DESIGN, SYNTHESIS AND APPLICATIONS OF FLUORESCENT AND ELECTROCHEMICAL PROBES

“Seeing is believing” the proverb well suits for fluorescent imaging probes. Since we can selectively and sensitively visualize small biomolecules, organelles such as lysosomes, neutral molecules, metal ions, anions through cellular imaging, fluorescent probes can help shed light on the physiological and pathophysiological path ways. Since these biomolecules are produced in low concentrations in the biochemical pathways, general analytical techniques either fail to detect or are not sensitive enough to differentiate the relative concentrations. During my Ph.D. study, I exploited synthetic organic techniques to design and synthesize fluorescent probes with desirable properties such as high water solubility, high sensitivity and with varying fluorescent quantum yields. I synthesized a highly water soluble BOIDPY-based turn-on fluorescent probe for endogenous nitric oxide. I also synthesized a series of cell membrane permeable near infrared (NIR) pH activatable fluorescent probes for lysosomal pH sensing. Fluorescent dyes are molecular tools for designing fluorescent bio imaging probes. This prompted me to design and synthesize a hybrid fluorescent dye with a functionalizable chlorine atom and tested the chlorine re-activity for fluorescent probe design. Carbohydrate and protein interactions are key for many biological processes, such as viral and bacterial infections, cell recognition and adhesion, and immune response. Among several analytical techniques aimed to study these interactions, electrochemical bio sensing is more efficient due to its low cost, ease of operation, and possibility for miniaturization. During my Ph.D., I synthesized mannose bearing aniline molecule which is successfully tested as electrochemical bio sensor. A Ferrocene-mannose conjugate with an anchoring group is synthesized, which can be used as a potential electrochemical biosensor.

Screening, optimization and extraction of polyhydroxyalkanoates and peptidoglycan from Bacillus megaterium

Bioplastics are polymers (such as polyesters) produced from bacterial fermentations that are biodegradable and nonhazardous. They are produced by a wide variety of bacteria and are made only when stress conditions allow, such as when nutrient levels are low, more specifically levels of nitrogen and oxygen. These stress conditions cause certain bacteria to build up excess carbon deposits as energy reserves in the form of polyhydroxyalkanoates (PHAs). PHAs can be extracted and formed into actual plastic with the same strength of conventional, synthetic-based plastics without the need to rely on foreign petroleum. The overall goal of this project was to select for a bacteria that could grow on sugars found in the lignocellulosic biomass, and get the bacteria to produce PHAs and peptidoglycan. Once this was accomplished the goal was to extract PHAs and peptidoglycan in order to make a stronger more rigid plastic, by combing them into a co-polymer. The individual goals of this project were to: (1) Select and screen bacteria that are capable of producing PHAs by utilizing the carbon/energy sources found in lignocellulosic biomass; (2) Maximize the utilization of those sugars present in woody biomass in order to produce optimal levels of PHAs. (3) Use room temperature ionic liquids (RTILs) in order to separate the cell membrane and peptidoglycan, allowing for better extraction of PHAs and more intact peptidoglycan. B. megaterium a Gram-positive PHA-producing bacterium was selected for study in this project. It was grown on a variety of different substrates in order to maximize both its growth and production of PHAs. The optimal conditions were found to be 30°C, pH 6.0 and sugar concentration of either 30g/L glucose or xylose. After optimal growth was obtained, both RTILs and enzymatic treatments were used to break the cell wall, in order to extract the PHAs, and peptidoglycan. PHAs and peptidoglycan were successfully extracted from the cell, and will be used in the future to create a new stronger co-polymer. Peptidoglycan recovery yield was 16% of the cells’ dry weight.

A THEORETICAL STUDY OF INTERACTION OF NANOPARTICLES WITH BIOMOLECULE

Many types of materials at nanoscale are currently being used in everyday life. The production and use of such products based on engineered nanomaterials have raised concerns of the possible risks and hazards associated with these nanomaterials. In order to evaluate and gain a better understanding of their effects on living organisms, we have performed first-principles quantum mechanical calculations and molecular dynamics simulations. Specifically, we will investigate the interaction of nanomaterials including semiconducting quantum dots and metallic nanoparticles with various biological molecules, such as dopamine, DNA nucleobases and lipid membranes. Firstly, interactions of semiconducting CdSe/CdS quantum dots (QDs) with the dopamine and the DNA nucleobase molecules are investigated using similar quantum mechanical approach to the one used for the metallic nanoparticles. A variety of interaction sites are explored. Our results show that small-sized Cd4Se4 and Cd4S4 QDs interact strongly with the DNA nucleobase if a DNA nucleobase has the amide or hydroxyl chemical group. These results indicate that these QDs are suitable for detecting subcellular structures, as also reported by experiments. The next two chapters describe a preparation required for the simulation of nanoparticles interacting with membranes leading to accurate structure models for the membranes. We develop a method for the molecular crystalline structure prediction of 1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1,2-Dimyristoyl-sn-glycero-3-phosphorylethanolamine (DMPE) and cyclic di-amino acid peptide using first-principles methods. Since an accurate determination of the structure of an organic crystal is usually an extremely difficult task due to availability of the large number of its conformers, we propose a new computational scheme by applying knowledge of symmetry, structural chemistry and chemical bonding to reduce the sampling size of the conformation space. The interaction of metal nanoparticles with cell membranes is finally carried out by molecular dynamics simulations, and the results are reported in the last chapter. A new force field is developed which accurately describes the interaction forces between the clusters representing small-sized metal nanoparticles and the lipid bilayer molecules. The permeation of nanoparticles into the cell membrane is analyzed together with the RMSD values of the membrane modeled by a lipid bilayer. The simulation results suggest that the AgNPs could cause the same amount of deformation as the AuNPs for the dysfunction of the membrane.