WO2011151953A1

WO2011151953A1 - Method for measuring substance

Info

Publication number: WO2011151953A1
Application number: PCT/JP2011/001171
Authority: WO
Inventors: 秀明大江; 真理子谷川; 淳典平塚; 典子佐々木; 信行吉田; 憲二横山
Original assignee: 株式会社村田製作所
Priority date: 2010-06-03
Filing date: 2011-03-01
Publication date: 2011-12-08

Abstract

Provided is a technique whereby, in the case of quantifying a target substance contained in a specimen, the measurement accuracy can be improved by reducing the influence of a current component, which differs from an oxidation current produced by the oxidation of a reducing substance that is formed by a reaction between the target substance in the specimen and an enzyme, from among current components contained in a response current obtained by applying a potential, based on a counter electrode, to a working electrode. A target substance is quantified based on a difference between a first current and a second current which are obtained respectively by applying different potentials E₁ and E₂ on a working electrode. Thus, the influence of a background current, which is a current component differing from an oxidation current produced by the oxidation of a reducing substance that is formed by an enzymatic reaction of the target substance, from among current components contained in a response current, can be reduced and the measurement accuracy can be improved in quantifying the target substance contained in a specimen.

Description

Method for measuring substances

The present invention relates to a method for measuring a substance for quantifying a measurement target substance contained in a specimen using a biosensor.

Conventionally, as shown in FIG. 6, using a biosensor 500 having an electrode system including a working electrode 501 and a counter electrode 502 and a reaction layer containing an enzyme that specifically reacts with a measurement target substance, measurement included in a specimen Quantification of the measurement target substance by measuring an oxidation current obtained by applying a voltage between the working electrode 501 and the counter electrode 502 to oxidize the reducing substance generated by the reaction between the target substance and the reaction layer. There is known a method for measuring a substance that performs the above (see Patent Document 1, for example).

A biosensor 500 shown in FIG. 6 is a sensor for quantifying glucose contained in a specimen, and includes an electrode layer formed by providing an electrode on an insulating substrate made of polyethylene terephthalate, a cover layer, an electrode layer, A spacer layer disposed between the cover layers is laminated and formed. In addition, the spacer layer is provided with a slit for forming a cavity to which the specimen is supplied, and the electrode layer and the spacer layer are bonded to the electrode layer by laminating the cover layer via the spacer layer. And the slit portion of the cover layer form a cavity for supplying the specimen, and the specimen is supplied to the cavity from the specimen inlet formed on the side surface of the biosensor 500. The cover layer is formed with an air hole communicating with the end portion of the formed cavity.

The electrode layer is provided with a working electrode 501, a counter electrode 502, and a specimen detection electrode 503, and electrode patterns 501 a, 502 a, and 503 a that are electrically connected to these

electrodes

501, 502, and 503, respectively. Thus, an electrode system is formed in the electrode layer. A reaction layer (not shown) is provided on the working electrode 501 and the counter electrode 502, and the working electrode 501, the counter electrode 502, and the specimen detection electrode 503 are exposed to cavities formed in the biosensor 500, respectively. So as to be provided on the electrode layer. Therefore, when a specimen made of a liquid is supplied to the cavity from the specimen inlet, each

electrode

501, 502, 503 exposed to the cavity and the reaction layer come into contact with the specimen, and the reaction layer is dissolved in the specimen.

Further, the reaction layer provided on the working electrode 501 and the counter electrode 502 contains glucose oxidase that specifically reacts with glucose contained in the specimen and potassium ferricyanide as a mediator (electron acceptor). The ferricyanide ions produced by the dissolution of potassium ferricyanide in the specimen are reduced to ferrocyanide ions, which are reductants, by electrons released when glucose is oxidized to gluconolactone by reacting with glucose oxidase. The Therefore, when a sample containing glucose is supplied to the cavity formed in the biosensor 500 from the sample introduction port, ferricyanide ions are reduced by electrons released by the oxidation of glucose, and thus included in the sample. Thus, ferrocyanide ions, which are reduced forms of ferricyanide ions, are generated in an amount corresponding to the concentration of glucose oxidized by the enzyme reaction.

In the biosensor 500 configured as described above, the oxidation current obtained by oxidizing the reduced form of the mediator generated as a result of the enzyme reaction on the working electrode 501 has a magnitude depending on the glucose concentration in the specimen. By measuring the current, glucose contained in the specimen can be quantified.

Next, an example of a method for quantifying glucose as a measurement target substance contained in a specimen using the biosensor 500 will be described with reference to FIG. First, when the control unit 601 detects that the biosensor 500 is set at a predetermined position of the measuring instrument 600, a voltage (for example, 500 mV) is applied between the specimen detection electrode 503 and the counter electrode 502 by the DC power supply 602. Applied. The current flowing between the specimen detection electrode 503 and the counter electrode 502 is converted into a voltage by the current / voltage conversion circuit 603 and input to the control unit 601 via the A / D conversion circuit 604.

When, for example, 3 μl of an aqueous glucose solution is supplied as a specimen from the specimen inlet to the cavity provided in the biosensor 500, the specimen reaches the air hole through the cavity by capillary action and enters the electrode system of the electrode layer. The provided reaction layer is dissolved in the specimen. At this time, when the specimen detection electrode 503 and the counter electrode 502 are in liquid junction, the voltage input to the control unit 601 via the A / D conversion circuit 604 increases, so that the gap between the specimen detection electrode 503 and the counter electrode 502 is increased. The change of the resistance value is detected, and it is detected that the specimen is supplied to the cavity based on the detected change of the resistance value, and the control unit 601 starts the measurement timer.

Subsequently, after a predetermined time (for example, 55 seconds) has elapsed since the specimen supplied to the cavity is detected, the switch 605 is switched, and a potential (for example, 500 mV) based on the counter electrode 502 is applied to the working electrode 501. . When a predetermined potential is applied to the working electrode 501, a current flowing between the working electrode 501 and the counter electrode 502 is converted into a voltage by the current / voltage conversion circuit 603, and the control unit 601 via the A / D conversion circuit 604. Is measured as a response current.

At this time, as a result of the enzyme reaction, ferrocyanide ions in an amount corresponding to the glucose concentration are generated in the specimen, and the generated reductant ferrocyanide ions are oxidized in the measured response current. Oxidation current due to being included. Therefore, for example, by measuring the value of the current flowing between the working electrode 501 and the counter electrode 502 five seconds after the potential based on the counter electrode 502 is applied to the working electrode 501, the concentration of glucose contained in the specimen is determined. A response current having a correlated magnitude can be obtained, and glucose contained in the specimen is quantified based on the measured response current and a conversion formula stored in advance in the measuring device 600. In addition, FIG. 6 is a figure for demonstrating an example of the measuring method of the conventional substance.

Japanese Patent No. 3106627 (paragraphs [0011] to [0016], [0026] to [0028], FIG. 8, etc.)

In the conventional method for measuring a substance described above, the measurement target substance contained in the specimen is quantified by measuring the response current flowing between the working electrode 501 and the counter electrode 502 by applying a predetermined potential to the working electrode 501. Is called. However, the measured response current includes, as a current component, an electric current formed between the working electrode 501 and the counter electrode 502 in addition to an oxidation current caused by oxidation of a reduced form of the mediator generated by the enzyme reaction. A background current such as a charging current (non-Faraday current) for charging the multilayer and a current based on an oxidation-reduction reaction of impurities other than the measurement target substance contained in the specimen is also included.

Such background current depends on the amount of various ionic components contained in the specimen and the amount of impurities contained in the specimen, and is a current component that has no correlation with the amount of the measurement target substance contained in the specimen. In addition, since the amounts of various ion components and impurities in the specimen are different for each specimen, the background current that flows when the measurement target substance contained in the specimen is quantified has a different magnitude for each specimen. Therefore, the response current includes current components due to background currents of different magnitudes for each specimen, such as the charging current of the electric double layer and the current based on the redox reaction of impurities. On the basis of this, there is a concern that the measurement accuracy may deteriorate when the measurement target substance contained in the specimen is quantified.

The present invention has been made in view of the above problems, and among the current components included in the response current obtained by applying a potential based on the counter electrode to the working electrode, the measurement target substance and the enzyme in the sample The measurement accuracy when quantifying the measurement target substance contained in the specimen can be improved by reducing the influence of the current component different from the oxidation current due to the oxidation of the reducing substance produced by the reaction of The purpose is to provide technology.

In order to achieve the above-described object, the present inventor has repeatedly conducted various experiments and measurements, and as a result, among the current components included in the measured response current, the charging current of the electric double layer and the redox reaction of the impurity The background current, which is different from the oxidation current caused by the oxidation of the reducing substance generated by the reaction between the analyte in the sample and the enzyme, such as the current based on the oxidization, oxidizes the reducing substance to be measured. It was found that the fluctuation due to the change in the magnitude of the potential applied to the working electrode was small compared with the oxidation current due to the above. That is, the change in the magnitude of the background current is small compared to the change in the magnitude of the oxidation current of the reducing substance to be measured due to the fluctuation of the potential applied to the working electrode. The present invention was completed by paying attention to. This is because the amount of various ionic components and impurities contained in the specimen is small compared to the amount of the reducing substance, so that the current based on the oxidation-reduction reaction of the impurity is smaller than the oxidation current of the reducing substance to be measured. This is because the electric capacity of the electric double layer formed by applying a potential to the working electrode is not so large that fluctuations in the applied potential to the working electrode have a large effect on the charging current. It is done.

The method for measuring a substance according to the present invention includes a biosensor having an electrode system including a working electrode and a counter electrode, and a reaction layer including an enzyme that specifically reacts with the target substance, and the target substance included in the specimen. Of the substance to be measured by measuring an oxidation current obtained by oxidizing a reducing substance produced by the reaction between the reaction layer and the reaction layer by applying a voltage between the working electrode and the counter electrode In the method of measuring a substance to be performed, a first measurement step of measuring a first current obtained when a first potential based on the counter electrode is applied to the working electrode, and a reference of the counter electrode to the working electrode Based on a second measurement step of measuring a second current obtained when a second potential different from the first potential is applied, and a difference value between the first current and the second current Quantify the substance to be measured It is characterized by comprising a quantitative step (claim 1).

In the invention configured as described above, in the first measurement step, the first current obtained when the first potential based on the counter electrode is applied to the working electrode is measured, and in the second measuring step, the working electrode is measured. A second current obtained when a second potential different from the first potential with respect to the counter electrode is applied to the first and second currents is measured. The background of oxidation current due to oxidation of the reducing substance produced by the reaction between the measurement target substance and the reaction layer contained in the electrode, and the electric current based on the charge current of the electric double layer and the oxidation-reduction reaction of impurities Current and are included.

However, the change in the magnitude of the background current is compared with the change in the magnitude of the oxidation current of the reducing substance to be measured due to the potential applied to the working electrode changing from the first potential to the second potential. Since the difference between the first current and the second current is taken, the ratio of the background current included in the difference value is very small compared to the ratio of the oxidation current to be measured. It becomes. Therefore, in the quantification step, the measurement target substance is quantified based on the difference value between the first current and the second current, so that a response obtained by applying a potential based on the counter electrode to the working electrode. Reduces the influence of the background current, which is a current component different from the oxidation current caused by the oxidation of the reducing substance produced by the reaction between the measurement target substance in the sample and the enzyme, among the current components included in the current. It is possible to improve the measurement accuracy when the measurement target substance contained in the specimen is quantified.

In addition, the conventional measurement method of measuring the response current after applying a potential to the working electrode and waiting until the charging current of the electric double layer and the oxidation-reduction reaction of the reducing substance and the impurities converge appropriately. In contrast, according to the measurement method of the present invention, the influence of the background current can be reduced by taking the difference between the first current and the second current, so that the response current can be measured at an earlier timing. Therefore, it is possible to shorten the measurement time when quantifying the measurement target substance contained in the specimen.

Further, each of the first potential and the second potential may be greater than or equal to an oxidation potential at which the reducing substance is oxidized (claim 2).

If comprised in this way, since each 1st electric potential and 2nd electric potential are magnitude | sizes more than the oxidation potential in which the reducing substance by the enzyme reaction of a measuring object substance is oxidized, the reduction | restoration by applying an electric potential to a working electrode. The amount of reducing substance contained in the sample does not increase due to the reaction, fluctuations in the concentration of the reducing substance in the sample can be suppressed, and the oxidation current due to oxidation of the reducing substance can be stabilized. Thus, it is possible to improve the measurement accuracy when quantifying the measurement target substance.

In addition, the magnitude of the charging current of the electric double layer is proportional to the time change of the potential applied to the working electrode and the electric capacity of the electric double layer, and the background current due to the charging current of the electric double layer is accurately determined. In order to make a measurement, it is necessary to apply a potential of the same magnitude to the working electrode as when the measurement target substance contained in the specimen is actually quantified. In addition, in order to accurately measure the background current due to the current based on the oxidation-reduction reaction of the impurity, it has the same potential as when the substance to be measured is actually quantified and is generated by the enzyme reaction of the substance to be measured. The potential to oxidize the reducing substance must be applied to the working electrode.

That is, for example, one of the first potential and the second potential is a potential lower than the oxidation potential of the reducing substance generated by the enzymatic reaction of the measurement target substance, and the other potential is the reducing substance to be measured. The fluctuation of the background current when the potential applied to the working electrode changes between one potential and the other potential when the potential is such that the oxidation current can be obtained. When both potentials are greater than or equal to the oxidation potential of the reducing substance, the potential applied to the working electrode is greater than the fluctuation of the background current when the potential changes between the first potential and the second potential. It will be big. However, by setting both the first potential and the second potential to be greater than or equal to the oxidation potential of the reducing substance, the first current and the second current generated by applying the first potential and the second potential can be reduced. Since the background currents included are approximated, the influence of the background currents included in the second current and the difference value between the second currents can be more reliably removed.

At this time, each of the first potential and the second potential is applied to the working electrode for at least 100 ms, and the first measurement step includes the first current after the first potential has been applied for 100 ms or more. Preferably, in the second measuring step, the second current is measured after 100 ms or more has elapsed since the second potential was applied.

With this configuration, the influence of the charging current (non-Faraday current) of the electric double layer is reduced when 100 ms or more has elapsed since the application of the first potential and the second potential, but the first potential and the second potential respectively. Is applied to the working electrode for at least 100 ms. In the first measurement step, the first current is measured after 100 ms or more has elapsed since the first potential is applied. In the second measurement step, the second potential is applied. Since the second current is measured after 100 ms or more has passed since the application, the influence of the charging current of the electric double layer can be further reduced, and the measurement accuracy can be further improved.

In the first measurement step and the second measurement step, the first current and the second current are measured at the timing when the same time has elapsed after the first potential and the second potential are applied to the working electrode, respectively. desirable. In this case, the first current and the second current are measured at the timing when the same time has elapsed after the potential is applied to the working electrode, that is, after the applied potential to the working electrode fluctuates. The charging current of the electric double layer included in each of the first current and the second current is approximately the same, and the background current included in the difference value between the first current and the second current The current component for charging the electric double layer can be further reduced.

The first potential and the second potential may be continuously and repeatedly applied to the working electrode (claim 4).

With this configuration, the first potential and the second potential are continuously and repeatedly applied to the working electrode without opening the working electrode and the counter electrode, thereby reducing the potential applied to the working electrode. Since rapid increase and decrease can be suppressed, it is possible to suppress the generation of a large charging current (non-Faraday current) to charge the electric double layer, and to stabilize the response current, so measurement The accuracy can be further improved.

Furthermore, it is desirable that the reaction layer further includes a mediator that is reduced by electrons generated by a reaction between the substance to be measured and the enzyme to become the reducing substance (Claim 5).

According to this structure, the reaction layer includes a mediator that is reduced by electrons generated by the reaction between the measurement target substance and the enzyme and becomes a reduced substance. Therefore, the measurement target substance is converted to the enzyme via the mediator. Electrons emitted by reaction can be transmitted to the working electrode, and the ratio of the oxidation current of the reducing substance to be measured included in each of the first current and the second current to be measured is increased. Therefore, the detection sensitivity and detection accuracy of the measurement target substance of the biosensor can be improved.

As the mediator, any one of potassium ferricyanide, ferrocene, ferrocene derivatives, benzoquinone, quinone derivatives, osmium complexes, and ruthenium complexes may be used (Claim 6).

According to the first aspect of the invention, in the first measurement step, the first current obtained when the first potential based on the counter electrode is applied to the working electrode is measured, and in the second measurement step, the working electrode is measured. A second current obtained when a second potential different from the first potential with respect to the counter electrode is applied to is measured, and based on a difference value between the first current and the second current in the determination step. By quantifying the measurement target substance, the ratio of the background current in the current component included in the difference value is much smaller than the ratio of the oxidation current of the reducing substance to be measured. Among the current components included in the response current obtained by applying a potential with the counter electrode as a reference to the electrode, the reducing substance produced by the reaction between the analyte in the sample and the enzyme is oxidized Acid by It is possible to reduce the influence of background current current to be different current components, it is possible to improve the measurement accuracy in quantifying the analyte contained in the specimen.

According to the invention of claim 2, since each of the first potential and the second potential is larger than the oxidation potential at which the reducing substance by the enzymatic reaction of the measurement target substance is oxidized, the potential is applied to the working electrode. The amount of reducing substance contained in the sample does not increase due to the reduction reaction caused by this, the fluctuation of the concentration of the reducing substance in the sample can be suppressed, and the oxidation current due to oxidation of the reducing substance can be stabilized. Therefore, it is possible to improve the measurement accuracy when quantifying the measurement target substance.

According to the invention of claim 3, each of the first potential and the second potential is applied to the working electrode for at least 100 ms, and in the first measurement step, the first potential is applied after the first potential is applied for 100 ms or more. In the second measurement step, the second current is measured after 100 ms or more has elapsed since the second potential was applied, so the electric double layer included in the first current and the second current is measured. The influence of the charging current can be further reduced, and the measurement accuracy can be further improved.

According to the invention of claim 4, since the first potential and the second potential are continuously and repeatedly applied to the working electrode, it is possible to suppress a rapid increase and decrease in the applied potential to the working electrode. Further, since it is possible to suppress the generation of a large charging current (non-Faraday current) for charging the electric double layer and to stabilize the response current, it is possible to further improve the measurement accuracy.

According to the invention of claim 5, since the reaction layer contains a mediator that is reduced by electrons generated by the reaction between the substance to be measured and the enzyme and becomes a reducing substance, the substance to be measured is interposed via the mediator. Electrons released by the enzyme reaction can be transmitted to the working electrode, and the ratio of the oxidation current of the reducing substance to be measured included in each of the first current and the second current to be measured is increased. Therefore, the detection sensitivity and detection accuracy of the measurement target substance of the biosensor can be improved.

It is a figure which shows an example of the biosensor system used in the measuring method of the substance of this invention. It is a figure which shows an example of a biosensor. It is a flowchart which shows an example of a measurement process. It is a figure which shows an example of the electric potential applied to a working electrode on the basis of a counter electrode. It is a figure which shows an example of the difference value of the 1st electric current and 2nd electric current which are obtained by applying the 1st electric potential and the 2nd electric potential to a working electrode. It is a figure for demonstrating an example of the measuring method of the conventional substance.

An embodiment of the method for measuring a substance of the present invention will be described with reference to FIGS.

FIG. 1 is a diagram showing an example of a biosensor system 1 used in the method for measuring a substance of the present invention. 2A and 2B are diagrams illustrating an example of the biosensor 100, where FIG. 2A is an exploded perspective view and FIG. 2B is a perspective view. FIG. 3 is a flowchart showing an example of the measurement process. 4A and 4B are diagrams illustrating an example of a potential applied to the working electrode 101 with reference to the counter electrode 102. FIG. 4A illustrates a potential applied to the working electrode 101, and FIG. 4B illustrates a time t2 in FIG. It is a subsequent partial enlarged view. FIG. 5 is a diagram illustrating an example of a difference value ΔI between the first current I ₁ and the second current I ₂ obtained by applying the first potential E ₁ and the second potential E ₂ to the working electrode 101. .

<Biosensor system>
As shown in FIG. 1, the biosensor system 1 includes an electrode system including a working electrode 101 and a counter electrode 102, a biosensor 100 having a reaction layer (not shown) including an enzyme that specifically reacts with a measurement target substance, And a measuring instrument 2 to which the biosensor 100 is detachably attached. Then, the biosensor system 1 includes a measurement target substance such as glucose contained in a specimen such as blood supplied to the cavity 103 provided on the distal end side of the biosensor 100 attached to the measuring device 2, and the biosensor 100. Included in the specimen by measuring the oxidation current obtained by applying a voltage between the working electrode 101 and the counter electrode 102 to oxidize the reducing substance produced by the reaction with the reaction layer provided. Quantify the target substance to be measured.

The measuring instrument 2 is automatically turned on when the attachment of the biosensor 100 is detected. When a specimen such as blood is supplied to the biosensor 100, the measuring instrument 2 measures a measurement target substance such as glucose in the specimen. Start. When the determination of the measurement target substance in the sample is completed, the measurement result is displayed on the display unit 3 formed by a display means such as an LCD, and an alarm for signaling the end of the measurement is output from the speaker 4. The measurement result is stored in the storage unit 5 formed by a storage medium such as a memory.

The measuring instrument 2 includes an operation unit 6 formed by operation switches and the like, and various initial settings are executed by operating the operation unit 6 or past measurements stored in the storage unit 5 are performed. Results and the like are displayed on the display unit 3.

Further, the measuring instrument 2 includes a serial interface 7 (I / F), and can transmit and receive data such as measurement results to and from an external personal computer connected via the I / F 7. The storage unit 5 is used for quantifying the measurement target substance contained in the specimen based on the past measurement results and the response current measured by applying a predetermined potential to the working electrode 101 of the biosensor 100. A conversion formula, a program that realizes various functions by being executed by the CPU 8, and the like are stored.

The measuring instrument 2 includes a voltage output unit 9, a current-voltage conversion unit 10, and an A / D conversion unit 11. The voltage output unit 9 has a digital-analog conversion function (D / A conversion function), and applies a constant reference potential to the counter electrode 102 of the biosensor 100 attached to the measuring device 2 based on a control command from the CPU 8. In addition to outputting, a predetermined potential based on the reference potential applied to the counter electrode 102 is output to the working electrode 101.

The current-voltage conversion unit 10 includes a general current-voltage conversion circuit formed by an operational amplifier or a resistance element, and the working electrode 101 is applied by applying a predetermined potential to the working electrode 101 of the biosensor 100 by the voltage output unit 9. And the counter electrode 102 are converted into a voltage signal so that the CPU 8 can capture the current. The A / D converter 11 converts the voltage signal converted by the current-voltage converter 10 into a digital signal. Then, the digital signal converted by the A / D conversion unit 11 is taken into the CPU 8, and a predetermined calculation is performed in the CPU 8, whereby the voltage signal is converted into a current signal.

The CPU 8 has the following functions by executing various programs stored in the storage unit 5 for quantifying the measurement target substance contained in the specimen.

The detection unit 8a monitors the value of the current flowing between the working electrode 101 and the counter electrode 102 input to the CPU 8 via the A / D conversion unit 11, so that the specimen between the working electrode 101 and the counter electrode 102 is made of a liquid. The change of the resistance value due to the short circuit is detected by this, and thereby, it is detected that the specimen is supplied to the cavity 103 provided in the biosensor 100. Based on a clock signal output from a clock circuit (not shown), the time measuring unit 8b, for example, an elapsed time after the detection unit 8a detects the supply of the specimen to the cavity 103, and the action of the voltage output unit 9 are used. Time for applying a predetermined potential to the pole 101 is measured.

The measuring unit 8 c measures the current flowing between the working electrode 101 and the counter electrode 102 when a predetermined potential with reference to the counter electrode 102 is applied to the working electrode 101 by the voltage output unit 9. In this embodiment, in the first measurement step, the first current I ₁ obtained when the first potential E _{1 based} on the counter electrode 102 is applied to the working electrode 101 by the voltage output unit 9 is obtained by the measurement unit 8c. In the second measurement step, the second current I ₂ obtained when a second potential E ₂ different from the first potential E ₁ with the counter electrode 102 as a reference is applied to the working electrode 101 is measured by the measuring unit 8c. It is measured by.

Determination unit 8d, in the quantitative process, the determination of analyte carried out on the basis of the first current I ₁ and the second current I ₂ of the difference value △ I measured by the measuring unit 8c. Specifically, the relationship between the difference value ΔI between the first current I ₁ and the second current I ₂ and the concentration of the measurement target substance contained in the sample is measured in advance, so that the difference value ΔI A conversion formula for converting the concentration is derived and stored in the storage unit 5 in advance. Then, the measurement target substance is quantified based on the conversion formula stored in the storage unit 5 and the actually measured difference value ΔI.

The notification unit 8e performs notification by displaying the quantification result by the quantification unit 8d on the display unit 3 or by outputting an alarm indicating that the measurement is completed from the speaker 4.

<Biosensor>
As shown in FIG. 2, the biosensor 100 is an electrode provided with a working electrode 101 and a counter electrode 102 formed of an insulating material such as ceramic, glass, plastic, paper, biodegradable material, and polyethylene terephthalate. As shown in FIG. 2A, the front side of the layer 110, the spacer layer 120 in which the slits 104 for forming the cavities 103 are formed, and the cover layer 130 in which the air holes 105 are formed are aligned. It is formed by laminating and adhering. Then, the biosensor 100 is attached to the measuring device 2 by being inserted and attached to a predetermined insertion port of the measuring device 2 from the rear end side.

In this embodiment, the electrode layer 110 is formed of a substrate made of polyethylene terephthalate, and a conductive material such as platinum, gold, palladium, or other noble metal or carbon formed on the substrate by screen printing or sputtering deposition. Patterning by laser processing is performed on the electrode film composed of the working electrode 101 and the counter electrode 102, and when the biosensor 100 is mounted on the measuring device 2, the working electrode 101 and the counter electrode 102, respectively, and the measuring device 2. Are provided with electrode patterns 101a and 102a.

In addition, the spacer layer 120 is formed of a substrate made of polyethylene terephthalate, and a slit 104 for forming the cavity 103 is formed at substantially the center of the front edge portion of the substrate, as shown in FIG. The electrode layer 110 and the electrode layer 110 are laminated and bonded together with their tips aligned.

The reaction layer is a thickener such as carboxymethyl cellulose or gelatin before the cover layer 130 is laminated on the working electrode 101 and the counter electrode 102 exposed to the cavity 103 formed by laminating the spacer layer 120 on the electrode layer 110. It is formed by dropping a reagent containing an additive such as an enzyme, mediator, amino acid or organic acid. Further, a hydrophilic agent such as a surfactant or phospholipid is applied to the inner wall of the cavity 103 in order to smoothly supply a specimen such as blood to the cavity 103.

Enzymes include glucose oxidase, lactate oxidase, cholesterol oxidase, alcohol oxidase, sarcosine oxidase, fructosylamine oxidase, pyruvate oxidase, glucose dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, hydroxybutyrate dehydrogenase, cholesterol esterase, creatininase, creatinase DNA polymerase or the like can be used, and various sensors can be formed by selecting these enzymes according to the substance to be measured.

For example, glucose oxidase or glucose dehydrogenase can be used to form a glucose sensor that detects glucose in a specimen, alcohol oxidase or alcohol dehydrogenase can be used to form an alcohol sensor that detects ethanol in a specimen, and lactate oxidase can be used to form a specimen. A lactic acid sensor for detecting lactic acid therein can be formed, and a total cholesterol sensor can be formed by using a mixture of cholesterol esterase and cholesterol oxidase.

As the mediator, potassium ferricyanide, ferrocene, ferrocene derivatives, benzoquinone, quinone derivatives, osmium complexes, ruthenium complexes and the like can be used.

The cover layer 130 is formed of a substrate made of polyethylene terephthalate, and an air hole 105 communicating with the cavity 103 when formed on the spacer layer 120 is formed in the substrate. Then, after the cover layer 130 is formed on the working electrode 101 and the counter electrode 102 where the reaction layer is exposed to the cavity, the cover layer 130 is laminated and adhered to the spacer layer 120, whereby the sample inlet for supplying the sample to the cavity 103. A biosensor 100 having 103a formed at the tip is formed.

In this embodiment, the biosensor system 1 is formed for the purpose of quantifying glucose in blood, and includes glucose oxidase as an enzyme that specifically reacts with glucose as a measurement target substance. A reaction layer containing potassium ferricyanide as a mediator that is reduced by electrons generated by the reaction between glucose and glucose oxidase, which is a reducing substance, is provided on the working electrode 101 and the counter electrode 102 exposed to the cavity 103.

In the biosensor 100 configured as described above, a specimen made of blood is brought into contact with the specimen introduction port 103a at the tip, whereby the specimen is sucked toward the air hole 105 by a capillary phenomenon and supplied to the cavity 103. . Then, when the reaction layer is dissolved in the specimen supplied to the cavity 103, electrons are released by the enzymatic reaction between glucose and glucose oxidase, which are measurement target substances in the specimen, and ferricyanide ions are released by the emitted electrons. By being reduced, ferrocyanide ions, which are reducing substances, are generated. The measuring device 2 electrochemically oxidizes the reducing substance generated by the oxidation-reduction reaction caused by the reaction layer being dissolved in the specimen by applying a voltage between the working electrode 101 and the counter electrode 102 of the biosensor 100. Thus, the glucose in the specimen is quantified by measuring the oxidation current flowing between the working electrode 101 and the counter electrode 102.

In this embodiment, the biosensor 100 is formed in a bipolar electrode structure having a working electrode 101 and a counter electrode 102. However, the biosensor 100 is formed in a tripolar electrode structure by further providing a reference electrode. Also good. In this case, a predetermined potential based on the counter electrode 102 may be applied to the working electrode 101 in a state where the counter electrode 102 is grounded and a reference potential is applied to the reference electrode by the voltage output unit 9.

In this embodiment, the specimen is supplied to the cavity 103 by monitoring a current flowing between the working electrode 101 and the counter electrode 102 by applying a predetermined voltage between the working electrode 101 and the counter electrode 102. However, a sample detection electrode is further provided, and a predetermined voltage is applied between the counter electrode 102 and the sample detection electrode to flow between the counter electrode 102 and the sample detection electrode. By monitoring the current, it may be detected that the sample is supplied to the cavity 103. Further, of the electrode layer 110, the spacer layer 120, and the cover layer 130 that form the biosensor 100, at least the cover layer 130 is formed of a transparent member so that it can be visually recognized that the specimen is supplied to the cavity 103. desirable.

<Measurement process>
Next, an example of measurement processing executed in the biosensor system 1 will be described. When it is detected by a detection circuit (not shown) that the biosensor 100 is attached to the measuring instrument 2, a blood sample is supplied to the cavity 103 of the biosensor 100 between the working electrode 101 and the counter electrode 102. A specimen detection voltage for detecting the detection is applied (step S1). Then, when the specimen is supplied to the cavity 103 and the working electrode 101 and the counter electrode 102 are liquid-contacted by the specimen, the current flowing between the working electrode 101 and the counter electrode 102 is increased, so a change in resistance value is detected. Thus, the detection unit 8a detects that the specimen is supplied to the cavity 103 (step S2).

When the detection unit 8a detects that the sample is supplied to the cavity 103, the application of the potential to the working electrode 101 by the voltage output unit 9 is stopped, and the circuit connected to the working electrode 101 and the counter electrode 102 is opened. (See time t0 in FIG. 4A, step S3). Then, after a predetermined time has elapsed since the circuit was opened, in this embodiment, at the time t1 when about 1.5 s has passed since the circuit was opened, the first potential E ₁ with respect to the working electrode 101 and the counter electrode 102 as a reference. When a second potential _{E 2} is continuously applied repeatedly by 500 ms (step S4).

Subsequently, as shown in FIG. 4 (b), after a predetermined time has elapsed since it was detected that the specimen was supplied to the cavity 103, in this embodiment, first after time t2 when about 5 s has elapsed. first current _{I 1} at a timing of time t3 _{E 1} is the first potential is applied to the working electrode 101 a predetermined time ts (in this embodiment approximately 300 ms) has passed is measured (first measurement step, step S5 ). Subsequently, a second current I ₂ is measured at time t4 when the second potential has elapsed the predetermined time ts is applied to the working electrode 101 (second measurement step, Step S6).

Then, based on the difference value ΔI between the first current I ₁ and the second current I ₂ measured in the first measurement step and the second measurement step, and the conversion formula stored in the storage unit 5, The contained glucose is quantified, and the measurement result is notified by the notification unit 8e, thereby terminating the process (quantification step, step S7).

In this embodiment, each of the first potential E ₁ and the second potential E ₂ is set to a magnitude equal to or higher than the oxidation potential at which ferrocyanide ions, which are reducing substances generated as a result of the enzymatic reaction of glucose, are oxidized. E ₁ is set to about 500 mV, and E ₂ is set to about 300 mV.

Further, each of the first potential E ₁ and the second potential E ₂ is applied to the working electrode 101 for 100 ms or longer, and after the first potential E ₁ is applied and 100 ms or longer has elapsed, the first current I ₁ is measured. The second current I ₂ is measured after 100 ms or more has elapsed since the second potential E ₂ is applied, but the same time after the first potential E ₁ and the second potential E ₂ are applied to the working electrode 101. the ts is the first current I ₁ and the second current I ₂ at the timing has elapsed is measured is preferred.

With this configuration, the first current I ₁ and the second current are applied at the timing when the same time ts has elapsed since the potential was applied to the working electrode 101, that is, after the applied potential to the working electrode 101 fluctuated. Since I ₂ is measured, the magnitude of the charging current of the electric double layer included in each of the first current I ₁ and the second current I ₂ is approximately the same, and the first current I ₁ and the second current I 2 Among the background currents included in the difference value ΔI of the current I ₂ , the current component for charging the electric double layer can be reduced.

As described above, according to this embodiment, the first current I ₁ to be measured in the first measurement step, in each of the second current I ₂ which is measured in the second measurement step, the specimen Oxidation current due to oxidation of reducing substance (ferrocyanide ion) produced by the reaction of glucose, which is the substance to be measured, with the enzyme and mediator contained in the reaction layer, and charging current of the electric double layer And a background current such as a current based on an oxidation-reduction reaction of impurities. However, when compared with the magnitude of the change in the oxidation current of the reduced substance to be measured due to variation from a potential first potential E ₁ applied to the working electrode 101 to the second potential E _2, the background current Since the change in magnitude of the current is small, the ratio of the background current included in the difference value ΔI between the _first current I ₁ and the second current I ₂ is the ratio of the oxidation current to be measured. It will be very small compared.

Accordingly, in the quantification step, the measurement target substance (glucose) is quantified based on the difference value ΔI, so that it is included in the response current obtained by applying a potential based on the counter electrode 102 to the working electrode 101. Can reduce the influence of the background current, which is a current component different from the oxidation current caused by oxidation of the reducing substance produced by the reaction between the analyte in the sample and the enzyme. The measurement accuracy when quantifying the measurement target substance contained in the specimen can be improved.

In addition, each of the first potential E ₁ and the second potential E ₂ has a magnitude equal to or higher than an oxidation potential at which the reducing substance by the enzymatic reaction of the measurement target substance is oxidized, and therefore, the potential is applied to the working electrode 101. The amount of reducing substance (ferrocyanide ion) contained in the specimen does not increase due to the reduction reaction, fluctuations in the concentration of the reducing substance in the specimen can be suppressed, and the oxidation current due to oxidation of the reducing substance since can be stabilized, as shown by a in FIG. 5, the magnitude of the first current I ₁ and the second current I ₂ of the difference value △ I is measured after the time t2 is stable, measured The measurement accuracy when quantifying the target substance can be improved.

As indicated by B in FIG. 5, the oxidation potential above about 500mV as a first potential _{E 1} was applied to the working electrode 101, applies a small about 100mV than the oxidation potential to the working electrode 101 as a second potential _{E 2} the case, when the second potential E ₂ is applied to the working electrode 101, the reducing substance in a sample reduction to ferrocyanide ions ferricyanide ions generated is increased, after time t2 The magnitude of the difference value ΔI between the first current I ₁ and the second current I ₂ measured at 1 is not stable.

Further, when 100 ms or more elapses after the first potential E ₁ and the second potential E ₂ are applied to the working electrode 101, the influence of the electric double layer charging current (non-Faraday current) is reduced, but the first potential E ₁ Each of the second electric potential E ₂ is applied to the working electrode 101 for at least 100 ms, and in the first measurement step, the first electric current I ₁ is measured after 100 ms or more have passed since the first electric potential E ₁ was applied. In the second measurement step, since the second current I ₂ is measured after 100 ms or more has elapsed since the second potential E ₂ is applied, the influence of the charging current of the electric double layer can be reduced. Measurement accuracy can be improved.

Further, the first potential E ₁ and the second potential E ₂ are continuously and repeatedly applied to the working electrode 101 without opening the circuit between the working electrode 101 and the counter electrode 102, so that the working electrode 101 can be used. Therefore, it is possible to suppress a rapid increase and decrease in the potential applied to the electrode, and thus to prevent a large charging current (non-Faraday current) from being generated for charging the electric double layer. Since the response current flowing between the two terminals 102 can be stabilized, the measurement accuracy can be further improved.

In addition, since the reaction layer contains a mediator that is reduced by electrons generated by the reaction between the substance to be measured and the enzyme and becomes a reducing substance, the substance to be measured is released through an enzyme reaction via the mediator. To increase the ratio of the oxidation current of the reducing substance to be measured included in each of the first current I ₁ and the second current I ₂ to be measured. Therefore, the detection sensitivity and detection accuracy of the measurement target substance of the biosensor 100 can be improved.

Note that the present invention is not limited to the above-described embodiment, and various modifications other than those described above can be made without departing from the spirit thereof. For example, the reaction of the above-described biosensor 100 is possible. You may form an ethanol sensor, a lactic acid sensor, etc. by changing the combination of the enzyme and mediator which are contained in a layer. The reaction layer does not necessarily include a mediator. In this case, the oxidation current due to oxidation of reducing substances such as hydrogen peroxide and enzyme reducts generated by the enzymatic reaction of the measurement target substance such as glucose is measured. do it.

Further, if the first potential E ₁ and the second potential E ₂ is applied to the working electrode 101 is different potentials may be either a high potential. In addition, the first potential E ₁ and the second potential E ₂ do not necessarily need to be continuously applied to the working electrode 101, and after the first potential E ₁ is applied and before the second potential E ₂ is applied, The circuit may be opened, or a potential different from the first potential E ₁ and the second potential E ₂ such as 0 V may be applied to the working electrode 101.

Further, the first potential _{E 1} and the second potential _{E 2,} may not be applied necessarily repeated working electrode 101. In addition, while the first potential E ₁ and the second potential E ₂ were applied to the working electrode 100, the response current was measured a plurality of times at the same timing from the start of application of the respective potentials. the average value of the response current of the first current I ₁ and the second may be a current I _2, respectively.

Further, the measurement timing and the magnitude of the applied potential in the above-described embodiment are all examples, and optimal values are appropriately set according to the type of the substance to be measured, the type of enzyme or mediator included in the reaction layer, and the like. What is necessary is to measure the first current I ₁ and the second current I ₂ which are obtained by applying different potentials to the working electrode 101 and sufficiently include the oxidation current and the background current to be measured. You can do that.

Further, the present invention can be applied to a measuring instrument that performs measurement using various biosensors.

Using a biosensor that has an electrode system that includes a working electrode and a counter electrode, and a reaction layer that includes an enzyme that specifically reacts with the target substance, the target substance and the reaction layer contained in the sample react to generate. The present invention is widely applied to a method for measuring a substance for quantifying a measurement target substance by measuring an oxidation current obtained by oxidizing a reduced substance to be measured by applying a voltage between a working electrode and a counter electrode. Can do.

100 Biosensor 101 Working Electrode 102 Counter Electrode E ₁ First Potential E ₂ Second Potential I ₁ First Current I ₂ Second Current ΔI Difference Value

Claims

Using the biosensor having an electrode system including a working electrode and a counter electrode, and a reaction layer containing an enzyme that specifically reacts with the measurement target substance, the measurement target substance contained in the sample reacts with the reaction layer. In the method for measuring a substance for quantifying the substance to be measured by measuring an oxidation current obtained by oxidizing a reducing substance produced in step 1 by applying a voltage between the working electrode and the counter electrode,
A first measurement step of measuring a first current obtained when a first potential based on the counter electrode is applied to the working electrode;
A second measurement step of measuring a second current obtained when a second potential different from the first potential with respect to the counter electrode is applied to the working electrode;
And a quantification step of quantifying the substance to be measured based on a difference value between the first current and the second current.
The method for measuring a substance according to claim 1, wherein each of the first potential and the second potential has a magnitude equal to or greater than an oxidation potential at which the reducing substance is oxidized.
Each of the first potential and the second potential is applied to the working electrode for at least 100 ms,
The first measuring step measures the first current after 100 ms or more has elapsed since the first potential was applied, and the second measuring step comprises measuring the first current after 100 ms or more has elapsed since the second potential was applied. The method for measuring a substance according to claim 1, wherein the second current is measured.
4. The method for measuring a substance according to claim 1, wherein the first potential and the second potential are continuously and repeatedly applied to the working electrode.
The substance according to any one of claims 1 to 4, wherein the reaction layer further includes a mediator that is reduced by electrons generated by a reaction between the substance to be measured and the enzyme to become the reducing substance. Measuring method.
6. The method for measuring a substance according to claim 5, wherein any one of potassium ferricyanide, ferrocene, ferrocene derivatives, benzoquinone, quinone derivatives, osmium complexes, and ruthenium complexes is used as the mediator.