Biological and pharmaceutical applications of Raman spectroscopy
Abstract
This thesis describes the development of new Raman spectroscopy methods for the analysis of biological (proteomic) and pharmaceutical samples. Chapter One details the synthesis and analytical testing of new isotopically encoded dye molecules to be used for relative quantitative Surface Enhanced Raman Spectroscopy. The five dye pairs all demonstrated deuterium induced peak shifts and demonstrated high reliability when used for relative concentration determination. Chapter Two expands the use of the isotope encoded dyes, rhodamine 6G-d0 and rhodamine 6G-d4 to protein labeling. Five proteins were covalently tagged and relative quantitation ability was assessed down to a total dye concentration 300 pM. Chapter Two also contains the first results demonstrating the ability to detect Surface Enhanced Resonance Raman signal from a dye labeled protein species on a silver stained one-dimensional gel electrophoresis matrix. Chapter Three describes the use of the inherent water Raman band to quantify protein spot fluorescence in two-dimensional electrophoresis gels. A set of protein standards was covalently tagged with TAMRA (rhodamine dye) or passively stained with Sypro Ruby, and imaged with a commercial fluorescence scanner and a home-built Raman microscope for comparison. The water Raman internal standard method allowed dye label quantification and estimation of protein concentration. Chapter Four describes the application of SERRS signal detection onto a two-dimensional electrophoresis gel format. The results show an increased relative quantitative ability (%RSD ≈ 16%) over previous methods such as difference gel electrophoresis (DIGE). Chapter Five details the thorough comparison of various Raman sampling methodologies to the problem of detecting subsurface Raman signal in compressed powers/coated pharmaceutical tablets. The methodologies are based on either Raman depth scanning or various implementations of the recently developed Spatial Offset Raman Spectroscopy (SORS) method. The results show that samples with transparent surfaces/coatings are just as effectively analyzed with Raman depth scanning and spatial offset detection. However, better subsurface signal collection is obtained with the fiber ring bundle collection method when the surface/coating is also a compressed powder.
Degree
Ph.D.
Advisors
Ben-Amotz, Purdue University.
Subject Area
Analytical chemistry|Biochemistry
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