| Infectious agents are microorganisms that cause infectious diseases and can be employed as potential biological warfare agents (BWAs) during a bioterrorism attack. Detection of infectious agents allows one to develop methods to study the etiology of infectious diseases as well as finding therapeutic treatment to victims. Timely and effective detection of released BWAs are crucial for an appropriate response to mitigate casualties caused by a bioterrorism attack.; DNA microarray technology has provided a promising high-throughput platform for rapid, sensitive, specific, and reliable detection of BWAs and other infectious agents of interest due to its capability of large-scale parallel DNA analysis. This thesis describes the development of a fiber-optic bead-based array for simultaneous detection of BWAs. Parallel identification of target BWAs in pure culture samples and spiked environmental samples after multiplex PCR amplification was demonstrated using a direct hybridization methodology on the multiplexed array with high sensitivity and specificity. The protocols and sequences developed on the in-house fabricated DNA array were also tested on a customized commercial array from Illumina, Inc. fabricated with 96 optical fiber bundles assembled into a Sentrix(TM) Array Matrix platform that can interrogate up to 1,500 different probes per array with a high sample throughput.; An alternative detection scheme to the direct hybridization of PCR amplified BWAs was developed as well for parallel detection of unlabeled target DNA using a species-specific probe extension reaction on the bead array without PCR amplification. Although with relatively higher detection limits, this assay format is promising for simultaneous identification of a broad range of infectious agents with higher multiplexing capabilities as the multiplexing level is not limited by the PCR.; In order to achieve ultra-high array density that would enable high information content, two optical fiber-based nanoarrays were fabricated, each having 700 nm and 300 nm feature sizes respectively. This thesis describes the fabrication and characterization of these nanoarrays, and demonstrates the possibility of using these two types of optical nanoarrays for DNA analysis. In addition to high array density, optical nanoarrays are also advantageous in terms of reduced sample consumption, improved array sensitivity, and large scale screening of biomolecular recognitions. |
