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- All Subjects: Microfluidics
- All Subjects: Genomics
- Genre: Academic theses
- Creators: Borges, Chad
- Member of: Theses and Dissertations
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
Cancer is a heterogeneous disease with discrete oncogenic mechanisms. P53 mutation is the most common oncogenic mutation in many cancers including breast cancer. This dissertation focuses on fundamental genetic alterations enforced by p53 mutation as an indirect target. p53 mutation upregulates the mevalonate pathway genes altering cholesterol biosynthesis and prenylation. Prenylation, a lipid modification, is required for small GTPases signaling cascades. Project 1 demonstrates that prenylation inhibition can specifically target cells harboring p53 mutation resulting in reduced tumor proliferation and migration. Mutating p53 is associated with Ras and RhoA activation and statin prevents this activity by inhibiting prenylation. Ras-related pathway genes were selected from the transcriptomic analysis for evaluating correlation to statin sensitivity. A gene signature of seventeen genes and TP53 genotype (referred to as MPR signature) is generated to predict response to statins. MPR signature is validated through two datasets of drug screening in cell lines. As advancements in targeted gene modification are rising, the CRISPR-Cas9 technology has emerged as a new cancer therapeutic strategy. One of the important risk factors in gene therapy is the immune recognition of the exogenous therapeutic tool, resulting in obstruction of treatment and possibly serious health consequences. Project 2 describes a method development that can potentially improve the safety and efficacy of gene-targeting proteins. A cohort of 155 healthy individuals was screened for pre-existing B cell and T cell immune response to the S. pyogenes Cas9 protein. We detected antibodies against Cas9 in more than 10% of the healthy population and identified two immunodominant T cell epitopes of this protein. A de-immunized Cas9 that maintains the wild-type functionality was engineered by mutating the identified T cell epitopes. The gene signature and method described here have the potential to improve strategies for genome-driven tumor targeting.
ContributorsRoshdi Ferdosi, Shayesteh (Author) / Anderson, Karen S (Thesis advisor) / LaBaer, Joshua (Thesis advisor) / Woodbury, Neel (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2017
Description
Dielectrophoresis (DEP) is a technique that influences the motion of polarizable particles in an electric field gradient. DEP can be combined with other effects that influence the motion of a particle in a microchannel, such as electrophoresis and electroosmosis. Together, these three can be used to probe properties of an analyte, including charge, conductivity, and zeta potential. DEP shows promise as a high-resolution differentiation and separation method, with the ability to distinguish between subtly-different populations. This, combined with the fast (on the order of minutes) analysis times offered by the technique, lend it many of the features necessary to be used in rapid diagnostics and point-of-care devices.
Here, a mathematical model of dielectrophoretic data is presented to connect analyte properties with data features, including the intercept and slope, enabling DEP to be used in applications which require this information. The promise of DEP to distinguish between analytes with small differences is illustrated with antibiotic resistant bacteria. The DEP system is shown to differentiate between methicillin-resistant and susceptible Staphylococcus aureus. This differentiation was achieved both label free and with bacteria that had been fluorescently-labeled. Klebsiella pneumoniae carbapenemase-positive and negative Klebsiella pneumoniae were also distinguished, demonstrating the differentiation for a different mechanism of antibiotic resistance. Differences in dielectrophoretic behavior as displayed by S. aureus and K. pneumoniae were also shown by Staphylococcus epidermidis. These differences were exploited for a separation in space of gentamicin-resistant and -susceptible S. epidermidis. Besides establishing the ability of DEP to distinguish between populations with small biophysical differences, these studies illustrate the possibility for the use of DEP in applications such as rapid diagnostics.
Here, a mathematical model of dielectrophoretic data is presented to connect analyte properties with data features, including the intercept and slope, enabling DEP to be used in applications which require this information. The promise of DEP to distinguish between analytes with small differences is illustrated with antibiotic resistant bacteria. The DEP system is shown to differentiate between methicillin-resistant and susceptible Staphylococcus aureus. This differentiation was achieved both label free and with bacteria that had been fluorescently-labeled. Klebsiella pneumoniae carbapenemase-positive and negative Klebsiella pneumoniae were also distinguished, demonstrating the differentiation for a different mechanism of antibiotic resistance. Differences in dielectrophoretic behavior as displayed by S. aureus and K. pneumoniae were also shown by Staphylococcus epidermidis. These differences were exploited for a separation in space of gentamicin-resistant and -susceptible S. epidermidis. Besides establishing the ability of DEP to distinguish between populations with small biophysical differences, these studies illustrate the possibility for the use of DEP in applications such as rapid diagnostics.
ContributorsHilton, Shannon (Author) / Hayes, Mark A. (Thesis advisor) / Borges, Chad (Committee member) / Herckes, Pierre (Committee member) / Arizona State University (Publisher)
Created2019
Description
In species with highly heteromorphic sex chromosomes, the degradation of one of the sex chromosomes can result in unequal gene expression between the sexes (e.g., between XX females and XY males) and between the sex chromosomes and the autosomes. Dosage compensation is a process whereby genes on the sex chromosomes achieve equal gene expression which prevents deleterious side effects from having too much or too little expression of genes on sex chromsomes. The green anole is part of a group of species that recently underwent an adaptive radiation. The green anole has XX/XY sex determination, but the content of the X chromosome and its evolution have not been described. Given its status as a model species, better understanding the green anole genome could reveal insights into other species. Genomic analyses are crucial for a comprehensive picture of sex chromosome differentiation and dosage compensation, in addition to understanding speciation.
In order to address this, multiple comparative genomics and bioinformatics analyses were conducted to elucidate patterns of evolution in the green anole and across multiple anole species. Comparative genomics analyses were used to infer additional X-linked loci in the green anole, RNAseq data from male and female samples were anayzed to quantify patterns of sex-biased gene expression across the genome, and the extent of dosage compensation on the anole X chromosome was characterized, providing evidence that the sex chromosomes in the green anole are dosage compensated.
In addition, X-linked genes have a lower ratio of nonsynonymous to synonymous substitution rates than the autosomes when compared to other Anolis species, and pairwise rates of evolution in genes across the anole genome were analyzed. To conduct this analysis a new pipeline was created for filtering alignments and performing batch calculations for whole genome coding sequences. This pipeline has been made publicly available.
In order to address this, multiple comparative genomics and bioinformatics analyses were conducted to elucidate patterns of evolution in the green anole and across multiple anole species. Comparative genomics analyses were used to infer additional X-linked loci in the green anole, RNAseq data from male and female samples were anayzed to quantify patterns of sex-biased gene expression across the genome, and the extent of dosage compensation on the anole X chromosome was characterized, providing evidence that the sex chromosomes in the green anole are dosage compensated.
In addition, X-linked genes have a lower ratio of nonsynonymous to synonymous substitution rates than the autosomes when compared to other Anolis species, and pairwise rates of evolution in genes across the anole genome were analyzed. To conduct this analysis a new pipeline was created for filtering alignments and performing batch calculations for whole genome coding sequences. This pipeline has been made publicly available.
ContributorsRupp, Shawn Michael (Author) / Wilson Sayres, Melissa A (Thesis advisor) / Kusumi, Kenro (Committee member) / DeNardo, Dale (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
Description
There is increasing interest and demand in biology studies for identifying and characterizing rare cells or bioparticle subtypes. These subpopulations demonstrate special function, as examples, in multipotent proliferation, immune system response, and cancer diagnosis. Current techniques for separation and identification of these targets lack the accuracy and sensitivity needed to interrogate the complex and diverse bioparticle mixtures. High resolution separations of unlabeled and unaltered cells is an emerging capability. In particular, electric field-driven punctuated microgradient separations have shown high resolution separations of bioparticles. These separations are based on biophysical properties of the un-altered bioparticles. Here, the properties of the bioparticles were identified by ratio of electrokinetic (EK) to dielectrophoretic (DEP) mobilities.
As part of this dissertation, high-resolution separations have been applied to neural stem and progenitor cells (NSPCs). The abundance of NSPCs captured with different range of ratio of EK to DEP mobilities are consistent with the final fate trends of the populations. This supports the idea of unbiased and unlabeled high-resolution separation of NSPCs to specific fates is possible. In addition, a new strategy to generate reproducible subpopulations using varied applied potential were employed for studying insulin vesicles from beta cells. The isolated subpopulations demonstrated that the insulin vesicles are heterogenous and showed different distribution of mobility ratios when compared with glucose treated insulin vesicles. This is consistent with existing vesicle density and local concentration data. Furthermore, proteins, which are accepted as challenging small bioparticles to be captured by electrophysical method, were concentrated by this technique. Proteins including IgG, lysozyme, alpha-chymotrypsinogen A were differentiated and characterized with the ratio factor. An extremely narrow bandwidth and high resolution characterization technique, which is experimentally simple and fast, has been developed for proteins. Finally, the native whole cell separation technique has also been applied for Salmonella serotype identification and differentiation for the first time. The technique generated full differentiation of four serotypes of Salmonella. These works may lead to a less expensive and more decentralized new tool and method for transplantation, proteomics, basic research, and microbiologists, working in parallel with other characterization methods.
As part of this dissertation, high-resolution separations have been applied to neural stem and progenitor cells (NSPCs). The abundance of NSPCs captured with different range of ratio of EK to DEP mobilities are consistent with the final fate trends of the populations. This supports the idea of unbiased and unlabeled high-resolution separation of NSPCs to specific fates is possible. In addition, a new strategy to generate reproducible subpopulations using varied applied potential were employed for studying insulin vesicles from beta cells. The isolated subpopulations demonstrated that the insulin vesicles are heterogenous and showed different distribution of mobility ratios when compared with glucose treated insulin vesicles. This is consistent with existing vesicle density and local concentration data. Furthermore, proteins, which are accepted as challenging small bioparticles to be captured by electrophysical method, were concentrated by this technique. Proteins including IgG, lysozyme, alpha-chymotrypsinogen A were differentiated and characterized with the ratio factor. An extremely narrow bandwidth and high resolution characterization technique, which is experimentally simple and fast, has been developed for proteins. Finally, the native whole cell separation technique has also been applied for Salmonella serotype identification and differentiation for the first time. The technique generated full differentiation of four serotypes of Salmonella. These works may lead to a less expensive and more decentralized new tool and method for transplantation, proteomics, basic research, and microbiologists, working in parallel with other characterization methods.
ContributorsLiu, Yameng (Author) / Hayes, Mark A. (Thesis advisor) / Wang, Xu (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2020
Description
Novel electric field-assisted microfluidic platforms were developed to exploit unique migration phenomena, particle manipulation, and enhanced droplet control. The platforms can facilitate various analytical challenges such as size-based separations, and delivery of protein crystals for structural discovery with both high selectivity and sensitivity. The vast complexity of biological analytes requires efficient transport and fractionation approaches to understand variations of biomolecular processes and signatures. Size heterogeneity is one characteristic that is especially important to understand for sub-micron organelles such as mitochondria and lipid droplets. It is crucial to resolve populations of sub-cellular or diagnostically relevant bioparticles when these often cannot be resolved with traditional methods. Herein, novel microfluidic tools were developed for the unique migration mechanism capable of separating sub-micron sized bioparticles by size. This based on a deterministic ratchet effect in a symmetrical post array with dielectrophoresis (DEP) for the fast migration allowing separation of polystyrene beads, mitochondria, and liposomes in tens of seconds. This mechanism was further demonstrated using high throughput DEP-based ratchet devices for versatile, continuous sub-micron size particle separation with large sample volumes. Serial femtosecond crystallography (SFX) with X-ray free-electron lasers (XFELs) revolutionized protein structure determination. In SFX experiments, a majority of the continuously injected liquid crystal suspension is wasted due to the unique X-ray pulse structure of XFELs, requiring a large amount (up to grams) of crystal sample to determine a protein structure. To reduce the sample consumption in such experiments, 3D printed droplet-based microfluidic platforms were developed for the generation of aqueous droplets in an oil phase. The implemented droplet-based sample delivery method showed 60% less sample volume consumption compared to the continuous injection at the European XFEL. For the enhanced control of aqueous droplet generation, the device allowed dynamic triggering of droplets for further improvement in synchronization between droplets and the X-ray pulses. This innovative technique of triggering droplets can play a crucial role in saving protein crystals in future SFX experiments. The electric field-assisted unique migration and separation phenomena in microfluidic platforms will be the key solution for revolutionizing the field of organelle separation and structural analysis of proteins.
ContributorsKim, Dai Hyun (Author) / Ros, Alexandra (Thesis advisor) / Hayes, Mark (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2020