The objective of my PhD study was to develop and characterize new methods and sensors based on fluorescence resonance energy transfer (FRET) for bioanalysis. Chapter 3 describes the use of FRET between donor fluorophores and acceptor labeled murine macrophage cells. FRET microscopy was used to determine whether the donor molecules truly permeate through the cell membrane or only adsorb to the cell surface. This method was found to be partially successful since the donor red tail fluorescence overlapped with the sensitized acceptor fluorescence and led to false reading of FRET. We found that is easier to monitor delivery of acceptor molecules into donor-labeled cells. Using donor labeled cells it was possible to determine whether the acceptor molecules were actually delivered into cells. However, a relatively high acceptor concentration in the hundreds of micromolar level was needed to obtain measurable FRET signals in the 3-D cellular system. The results underscored the need to reduce the dimensionality of FRET systems in order to increase the FRET efficiency between donor and acceptor molecules. Chapter 4 describes the development of FRET sensing lipobeads labeled with donors and their use to evaluate the interactions of acceptor molecules with the phospholipid membrane of FRET sensing lipobeads. The change in the dimensionality of the system in which FRET occurs, improved the sensitivity of our measurements by 3-folds compared to FRET measurements in solution. We concluded that a molecular recognition component had to be added to the sensing particles to further increase their selectivity and sensitivity. Chapter 5 describes the development of FRET trap sensing beads and their use for screening nonfluorescent carbohydrates and glycoproteins. The FRET sensing technique was based on binding between dextran molecules labeled with Texas Red (Dextran-TR) and polystyrene microparticles labeled with Fluorescein tagged Concanavalin A (FITC-ConA). It was found that carbohydrates and glycoproteins inhibit the binding between dextran-TR and FITC-ConA labeled particles. The inhibition effect was concentration dependent thus enabled screening carbohydrates and glycoproteins based on their inhibition potency. The dissertation critically evaluates the performance of FRET microscopy and FRET based sensors in delivery and screening applications.

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.