BIODETECTION TECHNOLOGIES 4th Edition - Technological Responses to Biological Threats

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Overview

With an emphasis on rapid detection and low-cost identification, this publication provides solutions to the challenges presented by the evolving pattern of complex biological threat agents of various origins. Important topics addressed include:

• Biothreat Sensing, Detection and Identification
• Biomarker Discovery
• PCR and non-PCR Detection Technologies
• Micro- and Nanoscale Technologies
• On-A-Chip Detection Technologies
• Fluorescence-Based Detection Systems

Table of Contents

Chapter 1
Requirements of Biosensor Technologies by Municipal Water Laboratories: Pathogens of Interest and Hardware Requirements
Tammy A. Spain, PhD, Senior Chemist/Technical Director of Microbiology, Analytical Division, Pinellas County Utilities Laboratory
Most of the biosensor technologies available on the market have the potential to test water for pathogens. Yet municipal water systems have been extremely challenging markets for biosensors. This presentation will provide an overview of biosensor technologies that might be useful to municipal water facilities, the types of pathogens and indicators that water laboratories are generally interested in detecting and hardware requirements for biosensors that may potentially be used in a municipal water laboratory.

Chapter 2
Biosensor Format and Technological Backbone for a “National Biodefense & Pandemie Immune System”
Peer F. Stähler, PhD, Vice President and Chief Scientific Officer, febit biotech GmbH, Germany
The unforeseeable nature of biodefense and pandemic threats suggest the development of a “National biodefense & pandemie immune system”. The technological backbone we propose provides: Fast and sensitive detection, ability to rapidly adapt and distribute tests, and decentralized test production to reduce vulnerability. A network of production sites feeds another network of analyzers. The components of this system are with units for flexible synthesis and instruments for analysis of high-speed microfluidic biochips equipped with microarrays of superb quality and 3’-oligos to enable polymerase based signaling.

Chapter 3
Current Advances in HTP DNA Sequencing Technologies
Kenton L. Lohman, PhD, Senior Biotechnology Advisor, MRI Mid Atlantic Operations, Midwest Research Institute
HTP sequencing technologies have begun to reach commercial fruition and to accelerate the discovery of biodiversity across many species. Technological approaches vary from conventional amplification-based sample preparations to single molecule microchip platforms. The application of these new technologies to Biodefense is driving biomarker discovery for diagnostics, vaccines, and therapeutics to defend against natural or engineered biological threats.

Chapter 4
Development of Assays and Qualified Reagents in Support of Biothreat Agent Detection
Kurt J. Langenbach, PhD, Assay Development Scientist, ATCC/Biodefense & Emerging Infections Research Resources Repository
Our program focuses on implementing increasingly rigorous levels of authentication and characterization to help establish standard reference biothreat organisms and nucleic acids. Assays and techniques that establish specimen identification, concentration, purity, functional activity and preservation/viability are evaluated. These may include biochemical profiling (using traditional and automated platforms), genetic profiling (such as sequencing, genome fingerprinting or PCR) and functional profiling (such as antibody reactivity/neutralization, or CPE). Our goal for these products and techniques is to extend the researcher’s ability to develop new technologies by enhancing the accuracy of pathogen identification.

Chapter 5
Host Responses to Biowarfare Agents: Exposure to Aerosol and Development of Immunity
Luis M. DaSilva, PhD, Microbiologist, Center for Aerobiological Sciences, USAMRIID - The United States Army Medical Research Institute for Infectious Diseases
Changes in host gene expression may provide early evidence of exposure as well as information on toxicity and host immunity against an infectious agent. The effects on gene expression in humans vaccinated with a live-attenuated bacterial vaccine will be discussed and compared to vaccination with a live-attenuated virus. When compared to aerosol exposure experiments using non-attenuated bacterial and viral strains in animal models, this analysis should reveal valuable information on infectivity and host immunity to bacterial and viral biowarfare agents.

Chapter 6
Biomarker Discovery using Bio-Rad’s SELDI-Based Technology
Amanda Bulman, PhD, Application Scientist, Bio-Rad Laboratories
Bio-Rad’s SELDI-based ProteinChip systems offer a single, unified platform for discovery and characterization of biomarkers of exposure, toxicity, and disease. SELDI facilitates rapid, high-throughput analysis of diverse sample types, including serum, urine, saliva, CSF, and cell and tissue lysates. Bio-Rad’s Biomarker Research Centers provide collaborative research services, allowing clients to access Bio-Rad’s internal expertise in study design and data analysis as well as novel technologies for enriching low abundance biomarkers.

Chapter 7
One-Shot Kinetics Measurement with ProteOn XPR36
Sergei Bibikov, PhD, Application Scientist, Bio-Rad Laboratories
Bio-Rad presents a new type of SPR instrument with multiplexing capabilities for high precision measurements of kinetic and thermodynamic parameters of bio-molecular interactions in non-labeled samples. ProteOn XPR36 system offers a fast parallel processing of 36 bio-molecular interactions with its unique 6x6 system of microfluidic channels. Diversified surface chemistry helps to preserve high level of protein binding activity on the surface. Interspot reference adds to the precision of kinetic measurements.

Chapter 8
RSK Inhibitors as Anti-Yersina Agents
Jeffrey A. Smith, PhD, Senior Scientist, Luna Innovations, Inc.
Subverting the host cell signaling events is a common strategy for survival of intracellular pathogens. Because the host cellís signaling events are as essential to establishing and maintaining infection as is the pathogenís own signaling machinery, they can be considered ìreciprocalî virulence factors. Microbial virulence factors have been identified as intriguing targets for novel classes of anti-microbial agents that should interfere with the process of infection rather than with bacterial viability. Following the same line of reasoning, the host cellís reciprocal virulence factors should also present attractive targets for anti-infective agents. We present evidence that targeting the host cellís reciprocal virulence factors interferes with the ability of Yersinia pseudotuberculosis to evade the microbicidal functions of the macrophage thereby limits the ability of the pathogen to survive inside the host cell.

Chapter 9
Field Testing of a Fully Automated Real-Time PCR Device for the Detection of an RNA Virus
Martin A. Lee, PhD, Technical Manager, Enigma Diagnostics Ltd., United Kingdom*
This presentation describes the functioning and field trialling of equipment that enables nucleic acid-based testing for the detection of biological agents to be carried out by non-technical operators in non-laboratory settings. The equipment is portable, fully automated and can be operated from a battery, a vehicle or mains supply. Tests can be performed for either specific DNA sequences or RNA sequences which means that both bacterial and viral agents can be rapidly confirmed to “gold-standard” levels using a single, portable instrument in less than 45 minutes. *In collaboration with: D.Squirrell. Enigma Diagnostics Ltd.; P.Wakeley, Veterinary Laboratory Agency

Chapter 10
Reliable Sample Preparation with Isothermal Real-Time Nucleic Acids Amplification for Rapid Detection of Microorganisms
George Hong, PhD, R&D Manager, Technology and Pre-Development, Millipore Corporation
Here, we present a simple and innovative approach to concentrate organisms from liquid samples and detecting them by using the real-time transcription-mediated amplification technology (Real-Time TMA, Gen-Probe Incorporated). The combined technologies can potentially be applied to detecting a wide variety of microorganisms, including bacteria, viruses, mycoplasma, and fungi and has the potential sensitivity to detect as low as 1-5 CFU.

Chapter 11
Microtransponder-Based Biodetection System
Wlodek Mandecki, PhD, President, PharmaSeq, Inc.
PharmaSeq provides ultra-small electronic chips, microtransponders, and a fluidics-based, bench-top flow reader as tools to perform complex bioassays. The assay involves three phases, conjugation or synthesis of capture molecules (probes, antibodies) on microtransponders, the binding reaction with reporter molecules (targets) labeled with a fluorescent dye, and the readout of the fluorescence and the microtransponder’s ID by the flow reader. The system has been used in genotyping, protein and cell-based assays.

Chapter 12
Nanoparticle Probes for Theranostic Applications
Arnold Kell, PhD, and Benoit Simard, PhD, Principal Research Officer, Steacie Institute for Molecular Sciences, National Research Council, Canada
Our group has synthesized a number of different nanoparticles over the past several years that exhibit unique optical (quantum dots, gold nanoparticles and dye-doped silica nanoparticles) and magnetic properties (superparamagnetic iron oxide nanoparticles). These properties lend themselves nicely to bioanalysis applications, where they can aid in the detection and identification of cells and biomarkers. This presentation will highlight our group’s efforts to develop surface-modified quantum dot, dye-doped silica and superparamagnetic iron oxide nanoparticle probes to aid in the detection and identification of a number of pathogenic species from biological samples.

Chapter 13
Biosensing with Semiconductor Quantum Dot Conjugates
Igor L. Medintz, PhD, Center for Bio/Molecular Science and Engineering, Code 6900, Naval Research Laboratory
Luminescent semiconductor nanocrystals or quantum dots (QDs) are a prototypical nanoparticle with unique electronic and photophysical properties including broad absorption spectra coupled to size-tunable narrow symmetrical photoluminescent emission, high quantum yields and exceptional resistance to photobleaching and chemical degradation. These properties are increasingly being applied to numerous biosensing and probing configurations. We have already demonstrated QD-bioconjugate based sensors specific for nutrients, explosives and enzymatic activity. The unique potential of QDs suggests they will find use in many biodetection configurations.

Chapter 14
Integration of Biology with Silicon Devices for Biological Detection: Opportunities and Future Prospects
Rashid Bashir, PhD, Professor, Birck Nanotechnology Center, Purdue University
Integration of biology with silicon devices and silicon-inspired fabrication promises to enable a wide range of applications in diagnostics, therapeutics, and tissue engineering. In this talk, we will present an overview of a range of projects in our group integrating micro-systems engineering & fabrication with biology, focused towards detection and characterization of biological entities using electrical or mechanical phenomenon at the micro and nano scale. Towards this end, we will present our work on developing silicon-based petri dishes-on-a-chip, nanomechanical cantilever sensors for detection of viruses, silicon based nano-pores for detection of DNA, and related projects.

Chapter 15
Printed Reader on Chip
Max Sonnleitner, PhD, Chief Technology Officer, BioIdent Technologies, Inc.
Easy and cost-efficient integration of miniaturized components for optical readout still represents a major obstacle in developing compact mobile environmental monitoring systems. BioIdent solves this by printing organic photonics directly onto lab on a chip platforms. The novel concept allows for the first time to integrate complete illumination and detection capabilities onto microfluidic-based devices. Absorption and chemiluminescence readout data show the high sensitivity and dynamic range of the integrated photonics.

Chapter 16
Standoff Spectral Detection of Bioaerosols by Laser-Induced Fluorescence
Jean-Robert Simard, PhD, Group Head, Optronic Surveillance Section, Defense Research and Development Canada (DRDC), Canada*
Defense Research and Development Canada (DRDC) has demonstrated a methodology based on Laser Induced Fluorescence (LIF) and intensified range-gated spectrometric detection to investigate standoff bioaerosol detection. The SINBAHD (for Standoff Integrated Bioaerosol Active Hyperspectral Detection) sensor, built for that purpose, has demonstrated impressive bioaerosol sensitivity and classification capabilities. In this presentation, the results associated with this innovative sensor to measure the spectral characteristics of various biological agent simulants, interferants and ambient bio-aerosols of natural and anthropogenic origins at multi-kilometre ranges will be presented and discussed. *In collaboration with: P.Lahaie, G.R.P. Mathieu, B.Déry, H.Lavoie, J.McFee, and J.Ho

Chapter 17
TIRF-EC Biosensors - Massively Parallel Dynamic DNA and Protein Microarrays for Accurate and Rapid Detection of Pathogens
Alexander N. Asanov, PhD, President, TIRF Technologies, Inc.
Total internal reflection fluorescence (TIRF) combined with electrochemistry and electric field control (TIRF-EC) is a platform technology, which is capable of detecting thousands of DNA/RNA and protein molecular markers in a matter of several seconds or a few minutes. The detection limit of TIRF-EC is at the level of single molecules. TIRF-EC biosensors are well suited for point-of-care and field applications, since they require no or minimum sample preparation stages. Cartridges of the TIRF-EC biosensors carry microarrays of assays for simultaneous detection of multiple DNA/RNA and proteins. In contrast to traditional DNA and protein arrays, TIRF-EC sensors monitor dynamics of association and dissociation, which allows for discriminating SNP in DNA/RNA targets and distinguishing between close homologs of proteins. In this presentation we report data on sensitivity, selectivity, and rate of responses for autonomous portable TIRF-EC biosensor.

Chapter 18
Rapid Trace Level Detection by Surface- Enhanced Raman Scattering (SERS) Substrates
Caterina Netti, PhD, Applications Manager, Mesophotonics Ltd, United Kingdom
Nano-texturing metal surfaces by photonic crystals results in highly reproducible SERS substrates with a 10⁴-10⁶ enhancement of the Raman scattering. The high sensitivity and reproducibility is key for the rapid and reliable detection of ppm-ppb of molecules adsorbed at the surface with portable Raman instruments. The developments in SERS substrate engineering will be reviewed. Finally, the applications to trace level detection for Homeland Security will be given highlighting the potential of SERS as routine analytical technique.

Chapter 19
Multi-Orthogonal Biothreat Assays Using the Luminex xMAP System
Kerry G. Oliver, PhD, President, Radix BioSolutions, Ltd.
The Luminex® xMAP® system is a multiplexed assay platform that permits detection and quantification of up to 100 unique threat agents, simultaneously. Radix BioSolutions and Tetracore have developed alternative applications for the multiplexing capabilities of the xMAP system. These applications can be employed universally to enhance data surety and greatly reduce the probability of false positive or false negative sample calls. Firstly, by utilizing Tetracore’s vast array of antibodies specific for different threat agent epitopes we have developed multi-orthogonal immunoassay arrays against various biothreat agents. In addition, we have developed a number of internal control microsphere sets that permits the user to track proper execution and performance of each step of an immunoassay as it is completed. Combined, these two additional applications for the xMAP system, generates an assay platform with unparalleled data quality, flexibility, and breadth.

Chapter 20
Organic Electronics for Monitoring Interactions between Biological Compounds
Kalle Levon, PhD, Professor, Polymer Research Institute, Polytechnic University
Electrically conducting polymers can be processed with gravure, flexo or ink jet printing for high throughput screening purposes. These ion sensitive electrodes can be further modified to be ion selective without any additional labeling as in electronic detection the binding event is coupled with the transduction of the binding signal. We shall present our surface imprinting approach for protein detection and binding results with Ab-Ag pair, hybridization including SNP data and also presumptive identification of bacterial spore proteins.

Chapter 21
Rapid Testing for Over 20 Respiratory Pathogens Simultaneously in Clinical Samples Using Resequencing Arrays
Baochuan Lin, PhD, Research Biologist, and David A. Stenger, PhD, Head, Laboratory for Biosensors and Biomaterials, Center for Bio/Molecular Science and Engineering, Naval Research Laboratory
A broad-spectrum modern diagnostic assay that can simultaneously test for 27 respiratory pathogens was developed by our group. By using the multiplexed RT-PCR/PCR amplification method, we demonstrated this assay can simultaneously detect 7 pathogens with high sensitivity and specificity, allows reproducible strain identification for pathogens represented with adequate prototype sequences, and retains the ability to detect close genetic neighbors. This is the first demonstration of such an assay which can produce direct sequence-based identification of multiple pathogens.

Chapter 22
Optical Biodetection Using Biomimetic Receptors in Real-Time
H. James Harmon, PhD, Director, Center for Sensors and Sensor Technology, Oklahoma State University*
Using immobilized commercially available biomimetic receptors we are able to detect, by visible light (non-laser origin) absorbance spectroscopy, the presence of Cholera toxin (CT), influenza virus, and T-antigen [b-D-Gal-(1,3)-a-D-GalNAc (1,O)-Me disaccharide linked to serine or threonine residues in cell surface glycoproteins expressed in >85% of human carcinomas. Isolated neuraminidase located on the influenza virus surface has been detected at 0.02 parts per trillion or approximately 3 X 10^-17 M; this assay has also been tested against human influenza vaccine at 0.03 parts per trillion as well as attenuated whole virus and is being tested against H5N1 Influenza virus surface proteins. The biosensor will be useful in homeland security, medical/diagnostic applications and for surveillance of plant/animal/food security and safety. *In collaboration with: A.Oliver, OSU

Chapter 23
A Protein Microarray ELISA for the Detection of Botulinum Neurotoxin A
Susan Varnum, PhD, Senior Research Scientist, Pacific Northwest National Laboratory
An enzyme-linked immunosorbent assay (ELISA) microarray was developed for the specific and sensitive detection of botulinum neurotoxin A (BoNT/A), using high-affinity recombinant monoclonal antibodies against the receptor binding domain of the heavy chain of BoNT/A. The ELISA microarray assay, because of its sensitivity, offers a screening test with detection limits comparable to the mouse bioassay, with results available in hours instead of days.

Chapter 24
Use of New Technologies for Pathogen Detection and On-Site Confirmation of Outbreaks of Avian Influenza in Migratory Birds
John Y. Takekawa, PhD, Research Scientist, Western Ecological Research Center,U.S. Geological Survey
The emerging threat posed by highly pathogenic avian influenza virus H5N1 has raised concerns about the role that migratory birds play in the maintenance and geographical spread of the disease. Many basic questions relating to avian influenza virus subtype identification, prevalence within populations, and the risk of spread at local and continental scales remain poorly understood. Technological advancements in the systems used to isolate and identify avian influenza viruses in wild birds and to track the movements of affected species are urgently needed. We will discuss the use of state-of-the art real time PCR pathogen detection and rapid analysis technologies for the field identification, as well as the use of satellite telemetry technology to determine movement patterns and predict spread by infected wild birds.