Enzyme discoloration agent for detection of persistent chemical agent

Abstract

The present invention provides an enzymatic chromosome for the detection of persistent chemical agents capable of detecting both mustard gas and VX by deriving an optimal combination of chloroperoxidase and its substrates, an azo dye and a colorimetric indicator, a pH buffer and a surfactant.

Description

Technology Field

[0001] The present invention relates to an enzymatic chromosome capable of detecting both mustard gas and VX, which are persistent chemical agents, and more specifically, to an enzymatic chromosome comprising chloroperoxidase. Background Technology

[0002] Chemical agents are classified into nerve, blister, and blood agents, and can have fatal effects on the human body even at very low concentrations. Since chemical agents are used for military purposes, terrorism, and assassination via missiles, airbombs, artillery shells, and spray dispersals to cause airborne dispersion and widespread surface contamination, developing technologies capable of detecting them within a short timeframe is crucial for establishing defense and response strategies against chemical agents.

[0003] Each chemical agent possesses specific volatility; for example, the neurochemical agent Sarin (GB) has a volatility of 22,000 mg / m² at 25°C. 3 It has very high volatility, spreads easily in gaseous form, and rapidly becomes toxic to the human body through inhalation. In contrast, the neurochemical agent VX has 75 mg / m² at 25°C. 3 , mustard gas HD, a blister chemical agent, is 610 mg / m² at 25℃ 3 It has very low volatility, can persist for a long time on contaminated surfaces, and is toxic to the human body through skin contamination. Therefore, detection technology must be developed considering the air diffusion ability and persistence of chemical agents.

[0004] To date, chemical agent detection technology has required rapid, high-sensitivity, and high-reliability capabilities, leading to the development of detection equipment based on Ion Mobility Spectrometry, Gas Chromatography, and Mass Spectrometry. While these devices are effective for the rapid and high-sensitivity detection of airborne chemical agents, they exhibit limited performance when detecting persistent agents, as they require a short distance from the contaminated area. For the detection of persistent agents, paper containing chemicals that change color upon contact with specific agents has been developed and is being utilized. However, this method using detection paper carries the risk of requiring the user to physically apply the paper to the contaminated surface, and detecting extensively contaminated areas requires a significant amount of time and manpower.

[0005] Recently, liquid spray-type chromosomes based on biological enzymatic reactions for the detection of chemical agents have been developed. Teledyne FLIR Detection commercialized Agentase C2, an enzyme-based chemical agent detection kit (US9556471B2 (2006)). This detection kit is a chemical agent detection spray system used to locate trace amounts of chemical agents by spraying it directly onto a surface. In particular, it can detect contamination by G-series and V-series nerve agents as well as mustard gas within 5 minutes and visually demonstrates the efficiency of decontamination. Furthermore, as it is a spray system, it does not require direct contact with the contaminated surface, thus posing a low risk, and is also suitable for detecting large areas of contamination. Meanwhile, although this enzyme chromosome offers high selectivity and a short detection time by changing color within 5 minutes, it has the disadvantage of requiring separate use for each source of contamination, as it is a detection system divided into nerve and blister chemical agents.

[0006] Therefore, an enzymatic chromosome was devised that can detect both mustard gas and VX, which are persistent chemical agents. Prior art literature

[0007] (Patent Document 0001) US 9556471 B2 The problem to be solved

[0008] The present invention aims to provide an enzymatic chromosome for detecting persistent chemical agents capable of detecting both mustard gas and VX by deriving an optimal combination of substrates, colorimetric indicators, etc.

[0009] Furthermore, the present invention, which aims to solve the conventional problems as described above, comprises chloroperoxidase (CPO) and a substrate chloride ion (Cl - Hypochlorous acid (OCl) is the product produced through the reaction of - The purpose is to provide an enzymatic chromogenic agent for detecting persistent chemical agents by introducing the enzymatic oxidation reaction mechanism of mustard gas and VX in a reaction that decolorizes azo dyes by generating ) as a detection system through colorimetric identification based on the presence or absence of persistent chemical agents.

[0010] The problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0011] To achieve the above objective, the enzyme chromosome composition for detecting persistent chemical agents according to the present invention is,

[0012] It includes a reaction mixture comprising a persistent chemical agent, an effective amount of chloroperoxidase for detoxification, an oxidizing agent, chloride ions, an azo dye, and a pH buffer.

[0013] In addition, the above-mentioned persistent chemical agent is either mustard gas or VX, and the concentration of the above-mentioned persistent chemical agent is 1.0 to 10 mg / mL.

[0014] In addition, the above-mentioned effective amount of chloroperoxidase is characterized by being 0.5 to 2.0 U / mL.

[0015] In addition, the oxidizing agent is hydrogen peroxide, and the concentration of the hydrogen peroxide is 0.04 to 0.08 w / w%.

[0016] In addition, the chloride salt of the chloride ion is either sodium chloride or potassium chloride, and the concentration of the chloride salt is 350 to 450 mM.

[0017] In addition, the above azo dye is characterized by being one or more selected from the group consisting of Orange-G, Sunset Yellow, Congo-Red, Alizarin Yellow, and Safranin-O.

[0018] In addition, the concentration of the azo dye is characterized as being 25 to 50 μM.

[0019] In addition, the pH buffer is characterized by being one or more selected from the group consisting of phosphate solution, phthalate solution, and citrate solution.

[0020] In addition, the above reaction mixture further comprises an indicator, and the indicator is characterized by being one or more selected from the group consisting of thymol blue, methyl orange, and bromophenol blue.

[0021] In addition, the above enzyme color changer composition further comprises a surfactant, and

[0022] The above surfactant is characterized by being one or more selected from the group consisting of sodium dodecyl sulfate (SDS), Tween-40, Triton X-100, and NP-40.

[0023] In addition, the concentration of the surfactant is characterized as being 1 to 5%.

[0024] The present invention provides an enzyme color changer comprising the aforementioned enzyme color changer composition. Effects of the invention

[0025] According to the present invention, an enzyme chromosome for detecting persistent chemical agents having an optimal combination of a substrate, a colorimetric indicator, etc., has the effect of detecting a surface contaminated with persistent chemical agents as a single enzyme chromosome rather than a bifurcated detection method, and detecting both mustard gas and VX.

[0026] In addition, according to the present invention, since the enzymatic colorimetric agent for detecting persistent chemical agents is a liquid solvent, it can be introduced into a spray system and utilized in extensive contaminated areas. Furthermore, in terms of the operation method of detection equipment and detection paper, which are surface chemical agent detection technologies, this invention has the effect of reducing the risk of a user being in close proximity to a chemical agent-contaminated area or coming into contact with a contaminated surface.

[0027] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description of the invention or the claims. Brief explanation of the drawing

[0028] FIG. 1 is a schematic diagram of the chloroperoxidase reaction mechanism of an enzyme chromosome composition for detecting persistent chemical agents according to one embodiment of the present invention. Figure 2 is a photograph of the decolorization reaction of safranin-O by chloroperoxidase according to mustard gas concentration of an enzyme chromogenic agent composition for detecting persistent chemical agents according to one embodiment of the present invention. Figure 3 is the absorbance result of the decolorization reaction of safranin-O by chloroperoxidase according to mustard gas concentration of an enzyme chromogenic agent composition for detecting persistent chemical agents according to one embodiment of the present invention. Figure 4 is a photograph of the decolorization reaction of safranin-O by chloroperoxidase according to the VX concentration of an enzyme chromogenic agent composition for detecting persistent chemical agents according to one embodiment of the present invention. Figure 5 is the absorbance result of the decolorization reaction of safranin-O by chloroperoxidase according to the VX concentration of an enzyme chromogenic agent composition for detecting persistent chemical agents according to one embodiment of the present invention. Figure 6 is a schematic diagram of the color change reaction of an enzyme colorimeter for detecting persistent chemical agents presented in the present invention. FIG. 7 is a photograph showing the detection of liquid and surface contamination of chemical agents by changing color using an enzyme colorimetric agent for detecting persistent chemical agents according to one embodiment of the present invention. Specific details for implementing the invention

[0029] Before describing the present invention in detail, it should be understood that the terms and words used in this specification should not be interpreted as being limited to their ordinary or dictionary meanings, and that the inventor of the present invention may appropriately define and use the concepts of various terms to best describe their invention, and furthermore, that these terms and words should be interpreted in a meaning and concept consistent with the technical spirit of the present invention.

[0030] In other words, it should be understood that the terms used in this specification are used merely to describe preferred embodiments of the present invention and are not intended to specifically limit the content of the present invention, and that these terms are defined in consideration of the various possibilities of the present invention.

[0031] In addition, it should be noted that in this specification, singular expressions may include plural expressions unless the context clearly indicates a different meaning, and that even if they are expressed in a similarly plural form, they may include the meaning of the singular.

[0032] Throughout this specification, where it is stated that a component "includes" another component, unless specifically stated otherwise, this may mean that it does not exclude any other component but may include any other component.

[0033] In addition, in describing the present invention below, detailed descriptions of components, such as prior art and known technology, that are deemed to unnecessarily obscure the essence of the invention may be omitted.

[0035] The present invention will be described in detail below.

[0036] Recently, liquid spray-type chromosomes based on biological enzymatic reactions for the detection of chemical agents have been developed. Teledyne FLIR Detection commercialized Agentase C2, an enzyme-based chemical agent detection kit (US9556471B2 (2006)). This detection kit is a chemical agent detection spray system used to locate trace amounts of chemical agents by spraying it directly onto a surface. In particular, it can detect contamination by G-series and V-series nerve agents as well as mustard gas within 5 minutes and visually demonstrates the efficiency of decontamination. Furthermore, as it is a spray system, it does not require direct contact with the contaminated surface, thus posing a low risk, and is also suitable for detecting large areas of contamination. Meanwhile, although this enzyme chromosome offers high selectivity and a short detection time by changing color within 5 minutes, it has the disadvantage of requiring separate use for each source of contamination, as it is a detection system divided into nerve and blister chemical agents.

[0037] In order to overcome the conventional dual chemical agent enzyme discoloration detection system, the present invention devised an enzyme discoloration agent capable of detecting both persistent chemical agents, mustard gas and VX.

[0038] According to the present invention, an enzymatic chromogenic agent composition for detecting persistent chemical agents is provided, comprising a reaction mixture including a persistent chemical agent, an effective amount of chloroperoxidase for detoxification, an oxidizing agent, chloride ions, an azo dye, and a pH buffer, wherein the reaction mixture has a pH value of 3 to 4.

[0039] Chloroperoxidase

[0040] Chloroperoxidase (CPO) is a heme-thiolate protein derived from fungi and is a multifunctional enzyme possessing haloporoxidase, peroxidase, catalase, and cytochrome P450-like activities, among which the halogenation of organic molecules is known as the primary reaction. Research has reported on the potential for chloroperoxidase to be utilized in various application fields, such as organic synthesis, environmental remediation, biomimetic catalysts, and the development of biosensors.

[0041] Chlorperoxidase reaction mechanism

[0042] Chloroperoxidase is a powerful oxidizing agent, hypochlorous acid (OCl). - It promotes the decolorization of azo dyes by generating ) Specifically, the heme (Heme, Fe) of chloroperoxidase 3+ The center reacts with hydrogen peroxide (H2O2) to form an active intermediate (Compound I, Fe 4+ =O intermediate) forms, and chloride ions (Cl - It reacts with ) to produce a ferric-hydrochloride intermediate and reacts with water (H2O) to produce hypochlorous acid as a final product. The generated hypochlorous acid converts the azo group (-N=N-) of the azo dye into a nitroso compound (-NO), causing discoloration in which the dye color disappears (Xuelian Li et.al. Industrial & Engineering Chemistry Research (2013) 52(38),13572-13579, Juan Zhang et.al. Chemical Engineering Journal (2012) 191, 236-242).

[0043] In addition, chloroperoxidase reduces the toxicity of chemical agents such as mustard gas HD and VX by oxidizing them. Specifically, the heme center of chloroperoxidase reacts with hydrogen peroxide to produce an active intermediate (Compound I), reacts with chloride ions to produce a hypochlorite intermediate, and the thioether group (RSR) of mustard gas (Cl-CH2-CH2-S-CH2-CH2-Cl) is oxidized to sulfoxide (R2-S=O) to produce sulfoxide mustard (HDO) as a final product (QungHao He et.al. Ecotoxicology and Environmental Safety (2021) 225, 112715).

[0044] FIG. 1 is a schematic diagram of the chloroperoxidase reaction mechanism of an enzyme chromosome composition for detecting persistent chemical agents according to one embodiment of the present invention.

[0045] Referring to FIG. 1, in the enzyme colorimetric agent composition for detecting persistent chemical agents of the present invention, the enzyme is chloroperoxidase and includes chloride ions and hydrogen peroxide, which is an oxidizing agent, for the enzymatic reaction.

[0046] Here, chloroperoxidase has a heme center and, in the absence of a persistent chemical agent, reacts with hydrogen peroxide to produce an active intermediate (Compound I), reacts with chloride ions to produce a hypochlorite intermediate, and reacts with water to produce the final product, hypochlorous acid. In the presence of a persistent chemical agent, the heme center reacts with hydrogen peroxide to produce an active intermediate (Compound I), reacts with chloride ions to produce a hypochlorite intermediate, and oxidizes the persistent chemical agent.

[0047] Persistent chemical agents

[0048] In the enzyme chromosome composition for detecting persistent chemical agents of the present invention, the persistent chemical agent is either mustard gas or VX, and the concentration of the persistent chemical agent may be 1.0 to 10 mg / mL.

[0049] Here, when the concentration of the above-mentioned persistent chemical agent is within the above range, the decolorization rate of the azo dye is very low so that decolorization does not occur, and thus both mustard gas and VX can be detected with the single enzyme decolorizing agent intended in the present invention.

[0050] If the concentration of the above-mentioned persistent chemical agent is less than 1.0 mg / mL, for example, 0.625 mg / mL, for example, 0.313 mg / mL, for example, 0.156 mg / mL, the decolorization of the azo dye proceeds by more than 50%, and furthermore, most of the azo dye may be decolorized, and the oxidation reaction of mustard gas and VX is minimal, so it is not desirable to detect both mustard gas and VX with a single enzyme chromosome.

[0051] Effective amount of chloroperoxidase for detoxification

[0052] The above effective amount of chloroperoxidase for detoxification may be 0.5 to 2.0 U / mL.

[0053] The compositional unit U / mL used in the present invention is an abbreviation for 'Unit per milliliter,' and 1 U represents the amount of chloroperoxidase enzyme required to catalyze the oxidation reaction of a persistent chemical agent and the production reaction of hypochlorous acid for 1 minute under specified conditions.

[0054] Here, when the effective amount of chloroperoxidase for detoxification is within the above range, the chloroperoxidase in the reaction mixture can effectively cause the oxidation reaction of the persistent chemical agent and the generation reaction of hypochlorous acid, and thus, both mustard gas and VX can be detected by the single enzyme chromogenic agent intended in the present invention.

[0055] Oxidizing agent

[0056] In the enzyme chromogenic agent composition for detecting persistent chemical agents of the present invention, the oxidizing agent is hydrogen peroxide, and the concentration of the hydrogen peroxide may be 0.04 to 0.08 w / w%.

[0057] Here, when the concentration of hydrogen peroxide is within the above range, chloroperoxidase in the reaction mixture can effectively cause the oxidation reaction of the persistent chemical agent and the generation reaction of hypochlorous acid, and thus, both mustard gas and VX can be detected as a single enzyme chromogen intended in the present invention.

[0058] chloride ions

[0059] Chloride ions play an important role in the oxidation reaction of persistent chemical agents facilitated by chloroperoxidase.

[0060] In the enzyme colorimetric agent composition for detecting persistent chemical agents of the present invention, chloride ions can be formed using chloride salts.

[0061] The chloride salt of the chloride ion above is either sodium chloride or potassium chloride, and the concentration of the chloride salt may be 350 to 450 mM.

[0062] Here, when the concentration of the chloride salt is within the above range, the chloroperoxidase in the reaction mixture can effectively cause the oxidation reaction of the persistent chemical agent and the generation reaction of hypochlorous acid, and thus both mustard gas and VX can be detected as a single enzyme chromogen intended in the present invention.

[0063] azo dyes

[0064] Azo dyes are decolorized by hypochlorous acid, a product of chloroperoxidase.

[0065] In the enzymatic colorimetric agent composition for detecting persistent chemical agents of the present invention, the azo dye may be one or more selected from the group consisting of Orange-G, Sunset Yellow, Congo-Red, Alizarin Yellow, and Safranin-O.

[0066] The concentration of the above azo dye may be 25 to 50 μM.

[0067] Here, when the concentration of the azo dye is within the above range, chloroperoxidase in the reaction mixture can effectively cause the oxidation reaction of the persistent chemical agent and the generation reaction of hypochlorous acid, and thus both mustard gas and VX can be detected as a single enzyme chromogen intended in the present invention.

[0068] pH buffer

[0069] The pH buffer serves to maintain the pH of the reaction mixture at 3 to 4 in the enzyme chromosome composition for detecting persistent chemical agents according to the present invention, so that chloroperoxidase can oxidize the persistent chemical agent and produce hypochlorous acid.

[0070] In the enzyme colorimetric agent composition for detecting persistent chemical agents of the present invention, the pH buffer may be one or more selected from the group consisting of phosphate solution, phthalate solution, and citrate solution.

[0071] Here, when the above pH buffer is used, the reaction mixture in the enzyme chromosome composition for detecting persistent chemical agents of the present invention maintains a pH of 3 to 4, so that chloroperoxidase can effectively cause the oxidation reaction of the persistent chemical agent and the production reaction of hypochlorous acid, and thus both mustard gas and VX can be detected by the single enzyme chromosome intended in the present invention.

[0072] In the case of a reaction in which chloroperoxidase enzymes and halogen ions react with bromide ions, etc., to produce a ferric hypochlorite intermediate, since the reaction does not proceed within the operating pH range of the reaction mixture in the enzyme color changer composition of the present invention, it is essential for the user to prepare the reaction mixture described above.

[0073] indicator

[0074] The indicator that can be used as a background color serves to clearly detect the presence or absence of persistent chemical agents and to confirm that the pH of the reaction mixture in the enzyme colorimetric agent composition for detecting persistent chemical agents of the present invention is 3 to 4.

[0075] In the enzyme colorimetric agent composition for detecting persistent chemical agents of the present invention, the reaction mixture further comprises an indicator, and the indicator may be one or more selected from the group consisting of thymol blue, methyl orange, and bromophenol blue.

[0076] Here, when the above indicator is used, it can be clearly confirmed that the reaction mixture in the enzyme chromosome composition for detecting persistent chemical agents of the present invention has a pH of 3 to 4, and thus it is confirmed that the oxidation reaction of the persistent chemical agent and the generation reaction of hypochlorous acid by chloroperoxidase occur well, so the user can easily confirm that mustard gas and VX are clearly detected by a single enzyme chromosome.

[0077] surfactants

[0078] Surfactants lower surface tension, helping the liquid enzyme colorant spread well and disperse into uniform particles.

[0079] The enzyme chromosome composition for detecting persistent chemical agents of the present invention further comprises a surfactant, and said surfactant may be one or more selected from the group consisting of sodium dodecyl sulfate (SDS), Tween-40, Triton X-100, and NP-40.

[0080] Here, when the above surfactant is used, the liquid enzyme color changer of the present invention spreads well and is dispersed into uniform particles, making it possible to introduce it into a spray system and utilize it in extensive contaminated areas. Furthermore, in the operation method of detection equipment and detection paper, which are surface chemical agent detection technologies, this has the effect of reducing the risk of the user being in close proximity to the chemical agent contaminated area or coming into contact with the contaminated surface.

[0081] The concentration of the above surfactant may be 1 to 5%.

[0082] If the concentration of the surfactant exceeds 5%, chloroperoxidase is not activated, which can significantly reduce the oxidation reaction of the persistent chemical agent and the reaction rate of hypochlorous acid production, and increase the turbidity of the enzyme colorant, which can cause errors in absorbance measurement.

[0083] The enzyme color changer of the present invention may include the aforementioned enzyme color changer composition as a raw material.

[0084] The present invention will be explained in more detail below through examples and comparative examples.

[0085] However, the following examples are merely for illustrating the present invention, and the content of the present invention is not limited to the following examples.

[0087] Preparation Examples and Comparative Examples

[0088] A liquid enzyme color changer was prepared by mixing according to the combinations in Table 1 below, and the absorbance of the agent was measured.

[0089] ingredient Preparation Example 1 Preparation Example 2 Preparation Example 3 Preparation Example 4 Comparative Example 1 Comparative Example 2 A solvent Citrate buffer 50 mM 50 mM 50 mM 50 mM 50 mM 50 mM CPO 0.5 U / mL 0.5 U / mL 0.5 U / mL 0.5 U / mL 0.5 U / mL 0.5 U / mL B Solvent Citrate buffer 50 mM 50 mM 50 mM 50 mM 50 mM 50 mM sodium chloride 400 mM 400 mM 400 mM 400 mM 400 mM 400 mM hydrogen peroxide 0.06 w / w% 0.06 w / w% 0.06 w / w% 0.06 w / w% 0.06 w / w% 0.06 w / w% Safranin-0 25 μM 25 μM 25 μM 25 μM 25 μM 25 μM Cymol Blue - 160 μM 160 μM 160 μM 160 μM 160 μM Sodium dodecyl sulfate - 2% 3% - 7% - Twin-40 - 2% 7% A:B mixing ratio 1:1 1:1 1:1 1:1 1:1 1:1 1:1 Whether absorbance measurement is possible O O O O X X

[0091] Referring to [Table 1] above, the absorbance of the reaction mixture prepared in Preparation Example 1, in which solvent A and solvent B were mixed in a 1:1 ratio, was measured. Also, in Preparation Examples 2 and 3, when thymol blue was used as a background indicator and sodium dodecyl sulfate was used as a surfactant at concentrations of 2% and 3%, respectively, the absorbance was measured, and in Preparation Example 4, when thymol blue was used as a background indicator and Tween-40 was used as a surfactant at a concentration of 2%, the absorbance was measured.

[0092] On the other hand, in Comparative Example 1, when thymol blue was used as a background indicator and sodium dodecyl sulfate was used as a surfactant at a concentration of 7%, absorbance measurement was impossible, and in Comparative Example 2, when thymol blue was used as a background indicator and Tween-40 was used as a surfactant at a concentration of 7%, absorbance measurement was impossible.

[0094] Examples

[0095] As an example of the present invention, a chemical agent detection test was conducted by designing combination conditions of enzyme, azo dye, pH buffer, background indicator, and surfactant based on the actual agents mustard gas HD and VX.

[0097] Example: Decolorization analysis according to mustard gas HD concentration

[0098] In Examples 1 to 4 and Comparative Examples 3 to 5, the decolorization of azo dyes according to the concentration of mustard gas HD, a blistering chemical agent, was analyzed. For the test, solvent A and solvent B were prepared separately.

[0099] Solvent A was prepared by adding 0.5 U / ml of chloroperoxidase to a citrate buffer at pH 4 and 50 mM. Solvent B was prepared by combining 400 mM of sodium chloride, 0.06% (mass percentage, w / w) of hydrogen peroxide, and 25 μM of safranin-O to a citrate buffer at pH 4 and 50 mM. The prepared solvents A and B were mixed in a mixing ratio of 1:1, and mustard gas at concentrations of 0.156, 0.313, 0.625, 1.25, 2.5, 5, and 10 mg / mL was added to prepare Examples 1 to 4 and Comparative Examples 3 to 5, which were then irradiated at 520 nm for 5 minutes using a spectrophotometer.

[0100] Figure 2 is a photograph of the decolorization reaction of safranin-O by chloroperoxidase according to mustard gas concentration of an enzyme chromogenic agent composition for detecting persistent chemical agents according to one embodiment of the present invention.

[0101] Figure 3 is the absorbance result of the decolorization reaction of safranin-O by chloroperoxidase according to mustard gas concentration of an enzyme chromogenic agent composition for detecting persistent chemical agents according to one embodiment of the present invention.

[0102] In Figure 2, the color change of the reaction solution after 5 minutes was analyzed visually, and in Figure 3, the absorbance after 5 minutes was graphed. The decolorization rate (%) was analyzed according to [Equation 1] and summarized in [Table 2].

[0103] [Equation 1] Decolorization rate

[0104]

[0105] HD (mg / mL) Discoloration rate (%) Example 1 10 2% Example 2 5 5% Example 3 2.5 2% Example 4 1.25 6% Comparative Example 3 0.625 53% Comparative Example 4 0.313 82% Comparative Example 5 0.156 90%

[0107] Referring to Figures 2 and 3 and [Table 2], it was confirmed that in Examples 1 to 4, under conditions where the concentration of the blistering agent HD was 1.25 mg / mL or higher, the discoloration rate was approximately 2 to 6% and no discoloration occurred, while in Comparative Examples 3 to 5, where the contamination concentration was at a μg level lower than that, discoloration proceeded at 0.625 mg / mL and more than 50%, and finally, most discoloration occurred at 0.156 mg / mL.

[0109] Example: Decolorization analysis according to VX concentration

[0110] In Examples 5 to 8 and Comparative Examples 6 and 7, the decolorization of azo dyes according to the concentration of the neurochemical agent VX was analyzed. For the test, solvent A and solvent B were prepared separately.

[0111] Solvent A was prepared by adding 0.5 U / ml of chloroperoxidase to a citrate buffer at pH 4 and 50 mM. Solvent B was prepared by combining 400 mM of sodium chloride, 0.06% (mass percentage, w / w) of hydrogen peroxide, and 25 μM of safranin-O to a citrate buffer at pH 4 and 50 mM. The prepared solvents A and B were mixed in a mixing ratio of 1:1, and VX at concentrations of 0.313, 0.625, 1.25, 2.5, 5, and 10 mg / mL was added to prepare Examples 5 to 8 and Comparative Examples 6 and 7, which were then irradiated at 520 nm for 5 minutes using a spectrophotometer.

[0112] Figure 4 is a photograph of the decolorization reaction of safranin-O by chloroperoxidase according to the VX concentration of an enzyme chromogenic agent composition for detecting persistent chemical agents according to one embodiment of the present invention.

[0113] Figure 5 is the absorbance result of the decolorization reaction of safranin-O by chloroperoxidase according to the VX concentration of an enzyme chromogenic agent composition for detecting persistent chemical agents according to one embodiment of the present invention.

[0114] In Figure 4, the color change of the reaction solution after 5 minutes was analyzed visually, and in Figure 5, the absorbance after 5 minutes was graphed. The decolorization rate (%) was analyzed according to [Equation 1] and summarized in [Table 3].

[0115] VX (mg / mL) Discoloration rate (%) Example 5 10 50% Example 6 5 55% Example 7 2.5 50% Example 8 1.25 59% Comparative Example 6 0.625 80% Comparative Example 7 0.313 90%

[0117] Referring to Figures 4 and 5 and [Table 3], in Examples 5 to 8, the decolorization rate was approximately 50–59% under conditions where the concentration of VX, a blister chemical agent, was 1.25 mg / mL or higher. In Comparative Examples 6 and 7, where the contamination concentration was at a level of μg lower than that, decolorization proceeded at 0.625 mg / mL or higher, and it was confirmed that most of the decolorization occurred at 0.313 mg / mL.

[0119] Example: Verification of enzymatic discoloration agent against mustard gas and VX

[0120] In Examples 9 to 12, enzyme colorimetric agents were prepared and detection tests for mustard gas and VX were conducted. To make the color change judgment more distinct, a background indicator was added, and the enzyme colorimetric agents were designed by increasing the concentrations of the indicators. The overall color change ability of the enzyme colorimetric agents of the present invention is shown in Fig. 6.

[0121] For the test, solvents A and B were prepared separately. Solvent A was prepared by adding 2 U / ml of chloroperoxidase to a pH 4, 50 mM citrate buffer. Solvent B was prepared by combining 400 mM sodium chloride, 0.06% (mass percentage, w / w) hydrogen peroxide, 50 μM safranin-O, 160 μM thymol blue, and 2% sodium dodecyl sulfate to a pH 4, 50 mM citrate buffer. The prepared solvents A and B were mixed in a mixing ratio of 1:1, and VX at a concentration of 10 mg / mL was added to prepare Examples 9 to 12.

[0122] In Example 9 (Bottle), to simulate a solution contaminated with a chemical agent, prepared solvent A and solvent B were mixed in a mixing ratio of 1:1, and VX at a concentration of 10 mg / mL was added to this, and the color of the solution was examined after 5 minutes.

[0123] In Example 10 (Paper), to simulate a contaminated environment where chemical agents are absorbed, 1 g / mL of VX and 10 μL of mustard gas were dropped onto the surface of filter paper, and solvents A and B prepared were applied in a mixing ratio of 1:1 and discoloration was investigated.

[0124] In Examples 11 (Glass) and 12 (Tile), 1 g / mL of VX and 10 μL of mustard gas were dropped onto glass and tile to simulate a contaminated environment where chemical agents do not absorb moisture but form water droplets on the surface, and solvents A and B prepared were applied in a mixing ratio of 1:1 and discoloration was investigated.

[0125] FIG. 7 is a photograph showing the detection of liquid and surface contamination of chemical agents by changing color using an enzyme colorimetric agent for detecting persistent chemical agents according to one embodiment of the present invention.

[0126] As shown in Fig. 7, clear detection by the enzyme chromosome was confirmed in Examples 9 (Bottle), 11 (Glass), and 12 (Tile), and in Example 10 (Paper), detection of the chemical agent over a wide area was confirmed through the diffusion of the chemical agent as it was absorbed.

[0128] Although various preferred embodiments of the present invention have been described above with some examples, the descriptions of various embodiments described in the "Specific details for carrying out the invention" section are merely illustrative, and those skilled in the art to which the present invention pertains will understand that the present invention can be carried out with various modifications or equivalent embodiments from the above description.

[0129] In addition, since the present invention can be implemented in various other forms, the present invention is not limited by the description above. The above description is provided merely to make the disclosure of the present invention complete and to fully inform those skilled in the art of the scope of the present invention, and it should be understood that the present invention is defined only by each claim of the claims.

Claims

Claim 1 An enzymatic chromogenic agent composition for detecting persistent chemical agents, comprising a reaction mixture comprising a persistent chemical agent, 0.5 to 2.0 U / mL of chloroperoxidase, hydrogen peroxide, chloride ions, an azo dye, and a pH buffer, wherein the reaction mixture has a pH value of 3 to 4. Claim 2 An enzyme colorimetric agent composition for detecting persistent chemical agents, characterized in that, in claim 1, the persistent chemical agent is either mustard gas or VX, and the concentration of the persistent chemical agent is 1.0 to 10 mg / mL. Claim 3 delete Claim 4 An enzyme colorimetric agent composition for detecting persistent chemical agents, characterized in that, in claim 1, the concentration of the hydrogen peroxide is 0.04 to 0.08 w / w%. Claim 5 An enzyme colorimetric agent composition for detecting persistent chemical agents, characterized in that, in claim 1, the chloride salt of the chloride ion is either sodium chloride or potassium chloride, and the concentration of the chloride salt is 350 to 450 mM. Claim 6 An enzymatic colorimetric composition for detecting persistent chemical agents, characterized in that, in claim 1, the azo dye is one or more selected from the group consisting of Orange-G, Sunset Yellow, Congo-Red, Alizarin Yellow, and Safranin-O. Claim 7 An enzyme colorimetric agent composition for detecting persistent chemical agents, characterized in that, in claim 1, the concentration of the azo dye is 25 to 50 μM. Claim 8 An enzymatic chromogenic agent composition for detecting persistent chemical agents, characterized in that, in claim 1, the pH buffer is one or more selected from the group consisting of a phosphate solution, a phthalate solution, and a citrate solution. Claim 9 An enzymatic colorimetric composition for detecting persistent chemical agents, wherein, in claim 1, the reaction mixture further comprises an indicator, and the indicator is one or more selected from the group consisting of thymol blue, methyl orange, and bromophenol blue. Claim 10 An enzyme chromosome composition for detecting persistent chemical agents according to claim 1, wherein the enzyme chromosome composition further comprises a surfactant, and the surfactant is one or more selected from the group consisting of sodium dodecyl sulfate (SDS), Tween-40, Triton X-100, and NP-40. Claim 11 delete Claim 12 Enzyme color changer comprising the composition of claim 1.

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