Detection Of Biological Warfare Agents Research Articles

Portable 24-analyte surface plasmon resonance instruments for rapid, versatile biodetection

Field use of surface plasmon resonance (SPR) biosensors for environmental and defense applications such as detection and identification of biological warfare agents has been hampered by lack of rugged, portable, high-performance instrumentation. To meet this need, we have developed compact multi-analyte SPR instruments based on Texas Instruments’ Spreeta sensing chips. The instruments weigh 3 kg and are built into clamshell enclosures measuring 28 cm × 22 cm × 13 cm. Functions are divided between an electronics unit in the base of the box and a fluidics assembly in the lid. Automated valves and pumps implement an injection loop flow system that allows sensors to be exposed to sample, rinsed, and treated with additional reagents (such as secondary antibodies) under computer control. Injected samples flow over the surfaces of eight sensor chips fastened into a temperature-controlled silicone flowcell. Each chip has 3 sensing regions, for a total detection of 24 areas that can be simultaneously monitored by SPR. Coating these areas with appropriate antibodies or other receptors allows a sample to be screened for up to 24 different substances simultaneously. The instruments report refractive index (RI) values every second, with a typical noise level of 1–3 × 10 −6 RI units. The design of the device is described, and performance is illustrated with detection of six distinct analytes ranging from small molecules to whole microbes during the course of a single experiment.

Technological advancements for the detection of and protection against biological and chemical warfare agents

There is a growing need for technological advancements to combat agents of chemical and biological warfare, particularly in the context of the deliberate use of a chemical and/or biological warfare agent by a terrorist organization. In this tutorial review, we describe methods that have been developed both for the specific detection of biological and chemical warfare agents in a field setting, as well as potential therapeutic approaches for treating exposure to these toxic species. In particular, nerve agents are described as a typical chemical warfare agent, and the two potent biothreat agents, anthrax and botulinum neurotoxin, are used as illustrative examples of potent weapons for which countermeasures are urgently needed.

Integrated Microfluidic CustomArray Device for Bacterial Genotyping and Identification

The ongoing threat of the potential use of biothreat agents (such as Bacillus anthracis) as a biochemical weapon emphasizes the need for a rapid, miniature, fully automated, and highly specific detection assay. An integrated and self-contained microfluidic device has been developed to rapidly detect B. anthracis and many other bacteria. The device consists of a semiconductor-based DNA microarray chip with 12,000 features and a microfluidic cartridge that automates the fluid handling steps required to carry out a genotyping assay for pathogen identification. This fully integrated and disposable device consists of low-cost microfluidic pumps, mixers, valves, fluid channels, reagent storage chambers, and DNA microarray silicon chip. Microarray hybridization and subsequent fluid handling and reactions were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. The genotyping results showed that the device was able to identify and distinguish B. anthracis from the other members of the closely related Bacillus cereus group, demonstrating the potential of integrated microfluidic and microarray technology for highly specific pathogen detection. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluid handling steps and allows the detection and identification of biological warfare agents in a rapid and automated fashion.

Continuous flow microfluidic device for cell separation, cell lysis and DNA purification

A novel integrated microfluidic device that consisted of microfilter, micromixer, micropillar array, microweir, microchannel, microchamber, and porous matrix was developed to perform sample pre-treatment of whole blood. Cell separation, cell lysis and DNA purification were performed in this miniaturized device during a continuous flow process. Crossflow filtration was proposed to separate blood cells, which could successfully avoid clogging or jamming. After blood cells were lyzed in guanidine buffer, genomic DNA in white blood cells was released and adsorbed on porous matrix fabricated by anodizing silicon in HF/ethanol electrolyte. The flow process of solutions was simulated and optimized. The anodization process of porous matrix was also studied. Using the continuous flow procedure of cell separation, cell lysis and DNA adsorption, average 35.7 ng genomic DNA was purified on the integrated microfluidic device from 1 μL rat whole blood. Comparison with a commercial centrifuge method, the miniaturized device can extract comparable amounts of PCR-amplifiable DNA in 50 min. The greatest potential of this integrated miniaturized device was illustrated by pre-treating whole blood sample, where eventual integration of sample preparation, PCR, and separation on a single device could potentially enable complete detection in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

Special Issue of Photonic Sensors for Chemical, Biological, and Physical Parameter Detection

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Biomimetic S-Layer Supported Lipid Membranes

The present review focuses on a unique biomolecular construction kit including surface-layer (S-layer) proteins as basic building blocks and patterning elements, but also major classes of biological molecules such as (membrane) proteins, lipids, and heteropolysaccharides for the design of functional S-layer supported lipid membranes. The biomimetic approach copying the supramolecular building principle of most archaeal cell envelopes composed of a plasma membrane and a closely associated S-layer lattice has resulted in robust lipid membranes. Reconstitution of responsive transmembrane proteins has been demonstrated in lipid membranes generated on S-layer-coated substrates and electrodes. This is a particular challenge as one-third of all proteins are membrane proteins such as pore-forming proteins, ion channels or receptors. Hence membrane proteins are a preferred target for pharmaceuticals (currently more than 60% of all consumed drugs). This novel type of supported lipid membrane is seen as one of the most innovative strategies in membrane protein-based nanobiotechnology with potential applications that range from pharmaceutical (high-throughput) drug screening over lipid (bio)chips to the detection of biological warfare agents.

Fiber-Optic Microsphere-Based Arrays for Multiplexed Biological Warfare Agent Detection

We report a multiplexed high-density DNA array capable of rapid, sensitive, and reliable identification of potential biological warfare agents. An optical fiber bundle containing 6000 individual 3.1-mum-diameter fibers was chemically etched to yield microwells and used as the substrate for the array. Eighteen different 50-mer single-stranded DNA probes were covalently attached to 3.1-mum microspheres. Probe sequences were designed for Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella melitensis, Clostridium botulinum, Vaccinia virus, and one biological warfare agent (BWA) simulant, Bacillus thuringiensis kurstaki. The microspheres were distributed into the microwells to form a randomized multiplexed high-density DNA array. A detection limit of 10 fM in a 50-microL sample volume was achieved within 30 min of hybridization for B. anthracis, Y. pestis, Vaccinia virus, and B. thuringiensis kurstaki. We used both specific responses of probes upon hybridization to complementary targets as well as response patterns of the multiplexed array to identify BWAs with high accuracy. We demonstrated the application of this multiplexed high-density DNA array for parallel identification of target BWAs in spiked sewage samples after PCR amplification. The array's miniaturized feature size, fabrication flexibility, reusability, and high reproducibility may enable this array platform to be integrated into a highly sensitive, specific, and reliable portable instrument for in situ BWA detection.

Colorimetric Biosensors Based on Polydiacetylene (PDA) and Polyamidoamine (PAMAM) Dendrimers

Abstract Biosensing techniques are crucially important in a wide variety of applications from the detection of biological warfare agents to analysis for pesticide residues in crops or determination of freshness of food products. The intense search for better and more accurate biosensors has recently drawn attention to polyamidoamine (PAMAM) dendrimers. The unique properties of globular PAMAM dendrimers, with large numbers of exo-presented functional end-groups, provide a very promising platform for biological sensor elements. Polydiacetylene (PDA), on the other hand, is a macromolecule that exhibits a colorimetric response to a variety of stimuli, and is therefore a strong candidate for sensor elements. Consequently, an ensemble of PAMAM dendrimers and PDA segments provides for a novel type of molecular biosensor, which is expected to have highly pronounced sensitivity and widely applicable versatility. In this article, an overview of recent experimental work and progress achieved on biosensors from PAMAM...

Remote atmospheric breakdown for standoff detection by using an intense short laser pulse

A remote atmospheric breakdown is a very rich source of UV and broadband visible light that could provide an early warning of the presence of chemical-biological warfare agents at extended standoff distances. A negatively chirped laser pulse propagating in air compresses in time and focuses transversely, which results in a rapid laser intensity increase and ionization near the focal region that can be located kilometers away from the laser system. Proof-of-principle laboratory experiments are performed on the generation of remote atmospheric breakdown and the spectroscopic detection of mock biological warfare agents. We have generated third harmonics at 267 nm and UV broadband radiation in air from the compression and focusing of femtosecond laser pulses. Fluorescence emission from albumin aerosols as they were illuminated by the femtosecond laser pulse has been observed.

Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection.

A fully integrated biochip device that consists of microfluidic mixers, valves, pumps, channels, chambers, heaters, and DNA microarray sensors was developed to perform DNA analysis of complex biological sample solutions. Sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis), polymerase chain reaction, DNA hybridization, and electrochemical detection were performed in this fully automated and miniature device. Cavitation microstreaming was implemented to enhance target cell capture from whole blood samples using immunomagnetic beads and accelerate DNA hybridization reaction. Thermally actuated paraffin-based microvalves were developed to regulate flows. Electrochemical pumps and thermopneumatic pumps were integrated on the chip to provide pumping of liquid solutions. The device is completely self-contained: no external pressure sources, fluid storage, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Pathogenic bacteria detection from approximately milliliters of whole blood samples and single-nucleotide polymorphism analysis directly from diluted blood were demonstrated. The device provides a cost-effective solution to direct sample-to-answer genetic analysis and thus has a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

Surface-attached molecular beacons light the way for DNA sequencing

Rapid testing of DNA and RNA nucleotide sequences is required for various research protocols including wide-scale genetic testing, diagnostics, fast detection of biological warfare agents, environmental testing and forensic medicine. At present many laboratories are interested in research and development of an inexpensive, easy-to-use, fast-response device for this purpose. Various methods based on acoustic, electronic and optical detection of the DNA hybridization event have been reported.

Fluorescence-based array biosensors for detection of biohazards.

Total internal reflection fluorescence (TIRF) is a process whereby fluorophores that are either attached to or are in close proximity with the surface of a waveguide are selectively excited via an evanescent wave. Planar waveguides provide the possibility of immobilizing multiple capture biomolecules onto a single surface and therefore, offer the exciting prospect of multi-analyte detection. The production of arrays and the results of various groups which use TIRF to interrogate such surfaces is reviewed, along with a look at how far the field has advanced toward the production of an automated, portable, multi-analyte array biosensor for real-time biohazard detection. In particular, a miniaturized, fully automated, stand-alone array biosensor developed at the Naval Research Laboratory is reported that monitors interactions between binding partners either as the final image or in real-time. A variety of analytes including toxins, bacteria and viruses have been detected both in buffer and complex matrices, such as blood and soil suspensions, with comparable detection limits. A number of developments have led to a TIRF array biosensor weighing only 5.5 kg which is automated for environmental, clinical and food monitoring or for detection of bioterrorist agents.

Nucleic acid approaches for detection and identification of biological warfare and infectious disease agents

Biological warfare agents are the most problematic of the weapons of mass destruction and terror. Both civilian and military sources predict that over the next decade the threat from proliferation of these agents will increase significantly. In this review we summarize the state of the art in detection and identification of biological threat agents based on PCR technology with emphasis on the new technology of microarrays. The advantages and limitations of real-time PCR technology and a review of the literature as it applies to pathogen and virus detection are presented. The paper covers a number of issues related to the challenges facing biological threat agent detection technologies and identifies critical components that must be overcome for the emergence of reliable PCR-based DNA technologies as bioterrorism countermeasures and for environmental applications. The review evaluates various system components developed for an integrated DNA microchip and the potential applications of the next generation of fully automated DNA analyzers with integrated sample preparation and biosensing elements. The article also reviews promising devices and technologies that are near to being, or have been, commercialized.

Immunological methods for detection and identification of infectious disease and biological warfare agents.

BW agents.The release of a biological weapon (BW) agent by a terrorist group or military force would likely be silent and undetectable or nearly so. As shown by anthrax attack during the fall of 2001 in the eastern United States, patients would begin appearing at hospitals and clinics within several

Electrochemical Biosensors for Detection of Biological Warfare Agents

AbstractThis review discusses current development in electrochemical biosensors for detection of biological warfare agents. This could include bacteria, viruses and toxins that are aerosoled deliberately in air, food or water to spread terrorism and cause disease or death to humans, animals or plants. The rapid and unequivocal detection and identification of biological warfare agents is a major challenge for any government including military, health and other government agents. Reliable, specific characterization and identification of the microorganism from sampling location, either air, water, soil or others is required. This review will survey different types of electrochemical biosensors has been developed based on the following: i) Immunosensors ii) PCR (DNA base Sensor) iii) Bacteria or whole cell sensor and iv) Enzyme sensor. This article gives an overview of electrochemical biosensor for detection of biological warfare agents. Electrochemical biosensors have the advantages of sensitivity, selectivity, to operate in turbid media, and amenable to miniaturization. Recent developments in immunofiltration, flow injection, and flow‐through electrochemical biosensors for bacteria, viruses, and toxin detection are reviewed. The current research and development in biosensors for biological warfare agents detection is of interest to the public as well as to the defense is also discussed.

Separation of Simulants of Biological Warfare Agents from Blood by a Miniaturized Dielectrophoresis Device

Separation of simulants of biological warfare agents from blood using dielectrophoresis (DEP) was demonstrated in a miniaturized DEP device. The device was fabricated by laminating five different layers (all 40 mm×40 mm) including a polycarbonate substrate, a pressure sensitive acrylic adhesive (PSA) layer, a patterned polyimide layer with a flip-chip bonded dielectrophoresis chip (DEP chip), a PSA layer with microfluidic channel, and a glass cover plate. The DEP chip consisted of repetitive interdigitated electrodes with characteristic dimension of 50 μm. This device was employed to separate different simulants of biological warfare agents (BWA), namely Bacillus cereus (B. cereus), Escherichia coli (E. coli) and Listeria monocytogenes (L. monocytogenes), from blood, individually or simultaneously. PCR amplification, which was inhibited by blood components in pre-separation samples, successfully revealed bands in post-separation samples containing single or multiple BWA. Up to 97% efficiency of separation was achieved as demonstrated by culturing post-separation E. coli cells. The DEP device described here can potentially be used to reduce sample complexity for detection of infectious disease pathogens and biological warfare agents.

Chemical and biological warfare: a comprehensive survey for the concerned citizen

1: Chemical warfare (CW) history 2: Defenses against chemical weapons 3: Incentives and Disincentives to Acquire Chemical Weapons 4: The Geopolitics of Proliferation 5: Strategies for Control and Disarmament 6: Definition of a biological warfare (BW) agent 7: Brief History of Biological Weapons 8: Motivations for Proliferation 9: Biological warfare (BW) agent detection 10: Biological warfare (BW) and terrorism 11: Biological and Toxin Weapons Convention of 1972

Rapid and sensitive detection of biological warfare agents using time-resolved fluorescence assays

We have achieved sensitive, rapid and reproducible detection of three biological threat agents in a variety of biological and environmental matrices using the DELFIA time-resolved fluorometry (TRF) assay system (Perkin-Elmer Life Sciences, Akron, OH). Existing ELISA assays for the detection of Francisella tularensis, Clostridium botulinum A/B neurotoxin (BotNT A/B), and Staphylococcus aureus enterotoxin B (SEB) were converted to TRF assays. They use 100 μl of positive control or unknown per test well and require just over 2 h to run. Fluorescent signal read time is a fraction of a second per well. The assay format consists of a capture ELISA utilizing a biotinylated capture antibody, prebound to a streptavidin-coated 96-well plate and a lanthanide (Europium, Eu 3+)-labeled detector antibody. The bound Eu-labeled detector antibody produces a fluorescent signal upon the addition of an enhancement solution. The signal results from the dissociation of the Europium from the antibody, creating a micelle, thus amplifying the signal nearly one million-fold. Sensitivities achieved by these assays were between 4 and 20 pg/ml in buffer. Additionally, we have tested this system in different matrices such as serum, urine, dirt, and sewage. Concentration curves generated from standard solutions produced a wide linear range making serial dilutions of unknown samples unnecessary. DELFIA TRF assays are significantly better in terms of sensitivity, linear range, and run time than standard capture ELISAs and should facilitate early detection of potential biological warfare agents in clinical and environmental samples.

Stochastic sensors inspired by biology.

Sensory systems use a variety of membrane-bound receptors, including responsive ion channels, to discriminate between a multitude of stimuli. Here we describe how engineered membrane pores can be used to make rapid and sensitive biosensors with potential applications that range from the detection of biological warfare agents to pharmaceutical screening. Notably, use of the engineered pores in stochastic sensing, a single-molecule detection technology, reveals the identity of an analyte as well as its concentration.

The application of the bioelectric recognition assay for the detection of human and plant viruses: definition of operational parameters

The bioelectric recognition assay (BERA) is a novel biosensory method based on a unique combination of a group of cells, their immobilization in a matrix that preserves their physiological functions and the expression of the cell interaction with viruses as a change in electrical properties. A BERA sensor consists of an electroconductive, tube-like probe containing components of immobilized cells in a gel matrix. Cells are selected to specifically interact with the virus under detection. In this way, when a positive sample is added to the probe, a characteristic, ‘signature-like’ change in electrical potential occurs upon contact between the virus and the gel matrix. In the present study, we demonstrate that BERA can be used for the detection of viruses in humans (hepatitis C virus) and plants (tobacco and cucumber viruses) in a remarkably specific, rapid (1–2 min), reproducible and cost-efficient fashion. The sensitivity of the virus detection with BERA (0.1 ng) is equal or even better than with advanced immunological, cytological and molecular techniques, such as the reverse transcription polymerase chain reaction. Moreover, a good storability of the sensors can be achieved without affecting their performance. The potential use of portable BERA biosensors in medicine, for mass screening purposes, as well as for the detection of biological warfare agents without prior knowledge of a specific receptor-molecule interaction is discussed.