Contents:

Conference Topic Thursday, June 17, 2004 1:55 Chairperson’s Opening Remarks H. James Harmon, PhD, Professor of Physics, Center for Sensors and Sensor Technologies, Oklahoma State University 2:00 KEY NOTE ADDRESS Establishing an Enduring Biodetection Capability Lawrence D. Kerr, PhD, Assistant Director for Homeland Security, Office of Science and Technology Policy, Executive Office of the President The foundation of our national defense against biological threat agents lies in the recognition that these agents have characteristics that are unique among weapons of mass destruction and that the use of biological threat agents holds the potential for catastrophic consequences. These unique properties of biological threat agents create both challenges and opportunities for successful biodetection efforts, and thus early detection is a key pillar of our Nation's comprehensive strategy for biodefense. The development of biodetection capabilities against high consequence biological threats for our federal, state and local operational end-users will be discussed. 2:30 Real-Time Monitoring System for Water Security and Environmental Applications Kevin Montgomery, PhD, Technical Director, National Biocomputation Center, Stanford University The water distribution system is a highly vulnerable target for potential bioterror attack. We have developed a system of small, inexpensive, self-powered, wireless sensors for water (physicochemical and bioassay) and air quality (physical and biological particulate) measurement and surveillance. These sensors transmit their data wirelessly to a central server that integrates disparate data streams and performs advanced analysis. The system automatically monitors for changes in environmental quality and security and, should an event occur, issues alerts to pagers/cell phones to allow for rapid and easy dissemination of vital information to local, state, and federal authorities via a secure, GIS-based website. As part of this presentation, we will present this real, deployed, functioning system, demonstrate the technology, discuss the deployment locations and lessons learned, and future plans for further development. 3:00 Electronic Taste Chip Sensors for Rapid Detection of Chemical and Biological Targets: Development of Customized Arrays for Bio-Defense and Civilian Applications John T. McDevitt, PhD, Professor, Dept of Chemistry & Biochemistry, The University of Texas at Austin Recent work from The University of Texas at Austin has led to the development of a powerful new "electronic taste chip" technology. By mimicking the chemical features of the human taste bud, the chip has the capacity to analyze rapidly the chemical and biochemical content of complex fluids such as human blood, environmental samples, and bioaerosol specimens. This technology is extremely versatile, making it suitable for the measurement of electrolytes, protein antigens, antibodies, whole bacteria and DNA/RNA. While these chips exhibit impressive analytical and diagnostic capabilities as compared with gold standards, their compact design and low cost also allows for their use in numerous important civilian and military applications which require autonomous operation. Moreover, because molecular detection is confined to a miniaturized chamber etched into a silicon chip, multiple tests can be performed simultaneously. Most recently, microbead arrays have been fashioned specifically for the detection of chemical weapons precursors and degradation products as well as for the identification of bacterial spores from the bacillus family. 4:00 Rapid Change – A Requirement for Biodetection David L. Danley, PhD, Director, Homeland Security and Defense Programs, CombiMatrix Corporation Until 9/11, the biothreat agent list included about 12 agents, based on the old U.S. offensive program. NIAID and CDC subsequently developed expanded lists that included potential agents from toxins to protozoa. Rapid emergence of new pathogens demonstrates that protection by extant detectors is effected only with a rapid change in agent identification. Microarray technology and electrochemical detection can be tailored for a wide variety of agents. Changing detection requirements can be rapidly implemented without changing detection strategy. 4:30 Portable Array Biosensor for Multi-Threat Detection Frances S. Ligler, DPhil, DSc, USN Senior Scientist for Biosensors & Biomaterials, Naval Research Laboratory A portable biosensor detects multiple targets simultaneously in multiple samples. Pathogens and toxins can be detected in clinical fluids, environmental samples, and homogenized foods with little if any sample preparation. Detection limits are generally in the 0.1-1.0 ng/mL for toxins and 1,000-10,000 cfu/mL for bacteria using antibodies for threat capture. Other capture molecules are being tested for the capability to recognize unanticipated threat agents. 5:00 Microarray Approaches to Detection of Emerging Pathogens Konstantin Chumakov, PhD, DSci, Chief, Laboratory of Methods Development, Center for Biologics Evaluation and Research, Food and Drug Administration New techniques based on hybridization with oligonucleotide microarrays were developed and used for identification and fine genotyping of a number of human pathogens. The talk will focus on attempts to characterize details of genomic structure of rapidly evolving viruses and outline new ways of screening for emerging pathogens. 5:30 Handheld PCR Instrumentation John W. Czajka, PhD, Deputy Director of Research and Product Development, Smiths Detection There is a growing need for portable, battery-powered instruments capable of detecting and identifying biological agents that may be used against military and civilian targets. The Bio-Seeq® detection system is designed to address this need. The instrument weighs 6.5 lbs. and can independently process up to six samples at one time and will operate for up to 2.5 hours with a rechargeable battery. The consumable developed by Smiths Detection is completely self-contained with all necessary reagents, filtering, and reconstitution fluids. Friday, June 18, 2004 8:55 Chairperson’s Remarks John T. McDevitt, PhD, Professor, Dept of Chemistry & Biochemistry, The University of Texas at Austin 9:00 Biodefense Research & Development: Strategic Opportunities Brad Smith, PhD, Fellow, Center for Biosecurity of UPMC, Assistant Professor, University of Pittsburgh The threat posed by biological weapons is real and growing. Currently, there are only limited tools and countermeasures available to prevent the use of bio-weapons or to mitigate their effects if prevention fails. As result, there is a tremendous opportunity for the science and engineering communities to develop new diagnostics, therapeutics, and other biodefense technologies. The Bush Administration has recognized the vital role that biomedical research will play in the fight against bioterrorism, as illustrated by the dramatic increases in biodefense R&D funding at NIH, and with the creation of HSARPA in the Department of Homeland Security. The unprecedented magnitude of these investments underlines the critical role that life sciences must now assume in the defense of the nation. Given these new opportunities, it is critical that biodefense R&D investments are made intelligently, and are driven by a strategic process that reflects both short and long-term needs. 9:30 Detection Technologies and Crop Biosecurity Philip H. Berger, PhD, National Science Program Leader - Molecular Diagnostics & Biotechnology USDA-APHIS-PPQ Center for Plant Health Science and Technology Our agricultural and plant resources are vulnerable to intentional or unintentional introduction of pest and pathogens. The responsibility for protecting US plant resources lies with APHIS and DHS, but involves many universities, state agencies, and private organizations. Effective detection and identification requires both field-deployable and laboratory-based diagnostics that are rapid, sensitive, and inexpensive. We face significant challenges, however, in meeting our safeguarding mission. Unlike the analogous situation for medical or veterinary diagnostics, we require diagnostics for thousands of pathogens and pests, affecting hundreds of hosts. There are nearly 400 species of pest or pathogen on the USDA-APHIS regulated pest list, and hundreds more that are reportable or actionable. At times identification to species is adequate, but frequently we require identification to race, biovar or strain. At times determining geographic origin of an agent is important. We use methods based on detection of nucleicacids, immunological methods, and 'classical' methods. Two case studies will be presented to illustrate the successes and challenges of integrating technology and regulatory sciences. 10:00 Challenges for Optically-Based Biological Agent Sensors LTC John C. Carrano, PhD, Program Manager, DARPA/MTO After the anthrax attacks during the fall of 2001, much focus has been placed on developing effective means to detect biological pathogens. Unfortunately, practical solutions still allude the technical community. DARPA’s Semiconductor Ultraviolet Optical Sources program has met with success in developing deep ultraviolet laser diodesand LED’s, and integrating new sources into a new generation of compact and sensitive bio-detectors. In this talk, we will describe two spectroscopic techniques useful for bio sensing: laser-induced fluorescence, and UV resonance-enhanced Raman. We will also detail advances in optoelectronic devices that now enable these techniques to become practical as effective early-warning bio-sensors capable of continuous monitoring without consumables. In particular, we will describe innovations that have resulted in UV LED’s operating at wavelengths as short as 275 nm with milliwatt class CW optical power, and laser diodes operating in the range of 340 nm to 365 nm. These devices are fabricated from epitaxial layers of AlInGaN grown on a variety of substrates. We will conclude with a description of the remaining key challenges for optically based biological agent sensors, and a vision of how to address these critical issues. 11:00 Bioaerosol Collection and Analysis Using Diffuse Reflectance Infrared Spectroscopy Alan C. Samuels, PhD, Research Chemist, Passive Standoff Detection Team, US Army Edgewood Chemical Biological Center Real time warning of the presence of a biological aerosol threat is a critical enabling technology for homeland defense and force protection. Fluorescence methods that are used now lack the specificity to indicate the nature of a biomaterial present, and the ubiquitous nature of ambient airborne particulates of biological origin leads to high false alarm rates. We describe proof-of-principle experimental data that demonstrates the facility with which aerosols can be collected and analyzed using infrared spectroscopy. We performed several experiments in which particulate matter is collected and analyzed in seconds to minutes to afford some discrimination and can be readily adapted to an in-situ approach to provide early warning of the appearance of a threat biological warfare agent. 11:30 Detection of Cellular Activation Using Mid Infrared Spectroscopy M. Kathleen Alam, PhD, Principal Member Technical Staff, Sandia National Laboratories Current methods of disease detection rely primarily on replication technology to reproduce the virus to the point it can be identified. An alternative to directly detecting the invading virus is to detect the large-scale secondary responses that occur upon infection. We will present data from ATR-IR experiment in which live cells were examined after activation using two different activating agents. Comparison with control samples shows good separation of the spectra. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United Stated Department of Energy under contract DE-AC04-AL85000. 12:00 Assay Method Validation for Biodetectors George A. Robertson, PhD, Vice President Science & Technology, PDA - An International Association for Pharmaceutical and Biopharmaceutical Science and Technology The pharmaceutical industry has a long history of regulations and guidelines for analytical methods for the quality of products, control of processes or the performance of medical devices. Although these standards may not have statutory applicability for biodetector systems in military or homeland defense applications, the scientific basis of these requirements go to the heart of system suitability and reliability. This talk will review the scientific basis of requirements for specificity, accuracy, precision, sensitivity, linearity, range and robustness and how they can apply to biodetector systems. 1:55 Chairperson’s Remarks Konstantin Chumakov, PhD, DSci, Chief, Laboratory of Methods Development, Center for Biologics Evaluation and Research, Food and Drug Administration 2:00 Update on Mass Spectrometry-Based Detection for Mixtures of Microorganisms Catherine Fenselau, PhD, Professor of Chemistry and Biochemistry, University of Maryland The promise of mass spectrometry is being realized by cross-disciplinary team efforts, to provide specificity, speed, sensitivity and broadband sensing without preselection of targets. Approaches will be reviewed for the identification of components of mixtures and of cluttered samples. In particular, the implementation of bioinformatics will be discussed, which obviates the requirement for reproducible spectra imposed by library searching. 2:30 The Autonomous Pathogen Detection System (APDS) Mary T. McBride, PhD, Group Leader, Biomedical Sciences and Engineering, Medical Physics and Biophysics Division, Lawrence Livermore National Laboratory We have developed and tested a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents. The APDS is completely automated, offering continuous aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and nucleic-acid based polymerase chain reaction (PCR) amplification and detection. This work was performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48. 3:00 Devices and Protocols for the Recovery of Organisms and Nucleic Acids from Complex Samples Lalitha Parameswaran, PhD, Staff Scientist, MIT Lincoln Laboratory Forensic, biodefense, clinical and food analysis applications require the ability to extract nucleic acids from varied sample types, while eliminating inhibitors that may adversely affect PCR. To address these needs, we have developed the Lincoln Nucleic-acid Kit (LiNK) cartridge, enabling fast, easy purification and stabilization of nucleic acids, and the Affinity Magnet (AM) protocol, which uses customized magnetic beads to achieve target concentration from raw samples. We are also implementing the AM protocol into a fieldable cartridge format. 4:00 Electrochemiluminescence Assays for Biological Agents George Sigal, PhD, Scientist, Meso Scale Diagnostics, LLC Electrochemiluminescence (ECL) is a proven technology with significant commercial success in clinical, life science research, industrial, environmental and defense applications. MSD’s Multi-ArrayTM technology allows ECL measurements to be carried out in a multi-well plate format using disposable plates with integrated electrodes. Tetracore specializes in the development of antibodies and kits for detecting biological agents. We demonstrate the use of MSD instrumentation and Tetracore reagents for the rapid, highly sensitive detection of multiple agents including bacteria, viruses and toxins. 4:30 Automated Immunoassay-Based Biosensors Timothy A. Postlethwaite, PhD, Chief Scientist, Constellation Technology Corporation Constellation Technology has developed several automated biosensor systems that perform immunoassays for the detection and identification of biological agents in environmental samples. The immunoassay approach was chosen because it is amenable to complete automation and it allows for the rapid, sensitive detection of a wide range of analytes including bacteria, viruses, and toxins. The biosensor systems to be described have been integrated into both man portable and fixed location configurations. This presentation will focus on the design, development and performance of these systems.