing fluorescently barcoded reporter phages with fluorogenic RNA aptamers. PhADS addresses the main short coming of MultiPhlex, not all phages are amenable to engineering, through indirect detection and provides a high throughput massively parallel phage screening sys (open full item for complete abstract)

The full impact of personalized medicine can only be realized through on-demand and convenient access to individual biomarker data both at home and in the clinic. Biosensors that enable on-demand quantification of drugs, hormones, and other clinically relevant analytes have long been promised as the next breakthrough for facilitating precision medicine. While point-of-care and continuous glucose monitors have drastically improved outcomes and quality of life for diabetic patients, there has been virtually no clinical adoption of biosensors for other target analytes beyond glucose. This is unfortunate, as there are numerous other opportunities where the potential for improved patient outcomes through on-demand access to patient-specific biomarker data is well-documented, such as in the precision dosing of narrow therapeutic index drugs (e.g., warfarin, digoxin, cyclosporine), patients requiring postoperative monitoring (e.g., troponin, cystatin C, anticoagulation status), and in tracking longitudinal changes of biomarkers indicative of disease progression (e.g., NT-proBNP, PSA). The technology behind glucose monitoring, i.e., enzymatic biosensing, is unfortunately not generalizable as it is limited to high concentration analytes (~mM) and requires a readily available enzyme that can oxidize/reduce analytes of interest. Beyond enzymatic glucose sensors, electrochemical aptamer sensors comprise the only other biosensing modality that has been broadly validated in vivo. Despite demonstration of continuous sensing for over a dozen different analytes in animal models, no electrochemical aptamer sensor has seen commercial viability due to lack of integration into feasibly deployable devices. Thus, described herein are several key advancements in translating electrochemical aptamer sensors into point-of-care and wearable devices as enabled by the fundamental insights and innovations put forth through this work. Specifically, we demonstrate sensors integrated into feasibly d (open full item for complete abstract)

Advancing diagnostic ability is imperative for improving patient outcomes. Wearable sensors, such as smart watches and continuous glucose monitors, have opened the door to continuous biosensing, which can provide critical information to clinical practitioners at minimal inconvenience to the patient. Electrochemical aptamer based (EAB) sensing can provide rapid and precise quantitative results for a broad range of potential sensing targets, making it a promising option for continuous, minimally invasive sensing. Another benefit to EAB sensors is that they can be easily interchanged on the same platform. Therefore, this project focused on surface modification techniques to improve the sensing platform for all EAB sensors, with specific emphasis on sensor longevity. First, acupuncture needles were used as a base to improve mechanical stability and provide easy insertion. Physical vapor deposition was used to deposit 10 nm of titanium and 50 nm of gold before either gold nanoparticles were electrochemically deposited or a 300 nm gold-silver (1:3) alloy was sputtered onto the surface. The needles with the gold-silver alloy were then etched in nitric acid to remove the silver and reveal a porous gold surface. Scanning electron microscopy was used to examine the resulting nanostructures. Mechanical and chemical roughening were employed to improve the adhesion between the stainless steel and the deposited layers. Cyclic voltammetry (CV) was utilized to examine the effects of these methods on surface adhesion. The second electrode base that was modified was 250 μm diameter gold wires. Previous studies have shown the benefits on surface gain of roughened gold wires. Building upon these advances, the effect of thermal annealing on the chemical stability of roughened gold wire was examined. A frequency sweep in combination with a concentration “titration” was used to compare the signal gain of annealed and unannealed roughened wires and to identify the ideal scanning par (open full item for complete abstract)

Electrochemical aptamer-based (E-AB) sensors are a class of biosensors that employ single-stranded DNA or RNA oligonucleotides as recognition elements. Signal transduction for this class of sensor relies on a conformation change of these aptamers in the presence of a target that alters the collisional frequency of a 3'-redox reporter. The E-AB sensor platform employing the tethered modified oligonucleotide affords dynamic measurements of analytes of interest that can be used for innovative, relevant detection of analytes. As such, electrochemical biosensors that employ oligonucleotides as recognition elements, require integration into other sensing platforms like microfluidics to exploit the dynamic, reagentless measurements afforded by this class of sensors, further optimization of the sensor in changing temperatures, and applications of the sensors in a useful way which will allow them to transcend from the lab to point-of-care (POC) or medical diagnostics. This dissertation describes several ways in which the dynamic measurements of these sensors can be used to help them transcend from the lab to POC or medical diagnostics. The integration of this class of sensors into a microfluidic device using 3D printing to make microfluidic molds affords rapid prototyping of different microfluidic architectures, coupled with epoxy-embedded electrodes that use a three-electrode setup, and fabricating E-AB sensors under flow conditions to exploit the dynamic measurements afforded by E-AB sensors. Additionally, E-AB sensor signaling was characterized at different temperatures to better understand how temperature changes affect sensor response. The sensors were interrogated in the absence of and with target analyte within a temperature window of 1°C to 37°C. The chapter looks into how temperature affects sensor signaling, signal polarity, and binding affinity within the chosen temperature range. Finally, the last half of this dissertation demonstrates the capability of a (open full item for complete abstract)

Electrochemical aptamer-based (E-AB) sensors are a class of biosensors that incorporate single-stranded DNA or RNA oligonucleotides as recognition elements. Signal transduction for this class of sensor relies on a conformation change of these aptamers in the presence of target that alters the collisional frequency of a 3'-redox reporter. As such, electrochemical sensors that employ nucleic acids as recognition elements, require an understanding of the biophysics of the surface-bound nucleic acid to aid in the rational design of sensors and control over the analytical performance. The work in this dissertation aims to understand how nucleic acid structure and flexibility affect the resulting electrochemical signal observed when either change. This dissertation describes, the extensively characterized electrochemical response of short (<60 nucleotides) tethered nucleic acids utilizing cation-condensation induced collapse to predictably control the structure of the nucleic acids on the electrode surface. Moreover, I discuss how the length of the nucleic acids and the packing density on the electrode surface affect signaling upon inducing collapse. By inducing collapse, I observe subtle differences in electrochemical signal that are dictated by the biophysical parameters of the nucleic acids including the axial charge spacing, the periodicity of the helix when duplexed, and the structural motif (e.g., stem-loop, linear, pseudoknot). With the new knowledge of how these various parameters affect electrochemical signaling, I am able to predict responses as well as determine what structures exist on a heterogenous modified electrode surface. Finally, the last half of this dissertation provides a fundamental electrochemical study of our electrode interfaces that employ nucleic acids by utilizing the change in peak splitting via cyclic voltammetry. More specifically, this approach provides a quantifiable way to determine the observed electrochemical regimes (reversible, (open full item for complete abstract)

Microbial antibiotic resistance is a contemporary challenge threatening the health system worldwide. According to World Health Organization, previous cases of bacterial infections - once treatable with antibiotics - can now be lethal due to the uncontrolled misuse of these agents. One of the main triggers of bacterial resistance is the over-expression of multi-drug resistant (MDR) efflux pumps. These pumps allow the bacterium to pump antibiotics out of the cell and therefore desensitizes the cells to the antibacterial inhibitory effect. In this thesis, I evaluated the efflux pump inhibitory activity of eight different plant extracts using the Gram negative bacterium, E. coli. I also described the development of synthetic nucleic acids called aptamers to bind to and block the outer membrane channel of the efflux pump TolC as another effective way to impede antibiotic resistant bacteria from effluxing antibiotics. To generate the DNA aptamers exhibiting a binding specificity to E. coli cells, the method of a whole-cell Systemic evolution of Ligands by Exponential enrichement (SELEX) was applied to a random single-stranded DNA library. The efflux pump inhibiting activity of the plant extracts and the DNA aptamers was evaluated using an in-vivo efflux assay.

The interaction between nucleic acids and small molecule ligands is continuously generating significant interest due to their widespread biological and bioanalytical applications. We investigated the mechanical property of the binding between aptamers and small molecules. Using an ATP binding aptamer as an example, we observed that the mechanical stability of the aptamers that are bound with ATP is higher than those without a ligand. Therefore, a force-based sensor can be developed to detect small molecules using aptamers as a platform. We determined the dissociation constant, Kd, for aptamer-ligand interactions at the single-molecule level by applying a Hess-like cycle. Our experiments allow the Kd determination from only one ligand concentration which was further validated by our capillary electrophoresis (CE) method. By using only one ligand concentration, such a method not only saves time and material, but also is less susceptible to reduced reproducibility due to run-to-run fluctuations. G-quadruplex forming sequences which are wide spread in the genome particularly in telomeres and promoter regions have been shown to be therapeutic targets. G-quadruplex structures have been extensively studied using mostly conventional methods. However, the mechanical stability, thermodynamics, kinetics properties of these interactions at the single-molecule level, remains to be fully understood. While the formation of these G-quadruplex structures is highly dynamic, they are mechanically stabilized upon ligand binding which may affect their biological functions. Small molecules which bind to nucleic acid structures may interfere with vital cellular processes such as transcription and protein translation during cell division by acting as energy barriers or mechanical blockage. Therefore, understanding stability of nucleic structures from a mechanical stability stand point is critical to fully explore their therapeutic potentials. We have investigated the human te (open full item for complete abstract)

The concentration ratio of glycated to non-glycated forms of various blood proteins can be used as a diagnostic measure in diabetes to determine a history of glycemic compliance. Depending on a protein's half-life in blood, compliance can be assessed from a few days to several months in the past, which can be used to provide additional therapeutic guidance. The most commonly studied glycated protein for assessing glycemic compliance is glycated hemoglobin. Current glycated protein concentration detection methods are limited in their ability to measure multiple proteins, and are susceptible to interference from other blood pathologies. In this study, DNA aptamers were developed and characterized for use in Surface Plasmon Resonance (SPR) sensors to assess the percentage of hemoglobin that is glycated in a patient. The aptamers were developed by way of a modified Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process which selects DNA sequences that have a high binding affinity to a specific protein. DNA products resulting from this process were sequenced, and identified aptamers were synthesized. The SELEX process was performed multiple times. One process produced aptamers specific to a glycated form of hemoglobin, another produced aptamers specific to non-glycated hemoglobin and aptamers specific to all forms of hemoglobin. A final SELEX process produced aptamers specific to fibrinogen. Equilibrium dissociation constants for the affinity of the identified aptamers to glycated hemoglobin, hemoglobin, and fibrinogen were calculated from fitting a Langmuir binding model to experimental, real time, binding data obtained through SPR. It was determined that two aptamers, GHA1 and GHA2, were selective to glycated hemoglobin, with equilibrium dissociation constants of 11.5 nM and 51.8 nM, respectively; one aptamer, HA2, was selective to non-glycated hemoglobin, with an equilibrium dissociation constant of 187 nM; and one aptamer was selective to bot (open full item for complete abstract)

Prion diseases are a group of fatal, transmissible neurodegenerative diseases found in both animals and humans. Prion diseases are thought to arise when the normal cellular prion protein, PrPC, undergoes a conformational change from a primarily α-helical form into a form rich in β-sheets and resistant to protease, called PrPSc. Animal forms of prion disease include scrapie in sheep, chronic wasting disease (CWD) in cervids, and bovine spongiform encephalopathy (BSE) in cattle. In humans, the diseases can take a sporadic, genetic or acquired form. One acquired form of human prion disease is variant Creutzfeldt-Jakob disease (vCJD), which emerged in the United Kingdom in 1995, and is thought to be caused by the consumption of BSE contaminated beef. The only definitive diagnostic techniques available at the present time use protease digested brain tissue.This thesis sought to develop a panel of diagnostic reagents for prion disease that enable the detection of PrPSc, but not PrPC, without protease digestion. We characterized two short peptide sequences, based on the Kringle domains of the serine protease plasminogen, for binding to PrPSc, following reports that plasminogen has PrPSc binding ability. The short peptides bound to all forms of PrPSc, both animal and human, and binding was retained in PrPSc-spiked human plasma. In addition, a panel of PrP binding DNA oligonucleotides, called aptamers, isolated through the SELEX technique, was used in a capture assay. The aptamers bound to full length and PK treated PrPSc from hamster scrapie, as well as from sporadic CJD, vCJD, mouse scrapie, sheep scrapie, and white-tailed deer derived CWD. Binding was not observed to PrPSc from mule deer CWD, or from BSE, making these reagents among the first to show species or strain specificity. Strikingly, these aptamers were able to distinguish buffy coat samples derived from scrapie afflicted sheep from those of healthy animals in an electrophoretic mobility shift assay with 96% sens (open full item for complete abstract)