In diagnostics, the ability to detect small quantities of molecules with high sensitivity and specificity is crucial for a quick and accurate diagnosis of the presence of target antigens. The ELISA assay, which uses fluorescence to detect the presence of target molecules, is a standard diagnostic and research method for antibody-antigen detection. However, current ELISA methods do not have fast binding kinetics at low target antigen concentrations, requiring several hours for complete results. Therefore, there is a significant need for development of a simple and inexpensive method to improve the molecular binding kinetics in ELISA assays. My hypothesis was that rotational stimulation during enzyme incubation would greatly increase the sensitivity of the assay in a much shorter amount of time. To test this hypothesis, I compared the measured fluorescence of two different types of incubations – the traditional static incubation and a rotational incubation method. I measured the fluorescence generated by each set of assays at varying enzyme concentrations to determine the limit of detection, the minimum concentration that can be detected in a sample, for each method. I also analyzed the binding kinetics of each method using time-based data of each method. It is expected that the rotational method increases the sensitivity of the assay using half the concentration of target molecules compared to that of the static method. It also significantly decreases the time required to reach a binding equilibrium state by several hours. Promising results would suggest that rotational stimulation could significantly improve the molecular binding kinetics in diagnostic assays and could be implemented in future protocols to produce a more efficient diagnostic assay procedure.

Current drug delivery methods lack the ability to effectively deliver a drug to a targeted region and fail to escape from the endosome. The ability to gain greater control over a drug’s effective delivery would lead to an enhanced efficacy of existing drugs, decrease the amount administration sessions, and reduce the number of side effects. Nanotechnology is a means to address these issues as the unique quantum properties witnessed on this scale allow for enhanced targeting and greater control over the delivery of the targeted drug. However, biocompatibility and toxicity are concerns with many developments within nanotechnology. To address these concerns, we have designed a siRNA-aptamer chimera based on protein biosynthesized calcium phosphate (CaP) nanoparticles. This was done due to CaP’s well-known biocompatibility and biodegradability as the calcium phosphate dissolves within the endosome, therefore releasing the siRNA into the cytoplasm of the cell. Within this project, we have targeted cancer cells as a means to quantify the efficacy of our nanoparticles. Through engineering a dual-functional protein expressed in E.coli, we were capable of 1) biosynthesizing CaP nanoparticles utilizing the protein’s affinity to calcium phosphate, and 2) binding siRNA onto the designed protein for delivery, which is utilized to inhibit the translation of the targeted cancer cells. The biomineralized nanoparticles were characterized via DLS and TEM to reveal that we had synthesized uniform 100nm nanoparticles, a promising result which supports our goal of designing nanoparticles capable of establishing greater control over drug delivery. The completion of this project would entail the successful design of biocompatible nanoparticles that could be effectively used to silence cancer cells. As the protein’s affinity for a substrate can be manipulated, these siRNA-aptamer chimeras could see use with other drugs as well; effectively leading to a powerful and versatile drug delivery system.

Vessels of different organs have heterogeneous permeability to serve distinct physiological functions. Differences in basal vascular permeability may result from heterogeneity in the endothelial cells that line the vessel walls, which are influenced by many factors—including differences in the complexity of cell-cell junctions and the expression of various diaphragms that span the cell body. In addition, many diseased states are often associated with elevated levels of permeability, including viral hemorrhagic fever and tumor-induced angiogenesis. To understand such fundamental heterogeneity of the blood vessels and further investigate the change of vascular structure and function in disease scenarios, we characterized the permeability of endothelial cells harvested from different areas of the body. Human primary cells—including endothelial cells from various organs and epithelial cells from the proximal tubules of the kidney (PTECs)—were seeded on gelatin-coated transwells. We measured transendothelial electrical resistance (TEER) using a Millicell ERS Voltohmmeter, and determined solute permeability by tracking the perfusion of fluorescent dextran across the cell monolayer over time. Immunostaining was performed to observe monolayer formation, while transmission electron microscopy (TEM) was utilized to observe ultrastructure. Preliminary TEM images demonstrate distinct morphological differences between endothelial cell types, specifically in the complexity of cell-cell junctions. Investigations into the impact of these morphological differences on vascular permeability are ongoing, and a definitive conclusion will be made upon the study’s completion.