JP3267933B2 - Substrate quantification method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a quantification method for quickly and accurately quantifying a substrate in a sample.

[0002]

2. Related Background Art As a quantitative analysis method of saccharides such as sucrose and glucose, a photometer method, a colorimetric method, a reduction titration method, a method using various types of chromatography, and the like have been developed.
However, all of these methods are not accurate because the specificity for saccharides is not very high. According to the photometer method among these methods, although the operation is simple, it is greatly affected by the temperature during the operation. Therefore, the photometer method is not suitable as a method for ordinary people to easily determine saccharides at home or the like. In recent years, various types of biosensors utilizing the specific catalytic action of enzymes have been developed. Hereinafter, a method for quantifying glucose will be described 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).

[0003] 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. The amount of decrease in oxygen and the amount of increase in hydrogen peroxide
Since it is proportional to the content of glucose in the sample liquid, glucose can be determined from the amount of decrease in oxygen or the amount of increase in hydrogen peroxide. This method has a drawback that the measurement result is greatly affected by the concentration of oxygen contained in the sample solution, as can be estimated from the reaction process. Further, when oxygen does not exist in the sample liquid, the measurement becomes impossible.

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, it is possible to form a reaction layer 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 reaction layer can be integrated with the electrode system in a state close to a dry state, a disposable glucose sensor based on this technology has been receiving much attention in recent years. In a disposable glucose sensor, 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.

[0005]

According to the measurement using the sensor as described above, the reduced electron carrier is oxidized on the working electrode, and the substrate concentration is determined based on the value of the oxidation current flowing at that time. However, when blood, fruit juice or the like is used as a sample, oxidizable substances such as ascorbic acid and uric acid contained in the sample are oxidized on the working electrode simultaneously with the reduced electron carrier. The oxidation reaction of the easily oxidizable substance may affect the measurement result.

In the measurement using the above-described sensor, the reaction of generating hydrogen peroxide using dissolved oxygen as an electron carrier proceeds simultaneously with the reduction of the electron carrier carried on the reaction layer. Further, the hydrogen peroxide generated by this reaction reoxidizes the reduced electron carrier. as a result,
When measuring the substrate concentration based on the oxidation current of the reduced electron carrier, dissolved oxygen may give a negative error to the measurement result.

In the above method, before applying a voltage between the working electrode and the counter electrode in order to obtain a current response, a voltage is applied between the working electrode and the counter electrode, and an electrical change between the working electrode and the counter electrode is applied. In many cases, liquid junction detection is performed based on At this time, before a sufficient amount of the sample liquid is supplied to the electrode system, the resistance value between the working electrode and the counter electrode may change and the measurement may start, which may affect the measurement result. In addition, the interface state of the working electrode may change, which may affect the measurement result. Further, in the two-electrode measurement method, the counter electrode is used as a reference electrode. For this reason, the reference potential of the reference electrode fluctuates with the oxidation-reduction reaction at the working electrode, which may affect the measurement result.

[0008] The present invention eliminates the above disadvantages,
It is an object of the present invention to provide a quantitative method capable of accurately measuring the concentration of a substrate by removing the influence of an oxidizable substance. Another object of the present invention is to provide a method for quantifying a substrate with reduced response variability.

[0009]

SUMMARY OF THE INVENTION In order to solve the above-mentioned problems, the present invention provides an electrically insulating substrate, and a working electrode, a counter electrode, and an interfering substance detection electrode formed on the substrate. an electrode system having a third electrode, the provided on said electrode system, except the third electrode, and a reaction layer containing at least an oxidoreductase and an electron mediator, and the substrate
And a cover member that forms a sample liquid supply path between
The third electrode is located above the sample liquid supply path than the reaction layer.
Disposed on the flow side, and wherein the working electrode is
Is also arranged upstream of the sample liquid supply path.
A biosensor is provided.

The present invention also provides an electrode system having an electrically insulating substrate, a working electrode formed on the substrate, a counter electrode, and a third electrode used as an interfering substance detection electrode. electrodes provided on the electrode system, except for a reaction layer containing at least an oxidoreductase and an electron mediator, consists of a cover member for forming a sample solution supply pathway between the substrate, the third than the electrodes the reactive layer is disposed on the upstream side of the sample solution supply pathway, and wherein the working electrode is the
Using a biosensor disposed on the upstream side of the sample liquid supply path from the counter electrode , reducing the electron carrier by electrons generated when reacting the substrate and the oxidoreductase contained in the sample liquid, Provided is a method for quantifying the concentration of a substrate in a sample solution by electrochemically measuring the amount of reduction of an electron carrier, which method includes the following steps. (1) a step of applying a voltage between the counter electrode and the third electrode; (2) a step of supplying the sample liquid to the reaction layer; and (3) a step of applying the sample liquid to the reaction layer. Detecting the electrical change between the third electrode and the third electrode; (4) measuring the current generated between the counter electrode and the third electrode after the detecting step (3); After the measuring step (4), a step of releasing the voltage application between the counter electrode and the third electrode, (6) a step of applying a voltage between the working electrode and the counter electrode, and (7) thereafter Measuring the current generated between the counter electrode and the working electrode.

In the quantification method of the present invention, it is preferable to use the third electrode as a reference electrode. That is, in the above step (6), a voltage is also applied between the working electrode and the third electrode. When a biosensor in which a cover member is combined with the substrate is used, it is preferable to arrange a lecithin-supporting layer on the wall surface of the cover member exposed to the sample liquid supply path. Further, the reaction layer preferably contains a hydrophilic polymer.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The configuration of a biosensor used in the quantification method of the present invention will be described. First, a first type of biosensor will be described with reference to FIG. In this sensor, a counter electrode 6, a working electrode 7, and a third electrode 8 are provided on an insulating substrate 1 made of polyethylene terephthalate or the like, and leads 2, 3, and 4 electrically connected thereto are provided. Have been. The carbon layer 9 is a layer provided for facilitating the production of the reaction layer, and does not function as an electrode. A circular reaction layer (not shown) containing an oxidoreductase and an electron carrier is provided on the counter electrode 6, the working electrode 7, and the carbon layer 9 except for the third electrode 8. In the figure,
5 is an insulating layer.

Next, a second type of biosensor is shown in FIG.
This will be described below. This sensor is a combination of the substrate 1 of FIG. 1 and a cover member including a cover 10 and a spacer 11. These are adhered in a positional relationship as shown by a dashed line in FIG. 2 to constitute a sensor. The spacer 11 has a slit 12 for forming a sample liquid supply path, and the cover 10 has an air hole 13. When the cover 10 is laminated and adhered on the substrate 1 via the spacer 11, a space serving as a sample liquid supply path is formed in the slit 12 of the spacer 11 by the substrate 1, the spacer 11 and the cover 10. The end of this space communicates with the air hole 13. In this bisensor, the working electrode 7 is arranged at a position closer to the sample liquid supply port 12a (corresponding to the open end of the slit 12) than the half-moon-shaped counter electrode 6, and the third electrode 8 is connected to the working electrode 7 Further, it is arranged at a position closer to the sample liquid supply port 12a. These electrodes 6, 7, and 8 are respectively exposed to the space.

In order to measure the substrate concentration using the above biosensor, first, the end of the sensor having the leads 2, 3 and 4 is set on a measuring instrument, and the third electrode is set with reference to the counter electrode 6. 8 is applied with a predetermined potential. In a state where this potential is applied, the reaction layer is dropped on a sample liquid reaction layer containing, for example, ascorbic acid as an interfering substance, and the reaction layer is dissolved in the sample liquid. Simultaneously with the supply of the sample liquid, the system for detecting the supply of the liquid based on the electrical change between the counter electrode 6 and the third electrode 8 of the electrode system operates, and thus the measurement timer starts.
At this time, a potential is continuously applied between the counter electrode 6 and the third electrode 8, and after a lapse of a predetermined time from the detection of the supply of the sample solution, the potential between the counter electrode 6 and the third electrode 8 is changed. Measure the current value. Since no reaction layer is disposed on the third electrode 8, it takes some time for the reduced form of the electron carrier generated as a result of the enzyme reaction to reach the vicinity of the third electrode 8. . Therefore, the above current value is caused by the oxidation reaction of ascorbic acid contained as an interfering substance.

Next, the voltage application between the counter electrode 6 and the third electrode 8 is released. Then, to the working electrode 7 based on the counter electrode 6,
A potential for oxidizing the reduced form of the electron carrier is applied, and a current value between the counter electrode 6 and the working electrode 7 is measured. This current results from the oxidation of the reduced form of the electron carrier and the pre-existing interfering substance ascorbic acid. That is, ascorbic acid gives a positive error to the measurement result. Since the current value between the counter electrode 6 and the third electrode 8 mainly reflects only the concentration of ascorbic acid, the influence of ascorbic acid is removed by correcting the measurement result based on this current value. In addition, an accurate substrate concentration can be obtained. In the second type of sensor, the counter electrode 6 and the third electrode 8
Since the supply of the sample liquid is detected in the interval, the entire exposed portion of the working electrode 7 is reliably filled with the sample liquid. This makes it possible to more reliably determine the supply of the sample liquid.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to embodiments. << Example 1 >> A method for quantifying glucose will be described. The substrate shown in FIG. 1 was used for the substrate of the glucose sensor. This glucose sensor was manufactured as follows. Silver paste was printed on the insulating substrate 1 made of polyethylene terephthalate by screen printing to form leads 2, 3, and 4, respectively. Next, a conductive carbon paste containing a resin binder was printed on the substrate 1 to form the working electrode 7 and the third electrode 8. The working electrode 7 and the third electrode 8 are in electrical contact with the leads 3 and 4, respectively. Next, an insulating paste was printed on the substrate 1 to form an insulating layer 5. The insulating layer 5 covers the outer peripheral portions of the working electrode 7 and the third electrode 8, so that the exposed areas of the working electrode 5 and the third electrode 8 are kept constant.
The insulating layer 5 covers a part of the leads 2, 3 and 4.

Next, an aqueous solution of carboxymethylcellulose (hereinafter abbreviated as CMC) is dropped on the counter electrode 6, the working electrode 7, and the carbon layer 9 except for the third electrode 8, and dried to form a CMC layer. did. When an aqueous solution containing GOD as an enzyme and potassium ferricyanide as an electron carrier is dropped on this CMC layer, the CMC layer made of a hydrophilic polymer is dissolved once, and then mixed with the enzyme and the like in a subsequent drying process. Form a reaction layer. However, since it does not involve stirring or the like, it does not become a completely mixed state,
The electrode system surface is in a state covered with only CMC. That is, since the enzyme, the electron carrier, and the like do not contact the electrode system surface, it is possible to prevent the adsorption of the protein to the electrode system surface.

In order to measure the glucose concentration using this sensor, first, the end having the leads 2, 3 and 4 is set on a measuring instrument, and 500 mV is applied to the third electrode 8 with respect to the counter electrode 6. An electric potential was applied. With this potential applied, 30 μl of a glucose aqueous solution containing ascorbic acid as an interfering substance was dropped on the reaction layer as a sample solution. The reaction layer on the electrode system was dissolved in the dropped sample solution. Simultaneously with the supply of the sample liquid, a system for detecting the supply of the liquid based on an electrical change between the counter electrode 6 of the electrode system and the third electrode 8 operates. This started the measurement timer. At this time, a potential is continuously applied between the counter electrode 6 and the third electrode 8, and after a lapse of a predetermined time from the detection of the supply of the sample solution, the potential between the counter electrode 6 and the third electrode 8 is changed. The current value was measured. This current value was caused by the oxidation reaction of ascorbic acid contained as an interfering substance, and gave a proportional relationship to the concentration. After the current value between the counter electrode 6 and the third electrode 8 was measured, the voltage application between both electrodes was released.

As described above, no reaction layer is disposed on the third electrode 8. Therefore, it takes some time for the ferrocyanide ions generated as a result of the enzyme reaction to reach the vicinity of the third electrode 8. That is, the counter electrode 6 and the third electrode in the time until the arrival of the ferrocyanide ion are reached.
The current value between the electrodes 8 mainly reflects only the concentration of ascorbic acid. Further, 25 seconds after the detection of the sample liquid, the counter electrode 6
Was applied, 500 mV was applied to the working electrode 7, and the current value after 5 seconds between the counter electrode 6 and the working electrode 7 was measured. The ferricyanide ion, glucose, and GOD in the liquid react,
As a result, glucose is oxidized to gluconolactone,
Ferricyanide ions are reduced to ferrocyanide ions. The concentration of this ferrocyanide ion is proportional to the concentration of glucose. The current between the counter electrode 6 and the working electrode 30 after 30 seconds from the detection of the sample liquid is caused by an oxidation reaction between ferrocyanide ions and ascorbic acid which already exists. That is, ascorbic acid gives a positive error to the measurement result. However, as described above, the current value between the counter electrode 6 and the third electrode 8 mainly reflects only the concentration of ascorbic acid. Therefore, by correcting the measurement result based on the result, the influence of ascorbic acid can be removed, and an accurate glucose concentration can be obtained.

Example 2 Similarly to Example 1, electrodes 6, 7, 8 and a carbon layer 9 were formed on a substrate 1. Next, a CMC aqueous solution is dropped on the counter electrode 6, the working electrode 7, and the carbon layer 9 except for the third electrode 8, and dried to form a CMC layer. On this CMC layer, GOD as an enzyme is formed. Then, an aqueous solution containing potassium ferricyanide as an electron carrier was dropped and dried to form a reaction layer. Next, on the reaction layer, in order to further smoothly supply the sample solution to the reaction layer, an organic solvent solution of lecithin, for example, a toluene solution, is spread from the sample solution supply port 12a over the reaction layer, and dried. By doing so, a lecithin layer was formed. Next, the cover 10 and the spacer 11 were adhered to the substrate 1 in a positional relationship as indicated by a dashed line in FIG. 2 to produce a glucose sensor.

This sensor was set on a measuring instrument, and a potential of 500 mV was applied to the third electrode 8 with reference to the counter electrode 6. With this potential applied, 3 μl of an aqueous glucose solution containing ascorbic acid as an interfering substance was supplied as a sample liquid from the sample liquid supply port 12a. The sample liquid reached the air holes 13 through the sample liquid supply path, and the reaction layer on the electrode system was dissolved. Simultaneously with the supply of the sample liquid, the system for detecting the supply of the liquid based on the electrical change between the counter electrode 6 and the third electrode 8 of the electrode system was operated, and the measurement timer was started. At this time, the potential is continuously applied between the counter electrode 6 and the third electrode 8, and after a lapse of a certain time from the detection of the sample liquid supply,
The current value between the counter electrode 6 and the third electrode 8 was measured. This current value was caused by the oxidation reaction of ascorbic acid contained as an interfering substance, and gave a proportional relationship to the concentration. After the current value between the counter electrode 6 and the third electrode 8 was measured, the voltage application between both electrodes was released.

As described above, no reaction layer is disposed on the third electrode 8. Therefore, it takes some time for the ferrocyanide ions generated as a result of the enzyme reaction to reach the vicinity of the third electrode 8. That is, the counter electrode 6 and the third
The current value between the electrodes 8 mainly reflects only the concentration of ascorbic acid. Further, 25 seconds after the detection of the sample liquid, the counter electrode 6
Was applied, 500 mV was applied to the working electrode 7, and the current value after 5 seconds between the counter electrode 6 and the working electrode 7 was measured. The ferricyanide ion, glucose, and GOD in the liquid react,
As a result, glucose is oxidized to gluconolactone,
Ferricyanide ions are reduced to ferrocyanide ions. The concentration of this ferrocyanide ion is proportional to the concentration of glucose. The current between the counter electrode 6 and the working electrode 30 after 30 seconds from the detection of the sample liquid is caused by an oxidation reaction between ferrocyanide ions and ascorbic acid which is present in advance. That is, ascorbic acid gives a positive error to the measurement result. However, as described above, the current value between the counter electrode 6 and the third electrode 8 mainly reflects only the concentration of ascorbic acid. Therefore, by correcting the measurement result based on the result, the influence of ascorbic acid can be removed, and an accurate glucose concentration can be obtained.

In this embodiment, since the supply of the sample liquid between the counter electrode 6 and the third electrode 8 is detected, the entire exposed portion of the working electrode 7 is reliably filled with the sample liquid. This makes it possible to more reliably determine the supply of the sample liquid.

Example 3 A glucose sensor was manufactured in the same manner as in Example 2. Set the sensor on the measuring instrument and set the counter electrode 6
, A potential of 500 mV was applied to the third electrode 8. With this potential applied, 3 μl of an aqueous glucose solution containing ascorbic acid as an interfering substance was supplied as a sample liquid from the sample liquid supply port 12a. The sample liquid reached the air holes 13 through the sample liquid supply path, and the reaction layer on the electrode system was dissolved. Simultaneously with the supply of the sample liquid, the system for detecting the supply of the liquid based on the electrical change between the counter electrode 6 and the third electrode 8 of the electrode system was operated, and the measurement timer was started. At this time, the potential is continuously applied between the counter electrode 6 and the third electrode 8, and after a lapse of a certain time from the detection of the sample liquid supply,
The current value between the counter electrode 6 and the third electrode 8 was measured. This current value was caused by the oxidation reaction of ascorbic acid contained as an interfering substance, and gave a proportional relationship to the concentration. After the current value between the counter electrode 6 and the third electrode 8 was measured, the voltage application between both electrodes was released.

As described above, no reaction layer is disposed on the third electrode 8. Therefore, it takes some time for the ferrocyanide ions generated as a result of the enzyme reaction to reach the vicinity of the third electrode 8. That is, the counter electrode 6 and the third
The current value between the electrodes 8 mainly reflects only the concentration of ascorbic acid. Further, 25 seconds after the detection of the sample liquid, 500 mV was applied to the working electrode 7 with reference to the third electrode 8, and the current value between the counter electrode 6 and the working electrode 7 after 5 seconds was measured. The ferricyanide ion, glucose, and GOD in the liquid react, and as a result, glucose is oxidized to gluconolactone, and the ferricyanide ion is reduced to ferrocyanide ion. The concentration of this ferrocyanide ion is proportional to the concentration of glucose. The current between the counter electrode 6 and the working electrode 30 after 30 seconds from the detection of the sample liquid is expressed as ferrocyanide ion,
It is caused by the oxidation reaction of pre-existing ascorbic acid. That is, ascorbic acid gives a positive error to the measurement result. However, as described above, the current value between the counter electrode 6 and the third electrode 8 mainly reflects only the concentration of ascorbic acid. Therefore, by correcting the measurement result based on the result, the influence of ascorbic acid can be removed, and an accurate glucose concentration can be obtained. When the potential of the third electrode 8 was measured with the silver / silver chloride electrode as a reference when the potential was applied to the working electrode 7, the working electrode 7 was measured.
However, despite the occurrence of the oxidation reaction, almost no change in the potential at the third electrode 8 was observed. In addition, the variation in response was reduced as compared with the conventional method in which liquid junction detection was performed based on a change in resistance between the working electrode and the counter electrode.

Example 4 In the same manner as in Example 2, a reaction layer was formed on the counter electrode 6, the working electrode 7, and the carbon layer 9 except for the third electrode 8. Next, in a groove formed on a cover member for forming a sample liquid supply path, an organic solvent solution of lecithin, for example, a toluene solution is spread to further smoothly supply the sample liquid to the reaction layer. Then, a lecithin layer was formed by drying. Next, cover 1
0 and the spacer 11 were adhered in a positional relationship as shown by a dashed line in FIG. 2 to produce a glucose sensor. When the lecithin layer is disposed from the reaction layer to the third electrode 8, the surface of the third electrode 8 may change due to the lecithin layer, and the response characteristics may vary widely. When the lecithin layer is disposed on the cover member side as described above, such variations are suppressed, and the response characteristics are improved.

Example 5 A glucose sensor was manufactured in the same manner as in Example 2 except that the CMC layer was removed from the reaction layer. Then, as a result of performing the measurement in the same manner as in Example 2, although the variation in the response was increased as compared with the case where the CMC layer was arranged, the concentration dependency on ascorbic acid and glucose was observed.

Example 6 A glucose sensor was manufactured in the same manner as in Example 4. Set the sensor on the measuring instrument and set the counter electrode 6
A potential of -1300 mV was applied to the third electrode 8 with reference to. With this potential applied, 3 μl of an air-saturated aqueous glucose solution was used as a sample liquid, and the sample liquid supply port 1 was used.
2a. The sample liquid reached the air holes 13 through the sample liquid supply path, and the reaction layer on the electrode system was dissolved. Simultaneously with the supply of the sample liquid, the system for detecting the supply of the liquid based on the electrical change between the counter electrode 6 and the third electrode 8 of the electrode system was operated, and the measurement timer was started. At this time, the potential was continuously applied between the counter electrode 6 and the third electrode 8, and the current value between the counter electrode 6 and the third electrode 8 was measured after a lapse of a predetermined time from the detection of the sample liquid supply. This current value was caused by a reduction reaction of dissolved oxygen, and when a glucose solution degassed with argon was supplied, the reduction current decreased sharply. After the current value between the counter electrode 6 and the third electrode 8 was measured, the voltage application between both electrodes was released.

As described above, no reaction layer is disposed on the third electrode 8. Therefore, it takes some time for the ferricyanide ions contained in the reaction layer to reach the vicinity of the third electrode 8. That is, the current value between the counter electrode 6 and the third electrode 8 during the time until the ferricyanide ion reaches the third electrode 8 mainly reflects only the dissolved oxygen concentration. Furthermore, 25 seconds after the detection of the sample liquid,
500 mV was applied to the working electrode 7 with reference to the third electrode 8, and the current value between the counter electrode 6 and the working electrode 7 after 5 seconds was measured.
Ferricyanide ion, glucose, and GO in liquid
D reacts and, as a result, glucose is oxidized to gluconolactone, and with this reaction, ferricyanide ions are reduced to ferrocyanide ions. On the other hand, as a competitive reaction of this reaction, dissolved oxygen in the sample solution acts as an electron carrier, and as glucose is oxidized to gluconolactone, a reaction in which dissolved oxygen is reduced to hydrogen peroxide simultaneously proceeds. . Hydrogen peroxide generated by this reaction reoxidizes ferrocyanide ions to ferricyanide ions. Therefore, when measuring the glucose concentration based on the oxidation current value of the ferrocyanide ion, the dissolved oxygen gives a negative error to the measurement result. However, as described above, the current value between the counter electrode 6 and the third electrode 8 mainly reflects only the dissolved oxygen concentration. Then, by correcting the measurement result based on the result, the influence of dissolved oxygen can be removed and an accurate glucose concentration can be obtained.

Example 7 A glucose sensor was manufactured in the same manner as in Example 4. Set the sensor on the measuring instrument and set the counter electrode 6
, A potential of 500 mV was applied to the third electrode 8. With this potential applied, 3 μl of an aqueous glucose solution containing ascorbic acid as an interfering substance was supplied as a sample liquid from the sample liquid supply port 12a. The sample liquid reached the air holes 13 through the sample liquid supply path, and the reaction layer on the electrode system was dissolved. Simultaneously with the supply of the sample liquid, the system for detecting the supply of the liquid based on the electrical change between the counter electrode 6 and the third electrode 8 of the electrode system was operated, and the measurement timer was started. At this time, the potential is continuously applied between the counter electrode 6 and the third electrode 8. Two seconds after the detection of the sample liquid supply,
The potential applied to the third electrode 8 was stepped to -1300 mV. Current values between the counter electrode 6 and the third electrode 8 at two points were measured immediately before stepping the potential to -1300 mV and three seconds after stepping to -1300 mV. −
The current value immediately before stepping the potential to 1300 mV mainly depends on the ascorbic acid concentration. On the other hand, -130
The current value 3 seconds after stepping to 0 mV mainly depends on the concentration of dissolved oxygen contained in the sample solution. After the current value between the counter electrode 6 and the third electrode 8 was measured 2 seconds and 5 seconds after the supply of the sample liquid, the application of the voltage between the electrodes was released. Further, 25 seconds after the detection of the sample liquid, 500 mV is applied to the working electrode 7 with reference to the third electrode 8, and 5 m between the counter electrode 6 and the working electrode 7 is applied.
The current value after 2 seconds was measured.

As described above, since the current value between the counter electrode 6 and the third electrode 8 mainly reflects the concentrations of ascorbic acid and dissolved oxygen, the concentrations of both substances can be obtained based on the current values. Therefore, by correcting the measurement result based on the result, the influence of ascorbic acid and dissolved oxygen can be removed, and an accurate glucose concentration can be obtained.

In the above embodiment, the detection of the sample liquid supply,
The applied potential to the third electrode 8 for detecting ascorbic acid or dissolved oxygen is set to 500 mV or -1300.
mV, but is not limited thereto. The potential applied to the working electrode 7 for obtaining a response current is 500 mV.
However, the present invention is not limited to this, and any potential may be used as long as the reduced form of the electron carrier generated as a result of a series of reactions can be oxidized. The time for measuring the current value is not limited to the above embodiment.

In the above embodiment, carboxymethylcellulose was used as the hydrophilic polymer, but various hydrophilic polymers can be used as the hydrophilic polymer forming the hydrophilic polymer layer. For example, hydroxyethylcellulose, hydroxypropylcellulose, methylcellulose, ethylcellulose, ethylhydroxyethylcellulose, carboxymethylethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids such as polylysine, polystyrenesulfonic acid, gelatin and its derivatives, polyacrylic acid and its salts, poly Examples include polymers of methacrylic acid and its salts, starch and its derivatives, maleic anhydride and its salts. Among them, carboxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose are preferred. The oxidoreductase contained in the reaction layer is selected according to the substrate contained in the sample solution. Examples of the oxidoreductase include fructose dehydrogenase, glucose oxidase, alcohol oxidase, lactate oxidase, cholesterol oxidase, xanthine oxidase, amino acid oxidase and the like.
Examples of the electron carrier include potassium ferricyanide, p-benzoquinone, phenazine methosulfate, methylene blue, and a ferrocene derivative. One or more of these electron carriers are used.

The enzyme and the electron carrier may be dissolved in a sample solution, or may not be dissolved in the sample solution by fixing the reaction layer to a substrate or the like. When the enzyme and the electron carrier are immobilized, the reaction layer preferably contains the hydrophilic polymer. In the above embodiment, an example of a specific electrode system is illustrated, but the shape of the electrode, the arrangement of the electrode and the lead, and the like are not limited thereto. In the above embodiment, carbon was described as the electrode material of the third electrode. However, the present invention is not limited to this, and other conductive materials and silver / silver chloride electrodes can be used.

[0035]

As described above, according to the present invention, highly reliable quantification of a substrate can be performed.

[Brief description of the drawings]

FIG. 1 is a plan view of a glucose sensor according to an embodiment of the present invention with a reaction layer removed.

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

[Explanation of symbols]

 REFERENCE SIGNS LIST 1 Insulating substrate 2, 3, 4 lead 5 Insulating layer 6 Counter electrode 7 Working electrode 8 Third electrode 9 Carbon layer 10 Cover 11 Spacer 12 Slit for forming sample liquid supply path 12 a Sample liquid supply port 13 Air hole

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-5-340915 (JP, A) JP-A-8-320304 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 27/327 G01N 27/416

Claims (5)

(57) [Claims] And 1. A electrically insulating substrate, effects formed on the substrate electrode, a counter electrode, and an electrode system and a third electrode used as an interfering substance detecting electrode, the third electrode provided on the electrode system, excluding a reaction layer containing at least an oxidoreductase and an electron mediator, and the substrate
And a cover member that forms a sample liquid supply path between
The third electrode is located above the sample liquid supply path than the reaction layer.
Disposed on the flow side, and wherein the working electrode is
Is also arranged upstream of the sample liquid supply path.
Biosensor 2. The method according to claim 1, wherein said cover member is exposed to a sample liquid supply passage.
That a layer containing lecithin as the main component was
The biosensor according to claim 1, which is a feature. 3. The reaction layer further comprises a hydrophilic polymer.
3. A bag according to claim 1, wherein
Io sensor. 4. A electrically insulating substrate, effects formed on the substrate electrode, a counter electrode, and an electrode system and a third electrode to be used as an interfering substance detecting electrodes, the third provided on the electrode system except the electrode consists of a reaction layer containing at least an oxidoreductase and an electron mediator, a cover member for forming a sample solution supply pathway between the substrate, the third electrode the trial but than the reaction layer is disposed on the upstream side of the sample solution supply path, and the working electrode than the counter electrode
Using a biosensor arranged on the upstream side of the sample liquid supply path, the electron carrier is reduced by electrons generated when reacting the substrate contained in the sample liquid with the oxidoreductase, and the electron carrier of the electron carrier is reduced. A method for quantifying the concentration of a substrate in a sample liquid by electrochemically measuring the amount of reduction, wherein a voltage is applied between a counter electrode and a third electrode, and the sample liquid is supplied to a reaction layer. A step of detecting an electrical change between the counter electrode and the third electrode caused by the supply of the sample solution to the reaction layer; after the detecting step, between the counter electrode and the third electrode. measuring the current produced, after the step of measuring the step of releasing voltage application between the counter electrode and the third electrode, applying a voltage between the working electrode and the counter electrode, and Measuring the current generated between the counter electrode and the working electrode thereafter. A method for quantifying a substrate, comprising: 5. An electrically insulating substrate and a substrate formed on the substrate.
Working electrode, counter electrode and interfering substance detection electrode or reference
An electrode system having a third electrode used as a pole;
At least oxidation provided on the electrode system except for the electrode 3
A reaction layer containing a reductase and an electron carrier, and the substrate
And a cover member forming a sample liquid supply path between,
The third electrode is closer to the sample liquid supply path than the reaction layer.
The working electrode is located on the upstream side and the working electrode is closer to the counter electrode.
Biomass disposed upstream of the sample liquid supply path
Substrate and oxidoreductase contained in sample solution using sensor
The electron carrier is generated by the electrons generated when reacting
And the amount of reduction of the electron carrier is measured electrochemically.
Is used to determine the concentration of the substrate in the sample solution.
A step of applying a voltage between the counter electrode and the third electrode , a step of supplying a sample liquid to the reaction layer, and a step of applying the sample liquid to the reaction layer .
Detecting an electrical change between the electrode and the electrode, and after the detecting step, a voltage is generated between the counter electrode and the third electrode.
Measuring the same current, after the measuring step, the voltage between the counter electrode and the third electrode.
Canceling the pressure application, applying a voltage between the working electrode, the third electrode and the counter electrode
Process, and then the current generated between the counter and working electrodes
Determination of the substrate, characterized by comprising the step, of measuring.