Date of Award

8-2019

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Dr. Ramakrishna Guda

Second Advisor

Dr. Ekkehard Sinn

Third Advisor

Dr. Sherine Obare

Fourth Advisor

Dr. Pamela Hoppe

Keywords

Organized self-assemblies, two-photon spectroscopy, protein aggregation, DNA stability, DNA interaction, polyelectrolytes

Abstract

Organized self-assemblies are the cornerstones for countless biological processes and are integral parts of lipids, proteins, carbohydrates, nucleic acids, and cell membranes. Several man-made organized assemblies also play a vital role in interdisciplinary sciences that include micelles, reverse micelles, polymers, polyelectrolytes etc. Weak chemical interactions such as hydrogen bonding, p-p stacking, hydrophilic-hydrophobic and electrostatics often result in interesting organized self-assemblies. Understanding organized self-assemblies provides a huge opportunity to mimic naturally occurring biological macromolecules, design materials and develop strategies for specific applications. Several techniques are routinely used to understand the organized self-assemblies, and optical techniques play an important role in most of them. However, the search for novel optical techniques to monitor organized self-assemblies is on-going, which is the main motivation behind the investigations carried out in this study. The research approach is to use the power of two-photon absorption (2PA) spectroscopy to monitor the organized assemblies.

The hypothesis is that specific non-canonical forms of organized self-assembly provide unique local electric fields and the 2PA cross-sections of chromophores solubilized in this environment can be altered by these local electric fields providing an efficient probe to study them. To test the hypothesis, investigations were carried out using known DNA binders with two non-canonical structures of DNA that were implicated for cancer, G-Quadruplex and G-Triplex DNA, in an attempt to use the 2PA cross-sections of the binders to monitor the melting and stability of these organized self-assemblies. The results have conclusively shown that 2PA cross-sections of the chromophores were sensitive to monitor the melting transitions in G-Quadruplex and G-Triplex with different enhancements that can be assigned to electrostatic and aromatic interactions of the drug binders with DNA. These results have enabled us to use the power of 2PA spectroscopy to monitor protein folding and aggregation of a model protein, bovine-serum albumin (BSA), and a protein implicated in Amyotrophic Lateral Sclerosis (ALS) disease superoxide dismutase (SOD1). The results have shown that the 2PA cross-sections of fluorescamine bound to these proteins is a valuable probe to monitor the folding and unfolding as well as follow the early onset of aggregation in these proteins. To build on understanding the organized self-assemblies using two-photon fluorescence spectroscopy, a novel one- and two-photon fluorescence-based biosensor with a specific oligonucleotide that forms a G-Quadruplex was developed to selectively and with sensitivity detect toxic metal ions such as Pb2+ and Hg2+. Guanine-rich nucleotide T30695 was designed to bind to Pb2+ and Hg2+ selectively and used the one- and two-photon fluorescence of cyanine chromophore to detect different levels of these toxic metal ions, and a detection limit of 4.5 ppb for Pb2+ and 5.0 ppb for Hg2+ was realized from the studies. We have also used the power of 2PA spectroscopy to monitor the local environment in synthetic organized self-assemblies like polyelectrolytes. The electrostatic interactions between the chromophore and polyelectrolytes were able to enhance the 2PA cross-sections of chromophores and have shown that this technique is quite universal and can be used to monitor several organized self-assemblies.

Access Setting

Dissertation-Open Access

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