Synthesis and Characterization of Miniaturized Fluorescence Sensors for Aqueous and Cellular Measurements

The objective of this Ph.D. study was to develop new and improved miniaturized particle-based optochemical sensors for the analysis of biological fluid and cellular components. This is highly important because current sensing systems can be biologically toxic and incompatible, invasive, and have limited responsiveness. To accomplish this goal we defined three tasks. The first was to develop lipobead-based sensors for chloride. The halide-specific fluorescence dye, lucigenin, was immobilized into the phospholipid membrane of the lipobeads to enable chloride ion detection. The fluorescence intensity of lucigenin decreases with increasing chloride ion concentration due to dynamic quenching. To stabilize the lipobeads we co-immobilized hexadecanesulfonate molecules into the phospholipid membrane. We also immobilized the chloride ionophore [9] mercuracarborand-3 (MC-3) into the lipobeads membrane. The study resulted in a unique submicrometric chloride ion sensor, which is suitable for chloride ion measurements in biological fluids. The second task was to develop for the first time lipobeadbased biosensors. Urea was chosen as a model substance since the urea/urease biosensing system is well known. Fluorescence sensing lipobeads were characterized by coating carboxylfunctionalized silica microspheres with phospholipids for the measurement of urea in aqueous samples. The enzyme urease and the pH indicator Fluorescein-5-thiosemicarbazide were attached covalently to the phospholipid membrane of the lipobeads. We prepared improved fluorescence sensing lipobeads by utilizing covalent chemistry to bind the phospholipid membrane to the silica particles and the fluorophores to the membrane. It led to improvement in the stability of the newly developed urea sensing lipobeads compared to previously developed micrometric fluorescence sensors. The final task of this study was to coat particle-based sensors with cell penetrating peptides to enable their permeation into cells. This step is essential for the use of particles as intracellular sensors. Streptavidin coated microspheres were modified by the strongest noncovalent interaction between avidin and biotin. Tat peptide and nonfluorescence indicator flubida were attached to the surface of the microspheres. These nanoparticles were delivered into MCF7 and Hela cancer cells for pH measurement. Before penetrating into the cells, flubida did fluoresce in cell medium; however it did not convert to fluorecein in Phosphate Buffered Saline (PBS) buffer