CZ117993A3 - Detection tube of choline esterases inhibitors in air and water - Google Patents

Abstract

The invention concerns a detection tube for cholinesterase inhibitors in air and water, which contains two ampoules with a buffer solution of pH 6.0 to 9.0, a comparative level of cullet impregnated with mixture of substrate and a chromogenic reagent or chromogenic substrate and indication layer of granulated bleached cellulose with immobilised cholinesterase. The cullet comparative level contains acetylthiocholine or butylrylthiocholine as a substrate and 5,5'-dithio-bis(2-nitobenzoic) acid as a chromogenic agent. Alternatively, the cullet comparative level contains acetylthiocholine or butylrylthiocholine as a substrate and N-ethyl-N-(4-(4-ethyl-3-sulfophenyl(methylaminophenyl)phenylmethylene-2,5-cyklohexadiene)-1-ylidene)-3-sulfobenzenmethanamine-umnatriumhydroxide internal salt as a chromogenic agent. Alternatively the comparative layer contains a chromogenic substrate and 1-(thia-zolylazo)-2-acetoxy-5-methylbenzendimethylsulphate. The indication layer preferably contains beef brain acetyl cholinesterase or horse plasma butyryl cholinesterase.<IMAGE>

Description

The invention relates to a tube for detecting cholinesterase inhibitors in air and water. The invention solves the construction of a means for the determination of extremely toxic compounds important in the fields of military and industrial chemistry, agrochemistry, environmental monitoring, civil defense and national security.

BACKGROUND OF THE INVENTION

For the detection and determination of a large set of cholinesterase inhibitors [ΑΜΜΟΝ P. - VOSS G., Pflijgers Arch. ges. Phy1OJ: 35 (1935),

393; MICHEL O., H. Lab. clin. Med., 34 (1949), 1564]. Biochemical cholinesterase reaction is widely used in many modifications and applications as a very sensitive and selective method. Various types of technical means for the detection and determination of cholinesterase inhibitors are based on cholinesterase reactions, ranging from complicated automatic air and water analyzers to simpler designs such as detection tubes [LEICHNITZ, K .: Priif rbhrchen-Ta.schenbuch. 5. Ausgabe, Drágenwerk AG, Lubeck 1982, p. 131], to the simplest, but all the more used detection papers [WP 85/04423, AO ČSFR 2701].

The known types of tubes based on the biochemical cholinesterase reaction have, regardless of the design and similar sensitivity and selectivity.

Commercially available tubes are structurally sealed glass tubes containing spaced layers of inert carrier and reagent sorbents and liquid reagent ampoules.

necessary for the biochemical enzyme reaction. Usually, the tubes have a sorption layer, most often silica gel, on which inhibitors are absorbed as air is drawn through the tube. The enzyme is then transferred to this layer, and after incubation of the enzyme with the inhibitor substrate, either by sequentially breaking the vials with reagent solutions or flushing the reagents from inert carriers.

- 2 or by dissolving dry Reagents after breaking the ampoule with buffer. Despite the complicated embodiment, this biochemical reaction principle is the only one that is sufficiently sensitive to the presence of extremely toxic cholinesterase inhibitors in the air and still used to construct simple technical means. The closest known solution for the determination of cholinesterase inhibitors in the air is the biochemical tube used in the military and civil defense of the Czech Republic and Slovakia. The tube contains an ampoule with a clarified butyryl choline esterase solution and an ampoule with a butyrylcholine iodide substrate solution and a phenolic red acid-base indicator. After breaking the ampoule with the enzyme and passing the analyzed air through a layer of crushed glass wetted with the butyryolinesterase solution and breaking the ampoule with the substrate solution and acid-base indicator, the color change of the test tube and comparison tube is visually compared. The disadvantage of such tubes is the short expiration time due to the fact that both the enzyme and the substrate are in aqueous solution, as well as the principle dependence of detection on acidic and basic substances trapped from the analyzed air in the tube. A more preferred solution of a biochemical vial with good storage stability is a vial according to NL 1568383. The vial contains one ampoule of buffer and lyophilized enzyme and chromogenic substrate preparations with addition of reagent dissolution enhancers in buffer and indicator zone to which inhibitors are absorbed. After opening the tube and sifting the air, by breaking the ampoule with buffer, both the soluble preparations, the enzyme and the chromogenic substrate, are washed off to the indicator layer with the sorbed inhibitors and the color of this layer is evaluated after the enzyme reaction has taken place.

The tubes used do not allow analysis of water and aqueous solutions.

Said drawbacks, in particular low tube life, acid and base disturbance, lower sensitivity, impossibility of water analysis, at high cost of production, are eliminated by the detection tube of cholinesterase inhibitors in air and water according to the invention.

SUMMARY OF THE INVENTION The present invention is a detection tube of cnolinesterase inhibitors in air and water, which comprises ampoules with a buffer, a color matching layer with a substrate and a chromogenic reagent or chromogenic substrate, and an indicator layer with immobilized shaverase esterase.

The use of the tube consists of suctioning air containing vapors or aerosol inhibitors of cholinesterase indicating

Substance and pouring solution with ro:

at.lm with analyzed water.

The pulses are not terminated at the time of incubation with the imodium humidified by wetting the screening layer of the screen or after removal of the crosslinked enzyme on the sorbent inhibitor indicator layer, the substrate is washed off the reference layer from the reference layer by chromogenic reagent or chroraogenic substrate. eni i η ai r.ači buffer vials and equilibrium civrst vy.

The tube according to the invention has a higher sensitivity than hitherto used tubes in the detection of choiinesterase inhibitors due to the use of a specific enzyme and substrate and the possibility of further increasing the sensitivity by prolonging incubation with inhibitor, longer expiration and allowing inhive aqueous and aqueous extracts from enzyme with bitor. i span

The pH of the sample in a broad indication and comparison of the staining rate and the comparison layer allows a semi-quantitative evaluation of the inhibitor concentration.

The attached drawing shows a tube model consisting of two ampoules of buffer 1 and 2, a comparative layer 3 impregnated with a substrate and a chromogenic reagent or a chromogenic substrate, an indicator layer 4 with immobilized cholinesterase. The layers are separated and sealed in the tube with polyethylene stars 5, stars 5 are separated from the layers by polyamide meshes

4 to 1 embodiment of the invention> example 1

The tube of Figure 1 contains 2 ampoules with phosphate buffer pH 8.5, the comparative layer being 0.1 <3 yellow glass chips (.lambda., Max. 412 nm) of grain size 1.0 to 1.2 mm impregnated etha η 1% of the salt containing 1% hioc holinjodide and 0.3% 5,5'-dithio-bis (2-dinitrobenzoic acid). The indicator layer is 0.05 g of white granular cellulose of a grain size of 0.8 to 1.2 mm with immobilized acetylcholinesterase having a specific activity of 1.5 nkat per 100 mg of cellulose. After breaking the ends of the tube and breaking the vial under the indicator layer so that the solution from the vial wetted the indicator layer, the analyzed air is drawn through the tube. After the sieving is complete, the buffer vial is broken above the reference layer and the solution is shaken to the indicator layer. In the presence of inhibitors in the analyzed air, the indicator layer remains white, and the uninhibited indicator layer turns yellow within 1 minute. When blow-through 1.4 1 of air per minute in the course of 2 minutes can be mg.I ^-1 isopropyimethy1fosfonof1uoridatu, pinakolyimethy1íosíonoíluoridatu, 2.10 ^-7 mg. 1 ^{1 _} 0-ethy1-S- (2-diisopropylamino 1 1 aminoethyl) methylamino thiofosfonatu 1 and 1.10 ^-7 mg.l ^-1-2 dimethylaminoethyldimethylamidoíosíofluoridatu.

find out 5.10 ~ 3. 10- ⁷ mg. if

Example 2

The tube of Figure 1 contains 2 ampoules with phosphate buffer pH 7.6, the reference layer is 0.1 g of blue glass pulp (Xmax 580 nm) of 1.0 to 1.2 mm grain size impregnated by lyophilization with an ethanol solution containing 0.6% The 1- [2-thiazolylazo] -2-acetoxy-5-methylbenzene dimethylsulphate indicator layer is 0.05 g of bleached granulated cellulose of 1 mm grain size with immobilized butyrylcholinesterase having a specific activity of 3 nkat per 100 mg cellulose. After breaking the ends

Claims (2)

1. Detection tube. Cholinesterase inhibitors in air and water, characterized in that they comprise ampoules with buffer, a color comparative layer with the substrate applied and a chromogenic reagent. It is an indicator layer with or a chromogenic substrate and a mob of water. Detection tube according to claim 1, characterized in that it contains two ampoules with a buffer solution of pH 6.0 to 9.0, a color comparative layer as an inert carrier impregnated with a substrate and a chronogenic reagent, preferably acetylthiocholine and 5,5'-dithio-bis acid. (2-dinitrobenzoic) or a chromogenic substrate, preferably 1- [2-thiazolylazo] -2-acetoxy-5-methylbenzene dimethylsulfate and an indicator layer, preferably a white cellulose sorbent with immobilized cholinesterase, preferably acetylcholinesterase or butyrylcholinesterase.