The cellular environment is characterized by confinement and macro-molecular crowding: both concepts that have been studied separately. To understand kinetics of enzymatic reactions, there is a need to understand how the diffusional encounters of enzyme and substrate proceed in an environment
that is confined and crowded simultaneously. The project carried out in this thesis is the first step towards achieving the ultimate goal of studying biochemical reactions in native cellular environment - Understanding diffusion in confinement. Despite multiple investigations of diffusion of analytes in confinement, there exists a research gap. There is inconsistency in the interpretation of the results and in the dependence of diffusion properties of analytes of different sizes in channels of different dimensions. Hence, to bridge the research gap, the main goal of this thesis project was to find the diffusion coefficient of 100 nm polystyrene beads in microchannel (200 µm wide and 4.5 µm high) and nanochannel (5 or 10 µm wide and 300 nm high). The Brownian motion of particles was observed using Confocal Laser Scanning Microscope. A preliminary study first confirmed the reliability and optimization of the particle tracking method of finding diffusion coefficient. Diffusion coefficient of the particles determined experimentally in microchannel (bulk system) was in agreement with the theoretical estimate and statistically significant. Experiments in the nanochannel revealed a reduction in the particle diffusion coefficient of about ∼52% compared to bulk, due to interactions with the confining depth
(300 nm) of nanochannel. An interesting behaviour was also exhibited by particles diffusing close to the side wall along the width of nanochannels, which was not confining (5 or 10 µm). The diffusion coefficient in such a case reduced by around ∼90% relative to bulk. The reduction in both cases can be mainly attributed to hydrodynamic interactions. The experimental investigations of diffusion coefficient carried out in this study were in agreement with long standing theoretical predictions. However, the research gap could not be fully expelled.