[go: up one dir, main page]

WO1993002208A1 - Method, apparatus and indicator for indicating evaporable hydrocarbons or environmental poisons in water or liquids - Google Patents

Method, apparatus and indicator for indicating evaporable hydrocarbons or environmental poisons in water or liquids Download PDF

Info

Publication number
WO1993002208A1
WO1993002208A1 PCT/FI1992/000211 FI9200211W WO9302208A1 WO 1993002208 A1 WO1993002208 A1 WO 1993002208A1 FI 9200211 W FI9200211 W FI 9200211W WO 9302208 A1 WO9302208 A1 WO 9302208A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
indicator
studied
procedure
halocarbons
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.)
Ceased
Application number
PCT/FI1992/000211
Other languages
French (fr)
Inventor
Rolf Kroneld
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.)
Individual
Original Assignee
Individual
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 Individual filed Critical Individual
Publication of WO1993002208A1 publication Critical patent/WO1993002208A1/en
Anticipated expiration legal-status Critical
Ceased 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
    • 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
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity

Definitions

  • the invention comprises a method and a technical device in the form of an apparatus or a biosensor to analyse of environmental pollutants such as halo ⁇ carbons, alkanes, alkenes, methanes, chlorophenols and benzene derivatives found in water and other solutions.
  • environmental pollutants such as halo ⁇ carbons, alkanes, alkenes, methanes, chlorophenols and benzene derivatives found in water and other solutions.
  • volatile halocarbons have received a great deal of attention recently. They are found in water, air, food and medical solutions and they accumulate in patients receiving medical treatment such as dialysis. These compounds could also be found in the tissues of healthy persons.
  • bioluminescens reactions are systems of biomaterial including proteins such as enzymes.
  • Luciferase catalyzes the oxidation of the reduced form of FMN which is FMNH 2 to an oxidized form FMN in the presence of an aldehyde and oxygen. The emitted light is directly proportional to the number of molecules in the reaction.
  • the bacterial luciferase system could easily be connected to oxidoreductase reactions, especially such bacterial systems which contain nicotinann ⁇ eadenini- nucleotide (NAD + ) and/or nicotinamideadenininucleotidephosphate (NADP + ). NADH and NADPH are reduced forms of NAD and NADP.
  • NAD + nicotinann ⁇ eadenini- nucleotide
  • NADP + nicotinamideadenininucleotidephosphate
  • the bacterial luciferase reaction could be depicted as follows:
  • Reaction (3) The light from Reaction (3) is estimated when bioluminescent reactions are used to analyse a compound.
  • Reaction (2) is catalyzed by NAD.FMN and is produced through known methods with bioluminescent bacteria.
  • cell enzyme mediated activities of bioluminescens are used to detect metals.
  • the emitted light is estimated by a photometer.
  • the European Patent numbered 0019786 uses cell activities in a layer such that a product gives rise to a bioluminescent reaction in a layer above.
  • the American Patent numbered 4,581,335 uses a microorganism cloned to synthesize luciferase.
  • the American Patent numbered WO 88/00617 uses luciferase genes in a microorganism to emit energy which can be estimated with the aid of a sensitive film.
  • Toxicity Assessment (5:337-350, 1990) a research group including the applicant explains how bioluminescence could be used to detect not only metals but also organic solvents and volatile halocarbons. The light could be detected by means of a photometer and film which is sensitive to specific compounds.
  • the method underlying the invention is characterized by the fact that cells cultured and/or cell aggregates and/or parts of cells handled so that they have bioluminescent characteristics and/or with a compound which produce a colour change could be analysing volatile pollutants.
  • the cells are freeze dried and put on a layer which is eventually surrounded with a shield for the cells.
  • the cells are brought in contact with the solution to be investigated and the possible occurrence of volatile halocarbons if a colour change or a bioluminescent reaction results.
  • the indicator is in this invention characterized by the fact that freeze dried cells and/or cell aggregates and/or parts of cells that undergo a colour change and/or give rise to electromagnetic emissions in the presence of halocarbons could be detected.
  • the invention could be used in an apparatus where the indicator is used in a specially developed "pocket" prepared with a closing mechanism through which sampling could easily be made while the indicator is in contact with the solution to be studied.
  • Freeze dried cells or cell aggregates can be prepared such that their activity and sensitivity to halocarbons on microgram per litre is achieved.
  • the changes in activity could be estimated either by means of a colour reaction (TB) or through light emissions by means of bioluminescence.
  • Oxygen is transformed with NADH dehydrogenase into superoxide, and oxidation occurs
  • the invention is built on the fact that the cells or cell aggregates used could be freeze dried and stored on an inert material for a long period so that they maintain their characteristics and sensitivity.
  • This cell material could, alternatively, be placed in a pocket or on a strip or be saved in a bag. It would be favourable if it would be covered by a net-formed shield, which is constructed to heltp the diffusion of the compounds to be studied.
  • the method is based on a colour indication with tryphofluorescine, for example, or luciferase.
  • bioluminescence method Another advantage of the bioluminescence method is that the cells deliver energy only when they come into contact with the toxic compounds through induction.
  • the colour-changing method employs a reaction not earlier used for this purpose.
  • tissue aggregates Different characteristics of tissue aggregates are used so that their sensitivity is affected and could be used in analyses in the presence of actual volatile halo ⁇ carbons.
  • the luminescent reaction described could be kept intact and activated in the presence of toxic compounds at concentrations according to maximum limit values.
  • Analyses and identification of the products of such analyses start immediately upon penetration of the covering net.
  • the semiquantitative result could be read through the indicator, or, alternatively the energy could be estimated with instruments presently on the market such as so-called photometers or lurnino- meters.
  • the indicator has certain advantages when analysing volatile halocarbons in water and other solutions and in the control of final products received through distillation particularly for the medical industry.
  • the inert base material and the freeze drying of cells and cell aggregates makes it possible to store the indicator for a long period in a state from which it could later be activated.
  • the indicator has a high degree of sensitivity at microgram per litre concentrations in detecting the pollutants already mentioned.
  • the indicator is based on research work on known effects of volatile halocarbons on membranes and different cell mechanisms related to protein synthesis.
  • the invention is related to an earlier patent of the applicant (Number 77380) concerned with the elimination of volatile pollutants, but the invention could even be used independently as a separate apparatus.
  • Cells with the capacity to synthesize protein, transport messenger nuclem acids and metabolize for example, BW 322, ATCC and JM 103 are used for the colour change method and as an example a plasmide PCSS 18 encoding lac- promoter or lac-repressor controlling luciferase according to the luminescence method.
  • a plasmide PCSS 18 encoding lac- promoter or lac-repressor controlling luciferase according to the luminescence method.
  • the luminescence method it is possible to use bacteria into which a suitable plasmid with luciferase characteristics could be incorporated.
  • a cell concentration of 10 "2 mol/1 has been found to be an optimal cell concentration with regard to sensitivity, cell number, freeze drying and analyses of volatile halocarbons such as trichloromethane, 1,1,1-trichloromethane, tetrachloro- methane,l,l,2-trichloroethylene,bromodichloromethane,bromotrichloromethane andchlorodibromomethane, 1,2-dibromomethane, tetrachloroethylene, tribromo- methane, 1,3-dimethylbenzene, 1,3-dimethylbenzene, 1,2-dimethylbenzene and methylbenzene.
  • volatile halocarbons such as trichloromethane, 1,1,1-trichloromethane, tetrachloro- methane,l,l,2-trichloroethylene,bromodichloromethane,bromotrichloromethane andchlorodibromomethane, 1,2-
  • Cells are then cultured in mediums consisting of 1% bacto- tryptone, 0.5% yeast extract, 0.5% sodium chloride and 0.1 mg/1 ampillicin. The cells are washed with Hank's balanced solution HBSS and 0.1% gelatine.
  • Toxic volatile halocarbons in decreasing concentrations (100, 10, 1.0, 0.1, 0.001, 0.0001 ⁇ g/1) are then added to the medium in order to make the cells sensitive to different concentrations of the compounds.
  • the cells are harvested and further cultured for the testing of specific compounds.
  • Dinitrophenol 100 ⁇ g/1 DNP and TB (tryptoflurescineblue) are then added to the cell culture according to Method one or L (luciferase) according to Method two.
  • the cells are harvested after four hours of culturing which is, based on the test results, the optimal time with reference to toxic effects.
  • Cells and cell aggregates can now be separated by means of ultracentrifugation (rpm 300 R, fifteen minutes) and fractionated against a sugar gradient.
  • the cells or aggregates used for Method two are given n-decylaldehyde (a substrate that could be added to the system also at the analysis stage).
  • Cells are freeze dried and put in the pockets or on strips depending on which of the methods is to be used. Freeze drying is with, for example GmbH apparatus, BRD.
  • the cells have been prepared in accordance with both methods so that a colour reaction as well as a release of electrochemical energy takes place upon contact with or in the presence of the toxic compounds studied.
  • the toxic substances do not produce changes in colour or energy emissions as was the case in earlier methods but directly induces such a reaction.
  • the indicator is now ready to be used. It is recommended that it be surrounded by a cover of a net-like material through which the solution to be studied can pass.
  • the indicator could be used smoothly in connection with the EVP-apparatus which is also a part of the invention.
  • Figure 1 is a schematic representation of a distillation apparatus to which an eliminator of water pollution is connected.
  • Figure 2 is a schematic representation of an EVP apparatus connected to a distiller and to an apparatus to control the result of the purification process.
  • Figure 3 is a schematic representation of the apparatus connected to the purifier.
  • Figure 4 is a schematic representation of the indicator.
  • the distiller apparatus is as can be seen in the picture, connected to a purifier, B, with which compounds are eliminated from the feed water in order to produce water for medical infusion, injection and dialysis solutions.
  • a purifier B
  • Figure 3 is a detail of the equipment. It has the form of a pocket, 6, with a closing device, 7, which regulates the in-flow of the medium to be studied or in this case the purified water from the purifier.
  • Indicators 8a and 8b are dipped into the medium to be studied. The compounds in the medium then give a notation on indicator 8a or 8b in the form of a colour change or luminescence.
  • Figures 4a and 4b are examples of how the indicator could be formed or designed to be used in connection with the apparatus.
  • the freeze dried cells have been applied to the border and surrounded with a net-like cover, which allows for the penetration of toxic compounds and also protects the cells (4a).
  • Figure 4b has differently prepared cells in different places 1,2,3 of the indicator to allow for analyses of different concentrations of the toxic compounds. In this way the indicator is sensitive to specific toxic substances and indicates then- presence in specific concentrations.
  • Cells of BW 322 into which PCSS 18 plasmid has been incorporated were cultured at a concentration of 10 "2 mol/1 in a medium with 1% bactotryphon, 0.5% yeast extract, 0.5% sodium chloride and 0.1 ml/1 ampicillin.
  • the cells were washed with Hank's balanced solution HBSS with 0.1% gelatin.
  • the medium was split into six equal parts to which toxic volatile halocarbons were introduced in decreasing concentrations (100, 10, 1.0, 0.1, 0.001, 0.0001 ⁇ g/1) to make the cells sensitive.
  • the cells were harvested and re-cultured. Then, 100 mg/1 DNP and luciferase were then infused into the cells.
  • the cells were harvested after four hours of growth, then separated and n-decyl- aldehyde was added.
  • Cells were then freeze dried by means of the GmbH technique and placed in pockets on strips of celluloid or on different parts of the strips according to the concentrations of the toxic compounds.
  • the indicator was shielded by a net-like cover.
  • the indicator was dipped into the medium to be analysed through an opening in pocket 6 and the luminescence reaction was read directly or by means of a luminometer.
  • a concentration of volatile halocarbons of 0.0001-1000.0 ⁇ g/1 could be analysed by means of its the bioluminescence as the cell by the earlier DNP- treatment has been prepared to give the indicator or reaction when the limit value, for example, 0.1 ⁇ g/1 for CHC1 3 (suggested by WHO, cp table 3, p. 3) is exceeded in the medium to be studied.
  • Example 1 The analysis was carried out as in Example 1. A colour change from white to light violet or light "pink” in the indicator showed the presence of toxic compounds.
  • concentration of the toxins was ca 0.001- 1000.0 ⁇ g/1 and a reaction was obtained depending on which concentration the cell had been prepared to react for (for example 0.1 ⁇ g/1 for CHC1 3 ).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Toxicology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Urology & Nephrology (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention comprises a method and an apparatus to indicate the presence of volatile hydrocarbons or pollutants in water and other solutions for medical use alone or in connection with an eliminator of volatile hydrocarbons, through distillation. Freeze dried cellls or cell aggregates which have been cultured by maintaining their activity to react to the presence of volatile halocarbons in solutions of microgram per litre concentrations on an inert base material either in special pockets or on strips or in bags covered by a net-like shield which can be penetrated by volatile halocarbons. In connection with application of the indicator in an eliminator of volatile hydrocarbons in connection with distillation the indicator is used in a pocket (6) with a closing mechanism (7) which makes sampling easier and the indicator comes in contact with the medium to be studied.

Description

METHOD, APPARATUS AND INDICATOR FOR INDICATING EVAPORABL HYDROCARBONS OR ENVIROMENTAL POISONS IN WATER OR LIQUIDS
The invention comprises a method and a technical device in the form of an apparatus or a biosensor to analyse of environmental pollutants such as halo¬ carbons, alkanes, alkenes, methanes, chlorophenols and benzene derivatives found in water and other solutions.
The formation and occurrence of volatile halocarbons have received a great deal of attention recently. They are found in water, air, food and medical solutions and they accumulate in patients receiving medical treatment such as dialysis. These compounds could also be found in the tissues of healthy persons.
These compounds are toxic, mutagenic, carcinogenic and teratogenic. Due to their lipohilic nature, they accumulate in tissues.
The World Health Organisation (WHO) has for many years tried to develop maximum limit values in order to decrease the exposure of humans to such compounds.
Examples of maximum limit values given by the WHO for volatile halocarbons as well as examples of compounds studied and their physical properties are given in the figures which follow. Tabelle 1. Ziisammenfassung der Resultate Table 1. Summary of the results
Quell- Milch wasser
IMS/1]
Figure imgf000004_0001
l g/ll
300 2XXX) 2000 2000 100 100 100
CHCl, ND 1,10 42,30 103,5 3,2 0,9 2,2
ND-1,30 ND-122,40 ND-129,40 ND-5,5 ND-1,5 ND-3,1
CHCC1, ND 0,007 0,009 0,008 ND-0,01 ND-0,02 ND-0,03
CCL ND 0,03 0,04 0,007 0,03 0,06 0,07
ND-0,05 ND-0,05 ND-0,02 ND-0,07 ND-0,1 ND-0,2
C1CH = ND 0,07 0,28 0,08 CO, ND-0,10 ND-0,42 ND-0,50
CHBrCl, ND 0,13 11,3 9,11 1,2 1,1 0,008
ND-0,20 ND-17,6 ND-19,80 ND-2,1 ND-2,1 ND-0,07
CBrCl, ND 0,05 0,06 + ND-0,07 ND-0,12
CHBrCl ND 0,05 4,1 5,10 0,4 0,3 0,04
ND-0,07 ND-5,6 ND-6,40 ND-0,9 ND-0,6 ND-0,3
BrCH2- ND 0,007 0,007 0,01 CH2BΓ ND-0,01 ND-0,01 ND-0,02
C12C = ND 0,08 0,01 0,01
CC1, ND-0,10 ND-0,02 ND-0,02
CHBr, ND 0,09 0,03 0,06 0,06
ND-0,12 ND-0,06 ND-0,08 ND-0,01 toluene ND 0,007 0,008 0,01 0,9 1,3
ND-0,01 ND-0,01 ND-0,02 ND-1,1 ND-2,3 m + p ND 0,005 0,006 0,01 0,06 0,04 + xylene ND-0,01 ND-0,02 ND-0,02 ND-0,1 ND-0,1 o-xylene ND + 0,004 0,04 0,07 0,05
ND-0,06 ND-0,08 ND-0,1 ND-0,2
+ = Spuren, traces
ND nicht gefunden, not detected Flϋchtige organische Halogenverbindungen in Lebensmitteln
Figure imgf000005_0001
0,09 0,07 0,03 0,006 2,3 7,1 ND-0,2 ND-0,8 ND-0,07 ND-0,04 ND-7,50 ND-14,20 ι+ 1 + + 1 ND ND + i + 1
0,6 0,6 0,006 0,007 0,62 0,81
ND-1,2 ND-1,3 ND-0,01 ND-0,08 ND-1,40 ND-1,60
+ 0,01 ND ND ND-0,07 ND-0,07
ND ND 0,002 +
ND-0,05
0,04 ND 0,06 0,008 ND ND-0,11 ND-0,15 ND-0,01
0,7 1,71 + ND ND 0,08 + ND
ND-1,3 ND-4,27 ND-0,03
0,09 0,09 0,007 ND ND 0,006 0,007 +
ND-0,2 ND-0,2 ND-0,02 ND-0,03 ND-0,01
0,06 0,06 0,006 ND ND + 0,006 +
ND-0,1 ND-0,1 ND-0,02 ND-0,03
Figure imgf000006_0001
WORLD HEALTH ORGANIZAΗON EEP/8235
ORGANISATION MONDIALE DE LA SANTE page 9.
Figure imgf000007_0001
Figure imgf000007_0002
Chlorophenols
Pentachlorophenol 10
2,4,6-Trichlorophenol 10
Polvnuclear Aromatic Hydrocarbons Benzo' (a) pyrene 0.01
Trihalomethanes Chloroform 30
Pesticides
Aldrin/Dieldrin 0.03
Chlordane 03
2,4 D 100
DDT 1
Heptachlor and Heptachlor Epoxide 0.1
Hexachlorobenzene 0.01
Lindane 3
Methoxyclor 30 Examples are given for tested solutions according to A) efficient concentrations in solutions, B) linearity during the emission of energy, C) effects of volatile halocarbons on cell aggregates, 1 = trichloromethane, 6 = chlorodibromo- methane, 10 = dichlorobromomethane, 15 = bromotrichloromethane, 19 •*■= toluene and D) the inhibitive effect on the energy flow of volatile halocarbons using trichloromethane, for example.
a) , Y / Y
Figure imgf000008_0002
b)
Figure imgf000008_0001
Time /h)
Figure imgf000009_0001
FIGURE FROM EXPERIMENT
Figure imgf000010_0001
Figure imgf000010_0002
te volatile edical use
e purity of tor, being
Figure imgf000010_0003
in routine check-ups so that the use of mass spectrometric and more expensive analyses
estimated and another compound has been used. These systems of biomaterial including proteins such as enzymes are called bioluminescens reactions.
A variety of bacteria produce visible light through luciferase with a co-factor flavine mononucleotide (FMN). Luciferase catalyzes the oxidation of the reduced form of FMN which is FMNH2 to an oxidized form FMN in the presence of an aldehyde and oxygen. The emitted light is directly proportional to the number of molecules in the reaction.
The bacterial luciferase system could easily be connected to oxidoreductase reactions, especially such bacterial systems which contain nicotinann^eadenini- nucleotide (NAD + ) and/or nicotinamideadenininucleotidephosphate (NADP + ). NADH and NADPH are reduced forms of NAD and NADP.
The bacterial luciferase reaction could be depicted as follows:
(1) Compound to be analysed + NAD (or NADP) enzyme <— -> NADH (or NADHP) + product
(2) NADH (or NADPH) + FMN
NAD.FMN oxidoreductase ^ FMNH2 + NAD (or NADP)
(3) RCHO + FMNH2 + 02 immobilized bacterial luciferase^
FMN + RCCOH + H202 + light
The light from Reaction (3) is estimated when bioluminescent reactions are used to analyse a compound. Reaction (2) is catalyzed by NAD.FMN and is produced through known methods with bioluminescent bacteria.
According to the American patent numbered 4,234,681, cell enzyme mediated activities of bioluminescens are used to detect metals. The emitted light is estimated by a photometer. The European Patent numbered 0019786 uses cell activities in a layer such that a product gives rise to a bioluminescent reaction in a layer above. The American Patent numbered 4,581,335 uses a microorganism cloned to synthesize luciferase. The American Patent numbered WO 88/00617 uses luciferase genes in a microorganism to emit energy which can be estimated with the aid of a sensitive film. Biotechnology Newswatch (10:5, 1989) reports how bioluminescence could be used to study waste water at Biosyne Corp., Houston, Texas. An article in the Journal of Bioluminescence and Chemiluminescence (4:551-554, 1989) explains how an automatic biosensor system could be developed to detect metals and that the problem is to develop an inducable light-producing system based on bioluminescence. In Toxicity Assessment (5:337-350, 1990) a research group including the applicant explains how bioluminescence could be used to detect not only metals but also organic solvents and volatile halocarbons. The light could be detected by means of a photometer and film which is sensitive to specific compounds.
The method underlying the invention is characterized by the fact that cells cultured and/or cell aggregates and/or parts of cells handled so that they have bioluminescent characteristics and/or with a compound which produce a colour change could be analysing volatile pollutants. The cells are freeze dried and put on a layer which is eventually surrounded with a shield for the cells. Thus the cells are brought in contact with the solution to be investigated and the possible occurrence of volatile halocarbons if a colour change or a bioluminescent reaction results.
The indicator is in this invention characterized by the fact that freeze dried cells and/or cell aggregates and/or parts of cells that undergo a colour change and/or give rise to electromagnetic emissions in the presence of halocarbons could be detected.
The invention could be used in an apparatus where the indicator is used in a specially developed "pocket" prepared with a closing mechanism through which sampling could easily be made while the indicator is in contact with the solution to be studied.
These favourable characteristics of the invention are characterized in the subclaims of the invention.
Freeze dried cells or cell aggregates can be prepared such that their activity and sensitivity to halocarbons on microgram per litre is achieved. The changes in activity could be estimated either by means of a colour reaction (TB) or through light emissions by means of bioluminescence.
The theory on which these reactions are based is presented in the following text: The compound to be analysed reacts with cell products leading to a reactive radical which gives rise to the indicative reaction in the cell. Oxygen participates and is transformed into superoxide leading to oxidization.
(1) NADP+ NADPH
(2) DNPH - DNP+
(3) TB2+ > > TB+ TB is reduced to a stable form, but the oxygen-sensitive cation radical can be changed
(4) 02 \_- O, Oxygen is transformed with NADH dehydrogenase into superoxide, and oxidation occurs
(5) CXC1_ /- , o- leading to an unknown halocarbon (CXC1_), which reacts with the cell products, leading to a reactive radical (CJJC^OO* which leads to an indicative reaction in the cell (Method one or two)
(6) <— -> L-NADH-OOH
The invention is built on the fact that the cells or cell aggregates used could be freeze dried and stored on an inert material for a long period so that they maintain their characteristics and sensitivity.
This cell material could, alternatively, be placed in a pocket or on a strip or be saved in a bag. It would be favourable if it would be covered by a net-formed shield, which is constructed to heltp the diffusion of the compounds to be studied. The method is based on a colour indication with tryphofluorescine, for example, or luciferase.
It is, then only the apparatus of the invention which is new, but also the fact that cells or cell aggregates could be freeze dried maintaining their qualities and that the biosensor could be made sensitive to specific compounds at certain limit values taking into consideration maximum limit values for each compound.
Another advantage of the bioluminescence method is that the cells deliver energy only when they come into contact with the toxic compounds through induction. The colour-changing method, on the other hand, employs a reaction not earlier used for this purpose.
Different characteristics of tissue aggregates are used so that their sensitivity is affected and could be used in analyses in the presence of actual volatile halo¬ carbons.
The luminescent reaction described could be kept intact and activated in the presence of toxic compounds at concentrations according to maximum limit values.
Analyses and identification of the products of such analyses start immediately upon penetration of the covering net. The semiquantitative result could be read through the indicator, or, alternatively the energy could be estimated with instruments presently on the market such as so-called photometers or lurnino- meters.
The indicator has certain advantages when analysing volatile halocarbons in water and other solutions and in the control of final products received through distillation particularly for the medical industry.
The inert base material and the freeze drying of cells and cell aggregates makes it possible to store the indicator for a long period in a state from which it could later be activated.The indicator has a high degree of sensitivity at microgram per litre concentrations in detecting the pollutants already mentioned.
The indicator is based on research work on known effects of volatile halocarbons on membranes and different cell mechanisms related to protein synthesis.
The invention is related to an earlier patent of the applicant (Number 77380) concerned with the elimination of volatile pollutants, but the invention could even be used independently as a separate apparatus.
Cells with the capacity to synthesize protein, transport messenger nuclem acids and metabolize, for example, BW 322, ATCC and JM 103 are used for the colour change method and as an example a plasmide PCSS 18 encoding lac- promoter or lac-repressor controlling luciferase according to the luminescence method. In the luminescence method it is possible to use bacteria into which a suitable plasmid with luciferase characteristics could be incorporated. A cell concentration of 10"2 mol/1 has been found to be an optimal cell concentration with regard to sensitivity, cell number, freeze drying and analyses of volatile halocarbons such as trichloromethane, 1,1,1-trichloromethane, tetrachloro- methane,l,l,2-trichloroethylene,bromodichloromethane,bromotrichloromethane andchlorodibromomethane, 1,2-dibromomethane, tetrachloroethylene, tribromo- methane, 1,3-dimethylbenzene, 1,3-dimethylbenzene, 1,2-dimethylbenzene and methylbenzene. Cells are then cultured in mediums consisting of 1% bacto- tryptone, 0.5% yeast extract, 0.5% sodium chloride and 0.1 mg/1 ampillicin. The cells are washed with Hank's balanced solution HBSS and 0.1% gelatine.
Toxic volatile halocarbons in decreasing concentrations (100, 10, 1.0, 0.1, 0.001, 0.0001 μg/1) are then added to the medium in order to make the cells sensitive to different concentrations of the compounds. The cells are harvested and further cultured for the testing of specific compounds. Dinitrophenol 100 μg/1 DNP and TB (tryptoflurescineblue) are then added to the cell culture according to Method one or L (luciferase) according to Method two.
The cells are harvested after four hours of culturing which is, based on the test results, the optimal time with reference to toxic effects. Cells and cell aggregates can now be separated by means of ultracentrifugation (rpm 300 R, fifteen minutes) and fractionated against a sugar gradient. The cells or aggregates used for Method two are given n-decylaldehyde (a substrate that could be added to the system also at the analysis stage). Cells are freeze dried and put in the pockets or on strips depending on which of the methods is to be used. Freeze drying is with, for example GmbH apparatus, BRD.
The cells have been prepared in accordance with both methods so that a colour reaction as well as a release of electrochemical energy takes place upon contact with or in the presence of the toxic compounds studied. The toxic substances do not produce changes in colour or energy emissions as was the case in earlier methods but directly induces such a reaction.
The indicator is now ready to be used. It is recommended that it be surrounded by a cover of a net-like material through which the solution to be studied can pass.
The indicator could be used smoothly in connection with the EVP-apparatus which is also a part of the invention.
The use of the invention is hence described.
Figure 1 is a schematic representation of a distillation apparatus to which an eliminator of water pollution is connected.
Figure 2 is a schematic representation of an EVP apparatus connected to a distiller and to an apparatus to control the result of the purification process.
Figure 3 is a schematic representation of the apparatus connected to the purifier.
Figure 4 is a schematic representation of the indicator.
The distiller apparatus is as can be seen in the picture, connected to a purifier, B, with which compounds are eliminated from the feed water in order to produce water for medical infusion, injection and dialysis solutions. For further details, see the text of the applicants patent number 77380.
In Figure 2 the device, C, is connected to output, 32 of the purificator, B. After the water being purified in purificator B, which has been described in the FI- patent text number 77380, the water continues to flow from the purifier along tube 32.
Figure 3 is a detail of the equipment. It has the form of a pocket, 6, with a closing device, 7, which regulates the in-flow of the medium to be studied or in this case the purified water from the purifier. Indicators 8a and 8b are dipped into the medium to be studied. The compounds in the medium then give a notation on indicator 8a or 8b in the form of a colour change or luminescence. Figures 4a and 4b are examples of how the indicator could be formed or designed to be used in connection with the apparatus. The freeze dried cells have been applied to the border and surrounded with a net-like cover, which allows for the penetration of toxic compounds and also protects the cells (4a). Figure 4b has differently prepared cells in different places 1,2,3 of the indicator to allow for analyses of different concentrations of the toxic compounds. In this way the indicator is sensitive to specific toxic substances and indicates then- presence in specific concentrations.
Examples of both methods are thus given in order to present the invention but not to in anyway to limit the invention to these examples.
EXAMPLE 1
The luminescence Method
a) Preparation of the indicator
Cells of BW 322 into which PCSS 18 plasmid has been incorporated, were cultured at a concentration of 10"2 mol/1 in a medium with 1% bactotryphon, 0.5% yeast extract, 0.5% sodium chloride and 0.1 ml/1 ampicillin. The cells were washed with Hank's balanced solution HBSS with 0.1% gelatin. The medium was split into six equal parts to which toxic volatile halocarbons were introduced in decreasing concentrations (100, 10, 1.0, 0.1, 0.001, 0.0001 μg/1) to make the cells sensitive. The cells were harvested and re-cultured. Then, 100 mg/1 DNP and luciferase were then infused into the cells.
The cells were harvested after four hours of growth, then separated and n-decyl- aldehyde was added.
Cells were then freeze dried by means of the GmbH technique and placed in pockets on strips of celluloid or on different parts of the strips according to the concentrations of the toxic compounds. The indicator was shielded by a net-like cover.
b) Analyses of water with the indicator
Water from a purifier according to Figure 2 was analysed.
The water was allowed to continue to an apparatus according to the invention described in Figure 3.
The indicator was dipped into the medium to be analysed through an opening in pocket 6 and the luminescence reaction was read directly or by means of a luminometer. A concentration of volatile halocarbons of 0.0001-1000.0 μg/1 could be analysed by means of its the bioluminescence as the cell by the earlier DNP- treatment has been prepared to give the indicator or reaction when the limit value, for example, 0.1 μg/1 for CHC13 (suggested by WHO, cp table 3, p. 3) is exceeded in the medium to be studied.
EXAMPLE 2
Colour Change Method
a) Preparation of the indicator
Preparation began as it did in Example 1, but no plasmide or substrate was added to the cells. TB (tryphofluorescinblue) was hence added, instead of luciferase.
b) Analyses of water by means of the indicator
The analysis was carried out as in Example 1. A colour change from white to light violet or light "pink" in the indicator showed the presence of toxic compounds. The concentration of the toxins was ca 0.001- 1000.0 μg/1 and a reaction was obtained depending on which concentration the cell had been prepared to react for (for example 0.1 μg/1 for CHC13).
The patent claims are presented in the following. The details of the invention may be varied within the scope of the invention presented in those.

Claims

Patent claims
1. A procedure to study the occurrence of volatile halocarbons in water and solutions by estimating changes in the characteristics of different cells and cell aggregates when they are exposed to pollutants, characterized in that
cultured cells and/or cell aggregates and/or part of cells prepared with lumi¬ nescent characteristics and/or with a substance allowing for colour changes in the presence of the halocarbons to be studied are freeze dried and applied to a border and protected by a net-like covering,
the border with cells is brought into contact with the medium to be studied and the occurrence of halocarbons is measured if a colour change or luminescence occurs.
2. A procedure based on claim 1, characterized in that cells or cell aggregates are given luminescence characteristics by means of the incorporation of a plasmide with luciferase for example, a plasmid from Vibrio bacteria.
3. A procedure based on claim 1, characterized in that the colour change is produced by adding tryphofluorescinblue, TB, the cells and/or cell aggregates and/or parts of the cells.
4. A procedure based on any of the claims 1-3, characterized in that a substance with oxido-reductive characteristics is added to the cells and/or cell aggregates and/or parts of the cells, for example, dinitrophenol (DNP), to make them more sensitive to the toxic compounds to be studied.
5. A procedure based on claim 4, characterized in that DNP is added at a concentration of 0.001-1000.0 μg/1, optimally 100 μg/1.
6. A procedure according to any of claims 1-5, characterized in that the cells can be cultured, for example, BW 322, ATCC or SM 103.
7. A procedure according to claims 2 or 4-5, characterized in that the plasmide is from PCSS 18 plasmide or any other plasmide containing lac-promotor or lac- repressor.
8. A procedure of any of claims 2 or 4-5, characterized in that the substrate n- decyl-aldehyde or other long-chained aldehyde is added.
9. A procedure according to claims 1-8, characterized in that the activity of the cells could be regulated during the culturing phase by adding to the medium the volatile halocarbons to be studied in different concentration.
10. A procedure according to claims 1-9, characterized in that the cells are cultured in concentrations, normally 10~2 mol/1 based on regulations for different limit values for which they are intended to be indicators for.
11. A procedure according to claims 1-10, characterized in that volatile halo¬ carbons could be analysed such as the analyses of volatile halocarbons such as trichloromethane, 1,1,1-trichloroethane, tetrachloromethane, 1,1,2- trichloroethylene, bromodichloromethane, bromotrichloromethane and chlorodi- bromomethane, l,2-dibromomethane,tetrachloroethylene, tribromomethane, 1,3- dimethylbenzene, 1,3-dimethylbenzene, 1,2-dimethylbenzene andmethylbenzene.
12. A procedure according to claims 1-11, characterized in that a colour change or bioluminescence could be directly, by means of a luminometer, with the aid of film or through a fluorescence microscope.
13. Use of a procedure of claims 1-11 in a device in which the indicator is used in a pocket (6) with a closing mechamsm (7) which makes sampling easier and the indicator comes in contact with the medium to be studied.
14. An indicator to analyse volatile hydrocarbons for example halohydrocarbons in water and solutions, characterized in that it consists of freeze dried cells and/or cell aggregates and/or parts of cells used with cells that go under colour changes and/or produce electromagnetic emissions in the presence of the hydrocarbon toxins to be studied.
15. An indicator according to claim 14, characterized in that the freeze dried cells and/or aggregates and/or the parts of the cells are stored on a plate and protected by a net-like shield.
16. An indicator according to claims 14 or 15, characterized in that it contains luciferase.
17. An indicator according to claims 14 or 15, characterized in that it contains tryphoflurescinblue TB.
18. An indicator according to claims 14 or 15, characterized in that it contains dinitrophenol DNP.
19. An indicator according to any of claims 14-18, characterized in that the cells or the like have the origin in cells which can be cultured, for example, BW 322, ATCC or SM 103.
20. An indicator according to claims 14-16 or 18-19, characterized in that PCSS 18 plasmide is added to the cells or the like.
21. An indicator according to claims 14-20, characterized in that it contains one or several layers of cell aggregates, optimally only one.
22. An indicator according to claims 16-19 and 21, characterized in that it contains substrate n-dekyl-aldehyde or some other long chained aldehyde.
23. A indicator according to claims 14-22, characterized in that it contains volatile halocarbons to be studied in different concentrations in different places of the indicator with different activities for different halocarbons to be studied.
PCT/FI1992/000211 1991-07-17 1992-07-09 Method, apparatus and indicator for indicating evaporable hydrocarbons or environmental poisons in water or liquids Ceased WO1993002208A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FI913443 1991-07-17
FI913443A FI913443A0 (en) 1991-07-17 1991-07-17 METHOD, APPARAT OCH INDIKATOR FOER ATT INDIKERA FLYKTIGA KOLVAETEN ELLER MILJOEGIFTER I VATTEN OCH VAETSKOR.

Publications (1)

Publication Number Publication Date
WO1993002208A1 true WO1993002208A1 (en) 1993-02-04

Family

ID=8532903

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/FI1992/000211 Ceased WO1993002208A1 (en) 1991-07-17 1992-07-09 Method, apparatus and indicator for indicating evaporable hydrocarbons or environmental poisons in water or liquids

Country Status (2)

Country Link
FI (1) FI913443A0 (en)
WO (1) WO1993002208A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4332165A1 (en) * 1993-09-22 1995-03-23 Kolibri Umweltanalytik Und On Method and apparatus for pollutant analysis of water samples
DE19621312A1 (en) * 1996-05-28 1997-12-04 Bayer Ag Masking of background fluorescence and signal amplification in the optical analysis of biological medical assays
JP3481212B2 (en) 2000-03-03 2003-12-22 サンジョン キム Luminescent bacterial strain for toxic substance detection, toxic substance inspection method and toxic substance analyzer

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4581335A (en) * 1982-12-01 1986-04-08 Texas A&M University System Process for producing a cloned luciferase-synthesizing microorganism
WO1990004037A1 (en) * 1988-10-03 1990-04-19 Genlux Forschungsgesellschaft Für Biologische Verfahren Mbh Process for detecting and identifying toxic substances by means of cloned micro-organisms
WO1990004645A1 (en) * 1988-10-21 1990-05-03 Molecular Devices Corporation Methods and apparatus for detecting the effect of cell affecting agents on living cells
US4927752A (en) * 1985-06-26 1990-05-22 Jose Remacle Support used in bioluminescent dosing of enzymes, substrates or enzymatic inhibitors

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4581335A (en) * 1982-12-01 1986-04-08 Texas A&M University System Process for producing a cloned luciferase-synthesizing microorganism
US4927752A (en) * 1985-06-26 1990-05-22 Jose Remacle Support used in bioluminescent dosing of enzymes, substrates or enzymatic inhibitors
WO1990004037A1 (en) * 1988-10-03 1990-04-19 Genlux Forschungsgesellschaft Für Biologische Verfahren Mbh Process for detecting and identifying toxic substances by means of cloned micro-organisms
WO1990004645A1 (en) * 1988-10-21 1990-05-03 Molecular Devices Corporation Methods and apparatus for detecting the effect of cell affecting agents on living cells

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICES, File 156, Toxline 1965-92, Dialog Accession No. 02412562/5, Subfile Biosis 91-03820, LAMPINEN J et al.: "Use of Escherichia Coli Cloned with Genes Encoding Bacterial Luciferase for Evaluation of Chemical Toxicity"; & TOXIC ASSESS, 5 (4), 1990, 337-350. *
DIALOG INFORMATION SERVICES, File Biosis 69-92, Dialog Accession No. 8113050/7, Biosis Accession No. 91034050, RIBO J M et al.: "Toxicity of Mixtures of Auatic Contaminants Using the Luminescent Bacteria Bioassay"; & TOXIC ASSESS 5 (2), 1990, 135-152. *
JOURNAL OF BIOLUMINESCENCE AND CHEMILUMINESCENCE, Vol. 4, 1989, MATTI KORPELA et al.: "Stable-Light-Emmiting Escherichia Coli as a Biosensor", pp. 551-554. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4332165A1 (en) * 1993-09-22 1995-03-23 Kolibri Umweltanalytik Und On Method and apparatus for pollutant analysis of water samples
DE19621312A1 (en) * 1996-05-28 1997-12-04 Bayer Ag Masking of background fluorescence and signal amplification in the optical analysis of biological medical assays
US6420183B1 (en) 1996-05-28 2002-07-16 Bayer Aktiengesellschaft Masking background fluorescence and luminescence in optical analysis of biomedical assays
US7063952B2 (en) 1996-05-28 2006-06-20 Bayer Healthcare Ag Masking background flourescence and luminescence in optical analysis of biomedical assays
US7138280B2 (en) 1996-05-28 2006-11-21 Bayer Healthcare Ag Masking of the background fluorescence and luminescence in the optical analysis of biomedical assays
US7615376B2 (en) 1996-05-28 2009-11-10 Bayer Schering Pharma Aktiengesellschaft Masking background fluorescence and luminescence in the optical analysis of biomedical assays
US8178359B2 (en) 1996-05-28 2012-05-15 Bayer Healthcare Ag Masking of the background fluorescence and luminescence in the optical analysis of biomedical assays
JP3481212B2 (en) 2000-03-03 2003-12-22 サンジョン キム Luminescent bacterial strain for toxic substance detection, toxic substance inspection method and toxic substance analyzer

Also Published As

Publication number Publication date
FI913443A0 (en) 1991-07-17

Similar Documents

Publication Publication Date Title
Stringfellow et al. Competitive metabolism of naphthalene, methylnaphthalenes, and fluorene by phenanthrene-degrading pseudomonads
Grundy et al. Phagocytic reduction and effects on lysosomal membranes by polycyclic aromatic hydrocarbons, in haemocytes of Mytilus edulis
Huang et al. Mechanisms of photoinduced toxicity of photomodified anthracene to plants: inhibition of photosynthesis in the aquatic higher plant Lemna gibba (duckweed)
McConkey et al. Toxicity of a PAH photooxidation product to the bacteria Photobacterium phosphoreum and the duckweed Lemna gibba: Effects of phenanthrene and its primary photoproduct, phenanthrenequinone
Potts et al. The 4-hydroxylation of cinnamic acid by sorghum microsomes and the requirement for cytochrome P-450
Tarasova et al. Bioluminescence as a tool for studying detoxification processes in metal salt solutions involving humic substances
Kudryasheva et al. Development of bioluminescent bioindicators for analysis of environmental pollution
Newland et al. Degradation of γ-BHC in simulated lake impoundments as affected by aeration
Zare et al. Resazurin reduction assay, a useful tool for assessment of heavy metal toxicity in acidic conditions
CA1268407A (en) Bioassay for toxic substances
Xu et al. Effect of ibuprofen on the sulfur autotrophic denitrification process and microbial toxic response mechanism
Gavlasova et al. Whole cell biosensor for polychlorinated biphenyl analysis based on optical detection
Fedorova et al. Bioluminescent monitoring of detoxification processes: Activity of humic substances in quinone solutions
Allard et al. Dechlorination of chlorocatechols by stable enrichment cultures of anaerobic bacteria
Bouwer et al. Ethylene dibromide transformation under methanogenic conditions
Van Vlaardingen et al. Property‐toxicity relationships of azaarenes to the green alga Scenedesmus acuminatus
WO1993002208A1 (en) Method, apparatus and indicator for indicating evaporable hydrocarbons or environmental poisons in water or liquids
Biagini et al. The antioxidant potential of pyruvate in the amitochondriate diplomonads Giardia intestinalis and Hexamita inflata
Gu et al. The continuous monitoring of field water samples with a novel multi-channel two-stage mini-bioreactor system
Huuskonen et al. Comparison of two bioassays, a fish liver cell line (PLHC-1) and a midge (Chironomus riparius), in monitoring freshwater sediments
Lee et al. Toxicological characterization of sediments from Baie des Anglais on the St. Lawrence Estuary
Kim et al. Alterations in fatty acid composition and fluidity of cell membranes affect the accumulation of PCB congener 2, 2′, 5, 5′‐tetrachlorobiphenyl by Ralstonia eutropha H850
Keane et al. Use of a whole‐cell biosensor to assess the bioavailability enhancement of aromatic hydrocarbon compounds by nonionic surfactants
Yamada et al. Oxidative stress by biphenyl metabolites induces inhibition of bacterial cell separation
Spratt Jr et al. Microbial manganese oxidation in saltmarsh surface sediments using a leuco crystal violet manganese oxide detection technique

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU MC NL SE

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA