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.

The nano/micro particles have been widely used as a carrier of therapeutic and contrast imaging agents. The nano/micro particles have many advantages, such as, specificity, controlled release, multifunctionality and engineerability. By tuning the chemical, physical and geometrical properties, the efficacy of delivery of nano/micro particle can be improved. In this study, by analyzing the effect of physical and geometrical properties of particle, such as, size, shape, material property and flow condition, the optimal condition for particle delivery will be explored. The objectives of this study are (1) to develop predictive mathematical models and (2) experimental models for particle margination and adhesion, and (3) to find optimal particle geometry in terms of size and shape to enhance the efficiency of its delivery. The effect of particle size expressed in terms of Stokes number and shape, namely, spherical, ellipsoidal, hemispherical, discoidal and cylindrical particle on the particle trajectory is investigated. For discoidal and cylindrical particles, the effect of aspect ratio is also considered. To calculate particle trajectory in the linear shear flow near the substrate, Newton's law of motion is decomposed into hydrodynamic drag and resistance induced by particle motion. The drag and resistance is estimated through finite volume formulation using Fluent v6.3. Particle behavior in the linear shear flow does strongly depend on Stokes number. Spherical particle is transported following the streamline in the absence of external body force. However, non-spherical particles could across the streamline and marginate to the substrate. For non-spherical particles, the optimal [Stokes number] in terms of particle margination is observed; [Stokes number almost equal to] 20 for ellipsoidal, hemispherical and discoidal particle; [Stokes number almost equal to] 10 for cylindrical particle. For discoidal particle with [gamma subscript d]=0.2 shows fastest margination to the substrate. The effect of gravitational force is also considered with respect to the fluid direction. When the gravitational force is applied, mostly, gravitational force plays a dominant role for particle margination. However, using small particle aspect ratio ([gamma subscript d]=0.2 and 0.33), spontaneous drift induced by particle-fluid-substrate interaction could overcome gravitational effect in some cases ([Stokes number]=10, G=0.1). In addition the adhesion characteristic of spherical particle has been studied using in vitro micro fluidic chamber system with different particle size and flow condition. The experimental results are compared to the mathematical model developed by Decuzzi and Ferrari (Decuzzi and Ferrari, 2006) and in vivo test (Decuzzi et al., 2010). The optimal particle size for S=75 and 90 is found to be 4-5 [micrometer] through the in vitro non-specific interaction of spherical particle on the biological substrate. The suggested mathematical model has proven to be valid for current experimental condition. At the end, the mathematical model, in vitro flow chamber results and in vivo test have been compared and the scaling law for particle adhesion on the vessel wall has been confirmed. / text

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