Theses and Dissertations

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    Solid-state NMR (¹³C, ²⁷Al and ²⁹Si) study of the reaction between AlMe₃ and the silica gel surface
    (Colorado State University. Libraries, 2007) Li, Jianhua, author; Maciel, Gary E., advisor
    The reaction between the silica gel surface and trimethylaluminum (AlMe3) has been studied in this thesis research. We have examined the AlMe3/silica reaction in the following stages: the initial AlMe3-reacted silica surface after it had been treated with AlMe3; the AlMe3-treated surface after it was washed with dry diethyl ether; the ether-washed surface after it was treated, in steps, with limited amounts of H2O; and finally the H2O-reacted surface after an excess-H2O workup. Solid-state NMR (13C, 27 Al and 29Si) have been used to elucidate the structures of moieties generated on the silica gel surface at each of the stages listed above. Solid-state 13C NMR showed that Al(Me)n is the major type of moieties generated on the surface in the initial AlMe3/silica reaction and Si-OMe is the second most important moiety generated. After the sample has been washed with dry diethyl ether, strong ether signals were observed by 13C NMR, which implies that diethyl ether is strongly attached to the surface, even after evacuation. There are no significant changes for the other surface moieties after the diethyl ether treatment. In the series of limited-amount H2O treatments that followed, the AlMen signal intensity decreased as more H2O was added to the surface. In the sample resulting from the final (excess H2O) work-up, AlMen and Si-OMe moieties are completely gone and peaks corresponding to Si-Me and Si(Me)2 are the only signals left in the 13C NMR spectrum. In the 29Si NMR spectra, the signal intensity of the (SiO)3Si(OH) (Q3) peak typical of silica dropped after the AlMe3 treatment. Q3 signal intensity was replaced with a broad peak centered at about-104 ppm, as expected for a conversion in which most of the Si-OH groups on the silica surface have reacted with AlMe3 and turned into Si-O-Al moieties. The formation of Si-Me, Si(Me)2 and Si(Me)3 moieties were also observed in the 29Si spectra. 29Si spectra didn't show significant changes in the sample-treatment stages that follow the initial AlMe3/SiO2 reaction. In the 27 Al spectra of AlMe3-treated silica samples, 4-, 5- and 6-coordinate Al moieties were observed. In the initial reacted sample, 5-coordinate Al moieties are the major initial products from the reaction. After the samples were washed with diethyl ether, the 5-coordinate Al moieties are still the major moieties. With limited amounts of H2O introduced onto the surface, the AlMen moieties reacted with H2O, as shown by the 13C spectra; in 27 Al NMR spectra, signal intensity of 5- coordinate Al moieties decreased, while that of 4- and 6-coordinate Al moieties increased, which implies that the 5-coordinate Al moieties turned into 4- and 6- coordinate Al moieties as a result of reaction with H2O. On the final work-up surface, the 4- and 6-coordinate Al moieties are the major Al structures remaining on the surface. This is the first observation of this kind of change of Al atom coordination on a AlMe3-reacted silica surface. The structures of surface Al moieties are much more complicated than those proposed in previous publications on AlMe3/silica reactions. In the initial reaction between AlMe3 and silica gel, we also made quantitative measurements aimed at tracking the route of methyl groups in the whole system. The methane generated during the reaction was trapped in a N2(I)-cooled trap and the volume of trapped methane was measured as a gas with the water-displacement method. Unreacted Al-Me groups in the supernatant liquid were measured by the liquid-sample 13C NMR spin-counting method. The amount of methyl groups attached on the silica surface were measured by the solid-state 13C NMR spin-counting method. The total amount of methyl groups tracked in the AlMe3/silica/toluene system is about 108% of the amount of methyl groups present in the initial AlMe3 and is about 90% for the AlMe3/silica/cyclohexane system. Relaxation studies were carried out on both the initial AlMe3-reacted and ether-washed AlMe3/silica samples using 13C CP/MAS NMR. The methyl-group proton T1 values were measured by the saturation-recovery technique and the cross polarization relaxation time (TCH) and rotating-frame proton spin-lattice relaxation time (T1p) were measured using variable-contact-time experiments. The AlMen moieties in the initial AlMe3-reacted sample showed very long (5 s ~ 7 s) proton T1 values, which implies that the AlMen moieties may be in a very restrained environment. This result supports the existence of 5-coordination Al structures indicated from 27Al results; in these structures methyl groups are bridged/shared between adjacent AlMen moieties. After the initial AlMe3-reacted silica sample was washed with diethyl ether, methyl-group proton T1 values were reduced by half, which may be due to replacing the methyl bridges with electron-rich centers consisting of the O atoms of the ether molecules introduced by the washing. This interpretation also explains why we have strong ether signals in 13C NMR spectra of the ether-washed sample and in the H2O-treated samples that followed. Overall, the moieties generated in the AlMe3/silica reaction have been characterized by solid-state NMR methods in this thesis work. And, methods were developed which quantitatively characterize the fate of all the Al-Me groups added into the reaction system.
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    Modeling conformational heterogeneity in biomolecules
    (Colorado State University. Libraries, 2023) Klem, Heidi, author; Paton, Robert, advisor; McCullagh, Martin, advisor; Levinger, Nancy, committee member; Kennan, Alan, committee member; Geiss, Brian, committee member
    Regulation of biocatalytic cascades is essential for biological processes but has yet to be exploited in real-world applications. Allostery is a prime example, where binding of an effector molecule alters function in a remote location of the same biomolecule. V-type allostery is especially fascinating, as the reaction rate can be either increased or decreased in response to effector binding. Determining how conformational changes affect the reaction rate is challenging due to the disparity of timescales between the underlying molecular processes. Experimental methods, such as X-ray crystallography, can help to capture large-scale conformational change. However, the resulting structures are not guaranteed to correspond to the biophysical state relevant to the research questions being addressed. Structural changes that occur during the chemical reaction are particularly elusive to this approach. To understand the connection between conformational change and catalytic consequence, a description of the reaction mechanism and relevant configurations is needed. Quantum mechanical (QM) methods can be used to propose enzyme reaction mechanisms by modeling femtosecond motions of forming and breaking bonds. Large-scale conformational changes take place over much longer timescales that cannot be simulated at the QM level, therefore requiring classical simulation techniques. This dissertation focuses on the challenges posed by conformational change in the field of computational biocatalysis. The first chapter examines the prevalence of conformational change in enzymes, its relationship to catalysis, and the difficulties it presents. The second chapter looks at the influence of active site structural features on reaction rates in the allosteric enzyme IGPS using QM approaches and energy decomposition schemes. The third chapter covers the development of methods that use molecular dynamics (MD) simulations to analyze relevant structural states from simulation data and identify long-range communication pathways in biomolecules. The fourth chapter presents a Python code, enzyASM, that automates the generation of QM-based truncated active site models and discusses ongoing developments that will aid reproducibility and standardization in this field of research. The fifth and final chapter summarizes the implications of this Thesis work in computational biocatalysis and envisions how remaining challenges can be addressed to maximize potential to solve real-world problems.
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    Synthetic control of magnetic resonance properties towards metal-based electron paramagnetic resonance imaging
    (Colorado State University. Libraries, 2023) Campanella, Anthony John, author; Zadrozny, Joseph, advisor; Shores, Matthew, committee member; Bandar, Jeff, committee member; Wu, Mingzhong, committee member
    Electron paramagnetic resonance imaging (EPRI) is the electron-spin analogue to conventional biological (nuclear) magnetic resonance imaging (MRI) whereby unpaired electron spins are probed in order to generate an image. The greater sensitivity of electron spins to their environment can thus be leveraged to capture detailed chemical information from the surroundings, producing an image of the local physiology that adds an extra dimension to the already powerful anatomical information gained from MRI. To move EPRI a step closer to common utilization, paramagnetic probes must be developed to sense the local environment using safe low-frequency microwaves at high (ca. 1.5 T) magnetic fields. Paramagnetic metal complexes are ideal candidates due to their electronic structures but have not been investigated for such purposes. The goal of this dissertation is to develop fundamental design principles to improve the utility of metal complexes as EPRI probes. Presented herein is the first comprehensive collection of experimental investigations to this end. Firstly, a method for improving signal sharpness is investigated, where exhaustive spectroscopic and computational studies suggest differences in relaxation dynamics as being a key factor in spectral linewidth (Chapters 2 and 3). A highly tunable clathrochelate structure is developed, inducing an unusual coordination geometry around the Ni(II) ion affording an 11 cm−1control of zero-field splitting (Chapter 4). The temperature dependence of zero-field splitting is examined in a series of Mn(II) complexes where an unusually high temperature sensitivity is found in the solid state (Chapter 5). Finally, the utility of metal complexes as environmental sensors is demonstrated with a pair of Mn(II) complexes showing that increasing magnetic anisotropy is a design strategy for enhancing microviscosity sensitivity (Chapter 6). The learned design principles will serve as a foundation for the design of metal-based EPRI agents towards improving the non-invasive diagnostic capabilities.
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    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.
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    Investigating the origins of slow magnetic relaxation of S = ½ Ni(III) cyclams
    (Colorado State University. Libraries, 2023) Morrison, Thomas L., author; Shores, Matthew P., advisor; Zadrozny, Joe, committee member; Kennan, Alan, committee member; Gelfand, Martin, committee member
    This dissertation describes the syntheses and characterizations of several Ni(III) and Ni(II) complexes in an attempt to better understand the origin of slow magnetic relaxation, or spin reversal, in S = ½ systems by utilizing Ni(III) cyclam (1,4,8,11-tetraazacyclotetradecane) as a toy model system. The content is organized as follows: Chapter 1 provides the historical context and theory surrounding the class of materials called single molecule magnets (SMMs). Therein I describe the prototypical SMM and its primary figures of merit and characteristics, such as S and D, followed by the observation of how S = ½ systems, which have previously been shown to act as SMMs, do not fit within the context currently provided by the literature. The choice of using the Ni(III) cyclam system is then elaborated upon, along with its quirks and foibles. In Chapter 2 I describe the synthesis and magnetic characterization of three Ni(III) cyclams. The first two contain halides in the axial positions, which are 100% abundant in isotopes containing nuclear spin, and the third complex has perchlorate bound in the axial position, where oxygen is nearly nuclear spin free. Neither halide systems showed slow magnetic relaxation, but it was not clear whether it was due to the superhyperfine coupling between the nuclear and electronic spins or due to the antiferromagnetic interactions present at low temperatures. The perchlorate containing complex did show slow magnetic relaxation, consistent with the literature and our predictions. Chapter three describes the crystallographic tuning tools and corresponding magnetic properties of novel S = ½ Ni(III) cyclam complex salts: strong antiferromagnetic coupling in sulfate-bridged chain {[Ni(cyclam)(µ2-SO4)]ClO4·H2O}n and field-, temperature-, and size-dependent slow magnetic relaxation in molecular [Ni(cyclam)(HSO4)2]HSO4. I have reported two methods of manipulating the dynamic magnetic response of these coordination molecules: particle size selection and deuteration. I find that particle size dependency, which I attribute to the phonon bottleneck effect, for the magnetic dynamics in the parent protiated compound is removed in deuterated isotopologue, revealing only the faster molecular relaxation mode(s). Chapter 4 describes the synthesis and characterization of four novel Ni(III) cyclams utilizing neutral ligands in the axial positions as opposed to the anionic ones considered previously, namely [Ni(cyclam)(acetonitrile)2]X3 (X = OTf, ClO4, BF4) and [Ni(cyclam)(butyronitrile)2]OTf3. Through these complexes we probe the role of ligand charge, identity, and subtle differences in the hydrogen-bonding network on the slow magnetic relaxation of the Ni(III) ion. Chapter 5 describes the solution phase studies of [Ni(cyclam)(MeCN)2]OTf3 and [Ni(cyclam)(butyronitrile)2]OTf3 in glassy and non-glassy solvents, as well as their suitability for studying other novel species in situ that may not be able to be synthesized and measured traditionally. We find that there are significant differences in the magnetic relaxation of the Ni(III) cyclams between glassy and non-glassy solutions and discuss the possibilities these findings present. In Chapter 6 I summarize the key findings from Chapters 2-5 and propose new avenues of research for further investigating this phenomenon. Finally, in Chapter 7 I describe a different ligand involving intra-ligand π-π interactions and explore the feasibility of using such interactions for intelligently controlling and tuning the first coordination sphere geometry and electronic structure. By introducing new substituents, changes to the aromaticity, and oxidation of the ligand we are able to exhibit rational control over the crystallographic and electronic structure of the metal center.