Date of Award
Doctor of Philosophy
Marc D. Porter
This dissertation explores the use of novel detection methods for biological and chemical components commonly found in the environment. It encompasses two techniques: surface-enhanced Raman scattering (SERS) and electrochemically modulated liquid chromatography (EMLC).;Immunoassays using SERS as a readout tool have been developed in this laboratory and have shown low levels of detection (i.e., pico- to femtomolar and single binding event detection) for disease and biowarfare agents. This thesis seeks to further the performance of this platform for bacteria detection and explore strategies to increase the SERS response. Specifically, the first section of this dissertation focuses on the detection of a common, economically devastating, bovine bacterium. By the judicious design of the assay platform, a selective assay for the bacteria was developed, and low levels of detection (∼500 bacilli/mL) were achieved. Further examination of these results led to the exciting discovery of an amplification phenomenon based on protein shedding from the surface of the bacteria. The last portion of the SERS readout immunoassay research focuses on fundamental studies employing resonant, dye molecules to create enhanced SERS signals. Full immunoassay results for four dyes, when compared with our standard, non-resonant reporter, yielded SERS signals ∼300 times more intense. Implications of signal enhancement with respect to limits of detection are elucidated and future work towards decreasing nonspecific binding briefly introduced.;The second part of this dissertation introduces research development in EMLC, specifically the use of mobile phase pH regulation and incorporation of novel stationary phases. By expanding upon current EMLC techniques, novel separations of weakly basic/acidic compounds were achieved. These studies revealed the potential power of EMLC with mobile phase pH control to improve resolution while simultaneously reducing elution time for seven compounds. These results, which are in contrary to other reversed phase LC systems, are based on the ability to "pull apart" a chromatogram. In addition, the capability to perform a titration with EMLC, and thus determine the pKa of a compound, is discussed in context of acid-base equilibria. This dissertation also introduces work underway for a redesigned column for testing monolithic carbon materials as an EMLC stationary phase. Finally, insights gained during this project are used to formulate further column redesign and in-situ monolith formation for improved EMLC separations.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Betsy Jean Yakes
Yakes, Betsy Jean, "Advanced detection and separation methods: developments in surface-enhanced Raman scattering readout immunoassays and electrochemically modulated liquid chromatography" (2007). Retrospective Theses and Dissertations. 15992.