JP2001330581A - Substrate concentration determination method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a substrate concentration determination method capable of determining the supply state of sample liquid easily and surely, and having high precision and little dispersion. SOLUTION: This substrate concentration determination method includes a process for judging the necessity of execution of a substrate concentration determination process, based on a period of time from the contact of the sample liquid with a working electrode 5 and a counter electrode 8 until the contact thereof with a liquid junction detection electrode 7, by using a biosensor in which the working electrode 5, the counter electrode 8, the liquid junction detection electrode 7, a reagent layer and a sample liquid supply opening 11 are provided and the liquid junction detection electrode 7 is arranged on the position separated from the sample liquid supply opening 11 furthermore than the working electrode 5 and the counter electrode 8.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for quantifying a substrate concentration using a biosensor.

[0002]

2. Description of the Related Art As a quantitative analysis method for saccharides such as sucrose and glucose, a photometric method, a colorimetric method, a reductive titration method, and a method using various types of chromatography have been developed. However, all of these methods are not accurate because the specificity for saccharides is not very high. Of these methods, the photometer method is easy to operate, but is greatly affected by the temperature during operation. Therefore, the photometer method is not suitable as a method for ordinary people to easily determine saccharides at home or the like.

[0003] In recent years, various types of biosensors utilizing the specific catalytic action of enzymes have been developed.

[0004] A method for quantifying glucose will be described below as an example of a method for quantifying a substrate in a sample solution. As an electrochemical glucose quantification method, a method using glucose oxidase (EC 1.1.3.4; hereinafter abbreviated as GOD) and an oxygen electrode or a hydrogen peroxide electrode is generally known (eg, for example). , Shuichi Suzuki, "Biosensor" Kodansha).

GOD selectively oxidizes β-D-glucose as a substrate to D-glucono-δ-lactone using oxygen as an electron carrier. In the presence of oxygen, oxygen is reduced to hydrogen peroxide during the oxidation reaction process by GOD. The oxygen electrode measures the decrease in oxygen, or the hydrogen peroxide electrode measures the increase in hydrogen peroxide. Since the amount of decrease in oxygen and the amount of increase in hydrogen peroxide are proportional to the content of glucose in the sample solution, the amount of glucose is determined from the amount of decrease in oxygen or the amount of increase in hydrogen peroxide.

[0006] In the above method, as can be inferred from the reaction process, the measurement result has a drawback that it is greatly affected by the concentration of oxygen contained in the sample solution, and the measurement cannot be performed if oxygen does not exist in the sample solution. Becomes

Therefore, without using oxygen as an electron carrier,
A new type of glucose sensor using an organic compound such as potassium ferricyanide, a ferrocene derivative, a quinone derivative or a metal complex as an electron carrier has been developed.
In this type of sensor, the concentration of glucose contained in the sample solution is obtained from the amount of oxidation current by oxidizing the reduced form of the electron carrier generated as a result of the enzyme reaction on the electrode. By using such an organic compound or metal complex as an electron carrier instead of oxygen, a reagent layer can be formed by accurately supporting a known amount of GOD and the electron carrier in a stable state on an electrode. Becomes possible. In this case, since the reagent layer can be integrated with the electrode system in a state close to a dry state, a disposable biosensor based on this technology has attracted much attention in recent years. A typical example is a biosensor disclosed in Japanese Patent No. 2517153. In this biosensor, the glucose concentration can be easily measured by the measuring device simply by introducing the sample liquid into the sensor detachably connected to the measuring device. Such a technique is applicable not only to the quantification of glucose but also to the quantification of other substrates contained in a sample solution.

[0008]

In the above-described biosensor, the supply of a sample liquid is detected based on a change in resistance between two electrodes (between a working electrode and a counter electrode), and the detection is used as a trigger to measure a substrate. Often start. However, in the case of such a detection method, if the sample liquid reaches between the two electrodes, the supply of the sample liquid is insufficient, and the measurement of the substrate may start even when both electrodes are not sufficiently in contact with the sample liquid. In some cases, the measurement results varied.

Further, when the working electrode and the counter electrode are arranged at positions facing each other via the space, it is difficult to visually determine the supply of the sample liquid to the sensor, and the same problem as described above occurs. May have occurred.

In order to solve these problems, in addition to the working electrode and the counter electrode, a third electrode used as a liquid-junction detecting electrode is provided at a position farther from the sample liquid supply port than the working electrode and the counter electrode. A biosensor and a method for quantifying a substrate using the same are disclosed in JP-A-8-320304. According to this, the supply of the sample liquid can be reliably detected by detecting the electrical change between the counter electrode and the third electrode caused by the supply of the sample liquid to the reaction layer.

However, in this biosensor and the method for quantifying a substrate using the biosensor, the measurement is started even if the sample is supplied again to compensate for the shortage of the sample.
In some cases, accurate measurement results could not be obtained.

Further, it has been desired to suppress the influence of interfering substances and the perturbation of the applied voltage caused by not using a reference electrode.

In view of the above problems, an object of the present invention is to provide a method for quantitatively determining the concentration of a substrate, which can easily determine the supply state of a sample solution, and has high accuracy and little variation.

[0014]

Means for Solving the Problems To solve the above problems, the method for determining the concentration of a substrate according to the present invention comprises a working electrode, a counter electrode,
A liquid-junction detecting electrode, a reagent layer containing at least an enzyme and an electron carrier, and a sample liquid supply port, wherein the liquid-junction detection electrode is located farther from the sample liquid supply port than the working electrode and the counter electrode. Using the placed biosensor,
The current value obtained in the substrate concentration quantification step including a step (A) of applying a potential for oxidizing the electron carrier to the working electrode and a step (B) of measuring a current value between the working electrode and the counter electrode. A substrate concentration quantifying method for quantifying a substrate concentration in a sample liquid based on the method (C) of supplying the sample liquid from the sample liquid supply port at least, wherein the sample liquid is supplied to the working electrode and the counter electrode. Detecting the contact (D), detecting that the sample liquid has contacted the liquid junction detection electrode (E), and contacting the sample liquid with the working electrode and the counter electrode; A step (F) of measuring a time (T1) until the liquid comes into contact with the liquid junction detection electrode, a step (G) of determining whether or not to perform the substrate concentration quantification step based on the T1 (G); The substrate concentration based on Characterized in that it comprises a step (H) to implement or discontinue extent.

Here, in the step (D), it is detected that the sample liquid has contacted the working electrode and the counter electrode by a change in an electric signal between the working electrode and the counter electrode. A change in an electrical signal between the electrode and the liquid-junction detecting electrode or between the counter electrode and the liquid-junction detecting electrode detects that the sample liquid has contacted the liquid-junction detecting electrode, and in step (F), From detecting a change in the electric signal between the working electrode and the counter electrode, and then detecting a change in the electric signal between the working electrode and the liquid-junction detection electrode or between the counter electrode and the liquid-junction detection electrode. Time (T2) is measured, and T
2 is preferably T1.

In addition, at least before the step (D), a step of applying a voltage between the working electrode and the counter electrode, and at least before the step (E), between the working electrode and the liquid-junction detecting electrode, or It is preferable that the method further includes a step of applying a voltage between the counter electrode and the liquid junction detection electrode.

At least before the step (D), a step of applying a potential to the working electrode, the counter electrode and the liquid-junction detecting electrode may be provided.

Further, a biosensor in which the working electrode, the counter electrode and the liquid-junction detecting electrode are arranged on the same substrate may be used.

Further, a biosensor may be used in which the working electrode and the counter electrode are arranged at positions facing each other via a space.

Further, by using a biosensor in which the reagent layer and the liquid-junction detection electrode face each other via a space, a current value between the counter electrode and the liquid-junction detection electrode is measured. It is preferable to include a step of detecting an interfering substance.

Further, it is preferable that the liquid junction detecting electrode functions as a reference electrode.

[0022]

Embodiments of the present invention will be described below.

In order to solve the above-mentioned problems, a method for quantifying a substrate concentration according to the present invention comprises a working electrode, a counter electrode, a liquid junction detecting electrode, a reagent layer containing at least an enzyme and an electron carrier, and a sample liquid supply port. Using a biosensor in which the liquid junction detection electrode is disposed farther from the sample liquid supply port than the working electrode and the counter electrode, and applying a potential for oxidizing the electron carrier to the working electrode. A sample is prepared based on the response current value obtained in the substrate concentration determination step including the step (A) and the step (B) of measuring a current value between the working electrode and the counter electrode (hereinafter, abbreviated as a response current value). A substrate concentration determination method for determining a substrate concentration in a liquid, wherein at least a step (C) of supplying the sample liquid from the sample liquid supply port, the sample liquid being in contact with the working electrode and the counter electrode. Detect Step (D): detecting that the sample liquid has contacted the liquid junction detection electrode (E), and after the sample liquid has contacted the working electrode and the counter electrode, the sample liquid is detected by the liquid junction detection. A step (F) of measuring a time (T1) until contact with the electrode, a step (G) of determining whether or not to perform the substrate concentration determination step based on the T1, and a determination of the substrate concentration based on the determination A step (H) of performing or stopping the step.

According to this method, the supply state of the sample liquid can be easily and reliably determined based on T1, and the substrate concentration quantification step is performed only when the sample liquid is appropriately supplied. Measurements can be performed with high accuracy and little variation.

In a preferred embodiment of the present invention, in the step (D), it is detected that the sample solution has contacted the working electrode and the counter electrode by detecting a change in an electric signal between the working electrode and the counter electrode. ), Detecting that the sample liquid has contacted the liquid-junction detecting electrode by detecting a change in an electrical signal between the working electrode and the liquid-junction detecting electrode or between the counter electrode and the liquid-junction detecting electrode; In F), after detecting a change in an electric signal between the working electrode and the counter electrode, a change in an electric signal between the working electrode and the liquid-junction detection electrode or between the counter electrode and the liquid-junction detection electrode is detected. (T2) is measured until T is detected, and T2 is defined as T1.

According to this method, the contact of the sample liquid with each electrode can be easily detected from a change in the electric signal, and T2 is used as T1, and the supply state of the sample liquid is determined based on T2. Can be easily and reliably determined,
Only when the sample liquid is appropriately supplied, the substrate concentration quantification step is performed, so that measurement with high accuracy and little variation can be performed.

Further, the method for determining a substrate concentration according to the present invention has a step of applying a voltage between the working electrode and the counter electrode at least before the step (D), and at least before the step (E).
It is preferable that the method further includes a step of applying a voltage between the working electrode and the liquid-junction detecting electrode or between the counter electrode and the liquid-junction detecting electrode.

At least before the step (D), a step of applying a potential to the working electrode, the counter electrode and the liquid-junction detecting electrode may be provided. In this case, the number of steps for applying a voltage or a potential is reduced, and the number of steps can be reduced.

In the substrate concentration determination method of the present invention, a biosensor in which a working electrode, a counter electrode, and a liquid-junction detecting electrode are arranged on the same substrate may be used.

Further, a biosensor may be used in which the working electrode and the counter electrode are arranged at positions facing each other via a space.

Further, by using a biosensor in which the reagent layer and the liquid-junction detecting electrode are arranged opposite to each other via a space, the current value between the counter electrode and the liquid-junction detecting electrode is measured. It is preferable to include a step of detecting an interfering substance. Here, the interfering substance is a substance that causes an error in the measurement result of the substrate concentration by affecting the response current value by electrochemically reacting on the working electrode or the counter electrode in the substrate concentration determination step. Examples thereof include easily oxidizable substances such as ascorbic acid and uric acid. In this case, the sample liquid comes into contact with the liquid junction detection electrode before the reactant generated by the reaction between the substrate in the sample liquid and the enzyme and the electron carrier in the reagent layer reaches the liquid junction detection electrode. By measuring the current value between the counter electrode and the liquid-junction detection electrode between the time when the sample liquid comes into contact with the liquid-junction detection electrode and the time when the reactant reaches the liquid-junction detection electrode, the interference substance can be easily detected. Can be detected. Therefore, the effect of the interfering substance can be removed from the measurement result without adding a new electrode for detecting the interfering substance in the reagent layer, so that high accuracy can be achieved without complicating the sensor configuration or complicating the manufacturing process. Quantitative determination of the substrate concentration can be performed.

It is preferable that the liquid junction detecting electrode functions as a reference electrode. In this way, the potential of the working electrode can be stabilized without adding a new electrode for the reference electrode, so that the sensor concentration can be reduced without complicating the sensor configuration or complicating the manufacturing process. Since a response current value having good linearity can be obtained, it is possible to quantitatively determine the substrate concentration with high accuracy.

In the present invention, as the enzyme contained in the reagent layer, an appropriate enzyme is selected according to the substrate contained in the sample solution. Examples of the enzyme include fructose dehydrogenase, glucose oxidase, alcohol oxidase, lactate oxidase, cholesterol oxidase, xanthine oxidase, and amino acid oxidase.

Examples of the electron carrier include potassium ferricyanide, p-benzoquinone, phenazine methosulfate, methylene blue, and a ferrocene derivative. Also, a current response can be obtained when oxygen is used as the electron carrier. One or more of these electron carriers are used.

[0035]

Embodiments of the present invention will be described below with reference to FIGS. 1 to 3 are exploded perspective views of a glucose sensor according to an embodiment of the present invention in a state where a reagent layer is removed.

Example 1 As an example of a biosensor,
The glucose sensor will be described. In this embodiment, FIG.
Was used. This glucose sensor has an insulating substrate 1 made of polyethylene terephthalate, a cover 9, and a spacer 10 sandwiched between the substrate 1 and the cover 9. These are adhered in a positional relationship as shown by a dashed line in FIG. 1 to constitute a glucose sensor.

The spacer 10 has a sample liquid supply port 11 formed therein, and the cover 9 has an air hole 12 formed therein. When the cover 9 is laminated and adhered on the substrate 1 with the spacer 10 interposed therebetween, a space (not shown) communicating with the sample liquid supply port 11 is formed by the substrate 1, the spacer 10, and the cover 9. It communicates with 12.

The substrate 1 is provided with a working electrode 5, a counter electrode 8, and a liquid-junction detecting electrode 7, and further provided with leads 2, 3, and 4 electrically connected to these. The working electrode 5 is arranged inside the ring-shaped counter electrode 8. Liquid junction detection electrode 7
Is arranged at a position farther from the sample liquid supply port 11 than the working electrode 5 and the counter electrode 8.

The glucose sensor was manufactured as follows.

A silver paste was printed by screen printing on an insulating substrate 1 made of polyethylene terephthalate to form leads 2, 3, and 4, respectively. Next, the working electrode 5 was formed by printing a conductive carbon paste containing a resin binder on the substrate 1. The working electrode 5 is in contact with the lead 2.

Next, an insulating paste was printed on the substrate 1 to form an insulating layer 6. The insulating layer 6 covers the outer periphery of the working electrode 5, whereby the area of the exposed portion of the working electrode 5 is kept constant. The insulating layer 6 includes the leads 2, 3,
4 is partially covered. The liquid junction detecting electrode 7 was formed by exposing the tip of the lead 3.

Next, a conductive carbon paste containing a resin binder was printed on the substrate 1 so as to be in contact with the leads 4 to form a counter electrode 8.

Next, an aqueous solution of carboxymethylcellulose (hereinafter abbreviated as CMC) was dropped on the working electrode 5 and the counter electrode 8 and dried to form a CMC layer. Further, an aqueous solution containing GOD as an enzyme and potassium ferricyanide as an electron carrier is dropped on the CMC layer,
The reagent layer was formed by drying.

Next, to further smoothly supply the sample solution to the reagent layer on the reagent layer, a toluene solution of lecithin is spread from the sample solution supply port 11 over the reagent layer and dried. To form a lecithin layer. In addition, although toluene was used for forming the lecithin layer, another organic solvent may be used. Next, the cover 9 and the spacer 10 were adhered to the substrate 1 in a positional relationship as shown by a dashed line in FIG. 1 to produce a glucose sensor.

This glucose sensor was mounted on a measuring instrument (not shown), and a 500 mV voltage was first applied between the working electrode 5 and the counter electrode 8.
Was applied. Next, a sufficient amount of blood for quantifying the glucose concentration as a sample liquid is supplied to the sensor through the sample liquid supply port 11.
Supplied more. When blood is introduced into the space by capillary action and the blood reaches the working electrode 5 and contacts the working electrode 5,
The measuring instrument detected a change in the electric resistance value as a change in the electric signal between the working electrode 5 and the counter electrode 8. The measurement timer was started simultaneously with the detection of this change, and then a voltage of 500 mV was applied between the working electrode 5 and the liquid-junction detecting electrode 7. When blood reaches the liquid junction detection electrode 7 and contacts the liquid junction detection electrode 7 following the working electrode 5 and the counter electrode 8, a change occurs in the electrical resistance value between the working electrode 5 and the liquid junction detection electrode 7. The change was detected with a measuring instrument. Here, the time (hereinafter, abbreviated as T2) from the start of the measurement timer to the change in the electrical resistance value between the working electrode 5 and the liquid junction detection electrode 7 was measured by the measurement timer.

Subsequent to the sample supply detection step, a substrate concentration determination step was performed. A potential of 500 mV is applied to the working electrode 5 25 seconds after the detection of the change in the electric resistance value between the working electrode 5 and the liquid-junction detecting electrode 7, and the current value between the working electrode 5 and the counter electrode 8 is changed 5 seconds later. It was measured. Glucose in blood, ferricyanide ion dissolved from reagent layer and GO
D reacts, resulting in the oxidation of glucose to gluconolactone and the reduction of ferricyanide to ferrocyanide. A current response is obtained by oxidizing the ferrocyanide ions at the working electrode 5. As a result, a response current value depending on the glucose concentration in the sample solution was obtained.

When a sufficient amount of blood is supplied, depending on the electrode arrangement, measurement environment (temperature, humidity, etc.), blood characteristics (hematocrit value, viscosity, etc.), etc. The time from contact with the counter electrode to contact of the sample liquid with the liquid junction detection electrode (hereinafter, abbreviated as T1) is at most about 1 to 2 seconds. Here, T2 can be regarded as substantially equal to T1. Therefore, when the quantitative results obtained when T2 is 2 seconds or less and the quantitative results obtained when T2 exceeds 2 seconds are compared, the results obtained when T2 is 2 seconds or less are as follows. In addition, the accuracy and dispersion of the measured values were greatly suppressed. If T2 exceeds 2 seconds, 1
It is conceivable that a sufficient amount of sample was not supplied in each sample supply, or that the sample was supplied again in order to compensate for the shortage of the sample amount, thereby affecting the quantitative result.

In order to reduce such a measurement error due to the shortage of the sample, after the step of measuring T2, if T2 is 2 seconds or less, the subsequent substrate concentration determination step is performed.
When the value was longer than 2 seconds, a step of determining to stop the measurement was incorporated in the measuring instrument, and the same measurement was performed. As a result, the measurement is performed only when a sufficient amount of the sample is supplied at one time, so that the measurement error is greatly reduced.

Example 2 In this example, the same glucose sensor as in Example 1 was used.

This glucose sensor was attached to a measuring instrument,
500 m between working electrode 5 and counter electrode 8 with reference to counter electrode 8
V, a voltage of 500 mV was applied between the counter electrode 8 and the liquid junction detecting electrode 7. Next, a sufficient amount of blood for quantifying glucose concentration was supplied to the sensor from the sample liquid supply port 11 as a sample liquid. When the blood was introduced into the space by capillary action and the blood reached the working electrode 5, the measuring instrument detected a change in the electric resistance between the working electrode 5 and the counter electrode 8, and at the same time the measurement timer was started. When blood reaches the liquid junction detection electrode 7 and contacts the liquid junction detection electrode 7 following the working electrode 5 and the counter electrode 8, a change occurs in the electrical resistance value between the working electrode 5 and the liquid junction detection electrode 7. The change was detected with a measuring instrument. Here, the time (T2) from when the measurement timer was started to when the electric resistance value between the working electrode 5 and the liquid junction detection electrode 7 changed was measured by the measurement timer.

Subsequent to this sample supply detection step, a substrate concentration determination step was performed. 25 seconds after the detection of a change in the electric signal between the counter electrode 8 and the liquid-junction detection electrode 7,
A potential of 00 mV is applied to the working electrode 5, and after 5 seconds, the working electrode 5
And the current value between the counter electrode 8 was measured. As a result, as in Example 1, a response current value depending on the glucose concentration in the sample solution was obtained.

Similar to Example 1, when the quantitative results obtained when T2 was 2 seconds or less and the quantitative results obtained when T2 exceeded 2 seconds were compared, T2 was 2 seconds or less. In the results obtained in the case of the above, the accuracy and variation of the measured values were significantly suppressed.

Therefore, after the step of measuring T2, T2
When T2 was 2 seconds or less, the subsequent substrate concentration quantification step was performed, and when T2 was longer than 2 seconds, a step of judging to stop was incorporated in a measuring instrument, and the same measurement was performed.
As a result, since the measurement was performed only when a sufficient amount of the sample was supplied at one time, the measurement error was significantly reduced as in the first embodiment.

Example 3 In this example, the glucose sensor shown in FIG. 2 was used.

An electrode and a lead made of palladium were formed on an insulating substrate 1 made of polyethylene terephthalate. Next, the spacer 10 having the sample supply port 11
Was adhered to the substrate 1 in a positional relationship as shown by a dashed line in FIG. 2 to partition the working electrode 5, the counter electrode 8, the liquid-junction detecting electrode 7, and the leads 2, 3, and 4. After forming a reagent layer in the compartment in the same manner as in Example 1, the glucose sensor was manufactured by attaching a cover 9 having an air hole 12 thereto.

As in Example 1, the glucose concentration in blood was measured by the sample supply detecting step and the substrate concentration quantifying step. As a result, the same effect as in Example 1 was obtained.
By performing the discrimination based on the above, the measurement error due to the insufficient sample amount was greatly suppressed.

In the case of the glucose sensor in this embodiment, the sample liquid supplied from the sample supply port 11 passes through the counter electrode 8 and the working electrode 5 in order, and finally reaches the liquid junction detecting electrode 7. Are arranged, but the present invention is not limited to this. The arrangement of the counter electrode 8 and the working electrode 5 is reversed, and the working electrode, the counter electrode,
Similar results were obtained when the sample arrived in the order of the liquid junction detection electrodes.

Example 4 In this example, the glucose sensor shown in FIG. 3 was used.

A lead 2 was formed by printing a silver paste on the insulating working electrode substrate 13 made of polyethylene terephthalate by screen printing. Then, a conductive carbon paste containing a resin binder is applied to the working electrode substrate 13.
Printing was performed thereon to form a working electrode 5. This working electrode 5 is in contact with the lead 2. Further, on the working electrode substrate 13,
The insulating paste was printed to form the insulating layer 6. The insulating layer 6 covers the outer periphery of the working electrode 5, thereby keeping the exposed area of the working electrode 5 constant.

In the same procedure, the counter electrode 8 and the liquid-junction detecting electrode 7 were formed on the insulating counter electrode substrate 14.

After a reagent layer is formed on the working electrode 5 in the same manner as in Example 1, the working electrode substrate 13, the counter electrode substrate 14 having the air holes 12, and the spacer 10 are arranged as shown by a dashed line in FIG. The biosensor was fabricated by bonding with a positional relationship. The spacer 10 is provided with a slit for forming a sample liquid supply path between both substrates. The sample supply port 11 is
It corresponds to the opening of the sample supply path.

Next, the glucose in the blood was measured. First, a voltage of 500 mV was applied between the working electrode 5 and the counter electrode 8. Next, blood was supplied from the sample liquid supply port 11 to the sensor in a sufficient amount as a sample liquid for determining the concentration of glucose containing ascorbic acid as an interfering substance. Blood was introduced into the space by capillary action, and when the blood reached the working electrode 5 and came into contact with the working electrode 5, the measuring instrument detected a change in the electrical resistance value between the working electrode 5 and the counter electrode 8. The measurement timer was started simultaneously with the detection of the change in the electric resistance value, and then a voltage of 500 mV was applied between the working electrode 5 and the liquid-junction detecting electrode 7.
When blood reaches the liquid junction detection electrode 7 and contacts the liquid junction detection electrode 7 following the working electrode 5 and the counter electrode 8, a change occurs in the electrical resistance value between the working electrode 5 and the liquid junction detection electrode 7. The change was detected with a measuring instrument. Here, the time (T2) from when the measurement timer was started to when the electric resistance value between the working electrode 5 and the liquid junction detection electrode 7 changed was measured by the measurement timer. Next, since this T2 was 2 seconds or less, it was decided to perform the subsequent substrate concentration quantification step.

Next, almost simultaneously with detecting the change in the electric resistance value between the working electrode 5 and the liquid-junction detecting electrode 7, the liquid-junction detecting electrode 7
A voltage of 500 mV was applied between the first and second electrodes 8 for 100 milliseconds, and a current value between the two electrodes (hereinafter, abbreviated as I1) was measured. I1 was caused by the oxidation reaction of ascorbic acid contained as an interfering substance, and gave a proportional relationship to the concentration. After the measurement of I1, the voltage application between the liquid junction detection electrode 7 and the counter electrode 8 was released.

As described above, the liquid junction detecting electrode 7 is disposed on the counter electrode substrate 14 on which no reagent layer is disposed. Therefore, it takes some time for the ferrocyanide ions generated as a result of the enzyme reaction to reach the vicinity of the liquid junction detection electrode 7. That is, the current value I1 between the liquid-junction detecting electrode 7 and the counter electrode 8 during the time until the arrival of the ferrocyanide ion mainly reflects only the concentration of ascorbic acid.

About 25 seconds after the measurement of I1, 5
A potential of 00 mV was applied to the working electrode 5, and the response current value I2 after 5 seconds was measured. This response current value I2 is caused by an oxidation reaction between ferrocyanide ions, which are proportional to the glucose concentration in the sample, and ascorbic acid that is present in the sample in advance. That is, ascorbic acid gives a positive error to the measurement result. Therefore, the current value I obtained by correcting the response current value I2 based on the current value I1
From 3, it is possible to remove the influence of ascorbic acid and obtain an accurate glucose concentration.

According to this embodiment, the influence of the interfering substance can be corrected by the function of the liquid-junction detecting electrode 7 also as the interfering substance detecting electrode. Therefore, a new electrode is added for detecting the interfering substance. Without this, it became possible to measure the substrate with higher accuracy.

Example 5 Using the same glucose sensor as in Example 4, glucose in blood was measured in the same procedure as in Example 4. However, when applying a potential to obtain a response current value between the working electrode 5 and the counter electrode 8, a potential of 700 mV is applied to the working electrode 5 with respect to the liquid-junction detecting electrode 7, and a potential of 200 mV is applied to the counter electrode 8. Was applied. That is,
The measurement was performed by a three-electrode system using the liquid-junction detecting electrode 7 as a reference electrode.

As a result, since the potential of the working electrode 5 was stabilized, the influence of the potential perturbation was suppressed, and the linearity of the response current value with respect to the glucose concentration was improved.

According to this embodiment, the liquid junction detecting electrode 7 can be used as both an interfering substance detecting electrode and a reference electrode.
It is possible to measure a substrate with higher accuracy without adding a new electrode for an interfering substance detection electrode or a reference electrode.

In the above embodiment, the potential or voltage applied to the electrode system for detecting the sample solution, detecting the interfering substance, and quantifying the substrate concentration is described, but the present invention is not limited to this. The potential or voltage at which a change in the electrical signal is observed when detecting a sample liquid, the potential or voltage at which an interfering substance reacts on an electrode when detecting an interfering substance, or a series of reactions when quantifying a substrate concentration The potential or the voltage may be such that the reduced form of the electron carrier resulting from the above is oxidized.

Further, in the step of judging whether to carry out or stop the substrate concentration determination step, the time for comparison with T2 is set to 2 seconds, but the present invention is not limited to this. T2
Is a value with factors such as the distance between the electrodes, the viscosity of the sample, and the temperature, and an appropriate time is set in accordance with these factors.
Also, the time for measuring the current value is not limited to the specific value described in the embodiment.

In Examples 1 and 3 to 5, a voltage was applied between the working electrode and the liquid-junction detecting electrode to confirm that the sample liquid was in contact with the liquid-junction detecting electrode. A change in the electrical signal between the detection electrodes was detected, but instead,
A voltage may be applied between the counter electrode and the liquid-junction detecting electrode to detect a change in an electric signal between the counter electrode and the liquid-junction detecting electrode. Further, in the second embodiment, in order to confirm that the sample liquid has reached the liquid junction detection electrode, a change in the electric signal between the working electrode and the liquid junction detection electrode is detected. A change in an electrical signal between the liquid junction detection electrodes may be detected.

In the above embodiment, a change in the electric resistance was detected as an electric signal in order to confirm that the sample liquid was in contact with each electrode. However, the present invention is not limited to this. Changes in potential, capacitance component, etc. may be detected.

In the above embodiment, an example of the sensor structure is shown, but the shapes of the electrodes and leads, the arrangement of the electrodes and leads,
The combination method of the sensor members is not limited to these.

In the above embodiment, carbon or palladium is described as an electrode material, but the present invention is not limited to this. As the working electrode material, any conductive material that does not oxidize itself when oxidizing the electron carrier can be used. As the counter electrode material, any commonly used conductive material such as silver or platinum can be used. Further, the method for manufacturing the electrode system is not limited to the screen printing method or the sputtering method, and an electrode system manufactured by another method such as an evaporation method can be used.

Further, the enzyme and the electron carrier may be insolubilized by fixing the reagent layer to the working electrode. In the case of immobilization, a cross-linking immobilization method or an adsorption method is preferred.
Further, an enzyme or an electron carrier may be mixed in the electrode material.

[0077]

As described above, according to the present invention, the supply state of the sample solution can be easily and reliably determined, and a highly accurate and low-variation substrate concentration determination method can be provided.

[Brief description of the drawings]

FIG. 1 is an exploded perspective view of a glucose sensor according to an embodiment of the present invention with a reagent layer removed.

FIG. 2 is an exploded perspective view of a glucose sensor according to another embodiment of the present invention with a reagent layer removed.

FIG. 3 is an exploded perspective view of a glucose sensor according to still another embodiment of the present invention with a reagent layer removed.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Substrate 2, 3, 4 lead 5 Working electrode 6 Insulating layer 7 Liquid-junction detecting electrode 8 Counter electrode 9 Cover 10 Spacer 11 Sample liquid supply port 12 Air hole 13 Working electrode substrate 14 Counter electrode substrate

Claims (8)

[Claims] A working electrode, a counter electrode, a liquid-junction detecting electrode, a reagent layer containing at least an enzyme and an electron carrier, and a sample liquid supply port, wherein the liquid-junction detecting electrode is higher than the working electrode and the counter electrode. A step (A) of applying a potential for oxidizing the electron carrier to the working electrode by using a biosensor arranged at a position distant from the sample liquid supply port, and determining a current value between the working electrode and the counter electrode. A substrate concentration quantification method for quantifying a substrate concentration in a sample liquid based on the current value obtained in the substrate concentration quantification step having the measuring step (B), wherein at least the sample liquid is supplied from the sample liquid supply port. (C), detecting that the sample liquid has contacted the working electrode and the counter electrode (D), and detecting that the sample liquid has contacted the liquid junction detection electrode (E). The sample solution is A step (F) of measuring a time (T1) from the time when the sample liquid comes into contact with the liquid junction detection electrode after contact with the working electrode and the counter electrode, and the step of measuring the substrate concentration based on the T1 or A method for quantifying a substrate concentration, comprising: a step (G) for determining a stop; and a step (H) for performing or stopping the substrate concentration quantification step based on the determination. 2. In the step (D), it is detected that the sample liquid has contacted the working electrode and the counter electrode by a change in an electric signal between the working electrode and the counter electrode. A contact between the sample liquid and the liquid-junction detecting electrode is detected based on a change in an electric signal between the liquid-junction detecting electrode and the counter electrode and between the counter electrode and the liquid-junction detecting electrode. Time from detecting a change in an electrical signal between a pole and the counter electrode to detecting a change in an electrical signal between the working electrode and the liquid-junction detection electrode or between the counter electrode and the liquid-junction detection electrode. The method according to claim 1, wherein (T2) is measured, and T2 is defined as T1. 3. A step of applying a voltage between the working electrode and the counter electrode at least before the step (D), and at least before the step (E), between the working electrode and the liquid-junction detecting electrode, or 3. The method according to claim 2, further comprising a step of applying a voltage between said counter electrode and said liquid junction detection electrode. 4. The method according to claim 2, further comprising the step of applying a potential to the working electrode, the counter electrode and the liquid-junction detecting electrode at least before step (D). 5. The method according to claim 1, wherein a working electrode, a counter electrode, and a liquid-junction detecting electrode are disposed on the same substrate using a biosensor. 6. The method according to claim 1, wherein the working electrode and the counter electrode use biosensors arranged at positions facing each other via a space. Law. 7. A biosensor in which a reagent layer and a liquid-junction detecting electrode are arranged to face each other via a space, and measures a current value between a counter electrode and the liquid-junction detecting electrode. The method according to any one of claims 1 to 6, further comprising a step of detecting an interfering substance. 8. The method according to claim 1, wherein the liquid-junction detecting electrode functions as a reference electrode.