JP3063352B2 - Concentration measuring device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a concentration measuring device for measuring the concentration of a substance to be measured based on a biochemical reaction between the substance to be measured and a biological substance in a solution to be measured.

[0002]

2. Description of the Related Art A biosensor converts the progress of a biochemical reaction between a biological substance such as an enzyme or a microorganism and a substance to be measured into an electric quantity, and measures the substance to be measured in a solution to be measured such as urine. And various things are known. For example, a potential value is measured based on a change in ion concentration of various substances involved in a biochemical reaction, or a current value obtained by an electrode reaction by a chemical substance (electrode active substance) generated or consumed in the reaction is measured. In addition to electrode-type biosensors, biosensors that measure thermal changes due to biochemical reactions with a thermal measurement device, and biosensors that guide biochemical reactions into chemiluminescence and measure the amount of light emitted by a photocounter and so on.

[0003] By using various kinds of enzymes and microorganisms as the biological substance to be used, a substance to be measured which reacts with the enzyme can be selectively detected. For example, when glucose oxidase is used as a biological substance, it becomes a biosensor that detects glucose in urine. Also, when ascorbate oxidase is used as a biological substance, it becomes a biosensor for detecting ascorbic acid in urine. That is, the biological substance to be used is appropriately selected according to the substance to be measured.

In order to determine the concentration of a substance to be measured using a biosensor, a concentration measuring device that processes a sensor output from the biosensor is used. This concentration measuring device includes a microcomputer including a CPU, a ROM, a RAM, and the like, in addition to the biosensor. Then, when the biosensor is brought into contact with the solution to be measured whose concentration is unknown, the concentration measuring device converts the amount of electricity (sensor output) inputted from the sensor into the concentration of the substance to be measured. The substance concentration can be determined. Note that a calibration curve previously stored in a ROM is generally used for conversion from the amount of electricity to the concentration.

When the concentration of a substance to be measured is measured by a concentration measuring device, there are various methods for bringing the solution to be measured into contact with the biosensor, and an appropriate method is selected. For example, a biosensor is immersed in a solution to be measured, or a solution to be measured is injected into the biosensor.

[0006]

However, when the concentration of the substance to be measured in the solution to be measured is determined, the concentration may not be measured correctly. This is for the following reasons. Since the solution to be measured is urine as described above, various substances other than the substance to be measured exist in the solution. And, although a certain kind of substance is not a substance to be measured, it causes a biochemical reaction with a biological substance used to convert the concentration of the substance to be measured into an electric quantity, and the electric quantity conversion of the concentration of the substance to be measured is performed. It becomes an interfering substance that inhibits.
For example, when glucose concentration in urine is measured using glucose oxidase, ascorbic acid, uric acid, bilirubin, etc. in urine become an interfering substance, so that glucose oxidase starts with glucose, and this ascorbic acid, etc. Cause a biochemical reaction. Therefore, the converted amount of electricity does not depend on the concentration of glucose alone, and the glucose concentration cannot be measured accurately.

Therefore, a technique has been proposed for oxidizing such an interfering substance or keeping it from approaching glucose oxidase (Japanese Patent Application Laid-Open Nos. 2-245650 and 58-5642). However, since the chemical structure of perchloric acid such as NaClO ₄ used for oxidizing the interfering substance is unstable, the reliability of the oxidizing of the interfering substance is low. No improvement can be expected. On the other hand, if the interfering substance is not
Since ascorbic acid or the like has a negative charge in the solution, a negative charge is applied around the electrode, and ascorbic acid or the like is electrically repelled so as to be kept away from the electrode. However, in order to keep a negative charge around the electrode at all times, its electrical configuration becomes complicated, which is not preferable.

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and has as its object to improve the measurement accuracy of the concentration of a substance to be measured by eliminating the influence of an interfering substance with a simple configuration.

[0009]

In order to achieve the above object, a means adopted by the present invention is to reduce the concentration of a substance to be measured based on a biochemical reaction between the substance to be measured and a biological substance in a solution to be measured. A concentration measuring device for measuring, comprising a discriminating layer carrying a biological substance causing a biochemical reaction with the substance to be measured, converting the progress of the biochemical reaction proceeding in the discriminating layer into an electric quantity, and outputting the measured object. A substance sensor element portion, and an identification layer carrying a biological substance that causes a biochemical reaction with an interfering substance that inhibits the conversion of electricity in the substance sensor element portion to be measured, and a progress state of the biochemical reaction that proceeds in the identification layer Is converted into an electric quantity, and an interfering substance sensor element section that outputs the electric quantity, and a sensor output from the measurement target substance sensor element section based on the sensor output from the interfering substance sensor element section is corrected. Or the sensor output from the interfering substance sensor element unit is converted into a sensor output when a biological substance carried on the identification layer in the measurement target substance sensor element unit has caused a biochemical reaction with the interfering substance. The gist of the present invention is to include a means and a concentration calculating means for calculating the concentration of the substance to be measured based on a sensor output obtained by subtracting the converted sensor output from the sensor output of the sensor element for the substance to be measured.

[0010]

The concentration measuring device having the above-mentioned structure includes a sensor element for measuring a substance to be measured and a sensor element for interfering substances. Each sensor element converts the following electric quantity and outputs the converted electric quantity as a sensor output. . In the measurement target substance sensor element, the biological substance carried on the identification layer causes a biochemical reaction between the measurement target substance and the interfering substance in the identification layer. The progress of this dependent biochemical reaction is converted into an electric quantity. On the other hand, in each interfering substance sensor element, the biological substance carried on the discriminating layer of each interfering substance sensor element causes a biochemical reaction with the interfering substance in this discriminating layer. The progress of this biochemical reaction is converted into an electric quantity.

Since the biological substance carried on the identification layer in the sensor element of the substance to be measured causes a slight biochemical reaction with the interfering substance, the amount of electricity (sensor output) converted by the sensor element of the substance to be measured has this interference. It includes the quantity of electricity (sensor output) based on the biochemical reaction with the substance. By the way, in the concentration measuring device of the present invention, the sensor output from the interfering substance sensor element is converted by the sensor output conversion means into a biological substance carried on the identification layer in the sensor element of the substance to be measured, causing a biochemical reaction with the interfering substance. Is converted to the sensor output in the case of Further, the concentration calculating means calculates the concentration of the substance to be measured based on the sensor output obtained by subtracting the converted sensor output from the sensor output of the sensor element for the substance to be measured. As a result, the concentration is calculated by excluding the quantity of electricity based on the biochemical reaction with the interfering substance from the quantity of electricity converted by the measurement target substance sensor element unit. The concentration of the substance to be measured can be determined based on the amount.

[0012]

Next, a concentration measuring apparatus according to an embodiment of the present invention will be described with reference to the drawings. FIG. 1 is a schematic perspective view of a biosensor 1 which is a main component of a concentration measuring device according to an embodiment. FIG. 2 is a concentration measuring device for obtaining a concentration of a substance to be measured based on a sensor output from the biosensor 1. FIG. 2 is a block diagram illustrating an electrical configuration of a first embodiment. This concentration measuring device 20 is used when measuring glucose in urine containing a solution to be measured, for example, glucose and ascorbic acid or the like which is an interfering substance for glucose measurement.

First, the biosensor 1 will be described.
As shown in FIG.
Used while being attached to and detached from the attached connector 35,
As shown in FIG. 1, this is a flat-plate electrode-type biosensor and has the following configuration.

As shown in FIG. 1, a biosensor 1 comprises an insulating substrate 3 having a thickness of 0.7 mm made by sintering alumina.
(20 mm × 30 mm), a sensor element 5 for measuring glucose, a sensor element 7 for measuring ascorbic acid,
It comprises a sensor element 9 for measuring uric acid and a sensor element 11 for measuring bilirubin.

The sensor element 5 for measuring glucose comprises a working electrode 5 a and a reference electrode 5 b formed on the insulating substrate 3.
It includes a counter electrode 5c, an identification layer 5d in which glucose oxidase (GOD) is supported and fixed on the working electrode 5a, and terminal portions 5e, 5f, and 5g of the electrodes such as the working electrode 5a. Similarly, the sensor element unit 7 for measuring ascorbic acid, the sensor element unit 9 for measuring uric acid, and the sensor element unit 11 for measuring bilirubin also include a working electrode, a reference electrode, a counter electrode, and the like. In addition,
Working electrodes and the like in each sensor element are denoted by reference numerals a to g, similarly to the case of the sensor element 5 for measuring glucose.

The sensor element 7 for measuring ascorbic acid has a discriminating layer 7d on which the ascorbate oxidase is supported and immobilized on the working electrode 7a, and the sensor element 9 for measuring uric acid is provided on the working electrode 9a. Uricase, which is uric acid oxidase, is immobilized on the identification layer 9d, and the sensor element 11 for measuring bilirubin is provided with an identification layer 11d, which carries and immobilizes bilirubin oxidase on the working electrode 11a. .

Each electrode such as a working electrode in each of the sensor element portions is insulated and covered with an insulating layer 13, and a side on which each identification layer is formed serves as a sensitive portion 15.

When the solution to be measured comes into contact with the sensitive portion 15, a biochemical reaction occurs between the above-mentioned biological material carried in the identification layer on the upper surface of each working electrode and the substance to be measured in the solution to be measured, and the biosensor 1 Outputs a current value reflecting the biochemical reaction to the apparatus main body 30 via the connector 35. The apparatus body 30 processes the current value as a sensor output value, and obtains a concentration as described later.

Next, the manufacturing process of the biosensor 1 will be described. First, an electrode in each sensor element unit is manufactured. That is, the working electrodes 5a to 11a and the subsequent terminals 5e to 11e are formed through screen printing of a platinum paste on the upper surface of the insulating substrate 3 and drying at 50 ° C. for 1 hour. At this time, as the platinum paste, a dispersion paste obtained by kneading 90 wt% of platinum fine powder and 10 wt% of butylcellulose was used. Next, each reference pole 5
The terminals b to 11b and the counter electrodes 5c to 11c and the terminals 5f to 11f and 5g to 11g following them are formed through screen printing of a graphite paste on the upper surface of the insulating substrate 3 and drying at 50 ° C. × 1 hour. . At this time, as the graphite paste, 60 wt% of graphite fine powder and 40 wt% of liquid paraffin were used.
And a dispersion paste obtained by kneading the mixture was used. In addition,
Each of the electrodes is formed to a thickness of 10 μm.
Further, after a plurality of sets of each sensor element section are formed, the insulating substrate 3 is cut into the above-described dimensions with the above four sensor element sections including the sensor element section 5 for glucose measurement as one set.

Thereafter, the insulating layer 13 is formed over each working electrode, reference electrode and counter electrode by printing and drying an appropriate insulating agent, for example, an epoxy resin.

Next, glucose oxidase (GO
The identification layer supporting each of the above-mentioned biological substances such as D) is immobilized on the upper surface of each working electrode as described below. A collagen GOD solution is prepared by kneading 94 wt% of collagen, 5 wt% of GOD, and 1 wt% of ferrocene, which is an electron acceptor (mediator). Then, this GOD solution was applied to a thickness of about 20 μm on the upper surface of the working electrode 5a on the sensitive part 15 side with a micropipette, and then the identification layer 5d was fixed by natural drying at room temperature for 2 hours.

Instead of the above 5 wt% GOD, 5 wt%
Each assortment of ascorbate oxidase, 5% by weight of uricase, and 5% by weight of bilirubin oxidase is used to knead each of the biological materials, the collagen and ferrocene to separately prepare a gel-like biological material solution. Then, each of the biological substance solutions is GOd onto the corresponding working electrodes 7a to 11a.
D solution and applied in the same manner as in the case of the D solution.
d was immobilized. Thus, the biosensor 1 integrally including the four sensor element portions on the insulating substrate 3 is completed.

Next, the apparatus main body 30 will be described. As shown in FIG. 2, the concentration measuring device 20 includes the above-described biosensor 1 and a device main body 30. Device body 30
Is an electronic control unit 40 that performs density calculation as described later.
And a display device 50 for displaying the converted concentration, an operation panel 60 having a power-on switch and the like, and a connector 35 to which the biosensor 1 is attached and detached.

The electronic control unit 40 includes a CPU 41, R
The OM 42, the RAM 43, and the timer 44 are mainly configured as a logical operation circuit, and input / output with the outside is performed by an input / output port 46 connected to the OM 42, the RAM 43, and the timer 44 via a common bus 45. The display device 50, the operation panel 60, and the connector 35 are connected to the input / output port 46 of the electronic control device 40. The display device 50 displays a concentration or the like based on a control signal from the electronic control device 40, and
Receives the application of the weak voltage for measurement via the connector 35 and outputs the current value output from each of the sensor element units 5 to 11 to the electronic control unit 40. Further, the operation panel 60 outputs on / off signals of various switches to the electronic control device 40.

Next, the density measurement control (routine) performed by the density measurement device 20 of the present embodiment having the above-described configuration will be described with reference to the flowchart of FIG. The flowchart shown in FIG. 3 shows a concentration measurement routine that is repeatedly performed from when a power switch (not shown) of the operation panel 60 is pressed and the power is turned on to the concentration measuring device 20 until the power is turned off. . As shown in FIG. 3, this routine is sequentially executed after initial processing executed only when the power is turned on, that is, after clearing an internal register of the CPU. Subsequent to the initial processing, first, it is determined whether or not to start measuring the glucose concentration based on the operation state of a measurement start switch (not shown) on the operation panel 60 (step 200; hereinafter, this step is simply referred to as S). ),
Wait until the measurement start switch is turned on.

If it is determined in step S200 that the concentration of the solution to be measured whose concentration is unknown, for example, the concentration of glucose in urine, is to be measured, the sensor outputs (current values) of the sensor elements 5 to 11 are read (S210). Of the read sensor outputs, the sensor outputs read from sensor element sections for interfering substances such as ascorbic acid measurement sensor element section 7, uric acid measurement sensor element section 9, and bilirubin measurement sensor element section 11 are used for glucose measurement. Is converted into a sensor output based on the biochemical reaction between glucose oxidase and each interfering substance in the identification layer 5d of the sensor element unit 5 for use (S220). The reason why the sensor output of each sensor element for measuring an interfering substance is converted into the sensor output of the sensor element for measuring glucose 5 is as follows.

Glucose oxidase in the identification layer 5d of the sensor element 5 for glucose measurement slightly reacts with each interfering substance present in urine. For this reason, the sensor output from the sensor element unit 5 for glucose measurement is based on the biochemical reaction between glucose and glucose oxidase in the discriminating layer 5d, and the biochemical reaction between each interfering substance and glucose oxidase in the discriminating layer 5d. And those based on. Therefore, if the sensor output based on the biochemical reaction between each interfering substance and glucose oxidase among the sensor outputs from the sensor element unit 5 for measuring glucose is found, the sensor output based only on the biochemical reaction between glucose and glucose oxidase is obtained. Is obtained. However, the reactivity of glucose oxidase to each interfering substance is naturally inferior to the reactivity to glucose, and each biological substance (ascorbic acid oxidase to ascorbic acid, uricase to uric acid,
The reactivity of bilirubin oxidase to bilirubin is also poor. Therefore, while taking into account the reactivity of glucose oxidase to each interfering substance, each sensor output of the sensor element section for interfering substance measurement is compared with each interfering substance and glucose oxidase in the identification layer 5d of the sensor element section 5 for glucose measurement. Is converted into a sensor output based on the biochemical reaction.

This conversion was performed by multiplying each sensor output read from the sensor element section for each interfering substance by a conversion coefficient determined according to each interfering substance. Note that this conversion coefficient is determined in advance and R
It is stored in the OM 42 and is read out for conversion. For example, if the sensor output (current value) from the ascorbic acid measurement sensor element section 7 is Ia, Ka · Ia obtained by multiplying the sensor output Ia by the conversion coefficient Ka for ascorbic acid is used as the glucose measurement sensor element section. The sensor output based on the biochemical reaction between ascorbic acid and glucose oxidase in the identification layer 5d of No. 5 was used. Similarly, Ku · Iu obtained by multiplying the sensor output Iu from the uric acid measurement sensor element unit 9 by the conversion coefficient Ku for uric acid is used to calculate the biochemistry of uric acid and glucose oxidase in the identification layer 5 d of the glucose measurement sensor element unit 5. The sensor output based on the reaction was used. The sensor output Ib from the bilirubin measurement sensor element 11 multiplied by the conversion coefficient Kb for bilirubin is calculated as Kb · Ib, and the biochemical reaction between bilirubin and glucose oxidase in the identification layer 5 d of the glucose measurement sensor element 5 is calculated. Was used as the sensor output.

The conversion coefficients Ka, Ku, and Kb are stored in the ROM 42 in advance in association with at least the concentrations of the interfering substances (sensor outputs from the sensor element units for measuring the interfering substances). Further, the above conversion coefficients were determined through experiments described below.

A test biosensor comprising only the sensor element 5 for measuring glucose in this embodiment is immersed in a glucose reagent adjusted to a predetermined concentration. First, a sensor in the case where only the adjusted concentration of glucose is present in the solution to be measured. An output initial value IG0 is obtained. Next, ascorbic acid is gradually added to the glucose reagent, and the transition of the sensor output of the test biosensor is observed. Then, the relationship between the amount of ascorbic acid added, that is, the ascorbic acid concentration and the variation ΔIG of the sensor output (deviation from the sensor output initial value IG0) is determined. The amount of change in the sensor output is based on the biochemical reaction between ascorbic acid and glucose oxidase. On the other hand, the ascorbic acid concentration can be converted into a sensor output IA of a biosensor using ascorbate oxidase by a calibration curve. Therefore,
The conversion coefficient Ka for ascorbic acid is the value of the ratio of the sensor output variation △ IG to the sensor output IA (△ IG /
IA), and the obtained conversion coefficient Ka is stored in the ROM 42 in association with the ascorbic acid concentration (sensor output IA). Similarly, conversion coefficients Ku and Kb for uric acid and bilirubin are determined and stored.

The above experiment was repeated with respect to glucose reagents having different adjusted concentrations, and the above conversion coefficients Ka were calculated.
, Ku, and Kb can be stored in the ROM 42 in association with the glucose concentration (sensor output) and each interfering substance concentration (sensor output). With this configuration, the conversion of the sensor output in S220 can be performed using the conversion coefficient that also reflects the glucose concentration (sensor output) in the solution to be measured, which is preferable because the conversion accuracy is improved.

After converting the sensor output of each sensor element for measuring interfering substances into the sensor output of the sensor element 5 for measuring glucose in S220, the sensor output of the sensor element 5 for measuring glucose read in S210 is corrected. And calculating the glucose concentration in the urine based on the corrected sensor output (S230). That is, the converted sensor output (Ka) based on the biochemical reaction between glucose oxidase and an interfering substance such as ascorbic acid in the identification layer 5d of the glucose measuring sensor element section 5 is obtained from the read sensor output of the glucose measuring sensor element section 5.・ Ia, K
u · Iu and Kb · Ib) are subtracted to perform the above correction.
Thereby, the sensor output of the glucose measuring sensor element unit 5 based on only the biochemical reaction between glucose and glucose oxidase is obtained. Then, the glucose concentration of the glucose reagent is calculated from the obtained sensor output (corrected sensor output) and the glucose calibration curve Kgcal shown in FIG. The calibration curve Kgcal is stored in the ROM 42 in advance as calibration curve map data in which the sensor output and the glucose concentration are associated with each other.

Thereafter, a control signal corresponding to the calculated value of the glucose concentration is sent from the electronic control unit 40 to the display device 50.
To display the glucose concentration value (S240),
The processing of this routine is once ended.

The glucose concentration in the glucose reagent was measured using the concentration measuring device 20 of the present embodiment having such a configuration. The prepared concentrations of glucose, ascorbic acid, uric acid, and bilirubin in the glucose reagent used for the measurement are as shown in Table 1 and are prepared in advance before the measurement. Further, the element units 5 to 11 in Table 1
Are the abbreviations for the sensor element for glucose measurement 5, the sensor element for ascorbic acid measurement 7, the sensor element for uric acid 9, and the sensor element for bilirubin 11 respectively.

The measured glucose concentration in Table 1 was determined as follows according to the described flowchart. In addition,
This description is made for the case where the prepared glucose concentration is 17 mg / dl. That is, among the sensor outputs read from the sensor element units 5 to 11, the sensor output Ia (10 × 10 ⁻⁶ A) of the ascorbic acid measurement sensor element unit 7,
The sensor output Iu of the uric acid measurement sensor element 9 (15 × 1
0 ⁻⁶ A), the sensor output Ib (8 × 10 ⁻⁶ A) of the bilirubin measuring sensor element section 11 is converted into a conversion coefficient Ka in the table.
By multiplying by (0.2), Ku (0.01), and Kb (0.5), a sensor based on a biochemical reaction between glucose oxidase and each interfering substance in the identification layer 5d of the glucose measuring sensor element unit 5 is obtained. Convert to output (S22
0). As a result, the converted sensor output is 2 × 10 ⁻⁶
A (Ka · Ia), 0.15 × 10 ⁻⁶ A (Ku · Iu)
), 4 × 10 ⁻⁶ A (Kb · Ib).

Thereafter, the glucose measuring sensor element 5
The sensor output Ig of the sensor element unit 5 is corrected by subtracting each converted sensor output from the sensor output Ig (19 × 10 ⁻⁶ A) read from the sensor output Ig.
gh (12.85 × 10 ⁻⁶ A = 19 × 10 ⁻⁶ A−2 × 1
A measured glucose concentration of 16.06 mg / dl is calculated from 0 ^-6 A-0.15 × 10 ^-6 A-4 × 10 ^-6 A) and the glucose calibration curve Kgcal shown in FIG. 4 (S230).

On the other hand, the sensor output Ig (19 × 10
^-6 A) was used to calculate the glucose concentration, the glucose concentration was 23.25 m as shown in FIG.
g / dl.

That is, as is clear from Table 1, according to the concentration measuring device 20 of the present embodiment, it is possible to measure the concentration approximate to each prepared glucose concentration, excluding the influence of the interfering substance. Measurement accuracy of glucose concentration can be improved. For this reason, when measuring the glucose concentration in urine which inevitably contains ascorbic acid, uric acid, and bilirubin, which are obstructive substances for the measurement of glucose concentration, the concentration measuring device 20 of the present embodiment accurately measures glucose concentration. The concentration can be measured.

Further, in the concentration measuring device 20 of this embodiment,
Since ferrocene is supported as an electron acceptor (mediator) together with the above-described biological substance on the discrimination layer in each sensor element portion, the glucose concentration can be measured more accurately even when the solution to be measured is urine with little dissolved oxygen.

Next, a description will be given of a concentration measuring apparatus used for measuring lactic acid in sweat containing lactic acid and urea which is an interfering substance to lactic acid measurement. Since the biosensor used in this concentration measuring device for measuring lactic acid is different from that of the concentration measuring device 20 of the above-described embodiment, the description of the device main body is omitted.

As shown in FIG. 5, the biosensor 70 used in the concentration measuring device for measuring lactic acid is a plate-type electrode-type biosensor like the above-described biosensor 1, and has the following configuration. . Since the configuration is similar to that of the biosensor 1, in the description of the biosensor 70, the description of the same configuration and the manufacturing process will be simplified.

The biosensor 70 has a sensor element 73 for measuring lactic acid and a sensor element 75 for measuring urea on an insulating substrate 71 (10 mm × 30 mm) made of polyethylene having a thickness of 1.0 mm.

The sensor element 73 for measuring lactic acid comprises a working electrode 73 a and a reference electrode 73 formed on the insulating substrate 71.
b, discrimination layer 7 in which lactate dehydrogenase lactate oxidase is carried and immobilized on counter electrode 73c and working electrode 73a
3d and terminal portions 73e and 73 of each electrode such as the working electrode 73a.
f, 73g. Urea sensor element 75
Is provided with a discriminating layer 75d that carries and immobilizes urease, which is a urea oxidase, on a working electrode 75a in addition to a working electrode, a reference electrode, and a counter electrode.

Each electrode such as a working electrode in each sensor element portion is insulated and coated with an insulating layer 77, and the side on which each identification layer is formed serves as a sensitive portion 79.

When the solution to be measured comes into contact with the sensitive portion 79, a biochemical reaction occurs between the above-mentioned biological material carried in the identification layer on the upper surface of each working electrode and the substance to be measured in the solution to be measured, and the biosensor 70 Outputs a current value reflecting the biochemical reaction to the apparatus main body 30 via the connector 35. The device body 30 processes the current value as a sensor output value to determine the concentration.

The electrodes including the working electrodes and the respective terminals in the biosensor 70 are formed by screen etching after plasma etching the surface of the insulating substrate 71 and roughening the surface. The paste used was a dispersion paste obtained by kneading 90% by weight of graphite fine powder and 10% by weight of butylcellulose. After screen printing, a drying treatment was performed at 50 ° C. × 1 hour. The thickness of each electrode was adjusted to 20 μm. After that, an insulating layer 77 was formed.

The discriminating layer 73d on the working electrode 73a is formed by kneading 10 wt% of the above-mentioned lactate oxidase and 90 wt% of albumin with a microsyringe to a thickness of about 20 μm.
And then fixed in glutaraldehyde saturated air for 30 minutes. The discriminating layer 75d on the working electrode 75a was similarly immobilized using a gel-like solution in which 10% by weight of urease and 90% by weight of albumin were kneaded. The immobilization of both discriminating layers is performed simultaneously in glutaraldehyde saturated air.

Next, an evaluation test regarding lactic acid measurement by the concentration measuring device 20 using the biosensor 70 will be described. The lactic acid reagent used for the measurement was prepared as follows, assuming that lactic acid in sweat was measured. Lactic acid reagent A: Reagent assuming sweat sweated in daily life Prepared lactic acid concentration → 80 mg / dl Prepared urea concentration → 13 mg / dl Lactic acid reagent B: Reagent assuming sweat sweated during continuation of exercise Prepared lactate concentration → 182 mg / Dl Prepared urea concentration → 28mg / dl

The measured lactic acid concentration of each of the above reagents was determined as follows in accordance with the described flowchart. The sensor output and the like at that time are as follows. Lactic acid reagent A Sensor output Il from the lactic acid measurement sensor element 73 → 5.
75 × 10 ^-6 A Sensor output Iu from the urea measurement sensor element 75 → 6.
5 × 10 ^-6 A Conversion coefficient used Ku → 0.25 Converted sensor output Ku · Iu → 1.63 × 10 ^-6 A Corrected sensor output Ilh → 4.12 × 10 ^-6 A (= 5.75 × 10 ^-6 A-1.63 × 10 ^-6 A) The corrected sensor output Ilh and the lactic acid calibration curve K shown in FIG.
From lcal, 72 mg / dl was calculated as the measured lactic acid concentration. When the lactic acid concentration was calculated using the sensor output Il (5.75 × 10 ⁻⁶ A) from the lactic acid measurement sensor element unit 73 as it was, as shown in FIG.
g / dl.

Lactic acid reagent B Sensor output Il from lactic acid measuring sensor element 73 → 1
3.85 × 10 ^-6 A Sensor output Iu from urea measurement sensor element 75 → 14
× 10 ⁻⁶ A Conversion coefficient used Ku → 0.25 Converted sensor output Ku · Iu → 3.5 × 10 ⁻⁶ A Corrected sensor output Ilh → 10.35 × 10 ⁻⁶ A (= 13.85 × 10 ⁻⁶ A−3.5 × 10 ^{−) 6} A) The corrected sensor output Ilh and the lactic acid calibration curve K shown in FIG.
From the lcal, 180 mg / dl was calculated as the measured lactic acid concentration. When the lactic acid concentration was calculated using the sensor output Il (13.85 × 10 ⁻⁶ A) from the lactic acid measurement sensor element unit 73 as it was, the lactic acid concentration was calculated as shown in FIG.
It becomes 1 mg / dl.

As described above, the concentration measuring device 20 of this embodiment
According to this, even if the measurement target substance is lactic acid, the lactic acid concentration can be accurately measured by eliminating the influence of urea, which is an interfering substance, and the measurement accuracy of the lactic acid concentration can be improved. Therefore, the concentration of lactic acid in sweat can be accurately measured.

The present invention is not limited to the above embodiment, but can be implemented in various modes without departing from the gist of the invention, and the following modifications are possible. For example, although the biosensor 1 has the sensor element portions provided on one surface of the insulating substrate 3, the biosensor 1 may have two sensor devices on both surfaces of the insulating substrate.
This is preferable because the sensor becomes compact. In addition, since a sensor output detecting ascorbic acid or the like is obtained, the concentration of ascorbic acid, uric acid, or the like, which is an interfering substance, may be displayed using a calibration curve.

Further, instead of glucose oxidase in a glucose measuring biosensor, enzymes such as pyranose oxidase and mutarotase, or Pseudomo
A microorganism such as nasfluorescens may be used. The substance to be measured is not limited to the above glucose and lactic acid. In addition to sugars such as fructose and galactose, vitamins such as ascorbic acid, metabolites such as urobilinogen, bilirubin, creatine, creatinine, albumin, globulin, and the like. Proteins such as hemoglobin, hormones such as human chorionic gonadotropin (hCG), steroids such as 17KS and 17OHCS, and acids such as amino acids, hippuric acid, glucuronic acid, uric acid, pyruvic acid, ketoglutaric acid, oxalic acid, and β-oxybutyric acid. There may be. It is needless to say that the biological substance may be appropriately selected according to the measurement target substances, and the biological substance which causes a biochemical reaction with respect to the interfering substance with respect to the measurement of each measurement target substance.

The biosensor to be used is not limited to a plate-type electrode-type biosensor, and is a so-called potentiometric sensor that measures a potential value based on a change in ion concentration of various substances involved in a biochemical reaction. Alternatively, a biosensor of a type in which a thermal change due to a biochemical reaction is measured by a thermal measurement device, or a sensor of a type in which a biochemical reaction is led to chemiluminescence and the amount of the emitted light is measured by a photo counter may be used.

[0055]

As described in detail above, according to the concentration measuring apparatus of the present invention, the amount of electricity converted by the sensor element for measuring a substance to be measured is converted into the distance between the biological substance and the interfering substance in the sensor element. Excluding the quantity of electricity based on the biochemical reaction, the concentration of the substance to be measured can be obtained based on the quantity of electricity reflecting only the concentration of the substance to be measured. As a result, the influence of the interfering substance can be eliminated, and the concentration of the substance to be measured can be accurately obtained, and the measurement accuracy can be improved.

[Brief description of the drawings]

FIG. 1 is a schematic perspective view of a biosensor 1 as a main component of a concentration measuring device 20 according to an embodiment.

FIG. 2 is a block diagram showing an electrical configuration of a concentration measuring device 20 according to the embodiment.

FIG. 3 is a flowchart illustrating a concentration measurement routine performed by the concentration measurement device 20.

FIG. 4 is a graph showing a glucose calibration curve Kgcal in which a glucose concentration and a sensor output current value are associated with each other.

FIG. 5 is a schematic perspective view of a biosensor 70 used for measuring a lactic acid concentration.

FIG. 6 is a graph showing a lactic acid calibration curve Klcal in which a lactic acid concentration is associated with a sensor output current value.

[Explanation of symbols]

 Reference Signs List 1,70 Biosensor 5 Glucose measurement sensor element 7 Ascorbic acid measurement sensor element 9 Uric acid measurement sensor element 11 Bilirubin measurement sensor element 20 Concentration measurement device 30 Device body 35 Connector 40 Electronic control device 5a to 11a , 73a-75a Working electrode 5b-11b, 73b-75b Reference electrode 5c-11c, 73c-75c Counter electrode 5d-11d, 73d-75d Identification layer 70 Biosensor 73 Lactate sensor element 75 Sensor element for urea measurement

[Table 1]

────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI G01N 27/327 G01N 27/46 336G 338 27/30 353U (56) References JP-A-60-211350 (JP, A) JP-A-61-270650 (JP, A) JP-A-63-50748 (JP, A) JP-A-58-5542 (JP, A) JP-A-2-245650 (JP, A) JP-A 64-10165 (JP) , A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 27/416 G01N 25/48 G01N 27/00 G01N 27/26 371 G01N 27/27 G01N 27/327

Claims (1)

(57) [Claims] 1. A concentration measuring apparatus for measuring the concentration of a substance to be measured based on a biochemical reaction between the substance to be measured and a biological substance in a solution to be measured, wherein the biological substance causing a biochemical reaction with the substance to be measured An identification layer that carries the same, converts the progress state of the biochemical reaction that proceeds in the identification layer into an electric quantity, and outputs the electric quantity, and inhibits the electric quantity conversion in the measurement target substance sensor element section. A discriminating layer carrying a biological substance causing a biochemical reaction with an interfering substance to be generated, an interfering substance sensor element unit for converting the progress of the biochemical reaction progressing in the discriminating layer into an electric quantity and outputting the same, In correcting the sensor output from the target substance sensor element section based on the sensor output from the sensor element section, the sensor output from the interfering substance sensor element section A sensor output converting means for converting the biological substance carried on the discriminating layer in the substance sensor element portion into a sensor output when a biochemical reaction has occurred with the interfering substance; and a conversion from the sensor output of the measurement target substance sensor element section. A concentration calculating means for calculating the concentration of the substance to be measured based on the sensor output obtained by subtracting the sensor output.