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CHAPTER 1 Real-Time Reagentless Detection of CBW Agents H. James Harmon, PhD, Professor of Physics, Center for Sensors and Sensor Technologies, Oklahoma State University Porphyrin monolayers and monolayers of porphyrin-enzyme complexes can optically detect CWA/TICs at or near PEL levels in seconds without added reagents using a hand-held device. These solid-state surfaces have been used to detect cholinesterase inhibitors down to 7 pptrillion levels in aqueous solution and can detect dipicolinic acid and sugars found in the spores of Bacillus and Chlostridium. Biodetection involves the ability to identify specific marker components of the target agents. Spectral changes in the porphyrins are measured evanescently with VCOTS using the immobilized monolayers as a planar waveguide; alternatively the surface can be used as a ÒwipeÓ test on exposed surfaces. Reagents, solutions, etc other than the sample are not needed. This detection technology can be useful to first responders and others where a rapid test is needed to discriminate ÒsafeÓ from ÒunsafeÓ materials, the latter indicating that further confirmatory tests are warranted. CHAPTER 2 Personnel and Environment Secure Ways for Analysis of a Bio-Terror Weapon in a Mobile Laboratory Didier Meyer, Marketing Manager, La Calhene, France Bio-terror weapons are by definition unpredictable. Their biological, physical & chemical analysis has to be done as quickly as possible in the nearest location where they are found. The combined use of dedicated isolators and DPTE transfer systems installed in a mobile clean-room fulfills the safety of operators, the protection of the environment and help to give the results of analysis in the best delays. This presentation highlights on physical and biological features and figures of leak tight isolators with proper gloving and the use of DPTE transfer system for ingress of components and samples and for the egress of waste. CHAPTER 3 Rapid Change Ð 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. CHAPTER 4 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. CHAPTER 5 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 nucleic acids, immunological methods, and 'classical' methods. Two case studies will be presented to illustrate the successes and challenges of integrating technology and regulatory sciences. CHAPTER 6 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. CHAPTER 7 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. Chapter 8 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. Chapter 9 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. Chapter 10 Utilization of Biodetection Systems by First Responders: Screening Field Samples vs. Lab Analysis David P. Trudil, PhD, Executive Vice President, New Horizons Diagnostics Corporation Chapter 11 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 developmen Copyright 2000 - 2005