JP6713364B2 - Ascorbic acid responsive electrode and biosensor - Google Patents

Description

The present invention relates to electrodes and biosensors for measuring ascorbic acid concentration in a sample.

Ascorbic acid (vitamin C) is ingested by diet, absorbed through the small intestine, and widely distributed to organs and tissues in the body. Biochemically, it is involved in collagen synthesis, carnitine synthesis, adrenocortical hormone synthesis, catecholamine synthesis, lipid peroxide degradation, active oxygen degradation, etc., and plays an important role in vivo. In recent years, due to the strong reducing property of vitamin C, anticancer action, immunity enhancement, and beautiful skin and whitening effects have been noted, and it has been applied to high-concentration vitamin C drip therapy.

Examples of the measurement of ascorbic acid include measurement for diagnosis of ascorbic acid deficiency and measurement of ascorbic acid in food. In addition, in high-concentration vitamin C drip therapy, immediate monitoring of vitamin C concentration in blood is essential, and in autologous blood glucose measurement in diabetes, since ascorbic acid in blood influences the glucose measurement value, There is a need to correct the glucose level based on the measured ascorbic acid concentration.

Patent Document 1 discloses a method for measuring ascorbic acid as a micronutrient in food by a high performance liquid chromatography (HPLC) method.
Patent Document 2 discloses, as an example of ascorbic acid measurement, a biosensor having an electrode including a detection layer containing an electron mediator such as ascorbate oxidase and a ferricyanide compound as a constituent element.

Special table 2005-517926 JP 09-243591

Regarding the ascorbic acid measurement by the high performance liquid chromatography (HPLC) method of the prior art document 1, there is a cost problem because the device becomes expensive. In addition, the above-mentioned device is often installed in a large-scale medical facility or inspection center, and in a facility where the device is not installed, since the inspection is outsourced, real-time measurement is not possible, but it is particularly high. Concentrated vitamin C drip therapy is a problem because it requires real-time measurement in consideration of the effects of metabolism and excretion of vitamin C.
In prior art document 2, a biosensor enables simple and immediate (real-time) measurement, but the use of an enzyme catalyst such as ascorbate oxidase causes a problem of deterioration of measurement accuracy due to nonspecific catalytic reaction. there were. Further, since the storage stability of the sensor is poor due to the denaturation of the enzyme, it is necessary to increase the prescription amount of the enzyme, which causes a problem of high cost.
In view of the above, an object of the present invention is to perform ascorbic acid immediate measurement accurately, at low cost, and easily.

As a result of extensive studies conducted by the inventor to solve the above problems, an ascorbic acid-responsive electrode capable of simply measuring ascorbic acid without using an enzyme catalyst such as conventional HPLC or ascorbic acid oxidase was developed. I found it. That is, the present invention includes an electrode and a detection layer that is in contact with the electrode and includes a non-catalytic electron acceptor that exchanges electrons from ascorbic acid, and in the detection layer, an oxidation reaction occurs between the electron acceptor and the electrode. An ascorbic acid responsive electrode is provided which is characterized by being performed.
The present invention also provides a biosensor including the ascorbic acid responsive electrode.
The present invention is also characterized by reacting an ascorbic acid-containing sample with the biosensor, applying an oxidation potential to the ascorbic acid-responsive electrode to measure a response current, and calculating an ascorbic acid concentration based on the response current. And a method for measuring ascorbic acid concentration.
The present invention is also a biosensor, controlling the voltage application to the biosensor, a control unit, obtained by applying a voltage to the biosensor, detecting an ascorbic acid response current, a detection unit, and from the current value. Provided is a measuring device including an arithmetic unit that calculates the concentration of ascorbic acid and an output unit that outputs the calculated concentration of ascorbic acid.
The “electron acceptor” is a compound capable of donating and accepting an electron from a compound having a function of an electron donor capable of donating an electron. The "non-catalyst" means that the biocatalyst such as an enzyme or an organelle does not have a function of a biocatalyst that catalyzes a chemical reaction, or the biocatalyst has a small function. Ascorbic acid has a strong electron donor function as a reducing substance. Utilizing this property, we have realized the transfer of electrons from ascorbic acid to electron acceptors. Furthermore, since the electron acceptor that has exchanged electrons from the electron donor becomes a reductant, it is possible to cause an electrochemical oxidation reaction on the electrode. The present invention has been completed by these various functions.

Since the ascorbic acid-responsive electrode of the present invention does not use an enzyme, it is possible to measure ascorbic acid with high accuracy while suppressing deterioration of measurement accuracy due to nonspecific catalytic reaction. Further, since there is no problem of storage stability due to denaturation of the enzyme protein, the cost can be suppressed. Therefore, according to the present invention, it becomes possible to perform an immediate measurement of ascorbic acid with high accuracy, at low cost, and easily.

FIG. 1 is a process diagram showing an example of a method for manufacturing a biosensor according to an embodiment of the present invention, and (A) to (E) show schematic diagrams of the biosensor in each process. FIG. 2 is a schematic diagram showing an aspect of the measuring apparatus of the present invention. FIG. 3 is a flowchart showing one aspect of a measurement program using the measurement device of the present invention. FIG. 4 is a graph showing the response current for 10 seconds after reacting samples containing various concentrations of ascorbic acid with the biosensor of the present invention and applying a voltage. FIG. 5 is a graph showing the results of plotting the ascorbic acid concentration and the bias (%) of the response current value of each concentration with respect to the response current value of the ascorbic acid concentration of 0 mg/dL after voltage application of 2, 4 or 8 seconds. Is.

(Composition of electrodes)
The ascorbic acid responsive electrode of the present invention, an electrode,
A sensing layer containing a non-catalytic electron acceptor that is in contact with the electrode and transfers and receives electrons from ascorbic acid;
In the detection layer, an oxidation reaction is performed between the electron acceptor and the electrode.
Note that, unlike the conventional enzyme electrode in which the detection layer contains an enzyme and an electron acceptor, in the ascorbic acid-responsive electrode of the present invention, the detection layer contains the electron acceptor but does not contain the enzyme.

(electrode)
The electrode is not particularly limited as long as it is a conductive material. For example, a metal material such as gold (Au), platinum (Pt), silver (Ag) and palladium (Pd), or graphite, carbon nanotube, graphene. , A carbon material typified by carbon such as mesoporous carbon. The electrodes are formed, for example, on an insulating substrate. The insulating substrate is made of thermoplastic resin such as polyetherimide (PEI), polyethylene terephthalate (PET), polyethylene (PE), various resins (plastics) such as polyimide resin and epoxy resin, glass, ceramic, paper. It is formed of such an insulating material. The size and thickness of the electrodes and the insulating substrate can be set appropriately.

(Electronic acceptor)
The electron acceptor may be any compound that receives an electron from ascorbic acid, is reduced, and is reoxidized at the electrode and has no catalytic action. Examples thereof include ruthenium compounds, potassium ferricyanide, cytochrome C, and pyrroloquinoline quinone (PQQ). , NAD ⁺ , NADP ⁺ , a copper complex, phenazine methosulfate and its derivatives, an osmium complex, and the like, and these may be used alone or in combination of two or more.
Among these, a complex composed of an oxidized metal atom and a ligand is preferable, and an oxidized ruthenium complex composed of trivalent ruthenium (Ru(III)) and a ligand is more preferable.

(Oxidized ruthenium complex)
Examples of the ligand of the oxidized ruthenium complex include nitrogen-containing ligands such as ammonia, bipyridine, imidazole, amino acids, phenanthroline, and ethylenediamine, and halogen ligands. Note that these may be combined to form a mixed ligand.

The ruthenium ammonia complex preferably includes the following compounds.
[Ru(NH ₃ ) ₅ X] ⁿ⁺
Here, examples of X include NH ₃ , halogen ions, CN, pyridine, nicotinamide, bipyridine, and H ₂ O, and among these, NH ₃ or halogen ions (for example, Cl ⁻ , F ⁻ , Br ⁻ , I ^- ) is preferred. N+ in the above chemical formula represents the valence of the oxidized ruthenium(III) complex and is appropriately determined depending on the type of X.

Examples of the bipyridine ruthenium complex include, for example, [Ru(bipyridine) ₃ ].
, [Ru(4,4′-dimethyl-2,2′-bipyridine) ₃ ], [Ru
(4,4'-diphenyl-2,2'-bipyridine) ₃ ], [Ru(4,4'-diamino-2,2'-bipyridine) ₃ ], [Ru(4,4'-dihydine) ₃ ].
Droxy-2,2'-bipyridine) ₃ ], [Ru(4,4'-dicarbo
xy-2,2'-bipyridine) ₃ ], [Ru(4,4'-dibromo-2,
2'-bipyridine ₃ ], [Ru(5,5'-dimethyl-2,2'-bipyridine) ₃ ], [Ru(5,5'-diphenyl-2,2'-bipyriri).
Dine) ₃ ], [Ru(5,5'-diamino-2,2'-bipyridine) ₃ ], [Ru(5,5'-dihydroxy-2,2'-bipyridine) ₃ ], [
Ru(5,5'-dicarbox-2,2'-bipyridine) ₃ ], [Ru(
5,5'-dibromo-2,2'-bipyridine) ₃ ] and the like.

Examples of the imidazole ruthenium complex include, for example, [Ru(imidazole) ₆ ].
, [Ru(4-methyl-imidazole) ₆ ], [Ru(4-phenyl-
[imidazole) ₆ ], [Ru(4-amino-imidazole) ₆ ], [Ru(4-hydroxy-imidazole) ₆ ], [Ru(4-carboxy-im).
idazole) ₆ ], [Ru(4-bromo-imidazole) ₆ ] and the like.

The content of the electron acceptor in the detection layer can be appropriately determined depending on the type of the measurement sample, etc., but is preferably 10 mmol to 100 mol, more preferably 10 mmol to 50 mmol, and particularly preferably 15 mmol to 20 mmol per 1 cm ² of the surface area of the detection layer. Is.

In addition, the sensing layer can contain a binder in addition to the electron acceptor. In the electrode manufacturing process, a sensing layer preparation liquid can be prepared as a screen printing compatible ink containing an electron acceptor, a binder and a solvent or a dispersion medium, and patterning printing is performed on a substrate such as a resin by a screen printing method. By forming the electrodes, the electrode production efficiency can be improved and the difference between the sensors can be reduced.

As the binder that can be used in the detection layer, for example, a resin binder such as a butyral resin type or a polyester resin type may be used, but it is possible to use the layered inorganic compound disclosed in re-table 2005/043146. preferable. Examples of the layered inorganic compound include compounds in which polyhedrons of an inorganic material are connected in a plane to form a sheet structure, and the sheet structures are further layered to form a crystal structure. Examples of the shape of the polyhedron include a tetrahedron sheet and an octahedron sheet, and specific examples thereof include a Si tetrahedron and an Al octahedron. Examples of such layered inorganic compounds include layered clay minerals, hydrotalcite, bentonite, smectite, vermiculite, halloysite, kaolin minerals and mica. As such a layered inorganic compound, for example, a commercially available product can be used, and trade name LUCENTITE SWN, trade name LUCENTITE SWF (synthetic hectorite), trade name ME (fluorine mica), Kunimine manufactured by Coop Chemical Co., Ltd. Smoketon SA (synthetic saponite) manufactured by Kogyo Co., Ltd., Tixopy W (synthetic hectorite) manufactured by Kyowa Chemical Industry Co., Ltd., Kyoward 500 (synthetic hydrotalcite) manufactured by Lapo, a product manufactured by Lapo Laponite (synthetic hectorite), natural bentonite manufactured by Nacalai Tesque, Inc., and multi-gel (bentonite) manufactured by Toyoshun Mining Co., Ltd. can be used. The content of the binder in the detection layer is, for example, in the range of 0.003 to 30 mg per 1 cm ² of the area of the detection layer.

Further, the detection layer may additionally contain additives such as a buffering agent and a surfactant, and the content thereof can be set appropriately.
The buffer is preferably an amine-based buffer, and examples thereof include Tris, ACES, CHES, CAPSO, TAPS, CAPS, Bis-Tris, TAPSO, TES, Tricine and ADA, and preferably ACES and Tris. More preferably it is ACES. These substances may be used alone or in combination of two or more. Further, as the buffer, a buffer having a carboxyl group is also preferable, and examples thereof include acetic acid-Na acetate buffer, malic acid-Na acetate buffer, malonic acid-Na acetate buffer, succinic acid-Na acetate buffer, and the like. Among these, succinic acid-Na acetate buffer is preferred.
Surfactants may include Triton X-100, sodium dodecyl sulfate, perfluorooctane sulfonic acid or sodium stearate. Examples thereof include alkylaminocarboxylic acid (or a salt thereof), carboxybetaine, sulfobetaine, phosphobetaine and the like.

(Method of manufacturing electrode)
The ascorbic acid-responsive electrode of the present invention is produced, for example, as follows. That is, a metal layer that functions as an electrode is formed on one surface of the insulating substrate. For example, by depositing a metal material by screen printing, physical vapor deposition (PVD, for example, sputtering), or chemical vapor deposition (CVD) on one surface of a film-like insulating substrate having a predetermined thickness (for example, about 100 μm), A metal layer having a desired thickness (for example, about 30 nm) is formed. Instead of the metal layer, an electrode layer made of a carbon material can be formed. Next, the sensing layer can be disposed on the electrode by coating the electrode with a sensing layer preparation liquid containing an electron acceptor and drying.

The ascorbic acid-responsive electrode of the present invention can be used in a biosensor for measuring the concentration of ascorbic acid contained in a sample. The sample is not particularly limited as long as it contains the substance to be measured, but a biological sample is preferable and examples thereof include blood and urine.

(Biosensor)
The biosensor as an ascorbic acid sensor includes an electrode serving as a counter electrode together with the ascorbic acid responsive electrode (working electrode) of the present invention. The counter electrode may be any one that can be generally used as a counter electrode of a biosensor, and for example, a carbon electrode formed by screen printing, physical vapor deposition (PVD, for example, sputtering), or chemical vapor deposition (CVD). A filmed metal electrode or a screen printed silver/silver chloride electrode can be used. Alternatively, a three-electrode system having a silver/silver chloride electrode, a carbon electrode formed by screen printing, or a metal electrode formed by physical vapor deposition or chemical vapor deposition as a reference electrode may be used.

When a sample containing ascorbic acid is brought into contact with the surface (working electrode) of the ascorbic acid-responsive electrode of the present invention, the electrons generated by the oxidation reaction of ascorbic acid are transferred to the electron acceptor, and the electron acceptor is reduced. Then, by applying an oxidation potential to the working electrode, the reduced type electron acceptor is oxidized on the surface of the working electrode, thereby generating an oxidation current. The ascorbic acid concentration in the sample can be measured based on this current value.

Hereinafter, an example of the biosensor of the present invention will be described based on FIG. 1A to 1E are perspective views showing a series of steps for manufacturing a biosensor. The biosensor of the present invention is not limited to the following modes.

As shown in FIG. 1(E), the biosensor A includes a substrate 11, an electrode system including a working electrode 12 having a lead portion 12a and a counter electrode 13 having a lead portion 13a, an insulating layer 14, and an electron acceptor. It is provided with a detection layer 16 containing a binder and a buffer solution, a spacer 18 having an opening, and a cover 19 having a through hole 20. As shown in FIG. 1B, a detection unit 15 is provided on the substrate 11, and the working electrode 12 and the counter electrode 13 are arranged on the detection unit 15 in parallel in the width direction of the substrate 11. Has been done. One end of each of the electrodes serves as a lead portion 12a, 13a, respectively, and the other end of the detection portion 15 is arranged to be vertical (FIG. 1A). Further, an insulating portion is provided between the working electrode 12 and the counter electrode 13. As shown in FIG. 1B, an insulating layer 14 is laminated on the substrate 11 having such an electrode system except for the lead portions 12a and 13a and the detecting portion 15, and the insulating layer 14 is provided. A detection layer 16 is laminated on the detection unit 15 where is not laminated. Then, on the insulating layer 14, as shown in FIG. 1D, a spacer 18 having an opening at a portion corresponding to the detecting portion 15 is arranged. Further, a cover 19 having a through hole 20 in a part corresponding to the opening is arranged on the spacer 18 (FIG. 1(E)). In this biosensor A, the space portion of the opening portion and the space portion sandwiched between the detection layer 16 and the insulating layer 14 and the cover 19 serves as a sample supply portion 17 having a capillary structure. Then, the through hole 20 becomes an air hole for inhaling the sample by the capillary phenomenon.

Such a biosensor can be manufactured as follows, for example.

First, as shown in FIG. 1A, an electrode system including a working electrode 12 having a lead portion 12a and a counter electrode 13 having a lead portion 13a is formed on a substrate 11.

Subsequently, as shown in FIG. 1B, an insulating layer 14 is formed on the substrate 11 on which the electrode systems 12 and 13 are formed. This insulating layer is formed on the substrate 11 excluding the lead portions 12a and 13a of the electrodes and the detection portion 15 that forms an electron acceptor and the like. The insulating layer 14 can be formed, for example, by printing an insulating paste in which an insulating resin is dissolved in a solvent on the substrate 11 and subjecting the insulating paste to heat treatment or ultraviolet treatment. Examples of the insulating resin include polyester, butyral resin, phenol resin, and the like, and examples of the solvent include carbitol acetate, a dibasic acid ester mixed solvent (DBE solvent), and the like.

Next, as shown in FIG. 1C, the detection layer 16 is formed on the substrate 11 and the electrodes 12 and 13 in the detection unit 15 in which the insulating layer 14 is not formed. The detection layer 16 can be formed, for example, by preparing a dispersion liquid in which an electron acceptor, a buffer and a binder are dispersed, dispensing the dispersion liquid into the detection unit 15, and drying the dispersion liquid. As the solvent used for preparing the dispersion liquid, for example, water, buffer alcohol, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO) and the like can be used.

Next, as shown in FIG. 1D, a spacer 18 is arranged on the insulating layer 14. As shown, the spacer 18 has an opening at a portion corresponding to the detection layer 16. As the material of the spacer 18, for example, a resin film or tape can be used. Further, if it is a double-sided tape, not only the adhesion with the insulating film 14 but also the cover 19 described later can be easily adhered. In addition to this, for example, the spacer may be formed by means such as resist printing.

Next, as shown in FIG. 1(E), a cover 19 is placed on the spacer 18. The material of the cover 19 is not particularly limited, but various plastics can be used, for example, and transparent resin such as PET is preferable.

The method of using the biosensor A will be described with reference to an example in which the sample is whole blood, the measurement target is ascorbic acid, and the electron acceptor is a ruthenium (III) compound.

First, the whole blood sample is brought into contact with one end of the opening 17 of the biosensor A. The opening 17 has the capillary structure as described above, and the cover 19 corresponding to the other end is provided with the air hole 20, so that the sample is sucked into the inside by the capillary phenomenon. The aspirated sample reaches the surface of the detection layer 16 provided on the detection unit 15. Then, the ascorbic acid in the sample reaching the surface reacts with the ruthenium (III) compound in the detection layer 16. Specifically, the ruthenium (III) compound is reduced by the electrons transferred by the oxidation reaction of ascorbic acid, which is the measurement target, to generate the ruthenium (II) compound. By applying a positive potential to the electrode, electrons are exchanged between the ruthenium (II) compound present in the detection layer 16 and the electrode located under the detection layer 16, and an oxidation current flows. .. Based on this, the ascorbic acid concentration can be measured.

Specifically, after a lapse of a certain time from the supply of the whole blood sample, a voltage is applied between the counter electrode 13 and the working electrode 12 by the means for applying the voltage, and the reduction type in the detection layer in contact with the electrode is applied. The above ruthenium (II) compound is electrochemically oxidized to the ruthenium (III) compound, and the oxidation current at that time is detected by means of measuring the electric signal via the lead portion 12a of the working electrode 12. Since the value of this oxidation current is proportional to the ascorbic acid concentration in the sample, the ascorbic acid concentration in the sample can be obtained by calculating this as the ascorbic acid concentration in the calculating means.
The voltage applied to the working electrode 12 may be a positive voltage with respect to the counter electrode, but is preferably 10 to 700 mV, 50 to 500 mV, or 100 to 400 mV.
After the sample is brought into contact with the electrode system, the voltage may be applied to the electrode system after being kept in a non-application state for a predetermined time, or the voltage may be applied to the electrode system at the same time when the sample is contacted. The holding time in the non-application state is, for example, 30 seconds or less, or 10 seconds or less.

(apparatus)
Next, the measuring device of the present invention will be described with reference to the drawings. Here, one example of the ascorbic acid measuring device has been illustrated, but the measuring device of the present invention is not limited to the following embodiments. FIG. 2 shows a configuration example of main electronic components housed in the measuring device B. A control computer 28, a potentiostat 29, and a power supply device 31 are provided on a board 30 housed in the housing.
In terms of hardware, the control computer 28 includes a processor such as a CPU (central processing unit), a recording medium such as a memory (RAM (Random Access Memory), ROM (Read Only Memory)), and a communication unit. By including the program stored in the recording medium (for example, ROM) in the RAM and executing the program, the processor functions as a device including the output unit 21, the control unit 22, the calculation unit 23, and the detection unit 24. .. The control computer 28 may include an auxiliary storage device such as a semiconductor memory (EEPROM, flash memory) or a hard disk.

The control unit 22 controls the timing of voltage application, the applied voltage value, and the like.
The power supply device 31 has a battery 26 and supplies power for operation to the control computer 28 and the potentiostat 29. The power supply device 31 can be placed outside the housing.
The potentiostat 29 is a device that keeps the potential of the working electrode constant with respect to the reference electrode, and is controlled by the control unit 22 to use the terminals CR and W between the counter electrode and the working electrode of the ascorbic acid sensor 27. A predetermined voltage is applied, the response current of the working electrode obtained at the terminal W is measured, and the measurement result of the response current is sent to the detection unit 24.

The calculator 23 calculates the concentration of the substance to be measured from the detected current value and stores it. The output unit 21 performs data communication with the display unit 25, and transmits the calculation result of the concentration of the measurement target substance by the calculation unit 23 to the display unit 25. The display unit 25 can display, for example, the calculation result of the ascorbic acid concentration received from the measuring device B on the display screen in a predetermined format.

FIG. 3 is a flowchart showing an example of ascorbic acid concentration measurement processing by the control computer 28. When the CPU (control unit 22) of the control computer 28 receives the start instruction of the ascorbic acid concentration measurement, the control unit 22 controls the potentiostat 29 to apply a predetermined voltage to the working electrode, and then the working electrode The measurement of the response current is started (step S01). The detection of the mounting of the sensor on the measuring device may be an instruction to start the concentration measurement.

Next, the potentiostat 29 receives a response current obtained by applying a voltage, that is, a charge transfer rate-determining current based on the transfer of electrons derived from the substance to be measured (ascorbic acid) in the sample to the electrode, for example, 1 from voltage application. The steady current after 20 seconds is measured and sent to the detector 24 (step S02).

The calculation unit 23 performs a calculation process based on the current value to calculate the ascorbic acid concentration (step S03). For example, the calculation unit 23 of the control computer 28 holds in advance a calculation formula of the ascorbic acid concentration or calibration curve data of the ascorbic acid concentration, and calculates the ascorbic acid concentration using these calculation formulas or calibration curves.

The output unit 21 transmits the calculation result of the ascorbic acid concentration to the display unit 25 through the communication link formed with the display unit 25 (step S04). After that, the control unit 22 detects the presence or absence of a measurement error (step S05), and if there is no error, ends the measurement,
The ascorbic acid concentration is displayed on the display. If there is an error, the error is displayed, and then the process according to the flow of FIG. It is also possible to store the calculation result in the calculation unit 23, call the calculation result later, and display it on the display unit for confirmation. Although the measurement error detection (step S05) is performed by the control unit 22 after the calculation result is transmitted to the display unit 25 (step S04), the order of these steps can be changed.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to the following embodiments.

(1) Preparation of biosensor As shown in FIG. 1, the biosensor was prepared by the following procedure. First, as shown in FIG. 1A, a PET substrate (length 50 mm, width 6 mm, thickness 250 μm) was prepared as the insulating substrate 11 of the ascorbic acid sensor, and one surface thereof was screen-printed to A carbon electrode system including a working electrode 12 and a counter electrode 13 each having a lead portion was formed.

Next, as shown in FIG. 1B, an insulating layer 14 was formed on the electrodes. First, the insulating resin polyester was dissolved in a solvent carbitol acetate so as to be 75% (wt) to prepare an insulating paste, which was screen-printed on the electrode. The printing conditions were a 300 mesh screen and a squeegee pressure of 40 kg, and the printing amount was 0.002 mL per 1 cm ² of the electrode area. Screen printing was not performed on the detection unit 15 and the lead units 12a and 13a. Then, heat treatment was performed at 155° C. for 20 minutes to form the insulating layer 14.

Further, as shown in FIG. 1C, a ruthenium compound layer 16 was formed on the detection portion 15 where the insulating layer 14 was not formed. First, synthetic smectite of 0.3% (wt) (trade name "LUCENTITE SWN" manufactured by Coop Chemical Co., Ltd.), ruthenium compound of 6.0% (wt) ([Ru (NH 3) 6] Cl 3, Dojin A ruthenium compound liquid (pH 7.5) containing sodium acetate and succinic acid was prepared. 1.0 μL of the ruthenium compound solution was dispensed to the detection unit 15. The surface area of the detection unit 15 was about 0.6 cm ² , and the surface area of the electrodes 12 and 13 in the detection unit 15 was about 0.12 cm ² . Then, this was dried at 30° C. to form the ruthenium compound layer 16.

As shown in FIG. 1D, a spacer 18 having an opening was arranged on the insulating layer 14. Further, as shown in FIG. 1(E), a cover 19 having a through hole 20 to be an air hole was arranged on the spacer 18 to manufacture a biosensor. Since the space of the opening of the spacer 18 sandwiched between the cover 19 and the insulating layer 14 has a capillary structure, this is used as the sample supply unit 17.

(2) Measurement of target component Next, ascorbic acid having a hematocrit (Ht) value of 42%, containing ascorbic acid (ASA) so as to have a predetermined concentration (0, 3, 6, 100, 300, or 600 mg/dL) Sample 1 of whole blood was prepared.

Then, each ascorbic acid sample was supplied from the sample supply unit of the biosensor and reacted, then a voltage of 200 mV was applied for 10 seconds, and the current value was measured. The current value at 2, 4, or 8 seconds was sampled with the voltage applied as the reference, and the bias (%) of each concentration was plotted against the response current value of the ascorbic acid concentration and the ascorbic acid concentration of 0 mg/dL. The acid (ASA) concentration dependence was confirmed.

(3) Results “Response characteristics of ascorbic acid”
FIG. 4 shows the relationship between current and voltage application time in the electrochemical measurement of ascorbic acid. It was confirmed that a current profile with an increased response current was obtained as the concentration of ascorbic acid increased. Further, in the plot of the bias (%) of each concentration with respect to the response current value of the ascorbic acid concentration and the ascorbic acid concentration of 0 mg/dL in FIG. 5, good ascorbic acid concentration dependency was confirmed at each sampling time. Although no data is shown, the same tendency can be confirmed even when the vertical axis of the graph is the current value. Although whole blood is used for the measurement of ascorbic acid, reducing substances such as uric acid and glutathione are present in whole blood. In the present invention, even in the presence of the reducing substance, ascorbic acid can be measured in a wide concentration range, and it has been confirmed that the measurement has specificity under the present measurement conditions.

A... Biosensor 11... Substrate 12... Working electrode 12a... Lead portion 13... Counter electrode 13a... Lead portion 14... Insulating layer 15... Detection portion 16... Detecting layer 17... Opening 18... Spacer 19... Cover 20... Air hole B... Measuring device 21... Output unit 22... Control unit 23... Computing unit 24... ..Detection unit 25...display unit 26...battery 27...ascorbic acid sensor 28...control computer 29...potentistat 30...substrate 31...power supply device CR, W ...Terminals

Claims (3)

The sensor has an electrode, a voltage used to oxidize a reduced electron acceptor is applied to measure a response current, and the sensor used in a measuring device for quantifying ascorbic acid contained in a sample based on the response current,
A sample supply unit to which the sample is supplied,
The electron acceptor, which is arranged in the sample supply unit so as to come into contact with the sample, exchanges electrons from the ascorbic acid to be reduced,
The electrode is arranged in the sample supply unit so as to be in contact with the electron acceptor reduced by the ascorbic acid, and has the electrode that oxidizes the electron acceptor reduced by a voltage applied from the measurement device,
The sensor in which the electron acceptor is a ruthenium (III) complex represented by the following chemical formula.
[Ru(NH ₃ ) ₅ X] ⁿ⁺
Here, X represents NH ₃ , halogen ion, CN, pyridine, nicotinamide, bipyridine or H ₂ O, and n+ represents the valence of the ruthenium (III) complex . A method of measuring the concentration of ascorbic acid contained in a sample,
Contacting the sample with an electron acceptor that transfers and receives an electron from ascorbic acid, and reducing the electron acceptor;
A response current is measured by applying a voltage that oxidizes the reduced electron acceptor, and the ascorbic acid concentration contained in the sample is quantified based on the response current,
Measurement method,
The said electron acceptor is a measuring method which is a ruthenium (III) complex shown by the following chemical formula.
[Ru(NH ₃ ) ₅ X] ⁿ⁺
Here, X represents NH ₃ , halogen ion, CN, pyridine, nicotinamide, bipyridine or H ₂ O, and n+ represents the valence of the ruthenium (III) complex . A sample supply unit to which a sample is supplied,
An electron acceptor which is arranged in the sample supply unit so as to come into contact with the sample, and which transfers and receives an electron from the ascorbic acid to be reduced,
A sensor having an electrode arranged in the sample supply unit so as to come into contact with the electron acceptor reduced by the ascorbic acid;
A means for applying a voltage that oxidizes the electron acceptor to the electrode to measure a response current, and quantifying ascorbic acid contained in a sample based on the response current,
A measuring device having
The electron acceptor is a ruthenium (III) complex represented by the following chemical formula.
[Ru(NH ₃ ) ₅ X] ⁿ⁺
Here, X represents NH ₃ , halogen ion, CN, pyridine, nicotinamide, bipyridine or H ₂ O, and n+ represents the valence of the ruthenium (III) complex .

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