EXP-SA: Explosives Tracking: A Microsystem for Detection of Bacterial Endospores as Self-Replicating Nucleic Acid Taggants

This proposal presents an integrated research and educational plan directed toward the production, detection, and identification of bacterial endospore taggants for explosive tracking. While the most immediate application of the research is related to stemming the activities of bioterrorists, the anticipated fundamental advances in bioengineering and sensor science and engineering will have significant societal relevance to other applications, including first-responder activities, healthcare, food safety, and pollution avoidance and mitigation. Intellectual Merit The investigators propose to combine bioengineering of Bacillus stearothermophilus endospores with microdevices for sample processing and taggant identification. A surface acoustic wave (SAW) microdroplet mixing/transport/incubator system will be coupled with molecular padlock probe technology for sensitive identification of bioengineered endospores. The specific research tasks are to: (i) Generate a number of different Bacillus spores, each with a unique DNA sequence or sequences spliced into its genome; (ii) Investigate and identify the optimal SAW device designs needed to germinate spores, lyse vegetative bacteria, transport, mix, and heat microdroplet samples; (iii) Design subsystems for DNA isolation; (iv) Develop a fluorescence-based molecular padlock probe system for DNA identification that can operate effectively in conjunction with the SAW fabrication microsystem platform; (v) Fabricate and test the proposed prototype identification system. Broader Impacts Broader impacts will be achieved through the following programs and activities to: (i) Train and interact with high school audiences through two major ongoing programs at University of Maine (UMaine), NSF Research Experiences for Teachers (RET) and the GK-12 Sensors; (ii) Involve undergraduates from Maine and other institutions directly into the research project under the umbrella of the ongoing NSF Research Experience for Undergraduates (REU) program at the UMaine; (iii) Identify appropriate Capstone projects for undergraduates involving cross-disciplinary research and design projects; (iv) Enhance existing graduate level courses (1) Microscale Bioengineering and (2) Design and Fabrication of Acoustic Wave Devices by incorporating research results into each course; (v) Contribute to the interdisciplinary multi-institutional NSF Integrative Graduate Education and Research Traineeship (IGERT) program in functional genomics, which involves UMaine, the Jackson Laboratory, and the Maine Medical Center Research Institute; (vi) Provide thesis topics for M.S. and Ph.D. students; (vii) Disseminate the research and educational material on a project website, and through conferences and printed literature. Project Outcomes ReportNew investigative tools are desperately needed to determine the origin and transit routes of contraband explosive materials, and the individuals who transport them. A powerful strategy for tracking and identifying specific lots of explosives is the incorporation or labeling with pre-and post-detonation identification tags, or taggants. This project involves the production, detection, and identification of bacterial endospore taggants for explosive tracking. It combines bioengineering of environmentally resistant Geobacillus thermoglucosidasius endospores with development of microdevices for sample processing and taggant identification. A surface acoustic wave (SAW) bacterial lysis system is coupled with on-chip fluorescence-based quantitative polymerase chain reaction (PCR) for identification of bioengineered endospores.Geobacillus spores with a unique DNA sequence encoded in well-retained plasmids have been generated. Optimal SAW device structures have been designed, fabricated and tested for lysis of the vegetative bacteria. A number of on-chip structures for multiplex PCR analysis have been created and tested. DNA release and fluorescence-based PCR analysis for identification of specific genomic DNA sequences can now be interfaced to the SAW microsystem platform to comprise an important part of the overall detection system. We anticipate that aspects of this technology will be useful for tracking contraband materials such as explosives, environmental monitoring, and potentially medical diagnostic applications. This project has fostered the multidisciplinary training of numerous undergraduate and graduate students in molecular biology, microbiology, biochemistry, and bioengineering