Test strip and method for detecting alcohol using the same

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]

[0001] The present invention relates to a test strip and a method for detecting alcohol using the test strip. [Background technology]

[0002] Accurate quantification of alcohol concentration is performed using analytical instruments such as gas chromatography and high-performance liquid chromatography. However, these instruments are expensive, require skilled operation, and take a long time to obtain results, resulting in drawbacks in terms of cost-effectiveness and speed. For this reason, efforts have been made to develop methods for detecting alcohol easily and inexpensively at any location without requiring special equipment or technology. For example, alcohol test strips have been developed in which reagents such as enzymes are immobilized on a carrier and then placed on a base support (see, for example, Patent Documents 1 to 4 listed below).

[0003] Reagents immobilized on a support can be broadly classified into two types. The first group consists of (a) alcohol dehydrogenase, (b) nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), (c) diaphorase, and (d) color change agents (Patent Documents 1 and 2). Examples of color change agents include formazan dyes and methylene blue. Figure 1 shows the principle by which ethanol changes color using (a)-(d). If trace amounts of NADH derived from the raw materials are problematic, an oxidizing agent (e) such as potassium persulfate may be added to eliminate it. The second group consists of (a) alcohol oxidase, (b) peroxidase, and (c) color change agents (Patent Documents 3 and 4). Examples of color-changing agents include 3,3'-diaminobenzidine, 3,3',5,5'-tetramethylbenzidine, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), which can produce color by themselves, and the so-called Trinder's reagent, which produces color by oxidative condensation with 4-aminoantipyrine. Figure 2 shows the principle by which ethanol produces color using (a)'-(c)'. If trace amounts of hydrogen peroxide from the raw materials are a problem, a (d)' reducing agent such as ascorbic acid can be added to eliminate it.

[0004] Although all of the above methods are capable of detecting ethanol, the storage stability of alcohol test papers is an issue because both alcohol dehydrogenase and alcohol oxidase are unstable.

[0005] In order to improve the storage stability of alcohol test strips, examples have been reported in which polyhydric alcohols such as mannitol or sugars such as cyclodextrin have been added to the carrier (Patent Documents 5 and 6). However, in all cases, storage stability at 37°C is only demonstrated for up to 20 days, and no quantitative comparison of sensitivity between the start of storage and after the storage period has been investigated. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 60-164498 [Patent Document 2] Japanese Patent Application Publication No. 59-166098 [Patent Document 3] Japanese Patent Application Publication No. 172298 / 1983 [Patent Document 4] Japanese Patent Application Publication No. 03-004799 [Patent Document 5] Japanese Patent Publication No. 61-212300 [Patent Document 6] Japanese Patent Application Publication No. 60-075299 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a test strip with excellent storage stability, in which sensitivity to alcohol at concentrations of 0.01% to 0.64% does not change over time. 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 producing the test strip. [Means for solving the problem]

[0008] In the course of intensive research to solve the above problems, the inventors discovered that by storing a test piece containing a carrier that has been impregnated with a reagent that changes color in response to alcohol and then dried under temperature conditions of 25°C to 40°C for at least 24 hours after drying, the sensitivity to alcohol at concentrations of 0.01% to 0.64% can be stabilized over a long period of time. As a result of further research, the present inventors have completed the present invention.

[0009] That is, the present invention relates to the following: [1] A test strip containing a carrier impregnated with a reagent that changes color in response to alcohol and dried, The test strip is for detecting alcohol at a concentration of 0.01% to 0.64% after being kept at a temperature of 25°C to 40°C for 24 hours or more after drying. [2] The test strip according to [1], wherein the reagent that changes color in response to alcohol contains alcohol oxidase. [3] The test strip according to [2], wherein the concentration of alcohol oxidase in the solution impregnated into the carrier is 0.1 U / mL to 100 U / mL.

[0010] [4] A test strip according to any one of [1] to [3], for detecting alcohol at a concentration of 0.01% to 0.64% after being kept at a temperature of 25°C to 40°C for 24 to 240 hours from drying. [5] A test strip according to any one of [1] to [4] for detecting the alcohol concentration in saliva. [6] A test strip according to any one of [1] to [5], which contains two or more carriers each having a different concentration of alcohol required for color change.

[0011] [7] A test piece obtained by keeping the test piece according to any one of [1] to [6] at a temperature of 25°C to 40°C for 24 hours or more after drying. [8] A method for detecting alcohol using the test strip according to any one of [1] to [7]. [9] The method according to [8], in which the alcohol concentration is measured based on the number of discolored carriers and / or the color intensity.

[10] The method according to [8] or [9], which comprises scoring the number and / or color intensity of discolored carriers.

[0012]

[11] A method for producing the test piece according to [7], The method comprises, in this order, a step of impregnating a carrier with a reagent that changes color in reaction with alcohol, a step of drying the carrier, and a step of maintaining the carrier at a temperature of 25°C to 40°C for 24 hours or more after drying.

[12] A detection kit comprising the test strip according to any one of [1] to [7] and a color sample showing the correspondence between the alcohol concentration and the color change of the carrier. [Effects of the Invention]

[0013] The test strip of the present invention shows less deterioration in alcohol detection sensitivity over long-term storage than conventional test strips, and can be used without problems even after storage for 60 days or more at temperatures below 40°C. This property is very important when using alcohol test strips commercially. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a diagram showing the principle of the ethanol coloration method using alcohol dehydrogenase. [Figure 2] FIG. 2 is a diagram showing the principle of the ethanol coloration method using alcohol oxidase. [Figure 3] FIG. 3 is a diagram showing a schematic diagram of a test strip according to one embodiment of the present invention for measuring the alcohol concentration based on the number of discolored carriers and / or the color intensity. [Figure 4] FIG. 4 is a diagram showing a schematic diagram of a test strip according to one embodiment of the present invention for measuring the alcohol concentration by scoring the number of discolored carriers and / or the color intensity. [Figure 5] FIG. 5 shows the results of applying 0.08% ethanol to the carriers of (A) the invention product and (B) the control product, and observing the appearance after 5 minutes. [Figure 6] Figure 6 shows the results of applying 0.08% ethanol to the carrier of test pieces of (A) the invention product and (B) the control product stored at 25°C for 62 days, and observing the appearance after 5 minutes. [Figure 7] Figure 7 shows the results of applying 0.08% ethanol to the carrier of test pieces of (A) the invention product and (B) the control product stored at 37°C for 62 days, and observing the appearance after 5 minutes. DETAILED DESCRIPTION OF THE INVENTION

[0015] The test strip of the present invention includes a carrier that has been impregnated with a reagent that changes color in response to alcohol and dried, and is intended for use in detecting alcohol at a concentration of 0.01% to 0.64% after being kept at a temperature of 25°C to 40°C for 24 hours or more after drying.

[0016] When a carrier is impregnated with a reagent that changes color in response to alcohol, dried, and then stored at temperatures between 25°C and 40°C, the sensitivity of a test piece containing such a carrier to alcohol drops sharply between immediately after drying (before storage) and 24 hours later, because the reagent that changes color in response to alcohol is inactivated due to its poor stability in the solid phase. However, surprisingly, after 24 hours from drying (after storage), there is no significant change in sensitivity even when stored under the above temperature conditions, and the sensitivity to alcohol stabilizes. The reason why sensitivity to alcohol stabilizes after 24 hours from drying is unclear, and it is difficult to directly identify the changes in the reagent that changes color in response to alcohol based on its structure or properties. While not intending to constrain the present invention by theory, one hypothesis is that certain components contained in the reagent that changes color in response to alcohol exist, some of which are heat-resistant and some of which are not. As a result, under temperature conditions of 25°C to 40°C, those that are not heat-resistant are inactivated within 24 hours of drying, and only those that are heat-resistant remain. In this case, there is no risk of the sensitivity to alcohol changing over time after storage, and it is thought that alcohol detection with stable sensitivity is possible even on test strips that have been stored for a longer period of time by the time of alcohol detection.

[0017] The temperature condition for holding the carrier after impregnating it with a reagent that changes color in reaction with alcohol and drying it is 25°C to 40°C, preferably 27°C to 35°C, and more preferably 28°C to 32°C. Holding temperatures lower than 25°C are not preferred because they may take too long for the test strip's sensitivity to alcohol to stabilize or may result in a test strip whose sensitivity to alcohol is not sufficiently stabilized. Holding temperatures higher than 40°C are also not preferred because they may result in the reagent that changes color in response to alcohol being inactivated more than necessary, preventing the sensitivity required for detecting alcohol at concentrations of 0.01% to 0.64%.

[0018] The time for which the carrier is maintained at a temperature of 25°C to 40°C is not particularly limited, as long as it is 24 hours or more after the carrier is impregnated with a reagent that changes color in response to alcohol and dried, but is preferably in the range of 24 hours to 240 hours, more preferably in the range of 24 hours to 200 hours, and even more preferably in the range of 24 hours to 150 hours. If the retention time is less than 24 hours, the reagent that changes color in response to alcohol will not be sufficiently deactivated, and there will be room for further deactivation, which is undesirable as it may result in a change in the sensitivity of alcohol detection over time. The test specimen is preferably stored under dry conditions, for example, by storing the test specimen together with a desiccant, and is preferably stored under light-shielding conditions, for example, by sealing the test specimen in an aluminum bag.

[0019] There are no limitations on the manner of holding the dried carrier under the temperature conditions of 25° C. to 40° C., as long as the dried carrier is held under the same conditions. For example, the dried carrier may be held alone, or a test piece formed by combining the dried carrier with other components such as a substrate may be held.

[0020] There are no particular limitations on the drying method, as long as the solvent described below is sufficiently removed from the carrier. Examples of drying methods include reduced-pressure drying using a vacuum pump and freeze-drying. To prevent inactivation of the reagent, which changes color in response to alcohol, drying is preferably performed at a temperature below room temperature. The temperature range for drying is preferably 1°C to 40°C, more preferably 4°C to 30°C, and even more preferably 4°C to 25°C. The less solvent remaining after drying, the better. For example, when water is used as the solvent, a typical amount that can be achieved by drying under reduced pressure overnight at 25° C. is sufficient.

[0021] In the test strip of the present invention, the alcohol detection sensitivity after being impregnated into a carrier with a reagent that changes color in response to alcohol under temperature conditions of 25°C to 40°C, drying, and then storing for 24 hours or more is generally thought to be approximately 30% to 90% of the sensitivity before storage.

[0022] The reagent used in the present invention that changes color in response to alcohol is not particularly limited as long as it changes color when the alcohol concentration exceeds a certain level. In this specification, color change includes coloring and fading. The reagent used in the present invention that changes color in response to alcohol may be a reagent based on the principles of the inventions described in, for example, Patent Documents 1 to 6.

[0023] In a preferred embodiment of the present invention, the reagent that changes color in response to alcohol contains alcohol oxidase, and in a more preferred embodiment, it contains alcohol oxidase, peroxidase, a color change agent, and a reducing agent, and even more preferably it contains alcohol oxidase, peroxidase, a color change agent, a reducing agent, and a stabilizing agent. In a preferred embodiment of the present invention, the alcohol oxidase is classified as EC 1.1.3.13, for example, derived from yeast. In a preferred embodiment of the present invention, the peroxidase is classified as EC 1.11.1.7, such as that derived from horseradish.

[0024] In a preferred embodiment of the present invention, the color changer may be used alone or in combination of two or more. Examples of a single color changer include 3,3'-diaminobenzidine or a salt thereof, 3,3',5,5'-tetramethylbenzidine or a salt thereof, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) or a salt thereof. Examples of a combination of two or more color changers include a combination of 4-aminoantipyrine or 3-methyl-2-benzothiazolinone hydrazone with a phenolic compound or an aniline derivative, and a preferred combination is 4-aminoantipyrine with TOOS (Sodium 3-(N-ethyl-3-methylanilino)-2-hydroxypropanesulfonate).

[0025] 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 sample, such as from the autoxidation of alcohol oxidase. Examples of the reducing agent include ascorbic acid, reduced glutathione, and cysteine, with ascorbic acid being preferred.

[0026] In a preferred embodiment of the present invention, the stabilizer is, for example, a polysaccharide, an oligosaccharide, a disaccharide, a monosaccharide, or a derivative thereof, preferably a non-reducing sugar alcohol, cyclodextrin, trehalose, or sucrose, and most preferably sucrose.

[0027] The range of alcohol concentrations that can be detected by the test strip of the present invention can be adjusted, for example, by varying the number of components or carriers in the reagent. The alcohol concentration detected by the test strip of the present invention is in the range of 0.01 to 0.64%, and preferably in the range of 0.02 to 0.4%. In this specification, "%" indicating the alcohol concentration means volume % (vol%). By setting the alcohol concentration to be detected within this range, the test strip can be used for a specific purpose, for example, to crack down on drunk driving by drivers.

[0028] In a preferred embodiment of the present invention, the reagent that changes color in response to alcohol is impregnated into the carrier in the form of a solution combined with a solvent, and the 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 in reaction with alcohol and can be removed by drying. Typically, water is used as the solvent.

[0029] In a preferred embodiment of the present invention, when preparing an aqueous solution of a reagent that changes color in response to alcohol using water as a solvent, the pH range of the aqueous solution is not particularly limited. From the viewpoint of stabilizing the reagent that changes color in response to alcohol, particularly from the viewpoint of stably obtaining a good color-developing reaction due to the action of the enzyme, in the case of a reaction method using alcohol oxidase, the pH of the aqueous solution is preferably in the range of pH 7.0 to 9.0, more preferably in the range of pH 7.0 to 8.5, and even more preferably in the range of pH 7.5 to 8.5. To stabilize the pH of the aqueous solution, a buffer solution containing water as a solvent and a buffering agent may be used. Examples of such buffer solutions include acetate buffer, phosphate buffer, citrate buffer, carbonate buffer, borate buffer, Tris (trishydroxymethylaminomethane) buffer, and various Good's buffer solutions. Among these, phosphate buffer and Tris (trishydroxymethylaminomethane) buffer, which have a buffer range of neutral to weakly alkaline where the enzyme is active, as well as Good's buffer solutions such as 3-morpholinopropanesulfonic acid (MOPS) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) are more preferred.

[0030] In a preferred embodiment of the present invention, the concentration of alcohol oxidase in the solution impregnated into the carrier is in the range of 0.1 U / mL to 100 U / mL, more preferably 0.5 U / mL to 50 U / mL, even more preferably 4 to 30 U / mL, and particularly preferably 4 to 20 U / mL. This concentration range is preferred because a test strip kept at 25°C to 40°C for 24 hours or more after drying exhibits appropriate sensitivity for detecting alcohol at a concentration of 0.01% to 0.64%, which is desirable.

[0031] In a preferred embodiment of the present invention, the concentration of peroxidase in the solution impregnated into the carrier is in the range of 0.1 U / mL to 1000 U / mL, more preferably in the range of 1 U / mL to 500 U / mL, and even more preferably in the range of 10 U / mL to 100 U / mL, taking into consideration the concentration of alcohol to be detected.

[0032] In a preferred embodiment of the present invention, the concentration of the discolorant in the solution impregnated into the carrier is in the range of 0.01 mmol / L to 100 mmol / L, more preferably in the range of 0.1 mmol / L to 10 mmol / L, and even more preferably in the range of 0.5 mmol / L to 5 mmol / L, taking into consideration the concentration of the alcohol to be detected.

[0033] In a preferred embodiment of the present invention, the concentration of the reducing agent in the solution to be impregnated into the carrier is in the range of 0 mmol / L to 100 mmol / L, more preferably in the range of 0.1 mmol / L to 10 mmol / L, and even more preferably in the range of 0.5 mmol / L to 5 mmol / L, taking into consideration the concentration of the alcohol to be detected.

[0034] In a preferred embodiment of the present invention, the concentration of the stabilizer in the solution to be impregnated into the carrier is in the range of 0% to 30%, more preferably in the range of 1% to 20%, and even more preferably in the range of 5% to 15%. In this specification, "%" indicating the concentration of the stabilizer means mass volume % (g / 100 mL).

[0035] The specimen to be used in the test strip of the present invention may contain any substance other than alcohol, but is preferably alcohol in an aqueous solution or alcohol in saliva. The alcohol sample to be used in the test strip of the present invention is not particularly limited as long as it undergoes a color change due to the reagent of the present invention, but is preferably a primary alcohol, more preferably methanol or ethanol, and most preferably ethanol.

[0036] The carrier contained in the test strip of the present invention is not limited as long as it is insoluble or poorly soluble in water, saliva, and alcohol at room temperature. For example, natural low-molecular-weight to high-molecular-weight materials such as gelatin, agar, cyclodextrin, cellulose, paper, chitin, and glucan may be used, as well as semi-synthetic and synthetic polymers such as rayon, vinylon, polyester, polyvinyl alcohol, nylon, and acrylic. From the viewpoint of water absorption and water retention, polyvinyl alcohol is preferred. There are no particular limitations on the size, thickness, shape, etc. of the carrier.

[0037] The test strip 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, etc. The shape of the substrate is also not particularly limited, and may be rod-like, circular, polygonal, etc.

[0038] The test strip of the present invention can contain two or more carriers, each with a different concentration required for color change. By combining a plurality of test strips of this type, the alcohol concentration can be measured and detected from two perspectives: the number of carriers that have changed color and the color intensity of each carrier, as shown in Figure 3. This is preferable because it allows the alcohol concentration to be measured without relying on the subjective judgment of the person measuring. When the test strip contains multiple carriers, the number of carriers is preferably 2 to 10, more preferably 2 to 5, even more preferably 2 to 4, and particularly preferably 2 to 3, from the viewpoints of the measurable alcohol concentration range and ease of manufacturing the test strip and carrier.

[0039] The present invention also relates to a test strip for use in detecting alcohol at a concentration of 0.01% to 0.64%, which includes a carrier impregnated with a reagent that changes color in response to alcohol and is dried, and which has been kept at a temperature of 25°C to 40°C for at least 24 hours after drying. The use of such a test strip is preferable because it provides good sensitivity that is stable over time for detecting alcohol at concentrations of 0.01% to 0.64%. When the test strips are further stored as described above, they are preferably stored under low temperature conditions to prevent inactivation of the reagent, which discolors in response to alcohol. However, if the test strips are stored at temperatures below 40°C, they can be stored for at least 60 days without deterioration in performance. The test specimen is preferably stored under dry conditions, for example, by storing the test specimen together with a desiccant, and is also preferably stored under light-shielding conditions, for example, by sealing the test specimen in an aluminum bag.

[0040] Furthermore, the present invention relates to a method for detecting alcohol using the above-mentioned test strip. The method for detecting alcohol using the test strip according to the present invention is not particularly limited as long as the sample is applied to a carrier, and examples of the method for applying the sample to the carrier include a method in which the sample is dropped onto a carrier and a method in which the test strip is immersed in the sample.

[0041] The alcohol detection method using the test strip of the present invention may be performed by measuring the alcohol concentration based on the number and / or color intensity of discolored carriers. For example, as shown in FIG. 4, the alcohol concentration may be measured by assigning a score to the number and / or color intensity of discolored carriers. Scoring may be performed based on the number of discolored carriers. For example, a score of 0 is assigned if 0 carriers on the test strip are discolored, a score of 1 is assigned if 1 carrier is discolored, and a score of 2 is assigned if 2 carriers are discolored, so that the score is either monotonically decreasing or monotonically increasing. Furthermore, scoring may be performed based on the color intensity of each carrier, so that a score of 0 is assigned if no discoloration occurs, a score of 1 is assigned if a slight discoloration occurs, and a score of 2 is assigned if a slight discoloration occurs. Scoring based on the color intensity of each carrier may be performed in any number of stages, but is preferably performed in two to four stages, preferably three stages, from the viewpoint of ease of determining the color intensity. Furthermore, scoring may be performed based on a combination of both the number of carriers and the color intensity of each carrier.

[0042] One embodiment of the method for producing a test strip of the present invention includes the steps of impregnating a carrier with a reagent that changes color in response to alcohol, drying the carrier, and maintaining the carrier at a temperature of 25° C. to 40° C. for 24 hours or more after drying. Preferred embodiments of the method for producing such a test strip are the same as those described above for the test strip.

[0043] The test strip of the present invention may be combined with a color sample showing the correspondence between the alcohol concentration and the color change of the carrier to form a detection kit. A colorimetric method that can be easily performed on-site involves preparing an evaluation sample in which multiple color samples corresponding to alcohol concentrations are assigned scores and arranged, and comparing the color change caused by the sample to the corresponding color on the color sample, thereby enabling detection of a predetermined concentration of alcohol with higher accuracy. The evaluation sample may be a photograph or a printed matter. [Example]

[0044] The present invention will be described in more detail below with reference to examples, which illustrate specific examples of the present invention and are not intended to limit the present invention.

[0045] 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, solidified by drying under reduced pressure at 25°C overnight, sealed in an aluminum bag with a desiccant, and stored at 30°C for 5 days to form the invention product. Reagent 2 was impregnated into a polyvinyl alcohol carrier, solidified by drying under reduced pressure at 25°C overnight to form the control product.

[0046] [Table 1]

[0047] [Table 2]

[0048] (Evaluation 1: Evaluation of test specimens before long-term storage) 0.08% ethanol was applied to the carriers of the invention product and the control product, and the appearance was observed after 5 minutes (number of samples: 2 for each). As shown in Figure 5, both (A) the invention product and (B) the control product showed a good color reaction.

[0049] (Evaluation 2-1: Evaluation 1 of test specimens after long-term storage) The invention product and the control product were sealed in aluminum bags with a desiccant and stored at 25°C for 62 days. 0.08% ethanol was applied to the carrier of each test piece, and the appearance was observed after 5 minutes (two samples per sample). As shown in Figure 6, (A) the invention product showed a color reaction with the same sensitivity as before long-term storage. On the other hand, (B) the control product showed a decrease in sensitivity compared to before long-term storage, and only showed a very weak color reaction.

[0050] (Evaluation 2-2: Evaluation 2 of test specimens after long-term storage) The invention product and the control product were sealed in aluminum bags with a desiccant and stored at 37°C for 62 days. 0.08% ethanol was applied to the carrier of each test piece, and the appearance was observed after 5 minutes (two samples per sample). As shown in Figure 7, (A) the invention product showed a color reaction with the same sensitivity as before long-term storage. On the other hand, (B) the control product showed a drastic decrease in sensitivity compared to before long-term storage, and the color reaction was barely detectable.

[0051] From the above results, it was found that the sensitivity to alcohol concentrations of 0.01% to 0.64% was stabilized by keeping the test piece under temperature conditions of 25°C to 40°C for 24 hours or more after drying.

Claims

1. A method for producing a test strip for detecting 0.01 to 0.64% alcohol, comprising: a step of impregnating a carrier with a reagent that changes color in response to alcohol, the reagent containing 4 U / mL to 30 U / mL of alcohol oxidase; a step of drying the mixture; and a step of keeping the carrier at a temperature of 25°C to 40°C for 24 hours or more after drying.

2. The method described in claim 1, which includes a step of holding the carrier under temperature conditions of 25°C to 40°C for 24 hours to 240 hours after drying.

3. A method according to claim 1 or 2, wherein the test strip is for detecting the alcohol concentration in saliva.

4. A method according to any one of claims 1 to 3, wherein the test piece comprises two or more carriers, each of which has a different concentration of alcohol required for discoloration.

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