JPWO2005073399A1 - Phosphate ion qualitative and quantitative methods and phosphate ion sensor - Google Patents

Abstract

(1) An enzyme-catalyzed reaction occurs due to the presence of phosphate ions, using a substrate that generates an oxide, an oxidoreductase, other substances required for the reaction, and an oxidized mediator. A method of measuring phosphate ions in a sample solution by measuring the resulting oxidation current, or (2) by reacting phosphate ions with phosphate ions and finally β- A β-D-glucose is produced by reacting in the presence of a substrate that produces D-glucose and an enzyme that catalyzes each reaction for producing this β-D-glucose. And a method of measuring the current generated by further oxidizing the resulting reduced mediator by reacting with the oxidized mediator in the presence of oxidoreductase and ( ) Acting on the substrate electrode, in a sensor having a counter electrode, phosphate ion sensor which these enzymes and reagents are placed on the working electrode and / or near.

Description

  The present invention relates to a qualitative and quantitative method for phosphate ions and a phosphate ion sensor. More specifically, a qualitative / quantitative method for quantifying phosphate ions that can be qualitatively or quantitatively detected by electrochemically detecting phosphate ions in a sample solution using a mediator utilizing electron transfer, and a phosphate ion sensor used therefor About.

Conventionally, measurement methods such as an absorptiometry, a volumetric method, an atomic absorption method, a gravimetric method, and a biosensor method are known as a quantification method for phosphate ions. Among the spectrophotometric methods, there are a molybdophosphoric acid method, a molybdenum blue method, a vanad molybdophosphoric acid method, and the like. These measurement methods are methods in which each reagent is reacted with phosphoric acid, and then the absorbance is measured by irradiating absorption light having a wavelength specific to each reactant, and the phosphate ion content is measured from the result. is there. The volume method is a method of measuring the content of phosphate ions by adding a base having a known concentration until the end of the reaction, and measuring the volume, and includes neutralization titration and chelate titration. Furthermore, the atomic absorption method is a method in which phosphoric acid is once precipitated as ammonium molybdate, filtered, dissolved again, and molybdenum is quantified by atomic absorption. The gravimetric method is a method in which magnesium and precipitate are precipitated in a magnesia mixture. This is a method of weighing and Mg ₂ P ₂ O ₇ after firing.

  However, the absorptiometric method has a problem that it is difficult to obtain an accurate measurement value because it is affected by another substance having a wavelength approximate to the intrinsic wavelength of the measurement substance. On the other hand, the volumetric method, atomic absorption method, gravimetric method, etc. can be used for accurate quantification, but because of the skill required for operation, there are restrictions on the user, and since it is not portable, it can be carried onsite such as on-site monitoring (on-site measurement). However, there is a problem that simple and rapid measurement is impossible. Furthermore, each of the above methods has a problem in that a substance that gives load to the environment, such as heavy metal ions, is used in a chemical reaction for measuring phosphate ions.

  In order to solve these problems, biosensors have attracted attention. The measurement of phosphate ions using a biosensor is an extremely excellent method because many biological materials such as biological recognition elements are used as the material, and the influence on the environment is small. Therefore, many biosensors have been proposed so far. And the desktop type automatic measuring device for phosphate ion measurement is researched and developed, and the result is reported.

この酵素反応系では、試料液中のリン酸イオンによってアルカリホスファターゼ反応（１）が阻害される。この阻害の度合いが、グルコースオキシダーゼ反応（２）で消費される酸素量として求められる。これらの酵素は、白金電極上に膜を使用して固定化されており、酸素消費量からリン酸イオン濃度が求められる。There have been many reports on quantification of phosphate ions by the biosensor method. First, a phosphate ion sensor combining alkaline phosphatase and glucose oxidase was first reported.

In this enzyme reaction system, the alkaline phosphatase reaction (1) is inhibited by phosphate ions in the sample solution. The degree of inhibition is determined as the amount of oxygen consumed in the glucose oxidase reaction (2). These enzymes are immobilized on a platinum electrode using a membrane, and the phosphate ion concentration is determined from the oxygen consumption.

Subsequently, a biosensor for measuring phosphate ions using microalgae and plants was proposed. In this sensor, Chlorella vulgaris , a kind of green algae, was used as a recognition element, and phosphate ions were measured by a microbial sensor in combination with an oxygen electrode. This microbial sensor utilizes the fact that Chlorella vulgaris significantly increases the amount of oxygen generated by photosynthesis in the presence of phosphate ions. Chlorella vulgaris is adsorbed and immobilized on a light permeable membrane on an oxygen electrode. This is based on the principle of combining an electrode and a reflective lamp and electrochemically measuring the amount of oxygen increase.

In addition, as a method using plants, a sensor that electrochemically measures phosphate ion concentration by adding glucose oxidase to a tissue section of Solanum tuberosum that contains a lot of acid phosphatase and using the principle of inhibitory action by phosphate ions There is a system. Furthermore, a method using inhibition of acid phosphatase reaction on the same principle has been carried out using potato tissue sections.

これらの酵素反応系を用いた最初のセンサは、２つの酵素反応（３）、（４）によって生成した過酸化水素を電極で測定して、リン酸イオン濃度を計測するものである。On the other hand, enzyme sensors that combine a nucleoside phosphorylase and a xanthine oxidase reaction have been widely developed.

The first sensor using these enzyme reaction systems measures the phosphate ion concentration by measuring the hydrogen peroxide generated by the two enzyme reactions (3) and (4) with an electrode.

  A sensor that measures two reaction products of uric acid and vortex hydrogen oxide at the same time has also been proposed and succeeded in achieving high sensitivity. In addition, the alkaline phosphatase reaction is used to amplify phosphate ions to increase sensitivity, and the phosphate ions are highly sensitive by flow injection analysis (FIA) method or by combining FIA method and chemiluminescence method. Can be measured. Thus, many sensors that combine the reactions of (3) and (4) have been reported.

この酵素反応ではこれまで開発されたセンサとは異なり、１種類の酵素のみが反応に寄与する特徴を有している。しかし、電極法を用いた電流測定型センサは、ピルビン酸オキシダーゼ反応（５）で消費された酸素の濃度を測定して、リン酸イオン濃度を計測している。A phosphate ion sensor using a pyruvate oxidase reaction, an electrode method, and a chemiluminescence method has also been proposed. The reaction formula of pyruvate oxidase is as follows.

Unlike the sensors developed so far, this enzyme reaction has a characteristic that only one kind of enzyme contributes to the reaction. However, the amperometric sensor using the electrode method measures the concentration of phosphate ions by measuring the concentration of oxygen consumed in the pyruvate oxidase reaction (5).

  Next, focusing on the chemiluminescence method, an FIA type enzyme sensor has been developed, and this sensor is composed of a liquid feed pump and an injector, an enzyme-immobilized column, a mixing joint, a chemiluminescence detector, and a recorder. In this sensor, pyruvate oxidase is immobilized on porous glass beads using a covalent bond method with glutaraldehyde, and this is packed in a column. As a luminescent reagent solution, luminol is used as a luminescent material, and the reaction is catalyzed. Peroxidase is used.

  However, since the biosensor for measuring phosphate ions is limited to the method of measuring the oxygen concentration that is reduced by the enzyme reaction or the hydrogen peroxide concentration that is generated, the influence of the dissolved oxygen concentration is inevitable. Furthermore, when measuring the oxygen concentration or the hydrogen peroxide concentration with electrodes, it is necessary to increase the setting of the applied voltage between the electrodes. Increasing the applied voltage can be achieved by, for example, environmental water, foods, body fluids containing a large amount of contaminants. There is a problem that accurate measurement is difficult because it is easily affected by reducing substances contained in the above. Similarly, by measuring the hydrogen peroxide concentration by the luminol chemiluminescence method, even in sensors that measure the phosphate ion concentration, by using a chemiluminescence method that is highly sensitive but inferior in specificity, it is possible to reduce reductivity. The influence of various contaminants including substances cannot be avoided.

  Furthermore, in order to improve the measurement sensitivity and the reproducibility of the signal obtained, it is necessary to immobilize a large amount of the enzyme to be used on the beads packed in the column. There were problems such as requiring skill.

  All of the phosphate ion measurement methods and phosphate ion sensors described above are unavoidable by the influence of contaminants and dissolved oxygen in the sample solution, and the measurement requires a household power supply and the device is not portable. In addition, since it is not a disposable type, the enzyme immobilized on the surface of the column or electrode has many common problems, such as that the stability required to withstand repeated use is required.

  Recently, a biosensor for measuring phosphate ions that combines chemiluminescence using a new enzyme reaction system and an FIA system has been proposed. In this reaction system, a maltose phosphorylase reaction using maltose and phosphate ions as substrates is started, and the product α-D-glucose is converted to β-D-glucose by mutarotase, and hydrogen peroxide by glucose oxidase reaction. Therefore, the phosphoric acid ion concentration is measured by measuring the amount of chemiluminescence produced by mixing a peroxidase / luminol detection reagent with a contact solution containing hydrogen peroxide. The lower limit of detection of phosphate ions of this sensor was 10 nM (0.001 ppm), and the sensitivity was sufficient for environmental measurement. However, since hydrogen peroxide is only measured, there is a problem that it is easily affected by interfering substances. In addition, since the FIA method is used, it is difficult to carry the sensor.

Japanese Patent Publication No. 08-020401 JP 05-093692 A (Patent No. 2,595,141) Japanese Patent Laid-Open No. 09-220085 JP 11-290096 A Taanta 50, 799-807 (1999)

  An object of the present invention is to provide a method for measuring a phosphate ion concentration capable of accurately measuring a phosphate ion without being affected by dissolved oxygen concentration, salinity, contaminants, etc. in a sample liquid to be inspected, and to be used for it. An object of the present invention is to provide a portable disposable ion sensor.

  The object of the present invention is as follows: (1) When an oxidoreductase that reacts with a phosphate ion in the presence of an oxidoreductase that catalyzes a reaction for producing this oxide, an oxidized mediator is used. A method of measuring a current generated by further oxidizing a reduced mediator generated by coexistence, or (2) a substrate which finally reacts with phosphate ions to generate β-D-glucose And β-D-glucose by reacting in the presence of an enzyme that catalyzes each reaction for generating β-D-glucose, and the generated β-D-glucose is converted into the presence of oxidoreductase. And a method of measuring the current generated by further oxidizing the reduced mediator produced by reacting with the oxidized mediator and (3) providing a working electrode and a counter electrode on the substrate. In example was sensors, these enzymes and reagents are achieved working electrode and / or by phosphate ion sensor arranged in vicinity.

  By using the mediator according to the method of the present invention, it is possible to obtain a large output value as compared with, for example, the hydrogen peroxide system, without being affected by contaminants, salt or dissolved oxygen in the sample solution. It becomes possible to accurately measure various substances to be measured such as. In addition, since the method (1) of the present invention uses a simple enzyme reaction system, the number of enzymes to be used is small, a sensor can be easily manufactured and used, and it is advantageous in terms of manufacturing and operating costs. It is. Furthermore, it is possible to construct a sensor with high repeatability.

  Further, by applying the method of the present invention to a disposable biosensor that realizes on-site monitoring, a disposable phosphate ion sensor that is portable (on-site monitoring) is provided. Furthermore, in the methods (1) and (2) of the present invention, when a substrate other than phosphate ions is sufficiently present, the acid phosphatase activity can be measured and can also be used for detection of prostate tumor markers. .

  In the method (1) of the present invention, an enzyme-catalyzed reaction occurs due to the presence of phosphate ions, and a substrate that generates an oxide, an oxidoreductase, other substances required for the reaction, and an oxidized mediator are used. Phosphate ions in the sample solution are measured by generating a reduced mediator from the acid, further oxidizing it, and measuring the resulting oxidation current.

この反応は、リン酸イオンを酸化還元酵素であるピルビン酸オキシダーゼと基質であるピルビン酸、補酵素としてのフラビンアデニンジヌクレオチド（ＦＡＤ）、チアミンピロリン酸（ＴＰＰ）および活性化剤としてのマグネシウムイオンの存在下で反応させるものである。ここで反応に際し、電子伝達体としての酸化型メディエーターを共存させると、還元型メディエーターが発生するため、この還元型メディエーターをさらに酸化することにより生じた電流を測定することで基質であるリン酸イオンの定性・定量が可能となる。Examples of the catalytic reaction system that generates an oxide include those represented by the above formula (5).
(1) Pyruvate oxidase system

This reaction involves the conversion of phosphate ions from pyruvate oxidase, which is an oxidoreductase, and pyruvate, which is a substrate, flavin adenine dinucleotide (FAD), thiamine pyrophosphate (TPP) as a coenzyme, and magnesium ion as an activator. It reacts in the presence. Here, when an oxidized mediator as an electron carrier is coexisted in the reaction, a reduced mediator is generated. Therefore, by measuring the current generated by further oxidizing this reduced mediator, a phosphate ion as a substrate is measured. Can be qualitatively and quantitatively determined.

この際発生したフェロシアンイオンは、作用極で酸化されて酸化電流を生ずる。

As the mediator, potassium ferricyanide, ferrocene, parabenzoquinone, or the like is used. When potassium ferricyanide K ₃ Fe (CN) ₆ is used as a mediator, the reaction with pyruvic acid or the like proceeds as follows.

The ferrocyan ion generated at this time is oxidized at the working electrode to generate an oxidation current.

In addition, when parabenzoquinone is used instead of potassium ferricyanide as a mediator, hydroquinone is produced by the reaction of pyruvate and parabenzoquinone in the presence of pyruvate oxidase, and the hydroquinone produced at this time is oxidized at the working electrode. Since an oxidation current is generated, its value is measured.

Pyruvate is oxidized in the presence of an enzyme such as pyruvate oxidase to produce acetyl phosphate, H ₂ O ₂ generated at that time is oxidized on the working electrode, and the oxidation current value at that time is measured, The method for indirectly determining the phosphate ion concentration is as described in the prior art. However, in such a method, when the measurement liquid contains an interfering substance such as seawater, it is not possible to measure an accurate phosphoric acid concentration. However, when the mediator is used together with pyruvate oxidase or the like, the phosphate concentration can be accurately measured without being affected by the interfering substance.

  For the measurement of phosphate ions, a sensor is preferably used in which a working electrode and a counter electrode are provided on a substrate, and a reference electrode is provided if necessary, and enzymes and reagents necessary for the measurement are arranged on and / or in the vicinity of the working electrode. . Examples of such sensors include platinum, gold, and carbon by an insulating substrate such as ceramics, glass, plastic, paper, and biodegradable materials (for example, microbial production polyester) by screen printing, vapor deposition, sputtering, etc. Each of the electrodes is formed on the working electrode and / or in the vicinity thereof, and an enzyme and reagent are immobilized by a crosslinking method, a covalent bonding method, an ionic bonding method, or the like.

  In measuring the phosphoric acid concentration, biosensors using off-the-shelf mediators such as glucose oxidase (EC 1.1.3.4.), Glucose dehydrogenase (EC 1.1.1.47, 1.1.1) are used. .118, 1.1.1.119, 1.1.9.10.10) or pyrroloquinoline quinone glucose dehydrogenase and other blood glucose sensors, blood lactate sensors and food glucose sensors using mediators, etc. These are commercially available, for example, ARKRAY product Glutest PRO R (using glucose oxidase and potassium ferricyanide), Glutest Neo (using glucose dehydrogenase and potassium ferricyanide), The, as a blood glucose sensor, The aSense product freestyle (uses pyrroloquinoline quinone glucose dehydrogenase and osmium complex) NOK product glucose meter GM1000 (for food, using glucose oxidase and potassium ferricyanide) or the like can be used as it is. That is, by using a mediator, an electrode, and the like arranged in a commercially available blood glucose sensor, it is possible to easily qualitatively and quantitatively determine phosphate ions. However, in this case, it is necessary to erase glucose in the sample solution in advance using glucose oxidase or glucose dehydrogenase.

  The blood glucose sensor is intended to measure glucose. In general, each electrode made of platinum, gold, carbon, or the like is formed on the insulating substrate as described above, and on the working electrode and / or its electrode. An oxidoreductase such as glucose oxidase and glucose dehydrogenase and a mediator are arranged in the vicinity. The commercially available blood glucose sensor is convenient to carry because the sensor itself is stored in a state where the influence of moisture, light, etc. is eliminated by packaging, and the electrode surface in the sensor's reaction detection part is protected by a reagent layer When it is used, a new feature of the biosensor that it can be measured easily without selecting a place is exhibited. Such a blood glucose sensor has been improved year by year in terms of measurement accuracy and reproducibility due to intensifying development competition in recent years. The blood glucose sensor uses glucose oxidase or glucose dehydrogenase, which is an enzyme with excellent substrate specificity, to selectively measure glucose slightly dissolved in blood (whole blood) rich in contaminants. In this mechanism, the catalyst reaction proceeds, and the electrons generated at that time are transmitted to the electrode via the electron mediator (Mediator). Compared to conventional glucose sensors that measure dissolved oxygen and hydrogen peroxide. It has an extremely excellent feature that it is not affected by dissolved oxygen slightly dissolved in blood, interfering substances, and salt (0.9% NaCl). Furthermore, by using a commercially available blood glucose sensor, unlike the case of using a normal electrode chip for measurement, it is not necessary to treat the electrode surface before use, that is, when applying a normal electrode to a biosensor. , The electrode surface was washed and some kind of treatment was necessary to remove the oxide film, but it was not necessary if using a commercially available blood glucose sensor in recent years. Have both.

  A commercially available blood glucose sensor is composed of a combination of a disposable biosensor (blood glucose sensor) main body and a measuring device that processes an electrical signal from the blood glucose sensor. A portable blood glucose sensor is usually a disposable type, and two or three electrodes patterned with a conductive material are arranged on a flat substrate, and an enzyme is mainly formed on the surface of the electrode constituting the detection portion. And mediators are deployed.

  The blood glucose sensor is connected to a measuring device, and a blood sample is introduced into the detection unit from the sample introduction port of the blood glucose sensor to start an enzyme reaction, and after a predetermined time, electrochemical measurement is performed. Thus, the glucose concentration in the sample solution can be measured. In the case of a commercially available blood glucose sensor, it is only necessary to be able to measure the range of blood glucose concentration in the human body. Therefore, the glucose measurement range in the blood glucose sensor is approximately 2 mM to 30 mM. On the other hand, some blood glucose sensors require only 0.3 μl of sample liquid and others require a measurement time of only 5 seconds.

  If such a blood glucose sensor is applied to the reaction system of the present invention, phosphate ions can be measured while maintaining the same performance as the sensor. Further, if a high-performance or downsized biosensor is commercially available in the future, the biosensor can be effectively applied to phosphate ion detection. By utilizing these features, for example, it will be described later. It is also possible to quickly detect SNPs in such a very limited sample solution. Conversely, the presence of a sufficient substrate such as phosphate ion makes it possible to measure the pyruvate oxidase activity.

  For such a blood glucose sensor, pyruvate oxidase 0.1 to 100 U, preferably 0.2 to 20 U, desalted if necessary, and final concentrations of sodium pyruvate 1 to 5000 mM, preferably 50 to 1000 mM, respectively. Magnesium ion 0.1-100 mM, preferably 1-50 mM, FAD 0-1000 μM, preferably 1-50 μM, TPP 1-5000 μM, preferably pH 7.0, 100 mM sodium citrate buffer solution 5 μl of the reagent mixture solution dissolved in 5 μl of phosphoric acid measurement sample solution is mixed well, then introduced into the blood glucose sensor, reacted exactly 0 to 20 minutes, preferably 1 to 10 minutes, and then controlled by a PC, for example Using an electrochemical analyzer (ALS701, a product of BAS) For example, in the case of a cyclic voltammetry method, a sweep rate of 1 to 500 mV / sec, preferably 10 to 100 mV / sec, an initial potential is used, for example, a cyclic voltammetry method, a potential step chronoamperometry method or a coulometry method 0 V, maximum negative potential −1 to 0 V, preferably −0.5 to 0 V, maximum positive potential +0.1 to +1 V, preferably +0.15 to +0.7 V, reaction (standby) time 0 to 20 minutes, Preferably, it is carried out by confirming the response to the reduced mediator produced by the catalytic reaction of the phosphate ion of pyruvate oxidase in 0 to 10 minutes.

  When measurement is performed using the biosensor as described above, the biosensor is attached to a measurement device, and an electrical value generated in the biosensor is measured. This measuring apparatus includes a measuring unit that measures an electrical value at the electrode of the biosensor and a display unit that displays the measured value. As a measuring method in the measuring unit, as described above, a potential step chronoamperometry method, a coulometry method, a voltammetry method, or the like can be used.

  The apparatus can also be equipped with a memory for storing measured values. Furthermore, when managing the measured values remotely, wireless means for transmitting measurement data to the measurement unit of the biosensor, preferably a non-contact type IC card or short-range wireless communication (for example, Bluetooth; registered trademark), etc. Wireless means can also be installed.

  On the other hand, the phosphate ion concentration contained in environmental water such as fresh water and seawater is extremely low compared to urine (about 20 mM), food (<20 mM), etc., and in the case of an oligotrophic lake, 0.06-0. Even in the case of 6 μM Pi, lake water that has been eutrophied, it is over 20 μM Pi. In this case, it is possible to perform highly sensitive measurement of a measurement target substance by using the following (2) substrate amplification reaction system together with (1) pyruvate oxidase system.

(2) Substrate amplification reaction system:
(I) pyruvate oxidase-phosphatase [acid phosphatase (EC 3.1.3.2.) Or alkaline phosphatase (EC 3.1.3.1.)] System;

  In the above reaction (5), one of the products, acetyl phosphate, is produced by the action of pyruvate oxidase in the presence of Pi and magnesium ions, flavin adenine dinucleotide, thiamine pyrophosphate, pyruvate and oxidized mediator. Then, acetic acid and Pi are produced by the reaction (9). In the substrate amplification reaction system referred to here, the phosphate ion to be measured is phosphorylated into the degradation product of one substrate by the first-stage reaction and then regenerated by the next reaction, and the substrate of the original first-stage reaction It becomes. Since this reaction is repeated, it is also called a cycling reaction. When this reaction system is used, if there is sufficient pyruvic acid as one of the substrates, the amplification reaction can proceed even in the presence of a very small amount of phosphate ions, which enables highly sensitive detection of phosphate ions. It becomes possible. Conversely, the presence of a sufficient substrate other than phosphate ions enables measurement of blood acid phosphatase activity, which is a prostate tumor marker.

On the other hand, pyrophosphate ions can be detected by combining the catalytic reaction of the following formula (10) with the catalytic reaction of the phosphate ion detection system of (1) to (2).
(3) Inorganic pyrophosphatase (EC 3.6.1.1.) System:

  Various nucleic acids (DNA and RNA) can be detected by application to the phosphate ion detection reaction system of the above formula (10). An example is shown below.

上記反応系（１１）では、長鎖側の１本鎖ＤＮＡがＳＮＰの検出対象であり、その塩基配列の中心部分にＳＮＰ（変異型とする；●）が含まれている。その配列に、相補的な２種類の１本鎖ＤＮＡ（プライマー）を左辺に示すように二本鎖形成（ハイブリダイズ）させたものに、連結に必要なエネルギーの供与体であるアデノシン５′−三リン酸（ＡＴＰ）の存在下で、連結酵素として知られるＤＮＡリガーゼを作用させると、その反応は右辺に進む。この場合、２種のプライマーが測定対象の塩基配列上で５′−末端と３′−末端が連結されるには、長鎖側の１本鎖ＤＮＡの５′−末端側に相補的にハイブリダイズするプライマーの５′−末端がリン酸化されている必要がある。この反応により生成したピロリン酸イオンを、上記の方法により測定することで、ＳＮＰの検出ができる。一方、測定対象である長鎖側の１本鎖ＤＮＡが野生型であれば、この場合ＤＮＡリガーゼ反応は進行しない。この方法でＳＮＰを検出する場合、２種のプライマーの内、一方のＳＮＰ部分に相補的な塩基配列、すなわち３′−末端の塩基の種類を変えることによって、上記の方法とは反対に、野生型の場合にＤＮＡリガーゼ反応を進行させることもできる。このＳＮＰ検出反応に使用するＤＮＡリガーゼとしては、Ｔ４ ＤＮＡリガーゼまたは大腸菌リガーゼが好適である。(4) SNP detection reaction system:
(I) DNA ligase (EC 6.5.1.1., 6.5.1.2.) System;

In the reaction system (11), a single-stranded DNA on the long chain side is an SNP detection target, and SNP (which is a mutant type; ●) is included in the central portion of the base sequence. A double-stranded DNA (primer) complementary to the sequence was double-stranded (hybridized) as shown on the left side, and adenosine 5'- which is a donor of energy necessary for ligation When a DNA ligase known as a ligating enzyme is allowed to act in the presence of triphosphate (ATP), the reaction proceeds to the right side. In this case, in order for the two primers to be linked at the 5′-end and the 3′-end on the base sequence to be measured, the primers hybridize complementary to the 5′-end side of the long-side single-stranded DNA. The 5'-end of the soybean primer needs to be phosphorylated. SNP can be detected by measuring pyrophosphate ions generated by this reaction by the above method. On the other hand, if the long-stranded single-stranded DNA to be measured is a wild type, the DNA ligase reaction does not proceed in this case. When detecting SNP by this method, by changing the base sequence complementary to one SNP portion of the two primers, that is, by changing the type of the 3′-terminal base, In the case of a mold, the DNA ligase reaction can be allowed to proceed. As the DNA ligase used for this SNP detection reaction, T4 DNA ligase or E. coli ligase is suitable.

上記１塩基対伸長反応系（１２）においても、長鎖側の１本鎖ＤＮＡがＳＮＰの検出対象であり、その塩基配列の中心部分にＳＮＰ（変異型とする；●）が含まれている。その長鎖の５′−末端からＳＮＰの塩基配列の１塩基手前までの配列に、相補的な１本鎖ＤＮＡプライマーを左辺に示すように長鎖とハイブリダイズさせたものに、基質であるデオキシリボヌクレオチド５′−三リン酸（ｄＮＴＰ）の存在下で、ＤＮＡポリメラーゼを作用させると、その反応は右辺に進む。この反応により生成したピロリン酸イオンを、上記の方法により測定することで、ＳＮＰの検出ができる。一方、測定対象である長鎖側の１本鎖ＤＮＡが野生型であれば、この場合にはＤＮＡポリメラーゼ反応は進行しない。この方法でＳＮＰを検出する場合、プライマーのＳＮＰ部分に相補的な塩基配列、すなわち５′−末端の塩基の種類を変えることによって、上記の方法とは反対に、野生型の場合にＤＮＡポリメラーゼ反応を進行させることもできる。このＳＮＰ検出反応に使用するＤＮＡポリメラーゼとしては、クレノーＤＮＡポリメラーゼ（ＤＮＡポリメラーゼＩラージフラグメント）およびＴ４ ＤＮＡポリメラーゼ、Ｔ７ ＤＮＡポリメラーゼ、好ましくは３’→５’エキソヌクレアーゼ活性を喪失させたクレノーＤＮＡポリメラーゼが好適である。(Ii) DNA polymerase (EC 2.7.7.7.) System A;

Also in the 1 base pair extension reaction system (12), the single-stranded DNA on the long side is the target of SNP detection, and the SNP (mutated type; ●) is included in the center of the base sequence. . To the sequence from the 5′-end of the long chain to the sequence one base before the SNP base sequence, a complementary single-stranded DNA primer was hybridized with the long chain as shown on the left side. When DNA polymerase is allowed to act in the presence of nucleotide 5'-triphosphate (dNTP), the reaction proceeds to the right side. SNP can be detected by measuring pyrophosphate ions generated by this reaction by the above method. On the other hand, if the long-stranded single-stranded DNA to be measured is a wild type, the DNA polymerase reaction does not proceed in this case. When SNP is detected by this method, by changing the base sequence complementary to the SNP part of the primer, that is, the kind of the 5′-terminal base, the DNA polymerase reaction is performed in the wild type, as opposed to the above method. Can also be advanced. As the DNA polymerase used in this SNP detection reaction, Klenow DNA polymerase (DNA polymerase I large fragment) and T4 DNA polymerase, T7 DNA polymerase, and preferably Klenow DNA polymerase having lost 3 ′ → 5 ′ exonuclease activity are suitable. It is.

上記多塩基対伸長反応系（１３）において、長鎖側の１本鎖ＤＮＡがＳＮＰの検出対象であり、その塩基配列の中心部分にＳＮＰ（変異型とする；●）が含まれている。その長鎖の５′−末端からＳＮＰの塩基配列までが相補的なＤＮＡプライマーを左辺に示すように長鎖とハイブリダイズさせたものに、基質であるｄＮＴＰｓの存在下でＤＮＡポリメラーゼを作用させると、その反応は右辺に進む。この反応により生成したピロリン酸イオンを上記の方法により測定することで、ＳＮＰの検出ができる。一方、測定対象である長鎖側の１本鎖ＤＮＡが野生型であれば、この場合はＤＮＡポリメラーゼ反応は進行しない。この方法でＳＮＰを検出する場合、プライマーのＳＮＰ部分に相補的な塩基配列、すなわち５′−末端の塩基の種類を変えることによって、上記の方法とは反対に、野生型の場合にＤＮＡポリメラーゼ反応を進行させることもできる。この反応系の場合、測定対象である長鎖側の１本鎖ＤＮＡのＳＮＰ部位から３′−末端の間の塩基配列を長くすることで、反応系（１２）よりも少ない量の測定対象ＤＮＡから、多くのピロリン酸イオンを生成することができるという特徴がある。このＳＮＰ検出反応に使用するＤＮＡポリメラーゼとしては、反応系（１２）の場合と同様にクレノーＤＮＡポリメラーゼおよびＴ４ ＤＮＡポリメラーゼ、Ｔ７ ＤＮＡポリメラーゼ、好ましくは３’→５’エキソヌクレアーゼ活性を除去してあるクレノーＤＮＡポリメラーゼが好適である。(Iii) DNA polymerase system B;

In the multi-base pair extension reaction system (13), a single-stranded DNA on the long chain side is an SNP detection target, and SNP (mutated type; ●) is included at the center of the base sequence. When a DNA primer that is complementary from the 5'-end of the long chain to the base sequence of SNP is hybridized with the long chain as shown on the left side, DNA polymerase is allowed to act in the presence of dNTPs as a substrate. The reaction goes to the right side. SNP can be detected by measuring pyrophosphate ions generated by this reaction by the above method. On the other hand, if the long-stranded single-stranded DNA to be measured is a wild type, the DNA polymerase reaction does not proceed in this case. When SNP is detected by this method, by changing the base sequence complementary to the SNP part of the primer, that is, the kind of the 5′-terminal base, the DNA polymerase reaction is performed in the wild type, as opposed to the above method. Can also be advanced. In the case of this reaction system, the amount of DNA to be measured is smaller than that in the reaction system (12) by lengthening the base sequence between the SNP site of the long-side single-stranded DNA to be measured and the 3′-end. Therefore, a feature is that many pyrophosphate ions can be generated. The DNA polymerase used for this SNP detection reaction is the same as in the reaction system (12), Klenow DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, preferably Klenow from which 3 ′ → 5 ′ exonuclease activity has been removed. DNA polymerase is preferred.

上記多塩基対伸長反応系（１４）において、長鎖側の１本鎖ＤＮＡがＳＮＰの検出対象であり、その塩基配列の中心部分にＳＮＰ（変異型とする；●）が含まれている。その長鎖の５′−末端からＳＮＰの塩基配列までが相補的で、続く１塩基または２塩基が長鎖側の塩基配列に対して相補的でないＤＮＡプライマーを左辺に示すように長鎖とハイブリダイズさせたものに、基質であるｄＮＴＰｓの存在下でＤＮＡポリメラーゼを作用させると、その反応は右辺に進む。この反応により生成したピロリン酸イオンを上記の方法により測定することで、ＳＮＰの検出ができる。一方、測定対象である長鎖側の１本鎖ＤＮＡが野生型であれば、この場合はＤＮＡポリメラーゼ反応は進行しない。この方法でＳＮＰを検出する場合、プライマーのＳＮＰ部分に相補的な塩基配列、すなわち５′−末端の塩基の種類を変えることによって、上記の方法とは反対に、野生型の場合にＤＮＡポリメラーゼ反応を進行させることもできる。この反応系の場合、測定対象である長鎖側の１本鎖ＤＮＡのＳＮＰ部位から３′−末端の間の塩基配列を長くすることで、反応系（１２）よりも少ない量の測定対象ＤＮＡから、多くのピロリン酸イオンを生成することができるという特徴がある。このＳＮＰ検出反応に使用するＤＮＡポリメラーゼとしては、反応系（１２）の場合と同様にクレノーＤＮＡポリメラーゼおよびＴ４ ＤＮＡポリメラーゼ、Ｔ７ ＤＮＡポリメラーゼ、好ましくはエンドヌクレアーゼ活性および３’→５’エキソヌクレアーゼ活性を除去してあるクレノーＤＮＡポリメラーゼが好適である。(Iv) DNA polymerase system C;

In the multi-base pair extension reaction system (14), a single-stranded DNA on the long chain side is an SNP detection target, and SNP (mutated type; ●) is included at the center of the base sequence. A DNA primer that is complementary from the 5'-end of the long chain to the base sequence of the SNP, and whose subsequent one or two bases are not complementary to the base sequence on the long chain side, hybridizes with the long chain as shown on the left side. When a DNA polymerase is allowed to act on soybeans in the presence of the substrate dNTPs, the reaction proceeds to the right side. SNP can be detected by measuring pyrophosphate ions generated by this reaction by the above method. On the other hand, if the long-stranded single-stranded DNA to be measured is a wild type, the DNA polymerase reaction does not proceed in this case. When SNP is detected by this method, by changing the base sequence complementary to the SNP part of the primer, that is, the kind of the 5′-terminal base, the DNA polymerase reaction is performed in the wild type, as opposed to the above method. Can also be advanced. In the case of this reaction system, the amount of DNA to be measured is smaller than that in the reaction system (12) by lengthening the base sequence between the SNP site of the long-side single-stranded DNA to be measured and the 3′-end. Therefore, a feature is that many pyrophosphate ions can be generated. As the DNA polymerase used for this SNP detection reaction, the Klenow DNA polymerase, T4 DNA polymerase, and T7 DNA polymerase, preferably the endonuclease activity and 3 ′ → 5 ′ exonuclease activity are removed as in the reaction system (12). The Klenow DNA polymerase is preferred.

上記反応系（１５）、（１６）は、１本鎖のゲノムを持つウィルスや細菌などの検出に好適である。反応式（１５）は、ランダムな塩基配列からなる複数のプライマーを測定対象である一本鎖ＤＮＡとハイブリダイズさせる反応である。反応式（１６）では、測定対象の１本鎖ゲノムと部分的にハイブリダイズしたプライマー同士をＤＮＡポリメラーゼによって連結することで、完全な２本鎖ＤＮＡが合成される。このとき、ｄＮＴＰｓがＤＮＡ鎖の構成材料として取り込まれ、ピロリン酸イオンが生成される。この反応系で使用するＤＮＡポリメラーゼとしては、大腸菌由来のＤＮＡポリメラーゼＩまたはクレノーＤＮＡポリメラーゼ、Ｔ４ ＤＮＡポリメラーゼ、Ｔ７ ＤＮＡポリメラーゼ、好ましくはクレノーＤＮＡポリメラーゼなどが好適である。さらに、この方法では、ゲノムに１本鎖ＲＮＡをもつウィルスなどの検出も逆転写酵素（ＲＮＡ依存性ＤＮＡポリメラーゼ）とＤＮＡのランダムプライマー、ｄＮＴＰｓの使用、またはＲＮＡポリメラーゼ（ＥＣ２．７．７．６．）、ＲＮＡのランダムプライマー、ＮＴＰｓの使用により可能である。(5) Single-stranded genome detection reaction system:
DNA polymerase system (random primer method);

The reaction systems (15) and (16) are suitable for detecting viruses and bacteria having a single-stranded genome. Reaction formula (15) is a reaction in which a plurality of primers having a random base sequence are hybridized with the single-stranded DNA to be measured. In reaction formula (16), complete double-stranded DNA is synthesized by ligating primers partially hybridized with the single-stranded genome to be measured with a DNA polymerase. At this time, dNTPs are incorporated as a constituent material of the DNA chain, and pyrophosphate ions are generated. As the DNA polymerase used in this reaction system, DNA polymerase I derived from Escherichia coli or Klenow DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, preferably Klenow DNA polymerase is suitable. Further, in this method, detection of viruses having single-stranded RNA in the genome is also performed using reverse transcriptase (RNA-dependent DNA polymerase) and random primers for DNA, dNTPs, or RNA polymerase (EC 2.7.7.6). )), Random RNA primers, and NTPs.

上記の反応系（１７）〜（１９）は、ゲノムに二本鎖ＤＮＡを持つウィルスや細菌などの検出に好適である。反応式（１７）では、デオキシリボヌクレアーゼ（ＤＮアーゼ）により、二本鎖ＤＮＡ上に無数の切れ目（ニック）が入る。この場合、ＤＮアーゼＩを使用することにより、二本鎖ＤＮＡの１本鎖部分のみにニックが入るため、二本鎖が切断されることはない。続いて、反応式（１８）では、二本鎖ＤＮＡ上に入ったニックをＤＮＡポリメラーゼが認識し、ＤＮＡポリメラーゼが持つエキソヌクレアーゼ活性によって、ニックの周囲の数十塩基を削除する。さらに、反応式（１９）では、同酵素のポリメラーゼ活性によって、二本鎖ＤＮＡは元の状態に修復される。この場合のＤＮアーゼとしてはＤＮアーゼＩ（ＥＣ３．１．２１．１）が、またＤＮＡポリメラーゼとしては大腸菌由来のＤＮＡポリメラーゼＩが好適である。この反応系では、例えば１個のウィルス由来のゲノムでも存在すれば、その反応系の一方の基質であるｄＮＴＰｓが消費されるまでこの反応は進むため、原理的にウィルスの検出は可能である。さらに、二本鎖ゲノムは熱変性またはアルカリ変性などにより、１本鎖を形成させることで、前記ランダムプライマー法による検出も可能である。この方法によればゲノムに二本鎖ＲＮＡをもつウィルスなどの検出も可能である。(6) Double-stranded genome detection reaction system:
DNA polymerase-DNase system (nick translation method);

The reaction systems (17) to (19) are suitable for detecting viruses or bacteria having double-stranded DNA in the genome. In the reaction formula (17), countless nicks are formed on the double-stranded DNA by deoxyribonuclease (DNase). In this case, by using DNase I, a nick is introduced only in the single-stranded part of the double-stranded DNA, so that the double strand is not cleaved. Subsequently, in reaction formula (18), the DNA polymerase recognizes the nick that has entered the double-stranded DNA, and tens of bases around the nick are deleted by the exonuclease activity of the DNA polymerase. Furthermore, in the reaction formula (19), the double-stranded DNA is restored to the original state by the polymerase activity of the enzyme. In this case, DNase I (EC 3.1.21.1) is preferable as the DNase, and DNA polymerase I derived from E. coli is preferable as the DNA polymerase. In this reaction system, for example, if a genome derived from a single virus is present, this reaction proceeds until dNTPs, which is one substrate of the reaction system, is consumed, and thus the virus can be detected in principle. Furthermore, the double-stranded genome can be detected by the random primer method by forming a single strand by heat denaturation or alkali denaturation. According to this method, it is possible to detect viruses having double-stranded RNA in the genome.

  The phosphate ion reaction system, the pyrophosphate ion reaction system, and these reaction systems can be applied to the biosensor of the present invention as a series of biologically related reactions involving phosphate ions such as nucleic acids and pyrophosphate ions. Is possible. In addition to the above-described application examples, nucleic acid sequencing (such as pyrosequencing), nucleic acid amplification reaction (PCR), RAC (rolling cycle amplification), SDA (Standard displacement amplification), NASBA (nucleic acid sequencing) For example) and receptor behavior (such as the behavior of G protein that consumes GTP to produce pyrophosphate ions), and other applications such as the measurement of the activity of enzymes that involve phosphate and pyrophosphate ions in catalytic reactions. Is possible. In this case, if the enzyme used is crudely purified or dissolved in a suspension containing phosphate, pretreatment such as desalting by ultrafiltration and purification by column chromatography should be performed. Is desirable. In particular, in an amplification reaction system using phosphate ions as a substrate cycle, it is desirable that no phosphate is present in the reaction detection portion of a commercially available blood glucose sensor.

  In the method (2) of the present invention, an enzyme-catalyzed reaction is caused by the presence of a phosphate ion, and a redox represented by a substrate and an enzyme, an oxidized mediator, and glucose oxidase that finally produce β-D-glucose. Phosphate ions in the sample solution can also be measured by generating a reduced mediator from phosphoric acid using an enzyme, further oxidizing it, and measuring the resulting oxidation current. If glucose is present in the sample solution, it is necessary to erase glucose in the sample solution in advance using glucose oxidase or the like.

（ｉｉ）スクロースホスホリラーゼ（ＥＣ２．４．１．７．）−グルコースイソメラーゼ（ＥＣ５．３．１．１８．）系；

Examples of the catalytic reaction system group that finally generates β-D-glucose include the following.
(7) Simple reaction system:
(I) Maltose phosphorylase (EC 2.4.1.8.)-Mutarotase (EC 5.1.3.3.) System;

(Ii) the sucrose phosphorylase (EC 2.41.7.)-Glucose isomerase (EC 5.3.1.18.) System;

β-D-glucose generates a reduced mediator by the following reaction or the like.

この際発生したフェロシアンイオンは、作用極で酸化されて酸化電流を生ずる。

Examples of the oxidoreductase include glucose oxidase (EC 1.1.3.4.) And glucose dehydrogenase (EC 1.1.1.147, 1.1.1.118, 1.1.1.119, 1.1). 99.10.), Pyrroloquinoline quinone, glucose dehydrogenase and the like are used, and as the mediator, potassium ferricyanide, ferrocene, parabenzoquinone and the like are used. When potassium ferricyanide K ₃ Fe (CN) ₆ is used as a mediator, the reaction with glucose or the like proceeds as follows.

The ferrocyan ion generated at this time is oxidized at the working electrode to generate an oxidation current.

In addition, when parabenzoquinone is used instead of potassium ferricyanide as a mediator, hydroquinone is produced by the reaction of glucose and parabenzoquinone in the presence of GOD, and the hydroquinone produced at this time is oxidized at the working electrode, resulting in an oxidation current. This value is measured.

β-D-glucose is oxidized in the presence of an enzyme by the action of glucose oxidase and the like to produce gluconolactone, H ₂ O ₂ generated at that time is oxidized on the working electrode, and the oxidation current value at that time is measured Thus, the method for indirectly determining the glucose concentration is as described in the prior art. However, according to such a method, when the measurement solution contains a disturbing substance such as seawater, it is not possible to measure an accurate phosphoric acid concentration. However, when the mediator is used together with glucose oxidase or the like, the phosphate concentration can be accurately measured without being affected by the interfering substance.

  For the measurement of phosphate ions, a sensor in which a working electrode and a counter electrode are provided on a substrate, and a reference electrode as necessary, and enzymes and reagents necessary for the measurement are arranged on the working electrode is preferably used. As such a sensor, those described above are used.

  In measuring the phosphoric acid concentration, a ready-made blood glucose sensor or a glucose sensor for foods can be used, and commercially available ones can be used as they are. For example, as the blood glucose sensor and glucose sensor, commercially available products as described above can be used as they are.

  Similarly to the method (1) of the present invention, in the method (2) of the present invention, a blood glucose sensor is connected to a measuring device, and a blood sample or the like is introduced from the sample inlet of the blood glucose sensor into the detection unit, thereby By measuring electrochemically after the reaction has started and a predetermined time has elapsed, the glucose concentration in the sample solution can be measured. If such a blood glucose sensor is applied to the above (7) simple reaction system of the method (2) of the present invention, phosphate ions in the same concentration range as this sensor can be measured. Moreover, if it exists in such a measurement range, it can be used for the measurement of the phosphate ion contained in blood, urine, foodstuffs, etc.

  In such a blood glucose sensor, 0.01 to 100 U, preferably 0.1 to 20 U, of maltose phosphorylase and mutarotase or sucrose phosphorylase and glucose isomerase, respectively desalted as necessary, are dissolved in a buffer solution. And a phosphoric acid measurement sample solution and 25 nmole or more, preferably 125 nmole to 5 μmole of a substrate solution other than phosphoric acid, that is, maltose or scroll solution, are thoroughly mixed and then introduced into the blood glucose sensor for exactly 0 to 20 minutes, preferably 1 After reacting for 10 minutes, for example, using an electrochemical analyzer (BAS product ALS701) controlled by a PC, electrochemical measurement methods such as cyclic voltammetry, potential step chronoamperometry, coulometry For example, in the case of cyclic voltammetry, the sweep rate is 1 to 500 mV / sec, preferably 10 to 100 mV / sec, the initial potential is 0 V, the maximum negative potential is −1 to 0 V, preferably −0.5 to 0 V, The maximum positive potential is +0.1 to +1 V, preferably +0.15 to +0.7 V, and the reaction (standby) time is 0 to 20 minutes, preferably 0 to 10 minutes, and the reaction to glucose is confirmed.

  When measurement is performed using the biosensor as described above, the biosensor is attached to the measurement device, the electrical value generated in the biosensor is measured, the details of the measurement device used and the measurement method are described above. It is as follows.

  On the other hand, the concentration of phosphate ions contained in environmental water such as fresh water and seawater is extremely low compared to food (<20 mM), urine (about 20 mM), etc., and in the case of an oligotrophic lake, 0.06-0. Even in the case of 6 μM Pi, lake water that has been eutrophied, it is over 20 μM Pi. In this case, instead of the (7) simple reaction system, the following (8) substrate amplification reaction system and (9) complex reaction system can be used to enable highly sensitive measurement of the measurement target substance.

(8) Substrate amplification reaction system:
(I) Maltose phosphorylase-phosphatase [acid phosphatase (EC 3.1.3.2.) Or alkaline phosphatase (EC 3.1.3.1.)] System;

  In the above reaction (20), β-D-glucose-1-phosphate, which is one of the products, is produced by the action of maltose phosphorylase in the presence of Pi and maltose. From this, β-D-glucose-1-phosphate is produced by reaction (29). D-glucose and Pi are produced. In the substrate amplification reaction system referred to here, the phosphate ion to be measured is phosphorylated into the degradation product of one substrate by the first-stage reaction and then regenerated by the next reaction, and the substrate of the original first-stage reaction It becomes. Since this reaction is repeated, it is also called a substrate recycling reaction. When this reaction system is used, if there is sufficient maltose as one of the substrates, the amplification reaction can proceed even in the presence of a very small amount of phosphate ions, enabling highly sensitive detection of phosphate ions. It becomes. Conversely, the presence of a substrate other than phosphate ions at a concentration of 10 mM or more, preferably 50 to 2000 mM, enables measurement of blood acid phosphatase activity, which is a tumor marker for prostate.

上記反応も、一段目のスクロースホスホリラーゼ反応とそれに続くホスファターゼ反応との組み合わせによって、（ｉ）のマルトースホスホリラーゼ−ホスファターゼ系と同様の効果を得ることができる。(Ii) a sucrose phosphorylase-phosphatase (acid phosphatase or alkaline phosphatase) -mutarotase system;

In the above reaction, the same effect as the maltose phosphorylase-phosphatase system of (i) can be obtained by a combination of the first-stage sucrose phosphorylase reaction and the subsequent phosphatase reaction.

（ｉｉ）スクロースホスホリラーゼ−ホスファターゼ（酸性ホスファターゼまたはアルカリ性ホスファターゼ）−ムタロターゼ−グルコースイソメラーゼ系；

上記（９）複合反応系における２種（ｉ）〜（ｉｉ）の反応系は（７）単純反応系と（８）基質増幅反応系を組み合わせたもので、基質増幅反応系よりも高感度なリン酸イオンの検出が可能である。(9) Complex reaction system (i) Maltose phosphorylase-phosphatase (acid phosphatase or alkaline phosphatase) -mutarotase system;

(Ii) a sucrose phosphorylase-phosphatase (acid phosphatase or alkaline phosphatase) -mutarotase-glucose isomerase system;

The two types (i) to (ii) of the reaction system (9) are combined with (7) a simple reaction system and (8) a substrate amplification reaction system, and are more sensitive than the substrate amplification reaction system. Phosphate ions can be detected.

On the other hand, pyrophosphate ions can be detected by combining the catalytic reactions of the phosphate ion detection systems (7) to (9) with the catalytic reaction of the following formula (10).
(10) Inorganic pyrophosphatase (EC 3.6.1.1.) System:

  Further, (10) inorganic pyrophosphatase system is changed to phosphate ion detection reaction system such as (4) SNP detection reaction system, (5) single-stranded genome detection reaction system and (6) double-stranded genome detection reaction system described above. By applying it, various nucleic acids (DNA and RNA) can be detected.

  Next, the method of the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples unless it exceeds the gist.

Reference example 1
Two platinum foils with a width of 1 mm are arranged in parallel at an interval of 0.5 mm on an insulating plastic substrate (7 mm × 22 mm), and glucose oxidase and potassium ferricyanide are disposed on the working electrode not containing phosphate. A sample solution containing glucose (pH 7.0, 100 mM sodium citrate buffer solution) is allowed to reach the working electrode by capillary action, and an electrical signal for glucose in the sample solution is transmitted to the biosensor in which the biosensor is disposed. It detected using the electrochemical analyzer (ALS701 by BAS) controlled by PC through the connector for exclusive use connected to the terminal. Cyclic voltammetry was used as the electrochemical measurement method, and the response to glucose was confirmed with a sweep rate of 50 mV / sec, an initial potential of 0 V, a maximum potential of +0.7 V, and a reaction (standby) time of 60 seconds. As a result, a peak of the current value peculiar to the mediator appeared in the vicinity of +0.2 V in the range of 2 mM to 100 mM glucose. As shown in FIG. 1, the peak value is almost proportional to the glucose concentration, and it was found that this biosensor has a wide measurement range for glucose and is suitable for the implementation according to the present invention.

[Example 1]
Using the biosensor used in Reference Example 1, (1) phosphate ions were measured using a pyruvate oxidase system. 200 units of pyruvate oxidase (from Aerococcus viridans) was dissolved in 250 μl of reaction reagent solution I in ice-cooling, that is, pH 7.0, 100 mM sodium citrate buffer solution containing 40 mM magnesium ions and 48 μM FAD. As reaction reagent solution II, 800 mM sodium pyruvate and 0.96 mM TPP dissolved in 250 μl of pH 7.0, 100 mM sodium citrate buffer solution in ice-cooling were used. As a measurement sample solution, a pH 7.0, 100 mM sodium citrate buffer solution containing 400 mM potassium dihydrogen phosphate was prepared and used. The measurement was performed by introducing 12.5 μL of a reaction reagent solution containing 2 units of pyruvate oxidase, 2.5 μL of a reaction reagent solution II, and 5 μl of a measurement sample solution into a blood glucose sensor and reacting them accurately for 3 minutes. The phosphate ion was measured by the cyclic voltammetry method under the same conditions as the measurement of glucose.

Comparative Example 1
In Example 1, TPP was not used.

Comparative Example 2
In Example 1, FAD was not used.

Comparative Example 3
In Example 1, no magnesium ion was used.

Comparative Example 4
In Example 1, a pH 7.0, 100 mM sodium citrate buffer solution not containing 100 mM potassium dihydrogen phosphate was used as a measurement sample solution.

  The results obtained in Example 1 and Comparative Examples 1 to 4 are shown in FIG. Each numerical value represents an average value of n = 3. The response of this biosensor to phosphate ions was not obtained except for the case where FAD was not included as a component of the reaction reagent solution. On the other hand, when FAD was excluded, the response was about half of the response to phosphate ions obtained with the usual reaction reagent composition. This is presumably because pyruvate oxidase originally has FAD, but the pyruvate oxidase used contained those from which FAD was released during the purification process. From the above, it was confirmed that magnesium ion, FAD, and TPP are required in the pyruvate oxidase system.

[Example 2]
Using the biosensor used in Reference Example 1, the relationship between TPP concentration and sensor response in (1) pyruvate oxidase system was examined. Reaction reagent solution I containing 100 units of pyruvate oxidase (from Aerococcus viridans), 20 mM magnesium ions and 24 μM FAD in 250 μl of 100 mM sodium citrate buffer solution at pH 7.0 in ice-cooling, The reaction reagent solution II containing 400 mM potassium dihydrogen phosphate, 400 mM sodium pyruvate, and a predetermined concentration of TPP is thoroughly mixed with 250 μl of pH 7.0, 100 mM sodium citrate buffer solution (2.5 μl). Was introduced into a blood glucose sensor and allowed to react accurately for 10 minutes, and then the current value was measured by the cyclic voltammetry method under the same conditions as when measuring glucose.

  The results obtained are shown in FIG. Each numerical value represents an average value of n = 3. The biosensor response to TPP was obtained in the range of 48-240 μM. From the above, it was shown that when the concentration of TPP is lower than 48 μM, the response of the present biosensor to phosphate ions is significantly reduced.

[Example 3]
Using the biosensor used in Reference Example 1, (1) the relationship between pyruvate concentration and sensor response in the pyruvate oxidase system was examined. Reaction reagent solution I used in Example 2, pH 7.0 in ice-cooling, 250 μl of 100 mM sodium citrate buffer solution, 400 mM potassium dihydrogen phosphate, 0.48 mM TPP, and sodium pyruvate at a predetermined concentration were added. Each reaction reagent solution II containing 2.5 μl was sufficiently mixed, this mixture solution was introduced into a blood glucose sensor, reacted for exactly 10 minutes, and then subjected to a current by cyclic voltammetry under the same conditions as when measuring glucose. A value measurement was made.

  The results obtained are shown in FIG. Each numerical value represents an average value of n = 3. The response of this biosensor to pyruvate was obtained in the range of 4 to 400 mM. From the above, it was shown that the present sensor can be applied to the measurement of pyruvic acid in the range of 4 to 400 mM.

[Example 4]
Using the biosensor used in the reference example, (1) the relationship between pyruvate oxidase activity and sensor response in the pyruvate oxidase system was examined. Reaction reagent solution I containing a predetermined unit of pyruvate oxidase (derived from Aerococcus viridans), 20 mM magnesium ions and 24 μM FAD in 250 μl of 100 mM sodium citrate buffer solution at pH 7.0 in ice cooling, In a pH 7.0, 100 mM sodium citrate buffer solution (250 μl), 400 μm potassium dihydrogen phosphate, 400 mM sodium pyruvate and 0.48 mM TPP reaction reagent solution II were mixed thoroughly and 2.5 μl each. After being introduced into the sensor and allowed to react for exactly 10 minutes, the current value was measured by the cyclic voltammetry method under the same conditions as when measuring glucose.

  The results obtained are shown in FIG. Each numerical value represents an average value of n = 3. The response of this biosensor to pyruvate oxidase activity was highly correlated (r = 0.9991) in the range of 6 to 200 munits / μl. From the above, it was shown that the present sensor can be applied to the measurement of pyruvate oxidase activity in the range of 6 to 200 m units / μl.

[Example 5]
Using the biosensor used in Reference Example 1, (1) the relationship between phosphate ions and sensor response in the pyruvate oxidase system was examined. The reaction reagent solution I and reaction reagent solution II used in Example 1 were used, and a pH 7.0, 100 mM sodium citrate buffer solution containing potassium dihydrogen phosphate having a predetermined concentration was prepared as a measurement sample solution. Used. The measurement was performed by introducing a mixture of reaction reagent solution I2.5 μL containing 2 units of pyruvate oxidase, reaction reagent solution II 2.5 μL, and measurement sample solution 5 μl into a blood glucose sensor and reacting accurately for 10 minutes. The current value was measured by the cyclic voltammetry method under the same conditions as when measuring glucose.

  The results obtained are shown in FIG. Each numerical value represents an average value of n = 3. The response of this biosensor to phosphate ions was obtained in the range of about 1 mM to 100 mM, and was confirmed to be suitable for the measurement of phosphate contained in foods, urine and the like.

[Example 6]
Using the biosensor used in Reference Example 1, (1) a phosphate ion dissolved in environmental water was measured using a pyruvate oxidase reaction system. The reaction reagent solution I and reaction reagent solution II used in Example 1 were used, and the measurement sample solution was placed in environmental water so that the concentration of potassium dihydrogen phosphate to be added was 5, 10, 15, 20 mM. The dissolved one was used. Environmental water was collected from Japanese gardens, cascades, and reservoirs on the Tokyo University of Technology site. The pH of the environmental water is 7.65, 7.64, 7.93, and the phosphate ion concentrations are 0.073 μM, 0.67 μM, and 5.4 μM, respectively (according to JIS K 0102: molybdenum blue absorption by ascorbic acid reduction) Photometric method). In the biosensor method of the present invention, first, a sensor response to a pH 7.0, 100 mM sodium citrate buffer solution and a solution to which 25 mM phosphate ions are added is obtained as a calibration curve value, and then phosphate ions are added. The response of each environmental water was applied to the calibration curve to determine the phosphate ion concentration in each environmental water. Similarly from the molybdenum blue absorptiometric method by ascorbic acid reduction, the phosphate ion concentration in each environmental water was calculated | required, and the value was made into the value calculated | required by the conventional method.

  The results obtained are shown in FIGS. 7a, b, c. Each numerical value represents an average value of n = 3. The average variation coefficient of biosensor response at this time was 7.6% for Japanese gardens, 9.1% for cascades, and 8.8% for Tameike. In the order of the Japanese garden a, the cascade b, and the basin c, the environmental water with a higher concentration of phosphate ions contained a higher sensor response value than the conventional method. As this cause, it can be considered that the environmental water having a higher concentration of phosphate ions contains more impurities and interacts with the phosphate ions. In other words, the conventional method requires the solution to be prepared to be strongly acidic in order to form a phosphorus-molybdenum complex, so that phosphate ions are less susceptible to interaction with contaminants, but pyruvate oxidase can catalyze. Since it is only phosphate ions that are truly in a dissolved state, it is considered that this is connected to such a result.

[Example 7]
Using the biosensor used in Reference Example 1, the relationship between the pH and the sensor response in (2) the amplification reaction system (i) pyruvate oxidase-acid phosphatase system was examined. 250 μl of an ice-cooled buffer solution having a predetermined pH, 100 units of pyruvate oxidase (from Aerococcus viridans), a reaction reagent solution I containing 40 mM magnesium ions and 48 μM FAD, and 250 μl of an ice-cooled buffer solution having a predetermined pH value. Reaction reagent II containing 400 mM potassium dihydrogen phosphate, 400 mM sodium pyruvate and 0.48 mM TPP, reaction reagent containing 20 units of acid phosphatase (derived from potato) in 50 μl of an ice-cooled buffer solution having a predetermined pH Liquid III was mixed thoroughly at 2.5 μl, 5 μl, and 2.5 μl, respectively, introduced into the blood glucose sensor, allowed to react accurately for 60 seconds, and then subjected to cyclic voltammetry under the same conditions as when measuring glucose. The current value was measured.

  The results obtained are shown in FIG. Each numerical value represents an average value of n = 3. As shown in this figure, it was found that the response to phosphate ions was the highest near the optimum pH (pH 7.0) in pyruvate oxidase activity than on the acid side where the optimum pH in acid phosphatase activity was present. In addition, from this result, compared with (1) simple reaction system of pyruvate oxidase alone, (2) the response to phosphate ions is much better in the pyruvate oxidase-acid phosphatase system which is a substrate amplification reaction system. It was shown to be.

Reference example 2
Two platinum foils with a width of 1 mm are arranged in parallel at an interval of 0.5 mm on an insulating plastic substrate (7 mm × 22 mm), and glucose oxidase and potassium ferricyanide are disposed on the working electrode not containing phosphate. Is placed on the working electrode by capillary action with pH 6.5 containing 20 mM sodium citrate buffer solution containing a predetermined concentration of glucose, and an electrical signal for glucose in the sample solution is Detection was performed using an electrochemical analyzer (ALS701) controlled by a PC via a dedicated connector connected to the sensor terminal. Cyclic voltammetry was used as the electrochemical measurement method, and the response to glucose was confirmed with a sweep rate of 50 mV / sec, an initial potential of 0 V, a maximum potential of +0.7 V, and a reaction (standby) time of 60 seconds. As a result, a peak of the current value peculiar to the mediator appeared in the vicinity of +0.2 V in the range of 2 mM to 100 mM glucose. The peak value is almost proportional to the glucose concentration as shown in FIG. 9, and it was found that this biosensor has a wide measurement range for glucose and is suitable for the implementation according to the present invention.

[Example 8]
The (7) simple reaction system (i) maltose phosphorylase-mutarotase system was applied to the biosensor used in Reference Example 2, and the response to the phosphate ion concentration was examined. Maltose phosphorylase (derived from Lactobacillus brevis), mutarotase (derived from porcine kidney) and glucose oxidase (derived from Aspergillus niger) were desalted using an ultrafiltration filter with a molecular weight cut off of 10,000, and then ice-cooled. Was dissolved in a 20 mM sodium citrate buffer solution at pH 6.5 and 0.4 unit / μl. Although glucose oxidase is pre-arranged in the biosensor as described in Reference Example 2, since the biosensor used varies from lot to lot, this example is used to ensure reproducibility. It was decided to use it after that. The substrate solution is a pH 6.5, 20 mM sodium citrate buffer solution containing 400 mM maltose, and the measurement sample solution is a pH 6.5, 20 mM sodium citrate buffer solution containing a predetermined concentration of potassium dihydrogen phosphate. Used. Next, after thoroughly mixing 2.5 μl of each enzyme-containing solution, 2.5 μl of the substrate solution and 5 μl of the sample solution for measurement into the biosensor and reacting accurately for 3 minutes, The current value was measured by cyclic voltammetry under the same conditions.

  The results obtained are shown in FIG. Each numerical value represents an average value of n = 3. The response of this biosensor to phosphate ions was obtained in the range of about 0.2 mM to 20 mM, and was confirmed to be suitable for the measurement of phosphate contained in food, urine and the like.

[Example 9]
In Example 8, acid phosphatase (derived from potato) was used in the same amount in place of mutarotase, and the reaction time was changed to 10 minutes to measure the current value. In (8) the substrate amplification reaction system, (I) The response to the phosphate ion concentration in the maltose phosphorylase-acid phosphatase system was examined. The reason why acid phosphatase was used instead of alkaline phosphatase is that glucose oxidase is optimal under weak acid conditions.

  The obtained results are shown in FIG. Each numerical value represents an average value of n = 3. The response of this biosensor to phosphate ions can be obtained in the range of 20 μM to 2 mM, which is about 10 times that of the simple reaction system shown in Example 8. It was confirmed that phosphate ions could be quantified with this sensor.

Comparative Example 5
In Example 9, acid phosphatase was not used. As a result, the response of this biosensor to various concentrations of phosphate ions was about 15 μA. Since this value is equal to the sensor response value in the absence of phosphate ions, it was confirmed that the glucose oxidase reaction does not proceed with maltose phosphorylase alone even if a sufficient amount of substrate is present for the reaction.

[Example 10]
The (9) complex reaction system (i) maltose phosphorylase-acid phosphatase-mutarotase system was applied to a blood glucose sensor, and the relationship between pH and sensor response was examined with a constant phosphate ion concentration. The maltose phosphorylase, mutarotase, glucose oxidase used in Example 8 and the acid phosphatase used in Example 9 were desalted using an ultrafiltration filter having a molecular weight cut off of 10,000, and then ice-cooled. Was dissolved in a 20 mM sodium citrate buffer solution at pH 6.5 and 0.4 unit / μl. The substrate solution is a pH 6.5, 20 mM sodium citrate buffer solution containing 400 mM maltose, the measurement sample solution is a pH 4.0 phthalate buffer solution containing 1 mM potassium dihydrogen phosphate, and each pH is 5 0.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 0.2M Tris-maleic acid buffer solutions were used, respectively. Next, 2.5 μl of each enzyme-containing solution, 2.5 μl of the substrate solution and 5 μl of the sample solution to be mixed are introduced into the biosensor, reacted for 10 minutes, and then subjected to cyclic voltammetry to determine the current value. Measurements were made.

  The results obtained are shown in FIG. Each numerical value represents an average value of n = 3. Here, the value shown on the horizontal axis is the pH in the reaction detection part of the blood glucose sensor in a state where the solution containing each enzyme, the maltose solution, and the measurement sample solution are mixed. Thereby, when the influence of pH was investigated, it was shown that the optimal pH in this composite reaction system is around 6.5. In addition, the sensor response was remarkably lowered when the pH was around 8. This is considered due to a decrease in the activity of acid phosphatase that performs the amplification reaction.

[Example 11]
In Example 10, a pH 6.5, 20 mM sodium citrate buffer solution containing 2 mM potassium dihydrogen phosphate was used as a measurement sample solution, and the current value when reacted for a predetermined time was measured. The relationship between reaction time and sensor response was investigated.

  The obtained results are shown in FIG. Each numerical value represents an average value of n = 3. The response of this biosensor to phosphate ions increases in proportion to the length of the reaction time. Under the reaction conditions in which maltose is sufficiently present, this complex reaction system can perform an amplification reaction due to the presence of phosphate ions. It was shown to wake up.

[Example 12]
In Example 10, a pH 6.5, 20 mM sodium citrate buffer solution containing 400 mM potassium dihydrogen phosphate is used as a substrate solution, and a pH 6.5, 20 mM sodium citrate solution containing a predetermined concentration of maltose is used as a measurement sample solution. A buffer solution was used to examine the response to maltose concentration.

  The results obtained are shown in FIG. Each numerical value represents an average value of n = 3. Responses of the present biosensor to maltose were obtained in the range of 1-300 mM.

[Example 13]
In Example 8, in addition to maltose phosphorylase, mutarotase and glucose oxidase, the acid phosphatase used in Example 9 was used, and the reaction time was changed to 10 minutes, and the current value was measured. (9) The response to the phosphate ion concentration in the (i) maltose phosphorylase-acid phosphatase-mutarotase system of the complex reaction system was examined.

  The obtained results are shown in FIG. Each numerical value represents an average value of n = 3. The response of the present biosensor to phosphate ions is about 20 times that in the range of 10 μM to 2 mM compared to the result of the simple reaction system shown in Example 8, and the substrate amplification shown in Example 9 The response of the reaction system was about twice as high as that of the reaction system, and it was confirmed that phosphate water can be quantified with this sensor if the eutrophication is advanced.

[Example 14]
In Example 13, instead of maltose phosphorylase, desalted sucrose phosphorylase (derived from Luconostock Mecentroides) was used in the same amount of activity, and (ii) sucrose phosphorylase- The response to phosphate ion concentration in the acid phosphatase-mutarotase system was investigated.

  The obtained results are shown in FIG. Each numerical value represents an average value of n = 3. The response of this biosensor to phosphate ions is obtained in the range of 0.1 to 30 mM, and it is confirmed that phosphate ions can be quantified with this sensor in the case of environmental water with enhanced eutrophication. It was done.

[Example 15]
In Example 14, a pH 6.5, 20 mM sodium citrate buffer solution containing 400 mM potassium dihydrogen phosphate as a substrate solution, and a pH 6.5, 20 mM sodium citrate buffer containing sucrose at a predetermined concentration as a measurement sample solution. Solutions were used to examine the response to sucrose concentration.

  The obtained results are shown in FIG. Each numerical value represents an average value of n = 3. Responses of this biosensor to sucrose were obtained in the range of 0.3-30 mM.

[Example 16]
In Example 13, a sample solution obtained by dissolving the following substances in ultrapure water was used.
Control: 2 mM phosphate ion 10 mM acetaminophen and 2 mM phosphate ion

The obtained results are shown in the following Table 1 and FIG.

Comparative Example 6
In Example 16, a biosensor in which the reagent layer containing the mediator on the biosensor was washed away with water was used.

The obtained results are shown in the following Table 2 and FIG.

  As shown in the above results, in the hydrogen peroxide system (Comparative Example 6), a significant decrease in response was observed compared to the mediator system (Example 16). In addition, it was confirmed that by using potassium ferricyanide as a mediator, a larger output value can be obtained compared to a hydrogen peroxide system using dissolved oxygen in the sample solution as a mediator, and the measurement accuracy is improved.

[Example 17]
In Example 13, a measurement sample solution in which the following substances were dissolved in ultrapure water was used, and the influence of contaminants contained in environmental water, foods, body fluids, and the like on the sensor response was examined.
Control: 2mM phosphate artificial _{seawater: 2.65% NaCl, 0.326% MgCl} 2, 0.207% MgSO 4, 0.136% CaSO 4, 0.0714% KCl and 2mM phosphate ion 0 3 mM ascorbic acid and 2 mM phosphate ion 1 ppm uric acid and 2 mM phosphate ion 1 ppm ammonium ion and 2 mM phosphate ion 1 ppm sodium dodecyl sulfate and 2 mM phosphate ion 1 ppm benzalkonium chloride and 2 mM phosphate ion 1 ppm lignin and 2mM phosphate · 4 ppm inorganic ^{_{anions: 1ppmF -, 1ppmNO 2 -,}} 1ppmNO 3 -, 1ppmSO 4 2- and 2mM phosphate · 5 ppm heavy ^{metals: 1ppmCu} 2+, ^1ppmZn 2+, 1 ^{pmMn 2+,} 1ppmCr ^3+, 1ppmFe 3+ and 2mM phosphate ions

The obtained results are shown in the following Table 3 and FIG. As shown in these results, sensor responses to heavy metal ions and phosphate ions contained in artificial seawater are slightly lower than those of the control, but their response ratios are both about 90%. It was. From this result, it was shown that the influence of contaminants can be reliably suppressed as compared with the conventional electrochemical method of dissolved oxygen and hydrogen peroxide detection system and the method using chemiluminescence.

[Example 18]
Subsequently, by using this biosensor, the (8) substrate amplification reaction system (i) maltose phosphorylase-acid phosphatase system was applied to the biosensor used in Reference Example 2, and the substrates maltose and phosphate were used. The relationship between the acid phosphatase activity and the sensor response value was investigated under conditions where ions were sufficiently present. Desalted maltose phosphorylase and glucose oxidase as in Example 8 were dissolved in ice-cooled pH 6.5, 20 mM sodium citrate buffer solution to a concentration of 0.4 unit / μl. In addition, as a substrate solution, pH 6.5, 20 mM sodium citrate buffer solution containing 400 mM maltose and 400 mM potassium dihydrogen phosphate, and as a measurement sample solution, desalted acid phosphatase as in Example 9 was used. What was melt | dissolved in the pH 6.5 and 20 mM sodium citrate buffer solution in ice-cooling was used so that it might become a predetermined density | concentration. Next, 2.5 μl of a solution containing maltose phosphorylase and glucose oxidase and 2.5 μl of a reaction substrate solution are mixed well, and then a mixture of 5 μl of a measurement sample solution is introduced into a biosensor and allowed to react accurately for 10 minutes. Thereafter, the current value was measured by a cyclic voltammetry method.

  The obtained results are shown in FIG. Each numerical value represents an average value of n = 3. The relationship between the acid phosphatase activity and the sensor response value showed a linear response when the enzyme activity in the sample solution was in the range of 0.02 to 0.2 unit / l. From these results, it was confirmed that it can be used for measurement of blood acid phosphatase activity, which is a prostate tumor marker, by applying the phosphate ion quantification method of this sensor.

[Example 19]
In Example 8, pH 6.5 containing pyrophosphate ions at a predetermined concentration as a measurement sample solution, 2.5 μl of 20 mM sodium citrate buffer solution and desalted inorganic pyrophosphatase (derived from baker's yeast) at 0.4 unit / μl. A solution prepared by mixing 2.5 μl of a solution dissolved in a pH 6.5, 20 mM sodium citrate buffer solution and reacting for 5 minutes is used. (10) Response of the inorganic pyrophosphatase system to pyrophosphate ion concentration I investigated.

  The obtained results are shown in FIG. Each numerical value represents an average value of n = 3. Responses of this biosensor to pyrophosphate ions were obtained in the range of 1-100 mM.

[Example 20]
In Example 19, pH 6.5 containing 10 mM pyrophosphate ion, 10 mM ATP or 10 mM dNTPs as a measurement sample solution, 2.5 μl of 20 mM sodium citrate buffer solution, and desalted inorganic pyrophosphatase to 0.4 unit / μl. As described above, a solution obtained by mixing 2.5 μl of a solution dissolved in a pH 6.5, 20 mM sodium citrate buffer solution and reacting for 5 minutes was used.

The obtained results are shown in Table 4 and FIG. This confirmed that inorganic pyrophosphatase did not react with ATP and dNTP required as nucleic acid detection substrates.

  By using the phosphate ion concentration measurement method according to the present invention, for example, phosphate ions contained in seawater can be measured without being affected by interfering substances. It can be used effectively in each field. In addition, the blood glucose sensor can be used to produce a sensor for the purpose of measuring phosphate ions in environmental water, and by measuring pyrophosphate ions, it can be used for portable SNP diagnosis and ultrasensitive virus detection. Can also be used effectively. Furthermore, when a substrate other than phosphate ions is sufficiently present, it is possible to measure the activity of acid phosphatase, and it can be applied to diagnosis by detecting a tumor marker of the prostate.

FIG. 1 is a graph showing the responsiveness of a blood glucose sensor to glucose. FIG. 2 is a graph showing the results of examining the necessity of TPP, FAD and magnesium ions in a pyruvate oxidase system. FIG. 4 is a graph showing the response of the biosensor to TPP in the oxidase system. [FIG. 4] A graph showing the response of the biosensor to pyruvate in the pyruvate oxidase system. [FIG. 5] The bio in the pyruvate oxidase system. FIG. 6 is a graph showing the response of the sensor to pyruvate oxidase activity. [FIG. 6] This is a graph showing the response of the biosensor to phosphate ions in the pyruvate oxidase system. A graph showing a comparison between Is (a) Japanese garden, b) cascade c) pond)
FIG. 8 is a graph showing the response of the biosensor to pH in the pyruvate oxidase-acid phosphatase system, which is a substrate amplification reaction system. FIG. 9 is a graph showing the response of the blood glucose sensor to glucose. FIG. 11 is a graph showing the response of the present biosensor to phosphate ions in a simple reaction (maltose phosphorylase-mutarotase) system. FIG. 11 shows the response to phosphate ions in a substrate amplification reaction (maltose phosphorylase-acid phosphatase) system. FIG. 12 is a graph showing the effect of pH of the present biosensor in a complex reaction (maltose phosphorylase-acid phosphatase-mutarotase) system. [FIG. 13] FIG. 14 is a graph showing the response of the biosensor to maltose in a complex reaction (maltose phosphorylase-acid phosphatase-mutarotase) system. 15] A graph showing the response of the biosensor to phosphate ions in a complex reaction (maltose phosphorylase-acid phosphatase-mutarotase) system. [FIG. 16] This bio in a substrate amplification reaction (sucrose phosphorylase-acid phosphatase-mutarotase) system. FIG. 17 is a graph showing the responsiveness of the biosensor to sucrose in a substrate amplification reaction (sucrose phosphorylase-acid phosphatase-mutarotase) system. FIG. 18 is a graph showing the effect of acetaminophen of this biosensor in a complex reaction (maltose phosphorylase-acid phosphatase-mutarotase) system. FIG. 19 is a graph showing the effect of acetaminophen in a hydrogen peroxide system. FIG. 20 is a graph showing the influence of various contaminants of this biosensor in a complex reaction (maltose phosphorylase-acid phosphatase-mutarotase) system. FIG. 21: Acid phosphatase activity in a substrate amplification reaction (maltose phosphorylase-acid phosphatase) system. [FIG. 22] A graph showing the response of the present biosensor to pyrophosphate ions in an inorganic pyrophosphatase system. [FIG. 23] ATP of the present biosensor in an inorganic pyrophosphatase system. It is a graph which shows the responsiveness with respect to dNTP

Such object of the present invention, (1) ready for the sample solution was used oxidized mediator with a redox enzyme that catalyzes a reaction for producing by reacting with phosphate ions oxides become substrates and the oxide after reaction in the sensor, the method or (2) the sample solution to measure the current generated by applying a voltage, pyruvate oxidase, magnesium ions, oxidized mediator with flavin adenine dinucleotide and thiamine pyrophosphate This is accomplished by a method of measuring the current produced by applying a voltage after reacting in a ready-made sensor used .

In the method (1) of the present invention, an enzyme-catalyzed reaction occurs due to the presence of phosphate ions, and is used for substrates and oxidoreductases that generate oxides, other substances required for the reaction, and off-the-shelf sensors. The oxidized mediator is used to generate a reduced mediator from phosphoric acid, which is further oxidized, and the resulting oxidation current is measured to measure phosphate ions in the sample solution.

For the measurement of phosphate ions, a sensor is used in which a working electrode and a counter electrode are provided on a substrate, and a reference electrode is provided if necessary, and enzymes and reagents necessary for the measurement are arranged on and / or near the working electrode. Examples of such sensors include platinum, gold, carbon, and the like by screen printing, vapor deposition, sputtering, etc. on an insulating substrate such as ceramics, glass, plastic, paper, biodegradable material (for example, microbial production polyester). Each of the electrodes is formed on the working electrode and / or in the vicinity thereof, and an enzyme and reagent are immobilized by a crosslinking method, a covalent bonding method, an ionic bonding method, or the like.

In the above reaction (5), one of the products is produced by the action of pyruvate oxidase in the presence of Pi and magnesium ions, flavin adenine dinucleotide, thiamine pyrophosphate, pyruvate and the oxidized mediator used in the ready-made sensor. Acetic acid and Pi are produced by reaction (9). In the substrate amplification reaction system referred to here, the phosphate ion to be measured is phosphorylated into the degradation product of one substrate by the first-stage reaction and then regenerated by the next reaction, and the substrate of the original first-stage reaction It becomes. Since this reaction is repeated, it is also called a cycling reaction. When this reaction system is used, if there is sufficient pyruvic acid as one of the substrates, the amplification reaction can proceed even in the presence of a very small amount of phosphate ions, which enables highly sensitive detection of phosphate ions. It becomes possible. Conversely, the presence of a sufficient substrate other than phosphate ions enables measurement of blood acid phosphatase activity, which is a prostate tumor marker.

In the method (2) of the present invention, an enzyme-catalyzed reaction is caused by the presence of phosphate ions, and finally a substrate and an enzyme that produce β-D-glucose, an oxidized mediator used in an off-the-shelf sensor, and Phosphate ions in a sample solution can be measured by using a redox enzyme typified by glucose oxidase to generate a reduced mediator from phosphoric acid, further oxidizing it, and measuring the resulting oxidation current. . If glucose is present in the sample solution, it is necessary to erase glucose in the sample solution in advance using glucose oxidase or the like.

For the measurement of phosphate ions, a working electrode and a counter electrode are provided on a substrate, and a reference electrode is provided if necessary, and a sensor in which enzymes and reagents necessary for the measurement are arranged on the working electrode is used . As such a sensor, those described above are used.

Claims (21)

When phosphate ions are reacted with phosphate ions in the presence of a substrate that becomes an oxide and an oxidoreductase that catalyzes the reaction for producing this oxide, it is produced by the coexistence of an oxidized mediator. A method for qualitative and quantitative determination of phosphate ions, characterized by measuring the current generated by further oxidizing the reduced mediator. Produced by reacting phosphate ion with pyruvate in the presence of pyruvate oxidase, magnesium ion, flavin adenine dinucleotide, thiamine pyrophosphate and oxidized mediator, and further oxidizing the reduced mediator produced with acetyl phosphate A qualitative and quantitative method for phosphate ions, characterized by measuring the measured current. The method for qualitative and quantitative determination of phosphate ions according to claim 2, wherein acid phosphatase or alkaline phosphatase is allowed to act on acetyl phosphate to regenerate phosphate. Phosphate ions are reacted with phosphate ions to finally produce β-D-glucose, and in the presence of an enzyme that catalyzes each reaction for producing β-D-glucose. β-D-glucose is generated, and the generated β-D-glucose is reacted with an oxidized mediator in the presence of an oxidoreductase, and a current generated by further oxidizing the resulting reduced mediator is measured. This is a qualitative and quantitative method for phosphate ions. Phosphate ions are reacted with maltose in the presence of maltose phosphorylase to produce β-D-glucose-1-phosphate, and the produced β-D-glucose-1-phosphate is converted to acid phosphatase or alkaline phosphatase. And producing β-D-glucose and phosphate, and reacting the obtained β-D-glucose with an oxidized mediator in the presence of glucose oxidase and / or glucose dehydrogenase, and further oxidizing the resulting reduced mediator A method for qualitative and quantitative determination of phosphate ions, characterized in that the current generated by the measurement is measured. Phosphate ions are reacted with sucrose in the presence of sucrose phosphorylase with or instead of maltose phosphorylase and maltose to produce α-D-glucose-1-phosphate, and the produced α-D-glucose- 1-phosphate is produced using acid phosphatase or alkaline phosphatase to produce α-D-glucose and phosphate, and further, mutarotase is allowed to act on the produced α-D-glucose to produce β-D-glucose. Wherein β-D-glucose obtained is reacted with an oxidized mediator in the presence of glucose oxidase and / or glucose dehydrogenase, and a current generated by further oxidizing the resulting reduced mediator is measured. Ion qualitative and quantitative methods. Acid phosphatase or alkaline phosphatase is used to produce or substitute β-D-glucose and phosphate with α-D-glucose obtained with β-D-glucose-1-phosphate. The qualitative / quantitative method for phosphate ions according to claim 5, wherein β-D-glucose is produced by acting. Acid phosphatase or alkaline phosphatase is used to produce α-D-glucose and phosphate, or alternatively, glucose isomerase is allowed to act on fructose obtained with α-D-glucose-1-phosphate. The qualitative and quantitative method for phosphate ions according to claim 6, wherein β-D-glucose is produced. 7. The method for detecting phosphate ions according to claim 3, 5 or 6, wherein the phosphate ions which are substrates for the enzyme reaction are detected by a combination that causes a substrate recycling reaction. The qualitative / quantitative method for phosphate ions according to any one of claims 1 to 9, wherein potassium ferricyanide, ferrocene or parabenzoquinone is used as the oxidized mediator. The qualitative / quantitative method for phosphate ions according to any one of claims 1 to 10, wherein a phosphate ion obtained by allowing an inorganic pyrophosphatase to act on pyrophosphate ion is used. 12. The method according to claim 1, which is used for nucleic acid detection, nucleic acid-specific sequence or nucleic acid sequence, nucleic acid amplification reaction and receptor behavior monitoring, and activity measurement of an enzyme involved in a catalytic reaction of phosphate ion or pyrophosphate ion. A method for qualitative and quantitative determination of phosphate ions according to the above. The qualitative / quantitative method for phosphate ion according to claim 12, wherein the enzyme involved in the catalytic reaction of phosphate ion is pyruvate oxidase or blood acid phosphatase which is a tumor marker of prostate. The qualitative and quantitative method for phosphate ions according to claim 13, wherein a substrate other than phosphate ions is used at a concentration of 10 mM or more. The qualitative and quantitative method for phosphate ions according to claim 11, which enables detection of gene polymorphisms (SNPs) by detecting pyrophosphate ions generated by the catalytic action of a ligase. The qualitative and quantitative method for phosphate ions according to any one of claims 1 to 15, wherein a ready-made sensor using an oxidized mediator is used for detection of phosphate ions. The qualitative / quantitative method for phosphate ions according to claim 16, wherein the off-the-shelf sensor using an oxidized mediator is a blood glucose sensor. A sensor having a working electrode and a counter electrode on a substrate, wherein the enzyme and the reagent according to any one of claims 1 to 15 are arranged on and / or in the vicinity of the working electrode. 19. A biosensor comprising: the biosensor according to claim 18; a measurement unit that measures an electrical value at an electrode of the biosensor; a display unit that displays a measurement value in the measurement unit; and a memory unit that stores the measurement value. apparatus. The biosensor device according to claim 19, wherein a potential step chronoamperometry method, a coulometry method, or a cyclic voltammetry method is applied as a measurement method in the measurement unit. 21. The biosensor device according to claim 19 or 20, further comprising wireless means for transmitting measurement data to a measurement unit of the biosensor, wherein the wireless means is a non-contact type IC card or short-range wireless communication.

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