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Transition metal oxynitrides are of growing interest for their use as electrocatalyst for nitrogen reduction reaction. The metals in the oxynitride used for catalytic process are stabilized in intermediate state for effective activation of nitrogen. Therefore, studying the interaction of metal oxynitrides films to ambient exposure is necessary. Here, sputter deposited vanadium oxynitride is compared to cobalt oxynitride using insitu Auger electron spectroscopy (AES), ex situ X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and scanning electron microscopy (SEM). After deposition in Ar/N2 environment, in situ AES spectra indicate that film is vanadium oxynitride despite oxygen is not the reactive gas. In contrast, in situ AES indicate film is pure cobalt nitride at the same base pressure and deposition condition (as vanadium). For ambient exposure, in situ AES indicate the incorporation of oxygen in the cobalt nitride film to form cobalt oxynitride. Ex situ XPS indicate both films get more oxidized but uniformly distributed as there is only slight difference in grazing and normal emission XPS. XRD and SEM also indicate how homogeneously distributed both films are. These finding confirms how important it is that transition metal centers are kept in intermediate oxidation state for the activation of nitrogen bond.

Synthetic cannabinoids are recreational drugs designed to mimic the effects of Δ9-tetrahydrocannabinol (THC), the main psychoactive component present in cannabis. These drugs exhibit severe toxic effects upon consumption due to their high binding affinity and potency at the cannabinoid receptors (CB1 and CB2). Synthetic cannabinoids have proliferated over the last decade and become a major public health and analytical challenge, critically impacting the clinical and forensic communities. Indazole carboxamide and indole carboxamide class synthetic cannabinoids have been particularly rampant, and are the compound classes most frequently reported to governmental agencies worldwide. However, the metabolic and pharmacological properties of many of these compounds remains unknown. Elucidating these characteristics allows members of the clinical and forensic communities to identify causative agents in patient samples, as well as render conclusions regarding their toxic effects. The aim of this research study was to assess the in vitro Phase I metabolic profile of five synthetic cannabinoids and report the major metabolites identified; compounds evaluated included MDMB-CHNINACA; APP-CHMINACA (PX-3); 5F-APP-PICA (PX-1); 5F-MDMB-PINACA (5F-ADB); and FUB-AMB. These analytes were incubated for 120 minutes with human liver microsomes, followed by analysis of the extracts via ultra high performance liquid chromatography – tandem mass spectrometry (UHPLC-MS/MS). The high-resolution mass spectrometry tool utilized (quadrupole-time of flight mass spectrometry, QTOF) allowed for a thorough characterization of the metabolites, including the assignment of a chemical formula and structure, and accurate mass. The metabolic stability and kinetic profiles of 5F-ADB and FUB-AMB were evaluated by aliquoting the incubation samples at various time points throughout the procedure. It was observed that these compounds were metabolized rapidly, resulting in short half-lives and relatively elevated metabolic clearances. A variety of metabolites were identified for most of the species studied, and this was dependent on the chemical structure of the parent molecule. The major metabolites identified overall for the species were products of amide or ester hydrolysis; hydroxylation (including polyhydroxylation) of the pentyl side chain or cyclohexylmethyl moiety; and oxidative defluorination. It is proposed that these metabolites (especially analyte-specific metabolite) be included in laboratory assay panels to facilitate unequivocal identification of the synthetic cannabinoid agent of interest. For select compounds (5F-ADB and FUB-AMB), authentic forensic human blood samples which screened positive for these analytes were provided by a renowned forensic toxicology laboratory. These samples were tested to verify that the major metabolites identified in the in vitro studies were also present in blood in vivo; the resultant data from the 5F-ADB and FUB-AMB samples showed that the major hydroxylated and hydrolysis metabolite, respectively, were present in greater abundance than the parent molecule, which was most often absent or not present in an appreciable quantity. Additionally, it was observed in the time studies of 5F-ADB and FUB-AMB that the metabolites containing carboxylic acid functional groups were detected in incubation samples longer than the hydroxylated metabolites, potentially indicative of longer detection windows in human samples. These findings have important toxicological implications; many synthetic cannabinoid metabolites, including those identified in this study may have pharmacological activity and contribute to a drug user’s overall impairment profile; identifying them in blood in the absence of parent compound can point to the causative agent. The results demonstrate that it is imperative that synthetic cannabinoid assays screen for known pharmacologically active metabolites; this is particularly important for drugs with short half-lives. The results of this research can be applied to the prediction of metabolic pathways for synthetic cannabinoids as well as non-drug substances with similar structural elements whose metabolic profile has not yet been elucidated, and whose pharmacological activity is currently unknown. Additionally, the results provide reference standard manufacturers and research scientists with further insight into the metabolic products of synthetic cannabinoids and related compounds for the synthesis of materials for the development of laboratory assays. / Chemistry

Master of Science / Department of Chemistry / Christopher T. Culbertson / A microfluidic device integrated with valves and a peristaltic pump was fabricated using multilayer soft lithography to analyze single cells. Fluid flow was generated and mammalian cells were transported through the channel manifold using the peristaltic pump. A laser beam was focused at the cross-section of the channels so fluorescence of individual labeled intact cells could be detected. Triggered by the fluorescence signals of intact cells, valves could be actuated so fluid flow was stopped and a single cell was trapped at the intersection. The cell was then rapidly lysed through the application of large electric fields and injected into a separation channel. Various conditions such as channel geometry, pumping frequency, control channel size, and pump location were optimized for cell transport. A Labview program was developed to control the actuation of the trapping valves and a control device was fabricated for operation of the peristaltic pump. Cells were labeled with a cytosolic dye, Calcein AM or Oregon Green, and cell transport and lysis were visualized using epi-fluorescent microscope. The cells were transported at rates of [simular to] 1mm/s. This rate was optimized to obtain both high throughput and single cell trapping. An electric field of 850-900 V/cm was applied so cells could be efficiently lysed and cell lysate could be electrophoretically separated. Calcein AM and Oregon Green released from single cells were separated and detected by laser-induced fluorescence. The fluorescence signals were collected by PMT and sampled with a multi-function I/O card. This analyzing method using microchip may be applied to explore other cellular contents from single cells in the future.

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / Microfluidic devices have a wide variety of biological applications. My Ph.D. dissertation focuses on three major projects. A) culturing a non-adherent immortal cell line within a microfluidic device under static and dynamic media flow conditions; B) designing and fabricating novel microfluidic devices for electrokinetic injecting analytes from a hydrodynamic fluid; and C) using this novel injection method to lyse single non-adherent cells by applying a high electric field across the cell at a microfluidic channel intersection. There are several potential advantages to the use of microfluidic devices for the analysis of single cells: First, cells can be handled with care and precision while being transported in the microfluidic channels. Second, cell culturing, handling, and analysis can be integrated together in a single, compact microfluidic device. Third, cell culturing and analysis in microfluidic devices uses only extremely small volumes of culturing media and analysis buffer. In this dissertation a non-adherent immortal cell line was studied under static media flow conditions inside a CO[subscript]2 incubator and under dynamic media flow conditions in a novel portable cell culture chamber. To culture cells they must first be trapped on a microfluidic device. To attempt to successfully trap cells, three different types of cellular traps were designed, fabricated and tested in polydimethylsiloxane (PDMS)-based microfluidic devices. In the first generation device, cubic-shaped traps were used. After 48 h of culturing in these devices the cell viability of 79 [plus or minus] 6 % (n = 3). In the second generation device, circular wells with narrow connecting channels were employed. However, after 12 h of culturing, no viable cells were found. While the second generation device was not capable of successfully culturing cells, it did demonstrate the importance of culturing under dynamic conditions which lead to next design. The third generation microfluidic device consisted of hydrodynamic shaped traps that were used to culture the cells in a less confined environment. The cell viability after 12 h in this design was 29 [plus or minus] 41% (n = 3). In addition to cell trapping, a novel electrokinetic injection method was developed for injecting analytes from a hydrodynamic flow into a separation channel that was followed by an electrokinetic separation. As the hydrodynamic flow could introduce some excess band broadening in the separation, the actual band broadening of an analyte was measured for different channel depths and hydrodynamic fluid flow rates. The results consistently showed that the separations performed on these devices were diffusion limited. Finally, using this novel injection method, single cell lysis was performed by applying a high voltage at the microfluidic channel intersection. The results of these studies may eventually be applied to help answer some fundamental questions in the areas of biochemistry and pharmaceutical science.

Master of Science / Department of Chemistry / Christopher T. Culbertson / The study of aphid salivary secretome has practical importance on understanding interactions of aphids and their host plants. Around 250 species of aphids out of the identified 4000 aphid species are considered as serious pests. The experiments were performed with pea aphids (Acyrthosiphon pisum) that were feeding on bean plants (Vivia fabe). Pea aphids feed on plant phloem sap by probing their stylet into the sieve elements of the plant and secreting saliva for external digestion. In order to collect aphid salivary proteins from the secreted saliva, small scale and large scale sample collection methods were carried out. The small scale sample method was performed in microfluidic devices using 10-25 aphids. Aphids were able to feed on the artificial diet by probing through a stretched ParafilmTM and survived for 2-3 days in the microfluidic devices. The experiments proved that the aphid survival and feeding rate could be improved with the factors such as ventilation, light intensity and increasing diet volume. However it was difficult to collect sufficient amounts of aphid saliva for detection using small scale devices. The large scale sample collection method was performed by feeding 8000 aphids in large screened chamber for 24/48h. The collected salivary samples after undergone a concentration process was capable of collecting detectable aphid salivary secretions. The experimental conditions were adjusted to obtain optimized HPLC separations. Finally, LC/MS/MS followed by peptide sequence database searching were able to identify potential aphid salivary proteins.

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / Chemical separations are a necessary component in many scientific analyses. Microfluidics, the use of micron-sized fluidic channels defined in glass or polymer blends, is a powerful branch of separation science that is developing rapidly. Miniaturized analytical devices offer important advantages compared to traditional bench-top techniques, most notably capillary electrophoresis (CE). This dissertation was focused on developing several novel methods to improve microfluidic based separations and techniques. The electrophoretic separation of small similarly charged analytes can be very difficult. Chapter 2 discusses a new buffer that has been developed for fast, high efficiency separations of amino acids by micellar electrokinetic chromatography (MEKC). This buffer is more environmentally friendly than the most commonly used surfactant containing buffers for MEKC separations. It uses a commercially available dish washing soap by Seventh Generation™ Inc. that contains three micelle forming agents; sodium lauryl ether sulfate (anionic), cocamidopropyl betaine (zwitterionic), and cocamide monoethanolamine (MEA) (non-ionic), and is completely void of organic solvents. Many biological samples contain analytes below the limit of detection of traditional detection systems; therefore, chapter 3 reports the fabrication of nanoporous membranes on microfluidic devices that are capable of analyte concentration enrichment. Donnan exclusion is responsible for the preconcentration of fluorescent dyes near a charged, porous titania membrane. The level of analyte enrichment was monitored, and enrichment factors greater than 4000 in 400 s were obtained for 2,7-Dichlorofluorescein. Chapter 4 describes the fabrication and characterization of paper based microfluidic devices. Mixtures of acrylate modified photocurable polymers were used to photolithographically define channels on multiple paper substrates. Flow characteristics are described and their use for monitoring complications associated with type 1 diabetes is demonstrated. Finally in Chapter 5, Sol-gel modified gold surfaces for preventing protein adsorption during surface plasmon resonance (SPR) detection are also presented.

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / During the last five years, great advancements in microfluidics have been achieved with the development of “sample-in-answer-out” systems. Such systems have begun to realize the true potential of analytical miniaturization since the concept of the “micro-Total Analysis System” was first envisioned. These systems are characterized by the elegant integration of multiple fluid-handling channel architectures that enable serial execution of sample preparation, separation and detection techniques on a single device. While miniaturization and portability are often identified as key advantages for microfluidics, these highly integrated systems are heavily reliant upon large off-chip equipment, i.e. the microchip is often tethered to the laboratory via multiple syringe pumps, vacuum pumps, solenoid valves, gas cylinders and high voltage power supplies. In this dissertation, a procedure for the facile integration of dielectric elastomer (DE) actuators (called IDEAs) onto microfluidic devices is described. Poly(dimethylsiloxane) (PDMS) is commonly used as a microchip substrate because it is cheap and easy to fabricate, mechanically robust and optically transparent. The operation of an IDEA exploits the ability of PDMS to behave as a smart material and deform in the presence of an electric field. In Chapter 2, the fabrication of IDEA units on a standard microchip electrophoresis device is described. IDEA-derived injections were used to evaluate the physical performance of this novel actuator configuration. In Chapter 3, the analytical merits of IDEA-derived injections were evaluated. Sampling bias caused by electokinetic injection techniques has been problematic for conventional microchip electrophoresis systems due to the lack of fluid access. The hydrodynamic injections created by IDEA operation were found to be highly reproducible, efficient, and possess a negligible degree of sampling bias. In Chapter 4, the spatial characteristics of microchannel deformation due to IDEA actuation have been investigated using fluorescence microscopy. It was determined that the DE compresses more along the edge of the channel than in the middle of the channel. This information can be used to design a new generation of more efficient IDEAs.

Doctor of Philosophy / Department of Chemistry / Daniel A. Higgins / This doctoral thesis covers the synthesis, preparation, and characterization of a series of four perylene diimide derivatives, and the nanofibrous composite materials formed by these perylene diimides when complexed with oppositely charged polyelectrolytes. The perylene diimides include a symmetric dication (TAPDI2+), a symmetric dianion (PDISO32-), and two singly charged asymmetric varieties (C11OPDI+ and C7OPDI+) that contain a hydrophilic head group and hydrophobic ether tail. For all studies presented in the following chapters, poly(acrylate) (PA-) or poly(diallyldimethylammonium) chloride (PDDA+) are used as the polyelectrolytes (PEs). The patterned deposition of sheer aligned, nanofibrous material within a fluidic device is conclusively demonstrated. Thin films of the nanofibrous composite are prepared from aqueous solutions of the semiconducting perylene diimides and oppositely charged polyelectrolyte precursors. By sequentially exposing a clean glass substrate to the cationic and anionic precursor solutions, a thin film of composite material is deposited in a layer-by-layer fashion. By utilizing electrostatic self-assembly (ESA) and layer-by-layer (LbL) procedures, precise control of film thickness and optical density are obtained. The effect of perylene diimide structure and charge on resultant composite film morphology is explored. Through spectroscopic and microscopic studies of bulk perylene diimide solutions and composite thin films, it was determined that the formation of these fibrous materials is dependent on the aggregation of the PDI within the precursor solutions. The molecular orientation of the perylene diimide within the composite nanofiber was determined to be perpendicular to the fiber long axis. For the special case of C7OPDI+/PA- composite, flow induced fiber alignment was observed for both dip coated and flow coated samples. The influence of solution flow profile, PE molecular weight (MW), and PDI structure on deposition efficiency, macroscopic and microscopic morphology, and the potential for nanofiber alignment are investigated. Film formation mechanisms involving two unique routes are also presented.

<p> X-ray absorption spectromicroscopy combines microscopy and spectroscopy to provide rich information about the chemical organization of materials down to the nanoscale. But with richness also comes complexity: natural materials such as biological or environmental science specimens can be composed of complex spectroscopic mixtures of different materials. The challenge becomes how we could meaningfully simplify and interpret this information. Approaches such as principal component analysis and cluster analysis have been used in previous studies, but with some limitations that we will describe. This leads us to develop a new approach based on a development of non-negative matrix approximation (NNMA) analysis with both sparseness and spectra similarity regularizations. We apply this new technique to simulated spectromicroscopy datasets as well as a preliminary study of the large-scale biochemical organization of a human sperm cell. NNMA analysis is able to select major features of the sperm cell without the physically erroneous negative weightings or thicknesses in the calculated image which appeared in previous approaches.</p>