Browsing by Author "Kipper, Matt, committee member"

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Open Access
Advancements in organocatalyzed atom transfer radical polymerization by investigation of key mechanistic steps
(Colorado State University. Libraries, 2022) Corbin, Daniel Andreas, author; Miyake, Garret, advisor; Finke, Richard, committee member; Rappé, Anthony, committee member; Kipper, Matt, committee member
Organocatalyzed atom transfer radical polymerization (O-ATRP) is a controlled radical polymerization method employing organic photoredox catalysts to mediate the synthesis of well-defined polymers. The success of this method derives from its reversible-deactivation mechanism, where polymers are activated by reduction of a chain-end C-Br bond to generate a reactive radical for chain growth, followed by deactivation of the polymer by reinstallation of the dormant bromide chain-end group. As a result, the polymer chain can be grown by reaction of the polymer radical with alkene-based monomers, but undesirable termination and side reactions can be suppressed by minimization of the radical concentration through deactivation. In this work, key mechanistic steps of O-ATRP are investigated to understand the fundamental limitations of this method and improve upon them. When N,N-diaryl dihydrophenazines were investigated, side reactions were identified in which alkyl radicals add to the phenazine core, leading to new core-substituted PC derivatives with non-equivalent catalytic properties. Employing these core-substituted PCs in O-ATRP showed these side reactions can be eliminated to improve polymerization control. In addition, the deactivation step of O-ATRP and related intermediates were studied, which revealed new side reactions that can limit polymerization efficiency as well as influences on the rate of deactivation. Finally, methods to exert control over the deactivation process were developed as a means of improving polymerization outcomes in challenging systems. For example, the intermediate responsible for deactivation was isolated and added to a polymerization to increase the rate of deactivation and limit side reactions in O-ATRP. Alternatively, a similar outcome could be achieved through in-situ electrolysis to increase the concentration of the desired intermediate during the polymerization. Ultimately, this work has yielded insight into important mechanistic processes in O-ATRP that will continue to benefit the development of this method.
Embargo
Analysis of LEAC biosensor for scalable manufacturing using BPM and FDTD simulation methods
(Colorado State University. Libraries, 2024) Holmes, Cameron Dane, author; Lear, Kevin L., advisor; Nikdast, Mahdi, committee member; Kipper, Matt, committee member
The increasing demand for rapid, scalable, and accurate diagnostic tools has driven the development of optical biosensing technologies. LEAC (Local Evanescent Array-Coupled) biosensors, which leverage the evanescent field generated by optical waveguides, are particularly well-suited for applications in biomedical diagnostics, environmental monitoring, and point-of-care testing. LEAC biosensors have previously been fabricated in incomplete and unoptimized near-commercial CMOS processes and fully custom processes in a university cleanroom but have not been implemented in suitable high-volume processes such as commercial silicon photonics. A primary motivation for the research presented in this thesis is to evaluate the ability to fabricate LEAC biosensors operating at 1550 nm wavelengths in the commercial AIM Photonics' active silicon photonics process. This thesis presents a comprehensive tolerance analysis of LEAC sensors for both bulk sample layers (400 nm thick) and protein monolayers (10 nm thick) in AIM's process, focusing on the impact of variations in key design parameters—specifically waveguide core thickness, cladding layers, and photodetector placement—on sensor sensitivity. Beam Propagation Method (BPM) and Finite-Difference Time-Domain (FDTD) simulation techniques are employed to assess how these tolerances affect optical field propagation, power dissipation, and flux into the photodetector, serving as proxies for sensor performance. Additionally, the study examines crosstalk between multiple sensing regions, evaluating how refractive index variations in one region influence adjacent regions—an important consideration for multi-region sensors. Results show that sensor sensitivity increases with cladding thickness and decreases with waveguide core thickness. A 25 nm manufacturing error in core thickness resulted in less than a 10% sensitivity shift, and a 300 nm cladding thickness error had a similarly small effect. Resonant absorption between the core and photodetector was observed across both bulk and monolayer samples. Sensitivity depends heavily on proximity to resonance; a 10% error in photodetector thickness at resonance caused a 600% change in sensitivity, while off-resonance, the same error had minimal impact. Coupled Mode Theory (CMT) explained these energy transfers and power fluctuations. ANOVA analysis of full-device FDTD simulations quantified forward crosstalk due to modulated absorption from sample regions closer to the optical source (upstream). Forward crosstalk was found to be negligible for protein monolayer samples but could be significant in bulk samples. However, even in bulk samples, forward crosstalk was largely mitigated using photocurrent ratios with a reference region. A crosstalk ration was used as a metric to determine the influence of each refractive index (n1, n3) on the photocurrent ratio. In the forward crosstalk direction, the use of photocurrent ratios decreased the magnitude of the forward crosstalk ratio; however, the use of photocurrents inherently introduce dependance on downstream indices (reverse crosstalk). Reverse crosstalk, caused by reflections at the dielectric boundary between sensing regions, was found to be negligible using photocurrent ratios with bulk analytes; however, with monolayers, the use of photocurrent ratios introduced a slight dependence on the downstream region, indicating minor backward crosstalk. This can be mitigated by using raw current values rather than current ratios. Raw currents eliminate backward crosstalk in region 1, while photocurrent ratios effectively eliminate forward crosstalk in region 3.
Open Access
Bioelectrochemical production of graphene oxide using bacteria as biocatalysts
(Colorado State University. Libraries, 2019) Nunez Hernandez, Diana Marcela, author; De Long, Susan, advisor; Kipper, Matt, committee member; Sambur, Justin, committee member
The demand for production of graphene oxide (GO), which is a precursor for large-scale production of graphene, has been increasing due to the broad array of uses of both nanomaterials. Due to the unique electrical and mechanical properties of these 2D nanomaterials, applications in composites have shown enhancements by contributing a tunable energetic band gap, high strength, and high transparency among other features. The tunable band gap of the graphene derivatives is one of the key properties of these nanomaterials. By varying the size of the energetic band gap (in eV) between the conduction and valence bands, resistance can be decreased to promote electron flow in the material lattice. A large energetic band gap (insulators) means more resistance for electron flow. Being able to control the band gap of a nanomaterial, allows for many applications in batteries, supercapacitors, and semiconductors being the most promising applications for these nanomaterials. Other applications include flexible electronics, renewable energy, drug delivery, contaminant removal, sensors, and more. Unfortunately, large-scale production of graphene using current methods is challenging due to low yield, impurities, high cost, high energy input, slow production rates and/or hazardous chemical reactants and wastes. For this study, the focus was on the bioelectrochemical production of GO (BEGO) as a novel technology for producing these nanomaterials with low energy input, inexpensive and non-hazardous reagents at standard conditions, and using microbes as biocatalysts. The BEGO process consists of a single-chamber microbial electrosynthesis cell (MES) that uses a graphite rod anode and a cathode (carbon cloth or stainless steel) to drive redox reactions. This MES can be operated at low voltage in a three-electrode (-0.8-1.4V vs. Ag/AgCl), or two-electrode system (~3.1V DC), with bacteria inoculated in a phosphate media solution. During this study, the BEGO process was investigated to advance understanding of the production process and the properties of the BEGO nanomaterial produced. To achieve this, the objectives established include: 1) developing methods for purifying and quantifying the nanomaterial during the production process in the complex aqueous-phase reactor matrix, 2) identifying key physical and chemical properties of the nanomaterial product using various spectroscopy and microscopy techniques, and 3) analyzing the microbial communities present in the reactors and in the graphite anode biofilm. To quantify the BEGO and estimate production rates, different spectrophotometric and gravimetric methods were used. Ultraviolet-visible spectroscopy (UV-Vis) at 229 nm was found to be the best method. This wavelength is specific to GO as it corresponds to the π → π * transitions of aromatic C-C bonds comprising the majority of the molecule, regardless of the oxidation state. Different centrifugation and filtration protocols were compared to purify the BEGO out of the complex matrix. For quantification methods in solution, centrifugation at 10,000 x g for 15 minutes was found to be the most effective method for removal of large particles and biological material, with BEGO remaining in solution. For material characterization, various techniques were used to identify the functional groups present and the morphology of the BEGO sheets. It was found through Fourier transform infrared spectroscopy (FT-IR) and UV-Vis, that the nanomaterial contained less carboxyl/carbonyl groups than GO produced by the traditional Hummers' method. Raman spectroscopy and thermogravimetric analysis (TGA) showed high disorder and weight loss events consistent with known GO spectra. Microscopy analysis revealed the BEGO process yields sheet sizes of a few hundred nm to 1-2 µm in lateral dimensions. Transparency and Fast Fourier transform (FFT) images indicate the BEGO consists of only single-layered to few-layered structures, which are needed for downstream applications. The microbial analysis was done on bioreactors with different inocula sources. DNA and RNA were extracted from both the bulk liquid media and the rod biofilm. At the end of the operation period, microbial communities in the bioreactors had diverged from the inoculum source. Microbial communities in the BEGO producing reactors consisted of both aerobic and anaerobic microorganisms. The most abundant genera on the rod biofilm were the unknown Comamonadaceae (10-11%), Hydrogenophaga (9-21%), Methyloversatilis (15-22%), and Pseudomonas (11-36%) all from the Proteobacteria phylum. Thus, these microbial phylotypes may play a key role in catalyzing BEGO production, enabling this novel and sustainable approach to nanomaterial synthesis.
Open Access
Cellulose nanocrystals extracted from hemp agro-waste as a potential coating for titanium medical devices
(Colorado State University. Libraries, 2024) Heacock, Jesse Andrew, author; Li, Yan Vivian, advisor; Kipper, Matt, committee member; Liu, James, committee member
Increases in biowaste worldwide have created a unique opportunity to extract natural polymers for a variety of uses. Over the last few decades, hemp has increased in popularity as a desirable industrial agricultural plant. Increases in hemp production for commercial product use have increased the amount of hemp agro waste (HAW). This agro-waste is a potentially great source for extraction of the natural polymer cellulose and the creation of a circular economy. Cellulose can be found ubiquitously in plants and has gained great interest as an alternative to synthetic polymers. In this work, cellulose nanocrystals (CNCs) were extracted from HAW using a one-step ammonium persulfate (APS) oxidation method. APS oxidation was used due to its reduction in hazardous wastes, making it a more environmentally friendly method for CNC extraction compared to other chemical methods. HAW, specifically the woody core of hemp known as hurd, underwent CNC extraction and the properties of the final product were analyzed. Depending on the initial source and method of extraction, CNCs properties have been shown to vary, creating a potential for selectivity when applying CNCs for different uses. It was found that changes in reaction time directly impact CNCs size, surface properties, final product mass, and hydrophilicity. Of note, as reaction time increased from 8 hours to 48 hours, the size of the nanocrystals significantly decreased in length and width. While other properties, such as mechanical strength, morphology, surface charge, and cytotoxicity, underwent no statistically significant changes due to increases in reaction time. The results suggest that HAW is a good source for CNC extraction and that changes in APS oxidation can allow for selective tuning of some CNC properties.
Open Access
Development and application of electrochemical dithiothreitol (DTT) assay for analysis of particulate matter
(Colorado State University. Libraries, 2017) Turner, Laurelle Rose, author; Henry, Charles, advisor; Kipper, Matt, committee member; Volckens, John, committee member
Particulate matter (PM) in air pollution, known to have a negative impact on biological systems, is regulated in many countries across the globe. The generation of PM from energy and mining industries is monitored in an effort to minimize its contributions to diminished human health. And although quantifying total PM generation (mass and number) and exposure can help track health risks, ultimately there exists a need to develop rapid, efficient, accurate methods for analyzing PM composition and health effects. Leading hypotheses over the last decade have theorized that PM, once absorbed into bodily tissues, generate reactive oxygen species (ROS), leading to oxidative stress, thus catalyzing cellular damage. A recently developed analytical electrochemical protocol has shown great potential for investigating the potential for PM to cause oxidative stress. The work discussed within this thesis focuses on the development of the electrochemical assay and its application to real world PM samples. Herein, the development of an electrochemical version of the well-studied dithiothreitol (DTT) absorbance spectroscopy assay is presented as a platform for the analysis of PM in air samples. Flow injection amperometry was used as the primary electrochemical method. Amperometry was performed using a CH Instruments potentiostat and commercially available DropSens modified carbon screen printed electrodes (SPE's) with a DropSens impinging jet flow cell, granting the assay flexibility to be performed in any lab with commercially available components. Previous examples used homemade components restricting accessibility to the field. The ability to use inexpensive, purchasable components eases trouble shooting, training, and allows for the potential to make the assay more mobile. The use of a flow cell also allows for the possibility for linking the assay to other analytical methods to further analyze PM which may not be reactive in the assay. Assay development focused on optimizing the assay temporally, as well as investigating its precision, detection limits, fluid dynamics, reproducibility, and relative accuracy. The electrochemical assay uses shorter reaction times and avoids the need for additional chemical quenching agents used in the absorbance assay, allowing for batch processing. Assay performance was compared to literature with a model oxidant quinone, 1,4-naphthoquinone, and trace metal, Cu(II). The assay was then applied to real exposure samples collected in Fresno, California and Honduras. Data from these samples were correlated against data obtained by the traditional DTT assay to investigate accuracy. Analyses of the Honduras samples will be correlated against health data as part of the Honduras Cookstove Project, moving one step closer to directly connecting PM reactivity to health effects. Although the traditional absorbance DTT assay is the standard in assessing PM reactivity in air samples, and has been used for the last 15 years, the electrochemical assay is a robust, quick, and precise alternative method that can be readily performed using readily available components.
Open Access
Dual nickel- and photoredox-catalyzed enantioselective desymmetrization of meso anhydrides and C-O bond activation via phosphines and photoredox catalysis
(Colorado State University. Libraries, 2018) Stache, Erin Elizabeth, author; Rovis, Tomislav, advisor; Doyle, Abigail G., advisor; Chen, Eugene, committee member; McNally, Andy, committee member; Reynolds, Melissa, committee member; Kipper, Matt, committee member
Described herein is the application of photoredox catalysis in the development of new synthetic methods. A dual nickel- and photoredox catalyzed desymmetrization of meso succinic anhydrides was developed to generate stereodefined cis keto-acids in high enantioselectivity and diastereoselectivity. The approach employed benzylic radicals as a coupling partner, generated from a photoredox catalyzed single-electron oxidation of benzylic trifluoroborates using an inexpensive organic dye. A unique epimerization event was discovered and the degree of epimerization was rendered tunable by changing catalyst loadings to ultimately form the trans diastereomer preferentially in high enantioeselectivity. A method for the C–O bond activation of aliphatic alcohols and carboxylic acids was developed using phosphines and photoredox catalysis. This novel reaction platform was used to generate aliphatic or acyl radicals directly from benzylic alcohols and aliphatic and aromatic acids, and with terminal hydrogen atom transfer, afforded the desired deoxygenated alkanes and aldehydes. Additionally, the intermediate acyl radicals could be intercepted in an intramolecular cyclization reaction to generate new lactones, amides and ketones.
Open Access
Effects of dexamethasone and oxidative environment on chondrogenesis of bone marrow-derived mesenchymal stem cells
(Colorado State University. Libraries, 2017) Tangtrongsup, Suwimol, author; Kisiday, John, advisor; Frisbie, David, committee member; Goodrich, Laurie, committee member; Kipper, Matt, committee member
Bone marrow-derived mesenchymal stem cell (MSCs) have received extensive consideration for applications to musculoskeletal tissue engineering based on their ability to differentiate into multiple skeletal lineage. For cartilage, MSCs-based therapies evaluated in vivo and in clinical studies have shown that MSCs can produce repair tissue that integrates with native tissue; however, defects remain partially cover, and the neotissue can contain fibrocartilage or evidence of hypertrophy. It is anticipated that a greater understanding of conditions that support MSCs chondrogenesis will lead to better results in cartilage tissue engineering. In chapter 1, fundamental aspects of MSCs including chondrogenesis and uses in tissue engineering are reviewed. Further, information regarding reactive oxygen species, and their involvement in the functioning of MSCs and chondrogenesis are presented. In chapter 2, MSC chondrogenesis was explored as a function of exposure to dexamethasone, anti-inflammatory glucocorticoid. Dexamethasone is known to support MSC chondrogenesis in vitro, although the effects of dose and timing of exposure are not well understood. Therefore, this study investigated these variables using a laboratory model of MSC chondrogenesis. In vitro MSCs chondrogenesis is conventionally induced in the presence of 100 nM dexamethasone; however, our result suggested that 1 nM dexamethasone was sufficient to supported robust cartilage-like ECM accumulation. By evaluating temporal exposure of MSCs to dexamethasone, we determined that exposure to dexamethasone during the first two days of culture was not critical, and that sustained exposure of at least a week appears to be necessary to maximize ECM accumulation. In chapter 3, we studied the oxidative environment associated with chondrogenic culture of MSCs. In conventional serum-free chondrogenic medium we noted that the concentration of intracellular reactive oxygen species (ROS) increased with time in culture. Previously, serum-free culture has been associated with increased ROS. Consistent with these reports, we found that supplementing chondrogenic cultures of MSCs with 5% fetal bovine serum reduced levels of intracellular ROS. Further, serum-supplementation increased the accumulation of collagen, a major component of cartilage extracellular matrix. Similar results were obtained using adult equine serum, which is as important as xenogeneic materials may be problematic for clinical applications. In summary, this study identified changes in the oxidative environment during MSC chondrogenesis, and suggested that lowering ROS may be an effective approach to increase collagen accumulation. In chapter 4, the extent to which reducing intracellular ROS can improve chondrogenesis was evaluated in a more precise fashion using antioxidants. To do so, we tested the effects of N- acetylcysteine (NAC), glutathione ethyl ester (GSHEE), or ammonium pyrrolidine dithiocarbamate (PDTC). First, we evaluate the effect of each antioxidant on intracellular ROS using DCFDA staining. We found that NAC and GSHEE were not effective in reducing intracellular ROS over time in our MSCs chondrogenic cultures. In contrast, PDTC decreased intracellular ROS and evidence of oxidative damage, while modestly increasing GAG accumulation. However, PDTC also moderately decreased the compressive stiffness of the MSC-seeded hydrogels. In summary, this study indicated that lowering ROS with specific antioxidants could enhance MSCs chondrogenesis, although loss of mechanical integrity is a major concern. The research described in this dissertation add to the knowledge of MSC chondrogenesis and the influences of dexamethasone and oxidative environment. We established that conventional doses of dexamethasone are at least a 100-fold higher than is necessary to support MSC chondrogenesis, which may be used to design dexamethasone delivery strategies to support MSCs chondrogenesis in vivo. During chondrogenesis, lowering levels of ROS that are encountered with conventional serum-free culture leads to higher levels of extracellular matrix accumulation. This information can be used to design in vitro or in vivo approaches to modulate the oxidative environment for optimal MSC chondrogenesis.
Open Access
Evaluating filtration membranes and detection systems for use with virus surrogates
(Colorado State University. Libraries, 2010) Stump, Emily D., author; Wickramasinghe, Sumith Ranil, advisor; Quackenbush, Sandra Lynn, committee member; Kipper, Matt, committee member; Pellegrino, John, committee member
Virus filtration membranes are used to provide size exclusion removal of viruses during the purification of biopharmaceutical products. This viral clearance is required by regulatory agencies to ensure the safety of patients by preventing contamination of product by adventitious or endogenous virus. Viral clearance studies are often laborious, expensive, and require highly trained personnel. Detection and quantification of virus using standard assays has restrictions in terms of limit of detection, extraneous contamination and false positives. Moreover, biosafety for personnel and the environment is always a concern when working with live virus. In order to avoid the hazards of live, adventitious virus, bacteriophages have been used previously as virus surrogates (Aranha-Creado & Brandwein, 1999). While the health threat associated with using live viruses is eliminated using bacteriophages as surrogates, the detection systems and quantitative assays are still laborious and difficult. Development of a non-biological system to simulate and quantify virus particles could reduce the time taken to perform viral clearance tests; reduce development costs; reduce the risk to personnel performing the tests; and lead to more reliable data, since a non-biological system will reduce variability in assays. Here we develop a prototype of a novel, gigantic magnetoresistive (GMR) detection system for magnetic virus surrogates. In addition, we investigate various polymeric membranes for their ability to reject virus. Results will be used as a benchmark for evaluating the behavior of a future, superparamagnetic virus surrogate. GMR-based technology has increasingly been on the rise since the 2007 Nobel Prize in physics was awarded to Albert Fert and Peter Grünberg for its discovery. GMR sensors show potential for being extremely sensitive, inexpensive, and flexible devices for use in biodetection assays. Compared to the current magnetic detection technology of the superconducting quantum interference device (SQUID), which requires complex instrumentation and qualified users, GMR technologies can be fabricated in such a manner so as to be applied to lab-on-chip systems. Here we discuss the sensor design and fabrication. Initial measurements indicate that 104 iron oxide nanoparticles, approximately 20nm in diameter, can be detected in 0.5μl of solution. Various virus filters as well as ultrafiltration membranes were challenged with feed streams spiked with high concentrations of minute virus of mice (MVM) in the presence and absence of 1% bovine serum albumin (BSA) (w/v). Changes in permeate flux with filtrate volume were determined in conjunction with changes in rejection of parvovirus. Decrease in permeate flux resulting from fouling of BSA was evaluated for its effect on virus rejection. The results, which compare the performance of virus filtration and similar ultrafiltration membranes, provide insights into the comparison of live virus with future virus surrogates.
Open Access
Evaluation of nitric oxide releasing polymers for wound healing applications
(Colorado State University. Libraries, 2015) Wold, Kathryn A., author; Reynolds, Melissa, advisor; Henry, Charles, committee member; Kipper, Matt, committee member; Popat, Ketul, committee member; Williams, John, committee member
Chronic, non-healing wounds afflict millions of Americans and represent a costly burden to the healthcare industry. In addition, the overuse and misuse of antibiotics has triggered the widespread emergence of drug-resistant bacteria, making the treatment of infected wounds more challenging. As a result, improved methods for wound care incorporating antibiotic-alternative bactericidal agents are in high demand. Recent wound care advances have focused on the development of dressings incorporating physical structures and biological components which mimic those encountered in a natural wound environment. Nitric oxide (NO), an endogenously produced molecule upregulated to promote cellular function and bactericidal activity during wound healing, has been harnessed in material systems and studied for wound healing potential. This work describes the characterization, bactericidal activity, cell functionality and processing of two NO-releasing polymer systems, one water-soluble and another water-insoluble. The results of this work demonstrate the capability of these polymeric NO-releasing materials to promote high log reductions of planktonic bacteria. Additionally, polymer dosages that promote cell survival and induce cytotoxicity in eukaryotic cells have been determined and nano-scale polymer fibers that maintain NO release properties have been processed. These results represent qualities beneficial towards the development of enhanced materials for the treatment of chronic infected wounds.
Open Access
Evaluation of osteogenic design factors in electrospun poly(ε-caprolactone) nanofiber scaffolds
(Colorado State University. Libraries, 2010) Ruckh, Timothy T., author; Popat, Ketul, advisor; James, Susan, committee member; Kipper, Matt, committee member; Ryan, Stewart, committee member
Biodegradable bone tissue scaffolds have the potential to impact patients with numerous ailments. Starting with fabrication techniques that produce nano-scale features, the ability to manipulate architecture, alter surface chemistry, and deliver biological molecules allows for the design of elegant and highly effective bone scaffolds. This work aimed to develop a porous, nanofiber scaffold with osteogenic design features the capability to deliver an antibiotic molecule from within the nanofibers. Two osteogenic design factors with unique mechanisms of action were selected; hydroxyapatite nanoparticles and oleic acid. Hydroxyapatite (HAp) is the primary inorganic phase of natural bone tissue and has been used to more closely mimic the extracellular environment of synthetic bone tissue scaffolds. Oleic acid (OLA) is an ω-9 fatty acid with suspected osteogenic effects due to activation of peroxisome proliferator-activator receptors (PPARs). In separate in vitro evaluations, OLA significantly increased osteoblast phenotypic behaviors and led to differential expression of the three PPAR isoforms, suggesting that the OLA is activating its anticipated receptor. HAp produced mixed results by inducing a small increase in alkaline phosphatase activity, but decreasing expression levels of bone matrix proteins. An in vivo evaluation of biocompatibility revealed that neither design factor increased the inflammatory response over control nanofiber scaffolds in paravertebral muscle pouches. However, both factors separately increased new osteoid production. Scaffolds with both HAp and OLA elicited the greatest osteogenic response in vivo, suggesting positive synergy between the two design factors. Finally, rifampicin (RIF), an antibiotic molecule was loaded into the nanofibers, and its release into static bacterial culture was effective in inhibiting bacterial population growth for both a Gram-positive and Gram-negative bacterial strain, separately. Overall, these nanofiber scaffolds were demonstrated to be effective carriers of soluble (OLA, RIF) and insoluble signals (HAp) which can modulate cell behaviors. Future work will aim to incorporate additional osteogenic features into the scaffolds and to develop multiple antibiotic release mechanisms from the nanofibers.
Open Access
Exploring model chemical systems through a new lens: combining novel microfluidic technology with infrared analysis techniques
(Colorado State University. Libraries, 2016) Barich, Michael, author; Krummel, Amber T., advisor; Levinger, Nancy, committee member; Strauss, Steve, committee member; Kipper, Matt, committee member; Bartels, Randy, committee member
Multiple designer peptides, such as RADA-16, have been used as model systems to investigate the chemical parameters that influence protein folding and self-assembly processes. As such, the cause and effect relationship between folding outcomes and folding environmental factors have been extensively investigated. However, the mechanism of the folding process is largely unexplained due to the lack of an analysis technique that can capture structural changes on the time scale of the folding process. This thesis is the first step towards the ability to monitor the protein folding process with atomic structural resolution in real time. In this work, the sample handling capabilities of microfluidic devices are used to expand the experimental range of both infrared (IR) and two dimensional infrared (2D IR) measurement techniques. This includes the development of novel channel designs, overcoming IR compatibility issues, and setting precedent in monitoring chemical processes within microfluidic devices. Microfluidic channel geometries that perform microsecond mixing were developed to allow access to early reaction kinetics. A novel fabrication technique was developed to afford IR analysis methods to be utilized in microfluidic detection schemes. Lastly, model chemical reactions were studied in both Fourier transform IR microspectroscopy (FTIR microspectroscopy) and 2D IR spectroscopy experiments to highlight the applicability of the technology towards a broad range of chemical and biological systems, including the protein folding and self assembly processes.
Open Access
Influence of polymeric nanowire topography on the differentiation of adipose-derived stem cells
(Colorado State University. Libraries, 2014) Trujillo, Nathan Anthony, author; Popat, Ketul, advisor; Williams, John, committee member; Dasi, Lakshmi Prasad, committee member; Kipper, Matt, committee member
Considering the many advances in tissue engineering, there are still significant challenges associated with restructuring, repairing, or replacing damaged tissue in the body. Recently biodegradable synthetic scaffolds have shown to be a promising alternative. However, based on the application, it is essential that the scaffold possess specific surface properties that promote cell-scaffold interactions and aid in extracellular matrix deposition. In this work, we present a novel solvent-free, template-synthesis technique for creating substrate-bound nanowire scaffolds from polycaprolactone, a biocompatible and biodegradable polymer. Nanowire surfaces were also fabricated from polycaprolactone that included 1 wt% hydroxyapatite nanoparticles for osteogenic studies. The fundamental concept behind successful synthetic tissue-engineered scaffolds is to promote progenitor cell migration, adhesion, proliferation, and induce differentiation, extracellular matrix synthesis, and finally integration with host tissue. Recently, lipoaspirate tissue has been identified as a viable alternative source for mesenchymal stem cells because it contains a supportive stroma that can easily be isolated. Adipose derived stem cells can differentiate into a variety of mesodermal lineages including the osteogenic, chondrogenic, and adipogenic phenotypes. The results indicated that during the growth period i.e., initial 7 days of culture, the nanowire surfaces supported adhesion, proliferation, and viability of the cells in addition to morphological changes. Osteogenic, chondrogenic, and adipogenic differentiation potential of adipose derive stem cells was evaluated with and without differentiation supplements to determine the influence of nanowire architecture on mechanotrasnduction. It was determined that nanowire topography stimulated the expression of osteogenic marker proteins, osteocalcin and osteopontin, as well as mineralization and alkaline phosphatase activity.
Open Access
Nitric oxide-releasing or generating surfaces for blood-contacting medical devices
(Colorado State University. Libraries, 2020) Zang, Yanyi, author; Reynolds, Melissa, advisor; Kipper, Matt, committee member; Li, Yan Vivian, committee member; Zabel, Mark, committee member
Medical device-induced thrombosis is a major complication that impairs the expected performance of blood-contacting medical devices. Traditional anticoagulation therapies are used to reduce thrombus formation; however, systemic anticoagulants such as heparin increase the risk of thrombocytopenia or even bleeding, which are detrimental to patients who already have injuries. To address these issues, surface modification has been widely studied to improve the performance of blood-contacting medical devices, ranging from biopassive surfaces to biomimetic surfaces. To date, such modifications are not sufficient to prevent blood clotting alone. Supplementary anticoagulation remains necessary to maintain clot-free surfaces. Nitric oxide (NO) is a well-known signaling molecule that has antiplatelet properties. Our approach is to use surfaces that can either release NO via NO donors or promote NO production via an NO catalyst. In this work, a NO-releasing polyelectrolyte multilayer coating effectively reduces platelet adhesion, platelet activation and delay blood clotting on titania nanotube array surfaces. In addition, NO-releasing polymeric surfaces mediate blood serum protein deposition in a manner that prevents platelet adhesion and platelet activation. However, the NO donors used in these two coatings are photo- and thermo- sensitive, and the NO release is limited by the amount of NO donor added to the coating. To overcome these shortcomings, a copper-based metal organic framework (MOF) was used to infinitely promote NO production from NO donors in the blood. The copper-based MOF polymer coating was successfully applied to the surfaces of extracorporeal life support catheters and circulation tubing via custom coating systems. These copper-based MOF-coating also exhibited inherent antibacterial properties under both static and dynamic flow conditions.
Open Access
Protein crystals as nanotemplating materials
(Colorado State University. Libraries, 2019) Kowalski, Ann, author; Snow, Christopher, advisor; Kipper, Matt, committee member; Peebles, Christie, committee member; Sambur, Justin, committee member
The advancement of nanomaterial development depends on the reliable and scalable synthesis of three dimensional nanostructures and devices. Applications for these materials range from catalysis and energy storage to biomedicine and imaging. Towards the goals of shape-controlled immobilization and synthesis, templating is arising as a promising manufacturing method. With the rise of bionanotechnology, DNA and protein scaffolds can be designed, synthesized, and functionalized to coordinate nanoparticles, enzymes, and other guests in three dimensions, or act as molds for the synthesis of anisotropic nanostructures. Inherently, protein crystals are an attractive target, as they have nearly unlimited designability, intrinsic functionality for a variety of useful materials, and mild reaction conditions. The overarching goal of this work is to explore the feasibility of protein crystals as templates for the creation of biohybrid materials. We show that protein crystals with large solvent channels can strongly adsorb and immobilize gold nanoparticles by reversible metal affinity interactions and that these nanoparticles can serve as nucleation sites for the growth of nanorods within the pores of protein crystals by a variety of gold growth methods. We show that, depending on the method used, gold nanorod synthesis within the crystals can be dependent on the presence of a seed particle. Despite their stability, these crystals can be dissolved to release the gold structures, which can be analyzed by electron microscopy and elemental analysis. A variety of gold nanorod products are formed, from highly anisotropic individual rods, to interconnected rod bundles, to parallel rods embedded within a protein matrix. Additionally, we show that protein crystal pores can be used for the long-term capture of multiple enzymes and that these enzymes retain their activity within the crystal. Product can be separated by a simple washing step, and the immobilized two-enzyme pathway can be used for multiple cycles over several weeks. Rates of product formation are higher for enzymes immobilized within crystals of a high surface-to-volume ratio; thus, the use of micron-sized crystals minimizes transport limitations typically associated with enzyme immobilization. Preliminary work suggests the crystals may also impart significant thermal stability to the embedded enzymes. Porous protein crystals may provide a superior templating method for the development of nanomaterials. Here we further demonstrate the wide variety of applications for protein crystals by revealing their success as scaffolds for immobilization, synthesis, and catalysis.
Open Access
Rhodium(III)-catalyzed amide-directed C-H activation and [4+2] cycloaddition for modular assembly of nitrogen heterocycles
(Colorado State University. Libraries, 2017) Semakul, Natthawat, author; Rovis, Tomislav, advisor; Kennan, Alan, committee member; Shi, Yian, committee member; McCullagh, Martin, committee member; Kipper, Matt, committee member
This dissertation describes the ligand and reaction developments by amide-directed rhodium(III)-catalyzed C(sp2)-H bond activation followed by amidoannulation with alkenes to form nitrogen-containing heterocycles. Chapter 1 details the ligand development for stereoselective synthesis of [4.1.0] dihydroisoquinolones through benzamidation of cyclopropenes mediated by Rh(III) catalysis. Quantum chemical calculations revealed the important role of heptamethylindenyl (Ind*) ligand and O-substituted ester of benzhydroxamate for achieving high diastereoselectivity in cyclopropene insertion. Efforts toward stereoselective synthesis of [4.1.0] dihydroisoquinolones have been also studied by streptavidin-based artificial metalloenzyme. Chapter 2 presents the stereoselective synthesis of [4.2.0] dihydroisoquinolones via the benzamidation of cyclobutenes. The transformation proved to have a broad substrate scope and functional group tolerance that generates the cyclobutane-fused azacycles with excellent diastereoselectivity. The artificial metalloenzymes can render this reaction asymmetric furnishing the dihydroisoquinolone products in moderate enantioselectivity. Chapter 3 communicates Rh(III)-catalyzed C-H activation and [4+2] annulation reaction of N-pivaloyloxy acrylamides with alkenes for an efficient synthesis of α,β-unsaturated-δ-lactams. This process offers a platform for the rapid assembly of a diverse set of delta-lactams from simple and abundant precursors. These lactams could serve as useful building blocks to access substituted piperidines.