Exploration of simple analytical approaches for rapid detection of pathogenic bacteria

Abstract

<p>Many of the current methods for detection of pathogenic bacteria require long sample-preparation and analysis time, as well as complex instrumentation. This dissertation explores simple analytical approaches that may be applied towards requirements of a microbial detection system, through development of methods and instrumentation. At the beginning, a simple photolithography-based technique for fabrication of a miniaturized immunosensing platform that was comprised of micrometer-sized antibody addresses capable of capturing specific bacteria in predetermined locations is demonstrated. The resulting array of thousands of addresses for the target analyte on a small chip will potentially yield advances by increases in sample throughput, reductions in sample volume, low levels of detection, and large scale multiplexing. The monolayers used to create the immunosensing platform are also structurally characterized employing infrared reflection spectroscopy (IRS), X-ray photoelectron spectroscopy (XPS), and electrochemical quartz crystal microbalance (EQCM). These results serve as a basis for ongoing efforts in the construction and application of a wide range of immunosensing platforms;A new methodology for the rapid identification and quantification of pathogenic bacteria by combining the selectivity of dye-labeled antibodies with the sample concentration capability of solid phase extraction, and the facile readout of the extracted, dye-labeled microorganisms by diffuse reflectance spectroscopy is presented. This technique features a small, inexpensive, easy-to-use, and portable instrument for detection and rapid sample workup time. Finally, the research efforts in the construction and evaluation of a prototype flow cytometry based rapid cell detector and enumerator are described. This proof of concept study demonstrated the ability of the co-localization of two dyes on an organism to provide a high level of confidence in identification of the pathogen.</p>