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Citrate-Capped Iridium Nanoparticles as Peroxidase Mimics with High Catalytic EfficiencyYishay, Tamar 01 January 2021 (has links) (PDF)
Over time, noble-metal nanostructures have sparked interest as peroxidase mimics for in-vitro diagnostics, with emphasis on inflammatory pathogenesis such as cancer. Recent advancements focusing on improving the catalytic efficiency of currently used synthetic and natural peroxidases towards future clinical applications such as Point of Care (POC) settings are being studied. Efforts in exploiting the size-dependent and physicochemical properties of noble metal nanoparticles to achieve superior catalytic performance may serve as excellent alternatives to traditional peroxidases. Here, we introduce a facile protocol to engineer citrate-capped iridium nanoparticles (Ir NPs) to study their efficacy as peroxidase mimics towards future applications as secondary antibody-labels in in-vitro diagnostic (IVD) techniques. Our inspiration for choosing to explore Ir NPs stems from the following (i) recent studies demonstrating Ir-based nanostructures with excellent catalytic performance (ii) the successful catalytic decomposition of hydrogen peroxide in the presence of Ir, revealing peroxidase-like behavior, (iii) activity of Ir NPs at high temperatures, rendering them stable and promising for hydrothermal synthesis. In this work, we successfully engineered citrate-capped Ir NPs with superior catalytic efficiency at the level of 106 – 107 s-1. We hope this work serves as an inspiration to explore the implementation of Ir NPs in practical applications.
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Investigating the Effects of Glycerol Administration on Glial Cell CultureScheller, Stephen 01 January 2020 (has links) (PDF)
The chemical compound glycerol was first discovered in 1779 by the Swedish chemist Carl Wilhelm Scheele when he washed out glycerol from heating a mixture of lead oxide and olive oil. Many industries have found glycerol to be useful in the manufacturing of a variety of products due to its unique chemical properties. One such industry, pharmaceuticals, has found glycerol to be useful in the preparation of many medications. However, glycerol administration alone has been proven to treat medical conditions such as trigeminal neuralgia. Trigeminal Neuralgia (TN) is a unilateral electric shock-like facial pain often triggered by non-painful stimuli such as washing, shaving the face or talking. In some patients with TN, administration of glycerol into the trigeminal ganglion can alleviate the pain but the exact mechanism of pain relief is not understood. Additionally, glycerol administration in patients suffering from oral or thyroid cancer has been shown to reduce the spread and growth of these cancers. In this study, experiments utilizing various concentrations of glycerol administration were conducted on various glial cell cultures to determine glycerol's effects. Morphology studies were conducted on the glial cell culture types to determine the effects of various glycerol concentrations on overall cell structure. Additionally, traction force microscopy studies were performed on each glial cell culture type to determine the effects the various glycerol concentrations had on the forces each cell culture type applied to their respective underlying substrates. This study shows administration of moderate concentrations of glycerol to glial cell culture leads to shrinking of the astrocytes and changes in their traction forces.
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Modulation of Actin Filament Severing and Mechanics by Gelsolin in Varying pH ConditionsToland, Claire 01 January 2021 (has links) (PDF)
Actin is an essential cytoskeletal protein that plays a critical role in cell mechanics, structure and organization with the help of actin binding proteins (ABPs). Gelsolin is a calcium-dependent ABP that severs actin filaments and caps them at their barbed end, regulating cell motility and signaling through dynamic actin cytoskeleton remodeling. A recent study has indicated that low pH stabilizes the active conformations of gelsolin. Additionally, the binding of gelsolin to the barbed end of an actin filament induces a conformational change that propagates along the actin filament. However, it has not been well understood how the complex intracellular environments involving variations in pH affect gelsolin-mediated actin filament severing and mechanics at the molecular level. In this thesis, we investigate how binding of gelsolin modulates actin filament severing and mechanics with changes in solution pH using total internal reflection fluorescence (TIRF) microscopy imaging. Furthermore, we explore how changes in intra-filament structure and dynamics occur upon gelsolin binding through the use of atomic force microscopy (AFM) imaging. Taken together, this work will elucidate a mechanism to control actin filament severing and mechanics modulated by gelsolin in the pH fluctuating intracellular environment.
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Micro/nanofabrication Process Development and Device Characterization Towards Tri-Modal (Optical, Electrical, and Microfluidic) 3D Microelectrode Arrays (3D MEAs)Freitas Orrico, Julia 01 January 2021 (has links) (PDF)
A polymer and metal-based microfabrication technology were fabricated toward 3D Microelectrode Arrays (3D MEAs) with tri-modal functionality for obtaining simultaneous data sets comprising electrical, optical, and microfluidic markers from a variety of electrogenic cellular constructs. 3D MEAs are the next-generation interfaces to transduce multi-modal data sets from the burgeoning field of "organ-on-a-chip" in vitro modeling of biological functions. The microfabrication process is fully characterized, including key processes of microdrilling/micromilling for low and higher density 3D electrodes/ microfluidic (μF) ports along with full spectrum impedance and RMS noise showcasing the ability to control the 3D microelectrode size. Further the material set used in the microfabrication process is characterized for biological metrics through both a novel transparency assay along with a biocompatibility assay with multiple electrogenic cell culture systems. Impedance metrics showcasing morphology and spread of electrogenic cells are further analyzed. Lastly, rapid neuronal spheroid attachment to the 3D microfluidic ports of the tri-modal 3D MEA is demonstrated successfully.
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Antimicrobial and Cytotoxicity Studies of Nano-Zinc OxideHeetai, Ryan 01 January 2021 (has links) (PDF)
Properties of nanoparticles can be engineered to exhibit desired properties for certain applications. In general, the surface area to volume ratio increases with the reduction of particle size. In some cases, this contributes to increase of surface defects available to the surrounding environment and hence reactivity. Changes in size, shape or coating of a nanoparticle can affect its properties. In this thesis, work was split into two main sections. The first part is an investigation into the antimicrobial and cytotoxicity effects of nano formulated N-acetyl cysteine coated zinc oxide (NAC-ZnO) as it can be encountered throughout the environment. NAC was used as a coating agent for its antimicrobial properties in terms of fighting against biofilm formation and its antioxidant properties. In this study, a comparative antimicrobial study of nano-size NAC-ZnO, nano-size NAC-ZnS, bulk ZnO (CR-41), and Zn(NO3)2 were conducted to understand the toxicity of these Zn based antimicrobials in the environment. The second part is a comprehensive investigation of Zinkicide®, a systemic nano formulated ZnO based antimicrobial for use in citrus trees to fight against bacterial diseases. Antimicrobial assays were performed for Zinkicide® on X. alfalfae, a gram-negative citrus phytopathogen surrogate, in efforts to find a solution to the citrus greening pandemic in Florida. Tests were also done to evaluate antimicrobial efficacy over time, to ensure that efficacy was not lost when stored or when used in the field by growers in their tank mixes. Hopefully, these results may help shed some light on how ZnO nanoparticles may react in the environment. This could lead to more nanotechnology-based products moving forward through the EPA & FDA pipeline to effectively make nanoparticle-based products more common place in agriculture.
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Impact of Crowder Size on Actin Filament Assembly KineticsDemosthene, Bryan 01 January 2021 (has links) (PDF)
Actin is an abundant and essential cytoskeletal protein that plays a central role in eukaryotic cell structure and motility. The intracellular environment where actin assembly occurs is crowded with various organelles, proteins, and macromolecules that limit the accessible volume for biomolecular reactions. Macromolecular crowding induces excluded volume effects influencing the activity of biological molecules as well as the shape and conformation of proteins. Crowding agents, such as polysaccharides or inert polymeric molecules, are used to mimic the conditions present in intracellular spaces and provide a better understanding of interactions inside the cell. Macromolecular crowding has been shown to affect actin filament assembly, however, how crowder size impacts actin assembly dynamics and kinetics is not well understood. In this thesis, we investigate how the excluded volume effects caused by crowding influences actin filament assembly kinetics by using synthetic polymeric crowder, polyethylene glycol (PEG), of various molecular weights. Using total internal reflection fluorescence (TIRF) microscopy, we directly visualized the assembly of individual actin filaments in various sizes of PEG crowded conditions. We quantified actin filament growth rates that depend on the size of crowder. Bulk fluorescence intensity was monitored to evaluate the effect of crowder size on actin assembly kinetics. These results demonstrate that the size of macromolecular crowding agents can modulate actin filament assembly kinetics, possibly by controlling the volume fractions. This work provides a foundation for a mechanism of how the dynamic cytoskeletal assembly occurs in living cells.
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The synthesis of Palladium-Platinum Core-shell Nanoparticles with High Catalytic Peroxidase Activities for Biosensing ApplicationsDavidson, Edwin 01 January 2020 (has links) (PDF)
In recent years, peroxidase mimic nanostructures have attracted special interest due to their low cost, high stability, and high catalytic activity. Herein, we demonstrate the use of Pd-Pt nanocubes (NCs) to achieve the rapid and sensitive colorimetric detection of ascorbic acid (AA), commonly known as vitamin C. The colored signal generated by the catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by the decomposition of hydrogen peroxide (H2O2) is quenched in the presence of AA based on its antioxidant property. This colorimetric method attains a detection limit of 0.40 µM and a linear range from 0 to 15 µM, with good linearity. To the best of our knowledge, the method proposed in this work presents the fastest AA detection among all the other colorimetric methods, with only a 3-minute reaction time. Furthermore, this method offers a simple procedure, cost-effectiveness, room temperature conditions and stability. As a proof-of-concept demonstration, the Pd-Pt NCs detection of AA was applied in human serum to corroborate its applicability in the biomedical field with the analysis of biological samples.
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Mechanism of Actin Bundle Assembly, Mechanics and Structure by Ion InteractionCastaneda, Nicholas 01 January 2017 (has links)
The assembly of actin filaments into bundles plays an essential role in mechanical strength and dynamic reorganization of cytoskeleton. Divalent counterions at high concentrations promote bundle formation through electrostatic attraction between charged filaments. Although it has been hypothesized that specific cation interactions may contribute to salt-induced bundling, molecular mechanisms of how salt modulates bundle assembly and mechanics are not well established. Here we determine the mechanical and dynamic properties of actin bundles with physiologically relevant cations. Using total internal reflection fluorescence (TIRF) microscopy, we measure the bending stiffness of actin bundles determined by persistence length analysis. We characterize real-time formation of bundles by dynamic light scattering intensity and direct visualization using TIRF microscopy. Our results show that divalent cations modulate bundle stiffness as well as time-dependent average bundle size. Furthermore, molecular dynamic simulations propose specificity for cation binding on actin filaments to form bundles. The work suggests that cation interactions serve a regulatory function in bundle assembly dynamics, mechanics, and structure.
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Nanofabrication and Characterization of an Enzyme-Less Electrochemical Biosensor for Creatinine DetectionBelharsa, Anas 01 January 2020 (has links)
This study will reveal the fabrication and development of an enzyme-less biosensor for creatinine detection. The biosensor involves a periodically patterned nano-porous TiO2 deposited with Au nanoparticles via e-beam evaporation and a layer of Imprinted Polymer (IP) of acrylamide and bis-acrylamide to obtain a heterostructure of I-Au-TiO2. The detection methods of creatinine are based on electrochemical measurements using Cyclic Voltammetry (CV), Electrochemical Impedance Spectroscopy (EIS) and Differential Pulse Voltammetry (DPV). The IP-Au-TiO2 sensor shows a detection LOD of 0.0949ng/mL and 0. 0.218ng/mL for EIS and DPV measurements, respectively. The nanofabricated biosensor was tested in the presents of urea, glucose, lactose, L-valine, and Glutamic acid and shows high specificity for creatinine due to the specific binding of the analyte to the imprinted polymer on the electrode. A comparison test was performed between the imprinted IP-Au-TiO2 versus Non-Imprinted (NI) NI-Au-TiO2 biosensors. the results show no specificity for the creatinine using NI-Au-TiO2 biosensor for the varied concentration from 0.1ng/ml to 1µg/ml compared to the I-Au-TiO2. However, The N-Au-TiO2 show enhanced specificity for creatinine in the presence of Localized Surface Plasmon Resonance (LSPR) at the interface of the Au nanoparticles and TiO2. The generated LSPR on the surface of the biosensor increased the sensitivity for creatinine due to charge separation and solution resistance between the sensor and mixture. This detection platform provided a promising result which can be easily expanded to detecting a variety of biomarkers linked to human diseases or pathogens such as bacteria or viruses for point of care detection.
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Synthesis of Ruthenium Bipyridine Conjugated Antibiotic for Fluorescence Lifetime Imaging and Spectroscopy Aided Tracking in CitrusParente, Ryan 01 January 2020 (has links)
Antibiotic treatment of systemic bacterial plant pathogens is an established practice in many crops; however, in citrus it has only recently become available for growers to utilize against Huanglongbing disease. The preliminary efficacy of these treatments is uncertain due to the inability to track their presence in phloem. The need to monitor their movement in planta, especially their presence and translocation in vascular tissue, is a necessary step in clarifying their effectiveness. Previous work has shown the value of Fluorescence Lifetime Imaging (FLIM) in distinguishing between fluorescent probes and plant tissue autofluorescence, which is normally a barrier in photochemical studies in plants. Our aim in this thesis was the synthesis and characterization of a fluorescent antibiotic conjugate that could be utilized for tracking in citrus tissue, with the specific goal of identifying movement through citrus plant phloem. Conjugation of streptomycin sulfate, a commercially available antibiotic, to a modified tris(bipyridine) ruthenium (II) chloride, a dye with desirable photophysical properties, was achieved via EDC:NHS coupling. Further studies were performed illustrating the characteristics and kinetics of this conjugate in planta, which showed that the conjugate had an increase in excited state lifetime upon introduction to a biological environment. Subsequent translocation experiments yielded results indicating that the conjugated antibiotic moves systemically upwards after 48 hours but fails to move down towards the root system of the plant after 168 hours.
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