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- Creators: Arizona State University
Description
In the realm of biosensors and nanotechnology, deoxyribonucleic acid (DNA) nanosensors have demonstrated tremendous potential across diverse real-world applications, from environmental monitoring to healthcare diagnostics. Fabrication of nanosensors allows assembling and designing of DNA molecules at nanoscale with high precision and versatility. Such fabricating DNA nanosensors are quite time consuming. Hence it is important to store them in batches. However synthetic DNA molecules can be prone to degradation over time, especially when exposed to various environmental factors like light, heat, or any other chemical contaminants. To address this issue, a shelf life study of DNA nanosensors using various lyoprotectant conditions was carried out to determine the long term stability of such sensors. This study involves fabrication of the dendritic, double - stranded DNA nanosensors involving five strands L1 through L5 conjugated with pHAb fluorophores via N-hydroxysuccinimide ester reaction and acetylcholinesterase (AChE) enzyme, a core component of the sensor. This sensor was originally a fluorescent ACh-selective nanosensors designed to accommodate the BTX ligand, AChE to image the ACh release in the submandibular region of the living mice to report real time quantitative endogenous ACh release triggered by electrical stimulation. AChE enzyme is a good receptor to detect acetylcholine release in the Peripheral Nervous System (PNS). The primary objective of the study was to assess DNA nanosensors with AChE, however due to the intricate interactions, non-specific binding and cost-effectiveness, the shelf life study was carried out separately. The shelf study includes observing DNA nanosensors with different disaccharide lyoprotectants like trehalose and sucrose that were analyzed under different temperature conditions: room temperature (25ºC) and at 50 ºC for different time intervals, over a week time. Also, Observing AChE with various protectants under 50 ºC with and without lyoprotectants for various time intervals like 24 hours and 48 hours. To replicate the real-world transit scenarios, the study also involves test-shipment of the samples with lyoprotectants for 2-3 days to both cross-country and local (in-state). As a result, the use of lyoprotectants, particularly trehalose, has proven to be more resilient and effective in preserving the stability and integrity of DNA nanosensors and Acetylcholinesterase (AChE) enzymes
ContributorsSrinivasan, Nikita (Author) / Clark, Heather A (Thesis advisor) / Ma, Kristine Y (Committee member) / Beeman, Scott (Committee member) / Arizona State University (Publisher)
Created2023
Description
Urease, an amidohydrolase, is an essential ingredient in the emerging engineering technique of biocementation. When free urease enzyme is used this carbonate precipitation process is often referred to as enzyme induced carbonate precipitation (EICP). To date, most engineering applications of EICP have used commercially available powdered urease. However, the high cost of commercially available urease is a major barrier to adoption of engineering applications of EICP in practice. The objective of this dissertation was to develop a simple and inexpensive enzyme production technique using agricultural resources. The specific objectives of this dissertation were (i) to develop a simple extraction process to obtain urease from common agricultural resources and identify a preferred agricultural resource for further study, (ii) to reduce the cost of enzyme production by eliminating the use of a buffer, centrifugation, and dehusking of the beans during the extraction process, (iii) investigate the stability of the extracted enzyme both in solution and after reduction to a powder by lyophilization (freeze-drying), and (iv) to study the kinetics of the extracted enzyme.
The results presented in this dissertation confirmed that inexpensive crude extracts of urease from agricultural products, including jack beans, soybeans, and watermelon seeds, are effective at catalyzing urea hydrolysis for carbonate precipitation. Based upon unit yield, jack beans were identified as the preferred agricultural resource for urease extraction. Results also showed that the jack bean extract retained its activity even after replacing the buffer with tap water and eliminating acetone fractionation, centrifugation, and dehusking. It was also found that the lyophilized crude extract maintained its activity during storage for at least one year and more effectively than either the crude extract solution or rehydrated commercial urease. The kinetics of the extracted enzyme was studied to gain greater insight into the optimum concentration of urea in engineering applications of EICP. Results showed higher values for the half-saturation coefficient of the crude extract compared to the commercial enzymes.
The results presented in this dissertation demonstrate the potential for a significant reduction in the cost of applying EICP in engineering practice by mass production of urease enzyme via a simple extraction process.
ContributorsJavadi, Neda (Author) / Kavazanjian, Edward (Thesis advisor) / Khodadadi Tirkolaei, Hamed (Committee member) / Hamadan, Naser (Committee member) / Delgado, Anca (Committee member) / Arizona State University (Publisher)
Created2021
Description
The complex network of the immune system defends the human body against infection, providing protection from pathogens. This work aims to improve preparation and structural knowledge of two proteins on opposite sides of the immune system spectrum. The first protein, secreted autotransporter toxin (Sat) is a class I serine protease autotransporter of Enterobacteriaceae (SPATE) that has cytotoxic and immunomodulatory effects on the host. Previous studies on Sat show its ability to aid in bacterial colonization and evasion of the immune system. This work improves the stability of Sat by making mutations to the active serine protease motif (GDSGS) while inhibiting remaining activity with reversible and irreversible serine protease inhibitors. Characterization of Sat by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and size-exclusion chromatography led to the first structural studies of Sat by x-ray crystallography and cryo-EM. Human leukocyte antigen class I proteins play an important role in the adaptive immune system by presenting endogenous viral peptides at the cell surface for CD8+ T cell recognition. In vitro production of HLA-I proteins is a difficult task without endoplasmic reticulum chaperones as present in vivo. Disulfide bond formation, folded light chain and a peptide bound are all key to refolding the HLA-I heavy chain for complex formation. The work presented in this dissertation represents systematic studies aimed at improving the production of HLA-I proteins in vitro in bacterial expression systems. Optimization of every step of the preparation was investigated providing higher expression yields, quality of inclusion bodies, and refolding improvements. With further improvements in the future, this work forms the basis for a more efficient small and large-scale production of HLA-I molecules for functional and structural studies.
ContributorsKiefer, Dalton (Author) / Anderson, Karen (Thesis advisor) / Fromme, Petra (Thesis advisor) / Chiu, Po-Lin (Committee member) / Mazor, Yuval (Committee member) / Arizona State University (Publisher)
Created2024
Description
The thylakoid membranes of oxygenic photosynthetic organisms contain four large membrane complexes vital for photosynthesis: photosystem II and photosystem I (PSII and PSI, respectively), the cytochrome b6f complex and ATP synthase. Two of these complexes, PSII and PSI, utilize solar energy to carry out the primary reaction of photosynthesis, light induced charge separation. In vivo, both photosystems associate with multiple antennae to increase their light absorption cross section. The antennae, Iron Stress Induced A (IsiA), is expressed in cyanobacteria as part of general stress response and forms a ring system around PSI. IsiA is a member of a large and relatively unexplored antennae family prevalent in cyanobacteria. The structure of the PSI-IsiA super-complex from the cyanobacteria Synechocystis sp. PCC 6803 was resolved to high resolution, revealing how IsiA interacts with PSI as well as the chlorophyll organization within this antennae system. Despite these structural insights, the basis for the binding between 18 IsiA subits and PSI is not fully resolved. Several IsiA mutants were constructed using insights from the atomic structure of PSI-IsiA, revealing the role of the C-terminus of IsiA in its interaction with PSI.
ContributorsLi, Jin (Author) / Mazor, Yuval (Thesis advisor) / Chiu, Po-Lin (Committee member) / Mills, Jeremy (Committee member) / Arizona State University (Publisher)
Created2024