Sensor for detecting ATP (adenosine triphosphate) related substances and application of sensor

Abstract

The invention provides a sensor for detecting ATP (adenosine triphosphate) related substances and application of the sensor. The sensor for detecting ATP-related substances has, on the same support, at least one of a reaction unit (1) in which a combination of enzymes capable of converting ATP, ADP, and AMP into xanthine is held on a solid-phase carrier, and a reaction unit (2) in which a combination of enzymes capable of converting inosinic acid into xanthine is held on a solid-phase carrier. And a reaction unit (3) in which a combination of enzymes capable of converting inosine into xanthine is held on a solid-phase carrier, wherein the combination of enzymes in each reaction unit contains xanthine oxidase. According to the present invention, the ATP-related substance can be easily and quickly detected even at the food manufacturing site, and the freshness of fresh food such as aquatic products and meat can be evaluated. In addition, not only K value and Ki value can be measured, but also the total concentration of ATP, ADP, and AMP and the concentration of inosinic acid can be measured.

Description

Technical Field

[0001] This invention relates to a sensor for detecting ATP-related substances, a method for using the sensor to detect ATP-related substances, and a kit and system for detecting ATP-related substances comprising the sensor. Background Technology

[0002] The freshness of fresh aquatic products is usually determined by the ratio of ATP to its decomposition products, i.e., the K value and Ki value. High-performance liquid chromatography (HPLC) is a known method for determining the K and Ki values ​​(Japanese Agricultural and Forestry Standard (JAS) 0023:2022). In this method, perchloric acid dilution is added to the sample to be tested for freshness, inactivating the endogenous enzymes that decompose ATP-related substances, while simultaneously extracting the ATP-related substances, which are then measured by HPLC. This method is unsuitable for simple and rapid on-site testing due to the complexity of the perchloric acid extraction and subsequent neutralization processes, the need for an HPLC analytical apparatus, and the approximately one-hour measurement time. Furthermore, freshness sensors using immobilized enzyme electrodes, reactors, and FIA (flow injection analysis) devices have been developed (e.g., Patent Document 1), but these suffer from the same problems as HPLC.

[0003] As a relatively simple method for determining K and Ki values, Patent Document 2 discloses a method for determining the freshness of aquatic products. Its characteristic is that multiple enzymes that convert ATP and other substances into nucleosides are reacted in a system containing a sample solution for freshness determination, a phosphate-free buffer solution, and a colorimetric reagent that develops color through oxidation. The degree of color development of each reaction solution is then measured. This method requires continuous execution of multiple enzyme reactions, making it complex to operate. In addition, methods using optical analysis devices such as cuvettes, spectrophotometers, and microplate readers (e.g., NovoCIB's PRECICE) are also known. R NucleotidesAssay Kits (non-patent literature 1, etc.) exist, but they have problems such as complicated operation and expensive analytical devices.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent Application Publication No. 8-205891

[0007] Patent Document 2: Japanese Patent No. 2879351

[0008] Non-patent literature

[0009] Non-patent literature 1: NovoCIB, PRECICER Nucleotides Assay Kit (IMP, Ino, Hypoxanthine) - User Manual - v.161001, "PRECICER Nucleotides Assay Kit for detecting three major ATP degradation products (IMP, Inosine, and Hypoxanthine) in seafood. Ref. K0700-003-22 (for microplate readers)" Summary of the Invention

[0010] The problem that the invention aims to solve

[0011] The purpose of this invention is to provide a simple and rapid method for detecting ATP-related substances.

[0012] Methods for solving problems

[0013] The inventors have discovered that by using a sensor having at least three reaction sections having an enzyme combination with ATP-related substances as substrates held on a solid support, it is possible to easily and rapidly detect ATP-related substances and evaluate their concentration.

[0014] This disclosure provides the following content.

[0015] Item 1.

[0016] A sensor for detecting ATP-related substances has, on the same support, at least one of a reaction section 1 containing a combination of enzymes capable of converting ATP, ADP, and AMP into xanthine held on a solid support, a reaction section 2 containing a combination of enzymes capable of converting inosine nucleotide into xanthine held on a solid support, and a reaction section 3 containing a combination of enzymes capable of converting inosine into xanthine held on a solid support, wherein the combination of enzymes in each reaction section includes xanthine oxidase.

[0017] Item 1A.

[0018] According to the sensor described in item 1, the reaction section 2 does not hold a combination of enzymes capable of converting ATP, ADP, AMP and adenosine into inosine, nor a combination of enzymes capable of converting ATP, ADP and AMP into inosine nucleotides, and the reaction section 3 does not hold a combination of enzymes capable of converting ATP, ADP, AMP, adenosine and inosine nucleotides into inosine.

[0019] Item 2.

[0020] The sensor according to item 1 or 1A further comprises a reaction section 4 in which xanthine oxidase is held on a solid support.

[0021] Item 2A.

[0022] According to the sensor described in item 2, the reaction section 4 does not hold a combination of enzymes capable of converting ATP, ADP, AMP, adenosine, inosine acid, and inosine into hypoxanthine.

[0023] Item 3.

[0024] The sensor according to any one of items 1, 1A, 2 and 2A, wherein each reaction section further retains peroxidase on a solid support.

[0025] Item 4.

[0026] According to any one of items 1, 1A, 2, 2A and 3, the sensor wherein the reaction section 1 holds alkaline phosphatase, adenosine deaminase, purine nucleoside phosphorylase and xanthine oxidase on a solid support.

[0027] Item 4A.

[0028] According to any one of claims 4, the sensor in the reaction section 1 does not retain enzymes that use ATP-related substances as substrates, except for alkaline phosphatase, adenosine deaminase, purine nucleoside phosphorylase and xanthine oxidase.

[0029] Item 5.

[0030] The sensor according to any one of items 1, 1A, 2, 2A, 3, 4 and 4A, wherein the reaction section 2 holds alkaline phosphatase, purine nucleoside phosphorylase and xanthine oxidase on a solid support.

[0031] Item 5A.

[0032] According to the sensor described in item 5, the reaction section 2 does not maintain enzymes that use ATP-related substances as substrates, except for alkaline phosphatase, purine nucleoside phosphorylase and xanthine oxidase.

[0033] Item 6.

[0034] The sensor according to any one of items 1, 1A, 2, 2A, 3, 4, 4A, 5 and 5A, wherein the reaction section 3 holds purine nucleoside phosphorylase and xanthine oxidase on a solid support.

[0035] Item 6A.

[0036] According to the sensor described in item 6, the reaction section 3 does not maintain enzymes that use ATP-related substances as substrates, except for purine nucleoside phosphorylase and xanthine oxidase.

[0037] Item 7.

[0038] A method for detecting ATP-related substances, comprising:

[0039] (A) The step of contacting the sensor for detecting ATP-related substances according to any one of items 1, 1A, 2, 2A, 3, 4, 4A, 5, 5A, 6, and 6A with the sample fluid.

[0040] (B) The step of detecting the signal from hydrogen peroxide in each reaction section of the above sensor.

[0041] Item 8.

[0042] According to the method described in item 7, in step (A), the sensor is brought into contact with the sample fluid by immersing it in the sample fluid.

[0043] Item 9.

[0044] According to the method described in item 7 or 8, in step (B), the signal is detected while the sensor is immersed in the sample fluid.

[0045] Item 10.

[0046] The method according to any one of items 7 to 9, wherein the pH of the sample solution is 8 to 10.

[0047] Item 11.

[0048] According to any one of items 7 to 10, the sample solution is prepared by immersing the test sample in a buffer solution with a pH of 8 to 10.

[0049] Item 12.

[0050] According to any one of items 7 to 11, in the method, each reaction section of the sensor further retains peroxidase on a solid support, and the sample fluid contains a compound that exhibits color development or luminescence in the presence of peroxidase and hydrogen peroxide.

[0051] Item 13.

[0052] The method according to any one of items 7 to 12 further includes: (C) the step of evaluating the concentration of ATP-related substances contained in the sample fluid based on the intensity of the signals detected in each reaction section.

[0053] Item 14.

[0054] According to the method described in item 13, the K value is calculated using the following formula 1 based on the intensity of the signal detected in reaction section 1 and reaction section 3.

[0055] [Number 1]

[0056]

[0057] Item 15.

[0058] According to the method described in item 13, the Ki value is calculated using the following formula 2 based on the intensity of the signal detected in reaction section 2 and reaction section 3.

[0059] [Number 2]

[0060]

[0061] Item 16.

[0062] According to any one of claims 13 to 15, the concentration of ATP-related substances contained in the sample fluid is evaluated based on the intensity of the signal detected within 2 to 5 minutes after the sensor is brought into contact with the sample fluid.

[0063] Item 17.

[0064] According to any one of claims 13 to 15, the concentration of ATP-related substances contained in the sample fluid is evaluated based on the intensity of the signal detected after a time longer than 5 minutes following contact between the sensor and the sample fluid.

[0065] Item 18.

[0066] According to any one of items 13 to 17, in step (A), each of the plurality of sensors is brought into contact with a plurality of sample solutions with different dilution ratios; in step (B), a signal from hydrogen peroxide is detected in each reaction section of the plurality of sensors; and in step (C), a sensor among the plurality of sensors whose detected signal weakens as the reaction section number increases is selected, and the concentration of ATP-related substances contained in the sample solution is evaluated based on the intensity of the signal detected in each reaction section of that sensor.

[0067] Item 19.

[0068] According to any one of items 13 to 17, the total concentration of ATP, ADP and AMP in the sample fluid is evaluated based on the difference in intensity of the signals detected in each of the reaction section 1 and the reaction section 2.

[0069] Item 20.

[0070] According to any one of items 13 to 17, the concentration of inosinic acid in the sample fluid is evaluated based on the difference in intensity of the signals detected in each of the reaction section 2 and the reaction section 3.

[0071] Item 21.

[0072] A kit for detecting ATP-related substances, comprising one or more sensors for detecting ATP-related substances according to any one of items 1, 1A, 2, 2A, 3, 4, 4A, 5, 5A, 6 and 6A.

[0073] Item 22.

[0074] A kit for detecting ATP-related substances, comprising a sensor for detecting ATP-related substances as described in any one of claims 1, 1A, 2, 2A, 3, 4, 4A, 5, 5A, 6 and 6A, and a reaction container for receiving the sensor and the sample solution.

[0075] Item 23.

[0076] According to the kit described in item 22, the reaction vessel has a portion made of a visible light-transmitting material for observing the reaction section of the sensor from the outside.

[0077] Item 24.

[0078] A system for detecting ATP-related substances, comprising: a sensor for detecting ATP-related substances, an imaging unit, and an image processing unit as described in any one of claims 1, 1A, 2, 2A, 3, 4, 4A, 5, 5A, 6, and 6A.

[0079] Item 25.

[0080] The system according to item 24 further comprises a reaction vessel for receiving the sensor and the sample fluid.

[0081] Item 26.

[0082] The kit according to any one of items 21 to 23 is used in the method according to any one of items 7 to 20.

[0083] Item 27.

[0084] The system according to item 24 or 25 is used in any one of items 7 to 20.

[0085] Invention Effects

[0086] According to the present invention, ATP-related substances can be easily and quickly detected even at the food manufacturing site, enabling the evaluation of the freshness of fresh foods such as aquatic products (fish, mollusks, shellfish, crustaceans, etc.) and meat. Furthermore, it can determine not only K and Ki values, but also the combined concentrations of ATP, ADP, and AMP, and the inosinic acid concentration. Attached Figure Description

[0087] [ Figure 1 [ ] is a diagram showing the decomposition process of ATP-related substances in fresh foods such as aquatic products.

[0088] [ Figure 2 [Illustration] is a diagram illustrating a scheme for the breakdown and detection of ATP-related substances in this disclosure using an example of available enzymes (ALP, ADA, PNP, and XOD).

[0089] [ Figure 3 [Image showing an example of a sensor used to detect ATP-related substances]

[0090] [ Figure 4 [Image] is an image of a sensor used for detecting ATP-related substances after being immersed in standard solutions of IMP, HxR, and Hx and then removed.

[0091] [ Figure 5 The image (top) shows the sensor used for detecting ATP-related substances after it has been immersed in the mixed standard solution and removed, and the graph (bottom) shows the Ki value calculated from the signal intensity.

[0092] [ Figure 6 The image (top) shows the sensor used for detecting ATP-related substances after it has been immersed in the mixed standard solution and removed, and the graph (bottom) shows the Ki value calculated from the signal intensity.

[0093] [ Figure 7 The image (top) shows the sensor used for detecting ATP-related substances after it has been immersed in the mixed standard solution and removed, and the graph (bottom) shows the time changes of the K and Ki values ​​calculated from the signal intensity.

[0094] [ Figure 8 The graph is a scatter plot of the K value (upper segment) and Ki value (lower segment) calculated based on the signal intensity at 1 minute, 2 minutes, 3 minutes, 5 minutes, and 10 minutes after the start of the reaction of the ATP-related substance detection sensor immersed in the mixed standard solution and then removed, relative to the theoretical value.

[0095] [ Figure 9 The image shows the kit (top left) when the ATP-related substance detection sensor is immersed in the mixed standard solution for color development, an image of the ATP-related substance detection sensor taken while immersed in the mixed standard solution (bottom), and a graph showing the time change of the K value calculated from the signal intensity (top right).

[0096] [ Figure 10 [Image of a sensor for detecting ATP-related substances, taken while the sensor is immersed in a fish (bream) extract.]

[0097] [ Figure 11A ] is with Figure 10 The image corresponds to the image of component G (1-8 minutes after the start of the reaction). The numbers on the reaction section indicate the absorbance (signal intensity) of component R.

[0098] [ Figure 11B ] is with Figure 10The image corresponds to the image of component G (9-15 minutes after the start of the reaction). The numbers on the reaction section indicate the absorbance (signal intensity) of component R.

[0099] [ Figure 12 ] is by Figure 11A and Figure 11B The graph shows the K and Ki values ​​calculated from the signal strength.

[0100] [ Figure 13 [Image of a sensor for detecting ATP-related substances taken while immersed in a fish (yellowtail) extract.]

[0101] [ Figure 14A ] is with Figure 13 The image corresponds to the image of component G (1-8 minutes after the start of the reaction). The numbers on the reaction section indicate the absorbance (signal intensity) of component R.

[0102] [ Figure 14B ] is with Figure 13 The image corresponds to the image of component G (9-15 minutes after the start of the reaction). The numbers on the reaction section indicate the absorbance (signal intensity) of component R.

[0103] [ Figure 15 ] is by Figure 14A and Figure 14B The graph shows the K and Ki values ​​calculated from the signal strength.

[0104] [ Figure 16 The image is a sensor used for detecting ATP-related substances, taken while the sensor is immersed in fish meat (six-lined rockfish) extract.

[0105] [ Figure 17 The above plots are scatter plots of K and Ki values ​​based on HPLC, calculated from the signal intensity of the sensor used to detect ATP-related substances, taken while the sensor is immersed in fish meat (hexagrammia) extract.

[0106] [ Figure 18 This is an image taken by a sensor used to detect ATP-related substances after fish meat (six-lined rockfish) extract was added.

[0107] [ Figure 19 The K value (upper segment) and Ki value (lower segment) of the sensor used for detecting ATP-related substances were calculated based on the signal intensity of the sensor after the extract of fish meat (six-lined fish) was added. A scatter plot of the K value and Ki value based on HPLC was then plotted.

[0108] [ Figure 20The Ki value is calculated from the signal intensity of the sensor used to detect ATP-related substances after the fish meat (coho salmon) extract was added, and a scatter plot of the Ki value based on HPLC is plotted.

[0109] [ Figure 21 The Ki value is calculated from the signal intensity of the sensor used to detect ATP-related substances after the extract of fish meat (six-lined fish) is added, and a scatter plot of the Ki value based on HPLC is plotted.

[0110] [ Figure 22 The Ki value is calculated from the signal intensity of the sensor used to detect ATP-related substances after the extract of yellowtail fish is added. A scatter plot of the Ki value based on HPLC is then plotted.

[0111] [ Figure 23 ] is to Figures 20-22 A scatter plot showing the Ki values ​​of all the fish meat.

[0112] [ Figure 24 [Image of a sensor used for detecting ATP-related substances after being immersed in an extract prepared from a swab on the surface of fish (salmon) and then removed.]

[0113] [ Figure 25 [Image of a sensor for detecting ATP-related substances after being immersed in a mixed standard solution with altered concentrations of chromogenic compounds and then removed.]

[0114] [ Figure 26 [This is a graph of Ki values ​​calculated from the signal intensity of a sensor used for detecting ATP-related substances after it has been immersed in a mixed standard solution with altered concentrations of chromogenic compounds and then removed.] Detailed Implementation

[0115] The following descriptions are sometimes based on representative embodiments or specific examples, but the present invention is not limited to such embodiments or specific examples. Unless otherwise specified, the numerical ranges indicated by "~" or "-" in this specification refer to the range including the values ​​at both ends as upper and lower limits. The upper and lower limits of the various numerical ranges shown in this specification can be arbitrarily combined.

[0116] [definition]

[0117] In this disclosure, ATP-related substances refer to adenosine triphosphate (ATP) and its breakdown products, namely adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine (AdR), inosine monophosphate (IMP), inosine (HxR), hypoxanthine (Hx), and xanthine (X). In the muscle of aquatic animals, etc., after several hours to several days post-mortem, ATP is sequentially broken down into ADP, AMP, IMP, HxR, Hx, and xanthine. It is known that in this series of ATP breakdown processes, the breakdown from IMP to HxR is the bottleneck (see [reference]). Figure 1 ).

[0118] The K and Ki values ​​are indicators of ATP breakdown that focus on this aspect, and are calculated using the following formula.

[0119] [Number 3]

[0120]

[0121] In any formula, the molecules represent the total of HxR and Hx, which are the decomposition products after the bottleneck stage has been exceeded. High K and Ki values ​​indicate that the decomposition of ATP has progressed beyond the bottleneck stage, meaning that its freshness has decreased.

[0122] [Sensor for detecting ATP-related substances]

[0123] In one aspect, this disclosure provides a sensor for detecting ATP-related substances (hereinafter also simply referred to as a sensor), which has on the same support at least one of a reaction section 1 containing a combination of enzymes capable of converting ATP, ADP, and AMP into xanthine, a reaction section 2 containing a combination of enzymes capable of converting IMP into xanthine, and a reaction section 3 containing a combination of enzymes capable of converting HxR into xanthine, wherein the combination of enzymes in each reaction section includes xanthine oxidase (XOD).

[0124] The sensor of this type can also be represented as a sensor having at least one of the following on the same support: a reaction section 1 containing an enzyme combination capable of converting ATP, ADP, and AMP to Hx and an XOD reaction section 2 containing an enzyme combination capable of converting IMP to Hx and an XOD reaction section 3 containing an enzyme capable of converting HxR to Hx and an XOD reaction section 3 on the same support.

[0125] The sensor of this type can also be represented as a sensor having at least one of the following on the same support: a reaction section 1 containing a combination of enzymes capable of converting ATP, ADP, and AMP into uric acid and hydrogen peroxide on a solid support; a reaction section 2 containing a combination of enzymes capable of converting IMP into uric acid and hydrogen peroxide on a solid support; and a reaction section 3 containing a combination of enzymes capable of converting HxR into uric acid and hydrogen peroxide on a solid support.

[0126] The sensor has a configuration comprising at least two reaction sections (at least one of reaction section 1 and reaction section 2, and reaction section 3) on a support. That is, the sensor may include at least reaction section 1 and reaction section 3, or at least reaction section 2 and reaction section 3, or all of reaction sections 1 to 3. The reaction section is a solid-phase carrier holding one or more enzymes with ATP-related substances as substrates. As detailed later, when the sensor is brought into contact with a sample solution containing ATP-related substances, the enzyme held by the solid-phase carrier of the reaction section reacts with the ATP-related substances in the sample to produce hydrogen peroxide.

[0127] The "combination of enzymes capable of converting ATP, ADP, and AMP into xanthine" in Reaction Section 1 refers to a combination of enzymes that, when combined, can break down ATP, ADP, and AMP into xanthine. The enzyme combination in Reaction Section 1 includes XOD.

[0128] One example of the enzyme combination in reaction section 1 is four enzymes: alkaline phosphatase (ALP), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and XOD. ALP hydrolyzes the phosphate ester bonds of ATP, ADP, and AMP to convert them to AdR, and hydrolyzes the phosphate ester bonds of IMP to convert them to HxR. ADA deaminates AdR to convert it to HxR. PNP removes ribose from HxR to convert it to Hx. XOD oxidizes Hx to convert it to xanthine and hydrogen peroxide, and then oxidizes xanthine to convert it to uric acid and hydrogen peroxide. Therefore, by maintaining this enzyme combination in reaction section 1, all ATP-related substances can be converted to xanthine, and xanthine can be further converted to uric acid, generating hydrogen peroxide in the process (see reference). Figure 2 ).

[0129] Another example of the enzyme combination in reaction section 1 is five enzymes: adenosine triphosphate (ATP) diphosphatase, 5'-nucleotidase, ADA, PNP, and XOD. ATP diphosphatase hydrolyzes the phosphate ester bonds of ATP and ADP to convert them to AMP. 5'-nucleotidase hydrolyzes the phosphate ester bonds of AMP to convert it to AdR, and hydrolyzes the phosphate ester bonds of IMP to convert it to HxR. ADA deamination of AdR to convert it to HxR. PNP removes ribose from HxR to convert it to Hx. XOD oxidizes Hx to convert it to xanthine and hydrogen peroxide, and then oxidizes xanthine to convert it to uric acid and hydrogen peroxide. Therefore, by maintaining this enzyme combination in reaction section 1, all ATP-related substances can be converted to xanthine, and xanthine can be further converted to uric acid, generating hydrogen peroxide in the process.

[0130] In one embodiment, reaction unit 1 does not retain enzymes that use ATP-related substances as substrates, except for a combination of enzymes capable of converting ATP, ADP, and AMP into xanthine. In another embodiment, reaction unit 1 does not retain enzymes other than a combination of enzymes capable of converting ATP, ADP, and AMP into xanthine and peroxidase.

[0131] The "combination of enzymes capable of converting IMP into xanthine" in Reaction Section 2 refers to a combination of multiple enzymes that can break down IMP into xanthine when combined. The enzyme combination in Reaction Section 2 includes XOD.

[0132] Reaction section 2 is configured such that ATP, ADP, AMP, and AdR cannot be converted into HxR, and ATP, ADP, and AMP cannot be converted into IMP. Reaction section 2 does not maintain a combination of enzymes capable of converting ATP, ADP, AMP, and AdR into HxR, nor does it maintain a combination of enzymes capable of converting ATP, ADP, and AMP into IMP.

[0133] One example of the enzyme combination in reaction section 2 is the combination of three enzymes: ALP, PNP, and XOD. As mentioned above, ALP can convert IMP to HxR, PNP can convert HxR to Hx, and XOD can convert Hx to xanthine and hydrogen peroxide, and then convert xanthine to uric acid and hydrogen peroxide. Therefore, based on this enzyme combination, IMP, HxR, and Hx can be converted to xanthine, and xanthine can be further converted to uric acid, generating hydrogen peroxide in the process.

[0134] Another example of an enzyme combination in reaction section 2 is the combination of three enzymes: 5'-nucleotidase, PNP, and XOD. As mentioned above, 5'-nucleotidase can convert IMP to HxR, PNP can convert HxR to Hx, and XOD can convert Hx to xanthine and hydrogen peroxide, and then convert xanthine to uric acid and hydrogen peroxide. Therefore, by using this combination of enzymes, it is also possible to convert IMP, HxR, and Hx to xanthine, and then convert xanthine to uric acid, generating hydrogen peroxide in the process.

[0135] In one embodiment, reaction unit 2 does not retain enzymes that use ATP-related substances as substrates, except for a combination of enzymes capable of converting IMP to xanthine. In another embodiment, reaction unit 2 does not retain enzymes other than a combination of enzymes capable of converting IMP to xanthine and peroxidase.

[0136] The "combination of enzymes capable of converting HxR into xanthine" in reaction section 3 refers to a combination of multiple enzymes that can break down HxR into xanthine when combined. The enzyme combination in reaction section 3 includes XOD.

[0137] Reaction section 3 is configured such that ATP, ADP, AMP, AdR, and IMP cannot be converted into HxR. Reaction section 3 does not maintain a combination of enzymes capable of converting ATP, ADP, AMP, AdR, and IMP into HxR.

[0138] One example of the enzyme combination in reaction section 3 is the combination of PNP and XOD. As mentioned above, PNP can convert HxR to Hx, and XOD can convert Hx to xanthine and hydrogen peroxide, and then convert xanthine to uric acid and hydrogen peroxide. Therefore, through this combination of enzymes, HxR and Hx can be converted to xanthine, and xanthine can be further converted to uric acid, generating hydrogen peroxide in the process.

[0139] In one embodiment, reaction unit 3 does not retain enzymes that use ATP-related substances as substrates, except for a combination of enzymes capable of converting HxR to xanthine. In another embodiment, reaction unit 3 does not retain enzymes other than a combination of enzymes capable of converting HxR to xanthine and peroxidase.

[0140] The enzyme combination in reaction section 1 only needs to be able to convert ATP, ADP, and AMP into xanthine and contain XOD. The enzyme combination held in reaction section 2 only needs to be able to convert IMP into xanthine and contain XOD. The enzyme combination held in reaction section 3 only needs to be able to convert HxR into xanthine and contain XOD. The enzyme combinations in reactions 1 to 3 are not limited to the combinations shown above.

[0141] 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.

[0142] The sensor may further have a reaction section 4 on the same support, in which XOD is held on a solid support. As described above, XOD can convert Hx into xanthine and hydrogen peroxide, and then convert xanthine into uric acid and hydrogen peroxide.

[0143] Reaction section 4 is configured such that ATP, ADP, AMP, AdR, IMP, and HxR cannot be converted into Hx. Reaction section 4 does not maintain a combination of enzymes capable of converting ATP, ADP, AMP, AdR, IMP, and HxR into Hx.

[0144] In one embodiment, reaction section 4 does not hold enzymes that use ATP-related substances as substrates, except for XOD. In another embodiment, reaction section 4 does not hold enzymes other than XOD and peroxidase.

[0145] The enzymes held on the solid support in the reaction section have no restrictions on their source or manufacturing method, as long as they possess the necessary activity. For example, ALP can be derived from calf intestine or Escherichia coli, ADA can be derived from calf intestine, bovine spleen, or Escherichia coli, PNP can be derived from Escherichia coli, XOD can be derived from cow's milk, and adenosine triphosphate diphosphatase can be derived from potatoes. The enzymes exemplified here can exert their activity in a weakly alkaline environment.

[0146] In addition to the reaction section, the sensor may further have a control section on the same support. The control section is a solid-phase carrier that does not retain the enzyme.

[0147] Each reaction section can further retain the peroxidase on a solid support. Furthermore, in this embodiment, the sensor can further include a reaction section 5 for retaining the peroxidase on the same support. Examples of peroxidases include horseradish peroxidase (HRP), formyl peroxidase, cytochrome C peroxidase, glutathione peroxidase, and lipid peroxidase. HRP is a preferred example of a peroxidase.

[0148] The reaction section 5 does not hold enzymes that use ATP-related substances as substrates. In one embodiment, the reaction section 5 does not hold enzymes other than peroxidase.

[0149] The solid-phase carrier that holds the enzyme in the reaction section only needs to be able to maintain the enzyme's solid phase at least during the enzyme reaction while the sensor is immersed in the sample solution. Examples of such solid-phase carriers include thin plates or sheets made of natural polymers such as cellulose, chitosan, collagen, and alginate, or synthetic polymers such as polypropylene, polyester, polystyrene, nitrocellulose, nylon, polyethersulfone, PVDF, glass fiber, polyacrylamide, and acrylic copolymers. Preferred examples of solid-phase carriers include chromatographic paper, filter paper, and absorbent pads.

[0150] The size and shape of the reaction section are not limited as long as they can detect the signal from hydrogen peroxide generated upon contact with the sample liquid. For example, in the case of signal detection by visual inspection, the reaction section only needs to be able to visually identify the size of the signal. The shape of the reaction section can be circular, triangular, quadrilateral, or a polygon of more than one shape, or it can be irregular. In addition, the shapes of the reaction sections in a single sensor can be the same or different from each other. Regarding the spacing of the solid phase carrier, it is only necessary to ensure that the signal from hydrogen peroxide is not affected by liquid mixing and contamination between the reaction sections. For example, if it is circular, a spacing greater than its radius is preferred; if it is triangular or quadrilateral, a spacing greater than half the length of its longer side is preferred. However, if the raw material of the reaction section is used to maintain the strong water absorption of the aqueous solution, as long as a slight spacing is ensured between the reaction sections, the possibility of liquid mixing and contamination between the reaction sections is low, so it is not necessary to limit it in this way.

[0151] Enzymes can be retained on solid supports using methods commonly used in enzyme immobilization. For example, this can be achieved by immersing an aqueous solution or aqueous suspension containing the enzyme in the solid support and then drying it; by gelling an aqueous solution containing a substance capable of gelling with the enzyme (alginic acid, acrylamide, etc.) on a support to create a solid support; or by immobilizing the enzyme through covalent bonds, ionic bonds, or adsorption with the modified functional groups of the solid support.

[0152] The concentration of the enzyme held on the solid-phase carrier can be appropriately adjusted according to the concentration and ratio of ATP-related substances in the sample fluid. For example, when the enzyme is held on the solid-phase carrier by immersing 6.5 μl of an aqueous solution or aqueous suspension containing the enzyme in a solid-phase carrier with a length of 4 mm, a width of 4 mm, and a thickness of 0.7 mm, the concentration of ALP in the aqueous solution or aqueous suspension can be 200~600 U / ml, preferably 300~500 U / ml; the concentration of ADA can be 60~180 U / ml, preferably 90~150 U / ml; the concentration of PNP can be 60~180 U / ml, preferably 90~150 U / ml; the concentration of XOD can be 0.16~0.48 U / ml, preferably 0.24~0.40 U / ml; and the concentration of HRP can be 0.8~2.4 U / ml, preferably 1.2~2.0 U / ml.

[0153] The concentration of enzymes held on solid supports in each reaction section of a sensor is preferably kept constant according to the type of enzyme. For example, for a sensor in which ALP, ADA, PNP, and XOD are held on the solid support in reaction section 1, ALP, PNP, and XOD are held on the solid support in reaction section 2, PNP and XOD are held on the solid support in reaction section 3, and XOD is held on the solid support in reaction section 4, the concentration of ALP in reaction sections 1 and 2 is preferably the same, the concentration of PNP in reaction sections 1 to 3 is preferably the same, and the concentration of XOD in reaction sections 1 to 4 is preferably the same. In addition, in this example of the sensor, the concentration ratio of enzymes held on solid supports, calculated per unit volume, can be ALP:ADA:PNP:XOD = 600~2000:180~600:180~600:1, preferably 900~1600:250~500:250~500:1.

[0154] The enzyme combination in reaction section 1 uses all ATP-related substances as substrates. Therefore, after the sensor comes into contact with the sample solution and the reaction proceeds fully, the signal intensity detected in reaction section 1 is the highest among the hydrogen peroxide signals detected in each reaction section. Therefore, the concentration of each enzyme held in the solid-phase carrier of the reaction section is preferably set such that the concentration of the hydrogen peroxide signal detected in reaction section 1 falls within the dynamic range of the signal detection unit, preferably at or near the upper limit of the dynamic range.

[0155] Furthermore, even for sample solutions with the same total concentration of ATP-related substances, the intensity of the signal detected after reaction section 2 may differ depending on the concentration of each ATP-related substance, but the intensity of the signal detected in reaction section 1 will be the same. Therefore, the intensity of the signal detected in reaction section 1 can be used for signal detection control and also for sensor accuracy management. For example, if, when a sample solution is brought into contact with multiple identical sensors, the intensity of the signal detected in reaction section 1 of one sensor is weaker than the intensity of the signal detected in reaction section 1 of other sensors, it can be determined that an anomaly has occurred in that sensor. Specifically, an anomaly is considered to be due to deterioration of the activity of the supported enzyme, deterioration of the solid-phase carrier, etc., which reduces the performance of the sensor. In addition, since the sensor is for single use, it is necessary to ensure that the predetermined reaction for the purpose is carried out in each reaction section and that an appropriate signal intensity is obtained. However, reaction section 1 also reflects the interpretation of differences between sensors, small differences in the time of immersion in the sample solution during measurement, and differences in the concentration of the sample solution. Therefore, it is also useful in terms of correcting measurement discrepancies based on the method of use and in confirming that the sensor was used correctly for the measurement.

[0156] There are no limitations on the size, shape, and material of the support constituting the sensor, nor on the arrangement of the reaction parts on the support. Those skilled in the art can appropriately determine these factors by considering factors such as operability and ease of detection of ATP-related substances. Examples of materials for the support include glass, plastic, metal, and wood. Furthermore, the reaction parts are preferably arranged in a row on the same surface of the support in descending or ascending order.

[0157] In one embodiment, the sensor has the following configuration: on one surface of a support body that is a rectangular thin plate, at least two reaction parts (at least one of reaction part 1 and reaction part 2, and reaction part 3) are provided along the long axis from one end to the other. Figure 3 In the illustrated sensor, the reaction parts 1 to 5 are fixed in a manner arranged sequentially from one end of the support to the other end, and a gripping part is provided on the other end side so that the sensor can be held using fingers or tweezers.

[0158] [Detection methods for ATP-related substances]

[0159] This disclosure also provides a method for detecting ATP-related substances, comprising: (A) contacting an ATP-related substance detection sensor with a sample solution; and (B) detecting a signal caused by hydrogen peroxide in each reaction section of the sensor.

[0160] The sample solution is an aqueous solution or aqueous suspension of substances intended for detection of ATP-related substances. The pH of the sample solution can be appropriately adjusted within the range where the enzymes used in the sensor can exert their activity. The pH of the sample solution can be weakly alkaline, for example, 7-10, preferably 7.5-10, and more preferably 8-9. The enzymes specifically exemplified in the sensor specifications above can exert their activity in a weakly alkaline environment.

[0161] In one embodiment, the sample fluid is an extract of the test sample, such as fresh seafood (fish, mollusks, shellfish, crustaceans, etc.), fresh meat, or other fresh foods containing animal muscle tissue. The extract can be prepared by immersing the test sample in a suitable aqueous medium. For example, fragmented test samples can be immersed in an aqueous medium, stirred or homogenized, and the supernatant can be recovered, or the sample can be filtered and the filtrate recovered, thereby preparing the extract. Alternatively, the extract can be prepared by immersing the surface of the test sample in a suitable aqueous medium using a suitable instrument (plastic rod, glass rod, plastic swab, glass swab, wooden swab, metal swab, nylon swab, cotton swab, polyester swab, etc.) by swabbing or scraping.

[0162] like Figure 1 As shown, fresh food is preferably food in which ATP is broken down via IMP, such as fish, mollusks (squid / octopus), arthropods (shrimp / crab), and echinoderms (crab nests).

[0163] The aqueous medium used for extraction can 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 and Forestry Standard (JAS) 0023:2022. In the case of using an extract in a strongly acidic aqueous medium, the pH is adjusted to a level that allows the enzyme used in the sensor to exert its activity before being used as the sample solution.

[0164] The aqueous medium used for extraction can be a buffer solution obtained by dissolving a buffer in water. The pH of the buffer solution is preferably weakly alkaline, for example, 7 to 10, more preferably 7.5 to 10, and even more preferably 8 to 9. Examples of buffer solutions include Tris, HEPES, and MOPS. HEPES is a preferred example of a buffer solution.

[0165] In a preferred embodiment, the sample solution is an extract prepared by immersing the test sample in a weakly alkaline buffer solution. The test sample sometimes contains endogenous enzymes that use ATP-related substances as substrates, but the optimal pH for these endogenous enzymes is 5-8. Therefore, by making the pH of the sample solution weakly alkaline, preferably 7.5-10, and more preferably 8-9, the activity of the enzymes on the sensor can be inhibited while maintaining the activity of the endogenous enzymes. Furthermore, by using a weakly alkaline buffer solution during extraction, the pH adjustment required when using a strongly acidic aqueous medium is eliminated, simplifying the operation.

[0166] When the various reaction parts of the sensor contain peroxidase on a solid support, the sample fluid may contain compounds that serve as substrates for the peroxidase.

[0167] In step (A), the method of bringing the sensor into contact with the sample fluid is not limited as long as all the reaction parts on the sensor are in contact with the sample fluid. For example, it can be done by dripping, spraying, or coating the sample fluid onto each reaction part of the sensor, or by immersing all the reaction parts of the sensor in the sample fluid. If the sensor has a control part, it is preferable that the control part, in addition to the reaction part, is also in contact with the sample fluid.

[0168] Upon contact with the sample fluid, an enzymatic reaction using ATP-related substances in the sample fluid as substrates begins in each reaction section of the sensor, generating hydrogen peroxide. In step (B), the signal from the generated hydrogen peroxide is detected.

[0169] Signals from hydrogen peroxide can be generated and detected using methods known to those skilled in the art. Examples of such methods include: an optical detection method that uses a compound that exhibits color development (coloration) or luminescence in the presence of hydrogen peroxide and detects the intensity of the color development or luminescence of the compound corresponding to the amount of hydrogen peroxide as a signal; and an electrochemical detection method that uses an enzyme electrode with immobilized peroxidase and detects the change in current corresponding to the amount of hydrogen peroxide as a signal.

[0170] The compounds used in optical detection methods can be those that produce color or light by being oxidized by peroxidase in the presence of hydrogen peroxide. Amplite is an example of such a compound. TM Red (10-acetyl-3,7-dihydroxyphenoxazine), 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), 2,2'-azidobis(3-ethylbenzothiazoline-6-sulfonic acid, ABTS), diaminobenzidine (DAB), cytochrome C, luminol, etc. TMRed is oxidized by peroxidase in the presence of hydrogen peroxide to form halogen (7-hydroxy-2H-phenoxazine-3-one). Halogen can be detected by fluorescence (excitation wavelength 530-570 nm, emission wavelength 580-590 nm) or by a purple-red color development.

[0171] The compounds used in optical detection methods can also be compounds that react with hydrogen peroxide to produce color or light, such as Ti-porphyrin reagents.

[0172] In optical detection methods utilizing compounds that exhibit color development or luminescence upon oxidation by peroxidase in the presence of hydrogen peroxide, the signal from hydrogen peroxide can also be generated by contacting the sensor with the sample solution, thereby allowing the peroxidase to contact the compound that serves as its substrate. However, for rapid signal detection, it is preferable to maintain at least one of the peroxidase or the substrate compound in each reaction section of the sensor, while pre-mixing the other in the sample solution. This allows for immediate detection of the signal from hydrogen peroxide upon contact between the sensor and the sample solution.

[0173] In a preferred embodiment, the reaction portions of the sensor hold peroxidase on a solid support, and the sample solution contains a compound that serves as a substrate for the peroxidase. In this embodiment, the sample solution is preferably an extract prepared by immersing the test sample in a buffer solution containing the compound that serves as a substrate for the peroxidase and having a weakly alkaline pH. The compound that serves as a substrate for the peroxidase can be added to the buffer solution used to immerse the test sample or to the extract solution after immersion.

[0174] Signals from hydrogen peroxide can be detected using detection methods appropriate to the type of signal. In cases where the signal is colorimetric or luminescent in the visible light region, it can also be detected visually using optical detection units such as spectrophotometers or imaging devices.

[0175] In one embodiment, the signal from hydrogen peroxide is detected by acquiring images of each reaction unit using an imaging device. Regarding the signal intensity, an appropriate region of interest can be defined within the image of each reaction unit, and the intensity can be represented as the brightness value obtained by converting the RGB values ​​of each pixel within that region to grayscale. Alternatively, the signal intensity can also be represented as the value of the RGB component that most closely approximates the color or light emission of the signal. For example, if the signal is a purplish-red color, the signal intensity can be obtained as the R component value of each pixel within the region of interest.

[0176] Furthermore, the signal intensity can be represented by the absorbance of the component in the RGB composition that is closest to the color of the signal. Absorbance can be obtained using images of each reactive region as follows: First, extract the image of the closest color component (e.g., component G in the case of a reddish-purple color) that is the opposite color to the signal from the images of each reactive region. Set an appropriate region of interest within the extracted image and obtain the luminance value of each pixel within that region. Then, substitute the obtained luminance values ​​into the following formula to calculate the absorbance.

[0177] [Number 4]

[0178]

[0179] By using absorbance as the signal intensity, the influence of external interference light during shooting can be reduced.

[0180] In step (A), when the sensor is in contact with the test fluid by immersing the entire reactive part of the sensor in the test fluid, the signal detection can be performed either after the immersed sensor is removed from the test fluid or while the sensor is immersed in the test fluid.

[0181] In this disclosure, the method for detecting ATP-related substances may further include: (C) evaluating the concentration of ATP-related substances contained in the sample fluid based on the intensity of the signals detected in each reaction section. In step (C), the concentration of ATP-related substances is evaluated based on the intensity of the signals detected in each reaction section.

[0182] In one embodiment, step (C) is to calculate the value of K based on the intensity of the signal detected in reaction section 1 and reaction section 3 using the following formula 1.

[0183] [Number 5]

[0184]

[0185] In another embodiment, step (C) is a step of calculating the Ki value based on the intensity of the signals detected in reaction section 2 and reaction section 3 using the following formula 2.

[0186] [Number 6]

[0187]

[0188] The enzyme combination in reaction section 1 can ultimately convert all ATP-related substances into uric acid and hydrogen peroxide. Therefore, the intensity of the signal detected in reaction section 1 corresponds to the total concentration of ATP-related substances contained in the sample fluid and can be used as the denominator when calculating the K value.

[0189] The enzyme combination in reaction section 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 section 2 corresponds to the total concentration of IMP, HxR, and Hx contained in the sample fluid, and can be used as the denominator when calculating the Ki value.

[0190] The enzyme combination in reaction section 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 section 3 corresponds to the total concentration of HxR and Hx contained in the sample fluid, and can be set as the molecule for calculating K and Ki values.

[0191] In one embodiment, step (C) is a step of evaluating the total concentration of ATP, ADP, and AMP in the sample fluid based on the difference in the intensity of the signals detected in reaction section 1 and reaction section 2 respectively. The value obtained by subtracting the signal intensity detected in reaction section 2 from the signal intensity detected in reaction section 1 corresponds to the total concentration of ATP, ADP, and AMP in the sample fluid. Therefore, using this value, even fresh test samples with good freshness where the breakdown of ATP into IMP has not progressed can be evaluated.

[0192] In one embodiment, step (C) is a step of evaluating the concentration of IMP in the sample fluid based on the difference in intensity of the signals detected in reaction section 2 and reaction section 3 respectively. The value obtained by subtracting the signal intensity detected in reaction section 3 from the signal intensity detected in reaction section 2 corresponds to the concentration of IMP in the sample fluid. Therefore, using this value, the concentration of IMP in the test sample can be evaluated, for example, umami can also be evaluated.

[0193] It should be noted that in the method for detecting ATP-related substances disclosed herein, the intensity of the signal measured in the control section can be used as a background value. Furthermore, in the method for detecting ATP-related substances disclosed herein, the signal detected in the reaction section 5 is considered to be an endogenous signal introduced from the sample, such as the signal from hydrogen peroxide contained in the sample fluid. Therefore, the intensity of the signal detected in the reaction section 5 can also be used as a background value for calculating the K and Ki values.

[0194] For example, the intensity of the signal detected in the control section or the reaction section 5 can be used as a background value by subtracting the intensity of the signal detected in each of the reaction sections 1 to 4. Furthermore, when the signal intensity is expressed as absorbance, the intensity of the signal detected in the control section or the reaction section 5 can also be used as a "background value" in the aforementioned formula for calculating absorbance.

[0195] One embodiment of the method for detecting ATP-related substances disclosed herein is as follows: In step (A), multiple sensors are each brought into contact with multiple sample solutions of different dilution ratios; in step (B), a signal from hydrogen peroxide is detected in each reaction section of the multiple sensors; in step (C), a sensor among the multiple sensors whose detected signal weakens as the reaction section number increases is selected, and the concentration of ATP-related substances contained in the sample solution is evaluated based on the intensity of the signal detected in each reaction section of that sensor.

[0196] In this embodiment, multiple sensors with the same concentration of each enzyme in the reaction section are each contacted with multiple sample solutions of different dilution ratios. The sensor whose signal intensity weakens as the reaction section number increases is selected as the evaluation target. A sensor whose signal intensity weakens as the reaction section number increases refers to a sensor whose signal intensity exhibits a gradient; for example, the signal intensity of reaction section 2 is weaker than that of reaction section 1, and the signal intensity of reaction section 3 is weaker than that of reaction section 2. This gradient of signal intensity is observed when the sample solution contains an amount of ATP-related substances that is neither too high nor too low relative to the enzyme concentration in the sensor's reaction section—that is, when the sample solution is contacted at a dilution ratio within the dynamic range of the sensor. Therefore, by selecting the sensor whose signal intensity weakens as the reaction section number increases from multiple sensors contacted with sample solutions of different dilution ratios as the evaluation target, the concentration of ATP-related substances can be evaluated with high precision, even for extracts with unknown extraction efficiency or unknown concentrations of ATP-related substances.

[0197] Another embodiment of the method for detecting ATP-related substances disclosed herein is as follows: In step (A), multiple sensors, whose concentrations of each enzyme in the reaction section are changed in stages, are each brought into contact with the same sample solution; in step (B), a signal from hydrogen peroxide is detected in each reaction section of the multiple sensors; in step (C), a sensor whose detected signal weakens as the number of the reaction section increases is selected, and the concentration of ATP-related substances contained in the sample solution is evaluated based on the intensity of the signal detected in each reaction section of that sensor.

[0198] This embodiment differs from the embodiment using multiple sample solutions with different dilution ratios in that, for a single sample solution, multiple sensors are used to progressively change the enzyme concentration in the reaction section. By using multiple sensors to progressively change the enzyme concentration in the reaction section, it is possible to select sensors that maintain an enzyme concentration that is neither too high nor too low relative to the concentration of ATP-related substances contained in the sample solution as the evaluation target. Therefore, even for extracts with unknown extraction efficiency and unknown concentrations of ATP-related substances, the concentration of ATP-related substances can be evaluated with high precision.

[0199] In the method for detecting ATP-related substances disclosed herein, step (C) can be performed by comparing the intensity of color development or luminescence observed visually at each reaction site. If color development or luminescence is observed only at the lower-numbered reaction sites, the ATP decomposition of the test sample is minimal, indicating good freshness. If the same intensity of color development or luminescence is observed in the higher-numbered reaction sites, the test sample has undergone ATP decomposition, indicating reduced freshness.

[0200] Specifically, for example, if there is a significant difference in the intensity of color development or luminescence between reaction section 2 and reaction section 3, it can be determined that the ATP decomposition of the test sample has proceeded to IMP, but not to HxR. On the other hand, if there is no significant difference in the intensity of color development or luminescence from reaction section 1 to reaction section 3, it can be determined that the ATP decomposition of the test sample has proceeded to at least HxR.

[0201] In the method for detecting ATP-related substances disclosed herein, the time from contacting the sensor with the sample solution to detecting a signal is not limited except for the time immediately following contact between the sensor and the sample solution. To accurately evaluate the concentration of ATP-related substances, it is preferable to detect the signal after the enzyme reaction in each reaction section has fully proceeded and the generation of hydrogen peroxide has stabilized. In this case, signal detection can be performed after, for example, more than 5 minutes, preferably more than 10 minutes, more preferably more than 15 minutes, and even more preferably more than 20 minutes after contacting the sensor with the sample solution. The embodiments described above for evaluating the total concentration of ATP, ADP, and AMP in the sample solution, evaluating the concentration of IMP in the sample solution, contacting multiple sensors with the same enzyme concentration with multiple sample solutions of different dilution ratios, and contacting multiple sensors with progressively altered enzyme concentrations with the same sample solution, preferably use the intensity of the signal detected during that time period for evaluation.

[0202] Furthermore, enzyme reactions in each reaction section tend to have a rapid initial reaction rate, resulting in a stronger detected signal intensity. Therefore, to evaluate the concentration of ATP-related substances with high sensitivity, it is preferable to detect the signal at the initial stage of the enzyme reaction. In this case, signal detection can be performed within a range of, for example, 2 to 10 minutes, preferably 2 to 5 minutes, and more preferably 2 to 3 minutes after the sensor comes into contact with the sample solution. The above-described embodiments for calculating the K value and Ki value can also detect subtle differences between the K and Ki values ​​with high sensitivity by evaluating the intensity of the signal detected during this time period.

[0203] Furthermore, it has been reported that ATP is decomposed via AdR in some fresh foods, and the extracts of such fresh foods contain AdR. The sensor disclosed herein, by setting the "combination of enzymes capable of converting ATP, ADP, and AMP into xanthine" in reaction section 1 to either the four examples above (ALP, ADA, PNP, and XOD) or the five examples above (ATP-ADP-bisphosphatase, 5'-nucleotidase, ADA, PNP, and XOD), can detect AdR contained in the sample solution along with ATP, ADP, and AMP in reaction section 1. Therefore, even in sample solutions containing AdR, the total concentration of ATP, ADP, AMP, and AdR in the sample solution can be evaluated based on the difference in signal intensity detected in reaction section 1 and reaction section 2 respectively. Furthermore, the degree of ATP decomposition can be evaluated based on the values ​​calculated using Equations 1 and 2 above.

[0204] [ATP-related substance detection kits and systems]

[0205] This disclosure also provides a kit for detecting ATP-related substances, which includes one or more sensors for detecting ATP-related substances.

[0206] This disclosure also provides an ATP-related substance detection kit, which includes one or more sensors for detecting ATP-related substances and a reaction container for receiving the sensors and sample solutions. As described above, when multiple sensors with the same enzyme concentration are contacted with multiple sample solutions of different dilution ratios, each sample solution can be placed into each compartment of the reaction container having multiple compartments, and the sensor can be contacted with the sample solution within that compartment.

[0207] The reaction vessel is preferably configured such that the reaction section of the sensor can be visually identified from outside the vessel. Furthermore, the reaction vessel as a whole, or the portion corresponding to the reaction section when the sensor is placed within the compartment, is preferably made of a visible light-transmitting material, thereby allowing the sensor's reaction section to be observed from the outside. Examples of visible light-transmitting materials include glass, polycarbonate resin, polyethylene terephthalate resin, polyacrylic acid resin, and polyvinyl chloride resin.

[0208] This disclosure also provides a system for detecting ATP-related substances, which includes a sensor for detecting ATP-related substances, an imaging unit, and an image processing unit. The imaging unit can be any conventional imaging device, such as a CCD camera or a CMOS camera. The image processing unit can be any computer capable of calculating the RGB values ​​or brightness values ​​of each pixel contained in the acquired image. Typically, the computer includes: an input unit for inputting instructions or information for the computer; an image input unit for inputting image data from the imaging device; a recording unit; a processing unit for processing the calculation of the RGB values ​​or brightness values ​​of each pixel based on the captured image data; an output unit for outputting the image and the processing results; and a control unit for controlling the operation of the above units.

[0209] In the system, the camera unit and image processing unit can be mobile devices equipped with a shooting device and a computer, such as smartphones and tablets. Additionally, the system may further include a recording medium containing a program for image processing in a computer-readable format.

[0210] These kits and systems can be appropriately used in the above-mentioned methods for detecting ATP-related substances.

[0211] These kits and systems may include further reagents for signal generation and detection (e.g., buffers or buffers for extracting test samples, compounds that produce color or light in the presence of peroxidase and hydrogen peroxide, etc.), instruments for collecting test samples (e.g., rods, swabs, etc. for sampling or scraping the surface of test samples), and manuals for using these kits and systems in the methods for detecting ATP-related substances described above.

[0212] The present invention is described in more detail below with examples, but the present invention is not limited to these examples. It should be noted that, in the examples, unless otherwise specified, the preparation and storage of the sample solution are carried out under ice-cold conditions, while other operations are carried out at room temperature.

[0213] Example

[0214] [Example 1. Fabrication of a sensor for detecting ATP-related substances]

[0215] As shown in Table 1, up to five enzymes were added to 1M Tris-HCl at pH 8.5 to prepare enzyme solutions 1-5. 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 E. coli, optimal pH 7.5), and xanthine oxidase (XOD, derived from cow's milk, optimal pH 8.5-9.0) were used for the breakdown of ATP-related substances, and horseradish peroxidase (HRP, optimal pH 7.0) was used for hydrogen peroxide detection. The optimal concentrations of each enzyme in the enzyme solutions were determined beforehand and set 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.

[0216] [Table 1]

[0217]

[0218] Six absorbent filter papers (4mm width × 4mm length × 0.7mm thickness, ADVANTEC, No. 26-WA) made of cellulose fibers (TAPPI (Technical Association of the Pulp and Paper Industry) T205) were spaced approximately 1mm apart and adhered to an acrylic support (4mm width, 80mm length) using double-sided adhesive. 1-5 μL of enzyme solution and 6.5 μL each of 1M Tris-HCl and pH 8.5 (as controls) were added to each filter paper. Each test strip was independently placed in an aluminum bag containing a silicone bag and stored at 4°C for 2 days. This fabricated a sensor for detecting ATP-related substances, which was used in the following examples.

[0219] [Example 2. Determination of ATP-related substances using standard solutions]

[0220] (1) Detection of IMP, HxR, and Hx

[0221] Amplite TM Red (Amplex) TM One vial of Red Xanthine Assay Kit (Cosmo Bio Co., Ltd. ABD) powder was diluted with 100 μL of DMSO to prepare Amplite. TMRed stock solution. Dilute this stock solution 100-fold with 1M Tris-HCl, pH 8.5. Dissolve IMP, HxR, or Hx in the diluent to prepare three standard solutions (200 μM IMP standard solution, 200 μM HxR standard solution, and 200 μM Hx standard solution). Additionally, Amplite... TM Red is usually colorless in solution, but it turns purple-red when reduced to halogenated halogenated by HRP in the presence of hydrogen peroxide. Therefore, by making it contain Amplite TM The red liquid (standard solution, sample solution, etc.) is in contact with the reaction section of the HRP, and the presence of hydrogen peroxide can be detected by color development.

[0222] The enzyme reaction was initiated by immersing the sensor in the aforementioned standard solutions for 1 to 2 seconds, and then immediately removed. The removed sensor was quickly placed horizontally in a white imaging box, and the reaction section was photographed with a digital camera at 1-minute intervals from the start of immersion. Reaction sections 1 and 2 contained all three standard solutions, reaction section 3 contained HxR and Hx standard solutions, and reaction section 4 contained Hx standard solution. A purplish-red color development was observed in reaction section 4. However, in contrast to reaction section 5, no color development was observed. Figure 4 Additionally, in the embodiments, unless otherwise specified, images captured by a digital camera are displayed by monochrome binarizing or grayscale converting RGB color images.

[0223] (2) Determination of K value and Ki value

[0224] Amplite TM The Red stock solution was diluted 100-fold with 1M Tris-HCl at pH 8.5. ATP, ADP, AMP, IMP, HxR, and Hx were dissolved in the diluent to prepare the three mixed standard solutions shown in Table 2. In Table 2, the K and Ki values ​​for each mixed standard solution are theoretical values.

[0225] [Table 2]

[0226]

[0227] Immerse the sensor in each of the mixed standard solutions with theoretical Ki values ​​of 33.3 and 57.1 for 1-2 seconds, then immediately remove it. Quickly place the removed sensor horizontally in a white imaging box and immerse it for 10 minutes. Then, photograph the reaction section with a digital camera at 5-minute intervals. For the green channel image extracted from the photographed images, set a region of interest at a position corresponding to the central part of each reaction section, obtain the brightness value of the pixels contained within the region of interest, and calculate the average value for each reaction section. Treating the average brightness value of reaction section 5 as the background, calculate the absorbance of the R component in each reaction section using the following formula, which is taken as the signal intensity of each reaction section.

[0228] Signal intensity of reaction section n = absorbance of component R in reaction section n = -100 × log 10 (Average brightness value of component G in reaction section n / Average brightness value of component G in reaction section 5)

[0229] Next, using the signal strength of reaction units 2 and 3, the Ki value (referred to as the measured Ki value) is calculated using the following formula.

[0230] Ki value (%) = Signal strength of reaction section 3 / Signal strength of reaction section 2 × 100

[0231] The captured images and measured Ki values ​​are shown below. Figure 5 For any mixed standard solution, the calculated Ki value did not change significantly after 10 minutes of reaction, and no large deviation from the theoretical value was found.

[0232] Next, the experiment was conducted using all three mixed standard solutions in the same manner, and the Ki values ​​were calculated. Two sensors were used for the mixed standard solutions with theoretical Ki values ​​of 18.1 and 33.3, respectively. The reaction section was photographed at 5-minute intervals starting from the start of immersion.

[0233] The captured images and measured Ki values ​​are shown below. Figure 6 The graph of Ki values ​​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 deviations were identified between the two sensors immersed in the mixed standard solution with theoretical Ki values ​​of 18.1 and 33.3, the overall trend is the same as in previous experiments: the Ki values ​​did not change significantly after 5 minutes of reaction, and no large deviations from the theoretical values ​​were identified.

[0234] (3) High sensitivity measurement

[0235] Amplite TM The Red stock solution was diluted 100-fold with 1M Tris-HCl at pH 8.5. ATP, ADP, AMP, IMP, HxR, and Hx were dissolved in the diluent to prepare the four mixed standard solutions shown in Table 3. In Table 3, the K and Ki values ​​for each mixed standard solution are theoretical values.

[0236] [Table 3]

[0237]

[0238] Using the four mixed standard solutions, the experiment was conducted in the same manner as described in (2) above, and the Ki value was calculated. The reaction section was photographed at 1-minute intervals starting from the immersion. In addition to the Ki value, the signal intensity of reaction sections 1 and 3 was also used to calculate the K value (referred to as the measured K value).

[0239] K value (%) = Signal strength of reaction section 3 / Signal strength of reaction section 1 × 100

[0240] The captured images and the time variations of the measured K and Ki values ​​are shown in the figure. Figure 7 The measured K and Ki values ​​at 1, 2, 3, 5, and 10 minutes after the reaction started were plotted as scatter plots relative to the theoretical values. Figure 8 Except for the mixed standard solution with the highest theoretical K value of 0.25, both K and Ki values ​​reached their highest values ​​2 minutes after the start of the reaction, and then gradually decreased and stabilized over time. The slope of the regression line for K was 1.93 times greater at 2 minutes and 1.61 times greater at 3 minutes compared to 10 minutes after the start of the reaction. The same trend was confirmed for Ki. This indicates that by detecting color development in a shorter reaction time than 10 minutes, especially around 2-3 minutes, K and Ki values ​​can be detected with higher sensitivity.

[0241] (4) Signal detection in the detection solution

[0242] Except for changing the order of the reaction sections, the sensor was fabricated using the same method as in Example 1. The concentrations of each enzyme in the enzyme solution were also set 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.

[0243] Additionally, Amplite TM The Red stock solution was diluted 100 times with 1M Tris-HCl at pH 8.5. ATP, IMP, HxR and Hx were dissolved in the diluent to prepare a mixed standard solution (ATP 20 μM, IMP 60 μM, HxR 10 μM, Hx 10 μM).

[0244] The mixed standard solution was dispensed into an acrylic reaction vessel with 3 compartments (the dimensions of each compartment are: 50 mm in height × 5 mm in width × 2 mm in thickness). Then, one sensor was immersed in each compartment.

[0245] The sensor was immersed in a mixed standard solution and placed in a white imaging box. Images of the reaction section were taken at 1-minute intervals starting from the start of immersion. The signal intensity of each reaction section was calculated, and the K-value was calculated using the signal intensities of reaction sections 1 and 3.

[0246] The captured images and the time variation of the measured K value are shown in... Figure 9And Table 4. The K value is the same as the result observed in (3) above, gradually decreasing and stabilizing over time. It was confirmed that even when the sensor is immersed in liquid, it can be analyzed in the same way as when it is removed from liquid.

[0247] [Table 4]

[0248]

[0249] [Example 3. Determination of ATP-related substances using fish extract]

[0250] (1) Simultaneous determination of dilution series using multiple sensors (cut sea bream pieces)

[0251] In Amplite TM Finely chopped sea bream pieces were added to a diluent prepared by diluting the Red stock solution 100-fold with 1M Tris-HCl at pH 8.5, at concentrations of 6.3, 12.5, 25, 50, 100, and 200 mg / mL, respectively, and mixed using a vortex mixer for approximately 10 seconds. The supernatant was then collected as the extract. The sensor was placed in a white imaging chamber with the sensor immersed in the extract, and images of the reaction zones were captured at 1-minute intervals starting from the start of immersion. The signal intensity of each reaction zone was calculated; the K value was calculated using the signal intensity of reaction zones 1 and 3, and the Ki value was calculated using the signal intensity of reaction zones 2 and 3.

[0252] Show the captured images Figure 10 The image showing its G component will be displayed. Figure 11A and Figure 11B The measured K and Ki values ​​are shown in Figure 12 and Table 5. In Figure 11A and Figure 11B The absorbance of component R within the region of interest (represented by circles in the figure) set on the reaction section is also displayed. Compared with extracts with a concentration of less than 50 mg / mL, there is no significant difference in signal intensity between reaction sections 1 and 2 in extracts with a concentration of more than 100 mg / mL. The signal intensity saturates 6 minutes after the start of the reaction. Figure 10 (within the dashed box). Furthermore, in extracts with concentrations below 50 mg / mL, the K and Ki values ​​are similar, while in extracts with concentrations above 100 mg / mL, both K and Ki values ​​are high. It is speculated that signal intensity saturation prevents accurate calculation of the K and Ki values, resulting in calculated values ​​that are higher than the actual values.

[0253] [Table 5]

[0254]

[0255] (2) Simultaneous determination of dilution series using multiple sensors (yellowfish chunks)

[0256] In Amplite TM Finely chopped yellowtail pieces were added to a diluent prepared by diluting the Red stock solution 100-fold with 1M Tris-HCl at pH 8.5, at concentrations of 230, 110, 57, 74, 37, and 19 mg / mL, respectively, and vortexed for approximately 10 seconds. The supernatant was then collected as the extract. The sensor was placed in a white imaging chamber with the extract immersed in it, and images of the reaction zones were taken at 1-minute intervals starting from the start of immersion. The signal intensity of each reaction zone was calculated; the K value was calculated using the signal intensity of reaction zones 1 and 3, and the Ki value was calculated using the signal intensity of reaction zones 2 and 3.

[0257] Show the captured images Figure 13 The image showing its G component will be displayed. Figure 14A and Figure 14B The measured K and Ki values ​​are shown in Figure 15 and Table 6. In Figure 14A and Figure 14B In the figure, the absorbance of component R within the region of interest (represented by circles) set on the reaction section is also displayed. Compared with extracts with a cut concentration of less than 57 mg / mL, there is no significant difference in signal intensity between reaction sections 1 and 2 in extracts with a cut concentration of more than 74 mg / mL. The signal intensity saturates 7 minutes after the start of the reaction. Figure 13 (within the dashed box). Furthermore, in extracts with concentrations below 57 mg / mL, the K and Ki values ​​were similar, while in extracts with concentrations above 74 mg / mL, both K and Ki values ​​were high. It is speculated that signal intensity saturation prevented accurate calculation of the K and Ki values, resulting in calculated values ​​that are higher than the actual values.

[0258] [Table 6]

[0259]

[0260] These results demonstrate that by performing staged dilutions of the sample solution, allowing the sensor to react with each dilution series, and selecting sensors with gradient signal intensities in each reaction zone—specifically, selecting sensors whose signal intensity decreases as the reaction zone number increases—it is possible to evaluate ATP-related substances in a single analysis, even in sample solutions with unknown concentrations. Therefore, for example, it is possible to calculate K and Ki values ​​in a single measurement without considering the influence of sample solution extraction efficiency.

[0261] Furthermore, these results indicate that as long as the signal intensity is not saturated, the calculated K and Ki values ​​remain constant even with different fish meat concentrations. Therefore, the K and Ki values ​​can be calculated independently of the amount of fish meat used to prepare the sample solution.

[0262] [Example 4. Comparison with HPLC]

[0263] (1) Determination of ATP-related substances using hexagram extract

[0264] Quantitative analysis using HPLC

[0265] Quantitative analysis of ATP-related substances was performed according to the JAS assay (JAS0023). Specifically, six rockfish were purchased from a fish market. The central portion of the back muscle from the side of the body was cut off, and the bloodline was removed to obtain the ordinary meat. Approximately 2 grams of the meat was crushed with a knife and weighed into a disposable test tube. 30 mL of cold 5% perchloric acid solution was added for homogenization. While measuring the pH with pH paper, 0.5 N and 0.1 N sodium hydroxide solutions were added to the homogenate and mixed to adjust the pH to 2.5–3.5. Water was added to bring the total volume to 50 mL, and the mixture was ice-cooled for at least 30 minutes. The filtrate was then filtered through a 0.45 μm PTFE membrane filter. This process was performed simultaneously with the ice-cooling of the sample.

[0266] The ATP-related substances in fish meat vary depending on freshness. Therefore, as a standard addition area containing all ATP-related substances, 1 mL of a standard solution containing each ATP-related substance at a concentration of approximately 800 μM was added to 9 mL of the above filtrate and mixed. Then, 1 M sodium hydroxide solution was added to adjust the pH to 7. At this point, the volume of sodium hydroxide solution added was recorded so that the proportion of filtrate contained in the pH-adjusted solution could be calculated. For the DW addition area, water was added instead of the standard solution, and the same operation was performed.

[0267] The quantification of various ATP-related substances was performed using HPLC equipped with a silica-based column via the following method. A solution adjusted to pH 7 was mixed with 0.25 mol / L phosphate buffer (pH 7.0) at a ratio of 4:1 (volume) to form the sample solution. The HPLC apparatus conditions were as follows.

[0268] Column: Capcelpak C18 AQ 5s 4.6 ID×250mm

[0269] Mobile phase: 0.05 mol / L phosphate buffer (pH 7.0), flow rate: 1.0 mL / min

[0270] Column temperature: 40℃

[0271] Measurement wavelength: 260nm

[0272] Injection volume: 10 μL

[0273] The results of quantification of ATP-related substances in each sample fluid by HPLC and the measured K and Ki values ​​calculated from the quantitative values ​​are shown in Table 7.

[0274] [Table 7]

[0275]

[0276] • Quantitative analysis using sensors (immersion in sample fluid)

[0277] A liquid was prepared by diluting the above 12 sample solutions with 1M Tris-HCl at pH 8.5 to a total concentration of 200 μM. An Amplex solution was added at 1% relative to the prepared 12 sample solutions. TM Red stock solution was used to quantify ATP-related substances using a sensor. The sensor was immersed in the sample solution and placed in a white imaging box. Reaction zones were imaged at 5-minute intervals starting from the start of immersion. The signal intensity of each reaction zone was calculated. 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.

[0278] Show the captured images Figure 16 The measured K and Ki values ​​are shown in Table 8. A scatter plot of HPLC-based K and Ki values ​​relative to the sensor-based measured K and Ki values ​​is also presented. Figure 17 Five minutes after the reaction started, deviations were observed in a portion of the measured K and Ki values. Furthermore, although the correlation with HPLC-based K and Ki values ​​was low, the deviations decreased at 15 and 20 minutes after the reaction started, and the correlations with HPLC-based Ki and K values ​​became high. These results indicate that by extending the sensor's reaction time, K and Ki values ​​can be quantified with the same accuracy as conventional HPLC methods.

[0279] [Table 8]

[0280]

[0281] • Quantitative analysis using sensors (addition of sample fluid)

[0282] Except for changing the shape of the test strip from a 4mm × 4mm square to an 8mm diameter circle, the sensor was fabricated using the same method as in Example 1. 3μL of the sample solution used for the sensor was added to each reaction section of the sensor. The sensor was placed in a white imaging box, and images of the reaction sections were taken at 5-minute intervals starting from the time of addition. The signal intensity of each reaction section was calculated; the K value was calculated using the signal intensity of reaction sections 1 and 3, and the Ki value was calculated using the signal intensity of reaction sections 2 and 3.

[0283] Images of representative examples of sensors that react with sample fluids are shown below. Figure 18 A scatter plot of HPLC-based K and Ki values ​​was generated relative to the sensor-based measured K and Ki values ​​at 20 minutes after the start of the reaction. Figure 19 Similar to the case where the reaction is carried out by immersion in the sample solution, a high correlation was confirmed with the Ki and K values ​​based on HPLC.

[0284] (2) Determination of ATP-related substances using extracts from various fish species

[0285] Using the same method as described in (1) above, a sample solution (standard solution addition area and DW addition area) for HPLC analysis was prepared from the common meat of coho salmon, rockfish, and yellowtail. ATP-related substances were quantified using HPLC, and the Ki value was calculated. Additionally, using the same method as described in (1) above, the sample solution for HPLC analysis was diluted to prepare a sample solution for the sensor. The sample solution was added to the sensor, and the Ki value was calculated from the signal intensity of each reaction section captured 5 minutes after addition.

[0286] A scatter plot of Ki values ​​based on the HPLC method was generated relative to the sensor-based Ki values. Figure 20 (Silver salmon) Figure 21 (Six-lined fish) Figure 22 (Yellowtail), a scatter plot summarizing the data of silver salmon, rockfish, and yellowtail will be created. Figure 23 Even in scatter plots summarizing the three fish species, sensor-based Ki values ​​showed a high correlation with HPLC-based Ki values. These results demonstrate that the sensor method can achieve the same quantification as the conventional HPLC method, regardless of the fish species.

[0287] [Example 5. Determination of ATP-related substances using fish extract]

[0288] In Example 3, Amplite is used. TM Red buffer (1M Tris-HCl, pH 8.5) was used to prepare fish meat extracts for use as sample solutions, but even when using solutions that do not contain Amplite... TMRed buffer was used to prepare the fish extract, by mixing it with a solution containing Amplite. TM The sample solution prepared with Red buffer can also be used to measure ATP-related substances. An example of such sample solution preparation is as follows: Place a few mm square pieces of fish meat into a 1.5 mL volumetric microtube containing 0.5 mL of buffer (1 M Tris-HCl, pH 8.5), cap, and shake for 20-30 seconds. Collect the liquid portion with a dropper and add it to Amplite. TM Add 1 drop to 0.25 mL of the diluted Red stock solution obtained by diluting it 100 times with buffer (1M Tris-HCl, pH 8.5) and mix to prepare the sample solution.

[0289] Furthermore, even instead of immersing the fish in the buffer solution, a sample solution prepared by repeatedly contacting a rod-shaped instrument with the surface of the fish in the buffer solution followed by immersion of the instrument in the buffer solution can still be used to measure ATP-related substances. An example of such a sample solution preparation method is as follows: After repeatedly swabbing the surface of the fish with a plastic rod (approximately 3 mm in diameter), the sample is immersed in a solution containing Amplite... TM The Red stock solution was diluted 100-fold with buffer solution (1M Tris-HCl, pH 8.5) to prepare a 0.25 mL volumetric tube (7.8 mm outer diameter × 40 mm length × 6 mm inner diameter). The sample solution was prepared by immersing the sensor in this sample solution for 1-2 seconds and then immediately removing it. After soaking for 5 minutes, the reaction area was photographed using a digital camera with a built-in smartphone. The captured image is shown below. Figure 24 It should be noted that in this experiment, a sensor with three reaction parts, namely reaction part 2, reaction part 3 and reaction part 5, was fabricated and used in the same manner as in Example 1.

[0290] [Example 6. Quantification of ATP-related substances with varying concentrations of chromogenic compounds]

[0291] The Amplite prepared in Example 2(1) TM The Red stock solution was diluted 100-fold with 1M Tris-HCl at pH 8.5 to prepare a 100% solution. This 100% solution was further diluted 7 / 10, 5 / 10, and 3 / 10 times with 1M Tris-HCl at pH 8.5 to prepare 70%, 50%, and 30% solutions, respectively. ATP, ADP, AMP, IMP, HxR, and Hx were dissolved in each of the 100%, 70%, 50%, and 30% solutions to prepare Amplite with a theoretical Ki value of 20. TMFour 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). Using these mixed standard solutions, the sensor was immersed in the sample solution in the same way as in Example 2 (2). After removal, the Ki value was calculated from the signal intensity of each reaction part.

[0292] Images taken after 5 minutes of immersion are shown. Figure 25 The Ki value, calculated based on images taken at 2-minute intervals until 14 minutes of immersion, is shown below. Figure 26 Compared to the 30% and 50% solutions, the Ki values ​​in the 70% and 100% solutions showed smaller deviations. (Amplite) TM Red may deteriorate depending on storage conditions, but by using Amplite at the same concentration as 100% liquid... TM Red, even Amplite TM Even with slight degradation of red, stable signal detection is still possible.

[0293] Explanation of reference numerals in the attached figures

[0294] 1. Sensor for detecting ATP-related substances

[0295] 2. Support

[0296] 3. Grip section

[0297] 11 Reaction Section 1

[0298] 12 Reaction Section 2

[0299] 13 Reaction Section 3

[0300] 14 Reaction Section 4

[0301] 15. Reaction section 5.

Claims

1. A sensor for ATP-related substance detection, having at least one of a reaction section 1 having a combination of enzymes capable of converting ATP, ADP and AMP into xanthine, and a reaction section 2 having a combination of enzymes capable of converting inosinic acid into xanthine, both held on a solid phase carrier, and a reaction section 3 having a combination of enzymes capable of converting inosine into xanthine, held on a solid phase carrier, on the same support. The combination of enzymes in each reaction section includes xanthine oxidase.

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

3. The sensor of claim 1, wherein, Each reaction section further has peroxidase held on a solid phase carrier.

4. The sensor of claim 1, wherein, The reaction section 1 has alkaline phosphatase, adenosine deaminase, purine nucleoside phosphorylase and xanthine oxidase held on a solid phase carrier.

5. The sensor of claim 1, wherein, The reaction section 2 has alkaline phosphatase, purine nucleoside phosphorylase and xanthine oxidase held on a solid phase carrier.

6. The sensor of claim 1, wherein, The reaction section 3 has purine nucleoside phosphorylase and xanthine oxidase held on a solid phase carrier.

7. A method for ATP-related substance detection, comprising: (A) a step of bringing the sensor for ATP-related substance detection according to any one of claims 1 to 6 into contact with a test liquid; (B) a step of detecting a signal from hydrogen peroxide in each reaction section of the sensor.

8. The method of claim 7, wherein, In step (A), the sensor is brought into contact with the test liquid by being immersed in the test liquid.

9. The method of claim 7, wherein, In step (B), the signal is detected while the sensor is immersed in the test liquid.

10. The method of claim 7, wherein, The test liquid has a pH of 8 to 10.

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

12. The method of claim 7, wherein, Each reaction section of the sensor further has peroxidase held on a solid phase carrier, and the test liquid contains a compound that develops color or luminescence in the presence of peroxidase and hydrogen peroxide.

13. The method of claim 7, further comprising: (C) a step of evaluating the concentration of ATP-related substance contained in the test liquid based on the intensity of the signal detected in each reaction section.

14. The method of claim 13, wherein, The K value is calculated from the intensities of the signals detected in the reaction section 1 and the reaction section 3 according to the following formula 1, [Formula 1] 。 15. The method of claim 13, wherein, The Ki value is calculated from the intensities of the signals detected in the reaction section 2 and the reaction section 3 according to the following formula 2, [Formula 2] 。 16. The method of claim 13, wherein, The concentration of ATP-related substance contained in the test liquid is evaluated based on the intensity of the signal detected within 2 to 5 minutes after the sensor is brought into contact with the test liquid.

17. The method of claim 13, wherein, The concentration of ATP-related substance contained in the test liquid is evaluated based on the intensity of the signal detected after a time longer than 5 minutes has elapsed after the sensor is brought into contact with the test liquid.

18. The method according to claim 13, wherein In step (A), a plurality of sensors are each brought into contact with a plurality of test liquids each having a different dilution factor, In step (B), a signal from hydrogen peroxide is detected in each reaction section of the plurality of sensors, In step (C), a sensor in which the signal detected in each reaction section becomes weaker as the number of the reaction section becomes larger is selected from among the plurality of sensors, and the concentration of ATP-related substance contained in the test liquid is evaluated based on the intensity of the signal detected in each reaction section of the sensor.

19. The method of claim 17, wherein, The total concentration of ATP, ADP, and AMP in the test liquid is evaluated based on the difference between the intensities of the signals detected in each of the reaction section 1 and the reaction section 2.

20. The method of claim 17, wherein, The concentration of inosinic acid in the test liquid is evaluated based on the difference between the intensities of the signals detected in each of the reaction section 2 and the reaction section 3.

21. A kit for ATP-related substance detection, comprising one or more of the ATP-related substance detection sensors according to any one of claims 1 to 6.

22. A kit for ATP-related substance detection, comprising the ATP-related substance detection sensor according to any one of claims 1 to 6, and a reaction container for housing the sensor and a test liquid.

23. The kit of claim 22, wherein, The reaction container has a portion composed of a visible light-transmissive material for observing the reaction section of the sensor from the outside.

24. A system for detecting an ATP-related substance, comprising: The ATP-related substance detection sensor according to any one of claims 1 to 6, the imaging unit, and the image processing unit.

25. The system according to claim 24, further comprising a reaction container for housing the sensor and a test liquid.

Images