Sensor for detecting ATP-related substance and use thereof

Abstract

The present invention provides an ATP-related substance detection sensor as well as a use thereof, the ATP-related substance detection sensor comprising, on the same support: at least one of a reaction section 1 in which a combination of enzymes capable of converting ATP, ADP, and AMP to xanthine is held on a solid-phase carrier and a reaction section 2 in which a combination of enzymes capable of converting inosinic acid to xanthine is held on a solid-phase carrier; and a reaction section 3 in which a combination of enzymes capable of converting inosine to xanthine is held on a solid-phase carrier. The combination of enzymes in each of the reaction sections includes xanthine oxidase. The present invention allows easy and rapid detection of an ATP-related substance at places including food production sites, for evaluation of the freshness of perishable food such as seafood and meat. Furthermore, the present invention allows, not only the measurement of the K value and the Ki value, but also the measurement of the total concentration of ATP, ADP and AMP, as well as inosinic acid concentration.

Description

Sensor for detecting ATP-related substances and use thereof

[0001] The present invention relates to a sensor for detecting an ATP-related substance, a method for detecting an ATP-related substance using the sensor, and a kit and system for detecting an ATP-related substance that include the sensor.

[0002] The freshness of fresh seafood is generally determined using the K value and Ki value, which are the ratio of the amount of ATP and its degradation products, as indicators. High-performance liquid chromatography (Japan Agricultural Standards (JAS) 0023:2022) is a known method for measuring the K value and Ki value. In this method, a diluted solution of perchloric acid is added to a sample requiring freshness testing to inactivate endogenous enzymes that degrade ATP-related substances and extract the ATP-related substances, which are then measured by high-performance liquid chromatography (HPLC). This method is not suitable for simple and rapid on-site testing due to the complicated procedures of extraction with perchloric acid and subsequent neutralization, the need for an HPLC analyzer, and the measurement time of approximately one hour. Freshness sensors and flow injection analysis (FIA) devices using immobilized enzyme electrodes or reactors have also been developed (e.g., Patent Document 1, etc.), but they suffer from similar issues as the high-performance liquid chromatography method.

[0003] As a relatively simple method for measuring K and Ki values, Patent Document 2 discloses a method for measuring the freshness of seafood, which comprises reacting a system containing a sample solution for measuring freshness, a phosphate-free buffer solution, and a color-developing reagent that develops color by oxidation with a plurality of enzymes that convert ATP and the like into nucleosides, and measuring the degree of color development in the solution after each reaction. This method requires multiple enzyme reactions to be carried out consecutively, making the operation complicated. In addition to this, there are also measurement methods using optical analysis devices such as cuvettes, spectrophotometers, and microplate readers (for example, NovoCIB's PRECICE Ｒ Nucleotides Assay Kits (Non-Patent Document 1, etc.) are also known, but have problems such as complicated operations and expensive analytical equipment.

[0004] JP 8-205891 Patent No. 2879351

[0005] NovoCIB, PRECICER Nucleotides Assay Kit (IMP, Ino, hypoxanthine)-User Manual-v. 161001, “PRECICER Nucleotides Assay Kit, For measurement of three major ATP breakdown products (IMP, inosine and hypoxanthine) in seafood. Ref. K0700-003-22 (for microplate reader)

[0006] An object of the present invention is to provide a means for enabling simple and rapid detection of ATP-related substances.

[0007] The inventors have discovered that by using a sensor having at least three reaction sites on a solid support, in which a combination of enzymes that use ATP-related substances as substrates is held, it is possible to easily and quickly detect ATP-related substances and evaluate their concentrations.

[0008] The present disclosure provides the following: Item 1. A sensor for detecting an ATP-related substance, comprising: reaction unit 1, which holds on a solid support a combination of enzymes capable of converting ATP, ADP, and AMP to xanthine; reaction unit 2, which holds on a solid support a combination of enzymes capable of converting inosinic acid to xanthine; and reaction unit 3, which holds on a solid support a combination of enzymes capable of converting inosine to xanthine, wherein the combination of enzymes in each reaction unit includes xanthine oxidase. Item 1A. The sensor according to Item 1, wherein reaction unit 2 does not hold a combination of enzymes capable of converting ATP, ADP, AMP, and adenosine to inosinic acid, or a combination of enzymes capable of converting ATP, ADP, and AMP to inosinic acid, and reaction unit 3 does not hold a combination of enzymes capable of converting ATP, ADP, AMP, adenosine, and inosinic acid to inosine. Item 2. Item 1 or 1A, further comprising a reaction unit 4 having xanthine oxidase supported on a solid support. Item 2A. The sensor according to Item 2, wherein reaction unit 4 does not support a combination of ATP, ADP, AMP, adenosine, inosinic acid, and enzymes capable of converting inosine to hypoxanthine. Item 3. The sensor according to any one of Items 1, 1A, 2, and 2A, wherein each reaction unit further supports peroxidase on the solid support. Item 4. The sensor according to any one of Items 1, 1A, 2, 2A, and 3, wherein reaction unit 1 supports alkaline phosphatase, adenosine deaminase, purine nucleoside phosphorylase, and xanthine oxidase on the solid support. Item 4A. The sensor according to any one of Items 4, wherein reaction unit 1 does not support any enzymes that use ATP-related substances as substrates other than alkaline phosphatase, adenosine deaminase, purine nucleoside phosphorylase, and xanthine oxidase. Item 5. The sensor according to any one of Items 1, 1A, 2, 2A, 3, 4, and 4A, wherein reaction unit 2 retains alkaline phosphatase, purine nucleoside phosphorylase, and xanthine oxidase on a solid support.Item 5A. The sensor according to Item 5, wherein reaction unit 2 does not retain any enzymes that use ATP-related substances as substrates other than alkaline phosphatase, purine nucleoside phosphorylase, and xanthine oxidase. Item 6. The sensor according to any one of Items 1, 1A, 2, 2A, 3, 4, 4A, 5, and 5A, wherein reaction unit 3 retains purine nucleoside phosphorylase and xanthine oxidase on a solid support. Item 6A. The sensor according to Item 6, wherein reaction unit 3 does not retain any enzymes that use ATP-related substances as substrates other than purine nucleoside phosphorylase and xanthine oxidase. Item 7. A method for detecting an ATP-related substance, comprising the steps of: (A) contacting an ATP-related substance detection sensor defined in any one of Items 1, 1A, 2, 2A, 3, 4, 4A, 5, 5A, 6, and 6A with a sample liquid; and (B) detecting a signal derived from hydrogen peroxide in each reaction unit of the sensor. Item 8. Item 7. The method of Item 7, wherein in step (A), the sensor is brought into contact with the sample liquid by immersing it in the sample liquid. Item 9. The method of Item 7 or 8, wherein in step (B), the signal is detected while the sensor is immersed in the sample liquid. Item 10. The method of any one of Items 7 to 9, wherein the pH of the sample liquid is 8 to 10. Item 11. The method of any one of Items 7 to 10, wherein the sample liquid is prepared by immersing a test sample in a buffer solution having a pH of 8 to 10. Item 12. The method of any one of Items 7 to 11, wherein each reaction zone of the sensor further carries peroxidase on a solid phase carrier, and the sample liquid contains a compound that develops color or emits light in the presence of peroxidase and hydrogen peroxide. Item 13. The method of any one of Items 7 to 12, further comprising the step of (C) evaluating the concentration of an ATP-related substance contained in the sample liquid from the intensity of the signal detected in each reaction zone. Item 14. From the intensities of the signals detected in Reaction Zone 1 and Reaction Zone 3, the following formula 1 is calculated: Item 15. The method according to Item 13, wherein the K value is calculated from the intensities of the signals detected in reaction zones 2 and 3 by the following equation 2: Item 16. The method of any one of Items 13 to 15, wherein the concentration of an ATP-related substance contained in the sample liquid is evaluated from the intensity of a signal detected within 2 to 5 minutes after contacting the sensor with the sample liquid. Item 17. The method of any one of Items 13 to 15, wherein the concentration of an ATP-related substance contained in the sample liquid is evaluated from the intensity of a signal detected more than 5 minutes after contacting the sensor with the sample liquid. Item 18. The method of any one of Items 13 to 17, wherein in step (A), each of a plurality of sensors is contacted with each of a plurality of sample liquids having different dilution ratios, in step (B), a signal derived from hydrogen peroxide is detected in each reaction zone of the plurality of sensors, and in step (C), a sensor that detects a signal that becomes weaker as the reaction zone number increases is selected from the plurality of sensors, and the concentration of an ATP-related substance contained in the sample liquid is evaluated from the intensity of the signal detected in each reaction zone of the sensor. Item 19. Item 20. The method according to any one of Items 13 to 17, wherein the total concentration of ATP, ADP, and AMP in the sample solution is evaluated from the difference in the intensity of the signal detected in Reaction Unit 1 and Reaction Unit 2. Item 21. A kit for detecting an ATP-related substance, comprising one or more sensors for detecting an ATP-related substance defined in any one of Items 1, 1A, 2, 2A, 3, 4, 4A, 5, 5A, 6, and 6A. Item 22. A kit for detecting an ATP-related substance, comprising a sensor for detecting an ATP-related substance defined in any one of Items 1, 1A, 2, 2A, 3, 4, 4A, 5, 5A, 6, and 6A, and a reaction vessel for containing the sensor and the sample solution. Item 23. Item 23. A kit according to Item 22, wherein the reaction vessel has a portion made of a visible light-transmitting material for externally observing the reaction portion of the sensor. Item 24. A system for detecting an ATP-related substance, comprising the ATP-related substance detection sensor defined in any one of Items 1, 1A, 2, 2A, 3, 4, 4A, 5, 5A, 6, and 6A, an imaging means, and an image processing means.Item 25. The system according to Item 24, further comprising a sensor and a reaction vessel for containing a sample liquid. Item 26. The kit according to any one of Items 21 to 23, for use in the method according to any one of Items 7 to 20. Item 27. The system according to Item 24 or 25, for use in the method according to any one of Items 7 to 20.

[0009] According to the present invention, ATP-related substances can be detected simply and quickly even at food manufacturing sites, and the freshness of fresh foods such as seafood (fish, mollusks, shellfish, crustaceans, etc.), meat, etc. can be evaluated. Furthermore, in addition to measuring the K value and Ki value, the total concentration of ATP, ADP, and AMP, and the concentration of inosinic acid can also be measured.

[0010] 1 is a diagram showing the decomposition process of ATP-related substances in fresh foods such as seafood. FIG. 2 is a diagram explaining the scheme for decomposition and detection of ATP-related substances in the present disclosure using examples of usable enzymes (ALP, ADA, PNP, and XOD). FIG. 3 is a diagram showing an example of a sensor for detecting ATP-related substances. FIG. 4 is an image of a sensor for detecting ATP-related substances after being immersed in a standard solution of IMP, HxR, and Hx and then removed. FIG. 5 is an image (top row) of a sensor for detecting ATP-related substances after being immersed in a mixed standard solution and then removed, and a graph (bottom row) of Ki values ​​calculated from signal intensity. FIG. 6 is an image (top row) of a sensor for detecting ATP-related substances after being immersed in a mixed standard solution and then removed, and a graph (bottom row) of Ki values ​​calculated from signal intensity. FIG. 7 is an image (top row) of a sensor for detecting ATP-related substances after being immersed in a mixed standard solution and then removed, and a graph (bottom row) showing the time changes in K and Ki values ​​calculated from signal intensity. This is a scatter plot of the K value (top row) and Ki value (bottom row) calculated from the signal intensity of an ATP-related substance detection sensor immersed in a mixed standard solution and then removed at 1, 2, 3, 5, and 10 minutes after the start of the reaction, plotted against the theoretical values. This shows the cartridge (top left) used to detect color development with the ATP-related substance detection sensor immersed in the mixed standard solution, an image of the ATP-related substance detection sensor taken while immersed in the mixed standard solution (bottom row), and a graph (top right) showing the time change in the K value calculated from the signal intensity. This is an image of the ATP-related substance detection sensor taken while immersed in a fish meat (sea bream) extract. This is an image corresponding to the image in Figure 10, showing the G component (1 to 8 minutes after the start of the reaction). The numbers above the reaction area represent the absorbance (signal intensity) of the R component. This is an image corresponding to the image in Figure 10, showing the G component (9 to 15 minutes after the start of the reaction). The numbers above the reaction area represent the absorbance (signal intensity) of the R component. 11A and 11B. This is a graph of the K value and Ki value calculated from the signal intensities of FIGS. 11A and 11B. This is an image of the ATP-related substance detection sensor taken while immersed in a fish meat (yellowtail) extract. This is an image showing the G component (1 to 8 minutes after the start of the reaction) corresponding to the image of FIG. 13. The numbers above the reaction area represent the absorbance (signal intensity) of the R component. This is an image showing the G component (9 to 15 minutes after the start of the reaction) corresponding to the image of FIG. 13. The numbers above the reaction area represent the absorbance (signal intensity) of the R component.14A and 14B. This is a graph of the K value and Ki value calculated from the signal intensity of FIGS. 14A and 14B. This is an image of an ATP-related substance detection sensor photographed while immersed in a fish meat (greenling) extract. This is a scatter plot in which the K value and Ki value by HPLC are plotted against the K value (upper row) and Ki value (lower row) calculated from the signal intensity of an ATP-related substance detection sensor photographed while immersed in a fish meat (greenling) extract. This is an image of an ATP-related substance detection sensor photographed after dripping of a fish meat (greenling) extract. This is a scatter plot in which the K value and Ki value by HPLC are plotted against the K value (upper row) and Ki value (lower row) calculated from the signal intensity of an ATP-related substance detection sensor photographed after dripping of a fish meat (greenling) extract. This is a scatter plot in which the Ki value by HPLC is plotted against the Ki value calculated from the signal intensity of an ATP-related substance detection sensor photographed after dripping of a fish meat (coho salmon) extract. 20 to 22. This is a scatter diagram in which the Ki values ​​calculated by HPLC are plotted against the Ki values ​​calculated from the signal intensity of the ATP-related substance detection sensor photographed after dripping of a fish meat (greenling) extract. This is a scatter diagram in which the Ki values ​​calculated by HPLC are plotted against the Ki values ​​calculated from the signal intensity of the ATP-related substance detection sensor photographed after dripping of a fish meat (yellowtail) extract. This is a scatter diagram in which the Ki values ​​of all the fish meat shown in FIGS. 20 to 22 are plotted together. This is an image of the ATP-related substance detection sensor after immersion in an extract prepared by swabbing the surface of fish meat (salmon) and then removed. This is an image of the ATP-related substance detection sensor after immersion in a mixed standard solution containing varying concentrations of color-forming compounds and then removed. This is a graph of Ki values ​​calculated from the signal intensity of the ATP-related substance detection sensor after immersion in a mixed standard solution containing varying concentrations of color-forming compounds and then removed.

[0011] The following description may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments or specific examples. In this specification, numerical ranges expressed using "to" or "-" mean ranges that include the numerical values ​​at both ends as upper and lower limits, unless otherwise specified. The upper and lower limits of each numerical range shown in this specification can be combined in any way.

[0012] [Definition] In the present disclosure, ATP-related substances refer to adenosine triphosphate (ATP) and its decomposition products, adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine (AdR), inosinic acid (IMP), inosine (HxR), hypoxanthine (Hx), and xanthine (X). In the muscles of seafood and other animals, ATP is sequentially decomposed into ADP, AMP, IMP, HxR, Hx, and xanthine over a period of several hours to several days after death. It is known that in this series of ATP decomposition processes, the decomposition of IMP to HxR is rate-limiting (see FIG. 1).

[0013] The K value and Ki value are indicators of ATP decomposition that take this into account, and are calculated by the following formula. In both equations, the numerator is the sum of HxR and Hx, which are the degradation products after the rate-limiting step has been reached. High K and Ki values ​​indicate that ATP decomposition has progressed to the point where the rate-limiting step has been reached, i.e., freshness has decreased.

[0014] [Sensor for detecting ATP-related substances] In one aspect, the present disclosure provides a sensor for detecting ATP-related substances (hereinafter also simply referred to as sensor) having, on the same support, at least one of reaction section 1 carrying on a solid phase carrier a combination of enzymes capable of converting ATP, ADP, and AMP to xanthine and reaction section 2 carrying on a solid phase carrier a combination of enzymes capable of converting IMP to xanthine, and reaction section 3 carrying on a solid phase carrier a combination of enzymes capable of converting HxR to xanthine, wherein the combination of enzymes in each reaction section includes xanthine oxidase (XOD).

[0015] The sensor of this embodiment can also be described as a sensor having, on the same support, at least one of reaction section 1 in which a combination of enzymes capable of converting ATP, ADP, and AMP to Hx and XOD are supported on a solid support, and reaction section 2 in which a combination of enzymes capable of converting IMP to Hx and XOD are supported on a solid support, and reaction section 3 in which an enzyme capable of converting HxR to Hx and XOD are supported on a solid support.

[0016] The sensor of this embodiment can also be described as a sensor having, on the same support, at least one of reaction unit 1, which carries on a solid phase carrier a combination of enzymes capable of converting ATP, ADP, and AMP to uric acid and hydrogen peroxide, and reaction unit 2, which carries on a solid phase carrier a combination of enzymes capable of converting IMP to uric acid and hydrogen peroxide, and reaction unit 3, which carries on a solid phase carrier a combination of enzymes capable of converting HxR to uric acid and hydrogen peroxide.

[0017] The sensor has a configuration comprising at least two reaction regions (reaction region 1 and / or reaction region 2, and reaction region 3) on a support. That is, the sensor includes at least reaction region 1 and reaction region 3, or at least reaction region 2 and reaction region 3, and may include all of reaction regions 1 to 3. The reaction region is a solid-phase carrier on which one or more enzymes that use an ATP-related substance as a substrate are supported. As will be described in detail later, when the sensor is brought into contact with a sample solution containing an ATP-related substance, an enzymatic reaction occurs between the enzyme supported on the solid-phase carrier in the reaction region and the ATP-related substance in the sample, generating hydrogen peroxide.

[0018] The "combination of enzymes capable of converting ATP, ADP, and AMP to xanthine" in Reaction Unit 1 refers to multiple enzymes that, when combined, can decompose ATP to xanthine, ADP to xanthine, and AMP to xanthine. The combination of enzymes in Reaction Unit 1 includes XOD.

[0019] An example of an enzyme combination in Reaction Unit 1 is alkaline phosphatase (ALP), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and XOD. ALP can hydrolyze the phosphate ester bonds of ATP, ADP, and AMP to convert them to AdR, and can also hydrolyze the phosphate ester bond of IMP to convert it to HxR. ADA can deaminate AdR to convert it to HxR. PNP can remove ribose from HxR to convert it to Hx. XOD can oxidize Hx to convert it to xanthine and hydrogen peroxide, and further oxidize xanthine to convert it to uric acid and hydrogen peroxide. Therefore, if Reaction Unit 1 contains this enzyme combination, all ATP-related substances can be converted to xanthine, and xanthine can be further converted to uric acid, producing hydrogen peroxide in the process (see Figure 2).

[0020] Another example of an enzyme combination in Reaction Unit 1 is five enzymes: apyrase, 5'-nucleotidase, ADA, PNP, and XOD. Apyrase can hydrolyze the phosphate ester bond of ATP and ADP to convert them to AMP. 5'-nucleotidase can hydrolyze the phosphate ester bond of AMP to convert it to AdR, and can hydrolyze the phosphate ester bond of IMP to convert it to HxR. ADA can deaminate AdR to convert it to HxR. PNP can remove ribose from HxR to convert it to Hx. XOD can oxidize Hx to convert it to xanthine and hydrogen peroxide, and further oxidize xanthine to convert it to uric acid and hydrogen peroxide. Therefore, even if Reaction Unit 1 contains this enzyme combination, all ATP-related substances can be converted to xanthine, and xanthine can be further converted to uric acid, producing hydrogen peroxide in the process.

[0021] In one embodiment, reaction unit 1 does not contain any enzymes that use ATP-related substances as substrates other than the combination of enzymes that can convert ATP, ADP, and AMP to xanthine. In one embodiment, reaction unit 1 does not contain any enzymes other than the combination of enzymes that can convert ATP, ADP, and AMP to xanthine and peroxidase.

[0022] The "enzyme combination capable of converting IMP to xanthine" in reaction section 2 refers to a plurality of enzymes that, when combined, can degrade IMP to xanthine. The enzyme combination in reaction section 2 includes XOD.

[0023] Reaction unit 2 is configured so that none of ATP, ADP, AMP, and AdR can be converted to HxR, and none of ATP, ADP, and AMP can be converted to IMP. Reaction unit 2 does not contain a combination of enzymes that can convert ATP, ADP, AMP, and AdR to HxR, and does not contain a combination of enzymes that can convert ATP, ADP, and AMP to IMP.

[0024] An example of a combination of enzymes in the reaction section 2 is three types of enzymes: ALP, PNP, and XOD. As described above, ALP converts IMP to HxR, PNP converts HxR to Hx, and XOD converts Hx to xanthine and hydrogen peroxide, and further converts xanthine to uric acid and hydrogen peroxide. Therefore, with this enzyme combination, IMP, HxR, and Hx can be converted into xanthine, and then xanthine can be converted into uric acid, producing hydrogen peroxide in the process.

[0025] Another example of a combination of enzymes in reaction section 2 is three enzymes: 5'-nucleotidase, PNP, and XOD. As described above, 5'-nucleotidase converts IMP to HxR, PNP converts HxR to Hx, and XOD converts Hx to xanthine and hydrogen peroxide, and then converts xanthine to uric acid and hydrogen peroxide. Therefore, this enzyme combination also converts IMP, HxR, and Hx to xanthine, and then converts xanthine to uric acid, producing hydrogen peroxide in the process.

[0026] In one embodiment, reaction unit 2 does not contain any enzymes that use ATP-related substances as substrates other than the combination of enzymes that can convert IMP to xanthine. In one embodiment, reaction unit 2 does not contain any enzymes other than the combination of enzymes that can convert IMP to xanthine and peroxidase.

[0027] The "enzyme combination capable of converting HxR to xanthine" in reaction section 3 refers to a plurality of enzymes that, when combined, can decompose HxR into xanthine. The enzyme combination in reaction section 3 includes XOD.

[0028] Reaction unit 3 is configured so that none of ATP, ADP, AMP, AdR, and IMP can be converted to HxR. Reaction unit 3 does not contain a combination of enzymes that can convert ATP, ADP, AMP, AdR, and IMP to HxR.

[0029] An example of a combination of enzymes in the reaction section 3 is two types of enzymes, PNP and XOD. As described above, PNP converts HxR to Hx, and XOD converts Hx to xanthine and hydrogen peroxide, and further converts xanthine to uric acid and hydrogen peroxide. Therefore, with this enzyme combination, HxR and Hx can be converted to xanthine, and then xanthine can be converted to uric acid, producing hydrogen peroxide in the process.

[0030] In one embodiment, reaction unit 3 contains no enzymes that use ATP-related substances as substrates other than the combination of enzymes that can convert HxR to xanthine. In another embodiment, reaction unit 3 contains no enzymes other than the combination of enzymes that can convert HxR to xanthine and peroxidase.

[0031] The combination of enzymes in reaction unit 1 may be any combination capable of converting ATP, ADP, and AMP to xanthine and containing XOD, the combination of enzymes held in reaction unit 2 may be any combination capable of converting IMP to xanthine and containing XOD, and the combination of enzymes held in reaction unit 3 may be any combination capable of converting HxR to xanthine and containing XOD. The combinations of enzymes in reaction units 1 to 3 are not limited to the combinations exemplified above.

[0032] In a preferred embodiment, the enzyme combination in reaction section 1 is ALP, ADA, PNP, and XOD. In a preferred embodiment, the enzyme combination in reaction section 2 is ALP, PNP, and XOD. In a preferred embodiment, the enzyme combination in reaction section 3 is PNP and XOD.

[0033] The sensor may further comprise a reaction section 4 on the same support, in which XOD is supported on a solid phase carrier. As described above, XOD can convert Hx into xanthine and hydrogen peroxide, and further convert xanthine into uric acid and hydrogen peroxide.

[0034] Reaction unit 4 is configured so that none of ATP, ADP, AMP, AdR, IMP, and HxR can be converted to Hx. Reaction unit 4 does not contain a combination of enzymes that can convert ATP, ADP, AMP, AdR, IMP, and HxR to Hx.

[0035] In one embodiment, the reaction section 4 does not contain any enzymes other than XOD that use ATP-related substances as substrates. In another embodiment, the reaction section 4 does not contain any enzymes other than XOD and peroxidase.

[0036] The enzymes supported on the solid support in the reaction zone may be of any origin or production method, as long as they have the required activity. For example, ALP may be derived from calf intestine or Escherichia coli, ADA may be derived from calf intestine, bovine spleen, or Escherichia coli, PNP may be derived from Escherichia coli, XOD may be derived from bovine milk, and apyrase may be derived from potato. Each of the enzymes exemplified here can exert its activity in weakly alkaline conditions.

[0037] In addition to the reaction zone, the sensor may further have a control zone on the same support. The control zone is a solid phase carrier that does not carry the enzyme.

[0038] Each reaction zone may further have peroxidase supported on the solid phase carrier. In this embodiment, the sensor may further have a reaction zone 5 supporting peroxidase on the same support. Examples of peroxidase include horseradish peroxidase (HRP), formyl peroxidase, cytochrome C peroxidase, glutathione peroxidase, and lipid peroxidase. One preferred example of peroxidase is HRP.

[0039] The reaction zone 5 does not retain any enzymes that use ATP-related substances as substrates. In one embodiment, the reaction zone 5 retains no enzymes other than peroxidase.

[0040] The solid phase carrier that holds the enzyme in the reaction zone may be any carrier capable of holding the enzyme at least while the enzymatic reaction is proceeding when the sensor is immersed in the sample solution. Examples of such solid phase carriers include thin plates or sheets made of natural polymeric materials such as cellulose, chitosan, collagen, and alginic acid, or synthetic polymeric materials such as polypropylene, polyester, polystyrene, nitrocellulose, nylon, polyethersulfone, PVDF, glass fiber, polyacrylamide, and acrylic copolymers. Preferred examples of solid phase carriers include chromatography paper, filter paper, and absorbent pads.

[0041] The size and shape of the reaction zone are not limited as long as the signal derived from hydrogen peroxide generated after contact with the sample liquid can be detected. For example, when signal detection is performed visually, the reaction zone may be large enough to visually recognize the signal. The shape of the reaction zone may be circular, triangular, rectangular, or polygonal with higher polygonal shapes, or may be irregular. Furthermore, the shapes of the reaction zones within a single sensor may be the same or different from each other. The spacing between the solid phase carriers is sufficient as long as the distance is sufficient to prevent the signal derived from hydrogen peroxide from being affected by contamination due to liquid mixing between the reaction zones. For example, for a circle, the spacing should be equal to or greater than the radius of the reaction zone, and for a triangle or rectangle, the spacing should be equal to or greater than half the length of the longer side. However, if the material of the reaction zone has a high water absorption capacity to retain the aqueous solution, ensuring even a small spacing between the reaction zones reduces the possibility of liquid mixing and contamination between the reaction zones, so this is not necessarily the case.

[0042] The enzyme can be retained on the solid phase carrier by a method commonly used for immobilizing enzymes, such as a method of impregnating a solid phase carrier with an aqueous solution or aqueous suspension containing the enzyme and then drying it, a method of gelling an aqueous solution containing the enzyme and a gellable substance (alginic acid, acrylamide, etc.) on a support to form a solid phase carrier, or a method of immobilizing the enzyme by covalent bonding, ionic bonding, or adsorption with a modified functional group on the solid phase carrier.

[0043] The concentration of the enzyme retained on the solid phase carrier can be adjusted appropriately depending on the concentration and ratio of the ATP-related substance in the sample solution. For example, when 6.5 μl of an aqueous solution or aqueous suspension containing the enzyme is impregnated into a solid phase carrier having a length of 4 mm, a width of 4 mm, and a thickness of 0.7 mm, the ALP concentration in the aqueous solution or suspension may be 200 to 600 U / ml, preferably 300 to 500 U / ml, the ADA concentration may be 60 to 180 U / ml, preferably 90 to 150 U / ml, the PNP concentration may be 60 to 180 U / ml, preferably 90 to 150 U / ml, the XOD concentration may be 0.16 to 0.48 U / ml, preferably 0.24 to 0.40 U / ml, and the HRP concentration may be 0.8 to 2.4 U / ml, preferably 1.2 to 2.0 U / ml.

[0044] The concentrations of enzymes held on the solid phase carriers in each reaction zone within a single sensor are preferably constant for each type of enzyme. For example, in the case of a single sensor in which ALP, ADA, PNP, and XOD are held on the solid phase carrier in reaction zone 1, ALP, PNP, and XOD are held on the solid phase carrier in reaction zone 2, PNP and XOD are held on the solid phase carrier in reaction zone 3, and XOD is held on the solid phase carrier in reaction zone 4, the ALP concentrations in reaction zones 1 and 2 are preferably the same, the PNP concentrations in reaction zones 1 to 3 are preferably the same, and the XOD concentrations in reaction zones 1 to 4 are preferably the same. In this example of the sensor, the concentration ratio of the enzymes retained on the solid phase carrier can be, in terms of units per unit volume, ALP:ADA:PNP:XOD=600-2000:180-600:180-600:1, preferably 900-1600:250-500:250-500:1.

[0045] Because the combination of enzymes in reaction unit 1 uses all ATP-related substances as substrates, after the sensor is brought into contact with the sample liquid and the reaction has progressed sufficiently, of the signals derived from hydrogen peroxide detected in each reaction unit, the signal detected in reaction unit 1 will have the greatest intensity. Therefore, it is desirable to set the concentration of each enzyme retained on the solid phase carrier in the reaction unit so that the concentration of the hydrogen peroxide-derived signal detected in reaction unit 1 falls within the dynamic range of the signal detection means, preferably at or near the upper limit of the dynamic range.

[0046] Furthermore, even if the total concentration of ATP-related substances is the same in a sample solution, the signal intensity detected in reaction unit 2 and subsequent units may vary depending on the concentration of each ATP-related substance, but the signal intensity detected in reaction unit 1 will be the same. Therefore, the signal intensity detected in reaction unit 1 can be used as a signal detection control and can also be used to control the accuracy of the sensor. For example, when one type of sample solution is contacted with multiple identical sensors, if the signal intensity detected in reaction unit 1 of one sensor is weaker than the signal intensity detected in reaction unit 1 of the other sensors, it can be determined that some abnormality has occurred in that sensor. Specifically, an abnormality may be a deterioration in the performance of the sensor due to deterioration of the enzyme activity supported on the sensor or deterioration of the solid phase carrier. Furthermore, because sensors are disposable, it is necessary to ensure that the desired reaction proceeds and appropriate signal intensity is obtained in each reaction unit. However, reaction unit 1 also reflects differences between sensors, slight differences in the time of immersion in the sample solution during measurement, differences in the concentration of the sample solution, etc. Therefore, it is useful for correcting differences between measurements made using manual methods and for confirming that measurements were made correctly using the sensor.

[0047] There are no limitations on the size, shape, and material of the support constituting the sensor, nor on the arrangement of the reaction sites on the support, and these can be appropriately determined by a person skilled in the art, taking into consideration factors such as operability and ease of detection of ATP-related substances. Examples of materials for the support include glass, plastic, metal, wood, etc. Furthermore, it is preferable that the reaction sites are arranged in a row in descending or ascending order on the same surface of the support.

[0048] In one embodiment, the sensor has a configuration in which at least two reaction regions (reaction region 1 and / or reaction region 2, and reaction region 3) are provided on one surface of a rectangular thin plate support from one end to the other in the longitudinal direction. In the sensor illustrated in Figure 3, reaction regions 1 to 5 are immobilized and aligned in this order from one end to the other of the support, and a gripping portion is provided at the other end that allows the sensor to be held with fingers, tweezers, or the like.

[0049] [Method for detecting ATP-related substances] The present disclosure also provides a method for detecting ATP-related substances, comprising the steps of (A) contacting a sensor for detecting ATP-related substances with a sample liquid, and (B) detecting a signal caused by hydrogen peroxide in each reaction site of the sensor.

[0050] The sample solution is an aqueous solution or aqueous suspension in which detection of an ATP-related substance is desired. The pH of the sample solution can be adjusted appropriately within a range in which the enzyme used in the sensor can exhibit its activity. The pH of the sample solution can be weakly alkaline, for example, 7 to 10, preferably 7.5 to 10, and more preferably 8 to 9. Each of the enzymes specifically exemplified in the sensor section above can exhibit their activity in weakly alkaline conditions.

[0051] In certain embodiments, the specimen solution is an extract of a test sample, and examples of the test sample include fresh seafood (fish, mollusks, shellfish, crustaceans, etc.) and fresh meat and other fresh foods containing animal muscle tissue. The extract of a test sample can be prepared by immersing the test sample in an appropriate aqueous medium. For example, the extract can be prepared by immersing a finely chopped test sample in an aqueous medium, stirring or homogenizing it, and then recovering the supernatant or filtering the sample to recover the filtrate. Alternatively, the extract can be prepared by swabbing or scraping the surface of the test sample with an appropriate tool (e.g., a plastic rod, glass rod, plastic swab, glass swab, wooden swab, metal swab, nylon swab, cotton swab, polyester swab, etc.), and then immersing the tool in an appropriate aqueous medium.

[0052] As shown in Figure 1, fresh foods are preferably those in which ATP is decomposed via IMP, such as fish, mollusks (squids and octopuses), arthropods (shrimps and crabs), and echinoderms (sea urchin gonads).

[0053] The aqueous medium used for extraction may be a strongly acidic aqueous medium such as a diluted perchloric acid solution. Extraction using a diluted perchloric acid solution can be performed as described in Japanese Agricultural Standards (JAS) 0023:2022. In the case of an extract using a strongly acidic aqueous medium, the pH is adjusted before use as a sample solution so that the enzyme used in the sensor can be activated.

[0054] The aqueous medium used for extraction may be a buffer solution prepared by dissolving a buffering agent in water. The pH of the buffer solution is preferably weakly alkaline, for example, 7 to 10, preferably 7.5 to 10, and more preferably 8 to 9. Examples of the buffer solution include Tris, HEPES, and MOPS. One preferred example of the buffer solution is HEPES.

[0055] In a preferred embodiment, the specimen solution is an extract prepared by immersing a test sample in a buffer solution having a weak alkaline pH. The test sample may contain endogenous enzymes that use ATP-related substances as substrates. Since the optimal pH for these endogenous enzymes is between 5 and 8, adjusting the pH of the specimen solution to a weak alkaline range, preferably between 7.5 and 10, and more preferably between 8 and 9, allows the activity of the enzymes on the sensor to be exerted while suppressing the activity of the endogenous enzymes. Furthermore, using a weak alkaline buffer solution during extraction eliminates the need for pH adjustment, which is required when using a strongly acidic aqueous medium, thereby simplifying the procedure.

[0056] When each reaction portion of the sensor holds peroxidase on a solid phase carrier, the sample liquid may contain a compound that serves as a substrate for the peroxidase.

[0057] In step (A), the method for contacting the sensor with the sample liquid is not limited as long as it allows all reaction sites on the sensor to be in contact with the sample liquid, and may be, for example, by dropping, spraying, or applying the sample liquid to each reaction site of the sensor, or by immersing all reaction sites of the sensor in the sample liquid. When the sensor has a control site, it is preferable to contact the control site as well as the reaction site with the sample liquid.

[0058] Upon contact with the sample solution, an enzymatic reaction using ATP-related substances in the sample solution as substrates is initiated in each reaction site of the sensor, producing hydrogen peroxide. In step (B), a signal derived from the produced hydrogen peroxide is detected.

[0059] A signal derived from hydrogen peroxide can be generated and detected by methods known to those skilled in the art, such as an optical detection method that uses a compound that develops color or emits light in the presence of hydrogen peroxide and detects the intensity of the color or luminescence of the compound as a signal corresponding to the amount of hydrogen peroxide, or an electrochemical detection method that uses an enzyme electrode to which peroxidase is immobilized and detects the change in current corresponding to the amount of hydrogen peroxide as a signal.

[0060] The compound utilized in the optical detection method may be a compound that is oxidized by peroxidase in the presence of hydrogen peroxide to produce color or light. Examples of such compounds include Amplite ＴＭ Examples of suitable dyes include Red (10-acetyl-3,7-dihydroxyphenoxazine), 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), 2,2'-azinodi(3-ethylbenzothiazoline-6-sulfonic acid, ABTS), diaminobenzidine (DAB), cytochrome C, and luminol. ＴＭ Red is oxidized by peroxidase in the presence of hydrogen peroxide to resorufin (7-hydroxy-2H-phenoxazin-3-one), which can be detected both as fluorescent light (excitation wavelength 530-570 nm, emission wavelength 580-590 nm) and as a reddish-purple color.

[0061] The compound utilized in the optical detection method may also be a compound that reacts with hydrogen peroxide to produce color or light, such as a Ti-porphyrin reagent.

[0062] In optical detection methods that utilize a compound that develops color or light when oxidized by peroxidase in the presence of hydrogen peroxide, a signal derived from hydrogen peroxide can be generated by contacting a sensor with a sample solution and then contacting the sensor with peroxidase and its substrate compound. However, for rapid signal detection, it is preferable to retain either peroxidase or the substrate compound in each reaction zone of the sensor and mix the other compound with the sample solution in advance. This allows the signal derived from hydrogen peroxide to be detected immediately after contacting the sensor with the sample solution.

[0063] In a preferred embodiment, each reaction portion of the sensor holds peroxidase on a solid phase carrier, and the sample solution contains a compound that serves as a substrate for peroxidase. In this embodiment, the sample solution is preferably an extract prepared by immersing a test sample in a weakly alkaline buffer solution containing the compound that serves as a substrate for peroxidase. The compound that serves as a substrate for peroxidase may be added to the buffer solution in which the test sample is immersed, or may be added to the extract solution after immersion.

[0064] The signal derived from hydrogen peroxide can be detected by a detection means appropriate for the type of signal. When the signal is color development or luminescence in the visible light region, the signal can be detected by visual observation or by optical detection means such as a spectrophotometer or an imaging device.

[0065] In one embodiment, the signal derived from hydrogen peroxide is detected by acquiring an image of each reaction site using an imaging device. The signal intensity can be expressed as a brightness value obtained by setting an appropriate region of interest within the image of each reaction site and converting the RGB values ​​of each pixel within the region into grayscale. The signal intensity can also be expressed as the value of the RGB component that is closest to the color or light emission of the signal. For example, if the signal is reddish-purple in color, the signal intensity can be obtained as the value of the R component of each pixel included in the region of interest.

[0066] Furthermore, the signal intensity may be represented by the absorbance of the RGB component that is closest to the color of the signal. The absorbance can be obtained using the acquired image of each reaction site as follows: First, an image of the color component closest to the color of the signal (e.g., the G component if the signal is reddish-purple) that is the opposite color to the color of the signal is extracted from the acquired image of each reaction site. An appropriate region of interest is set within the extracted image, and the brightness value of each pixel within the region is obtained. Next, the absorbance can be calculated by applying the acquired brightness value to the following equation: By using absorbance as the signal intensity, the influence of ambient light during imaging can be reduced.

[0067] In step (A), when the sensor is brought into contact with the sample liquid by immersing the entire reaction area of ​​the sensor in the sample liquid, the detection of the signal may be performed after the immersed sensor is removed from the sample liquid, or may be performed while the sensor is still immersed in the sample liquid.

[0068] In the present disclosure, the method for detecting an ATP-related substance may further include a step (C) of evaluating the concentration of the ATP-related substance contained in the sample solution from the intensity of the signal detected in each reaction portion. In step (C), the concentration of the ATP-related substance is evaluated based on the intensity of the signal detected in each reaction portion.

[0069] In one embodiment, step (C) is carried out by calculating a signal intensity detected in reaction zone 1 and reaction zone 3 using the following formula 1: In one embodiment, step (C) is a step of calculating the K value from the intensities of the signals detected in reaction zones 2 and 3 using the following formula 2: This is the step of calculating the Ki value by the following formula.

[0070] The combination of enzymes in reaction unit 1 can ultimately convert all of the ATP-related substances into uric acid and hydrogen peroxide. Therefore, the intensity of the signal detected in reaction unit 1 corresponds to the total concentration of the ATP-related substances contained in the sample solution, and can be used as the denominator when calculating the K value.

[0071] The combination of enzymes in reaction unit 2 can ultimately convert IMP, HxR, and Hx into uric acid and hydrogen peroxide, but cannot convert ATP, ADP, AMP, and AdR. Therefore, the intensity of the signal detected in reaction unit 2 corresponds to the total concentration of IMP, HxR, and Hx contained in the sample solution, and can be used as the denominator when calculating the Ki value.

[0072] The combination of enzymes in reaction unit 3 can ultimately convert HxR and Hx into uric acid and hydrogen peroxide, but cannot convert ATP, ADP, AMP, AdR, and IMP. Therefore, the intensity of the signal detected in reaction unit 3 corresponds to the total concentration of HxR and Hx contained in the sample solution, and can be used as the numerator when calculating the K value and Ki value.

[0073] In one embodiment, step (C) is a step of evaluating the total concentration of ATP, ADP, and AMP in the sample solution from the difference in the intensity of the signals detected in reaction unit 1 and reaction unit 2. The value obtained by subtracting the signal intensity detected in reaction unit 2 from the signal intensity detected in reaction unit 1 corresponds to the total concentration of ATP, ADP, and AMP in the sample solution. Therefore, this value can be used to evaluate the freshness of even a test sample that is relatively fresh and in which decomposition of ATP to IMP has not progressed.

[0074] In one embodiment, step (C) is a step of evaluating the concentration of IMP in the sample solution from the difference in the intensities of the signals detected in reaction zone 2 and reaction zone 3. The value obtained by subtracting the signal intensity detected in reaction zone 3 from the signal intensity detected in reaction zone 2 corresponds to the concentration of IMP in the sample solution. Therefore, this value can be used to evaluate the concentration of IMP in the test sample, and it is also possible to evaluate, for example, umami taste.

[0075] In the method for detecting an ATP-related substance of the present disclosure, the signal intensity measured in the control section can be used as a background value. Furthermore, in the method for detecting an ATP-related substance of the present disclosure, the signal detected in the reaction section 5 is considered to be an endogenous signal brought in from the sample, such as that derived from hydrogen peroxide contained in the sample solution. Therefore, the signal intensity detected in the reaction section 5 can also be used as a background value for calculating the K value and Ki value.

[0076] For example, the signal intensity detected in the control zone or reaction zone 5 can be used as a background value by subtracting it from the signal intensity detected in each of reaction zones 1 to 4. Furthermore, when the signal intensity is expressed as absorbance, the signal intensity detected in the control zone or reaction zone 5 can also be used as the "background value" in the above-mentioned formula for calculating absorbance.

[0077] One embodiment of the method for detecting ATP-related substances disclosed herein is a method in which, in step (A), each of multiple sensors is contacted with each of multiple sample liquids having different dilution ratios, in step (B), a signal derived from hydrogen peroxide is detected in each reaction zone of the multiple sensors, and in step (C), a sensor is selected from the multiple sensors that detects a weaker signal as the reaction zone number increases, and the concentration of the ATP-related substance contained in the sample liquid is evaluated from the intensity of the signal detected in each reaction zone of the sensor.

[0078] In this embodiment, multiple sensors with the same enzyme concentration in the reaction zone are contacted with multiple sample solutions with different dilution ratios, and the sensor whose detected signal intensity decreases with increasing reaction zone number is selected and used as the evaluation target. A sensor whose detected signal intensity decreases with increasing reaction zone number is a sensor whose detected signal intensity has a gradient, where reaction zone 2 has a weaker signal intensity than reaction zone 1, and reaction zone 3 has a weaker signal intensity than reaction zone 2. This signal intensity gradient is observed when the sensor is contacted with a sample solution containing an ATP-related substance in an amount that is neither too high nor too low relative to the enzyme concentration in the reaction zone of the sensor, i.e., when the sensor is contacted with a sample solution whose dilution ratio falls within the dynamic range of the sensor. Therefore, by selecting and using as the evaluation target a sensor whose detected signal intensity decreases with increasing reaction zone number from multiple sensors contacted with sample solutions with different dilution ratios, the concentration of an ATP-related substance can be accurately evaluated even for extract solutions with unknown extraction efficiency and unknown ATP-related substance concentrations.

[0079] Another embodiment of the method for detecting ATP-related substances disclosed herein is a method in which, in step (A), each of a plurality of sensors in which the concentration of each enzyme in the reaction zone is gradually changed is brought into contact with the same sample liquid, in step (B), a signal derived from hydrogen peroxide is detected in each reaction zone of the plurality of sensors, and in step (C), a sensor is selected from the plurality of sensors that detects a signal that becomes weaker as the reaction zone number increases, and the concentration of the ATP-related substance contained in the sample liquid is evaluated from the intensity of the signal detected in each reaction zone of the sensor.

[0080] This embodiment differs from the aforementioned embodiment using multiple sample solutions with different dilution ratios in that it uses multiple sensors with gradually changing enzyme concentrations in the reaction zone for one type of sample solution. By using multiple sensors with gradually changing enzyme concentrations in the reaction zone, it is possible to select and evaluate a sensor that retains an amount of enzyme that is neither too high nor too low relative to the concentration of ATP-related substances contained in the sample solution. This makes it possible to accurately evaluate the concentration of ATP-related substances even for extract solutions with unknown extraction efficiency and unknown concentrations of ATP-related substances.

[0081] In the method for detecting an ATP-related substance of the present disclosure, step (C) may be performed by visually comparing the intensity of color or luminescence of each reaction zone. If color or luminescence is observed only in the reaction zone with a lower number, it can be determined that ATP decomposition of the test sample has not progressed much and that the test sample is of good freshness. If color or luminescence of similar intensity is observed in the reaction zone with a higher number, it can be determined that ATP decomposition of the test sample has progressed and that the freshness has decreased.

[0082] Specifically, for example, if there is a large difference in color or luminescence intensity between reaction zone 2 and reaction zone 3, it can be determined that ATP decomposition in the test sample has progressed to IMP but not to HxR. On the other hand, if there is no large difference in color or luminescence intensity between reaction zone 1 and reaction zone 3, it can be determined that ATP decomposition in the test sample has progressed at least to HxR.

[0083] In the method for detecting an ATP-related substance disclosed herein, the time from contacting the sensor with the sample solution to detecting the signal is not limited, except for the time immediately after contacting the sensor with the sample solution. To accurately evaluate the concentration of an ATP-related substance, it is desirable to detect the signal after the enzyme reaction in each reaction zone has progressed sufficiently and the production of hydrogen peroxide has stabilized. In this case, signal detection can be performed, for example, after more than 5 minutes, preferably more than 10 minutes, more preferably more than 15 minutes, and even more preferably more than 20 minutes have elapsed since contacting the sensor with the sample solution. In the above-mentioned embodiments for evaluating the total concentration of ATP, ADP, and AMP in the sample solution, the embodiment for evaluating the concentration of IMP in the sample solution, the embodiment in which multiple sensors with the same enzyme concentration are contacted with multiple sample solutions with different dilution ratios, and the embodiment in which multiple sensors with gradually changing enzyme concentrations are contacted with the same sample solution, it is preferable to use the signal intensity detected during this time period for evaluation.

[0084] Furthermore, the enzymatic reaction in each reaction zone tends to have a fast initial reaction rate and detect a stronger signal intensity. Therefore, to evaluate the concentration of an ATP-related substance with high sensitivity, it is desirable to detect the signal at the beginning of the enzymatic reaction. In this case, the signal can be detected, for example, within 2 to 10 minutes, preferably within 2 to 5 minutes, and more preferably within 2 to 3 minutes, after contacting the sensor with the sample solution. The above-described embodiment for calculating the K value and the embodiment for calculating the Ki value perform evaluation using the signal intensity detected during this time period, allowing even subtle differences in the K value and Ki value to be detected with high sensitivity.

[0085] It has been reported that ATP is decomposed via AdR in some fresh foods, and extracts of such fresh foods contain AdR. In the sensor of the present disclosure, the "combination of enzymes capable of converting ATP, ADP, and AMP to xanthine" in reaction unit 1 is the four types of enzymes exemplified above (ALP, ADA, PNP, and XOD) or the five types (apyrase, 5'-nucleotidase, ADA, PNP, and XOD), so that AdR contained in the sample solution can be detected in reaction unit 1 along with ATP, ADP, and AMP. This allows the total concentrations of ATP, ADP, AMP, and AdR in the sample solution to be evaluated from the difference in signal intensity detected in reaction unit 1 and reaction unit 2, even for sample solutions containing AdR. Furthermore, the degree of ATP decomposition can be evaluated based on the values ​​calculated using the above-described formulas 1 and 2.

[0086] [ATP-related substance detection kit, ATP-related substance detection system] The present disclosure also provides an ATP-related substance detection kit including one or more ATP-related substance detection sensors.

[0087] The present disclosure further provides a kit for detecting an ATP-related substance, which kit includes one or more sensors for detecting an ATP-related substance and a reaction vessel for containing the sensors and a sample liquid. As described above, when multiple sensors having the same enzyme concentration are to be brought into contact with multiple sample liquids having different dilution ratios, each sample liquid can be placed in a compartment of a reaction vessel having multiple compartments, and the sensor can be brought into contact with the sample liquid in that compartment.

[0088] The reaction vessel is preferably configured so that the reaction site of the sensor is visible from outside the vessel. Furthermore, the entire reaction vessel, or a portion that corresponds to the reaction site when the sensor is placed in the compartment, is preferably made of a visible light-transmitting material, thereby allowing the reaction site of the sensor to be observed from outside. Examples of visible light-transmitting materials include glass, polycarbonate resin, polyethylene terephthalate resin, polyacrylic resin, and polyvinyl chloride resin.

[0089] The present disclosure also provides a system for detecting ATP-related substances, including an ATP-related substance detection sensor, an imaging means, and an image processing means. The imaging means may be a general imaging device, such as a CCD camera or a CMOS camera. The image processing means may be a computer capable of calculating the RGB values ​​or luminance values ​​of each pixel included in the acquired image. The computer typically includes an input unit that inputs instructions and information to the computer, an image input unit that inputs image data from the imaging device, a recording unit, a calculation unit that performs processing to calculate the RGB values ​​or luminance values ​​of each pixel from the captured image data, an output unit that outputs images and calculation results, and a control unit that controls the operation of each of the above units.

[0090] In the system, the imaging means and image processing means may be a mobile device equipped with an imaging device and a computer, such as a smartphone, a tablet terminal, etc. The system may further include a recording medium on which a program for image processing is recorded in a computer-readable manner.

[0091] These kits and systems can be suitably used in the above-mentioned method for detecting ATP-related substances.

[0092] These kits and systems may include additional reagents for generating and detecting signals (e.g., buffers or buffering agents for extracting the test sample, compounds that develop color or light in the presence of peroxidase and hydrogen peroxide, etc.), instruments for collecting the test sample (e.g., sticks, swabs, etc. for swabbing or scraping the surface of the test sample), and may include manuals for using these kits and systems in the above-mentioned methods for detecting ATP-related substances.

[0093] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these. In the examples, unless otherwise specified, sample solutions were prepared and stored on ice, and other operations were carried out at room temperature.

[0094] Example 1 Preparation of Sensor for Detecting ATP-Related Substances As shown in Table 1, up to five types of enzymes were added to 1 M Tris-HCl, pH 8.5, and mixed to prepare enzyme solutions 1 to 5. Alkaline phosphatase (ALP, derived from calf intestine, optimal pH 8.0-10.0), adenosine deaminase (ADA, derived from calf intestine, optimal pH 7.0-7.4), purine nucleoside phosphorylase (PNP, derived from Escherichia coli, optimal pH 7.5), and xanthine oxidase (XOD, derived from bovine milk, optimal pH 8.5-9.0) were used to decompose ATP-related substances, and horseradish peroxidase (HRP, optimal pH 7.0) was used to detect hydrogen peroxide. The optimal concentrations of the enzymes in the enzyme solution were determined in advance as follows: ALP 400 U / ml, ADA 120 U / ml, PNP 120 U / ml, XOD 0.32 U / ml, and HRP 1.6 U / ml.

[0095] Six absorbent filter papers (4 mm wide x 4 mm long x 0.7 mm thick, ADVANTEC, No. 26-WA) made of cellulose fiber (TAPPI (Technical Association of the Pulp and Paper Industry) T205) were attached to an acrylic support (4 mm wide x 80 mm long) with approximately 1 mm spacing using a double-sided adhesive sheet. Enzyme solutions 1 to 5 and 1 M Tris-HCl (pH 8.5) were added in 6.5 μL each to each filter paper strip, and the strips were placed in an aluminum pouch containing a silica gel bag and stored at 4°C for two days to prepare sensors for detecting ATP-related substances, which were used in the following examples.

[0096] Example 2: Measurement of ATP-related substances using standard solutions (1) Detection of IMP, HxR, and Hx Amplite ＴＭ Red(Amplex ＴＭ One vial of powder from Red Xanthine Assay Kit (Cosmo Bio Co., Ltd. ABD) was diluted with 100 μL of DMSO to prepare Amplify. ＴＭThis stock solution was diluted 100-fold with 1 M Tris-HCl, pH 8.5, and IMP, HxR, or Hx was dissolved in the diluted solution to prepare three types of standard solutions (200 μM IMP standard solution, 200 μM HxR standard solution, and 200 μM Hx standard solution). ＴＭ Red is normally colorless in solution, but in the presence of hydrogen peroxide, it is reduced to resorufin by HRP, which turns reddish purple. ＴＭ By contacting a liquid containing Red (standard solution, sample solution, etc.), the presence of hydrogen peroxide can be detected by color development.

[0097] The enzyme reaction was initiated by immersing the sensor in each of the above standard solutions for 1 to 2 seconds and then immediately removed. The removed sensor was quickly placed horizontally in a white photography box, and the reaction zone was photographed with a digital camera at 1-minute intervals from the start of immersion. Reddish-purple color development was observed in reaction zones 1 and 2 with all three standard solutions, in reaction zone 3 with the HxR standard solution and Hx standard solution, and in reaction zone 4 with the Hx standard solution, while no color development was observed in reaction zone 5 and the control (Figure 4). In the examples, unless otherwise specified, images taken with a digital camera are displayed as monochrome binary or grayscale RGB color images. (2) Measurement of K value and Ki value Amplite ＴＭ The Red stock solution was diluted 100-fold with 1 M Tris-HCl, pH 8.5, and ATP, ADP, AMP, IMP, HxR, and Hx were dissolved in the diluted solution to prepare three types of mixed standard solutions shown in Table 2. In Table 2, the K value and Ki value of each mixed standard solution are theoretical values.

[0098] The sensor was immersed for 1-2 seconds in each of the mixed standard solutions with theoretical Ki values ​​of 33.3 and 57.1, and then immediately removed. The removed sensor was quickly placed horizontally in a white photography box, and the reaction zone was photographed with a digital camera at 5-minute intervals starting 10 minutes after immersion. For the Green channel image extracted from the photographed image, a region of interest was set at a position corresponding to the center of each reaction zone, and the brightness values ​​of the pixels contained within the region of interest were obtained, and the average value was calculated for each reaction zone. The average brightness value of reaction zone 5 was considered the background, and the absorbance of the R component in each reaction zone was calculated using the following formula, which was used as the signal intensity of each reaction zone. Signal intensity of reaction zone n = absorbance of R component in reaction zone n = -100 × log １０ (average brightness value of G component in reaction unit n / average brightness value of G component in reaction unit 5)

[0099] Next, the Ki value (referred to as the actual Ki value) was calculated using the signal intensities of reaction zones 2 and 3 according to the following formula: Ki value (%) = signal intensity of reaction zone 3 / signal intensity of reaction zone 2 × 100

[0100] The photographed images and the measured Ki values ​​are shown in Figure 5. For all mixed standard solutions, the calculated Ki values ​​did not change significantly after 10 minutes from the start of the reaction, and no significant deviation from the theoretical value was observed.

[0101] Next, the same test was performed using all three mixed standard solutions, and the Ki values ​​were calculated. Two sensors were used for each mixed standard solution with theoretical Ki values ​​of 18.1 and 33.3. Photographs of the reaction zone were taken at 5-minute intervals from the start of immersion.

[0102] The photographed images and the measured Ki values ​​are shown in Figure 6. The Ki value graph also shows the coefficient of variation (CV) of the measured Ki values ​​calculated from two sensors immersed in a mixed standard solution with theoretical Ki values ​​of 18.1 and 33.3. Although some variation was observed between the two sensors immersed in the mixed standard solution with theoretical Ki values ​​of 18.1 and 33.3, the overall trend was similar to that of the previous test, with the Ki values ​​not changing significantly after 5 minutes from the start of the reaction, and no significant deviation from the theoretical value was observed.

[0103] (3) High-sensitivity measurement: AmpliteＴＭ The Red stock solution was diluted 100-fold with 1 M Tris-HCl, pH 8.5, and ATP, ADP, AMP, IMP, HxR, and Hx were dissolved in the diluted solution to prepare the four types of mixed standard solutions shown in Table 3. In Table 3, the K value and Ki value of each mixed standard solution are theoretical values.

[0104] Using these four types of mixed standard solutions, a test was performed in the same manner as in (2) above, and the Ki value was calculated. Photographs of the reaction zone were taken at 1-minute intervals from the start of immersion. In addition to the Ki value, the K value (referred to as the actual K value) was calculated using the signal intensities of reaction zones 1 and 3. K value (%) = signal intensity of reaction zone 3 / signal intensity of reaction zone 1 × 100

[0105] Figure 7 shows the captured images and the changes in the measured K and Ki values ​​over time. Figure 8 shows a scatter plot of the measured K and Ki values ​​at 1, 2, 3, 5, and 10 minutes after the start of the reaction against the theoretical values. With the exception of the mixed standard solution with the highest theoretical K value of 0.25, both the K and Ki values ​​reached their highest values ​​2 minutes after the start of the reaction, then gradually decreased and stabilized over time. The slope of the regression line for the K values ​​was 1.93 times larger at 2 minutes and 1.61 times larger at 3 minutes than at 10 minutes after the start of the reaction, and a similar trend was observed for the Ki values. This indicates that detecting color development in reaction times shorter than 10 minutes, especially within 2 to 3 minutes, allows for more sensitive detection of K and Ki values.

[0106] (4) Signal detection in detection solution A sensor was prepared in the same manner as in Example 1, except that the order of the reaction sections was changed. The concentrations of the enzymes in the enzyme solution were also the same as in Example 1: ALP 400 U / ml, ADA 120 U / ml, PNP 120 U / ml, XOD 0.32 U / ml, and HRP 1.6 U / ml.

[0107] Also, Amplite ＴＭ The Red stock solution was diluted 100-fold with 1 M Tris-HCl, pH 8.5, and ATP, IMP, HxR, and Hx were dissolved in the diluted solution to prepare a mixed standard solution (ATP 20 μM, IMP 60 μM, HxR 10 μM, Hx 10 μM).

[0108] The mixed standard solution was dispensed into an acrylic reaction vessel with three compartments (size of each compartment: height 50 mm x width 5 mm x thickness 2 mm), and one sensor was then immersed in each compartment. The sensor was placed in a white photography box while immersed in the mixed standard solution, and the reaction zone was photographed at 1-minute intervals from the start of immersion. The signal intensity of each reaction zone was calculated, and the K value was calculated using the signal intensities of reaction zones 1 and 3.

[0109] The photographed images and the changes in the measured K value over time are shown in Figure 9 and Table 4. Similar to the results observed in (3) above, the K value gradually decreased over time and then stabilized. It was confirmed that analysis was possible even when the sensor was immersed in the liquid, in the same way as when it was removed from the liquid.

[0110] Example 3: Measurement of ATP-related substances using fish meat extract (1) Simultaneous measurement of dilution series using multiple sensors (sea bream fillet) Amplite ＴＭ Finely chopped sea bream fillets were added to the diluted solutions prepared by diluting the Red stock solution 100-fold with 1 M Tris-HCl, pH 8.5, to concentrations of 6.3, 12.5, 25, 50, 100, and 200 mg / mL, respectively, and mixed by vortexing for approximately 10 seconds. The supernatant was then collected and used as the extract. The sensor was immersed in the extract and placed in a white photography box, and images of the reaction zone were taken at 1-minute intervals from the start of immersion. The signal intensity of each reaction zone was calculated, and the K value was calculated using the signal intensities of reaction zones 1 and 3, and the Ki value was calculated using the signal intensities of reaction zones 2 and 3.

[0111] The captured image is shown in Figure 10, the image showing the G component is shown in Figures 11A and 11B, and the measured K and Ki values ​​are shown in Figure 12 and Table 5. Figures 11A and 11B also show the absorbance of the R component within the region of interest (indicated by a circle in the figure) set on the reaction zone. Compared to the extract of fillets at 50 mg / mL or less, the extract of fillets at 100 mg / mL or more showed no significant difference in signal intensity between reaction zones 1 and 2, and the signal intensity saturated after 6 minutes after the start of the reaction (dotted frame in Figure 10). Furthermore, the K and Ki values ​​were similar for the extract of fillets at 50 mg / mL or less, whereas both the K and Ki values ​​were higher for the extract of fillets at 100 mg / mL or more. It was presumed that the K and Ki values ​​could not be accurately calculated due to saturation of signal intensity, resulting in the calculated values ​​being higher than the true values.

[0112] (2) Simultaneous measurement of dilution series using multiple sensors (yellowtail fillets) Amplite ＴＭ Finely chopped yellowtail fillets were added to the diluted solutions prepared by diluting the Red stock solution 100-fold with 1 M Tris-HCl, pH 8.5, to concentrations of 230, 110, 57, 74, 37, and 19 mg / mL, respectively, and the solutions were vortexed and mixed for approximately 10 seconds. The supernatant was then collected and used as the extract. The sensor was immersed in the extract and placed in a white photography box, and images of the reaction zone were taken at 1-minute intervals from the start of immersion. The signal intensity of each reaction zone was calculated, and the K value was calculated using the signal intensities of reaction zones 1 and 3, and the Ki value was calculated using the signal intensities of reaction zones 2 and 3.

[0113] The captured image is shown in Figure 13, the image showing the G component is shown in Figures 14A and 14B, and the measured K and Ki values ​​are shown in Figure 15 and Table 6. Figures 14A and 14B also show the absorbance of the R component within the region of interest (indicated by a circle in the figure) set on the reaction zone. Compared to the extract of fillets at 57 mg / mL or less, the extract of fillets at 74 mg / mL or more showed no significant difference in signal intensity between reaction zones 1 and 2, and the signal intensity saturated after 7 minutes after the start of the reaction (dotted frame in Figure 13). Furthermore, the K and Ki values ​​were similar for the extract of fillets at 57 mg / mL or less, whereas both the K and Ki values ​​were higher for the extract of fillets at 74 mg / mL or more. It was presumed that the K and Ki values ​​could not be accurately calculated due to saturation of signal intensity, resulting in the calculated values ​​being higher than the true values.

[0114] These results show that by serially diluting a sample solution, reacting a sensor with each dilution series, and selecting a sensor with a gradient in signal strength at each reaction site, specifically a sensor with a weaker signal strength as the reaction site number increases, it is possible to evaluate ATP-related substances in a single measurement, even for sample solutions whose concentrations are unknown. This makes it possible to calculate the K value and Ki value in a single measurement, for example, without considering the influence of the extraction efficiency of the sample solution.

[0115] Furthermore, these results showed that unless the signal intensity was saturated, the calculated K and Ki values ​​were constant even if the fish meat concentration was different, and therefore it was possible to calculate the K and Ki values ​​regardless of variations in the amount of fish meat used to prepare the sample solution.

[0116] Example 4. Comparison with HPLC Method (1) Measurement of ATP-Related Substances Using Fat Greenling Extract - Quantification by HPLC Method Quantification of ATP-related substances was performed according to the JAS test method (JAS0023). Six fat greenlings were purchased from a fishmonger, and the central portion of the back meat of the lateral muscle was excised and the dark muscle removed to obtain normal meat. After crushing with a knife, approximately 2 grams were weighed into a disposable test tube and homogenized by adding 30 mL of cold 5% perchloric acid solution. While measuring the pH with test paper, 0.5 N and 0.1 N sodium hydroxide solutions were added to the homogenized solution and mixed to adjust the pH to 2.5 to 3.5. Water was added to bring the total volume to 50 mL, and the solution was cooled on ice for at least 30 minutes. The solution was then filtered through a PTFE membrane filter with a pore size of 0.45 μm to obtain the filtrate. This procedure was performed while the sample was cooled on ice.

[0117] Since the content of ATP-related substances in fish meat varies depending on freshness, a standard addition group containing all ATP-related substances was prepared by adding 1 mL of a standard solution containing each ATP-related substance at a concentration of approximately 800 μM to 9 mL of the above filtrate and mixing. Then, 1 M sodium hydroxide solution was added to adjust the pH to 7. The volume of the added sodium hydroxide solution was recorded so that the proportion of filtrate contained in the solution after adjusting to pH 7 could be calculated. For the DW addition group, the same procedure was performed, except that water was added instead of the standard solution.

[0118] The quantification of each ATP-related substance was carried out using an HPLC equipped with a silica column according to the following method. The solution adjusted to pH 7 and 0.25 mol / L phosphate buffer (pH 7.0) were mixed at a volume ratio of 4:1 to prepare a sample solution. The HPLC conditions were as follows: Column: Capcelpak C18 AQ 5s 4.6 ID x 250 mm; Mobile phase: 0.05 mol / L phosphate buffer (pH 7.0); Flow rate: 1.0 mL / min; Column temperature: 40°C; Measurement wavelength: 260 nm; Injection volume: 10 μL.

[0119] Table 7 shows the results of quantification of ATP-related substances in each sample solution by HPLC, as well as the measured K values ​​and Ki values ​​calculated from the quantified values.

[0120] Quantitative determination using a sensor (immersed in a sample solution) The 12 types of sample solutions were diluted with 1 M Tris-HCl, pH 8.5, so that the total concentration was 200 μM, and the diluted solution was used as a sample solution for the sensor. ＴＭ Red stock solution was added and ATP-related substances were quantified using the sensor. The sensor was immersed in the sample solution and placed in a white photography box, and the reaction zone was photographed every 5 minutes from the start of immersion. The signal intensity of each reaction zone was calculated, and the K value was calculated using the signal intensities of reaction zones 1 and 3, and the Ki value was calculated using the signal intensities of reaction zones 2 and 3.

[0121] The photographed image is shown in Figure 16, the measured K values ​​and Ki values ​​are shown in Table 8, and a scatter diagram plotting the K values ​​and Ki values ​​measured by HPLC against the K values ​​and Ki values ​​measured by the sensor is shown in Figure 17. Five minutes after the start of the reaction, some of the measured K values ​​and Ki values ​​showed variability, and the correlation with the K values ​​and Ki values ​​measured by HPLC was also low. However, 15 and 20 minutes after the start of the reaction, the variability became smaller, and the correlation with the Ki values ​​and K values ​​measured by HPLC was also high. These results demonstrate that by extending the sensor reaction time, it is possible to quantify K values ​​and Ki values ​​at a level equivalent to that of conventional HPLC methods.

[0122] Quantitation by sensor (dropping of sample solution) A sensor was prepared in the same manner as in Example 1, except that the shape of the test paper was changed from a 4 mm x 4 mm square to an 8 mm diameter circle. 3 μL of the above-mentioned sample solution for the sensor was dropped onto each reaction site of the sensor. The sensor was placed in a white photography box, and the reaction sites were photographed at 5-minute intervals from the start of the drop. The signal intensity of each reaction site was calculated, and the K value was calculated using the signal intensities of reaction sites 1 and 3, and the Ki value was calculated using the signal intensities of reaction sites 2 and 3.

[0123] An image of a representative example of a sensor reacted with a sample solution is shown in Figure 18, and a scatter diagram in which the K and Ki values ​​measured by HPLC are plotted against the K and Ki values ​​actually measured by the sensor 20 minutes after the start of the reaction is shown in Figure 19. As with the case where the reaction was carried out by immersion in the sample solution, a high correlation was observed between the Ki and K values ​​measured by HPLC.

[0124] (2) Measurement of ATP-related substances using extracts from multiple fish species. Using the same method as in (1) above, sample solutions for HPLC analysis (standard and DW added) were prepared from the normal flesh of coho salmon, fat greenling, and yellowtail. ATP-related substances were quantified by HPLC, and the Ki value was calculated. Also, using the same method as in (1) above, the sample solution for HPLC analysis was diluted to prepare a sample solution for the sensor. The sample solution was then dropped onto the sensor, and the Ki value was calculated from the signal intensity of each reaction site photographed 5 minutes after the drop.

[0125] Scatter diagrams plotting Ki values ​​by the HPLC method against Ki values ​​by the sensor method are shown in Figure 20 (Coho salmon), Figure 21 (Greenling), and Figure 22 (Yellowtail), and a scatter diagram plotting data for Coho salmon, Greenling, and Yellowtail together is shown in Figure 23. Even in the scatter diagram plotting the three fish species together, there was a high correlation between the Ki values ​​by the sensor and the Ki values ​​by HPLC. These results demonstrated that the sensor method allows quantification similar to that of conventional HPLC methods, regardless of fish species.

[0126] Example 5. Measurement of ATP-related substances using fish meat extract. ＴＭ A fish meat extract was prepared using a buffer solution (1 M Tris-HCl, pH 8.5) containing Red and used as a test solution. ＴＭ A fish meat extract was prepared using a buffer solution containing no red. ＴＭ It was also possible to measure ATP-related substances using a sample solution prepared by mixing the fish meat with a buffer solution containing Red. An example of a method for preparing such a sample solution is as follows. A fish meat measuring several mm square was placed in a 1.5 mL microtube containing 0.5 mL of buffer solution (1 M Tris-HCl, pH 8.5), the tube was capped, and the tube was shaken for 20 to 30 seconds. The liquid portion was collected with a dropper and diluted with Amplite. ＴＭ One drop of the Red stock solution was added to 0.25 mL of a 100-fold diluted solution with a buffer solution (1 M Tris-HCl, pH 8.5), and the mixture was mixed to prepare a sample solution.

[0127] Furthermore, instead of immersing the fish meat in a buffer solution, a rod-shaped instrument was rubbed against the surface of the fish meat several times, and then the instrument was immersed in the buffer solution to prepare a sample solution, which also enabled measurement of ATP-related substances. An example of a method for preparing such a sample solution is as follows: The surface of the fish meat was swabbed several times with a plastic rod (approximately 3 mm in diameter), and then the sample solution was immersed in Amplite ＴＭ A sample solution was prepared by immersing the Red stock solution in a 0.5 mL tube (outer diameter 7.8 mm x length 40 mm x inner diameter 6 mm) containing 0.25 mL of a diluted solution prepared by diluting the Red stock solution 100-fold with a buffer solution (1 M Tris-HCl, pH 8.5). The sensor was immersed in this sample solution for 1-2 seconds and immediately removed. Five minutes after immersion, the reaction zone was photographed with a smartphone's built-in digital camera. The photographed image is shown in Figure 24. In this test, a sensor having three reaction zones, reaction zone 2, reaction zone 3, and reaction zone 5, was prepared in the same manner as in Example 1 and used.

[0128] [Example 6. Quantification of ATP-related substances with different concentrations of color-forming compounds] Amplite prepared in Example 2 (1) ＴＭ The Red stock solution was diluted 100-fold with 1 M Tris-HCl, pH 8.5 to make a 100% solution. The 100% solution was further diluted 7 / 10-fold, 5 / 10-fold, and 3 / 10-fold with 1 M Tris-HCl, pH 8.5 to make a 70%, 50%, and 30% solution. ATP, ADP, AMP, IMP, HxR, and Hx were dissolved in each of the 100%, 70%, 50%, and 30% solutions, and the theoretical Ki value was 20. ＴＭ Four mixed standard solutions with different Red concentrations (ATP 0 μM, ADP 0 μM, AMP 0 μM, IMP 80 μM, HxR 0 μM, Hx 20 μM) were prepared. Using these mixed standard solutions, the sensor was immersed in the sample solution and removed in the same manner as in Example 2(2), and the Ki value was calculated from the signal intensity of each reaction site.

[0129] The image taken after 5 minutes of immersion is shown in Figure 25, and the Ki values ​​calculated based on images taken at 2-minute intervals up to 14 minutes after immersion are shown in Figure 26. The Ki values ​​for the 70% and 100% solutions showed smaller variations than those for the 30% and 50% solutions. ＴＭRed may deteriorate depending on storage conditions, but Amplite, which has the same concentration as a 100% solution, ＴＭ By using Red, Amplite ＴＭ It was shown that stable signal detection was possible even if Red deteriorated to some extent.

[0130] REFERENCE SIGNS LIST 1 ATP-related substance detection sensor 2 Support 3 Holding part 11 Reaction part 1 12 Reaction part 2 13 Reaction part 3 14 Reaction part 4 15 Reaction part 5

Claims

1. A sensor for detecting ATP-related substances, comprising at least one of reaction section 1 in which a combination of enzymes capable of converting ATP, ADP and AMP to xanthine is held on a solid support, reaction section 2 in which a combination of enzymes capable of converting inosinic acid to xanthine is held on a solid support, and reaction section 3 in which a combination of enzymes capable of converting inosine to xanthine is held on a solid support, wherein the combination of enzymes in each reaction section includes xanthine oxidase.

2. The sensor according to claim 1, further comprising a reaction section 4 having xanthine oxidase supported on a solid phase carrier.

3. The sensor according to claim 1, wherein each reaction portion further carries peroxidase on a solid phase carrier.

4. The sensor according to claim 1, wherein reaction part 1 carries alkaline phosphatase, adenosine deaminase, purine nucleoside phosphorylase, and xanthine oxidase on a solid support.

5. The sensor according to claim 1, wherein reaction part 2 carries alkaline phosphatase, purine nucleoside phosphorylase, and xanthine oxidase on a solid support.

6. The sensor according to claim 1, wherein the reaction part 3 carries purine nucleoside phosphorylase and xanthine oxidase on a solid support.

7. A method for detecting an ATP-related substance, comprising: (A) a step of contacting an ATP-related substance detection sensor defined in any one of claims 1 to 6 with a sample liquid; and (B) a step of detecting a signal derived from hydrogen peroxide in each reaction portion of the sensor.

8. The method of claim 7, wherein in step (A), the sensor is contacted with the sample liquid by immersion in the sample liquid.

9. The method according to claim 7, wherein in step (B), the signal is detected while the sensor is immersed in the sample liquid.

10. The method according to claim 7, wherein the pH of the sample solution is 8 to 10.

11. The method according to claim 7, wherein the specimen solution is prepared by immersing the test sample in a buffer solution having a pH of 8 to 10.

12. The method according to claim 7, wherein each reaction portion of the sensor further carries peroxidase on a solid phase carrier, and the sample liquid contains a compound which develops color or emits light in the presence of peroxidase and hydrogen peroxide.

13. The method according to claim 7, further comprising the step of (C) evaluating the concentration of an ATP-related substance contained in the sample liquid from the intensity of the signal detected in each reaction area.

14. From the signal intensities detected in reaction zones 1 and 3, the following formula 1 is calculated. The method of claim 13, wherein the K value is calculated by:

15. From the signal intensities detected in reaction zones 2 and 3, the following formula 2 is calculated. The method of claim 13, wherein the Ki value is calculated by:

16. The method according to claim 13, wherein the concentration of an ATP-related substance contained in the sample liquid is evaluated from the intensity of a signal detected within 2 to 5 minutes after contacting the sensor with the sample liquid.

17. The method according to claim 13, wherein the concentration of an ATP-related substance contained in the sample liquid is evaluated from the intensity of the signal detected more than 5 minutes after contacting the sensor with the sample liquid.

18. The method according to claim 13, wherein in step (A), each of a plurality of sensors is contacted with each of a plurality of sample liquids having different dilution ratios; in step (B), a signal derived from hydrogen peroxide is detected in each reaction site of the plurality of sensors; and in step (C), a sensor is selected from the plurality of sensors that detects a weaker signal as the reaction site number increases, and the concentration of an ATP-related substance contained in the sample liquid is evaluated from the intensity of the signal detected in each reaction site of the sensor.

19. The method according to claim 17, wherein the total concentration of ATP, ADP and AMP in the sample solution is evaluated from the difference in the intensity of the signals detected in reaction zone 1 and reaction zone 2.

20. The method according to claim 17, wherein the concentration of inosinic acid in the sample solution is evaluated from the difference in the intensity of the signals detected in reaction zones 2 and 3.

21. A kit for detecting an ATP-related substance comprising one or more sensors for detecting an ATP-related substance defined in any one of claims 1 to 6.

22. A kit for detecting an ATP-related substance comprising an ATP-related substance detection sensor defined in any one of claims 1 to 6 and a reaction vessel for containing the sensor and a sample liquid.

23. The kit according to claim 22, wherein the reaction vessel has a portion made of a visible light-transmitting material for externally observing the reaction portion of the sensor.

24. A system for detecting an ATP-related substance comprising an ATP-related substance detection sensor defined in any one of claims 1 to 6, an imaging means, and an image processing means.

25. The system of claim 24, further comprising a sensor and a reaction vessel for containing a sample fluid.

Images