JP5344665B2 - Reactor for amplifying adenosine triphosphate, adenosine triphosphate amplification method, and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reactor for amplifying ATP and a method for amplifying ATP each of which is utilizable for the quantitative determination of trace ATP and to provide utilization thereof. <P>SOLUTION: Along a flow passage provided with a first region on which adenylate kinase is immobilized and a second region on which an ADP phosphorylase which converts ADP into ATP is immobilized, ATP, AMP, and a substrate on which ADP phosphorylase acts are passed in the order of the first region and the second region. In this way, it is possible to quantitatively amplify ATP. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to a reactor for amplifying adenosine triphosphate, a method for amplifying adenosine triphosphate, and use thereof, and in particular, adenosine triphosphate usable for quantifying a trace amount of adenosine triphosphate. The present invention relates to a reactor for amplifying phosphoric acid, a method for amplifying adenosine triphosphate, and use thereof.

  Currently, as a method for measuring adenosine triphosphate (hereinafter, also referred to as “ATP”), a bioluminescent measurement method using firefly luciferase is generally used (see Non-Patent Document 1). The measurement method is a standard method for hygiene inspection using HATP (Hazard Analysis Critical Control Point) as an index of ATP contained in a living organism.

  According to the measurement method, ATP can be quantitatively detected in a wide concentration range. However, when the detection target is an extremely small amount of ATP or a small amount of microorganisms of 1,000 or less individuals, the luminescence generated from such a small amount of ATP is far below the detectable range of a commercially available luminometer. There is a problem that is insufficient.

  In order to solve such a problem, the present inventors have heretofore used a very small amount of ATP, adenylate kinase (hereinafter also referred to as “ADK”) and polyphosphate kinase (hereinafter also referred to as “PPK”). An ATP amplification method has been developed in which ATP is artificially amplified by a conjugation reaction (chain ATP amplification reaction) using an ATP (see Patent Document 3).

  Specifically, in the ATP amplification method of Patent Document 3, two molecules of adenosine diphosphate (hereinafter also referred to as “ADP”) from ATP and adenylic acid (hereinafter also referred to as “AMP”) under the catalyst of ADK. Reaction (hereinafter also referred to as “ADK reaction”) and reaction that generates two molecules of ATP from the two molecules of ADP and polyphosphoric acid (hereinafter also referred to as “Pn”) under the catalyst of PPK (hereinafter referred to as “ADK reaction”). , Also referred to as “PPK reaction”) in one reaction system.

In the ATP amplification method, when the ADK reaction and the PPK reaction are one cycle and the reaction is performed for n cycles, ATP can be amplified 2 ⁿ times.

  Further, the present inventors have found that in the ATP amplification method disclosed in Patent Document 1, only exogenous ATP can be amplified by using an enzyme that does not contain ADP as an impurity (see Non-Patent Document 2). reference).

  Further, in Non-Patent Document 2, according to the ATP amplification method, the ATP detection sensitivity is improved by 10,000 times or more compared to the conventional method in which ATP is not amplified, and ATP of several amol level is obtained. It is described that it can be detected. Furthermore, Non-Patent Document 2 describes that according to the ATP amplification method, ATP of microorganisms at the level of one cell can be amplified and detected.

Furthermore, the present inventors separated the target microorganism only from the microorganisms contained in the sample using the target microorganism-specific antibody, and then performed the ATP amplification method disclosed in Non-Patent Document 2, thereby It has been found that detection and identification of microbial species can be performed simultaneously (see Non-Patent Document 3 and Patent Document 2). Specifically, in Non-Patent Document 3 and Patent Document 2, according to such a method, Escherichia coli, Staphylococcus aureus in milk, wiping test or microorganisms contained in environmental water are detected at the level of one individual. It describes what you can do.
JP 2001-299390 A (published October 30, 2001) JP 2006-81506 A (published March 30, 2006) JP 2006-223163 A (published August 31, 2006) DeLuca, M. and McElroy, WD Kinetics of the firefly luciferase catalyzed reactions.Biochemistry, 26, 912-925 (1974) T. Satoh, J. Kato, N. Takiguchi, H. Ohtake, A. Kuroda ATP amplification for ultrasensitive bioluminescence assay: detection of a single bacterial cell Biosc. Biotechnol. Biochem. 68, 1216-1220 (2004). Y. Asami, T. Satoh, A. Kuroda Polyphosphate-ATP amplification technology: principle and its application to specific bacteria detection J. Environ. Biotechnol. 6, 2, 105-108 (2006).

  As described above, the ATP amplification methods disclosed in Patent Documents 1 and 2 and Non-Patent Documents 2 and 3 are effective for primary screening for testing the presence or absence of microorganisms.

  In the ATP amplification method, since the chain ATP amplification reaction is carried out in a batch, the chain ATP amplification reaction continues to be amplified until AMP is consumed, and finally converges to a certain amount of ATP (see FIG. However, since the amplification rate varies depending on the amount of ATP added to the reaction system, relative ATP measurement can be performed by dividing the reaction rate by a certain reaction time.

  Specifically, the present inventors have developed a technique disclosed in Patent Document 3 as an ATP quantification method using an ATP amplification method. In the method disclosed in Patent Document 3, ATP amplified by the ATP amplification method as described above can be quantitatively detected by the hexokinase method.

  However, in ATP quantification using the conventional ATP amplification method including Patent Document 3, as shown in FIG. 2, since the chain ATP amplification reaction is performed in batches, ATP is amplified exponentially. Therefore, a large error is caused only by a slight difference in enzyme activity (or enzyme amount) between the two compared. As a result, when a small amount of ATP is quantified, it is possible to perform a certain amount of quantification, but there is a problem that high-precision quantification is difficult.

  The present invention has been made in view of the above problems, and an object thereof is to provide a reactor for amplifying ATP that can be used for quantifying a small amount of ATP, an ATP amplification method, and use thereof. There is to do.

  As a result of intensive studies in view of the above problems, the present inventors have determined that the number of ATP amplifications is desired according to the flow system in which an adenylate kinase and an enzyme that converts adenosine diphosphate to adenosine triphosphate are immobilized. The inventors have found that they can be controlled, and have completed the present invention. That is, the present invention includes the following industrially useful inventions.

  (1) A reactor for amplifying adenosine triphosphate, a first region in which adenylate kinase is immobilized, and a second region in which an enzyme that converts adenosine diphosphate into adenosine triphosphate is immobilized And a flow path provided with a reactor.

  (2) The reactor according to (1), wherein the enzyme that converts adenosine diphosphate into adenosine triphosphate is pyruvate kinase, polyphosphate kinase, acetate kinase, creatine kinase, or hexokinase. .

  (3) The reactor according to (1), wherein a plurality of the flow paths are connected, and the first region and the second region are alternately arranged.

  (4) Liquid is sent along the flow path from the first region to the second region, and the liquid passes through the first region and the second region in order. The reactor according to (1), further comprising a solution circulation mechanism that enables liquid feeding again from the upstream of the first region toward the second region.

  (5) Adenosine triphosphate along a flow path provided with a first region in which adenylate kinase is immobilized and a second region in which an enzyme that converts adenosine diphosphate to adenosine triphosphate is immobilized A method for amplifying adenosine triphosphate, wherein adenylic acid and a substrate for the enzyme are passed in the order of the first region and the second region.

  (6) The combination of the enzyme that converts adenosine diphosphate to adenosine triphosphate and the substrate of the enzyme includes a combination of pyruvate kinase and phosphoenolpyruvine, a combination of polyphosphate kinase and polyphosphate, acetate kinase The method for amplifying adenosine triphosphate according to (5), which is a combination of acetylphosphate and creatine kinase and phosphocreatine, or a combination of hexokinase and hexose phosphate.

  (7) An adenosine triphosphate amplification kit comprising at least one of a carrier on which adenylate kinase is immobilized and a carrier on which an enzyme that converts adenosine diphosphate to adenosine triphosphate is immobilized.

  As described above, the reactor according to the present invention is a reactor for amplifying ATP, and includes a first region in which ADK is fixed, and a second region in which an enzyme that converts ADP to ATP is fixed. Are provided with a channel provided one by one.

  Therefore, ATP can be amplified twice by passing ATP, AMP, and the substrate of the enzyme along the flow path in the order of the first region and the second region. Furthermore, ATP can be amplified to a power of 2 by repeatedly performing the same operation.

  That is, according to the above configuration, the number of amplifications (amplification magnification) of ATP contained in the sample can be controlled as desired. Therefore, there is an effect that ATP contained in the sample can be quantitatively amplified.

  An embodiment of the present invention will be described below with reference to FIGS. 1 and 2, but the present invention is not limited to this.

<I. ATP amplification reactor>
The reactor according to the present invention is a reactor for amplifying ATP (hereinafter also referred to as “ATP amplification reactor”). Specifically, as shown in FIG. 1, the ATP amplification reactor has a first region where ADK is immobilized and an enzyme that converts ADP to ATP (hereinafter also referred to as “ADP kinase”). And a flow path provided with the second region.

  The ADP kinase is not particularly limited. Specifically, for example, pyruvate kinase (hereinafter also referred to as “PK”), polyphosphate kinase (hereinafter also referred to as “PPK”), Examples thereof include acetate kinase, creatine kinase, and hexokinase.

  According to the above configuration, ATP is amplified twice by passing ATP, AMP, and the substrate of the ADP phosphorylase along the flow path in the order of the first region and the second region. can do.

  This point will be described in more detail with reference to FIG. In FIG. 2, PK is used as the ADP kinase, but the present invention is not limited to this.

  ATP, AMP, and phosphoenolpyruvate (hereinafter also referred to as “PEP”), which is a substrate of PK, are allowed to pass through the first region along the flow path. Since ADK is fixed in the first region, one molecule of AMP and one molecule of ATP react to generate two molecules of ADP (see FIG. 2).

  The two molecules of ADP generated in the first region are allowed to pass through the second region along the upper flow path together with the PEP. Since PK is immobilized in the second region, two molecules of ADP and two molecules of PEP react to generate two molecules of ATP and two molecules of pyruvic acid (see FIG. 2).

  By such a mechanism, ATP that has passed through the flow path is amplified twice. This series of reactions is also called a chain ATP amplification reaction. By repeatedly performing this chain ATP amplification reaction, ATP can be amplified to a power of two.

  Thus, according to the ATP amplification reactor according to the present invention, the number of amplifications (in other words, amplification magnification) of ATP contained in the sample can be controlled as desired. Therefore, ATP contained in the sample can be quantitatively amplified.

  The method for forming the first region and the second region on the flow path is not particularly limited. Specifically, for example, a carrier on which ADK and ADP phosphorylase are respectively immobilized is placed on the flow path. By arranging (fixing), the first region and the second region can be formed.

  More specifically, the first region and the second region can be formed by a hollow column or a packed column produced using a carrier on which ADK and ADP phosphorylase are respectively immobilized.

  The method for immobilizing ADK and ADP kinase on the carrier is not particularly limited. For example, methods such as adsorption method, chemical modification method, and comprehensive immobilization method, such as those exemplified in “Immobilized biocatalyst” (Ichiro Chibata, Kodansha, 1986, ISBN-10: 4061396730), polyion complex, antigen A method using an antibody reaction, a self-assembled film method, or the like may be selected and used. Moreover, these methods may be used independently and may be used in combination of multiple.

  Among them, a method of immobilizing ADK or ADP phosphorylase on a carrier by a strong immobilization method typified by a covalent bond method is preferable. According to such a method, it is possible to prevent the enzyme from dissociating from the carrier and leaking during the chain ATP amplification reaction. Therefore, the number of ATP by the chain ATP amplification reaction can be controlled more precisely.

  The carrier is not particularly limited, and any conventionally known carrier can be used as long as it can immobilize proteins.

  For example, in the embodiment in which the first region and the second region are formed by a packed column, the immobilization carrier includes various members known as fillers for the packed column, such as resin, glass, ceramics, metal, and polysaccharide. Can be used.

  In the embodiment in which the first region and the second region are formed by the packed column, it is important that the immobilization support does not leak from the flow path. Therefore, it is preferable that the carrier on which ADK or ADP phosphorylase is immobilized is firmly blocked toward at least the downstream side of the liquid feeding method of the flow path.

  In the embodiment in which the first region and the second region are formed by a packed column, the immobilization carrier may be filled after enzyme immobilization, or the enzyme may be immobilized after filling. Furthermore, in the present invention, the concentration distribution of the immobilized enzyme in the column is not particularly limited.

  In the ATP amplification reactor according to the present invention, a plurality of the flow paths may be connected. At this time, the first region and the second region are connected so as to be alternately arranged. According to such a configuration, along this connected flow path, ATP, AMP, and the substrate for the ADP phosphorylase are mixed in the first region, the second region, the first region, the second region, .., And ATP can be amplified to a power of 2 in one operation according to the number of connected flow paths.

  The ATP amplification reactor according to the present invention may include a member other than the above-described flow path. Specifically, for example, the liquid is fed along the flow path from the first region to the second region, and sequentially passes through the first region and the second region. You may further provide the solution circulation mechanism which enables the liquid which passed through again to be sent to the direction of a 2nd area | region from the upstream of the said 1st area | region.

  Specifically, as the solution circulation mechanism, for example, the flow path is formed in a loop shape, the liquid that has passed through the second region is returned to the upstream of the first region again, and the liquid is again discharged from the first region. A mechanism for feeding liquid in the direction of the second region can be mentioned.

  Further, as another embodiment, the liquid that has passed through the second region is caused to flow backward from the second region to the first region to pass through the second region and the first region, and the liquid that has passed through the first region. Again, there is a mechanism for feeding liquid in the direction from the first region to the second region. Since the reverse reaction of the chain ATP amplification reaction is substantially negligible, even if the sample that has passed through the second region flows backward on the same channel, the chain ATP amplification reaction that is repeatedly performed is affected. Never give.

  According to such a configuration, the chained ATP amplification reaction can be repeatedly performed without being limited by the number of connected flow paths. In addition, since the number of connections of the flow paths can be reduced, the ATP amplification reactor can be reduced in size.

  Further, the ATP amplification reactor according to the present invention has passed through the second region, the liquid feeding portion for feeding a liquid into the flow channel, the injection portion for injecting a substance such as ATP into the flow channel, and the second region. You may provide the collection | recovery part which collect | recovers a liquid, the temperature control part for controlling the temperature of the said flow path, etc.

  The liquid feeding unit may include a pump, a pipe, an injector, a connector, a column, a column filler, a fraction collector, a detector, and the like, but these preferably have high pressure resistance. Specifically, it is preferable to have a pressure resistance performance of 0.1 MPa or more.

  In the configuration in which a plurality of the flow paths are connected, the total length of the flow paths becomes long and the pressure increases during liquid feeding, but according to the above configuration, even when a plurality of flow paths are connected in that way, Liquid can be sent.

  Since the ATP amplification reactor according to the present invention has the above-described configuration, ATP can be efficiently amplified. Furthermore, the ATP amplification reactor according to the present invention can amplify ATP contained in an arbitrary sample with high quantitativeness. Therefore, the ATP amplification reactor according to the present invention can be suitably used for applications in which a trace amount of ATP contained in an arbitrary sample is detected or quantified.

  Here, a method for amplifying ATP using the ATP amplification reactor according to the present invention (that is, the ATP amplification method according to the present invention) will be described.

  In the ATP amplification method according to the present invention, ATP, AMP, and a substrate of ADP phosphorylase are passed in the order of the first region and the second region along the flow path of the ATP amplification reactor.

  The specific means is not particularly limited. Specifically, for example, a sample solution containing ATP, AMP, and a substrate for ADP kinase is placed upstream of the first region of the channel. ATP, AMP, and ADP phosphorylase are sent in the direction from the first region to the second region by sending a buffer solution that does not contain any of the ATP, AMP, and ADP kinase enzymes. The substrate of the oxidase can be passed through the first region and the second region in this order.

  As another embodiment, for example, a sample solution containing ATP is provided upstream of the first region of the flow path, and a buffer solution containing a substrate of AMP and ADP phosphorylase is directed from the first region to the second region. In this case, ATP, AMP, and the substrate for ADP phosphorylase can be passed through the first region and the second region in this order along the flow path.

  According to the above configuration, two molecules of ADP can be generated by reacting one molecule of AMP and one molecule of ATP in the first region. Furthermore, when these two molecules of ADP pass through the second region, they can react with an ADP kinase enzyme to generate two molecules of ATP. That is, ATP can be amplified twice by carrying out the above process once. Further, ATP can be amplified to a power of 2 by repeating this operation a plurality of times.

  In the embodiment in which the sample solution introduced into the ATP amplification reactor is introduced into the ATP amplification reactor after mixing the sample (ATP-containing sample) and another reaction substrate (a substrate of AMP and ADP kinase) in advance. Preferably, a sufficiently large amount of sample is introduced so that a portion of the sample segment is not diluted until detected or recovered. Such a necessary amount can be experimentally known by, for example, introducing a dye as a dummy into an ATP amplification reactor and feeding it to detect and recover the dye.

  On the other hand, in the embodiment in which the sample (ATP-containing sample) is mixed with other substrates (AMP and ADP phosphorylase substrates) before and after the flow direction of the sample segment in the ATP amplification reactor, It is preferable to introduce a sufficiently small amount of sample so that it can be mixed before reaching. Such a necessary amount can be known experimentally, for example, by introducing a dye that mixes with a dummy to develop a color into the ATP amplification reactor and feeding it to detect and collect it before the first region. it can. In an embodiment in which a sample (ATP-containing sample) runs in parallel with another substrate (a substrate for AMP and ADP phosphorylase) in the ATP amplification reactor, and the two are mixed by feeding and joining. The amount is arbitrary.

  The substrate of the ADP kinase is different depending on the type of ADP kinase immobilized on the second region. For example, when the ADP kinase is PK, the substrate is PEP. When the ADP kinase is PPK, the substrate is polyphosphate (hereinafter also referred to as “Pn”). Further, when the ADP kinase is acetate kinase, the substrate is acetyl phosphate. When the ADP kinase is creatine kinase, the substrate is phosphocreatine. When the ADP kinase is hexokinase, the substrate is hexose phosphate.

  The Pn is not particularly limited, and Pn or a salt thereof can be preferably used. Preferably, 10 to 1000, more preferably 10 to 100 phosphoric acids polymerized in a linear form are used. Pn may be derived from bacteria or may be obtained by chemical synthesis. Alternatively, it may be synthesized from ATP using polyphosphate synthase.

  It is preferable that the substrate of AMP and ADP kinase used in the ATP amplification method according to the present invention does not contain ATP. Since AMP, PEP, and Pn are phosphoric acid substances, usually, commercially available products contain a very large amount of ATP. Therefore, it is preferable to purify the substrate of AMP and ADP kinase and remove ATP. More specifically, the concentration of AMP and the ATP derived from the substrate in the sample solution or buffer containing the substrate of AMP and ADP kinase is preferably 1 mM or less, and is 1 μM or less. It is more preferable to do so.

  The buffer solution to be sent to the flow path and the buffer solution used for preparing the sample solution containing ATP are not particularly limited, and suitable buffers such as Tris buffer solution, HEPES buffer solution, and phosphate buffer solution are not particularly limited. A liquid may be used. Moreover, it is preferable that the said buffer solution contains bivalent metal ions, such as Mg ion, for example.

  As the buffer solution, it is preferable to select an appropriate buffer solution according to the method for detecting the amplified ATP. When the most commonly used luciferase luminescence method is used as an ATP detection method, the buffer solution preferably contains as little an inhibitor of the luciferase reaction as possible.

  The luciferase reaction is inhibited by, for example, phosphate ions or chloride ions, as shown in Examples described later. Therefore, when detecting ATP amplified by the ATP amplification method according to the present invention by the luciferase luminescence method, it is preferable that the phosphate ion and chloride ion concentrations in the buffer solution be as low as possible.

  Specifically, the phosphate ion concentration is preferably 1 M or less, and more preferably 1 mM or less. Further, the chloride ion concentration is preferably 1 M or less, and more preferably 1 mM or less.

  Further, the temperature at which the ATP amplification method according to the present invention is performed is not particularly limited, and may be performed within a range not exceeding the optimum temperature of the enzyme used for the chain ATP amplification reaction. Specifically, it is preferable to carry out in a temperature range of 4 ° C to 98 ° C. If the temperature is lower than the optimum temperature of the enzyme used for the chain ATP amplification reaction, the enzyme activity tends to be low and ATP cannot be sufficiently amplified. On the other hand, if the temperature exceeds the optimum temperature of the enzyme used for the chain ATP amplification reaction, the enzyme is deactivated and ATP tends not to be sufficiently amplified.

  Therefore, in the configuration in which the ATP amplification reactor according to the present invention includes the temperature control unit, the temperature control unit can control the flow path within a temperature range from room temperature to the optimum temperature of the enzyme used for the chain ATP amplification reaction. It is preferable.

  The liquid feeding method for feeding the buffer solution along the flow path is not particularly limited, and may be a continuous liquid feeding method or a stopped flow method. In addition, the flow rate at that time is not particularly limited, and may be a flow rate at which the chain ATP amplification reaction can sufficiently proceed in the first region and the second region. The flow rate depends on the amount of enzyme and the enzyme activity immobilized on the first region and the second region, and is preferably set appropriately according to the flow path used. Specifically, an appropriate flow rate can be set by a method as shown in an example described later.

  In particular, it is preferable to select a flow rate as high as possible within an appropriate flow rate range. The part of the flow system that does not contribute to the reaction is called dead volume. As the flow rate is reduced, the time required to feed the dead volume increases in inverse proportion to the flow rate. Therefore, in some cases, it is preferable to adopt a stopped flow method in order to ensure a sufficient residence time in the first region and the second region.

  It is preferable that the reactions in the first region and the second region reach an almost equilibrium state. Thereby, ATP can be amplified with good reproducibility. Specifically, it is preferable that the analyte-side substrate in the reaction in the first region and the second region, that is, ATP is consumed in the first region and ADP is consumed in the second region. . Note that this is not necessary as long as ATP can be amplified with good reproducibility.

  In the ATP amplification reactor according to the present invention, the higher the amount of immobilized enzyme and the higher the specific activity of the enzyme, the faster the equilibrium state is reached. Further, if the residence time in the first region and the second region is long, the equilibrium state is more reliably reached. The length of the first region and the second region (in other words, the column length) is long, and the larger the cross-sectional area perpendicular to the axial direction of the flow in the first region and the second region, the residence time is the same. become longer. Moreover, the residence time becomes longer as the flow rate of liquid feeding is slower. These flow system design conditions and implementation conditions can be determined experimentally based on the pressure resistance conditions of selectable flow system members and constraints such as enzyme activity.

  In addition, in the ATP amplification method according to the present invention, before the ATP is supplied to the flow path (in other words, before the chain ATP amplification reaction is performed), the buffer solution containing the ADP phosphorylase substrate is added to the flow path. It is preferable to send the solution to the path.

  Many of the above ADP kinases form a complex with ADP which is a reaction substrate. Specifically, it is known that both PK and PPK, which are ADP kinases, form a complex with ADP.

  When ADP phosphorylase forms such a complex with ADP, it is difficult to perform quantitative ATP amplification because the ADP is converted to ATP in the chain ATP amplification reaction.

  However, even if the ADP phosphorylase immobilized in the second region forms a complex with ADP by feeding a buffer containing the substrate for the ADP phosphorylase in advance into the channel, ADP can be removed. That is, according to the above configuration, ATP can be amplified quantitatively.

  In addition, after using the ATP amplification reactor according to the present invention, a buffer solution containing the ADP phosphorylase substrate is sent to the flow path to easily remove ADP bound to ADP phosphorylase. can do. Therefore, the ATP amplification reactor according to the present invention can be used repeatedly by an easy maintenance process.

  In the ATP amplification method according to the present invention, it is further preferable to send phosphoric acid to the channel before supplying ATP to the channel (in other words, before performing the chain ATP amplification reaction).

  According to the said structure, even if ATP has adhered (stagnation) in the flow path, this ATP can be washed away. Therefore, ATP can be amplified quantitatively.

  Moreover, after using the ATP amplification reactor according to the present invention, ATP on the flow path can be easily removed by feeding phosphoric acid into the flow path. Therefore, the ATP amplification reactor according to the present invention can be used repeatedly by an easy maintenance process.

  Since the ATP amplification method according to the present invention has the above-described configuration, it is possible to amplify only ATP supplied to the flow path with high quantitativeness. Therefore, the ATP amplification reactor according to the present invention and the ATP amplification method according to the present invention can be suitably used for high-sensitivity detection and high-sensitivity quantification of a trace amount of ATP contained in a sample. Of course, it can be used for the purpose of synthetic production of ATP.

  Therefore, the present invention includes a method for detecting ATP using the ATP amplification reactor according to the present invention (hereinafter also referred to as “ATP detection method”) and a method for quantifying ATP (hereinafter referred to as “ATP quantification method”). Is also included. The ATP detection method and ATP quantification method will be described later.

  The present invention also includes an ATP amplification kit, an ATP detection kit, an ATP quantification kit (hereinafter, referred to as “ATP amplification kit”, “ATP detection method”, and “ATP quantification kit”). These kits are also collectively referred to as “kits according to the present invention”).

  The kit according to the present invention only needs to contain at least one of a carrier on which ADK is immobilized and a carrier on which ADP phosphorylase is immobilized, and other specific configurations are not particularly limited.

  Further, the carrier on which ADK is immobilized and the carrier on which ADP phosphorylase is immobilized are included in the kit according to the present invention in a state where ADK and ADP phosphorylase are already immobilized on the carrier. However, it may be included as a reagent set for immobilizing each of ADK and ADP kinase on the carrier.

  The kit according to the present invention preferably further contains a reagent used for the chain ATP amplification reaction and a reagent used for the detection of ATP.

  Specific examples of the reagent used for the chain ATP amplification reaction include PEP, polyphosphate compound, and AMP.

  Examples of the reagent used for the ATP detection include luciferase and luciferin.

  The kit according to the present invention includes, in addition to those exemplified above, a buffer solution for dissolving the above compounds, an ATP standard solution, a reagent for extracting ATP from a sample, a plate used for the reaction, and the like. Also good.

<II. ATP detection method and ATP quantification method>
The ATP detection method according to the present invention is a method for detecting ATP in the sample by amplifying ATP contained in the sample by the ATP amplification method according to the present invention and detecting the amplified ATP.

  On the other hand, the ATP quantification method according to the present invention is a method in which ATP contained in a sample is amplified by the ATP amplification method according to the present invention, and the ATP in the sample is quantified by quantifying the amplified ATP. .

  A sample to be detected or quantified by the ATP detection method and ATP quantification method according to the present invention is not particularly limited as long as it may contain ATP. Since ATP is an energy substance present in all living organisms such as animals, plants, and microorganisms, biological samples can be the target sample of the present invention.

  Since this method can detect and quantify an extremely small amount of ATP, this method can be applied to, for example, as a medicinal effect test of an anticancer agent, detection of cells that have died due to apoptosis, and traces of living organisms. Detection of remaining ATP, inspection of food residues that future microorganisms will inhabit at a hygiene inspection site, detection of extremely few microorganisms, and the like. In particular, if it is applied to detection of microorganisms, the presence of a very small number of microorganisms can be confirmed very simply, and therefore a sample that may contain microorganisms is suitable as the target sample of the present invention.

  The method for extracting ATP from the sample is not particularly limited, and may be appropriately selected from known methods according to the type of the sample. For example, when extracting ATP from a sample containing cells or a sample containing microorganisms, a method of crushing cells or microorganisms by physical means (for example, a homogenizer, ultrasonic wave, etc.), a cell or microorganisms by an enzyme (for example, lysozyme, etc.) And a method of crushing a membrane of cells or microorganisms with a surfactant (for example, TritonX-100 (trade name), Tween 20 (trade name), etc.) can be used.

  However, if only cells are disrupted, enzymes such as pyruvate kinase, polyphosphate kinase, and adenylate kinase contained in the cells may affect the chain ATP amplification reaction. Therefore, it is preferable to use a method in which ATP is extracted by crushing cells or the like to extract ATP and then heat-treating to deactivate the enzyme or heat-treating the cells.

  In the ATP detection method and ATP quantification method according to the present invention, the method for detecting and quantifying ATP amplified by the ATP amplification method according to the present invention is not particularly limited, and is conventionally known as an ATP detection method and an ATP quantification method. Any method can be used.

  The most common method used for detection and quantification of ATP includes measurement of the amount of luminescence by the reaction of luciferase and ATP. In the present invention, the method can be preferably used. When ATP is measured by measuring the amount of luminescence, a commercially available ATP measurement kit using luciferase may be used.

  The present invention is not limited to the configurations described above, and various modifications can be made within the scope of the claims, and the technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention.

  Although this invention is demonstrated more concretely based on an Example and FIGS. 3-11, this invention is not limited to this. Those skilled in the art can make various changes, modifications, and alterations without departing from the scope of the present invention.

[Example 1: ATP amplification using enzyme-immobilized column]
(1) Production of enzyme-immobilized column An ADK-immobilized column and a PK-immobilized column were produced by the following procedure.

  First, 6 ml of 1 mM HCl cooled on ice in advance was passed through a coupling column for ligand immobilization (trade name: HiTrap NHS-activated HP column 1 ml (GE Healthcare)) with a syringe.

Immediately, for the ADK-immobilized column, a chicken muscle-derived myokinase (= ADK, SIGMA) 30 μg / ml enzyme solution prepared with a binding buffer (0.2 M NaHCO ₃ -NaOH, 0.5 M NaCl, pH 8.4) was added to the PK. About the immobilization column, 1 ml each of the rabbit muscle-derived Pyruvate kinase (SIGMA) 144 μg / ml enzyme solution prepared with the above binding buffer was passed through, and incubated at 25 ° C. for 30 minutes.

Next, 6 ml of a binding stop solution (0.5 M ethanolamine, 0.5 M NaCl, pH 8.0) was passed through each column and kept at 25 ° C. for 30 minutes. Finally, 6 ml of transfer buffer (50 mM HEPES-NaOH, 5 mM MgCl ₂ , pH 7.4) was passed through each column. As a result, an ADK-immobilized column and a PK-immobilized column were obtained.

(2) Purification of substrate AMP and PEP used as substrates in the chain ATP amplification reaction were purified by the following procedure.

  First, 10 mM AMP aqueous solution and 500 mM PEP aqueous solution were prepared using commercially available AMP (Tokyo Chemical Industry) and PEP (SIGMA). 100 μl each of the prepared AMP aqueous solution and PEP aqueous solution was applied to an anion exchange column (trade name: DEAE-2SW (Tosoh Corporation)) equilibrated with buffer A (10 mM citric acid, 5 mM NaCl, pH 2.85). .

  Thereafter, AMP and PEP were eluted with a linear gradient with a buffer B concentration of 0% to 100% using buffer A and buffer B (10 mM citrate, 1M NaCl, pH 4.40), respectively. In addition, the flow rate sent to the column was 1.0 ml / min.

As for AMP, the absorbance at 254 nm (A ₂₅₄ ) of the eluted solution was monitored, and fractions having peak absorbance of 2.5 or more were collected. On the other hand, for PEP, the absorbance at 250 nm (A ₂₅₀ ) of the eluted solution was monitored, and the fraction having a peak absorbance of 2.0 or more was fractionated to obtain purified AMP and PEP.

  The AMP and PEP fractions thus collected were subjected to a concentration test. The concentration of the AMP fraction was 2.8 mM, and the concentration of the PEP fraction was 25.8 mM.

(3) Examination of flow rate of substrate solution sent to enzyme-immobilized column When substrate solution is sent to ADK-immobilized column and PK-immobilized column prepared in (1) above, the reaction should proceed sufficiently. A possible flow rate was examined by the following procedure.

a) Examination of flow rate of substrate solution sent to PK immobilization column The PK immobilization column prepared in (1) above was connected to an automatic liquid delivery device (trade name: AKTA explorer 100: GE Healthcare). Transfer buffer (50 mM HEPES-NaOH, 5 mM MgCl ₂ , pH 7.4) was passed through at least 2 ml at 0 ml / min to equilibrate.

After equilibration, 1 ml of PK reaction solution (50 nM ATP, 1.0 mM PEP, 50 mM HEPES-NaOH, 5 mM MgCl ₂ , pH 7.4) was added at a flow rate of 0.1, 0.2, 0.5, 1.0, ₂ 0.0 or 4.0 ml / min was passed through, and a total of 5 ml was taken in a 96-well microplate using a fraction collector. Next, the ATP amount and ADP amount of each fraction collected were measured.

  The amount of ATP is measured with respect to 20 μl of a sample by using a luciferase solution (luciferase (trade name: Luciferase FM (Bio-Enex)) 0.025 mg / ml, luciferin potassium 2 mM, 50 mM Tricin-NaOH, 1 mM magnesium acetate, 5% trehalose. , PH 7.8) 20 μl was added, and the luminescence value was measured with a plate reader (trade name: Wallac 1420 Arvo SX (Perkin Elmer)).

On the other hand, in the measurement of the amount of ADP, first, 20 μl of a PK enzyme solution (rabbit muscle-derived PK 10 U / ml, 50 mM HEPES-NaOH, 5 mM MgCl ₂ , pH 7.4) is added to 40 μl of a sample, and then at 37 ° C. for 60 minutes. Keep warm. Thereafter, 20 μl of the luciferase solution was added to 40 μl of the reaction solution, and the luminescence value was measured with a plate reader (trade name: Wallac 1420 Arvo SX (Perkin Elmer)). Thus, the ADP amount was calculated by subtracting the ATP amount obtained by the measurement of the ATP amount from the total ATP amount obtained by adding all the ADP and ATP of each fraction to the ATP.

  The reaction rate when the PK reaction solution was fed at each flow rate was determined from the ATP amount and ADP amount of each fraction measured by the above method.

  As a result, as shown in FIG. 3, when the PK reaction solution was fed at a flow rate of 1.0 ml / min or more, a decrease in the reaction rate was observed as the flow rate increased. This decrease in the reaction rate occurs when the PK reaction solution is fed at a flow rate of 1.0 ml / min or more when the enzyme amount is immobilized on the PK immobilization column prepared in (1) above. This is probably because the reaction time sufficient for the reaction to sufficiently proceed cannot be secured.

b) Examination of flow rate of substrate solution sent to ADK-immobilized column The ADK-immobilized column prepared in (1) above was connected to an automatic liquid feeder, and at least 2 ml of the above moving buffer was flowed at a flow rate of 1.0 ml / min. The solution was passed through and equilibrated. After equilibration, 1 ml of ADK reaction solution (100 nM ADP, 0.1 mM AMP, 50 mM HEPES-NaOH, 5 mM MgCl ₂ , pH 7.4) was added at a flow rate of 0.1, 0.2, 0.5, 1.0, 2. The solution was allowed to flow through at 0 or 4.0 ml / min, and 100 ml was dispensed into a 96-well plate with a fraction collector for a total of 5 ml. Next, the amount of ATP and the amount of ADP of each fraction collected were measured by the same method as described above.

  Then, the reaction rate when the ADK reaction solution was fed at each flow rate was determined from the measured ATP amount and ADP amount of each fraction.

  As a result, as shown in FIG. 4, when the ADK reaction solution was fed at a flow rate of 1.0 ml / min or more, a decrease in the reaction rate was observed as the flow rate increased. This decrease in the reaction rate occurs when the ADK reaction solution is fed at a flow rate of 1.0 ml / min or more when the enzyme amount immobilized on the ADK immobilization column prepared in (1) above is passing through the column. This is considered to be because the reaction time sufficient for the reaction to sufficiently proceed cannot be secured.

  Thus, in both the PK-immobilized column and the ADK-immobilized column, when the substrate solution was fed at a flow rate of 1.0 ml / min or more, the reaction could not be sufficiently performed. On the other hand, when the substrate solution was fed at a flow rate of 0.5 ml / min or less, as shown in FIGS. 3 and 4, the reaction rate was almost 100%. Therefore, in the following experiment, the flow rate for feeding the liquid to the PK-immobilized column and the ADK-immobilized column was set to 0.5 ml / min.

(4) Chain ATP amplification reaction using enzyme-immobilized column Four ADK-immobilized columns and four PK-immobilized columns prepared by the method of (1) above were prepared.

  Using these four columns, (a) a linking column composed of an ADK-immobilized column-PK-immobilized column, and (b) an ADK-immobilized column-PK-immobilized column-ADK-immobilized column-PK-immobilized column. (C) ADK-immobilized column-PK-immobilized column-ADK-immobilized column-PK-immobilized column-ADK-immobilized column-PK-immobilized column, (d) ADK-immobilized column-PK-immobilized A linking column composed of a column-ADK-immobilized column-PK-immobilized column-ADK-immobilized column-PK-immobilized column-ADK-immobilized column-PK-immobilized column was prepared.

Each of the above connection columns (a) to (d) is attached to an automatic liquid feeder, and 2 ml or more of a moving buffer (50 mM HEPES-NaOH, 5 mM MgCl ₂ , pH 7.4) is passed at a flow rate of 1.0 ml / min. And equilibrated. In addition, when measuring several times using the same connection column, equilibration on the said conditions was implemented between all the measurements.

After equilibration, 2 ml of amplification reaction solution (65 nM ATP, 1.0 mM PEP, 0.1 mM AMP, 50 mM HEPES-NaOH, 5 mM MgCl ₂ , pH 7.4) was passed through each ligation column at a flow rate of 0.5 ml / min. A total of 20 ml was taken in a 96-well microplate using a fraction collector. In addition, AMP and PEP refine | purified by the purification method 1 of said (2) were used for preparation of the said amplification reaction liquid.

  Next, the amount of ATP in each fraction was measured. The amount of ATP was measured with respect to 20 μl of sample using a luciferase solution (luciferase (trade name: Luciferase FM (Bio-Enex)) 0.025 mg / ml, luciferin potassium 2 mM, 50 mM Tricin-NaOH, 1 mM magnesium acetate, 5% trehalose, pH 7 .8) 20 μl was added, and the luminescence value was measured by a plate reader (trade name: Wallac 1420 Arvo SX (Perkin Elmer)).

  Further, an amplification reaction solution (background) not containing ATP was applied to each linking column, and the same operation was performed. For each fraction collected, the amount of ATP is measured using the above method, and the measured value is subtracted from the measured value when the amplification reaction solution containing ATP is used. The amount of ATP was calculated.

  As a result, as shown in FIG. 5, by passing the amplification reaction solution through each linking column (amplification column), 2 ml of the reaction solution was diluted and flowed out with a peak having an ATP concentration gradient of about 5 ml.

  Moreover, about 1.5 ml of the peak top was converged to a fixed numerical value in any number of amplifications of 1 to 4. From this, it was considered that this fraction was a fraction of the solution that reacted without being influenced by dilution.

  In addition, the peak ATP concentration increased as compared with the ATP concentration before the reaction at any number of amplifications of 1 to 4. From this, it was confirmed that the chain ATP amplification reaction proceeds at any number of amplifications.

  In addition, an increase in ATP concentration was observed with an increase in the number of columns connected. Therefore, a semi-log plot of the relationship between the average value and the number of amplifications of the samples amplified at each amplification number (2 fractions before and after the peak top, a total of 5 fractions).

As a result, as shown in FIG. 6, there was a tendency to amplify at 1.9 ^N and exponentially amplify at a value close to the theoretical value of 2 ^N. From the above results, it became clear that the number of amplifications was controlled by the number of connections between the ADK-immobilized column and the PK-immobilized column, and as a result, ATP amplification could be controlled.

  That is, in the conventional ATP amplification reaction by the batch method, since the control of the number of amplifications depends on the reaction time, the degree of ATP amplification greatly fluctuates due to a subtle difference in enzyme activity, making quantitative high-sensitivity measurement difficult. . However, in the ATP amplification method according to the present invention, a chain ATP amplification reaction is performed in a flow system using an immobilized enzyme. In this ATP amplification method, the number of ATP amplifications depends on the number of enzyme-immobilized columns to be linked. For this reason, it has become clear that the number of amplifications (in other words, the degree of amplification of ATP) can be strictly controlled by the number of amplification columns.

[Example 2: Sensitive determination of ATP using an enzyme-immobilized column]
It was verified whether ATP could be quantified with high sensitivity by ATP amplification using an enzyme-immobilized column. (D) ADK-immobilized column-PK-immobilized column-ADK-immobilized column-PK-immobilized column-ADK-immobilized column-PK-immobilized column-ADK-immobilized prepared in (4) of Example 1 as a linking column A linked column consisting of a column-PK immobilized column was used. That is, in Example 2, the number of amplification of ATP by the chain ATP amplification reaction was set to 4 times.

  The above-mentioned connected column was attached to an automatic liquid feeder, and buffer C (10 mM citric acid, 5 mM NaCl, pH 2.85) was passed at 2 CV or more at a flow rate of 1.0 ml / min to equilibrate. In addition, equilibration was carried out under the same conditions between all measurements thereafter.

After equilibration, 2 ml of ATP diluted reaction solution (ATP, 1.0 mM PEP, 0.1 mM AMP, 50 mM HEPES-NaOH, 5 mM MgCl ₂ , pH 7.4) was passed at a flow rate of 0.5 ml / min. A total of 20 ml was dispensed into a 96-well microplate at 200 μl. In addition, AMP and PEP refine | purified in Example 1 were used for preparation of the said ATP dilution reaction liquid. The ATP concentration of the ATP diluted reaction solution was 500 fM, 5 pM, 50 pM, 500 pM, 5 nM, and 50 nM.

  Next, the amount of ATP in each fraction was measured. As for ATP, 20 μl of the above luciferase solution was added to 5 μl of sample, and the luminescence value was measured with a plate reader (trade name: Wallac 1420 Arvo SX (Perkin Elmer)), and the amount of ATP was measured.

  Further, the same operation was performed for the ATP diluted reaction solution (background) not containing ATP. And the measured value obtained from the background was subtracted from the measured value obtained using the ATP diluted reaction solution, and the ATP amount after the chain ATP amplification reaction was calculated.

  Further, as a comparison control (control in which chain ATP amplification reaction was not performed), 20 μl of the luciferase solution was added to 5 μl of the ATP diluted reaction solution, and the luminescence value was measured using a plate reader (trade name: Wallac 1420 Arvo SX (trade name: Perkin Elmer))), and the luminescence value was measured.

  As a result, as shown in FIG. 7, ATP contained in each ATP dilution reaction solution was amplified by four times amplification by the chain ATP amplification reaction, and the luminescence value increased at each ATP concentration of 50 pM or more. More specifically, a proportional relationship was established between the luminescence value and the ATP concentration when the ATP concentration of the ATP-diluted reaction solution was in the range of 50 pM to 50 nM, which was a quantifiable range.

  In contrast, when the ATP diluted reaction solution before amplification was measured by a luciferase assay, a proportional relationship was established between the luminescence value and the ATP concentration in the range of ATP concentration of 500 pM to 50 nM, and it was within the quantifiable range. However, at an ATP concentration of 50 pM or less, there was no difference in the luminescence value because the plate reader (trade name: Wallac 1420 Arvo SX (Perkin Elmer)) was below the detection limit.

  In other words, when ATP is amplified under the above conditions, the ATP concentration is amplified about 16 times by amplification four times. Even when the ATP concentration of the ATP dilution reaction solution is 50 pM, ATP is amplified and is generated from the amplified ATP. The luminescence value was within the detection range.

  As a result, it became clear that the quantitative sensitivity of the ATP luciferase assay can be improved by ATP amplification using an immobilized enzyme. Specifically, the four-time amplification of ATP was able to improve the sensitivity 10 times compared to the ATP quantification by the conventional luciferase assay.

  Further, according to the ATP amplification method of the present invention, the number of ATP amplifications can be controlled as desired, so that the quantitative sensitivity of the ATP luciferase assay is further improved by increasing the number of ATP amplifications. be able to.

[Example 3: Repeated ATP amplification using an enzyme-immobilized column]
Rather than connecting multiple sets of ADK-immobilized columns and PK-immobilized columns, the sample is passed multiple times by passing the sample multiple times through a concatenated column in which ADK-immobilized columns and PK-immobilized columns are connected one by one. ATP amplification was performed.

Specifically, a linking column in which an ADK-immobilized column and a PK-immobilized column are connected one by one is attached to an automatic liquid feeder, and a running buffer (1 mM PEP, 0.1 μM AMP, 5 mM) at a flow rate of 1.0 ml / min. _{MgCl 2, 50mM HEPES-NaOH,} pH7.4) was passed through, and equilibrated. Note that unpurified AMP and PEP were used for the preparation of the running buffer.

  After equilibration, 1 ml of 50 pmol ATP solution prepared with the above running buffer was injected, and 0.1 ml each was sampled for a total of 5 ml.

  Next, 5 ml of the sampled sample was again injected into the same connected column, and 10 ml was sampled in 0.1 ml increments by the same method.

  Furthermore, 10 ml of the sampled sample was again injected into the same connecting column, and a total of 15 ml was sampled by 0.1 ml in the same manner.

  As a result, a sample obtained by amplifying 3 ml of 1 ml of ATP with 50 pmol of ATP solution was obtained. Further, the same treatment was performed for the sample (background) not containing ATP, and the amount of ATP after the first amplification, the second amplification, and the third amplification was determined using the same method as in Examples 1 and 2. It was measured.

  As a result, as shown in FIG. 8, it was clarified that ATP can be amplified by passing a sample through a set of connected columns a plurality of times as in the case of a connected column in which a plurality of sets are connected.

  Moreover, in Example 1 and Example 2, AMP and PEP, which are substrates for the chain ATP amplification reaction, were added to the ATP diluted reaction solution and the amplification reaction solution containing ATP. In this regard, in Example 3, AMP and PEP were not added to the ATP dilution reaction solution or the amplification reaction solution, but were added to the running buffer. Even in this case, it was confirmed that the chain ATP amplification reaction proceeded.

[Example 4: Inhibition of luciferase activity by KCl]
In the ATP quantification by the luciferase assay, the effect of KCl (in other words, chloride ion) on the luciferase activity was verified by the following method.

Reaction solutions (50 mM Hepes (pH 7.4), 5 mM MgCl ₂ , 100 nM ATP) each containing 100, 10, 1, 0.1, and 0 mM KCl were prepared.

  Next, 20 μl of a 25 μg / ml luciferase solution and 40 μl of the reaction solution were mixed, and the luminescence value was measured with a luminometer (trade name: Lumitester C-100 (Kikkoman)).

  As a result, as shown in FIG. 9, when the reaction solution contains 10 mM or more of KCl (in other words, chloride ions), the luciferase activity is remarkably inhibited, and when 100 mM KCl is contained, It was found that the luminescence value decreased to about 20% when no is contained.

[Example 5: Inhibition of luciferase activity by sodium hydrogen phosphate]
In the ATP quantification by the luciferase assay, the influence of sodium hydrogen phosphate (in other words, phosphate ion) on the luciferase activity was verified by the following method.

Reaction solutions (50 mM Hepes (pH 7.4), 5 mM MgCl ₂ , 100 nM ATP) each containing 75, 7.5, 0.75, and 0 mM sodium hydrogen phosphate were prepared.

  Next, 20 μl of a 25 μg / ml luciferase solution and 40 μl of the reaction solution were mixed, and the luminescence value was measured with a luminometer (trade name: Lumitester C-100 (Kikkoman)).

  As a result, as shown in FIG. 10, when sodium hydrogen phosphate (in other words, phosphate ion) is contained in the reaction solution, luciferase activity is inhibited and 7.5 mM sodium hydrogen phosphate is contained. It was revealed that the luminescence value decreased to about 27% when no sodium hydrogen phosphate was contained, and almost no light was emitted when 75 mM sodium hydrogen phosphate was contained.

[Example 6: Examination of purification method of AMP and PEP]
(1) Measurement of ADP and ATP amounts in each AMP Commercially available AMP, AMP obtained by recrystallizing commercially available AMP, and AMP purified in Example 1 were each prepared to be 1.0 mM. 90 μl of PK enzyme solution (rabbit muscle-derived PK 10 U / ml, 50 mM HEPES-NaOH, 5 mM MgCl ₂ , pH 7.4) was added to 10 μl of each AMP sample, and the mixture was incubated at 37 ° C. 0, 10, 30, and 60 minutes after the start of the reaction, 45 μl of a 25 μg / ml luciferase solution was added to 5 μl of the reaction solution, and the luminescence value was measured with a luminometer (trade name: Lumitester C-100 (Kikkoman)). did.

  As a result, as shown in FIG. 11, the ATP contained in the AMP purified in Example 1 (described as “HPLC” in FIG. 11) and the amount of ADP were described as commercially available AMP (in FIG. 11, “no purification”). ), Compared with AMP obtained by recrystallizing commercially available AMP (described as “recrystallization method” in FIG. 11), it was suggested that purification by HPLC can effectively remove ATP and ADP.

(2) Changes in ATP and ADP amounts before and after purification of AMP and PEP by the method of Example 1 The amounts of ATP and ADP contained in the commercially available AMP and PEP and the AMP and PEP purified in Example 1 were measured. The amount of ATP was adjusted to 1.0 mM each of commercially available AMP and PEP, and AMP and PEP purified in Example 1, and 45 μl of a 25 μg / ml luciferase solution was added to 5 μl of each sample, and the luminescence value was Was measured with a luminometer (trade name: Lumitester C-100 (Kikkoman)), and the ATP amount was determined from the luminescence value.

The amount of ADP is adjusted so that each sample is 10 mM, and 90 μl of PK enzyme solution (rabbit muscle-derived PK 10 U / ml, 50 mM HEPES-NaOH, 5 mM MgCl ₂ , pH 7.4) is added to each sample 10 μl, After incubation at 37 ° C. for 60 minutes, 45 μl of a 25 μg / ml luciferase solution was added to 5 μl of the reaction solution, and the luminescence value was measured with a luminometer (trade name: Lumitester C-100 (Kikkoman)).

  The amounts of ATP and ADP were determined from the luminescence values, and the amount of ATP determined by the above method was subtracted to determine the amount of ADP.

  As a result, as shown in Table 1, ATP and ADP contained in AMP and PEP obtained in Example 1 were effectively removed, and each was 1 pM or less.

Note that the present invention is not limited to the configurations described above, and various modifications are possible within the scope of the claims, and technical means disclosed in different embodiments and examples respectively. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention.

  As described above, in the present invention, when ATP is artificially amplified, the number of amplifications can be controlled as desired, so even if the amount of ATP contained in the sample is very small, the ATP can be quantitatively amplified. it can. Therefore, the present invention can be widely used in all industrial fields where it is necessary to quantify ATP contained in a sample. In particular, it can be suitably used in the food industry, the pharmaceutical industry, the clinical examination field, the environmental hygiene field, and the like that require microbial testing.

FIG. 1 is a diagram showing a main part of an ATP amplification reactor according to an embodiment of the present invention. FIG. 2 is a diagram showing the principle of the ATP amplification method according to the present invention. FIG. 3 is a graph showing the relationship between the flow rate of the PK-immobilized column and the reaction rate. FIG. 4 is a graph showing the relationship between the flow rate of the ADK-immobilized column and the reaction rate. FIG. 5 is a diagram showing the relationship between the flow rate and the ATP concentration at each number of amplifications using the ATP amplification method according to the present invention. FIG. 6 is a diagram showing the relationship between the average value of the peak top of the ATP concentration of the sample discharged from the ATP amplification reactor according to one embodiment of the present invention and the number of amplifications. FIG. 7 shows the relationship between the average luminescence value of the ATP concentration peak top 5 samples after amplification of each ATP diluted solution and the ATP concentration before amplification. FIG. 8 is a diagram illustrating a result of amplifying ATP in a sample by passing through a flow path formed by connecting an ADK-immobilized column and a PK-immobilized column a plurality of times. FIG. 9 shows inhibition of luciferase activity by KCl. FIG. 10 shows inhibition of luciferase activity by phosphate. FIG. 11 is a diagram showing the results of measuring the amounts of ADP and ATP in commercially available AMP, AMP obtained by recrystallizing commercially available AMP, and AMP purified in Example 1. FIG. 12 is a diagram showing the relationship between reaction time and ATP amplification in the chain ATP amplification reaction by the batch method, which is a conventional technique.

Claims (6)

A reactor for amplifying adenosine triphosphate,
A flow path provided with a first region in which adenylate kinase is fixed and a second region in which an enzyme that converts adenosine diphosphate to adenosine triphosphate is fixed ;
The flow path allows the adenosine triphosphate, adenylic acid, and an enzyme substrate that converts the adenosine diphosphate to adenosine triphosphate to pass through the first region and the second region in this order. Yes,
The first region consumes all of the adenosine triphosphate,
The reactor in which the second region consumes all of the adenosine diphosphate .   The reactor according to claim 1, wherein the enzyme that converts adenosine diphosphate into adenosine triphosphate is pyruvate kinase, polyphosphate kinase, acetate kinase, creatine kinase, or hexokinase. A plurality of the flow paths are connected,
The reactor according to claim 1, wherein the first region and the second region are alternately arranged. Liquid is sent in the direction from the first region to the second region along the flow path, and the liquid passes through the first region and the second region in order,
The liquid circulation mechanism that enables the liquid that has passed through the second region to be fed again in the direction of the second region from the upstream side of the first region. The reactor described. Adenosine triphosphate and adenylate are formed along a channel provided with a first region to which adenylate kinase is immobilized and a second region to which an enzyme that converts adenosine diphosphate to adenosine triphosphate is immobilized. And the substrate of the enzyme are passed through the first region and the second region in this order ,
In the first region, all of the adenosine triphosphate is consumed,
The method for amplifying adenosine triphosphate, wherein all the adenosine diphosphate is consumed in the second region .   The combination of the enzyme that converts adenosine diphosphate into adenosine triphosphate and the substrate of the enzyme includes a combination of pyruvate kinase and phosphoenolpyruvine, a combination of polyphosphate kinase and polyphosphate, acetate kinase and acetyl phosphate. 6. The method for amplifying adenosine triphosphate according to claim 5, which is a combination of an acid, a combination of creatine kinase and phosphocreatine, or a combination of hexokinase and hexose phosphate.