CA2143618A1 - Biosensor for detection of and distinguishing between cholinesterases inhibitors - Google Patents
Biosensor for detection of and distinguishing between cholinesterases inhibitorsInfo
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- CA2143618A1 CA2143618A1 CA 2143618 CA2143618A CA2143618A1 CA 2143618 A1 CA2143618 A1 CA 2143618A1 CA 2143618 CA2143618 CA 2143618 CA 2143618 A CA2143618 A CA 2143618A CA 2143618 A1 CA2143618 A1 CA 2143618A1
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- cholinesterase
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- 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
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
- C12Q1/46—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase involving cholinesterase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
- C12N11/12—Cellulose or derivatives thereof
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- 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
- C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
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- Biomedical Technology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a biosensor of cholinesterase inhibitors in air, water, foodstuffs, soil, at surfaces and in extracts from samples, comprised of cellulose textile with bioafinity immobilized and stabilized cholinesterase, colour standard and of a carrier from substrate and chromogenic agent and its application to the detection of and distinguishing between cholinesterase in-hibitors. In distinguishing between the inhibitors by a biosensor in case of cholinesterase immobilized at cellulose textile of the biosensor after inhibition by organophosphorous or carbamate inhibitor, hydrolytic activity restores the activity of nucleophilic agents. According to the level of the restored hydrolytic activity of the immobilized cholinesterase in relation to the exposition time to reactivators, e.g. to quaternary aldoximes or water, and according to the activity change of the immobilized cholinesterase exposed in the inhibitor solution and in the inhibitor solution containing ions, e.g. natrium, calcium or ammonium ions, the type of the organophosphorous and carbamate inhibitor is determined.
Description
21~3618 BIOSENSOR FOR DETECTION OF AND FOR DISTINGUISHING BETWEEN
INHIBITORS OF CHOLINESTERASES
FIELD OF THE ART
The invention relates to the analysis of biologically very effective organophosphorous and carbamate inhibitors of cholinesterases by a simple biosensor. The invention makes possible to use the sensitive biochemical cholinesterase reaction to detect and distinguish between the biologically extremely effective organophosphates and carbamates that are permanently used in the field of the industrial chemistry, agrochemistry and in the military sector.
STATE OF THE ART
The biochemical reaction of cholinesters is a-very sensitive and selective method that is widely used for a long time in a number of modifications and applications to detect and determine a numerous group of agents that are cholinesterases inhibitors [AMMON, R. - VOSS, G., Pflugers, Arch. ges. Physiol., 235 (1935), p. 393; MICHEL, 0., H., J.
Lab. clin. Med., 34 (1949), p. 1564]. Technical means of various design used to detect and determine cholinesterases inhibitors, starting with the complicated automatic atmosphere and water analysers, continuing with more simple designs, e.g. tube detectors and ending with the most simple but therefore more used detection test papers, are based on the cholinesterase reaction. [FRANKE, S.: Lehrbuch der Militarchemie, Militarverlag DDR, part II, Berlin 1977, p.
481; U.S. patent No. 4.324,858; ~O 8504424]. Regardless of AMENDEO StlEEr _ - 2 _ 21~ 361 the total complexity of the design and financial costs all categories of means maintain a similar sensibility and selectivity owing to the same reaction principle.
Consequently, in the absolute majority of cases the most simple and therefore also the cheapest design, i.e. the detection paper is preferred. The individual detection papers differ in two ways. At first, in the used substrate and consequently in the relevant indication reaction. This part of the cholinesterase reaction materialization in the test paper is not essential as it cannot have an influence on the applicability for determinations in water and atmosphere, but it can influence the determination sensitivity. The basic problem is the method how to fix and stabilize the enzyme. Some existing methods are based on the enzyme adsorption in the filtration paper [AO CSFR 269267].
Another solution of this problem is to immobilize cholinesterase in the carrier by a chemical bond, what is accompanied by a significant hydrolytic activity loss of the enzyme. Additionally, it requires to use a highly purified and therefore expensive enzyme for the immobilization, whereby prolongation of the enzyme paper expiration time is not very high [RO 105190].
The nearest disclosed design to the one according to this invention is to prepare the enzyme detection paper by immobilization of cholinesterase in a special chromatographic paper with ion exchange function in mixture with dextrane gel and to make indication by a chromogenic substrate [~O 85/04423]. However, in this case the required sensitivity of the detection paper was not o~tained in case of increased storage time. In spite of a great number of various modifications relating to the method of evaluation of the used enzyme, substrate, various environments or to modification of the other workmanship conditions, all such modifications are limited by the fact that the biochemical cholinesterase reaction is a group reaction for a very AMEND~D SHEET
INHIBITORS OF CHOLINESTERASES
FIELD OF THE ART
The invention relates to the analysis of biologically very effective organophosphorous and carbamate inhibitors of cholinesterases by a simple biosensor. The invention makes possible to use the sensitive biochemical cholinesterase reaction to detect and distinguish between the biologically extremely effective organophosphates and carbamates that are permanently used in the field of the industrial chemistry, agrochemistry and in the military sector.
STATE OF THE ART
The biochemical reaction of cholinesters is a-very sensitive and selective method that is widely used for a long time in a number of modifications and applications to detect and determine a numerous group of agents that are cholinesterases inhibitors [AMMON, R. - VOSS, G., Pflugers, Arch. ges. Physiol., 235 (1935), p. 393; MICHEL, 0., H., J.
Lab. clin. Med., 34 (1949), p. 1564]. Technical means of various design used to detect and determine cholinesterases inhibitors, starting with the complicated automatic atmosphere and water analysers, continuing with more simple designs, e.g. tube detectors and ending with the most simple but therefore more used detection test papers, are based on the cholinesterase reaction. [FRANKE, S.: Lehrbuch der Militarchemie, Militarverlag DDR, part II, Berlin 1977, p.
481; U.S. patent No. 4.324,858; ~O 8504424]. Regardless of AMENDEO StlEEr _ - 2 _ 21~ 361 the total complexity of the design and financial costs all categories of means maintain a similar sensibility and selectivity owing to the same reaction principle.
Consequently, in the absolute majority of cases the most simple and therefore also the cheapest design, i.e. the detection paper is preferred. The individual detection papers differ in two ways. At first, in the used substrate and consequently in the relevant indication reaction. This part of the cholinesterase reaction materialization in the test paper is not essential as it cannot have an influence on the applicability for determinations in water and atmosphere, but it can influence the determination sensitivity. The basic problem is the method how to fix and stabilize the enzyme. Some existing methods are based on the enzyme adsorption in the filtration paper [AO CSFR 269267].
Another solution of this problem is to immobilize cholinesterase in the carrier by a chemical bond, what is accompanied by a significant hydrolytic activity loss of the enzyme. Additionally, it requires to use a highly purified and therefore expensive enzyme for the immobilization, whereby prolongation of the enzyme paper expiration time is not very high [RO 105190].
The nearest disclosed design to the one according to this invention is to prepare the enzyme detection paper by immobilization of cholinesterase in a special chromatographic paper with ion exchange function in mixture with dextrane gel and to make indication by a chromogenic substrate [~O 85/04423]. However, in this case the required sensitivity of the detection paper was not o~tained in case of increased storage time. In spite of a great number of various modifications relating to the method of evaluation of the used enzyme, substrate, various environments or to modification of the other workmanship conditions, all such modifications are limited by the fact that the biochemical cholinesterase reaction is a group reaction for a very AMEND~D SHEET
2 1 J~ 3 6 1 8 r numerous group of the cholinesterases inhibitors. The most important of them are the organophosphorous compounds and the carbamates. To make possible to identify what inhibitor is present, it is necessary to carry out some detection reactions or physicaZ chemistry measurements. However, this requires a greater amount of inhibitor or by several orders higher inhibitor concentration in the sample solution than as it is necessary in case of the very sensitive biochemical cholinesterase method. In such case it is necessary to carry out difficult, complex and lengthy sample concentration preceding the proper chemical or physical chemistry identification. Such methods with the exception of some more simple separation and identification techniques, as is the thin-layer chromatography, are not only time consuming but also require further operations connected with performing the analytical detection reactions or with the use of expensive instruments, that can not be operated in the field. Moreover, considering the extreme biological effectiveness~ a number of the organophosphorous and carbamate cholinesterases inhibitors constitutes also a substantial risk with regard to labour safety if we consider the duration of the necessary operations with concentrated solution of the compound which is to be identified.
The nearest disclosed solution of the analysis method with regard to this invention is the method of the organophosphorous inhibitors identification which is based on the formation of two types of complexes with metal ions and the detection by ~he cholinesterase reaction [CEBOTARJOV, 0., V.: Vojskovaja indikacia, Izdatelstvo akademii Moscow 1978] and the method of distinguishing between the organic inhibitors by the cholinesterase reaction after cholinesterase was modified by metal ions.
The disadvantages mentioned consisting in the group nature of the biochemical cholinesterase reaction require that AMENDED SIIEE~
- ~ 4 ~ 219 361 8 ' "'"
laborious and lengthy inhibitor sample concentration is necessary and that expensive and complicated instruments are to be used for identification. The above disadvantages are removed by detection of organophosphorous and carbamate cholinesterases inhibitors according to this invention.
THE EMBODIMENT OF THE INVENTION
The invention consists in a biosensor used to detect and identify cholinesterases inhibitors in atmosphere, water, foodstuffs, soil, surfaces and extracts from samples. The biosensor in the form of test strips consists of a cellulose cloth zone made of cotton fibres containing immobilized cholinesterase, of a colour standard and of an inert carrier zone, e.g. of a filter paper with substrate and chromogenic agent. During the cholinesterase inhibitors detection, e. g. the acetylthiocholiniodide substrate and the 5,5'-di-thiobis(2-nitrobenzoic) acid as chromogenic agent, are transferred to a wet cellulose cloth cont~;n;ng immobilized cholinesterase exposed in a controlled environment, by pressing filter paper cont~;n;ng them towards it. The chromogenic agent reacts by colour change to products of the substrate hydrolysis. The hydrolytic activity of the immobilized cholinestarase is visually evaluated by comparison with the colour standard as a change of the cellulose cloth colour. Application of this biosensor to distinguishing between inhibitors consists in that in case of cholinesterases immobilized on cellulose cloth, after inhibition by organophosphate or carbamate inhibitor, the hydrolytic activity is restored by induced reactivation by quaternary aldoximes, e.g. by l-(2-hydroxyiminomethylpyri-dinum)-3-(4-carba-moylpyridinum-2-oxapropan)dichloride (further only HI-6) and N,N'-tri-methylenbis(4-pyridinium-adoxim)dichloride (further only TMB-4) or by spontaneous reactivation by water. Simultaneously, the samples of cholinesterase immobilized on cellulose cloth of the AMENDED S~IEET
r ~ ~ r - s -_ 21~3618 biosensor are exposed in water solution of inhibitor and in the water solution of inhibitor with the addition of ionic compounds, e.g. of natrium, calcium or ammonium chloride. By evaluation of the hydrolytic activity of the reactivated cholinesterase and of the activity of the cholinesterase exposed in the inhibitor solution and in the inhibitor solution with the added sodium, calcium or ammonium chloride, a characteristic difference in activity is indicated that makes possible to identify which inhibitor it is.
In comparison to the prior art the present invention is distinguished by the carrier nature and carrier treatment that makes an easy immobilization of enzyme and further by possibility of distinguishing inhibitors by the biosensor.
By the use of 100 ~ cotton cloth for immobilization of cholinesterase without further treatment and additives a composite material having new biochemical, mechanical and optical characteristics is formed.
The above can be documented by the following characteristics:
The used carrier - the cotton cloth can be used in a simple immobilization of enzymes of different etiology and quality and the obtained cellulose - cholinesterase composite is very stable.
The used carrier containing immobilized enzyme is of such superior mechanical properties that it allows repeated, up to 60 times, use of the detection cloth, long time exposition in running water or long time exposition in untreated complex samples, e.g. in food homogenizates.
U- a S~
~ 1 ~ 3 6 lr8 ~ ~ ~ ; r e The detection cloth makes possible to wash out perfectly the co-extracted impurities and colours that would interfere with the visual evaluation of the reaction course.
All said above is not possible in case of the disclosed carriers.
Methods of simple and effective immobilization of cholinesterases of various etiology and purity giving products stable at usual and elevated (up to 60 ~C) temperature are not known.
Research of the inhibited enzymes is in reality limited by the possibility to isolate them from the analyzed environment. An effective isolation from the environment is ensured by preparation a stable enzyme composite cotton cloth - cholinesterase and the difference in structure of the isolated enzyme-inhibitor complexes can by studied individually.
AMEN~)ED SHEET
7 ~ 2 1 4 3 6 1 8 EXAMPLES
Example 1 Preparation of acetylcholinesterase solution The prepared acetylcholinesterase is used to prepare a solu-tion having specific activity 7 to 15 nkat/ml by dissolving it in a solution buffered of pH 5 to 9.
Example 2 Modification of cellulose cloth.
A cellulose cloth woven from cotton fibres is modified by alkaline oxidative bleaching by boiling the cloth in a solution cont~inlng 7.9 g/l of hydrogen peroxide (35 %), 0.2 g/l of magnesium chloride, 0.2 g/l of non-ionogenic tenside SLOVASOL SF-10, 2.1 g/l of sodium hydroxide and 2.5 g/l of water glass.
Example 3 Immobilization of acetylcholinesterase on bleached cellulose cloth The cloth modified according to Example 2 is washed by water and dried at 50 C and thereafter immersed in the solution prepared according to Example 1 for 30 minutes. Thereafter, it is taken out of the solution and dried at 20 C.
Example 4 Preparation of the carrier with substrate and chromogenic agent A filter paper is immersed in a solution of 0.5 % of acetyl-thiocholiniodide and 0.15 % of 5,5'-dithio-bis(2-nitro-benzoic) acid in ethanol for 10 minutes. Thereafter, thepaper is dried at 20 C.
AMENDED ~!!EET
21:4,~618 ' - ' Example 5 Detection of inhibitors in water and in samples of water homogenizates The detection cloth prepared according to Example 3 is immersed into water or water extract for 30 minutes.
Thereafter, the cloth is taken out and for 1 minute paper with substrate and chromogenic agent prepared according to Example 4 is pressed towards it. After the paper is removed the cloth is yellow coloured in case of acetylcholinesterase inhibitors absence. Unchanged colour of the cloth is an evidence that inhibitors are present.
Sensitivity to organophosphate and carbamate insecticides detection in water and water extracts from foodstuffs Insecticide Detection Limit (mg/l) CARBOFURAN 2.10-2 METHOMYL 1.10-DICHLORVOS 1.10-1 MEVINPHOS 1.10-2 PARATHION - 2.10-2 ~ETHYLPARATHION 4.10-2 Example 6 Inhibitors detection in air Detection cloth prepared according to example 3 is made wet by water and left in a controlled atmosphere minimally 1 minute. Thereafter, paper with substrate and chromogenic agent prepared according to Example 4 is pressed towards the cloth. After the paper is removed the cloth is coloured yellow if inhibitors of cholinesterase are not present. An evidence of inhibitors is the unchanged colour of the textile.
AM~ND'a ~' 2 1 ~
Detection sensitivity to nerve agents detection in atmosphere nerve agent detection limit (mg/l) after exposition 2 min 20 min isopropylmethylfluorophosohonate 5 7 SARIN GB 1.10- 4.10-pinakolylmethylfluorophosohonate 6 7 SOMAN GD 8.10- 2.10-0-etthyl-s-(2-diisopropylamino-ethyl)methylthiophosphonate 5 7 AGENT VX 5.10- 5.10-2-dimethylamionoethyldimethyl-amidofluorophosphate ACENT GP 5.10-6 2.10-7 ethyldimethylamidocyanophosphate 5 TABUN GA 8.10- 1.10-6 cyclohexylmethylfluorophossphonate 6 7 CYKLOSIN GF 3.10- 1.10-Example 7 --Use of biosensor to distinguish between GD and GF compounds Cloth samples containing the immobilized acetylcholin-esterase inhibited to 100% by 2 to 5 minutes of incubation in a solution containing organophosphate inhibitor are reac-tivated for 5 minutes in 0.02 mg/ml of ~I-6 and 0.05 mg/ml of TMB-4. After reactivation, hydrolytic activity of the enzyme in both samples containing inhibited cholinesterase is evaluated. If the hydrolytic activity of the inhibited enzyme was restored only by reactivation in 0.02 mg/ml of HI-6, the solution contains the GD or GF compound. The reactivation is repeated after the inhibition of a sample of immobilized acetylcholinesterase by 30 minutes of incubation. ~hen the enzyme hydrolytic activity is restored AMENOED SHE~T
- 1 0 - ' ' ' ' ' ' by the reactivation the solution contains the GF compound.
Example 8 Use of biosensor to distinguish between VX and GB compounds Cloth samples containing the immobilized acetylcholin-esterase inhibited to 100% by 2 to 5 minutes of incubation in the solution containing organophosphate inhibitor are reactivated for 5 minutes in 0.02 mg/ml of HI-6 and 0.05 mg/ml of TMB-4. After reactivation, the enzyme hydrolytic activity in both samples of inhibited cholinesterase is evalua~ed. If the hydrolytic activity of the inhibited enzyme was restored by reactivation in both reactivators the solution contains the compound VX or GB. The solution is diluted ten-times and divided to two parts. To one part of the diluted solution natrium chloride is added in surplus and in both solutions samples of immobilized acetylcholin-esterase are simultaneously incubated for 2, 5, 10, and 15 minutes till different hydrolytic activities of samples are reached. In case of an increase of the sample hydrolytic activity which was incubated in the solution with natrium chloride in comparison to the activity of the sample incubated in the solution without natrium chloride the analyzed sample contains the VX compound.
Example 9 Use of biosensor to distinguish between GA and GP compounds Samples of the immobilized acetylcholinesterase inhibited to 100% by 2 to 5 minutes of incubation in the solution containing organophosphate inhibitor are reactivated for 5 minutes in 0.02 mg/ml of HI-6 and 0.05 mg/ml of TMB-4. After reactivation, the enzyme hydrolytic activity is evaluated in both samples of inhibited cholinesterase. If the hydrolytic AME~IDEDSHEET
- 11 219~618 activity of the inhibited enzyme is not restored in both reactivators the sample contains solution of the GA or GP
compound. The solution will be ten-times diluted and divided to two parts. Natrium chloride is added in surplus to one part of the diluted solution and simultaneously in both solutions the samples of immobilized acetylcholinesterase will be incubated for 2, 5, 10 and 15 minutes till a different hydrolytic sample activity is obtained. In case the hydrolytic activity of the sample incubated in the solvent with natrium chloride is higher than that of the solution without natrium chloride, the analyzed sample contains the GP compound.
Example 10 Use of biosensor to distinguish organophosphorous and carbamate insecticides .
Samples of the immobilized acetylcholinesterase inhibited to 100% by 2 to S minutes of incubation in the solution containing organophosphate or carbamate inhibitor are reactivated for 5 minutes in 0.02 mg/ml of HI-6 and 0.05 mg/ml of TMB-4. After reactivation, the enzyme hydrolytic activity in both samples of inhibited cholinesterase will be evaluated. If hydrolytic activity of the inhibited enzyme is not restored after 5 minutes of reactivation in both reacti-vators, the sample is immersed for 30 to 120 minutes into distilled water. If the hydrolytic activity is restored after 60 minutes of reactivation in water, the solution contains l-naftyl-~-methylcarbamate. If the hydrolytic activity is restored after 120 minutes of reactivation in water, the solution contains 2,3-dihydro-2,2-dimethylbenzo-furane-7-yl-~-methylcarbamate.
A~ !DE~
21436I8 .
Example 11 Biosensor use in identification of nerve paralyzing agents Two samples of the detection cloth are again immersed into the analyzed solution containing an unspecified nerve gas in which the detecting cloth sample containing immobilized acetylcholinesterase remained white after 2 minutes of exposition. Thereafter, the samples are immersed into the reactivator solution for S minutes, one sample into 0.05 mg.ml~l of TMB-4 solution and the other into 0.02 mg.ml~l of the HI-6 solution. ~hen the cloth is removed from the reactivators solutions three alternative possibilities can be observed with regard to the cloth activity:
a) Both cloth samples remain white. In this case the solution contains the compound GA or GP.
b) Both cloth samples become yellos. In this case the solution contains the compound VX or GP.
c) The cloth reactivated in TMB-4 remains white. The cloth which has been reactivated in HI-6 becomes yellow. In this case the solution contains the compound GF or GD.
Identification of the individual compounds from the alterna-tives a) or b) is carried out so that the solution is diluted by water in ratio 1:1 and divided in two parts. NaCl is added to one part so that a saturated solution or a solution with surplus of undissolved NaCl is obtained.
Thereafter, inhibition in both solutions is compared. If the cloth inhibited by the solution with NaCl becomes less yellow and the cloth from the solution without NaCl remains white, the solution contains the VX or GP compounds. In the c) case it is possible to identify GF from GD so that a sample of the detection cloth is exposed in the original solution for 30 minutes. ~hen the exposition is inished the AMEN~)~O SHE~
_ - 13 _ 2 1.4 3 6 1 8 cloth sample is immersed into the solution of ~I-6 for 5 minutes and if after reactivation the cloth becomes yellow the solution contains GF. In case the cloth remains inhibited even after the reactivation the solution contains the GD compound.
The invention can be used in qualitative and quantitative determination of organophosphorous and carbamate inhibitors of cholinesterase. Such determination is carried out in many industries and agriculture, but also by medical and veterinary institutions. As a method of detection and determination of organophosphorous and carbamate poisons the modified cholinesterase reaction according to the invention can be used also in the field of military chemistry if chemical reconnaissance or tests are carried out, in civil defense and state safety matters and in monitoring environment contamination by inhibitors of cholinesterases.
The biosensor according to this invention is applicable in testing contaminated samples of various etiology and composition. Rapid field checking without usual laboratory equipment is possible.
AM~NDED~E~
The nearest disclosed solution of the analysis method with regard to this invention is the method of the organophosphorous inhibitors identification which is based on the formation of two types of complexes with metal ions and the detection by ~he cholinesterase reaction [CEBOTARJOV, 0., V.: Vojskovaja indikacia, Izdatelstvo akademii Moscow 1978] and the method of distinguishing between the organic inhibitors by the cholinesterase reaction after cholinesterase was modified by metal ions.
The disadvantages mentioned consisting in the group nature of the biochemical cholinesterase reaction require that AMENDED SIIEE~
- ~ 4 ~ 219 361 8 ' "'"
laborious and lengthy inhibitor sample concentration is necessary and that expensive and complicated instruments are to be used for identification. The above disadvantages are removed by detection of organophosphorous and carbamate cholinesterases inhibitors according to this invention.
THE EMBODIMENT OF THE INVENTION
The invention consists in a biosensor used to detect and identify cholinesterases inhibitors in atmosphere, water, foodstuffs, soil, surfaces and extracts from samples. The biosensor in the form of test strips consists of a cellulose cloth zone made of cotton fibres containing immobilized cholinesterase, of a colour standard and of an inert carrier zone, e.g. of a filter paper with substrate and chromogenic agent. During the cholinesterase inhibitors detection, e. g. the acetylthiocholiniodide substrate and the 5,5'-di-thiobis(2-nitrobenzoic) acid as chromogenic agent, are transferred to a wet cellulose cloth cont~;n;ng immobilized cholinesterase exposed in a controlled environment, by pressing filter paper cont~;n;ng them towards it. The chromogenic agent reacts by colour change to products of the substrate hydrolysis. The hydrolytic activity of the immobilized cholinestarase is visually evaluated by comparison with the colour standard as a change of the cellulose cloth colour. Application of this biosensor to distinguishing between inhibitors consists in that in case of cholinesterases immobilized on cellulose cloth, after inhibition by organophosphate or carbamate inhibitor, the hydrolytic activity is restored by induced reactivation by quaternary aldoximes, e.g. by l-(2-hydroxyiminomethylpyri-dinum)-3-(4-carba-moylpyridinum-2-oxapropan)dichloride (further only HI-6) and N,N'-tri-methylenbis(4-pyridinium-adoxim)dichloride (further only TMB-4) or by spontaneous reactivation by water. Simultaneously, the samples of cholinesterase immobilized on cellulose cloth of the AMENDED S~IEET
r ~ ~ r - s -_ 21~3618 biosensor are exposed in water solution of inhibitor and in the water solution of inhibitor with the addition of ionic compounds, e.g. of natrium, calcium or ammonium chloride. By evaluation of the hydrolytic activity of the reactivated cholinesterase and of the activity of the cholinesterase exposed in the inhibitor solution and in the inhibitor solution with the added sodium, calcium or ammonium chloride, a characteristic difference in activity is indicated that makes possible to identify which inhibitor it is.
In comparison to the prior art the present invention is distinguished by the carrier nature and carrier treatment that makes an easy immobilization of enzyme and further by possibility of distinguishing inhibitors by the biosensor.
By the use of 100 ~ cotton cloth for immobilization of cholinesterase without further treatment and additives a composite material having new biochemical, mechanical and optical characteristics is formed.
The above can be documented by the following characteristics:
The used carrier - the cotton cloth can be used in a simple immobilization of enzymes of different etiology and quality and the obtained cellulose - cholinesterase composite is very stable.
The used carrier containing immobilized enzyme is of such superior mechanical properties that it allows repeated, up to 60 times, use of the detection cloth, long time exposition in running water or long time exposition in untreated complex samples, e.g. in food homogenizates.
U- a S~
~ 1 ~ 3 6 lr8 ~ ~ ~ ; r e The detection cloth makes possible to wash out perfectly the co-extracted impurities and colours that would interfere with the visual evaluation of the reaction course.
All said above is not possible in case of the disclosed carriers.
Methods of simple and effective immobilization of cholinesterases of various etiology and purity giving products stable at usual and elevated (up to 60 ~C) temperature are not known.
Research of the inhibited enzymes is in reality limited by the possibility to isolate them from the analyzed environment. An effective isolation from the environment is ensured by preparation a stable enzyme composite cotton cloth - cholinesterase and the difference in structure of the isolated enzyme-inhibitor complexes can by studied individually.
AMEN~)ED SHEET
7 ~ 2 1 4 3 6 1 8 EXAMPLES
Example 1 Preparation of acetylcholinesterase solution The prepared acetylcholinesterase is used to prepare a solu-tion having specific activity 7 to 15 nkat/ml by dissolving it in a solution buffered of pH 5 to 9.
Example 2 Modification of cellulose cloth.
A cellulose cloth woven from cotton fibres is modified by alkaline oxidative bleaching by boiling the cloth in a solution cont~inlng 7.9 g/l of hydrogen peroxide (35 %), 0.2 g/l of magnesium chloride, 0.2 g/l of non-ionogenic tenside SLOVASOL SF-10, 2.1 g/l of sodium hydroxide and 2.5 g/l of water glass.
Example 3 Immobilization of acetylcholinesterase on bleached cellulose cloth The cloth modified according to Example 2 is washed by water and dried at 50 C and thereafter immersed in the solution prepared according to Example 1 for 30 minutes. Thereafter, it is taken out of the solution and dried at 20 C.
Example 4 Preparation of the carrier with substrate and chromogenic agent A filter paper is immersed in a solution of 0.5 % of acetyl-thiocholiniodide and 0.15 % of 5,5'-dithio-bis(2-nitro-benzoic) acid in ethanol for 10 minutes. Thereafter, thepaper is dried at 20 C.
AMENDED ~!!EET
21:4,~618 ' - ' Example 5 Detection of inhibitors in water and in samples of water homogenizates The detection cloth prepared according to Example 3 is immersed into water or water extract for 30 minutes.
Thereafter, the cloth is taken out and for 1 minute paper with substrate and chromogenic agent prepared according to Example 4 is pressed towards it. After the paper is removed the cloth is yellow coloured in case of acetylcholinesterase inhibitors absence. Unchanged colour of the cloth is an evidence that inhibitors are present.
Sensitivity to organophosphate and carbamate insecticides detection in water and water extracts from foodstuffs Insecticide Detection Limit (mg/l) CARBOFURAN 2.10-2 METHOMYL 1.10-DICHLORVOS 1.10-1 MEVINPHOS 1.10-2 PARATHION - 2.10-2 ~ETHYLPARATHION 4.10-2 Example 6 Inhibitors detection in air Detection cloth prepared according to example 3 is made wet by water and left in a controlled atmosphere minimally 1 minute. Thereafter, paper with substrate and chromogenic agent prepared according to Example 4 is pressed towards the cloth. After the paper is removed the cloth is coloured yellow if inhibitors of cholinesterase are not present. An evidence of inhibitors is the unchanged colour of the textile.
AM~ND'a ~' 2 1 ~
Detection sensitivity to nerve agents detection in atmosphere nerve agent detection limit (mg/l) after exposition 2 min 20 min isopropylmethylfluorophosohonate 5 7 SARIN GB 1.10- 4.10-pinakolylmethylfluorophosohonate 6 7 SOMAN GD 8.10- 2.10-0-etthyl-s-(2-diisopropylamino-ethyl)methylthiophosphonate 5 7 AGENT VX 5.10- 5.10-2-dimethylamionoethyldimethyl-amidofluorophosphate ACENT GP 5.10-6 2.10-7 ethyldimethylamidocyanophosphate 5 TABUN GA 8.10- 1.10-6 cyclohexylmethylfluorophossphonate 6 7 CYKLOSIN GF 3.10- 1.10-Example 7 --Use of biosensor to distinguish between GD and GF compounds Cloth samples containing the immobilized acetylcholin-esterase inhibited to 100% by 2 to 5 minutes of incubation in a solution containing organophosphate inhibitor are reac-tivated for 5 minutes in 0.02 mg/ml of ~I-6 and 0.05 mg/ml of TMB-4. After reactivation, hydrolytic activity of the enzyme in both samples containing inhibited cholinesterase is evaluated. If the hydrolytic activity of the inhibited enzyme was restored only by reactivation in 0.02 mg/ml of HI-6, the solution contains the GD or GF compound. The reactivation is repeated after the inhibition of a sample of immobilized acetylcholinesterase by 30 minutes of incubation. ~hen the enzyme hydrolytic activity is restored AMENOED SHE~T
- 1 0 - ' ' ' ' ' ' by the reactivation the solution contains the GF compound.
Example 8 Use of biosensor to distinguish between VX and GB compounds Cloth samples containing the immobilized acetylcholin-esterase inhibited to 100% by 2 to 5 minutes of incubation in the solution containing organophosphate inhibitor are reactivated for 5 minutes in 0.02 mg/ml of HI-6 and 0.05 mg/ml of TMB-4. After reactivation, the enzyme hydrolytic activity in both samples of inhibited cholinesterase is evalua~ed. If the hydrolytic activity of the inhibited enzyme was restored by reactivation in both reactivators the solution contains the compound VX or GB. The solution is diluted ten-times and divided to two parts. To one part of the diluted solution natrium chloride is added in surplus and in both solutions samples of immobilized acetylcholin-esterase are simultaneously incubated for 2, 5, 10, and 15 minutes till different hydrolytic activities of samples are reached. In case of an increase of the sample hydrolytic activity which was incubated in the solution with natrium chloride in comparison to the activity of the sample incubated in the solution without natrium chloride the analyzed sample contains the VX compound.
Example 9 Use of biosensor to distinguish between GA and GP compounds Samples of the immobilized acetylcholinesterase inhibited to 100% by 2 to 5 minutes of incubation in the solution containing organophosphate inhibitor are reactivated for 5 minutes in 0.02 mg/ml of HI-6 and 0.05 mg/ml of TMB-4. After reactivation, the enzyme hydrolytic activity is evaluated in both samples of inhibited cholinesterase. If the hydrolytic AME~IDEDSHEET
- 11 219~618 activity of the inhibited enzyme is not restored in both reactivators the sample contains solution of the GA or GP
compound. The solution will be ten-times diluted and divided to two parts. Natrium chloride is added in surplus to one part of the diluted solution and simultaneously in both solutions the samples of immobilized acetylcholinesterase will be incubated for 2, 5, 10 and 15 minutes till a different hydrolytic sample activity is obtained. In case the hydrolytic activity of the sample incubated in the solvent with natrium chloride is higher than that of the solution without natrium chloride, the analyzed sample contains the GP compound.
Example 10 Use of biosensor to distinguish organophosphorous and carbamate insecticides .
Samples of the immobilized acetylcholinesterase inhibited to 100% by 2 to S minutes of incubation in the solution containing organophosphate or carbamate inhibitor are reactivated for 5 minutes in 0.02 mg/ml of HI-6 and 0.05 mg/ml of TMB-4. After reactivation, the enzyme hydrolytic activity in both samples of inhibited cholinesterase will be evaluated. If hydrolytic activity of the inhibited enzyme is not restored after 5 minutes of reactivation in both reacti-vators, the sample is immersed for 30 to 120 minutes into distilled water. If the hydrolytic activity is restored after 60 minutes of reactivation in water, the solution contains l-naftyl-~-methylcarbamate. If the hydrolytic activity is restored after 120 minutes of reactivation in water, the solution contains 2,3-dihydro-2,2-dimethylbenzo-furane-7-yl-~-methylcarbamate.
A~ !DE~
21436I8 .
Example 11 Biosensor use in identification of nerve paralyzing agents Two samples of the detection cloth are again immersed into the analyzed solution containing an unspecified nerve gas in which the detecting cloth sample containing immobilized acetylcholinesterase remained white after 2 minutes of exposition. Thereafter, the samples are immersed into the reactivator solution for S minutes, one sample into 0.05 mg.ml~l of TMB-4 solution and the other into 0.02 mg.ml~l of the HI-6 solution. ~hen the cloth is removed from the reactivators solutions three alternative possibilities can be observed with regard to the cloth activity:
a) Both cloth samples remain white. In this case the solution contains the compound GA or GP.
b) Both cloth samples become yellos. In this case the solution contains the compound VX or GP.
c) The cloth reactivated in TMB-4 remains white. The cloth which has been reactivated in HI-6 becomes yellow. In this case the solution contains the compound GF or GD.
Identification of the individual compounds from the alterna-tives a) or b) is carried out so that the solution is diluted by water in ratio 1:1 and divided in two parts. NaCl is added to one part so that a saturated solution or a solution with surplus of undissolved NaCl is obtained.
Thereafter, inhibition in both solutions is compared. If the cloth inhibited by the solution with NaCl becomes less yellow and the cloth from the solution without NaCl remains white, the solution contains the VX or GP compounds. In the c) case it is possible to identify GF from GD so that a sample of the detection cloth is exposed in the original solution for 30 minutes. ~hen the exposition is inished the AMEN~)~O SHE~
_ - 13 _ 2 1.4 3 6 1 8 cloth sample is immersed into the solution of ~I-6 for 5 minutes and if after reactivation the cloth becomes yellow the solution contains GF. In case the cloth remains inhibited even after the reactivation the solution contains the GD compound.
The invention can be used in qualitative and quantitative determination of organophosphorous and carbamate inhibitors of cholinesterase. Such determination is carried out in many industries and agriculture, but also by medical and veterinary institutions. As a method of detection and determination of organophosphorous and carbamate poisons the modified cholinesterase reaction according to the invention can be used also in the field of military chemistry if chemical reconnaissance or tests are carried out, in civil defense and state safety matters and in monitoring environment contamination by inhibitors of cholinesterases.
The biosensor according to this invention is applicable in testing contaminated samples of various etiology and composition. Rapid field checking without usual laboratory equipment is possible.
AM~NDED~E~
Claims (9)
1. Biosensor for detection of and distinguishing between cholinesterase inhibitors in the form of test strips containing immobilized cholinesterase zone, colour standard and inert carrier zone containing chromogenic agent, characterized in that the carrier of the immobilized cholinesterase is cellulose cloth, preferably made of cotton fibres.
2. Biosensor for detection of and distinguishing between cholinesterase inhibitors according to claim 1, characterized in that the cellulose cloth is modified by bleaching, preferably by alkaline oxidative bleaching.
3. Biosensor for detection of and distinguishing between cholinesterase inhibitors according to claim 1 or 2 characterized in that the inert carrier, preferably a filter paper contains a substrate, preferably acetylthiocholine, and the chromogenic agent reacting by colour change to products of the substrate hydrolysis, preferably 5,5'-dithiobis(2-nitrobenzoic) acid.
4. Biocensor for detection of and distinguishing between cholinesterase inhibitors according to anyone of claims 1 - 3 characterized in that the inert carrier, preferably filter paper contains the chromogenic substrate, preferably 1-[2-thiazoylazo]-2-acetoxy-5-methylbenzene-dimethylsulphate.
5. Method of producing the biosensor for detection of and distinguishing between cholinesterase inhibitors according to anyone of claims 1 - 4, characterized in that the bleached cellulose cloth contains cholinesterase immobilized by immersing the cloth into a buffered solution of the cholinesterase, preferably the brain acetylcholinesterase or the plasma butyrylcholnesterase.
6. Use of the biosensor for detection of and distinguishing between cholinesterase inhibitors according to anyone of claims 1 - 4, characterized in that the chromogenic agent is deposited at the wet cellulose cloth that has been exposed in the tested environment and the hydrolytic activity of the cholinesterase is visually evaluated according to the colour standard as the cellulose cloth colour change.
7. Use of the biosensor for detection of and distinguishing between cholinesterase inhibitors according to claim 6 characterized in that the chromogenic agent is deposited at wet cellulose textile by pressing the inert carrier saturated with the chromogenic agent towards it.
8. Use of the biosensor for detection of and distinguishing between cholinesterase inhibitors according to claim 6 or 7 characterized in that the hydrolytic activity of the inhibited cholinesterase immobilized at the cellulose cloth is evaluated after an induced reactivation, preferably by quarternary aldoximes and after spontaneous reactivation by water.
9. Use of the biosensor for detection of and distinguishing between cholinesterase inhibitors according to anyone of claims 6 - 8 characterized in that to detect and identify nerve paralytic compounds the change in the hydrolytic activity of inhibited cholinesterase immobilized at the cellulose cloth is evaluated after a reactivation induced by agents, preferably by quaternary aldoximes or after reactivation by water and after the action of ions, preferably ions of alkaline metals and earths.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CZPV2735-92 | 1992-09-01 | ||
| CS19922735A CZ288576B6 (en) | 1992-09-01 | 1992-09-01 | Biosensor for detecting and recognizing cholinesterase inhibitors, process for preparing biosensor immobilized cholinesterase zone, detection method of cholinesterase inhibitors and recognition method of cholinesterase inhibitors |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2143618A1 true CA2143618A1 (en) | 1994-03-17 |
Family
ID=5365284
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2143618 Abandoned CA2143618A1 (en) | 1992-09-01 | 1993-09-01 | Biosensor for detection of and distinguishing between cholinesterases inhibitors |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0672169A1 (en) |
| AU (1) | AU4941393A (en) |
| CA (1) | CA2143618A1 (en) |
| CZ (1) | CZ288576B6 (en) |
| SK (1) | SK281395B6 (en) |
| WO (1) | WO1994005808A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE340266T1 (en) * | 1999-04-26 | 2006-10-15 | Us Army Med Res Mat Command | IMMOBILIZED ENZYMES AS BIOSENSORS FOR CHEMICAL TOXINS |
| SG143093A1 (en) * | 2006-11-27 | 2008-06-27 | Dso Nat Lab | Rapid detection of cholinesterase inhibitors |
| EP3196315B1 (en) | 2016-01-19 | 2020-02-05 | ORITEST spol. s r.o. | Spherical pellets, manufacturing process of such pellets, use, and a detection tube comprising such pellets |
| CN109187386A (en) * | 2018-08-23 | 2019-01-11 | 成都众粒生物科技有限公司 | For detecting gel and its application of Organophosphorus and carbamate pesticides class pesticide concentration |
| CN112816592B (en) * | 2020-12-31 | 2023-03-24 | 兰州大学 | Preparation of immobilized acetylcholinesterase and application thereof in screening and identifying enzyme inhibitor |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3624095A (en) * | 1962-09-11 | 1971-11-30 | Us Army | 4-cyanoformyl-1-alkyl pyridinium halide oxime and its derivatives |
| NL7013950A (en) * | 1970-09-21 | 1972-03-23 | ||
| US4324858A (en) * | 1980-06-16 | 1982-04-13 | Midwest Research Institute | Stabilization of cholinesterase, detector kit using stabilized cholinesterase, and methods of making and using the same |
| SE454597B (en) * | 1984-04-02 | 1988-05-16 | Erik Dahlgren | ENZYM PAPERS FOR INDICATING CHOLINE ESTIMATE INHIBITORS WHICH ARE ENZYMED IMMOBILIZED BY AN ANION EXCHANGE, PROCEDURE FOR ITS PREPARATION AND USE OF PAPER WITH A SUBSTRATE |
| DD301358A7 (en) * | 1985-12-19 | 1992-12-10 | Bundesamt Fuer Sicherheit In D | SEMI-QUANTITATIVE FAST DETERGENT FOR CHOLINESTERASE ACTIVITY IN SERUM AND WHOLE BLOOD |
| FR2677373B1 (en) * | 1991-06-07 | 1995-10-06 | Anadiag | DETECTION OF PESTICIDES FROM THE ORGANOPHOSPHORUS AND / OR CARBAMATE FAMILY, IN AQUEOUS MEDIA. |
-
1992
- 1992-09-01 CZ CS19922735A patent/CZ288576B6/en not_active IP Right Cessation
- 1992-09-01 SK SK2735-92A patent/SK281395B6/en unknown
-
1993
- 1993-09-01 WO PCT/CZ1993/000021 patent/WO1994005808A1/en not_active Application Discontinuation
- 1993-09-01 CA CA 2143618 patent/CA2143618A1/en not_active Abandoned
- 1993-09-01 AU AU49413/93A patent/AU4941393A/en not_active Abandoned
- 1993-09-01 EP EP93918876A patent/EP0672169A1/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| AU4941393A (en) | 1994-03-29 |
| WO1994005808A1 (en) | 1994-03-17 |
| EP0672169A1 (en) | 1995-09-20 |
| SK273592A3 (en) | 1995-05-10 |
| SK281395B6 (en) | 2001-03-12 |
| CZ288576B6 (en) | 2001-07-11 |
| CZ273592A3 (en) | 1994-03-16 |
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