Method for detecting an acetylcholinesterase inhibitor in a water sample

Abstract

A known method for detecting an AChE inhibitor in a water sample involves the following steps: • Addition of AChE to the water sample, wherein an AChE inhibitor contained in the water sample inhibits the AChE. • Detection of AChE inhibition via a color reaction. The number of false-positive test results from enzyme-based AChE rapid tests should be reduced. For this purpose, any chlorine present and used for germicidal purposes is neutralized with a reducing agent prior to the addition of AChE to the water sample, wherein the reducing agent consists of 90 to 100% by weight of a reducing agent selected from the group consisting of sodium thiosulfate, sodium dithionite, sodium disulfite, sodium sulfite, sodium hypophosphite and sodium formate.

Description

[0001] The invention relates to a method for detecting an acetylcholinesterase (AChE) inhibitor in a water sample.

[0002] A generic method for detecting an AChE inhibitor in a water sample is known from EP 4 141 125 A1. The steps of this method are: • Addition of AChE to the water sample, wherein an AChE inhibitor contained in the water sample inhibits the AChE. • Detection of AChE inhibition via a color reaction.

[0003] Drinking water typically contains chlorine, which, through oxidation, leads to the inactivation of the enzyme AChE. Analytically, this inactivation behaves identically to the inhibition of the enzyme by a nerve agent, so chlorinated drinking water leads to a false-positive test result.

[0004] The object of the invention is to reduce the number of false-positive test results of enzyme-based AChE rapid tests, starting from a generic method.

[0005] This problem is solved according to the invention by the features of claim 1.

[0006] The advantages of the invention are based on the fact that, prior to the step of adding AChE to the water sample, any chlorine present and used for germicidal purposes is neutralized with a reducing agent, wherein the reducing agent consists of 90 to 100% by weight of a reducing agent selected from the group consisting of sodium thiosulfate, sodium dithionite, sodium disulfite, sodium sulfite, sodium hypophosphite and sodium formate.

[0007] The addition of a reducing agent to neutralize chlorine eliminates cross-sensitivity of AChE rapid tests to chlorine. Neutralizing chlorine and other oxidizing agents (disinfectants) reduces false-positive test results, particularly in operational settings, with AChE rapid tests.

[0008] According to an advantageous embodiment of the invention, a reducing agent is used which consists of 90 to 100% by weight of sodium thiosulfate. Sodium thiosulfate combines the following positive properties with other reducing agents: it exists as a solid under operating conditions, is readily soluble in water, is colorless, odorless, and has good market availability.

[0009] According to an advantageous embodiment of the invention, a reducing agent is used which is present as a frost-resistant solid at the beginning of the process. This facilitates handling.

[0010] Exemplary embodiments of the invention are described in more detail below.

[0011] The following steps form the basis of a method for detecting an AChE inhibitor in a water sample: • Addition of AChE to the water sample, wherein an AChE inhibitor contained in the water sample inhibits the AChE, • Detection of AChE inhibition via a color reaction.

[0012] Before adding AChE to the water sample, any chlorine present, which is used for germicidal purposes, is neutralized with a reducing agent.

[0013] The reducing agent used consists of 100% by weight of sodium thiosulfate. Alternatively, the embodiment could also consist of only 90% by weight of sodium thiosulfate and 10% by weight of sodium disulfite. In principle, the reducing agent could consist of 90% to 100% by weight of a reducing agent selected from the group consisting of sodium thiosulfate, sodium dithionite, sodium disulfite, sodium sulfite, sodium hypophosphite, and sodium formate.

[0014] The reducing agent used is present as a frost-resistant solid at the beginning of the process.

[0015] The procedure, in more detail, comprises the following steps: 1) The salts of a buffer are placed in a sample tube. The amounts are chosen so that, after the subsequent addition of a water sample to be tested (2 mL, step 3), a pH value in the range of pH 7.3 to pH 7.5 is established. The buffer is a phosphate buffer. The salt amounts are: 16 mg sodium chloride, 0.4 mg potassium chloride, 2.8 mg disodium hydrogen phosphate (or alternatively: 3.6 mg disodium hydrogen phosphate dihydrate), and 0.5 mg potassium dihydrogen phosphate. 2) 10 mg of the reducing agent sodium thiosulfate are added to the salts of the buffer. 3) 2 mL of the water sample to be tested are added to the sample tube. The reducing agent from step 2) reduces any chlorine present in the water sample. 4) A first lid, which contains the first reactive component in the form of human AChE, is screwed onto the sample tube and shaken for one minute. 5) The first cap is replaced with a second cap. The second cap contains the second reactive component, 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), and the third reactive component, acetylthiocholine. The sample tube is shaken for another minute. Depending on the activity of the AChE contained in step 4), an intense yellow dye, 2-nitro-5-thiobenzoate (TNB), is formed. If an AChE inhibitor is present in the water sample, no yellow coloration occurs. The intensity of the yellow coloration is compared using a color scale.

[0016] The aforementioned steps 1) and 2) could alternatively be replaced by the following steps: Step 1 (alternative): 0.5 mL of a phosphate buffer solution with a pH value in the pH range of pH 7.3 to pH 7.5 is placed in a sample tube. Step 2 (alternative): 0.5 mL of a reducing agent solution with a concentration of 20 mg / mL is added to the buffer solution. QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited patent literature

[0000] EP 4 141 125 A1

[0002]

Claims

[1] Method for detecting an acetylcholinesterase (AChE) inhibitor in a water sample, comprising the steps: • Addition of AChE to the water sample, wherein an AChE inhibitor contained in the water sample inhibits the AChE, • Detection of AChE inhibition via a color reaction, characterized by , that any chlorine present and used for germicidal purposes is neutralized with a reducing agent prior to the step of adding AChE to the water sample, wherein the reducing agent consists of 90 to 100% by weight of a reducing agent selected from the group consisting of sodium thiosulfate, sodium dithionite, sodium disulfite, sodium sulfite, sodium hypophosphite and sodium formate. [2] Method according to claim 1, characterized by that a reducing agent is used which consists of 90 to 100 percent by weight of sodium thiosulfate. [3] Method according to claim 1 or 2, characterized bythat a reducing agent is used which is present as a frost-resistant solid at the beginning of the process.