Theses and Dissertations

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Open Access
Advancing point-of-need bacteria detection using microfluidic paper-based analytical devices
(Colorado State University. Libraries, 2018) Boehle, Katherine Elizabeth, author; Henry, Charles S., advisor; Krummel, Amber T., committee member; Geiss, Brian J., committee member; Ackerson, Christopher J., committee member
Bacteria are responsible for more hospitalizations and deaths than any other foodborne contaminant, making the detection of these pathogens of utmost importance. To further complicate bacteria detection, the overuse of antibiotics and genetic plasticity of bacteria has caused antimicrobial resistant (AMR) bacteria to become a more prevalent issue that threatens to be the number one cause of death worldwide by 2050 unless significant innovations are made. Although bacteria detection in the field is ideal, the current gold standards for detection require trained personnel and a central laboratory. The primary work in this dissertation acts to improve upon current bacteria detection methods by designing, developing, and optimizing inexpensive user-friendly tests that detect bacteria at the point-of-need without trained personnel or expensive equipment. These goals are accomplished using microfluidic paper-based analytical devices (μPADs), a growing field for point-of-need detection that have been used for a variety of analytes and applications. Using paper as a platform has allowed for the simple development of user-friendly devices because of their easily designed and modifiable material that typically costs <$0.01 USD per device and allows for multiple tests to be completed from one sample addition. Devices that will be described include colorimetric spot tests that detect common fecal indicator bacteria (FIB) species Escherichia coli and Enterococci spp. based on enzymes that are naturally produced by the bacteria. Utilizing these enzymes, a test was developed that turns from clear to yellow as an indication of live bacteria. These tests were successfully used in the detection of bacteria in food and water samples to demonstrate its efficacy in food safety applications. To improve specificity and sensitivity of bacteria detection, a second spot test was developed that utilizes immunomagnetic separation (IMS) and an enzymatic sandwich immunoassay in the detection of another common foodborne pathogen, Salmonella typhimurium. This assay was developed specifically for detecting pathogens in complex matrices, such as one of the most common causes of pathogen contamination: animal feces. Because AMR bacteria are becoming a more prevalent problem, devices were developed to specifically detect bacteria resistant to β-lactam antibiotics, the most common case of antimicrobial resistance observed in bacteria. The first generation of devices were developed to detect β-lactamase activity, an enzyme that facilitates resistance against β-lactam antibiotics. These devices were successful in detecting AMR in different species of bacteria isolated from environmental samples, and in the detection of AMR in sewage water. The second generation of devices enables detection of resistance against specific antibiotics through hydrolysis of the antibiotic and detecting a change in pH. Although not yet demonstrated, these devices will eventually be used to determine if bacteria are resistant against specific classes of β-lactam antibiotics, including a commonly used class of last resort antibiotics, carbapenems. Beyond bacteria detection, this dissertation also explores developing a field-ready device to identify falsified and substandard antibiotics. Because antibiotics are most commonly counterfeited in resource-limited settings, it is imperative to develop user-friendly point-of-need devices that can quantify the amount of active pharmaceutical ingredient in antibiotics. This was accomplished using enzyme competition, a method that had not been demonstrated paper-based devices. Finally, all devices that have been developed and optimized in this dissertation utilized colorimetric detection. While a user-friendly and easily implemented method of detection, it does suffer from drawbacks such as sensitivity and user subjectivity when using the devices. To eliminate subjectivity, a portable system using a Raspberry Pi computer and 3D-printed light box and device holder have been optimized. Although the system has been demonstrated by automatically analyzing images and calculating Michaelis-Menten enzyme kinetic values, this system has limitless possibilities in automatically analyzing colorimetric paper-based devices for truly objective colorimetric readouts and quantitative infield detection of pathogens or other analytes.
Open Access
Combining fundamental studies with advanced characterization for analyzing nitric oxide polymer systems
(Colorado State University. Libraries, 2014) Joslin, Jessica Marie, author; Reynolds, Melissa M., advisor; Ladanyi, Branka M., committee member; Krummel, Amber T., committee member; Ackerson, Christopher J., committee member; Williams, John D., committee member
Nitric oxide (NO) releasing materials have been investigated over the past couple of decades as potential biomaterials. A multitude of NO releasing platforms have been reported with different NO release properties indicating use in various bioapplications. Despite the positive implications associated with these materials, the field is currently limited by a couple of major issues. First, the reservoirs of NO stored in current systems do not allow the prolonged and controllable release necessary for long-term usage. Additionally, the field has experienced a lack of complete characterization of both the NO loading and release processes associated with these systems. To develop NO releasing platforms with enhanced NO reservoirs and controllable NO release profiles, fundamental studies are required to probe the physical processes that occur in these polymers. To enhance NO reservoirs in polymer systems, it is critical to probe the efficiency of the NO loading process. Systematic studies are presented where the efficiency and nature of S-nitrosothiol NO donor formation is investigated in a polymer environment. The nitrosating agent and polymer presence have a significant impact on the kinetics of S-nitrosation. Also, due to the versatile nature of nitrosation, NO byproducts that form competitively with S-nitrosothiols are characterized. By tuning the polymer functional groups, competitive nitrosation products can be eliminated and NO recoveries enhanced. Another critical obstacle towards understanding NO materials involves probing NO donor behavior in conjunction with NO release. For model polymers containing covalently linked S-nitrosothiol moieties, spectroscopy is coupled to direct NO detection to fully characterize the NO loading and release stages. Decomposition of the S-nitrosothiol moiety is directly correlated to NO release. S-nitrosothiol blended films are also investigated to determine the spatial distribution of NO release, which is critical to ensure a localized NO effect at the material surface. Finally, NO releasing polymer substrates are exposed to water plasma processing conditions. Surface wettability is significantly enhanced, while the NO release kinetics are maintained, suggesting that these materials can withstand processing towards tunable surface properties. Overall, fundamental and systematic studies of model NO releasing materials are presented that have not been formerly considered. Only by characterizing these materials completely can this class of biomaterial be better understood towards the ability to control the therapeutic and surface properties.
Embargo
Development of low-cost capillary driven immunoassays for improved medical diagnostics
(Colorado State University. Libraries, 2023) Link, Jeremy S., author; Henry, Charles S., advisor; Van Orden, Alan, committee member; Ackerson, Christopher J., committee member; Kipper, Matt J., committee member
Rapid medical diagnostics are a crucial part of an effective healthcare system. While traditional laboratory diagnostic methods are well established and sensitive, they are also time consuming and expensive. Point of care (POC) diagnostics offer an attractive alternative to traditional testing for more affordable, fast results. Their simplicity allows for POC devices to be run quickly by untrained personnel, but the simplicity often limits their detection range and sensitivity. In this dissertation I discuss affordable, capillary-driven immunoassay devices that are capable of passively delivering reagents associated with a traditional well-plate enzyme linked immunosorbent assay (ELISA) to test strips. These devices are made of patterned and laser cut double-sided adhesive. When stacked and laminated together, the patterns cut from the layers form hollow microfluidic channels that can passively transport fluids through capillary action. The devices in this dissertation require only a single end-user step to perform a sandwich immunoassay, and signal from the enzyme/substrate reaction is detectable in under 30 min. Chapter 2 discusses the first application for visual detection of SARS-CoV-2 in these affordable capillary-driven immunoassay devices. The device in this chapter uses the enzyme horseradish peroxidase (HRP) and the substrate 3,3',5,5'-tetramethylbenzydine (TMB) to produce signal at the test line. Upon sample addition, the device channels fill, rehydrating the detection antibody and substrate dried on conjugate release pads that are stored in the channels of the device. Within 20 min, target, reagents, and washing steps are passively delivered to a nitrocellulose test strip containing a capture antibody test line. The device performance was compared to a well-plate ELISA, and the detection limits for inactivated SASR-CoV-2 were 86 PFU/mL and 8 PFU/mL for the device and ELISA respectively. A dose response curve was also generated for spiked nasal swab samples with a detection limit of 222 PFU/mL, demonstrating the device's use with complex biological samples. Chapter 3 expands on the work in Chapter 2 by demonstrating an alternative detection method. Chemiluminescent immunoassays are highly sensitive assays that rely on the energy provided by a chemical reaction to excite electrons. When the electrons move back to the ground state, they produce light that can be detected with an imager. In Chapter 3, I demonstrate the first example of a one-step, capillary driven immunoassay for chemiluminescent detection. The device is similar to that in Chapter 2, but the detection system relies on the reaction between HRP and a luminol based substrate to detect SARS-CoV-2 antigen. This work was done in collaboration with Burst Diagnostics Inc. and will be published when the appropriate patents and protections are in place. Chapter 4 introduces the first capillary driven enzyme-linked immunoassay for the simultaneous detection of multiple biomarkers. This multiplexed device is made of the same inexpensive materials as the previous chapters, but the microfluidic channels are designed in such a way that reagents are delivered to two, spatially separated test strips. This separation allows for simultaneous detection of two targets without cross-reactivity between reagents, reducing the chance of false positives. To demonstrate the purpose of this device, they were used to detect SARS-CoV-2 antigen on one test strip, and influenza antigen on the other. The illnesses caused by these two viruses lead to very similar symptoms, so distinguishing between the two illnesses from a single device would be beneficial. Dose response curves were gathered for both antigens, and the device was able to detect both diseases visually without false positives on the other test strip. Another form of multiplexed detection is simultaneous detection of two targets. To demonstrate this, SARS-CoV-2 and influenza antigen were detected simultaneously. Additionally, SARS-CoV-2 virus and c-reactive protein (CRP), a biomarker that can be used to determine the severity of COVID-19 cases, were detected simultaneously. This multiplexed assay has the potential to tell a healthcare provider 1) if an infection is or is not SARS-CoV-2, and 2) what level of care might be needed. This dissertation introduces three capillary driven immunoassay devices primarily for the use of detecting communicable diseases. The devices all run from a single end-user step, and fully automate the steps required for a more time consuming and expensive ELISA. Although the focus of this dissertation was on detecting communicable diseases, these devices can (and are) being further developed for chronic illnesses. In the future, by swapping the antibodies used in the immunoassay, the applications of these devices are innumerable. Additionally, different detection methods, such as fluorescent, electrochemical, and further chemiluminescent work could continue to push the detection limit down, widening the application of these devices even further.
Open Access
Synthesis and characterization of sterically and electronically tuned ligands toward magnetic control of iron and cobalt complexes
(Colorado State University. Libraries, 2015) Klug, Christina M., author; Shores, Matthew P., advisor; Rappé, Anthony K., committee member; Ackerson, Christopher J., committee member; Levinger, Nancy E., committee member; Wu, Mingzhong, committee member
Presented within this dissertation are the syntheses and characterizations of iron and cobalt complexes featuring ligands designed to tune the magnetic properties. Two key magnetic phenomena are of interest: spin crossover and single-molecule magnetism. Both of these topics are known to be significantly influenced by subtle changes in coordination and inter- and intramolecular interactions. The overarching goal is to understand how the magnetic properties of the metal center can be controlled via electronic and steric modifications. In Chapter 1, I offer a brief introduction into the background and motivation of the works presented in this dissertation in the realm of spin crossover and single-molecule magnetism. The first section of this chapter is focused on spin crossover and how host:guest interactions can be exploited to alter the magnetic behavior of first-row transition metals. Examples of Fe(II) complexes that display anion-dependent spin state behaviors in both the solid-state and in solution are discussed. Functionalized tripodal Schiff-base ligands are placed into context as an extension of previous research into tripodal ligands for use as metal-based anion-receptors and tripodal spin crossover complexes. The second section of Chapter 1 gives a brief introduction into single-molecule magnetism. An examination of mononuclear Co(II) complexes displaying slow magnetic relaxation and application of acetylide-bridged metal centers to enhance magnetic communication are also given. In Chapter 2, I discuss the preparation and characterizations of a Fe(II) complex coordinated by the alcohol functionalized hexadentate tripodal iminopyridine L6-OH with varying anions. Solid-state magnetic susceptibility measurements of [FeL6-OH]X2 (X = OTf-, Br-, I-, or BPh4-) reveal an anion-dependence on the magnetic behavior. Magnetostructural correlations indicate that stronger hydrogen-bonding interactions are achieved with larger anions, which are better able to undergo bifurcated interactions with the hydroxyl groups from two of the arms. Removal of the tether between the ligand arms leads to the formation of [Fe(L2)2](OTf)2, a bis(tridentate) complex that remains high spin at all temperatures. Variable temperature magnetic measurements in d3-methanol reveal that the high spin state of [FeL6-OH]2+ persists regardless of the anion down to 183 K. In Chapter 3, attempts towards synthesizing the heteroarmed tris(imine) [FeL556]2+ and analogous bis(imine)-mono(amine) [FeL556-NH]2+ complexes are discussed. Several routes are attempted to synthesize the tris-iminopyridine species including selective deprotonation of tris(2-aminoethyl)amine*3 HCl, in situ complex formation via metal-templated self-assembly, and use of presynthesized ligands. Analyses of the reaction mixtures by mass spectrometry suggest that mixtures of products are formed regardless of the method. An anion and solvent dependence leads to preferential formation of the low-spin species [FeL5-ONHtBu]2+, while using solvents such as acetonitrile and ethanol lead to increased production of the desired [FeL556]2+. To test if anion-dependent magnetic behavior can be observed with this ligand type, the comparable complex [FeL556-NH]2+ was synthesized and characterized. Variable temperature solution measurements in d3-acetonitirile suggest that host:guest interactions in solution induce a stabilization of the low-spin state for [FeL556-NH]2+ as indicated by a decrease in susceptibility at lower temperatures for the Cl- salt. In Chapter 4, the preparation, structural, and magnetic characterizations for a family of Fe(II) complexes of tripodal ligands based on L5-ONHtBu are presented. The series of ligands aim to tune the ligand field by selectively reducing imines to amines, producing the ligands L5-(NH)x (x = 1 - 3, number of amines). In the solid state, the three Fe(II) complexes formed are high spin, but significant differences in the structural distortion of both the coordination environment of the Fe(II) center as well as the anion-binding pocket of the amides are noted. In solution, the complexes [FeL5-(NH)3]2+ and [FeL5-NH]2+ are high spin between 183 and 308 K in d6-acetone but interestingly, [FeL5-(NH)2]2+ undergoes a spin-state change with decreasing temperature. Variable temperature studies in d6-acetone and anion titrations in d3-acetonitrile at room temperature monitored by Evans' method of [FeL5-(NH)2]2+ show host:guest interactions stabilize the high spin state. These studies suggest a viable method of ligand tuning for spin-state control by host:guest interactions. In Chapter 5, I discuss the structural and magnetic properties of [Co5-ONHtBu]X2 (X = Cl-, Br-, I-, and ClO4-). These hexadentate Co(II) complexes vary only in the charge-balancing anion, but marked differences in their magnetic properties are observed. Investigation of the magnetic anisotropy of the various salts reveal that the chloride salt possesses the most axial anisotropy, which manifests as an exhibition of slow magnetic relaxation under application of an external field. To my knowledge this is the first example of anion-binding influencing the magnetic anisotropy and 'turning on' single-molecule magnet-like behavior. Lastly, Chapter 6 describes the syntheses and magnetic properties of a series of mono-and dinuclear Fe(III) complexes bridged by ethynylmesitylene ligands. Inclusion of steric bulk onto the bridging-aryl ligand is predicted to increase orbital overlap between the singly-occupied molecular orbital of the metal center and the π-system of the aryl linker. The addition of methyl groups to the aryl ring cements the desired equatorial ligand orientation with respect to the π-system. This leads to an increase in ferromagnetic coupling between the metal centers.
Open Access
Understanding the amide-assisted synthesis and olivine structure-directed twinning of Fe₂GeS₄ nanoparticles
(Colorado State University. Libraries, 2020) Miller, Rebecca Caroline, author; Prieto, Amy L., advisor; Shores, Matthew P., committee member; Sites, James R., committee member; Ackerson, Christopher J., committee member
The reality of detrimental anthropogenic effects on the environment requires the development of a number of sustainable practices and technologies. The Prieto Group strives to advance the synthesis and understanding of materials for use in energy conversion and storage. Advances in computational solid-state chemistry have resulted in the identification of a number of earth-abundant, relatively non-toxic compounds as promising photovoltaic absorber materials. However, the synthesis of solids remains a step behind, requiring empirical exploration of precursors and conditions. As reaction intermediates and mechanisms are discovered, general synthetic strategies can be translated from one material system to the next. Inorganic nanoparticle (NP) syntheses rely on the interdisciplinary expertise of solid-state, organometallic, and organic chemistry and show interesting complexity. The work herein has advanced the understanding of amide-assisted NPs syntheses and examined the microstructure of twinned Fe2GeS4 NPs. Chapter 1 presents a history of solution-based, amide-assisted NP reactions. As scientists understand the in situ speciation of precursors, more efficient reactions can be designed. This understanding allows the use of more benign and safe (both in terms of human and environmental) precursors and provides higher synthetic control over the end products. The presence of amide bases has generally provided access to higher NP nucleation rates and accessed smaller, more monodisperse particles. The increased monomer reactivity has also allowed the formation of ternary NPs free from binary or unary impurities by balancing the reactivity of cations of different valency. The most common amide base is LiN(SiMe3)2, and I relate this field to the use of its conjugate acid, hexamethyldisilazane or HMDS, in NP syntheses. Its addition has aided the production of NPs, but its chemical role remains unclear. This chapter was written utilizing a portion of an invited review paper written by myself, Jennifer M. Lee, Lily J. Moloney, and Amy L. Prieto in the Journal of Solid State Chemistry (2019, 273, 243-286.). Section 2.2 of the review outlined the evolution of understanding of amide-assisted NP syntheses and was adapted and expanded upon herein. In Chapter 2, I report the redesign of a Fe2GeS4 NP synthesis. In 2013, the Prieto group was the first to report a NP synthesis for the compound, which had been predicted to be a promising photovoltaic absorber material in 2011. The original reaction relied on HMDS as an additive and employed the highly-reactive S precursor, hexamethyldisilathiane. Herein, I speculate on these precursors' roles and exchange their use for LiN(SiMe3)2 and S powder, eliminating the formation of an Fe1–xS intermediate and reducing the growth time from 24 h to 10 min. I thoroughly map the reaction landscape of this system and provide structural, compositional, and optical characterization of the particles. This work was published in the Journal of the American Chemical Society (J. Am. Chem. Soc. 2020, 142 (15), 7023–7035.). The Fe2GeS4 NPs show an interesting star-shaped morphology, so I examine the microstructure via electron microscopy and identify the presence of crystal twinning in Chapter 3. The particles exist as three sets of stacked nanoplates intersecting at 60˚ angles, which forms a triplet of twins or trillings. In the products, 98% of the particles are twinned. Because crystal twinning, and especially trilling formation, in macroscopic crystals is rare, a synthetic route to a massive collection of twinned particles stands as a valuable resource for understanding the fundamentals of crystal twinning in olivine compounds. I relate the twinning to the underlying hexagonal pseudosymmetry of the orthorhombic, olivine crystal structure. Because of the ratio of the unit cell dimensions (a_Pnma/b_(Pnma )≈√3), the compound is susceptible to forming twins with growth of the [010] direction off the {310} faces. This can occur for other olivine compounds of similar unit cell dimension ratios, so I rank all of the olivine compounds listed in the Inorganic Crystal Structure Database according to this metric in Appendix A. This chapter is a manuscript prepared for submission. Finally, Chapter 4 outlines our recommendations for future work to advance the understanding of amide-assisted NP syntheses and translate this synthetic system to other compounds. I suggest the systematic development of SnS NP reactions utilizing each of the precursors: Sn silylamide, alkali silylamides, and HMDS. I outline a set of complementary techniques to characterize the reaction intermediates and mechanisms. This type of investigation has been done by the Kovalenko group for the formation of unary Sn0 NPs, but the interaction of the chalcogen species remains unknown. Further, no systematic mechanistic study exists for the use of HMDS in NP synthesis. This work would advance the understanding and use of amide-assisted syntheses for all metal chalcogenide compounds. In addition, I present preliminary data in our extrapolation of the Fe2GeS4 NP synthesis to the following solid solutions: Fe2GeS4–xSe (including the end member Fe2GeSe4) and Fe2–xMnxGeS4. One composition of each solid solution was formed and characterized by powder X-ray diffraction, and I present electron microscopy to show twinning in the Fe2GeS4–xSex (x = 0.96, 24 mol% Se) NPs. Lastly, I consider the possibility for twinning in an important olivine compound for battery science, LiFePO4, which is a common cathode material. The crystal structure shows a high degree of hexagonal pseudosymmetry, indicating that the energetics of forming twin domains may be favorable. I discuss the possible ramifications this may have on battery cycling performance. Thus, the scope of this work focuses on one compound, Fe2GeS4, but investigation into its synthesis and microstructure has opened a number of avenues for promising research. This compound itself presents a promising material for both photovoltaic and thermoelectric energy conversion, and the syntheses herein provide a launching point for property measurement and application evaluation. Further, the general examination of twinning in olivine compounds identifies questions for evaluating the function of other compounds useful for a number of applications. Lastly, analogous calculations to the geometrical evaluation done for orthorhombic olivine compounds could be carried out for other crystal structure types with unit cells that exist close to higher orders of symmetry. The advances presented herein on understanding the reactivity and roles of NP precursors are fundamental for progressing the field of NP synthesis. The reproducible formation and structural characterization of these twinned NPs provide a promising system for future explorations in crystal twinning and its effect on material properties.

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