JP2023007799A - Test piece and alcohol detection method using the same - Google Patents

Abstract

To provide a test piece with superior storage stability that keeps sensitivity thereof to alcohol of 0.01-0.64% concentration unchanged throughout long-term storage.SOLUTION: A test piece is provided, comprising a carrier that has been impregnated with a reagent designed to discolor in reaction with alcohol and dried, the test piece being designed to detect alcohol of 0.01-0.64% concentration after being kept for 24 hours or longer from drying under a temperature condition of 25-40°C.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to a test strip and a method for detecting alcohol using the same.

Accurate determination of alcohol concentration is performed by analytical instruments such as gas chromatography and high performance liquid chromatography. However, these devices are expensive, require skill to operate, and take a long time to obtain results. Therefore, methods for detecting alcohol simply and inexpensively at any place have been developed without requiring special devices or techniques. For example, an alcohol test paper has been developed in which a group of reagents such as enzymes is immobilized on a carrier and placed on a support as a base (see, for example, Patent Documents 1 to 4 below).

Groups of reagents immobilized on carriers are roughly classified into two types. The first is the group consisting of (a) alcohol dehydrogenase, (b) nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), (c) diaphorase, (d) color change agents ( Patent Documents 1 and 2). Examples of discoloring agents include formazan dyes and methylene blue. FIG. 1 shows the principle of coloration of ethanol by (a)-(d). If a minute amount of NADH derived from the raw material becomes a problem, (e) an oxidizing agent such as potassium persulfate may be added for the purpose of eliminating it. The second is a group consisting of (a)' alcohol oxidase, (b)' peroxidase, and (c)' discoloration agent (Patent Documents 3 and 4). Examples of color change agents include 3,3'-diaminobenzidine, 3,3',5,5'-tetramethylbenzidine, 2,2'-azino-bis(3-ethylbenzothiazoline-6- sulfonic acid), and the so-called Trinder's reagent that develops color by oxidation condensation with 4-aminoantipyrine. FIG. 2 shows the principle of coloration of ethanol by (a)'-(c)'. If a minute amount of hydrogen peroxide originating from the raw material poses a problem, a (d)' reducing agent such as ascorbic acid may be added for the purpose of eliminating it.

All of the above methods are capable of detecting ethanol, but since both alcohol dehydrogenase and alcohol oxidase are unstable, the storage stability of the alcohol test paper is a problem.

Examples of adding polyhydric alcohols such as mannitol and sugars such as cyclodextrins to carriers for the purpose of improving the storage stability of alcohol test papers have been reported (Patent Documents 5 and 6). However, all of them show storage stability at 37° C. for only 20 days or less, and no quantitative comparison of sensitivity between the start of storage and after the storage period has elapsed has been made.

JP-A-60-164498 JP-A-59-166098 JP-A-60-172298 JP-A-03-004799 JP-A-61-212300 JP-A-60-075299

SUMMARY OF THE INVENTION An object of the present invention is to provide a test strip having excellent storage stability, in which the sensitivity to alcohol of 0.01% to 0.64% concentration is not changed by long-term storage. Another object of the present invention is to provide a method for detecting alcohol using the test strip, a detection kit including the test strip, and a method for manufacturing the test strip.

In intensive research to solve the above problems, the present inventors have found that a test piece containing a carrier impregnated with a reagent that changes color by reacting with alcohol and dried is dried under a temperature condition of 25 ° C. to 40 ° C. The present inventors have found that the sensitivity to alcohol with a concentration of 0.01% to 0.64% is stabilized over a long period of time by holding for 24 hours or longer, and have completed the present invention as a result of further research.

That is, the present invention relates to the following.
[1] A test piece containing a carrier impregnated with a reagent that discolors in response to alcohol and dried,
The above-mentioned test piece for detecting alcohol with a concentration of 0.01% to 0.64% after being kept for 24 hours or more after drying under temperature conditions of 25°C to 40°C.
[2] The test piece according to [1], wherein the reagent that changes color in response to alcohol contains alcohol oxidase.
[3] The test piece according to [2], wherein the concentration of alcohol oxidase in the solution with which the carrier is impregnated is 0.1 U/mL to 100 U/mL.

[4] For detecting alcohol with a concentration of 0.01% to 0.64% after holding for 24 hours to 240 hours after drying under a temperature condition of 25 ° C. to 40 ° C., [1] to [3] A test piece according to any one of the above.
[5] The test strip according to any one of [1] to [4], for detecting alcohol concentration in saliva.
[6] The test piece according to any one of [1] to [5], comprising two or more carriers having different alcohol concentrations necessary for discoloration.

[7] A test piece obtained by drying the test piece according to any one of [1] to [6] at a temperature of 25°C to 40°C for 24 hours or longer.
[8] A method for detecting alcohol, using the test piece according to any one of [1] to [7].
[9] The method according to [8], wherein the alcohol concentration is measured by the number and/or color depth of discolored carriers.
[10] The method of [8] or [9], which comprises scoring the number and/or color intensity of discolored carriers.

[11] A method for manufacturing the test piece according to [7],
A step of impregnating the carrier with a reagent that reacts with alcohol to change color, a step of drying this, and a step of holding the carrier at a temperature of 25 ° C. to 40 ° C. for 24 hours or more after drying, in this order. the aforementioned method.
[12] A detection kit comprising the test piece according to any one of [1] to [7] and a color sample showing the correspondence between alcohol concentration and discoloration of the carrier.

The test piece of the present invention shows less decrease in alcohol detection sensitivity due to long-term storage than the test piece of the prior art, and can be used without problems even after being stored under temperature conditions of 40° C. or less for 60 days or more. This property is of great importance in the commercial use of alcohol test papers.

FIG. 1 is a diagram showing the principle of coloration of ethanol using alcohol dehydrogenase. FIG. 2 is a diagram showing the principle of an ethanol coloration method using alcohol oxidase. FIG. 3 is a schematic representation of a test strip according to one embodiment of the present invention for measuring alcohol concentration by the number and/or color intensity of discolored carriers. FIG. 4 is a schematic representation of a test strip according to one embodiment of the present invention for measuring alcohol concentration by scoring the number and/or color intensity of discolored carriers. FIG. 5 shows the results of applying 0.08% ethanol to the carrier of (A) the invention product and (B) the control product, and observing the appearance after 5 minutes. FIG. 6 is a diagram showing the results of observing the appearance after 5 minutes after applying 0.08% ethanol on the carrier of the test piece in which (A) the invention product and (B) the control product were stored at 25° C. for 62 days. is. FIG. 7 is a diagram showing the results of observing the appearance after 5 minutes after applying 0.08% ethanol on the carrier of the test pieces of (A) the invention product and (B) the control product stored at 37° C. for 62 days. is.

The test piece of the present invention contains a dry carrier impregnated with a reagent that reacts with alcohol and changes color, and after holding for 24 hours or more after drying under a temperature condition of 25 ° C. to 40 ° C., the concentration is 0.01% to It is a test strip for the detection of 0.64% alcohol.

When the carrier is impregnated with a reagent that reacts to discoloration with alcohol, dried, and then held at a temperature of 25° C. to 40° C., the sensitivity to alcohol of the test piece containing such a carrier changes color in response to alcohol. Since the stability of the reagent in the solid phase state is poor and the reagent is deactivated, it drops sharply during the period from immediately after drying (before retention) to 24 hours. However, unexpectedly, after 24 hours from drying (after holding), the sensitivity does not change significantly even if the film is held under the above temperature conditions, and the sensitivity to alcohol is stabilized.
It is not clear why the sensitivity to alcohol stabilizes after 24 hours have passed since drying, and it is also difficult to directly identify the change in the reagent that reacts with alcohol and changes color from its structure or properties. While not intending to bind the present invention by theory, one hypothesis is that some of the specific components contained in the reagent that reacts with alcohol to change color are heat resistant and some are not, As a result, it is conceivable that under a temperature condition of 25° C. to 40° C., those having no heat resistance are deactivated within 24 hours after drying, and only those having heat resistance remain thereafter. In this case, there is no fear that the sensitivity to alcohol after retention will change over time, and alcohol can be detected with stable sensitivity even with test pieces that have been stored for a long time (test pieces after storage). It is considered possible.

The temperature condition for holding after impregnating the carrier with a reagent that reacts with alcohol and discoloring and drying is 25°C to 40°C, preferably 27°C to 35°C, more preferably 28°C to 32°C. be.
If the temperature condition for holding is lower than 25°C, the time required for the sensitivity of the test piece to alcohol to stabilize may become too long, or the test piece may give a test piece whose sensitivity to alcohol is not sufficiently stabilized. I don't like it. In addition, when the temperature condition for holding is higher than 40 ° C., the reagent that reacts with alcohol and discolors is deactivated more than necessary, and the sensitivity necessary for detecting alcohol at a concentration of 0.01% to 0.64% is sufficient. It is not preferable because there is a risk that the

The time for holding under temperature conditions of 25° C. to 40° C. is not particularly limited as long as it is 24 hours or more after impregnating the carrier with a reagent that reacts with alcohol and discoloring and drying, but preferably 24 hours to 240 hours. , more preferably 24 hours to 200 hours, more preferably 24 hours to 150 hours.
If the retention time is less than 24 hours, the deactivation of the reagent that reacts with alcohol and discolors does not proceed sufficiently, leaving room for further deactivation, so the sensitivity of alcohol detection changes over time. I am afraid and I don't like it.
Holding the specimen is preferably under dry conditions, such as holding the specimen with a desiccant. Moreover, it is preferable to hold the test piece under light-shielding conditions such as closing the test piece in an aluminum bag.

There are no restrictions on the mode of holding under temperature conditions of 25° C. to 40° C. as long as the dried carrier is held under the same conditions. For example, only the dried carrier may be held, or a test piece in which the dried carrier and other components such as a substrate are combined may be held.

There is no particular limitation on drying, as long as the solvent described below is sufficiently removed from the carrier. One mode of drying includes vacuum drying and freeze-drying using a vacuum pump. In order to prevent deactivation of the reagent that reacts with alcohol and discolors, it is preferable to dry at room temperature or below. The temperature range for drying is preferably 1°C to 40°C, more preferably 4°C to 30°C, even more preferably 4°C to 25°C.
The smaller the amount of solvent remaining after drying, the better. For example, when water is used as a solvent, a general range that can be achieved by drying under reduced pressure overnight at 25°C is sufficient.

In the test piece of the present invention, the alcohol detection sensitivity after holding for 24 hours or more after impregnating the carrier with a reagent that reacts with alcohol and discoloring under temperature conditions of 25 ° C. to 40 ° C. and drying is generally is considered to be about 30% to 90% before holding.

The reagent that reacts with alcohol and changes color to be used in the present invention is not particularly limited as long as it changes color when the concentration of alcohol exceeds a certain level. Color change, as used herein, includes color change and fading. The reagent that reacts with alcohol to change color used in the present invention may be a reagent based on the principles of the invention described in Patent Documents 1 to 6, for example.

In a preferred embodiment of the present invention, the reagent that changes color in response to alcohol contains alcohol oxidase, in a more preferred embodiment alcohol oxidase, peroxidase, a color changing agent and a reducing agent, and even more preferably alcohol oxidase. , peroxidase, color change agents, reducing agents, and stabilizers.
In a preferred embodiment of the invention, the alcohol oxidase is classified under EC 1.1.3.13, eg from yeast.
In a preferred embodiment of the invention, the peroxidase is classified under EC 1.11.1.7, eg from horseradish.

In a preferred embodiment of the present invention, discoloring agents may be used alone or in combination of two or more. Single discoloration agents are, for example, 3,3′-diaminobenzidine or its salts, 3,3′,5,5′-tetramethylbenzidine or its salts, 2,2′-azino-bis(3-ethylbenzothiazoline- 6-sulfonic acid) or salts thereof, and the like. Examples of discoloration agents in combination of two or more types include combinations of 4-aminoantipyrine, 3-methyl-2-benzothiazolinone hydrazone, etc., and phenolic compounds, aniline derivatives, etc., preferably 4-amino It is a combination of antipyrine and TOOS (Sodium 3-(N-ethyl-3-methylanilino)-2-hydroxypropanesulfonate).

In a preferred embodiment of the present invention, the reducing agent is not particularly limited as long as it can reduce hydrogen peroxide generated before application of the specimen, which is derived from autooxidation of alcohol oxidase, etc. Examples include ascorbic acid and reduced glutathione. , cysteine, etc., preferably ascorbic acid.

In preferred embodiments of the present invention, stabilizers are, for example, polysaccharides, oligosaccharides, disaccharides, monosaccharides, and derivatives thereof, preferably non-reducing sugar alcohols, cyclodextrins, trehalose, and sucrose. and most preferably sucrose.

The concentration range of alcohol detected by the test strip of the present invention can be adjusted, for example, by changing the components of the reagent or the number of carriers.
The concentration of alcohol detected by the test strip of the present invention is in the range of 0.01-0.64%, preferably in the range of 0.02-0.4%. In this specification, "%" indicating alcohol concentration means volume percent (vol%).
By setting the concentration of alcohol to be detected in such a range, the test strip can be used for a specific purpose, for example, the purpose of cracking down on drunk driving by drivers.

In a preferred embodiment of the present invention, the reagent that reacts with alcohol to change color is impregnated into the carrier in the form of a solution in combination with a solvent. Solvent is removed by drying.
In a preferred embodiment of the present invention, the solvent is not particularly limited as long as it can dissolve the reagent that changes color upon reaction with alcohol and can be removed by drying. Water is typically used as the solvent.

In a preferred embodiment of the present invention, when water is used as a solvent to prepare an aqueous solution of a reagent that reacts with alcohol to change color, the pH range of the aqueous solution is not particularly limited. From the viewpoint of stabilizing a reagent that reacts with alcohol to discolor, particularly from the viewpoint of stably obtaining a good color-developing reaction due to the action of an enzyme, in the case of a reaction mode using alcohol oxidase, the pH of the aqueous solution is preferably pH 7.0 to 7.0. 9.0, more preferably pH 7.0-8.5, still more preferably pH 7.5-8.5.
In order to stabilize the pH of the aqueous solution, a buffer solution obtained by adding a buffer to the solvent water may be used. Examples of such buffers include acetate buffers, phosphate buffers, citrate buffers, carbonate buffers, borate buffers, Tris (trishydroxymethylaminomethane) buffers, various Good's buffers, and the like. be done. Among these, a phosphate buffer having a neutral to weakly alkaline buffer range in which the enzyme works, a Tris (trishydroxymethylaminomethane) buffer, and 3-morpholinopropanesulfonic acid (MOPS) or 4-( Good's buffers such as 2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) are more preferred.

In a preferred embodiment of the present invention, the concentration of alcohol oxidase in the solution with which the carrier is impregnated ranges from 0.1 U/mL to 100 U/mL, more preferably from 0.5 U/mL to 50 U/mL, and still more It is preferably in the range of 4-30 U/mL, particularly preferably in the range of 4-20 U/mL. By adopting such a concentration range, a test piece kept for 24 hours or more after drying under a temperature condition of 25 ° C. to 40 ° C. has an appropriate sensitivity for detecting alcohol at a concentration of 0.01% to 0.64%. is preferred because it shows

In a preferred embodiment of the present invention, the concentration of peroxidase in the solution with which the carrier is impregnated is in the range of 0.1 U/mL to 1000 U/mL, more preferably 1 U/mL, from the viewpoint of the alcohol concentration to be detected. in the range of -500 U/mL, even more preferably in the range of 10 U/mL to 100 U/mL.

In a preferred embodiment of the present invention, the concentration of the discoloring agent in the solution with which the support is impregnated is in the range of 0.01 mmol/L to 100 mmol/L, more preferably 0.01 mmol/L, from the viewpoint of the alcohol concentration to be detected. It ranges from 1 mmol/L to 10 mmol/L, even more preferably from 0.5 mmol/L to 5 mmol/L.

In a preferred embodiment of the present invention, the concentration of the reducing agent in the solution with which the carrier is impregnated is in the range of 0 mmol/L to 100 mmol/L, more preferably 0.1 mmol/L, from the viewpoint of the alcohol concentration to be detected. It is in the range of L to 10 mmol/L, even more preferably in the range of 0.5 mmol/L to 5 mmol/L.

In a preferred embodiment of the invention, the concentration of stabilizer in the solution in which the support is impregnated ranges from 0% to 30%, more preferably from 1% to 20%, even more preferably from 5% to 15%. is in the range of In this specification, "%" indicating the concentration of the stabilizer means mass volume % (g/100 mL).

The specimen applied to the test strip of the present invention may contain any substance other than alcohol, but is preferably alcohol in aqueous solution or alcohol in saliva.
The alcohol of the specimen applied to the test piece of the present invention is not particularly limited as long as it exhibits discoloration by the reagent of the present invention, preferably primary alcohol, more preferably methanol or ethanol, most preferably ethanol. .

The carrier contained in the test piece of the present invention is not limited as long as it is insoluble or sparingly soluble in water, saliva, and alcohol at room temperature. For example, from low-molecular to high-molecular weight natural products such as gelatin, agar, cyclodextrin, cellulose, paper, chitin, and glucan, as well as synthetic materials such as rayon, vinylon, polyester, polyvinyl alcohol, nylon, and acrylic. A synthetic polymer or the like may also be used. Polyvinyl alcohol is preferable from the viewpoint of water absorption and water retention.
There are no particular restrictions on the size, thickness, shape, etc. of the carrier.

The test piece of the present invention may be constructed by providing a carrier on a substrate. The material of the substrate is not particularly limited, and may be, for example, paper, plastic, rubber, resin, or the like. Moreover, the shape of the base material is not particularly limited and may be rod-like, circular, polygonal, or the like.

The test strips of the present invention can contain two or more carriers each having a different concentration required for color change. By using a plurality of such test pieces in combination, the concentration of alcohol can be measured and detected from the two viewpoints of the number of discolored carriers and the color depth of each carrier, as shown in FIG. This is preferable because the concentration of alcohol can be measured regardless of subjectivity of the measurer.
When the test piece contains a plurality of carriers, the number of carriers is preferably 2 to 10, more preferably 2, from the viewpoint of the measurable concentration range of alcohol and the ease of manufacturing the test piece and the carrier. to 5, more preferably 2 to 4, and particularly preferably 2 to 3.

In addition, the present invention includes a carrier impregnated with a reagent that reacts with alcohol and discolored and dried, and kept at a temperature of 25 ° C. to 40 ° C. for 24 hours or more after drying, with a concentration of 0.01% to 0.64. It relates to a test strip for the detection of % alcohol.
By adopting such a test piece, it is possible to obtain good sensitivity for detection of alcohol at a concentration of 0.01% to 0.64%, which is stable over time, and thus is preferable.
When the test piece held as described above is further stored, it is preferable to store it under low temperature conditions from the viewpoint of preventing deactivation of the reagent that discolors by reacting with alcohol. It can be stored for days without deterioration in performance.
Storage of the test piece is preferably carried out under dry conditions, such as storage of the test piece with a desiccant. Moreover, it is preferable to store the test piece under light-shielding conditions such as closing the test piece in an aluminum bag.

Furthermore, the present invention relates to a method for detecting alcohol using the test strip described above.
The method for detecting alcohol using the test piece according to the present invention is not particularly limited as long as the sample is provided to the carrier. The method of applying the sample to the carrier includes, for example, a method of dropping the sample onto the carrier and a method of immersing the sample together with the test piece.

The alcohol detection method using the test piece according to the present invention may be performed by measuring the concentration of alcohol based on the number of discolored carriers and/or the color intensity. For example, as shown in FIG. Alternatively, the concentration of alcohol may be measured by scoring the number and/or the color depth of the carriers. Scoring can be done by the number of discolored carriers. For example, if 0 carriers of the test piece are discolored, the score is 0; if 1 carrier is discolored, 1 point; if 2 carriers are discolored, 2 points; Further, the scoring is performed such that, for example, depending on the color depth of each carrier, it monotonically decreases or increases, such as 0 points for no discoloration, 1 point for light discoloration, and 2 points for light discoloration. Scoring according to the color depth of each carrier may be performed at any stage for each carrier, but from the viewpoint of ease of judgment of color depth, etc., 2 to 4 stages, preferably 3 stages is done in Furthermore, scoring may be done by a combination of both the number of carriers and the color depth of each carrier.

As one aspect of the test piece manufacturing method of the present invention, a step of impregnating a carrier with a reagent that reacts with alcohol and discoloring, a step of drying this, and drying the carrier under a temperature condition of 25 ° C. to 40 ° C. and a step of holding for 24 hours or more in this order. Preferred embodiments of the method for manufacturing such test strips are the same as the preferred embodiments described for the test strips.

The test strip of the present invention may be used as a detection kit in combination with a color sample showing the correspondence between the concentration of alcohol and the discoloration of the carrier. As a colorimetric method that can be easily performed at the site of detection, a sample for evaluation is prepared by scoring multiple color samples corresponding to the concentration of alcohol and arranging them, and comparing which color of the color sample corresponds to the color change due to the sample. Therefore, alcohol of a predetermined concentration can be detected with higher accuracy. The evaluation sample may be a photograph, a printed matter, or the like.

EXAMPLES The present invention will be described in more detail below with reference to examples, which illustrate specific embodiments of the invention and are not intended to limit the invention.

Reagents 1 and 2 were prepared according to the formulations shown in Tables 1 and 2. Reagent 1 was impregnated into a polyvinyl alcohol carrier, dried overnight at 25°C under reduced pressure to form a solid phase, sealed in an aluminum bag together with a desiccant, and held at 30°C for 5 days. Reagent 2 was impregnated into a polyvinyl alcohol carrier and dried overnight at 25° C. under reduced pressure to obtain a solid phase, which was used as a control product.

(Evaluation 1: Evaluation of test piece before long-term storage)
0.08% ethanol was applied to the carriers of the invention product and the control product, and the appearance after 5 minutes was observed (the number of each sample: 2). As shown in FIG. 5, both (A) the invention product and (B) the control product showed good color development reaction.

(Evaluation 2-1: Evaluation 1 of test piece after long-term storage)
The invention product and the control product were closed in an aluminum bag together with a desiccant, and 0.08% ethanol was applied to the carrier of the test piece stored at 25 ° C. for 62 days, and the appearance after 5 minutes was observed (each sample number: 2 ). As shown in FIG. 6, the invention product (A) showed a color development reaction with the same sensitivity as before long-term storage. On the other hand, the control product (B) had lower sensitivity than before long-term storage, and showed only a very weak coloring reaction.

(Evaluation 2-2: Evaluation 2 of test piece after long-term storage)
The inventive product and the control product were closed in an aluminum bag together with a desiccant, and 0.08% ethanol was applied to the carrier of the test piece stored at 37 ° C. for 62 days, and the appearance after 5 minutes was observed (the number of each sample: 2 ). As shown in FIG. 7, the invention product (A) showed a color development reaction with the same sensitivity as before long-term storage. On the other hand, the sensitivity of the control product (B) was extremely low compared to before long-term storage, and almost no coloring reaction could be observed.

From the above results, it was found that the sensitivity to alcohol with a concentration of 0.01% to 0.64% was stabilized by keeping the test piece under a temperature condition of 25°C to 40°C for 24 hours or more after drying.

Claims (12)

A test strip containing a dried carrier impregnated with a reagent that reacts with alcohol and discolors,
The above-mentioned test piece for detecting alcohol with a concentration of 0.01% to 0.64% after being kept for 24 hours or more after drying under temperature conditions of 25°C to 40°C. The test piece according to claim 1, wherein the reagent that changes color in response to alcohol contains alcohol oxidase. The test piece according to claim 2, wherein the concentration of alcohol oxidase in the solution with which the carrier is impregnated is 0.1 U/mL to 100 U/mL. For detecting alcohol at a concentration of 0.01% to 0.64% after holding for 24 hours to 240 hours after drying under temperature conditions of 25 ° C. to 40 ° C., according to any one of claims 1 to 3 Specimen as described. The test strip according to any one of claims 1 to 4, for detecting alcohol concentration in saliva. The test strip according to any one of claims 1 to 5, comprising two or more carriers each having a different concentration of alcohol required for discoloration. A test piece obtained by keeping the test piece according to any one of claims 1 to 6 under temperature conditions of 25° C. to 40° C. for 24 hours or more after drying. A method for detecting alcohol using the test piece according to any one of claims 1 to 7. 9. The method according to claim 8, wherein the concentration of alcohol is determined by the number and/or color intensity of discolored carriers. 10. A method according to claim 8 or 9, comprising scoring the number and/or color intensity of discolored carriers. A method of manufacturing a test piece according to claim 7,
A step of impregnating the carrier with a reagent that reacts with alcohol to change color, a step of drying this, and a step of holding the carrier at a temperature of 25 ° C. to 40 ° C. for 24 hours or more after drying, in this order. the aforementioned method. A detection kit comprising the test piece according to any one of claims 1 to 7 and a color sample showing the correspondence between alcohol concentration and discoloration of the carrier.

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