EP0153366A1 - Bioluminescent chemical system and method for detecting the presence of chemical agents in a medium - Google Patents

Bioluminescent chemical system and method for detecting the presence of chemical agents in a medium

Info

Publication number
EP0153366A1
EP0153366A1 EP19840903126 EP84903126A EP0153366A1 EP 0153366 A1 EP0153366 A1 EP 0153366A1 EP 19840903126 EP19840903126 EP 19840903126 EP 84903126 A EP84903126 A EP 84903126A EP 0153366 A1 EP0153366 A1 EP 0153366A1
Authority
EP
European Patent Office
Prior art keywords
substrain
sensitive
resistant
chemical
substrains
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19840903126
Other languages
German (de)
French (fr)
Inventor
Harvey Drucker
Michael A. Lind
Gordon L. Williams
Anthony M. Sutey
Richard A. Lindberg
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Battelle Development Corp
Original Assignee
Battelle Development Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Battelle Development Corp filed Critical Battelle Development Corp
Publication of EP0153366A1 publication Critical patent/EP0153366A1/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G01N21/763—Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2520/00—Use of whole organisms as detectors of pollution

Definitions

  • This invention relates to the detection of chemical agents, especially toxic substances, in a vapor, aerosol, or liquid medium or on solid surfaces. More particularly, this invention • concerns the use of genetically selected sensitive and resistant biolumi- nescent microorganism substrains to indicate the pres ⁇ ence of specific chemical agents by comparing the lumi ⁇ nescence of the sensitive and resistant microorganism substrains.
  • the present invention obviates these inherent problems contained in prior detection methods by pro ⁇ viding a bioluminescent chemical system capable of detecting a plurality of chemical agents in either a vapor, aerosol, or liquid medium.
  • the bioluminescent chemical system of the present invention is capable of detecting relatively minuscule levels of toxic agents even when relatively high concentrations of similar chemical agents or other chemical agents are present in the medium. Since the sensitive and resistant mutant substrains used in the present invention are geneti ⁇ cally derived from the same parental strain of biolumi ⁇ nescent microorganisms, the presence of other chemical agents does not affect the differences in light output of the selective and resistant substrains when exposed to the chemical agents the detection of which is sought.
  • the bioluminescent chemical system of the present invention is capable of detecting the presence of one or more chemical agents in a vapor, aerosol, or liquid medium or on solid surfaces.
  • the system includes one or more pairs of strains of photolumi- nescent microorganisms. Each pair of photoluminescent
  • OMPI microorganisms comprises a genetically-selected sensi ⁇ tive substrain and a genetically-selected resistant substrain.
  • the sensitive and resistant substrains are derived by mutation from a single parent strain.
  • a comparator means such as a self-normalizing difference circuit or an electro-optic sensor, observes the dif ⁇ ferences in photoluminescence between the sensitive substrain and resistant substrain of each parent strain.
  • An indicator means such.as an alarm-display circuit, registers the progressive change in the rela ⁇ tive light output of the sensitive and resistant sub ⁇ strains derived from the parent strain as observed by the comparator means.
  • the sensitive sub ⁇ strain and resistant substrain of each parent are lyo- philized after mutation with the lyophilized substrains being activated just prior to use in detecting the presence of the chemical agents.
  • a plurality of mutant pairs, each having a sensitive substrain sensitive to a specific chemical agent and a resistant substrain resistant to a specific chemical agent, may be used in the bioluminescent chemical system to detect the pres ⁇ ence of a plurality .of chemical agents contained in the medium.
  • a storage and activation system for the bioluminescent detection of the presence of one or more chemical agents in a vapor, aerosol, or liquid medium is pro ⁇ vided and includes a sheet unit with one or more cap ⁇ sules formed within the sheet unit. Each capsule is divided into a microorganism compartment and an activa ⁇ tion solution compartment. Lyophilized microorganisms, which are capable of being photoluminescent when acti ⁇ vated, are encapsulated within each microorganism com ⁇ partment and an activation solution is encapsulated within each activation solution compartment.
  • Crushing means such as a pair of rollers, are provided for breaking the capsules to allow the activation solution to come into contact with the lyophilized microorgan ⁇ isms contained within the microorganism compartment.
  • the activation solution contacts the lyophilized microorganisms, the microorganisms are activated into a state of photoluminescence.
  • the present invention provides a method of detecting one or more chemical agents in a vapor, aerosol, or liquid medium by the use of a photo ⁇ luminescent chemical system which includes the steps of deriving by mutation a genetically selected sensitive substrain and a genetically resistant substrain from a parent strain of a photoluminescent microorganism.
  • the sensitive and resistant substrains are separately grown and then lyophilized.
  • the lyophilized substrains are stored dry in capsules which protect the substrains from oxygen, water, and light.
  • the lyophilized substrains are activated by adding an activation solution to the lyo ⁇ philized substrains.
  • the sensitive substrain and resistant substrain are exposed to the vapor, aerosol, or liquid medium or solid surface containing the chemi ⁇ cal agents so that the luminescence of the sensitive substrains can be compared to the luminescence of the resistant substrains to detect and identify specific chemical agents.
  • Figure 1 is a block diagram representing the present invention.
  • Figure 2 is a perspective view of a preferred- embodiment of the storage and activation system of the present invention.
  • O PI Figure 2a is a sectional view taken along 2-2 of Figure 2.
  • Figure 3 is a side schematic view of the cap ⁇ sules containing lyophilized microorganisms used in the storage and activation system.
  • Figure 4 is a side view of an alternative embodiment of the capsules used in the storage and activation system.
  • Figure 5 is a perspective view of an alternative embodiment of the storage and activation system.
  • Figure 6 is the schematic diagram of a simplified circuit system of the present invention.
  • FIG. 1 is a block diagram of a biolumines ⁇ cent chemical system 10 in accordance with the present invention.
  • the bioluminescent chemical system 10 includes a transportable unit 11 containing a sensitive substrain 12 and a resistant substrain 13.
  • the sensi ⁇ tive substrain 12 and resistant substrain 13 are genet ⁇ ically selected and derived by mutation from the same parent strain of photoluminescent microorganisms.
  • the sensitive substrain 12 is sensitive to the specific chemical agent contained in the aerosol, vapor, or liquid medium. This sensitivity is exhibited by the decreased luminescence of the sensitive substrain upon exposure to the specific chemical agent.
  • the resistant substrain 13 remains relatively unaf- fected by exposure to the specific chemical agent to which the sensitive substrain is sensitive and, accord ⁇ ingly, the photoluminescence of the resistant substrain 13 does not significantly decrease upon exposure to that specific chemical agent.
  • the detec- tion unit 11 has a power pack 14 which periodically
  • O PI advances a fresh unit of sensitive substrain 12 and resistant- substrain 13 in the detection unit 11 to an operation position for exposure to the chemical agents in the medium.
  • Electro-optic sensors 15 observe the differ ⁇ ences in light output between the sensitive substrain 12 and the resistant substrain 13, when both are exposed to the same chemical agents contained within the same medium. The differences in light output between the sensitive and resistant substrains are quantitatively correlated with the concentration of the specific chemical agent in the medium.
  • Differences in light output due to the presence of other toxic or non- toxic agents are generally not significant and are not detected by the electro-optic sensor 15 because the sensitive substrain 12 and resistant " substrain 13 are derived from the same parent strain of photoluminescent microorganism, and they have the same sensitivity to other toxic or non-toxic agents.
  • the measured light output from the electro- optic sensor 15 is conditioned and compared by the signal conditioning electronics 20.
  • the signal conditioning electronics are a self-normalizing difference circuit.
  • the self-normalizing difference circuit adjusts the readings of the electro-optic sen ⁇ sors 15 for the decrease of photoluminescence from the microorganisms due to the lapse of time, and can be used to adjust for temperature and humidity variations.
  • the signal from the signal conditioning electronics 20 is recorded by the logic control electronics 21, which evaluate the signals to determine the presence of the specific chemical agent that caused the particular decrease in luminescence of the sensitive substrain 12.
  • the logic control electronics 21 also can be programmed to indi ⁇ cate when a threshold concentration of a chemical agent is present so as to activate the alarm display elec ⁇ tronics 22.
  • the logic control electronic control 21 also compiles the differences in photoluminescence between the sensitive and resistant substrains for two or more pairs of substrains in which each pair of sen- sitive and resistant substrains is derived from a parent strain based upon selection of mutants sensitive and resistant to the chemical agent, so as to distin ⁇ guish among a number of chemical agents.
  • the alarm-display electronics 22 provides a visual reading of the various operational parameters and concentration levels. Various types of visual, auditory, and tactile alarms can be incorporated in the bioluminescent chemical system to warn of the presence of . toxic levels of a chemical agent.
  • An environmental control module 23 monitors and controls the various external parameters to insure a reliable operation of the unit 11.
  • all of the electronic cir ⁇ cuitry of the bioluminescent system, with the exception of the alarm-display electronics 22 and the electro- optic sensors 15, can be incorporated into a single hybrid chip.
  • the parent strain of microorganism used in the mutant derivation of each pair of sensitive and resistant substrains may be any photoluminescent micro- organism such as bacteria or fungi.
  • Bacteria such as Photobacteriu fischeri, Photobacterium phosphoreum, Photobacterium leioqnathi, and Vibrio harveyi may be employed.
  • Bioluminescent fungi such as Armillarin mellea, Panus stipticus.
  • Mycelium X, and members of the genus Mycena are also useful for this purpose.
  • any photoluminescent organism manipulable through established microbial genetic methods, serves as a basis for the development of a specific detector system in which sensitive and resistant substrains are mutationally derived or derived by direct manipulation of genetic material.
  • Sensitive and resistant substrains which either are genetically altered or derived from parent strains so as to have different emission spectra in the presence of a specific chemical agent, may be used in the pres ⁇ ent invention. Mixtures of substrains with demon- strated sensitivity and resistance relative to light output and/or emission spectra may be used to increase the system's specificity for a given specific agent, or to detect more than one. specific agent in a given milieu of chemical agents through the use of appro- priate logic control circuitry 21.
  • sub ⁇ strains of Photobacterium leioqnathi that are sensitive or resistant to acetone can be derived by two processes which differ only in the method of final selection of the substrain.
  • a viable, actively grow ⁇ ing culture of the microorganism is exposed for defined periods of time to either nitrosoguanidine or ultra ⁇ violet light under conditions such that approximately 98 percent of the cells in the culture are killed.
  • the remaining viable cells are rescued and shown to be a random assortment of mutant types, with the nature of genetic lesion a function of mutagen employed.
  • single colony isolates of the mutant sub- strains are screened by the addition of acetone to the
  • OMPI liquid cultures while a determination of the changes in light output of the microorganisms is made. This technique results in a pure culture isolation of ace ⁇ tone sensitive and resistant substrains. The same methodology can be used for the isolation of substrains sensitive or resistant to benzene.
  • the ultra ⁇ violet mutated cultures of Photobacterium leioqnathi are designed to expose evenly a large number of bacter- ial colonies to a specific chemical agent. An immedi ⁇ ate comparison of luminescence of cultures is per ⁇ formed.
  • air is pumped through a sparger submerged in acetone, and a petri dish containing the colonies of mutated bacteria are then exposed to the acetone saturated air.
  • the petri dishes are illumi-*- nated from the side with a red light which makes lumi ⁇ nescent colonies appear blue and nonluminescent colo- ' nies appear red.
  • the last colony to turn red is selected as a resistant sub ⁇ strain.
  • This selection technique is used to derive sensitive and resistant substrains capable of detecting acetone at lower concentrations than substrains derived by the first methodology.
  • the second screening technique can be used to derive sensitive and resistant substrains of Photobacterium phosphoreu capable of detecting chloroform or formaldehyde.
  • the genetically derived and screened sub ⁇ strains can be preserved by lyophilization so that they may be stored until needed. As a first step in the lyophilization process, the microorganism cells are grown in the appropriate liquid medium until lumines- cence is maximum.
  • the cells are then harvested by centrifugation and resuspended in a cryoprotective agent.
  • a skim milk solution is preferably used as a cryoprotective agent for the Photobacteria species.
  • a sucrose solution in a one-fourth strength artificial seawater medium, is used for the Vibrio harveyi spe ⁇ cies.
  • the suspensions are then frozen and dried to a powder, which is the stored form of the lyophilized bacteria.
  • the lyophilized cultures of many microorgan ⁇ isms have been shown to be capable of reconstitution, upon the addition of an appropriate activation solu ⁇ tion, after years of storage if the lyophilized culture is protected from water, oxygen, and light. Longevity tests on luminescent microorganisms that have been lyo ⁇ philized by the above techniques have shown that stor- age up to one month does not significantly alter the culture luminescence upon culture reactivation with an activation solution.
  • the lyophilized cultures when activated with an"appropriate activation solution, emit light which is comparable to that observed before lyophilization. Depending upon the strain of microorganisms, the light emission continues for several hours and as long as 24 hours. Although the quantity of light decreases with time, the electronics of the detection system automati- cally adjusts to this decrease in luminescence. Accordingly, a single lyophilized packet of photolumi ⁇ nescent bacteria, after reconstitution with an activa ⁇ tion solution, could be used in the detection device of the present invention for periods of 24 hours even as the photoluminescence of the bacteria naturally decreases.
  • the bioluminescent chemical system 10 was evaluated for its ability to detect and quantify
  • Substrains of Photobacterium leiognathi which were sensitive or resistant to ace ⁇ tone, were genetically derived by exposing the growing cultures of the microorganisms to either nitrosoguani- dine or. ultraviolet light under conditions such that approximately 98 percent of the cells in the culture were killed. Some of the mutant substrains were screened by the addition of acetone to the liquid cul ⁇ ture, while determining the changes in light output of the microorganisms. The remainder of the mutant sub ⁇ strains were screened by pumping air through a sparger submerged in acetone, and then exposing petri dishes containing the colonies to the acetone saturated air. The petri dishes were illuminated from the side with a red light.
  • the genetically selected substrains were lyo ⁇ philized by growing the cells until luminescence was maximum, and the cells were harvested by centrifuga ⁇ tion.
  • the substrains were suspended in a skim milk cryoprotective solution. The suspensions were frozen, dried under a vacuum, and stored until needed. The cultures were protected from oxygen, water, and light.
  • the cells were activated just prior to use. After activation, the activated cells were exposed, in an aerosol chamber, to defined concentrations of acetone or benzene.
  • the benzene detection system was evaluated for its response to the structurally similar chemical toluene. The results of these procedures are summarized in the following table:
  • ' "detection” refers to a concen ⁇ tration of chemical agent sufficient to be sensed as indicated by the lighting of the first light of the detector (approximately a 1% light level change).
  • concentrations of the specific chemical agents increase, the signal generated by the light output dif ⁇ ference between the sensitive and resistant substrains increases monotonically. Therefore, quantification of response over a given concentration range of specific agents is straightforward.
  • higher concen ⁇ trations of acetone and benzene would trigger the next optical indicators and the audible alarm system. The response of these functions was dependent upon the sen ⁇ sitive and resistant substrains employed and the sensi ⁇ tivity setting of the detection device.
  • the luminescence from the microbial cells depends upon the maintenance of the cells in an optimal physiological state. An adequate supply of oxygen is required, and dehydration of the cultures should be avoided. Nutrients and an appropriate transparent matrix .material for support of the cells should be pro ⁇ vided.
  • FIGS 2 and 2a illustrate a storage and activation system.
  • the lyophilized cells are sealed in a clear, semipermeable plastic capsule 25.
  • Each cap- _ sule 25 is divided into a microorganism compartment 26 and an activation solution compartment 27.
  • the lyo ⁇ philized microorganisms are enclosed in the microorgan ⁇ ism compartment 26, and the activation solution is enclosed in the activation solution compartment 27.
  • each compartment of capsule 25 is ruptured so that the activation solution in the activation solution compart ⁇ ment 27 enters the microorganism compartment 26 to activate the lyophilized microorganisms contained therein.
  • a single membrane could be used to separate the microorganisms from the activation solution in capsule 25.
  • activated microorganism mixture may be spread over the surface of a thin
  • agar sorbent or hydrophilic polymer The spread of activated material occurs between the top 29 and bottom 30 layers formed from a transparent, semi- permeable membrane substance.
  • the activated microorganisms can be spread over sorbent surface 31 which is not covered with a top layer 29. This latter technique would allow for the measurement of chemical agents which might not be capable of penetrating a semipermeable membrane.
  • a chamber 32, enclosing a sorbent layer 31, is formed from the bottom layer 30 and the top layer 29 to allow the luminescent microorganisms to form a defined layer.
  • oxygen, other gases, and solutes trapped in aerosols, along with the specific chemical agents to be detected, will pass through the polymer membranes 29 and 30 and readily contact the microorganism.
  • the membrane pre ⁇ vents the premature desiccation of the culture without need for humidification, while preventing the direct contact of the microorganisms, media, or other liquids with the internal parts of the detection device.
  • the lyophilized microorganisms Prior to activation, the lyophilized microorganisms remain viable and packaged in a potent state for a period of months to years.
  • a plurality of capsules 25 can be formed in a sheet unit 35, as shown in Figure 2, with a sprocket 41 advancing the capsules 25 from a supply roll 42 to an expended roll 43 in a manner similar to the advancement of a film roll in a camera.
  • the sheet unit 35 is pref ⁇ erably made of a clear, semipermeable plastic so that the capsules 25 can be easily formed therein.
  • the storage and activation system shown in Figure 2 pro ⁇ vides a number of ready-to-activate sheet units 35 which can be located sequentially along a roll.
  • each sheet unit has three capsules 25, which are advanced together. Three capsules are used to simultaneously detect three chemical agents, as shown in Table 2. The number of capsules may vary depending upon the number of agents being detected.
  • a light sensor 36 observes the relative differences in total luminescence between the sensitive and resistant sub ⁇ strains as the capsules 25 are advanced over the light sensor 36.
  • Various other means may be used to simul- taneously expose a number of substrains to the aerosol, vapor, or liquid medium so that one or more chemical agents may be detected.
  • a pack 37 including a plurality of detection units 38, having its own battery 40 and electronic system 39, can be used.
  • a new detection unit 38 is automatically
  • the battery pack 40 is capable of providing power sufficient for device operation over the lifetime of the photolumines ⁇ cence detection units 38 included in pack 37. This fail-safe feature alleviates concern about battery failure as a maintenance function separate from the microbial lifetime.
  • the material which encapsulates the lyophil ⁇ ized microorganisms and solution in the microorganism compartment 26 and the activation solution compartment 27, respectively, is preferably made from a material, such as polylactate, which dissolves slowly and at a controlled rate.
  • a material such as polylactate
  • the microorganism cells are released and activated to exhibit lumines- cence.
  • the rate of matrix dis ⁇ solution and cell replacement is equivalent to the rate of loss of cell luminescence.
  • an electronic logic con ⁇ trol circuit 21 is utilized. For example, if one is interested in determining the presence or concentration of three agents A, B, and C, and if it was possible to develop microbial substrains sensitive or resistant to any two of the agents but not all three simultaneously (for example, abC is a strain that is resistant to agents A and B but sensitive to agent C), then three strains are needed, namely, abC, aBc, and Abe. In the following, small letters refer to resistance to the ' chemical agent and capital letters refer to sensitivity to the chemical agent. Using these three strains (abC, aBc, and Abe), the following truth table is generated:
  • OMPI agents can be defined with the use of " only three substrains.

Abstract

Le système chimique bioluminescent décrit dans la présente invention est capable de détecter la présence d'un ou plusieurs agents chimiques dans un milieu liquide, de vapeur ou aérosol en utilisant des sous-souches de micro-organismes photoluminescents dérivées par mutation génétique à partir d'une souche parentale. Pour la détection d'un agent chimique spécifique, des sous-souches sensibles et résistantes sont sélectionnées génétiquement à partir de la même souche parentale de micro-organismes photoluminescents. L'émission lumineuse de la sous-souche sensible décroît en présence de l'agent chimique spécifique. Par opposition, l'émission ou puissance lumineuse de la sous-souche résistante ne change pas ou ne change pas parallèlement à celle de la sous-souche sensible, lorsqu'elle est exposée au même agent chimique. La différence relative de photoluminescence entre la sous-souche sensible et la sous-souche résistante est comparée de manière à indiquer la quantité ou la présence de l'agent chimique. De manière à permettre le stockage des sous-souches sélectionnées génétiquement pendant de longues périodes de temps entre le moment de la sélection génétique et le moment où elles sont finalement utilisées, les sous-souches sont lyophilisées. Immédiatement avant leur utilisation, les sous-souches sont activées en mélangeant les micro-organismes avec une solution d'activation de manière à leur redonner leur luminescence.The bioluminescent chemical system described in the present invention is capable of detecting the presence of one or more chemical agents in a liquid, vapor or aerosol medium by using sub-strains of photoluminescent microorganisms derived by genetic mutation from a parental strain. For the detection of a specific chemical agent, sensitive and resistant sub-strains are genetically selected from the same parental strain of photoluminescent microorganisms. The light emission of the sensitive sub-strain decreases in the presence of the specific chemical agent. In contrast, the emission or light power of the resistant sub-strain does not change or does not change parallel to that of the sensitive sub-strain, when it is exposed to the same chemical agent. The relative difference in photoluminescence between the sensitive sub-strain and the resistant sub-strain is compared to indicate the amount or the presence of the chemical agent. In order to allow the storage of genetically selected sub-strains for long periods of time between the time of genetic selection and the time when they are finally used, the sub-strains are lyophilized. Immediately before their use, the sub-strains are activated by mixing the microorganisms with an activation solution so as to restore their luminescence.

Description

BIOLUMINESCENT CHEMICAL SYSTEM AND
METHOD FOR DETECTING THE PRESENCE
OF CHEMICAL AGENTS IN A MEDIUM
Summary of the Invention
This invention relates to the detection of chemical agents, especially toxic substances, in a vapor, aerosol, or liquid medium or on solid surfaces. More particularly, this invention • concerns the use of genetically selected sensitive and resistant biolumi- nescent microorganism substrains to indicate the pres¬ ence of specific chemical agents by comparing the lumi¬ nescence of the sensitive and resistant microorganism substrains.
Environmental and health considerations require a quick, reliable, and easy detection of toxic chemicals contained in various media, such as vapor, aerosols, and liquid so as to avoid unnecessary expo- sure to the toxic agent. Various systems using bio- luminescent organisms for the detection of toxic agents have been proposed. For example, A.L. Jordan et al. in United States Patent No. 3,370,175 provides a system for using a single culture of luminescent microorgan- isms as sensors for a- toxic chemical agent in an aero¬ sol or gas. Y.A. Sakaida et al. in U.S. Patent No. 3,849,653 discloses a system for detecting a specific vapor in an atmosphere by using various strains of bio- luminescent microorganisms which react differently when exposed to a specific vapor.
These prior art methods, however, are gener¬ ally not usable in liquid or suspended particle media, and they are unable to detect the presence of a multi¬ plicity of chemical agents within the same medium. Additionally, the photoluminescent microorganisms used in these methods often do not detect relatively low concentrations of the chemical agents being determined when relatively high concentrations of similar chemical agents or other chemical agents are present within the media. The primary constraint for making practical use of any previously developed bioluminescent detec¬ tion system, such as Jordon et al. or Sakaida et al., has been the problem of maintaining viable luminescent cultures of microorganisms under conditions compatible with storage requirements and the design constraints of the detector device. Various approaches have attempted to resolve this difficulty arising from luminescent microorganisms and the physiological requirements for developing and maintaining luminescence. For example, Edward E. Elson et al. in U.S. Patent No. 3,728,227 suggests an elaborate device for the storage, inocula¬ tion, and utilization of the microorganisms. Douglas R. Doonan et al. in U.S. Patent No. 3,958,-938 dis¬ closes a detector system which provides humidity to the culture testing area to prevent culture dehydration while the sample air is passed over the culture in the detector.
However, in all of these methods, viable growing cultures are exposed to chemical agents on an agar medium or in a liquid broth resulting in severe physiological constraints relative to the applicability and versatility of the detector devices. Accordingly, while photoluminescent bacteria have been suggested during the past two decades as a basis for the detec- tion of specific chemical agents, the inability to solve the key problems of effective long term storage and a reconstitution capable of maintaining the lumi¬ nescent potential has impeded the widespread use of this technique to detect toxic agents.
O The present invention obviates these inherent problems contained in prior detection methods by pro¬ viding a bioluminescent chemical system capable of detecting a plurality of chemical agents in either a vapor, aerosol, or liquid medium. The bioluminescent chemical system of the present invention is capable of detecting relatively minuscule levels of toxic agents even when relatively high concentrations of similar chemical agents or other chemical agents are present in the medium. Since the sensitive and resistant mutant substrains used in the present invention are geneti¬ cally derived from the same parental strain of biolumi¬ nescent microorganisms, the presence of other chemical agents does not affect the differences in light output of the selective and resistant substrains when exposed to the chemical agents the detection of which is sought.
When the bioluminescent microorganisms of the present invention are lyophilized after mutant deriva- tion and stored until needed, the lyophilized cells are capable of returning to normal physiological and lumi¬ nescent potential by the addition of the appropriate activation fluid. During storage, the lyophilized microorganisms will remain viable and capable of detecting the toxic chemical agents when activated months, or even years later. In the present invention, by storing the genetically selected substrains in a lyophilized state, the problems of storage and recon- stitution found in the prior art are solved. The bioluminescent chemical system of the present invention is capable of detecting the presence of one or more chemical agents in a vapor, aerosol, or liquid medium or on solid surfaces. The system includes one or more pairs of strains of photolumi- nescent microorganisms. Each pair of photoluminescent
OMPI microorganisms comprises a genetically-selected sensi¬ tive substrain and a genetically-selected resistant substrain. The sensitive and resistant substrains are derived by mutation from a single parent strain. A comparator means, such as a self-normalizing difference circuit or an electro-optic sensor, observes the dif¬ ferences in photoluminescence between the sensitive substrain and resistant substrain of each parent strain. An indicator means, such.as an alarm-display circuit, registers the progressive change in the rela¬ tive light output of the sensitive and resistant sub¬ strains derived from the parent strain as observed by the comparator means. Preferably, the sensitive sub¬ strain and resistant substrain of each parent are lyo- philized after mutation with the lyophilized substrains being activated just prior to use in detecting the presence of the chemical agents. A plurality of mutant pairs, each having a sensitive substrain sensitive to a specific chemical agent and a resistant substrain resistant to a specific chemical agent, may be used in the bioluminescent chemical system to detect the pres¬ ence of a plurality .of chemical agents contained in the medium.
In one embodiment of the present invention, a storage and activation system for the bioluminescent detection of the presence of one or more chemical agents in a vapor, aerosol, or liquid medium is pro¬ vided and includes a sheet unit with one or more cap¬ sules formed within the sheet unit. Each capsule is divided into a microorganism compartment and an activa¬ tion solution compartment. Lyophilized microorganisms, which are capable of being photoluminescent when acti¬ vated, are encapsulated within each microorganism com¬ partment and an activation solution is encapsulated within each activation solution compartment. Crushing means, such as a pair of rollers, are provided for breaking the capsules to allow the activation solution to come into contact with the lyophilized microorgan¬ isms contained within the microorganism compartment. When the activation solution contacts the lyophilized microorganisms, the microorganisms are activated into a state of photoluminescence.
Additionally, the present invention provides a method of detecting one or more chemical agents in a vapor, aerosol, or liquid medium by the use of a photo¬ luminescent chemical system which includes the steps of deriving by mutation a genetically selected sensitive substrain and a genetically resistant substrain from a parent strain of a photoluminescent microorganism. The sensitive and resistant substrains are separately grown and then lyophilized. The lyophilized substrains are stored dry in capsules which protect the substrains from oxygen, water, and light. When it is time to use the detection system, the lyophilized substrains are activated by adding an activation solution to the lyo¬ philized substrains. The sensitive substrain and resistant substrain are exposed to the vapor, aerosol, or liquid medium or solid surface containing the chemi¬ cal agents so that the luminescence of the sensitive substrains can be compared to the luminescence of the resistant substrains to detect and identify specific chemical agents.
Brief Description of the Drawings Figure 1 is a block diagram representing the present invention.
Figure 2 is a perspective view of a preferred- embodiment of the storage and activation system of the present invention.
Γ EX
O PI Figure 2a is a sectional view taken along 2-2 of Figure 2.
Figure 3 is a side schematic view of the cap¬ sules containing lyophilized microorganisms used in the storage and activation system.
Figure 4 is a side view of an alternative embodiment of the capsules used in the storage and activation system.
Figure 5 is a perspective view of an alternative embodiment of the storage and activation system.
Figure 6 is the schematic diagram of a simplified circuit system of the present invention.
Detailed Description of the Invention
Figure 1 is a block diagram of a biolumines¬ cent chemical system 10 in accordance with the present invention. The bioluminescent chemical system 10 includes a transportable unit 11 containing a sensitive substrain 12 and a resistant substrain 13. The sensi¬ tive substrain 12 and resistant substrain 13 are genet¬ ically selected and derived by mutation from the same parent strain of photoluminescent microorganisms. The sensitive substrain 12 is sensitive to the specific chemical agent contained in the aerosol, vapor, or liquid medium. This sensitivity is exhibited by the decreased luminescence of the sensitive substrain upon exposure to the specific chemical agent. In contrast, the resistant substrain 13 remains relatively unaf- fected by exposure to the specific chemical agent to which the sensitive substrain is sensitive and, accord¬ ingly, the photoluminescence of the resistant substrain 13 does not significantly decrease upon exposure to that specific chemical agent. Preferably, the detec- tion unit 11 has a power pack 14 which periodically
O PI advances a fresh unit of sensitive substrain 12 and resistant- substrain 13 in the detection unit 11 to an operation position for exposure to the chemical agents in the medium. Electro-optic sensors 15 observe the differ¬ ences in light output between the sensitive substrain 12 and the resistant substrain 13, when both are exposed to the same chemical agents contained within the same medium. The differences in light output between the sensitive and resistant substrains are quantitatively correlated with the concentration of the specific chemical agent in the medium. Differences in light output due to the presence of other toxic or non- toxic agents are generally not significant and are not detected by the electro-optic sensor 15 because the sensitive substrain 12 and resistant" substrain 13 are derived from the same parent strain of photoluminescent microorganism, and they have the same sensitivity to other toxic or non-toxic agents. The measured light output from the electro- optic sensor 15 is conditioned and compared by the signal conditioning electronics 20. Preferably, the signal conditioning electronics are a self-normalizing difference circuit. The self-normalizing difference circuit adjusts the readings of the electro-optic sen¬ sors 15 for the decrease of photoluminescence from the microorganisms due to the lapse of time, and can be used to adjust for temperature and humidity variations. This natural diminution of light output is unrelated to the exposure of the microorganisms to the specific chemical agent but, rather, relates to the useful life of the photoluminescent microorganisms. The signal from the signal conditioning electronics 20 is recorded by the logic control electronics 21, which evaluate the signals to determine the presence of the specific chemical agent that caused the particular decrease in luminescence of the sensitive substrain 12. The logic control electronics 21 also can be programmed to indi¬ cate when a threshold concentration of a chemical agent is present so as to activate the alarm display elec¬ tronics 22. The logic control electronic control 21 also compiles the differences in photoluminescence between the sensitive and resistant substrains for two or more pairs of substrains in which each pair of sen- sitive and resistant substrains is derived from a parent strain based upon selection of mutants sensitive and resistant to the chemical agent, so as to distin¬ guish among a number of chemical agents.
The alarm-display electronics 22 provides a visual reading of the various operational parameters and concentration levels. Various types of visual, auditory, and tactile alarms can be incorporated in the bioluminescent chemical system to warn of the presence of .toxic levels of a chemical agent. An environmental control module 23 monitors and controls the various external parameters to insure a reliable operation of the unit 11. Preferably, all of the electronic cir¬ cuitry of the bioluminescent system, with the exception of the alarm-display electronics 22 and the electro- optic sensors 15, can be incorporated into a single hybrid chip.
The parent strain of microorganism used in the mutant derivation of each pair of sensitive and resistant substrains may be any photoluminescent micro- organism such as bacteria or fungi. Bacteria, such as Photobacteriu fischeri, Photobacterium phosphoreum, Photobacterium leioqnathi, and Vibrio harveyi may be employed. Bioluminescent fungi such as Armillarin mellea, Panus stipticus. Mycelium X, and members of the genus Mycena are also useful for this purpose. In
OMFI principle, any photoluminescent organism, manipulable through established microbial genetic methods, serves as a basis for the development of a specific detector system in which sensitive and resistant substrains are mutationally derived or derived by direct manipulation of genetic material.
In addition to changes in light output, other parameters relative to photoemission may be employed to increase the sensitivity and specificity of detection. Sensitive and resistant substrains, which either are genetically altered or derived from parent strains so as to have different emission spectra in the presence of a specific chemical agent, may be used in the pres¬ ent invention. Mixtures of substrains with demon- strated sensitivity and resistance relative to light output and/or emission spectra may be used to increase the system's specificity for a given specific agent, or to detect more than one. specific agent in a given milieu of chemical agents through the use of appro- priate logic control circuitry 21.
In one example of the present invention, sub¬ strains of Photobacterium leioqnathi that are sensitive or resistant to acetone can be derived by two processes which differ only in the method of final selection of the substrain. In both cases, a viable, actively grow¬ ing culture of the microorganism is exposed for defined periods of time to either nitrosoguanidine or ultra¬ violet light under conditions such that approximately 98 percent of the cells in the culture are killed. The remaining viable cells are rescued and shown to be a random assortment of mutant types, with the nature of genetic lesion a function of mutagen employed.
In one method of final selection of the sub¬ strains, single colony isolates of the mutant sub- strains are screened by the addition of acetone to the
OMPI liquid cultures, while a determination of the changes in light output of the microorganisms is made. This technique results in a pure culture isolation of ace¬ tone sensitive and resistant substrains. The same methodology can be used for the isolation of substrains sensitive or resistant to benzene.
In the second selection technique, the ultra¬ violet mutated cultures of Photobacterium leioqnathi are designed to expose evenly a large number of bacter- ial colonies to a specific chemical agent. An immedi¬ ate comparison of luminescence of cultures is per¬ formed. To screen the mutants for sensitivity or resistance to acetone, air is pumped through a sparger submerged in acetone, and a petri dish containing the colonies of mutated bacteria are then exposed to the acetone saturated air. The petri dishes are illumi-*- nated from the side with a red light which makes lumi¬ nescent colonies appear blue and nonluminescent colo- ' nies appear red. As exposure continues, if one colony changes from blue to red before the other colonies, it is selected as a sensitive mutant substrain. The last colony to turn red is selected as a resistant sub¬ strain. This selection technique is used to derive sensitive and resistant substrains capable of detecting acetone at lower concentrations than substrains derived by the first methodology. Additionally, the second screening technique can be used to derive sensitive and resistant substrains of Photobacterium phosphoreu capable of detecting chloroform or formaldehyde. The genetically derived and screened sub¬ strains can be preserved by lyophilization so that they may be stored until needed. As a first step in the lyophilization process, the microorganism cells are grown in the appropriate liquid medium until lumines- cence is maximum. The cells are then harvested by centrifugation and resuspended in a cryoprotective agent. A skim milk solution is preferably used as a cryoprotective agent for the Photobacteria species. A sucrose solution, in a one-fourth strength artificial seawater medium, is used for the Vibrio harveyi spe¬ cies. The suspensions are then frozen and dried to a powder, which is the stored form of the lyophilized bacteria. The lyophilized cultures of many microorgan¬ isms have been shown to be capable of reconstitution, upon the addition of an appropriate activation solu¬ tion, after years of storage if the lyophilized culture is protected from water, oxygen, and light. Longevity tests on luminescent microorganisms that have been lyo¬ philized by the above techniques have shown that stor- age up to one month does not significantly alter the culture luminescence upon culture reactivation with an activation solution.
The lyophilized cultures, when activated with an"appropriate activation solution, emit light which is comparable to that observed before lyophilization. Depending upon the strain of microorganisms, the light emission continues for several hours and as long as 24 hours. Although the quantity of light decreases with time, the electronics of the detection system automati- cally adjusts to this decrease in luminescence. Accordingly, a single lyophilized packet of photolumi¬ nescent bacteria, after reconstitution with an activa¬ tion solution, could be used in the detection device of the present invention for periods of 24 hours even as the photoluminescence of the bacteria naturally decreases.
Example
The bioluminescent chemical system 10 was evaluated for its ability to detect and quantify
OMPI specific chemical agents. Substrains of Photobacterium leiognathi, which were sensitive or resistant to ace¬ tone, were genetically derived by exposing the growing cultures of the microorganisms to either nitrosoguani- dine or. ultraviolet light under conditions such that approximately 98 percent of the cells in the culture were killed. Some of the mutant substrains were screened by the addition of acetone to the liquid cul¬ ture, while determining the changes in light output of the microorganisms. The remainder of the mutant sub¬ strains were screened by pumping air through a sparger submerged in acetone, and then exposing petri dishes containing the colonies to the acetone saturated air. The petri dishes were illuminated from the side with a red light. As exposure to the red light continued, the colonies that first changed from blue to red in photo¬ luminescence were selected as the sensitive mutant sub¬ strain. The last colony to turn red was selected as the resistant substrain. These techniques were also followed with Photobacterium leiognathi to derive genetically sensitive and resistant substrains to ben¬ zene.
The genetically selected substrains were lyo¬ philized by growing the cells until luminescence was maximum, and the cells were harvested by centrifuga¬ tion. The substrains were suspended in a skim milk cryoprotective solution. The suspensions were frozen, dried under a vacuum, and stored until needed. The cultures were protected from oxygen, water, and light. The cells were activated just prior to use. After activation, the activated cells were exposed, in an aerosol chamber, to defined concentrations of acetone or benzene. To demonstrate the specificity of the sub¬ strains to a specific chemical agent, the benzene detection system was evaluated for its response to the structurally similar chemical toluene. The results of these procedures are summarized in the following table:
TABLE NO. 1
Chemical Sensitive/Resistant Lower Limit- for
Aqent Substrain System Detection (10%) ppm
Acetone Al 2
Acetone A 2
Acetone A3 4
Acetone A4 7
Acetone A5 5
Benzene Bl 27
Benzene B 2
Toluene B2 5000
. In the above,' "detection" refers to a concen¬ tration of chemical agent sufficient to be sensed as indicated by the lighting of the first light of the detector (approximately a 1% light level change). As the concentrations of the specific chemical agents increase, the signal generated by the light output dif¬ ference between the sensitive and resistant substrains increases monotonically. Therefore, quantification of response over a given concentration range of specific agents is straightforward. In practice, higher concen¬ trations of acetone and benzene would trigger the next optical indicators and the audible alarm system. The response of these functions was dependent upon the sen¬ sitive and resistant substrains employed and the sensi¬ tivity setting of the detection device.
The results of the tests suggest that the detection system can quantitatively measure specific agents over ranges of concentrations depending upon the substrain employed and the detection system's optical- electronic response circuitry. The data indicates that
-£TJRE_C OMPI a substrain system chosen for the detection of benzene does not respond to a three order of magnitude or high¬ er concentration of the structurally similar chemical toluene. The tests show that icrobial mutant sub- strains, sensitive or resistant in terms of light emis¬ sion to specific chemical agents, can be genetically derived, stored for long periods of time with minimal loss of luminescence, and used to monitor and detect concentrations of specific chemical agents with high sensitivity and specificity.
The luminescence from the microbial cells depends upon the maintenance of the cells in an optimal physiological state. An adequate supply of oxygen is required, and dehydration of the cultures should be avoided. Nutrients and an appropriate transparent matrix .material for support of the cells should be pro¬ vided.
Figures 2 and 2a illustrate a storage and activation system. The lyophilized cells are sealed in a clear, semipermeable plastic capsule 25. Each cap- _ sule 25 is divided into a microorganism compartment 26 and an activation solution compartment 27. The lyo¬ philized microorganisms are enclosed in the microorgan¬ ism compartment 26, and the activation solution is enclosed in the activation solution compartment 27. By passing the capsules 25 through a pair of rollers 28, each compartment of capsule 25 is ruptured so that the activation solution in the activation solution compart¬ ment 27 enters the microorganism compartment 26 to activate the lyophilized microorganisms contained therein. Alternately, a single membrane (not shown) could be used to separate the microorganisms from the activation solution in capsule 25.
As shown in Figure 3, activated microorganism mixture may be spread over the surface of a thin
' surface 31 of agar sorbent or hydrophilic polymer. The spread of activated material occurs between the top 29 and bottom 30 layers formed from a transparent, semi- permeable membrane substance. Alternatively, as indi- cated in Figure 4, the activated microorganisms can be spread over sorbent surface 31 which is not covered with a top layer 29. This latter technique would allow for the measurement of chemical agents which might not be capable of penetrating a semipermeable membrane. In Figure 3, a chamber 32, enclosing a sorbent layer 31, is formed from the bottom layer 30 and the top layer 29 to allow the luminescent microorganisms to form a defined layer.
With respect to the embodiment of Figure 3, oxygen, other gases, and solutes trapped in aerosols, along with the specific chemical agents to be detected, will pass through the polymer membranes 29 and 30 and readily contact the microorganism. The membrane pre¬ vents the premature desiccation of the culture without need for humidification, while preventing the direct contact of the microorganisms, media, or other liquids with the internal parts of the detection device. Prior to activation, the lyophilized microorganisms remain viable and packaged in a potent state for a period of months to years.
A plurality of capsules 25 can be formed in a sheet unit 35, as shown in Figure 2, with a sprocket 41 advancing the capsules 25 from a supply roll 42 to an expended roll 43 in a manner similar to the advancement of a film roll in a camera. The sheet unit 35 is pref¬ erably made of a clear, semipermeable plastic so that the capsules 25 can be easily formed therein. The storage and activation system shown in Figure 2 pro¬ vides a number of ready-to-activate sheet units 35 which can be located sequentially along a roll. In
OMPI Figure 2, each sheet unit has three capsules 25, which are advanced together. Three capsules are used to simultaneously detect three chemical agents, as shown in Table 2. The number of capsules may vary depending upon the number of agents being detected. A light sensor 36 observes the relative differences in total luminescence between the sensitive and resistant sub¬ strains as the capsules 25 are advanced over the light sensor 36. Various other means may be used to simul- taneously expose a number of substrains to the aerosol, vapor, or liquid medium so that one or more chemical agents may be detected.
Alternatively, as depicted in Figure 5, a pack 37 including a plurality of detection units 38, having its own battery 40 and electronic system 39, can be used. A new detection unit 38 is automatically
» advanced and activated into a detection position, and the previous expended unit is ejected from the sensor system by an electronics system 39, which responds to a reduction in luminescence from the cells. The battery pack 40 is capable of providing power sufficient for device operation over the lifetime of the photolumines¬ cence detection units 38 included in pack 37. This fail-safe feature alleviates concern about battery failure as a maintenance function separate from the microbial lifetime.
The material, which encapsulates the lyophil¬ ized microorganisms and solution in the microorganism compartment 26 and the activation solution compartment 27, respectively, is preferably made from a material, such as polylactate, which dissolves slowly and at a controlled rate. As the encapsulation matrix is dis¬ solved by the activation solution, the microorganism cells are released and activated to exhibit lumines- cence. This activation .results in a stable light base for the detector system to be used over an extended period of time, since new luminescent microorganisms are activated by the dissolution of the encapsulation matrix to replace the old cells, which are diminishing in light production. Ideally, the rate of matrix dis¬ solution and cell replacement is equivalent to the rate of loss of cell luminescence.
When the detection device is used for the simultaneous detection of a number of chemical agents or for discrimination among a number of chemically or structurally similar agents, an electronic logic con¬ trol circuit 21 is utilized. For example, if one is interested in determining the presence or concentration of three agents A, B, and C, and if it was possible to develop microbial substrains sensitive or resistant to any two of the agents but not all three simultaneously (for example, abC is a strain that is resistant to agents A and B but sensitive to agent C), then three strains are needed, namely, abC, aBc, and Abe. In the following, small letters refer to resistance to the' chemical agent and capital letters refer to sensitivity to the chemical agent. Using these three strains (abC, aBc, and Abe), the following truth table is generated:
4 ΓABLE NO. 2
Substrain A B C AB AC BC AB abC - - + + + + aBc - + + - +
Abe + — + + — +
where "+" means the strain is affected, and "-" means that it is not. There is a unique combination of "+" and "-" for each chemical agent or combination of agents (ver¬ tical columns). Accordingly, seven combinations of
- RE
OMPI agents can be defined with the use of" only three substrains.
Similarly, more complex logic circuits or truth tables can be derived to detect or distinguish among a large number of agents. As a matter of practi¬ cal implementation, one might use a sheet unit 35 having a multiplicity of capsules 25 containing a vari¬ ety of sensitive and resistant substrain pairs enclosed in the capsules 25. This multiple unit would permit the simultaneous detection of a variety of chemical agents.

Claims

What is Claimed is:
1. A bioluminescent chemical system for detect¬ ing the presence of one or more chemical agents in a vapor, aerosol, or liquid medium or on a solid surface comprising: (a) one or more pairs ' of photoluminescent microorganisms comprising a genetically selected sensi¬ tive substrain and a genetically selected resistant substrain, the sensitive and resistant substrains being derived by the mutation of the parent strain: (b) comparator means for observing the rela¬ tive differences in photoluminescence between the sen¬ sitive substrain and the resistant substrain of each parent strain; and
(c) indicator means to register a progressive change in the relative output of the sensitive sub¬ strain and the resistant substrain of each parent strain as observed by the comparator means.
2. The bioluminescent chemical system of claim 1, wherein the sensitive and resistant substrains of each parent strain are lyophilized after mutation, the lyophilized substrains being activated prior to use in detecting the presence of the chemical' agents.
3. The bioluminescent chemical system of claim 1, wherein the photoluminescent microorganisms are selected from the group consisting of bacteria and fungi.
4. The bioluminescent chemical system in claim 3, wherein the bacteria are selected from the group consisting of Photobacterium fischeri, Photobacterium phosphoreum, ' Photobacterium leiognathi, and Vibrio harveyi.
5. The bioluminescent chemical system of claim 3, wherein the fungi are selected from the group con¬ sisting of Armillarin mellea, Panus stipticus. Mycelium X, and the genus Mycena.
6. The bioluminescent chemical system of claim 1, wherein the photoluminescence of the sensitive sub¬ strain decreases as the sensitive substrain is exposed to one of the chemical agents while the photolumines¬ cence of the resistant substrain remains unchanged as the resistant substrain is exposed to the same chemical agent.
7. The bioluminescent chemical system of claim 1, wherein the sensitive and resistant substrains of each parent strain differ in their emission spectra when exposed to the same chemical agent.
8. The bioluminescent chemical system of claim 7, wherein the comparator means observes the relative differences in emission spectra between the sensitive and resistant substrains.
9. The bioluminescent chemical system of claim 1, wherein the comparator means includes one or more electro-optic sensors for quantitatively detecting the relative difference in photoluminescence between the sensitive and resistant substrains.
10. The bioluminescent chemical system of claim 1, wherein the comparator means includes a self- normalizing difference circuit to adjust for the dimi¬ nution of the photoluminescence of the substrains over time.
11. The bioluminescent chemical system of claim 1, further comprising an alarm means to warn when a preselected concentration level of one or more of the chemical agents is shown by the indicator means.
12. The bioluminescent chemical system of claim 1, further comprising a logic control means for
0MH compiling the difference in photoluminescence between pairs of sensitive and resistant substrains, each pair of substrains being derived from a different parent strain based on mutation and selection of sensitive or resistant substrains so as to distinguish among a number of chemical agents in the media.
13. The bioluminescent chemical system of claim 1, further comprising exposure means for simultaneously exposing the sensitive and resistant substrains to the vapor, aerosol, or liquid medium.
14. A storage and activation system for the bio¬ luminescent detection of the presence of one or more chemical agents in a vapor, aerosol, or liquid medium or on a solid surface comprising: (a) a sheet unit
(b) one or more capsules formed within the sheet units, each capsule being divided into a micro¬ organism compartment and an activation solution com¬ partment; (c) lyophilized microorganisms encapsulated within each microorganism compartment, the lyophilized microorganisms capable of being photoluminescent when activated;
(d) an activation solution encapsulated within each activation solution compartment; and
(e) crushing means for breaking the encapsu¬ lated activation solution compartment and the micro¬ organism compartment to allow the activation solution to come into contact with the lyophilized microorgan- isms contained within the microorganism compartment so as to activate the microorganisms into a state of photoluminescence.
15. The storage and activation system of claim 14, wherein each capsule has a top layer and a bottom layer, the top layer and the bottom layer being formed from a transparent, semipermeable plastic material to allow for the passage of the chemical agents . into the activated microorganisms.
16. The storage and activation system of claim 15, wherein an uncovered sorbent layer covers a portion of the bottom layer of the capsule, the sorbent layer being capable of receiving a flow of microorganisms after activation.
17. The storage and activation system of claim 16, wherein the sorbent layer is agar.
18. The storage and activation system of claim 14, wherein an encapsulation matrix separates the microorganism compartment and the activation compart¬ ment, the matrix being composed of a material which is dissolved by the activation medium at a controlled rate.
19. The storage and activation system of claim 18, wherein the rate of dissolution of the encapsula¬ tion matrix is approximately equal to the rate of loss of microorganism luminescence.
20. The storage and activation system of claim 14, wherein the matrix material is polylactate.
21. The storage and activation system of claim 14, wherein the lyophilized microorganisms in each cap- sule are all of the same genetically selected substrain of a parent strain of photoluminescent microorganisms.
22. The storage and activation system of claim 21, wherein the substrains are derived by the mutation of the parent strain.
23. The storage and activation system of claim 14, further comprising an advance means for advancing the sheet unit into a detection position from a storage position.
O PI
24. The storage and activation system of claim 23, wherein the advance means responds to the reduction in luminescence of the activated microorganisms.
25. The storage and activation system of claim 23, wherein the advance means is a battery power pack.
26. The storage and activation system of claim 14, wherein the crushing means is a pair of rollers.
27. The bioluminescent chemical system of claim 14, further comprising exposure means for simultane- ously exposing the sensitive and resistant substrains to the vapor, aerosol, or liquid medium.
28. A method of detecting one or more chemical agents in a vapor, aerosol, or liquid medium or on a solid surface by a bioluminescent chemical system com- prising the steps of:
(a) deriving by mutation a genetically selected sensitive substrain and a genetically resis¬ tant substrain from a parent strain of photoluminescent microorganisms; (b) . separating the sensitive substrain from the resistive substrain;
(c) exposing the sensitive substrain and the resistant substrain to a vapor, aerosol, or liquid medium or a solid surface containing the chemical agent; and
(d) comparing the luminescence of the sensi¬ tive substrain and the resistant substrain to detect the presence or identity of the chemical agent.
29. A method of detecting one or more chemical agents in a vapor, aerosol, or liquid medium or on a solid surface by a bioluminescent chemical system com¬ prising the steps of:
(a) deriving by mutation a genetically selected sensitive substrain and a genetically resis- tant substrain from a parent strain or photoluminescent microorganisms; Cb) separating the sensitive substrain from the resistant substrain;
(c) lyophilizing the sensitive and the resistant substrain; (d) storing the lyophilized substrains to protect the substrains from oxygen, water, and light;
(e) activating the lyophilized substrains by adding an activation solution to the lyophilized sub¬ strains; (f) exposing the sensitive substrain and the resistant substrain to a vapor, aerosol, or liquid medium or a solid surface containing the chemical agent; and
(g) comparing the luminescence of the sensi- tive substrain to the resistant substrain to detect the presence or identity of the chemical agent.
-f J E
OrV.PI
EP19840903126 1983-08-16 1984-07-30 Bioluminescent chemical system and method for detecting the presence of chemical agents in a medium Withdrawn EP0153366A1 (en)

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