JP5301101B2 - Electrochemical sensor and electrochemical sensor system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an electrochemical sensor capable of measuring the concentration of a bio-related substance such as glucose, uric acid, etc. with high precision while using an inexpensive material. <P>SOLUTION: The electrochemical sensor 1 is equipped with a support 21, the acting electrode 2 comprising a conductive DLC film formed on the support 21 and counter electrode 3. The electrochemical sensor system is equipped with the electrochemical sensor 1 and a control part for controlling the electrochemical sensor 1 so as to detect the oxidation-reduction current of a specimen. <P>COPYRIGHT: (C)2008,JPO&amp;INPIT

Description

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

Conventionally, biological substances such as glucose and uric acid are generally detected by a method using a sensor using an enzyme. As a method that does not use an enzyme, a method of electrochemically measuring a glucose concentration using a diamond electrode imparted with conductivity by mixing boron or the like has been proposed.
JP 2002-310977 A

  Conventional sensors using enzymes or diamond electrodes are expensive, and sensors using cheaper materials are desired. Moreover, according to the study by the present inventors, when the glucose concentration of blood is measured using an enzyme or a diamond electrode, platelets contained in the blood coagulate on the oxygen or diamond electrode, so that the sensor is washed. It is extremely difficult to reuse. Since the material is expensive, it is difficult to reuse and there is room for improvement.

  Therefore, an object of the present invention is to provide a sensor capable of measuring the concentration of biologically related substances such as glucose and uric acid with high accuracy while using an inexpensive material.

  In one aspect, the invention relates to an electrochemical sensor. The electrochemical sensor according to the present invention includes a support, a working electrode made of a conductive DLC film formed on the support, and a counter electrode.

  According to the electrochemical sensor of the present invention, it is possible to measure the concentration of a biological substance such as glucose and uric acid with high accuracy while using an inexpensive material. A current value at a predetermined potential between the working electrode and the counter electrode is measured while the working electrode and the counter electrode of the electrochemical sensor are brought into contact with a sample containing a biological substance as a specimen. At this time, the redox current value of the specimen is measured by setting the potential of the working electrode to a potential at which the redox current of the specimen is appropriately observed. In general, the concentration of the sample correlates with the oxidation-reduction current value. If a calibration curve representing these correlations is created in advance, the concentration of the sample is calculated based on this calibration curve from the measured oxidation-reduction current value. can do.

  The DLC film preferably contains a transition metal element. In addition, the electrochemical sensor according to the present invention preferably further includes a biological substance fixed on the surface of the DLC film. Thereby, the detection sensitivity of the specimen is further increased.

  The electrochemical sensor according to the present invention includes a compound that is immobilized on the surface of a DLC film and that captures an analyte and quantitatively changes the oxidation-reduction current of the oxidation-reduction substance in accordance with the amount of the captured analyte. Furthermore, you may provide. This makes it possible to detect the concentration of a specimen that does not directly contribute to the electrochemical reaction based on the redox current of another redox substance.

The support is preferably a plastic substrate . Also, the plastic substrate specimen is relatively hardly adsorbed, there is an advantage that cleaning after the measurement is easy. In order to form a conductive diamond thin film, it is necessary to heat to a high temperature, and it was extremely difficult to form a diamond thin film on a plastic substrate, whereas a DLC film has a relatively low temperature. A plastic substrate can be employed as the support.

  The electrochemical sensor may further include a gel electrolyte membrane covering the working electrode and the counter electrode. As a result, it becomes possible to measure the oxidation-reduction current and concentration using a gas such as ammonia or acetone as a specimen.

  In another aspect, the present invention relates to an electrochemical sensor system. The electrochemical sensor according to the present invention includes the electrochemical sensor according to the present invention and a control unit that detects an oxidation-reduction current generated in the electrochemical sensor. According to this system, it is possible to measure the concentration of biological substances such as glucose and uric acid with high accuracy while using an inexpensive material.

  According to the electrochemical sensor of the present invention, it is possible to measure the concentration of a biological substance such as glucose and uric acid with high accuracy while using an inexpensive material.

  In addition, the DLC film hardly adheres to glucose or the like on the surface, and can be easily washed after measurement. Therefore, the sensor can be used easily and repeatedly.

  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 includes an electrochemical sensor 1 having a working electrode 2, a counter electrode 3, and a reference electrode 5, and a control unit 10 that controls the electrochemical sensor 1 so that a redox current of a specimen is detected. And comprising. 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. 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. When referring to the composition of the DLC film in CH _n, it is 0.05 ≦ n ≦ 0.7. 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

  According to the knowledge of the present inventors, when a DLC membrane is used as a working electrode, the oxidation potential of components in urine such as ascorbic acid is shifted to a high potential side. As a result of this potential shift, the oxidation current of glucose or urea is observed separately from the oxidation current of ascorbic acid or the like at a predetermined voltage. As a result, it becomes possible to measure the glucose and urea concentrations in blood containing mixed components such as ascorbic acid with high accuracy.

  In addition, the surface of the DLC film is smoother than that of a diamond film or the like, and has an advantage that the sample liquid does not easily remain on the surface after measurement by immersing in a sample liquid containing a specimen and is easy to clean. For example, in the case of a diamond film, when used for measuring the glucose concentration of serum, it is difficult to sufficiently wash the serum remaining on the film surface. When used for blood testing, the plasma will coagulate on the diamond membrane, making it virtually impossible to reuse as a working electrode. On the other hand, in the case of a DLC film, there is clearly little sticking due to residual sample liquid and coagulation of plasma and it can be easily reused as a working electrode.

  For example, the surface roughness Ra measured by a method defined in JIS B0601 of a DLC film formed on a Si wafer is 0.1 μm or less. On the other hand, the surface roughness Ra of the diamond film is usually about 0.5 to 3 μm.

The DLC film as the working electrode 2 has a conductivity sufficient to function as an electrode. Specifically, the resistivity of the DLC film is preferably 10 ⁸ Ωcm or less, more preferably 10 ⁶ Ωcm or less, and still more preferably 10 ² Ωcm or less. The lower limit of the resistivity is not particularly limited, but is usually about 10 ⁻⁴ Ωcm.

  The DLC film is often doped with at least one element selected from the group consisting of nitrogen, phosphorus, arsenic, antimony, bismuth, boron, aluminum, gallium, indium, and thallium in order to increase the conductivity.

  As understood by those skilled in the art, the conductive DLC film can be formed by a method usually employed as a method of forming a DLC film, such as an ionization vapor deposition method or a high-frequency plasma CVD method. 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 thickness of the DLC film is preferably 0.01 to 20 μm. If this film thickness is less than 0.01 μm, the resistance value of the DLC film tends to increase, and if it exceeds 20 μm, the substrate as a support tends to warp or the substrate tends to crack. From the same viewpoint, the thickness of the DLC film is more preferably 0.05 to 10 μm, and further preferably 0.1 to 5 μm.

  An intermediate layer such as an amorphous mixed layer composed of silicon and carbon for improving the adhesion may be provided between the first substrate 21 and the DLC film (working electrode 2).

  The DLC film preferably contains a transition metal element. The transition metal element may be added to the DLC film in the form of metal fine particles, or added to the DLC film as a metal complex with an organic substance such as an oxide, nitride, chloride, bromide, iodide, or phthalocyanine. It may be. Specifically, the transition metal elements are scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, hafnium, tantalum, Tungsten, rhenium, osmium, iridium, platinum, gold, lanthanum, cerium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, europium, thulium, ytterbium, lutetium, actinium, thorium, protactinium, uranium , Neptunium, Americium, Curium, Barium, Calfornium, Einsteinium, Fermium, Mendelevium, Nobelium and Lorenci It is selected from the group consisting of beam. Since these transition metal elements have d vacancy orbitals, they have a catalytic function to promote the redox reaction of the specimen. Thereby, the detection sensitivity of the specimen is improved. The transition metal element is preferably present on the surface of the DLC film in order to easily develop a function as a catalytic function.

  Examples of the method of adding the transition metal element to the DLC film include vacuum deposition, ion plating, RF sputtering, chemical vapor deposition (CVD), ion implantation, electrochemical electrodeposition, mechanochemical reaction, thermal diffusion, or The method by application | coating is mentioned. You may introduce | transduce simultaneously as source gas in the film-forming of a DLC film.

  A halogen such as fluorine, chlorine and iodine, or oxygen may be bonded to the surface of the DLC film. Thereby, the wettability of a DLC film, the adhesiveness of plasma, etc. can be controlled. In addition, chemical modification can be performed by bonding various compounds to the surface of the DLC film. By chemical modification, for example, functional groups such as hydrogen, hydroxyl groups, carboxyl groups and amino groups are introduced on the surface. Alternatively, ferrocene, nickelocene, ruthenium carbonyl complex, ruthenium Prussian blue analog, osmium 2,2′-bipyridine complex, phthalocyanine-based cobalt porphyrin complex, and derivatives thereof (eg, 4- (4-ferrocenephenyliminomethyl) phenol) Complex catalysts such as, cytochrome oxidase, D-amino acid oxidase, heme oxidase, NADH oxidase, copper amine oxidase, galactose oxidase, HIF-1 proline hydroxylase, kynurenine oxidase, ubiquinol oxidase, lipoxygenase, catalase, Peroxidase, glutathione peroxidase, glucose oxidase, phenol oxidase, kaurenoic acid oxidase, superoxide dismutase, xanthine Sidase, cholesterol oxidase, cholesterol esterase, lipase, alkaline phosphatase, phospholipase C, acyl-CoA synthase, urease, glutamate dehydrase, uricase, creatininase, creatinase, pyruvate oxidase, methionine loriase, lactate dehydrogenase L-amino acid oxidase, citrate lyase, glycogen phosphorylase, cholinesterase, penicillinase, hypoxanthine guanine phosphoribosyltransferase, cholinesterase, aminoacylase, aminotransferase, glucose isomerase, aspartase, penicillin amylase, fumarase, lactase, L-Asp b-decarboxylase, nitrile hydratase, a-glucosyltransferase Redox reactions of analytes such as glycerol, b-fructofuranosidase, lipase, maltooligosaccharide-forming enzyme, brewer's yeast, naringinase, papain, dehydrogenase (cells), acetate kinase, phosphodiesterase, creatinase and sarcosine oxidase A bio-related substance that promotes may be immobilized on the surface of the DLC film.

  A compound that captures a sample by electrostatic force, van der Waals force, coordination bond, hydrogen bond, insertion, etc., and quantitatively changes the redox current of the redox substance according to the amount of the captured sample The compound may be fixed on the surface of the DLC film. In this case, when the sample is captured by the compound that captures the sample, the oxidation-reduction reaction of a redox substance such as ferrocene is promoted or suppressed. The oxidation-reduction current value of the oxidation-reduction substance changes quantitatively according to the amount of analyte to be captured. The amount of the sample to be captured is proportional to the concentration of the sample in the sample. Therefore, even if the specimen does not directly contribute to the electrochemical reaction, if a calibration curve representing the correlation between the oxidation-reduction current value of the predetermined redox substance and the concentration of the specimen is created in advance, It becomes possible to detect the concentration in the sample in the presence of the redox substance.

  In the case of a DLC film, a compound that captures a specimen can be easily fixed on the surface by an organic chemical reaction. In addition, the DLC film has an advantage that the residual current is small and the detection sensitivity is higher than that of a metal electrode, a glassy carbon electrode or a highly oriented pyrolytic carbon electrode.

  Specific examples of the compound that captures the specimen include crown ether, thiourea, cyclic polyamide, α, α-Bis (N′-butylthioureylene) -m-xylene, α, α-Bis (N′-1-naphthylthioylene)- m-xylene, 2,7-Di-tert-butyl-9,9-dimethyl-4,5xanthenedylbiscarbamic acid dibenzyl ester, 2,7-Di-tert-butyl-9,9-dimethyl-4,5-bis (N '-Butylthioureylene) -xanthene, 2,7-Di-tert-butyl-9,9-dimethyl-4,5-bis (N'-phenylthioureylene)- Anthene, and 2,7-Di-tert-butyl-9,9-dimethyl-4,5-bis (N'-phenylthioureylene) -xanthene and DNA and the like.

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. Specifically, for example, a substrate formed of metal, plastic, ceramics, paper, or the like can be used as the first substrate 21 (support) . It is preferable to use a plastic substrate formed of plastic. Among the plastic substrates, a plastic substrate made of a plastic selected from olefinic polymers such as polyethylene and polypropylene, polycarbonate, and polyethylene terephthalate is preferable. The plastic substrate may be a film or a non-woven fabric.

  The counter electrode 3 is comprised from the electroconductive material for electrodes normally used in an electrochemical reaction. Preferably, the counter electrode 3 is composed of a noble metal such as platinum and gold, silver-silver chloride, or mercury-mercury chloride.

  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 electrochemical sensor according to the present invention is not limited to the embodiment described above. For example, a gel electrolyte membrane that covers the working electrode, the counter electrode, and the reference electrode may be formed. As a result, it becomes possible to measure the oxidation-reduction current and concentration using a gas such as acetone or ammonia as a specimen. In this case, the gel electrolyte only needs to dissolve the specimen. For example, a buffer solution of pH 7.4 with disodium hydrogen phosphate-potassium dihydrogen phosphate, or a buffer of pH 8.4 with boric acid-borax. What added agar or gelatin to the solution is mentioned. The thickness of the gel electrolyte membrane is preferably about 0.1 to 20 μm.

  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. And a potentiostat and a processor for controlling the ammeter. The processor (CPU = central processing unit) controls the potentiostat and the ammeter by executing predetermined 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 specimen is appropriately detected. In addition, the processor reads a calibration curve relating to the relationship between the concentration of the specimen at a predetermined potential and the oxidation current value, and calculates the concentration of the specimen based on the calibration curve.

FIG. 5 is a flow diagram illustrating one embodiment of a method for measuring the concentration of an analyte. 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 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 specimens can be separated and detected by potential setting using a redox potential unique to 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 specimens.

  The specimen detected by the system according to the present embodiment is not particularly limited as long as it is a chemical substance that causes an oxidation-reduction reaction, but various organic or inorganic substances in biological samples, environmental samples, agricultural and industrial samples, or food samples are specimens. It can be. Specific examples of the sample include glucose, cholesterol, lactic acid, creatinine, protein, hydrogen peroxide, alcohol, cholesterol, glutamic acid, alcohol amino acid, ammonia, trimethylamine, acetone, ethane, pentane, hydrogen, oxygen, methane, propane, butane, Isoprene, mercaptans, ferrocene, cytochrome, D-amino acid, heme, amine, galactose, HIF-1 proline, quinurenin, ubiquinol, linoleic acid and other unsaturated fatty acids, glutathione, phenol, kaurene, hydrogen peroxide, cholesterol, cholesterol ester , Triglycerides, urea, ammonia, uric acid, creatinine, creatine, bilirubin, methionine, lactic acid, pyruvate, citric acid, xanthine, hypoxanthine and guanine phosphoribo Transferases, cholinesterase, acetyl -DL- amino, fumaric acid, penicillin G, L-asparagine, acrylonitrile, sucrose, DL-aspartic acid, liquefied starch, ADP and sarcosine. Among these, the system according to the present embodiment is particularly useful when used to determine the concentration of a biological substance such as glucose in blood or serum.

  Other specific examples of the specimen include biological related substances other than the above, alkaloid substances such as caffeine and theophylline, serotonin, histamine, glutathione, theophylline (asthma drug), homocysteine, glutathione, 2-mercapto Examples include ethanesulfonic acid, cephalexin, naprocene, NADH and derivatives thereof. Furthermore, diols such as bisphenol A, which is an environmental hormone, chlorophenol, which is a dioxin precursor, polyethylene glycol (PEG) and bis3-sulfopropyl disulfide (SPS), which are plating solution additives, saccharin, and butynediol. It is done.

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

1. Example 1 Production of electrochemical sensor and measurement of analyte concentration using the same
Using Si ₂ H ₆ and CH ₄ as source gases, an amorphous layer (thickness 3 μm) composed of silicon and carbon was formed on a 0.25 cm ² aluminum plate using an ionized vapor deposition apparatus. This is placed in a vacuum film forming apparatus having a pair of electrodes arranged opposite to each other, and a voltage having a frequency of 13.56 MHz is applied between the electrodes while supplying ethane and pyridine as the raw material gas. Was turned into plasma. Carbon generated from the plasma-generated source gas was deposited on the amorphous layer, and a conductive DLC film as a working electrode was formed. Pyridine was introduced using hydrogen gas as the carrier gas. The resistivity of the formed working electrode was 0.3 Ωcm ⁻¹ . A DLC film as a counter electrode was also formed under the same conditions as the working electrode. A silver-silver chloride electrode as a reference electrode was also formed. An electrochemical sensor was produced as described above.

  The electrochemical sensor system constructed as described above was connected to a potentiostat controlled by a processor loaded with driving software, and the current value flowing between the working electrode and the counter electrode was measured. .

A sample in which uric acid and ascorbic acid are dissolved in a buffer solution of pH 7.4 with 0.05M disodium hydrogen phosphate-potassium dihydrogen phosphate, the concentration of uric acid is 0.500 mM, and the concentration of ascorbic acid is 1.0 mM A solution was prepared. The working electrode, reference electrode, and counter electrode of the electrochemical sensor to which the potentiostat was connected were immersed in the sample solution, and the potential of the working electrode with respect to the reference electrode was set to 0.8V. The current between the working electrode and the counter electrode at that time was regarded as the oxidation current of uric acid, and the current value was measured. The measured oxidation current value of uric acid was 0.080 mA · cm ⁻¹ . From this oxidation current value, the uric acid concentration of the sample solution was calculated to be 0.501 mM by a processor reading a calibration curve regarding the relationship between the uric acid concentration and the oxidation current value. The error of the calculated density with respect to the actual density was 0.2%.

Comparative Example 1
The oxidation current value of uric acid was measured by the same method as in Example 1 except that a platinum plate was used as the working electrode. As a result, it was 0.1322 mA · cm ⁻¹ and the uric acid concentration was calculated to be 0.8248 mM. The error was 64.8%.

  When a platinum plate is used as the working electrode as in Comparative Example 1, the oxidation potentials of uric acid and ascorbic acid are close to each other, and current peaks based on these oxidation currents are observed to overlap. Therefore, the observed oxidation current includes not only uric acid but also ascorbic acid, and the concentration required from this also includes a large error. On the other hand, in the case of Example 1 using the DLC membrane as the working electrode, the oxidation potential of ascorbic acid shifts to the high potential side, and the oxidation current of uric acid can be observed alone, so the uric acid concentration is high. Quantified with accuracy.

Example 2
When measurement was performed in the same manner as in Example 1 except that ascorbic acid was not added to the sample solution, the uric acid concentration was calculated to be 0.501 mM. The error of the calculated density with respect to the actual density was 0.2%.

Comparative Example 2
Measurement was performed in the same manner as in Example 2 except that a platinum plate was used as the working electrode, and the uric acid concentration was calculated to be 0.462 mM. The error of the calculated concentration with respect to the actual concentration was 7.6%. In the case of a platinum plate, an electrochemical reaction due to the adsorption of water occurred, and this was detected as a residual current. On the other hand, in the case of the DLC film as in Example 2, since the residual current is smaller than that of the platinum plate, high detection accuracy is achieved. When a similar experiment was performed using a glassy carbon electrode or a highly oriented pyrolytic carbon electrode, the error was larger than that of the DLC film.

Example 3
Under the same conditions as in Example 1, a DLC film as a working electrode or a counter electrode was formed on a polycarbonate substrate. Copper fine particles having a nanometer-scale particle size were deposited on the formed DLC film by RF sputtering. A reference electrode was formed by printing a paste containing silver and silver chloride. Further, three copper wiring patterns as lead wires were formed using a metal mask so as to be connected to the working electrode, the counter electrode, or the reference electrode, respectively. And the electrochemical film which has the structure similar to FIG. 3 was adhere | attached by adhere | attaching the polycarbonate film in which the window in which the edge part of a working electrode, a counter electrode, a reference electrode, and a copper wiring pattern was exposed was adhere | attached. A potentiostat was connected to this in the same manner as in Example 1 to constitute an electrochemical sensor system.

Glucose was dissolved in a buffer solution of pH 7.4 with 0.05 M disodium hydrogen phosphate-potassium dihydrogen phosphate to prepare a sample solution having a glucose concentration of 5 mM. The working electrode, counter electrode, and reference electrode of the electrochemical sensor prepared above were immersed in the sample solution, and the potential of the working electrode with respect to the reference electrode was set to 0.7V. The current between the working electrode and the counter electrode at that time was regarded as the oxidation current of glucose, and the current value was measured. The measured glucose oxidation current value was 14.2 mA · cm ⁻¹ . From this oxidation current value, the glucose concentration of the sample solution was calculated to be 4.98 m by a processor reading a calibration curve relating to the relationship between the glucose concentration and the oxidation current value. The error of the calculated density with respect to the actual density was 0.4%.

Comparative Example 3
The glucose oxidation current in the sample solution was measured in the same manner as in Example 3 except that a copper plate was used as the working electrode, and it was 3.2 mA. From this oxidation current value, the glucose concentration was calculated to be 4.69 mM, and the error was 6.2%.

Example 4
A conductive DLC film (thickness 0.5 μm) was formed on a polycarbonate substrate by plasma CVD, and copper fine particles having a nanometer-scale particle size were applied thereto by RF sputtering. Otherwise, the oxidation current was measured for the sample solution having a glucose concentration of 5 mM in the same manner as in Example 3, and the glucose concentration was calculated to be 4.99 mM with an error of 0.3%.

Comparative Example 4
A vaporized raw material prepared by dissolving boron oxide in a mixed solvent of methanol: acetone = 1: 9 (volume ratio) is introduced into the reactor together with a hydrogen carrier gas, and a voltage is applied at a frequency of 13.56 MHz to form plasma. Thus, an attempt was made to form a conductive boron-doped diamond thin film on a polycarbonate substrate. However, when the temperature of the polycarbonate substrate is 230 ° C. or lower, which is the melting point of polycarbonate, a conductive boron-doped diamond thin film cannot be formed.

Comparative Example 5
A conductive boron-doped diamond thin film (thickness 0.5 μm) was formed in the same manner as in Comparative Example 4 except that a p-type (111) plane Si wafer was used instead of the polycarbonate substrate, and the nanometer scale was formed there. Copper fine particles having a particle size of 5 mm were applied by RF sputtering. . Otherwise, the oxidation current was measured for the sample solution having a glucose concentration of 5 mM in the same manner as in Example 3, and the glucose concentration was calculated to be 4.98 mM with an error of 0.5%.

Example 5
The glucose concentration of the sample solution was calculated in the same manner as in Example 3 except that ruthenium oxide fine particles were applied to the DLC film by RF sputtering instead of the copper fine particles.

Example 6
An electrochemical sensor having the same configuration as in FIG. 4 was prepared without forming the reference electrode, and the sample solution was prepared in the same manner as in Example 3 except that the working electrode was controlled by the potential with respect to the counter electrode instead of the reference electrode. The glucose concentration was measured and found to be 4.98 mM, with an error of 0.4%.

Example 7
An electrochemical sensor system similar to that of Example 3 was configured except that the DLC film was treated with oxygen plasma and further aminopropyltriethoxysilane was reacted at 80 ° C. to fix the phthalocyanine-modified cobalt on the DLC film. . It was 5.01 mM when the glucose concentration of the sample solution was measured using this.

Example 8
An electrochemical sensor system was constructed in the same manner as in Example 3 except that glucose oxidase was immobilized on the DLC film. It was 5.00 mM when the glucose concentration of the sample solution was measured using this.

Example 9
The surface of the DLC film formed in the same manner as in Example 1 is treated with a mixed acid (concentrated nitric acid + concentrated sulfuric acid) to introduce a nitro group, and the nitro group is reduced to an amino group in ethanol using tin chloride as a catalyst. did. Subsequently, 2,7-Di-tert-butyl-9,9-dimethyl-4,5-bis) N'-phenylthiourelene) -xanthene is bound to the amino group, and a DLC film having a function of capturing the sample is added. was gotten. Thereafter, gelatin and ferrocene were dissolved in a buffer solution of pH 7.4 using 0.05 M disodium hydrogen phosphate-potassium dihydrogen phosphate to prepare a solution, and a thickness of 50 μm was formed on the surface of the heated solution DLC film. It was applied to. After cooling, a gel electrolyte film containing ferrocene as a redox substance was formed on the DLC film.

  The electrochemical system similar to Example 1 was comprised using the obtained DLC film. When the DLC film was brought into contact with air containing acetone gas at a concentration of 10 to 100 ppm and the oxidation current of ferrocene was measured, the oxidation-reduction current value of ferrocene decreased in proportion to the concentration of acetone gas. That is, it was confirmed that the concentration of acetone gas could be quantified by creating a calibration curve showing the correlation between the concentration of acetone gas and the oxidation-reduction current value of ferrocene.

2. Examination of Washability of Electrochemical Sensor Using the electrochemical sensors produced in Example 4 and Comparative Example 5, respectively, measurement of glucose concentration and washing with pure water after measurement were repeated four times. In the case of Example 4, the measured values of glucose concentration were 4.99, 5.01, 4.98, and 5.02 mM in order, and were stable. On the other hand, in the case of the comparative example 5 using a diamond thin film, it is 5.02, 5.03, 5.05, and 5.06 mM in order, and a measurement error becomes large clearly as measurement and washing are repeated. A trend was observed. This is considered to be because the sample solution could not be sufficiently removed from the surface of the diamond thin film by washing.

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 flow figure showing one embodiment of a method of measuring a concentration of 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 (8)

A support;
A working electrode made of a conductive DLC film formed on the support;
A counter electrode, and
In the Raman spectrum of the DLC film, a peak is observed in the Disordered band near 1350 cm ⁻¹ , and the resistivity of the DLC film is 10 ⁻⁴ Ωcm to 10 ² Ωcm,
The DLC film is a DLC film formed by ionized vapor deposition or high-frequency plasma CVD,
Said support Ri plastic substrate der,
An electrochemical sensor that is cleaned and used repeatedly after measurement .   The DLC film contains a transition metal element, and the transition metal element is added to the DLC film as a metal complex with an oxide, nitride, chloride, bromide, iodide, or organic substance. The described electrochemical sensor.   The electrochemical sensor according to claim 1 or 2, further comprising a biological substance immobilized on the surface of the DLC film that promotes a redox reaction of the specimen.   The bio-related substances are ferrocene, nickelocene, ruthenium carbonyl complex, ruthenium Prussian blue analog, osmium 2,2′-bipyridine complex, phthalocyanine-based cobalt porphyrin complex, 4- (4-ferrocenephenyliminomethyl) phenol, cytochrome oxidase , D-amino acid oxidase, heme oxidase, NADH oxidase, copper amine oxidase, galactose oxidase, HIF-1 proline hydroxylase, kynurenine oxidase, ubiquinol oxidase, lipoxygenase, catalase, peroxidase, glutathione peroxidase, glucose oxidase, Phenol oxidase, kaurenoic acid oxidase, superoxide dismutase, xanthine oxidase, cholesterol oxidase, Lesterol esterase, lipase, alkaline phosphatase, phospholipase C, acyl CoA synthase, urease, glutamate dehydrogenase, uricase, creatininase, creatinase, pyruvate oxidase, methionine loriase, lactate dehydrogenase, L-amino acid oxidase Citrate lyase, glycogen phosphorylase, cholinesterase, penicillinase, hypoxanthine guanine phosphoribosyltransferase, cholinesterase, aminoacylase, aminotransferase, glucose isomerase, aspartase, penicillin amylase, fumarase, lactase, L-Asp b-decarboxylase, Nitrile hydratase, a-glucosyltransferase, b-fructofuranosidase The electrochemical sensor according to claim 3, selected from lipase, maltooligosaccharide-forming enzyme, brewer's yeast, naringinase, papain, dehydrogenase (bacteria), acetate kinase, phosphodiesterase, creatinase and sarcosine oxidase.   The compound further comprising a compound that is immobilized on the surface of the DLC film and that captures the analyte and that quantitatively changes the oxidation-reduction current of the oxidation-reduction substance in accordance with the amount of the captured analyte. The electrochemical sensor according to 1.   The compound supplementing the specimen is crown ether, thiourea, cyclic polyamide, α, α-Bis (N′-butylthioureene) -m-xylene, α, α-Bis (N′-1-naphthylthioylene) -m-xylene. , 2,7-Di-tert-butyl-9,9-dimethyl-4,5xanthenediylbiscarbamic acid dibenzyl ester, 2,7-Di-tert-butyl-9,9-dimethyl-4,5-bis (N'-butylethyl) ) -Xanthene, 2,7-Di-tert-butyl-9,9-dimethyl-4,5-bis (N'-phenylthioureylene) -xanth ne, and 2,7-Di-tert-butyl-9,9-dimethyl-4,5-bis (N'-phenylthioureylene) selected from -Xanthene, electrochemical sensor of claim 5, wherein.   The electrochemical sensor according to any one of claims 1 to 6, further comprising a gel electrolyte membrane covering the working electrode and the counter electrode.   An electrochemical sensor system comprising: the electrochemical sensor according to any one of claims 1 to 7; and a control unit that detects a redox current generated in the electrochemical sensor.

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