JP5245388B2 - Electrochemical sensor and electrochemical sensor system - Google Patents

Description

  The present invention relates to an electrochemical sensor and an electrochemical sensor system.

Since an electrochemical sensor using an electrochemical reaction can detect a very small amount of current, it is suitable in principle for detecting a very small amount of a chemical substance that causes a redox reaction. Conventionally, it has been proposed to use conductive diamonds formed on a substrate by a vapor phase method as an electrode for electrochemical analysis (Patent Document 1). It has also been proposed to use a platinum electrode as an electrode for electrochemical analysis (Patent Document 2).
Japanese Patent No. 2767124 Japanese Patent Laid-Open No. 61-50054

  However, the conventional electrochemical sensor has a problem that the residual current is large. It is considered that the residual current is caused by an inner sphere reaction including a redox reaction caused by adsorption of a solvent or a solute on the electrode. When this residual current is large, a minute current based on the oxidation-reduction reaction of the detected species is buried in the residual current, so that sufficient detection sensitivity cannot be obtained.

Although platinum and palladium are chemically stable and excellent in catalytic activity, the residual current caused by the inner sphere reaction tends to increase because the free electrons of the metal serve as adsorption sites for water molecules and electrolytes. Further, in the case of a conventional DLC mainly composed of a graphite electrode such as glassy carbon or sp ² bond, the residual current tends to increase because the π bond serves as an adsorption site for water molecules.

  Therefore, an object of the present invention is to further improve the sensitivity of the electrochemical sensor.

  The present invention relates to an electrochemical sensor for measuring the concentration of a chemical substance. The electrochemical sensor according to the present invention includes a working electrode having a conductive DLC film containing 10 to 40 atomic% hydrogen and 30 to 85 atomic% carbon.

  According to the knowledge of the present inventors, it is possible to dramatically increase the sensitivity of the electrochemical sensor by using the conductive DLC film having the specific composition as a working electrode.

  The DLC film may be an n-type semiconductor film further containing 5 to 30 atomic% of an element selected from the group consisting of nitrogen, phosphorus, arsenic, antimony, and bismuth. In this case, the electrochemical sensor preferably further includes an n-type crystalline silicon substrate that supports the DLC film. As a result, even if the DLC film is thinned, its resistivity is kept low and good sensitivity can be obtained.

  The DLC film may be a p-type semiconductor film further containing 5 to 30 atomic% of an element selected from the group consisting of boron, gallium, and indium. In this case, the electrochemical sensor preferably further includes a p-type crystalline silicon substrate that supports the DLC film. As a result, even if the DLC film is thinned, its resistivity is kept low and good sensitivity can be obtained.

  It is preferable that the DLC film further contains 0.01 to 20 atomic% oxygen.

From the viewpoint of ensuring good sensitivity more reliably, the resistivity of the DLC film is preferably 10 ⁻³ to 10 ⁶ Ωcm.

  The working electrode may have a functional film formed on the DLC film, and the functional film may contain an enzyme that selectively causes an electrochemical reaction of a chemical substance. This makes it possible to selectively detect a specific chemical substance with high sensitivity. In this case, it is preferable that the functional film further includes a mediator.

  The enzyme is at least one selected from the group consisting of glucose oxidase, glucose dehydrogenase, cholesterol oxidase, cholesterol dehydrogenase, alcohol oxidase, alcohol dehydrogenase, xanthine oxidase, lactate oxidase, lactate dehydrogenase, fructose dehydrogenase, ascorbate oxidase and bilirubin oxidase. Preferably there is.

  The electrochemical sensor system according to the present invention includes the electrochemical sensor and a control unit that detects an oxidation-reduction current generated in the electrochemical sensor. According to this system, it is possible to detect chemical substances with high sensitivity.

  According to the present invention, the sensitivity of the electrochemical sensor is improved. Since the sensitivity is good, it can be used as, for example, a noninvasive glucose sensor for measuring the glucose concentration of extracellular fluid. Moreover, in the electrochemical sensor of this invention, since a residual current is small, the drift of an electric signal is suppressed. Therefore, calibration for ensuring accuracy is not necessarily required, and measurement workability is also good. Furthermore, it has a smooth surface compared to conventional electrodes such as boron-doped diamond electrodes, and can be manufactured at low cost.

  Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

  FIG. 1 is a block diagram showing an outline of an embodiment of an electrochemical sensor system. An electrochemical sensor system 100 shown in FIG. 1 has an electrochemical sensor 1 having a working electrode 2, a counter electrode 3, and a reference electrode 5, and the electrochemical sensor 1 detects a redox current of an analyte chemical substance in a specimen. And a control unit 10 for controlling. The working electrode 2, the counter electrode 3, and the reference electrode 5 are each electrically connected to the control unit 10. The working electrode 2 is controlled by the control unit 10 so as to be maintained at a predetermined potential with respect to the reference electrode 5. Then, a current between the working electrode 2 and the counter electrode 3 is detected by the control unit 10.

  The electrochemical sensor system according to the present invention may not include the reference electrode as in the embodiment shown in FIG. In this case, the potential of the working electrode 2 is controlled with reference to the counter electrode 3.

  FIG. 3 is a plan view showing an embodiment of an electrochemical sensor. The electrochemical sensor 1 shown in FIG. 3 includes a first substrate 21, a working electrode 2, a counter electrode 3, a reference electrode 5, and a working electrode 2 provided near one end on one surface of the first substrate 21. Lead wires 2a, 3a and 5a formed on the first substrate 21 so as to be connected to the counter electrode 3 and the reference electrode 5 and to extend to the other end of the first substrate 21, respectively. And a second substrate 22 formed with a window 25 that is exposed to the working electrode 2, the counter electrode 3, and the reference electrode 5. The second substrate 22 covers portions of the lead wires 2a, 3a, and 5a other than the vicinity of the other end of the first substrate 21.

The working electrode 2 is a conductive DLC film formed on the first substrate 21. The DLC film is generally called a diamond-like carbon film, a diamond-like carbon film, a diamond-like carbon film, or an i-carbon film. The DLC film is an amorphous carbon film mainly composed of carbon and hydrogen and having a mixture of sp ³ bonds and sp ² bonds. The diamond film is a film containing a crystal having a diamond structure, and the structure is different from that of the DLC film. It is known that a DLC film and a diamond film can be clearly distinguished by Raman spectroscopy. In the Raman spectrum, a clear peak is observed at 1333 cm ⁻¹ in the case of the diamond film, whereas a broad peak is observed in the Disordered band near 1350 cm ⁻¹ and the graphic band near 1550 cm ^{−1 in} the case of the DLC film. The

The DLC film contains 10 to 40 atomic% hydrogen and 30 to 85 atomic% carbon. When the DLC film has a composition in a specific range, the sensitivity as an electrochemical sensor is improved while maintaining an appropriate resistivity. When hydrogen is contained in this range, the proportion of carbon atoms in the film that form sp ³ bonds including dangling bonds terminated with hydrogen increases, and the film becomes amorphous. An amorphous DLC film is advantageous for industrial production because the film formation temperature is low, film formation with uniform quality is easy, and the film formation rate is high.

In addition, a DLC film mainly composed of sp ³ -bonded carbon has a very small process of adsorbing chemical substances that are oxidized or reduced by an electrochemical reaction. The inner sphere redox reaction through adsorption to the electrode is extremely difficult to occur. As a result, a noise current called a residual current becomes extremely small, so that an electrochemical reaction of a chemical substance to be detected can be detected with a high SN ratio.

  The DLC film may contain at least one element selected from the group consisting of nitrogen, phosphorus, arsenic, antimony, and bismuth. By containing 5 to 30 atomic% of these elements, the DLC film becomes an n-type semiconductor film. Alternatively, the DLC film may further contain an element selected from the group consisting of boron, gallium and indium. By containing 5 to 30 atomic% of these elements, the DLC film becomes a p-type semiconductor film.

  The DLC film preferably further contains 0.01 to 20 atomic% oxygen. The DLC film preferably further contains 0.002 to 1 atom% of chlorine. If the proportion of chlorine is 0.002 atomic% or less, insulation tends to be difficult to occur, and if it exceeds 1 atomic%, the conductivity tends to decrease.

In order to obtain the sensitivity improvement effect particularly remarkably, the resistivity of the DLC film is preferably 10 ⁻³ to 10 ⁶ Ωcm, more preferably 10 ⁻³ to 10 ³ Ωcm, and further preferably 10 ⁻³ to 10 ² Ωcm. is there.

  The thickness of the DLC film is desirably 0.01 to 20 μm. If the thickness of the DLC film is less than 0.01 μm, the resistivity of the DLC film tends to increase or pinholes tend to occur. If many pinholes occur, the sensor electrode may not function normally. On the other hand, if the thickness of the DLC film exceeds 20 μm, the first substrate 21 as the base tends to be warped or cracked. Further, when the DLC film is thick, a voltage drop generated in proportion to the product of the current and the resistance between the first substrate 21 and the solution increases, and the sensitivity tends to decrease. Further, it is desirable that the thickness of the DLC film is as small as possible from the viewpoint of miniaturization of the sensor. From the same viewpoint, the thickness of the DLC film is more desirably 0.05 to 10 μm, and further desirably 0.1 to 5 μm.

  The conductive DLC film can be formed by, for example, a vapor phase method using a source gas containing a hydrocarbon gas while heating the first substrate 21. As the vapor phase method, an ionized vapor deposition method and a high-frequency plasma CVD method are suitable. By adjusting the composition of the source gas, the heating temperature of the first substrate 21 during film formation, and the plasma conditions, a DLC film having the above-described specific composition can be formed. Specifically, for example, a DLC film containing 10 to 40 atomic% hydrogen and 30 to 85 atomic% carbon by forming the film while heating the temperature of the first substrate 21 to 150 to 450 ° C. Is often formed.

  Nitrogen gas, ammonia gas, nitrogen-containing organic substances such as pyridine, boron oxide and alcohols or keto, or a gas such as boron alkoxide, together with hydrocarbon gas as a carbon source during film formation, nitrogen is used. Then, a DLC film doped with boron or the like is formed. For example, a mixed gas containing hydrocarbon gas, nitrogen gas, and optionally at least one additive gas selected from the group consisting of hydrogen, neon, helium, argon and oxygen is introduced as a source gas into the vacuum film forming apparatus. However, the conductive DLC film can be formed by applying a voltage of 13.56 MHz to the raw material gas to form plasma and depositing hydrocarbons generated from the plasmaized raw material gas.

  The working electrode 2 may have a functional film formed on the DLC film. This functional film contains an enzyme that selectively causes an electrochemical reaction of a chemical substance to be analyzed in a specimen. Specific examples of enzymes include glucose oxidase, glucose dehydrogenase, cholesterol oxidase, cholesterol dehydrogenase, alcohol oxidase, alcohol dehydrogenase, xanthine oxidase, lactate oxidase, lactate dehydrogenase, fructose dehydrogenase, ascorbate oxidase, creatine phosphokinase, glycerol oxidase, acyl- Coenzyme A oxidase, tyramine oxidase, amino acid oxidase, glycolate oxidase, pyridoxal-4-oxidase, sorbose oxidase, glonolactooxidase, galactose oxidase, pyranose oxidase, uricase, pyruvate dehydrogenase, pyruvate oxidase Fine bilirubin oxidase and the like.

  By the action of glucose oxidase, when a necessary potential is applied to the electrode, glucose in the sample solution is oxidized to gluconic acid to generate hydrogen peroxide. At this time, ferrocene derivatives such as potassium ferricyanide, ferrocene, 1,1′-dimethylferrocene, ferrocenecarboxylic acid and ferrocenecarboxaldehyde, quinones such as hydroquinone, chloranil and bromanyl, or ferricyan ion, octacyanotungstate ion and octane. When a mediator such as a metal complex ion such as cyanomolybdate ion is used, a current proportional to the glucose concentration can be detected selectively and with high sensitivity by the DLC film.

  The functional film containing an enzyme can be formed, for example, by forming a film of a solution containing an enzyme and, if necessary, other components such as a mediator on the DLC film and drying it.

  The first substrate 21 is a support that supports the working electrode 2, the counter electrode 3, and the reference electrode 5. The first substrate 21 only needs to have a physical strength that can withstand use as an electrochemical sensor.

  When the DLC film is an n-type semiconductor film, the first substrate 21 is preferably an n-type crystalline silicon substrate. This makes it difficult for interface resistance such as a Schottky barrier to occur between the first substrate 21 and the DLC film. Since the detection current of the electrochemical sensor is usually a minute current of about 1 mA or less, the occurrence of IR drop is sufficiently prevented. From the same viewpoint, when the DLC film is a p-type semiconductor film, the first substrate 21 is preferably a p-type crystalline silicon substrate.

  The counter electrode 3 is comprised from the electroconductive material for electrodes normally used in an electrochemical reaction. Preferably, the counter electrode 3 is made of a noble metal such as platinum and gold, or a conductive DLC film similar to the working electrode 2.

  The reference electrode 5 is typically composed of silver-silver chloride or mercury-mercury chloride. The reference electrode 5 can be formed by a method using a paste.

  The lead wires 2a, 3a, 5a are made of a conductive material such as copper and can be formed by a normal method using a metal mask.

  As the second substrate 22, a substrate similar to the first substrate 21 is used. The first substrate 21 and the second substrate 22 are bonded by an adhesive with the working electrode 2 and the like interposed therebetween.

  FIG. 4 is a plan view illustrating an embodiment of an electrochemical sensor that does not include a reference electrode. The electrochemical sensor 1 shown in FIG. 4 has the same configuration as the electrochemical sensor 1 of FIG. 3 except that the reference electrode 5 and the lead wire 5a connected thereto are not formed.

  The controller 10 constituting the electrochemical sensor system 100 includes, for example, a potentiostat that controls the potential of the working electrode 2 with respect to the reference electrode 5, and an ammeter that measures a current flowing between the working electrode 2 and the counter electrode 3. , A processor for controlling the potentiostat and the ammeter, and software for controlling the potentiostat and the ammeter. The processor (CPU = central processing unit) controls the potentiostat and the ammeter by executing the software. The potentiostat is controlled so that the potential of the working electrode 2 is maintained at a predetermined potential such that the redox current inherent to the chemical substance to be analyzed in the sample is appropriately detected. Further, the processor reads a calibration curve relating to the relationship between the concentration of the chemical substance to be analyzed in the sample at a predetermined potential and the oxidation current value, and calculates the concentration of the chemical substance to be analyzed in the sample based on this calibration curve. The electrochemical sensor system 100 preferably includes a monitor that displays the calculated results.

FIG. 5 is a flowchart showing an embodiment of a method for measuring the concentration of a chemical substance to be analyzed in a sample. In step S1, calibration curve data created in advance is read into the processor. The working electrode in step S2 is held at a predetermined potential E _n, the working electrode in the step S3 - measuring a current value I _n at a given time t _n seconds after the start of the voltage application between the counter electrode. Reads the current value I _n in step S4, the concentration C of the analyte chemical substance in a sample is calculated based on interpolation from the calibration curve in step S5. In step S6, the validity of the calculation result is determined. If it is valid, the result is displayed in step S7. If it is determined that the calculation result is not valid, the process returns to step S2.

  According to such an electrochemical sensor system, a plurality of types of chemical substances can be separated and detected by setting a potential using a redox potential specific to the chemical substance to be analyzed in the specimen. In this respect, the system according to this embodiment is superior to a semiconductor sensor that cannot distinguish and detect a plurality of types of chemical substances.

  The electrochemical system according to the present embodiment can measure a metabolome contained in a body fluid selected from blood, saliva, urine, sweat, tears, extracellular fluid and lymph fluid. Metabolome generally refers to a metabolite. Specific examples of the metabolome include sugars (such as glucose), amino acids, and proteins. Among these, in addition to basic metabolites, substances that can be treated as oxidative stress markers and mental stress markers are included, and detection of these metabolome concentrations has clinical significance. .

  An oxidative stress marker is a substance that serves as an indicator of cell aging, DNA damage, arteriosclerosis, and the like. Oxygen stress markers are, for example, oxidative reactions caused by superoxide generated from mitochondria, nitric oxide, peroxynitride, hypochlorous acid or their free radicals, metal ions, lipoxygenase, and myeloperoxidinase. Generate. Representative oxidative stress markers, 8-hydroxy-2'-deoxysideguanosine (8-OHdG), 8-hydroxyguanine (8-OH-Gua), 8-oxo-7,8-dihydro-2'-deoxy DNA damage markers such as guanosine (8-oxodG) and 8-nitroguanosine are caused by oxidative stress damage of DNA. Other oxidative stress markers include 8-isoplastane, a free radical oxidation product of arachidonic acid, and hydroxyleucine, hydroxyvaline, nitrotyrosine, carboxymethyllysine, pentosidine, nitrotyrosine, which are amino acid / protein oxidation disorder markers. And thymine glycol, α-tocophenol which is an in-vivo oxidizing component, biopyrine, oleic acid, thyrodoxine, oxidized coenzyme Q10, carboxymethyllysine, peroxynitrite, nitrogen oxides such as nitric oxide, nitrosothiol reactant , Lipid peroxide (LPO), malondialdehyde, oxidized LDL and oxidized LP (a), creatol MDA-LDL, and the like.

  Examples of mental stress markers include cortisol, norepinephrine, chromogranin A, IgA, and β-endorphin.

  Other chemical substances to be analyzed in the specimen are not particularly limited as long as they are chemical substances that are metabolomes that cause a redox reaction. For example, cholesterol, lactic acid, creatinine, protein, hydrogen peroxide, alcohol, glutamic acid, alcohol amino acid, fructosamine , Glycerol, acyl-coenzyme A, tyramine, amino acid, glycolate, pyridoxal-4-, sorbose, glonolactos, galactose, pyranose, uric acid, pyruvic acid, ascorbic acid, ammonia, trimethylamine, acetone, ethane, pentane, hydrogen, There are oxygen, methane, propane, butane, isoprene, and mercaptans. Other fields include various organic or inorganic substances in agricultural or industrial samples or food samples.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples.

Example 1
An n-type (100) plane single crystal silicon wafer was placed in a vacuum film forming apparatus provided with electrodes arranged to face each other. Then, while heating the silicon wafer to 200 ° C., ethylene and nitrogen are introduced as source gases at 5: 5 sccm, and the inside of the vacuum apparatus is turned into plasma with a power of 500 W at a frequency of 13.56 MHz. A conductive DLC film was formed. The resistivity of the formed DLC film was 0.3 Ωcm, and its composition was 10 atomic% hydrogen, 5 atomic% nitrogen, and 85 atomic% carbon. The silicon wafer on which the DLC film was formed was cut into 5 mm square to obtain a chip in which the DLC film was formed on the n-type single crystal silicon substrate.

  As shown in FIG. 1, a DLC film is used as a working electrode, a silver-silver chloride electrode is formed as a reference electrode by printing a silver / silver chloride paste, and a DLC film as a counter electrode is formed in the same manner as described above. An electrochemical sensor having a simple circuit was constructed.

  The produced electrochemical sensor was connected to a self-made potentiostat controlled by a processor loaded with driving software, and an electrochemical sensor system was configured to measure the current value flowing between the working electrode and the counter electrode. The driving software was set such that the detection potential was 1.0 V with respect to the reference electrode.

When a buffer solution of pH 7.2 with 0.05 M disodium hydrogen phosphate-potassium dihydrogen phosphate was measured with an electrochemical sensor as a blank, the current value was observed to be 4 μA · cm ⁻¹ .

1.0 μM 8-OHdG as a detection species was dissolved in a buffer solution of pH 7.2 with 0.05 M disodium hydrogenphosphate-potassium dihydrogenphosphate. When an electrochemical sensor was immersed therein, the current value was measured to be 78 nA · cm ⁻¹ . From this current value, the concentration of 8-OHdG was calculated to be 0.95 μM when calculated by a processor that read out a calibration curve prepared in advance. The measurement error was 5%.

Further, when the residual current of the electrochemical sensor system was measured, it was 1 μA / cm ² or less.

Examples 2-5
By changing the introduction ratio of ethylene and nitrogen gas in the same manner as in Example 1, conductive DLC films having different compositions were formed on the silicon substrate. About each DLC film | membrane, the composition, film thickness, and resistivity were measured. Furthermore, an electrochemical sensor system was configured in the same procedure as in Example 1, and the concentrations of the blank and 8-OHdG were measured. The results are shown in Table 1. Moreover, the residual current of any electrochemical sensor system was 1 μA / cm ² or less.

Comparative Example 1
An electrochemical sensor system was prepared in the same manner as in Example 1 except that a platinum plate was used as the working electrode. Using the obtained electrochemical sensor system, the blank and 8-OHdG concentrations were measured in the same procedure as in Example 1. As a result, it was calculated to be 0.23 μM. The measurement error was 77%. The residual current was 48 μA / cm ² .

Comparative Example 2
An electrochemical sensor system was prepared in the same manner as in Example 1 except that glassy carbon was used as the working electrode. Using the obtained electrochemical sensor system, an attempt was made to measure the concentration of the blank and 8-OHdG in the same procedure as in Example 1. As a result, the residual current was as large as 98 μA / cm ² , so that the measurement was performed effectively. I couldn't.

Comparative Example 3
A conductive DLC film was formed on a silicon substrate in the same manner as in Example 1 except that a conductive DLC film formed by sputtering was used as the working electrode. An electrochemical sensor system was prepared. This DLC film is formed by sputtering in plasma using graphite as a target, and is an amorphous film mainly composed of sp2 bonds. Using the obtained electrochemical sensor system, an attempt was made to measure the concentration of the blank and 8-OHdG in the same procedure as in Example 1. As a result, the residual current was as large as 98 μA / cm ² , so that the measurement was performed effectively. I couldn't.

Comparative Example 4
An electrochemical system was prepared in the same manner as in Example 1 except that the silicon wafer was heated to 80 ° C. during film formation. About the formed DLC film, the composition, film thickness, and resistivity were measured. Furthermore, when an electrochemical sensor system was configured in the same procedure as in Example 1 and an attempt was made to measure the concentration of the blank and 8-OHdG, the resistivity of the DLC film was as large as 1.5 × 10 ⁶ Ωcm. In this case, an effective current could not be passed, and measurement could not be performed.

Example 7
In the same manner as in Example 1, a chip having a DLC film formed on an n-type single crystal silicon substrate was obtained. The resistivity of the formed DLC film was 0.3 Ωcm, and its composition was 10 atomic% hydrogen, 5 atomic% nitrogen, and 85 atomic% carbon.

  Next, 2 U glucose dehydrogenase as an enzyme, 0.025 mg flavin adenine dinucleotide as a coenzyme, and 0.025 mg potassium ferricyanide as a mediator were dissolved in 5 μL of a 0.05 M phosphate buffer solution at pH 7.2. Appropriately apply the obtained solution onto the conductive DLC membrane, and then apply 1 μL of 1 wt% bovine albumin solution and 1 μL of 20 wt% glutaraldehyde solution prepared using 0.05 M phosphate buffer solution of pH 7.2 in order. did. Thereafter, the film was dried in an environment of 30 ° C. and a humidity of 10% for 30 minutes to form a functional film on the DLC film.

  An electrochemical sensor system was configured in the same manner as in Example 1 except that the functional film and the DLC film were used as working electrodes. The driving software was set so that the detection potential was 1.2 V with respect to the reference electrode.

  A glucose solution in which glucose was dissolved at a concentration of 3 mM in a buffer solution of pH 7.2 with 0.05 M disodium hydrogen phosphate-potassium dihydrogen phosphate was prepared, and an electrochemical sensor was immersed therein to measure the current value. Moreover, the electric current value of the buffer solution which did not melt | dissolve glucose as a blank was also measured. Table 3 shows the glucose concentration calculated from the measurement results together with the SN ratio.

Example 8-10
By changing the introduction ratio of ethylene and nitrogen gas in the same manner as in Example 1, conductive DLC films having different compositions were formed on the silicon substrate. Except this, it carried out similarly to Example 7, and prepared the electrochemical sensor and the electrochemical sensor system. The glucose concentration was measured in the same manner as in Example 7 using the obtained electrochemical sensor. The results are summarized in Table 3.

Comparative Example 5
An electrochemical sensor system was prepared in the same manner as in Example 7 except that a platinum plate was used as the working electrode, and the glucose concentration was measured. The calculated glucose concentration was 2.8 mM and the SN ratio was 6.7.

Comparative Example 6
An electrochemical sensor system was prepared and prepared in the same manner as in Example 7 except that glassy carbon was used as the working electrode, and the glucose concentration was measured. The calculated glucose concentration was 2.7 mM, and the SN ratio was 7.7.

Comparative Example 7
An electrochemical sensor system was prepared in the same manner as in Example 7 except that a conductive DLC film formed by sputtering was used as the working electrode. This DLC film is formed by sputtering in plasma using graphite as a target, and is an amorphous film mainly composed of sp ² bonds. When the glucose concentration was measured using the obtained electrochemical sensor system in the same manner as in Example 7, the calculated value of the glucose concentration was 2.7 mM, and the SN ratio was 7.8.

Glucose concentration in plasma, tears, saliva or skin extracellular fluid Plasma, tears, saliva and skin extracellular fluid were prepared as body fluids containing glucose. These glucose concentrations were measured using the electrochemical sensor systems of Example 7 and Comparative Example 5 above. For comparison, the glucose concentration was measured for the same sample by the hexonase method, which is a highly accurate measurement method.

  As shown in the above table, Example 7 well reproduced the measurement value equivalent to that of the hexonase method, but Comparative Example 5 had a large error.

Example 12
A functional film for detecting cholesterol was formed on the conductive DLC film formed in the same manner as in Example 1 by the following procedure. Except for this, an electrochemical sensor system was prepared in the same manner as in Example 1.

  2U cholesterol dehydrogenase as an enzyme, 0.025 mg diaphorase as a coenzyme, and 0.025 mg potassium ferricyanide as a mediator were dissolved in 5 μL of a 0.05 M phosphate buffer solution at pH 7.2. The obtained solution was appropriately applied onto the DLC film, and further 1 μL of 20% glutaraldehyde was appropriately applied. Thereafter, the film was dried in an environment of 30 ° C. and a humidity of 10% for 30 minutes to form a functional film.

Example 13
A functional film for detecting alcohol was formed on the conductive DLC film formed in the same manner as in Example 1 by the following procedure. Except for this, an electrochemical sensor system was prepared in the same manner as in Example 1.

  2 U alcohol dehydrogenase as an enzyme and 0.025 mg potassium ferricyanide as a mediator were dissolved in 5 μL of a McKilpain buffer solution at pH 5.5. The obtained solution was appropriately applied onto the DLC film, and further 1 μL of 20% glutaraldehyde was appropriately applied. Thereafter, the film was dried in an environment of 30 ° C. and a humidity of 10% for 30 minutes to form a functional film.

Example 14
On the conductive DLC film formed in the same manner as in Example 1, a functional film for detecting xanthine was formed by the following procedure. Except for this, an electrochemical sensor system was prepared in the same manner as in Example 1.

  2 U xanthine oxidase as an enzyme, 0.025 mg nicotinamide adenine dinucleotide as a coenzyme, and 0.025 mg potassium ferricyanide as a mediator were dissolved in 5 μL of a 0.05 M phosphate buffer solution at pH 7.2. The obtained solution was appropriately applied onto the DLC film, and further 1 μL of 20% glutaraldehyde was appropriately applied. Thereafter, the film was dried in an environment of 30 ° C. and a humidity of 10% for 30 minutes to form a functional film.

Comparative Examples 8-10
The electrochemical sensor systems of Comparative Examples 8 to 10 were prepared in the same manner as in Examples 12 to 14 except that a platinum plate was used as the working electrode.

  A solution was prepared by dissolving glucose, cholesterol, ethanol, and xanthine at 3 mM in a 0.05 M phosphate buffer solution of pH 7.2. The electrochemical sensors of Examples 7 and 12 to 14 and Comparative Examples 5 and 8 to 10 were immersed in these solutions, and the concentration of each detection species was measured in the same manner as in Example 1.

  The following table shows the measured concentration and SN ratio. As shown in the table, the selectivity of each sensor and a high S / N ratio relative to the comparative example could be confirmed.

It is a block diagram showing one embodiment of an electrochemical sensor system. It is a block diagram showing one embodiment of an electrochemical sensor system. It is a top view which shows one Embodiment of an electrochemical sensor. It is a top view which shows one Embodiment of an electrochemical sensor. It is a flowchart which shows one Embodiment of the method of measuring the density | concentration of the chemical substance to be analyzed in a sample.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Electrochemical sensor, 2 ... Working electrode, 3 ... Counter electrode, 5 ... Reference electrode, 10 ... Control part, 21 ... 1st board | substrate (support body), 22 ... 2nd board | substrate, 100 ... Electrochemical sensor system.

Claims (7)

A working electrode having a conductive DLC film containing 10 to 40 atomic% hydrogen and 30 to 85 atomic% carbon;
The DLC film, nitrogen, phosphorus, arsenic, n-type semiconductor film Der further containing 5-30 atomic% of an element selected from the group consisting of antimony and bismuth is,
The DLC film has a resistivity of 10 ^{−3 to} 0.5 Ωcm,
The electrochemical sensor which measures the density | concentration of a chemical substance further provided with the n-type crystalline silicon substrate which supports the said DLC film . A working electrode having a conductive DLC film containing 10 to 40 atomic% hydrogen and 30 to 85 atomic% carbon;
The DLC film, Ri p-type semiconductor film Der further comprising boron, 5-30 atomic% of an element selected from the group consisting of gallium and indium,
The DLC film has a resistivity of 10 ^{−3 to} 0.5 Ωcm,
An electrochemical sensor for measuring the concentration of a chemical substance, further comprising a p-type crystalline silicon substrate supporting the DLC film . The DLC film further comprises an oxygen of 0.01 to 20 atomic%, an electrochemical sensor according to claim 1 or 2, wherein. The working electrode has the function film formed on the DLC film, comprising an enzyme the function film causes selective electrochemical reaction of the chemical substance, any one of claims 1 to 3 The electrochemical sensor according to one item. The electrochemical sensor according to claim 4 , wherein the functional film further comprises a mediator. The enzyme is at least one selected from the group consisting of glucose oxidase, glucose dehydrogenase, cholesterol oxidase, cholesterol dehydrogenase, alcohol oxidase, alcohol dehydrogenase, xanthine oxidase, lactate oxidase, lactate dehydrogenase, fructose dehydrogenase, ascorbate oxidase and bilirubin oxidase. The electrochemical sensor according to claim 4 or 5 . An electrochemical sensor system comprising: the electrochemical sensor according to any one of claims 1 to 6 ; and a control unit that detects an oxidation-reduction current generated in the electrochemical sensor.

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