9
Function-Based Detection

For the purposes of this report, a function-based detector is defined as a naturally occurring biological organism or portion of that organism (whether organ, tissue, cell, or receptor) that reacts in a measurable way when exposed to a range of chemical or biological toxic material.

Historically, many sentinel animals have been used as detectors, indicators, and alarms for the presence of toxic agents. The use of caged canaries by miners to detect methane in coal mines is well known. The canary is highly sensitive to methane, an odorless gas that is found in mines. If the caged canary died it provided a visible functional alarm, warning the miners that they should leave the area. Similarly, Japanese police used caged canaries to detect sarin during raids on the Aum Shin Rikyo enclaves. Chickens are used as sentinels on the Canadian border to detect encephalitis virus.

There has been widespread use of biological systems in environmental quality testing to provide an integrated picture of overall toxicity of an effluent or a sample of water, sediment, or soil from a contaminated site.1 Fathead minnows, various aquatic invertebrates, earthworms, protozoans, and seeds are all used for bioassays of aquatic samples.2 Daphnia (small freshwater crustaceans) are also used to provide an indication of water quality. They are sensitive to changes in water chemistry and their response to toxicants can be readily monitored.3 The Center for Environmental Research at Fort Detrick uses bluegill fish as sensing systems. During Operation Freedom in Iraq in the spring of 2003, soldiers were reported to carry chickens in cages on the hoods of their vehicles to serve as chemical weapon sentries. The many unknowns surrounding Gulf War Syndrome have prompted soldiers to act independently in order to protect themselves from compounds that may not be detected by existing equipment. The ad hoc employment of an uncharacterized function-based detection system in the front lines indicates the need for more research in this area.

Function-based detection systems are similar to the structure-based biosensors discussed in Chapter 7 in that they do not measure the concentration of biological agent directly but rely on some form of transduction device to measure response. However, the two methods differ greatly in their specificity. Structure-based identifiers (examples include nucleic acid primers and antibodies) are highly specific but

1  

F. Botre, E. Podesta, B. Silvestrini, and C. Botre. 2001. Toxicity testing in environmental monitoring: The role of enzymatic biosensors. Ann. Ist. Super. Sanita. 37:607-613.

2  

C.J. Keddy, J.C. Green, and M.A. Bonnell. 1995. Review of the whole organism bioassays: Soil freshwater sediment and freshwater assessment in Canada. Ecotoxicity and Environmental Safety 30:221-251.

3  

U.S. Environmental Protection Agency. 1991. Methods for Measuring the Acute Toxicity of Effluents to Aquatic Organisms. Cincinnati, Ohio: Environmental Protection Agency.



The National Academies | 500 Fifth St. N.W. | Washington, D.C. 20001
Copyright © National Academy of Sciences. All rights reserved.
Terms of Use and Privacy Statement



Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.

OCR for page 149
Sensor Systems for Biological Agent Attacks: Protecting Buildings and Military Bases 9 Function-Based Detection For the purposes of this report, a function-based detector is defined as a naturally occurring biological organism or portion of that organism (whether organ, tissue, cell, or receptor) that reacts in a measurable way when exposed to a range of chemical or biological toxic material. Historically, many sentinel animals have been used as detectors, indicators, and alarms for the presence of toxic agents. The use of caged canaries by miners to detect methane in coal mines is well known. The canary is highly sensitive to methane, an odorless gas that is found in mines. If the caged canary died it provided a visible functional alarm, warning the miners that they should leave the area. Similarly, Japanese police used caged canaries to detect sarin during raids on the Aum Shin Rikyo enclaves. Chickens are used as sentinels on the Canadian border to detect encephalitis virus. There has been widespread use of biological systems in environmental quality testing to provide an integrated picture of overall toxicity of an effluent or a sample of water, sediment, or soil from a contaminated site.1 Fathead minnows, various aquatic invertebrates, earthworms, protozoans, and seeds are all used for bioassays of aquatic samples.2 Daphnia (small freshwater crustaceans) are also used to provide an indication of water quality. They are sensitive to changes in water chemistry and their response to toxicants can be readily monitored.3 The Center for Environmental Research at Fort Detrick uses bluegill fish as sensing systems. During Operation Freedom in Iraq in the spring of 2003, soldiers were reported to carry chickens in cages on the hoods of their vehicles to serve as chemical weapon sentries. The many unknowns surrounding Gulf War Syndrome have prompted soldiers to act independently in order to protect themselves from compounds that may not be detected by existing equipment. The ad hoc employment of an uncharacterized function-based detection system in the front lines indicates the need for more research in this area. Function-based detection systems are similar to the structure-based biosensors discussed in Chapter 7 in that they do not measure the concentration of biological agent directly but rely on some form of transduction device to measure response. However, the two methods differ greatly in their specificity. Structure-based identifiers (examples include nucleic acid primers and antibodies) are highly specific but 1   F. Botre, E. Podesta, B. Silvestrini, and C. Botre. 2001. Toxicity testing in environmental monitoring: The role of enzymatic biosensors. Ann. Ist. Super. Sanita. 37:607-613. 2   C.J. Keddy, J.C. Green, and M.A. Bonnell. 1995. Review of the whole organism bioassays: Soil freshwater sediment and freshwater assessment in Canada. Ecotoxicity and Environmental Safety 30:221-251. 3   U.S. Environmental Protection Agency. 1991. Methods for Measuring the Acute Toxicity of Effluents to Aquatic Organisms. Cincinnati, Ohio: Environmental Protection Agency.

OCR for page 149
Sensor Systems for Biological Agent Attacks: Protecting Buildings and Military Bases are only able to recognize certain structural elements that have previously been characterized and prepared for. Function-based detectors, on the other hand, can react to previously uncharacterized structures that affect the monitored biological functions. Thus, function-based detectors are less specific or selective but have the potential for detecting the presence of unknown chemical and biological agents. Initially, research in this area focused on chemical toxicants; it is only recently that function-based techniques have been applied to the detection of biological warfare agents. Currently, there are a wide variety of biosensing systems that could fall under the banner of functional. These include enzyme systems, ion channel/receptors, cells, and whole organisms.4 Table 9.1 provides a basic comparison of various recognition elements and the level of information they can provide. Antibodies and synthetic ligands can be generated to recognize a specific epitope (structural element). These recognition elements can be highly specific or common to various target organisms. For example, a given monoclonal antibody may identify a specific Bacillus species, while another may recognize all Bacillus species. Similarly, nucleic acid-based identification sequences can be specific to the strain level, or they can be directed to conserved regions of particular genomes. Enzymes and cellular chemistries are modulated by compounds that affect their active site and thus modulate their function. Since modulation of most enzyme activities is accomplished by the natural substrate as well as structural analogs, the information generated is rather generic; e.g., inhibition of cholinesterase activity by various nerve agents. Similar restrictions of specific identification apply to receptors, cells, and whole organisms. These systems are valuable for providing functional information regarding potentially harmful material in the environment. While the ability to identify harmful material for which no specific assay exists is important, one must also consider the fact that the response of an isolated cell may or may not model the response of humans to the same challenge. Differential responses among multiple function-based sensing elements (e.g., different cell types) may provide fingerprints of various classes of compounds in much the same way that surface acoustic wave devices identify chemicals (see Chapter 8). There is a very fine line dividing structure-based and function-based detection systems. In the following examples, hybrid systems exploit part of the functional process within a cell-based response system, even as they are targeted at a specific characterized function. The canary-on-a-chip concept,5 which uses genetically engineered B cells or B lymphocytes with the appropriate antibody incorporated into the genome and displayed on the cell surface, is an example of a biosensing system that exhibits characteristics of a functional detector but is reliant on having a specific TABLE 9.1 Type of Information Provided by Various Recognition Systems Recognition Element Functional Information Specific Identificationa Generic Detection and Classification Antibodies   Nucleic acids   Synthetic ligands   Enzymes/cellular chemistry   Ion channel/receptors   Cell   Whole organisms   a Relies on a specific binding event (e.g., gene probe primer sets for a given organism or antibody antigen binding). 4   J. Pancrazio, Naval Research Laboratory. Presentation to the committee on September 26, 2002. 5   J.D. Harper, MIT Lincoln Laboratory. Discussions with the committee on June 13, 2002.

OCR for page 149
Sensor Systems for Biological Agent Attacks: Protecting Buildings and Military Bases antibody for the recognition event. Similarly, an ion channel system6 is based on an approach that consists of ion channels embedded in lipids that have antibodies bound to the lipid layers. Again, this approach is dependent on having a specific targeting antibody. Both of these types are discussed in detail in Chapter 7. A well-known example of an enzyme inhibition system is the U.K.'s NAIAD, a nerve agent detector used for a military application.7 The bioreceptor in this case is the enzyme butyrylcholinesterase, which exhibits the same enzyme activity as human acetylcholinesterase. The enzyme is immobilized onto a temperature-controlled pad that is continually washed with butyrylthiocholine methane sulfonate in an aqueous phosphate buffer. The enzyme catalyses the hydrolysis of the ester, producing butrylthiocholine, and the concentration is monitored by an electrochemical cell arrangement (this is the transduction system). If nerve agent is present, it inhibits the butyrylcholinesterase, which in turn causes an alteration in the electrical potential within the electrochemical cell, thus triggering an alarm at a preset level. This type of detector is extremely sensitive to low levels of nerve agents such as tabun and sarin. Enzymes have been used in assay systems for some time. A good example is the bioluminescence assay using the enzyme luciferase (present in fireflies). This assay is used widely to measure bacterial contamination in a number of sectors—e.g., food industry, defense, and health care. It is based on the level of adenosine triphosphate (ATP) present within a bacterial cell; however, the amount of ATP present varies depending on the bacterial species and the cell state. An example of an enzyme amplification system that improves sensitivity levels is the use of adenylate kinase (AK), an enzyme present in the cell that converts adenosine diphosphate (ADP) to ATP. By exploiting this reaction, the bioluminescence assay sensitivity can theoretically be increased 40,000-fold. A bioluminescence assay method has been developed8 in which the AK is extracted from the bacterial cell and ADP is added in excess. The ADP is converted to ATP and then the conventional ATP bioluminescent assay is carried out. This method significantly increases the detection sensitivity by orders of magnitude. CELL-BASED RESPONSE SYSTEMS A functional detection system relies on the cell to respond in a measurable way to a toxic agent. For example, fish are sensitive indicators of their environment. Fish kills are frequent indicators of toxins as well as low oxygen levels in their environments.9 The U.S. military is using whole fish as sensing agents for environmental monitoring. The whole organism can be monitored remotely to look for changes in behavior that are indicators of changes in the environment. Isolated fish chromatophore cells can also be used as a detection system.10 This approach exploits the ability of living chromatophores to respond to many active substances such as bacterial toxins; the response of the chromatophore is measured as changes in the appearance of the cell due to intracellular activity of the colorants.11 Tissue biosensors made from immobilized whole-cell photosynthetic microorganisms have been developed for airborne chemical warfare agents and simulants. This is based on fluorescence induction by living photosynthetic tissue. Photosynthetic prokaryotes and eukaryotes fluoresce when illuminated by light following a period of darkness. Structural differences between the two types of organisms affect the fluorescence signal produced, and their sensitivity to perturbing factors such as toxicants alters the 6   B.A. Cornell, V.L.B. Braach-Maksvytis, L.G. King, P.D.J. Osman, B. Raguse, L. Wieczoreck, and R.J. Pace. 1997. A biosensor that uses ion-channel switches. Nature 387:580-583. 7   R.J. Powell. 1988. Detectors in battle. Chemistry in Britain July:665-669. 8   D.J. Squirrell and M.J. Murphy. 1994. Adenylate kinase as a cell marker in bioluminescence assays. Bioluminescence and Chemiluminescence—Fundamentals and Applied Aspects. A.K. Cambell, L.J. Kricka, and P.E. Stanley, eds. Chichester: John Wiley, pp. 486-489. 9   U.S. Environmental Protection Agency. 1991. Methods for Measuring Acute Toxicity of Effluents of Receiving Water to Freshwater and Marine Organisms. Washington, D.C.: Environmental Protection Agency. 10   F.W. Chaplen, R.H. Upson, P.N. McFadden, and W. Kolodziej. 2002. Fish chromatophores as cytosensors in a microscale device: Detection of environmental toxins and bacterial pathogens. Pigment Cell Res. 15:19-26. 11   R.R. Preston and P.N. McFadden. 2001. A two-cell biosensor that couples neuronal cells to optically monitored fish chromatophores. Biosens. Bioelectron. 16:447-455.

OCR for page 149
Sensor Systems for Biological Agent Attacks: Protecting Buildings and Military Bases characteristic fluorescence pattern of healthy photosynthetic tissue. Whole cell microorganisms such as Chlorella vulgaris, a unicellular green alga, and Nostoc commune, a cyanobacterium, have been used to detect nerve agents. Exposure to toxicants causes changes in their characteristic fluorescence induction curves with resultant changes in photochemical yields.12 Neuronal tissue is the target of many toxins—specifically, nerve agents—and represents an information-rich source of material for the development of sensing systems. Cultured neuronal networks based on murine spinal cord, frontal cortex, and auditory cortex tissues have been cultured on microelectrode arrays.13 The system contains all the metabolic and electrophysiological mechanisms of the parent tissue. The response of the cell can be characterized by recording the extracellular action potentials.14 This type of system has been shown to detect a wide variety of neuroactive compounds. Laboratory and prototype portable systems have been developed for conducting extracellular recordings of electrically active cells.15 These systems have the potential to provide some discrimination whereby different cells produce differential response patterns such that an array of different cell types may characterize the toxin to a particular class. A gene-based optical activity detection system is being developed that measures gene expression and cellular activity of the lung cells with an optical lattice/scaffold arrangement. Lung cells are grown on optical fibers, and spectroscopic signatures of the cells are then monitored.16 The objective of this approach is to mimic the lung's in vivo environment by the utilization of a scaffolding technique so that the responses to toxicants reflect actual events within the organism producing a visual signal. Conducting in vitro assays as a predictor of in vivo effects is always subject to challenge. Cells and tissues outside their natural environment can behave quite differently than if they remained in the host. In an attempt to preclude this potential for artifact, investigators are seeking noninvasive, whole organism measurements that may provide information relative to the health of the organism. Continuous noninvasive monitoring of blood chemistry and other metabolic diagnostic indicators are being explored to monitor changes due to toxicant effect on the metabolic processes.17 Current emphasis is on monitoring the spectral changes in the blood chemistry in veins near the surface of the skin. A remote system for monitoring an individual's health state has been claimed; if this claim is correct, it has the potential to measure blood chemistry and other vital functions noninvasively and to communicate this information via telemetry.18 12   C.A. Sanders, M. Rodriguez, Jr., and E. Greenbaum. 2001. Stand-off tissue-based biosensors for the detection of chemical warfare agents using photosynthetic fluorescence induction. Biosens. Bioelectron. 16:439-446. M. Rodriguez, C. Sanders, and E. Greenbaum. 2001. Biosensors for rapid monitoring of primary-source drinking water using naturally occurring photosynthesis. Biosens. Bioelectron. 17:843. 13   S.I. Morefield, E.W. Keefer, K.D. Chapman, and G.W. Gross. 2000. Drug evaluations using neuronal networks cultured on microelectrode arrays. Biosens. Bioelectron. 15:383-396. A.M. Aravanis, B.D. DeBusschere, A.J. Chruscinski, K.H. Gilchrist, B.K. Kobilka, and G.T. Kovacs. 2001. A genetically engineered cell-based biosensor for functional classification of agents. Biosens. Bioelectron. 16:571-577. J. Pancrazio, P.P. Bey, Jr., D.S. Cuttino, J.K. Kusel, D.A. Borkholder, K.M. Shaffer, G.T.A. Kovacs, and D.A. Stenger. 1998. Portable cell-based biosensor system for toxin detection. Sensors and Actuators B 53:179-185. 14   D.A. Stenger, G.W. Gross, E.W. Keefer, K.M. Shaffer, J.D. Andreadis, W. Ma, and J.J. Pancrazio. 2001. Detection of physiologically active compounds using cell-based biosensors. Trends in Biotechnology 19:304-309. E.W. Keefer, A. Gramowski, and G.W. Gross. 2001. NMDA receptor-dependent periodic oscillations in cultured spinal cord networks. Journal of Neurophysiology 86:3030-3042. B.D. DeBusschere and G.T. Kovacs. 2001. Portable cell-based biosensor system using integrated CMOS cell-cartidges. Biosens. Bioelectron. 16:543-556. 15   DeBusschere and Kovacs, 2001. See note 14 above. 16   A. Rudolph, DARPA Defense Sciences Office. Presentation to the committee on September 26, 2002. 17   R. Coifman, I.S. Dalbosco, E.M. Russo, and R.S. Moises. 1999. Specific insulin and proinsulin in normal glucose tolerant first-degree NIDDM patients. Braz. J. Med. Biol. Res. 32:67-72. F. Torella, R. Cowley, M.S. Thorniley, and C.N. McCollum. 2002. Monitoring blood loss with near infrared spectroscopy. Com. Biochem. Physiol. A Mol. Integr. Physiol. 132:199-203. 18   P.D.E. Biggins, C.S. Cox, and K.L. Martin. 2001. Non invasive remote patient monitoring patent. Dstl Report GB01/02450. Wiltshire, U.K.: Defense Science and Technology Laboratory.

OCR for page 149
Sensor Systems for Biological Agent Attacks: Protecting Buildings and Military Bases RESEARCH ISSUES For most biological agents of interest, functional systems do not currently have the speed required to meet the detect-to-warn goal of a 1-minute response. Rather, their system response times are on the order of minutes to hours. The reason for this temporal delay is often found in the mechanism of action of the particular toxic agent. While measurable responses in the laboratory can be demonstrated for the various systems, they have yet to be tested under military field conditions. For cell-based assays in their current format, it is likely that existing environmental pollutants in the atmosphere will poison them, and that naturally occurring nonpathogenic microorganisms will break down the sensor cells over time. Similarly, the selectivity, specificity, and sensitivity of the systems have yet to be fully characterized, so that they are not prone to false alarms. These systems, while promising, are still in the prototype stage and hence require skilled specialists to operate them and interpret the data. Interfacing with Sampling Systems As already mentioned, cell-based systems are currently in the laboratory and prototype phases, and the issue of how to sample the air and present the collected sample to the cell needs considerable attention. The preparation of the sample for exposure to the cell-based response system will add to the overall system response time. Water sampling systems, though complex, are less challenging than air sampling. DARPA is currently funding an effort to generate a standardized system for water testing that will monitor and/or adjust critical parameters such as pH, temperature, and osmolality. Operational Deployment Operational deployment of function-based sensing systems provides some unique challenges. Any system that utilizes living material will have to have an accompanying life support system, unless methods can be developed for lyophilization of the cells and rapid rehydration before use. The complexity of this system will be determined by the element(s) requiring support. A further challenge arises from the fact that cell-based detection systems may require a sterile environment, depending on the cell type(s) incorporated, if operated in a continuous, reusable mode. However, sterilization of the sample before it is introduced to the cells may in some cases destroy the biological effects one is trying to detect. While this would not affect the ability of function-based systems to detect toxins (unless the toxins are significantly denatured by the sterilization process), it may limit the types of intact organisms that can be detected without additional sample preparation.19 Conclusion In summary, with advances in the understanding of cellular processes and how they are inhibited, it should be possible to develop artificial sentinel systems that can detect exposure to a wide range of unknown toxic materials. Currently it is not clear what role these systems may play in a detect-to-warn sensor architecture. Response times so far reported vary from minutes to hours. Detection sensitivities for the systems have yet to be determined, and the targets that are being disrupted in the cellular chemistry need to be elucidated. FINDINGS AND RECOMMENDATIONS Finding 9-1: Functional detection systems currently do not have the sensitivities or response times needed for detect-to-warn applications; further, they have not been demonstrated against most of the biological agents. However, they offer considerable potential for filling the gap that is not currently being addressed by specific identifier systems—that is, they could provide a generic detection capability that 19   The severity of this limitation is not clear. For example, sentinel cells can have a strong reaction to bacterial cell walls, even though the cells themselves have been fragmented by sterilization.

OCR for page 149
Sensor Systems for Biological Agent Attacks: Protecting Buildings and Military Bases can indicate the presence of unknown toxic agents. Recommendation 9-1: Research should be conducted to explore how far functional detection systems can progress in satisfying detect-to-warn requirements for low response time, high sensitivity, low false alarm rate, ease of sample preparation, and acceptable logistics for deployment. The research should identify systems that offer a broad spectrum of sensitivities as well as the potential for providing classification of toxic materials. The research should address issues such as sensitivity, false alarm rate, sample preparation, and logistical requirements for deployment. Finding 9-2: Because of the requirement for sterility when using biological materials as detection elements, function-based detectors that utilize cells and operate continuously may have limitations if intact threat organisms are required for detection. Recommendation 9-2: Research is needed to explore the limits of cell-based detection systems, including requirements for sample collection and sterilization as well as methodologies for extending the functional life of the sensing elements, both in operation and storage of reagents (cells) for future use. Development of cell lyophilization and rapid rehydration technologies is also needed. Finding 9-3: The use of sentinel animals has been, and continues to be, an effective method of detection of the introduction of harmful material into a population. The function-based detection system is an extension of this approach that may provide increased sensitivity and decreased detection time. Recommendation 9-3: Research should be conducted to develop more sophisticated, noninvasive methods (e.g., spectroscopic analysis of blood chemistry close to the skin surface) for detecting rapid biological changes in sentinel animals that result from exposure to a toxic agent.