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WO2024257799A1 - Procédé de détection d'enzymes et kit de détection d'enzymes - Google Patents

Procédé de détection d'enzymes et kit de détection d'enzymes Download PDF

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WO2024257799A1
WO2024257799A1 PCT/JP2024/021356 JP2024021356W WO2024257799A1 WO 2024257799 A1 WO2024257799 A1 WO 2024257799A1 JP 2024021356 W JP2024021356 W JP 2024021356W WO 2024257799 A1 WO2024257799 A1 WO 2024257799A1
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enzyme
detection
adp
detecting
atp
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Japanese (ja)
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博行 野地
博史 上野
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University of Tokyo NUC
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University of Tokyo NUC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/50—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving creatine phosphokinase

Definitions

  • 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.
  • Immunochemical techniques 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.
  • a multiplexed single enzyme assay technology has been developed to examine the isozymes of enzymes. 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 source of the disease.
  • isozymes that form multimers may not have a uniform activity distribution of one molecule, and by investigating the shape of this distribution, it is possible to investigate which disease is affected.
  • a method that automatically performs detection and analysis has also been developed, and these digital bioanalyses (digital ELISAs) are also attracting attention as a method for detecting biomarkers with high sensitivity.
  • Patent Document 1 discloses a detection device and a detection method for detecting the enzyme activity of a large number of trace samples, which includes 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 and a detection method using the same 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.
  • 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 part of the substrate is modified so that it can be detected by the reaction.
  • fluorescent substrates enzymes for which such specific substrates (substrates that can be used for fluorescence detection, hereinafter sometimes referred to as fluorescent substrates) have not been found, for example, 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.
  • 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.
  • mutations in Raf kinase and activity of thymidine kinase in blood are biomarkers closely related to cell proliferation, and are expected to be used as tools for monitoring the prognosis of cancer treatment.
  • an increase in thymidine kinase activity in serum is associated with neoplastic disease, malignant disease, vitamin B12 deficiency, and viral infection.
  • thymidine kinase 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 blood increases in blood concentration after the onset of myocardial infarction. Therefore, it is widely used in diagnosing myocardial infarction.
  • CK contains 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 in clinical diagnosis, but their enzymatic activity catalyzes the conversion (synthesis) of ATP and ADP.
  • thymidine kinase catalyzes the reaction of thymidine + ATP by adding a phosphate to thymidine to form thymidylic acid (TMP) + ADP.
  • TMP thymidylic acid
  • the present invention has been made in consideration of the above circumstances, and its purpose is to provide an enzyme detection method and enzyme detection kit for use therein that can detect enzymes that synthesize ATP or ADP, and that can detect these enzymes that do not have a fluorescent substrate with high sensitivity at the single molecule level, enabling highly sensitive measurement of disease biomarkers, etc.
  • 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 according to [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.
  • the detection reagent comprises resazurin and diaphorase.
  • [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.
  • 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 prior to the sample addition step, to allow the reaction to proceed.
  • 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:
  • the present invention makes it possible to detect enzymes that synthesize ATP or ADP, and enables highly sensitive detection at the single molecule level for these enzymes that do not have a fluorescent substrate, thereby providing an enzyme detection method and an enzyme detection kit for use therein that enable highly sensitive measurement of disease biomarkers, etc.
  • FIG. 2 is an exploded partial enlarged view of the detection device according to the embodiment.
  • 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 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. 2 is a photograph of the fluorescent signal of Test Example 1 of this embodiment taken from the bottom direction.
  • FIG. 1 shows the single molecule activity distribution per ⁇ in Test Example 1 of this embodiment.
  • FIG. 2 is a photograph of the fluorescent signal of Test Example 2 of this embodiment taken from the bottom direction.
  • FIG. 1 shows the single molecule activity distribution per ⁇ in Test Example 1 of this embodiment.
  • FIG. 13 is a diagram showing the single molecule activity distribution per ⁇ in Test Example 2 of this embodiment.
  • FIG. 1 is a photograph of the fluorescent signal of Test Example 3 of this embodiment taken from the bottom direction.
  • FIG. 13 is a diagram showing the single molecule activity distribution per ⁇ in Test Example 3 of this embodiment.
  • FIG. 1 is a photograph of the fluorescent signal from Test Example 4 of this embodiment taken from the bottom.
  • FIG. 13 is a diagram showing the single molecule activity distribution per ⁇ in Test Example 4 of this embodiment.
  • 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 into which a detection liquid can be filled are arranged, and 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 prepare the detection liquid, and a detection step of detecting a signal due to the synthesis of ADP or ATP from the detection liquid.
  • the enzyme sample broadly includes samples that may contain enzymes.
  • 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.
  • 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.
  • 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 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.
  • the target enzyme that synthesizes ADP or ATP broadly refers to an enzyme that catalyzes a reaction in which ADP or ATP is synthesized through its enzymatic activity.
  • an enzyme that catalyzes a reaction in which ADP or ATP is synthesized 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.
  • the target enzyme of this embodiment is preferably a kinase.
  • Kinases are enzymes that transfer phosphate groups from molecules having 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.
  • therapeutic drugs e.g., molecular targeted drugs
  • CKs have traditionally been identified by absorbance-based measurement and isozyme-specific inhibitory antibodies, and therefore, analysis methods using batch detection methods are 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.
  • Examples of enzymes that synthesize ADP include F 1 -ATPase, ie, F 1 from thermophilic Bacillus PS3 (TF 1 ), which has ATPase activity.
  • PK pyruvate kinase
  • the detection device 1 is an exploded partial 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 containers 31 capable of being filled with a detection liquid 20 are arranged.
  • the microchamber array member 30 is made of a plate-like member formed of, for example, glass, silicon, polymer resin, or the like.
  • the microchamber array member 30 has a plurality of cylindrical holes arranged (arrayed) that are connected from the flat portion to the bottom portion of the plate-like member, and the holes serve as a plurality of storage sections 31 that can be filled with detection liquid.
  • 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 this bottom surface member 32, so that the storage section 31 can be filled with 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.
  • the bottom surface member 32 is made of a light-transmitting constituent material such as glass or polymer resin.
  • the storage sections 31 are arranged with a distance I between them. This distance 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 distance I. In this embodiment, the depth T is 3 ⁇ m and the diameter D is 4 ⁇ m.
  • 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 of the storage section 31.
  • detection is performed using an sCMOS camera (not shown) attached to a fluorescent microscope for observing the microchamber array member 30.
  • 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.
  • 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.
  • a partition that can be broken 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 can be elastically deformed 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.
  • a sensor member having sensors respectively corresponding to the positions of the plurality of storage sections 31 may be provided.
  • 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.
  • the detection device of this embodiment may be, for example, that described in Patent Document 1.
  • This detection device is a detection device that includes a microchamber array (microchamber array member) that is disposed inside a main body and has a plurality of storage units filled with a hydrophilic solvent containing a biological sample, and an image sensor (sensor member) in which pixels are provided corresponding to the storage units, in which the main body has a flow passage that communicates with an opening of the storage unit, a hydrophobic solvent supply unit that is connected to the flow passage, and a through hole that allows the hydrophilic solvent to 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.
  • a microchamber array microchamber array member
  • the main body has a flow passage that communicates with an opening of the storage unit, a hydrophobic solvent supply unit that is connected to the flow passage, and a through hole that allows the hydrophilic solvent to enter and exit the flow passage
  • the microchamber array has
  • sample Addition Step each step of the enzyme detection method of the present embodiment will be described.
  • 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.
  • 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.
  • the reagent when detecting a target enzyme that synthesizes ADP, may contain a reagent for proceeding to a cascade reaction of enzyme reactions that uses the ADP synthesized by the target enzyme to phosphorylate glucose by an ADP-dependent hexokinase reaction, and then proceeds to an enzyme reaction that finally emits a fluorescent signal.
  • the target enzyme is an enzyme that synthesizes ADP
  • the hexokinase enzyme is preferably ADP-dependent hexokinase.
  • the reagent when detecting a target enzyme that synthesizes ATP, 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.
  • the target enzyme is an enzyme that synthesizes ATP
  • the hexokinase enzyme is preferably an ATP-dependent hexokinase.
  • 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 a reaction in a sample addition step when a target enzyme contained in an enzyme sample is an enzyme that synthesizes ADP.
  • the enzyme that synthesizes ADP includes various kinases and ATPases.
  • 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.
  • the detection reagent When the detection reagent is added to the enzyme sample containing the target enzyme, ATP is first catalyzed by the target enzyme, and ADP is synthesized.
  • the target enzyme is a kinase
  • the phosphorylated substrate that is the target of phosphorylation by the kinase may be added together with the detection reagent.
  • the phosphorylated substrate is choline.
  • ADP and choline phosphate are synthesized from ATP.
  • glucose is then catalyzed by ADP-dependent hexokinase to synthesize AMP and glucose-6-phosphate.
  • Glucose-6-phosphate is synthesized, and then NADP and glucose-6-phosphate are 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.
  • 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.
  • the enzyme that synthesizes ADP includes various kinases as well as ATP synthase. When various kinases are used, the reverse reaction can be detected from the case of the enzyme that synthesizes ADP.
  • 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.
  • ADP is first catalyzed by the target enzyme to synthesize ATP.
  • the target enzyme is a kinase
  • a phosphorylated substrate may be added together with the detection solution.
  • the kinase is pyruvate kinase
  • ATP and pyruvate are synthesized from the phosphorylated substrate phosphoenolpyruvate (PEP) and ADP.
  • PEP phosphoenolpyruvate
  • glucose is then catalyzed by ATP-dependent hexokinase to synthesize ADP and glucose-6-phosphate.
  • Glucose-6-phosphate is synthesized, and then NADP and glucose-6-phosphate are 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.
  • the detection reagent may be configured to contain the above components in separate substrate liquids containing the above-mentioned reaction substrates and enzyme liquids containing the above-mentioned reaction enzymes, and to mix the substrate liquid and enzyme liquid in the sample addition step. By mixing the substrate liquid and enzyme liquid immediately before the test, the above reaction can occur during the test.
  • 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.
  • 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.
  • examples of amine compounds not included in the detection reagent of this embodiment include Tris (trishydroxymethylaminomethane).
  • 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.
  • 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.
  • the diaphorase and hexokinase enzymes are recombinant enzymes derived from thermophilic bacteria.
  • 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.
  • 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.
  • thermophilic bacteria an expression system for a recombinant enzyme derived from thermophilic bacteria is constructed, and while digital measurements are performed to check for false positives, careful heat treatment and purification are performed to reduce false positive signals by about 30-40 times.
  • kits containing a detection reagent that can be used to detect the enzymes that synthesize ADP and ATP As a kit containing a detection reagent that can be used to detect the enzymes that synthesize ADP and ATP, the Fluorospark(R) Kinase/ADP Multi-Assay Kit (Fujifilm Wako Pure Chemical Industries, Ltd.) or a detection kit containing a similar composition can be used.
  • 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 it before the sample addition step.
  • This pretreatment is performed when detecting an enzyme that synthesizes ADP.
  • 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.
  • the pretreatment solution can be one containing phosphocreatine as the phosphorylated substrate and creatine kinase as the kinase.
  • ADP removal process a pretreatment solution containing creatine kinase and phosphocreatine is added to the enzyme sample and reacted to remove ADP from the enzyme sample.
  • Creatine kinase causes a phosphoryl transfer reaction from phosphocreatine to ADP, which results in the conversion of ADP to ATP, resulting in a reduction in ADP.
  • this process reduces ADP to 0.1% or less. If ADP is contained in the enzyme sample before it is synthesized by the target enzyme, it will become a background signal when detecting ADP synthesis, and the data and calibration curve of the control sample for ADP synthesis will be inaccurate. By carrying out this ADP removal treatment step, these problems can be prevented.
  • the ADP removal treatment step is particularly effective in increasing the accuracy of detection.
  • an enzyme and a fluorescent substrate dedicated to that enzyme are introduced into a microchamber array, sealed in oil, and the fluorescence associated with the reaction is imaged using a microscope or the like.
  • an assay solution conjugated with three enzyme reactions is added, and the ADP and ATP produced in the enzyme reaction are converted into fluorescent substances, and the fluorescence is imaged.
  • 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 level of a single enzyme molecule, rather than using an artificial fluorescent substrate.
  • 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.
  • 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.
  • the enzyme detection kit of this embodiment can suitably detect a target enzyme using the enzyme detection method.
  • the enzyme detection kit of this embodiment contains the above-mentioned components and may also contain optional components described below.
  • the reaction may contain a reaction terminating solution for terminating the enzyme reaction.
  • the reaction terminating solution may contain, for example, a reducing agent.
  • a reagent containing a substrate, a control, or ATP or ADP for a calibration curve may be included in addition to the substrate solution and enzyme solution.
  • 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 makes it possible to perform digital bioanalysis on enzymes that do not have a fluorescent substrate.
  • bioassays in conventional technology have a one-to-one correspondence between substrate types and enzyme types, it has become possible to construct a system that allows digital measurement of multiple enzymes on a single assay platform.
  • the technology of this embodiment can be applied 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 the detection of disease biomarkers using enzyme activity patterns.
  • kinases and enzymes that synthesize ATP and ADP may be useful as biomarkers for diseases such as cancer.
  • 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.
  • ATP, ADP, and AMP hydrolysis activities in serum are significantly higher than in healthy subjects.
  • Patients with lower clinical stages (CS-IIA) showed increased ATP hydrolysis activity compared to patients with more advanced clinical stages (CS-IIB and CS-III). It has been reported.
  • the method of this embodiment for detecting enzymes that synthesize ATP and ADP may be applicable to methods for obtaining information about cancer.
  • 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.
  • hChK human choline kinase
  • the detection apparatus described in the above embodiment (5 million containers (reactors) per apparatus) was used.
  • An enzyme sample was prepared in which the hChK concentration was adjusted so that the ratio of the number of enzymes used to the number of containers was ⁇ .
  • Resazurin solution and reaction stop solution were prepared for adjusting the detection solution.
  • the substrate solution and the above solutions were used at concentrations that did not produce background when used.
  • ATP solution and ADP solution were prepared for the kinase substrate and calibration curve.
  • a phosphate buffer using potassium phosphate without containing an amine compound or HEPES 50 mM potassium phosphate, pH 7.5, 100 mM KCL, 2 mM MgCl 2 , 0.02% Tween 20, 1 mM glucose, 100 ⁇ M NADP+, 100 ⁇ M resazurin
  • the diaphorase enzyme and the hexokinase enzyme a recombinant enzyme derived from a thermophile was produced, and heat-treated and purified until the false positive signal was reduced by about 30-40 times.
  • 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 to ATP through a phosphorylation transfer reaction from phosphocreatine to ADP, and the amount of residual ADP was reduced to 0.1% or less.
  • the number of molecules of enzyme contained in a container can be calculated from the number of enzymes dispensed into the container relative to the number of containers.
  • the fluorescent signal of the enzyme is detected in the storage section containing one enzyme molecule.
  • FIG. 5 shows the single molecule activity distribution per ⁇ .
  • the vertical axis (Counts) is the number of containers
  • the horizontal axis (Slope) is the activity (au/min). For example, a high peak appears near the left end of each graph, which indicates that a large number of empty containers with no activity (no enzyme molecules inside) were detected.
  • a graph (lower left) with the vertical axis of 8000 to 12 ⁇ 10 3 and the horizontal axis of 10 to 40 is shown superimposed with a partially enlarged view (upper right).
  • the peak of the active compartment was confirmed in the figure of the test in which choline was added (Choline +), and the peak was seen on the active side (more to the right) in the figure of the test in which choline was not added.
  • the peak was observed in a choline-dependent manner, the activity of one molecule of hChK could be detected.
  • the distribution shape was not a single clear peak (heterogeneous). This may be because there may be inactive molecular species derived from multimers, which may have been detected in this test.
  • Test Example 2 Detection of ATPase
  • F 1 -ATPase which is an ATPase
  • the detection device and detection reagent were the same as in Test Example 1.
  • 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 could be detected among the arranged storage sections.
  • FIG. 7 is a diagram showing the single molecule activity distribution per ⁇ .
  • the explanation in the figure is the same as in Test Example 1.
  • a peak of activity was confirmed in the test graph for each ⁇ , indicating that the activity of each F 1 -ATPase molecule could be detected.
  • Test Example 3 Detection of pyruvate kinase
  • PK pyruvate kinase
  • 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 thawing system (PEP+ADP to Pyruvate+ATP).
  • PK works as a tetramer.
  • 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.
  • 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 where the fluorescent signal of the enzyme could be detected among the arranged storage sections.
  • FIG. 9 is a diagram showing the single molecule activity distribution per ⁇ .
  • the explanation in the figure is the same as in Test Example 1.
  • a peak of activity was confirmed in the test graph for each ⁇ . In other words, it was demonstrated that the activity of each pyruvate kinase molecule could be detected.
  • Test Example 4 Detection of alkaline phosphatase
  • ALP alkaline phosphatase
  • 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.
  • 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 could be detected among the arranged storage sections.
  • FIG. 11 is a diagram showing the single molecule activity distribution per ⁇ .
  • the explanation in the figure is the same as in Test Example 1.
  • a peak of the active storage portion was confirmed in the graph of each ⁇ test.
  • alkaline phosphatase has a heterogeneous distribution in which two activity peaks occur depending on whether two subdomains show activity.
  • 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.
  • the present invention makes it possible to detect enzymes that synthesize ATP or ADP, and enables highly sensitive detection at the single molecule level for these enzymes that do not have a fluorescent substrate, thereby providing an enzyme detection method and an enzyme detection kit for use therein that enable highly sensitive measurement of disease biomarkers, etc.

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Abstract

La présente invention concerne : un procédé de détection d'enzymes permettant de détecter les enzymes synthétisant l'ATP ou l'ADP, de réaliser une détection très sensible à un niveau moléculaire pour une enzyme ne présentant pas de substrat fluorescent parmi les enzymes, et de réaliser une mesure très sensible d'un biomarqueur de maladie ou autre ; et un kit de détection d'enzymes utilisé dans ce procédé. La présente invention concerne un procédé de détection d'enzymes pour détecter, à partir d'un échantillon enzymatique, une enzyme cible de synthèse d'ADP ou d'ATP, ainsi qu'un kit de détection d'enzymes utilisé dans ce procédé, le procédé comportant : une étape d'ajout d'échantillon pour préparer un dispositif de détection comprenant un élément de réseau de microchambres dans lequel sont agencées plusieurs parties de stockage pouvant être remplies d'un liquide de détection, et le remplissage des parties de stockage du dispositif de détection avec un liquide de détection contenant l'échantillon enzymatique et une enzyme hexokinase ; et une étape de détection pour détecter, à partir du liquide de détection, un signal obtenu par la synthèse de l'ADP ou de l'ATP.
PCT/JP2024/021356 2023-06-16 2024-06-12 Procédé de détection d'enzymes et kit de détection d'enzymes Pending WO2024257799A1 (fr)

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WO2015076361A1 (fr) * 2013-11-21 2015-05-28 国立大学法人東京大学 Procédé de détection de fluorescence ou d'absorbance, procédé de suppression du fond, procédé de mesure d'adp, procédé de mesure de l'activité d'une enzyme synthétisant l'adp, et procédé de mesure de l'activité de la glucosyltransférase
JP2021072837A (ja) * 2016-04-27 2021-05-13 ソシエテ・デクスプロワタシオン・デ・プロデュイ・プール・レ・アンデュストリー・シミック・セピックＳｏｃｉｅｔｅ Ｄ’Ｅｘｐｌｏｉｔａｔｉｏｎ Ｄｅ Ｐｒｏｄｕｉｔｓ Ｐｏｕｒ Ｌｅｓ Ｉｎｄｕｓｔｒｉｅｓ Ｃｈｉｍｉｑｕｅｓ Ｓｅｐｐｉｃ 組成物の筋損傷および筋疲労を予防する能力を評価する方法；食品補助剤および医薬品
JP2023501002A (ja) * 2019-11-11 2023-01-17 バイオクルーシブル リミテッド 生化学反応方法及び天然変性領域を含む試薬

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JP2021072837A (ja) * 2016-04-27 2021-05-13 ソシエテ・デクスプロワタシオン・デ・プロデュイ・プール・レ・アンデュストリー・シミック・セピックＳｏｃｉｅｔｅ Ｄ’Ｅｘｐｌｏｉｔａｔｉｏｎ Ｄｅ Ｐｒｏｄｕｉｔｓ Ｐｏｕｒ Ｌｅｓ Ｉｎｄｕｓｔｒｉｅｓ Ｃｈｉｍｉｑｕｅｓ Ｓｅｐｐｉｃ 組成物の筋損傷および筋疲労を予防する能力を評価する方法；食品補助剤および医薬品
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