JP2013242171A - Concentration measuring apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a concentration measuring apparatus capable of accurately measuring a concentration of a target substance while considering existence of a reducing substance even in a sample including a reducing substance of a high concentration, and having a simple constitution.SOLUTION: A concentration measuring apparatus 1 includes a first sensor 10 having a first electrode 101 and a reaction film 102 formed on the first electrode 101 and a second sensor 20 having a second electrode 201. The reaction film 102 includes an oxidation enzyme for oxidizing a target substance. The first sensor 10 detects a first current including an oxidation current generated by action of the oxidation enzyme to a target substance and an electrolytic current generated by electrode reaction between the reducing substance and the first electrode 101 when a liquid sample contains the reducing substance. The second sensor 20 detects a second current generated by electrode reaction between the reducing substance and the second electrode 201 when the liquid sample contains the reducing substance.

Description

  The present invention relates to a concentration measuring apparatus.

  An example of the concentration measuring device is described in Patent Document 1. Patent Document 1 describes a method of measuring glucose and / or ascorbic acid concentration in a sample containing glucose and ascorbic acid. According to Patent Document 1, this method is characterized in that the current measurement by the enzyme electrode method and the measurement of the luminescence intensity are performed simultaneously, so that even if glucose and ascorbic acid coexist in the sample. It is said that it is possible to accurately measure the glucose concentration by eliminating the influence of ascorbic acid, and simultaneously measure the ascorbic acid concentration in addition to glucose.

JP 2005-140600 A

  However, the technique described in the above-mentioned document measures ascorbic acid concentration by measuring luminescence intensity using luminol, and it is necessary to provide a current measuring instrument and an absorptiometer and two different measuring instruments. Therefore, there exists a problem that the structure of the whole measuring apparatus becomes complicated. In addition, if the concentration of ascorbic acid in the sample is too high compared to the amount of luminol, the emission intensity will be below the detection limit. Therefore, a sample containing a high concentration of ascorbic acid cannot be measured with high accuracy, and the measurement range is limited.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to accurately control the concentration of a target substance in consideration of the presence of the reducing substance even in a sample containing a high concentration of the reducing substance. An object of the present invention is to provide a concentration measuring apparatus that can measure and has a simple configuration.

According to the present invention,
A concentration measuring device for measuring a concentration of the target substance in a liquid sample containing the target substance to be measured,
A first sensor having a first electrode and a reaction film formed on the first electrode;
A second sensor having a second electrode;
Have
The reaction film includes an oxidase that oxidizes the target substance,
The first sensor includes an oxidation current generated by the oxidase acting on the target substance, and an electrolytic current generated by an electrode reaction between the reduction substance and the first electrode when the liquid sample contains a reduction substance. And detecting a first current including
The second sensor is provided with a concentration measuring device for detecting a second current generated by an electrode reaction between the reducing substance and the second electrode when the liquid sample contains the reducing substance.

  According to this invention, the first sensor has two types of sensors (first and second sensors) for detecting the current in the sample, and the first sensor includes the oxidase that oxidizes the target substance. The sensor detects a first current including an oxidation current generated when the oxidase acts on the target substance and an electrolysis current of the reducing substance, and a second sensor detects the electrolysis current of the reducing substance. Accordingly, the concentration of the target substance in the sample can be measured in consideration of the measurement error of the oxidation current affected by the reducing substance. Thereby, since the concentration of the reducing substance can be directly measured from the current generated by the electrode reaction, the target substance can be measured in consideration of the presence of the reducing substance even when a high concentration of the reducing substance is present in the sample. Further, in this configuration, since the concentrations of the target substance and the reducing substance can be detected as currents, the apparatus configuration can be simplified. Therefore, even if the sample contains a high concentration of reducing substance, it is possible to accurately measure the target substance in consideration of the presence of the reducing substance and realize a concentration measuring apparatus having a simple configuration.

  According to the present invention, there is provided a concentration measuring apparatus that can accurately measure the concentration of a target substance in consideration of the presence of the reducing substance even in a sample containing a high concentration of the reducing substance and has a simple configuration. Provided.

It is the top view which showed typically the density | concentration measuring apparatus which concerns on embodiment. It is sectional drawing which showed typically the density | concentration measuring apparatus which concerns on 1st Embodiment. It is a figure explaining the manufacturing method of the density | concentration measuring apparatus which concerns on 1st Embodiment. It is the top view which showed typically the density | concentration measuring apparatus which concerns on embodiment. It is sectional drawing which showed typically the density | concentration measuring apparatus which concerns on 2nd Embodiment. It is a figure explaining the manufacturing method of the density | concentration measuring apparatus which concerns on 2nd Embodiment. It is a figure which shows an example of the output current of the density | concentration measuring apparatus which concerns on 1st Embodiment. It is a figure which shows an example of the output current of the density | concentration measuring apparatus which concerns on 1st Embodiment. It is a figure which shows an example of the output current of the density | concentration measuring apparatus which concerns on 2nd Embodiment.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In all the drawings, the same reference numerals are given to the same components, and the description will be omitted as appropriate.

(First embodiment)
The present embodiment is a concentration measuring device that measures the concentration of a target substance in a liquid sample containing the target substance to be measured. 1 and 2 are diagrams illustrating an example of the concentration measuring apparatus according to the present embodiment. 1 is a plan view of the concentration measuring apparatus 1, and FIG. 2 is a cross-sectional view taken along the line AA ′ of FIG. The concentration measuring apparatus 1 includes a first sensor 10 having a first electrode 101, a reaction film 102 formed on the first electrode 101, a second sensor 20 having a second electrode 201, Have The reaction film 102 includes an oxidase that oxidizes the target substance, and the first sensor 10 includes an oxidation current generated by the action of the oxidase on the target substance, and the reducing substance and the first when the liquid sample contains the reducing substance. A first current including an electrolysis current generated by an electrode reaction with the electrode 101 is detected. The second sensor 20 detects a second current generated by an electrode reaction between the reducing substance and the second electrode 201 when the liquid sample contains the reducing substance.

  The liquid sample is not limited as long as it contains a substance to be measured and a reducing substance that are oxidized by an oxidase, but fruit juice, beverages, or seasonings contain a reducing substance in a high concentration, and therefore the concentration measurement apparatus 1 measures Preferred as a subject. Beverages include soft drinks and alcoholic drinks. Examples of the seasoning include liquid seasonings such as sake, soy sauce and mirin. Further, the liquid sample may be a biological sample such as urine or blood.

  The target substance to be measured includes glucose or glutamic acid. In the liquid sample, the concentration of the substance to be measured is preferably 3 mmol / L or more, and more preferably 5 to 100 mmol / L. For example, when glucose is the target substance, the concentration of glucose in the liquid sample is preferably 54 mg / dL or more, and more preferably 90 mg / dL to 1800 mg / dL. When glutamic acid is used as a target substance, the concentration of glutamic acid is preferably 44 mg / dL or more, more preferably 74 to 1470 mg / dL in the liquid sample.

  Examples of the reducing substance include ascorbic acid, acetaminophen, or a mixture thereof, and those containing at least ascorbic acid are preferable. From the viewpoint of measuring the concentration of the target substance with higher accuracy, the concentration measuring apparatus 1 can preferably measure a liquid sample containing a reducing substance in a range of 0 mmol / L to 60 mmol / L, and a reducing substance in an amount of 2 mmol. / L or more may be included. For example, when ascorbic acid is contained as a reducing substance, the liquid sample preferably contains 0 mg / dL or more and 1000 mg / dL or less of ascorbic acid, and may contain 35 mg / dL or more.

  The first sensor 10 and the second sensor 20 may be members that can be separated separately, but are preferably formed on the same substrate as shown. The substrate 30 is an insulating substrate, and ceramic, plastic, silicon, glass, or a polymer material can be used. Examples of the polymer material include synthetic resins such as polyethylene terephthalate, polyvinyl chloride, and polycarbonate.

  The first sensor 10 includes a first electrode 101, and the second sensor 20 includes a second electrode 201. The first electrode 101 and the second electrode 201 may be either a bipolar electrode or a tripolar electrode, but from the viewpoint of stably obtaining a response current and stabilizing measurement accuracy, Tripolar electrodes are preferred. More preferably, as shown in FIG. 1, the first electrode 101 can be composed of a working electrode W1, a reference electrode R1, and a counter electrode C1. Similarly, the second electrode 201 can be composed of a working electrode W2, a reference electrode R2, and a counter electrode C2. The first electrode 101 and the second electrode 201 can be formed using a conductive material, and examples of the conductive material include carbon, palladium, silver, platinum, gold, copper, nickel, and alloys thereof. A metal material is mentioned. The first electrode 101 and the second electrode 201 may be formed of one type of conductive material, or may be formed of two or more types of conductive material. From the viewpoint of improving measurement accuracy, The first electrode 101 and the second electrode 201 are preferably formed from the same conductive material. Specifically, the working electrodes W1, W2 and the counter electrodes C1, C2 are preferably platinum electrodes, and the reference electrodes R1, R2 are preferably silver-silver chloride electrodes. Thereby, a more stable response current can be obtained, and the measurement accuracy can be further stabilized.

  The first sensor 10 includes a reaction film 102 on the first electrode 101. The reaction film 102 preferably covers part or the entire surface of the first electrode 101. The oxidase included in the reaction film 102 may be any oxidase that can oxidize the target substance in the liquid sample, and is preferably glucose oxidase or glutamate oxidase. When measuring the glucose concentration in a measurement sample, glucose oxidase (GOX) can be used. Moreover, when measuring the glutamate oxidase concentration in a measurement sample, glutamate oxidase can be used.

  In the reaction membrane 102, a fixed GOX can be used. For immobilization of GOX, an entrapment method, a carrier binding method, a crosslinking method, or a composite method combining these can be used. When immobilizing GOX by the inclusion method, GOX can be wrapped with an organic gel or the like. In addition, when GOX is immobilized by the carrier binding method, GOX may be bound to a resin or the like and insolubilized. Among these, those fixed with a crosslinking agent or those fixed by inclusion with an albumin cross-linked membrane are more preferable.

  As the cross-linking agent, a polyfunctional aldehyde compound or a polyfunctional epoxy compound can be used. Can be used. Examples of the multifunctional epoxy compound include sorbitol polyglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, polyethylene glycol diglycidyl ether, and the like. Of these, glutaraldehyde is preferred from the viewpoint of ease of use and ease of control of the film thickness.

  The albumin crosslinked membrane is obtained by crosslinking ovalbumin or animal-derived serum albumin, preferably albumin of bovine calf serum (BSA: Bovine Serum Albumin) using a crosslinking agent. As the cross-linking agent for albumin, the same GOX cross-linking agents as those exemplified above can be used.

  As a method for immobilizing GOX, a known immobilization enzyme production technique can be appropriately employed. For example, GOX can be immobilized by mixing GOX, BSA, and glutaraldehyde in an arbitrary solvent. In this case, examples of the solvent include water, alcohol, HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), TES (N- [tris (hydroxymethyl) -methyl] -2-aminoethane). Good buffers such as sulfonic acid) and PIPES (4-piperazinediethanesulfonic acid) can be used. In this method, an immobilized GOX composed of a mixture of glutaraldehyde-crosslinked GOX in which glutaraldehyde is directly cross-linked to GOX and BSA-encapsulated GOX encapsulated by a crosslinked product of BSA and glutaraldehyde can be obtained.

  In the second sensor 20, an organic film 202 is preferably formed on the second electrode 201. The organic film 202 may be provided with no oxidase, but it is more preferable that the components other than the oxidase are the same as those of the reaction film 102. For example, when the reaction film 102 includes GOX immobilized with BSA and glutaraldehyde, the organic film 202 preferably does not include GOX but includes BSA and glutaraldehyde.

  The first sensor 10 and the second sensor 20 can be integrally covered with a resin film 40 as shown. The resin film 40 can be preferably formed using a resin selected from a fluororesin and a silicon resin, and more preferably formed using a fluororesin. Examples of the fluororesin include polyfluoroperfluorodecyl methacrylate. By covering the first sensor 10 and the second sensor 20 integrally with the resin film 40, the liquid sample is permeated and oxygen necessary for the oxidation reaction is permeated. Further, insoluble components in the liquid sample and excess The reducing agent can be removed.

  Subsequently, a manufacturing method of the concentration measuring apparatus 1 according to the present embodiment will be described using a glucose concentration measuring apparatus as an example with reference to FIG. First, a conductive material is applied on an insulating substrate 30 such as silicon or glass by screen printing, sputtering, or vapor deposition to form the first electrode 101 and the second electrode 201 (FIG. 3). (A)). The film thicknesses of the first electrode 101 and the second electrode 201 can be, for example, 2 μm or less, preferably 1 μm or less, and more preferably 0.5 μm or less. Then, you may form the limiting permeable film (not shown) which permeate | transmits hydrogen peroxide so that the 1st electrode 101 and the 2nd electrode 201 may be covered. The restricted permeable membrane may be selectively formed on the first electrode 101.

  Next, a resist P1 is applied on the first electrode 101 and the second electrode 201 and patterned to selectively expose a formation region of the second sensor 20 (FIG. 3B).

  Next, a solution not containing GOX is applied so as to cover the entire second electrode 201 (FIG. 3C). Examples of the solution not containing GOX include a mixture of BSA and glutaraldehyde and a good buffer such as TES. The thickness of the coating film 202a can be about 0.5 to 1 μm.

  Thereafter, the resist P1 is removed, and unnecessary portions are removed to form the organic film 202 (FIG. 3D).

  Next, a resist P2 is applied on the first electrode 101 and the organic film 202 and patterned to selectively expose the formation region of the first sensor 10 (FIG. 3E).

  Next, a solution containing GOX is applied so as to cover the entire first electrode 101 (FIG. 3F). Examples of the solution containing GOX include a mixed solution of GOX, BSA and glutaraldehyde and a Good buffer such as TES. The thickness of the coating film 102a is preferably the same as the thickness of the coating film 202a, and can be, for example, about 0.5 to 1 μm.

  Thereafter, the resist P2 is peeled off, and unnecessary portions are removed to form the reaction film 102 (FIG. 3G).

  And the resin film 40 is formed so that the 1st sensor 10 and the 2nd sensor 20 may be covered, and the density | concentration measuring apparatus 1 is obtained.

  As shown in FIG. 4, the concentration measuring apparatus 1 thus obtained can further include amplifier circuits 51 and 52, an arithmetic circuit 53, and a display unit 54.

  The amplifier circuit 51 receives the first current detected by the first sensor 10 from the first sensor 10 and amplifies it.

  The amplifier circuit 52 receives the second current detected by the second sensor 20 from the second sensor 20 and amplifies it.

  The arithmetic circuit 53 receives the first current from the amplifier circuit 51, receives the second sensor from the amplifier circuit 52, compares the first current and the second current, and compares the concentration of the target substance in the liquid sample. Is calculated.

  The display unit 54 displays the concentration of the target substance calculated by the arithmetic circuit 53 to the user.

  The arithmetic circuit 53 may calculate the concentration of the reducing substance in the liquid sample from the second current, and the display unit 54 may display this together with the concentration of the target substance.

  The arithmetic circuit 53 may output the calculated result to a printer or the like, or may transmit the result to a terminal via a LAN or a network.

  Subsequently, with respect to the measuring method using the concentration measuring apparatus 1 shown in FIG. 4, measurement of glucose concentration using a liquid sample containing glucose as a target substance to be measured and ascorbic acid as a reducing substance is taken as an example. I will explain.

  When the liquid sample is added to the first sensor 10 and the second sensor 20, in the first sensor 10, glucose in the liquid sample acts on GOX in the reaction film 102, and reacts with dissolved oxygen to generate hydrogen peroxide. Occur. The generated hydrogen peroxide is oxidized on the first electrode 101 to give an oxidation current. In addition, the reducing substance in the liquid sample causes an electrolytic reaction on the first electrode 101 and gives an electrolytic current. The oxidation current and the electrolysis current are amplified as the first current by the amplifier circuit 1 (51), and this is received by the arithmetic circuit 53.

  On the other hand, since GOX does not exist in the second sensor 20, the reaction between glucose and dissolved oxygen does not occur, and the oxidation current of hydrogen peroxide is not detected on the second electrode 201. The reducing substance in the liquid sample causes an electrolytic reaction on the second electrode 201 and gives a second current. This second current is amplified by the amplifier circuit 2 (52), and this is received by the arithmetic circuit 53.

The arithmetic circuit 53 uses the output S ₁ of the first sensor 10 received from the amplifier circuit 51 and the output S ₂ of the second sensor 20 received from the amplifier circuit 52, for example, From (2), the glucose concentration C _glu and the ascorbic acid concentration C _asc are calculated.
S ₁ = (C _glu + C _asc ) * (1-α * C _glu ^a * C _asb ^b ) (1)
_{S 2 = C asc * (1-1} -α * C glu a * C asc b) (2)

  In equations (1) and (2), α is a sensor constant, and a and b are powers of the reaction rate. In the present embodiment, both a and b are in the range of 0.5 to 1.0. a and b are almost constant depending on the structure of the sensor, and α is a number specific to each sensor and can be obtained when it is composed of a single or mixed standard solution. For example, a range of 0.6 to 2.5 It is.

  The calculation result of the arithmetic circuit 53 is output to the display unit 54 and can be displayed. FIG. 4 shows an example in which the glucose concentration is 0.5% and the ascorbic acid concentration is 0.2%.

  Then, the effect of the density | concentration measuring apparatus 1 is demonstrated. According to the concentration measuring apparatus 1, the first sensor 10 includes the first sensor 10 and the second sensor 20, and the first sensor 10 includes the oxidase that oxidizes the target substance. A first current including an oxidation current generated by the enzyme acting on the target substance and an electrolytic current of the reducing substance is detected, and the second sensor 20 detects an electrolytic current of the reducing substance. Accordingly, the concentration of the target substance in the sample can be measured in consideration of the measurement error of the oxidation current affected by the reducing substance. In this configuration, since both the concentration of the target substance and the reducing substance can be detected as current, the apparatus configuration can be simplified. Therefore, a concentration measuring apparatus having a simple configuration can be realized.

  On the other hand, in the concentration measuring apparatus 1, the first sensor 10 and the second sensor 20 can be formed using the same material, except that the reaction film 102 of the first sensor 10 contains an oxidase. preferable. Thereby, both the relationship between the oxidation current of the measurement sample and the concentration of the measurement sample, and the relationship between the electrolytic current of the reducing substance and the concentration of the reducing substance can be linearized. Therefore, it is possible to realize a concentration measuring device with no limitation on the measurement range. For example, the concentration of the target substance can be accurately measured even for a sample containing 2 mmol / L or more of a reducing substance (35 mg / dL or more in the case of ascorbic acid).

(Second Embodiment)
This embodiment is also a concentration measuring device that measures the concentration of a target substance in a liquid sample containing a target substance to be measured and a reducing substance. FIG. 5 is a cross-sectional view showing an example of the concentration measuring apparatus according to the present embodiment. The plan view of the concentration measuring device 2 is the same as FIG. 1, and FIG. 5 is a cross-sectional view taken along the line AA ′ of FIG. The concentration measuring device 2 has the same configuration as the concentration measuring device 1 of the first embodiment except that the first sensor 10 includes an ion exchange membrane 103 on the reaction membrane 102.

  Examples of the ion exchange membrane 103 include a cation exchange membrane and an anion exchange membrane, and a cation exchange membrane is preferable. For example, Nafion (registered trademark) can be used.

  A manufacturing method of the concentration measuring device 2 will be described with reference to FIG. 6 taking a glucose concentration measuring device as an example. First, similarly to the method described in the first embodiment, the first electrode 101 and the second electrode 201 are formed on the insulating substrate 30 (FIG. 6A).

  Next, a resist P3 is applied on the first electrode 101 and the second electrode 201 and patterned to selectively expose the formation region of the first sensor 10 (FIG. 6B).

  Next, a solution containing GOX is applied so as to cover the entire first electrode 201 (FIG. 6C). Examples of the solution containing GOX include a mixed solution of GOX, BSA and glutaraldehyde and a Good buffer such as TES. The thickness of the coating film 102b can be, for example, about 0.5 to 1 μm.

  Thereafter, the resist P3 is peeled off, and unnecessary portions are removed, thereby forming the reaction film 102 (FIG. 6D).

  Next, a resist P4 is applied on the reaction film 102 and the second electrode 201 and patterned to selectively expose the formation region of the second sensor 20 (FIG. 6E).

  Next, a solution not containing GOX is applied so as to cover the entire second electrode 201 (FIG. 6F). Examples of the solution not containing GOX include a mixture of BSA and glutaraldehyde and a good buffer such as TES. The thickness of the coating film 202b is preferably the same as the thickness of the coating film 102a, and can be, for example, about 0.5 to 1 μm.

  Thereafter, the resist P4 is peeled off, and unnecessary portions are removed to form the organic film 202 (FIG. 6G).

  Further, a resist P5 is applied on the reaction film 102 and the organic film 202 and patterned to selectively expose the formation region of the first sensor 10 (FIG. 6H).

  Next, an ion exchange membrane 103b is laminated so as to cover the surface of the reaction membrane 102 (FIG. 6 (i)), and then the resist P5 is removed and unnecessary portions are removed to form the ion exchange membrane 103 (FIG. 6). 6 (j)).

  And the resin film 40 is formed so that the 1st sensor 10 and the 2nd sensor 20 may be covered, and the density | concentration measuring apparatus 2 is obtained.

  Similarly to the concentration measuring apparatus 1, the concentration measuring apparatus 2 obtained in this way can be configured as shown in FIG. 4 further including amplification circuits 51 and 52, an arithmetic circuit 53, and a display unit 54.

The measurement of the sample concentration using the concentration measuring device 2 can be performed in the same manner as the measurement of the sample concentration using the concentration measuring device 1 described in the first embodiment. In this embodiment, when measuring a sample containing glucose as a target substance and ascorbic acid as a reducing substance, the following formulas (3) and (4) are used instead of formulas (1) and (2). Is preferably adopted. That is, in the concentration measuring apparatus 2, the arithmetic circuit 53 uses the output S ₁ of the first sensor 10 received from the amplifier circuit 51 and the output S ₂ of the second sensor 20 received from the amplifier circuit 52, for example. From the following formulas (3) and (4), the glucose concentration C _glu and the ascorbic acid concentration C _asc are calculated.
_{S 1 = C glu * (1} -β * C glu c * C asc d) (3)
_{S 2 = C asc * (1} -β * C glu c * C asc d) (4)

  In equations (3) and (4), β is a sensor constant, and c and d are powers to the reaction rate. In the present embodiment, c can be in the range of 0 to 0.1, and d can be in the range of 0.1 to 1.5. c and d are substantially constant depending on the structure of the sensor, and β is a number unique to each sensor and can be obtained when it is constituted by a single or mixed standard solution. For example, a range of 0.5 to 0.8 It is.

  In the concentration measuring apparatus 2, since components other than the measurement target substance and the reducing substance can be removed by the ion exchange membrane 103, the current caused by the measurement target substance and the reducing substance can be selectively detected. Therefore, the concentration of the target substance can be obtained with higher accuracy. In the case of the concentration measuring apparatus 2 as well, from the viewpoint that the target substance can be measured with higher accuracy, preferably a liquid sample containing a reducing substance of 0 mmol / L or more and 60 mmol / L or less can be measured. / L or more may be included. For example, when ascorbic acid is contained as a reducing substance, the liquid sample preferably contains 0 mg / dL or more and 1000 mg / dL or less of ascorbic acid, and may contain 35 mg / dL or more.

  As mentioned above, although embodiment of this invention was described with reference to drawings, these are the illustrations of this invention, Various structures other than the above are also employable.

Example 1
Using the configuration shown in FIGS. 1 and 2, a glucose concentration measuring apparatus was produced by the method shown in FIG. The substrate 30 was a glass substrate. The first electrode 101 and the second electrode 201 were tripolar electrodes, the working electrodes W1, W2 and the counter electrodes C1, C2 were platinum electrodes, and the reference electrodes R1, R2 were silver-silver chloride electrodes. The reaction film 102 was formed by applying a mixture of GOX and TES buffer fixed with BSA and glutaraldehyde, and the organic film 202 was formed from a mixture of BSA, glutaraldehyde and TES buffer. The resin film 40 was formed using polyfluorofluoro perfluorodecyl methacrylate.

  Using the glucose concentration measurement apparatus of Example 1, the output from the first sensor 10 was examined using an aqueous solution having a glucose concentration of 100, 500 mg / dL and an ascorbic acid concentration of 0, 40, 200 mg / dL as a measurement sample. It was. As a result, the output was linear as shown in FIG. 7, and the output current increased as the glucose concentration increased. In addition, as the ascorbic acid content increased, the output from the first sensor 10 also increased. In FIG. 7, “Glu (Asc0)” indicates a glucose aqueous solution with an ascorbic acid concentration of 0 mg / dL, “Glu (Asc40)” indicates a glucose aqueous solution with an ascorbic acid concentration of 40 mg / dL, and “Glu “(Asc200)” indicates an aqueous glucose solution having an ascorbic acid concentration of 200 mg / dL.

  Using the glucose concentration measuring apparatus of Example 1, the output from the second sensor 20 was examined using 40, 200 mg / dL ascorbic acid aqueous solution as a measurement sample. As a result, the output was linear as shown in FIG. It has been shown that the output current increases as the concentration of is increased.

Example 2
A glucose concentration measuring device was manufactured by the method shown in FIG. 6 using the configuration shown in FIGS. Example 1 was repeated except that the ion exchange membrane 103 was Nafion (registered trademark) manufactured by Sigma-Aldrich.

  Using the glucose concentration measuring apparatus of Example 2, the output from the first sensor 10 was examined using an aqueous solution having a glucose concentration of 100, 500 mg / dL and an ascorbic acid concentration of 0, 40, 200 mg / dL as a measurement sample. It was. As a result, the output was linear as shown in FIG. 9, and the output current decreased as the glucose concentration increased. As the ascorbic acid content increased, the output from the first sensor 10 decreased. In FIG. 9, “Glu (Asc0)” indicates an aqueous glucose solution with an ascorbic acid concentration of 0 mg / dL, “Glu (Asc40)” indicates an aqueous glucose solution with an ascorbic acid concentration of 40 mg / dL, and “Glu “(Asc200)” indicates an aqueous glucose solution having an ascorbic acid concentration of 200 mg / dL.

<Evaluation>
Using the glucose concentration measuring apparatus of Examples 1 and 2, the glucose concentration and ascorbic acid concentration of various beverages were measured. The true glucose concentration was measured using a fully automatic glucose measuring device (glucose ord and stat, manufactured by Arkray), and compared with the glucose concentration calculated by the glucose concentration measuring device of Examples 1 and 2 using this as a reference value. . The glucose concentrations of the glucose concentration measuring apparatuses of Examples 1 and 2 are the output current S ₁ from the first sensor 10 and the output current S ₂ from the second sensor 20. The glucose concentration was calculated by substituting into equations (1) and (2). In the formulas (1) and (2), α = 1.24, a = 1, and b = 1. Moreover, in Example 2, the glucose concentration was calculated by substituting into the above formulas (3) and (4). In formulas (3) and (4), β = 0.65, c = 0.08, and d = 0.3.
Further, ascorbic acid concentration was determined by calculating the output current S ₂ when the ascorbic acid concentration was determined known standard solution (aqueous solution of ascorbic acid) as a reference.
As a comparative example, a glucose measuring device (UG-201, manufactured by Tanita Co., Ltd.) including only the first sensor 10 of Example 2 was used.
The measurement results of the glucose concentration and ascorbic acid concentration of Examples 1 and 2 and Comparative Example are shown in Table 1, and the results of comparing the glucose concentration of Examples 1 and 2 and Comparative Example with the true glucose concentration are shown in Table 2. Shown in

In Tables 1 and 2, * 1 to 6 are as follows.
* 1 Sake: Yamada Nishiki, Sawa no Tsuru Co., Ltd.
* 2 Coke: Coca-Cola and Coca-Cola Company.
* 3 Lemon flavor soft drink: C1000, manufactured by House Wellness Foods.
* 4 Lemon-flavored soft drink (3-fold dilution): C1000 (House Wellness Foods) diluted 3-fold with water.
* 5 Lemon-flavored soft drink (5-fold dilution): C1000 (House Wellness Foods) diluted 5 times with water.
* 6 Fully automatic glucose measuring device (glucose ord and stat, manufactured by ARKRAY)

  As shown in Tables 1 and 2, even in samples containing a high concentration of ascorbic acid such as lemon-flavored soft drinks, the devices of Examples 1 and 2 were able to accurately measure the glucose concentration. In addition, as shown in Table 2, the conventional glucose concentration measuring device including a pair of electrodes has an error of 10% or more with respect to the true glucose concentration, whereas the ascorbic acid concentration measuring electrode is included. In the glucose concentration measuring devices of Examples 1 and 2, the error range was within 6% of the true glucose concentration regardless of the ascorbic acid concentration.

DESCRIPTION OF SYMBOLS 1 Concentration measuring device 2 Concentration measuring device 10 1st sensor 20 2nd sensor 30 Substrate 40 Resin film 51 Amplifying circuit 52 Amplifying circuit 53 Arithmetic circuit 54 Display part 101 1st electrode 102 Reaction film 102a Coating film 102b Coating film 103 Ion exchange membrane 103b Ion exchange membrane 201 Second electrode 202 Organic membrane 202a Coating film 202b Coating film C1 Counter electrode C2 Counter electrode P1 Resist P2 Resist P3 Resist P4 Resist P5 Resist R1 Reference electrode R2 Reference electrode W1 Working electrode W2 Working electrode

Claims (11)

A concentration measuring device for measuring a concentration of the target substance in a liquid sample containing the target substance to be measured,
A first sensor having a first electrode and a reaction film formed on the first electrode;
A second sensor having a second electrode;
Have
The reaction film includes an oxidase that oxidizes the target substance,
The first sensor includes an oxidation current generated by the oxidase acting on the target substance, and an electrolytic current generated by an electrode reaction between the reduction substance and the first electrode when the liquid sample contains a reduction substance. And detecting a first current including
The second sensor is a concentration measuring device that detects a second current generated by an electrode reaction between the reducing substance and the second electrode when the liquid sample contains the reducing substance.   The concentration measuring apparatus according to claim 1, wherein the first sensor and the second sensor are formed on the same substrate.   The concentration measuring apparatus according to claim 1, wherein the first electrode and the second electrode are made of the same material.   The concentration measuring device according to claim 1, wherein the first sensor has an ion exchange membrane on the reaction membrane. The second sensor has an organic film on the second electrode,
5. The concentration measuring apparatus according to claim 1, wherein the organic film is composed of components excluding the oxidase among the components of the reaction film.   The concentration measuring apparatus according to claim 1, wherein the reducing substance includes ascorbic acid or acetaminophen.   The concentration measuring apparatus according to any one of claims 1 to 6, wherein the oxidase is glucose oxidase or glutamate oxidase.   The concentration measuring apparatus according to claim 1, wherein the target substance is glucose or glutamic acid.   The concentration measuring apparatus according to any one of claims 1 to 8, wherein the liquid sample is a biological sample containing fruit juice, beverage, seasoning, urine or blood. The first current detected by the first sensor is received from the first sensor, the second current detected by the second sensor is received from the second sensor, and the first current is detected. And the second current to calculate the concentration of the target substance in the liquid sample,
An output unit that outputs the concentration of the target substance calculated by the calculation unit;
The concentration measuring device according to claim 1, comprising: The calculation unit calculates the concentration of the reducing substance in the liquid sample from the second current when the liquid sample contains a reducing substance,
The concentration measuring apparatus according to claim 10, wherein the output unit outputs a concentration of the reducing substance.

Images