BIOSENSOR FOR DETECTION OF AND DISTINGUISHING BETWEEN CHOLINESTERASES INHIBITORS

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 inhibitors. 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: BIOSENSOR FOR DETECTION OF AND DISTINGUISHING BETVEEN CHOLINESTERASES INHIBITORS FIELD OF THE ART The invention relates to analysis of biologically very effective organophosphate and carbamate cholinesterases inhibitors by a simple biosensor. The invention makes possible to use sensitive biochemical cholinesterase reaction as a sensitive determination method of the biologically extremely effective organophosphates and carbamates permanently used in the field of industrial chemistry, agrochemistry and the military sector. STATE OF THE ART Biochemical cholinesteras reaction is a very sensitive and selective method widely used for a long time in a number of modifications and applications for detection and determination tion of a numerous set of cholinesteras inhibitors [AMMON, R. - VOSS, G. , Pflϋgers, Arch, ges. Physiol., 235 (1935), p. 393; MICHEL, 0., H. , J. Lab. clin. Med. , 34 (1949), p. 1564]. Various types of technical means to detect and determine cholinesteras inhibitors are based on the cholinesteras reaction, starting with the complicated automatic atmosphere and water analysers, continuing with more simple workmanships, e.g. tube detectors and ending with the most simple but the more used detection test papers [FRANKE, S. : Lehrbuch der Militarchemie, Militarverlag DDR, part II, Berlin 1977, p. 481; U.S. patent No. 4.324,858; VO 8504424] . Regardless of the total complexity of the design and financial costs all categories of means maintain similar sensibility and selectivity owing to the same reaction principle with the consequence that in the absolute majority of cases the most simple and therefore also the cheapest workmanship i.e. detection paper is preferred. The individual detection papers are different in two ways. At first, in the used substrate and then in the indication reaction related to it. This part of the cholinesterase reaction materialization in the test paper is not essential as it cannot have an influence on the application for determination in water and atmosphere, but it can influence the sensitivity of determination. The basic problem is the method how to fix and stabilize the enzyme. Some existing methods are based on the enzyme adsorption in the filtering paper [AO CSFR 269267]. The other solution of this problem is to immobilize cholinesterase in the carrier by a chemical bond, what is accompanied by a significant loss of the hydrolytic activity of the enzyme and additionally it requires to use a highely purified and therefore expensive enzyme for the immobilization, whereby the prolongation of the expiration time of the enzyme paper is not very high [RO 105190] . The most similar solution to that according to this invention is to prepare the enzyme detection paper by immobilization of cholinesterase in a special chromatographic paper with the ion exchanging function in mixture with dextrane gel and by indication with a chromogenic substrate [VO 85/04423] where in case of increased storage life the required sensitivity of the detection paper was not obtained. In spite of a great number of various modifications consisting in the method of evaluation of the used enzyme, substrate, various environments or in modification of the other workmanship conditions all such modifications are determined by the fact that the biochemical cholinesterase reaction is a group reaction for a very numerous set of the cholinesterase inhibitors, the most important of which are organophosphorous compounds and carbamates. To make possible to determine what inhibitor it is, it is necessary to carry out some detection reactions or physico-chemical measurements. However, this requires a greater amount of inhibitor or by several orders higher concentration of the sample solution than as it is in the case of the very sensitive biochemical cholinesterase method. In such case it is necessary to carry out difficult, complex and lengthy concentration of the sample, preceding the proper chemical or physico-chemical 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 analytical detection reactions or with use of expensive instruments, that can not be operated in the field. Moreover, a number of the organophosphate and carbamate cholinesterases inhibitors constitutes also a substantial risk with regard to labour safety, considering the duration of the necessary operations with concentrated solution of the determined compound. The nearest known solution of the analysis method according to invention is the method how to distinguish organophosphate inhibitors based on formation of complexes of two types with metal ionts and detection by cholinesterase reaction [CEBOTARJOV, 0. , Y. :Vo skovaja indikacia, Izdatelstvo akademii Moscow 1978] and the method of distinguishing between organic inhibitors by cholinesterase reaction after modification of the cholinesteras by metal ionts [Certificate of Authorship CSFR 2699]. The mentioned disadvantages consisting in the group nature of the biochemical cholinesterase reaction require that laborous and lengthy inhibitor sample concentration is necessary and that complicated apparatuses are to be used. The above disadvantages are removed by detection of organophosphate and carbamate cholinesterase inhibitors according to the invention. THE EMBODIMENT OF THE INVENTION The invention consists in a biosensor of cholinesterases inhibitors in atmosphere, water, foodstuffs, soil, surfaces and extracts from samples and in its application for detection of and distinguishing between cholinesterase inhibitors. The biosensor consists of a cellulose textile with cholinesterase immobilized by bioaffinity and stabilized, a colour standard and a carrier, e.g. filtering paper with substrate and chromogenic agent. During the detection of cholinesteras inhibitors e. g. the acetylthiocholinjodid substrate and chromogenic agent are put on wet cellulose textile with immobilized cholinesterase exposed in a controlled environment by pressing filtering paper containing them to it. The paper reacts by colour change to products of substrate hydrolysis, the 5,5' -di- thiobis(2-nitrobenzoic) acid and the hydrolytic activity of the immobilized cholinestarase is visually evaluated according to a colour standard as a colour change of the cellulose textile. Application of this biosensor to distiguishing between inhibitors consists in that in case of cholinesterases immobilized on cellulose textile hydrolytic activity induced by reactivation of quartery aldoxi es, e.g. 1- (2-hydroxyiminomethylpyridinum) -3- (4-carbamoylpyridi- num-2-oxapropan)dichloride and N,N' -trimethylen-bis(4-pyri- diniumadoxim)dichloride, or by spontaneous reactivation by water, is restored after inhibition by organophosphate or carbamate inhibitor. Simultaneously, samples of cholinesterase immobilized on cellulose textile of the biosensor are exposed in water solution of inhibitor and in water solution of inhibitor under addition of ionic compounds, e.g. natrium, calcium or ammonium chloride. By evaluation of 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, characteristic difference in activity is established that makes possible to determine what inhibitor it is. EXAMPLES Example 1 Preparation of acetylcholinesterase solution The prepared acetylcholinestearase is used to prepare a solution having specific activity 7 to 15 nkat/ml by dissolving it in a buffer solution of pH 5 to 9. Example 2 Modification of cellulose textile Cellulose textile woven from coton fibres is modified by alkaline oxidative bleaching by boiling the textile in a solution containing 7.9 g/1 of hydrogen peroxide (35 %) , 0.2 g/1 of magnesium chloride, 0.2 g/1 of non-ionogenic tenside SLOVASOL SF-10, 2.1 g/1 of sodium hydroxide and 2.5 g/1 of water glass. Example 3 Immobilization of acetylcholinesterase on bleached cellulose textile The cellulose textile modified according to Example 2 is immersed in the solution prepared according to Example 1 for 30 minutes. Thereafter, it is taken out and dried in air at 20 °C. Example 4 Praparation of the carrier with substrate and chromogenic agent The filtering paper is immersed in a solution of 0.5 % of acetylthiocholinjodide and 0.15 % of 5,5' -dithio-bis(2- nitrobenzoic) acid in ethanol. Thereafter, the paper is dried in air at 20 "C. Example 5 Detection of inhibitors in water and in water homogenizates of samples The detection textile prepared according to Example 3 is immersed in water or water extract for 30 minutes. There¬ after, the textile is taken out and for 1 minute paper with substrate and chromogenic agent prepared according to Example 4 is pressed to it. After the paper is removed the textile is yellow coloured in case of absence of acetylcholinesterase inhibitors. Unchanged colour of the textile 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/1) CARBOFURAN 2.10 "2 METHOMYL 1.10 "1 DICHLORVOS 1.10 '1 MEVINPHOS 1.10 "2 PARATHIONMETHYL 2.10 "2 Example 6 Inhibitor detection in air Detection textile prepared according to example 3 is made wet by water and left in controlled atmosphere minimally 1 minute. Thereafter, paper with substrate and chromogenic agent prepared according to Example 4 is pressed to the textile. After the paper is removed the textile is colloured yellow if inhibitors of cholinesteras are not present. An evidence of inhibitors is the unchanged colour of the textile. Sensitivity to nerves agents detection in air nerve agent detection limit (mg/1) after exposition 2 min 20 min SARIN GB SOMAN GD AGENT VX AGENT GP TABUN GA CYKLOSIN GF Example 7 Use of biosensor to distinguish between pinakolylmethyl- fluorophosphonate and cyclohexylmethylfluorophosphonate Samples of the immobilized acetylcholinestearase inhibited to 100 % by 2 to 5 minutes of incubation in solution containing organophosphate inhibitor are reactivated for 5 minutes in 0.02 mg/ml of 1- (2-hydroxyiminomethylpyridinium) -3- (4-karba- moylpyridinium) -2-oxapropandichloride and 0.05 mg/ml of N, N' -trimethylene-bis(4-pyridiniumaldoxim)dichloride. After reactivation, hydrolytic activity of the enzyme in both samples of inhibited cholinesterase is evaluated. If hydrolytic activity of the inhibited enzyme was restored only by reactivation in 0.02 mg/ml of 1- (2-hydroxyiminome- thyl-pyridinium)-3- (4-carbamoylpyridinium)-2-oxapropandi- chloride, the solution contains pinakolylmethylfluorophos¬ phonate or cyclohexylmethylfluorophosphonate. The reactiva¬ tion is repeated after the inhibition of a sample of immobi¬ lized acetylcholinesterase by 30 minutes of incubation. Vhen hydrolytic activity of the enzyme is restored by reactiva¬ tion the solution contains cyclohexylmethylfluorophospho¬ nate. Example 8 Use of biosensor to distinguish between O-ethyl-S- (2-diiso- propylaminoethyl)methylthiophosphonate and isopropylmethyl- fluorophosphonate Samples of immobilized acetylcholinestarase inhibited to 100 % by 2 to 5 minutes of incubation in the solution con¬ taining organophosphorous inhibitor are reactivated for 5 minutes in 0.02 mg/ml of 1- (2-hydroxyiminomethylpyridinium)- 3- (4-carbamoylpyridinuium) -2-oxapropandichloride and 0.05 mg/ml of N,N' -trimethylene-bis(4-pyridiniumaldoxim)dichlori- de. After reactivation hydrolytic activity of the enzyme of both samples of inhibited cholinesterases is evaluated. If hydrolytic activity of the inhibited enzyme was restored by ractivation in both reactivators the solution contains O-ethyl-S- (2-diisopropylaminoethyl)methylthiophosphonate or isopropylmethyIfluorophosphonate. The solution is dissolved ten-times and divided to two parts. To one part of the dis¬ solved solution natrium chloride is added in surplus and in both solutions are paralelly incubated samples of immobi¬ lized acetylcholinesterase for 2, 5, 10, 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 O-ethyl-S- (2-diisopropylaminoethyl)methylthiophos- phonate. Example 9 Use of biosensor to distinguish between ethyldimethylamido- cyanophosphate and 2-dimethylaminoethyldimethylamidofluoro- phosphate Samples of immobilized acetylcholinesterase inhibited to 100 % by 2 to 5 minutes of incubation in the solution containing organophosphorous inhibitor are reactivated for 5 minutes in 0.02 mg/ml of 1- (2-hydroxyiminomethylpyridinium) - 3- (4-carbamoylpyridinium)-2-oxapropandichloride and 0.05 mg/ml of N,N' -tri ethylenebis- (4-pyridiniumaldoxim)dichlori- de. After reactivation the hydrolytic activity of enzyme is evaluated in both samples of the inhibited cholinesterase. If the hydrolytic activity of the inhibited enzyme is not restored in both reactivators, the sample contains solution of ethyldimethylamidocyanophthate or 2-dimethylaminoethyldi- methylamidofluorophosphate. 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 paralelly in both solutions will be incubated samples of immobilized acetylcholinestearase for 2, 5, 10, and 15 minutes till a different hydrolytic sample activity is obtained. In case 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 2-dimethylamino-ethyldimethylamidofluorophosphate. Example 10 Use of biosensor to distinguish organophosphorous and carbamate insecticides Samples of immobilized acetylcholinesterase inhibited to 100 % by 2 to 5 minute of incubation in the solution contai¬ ning organophosphorous or carbamate inhibitor are reacti¬ vated for 5 minutes in 0.02 mg/ml of 1- (2-hydroxyiminome- thylpyridinum) -3- (4-carbamoylpyridinium) -2-oxapropandichlo- ride and 0.05 mg/ml of N,N' -trimethylenebis(4-pyridinium- aldoxim)dichloride. After reactivation hydrolytic activity of enzyme 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 reactiva¬ tors, the sample is immersed for 30 to 120 minutes in distilled water. If hydrolytic activity is restored after 60 minutes of reactivation in water, the solution contains 1-naftyl-N-methylcarbamate. If hydrolytic activity is restored after 120 minutes of reactivation in water, the solution contains 2,3-dihydro-2,2-dimethylbenzofurane-7-yl- -N-methylcarbamate. 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 in medical and veteri¬ nary institutions. As a method of detection and determina¬ tion 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 defence 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.

Claims: CLAIMS 1. Biosensor for detection of and distinguishing between cholinesteras inhibitors characterized in that it is comprised of cellulose textile with bioaffinity immobi¬ lized and stabilized cholinesterase, colour standard and chromogenic agent for visual evaluation. 2. Biosensor according to claim 1, characterized in that the cellulose textile is preferably produced from coton fibres. 3. Biosensor according to claims 1 and 2 characterized in that the cellulose textile is modified by bleaching, preferably by alkaline oxidative bleaching. 4. Method of making cellulose textile with by bioaffinity immobilized cholinestearase, characterized in that at the cellulose textile according to claims 2 and 3 cholinestearase is immobilized by immersing the textile in the buffer solution of cholinestearas, preferably acetylcholinestearase or butyrylcholinestearase and after the cellulose textile is taken out of the solution it is dried. 5. Application of the biosensor according to claims 1 to 4 characterized in that chromogenic agent is deposited at wet cellulose textile exposed in controlled environ¬ ment and the hydrolytic activity of cholinesterase is visually evaluated according to a colour standard as the cellulose textile colour change. 6. Application of the biosensor according to claims 1 to 5 characterized in that chromogenic agent is deposited at wet cellulose textile by pressing of an inert carrier saturated with the chromogenic agent to it. 7. Application of the biosensor according to claims 1 to 6 characterized in that the inert carrier, preferably filtering paper contains a substrate, preferably acetylthiocholine, and chromogenic agent reacting by colour change to products of the substrate hydrolysis preferably 5,5' -dithiobis(2-nitrobenzoic) acid. 8. Application of the biosensor according to claims 1 to 6 characterized in that the inert carrier, preferably filtering paper contains chromogenic substrate, prefe¬ rably 1- [2-thiazoylazo] -2-acetoxy-5-methylbenzene- dimethylsulphate. 9. Application of the biosensor according to claims 1 to 8 to distinguish organophosphorous and carbamate inhi¬ bitors characterized in that the hydrolytic activity of the inhibited cholinesterase immobilized at cellulose textile is evaluated after induced reactivation prefe¬ rably by quarternary aldoximes and after spontaneous reactivation by water. 10. Application of the biosensor according to claims 1 to 8 to distinguish nerve paralytic compouds, characteri¬ zed in that change in hydrolytic activity of inhibited cholinesterase immobilized at cellulose textile is eva¬ luated after induced reactivation, preferably by quarternary aldoximes and after the action of ionts, pre ferably ionts of alcaline metals.

Inventors: HALAMEK EMIL, TUSAROVA IVANA, HALAMEK, EMIL, TUSAROVA, IVANA
Applicants: HALAMEK EMIL, ORITEST S S R O, TUSAROVA IVANA, ORITEST S. S R.O.
Application year: 1993
Main classification: C12Q1
IPC codes: C12N11, C12Q1