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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

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
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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

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.
ContributorsKeebaugh, Michael (Author) / Hayes, Mark (Thesis advisor) / Ros, Alexandra (Committee member) / Buttry, Daniel (Committee member) / Arizona State University (Publisher)
Created2015
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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

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.
ContributorsLuo, Jinghui (Author) / Ros, Alexandra (Thesis advisor) / Hayes, Mark (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2015
Description
DNA and DNA nanoassemblies such as DNA origamis have large potential in biosensing, drug delivery, nanoelectronic circuits, and biological computing requiring suitable methods for migration and precise positioning. Insulator-based dielectrophoresis (iDEP) provides an efficient and matrix-free approach for manipulation of micro-and nanometer-sized objects. In order to exploit iDEP for naturally

DNA and DNA nanoassemblies such as DNA origamis have large potential in biosensing, drug delivery, nanoelectronic circuits, and biological computing requiring suitable methods for migration and precise positioning. Insulator-based dielectrophoresis (iDEP) provides an efficient and matrix-free approach for manipulation of micro-and nanometer-sized objects. In order to exploit iDEP for naturally formed DNA and DNA nanoassemblies, a detailed understanding of the underlying polarization and dielectrophoretic migration is essential. The shape and the counterion distribution are considered two essential factors in the polarization mechanism. Here, the dielectrophoretic behavior of 6-helix bundle (6HxB) and triangle DNA origamis with identical sequences but substantial topological differences was explored. The polarizability models were discussed for the two species according to their structural difference. The experimental observations reveal distinct iDEP trapping behavior in low frequency AC electric fields in addition to numerical simulations showing a considerable contribution of the electrophoretic transport of the DNA origami species in the DEP trapping regions. Furthermore, the polarizabilities of the two species were determined by measuring the migration times through a potential landscape exhibiting dielectrophoretic barriers. The resulting migration times correlate to the depth of the dielectrophoretic potential barrier and the escape characteristics of the DNA origamis according to an adapted Kramer’s rate model. The orientations of both species in the escape process were studied. Finally, to study the counterion distribution around the DNA molecules, both λ-DNA and 6HxB DNA were used in a phosphate buffer containing magnesium, revealing distinctive negative dielectrophoretic trapping behavior as opposed to positive trapping in a sodium/potassium phosphate buffer system.
ContributorsGan, Lin (Author) / Ros, Alexandra (Thesis advisor) / Buttry, Daniel (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2015
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Description
Microfluidics has shown great potential in rapid isolation, sorting, and concentration of bioparticles upon its discovery. Over the past decades, significant improvements have been made in device fabrication techniques and microfluidic methodologies. As a result, considerable microfluidic-based isolation and concentration techniques have been developed, particularly for rapid pathogen detection. Among

Microfluidics has shown great potential in rapid isolation, sorting, and concentration of bioparticles upon its discovery. Over the past decades, significant improvements have been made in device fabrication techniques and microfluidic methodologies. As a result, considerable microfluidic-based isolation and concentration techniques have been developed, particularly for rapid pathogen detection. Among all microfluidic techniques, dielectrophoresis (DEP) is one of the most effective and efficient techniques to quickly isolate and separate polarizable particles under inhomogeneous electric field. To date, extensive studies have demonstrated that DEP devices are able to precisely manipulate cells ranging from over 10 μm (mammalian cells) down to about 1 μm (small bacteria). However, very limited DEP studies on manipulating submicron bioparticles, such as viruses, have been reported.

In this dissertation, rapid capture and concentration of two different and representative types of virus particles (Sindbis virus and bacteriophage M13) with gradient insulator-based DEP (g-iDEP) has been demonstrated. Sindbis virus has a near-spherical shape with a diameter ~68 nm, while bacteriophage M13 has a filamentous shape with a length ~900 nm and a diameter ~6 nm. Under specific g-iDEP experimental conditions, the concentration of Sindbis virus can be increased two to six times within only a few seconds, using easily accessible voltages as low as 70 V. A similar phenomenon is also observed with bacteriophage M13. Meanwhile, their different DEP behavior predicts the potential of separating viruses with carefully designed microchannels and choices of experimental condition.

DEP-based microfluidics also shows great potential in manipulating blood samples, specifically rapid separations of blood cells and proteins. To investigate the ability of g-iDEP device in blood sample manipulation, some proofs of principle work was accomplished including separating two cardiac disease-related proteins (myoglobin and heart-type fatty acid binding protein) and red blood cells (RBCs). Consistent separation was observed, showing retention of RBCs and passage of the two spiked protein biomarkers. The numerical concentration of RBCs was reduced (~70 percent after one minute) with the purified proteins available for detection or further processing. This study explores and extends the use of the device from differentiating similar particles to acting as a sample pretreatment step.
ContributorsDing, Jie (Author) / Hayes, Mark A. (Thesis advisor) / Ros, Alexandra (Committee member) / Buttry, Daniel A (Committee member) / Arizona State University (Publisher)
Created2017
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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

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
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Description
Single-cell proteomics and transcriptomics analysis are crucial to gain insights of

healthy physiology and disease pathogenesis. The comprehensive profiling of biomolecules in individual cells of a heterogeneous system can provide deep insights into many important biological questions, such as the distinct cellular compositions or regulation of inter- and intracellular signaling pathways

Single-cell proteomics and transcriptomics analysis are crucial to gain insights of

healthy physiology and disease pathogenesis. The comprehensive profiling of biomolecules in individual cells of a heterogeneous system can provide deep insights into many important biological questions, such as the distinct cellular compositions or regulation of inter- and intracellular signaling pathways of healthy and diseased tissues. With multidimensional molecular imaging of many different biomarkers in patient biopsies, diseases can be accurately diagnosed to guide the selection of the ideal treatment.

As an urgent need to advance single-cell analysis, imaging-based technologies have been developed to detect and quantify multiple DNA, RNA and protein molecules in single cell in situ. Novel fluorescent probes have been designed and synthesized, which targets specifically either their nucleic acid counterpart or protein epitopes. These highly multiplexed imaging-based platforms have the potential to detect and quantify 100 different protein molecules and 1000 different nucleic acids in a single cell.

Using novel fluorescent probes, a large number of biomolecules have been detected and quantified in formalin-fixed paraffin-embedded (FFPE) brain tissue at single-cell resolution. By studying protein expression levels, neuronal heterogeneity has been revealed in distinct subregions of human hippocampus.
ContributorsMondal, Manas (Author) / Guo, Jia (Thesis advisor) / Gould, Ian (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2018
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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

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.
ContributorsJones, Paul Vernon (Author) / Hayes, Mark (Thesis advisor) / Ros, Alexandra (Committee member) / Herckes, Pierre (Committee member) / Arizona State University (Publisher)
Created2015
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Description
Biological fluids, in particular blood plasma, provide a vital source of information on the state of human health. While specific detection of biomarker species can aid in disease diagnostics, the complexity of plasma makes analysis challenging. Despite the challenge of complex sample analysis, biomarker quantification has become a primary interest

Biological fluids, in particular blood plasma, provide a vital source of information on the state of human health. While specific detection of biomarker species can aid in disease diagnostics, the complexity of plasma makes analysis challenging. Despite the challenge of complex sample analysis, biomarker quantification has become a primary interest in biomedical analysis. Due to the extremely specific interaction between antibody and analyte, immunoassays are attractive for the analysis of these samples and have gained popularity since their initial introduction several decades ago. Current limitations to diagnostics through blood testing include long incubation times, interference from non-specific binding, and the requirement for specialized instrumentation and personnel. Optimizing the features of immunoassay for diagnostic testing and biomarker quantification would enable early and accurate detection of disease and afford rapid intervention, potentially improving patient outcomes. Improving the limit of quantitation for immunoassay has been the primary goal of many diverse experimental platforms. While the ability to accurately quantify low abundance species in a complex biological sample is of the utmost importance in diagnostic testing, models illustrating experimental limitations have relied on mathematical fittings, which cannot be directly related to finite analytical limits or fundamental relationships. By creating models based on the law of mass action, it is demonstrated that fundamental limitations are imposed by molecular shot noise, creating a finite statistical limitation to quantitative abilities. Regardless of sample volume, 131 molecules are necessary for quantitation to take place with acceptable levels of uncertainty. Understanding the fundamental limitations of the technique can aid in the design of immunoassay platforms, and assess progress toward the development of optimal diagnostic testing. A sandwich-type immunoassay was developed and tested on three separate human protein targets: myoglobin, heart-type fatty acid binding protein, and cardiac troponin I, achieving superior limits of quantitation approaching ultimate limitations. Furthermore, this approach is compatible with upstream sample separation methods, enabling the isolation of target molecules from a complex biological sample. Isolation of target species prior to analysis allows for the multiplex detection of biomarker panels in a microscale device, making the full optimization of immunoassay techniques possible for clinical diagnostics.
ContributorsWoolley, Christine F (Author) / Hayes, Mark A. (Thesis advisor) / Ros, Alexandra (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2015
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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

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