Browsing by Author "Edmond Bowden, Committee Member"

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Activators Generated by Electron Transfer for Atom Transfer Radical Polymerization in Polymerization-based DNA Sensing.
(2010-12-16) Qian, Hong; Lin He, Committee Chair; Morteza Khaledi, Committee Member; Bruce Novak, Committee Member; Edmond Bowden, Committee Member
Binding and Local Dynamics of Iso-1-cytochrome c to Substrate-Supported Lipid Nanotube Arrays
(2004-01-15) Smith, Shani Jomella; Edmond Bowden, Committee Member; Alex Smirnov, Committee Chair; Tatyana Smirnova, Committee Member
A study of the binding and local dynamics of iso-1-cytochrome c to lipid nanotube arrays composed of 1,2-Diheptadecanoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DOPG), an anionic phospholipid on an nanoporous anodic aluminum oxide (AAO) substrate was undertaken to gain an understanding of the effects of nanoscale confinement on protein binding. Iso-1-cytochrome c was spin-labeled with 1-oxyl-2,2,3,3-tetramethylpyrolline-3-methylmethanesulfonate (MTSL) at cysteine 102. The binding and local dynamics of the spin-labeled iso-1-cytochrome c bound to multilamellar liposomes was compared with the binding to lipid nanotube arrays. Location of spin-labeled cysteine 102 of iso-1-cytochrome c with respect to the bilayer was probed using an EPR oxygen accessibility experiment. CD monitored changes in the heme coordination in iso-1-cytochrome c upon binding to DOPG.
Characterization and analytical applications of dye-encapsulated zwitterionic liposomes
(2010-02-16) Yitbarek, Emnet; Morteza Khaledi, Committee Chair; Edmond Bowden, Committee Member; Tatyana Smirnova, Committee Member; Alexander Nevzorov, Committee Member
The aim of this project was to use marker encapsulated liposomes as biomembrane mimicking entities in order to study membrane properties like permeability and to better understand the interaction of biological lipid bilayers with membrane-active molecules, like beta blocker drugs and antimicrobial peptides (AMP). The physical characteristics of liposomes, such as size, surface charge and encapsulation capacity were also studied using electrophoretic, fluorescence and light scattering techniques. In addition, marker-encapsulated and self-lysing liposomes were used to study antigen-antibody binding. The immunoassay application of these self-lysing liposomes was also investigated. The first area of research is focused on investigating the effect of the liposome lipid composition on the size and the electrical properties of zwitterionic liposomes. The cholesterol composition of phosphatidylcholine (PC) and sphingomyelin (Sph) liposomes is varied and the effect on their size, zeta potential and electrophoretic mobility is monitored using dynamic light scattering (DLS), laser doppler velocimetry (LDV), and capillary zone electrophoresis (CZE) techniques, respectively. In addition, the permeability and the encapsulation capacity of large unilamellar vesicles (LUV), or liposomes that are made by extrusion, were compared as their lipid and cholesterol composition varied. The size and electrophoretic mobility of zwitterionic liposomes was found to increase with the cholesterol composition. The interaction of indolicidin, a 13-mer cationic AMP, with (dye-encapsulated) liposomes that were made of different lipid and cholesterol composition was investigated by DLS, fluorescence and capillary electrophoresis (CE) methods. DLS results show a change in liposome size, and size distribution index (PI), after indolicidin interaction. Fluorescence leakage experiments show the extent of membrane perturbation caused by the AMP and the AMPÃ¢â‚¬â„¢s innate tryptophan fluorescence provided qualitative information regarding the type (polar/non-polar) and nature of the liposome-AMP interaction, as lipid composition of the liposomes varied. In addition, CZE and liposome electrokinetic chromatography (LEKC) techniques were also used to further probe the (polar/non-polar/electrostatic) nature of this interaction. The immunoassay application of the marker encapsulated liposomes was investigated using a combination of fluorescence, DLS, and CE-LIF (capillary electrophoresis with laser induced fluorescence detector) techniques. The liposomes were made from a non-lamellar lipid DOPE (dioleoylphosphatidylethanolamine) that was stabilized with a 20% bilayer lipid DPPC (dipalmitoylphosphatidylcholine) and a 1% hapten-attached DPPE lipid. Small hapten molecules, like biotin and DNP (dinitrophenyl), were attached to the liposome surface via the DPPE lipid, and used to detect their conjugate molecules (avidin and anti-DNP antibody) in a homogeneous solution. The biotin-attached DOPE liposomes aggregate and leaked their marker content in standard avidin solution. The extent of liposome aggregation and the fluorescence intensity of the leaked dye are dependent on the concentration of avidin present in solution. The different parameters that affect the quality of the assay were also investigated.
The Development and Utilization of Aerodynamic Devices in Biological Mass Spectrometry
(2009-09-03) Dixon, Robert Brent; Edmond Bowden, Committee Member; Jack R. Edwards, Committee Member; Kenneth Hanck, Committee Member; David C. Muddiman, Committee Chair
The role of mass spectrometry in the chemical and biological sciences is ever evolving as there is an increasing interest to understand the biomolecular structure and interaction of proteins and their potential impact on human health. To this end, emerging techniques that complement or enhance mass spectrometric capabilities are of significant relevance. While the impact of mass spectrometry in every field (e.g. proteomics, drug development, environmental, crop science, or petrochemical) has varied, increased accessibility of instrumentation and expertise to address relevant issues across disciplines will result in even greater acceptance of this powerful technology. Some compounds may not ionize by a predominant method, electrospray ionization (ESI) or matrix assisted laser desorption ionization (MALDI), but recently a number of ionization sources have been developed that further encompass more classes of molecules for mass spectrometric analysis. Limited sample availability is a practical issue which demands lower limits-of-detection. In efforts to address this, devices such as the ion funnel and the air amplifier are being developed to more efficiently transport ions within or into the mass spectrometer (respectively). With continued ionization source and instrumentation improvements, there will undoubtedly be additional physicians, scientists, and engineers who realize the great potential and benefit of using mass spectrometry to address questions related to their particular field of study. This dissertation describes the utilization of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), which affords high resolving power and mass measurement accuracy, for the majority of ionization source developments. These ionization source efforts have been directed at improving current and emerging ionization techniques. A method for transporting neutral species for subsequent ionization termed remote analyte sampling, transport and ionization relay (RASTIR) has been developed and is described herein. Improvements for electrospray ionization have been realized using the air amplifier as it focuses and desolvates electrospray droplets; these mechanisms for improved ion abundance are investigated. An acoustic method for direct analysis of proteins and peptides from an oscillating surface is the premise of the radio frequency acoustic desorption ionization source. Collectively, this work aims to improve existing ionization techniques while investigating new avenues for biological mass spectrometry.
The Development of Analytical Strategies for the Quantitative Proteomic Analysis of Biological Systems Impacting Agriculture and Human Health.
(2010-10-22) Collier, Timothy; David Muddiman, Committee Chair; Gary Payne, Committee Member; Edmond Bowden, Committee Member; Morteza Khaledi, Committee Member
The Development of Desorption Electrospray Ionization and Nano Flow Liquid Chromatography Mass Spectrometric Methods for Glycan Analysis: Applications for Biomarker Discovery in Epiethelial Ovarian Cancer
(2009-10-02) Bereman, Michael Seth; David Muddiman, Committee Chair; Lin He, Committee Member; Chris Gorman, Committee Member; Edmond Bowden, Committee Member
Ovarian cancer is often referred to as the Ã¢â‚¬Å“silent killerÃ¢â‚¬Â due to the deadly nature of this malignancy and its asymptomatic nature. The disease will affect an estimated 24,000 women in the United States in 2009. If the disease is caught in its early stages over 90 % of patients survive longer than 5 years; however, 7 out of 10 patients are diagnosed in the late stages, after metastasis, where 1 in 5 people meet this 5-year survival mark. Advancements in early diagnosis are critical for both early intervention as well as a more in-depth understanding of this cancer such that therapeutic targets can be elucidated. One of the major limitations for biomarker discovery research is the massive amount of time that must be allocated for preparation and analysis of large sample sets. The recent development of hybrid ionization techniques which combine ambient analysis with limited sample preparation and lead to higher sample throughput could help circumvent this current limitation. Herein, the development of a newly introduced ionization technique termed desorption electrospray ionization (DESI) is characterized for various biomarker discovery applications including proteomics and glycomics. Glycomics is an emerging field for biomarker discovery research. Herein, a method for the analysis of N-linked glycans is provided. This method is then utilized to compare the N-linked glycan profile between 48 plasma samples derived from epithelial ovarian cancer patients, 48 controls samples and 8 healthy samples. Three glycans were evaluated for their ability to differentiate between EOC and control and these results were compared to the gold standard in EOC detection, CA 125. Results indicated limited diagnostic value for three glycans in distinguishing control and EOC patients and moderate diagnostic value in differentiating EOC and healthy samples.
Experimentation in Replacement Lithography, Reductive Desorption, and Scanning Probe Imaging on Various Metal Substrates.
(2010-06-28) Williams, James Alfred; Christopher Gorman, Committee Chair; Edmond Bowden, Committee Member; Stefan Franzen, Committee Member; Lin He, Committee Member; Jack Odle, Committee Member
Impact of Analyte-Surface Spacing on the Performance of Desorption/Ionization on Porous Silicon Mass Spectrometry (DIOS-MS)
(2009-08-08) Shah, Abhilasha Parag; Morteza Khaledi , Committee Member; Edmond Bowden, Committee Member; Lin He , Committee Chair
Desorption/ionization on porous silicon (DIOS), a matrix free-laser desorption mass spectrometric technique shows potential for direct analysis of biological compounds in the low mass region. However, the geometric scale of the porous silicon is significantly smaller than the biological samples used in MS imaging and suspect to limit an effective direct analysis of analyte. Herein, I have investigated the effect of the spacing distance between analytes and a porous substrate in DIOS-MS. In particular, organic polymer films were used to simulate the biological tissue between analyte and porous substrate. These films circumvent biological complexity that simplifies data analysis. The thicknesses of polymer film on the substrate controlled by varying spin coating condition. Different classes of analytes were used as standard molecules to evaluate the analyte-substrate spacing effect. In all cases, the standard molecules were successfully detected atop of the polymer film in DIOS-MS. The insulating layer of polymer shift the laser threshold compare to bare DIOS. Relatively stable signal-to-background (S/B) ratios across the tested spacing distances suggest that the analyte detection is less dependent of the distance between analyte and porous surface. The total ion current (TIC) of the analytes however, decreases as the distance increases suggesting distance effect on desorption/ionization. Moreover, the TIC was limited by the amount of analyte accessible for detection. In addition, the ultra-thin SiOx film showed improvement in analyte detectability over the tested thickness. Analyte detection on the DIOS surface greatly influence by surface chemical functionality; oxidized surface is advantageous for positive mode detection whereas the amine derivatized surface showed improvement in negative mode detection.
Investigations of Liposome/Water Partitioning Using Electrokinetic Chromatography
(2005-08-03) Barker, Jason Alan; Charles Boss, Committee Member; Morteza Khaledi, Committee Chair; Edmond Bowden, Committee Member; Dan Feldheim, Committee Member
In this two-part investigation, Linear Solvation Energy Relationship (LSER) models were applied to liposome/water partitioning data determined by liposome electrokinetic chromatography (LEKC). In the first part of the study, LEKC retention factors were determined for a set of 71 solutes including 50 neutral aromatic solutes and 21 neutral drugs using the liposome phases mM DPPG20DPPC80, 15 mM DPPG20DPPC80/3 mM cholesterol, and 15 mM DPPG20DPPC80/6 mM cholesterol. These retention factors were used to determine linear solvation energy relationships (LSER) models for each of the three lipid phases for the set of 50 neutral aromatic solutes and the entire set of 71 solutes. The predictive ability of the LSER model was tested by using the model generated for the set of 50 neutral aromatic solutes to predict retention factors for the 21 neutral drugs. The descriptive ability of the LSER model was tested and used to examine the interactions that control liposome/water partitioning for the set of 50 neutral aromatic solutes and the set of 71 solutes including 21 neutral drugs. In the second part of the study, wo liposome electrokinetic chromatography (LEKC) lipid phases were introduced that approximate the lipid composition of the cell membrane of monkey intestinal epithelial cells. 15 mM PI10DPPS10DPPC30DPPE30SPM20/9.75 mM cholesterol most nearly approximates the lipid composition of the cell membrane of monkey intestinal epithelial cells. 15 mM DPPG20DPPC80/9.75 mM cholesterol replaces the specific lipids contained in the monkey intestinal epithelial cell membranes with the zwitterioninc lipid DPPC and DPPG. The retention factors determined for a set of 50 neutral aromatic solutes using these two LEKC phases are highly correlated (r2=0.99), with a slope near unity (m=0.91) and an intercept near zero (b=0.05). The normalized LSER system coefficients for these lipid phases are also very similar. The LSER system coefficients determined for 15 mM PI10DPPS10DPPC30DPPE30SPM20/9.75 mM cholesterol and 15 mM DPPG20DPPC80/9.75 mM cholesterol were compared with an LSER model generated by Abraham, et al. for human intestinal absorption.
Issues in Patterning Self-Assembled Monolayers by Molecular Replacement: Comparison of Adventitious and Directed Replacement of One Molecule in a SAM with Another.
(2010-12-20) Tucker, Eric; Christopher Gorman, Committee Chair; David Muddiman, Committee Member; Edmond Bowden, Committee Member; Gregory Parsons, Committee Member
Label-free, Multiplexed Bioassay on Au/Ag Striped Nanorods Using Fluorescent Conjugated Polymers.
(2010-08-16) Zheng, Weiming; Lin He, Committee Chair; Edmond Bowden, Committee Member; Bruce Novak, Committee Member; Matthew Breen, Committee Member
Materials Science and Sensing Applications of Surface Plasmon Resonance in Conducting Metal Oxides
(2008-06-15) Rhodes, Crissy Lynette; Jan Genzer, Committee Member; Stefan Franzen, Committee Chair; Lin He, Committee Member; Edmond Bowden, Committee Member
Poly(lactic acid) Polymer Brushes as Dynamic Surfaces.
(2011-01-05) Xu, Lebo; Christopher Gorman, Committee Chair; Jan Genzer, Committee Member; Edmond Bowden, Committee Member; Stefan Franzen, Committee Member; Lin He, Committee Member
Polymerization in Confined Geometries
(2006-08-09) Petrie, Randall James; Jan Genzer, Committee Co-Chair; Chris Gorman, Committee Co-Chair; Kenneth Hanck, Committee Member; Edmond Bowden, Committee Member
The work presented in this PhD thesis is centered on nanometer-sized pores. Two detailed objectives include: 1) investigating the confinement effect on "grafting from" polymerization carried out directly inside the pore, and 2) using porous silicon as a novel platform for controlled motion of liquid drops moving along wettability gradients created on the pores. In chapter 2 we investigate the confinement effect of the pore (< 50 nm) on the polymerization of poly methyl methacryrlate (PMMA) in porous silicon. Porous silicon has the unique quality of acting as a replacement for the conventional organic matrix used in matrix assited laser desorption ionization (MALDI). To this end, porous silicon not only acts as the substrate in which the polymerization takes place, but also serves as an in situ platform for the direct molecular weight analysis of the pore-grown polymer. We also report on the efficiency of porous silicon to produce MALDI spectra of PMMA as compared to MALDI spectra obtained using a conventional organic matrix. Chapter 3 focuses upon the use of an alternative substrate, anodic aluminum oxide (AAO), for the in-pore polymerization of PMMA. AAO is attractive for its homogeneous pore distribution and commercial availability. Although AAO does not serve as an organic matrix replacement for MALDI like porous silicon, a procedure for the ex situ characterization of the pore grown PMMA via MALDI is discussed. In chapter 4 we report on the motion of water droplets on porous and flat silicon surfaces decorated with molecular gradients comprising semifluorinated (SF) organosilanes. SF molecular gradients deposited on flat silica substrates facilitate faster motion of water droplets relative to the specimens covered with an analogous hydrocarbon gradient. Further increase in the drop speed is achieved by advancing it along porous substrates coated with the SF wettability gradients. The results of our experiments are in quantitative agreement with a simple scaling theory that describes the faster liquid motion in terms of reduced friction at the liquid/substrate interface.
Quantification via Inductively-Coupled Plasma Optical Emission Spectroscopy (ICP-OES) of the Cellular Internalization and Nuclear Localization of Gold Nanoparticles Passivated with BSA-SV40 Large T NLS Conjugates after Incubation with Human Cervical Cancer (HeLa) Cells
(2009-06-22) Ryan, Joseph Anthony; Stefan Franzen, Committee Chair; Edmond Bowden, Committee Member; Kenneth Hanck, Committee Member; Wayne Robarge, Committee Member
Rhodamine-labeled, cysteine-modified SV40 large T NLS peptide sequences were conjugated in varying amounts (~ 3 to 15 molar ratio) with bovine serum albuin (BSA) via the heterobifunctional linker succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). These conjugates were then used to passivate nanomolar aliquots of citrate-coated gold nanoparticles of varying diameter (5, 10, 15, and 20 nm), and the stability of these nanoparticle complexes were evaluated with respect to: 1) amount of large T-BSA per nanoparticle (found to be stable under the following BSA:nanoparticle ratios: 5 nm diameter = 125:1, 10 nm diameter = 250:1, 15 nm diameter = 250:1, and 20 nm diameter = 500:1), 2) ionic strength of solution (critical coagulation concentration values above 0.85 M), and 3) temperature (found to be stable at 4, 25, and 37 degrees Celsius). A robust method was developed using inductively-coupled plasma optical emission spectroscopy (ICP-OES) as a means to quantify the internalization of nanoparticle complexes after incubation with human cervical cancer cells (HeLa) under many differing conditions. It was determined cellular internalization increased as a function of: 1) increasing amount of large T per nanoparticle complex, 2) longer incubation times, 3) temperature (which is to say incubations at 4 degrees Celsius afforded nearly no internalization), and 4) increasing nanoparticle diameter. Additionally, data from a pulse-chase experiment demonstrated that these nanoparticle complexes tend to remain associated with HeLa cells in similar concentration up to twelve hours after initial exposure. Lastly, a sub-cellular fractionation kit was used to extract nuclei from HeLa cells post-incubation with 5 nm diameter gold nanoparticle complexes. It was observed that nuclear localization of these nanoparticle complexes increased as a function of large T:nanoparticle ratio, but that resulting cell viability decreased dramatically at the highest large T:nanoparticle ratio.
The role of surface chemistry in defining the energetics and kinetics of single electron tunneling through individual gold nanoparticles
(2003-06-24) McConnell, Wyatt Parks; Orlin Velev, Committee Member; Daniel L. Feldheim, Committee Chair; Christopher Gorman, Committee Member; Edmond Bowden, Committee Member; Charles Boss, Committee Member
The properties of nanoscale materials are often dominated by their surface chemistry due to their increased surface-to-volume ratio. Metal nanoparticles with diameters smaller than ~12nm show a technologically relevant non-linear current-voltage response known as single electron tunneling. Gold nanoparticles offer an excellent platform for understanding how the surface chemistry of the metal island in a single electron-tunneling device can affect the current response of the structure. This is because the surfaces of these particles can be custom tailored using thiol-based self-assembled monolayer protocols that have been extensively developed for planar gold substrates. This dissertation describes STM measurements of single electron tunneling through individual gold nanoparticles of various sizes and surface chemistries in both air and solvent at room temperature and pressure. The voltage step-width of the resulting coulomb staircase was shown to be dependent on particle size. Solution experiments show that the energetics of single electron tunneling of particles with certain surface chemistries is dependent on the composition of the solution, while other surface chemistries can produce particles that give a stable single electron tunneling response in a wide variety of local chemical environments. Using acid and base terminated surface chemistries, particles were made that showed a defined response to a specific change in the local solution pH. The kinetics of single electron tunneling was also shown to be highly dependent on the chemical bond formed between the capping ligand and the metal particle.
Simple Molecule Mercury Sensor
(2008-11-13) Wan, Yanjun; Edmond Bowden, Committee Member; Christopher Gorman, Committee Chair; David Shultz, Committee Member; Stefan Franzen, Committee Member
Several molecules previously produced from a nitrile-based cascade cyclization were examined as potential mercury sensors. Various analytical parameters, including fluorescence quantum yield, UV shift, fluorescence quenching, binding constant, binding ratio, and lowest detection limit, were measured. The best mercury sensor molecule was found to be molecule 3c, which could be easily synthesized in gram quantities (3 steps, 55% overall yield). This molecule has a very high fluorescence quantum yield (Î¦ = 0.87), high sensitivity and selectivity towards mercury ion in both organic and aqueous media. The overall performance of molecule 3c is as good as, or better than, the majority of organic dye based mercury sensors reported to date.
Simplified Plant Sample Preparation for use in Gas Chromatography-Mass Spectrometry (GC-MS) Based Metabolomic Profiling and Targeted Analyte Quantitation
(2008-02-25) Jaeger, Frederick Howard; Edmond Bowden, Committee Member; David Muddiman, Committee Member; Lin He, Committee Chair
A simple, fast, reproducible and less laborious sample preparation protocol was developed for the analysis of Arabidopsis thaliana using Gas chromatography coupled with mass spectrometry (GC-MS). In particular, a semi-automated machine tool is used to replace the traditional mortar-pestle method in tissue grinding. One-pot chemical extraction-derivatization is used to provide simplified sample preparation over the conventional multi-step liquid-liquid extraction protocol. Wild-type and transgenic Arabidopsis thaliana seedlings were used as the model system to evaluate performance of this newly developed method for use in metabolic profiling and also targeted quantitative analysis of salicylic acid for the study of systemic acquired resistance.
Solution and Surface Characterization of DNA and Proteins
(2005-12-01) Brewer, Scott Harmon; Stefan Franzen, Committee Chair; Edmond Bowden, Committee Member; Daniel Feldheim, Committee Member; Jan Genzer, Committee Member
A variety of experimental spectroscopic techniques complemented by theoretical calculations when appropriate were used to investigate DNA adlayers on surfaces and proteins in solution. The formation and characterization of DNA adlayers on gold and indium tin oxide surfaces were characterized along with subsequent surface DNA hybridization. Prior to the modification of indium tin oxide surfaces with DNA, the optical and electronic properties of this metal oxide, fluorine doped tin oxide and iridium oxide were investigated. The detection of DNA hybridization on indium tin oxide surfaces utilized gold nanoparticle labeled target ssDNA. Further work on gold surfaces was performed using infrared spectroscopy to detect sugar binding to a phenylboronic acid terminated self-assembled monolayer. The binding properties and stability of gold nanoparticles were investigated by characterizing citrate and bovine serum albumin binding to gold surfaces and the stability of particles stabilized by these molecules. The stability and folding kinetics of the three helix bundle protein, villin headpiece subdomain was also investigated in addition to a theoretical investigation of the vibrational Stark effect. Finally, a time resolved step scan FTIR spectrometer was implemented with five microsecond time resolution.
Spectroscopic Characterization of the Function and Mechanism of Dehaloperoxidase
(2004-12-01) Belyea, Jennifer Lee; Edmond Bowden, Committee Member; Steven Lommel, Committee Member; Stefan Franzen, Committee Chair
The research presented in this thesis focuses on three main areas of work with dehaloperoxidase, DHP. The development of a viable source of recombinant DHP is discussed first. Enzymatic activity of DHP is the second area of research discussed followed by ligand binding studies. All of this research is done in the attempt to understand the structure function relationship within DHP. DHP's globin structure and peroxidase activity is the source of our interest in studding the structure function relationship in DHP. Typically, globins are not peroxidases. In DHP's case a unique heme active site, that is not common for globins or peroxidases, alters the ligand binding and the Poulos-Kraut push/pull mechanism of compound I formation. Specifically DHP has a distal valine residue that is unable to function as the residue that is responsible for the abstraction of a proton. Typically, the distal residue in both globins and peroxidases is a histidine. The distal histidine is credited with the abstraction of the proton in the pull step of the Poulous-Kraut push/pull mechanism.