WO2012123936A2

WO2012123936A2 - Devices and methods for real time detection of chemical and biological gaseous hazards

Info

Publication number: WO2012123936A2
Application number: PCT/IL2012/000118
Authority: WO
Inventors: Raphael Zvi MAYER; Avishai Levy; Ofra Levy-Hacham
Original assignee: IDS Innovative Defence Systems ltd.
Priority date: 2011-03-17
Filing date: 2012-03-14
Publication date: 2012-09-20
Also published as: WO2012123936A3

Abstract

Systems, methods and devices are provided for real-time monitoring of a chemical or pathogen presence and/or level in a gaseous environment. The system comprises at least one fixed or portable device adapted to monitor conformational changes of a crystal composed of protein receptor or antibody detected optically. In one embodiment, the system may comprise an air pump, at least one crystalline protein, a light source, a light monitoring device, an energy source, a processor and alarm.

Description

DEVICES AND METHODS FOR REAL TIME DETECTION OF CHEMICAL AND BIOLOGICAL GASEOUS HAZARDS

FIELD OF THE INVENTION

The present invention relates generally to systems and methods for detecting chemical and biological hazards, and more specifically to methods and apparatus for real-time detection of gaseous-phase borne hazards or pathogens.

BACKGROUND OF THE INVENTION

There is a growing concern that gaseous-phase borne hazards may harm people by means of accident, war or bioterrorism. These hazards may include chemical hazards such as gases including, but not limited to pesticides, nerve gases, and cyanides. Moreover the gaseous-phase borne hazards may include biohazards such as, but not limited to pathogen-like viruses and virulent bacteria in an aerosol or other gaseous phase. There is therefore a need to provide a device, which can be placed in any place where such a device may be required, such as, but not limited to, crowded places, and which is adapted to provide real-time alerts and/ or inputs about the existence and possibly the identity and/or the concentration of the gaseous-phase borne hazards. Due to the increasing tendency of terror organizations to execute mass-terror attacks, and the increase in pollutants, pesticides, herbicides on the one hand, and on the other hand, there is a growingly urgent need to detect these gaseous-phase borne hazards.

There are many known techniques to detect and quantify chemicals, such as nerve gas or pollutants in liquid-phase samples or in a gas phase. Most present realtime detectors for gas phase analysis are based on different physical properties of the target molecule /using diverse methodologies, which often lack selectivity and further suffer from different signal to noise malfunctions and relatively high prices for a unit or a full system. Several detectors are known in the art, such as biosensors. Several attempts were recorded for the use of biosensors to detect organophosphates ([Turkish Journal of Biochemistry-Turk J Biochem] 2010; 35 (1); 72-74.) including expression of acetylcholinesterases in vitro and use as a biosensor for detecting organophosphates and carbamate insecticides (10). For this purpose, enzyme expression has been made from bovine erythrocyte, Electrophorus electricus, Drosophilia melanogaster, Torpedo California and Caenorhabditis elegance. As a result, it has been found out that D. melanogaster is eight times more sensitive to the enzyme than E. electricus, and this sensitivity may increase up to twelve times due to a mutation in 408th position. (Villatte F, Marcel V, Estrada-Mondaca S, Fournier D. (1998) Engineering sensitive acetylcholinesterase for detection of organophosphate and carbamate insecticides, Biosens Bioelectron. 13(2):157-64.)

Similarly, a biosensor has been developed, based on the method of potentiometric enzyme electrodes to provide direct measurements of organophosphorus nerve agents. In this biosensor system, organophosphor^ous (OP) hydrolase is immobilized via cross-links with bovine serum albumin and glutaraldehyde and bound to a pH-electrode. It is based on the measurement of the amount of protons resulting from the hydrolysis of the enzyme in proportion to the concentration of enzyme substrate. The best sensitivity and response time in this method has been achieved by the sensor operating in 1- mM HEPES buffer, at a pH of 8.5, which has been developed with 500 IU organophosphorus hydrolase. Similarly, it has been found that such organophosphoric compounds including 2 μΜ paraoxon, ethyl parathion, methyl parathion and diazinon have been detected/measured under similar conditions (Mulchandani P, Mulchandani A, Kaneva I, Chen W. (1999) Biosensor for direct determination of organophosphate nerve agents.1.Potentiometric enzyme electrode. Biosens Bioelectron. 14(1): 77-85). In addition, for detecting OP nerve agents, a fiber-optic microbial biosensor has been developed by using recombinant E. coli cells. It is based on the detection of end products obtained as the result of organophosphorus hydrolysis catalyzed by OP hydrolase expressed in the cell surface. It has been pointed out that this system is advantageous in comparison to traditional microbial sensors and enzyme-based sensors because of the fact that such cells containing metabolic enzymes on the surfaces are used, and further that analytes and products do not show any resistance during the transition through cell membrane. Such a sensor may be cost effective due to the elimination of the requirement for enzyme purification (13= Mulchandani A, Kaneva I, Chen W. (1998) Biosensor for direct determination of organophosphate nerve agents using recombinant Escherichia coli with surface-expressed organophosphor^ous hydrolase. 2. Fiber-optic microbial biosensor. Anal Chem. 70(23): 5042-6.).

Additionally, in another biosensor developed, OP pesticides and recombinant E. coli cells have been used to directly identify OP neurotoxins and to hydrolyze chemical warfare agents having this structure. This biosensor incorporates a potentiometric system, which has been used as transducer (13).

US patent 6,821,738 discloses a method for rapidly detecting nerve agents, and organophosphates analytes that can be detected by monitoring optical changes in the absorbance and/or fluorescence of heterocyclic compounds. The invention pertains generally to a method and apparatus for rapidly detecting nerve agents, organophosphates, and other chemical warfare agents. A sensor has been developed that can be used to rapidly detect multiple analytes such as organic compounds. Analytes can be detected by monitoring changes in the optical properties of the absorbance and/or fluorescence spectra of highly colored heterocyclic compounds such as porphyrins or related compounds such as phthalocyanines. The result is a real- time monitor that is suitable for use in situations where encounter with chemical warfare agents is possible. The technological and scientific principles of US patent 6,821,738 are significantly different from the technology and scientific principles that form the basis of this invention. The detection method and monitoring of the present invention is advantageous over US patent 6,821,738, inter alia, with respect to its specificity and accuracy.

To date, enzymatic tests are non-continuous and thus can perform detection but not real-time monitoring of acetylcholinesterase inhibitors. SUMMARY OF THE INVENTION

It is an object of some aspects of the present invention to provide devices and methods for real time detection of chemical and biological gaseous hazards. The present invention described herein aims to utilize the binding capacity of proteins in a solid phase for both detection and monitoring of a ligand of interest. The monitoring device enables real-time monitoring. The real-time monitoring is a continuous operation that saves the need to perform detection.

In some embodiments of the present invention, improved methods and apparatus are provided for detecting gaseous-phase borne hazards or pathogens.

In some embodiments of the present invention, improved methods and apparatus are provided for real-time monitoring of gaseous-phase borne hazards or pathogens.

Furthermore, according to some embodiments of the present invention there is provided a method for real-time detection of a gaseous-phase-borne hazard, said method comprising: a. providing incident light onto at least one crystalline protein; b. receiving at least one signal associated with a light output responsive to at least one change in said least one crystalline protein; and c. analyzing said at least one signal indicative of said gaseous-phase- borne hazard thereby enabling real-time detection of said gaseous- phase-borne hazard.

In one embodiment, the invention provides a device for real-time monitoring of a chemical in a gaseous environment, which is detected at a level of at least a harm level. The device comprises of at least one crystalline protein, at least one light source and at least one light monitoring device. The device may further include a processor. The device may further include an energy source.

In one embodiment of the present invention, the device further comprises an air pump that pushes air from the surrounding environment to the direction of detector which also termed herein a "sniffer". In yet another preferred embodiment the device further comprises an alarm.

Some embodiments of said present invention provide a system for real-time detection of a gaseous-phase-borne hazard, said system comprising: a) at least one crystalline protein;

b) at least one light source adapted to provide incident light onto said at least one crystalline protein; and

c) at least one light monitoring device adapted to receive a light output from said at least one crystalline protein, wherein said at least one light monitoring device is adapted to provide at least one signal relating to said light output responsive to said at least one change to said least one crystalline protein indicative of said gaseous-phase- borne hazard thereby enabling real-time detection of said gaseous- phase-borne hazard.

The present invention will be more fully understood from the following detailed description of the preferred embodiments thereof, taken together with the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in connection with certain preferred embodiments with reference to the following illustrative figures so that it may be more fully understood.

With specific reference now to the figures in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

In the drawings:

Fig. 1 is a simplified pictorial illustration showing a system for real time detection of chemical and biological gaseous hazard, in accordance with an embodiment of the present invention.

Fig. 2 is a simplified flow chart of a method for real time detection of chemical and biological gaseous hazards, in accordance with an embodiment of the present invention.

Fig. 3 is a simplified flow chart of a method for preparing a crystalline protein as a prism in the system of Fig. 1.

In all the figures similar reference numerals identify similar parts.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In the detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that these are specific embodiments and that the present invention may be practiced also in different ways that embody the characterizing features of the invention as described and claimed herein.

The current invention is partially based on a new and inventive application of the fact that crystals of proteins including antibodies, like other crystals in nature can be built in a way to serve as an optical prism. The protein/antibody crystals may be constructed and configured to form transparent optical elements. These optical elements may comprise one or more of a flat surface, constructed and configured to emit and/or reflect light (Acetylcholinesterase, Ii. Crystallization, Absorption Spectra, Isoionic Point. Walo Leuzinger, A. L. Baker, T And Elsa Cauvin. Biochemistry 1968. 59:620-623, Structural Studies On Acetylcholinesterase And Paraoxonase Directed Towards Development Of Therapeutic Biomolecules For The Treatment Of Degenerative Diseases And Protection Against Chemical Threat Agents Joel L. Sussmanl,2*, Israel Silman2,3, From Molecules To Medicines, 18:183-199 © Springer Science + Business Media B.V. 2009. Another type of molecule relevant for detection purposes are immunoglobulins. From biological point of view, antibodies has a crucial role in protection human and animals against invading bacteria and viruses and it is therefore easy to develop antibodies capable of recognizing specifically different types of bacteria and viruses. Indeed over the years many such monoclonal antibodies were developed for diagnostics purposes (Monoclonal antibodies against viruses and bacteria: a survey of patents. Xiao X, Dimitrov DS. Recent Pat Antiinfect Drug Discov. 2007 Nov;2(3): 171-7.). These antibodies are widely used and supplied by many companies and are also used by others to detect bio-terror agent (http://www.ncbi.nlm.nih.gov/books/NBK36641/; Bioterrorism Preparedness and Response: Use of Information Technologies and Decision Support Systems. Evidence

Reports/Technology Assessments, No. 59. Bravata DM, McDonald K, Owens DK, et al. Rockville (MD): Agency for Healthcare Research and Quality (US); 2002 Jun.). The ability to crystallize such antibodies was well established (Human antibody-Fc receptor interactions illuminated by crystal structures. Woof JM, Burton DR. Nat Rev Immunol. 2004 Feb;4(2):89-99.). This, in principle, (there is a huge difference between this poposed concept and the previous on due to the size difference of small moeMecule and bacteria) enables the use of these antibodies methodologies by incorporating the crystalline antibodies into the device of the present invention and using it to detect a bacterial or viral threat in real-time with use of no other reagent.

Yet another issue is how to detect the binding of the ligand to a pre- crystallized protein or antibody. One possibility documented in scientific literature is to preload the crystal with a fluorescent ligand and thus to monitor competition between present ligand and other competing compounds. Another possibility is to monitor changes in the crystal morphology and phenotypes due to the chemical/physical interaction with the specific ligand. It is well known in the literature, that binding of a ligand to protein cause a conformational change due to the chemical interaction in the binding site. The conformational changes were detected in crystal structure study were the X-ray analysis of the bound protein revealed different conformation (PLoS One. 2010 Feb 19;5(2):e9330.Roles of electrostatics and conformation in protein-crystal interactions Azzopardi PV, O'Young J, Lajoie G, Karttunen M, Goldberg HA, Hunter GK).

Due to the increasing tendency of terror organizations to execute mass terror attacks, on the one hand, and the increase in pollutants, pesticides and herbicides, on the other hand, the need for efficient solution for real-time detection of chemical agents and pathogens in a gaseous environment is constantly growing. Reference is now made to Fig. 1, which is a simplified pictorial illustration showing a system 100 for real time detection of chemical and/or biological gaseous hazard, in accordance with an embodiment of the present invention.

The system comprises a light source 102, which impinges light rays 104 onto a optic unit 108. Optic unit 108 houses a protein crystal 110. The crystal may receive light 104 directly or indirectly via second light rays 106. The crystal is constructed and configured to form output light rays 112, which may be detected directly by optical detector 114. Additionally or alternatively, the rays may be transformed in unit 108 into secondary rays 113, which are detected by the detector.

In one embodiment of the present invention, system 100 further comprises an air pump 118, which pushes air 121 from the surrounding environment to the direction of receiving unit. The pump is also termed herein, a "sniffer".

The optical detector passes output signals 115 to a processor 116. The processor is constructed and configured to analyze the signals and to determine whether a hazard is present.

The processor sends outputs 117, 119 to an alarm 120 and to a display 122, respectively. These outputs are indicative of the hazard status.

Reference is now made to Fig. 2, which shows a simplified flow chart 200 of a method for real time detection of chemical and biological gaseous hazards, in accordance with a embodiment of the present invention. This flowchart is explained with reference to system 100 of Fig. 1.

In an impinging step 202, energy /light 104 is impinged onto protein crystal prism 110. This step may include sub-steps such as filtering, concentrating, and deflecting, refracting, collimating or other optical process steps as are known in the art.

In a light passing step 204, energy/light passes through and/or reflects off crystal 110, thereby forming at least one of output light rays 112 and secondary rays 113. In parallel air pump 118 passes air 121 from the environment into receiving unit.

In a detection step 206, optical detector 114 detects light rays 112 and or 113. It should be understood that this step may also include sub-steps such as filtering, concentrating, deflecting, refracting, collimating or other optical process steps as are known in the art.

If there is a hazard present, the structure of crystal 110, which is sensitive to the hazard, will change. This, in turn will result in a change in at least one of the properties of rays 112, 113. Thus these changes will be detected by optical detector 114. The detection may be a quantitative and or qualitative detection, as is known in the art (see references listed herein below, which are incorporated herein by reference). In a checking step 212, the processor is constructed and configured to analyze output signals 115 from the detector to see if they are outputs in safe range. For example if the crystal conformation, in the absence of a hazard, provides certain ray/beam wavelengths 112, 113. If the detected values by detector 114 fall in this range, then output signals 115 will be within a predefined safe range.

In some embodiments, if the outputs are within the safe range, a waiting step 214 will ensue, and then steps 202-212 will be repeated.

If the conformation of the crystal is deformed/changed due to the presence of one or more hazards then output signals 115 will fall out of the predefined safe range.

In the latter case, an activating step 216 will ensue and the processor will provide at least one of a display instruction 119 to display 122 and an alarm signal 117 to alarm 120. This activating step may further include pumping fresh air into the region of the system via air pump 118 and other steps, such as dialing to emergency services. It should be understood that the air pump may be air pump 118 (Fig. 1) and or any other ventilation systems in the vicinity of system 100. Additionally or alternatively, suction systems may be activated to remove air from the vicinity of system 100 and concurrently introduce new filtered air to that vicinity.

Turning to Fig. 3, there is shown a simplified flow chart 300 of a method for preparing a crystalline protein as prism 110 in system 100 as shown in Fig. 1.

In a protein/enzyme/antibody (P/E/A) obtaining step 302, a P/E/A is obtained which has predefined reaction kinetics with a biohazard 101. For example, the biohazard may be a competitive or non-competitive inhibitor of an enzyme (such as carbon monoxide and haemoglobin).Regardless of the mechanism, conformational changes are predicted to occur.

The protein obtained in the previous step is then crystallized under certain conditions in a crystallizing step 304 to form crystals of the P/E/A. In some examples, crystals are formed in solution under highly saturated conditions, as is known in the art.

The crystals are then isolated, such as by filtering, centrifuging, precipitating out of solution by any one or more methods, known in the art in an isolating step 306.

In some cases, further optional crystal processing steps 308 are performed to form optic unit 108. These steps may include, selecting a crystal size, selecting crystals without lattice defects, selecting crystals without other defects, finishing processes applied to the crystals, as is known in the art.

In a setting step 310 the optical device/crystal 108 as formed by the above steps is set in optic unit 110 (Fig. 1).

In one embodiment, the invention provides a device for real-time monitoring of a chemical level in a gaseous environment. The device composed of at least one crystalline protein, at least one light source and at least one light monitoring device. The device may further include a processor. The device may further include an energy source.

In one preferred embodiment the device further comprises an air pump, which is constructed and configured to push air from the surrounding environment to the direction of detector which also called a "sniffer".

In yet another preferred embodiment the device further comprises an alarm. The light source that target a light been towards the crystal may be of various different light generating instruments and a variety of wave length. Such light sources can be, without limitation, selected from the group consisting of a laser, a light- emitting diode, an organic light-emitting diode, a polymer light-emitting diode, a solid-state light, an LED lamp, an electroluminescent sheet, an electroluminescent wire, a carbon button lamp, a conventional incandescent light bulb, a flashlight, a halogen lamp, a globar, a fluorescent lamp, a phosphorescent lamp, an argon lamp, an argon flash, an acetylene/carbide lamp, a betty lamp, a butter lamp, a candle, a flash powder, a gas light, a gas mantle, a kerosene lamp, a lanterns, a limelight, an oil lamps, a rushlight, a safety lamps, a Davy lamp, a Geordie lamp, a torches, a common household incandescent light bulb, a filament of incandescent close-up light bulb, an Electron Stimulated Luminescence (ESL light bulbs), a cathode ray tube (CRT monitor), a Nixie tube, a gas discharge lamp, a fluorescent lamp, a compact fluorescent lamp, a black light, an inductive light, a hollow cathode lamp, a neon and argon lamp, a plasma lamps, a xenon flash lamp, a carbon arc lamp, a ceramic discharge metal halide lamp, a hydrargyrum medium-arc iodide lamp, a mercury- vapor lamp, a metal halide lamp, a sodium vapor lamp, a xenon arc lamp and combinations thereof.

According to some embodiments, the device may also include at least one filter to reduce noise from a non-specific reaction of one or more non-related particles with the crystal.

The invention also provides a method for monitoring a chemical level above threshold and potentially, a range of levels, in a gaseous environment. The method comprises monitoring conformational changes in a protein crystal or in an antibody crystal or in a macromolecule crystal as reflected in changes in the wave length, spectral, rotational or otherwise optical properties of the crystal following the binding of said at least one chemical molecule to the protein crystal. In another aspect of the invention the invention also provides a method for monitoring of a chemical level in a gaseous environment. According to some embodiments of the present invention, the method comprises: a) providing at least one protein crystal and at least one light source; b) directing a light beam from the at least one light source towards at least one protein crystal; and c) measuring optical changes in at least one of a reflected wave length, a spectral property, a rotational property and another optical property of the at least one protein crystal.

In yet another embodiment, the method further comprises analyzing at least one of the optical changes and other data by means of a processor. The method may further comprise activating an alarm responsive to the measured optical changes.

The method may further comprise activating an air pump responsive to the measured optical changes.

In a preferred embodiment, the method is a real-time method. In any of the methods and devices provided in the invention, the light source can be applied form different sources and origins and of different wave length. Such light sources can be, without limitation, selected from the group consisting of a laser, a light-emitting diode, an organic light-emitting diode, a polymer light-emitting diode, a solid-state light, an LED lamp, an electroluminescent sheet, an electroluminescent wire, a carbon button lamp, a conventional incandescent light bulb, a flashlight, a halogen lamp, a globar, a fluorescent lamp, a phosphorescent lamp, an argon lamp, an argon flash, an acetylene/carbide lamp, a betty lamp, a butter lamp, a candle, a flash powder, a gas light, a gas mantle, a kerosene lamp, a lanterns, a limelight, an oil lamps, a rushlight, a safety lamps, a Davy lamp, a Geordie lamp, a torches, a common household incandescent light bulb, a filament of incandescent close-up light bulb, an Electron Stimulated Luminescence (ESL light bulbs), a cathode ray tube (CRT monitor), a Nixie tube, a gas discharge lamp, a fluorescent lamp, a compact fluorescent lamp, a black light, an inductive light, a hollow cathode lamp, a neon and argon lamp, a plasma lamps, a xenon flash lamp, a carbon arc lamp, a ceramic discharge metal halide lamp, a hydrargyrum medium-arc iodide lamp, a mercury- vapor lamp, a metal halide lamp, a sodium vapor lamp, a xenon arc lamp and combinations thereof.

It is obvious to those skilled in the art that the method and devices of the invention can be applied for a variety of chemicals, pathogens and ligands, including, but not limited to, peptides, proteins, viruses and bacteria. For explanatory purposes and without limitation, the chemical agents detected by the system can be selected form the group consisting of organo phosphorus-derived nerve gas, cyanide-derived nerve gas, nitric oxide-derived gas, any biological agent in aerosol forms such as Butolinum, Anthrax- Bacillus anthracis, Clostridium botulinum and toxin, Yersinia pestis, Variola major, Francisella tularensis, filovirases [e.g., Ebola, Marburg] and arenaviruses [e.g., Lassa, Machupo], Brucella species, Epsilon toxin of Clostridium perfringens, Salmonella species, Escherichia coli 0157:H7, Shigella, Burkholderia mallei, Burkholderia pseudomallei, Chlamydia psittaci, Coxiella burnetii, Ricin toxin from Ricinus communis, Staphylococcal enterotoxin B, Rickettsia prowazekii, alphaviruses [e.g., Venezuelan equine encephalitis, eastern equine encephalitis, western equine encephalitis, Vibrio cholerae, Cryptosporidium parvum. Adenoviridae, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, Togaviridae, Mycobacterium tuberculosis, Campylobacter, Bordetella pertussis, Borrelia burgdorferi, Campylobacter jejuni, Chlamydia pneumonia, Chlamydia trachomatis, Clostridium tetani, Corynebacterium diphtheria, Enterococcus faecalis, Enterococcus faecium, Enterotoxigenic Escherichia coli, Haemophilus influenza, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycoplasma pneumonia, Neisseria gonorrhoeae, Neisseria meningitides, Pseudomonas aeruginosa, Rickettsia rickettsii, Shigella sonnei, Staphylococcus aureusa, Streptococcus agalactiae, Streptococcus pneumonia, Streptococcus pyogenes, Treponema pallidu and Yersinia pestis. In yet another embodiment of the invention any of the method and devices of the invention may also include a filter to reduce the possible non specific reactions.

In yet another embodiment a system is provided for monitoring of a chemical level in a gaseous environment. The system may comprise at least one fixed or portable device adapted to monitor changes in wave length, spectral, rotational or otherwise optical properties of a protein crystal following the attachment of a chemical to the protein crystal.

In another specific embodiment of the invention, a method of detecting and monitoring the presence of an amount of cholinesterase inhibitor/ligand in a sample comprising the steps of: (a) contacting the cholinesterase inhibitor/ligand with crystallized acetylcholinesterase; and (b) monitoring the cholinesterase inhibitor/ ligand by way of changes induced to the crystallized protein chemistry and/or geometry.

In a preferred embodiment of the invention, there is provided a device for realtime monitoring of a pathogen concentration in a gaseous environment, the device comprises at least one of crystallized antibody or crystallized biological macromolecule, at least one light source and at least one light monitoring device.

This device may further comprise at least one additional feature selected from the group consisting of: a processor, an energy source, an air pump, an alarm and a filter to reduce possible non-specific reactions. Similar to all the methods and devices provided in the invention the device can include and be operated by different types of light sources. Such light sources can be, without limitation, selected from the group consisting of a laser, a light-emitting diode, an organic light-emitting diode, a polymer light-emitting diode, a solid-state light, an LED lamp, an electroluminescent sheet, an electroluminescent wire, a carbon button lamp, a conventional incandescent light bulb, a flashlight, a halogen lamp, a globar, a fluorescent lamp, a phosphorescent lamp, an argon lamp, an argon flash, an acetylene/carbide lamp, a betty lamp, a butter lamp, a candle, a flash powder, a gas light, a gas mantle, a kerosene lamp, a lanterns, a limelight, an oil lamps, a rushlight, a safety lamps, a Davy lamp, a Geordie lamp, a torches, a common household incandescent light bulb, a filament of incandescent close-up light bulb, an Electron Stimulated Luminescence (ESL light bulbs), a cathode ray tube (CRT monitor), a Nixie tube, a gas discharge lamp, a fluorescent lamp, a compact fluorescent lamp, a black light, an inductive light, a hollow cathode lamp, a neon and argon lamp, a plasma lamps, a xenon flash lamp, a carbon arc lamp, a ceramic discharge metal halide lamp, a hydrargyrum medium-arc iodide lamp, a mercury-vapor lamp, a metal halide lamp, a sodium vapor lamp, a xenon arc lamp and combinations thereof.

In yet another embodiment, the invention provides a system for real-time monitoring of a pathogen concentration in a gaseous environment, the system comprising: a) at least one of an antibody crystal or a crystallized biological macromolecule forming an optical element; and b) at least one fixed or portable device adapted to monitor conformational changes reflected in said optical element; said at least one device being adapted to provide real-time detection of said pathogen concentration.

The system may further comprise at least one of a light source, a light monitoring device, an energy source, a processor, an alarm and combinations thereof.

In a preferred embodiment the invention provides a method of detecting and monitoring the presence of an amount of bio warfare agents or any pathogen in a gaseous environment comprising the steps of: (a) allowing the pathogen to ligand with crystallized antibody or crystallized macromolecule; (b) monitoring by way of changes induced to the crystallized protein chemistry and/or geometry in space.

In yet another embodiment the invention provides a method of measuring the changes in conformation of the prism of the crystallized antibody or crystallized macromolecule by way of monitoring the light diffraction, the light wave length, or the light intensity at a certain wave length by the crystal diffraction.

In yet another embodiment the invention provides a method of measuring the changes in conformation in the geometry of the prism of the crystallized protein by way of monitoring the light diffraction, the light wave length, or the light intensity at a certain wave length by the crystallized protein diffraction.

In yet another embodiment the invention provides a method of measuring the changes of the crystallized protein transition medium by way of monitoring the light diffraction, the light wave length, or the light intensity at a certain wave length by the crystallized protein diffraction. In another embodiment the invention provides a device composed of a crystal protein that has the ability to bind gas of certain chemical composition. For example, the crystal protein maybe acetylcholine esterase and the ligand gas maybe organophosphate with high affinity to acetyl cholinesterase. The crystal protein maybe a cytochrome and the ligand gas may be a cyanide derivative with high affinity for the cytochrome. The crystal protein maybe hemoglobin and the ligand gas maybe nitrogen oxide with a high affinity for hemoglobin.

In yet another embodiment the invention provides a method of detecting and monitoring the presence of an amount of a cytochrome inhibitor/ ligand in a sample comprising the steps of: (a) contacting the cytochrome ligand with a crystallized cytochrome; and (b) monitoring the cytochrome -ligand by way of changes induced to the crystallized protein chemistry and/or its geometry in space.

In yet another embodiment the invention provides a method of detecting and monitoring the presence of an amount of an antigen in a sample comprising the steps of: (a) contacting the antigen with a crystallized antibody; (b) monitoring an antigen- antibody recognition/interaction by way of changes inflicted to the crystallized protein chemistry and/or its geometry in space.

In yet another embodiment, the invention provides a device that include a unit that delivers an air stream air onto a crystal unit .The crystal unit may be termed herein "a sniffer". The crystal unit may further include filters to reduce non-specific reactions of non-related particles with the crystal unit.

In yet another embodiment, the crystal protein within the device retains ability to bind to protein ligands. In yet another embodiment of the invention, the binding of ligand to the protein crystal within the device induces conformational changes that alter in the protein crystal, thereby altering at least one optical characteristic of the protein crystal.

The device is adapted to monitor the changes in the optical properties of the crystallized protein as reflected for example in its prism, spectral or otherwise optical properties of a crystal composed of the chemical molecule protein receptor. The monitoring ability of the device may include light source such as laser or

LED, adapted to illuminate the crystal structure with light with defined optical properties such as specific wave length(s) and/ spectra with or without the use of specific filters. It also includes light detector such as PMT or CCD with or without specific filers able to detect by binding of the ligand to the crystal. The changes in the light maybe change in wave-length (shift) or intensity.

In yet another embodiment the invention, the light from the detector is translated into electrical signal by light detector such as, a PMT and transferred into the processor. The processor may be part of the device or a separate computer. If the processor identifies a predefined change in the signal, an alarm may be activated. The current invention is partially based on a new and inventive application of the fact that crystals of proteins and antibodies, like other crystals in nature, can be built in a way to serve as an optical prism. These crystals may be transparent optical elements with fiat, polished surfaces, which refract light. The exact angles between the surfaces depend on the protein. Since proteins are composed of amino acids, they are transparent to the wavelengths in the visible spectrum. The protein crystal prism can be used to break light up into its constituent spectral colors. Prisms can also be used to reflect light, or to split light into components with different polarizations. Similarly to other proteins, acetylcholine esterase (AChE) crystals may be in the form of prisms. They have regular hexagonal prisms and thus may be unique and/or have unique optical properties.

AChE binds a ligand after the protein is crystallized: In the last eighteen years after the 3D structure of TcAChE was elucidated, the structures of over forty complexes and conjugates of a broad repertoire of inhibitors with this enzyme, were elucidated. In most cases, the ligand-AcChE structures were obtained by soaking the inhibitor into native crystals. Based on this well-established phenomenon, a device has been configured and constructed, utilizing the physical phenomena associated with optical changes occurring when crystal proteins to bind their ligand.

Another type of molecules relevant for detection purposes are immunoglobulins. From biological point of view, antibodies have a crucial role in protection human and animals against invading bacteria and viruses. It is therefore easy to develop antibodies capable of recognizing specifically different types of bacteria and viruses. Indeed, over the years, many such monoclonal antibodies were developed for diagnostics purposes. Advantages of reaction of crystal protein:

For any chemical reaction, the rate law is an equation which links the reaction rate with concentrations of reactants. As the chemical reaction of the analyte is given in any detection system based on acetylcholinesterase, the rate is dependent on the acetylcholinesterase concentration. The ultimate highest concentration of a protein is in a form of organized crystal and thus, the device based on binding abilities of the crystal may possibly have the highest reaction rate.

The present invention includes the device illustrated in Figure 1.

In the basis of this invention is the claim that conformational changes in the protein leads to change in the omitted light properties such as wavelength, the same phenomena was observed in an organic crystal (Kossover et al.).

The device for real-time monitoring of a chemical or pathogen presence and/or concentration in a gaseous environment, may be fixed or portable.

The device may include a unit which provides an air stream onto the crystal unit (see Fig. 3 below).The unit is termed sometimes a "sniffer", which may also include filters to reduce non-specific reactions of non-related particles with the crystal.

The device comprises a protein crystal, which has the ability to bind gas of certain chemical compositions. For example, the crystal protein may be acetylcholine esterase, a cytochrome, a hemoglobin or any other suitable protein. The ligand gas maybe organophosphate, cyanide, nitrogen oxide or any other suitable gas, with a high affinity for the protein.

The device is adapted to monitor conformational changes reflected in prism, spectral or otherwise optical properties of a crystal composed of the chemical molecule protein receptor. The device includes light source, such as laser or LED, able to illuminate the crystal structure with light with defined optical properties such as specific wavelength(s) and/ spectra, with or without the use of specific filters. The device also includes a light detector such as a photomultiplier tube (PMT) or charge coupled device (CCD), with or without specific filers, the detector being able to detect by binding of the ligand to the crystal. The changes in the light maybe change in wave-length (shift) or intensity.

The light from the detector is translated into electrical signal by the PMT and transferred into the processor. The processor may be part of the device or a separate computer. If the processor identifies predefined change in the signal, an alarm may be activated.

A method of using the above described monitors/detectors to identify ligands of acetylcholinesterase threats such as nerve gases or other such ligands such as insecticides. A method is provided to measure the amount of the threat in terms of its effect on acetylcholine esterase. Here, we demonstrate a unique feature of our device. It measures the amount of threat rather then the molar amount or actual concentration of the threat. For example, the system is constructed and configured to discriminate between small amounts of insecticide - non dangerous to human and small amounts of nerve gas dangerous to humans. Large amount of the insecticide dangerous to humans will however, activate the alarm system. The invention further encompasses a method of connecting the device/monitor to a data processing unit, connected to alarm system.

DEFINITIONS

Protein - A sequence of amino acids links between each other by peptide bonds in a linear chain called polypeptide chains. Amino acids - this peptide bond is formed between the carboxyl and amino groups of neighboring amino acids. Proteins are formed by one or several polypeptide chains.

Macromolecule - A large molecule (above 1000 KD) created by some form of polymerization of repeated unit such as amino acid or a saccharide. The repeated unit can be one or more. For example polylysine has a repeated unit of lysine but most protein are composed of many different amino-acids polymerized together. Some Macromolecules consist of more than one type of monomer (the repeated unit of macromolecule, mono=single, meros=part). For example, glycoproteins are composed of monomers of amino acids and monomers of saccharides.

Crystal - Highly symmetrical and periodically repeated pattern of atoms or molecules in a solid form. "Protein Crystal" - Protein molecules aligned in a repeating series of unit cells in a solid form by adopting a consistent orientation. Under this definition the protein molecule maybe of any size from dimer (two amino acid molecules) to large molecular weight polypeptide composed of any number of repeated monomelic unit. The protein may be composed of one or more polypeptides termed, oligo polypeptide. The oligo polypeptide maybe either homopolypeptide were the protein is composed of several different polypeptides or hetero-polypeptide were the protein is composed of several different polypeptides.

"Antibodies Crystal" - Immunoglubulin protein molecules aligned in a repeating series of unit cells in a solid form by adopting a consistent orientation is termed antibodies crystal. The immunoglobulin can be of any biological origin including human and animal as well as recombinant. In most cases antibodies crystal are composed monoclonal antibodies but may be also composed from several similar monoclonal or even polyclonal antibodies.

1. Chemical warfare gases - Gases with toxic properties used as chemical weapons. A chemical used in warfare is called a chemical warfare agent (CWA). Many such chemicals exist but the most used in mass destruction are phosphorus-containing organic chemicals (organophosphates) termed nerve gases. They block and disrupt the mechanism by which nerves signal messages to cells. The disruption is caused by blocking acetyl cholinesterase, an enzyme that normally modulates the activity of the neurotransmitter acetylcholine. Another example of warfare guess is cyanide derived molecules, they block and disrupt the mechanism by which Cytochrome molecules are working in the cell signaling pathway. Another example of non classical warfare guess can be Botulinum or other toxins. Other examples are Selected from the list consisting of but not limited : Cyclosarin (GF), Sarin (GB), Soman (GD), Tabun (GA), VX, VR, Some insecticides, Novichok agents, Arsines, Cyanogen chloride, Hydrogen cyanide, Sulfur mustard (HD, H), Nitrogen mustard, Lewisite (L), Phosgene dxime, Chlorine, Hydrogen chloride, Nitrogen oxides, Phosgene, Tear gas, Pepper spray, Agent 15 (BZ), Ricin, Abrin.

2. Pathogens - An infectious biological agent, such as a virus, bacteria, toxin, prion, or fungus that causes disease to its host.

3. Bio- warfare agents— A pathogen used as a weapon.

4. Gaseous environment - Gases in certain geographical location, mainly air and other gases including hi humidity environment and aerosol. The references cited herein teach many principles that are applicable to the present invention. Therefore the full contents of these publications are incorporated by reference herein where appropriate for teachings of additional or alternative details, features and/or technical background.

It is to be understood that the invention is not limited in its application to the details set forth in the description contained herein or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Those skilled in the art will readily appreciate that various modifications and changes can be applied to the embodiments of the invention as hereinbefore described without departing from its scope, defined in and by the appended claims.

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Claims

1. A system for detection of a gaseous-phase-borne hazard, the system comprising:

a) at least one crystalline protein;

c) at least one light monitoring device adapted to receive a light output from said at least one crystalline protein, wherein said at least one light monitoring device is adapted to provide at least one signal responsive to said at least one change in at least one spectral property or conformation to said least one crystalline protein indicative of said gaseous-phase-borne hazard thereby enabling detection of said gaseous-phase-borne hazard.

2. The system of claim 1 wherein the detection is real- time detection.

3. The system of claim 1 wherein the at least one crystalline protein is a crystalline antibody.

4. A system according to claim 1, further comprising a processor constructed to process said at least one signal and to provide data indicative of said gaseous- phase-borne hazard responsive to said at least one signal.

5. A system according to either of claims 1 to 4, further comprising an energy source.

6. A system according to any one of claims 1 to 5, further comprising an air pump.

7. A system according to any one of claims 1 to 6, further comprising an alarm.

8. A system according to any one of claims 1 to 7, further comprising an air filtration system.

9. A system according to any one of claims 1 to 8, further comprising at least one filter configured to remove at least one non-specific agent associated with a non- specific reaction not associated with said gaseous-phase-borne hazard.

10. A system according to any one of claims 1 to 9, wherein said gaseous-phase- borne hazard is a pathogen.

11. A system according to any one of claims 1 to 9, wherein said gaseous-phase- bome hazard is a chemical hazard.

12. A system according to any of claims 1 to 11, wherein said at least one light source is selected from the group consisting of a laser, a light-emitting diode, an organic light-emitting diode, a polymer light-emitting diode, a solid-state light, an LED lamp, an electroluminescent sheet, an electroluminescent wire, a carbon button lamp, a conventional incandescent light bulb, a flashlight, a halogen lamp, a globar, a fluorescent lamp, a phosphorescent lamp, an argon lamp, an argon flash, an acetylene/carbide lamp, a betty lamp, a butter lamp, a candle, a flash powder, a gas light, a gas mantle, a kerosene lamp, a lanterns, a limelight, an oil lamps, a rushlight, a safety lamps, a Davy lamp, a Geordie lamp, a torches, a common household incandescent light bulb, a filament of incandescent close- up light bulb, an Electron Stimulated Luminescence (ESL light bulbs), a cathode ray tube (CRT monitor), a Nixie tube, a gas discharge lamp, a fluorescent lamp, a compact fluorescent lamp, a black light, an inductive light, a hollow cathode lamp, a neon and argon lamp, a plasma lamps, a xenon flash lamp, a carbon arc lamp, a ceramic discharge metal halide lamp, a hydrargyrum medium-arc iodide lamp, a mercury- vapor lamp, a metal halide lamp, a sodium vapor lamp, a xenon arc lamp and combinations thereof.

13. A method for detection of a gaseous-phase-borne hazard, the method comprising:

a) providing incident light onto at least one crystalline protein;

b) receiving at least one signal associated with a light output responsive to at least one change in said least one crystalline protein; and c) analyzing said at least one signal indicative of said gaseous-phase- borne hazard thereby enabling real-time detection of said gaseous- phase-borne hazard.

14. A method according to claim 13, wherein the detection is real-time detection.

15. A method according to claim 13, wherein the at least one crystalline protein is a crystalline antibody.

16. A method according to claim 13, wherein said at least one change in said least one crystalline protein is selected from the group consisting of: a change in a wavelength of said light output, a change in a spectrum of said light output, a rotational change of said light output, another optical property and combinations thereof.

17. A method according to claim 13, wherein said light output comprises at least one of reflected light, refracted light, diffracted light and returned light.

18. A method according to claim 13, wherein said at least one change is due to binding of at least one chemical molecule to the at least one crystalline protein/protein crystal.

19. A method according to claim 13, wherein said analyzing step comprises detecting at least one of a change in an emitted light spectrum and a change in an optical angle.

20. A method for monitoring of a chemical presence in a gaseous environment, the method comprising:

a) providing at least one protein crystal and at least one light source;

b) directing a light beam from at least one light source towards said at least one protein crystal; and

c) measuring over time at least one change in a reflected/refracted light from the at least one protein crystal, comprising at least one change in a wavelength, spectrum, rotational property, other optical property or combinations thereof .

21. A method according to claim 20, further comprising analyzing the optical changes and other data.

22. A method according to claim 20 or 21, further comprising activating at least one alarm responsive to said at least one change.

23. A method of any one of claims 20 to 22, further comprising activating an air pump responsive to said at least one change.

24. The method of any one of claims 20 to 23, further comprising activating ventilation responsive to said at least one change.

25. The method of any one of claims 20 to 24, wherein the monitoring is a real-time monitoring.

26. The method of any one of claims 20-25, wherein the light source is selected of a laser, a light-emitting diode, an organic light-emitting diode, a polymer light- emitting diode, a solid-state light, an LED lamp, an electroluminescent sheet, an electroluminescent wire, a carbon button lamp, a conventional incandescent light bulb, a flashlight, a halogen lamp, a globar, a fluorescent lamp, a phosphorescent lamp, an argon lamp, an argon flash, an acetylene/carbide lamp, a betty lamp, a butter lamp, a candle, a flash powder, a gas light, a gas mantle, a kerosene lamp, a lanterns, a limelight, an oil lamps, a rushlight, a safety lamps, a Davy lamp, a Geordie lamp, a torches, a common household incandescent light bulb, a filament of incandescent close-up light bulb, an Electron Stimulated Luminescence (ESL light bulbs), a cathode ray tube (CRT monitor), a Nixie tube, a gas discharge lamp, a fluorescent lamp, a compact fluorescent lamp, a black light, an inductive light, a hollow cathode lamp, a neon and argon lamp, a plasma lamps, a xenon flash lamp, a carbon arc lamp, a ceramic discharge metal halide lamp, a hydrargyrum medium-arc iodide lamp, a mercury-vapor lamp, a metal halide lamp, a sodium vapor lamp, a xenon arc lamp and combinations thereof.

27. The method of any one of claims 20-26, wherein the steps performed are performed with a computer-aided system.

28. The method of any one of claims 20-27, wherein the chemical is selected form the group consisting of organo phosphorus derived nerve gas, cyanide derived nerve gas, nitric oxide derived gas, any biological agent in aerosol forms such as butolinum, anthrax. Selected from the list consisting of: Cyclosarin (GF), Sarin (GB), Soman (GD), Tabun (GA), VX, VR, Some insecticides, Novichok agents, Arsines, Cyanogen chloride, Hydrogen cyanide, Sulfur mustard (HD, H), Nitrogen mustard, Lewisite (L), Phosgene oxime, Chlorine, Hydrogen chloride, Nitrogen oxides, Phosgene, Tear gas, Pepper spray, Agent 15 (BZ), Ricin, Abrin.

29. The method of any one of claims 18-26, further comprising activating at least one filter to reduce non specific reaction of non-related particles with the crystal.

30. A system for monitoring of a chemical level in a gaseous environment, the system comprising at least one fixed or portable device adapted to monitor changes in wave length, spectral, rotational or otherwise optical properties of a protein crystal following the attachment of a chemical to the protein crystal.

31. The system of claim 30 further comprising a processor to analyze signals coming from the at least one device.

32. The system of claim 30, further comprising a ventilation system.

33. A method of detecting and monitoring the presence of an amount of cholinesterase inhibitor/ ligand in a sample comprising the steps of: (a) contacting the cholinesterase inhibitor/ ligand with crystallized acetylcholinesterase; (b) monitoring the cholinesterase inhibitor/ ligand by way of changes induced to the crystallized protein chemistry, geometry in space.

34. A device for monitoring of a pathogen concentration in a gaseous environment, the device comprising at least one of crystallized antibody or crystallized biological macromolecule, at least one light source and at least one light monitoring device.

35. A device according to claim 34, wherein the monitoring is real time monitoring.

36. A device according to claim 34, further comprising a processor.

37. A device according to either of claims 34 and 35, further comprising an energy source.

38. A device according to any one of claims 34-37, further comprising an air pump.

39. A device according to any one of claims 34-38, further comprising an alarm.

40. A device according to any one of claims 34-39, wherein the light source is selected form the group consisting of a laser, a light-emitting diode, an organic light-emitting diode, a polymer light-emitting diode, a solid-state light, an LED lamp, an electroluminescent sheet, an electroluminescent wire, a carbon button lamp, a conventional incandescent light bulb, a flashlight, a halogen lamp, a globar, a fluorescent lamp, a phosphorescent lamp, an argon lamp, an argon flash, an acetylene/carbide lamp, a betty lamp, a butter lamp, a candle, a flash powder, a gas light, a gas mantle, a kerosene lamp, a lanterns, a limelight, an oil lamps, a rushlight, a safety lamps, a Davy lamp, a Geordie lamp, a torches, a common household incandescent light bulb, a filament of incandescent close-up light bulb, an Electron Stimulated Luminescence (ESL light bulbs), a cathode ray tube (CRT monitor), a Nixie tube, a gas discharge lamp, a fluorescent lamp, a compact fluorescent lamp, a black light, an inductive light, a hollow cathode lamp, a neon and argon lamp, a plasma lamps, a xenon flash lamp, a carbon arc lamp, a ceramic discharge metal halide lamp, a hydrargyrum medium-arc iodide lamp, a mercury- vapor lamp, a metal halide lamp, a sodium vapor lamp, a xenon arc lamp and combinations thereof.

41. A device according to any one of claims 34-40, further comprising at least one filter to reduce non specific reaction of non related particles with the crystal.

42. A system for monitoring of a pathogen concentration in a gaseous environment, the system comprises at least one fixed or portable device adapted to monitor conformational changes reflected in at least one of an antibody crystal or a crystallized biological macromolecule serves as a prism by spectral or optical means, a crystal of said antibody or macromolecule, a light source, a light monitoring device, an energy source, a processor, and an alarm.

43. The system of claim 42, wherein the monitoring is a real-time monitoring.

44. A method of detecting and monitoring the presence of an amount of bio warfare agents or any pathogen in a gaseous environment comprising the steps of: (a) allowing the pathogen to ligand with crystallized antibody or crystallized macromolecule; (b) monitoring by way of changes inflicted to the crystallized protein chemistry, geometry in space.

45. A method of measuring the changes in geometry or changes in conformation of the crystallized protein, crystallized antibody or crystallized macromolecule by way of monitoring the light diffraction by the crystallized protein diffraction, changes in light wave length by the crystallized protein diffraction, or changes in the light intensity at a certain wave length by the crystal diffraction.

46. A method of measuring the changes in geometry of the crystallized protein by way of monitoring the light diffraction, the light wave length, or the light intensity at a certain wavelength by the crystallized protein diffraction,