Enzyme detection method and enzyme detection kit

Abstract

To provide 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 for a disease biomarker or the like can be performed; and an enzyme detection kit used in the same.SOLUTION: 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 resulting from the synthesis of ADP or ATP.SELECTED DRAWING: None

Description

[Technical field]

[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. [Background technology]

[0002] Conventionally, a technique has been used to obtain information on biological samples and enzymes by detecting enzymes with specific activities from various biological samples. For example, by detecting an enzyme from a sample derived from a specific organ, it is possible to detect associations with disease or infection (disease / infection biomarkers). Kinases are considered as disease biomarkers.

[0003] Immunochemical techniques, particularly ELISA, can be used to detect enzymes in small amounts and large numbers of samples, and can be applied to the detection of disease and infection biomarkers. In addition, a multiplexed single enzyme assay technology has been developed to examine isozymes. Isozymes are enzymes that catalyze the same reaction, but have slightly different structures and different substrate specificities. This assay is a method to identify isozymes by measuring their substrate-specific activity at the single molecule level. Since there are various organ-specific isozymes in the blood, it is possible to investigate with high sensitivity which organ may be the cause of the disease. In addition, isozymes that form multimers may not have a uniform activity distribution of single molecules, and by investigating the shape of this distribution, it is possible to investigate which disease is affected. In addition, in the analysis of small amounts and many samples such as ELISA, methods that perform detection and analysis automatically have been developed, and these digital bioanalyses (digital ELISA) are also attracting attention as a method to detect biomarkers with high sensitivity.

[0004] As a detection device and a detection method for detecting the enzyme activity of a large number of trace samples, for example, Patent Document 1 discloses a detection device including a microchamber array having a plurality of storage sections filled with a hydrophilic solvent containing a biological sample, and an image sensor having pixels corresponding to the storage sections, in which the microchamber array has a flow passage communicating with the opening of the storage section, a hydrophobic solvent supply section connected to the flow passage, and a through hole through which the hydrophilic solvent can enter and exit the flow passage, and the hydrophobic solvent supply section causes the hydrophobic solvent to flow into the flow passage by a force applied from the outside, and a detection method using the same. This technology aims to obtain a detection device that can prevent a decrease in the concentration of the biological sample and reagent contained in the storage section and improve measurement sensitivity by sealing the opening of the storage section with a hydrophobic solvent and confining the hydrophilic solvent containing the biological sample in the storage section, and a detection method using the same. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2014 / 034781 Summary of the Invention [Problem to be solved by the invention]

[0006] As described above, conventional biomarker detection techniques are capable of detecting a large number of samples with high sensitivity, but are unable to detect various enzyme-type biomarkers. That is, in conventional biomarker detection techniques, a substrate that reacts with an enzyme of interest is prepared, and the enzyme reacts with the substrate (e.g., the substrate is partially cleaved by the enzyme reaction) to detect the enzyme. For this purpose, for example, a portion of the substrate is modified so that it can be detected by the reaction. However, in the case of enzymes for which such specific substrates (substrates that can be used for fluorescence detection may hereinafter be referred to as fluorescent substrates) have not been found, such as enzymes that catalyze reactions of general molecules in living organisms, such as ADP and ATP, they cannot be detected. Even if a substrate is found, it cannot be detected if it is difficult to modify it so that the reaction can be detected.

[0007] For example, kinases are involved in the proliferation and invasion of cancer cells, and it is believed that kinase mutations lead to cancer. Many kinase inhibitors have been developed as molecular targeting drugs for cancer. For example, mutations in Raf kinase and activity of thymidine kinase in the blood are biomarkers closely related to cell proliferation, and are expected to be used as tools for monitoring the prognosis of cancer treatment. For example, it has been reported that an increase in thymidine kinase activity in serum is associated with neoplastic diseases, malignant diseases, vitamin B12 deficiency, and viral infections. Activity of thymidine kinase is a biomarker closely related to cell proliferation, and is expected to be used as a tool for monitoring the prognosis of cancer treatment. Creatine kinase (CK) in the blood increases in blood concentration after the onset of myocardial infarction. Therefore, it is widely used in the diagnosis of myocardial infarction. CK has isozymes derived from the brain, smooth muscle, heart, and skeletal muscle, and by identifying the type, it is possible to infer which organ is damaged. Kinases are thus very important as target molecules for 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. As a result, no fluorescent substrates have been found for kinases, including thymidine kinase, and therefore 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-mentioned circumstances, and an object of the present invention 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 these enzymes that do not have a fluorescent substrate, thereby enabling highly sensitive measurement of disease biomarkers, etc. [Means for solving the problem]

[0009] Embodiments of the present invention have the following aspects. [1] A method for detecting a target enzyme that synthesizes ADP or ATP from an enzyme sample, comprising: A detection device is provided that includes a microchamber array member having an array of multiple storage sections that can be filled with a detection solution; a sample addition step of filling the container of the detection device with the enzyme sample and a detection reagent containing a hexokinase enzyme to prepare the detection solution; a detection step of detecting a signal due to synthesis of ADP or ATP from the detection solution; A method for detecting an enzyme comprising the steps of: [2] The method for detecting an enzyme according to [1], wherein the target enzyme is a kinase. [3] The method for detecting an enzyme described in [1] or [2], wherein when the target enzyme is an enzyme that synthesizes ADP, the hexokinase enzyme is ADP-dependent hexokinase, and when the target enzyme is an enzyme that synthesizes ATP, the hexokinase enzyme is ATP-dependent hexokinase. [4] The method for detecting an enzyme described in any one of [1] to [3], wherein the detection reagent contains resazurin and diaphorase. [5] The method for detecting an enzyme described in 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 a phosphate. [7] The method for detecting an enzyme described in any one of [1] to [6], wherein the detection reagent contains diaphorase, and the diaphorase and hexokinase enzyme are recombinant enzymes derived from thermophilic bacteria. [8] A method for detecting an enzyme described in 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 prior to the sample addition step, to allow reaction. [9] An enzyme detection kit for detecting a target enzyme that synthesizes ADP or ATP from an enzyme sample, comprising: A detection device including a microchamber array member in which a plurality of storage sections capable of being filled with a detection liquid in which an enzyme sample and a detection reagent are mixed are arranged; the detection reagent comprising a hexokinase enzyme; An enzyme detection kit comprising: Effect of the Invention

[0010] According to the present invention, it is possible to detect enzymes that synthesize ATP or ADP, and it is possible to detect these enzymes that do not have a fluorescent substrate 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. [Brief description of the drawings]

[0011] [Figure 1] FIG. 2 is an exploded partial enlarged view of the detection device according to the embodiment. [Diagram 2] FIG. 2 is a schematic diagram showing the reaction in the sample addition step when the target enzyme contained in the enzyme sample of this embodiment is an enzyme that synthesizes ADP. [Diagram 3] FIG. 2 is a schematic diagram showing the reaction in the sample addition step when the target enzyme contained in the enzyme sample of this embodiment is an enzyme that synthesizes ATP. [Figure 4] FIG. 2 is a photograph of the fluorescent signal in Test Example 1 of this embodiment taken from the bottom direction. [Diagram 5] FIG. 1 shows the single molecule activity distribution per λ in Test Example 1 of the present embodiment. [Figure 6] FIG. 2 is a photograph of the fluorescent signal from Test Example 2 of this embodiment taken from the bottom. [Figure 7] FIG. 1 shows the single molecule activity distribution per λ in Test Example 2 of this embodiment. [Figure 8] FIG. 2 is a photograph of the fluorescent signal in Test Example 3 of this embodiment taken from the bottom. [Figure 9] FIG. 1 shows the single molecule activity distribution per λ in Test Example 3 of this embodiment. [Figure 10] FIG. 1 is a photograph of the fluorescent signal from Test Example 4 of this embodiment taken from the bottom. [Figure 11] FIG. 1 shows the single molecule activity distribution per λ in Test Example 4 of the present embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Hereinafter, the enzyme detection method and the enzyme detection kit used therein of the present invention will be described 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 having a microchamber array member in which a plurality of storage sections into which a detection liquid can be filled is prepared, and the detection liquid is prepared by filling the storage sections of the detection device with the enzyme sample and a detection reagent containing a hexokinase enzyme, and a detection step of detecting a signal due to the synthesis of ADP or ATP from the detection liquid.

[0014] The enzyme sample broadly includes samples that may contain enzymes. Examples of samples that may contain enzymes include samples taken from living organisms, including tissues, various body fluids, processed products thereof, and extracts. Examples of such samples include serum. Examples of biological tissues include tissue samples obtained from various organs of living organisms. Living organisms include, inter alia, humans and non-human animals, Humans include, for example, healthy individuals and individuals suffering from various types of cancer.

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

[0016] The target enzyme that synthesizes ADP or ATP broadly refers to an enzyme that catalyzes a reaction in which ADP or ATP is synthesized by its enzymatic activity. (In this specification, the "enzyme that catalyzes a reaction in which ADP or ATP is synthesized" is also referred to as the "enzyme that synthesizes ADP or ATP.") The target enzyme can be any enzyme that has some activity of synthesizing 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. Kinases that can be used include the above-mentioned thymidine kinase, creatine kinase, Raf kinase, choline kinase (hChK), and other kinases. Kinases have the advantage that by obtaining information on their expression, exploration, and reaction mechanisms, the information can be applied to cancer diagnosis, treatment, and development of therapeutic drugs (e.g., molecular targeted drugs), etc. For example, CK has traditionally been identified by absorbance-based measurement and isozyme-specific inhibitory antibodies, and therefore, a batch detection method is known, making it possible to study the detection method of this embodiment. Kinase is an enzyme that synthesizes ADP, and can also function as an enzyme that synthesizes ATP through a reverse reaction. In this embodiment, either reaction can be detected.

[0018] An example of an enzyme that synthesizes ADP is F1-ATPase, i.e., F1from thermophilic Bacillus PS3 (TF1), which has ATPase activity.

[0019] An example of an enzyme that synthesizes ATP and has ATP synthase activity is pyruvate kinase (PK).

[0020] (Detection device) 1 is an exploded partial enlarged view of a detection device 100 of this embodiment. The detection device of this embodiment includes a microchamber array member 30 in which a plurality of storage sections 31 capable of being filled with a detection liquid 20 are arranged.

[0021] In this embodiment, the microchamber array member 30 is made of 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 are connected from the flat surface portion to the bottom surface portion of the plate-like member, and the holes serve as a plurality of storage sections 31 that can be filled with detection liquid. In this embodiment, the bottom surface of the microchamber array member 30 is provided with a bottom surface member 32. The bottom surface of the storage section 31 is blocked by the bottom surface member 32, so that the storage section 31 can be filled with the detection liquid 20. The bottom surface member 32 is made of a light-transmitting constituent material so as not to interfere with the optical detection of the detection liquid 20 by a member (not shown) for detecting a signal, which will be described later. In this embodiment, the bottom surface member 32 is made of a light-transmitting constituent 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 liquid 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 appropriately 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] As the member for detecting the signal, any member capable of detecting a fluorescent signal can be used appropriately. Fluorescence signals are widely used in combination with immunological detection means, and are effective because they can increase sensitivity and selectivity. In this embodiment, the fluorescent signal of resorufin produced by a detection solution described later can be detected.

[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 height inside the main body may be equal to or smaller than the size of an oil droplet of the hydrophobic solvent. In addition, a partition breakable by the force may be provided between the hydrophobic solvent supply unit and the flow channel, a deformation unit in the hydrophobic solvent supply unit that is elastically deformable by a force applied from the outside, the hydrophilic solvent not containing the sample concentrated in the flow channel, and an openable and closable lid unit that closes the through hole.

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

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

[0028] In the sample addition step, a reaction of a substance contained in the detection reagent is catalyzed by the target enzyme contained in the enzyme sample, and a signal is generated by the reaction in the detection solution. The detection reagent contains a substance that causes an enzymatic reaction or a cascade of enzymatic reactions by a hexokinase enzyme to proceed using ADP or ATP synthesized by the target enzyme, and ultimately emits a signal, i.e., the detection reagent contains the hexokinase enzyme and substrates, enzymes, and other reagents required for the enzymatic reaction or the cascade of enzymatic reactions. Specifically, for example, when detecting a target enzyme that synthesizes ADP, the reagent may contain a reagent for proceeding to a cascade reaction of enzyme reactions that proceeds from the ADP-dependent hexokinase reaction to phosphorylate glucose using the ADP synthesized by the target enzyme to an enzyme reaction that finally emits a fluorescent signal. That is, when the target enzyme is an enzyme that synthesizes ADP, the hexokinase enzyme is preferably ADP-dependent hexokinase. For example, when detecting a target enzyme that synthesizes ATP, the reagent may contain a reagent for proceeding to a cascade reaction of enzyme reactions, which uses the ATP synthesized by the target enzyme to phosphorylate glucose through an ATP-dependent hexokinase reaction, and then proceeds to an enzyme reaction that finally emits 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 described more specifically below, taking as an example a case in which the target sample is an enzyme that synthesizes ADP. 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, the enzyme that synthesizes ADP includes various kinases as well as ATPase.

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

[0032] The reaction in this sample addition step will now be described in more detail, taking as an example a case in which the target sample is an enzyme that synthesizes ATP. 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, the enzyme that synthesizes ADP includes various kinases as well as ATP synthase. When various kinases are used, the reverse reaction to that in the case of the 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 the detection reagent is added to an enzyme sample containing a target enzyme, ADP is first catalyzed by the target enzyme to synthesize ATP. In this case, if the target enzyme is a kinase, a phosphorylated substrate may be added together with the detection solution. If the kinase is pyruvate kinase, ATP and pyruvate are synthesized from the phosphorylated substrate phosphoenolpyruvate (PEP) and ADP. ATP is then synthesized, and glucose is then catalyzed by ATP-dependent hexokinase to synthesize ADP and glucose-6-phosphate. Glucose-6-phosphate is then synthesized, and NADP and glucose-6-phosphate are then catalyzed by glucose-6-phosphate dehydrogenase to synthesize NADPH and 6-phosphogluconolactone. NADPH is synthesized, and then NADPH and resazurin are catalyzed by diaphorase to synthesize NADP and resorufin. Resorufin emits fluorescence with a wavelength of 590 nm in response to incidence of excitation light with a wavelength of 540 nm. Therefore, when the detection reagent is added to an enzyme sample containing the target enzyme, a fluorescent signal can be detected in the detection solution, and 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 substrate solutions containing the above-mentioned reaction substrate and enzyme solutions containing the above-mentioned reaction enzyme, and to mix the substrate solution and enzyme solution in the sample addition step. By mixing the substrate solution and enzyme solution immediately before the test, the above reaction can occur during the test.

[0036] It is also preferred that the detection reagent comprises 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 preferred that the detection reagent does not contain an amine compound or HEPES, but contains a phosphate, where the amine compound, HEPES and phosphate refer to those that are added mainly as buffer compounds in a pH buffer solution. Specifically, examples of amine compounds not included in the detection reagent of this embodiment include Tris (trishydroxymethylaminomethane). In addition, examples of phosphates that are preferably contained in the detection reagent of this embodiment include potassium phosphate and sodium phosphate. It is more preferable that the detection solution of this embodiment contains potassium phosphate. That is, the detection reagent of this embodiment is preferably a phosphate buffer to which a phosphate has been added.

[0038] In conventional assay systems that detect fluorescence, detection reagents containing amine buffers, HEPES, etc., have been used as pH buffering compounds added to the buffer solution. When these amine buffers contain HEPES, the background reaction speed is fast in fluorescence microscopy observation, and the background light becomes bright rapidly (there is a possibility of photoreduction), making accurate detection difficult. In conventional techniques, in bulk measurements with a large volume of storage (solution containing an enzyme sample), the background light of the background reaction can be relatively reduced by increasing the enzyme concentration, so the influence can be reduced. However, when attempting to perform measurements on a small scale system with a small volume of storage (detection solution) as in this embodiment, a problem occurs because the signal of the activity of a small amount of molecules must be compared with the increase in background light. In this embodiment, the background light of these background reactions can be prevented by preparing the detection reagent as a buffer solution that does not contain an amine compound or HEPES but contains phosphate, for example, a phosphate buffer.

[0039] In addition, 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 enzyme sample does not contain the target enzyme, and may produce many bright spots during detection, i.e., a high false positive rate. For example, using commercially available diaphorase and ADP hexokinase (glucokinase) as is may result in a high false positive rate. In this embodiment, a recombinant enzyme derived from a thermophilic bacterium is expressed and purified so as to reduce false positives, and thus the false positives can be reduced.

[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 carrying out thorough heat treatment and purification, false positive signals are reduced by about 30-40 times.

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

[0042] The enzyme detection method of this embodiment preferably further includes an ADP removal treatment step of adding a pretreatment solution for removing ADP to the enzyme sample and reacting the sample before the sample addition step. This pretreatment is performed when detecting an enzyme that synthesizes ADP. As the pretreatment solution for removing ADP, for example, one containing a phosphorylated substrate and a kinase that catalyzes the dephosphorylation of the substrate can be used. Specifically, the pretreatment solution can be one containing phosphocreatine as the phosphorylated substrate and creatine kinase as the kinase. In the ADP removal process, 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 phosphoryl transfer reaction from phosphocreatine to ADP, which results in ADP becoming ATP and reducing ADP. As a guideline, it is thought that ADP will be reduced to 0.1% or less by this process. If ADP is contained in the enzyme sample before it is synthesized by the target enzyme, it can cause problems such as background when detecting ADP synthesis, and the data and calibration curve of the control sample for ADP synthesis can be inaccurate. These problems can be prevented by performing this ADP removal treatment step. Furthermore, in this embodiment, when a measurement is performed in a small-scale system or with a small capacity of the storage section, the effect of ADP contained in the enzyme sample becomes greater. Therefore, in this embodiment, the ADP removal treatment step is particularly effective in increasing the accuracy of detection.

[0043] As an effect of this embodiment, in conventional digital bioanalysis using a microchamber array, such as in Patent Document 1, an enzyme and a fluorescent substrate dedicated to that enzyme are introduced into a microchamber array, sealed with oil, and the fluorescence accompanying the reaction is imaged using a microscope or the like. In this embodiment, instead of a dedicated fluorescent substrate, an assay solution conjugated with three enzyme reactions is added, and ADP and ATP produced in the enzyme reaction are converted into fluorescent substances, and the fluorescence is imaged. In this way, a fluorescent substrate dedicated to the enzyme is no longer necessary, and in principle, all enzymes that produce ADP and ATP can be measured with this one system. Another advantage is that the decomposition reaction of the enzyme's original natural substrate can be measured at the enzyme single molecule level, rather than using an artificial fluorescent substrate.

[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 a plurality of storage sections 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.

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

[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 contains the above-mentioned components and may also contain optional components described below. For example, the reaction may contain a reaction terminating solution for terminating the enzyme reaction. The reaction terminating solution may contain, for example, a reducing agent. Furthermore, reagents containing substrates, controls, or ATP or ADP for calibration curves may be included separately from the substrate solution and enzyme solution.

[0048] [Effects of this embodiment] According to the present embodiment, 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.

[0049] This embodiment provides a new digital bioanalysis technology that does not require a fluorescent substrate to be 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 a one-to-one correspondence between substrate species and enzyme species, a system capable of digitally measuring multiple enzymes on a single assay platform has been constructed. The technology of this embodiment is applicable to various enzymes that produce ATP and ADP, and is expected to be applied to precise measurement of the activity of a single enzyme molecule, which was previously impossible to measure, and 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 activities in serum are significantly higher in colorectal cancer patients than in healthy subjects. It has been reported that these activities are significantly increased, especially in advanced cancer patient groups. It has been reported that ATP, ADP, and AMP hydrolysis activities in serum are significantly higher in prostate cancer patients than in 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 of this embodiment for detecting enzymes that synthesize ATP and ADP may be applicable to methods for obtaining information about cancer. According to this embodiment, it is possible to perform high-sensitivity activity measurement of small amounts of molecules, for example, single molecules, for kinases that are often used as biomarkers for diseases such as cancer, and it is also possible to detect activity distributions characteristic of each disease. It is expected that the use of such activity distributions will enable the discovery and diagnosis of enzyme biomarkers with higher sensitivity and selectivity.

[0051] (Other embodiments) In one embodiment, the present invention provides a method for diagnosing a disease using the method for detecting an enzyme. 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 producing a detection kit for the enzyme. In one embodiment, the present invention provides the use of said detection reagent for the manufacture of a kit for the detection of said enzyme. In one embodiment, the present invention provides the use of said detection device for the manufacture of a detection kit for said enzyme.

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

[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, the sensitivity of enzyme detection relative to the number of enzymes per enzyme sample was examined. hChK exists in three isoforms, α1, α2, and β, and is known to function as a dimer and catalyze the first step of phospholipid synthesis in vivo. hChKα1 is involved in tumorigenesis and is attracting attention as a new target molecule for cancer therapeutics.

[0055] The detection device described in the above embodiment (each device has 5 million containers (reactors)) was used. An enzyme sample was prepared in which the hChK concentration was adjusted so that the ratio of the number of enzymes to the number of containers was λ. Resazurin solution and reaction stop solution were prepared to adjust the detection solution. The substrate solution and the above solutions were used at concentrations that did not produce background when used. ATP and ADP solutions were prepared for the kinase substrate and the calibration curve. As for the detection reagent, a phosphate buffer using potassium phosphate without containing amine compounds or HEPES (50 mM potassium phosphate, pH 7.5, 100 mM KCL, 2 mM MgCl2, 0.02% Tween 20, 1 mM glucose, 100 μM NADP+, 100 μM resazurin) was used. As for the diaphorase enzyme and the hexokinase enzyme, recombinant enzymes derived from thermophiles were produced, and heat treatment and purification were performed until the false positive signal was reduced by about 30-40 times. As for the enzyme sample, a pretreatment solution containing creatine kinase and phosphocreatine was added to the enzyme sample before the sample addition step and reacted. That is, an ADP removal treatment step was performed to reduce ADP by converting ADP in the enzyme sample into ATP through phosphorylation transfer reaction from phosphocreatine to ADP, and the residual amount of ADP was reduced to 0.1% or less.

[0056] Samples were also prepared with and without the addition of choline molecules as a substrate. The reaction of human choline kinase synthesizes phosphorylated choline + ADP from choline + ATP.

[0057] Figure 4 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 could be detected among the arranged storage sections. The number of enzyme molecules contained in a container can be calculated from the number of enzymes dispensed into the container relative to the number of containers. Considering the case where a sample solution containing a fixed number of enzymes is dispensed into all containers, when the ratio of the number of enzymes in all sample solutions to the number of containers is λ, the probability P(n) that n molecules of enzyme are contained in one container is expressed by the Poisson distribution P(n, λ) = (λ n e -λ ) / n!, if λ=0.1 or less, then one molecule of enzyme or less can be accommodated in one storage compartment. At λ=0.1 and 0.01 in the figure, it is considered that there are some containers that contain 0 enzyme molecules and some that contain 1 enzyme molecule. From the figure, it is considered that the fluorescent signal is detected only in some containers. In other words, it is considered that the fluorescent signal of the enzyme is detected in the container that contains 1 enzyme molecule. On the other hand, at λ=0, no enzyme is detected even though none of the chambers contain enzyme, and at λ=1, excess signal is seen from all chambers even though all chambers contain one or more enzymes. From the example shown in the figure, it became clear that this enzyme detection method can detect the presence or absence of a molecule in each storage compartment when λ = 0.01 and 0.1, and can obtain a sensitivity that allows detection if one enzyme molecule is present per storage compartment.

[0058] Figure 5 shows the single molecule activity distribution per λ. In the figure, the vertical axis (Counts) is the number of storage compartments, and the horizontal axis (Slope) is the activity (au / min) value. For example, a high peak appears near the left end of each graph, close to the activity of 0, indicating that a large number of empty storage compartments with no activity (no enzyme molecules inside) were detected. In addition, for each test graph, the vertical axis is 8000 to 12 x 10 3 The graph (bottom left) shows the horizontal axis ranging from 10 to 40, and an enlarged portion of it (top right) is superimposed on it.

[0059] As shown in the figure, a peak in the active compartment was confirmed in the figure of the test with choline added (Choline+), while in the figure of the test without choline added, a peak was seen on the active side (further to the right). In other words, since a peak was observed in a choline-dependent manner, the activity of a single molecule of hChK could be detected. In addition, in the graph of the Choline+ test, where a peak was observed, the distribution shape was not a single clear peak (heterogeneous). This may be because there may be inactive molecular species derived from polymers, which may have been detected in this test.

[0060] (Test Example 2: Detection of ATPase) Using F1-ATPase, an ATPase, as the target enzyme, a test was carried out regarding the sensitivity of the enzyme detection with respect 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 of the detection device taken from the bottom. The white dots in the figure indicate the storage compartments where the fluorescent signal of the enzyme could be detected among the arranged storage compartments. At λ=0.3 in the figure, the fluorescent signal was detected only in some storage compartments, making it clear that this enzyme detection method can detect the presence or absence of molecules in each storage compartment at λ=0.3.

[0062] 7 is a diagram showing 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, 0.5, 0.3, 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 F1-ATPase molecule could be detected.

[0063] (Test Example 3: Detection of pyruvate kinase) Using pyruvate kinase (PK) that synthesizes ATP as a target enzyme, a test was conducted on the sensitivity of enzyme detection relative to the number of enzymes per enzyme sample. The detection device and detection reagent were the same as those in Test Example 1. Pyruvate kinase is widely known to catalyze the final reaction of the unfreezing system (PEP+ADP to Pyruvate+ATP). PK works 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 of the detection device taken from the bottom. The white dots in the figure indicate the storage compartments where the fluorescent signal of the enzyme could be detected among the arranged storage compartments. At λ=0.1 in the figure, the fluorescent signal was detected only in some storage compartments, making it clear that this enzyme detection method can detect the presence or absence of molecules in each storage compartment at λ=0.1.

[0065] 9 is a diagram showing 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 on the sensitivity of the enzyme detection to the number of enzymes per enzyme sample. The detection device and detection reagent were the same as those in Test Example 1. Alkaline phosphatase is widely known as an enzyme that breaks down 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 be used in conventional techniques. In other words, we investigated whether the method of this example can be used in place of conventional methods.

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

[0068] 11 is a diagram showing 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=0.1, 0.01, 0.001, and 0. As shown in the figure, a peak of the active storage portion was confirmed in the graph of each λ test. All of λ=0.1, 0.01, and 0.02 show a heterogeneous distribution (two peaks). It is known that alkaline phosphatase has a heterogeneous distribution in which there are two activity peaks depending on whether two subdomains show activity. In other words, it was shown that the activity of alkaline phosphatase can be detected by the method of this example. From this result, it is considered that the method of this example is also effective for detecting enzymes in the presence of 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 in the scope of the invention and its equivalents described in the claims, as well as in the scope and spirit of the invention. [Industrial Applicability]

[0070] According to the present invention, it is possible to detect enzymes that synthesize ATP or ADP, and it is possible to detect these enzymes that do not have a fluorescent substrate 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. [Explanation of symbols]

[0071] 20 Detection solution 30 Microchamber array component 31 Storage unit 32 Bottom member 100 Detection device D diameter I Interval T Depth

Claims

1. A method for detecting a target enzyme that synthesizes ADP or ATP from an enzyme sample, comprising: A detection device is provided that includes a microchamber array member having an array of multiple storage sections that can be filled with a detection solution; a sample addition step of filling the container of the detection device with the enzyme sample and a detection reagent containing a hexokinase enzyme to prepare the detection solution; a detection step of detecting a signal due to synthesis of ADP or ATP from the detection solution; A method for detecting an enzyme comprising the steps of:

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

3. 3. The 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. 3. The method for detecting an enzyme according to claim 1, wherein the detection reagent contains diaphorase, and the diaphorase and hexokinase enzymes are recombinant enzymes derived from thermophilic bacteria.

8. 3. The method for detecting an enzyme according to claim 1, further comprising an ADP removal treatment step of adding a pretreatment solution containing creatine kinase and phosphocreatine to the enzyme sample and allowing the reaction to proceed 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 including a microchamber array member in which a plurality of storage sections capable of being filled with a detection liquid in which an enzyme sample and a detection reagent are mixed are arranged; the detection reagent comprising a hexokinase enzyme; An enzyme detection kit comprising:

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