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Conference Topics Pre-Conference Workshop DETECTING, IDENTIFYING AND ANALYZING NEW CHEMICAL AND BIOLOGICAL THREATS: Case Studies and Practical Examples December 2, 2004 (morning) - Rapid detection of biological agents in water - Approaches to detecting threat agents in the water supply - Oligonucleotide microarray for biodefense applications: identification of microbial agents using MAGIChip™ - National guard civil support teams: mission, employment and equipment Pre-Conference Workshop DETECTING, IDENTIFYING AND ANALYZING NEW CHEMICAL AND BIOLOGICAL THREATS: Case Studies and Practical Examples Thursday, December 2, 2004 8:15 Registration, Exhibit/Poster Setup, Coffee and Pastries 9:00 Chairperson's Opening Remarks Tammy A. Spain, PhD, Principal Biochemist, Constellation Technology Corporation 9:10 Rapid Detection of Biological Agents in Water Rolf A. Deininger, PhD, Professor, Environmental Health Sciences, University of Michigan ATP (adenosine tri-phosphate) is a winning member of the cast.The procedure describes the test and the consequences. 10:00 Approaches to Detecting Threat Agents in the Water Supply James A. Higgins, PhD, Microbiologist, USDA-ARS Potable water supplies may represent a potential target for a bioterror attack using spores of the bacterium B. anthracis. Accordingly, we have developed a method, using successive filtrations, culture, and PCR, to detect spores of B. anthracis in tap water. The protocol is capable of detecting as few as 100 spores in 10 L water, and can be implemented using reagents and equipment found in most water quality testing laboratories. 10:40 Refreshments Break, Exhibits/Poster Viewing 11:00 Oligonucleotide microarray for biodefense applications: identification of microbial agents using MAGIChip™ Hoa N. Ho, PhD, Associate Research Scientist, Johns Hopkins Applied Physics Laboratory* Potential exposure to biological agents is a major concern in the United States. Significant efforts have been focused on the development of technologies which allow for the accurate and rapid detection and identification of the presence of select agents. The MAGIChip™ (Micro Arrays of Gel Immobilized Compounds on a Chip) is a promising microarray-based technology with capability to detect a broad spectrum of bacterial organisms in a relatively short period of time. This technology is based on the use of high capacity probes. The probes are uniquely designed to recognize highly amplified ribosomal RNA (rRNA) sequences, eliminating the need for polymerase chain reaction (PCR) amplification and, therefore, reducing the assay time. These 3D DNA microarrays, with high probe capacity, permit evaluation for identification at multiple genetic elements. The MAGIChip™ protocol is relatively straightforward and user-friendly. Total nucleic acids are released from isolated samples with the use of lysozyme, and immobilized onto a preparation column. Subsequent step of purification and concurrent fragmentation and labeling of the nucleic acids are all chemical, rather than enzymatic, reactions. The labeled nucleic acids are then hybridized onto the MAGIChip. Visualization and automatic analysis, aided by automatic algorithms, of the hybridization results occur in a matter of minutes. The whole process from cell lysis to the determination of the microorganism at the species level takes less than 2 hours. The ability to rapidly detect and identify the microorganisms is crucial in the decision making process for the course of action after a potential exposure to select agents. The high capacity of probes per chip makes the MAGIChip™ ideal for high throughput screening of a wide variety of organisms. This unique method has a great potential to enhance the counterproliferation response to the threat of bioterrorism. *In collaboration with: M.L.Theodore, N.Boggs, W.Bethea, and J.Jackman, JHAPL 11:40 National Guard Civil Support Teams: Mission, Employment and Equipment Major Brad A. Jackson, Executive Officer, 10th Civil Support Team, Washington Air National Guard Abstract not available at time of print. 12:20 Discussion Facilitator: Tammy A. Spain 12:40 End of Workshop Main Conference 1:50 Chairperson's Opening Remarks Willie L. Hinze, PhD, Professor, Dept of Chemistry, Wake Forest University 2:00 Chemical and Biodefense R&D in the Federal Budget for 2005 Kei Koizumi, Director, R&D Budget and Policy Program, American Association for the Advancement of Science Since the terrorist attacks of fall 2001, federal spending on biodefense R&D has quintupled to nearly $2 billion a year, and investments to counter chemical threats has also increased dramatically. The National Institutes of Health and the new Department of Homeland Security are responsible for a growing, diverse portfolio of research on all aspects of chemical and biodefense, including detection, fundamental science, countermeasures, and rapid response. The presentation will review the federal investment in chemical and biodefense R&D for 2005. 2:30 KEY NOTE ADDRESS The EPA and Rapid Detection Technologies Jafrul Hasan, PhD, MPH, Microbiologist, Office of Science and Technology, National Homeland Security Research Center, U.S. Environmental Protection Agency* The September 11 attacks in the U.S. have raised concerns that critical elements of the U.S. infrastructure such as the water distribution system might be vulnerable to terrorism or other types of deliberate attack. The public drinking water distribution systems serve 90% of Americans by providing safe drinking water. Water quality sensors were found to respond to a number of non-toxic test compounds selected to simulate possible chemical and biological threats to the water distribution system. Among all the parameters monitored by the sensors, chloride, free chlorine, oxidation reduction potential (ORP), specific conductance, total organic carbon (TOC), and turbidity exhibited a unique and consistent pattern of changes in signal upon injection of wastewater, potassium ferricyanide, malathion, glyphosate and groundwater into a drinking water distribution system simulator. The data suggest that the sensor system may provide information on the characteristics of unknown contaminant and facilitate the subsequent identification with more sophisticated instruments such as GC-MS. With further optimization, a system of sensors may be used as an early warning system. *In collaboration with: R.Haught, J.Goodrich, J.Hall, and J.Herrmann 3:00 Optical Biosensors for Rapid Biodetection Philippe M. Fauchet, PhD, Professor, Director, Center for Future Health, University of Rochester The availability of sensitive, easy-to-use, and reliable detection systems for harmful pathogens including biowarfare agents has become a national priority. The detection of pathogens typically relies on the amplification of nucleic acids coupled with fluorescence detection, a complex technology whose implementation requires highly trained laboratory technicians. This presentation reports the development of devices and systems that employ photonics and nanotechnology and can be operated by individuals with little or no training (for example, soldiers on the battlefield, civilians at home or at work) or robots for areas that are especially hazardous or difficult to reach. Novel biosensing strategies using silicon nanostructures or semiconductor nanocrystals will be discussed and progress toward the development of biosensor systems will be presented. 3:30 Enhanced Detection of Illicit Materials with Micromachined and MEMS Devices Andrew McGill, PhD, Functionalized Materials Devices Section Head, Naval Research Laboratory In order to provide miniaturized and inexpensive detection platforms for chemical agent and explosives detection, a range of micromachined approaches are being investigated to develop analytical system components in a silicon platform. These include sub system such as preconcentrators, gas chromatographs, and sensing transducers. In this presentation, recent work at the Naval Research Laboratory and elsewhere will be used to illustrate advances in these areas, and the progression towards an integrated micromachined detector. 4:15 BEADS: A Platform for Automated Sample Preparation and Detection of Biological Analytes Cindy Bruckner-Lea, PhD, Senior Research Scientist, Fundamental Science Directorate, Pacific Northwest National Laboratory* Methods for the automated purification and concentration of cells, nucleic acids, and proteins are critical to enable the trace detection of biological analytes in environmental samples. The BEADS platform (Biodetection Enabling Analyte Delivery System) utilizes derivatized microbeads to capture the analytes of interest, while washing away the sample matrix materials that can interfere with detection. This presentation will highlight two BEADS system configurations. The first system is configured for the automated sample preparation of nucleic acids for pathogen identification. This system includes automated cell capture from large sample volumes (milliliters and larger), followed by flow-through polymerase chain reaction for DNA amplification, and microarray detection. This configuration enables the detection of only 10 cells/mL in environmental samples. The second system configuration includes on-column fluorescence detection of a sandwich immunoassay designed for toxin detection. The automated on-column immunoassay enables the rapid detection of toxins in less than 5 minutes. *In collaboration with: B.Dockendorff, M.Warner, T.Straub, M.Feldhaus, and J.W.Grate 4:45 Challenges of Using Microtechnology-Based Instrumentation on Real-World Samples Raymond P. Mariella Jr., PhD, Director, Center for Micro- and Nanotechnology, Lawrence Livermore National Laboratory Although there has been significant progress made in creating microtechnology-based instruments to perform bioassays against pathogens, each of these instruments has in common the need for a sample that has been cleaned up and, probably, concentrated, along with the necessary reagents, into an analysis chamber that has dimensions of millimeters. In real-world cases, such sample collection and preparation remain difficult at the microscale. This work was performed under the auspices of the U.S. Department of Energy by University of California Lawrence Livermore National Laboratory under contract No. W-7405-Eng-48. 5:15 PathAlert™, LUX™, and RLS - Single and Multi-Agent Assay Technologies Bill Folkerts, Associate Director, Biodefense, and Eric Ubil, Senior Research Associate, Biological Defense Systems, an Invitrogen Company Invitrogen Corporation, through its subsidiary organization Biological Defense Systems, has developed three detection technologies, PathAlert™, LUX™, and RLS. Both PathAlert™ and LUX™ are PCR based assays with COTS availability for a number of biothreat agents. RLS (Resonance Light Scattering) is an immunosensor currently under development. All three technologies were included in a Technical Readiness Assessment sponsored by the JPEO conducted at Dugway Proving Ground in June 2004. PathAlert™ was also included in a recent Environmental Technology Verification assessment sponsored by EPA and performed by Battelle. These technologies and their assessment data will be presented. 5:45 Panel Discussion Creating Value with Front-End Technology and System Integration: Reagents • Sampling • Devices Facilitator: Raymond P. Mariella Jr. Panelists: Michael Connolly Larry Gold Michelle Hanna Jafrul Hasan Willie L. Hinze Brad A. Jackson 6:15 End of Day One Friday, December 3, 2004 8:55 Chairperson's Remarks Raymond P. Mariella Jr., PhD, Director, Center for Micro- and Nanotechnology, Lawrence Livermore National Laboratory 9:00 Performance Evaluation of the Array Biosensor for the Simultaneous Detection of Multiple Bio-Warfare Agents Tammy A. Spain, PhD, Principal Biochemist, Constellation Technology Corporation Constellation Technology and the Naval Research Laboratory have developed the Array Biosensor (ABS), an immunosensor as sensitive as the ELISA. A fully automated prototype has been built and tested at various sites including Dugway Proving Ground. Multiple assays were performed in parallel on multiple samples. No cross-reactivity or sensitivity-loss was observed during multiplex testing. This presentation will discuss the test scenarios and the performance of the ABS during these tests. 9:30 Aptamers as Capture Agents for Microbes and Chemicals Larry Gold, CSO and Chairman, SomaLogic Aptamers are single-stranded nucleic acids that function like antibodies (surprising though that is, even 15 years after the first aptamers were discovered). Aptamers have been prepared (by the SELEX process) against several pathogens (viral and bacterial), very many pure proteins, and many small organic molecules. In direct comparisons with other capture agents, even antibodies, aptamers have shown high affinity and remarkable specificity. SomaLogic holds an exclusive license to use aptamers for detection purposes, and builds aptamer arrays for simultaneous analyte measurements. Different formats for aptamer arrays will be described. Proteins have been measured, quantitatively and at low concentrations, in complex matrices (like serum), with provocative early clinical data. 10:00 The RiboMaker® Detection System Michelle Hanna, PhD, CEO and President, Ribomed Biotechnologies, Inc. The RiboMaker® Detection System is a robust, isothermal, PCR-free method for detecting and quantifying proteins and nucleic acids. The core signal-generation technology, Abscription™, utilizes the reiterative synthesis of short aborted RNA transcripts (Abscripts™) by RiboMaker® Abscriptase™ to generate a quantifiable signal. The RiboMaker® Detection System can be used for the detection of methylated DNA in silenced genes, inherited or sporadic disease-associated SNPs and other DNA mutations, pathogenic organisms associated with diseases (HPV and cervical cancer, Hepatitis B and liver cancer, etc), and disease-associated changes in RNA and protein expression levels. At present, Ribomed is collaborating on two contracts with Northrop Grumman using the RiboMaker® Detection System to detect blood-borne and airborne RNA viruses and protein toxins. 11:00 BioDetect™ - A Novel Electronic DNA Identification Technology Michael Connoly, PhD, Founder, President and CEO, Integrated Nano-Technologies, LLC Integrated Nano-Technologies (INT) is developing an electronic biosensor designed to rapidly and accurately provide DNA-based field detection of pathogenic organisms. The most significant feature of INT's biosensor is its ability to accurately and rapidly detect the presence of a particular DNA sequence within a complex mixture, avoiding the use of a prior DNA amplification method. INT is currently developing tests designed for the military and postal service markets. 11:30 Rapid Detection of Viable Pathogenic Organisms Using Microbiosensors Antje J. Baeumner, PhD, Assistant Professor, Dept of Biological and Environmental Engineering, Cornell University The rapid, highly specific and inexpensive detection of viable pathogenic organisms is becoming increasingly important for our safety, whether it relates to food safety, environmental and medical applications or biosecurity. Biosensors in combination with micro- and nanotechnology hold the promise of being the sought after analytical tool. We develop in our lab platform technologies of microbiosensors and micro-sample preparation modules that are designed for the detection of viable pathogens such as B. anthracis, C. parvum and Dengue virus at concentrations of 10 organisms per sample. 12:00 ViriChip™ System: A Virus Particle Capture and Detection System Saju R. Nettikadan, PhD, Project Director, BioForce Nanosciences, Inc. An assay ViriChip™ System for the rapid detection and identification of virus particles has been developed at BioForce Nanosciences Inc. This system utilizes a solid-phase substrate with multiplexed arrays of ultra-micro affinity capture domains (~10um) for virus particle capture. The captured virus particles are detected by monitoring the changes in the surface profile of the capture domain surface. The identity of the particles is confirmed based on their immunological and morphological properties. 1:55 Chairperson's Remarks Michael Connolly, PhD, Founder, President and CEO, Integrated Nano-Technologies, LLC 2:00 Featured Case Study Presentation Molecular Epidemiological Characterization of the Severe Acute Respiratory Syndrome (SARS) Coronavirus - Telling the Complete Story? Frederick C. Leung, Dean of Science, The University of Hong Kong Severe Acute Respiratory Syndrome (SARS) is a newly emerged disease caused by a novel coronavirus (SARS CoV) first noticed in March 2003 in Hong Kong and then spread globally within days affecting over 30 countries. Intense international collaboration was one of the reasons that the virus was brought under controlled in such a short time and therefore no pandemic outbreak was follow. This presentation aims at using molecular genetic sequencing data to analyze that case in great detail. 2:30 Development of a Portable SAW Based Sensor System for the Detection of Chemical Agents Timothy A. Postlethwaite, PhD, Chief Scientist, Constellation Technology Corporation* Constellation Technology, in collaboration with the Naval Research Laboratory, has been working towards the development of the CT-CAD, a new surface acoustic wave (SAW) based chemical agent detector. The CT-CAD is a portable sensor system for the detection of nerve and blister agents. Improvements have resulted in increased agent sensitivity and signal kinetics as well as decreased humidity and temperature sensitivity. Simulant and surety test results of the CT-CAD will be presented and discussed. This work was supported by the US Army Space and Missile Defense Command and the Defense Threat Reduction Agency. *In collaboration with: S.Barone, L.L.Manoosingh, R.D.Sorrells, B.A.Harmon, C.Traynor, M.V.Tesone, A.Guim, and J.E.Knighton, Constellation Technology; R.A.McGill, E.J.Houser, S.Stepnowski, and J.Stepnowski, NRL 3:00 Rapid and Sensitive Detection of Microbes and Toxins via Dynamic Surface Generation and Imaging Stuart Kushon, PhD, Bio-Defense Team Leader, Assay Development, QTL Biosystems QTL has developed multiple novel technologies for the generation of highly sensitive assays for Microbes and Toxins utilizing a Dynamic Surface Generation technology. This platform in combination with our handheld field biosensor allows for rapid, on-site detection of dangerous bio-agents for first responders, field and military personnel. The hand-held QTL Biosensor can very sensitively detect changes of fluorescence in a liquid sample that may contain dissolved or colloidal fluorescent materials. The assay is based upon capture of an analyte in the detector compartment and a level of fluorescence that provides a quantitative assay for the analyte. The assay has shown feasibility for Anthrax, Ricin, and SEB. 3:45 Separation and Identification of Microbes (Bacteria, Virus, Fungi, etc.) by Capillary Electrophoresis (CE): A Viable Approach? Willie L. Hinze, PhD, Professor, Dept of Chemistry, Wake Forest University* This presentation will provide a concise historical overview of the prior efforts related to the characterization, separation, identification and viability evaluation of microbes by CE. Whereas the early attempts employing a variety of electrophoretic based techniques suffered from poor efficiency, the recent incorporation of polymeric additives (such as polyethylene oxide (PEO), dextran, etc.) in the run buffer has allowed for highly efficient separation of intact microbes. Published applications include a rapid CE-based assay for the identification of bacteria (Staphylococcus saprophyticus or Escherichia coli) causing urinary tract infections as well as for assaying for food borne species (as Salmonella enteritidis) Stringent sample preparation/treatment steps are required in order to obtain reproducible CE results. Some preliminary results regarding the HPLC separation of bacteria will also be disclosed. *In collaboration with: M.Endo 4:15 Validation Issues for Molecular Based Diagnostic Assays Johnny Callahan, Scientist - Viral Assay Development, Tetracore, Inc. Abstract not available at time of print. Please come back soon for the latest program updates. 4:45 Selected Oral Poster Presentations, Discussion 5:00 Concluding Remarks, End of Conference