JP2000162176A - Measuring method and measuring device using biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To perform a highly precise measurement and to minimize the dispersion between sensors. SOLUTION: A DC voltage of 0.15 V is applied to the working electrode and reference electrodes of a biosensor, the applied voltage is changed to 0.6 V after a fixed time (t [seconds]) from the detection of supply of a blood sample, and the current carried between the working electrode and reference electrode is detected after 5 seconds from the change to measure the glucose quantity in the blood sample. Otherwise, the measurement may be performed by applying DC voltage of 0.1 V and 0.15 V before the supply of the blood sample and after the detection of the supply, respectively, changing the voltage to 0.6 V after a fixed time from the detection of the supply, and performing the measurement after 5 seconds after the change.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for measuring the concentration of a specific component in a liquid sample containing various substances using a biosensor. More specifically, the present invention relates to a method and an apparatus for measuring the concentration of a substance to be measured using a biosensor in which a method for applying a potential is devised, and to provide a more accurate measurement method and an apparatus.

[0002]

2. Description of the Related Art Hitherto, a biosensor has been employed to measure the concentration of a specific component from a liquid sample of a living body such as blood or urine. For example, glucose in blood (hereinafter referred to as "blood glucose") is used.
Has been used for concentration measurement. In recent years, blood glucose biosensors with easy operation and high measurement accuracy have been developed for individuals to check their own blood glucose levels, and can measure a small amount of blood in a short time without the need for pretreatment operations such as dilution. There is provided a blood glucose meter which has a low running cost and is equipped with a disposable type biosensor for each measurement. As an example, a biosensor disclosed in Patent No. 2517151 will be introduced. The biosensor of this example has an electrode system consisting of a measurement electrode and a counter electrode on an insulating substrate, and an enzyme reaction containing a redox enzyme, a hydrophilic polymer, and an electron acceptor on the surface of the electrode system. In the structure in which the layers are stacked, a change in the concentration of a substance generated by the reaction between the enzyme, the electron acceptor, and the liquid sample is electrochemically detected by an electrode system to measure the concentration of the substance to be measured. In the biosensor, the following two methods of applying a potential are proposed. One is to apply a constant DC voltage between a measurement electrode and a counter electrode (hereinafter, referred to as “between electrodes”) while applying a constant DC voltage on the enzyme reaction layer. Supply the liquid sample to the
The other method is to measure the flowing current, and the other is to supply a liquid sample onto the enzyme reaction layer while applying a constant DC voltage between the two electrodes.After detecting the supply of the liquid sample, once stop applying the voltage,
This is a method of applying a voltage again after a certain time and measuring the flowing current.

[0003]

However, such a conventional method of applying a potential to a biosensor has the following problems.

[0004] First, a method of supplying a liquid sample while applying a constant DC voltage between both electrodes and detecting the supply of the liquid sample is common (in the concentration measurement of a substance to be measured by a biosensor, it is commercially available). In the known example, which is also used in the apparatus described above, the above-mentioned example merely applies the well-known measurement method to a biosensor using an oxidoreductase and an electron transfer substance. Since the enzymatic reaction and the electrochemical phenomenon are proceeding simultaneously, the oxidized electron mediator is reduced to the reduced electron mediator with the enzymatic reaction. Even though the electron mediator is generated, it is electrolytically oxidized on the electrode. Therefore, there is no accumulation of the reduced electron mediator and a higher concentration of the reduced electron mediator cannot be supplied to the electrode surface, so that the obtained electrode output is small and the S / N ratio is not only bad, but also the measurement is not possible. There is a problem that the concentration range that can be obtained is narrow, and measurement was impossible when the concentration of the substance to be measured was particularly low. Further, since the electrode output is obtained from the supply point of the liquid sample, the measurement time can be shortened.However, in the case of a liquid sample having a high viscosity such as blood, a difference occurs in the supply state of the liquid sample, and the electrode is changed depending on the sensor. There is a problem that the difference in output is large and the reproducibility of the measurement result is poor. In addition, in such a voltage application pattern, a potential higher than the reaction voltage is continuously applied until the liquid sample is aspirated. Therefore, when this applied voltage is higher than the oxidation-reduction potential of the electron transfer substance. Under the condition that the reagents are dissolved in advance by the influence of the environment (humidity), the reduced electron transfer substance generated before the suction of the liquid sample is electrolytically oxidized on the electrode. A phenomenon occurs in which the total amount of reagents contributing to the reaction of the sample decreases. As a result, the number of reagent layers contributing to the reaction decreases, and the sensor output decreases. Under such conditions, measurement accuracy has been reduced.

[0005] The other method is to temporarily stop the applied voltage between the electrodes after detecting the supply of the liquid sample, apply the voltage again after a certain time, and measure the flowing current. Therefore, the reducing substance contained in the liquid sample in advance cannot be removed by electrolytic oxidation.
Therefore, in the measurement after the application of the voltage again, there was interference of the reducing substance, so that high measurement accuracy could not be achieved, and the variation between the sensors was large. It is not preferable to temporarily stop the application of the voltage between the two electrodes and then apply the voltage again for stabilizing the electrode surface. That is, when voltage application between the two electrodes starts, the electrode surface gradually becomes stabilized and has an aging effect. However, when the application is stopped, the stabilization process does not proceed, and after a certain period of time, the re-stabilization process does not proceed. The re-stabilization is started by the application, so that not only the efficiency of stabilization of the electrode surface is low but also the variation of the stabilization between sensors is increased.

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems of the prior art.
It is an object of the present invention to provide a method and an apparatus for measuring the concentration of a substance to be measured using a biosensor capable of performing high-accuracy measurement and having little variation between sensors.

[0007]

Means for Solving the Problems In order to achieve the above object, a first invention uses a biosensor having an electrode system including at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme. A method for detecting an electrochemical phenomenon caused by a reaction between a liquid sample supplied to the reaction layer and an enzyme with the electrode system and measuring a concentration of a specific component in the liquid sample, wherein the first potential is applied to the electrode system. The liquid sample is supplied to the reaction layer in a state where a predetermined DC potential is applied,
After a certain time from either the detection of the supply of the liquid sample or the supply of the liquid sample, the potential applied to the electrode system is changed to a measurement potential different from the first potential, and the measurement potential after the change is changed. The method is characterized in that the concentration of the specific component is measured based on a current detected by the electrode system in a state where the voltage is applied.

As described above, the potential applied to the electrode system is changed between the time of supplying the liquid sample and the time of the measurement to make the potential in two stages, thereby suppressing the variation in the measurement result between the sensors due to the difference in the supply state of the liquid sample. At the same time, a high-precision measurement can be performed by applying a potential that provides a sufficient output at the time of measurement. Further, since the application of the potential to the electrode system is not interrupted in the middle, the efficiency of stabilizing the surface of the electrode system is good, and the variation in stabilization between sensors can be suppressed.

According to a second aspect of the present invention, a liquid supplied to a reaction layer is provided by using a biosensor having an electrode system including at least a working electrode and a reference electrode, and a reaction layer including at least an enzyme and an electron transfer substance. In a measurement method for detecting the concentration of a specific component in the liquid sample by detecting an electrochemical phenomenon caused by a reaction between a sample and an enzyme with the electrode system, a state in which a predetermined potential is applied to the electrode system as a first potential. The liquid sample is supplied to the reaction layer, and after a certain time from either the detection of the supply of the liquid sample or the supply of the liquid sample, the potential applied to the electrode system is a measurement potential different from the first potential. And measuring the concentration of the specific component based on the current detected by the electrode system in a state where the changed measurement potential is applied.

As described above, the potential applied to the electrode system is changed between the time of supplying the liquid sample and the time of the measurement so as to have two stages, thereby suppressing the variation in the measurement result between the sensors due to the difference in the supply state of the liquid sample. At the same time, a high-precision measurement can be performed by applying a potential that provides a sufficient output at the time of measurement. Further, since the application of the potential to the electrode system is not interrupted in the middle, the efficiency of stabilizing the surface of the electrode system is good, and the variation in stabilization between sensors can be suppressed.

An electron transfer substance is a substance that transfers electrons responsible for an electrochemical phenomenon caused by an enzyme reaction between an enzyme and a specific component in a liquid sample, and can promote the enzyme reaction between the enzyme and the liquid sample.

A third aspect of the present invention includes an electrode system including at least a working electrode and a reference electrode, and a reaction layer including at least an enzyme and an electron transfer substance, wherein the reaction layer facilitates formation of the reaction layer. Using a biosensor containing a retaining agent to be measured, measuring the concentration of a specific component in the liquid sample by detecting the electrochemical phenomenon caused by the reaction between the liquid sample and the enzyme supplied to the reaction layer by the electrode system. In the method, the liquid sample is supplied to the reaction layer in a state where a predetermined DC potential is applied as a first potential to the electrode system, and the supply of the liquid sample is fixed from one of detection of the supply of the liquid sample and supply of the liquid sample. After a time, the potential applied to the electrode system is changed to a measurement potential different from the DC potential, and the concentration of the specific component is determined based on the current detected by the electrode system in a state where the changed measurement potential is applied. To Characterized in that it constant.

As described above, the potential applied to the electrode system is changed between the time of supplying the liquid sample and the time of the measurement so as to have two steps, thereby suppressing the variation in the measurement result between the sensors due to the difference in the supply state of the liquid sample. At the same time, a high-precision measurement can be performed by applying a potential that provides a sufficient output at the time of measurement. Further, since the application of the potential to the electrode system is not interrupted in the middle, the efficiency of stabilizing the surface of the electrode system is good, and the variation in stabilization between sensors can be suppressed.

According to a fourth aspect, in the third aspect, the retaining agent is a low molecular compound.

As described above, even in a biosensor using a low-molecular compound as a retaining agent, the same effect can be obtained by changing the potential applied to the electrode system between the supply of the liquid sample and the measurement at two stages. can get.

The low molecular weight compound has a molecular weight of about 100
Compounds of 0 or less can be appropriately selected according to the properties of the enzyme, liquid sample, and the like. By adding a low-molecular compound, it is easier to dissolve in the liquid state before solidifying and forming the reaction layer than in the case of adding a high-molecular compound. Dissolution is quick, and the time required for measurement can be reduced.

In a fifth aspect based on the first to fourth aspects, the measured potential is a potential higher than the first potential before the change.

By setting the measurement potential higher than the first potential in this manner, a sufficient output for measurement can be obtained from the sensor at the time of measurement, and high-precision measurement can be performed.

In a sixth aspect based on the second to fourth aspects, the measured potential is higher than the oxidation-reduction potential of an electron transfer substance contained in the reaction layer.

As described above, by making the measured potential higher than the oxidation-reduction potential of the electron mediator, the electron mediator is oxidized or reduced to promote the enzymatic reaction, thereby reducing the amount of the specific component in the liquid sample. Since a corresponding output is obtained, highly accurate measurement is possible.

In a seventh aspect based on the second, third, fourth or sixth aspect, the first potential is a potential lower than the oxidation-reduction potential of the electron transfer substance contained in the reaction layer. I do.

In this way, when the first potential is applied, even if the liquid sample is supplied to the reaction section, the reduced or oxidized electron transfer substance produced by the enzymatic reaction is oxidized or reduced. Since accumulation can be performed without consumption, a large output with a good S / N ratio can be obtained from the sensor, and measurement can be performed over a wide concentration range.

In an eighth aspect based on the seventh aspect, the first potential is 0 volt or a potential at which the potential of the working electrode is lower than the potential of the reference electrode.

The first potential may be appropriately set, and may be 0 volt or a potential at which the potential of the working electrode is lower than the potential of the reference electrode.

In a ninth aspect based on the seventh aspect, the liquid sample contains a reducing substance, and the first potential is a potential capable of oxidizing the reducing substance. .

In this way, when the liquid sample contains a reducing substance that affects the measurement, the reducing substance can be oxidized and removed when the first potential is applied, so Accuracy can be measured.

According to a tenth aspect of the present invention, there is provided a biosensor having an electrode system including at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme. In a measurement method for detecting the concentration of a specific component in the liquid sample by detecting an electrochemical phenomenon due to a reaction with the electrode system, the liquid sample is subjected to a predetermined DC potential as a first potential applied to the electrode system. A second potential for supplying to the reaction layer and applying a potential applied to the electrode system at a time of either the detection of the supply of the liquid sample or the supply of the liquid sample to wait for a chemical reaction in the electrode system. Is changed to a predetermined DC potential, and after a certain period of time from either one of the detection of the supply of the liquid sample and the supply of the liquid sample, the potential applied to the electrode system is changed to the first potential and the second potential. Position change to a different measurement potential and characterized by measuring the concentration of the particular component based on the current detected by the electrode system while applying the measurement potential after change.

As described above, the potential applied to the electrode system is changed between before and after the supply of the liquid sample, after the supply, and during the measurement so as to have three stages. A high-precision measurement can be performed by suppressing the variation and applying a potential that can provide a sufficient output at the time of measurement. Further, since the application of the potential to the electrode system is not interrupted in the middle, the efficiency of stabilizing the surface of the electrode system is good, and the variation in stabilization between sensors can be suppressed. The reaction layer may contain a holding agent composed of an electron transfer material and / or a low molecular compound.

According to an eleventh aspect, in the tenth aspect,
The measurement potential is a potential higher than the first potential and the second potential before the change.

By setting the measurement potential higher than the first potential and the second potential in this way, it is possible to obtain an output sufficient for measurement from the sensor at the time of measurement, and it is possible to perform highly accurate measurement.

According to a twelfth aspect, in the tenth aspect,
The reaction layer includes an electron transfer material, and the second potential is lower than an oxidation-reduction potential of the electron transfer material.

In this way, when the second potential is applied, the liquid sample is supplied to the reaction section to oxidize or reduce and consume the reduced or oxidized electron mediator generated by the enzyme reaction. Since it is possible to accumulate without a delay, a large output with a good S / N ratio can be obtained from the sensor, and the measurement can be performed over a wide concentration range.

According to a thirteenth aspect, in the tenth aspect,
The reaction layer includes an electron mediator, and the second potential is 0 volt or a potential lower than the oxidation-reduction potential of the electron mediator.

According to a fourteenth aspect, in the twelfth aspect,
The liquid sample contains a reducing substance, and the second potential is a potential capable of oxidizing the reducing substance.

In this way, when the liquid sample contains a reducing substance that affects the measurement, the reducing substance can be oxidized and removed when the second potential is applied, so that the liquid sample can be removed. Accuracy can be measured.

According to a fifteenth aspect, an electrode system including at least a working electrode and a reference electrode, a reaction layer including at least an enzyme,
A measuring device for detecting a concentration of a specific component in the liquid sample by detecting an electrochemical phenomenon caused by a reaction between the liquid sample supplied to the reaction layer and an enzyme with the electrode system, using a biosensor having A potential applying means for applying a predetermined DC potential to the electrode system; and a potential changing means for changing a DC potential applied to the electrode system, wherein the potential applying means comprises: Is supplied to the reaction layer, a predetermined DC potential is applied to the electrode system as a first potential, and after a certain time from either the detection of the supply of the liquid sample or the supply of the liquid sample, the change in the potential is performed. Means for changing the potential applied to the electrode system to a measurement potential different from the first potential, and applying the measured potential after the change based on the current detected by the electrode system. And measuring the concentration of the component.

As described above, the potential applied to the electrode system is changed by the potential changing means between the time of supply of the liquid sample and the time of measurement, so that the measurement result between the sensors due to the difference in the supply state of the liquid sample is obtained. And the measurement can be performed with high accuracy by applying a potential that can provide a sufficient output at the time of measurement. Further, since the application of the potential to the electrode system is not interrupted in the middle, the efficiency of stabilizing the surface of the electrode system is good, and the variation in stabilization between sensors can be suppressed.

According to a sixteenth aspect, in the fifteenth aspect,
The measurement potential is higher than the first potential.

By setting the measurement potential higher than the first potential in this way, a sufficient output for measurement can be obtained from the sensor at the time of measurement, and highly accurate measurement can be performed.

According to a seventeenth aspect, in the fifteenth aspect,
The reaction layer includes an electron transfer material, and the measured potential is higher than the oxidation-reduction potential of the electron transfer material included in the reaction layer.

As described above, by making the measured potential higher than the oxidation-reduction potential of the electron mediator, the oxidation or reduction of the electron mediator is performed to promote the enzymatic reaction, thereby reducing the amount of the specific component in the liquid sample. Since a corresponding output is obtained, highly accurate measurement is possible.

According to an eighteenth aspect, in the fifteenth aspect,
The reaction layer includes an electron transfer material, and the first potential is lower than an oxidation-reduction potential of the electron transfer material included in the reaction layer.

In this way, when the first potential is applied, even if the liquid sample is supplied to the reaction section, the reduced or oxidized electron transfer substance generated by the enzymatic reaction is oxidized or reduced. Since accumulation can be performed without consumption, a large output with a good S / N ratio can be obtained from the sensor, and measurement can be performed over a wide concentration range.

According to a nineteenth aspect, in the eighteenth aspect,
The first potential is 0 volt or a potential at which the potential of the working electrode is lower than the potential of the reference electrode.

The first potential may be appropriately set, and may be 0 volt or a potential at which the potential of the working electrode is lower than the potential of the reference electrode.

According to a twentieth aspect, in the eighteenth aspect,
The liquid sample contains a reducing substance, and the first potential is a potential capable of oxidizing the reducing substance.

In this way, when the liquid sample contains a reducing substance that affects the measurement, the reducing substance can be oxidized and removed when the first potential is applied, so that the liquid sample can be removed. Accuracy can be measured.

According to a twenty-first aspect, an electrode system including at least a working electrode and a reference electrode, a reaction layer including at least an enzyme,
A measuring device for detecting a concentration of a specific component in the liquid sample by detecting an electrochemical phenomenon caused by a reaction between the liquid sample supplied to the reaction layer and an enzyme with the electrode system, using a biosensor having A potential applying means for applying a predetermined DC potential to the electrode system; and a potential changing means for changing a DC potential applied to the electrode system, wherein the potential applying means comprises: Until a predetermined direct current potential is applied to the electrode system as the first potential until the supply of the liquid sample and the supply of the liquid sample are performed at any one time until the potential change means is supplied to the reaction layer. By changing the potential applied to the electrode system to a predetermined DC potential as a second potential for waiting for a chemical reaction in the electrode system, any of the detection of the supply of the liquid sample and the supply of the liquid sample After a certain period of time from one time point, the potential applied to the electrode system is changed to a measurement potential different from the first potential and the second potential by the potential changing means, and the measurement potential after the change is applied in a state where the changed measurement potential is applied. The concentration of the specific component is measured based on a current detected by the electrode system.

As described above, the potential applied to the electrode system is changed by the potential changing means before, after, and after the supply of the liquid sample and at the time of measurement, so that three stages are provided. In addition to suppressing variations in the measurement results, it is possible to perform high-precision measurement by applying a potential that can provide a sufficient output during measurement.
Further, since the application of the potential to the electrode system is not interrupted in the middle, the efficiency of stabilizing the surface of the electrode system is good, and the variation in stabilization between sensors can be suppressed.

[0050]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of a biosensor according to the present invention will be described with reference to the drawings.

(First Embodiment) FIG. 1 shows a first embodiment of the present invention.
FIG. 1 shows a biosensor according to the embodiment of FIG.
1A is an exploded perspective view, FIG. 1B is an external view, and FIG.
FIG. 2 is a sectional view taken along line XX of FIG.

In the biosensor 10 according to the present invention, a working electrode 2 and a reference electrode 3 as an electrode system and one of the electrode systems are provided on one surface of a substrate 1 made of an insulating film employing polyethylene terephthalate. The insulating film 4 and the reagent layer (reaction layer) 5 for covering the part are formed by lamination, and the spacer 6
And a cover 7 are attached. The working electrode 2 and the reference electrode 3 are both made of the same carbon-based material.
Lead portions 2b and 3b are covered by
a, 3a and the connection terminal portions 2c, 3c are exposed. A reagent layer 5 is formed on the electrode reaction sections 2a and 3a by applying and drying a solution containing at least an enzyme. A gap 10 a corresponding to the thickness of the spacer 6 is formed between the substrate 1 and the cover 7, and the structure has the reagent layer 5 in the gap 10 a.

At the time of measuring blood sugar, if the gap 10a at the tip of the biosensor 10 where the reagent layer 5 and the electrode reaction portions 2a, 3a are formed is brought into contact with a blood sample such as a fingertip, the blood sample is moved into the gap 10a by capillary action. And is introduced into the reagent layer 5.

The shape of the biosensor is not limited to the illustrated example. Materials and forming methods of the insulating film, the working electrode, the reference electrode, the insulating film, the spacer, and the cover include known materials,
A suitable method can be appropriately selected from the formation methods.
For example, the substrate, the spacer and the cover are made of a material other than polyethylene terephthalate, polyethylene naphthalate,
The resin sheet can be selected from a resin sheet made of polyethylene sulfide, polycarbonate, polyallylate, polyether sulfide, polyimide, and the like, as well as plastic, ceramics, a thin glass plate, and paper. Working electrode,
Although the reference electrode was formed by screen printing suitable for mass production, electrode materials such as platinum, gold, silver, silver chloride, iron, zinc, nickel, and palladium were deposited by vapor deposition, sputtering, plating,
It can also be manufactured by a thin film forming method such as ion plating.
Further, the electrode reaction section, the lead section, and the connection terminal section may be formed of different materials. The reagent layer contains at least an enzyme. The enzyme must be appropriately selected depending on the substance to be determined, and examples thereof include glucose oxidase, lactate oxidase, alcohol oxidase, cholesterol oxidase, uricase, and pyruvate oxidase. Further, depending on the substance to be determined, not only the enzyme but also an electron transfer substance such as potassium ferricyanide, a ferrocene compound, and p-benzoquinone may need to be added to the reagent layer. Further, a retaining agent for easily forming the reagent layer into a layer may be added, and the retaining agent is a low molecular compound, and is a compound or a compound selected from monosaccharides, disaccharides, and trisaccharides. It is a mixture of the above, one kind of compound selected from amino acids, or a mixture of two or more kinds. The formation of the reagent layer is generally performed by drying the dropped reagent solution, but a screen printing method or the like can be appropriately selected. Further, the spacer and the cover are adhered with an adhesive in the present embodiment, but other joining methods can be appropriately adopted. In short, in the present invention, various materials, shapes, thicknesses, and the like of the biosensor are not limited, and may be appropriately selected or selected according to a usage mode of the biosensor.

FIG. 2 is a block diagram showing a schematic structure of a main part of a measuring device for performing measurement using the biosensor 10, but the structure of the measuring device is not limited to this.

The connection terminal 3c of the working electrode 3 feeds the output back to the inverting input terminal via the resistor 21 and connects the non-inverting input terminal to the inverting input terminal 23a of the IV converter 23 composed of the operational amplifier 22 with the ground. Connected. The current detected by the working electrode is converted into a voltage by the IV converter 23. This voltage is converted into a digital signal by an AD conversion circuit 24 and input to a control circuit 25 including a CPU, a memory, and the like.

The connection terminal 4c of the reference electrode 4 is connected to an output terminal 27a of a buffer circuit 27 composed of an operational amplifier 26 whose output is fed back to an inverting input terminal. Terminal 28 at 28
8a can be changed by connecting to the terminal 28b or the terminal 28c. The switch 28 switches the voltage according to a signal from the control circuit 25. In the present embodiment, the potential application means includes a voltage supply source (not shown) that supplies a predetermined voltage to the IV conversion unit 23, the A / D conversion circuit 24, the control circuit 25, the buffer circuit 27, and the terminals 28b and 28c. The potential changing means includes a switch 28 and a control circuit 25. The control circuit 25 is connected to a sensor detection unit 29 for detecting whether or not the biosensor 10 is attached to the measurement device, and a display unit 30 for displaying information such as measurement results. A measuring method using the measuring device will be described.

The application of the voltage is performed in two stages.

In the first step, switch 28 is
Connected to the b side, a first potential is applied to the working electrode 2 and the reference electrode 3, and an enzymatic reaction is mainly performed, and a reduced type electron transfer substance accompanying the enzymatic reaction is accumulated. In the next stage, which is a higher potential than the first step, the switch 28 is switched to the 28c side, and the measured potential is applied to the working electrode 2 and the reference electrode 3,
Both the enzymatic reaction and the electrochemical reaction proceed, and the reduced electron mediator is oxidized. That is, in the first stage of the relatively low potential (first potential), a constant potential equal to or lower than the oxidation-reduction potential of the electron transfer substance contained in the reagent layer is applied for a fixed time, and the supply of the liquid sample is detected. The reducing substance contained in the liquid sample is removed by electrolytic oxidation, and the initial potential is lower than the oxidation-reduction potential of the electron transfer substance. The transmitter will be stored. In the second stage, the applied potential between the two electrodes is changed by switching, and a potential (measurement potential) higher than the potential before the change and higher than the oxidation-reduction potential of the electron transfer substance in the reagent layer is applied for a certain period of time. The concentration of the substance to be measured in the liquid sample is calculated from the subsequent electrode output. The first potential and the measured potential, the time from the supply of the liquid sample to the change of the potential and the time from the change of the potential to the sampling of the electrode output for the concentration measurement are appropriately determined according to the type and characteristics of the enzyme, the liquid sample and the electrode system Just set it.

In this manner, a method of measuring the concentration of a substance to be measured by a biosensor having a high measurement accuracy with little influence of a reducing substance and variation between sensors is possible. In addition, application of a low potential to a high potential is also preferable for stabilization of the electrode surface, and the electrode surface is rapidly stabilized, its stability is high, and variation between sensors is small.

In this embodiment, the electrode system is composed of two electrodes, the working electrode and the reference electrode. However, the electrode system may be composed of three electrodes by providing a counter electrode, or may be composed of more electrodes.
When a counter electrode is provided, it may be connected to the non-inverting input terminal side of the operational amplifier 26.

EXAMPLE As an example, a biosensor for measuring blood glucose will be described.

First, the substrate 1 was 150 mm × 150 m
m of polyethylene terephthalate (hereinafter referred to as “PET”) having a thickness of 188 μm (not shown).
The reference electrode 3 is formed by screen printing using a polymer type carbon paste using a thermosetting phenol resin as a binder. Further, the insulating film 4 is formed of a thermosetting resist.
Are formed, and the respective lead portions 2b, 2b,
3b, the electrode reaction portions 2a, 3a and the connection terminal portions 2c,
3c.

Next, the reagent solution 5 is placed on the electrode reaction sections 2a and 3a.
μl was added dropwise and dried at 50 ° C. for 1 hour to form a reagent layer 5. The composition of the reagent solution was the enzyme glucose oxidase 0.1.
1%, potassium ferricyanide which is an electron mediator
0% and the holding material trehalose 1.0%.

Finally, PET (100 μm in thickness) cut to a length of 10 mm and a width of 5 mm was adhered to the biosensor portion of the insulating film with an adhesive, and P
After bonding ET (thickness 188 μm, 150 mm × 150 mm) with an adhesive, the length per one biosensor is 20
Cut to a width of 6 mm.

The blood glucose biosensor 1 thus manufactured
0, as shown in FIG.
Is applied with a voltage of 0.15 volt (first potential)
When the tip of the biosensor 10 is brought into contact with blood such as a fingertip, the blood sample flows into the gap 10 a by capillary action and is introduced into the reagent layer 5. When the supply of the blood sample can be confirmed by the electrode output, a voltage (measurement potential) of 0.6 volt is applied to the working electrode 2 with respect to the reference electrode 3 after a certain time (t [sec]).
Is applied, and the blood glucose level is calculated from the electrode output 5 seconds after the application. Blood contains reducing substances ascorbic acid, bilirubin, creatine, and uric acid as interference substances.
The above-mentioned 0.15 volt is an oxidizable potential of these reducing substances, which is lower than the oxidation-reduction potential of potassium ferricyanide, which is an electron mediator, so that the reducing substances can be removed and the enzymatic reaction can be carried out. The resulting potassium ferrocyanide can accumulate.

In the present embodiment and the examples, the supply of the blood sample is detected by the electrode output. However, the supply of the blood sample is input using input means such as a button, and the voltage is changed after a predetermined time from the supply. (The same applies to the second embodiment and the example). If it is not necessary to detect the supply of the blood sample by the electrode output, the voltage (first potential) before the change may be set to 0V.

(Second Embodiment) Hereinafter, a second embodiment will be described. Since the configuration of the biosensor is the same as that of the first embodiment, the description is omitted. This embodiment is different from the first embodiment in a part of the configuration of the measuring apparatus and the measuring method. Therefore, the same components are denoted by the same reference numerals, and the description will be omitted, and different portions will be described.

FIG. 4 is a block diagram showing a schematic configuration of the measuring apparatus according to the present embodiment.

In this embodiment, the switch 31 is connected to the terminal 31
By connecting a to the terminals 31b, 31c, 31d, the voltage input to the non-inverting input terminal of the operational amplifier 26 can be changed. The switch 28 switches the voltage according to a signal from the control circuit 25. The potential application means is an IV conversion unit 23, an A / D conversion circuit unit 24, a control circuit 25, a buffer circuit 27, and terminals 31b, 31c, 3
1d, a voltage supply source (not shown) for supplying a predetermined voltage to the switch 1d.
Are the same as in the first embodiment.

Hereinafter, a measuring method using this measuring device will be described.

The application of the potential is performed in three stages.

In the first step, the switch 31 is set to 31
A preliminary potential (first potential) is applied to the working electrode 2 and the reference electrode 3 while being connected to the b-side. This step is a preliminary stage until the liquid sample is sucked by the sensor. In order to estimate the suction of the liquid sample from the current flowing through the sensor, it is necessary to continuously apply the voltage. The preliminary potential is set to a potential lower than the oxidation-reduction potential of the electron transfer substance contained in the reagent layer. By setting to such a potential, even if the reagent has previously melted out due to environmental factors such as humidity,
The reducing electron transfer material generated before the sample supply is not electrolytically oxidized on the electrode. Therefore, it is possible to prevent the total amount of reagents that contribute to the actual reaction from decreasing. In the second step, the switch 31 is connected to the side 31c, and a standby potential (second potential) is applied to the working electrode 2 and the reference electrode 3. This step is a stage in which an enzymatic reaction is mainly performed and a reduced electron transfer substance accompanying the enzymatic reaction is accumulated. The standby potential is set higher than the preliminary potential and equal to or lower than the oxidation-reduction potential of the electron transfer substance. By setting to such a potential, the reducing substance contained in the sample is electrolytically oxidized and removed, and the reduced electron transfer substance generated by the enzymatic reaction is stored and accumulated. In the third step, the switch 31 is connected to the 31d side, and the working electrode 2 and the reference electrode 3 have a potential higher than the potential in the second step and a potential lower than the oxidation-reduction potential of the electron transfer substance in the reagent layer. A high potential is applied, and the concentration of the substance to be measured in the sample is calculated from the electrode output after a certain period of time. In this way, a method of measuring a substance to be measured by a biosensor having a high measurement accuracy, which has a small influence of a reducing substance and variation between sensors, is possible. In addition, application of a low potential to a high potential is also preferable for stabilization of the electrode surface. The electrode surface is rapidly stabilized, its stability is high, and there is little variation between sensors.

(Example) As an example, a case where the same blood glucose measuring biosensor as that of the example of the first embodiment is used will be described. Since the measurement is performed by the same method except for the method of applying the voltage, only different portions will be described.

First, a voltage of 0.1 volt is applied to the working electrode 2 with respect to the reference electrode 3 in advance to the blood glucose biosensor 10 as shown in FIG.
When the leading end of the blood sample is in contact, the blood sample flows into the gap 10 a by capillary action and is introduced into the reagent layer 5. When the supply of the blood sample is confirmed by the electrode output, a voltage of 0.15 V is applied, and after a certain time (t [sec]), a voltage of 0.6 V is applied to the working electrode 2 with respect to the reference electrode 3. The blood glucose level is calculated from the electrode output 5 seconds after the voltage application.

In the present embodiment and the example, the second potential is set higher than the first potential, but may be set to 0 volt.

[0077]

As described above, as in the first, second or third invention, the potential applied to the electrode system is changed between the supply of the liquid sample and the measurement of the liquid sample in two stages. By
Variations in measurement results between sensors due to differences in the supply state of the liquid sample can be suppressed, and high-precision measurement can be performed by applying a potential that provides a sufficient output during measurement. Further, since the application of the potential to the electrode system is not interrupted in the middle, the efficiency of stabilizing the surface of the electrode system is good, and the variation in stabilization between sensors can be suppressed.

According to the fourth aspect, even in a biosensor using a low-molecular compound as a retaining agent, the potential applied to the electrode system is changed between the supply of the liquid sample and the measurement of the liquid sample in two steps. Thereby, the same effect as that of the third invention can be obtained.

By setting the measurement potential higher than the first potential as in the fifth invention, an output sufficient for measurement can be obtained from the sensor at the time of measurement, and high-precision measurement can be performed.

By making the measured potential higher than the oxidation-reduction potential of the electron mediator as in the sixth invention, the oxidation or reduction of the electron mediator is performed to promote the enzymatic reaction, and the specific component in the liquid sample is reduced. Output can be obtained according to the amount of
High-precision measurement becomes possible.

According to the seventh aspect, when the first potential is applied, even if the liquid sample is supplied to the reaction part, the reduced or oxidized electron transfer substance generated by the enzymatic reaction is oxidized or reduced. Since it is possible to accumulate data without consuming it, a large output with a good S / N ratio can be obtained from the sensor, and measurement can be performed over a wide concentration range.

As in the eighth invention, the same effect as in the seventh invention can be obtained even when the first potential is set to 0 volt or the potential at which the potential of the working electrode is lower than the potential of the reference electrode.

According to the ninth aspect, when the liquid sample contains a reducing substance that affects the measurement, the reducing substance can be oxidized and removed when the first potential is applied. , High-precision measurement becomes possible.

As in the tenth aspect, the potential applied to the electrode system is changed before and after the supply of the liquid sample and after the supply and at the time of measurement to make three stages, so that the sensor due to the difference in the supply state of the liquid sample is obtained. While reducing the variation in measurement results between
At the time of measurement, high-precision measurement can be performed by applying a potential that can provide a sufficient output. Also,
Since the application of the potential to the electrode system is not interrupted on the way, the efficiency of stabilizing the surface of the electrode system is high, and the variation in stabilization between sensors can be suppressed.

By making the measurement potential higher than the first potential and the second potential as in the eleventh aspect, an output sufficient for measurement can be obtained from the sensor during measurement, and high-precision measurement can be performed. .

According to the twelfth aspect, when the second potential is applied, the liquid sample is supplied to the reaction section to oxidize or reduce the reduced or oxidized electron transfer substance generated by the enzymatic reaction, thereby consuming it. Since it is possible to accumulate the data without performing the process, a large output with a good S / N ratio can be obtained from the sensor, and the measurement can be performed over a wide concentration range.

As in the thirteenth aspect, the same effect as in the tenth aspect can be obtained by setting the second potential to 0 volt or a potential lower than the oxidation-reduction potential of the electron transfer material contained in the reaction layer.

According to the fourteenth aspect, when the liquid sample contains a reducing substance that affects the measurement, the reducing substance can be oxidized and removed when the second potential is applied. , High-precision measurement becomes possible.

As in the fifteenth aspect, the potential applied to the electrode system is changed between the time of supply of the liquid sample and the time of measurement by the potential changing means so that the potential is changed into two stages, whereby the sensor based on the difference in the supply state of the liquid sample is obtained. In addition to suppressing variations in measurement results during the measurement, high-precision measurement can be performed by applying a potential that can provide a sufficient output during measurement. Further, since the application of the potential to the electrode system is not interrupted in the middle, the efficiency of stabilizing the surface of the electrode system is good, and the variation in stabilization between sensors can be suppressed.

By making the measurement potential higher than the first potential as in the sixteenth aspect, a sufficient output for measurement can be obtained from the sensor at the time of measurement, and high-precision measurement becomes possible.

By setting the measured potential higher than the oxidation-reduction potential of the electron mediator as in the seventeenth aspect, the electron mediator is oxidized or reduced to promote the enzymatic reaction,
Since an output corresponding to the amount of the specific component in the liquid sample is obtained, highly accurate measurement is possible.

According to the eighteenth aspect, when the first potential is applied, even if the liquid sample is supplied to the reaction part, the reduced or oxidized electron transfer substance generated by the enzymatic reaction is oxidized or reduced. Since it is possible to accumulate data without consuming it, a large output with a good S / N ratio can be obtained from the sensor, and measurement can be performed over a wide concentration range.

As in the nineteenth aspect, the same effect as in the eighteenth aspect can be obtained by setting the first potential at 0 volt or a potential at which the potential of the working electrode is lower than the potential of the reference electrode.

According to the twentieth aspect, when the liquid sample contains a reducing substance that affects the measurement, the reducing substance can be oxidized and removed when the first potential is applied. , High-precision measurement becomes possible.

As in the twenty-first aspect, the potential applied to the electrode system is changed by the potential changing means before, after, and during the supply of the liquid sample, and the measurement is performed in three stages, whereby the supply state of the liquid sample is changed. In addition to suppressing variations in measurement results between sensors due to the difference between the two, it is possible to perform high-precision measurement by applying a potential that provides a sufficient output during measurement. Also, since the potential application to the electrode system is not interrupted on the way, the efficiency of stabilizing the surface of the electrode system is high,
Variations in stabilization between sensors can also be suppressed.

[Brief description of the drawings]

FIG. 1A is an exploded perspective view of a biosensor according to a first embodiment of the present invention. FIG. 1B is a perspective view showing the appearance of the biosensor. FIG. 1 (c) is FIG.
It is XX sectional drawing of (b).

FIG. 2 is a block diagram showing a schematic configuration inside a measuring device using the biosensor according to the first embodiment of the present invention.

FIG. 3 is a graph showing a change in a voltage applied to the biosensor by the measuring device according to the first embodiment of the present invention.

FIG. 4 is a block diagram showing a schematic configuration inside a measuring device using a biosensor according to a second embodiment of the present invention.

FIG. 5 is a graph showing a change in a voltage applied to a biosensor by a measuring device according to a second embodiment of the present invention.

[Explanation of symbols]

 2 Working electrode 2a Electrode reaction part 2b Lead part 2c Connection terminal part 3 Reference electrode 3a Electrode reaction part 3b Lead part 3c Connection terminal part 5 Reagent layer 10 Biosensor 25 Control circuit 28, 31 Switch

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Masato Arai 24, Yamanouchi Yamanoshitamachi, Ukyo-ku, Kyoto, Kyoto Prefecture OMRON Life Science Laboratory Co., Ltd. OMRON Life Science Research Inc. (72) Inventor Tomomi Kitawaki 24, Yamanouchi Yamanoshitamachi, Ukyo-ku, Kyoto, Kyoto Prefecture OMRON Life Science Research Inc. (72) Inventor Yusaku Sakota Yamanouchi Yamanoshitacho, Kyoto, Kyoto Address: OMRON Life Science Research Inc.

Claims (21)

[Claims] 1. Using a biosensor having an electrode system including at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, electrochemical reaction by a reaction between a liquid sample supplied to the reaction layer and an enzyme. In a measuring method for detecting a phenomenon with the electrode system and measuring the concentration of a specific component in the liquid sample, the liquid sample is applied to the reaction layer while a predetermined DC potential is applied as a first potential to the electrode system. After a certain period of time from either the detection of the supply of the liquid sample or the supply of the liquid sample, the potential applied to the electrode system is changed to a measurement potential different from the first potential. A measurement method using a biosensor, wherein the concentration of the specific component is measured based on a current detected by the electrode system while a measurement potential is applied. 2. A liquid sample supplied to the reaction layer, an enzyme, and a biosensor having an electrode system including at least a working electrode and a reference electrode, and a reaction layer including at least an enzyme and an electron transfer substance. In the measurement method for detecting the electrochemical phenomenon due to the reaction of the reaction with the electrode system and measuring the concentration of a specific component in the liquid sample, the liquid sample is subjected to a predetermined potential as a first potential applied to the electrode system. Supply to the reaction layer, after a fixed time from either the detection of the supply of the liquid sample and the supply of the liquid sample, change the potential applied to the electrode system to a measurement potential different from the first potential, A measurement method using a biosensor, wherein the concentration of the specific component is measured based on a current detected by the electrode system in a state where the changed measurement potential is applied. 3. An electrode system including at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme and an electron transfer substance, wherein the reaction layer contains a retaining agent that facilitates formation of the reaction layer. Using a biosensor comprising: a measurement method for detecting an electrochemical phenomenon caused by a reaction between a liquid sample supplied to the reaction layer and an enzyme with the electrode system to measure the concentration of a specific component in the liquid sample; The liquid sample is supplied to the reaction layer in a state where a predetermined DC potential is applied as a first potential to the electrode system, and after a certain time from either the detection of the supply of the liquid sample or the supply of the liquid sample, The potential applied to the electrode system is changed to a measurement potential different from the DC potential, and the concentration of the specific component is measured based on the current detected by the electrode system with the changed measurement potential applied. Measuring method using a biosensor according to claim. 4. The measuring method using a biosensor according to claim 3, wherein the holding agent is a low molecular compound. 5. The method according to claim 1, wherein the measurement potential is higher than the first potential before the change. 6. The method according to claim 2, wherein the measurement potential is higher than an oxidation-reduction potential of an electron transfer substance contained in the reaction layer. 7. The biosensor according to claim 2, wherein the first potential is lower than an oxidation-reduction potential of an electron transfer substance contained in the reaction layer. Measuring method. 8. The measurement using the biosensor according to claim 7, wherein the first potential is 0 volt or a potential at which the potential of the working electrode is lower than the potential of the reference electrode. Method. 9. The biosensor according to claim 7, wherein the liquid sample contains a reducing substance, and the first potential is a potential capable of oxidizing the reducing substance. Measuring method. 10. An electrochemical reaction by a reaction between a liquid sample supplied to the reaction layer and an enzyme, using a biosensor having an electrode system including at least a working electrode and a reference electrode, and a reaction layer including at least an enzyme. In a measuring method for detecting a phenomenon with the electrode system and measuring the concentration of a specific component in the liquid sample, the liquid sample is applied to the reaction layer while a predetermined DC potential is applied as a first potential to the electrode system. Supply, at any one of the detection of the supply of the liquid sample and the supply of the liquid sample, a predetermined potential as a second potential for waiting for a chemical reaction in the electrode system at a potential applied to the electrode system. The potential is changed to a DC potential, and the potential applied to the electrode system is different from the first potential and the second potential after a certain time from either the detection of the supply of the liquid sample or the supply of the liquid sample. That was changed to the measured potential, a measuring method using a biosensor, which comprises measuring the concentration of the particular component based on the current detected by said electrode system while applying the measurement potential after change. 11. The measuring method using a biosensor according to claim 10, wherein the measurement potential is higher than the first potential and the second potential before the change. 12. The biosensor according to claim 10, wherein the reaction layer includes an electron transfer substance, and the second potential is lower than an oxidation-reduction potential of the electron transfer substance. Measuring method. 13. The biosensor according to claim 10, wherein the reaction layer includes an electron transfer material, and the second potential is 0 volt or a potential lower than an oxidation-reduction potential of the electron transfer material. Measurement method using. 14. The biosensor according to claim 12, wherein the liquid sample contains a reducing substance, and the second potential is a potential capable of oxidizing the reducing substance. Measuring method. 15. Using a biosensor having an electrode system including at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, electricity generated by a reaction between a liquid sample supplied to the reaction layer and an enzyme. A measuring device for detecting a concentration of a specific component in the liquid sample by detecting a chemical phenomenon with the electrode system, wherein a potential application unit for applying a predetermined DC potential to the electrode system, and a potential application unit for applying a predetermined DC potential to the electrode system A potential changing means for changing a DC potential, wherein the potential applying means applies a predetermined DC potential to the electrode system as a first potential when the liquid sample is supplied to the reaction layer. After a certain time from either the detection of the supply of the liquid sample or the supply of the liquid sample, the potential changing means changes the potential applied to the electrode system to a measurement potential different from the first potential. And, measuring apparatus using a biosensor and measuring the concentration of the particular component based on the current detected by the electrode system while applying the measurement potential after change. 16. The measuring device using a biosensor according to claim 15, wherein the measurement potential is higher than the first potential. 17. The measurement using a biosensor according to claim 15, wherein the reaction layer includes an electron transfer substance, and the measurement potential is higher than an oxidation-reduction potential of the electron transfer substance. apparatus. 18. The biosensor according to claim 15, wherein the reaction layer includes an electron transfer material, and the first potential is lower than a redox potential of the electron transfer material. measuring device. 19. The measurement using a biosensor according to claim 18, wherein the first potential is 0 volt or a potential at which the potential of the working electrode is lower than the potential of the reference electrode. apparatus. 20. The biosensor according to claim 18, wherein the liquid sample contains a reducing substance, and the first potential is a potential capable of oxidizing the reducing substance. measuring device. 21. Using a biosensor having an electrode system including at least a working electrode and a reference electrode, and a reaction layer containing at least an enzyme, electricity generated by a reaction between a liquid sample supplied to the reaction layer and an enzyme. A measuring device for detecting a concentration of a specific component in the liquid sample by detecting a chemical phenomenon with the electrode system, wherein a potential application unit for applying a predetermined DC potential to the electrode system, and a potential application unit for applying a predetermined DC potential to the electrode system. A potential changing means for changing a DC potential, wherein the potential applying means applies a predetermined DC potential to the electrode system as a first potential until the liquid sample is supplied to the reaction layer. A potential for applying a potential applied to the electrode system by the potential changing means at a time point of either the detection of the supply of the liquid sample or the supply of the liquid sample to wait for a chemical reaction in the electrode system. A predetermined DC potential as a potential, and after a fixed time from either one of the detection of the supply of the liquid sample and the supply of the liquid sample, the potential applied to the electrode system by the potential changing means is the It is characterized by changing to a measurement potential different from the first potential and the second potential, and measuring the concentration of the specific component based on a current detected by the electrode system in a state where the measurement potential after the change is applied. A measuring device using a biosensor.