WO2002025261A1

WO2002025261A1 - Diamond electrode for measuring glucose concentration, and measuring method and apparatus employing the same

Info

Publication number: WO2002025261A1
Application number: PCT/JP2001/008274
Authority: WO
Inventors: Akira Fujishima; Tata Narasinga Rao; Ryuji Uchikado
Original assignee: Center For Advanced Science And Technology Incubation, Ltd.
Priority date: 2000-09-21
Filing date: 2001-09-21
Publication date: 2002-03-28
Also published as: JP4619506B2; AU2001288106A1; JP2002310977A

Abstract

A diamond electrode with which the concentration of glucose can be specifically and easily measured in a short time. The diamond electrode comprises conductive diamond and fixed thereto at least one member selected from the group consisting of nickel, copper, gold, platinum, palladium, ruthenium, iridium, cobalt, and rhodium. This diamond electrode has the property of electrochemically and specifically oxidizing glucose on the surface thereof. The concentration of glucose can be measured based on this property.

Description

Description: Diamond electrode for measuring glucose concentration, and measuring method and apparatus using the same

[Background of the Invention]

Technical field

The present invention relates to a diamond electrode for measuring the concentration of glucose that is clinically or foodically important, a method for measuring the glucose concentration using the same, and a device therefor.

Background art

Diamond is inherently the resistivity is an insulating material of about ¹ 0 1 ³ Ω cm, obtaining conductivity by de one-flop traces impure product. This conductive diamond is expected to have various uses. One of them is utilization as an electrode for electrochemical use. When conductive diamond is viewed as an electrode for electrochemical use, it has the excellent features of having a wide potential window and extremely low background current. In addition, it is physically and chemically stable and has excellent durability. Electrodes having a conductive diamond (preferably a thin film thereof) have been commonly referred to as diamond electrodes.

Pioneering research on diamond electrodes was performed by Iwaki et al. (Iwaki et al., Nuclear Instruments and Methods, 209-210, 1129 (1983)). They studied the properties of single-crystal diamond with surface conductivity imparted by implanting argon-nitrogen ions as an electrically conductive material. At the same time, a cyclic voltammogram in the electrolyte solution was also shown. Subsequently, the properties of polycrystalline diamond electrodes synthesized by gas phase using hot filaments have been reported (Pleskov et al., J. Electroanal. Chem., 228, 19 (1993)).

Some of the present inventors have previously reported the reduction of nitrogen oxides using a diamond electrode synthesized in the vapor phase (Tenne et al., J. Electroanal. Chem. 347, 409 (1993)). In this study, we introduced a p-type semiconductor diamond with boron as a dopant. Monde was used as an electrode. After that, as the diamond electrode, the use of a p-type semiconductor using boron as a dopant or a metal-like conductive diamond having higher conductivity has become mainstream. In the 1990s, diamond electrode research was conducted by several groups, and from 1995 onwards, plasma electrodeposition (PCVD) equipment was used to obtain larger diamond thin films. Research on diamond electrodes has also been seen in the electrochemical field.

By the way, in recent years, the number of patients with diabetes has been steadily increasing. In the past, diagnosis of diabetes was generally performed by measuring blood glucose levels.However, it is a simple method because blood sampling is required and analysis is time-consuming. It was difficult.

On the other hand, in recent years, diabetes diagnosis based on urinary sugar level measurement has come into practical use. This measurement of urinary glucose does not require painful blood collection, and the number of users is expected to increase in the future because of the simplicity of using urine obtained by urination. As a sensor used for measuring such a urinary sugar level, a biosensor using an enzyme electrode such as glucose soxidase is currently known.

In addition, in the field of food as well as in the field of medicine and pharmacy, if glucose concentration can be easily measured, it is considered to be highly useful in food production and nutrition. Thus, if the amount of glucose in a living body or in a sample derived from the living body or in a food can be easily known, it is extremely significant in medicine, pharmaceuticals and food production.

[Summary of the Invention]

The present inventors have recently proposed that a diamond electrode carrying at least one selected from the group consisting of nickel, copper, gold, platinum, palladium, ruthenium, iridium, conoort, and rhodium is specific for glucose. It was found that the amount of the abundance could be quantified from the current value at the oxidation potential. The present invention is based on such findings.

Accordingly, an object of the present invention is to provide a diamond electrode capable of easily and specifically measuring the concentration of glucose in a short time. Another object of the present invention is to provide a method for measuring the concentration of glucose in a test sample using the diamond electrode and an apparatus therefor.

According to a first aspect of the present invention, there is provided a diamond electrode, which is preferably used for measuring glucose concentration. The diamond electrode comprises conductive diamond, nickel, copper , Gold, platinum, palladium, ruthenium, iridium, cobalt, and at least one selected from the group consisting of rhodium.

According to another aspect of the present invention, there is provided a method for measuring the concentration of glucose in a test sample using the above-mentioned diamond electrode.

Prepare the diamond electrode and the counter electrode,

The diamond electrode and the counter electrode are brought into contact with a test sample,

Applying a voltage at which an oxidation reaction occurs on the diamond electrode between the diamond electrode and the counter electrode; measuring a current value under the voltage;

Calculating the concentration of glucose in the test sample from the obtained current value.

Further, according to another aspect of the present invention, there is provided an apparatus for measuring the concentration of glucose in a test sample using the diamond electrode, wherein the apparatus comprises:

The diamond electrode,

A counter electrode,

Means for bringing the diamond electrode and the counter electrode into contact with a test sample,

Means for applying a voltage that causes an oxidation reaction on the diamond electrode between the diamond electrode and the counter electrode;

Means for measuring a current value under the applied voltage;

Means for calculating the concentration of glucose in the test sample from the obtained current value.

According to another aspect of the present invention, there is provided use of the above-mentioned diamond electrode for measuring a glucose concentration. [Brief description of drawings]

1A and 1B are diagrams showing the structure of a diamond electrode, and FIG. 1A is a cross-sectional view of a diamond electrode 1, which is a conductive diamond thin film 3 formed on a substrate 2. Consists of FIG. 1B is a perspective view of the diamond electrode 1 and includes a conductive diamond thin film 3 formed on a base 2 and a nickel thin film 4 formed thereon.

FIG. 2A is a diagram schematically showing a flow injection method used in the measurement method according to the present invention, and FIG. 2B is a diagram showing a basic structure of a flow cell used in the flow injection method.

FIG. 3 is a diagram showing a basic configuration of a measuring device according to the present invention.

FIG. 4 is a scanning electron microscope (SEM) image of the surface of the nickel-supported diamond electrode of the present invention produced in Example 1.

FIG. 5 is a calibration curve of a glucose concentration and a signal current value prepared using the nickel-supported diamond electrode shown in FIG.

FIG. 6 is a scanning electron microscope (SEM) image of the surface of the copper-supported diamond electrode of the present invention produced in Example 2.

7A and 7B are calibration curves of the glucose concentration and the signal current value, which were prepared using the copper-supported diamond electrode shown in FIG. 6, and FIG. 7A shows that the Darcos concentration was 0. FIG. 7B shows a calibration curve in the range of glucose concentration of 0-2 O mg / d1.

[Specific description of the invention]

Test compound

According to the method of the present invention, the concentration of glucose in a sample solution can be selectively known. The present inventors have found that, as described above, glucose is specifically oxidized electrochemically on a diamond electrode on which a catalyst metal such as nickel or copper is supported. It was also confirmed that the current value generated between the electrode and the counter electrode was directly proportional to the concentration of glucose in the system. As a result, it is possible to quantitatively detect glucose in the test sample. I got it.

The test sample may be blood, a body fluid, or the like derived from a living body that is thought to contain glucose, or a food or a diluted solution or suspension of the food. By using urine as a test sample, the diamond electrode according to the present invention can be used for diagnosing diabetes by measuring urine sugar level. In general, the following standards are known for urinary glucose (ie, glucose concentration in urine);

i) It is normal if the urinary sugar level is less than 5 Omg / d1 before meals and less than 10 Omg / d1 after meals,

ii) 100-500 mg / d1 after meals indicates impaired glucose tolerance (boundary type); iii) 5 mg / d1 or more before meals and 50 Omg after meals If / d 1 or more, it is highly possible that the patient has diabetes.

According to the diamond electrode of the present invention, since the urine sugar level (glucose concentration) can be known within the above concentration range, diagnosis of diabetes and impaired glucose tolerance (boundary type) can be performed without performing painful blood sampling. Can be performed easily in a short time. As described above, the diamond electrode according to the present invention, which can easily and easily measure the urine concentration of glucose in a short time, is useful for diagnosis and treatment thereof. Furthermore, glucose is contained in many foods, and it is significant in food production and nutrition to be able to easily know its concentration.

Diamond electrode and its production

Diamond is inherently a good insulator. However, by adding Group 3 or Group 5 impurities, it becomes semiconductor-like or metal-like conductive. In the present invention, diamond having a semiconductor-metal-like conductive property is used as an electrode. Examples of the substance added to impart such a conductive property include Group 3 and 5 elements as described above, more preferably include boron, nitrogen, and phosphorus, and most preferably include boron or nitrogen. is there. Amount of material that will be added to impart the conductivity, yo are suitably determined within the range that can impart conductivity to the diamond bur, for example ^{^{1 X 1 0- 2 ~1 0- 6}} Ω cm about It is preferable to add an amount that gives conductivity. Generally, the amount of the substance added to impart the conductivity is controlled by the amount added in the process of manufacturing the conductive diamond. The diamond electrode according to the present invention uses the conductive diamond as an electrode, and further supports a catalytic metal thereon. As the catalyst metal, nickel, copper, gold, platinum, palladium, ruthenium, iridium, cobalt, orifice, and combinations thereof are used, and preferably copper and / or nickel. The diamond electrode on which these catalytic metals are supported has an extremely interesting property that when an electrochemical reaction is carried out in water, oxygen is not generated by the electrolysis of water as an oxidation reaction, and the glucose oxidation reaction specifically occurs. Had. This property is considered to be obtained if the catalytic metal is supported on conductive diamond in any form. In addition, any one of the above catalyst metals may be supported alone on conductive diamond, or two or more of the above catalyst metals may be supported on conductive diamond as separate metals or as alloys. Is also good.

According to a preferred aspect of the present invention, nickel is supported on at least a part of the surface of the conductive diamond in the form of a thin film. The nickel thin film has a strong tendency to be formed only partially, not entirely, on the conductive diamond. Even if the nickel thin film is not formed on the entire surface of the conductive diamond electrode, there is no problem in detecting glucose. The weight of nickel per unit area is not particularly limited, but is preferably about 5 to 100 g / cm ² , and more preferably about ²⁰ to 30 g / cm ² .

According to another preferred embodiment of the present invention, copper can be supported on conductive diamond in a particulate state. The particle size of the copper particles is not particularly limited, but the particle size of each particle measured by a scanning electron microscope (SEM) is preferably from 10 to 50 nm, more preferably from 50 to 30 nm. O nm. Copper weight per unit area is not particularly limited, 5 is preferably 1 0 0〃G / cm ² or so, yo Ri is preferably 2 0 to 3 0 g / cm ² approximately.

Although the conductive diamond itself carrying a catalytic metal such as nickel or copper can be used as an electrode without depending on the support of the substrate, according to a preferred embodiment of the present invention, conductive diamond is provided on the substrate. It is preferable to form a thin film of the conductive diamond thin film and to carry a catalytic metal on the conductive diamond thin film and connect a conductive wire to form an electrode. Substrates include Si (eg, single crystal silicon), Mo, W, Nb, Ti, Fe, Au, Ni, Co, A 1 2 0 3, S i C, S i 3 N 4, Zr_〇 _2, MgO, black lead, single crystal diamond, cBN, quartz glass and the like, in particular monocrystalline silicon , Mo, W, Nb, Ti, SiC; and the use of single crystal diamond are preferred. The electrode of this embodiment will be further described with reference to FIG. FIG. 1A is a cross-sectional view of a nickel-supported diamond electrode 1, which comprises a conductive diamond thin film 3 formed on a base material 2 and a nickel thin film 4 supported thereon in a thin film form. Further, a conductive wire 6 is connected to the conductive diamond thin film 3 via, for example, a gold coating 5. FIG. 1B is a perspective view of the diamond electrode 1, which is composed of a conductive diamond thin film 3 formed on a base material 2 and a nickel thin film 4 supported thereon. The conducting wire 6 is connected via a gold coating 5 to be used as an electrode. Further, as shown by a dotted line in FIG. 1B, a protective film 7 may be formed on the outer edge and the side surface of the outermost surface of the diamond electrode 1 on the nickel thin film 4 side. The protective film 7 is preferably formed of an insulating resin such as an epoxy resin, thereby ensuring an electrochemically stable state on the side surface of the end of the diamond electrode 1, the gold coating 5, and the conductive wire 6. To enable more stable and accurate measurements.

The thickness of the conductive diamond thin film is not particularly limited, but is preferably about 1 to 100 111, more preferably about 5 to 5 Oim.

Further, according to a preferred embodiment of the present invention, the diamond electrode according to the present invention can take the form of a micro-hole electrode. The concept of a microelectrode is already known, and in the present invention, a diamond electrode in the form of a microelectrode is formed by sharply cutting the end of a fine wire such as Pt, W, Mo, etc. This means a structure in which a conductive diamond thin film is formed on the terminal surface.

According to a preferred embodiment of the present invention, the conductive diamond thin film is preferably produced by a chemical vapor deposition method. The chemical vapor deposition method is a method of synthesizing a substance by chemically reacting a gaseous raw material in a gas phase, and is generally called a CVD (Chemical Vapor Deposition) method. This method is widely used in the semiconductor manufacturing process, and can be used for the production of the conductive diamond thin film of the present invention with appropriate modification. Chemical vapor synthesis of diamond is performed by using a mixture of hydrogen and a carbon-containing gas such as methane as a raw material gas, exciting it with an excitation source, supplying it to a substrate, and depositing it.

Excitation sources include hot filament, microwave, high frequency, DC glow discharge, DC arc discharge, and combustion flame. It is also possible to adjust the nucleation density by combining a plurality of them, and to enlarge and uniform the area.

As a raw material, many types that contain a carbon, decomposition by the excitation source is excited, active carbon, such as CC _2, and the coal hydrocarbon radicals such as _{_{_{CH CH 2 CH 3 C 2 H}}} 2 The resulting compound is available. Specific preferred _{_{_{examples, CH 4 C 2 H 2 C2H4}}} , C .oH 16 s COCF 4, CH 3 OH C 2 H 5 OH (CHs) 2 CO as a liquid, graphite as a solid, fullerene and the like are found listed as a gas .

In the gas phase synthesis method, the addition of a substance that imparts conductivity to diamond is performed, for example, by placing a disk of the added substance in the system, exciting it in the same way as the carbon source material, and introducing the added substance into the carbon gas phase. The method can be carried out by adding an additive substance to a carbon source in advance, introducing the additive substance into the system together with the carbon source, exciting with an excitation source, and introducing the additive substance into the carbon gas phase. According to a preferred embodiment of the present invention, the latter method is preferred. In particular, when a liquid such as acetone or methanol is used as the carbon source,

(B ₂ 0 ₃₎ method dissolved to obtain an boron source is, is easy to control the concentration of boron preferred because either One is simple. For example, in the case of adding boron to a carbon source in a gas phase synthesis method, about 1012,000 ppm is generally used, and about 100,000 to 10,000 ppm is preferable.

According to a preferred embodiment of the present invention, the production of the conductive diamond thin film is preferably performed by a plasma chemical vapor deposition method. This plasma-enhanced chemical vapor synthesis method has the advantage that the activation energy for causing a chemical reaction is large and the reaction is fast. Furthermore, according to this method, it is possible to generate a chemical species that does not exist at a high temperature thermodynamically, and to perform a reaction at a low temperature. There have already been several reports on the production of conductive diamond thin films by plasma enhanced chemical vapor deposition, including some of the present inventors.

(For example, Yano et al. J. Electrochem. Soc 145 (1998) 1870) It is preferable to carry out according to the method described in the report.

In the present invention, the method of supporting the catalyst metal on the conductive diamond is not particularly limited, and can be performed by various methods.

According to a preferred aspect of the present invention, when nickel is supported on conductive diamond, a solution containing nickel ions (eg, nickel nitrate solution, nickel sulfate ammonium solution) is dropped on the conductive diamond and dried. This can be done by: The nickel thus supported on the conductive diamond is generally in the form of a partially formed thin film. Heating may be performed during drying. Also, nickel may be supported on conductive diamond by an electrodeposition method.o

According to a preferred embodiment of the present invention, when copper is supported on a conductive diamond, the conductive diamond is immersed in a solution containing copper ions, and a negative voltage is applied to the conductive diamond. This can be done by depositing copper on the top. As such a solution, a solution in which copper sulfate is dissolved in sulfuric acid is preferable. Thus, the copper supported on the conductive diamond is generally in the form of particles o

In order to increase the adhesion of the copper particles to the conductive diamond, it is preferable to oxidize the diamond by applying a positive voltage to the conductive diamond in sulfuric acid before depositing copper. . The anodic oxidation of such diamond is preferably carried out in sodium hydroxide of about 0.1 M at +2.5 V (vs. SCE) for 1 hour or more.

Measuring method and device therefor

According to the present invention, the diamond electrode electrochemically oxidizes glucose electrochemically, the diamond electrode is used as a working electrode, and the current value generated between the diamond electrode and the counter electrode is directly proportional to the glucose concentration in the system. Quantitatively detects glucose in the test sample using Since the measurement is performed by knowing the current value, the measurement time is short and simple, and the method according to the present invention is extremely advantageous in that the glucose concentration can be easily known in a short time.

Further, the diamond electrode of the present invention can oxidize compounds other than glucose. It is very sensitive to glucose. As far as the present inventors know, in many samples from organisms or foods where glucose is expected to be present, even if substances other than glucose are present, they do not react with them, and only glucose is used as a diamond electrode. Oxidizes. Therefore, the measuring method according to the present invention enables specifically measuring only glucose.

However, if a substance other than glucose that is responsive to the diamond electrode is present in the test sample or is likely to be present, perform a pretreatment to separate the responsive substance other than glucose prior to glucose measurement. Preferably, glucose is distinguished from other responsive substances and subjected to the measurement method according to the present invention. In particular, since the diamond electrode of the present invention may have a portion where the conductive diamond is exposed without supporting the catalytic metal, it is preferable to separate in advance the substance which responds to the conductive diamond. For example, when urine is used as a test sample, uric acid and ascorbic acid other than glucose can react with the diamond electrode. Therefore, it is desirable to separate uric acid and ascorbic acid from urine prior to glucose measurement. . The method of separating substances that can react with the diamond electrode other than glucose is not particularly limited, but a separation method using a separation column (for example, liquid chromatography, preferably high performance liquid chromatography (HPLC)) is rapid and rapid. This is preferable because the responsive substance can be easily separated. In addition, a coulometric cell, a pre-electrolysis treatment and the like can be preferably mentioned.

In addition, according to the measuring method using the diamond electrode according to the present invention, glucose is reduced to about 0.1 mg / d 1 to saturation concentration, preferably about 0.1 to; I 5 O mg / d 1, more preferably about 0.1 1 to: L 0 O mg / d 1, more preferably about 0.1 to 20 mg / dl, most preferably about 0.1 S mgZd!

In the glucose concentration measuring method according to the present invention, the electrochemical system for quantifying glucose can be a general electrochemical system except that a diamond electrode is used as a working electrode.

That is, in the glucose concentration measuring method according to the present invention, a diamond electrode is used as a working electrode and is brought into contact with a test sample together with a counter electrode. A voltage that causes an oxidation reaction is applied on the diamond electrode, and the current value under this voltage is measured. Then, the concentration of glucose in the test sample is calculated from the obtained current value. As described above, the current value generated between the diamond electrode and the counter electrode due to the oxidation of glucose is directly proportional to the concentration of glucose in the system. Therefore, once the relationship between the current value and the glucose concentration at a certain voltage value is determined, the glucose concentration in the test sample corresponding to the obtained current value can be easily known from the relationship. That is, in a preferred embodiment of the present invention, a calibration curve between the glucose concentration and the current value is created in advance, and the calibration curve is compared with the obtained current value to determine the glucose concentration. You can know.

In the present invention, as the counter electrode, platinum, carbon, stainless steel, gold, diamond,

S n 0 ₂ use of the like are preferable.

According to a preferred embodiment of the present invention, the surface area of the counter electrode is preferably at least 10 times the surface area of the diamond electrode, more preferably at least 100 times, and even more preferably at least 100 times. ,. Thereby, high measurement accuracy can be realized without using a reference electrode. Preferable examples of such a counter electrode having a large surface area include platinum black obtained by further plating platinum on a platinum electrode.In the present invention, a diamond electrode serving as a working electrode, a counter electrode The voltage applied during this period is not limited as long as an oxidation reaction of glucose occurs on the diamond electrode, but from the viewpoint of measurement efficiency and accuracy, this applied voltage is a voltage that gives a beak current for glucose oxidation. Preferably, there is. Here, the peak current can be determined as a voltage that gives a maximum current value by, for example, cyclic voltammetry. However, if the background current increases significantly near the peak current, the voltage closest to the beak current within a range where the background current does not adversely affect the measurement, that is, the background current and the oxidation It is preferable to select a voltage at which the difference from the current generated by the reaction is the largest, since a stable and highly sensitive measurement can be performed. Further, according to another preferred embodiment of the present invention, the voltage giving the maximum current value can be obtained by a rotating electrode method or a microelectrode method. The rotating electrode method or the microelectrode method is advantageous in that the possibility of measurement errors due to measurement conditions and the like can be further eliminated. Further, according to a preferred embodiment of the present invention, the reference electrode is brought into contact with the test sample, and the absolute value of a voltage at which an oxidation reaction occurs on the diamond electrode is controlled between the diamond electrode and the counter electrode. It is preferable from the viewpoint of measurement accuracy. A well-known reference electrode can be used, and a saturated calomel electrode (SCE), a standard hydrogen electrode, a silver-silver chloride electrode, a mercury-mercury chloride electrode, a palladium hydrogen electrode and the like can be used.

In the measuring method according to the present invention, the electrochemical system can be a general electrochemical system except that a diamond electrode is used as a working electrode. According to a preferred embodiment of the present invention, The solution is preferably flowed through the system as a carrier at a constant flow rate, and a test sample is injected into the carrier solution to perform the measurement, preferably by a flow injection method using a flow cell.

An outline of the flow injection method using a flow cell is as shown in FIG. 2A. A carrier solution is injected into the flow cell 21 from a carrier solution reservoir 8 by a pump 9. A test sample inlet 10 is provided between the pump 9 and the flow cell 21 so that the test sample can be injected into the carrier solution. The carrier solution passed through the flow cell 21 is collected in the waste liquid reservoir 11. If pretreatment for separating responsive substances other than glucose is performed prior to glucose measurement, use a separation column or pre-electrolyzer between the test sample inlet 10 and the flow cell 21. A device (not shown) is provided.

The basic structure of the flow cell 21 is as shown in FIG. 2B. The flow cell 21 is configured so that the diamond electrode 1, the counter electrode 22 and the reference electrode 23 are exposed in the flow path 24 through which the carrier solution and the test sample flow, and can be brought into contact with the test sample. In FIG. 2B, the counter electrode 22 extends in a direction perpendicular to the paper surface. The diamond electrode 21 basically has the structure shown in FIG. 1. The nickel thin film 4 in FIG. 1 is exposed in the flow path 24 and comes into contact with the carrier solution and the test sample. The carrier solution enters from the inlet 25 of the flow channel 24, flows as indicated by the arrow in the figure, and reaches the outlet 26. Between the wire A of the diamond electrode 1 and the wire B of the counter electrode 22, a voltage that causes glucose to oxidize on the diamond electrode 1, preferably, a peak current or a background current does not adversely affect the measurement. Apply the voltage closest to the peak current within the lower range. The measurement is performed as follows. First, the flow of only the carrier solution into which the test sample is not injected is passed to minimize and stabilize the so-called background current. The diamond electrode is characterized by a low background current. Next, the test sample is injected from the test sample inlet 10. This injection may be performed continuously, or an amount sufficient to measure the peak current may be injected at one time. When glucose is contained in the test sample, glucose is oxidized on the diamond electrode 1, and the current value associated with the oxidation reaction can be measured. From the current value, the concentration of glucose is determined. In the present invention, when nickel and Z or copper are used as the catalyst metal, nickel and / or Z at the time of measuring the current value is determined by the following equation. It is thought that the oxidation reaction with glucose occurs when the valence of copper is 3.

N i (II) → N i (III) + e- (OH-medium)

Ni (III) + glucose Ni (II) + product

Cu (II) → Cu (III) + e- (in OH-)

Cu (III) + glucose → Cu (II) + product

That is, it is considered that trivalent nickel, Z or copper, rather than divalent, contributes to the oxidation reaction with glucose. Therefore, when the valence of nickel, nickel, or copper is not 3 (for example, divalent) in measuring the current value, it is desirable to set the valence to trivalent.

In order to make nickel and / or copper trivalent, it is effective to set the pH of the test sample to be high, and the preferred pH is 10 to 14, more preferably pH 11 to 13. According to a preferred embodiment of the present invention, it is preferable to perform a step of adjusting the pH of the test sample to 10 to 14 before measuring the current value. The pH may be adjusted by using a solvent adjusted to pH 10 to 14 as a carrier of the flow injection.

According to another preferred embodiment of the present invention, it is also preferable that the diamond electrode 1 in the flow cell 21 is in the form of a microelectrode. According to another preferred embodiment of the present invention, the diamond electrode 1 may be a rotating electrode.

Further, according to another aspect of the present invention, there is provided an apparatus for measuring the concentration of glucose in a test sample. The basic configuration of this device is as shown in FIG. Figure 3 In the device, the power supply and ammeter 31 are connected to the conductor A connected to the diamond electrode 1, the conductor B connected to the counter electrode 22 and the reference electrode 23 from the flow cell shown in Fig. 2. Conductor C is connected. The power supply / ammeter 31 serves as a means for applying a voltage at which an oxidation reaction occurs on the diamond electrode between the diamond electrode and the counter electrode, and a means for measuring a current value under the applied voltage. is there. That is, a voltage that causes glucose to be oxidized on the diamond electrode 1 is applied between the diamond electrode 1 and the counter electrode 22 by the power supply / ammeter 31, and the glucose is oxidized on the diamond electrode 1. This current value is sent to the current value comparison / concentration calculation device 32. A calibration curve data 33 is sent to the device 32, and the current value sent from the power supply / ammeter 31 is compared with the calibration data data in the device to determine the glucose concentration. calculate. That is, a means is provided for calculating the concentration of glucose in the test sample from the obtained current value. The obtained glucose concentration is displayed on the display device 34.

[Example]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

In the following examples, either a saturated calomel electrode (SCE) or an Ag / AgC1 electrode was used as a reference electrode, but almost the same potential was obtained with any of these electrodes. Therefore, it is considered that there is no substantial difference in measurement.

Example 1: Measurement of glucose concentration using nickel-supported diamond electrode Preparation of nickel-supported diamond electrode

First, a conductive diamond thin film was prepared by a microwave plasma assisted CVD method using a microwave CVD film forming apparatus manufactured by AS TeX. Specifically, it is as follows.

The surface of a silicon substrate (Si (100)) was polished with 0.5 μm diamond powder, and then set on a substrate holder of a CVD film forming apparatus. A mixture of acetone and methanol (mixing ratio 9: 1 (volume ratio)) was used as the carbon source. Furthermore, this mixture Boron oxide (B ₂ 0 ₃₎ at the object, through a pure hydrogen gas to a mixture of boron / carbon ratio 10 ⁴ ppm and qs dissolved c carbon source, was replaced with dissolved gas. Meanwhile, pure hydrogen gas was flowed through the chamber at a flow rate of 532 cc / min, and the pressure was set at 115 torr (115 x 133.322 Pa). Next, a microwave of 2.45 GHz was injected into the chamber and discharged, so that the output was adjusted to 5 KW. After confirming that the device was stabilized, pure hydrogen gas was flowed as a carrier gas through the carbon source mixture at a flow rate of 15 ccZ, and introduced into the champer to form a film. The deposition rate was 1-4 m / hour. Film formation was performed to a thickness of about 30 zm. Although the substrate was not heated, it was observed that the temperature was about 850-950 ° C in the steady state.

When a Raman spectrum of the obtained conductive diamond thin film was taken, only a single peak was observed at 1333 cm- ¹ . The electric conductivity was about 10- ³ Ω cm. When the concentration of boron contained in the diamond thin film was measured, it was 10,000 ppm.

Next, nickel was carried on the obtained conductive diamond thin film as follows. The conductive diamond thin film, 1 nickel nitrate N i (N0 ₃₎ of Omm ₂₎ about 100 il dropped, and the mixture was dried for 2-3 hours at 100 ° C. Thereafter, the conductive diamond thin film is rinsed lightly with pure water, and then swept 200 times in a 0.2 M aqueous sodium hydroxide solution at a potential range of 0.0 to 0.8 V (vs. SCE) to obtain an electric current. The catalytic metal was activated chemically.

The surface of the obtained nickel-supported diamond electrode was observed with a scanning line electron microscope (SEM) and analyzed by electron optical spectroscopy (XPS). Figure 4 shows an SEM image of the diamond electrode surface obtained. In the figure, the white portion is nickel. As shown in FIG. 4, it was observed that nickel was deposited on the conductive diamond in a thin film form. In XPS measurement, a peak derived from nickel was observed at around 855 eV.

Selection of measurement conditions

In order to select the optimal glucose concentration measurement conditions for the fabricated nickel-supported diamond electrode, cyclic volummetry and hydro- We performed Namiki Bol evening measurement.

First, a cyclic voltammogram with a potential sweep rate of 10 OmVs- ¹ was measured in a 0.2 M sodium hydroxide solution (about pH 13.3). Specifically, the measurement was performed at room temperature using a Hokuto Denko HA-502 potentiostat, a Hokuto Denko HB-11 function generator 1, and a Riken Denshi xy recorder in a glass cell. At this time, the nickel-carrying diamond electrode obtained as described above was used as a working electrode, and a platinum foil was used as a counter electrode. A saturated calomel electrode (SCE) was used as a reference electrode. As a result, it was confirmed that the peak potential was around +0.5 V. In addition, no significant decrease in beak was observed after multiple cycles.

Next, the hydrodynamic voltammetry of 20 1 of a solution in which 1 mM of glucose was dissolved in a 0.2 M sodium hydroxide solution was measured at a flow rate of 1 ml / s. Specifically, the potential was fixed, and after the background current at that potential reached a steady state, a sample solution was injected and the signal current was measured. At this time, the potential was varied between 0.05 and 0.1 V between 0.0 and 10 V (against Ag / AgCl). As a result, the signal current increased with potential, but the background current significantly increased around +0.5 V (vs. AgZAgCl). Therefore, the optimal detection potential for glucose concentration measurement is +0.45 V (vs. Ag / AgCl), which provides the highest signal current within a range that is low enough that background current does not adversely affect the measurement. Selected.

Measurement of glucose concentration

Using the nickel-supported diamond electrode obtained as described above, the concentration of glucose in urine was measured. Specifically, it is as follows.

(a) Preparation of equipment

The apparatus shown in FIGS. 2 and 3 was prepared. At this time, in order to separate uric acid and ascorbic acid, which are responsive substances other than glucose, from urine, a high-performance liquid chromatography (H PLC) column (HAMILTON Company; RCX-10 (Part No. 79940)), Total column length; 2 40 mm, column diameter; 4. lmm). In addition, a BAS (LC-4C) potentiostat and Scientific Software, Inc. EZ Chrom Elite Client / Server were used as the measuring device shown in FIG. At this time, the diamond electrode obtained as described above was used as the working electrode 1, and a platinum foil was used as the counter electrode 22. An AgZAgC 1 electrode was used as the reference electrode 23.

(b) Preparation of calibration curve

First, in preparing the calibration curve, it is assumed that urine is diluted 10-fold with 30 mM sodium hydroxide (about pH 12.5) for measurement. It was set in the range of 10 Omg / d1. Here, the urine was diluted 10 times, assuming that the actual concentration in urine was 0 to 100 Omg / d1, and within the range of the preferable measurement concentration of the diamond electrode of the present invention. This is to enable measurement. Next, glucose solutions of various concentrations were prepared within a concentration range of 0 to 10 OmgZd1.

The resulting glucose solutions of each concentration were injected into the test sample inlet 10 and reacted at a detection potential of +0.45 V via the device shown in Fig. 3 while passing through the flow cell 21 via the HPLC column. Current was detected (averometric measurement). At this time, a 3 OmM aqueous sodium hydroxide solution (about pH 12.5) was used as the carrier solution. A calibration curve was created by obtaining the relationship between the obtained current value and the glucose concentration. The obtained calibration curve was as shown in FIG. As shown in FIG. 5, in the range of 0 to 10 Omg / d1, the detected current also changed in proportion to the concentration.

(c) Determination of glucose

First, the following three sample solutions were prepared:

i) Sample solution A: Sample solution obtained by diluting human urine 10-fold with 3 OmM sodium hydroxide (pH about 12.5)

ii) Sample solution B; a solution obtained by adding 1 Omg / d1 of glucose to the above sample solution A; iii) Sample solution C: A solution obtained by adding 5 Omg / d1 of glucose to the above sample solution A.

For each of the sample solutions A to C, a signal current value was determined by a flow injection method using the flow cell shown in FIG. Test sample solution The reaction current was detected at a detection potential of +0.45 V through the device shown in Fig. 3 while the liquid was injected into the sample inlet 10 and passed through the flow cell 21 through the HP LC column. Trick measurement). At this time, a 3 OmM sodium hydroxide solution was used as a carrier solution. The applied voltage was +0.45 V with the silver chloride electrode. The signal current value was measured after the time at which the signal current derived from glucose, which had been checked in advance, had elapsed.

The obtained signal current values were 4 nA for sample solution A, 14 nA for sample solution B, and 72 nA for sample solution C. The glucose concentration of each solution was calculated by applying these signal current values to the calibration curve shown in Fig. 5, and the results were as follows: 2 mg Zdl for sample solution A, 12 mg 7 d 1 for sample solution B, and 52 mg / d 1 for sample solution C. Met. From these results, 2 Omg / d1, which is 10 times the concentration of the sample solution A, was a normal value, and the subject was determined not to have diabetes. In addition, the result that the concentration of the sample solution B was higher than that of the sample solution A by the amount of glucose added, and the result that the concentration of the sample solution C was similarly higher than that of the sample solution A by the amount of glucose added was obtained.

Example 2: Preparation of calibration curve using copper-supported diamond electrode

Fabrication of copper-supported diamond electrode

First, in the same manner as in Example 1, a conductive diamond thin film was prepared by a microwave plasma assisted CVD method.

Next, copper was supported on the obtained conductive diamond thin film as follows. First, +2 V was applied to a conductive diamond thin film in a 0.1 M sodium hydroxide solution.

A voltage of (to SCE) was applied for 15 minutes to oxidize. This oxidized conductive diamond thin film is immersed in a solution of ImM copper sulfate dissolved in 5 OmM sulfuric acid to generate a current of 6 OA at a voltage of -0.12 V (vs. SCE). After flowing for 22 minutes, copper particles were deposited on the conductive diamond. The density of copper on the obtained conductive diamond film was 36 μg / cm ² . Then, the conductive diamond thin film is lightly rinsed with pure water, and then 0.2-0.8 V in 0.2 M aqueous sodium hydroxide solution.

The catalytic metal was electrochemically activated by sweeping 200 times in the potential range (vs. SCE). The surface of the obtained copper-supported diamond electrode was observed with a scanning electron microscope (SEM). Figure 6 shows an SEM image of the diamond electrode surface obtained. In the figure, the white particles are copper. As shown in FIG. 6, it was observed that copper was attached to the conductive diamond in the form of particles.

Selection of measurement conditions

Cyclic voltammetry and hydrodynamic voltammetry were performed on the fabricated copper-supported diamond electrode to select the optimal measurement conditions for glucose concentration.

First, a cyclic voltammogram at a potential sweep rate of 10 OmVs ¹ was measured in a 0.1 M sodium hydroxide solution (about pH 13.0). Specifically, the measurement was performed at room temperature using a Hokuto Denko HA-502 potentiostat, a Hokuto Denko HB-11 function generator, and a Riken Denshi xy recorder in a glass cell. At this time, the copper-supported diamond electrode obtained as described above was used as a working electrode, and a platinum foil was used as a counter electrode. An Ag / AgC1 electrode was used as a reference electrode. As a result, it was confirmed that the beak potential was around +0.65 V (vs. Ag / AgC 1). In addition, no significant decrease in beak was observed even when the cycle was repeated several times.

Next, the hydrodynamic voltammetry of a solution in which 1 mM glucose was dissolved in a 0.1 M sodium hydroxide solution (about pH 13.0) was measured. Specifically, the potential was fixed, and after the background current at that potential became a steady state, the sample solution was injected and the signal current was measured. At this time, the potential was changed between 0.0 and 0.1 V (against Ag / AgC 1) at intervals of 0.05 to 0.1 V. As a result, the signal current increased with the potential, but the background current increased markedly at +0.8 V (vs. Ag / AgCl). As described above, since the background current is very low at +0.65 V (vs. Ag / Ag C1) where a beak current is obtained, this +0.625 V is the optimum detection potential for glucose concentration measurement. V (vs. Ag / AgCl) was selected. Creating a calibration curve

Using the copper-supported diamond electrode obtained as described above, the concentration of glucose in urine was measured. Specifically, it is as follows.

(a) Preparation of equipment

An apparatus was prepared in the same manner as in Example 1, except that the above-mentioned copper-supported diamond electrode was used as the working electrode 1, the saturated calomel (SCE) electrode was used as the reference electrode 23, and no separation column was provided. did.

(b) Preparation of calibration curve

First, in preparing the calibration curve, the concentration range of glucose to be measured was set in the range of 0 to 2 O mg / d1. Next, glucose solutions of various concentrations were prepared within this concentration range.

The obtained glucose solutions of each concentration were injected into the test sample inlet 10 and passed through the flow cell 21 while being referred to as +0.625 V (vs. SCE) via the device shown in FIG. The reaction current was detected at the detection potential (amperometric measurement). At this time, a 0.1 M aqueous sodium hydroxide solution (about pH 13.0) was used as a carrier solution. The relationship between the obtained current value and the glucose concentration was obtained, and a calibration curve was created. The obtained calibration curves were as shown in FIGS. 7A and 7B. As shown in FIG. 7A, in the range of 0 to 0.2 mgZd1, the detected current also changed in proportion to the concentration. In addition, as shown in FIG. 7B, in the range of 0 to 2 Omg / d1, the detected current also changed almost in proportion to the concentration.

Thus, a calibration curve having a proportional relationship to the glucose concentration was obtained for the copper-supported diamond electrode as in the case of the nickel-supported diamond electrode. Therefore, it can be seen that the concentration of glucose can be measured very quickly and easily with a copper-supported diamond electrode, as well as with a nickel-supported diamond electrode.

Claims

The scope of the claims

1. Conductive diamond and

A diamond electrode comprising, supported thereon, at least one selected from the group consisting of nickel, copper, gold, platinum, palladium, ruthenium, iridium, conoreto, and rhodium.

2. The diamond electrode according to claim 1, wherein nickel, copper, or copper is supported on the conductive diamond.

3. The diamond electrode according to claim 1, wherein nickel is supported on at least a part of the surface of the conductive diamond in a thin film form.

4. The diamond electrode according to any one of claims 1 to 3, wherein copper is supported on the conductive diamond in the form of particles.

5. The diamond electrode according to any one of claims 1 to 4, wherein the conductive diamond is a diamond thin film made conductive by mixing a Group 3 or Group 5 element.

6. The conductive diamond according to any one of claims 1 to 5, wherein the conductive diamond is a diamond thin film made conductive by mixing one or more elements selected from the group consisting of boron, nitrogen, and phosphorus. Diamond electrode.

7. The diamond electrode according to claim 6, wherein the conductive diamond has a diamond thin film made conductive by mixing boron.

8. A glucose concentration sensor comprising the diamond electrode according to any one of claims 1 to 7, which is used for measuring glucose concentration.

9. A method for measuring the concentration of glucose in a test sample, comprising:

Providing a diamond electrode according to any one of claims 1 to 8 and a counter electrode,

Calculating the concentration of glucose in the test sample from the obtained current value Comprising the method.

10. The step of calculating the concentration of glucose in the test sample from the obtained current value is to compare the previously prepared calibration curve between the concentration of glucose and the current value with the obtained current value. The method according to claim 9, which is performed by:

11. The method according to claim 9 or 10, wherein the pH of the test sample is from 10 to 14.

12. The method according to any one of claims 9 to 11, further comprising a step of adjusting the pH of the test sample to 10 to 14 before measuring the current value.

13. The method according to any one of claims 9 to 12, wherein the nickel and / or copper has a valence of 3 when measuring the current value.

14. The method c according to any one of claims 9 to 13, further comprising, prior to the measurement of the current value, setting a valence of the nickel, nickel, or copper to 3.

15. The method according to any one of claims 9 to 14, wherein the test sample is urine.

16. The method of claim 15, further comprising the step of separating uric acid and ascorbic acid from the urine prior to the glucose measurement.

17. The method according to claim 16, wherein said separation is performed by a separation column.

18. The method according to any one of claims 9 to 17, wherein the voltage at which an oxidation reaction occurs on the diamond electrode is a voltage that gives a peak current.

19. The voltage according to any one of claims 9 to 18, wherein a voltage at which an oxidation reaction occurs on the diamond electrode is a voltage at which a difference between a background current and a current caused by an oxidation reaction is largest. The described method.

20. The method further comprising: bringing a reference electrode into contact with a test sample, and controlling an absolute value of a voltage at which an oxidation reaction occurs on the diamond electrode between the diamond electrode and the counter electrode. Item 10. The method according to any one of Items 9 to 19.

21. The method according to any one of claims 9 to 19, wherein a counter electrode having a surface area of at least 10 times the surface area of the diamond electrode is used.

22. An apparatus for measuring the concentration of glucose in a test sample,

A diamond electrode according to any one of claims 1 to 7, A counter electrode,

Means for bringing the diamond electrode and the counter electrode into contact with a test sample; means for applying a voltage causing an oxidation reaction on the diamond electrode between the diamond electrode and the counter electrode;

Means for measuring a current value under the applied voltage;

23. A reference electrode is further provided.

Means for bringing the reference electrode into contact with the test sample;

Means for controlling an absolute value of a voltage at which an oxidation reaction occurs on the diamond electrode, between the diamond electrode and the counter electrode;

The apparatus according to claim 22, comprising:

24. The apparatus according to claim 22, wherein the surface area of the counter electrode is at least 10 times the surface area of the diamond electrode.

25. Use of the diamond electrode according to any one of claims 1 to 7 for measuring glucose concentration.