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Scientific Research of the Union of Scientists in Bulgaria - Plovdiv, series G. Medicine, Pharmacy and Dental medicine, Vol. XVII, ISSN 1311-9427, International Conference of Young Scientists, 11 - 13 June 2015, Plovdiv
WEAPONIZED BIOLOGICAL AGENTS AN OVERVIEW Matthew Serkedjiev2, Iliyana Mitova1, Kiril Angelov1,
'Laboratory of Electron-Phonon Interaction, Institute of Solid State Physics "Georgi Nadjakov" Bulgarian Academy of Sciences, 72, Tzarigradsko Chaussee, Blvd., 1784 Sofia, Bulgaria 2Laboratory of Yeast Molecular Genetics, Institute of Molecular Biology "Roumen Tsanev", Bulgarian Academy of Sciences, "Acad. G. Bonchev", str., bl. 21, 1113 Sofia, Bulgaria
Abstract:
The aim of this overview is to elucidate the basic concerns involving biological weapons and their use in bioterrorism. The potential of biological weapons emerges from the fact that it is more technically accessible than either nuclear or chemical weapons. Biological weapons are different from any other means of destruction in that they are the only ones devised expressly to kill defenseless humans, with little real battlefield potential in modern warfare. Most governmental agencys predict that the method of deployment of a biological agent with the purpose of bioterrorist attack is in aerosolized state. This overview includes the statistically more likely to be weaponized and obtained by renegade groups or terrorist biological agents, methods for detection and simulants of weaponized biological agents. This overview will simplify the biology of these biological agents and give information for their implement.
Keywords: sensor, bioterrorism, detection, biological agent
What is constituted a Biological weapon?
Biological treat agents refer to biological agents (bacteria, virus, and toxin) intended for the use of creating biological weapons. Biological weapons refer to munitions, equipment or other means of delivery including bombs, aircraft spray tanks and other devices, intended for use in the dissemination of biological agents and toxins for hostile purposes. The principal means of dissemination are as an aerosol to be inhaled by a target population or as a spray to be deposited on crop plants. They are few intrinsic features of biological agents which influence their potential for use as weapon.(Chauhan, 2004)
Infectivity: Infectivity of a given agent reflects the relative ease by which the microorganisms establish themselves in a host species. Pathogens with high infectivity cause disease with relatively few organisms, while those with low infectivity require a large number.
Virulence: Virulence of a given agent reflects the relative severity of disease produced by the pathogen. Different biological agent and different strains of the same agent may cause diseases of different severity.
Toxicity: toxicity of a given agent reflects the negative effects on the physiology of the host produced by the toxin.
Pathogenicity: The capability of an infectious agent to cause disease in a susceptible host.
Incubation Period: A sufficient number of microorganisms or quantity of toxin must penetrate the body's natural defenses to initiate infection (the infection dose varies among agents), or intoxication (the intoxicating dose). Infectious agents must then multiply to produce disease. The time between exposure and the appearance of symptoms is known as the incubation period.
Transmissibility: Some biological agents can be transmitted from person- to-person. In the context of biological treat casualty management, the relative ease with which an agent is passed from person to person constitutes the principal concern.
Lethality: Lethality reflects the relative ease which an agent causes death in a susceptible population.
Stability: The viability of an agent is affected by various environmental factors like, humidity, atmospheric population and sunlight. A quantitative measure of stability is an agent's decay rate (aerosol decay rate).
Classification of biological warfare agents by mode of delivery (or spared):
Respiratory route (aerosols): Inhalation of agents aerosols, with resultant deposition of infectious or toxin particles within alveoli, provides a direct pathway into the systemic circulation. The natural process of breathing causes a continuing influx of biological agent to exposed individuals. From the bioterrorism point of view this is most important route to inflict mass casualty, and hence the most dangerous route. This route spreads most deadly agents like Smallpox, Anthrax, Hemorrhagic fevers (Ebola)
Alimentary route (ingestion): Alimentary exposure - food and water supplies may be contaminated during an aerosolized biological agent attack;
Dermal route: Dermal exposure (percutaneous) - intact skin provides an excellent barrier for most, but not all biological agents. However mucous membranes and damaged skin constitute breaches in this normal barrier trough which agents may readily pass.
Vector Borne: Attempts might be made to spread typical vector borne disease by release infected arthropods host such as mosquitoes, ticks or fleas. This live vector can be produced in large numbers and infected reservoirs. (Chauhan, 2004)
CDC categories:
This overview uses the CDC categorization of bio agents which is created under current US law declared by the US Department of Health and Human Services. CDC categorizes these agents (A, B or C).The purpose of this categorization is to inform for the high priority agents believed to pose a great risk to national security. It can be easily transmitted and disseminated which results in high mortality, potential major public health impact, may cause public panic, or require special actions for public health. Category B and C are not included in this overview because of their somewhat less or non-documented use; lack of mass dissemination protocol and in some case low mortality rate (category B, C)
Category A - Biological agents:
Tularemia: Tularemia is a zoonosis, a disease of animals transmissible to humans. The causative agent is Francisella tularensis. This bacteria is a non-motile, Gram-negative, facultatively intracellular, aerobic coccobacillus, measuring 0.2 microns x0.3-0.7 microns. Natural transmission to humans usually occurs, through the bite of a blood feeding arthropod (tick, mosquito), but ingestion of infected food or water or inhalation of the bacterium may also cause infection. F.tularensis is considered a potential biological threat agent. Clinical manifestation of Tularemia depends on the route of entry and the virulence of the agent. Infection by inhalation may produce primary pneumonia or tracheitis and bronchitis and infection through the skin or conjunctiva produces an ulcero-glandular form of the disease in the and infection resulting from ingestion produces painful pharyngitis and associated cervical lymphedema. In a bioterrorism event F.tulanesis would most likely be dispersed by aerosol dissemination. If effective infection occurs the incubation period varies from 1 to approximately 14 days averaging 3-5days.(Katz & Zilinskas, 2011)
Anthrax: The causative agent of anthrax is the vegetative form of Bacillus Anthracis. This bacterium is a non-motile, rod shaped, Gram-positive, aerobic or facultative anaerobic bacillus measuring 1-1.2 microns x 3-5 microns. The most important ability of this potential biological threat agent is its ability to form spores. Spore is the resting stage that enables the organism to endure adverse conditions, when those conditions improve e.i. (the human body) the spore transforms into its vegetative form which is highly virulent. B. Anthracis most commonly occurs in cattle, sheep, goats, but can also infect humans. Anthrax may be spread by different routes. Infection of humans can occurs in one of tree forms depending on the route of acquisition. Inhalational anthrax when bacteria or spores enter the blood through inhalation in order to induce infection. Cutaneous anthrax when bacteria enter through cut on the skin and last but not least intestinal anthrax in which bacteria contaminate food is ingested; Infection of humans can occur in one of tree forms depending on the route of acquisition. It's anticipated that anthrax is more likely to be used as an aerosol containing spores. Following an incubation period of 1 to 6 days after respiratory exposure, a nonspecific syndrome of fever, malaise, myalgia, fatigue, cough and chest pain develops. There is an interval of improvement over the next 2 to 3 days before the final phase of the disease is initiated by higher fever, dyspnea, and cyanosis, followed by septic shock and death within 36 h.(Katz & Zilinskas, 2011)
Smallpox: The causative agent of smallpox is Variola Major (vary rare cases V. Minor). The small pox virus, is a member of the Poxviridae family and the orthopoxvirus genus, it has a genome composed of DNA and an elaborate capsid with an enclosing envelop. The capsid has a rectangular form and resembles a brick, with the proteins of the capsid organized into a series of rod like structures. The virus measures about 0.400 microns in length and about 0.20 microns in width and depth. The smallpox virus is limited to human host, it's only equipped to enter and use the replication machinery of human cells which has been exploited by scientist to assure its eradication.
Infection occurs in nature - and presumably in the event of deliberate dissemination via the respiratory system. Most commonly, the virus spreads from person to person when viral particles (virions) are expelled from the mouth cavity of the infected individual and inhaled by a person. Upon inhalation, the virus particles(virion) becomes implanted in the lining of the mouth and lungs. From this area the virus is carried to the lymphnodes.
Incubation period is between 7 and 19 days after initial exposure with rash appearing 2-5 days afterward the
onset is sudden, with a 2-4 day period with influenza like syndrome (fever, malaise, headache, back pain) Fever may drop, and maculo-papular rash appears (on oral mucosa, face, hand and forearms, after a few days it progresses to the trunk). Lesions progress from macules to papules to pastule vescicals. From 8 to 14 days after the initial onset, postules for scabs which fall of after 3-4 weeks. Variola minor is accompanied by milder symptoms and case fatality rate is less than 1% while that of V.major is 20-40%.
Acquisition and weaponization:
• Former Soviet biological weapons program, which trough out the 1970s is said to have maintained 20 ton per year stock pile of the virus. Since the dissolution of the Soviet Union there is a possibility that stocks of certain biological threat agents including the smallpox virus have been illegally removed from the many repositories of the former soviet BW program.
• Vector Institute is a biological research center in Koltsovo, Novosibir Oblast, Russia. It is considered as one of the two official repositories for the now-eradicated smallpox virus.
• Second possible source of virus from the pre-eradication era is the bodies of small pox victims buried in permafrost grounds (Siberia, Alaska).The virus is preserved in deep cold.
• Production - accomplished the same way a vaccine is made. Inoculation of pathogenic seed strain in a living host. For example the 1970 Soviet Union BW program collected hundred
and thousands of eggs for inoculation with the virus and harvesting it later. Soviet scientist also developed effective techniques used to this day for growing viruses in cell cultures.
• Delivery - the most likely delivery system for small pox would be a simple aerosol device such as an atomizer, inhaler or handheld sprayer.
Bubonic plague: The causative agent of plague is Y.Pestis. The bacterium is a Gramnegative non motile, non-spore forming coccobacillus measuring approximately 1.5microns x 0.75 microns, capable of both aerobic and anaerobic growth. Y. Pesties is founds in every continent except Australia and Antarctica. The pathogen is present in animal reservoirs, particularly in rodents, it is transmitted from one animal host to another either directly or via a flea vector (often Xenopsylla Cheopis).Outbreaks of plague in humans are often associated with close contact with animal reservoirs. The most common of plague in humans is the bubonic, is spread mainly by the bite of fleas or by entry of the pathogen from infected fleas through a skin lesion. If the lungs become infected, as occasionally occurs in patients with the bubonic form, a much more virulent form. Pneumonic plague ensues and can be transited person to person by droplet infection. The incubation period is 2-6 days in bubonic plague and somewhat less for the pneumonic form. Initial symptoms may be nonspecific (fever, chills, malaise, myalgia, nausea, sore throat, and headache). In case of infection occurred by aerosol inhalation the disease will present as primary pneumonia. As the disease progresses, patients experience shock, delirium and coma. Untreated bubonic plague has a case-fatality rate as high as 60%, while untreated pneumonic plague is almost always fetal.
Acquisition and weaponization:
• Y. Pestis has long been an attractive biological agent for weaponization because of its high infectious potential. In the 1950s and 1960s, the US AND Soviet biological warfare programs developed techniques to directly aerosolize plague particles in order to cause the more dangerous pneumonic plague variant. Both programs managed to develop methodologies for suspending or dissolving optimal quantities of Y. Pestis in solutions containing preservatives, adjuvants, and antistatic chemicals.
• The end result was a large quantity of the agent suitable for pleasing into weapons. Plague formulations in both liquid and dry powder forms were developed for effective dissemination of bacteria by explosion or spraying. Further, Soviet research on genetically modified plague resulted in the creation of multidrug resistant strains (Orent, 2004).The weaponized plague is considered one of the most effective weapons ever created by the Soviet Union. The organism can be manufactured by large scale fermentation without affecting its property as a BW agent. Y. Pesties can be stored relatively easily because it can survive at low freezing temperatures for an extended periods as long as there is water available but its killed by 15 min of exposure to 55Oc.It can be freeze-dried and stored for up to several years without loss of viability.
• Plague bacteria are considered somewhat more effective than anthrax bacteria, but less effective than tularemia bacteria in causing infection. The dose of viable bacteria cells in aerosol an individual has to receive to have 50% chance of being infected is estimated to be about 10003000 cells. A 1970 WHO study concluded that, in a worse-case scenario, 50 kg of Y. Pestis released as an aerosol over a city of five million could result in 150.000 cases of pneumonic plague, with 80.000 to 100.00- cases require hospitalization and 36.000 deaths. The WHO study also found that the organism could remain viable for up to 1h after dispersal as an aerosol and be carried for a distance of up to 10km from point of release.(Katz & Zilinskas, 2011)
Hemorrhagic fever viruses: The Viral Hemorrhagic Fevers are a group of illnesses which are a diverse group of RNA viruses that present with common clinical characteristics known as the viral hemorrhagic fever syndrome. These illnesses range from a mild flu-like illness to endothelial damage resulting in increased vascular permeability and bleeding complications.
Notable agents
• Filoviridae. Ebola virus, Marburg virus;
• Arenaviridae. Junin virus (Argentine Hemorrhagic Fever, AHF), Machupo virus (Bolivian Hemorrhagic Fever, BHF), Sabia virus (Brazilian Hemorrhagic Fever), Lassa fever virus, Guanarito virus (Venezuelan Hemorrhagic Fever, VHF);
• Bunyaviridae. Crimean-Congo Hemorrhagic Fever (CCHF) virus, hantaviruses, Rift Valley Fever (RVF) virus;
• Flaviviridae. Omsk Hemorrhagic Fever (OHF) virus, Yellow Fever (YF) virus;
Acquisition and weaponization:
• Former biological warfare programs of both the United States and the former Soviet Union are known to have investigated hemorrhagic fever viruses for offensive applications. The chosen agent by the United States was Yellow-fever virus which was not successfully weaponized before the program was terminated in 1969.The former Soviet Union was successful in weaponizing Marburg virus, and is known to have researched Ebola virus.
• Some consider HFVs as a major choice for weaponization but the potential of HFV as BWA is outweighed by the fact that they are major difficulties in maintaining safety, propagation, storage, and delivery. From the point of view of parties who might seek to weaponize HFV, there are few vaccines to protect the workers and hardly any effective treatment if workers were to contract hemorrhagic fever.
• All of the HFV agents can only be grown in tissue cultures, eggs or animals. Most of this agents would not survive in the environment and there delivering by aerosol, water or food has proven tedious because of their fragile nature. Many are only transmissible through the bites of arthropods.
Botulinum Toxin: The causative agent of Botulism is the toxin produced by clostridium bacterium. The clostridium neurotoxins are among the most lethal toxins in the world, with median lethal doses (ld50) for humans in the nanogram/kilogram range. Botulinum toxin is produced by Tahe Bacterium in the form of a single polypeptide chain and subsequently altered to produce a heavy chain and light chain connected disulfide bounds.In case of severe Botulinum toxins intoxication the major target of the toxin are the neuromuscular junction and the muscarinic peripheral autonomic synapse. This essentially disrupts exocytosis or the release of neurotransmitters. This blocking of neurotransmitters causes weakness and autonomic dysfunction death occurs by paralysis of the respiratory muscles.
Acquisition and weaponization:
• First known development of botulinum toxin based weapons took place in Manchuria during the 1930s under Unit 731(a Japanese biological warfare research unit). During World War II the United States also developed methods to produce botulinum toxin (scientist branded it agent X).The Soviet Union have also experimented with techniques to weaponized botulinum toxin. In one point Iraq had the largest known of military botulinum toxin programs during which 19.000 liters of concentrated toxin was produced, much of which was loaded into missiles and artilleries shells.
• The Japanese cult Aum Shinriokyo disseminated botulinum toxin as a weapon on at least three occasions in the early 1996s.Their attempts on causing death did not come to fruition because of limited knowledge in the needed field and ineffective aerosolization procedure. The Aum Shinriokyo incidents demonstrated the potential for small groups to use botulinum toxin as an biological agent for bioterrorism.
• Botulinum toxin is potential target for weaponization in part because of its incapacitating effects and the way it delivers this effect. Further the expense and diversion or resources involved in trying to support a large number of patients with ventilator failure would itself constitute the
potential of the toxin.
Biological agents have some unique characteristics that make their weaponization quite attractive (Table 1.). We understand now that most biological weapons consist of living organism and, thus can replicate once disseminated. Any country having pharmaceutical, cosmetic or advanced food storage industries will have the potential to synthesize and stabilize biological weapons. The ability to disseminate the biological agent over a wide area would be limited to those countries having cruise missiles or advanced aircrafts. However, even the smallest country or a terrorist group has the capability to deliver small quantities of BW to a specific target.
Agents Type Disease Fatality Epidemic Lethal dose Vaccine Treat ability
Bacillus anthracis Bacteria Anthrax High No 10,000 cells Yes If detected early
Yersinia pestis Bacteria Plague High High 1000 Ineffective If detected early
Variola major Virus Small pox High Moderate 30 Yes No
Clostridium botulinum Toxin Botulism High No 0.1 lg Yes No
Francisella tularensis Bacteria Tularemia Moderate No 10-50 Ineffective Yes
Filovirindea Virus Ebola High Moderate 3 No No
Table 1. Potential Biological warfare agents.
Stabilization and dissemination are important issues because of the susceptibility of the biological agents to the environmental effects in storage and also in application. The loss of bioactivity can result from exposure to high physical and chemical stress such as high surface area at air.
Biological Simulants:
Simulants are less lethal or nonlethal lethal substitutes for biological warfare agents. Most simulants are themselves microorganisms, the vast majority being nonpathogenic species that exhibit properties similar to biological threat agents but are less dangerous. In addition to nonvirulent microbes, other compounds can also serve as simulants, such as isolated proteins or chemical compounds. Simulants of biological threat agents include: bacterial spores, Bacillus Globigii (BG) from Dugway Proving Grounds (DPG), bacterial vegetative cells; and proteins: albumin and ovalbumin (OV). In order to test the properties of a given biological threat agent the simulants are aerosolized. These aerosol particles are generated using a Collison nebulizer, with sufficient dilution to result in relatively small particles around 1.5 micron aerodynamic diameter.
Bacterial simulants: Bacillus Globigii (BG), also known as Bacillus Subtilis var. Niger, has frequently been used as a surrogate species for Bacillus Anthracis because of physical similarities between the two organisms. (BG) is a ubiquitous, naturally occurring, saprophytic (i.e., feeding on decaying matter) bacterium that is commonly recovered from soil, water, air, and decomposing plant material. Under most conditions, it exists in spore form and is not biologically active. BG is not known to be a human pathogen but does produce the proteolytic enzyme subtilisin, which has been implicated in cases of allergic asthma, hypersensitive skin reactions, and pulmonary inflammation upon repeated exposure. BG was one of the first simulants produced by the U.S. Army and has since seen widespread use as a BW agent stimulant. For example, in 1966, BG was released into the New York City subway system to model the dispersal of B. Anthracis spores in an enclosed system with significant airflow.The test was alarmingly successful in its results in terms of demonstrating the vulnerability of subways to biological attack, but no injuries or illnesses were reported to have resulted from the test. 196
Viral stimulant: Comparatively, simulants for viral agents have received little attention Bacteriophages (viruses that infect bacterial cells) have often been used as viral biological threat agent simulant, of which the Levi virus MS2 is a typical model.The bacteriophage MS2 is an icosahedral, positive-sense single-stranded RNA virus that infects the bacterium Escherichia coli. An MS2 virion (viral particle) is about 27 nm in diameter, as determined by electron microscopy). The total molecular weight of MS2 has been previously determined by classical light scattering (3.6 x 106 g/mol) and sedimentation velocity (3.87 x 106 g/mol).
Toxin simulants: Ovalbumin constitutes 54% of egg white's total protein and is its main protein. Ovalbumin is a monomer, globular phospoglycoprotein with molecular weight of 44.5 kDa,Ovalbumin contains 3.5% carbohydrates and has four free sulfhydrylic groups and a disulfide group. It can be denatured by heat exposure, by surface absorption, in films, through agitation, or by the action of several denaturant agents.For testing and development purposes, ovalbumin, is typically isolated from chicken eggs, and is commonly used as a simulant for botulinum toxin. Ovalbumin is similar to human serum albumin. Another candidate is bovine serum albumin (BSA) a serum albumin protein derived from cows. It is often used as a protein concentration standard in lab experiments. It has seen extensively used as a toxin simulant of botulinum toxin in numerous experiments. (Katz & Zilinskas, 2011)
Detecting biological warfare agents:
One of the necessities in dealing with a biological threat agent is actually detecting the biological agent and determining whether an attack has occurred. It is necessary to establish what kind of virulent agent is used because of the initial symptoms after infection from a possible biological threat agent may mimic the symptoms from an infection caused by a more benign biological agent. More importantly knowing the exact biological threat agent is of great importance because some biological agents are transmissible from host to host. The solution to the detection problem is the deployment of sensors, which can identify chemical markers from a given biological agent.A biosensor system must be able to provide a warning within minutes from, most frequently, an airborne sample with a minimal to none user intermission. This overview will only outline some of the more promising and commercially available and established biosensors for detecting biological threat agent. The emphasis will be on mobile hand held devises (RAPTOR) and stand-alone sensors (Air Sentinel). Before overviewing the methods of analysis and the actual biosensors we must establish the criteria by which the biological threat agents are detected.Categoiy A biological agents cover the main types of agents with potential for bioterrorism application: Anthrax, tularemia, botulinum toxin, plague (aerosol version only), small pox, hemorrhagic fever. Category-A biological threat agent will be treated as indicative analytes for the biosensor. To develop biosensors for these analytes the important consideration are the matrix in which the analyte will be found (air water, food, host) the form of the analyte (bacteria, spore, virus, and toxin) and the biological futures of the analyte which could be recognized with an appropriate bio recognition molecules. In order to identify such futures knowledge of the biological species and the forms in which they are found is required. In the case of bacteria and virus species, nucleic acid technics can be used to identify the organism or affinity molecules can be used to detect and bind to the surface architecture of the organism. (Gooding, 2006) An alternative method for bacterial species is the monitoring of the released toxins which is considered problematic The problem with detecting the toxins after release is that approaches such as immunoassays are too insensitive to detect toxins in infected patients unless the levels are so high that death will be the result.B. Anthracis is a possible bioterrorism threat that can be deployed as spores which are very resistant to degradation. Opportunities exist to detect either the spore or the toxin (edema toxin, lethal toxin) after infection the detection of the spores would be required if a biosensor was to be able to detect the presence of anthrax spore in the atmosphere. To detect anthrax spores there are essentially three options. Firstly to detect dipicolinic acid (DPA) which constitutes most bacterial spores
which is a drawback considering that DPA constitutes most spores meaning the detection informs that bacterial spores are present. The second option is to detect surface futures (epitopes) on the spores which are specific to anthrax spores using affinity molecules such as antibodies. Exemplary is the presence of glycoproteins, on the spore surface, which differs between species and hence can be used as the epitope which antibodies bind to.For Francisella tularensis an Immune and nucleic acid markers are the most likely recognition molecules. For toxins like botulinum neurotoxin the most important analyte of this type of biological threat we can use protein antibodies that bind to it as the basics of an immunoassay.In the case of botulinum toxin an alternative bio recognition molecules are gangliosides.Botulinum toxin penetrates the cell membrane by binding at ganglioside on the membrane which makes them a perfect candidate for incorporation in a multipurpose biosensors .For viral detection the options are to identify nucleic acid markers or to use affinity ligands binding to the protein coat of the virus.(Joshi, Kumar, Maini, & Sharma, 2013)
Detection Methods:
Optical interferometers (refractive-index sensors):
Optical interferometers have features that make them versatile platforms for bio molecular sensing. Because refractive index is a fundamental material property, the interferometer becomes a sensing platform capable of responding to almost any physical change, chemical species, or biological agent via a change to the selectivity of the surface layer (Fig.1). Because the sensor is measuring each binding event, this real-time detection is also inherently quantitative, which can be valuable in some diagnostic applications. In addition, the penetration depth of the evanescent field is approximately half the wavelength of the coupled light wave, allowing discrimination between solution- and surface-bound biomolecules. The sensitivity provides perhaps the most exciting feature of these devices their ability to provide real-time information about the presence of target bio agents without the prerequisite for either wash steps or secondary labels. In other words, the devices can perform easy, fast assays even in complex biological fluids such as whole blood. Such simplicity of operation is a key component for a real-world counter-terrorism biosensor in both armed forces and civilian use.
Spectro Sens - TM sensors: The Spectro Sens TM sensor is a microchip sensor system which is capable of real-time, label-free detection of different varieties of biological agents. It has been demonstrated to be suitable for the detection of biological agent simulants including: proteins (ovalbumin<10mm); viruses (MS2<100nm); bacterial cells (E. Coli) and spores (Bacillus Atropheus).The Spectro Sens chip contain multiple high-precision planar Bragg gratings which function as a refractive index sensors.
Fig.l. Optical interferometer scheme. The Bragg gratings act as sensitive wavelength filters reflecting light at precisely defined
wavelengths. The sensitivity for a biological agent is accomplished by functionalizing the sensors sensing surface with antibodies selected agents the antigen (biological agent) of interest. The sensing surface is metal oxide coated and the immobilization to it is accomplished by a modified Amino-terminated silane monolayer activated by glutaraldehyde cross-linkage enabling covalent attachment of recombinant protein to which agent specific antibodies can be immobilized. Binding of target antigen (Biological agent) to the surface immobilized antibodies results in localized changes in refractive index; upon laser-induced interrogation of the sensing region via optical fibers, this antibody-antigen interactions manifest as increases in wavelength of light reflected from the Bragg grating. The large size range of detection targets is attributed to a large penetration depth of the sensing light of >1um into the sample liquid using these sensors.(Bhatta, Stadden, Hashem, Sparrow, & Emmerson, 2010)
UV light-emitting diode based fluorescence sensor: This particular type of bio particle sensors employ laser induced Fluorescence (LIF) analysis combined with the examination of the particles size and sometimes shapes. Such fluorescence detection systems are sensitive enough for the interrogation of a single aerosol particle and even possible discrimination between biological species. The principal of (LIF) system is based on spectrally selected excitation and detection of the fluorescence caused by the bio agent's natural bio fluorophores like aromatic amino acids and coenzymes. Light with a wavelength of 260-290 nm is used for excitation of aromatic amino acids(tyrosine, tryptophan, phenylalanine) with characteristic emission band in the range of 300-400nm.
Scattered light intensity Fig.2. UV-LIF scheme
Tryptophan
Tyros i n e,
T \ \ NADH
Phenylalanin e 1 Y\ Ri bofld vl n
rW*v / K
■JOO 500
Wavelength [nm]
Fig.3. Fluorescence intensity of biomolecules for 266 nm excitation. Adapted from (Joshi
et al., 2013)
Light with longer wavelength 350-450nm is used for the excitation of coenzymes (NADH, biotin) with a emission centered at about 465nm and at 560nm. Aromatic amino acids are present in almost all proteins determining the fluorescent characteristics of the biological agent. Most sensing systems utilize dual modes of wavelength excitations, deep UV and UV blue spectral region. In order to increase the identification confidence of fluorescence sensors to acceptable levels, improved recognition schemes that involve additional dimensions of fluorescence are required. The most advanced LIF systems combine due-wave length excitation with multichannel analysis of the fluorescence spectrum, single particle size and velocity recognition, and sophisticated signal processing. In addition, information on the particle shape can be extracted from the pattern of scattered light.
Currently there are marketed UV fluorescence based sensors two of them named AirSentinel® 1000B and The Biological Aerosol Warning System (BAWS).(Ryskevic et al., 2010)
AirSentinel: The AirSentinal is a lightweight, continuously operating airborne particle sensor that monitors for potentially harmful biological agents. The sensor uses a combination of particle count information, particle fluorescence, and an algorithm to determine whether an event is a suspected biological treat is released. AirSentinel runs continuously, analyzing a new aerosol sample approximately every 30 s to 10 min and response time is 30sec to 2 min depending on userset parameters. If a sample emits sufficient fluorescence, a second air sampler located inside the unit and operating at a higher flow rate is activated to capture a second sample for further analysis. A separate system, a laboratory is required to analyze the sample and identify the specific biological agent. AirSentinel® 1000B can detect multiple biological agents simultaneously, but because it is non-specific, it cannot identify the specific biological agents. The pre-filter is recommended in particle-heavy environments (e.g., mailrooms). A complete self-diagnostic system identifies the need for service and maintenance. Routine maintenance requires 30 min every six months. Collection kits have a shelf life of one year.
The Biological Aerosol Warning System (BAWS): A UV-fluorescence-based bio aerosol detection system that was developed for the US Military. The BAWS is an array of point biological aerosol detectors networked to detect biological threat agents. The system is intended for a remote, early detection for biological attack and for perimeter monitoring of key areas. BAWS is comprised of a network of Remote Detection Stations, a Base Control Station, and a PC Analysis Workstation, the sensor provides an overall picture of a developing biological attack with detection, wind-speed and direction, and location data. In addition, BAWS is configured to allow easy integration with other types of sensors (i.e., motion detectors, IR sensors, etc.) in order
to create a customized overall perimeter monitoring system.(Fatah)
Fiber optic based biosensor: Portable automated fiber optic biosensor has been developed over the last decade in order to meet the needs for a small, portable and easily operated biosensor for detection of biological threats in the field. The analytical method for detection is based on fluorescent sandwich immunoassays on the surface of short polystyrene optical probes. A monolayer of capture antibodies is immobilized on optical fibers. When the sample flows over the fiber probes, the immobilized antibodies capture a complementary analyte. Fluorescently labelled reporter antibodies bound to the analyte forms a fluorescent complex or "sandwich" The sensor monitors the formed complex by evanescently exiting the surface-bounds fluorophores with a diode laser. The advantage of using evanescent electromagnetic radiation is that analytes can be detected in real time as only fluorophores near the surface of the optical fiber will be excited, thus relaxing the requirements for separation of bound from free reporter antibodies. The optical probe captures a portion of the emitted fluorescence which returns back up the fiber to the photodiode detection. Excitation intensity and efficiently or the fluorescence recovery falls exponentially with distance from the fiber probe surface, which makes the system highly discriminatory for the surface bound fluorophores. Currently there is a commercialized fiber optic based biosensor used by the US Marine Corps named RAPTOR.
RAPTOR: A portable automated fiber optic biosensor for detection of biological threat agents. It performs rapid (3 to 10 minute), fluorescent sandwich immunoassays on the surface of short polystyrene optical probes for up to four target analytes simultaneously. The optical probes can be reused up to forty times, or until a positive result is obtained, reducing the logistical burden for field operations. Numerous assays for toxins, such as SEB and ricin, and bacteria, such as Bacillus Anthracis and Francisella Tularensis, have been developed. Research International has commercialized the RAPTOR,fFig.4./) and development of a second-generation instrument, sponsored by the US Marine Corps, is now in progress.(Anderson & Nerurkar, 2002) A disadvantage is that it is operator-dependent.
Fig.4. RAPTOR schematics Flame Spectrophotometry: Ambient air is burned with hydrogen gas; the flame decomposes any substance present in the air, and substances that contain phosphorus and sulfur produce hydrogen, phosphorus, and oxygen (HPO), and elemental sulfur (S), respectively. At the high flame temperature, the phosphorus and sulfur emit light of specific wavelengths. A set of optical filters is used to selectively transmit only the light emitted from the presence of phosphorus and sulfur to a photomultiplier tube that produces an analog signal related to the concentration of
the phosphorus- and sulfur-containing compounds in the air. Since living organisms contain phosphorus and sulfur, they are detected by this technology. Currently Proengin USA has created a sensor that uses flame spectrophotometry technology.
Biological Alarm Monitor (MAB): Sensor especially adapted to the rigors of a military defense. The Biological Alarm Monitor(MAB) Is adapted to harsh environmental conditions giving continuous measurement and raises a real time alarm on all changes in the biological background (bacteria, virus, toxins).The sensor is remote controllable by a RS485 data link. The sensor samples particles from 2 to 10 microns the sampling flow rate is 16 liters per minute and the response time is commonly 10 to 15 seconds with 1 minute threshold level.(Fatah)
In conclusion, advancement in countering biological agent treats is greatly reliant on the ability to rapidly detect threats with a high degree of accuracy, specificity and least effort. The gold standard is achieving the very difficult task of producing a biosensor with the ability to detect-to protect meaning providing a warning within minutes of possible contamination with biological treat agent and being able to sample analytes from complex bio-aerosols, giving response within a minute or two and giving very few false positives. The devises shown in this overview are not far from fulfilling all these criteria but are still lacking in some part. These overview vanguards the possibility of creating multipurpose biosensor that is more than capable in meeting the necessary requirements for detection of bioaerosols. The proposed rout in engineering such a device is true the incorporation of surface photo-charge effect (SPCE) as the main principle for detection.
Surface Photo Charge Effect (SPCE)
Surface photo charge effect is a phenomenon exerting its effect true irradiation with electromagnetic field different matters (solid, liquid and gas) and in the process charging them with an alternating potential difference, which frequency is equal to the frequency incident electromagnetic wave.(O. Ivanov, A.Vaseashta & Mihailescu,2008) Shortly after irradiation the material generates a signal specific to its type.(Grimes, Dickey, & Pishko, 2006) The voltage is measured contactless between the irradiated material and a second material whose potential is assumed to be zero. The wave length of the irradiation is selected depending on the conditions in order to exclude the contribution of the external photoelectric effect. SPCE is a very fast effect. Irradiation for 20ns with a laser pulse creates a signal response which reproduces the waveform of the incident pulse. The main idea that is prospected is due to the strong susceptibility of this effect to the state of the irradiated matter, each change in the liquid or gas contacting surface would cause a change in observed signals.(O. IVANOV & KONSTANTINOV, 2000) Studies of SPCE revealed that the signal amplitude is strongly dependent on the properties of the illuminated surface. (O. Ivanov & M. Kuneva, 2011) This makes possible various practical applications like (1) visualization of implanted regions in solids (2) detection of mechanical imperfections of semiconductors and metal surfaces as well the presence of impurities in those surfaces (4) automatic visualization of the electron surface topography (5) investigation of surface electron states via the temperature dependence of the SPCE (6) possibility to monitor the octane factors of gasoline impurities in liquids and gases.(O. D. Ivanov & Konstantinov, 2002) The essence of the idea is to put the irradiated solid surface in contact with the investigated fluid or gas. Since the electron properties at a solid surface influence essentiality the interface with an overlying fluid or gas layer, one can expect that optically excited changes in such a system will provoke measurable signals. In this way, with all other conditions fixed, it is possible to detect changes in the fluid or gas properties. It is reasonable to expect that any kind of changes in the fluid or gas will cause corresponding changes at the exposed interface, thus changing the SPCE.(O. IVANOV & KONSTANTINOV, 2000). In contrast there is enough date collected which makes creation of SPCE based sensors feasible in the near future.
Acknowledgements:
This work has been funded by EU FP7 Security program under contract 312804. References:
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