Measurement device and measurement method

Abstract

This measurement device measures the concentration of a specimen by measuring a current corresponding to a temporal change in the concentration of a measurement substance generated as a result of a multi-step reaction occurring in the specimen in response to a solution containing at least the specimen being dropped onto a first electrode. The measurement device applies a voltage to the first electrode after a lapse of time from the drop of the solution onto the first electrode until the reaction reaches a steady state within a range in which the reaction does not affect the measurement accuracy (S13, S17). The measurement device measures the concentration of the specimen using the current flowing in response to the application of the voltage (S21). The measurement device measures the concentration of the specimen under a condition that allows an intermediate reaction in the multi-step reaction to proceed at a rate greater than or equal to a predetermined reaction rate. The predetermined reaction rate is a rate necessary for maintaining the accuracy of conversion of the current to the concentration of the measurement substance on the assumption that the initial concentration of the specimen and the rate of change of the measurement substance are linearly related.

Description

Measuring device and measuring method

[0001] The present invention relates to a measurement device and a measurement method.

[0002] There is known a device that measures the concentration of an analyte by measuring a current that flows based on an electrochemical reaction of the analyte. Patent Document 1 discloses a sensor system including a measurement device and a sensor strip. The sensor strip includes a working electrode and a counter electrode. A sample containing an analyte such as glucose is introduced into the sensor strip. The measurement device applies a pulse voltage to the working electrode and counter electrode of the sensor strip into which the sample has been introduced. The measurement device acquires a contour plot showing the relationship between the voltage value of the pulse voltage and the value of the current that flows when the pulse voltage is applied. The measurement device determines the concentration of the analyte in the sample based on the relationship between the acquired contour plot and contour plots previously measured for each analyte concentration.

[0003] Special Publication No. 2009-510434

[0004] The inventors of the present application have found that when an electrochemical reaction of an analyte occurs in multiple stages, if the reaction rate of the intermediate reaction in the multistage reaction is sufficiently fast, a linear relationship is maintained between the initial concentration of the analyte and the rate of change of the measured substance generated by the electrochemical reaction.

[0005] An object of the present invention is to provide a measuring device and a measuring method that are capable of measuring the concentration of an analyte when the electrochemical reaction of the analyte occurs in multiple stages due to the application of a voltage.

[0006] A measuring device according to a first aspect of the present invention is a measuring device that measures the concentration of an analyte by measuring a current corresponding to the change over time in the concentration of a measured substance that occurs as a result of a multi-stage reaction in the analyte in response to the dripping of a solution containing at least the analyte onto a first electrode, and that comprises: an application means that applies a voltage to the first electrode after a time has elapsed since the dripping of the solution onto the first electrode, when the reaction has reached a steady state within a range that does not affect the measurement accuracy; and a measurement means that measures the concentration of the analyte using the current that flows in response to the application of the voltage by the application means, wherein the measurement means measures the concentration of the analyte under conditions in which an intermediate reaction in the multi-stage reaction reacts at a rate equal to or greater than a predetermined reaction rate, and the predetermined reaction rate is a rate required to maintain the accuracy of converting a current into the concentration of the measured substance, assuming that the initial concentration of the analyte and the rate of change of the measured substance are linear.

[0007] The measuring device according to the first aspect can accurately measure the concentration of an analyte even when the electrochemical reaction of the analyte occurs in multiple stages by utilizing the fact that the initial concentration of the analyte and the rate of change of the measured substance maintain a linear relationship.

[0008] In a first aspect, an enzyme membrane containing a plurality of enzymes may be formed on the first electrode, and the multi-stage reaction may include at least a first reaction that occurs within the enzyme membrane as a result of a reaction between a first enzyme of the plurality of enzymes and the analyte, and a second reaction that occurs within the enzyme membrane as a result of a reaction between a second enzyme of the plurality of enzymes and a product of the first reaction. In this case, a user can measure the concentration of the analyte using the measurement device by dropping a solution containing at least the analyte onto the enzyme membrane containing the first enzyme and the second enzyme.

[0009] In a first aspect, the analyte may be inosinic acid, the first enzyme may be inosine phosphate dehydrogenase, and the second enzyme may be reduced nicotinamide adenine dinucleotide oxidase. In this case, the measuring device can accurately measure the concentration of inosinic acid by utilizing the fact that a multi-stage reaction occurs in inosinic acid.

[0010] In the first aspect, the first electrode is a working electrode, and the measuring device may further include a second electrode that is a counter electrode and a third electrode that is a reference electrode. In this case, the measuring device can accurately measure the concentration of the analyte by adopting a three-electrode method.

[0011] A second aspect of the present invention provides a measurement method for measuring the concentration of an analyte by measuring a current corresponding to a change in concentration of the analyte over time, which occurs as a result of a multistage reaction in the analyte in response to the dripping of a solution containing at least the analyte onto a first electrode, the method comprising: applying a voltage to the first electrode after a time has elapsed since the dripping of the solution onto the first electrode, when the reaction has reached a steady state within a range that does not affect measurement accuracy; and measuring the concentration of the analyte using the current that flows in response to the application of the voltage by the applying step, wherein the measurement step measures the concentration of the analyte under conditions in which an intermediate reaction in the multistage reaction reacts at a predetermined reaction rate, the predetermined reaction rate being a rate required to maintain accuracy in converting a current into the concentration of the analyte, assuming that the initial concentration of the analyte and the rate of change of the analyte are linear. The second aspect can achieve the same effects as the first aspect.

[0012] 1 is a schematic diagram showing a measurement device 3 and a block diagram showing an electrical configuration; FIG. 2 is an enlarged view of an electrode 32; FIG. 3 is a graph showing a simulation result regarding a first reaction; FIG. 4 is a graph showing a simulation result regarding a second reaction; FIG. 5 is a flowchart of a main process; FIG. 6 is a graph showing a change over time in voltage applied to an electrode 32A; and FIG. 7 is a table showing experimental results.

[0013] An embodiment of the present invention will be described below with reference to the drawings. The drawings are used to explain technical features that can be adopted by the present invention. The configurations of the described devices are merely illustrative examples and are not intended to be limiting.

[0014] <Summary> Inosinic acid (IMP) is one of the important components that creates the "umami" flavor in foods. IMP is found in abundance in animal foods, particularly meat and fish, and is produced during the aging and cooking process of ingredients. "Umami" affects the sense of taste and enriches the flavor of food. For this reason, it is desirable to accurately measure IMP in foods.

[0015] Furthermore, IMP is an important intermediate involved in the synthesis of nucleic acids in cells, and its metabolism plays an important role in intracellular biochemical processes.

[0016] First, IMP binds to the coenzyme nicotinamide adenine dinucleotide (NAD ＋ Under the influence of ATP, IMP is degraded by the enzyme IMP dehydrogenase (IMPDH). This reaction converts IMP to its oxidized form, inosine monophosphate (XOP), and simultaneously converts it to the coenzyme NAD. ＋ is reduced to produce reduced nicotinamide adenine dinucleotide (NADH). Hereinafter, this reaction will be referred to as the "first reaction." IMP + NAD ＋ + H ２ O → XOP + NADH + H ＋

[0017] The first reaction plays an important role in regulating IMP, a key intermediate in the nucleic acid synthesis pathway. By regulating IMP metabolism, cells can regulate the rate and amount of nucleic acid synthesis as needed.

[0018] NADH is then broken down by the enzyme reduced nicotinamide adenine dinucleotide oxidase (NOD). ＋ and at the same time hydrogen and oxygen (O ２ ) is consumed. Hereafter, this reaction is referred to as the "second reaction." NADH + O ２ + 2H ＋ →NAD ＋ + H ２ O ２

[0019] In the second reaction, NADH is converted back to NAD ＋converted to NAD ＋ The ratio of NAD / NADH is maintained. ＋ Regeneration of ATP is necessary for intracellular energy metabolism.

[0020] In other words, the first and second reactions occur in multiple steps, resulting in the production of NAD, which is necessary for regulating nucleic acid synthesis. ＋ The ratio of IMPDH to NADH is maintained. This process is important for maintaining the balance of metabolic pathways within the cell and maintaining normal cell function. The multi-step reaction system mediated by two enzymes (IMPDH and NOD) plays an important role in many biological processes.

[0021] In the above reaction system, one molecule of IMP is consumed in the first reaction, and O is consumed in the second reaction. ２ One molecule of IMP is consumed. ２ There is a correlation between the concentration of each and the amount of consumption.

[0022] <Outline and Electrical Configuration of Measuring Device 3> The measuring device 3 shown in FIG. 1 is a device for measuring O2 by a second reaction in association with the consumption of IMP by a first reaction. ２ The measuring device 3 is an apparatus for measuring the concentration of IMP by an electrochemical method, utilizing a multi-stage reaction in which IMP is consumed. The measuring device 3 includes a sensor chip 3A and a main body 3B.

[0023] The sensor chip 3A has a measuring section 31 and an electrode 32. A solution containing two enzymes (IMPDH and NOD) is applied to the surface of the electrode 32 and then dried to form a thin film (hereinafter referred to as "enzyme film 10"). The enzyme film 10 contains IMPDH and NAD. ＋ A solution containing IMP (hereinafter referred to as "containing solution 30") is dropped onto the electrode 32. The measuring unit 31 is capable of measuring the current value that flows when a voltage is applied to the electrode 32. The measured current value is output to the main body 3B. The main body 3B measures the concentration of IMP contained in the containing solution 30 based on the current value output from the sensor chip 3A.

[0024] The main body 3B includes a CPU 21, a storage device 22, a display device 23, an input device 24, and an interface device 25. The CPU 21 controls the entire measurement device 3. The storage device 22 stores programs executed by the CPU 21, various parameters, various tables, and measurement results. The display device 23 displays the measured IMP concentration. The input device 24 accepts input operations to the main body 3B. The interface device 25 communicates with the sensor chip 3A.

[0025] As shown in Figure 2, the electrodes 32 of the sensor chip 3A include an electrode 32A having an enzyme membrane 10 on its surface, as well as electrodes 32B and 32C. The electrode 32A is a working electrode, the electrode 32B is a counter electrode, and the electrode 32C is a reference electrode. When the containing solution 30 is dropped onto the enzyme membrane 10, the IMP contained in the containing solution 30 is oxidized by IMPDH in the enzyme membrane 10 and converted to XOP (arrow Y11). At the same time, NAD ＋ is reduced to produce NADH (arrow Y12). The produced NADH is then decomposed by NOD in the enzyme membrane 10 to produce NAD. ＋ At the same time, hydrogen and O in the containing solution 30 are converted into ２ is consumed to produce hydrogen peroxide (arrow Y22). Note that arrows Y11 and Y12 correspond to the first reaction, and arrows Y21 and Y22 correspond to the second reaction.

[0026] The CPU 21 (see FIG. 1) of the main body 3B outputs a signal specifying the voltage to be applied to the electrode 32 to the measurement unit 31 (see FIG. 1) of the sensor chip 3A. As shown in FIG. 2, the measurement unit 31 applies the specified voltage to the electrode 32A in response to the signal, using the electrodes 32B and 32C as references. At this time, the O remaining in the containing solution 30 is ２ The enzyme permeates the enzyme membrane 10 of the electrode 32A and reaches the electrode 32A. ２ The electrons combine with the electrons supplied from the electrode 32A and are consumed (arrow Y3). Hereinafter, the application of a voltage to the electrode 32A by the CPU 21 outputting a signal to the sensor chip 3A will be rephrased as "the CPU 21 applies a voltage to the electrode 32A."

[0027] The measuring unit 31 outputs a signal indicating the value of the current that flows in response to the application of a voltage to the electrode 32A to the main body 3B. The CPU 21 of the main body 3B calculates the amount of O consumed based on the current value indicated by the signal received from the sensor chip 3A. ２ In other words, the concentration of O remaining in the containing solution 30 ２ That is, the measurement device 3 can identify the concentration of O in the containing solution 30, which is generated as a result of the second reaction. ２ The change in concentration of O ２ This can be determined by measuring the current value of the current generated by the consumption of

[0028] In addition, O ２ The O in the containing solution 30 due to consumption of ２ The residual concentration of IMP decreases as the IMP contained in the IMP-containing solution 30 is decomposed by the first reaction. There is a correlation between the concentration of IMP contained in the IMP-containing solution 30 and the current value of the current that flows in response to the application of a voltage to the electrode 32A. Therefore, the CPU 21 can measure the concentration of IMP contained in the IMP-containing solution 30 based on the current value indicated by the signal received from the sensor chip 3A. Hereinafter, the CPU 21 obtaining the current value by receiving a signal from the sensor chip 3A will be rephrased as "the CPU 21 measuring the current value of the current that flowed through the electrode 32A."

[0029] <Basic equations> IMP, NAD ＋ , NADH, and O ２ In the following, I, N, A, and C are IMP, NAD, respectively. ＋ , NADH, and O ２ The molar concentrations of each are shown. Ｉ , D Ｎ , D Ａ , and D Ｃ are IMP and NAD, respectively. ＋ , NADH, and O ２ The diffusion coefficients of each of the Ｉ , k Ｎ , k Ａ , and k Ｃ are IMP and NAD, respectively. ＋ , NADH, and O ２where L is the thickness of the enzyme membrane 10. y is the height non-dimensionalized by the thickness of the enzyme membrane 10. The reference position of y, "0," is the portion of the enzyme membrane 10 that is in contact with the electrode 32A.

[0030] The basic equation that takes into account the diffusion of IMP is IMP and NAD ＋ Considering the reaction (first reaction) with, it can be expressed by the following formula (1-1).

[0031] NAD ＋ The basic equation considering the diffusion of IMP and NAD is ＋ Considering that the and decrease by the same amount, it is expressed by the following formula (1-2).

[0032] The basic equation considering the diffusion of NADH is that NADH is converted to IMP and NADH by the first reaction. ＋ The amount of NADH and O produced in the second reaction is equal to the amount of O ２ Considering the reaction, it is expressed by the following formula (1-3).

[0033] Also, O ２ The basic equation taking into account the diffusion of is expressed by the following equation (1-4).

[0034] Here, it is difficult to further simplify the equations (1-1) to (1-4), and it is also difficult to derive analytical solutions. For this reason, we will attempt to derive analytical solutions using a numerical method.

[0035] Considering that I, N, A, and C are functions of time t and height y, the boundary condition for I is defined as in equation (2-1).

[0036] Moreover, the boundary condition for N is defined as in equation (2-2).

[0037] The boundary condition for A is defined as follows by equation (2-3).

[0038] The boundary condition for C is defined as follows by equation (2-4).

[0039] The reason why the first derivatives of all variables are identically set to 0 when y=0 is that no mass transfer by diffusion occurs in the contact portion of the enzyme membrane 10 with the electrode 32A.

[0040] Next, we will attempt to derive a steady-state theoretical solution based on the equations defined above. When performing a steady-state approximation, i.e., an approximation without time change, the partial differential equation for I is differentiated as shown in equation (3-1).

[0041] Moreover, the partial differential equation for N is ordinary differentiated as in equation (3-2).

[0042] Moreover, the partial differential equation for A is differentiated into ordinary differential equations as shown in equation (3-3).

[0043] Moreover, the partial differential equation for C is differentiated into ordinary differential equations as shown in equation (3-4).

[0044] From equations (3-1), (3-3), and (3-4), k Ｉ IN and k Ａ By eliminating AC, equation (4-1) is derived.

[0045] Here, NADH to NAD ＋ If we make the approximation that the reaction converting NADH into NADH, i.e., the second reaction, is extremely fast and NADH is quickly consumed, so that no concentration distribution occurs, then equations (4-2) and (4-3) can be derived from equation (4-1).

[0046] Equation (4-3) can be simplified and expressed as equation (5-1), where g represents the diffusion coefficient ratio.

[0047] Here, C is replaced by C ０ and I is made dimensionless by I ０ When non-dimensionalized, it is expressed as equation (5-2). ０ Is O２ indicates the initial concentration of I ０ indicates the initial concentration of IMP.

[0048] By integrating both sides of the differential equation shown in equation (5-2) twice, equation (5-3) is derived, where S and B are integral constants.

[0049] Here, y=1, that is, the surface of the enzyme membrane 10 is normalized as C=1 and I=1 as described above, so the relationship of the following equation (5-4) holds.

[0050] Equation (5-4) can be transformed into equation (5-5).

[0051] By differentiating both sides of equation (5-5) with respect to y, equation (5-6) is derived. The boundary conditions are defined as in equation (5-7).

[0052] Substituting equation (5-7) into equation (5-6), equation (5-8) is derived.

[0053] From the above, equation (5-9) is derived.

[0054] When I is derived based on equation (5-9), it is expressed as equation (5-10).

[0055] Next, when A in equation (3-3), that is, the second derivative term of NADH, is approximated to 0, equation (6-1) is derived.

[0056] Furthermore, formula (6-1) is NAD ＋ If we assume that the concentration of IMP is in excess of that of IMP, then equation (6-2) can be derived.

[0057] Substituting equation (6-2) into equation (3-4), equation (6-3) is derived.

[0058] By substituting the equation (6-2) into the equation (6-3), the equation (6-4) is derived.

[0059] What is needed here is the oxygen concentration on electrode 32A. If we limit it to electrode 32A, the left side of equation (6-3) becomes 0. In other words, since diffusion does not occur under the boundary conditions, the relationship of equation (6-5) is satisfied.

[0060] By solving equation (6-5) for C, equation (6-6) is derived.

[0061] If g in equation (6-6) is redefined as an inverse number, it is expressed as equation (6-7).

[0062] From the above, we can derive equation (6-8) as the steady-state solution.

[0063] From equation (6-8), the initial concentration of IMP, I ０ and O ２ rate of change (C / C ０ ) is completely linear. This relationship is based on the approximation that the second reaction is extremely fast, NADH is consumed immediately, and no concentration distribution occurs. In other words, when a voltage is applied to the electrode 32A after a predetermined time has elapsed after the infusion of the solution 30 onto the enzyme membrane 10, and the concentration of IMP is measured using the current that flows at that time, the initial concentration of IMP I ０ and O ２ rate of change (C / C ０ Provided that the second reaction occurs fast enough that the relationship between current and IMP is linear, the current can be accurately converted to IMP concentration.

[0064] That is, even in a complicated multi-stage reaction, in the steady state solution on the electrode 32A, the initial concentration of IMP I ０ and O ２ rate of change (C / C ０ ) is linear, and the slope of the linear relationship is expressed by g, which is the diffusion coefficient ratio.

[0065] <Simulation Results> Figures 3 and 4 show the initial concentration of IMP, ０ and O ２ rate of change (C / C ０) for each reaction rate coefficient k. In this simulation, equations (1-1) to (1-4) were numerically solved to derive the oxygen concentration reduction ratio after a sufficient elapsed time (100 seconds or more). Figure 3 shows the reaction rate coefficient of the first reaction occurring in the presence of IMPDH, calculated as 7.00 × 10 －４ , 1.00 x 10 －３ , 3.00 x 10 －３ , 1.00 x 10 －２ 4 shows the results when the reaction rate coefficient of the second reaction occurring in the presence of NOD was set to 7.00 × 10 －４ , 1.00 x 10 －３ , 3.00 x 10 －３ , 1.00 x 10 －２ The results for each setting are shown below.

[0066] As shown in Figure 3, in the case of the first reaction, the curves corresponding to the multiple reaction rate coefficients overlapped with each other and showed almost the same tendency. This indicates that even if the reaction rate of the first reaction changes, the initial concentration of IMP, I ０ and O ２ rate of change (C / C ０ ) is almost maintained. On the other hand, as shown in Figure 4, in the case of the second reaction, the curves corresponding to the multiple reaction rate coefficients were different from each other and showed different trends.

[0067] From the results shown in Figures 3 and 4, the initial concentration of IMP I ０ and O ２ rate of change (C / C ０ ) also changed significantly. In particular, the reaction rate of the second reaction changed significantly with the initial concentration of IMP, I ０ It has become clear that this has a significant impact on the measurement accuracy of IMP. This is because the initial concentration IMP is not measured when the approximation in the process of deriving the above theory, i.e., the assumption that the reaction rate of the second reaction is sufficiently fast, is not established. ０ and O ２ rate of change (C / C ０ ) is nonlinear.

[0068] 5, a description will be given of the main processing executed by the CPU 21 of the measurement device 3. When the measurement device 3 is powered on, the CPU 21 starts the main processing by reading and executing a program stored in the storage device 22.

[0069] The CPU 21 determines whether the containing solution 30 has been dripped onto the electrode 32A by the user (S11). Here, when the user drips the containing solution 30 onto the electrode 32A, the user simultaneously performs an input operation via the input unit 24 to indicate that the dripping has been performed. If the CPU 21 does not detect an input operation via the input unit 24, it determines that the containing solution 30 has not been dripped onto the electrode 32A (S11: NO). In this case, the CPU 21 returns to S11 and continues to determine whether the containing solution 30 has been dripped onto the electrode 32A. On the other hand, if the CPU 21 detects an input operation via the input unit 24, it determines that the containing solution 30 has been dripped onto the electrode 32A (S11: YES). In this case, the CPU 21 proceeds to S13.

[0070] By dropping the containing solution 30 onto the electrode 32A, a first reaction occurs in the presence of IMPDH and a second reaction occurs in the presence of NOD on the enzyme membrane 10 of the electrode 32A. ０ and O ２ rate of change (C / C ０ ) is linear (hereinafter referred to as "predetermined reaction rate"). Note that, if the approximation assumed when deriving equations (4-2) and (4-3) from equation (4-1), that is, the second reaction is extremely fast and NADH is consumed immediately, so that no concentration distribution occurs, is valid, then the initial concentration I ０ and O ２ rate of change (C / C ０ ) shows a linear relationship.

[0071] Here, for example, an enzyme with a fast reaction rate may be selected so that the second reaction satisfies the above condition. Furthermore, since it is obvious that the reaction rate is proportional to the amount of enzyme, for example, the content ratio of the enzyme in the first reaction may be reduced and the content ratio of the enzyme in the second reaction may be increased so that the second reaction satisfies the above condition.

[0072] The CPU 21 applies a first voltage V(1) to the electrode 32A for a first application time Ta(1) at the timing when it is determined that the containing solution 30 has been dropped onto the electrode 32A, more specifically, after a first elapsed time Tp(1) has elapsed since the containing solution 30 was dropped onto the electrode 32A (S13, see FIG. 6). ２ is consumed by electrolysis. It takes time Tn for the voltage of the electrode 32A to change from 0 V to the first voltage V(1). The time Tn is set to the time when O2 consumed by electrolysis is reduced to a level that does not affect the measurement accuracy. ２ The amount will be adjusted to reduce consumption.

[0073] The first elapsed time Tp(1) is the time required for the reaction occurring after the dropping of the containing solution 30 onto the electrode 32A to reach a steady state within a range that does not affect measurement accuracy. This time is predetermined as a range of time within which the progress of the reaction occurring after the dropping of the containing solution 30 onto the electrode 32A over time does not affect measurement accuracy. More specifically, the first elapsed time Tp(1) is predetermined as the time after the dropping of the containing solution 30 onto the electrode 32A and the initiation of a first reaction occurring in the presence of IMPDH and a second reaction occurring in the presence of NOD on the enzyme membrane 10 of the electrode 32A.

[0074] The first voltage V(1) and the first application time Ta(1) are set to a value that is proportional to the amount of O consumed by electrolysis caused by application of a voltage to the electrode 32A. ２ The amount of ２ The value is previously defined as a range that does not affect the measurement accuracy of the concentration of IMP. ２ Only O is consumed, and the concentration gradient is formed, causing O to flow toward the electrode 32A. ２ is diffused, and the consumed O ２ More specifically, the first voltage V(1) and the first application time Ta(1) are set to recover the O supplied by diffusion. ２ The consumed O ２ is predefined as a value that can recover the

[0075] 8, the CPU 21 measures the current value of the current flowing through the electrode 32A while the first voltage V(1) is being applied to the electrode 32A in the process of S13 (S15). The CPU 21 calculates the integrated value of the current when the first voltage V(1) is applied to the electrode 32A or the current flowing through the electrode 32A while the first voltage V(1) is being applied as the first current i ０ and stores the measured first current i ０ is the O at the time of measurement ２ It is correlated with the concentration of

[0076] After applying the first voltage V(1) to the electrode 32A in the process of S13, the CPU 21 applies a voltage to the electrode 32A under the same conditions as the application of the first voltage V(1) after the second elapsed time Tp(2) has elapsed (S17, see FIG. 6). ２ is consumed by electrolysis. The second voltage V(2) and the second application time Ta(2) are equal to the first voltage V(1) and the first application time Ta(1) when the second voltage V(2) is applied to the electrode 32A by the process of S13. The time Tn required for the voltage of the electrode 32A to change from 0 V to the second voltage V(2) is also equal to that when the first voltage V(1) is applied to the electrode 32A by the process of S13 (see FIG. 6).

[0077] The second elapsed time Tp(2) is the time when O is generated by electrolysis during application of the first voltage V(1) to the electrode 32A (S13, see FIG. 5). ２ After the consumption of O in the containing solution 30 ２ O 2 is introduced from the periphery of the electrode 32A to the electrode 32A by a diffusion phenomenon based on the concentration gradient of ２ is supplied, and O is supplied to a degree that does not affect the measurement accuracy. ２ It is predefined as the time it takes for the concentration to recover.

[0078] 5, the CPU 21 measures the current value of the current flowing through the electrode 32A while the second voltage V(2) is being applied to the electrode 32A in the process of S17 (S19). The CPU 21 measures the current when the second voltage V(2) is applied to the electrode 32A, or the integrated value of the current flowing through the electrode 32A while the second voltage V(2) is being applied to the electrode 32A, as the second current i, and stores it in the storage device 22. The second current i is calculated based on the O ２ It is correlated with the concentration of

[0079] The CPU 21 calculates the first current i measured in the process of S15. ０ , and the second current i measured by the process of S19 are read out from the storage device 22 and acquired. ０ is calculated as the ratio Rc. ０ Yes, O ２ It is correlated with the rate of change of

[0080] The CPU 21 reads out a linear function f, which indicates the relationship between the concentration of IMP and the ratio Rc, from the storage device 22. As described above, of the first and second reactions that occur in the enzyme membrane 10 when the IMP-containing solution 30 is dropped onto the electrode 32A, the second reaction proceeds at a reaction rate equal to or higher than a predetermined reaction rate. The predetermined reaction rate is defined as the rate at which the initial concentration of IMP, I ０ and O ２ rate of change (C / C ０ ) is a reaction rate that is sufficiently high to show a linear relationship. Therefore, the linear function f pre-stored in the storage device 22 is ０ and O ２ rate of change (C / C ０ ) is linear. In this case, accuracy can be maintained when converting the current ratio Rc into the IMP concentration.

[0081] The CPU 21 determines the concentration of IMP by applying the calculated ratio Rc to the read linear function f (S21). The CPU 21 displays the determined concentration of IMP as a measurement result on the display unit 23. The CPU 21 then ends the main processing.

[0082] Experimental Example: The concentration of IMP was measured using the measuring device 3 and compared with the results of HPLC analysis based on the JAS method. The concentrations of HxR and Hx were also measured using a measuring device that utilizes the reaction in which IMP is decomposed in the presence of the enzyme inosinate hydrolase to produce inosine (HxR) and hypoxanthine (Hx). This reaction is a multi-step reaction that includes a first reaction in which the phosphate group of inosinate is cleaved in the presence of inosinate hydrolase to decompose IMP into inosine, and a second reaction in which inosine is decomposed into hypoxanthine.

[0083] Furthermore, the K values ​​calculated and compared were calculated based on the following formula. In the formula, the concentrations of adenosine triphosphate (ATP), adenosine monophosphate (ADP), adenosine triphosphate (AMP), IMP, HxR, and Hx were simply expressed as ATP, ADP, AMP, IMP, HxR, and Hx. K value (%) = (HxR + Hx) / (ATP + ADP + AMP + IMP + HxR + Hx) × 100

[0084] Measurement samples were prepared as follows. First, the fish meat samples (two types each of horse mackerel, mackerel, flounder, cod, and sea bream) were heated in a microwave oven for approximately 2 minutes to convert the ATP in the samples to HxR-Hx via ADP, AMP, and IMP. Next, the heated samples were diluted 10-50 times with phosphate buffer to remove coagulation proteins. The samples prepared in this manner are labeled "Horse Mackerel 1," "Horse Mackerel 2," "Mackerel 1," "Mackerel 2," "Flounder 1," "Flounder 2," "Cod 1," "Cod 2," "Sea Bream 1," and "Sea Bream 2" in Figure 7.

[0085] 7 shows the IMP concentration measured using the measurement device 3 and the IMP concentration measured by HPLC analysis. The results shown in FIG. 7 reveal that the IMP concentration measured by the measurement device 3 and the IMP concentration measured by HPLC analysis are similar. A similar tendency was also observed in the results for HxR, Hx concentration, and K value. These results reveal that the measurement device 3 can be used to accurately measure the IMP concentration, HxR, Hx concentration, and K value.

[0086] <Functions and Effects of the Present Embodiment> When the containing solution 30 is dropped onto the electrode 32A of the measuring device 3, a first reaction and a second reaction occur in the enzyme membrane 10 of the electrode 32A. Here, the second reaction occurs when the initial concentration of IMP, I ０ and O ２ rate of change (C / C ０ The reaction proceeds at a rate sufficient to show a linear relationship between the initial concentration of IMP, I ０ and O ２ rate of change (C / C ０ The measurement device 3 performs measurements under these conditions to obtain the ratio Rci / i ０ The initial concentration of IMP I is calculated by applying a linear function f. ０ The linear function f is calculated based on the initial concentration of IMP I ０ and O ２ rate of change (C / C ０ ) is derived in advance on the assumption that the relationship between the current ratio Rc and the IMP concentration is linear. In this case, the accuracy can be maintained when converting the current ratio Rc into the IMP concentration using the linear function f. Therefore, the measurement device 3 can calculate the initial IMP concentration I ０ and O ２ rate of change (C / C ０ ) maintains a linear relationship, the concentration of IMP can be measured with high accuracy even when the electrochemical reaction of IMP occurs in multiple stages.

[0087] The enzyme film 10 formed on the electrode 32A contains IMPDH and NOD as enzymes. When the containing solution 30 is dropped onto the electrode 32A, a first reaction occurs in which IMP is consumed in the presence of IMPDH, and NADH, which is a product of the first reaction, reacts with NOD to produce O ２ A second reaction occurs in which O is consumed. ２ The concentration of IMP can be measured from the rate of change of

[0088] The enzyme membrane 10 contains IMPDH and NOD as enzymes. The measuring device 3 can accurately measure the concentration of IMP when a multi-stage reaction occurs, including a first reaction that occurs in the presence of IMPDH and a second reaction that occurs in the presence of NOD.

[0089] The measurement device 3 has a working electrode 32A, a counter electrode 32B, and a reference electrode 32C as electrodes 32. The measurement device 3 employs a three-electrode method, thereby enabling accurate measurement of the IMP concentration.

[0090] <Modifications> The present invention is not limited to the above embodiment, and various modifications are possible. The measurement device 3 may be composed of only the sensor chip 3A. In this case, the measurement unit 31 may measure the current value that flows when a voltage is applied to the electrode 32, and detect the concentration of IMP contained in the sample in the containing solution 30. The sensor chip 3A may display the detected concentration on a display unit (not shown). Furthermore, the measurement device 3 may have a structure in which the sensor chip 3A and the main body unit 3B are integrated.

[0091] The measurement target by the measurement device 3 is not limited to IMP, and the concentrations of other substances that cause a multi-stage reaction may also be measured. For example, just as the concentrations of HxR and Hx were measured in the <Experimental Example>, the concentrations of HxR and Hx may be measured based on a multi-stage reaction including a first reaction in which the phosphate group of inosinic acid is cleaved in the presence of inosinate hydrolase to decompose it into inosine, and a second reaction in which inosine is decomposed into hypoxanthine.

[0092] The measuring device 3 calculates the second current i from the first current i ０ The concentration of IMP was measured based on the ratio Rc obtained by dividing the first current i ０ For example, the measurement device 3 may measure the concentration of IMP by normalizing the second current i using ０ and a plurality of second currents i may be acquired and statistically analyzed to measure the concentration of IMP.

[0093] The measuring device 3 determines the concentration of IMP by applying the calculated ratio Rc to the linear function f stored in the storage device 22 (S21). The measuring device 3 may determine the concentration of IMP from the ratio Rc by another method. For example, the measuring device 3 may store in the storage device 22 a table that associates multiple candidates for the ratio Rc with the IMP concentrations corresponding to each of the multiple candidates. The measuring device 3 may determine the IMP concentration associated with one of the multiple candidates that is closest to the calculated ratio Rc as the IMP concentration in the IMP-containing solution 30.

[0094] The measurement device 3 may have a dripping mechanism that drips the containing solution 30 onto the electrode 32. The measurement device 3 may start applying a first voltage V(1) to the electrode 32A at the timing when the dripping mechanism drips the containing solution 30 onto the electrode 32 (S13).

[0095] The electrode 32 may have only a working electrode and a counter electrode, and may not have a reference electrode. In this case, the measurement device 3 may measure the histamine concentration by applying a voltage to the electrodes and measuring the current based on the bipolar electrode method.

[0096] <Others> IMP is an example of the "subject" of the present invention. ２ are an example of a "measurement substance" of the present invention. Electrode 32A is an example of a "first electrode" of the present invention. The processes of S13 and S17 are an example of an "application means" or "application step" of the present invention. The processes of S15, S19, and S21 are an example of a "measurement means" or "measurement step" of the present invention. IMPDH is an example of a "first enzyme" of the present invention. NOD is an example of a "second enzyme" of the present invention. Electrode 32B is an example of a "second electrode" of the present invention. Electrode 32C is an example of a "third electrode" of the present invention.

Claims

1. A measuring device that measures the concentration of an analyte by measuring a current corresponding to the change over time in the concentration of a substance to be measured, which is generated as a result of a multistage reaction in the analyte in response to the dropping of a solution containing at least the analyte onto a first electrode, the measuring device comprising: an application means that applies a voltage to the first electrode after a period of time has elapsed since the dropping of the solution onto the first electrode, when the reaction has reached a steady state within a range that does not affect measurement accuracy; and a measuring means that measures the concentration of the analyte using the current that flows in response to the application of the voltage by the application means, wherein the measuring means measures the concentration of the analyte under conditions in which an intermediate reaction in the multistage reaction reacts at a rate equal to or greater than a predetermined reaction rate, and the predetermined reaction rate is a rate required to maintain the accuracy of converting current into the concentration of the substance to be measured, assuming that the initial concentration of the analyte and the rate of change of the substance to be measured are linear.

2. The measuring device described in claim 1, characterized in that an enzyme membrane containing a plurality of enzymes is formed on the first electrode, and the multi-stage reaction includes at least: a first reaction that occurs within the enzyme membrane as a result of a first enzyme of the plurality of enzymes reacting with the analyte; and a second reaction that occurs within the enzyme membrane as a result of a second enzyme of the plurality of enzymes reacting with a product of the first reaction.

3. The measuring device according to claim 2, wherein the analyte is inosinic acid, the first enzyme is inosine phosphate dehydrogenase, and the second enzyme is reduced nicotinamide adenine dinucleotide oxidase.

4. The measuring device according to claim 1, further comprising: a second electrode serving as a counter electrode; and a third electrode serving as a reference electrode.

5. A method for measuring the concentration of an analyte by measuring a current corresponding to the change over time in the concentration of a substance to be measured, which is generated as a result of a multistage reaction in the analyte in response to the dropping of a solution containing at least the analyte onto a first electrode, the method comprising: an application step of applying a voltage to the first electrode after a period of time has elapsed since the dropping of the solution onto the first electrode, when the reaction has reached a steady state within a range that does not affect measurement accuracy; and a measurement step of measuring the concentration of the analyte using the current that flows in response to the application of the voltage by the application step, wherein the measurement step measures the concentration of the analyte under conditions in which an intermediate reaction in the multistage reaction reacts at a predetermined reaction rate, and the predetermined reaction rate is a rate required to maintain the accuracy of converting a current into the concentration of the substance to be measured, assuming that the initial concentration of the analyte and the rate of change of the substance to be measured are linear.

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