Doctor of Philosophy / Department of Chemistry / Jun Li / Reduction in electrode size down to nanometers dramatically enhances the detection sensitivity and temporal resolution. Here we explore nanoelectrode arrays (NEAs) and nanoparticles in building high performance biosensors. Vertically aligned carbon nanofibers (VACNFs) of diameter ~100 nm were grown on a Si substrate using plasma enhanced chemical vapor deposition. SiO[subscript]2 embedded CNF NEAs were then fabricated using techniques like chemical vapor deposition, mechanical polishing, and reactive ion etching, with CNF tips exposed at the final step. The effect of the interior structure of CNFs on electron transfer rate (ETR) was investigated by covalently attaching ferrocene molecules to the exposed end of CNFs. Anomalous differences in the ETR were observed between DC voltammetry (DCV) and AC voltammetry (ACV). The findings from this study are currently being extended to develop an electrochemical biosensor for the detection of cancerous protease (legumain). Preliminary results with standard macro glassy carbon electrodes show a significant decrease in ACV signal, which is encouraging. In another study, NEA was employed to capture and detect pathogenic bacteria using AC dielectrophoresis (DEP) and electrochemical impedance spectroscopy (EIS). A nano-DEP device was fabricated using photolithography processes to define a micro patterned exposed active region on NEA and a microfluidic channel on macro-indium tin oxide electrode. Enhanced electric field gradient at the exposed CNF tips was achieved due to the nanometer size of the electrodes, because of which each individual exposed tip can act as a potential DEP trap to capture the pathogen. Significant decrease in the absolute impedance at the NEA was also observed by EIS experiments. In a final study, we modified gold nanoparticles (GNPs) with luminol to develop chemiluminescence (CL) based blood biosensor. Modified GNPs were characterized by UV-Vis, IR spectroscopy and TEM. We have applied this CL method for the detection of highly diluted blood samples, in both intact and lysed forms, which releases Fe[superscipt]3[superscript]+ containing hemoglobin to catalyze the luminol CL. Particularly, the lysed blood sample can be detected even after 10[superscript]8 dilution (corresponding to ~0.18 cells/well). This method can be readily developed as a portable biosensing technique for rapid and ultrasensitive point-of-care applications.

Doctor of Philosophy / Department of Mechanical and Nuclear Engineering / Kevin B. Lease / The overall goal of this study was to assess the effects of surface chemistry on adhesion of polymeric systems underwater. The adhesion is quantified by the thermodynamic work of adhesion (W) when two surfaces are approached and the energy release rate (G) when the surfaces are separated. For some polymeric systems there is a difference between W and G, referred to as adhesion hysteresis. For this study an experimental approach based upon Johnson-Kendall-Roberts (JKR) theory of contact mechanics was utilized to evaluate how surface chemistry affects the adhesion behavior (both W and adhesion hysteresis) in the presence of water. The interfacial interactions were also studied in air and contrasted to those obtained underwater. To accomplish the overall goal of this research, this study was divided into two phases where smooth model surfaces with disparate surface chemistries were used. The model surfaces in the first part included poly(dimethysiloxane) (PDMS), glass surfaces chemically functionalized to display hydrophilic to medium to hydrophobic characteristics, and thin films of wood-based biopolymers. The functionalities used to modify glass surfaces included polyethylene oxide (PEO) with hydrophilic nature; amine, carbomethoxy, and mercapto (thiol) with intermediate characteristics; cyclohexyl, fluorocarbon, and methyl with hydrophobic behavior. In addition to these surfaces, flat PDMS and clean glass surfaces were also used for means of comparison. The wood-derived polymers included two different cellulose types (natural cellulose and regenerated cellulose) as well as one lignin surface (from hardwood milled lignin). These surfaces were probed with native PDMS hemispheres, which are hydrophobic. The results showed that in air the value of W for all model surfaces was independent of the surface chemistry, except fluorocarbon which was lower. Underwater W was significantly affected by the surface hydrophilicity/ hydrophobicity. The adhesion hysteresis both in air and underwater was significantly dependent on the structure of the probed surface. For the second phase PDMS hemispheres were chemically modified with amine functionality to probe model surfaces with hydrophilic and intermediate behavior. These surfaces included glass surfaces functionalized with PEO and amine as well as PDMS sheets that were functionalized with amine. Native PDMS flat surfaces were also used for means of comparison. The results showed that for the selected surfaces both W and hysteresis were affected by the surface chemistry in both media.

Doctor of Philosophy / Department of Chemistry / Duy H. Hua / Three research projects are described in this dissertation and they consist of the discovery of norovirus protease inhibitors, modification of graphene oxides (GO) for the detection of norovirus, and design and fabrication of nanoelectronic device based on nanocarbon fibers for the detection of breast cancer proteases, legumain and cathepsin B. A novel class of tripeptidyl anti-noroviral compounds which strongly inhibit NV3CL[superscript]pro in enzyme and cell based assays was discovered. An example of one of the most active compounds is (1-{3-methyl-1-[2-oxo-1-(2-oxo-pyrrolidin-3-ylmethyl)-ethylcarbamoyl]-butylcarbamoyl}-2-naphthalen-1-yl-ethyl)-carbamic acid benzyl ester, which showed an IC₅₀ value of 0.14 ± 0.2 μM (enzyme assay) and EC₅₀ value of 0.04 ± 0.02 μM (cell based assay). This compound has an aldehyde warhead, a P1 glutamine surrogate, a P2 leucine, a P3 L-1-napthylalanine and an N-terminal carboxybenzyl cap. The corresponding bisulfite adduct, 2-[2-(2-benzyloxycarbonylamino-3-naphthalen-1-yl-propionylamino)-4-methyl-pentanoylamino]-1-hydroxy-3-(2-oxo-pyrrolidin-3-yl)-propane-1-sulfonic acid monosodium salt, has a comparable activity in enzyme and cell based assays (IC₅₀ 0.24 ± 0.1 μM; EC₅₀ 0.04 ± 0.03 μM). (1-{3-methyl-1-[2-oxo-1-(2-oxo-pyrrolidin-3-ylmethyl)-ethylcarbamoyl]-butylcarbamoyl}-2-naphthalen-1-yl-ethyl)-carbamic acid benzyl ester and its ketoamide derivative, (1-{1-[2-isopropylcarbamoyl-2-oxo-1-(2-oxo-pyrrolidin-3-ylmethyl)-ethylcarbamoyl]-3-methyl-butylcarbamoyl}-2-naphthalen-1-yl-ethyl)-carbamic acid benzyl ester, exhibited very good broad spectrum anti-viral activity, especially in human rhino virus and severe acute respiratory syndrome bioassays. We demonstrated that the surface of graphene oxide can be chemically modified with t-butylester and carboxylic acid functionalities. Fourier transform infrared spectroscopy, Raman spectroscopy and solid state nuclear magnetic resonance spectroscopy confirmed the presence of t-butylester and carboxylic acid functional groups. One sided oligonucleotide functionalized graphene oxide was synthesized using a solid state technique. A carboxylic acid functionalized graphene oxide was deposited onto the surface of electronic chips to bridge two gold electrodes, using a direct deposition technique. The carboxylic acid functionalized graphene oxide displayed semi-conductive properties and its use in an electronic biosensor device to detect noroviral RNA was investigated. Novel redox-active protease substrate peptides H₂N-(CH₂)₄CO-Ala-Ala-Asn-Leu-NHCH₂-ferrocene and H₂N-(CH₂)₄CO-Leu-Arg-Phe-Gly-NHCH₂-ferrocene were synthesized successfully and used in an alternating current voltammetry technique to facilitate the detection of the cancer related protease enzymes legumain and cathepsin B. After attachment of these peptides to the tips of carbon nanofiber nanoelectrode arrays, the presence of active protease enzymes could be detected as manifest by an exponential decay in current signal detect when monitored by alternating current voltammetry, at initial enzyme concentrations of 80.1 nM (legumain) and 30.7 nM (cathepsin B). The peptide cleavage sites were confirmed by analyses of the cleaved fragments using high performance liquid chromatography and mass spectrometry. Results showed that the cleavage of H₂N-(CH₂)₄CO-Ala-Ala-Asn-Leu-NHCH₂-ferrocene at the C-terminal side of asparagine residues by legumain and cleavage of H₂N-(CH₂)₄CO-Leu-Arg-Phe-Gly-NHCH₂-ferrocene at the C-terminal side of arginine residues by cathepsin B. Legumain exhibited a specificity constant (k[subscript]cat/K[subscript]m) of 11.3 x 10ᶟ M⁻¹S⁻¹ while cathepsin B exhibited a higher value of specificity constant (4.3 x 10⁴ M⁻¹S⁻¹) which agreed with the values obtained from fluorescence enzyme assay.

Doctor Of Philosophy / Department of Chemistry / Christer Aakeröy / Crystal engineering, or non-covalent synthesis in the solid state, requires an understanding of intermolecular forces, and the hydrogen bond has become a reliable non-covalent tool in the construction of supramolecular architectures. In the same way that synthetic chemists refer to a “yield” to quantify a desired product, crystal engineers typically determine the successful formation of a supramolecular product according to the frequency or occurrence of preferred intermolecular interactions between molecules under certain reaction conditions, thus, the supramolecular yield. These non-covalent reactions can be effectively carried out using supramolecular reagents (SR’s). A family of ditopic bis-imidazol-1-yl/benzimidazol-1-yl compounds were synthesized and used as SR’s in combination with a variety of dicarboxylic acids to produce binary solids in 100% yield through the primary acid···imidazol-1-yl/benzimidazol-1-yl synthons even in the presence of potentially disruptive intermolecular interactions. We furthermore noted that secondary C–H···O interactions within and between 1-D chains were of equal structural importance based upon an analysis of the metrics displayed by these interactions. The use of these SR’s as ligands with neutrally charged metal complexes was also investigated. SR’s containing benzimidazol-1-yl and carboxamide moieties were synthesized and combined with two different carboxylic acids to make ternary solids through acid···benzimidazol-1-yl and carboxamide···acid hydrogen bonds using a hierarchical approach – the best donor-best acceptor, second best donor-second best acceptor guidelines. These SR’s were also employed as ligands for high-yielding syntheses of linear metal complexes where neighboring complexes are linked via carboxamide···carboxamide hydrogen bonds. Asymmetric SR’s possessing two different N-heterocycles were synthesized and employed in the construction of ternary supermolecules with a high degree of structural selectivity and specificity when introduced to two different carboxylic acids. The stronger acid interacts at the more basic site, while the weaker acid hydrogen-bonds with the less basic nitrogen atom. Finally, an SR containing three different binding sites was designed and synthesized with the aim of producing quaternary co-crystals.

Doctor of Philosophy / Department of Chemistry / Takashi Ito / Self-organized nanoporous materials have drawn a lot of attention because the uniform, highly dense, and ordered cylindrical nanopores in these materials provide a unique platform for chemical separations and chemical sensing applications. Here, we explore self-organized nanopores of PS-b-PMMA diblock copolymer thin films and anodic gallium oxide for chemical separations and sensing applications. In the first study, cyclic voltammograms of cytochrome c on recessed nanodisk-array electrodes (RNEs) based on nanoporous films (11, 14 or 24 nm in average pore diameter; 30 nm thick) derived from polystyrene-poly(methylmethacrylate) diblock copolymers were measured. The faradic current of cytochrome c was observed on RNEs, indicating the penetration of cytochrome c (hydrodynamic diameter ≈ 4 nm) through the nanopores to the underlying electrodes. Compared to the 24-nm pores, the diffusion of cytochrome c molecules through the 11- and 14-nm pores suffered significantly larger hindrance. The results reported in this study will provide guidance in designing RNEs for size-based chemical sensing and also for controlled immobilization of biomolecules within nanoporous media for biosensors and bioreactors. In another study, conditions for the formation of self-organized nanopores of a metal oxide film were investigated. Self-organized nanopores aligned perpendicular to the film surface were obtained upon anodization of gallium films in ice-cooled 4 and 6 M aqueous H2SO4 at 10 V and 15 V. The average pore diameter was in the range of 18 ~ 40 nm, and the anodic gallium oxide was ca. 2 µm thick. In addition, anodic formation of self-organized nanopores was demonstrated for a solid gallium monolith incorporated at the end of a glass capillary. Nanoporous anodic oxide monoliths formed from a fusible metal will lead to future development of unique devices for chemical sensing and catalysis. In the final study, surface chemical property of self-organized nanoporous anodic gallium oxide is explored through potentiometric measurements. The nanoporous anodic and barrier layer gallium oxide structures showed slow potentiometric response only at acidic pH (≤ 4), in contrast to metallic gallium substrates that exhibited a positive potentiometric response to H⁺ over the pH range examined (3-10). The potentiometric response at acidic pH probably reflects some chemical processes between gallium oxide and HCl.

Doctor of Philosophy / Department of Biochemistry and Molecular Biophysics / John M. Tomich / Branched Amphiphilic Peptide Capsules (BAPCs) are peptide nano-spheres comprised of equimolar proportions of two branched peptide sequences bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK that self-assemble in water to form bilayer delimited poly-cationic capsules capable of trapping solutes. We examined the lipid-like properties of this system including assembly, fusion, solute encapsulation, and resizing by membrane extrusion as well as their capability to be maintained at a specific size by storage at 4˚C. These studies along with earlier work from the lab (Gudlur et al. (2012) PLOS ONE 7(9): e45374) demonstrated that the capsules, while sharing many properties with lipid vesicles, were much more robust. We next investigated the stability, size limitations of encapsulation, cellular localization, retention and, bio-distribution of the BAPCs. We demonstrated that the BAPCs are readily taken up by epithelial cells in culture, escape or evade the endocytotic pathway, and accumulate in the peri-nuclear region where they persist without any apparent degradation. The stability and persistence of the capsules suggested they might be useful in delivering radionuclides. The BAPCs encapsulated alpha particle emitting radionuclides without any apparent leakage, were taken up by cells and were retained for extended periods of time. Their potential in this clinical application is being currently pursued. Lastly we studied the temperature dependence of capsule formation by examining the biophysical characteristics of temperature induced conformational changes in BAPCs and examined the structural parameters within the sequences that contribute to their remarkable stability. A region in the nine-residue sequence was identified as the critical element in this process. The ability to prepare stable uniform nano-scale capsules of desired sizes makes BAPCs potentially attractive as delivery vehicles for various solutes/drugs.

Master of Science / Department of Chemistry / Christopher T. Culbertson / Capillary isoelectric focusing (cIEF) is one of several electrophoretic separation techniques for proteins and various other bio - molecules widely used in biochemistry laboratories. A wide range of analytes separable by the different modes of Capillary Electrophoresis (CE) includes from a small organic or an inorganic molecule to the complex bio-molecules such as protein, peptides, cell organelles, and live microorganisms (e.g. bacteria and viruses). Of the various modes of electrophoresis, Isoelectric focusing (IEF) is a good method for the separation of large amphoteric molecules such as peptides and proteins because of the attainment of overall surface charge depending up on its environment pH. This thesis mainly focuses on application of cIEF for proteins separation and viruses’ detection, which is one of the biggest concerns of human and animal health because of viral outbreak causing loss of thousands of lives and property every year. In chapter one of this thesis, the principles and mechanisms of separation of CE, cIEF, comparative advantages of dynamic coatings over static coating, and advantages of Whole Column Imaging Detection (WCID) over On - olumn Single Point Detection have been discussed. Chapter two includes experimental procedure and calculations for EOF determination. The results of cIEF experiments with standard proteins to develop calibration curve followed by UV absorbance detection of two bacteriophage viruses TR4 and T1 are presented in the chapter three. Final chapter four includes the conclusion and discussion on future direction for the project. The main motivation for this work was to develop a method which is less labor intensive and requires shorter detection time compared to traditional detection methods such as virus culture in serology (7days), polymerase chain reaction (PCR) and gel electrophoresis (6hrs to 2days). A commercially available dynamic coating reagent, EoTrol LN® copolymer used our CE experiments found to be more convenient and efficient than commonly used surface modifiers for example silane-based reagents. Preliminary determination of the pIs of these T1 and TR4 by cIEF was 3.1 ± 1.0 and 6.8 ± 1.0 respectively. The pI of viruses can differ by their strains and the phase of virus - growth. The viruses, though closely related, are easily distinguishable by their different pIs.

Master of Science / Department of Chemistry / Mark Hollingsworth / The crystal structures of the inclusion compounds 1,6-diisocyanohexane/urea and 1,6-dicyanohexane/urea were refined at several temperatures from X-ray data. These urea inclusion compounds are commensurate structures with host:guest ratios of 6:1. In contrast with the ordinary helical topology of the urea host, these structures have stacked loop topologies of the host hydrogen bonds and crystallize in space group P21/n. At room temperature, both structures are distorted along [001] from hexagonal metric symmetry. As in earlier studies of 1-chloro-6-cyanohexane/urea, cooling 1,6-diisocyanohexane/urea gives rise to an exothermic phase transition (Hº = -856 cal mol 1, Sº = -5 cal mol-1 K-1) at 175 K that results in the cooperative translation of guest molecules by 5.5 Å along the channel axis. In the low temperature form, 1,6-diisocyanohexane/urea is distorted along [100], much like X(CH2)6Y, where X, Y = Br, Cl. Although the crystal structure of 1,6-dicyanohexane/urea is essentially isomorphous with that of the high temperature form of 1,6-diisocyanohexane/urea, it does not undergo an equivalent phase transition at low temperatures. Both of these systems exhibit dynamic disorder between two gauche conformers of the guest, which have mean planes of the alkyl chains lying within 1º of [100] (major conformer) and approximately 14º from [001] (minor conformer). The temperature dependence of site occupancy factors for the disordered sites yielded enthalpy differences between major and minor sites in 1,6-diisocyanohexane/urea and 1,6-dicyanohexane/urea of 216 and 127 cal mol 1, respectively. Since the low temperature form of 1,6-diisocyanohexane/urea is distorted along [100], this is favored at low temperatures by an increased concentration of the major conformer, which predominates in 1,6-dibromohexane/urea and congeners. In 1,6-diisocyanohexane/urea, the phase transition to the low temperature form occurs at a threshold concentration for the major conformer of 67%. With its shallower temperature dependence, 1,6-dicyanohexane/urea should not reach this threshold population until approximately 92 K, at which temperature the system cannot overcome the barrier for cooperative translation of guests along the channel axis.

Doctor of Philosophy / Department of Chemical Engineering / Keith L. Hohn / Amorphous silica-alumina is widely used as a solid acid catalyst for various reactions in oil refining and the petrochemical industry. The strength and the number of the acid sites in the material are most often believed to arise from the alumina atoms inserted into the silica lattice. The existence of the acidity distribution across the framework is a result of the local composition or the short-range interactions on the silica-alumina surface. Conventional techniques used to characterize silica-alumina provide effective information on the average acidity, but may not reflect the heterogeneity of surface acidity within the material. Recently, it is possible to study individual catalytic sites on solid catalysts by single molecule fluorescence microscopy with high time and space resolution. Fluorophores can be chosen that emit at different wavelengths depending on the properties of the local environment. By doping these fluorophores into a solid matrix at nanomolar concentrations, individual probe molecules can be imaged. Valuable information can be extracted by analyzing changes in the fluorescence spectrum of the guest molecules within a host matrix. In this research, silica-alumina thin films were studied with single molecule fluorescence microscopy. The samples were prepared by a sol-gel method and a wide-field fluorescence microscope was used to locate and characterize the fluorescent behaviors of pH sensitive probes. In mesoporous thin films, the ratio of the dye emission at two wavelengths provides an effective means to sense the effective pH of the microenvironment in which each molecule resides. The goal of this work was to develop methods to quantify the acidity of individual micro-environments in heterogeneous networks. Pure silica films treated with external phosphate solutions of different pH values were used to provide references of the fluorescence signals from individual dye molecules. SM emission data were obtained from mesoporous Al-Si films as a function of Al content in films ranging from 0% to 20% alumina. Histograms of the emission ratio revealed that films became more acidic with increasing Al content. The acidity on interior surfaces in zeolite pores was also of interest in this work. A microfluidic device was built to isolate the interior surface from the exterior surface. Some preliminary results showed the potential of using SM fluorescence method to study the acidic properties inside the pores of zeolite crystals.

Doctor of Philosophy / Department of Chemistry / Viktor Chikan / The research work in this thesis aims at investigating the basic physic-chemical properties of magnetic and metal nanoparticles (NPs) for biomedical applications such as magnetic hyperthermia and controlled drug release. Magneto-plasmonic properties of magnetic NPs are important to evaluate potential applications of these materials. Magnetic property can be used to control, monitor and deliver the particles using a magnetic field while plasmonic property allows the tracking of the position of the particles, but aggregation of NPs could pose a problem. Here, the aggregation of NPs is investigated via the Faraday rotation of gold coated Fe[subscript]2O[subscript]3 NPs in alternating magnetic fields. In addition, the Faraday rotation of the particles is measured in pulsed magnetic fields, which can generate stronger magnetic fields than traditional inductive heaters used in the previous experiments. In the second project, the formation of protein-NPs complexes is investigated for hyperthermia treatment. The interactions between gold and iron-platinum NPs with octameric mycobacterial porin A from Mycobacterium smegmatis (MspA) and MspA[superscript])cys protein molecules are examined to assemble a stable, geometrically suitable and amphiphilic proteins-NPs complex. Magnetic NPs show promising heating effects in magnetic hyperthermia to eliminate cancer cells selectively in the presence of alternating magnetic field. As a part of investigation, the heating capacity of a variety of magnetic NPs and the effects of solvent viscosity are investigated to obtain insight into the heating mechanism of these particles. Finally, the controlled drug release of magnetic NPs loaded liposomes by pulsed magnetic field is investigated. The preliminary data indicate about 5-10% release of drug after the application of 2 Tesla magnetic pulses. The preliminary experiments will serve as the initial stage of investigation for more effective magnetic hyperthermia treatment with the help of short magnetic pulses.