Browsing by Subject "Electrophoresis"
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Item A micro-scale universal detector based on backscattering interferometry(Texas Tech University, 2003-05) Wang, ZhanlingAs the current trend in miniaturization of analytical instrument continues, there is an urgent requirement for sensitive, small volume detection methodologies. Refractive index (Rl) detection, affords the generation of a signal for essentially all analyses, further more, the analytical signal is concentration rather than mass sensitive, and the technique is nondestructive. In the first part of this thesis, a universal detector based on backscatter interferometry has been developed to perform nanoliter volume refractive index measurements for on-chip SDS gel electrophoresis of proteins. The on-chip backscattering interferometry (OCIBD) detection system consists of a simple, folded optical train, it is based on the interaction of a laser beam and an etched channel in the shape of half cylinder in a silica plate. The backscattered light from the channel takes on the form of a high contrast interference pattern that contains information related to the bulk properties of the fluid located within the detection volume of 2.32 x 10"^ L. Depending on CE injection method, the positional changes of the interference fringes allow for the quantification of unlabeled proteins at the levels ranging from 17 to 310 attomoles. Separation and detection of 5 label-free proteins was achieved in less than 100 seconds with detection limits ranging from 0.95 picograms (1.1 x 10^-16mol) of Calmodulin to 7.0 picograms (1.0 x 10^-16) of BSA without signal filtering or active thermal control. In the second part of this thesis, an ultra sensitive Dual capillary-Dual bicell (DCDB) MIBD system was also developed. DCDB MIBD system focuses a He-Ne laser through a half wave plate onto the cross section of two capillaries, one for reference and another for sensing analyte. The backscatering fringe patterns were detected with hwo bicell photodetectors. Because the two capillaries are located in identical thermal environment, the difference of the two bicell signals which represent the reference and probing capillary respectively, can cancel out the errors from the thermal drift, which is the major error in MIBD system. Thus gives an accurate detection of the specific signal of analyte without preciously controlling of the environment temperature. The characteristic of this system has been demonstrated by measuring glycerol concentration and real time monitoring protein folding/unfolding when temperature changed in a wild range. It has potential use as a real time nanoliter detector in |j,-HPLC, CE, FIA and scanning micro-calorimetry.Item A microfluidic device for continuous capture and concentration of pathogens from water(2009-05-15) Balasubramanian, Ashwin KumarA microfluidic device, based on electrophoretic transport and electrostatic trapping of charged particles, has been developed for continuous capture and concentration of microorganisms from water. A generic design, utilizing mobility and zeta potential measurements of various microorganisms exposed to different environmental conditions and physiological states, was employed. Water and buffer samples at pH values ranging from 5.2?7.0 were seeded with bacteria (E. coli, Salmonella, and Pseudomonas) and viruses (MS-2 and Echovirus). Negative control and capture experiments were performed simultaneously using two identical devices. Both culture based methods and real-time PCR analysis were utilized to characterize the capture efficiency as a function of time, flowrate, and applied electric field. Based on differences between the capture and negative control data, capture efficiencies of 90% to 99% are reported for E. coli, Salmonella, Pseudomonas, and MS-2, while the capture efficiency for Echovirus was around 75%. Overall, the device exhibits 16.67 fold sample volume reduction within an hour at 6 mL/hr. This results in a concentration factor of 15 at 90% capture efficiency. Direct quantification of capture on the anode of the prototype microfluidic device was also performed by particle tracking using fluorescent microscopy. Based on image processing, the capture data at different locations on the electrode surface is quantified as a function of the wall shear stress at these locations, which is calculated using CFD simulations. Finally, the Faradaic processes in the microchannel due to electrochemical reactions are studied to predict the amount of electrophoresis in the system. Scaling of the device to sample 5 L/hr can be achieved by stacking 835 identical microchannels. Power and wetted volume for the prototype and scaled devices are presented. The device can thus function either as a filtration unit or as a sample concentrator to enable the application of real-time detection sensor technologies. The ability to continuously sample water without chemical additives facilitates the use of this device in drinking water distribution systems. This work constitutes the first step in our development of a continuous, microbial capture and concentration system from large volumes of potable water.Item A thin film transistor driven microchannel device(Texas A&M University, 2005-02-17) Lee, Hyun HoNovel electrophoresis devices for protein and DNA separation and identification have been presented and studied. The new device utilizes a contact resistance change detection method to identify protein and DNA in situ. The devices were prepared with a microelectronic micromechanical system (MEMS) fabrication method. Three model proteins and six DNA fragments were separated by polyacrylamide gel microchannel electrophoresis and surface electrophoresis. The detection of the proteins or DNA fragments was accomplished using the contact resistance increase of the detection electrode due to adsorption of the separated biomolecules. Key factors for the success of these devices were the optimization of fabrication process and the enhancement of detection efficiency of the devices. Parameters, such as microchannel configuration, size of electrode, and affinity of protein or polyacrylamide gel to the microchannel sidewall and bottom surface were explored in detail. For DNA analysis, the affinity to the bottom surface of the channel was critical. The surface modification method was used to enhance the efficiency of the microchannel surface electrophoresis device. The adsorption of channel separated protein and DNA on the detection electrode was confirmed with the electron spectroscopy for chemical analysis (ESCA) method. The electrical current (I) from the protein microchannel electrophoresis was usually noisy and fluctuated at the early stage of the electrophoresis process. In order to remove the current perturbation, an amorphous silicon (a-Si:H) thin film transistor (TFT) was connected to the microchannel device. The self-aligned a-Si:H TFT was fabricated with a two-photomask process. The result shows that the attachment of the TFT successfully suppressed the current fluctuation of the microchannel electrophoresis process. In summary, protein and DNA samples were effectively separated and detected with the novel TFT-driven or surface microchannel electrophoresis device.Item An analysis of the patterns of environmental and electrophoretic variability in populations of Haplopappus gracilis (Compositae) and related species(Texas Tech University, 1978-12) Spohn, Ronald TerryNOT AVAILABLEItem Analysis of chromatographically and chemically fractionated native egg white using two acrylamide gel electrophoretic techniques(1980-08) Laney, James A. IIINot AvailableItem Characterization Of The Local Electrical Environment In An Electrically-guided Protein Patterning System Incorporating Antifouling Self-assembled Monolayer(2011-10-21) Park, JinseonIn earlier research in our lab, the manipulation of microtubules on gold patterned silicon wafers was achieved by E-beam lithography, Poly (ethylene glycol) self assembled monolayers (PEG-SAMs) and electrophoresis. To develop a technique for delicate single microtubule manipulation, further studies need to be done on PEG-SAMs and electrophoresis. As a foundation of this goal, we examined the electric field in an aqueous solution between two planar electrodes and the compatibility of the antifouling property of PEG-SAMs with the electric field. For this purpose, the distribution of microbeads was analyzed using a Boltzmann distribution. The amount of adsorbed microtubules on a PEG-SAM was examined to test the compatibility of the antifouling property of a PEG-SAM with concomitant exposure to electric field. It is shown that the product of the electric field and the effective charge of the microbead does not have a linear relation with the applied electric potential but an exponentially increasing function with respect to the potential. The antifouling property of the PEG-SAM was not retained after an exposure to the electric field.Item Cotton fiber proteins during development: an SDS-polyacrylamide gel electrophoresis study(Texas Tech University, 1980-08) Woodworth, Margaret JaneNot availableItem Electrophoretic protein differences between selected varieties and lines of upland cotton(Texas Tech University, 1970-12) Castleberry, Ronnie MackOne of these problems is caused by climatic conditions which dictate the use of stormproof varieties of cotton in the above mentioned area, Economic returns from cotton in this area would generally be greater if adapted longer staple non-stormproof varieties could be grouin (47), Genetic studies of stormproof character have been hampered by difficulties in classifying individual cotton plants as stormproof or non-stormproof (23,32). Consistent electrophorectic protein differences between stormproof and nonstormproof varieties could provide a criterion for classification and facilitate the incorporation of stormproof character into longer stapled varieties. The second problem involves the need for a protection or patenting program for new cotton varieties, To effectively protect new varieties criteria must be established that describe each variety precisely, rÃlorphological characteristics presently used are somewhat arbitrary and time consuming (28), The use of biochemical characteristics is a logical and promising way to describe new varieties. However, biochemical differences between varieties must be 'determined and cataloged before they can be used as criteria for varietal identification.Item Electrophoretic studies of relationships within the genus Artibeus (Chiroptera: Phyllostomidae)(Texas Tech University, 1982-08) Koop, Benjamin FNot availableItem Enhanced Purification of Platelet Beta-Lysin(Texas Tech University, 1982-05) Rickaby, Julia MarieExperimental alterations in the purification scheme established by Donaldson et al. for platelet B-lysin from rabbit serum were made with the objectives of enhanced bactericidal activity and increased stability. Either bovine serum albumin or reduced glutathione, when added to the filtrates in the purification scheme, increased bactericidal activity against Bacillus subtilis and Escherichia coli. In addition, ammonium bicarbonate was a more effective eluant than sodium chloride, which was used in the established procedure for removing 6-lysin from asbestos filter pads. The modified procedure, using glutathione and ammonium bicarbonate, yielded a stable 6-lysin preparation with increased bactericidal activity. Three UV absorbance peaks were obtained after filtration on a Sephadex G-50 column, with the third and largest peak showing substantial bactericidal activity. Bactericidal activity was also demonstrated in g-lysin samples after affinity chromatography on Affi-Gel blue. Analysis of the various 6-lysin preparations by polyacrylamide gel electrophoresis showed that the affinity chromatography step reduced albumin contamination in the samples of platelet 6-lysin.Item Item Microsecond electrophoresis(2006) Plenert, Matthew Lee; Shear, Jason B.Spectroscopy is a powerful tool for the investigation of chemical phenomena, but often is hindered in the analysis of species in chemical mixtures—in which chemical interferences can convolute the attainable spectroscopic information. Separation techniques such as liquid chromatography and capillary electrophoresis provide a means of deconvoluting such information but typically are performed over timescales much longer than the life span of highly unstable compounds. This dissertation describes the development of the world’s fastest separation technique, microsecond electrophoresis, and application of this technique to the study of transient species. Chapter 1 provides a discussion of issues involved in the performance of high speed electrophoretic separations. Chapter 2 describes development of multiphoton excited (MPE) optical injection and detection strategies which allow transient species to be analyzed using electrophoresis over low micron separation distances. Chapter 3 presents a method by which separation capillaries are modified such that electric field strengths exceeding 0.1 MV/cm can be used to perform separations (resulting in a ~ 100-fold reduction in analysis time over previous high speed separation techniques). Chapter 4 presents further instrumental developments which allow differently-charged species to be detected at different locations within the separation channel (allowing “infinite resolution’ microsecond separations to be performed). Chapter 5 details work towards application of low-timescale electrophoretic separations to the study of folding protein and peptide species.Item Multi-dimensional analysis of hdl: an approach to understanding atherogenic hdl(2009-05-15) Johnson, Jr., Jeffery DevoyneDensity gradient ultracentrifugation (DGU) is a powerful method for analyzing lipoprotein particles in great detail. It yields considerable amounts of information regarding the density distribution of these particles when coupled with fluorometric analysis and is an invaluable tool in determining their relative abundance. This union allows relationships between subclasses of lipoproteins to be established that gives researchers a more focused path to aid them in developing methods to predict the early onset of coronary artery disease (CAD). The research presented here focuses on the pairing of DGU with post-separatory techniques including matrix-assisted laser desorption mass spectrometry (MALDI-MS), liquid chromatography mass spectrometry (LC-MS), capillary electrophoresis (CE), isoelectric focusing (IEF) and apoptosis studies involving cell cultures. It is becoming clearer that cholesterol concentrations themselves do not provide sufficient data to assess the quality of cardiovascular health. As a result, research is becoming more focused on identifying better markers that may be indicative of development of CAD in a patient. Of specific interest is group of particles known as high density lipoproteins (HDL). Classically, this molecule is considered the ?good cholesterol?, but literature from the last decade suggests that there may be atherogenic variants to this group. By utilizing DGU as a preparatory method for secondary analyses, new dimensions can be added to the density distribution analysis to allow a better determination of markers of cardiovascular health. The aim of this work is to utilize the principles involved with these various techniques to develop a comprehensive set of methods to aid in the detection of potential risk markers. In this study, the properties of metal ion complexes of EDTA as solute systems for analysis of lipoproteins by DGU are analyzed. We show that by varying the complexing ion and counter-ion of these metal-ion complexes, we gain the ability to control the separation of lipoprotein subclasses for subsequent analyses. Qualitative and quantitative data is presented that describes the analysis of different density regions of HDL for apolipoprotein content. Trends between control and atherogenic samples are also described and a clinical link between the biological activity of these regions and the chemical analysis is discussed.Item Novel devices for analytical-scale isoelectric trapping separations(2009-05-15) Lim, Peniel JasonIsoelectric trapping (IET), has proven to be one of the most successful electrophoretic techniques used for separations of ampholytic compounds. IET is carried out in multicompartment electrolyzers (MCEs) in which adjacent compartments are joined through buffering membranes whose pH values bracket the pI of the ampholytic component to be trapped in the compartment. The present small-scale instruments use plastics as their structural materials, which causes poor Joule heat dissipation. The separation compartments have cylindrical or pear-shaped interiors with large internal diameters, which create long heat transfer paths. The long electrode distances yield low field strengths that lead to low electrophoretic velocities for the analytes. These factors interrelatedly limit the electric power that can be applied to the system, contributing to long separation times. Furthermore, these devices do not offer a realistic solution to the problems associated with the detection of low abundance proteins. To address these problems, two novel IET devices have been developed for small-scale IET separations. The first device, named MSWIFT, was constructed using thermally conductive, high-purity alumina as the structural material of the separation compartments. By creating narrow, 0.1- or 0.2-mL channels in thin alumina blocks, the heat transfer path from the center of the compartment to the wall was significantly decreased; and the distance between electrodes was greatly shortened. MSWIFT achieved 6 to 50 times faster IET separations compared to other MCEs. The second device, named ConFrac, was developed to simultaneously fractionate and concentrate ampholytic components from a complex sample into 0.1-mL collection compartments. By designing a system with a 2-dimensional pH gradient and allowing recirculation of the sample feed, the ConFrac demonstrated enrichment of analytes by a factor of 100 and greater.Item Study of electrophoretic variation of aromatic esterases and general proteins of Peromyscus difficilis comanche (Rodentia: cricetidae) and certain related taxa(Texas Tech University, 1969-05) Mize, Joyce AvaNOT AVAILABLEItem Supported Lipid Bilayer Electrophoresis: A New Paradigm in Membrane Biophysics and Separations(2012-11-28) Pace, Hudson 1982-The motivation of this work was to produce novel analytical techniques capable of probing the physical properties of the cell surface. Many researchers have used supported lipid bilayers (SLBs) as models to study the structure and function of the cell membrane. The complexity of these models is consistently increasing in order to better understand the myriad of physiologically relevant processes regulated by this surface. In order to aid researchers in studying such phenomenon, the following contributions were made. To manipulate components within the cell membrane, an electrophoretic flow cell was designed which can be used as a probe to study the effect of electrical fields on charged membrane components and for the separation of these components. This devise allows for the strict control of pH and ionic strength as species are observed in real-time using fluorescence microscopy. Additionally, advancements have been made to the production of patterned heterogeneous SLBs for use in separations and to probe the interactions of membrane components. The methodology to couple SLB separations and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) imaging was devised. This technology allows for the label-free mapping of the SLB surface post electrophoresis in order to observe naturally occurring species unperturbed by the addition of extrinsic tags. The final contribution, and perhaps the greatest, is the development of a procedure to create highly mobile SLBs from native membranes. These surfaces have vast potential in that they are no longer simple models of the cell surface, they are in fact the actual cell surface made planar. This advancement will be of great use to biophysicists and biochemists interested in using surface specific analytical methods to better understand physiological processes. These highly mobile native membrane surfaces have been coupled with the SLB electrophoresis technology to separate discrete bands of lipids and proteins, a proof of principle that will hopefully be further developed into a standard method for membrane proteomic studies. Collectively the tools and methodologies described herein show great potential in allowing researchers to further add to mankind?s understanding of the cellular membrane.Item Systematics of the Drosophila virilis species group as assessed by two-dimensional electrophoresis(Texas Tech University, 1985-08) Spicer, Gregory S.A primary problem that often faces an evolutionary biologist trying to determine a phylogeny is finding enough characters to build the tree. Increasingly, molecular biology is solving this problem, but many shortcomings and limitations still exist with the techniques currently in use. For example, allozymic studies are very useful, but they constitute a time consuming task that often does not produce enough characters to fully resolve the branching diagram. Immunological and DNA hybridization techniques can often discern phenetic relationships between taxa, but they cannot be used in a phylogenetic analyses, because they are based on overall similarity (Entingh, 1970). Approaches such as DNA, RNA, and protein sequencing are very powerful techniques for phylogenetic reconstruction, but they are extremely time consuming and effectively cost prohibitive, particularly when one is working on lower taxonomic levels. The development of the mitochondrial DNA technology may solve many of these problems, although recent studies seem to indidcate that it may not be more effective than the current electrophoretic studies at producing phylogenetic trees (Kessler and Avise, 1984). However, this research is still in its infancy, so it is difficult to assess its final impact.