Enzyme detection method and enzyme detection kit

Abstract

Provided are: an enzyme detection method by which enzymes for synthesizing ATP or ADP can be detected, highly sensitive detection at one molecular level can be performed for an enzyme not having a fluorescent substrate among the enzymes, and highly sensitive measurement of a disease biomarker or the like can be performed; and an enzyme detection kit used in the same. Provided are an enzyme detection method for detecting, from an enzyme sample, a target enzyme for synthesizing ADP or ATP, and an enzyme detection kit used in the same, the method comprising: a sample addition step for preparing a detection device including a microchamber array member in which a plurality of storage parts capable of being filled with a detection liquid are arranged, and filling the storage parts of the detection device with a detection liquid containing the enzyme sample and a hexokinase enzyme; and a detection step for detecting, from the detection liquid, a signal obtained by synthesizing ADP or ATP.

Description

Enzyme detection method and enzyme detection kit

[0001] The present invention relates to an enzyme detection method for detecting enzymes, including kinases, that serve as disease markers, and an enzyme detection kit used therefor. This application claims priority to Japanese Patent Application No. 2023-099051, filed on June 16, 2023, the contents of which are incorporated herein by reference.

[0002] Conventionally, techniques have been used to obtain information about biological samples and enzymes by detecting enzymes with specific activities from various biological samples. For example, detecting enzymes from samples derived from specific organs can detect associations with disease or infection (disease / infection biomarkers). Kinases are considered as disease biomarkers.

[0003] Immunochemical techniques, particularly ELISA, can detect enzymes in small amounts and large numbers of samples, making them applicable to the detection of disease and infection biomarkers. Multiplexed single enzyme assay technology has also been developed to examine enzyme isozymes. Isozymes are enzymes that catalyze the same reaction but have slightly different structures and thus different substrate specificities. This assay identifies isozymes by measuring their substrate-specific activity at the single-molecule level. Because various organ-specific isozymes exist in the blood, it is possible to highly sensitively investigate which organs may be responsible for the disease. Furthermore, isozymes that form multimers can exhibit nonuniform activity distribution per molecule, and examining this distribution can identify which diseases may be affected. Furthermore, in the analysis of a large number of small amounts of samples, such as by ELISA, methods have been developed that automate detection and analysis, and these digital bioanalyses (digital ELISAs) are also attracting attention as methods for detecting biomarkers with high sensitivity.

[0004] Patent Literature 1, for example, discloses a detection device and a detection method for detecting the enzyme activity of a large number of trace samples, comprising a microchamber array having a plurality of storage compartments filled with a hydrophilic solvent containing a biological sample, and an image sensor having pixels corresponding to the storage compartments, wherein the microchamber array has a flow channel communicating with the opening of the storage compartment, a hydrophobic solvent supply unit connected to the flow channel, and a through-hole through which the hydrophilic solvent can enter and exit the flow channel, and the hydrophobic solvent supply unit flows the hydrophobic solvent into the flow channel by an externally applied force, and a detection method using the same. This technology aims to provide a detection device and a detection method using the same, which can prevent a decrease in the concentration of the biological sample and reagent contained in the storage compartment by sealing the opening of the storage compartment with a hydrophobic solvent and confining the hydrophilic solvent containing the biological sample in the storage compartment, thereby preventing a decrease in the concentration of the biological sample and reagent contained in the storage compartment and improving measurement sensitivity.

[0005] International Publication No. 2014 / 034781

[0006] As described above, conventional biomarker detection techniques can detect a large number of samples with high sensitivity, but they cannot detect biomarkers of various enzyme types. That is, conventional biomarker detection techniques involve preparing a substrate that reacts with the target enzyme, and then detecting the enzyme by reacting with the substrate (e.g., by partially cleaving the substrate through the enzymatic reaction). To achieve this, for example, a portion of the substrate is modified to enable detection through the reaction. However, enzymes for which such specific substrates (substrates that can be used for fluorescent detection, hereinafter sometimes referred to as fluorescent substrates) have not been found, such as enzymes that catalyze reactions of common molecules in living organisms, such as ADP and ATP, cannot be detected. Furthermore, even if a substrate has been found, detection is not possible if it is difficult to modify the substrate in a way that allows for detection of the reaction.

[0007] For example, kinases are involved in the proliferation and invasion of cancer cells, and kinase mutations are thought to lead to carcinogenesis. Many kinase inhibitors have been developed as molecularly targeted cancer drugs. For example, Raf kinase mutations and thymidine kinase activity in the blood are biomarkers closely related to cell proliferation and are expected to serve as tools for monitoring the prognosis of cancer treatment. For example, elevated serum thymidine kinase activity has been reported to be associated with neoplastic diseases, malignant diseases, vitamin B12 deficiency, and viral infections. Thymidine kinase activity is a biomarker closely related to cell proliferation and is expected to serve as a tool for monitoring the prognosis of cancer treatment. Creatine kinase (CK) levels in the blood increase after myocardial infarction. Therefore, it is widely used in diagnosing myocardial infarction. CK isozymes exist in the brain, smooth muscle, heart, and skeletal muscle, and identifying the type can help predict which organs are damaged. Kinases are thus extremely important as target molecules in clinical diagnosis, but their enzymatic activity catalyzes the conversion (synthesis) of ATP and ADP. For example, thymidine kinase catalyzes the reaction of thymidine + ATP by adding a phosphate to thymidine to form thymidylic acid (TMP) + ADP. Therefore, no fluorescent substrates have been found for kinases, including thymidine kinase, and highly sensitive detection at the single-molecule level using digital bioanalysis has yet been successful.

[0008] The present invention has been made in consideration of the above circumstances, and its object is to provide an enzyme detection method and an enzyme detection kit for use therein, which are capable of detecting enzymes that synthesize ATP or ADP, and which enable highly sensitive detection at the single molecule level of enzymes that do not have fluorescent substrates, and which enable highly sensitive measurement of disease biomarkers, etc.

[0009] Embodiments of the present invention have the following aspects. [1] An enzyme detection method for detecting a target enzyme that synthesizes ADP or ATP from an enzyme sample, comprising: preparing a detection device equipped with a microchamber array member in which a plurality of storage compartments that can be filled with a detection solution are arranged; a sample addition step of filling the storage compartments of the detection device with the enzyme sample and a detection reagent containing a hexokinase enzyme to form the detection solution; and a detection step of detecting a signal resulting from the synthesis of ADP or ATP from the detection solution. [2] The enzyme detection method according to [1], wherein the target enzyme is a kinase. [3] The enzyme detection method according to [1] or [2], wherein if the target enzyme is an enzyme that synthesizes ADP, the hexokinase enzyme is an ADP-dependent hexokinase, and if the target enzyme is an enzyme that synthesizes ATP, the hexokinase enzyme is an ATP-dependent hexokinase. [4] The enzyme detection method according to any of [1] to [3], wherein the detection reagent includes resazurin and diaphorase. [5] The method for detecting an enzyme according to any one of [1] to [4], wherein the detection step detects a fluorescent signal. [6] The method for detecting an enzyme according to any one of [1] to [5], wherein the detection reagent does not contain an amine compound or HEPES but contains phosphate. [7] The method for detecting an enzyme according to any one of [1] to [6], wherein the detection reagent contains diaphorase, and the diaphorase and the hexokinase enzyme are recombinant enzymes derived from thermophilic bacteria. [8] The method for detecting an enzyme according to any one of [1] to [7], further comprising an ADP removal treatment step of adding a pretreatment solution containing creatine kinase and phosphocreatine to the enzyme sample and allowing it to react before the sample addition step. [9] An enzyme detection kit for detecting a target enzyme that synthesizes ADP or ATP from an enzyme sample, comprising: a detection device having a microchamber array member in which a plurality of storage compartments are arranged and capable of being filled with a detection solution containing a mixture of an enzyme sample and a detection reagent; and the detection reagent containing a hexokinase enzyme.

[0010] According to the present invention, it is possible to detect enzymes that synthesize ATP or ADP, and among these enzymes, enzymes that do not have fluorescent substrates can be detected with high sensitivity at the single molecule level, thereby providing an enzyme detection method that enables high-sensitivity measurement of disease biomarkers, etc., and an enzyme detection kit used therefor.

[0011] 1 is an exploded partial enlarged view of the detection device of this embodiment. FIG. 1 is a schematic diagram showing a reaction in a sample addition step when the target enzyme contained in the enzyme sample of this embodiment is an enzyme that synthesizes ADP. FIG. 2 is a schematic diagram showing a reaction in a sample addition step when the target enzyme contained in the enzyme sample of this embodiment is an enzyme that synthesizes ATP. FIG. 3 is a photograph of the fluorescent signal of Test Example 1 of this embodiment taken from the bottom. FIG. 4 is a diagram showing the single molecule activity distribution per λ of Test Example 1 of this embodiment. FIG. 5 is a photograph of the fluorescent signal of Test Example 2 of this embodiment taken from the bottom. FIG. 6 is a diagram showing the single molecule activity distribution per λ of Test Example 2 of this embodiment. FIG. 7 is a photograph of the fluorescent signal of Test Example 3 of this embodiment taken from the bottom. FIG. 8 is a diagram showing the single molecule activity distribution per λ of Test Example 3 of this embodiment. FIG. 9 is a photograph of the fluorescent signal of Test Example 4 of this embodiment taken from the bottom. FIG. 10 is a diagram showing the single molecule activity distribution per λ of Test Example 4 of this embodiment.

[0012] The enzyme detection method and enzyme detection kit used therein of the present invention will be described below with reference to embodiments, although the present invention is not limited to the following embodiments.

[0013] [Enzyme detection method] (Enzyme sample, target enzyme) The enzyme detection method of this embodiment is an enzyme detection method for detecting a target enzyme that synthesizes ADP or ATP from an enzyme sample, and includes a detection device equipped with a microchamber array member in which a plurality of storage sections that can be filled with a detection liquid are prepared, and the method includes a sample addition step of filling the storage sections of the detection device with the enzyme sample and a detection reagent containing a hexokinase enzyme to form the detection liquid, and a detection step of detecting a signal due to the synthesis of ADP or ATP from the detection liquid.

[0014] Enzyme samples broadly encompass samples that may contain enzymes. Examples of samples that may contain enzymes include samples collected from living organisms, including tissues, various body fluids, processed products, and extracts thereof. Such samples include serum. Examples of biological tissues include tissue samples obtained from various organs of living organisms. Examples of living organisms include humans and non-human animals. Examples of humans include healthy individuals and patients with various types of cancer.

[0015] In this embodiment, the number of enzyme molecules contained in the enzyme sample can be detected from as little as one molecule per enzyme sample (a liquid sample filled in one of the storage sections described below). For example, by detecting the fluorescent signal of the reaction described below and by setting conditions for reducing the background, the signal of the enzyme reaction of one molecule can be detected.

[0016] A target enzyme that synthesizes ADP or ATP broadly refers to an enzyme that catalyzes a reaction that synthesizes ADP or ATP through its enzymatic activity. (Herein, "an enzyme that catalyzes a reaction that synthesizes ADP or ATP" is also referred to as "an enzyme that synthesizes ADP or ATP.") The target enzyme can be any enzyme that has some activity that synthesizes ADP or ATP. These enzymes can be selected from, for example, various ATPases, various ATP synthases, or various kinases.

[0017] The target enzyme of this embodiment is preferably a kinase. Kinases are enzymes that transfer phosphate groups from molecules with high-energy phosphate bonds, such as ATP, to substrates or target molecules (phosphorylating the target molecule and synthesizing ADP from the ATP), and function as regulators of various intracellular signal transduction and metabolism. Examples of kinases that can be used include the aforementioned thymidine kinase, creatine kinase, Raf kinase, choline kinase (hChK), and other kinases. By obtaining information on their expression, discovery, and reaction mechanisms, kinases have the advantage of being able to be applied to cancer diagnosis, treatment, and the development of therapeutic drugs (e.g., molecularly targeted drugs). For example, CK types have traditionally been identified using absorbance-based measurements and isozyme-specific inhibitory antibodies, and batch detection methods for analysis are known, offering the advantage that the detection method of this embodiment can be considered. Kinases can synthesize ADP as well as ATP via the reverse reaction, and both reactions can be detected in this embodiment.

[0018] As an example of an enzyme that synthesizes ADP, an enzyme with ATPase activity is F １ -ATPase, i.e., F １ from thermophilic Bacillus PS3 (TF １ ) etc.

[0019] Examples of enzymes that synthesize ATP include pyruvate kinase (PK), which has ATP synthase activity.

[0020] 1 is an exploded, partially enlarged view of a detection device 100 according to this embodiment. The detection device according to this embodiment includes a microchamber array member 30 in which a plurality of storage sections 31 that can be filled with a detection solution 20 are arranged.

[0021] In this embodiment, the microchamber array member 30 is a plate-like member formed of, for example, glass, silicon, polymer resin, or the like. In this embodiment, the microchamber array member 30 has a plurality of cylindrical holes arranged (arrayed) that each communicate from the flat portion to the bottom portion of the plate-like member, and these holes form a plurality of storage sections 31 that can be filled with detection liquid. In this embodiment, a bottom member 32 is provided on the bottom surface of the microchamber array member 30. The bottom of the storage section 31 is closed by this bottom member 32, allowing the storage section 31 to be filled with detection liquid 20. The bottom member 32 is made of an optically transparent material so as not to interfere with optical detection of the detection liquid 20 by a signal detection member (not shown), which will be described later. In this embodiment, the bottom member 32 is made of an optically transparent material such as glass or polymer resin.

[0022] The storage sections 31 are arranged at intervals I from each other. This interval I is set to a length that allows the fluorescent signals from the array to be sufficiently separated without overlapping. The size of the storage section 31, i.e., the depth T (in this embodiment, the thickness of the microchamber array member 30) and diameter D, are appropriately selected based on the volume of the detection solution 20 to be detected and the interval I. In this embodiment, the depth T is 3 μm and the diameter D is 4 μm.

[0023] A member for detecting a signal due to synthesis of ADP or ATP may be provided as appropriate as long as it is in a form that allows detection in the storage section 31. In this embodiment, detection is performed using an sCMOS camera (not shown) attached to a fluorescence microscope for observing the microchamber array member 30.

[0024] The member for detecting the signal can be any member capable of detecting a fluorescent signal. Fluorescent signals are widely used in combination with immunological detection means, and are effective because they can increase sensitivity and selectivity. In this embodiment, the member can be used to detect the fluorescent signal of resorufin using a detection solution, which will be described later.

[0025] In this detection device, the hydrophobic solvent supply unit and the internal space of the flow channel may be connected, and the hydrophilic solvent may be held in the flow channel in a state separated from the hydrophobic solvent filled in the hydrophobic solvent supply unit. The internal height of the main body may be equal to or smaller than the size of an oil droplet of the hydrophobic solvent. Furthermore, the detection device may be provided with components such as a partition wall breakable by the force between the hydrophobic solvent supply unit and the flow channel, a deformation unit in the hydrophobic solvent supply unit that is elastically deformable by an externally applied force, the hydrophilic solvent not containing the sample filled in the flow channel, and an openable and closable lid that closes the through-hole.

[0026] Another embodiment that can be used for the detection of the above-described detection device may include a sensor member having sensors corresponding to the positions of the plurality of storage units 31. In this case, the sensor member is provided adjacent to the bottom surface of the microchamber array member. The sensor member is provided with position sensors corresponding to each of the plurality of storage units. As another embodiment, the detection device of this embodiment may be, for example, that described in Patent Document 1. This detection device includes a microchamber array (microchamber array member) disposed inside a main body and having a plurality of storage units filled with a hydrophilic solvent containing a biological sample, and an image sensor (sensor member) having pixels corresponding to the storage units. The main body has a flow channel communicating with an opening of the storage unit, a hydrophobic solvent supply unit connected to the flow channel, and a through-hole through which the hydrophilic solvent can enter and exit the flow channel. The microchamber array has a hydrophobic surface, and the hydrophobic solvent supply unit can flow the hydrophobic solvent into the flow channel by an externally applied force.

[0027] (Sample Addition Step) Next, each step of the enzyme detection method of this embodiment will be described. In the sample addition step, the enzyme sample and a detection reagent containing a hexokinase enzyme are filled into the storage unit of the detection device to prepare the detection solution.

[0028] In the sample addition step, the target enzyme contained in the enzyme sample catalyzes a reaction of a substance contained in the detection reagent, and the reaction generates a signal in the detection solution. The detection reagent contains a substance that uses ADP or ATP synthesized by the target enzyme to cause an enzymatic reaction or a cascade of enzymatic reactions by a hexokinase enzyme, ultimately generating a signal. That is, the detection reagent contains a hexokinase enzyme and substrates, enzymes, and other reagents required for the enzymatic reaction or cascade of enzymatic reactions. Specifically, for example, when detecting a target enzyme that synthesizes ADP, the detection reagent may contain reagents for a reaction that uses ADP synthesized by the target enzyme to phosphorylate glucose via an ADP-dependent hexokinase reaction, and then a cascade of enzymatic reactions that ultimately generates a fluorescent signal. That is, when the target enzyme is an enzyme that synthesizes ADP, the hexokinase enzyme is preferably an ADP-dependent hexokinase. For example, when detecting a target enzyme that synthesizes ATP, the reagent may include a reagent for a cascade reaction in which glucose is phosphorylated by an ATP-dependent hexokinase reaction using the ATP synthesized by the target enzyme, followed by an enzymatic reaction that ultimately produces a fluorescent signal. That is, when the target enzyme is an enzyme that synthesizes ATP, the hexokinase enzyme is preferably an ATP-dependent hexokinase.

[0029] The reaction in this sample addition step will be explained in more detail using the example of the target sample being an enzyme that synthesizes ADP. Figure 2 is a schematic diagram showing the reaction in the sample addition step when the target enzyme contained in the enzyme sample is an enzyme that synthesizes ADP. Here, examples of the enzyme that synthesizes ADP include various kinases and ATPases.

[0030] The detection reagent contains the compounds shown in the figure for this reaction, ATP, glucose, NADP, and resazurin as reaction substrates, and ADP-dependent hexokinase, glucose-6-phosphate dehydrogenase, and diaphorase as reaction enzymes.

[0031] When a detection reagent is added to an enzyme sample containing a target enzyme, ATP is first catalyzed by the target enzyme to synthesize ADP. At this time, when the target enzyme is a kinase, a phosphorylated substrate, which is the target of phosphorylation by the kinase, may be added together with the detection reagent. For example, when the kinase is choline kinase, the phosphorylated substrate is choline. In this case, ADP and choline phosphate are synthesized from ATP. After ADP is synthesized, glucose is then catalyzed by ADP-dependent hexokinase to synthesize AMP and glucose-6-phosphate. After glucose-6-phosphate is synthesized, NADP and glucose-6-phosphate are then catalyzed by glucose-6-phosphate dehydrogenase to synthesize NADPH and 6-phosphogluconolactone. After NADPH is synthesized, NADPH and resazurin are then catalyzed by diaphorase to synthesize NADP and resorufin. Resorufin emits fluorescence with a wavelength of 590 nm in response to excitation light with a wavelength of 540 nm. Therefore, when a detection reagent is added to an enzyme sample containing a target enzyme, a fluorescent signal can be detected in the detection solution. Thus, the target enzyme contained in the enzyme sample can be detected.

[0032] The reaction in this sample addition step will be explained in more detail below, taking as an example a case where the target sample is an enzyme that synthesizes ATP. Figure 3 is a schematic diagram showing the reaction in the sample addition step when the target enzyme contained in the enzyme sample is an enzyme that synthesizes ATP. Here, examples of the enzyme that synthesizes ADP include various kinases as well as ATP synthase. When various kinases are used, the reverse reaction to that in the case of an enzyme that synthesizes ADP can be detected.

[0033] The detection reagent contains the compounds shown in the figure for this reaction, ADP, glucose, NADP, and resazurin as reaction substrates, and ATP-dependent hexokinase, glucose-6-phosphate dehydrogenase, and diaphorase as reaction enzymes.

[0034] When a detection reagent is added to an enzyme sample containing a target enzyme, ADP is first catalyzed by the target enzyme to synthesize ATP. At this time, when the target enzyme is a kinase, a phosphorylated substrate may be added together with the detection solution. When the kinase is pyruvate kinase, ATP and pyruvate are synthesized from the phosphorylated substrate phosphoenolpyruvate (PEP) and ADP. After ATP synthesis, glucose is then catalyzed by ATP-dependent hexokinase to synthesize ADP and glucose-6-phosphate. After glucose-6-phosphate synthesis, NADP and glucose-6-phosphate are then catalyzed by glucose-6-phosphate dehydrogenase to synthesize NADPH and 6-phosphogluconolactone. After NADPH synthesis, NADPH and resazurin are then catalyzed by diaphorase to synthesize NADP and resorufin. Resorufin emits fluorescence with a wavelength of 590 nm in response to excitation light with a wavelength of 540 nm. Therefore, when a detection reagent is added to an enzyme sample containing a target enzyme, a fluorescent signal can be detected in the detection solution. Thus, the target enzyme contained in the enzyme sample can be detected.

[0035] The detection reagent may be configured to contain the above components in separate forms: a substrate solution containing the above-mentioned reaction substrate; and an enzyme solution containing the above-mentioned reaction enzyme, and the substrate solution and the enzyme solution may be mixed in the sample addition step. By mixing the substrate solution and the enzyme solution immediately before the test, the above reaction can occur during the test.

[0036] The detection reagent preferably contains resazurin and diaphorase. The reaction in which NADPH is converted to NADP and resazurin is synthesized into resorufin is catalyzed by diaphorase. Therefore, when the detection reagent contains resazurin and diaphorase, the fluorescence emitted by resorufin can be suitably detected.

[0037] It is also preferable that the detection reagent does not contain an amine compound or HEPES, but contains a phosphate. Here, the amine compound, HEPES, and phosphate mainly refer to compounds added as buffer compounds in pH buffer solutions. Specifically, an amine compound not included in the detection reagent of this embodiment includes Tris (trishydroxymethylaminomethane). Furthermore, examples of phosphates that are preferably included in the detection reagent of this embodiment include potassium phosphate and sodium phosphate. The detection solution of this embodiment more preferably contains potassium phosphate. That is, the detection reagent of this embodiment is preferably a phosphate buffer to which phosphate has been added.

[0038] In conventional assay systems for detecting fluorescence, detection reagents containing amine-based buffers, HEPES, etc., have been used as pH buffering compounds added to buffer solutions. When these amine-based buffers contain HEPES, the background reaction rate is rapid during fluorescence microscopy observation, causing the background light to rapidly brighten (potential for photoreduction), making accurate detection difficult. In conventional bulk measurements using a large-volume container (solution containing an enzyme sample), increasing the enzyme concentration can relatively reduce the background light from the background reaction, thereby reducing its impact. However, when attempting to perform measurements on a small-scale system with a small volume container (detection solution), as in this embodiment, the signal from the activity of a small number of molecules must be compared with the increase in background light, which poses a problem. In this embodiment, the background light from these background reactions can be prevented by using a buffer containing phosphate, such as a phosphate buffer, that does not contain an amine compound or HEPES.

[0039] Furthermore, when the detection reagent contains diaphorase and hexokinase enzymes, it is also preferable that the diaphorase and hexokinase enzymes are recombinant enzymes derived from thermophilic bacteria. Depending on the purification process, diaphorase and hexokinase enzymes may contain enzymes that react even when the target enzyme is not contained in the enzyme sample, resulting in many bright spots during detection, or a high rate of false positives. For example, using commercially available diaphorase or ADP hexokinase (glucokinase) as is can result in a high rate of false positives. In this embodiment, this false positive rate can be reduced by expressing a recombinant enzyme derived from thermophilic bacteria and using an enzyme that has been purified to reduce false positives.

[0040] In this embodiment, an expression system for a recombinant enzyme derived from thermophilic bacteria is constructed, and false positives are confirmed by digital measurement. By carefully heat-treating and purifying the enzyme, false positive signals are reduced by approximately 30-40 times.

[0041] As a kit containing a detection reagent that can be used to detect enzymes that synthesize ADP and ATP, Fluorospark® Kinase / ADP Multi-Assay Kit (Fujifilm Wako Pure Chemical Industries, Ltd.) or a detection kit containing an equivalent composition can be used.

[0042] The enzyme detection method of this embodiment preferably further includes an ADP removal treatment step, prior to the sample addition step, in which a pretreatment solution for removing ADP is added to the enzyme sample and reacted. This pretreatment is performed when detecting an enzyme that synthesizes ADP. The pretreatment solution for removing ADP can be, for example, one containing a phosphorylated substrate and a kinase that catalyzes the dephosphorylation of the substrate. Specifically, the pretreatment solution can be one containing phosphocreatine as the phosphorylated substrate and creatine kinase as the kinase. In the ADP removal treatment step, ADP contained in the enzyme sample can be removed by adding a pretreatment solution containing creatine kinase and phosphocreatine to the enzyme sample and reacting it. Creatine kinase causes a phosphorylation transfer reaction from phosphocreatine to ADP, resulting in the conversion of ADP to ATP, thereby reducing ADP. As a guideline, this step is expected to reduce ADP to 0.1% or less. If ADP is contained in an enzyme sample before it is synthesized by the target enzyme, it can become a background signal when detecting ADP synthesis, and can also lead to inaccurate data and calibration curves for ADP synthesis control samples. These problems can be prevented by performing this ADP removal treatment step. Furthermore, in this embodiment, when measurements are performed on a small-scale system or with a small capacity container, the influence of ADP contained in the enzyme sample becomes even greater. Therefore, in this embodiment, the ADP removal treatment step is particularly effective for increasing the accuracy of detection.

[0043] As an advantage of this embodiment, in conventional digital bioanalysis using a microchamber array, such as that described in Patent Document 1, enzymes and their dedicated fluorescent substrates are introduced into a microchamber array, encapsulated in oil, and the fluorescence associated with the reaction is then imaged using a microscope or other device. In this embodiment, instead of using dedicated fluorescent substrates, an assay solution conjugated with three enzyme reactions is added, converting the ADP and ATP produced in the enzyme reaction into fluorescent substances, and imaging their fluorescence. This eliminates the need for dedicated fluorescent substrates for the enzymes, and in principle, all enzymes that produce ADP and ATP can be measured using this single system. Another advantage is that the decomposition reaction of the enzyme's natural substrate, rather than an artificial fluorescent substrate, can be measured at the single enzyme molecule level.

[0044] [Enzyme detection kit] The enzyme detection kit of this embodiment is an enzyme detection kit for detecting a target enzyme that synthesizes ADP or ATP from an enzyme sample, and includes a detection device having a microchamber array member in which multiple storage sections are arranged that can be filled with a detection liquid that is a mixture of an enzyme sample and a detection reagent, and the detection reagent containing a hexokinase enzyme.

[0045] The enzyme sample, target enzyme, detection reagent, and detection device can be selected from the same ones as those described in the enzyme detection method described above.

[0046] According to the enzyme detection kit of this embodiment, the target enzyme can be suitably detected using the enzyme detection method.

[0047] The enzyme detection kit of this embodiment may contain the above-mentioned components and may also contain optional components described below. For example, it may contain a reaction stop solution for terminating the enzyme reaction. The reaction stop solution may contain, for example, a reducing agent. Furthermore, it may contain a reagent containing ATP or ADP for a substrate, control, or calibration curve, separate from the substrate solution and enzyme solution.

[0048] [Effects of this embodiment] This embodiment makes it possible to detect enzymes that synthesize ATP or ADP, and among these enzymes, enzymes that do not have fluorescent substrates can be detected with high sensitivity at the single molecule level, thereby providing an enzyme detection method that allows for high-sensitivity measurement of disease biomarkers, etc., and an enzyme detection kit used therefor.

[0049] This embodiment provides a new digital bioanalysis technology that does not require a fluorescent substrate recognized by the target enzyme. The technology of this embodiment enables digital bioanalysis of enzymes that do not have a fluorescent substrate. Furthermore, while bioassays in conventional technology have been based on a one-to-one correspondence between substrate species and enzyme species, it has become possible to construct a system that enables digital measurement of multiple enzymes using a single assay platform. The technology of this embodiment is applicable to various enzymes that produce ATP and ADP, and is expected to be applied to the precise measurement of the activity of single enzyme molecules, which was previously impossible to measure, and to the detection of disease biomarkers using enzyme activity patterns.

[0050] As mentioned above, it is believed that kinases and enzymes that synthesize ATP and ADP may be used as biomarkers for diseases such as cancer. For example, ATPase and AMPase activity in the serum of colorectal cancer patients is significantly higher than that of healthy subjects. It has been reported that these activities are significantly increased, particularly in patients with advanced cancer. It has also been reported that prostate cancer patients have significantly higher ATP, ADP, and AMP hydrolysis activity in the serum than healthy subjects. Patients with lower clinical stage (CS-IIA) showed elevated ATP hydrolysis activity compared to patients with more advanced clinical stage (CS-IIB and CS-III). These results suggest that ATPase may be used as a new biomarker for cancer risk assessment. Therefore, the method for detecting enzymes that synthesize ATP and ADP of this embodiment may be applicable to methods for obtaining information about cancer. According to this embodiment, it is possible to measure the activity of small amounts of molecules, for example, single molecules, of kinases that are often used as biomarkers for diseases such as cancer, with high sensitivity, and it is also possible to detect activity distributions that are characteristic of each disease. Utilizing such activity distributions is expected to enable more sensitive and selective discovery and diagnosis of enzyme biomarkers.

[0051] (Other Embodiments) In one embodiment, the present invention provides a method for diagnosing a disease using the enzyme detection method. In one embodiment, the present invention provides a method for diagnosing cancer or myocardial infarction using the enzyme detection method. In one embodiment, the present invention provides a method for diagnosing a disease using the enzyme detection kit. In one embodiment, the present invention provides a method for diagnosing cancer or myocardial infarction using the enzyme detection kit. In one embodiment, the present invention provides a method for manufacturing the enzyme detection kit. In one embodiment, the present invention provides use of the detection reagent for manufacturing the enzyme detection kit. In one embodiment, the present invention provides use of the detection device for manufacturing the enzyme detection kit.

[0052] In each of these embodiments, the configurations and conditions of use are the same as those described above.

[0053] The present embodiment will be described below with reference to examples, but the present invention is not limited to the following examples.

[0054] (Test Example 1: Detection of human choline kinase) Using human choline kinase (hChK) as the target enzyme, a test was conducted to determine the sensitivity of enzyme detection relative to the number of enzymes per enzyme sample. hChK exists in three isoforms, α1, α2, and β, and is known to function as a dimer, catalyzing the first step of phospholipid synthesis in vivo. hChKα1 is involved in tumorigenesis and has attracted attention as a new target molecule for cancer therapeutics.

[0055] The detection device described in the above embodiment (5 million storage units (reactors) per device) was used. An enzyme sample was prepared with an hChK concentration adjusted so that the ratio of the number of enzymes used to the number of storage units was λ. A resazurin solution and a reaction stop solution were prepared to adjust the detection solution. The concentrations of the substrate solution and each of the above solutions were determined based on the conditions under which no background was generated when used. An ATP solution and an ADP solution were prepared for the kinase substrate and the calibration curve. A phosphate buffer containing potassium phosphate (50 mM potassium phosphate, pH 7.5, 100 mM KCl, 2 mM MgCl) was used as the detection reagent, without containing an amine compound or HEPES. ２ , 0.02% Tween 20, 1 mM glucose, 100 μM NADP+, 100 μM resazurin) was used. For the diaphorase enzyme and hexokinase enzyme, recombinant enzymes derived from thermophilic bacteria were produced and heat-treated and purified until false-positive signals were reduced by approximately 30-40 times. For the enzyme sample, a pretreatment solution containing creatine kinase and phosphocreatine was added to the enzyme sample prior to the sample addition step, and the reaction was allowed to proceed. Specifically, an ADP removal treatment step was performed in which the ADP in the enzyme sample was converted to ATP by a phosphorylation transfer reaction from phosphocreatine to ADP, thereby reducing the ADP content, and the residual ADP level was reduced to 0.1% or less.

[0056] Samples were also prepared with and without the addition of choline molecules as a substrate. Phosphorylated choline and ADP are synthesized from choline and ATP by the reaction of human choline kinase.

[0057] Figure 4 is a photograph of the fluorescent signal of the detection device taken from the bottom. The white dots in the figure indicate the receptacles in which the fluorescent signal of the enzyme could be detected among the array of receptacles. The number of molecules of enzyme contained in a receptacle can be calculated from the number of enzymes dispensed into the receptacle relative to the number of receptacles. Considering the case where a sample solution containing a fixed number of enzymes is dispensed into all receptacles, when the ratio of the number of enzymes in all sample solutions to the total number of receptacles is λ, the probability P(n) that n enzymes will be contained in one receptacle is given by the Poisson distribution P(n, λ) = (λ ｎ ・e －λ ) / n!, it is believed that when λ = 0.1 or less, one enzyme molecule or less is contained in each container. At λ = 0.1 and 0.01 in the figure, it is believed that there are containers containing zero enzyme molecules and containers containing one enzyme molecule. From the figure, fluorescent signals are detected only in some containers. In other words, it is believed that the fluorescent signal of the enzyme is detected in containers containing one enzyme molecule. On the other hand, at λ = 0, no enzyme is present in any container, but no enzyme is detected. At λ = 1, all containers contain one or more enzymes, but excessive signals are observed in all containers. From the example shown in the figure, it was revealed that this enzyme detection method can detect the presence or absence of a molecule in each container at λ = 0.01 and 0.1, and can achieve sensitivity sufficient to detect if one enzyme molecule is present per container.

[0058] Figure 5 shows the single molecule activity distribution per λ. In the figure, the vertical axis (Counts) represents the number of containers, and the horizontal axis (Slope) represents the activity (a.u. / min). For example, a high peak near the left end of each graph, where activity is 0, indicates that many empty containers with no activity (no enzyme molecules contained) have been detected. In addition, for each test graph, the vertical axis ranges from 8000 to 12 x 10 ３The graph (bottom left) with the horizontal axis ranging from 10 to 40 is superimposed with an enlarged portion of it (top right).

[0059] As shown in the figure, a peak in the active compartment was observed in the test with choline added (Choline+), while a peak was observed on the active side (farther to the right) in the test without choline added. In other words, a choline-dependent peak was observed, allowing the activity of a single hChK molecule to be detected. Furthermore, in the test with Choline+, where a peak was observed, the distribution shape was not a single clear peak (heterogeneous). This may indicate the presence of inactive molecular species derived from multimers, which may have been detected in this test.

[0060] (Test Example 2: Detection of ATPase) F, which is an ATPase, was used as the target enzyme. １ Using ATPase, a test was conducted to determine the sensitivity of enzyme detection relative to the number of enzymes per enzyme sample. The detection device and detection reagent were the same as in Test Example 1.

[0061] Figure 6 is a photograph of the fluorescent signal from the detection device taken from the bottom. The white dots in the figure indicate the storage sections where the fluorescent signal of the enzyme was detected among the arrayed storage sections. At λ = 0.3 in the figure, the fluorescent signal was detected only in some storage sections, demonstrating that this enzyme detection method can detect the presence or absence of molecules in each storage section at λ = 0.3.

[0062] FIG. 7 shows the single molecule activity distribution per λ. The explanation in the figure is the same as in Test Example 1. The test was performed at λ = 1, 0.5, 0.3, and 0. As shown in the figure, a peak of activity was confirmed in the test graph for each λ. That is, F １ It was shown that the activity of each single ATPase molecule can be detected.

[0063] (Test Example 3: Detection of Pyruvate Kinase) Using pyruvate kinase (PK), which synthesizes ATP, as the target enzyme, a test was conducted to assess the sensitivity of enzyme detection relative to the number of enzymes per enzyme sample. The detection device and detection reagents were the same as in Test Example 1. Pyruvate kinase is widely known to catalyze the final reaction in the thawing system (PEP + ADP to Pyruvate + ATP). PK functions as a tetramer. There are four isoforms of PK: PKL, PKR, PKM1, and PKM2. PKM2 is involved in the metabolism specific to cancer cells and is attracting attention as a target molecule for cancer therapeutic drugs and as a blood biomarker for cancer.

[0064] Figure 8 is a photograph of the fluorescent signal from the detection device taken from the bottom. The white dots in the figure indicate the storage sections in which the fluorescent signal of the enzyme was detected. At λ = 0.1 in the figure, the fluorescent signal was detected only in some storage sections, demonstrating that this enzyme detection method can detect the presence or absence of molecules in each storage section at λ = 0.1.

[0065] FIG. 9 shows the single molecule activity distribution per λ. The explanation in the figure is the same as in Test Example 1. The experiment was performed at λ = 1, 0.1, and 0. As shown in the figure, a peak of activity was confirmed in the test graph for each λ. In other words, it was demonstrated that the activity of each pyruvate kinase molecule could be detected.

[0066] (Test Example 4: Detection of alkaline phosphatase) Using alkaline phosphatase (ALP) as the target enzyme, a test was conducted to determine the sensitivity of enzyme detection relative to the number of enzymes per enzyme sample. The detection device and detection reagents were the same as those used in Test Example 1. Alkaline phosphatase is widely known as an enzyme that decomposes phosphate compounds and is used, for example, as a criterion for diagnosing liver disease. Alkaline phosphatase is an enzyme for which a fluorescent substrate exists that can also be used with conventional techniques; therefore, it was also examined whether the method of this example could be used in place of conventional methods.

[0067] Figure 10 is a photograph of the fluorescent signal from the detection device taken from the bottom. The white dots in the figure indicate the storage sections where the fluorescent signal of the enzyme was detected among the arrayed storage sections. At λ = 0.1 in the figure, the fluorescent signal was detected only in some storage sections, demonstrating that this enzyme detection method can detect the presence or absence of molecules in each storage section at λ = 0.1.

[0068] FIG. 11 shows the single-molecule activity distribution per λ. The explanation in the figure is the same as in Test Example 1. Tests were performed at λ = 0.1, 0.01, 0.001, and 0. As shown in the figure, peaks of active compartments were confirmed in the graphs for each λ test. All of λ = 0.1, 0.01, and 0.02 show a heterogeneous distribution (two peaks). Alkaline phosphatase is known to have two activity peaks depending on whether two subdomains exhibit activity. In other words, it was demonstrated that the method of this example can detect alkaline phosphatase activity. Based on these results, it is believed that the method of this example is also effective for detecting enzymes that have conventional fluorescent substrates.

[0069] Although several embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, as well as within the scope of the invention and its equivalents as defined in the claims.

[0070] According to the present invention, it is possible to detect enzymes that synthesize ATP or ADP, and among these enzymes, enzymes that do not have fluorescent substrates can be detected with high sensitivity at the single molecule level, thereby providing an enzyme detection method that enables high-sensitivity measurement of disease biomarkers, etc., and an enzyme detection kit used therefor.

[0071] 20 Detection solution 30 Microchamber array member 31 Storage section 32 Bottom member 100 Detection device D Diameter I Spacing T Depth

Claims

1. An enzyme detection method for detecting a target enzyme that synthesizes ADP or ATP from an enzyme sample, comprising: preparing a detection device equipped with a microchamber array member in which a plurality of storage compartments capable of being filled with a detection liquid are arranged; a sample addition step in which the storage compartments of the detection device are filled with the enzyme sample and a detection reagent containing a hexokinase enzyme to prepare the detection liquid; and a detection step in which a signal due to the synthesis of ADP or ATP is detected from the detection liquid.

2. The method for detecting an enzyme according to claim 1, wherein the target enzyme is a kinase.

3. A method for detecting an enzyme according to claim 1 or 2, wherein when the target enzyme is an enzyme that synthesizes ADP, the hexokinase enzyme is an ADP-dependent hexokinase, and when the target enzyme is an enzyme that synthesizes ATP, the hexokinase enzyme is an ATP-dependent hexokinase.

4. The method for detecting an enzyme according to claim 1 or 2, wherein the detection reagent comprises resazurin and diaphorase.

5. The method for detecting an enzyme according to claim 1 or 2, wherein the detection step detects a fluorescent signal.

6. The method for detecting an enzyme according to claim 1 or 2, wherein the detection reagent does not contain an amine compound or HEPES, but contains a phosphate.

7. The method for detecting an enzyme according to claim 1 or 2, wherein the detection reagent contains diaphorase, and the diaphorase and hexokinase enzymes are recombinant enzymes derived from thermophilic bacteria.

8. The method for detecting an enzyme according to claim 1 or 2, further comprising an ADP removal treatment step of adding a pretreatment solution containing creatine kinase and phosphocreatine to the enzyme sample and allowing it to react prior to the sample addition step.

9. An enzyme detection kit for detecting a target enzyme that synthesizes ADP or ATP from an enzyme sample, comprising: a detection device having a microchamber array member in which a plurality of storage compartments are arranged in which a detection liquid containing a mixture of an enzyme sample and a detection reagent can be filled; and the detection reagent containing a hexokinase enzyme.

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