The present work utilizes single molecule methods and analysis to investigate soft and hard substrates. First, the effect of charged hard surfaces on charged probes were evaluated, as the soft surfaces were built upon such a structure. Then, selected polymers were selected according to their importance in smart surface technology. The extent of interaction of the selected probes with the array of soft surfaces gives insight to the potential tunability of these surfaces. The three distinct polymers range from ubiquitous usage to advancements in current technology. The studies presented here are needed to characterize, on the nanoscale, the Coulombic interactions of these polymers.

Luminescent conjugated polymers are widely used in organic optoelectronics. The device is fabricated by spin coating the polymer solutions into thin films. It is important to understand the chain conformation in the solution phase, which is mainly determined by the solubility properties of the solutes and the solvents. The purpose of this study is focused on the aggregate structures of MDMO-PPV polymer in the solution mixing of toluene, heptanes, and decahydronaphthalene. Compared to the polymer in toluene solution, the absorption and fluorescence spectra in the mixing solutions are red-shifted, which indicates the aggregation between polymer chains. In order to identify the aggregation is inter-chain or intra-chain effect, we perform concentration dependent measurements of the fluorescence spectra down to 10-10 M. Our results suggest that inter-chain aggregation is the major influence in the concentration. Since the intra-chain aggregation is strongly influenced by polymer concentration, we carry out the experiments to identify how the inter-chain effect influences at even lower concentrations. Fluorescence correlation spectroscopy (FCS) is used to determine the particle size at 10-12M concentration, which relates directly to the aggregation size. The results show that particle size in the poor solution is larger than that in the good solution. Therefore, we conclude that the change of the fluorescence spectra is caused by the inter-chain aggregation even at the concentration to 10-12M.

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The practical application of nanoparticles requires transitioning from well controlled experimental settings to highly variable "real-life" conditions. Understanding the resulting changes in the behavior and stability of nanoparticles is therefore of paramount importance. This thesis discusses the development and practical applications of tools to monitor the behavior of nanoparticles in real-time using intensity correlation spectroscopy techniques. I show how-correlation spectroscopy can be adapted to nanoparticle systems; and provide particular parameters and settings especially vital for heterogeneous systems. Oftentimes nanoparticles have to be labeled to be detected, which can complicate the system of study and can introduce systematic errors into the analysis. Intensity correlation spectroscopy was tested on dye-labeled magnetite nanocrystals. The fluorescence correlation spectroscopy results were surprisingly biased towards a low concentration of aggregates. Scattering and absorption cross-sections of gold nanoparticles are greatly enhanced near the plasmon resonance wavelength, providing strong intrinsic signals for directly visualizing nanoparticles. I show here how scattering and absorption scale with nanoparticle size; and how size heterogeneity within nanoparticle samples translates into the detected signals. One-photon luminescence of gold nanoparticles, an often neglected signal, was also considered. A comparison between one-photon luminescence and scattering correlation spectroscopy revealed that the former has a much smaller bias towards aggregates and therefore is advantageous in systems prone to aggregation. Overall, the work presented here describes the tools and methods that were developed towards better understanding of nanoparticle behavior in a liquid medium where they are to be employed for environmental and biological applications.

The motion of dye molecules in the solution is highly influenced by the Brownian motion caused by the stochastic collisions with the solvents, and it results the fluorescence intensity fluctuation. The thesis study the fluorescence intensity fluctuation of dilute dye molecule (Rhodamine B) in methanol solution ( - ), under confocal and total internal reflection (TIR) microscopy configurations. Five parameters are used to probe the fluorescence characteristics: (1) the difference between confocal and the TIR configurations. The configuration influences the laser focusing area and consequently the intensity distribution. The effective focusing area in confocal configuration is an ellipsoid shape, while that of TIR configuration is a disk shape around the interface with depth 100-200 nm. It results the TIR configuration less background and higher concentration capability. (2) concentration. We control the concentration from much less than one molecule to more than one molecule in the effective focusing area, and we observe the change of burst intensity distribution. (3) the focus position. By changing the focusing position, we study the effective focusing region changes. (4) excited intensity, and (5) fluorescence correlation spectroscopy (FCS). Our results indicate that TIR configuration exhibits lower background, and is suitable to higher concentration solution. In addition, when the dye concentration in the focusing area is much less than 1, the FCS amplitude is no longer follow 1/N, but rather be proportional to N, where N is the concentration.

Research in the field of non-viral gene delivery has demonstrated that a deeper understanding of the fundamental processes of nanoparticle assembly is required in order to improve their efficacy. While gemini nanoparticles (gemini NPs) and other non-viral delivery systems have been vigorously characterized using several techniques, our knowledge is still incomplete. The first objective of this study was the development of new methodology using fluorescence correlation spectroscopy (FCS) to investigate the stages of gemini NPs assembly. It was demonstrated that by labeling the plasmid, different stages of gemini NP assembly from the gemini-plasmid pre-complex (GP) to the final gemini nanoparticle (or gemini-plasmid-lipid complex; GPL), could be studied. Based on diffusion coefficients and particle numbers extrapolated from the autocorrelation function (ACF), FCS was able to determine that each phase of assembly had distinct characteristics. The FCS study using 12-3-12 gemini surfactant showed that both the diffusion coefficient and particle number of GPs (0.98??0.31 x 10-12 m2/s) was significantly lower than the final GPL (3.11??0.41 x 10-12 m2/s). Based on the Stokes-Einstein equation the particle size was calculated to be 300-500 nm for GP and 200-300 nm for GPLs. The raw intensity histograms showed that both GPs and GPLs are composed of multiple plasmids. Furthermore the study showed that the final GPLs contain fewer plasmids compared to the intermediate GP. FCS results were validated by using existing characterization methods including dynamic light scattering (DLS), zeta potential and dye exclusion assays. The second objective involved the detailed characterization of gemini NP. Nine different gemini surfactants and two different phospholipids were used in a systematic study to assess the effect of gemini surfactant and lipid structure on the final morphology of gemini NP. The study revealed that gemini surfactant structure had a strong effect on structure of GP intermediates, but addition of phospholipids resulted in the formation of uniform gemini NPs. Based on the results of this study a new model for GP and GPL assembly is proposed based on the formation of supramolecular aggregates of gemini-plasmids, governed by gemini surfactant chemical structure, and dispersed by phospholipids to form GPLs.

In the cancer research, it is important to understand protein dynamics which are involved in cell signaling. Therefore, particular protein detection and analysis of target protein behavior are indispensable for current basic cancer research. However, it usually performed by conventional biochemical approaches, which require long process time and a large amount of samples. We have been developed the new applications based on microfluidics and Raster image Correlation spectroscopy (RICS) techniques. A simple microfluidic 3D hydrodynamic flow focusing device has been developed for quantitative determinations of target protein concentrations. The analyte stream was pinched not only horizontally, but also vertically by two sheath streams by introducing step depth cross junction structure. As a result, a triangular cross-sectional flow profile was formed and the laser was focused on the top of the triangular shaped analyte stream. Through this approach, the target protein concentration was successfully determined in cell lysate samples. The RICS technique has been applied to characterize the dynamics of protein 53 (p53) in living cells before and after the treatment with DNA damaging agents. P53 tagged with Green Fluores-cent Protein (GFP) were incubated with and without DNA damaging agents, cisplatin or eptoposide. Then, the diffusion coefficient of GFP-p53 was determined by RICS and it was significantly reduced after the drug treatment while that of the one without drug treatment was not. It is suggested that the drugs induced the interaction of p53 with either other proteins or DNA. This result demonstrates that RICS is able to detect protein-protein or protein-DNA interactions in living cells and it may be useful for the drug screening. As another application of microfluidics, an integrated microfluidic platform was developed for generating collagen microspheres with encapsulation of viable cells. The platform integrated four automated functions on a microfluidic chip, (1) collagen solution cooling system, (2) cell-in-collagen microdroplet generation, (3) collagen microdroplet polymerization, and (4) incubation and extraction of the microspheres. This platform provided a high throughput and easy way to generate uniform dimensions of collagen microspheres encapsulating viable cells that were able to proliferate for more than 1 week.

Doctor Pharmaceuticae - DPharm / Natural products have been an important source of drugs and novel lead compounds in drug discovery. Their unique scaffolds have led to the synthesis of derivatives that continue to give rise to medicinally relevant agents. Thus, natural product-inspired drugs represent a significant proportion of drugs in the market and with several more in development. Cancer is among the leading public health problems and a prominent cause of death globally. Chemotherapy has been important in the management of this disease even though side effects that arise due to lack of selectivity is still an issue.

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