Method and kit for evaluating enzyme activity

Abstract

Disclosed is a method for evaluating the oxidation activity of a dehydrogenase, the method comprising causing the dehydrogenase and DT-diaphorase to catalyze an oxidation-reduction reaction in an aqueous solution containing one molecule of the dehydrogenase, a substrate of the dehydrogenase, a nucleoside diphosphate derivative, the DT-diaphorase, and a fluorescent probe, and measuring the fluorescence after the reaction.

Description

Methods and kits for assessing enzyme activity

[0001] The present disclosure relates to methods and kits for the assessment of enzyme activity.

[0002] Enzymes are biopolymers composed primarily of proteins that act as catalysts in chemical reactions within the body. There are over thousands of enzymes in the body, and metabolic reactions performed by these enzymes maintain homeostasis. Abnormalities in their function are known to be associated with many diseases. Enzyme activity in the body is regulated not only by its amino acid sequence but also by various factors, such as post-translational modifications and allosteric regulation by metabolites. Therefore, when evaluating enzymes derived from biological samples, it is extremely effective to assess their activity in addition to simply assessing their expression levels and changes at the gene level.

[0003] Enzymes in vivo exist as multiple subtypes that target the same substrate but whose activity varies depending on the tissue of origin. Some of these subtypes are expressed at extremely low levels, and it is speculated that some of these subtypes may exhibit increased activity only in specific diseases, such as cancer. However, conventional enzyme activity assessment methods, such as those using standard substrates, assess the total (bulk) activity of all enzymes present in a sample, thereby overshadowing the activity of subtypes with low levels of expression and preventing significant differences from being detected. To assess the activity of such subtypes with low levels of expression, an assessment method that measures enzyme activity at the single-molecule level is useful (Non-Patent Document 1). A microchamber that enables such single-molecule analysis has also been reported (Patent Document 1).

[0004] Dehydrogenase is an oxidase that oxidizes a substrate using an oxidized nucleoside diphosphate derivative, such as an oxidized nicotinamide adenine dinucleotide derivative or an oxidized flavin adenine dinucleotide derivative, as a coenzyme. Many dehydrogenases are present in enzymes that constitute metabolic pathways such as glycolysis. Many dehydrogenases have also been reported to be associated with diseases such as cancer.

[0005] The present inventors have developed a coupled assay system for detecting the activity of D-lactate dehydrogenase, a type of dehydrogenase (Non-Patent Document 2).

[0006] U.S. Patent No. 9,797,837

[0007] Yannick Rondelez et al., "Microfabricated arrays of femtoliter chambers allow single molecule enzymology", Nat. Biotech. 23(3), 361-365 (2005).Kouichi Yanagi et al., "Development of pathway-oriented screening to identify compounds to control 2-methylglyoxal metabolism in tumor cells", Commun.Chem. 6, 68 (2023).Narae Shin et al., "Development of an Azoreductase-based Reporter System with Synthetic Fluorogenic Substrates", ACS Chem. Biol. 12(2):558-563 (2017).Chengli Jia et al., "A fast-responsive fluorescent turn-on probe for nitroreductase imaging in living cells", RSC Advances 11(15):8516-8520 (2021).Kouichi Yanagi et al., “Establishment of "live-cell-based coupled assay system for identification of compounds to modulate metabolic activities of cells", Cell Reports., 36(1), 109311 (2021).

[0008] To date, there have been no known cases in which the oxidative activity of a dehydrogenase has been detected at the single-molecule level, nor have there been any known cases in which a coupled assay for detecting the activity of an enzyme to be evaluated has been performed under conditions in which the enzyme to be evaluated is present at only a single molecule level.

[0009] Furthermore, to directly evaluate the oxidative activity of dehydrogenases using fluorescent probes using conventional evaluation methods, it is necessary to design and develop a fluorescent probe for each dehydrogenase that serves as a substrate for the dehydrogenase and whose fluorescence intensity increases upon enzymatic reaction. Furthermore, in single-molecule activity detection, to detect and distinguish signals from multiple subtypes from the fluorescence pattern, it is necessary to develop multiple fluorescent probes that serve as substrates for a given dehydrogenase. However, many dehydrogenases have high substrate specificity, and it is extremely difficult to develop multiple fluorescent probes that serve as substrates for such enzymes.

[0010] An object of the present disclosure is to provide a method for evaluating the oxidative activity of a dehydrogenase, and a method for evaluating enzyme activity at the single molecule level.

[0011] The present inventors have found that oxidative activity of a dehydrogenase molecule can be evaluated at the single-molecule level by a coupled assay in an aqueous solution containing a single molecule of dehydrogenase, a substrate for the dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe. Furthermore, they have found that, even when a single type of fluorescent probe is used, different fluorescence patterns can be obtained from the same sample by using different dehydrogenase substrates and / or nucleoside diphosphate derivatives. This discovery has led to the completion of the present disclosure.

[0012] The present inventors have also found that the oxidative activity of a metabolic enzyme can be evaluated at the single molecule level by a coupled assay in an aqueous solution containing a single molecule of the metabolic enzyme, a substrate for the metabolic enzyme, a dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe.

[0013] The present disclosure relates to, for example, the following [1] to

[11] : [1] A method for evaluating the oxidizing activity of a dehydrogenase, comprising the following steps (A) to (C): step (A): preparing an aqueous solution containing one molecule of a dehydrogenase, a substrate for the dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe; step (B): allowing the dehydrogenase and the DT-diaphorase to catalyze an oxidation-reduction reaction in the aqueous solution prepared in step (A); and step (C): measuring the fluorescence of the aqueous solution obtained in step (B); wherein the dehydrogenase is capable of utilizing the oxidized nucleoside diphosphate derivative as a coenzyme, the DT-diaphorase is capable of utilizing the reduced nucleoside diphosphate derivative as a coenzyme, and the fluorescent probe is a compound whose fluorescence intensity increases when reduced by the DT-diaphorase. [2] The evaluation method according to [1], wherein the step (A) is a step of filling a plurality of storage units of a microchamber array having a hydrophobic surface with an aqueous solution containing the dehydrogenase, a substrate for the dehydrogenase, the nucleoside diphosphate derivative, the DT-diaphorase, and the fluorescent probe, by pouring the aqueous solution containing the dehydrogenase, the substrate for the dehydrogenase, the nucleoside diphosphate derivative, the DT-diaphorase, and the fluorescent probe through openings of the storage units so that the number-average molecular number of the dehydrogenase filled in each of the plurality of storage units is 0.0002 to 0.5, and closing the openings with a hydrophobic solvent. [3] The fluorescent probe is -CO ２ H, -PO ３ H ２ and -SO ３ [4] The evaluation method according to [1] or [2], wherein the nucleoside diphosphate derivative has at least one substituent selected from the group consisting of H. ... (In the formula, R １ is a monovalent group, and X １ is —H, —OH or —PO ３ H ２ and Y １is O or S.) [5] The evaluation method according to any one of [1] to [4], wherein the DT-diaphorase is at least one compound selected from the group consisting of compounds represented by the following formula:

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[10] The evaluation method according to any one of [1] to [5], wherein the DT-diaphorase is at least one compound selected from the group consisting of compounds represented by the following formula:

[0023]

[0024]

[0025]

[11] The evaluation method according to any one of [1] to [5], wherein the dehydrogenase is a dehydrogenase contained in at least one selected from the group consisting of plasma, serum, urine, saliva, tears, cerebrospinal fluid, tissue homogenate, cell homogenate, and extracts thereof. [7] A method for evaluating the oxidation activity of a dehydrogenase, comprising the following steps (A-1) to (C-1) and steps (A-2) to (C-2): step (A-1): preparing an aqueous solution containing one molecule of a dehydrogenase, a substrate for the dehydrogenase, a first nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe; step (B-1): allowing the dehydrogenase and the DT-diaphorase to catalyze an oxidation-reduction reaction in the aqueous solution prepared in step (A-1); step (C-1): measuring the fluorescence of the aqueous solution obtained in step (B-1); step (A-2): preparing an aqueous solution containing one molecule of the dehydrogenase, a substrate for the dehydrogenase, a second nucleoside diphosphate derivative, DT-diaphorase, and the fluorescent probe; step (B-2): allowing the dehydrogenase and the DT-diaphorase to catalyze an oxidation-reduction reaction in the aqueous solution prepared in step (A-2); and step (C-2): measuring the fluorescence of the aqueous solution obtained in step (B-2); wherein the first nucleoside diphosphate derivative is different from the second nucleoside diphosphate derivative, the dehydrogenase can utilize the oxidized first nucleoside diphosphate derivative and the oxidized second nucleoside diphosphate derivative as coenzymes, the DT-diaphorase can utilize the reduced first nucleoside diphosphate derivative and the reduced second nucleoside diphosphate derivative as coenzymes, and the fluorescent probe is a compound whose fluorescence intensity increases when reduced by the DT-diaphorase.[8] A method for evaluating the oxidation activity of a dehydrogenase, comprising the following steps (A-3) to (C-3) and steps (A-4) to (C-4): step (A-3): preparing an aqueous solution containing one molecule of a first dehydrogenase, a substrate for the first dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe; step (B-3): allowing the first dehydrogenase and the DT-diaphorase to catalyze an oxidation-reduction reaction in the aqueous solution prepared in step (A-3); step (C-3): measuring the fluorescence of the aqueous solution obtained in step (B-3); step (A-4): preparing an aqueous solution containing one molecule of a second dehydrogenase, a substrate for the second dehydrogenase, the nucleoside diphosphate derivative, DT-diaphorase, and the fluorescent probe; step (B-4): allowing the second dehydrogenase and the DT-diaphorase to catalyze an oxidation-reduction reaction in the aqueous solution prepared in step (A-4); and step (C-4): measuring the fluorescence of the aqueous solution obtained in step (B-4), wherein a substrate for the first dehydrogenase is different from a substrate for the second dehydrogenase, the first dehydrogenase and the second dehydrogenase can utilize the oxidized form of the nucleoside diphosphate derivative as a coenzyme, the DT-diaphorase can utilize the reduced form of the nucleoside diphosphate derivative as a coenzyme, and the fluorescent probe is a compound whose fluorescence intensity increases when reduced by the DT-diaphorase. [9] A kit for evaluating the oxidizing activity of a dehydrogenase, comprising: a dehydrogenase substrate, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe, wherein the dehydrogenase can use the oxidized nucleoside diphosphate derivative as a coenzyme, the DT-diaphorase can use the reduced nucleoside diphosphate derivative as a coenzyme, and the fluorescent probe is a compound whose fluorescence intensity increases when reduced by the DT-diaphorase.

[10] A method for evaluating the metabolic activity of a metabolic enzyme, comprising the following steps (A) to (C): step (A): preparing an aqueous solution containing one molecule of the metabolic enzyme, a substrate for the metabolic enzyme, a dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe; step (B): allowing the metabolic enzyme, the dehydrogenase, and the DT-diaphorase to catalyze an enzymatic reaction in the aqueous solution prepared in step (A); and step (C): measuring the fluorescence of the aqueous solution obtained in step (B); wherein the dehydrogenase is a dehydrogenase that utilizes, as a substrate, a metabolite obtained by metabolism of a substrate for the metabolic enzyme by the metabolic enzyme, the dehydrogenase can utilize the oxidized form of the nucleoside diphosphate derivative as a coenzyme, the DT-diaphorase can utilize the reduced form of the nucleoside diphosphate derivative as a coenzyme, and the fluorescent probe is a compound whose fluorescence intensity increases when reduced by the DT-diaphorase.

[11] A kit for evaluating the metabolic activity of a metabolic enzyme, comprising: a substrate for the metabolic enzyme; a dehydrogenase; a nucleoside diphosphate derivative; DT-diaphorase; and a fluorescent probe; wherein the dehydrogenase uses as a substrate a metabolite obtained by metabolizing the substrate for the metabolic enzyme by the metabolic enzyme; the dehydrogenase can use the oxidized form of the nucleoside diphosphate derivative as a coenzyme; the DT-diaphorase can use the reduced form of the nucleoside diphosphate derivative as a coenzyme; and the fluorescent probe is a compound whose fluorescence intensity increases when reduced by the DT-diaphorase.

[0014] According to the present disclosure, it is possible to provide a method for evaluating the oxidizing activity of a dehydrogenase, and also to provide a method for evaluating enzyme activity at the single molecule level.

[0015] According to one embodiment of the present disclosure, the oxidizing activity of a dehydrogenase can be evaluated (detected) at the level of a single enzyme molecule.

[0016] According to one embodiment of the present disclosure, even when one type of fluorescent probe is used, the oxidative activities of a plurality of types of dehydrogenases can be detected (evaluated) by using substrates for various dehydrogenases.

[0017] According to one embodiment of the present disclosure, even if one type of fluorescent probe is used, the oxidizing activity of multiple types of dehydrogenases can be detected (evaluated) by using various nucleoside diphosphate derivatives.

[0018] According to one embodiment of the present disclosure, whether a sample satisfies a specific condition can be evaluated by detecting the enzymatic activity of the dehydrogenase in the sample, utilizing a change in the oxidizing activity of a dehydrogenase derived from the sample. In this case, according to one embodiment of the present disclosure, even if a single type of fluorescent probe is used, a coupled assay system suitable for evaluating a sample that satisfies such a specific condition can be found by using various dehydrogenase substrates and various nucleoside diphosphate derivatives.

[0019] 1A is a diagram showing the absorption spectra of Q-sHMRG before and after the reduction reaction by NADH:quinone oxidoreductase in Reference Example 1. FIG. 1B is a diagram showing the fluorescence intensity derived from Q-sHMRG when the reduction reaction by NADH:quinone oxidoreductase was carried out in the presence of various concentrations of NADH in Reference Example 1. FIG. 1C is a diagram showing the fluorescence intensity derived from Q-sHMRG when the reduction reaction by NADH:quinone oxidoreductase was carried out in the presence of various concentrations of NADH in Reference Example 1. FIG. 2 is a diagram showing the fluorescence intensity derived from Q-sHMRG when the reduction reaction by NADH:quinone oxidoreductase was carried out in the presence of various concentrations of NADH in Example 1. ＋ 3 is a diagram showing a fluorescent image and a line plot of the fluorescent intensity when an evaluation method according to an embodiment of the present disclosure is performed using plasma derived from a healthy subject as a sample, using various dehydrogenase substrates and NADPH as a nucleoside diphosphate derivative in Example 2. ＋4 is a diagram showing a fluorescent image obtained when an evaluation method according to an embodiment of the present disclosure was performed using plasma derived from a healthy subject as a sample, using lactate, glucose-6-phosphate, isocitrate, or 6-phosphogluconate as a dehydrogenase substrate, and NADPH as a nucleoside diphosphate derivative in Example 3. ＋ , NADP ＋ , Thio-NAD ＋ or NDG ＋ 5 is a diagram showing a fluorescent image obtained when an evaluation method according to an embodiment of the present disclosure is performed using plasma derived from a healthy subject as a sample, using glucose-6-phosphate or 6-phosphogluconate as a dehydrogenase substrate, and NADPH as a nucleoside diphosphate derivative in Example 4. ＋ , NADP ＋ or Thio-NAD ＋8 is a diagram showing fluorescence images obtained when an evaluation method according to an embodiment of the present disclosure was performed using plasma samples derived from healthy subjects or pancreatic cancer patients, respectively. FIG. 6 is a diagram showing the results of evaluating the activity of metabolic enzymes contained in plasma derived from pancreatic cancer patients in Example 5. FIG. 7 is a diagram showing fluorescence images obtained when samples derived from mice not subjected to TAA treatment (control group) or mice subjected to TAA treatment (TAA group) were used under conditions in which glucose-6-phosphate was added in Example 6. FIG. 8 is a diagram showing fluorescence images obtained when samples derived from mice not subjected to TAA treatment (control group) or mice subjected to TAA treatment (TAA group) were used under conditions in which lactic acid was added in Example 6. FIG. 8 is a histogram showing the distribution of fluorescence intensity for each well in which fluorescence was detected in a fluorescence image obtained under the same conditions as in FIG. 7 and FIG. 8 in Example 6. 11 is a diagram showing fluorescence images obtained using CSF samples derived from PCNSL patients (PCNSL group) and subjects in which PCNSL had regressed (Regression group) in Example 7. FIG. 12 is a diagram showing the results of counting the number of wells in which fluorescence was observed in fluorescence images obtained using CSF samples derived from PCNSL patients (PCNSL group), subjects in which PCNSL had regressed (Regression group), patients in which PCNSL recurred (Recurrence group), and subjects in which PCNSL had gone into remission (Remission group) in Example 7, which were obtained under the same conditions as in FIG. 11. FIG. 13 is a diagram showing the rate of fluorescence increase ( / min) in the presence of substrates for various metabolic enzymes when plasma samples were added at 1000-fold or 100-fold dilution in Comparative Example 1. FIG. 14 is a diagram showing fluorescence images of the device 20 hours after addition of plasma in Example 8.

[0020] Hereinafter, embodiments for carrying out the present disclosure will be described in detail, but the present disclosure is not limited to the following embodiments.

[0021] In this disclosure, unless otherwise specified, amino acids refer to amino acids in the L-configuration. For example, in this disclosure, the terms "alanine" and "L-alanine" refer to alanine in the L-configuration, and the term "D-alanine" refers to alanine in the D-configuration.

[0022] <First Aspect> A first aspect of the present disclosure is a method for evaluating the oxidizing activity of a dehydrogenase, the method comprising the following steps (A) to (C): step (A): preparing an aqueous solution containing one molecule of a dehydrogenase, a substrate for the dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe (preparation step); step (B): allowing the dehydrogenase and the DT-diaphorase to catalyze an oxidation-reduction reaction in the aqueous solution prepared in step (A) (reaction step); and step (C): measuring the fluorescence of the aqueous solution obtained in step (B) (fluorescence measurement step); wherein the dehydrogenase is capable of utilizing the oxidized nucleoside diphosphate derivative as a coenzyme, the DT-diaphorase is capable of utilizing the reduced nucleoside diphosphate derivative as a coenzyme, and the fluorescent probe is a compound whose fluorescence intensity increases when reduced by the DT-diaphorase. That is, a first aspect of the present disclosure is a method for performing a coupled assay shown in the following reaction formula at the level of a single dehydrogenase molecule. In the present disclosure, a coupled assay refers to an enzymatic assay performed by a combination of multiple enzymatic reactions. In the coupled assay shown in the following reaction formula, the enzymatic reaction by the dehydrogenase is made the rate-limiting step of the entire coupled assay system, that is, the reduced nucleoside diphosphate derivative produced by the enzymatic reaction of the dehydrogenase is quickly consumed in the reduction reaction of the fluorescent probe by DT-diaphorase. This allows the oxidizing activity of the dehydrogenase to be evaluated using the fluorescence of the reduction product of the enzymatic reaction by DT-diaphorase as an indicator.

[0023] <Dehydrogenase> Dehydrogenase is an oxidase (oxidoreductase) that oxidizes a substrate using an oxidized nucleoside diphosphate derivative, such as an oxidized nicotinamide adenine dinucleotide derivative or an oxidized flavin adenine dinucleotide derivative, as a coenzyme. Many dehydrogenases are present in enzymes that constitute metabolic pathways such as glycolysis, and many dehydrogenases have been reported to be associated with diseases such as cancer.

[0024] In the present disclosure, the dehydrogenase is not particularly limited as long as it is an oxidase (oxidoreductase) that can oxidize a substrate using an oxidized nucleoside diphosphate derivative as a coenzyme. In a preferred embodiment of the present disclosure, the dehydrogenase is an oxidase (oxidoreductase) that can oxidize a substrate using an oxidized nicotinamide adenine dinucleotide derivative or an oxidized flavin adenine dinucleotide derivative as a coenzyme. In a preferred embodiment of the present disclosure, the dehydrogenase is an oxidase (oxidoreductase) that can oxidize a substrate using an oxidized nicotinamide adenine dinucleotide derivative or an oxidized flavin adenine dinucleotide derivative as a coenzyme. In addition, in a preferred embodiment of the present disclosure, the dehydrogenase is an oxidase (oxidoreductase) that can oxidize a substrate using an oxidized nicotinamide adenine dinucleotide derivative or an oxidized flavin adenine dinucleotide derivative as a coenzyme. In the EC7 EC classification established by the Committee on Biology in 2019, the enzyme may be classified as EC1.1.1, EC1.2.1, EC1.3.1, EC1.4.1, EC1.4.4, EC1.5.1, EC1.6 (e.g., EC1.6.1), EC1.7.1, EC1.8.1, EC1.10.1, EC1.12.1, EC1.14.12, EC1.14.13, EC1.14.21, EC1.17.1, EC1.18.1, EC1.19.1, EC1.20.1, EC1.21.1, or EC1.23.1. In a more preferred embodiment, the dehydrogenase may be an enzyme classified in EC7 as EC1.1.1, EC1.2.1, EC1.3.1, EC1.4.1, EC1.4.4, EC1.5.1, EC1.6 (e.g., EC1.6.1), EC1.7.1, EC1.8.1, EC1.10.1, EC1.12.1, EC1.14.12, EC1.14.13, EC1.14.21, EC1.18.1, EC1.19.1, or EC1.21.1. In a further preferred embodiment, the dehydrogenase may be an enzyme classified in EC7 as EC1.1.1 or EC1.2.1.Examples of dehydrogenases according to an embodiment of the present disclosure include lactate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, cytochrome P450, glutathione reductase, and HMG-CoA reductase.

[0025] In one embodiment of the present disclosure, the dehydrogenase may be a dehydrogenase contained in at least one selected from the group consisting of plasma, serum, urine, saliva, tears, cerebrospinal fluid, tissue homogenates, cell homogenates, and extracts thereof. In a preferred embodiment, the dehydrogenase may be a dehydrogenase contained in at least one selected from the group consisting of plasma, serum, urine, saliva, tears, cerebrospinal fluid, and extracts thereof, and in a more preferred embodiment, a dehydrogenase contained in at least one selected from the group consisting of plasma, serum, tears, cerebrospinal fluid, and extracts thereof. When a sample from which the dehydrogenase is derived is evaluated by evaluating the oxidative activity of the dehydrogenase using the evaluation method according to one embodiment of the present disclosure, the sample may be at least one selected from the group consisting of plasma, serum, urine, saliva, tears, cerebrospinal fluid, tissue homogenates, cell homogenates, and extracts thereof.

[0026] <Substrate for dehydrogenase> The substrate for the dehydrogenase according to the present disclosure is not particularly limited as long as it is a substance that serves as a substrate for the dehydrogenase and is oxidized by the dehydrogenase. The substrate for the dehydrogenase according to the present disclosure may be a natural substrate for the dehydrogenase, or may be an artificial substrate such as a derivative of such a natural substrate.

[0027] Substrates that can be used for the dehydrogenases according to the present disclosure include, for example, glycine, serine, D-serine, glutamic acid, lysine, leucine, glutathione, glucose-6-phosphate, lactic acid, D-lactic acid, isocitrate, malic acid, inosinic acid, 6-phosphogluconic acid, succinic semialdehyde, terephthalaldehyde acid, and HMG-CoA (hydroxymethylglutaryl CoA).

[0028] The concentration of the dehydrogenase substrate at the start of step (B) (reaction step) in the first aspect of the present disclosure is not particularly limited as long as it is a concentration at which fluorescence can be detected in the subsequent step (C) (fluorescence measurement step), and may be, for example, 10 μM to 1 M, 30 μM to 100 mM, 100 μM to 30 mM, or 300 μM to 10 mM.

[0029] <Nucleoside diphosphate derivative> The nucleoside diphosphate derivative according to the present disclosure is a nucleoside diphosphate derivative that can be used as a coenzyme by the dehydrogenase according to the first aspect of the present disclosure and can be used as a coenzyme by the DT-diaphorase according to the first aspect of the present disclosure. In one embodiment of the present disclosure, the nucleoside diphosphate derivative may be a nicotinamide adenine dinucleotide derivative or a flavin adenine dinucleotide derivative, for example, a nucleoside diphosphate derivative represented by the following formula (I) or (II): (In the formula, R １ is a monovalent group, and X １ is —H, —OH or —PO ３ H ２ and Y １ is O or S.) The compounds represented by the above formulas (I) and (II) may be at least one compound selected from the group consisting of compounds represented by the following formulas (I') and (II'), respectively, in reduced form: The nucleoside diphosphate derivatives according to the present disclosure may be in the form of a salt, a solvate, or a solvate of a salt, for example, a sodium salt, a hydrochloride salt, or a hydrate.

[0030] The nucleoside diphosphate derivative according to the present disclosure may be a compound represented by the above formula (I) or (II), and in a more preferred embodiment and a most preferred embodiment, may be a compound represented by the above formula (I). １ is —H, —OH or —PO ３ H ２ For example, —OH or —PO ３ H ２In the above formula (I) or (II), R １ In one preferred embodiment, the compound has the following structural formula: (In the formula, R ２ is —H, an alkyl group, an alkylamino group, or an (aminoalkyl)amino group. ３ is a monovalent group, preferably H, an alkyl group, an acyl group, a benzyl group, or a furfuryl group, and more preferably —H, a benzyl group, a furfuryl group, or —C(═O)R ３ａ and more preferably —H or —C(═O)R ３ａ In the formula, R ３ａ In these cases, the alkyl contained in the alkyl group, alkylamino group, and (aminoalkyl)amino group is preferably C １ ~C ６ alkyl, more preferably C １ ~C ４ and alkyl, more preferably methyl, ethyl, or n-propyl. In a more preferred embodiment, the substituent may be a monovalent group selected from the group consisting of substituents represented by the following structural formula: (In the formula, R ２ is —H or a methyl group, and R ３ is a monovalent group.) In a most preferred embodiment, the substituent may be a monovalent group selected from the group consisting of substituents represented by the following structural formula: That is, in a most preferred embodiment, R １ may be a group of adenine (A), guanine (G), cytosine (C), thymine (T), or uracil (U) in which one hydrogen atom has been removed from a position corresponding to the phosphate modification site in the monophosphate form (AMP, GMP, CMP, TMP, or UMP) of these bases present in vivo.

[0031] The concentration of the nucleoside diphosphate derivative at the start of step (B) (reaction step) in the first aspect of the present disclosure is not particularly limited as long as it is a concentration at which fluorescence can be detected in the subsequent step (C) (fluorescence measurement step), and may be, for example, 1 μM to 10 mM, 3 μM to 3 mM, 10 μM to 1 mM, 30 μM to 800 μM, or 100 μM to 500 μM.

[0032] <DT-Diaphorase> The DT-diaphorase according to the present disclosure is an enzyme capable of reducing the fluorescent probe according to the first aspect of the present disclosure using the reduced form of the nucleoside diphosphate derivative according to the first aspect of the present disclosure as a coenzyme, and is not particularly limited as long as it is such an enzyme. The DT-diaphorase according to the present disclosure may be, for example, an NADH:quinone oxidoreductase, an azoreductase, or a nitroreductase. In a preferred embodiment, it may be an NADH:quinone oxidoreductase or an azoreductase. In a more preferred embodiment, it may be an NADH:quinone oxidoreductase. The origin of the NADH:quinone oxidoreductase according to the present disclosure is not particularly limited. For example, it may be derived from a human (NOQ1, NAD(P)H:Quinone Oxidoreductase 1) or a microbial organism (DAD-311, DT-Diaphorase-311), and in a preferred embodiment, it may be derived from a human (NOQ1).

[0033] The concentration of DT-diaphorase at the start of step (B) (reaction step) in the first aspect of the present disclosure is not particularly limited as long as it is a concentration at which fluorescence can be detected in the subsequent step (C) (fluorescence measurement step), and may be, for example, 0.1 μg / mL to 100 μg / mL, 0.5 μg / mL to 60 μg / mL, or 1.0 μg / mL to 30 μg / mL.

[0034] the number of molecules of DT-diaphorase present in the aqueous solution at the start of step (B) (reaction step) of the first aspect of the present disclosure, and the metabolic turnover number k of DT-diaphorase ｃａｔThe reaction rate constant k [ / sec] of the enzymatic reaction catalyzed by DT-diaphorase in an aqueous solution, defined as the product of the concentration of DT-diaphorase and the concentration of DT-diaphorase (per second) in the aqueous solution, is not particularly limited as long as the concentration allows detection of fluorescence in the subsequent step (C) (fluorescence measurement step), and may be, for example, 0.001 or more, 0.003 or more, and preferably 0.01 or more, 0.03 or more, or 0.1 or more. When the reaction rate constant k of the enzymatic reaction catalyzed by DT-diaphorase in an aqueous solution in step (B) (reaction step) is equal to or greater than the above-mentioned lower limit, the rate-limiting step in the entire coupled assay tends to be the enzymatic reaction catalyzed by the dehydrogenase, making it easier to evaluate the oxidizing activity of the dehydrogenase.

[0035] <Fluorescent Probe> The fluorescent probe according to the first aspect of the present disclosure is a compound whose fluorescence intensity increases upon reduction by the DT-diaphorase according to the first aspect of the present disclosure. The fluorescent probe according to the first aspect of the present disclosure can be designed or selected to suit the substrate properties of the DT-diaphorase according to the first aspect of the present disclosure. The fluorescence intensity of the compound (reduction product) obtained by reducing the fluorescent probe with DT-diaphorase may be, for example, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 7 times or more, 10 times or more, 15 times or more, 20 times or more, 30 times or more, 50 times or more, 70 times or more, or 100 times or more, and in a preferred embodiment, 5 times or more, of the fluorescence intensity of the fluorescent probe. These fluorescence intensities may be values ​​measured in, for example, phosphate-buffered saline (PBS) at pH 7.4.

[0036] The product of the molar extinction coefficient [L / mol cm] at the maximum absorption wavelength and the fluorescence quantum yield of a compound (reduction product) obtained by reducing the fluorescent probe according to the first aspect of the present disclosure with DT-diaphorase may be, for example, 2,000 or more, 5,000 or more, 10,000 or more, 15,000 or more, 20,000 or more, 30,000 or more, 40,000 or more, or 50,000 or more, and in a preferred embodiment, 10,000 or more. For example, sHMRG represented by the following structural formula: has a molar absorption coefficient of 80,000 and a fluorescence quantum yield of 0.92, and the product thereof is 74,000. Also, for example, 7-amino-4-methylcoumarin has a molar absorption coefficient of 20,000 and a fluorescence quantum yield of 0.42, and the product thereof is 8,400. These fluorescence properties may be values ​​measured in, for example, phosphate buffered saline (PBS) at pH 7.4.

[0037] The maximum absorption wavelength of the compound (reduction product) obtained by reducing the fluorescent probe according to the first aspect of the present disclosure with DT-diaphorase may be, for example, 300 nm or more, 320 nm or more, 340 nm or more, 360 nm or more, 380 nm or more, 400 nm or more, 410 nm or more, 420 nm or more, 430 nm or more, 440 nm or more, 450 nm or more, 460 nm or more, 470 nm or more, or 480 nm or more, and in a preferred embodiment, it may be 420 nm or more. The maximum fluorescence wavelength of the compound (reduction product) obtained by reducing the fluorescent probe according to the first aspect of the present disclosure with DT-diaphorase may be, for example, 400 nm or more, 420 nm or more, 430 nm or more, 440 nm or more, 450 nm or more, 460 nm or more, 470 nm or more, 475 nm or more, 480 nm or more, 485 nm or more, 490 nm or more, 495 nm or more, or 500 nm or more, and in a preferred embodiment, 450 nm or more. For example, the maximum absorption wavelength of sHMRG is 498 nm and the maximum fluorescence wavelength is 517 nm, and the maximum absorption wavelength of 7-amino-4-methylcoumarin is 350 nm and the maximum fluorescence wavelength is 440 nm. These fluorescence properties may be values ​​measured in, for example, phosphate-buffered saline (PBS) at pH 7.4. When the fluorescence intensity of the compound obtained by reducing the fluorescent probe with DT-diaphorase has the fluorescence properties described above, the fluorescence can be easily measured in step (C), and the activity of the dehydrogenase can be easily evaluated.

[0038] In a preferred embodiment of the first aspect of the present disclosure, the fluorescent probe is —CO ２ H, -PO ３ H ２ and -SO ３and in a more preferred embodiment, has at least one substituent selected from the group consisting of —SO ３ The fluorescent probe may have at least one substituent represented by H. When the fluorescent probe has these substituents, the solubility of the fluorescent probe in the aqueous solvent in step (B) is improved, thereby ensuring the concentration and suppressing precipitation, thereby facilitating fluorescence detection in the subsequent step (C).

[0039] In a preferred embodiment of the first aspect of the present disclosure, the fluorescent probe according to the first aspect of the present disclosure and / or a compound obtained by reducing the fluorescent probe with DT-diaphorase may have high water solubility, and the fluorescent probe according to the first aspect of the present disclosure and a compound obtained by reducing the fluorescent probe with DT-diaphorase may also have high water solubility. High water solubility may mean, for example, that when 1 μM of the compound is added to a two-layer system of octanol and phosphate-buffered saline (PBS) at pH 7.4 in a 1:1 volumetric ratio, the proportion of the compound that distributes in the PBS layer is, for example, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more.

[0040] In a preferred embodiment of the first aspect of the present disclosure, the fluorescent probe according to the first aspect of the present disclosure may have high reactivity with DT-diaphorase. High reactivity with DT-diaphorase means, for example, high reactivity with DT-diaphorase in the presence of 1 U / mL of DT-diaphorase and a predetermined concentration of an oxidized nucleoside diphosphate derivative (e.g., NADPH). ＋ or NADP ＋ The predetermined concentration may be, for example, 0.03, 0.1 mM, 0.3 mM, or 1 mM, and preferably 0.1 mM.

[0041] In the present disclosure, the fluorescent probe may be in the form of a salt, solvate, or solvate of a salt. The counter ion in such a salt or solvate thereof is not particularly limited, and may be, for example, a sodium ion, a potassium ion, an ammonium ion, a triethylammonium ion, an N,N-diisopropylethylammonium ion, a tetra-n-butylammonium ion, a chloride ion, an acetate ion, or a trifluoroacetate ion. The solvating solvent in such a solvate or solvate of a salt is not particularly limited, and may be, for example, water, acetonitrile, dimethyl sulfoxide (DMSO), methanol, acetone, dimethylformamide (DMF), or triethylamine.

[0042] The fluorescent probe according to the first aspect of the present disclosure may be represented, for example, by the following formula (III): X-NH-Y ... (III) or formula (III'): X-N=Y' ... (III'), where in formula (III) and formula (III'), X represents a fluorescent group or a partial structure thereof, Y and Y' represent leaving groups removed by the enzymatic reaction of DT-diaphorase according to the first aspect of the present disclosure, and X-NH ２ The fluorescence intensity of the compound represented by the structural formula of X-NH-Y is greater than that of the compound represented by the structural formula of X-N=Y'. Hereinafter, the compound represented by the structural formula of X-NH-Y and the compound represented by the structural formula of X-N=Y' will also be referred to as "compounds before elimination", and X-NH ２The compound represented by the structural formula (i.e., the reduction product of the reduction reaction with DT-diaphorase) is also referred to as the "compound after elimination." In this case, with respect to the fluorescence intensity of the compound before and after elimination, for example, the fluorescence intensity of the compound after elimination at a pH similar to the pH of the aqueous solution in step (B) (e.g., within ±1.0, ±0.5, ±0.2, ±0.1, ±0.05, or ±0.01) is greater than the fluorescence intensity of the compound before elimination under the same conditions. The fluorescence intensity in these cases may be, for example, the fluorescence intensity at the maximum fluorescence wavelength of the compound after elimination when excited with excitation light having a wavelength within the wavelength range of the excitation light irradiated in step (C). Furthermore, the fluorescence intensity of the compound after elimination may be, for example, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 7 times or more, 10 times or more, 15 times or more, 20 times or more, 30 times or more, 50 times or more, 70 times or more, or 100 times or more, and in a preferred embodiment, 5 times or more, of the fluorescence intensity of the compound before elimination. The product of the molar extinction coefficient [L / mol cm] at the maximum absorption wavelength of the compound after elimination and the fluorescence quantum yield may be, for example, 2000 or more, 5000 or more, 10000 or more, 15000 or more, 20000 or more, 30000 or more, 40000 or more, or 50000 or more, and in a preferred embodiment, may be 10000 or more. The maximum absorption wavelength of the compound after elimination may be, for example, 300 nm or more, 320 nm or more, 340 nm or more, 360 nm or more, 380 nm or more, 400 nm or more, 410 nm or more, 420 nm or more, 430 nm or more, 440 nm or more, 450 nm or more, 460 nm or more, 470 nm or more, or 480 nm or more, and in a preferred embodiment, may be 420 nm or more. The maximum fluorescence wavelength of the compound after elimination may be, for example, 400 nm or more, 420 nm or more, 430 nm or more, 440 nm or more, 450 nm or more, 460 nm or more, 470 nm or more, 475 nm or more, 480 nm or more, 485 nm or more, 490 nm or more, 495 nm or more, or 500 nm or more, and in a preferred embodiment, may be 450 nm or more. When the compound after elimination has the fluorescence properties described above, fluorescence can be easily measured in step (C), facilitating evaluation of dehydrogenase activity.

[0043] When the DT-diaphorase according to the first aspect of the present disclosure is an NADH:quinone oxidoreductase, the fluorescent probe according to the first aspect of the present disclosure may be, for example, a fluorescent probe represented by the following formula (IV): (wherein X is the same as X in formula (III)), or a compound represented by the following formula (V): (In the formula, Z １ ~Z ６ each independently represents a monovalent group, and preferably each independently represents —H or —CO ２ H, -PO ３ H ２ , -SO ３ H or an alkyl group, and more preferably each independently represents —H or —CO ２ H, -PO ３ H ２ or -SO ３ H), and preferably a compound represented by formula (IV).

[0044] The compound represented by formula (IV) is converted to a molecule with high fluorescence intensity, X-NH, by an enzymatic reaction (reduction reaction) catalyzed by the following NADH:quinone oxidoreductase: ２ This results in:

[0045] An example of the compound represented by formula (V) is resazurin, which generates resorufin, a molecule with high fluorescence intensity, through an enzymatic reaction (reduction reaction) catalyzed by the following NADH:quinone oxidoreductase.

[0046] When the DT-diaphorase according to the first aspect of the present disclosure is an azoreductase, the fluorescent probe according to the first aspect of the present disclosure may be, for example, a fluorescent probe represented by the following formula (VI): (wherein X is the same as X in formula (III), and Z ７ and Z ８each independently represents a monovalent group.) As such a fluorescent probe, for example, the one described in Non-Patent Document 3 can be used. In addition, the structure of the fluorescent probe, particularly the portion other than X, can be designed or selected with reference to Non-Patent Document 3, for example. The compound represented by formula (VI) is converted to X-NH, a molecule with high fluorescence intensity, by an enzymatic reaction (reduction reaction) caused by azoreductase. ２ This results in:

[0047] Furthermore, the fluorescent probe according to the first aspect of the present disclosure may be a fluorescent probe in which a self-cleaving group, the terminal amino group of which is converted into a precursor by a moiety that is eliminated or reduced by the enzymatic reaction of DT-diaphorase, is bound to a fluorescent group via an ether bond or a carbamate bond. The self-cleaving group may be, for example, p-aminobenzyl, o-aminobenzyl, or aminomethyl, and in one embodiment may be p-aminobenzyl or o-aminobenzyl, and in a preferred embodiment may be p-aminobenzyl. The precursor of the terminal amino group of the self-cleaving group may be, for example, an azo group, a nitro group, or an amide group.

[0048] The fluorescent probe containing a self-cleaving group in which the terminal amino group has been converted into a precursor as described above may be, for example, a probe in which X-* in formula (IV) or formula (VI) is replaced with X-O-L-* or X-NH-C(=O)-O-L-* (L represents the portion of the self-cleaving group excluding the terminal amino group, and * represents a bond). For example, in a fluorescent probe in which X-* in formula (VI) is replaced with X-O-L-* and the self-cleaving group is p-aminobenzyl, as shown below, the azo group is reduced to an amino group in an enzymatic reaction (reduction reaction) by azoreductase, resulting in the elimination of p-aminobenzyl, the self-cleaving group formed as a partial structure, and generating X-OH, a molecule with high fluorescent intensity. In the formula, Z ７ and Z ８ and X is the same as in formula (VI).

[0049] The fluorescent probe containing a self-cleaving group in which the terminal amino group has been converted into a precursor as described above may be, for example, a fluorescent probe in which a group represented by X-O-* or X-NH-C(=O)-O-* is bound to p-nitrobenzyl or o-nitrobenzyl. For example, in a fluorescent probe in which a group represented by X-O-* is bound to p-nitrobenzyl, the nitro group is reduced to an amino group by an enzymatic reaction (reduction reaction) with nitroreductase, as shown below, and the self-cleaving group p-aminobenzyl generated as a partial structure is eliminated, generating X-OH, a molecule with high fluorescent intensity. Furthermore, in a fluorescent probe in which a group represented by X-NH-C(=O)-O-* is bound to nitrobenzyl, decarboxylation also occurs in addition to the elimination of aminobenzyl, generating X-NH, a molecule with high fluorescent intensity. ２ This results in:

[0050] When the DT-diaphorase according to the first aspect of the present disclosure is a nitroreductase, the fluorescent probe according to the first aspect of the present disclosure may be, for example, a compound represented by the following formula (VII): (In the formula, Z ７ ~Z １３ each independently represents a monovalent group; Z ７ , Z ８ and Z １０ ~Z １３ are preferably each independently —H, —CO ２ H, -PO ３ H ２ , -SO ３ H or an alkyl group, and more preferably each independently represents —H or —CO ２ H, -PO ３ H ２ or -SO ３ H, Z ９ is preferably —CF ３ represents R １ａ ~R １ｃ each independently represents a monovalent group, preferably represents —H or an alkyl group, more preferably represents an alkyl group, even more preferably represents a methyl group, an ethyl group, or an n-propyl group, and even more preferably represents a methyl group.) Examples of such fluorescent probes include NTR-NO２ and NTR-NO ２ is converted to NTR-NH, a molecule with high fluorescence intensity, by an enzymatic reaction (reduction reaction) caused by nitroreductase. ２ This results in (Non-Patent Document 4).

[0051] In a preferred embodiment of the first aspect of the present disclosure, X in formula (III), formula (III′), formula (IV) and formula (VI) is —CO ２ H, -PO ３ H ２ and -SO ３ H, and has at least one water-soluble substituent selected from the group consisting of: (In the formula, R ４ and R ５ are each independently a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a halogen atom, the water-soluble substituent, or an alkyl group having 1 to 6 carbon atoms and the water-soluble substituent, preferably a hydrogen atom, a methyl group, an ethyl group, or a halogen atom, more preferably a hydrogen atom or a halogen atom, and even more preferably a hydrogen atom; R ６ and R ７ are each independently a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a halogen atom, the water-soluble substituent, or an alkyl group having 1 to 6 carbon atoms and the water-soluble substituent, preferably a hydrogen atom, a methyl group, an ethyl group, or a halogen atom, more preferably a hydrogen atom or a halogen atom, and even more preferably a hydrogen atom; R ８ and R ９ are each independently a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted alkenyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms and having the above-mentioned water-soluble substituent, or an alkenyl group having 1 to 6 carbon atoms and having the above-mentioned water-soluble substituent, preferably a hydrogen atom or an unsubstituted alkyl group having 1 to 6 carbon atoms, more preferably a hydrogen atom, a methyl group or an ethyl group, and even more preferably a hydrogen atom, ８ and R ９ Let's get together and ８ and R９ may form a 4- to 7-membered heterocyclyl optionally having a substituent containing a nitrogen atom to which R is bonded, ８ or R ９ Alternatively, R ８ and R ９ are respectively R ５ or R ６ Together with ８ or R ９ may form a 5- to 7-membered heterocyclyl or heteroaryl containing a nitrogen atom to which R is bonded, and the heterocyclyl or heteroaryl may further have a substituent selected from the group consisting of alkyl having 1 to 6 carbon atoms, alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, aralkyl groups having 6 to 10 carbon atoms, and alkyl-substituted alkenyl groups having 6 to 10 carbon atoms, and the heterocyclyl and heteroaryl may have the water-soluble substituent bonded directly or via the substituent, and R ａ and R ｂ are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, or a substituted or unsubstituted aryl group, where R ａ and R ｂ The alkyl group and the aryl group may be substituted with the water-soluble substituent. In a more preferred embodiment, the alkyl group and the aryl group may be one selected from groups represented by formula (VIII) and formula (IX), and in a further preferred embodiment, the alkyl group and the aryl group may be one selected from groups represented by formula (VIII). In another further preferred embodiment, the alkyl group and the aryl group may be one selected from groups represented by formula (VIII) In a more preferred embodiment, the compound represented by formula (III) may be a group represented by formula (VIII'): The compound represented by the formula (I) is also referred to as Q-sHMRG. The product of the enzymatic reaction (reduction reaction) of Q-sHMRG by NADH:quinone oxidoreductase is the above-mentioned sHMRG.

[0052] The fluorescent probes described above can be organically synthesized according to methods commonly used by those skilled in the art.

[0053] The concentration of the fluorescent probe at the start of step (B) (reaction step) in the first aspect of the present disclosure is not particularly limited as long as it is a concentration at which fluorescence can be detected in the subsequent step (C) (fluorescence measurement step), and may be, for example, 1 μM to 1000 μM, 3 μM to 700 μM, 10 μM to 500 μM, or 30 μM to 300 μM.

[0054] <Evaluation System> A method for evaluating the oxidizing activity of a dehydrogenase according to a first aspect of the present disclosure comprises the following steps (A) to (C): step (A): preparing an aqueous solution containing one molecule of a dehydrogenase, a substrate for the dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe (preparation step); step (B): allowing the dehydrogenase and the DT-diaphorase to catalyze an oxidation-reduction reaction in the aqueous solution prepared in step (A) (reaction step); and step (C): measuring the fluorescence of the aqueous solution obtained in step (B) (fluorescence measurement step). The evaluation system in this evaluation method is not particularly limited, as long as it can form a closed reaction system using an aqueous solution containing one molecule of a dehydrogenase, a substrate for the dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe, and can quantitatively measure the fluorescence derived from the reduction product of the fluorescent probe produced in the closed reaction system. The closed reaction system may be spatially closed by, for example, a resin or oil, and the volume of the closed reaction system may be, for example, 5 fL to 100 fL. Alternatively, a plurality of such closed reaction systems may be present, and step (B) may be carried out in each of them. A more specific evaluation system may be, for example, an evaluation system using a microdevice provided with wells, each with a volume of about 5 fL to 100 fL. In this case, the surface of the aqueous solution (closed reaction system) in step (B) that is not covered by the wells may be covered by an organic solvent or a gas (e.g., air). When the surface of the aqueous solution in step (B) that is not covered by the wells is covered by a gas, the aqueous solution may remain in the wells due to, for example, gravity and / or surface tension. Another specific evaluation system may be, for example, an evaluation system using droplets (e.g., liposomes) present in an aqueous solution, each having a volume of approximately 5 fL to 100 fL. In this case, each droplet contains an aqueous solution containing one molecule of dehydrogenase, a substrate for the dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe, thereby enabling the evaluation of the oxidizing activity of each dehydrogenase.

[0055] In a preferred embodiment, the evaluation system for the method for evaluating the oxidative activity of dehydrogenases according to the first aspect of the present disclosure may be an evaluation system using a microdevice provided with wells, each with a volume of approximately 5 fL to 100 fL. In step (B), the surface of the aqueous solution (closed reaction system) not covered by the wells may be covered with an organic solvent. Hereinafter, this evaluation system will be referred to as an "oil-sealed chamber system." Such evaluation systems are described, for example, in Patent Document 1 and Non-Patent Document 1. In an oil-sealed chamber system, a closed reaction system can be achieved by utilizing the immiscibility of the hydrophobic solvent and the aqueous solution. The hydrophobic solvent is not particularly limited as long as it is immiscible with the aqueous solution. Suitable examples include, for example, at least one selected from the group consisting of saturated hydrocarbons, unsaturated hydrocarbons, aromatic hydrocarbons, silicone oils, perfluorocarbons, halogenated solvents, and hydrophobic ionic liquids, or a mixture containing such a solvent.

[0056] <Step (A): Preparation Step> In step (A), an aqueous solution containing one molecule of dehydrogenase, a substrate for the dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe is prepared. In step (A), typically, a solution (pre-encapsulation aqueous solution) containing the dehydrogenase, the substrate for the dehydrogenase, the nucleoside diphosphate derivative, DT-diaphorase, and the fluorescent probe is first prepared, and then the resulting pre-encapsulation aqueous solution is encapsulated in a microdevice, a droplet, or the like, so that the number of dehydrogenase molecules in a single closed reaction system is one. That is, in one embodiment, step (A) may include encapsulating the pre-encapsulation aqueous solutions containing the dehydrogenase, the substrate for the dehydrogenase, the nucleoside diphosphate derivative, DT-diaphorase, and the fluorescent probe in a space closed relative to the aqueous solutions, so that the number of dehydrogenase molecules in each aqueous solution is one.

[0057] When step (A) includes encapsulating pre-encapsulation aqueous solutions containing a dehydrogenase, a substrate for the dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe into a space closed relative to the aqueous solution so that each aqueous solution contains one molecule of dehydrogenase, and when the aqueous solutions are encapsulated into a large number of closed spaces (e.g., 1,000 or more), the number-average number of molecules of dehydrogenase contained per aqueous solution may be, for example, 0.0002 to 0.5, or may be 0.0005 to 0.1, 0.001 to 0.07, or 0.003 to 0.05. When the number-average number of molecules of dehydrogenase contained per aqueous solution is within the above range, the aqueous solution in which the dehydrogenase is encapsulated is likely to contain only one molecule of dehydrogenase, thereby making it easier to prevent the measurement of fluorescence derived from an aqueous solution in which two or more molecules of dehydrogenase are encapsulated in step (C). This makes it easier to evaluate the oxidizing activity of the dehydrogenase. The number-average number of dehydrogenase molecules contained in each of the above aqueous solutions may be, for example, the value obtained by multiplying the concentration of dehydrogenase in the aqueous solution before encapsulation [molecules / L] by the volume of the aqueous solution in step (B) (e.g., the volume of the well) [L].

[0058] The pre-encapsulation aqueous solution containing a dehydrogenase, a substrate for the dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe is typically prepared by adding the dehydrogenase, a substrate for the dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe to a solvent. The fluorescent probe and the nucleoside diphosphate derivative may be added in the form of, for example, a salt, a solvate, or a solvate of a salt. Furthermore, the nucleoside diphosphate derivative is typically added in its oxidized form, because the presence of a reduced form would result in a reduction reaction by DT-diaphorase, regardless of whether or not an oxidation reaction by the dehydrogenase occurs, resulting in a background signal. In the nucleoside diphosphate derivative to be added, the proportion by mass of the oxidized form to the total mass of the oxidized form and the reduced form is preferably 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, 98.5% or more, 99.0% or more, 99.5% or more, 99.8% or more, or 100%.

[0059] The concentration of the dehydrogenase in the aqueous solution before encapsulation is not particularly limited as long as it is a concentration at which fluorescence can be detected in the subsequent step (C) (fluorescence measurement step), and may be, for example, 5 fM to 20 pM, 10 fM to 10 pM, 30 fM to 3 pM, or 100 fM to 1 pM.

[0060] Furthermore, for example, in one embodiment of the present disclosure, when the dehydrogenase is a dehydrogenase contained in plasma, the dilution ratio of the plasma in the aqueous solution before encapsulation is not particularly limited as long as it is a dilution ratio at which fluorescence can be detected in the subsequent step (C) (fluorescence measurement step), and may be, for example, 10 to 1,000,000 times, 30 to 300,000 times, 100 to 100,000 times, 300 to 30,000 times, or 500 to 10,000 times.

[0061] Furthermore, the concentrations of the dehydrogenase substrate, the nucleoside diphosphate derivative, the DT-diaphorase, and the fluorescent probe in the aqueous solution before encapsulation are usually the same as the concentrations of the dehydrogenase substrate, the nucleoside diphosphate derivative, the DT-diaphorase, and the fluorescent probe, respectively, in the aqueous solution prepared in step (A), and the concentrations of the dehydrogenase substrate, the nucleoside diphosphate derivative, the DT-diaphorase, and the fluorescent probe, respectively, in the aqueous solution at the start of step (B).

[0062] The pH of the aqueous solution prepared in step (A) is not limited as long as it is a pH at which the enzymatic reaction of the dehydrogenase and DT-diaphorase occurs. For example, the aqueous solution may be a pH within the range of the optimum pH of the dehydrogenase ±3.0 and the optimum pH of the DT-diaphorase ±3.0; a pH within the range of the optimum pH of the dehydrogenase ±2.0 and the optimum pH of the DT-diaphorase ±2.0; a pH within the range of the optimum pH of the dehydrogenase ±1.5 and the optimum pH of the DT-diaphorase ±1.5; or a pH within the range of the optimum pH of the dehydrogenase ±1.0 and the optimum pH of the DT-diaphorase ±1.0. Specifically, the pH of the aqueous solution prepared in step (A) may be, for example, 3.5 or more, 4.0 or more, 4.5 or more, 5.0 or more, 5.5 or more, 6.0 or more, 6.5 or more, 7.0 or more, 7.5 or more, 8.0 or more, 8.5 or more, or 9.0 or more, or may be 9.5 or less, 9.0 or less, 8.5 or less, 8.0 or less, 7.5 or less, 7.0 or less, 6.5 or less, 6.0 or less, 5.5 or less, 5.0 or less, 4.5 or less, or 4.0 or less.

[0063] The aqueous solution prepared in step (A) may contain other components in addition to the dehydrogenase, the dehydrogenase substrate, the nucleoside diphosphate derivative, DT-diaphorase, and the fluorescent probe. Examples of other components include buffers (e.g., phosphate buffer, HEPES buffer, and Tris buffer), pH adjusters (e.g., hydrochloric acid, citric acid, sodium hydroxide, sodium carbonate, and sodium bicarbonate), ion concentration adjusters (e.g., calcium chloride and magnesium chloride), osmotic pressure adjusters (e.g., sodium chloride), antioxidants (e.g., dithiothreitol (DTT)), and surfactants (e.g., Triton (registered trademark) X-100, CHAPS, and Tween (registered trademark) 20).

[0064] In one embodiment of the first aspect of the present disclosure, when the evaluation system is an oil-sealed chamber system, step (A) may be a step of filling a plurality of storage compartments of a microchamber array having a hydrophobic surface with an aqueous solution containing the dehydrogenase, the dehydrogenase substrate, the nucleoside diphosphate derivative, the DT-diaphorase, and the fluorescent probe, the step of pouring the aqueous solution containing the dehydrogenase, the dehydrogenase substrate, the nucleoside diphosphate derivative, the DT-diaphorase, and the fluorescent probe into the opening of the storage compartment so that the number-average molecular number of the dehydrogenase filled in each of the plurality of storage compartments is 0.0002 to 0.5, and the step of blocking the opening with a hydrophobic solvent, in this order. Examples of such microchamber arrays include those described in Patent Document 1 and Non-Patent Document 1, and more specifically, for example, a Simoa (registered trademark) disk (Quanterx). The volume of the solution in each of the plurality of storage sections may be, for example, 1 fL to 500 fL, 5 fL to 100 fL, 10 fL to 75 fL, or 20 fL to 60 fL. The number-average molecular number of the dehydrogenase filled in each of the plurality of storage sections may be, for example, 0.0002 to 0.5, 0.0005 to 0.1, 0.001 to 0.07, or 0.003 to 0.05.

[0065] In one embodiment of the first aspect of the present disclosure, when the evaluation system is an oil seal chamber system, a fluorescent probe having a high water solubility in water and its reduction product can be preferably used as the fluorescent probe from the viewpoint of suppressing leakage into the hydrophobic solvent. ２ H, -PO ３ H ２ and -SO ３ and a fluorescent probe having at least one substituent selected from the group consisting of H, and a fluorescent probe in which, when 1 μM of the compound is added to a two-layer system of octanol and phosphate buffered saline (PBS) at pH 7.4 in a volume ratio of 1:1, 80% or more of the compound is distributed in the PBS layer.

[0066] In one embodiment of the first aspect of the present disclosure, when the evaluation system is an oil seal chamber system, from the viewpoint of making it easier to observe fluorescence, a fluorescent probe having a product of the molar extinction coefficient [L / mol cm] at the maximum absorption wavelength of the reduction product and the fluorescence quantum yield of 10,000 or more can be preferably used. From the same viewpoint, a fluorescent probe having a maximum fluorescence wavelength of the reduction product of 450 nm or more can be preferably used.

[0067] <Step (B): Reaction Step> In step (B), the dehydrogenase and DT-diaphorase are allowed to catalyze an oxidation-reduction reaction in the aqueous solution prepared in step (A).

[0068] The reaction temperature in step (B) is not particularly limited as long as it is a temperature at which the enzymatic reaction of the dehydrogenase and DT-diaphorase occurs, and may be, for example, from 20° C. to 50° C., or from 23° C. to 40° C. Specific examples of the reaction temperature in step (B) include 25° C. and 37° C.

[0069] The reaction time in step (B) is not particularly limited as long as it is a time that allows quantitative measurement of fluorescence in the subsequent step (C) (fluorescence measurement step), and may be, for example, 1 minute to 72 hours, 5 minutes to 48 hours, 10 minutes to 30 hours, 20 minutes to 24 hours, or 30 minutes to 8 hours. Specific examples of the reaction time in step (B) include 1 hour, 3 hours, 6 hours, and 12 hours.

[0070] <Step (C): Fluorescence Measurement Step> In step (C), the fluorescence of the aqueous solution obtained in step (B) is measured to obtain fluorescence data. Step (C) may be performed simultaneously with step (B), and in this case, step (C) may be performed multiple times during the course of step (B) (so-called time-lapse measurement may be performed). The method for measuring fluorescence is not particularly limited as long as it is a method that can measure the fluorescence of the aqueous solution obtained in step (B), and may be, for example, a method using a fluorescence microscope, and the fluorescence microscope may be, for example, an epi-illumination microscope, a confocal microscope, or a total internal reflection microscope.

[0071] <Evaluation of Oxidation Activity of Dehydrogenase> The oxidative activity of dehydrogenase can be evaluated based on the fluorescence data obtained by the method described above. The evaluation of the oxidative activity of dehydrogenase may be performed using as an index the number of aqueous solutions for which a fluorescence intensity equal to or greater than a certain threshold was observed among the aqueous solutions subjected to fluorescence measurement, the distribution (pattern) of the fluorescence intensities of the aqueous solutions subjected to fluorescence measurement, or some other index.

[0072] Specifically, for example, if it is known that an aqueous solution containing a dehydrogenase that satisfies a specific condition (e.g., a condition in which a genetic mutation has occurred or a condition in which a post-translational modification has occurred) will have a fluorescence intensity equal to or greater than a threshold, whereas an aqueous solution containing a dehydrogenase that does not satisfy a specific condition (e.g., a condition in which a genetic mutation has not occurred or a condition in which a post-translational modification has not occurred) will have a fluorescence intensity less than the threshold, the oxidizing activity of the dehydrogenase may be evaluated based on whether the fluorescence intensity is equal to or greater than the threshold, and a dehydrogenase that has a fluorescence intensity equal to or greater than the threshold can be evaluated as a dehydrogenase that satisfies a specific condition.

[0073] Specifically, for example, in a case where an average fluorescence intensity of an aqueous solution containing a dehydrogenase derived from a sample satisfying a specific condition is equal to or greater than a threshold, whereas an average fluorescence intensity of an aqueous solution containing a dehydrogenase derived from a sample not satisfying the specific condition is less than the threshold, the sample itself can be evaluated through evaluation of the oxidative activity of the dehydrogenase based on whether the average fluorescence intensity of the aqueous solution containing the dehydrogenase derived from the target sample is equal to or greater than the threshold. For example, a sample whose average fluorescence intensity is equal to or greater than the threshold can be evaluated as a sample satisfying the specific condition. In this case, for example, if the sample satisfying the specific condition is a specimen derived from a cancer patient and the sample not satisfying the specific condition is a specimen derived from a healthy subject, whether the average fluorescence intensity of the aqueous solution containing the dehydrogenase derived from the sample is equal to or greater than the threshold can be used as a criterion to assist in the diagnosis of cancer or to obtain data for use in determining whether a subject has cancer.

[0074] Furthermore, for example, in a case where a sample that satisfies a specific condition has a proportion of aqueous solutions whose fluorescence intensity is equal to or greater than a threshold equal to or greater than a predetermined value, whereas a sample that does not satisfy the specific condition has a proportion of aqueous solutions whose fluorescence intensity is equal to or greater than the threshold less than the predetermined value, the sample itself can be evaluated through evaluation of the oxidative activity of dehydrogenases based on whether or not the proportion of aqueous solutions whose fluorescence intensity is equal to or greater than the threshold in the target sample is equal to or greater than the predetermined value. For example, a sample whose proportion of aqueous solutions whose fluorescence intensity is equal to or greater than the threshold can be evaluated as satisfying the specific condition. In this case, for example, if the sample that satisfies the specific condition is a specimen derived from a cancer patient and the sample that does not satisfy the specific condition is a specimen derived from a healthy subject, whether or not the proportion of aqueous solutions whose fluorescence intensity is equal to or greater than the threshold in the sample is equal to or greater than the predetermined value can be used as a criterion to assist in the diagnosis of whether or not the subject has cancer, and also to obtain data for use in determining whether or not the subject has cancer.

[0075] Specifically, for example, when the distribution (pattern) of fluorescence intensity in an aqueous solution containing a dehydrogenase derived from a sample that satisfies certain conditions differs from the distribution (pattern) of fluorescence intensity in an aqueous solution containing a dehydrogenase derived from a sample that does not satisfy certain conditions, the sample itself can be evaluated through the evaluation of the oxidative activity of the dehydrogenase based on whether the distribution (pattern) of fluorescence intensity in the aqueous solution containing the dehydrogenase derived from the target sample is more similar to the other distributions. For example, when the p-value obtained when a Student's t-test is performed between a sample that satisfies certain conditions and the target sample is compared with the p-value obtained when a Student's t-test is performed between a sample that does not satisfy certain conditions and the target sample, if the p-value obtained when the Student's t-test is performed between the sample that satisfies certain conditions and the target sample is smaller, the sample can be evaluated as satisfying certain conditions. In this case, for example, if the sample that satisfies the specific conditions is a specimen derived from a cancer patient and the sample that does not satisfy the specific conditions is a specimen derived from a healthy individual, whether or not the p-value is larger when a Student's t-test is performed between the sample that satisfies the specific conditions and the target sample can be used as a criterion to assist in diagnosing whether or not the subject has cancer, and data can be obtained that can be used to determine whether or not the subject has cancer.

[0076] Furthermore, specifically, for example, if the fluorescence data is a fluorescence image (e.g., a fluorescence image obtained by evaluation in an oil seal chamber system), a machine learning model that has been trained to consider a fluorescence image obtained when a sample that satisfies certain conditions as a sample that satisfies the certain conditions, and a fluorescence image obtained when a sample that does not satisfy the certain conditions as a sample that does not satisfy the certain conditions, can be used as input to determine whether the target sample satisfies certain conditions, thereby evaluating the oxidative activity of dehydrogenases and thereby evaluating the sample itself.

[0077] <Functions and Other Embodiments> According to one embodiment of the present disclosure, the oxidizing activity of a dehydrogenase can be evaluated (detected) at the level of a single dehydrogenase molecule.

[0078] According to one embodiment of the present disclosure, even if one type of fluorescent probe is used, the oxidative activities of multiple types of dehydrogenases can be detected (evaluated) by using various nucleoside diphosphate derivatives. According to one embodiment of the present disclosure, even if one type of fluorescent probe is used, the oxidative activities of multiple types of dehydrogenases can be detected (evaluated) by using various nucleoside diphosphate derivatives. Another embodiment of the first aspect of the present disclosure is a method for evaluating the oxidizing activity of a dehydrogenase, comprising the following steps (A-1) to (C-1) and steps (A-2) to (C-2): step (A-1): preparing an aqueous solution containing one molecule of a dehydrogenase, a substrate for the dehydrogenase, a first nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe; step (B-1): allowing the dehydrogenase and the DT-diaphorase to catalyze an oxidation-reduction reaction in the aqueous solution prepared in step (A-1); step (C-1): measuring the fluorescence of the aqueous solution obtained in step (B-1); step (A-2): preparing an aqueous solution containing one molecule of the dehydrogenase, a substrate for the dehydrogenase, a second nucleoside diphosphate derivative, DT-diaphorase, and the fluorescent probe; step (B-2): allowing the dehydrogenase and the DT-diaphorase to catalyze an oxidation-reduction reaction in the aqueous solution prepared in step (A-2); and step (C-2): measuring the fluorescence of the aqueous solution obtained in step (B-2), wherein the first nucleoside diphosphate derivative is different from the second nucleoside diphosphate derivative (i.e., the first and second nucleoside diphosphate derivatives have different chemical structures), the dehydrogenase can utilize the oxidized first nucleoside diphosphate derivative and the oxidized second nucleoside diphosphate derivative as coenzymes, the DT-diaphorase can utilize the reduced first nucleoside diphosphate derivative and the reduced second nucleoside diphosphate derivative as coenzymes, and the fluorescent probe is a compound whose fluorescence intensity increases when reduced by the DT-diaphorase.According to the above embodiment, by using one type of fluorescent probe and two types of nucleoside diphosphate derivatives, i.e., a first nucleoside diphosphate derivative and a second nucleoside diphosphate derivative, the oxidative activity of multiple types of dehydrogenases that metabolize the same substrate can be detected and distinguished by utilizing the difference in utilization efficiency by dehydrogenases depending on the type of nucleoside diphosphate derivative. For example, if dehydrogenase A can utilize the first nucleoside diphosphate derivative as a coenzyme and dehydrogenase B can utilize the second nucleoside diphosphate derivative as a coenzyme, the result of measuring a certain sample under conditions in which the first nucleoside diphosphate derivative is added can be detected as the oxidative activity of dehydrogenase A, and the result of measuring the same sample under conditions in which the second nucleoside diphosphate derivative is added can be detected as the oxidative activity of dehydrogenase B.

[0079] According to one embodiment of the present disclosure, even when a single type of fluorescent probe is used, the oxidative activities of multiple types of dehydrogenases can be detected (evaluated) by using substrates for various dehydrogenases. According to one embodiment of the present disclosure, even when a single type of fluorescent probe is used, the oxidative activities of multiple types of dehydrogenases can be detected (evaluated) by using substrates for various dehydrogenases. Another embodiment of the first aspect of the present disclosure is a method for evaluating the oxidation activity of a dehydrogenase, comprising the following steps (A-3) to (C-3) and steps (A-4) to (C-4): step (A-3): preparing an aqueous solution containing one molecule of a first dehydrogenase, a substrate for the first dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe; step (B-3): allowing the first dehydrogenase and the DT-diaphorase to catalyze an oxidation-reduction reaction in the aqueous solution prepared in step (A-3); step (C-3): measuring the fluorescence of the aqueous solution obtained in step (B-3); step (A-4): preparing an aqueous solution containing one molecule of a second dehydrogenase, a substrate for the second dehydrogenase, the nucleoside diphosphate derivative, DT-diaphorase, and the fluorescent probe; step (B-4): allowing the second dehydrogenase and the DT-diaphorase to catalyze an oxidation-reduction reaction in the aqueous solution prepared in step (A-4); and step (C-4): measuring the fluorescence of the aqueous solution obtained in step (B-4), wherein the substrate of the first dehydrogenase is different from the substrate of the second dehydrogenase (i.e., the two have different chemical structures), the first dehydrogenase and the second dehydrogenase are capable of utilizing the oxidized form of the nucleoside diphosphate derivative as a coenzyme, the DT-diaphorase is capable of utilizing the reduced form of the nucleoside diphosphate derivative as a coenzyme, and the fluorescent probe is a compound whose fluorescence intensity increases when reduced by the DT-diaphorase. According to the above embodiment, by using one type of fluorescent probe and two types of dehydrogenase substrates, namely, a substrate for a first dehydrogenase and a substrate for a second dehydrogenase, it is possible to detect and distinguish the oxidizing activity of multiple types of dehydrogenases by utilizing the differences in substrate specificity of the dehydrogenases.For example, if dehydrogenase A can metabolize a substrate for a first dehydrogenase and dehydrogenase B can metabolize a substrate for a second dehydrogenase, the results of measuring a sample under conditions in which the substrate for the first dehydrogenase is added can be detected as the oxidative activity of dehydrogenase A, and the results of measuring the same sample under conditions in which the substrate for the second dehydrogenase is added can be detected as the oxidative activity of dehydrogenase B.

[0080] According to one embodiment of the present disclosure, whether a sample satisfies a specific condition can be evaluated by detecting the enzymatic activity of a dehydrogenase in the sample, utilizing a change in the oxidizing activity of a dehydrogenase derived from the sample that satisfies the specific condition. In this case, according to one embodiment of the present disclosure, even if a single type of fluorescent probe is used, a coupled assay system suitable for evaluating a sample that satisfies the specific condition can be found by using various dehydrogenase substrates and various nucleoside diphosphate derivatives.

[0081] <Kit> A second aspect of the present disclosure is a kit for evaluating the oxidative activity of a dehydrogenase, the kit comprising a dehydrogenase substrate, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe, wherein the dehydrogenase can use the oxidized form of the nucleoside diphosphate derivative as a coenzyme, the DT-diaphorase can use the reduced form of the nucleoside diphosphate derivative as a coenzyme, and the fluorescent probe is a compound whose fluorescence intensity increases upon reduction by the DT-diaphorase. The dehydrogenase substrate, nucleoside diphosphate derivative, DT-diaphorase, and fluorescent probe according to the second aspect of the present disclosure can be those described in the first aspect of the present disclosure, and the dehydrogenase to be evaluated can be the same as that described in the first aspect of the present disclosure. The kit according to the second aspect of the present disclosure allows the oxidative activity of a dehydrogenase to be evaluated according to the evaluation method according to the first aspect of the present disclosure. In one embodiment, the kit according to the second aspect of the present disclosure may further include a package insert describing that the oxidative activity of a dehydrogenase is to be evaluated, or may further include a package insert describing that the oxidative activity of a single molecule of dehydrogenase is to be evaluated, and the package insert may describe performing steps corresponding to steps (A) to (C) in the evaluation method according to one embodiment of the first aspect of the present disclosure. In one embodiment, the kit according to the second aspect of the present disclosure may further include a microchamber array, and the microchamber array may be usable in the evaluation method according to the first aspect of the present disclosure.

[0082] <Modification: Method for Evaluating Metabolic Activity of Metabolic Enzyme> A third aspect of the present disclosure is a method for evaluating metabolic activity of a metabolic enzyme, comprising the following steps (A) to (C): step (A): preparing an aqueous solution containing one molecule of a metabolic enzyme, a substrate for the metabolic enzyme, a dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe; step (B): allowing the metabolic enzyme, the dehydrogenase, and the DT-diaphorase to catalyze an enzymatic reaction in the aqueous solution prepared in step (A); and step (C): measuring the fluorescence of the aqueous solution obtained in step (B); The present disclosure provides an evaluation method comprising the steps of: (a) a dehydrogenase that utilizes as a substrate a metabolite obtained by metabolizing a substrate of the metabolic enzyme by the metabolic enzyme; (b) the dehydrogenase is capable of utilizing the oxidized form of the nucleoside diphosphate derivative as a coenzyme; (c) the DT-diaphorase is capable of utilizing the reduced form of the nucleoside diphosphate derivative as a coenzyme; and (d) the fluorescent probe is a compound whose fluorescence intensity increases upon reduction by the DT-diaphorase. That is, a third aspect of the present disclosure is a method for performing a coupled assay shown in the following reaction formula at the level of a single metabolic enzyme molecule. In the coupled assay shown in the following reaction formula, the enzymatic reaction by the metabolic enzyme is the rate-limiting step of the entire coupled assay system, i.e., the metabolite produced by the enzymatic reaction of the metabolic enzyme is rapidly consumed in the enzymatic reaction by the dehydrogenase and DT-diaphorase, thereby enabling the metabolic activity of the metabolic enzyme to be evaluated using the fluorescence of the reduction product of the enzymatic reaction by DT-diaphorase as an indicator.

[0083] As shown in the above reaction formula, "metabolite of metabolic enzyme = substrate of metabolic enzyme," the evaluation method according to the third aspect of the present disclosure can also be described as an evaluation method in which, in step (A), an aqueous solution containing one molecule of a metabolic enzyme and the metabolic enzyme's substrate is prepared instead of the dehydrogenase substrate, and in step (B), the metabolic enzyme is also allowed to catalyze an enzymatic reaction, where the dehydrogenase is a dehydrogenase that uses, as a substrate, a metabolite obtained by metabolism of the metabolic enzyme's substrate by the metabolic enzyme. As a result, the metabolite obtained by metabolism of the metabolic enzyme's substrate by the metabolic enzyme serves as the "dehydrogenase substrate" in the evaluation method according to the first aspect of the present disclosure.

[0084] The nucleoside diphosphate derivatives, DT-diaphorase, and fluorescent probes according to the third aspect of the present disclosure can be the same as those according to the first aspect of the present disclosure. Furthermore, the evaluation system and conditions for steps (A) to (C) according to the third aspect of the present disclosure can be the same as those according to the first aspect of the present disclosure, except as otherwise specifically described below. Furthermore, the evaluation of metabolic enzyme metabolic activity according to the third aspect of the present disclosure can be carried out using the same evaluation method as that used to evaluate the oxidative activity of dehydrogenases described in the first aspect of the present disclosure.

[0085] <Metabolic Enzymes> In the present disclosure, metabolic enzymes refer to enzymes present inside cells or on cell membranes, or secreted from cells, that have the function of converting one substance into another. Typical examples include enzymes that constitute energy production pathways, such as the glycolytic pathway, the citric acid cycle, and the electron transport chain, as well as enzymes involved in amino acid metabolism or lipid metabolism. Examples of metabolic enzymes according to one embodiment of the third aspect of the present disclosure include enzymes that constitute the glycolytic pathway, enzymes involved in glycogen metabolism, enzymes involved in gluconeogenesis, enzymes that constitute the citric acid cycle, enzymes that constitute the pentose phosphate pathway, enzymes involved in drug metabolism, enzymes involved in the metabolism of amino acids and their analogs, and enzymes involved in lipid metabolism. More specific examples include hexokinase, phosphofructokinase, aldolase, enolase, pyruvate decarboxylase, phosphoglucomutase, fructose bisphosphatase, aconitase, fumarate hydratase, 6-phosphonogluconolactonase, glycine decarboxylase, lipid desaturase, and phospholipase. Furthermore, examples of metabolic enzymes according to one embodiment of the third aspect of the present disclosure include metabolic enzymes that produce, as a metabolic product through a metabolic reaction, a substrate for the dehydrogenase according to the first aspect of the present disclosure (e.g., glycine, serine, D-serine, glutamic acid, lysine, leucine, glutathione, glucose-6-phosphate, lactic acid, D-lactic acid, isocitrate, malic acid, inosinic acid, 6-phosphogluconic acid, succinic semialdehyde, terephthalaldehyde acid, or HMG-CoA).

[0086] In one embodiment of the third aspect of the present disclosure, the metabolic enzyme may be a metabolic enzyme contained in at least one selected from the group consisting of plasma, serum, urine, saliva, tears, cerebrospinal fluid, tissue homogenates, cell homogenates, and extracts thereof. In a preferred embodiment, the metabolic enzyme may be a metabolic enzyme contained in at least one selected from the group consisting of plasma, serum, urine, saliva, tears, cerebrospinal fluid, and extracts thereof, and in a more preferred embodiment, it may be a metabolic enzyme contained in at least one selected from the group consisting of plasma, serum, tears, cerebrospinal fluid, and extracts thereof. When a sample from which the metabolic enzyme is derived is evaluated by evaluating the metabolic activity of the metabolic enzyme using the evaluation method according to one embodiment of the third aspect of the present disclosure, the sample may be at least one selected from the group consisting of plasma, serum, urine, saliva, tears, cerebrospinal fluid, tissue homogenates, cell homogenates, and extracts thereof.

[0087] <Substrate of Metabolic Enzyme> In the present disclosure, a substrate of a metabolic enzyme refers to a molecule that serves as a substrate for a metabolic enzyme according to one embodiment of the third aspect of the present disclosure. The substrate of a metabolic enzyme according to the present disclosure may be a natural substrate of the metabolic enzyme, or an artificial substrate, such as a derivative of such a natural substrate. The substrate of the metabolic enzyme can be selected according to the type of metabolic enzyme to be evaluated. For example, if the metabolic enzyme is hexokinase, the substrate of the metabolic enzyme may be at least one selected from the group consisting of hexoses such as D-glucose, D-mannose, and D-fructose. For example, if the metabolic enzyme is phosphoglucomutase, the substrate of the metabolic enzyme may be glucose-1-phosphate. For example, if the metabolic enzyme is aconitate hydratase, the substrate of the metabolic enzyme may be citric acid and / or cis-aconitic acid. For example, if the metabolic enzyme is 6-phosphogluconolactonase, the substrate of the metabolic enzyme may be 6-phosphoglucono-1,5-lactone.

[0088] The concentration of the metabolic enzyme substrate at the start of step (B) (reaction step) in the third aspect of the present disclosure is not particularly limited as long as it is a concentration at which fluorescence can be detected in the subsequent step (C) (fluorescence measurement step), and may be, for example, 10 μM to 1 M, 30 μM to 100 mM, 100 μM to 30 mM, or 300 μM to 10 mM.

[0089] <Dehydrogenase> The type of dehydrogenase according to the third aspect of the present disclosure can be the same as that described in the first aspect of the present disclosure. In addition, a dehydrogenase capable of using as a substrate a metabolic product obtained by the metabolic enzyme's metabolism of the metabolic enzyme's substrate can be selected in accordance with the metabolic enzyme and the metabolic enzyme's substrate. For example, if the metabolic enzyme is hexokinase and the metabolic enzyme's substrate is D-glucose, the dehydrogenase can be glucose-6-phosphate dehydrogenase (G6PDH), a dehydrogenase that uses glucose-6-phosphate, a metabolic product of D-glucose catalyzed by hexokinase, as a substrate. Furthermore, if the metabolic enzyme is phosphoglucomutase and the metabolic enzyme's substrate is glucose-1-phosphate, the dehydrogenase can be glucose-6-phosphate dehydrogenase (G6PDH), a dehydrogenase that uses glucose-6-phosphate, a metabolic product of glucose-1-phosphate catalyzed by phosphoglucomutase, as a substrate. For example, when the metabolic enzyme is aconitate hydratase and the substrate of the metabolic enzyme is citric acid, the dehydrogenase can be isocitrate dehydrogenase, which is a dehydrogenase that uses isocitrate, a metabolic product of citric acid by aconitate hydratase, as a substrate.For example, when the metabolic enzyme is 6-phosphogluconolactonase and the substrate of the metabolic enzyme is 6-phosphoglucono-1,5-lactone, the dehydrogenase can be 6-phosphogluconate dehydrogenase, which is a dehydrogenase that uses 6-phosphogluconate, a metabolic product of 6-phosphoglucono-1,5-lactone by 6-phosphogluconolactonase, as a substrate.

[0090] The concentration of dehydrogenase at the start of step (B) (reaction step) in the third aspect of the present disclosure is not particularly limited as long as it is a concentration at which fluorescence can be detected in the subsequent step (C) (fluorescence measurement step), and may be, for example, 0.1 μg / mL to 100 μg / mL, 0.5 μg / mL to 60 μg / mL, or 0.8 μg / mL to 30 μg / mL.

[0091] the number of dehydrogenase molecules present in the aqueous solution at the start of step (B) (reaction step) of the third aspect of the present disclosure, and the metabolic turnover number k of the dehydrogenase ｃａｔ The reaction rate constant k [ / sec] of the enzymatic reaction catalyzed by the dehydrogenase in an aqueous solution, defined as the product of k / sec and k / sec, is not particularly limited as long as the concentration is such that fluorescence can be detected in the subsequent step (C) (fluorescence measurement step), and may be, for example, 0.001 or more, 0.003 or more, and preferably 0.01 or more, 0.03 or more, or 0.1 or more. When the reaction rate constant k of the enzymatic reaction catalyzed by the dehydrogenase in an aqueous solution in step (B) (reaction step) is equal to or greater than the above-mentioned lower limit, the rate-limiting step in the entire coupled assay is likely to be the enzymatic reaction catalyzed by the metabolic enzyme, making it easier to evaluate the oxidative activity of the metabolic enzyme.

[0092] <Step (A) of the Third Aspect: Preparation Step> In step (A) according to the third aspect of the present disclosure, an aqueous solution containing one molecule of a metabolic enzyme, a substrate for the metabolic enzyme, a dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe is prepared. In step (A), typically, the metabolic enzyme, the substrate for the metabolic enzyme, the dehydrogenase, the nucleoside diphosphate derivative, DT-diaphorase, and the fluorescent probe are prepared, and the resulting pre-encapsulation aqueous solution is then encapsulated in a microdevice, a droplet, or the like, so that the metabolic enzyme molecule is present in a single closed reaction system. That is, in one embodiment, step (A) may include encapsulating the pre-encapsulation aqueous solution containing the metabolic enzyme, the substrate for the metabolic enzyme, the dehydrogenase, the nucleoside diphosphate derivative, DT-diaphorase, and the fluorescent probe in a space closed relative to the aqueous solution, so that the metabolic enzyme molecule is present in each aqueous solution.

[0093] When step (A) involves encapsulating pre-encapsulation aqueous solutions containing a metabolic enzyme, a substrate for the metabolic enzyme, a dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe in a space closed relative to the aqueous solution so that each aqueous solution contains one metabolic enzyme molecule, and when the aqueous solutions are encapsulated in a large number (e.g., 1,000 or more) of closed spaces, the number-average number of metabolic enzyme molecules contained in each aqueous solution may be, for example, 0.0002 to 0.5, or may be 0.0005 to 0.1, 0.001 to 0.07, or 0.003 to 0.05. When the number-average number of metabolic enzyme molecules contained per aqueous solution is within the above range, the aqueous solution in which the metabolic enzyme is encapsulated is likely to contain one metabolic enzyme molecule, thereby making it easier to prevent the measurement of fluorescence derived from an aqueous solution in which two or more metabolic enzyme molecules are encapsulated in step (C). This makes it easier to evaluate the oxidative activity of the metabolic enzyme. The number-average number of metabolic enzyme molecules contained in each of the above aqueous solutions may be, for example, the value obtained by multiplying the concentration of the metabolic enzyme in the aqueous solution before encapsulation [units / L] by the volume of the aqueous solution in step (B) (e.g., the volume of the well) [L].

[0094] The concentration of the metabolic enzyme in the aqueous solution before encapsulation is not particularly limited as long as it is a concentration at which fluorescence can be detected in the subsequent step (C) (fluorescence measurement step), but may be, for example, 5 fM to 20 pM, 10 fM to 10 pM, 30 fM to 3 pM, or 100 fM to 1 pM.

[0095] Furthermore, for example, in one embodiment of the present disclosure, when the metabolic enzyme is a metabolic enzyme contained in plasma, the dilution ratio of the plasma in the aqueous solution before encapsulation is not particularly limited as long as it is a dilution ratio at which fluorescence can be detected in the subsequent step (C) (fluorescence measurement step), and may be, for example, 10 to 1,000,000 times, 30 to 300,000 times, 100 to 100,000 times, 300 to 30,000 times, or 500 to 10,000 times.

[0096] Furthermore, the concentrations of the metabolic enzyme substrate, dehydrogenase, nucleoside diphosphate derivative, DT-diaphorase, and fluorescent probe in the aqueous solution before encapsulation are usually the same as the concentrations of the metabolic enzyme substrate, dehydrogenase, nucleoside diphosphate derivative, DT-diaphorase, and fluorescent probe, respectively, in the aqueous solution prepared in step (A), and the concentrations of the metabolic enzyme substrate, dehydrogenase, nucleoside diphosphate derivative, DT-diaphorase, and fluorescent probe, respectively, in the aqueous solution at the start of step (B).

[0097] Furthermore, the aqueous solution prepared in step (A) may contain other components in addition to the metabolic enzyme, the substrate for the metabolic enzyme, the dehydrogenase, the nucleoside diphosphate derivative, the DT-diaphorase, and the fluorescent probe. Examples of the other components include the other components listed in the first aspect of the present disclosure and components consumed by the metabolic enzyme in the metabolic reaction. Examples of the components consumed by the metabolic enzyme in the metabolic reaction include adenosine triphosphate (ATP) and adenosine diphosphate (ADP).

[0098] In one embodiment of the third aspect of the present disclosure, when the evaluation system is an oil-sealed chamber system, step (A) is a step of filling a plurality of storage compartments of a microchamber array having a hydrophobic surface with an aqueous solution containing the metabolic enzyme, a substrate for the metabolic enzyme, a dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe, the aqueous solution containing the metabolic enzyme, the substrate for the metabolic enzyme, the dehydrogenase, the nucleoside diphosphate derivative, DT-diaphorase, and the fluorescent probe is poured into each of the openings of the storage compartments so that the number-average molecular number of the metabolic enzyme filled in each of the plurality of storage compartments is 0.0002 to 0.5, and the openings may be blocked with a hydrophobic solvent, in this order. Examples of such microchamber arrays include those described in Patent Document 1 and Non-Patent Document 1, and more specifically, for example, a Simoa (registered trademark) disk (Quanterx). The volume of the solution in each of the plurality of storage sections may be, for example, 1 fL to 500 fL, 5 fL to 100 fL, 10 fL to 75 fL, or 20 fL to 60 fL. The number-average molecular number of the metabolic enzyme filled in each of the plurality of storage sections may be, for example, 0.0002 to 0.5, 0.0005 to 0.1, 0.001 to 0.07, or 0.003 to 0.05.

[0099] <Step (B) of the Third Aspect: Reaction Step> In step (B), an enzymatic reaction is caused to occur among the metabolic enzyme, the dehydrogenase, and the DT-diaphorase in the aqueous solution prepared in step (A). Conditions for step (B) of the third aspect of the present disclosure may be the same as those for the first aspect of the present disclosure.

[0100] <Step (C) of the Third Aspect: Fluorescence Measurement Step> In step (C), the fluorescence of the aqueous solution obtained in step (B) is measured to obtain fluorescence data. Conditions for step (C) of the third aspect of the present disclosure may be the same as those for the first aspect of the present disclosure.

[0101] <Kit for Measuring Metabolic Enzyme Activity> A fourth aspect of the present disclosure is a kit for evaluating the metabolic activity of a metabolic enzyme, comprising a metabolic enzyme substrate, a dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe, wherein the dehydrogenase is a dehydrogenase that utilizes a metabolite obtained by metabolizing the metabolic enzyme substrate by the metabolic enzyme, the dehydrogenase is capable of utilizing the oxidized nucleoside diphosphate derivative as a coenzyme, the DT-diaphorase is capable of utilizing the reduced nucleoside diphosphate derivative as a coenzyme, and the fluorescent probe is a compound whose fluorescence intensity increases upon reduction by the DT-diaphorase. The metabolic enzyme substrate, dehydrogenase, nucleoside diphosphate derivative, DT-diaphorase, and fluorescent probe according to the fourth aspect of the present disclosure can be those described in the third aspect of the present disclosure, and the metabolic enzyme to be evaluated can be the same as those described in the third aspect of the present disclosure. The kit according to the fourth aspect of the present disclosure can evaluate the oxidative activity of a metabolic enzyme according to the evaluation method according to the third aspect of the present disclosure. In one embodiment, the kit according to the fourth aspect of the present disclosure may further include a package insert describing that the oxidative activity of a metabolic enzyme is to be evaluated, or may further include a package insert describing that the oxidative activity of a single metabolic enzyme molecule is to be evaluated, and the package insert may describe performing steps corresponding to steps (A) to (C) in the evaluation method according to one embodiment of the third aspect of the present disclosure. In one embodiment, the kit according to the fourth aspect of the present disclosure may further include a microchamber array, and the microchamber array may be usable in the evaluation method according to the third aspect of the present disclosure.

[0102] The present disclosure will be described in more detail below using examples, but is not limited to the examples of the present disclosure.

[0103] Synthesis Example 1: Q-sHMRG The fluorescent probe Q-sHMRG used in the examples was synthesized as follows.

[0104] Synthesis Example 1-1: Preparation of fluorescein 5 / 6-sulfonic acid (Compound 1) An aqueous solution of 4-sulfophthalic acid (50%, 20 mL, 40.7 mmol) was mixed with resorcinol (8.95 g, 81.3 mmol), and methanesulfonic acid (2.5 mL) was added. The resulting reaction solution was stirred at 130°C for 18 hours. ２ 0 (50 mL) was added to the reaction mixture and the yellow precipitate was filtered and ２ The precipitate was dried to give a crude product of the 5 / 6-isomer of fluorescein sulfonic acid (15.2 g, 91%, Compound 1).

[0105] Synthesis Example 1-2: Preparation of 3-oxo-3',6'-bis(((trifluoromethyl)sulfonyl)oxy)-3H-spiro[isobenzofuran-1,9'-xanthene]-6-sulfonic acid (Compound 2) The 5 / 6-isomer of fluorescein sulfonic acid (1.7 g, 4.1 mmol) obtained in Synthesis Example 1-1 was dissolved in DMF (10 mL) and N,N-diisopropylethylamine (DIEA, 10.7 mL, 61.8 mmol). To the resulting mixture, N-phenylbis(trifluoromethanesulfonimide) (2.9 g, 8.3 mmol) was added in small portions as a solid on ice, and the reaction was stirred at 25° C. for 2 hours. After confirming complete consumption of the starting material, DIEA was removed under reduced pressure, and dichloromethane (DCM) was added to the remaining solution. The organic layer was washed twice with 2N aqueous HCl and once with saturated brine, and then extracted with Na ２ SO ４ The residue was purified by MPLC (15 min gradient A / B = 90 / 10 to 0 / 100, where A is 0.1% trifluoroacetic acid (TFA) and B is 80% acetonitrile (ACN)-20% H ２ The residue was purified by separation and filtration using 0.1% TFA containing 0.0 to give compound 2 (2.4 g, 86%).

[0106] Synthesis Example 1-3: Preparation of 3',6'-bis(((trifluoromethyl)sulfonyl)oxy)-3H-spiro[isobenzofuran-1,9'-xanthene]-5 / 6-sulfonic acid (Compound 3) Compound 2 (2.0 g, 3.0 mmol) obtained in Synthesis Example 1-2 was dissolved in dry THF (30 mL). The resulting solution was purged with an argon atmosphere and cooled to -20°C. A THF solution (2 M, 4.4 mL, 8.8 mmol) of lithium aluminum hydride (LAH) was added dropwise thereto, and the mixture was then heated to 25° C. and stirred for 2 hours. The reaction solution was slowly poured into ice-cooled 2N aqueous HCl solution (50 mL), and dichloromethane (100 mL) was added. The organic layer was recovered, washed with saturated brine, and then extracted with NaCl. ２ SO ４ The residue was purified by MPLC (15 min gradient A / B = 90 / 10 to 0 / 100, where A is 0.1% trifluoroacetic acid (TFA) and B is 80% acetonitrile (ACN)-20% H ２ The residue was purified by separation using 0.1% TFA containing 0.0 to give compound 3 (1.35 g, 69%).

[0107] Synthesis Example 1-4: Preparation of 3',6'-diamino-3H-spiro[isobenzofuran-1,9'-xanthene]-5 / 6-sulfonic acid (sHMRG) 6-isomer (3',6'-diamino-3H-spiro[isobenzofuran-1,9'-xanthene]-5 / 6-sulfonic acid (sHMRG) 6-isomer) (Compound 4) Compound 3 (500 mg, 0.74 mmol) obtained in Synthesis Example 1-3 was dissolved in toluene (30 mL) and triisopropyl orthoformate (1.6 mL, 7.4 mmol), and the reaction mixture was stirred at 25°C for 30 minutes. The solvent was removed under reduced pressure, and the residue was redissolved in 10 mL of toluene. Benzphenone imine (633 mg, 3.8 mmol), Pd ２ (dba) ３ -CHCl ３(117 mg, 0.11 mmol), xantphos (87 mg, 0.20 mmol) and Cs ２ CO ３ (2.46 g, 7.6 mmol) was added, and the reaction was refluxed (105°C) under an argon atmosphere for 18 hours. The solvent of the reaction solution was removed under reduced pressure, and saturated aqueous hydrochloric acid (10 mL) was added to the residue. After stirring for 30 minutes, the reaction was diluted with DMSO-H ２ The resulting mixture was diluted in 0 and directly subjected to preparative purification. Preparative purification was performed by MPLC (a 15-minute gradient of A / B = 99 / 1 to 0 / 100, where A is 0.1 M triethylamine acetate buffer and B is methanol) by collecting fractions having a peak absorbance band near 500 nm. The isomer eluted first was the 6-sulfonic acid isomer (Compound 4, 41 mg, 14%), and the isomer eluted later was the 5-sulfonic acid isomer (Compound 5, 36 mg, 12%).

[0108] Compound 4 1 H-NMR (DMSO-d6): delta 4.13 (s, 2H), 6.73 (d, 2H, J = 2.0 Hz), 6.83 (dd, 2H, J = 2.0, 9.2 Hz), 6.95 (d, 2H, J = 9.2 Hz), 7.38 (d, 1H, J = 1.6 HRMS (ESI + ): Calcd. for [M+H] + 397.0853, found 397.0840 (-1.3 mmu).

[0109] Synthesis Example 1-5: Preparation of Q-sHMRG (Compound 6) Compound 4 (36 mg, 0.091 mmol) obtained in Synthesis Example 1-4 was dissolved in dry DMF (2 mL). 3-Methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoic acid (23 mg, 0.091 mmol), HATU (34 mg, 0.091 mmol), and DIEA (110 mL, 0.63 mmol) were added, and the mixture was stirred at 25°C for 2 hours. The solvent was concentrated under reduced pressure, and the residue was directly subjected to preparative purification. Preparative purification was performed by collecting fractions with an absorption band peaking around 500 nm using MPLC (a 15-minute gradient of A / B = 99 / 1 to 0 / 100, where A is 0.1% trifluoroacetic acid and B is methanol). The obtained fraction was lyophilized to give Compound 6 (13 mg, 34%) as an orange solid.

[0110] Q-sHMRG (compound 6) 1 H-NMR (DMSO-d6): delta 1.36 (s, 6H), 1.87 (s, 3H), 1.89 (s, 3H), 2.03 (s, 3H), 2.94 (s, 2H), 5.22 (s, 2H), 6.45 (d, 1H, J = 8.2 Hz), 6.6-7.8 (m, 7H), 8.95 (m, 1H). HRMS (ESI + ): Calcd. for [M+H] + 629.1958, found 629.1966 (+0.8 mmu).

[0111] Reference Example 1: Reactivity of Q-sHMRG with DT-diaphorase and Increase in Fluorescence ２ (1mM), MgCl ２Q-sHMRG prepared in Synthesis Example 1 was dissolved in HEPES buffer (100 mM, pH 7.4) containing ATP (1 mM), DTT (dithiothreitol, 100 μM), and Triton X-100 (250 μM) to a final concentration of 1 μM, and the absorption spectrum was measured. NADH (100 μM) and NADH:quinone oxidoreductase (10 μg / mL, Toyobo Co., Ltd., DAD-311) were added to the solution, and the mixture was incubated at 25°C for 4 hours, after which the absorption spectrum was measured again. The results are shown in Figure 1(A).

[0112] Also, CaCl ２ (1mM), MgCl ２ Q-sHMRG prepared in Synthesis Example 1 was dissolved in HEPES buffer (100 mM, pH 7.4) containing ATP (1 mM), DTT (dithiothreitol, 100 μM), and Triton X-100 (250 μM) to a final concentration of 1 μM, and various concentrations of NADH and NADH:quinone oxidoreductase (10 μg / mL) were added thereto. After incubation at 25° C. for 30 minutes, the fluorescence intensity was measured. The results are shown in Figure 1 (B) and (C).

[0113] 1(A) to (C), the fluorescence intensity of Q-sHMRG increased by more than 200 times (235 times) due to the reduction reaction of NADH:quinone oxidoreductase. Furthermore, FIG. 1(B) and (C) show that the combination of Q-sHMRG and NADH:quinone oxidoreductase can detect trace amounts of NADH, such as 0.01 μM, demonstrating the extremely high reactivity of Q-sHMRG with NADH:quinone oxidoreductase.

[0114] Example 1: Detection of oxidative activity of lactate dehydrogenase in human plasma An investigation was conducted to determine whether the activity of lactate dehydrogenase in human plasma can be detected (evaluated) by an evaluation method according to an embodiment of the present disclosure.

[0115] CaCl ２ (1mM), MgCl ２In a HEPES buffer (100 mM, pH 7.4) containing (1 mM), DTT (dithiothreitol, 100 μM), and Triton X-100 (200 μM), Q-sHMRG (30 μM), Q-HMRG (30 μM), or resazurin (30 μM), lactate (1 mM), NAD ＋ (Oxidized nicotinamide adenine dinucleotide, 500 μM) and NADH:quinone oxidoreductase (50 μg / mL, Toyobo Co., Ltd., DAD-311) were added. Plasma obtained from a healthy human subject was added to the well so that it was diluted 3000 times (preparation step, step (A)). 40 μL of the solution thus prepared (pre-encapsulation aqueous solution) was introduced into a Simoa® disk (Quanterix), a microchamber array. Next, 80 μL of Fluorinert® FC-70 (Sigma-Aldrich) was introduced into the microchamber array as a hydrophobic solvent, and the aqueous solution that was not introduced into the well was removed, forming W / O droplets in the wells. After incubation at room temperature for 3 hours (reaction step, step (B)), a fluorescent image was acquired using an epi-illumination microscope Ti2 (Nikon) equipped with a 20x objective lens (Plan Apo 20x), an sCMOS camera (ORCA-Fusion C14440, Hamamatsu Photonics), a white light unit (X-Cite Xylis, Optoscience) and a motorized stage (fluorescence measurement step, step (C)).

[0116] The results are shown in Figure 2. In Figure 2, the arrows in the line plots of the results for Q-HMRG and resazurin indicate wells where the fluorescence intensity derived from the fluorescent dye produced by metabolism of the fluorescent probe appears to have increased. Figure 2 shows that the fluorescence intensity derived from the fluorescent dye produced by metabolism of the fluorescent probe increased in wells of the microchamber array where lactate dehydrogenase was presumed to have been stochastically encapsulated. This demonstrates that the evaluation method according to an embodiment of the present disclosure makes it possible to evaluate the oxidative activity of a single dehydrogenase molecule at the single-molecule level. Furthermore, when comparing the results between fluorescent dyes, the ratio (S / N ratio) of the fluorescence intensity of wells encapsulating lactate dehydrogenase relative to the fluorescence intensity of wells not encapsulating lactate dehydrogenase was greater when Q-sHMRG, which has a sulfo group and is highly water-soluble, was used. Furthermore, when a similar experiment was performed using SiRWST-3 (Non-Patent Document 5) or Q-dsAMC (Non-Patent Document 2) as the fluorescent probe (however, when SiRWST-3 was used, 100 μM of the electron mediator 1-methoxy-5-methylphenazinium methylsulfate (1-MeO-PMS) was added instead of NADH:quinone oxidoreductase), the S / N ratio was higher when Q-sHMRG, Q-HMRG, or resazurin was used, and in particular, when Q-sHMRG was used, the ratio (S / N ratio) of the fluorescence intensity in wells in which lactate dehydrogenase was encapsulated to the fluorescence intensity in wells in which lactate dehydrogenase was not encapsulated was higher.

[0117] Example 2: Detection of oxidation activity of various dehydrogenases in human plasma In an evaluation method according to an embodiment of the present disclosure, it was investigated whether it is possible to detect (evaluate) the activity of dehydrogenases corresponding to the respective substrates by using substrates for the various dehydrogenases.

[0118] CaCl ２ (1mM), MgCl ２ In a HEPES buffer (100 mM, pH 7.4) containing (1 mM), DTT (dithiothreitol, 100 μM) and Triton X-100 (200 μM), Q-sHMRG (30 μM), a substrate for dehydrogenase (1 mM), NAD ＋(100 μM) and NADH:quinone oxidoreductase (50 μg / mL) were added. Plasma obtained from a healthy human subject was added to the mixture so that it was diluted 1000 times (preparation step, step (A)). 40 μL of the solution thus prepared (pre-encapsulation aqueous solution) was introduced into a Simoa® disk (Quanterix), a microchamber array. Next, 80 μL of Fluorinert® FC-70 (Sigma-Aldrich) as a hydrophobic solvent was introduced into the microchamber array, and the aqueous solution that was not introduced into the wells was removed, forming W / O droplets in the wells. After 3 hours of incubation at room temperature (reaction step, step (B)), a fluorescent image was acquired using an epi-illumination microscope Ti2 (Nikon) set up similar to that in Example 1 (fluorescence measurement step, step (C)).

[0119] The dehydrogenase substrates used were glycine, serine, D-serine, glutamic acid, lysine, leucine, glutathione, glucose-6-phosphate, lactic acid, D-lactic acid, isocitrate, malic acid, inosinic acid, 6-phosphogluconic acid, succinic semialdehyde, terephthalaldehyde, and HMG-CoA (hydroxymethylglutaryl CoA). The dehydrogenases corresponding to these substrates are shown in FIG. 3.

[0120] The results are shown in Figure 3. As shown in Figure 3, simply changing the type of dehydrogenase substrate added resulted in various changes in the percentage of wells in which fluorescence was observed in the obtained fluorescent image and in the fluorescence intensity, demonstrating that the evaluation method according to an embodiment of the present disclosure makes it possible to detect (evaluate) the activity of multiple types of dehydrogenases at the single molecule level by changing the type of dehydrogenase substrate added, even when only one type of fluorescent probe is used.

[0121] Example 3: Detection of oxidative activity of dehydrogenase when various nucleoside diphosphate derivatives are used. In an evaluation method according to an embodiment of the present disclosure, it was investigated whether the oxidative activity of different subtypes of dehydrogenases targeting the same substrate can be detected (evaluated) by using various nucleoside diphosphate derivatives as coenzymes.

[0122] CaCl ２ (1mM), MgCl ２ Q-sHMRG (30 μM), dehydrogenase substrate (1 mM), nucleoside diphosphate derivative (500 μM), and NADH:quinone oxidoreductase (50 μg / mL) were added to a HEPES buffer (100 mM, pH 7.4) containing ATP (1 mM), DTT (dithiothreitol, 100 μM), and Triton X-100 (200 μM). Plasma obtained from a healthy human subject was added to the solution to a 1000-fold dilution (preparation step, step (A)). 40 μL of the solution thus prepared (pre-encapsulation aqueous solution) was introduced into a Simoa® disk (Quanterix), a microchamber array. Next, 80 μL of Fluorinert® FC-70 (Sigma-Aldrich) was introduced into the microchamber array as a hydrophobic solvent, and the aqueous solution that was not introduced into the wells was removed to form W / O droplets in the wells. After incubation at room temperature for 3 hours (reaction step, step (B)), a fluorescent image was acquired using an epi-illumination microscope Ti2 (Nikon) set up similarly to Example 1 (fluorescence measurement step, step (C)).

[0123] Lactic acid, glucose-6-phosphate, isocitrate, or 6-phosphogluconic acid was used as the substrate for the dehydrogenase. ＋ (oxidized nicotinamide adenine dinucleotide), NADP ＋ (oxidized nicotinamide adenine dinucleotide phosphate), Thio-NAD ＋ (oxidized thionicotinamide adenine dinucleotide) or NGD ＋ (oxidized nicotinamide guanine dinucleotide) was used.

[0124] The results are shown in Figure 4. As shown in Figure 4, the observed fluorescent image pattern (the percentage of wells in which strong fluorescence was observed and the fluorescence intensity) varied significantly depending on the type of nucleoside diphosphate derivative added, even when the same dehydrogenase substrate was used. This is presumably because the type of nucleoside diphosphate derivative that can be used as a coenzyme and its utilization efficiency vary significantly depending on the dehydrogenase subtype present in each well. Therefore, it was revealed that the evaluation method according to an embodiment of the present disclosure can detect (evaluate) the oxidative activity of different dehydrogenase subtypes that target the same substrate at the single enzyme molecule level by changing the type of nucleoside diphosphate derivative added, even when only one type of fluorescent probe is used. According to this evaluation method according to an embodiment of the present disclosure, the oxidative activity of minor subtypes of dehydrogenases that can highly efficiently utilize specific nucleoside diphosphate derivatives, which would be overlooked in a bulk evaluation system, can be detected (evaluated) at the single enzyme molecule level.

[0125] Example 4 Dependence of Fluorescence Images on Dehydrogenase Substrate and Nucleoside Diphosphate Derivative when Plasma Derived from Healthy Subjects and Pancreatic Cancer Patients is Used Whether differences in fluorescence image patterns are observed between plasma derived from healthy subjects and pancreatic cancer patients was investigated using the evaluation method according to an embodiment of the present disclosure demonstrated in Examples 1 to 3. In particular, whether differences in fluorescence image patterns occur between plasma derived from healthy subjects and pancreatic cancer patients depending on the type of dehydrogenase substrate and the type of nucleoside diphosphate derivative was investigated.

[0126] CaCl ２ (1mM), MgCl ２Q-sHMRG (30 μM), dehydrogenase substrate (1 mM), nucleoside diphosphate derivative (500 μM), and NADH:quinone oxidoreductase (50 μg / mL) were added to a HEPES buffer (100 mM, pH 7.4) containing ATP (1 mM), DTT (dithiothreitol, 100 μM), and Triton X-100 (200 μM). Plasma (sample) obtained from a healthy human subject or a human subject suffering from pancreatic cancer was added to the solution so as to be diluted 1000-fold (preparation step, step (A)). 40 μL of the solution thus prepared (pre-encapsulation aqueous solution) was introduced into a Simoa® disk (Quanterix), a microchamber array. Next, 80 μL of Fluorinert® FC-70 (Sigma-Aldrich) was introduced into the microchamber array as a hydrophobic solvent, and the aqueous solution that was not introduced into the wells was removed to form W / O droplets in the wells. After incubation at room temperature for 3 hours (reaction step, step (B)), a fluorescent image was acquired using an epi-illumination microscope Ti2 (Nikon) set up similarly to Example 1 (fluorescence measurement step, step (C)).

[0127] Glucose-6-phosphate or 6-phosphogluconic acid was used as the substrate for the dehydrogenase. ＋ , NADP ＋ or Thio-NAD ＋ was used.

[0128] The results are shown in Figure 5. As shown in Figure 5, even when the same fluorescent probe was used, the fluorescent image patterns in the plasma from healthy subjects and pancreatic cancer patients differed greatly depending on the type of dehydrogenase substrate and nucleoside diphosphate derivative. For example, when 6-phosphogluconic acid was used as the dehydrogenase substrate and Thio-NAD was used as the nucleoside diphosphate derivative, ＋When each of the above was used, no bright spots were observed in the plasma from healthy subjects, whereas bright spots were observed in the plasma from pancreatic cancer patients, allowing for a clear evaluation of the difference in the fluorescent image patterns between the two. From the above, it was demonstrated that, according to an evaluation method according to an embodiment of the present disclosure, an evaluation system that is likely to produce fluorescent image patterns between multiple groups to be distinguished can be found by changing the type of dehydrogenase substrate and the type of nucleoside diphosphate derivative, without relying on the type of fluorescent probe.

[0129] Example 5: Detection of metabolic enzyme activity in plasma from pancreatic cancer patients CaCl ２ (1mM), MgCl ２ Q-sHMRG (30 μM), glucose (1 mM), ATP (1 mM), NADPH (1 mM), ATP (1 mM), and ATP-100 (200 μM) were added to a HEPES buffer (100 mM, pH 7.4) containing ATP (1 mM), DTT (dithiothreitol, 100 μM), and Triton X-100 (200 μM). ＋ (500 μM), NADP ＋ (500 μM), glucose-6-phosphate dehydrogenase (G6PDH, 1 μg / mL, manufactured by R&D Systems), and NADH:quinone oxidoreductase (5 μg / mL) were added. Plasma (sample) obtained from a human subject with pancreatic cancer was added to the well so that it was diluted 1000 times (preparation step, step (A)). 40 μL of the solution thus prepared (pre-encapsulation aqueous solution) was introduced into a Simoa® disk (Quanterix), a microchamber array. Next, 80 μL of Fluorinert® FC-70 (Sigma-Aldrich) was introduced into the microchamber array as a hydrophobic solvent, and the aqueous solution that was not introduced into the well was removed, forming W / O droplets in the wells. After incubation at room temperature for 3 hours (reaction step, step (B)), a fluorescent image was acquired (fluorescence measurement step, step (C)) using an incident-light microscope Ti2 (Nikon Corporation) set up in the same manner as in Example 1. As a control experiment, a similar test was also carried out under conditions in which G6PDH was not added (G6PDH(-)).

[0130] The results are shown in Figure 6. As shown in Figure 6, under conditions where G6PDH was added, the metabolic reaction by hexokinase, the oxidation reaction by G6PDH, and the reduction reaction by DT-diaphorase all occurred in the coupled assay system, resulting in the observation of fluorescence derived from sHMRG, whereas under conditions where G6PDH was not added, no fluorescence was observed. This demonstrates that the metabolic activity of metabolic enzymes can be detected (evaluated) at the single metabolic enzyme molecule level by the method according to one embodiment of the third aspect of the present disclosure.

[0131] Example 6 Detection of Dehydrogenase Activity in Plasma from Acute Liver Injury Model Mice Thioacetamide (TAA)-treated mice were prepared as acute liver injury model mice according to the method described in the literature (Tatsuya Ukegawa et al., "Thioester-Based Coupled Fluorogenic Assays in Microdevice for the Detection of Single-Molecule Enzyme Activities of Esterases with Specified Substrate Recognition", Adv. Sci. 11, 2306559 (2024)). Blood was collected from the prepared acute liver injury model mice, and plasma (sample) was obtained from the blood. Using the plasma, dehydrogenase activity was detected according to the same method as in Example 2. Glucose-6-phosphate and lactate were used as dehydrogenase substrates.

[0132] FIG. 7 shows fluorescent images obtained when samples from mice not treated with TAA (control group) or mice treated with TAA (TAA group) were used under conditions in which glucose-6-phosphate was added. FIG. 8 shows fluorescent images obtained when samples from mice not treated with TAA (control group) or mice treated with TAA (TAA group) were used under conditions in which lactic acid was added. According to FIGS. 7 and 8, when samples from acute liver injury model mice were used, the number of wells in which fluorescence was detected was greater than when samples from control mice were used. These results reveal that the activity of G6PDH, a dehydrogenase that uses glucose-6-phosphate as a substrate, and lactate dehydrogenase (LDH), a dehydrogenase that uses lactic acid as a substrate, is enhanced in samples from acute liver injury model mice, and that this enhancement can be detected (evaluated) by a method according to one embodiment of the first aspect of the present disclosure.

[0133] 9 is a histogram showing the distribution of fluorescence intensity for each well in which fluorescence was detected, obtained under the same conditions as in FIG. 7. Interestingly, FIG. 9 shows that the samples derived from acute liver injury model mice had two populations of G6PDH activity, and the number of wells containing G6PDH active in both populations was increased in the samples derived from acute liver injury model mice. However, the rates of increase were different, with the number of wells containing G6PDH active in the low-activity population being higher. From these results, it was revealed that the method according to one embodiment of the first aspect of the present disclosure can evaluate and distinguish multiple populations having the same metabolic activity but different levels of activity, and can also detect the number of low-activity populations, which may be difficult to detect in bulk systems containing many enzyme molecules.

[0134] Figure 10 is a diagram showing the percentage of wells (lambda) in which fluorescence was detected in a fluorescence image acquired under the same conditions as in Figures 7 and 8. The results in Figure 10 confirm that the number of wells in which fluorescence was detected was high in the acute liver injury model mouse for all of the high G6PDH activity population (G6PDH high), low G6PDH activity population (G6PDH low), and LDH. This suggests that a method according to an embodiment of the first aspect of the present disclosure, using glucose-6-phosphate or lactate as a substrate, can be used to diagnose, assist in, and obtain data for the diagnosis of diseases such as acute liver injury.

[0135] Example 7: Detection of dehydrogenase activity in cerebrospinal fluid from patients with primary central nervous system lymphoma (PCSNL) We investigated whether dehydrogenase activity in cerebrospinal fluid (CSF) from patients with primary central nervous system lymphoma (PCSNL) can be detected using a method according to an embodiment of the first aspect of the present disclosure. CSF samples were collected from patients who had histologically confirmed PCSNL and who underwent surgery and high-dose methotrexate-based chemotherapy at the Department of Neuro-Oncology / Neurosurgery, Saitama Medical University International Medical Center, between March 2019 and December 2023. This study was approved by the Medical Ethics Review Board of Saitama Medical University International Medical Center (Approval Number: 2021-017) and the University of Tokyo (Approval Number: 5-2). Written informed consent was obtained from all patients. The study was conducted in accordance with the Declaration of Helsinki. CSF samples were collected from newly diagnosed PCNSL patients before treatment, and serially during and after chemotherapy. For recurrent PCNSL, CSF samples were collected when central nervous system recurrence was detected using contrast-enhanced MRI. All patients had normal immune function and were HIV-negative. All CSF samples were collected by lumbar puncture. For comparison, CSF samples were also collected from patients with glioblastoma or metastatic brain tumors. Pathological diagnosis was performed by a senior neuropathologist according to the 2016 World Health Organization (WHO) classification system. CSF samples were centrifuged at 500 × g for 10 minutes to remove cells, and the supernatant was stored at -80°C within 30 minutes of collection. Using these CSF samples, dehydrogenase activity was detected using the same method as in Example 2. Glucose-6-phosphate was used as the dehydrogenase substrate.

[0136] Fig. 11 shows fluorescence images of CSF samples derived from PCNSL patients (PCNSL group) and subjects with tumor regression (Regression group). Fig. 12 shows the results of counting the number of wells in which fluorescence was observed in fluorescence images of CSF samples derived from PCNSL patients (PCNSL group), subjects with tumor regression (Regression group), patients with recurrent PCNSL (Recurrence group), and subjects with remission of PCNSL (Remission group), acquired under the same conditions as Fig. 11. As shown in Figs. 11 and 12, the number of wells in which fluorescence was observed was greater in the CSF samples from the PCNSL group and Recurrence group, in which PCNSL symptoms were apparent, than in the CSF samples from the Regression group and Remission group, in which symptoms were improving. In particular, the number of wells in which fluorescence was observed in the PCNSL group was significantly higher than that in the regression group (P value of 0.0070 in the Mann-Whitney test). These results demonstrate that the activity of G6PDH, a dehydrogenase that uses glucose-6-phosphate as a substrate, is elevated in CSF samples from PCNSL patients, and that this elevation can be detected (evaluated) by a method according to an embodiment of the first aspect of the present disclosure. Furthermore, it was demonstrated that the method according to an embodiment of the first aspect of the present disclosure can detect dehydrogenase activity in cerebrospinal fluid. This also demonstrates that the biological samples to which the method according to an embodiment of the first aspect of the present disclosure can be applied are not limited to blood samples. Furthermore, it was suggested that the method according to an embodiment of the first aspect of the present disclosure, using glucose-6-phosphate as a substrate, can be used to diagnose, assist in, and obtain data for the diagnosis of diseases such as PCNSL.

[0137] Comparative Example 1: Detection of dehydrogenase activity using a bulk measurement system Dehydrogenase activity was detected using a bulk measurement system, and the results were compared with those of Example 2 to examine the advantages of performing the method according to an embodiment of the first aspect of the present disclosure at the single dehydrogenase molecule level. ２ (1mM), MgCl ２In a HEPES buffer (100 mM, pH 7.4) containing (1 mM), DTT (dithiothreitol, 100 μM) and Triton X-100 (200 μM), Q-sHMRG (10 μM), a dehydrogenase substrate (1 mM), NAD ＋ (500 μM), NADP ＋ To the solution, 500 μM of NADH:quinone oxidoreductase (50 μg / mL) was added. The solution was loaded into each well of a 384-well microplate (Greiner). The same plasma sample as in Example 2 was added to the well so as to be diluted 1000-fold or 100-fold. The rate of fluorescence increase in each well was measured at room temperature for 30 minutes using a microwell plate reader (2103 EnVision, PerkinElmer).

[0138] FIG. 13 shows the fluorescence increase rate ( / min) in the presence of various metabolic enzyme substrates when plasma samples were added at 1000-fold or 100-fold dilutions. The results in FIG. 13 are shown as mean ± standard deviation (Mean ± S.D.) for n=3. Blank (plasma-) represents a control to which no plasma sample or substrate was added, and Blank (plasma+) represents a control to which no substrate was added. As shown in FIG. 13 , when an evaluation using the same enzyme reaction as in one embodiment of the first aspect of the present disclosure was performed using a bulk system rather than a single-molecule evaluation system, dehydrogenase activity was only detected when lactate and malate were used as substrates at a 1000-fold dilution. This result contrasts with the results demonstrated in Example 2, where dehydrogenase activity was detected when multiple types of substrates were used by performing the method of one embodiment of the first aspect of the present disclosure using a single-molecule evaluation system. These results demonstrate that the method of one embodiment of the first aspect of the present disclosure uses a system containing a single dehydrogenase molecule, thereby enabling the detection of a wide range of dehydrogenases that would be difficult to detect in a bulk system because their signals would be buried in background signals.

[0139] Synthesis Example 2: Azo-rhodamine Azo-rhodamine was designed and synthesized as a fluorescent probe whose fluorescence intensity increases when metabolized by azoreductase, a type of DT-diaphorase. Azo-rhodamine is a fluorescent probe whose fluorescence intensity increases when metabolized by azoreductase, as shown in the following reaction formula:

[0140] 20 mg (0.055 mmol) of Rhodamine 110, a known fluorescent dye that can also be purchased from commercial suppliers, was added to H ２ After dissolving in 0.5% NaNO3 / MeCN / Conc. HCl (4 / 1 / 1 mL), the mixture was stirred on an ice bath under an argon atmosphere. ２ 3.65 mg (0.055 mmol) in 183 μL of H ２ The resulting solution was dissolved in 0 and added dropwise to the solution, followed by stirring for 2 hours. Aqueous ammonia was added to adjust the pH to 6-7, and 13.8 μL (0.11 mmol) of N,N-dimethylaniline dissolved in AcCN was added. The reaction solution was purified by MPLC (a 15-minute gradient of A / B = 95 / 5 to 0 / 100, where A is 0.1 M triethylamine acetate buffer, and B is AcCN containing 0.1 M triethylamine acetate). Fractions with an absorption band peaking around 500 nm were collected and lyophilized to yield 1.7 mg (6.7%) of compound.

[0141] Azo-rhodamine 1 H-NMR (400 MHz, CD3OD):δ 8.36 (d, J = 7.8 Hz, 1H), 8.05 (d, J = 1.4 Hz, 1H), 7.94-7.82 (m, 5H), 7.47 (d, J = 7.3 Hz, 1H), 7.32 (d, J = 8.7 Hz, 1H), 7.20 (d, J = 9.1 Hz, 1H), 6.98-6.93 (m, 2H), 6.88 (dd, J = 7.3, 2.3 Hz, 2H), 3.17 (s, 6H)

[0142] Example 8: Detection of dehydrogenase activity using azoreductase as DT-diaphorase CaCl２ (1mM), MgCl ２ Azo-rhodamine (30 μM), lactate (1 mM), NADPH (1 mM), DTT (100 μM), and Triton X-100 (250 μM) were added to HEPES buffer (100 mM, pH 7.4). ＋ (500 μM), flavin mononucleotide (FMN, 0.01 μM), and azoreductase (5 μg / mL, recombinant enzyme obtained by the method described in Non-Patent Document 3) were added. Plasma obtained from a healthy human subject was added to the solution so as to be diluted 3000 times (preparation step, step (A)). 40 μL of the solution thus prepared (aqueous solution before encapsulation) was introduced into a Simoa (registered trademark) disk (Quanterx), a microchamber array. Next, 80 μL of Fluorinert (registered trademark) FC-70 (Sigma-Aldrich) was introduced into the microchamber array as a hydrophobic solvent, and the aqueous solution that was not introduced into the wells was removed, forming W / O droplets in the wells. After incubation at room temperature for 20 hours (reaction step, step (B)), a fluorescent image was obtained using an incident-light microscope Ti2 (Nikon Corporation) set up in the same manner as in Example 1 (fluorescence measurement step, step (C)).

[0143] Figure 14 shows a fluorescent image of the device 20 hours after the addition of plasma. As shown in Figure 14, fluorescence was detected from wells where LDH was thought to have been stochastically encapsulated. This demonstrates that the method of one embodiment of the first aspect of the present disclosure can evaluate (detect) dehydrogenase activity even when azoreductase is used as DT-diaphorase. This demonstrates that the DT-diaphorase used in the method of one embodiment of the first aspect of the present disclosure is not limited to NADH:quinone oxidoreductase.

Claims

1. A method for evaluating the oxidizing activity of a dehydrogenase, comprising the following steps (A) to (C): step (A): preparing an aqueous solution containing one molecule of a dehydrogenase, a substrate for the dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe; step (B): allowing the dehydrogenase and the DT-diaphorase to catalyze an oxidation-reduction reaction in the aqueous solution prepared in step (A); and step (C): measuring the fluorescence of the aqueous solution obtained in step (B); wherein the dehydrogenase is capable of using the oxidized nucleoside diphosphate derivative as a coenzyme, the DT-diaphorase is capable of using the reduced nucleoside diphosphate derivative as a coenzyme, and the fluorescent probe is a compound whose fluorescence intensity increases when reduced by the DT-diaphorase.

2. The evaluation method according to claim 1, wherein the step (A) is a step of filling a plurality of storage sections of a microchamber array having a hydrophobic surface with an aqueous solution containing the dehydrogenase, a substrate for the dehydrogenase, the nucleoside diphosphate derivative, the DT-diaphorase and the fluorescent probe, comprising: pouring the aqueous solution containing the dehydrogenase, the substrate for the dehydrogenase, the nucleoside diphosphate derivative, the DT-diaphorase and the fluorescent probe through openings of the storage sections so that the number-average molecular number of the dehydrogenase filled in each of the plurality of storage sections is 0.0002 to 0.5; and blocking the openings with a hydrophobic solvent, in that order.

3. The fluorescent probe is -CO ２ H, -PO ３ H ２ and -SO ３ The evaluation method according to claim 1 or 2, wherein the compound has at least one substituent selected from the group consisting of H.

4. The nucleoside diphosphate derivative has the following formula (I) or (II) in an oxidized form: (In the formula, R １ is a monovalent group, X １ is -H, -OH or -PO ３ H ２ And Y １ The evaluation method according to claim 1 or 2, wherein the compound is at least one compound selected from the group consisting of compounds represented by the following formula:

5. The evaluation method according to claim 1 or 2, wherein the DT-diaphorase is NADH:quinone oxidoreductase, azoreductase or nitroreductase.

6. The evaluation method according to claim 1 or 2, wherein the dehydrogenase is a dehydrogenase contained in at least one selected from the group consisting of plasma, serum, urine, saliva, tears, cerebrospinal fluid, tissue homogenates, cell homogenates, and extracts thereof.

7. A method for evaluating the oxidation activity of a dehydrogenase, comprising the following steps (A-1) to (C-1) and steps (A-2) to (C-2): step (A-1): preparing an aqueous solution containing one molecule of a dehydrogenase, a substrate for the dehydrogenase, a first nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe; step (B-1): allowing the dehydrogenase and the DT-diaphorase to catalyze an oxidation-reduction reaction in the aqueous solution prepared in the step (A-1); step (C-1): measuring the fluorescence of the aqueous solution obtained in the step (B-1); step (A-2): preparing an aqueous solution containing one molecule of the dehydrogenase, a substrate for the dehydrogenase, a second nucleoside diphosphate derivative, the DT-diaphorase, and the fluorescent probe; step (B-2): allowing the dehydrogenase and the DT-diaphorase to catalyze an oxidation-reduction reaction in the aqueous solution prepared in the step (A-2); and step (C-2): measuring the fluorescence of the aqueous solution obtained in step (B-2), wherein the first nucleoside diphosphate derivative is different from the second nucleoside diphosphate derivative, the dehydrogenase can utilize the oxidized first nucleoside diphosphate derivative and the oxidized second nucleoside diphosphate derivative as coenzymes, the DT-diaphorase can utilize the reduced first nucleoside diphosphate derivative and the reduced second nucleoside diphosphate derivative as coenzymes, and the fluorescent probe is a compound whose fluorescence intensity increases when reduced by the DT-diaphorase.

8. A method for evaluating the oxidation activity of a dehydrogenase, comprising the following steps (A-3) to (C-3) and steps (A-4) to (C-4): step (A-3): preparing an aqueous solution containing one molecule of a first dehydrogenase, a substrate for the first dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe; step (B-3): allowing the first dehydrogenase and the DT-diaphorase to catalyze an oxidation-reduction reaction in the aqueous solution prepared in step (A-3); step (C-3): measuring the fluorescence of the aqueous solution obtained in step (B-3); step (A-4): preparing an aqueous solution containing one molecule of a second dehydrogenase, a substrate for the second dehydrogenase, the nucleoside diphosphate derivative, the DT-diaphorase, and the fluorescent probe; step (B-4): allowing the second dehydrogenase and the DT-diaphorase to catalyze an oxidation-reduction reaction in the aqueous solution prepared in the step (A-4); and step (C-4): measuring the fluorescence of the aqueous solution obtained in the step (B-4), wherein a substrate for the first dehydrogenase is different from a substrate for the second dehydrogenase, the first dehydrogenase and the second dehydrogenase are capable of using the oxidized nucleoside diphosphate derivative as a coenzyme, the DT-diaphorase is capable of using the reduced nucleoside diphosphate derivative as a coenzyme, and the fluorescent probe is a compound whose fluorescence intensity increases when reduced by the DT-diaphorase.

9. A kit for evaluating the oxidizing activity of a dehydrogenase, comprising: a dehydrogenase substrate, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe, wherein the dehydrogenase can utilize the oxidized nucleoside diphosphate derivative as a coenzyme, the DT-diaphorase can utilize the reduced nucleoside diphosphate derivative as a coenzyme, and the fluorescent probe is a compound whose fluorescence intensity increases when reduced by the DT-diaphorase.

10. A method for evaluating the metabolic activity of a metabolic enzyme, comprising the following steps (A) to (C): step (A): preparing an aqueous solution containing one molecule of the metabolic enzyme, a substrate for the metabolic enzyme, a dehydrogenase, a nucleoside diphosphate derivative, DT-diaphorase, and a fluorescent probe; step (B): allowing the metabolic enzyme, the dehydrogenase, and the DT-diaphorase to catalyze an enzymatic reaction in the aqueous solution prepared in step (A); and step (C): measuring the fluorescence of the aqueous solution obtained in step (B); wherein the dehydrogenase is a dehydrogenase that utilizes a metabolic product obtained by metabolizing a substrate for the metabolic enzyme by the metabolic enzyme, the dehydrogenase is capable of utilizing the oxidized nucleoside diphosphate derivative as a coenzyme, the DT-diaphorase is capable of utilizing the reduced nucleoside diphosphate derivative as a coenzyme, and the fluorescent probe is a compound whose fluorescence intensity increases when reduced by the DT-diaphorase.

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