Browsing by Author "Kenneth W. Hanck, Committee Member"

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Analysis of Reactive Dye Mixtures - Characterization of Products from Bis-Dichlorotriazine Dye Synthesis
(2007-08-22) Chen, Kangqin; Kenneth W. Hanck, Committee Member; Harold S. Freeman, Committee Chair; C. Brent Smith, Committee Co-Chair
The Application of Single-Pass Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy for Protein Analysis
(2003-04-07) Smith, Brandye Michelle; Daniel L. Feldheim, Committee Member; Kenneth W. Hanck, Committee Member; Paul Wollenzien, Committee Member; Stefan Franzen, Committee Chair
Application of single-pass attenuated total reflection Fourier-transform infrared (ATR-FT-IR) spectroscopy was investigated for the following; 1) secondary structure protein analysis, 2) protein secondary structure prediction, 3) amino acid classification, 4) peptide conformational, and 5) protein binding interactions. This research is the first reported application of single-pass ATR-FT-IR for protein analysis thus the method was validated using transmission FT-IR and multi-pass ATR-FT-IR as referee methods. The technique was advantageous since it permitted rapid secondary structure analysis on small volumes of protein in H2O solution without the use of demountable thin pathlength sample cells and without performing a H2O subtraction. Principle component regression (PCR) was applied to a spectral library of proteins in H2O solution acquired by single-pass ATR-FT-IR spectroscopy to predict the secondary structure content, principally a-helical and the b-sheet content, of proteins within a spectral library. An 'inside model space' bootstrap and a genetic algorithm (GA) were successfully used to improve prediction results. Three spectral libraries are presented and the validation results yielded an average absolute error of 1.6% for a-helix content prediction and an average absolute error of 1.7% for b-sheet content prediction. Single-pass ATR-FT-IR and normal mode analysis was employed to study amino acids and their role in the spectra of peptides. An amino acid identification model based upon Mahalanobis' distance yielded 100% correct classification for all 20 amino acids used in this study. Comparisons of peptides in free solution and those associated with proteins were made and it was established that the single-pass ATR method has a sufficient signal-to-noise ratio to obtain difference spectra for peptides in solution and peptides conjugated or bound to proteins. This study showed that protein-associated peptides differ in conformation from those in free solution. To investigate protein-binding interactions a Ge internal reflectance element was modified. Successful binding of his-tagged dehaloperoxidase (DHP) and his-tagged biotin via a nickel nitrilotriacetic acid (Ni-NTA) surface was monitored via single-pass ATR-FT-IR. The surface modification method presented had minimal non-specific binding, the surface was re-usable, and removal of the organic surface was achievable.
Development and Characterization of an Atmospheric Pressure Ionization Source Matrix-Assisted Laser Desorption Electrospray Ionization Coupled to Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Analysis of Biological Macromolecules
(2009-07-16) Sampson, Jason Scott; Jorge A. Piedrahita, Committee Member; Edmond F. Bowden, Committee Member; Kenneth W. Hanck, Committee Member; David C. Muddiman, Committee Chair
The field of mass spectrometry has grown tremendously over the past few decades due in large part to the continued application to new and interesting areas of exploration. The advent of soft ionization sources such as electrospray ionization and matrix-assisted laser desorption/ionization have dramatically increased the capabilities of mass spectrometry and increased the amount of information derived from biological samples. As a result, there has been tremendous growth in the development of new ionization source technology in recent years. One of the main driving forces behind the development of new ionization technology is for the reduction of sample preparation required prior to analysis, yielding high throughput sample analysis. Demonstrated herein is the development and characterization of an atmospheric pressure ionization source called matrix-assisted laser desorption electrospray ionization (MALDESI). MALDESI is a hybrid combination of MALDI and ESI which utilizes laser desorption with electrospray postionization for the generation of multiply-charged ions. Multiply-charged ions are of particular importance when using Fourier transform mass spectrometry, due to the increase in resolving power and mass accuracy with increasing charge on the molecule. Top-down characterization of intact polypeptides is demonstrated as well as high mass accuracy utilizing internal calibration. A newly designed highly robust and versatile atmospheric pressure ionization platform is developed and described in detail. Solid- and liquid state analysis of three out of the four classes of biological molecules is demonstrated utilizing the newly developed versatile ionization platform. MALDESI utilizing ultraviolet and infrared laser desorption is demonstrated at various wavelengths (UV, 337 nm and 349 nm and IR, 2.94 Ã‚Âµm and 10.6 Ã‚Âµm) with and without ESI postionization for the generation of multiply-charged ions. The characteristics of liquid-state samples are described as a macroscopic electrospray droplet and characterized. MALDESI direct analysis is demonstrated and applications for high throughput analysis of complex samples are described.
Exploring Fundamental Aspects of Proteomic Measurements: Increasing Mass Measurement Accuracy, Streamlining Absolute Quantification, and Increasing Electrospray Response
(2009-05-08) Williams, Dennis Keith Jr.; Kenneth W. Hanck, Committee Member; Edmond F. Bowden, Committee Member; David C. Muddiman, Committee Chair; Lin He, Committee Member
Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) offers unparalleled performance in terms of resolving power and mass measurement accuracy. In order to realize the highest achievable mass measurement accuracy for a given FT-ICR MS system, frequency shifts due to space-charge effects must be accounted for via external or internal calibration methods. Herein, a dual electrospray ionization source was coupled to a hybrid quadrupole Fourier transform ion cyclotron resonance mass spectrometer and utilized to incorporate internal calibrant to yield high mass measurement accuracy results. Frequency shifts from space-charge effects were also counteracted by accounting for total ion population and relative ion population on a MALDI-FT-ICR MS and a hybrid LTQ-FT-ICR MS equipped with automatic gain control. This was achieved through the use of multiple linear regression and artificial neural networks. These experiments resulted in mean mass measurement accuracies in the parts-per-billion range. C-reactive protein (CRP) is an important clinical marker for inflammation, atherosclerosis, and has also been observed to be upregulated in patients with epithelial ovarian cancer (EOC). This dissertation contains results from a protein cleavage isotope dilution mass spectrometry method developed for the absolute quantification of CRP from human plasma. A total of 110 human plasma samples were analyzed including 54 samples from patients with EOC. The results were compared to a CLIA certified ELISA test which showed high correlation but different absolute values, which suggested different reference ranges for different analytical techniques. In addition, a correlation was observed between the stage of diagnosis of cancer and CRP concentrations. Chemical tags have long been utilized with mass spectrometry for a multitude of purposes, extending the effectiveness of the measurements dramatically. Electrospray ionization has been shown to preferentially ionize more hydrophobic species. Four new iodoacetamide derivatives, which react with the amino acid cysteine, were reacted with three peptides to determine their ability to increase electrospray response. This resulted in increases up to 2000-fold compared to alkylation of peptides with iodoacetamide. The combination of these results will aid future studies to identify and quantify proteins and peptides which contain cysteine and can be expanded to all peptides using amine-specific chemistry.
The Interaction of Boronic Acid Based Self-Assembled Monolayers as a Potential Glucose Sensor
(2005-08-02) Allen, Angela M.; Christopher Gorman, Committee Chair; Charles B. Boss, Committee Member; Edmond F. Bowden, Committee Member; Kenneth W. Hanck, Committee Member
Recognition of sugars such as glucose with phenylboronic acid–terminated self assembled monolayers (SAMs) was the basis for the designing and fabricating a biosensor. Self-assembled monolayers were formed on gold surfaces as shown by infrared spectroscopy. The continuous, repeatable increase of the electrochemical impedance as the concentration of glucose was increased gave indication of binding. This binding could be attributed to the precedent formation of relatively stable esters between phenylboronic acids and sugars. This impedance change upon binding between glucose and a phenylboronic acid-terminated SAM showed promise in the development of a glucose biosensor.
Interfacial Electrochemical Investigation of Dehaloperoxidase.
(2004-04-14) Robinson, KeAndra R.; Charles B. Boss, Committee Member; Kenneth W. Hanck, Committee Member; Edmond F. Bowden, Committee Chair
The terebellid polychaete marine worm Amphitrite ornata contains an enzyme known as dehaloperoxidase (DHP). Dehaloperoxidase has unique characteristics that permit Amphitrite ornata to cohabit with other marine worms that secrete toxic haloaromatic compounds. Dehaloperoxidase has the capability to break down such compounds in the presence of hydrogen peroxide. For example, trihalophenols can be oxidized to dihaloquinones. Prior studies have described the structure, function, and mechanistic behavior of dehaloperoxidase. However, there have been no reports of the electrochemical analysis of dehaloperoxidase. The data in this thesis demonstrates the direct electrochemistry of dehaloperoxidase at bare indium tin oxide and at modified gold electrode surfaces. The gold electrodes were modified with HS(CH2)10COOH/HS(CH2)6OH mixed SAM. Not only diffusional voltammetry has been demonstrated, but also adsorption of electroactive dehaloperoxidase at the modified gold electrode surface. The successful electrochemistry of DHP is attributed to a cluster of lysines near a heme edge, analogous to cytochrome c. Using cyclic voltammetry, a formal reduction potential of the DHP FeIII/FeII heme couple in the presence of dioxygen was determined, which will provide insight into the enzymatic activity of the protein. This reduction potential was determined to be +440 mV v/s NHE. In the presence of tri-bromophenol, an organic substrate for DHP, the catalytic reduction was observed in the presence of dioxygen. In general, this thesis reports the first electrochemical investigation of dehaloperoxidase and explores its interfacial electrochemical properties at the indium tin oxide and modified gold electrode surfaces.
New Software for Chemistry Applications: Algorithms and Experiment Planners for Automated Chemistry, and an Interactive Program for Use in Photochemistry (PhotochemCAD)
(2004-11-15) Dixon, James M.; Edmond F. Bowden, Committee Member; Jonathan S. Lindsey, Committee Chair; Kenneth W. Hanck, Committee Member; Charles B. Boss, Committee Member
This dissertation is based on convergent projects that focus on helping integrate the use of computers in the chemical field. Project 1 entails development of experiment planners for automated chemistry workstations. The power of parallel workstations has become evident in their pervasive application in high-throughput screening and combinatorial chemistry. Adaptive systems make decisions automatically based on data collected from ongoing experiments. Early adaptive systems performed reactions serially and were quickly eclipsed by workstations capable of performing parallel operations but without decision-making capabilities (i.e., open-loop). Great effort has been put into developing software for automated workstations that enables parallel, adaptive experimentation. A suite of algorithms that accompany an experiment planning software package for use with automated chemistry workstations has been developed. The main objective of this research is reaction optimization, a very general problem in many areas of chemistry, but the approach is quite general. The algorithms developed for parallel, adaptive experimentation include the successive focused grid search (SFGS), multidirectional search (MDS), parallel multidirectional search (PMDS'), parallel simplex search (PSS), parallel screening each member of a set of catalysts across a range of concentration (Parascreen), and several two-tiered methods that enable broad screening followed by in-depth searches. Project 2 focuses on the growth and improvement of the PhotochemCAD software. Laboratory researchers often face the problem of organizing and managing large quantities of experimental data, as well as comparing their data with literature data. In the photochemical sciences, the core data include the spectral properties of diverse substances. One wants to know the absorption and emission properties of a compound, have pointers to the literature, and be able to perform calculations using such spectral data. A versatile software package (PhotochemCAD) has been developed that includes a database of spectral data with literature references for over 150 compounds of natural and synthetic origin. Features are available for performing diverse calculations of interest across the photochemical sciences, including Förster energy transfer, oscillator strength, multicomponent analysis, blackbody radiator, etc. This development effort has much in common with research aimed at creating versatile software programs that address the needs of experimentalists in laboratory settings.
RNA-Mediated Synthesis of Particles from Organometallic Palladium and Platinum Precursors
(2007-08-01) Gugliotti, Lina Ann; Tatyana I. Smirnova, Committee Member; Kenneth W. Hanck, Committee Member; Bruce E. Eaton, Committee Chair; Daniel L. Feldheim, Committee Member
RNA sequences have been discovered that mediate the growth of hexagonal, cubic and spherical palladium and platinum containing particles. In vitro selection techniques were used to evolve an initial library of ˜ 3 x 10¹³ unique RNA sequences through six to ten cycles of selection to yield several active sequence families. The particle growth occurred in aqueous solution at ambient temperature, without any endogenous reducing agent, and at low concentrations of metal precursor (10 μM — 400 μM). Relative to the metal precursor the RNA concentration was significantly lower (1 μM). RNA sequences that utilize the organometallic complex tris(dibenzylideneacetone) dipalladium(0) ([Pd₂(DBA)₃]) to form palladium containing particles were further characterized for their ability to control particle growth. These RNA sequences (Pdases) were found to form hexagonal and cubic palladium containing particles with a high degree of shape specificity. Replacing the pyridyl-modified RNA sequence with native RNA resulted in a complete loss of RNA function. Removing the 3′-fixed sequence region from the Pdase had little effect on particle growth; however, further truncations into the variable region resulted in a significant loss of activity and particle shape control. Changing the metal center and ligand of the group VIII organometallic precursor complex revealed a strong dependence of particle growth and shape on the DBA ligands. The Pdases were covalently immobilized on gold surfaces and evaluated for their activity toward particle synthesis. When coupled to gold via oligoethylene glycol linkers, both RNA isolates 17 and 34 were able to mediate the formation of Pd containing particles with the same shape control previously observed in solution. Finally, the use of surface-bound RNA as a tool for directing the orthogonal synthesis of materials on surfaces was demonstrated. Patterning the RNA sequence for hexagons next to the sequence for cubes, followed by incubation in a solution containing [Pd₂(DBA)₃], resulted in the spontaneous formation of spatially distinct spots of Pd containing hexagonal and cubic particles.
Structure-Property Relationships for Electron Transfer Kinetics in Metal Tris(bipyridine) Core Dendrimers
(2005-07-28) Hong, Young-Rae; Edmond F. Bowden, Committee Member; Christopher B. Gorman, Committee Chair; David A. Shultz, Committee Member; Kenneth W. Hanck, Committee Member
Structure property relationship in the redox-active core dendrimers were systematically studied by probing the rate and driving force for electron transfer. An isostructural series of redox-active, metal tris(bipyridine) core dendrimers were synthesized for this purpose. Various synthetic routes were attempted to introduce the bulky dendritic moieties to the bipyridine units with high yields. Heterogeneous electron transfer kinetics was studied by electrochemical methods. In the second generation of these dendrimers, a large attenuation of electron transfer rate was observed qualitatively. A newly designed thin layer electrode was constructed and utilized to study heterogeneous electron transfer kinetics in the second generation dendrimers. In the finite condition, the slow heterogeneous electron transfer kinetics in second generation dendrimers could be studied by computer simulation. Homogeneous electron self-exchange kinetics was studied by nuclear magnetic resonance spectroscopy. The rate attenuation of electron transfer with dendrimer generation was not the same as the behavior found in heterogeneous, electrochemical electron transfer rate determinations. While a large attenuation was observed between the zeroth and first generation, the attenuation of electron transfer between the first and second generation was insignificant. This was rationalized by the concept of core mobility. The redox core in slow exchange limit can move in a non-rate limiting fashion toward a neighboring redox core with the result that the structural effect of the dendrimer is reduced and electron transfer is facilitated in larger dendrimers. For further studies, thermodynamic activation parameters were also obtained by variable temperature nuclear magnetic resonance studies.