Matching Items (13)
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- All Subjects: Analytical Chemistry
- Genre: Academic theses
- Creators: Hayes, Mark
Description
In an effort to begin validating the large number of discovered candidate biomarkers, proteomics is beginning to shift from shotgun proteomic experiments towards targeted proteomic approaches that provide solutions to automation and economic concerns. Such approaches to validate biomarkers necessitate the mass spectrometric analysis of hundreds to thousands of human samples. As this takes place, a serendipitous opportunity has become evident. By the virtue that as one narrows the focus towards "single" protein targets (instead of entire proteomes) using pan-antibody-based enrichment techniques, a discovery science has emerged, so to speak. This is due to the largely unknown context in which "single" proteins exist in blood (i.e. polymorphisms, transcript variants, and posttranslational modifications) and hence, targeted proteomics has applications for established biomarkers. Furthermore, besides protein heterogeneity accounting for interferences with conventional immunometric platforms, it is becoming evident that this formerly hidden dimension of structural information also contains rich-pathobiological information. Consequently, targeted proteomics studies that aim to ascertain a protein's genuine presentation within disease- stratified populations and serve as a stepping-stone within a biomarker translational pipeline are of clinical interest. Roughly 128 million Americans are pre-diabetic, diabetic, and/or have kidney disease and public and private spending for treating these diseases is in the hundreds of billions of dollars. In an effort to create new solutions for the early detection and management of these conditions, described herein is the design, development, and translation of mass spectrometric immunoassays targeted towards diabetes and kidney disease. Population proteomics experiments were performed for the following clinically relevant proteins: insulin, C-peptide, RANTES, and parathyroid hormone. At least thirty-eight protein isoforms were detected. Besides the numerous disease correlations confronted within the disease-stratified cohorts, certain isoforms also appeared to be causally related to the underlying pathophysiology and/or have therapeutic implications. Technical advancements include multiplexed isoform quantification as well a "dual- extraction" methodology for eliminating non-specific proteins while simultaneously validating isoforms. Industrial efforts towards widespread clinical adoption are also described. Consequently, this work lays a foundation for the translation of mass spectrometric immunoassays into the clinical arena and simultaneously presents the most recent advancements concerning the mass spectrometric immunoassay approach.
ContributorsOran, Paul (Author) / Nelson, Randall (Thesis advisor) / Hayes, Mark (Thesis advisor) / Ros, Alexandra (Committee member) / Williams, Peter (Committee member) / Arizona State University (Publisher)
Created2011
Description
Obtaining local electrochemical (EC) information is extremely important for understanding basic surface reactions, and for many applications. Scanning electrochemical microscopy (SECM) can obtain local EC information by scanning a microelectrode across the surface. Although powerful, SECM is slow, the scanning microelectrode may perturb reaction and the measured signal decreases with the size of microelectrode. This thesis demonstrates a new imaging technique based on a principle that is completely different from the conventional EC detection technologies. The technique, referred to as plasmonic-based electrochemical imaging (PECI), images local EC current (both faradaic and non-faradaic) without using a scanning microelectrode. Because PECI response is an optical signal originated from surface plasmon resonance (SPR), PECI is fast and non-invasive and its signal is proportional to incident light intensity, thus does not decrease with the area of interest. A complete theory is developed in this thesis work to describe the relationship between EC current and PECI signal. EC current imaging at various fixed potentials and local cyclic voltammetry methods are developed and demonstrated with real samples. Fast imaging rate (up to 100,000 frames per second) with 0.2×3µm spatial resolution and 0.3 pA detection limit have been achieved. Several PECI applications have been developed to demonstrate the unique strengths of the new imaging technology. For example, trace particles in fingerprint is detected by PECI, a capability that cannot be achieved with the conventional EC technologies. Another example is PECI imaging of EC reaction and interfacial impedance of graphene of different thicknesses. In addition, local square wave voltammetry capability is demonstrated and applied to study local catalytic current of platinum nanoparticle microarray. This thesis also describes a related but different research project that develops a new method to measure surface charge densities of SPR sensor chips, and micro- and nano-particles. A third project of this thesis is to develop a method to expand the conventional SPR detection and imaging technology by including a waveguide mode. This innovation creates a sensitive detection of bulk index of refraction, which overcomes the limitation that the conventional SPR can probe only changes near the sensor surface within ~200 nm.
ContributorsShan, Xiaonan (Author) / Tao, Nongjian (Thesis advisor) / Chae, Junseok (Committee member) / Christen, Jennifer Blain (Committee member) / Hayes, Mark (Committee member) / Arizona State University (Publisher)
Created2011
Description
Mass spectrometric analysis requires that atoms from the sample be ionized in the gas phase. Secondary ion mass spectrometry achieves this by sputtering samples with an energetic primary ion beam. Several investigations of the sputtering and ionization process have been conducted. Oxygen is commonly used in secondary ion mass spectrometry (SIMS) to increase ion yields, but also can complicate the interpretation of SIMS analyses. An 18O implant in silicon has been used to quantify the oxygen concentration at the surface of sputtered silicon in order to study the dependence on oxygen of several sputtering and depth profile phenomena. The ion yield dependence of trace elements in silicon on the surface oxygen concentration is a function of the ionization potential of the element. The ion yield is high and unaffected by oxygen for elements with low ionization potential and ranges over several orders of magnitude for elements with high ionization potential. Depth resolution in sputter profiles has been shown to be degraded by the presence of oxygen, the mechanism of this effect has been investigated using an 18O implant to quantify oxygen levels and it is shown that the process does not appear to be a consequence of surface oxide formation. Molecular ions are a source of mass interference in SIMS analysis, and multiply charged atomic ion signals might be interference-free due to the possible instability of multiply-charged molecular ions. Sputtered SiH2+, AlH2+, BeH2+, Mo22+ and Mg22+ ions have been observed and appear surprisingly stable. The formation mechanism of some of these species has been explored.
ContributorsSobers, Richard Carlisle, Jr (Author) / Williams, Peter (Thesis advisor) / Hayes, Mark (Committee member) / Petuskey, William (Committee member) / Arizona State University (Publisher)
Created2012
Description
Electrophoretic exclusion is a counter-flow gradient focusing method that simultaneously separates and concentrates electrokinetic material at a channel entrance utilizing electric and fluid velocity fields. However, its effectiveness is heavily dependent on the non-uniform field gradients about the entrance. This work assesses the capability of electrophoretic exclusion to capture and enrich small molecules and examines the channel entrance region both quantitatively and qualitatively to better understand the separation dynamics for future design.
A flow injection technique is used to experimentally evaluate electrophoretic exclusion of small molecules. Methyl violet, a cationic dye, and visible spectroscopy are used to monitor flow and electrophoretic dynamics at the entrance region resulting in successful capture and simultaneous enrichment of methyl violet at the channel interface. Investigation of the entrance region is performed using both experiment data and finite element analysis modeling to assess regional flow, electric fields, diffusion, convection, and electrophoretic migration. Longitudinal fluid velocity and electric field gradient magnitudes near the channel entrance are quantified using Particle Tracking Velocimetry (PTV) and charged fluorescent microspheres. Lateral studies using rhodamine 123 concentration monitoring agree qualitatively with simulation results indicating decreased gradient uniformity for both electric and fluid velocity fields closer to the channel wall resulting in a localized concentration enhancement at lower applied voltages than previously observed or predicted. Resolution interrogation from both a theoretical assessment and simulation construct demonstrate resolution improvement with decreased channel width and placement of an electrode directly at the interface. Simulation resolution predictions are in general agreement with early experimental assessments, both suggesting species with electrophoretic mobilities as similar as 10-9 m2/(Vs) can be separated with the current design. These studies have helped evolve the understanding of the interface region and set the foundation for further interface developments.
A flow injection technique is used to experimentally evaluate electrophoretic exclusion of small molecules. Methyl violet, a cationic dye, and visible spectroscopy are used to monitor flow and electrophoretic dynamics at the entrance region resulting in successful capture and simultaneous enrichment of methyl violet at the channel interface. Investigation of the entrance region is performed using both experiment data and finite element analysis modeling to assess regional flow, electric fields, diffusion, convection, and electrophoretic migration. Longitudinal fluid velocity and electric field gradient magnitudes near the channel entrance are quantified using Particle Tracking Velocimetry (PTV) and charged fluorescent microspheres. Lateral studies using rhodamine 123 concentration monitoring agree qualitatively with simulation results indicating decreased gradient uniformity for both electric and fluid velocity fields closer to the channel wall resulting in a localized concentration enhancement at lower applied voltages than previously observed or predicted. Resolution interrogation from both a theoretical assessment and simulation construct demonstrate resolution improvement with decreased channel width and placement of an electrode directly at the interface. Simulation resolution predictions are in general agreement with early experimental assessments, both suggesting species with electrophoretic mobilities as similar as 10-9 m2/(Vs) can be separated with the current design. These studies have helped evolve the understanding of the interface region and set the foundation for further interface developments.
ContributorsKeebaugh, Michael (Author) / Hayes, Mark (Thesis advisor) / Ros, Alexandra (Committee member) / Buttry, Daniel (Committee member) / Arizona State University (Publisher)
Created2015
Description
Efficient separation techniques for organelles and bacteria in the micron- and sub-micron range are required for various analytical challenges. Mitochondria have a wide size range resulting from the sub-populations, some of which may be associated with diseases or aging. However, traditional methods can often not resolve within-species size variations. Strategies to separate mitochondrial sub-populations by size are thus needed to study the importance of this organelle in cellular functions. Additionally, challenges also exist in distinguishing the sub-populations of bio-species which differ in the surface charge while possessing similar size, such as Salmonella typhimurium (Salmonella). The surface charge of Salmonella wild-type is altered upon environmental stimulations, influencing the bacterial survival and virulence within the host tissue. Therefore, it is important to explore methods to identify the sub-populations of Salmonella.
This work exploits insulator-based dielectrophoresis (iDEP) for the manipulation of mitochondria and Salmonella. The iDEP migration and trapping of mitochondria were investigated under both DC and low-frequency AC conditions, establishing that mitochondria exhibit negative DEP. Also, the first realization of size-based iDEP sorting experiments of mitochondria were demonstrated. As for Salmonella, the preliminary study revealed positive DEP behavior. Distinct trapping potential thresholds were found for the sub-populations with different surface charges.
Further, DEP was integrated with a non-intuitive migration mechanism termed absolute negative mobility (ANM), inducing a deterministic trapping component which allows the directed transport of µm- and sub-µm sized (bio)particles in microfluidic devices with a nonlinear post array under the periodic action of electrokinetic and dielectrophoretic forces. Regimes were revealed both numerically and experimentally in which larger particles migrate against the average applied force, whereas smaller particles show normal response. Moreover, this deterministic ANM (dANM) was characterized with polystyrene beads demonstrating improved migration speed at least two orders of magnitude higher compared to previous ANM systems with similar sized colloids. In addition, dANM was induced for mitochondria with an AC-overlaid waveform representing the first demonstration of ANM migration with biological species. Thus, it is envisioned that the efficient size selectivity of this novel migration mechanism can be employed in nanotechnology, organelle sub-population studies or fractionating protein nanocrystals.
This work exploits insulator-based dielectrophoresis (iDEP) for the manipulation of mitochondria and Salmonella. The iDEP migration and trapping of mitochondria were investigated under both DC and low-frequency AC conditions, establishing that mitochondria exhibit negative DEP. Also, the first realization of size-based iDEP sorting experiments of mitochondria were demonstrated. As for Salmonella, the preliminary study revealed positive DEP behavior. Distinct trapping potential thresholds were found for the sub-populations with different surface charges.
Further, DEP was integrated with a non-intuitive migration mechanism termed absolute negative mobility (ANM), inducing a deterministic trapping component which allows the directed transport of µm- and sub-µm sized (bio)particles in microfluidic devices with a nonlinear post array under the periodic action of electrokinetic and dielectrophoretic forces. Regimes were revealed both numerically and experimentally in which larger particles migrate against the average applied force, whereas smaller particles show normal response. Moreover, this deterministic ANM (dANM) was characterized with polystyrene beads demonstrating improved migration speed at least two orders of magnitude higher compared to previous ANM systems with similar sized colloids. In addition, dANM was induced for mitochondria with an AC-overlaid waveform representing the first demonstration of ANM migration with biological species. Thus, it is envisioned that the efficient size selectivity of this novel migration mechanism can be employed in nanotechnology, organelle sub-population studies or fractionating protein nanocrystals.
ContributorsLuo, Jinghui (Author) / Ros, Alexandra (Thesis advisor) / Hayes, Mark (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2015
Description
Disease prevention and personalized treatment will be impacted by the continued integration of protein biomarkers into medical practice. While there are already numerous biomarkers used clinically, the detection of protein biomarkers among complex matrices remains a challenging problem. One very important strategy for improvements in clinical application of biomarkers is separation/preconcentration, impacting the reliability, efficiency and early detection. Electrophoretic exclusion can be used to separate, purify, and concentrate biomarkers. This counterflow gradient technique exploits hydrodynamic flow and electrophoretic forces to exclude, enrich, and separate analytes. The development of this technique has evolved onto an array-based microfluidic platform which offers a greater range of geometries/configurations for optimization and expanded capabilities and applications. Toward this end of expanded capabilities, fundamental studies of subtle changes to the entrance flow and electric field configurations are investigated. Three closely related microfluidic interfaces are modeled, fabricated and tested. A charged fluorescent dye is used as a sensitive and accurate probe to test the concentration variation at these interfaces. Models and experiments focus on visualizing the concentration profile in areas of high temporal dynamics, and show strong qualitative agreement, which suggests the theoretical assessment capabilities can be used to faithfully design novel and more efficient interfaces. Microfluidic electrophoretic separation technique can be combined with electron microscopy as a protein concentration/purification step aiding in sample preparation. The integrated system with grids embedded into the microdevice reduces the amount of time required for sample preparation to less than five minutes. Spatially separated and preconcentrated proteins are transferred directly from an upstream reservoir onto grids. Dilute concentration as low as 0.005 mg/mL can be manipulated to achieve meaningful results. Selective concentration of one protein from a mixture of two proteins is also demonstrated. Electrophoretic exclusion is also used for biomarker applications. Experiments using a single biomarker are conducted to assess the ability of the microdevice for enrichment in central reservoirs. A mixture of two protein biomarkers are performed to evaluate the proficiency of the device for separations capability. Moreover, a battery is able to power the microdevice, which facilitates the future application as a portable device.
ContributorsZhu, Fanyi (Author) / Hayes, Mark (Thesis advisor) / Ros, Alexandra (Committee member) / Buttry, Daniel (Committee member) / Arizona State University (Publisher)
Created2019
Description
Glycans are monosaccharide-based heteropolymers that are found covalently attached to many different proteins and lipids and are ubiquitously displayed on the exterior surfaces of cells. Serum glycan composition and structure are well known to be altered in many different types of cancer. In fact, glycans represent a promising but only marginally accessed source of cancer markers. The approach used in this dissertation, which is referred to as “glycan node analysis”, is a molecularly bottom-up approach to plasma/serum (P/S) glycomics based on glycan linkage analysis that captures features such as α2-6 sialylation, β1-6 branching, and core fucosylation as single analytical signals.
The diagnostic utility of this approach as applied to lung cancer patients across all stages as well as prostate, serous ovarian, and pancreatic cancer patients compared to certifiably healthy individuals, nominally healthy individuals and/or risk-matched controls is reported. Markers for terminal fucosylation, α2-6 sialylation, β1-4 branching, β1-6 branching and outer-arm fucosylation were most able to differentiate cases from controls. These markers behaved in a stage-dependent manner in lung cancer as well as other types of cancer. Using a Cox proportional hazards regression model, the ability of these markers to predict progression and survival in lung cancer patients was assessed. In addition, the potential mechanistic role of aberrant P/S glycans in cancer progression is discussed.
Plasma samples from former bladder cancer patients with currently no evidence of disease (NED), non-muscle invasive bladder cancer (NMIBC), and muscle invasive bladder cancer (MIBC) along with certifiably healthy controls were analyzed. Markers for α2-6 sialylation, β1-4 branching, β1-6 branching, and outer-arm fucosylation were able to separate current and former (NED) cases from controls; but NED, NMIBC, and MIBC were not distinguished from one another. Markers for α2-6 sialylation and β1-6 branching were able to predict recurrence from the NED state using a Cox proportional hazards regression model adjusted for age, gender, and time from cancer. These two glycan features were found to be correlated to the concentration of C-reactive protein, a known prognostic marker for bladder cancer, further strengthening the link between inflammation and abnormal plasma protein glycosylation.
The diagnostic utility of this approach as applied to lung cancer patients across all stages as well as prostate, serous ovarian, and pancreatic cancer patients compared to certifiably healthy individuals, nominally healthy individuals and/or risk-matched controls is reported. Markers for terminal fucosylation, α2-6 sialylation, β1-4 branching, β1-6 branching and outer-arm fucosylation were most able to differentiate cases from controls. These markers behaved in a stage-dependent manner in lung cancer as well as other types of cancer. Using a Cox proportional hazards regression model, the ability of these markers to predict progression and survival in lung cancer patients was assessed. In addition, the potential mechanistic role of aberrant P/S glycans in cancer progression is discussed.
Plasma samples from former bladder cancer patients with currently no evidence of disease (NED), non-muscle invasive bladder cancer (NMIBC), and muscle invasive bladder cancer (MIBC) along with certifiably healthy controls were analyzed. Markers for α2-6 sialylation, β1-4 branching, β1-6 branching, and outer-arm fucosylation were able to separate current and former (NED) cases from controls; but NED, NMIBC, and MIBC were not distinguished from one another. Markers for α2-6 sialylation and β1-6 branching were able to predict recurrence from the NED state using a Cox proportional hazards regression model adjusted for age, gender, and time from cancer. These two glycan features were found to be correlated to the concentration of C-reactive protein, a known prognostic marker for bladder cancer, further strengthening the link between inflammation and abnormal plasma protein glycosylation.
ContributorsRoshdiferdosi, Shadi (Author) / Borges, Chad R (Thesis advisor) / Woodbury, Neal (Committee member) / Hayes, Mark (Committee member) / Arizona State University (Publisher)
Created2018
Description
Biological fluids contain information-rich mixtures of biochemicals and particles such as cells, proteins, and viruses. Selective and sensitive analysis of these fluids can enable clinicians to accurately diagnose a wide range of pathologies. Fluid samples such as these present an intriguing challenge to researchers; they are packed with potentially vital information, but notoriously difficult to analyze. Rapid and inexpensive analysis of blood and other bodily fluids is a topic gaining substantial attention in both science and medicine. Current limitations to many analyses include long culture times, expensive reagents, and the need for specialized laboratory facilities and personnel. Improving these tests and overcoming their limitations would allow faster and more widespread testing for disease and pathogens, potentially providing a significant advantage for healthcare in many settings.
Both gradient separation techniques and dielectrophoresis can solve some of the difficulties presented by complex biological samples, thanks to selective capture, isolation, and concentration of analytes. By merging dielectrophoresis with a gradient separation-based approach, gradient insulator dielectrophoresis (g-iDEP) promises benefits in the form of rapid and specific separation of extremely similar bioparticles. High-resolution capture can be achieved by exploiting variations in the characteristic physical properties of cells and other bioparticles.
Novel implementation and application of the technique has demonstrated the isolation and concentration of blood cells from a complex biological sample, differentiation of bacterial strains within a single species, and separation of antibiotic-resistant and antibiotic-susceptible bacteria. Furthermore, this approach allows simultaneous concentration of analyte, facilitating detection and downstream analysis. A theoretical description of the resolving capabilities of g-iDEP was also developed. This theory explores the relationship between experimental parameters and resolution. Results indicate the possibility of differentiating particles with dielectrophoretic mobilities that differ by as little as one part in 100,000,000, or electrophoretic mobilities differing by as little as one part in 100,000. These results indicate the potential g-iDEP holds in terms of both separatory power and the possibility for diagnostic applications.
Both gradient separation techniques and dielectrophoresis can solve some of the difficulties presented by complex biological samples, thanks to selective capture, isolation, and concentration of analytes. By merging dielectrophoresis with a gradient separation-based approach, gradient insulator dielectrophoresis (g-iDEP) promises benefits in the form of rapid and specific separation of extremely similar bioparticles. High-resolution capture can be achieved by exploiting variations in the characteristic physical properties of cells and other bioparticles.
Novel implementation and application of the technique has demonstrated the isolation and concentration of blood cells from a complex biological sample, differentiation of bacterial strains within a single species, and separation of antibiotic-resistant and antibiotic-susceptible bacteria. Furthermore, this approach allows simultaneous concentration of analyte, facilitating detection and downstream analysis. A theoretical description of the resolving capabilities of g-iDEP was also developed. This theory explores the relationship between experimental parameters and resolution. Results indicate the possibility of differentiating particles with dielectrophoretic mobilities that differ by as little as one part in 100,000,000, or electrophoretic mobilities differing by as little as one part in 100,000. These results indicate the potential g-iDEP holds in terms of both separatory power and the possibility for diagnostic applications.
ContributorsJones, Paul Vernon (Author) / Hayes, Mark (Thesis advisor) / Ros, Alexandra (Committee member) / Herckes, Pierre (Committee member) / Arizona State University (Publisher)
Created2015
Description
Volcanic devolatilization is one of the major processes in the global nitrogen cycle. Past studies have often estimated the magnitude of this flux using volcanic emission measurements, which are limited to currently active systems and sensitive to atmospheric contamination. A different methodological approach requires appropriate analytical parameters for nitrogen analysis in silicate glasses by secondary ion mass spectrometry (SIMS), which have not yet been established. To this end, we analyze various ion implanted basaltic and rhyolitic glasses by SIMS. We demonstrate that water content significantly affects the ion yields of 14N+ and 14N16O−, as well as the background intensity of 14N+ and 12C+. Application of implant-derived calibrations to natural samples provide the first reported concentrations of nitrogen in melt inclusions. These measurements are from samples from the Bishop Tuff in California, the Huckleberry Ridge Tuff of the Yellowstone Volcanic Center, and material from the Okaia and Oruanui eruptions in the Taupo Volcanic Center. In all studied material, we find maximum nitrogen contents of less than 45 ppm and that nitrogen concentration varies positively with CO2 concentration, which is interpreted to reflect partial degassing trend. Using the maximum measured nitrogen contents for each eruption, we find that the Bishop released >3.6 x 1013 g of nitrogen, the Huckleberry Ridge released >1.3 x 1014 g, the Okaia released >1.1 x 1011 g of nitrogen, the Oruanui released >4.7 x 1013 g of nitrogen. Simple calculations suggest that with concentrations such as these, rhyolitic eruptions may ephemerally increase the nitrogen flux to the atmosphere, but are insignificant compared to the 4 x 1021 g of nitrogen stored in the atmosphere.
ContributorsRegier, Margo Elaine (Author) / Hervig, Richard L (Thesis advisor) / Roggensack, Kurt (Committee member) / Till, Christy B. (Committee member) / Arizona State University (Publisher)
Created2016
Description
Microfluidic platforms have been exploited extensively as a tool for the separation of particles by electric field manipulation. Microfluidic devices can facilitate the manipulation of particles by dielectrophoresis. Separation of particles by size and type has been demonstrated by insulator-based dielectrophoresis in a microfluidic device. Thus, manipulating particles by size has been widely studied throughout the years. It has been shown that size-heterogeneity in organelles has been linked to multiple diseases from abnormal organelle size. Here, a mixture of two sizes of polystyrene beads (0.28 and 0.87 μm) was separated by a ratchet migration mechanism under a continuous flow (20 nL/min). Furthermore, to achieve high-throughput separation, different ratchet devices were designed to achieve high-volume separation. Recently, enormous efforts have been made to manipulate small size DNA and proteins. Here, a microfluidic device comprising of multiple valves acting as insulating constrictions when a potential is applied is presented. The tunability of the electric field gradient is evaluated by a COMSOL model, indicating that high electric field gradients can be reached by deflecting the valve at a certain distance. Experimentally, the tunability of the dynamic constriction was demonstrated by conducting a pressure study to estimate the gap distance between the valve and the substrate at different applied pressures. Finally, as a proof of principle, 0.87 μm polystyrene beads were manipulated by dielectrophoresis. These microfluidic platforms will aid in the understanding of size-heterogeneity of organelles for biomolecular assessment and achieve separation of nanometer-size DNA and proteins by dielectrophoresis.
ContributorsOrtiz, Ricardo (Author) / Ros, Alexandra (Thesis advisor) / Hayes, Mark (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2021