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- All Subjects: Biomedical Engineering
- Creators: Arizona State University
- Creators: Greger, Bradley
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
Cellular metabolism is an essential process required for tissue formation, energy production and systemic homeostasis and becomes dysregulated in many disease states. In the context of human cerebral cortex development, there’s a limited understanding of how metabolic pathways, such as glycolysis, impacts proliferation and differentiation of cortical cells. The technical challenges of studying primary in vivo cortical tissue at a cellular and molecular level led to the development of human pluripotent stem cell (PSC) derived cortical organoids. Cortical organoids are a highly tractable model system that can be used for high-throughput investigation of early stages of development and corresponding glycolytic programs. Through transplantation of cortical organoids into the developing mouse cortex, human cortical cells can also be studied in an in vivo environment that more closely resembles endogenous development where the impact of metabolism in typical developmental programs and disease states can be studied. While current data is preliminary, initial observations suggest that cortical populations increase glucose uptake over time and regulation of glucose uptake rates occur in cell type-specific manner. Additionally, mouse transplantation data suggests that glycolytic activity is downregulated post-transplantation, suggesting that the in vitro environment contributes metabolic state. The more dynamic range of metabolic states in vivo may impact the rate of differentiation and maturation in cellular populations in the transplant model. I hypothesize that the more endogenous-like regulation of glycolysis may impact the proliferative window and expansion of key progenitor cell types in the human brain, particularly the intermediate progenitor cells.
ContributorsMorales, Alexandria (Author) / Andrews, Madeline (Thesis advisor) / Newbern, Jason (Committee member) / Stabenfeldt, Sarah (Committee member) / Arizona State University (Publisher)
Created2023