JP2006105615A - Electrochemical measuring method and measuring apparatus using it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an electrochemical measuring method which enables the simple measurement of a target component by neither providing a permselective membrane for obstructing an inhibition component nor especially measuring the response current due to the inhibition component, and a measuring apparatus using it. <P>SOLUTION: In the electrochemical measuring method for detecting the current signal associated with the electrochemical oxidation or reduction of the target component by applying potential to an acting electrode using an electrode system composed of the acting electrode, a reference electrode and a counter electrode, at least two potentials different in value selected so that the current signals due to the oxidation and reduction of the target component are different and the concentration of the target component contained in a sample is calculated from the difference between at least two obtained response currents different in value. The current signal produced by the oxidation or reduction of a substance other than the target component can be removed by this method. The electrochemical measuring apparatus is also disclosed. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to sample analysis, and more particularly to an electrochemical measurement method and apparatus for measuring a target component from a sample containing a plurality of components by a simple method.

  There has been known a method in which an electrode is used as a detection element, a current or potential change based on a reaction occurring at an electrode interface is detected, and various substances are electrochemically detected and measured. For example, there is a method of electrochemically determining the hydrogen peroxide concentration by measuring the oxidation current of hydrogen peroxide occurring on a platinum electrode, and an apparatus (hydrogen peroxide electrode) based on the method has been developed.

  The hydrogen peroxide electrode generally has a triode structure including a working electrode, a counter electrode, and a reference electrode. These three electrodes are connected to, for example, a potentiostat circuit, and a predetermined potential (for example, 0.6 V with respect to the Ag / AgCl reference electrode) is applied between the reference electrode and the working electrode in advance. The hydrogen peroxide that has reached the surface is oxidized by the reaction shown in “Chemical Formula 1”, and electrons having a mole number twice that of hydrogen peroxide are generated and converted into electronic information (current value). Electronic information is generally extracted by comparing the current value before and after contact with the surface of the working electrode (base current).

  In addition, a biosensor comprising a combination of a hydrogen peroxide electrode and a biocatalyst containing a hydrogen peroxide producing enzyme has been developed. A typical example is a glucose sensor comprising a glucose oxidase and a platinum electrode. That is, glucose is converted into hydrogen peroxide by “chemical formula 2”, and the converted hydrogen peroxide is detected by the hydrogen peroxide electrode.

  A hydrogen peroxide electrode, which is a type of current measuring transducer, responds to a reducing substance (hereinafter referred to as an interfering component) such as uric acid or ascorbic acid in addition to hydrogen peroxide, and generates a current. Therefore, when measuring components in a sample including a living body-related sample in which many of these interfering components are present, it is a problem to remove the influence of the interfering components in order to ensure the measurement accuracy of the enzyme electrode.

  The most common means taken to solve the above problems is to provide a permselective membrane that selectively or preferentially permeates hydrogen peroxide by eliminating coexisting substances on or near the electrode surface.

  FIG. 11 shows the basic structure of this type of biosensor. That is, a hydrogen peroxide selective permeable membrane is provided between the electrode and the enzyme membrane. Briefly explaining the principle, when the sensor is brought into contact with the medium to be measured, the target component, for example glucose, diffuses into the enzyme membrane and is oxidized by the enzyme contained in the enzyme membrane, for example glucose oxidase, to produce hydrogen peroxide. Is done. The generated hydrogen peroxide passes through the hydrogen peroxide selective permeable membrane and reaches the hydrogen peroxide electrode.

  On the other hand, the coexistence disturbing component diffuses to the enzyme membrane like glucose, but is excluded by the permselective membrane provided between the enzyme membrane and the hydrogen peroxide electrode, and most cannot reach the electrode surface. This ensures that the output current is derived from the target component and ensures measurement accuracy.

  One of the most important indicators that determine the performance of this type of biosensor is its selectivity for interfering components. Here, the selectivity for the disturbing component is defined as the ratio of the response current to the disturbing component of the same concentration and the response current to the target component. Hereinafter, it is also simply referred to as selectivity. For example, in the case of a glucose sensor, the selectivity for ascorbic acid (ASA), which is an interfering component (ASA / GLC selectivity), is the response current (IA) for unit concentration of ascorbic acid (IA) and the response current for the same glucose (GLC) (IG ) Ratio:

      ASA / GLC selection rate (%) = IA / IG * 100 1)

  Needless to say, the greatest factor that determines the selectivity is the permeation blocking performance of the permselective membrane against the disturbing components. A parameter that evaluates the performance of the permselective membrane is the ratio of the response current (IA) to ascorbic acid (IA) and the response current (IH) to hydrogen peroxide (HPO) (ASA / HPO selectivity). :

      ASA / HPO selection rate (%) = IA / IH * 100 2)

  Glucose in contact with the sensor surface enters the enzyme membrane and is converted to hydrogen peroxide by the enzyme contained within the membrane, in many cases the change is part of it. In addition, the converted hydrogen peroxide has a portion that diffuses and is lost to the outside (offshore) in contact with the enzyme membrane, rather than the portion that passes through the permselective membrane by diffusion and reaches the electrode surface. An indicator of the effective conversion rate of glucose to hydrogen peroxide is the GLC / HPO conversion rate:

      GLC / HPO conversion rate (%) = IG / IH * 100 3)

  Therefore, the sensor selectivity is determined by the product of the ASA / HPO selectivity and the GLC / HPO conversion rate.

  Interfering components are often small molecules similar to hydrogen peroxide, so permselective membranes often block the permeation of hydrogen peroxide. To achieve this, the optimum membrane material and manufacturing method should be used. Of course, in order to ensure a certain level of performance, it is necessary to make the membrane dense or thick to some extent, resulting in a decrease in the output against hydrogen peroxide, which is another indicator of sensor performance. Reduce. Further, the thicker the permselective membrane, the greater the resistance of hydrogen peroxide to diffuse to the electrode surface, which has the effect of simultaneously degrading sensor sensitivity and selectivity by reducing the effective conversion rate.

  Against this background, there have been few sensors that have been practically successful despite the fact that much research has been conducted on sensors based on the present principle, particularly on permselective membranes (see Non-Patent Document 1). In addition, in order to ensure the selectivity, it is necessary to establish a sophisticated and difficult mass production technique, resulting in an increase in the manufacturing cost of the sensor.

  Due to the high cost, this type of sensor is usually used repeatedly. However, there is another problem that stability is difficult, such as an increase in selectivity during use, and the service life is short. In general, the stability of bisensors using biocatalysts such as enzymes tends to depend on the stability of the enzyme. However, highly stable enzymes such as glucose oxidase exist in nature, and further advances in biotechnology Now that it has become possible to produce highly stable biocatalysts, the stability of the permselective membrane has become an impediment to determining sensor life.

  As another method to remove the influence of interfering components, another electrode that does not immobilize the enzyme can be provided at the same time, and correction can be made from the output difference between the two electrodes. Since it is difficult to stabilize and stabilize the system, the error becomes large and the system becomes complicated.

In Patent Document 2, using a working electrode made of gold, hydrogen peroxide and ascorbic acid react together (1.1V), and ascorbic acid reacts, but hydrogen peroxide does not react. A method has been proposed in which (0.3 V) is alternately applied and measured, the concentration of ascorbic acid is calculated from the result at 0.3 V, and the response current of ascorbic acid at 1.1 V is obtained and corrected. A method similar to this is also proposed for measurement using a diamond electrode (Patent Document 3). However, these methods require the presence of a potential that reacts only with interfering components, and it is necessary to apply two potentials that are far away from each other, so that electrode materials that can be used are limited. For example, platinum electrodes, which are best suited as hydrogen peroxide electrodes, can barely eliminate the effects of ascorbic acid with these methods, but if the potential is switched greatly, a very high dark current flows due to the oxidation and reduction of the electrodes themselves. ,I can not use it. In addition, the influence of other interfering components such as uric acid cannot be corrected. Furthermore, in these methods, it is necessary to create at least two calibration curves not only for the target component but also for the interfering component, so it is necessary to create a total of at least three calibration curves before measurement. There is. Further, when calibration is required to maintain the measurement accuracy, a plurality of calibration operations are performed each time, so that the measurement system becomes complicated.
ACS Symposium Series, Vol.487, p125-132 (1992) JP-A-7-103939 JP-A-11-83799

  The present invention has been made to solve the above problems, and the object of the present invention is to provide a target component without providing a permselective membrane for blocking the disturbing component, and without specifically measuring the response current due to the disturbing component. It is an object to provide an electrochemical measurement method and apparatus that makes it possible to easily measure the above.

  In order to achieve the above object, according to the electrochemical measurement method of the first aspect of the present invention, an electrode system comprising a working electrode, a reference electrode, and a counter electrode is used, and a potential is applied to the working electrode to perform electrochemical analysis of the target component. In an electrochemical measurement method for detecting a current signal associated with chemical oxidation or reduction, a value obtained by applying at least two potentials having different values selected such that the current signal due to oxidation or reduction of a target component is different There is provided an electrochemical measurement method characterized in that the concentration of one or more target components contained in a sample is determined from the difference between at least two different response currents. This method makes it possible to eliminate current signals generated by oxidation or reduction of substances other than the target component, and accurately measure target components even for samples containing coexisting interfering substances with multiple redox activities. can do.

  In order to achieve the above object, according to the electrochemical measurement method of the invention described in claim 2, an electrode system comprising a working electrode, a reference electrode and a counter electrode is combined with a biocatalyst containing a hydrogen peroxide-producing enzyme. An electrochemical measurement that uses a measurement system to apply a potential to the working electrode and detect a current signal that accompanies the electrode oxidation of hydrogen peroxide to measure a substance that is a substrate for the hydrogen peroxide-producing enzyme contained in the sample. In the method, at least two potentials having different values, which are selected so that current signals due to oxidation of hydrogen peroxide are different, are applied, and hydrogen peroxide contained in the sample is obtained from the difference between the obtained at least two response currents. There is provided an electrochemical measurement method characterized by determining the concentration of a substance that is a substrate for a produced enzyme. This method makes it possible to eliminate current signals generated by oxidation or reduction of reducing substances other than hydrogen peroxide, and even for samples containing coexisting substances with reducing activity, It is possible to accurately measure the target component without providing a countermeasure against the disturbing component.

  The electrochemical measurement method according to claim 3 is characterized in that, in claim 1 or claim 2, the material of the working electrode is mainly composed of platinum. A platinum electrode is most preferable as an electrode material used in the measuring method according to the present invention because it is excellent in stability and has a wide potential window.

The electrochemical measurement method according to claim 4 is characterized in that, in claim 1 or claim 2, the difference between two applied potentials having different values is in a range of 0.1 to 0.4V. Along with the potential change, in addition to the current due to the electrochemical reaction by the substance such as the target component, a current due to the change of the electric double layer or the electrode itself is temporarily generated, but the difference between the two applied potentials falls within this range. If present, such primary current can be minimized. At the same time, there is a sufficient difference in the current corresponding to each potential due to the target component, and a highly accurate measurement can be performed.
Furthermore, the electrochemical measurement apparatus according to claim 5 uses an electrode system including a working electrode, a reference electrode, and a counter electrode, and applies a potential (detection potential) to the working electrode to generate a current signal associated with electrode oxidation of the target component. An electrochemical measuring device that measures the potential of the working electrode at an arbitrary value relative to the reference electrode, a mechanism that changes the potential applied to the working electrode at an appropriate timing, and a different applied potential. Since it has a mechanism that calculates the concentration of the component to be measured contained in the sample from the difference between the multiple current signals, the target component can be accurately detected even for samples containing coexistence interference components. Can be measured.

Hereinafter, the present invention will be described in more detail with reference to the drawings.

  The electrochemical measurement system used in the present invention includes a working electrode, a reference electrode, and a counter electrode. Examples of the working electrode material include noble metals such as platinum, palladium, iridium, and gold, carbon, and diamond. Platinum is the best because of its stability and ease of use. Examples of the reference electrode include a silver / silver chloride electrode and a permeation electrode, but a silver / silver chloride electrode is most preferred because it is solid and easy to mold. In order for the silver / silver chloride electrode to function stably, it needs to be in contact with a certain concentration of chloride ion, for example, potassium chloride (KCl), and its reference potential (relative to the standard hydrogen electrode) depends on the concentration of KCl. (See FIG. 9). In the present invention, unless otherwise stated, the potential refers to saturated KCl. The same material as the working electrode may be used as the counter electrode, for example, platinum. These electrodes may be separately connected to the potentiostat or formed on a single insulating substrate. FIG. 10 shows a planar electrode system (planar electrode) made of a platinum working electrode, a platinum counter electrode, and silver / silver chloride formed on a ceramic substrate. In the case of a planar electrode, instead of providing the reference electrode with the internal solution, a method of keeping the potential of the reference electrode constant by bringing the electrode into contact with an aqueous solution containing a constant concentration of KCl, for example, a buffer solution may be used.

  When a platinum working electrode is used, reducing substances such as hydrogen peroxide, various amines, uric acid and ascorbic acid can be directly detected. Further, by immobilizing a biocatalyst containing hydrogen peroxide-producing enzyme or the like on the surface of the working electrode or in the vicinity of the surface, it can be used as a bisensor for detecting various components that are substrates for the hydrogen peroxide-forming enzyme.

  The electrochemical measurement method according to the present invention applies at least two potentials having different values selected so that current signals due to oxidation or reduction of a target component are different, and obtains at least two response currents having different values. From the difference, the concentration of the target component contained in the sample is obtained. For example, when measuring a sample containing one substance A as a target component to be measured, first, the potential E1 is applied, the obtained current is set to I1, then the potential E2 is applied, and the obtained current is set to I2. Then, the concentration CA of the target component A is obtained by the following formula:

      CA = (I2-I1) / K 4)

In the formula, K is a coefficient determined from a calibration curve obtained by measuring a standard sample.

  The basic principle will be described below with reference to the drawings.

  FIG. 1 is a steady-state current-potential curve of substance A. The steady-state current-potential curve is a current-potential curve that is not affected by time, and the current value at each potential is proportional to the concentration of the substance in the medium in contact with the electrode surface. If the concentration is constant, the current is determined by the potential, as shown. The steady-state current-potential curve can be obtained by scanning the potential slowly (for example, the scanning speed is 5 mV / s or less) or switching stepwise. The current value increases as the potential increases. However, when the potential reaches a certain potential, the current value becomes maximum and the increase stops. This maximum current value is called a limiting current. When the potential is fixed at an arbitrary value where the current value is larger than zero (that is, the substance A is oxidized) and measured at different concentrations, a curve indicating the relationship between the concentration and the current, that is, a calibration curve, is obtained. FIG. 2 schematically shows a calibration curve corresponding to the potentials E1 and E2 of FIG.

I1 = K1 * C 5)
I2 = K2 * C 6)

  If there is no current due to a substance other than substance A at the selected potential or can be ignored, the concentration can be determined based on which calibration curve by single-point measurement. When a coexisting component (interfering component) is present, the measured current value includes a contribution due to the disturbing component, and this method greatly reduces the measurement accuracy.

  FIG. 3 shows a current-potential curve when the disturbing component B coexists. The curve when the substance A and the interfering component B exist alone is IA and IB, but the current I when they coexist is the sum of IA and IB.

  In general, when two substances coexist, the current at each potential is the sum of the currents from both components:

I1 = KA1 * CA + KB1 * CB 7)
I2 = KA2 * CA + KB2 * CB 8)

  KA1, KA2, KB1, and KB2 are coefficients determined from calibration curves obtained from respective standard samples. Each concentration can be obtained by solving the simultaneous equations. For example, the concentration of the target substance A is obtained by the following formula:

    CA = (KB1 * I2-KB2 * I1) / (KB1 * KA2-KB2 * KA1) 9)

  As shown in FIG. 3, the disturbing component B shows a current-potential curve having a pattern different from that of the target component A. Therefore, the currents IA1 and IA2 generated by the target component A can be obtained at the potentials E1 and E2 by appropriately selecting the potential. The currents IB1 and IB2 due to the disturbing component B are substantially the same. In this case, since KB1 and KB2 in Equation 9) are substantially the same value, Equation 9) can be rewritten as follows:

    CA = (I2-I1) / (KA2-KA1) = (I2-I1) / K 10)

  That is, the concentration of the target component A is determined by the difference between the currents measured at the two potentials, and is not affected by the presence of the coexistence disturbing component B. Further, even when other disturbing components, for example, disturbing component C are present, the influence is removed in the same manner as in the case of disturbing component B by the potential appropriately selected so that the corresponding currents IC1 and IC2 are almost the same. can do.

  As can be seen from the above principle, for the method according to the present invention to be effective, it is necessary that the electrochemical properties are significantly different between the target component and the interfering component and that the applied potential is appropriately selected. is there. Here, the electrochemical properties are significantly different from each other, as specifically shown in FIG. 3, the current due to the target component differs depending on the pattern of the current-potential curve of both, but the current due to the disturbing component is substantially different. There are two different potentials that are the same.

  Here, “substantially the same” means that the difference between the current signals due to the disturbing components at both potentials is sufficiently small, and the influence of the contribution on the target component measurement is within an allowable error. Therefore, the actual allowable difference is the concentration relationship between the target component and the disturbing component, the coefficient in the sensitivity relationship between the target component and the disturbing component (specifically, Equation 7): KA1, KA2, KB1, KB2), and the tolerance It depends on the error. The higher the concentration of the coexistence disturbing component is relative to the target component, and the higher the sensitivity of the disturbing component, the smaller the difference in the current signal due to the disturbing component at both potentials allowed. For example, if the sensitivity of the disturbing component and the target component is the same, and the current due to the target component at the potential E2 is twice that at the potential E1, the difference in current due to the disturbing component at the same potential is 10%. Since the error is 10%, if the tolerance due to the disturbing component is 20%, the difference will be substantially the same even if the difference is 20%. On the other hand, if the allowable error is 10%, “substantially the same” means that the difference between the two becomes 10% or less. Also, if the current due to the target component at the potential E2 is 1.5 times that at the potential E1, if the difference in current due to the disturbing component at the same potential is 20%, the error due to the disturbing component will be 40%. If it is 10%, “substantially the same” means that the difference between them is 5% or less.

  Therefore, in the present invention, it is important to appropriately determine the applied potential. Hereinafter, a method for determining the applied potential will be described. In the case of one target component, at least two applied potentials are necessary, but generally two potentials are sufficient.

  First, it is investigated what components having redox properties other than the target component are contained in the sample to be measured. Among them, those whose contents have reached a level that cannot be ignored compared to the target component are listed. For example, when measuring glucose in the urine using an enzyme sensor that combines glucose oxidase and a platinum electrode, hydrogen peroxide becomes a substantial target component, so in the potential range where hydrogen peroxide undergoes an oxidation reaction, Interfering components that can coexist are uric acid, ascorbic acid, various biogenic amines, and acetaminophen, which is a component of wind medicine, but components that are present at a level that cannot be ignored regularly are uric acid and ascorbic acid.

  Next, the current-potential curve of the listed disturbing component and target component is measured to reveal its oxidation behavior.

  The measurement of the current-potential curve may be performed by using a general electrochemical method such as a method of continuously scanning the potential or a method of changing the potential stepwise. It is desirable to use an electrochemical material such as an electrode that conforms to the actual measurement. As for the electrode, it is desirable to use a material for the working electrode as well as a reference electrode and a counter electrode that are actually used.

Hereinafter, in the case of urine sugar measurement, the measurement of current curves of uric acid and ascorbic acid that become interference components will be exemplified. As the electrode, a planar type platinum electrode shown in FIG. 10 was used. The working electrode of the electrode is formed by screen printing using platinum paste as a raw material and sintered. The size of the working electrode is 1.7 × 1.8 mm, and the area is about 3 mm 2. This electrode was attached to the flow-through cell and incorporated in the measurement system shown in FIG. As an electrolytic solution, a 0.067 M phosphate buffer (pH 6.8) containing 0.05 M potassium chloride was added with predetermined components. Potassium chloride is added to stabilize the potential of the silver / silver chloride reference electrode. The standard potential of silver / silver chloride depends on the concentration of potassium chloride in contact with the reference electrode (see FIG. 9), and shows the relationship between the standard potential of the silver / silver chloride electrode and the concentration of potassium chloride. From FIG. 9, when the potassium chloride concentration is 0.05M, the reference potential is about 0.3V, and in the case of saturated potassium chloride, it is about 0.2V. Therefore, the potential in this measurement system is higher than that in the system using saturated potassium chloride. Is about 0.1V higher.
Electrolyte is fed at a rate of 1 ml / min (average linear velocity at the working electrode surface is about 30 cm / min), the initial potential is set to 0 V or 0.1 V, and the speed potential of 5 mV / sec is scanned to 0.8 V. Was recorded. The obtained current potential curve is shown in FIG. As can be seen from FIG. 4, the current increased with increasing applied potential for all three components, but the ascorbic acid oxidation current was 0.35 V or higher and the current almost did not increase. Uric acid did not increase at 0.5V or higher. On the other hand, the oxidation current of hydrogen peroxide tended to increase even when the potential reached 0.8v.

  From the above results, when the target component to be measured is hydrogen peroxide, select the potential E1 from 0.5V or more, for example, 0.5-0.6, and select the potential E2 from 0.55V, for example, 0.6-0.8V. Measurement can eliminate the effects of ascorbic acid and uric acid. Of course, the same applies to the case where a hydrogen peroxide-producing enzyme substrate, for example, glucose, is measured by combining a hydrogen peroxide-producing enzyme and an electrode.

  On the other hand, when the target component is uric acid, for example, uric acid is measured without being affected by ascorbic acid by selecting another potential on the condition that hydrogen peroxide is not contained in the medium to be measured. be able to. As an example, the potential E1 is 0.4V and the potential E2 is 0.5V.

  Note that the interval between the potentials E1 and E2 is not particularly specified if the two currents due to the disturbing components other than the target component are substantially the same and the two currents due to the target component are significantly different. In addition, it is desirable that there is a difference of 20% or more between the two currents due to the target component. If the difference is small, the denominator of Equation 8 becomes small, and the measurement error increases. In this sense, it seems better to separate E1 and E2 as much as possible, but if they are too far apart, E2 will become high, so that current will be generated due to oxidation of the electrode itself, and dark current due to large jump of current The measurement error may increase in the same manner due to the increase of. As a specific interval, it is desirable that the potential E2 is in a range higher by 0.1V-0.4V than the potential E1.

  As for the order of applying the two potentials, any potential may be applied first. However, the larger the shift from the natural potential, the more the dark current is generated due to the change of the electric double layer or the electrode itself. It is desirable to apply the potential E1 and then shift to E2. Examples of the method of switching from the potential E1 to E2 or from E2 to E1 include a method of switching instantaneously, a method of switching stepwise, or a method of scanning the potential at a constant speed. The method of switching the potential instantaneously has the advantage that the device is simple. However, since dark current due to changes in the electric double layer and the electrode itself is also included immediately after switching, it is desirable to read the current after a certain period of time as a signal. On the other hand, in the method of scanning the potential, the dark current due to the change of the electric double layer or the electrode itself can be suppressed to a small level by controlling the scanning speed to be moderately slow, so that the current immediately after reaching the potential E2 is signaled. It can be. However, the apparatus becomes a little complicated, such as the need for a potential operation mechanism.

  The measurement principle and method when there is one target component have been described above. In a similar manner, two target components can be measured. In this case, select at least three potentials and measure at three or more points. For example, when measuring urinary glucose and uric acid, the potential E1 is set to 0.4V, the potential E2 is set to 0.5V, the potential E3 is set to 0.7V, and the influence of urinary ascorbic acid is measured by measuring three currents. Without receiving, uric acid and glucose can be measured.

  Next, a specific measurement sequence will be described. As for the actual measurement method, a basic method is adopted in which two potentials are applied, two response currents corresponding to the two potentials are measured, and the concentration of the target component is determined from the difference between the two currents obtained. There is no particular limitation.

FIG. 5 shows an example of a procedure for measuring one target component by applying two potentials. In the standby state, the power is turned on or the measurement mode is entered by a measurement button or the like, and the potential E1 is applied. Next, a sample is introduced, and a current I1 corresponding to the potential E1 is measured and stored. Next, the applied potential is shifted to E2, the current I2 corresponding to the potential E2 is stored, and the measurement result is output and displayed at this stage.
Next, the sample is discharged, the system including the electrode is washed, and the system returns to the standby state to prepare for the next measurement.

  In this measurement sequence, no potential is applied to the standby electrode, but it is necessary to start measurement immediately with the user's excretion, such as a toilet installation type inspection device, and output the result in a short time. A method of applying a constant potential, for example, the first potential E1 during standby may be adopted. An example of the measurement sequence in this case is shown in FIG.

  As a method of introducing a sample, the sample may be introduced as it is onto the surface of the working electrode. However, when the concentration of the component contained in the sample is high or measurement accuracy is required, the sample is introduced with a base electrolyte having a constant component. The method of introducing after dilution is desirable. As a specific method, the sample is mixed with the base electrolyte in advance and mixed uniformly and then introduced to the electrode surface. The sample is injected in the middle of the flow path of the solution while the base electrolyte is being fed. Examples of such a method are as follows. An example of the latter is flow injection analysis. The flow injection analysis method does not require a special cleaning operation and has advantages such as quick measurement, but the sample passes through the electrode surface as a solution zone with a certain concentration distribution, so it matches the application timing of potentials E1 and E2. The sample needs to be injected twice.

  The principle of the method according to the present invention and the specific measurement method have been described above. Next, an electrochemical measurement apparatus according to the present invention will be described. Since the apparatus according to the present invention is characterized by a potential operation and a calculation method of the obtained current signal, the electrochemical circuit and the built-in control / calculation software are the points. Specifically, a mechanism for holding the potential of the working electrode at an arbitrary value with respect to the reference electrode, a mechanism for changing the potential applied to the working electrode at an appropriate timing, and a plurality of currents obtained from different applied potentials A mechanism for calculating the concentration of the component to be measured contained in the sample from the signal difference is included. Other systems such as a liquid feeding / sample introduction system, a cleaning system, and an output / display system may be conventionally used as necessary.

(Example) Measurement of glucose in the presence of ascorbic acid and uric acid

  Production of glucose sensor: The surface of the working electrode of the planar electrode shown in FIG. 10 contains glucose oxidase (2290 units / ml), bovine serum albumin (17.5 mg / ml), and glutaraldehyde (0.2%) as a cross-linking agent. A glucose sensor having a GOD enzyme film on the surface of the working electrode was prepared by dropping an aqueous solution (10 μl) in a spot shape and drying. FIG. 7 shows the film structure on the working electrode surface of the completed sensor. It is a sensor having a very simple structure in which an enzyme film is provided on the surface of a platinum working electrode.

  The resulting sensor was attached to the flow cell and set in the analyzer shown in FIG. First, the base electrolyte (composition: 0.033M disodium hydrogen phosphate, 0.033M potassium dihydrogen phosphate, 0.05M KCl) was pumped to fill the cell, and then 0.5V potential was applied to the working electrode surface. Applied. Next, it switched to the complex liquid mixed with the ratio of the base electrolyte solution 35 with respect to the glucose solution 1, and liquid-fed. After the response current was confirmed, the applied potential was switched to 0.7 V and the current was continuously recorded. As an example of the resulting current curve, a current-potential curve obtained by measuring 10 mM glucose is shown in FIG. When glucose is fed to the cell with a 0.5 V potential applied, the current increases and eventually reaches a steady value. This current signal is a response current I1 corresponding to the potential E1. Next, when the potential is shifted to 0.7 V, the current once suddenly rises, but then falls rapidly and becomes stable again at a level higher than I1. This stable current is a response current I2 corresponding to E2. From FIG. 8, it takes 2 minutes from the potential switching until it stabilizes, but as already explained, the current change due to the potential switching is basically due to the change of the electric double layer or the electrode itself, and the substance in the medium to be measured Since it is irrelevant to the concentration and can be predicted and corrected in advance, when it is necessary to further shorten the measurement time, for example, a current at 1 minute may be used. In this embodiment, the base current until contact with the sample is very low and can be ignored. However, when it cannot be ignored, I1 and I1 are values obtained by subtracting the base current.

  The glucose concentration was changed and measured, and uric acid and ascorbic acid were also measured. Each response current response value is shown in Table 1. A calibration curve was prepared for glucose. The results are shown in Table 1.

  From Table 1, it can be confirmed that the response current value for glucose differs greatly depending on the applied potential, but there was almost no change associated with the applied potential for uric acid and ascorbic acid.

Next, a mixed solution containing glucose, uric acid and ascorbic acid was prepared and measured in the same manner as a sample. Table 2 shows the obtained current response values and the calculation results of the glucose concentration.

  From Table 2, this sensor is able to accurately measure the glucose concentration even when both components coexist even though the response currents corresponding to uric acid and ascorbic acid were recorded at high values at two potentials. It was confirmed that

It is a figure which shows notionally the electric current potential curve with respect to a target component. It is a figure which shows the calibration curve with respect to the target component corresponding to an applied potential. It is a figure which shows the principle which measures the target component by this invention. It is a figure which shows the electric potential curve of ascorbic acid, uric acid, and hydrogen peroxide. FIG. 4 shows a measurement sequence of the method according to the invention. FIG. 6 shows another measurement sequence of the method according to the invention. It is a figure which shows the film | membrane structure of the glucose sensor made as an experiment in the Example. It is a figure which shows the electric current signal when measuring glucose in an Example. It is a figure which shows the relationship between the standard electric potential of a silver / silver chloride reference electrode, and potassium chloride concentration. It is a figure which shows the planar type electrode system formed on the ceramic substrate. It is a figure which illustrates the structure of the glucose sensor used for a prior art. It is a figure which illustrates the measuring apparatus used for the method by this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 electrode 2 ... Working electrode 7 ... Reference electrode 8 ... Counter electrode 10 ... Planar electrode system 26 ... Hydrogen peroxide selective permeable membrane 28 ... Enzyme membrane

Claims (5)

In an electrochemical measurement method that uses an electrode system composed of a working electrode, a reference electrode, and a counter electrode and applies a potential to the working electrode to detect a current signal accompanying electrochemical oxidation or reduction of the target component, One or a plurality of target components contained in the sample from the difference between at least two response currents obtained by applying at least two potentials having different values, the current signals resulting from the reduction being selected to be different A method for electrochemical measurement, characterized in that the concentration of lysine is determined. Using a measurement system that combines an electrode system consisting of a working electrode, a reference electrode and a counter electrode, and a biocatalyst containing a hydrogen peroxide-producing enzyme. In an electrochemical measurement method for detecting a signal and measuring a substance serving as a substrate for the hydrogen peroxide-producing enzyme contained in a sample, the current signal due to oxidation of hydrogen peroxide is selected to be different, and at least different values A method for electrochemical measurement, characterized in that a concentration of a substance serving as a substrate for a hydrogen peroxide-producing enzyme contained in a sample is determined from a difference between at least two response currents obtained by applying two potentials. The electrochemical measurement method according to claim 1, wherein the material of the working electrode contains platinum as a main component. The electrochemical measurement method according to claim 1 or 2, wherein a difference between two applied potentials having different values is in a range of 0.1 to 0.4V. An electrochemical measurement device that uses an electrode system composed of a working electrode, a reference electrode, and a counter electrode, applies a potential (detection potential) to the working electrode, and measures a current signal associated with electrode oxidation of the target component,
A mechanism that holds the potential of the working electrode at an arbitrary value with respect to the reference electrode, a mechanism that changes the potential applied to the working electrode at an appropriate timing, and a difference between multiple current signals obtained from different applied potentials, An electrochemical measurement apparatus having a mechanism for calculating a concentration of a component to be measured contained therein.

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