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Characterization and Development of an Enzymatically Signal-Enhanced Lateral Flow Assay Test for HIV Detection Using the P24 AntigenPankti Rajesh Thakkar (15354871) 28 April 2023 (has links)
<p>In 2021, an estimated 1.5 million people were diagnosed with HIV globally, increasing the total to 38.4 million people. Approximately 16% of this population were unaware of their infected status and required HIV testing, which is a critical first step in HIV prevention, treatment, and care. Hence, there is a need to develop a rapid, user-friendly, and cost-effective point-of-care test for HIV detection. The time between HIV infection and a detectable host HIV antibody concentration can extend up to 90 days. By incorporating more sensitive testing for the HIV p24 antigen on the virus, the diagnosis lag can be reduced to 17 days. This window could be further shortened by using horseradish peroxidase (HRP) enzyme as a signal enhancement technique. The work herein focuses on developing an enzymatically signal-enhanced lateral flow assay test for the p24 antigen to detect HIV during the acute phase of infection. Conjugation chemistry for the sandwich assay was characterized using DLS and UV-Vis. Dot blots were then used to assess and enhance the functionality of the individual components via a visual color gradient formed by the protein coupled with antibody-conjugated gold nanoparticles. A quantitative analysis was performed using ImageJ software through signal pixel intensity analysis. A limit of detection (LoD) of 6 ng/mL was obtained for the detection of the p24 antigen. This LoD was improved to 0.2 ng/mL by incorporating HRP signal enhancement with the diaminobenzidine substrate. This 30x signal improvement could drive down the LoD even further to improve the sensitivity of the commercial p24 antigen tests. Different fabrication and scalability studies were performed to produce a cost- efficient, fully functional prototype of a paper-based lateral flow device incorporating the signal- enhanced p24 assay. This study serves as a solid foundation to research focused on creating more efficient point-of-care tests that can be used in resource-limited settings to provide early detection of HIV for the 6 million individuals who are currently unaware of their HIV status. </p>
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ENGINEERING DESIGN OF NOVEL 3D MICROPHYSIOLOGICAL SYSTEM AND SENSOR FOR FUNCTIONAL ASSESSMENT OF PANCREATIC BETA-CELLSEmma Vanderlaan (15348208) 25 April 2023 (has links)
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<p>Diabetes, a chronic condition characterized by elevated blood glucose levels, arises when pancreatic β-cells lose capacity to produce a robust, dynamic glucose-stimulated insulin secretion (GSIS) response. Accurate measurement of β-cell health and function <em>ex vivo</em> is thus fundamental to diabetes research, including studies evaluating disease mechanisms, novel drug candidates, and replacement β-cell populations. However, present-day dynamic GSIS assays typically represent end-point measurements, involve expensive commercial perifusion machines, and require time-consuming enzyme-linked immunosorbent assays (ELISA) for insulin detection. Microfluidic devices developed as accessible, low-cost alternatives still rely on secondary ELISAs and suspend islets in liquid medium, limiting their survival <em>in vitro</em>. Here, we present a novel, 3D-printed microphysiological system (MPS) designed to recreate components of <em>in-vivo</em> microenvironments through encapsulation in fibrillar type I collagen and restoration of favorable molecular transport conditions. Following computational-informed design and rapid prototyping, the MPS platform sustained collagen-encapsulated mouse islet viability and cytoarchitecture for 5 days and supported <em>in-situ</em> measurements of dynamic β-cell function. To rapidly detect insulin secretion from β-cells in the MPS, we then developed a highly sensitive electrochemical sensor for zinc (Zn2+), co-released with insulin, based on glassy carbon electrodes modified with bismuth and indium and coated with Nafion. Finally, we validated sensor detection of Zn2+ released from glucose-stimulated INS-1 β-cells and primary mouse islets, finding high correlation with insulin as measured by standard ELISA. Together, the 3D MPS and Zn2+ sensor developed in this dissertation represent novel platforms for evaluating β-cell health and function in a low-cost, user-friendly, and physiologically-relevant manner. </p>
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<b>Reprogramming the Pancreatic Cancer Stroma by Targeting Coagulation at the Tumor Microenvironment</b>Sae Rome Choi (18392505) 17 April 2024 (has links)
<p dir="ltr">Pancreatic ductal adenocarcinoma (PDAC) remains one of the most deadliest cancer and despite advancements in cancer therapy, remain highly refractory to treatment, largely due to its desmoplastic tumor microenvironment (TME) characterized by complex interactions among cancer cells and stromal components. Particularly, the PDAC associated coagulation system due to leaky tumor vasculatures plays a pivotal role in reshaping the PDAC stroma and its pathogenesis. Understanding the intricate interplay between tumor cells, stromal cells, and the elevated coagulation pathway elements, including tissue factor, thrombin, and fibrin, is essential for developing effective therapeutic strategies. To address these challenges, this research proposes the engineering of a novel PDAC-associated coagulation system using a microfluidic technology, known as coagulation-on-tumor-microenvironment-on-chip (cT-MOC). The study aims to integrate key coagulation pathways in cT-MOC to investigate pivotal interactions in the PDAC stroma: <i>i)</i> thrombin-protease-activated receptors (PARs) mediated promotion of PDAC fibrosis via activation of cancer-fibroblast cross-talk; <i>ii)</i> in-depth analysis of transport and mechanical properties of collagen-fibrin microstructure; <i>iii)</i> inhibited drug delivery in reprogrammed PDAC stroma due to pronounced fibrin deposition on collagen. By leveraging innovative microfluidic technologies and comprehensive experimental approaches, the research endeavors to provide a novel platform that bridges traditional <i>in vitro</i> and <i>in vivo</i> models to overcome the challenges posed by the desmoplastic TME and enhance therapeutic strategies for treatment by targeting the coagulation at the PDAC TME.</p>
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