Design and Construction of Fluorescence Recovery after Photobleaching set up & Investigating Structure and Diffusive Dynamics Inside Random and Restricted Media

Abstract

Molecular transport is imperative to sustenance of life owing to movement of nutrients, genetic materials, ions across the cellular membrane. Besides aiding the smooth functioning of life processes molecular transport are crucial to stabilization of industrial products and enhancement of their shelf life and performance. Molecular transport as easy it may seem is not so considering the true environment of real life systems as they are severely restricted either by the presence of other molecules (macromolecular crowding) or due to their own inherent structural organization (gels). Apart from hindrance offered due to presence of obstacles molecular transport is also affected by the interactions present in the system. The environmental constraints make it extremely difficult to figure out the factors affecting molecular transport inside these random and restricted medium. Since the practical implications of molecular transport is monumental the studies on them cannot be ignored how much ever difficult it may be. Literature however indicates that a large number of studies that have been previously done are limited to dilute and neutral systems and because of these elaborate studies the theoretical background of these simpler systems is relatively well understood. Unfortunately the theory developed for dilute and neutral systems cannot be simply extended when crowding and interactions come into picture. On the other hand a direct study on these systems is marred by the simultaneous influence of numerous factors making it highly improbable to pinpoint the role of each one on the systems dynamics. This inadequacy in understanding physiologically relevant systems led to the adoption of alternate approaches relating to the use of “model systems” which imparts the freedom to precisely vary one parameter at a time. The studies on model systems should therefore be carefully designed to take into account the appropriate amount of crowding, interactions and structural organization of real life systems. This will help us to derive results in strong likeness to that of real systems. In addition these studies will help us to address the essential questions such as the influence of random and restricted medium on diffusion as well as factors affecting the architecture of gel systems.

Taking into consideration these facts the research work presented in current thesis is framed under two – fold objectives. The first objective is to investigate how the diffusive dynamics is influenced by the crowding, interaction and structural organization of the background. The second objective deals with the study of factors influencing the microstructure of hydrogels.

The effect of crowding and interaction is investigated by probing the molecular motion of green fluorescent protein (GFP) inside oppositely charged poly – L – lysine solutions as a function of host polymer concentration and solution conditions including ionic strength and solution pH. While the host concentration is expected to vary the degree of crowding alone, varying solution conditions tend to vary both crowding and interactions. We found that background concentration via viscosity decreases the diffusivity of the guest species which is in accordance to the obstruction theory. On the other hand a much more serious influence of interaction is observed on probe diffusion. The interacting charged monomeric units lead the chains to sufficiently expand so that the diffusing species encounters viscosity similar to that of the solvent viscosity. This results in ~ 200 – fold faster probe diffusion as against predicted from the theory. Moreover interaction between oppositely charged polymer – solute systems can lead to adsorption of one over the other giving rise to a slowly moving aggregated mass. Diffusion inside complex polyelectrolyte systems is found to be strongly influenced by its background, interactions between solute – polymer and chain flexibility of polymer chains. Our studies of probe diffusion inside polyacrylamide hydrogel matrices revealed that diffusion is considerably influenced by microstructural architecture of gel networks. At low constituent (monomer or crosslinker) concentrations gels formed are relatively dilute and homogeneous wherein dynamics is essentially governed by diffusion alone. However as the concentration of constituents in gels is raised the gel matrices becomes progressively heterogeneous with dense and loose regions. While the loose regions permit diffusion the dense regions tend to trap moving molecules within them. Thus the obstruction and hydrodynamic theories based on uniformity of matrices to explain diffusion are no longer valid at such high constituent concentrations.

Our rheological measurements suggest that the structural organization of gels not only depends on its monomer or crosslinker concentration but also the type of crosslinker used to synthesize it. Varying the crosslinker type varies the gel strength and its elasticity. Increasing crosslinker concentration leads to phase separation of hydrogels, irrespective of the type of crosslinker used. The mechanical stability tests of hydrogels synthesized in presence of ancillary materials exhibit loosening of gels which most probably might be due to poor crosslinking in the matrix in presence of these materials. Apart from altering their macroscopic properties the incorporated foreign species tend to modify the microstructural arrangement of hydrogels which is validated from our light scattering measurements.

The thesis work also involves the design and build up of an in – house Fluorescence Recovery After Photobleaching (FRAP) set up to study dynamics of random and restricted media including hydrogels and concentrated polymer solutions.

We believe that these studies will aid our fundamental understanding of real life systems and their functioning under severely constraint environment. This will open avenues to specifically design matrices needed for suitable applications including controlled release systems, separation matrices, and scaffolds for enzyme immobilization.