Matching Items (34)

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The Focusing of Proteins Using Dielectrophoresis in an Improved Microfluidic Device

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Dielectrophoresis is a separations strategy that has the potential to separate small amounts of different proteins from each other. The forces at play in the channel used for dielectrophoresis are

Dielectrophoresis is a separations strategy that has the potential to separate small amounts of different proteins from each other. The forces at play in the channel used for dielectrophoresis are electroosmotic flow (EOF), electrophoresis (EP), and dielectrophoresis (DEP). EOF is the force exerted on liquid from an applied potential (1). EP is the force exerted on charged particles in a uniform electric field (2). DEP is the force exerted on particles (charged and uncharged) in a non-uniform electric field (3). This experiment was focused on the testing of a new microfluidic device to see if it could improve the focusing of proteins in dielectrophoresis. It was predicted that the addition of a salt bridge would improve focusing by preventing the ions created by the electrolysis of water around the electrodes from interacting with the proteins and causing aggregation, among other problems. Control trials using the old device showed that electrolysis was likely occurring and was the causal agent for poor outcomes. After applying the electric potential for some time a pH front traveled through the channel causing aggregation of proteins and the current in the channel decreased rapidly, even while the voltage was held constant. The resistance in the channels of the control trials also slightly decreased over time, until the pH shift occurred, at which time it increased rapidly. Experimental trials with a new device that included salt bridges eliminated this pH front and had a roughly linear increase of current in the channel with the voltage applied. This device can now be used in future research with protein dielectrophoresis, including in the potential differentiation of different proteins. References: 1) Electroosmosis. Oxford Dictionary of Biochemistry and Molecular Biology. 2. Oxford University Press: Oxford, England. 2006. 2) Electrophoresis. Oxford Dictionary of Biochemistry and Molecular Biology. 2. Oxford University Press: Oxford, England. 2006. 3) Dielectrophoresis. Oxford Dictionary of Biochemistry and Molecular Biology. 2. Oxford University Press: Oxford, England. 2006.

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  • 2016-05

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Determining the Validity of Using Heavy-Isotope-Permethylated Glycans as Internal Standards for Glycan Node Analysis

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In this thesis, glycan nodes, the basic subunits of complex biological sugars, were studied to determine the reproducibility of gas chromatography-mass spectrometry (GC/MS) based methylation analysis of whole blood plasma

In this thesis, glycan nodes, the basic subunits of complex biological sugars, were studied to determine the reproducibility of gas chromatography-mass spectrometry (GC/MS) based methylation analysis of whole blood plasma by normalization using an internal standard of heavy permethylated glycans. Glycans are complex biological sugars that have a variety of applications in the human body and will display aberrant compositions when produced by cancerous cells. Thus an assay to determine their composition can be used as a diagnostic tool. It was shown that the assay may have potential use, but needs further refinement to become an improvement over current methods by analyzing the results of ratio-determination and replicate experiments.

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  • 2015-05

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Dielectrophoresis of Gold Nanoparticles

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Dielectrophoretic trapping is a separatory/analytical method that is capable of achieving high levels of analyte differentiation using a combination of electroosmotic flow, electrophoresis, and dielectrophoresis. The form of dielectrophoretic device

Dielectrophoretic trapping is a separatory/analytical method that is capable of achieving high levels of analyte differentiation using a combination of electroosmotic flow, electrophoresis, and dielectrophoresis. The form of dielectrophoretic device used in these trials was of a gradient insulator-based design that induced the non-uniform electric fields necessary for dielectrophoretic trapping to occur. Development of such microfluidic devices began in the early 2000s and has produced several successful trials and refinements since then. Improvements have led to the ability of these devices to separate analytes to extremely high degrees of resolution as was demonstrated by the simultaneous separation of antibiotic resistant and antibiotic susceptible strains of bacteria in other experiments. The majority of analytes examined with these microfluidic devices have been biological in nature and on the scale of micrometers in size. The objective of this experiment was to test the lower limit of the device's resolution by attempting to use dielectrophoresis to trap gold nanoparticles via the balancing point between electrophoretic and dielectrophoretic mobilities. Trials successfully captured 10 nm fluorophore tagged gold nanoparticles at a mobility ratio of 6.16 x 1011 V2/m3, 60 nm citrate-capped gold nanoparticles at approximately 3.61 x 1010 V2/m3, and bare 10 nm gold nanoparticle aggregates at both 1.63 x 1010 V2/m3 and 1.68 x 1010 V2/m3. The corresponding voltages that were applied to achieve trapping were -1500 V, -2000 V, and -1500 V respectively. These findings were promising but reproducibility of the results was very low, largely due to matters of contaminants entering the devices and preventing the even, continuous flow of the analyte solution. Refinement of the analytical process should be pursued.

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  • 2018-12

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Time-Lapse Large-Volume Light Scattering Imaging Cytometry

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Cytometry is a method used to measure and collect the physical and chemical characteristics of a population of cells. In modern medical settings, the trend of precision and personalized medicines

Cytometry is a method used to measure and collect the physical and chemical characteristics of a population of cells. In modern medical settings, the trend of precision and personalized medicines has imposed a need for rapid point-of-care diagnostic technologies. A rapid cytometric method, which aims at detecting and analyzing cells in direct patient samples, is therefore desirable. This dissertation presents the development of light-scattering-based imaging methods for detecting and analyzing cells and applies the technology in four applications. The first application is tracking phenotypic features of single particles, thereby differentiating bacterial cells from non-living particles in a label-free manner. The second application is a culture-free antimicrobial susceptibility test that rapidly tracks multiple, antimicrobial-induced phenotypic changes of bacterial cells with results obtained within 30 – 90 minutes. The third application is rapid antimicrobial susceptibility testing (AST) of bacterial cell growth directly in-patient urine samples, without a pre-culture step, within 90 min. This technology demonstrated rapid (90 min) detection of Escherichia coli in 24 clinical urine samples with 100% sensitivity and 83% specificity and rapid (90 min) AST in 12 urine samples with 87.5% categorical agreement with two antibiotics, ampicillin and ciprofloxacin. The fourth application is a multi-dimensional imaging cytometry system that integrates multiple light sources from different angles to simultaneously capture time-lapse, forward scattering and side scattering images of blood cells. The system has demonstrated capacity to detect red blood cell agglutination, assess red blood cell lysis, and differentiate red and white blood cells for potential implementation in clinical hematology analyses. These large-volume, light-scattering cytometric technologies can be used and applied in clinical and research settings to study, detect, and analyze cells. These studies developed rapid point-of-care diagnostic and imaging technologies for collectively advancing modern medicine and global health.

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Date Created
  • 2020

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High Resolution Identification of Bioparticle Subpopulations with Electrophysical Properties

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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

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.

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  • 2020

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Microanalysis for Oxygen Fugacity by Secondary Ion Mass Spectrometry

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Oxygen fugacity (ƒO2) is a thermodynamic variable used to represent the redox state of a material or a system. It is equivalent to the partial pressure of oxygen in a

Oxygen fugacity (ƒO2) is a thermodynamic variable used to represent the redox state of a material or a system. It is equivalent to the partial pressure of oxygen in a particular environment corrected for the non-ideal behavior of the gas. ƒO2 is often used to indicate the potential for iron to occur in a more oxidized or reduced state at a particular temperature and pressure in a natural system. Secondary ion mass spectrometry (SIMS) is a powerful analytical instrument that can be used to analyze elemental and isotopic compositional information about microscopic features within solid materials. SIMS analyses of the secondary ion energy distribution of semi-pure metals demonstrate that the energy spectrum of individual mass lines can provide information about alterations in its surface environment.

The application of high-resolution (see Appendix C) energy spectrum calibrations to natural ilmenite led to the investigation of zirconium (90Zr+) and niobium (93Nb+) as potential indicators of sample ƒO2. Energy spectrum measurements were performed on an array of ilmenite crystals from the earth’s upper mantle retrieved from kimberlites and from a reduced meteorite. In all studied materials, variability in the peak shape and width of the energy spectra has been correlated with inferred sample ƒO2. The best descriptor of this relationship is the full-width at half-maximum (FWHM; see Appendix C) of the energy spectra for each sample. It has been estimated that a 1eV change in the FWHM of 93Nb+ energy spectra is roughly equivalent to 1 log unit ƒO2. Simple estimates of precision suggest the FWHM values can be trusted to  1eV and sample ƒO2 can be predicted to ±1 log unit, assuming the temperature of formation is known.

The work of this thesis also explores the applicability of this technique beyond analysis of semi-pure metals and ilmenite crystals from kimberlites. This technique was applied to titanium oxides experimentally formed at known ƒO2 as well as an ilmenite crystal that showed compositional variations across the grain (i.e., core to rim chemical variations). Analyses of titanium oxides formed at known ƒO2 agree with the estimation that 1 eV change in the FWHM of 93Nb+ is equivalent to ~1 log unit ƒO2 (in all cases but one); this is also true for analyses of a natural ilmenite crystal with compositional variations across the grain.

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Date Created
  • 2019

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Targeted proteomics studies: design, development and translation of mass spectrometric immunoassays for diabetes and kidney disease

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

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.

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Date Created
  • 2011

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Migration for organelles and bacteria in insulator-based microfluidic devices

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

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.

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Date Created
  • 2015

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Charge transport in single molecules

Description

Studying charge transport through single molecules is of great importance for unravelling charge transport mechanisms, investigating fundamentals of chemistry, and developing functional building blocks in molecular electronics.

First, a study of

Studying charge transport through single molecules is of great importance for unravelling charge transport mechanisms, investigating fundamentals of chemistry, and developing functional building blocks in molecular electronics.

First, a study of the thermoelectric effect in single DNA molecules is reported. By varying the molecular length and sequence, the charge transport in DNA was tuned to either a hopping- or tunneling-dominated regimes. In the hopping regime, the thermoelectric effect is small and insensitive to the molecular length. Meanwhile, in the tunneling regime, the thermoelectric effect is large and sensitive to the length. These findings indicate that by varying its sequence and length, the thermoelectric effect in DNA can be controlled. The experimental results are then described in terms of hopping and tunneling charge transport models.

Then, I showed that the electron transfer reaction of a single ferrocene molecule can be controlled with a mechanical force. I monitor the redox state of the molecule from its characteristic conductance, detect the switching events of the molecule from reduced to oxidized states with the force, and determine a negative shift of ~34 mV in the redox potential under force. The theoretical modeling is in good agreement with the observations, and reveals the role of the coupling between the electronic states and structure of the molecule.

Finally, conclusions and perspectives were discussed to point out the implications of the above works and future studies that can be performed based on the findings.

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Date Created
  • 2017

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Foundational investigation of electrophoretic exclusion

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

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.

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Date Created
  • 2015