- Email: [email protected]
Chapter 20b Bioactivity detectors
Chapter 20b
Bioactivity detectors Sue M. Ford
20b.1 20b.1.1
BACKGROUND Needs for bioactivity detection
Routine monitoring of food and water for the presence of pathogens, toxins, and spoilage-causing microbes is a major concern for public health departments and the food industry. In the case of diseasecausing contamination, identification of the organism is critical to trace its source. The Environmental Protection Agency monitors drinking water, ambient water, and wastewater for the presence of organisms such as non-pathogenic coliform bacteria, which are indicators of pollution. Identification of a specific organism can aid in locating the source of the pollution by determining whether the organism is from humans, livestock, or wildlife [1]. Pharmaceutical companies monitor waterfor-injection for bacterial toxins called pyrogens, which are not removed by filter-sterilization methods. There is a need for methods to quickly detect biothreat agents that may be dispersed as bacteria, spores, viruses, rickettsiae, or toxins such as the botulism toxin or ricin [2,3]. 20b.2 20b.2.1
METHODS Traditional bioactivity methods
The FDA, USDA, and EPA have established methods and standards for detecting food- and water-borne pathogens; these methods are available on the agencies’ websites [4–8]. Many of the procedures for microbe identification rely on culturing the sample for subsequent identification by multiple tests such as colony characteristics, selective growth conditions, and biochemical assays for metabolites. Such traditional methods take upward of 24 h and remain the standard against which new Comprehensive Analytical Chemistry 47 S. Ahuja and N. Jespersen (Eds) Volume 47 ISSN: 0166-526X DOI: 10.1016/S0166-526X(06)47030-6 r 2006 Elsevier B.V. All rights reserved.
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tests are evaluated. Recently developed procedures for microbial detection include biochemical test kits, genetic and molecular biology techniques, and miniaturized instruments. New assays for bacteria are very sensitive, requiring fewer organisms for detection. Although an incubation period is still generally needed to expand the population above detection limits, these more sensitive methods shorten the incubation period. Testing for toxins such as the botulism toxin relied on observing the effects of a test compound injected in mice or the use of colorimetric [9] and immunoassays. 20b.2.2
Methods and instruments based on molecular recognition
Immunology-based methods have been the most important technology for detection and identification of microbes, viruses, and toxins for many years [10]. Traditional immunoassays rely on antibodies produced in immunized animals or in hybridoma cultures. The interaction of the antigen of interest (pathogen, toxin) with the antibody is combined with a detection protocol which may involve labeling the bound antigen/antibody complex with a reporter molecule that allows detection. The methods include latex agglutination, immunodiffusion, enzyme-linked immunoassays, immunoprecipitation, and immunochromatography, which are rapid, sensitive, and specific [5,11]. The reagents can be incorporated into a disposable device similar to a home pregnancy test kit so that the presence or absence of the test organism is indicated by a color change or development of a line. The devices have quality control mechanisms incorporated to increase reliability; nonetheless, some products have been reported to have high false-positive response rates [12]. The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13–15]. When the tag is 778
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electrically stimulated, it undergoes oxidation–reduction cycling with tripropylamine in the mixture. The excitation of the tag results in the emission of light. ECL has been used for detection of Staphylococcus enterotoxin B, ricin, Yersinia pestis antigen, anthrax PA antigen, and equine encephalitis virus [11]. ECL is particularly useful for detection of toxins that are not conveniently detected by molecular biology-based assays. BioVeris (Gaithersburg, MD) BV M-SERIESs instruments use ECL technology. A developing alternative to antibody-based molecular recognition is phage-display technology. Filamentous phages are thread-shaped viruses that attack bacteria [10,16]. Phage-display libraries are mixtures of phages containing foreign peptides encoded into their surface coat proteins. The number of phages containing different peptides in a population can exceed 106, providing a vast selection of possible antigenbinding domains. From such populations, phages can be selected for affinity for a particular antigen. The selected phage is then replicated in host bacteria and harvested. An advantage of phage display over traditional immunological methods is that these phage ‘‘antibodies’’ can be prepared against compounds such as botulism toxin or organisms that would kill the animals that would be used to produce antibodies, and for molecules with low antigenicity. A problem with phage display is the potential for false positives due to unpredictable non-specific interactions in a complex mixture [10]. 20b.2.3
Methods and instruments based on nucleotide analysis
Many modern instruments for bioactivity detection are based on analysis of nucleic acids subsequent to multiplication of the amount of unknown DNA of interest by the polymerase chain reaction (PCR). The basic process of PCR involves adding the unknown DNA to a reaction mixture which contains (1) nucleotides for DNA strand synthesis, (2) the heat-stable Taq DNA polymerase to catalyze the process, and (3) nucleotide primers chosen to initiate polymerase activity along specific regions of target DNA. In the instrument, the temperature is increased to 901C and the test DNA denatures into two single strands. After denaturation of DNA, the reaction is cooled to 551C to allow the primer to bind (anneal) to the corresponding region on the strands of test DNA and then the temperature is raised to 751C, the optimum for polymerase activity. At the end of the elongation reaction, the amount of target DNA has doubled. This process is repeated numerous times in the thermocycler, increasing the DNA exponentially. The resulting 779
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DNA is subsequently analyzed by various characterization techniques, such as molecular weight determination, electrophoresis, and hybridization to known probes. Using these methods, the presence of DNA unique to a particular species can be confirmed. Alternatively, with appropriate primers, a mixture of DNA fragments, which are uniquely characteristic of an individual or organism, will result in a fingerprint that can be used for identification. The conventional steps for PCR may take hours to days to obtain results. Numerous improvements and variations in the process have enhanced the sensitivity and speed of PCR for detection and identification of bacteria, spores, and viruses. The amplicons or the oligonucleotide probes can be tagged with fluorescent dyes so that the reaction can be followed as it progresses, a process referred to as real-time PCR (RT-PCR). The amplification process and detection are carried out in the same closed vessel [17]. This eliminates the need to use electrophoresis, for example, after PCR to determine if a sample is positive for an amplicon of interest [11] and also prevents contamination by nucleic acids during sample handling in the laboratory. Instrumentation for temperature cycling has been improved such that 30 cycles can be accomplished in 30 min, a process referred to as rapid-cycle, real-time PCR [17,18]. The use of reporter tags, such as fluorescent dyes that bind to double-stranded DNA or sequence-specific probes can be added to the reaction to allow quantitation of amplicons at the end of each cycle. Fluorescence resonance energy transfer (FRET) RT-PCR involves the transfer of energy between two dyes when they are in proximity to one another. An excited reporter fluorophore excites a nearby quencher fluorophore, which then emits a photon. This phenomenon is used to construct PCR probes with reporter and quencher components. FRET RT-PCR is a powerful technique that allows several strains of smallpox viruses, for example, to be detected simultaneously [19]. The temperature at which 50% of the DNA double strands separate is the melting point (Tm) [10]. Melting point profiles can also be used to identify DNA inasmuch as a single-base substitution can change the melting curve for DNA. The LightCyclerTM by Roche Applied Science (Indianapolis, IN) is a temperature-controlled microvolume fluorimeter with RT-PCR module and software to facilitate quantitation of DNA, as well as identification and genotyping by fluorescence detection or melting profiles analysis. RT-PCR assays are very sensitive, with limits of detection for smallpox virus being around 12 gene copies [20]. PCR is now the front-end process for other DNA profiling techniques such as pulse-field gel electrophoresis (PFGE), terminal restriction 780
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fragment length polymorphism (T-RFLP), dentaturing-gradient gel electrophoresis (DGGE), and others [1]. 20b.2.4 Methods and instruments based on chemical and physical analysis
Even the simplest bacterium is composed of innumerable lipids, proteins, and polysaccharides; however, few of these molecules are specific to a particular species. Similarly, the fluorescence of small metabolites such as ATP and nucleic acids may confirm the presence of biological organisms in aerosols or water [12], but cannot identify the agent nor indicate its virulence. A major exception is dipicolinic acid (DPA), which is found in the spores of bacteria, particularly the anthrax organism, Bacillus anthracis. Although non-pathogenic bacteria form endospores as well, DPA is characteristic enough to be a marker for anthrax spores. Coherent anti-Stokes Raman scattering (CARS) of DPA appears to be promising in detecting anthrax spore clumps less than 6 mm in diameter [21], which is in the range of bioweapons grade anthrax [22]. Protein toxins such as botulism, staphylococcal enterotoxin B, or ricin can be separated with gas or liquid chromatography, electrophoresis, or a combination. The mChemLabTM (Sandia National Laboratories Albuquerque, NM) series of instruments includes a hand-held Bio Detector. Proteins in the sample are labeled with fluorescent tags, and nanoliter volumes of samples are separated by microchannels etched into a glass chip. The separation occurs as the sample moves through the channels and identification is based on retention times. The analyses can be completed within 10 min. 20b.2.4.1 Fingerprint methods Analysis of individual microbial compounds is generally of limited use for distinguishing pathogens from benign organisms because of the lack of specificity. Instead, a new approach to identifying bacteria through chemical analysis is evolving. Chemotaxonomy refers to the process by which a whole organism is deconstructed into a chemical signature or fingerprint. This concept shows promise for rapid detection of microbes of interest. For example, various species of bacteria have distinct profiles of lipid composition, which may be used for identification [23], although this method may have limitations if the spectra are altered by the nutrients available during microbial growth. Proteins and other large biomolecules can be analyzed using MALDI (matrix-assisted laser desorption and ionization) with TOF 781
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(time-of-flight) mass spectrometry (see Chapter 11). The sample is mixed with a matrix of organic molecules, which absorbs energy from a laser and transfers it to the protein molecules, forming ions typically with a single positive charge. Whole, intact cells can be mixed with such a matrix, dried, and analyzed directly to produce a spectral fingerprint that can distinguish among various bacterial and viral species. However, for MALDI, as for all other fingerprint methods, the ability to identify pathogens mixed with non-pathogens requires further research into mathematical procedures to interpret the results (NRC [24]). Pyrolysis has been successfully used to prepare samples for lipid profiling by mass spectrometry [23]. Rather than a separate step in sample preparation, the methylation of fatty acids is done during the pyrolysis, a process referred to as in situ thermal hydrolysis methylation (THM). This reduces the sample preparation time to 30 s and the total analysis with MS is less than 10 min. The authors developed a portable ion-trap MS using this procedure which did not require pressurized gas; however, it required a large number of bacteria (107–108 cells) in order to distinguish among the four strains studied. Py-GC/MS has low resolving power and is susceptible to false positives; however, the portability and low cost make it attractive as a screening device [25]. Ion mobility spectroscopy (IMS) can also produce fingerprints to identify bacterial strains within 1 min [26]. Plasmagrams from microgram quantities of bacteria are sufficiently unique to distinguish among 200 strains or species of bacteria. The technique can be enhanced by stepped increases in the desorber temperature during desorption. Such programed temperature ramping improves peak detection and adds components to the fingerprints. This increases the complexity of the output, permitting greater discrimination among species. Pyrolysis can also be used with instruments combining short capillary GC columns with IMS for detection of bacterial biomarkers in suspect aerosols, data can be obtained within 4 min with a hand-held instrument [25]; interestingly, 3 min of that time is for collection of the aerosol. Another type of sensor relies on semiselective polymer films, which are fabricated to interact with multiple chemicals. When a chemical is absorbed onto a polymer, which contains carbon black for electronic conduction, a change in size of the polymer—and therefore change in electrical conductivity—occurs, initiating a signal to the transducer. The magnitude of the signal varies depending on the chemical and the polymer. Greater discrimination power can be achieved by using multiple sensors with varying chemical sensitivities, an arrangement called a sensor array. Individual chemicals interact differently with each of 782
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the polymer surfaces in the array, creating fingerprint signals. For example, the Cyranose 320 unit, which evolved from NASA’s E-Nose, has 32 sensors. The unit is able to identify certain classes of bacteria with 96–98% accuracy using combined statistical algorithms; however, the difficulty of this analysis [27] may preclude its use for identification. Measuring volatile metabolites with such sensors requires a highenough concentration of bacteria to produce sufficient material for detection. Although the method is rapid, it is not sensitive enough to detect small numbers of bacteria for early detection of a biothreat. Microcalorimetric spectroscopy-based detection uses vibrational photothermal spectra to identify organic molecules. It can be applied to the detection and identification of microorganisms. The specificity of the method can be enhanced by using a chemically selective layer to enhance trapping of the organisms on the thermal detector. This method can detect amounts of bacteria in an order of magnitude smaller than conventional IR and FTIR [28]. 20b.3 20b.3.1
BIOWEAPONS Characteristics of bioweapons
Particles containing bacterial and spore clumps to be used as weapons must fall within a certain size range. They must be small enough to remain suspended in the air for maximum distribution, yet large enough to be retained in the pulmonary tract; thus the sizes of such particles generally fall in the range of 0.5–20 mm [12]. Thus the size and uniformity of particulate sizes in a cloud can indicate the presence of bioweapons. The analytical problems in developing bioweapons detectors are substantially different from monitoring food and water for inadvertent contamination. First, time is often an issue as the need is to treat or warn individuals, who have been exposed to microbes that have been chosen for their ability to cause disease or death in as many people as possible. Second, the quantities of biological agent to be detected are very small. Biological agents are more potent on a mass basis than chemical agents because microbes and viruses multiply within the host. Third, aerosols are diluted as the cloud drifts [3]. In order to concentrate the agent, samplers need to draw in large amounts of air. The samples therefore contain dust and other solid materials that interfere with particulate analyses. Fourth, there are many types of biological agents that might be used as weapons (see charts in reference [3] for an extensive listing), which are diverse in physical and chemical nature 783
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and not amenable to a common analytical procedure. Fifth, there is a naturally occurring background of non-pathogenic microorganisms and chemicals. Consequently, the detection methods need to distinguish microbes from the non-biological particulates as well as to distinguish pathogenic microorganisms from benign ones in a mixture. Methods that rely on quantitation of a macromolecule such as DNA would be able to recognize biological agents in an aerosol or dust, but would not be able to determine whether the agent is a pathogen or benign microbe. Such a distinction depends on identification of the microbe, which in turn requires DNA sequencing.
20b.3.2
Detect-to-warn, detect-to-treat
Rapid identification of a specific bioagent is critical for assessing the level of threat. Ideally, detectors for pathogens and toxins would produce results that are rapid, specific for individual organisms, and sensitive; however, current technical limitations require compromises, which depend on the use of the instrument. Detectors for bioweapons may be divided into several categories based on urgency of results: detect-to-warn (DTW), detect-to-treat, and detection to monitor decontamination. The purpose of DTW is, upon a positive result, to immediately set in motion actions to protect personnel and limit the spread of the agent. Thus, time is the critical feature. The goal for technology is to develop DTW systems that provide reliable alerts within 1 min [24], i.e., sample collection, preparation, identification, and data reporting should be completed within that interval. The other phases of biological agent warning systems have less urgency but have increased need for specificity and sensitivity. Depending on the organism, the time frame for detect-to-treat emergencies may range from hours to days, in order to identify those who were exposed before symptoms appear and start prophylactic treatment with antibiotics. This approach would be suitable for agents such as Bacillus anthracis for which there is a reasonable window for treatment (up to 9 days) after exposure [29]. In this case, the goal is accurate and specific identification of potential agents [3]. For decontamination, time is less of a critical factor than sensitivity in the ability to monitor diminishing contamination levels. In order to facilitate detection, technologies for bioactivity analysis are often optimized either for rapid detection of biological agents or for identification.
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Stand-off detectors, point detectors, light scattering
For practical reasons, terrorists would discharge bioweapons as aerosols or particles, which may be mixed with dispersion aids or additional toxic materials [24]. The agents may be released as suspended aerosolized masses (clouds) in external environments such as military facilities or public arenas, may be transmitted by a more contained mechanism such as the HVAC systems of buildings, or may be dispersed through contaminated items such as mail. When suspicious clouds are observed, a first response might be to probe from a distance for the presence of biological agents such as spores or live bacteria mixed with inert material. Remotely operated instruments, referred to as stand-off detectors, detect biological material in a cloud by evaluating the general physical characteristics of the particulates. Methods to remotely detect aerosols of particles or droplets include Doppler radio detection and LIDAR (light detection and ranging) [12]. Spores or microbes have physical properties (characteristic shape, uniformity of size) and chemical properties—such as fluorescence due to ATP, NADH, tryptophan, riboflavin—that can be detected by such lightscattering instruments. In contrast to stand-off detectors, the sampling mechanisms for point detectors must directly take in the air to be tested so that the sample must be collected at or within the test area. Point detectors can be transported into large suspicious clouds on trucks or carried by personnel to the point of measurement. Stationary or hand-carried units are used for evaluating biological threats in interior areas or surfaces. For high-risk buildings or public gathering spots, the ideal for DTW would be to have continuously sampling sensors placed in the various places, which would activate an alarm when a positive result is found. Such devices are referred to as biological ‘‘smoke detectors’’ [24]. Another option is the Biological Aerosol Sentry and Information System (BASIS) developed by the Lawrence Livermore and Los Alamos National Laboratories. This is a detect-to-treat system in which individual air samplers are placed in high-risk areas. The units pull air through filters, which retain particulates for analysis at regular intervals. Mobile laboratory units use PCR methods to identify pathogens with a high degree of reliability. One type of point detector, the Aerosol Particle Sizer (APS), inhales air with a high-speed sampler, then counts and sizes the particulates. In the APS the counting and sizing of particles is done with a flow
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cytometer, which uses a precision fluidics system and a laser to count individual particles. The light scattered by the particle is gathered by multiple detectors. Flow cytometers have also been reduced in size to bench-top instruments, which can be easily transported to the field. Other configurations for point detectors include hand-held mobile units that can be used to scan contaminated internal or external environments, and units with tiered detection. In the latter case, the system contains four components—a trigger (and/or cue) which detects particulates in near real time, the collector, a detector, and an identifier [3]. The trigger is the sentinel unit. When it senses an increase in particulates and, in some cases, the cueing device senses the fluorescence of biomolecules such as tryptophan, ATP, NADH, or riboflavin, the rest of the detector components are activated. The collector filters air and concentrates the particulates in water. The detector is a flow cytometer that further examines the material for biological material, which is then passed to the identifier. The identifier, an immunoassay device, makes the final determination based on a set of pre-selected possibilities. These point detection systems are fairly large and are mobilized on armored trucks to enter suspected aerosol clouds. From trigger to detection takes 4 min, and the final immunoassay reports in 20 min [22]. Chemical sensors offer great flexibility and possibilities as bioactivity detectors, which can be distributed throughout buildings or vulnerable areas. The two basic components of such sensors are the substrate with sites that recognize and bind the specific analyte and a transducer that produces an electric signal in response to the binding. The variety of sensors and transducers provide flexibility in producing detectors. Common sensors include those which rely on biological recognition mechanisms such as antibody–antigen interactions and DNA hybridization to a probe. Such sensors will have a high degree of specificity for the target structure; however, possible fouling of the sensitive surface will create problems for continuous monitoring devices. Single-use detectors or those with renewable surfaces may be a better option inasmuch as the harsh conditions needed to dissociate the analyte from the antibody or DNA hybridization fragment may limit its lifetime.
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C.L. Meays, K. Broersma, R. Nordin and A. Mazumder, Source tracking fecal bacteria in water: a critical review of current methods, J. Environ. Manage., 73 (2004) 71–79.
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T. Cieslak and E.M. Eitzen, Bioterrorism: agents of concern, J. Pub. Health Manage. Prac., 6 (2000) 19–29. A.A. Fatah, J.A. Barrett, Jr., R.D. Arcilesi, K.J. Ewing, C.H. Lattin and T.F. Moshier, An introduction to biological agent detection equipment for emergency first responders. NIJ Guide, (2001) 7–12. US FDA, ITG subject: bacterial endotoxins/pyrogens. In: Office of Regulatory Affairs (Ed.), Inspector’s Technical Guide, Washington, DC, 1987. US FDA Center for Food Safety and Applied Nutrition, Bacteriological Analytical Manual Online, 2001. U.S. Environmental Protection Agency, Water science: analytical methods. EPA 821-B-03-004, 2005. USDA Food Safety and Inspection Service, Microbiology Laboratory Guidebook, 2004. USDA Microbiological Data Program, SOPs for laboratory activities, 2005. http://www.ams.usda.gov/science/MPO/SOPs.htm N. Bouaı¨cha, I. Maatouk, G. Vincent and Y. Levi, A colorimetric and fluorometric microplate assay for the detection of microcystin-LR in drinking water without preconcentration, Food Chem. Toxicol., 40 (2002) 1677–1683. S.S. Iqbal, M.W. Mayo, J.G. Bruno, B.V. Bronk, C.A. Batt and J.P. Chambers, A review of molecular recognition technologies for detection of biological threat agents, Biosens. Bioelectron., 15 (2000) 549–578. J.A. Higgins, M.S. Ibrahim, F.K. Knauert, G.V. Ludwig, T.M. Kijek, J.W. Ezzell, B.C. Courtney and E.A. Henchal, Sensitive and rapid identification of biological threat agents, Ann. NY Acad. Sci., 894 (1999) 130–148. M.E. Kosal, The basics of chemical and biological weapons detectors, 2003. http://cns.miis.edu/pubs/week/031124.htm P.E. Andreotti, G.V. Ludwig, A.H. Peruski, J.J. Tuite, S.S. Morse and L.F. Peruski Jr., Immunoassay of infectious agents, BioTechniques, 35 (2003) 850–859. J. Liu, D. Xing, X. Shen and D. Zhu, Electrochemiluminescence polymerase chain reaction detection of genetically modified organisms, Anal. Chim. Acta, 537 (2005) 119–123. X.B. Yin, S. Dong and E. Wang, Analytical applications of the electrochemiluminescence of tris(2,20 -bipyridyl) ruthenium and its derivatives, Trends Anal. Chem., 23 (2004) 432–441. V.A. Petrenko and V.J. Vodyanoy, Phage display for detection of biological threat agents, J. Microbiol. Meth., 53 (2003) 253–262. J.R. Uhl, C.A. Bell, L.M. Sloan, M.J. Espy, T.F. Smith, J.E. Rosenblatt and F.R. Cockerill, Application of rapid-cycle real-time polymerase chain reaction for the detection of microbial pathogens: the Mayo–Roche rapid anthrax test, Mayo Clin. Proc., 77 (2002) 673–680. M.J. Espy, J.R. Uhl, L.M. Sloan, J.E. Rosenblatt, F.R. Cockerill and T.F. Smith, Detection of Vaccinia virus, Herpes Simplex virus, Varicella-Zoster
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virus, and Bacillus anthracis DNA by lightcycler polymerase chain reaction after autoclaving: implications for biosafety of bioterrorism agents, Mayo Clin. Proc., 77 (2002) 624–628. M. Panning, M. Asper, S. Kramme, H. Schmitz and C. Drosten, Rapid detection and differentiation of human pathogenic orthopox viruses by a fluorescence resonance energy transfer real-time PCR assay, Clin. Chem., 50 (2004) 702–708. D.A. Kulesh, R.O. Baker, B.M. Loveless, D. Norwood, S.H. Zwiers, E. Mucker, C. Hartmann, R. Herrera, D. Miller, D. Christensen, L.P. Wasieloski Jr., J. Huggins and P.B. Jahrling, Smallpox and pan-orthopox virus detection by real-time 30 -minor groove binder TaqMan assays on the Roche lightcycler and the Cepheid smart cycler platforms, J. Clin. Microbiol., 42 (2004) 601–609. G. Beadie, J. Reintjes, M. Bashkansky, T. Opatrny and M.O. Scully, Towards a FAST-CARS anthrax detector: CARS generation in a DPA surrogate molecule, J. Mod. Opt., 50 (2003) 2361–2368. R.C. Spencer and N.F. Lightfoot, Preparedness and response to bioterrorism, J. Infect., 43 (2001) 104–110. F. Basile, M.B. Beverly, K.J. Voorhees and T.L. Hadfield, Pathogenic bacteria: their detection and differentiation by rapid lipid profiling with pyrolysis mass spectrometry, Trends Anal. Chem., 17 (1998) 95–108. National Research Council (NRC), Sensor Systems for Biological Agent Attacks: Protecting Buildings and Military Bases, National Academies Press, Washington, DC, 2005. J.P. Dworzanski, W.H. McClennen, P.A. Cole, S.N. Thornton, H.L.C. Meuzelaar, N.S. Arnold and A.P. Snyder, Field-portable, automated pyrolysis-GC/IMS system for rapid biomarker detection in aerosols: A feasibility study, Field Anal. Chem. Technol., 1 (1998) 295–305. R.T. Vinopal, J.R. Jadamec, P. deFur, A.L. Demars, S. Jakubielski, C. Green, C.P. Anderson and J.E.D.R.F. Dugas, Fingerprinting bacterial strains using ion mobility spectrometry, Anal. Chim. Acta, 457 (2005) 83–95. R. Dutta, E. Hines, J. Gardner and P. Boilot, Bacteria classification using Cyranose 320 electronic nose, BioMed. Eng. OnLine, 1 (2002) 4. E.T. Arakawa, N.V. Lavrik and P.G. Datskos, Detection of anthrax stimulants with microcalorimetric spectroscopy: Bacillus subtilis and Bacillus cereus spores, Appl. Opt., 42 (2003) 1757–1762. J.M. Teich, M.M. Wagner, C.F. Mackenzie and K.O. Schafer, The informatics response in disaster, terrorism, and war, J. Am. Med. Inform. Assoc., 9 (2002) 97–104.
REVIEW QUESTIONS 1. 2. 788
Describe some instruments based on molecular recognition. Describe some methods to detect bioweapons.
Bioactivity detectors Sue M. Ford
20b.1 20b.1.1
BACKGROUND Needs for bioactivity detection
Routine monitoring of food and water for the presence of pathogens, toxins, and spoilage-causing microbes is a major concern for public health departments and the food industry. In the case of diseasecausing contamination, identification of the organism is critical to trace its source. The Environmental Protection Agency monitors drinking water, ambient water, and wastewater for the presence of organisms such as non-pathogenic coliform bacteria, which are indicators of pollution. Identification of a specific organism can aid in locating the source of the pollution by determining whether the organism is from humans, livestock, or wildlife [1]. Pharmaceutical companies monitor waterfor-injection for bacterial toxins called pyrogens, which are not removed by filter-sterilization methods. There is a need for methods to quickly detect biothreat agents that may be dispersed as bacteria, spores, viruses, rickettsiae, or toxins such as the botulism toxin or ricin [2,3]. 20b.2 20b.2.1
METHODS Traditional bioactivity methods
The FDA, USDA, and EPA have established methods and standards for detecting food- and water-borne pathogens; these methods are available on the agencies’ websites [4–8]. Many of the procedures for microbe identification rely on culturing the sample for subsequent identification by multiple tests such as colony characteristics, selective growth conditions, and biochemical assays for metabolites. Such traditional methods take upward of 24 h and remain the standard against which new Comprehensive Analytical Chemistry 47 S. Ahuja and N. Jespersen (Eds) Volume 47 ISSN: 0166-526X DOI: 10.1016/S0166-526X(06)47030-6 r 2006 Elsevier B.V. All rights reserved.
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tests are evaluated. Recently developed procedures for microbial detection include biochemical test kits, genetic and molecular biology techniques, and miniaturized instruments. New assays for bacteria are very sensitive, requiring fewer organisms for detection. Although an incubation period is still generally needed to expand the population above detection limits, these more sensitive methods shorten the incubation period. Testing for toxins such as the botulism toxin relied on observing the effects of a test compound injected in mice or the use of colorimetric [9] and immunoassays. 20b.2.2
Methods and instruments based on molecular recognition
Immunology-based methods have been the most important technology for detection and identification of microbes, viruses, and toxins for many years [10]. Traditional immunoassays rely on antibodies produced in immunized animals or in hybridoma cultures. The interaction of the antigen of interest (pathogen, toxin) with the antibody is combined with a detection protocol which may involve labeling the bound antigen/antibody complex with a reporter molecule that allows detection. The methods include latex agglutination, immunodiffusion, enzyme-linked immunoassays, immunoprecipitation, and immunochromatography, which are rapid, sensitive, and specific [5,11]. The reagents can be incorporated into a disposable device similar to a home pregnancy test kit so that the presence or absence of the test organism is indicated by a color change or development of a line. The devices have quality control mechanisms incorporated to increase reliability; nonetheless, some products have been reported to have high false-positive response rates [12]. The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13–15]. When the tag is 778
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electrically stimulated, it undergoes oxidation–reduction cycling with tripropylamine in the mixture. The excitation of the tag results in the emission of light. ECL has been used for detection of Staphylococcus enterotoxin B, ricin, Yersinia pestis antigen, anthrax PA antigen, and equine encephalitis virus [11]. ECL is particularly useful for detection of toxins that are not conveniently detected by molecular biology-based assays. BioVeris (Gaithersburg, MD) BV M-SERIESs instruments use ECL technology. A developing alternative to antibody-based molecular recognition is phage-display technology. Filamentous phages are thread-shaped viruses that attack bacteria [10,16]. Phage-display libraries are mixtures of phages containing foreign peptides encoded into their surface coat proteins. The number of phages containing different peptides in a population can exceed 106, providing a vast selection of possible antigenbinding domains. From such populations, phages can be selected for affinity for a particular antigen. The selected phage is then replicated in host bacteria and harvested. An advantage of phage display over traditional immunological methods is that these phage ‘‘antibodies’’ can be prepared against compounds such as botulism toxin or organisms that would kill the animals that would be used to produce antibodies, and for molecules with low antigenicity. A problem with phage display is the potential for false positives due to unpredictable non-specific interactions in a complex mixture [10]. 20b.2.3
Methods and instruments based on nucleotide analysis
Many modern instruments for bioactivity detection are based on analysis of nucleic acids subsequent to multiplication of the amount of unknown DNA of interest by the polymerase chain reaction (PCR). The basic process of PCR involves adding the unknown DNA to a reaction mixture which contains (1) nucleotides for DNA strand synthesis, (2) the heat-stable Taq DNA polymerase to catalyze the process, and (3) nucleotide primers chosen to initiate polymerase activity along specific regions of target DNA. In the instrument, the temperature is increased to 901C and the test DNA denatures into two single strands. After denaturation of DNA, the reaction is cooled to 551C to allow the primer to bind (anneal) to the corresponding region on the strands of test DNA and then the temperature is raised to 751C, the optimum for polymerase activity. At the end of the elongation reaction, the amount of target DNA has doubled. This process is repeated numerous times in the thermocycler, increasing the DNA exponentially. The resulting 779
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DNA is subsequently analyzed by various characterization techniques, such as molecular weight determination, electrophoresis, and hybridization to known probes. Using these methods, the presence of DNA unique to a particular species can be confirmed. Alternatively, with appropriate primers, a mixture of DNA fragments, which are uniquely characteristic of an individual or organism, will result in a fingerprint that can be used for identification. The conventional steps for PCR may take hours to days to obtain results. Numerous improvements and variations in the process have enhanced the sensitivity and speed of PCR for detection and identification of bacteria, spores, and viruses. The amplicons or the oligonucleotide probes can be tagged with fluorescent dyes so that the reaction can be followed as it progresses, a process referred to as real-time PCR (RT-PCR). The amplification process and detection are carried out in the same closed vessel [17]. This eliminates the need to use electrophoresis, for example, after PCR to determine if a sample is positive for an amplicon of interest [11] and also prevents contamination by nucleic acids during sample handling in the laboratory. Instrumentation for temperature cycling has been improved such that 30 cycles can be accomplished in 30 min, a process referred to as rapid-cycle, real-time PCR [17,18]. The use of reporter tags, such as fluorescent dyes that bind to double-stranded DNA or sequence-specific probes can be added to the reaction to allow quantitation of amplicons at the end of each cycle. Fluorescence resonance energy transfer (FRET) RT-PCR involves the transfer of energy between two dyes when they are in proximity to one another. An excited reporter fluorophore excites a nearby quencher fluorophore, which then emits a photon. This phenomenon is used to construct PCR probes with reporter and quencher components. FRET RT-PCR is a powerful technique that allows several strains of smallpox viruses, for example, to be detected simultaneously [19]. The temperature at which 50% of the DNA double strands separate is the melting point (Tm) [10]. Melting point profiles can also be used to identify DNA inasmuch as a single-base substitution can change the melting curve for DNA. The LightCyclerTM by Roche Applied Science (Indianapolis, IN) is a temperature-controlled microvolume fluorimeter with RT-PCR module and software to facilitate quantitation of DNA, as well as identification and genotyping by fluorescence detection or melting profiles analysis. RT-PCR assays are very sensitive, with limits of detection for smallpox virus being around 12 gene copies [20]. PCR is now the front-end process for other DNA profiling techniques such as pulse-field gel electrophoresis (PFGE), terminal restriction 780
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fragment length polymorphism (T-RFLP), dentaturing-gradient gel electrophoresis (DGGE), and others [1]. 20b.2.4 Methods and instruments based on chemical and physical analysis
Even the simplest bacterium is composed of innumerable lipids, proteins, and polysaccharides; however, few of these molecules are specific to a particular species. Similarly, the fluorescence of small metabolites such as ATP and nucleic acids may confirm the presence of biological organisms in aerosols or water [12], but cannot identify the agent nor indicate its virulence. A major exception is dipicolinic acid (DPA), which is found in the spores of bacteria, particularly the anthrax organism, Bacillus anthracis. Although non-pathogenic bacteria form endospores as well, DPA is characteristic enough to be a marker for anthrax spores. Coherent anti-Stokes Raman scattering (CARS) of DPA appears to be promising in detecting anthrax spore clumps less than 6 mm in diameter [21], which is in the range of bioweapons grade anthrax [22]. Protein toxins such as botulism, staphylococcal enterotoxin B, or ricin can be separated with gas or liquid chromatography, electrophoresis, or a combination. The mChemLabTM (Sandia National Laboratories Albuquerque, NM) series of instruments includes a hand-held Bio Detector. Proteins in the sample are labeled with fluorescent tags, and nanoliter volumes of samples are separated by microchannels etched into a glass chip. The separation occurs as the sample moves through the channels and identification is based on retention times. The analyses can be completed within 10 min. 20b.2.4.1 Fingerprint methods Analysis of individual microbial compounds is generally of limited use for distinguishing pathogens from benign organisms because of the lack of specificity. Instead, a new approach to identifying bacteria through chemical analysis is evolving. Chemotaxonomy refers to the process by which a whole organism is deconstructed into a chemical signature or fingerprint. This concept shows promise for rapid detection of microbes of interest. For example, various species of bacteria have distinct profiles of lipid composition, which may be used for identification [23], although this method may have limitations if the spectra are altered by the nutrients available during microbial growth. Proteins and other large biomolecules can be analyzed using MALDI (matrix-assisted laser desorption and ionization) with TOF 781
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(time-of-flight) mass spectrometry (see Chapter 11). The sample is mixed with a matrix of organic molecules, which absorbs energy from a laser and transfers it to the protein molecules, forming ions typically with a single positive charge. Whole, intact cells can be mixed with such a matrix, dried, and analyzed directly to produce a spectral fingerprint that can distinguish among various bacterial and viral species. However, for MALDI, as for all other fingerprint methods, the ability to identify pathogens mixed with non-pathogens requires further research into mathematical procedures to interpret the results (NRC [24]). Pyrolysis has been successfully used to prepare samples for lipid profiling by mass spectrometry [23]. Rather than a separate step in sample preparation, the methylation of fatty acids is done during the pyrolysis, a process referred to as in situ thermal hydrolysis methylation (THM). This reduces the sample preparation time to 30 s and the total analysis with MS is less than 10 min. The authors developed a portable ion-trap MS using this procedure which did not require pressurized gas; however, it required a large number of bacteria (107–108 cells) in order to distinguish among the four strains studied. Py-GC/MS has low resolving power and is susceptible to false positives; however, the portability and low cost make it attractive as a screening device [25]. Ion mobility spectroscopy (IMS) can also produce fingerprints to identify bacterial strains within 1 min [26]. Plasmagrams from microgram quantities of bacteria are sufficiently unique to distinguish among 200 strains or species of bacteria. The technique can be enhanced by stepped increases in the desorber temperature during desorption. Such programed temperature ramping improves peak detection and adds components to the fingerprints. This increases the complexity of the output, permitting greater discrimination among species. Pyrolysis can also be used with instruments combining short capillary GC columns with IMS for detection of bacterial biomarkers in suspect aerosols, data can be obtained within 4 min with a hand-held instrument [25]; interestingly, 3 min of that time is for collection of the aerosol. Another type of sensor relies on semiselective polymer films, which are fabricated to interact with multiple chemicals. When a chemical is absorbed onto a polymer, which contains carbon black for electronic conduction, a change in size of the polymer—and therefore change in electrical conductivity—occurs, initiating a signal to the transducer. The magnitude of the signal varies depending on the chemical and the polymer. Greater discrimination power can be achieved by using multiple sensors with varying chemical sensitivities, an arrangement called a sensor array. Individual chemicals interact differently with each of 782
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the polymer surfaces in the array, creating fingerprint signals. For example, the Cyranose 320 unit, which evolved from NASA’s E-Nose, has 32 sensors. The unit is able to identify certain classes of bacteria with 96–98% accuracy using combined statistical algorithms; however, the difficulty of this analysis [27] may preclude its use for identification. Measuring volatile metabolites with such sensors requires a highenough concentration of bacteria to produce sufficient material for detection. Although the method is rapid, it is not sensitive enough to detect small numbers of bacteria for early detection of a biothreat. Microcalorimetric spectroscopy-based detection uses vibrational photothermal spectra to identify organic molecules. It can be applied to the detection and identification of microorganisms. The specificity of the method can be enhanced by using a chemically selective layer to enhance trapping of the organisms on the thermal detector. This method can detect amounts of bacteria in an order of magnitude smaller than conventional IR and FTIR [28]. 20b.3 20b.3.1
BIOWEAPONS Characteristics of bioweapons
Particles containing bacterial and spore clumps to be used as weapons must fall within a certain size range. They must be small enough to remain suspended in the air for maximum distribution, yet large enough to be retained in the pulmonary tract; thus the sizes of such particles generally fall in the range of 0.5–20 mm [12]. Thus the size and uniformity of particulate sizes in a cloud can indicate the presence of bioweapons. The analytical problems in developing bioweapons detectors are substantially different from monitoring food and water for inadvertent contamination. First, time is often an issue as the need is to treat or warn individuals, who have been exposed to microbes that have been chosen for their ability to cause disease or death in as many people as possible. Second, the quantities of biological agent to be detected are very small. Biological agents are more potent on a mass basis than chemical agents because microbes and viruses multiply within the host. Third, aerosols are diluted as the cloud drifts [3]. In order to concentrate the agent, samplers need to draw in large amounts of air. The samples therefore contain dust and other solid materials that interfere with particulate analyses. Fourth, there are many types of biological agents that might be used as weapons (see charts in reference [3] for an extensive listing), which are diverse in physical and chemical nature 783
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and not amenable to a common analytical procedure. Fifth, there is a naturally occurring background of non-pathogenic microorganisms and chemicals. Consequently, the detection methods need to distinguish microbes from the non-biological particulates as well as to distinguish pathogenic microorganisms from benign ones in a mixture. Methods that rely on quantitation of a macromolecule such as DNA would be able to recognize biological agents in an aerosol or dust, but would not be able to determine whether the agent is a pathogen or benign microbe. Such a distinction depends on identification of the microbe, which in turn requires DNA sequencing.
20b.3.2
Detect-to-warn, detect-to-treat
Rapid identification of a specific bioagent is critical for assessing the level of threat. Ideally, detectors for pathogens and toxins would produce results that are rapid, specific for individual organisms, and sensitive; however, current technical limitations require compromises, which depend on the use of the instrument. Detectors for bioweapons may be divided into several categories based on urgency of results: detect-to-warn (DTW), detect-to-treat, and detection to monitor decontamination. The purpose of DTW is, upon a positive result, to immediately set in motion actions to protect personnel and limit the spread of the agent. Thus, time is the critical feature. The goal for technology is to develop DTW systems that provide reliable alerts within 1 min [24], i.e., sample collection, preparation, identification, and data reporting should be completed within that interval. The other phases of biological agent warning systems have less urgency but have increased need for specificity and sensitivity. Depending on the organism, the time frame for detect-to-treat emergencies may range from hours to days, in order to identify those who were exposed before symptoms appear and start prophylactic treatment with antibiotics. This approach would be suitable for agents such as Bacillus anthracis for which there is a reasonable window for treatment (up to 9 days) after exposure [29]. In this case, the goal is accurate and specific identification of potential agents [3]. For decontamination, time is less of a critical factor than sensitivity in the ability to monitor diminishing contamination levels. In order to facilitate detection, technologies for bioactivity analysis are often optimized either for rapid detection of biological agents or for identification.
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Stand-off detectors, point detectors, light scattering
For practical reasons, terrorists would discharge bioweapons as aerosols or particles, which may be mixed with dispersion aids or additional toxic materials [24]. The agents may be released as suspended aerosolized masses (clouds) in external environments such as military facilities or public arenas, may be transmitted by a more contained mechanism such as the HVAC systems of buildings, or may be dispersed through contaminated items such as mail. When suspicious clouds are observed, a first response might be to probe from a distance for the presence of biological agents such as spores or live bacteria mixed with inert material. Remotely operated instruments, referred to as stand-off detectors, detect biological material in a cloud by evaluating the general physical characteristics of the particulates. Methods to remotely detect aerosols of particles or droplets include Doppler radio detection and LIDAR (light detection and ranging) [12]. Spores or microbes have physical properties (characteristic shape, uniformity of size) and chemical properties—such as fluorescence due to ATP, NADH, tryptophan, riboflavin—that can be detected by such lightscattering instruments. In contrast to stand-off detectors, the sampling mechanisms for point detectors must directly take in the air to be tested so that the sample must be collected at or within the test area. Point detectors can be transported into large suspicious clouds on trucks or carried by personnel to the point of measurement. Stationary or hand-carried units are used for evaluating biological threats in interior areas or surfaces. For high-risk buildings or public gathering spots, the ideal for DTW would be to have continuously sampling sensors placed in the various places, which would activate an alarm when a positive result is found. Such devices are referred to as biological ‘‘smoke detectors’’ [24]. Another option is the Biological Aerosol Sentry and Information System (BASIS) developed by the Lawrence Livermore and Los Alamos National Laboratories. This is a detect-to-treat system in which individual air samplers are placed in high-risk areas. The units pull air through filters, which retain particulates for analysis at regular intervals. Mobile laboratory units use PCR methods to identify pathogens with a high degree of reliability. One type of point detector, the Aerosol Particle Sizer (APS), inhales air with a high-speed sampler, then counts and sizes the particulates. In the APS the counting and sizing of particles is done with a flow
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cytometer, which uses a precision fluidics system and a laser to count individual particles. The light scattered by the particle is gathered by multiple detectors. Flow cytometers have also been reduced in size to bench-top instruments, which can be easily transported to the field. Other configurations for point detectors include hand-held mobile units that can be used to scan contaminated internal or external environments, and units with tiered detection. In the latter case, the system contains four components—a trigger (and/or cue) which detects particulates in near real time, the collector, a detector, and an identifier [3]. The trigger is the sentinel unit. When it senses an increase in particulates and, in some cases, the cueing device senses the fluorescence of biomolecules such as tryptophan, ATP, NADH, or riboflavin, the rest of the detector components are activated. The collector filters air and concentrates the particulates in water. The detector is a flow cytometer that further examines the material for biological material, which is then passed to the identifier. The identifier, an immunoassay device, makes the final determination based on a set of pre-selected possibilities. These point detection systems are fairly large and are mobilized on armored trucks to enter suspected aerosol clouds. From trigger to detection takes 4 min, and the final immunoassay reports in 20 min [22]. Chemical sensors offer great flexibility and possibilities as bioactivity detectors, which can be distributed throughout buildings or vulnerable areas. The two basic components of such sensors are the substrate with sites that recognize and bind the specific analyte and a transducer that produces an electric signal in response to the binding. The variety of sensors and transducers provide flexibility in producing detectors. Common sensors include those which rely on biological recognition mechanisms such as antibody–antigen interactions and DNA hybridization to a probe. Such sensors will have a high degree of specificity for the target structure; however, possible fouling of the sensitive surface will create problems for continuous monitoring devices. Single-use detectors or those with renewable surfaces may be a better option inasmuch as the harsh conditions needed to dissociate the analyte from the antibody or DNA hybridization fragment may limit its lifetime.
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T. Cieslak and E.M. Eitzen, Bioterrorism: agents of concern, J. Pub. Health Manage. Prac., 6 (2000) 19–29. A.A. Fatah, J.A. Barrett, Jr., R.D. Arcilesi, K.J. Ewing, C.H. Lattin and T.F. Moshier, An introduction to biological agent detection equipment for emergency first responders. NIJ Guide, (2001) 7–12. US FDA, ITG subject: bacterial endotoxins/pyrogens. In: Office of Regulatory Affairs (Ed.), Inspector’s Technical Guide, Washington, DC, 1987. US FDA Center for Food Safety and Applied Nutrition, Bacteriological Analytical Manual Online, 2001. U.S. Environmental Protection Agency, Water science: analytical methods. EPA 821-B-03-004, 2005. USDA Food Safety and Inspection Service, Microbiology Laboratory Guidebook, 2004. USDA Microbiological Data Program, SOPs for laboratory activities, 2005. http://www.ams.usda.gov/science/MPO/SOPs.htm N. Bouaı¨cha, I. Maatouk, G. Vincent and Y. Levi, A colorimetric and fluorometric microplate assay for the detection of microcystin-LR in drinking water without preconcentration, Food Chem. Toxicol., 40 (2002) 1677–1683. S.S. Iqbal, M.W. Mayo, J.G. Bruno, B.V. Bronk, C.A. Batt and J.P. Chambers, A review of molecular recognition technologies for detection of biological threat agents, Biosens. Bioelectron., 15 (2000) 549–578. J.A. Higgins, M.S. Ibrahim, F.K. Knauert, G.V. Ludwig, T.M. Kijek, J.W. Ezzell, B.C. Courtney and E.A. Henchal, Sensitive and rapid identification of biological threat agents, Ann. NY Acad. Sci., 894 (1999) 130–148. M.E. Kosal, The basics of chemical and biological weapons detectors, 2003. http://cns.miis.edu/pubs/week/031124.htm P.E. Andreotti, G.V. Ludwig, A.H. Peruski, J.J. Tuite, S.S. Morse and L.F. Peruski Jr., Immunoassay of infectious agents, BioTechniques, 35 (2003) 850–859. J. Liu, D. Xing, X. Shen and D. Zhu, Electrochemiluminescence polymerase chain reaction detection of genetically modified organisms, Anal. Chim. Acta, 537 (2005) 119–123. X.B. Yin, S. Dong and E. Wang, Analytical applications of the electrochemiluminescence of tris(2,20 -bipyridyl) ruthenium and its derivatives, Trends Anal. Chem., 23 (2004) 432–441. V.A. Petrenko and V.J. Vodyanoy, Phage display for detection of biological threat agents, J. Microbiol. Meth., 53 (2003) 253–262. J.R. Uhl, C.A. Bell, L.M. Sloan, M.J. Espy, T.F. Smith, J.E. Rosenblatt and F.R. Cockerill, Application of rapid-cycle real-time polymerase chain reaction for the detection of microbial pathogens: the Mayo–Roche rapid anthrax test, Mayo Clin. Proc., 77 (2002) 673–680. M.J. Espy, J.R. Uhl, L.M. Sloan, J.E. Rosenblatt, F.R. Cockerill and T.F. Smith, Detection of Vaccinia virus, Herpes Simplex virus, Varicella-Zoster
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REVIEW QUESTIONS 1. 2. 788
Describe some instruments based on molecular recognition. Describe some methods to detect bioweapons.