JP2017106913A - Method and device for measuring target component - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new device and a new method for measuring and analyzing a component.SOLUTION: The method for measuring a target component includes: a first application step of applying a first voltage (V) to an electrode system under the presence of a sample, an oxidation-reduction catalyst for a target component, and an electron conveying material; a second application step of applying a second voltage (V) to the electrode system; and a signal acquiring step of acquiring a signal by the electrode system in the second application step. The first voltage is not smaller than the oxidation potential (E) of the electron conveying material, and the second voltage is less than the oxidation potential (E).SELECTED DRAWING: Figure 3

Description

  The present invention relates to a target component measurement method and a target component measurement apparatus.

  As an electrochemical measurement method for blood glucose concentration, glucose oxidase or glucose dehydrogenase, an electron transfer substance and an electrode were used, and a voltage higher than the oxidation potential of the electron transfer substance was applied to the electrode. A method of calculating the glucose concentration based on the measured value is used.

  However, the glucose concentration calculated by the measurement method is affected by interfering substances such as hematocrit and ascorbic acid in blood. For this reason, it is necessary to measure interfering substances such as hematocrit and ascorbic acid with an electrode different from the electrode for measuring glucose, and to correct the amount of glucose from these measured values (Patent Documents 1 and 2).

  Therefore, a new glucose measurement method is required.

Japanese Patent No. 3106613 Japanese Patent No. 5239860

  Accordingly, an object of the present invention is to provide a new target component measurement method and target component measurement apparatus for measuring target components including glucose, for example.

In order to solve the above-described problems of the present invention, a method for measuring a target component of the present invention (hereinafter also referred to as “measurement method”) is performed in the presence of a sample, a redox catalyst for the target component, and an electron transfer substance. A first application step of applying a voltage of 1 (V ₁ ) to the electrode system;
A second application step of applying a _second voltage (V ₂ ) to the electrode system;
In the second application step, including a signal acquisition step of acquiring a signal by the electrode system,
The first voltage is equal to or higher than an oxidation potential (E) of the electron transfer material;
The second voltage is a voltage lower than an oxidation potential (E) of the electron transfer substance.

An apparatus for measuring a target component of the present invention (hereinafter also referred to as “measuring apparatus”) includes an electrode system,
Voltage application means for applying a _first voltage (V ₁ ) and a second voltage (V ₂ ) to the electrode system in the presence of a sample, a redox catalyst for the target component, and an electron transfer material;
Signal acquisition means for acquiring a signal from the electrode system in applying the second voltage (V ₂ ),
The first voltage is equal to or higher than an oxidation potential (E) of the electron transfer material;
The second voltage is a voltage less than an oxidation potential (E) of the electron transfer material;
It is used for the measuring method of the target component of the present invention.

  According to the present invention, glucose and other target components can be measured.

FIG. 1 is a perspective view showing a method for manufacturing a biosensor used in Example 1 of the present invention. FIG. 2 is a cyclic voltammogram showing the current value under the oxidation potential measurement conditions of Example 1 of the present invention. FIG. 3 is a graph showing the current value obtained with the glucose sample in Example 1 of the present invention. FIG. 4 is a graph showing the current value obtained with the glucose sample in Example 1 of the present invention. FIG. 5 is a graph showing the degree of influence of the Ht value in Example 2 of the present invention. FIG. 6 is a graph showing the degree of influence of the Ht value in Example 3 of the present invention. FIG. 7 is a graph showing the degree of influence of the Ht value in Example 3 of the present invention. FIG. 8 is a graph showing the degree of influence of ascorbic acid in Example 4 of the present invention. FIG. 9 is a graph showing CV values in Example 4 of the present invention.

In the measurement method and the measurement apparatus of the present invention, for example, the first voltage (V ₁ ) is a voltage in a range of E ≦ V ₁ ≦ E + 1.15.

In the measurement method and the measurement apparatus of the present invention, for example, the second voltage (V ₂ ) is a voltage in a range of E−0.25 ≦ V ₂ <E.

  In the measurement method and the measurement apparatus of the present invention, for example, the signal is a signal acquired within 0.4 seconds with reference to the time when the second voltage is applied.

  In the measuring method and measuring apparatus of the present invention, for example, the signal is a signal acquired at one time point.

  In the measurement method and measurement apparatus of the present invention, for example, the signal is a signal obtained continuously.

  In the measurement method and measurement apparatus of the present invention, for example, the signal is a signal acquired at a plurality of time points.

  In the measuring method and measuring apparatus of the present invention, for example, the target component is glucose.

  In the measuring method and measuring apparatus of the present invention, for example, the redox catalyst is an oxidoreductase or a dehydrogenase.

  In the measuring method and measuring apparatus of the present invention, for example, the redox catalyst is glucose oxidase or glucose dehydrogenase.

  In the measurement method and measurement apparatus of the present invention, for example, the sample is a biological sample, preferably blood.

  The measurement method of the present invention includes, for example, a calculation step of calculating the target component amount in the sample from the signal.

The measurement method of the present invention includes, for example, a correction signal acquisition step of acquiring a correction signal by the electrode system,
A correction step of correcting the signal based on the correction signal,
In the calculation step, the target component amount in the sample is calculated from the corrected signal.

In the measurement method and the measurement apparatus of the present invention, for example, the first voltage (V ₁ ) is E <V ₁ .

  The measurement apparatus of the present invention includes, for example, a calculation unit that calculates the target component amount in the sample from the signal.

The measurement apparatus of the present invention, for example, a correction signal acquisition means for acquiring a correction signal by the electrode system,
Correction means for correcting the signal based on the correction signal,
In the calculation means, the target component amount in the sample is calculated from the corrected signal.

<Measurement method of target component>
As described above, the method for measuring the target component of the present invention is the first application in which the first voltage (V ₁ ) is applied to the electrode system in the presence of the sample, the redox catalyst for the target component, and the electron transfer material. A second application step of applying a _second voltage (V ₂ ) to the electrode system, and a signal acquisition step of acquiring a signal by the electrode system in the second application step, The voltage of 1 is a voltage equal to or higher than the oxidation potential (E) of the electron transfer substance, and the second voltage is a voltage lower than the oxidation potential (E) of the electron transfer substance. The measurement method of the present invention is characterized in that the first voltage is a voltage equal to or higher than the oxidation potential of the electron transfer material, and the second voltage is a voltage lower than the oxidation potential of the electron transfer material. The other steps and conditions are not particularly limited.

  As a result of intensive studies, the inventor has obtained a signal obtained when a second voltage lower than the oxidation potential of the electron transfer material is applied after applying the first voltage equal to or higher than the oxidation potential of the electron transfer material. Knowledge that the amount of the target component varies depending on the amount of the target component in the sample. Specifically, for example, the transient current obtained after the application of the first voltage and the start of the application of the second voltage is the target component in the sample. The present inventors have found the knowledge of showing a negative correlation with the amount and have established the present invention. Further, although the mechanism of the measurement method of the present invention is unknown, the influence of interfering substances such as hematocrit and ascorbic acid can be reduced in the measurement of the target component. For this reason, the measurement method of the present invention can be implemented even with a biosensor having a simple structure that does not include an electrode for measuring the interference substance, for example. Furthermore, since the influence of the interference substance is reduced, for example, the measurement method of the present invention can be performed without obtaining a correction signal for correcting the influence of the interference substance. For this reason, for example, the measurement method of the present invention can reduce the number of signal acquisitions and reduce the influence of measurement errors that occur when acquiring signals, so that the target component can be measured with good reproducibility. Moreover, although the said patent documents 1 and 2 acquire a signal in the state where the Cottrell current was stabilized after the application of the voltage to the said electrode system, before the said Cottrell current becomes stable, the measurement method of this invention is Can be implemented. For this reason, according to the measuring method of the present invention, for example, the measuring time can be shortened. Furthermore, the measurement method of the present invention is carried out with a biosensor including an electrode system used in a known electrochemical measurement method, for example, by adjusting the voltage applied to the electrode system and the acquisition time of the signal. it can. For this reason, according to the measuring method of the present invention, it can be introduced into a known biosensor without cost.

  In the present invention, “acquiring a signal” can also be referred to as “measuring a signal”, for example. Also, “acquiring or measuring“ signal ”means acquiring or measuring“ signal value ”representing, for example, the magnitude or intensity of the signal. Moreover, the fluctuation | variation of a signal means the fluctuation | variation of a signal value, for example.

  In the present invention, the “oxidation potential” is, for example, the potential of the electrode system in an oxidation reaction system including the electron transfer substance and the electrode system. The oxidation potential is obtained by using, for example, an oxidation potential measurement system including the electron transfer substance and an electrode system including a working electrode, a counter electrode and a reference electrode or an electrode system including the working electrode and the reference electrode. In the state where the working electrode and the reference electrode are connected, the measurement can be performed under the following measurement conditions for the oxidation potential, and can be measured as a potential indicating a positive current value in the positive direction in the obtained current value. The oxidation potential may be set based on, for example, a potential obtained by a single measurement, or may be set based on a potential obtained by a plurality of measurements. In the latter case, the oxidation potential may be, for example, an average, minimum or maximum potential in potentials obtained by a plurality of measurements, or a minimum potential to a maximum potential in potentials obtained by a plurality of measurements. It is good also as a range. The oxidation potential may be, for example, an oxidation potential measured in advance or an oxidation potential measured when measuring the target component. As a specific example, when the electron transfer substance is a ruthenium complex and the electrode system material is carbon, the oxidation potential is, for example, 30 to 50 mV.

(Oxidation potential measurement conditions)
Concentration of electron transfer substance in measurement system: 10 to 500 mmol / L
Sample used for measurement system: Blood sample measurement system temperature: 25 ° C
Starting potential: -800 mV
Folding potential: 800mV
Sweep speed: 20 mV / s

  The measurement method of the present invention may be, for example, a qualitative analysis that measures the presence or absence of the target component of the sample, or a quantitative analysis that measures the amount of the target component of the sample.

  In the present invention, the first application step, the second application step, and the signal acquisition step are performed in the presence of the sample, the redox catalyst, and the electron transfer substance. Therefore, the measurement method of the present invention includes, for example, the first application step, the second application step, and the signal acquisition for the reaction system including the sample, the redox catalyst, and the electron transfer material. It can also be said that a process is carried out. The reaction system is preferably a liquid system, for example, and can also be referred to as a reaction solution or a redox solution containing the sample, the redox catalyst, and the electron transfer substance.

  In the present invention, the sample is not particularly limited, and is, for example, a liquid sample. Examples of the liquid sample include a biological specimen (biological sample), and may be a diluted solution or a suspension of the biological sample. Examples of the biological sample include blood, urine, gastric fluid, sputum, amniotic fluid, peritoneal fluid, interstitial fluid, and other body fluids, large intestine, lung and other tissues, oral cells, germ cells, nail, hair and other cells. It is done. Examples of the blood include whole blood, plasma, serum, and hemolyzed blood. The sample may be, for example, a sample containing the target component or a sample in which it is unknown whether the target component is included.

  In the present invention, the target component is not particularly limited, and examples thereof include sugars such as glucose, C-reactive protein (CRP), HbA1c, thyroid stimulating hormone (TSH), FT3, FT4, hCG, HBs antigen, HBc antibody, HCV antibody, TY antigen, anti-streptolysin O (ASO), type IV collagen, matrix metalloproteinase-3 (MMP-3), PIVAK-II, α1 microglobulin, β1 microglobulin, amyloid A (SAA), elastase 1 , Basic fetoprotein (BFP), Candida antigen, granulocyte elastase in cervical mucus, digoxin, cystatin C, factor XIII, urinary transferrin, syphilis, hyaluronic acid, fibrin monomer complex (SFMC), von Willebrand factor (No. V Factor II-like antigen), protein S, rheumatoid factor (RF), IgD, α1 acid glycoprotein (α1AG), α1 antitrypsin (α1AT), α2 macroglobulin, albumin (Alb), ceruloplasmin (Cp), haptoglobin (Hp) ), Prealbumin, retinol binding protein (RBP), β1C / β1A globulin (C3), β1E globulin (C4), IgA, IgG, IgM, β lipoprotein (β-LP), apoprotein AI, apoprotein A -II, apoprotein B, apoprotein C-II, apoprotein C-III, apoprotein E, transferrin (Tf), urinary albumin, plasminogen (PLG) and lipoprotein (a) (LP (a)) Lactic acid, low density lipid, high density lipid, triglyceride and the like. In the present invention, the target component to be measured may be, for example, one type or two or more types.

  In the present invention, the redox catalyst is, for example, a catalyst that catalyzes a redox reaction with respect to the target component, and may catalyze one or both of the oxidation reaction and the reduction reaction. The oxidation-reduction catalyst is not particularly limited, and can be appropriately determined according to, for example, the target component. Examples of the redox catalyst include oxidase, reductase, oxidoreductase, dehydrogenase and the like. As a specific example, when the target component is glucose, examples of the oxidase include glucose oxidase, and examples of the dehydrogenase include glucose dehydrogenase. When the target component is lactic acid, examples of the oxidoreductase include lactate oxidoreductase, and examples of the dehydrogenase include lactate dehydrogenase. The redox catalyst (eg, oxidoreductase) may further contain a cofactor, for example. The cofactor is not particularly limited, and can be appropriately determined according to, for example, the type of the redox catalyst (for example, redox enzyme). Examples of the cofactor include flavin adenine dinucleotide (FAD), pyrroloquinoline quinone (PQQ), nicotinamide adenine dinucleotide (NAD), and nicotinamide adenine dinucleotide phosphate (NADP). As the redox catalyst (for example, redox enzyme), for example, one kind may be used alone, or two or more kinds may be used in combination.

  In the present invention, the electron transfer substance may be, for example, a substance that accepts an electron generated by a redox reaction between the target component and the redox catalyst and donates the electron to the electrode system. It may be a substance that accepts and donates to the redox reaction between the target component and the redox catalyst. The electron transfer substance is not particularly limited, and can be appropriately determined according to, for example, the type of the redox catalyst. Specifically, the electron transfer substance is, for example, a ruthenium complex, a ferricyan compound, an osmium complex, an iron complex, another organometallic complex, an organometallic complex polymer, a conductive ion salt, an organic compound such as benzoquinone, a conductive oxidation, or the like. Examples thereof include reduced polymers. For example, one type of the electron transfer substance may be used alone, or two or more types may be used in combination.

  In the present invention, the electrode system is, for example, an electrode that detects a signal due to a redox reaction of the target component and the redox catalyst. The electrode system includes, for example, at least one set of electrode groups including a working electrode and a counter electrode that are the signal detection sites. In the electrode system, the number of the electrode groups may be, for example, one set or two or more sets. In the electrode system, the working electrode may be one, for example, or two or more. In the latter case, examples of the two or more working electrodes include different working electrodes. Examples of the different working electrodes include those having different electrode materials and those having different target components to be detected. For example, the redox catalyst and the electron transfer substance may be disposed in advance on the working electrode, and the redox catalyst and the electron transfer substance may be coated on the working electrode. In this case, for example, the target component can be measured by bringing the electrode system into contact with the sample, and the target component can be measured more easily. In the electrode system, the counter electrode may be one, for example, or two or more. In the electrode system, each electrode group may have a separate counter electrode, for example, and a counter electrode in one electrode group may also serve as a counter electrode of another electrode group. The electrode system may include other electrodes such as a reference electrode, if necessary. The material of each electrode in the electrode system is not particularly limited, and can be appropriately determined according to, for example, the type of the target component, the redox catalyst, and the electron transfer substance.

  Preferably, the electrode system is further connected to, for example, signal acquisition means, and the signal acquisition means is connected to the working electrode. In this case, the working electrode may be coupled to the signal acquisition unit via, for example, a terminal. Examples of the signal acquisition means include a detector. The detector is not particularly limited, and examples thereof include a current detector, a potential detector, and a voltage detector. The detector may include, for example, a current / voltage converter, an A / D converter, an arithmetic unit, and the like.

  As the electrode system, for example, an electrode system of a biosensor used in a known electrochemical measurement method capable of measuring the target component may be used. When the biosensor is used as the electrode system, the biosensor includes, for example, an electrode system including at least one set of electrode groups including the working electrode and the counter electrode, and the detector. Are connected to the working electrode. In the biosensor, the detector may be disposed, for example, inside the biosensor or may be disposed outside the biosensor. In the latter case, for example, the biosensor and the detector may be connected in advance before acquisition of the signal, or the biosensor and the detector may be connected at the time of acquisition.

  The biosensor may further include, for example, an introduction unit that introduces the sample, a moving unit that moves the sample to the electrode system, and the like. In the biosensor, for example, the redox catalyst and the electron transfer substance may be arranged. Examples of the arrangement location of the redox catalyst and the electron transfer substance include the working electrode, the introduction unit, the moving unit, and the like. For example, the oxidation-reduction catalyst and the electron transfer substance may be disposed in one place or in two or more places. When arranged on the working electrode, the working electrode is coated, for example, on the redox catalyst and the electron transfer substance. When the biosensor has two or more working electrodes, the oxidation-reduction catalyst and the electron transfer substance disposed on each working electrode may be the same or different.

  In the present invention, the signal is a signal obtainable by the electrode system. The signal is not particularly limited, and is, for example, a signal based on a redox reaction of the target component and the redox catalyst, and specific examples thereof include an electric signal. The electric signal may be, for example, an electric current, a voltage converted from the electric current, an electric quantity calculated from the electric current and time, and a digital signal corresponding to these. These electric signals can be converted into each other by, for example, a known method. As a specific example, when a current is measured by the electrode system, the current can be converted into a voltage by, for example, a current / voltage converter, and a voltage that is an analog signal is, for example, A / D (analog / digital). ) Can be converted to a digital signal by a converter.

  In the first application step, as described above, the first voltage is applied to the electrode system in the presence of the sample, a redox catalyst for the target component, and the electron transfer substance. In the first application step, for example, the electrode system is brought into contact with a reaction system including the sample, the oxidation-reduction catalyst, and the electron transfer substance, and the first voltage is applied to the electrode system. Can be implemented. The reaction system can be prepared, for example, by adding the redox catalyst and the electron transfer substance to the sample. When the biosensor electrode system is used as the electrode system, the reaction system is prepared, for example, by bringing the sample into contact with the electrode system coated with the redox catalyst and the electron transfer material. May be. The application of the first voltage to the electrode system can be performed by, for example, a voltage applying unit. The voltage application means is not particularly limited, and may be any voltage as long as it can apply a voltage to the electrode system, and a voltage device or the like can be used as a known means.

In the first application step, the first voltage (V ₁ ) may be a voltage equal to or higher than the oxidation potential (E), that is, E ≦ V ₁ (V), and the upper limit is not particularly limited. . When the oxidation potential is set based on a potential obtained by a plurality of measurements, the oxidation potential is preferably a maximum potential among the potentials obtained by the plurality of measurements. The first voltage preferably satisfies E + 0.15 ≦ V ₁ (V) and E + 0.25 ≦ V ₁ (V), for example, because noise at the time of signal acquisition can be reduced. The upper limit of the first voltage is preferably, for example, the maximum value of the voltage at which hydrolysis of water does not occur in the reaction system, and the influence of the interference substance can be reduced. More preferably, V ₁ ≦ E + 1.15 ( V), V ₁ ≦ E + 1.05 (V), V ₁ ≦ E + 1 (V), and V ₁ ≦ E + 0.95 (V). The range of the first voltage is preferably a range of E ≦ V ₁ ≦ E + 1.15 (V), E + 0.15 because noise at the time of signal acquisition can be reduced and the influence of the interference substance can be reduced. ≦ V ₁ ≦ E + 1.05 (V), E + 0.25 ≦ V ₁ ≦ E + 0.95 (V). As a specific example, when the electron transfer substance is a ruthenium complex and the material of the electrode system is carbon, the first voltage is, for example, in the range of 50 to 1200 mV, in the range of 200 to 1100 mV, or in the range of 300 to 1000 mV. It is a range. For example, the first voltage may be constant or may vary, but is preferably the former.

  In the first application step, the application time of the first voltage is not particularly limited, and is, for example, 0.1 to 10 seconds, 0.5 to 8 seconds, or 1 to 5 seconds. The voltage application to the electrode system may be applied continuously or non-continuously, for example. In the first application step, the current of the electrode system can be appropriately set according to, for example, the reaction system.

  In the second applying step, as described above, the second voltage is applied to the electrode system. The second application step can be performed, for example, by applying the second voltage to the electrode system in contact with the reaction system. The application of the second voltage to the electrode system can be performed, for example, by the above-described voltage applying unit.

  The second application step may be performed continuously or discontinuously with the first application step, for example, but is preferably the former. In the former case, in the second application step, the second application step is performed simultaneously with the end of the first application step.

In the second application step, the second voltage (V ₂ ) may be a voltage lower than the oxidation potential (E), that is, V ₂ <E (V), and the lower limit thereof is not particularly limited. . When the oxidation potential is set based on a potential obtained by a plurality of measurements, the oxidation potential is preferably the minimum potential among the potentials obtained by the plurality of measurements. The second voltage is preferably a potential close to the oxidation potential because, for example, noise at the time of signal acquisition can be further reduced as the second voltage approaches the oxidation potential. The lower limit of the second voltage is preferably, for example, E-0.25 ≦ V ₂ (V), E−0.2 ≦ V ₂ (V), E− because the influence of the interference substance can be reduced. 0.15 ≦ _V 2 _(V), a _{E-0.1 ≦ V 2 (V} ). The range of the second voltage is preferably a range of E−0.25 ≦ V ₂ <E (V), for example, because noise at the time of signal acquisition can be reduced and the influence of the interference substance can be reduced. , E−0.2 ≦ V ₂ <E (V), E−0.15 ≦ V ₂ <E (V), E−0.1 ≦ V ₂ <E (V). . As a specific example, when the electron transfer substance is a ruthenium complex and the electrode system material is carbon, the second voltage is, for example, in a range of −200 mV or more and less than 50 mV, −100 mV or more and less than 50 mV. The range is -50 mV or more and less than 50 mV. For example, the second voltage may be constant or may vary, but is preferably the former.

  In the second application step, the application time of the second voltage is not particularly limited, and can be appropriately set according to the signal acquisition time in the signal acquisition step described later. The application time of the second voltage is, for example, 0.001 to 10 seconds, 0.001 to 2 seconds, 0.005 to 1.5 seconds, and 0.01 to 1 second. The voltage application to the electrode system may be applied continuously or non-continuously, for example. In the second application step, the current of the electrode system can be appropriately set according to, for example, the reaction system.

  In the signal acquisition step, as described above, in the second application step, a signal is acquired by the electrode system. The signal can be acquired by, for example, the signal acquisition means (for example, the detector or the like) connected to the electrode system. When the electrode system has a plurality of working electrodes, the signal may be acquired by the plurality of working electrodes.

  In the signal acquisition step, the number of acquisitions of the signal is not particularly limited, and may be, for example, once or a plurality of times. In the former case, the signal may be, for example, a signal acquired at one time point or a signal acquired continuously for a predetermined time. When the signal is a signal acquired continuously, the signal can also be referred to as an integral value of the signal, for example. In the latter case, the signal may be a signal acquired at a plurality of time points or a signal acquired at a plurality of working electrodes.

  The signals acquired at the plurality of time points mean, for example, signals acquired multiple times over time. The number of the time points, that is, the number of signal acquisitions may be a plurality of time points, for example, two or more time points, preferably 2 to 10 time points, or 2 to 5 time points.

  In the signal acquisition step, the timing for acquiring the signal is not particularly limited, and may be appropriately determined depending on, for example, the target component, the redox catalyst, the electron transfer substance, the first voltage, the second voltage, and the like. Can be determined. As a specific example, since the signal can reduce the influence of the interfering substance, it is preferable that the second voltage is applied within a period of 0.4 seconds, 0.2 seconds, 0. It is a signal acquired within 1 second, within 0.05 second, more preferably 0 to 0.4 second, 0.05 to 0.4 second, 0 to 0 with respect to the time of applying the second voltage. Signals acquired at 2 seconds, 0.05 to 0.2 seconds, 0 to 0.1 seconds, 0.05 to 0.1 seconds, and 0 to 0.05 seconds. When the signal is a signal acquired at one time point, the timing for acquiring the signal is, for example, within 0.2 seconds, within 0.15 seconds, and 0.1 seconds with respect to the time when the second voltage is applied. Preferably within 0 to 0.2 seconds, 0.05 to 0.2 seconds, 0 to 0.15 seconds, 0.05 to 0, based on the time of applying the second voltage. Signals acquired at 15 seconds, 0-0.1 seconds, 0.05-0.1 seconds. When the signal is an integral value of the signal, the timing to acquire the signal is, for example, 0 to 0.2 seconds, 0.05 to 0.2 seconds, 0 to 0 seconds when the second voltage is applied. Signals acquired at ˜0.15 seconds, 0.05 to 0.15 seconds, 0 to 0.1 seconds, and 0.05 to 0.1 seconds. When the signal is a signal acquired at the plurality of time points, the signal acquired first among the plurality of signals is acquired within 0.5 seconds, within 0.3 seconds, within 0.1 seconds, for example. A signal, preferably 0 to 0.2 seconds, 0.05 to 0.2 seconds, 0 to 0.15 seconds, 0.05 to 0.15 seconds, based on the second voltage application time, Signals acquired at 0 to 0.1 seconds and 0.05 to 0.1 seconds. The signal acquired last among the plurality of signals is, for example, a signal acquired within 0.5 seconds, within 0.3 seconds, or within 0.1 seconds, and preferably the second voltage application 0 to 0.2 seconds, 0.05 to 0.2 seconds, 0 to 0.15 seconds, 0.05 to 0.15 seconds, 0 to 0.1 seconds, 0.05 to 0. It is a signal acquired in 1 second. As a specific example, when the target component is glucose, the redox catalyst is glucose dehydrogenase, and the electron mediator is a ruthenium complex, the timing for acquiring the signal is, for example, the second voltage. 0 to 0.2 seconds, 0.05 to 0.2 seconds, 0 to 0.15 seconds, 0.05 to 0.15 seconds, 0 to 0.1 seconds, 0.05 to 0 with reference to the time of application The signal acquired in 1 second.

  When the signal is the signal of the plurality of times, the time interval for acquiring the signal is not particularly limited. For example, the type of the signal, the target component, the first voltage, the second voltage, etc. Can be determined as appropriate. As a specific example, the plurality of signals are signals acquired at intervals of, for example, 0.01 to 10 seconds, 0.05 to 5 seconds, and 0.1 to 3 seconds.

  The measurement method of the present invention may include a calculation step of calculating a target component amount in the sample from the signal. In the calculation step, the target component amount can be calculated based on, for example, the obtained signal and the correlation between the signal and the target component amount in the sample. The correlation can be represented by, for example, a calibration curve, a regression line, a calibration table, or the like. The correlation can be obtained, for example, by plotting the signal obtained by the measurement method of the present invention and the target component amount of the standard sample for a standard sample whose target component amount is known. . The standard sample is preferably a dilution series of the target component. By calculating in this way, reliable quantification is possible. The calculation step can be performed, for example, by calculation means such as a data processing apparatus that is hardware, and specifically, can be performed by the biosensor described above. The data processing device may include a CPU, for example.

  In the measurement method of the present invention, for example, the influence of the interfering substance is reduced, and more reliable measurement is possible. Therefore, the signal may be corrected with a correction signal. Specifically, the measurement method of the present invention includes a correction signal acquisition step of acquiring a correction signal by the electrode system, and a correction step of correcting the signal based on the correction signal. In the calculation step, the correction It is preferable to calculate the amount of the target component in the sample from the subsequent signal.

  The correction signal is, for example, a signal that corrects the influence of the interference substance. Examples of the interfering substance include hematocrit, ascorbic acid, sugar, and blood coagulation inhibitors such as EDTA (ethylenediaminetetraacetic acid). Specifically, the correction signal includes, for example, a signal derived from the interference substance acquired by the electrode system, a signal derived from the target component acquired by the electrode system, and more specifically, the electrode system The signal derived from the said target component acquired by the connected signal acquisition means is mention | raise | lifted.

  When the correction signal is a signal derived from the interference substance, the electrode group used for measuring the correction signal may be the same as or different from the electrode group for measuring the target component, for example. In the latter case, the electrode system includes, for example, an electrode group for measuring the interference substance in addition to the electrode group for measuring the target component. In addition, the reaction system may include, for example, a reagent necessary for obtaining a signal derived from the interference substance according to the type of the interference substance. The method for obtaining the correction signal by the electrode system is not particularly limited, and can be performed by a known method, for example, depending on the type of the interference substance. The method for correcting the signal using the signal derived from the interfering substance is not particularly limited, and examples thereof include a method of correcting based on the correlation between the signal derived from the interfering substance and the fluctuation of the signal. The correlation is, for example, the signal obtained by the measurement method of the present invention and the amount of the target component of the standard sample for a standard sample in which the amount of the target component is known and includes the different interference substance. It can be obtained by plotting.

  When the correction signal is a signal derived from the target component, the correction signal may be a plurality of signals acquired in the signal acquisition step, for example. For the plurality of signals, for example, the above description can be used. The signal correction method using the plurality of signals is not particularly limited, and examples thereof include a correction method based on the relationship between the target component amount and a plurality of signals corresponding thereto. As a specific example, US Pat. No. 8,760,178 can be referred to for the signal correction method. The correction step can be performed, for example, by correction means such as a data processing device that is the hardware described above, and specifically, can be performed by the biosensor described above.

  The target component amount can be calculated based on, for example, the corrected signal and the correlation between the signal and the target component amount in the sample.

<Measurement device for target components>
As described above, the measurement apparatus for a target component of the present invention includes a first voltage (V ₁ ) and a second voltage (in the presence of an electrode system, a sample, a redox catalyst for the target component, and an electron transfer substance ( V ₂ ) to the electrode system, and in the second voltage (V ₂ ) application, the signal acquisition means to acquire a signal from the electrode system, and the first voltage is The voltage is equal to or higher than the oxidation potential (E) of the electron transfer substance, and the second voltage is a voltage lower than the oxidation potential (E) of the electron transfer substance, and is used in the method for measuring the target component of the present invention. It is characterized by that.

  In the measuring apparatus of the present invention, the first voltage applied by the voltage applying means is a voltage equal to or higher than the oxidation potential (E) of the electron transfer substance, and the second voltage is an oxidation of the electron transfer substance. The voltage is less than the potential (E), and is used in the method for measuring a target component of the present invention. Other configurations and conditions are not particularly limited. According to the measuring apparatus of the present invention, for example, the measuring method of the present invention can be easily implemented. For example, the description of the measurement method of the present invention can be used for the measurement device of the present invention.

  Next, examples of the present invention will be described. In addition, this invention is not restrict | limited at all by the following Example.

[Example 1]
An oxidation potential was measured using a biosensor including the redox catalyst, the electron transfer substance, and the electrode system, and it was confirmed that the target component could be measured by the measurement method of the present invention.

(1) Production of Biosensor The biosensor was produced by the following procedure as shown in FIG. First, as shown in FIG. 1A, a PET (polyethylene terephthalate) substrate (length: 50 mm, width: 6 mm, thickness: 250 μm) is prepared as an insulating substrate 11 of a glucose sensor. A carbon electrode system including a working electrode 12 and a counter electrode 13 each having a lead portion was formed by printing.

Next, as shown in FIG. 1B, an insulating layer 14 was formed on the electrode. First, an insulating resin polyester was dissolved in a solvent carbitol acetate so as to be 75% (wt) to prepare an insulating paste, which was screen-printed on the electrode. The printing conditions were a 300 mesh screen and a squeegee pressure of 40 kg, and the amount printed was 0.002 mL per 1 cm ^{2 of} electrode area. Note that screen printing was not performed on the detection unit 15 and the lead units 12a and 13a. Then, the insulating layer 14 was formed by heat treatment at 155 ° C. for 20 minutes.

Further, as shown in FIG. 1C, an inorganic gel layer 16 was formed on the detection portion 15 where the insulating layer 14 was not formed. First, 0.3% (wt) synthetic smectite (trade name “Lucentite SWN”, manufactured by Corp Chemical Co.), 6.0% (wt) ruthenium compound ([Ru (NH ₃ ) ₆ ] Cl ₃ , Dojin) An inorganic gel forming solution (pH 7.5) containing sodium acetate and succinic acid was prepared. 1.0 μL of the inorganic gel forming solution was dispensed into the detection unit 15. The surface area of the detection unit 15 was about 0.6 cm ² , and the surface areas of the electrodes 12 and 13 in the detection unit 15 were about 0.12 cm ² . And this was dried at 30 degreeC and the inorganic gel layer 16 was formed.

  Next, an enzyme layer 17 was formed on the inorganic gel layer 16 as shown in FIG. First, 2.7 U of Aspergillus oryzae FAD-GDH (trade name “Glucosedehydrogenase (FAD-dependent) (GLD-351)”, manufactured by Toyobo Co., Ltd.), 25% (wt) of 1-methoxy PMS (Dojindo Laboratories) And an enzyme solution containing ACES buffer (pH 7.5). 1.0 μL of the enzyme solution was dispensed onto the inorganic gel layer 16 of the detection unit 15 and dried at 30 ° C. to form the enzyme layer 17.

  As shown in FIG. 1E, a spacer 18 having an opening is disposed on the insulating layer 14. Further, as shown in FIG. 1 (F), a biosensor was manufactured by arranging a cover 19 having a through hole 20 serving as an air hole on the spacer 18. Since the space of the opening of the spacer 18 sandwiched between the cover 19 and the insulating layer 14 has a capillary structure, this was used as the sample supply unit 21.

(2) Measurement of oxidation potential Whole blood (Ht value 42%) was introduced into the biosensor, and the current value was measured under the above-described measurement conditions of the oxidation potential. The current value was measured three times using a self-prepared electrochemical measuring instrument.

  The result is shown in FIG. FIG. 2 is a cyclic voltammogram showing the current value under the measurement conditions of the oxidation potential. In addition, in FIG. 2, the electric current value in -0.2-0.2V is shown. In FIG. 2, the horizontal axis represents the potential applied to the electrode system, and the vertical axis represents the response current value. As shown in FIG. 2, a peak was observed at 30 to 50 mV at a positive current value. For this reason, in the said biosensor, it turned out that the oxidation potential of the said electron transfer substance is 30-50 mV.

(3) Measurement of target component 1
Next, a glucose sample 1 having a hematocrit (Ht) value of 42% containing glucose so as to have a predetermined concentration (0, 67, 134, 336, or 600 mg / dL) was prepared.

  For each glucose sample, the first voltage and the first voltage are applied under the following voltage application conditions (1-1) to (1-6) using the biosensor and the electrochemical measuring instrument as the conditions of the examples. A voltage of 2 was applied, and the current value at 0.05 or 0.2 seconds was measured with the second voltage applied as a reference.

(Voltage application condition)
(1-1) First voltage application: 1 V 1 second, second voltage application: 0 V 5 seconds (1-2) First voltage application: 1 V 2 seconds, second voltage application: 0 V 5 seconds (1-3) first 1 voltage application: 350 mV 2 seconds, second voltage application: 0 V 5 seconds (1-4) first voltage application: 350 mV 2 seconds, second voltage application: 10 mV 5 seconds (1-5) first voltage application: 350 mV 2 Second, second voltage application: -100 mV for 5 seconds (1-6) first voltage application: 200 mV for 2 seconds, second voltage application: 0 V for 5 seconds

  These results are shown in FIG. FIG. 3 is a graph showing the current value obtained with the glucose sample. 3A to 3F show the results of the conditions (1-1) to (1-6), respectively. In FIG. 3, the horizontal axis indicates the glucose concentration, the vertical axis indicates the current value, the diamond (◇) in the figure indicates the result for 0.05 seconds, and the square (□) indicates 0.2 seconds. The results are shown. As shown in FIGS. 3A to 3F, it was found that the glucose concentration in the glucose sample and the current value showed a negative correlation under any voltage condition and signal acquisition time. Since the glucose concentration and the current value show a negative correlation, it can be said that the glucose concentration in the sample can be calculated from the current value obtained from the sample. Therefore, it was found that the target component including glucose can be measured by the measurement method of the present invention.

(4) Measurement of target component 2
A glucose sample 2 having a hematocrit (Ht) value of 42% containing glucose at a predetermined concentration (0, 67, 336, or 600 mg / dL) was prepared. And about each glucose sample, it replaced with said conditions (1-1)-(1-6) as conditions of an Example except having set it as the voltage application conditions of the following (2-1)-(2-3). In the same manner as in the above (3), the current value at 0.05, 0.2, or 0.4 seconds was measured.

(Voltage application condition)
(2-1) First voltage application: 1 V 2 seconds, second voltage application: 0 V 5 seconds (2-2) First voltage application: 1 V 2 seconds, second voltage application: -200 mV 5 seconds (2-3) First voltage application: 350 mV for 2 seconds, second voltage application: -200 mV for 5 seconds

  These results are shown in FIG. FIG. 4 is a graph showing the current value obtained with the glucose sample. 4A to 4C show the results of the conditions (2-1) to (2-3), respectively. In FIG. 4, the horizontal axis indicates the glucose concentration, the vertical axis indicates the current value, the diamond (◇) in the figure indicates the result of 0.05 seconds, and the square (□) indicates 0.2 seconds. The triangle (Δ) indicates the result for 0.4 seconds. As shown in FIGS. 4A to 4C, it was found that the glucose concentration in the glucose sample and the current value showed a negative correlation under any voltage condition and signal acquisition time. Since the glucose concentration and the current value show a negative correlation, it can be said that the glucose concentration in the sample can be calculated from the current value obtained from the sample. Therefore, it was found that the target component including glucose can be measured by the measurement method of the present invention.

[Example 2]
It was confirmed that the influence of hematocrit in the measurement of the target component was reduced by the measurement method of the present invention.

  A glucose sample 3 with a predetermined Ht value (0, 20, 42, or 70%) containing 336 or 600 mg / dL glucose was prepared. And, in place of the glucose sample 1, except that the glucose sample 3 was used, the same as in the condition (1-2) of Example 1 (3), with the second voltage applied as a reference, The current value at 0.2 seconds was measured. Then, based on the following formula (1), the degree of influence of the Ht value was calculated based on the current value of the glucose sample 3 having an Ht value of 42%. In addition, instead of the first voltage and the second voltage, the control is performed by applying a voltage of 200 mV to the biosensor as a comparative example condition for 5 seconds and using the voltage application start time as a reference. Except for measuring the current value in seconds, the degree of influence of the Ht value was calculated in the same manner.

E _h = (S _g / B _g ) × 100-100 (1)
E _h : Influence level of Ht value S _g : Current value of glucose sample B _g : Current value of glucose sample with 42% Ht value

  These results are shown in FIG. FIG. 5 is a graph showing the degree of influence of the Ht value. In FIG. 5, (A) shows the result of the glucose sample containing 336 mg / dL glucose in the example, (B) shows the result of the glucose sample containing 600 mg / dL glucose in the example, and (C ) Shows the result of the control. In FIG. 5, the horizontal axis indicates the Ht value, and the vertical axis indicates the degree of influence of the Ht value. As shown in FIG. 5C, when a constant voltage was applied, the degree of influence of the Ht value on the obtained current value increased as the Ht value moved away from 42%. On the other hand, as shown in FIGS. 5A and 5B, when a first voltage equal to or higher than the oxidation potential and a second voltage lower than the oxidation potential were applied, it was obtained at any Ht value. The degree of influence of the Ht value on the current value was low. From the above, it has been found that the measurement method of the present invention reduces the influence of hematocrit.

[Example 3]
When various first voltages and second voltages that satisfy the conditions of the first voltage and the second voltage in the measurement method of the present invention are applied, the influence of hematocrit in the measurement of the target component is reduced. It was confirmed.

  Using the glucose sample 3 containing 336 mg / dL of glucose, the first voltage and the second voltage were applied under the voltage application conditions of the following (3-1) to (3-11) as conditions of the example. The current value at 0.05 or 0.2 seconds was measured in the same manner as in Example 1 (3) except that the second voltage was applied. And about each measurement time, it carried out similarly to the said Example 2, and calculated the influence degree of Ht value. Further, the control calculated the degree of influence of the Ht value in the same manner except that the voltage was applied under the following conditions (3-12) to (3-14) as the conditions of the comparative example. In the conditions (3-4), (3-5), (3-13), and (3-14), a current value at 0.4 seconds is measured with the second voltage applied as a reference. Similarly, the degree of influence of the Ht value was calculated.

(Voltage application condition)
(3-1) First voltage application: 1 V 2 seconds, second voltage application: 0 V 2 seconds (3-2) first voltage application: 500 mV 2 seconds, second voltage application: 0 V 2 seconds (3-3) first 1 voltage application: 1 V 5 seconds, second voltage application: 0 V 2 seconds (3-4) first voltage application: 1 V 2 seconds, second voltage application: 0 V 7 seconds (3-5) first voltage application: 1 V 2 Second, second voltage application: 10 mV for 7 seconds (3-6) first voltage application: 1 V for 1 second, second voltage application: 0 V for 5 seconds (3-7) first voltage application: 1 V for 2 seconds, second voltage Application: 0 V 5 seconds (3-8) First voltage application: 350 mV 2 seconds, second voltage application: −100 mV 5 seconds (3-9) First voltage application: 350 mV 2 seconds, second voltage application: 0 V 5 seconds ( 3-10) First voltage application: 350 mV for 2 seconds, second voltage application: 10 mV for 5 seconds (3-11) first voltage mark : 200 mV for 2 seconds, second voltage application: 0 V for 5 seconds (3-12) first voltage application: 1 V for 2 seconds, second voltage application: 200 mV for 7 seconds (3-13) first voltage application: 1 V for 2 seconds, second Voltage application: 100 mV for 7 seconds (3-14) first voltage application: -200 mV for 2 seconds, second voltage application: 0 V for 7 seconds

  These results are shown in FIGS. 6 and 7 are graphs showing the degree of influence of the Ht value. 6, (A) to (K) show the results of the conditions (3-1) to (3-11), respectively. In FIG. 7, (A) to (C) respectively show the conditions. The results of (3-12) to (3-14) are shown. 6 and 7, the horizontal axis indicates the Ht value, the vertical axis indicates the degree of influence of the Ht value, the rhombus (◇) in the figure indicates the result for 0.05 seconds, and the square (□ ) Indicates a result of 0.2 seconds, and a triangle (Δ) indicates a result of 0.4 seconds. As shown in FIGS. 6A to 6K, in the case of conditions (3-1) to (3-11) where a first voltage equal to or higher than the oxidation potential and a second voltage lower than the oxidation potential are applied, The degree of influence of the Ht value on the obtained current value was small even at the Ht value and the measurement time. On the other hand, as shown in FIGS. 7A to 7C, conditions (3-12) and (3-13) in which the first voltage and the second voltage equal to or higher than the oxidation potential are applied, and the oxidation potential. In the case of the condition (3-14) in which the first voltage and the second voltage below were applied, the degree of influence of the Ht value on the obtained current value increased as the Ht value moved away from 42%. That is, it was greatly affected by the Ht value. Further, as shown in FIGS. 6 (I) and (K), when the first voltage is set to a voltage close to the oxidation potential of the electron transfer substance, the influence of hematocrit can be further reduced. Furthermore, as shown in FIGS. 6 (H) to (J), when the second voltage is set to a voltage close to the oxidation potential of the electron transfer substance, the influence of hematocrit can be further reduced. From the above, even when various first voltages and second voltages that satisfy the conditions of the first voltage and the second voltage in the measurement method of the present invention are applied, the hematocrit in the measurement of the target component is applied. It has been found that the influence can be reduced, and that the influence of hematocrit can be further reduced by making the first voltage and the second voltage closer to the oxidation potential of the electron transfer substance.

[Example 4]
It was confirmed that the influence of ascorbic acid in the measurement of the target component was reduced by the measurement method of the present invention.

  A blood sample containing 336 mg / dL glucose and 0 or 6 mg / dL ascorbic acid was prepared using venous blood collected on the day before the measurement. The oxygen partial pressure of the blood sample was 30-70 mmHg.

  Then, in place of the glucose sample 1, the blood sample was used, and the current value at 0.05 or 0.2 seconds was measured with reference to the time when the second voltage was applied. The current value was measured in the same manner as in the condition (1-2). The current value was measured for each blood sample by 10 samples. Based on the following formula (2), the degree of influence of ascorbic acid when the current value of the blood sample containing 0 mg / dL of ascorbic acid was used as a reference was calculated. Further, a coefficient of variation (CV value) was calculated based on the current values of the 10 samples. Furthermore, instead of the first voltage and the second voltage, the control applies a voltage of 200 mV to the biosensor for 20 seconds, and measures a current value at 5 seconds with reference to the voltage application start time. Except for the above, the degree of influence of ascorbic acid and the CV value were calculated in the same manner.

E _a = (S _a / B _a ) × 100-100 (2)
E _a : Influence level of ascorbic acid S _a : Current value of blood sample B _a : Current value of blood sample of ascorbic acid 0 mg / dL

  The results of the degree of influence of ascorbic acid are shown in FIG. FIG. 8 is a graph showing the degree of influence of ascorbic acid. In FIG. 8, (A) shows the results of the example, and (B) shows the results of the control. In FIG. 8, the horizontal axis indicates the concentration of ascorbic acid (AsA (mg / dL)), and the vertical axis indicates the degree of influence of ascorbic acid. As shown in FIG. 8A, when a first voltage equal to or higher than the oxidation potential and a second voltage lower than the oxidation potential are applied, the influence of ascorbic acid on the obtained current value regardless of the presence or absence of ascorbic acid. The degree was low. On the other hand, as shown in FIG. 8B, when ascorbic acid was included when a constant voltage was applied, the degree of influence of the ascorbic acid on the obtained current value increased.

  The result of CV value is shown in FIG. FIG. 9 is a graph showing the CV value. In FIG. 9, (A) shows the results of the example, and (B) shows the results of the control. In FIG. 9, the horizontal axis indicates the ascorbic acid concentration, and the vertical axis indicates the CV value (CV (%)). As shown in FIG. 9A, when a first voltage not lower than the oxidation potential and a second voltage lower than the oxidation potential are applied, the CV value of the obtained current value is constant regardless of the presence or absence of ascorbic acid. Met. In contrast, as shown in FIG. 9B, when a constant voltage was applied, the CV value of the obtained current value increased when ascorbic acid was included.

  From the above, it was found that the influence of ascorbic acid was reduced in the measurement method of the present invention. It was also found that the measurement method of the present invention can measure the target component with good reproducibility.

  As mentioned above, although this invention was demonstrated with reference to embodiment and an Example, this invention is not limited to the said embodiment and Example. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

  This application claims the priority on the basis of Japanese application Japanese Patent Application No. 2015-238533 for which it applied on December 7, 2015, and takes in those the indications of all here.

  According to the present invention, glucose and other target components can be measured. For this reason, it can be said that the present invention is extremely useful in the analysis field, the clinical field, and the like.

Claims (14)

A first application step of applying a _first voltage (V ₁ ) to the electrode system in the presence of a sample, a redox catalyst for the target component, and an electron transfer material;
A second application step of applying a _second voltage (V ₂ ) to the electrode system;
In the second application step, including a signal acquisition step of acquiring a signal by the electrode system,
The first voltage is equal to or higher than an oxidation potential (E) of the electron transfer material;
The method for measuring a target component, wherein the second voltage is a voltage lower than an oxidation potential (E) of the electron transfer substance. The method for measuring a target component according to claim 1, wherein the first voltage (V ₁ ) is a voltage in a range of E ≦ V ₁ ≦ E + 1.15. The method for measuring a target component according to claim 1, wherein the second voltage (V ₂ ) is a voltage in a range of E−0.25 ≦ V ₂ <E. The method for measuring a target component according to any one of claims 1 to 3, wherein the signal is a signal acquired within 0.4 seconds on the basis of the time when the second voltage is applied. The method for measuring a target component according to any one of claims 1 to 4, wherein the signal is a signal acquired at one time point. The method for measuring a target component according to any one of claims 1 to 4, wherein the signal is a signal obtained continuously. The method for measuring a target component according to any one of claims 1 to 4, wherein the signal is a signal acquired at a plurality of time points. The method for measuring a target component according to any one of claims 1 to 7, wherein the target component is glucose. The method for measuring a target component according to any one of claims 1 to 8, wherein the redox catalyst is an oxidoreductase or a dehydrogenase. The method for measuring a target component according to any one of claims 1 to 9, wherein the sample is a biological sample. The measuring method of the target component as described in any one of Claim 1 to 10 including the calculation process of calculating the target component amount in the said sample from the said signal. A correction signal acquisition step of acquiring a correction signal by the electrode system;
A correction step of correcting the signal based on the correction signal,
The method for measuring a target component according to claim 11, wherein, in the calculation step, a target component amount in the sample is calculated from the corrected signal. It said first voltage (V ₁₎ is a E <V _1, the measuring method of the target component according to any one of claims 1 to 12. An electrode system;
Voltage application means for applying a _first voltage (V ₁ ) and a second voltage (V ₂ ) to the electrode system in the presence of a sample, a redox catalyst for the target component, and an electron transfer material;
Signal acquisition means for acquiring a signal from the electrode system in applying the second voltage (V ₂ ),
The first voltage is equal to or higher than an oxidation potential (E) of the electron transfer material;
The second voltage is a voltage less than an oxidation potential (E) of the electron transfer material;
An apparatus for measuring a target component, which is used in the method for measuring a target component according to any one of claims 1 to 13.