Keynote Address: Building a Better Bug Trap: BioWatch Past, Current, and Future Technologie
Michael V. Walter, PhD, BioWatch Program Manager, Office of Health Affairs, U.S. Department of Homeland Security
Early detection of a bioterrorist attack has the potential to save thousands, if not millions, of lives. Since 2003, the BioWatch Program has quietly operated in more than 30 cities nationwide, monitoring the air for select pathogens. During the past seven years, much has been learned, including the importance of reliable, accurate detection technology. Dr. Walter will discuss lessons learned from technological problems, the current status of BioWatch Gen-3 sensor-type technologies, and plans for Gen-4 advanced systems.
Microfluidic Sensor Based on Hydrodynamic Flow Focusing with Tunable Sensitivity for Detection of Cells or Microparticles
Mansoor Nasir, PhD, Center for Bio/Molecular Science and Engineering, Naval Research Laboratory
A conductance-based microfluidic sensor utilizing hydrodynamic focusing of a conducting sample fluid by a non-conducting sheath fluid has been realized. The electric field is generated by the four-electrode system and is confined in the focused conducting stream. Both magnetic microspheres and antibody-captured bacteria cause a detectable change in impedance. The sensitivity is tuned using the sheath-to-sample flow-rate ratio. This technique allows for high detection sensitivity while preventing the clogging common to microfluidic detection systems.
Non-PCR Point of Care System for Pathogen Identification and Antibiotics Susceptibility Tests
Vincent Gau, PhD, Co-Founder, CEO, President and CTO, Genefluidics
A point-of-care detection system that integrates the multiplexed electrochemical sensor technology with a microfluidics cartridge has been developed and tested with clinical samples. The result is a revolution in molecular analysis - complete automated sample preparation and detection in a small, portable package. The system utilizes a modular microfluidics cartridge with configurable functional components. Application-specific components are inserted into the universal cartridge case. A single cartridge configuration can run a variety of assays. By interchanging the modular components, the possible assay configurations are limitless. The universal electrochemical sensor array allows detection of multiple targets from a single sample. The revolutionary sensor technology allows any combination of different RNA, DNA, and protein targets to be detected simultaneous in the same cartridge. Pathogen identification and antibiotics susceptibility tests can be accomplished within 60 minutes instead of overnight.
Rapid Prototyping of Microfluidics
Leanna M. Levine, PhD, President and CEO, ALine, Inc.
Rapid Prototyping is a necessary component of product development. There are few rapid prototyping technologies that support the use of materials and methods that provide sophisticated functional prototypes suitable for production quantities in the tens of thousands. We describe a novel polymer laminate technology platform, PLT, which uses a no tooling approach to rapidly create complex fluid structures with pumps and valves that have been used for early prototyping through to product launch. In this talk we demonstrate its application for product development of a molecular diagnostic product.
Microfluidic PCR Array for Parallel Genetic Analysis of Waterborne Pathogens
Thomas Haiqing Gong, PhD, Associate Professor, Thermal and Fluids Engineering Division, School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore
We developed a high throughput microfluidic PCR array card, pre-loaded with different primer pairs for simultaneous (parallel) detection of multiple waterborne pathogens. The PCR mixture loading into an array of reactors and subsequent sealing of the reactors were realized by a simple and automated microfluidic scheme. This PCR array greatly reduces the complexity of the microfabrication and fluidic operation process where individual valving or sealing of a PCR reactor is required. The card performance was successfully demonstrated by detecting DNA of a pool of waterborne pathogens.
Multiplexed Pathogen Detection Using a Micro Flow Cytometer with Integrated Sample Preparation
Li (Julie) Zhu, PhD, Bioanalytical Chemist, Chemistry Technology and Material Characterization, GE Global Research, General Electric Company
NRL and GE have developed a micro flow cytometer with integrated sample preparation for rapid and multi-analyte detection for point-of-care applications. The system consisted of disposable fluidic chips and compact instrument (including optical, fluidic and electronics controlling modules). The flow cytometer chip employed novel design to achieve hydrodynamic focusing for optical detection. The sample preparation chip employed multiple on-chip valves, magnetic trap, and pressure driven flow to fully automate the sample preparation protocol. The two microfluidic chips were interconnected for a complete analysis. Multiplexed detection of bacteria and toxins using the flow cytometer system will be presented.
Integrated Lab-On-A-Chip: A Combined Sample Preparation and PCR System as Ultrafast Analytical Tool for B-Agents
Claudia Gärtner, PhD, CEO, microfluidic ChipShop GmbH, Germany
The overall aim is the realization of a reliable, ultrafast, and portable tool for the identification of B-agents at the point of interest. PCR is the method to be used for the doubtless identification of e.g. bacteria, and viruses. Miniaturization is the way to include the overall analysis process, from sample preparation to detection, on a microtiterplate-sized consumable device and to allow to carry out the analysis without the need for an equipped biological laboratory. An innovative PCR concept with constant temperature zones allows also for the instrument to become portable, due to much reduced power consumption since no thermocycling is necessary. An overall concept to implement the biological reactions for the analysis on-chip, the methods of sample preparation on-chip, and the results of the ultrafast PCR with B-Agents on-chip, as well as the basic instrument will be presented.
VereThreat™ – Detection, Differentiation and Identification of: Anthrax, Plague, Small Pox and Tularemia in One Test
Rosemary Tan, PhD, CEO, Genecet Biotechnologies Pte Ltd.; CEO, Veredus Laboratories Pte Ltd., Singapore
We have developed a novel Lab-on-Chip (VereThreat™) that can rapidly detect and identify catastrophic pathogens. This fast, user-friendly and low cost device combines an ultra-fast miniaturised PCR for amplification of nucleic acids and a high quality microarray for detection in a single chip. Customised spotting on the chip means that the chip can be easily reconfigured to expand its range of detection to detect other pathogenic targets of interest. This device allows users to apply the full benefits of DNA testing in real-world conditions, at a fraction of the time, cost and complexity. Details of VereThreat™ will be presented.
TaqMan Copy Number Assays: New Tool for Linking CNVs to Human Diseases
Edward J. Enright, Technical Specialist-Gene Expression and Genetic Variation, Life Technologies Corporation
In the past few years, the importance of copy number variants (CNVs) has become apparent. CNVs are a class of structural genetic variants that involve a copy number change of DNA fragments from ~1 kb to several megabases. They can be as simple as a deletion or tandem duplication, or much more complex. It is now widely believed that CNVs are a major source of genetic variability in humans, with a significant impact on both normal and disease-related phenotypic variation. Life Technologies has developed a comprehensive product line of over 1.6 million pre-designed human TaqMan® Copy Number Assays which allow researchers to not only accurately detect and quantitate copy number changes but also characterize chromosomal breakpoints of a given CNV for fine-mapping studies.
Challenges of Biological Dosimetry in a Nuclear or Radiological Incident
Marcy Beth Grace, PhD, Project Officer, Biomedical Advanced Research Development Authority (BARDA), Office of the Assistant Secretary for Preparedness and Response, U.S. Department of Health & Human Services
Cutting-edge approaches to biodosimetry focus on finding molecular biomarkers. Promising biomarkers are being explored in proteomics, genomics, metabolomics, glycanomics, and lipidomics. Using a combination of funding mechanisms, HHS, in collaboration with interagency partners, is focused on improving public health emergency response for radiological or nuclear incidents through supporting the development. Despite challenges, great progress has been made in the development of minimally invasive, rapid, and high throughput biodosimetry tools
BT Detection: A View from the Field
Pete L. Estacio, PhD, MD, MPH, Lawrence Livermore National LaboratoryKathryn M. Hansen, Bioterrorism Response, Microbial Diseases Laboratory, California Department of Public Health
This presentation will look at the deployment of new technologies by civil response (Hazmat) teams: their needs, requirements, and the difficulties they face. It will look at current field testing recommendations (including CDC/FBI, AOAC, and State LRN Laboratories), current response protocols and how new technology is integrated into them, and the obstacles facing such adoption.
Panel Discussion:
The Way Forward: First Responder's Perspective and Requirements for the State-of-the-Art in Detection Technologies
Moderator:
Pete Estacio, MD, MPH., PhD
Director, Health Services, Lawrence Livermore National Laboratory
Panelists:
Lt. Shane Francisco
Intelligence Officer, San Francisco Fire Dept, Northern California Regional Intelligence Center
William "Will" So, PhD
Policy & Program Specialist, FBI Headquarters’ Weapons of Mass Destruction Directorate, Countermeasures Unit I - Bioterrorism Prevention Program
Since the anthrax letters following 9/11, field response to bioterrorism has been fractured. Not only have multiple agencies developed various, and at times conflicting, guidelines, but also companies manufacturing “field identification” kits market directly to responders who have little understanding of such tests’ application and limits. Currently no national guidelines or best practices directives exist. This moderated discussion will address the efforts to correct the situation. More specifically we will discuss:
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Various national working groups and their work to standardize field testing kit requirements, sampling protocols, and training and certification standards;
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FBI’s roles and responsibilities in terrorist events and how they differ from public safety needs;
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The needs of the first responders in the field; and
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The Laboratory Response Network (LRN) and its roles and responsibilities.
Short presentations by the panel members on these topics will be followed by a question and answer period.
Genomic Barcoding for Pathogen Detection and Surveillance Systems
Willy A. Valdivia-Granda, PhD, CEO, Orion Integrated Biosciences Inc.
Unless the world community acts decisively a terrorist attack using biological weapons will occur somewhere in the world by the end of 2013 [1]. The impact of such attack will depend on the use of pathogen genomic information not only as new regulatory strategy to prevent the proliferation and the illegal transfer of deadly biological agents, but to develop a new generation of countermeasures. While pathogen genome sequencing is generating vast amounts of information, the use of this data for the inclusion of an organism within the select agent list remains limited. Here we will present specific strategies to use genomic units that are specific for a genus, species or strain and the development of an international database compiling information about microbial collections. These initiatives can open new directions for verification of international treaties, forensics, attribution and the enhancement of intelligence judgment. [1] World at Risk; The Report of the Commission on the Prevention of WMD Proliferation and Terrorism.
Deployment of a Network of SmartBio Sensors for Special Events Bio-Monitoring
Ken Klein, Product Manager, Smiths Detection, Inc.
An important component to countering Bio-Terrorist threats is rapid detection of bio-releases. Vulnerable targets are large public gatherings at indoor and outdoor venues. We describe a pilot system currently deployed with a police department equipped with a wireless array of SmartBio Sensors communicating to a command and control vehicle. We will also present a protocol for using complementary Bio-Identifier technologies to confirm an alarm event and provide field presumptive identification of high threat BWAs.
ECIS for Synthetic Pathogen Detection
Douglas B. Chrisey, PhD, Professor, Dept of Materials Science and Engineering, Rensselaer Polytechnic Institut
Synthetic pathogens are believed to be likely future weapons of terrorists. Electric Cell-Impedance Sensing is a real-time system that can be used to detect both existing and synthetic pathogens by measuring the change in impedance of a confluent cell monolayer grown on interdigitated electrodes. Pathogen toxicity causes a change in the cell barrier function, i.e., the resistance to current flow caused by tight junction formation between the individual cells. Pathogen exposure results in an immediate drop in the measured resistance/capacitance.
Characterization and Optimization of Quantum Dot-Antibody Conjugates for Biological Detection, Identification, and Quantification
Michele D. Kattke and Larry D. Stephenson, PhD, U.S. Army Engineering Research and Development Center Construction Engineering Research Laboratory, U.S. Army Corps of Engineers
FRET-based quantum dot-antibody biosensors are capable of highly sensitive and specific detection. Sensitivity can be compromised by the presence of unconjugated antibodies, which compete for target antigen. Antibody aggregation may be responsible; this possibility was investigated after size exclusion chromatography and dialysis failed to adequately reduce the number of excess antibody fragments. SDS-PAGE was used to visualize the effect of unaggregated versus aggregated antibody on antibody fragmentation and purification efficiency.
Infrared Assembled Microfluidic Device Fabrication for Integrated Biological Analysis
Jay A.A. West, PhD, Chief Technology Officer, Founder, Arcxis Biotechnologies
As part of Arcxis’ efforts to develop a rapid integrated molecular sample to detection diagnostic system, we call BioPhalanxTM, the following studies were designed to develop a highly scalable polymer manufacturing process for microfluidic devices. Using an Infrared assembly method (IRAM) we demonstrate a fabrication process which enables a wide degree of flexibility in device fabrication, demonstrates high pressure standoffs, and has the ability to integrate several design elements into single devices using a variety of polymer substrates. With the developed fabrication technique we created a sample preparation device which was used for automated sample extraction of total RNA and viral DNA from plasma. Furthermore, these devices were utilized in conjunction with a porous polymer monolith column to enable integrated preparation and detection. These PPM columns were modified to enable hybridization capture detection using both Tentacle ProbesTM molecular diagnostic reagents as well as primer extension of a model pathogen target. This system demonstrated the ability to perform integrated filtering, mixing, capture and primer extension detection using the IRAM constructed sample preparation cards in a breadboard instrument format. The combination of IRAM device fabrication and integrated bioanalytical sample preparation and analysis will allow the ability to perform point of care analysis with clinically relevant sample sizes.
Hot Start dNTPs - A Novel Tool for Controlled Nucleotide Incorporation in PCR
Natasha Paul, PhD, TriLink BioTechnologies, Inc.
Magnetic Field Enhanced Immuno-Agglutination Technology for Rapid Detection of Environmental Pathogens: KIM Technology
Elodie Brient-Litzler, PhD, Chief Project Scientist, Bertin Technologies, France
Bertin Technologies has developed a patented technology dedicated to the detection of Bio Warfare Agents. Based on antibody-grafted magnetic colloids agglutination, this homogeneous assay enables the rapid and sensitive bioanalysis of a range of environmental samples. Within 10 minutes, typical limits of detection of 1 ng/ml for toxins and 1E5 CFU/ml for bacteria are achieved. Specific agents’ kits have been validated to be used with the KIM analyzer.
Using Stable Isotope Ratios to Match Bacterial Spore Samples
Derek B. Janszen, PhD, Biostatistician, Computational Biology & Bioinformatics, Pacific Northwest National Laboratory
The isotopic composition of microorganisms, measured as the stable isotope ratios (IRs)13C/12C, 2H/1H, 18O/16O and 15N/14N, are a function of the growth environment and culture conditions. IRs have been used in the forensic analysis of microorganisms to exclude nutrient or water sources as possible growth substrates for specific cultures. Another use of this analytical signature can be to determine whether two or more separate samples of the same biological agent could have been derived from the same source. We present an algorithm that can determine with high sensitivity and specificity whether two bacterial spore samples could have been grown under identical media and culture conditions.