JP5508312B2 - Method for producing electrode for electrochemical measurement - Google Patents

Description

The present invention relates to method for producing an electrochemical measurement electrodes used in electrochemical measurements.

  In the analysis and biosensor based on the electrochemical method, a technique for measuring a lower concentration target substance with high sensitivity is important. As a technique for performing high-sensitivity measurement, there is a technique that includes two electrodes (a generator electrode and a collector electrode) that are configured by arranging working electrodes close to each other on the order of μm (see Patent Document 1 and Non-Patent Document 1). In this technique, an electrochemical redox cycle is generated on the electrode, and the redox current is repeatedly extracted from one target molecule, thereby increasing the apparent current value and enabling highly sensitive measurement. . By using such an electrode structure, highly sensitive electrochemical detection is possible, and a highly sensitive biosensor using this can be realized.

Japanese Patent Laid-Open No. 01-272958

C.E.Chidsey et al., "Micrometer-Spaced Platinum Interdigitated Array Electrode: Fabrication, Theory, and Initial Use", Anal. Chem., Vol.58, pp.601-607, 1986. K. Hayashi et al., "Development of Nanoscale Interdigitated Array Electrode as Electrochemical Sensor Platform for Highly Sensitive Detection of Biomolecules", Journal of The Electrochemical Society, vol.155, no.9, pp.J240-J243, 2008. S. Yang et al., "Controllable Adsorption of Reduced Graphene Oxide onto Self-Assembled Alkanethiol Monolayers on Gold Electrodes: Tunable Electrode Dimension and Potential Electrochemical Applications", J. Phys. Chem. C, vol. 114, pp. 4389-4393 , 2010.

  By the way, as described above, in order to perform detection with higher sensitivity using two electrodes brought close to each other on the order of μm, it is important to generate a redox cycle on the electrodes without depleting the target substance. is there. For example, if the intermediate substance reacted at the generator electrode is inactivated before reaching the collector electrode by diffusion or transport, a sufficient redox cycle cannot be generated, and high sensitivity cannot be achieved.

  The degree of sensitivity increase is indicated by the capture rate represented by (reduction current value) / (oxidation current value) × 100. If this value is high, the intermediate substance reacted at the generator electrode reaches the collector electrode while maintaining the electrochemical activity, and the apparent current value increases due to the redox cycle. In order to increase the capture rate, it is effective to shorten the diffusion distance of the target substance or intermediate substance by making the electrode width smaller and making the electrode interval closer (see Non-Patent Document 2).

  However, it is not easy to form a finer electrode pattern, for example, smaller than the μm order, and the most advanced manufacturing technology is required. Moreover, it becomes difficult to obtain an electrode having a uniform size with good reproducibility due to miniaturization of the electrode pattern. Of course, there is a limit to the miniaturization of the electrode pattern. Thus, there is a problem that high sensitivity by miniaturization of the electrode pattern is not easy.

  The present invention has been made to solve the above-described problems, and can achieve high sensitivity without making the electrode pattern very small by electrochemical measurement using two electrodes brought close to each other. The purpose is to do so.

The method for producing an electrode for electrochemical measurement according to the present invention includes a first step of forming a metal layer on a resist pattern layer after forming a resist pattern layer on an insulating layer, and a carbon material made of graphene oxide . A second step of forming a carbon layer made of a carbon material on the metal layer by applying a dispersion solution in which small pieces are dispersed on the metal layer, and an oxidation constituting the carbon layer formed on the metal layer After reducing the graphene, at least a third step of patterning the metal layer and the carbon layer by a lift-off method for removing the resist pattern layer to form a carbon material layer on the two electrodes is provided.

  In the method for producing an electrode for electrochemical measurement, the two electrodes are, for example, two comb-shaped electrodes that alternately enter and are opposed to each other.

  As described above, according to the present invention, a carbon material layer is formed on two electrodes using a dispersion solution in which small pieces of carbon material are dispersed. In the measurement, it is possible to obtain an excellent effect that the sensitivity can be increased without making the electrode pattern very fine.

FIG. 1 is a flowchart for explaining a method for producing an electrode for electrochemical measurement according to Embodiment 1 of the present invention. FIG. 2A is a cross-sectional view schematically showing the configuration of the electrochemical measurement electrode in the step for explaining the method for producing the electrochemical measurement electrode in Embodiment 1 of the present invention. FIG. 2B is a cross-sectional view schematically showing the configuration of the electrochemical measurement electrode in the step for explaining the electrochemical measurement electrode manufacturing method in Embodiment 1 of the present invention. FIG. 2C is a cross-sectional view schematically showing a configuration of the electrochemical measurement electrode in a step for describing the method for manufacturing the electrochemical measurement electrode in Embodiment 1 of the present invention. FIG. 2D is a cross-sectional view schematically showing the configuration of the electrochemical measurement electrode in the step for explaining the method for producing the electrochemical measurement electrode in Embodiment 1 of the present invention. FIG. 2E is a plan view showing a partial configuration of the electrode for electrochemical measurement in a step for explaining the method for manufacturing the electrode for electrochemical measurement in Embodiment 1 of the present invention. FIG. 3 is a configuration diagram showing a configuration of an electrochemical measurement electrode chip. FIG. 4 is a perspective view showing a configuration of a chip of an electrode for electrochemical measurement. FIG. 5 is a characteristic diagram showing observation results of limit currents indicating the oxidation reaction and reduction reaction of pAP in the comb electrode of Example 1. FIG. 6 is a flowchart for explaining a method for producing an electrode for electrochemical measurement according to Embodiment 2 of the present invention. FIG. 7A is a cross-sectional view schematically showing a configuration of an electrode for electrochemical measurement in a step for explaining a method for manufacturing the electrode for electrochemical measurement in Embodiment 2 of the present invention. FIG. 7B is a cross-sectional view schematically showing the configuration of the electrochemical measurement electrode in the step for explaining the production method of the electrochemical measurement electrode in Embodiment 2 of the present invention. FIG. 7C is a cross-sectional view schematically showing a configuration of the electrode for electrochemical measurement in a step for explaining the method for manufacturing the electrode for electrochemical measurement in Embodiment 2 of the present invention. FIG. 7D is a cross-sectional view schematically showing the configuration of the electrochemical measurement electrode in the step for explaining the method for manufacturing the electrochemical measurement electrode in Embodiment 2 of the present invention. FIG. 8 is a characteristic diagram showing observation results of limiting currents indicating the oxidation and reduction reactions of pAP in the comb electrode of Example 2. FIG. 9 is a characteristic diagram showing observation results of responses indicating an oxidation reaction and a reduction reaction of Ru (NH ₃ ) ₆ ²⁺ in the comb electrode of Example 3. FIG. 10 is a cross-sectional view showing a configuration example of an electrode for electrochemical measurement according to the present invention.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

[Embodiment 1]
First, Embodiment 1 of the present invention will be described. FIG. 1 is a flowchart for explaining a method for producing an electrode for electrochemical measurement according to Embodiment 1 of the present invention. In the following, a case will be described in which two electrodes are composed of two comb-shaped electrodes that alternately enter and face each other.

  In this manufacturing method, first, in step S101, a metal layer is formed on an insulating layer. Next, in step S102, a dispersion solution in which small pieces of carbon material are dispersed is applied onto the metal layer to form a carbon layer made of the carbon material on the metal layer. Next, in step S103, the metal layer and the carbon layer are patterned to form a state in which a carbon material layer is formed on the two comb-shaped electrodes that are alternately arranged and face each other.

  According to this manufacturing method, the comb-shaped electrode used in the electrochemical measurement is formed with a carbon material layer thereon. The layer of carbon material is composed of small pieces of carbon material. Thus, by forming the carbon material layer on the comb-shaped electrode, high sensitivity can be achieved without changing (more miniaturizing) the dimensions of the comb-shaped electrode.

  It has been reported that high sensitivity can be achieved by forming (immobilizing) any one of single layer or 2 to 10 layers of multilayer graphene, graphite, and their reduced derivatives on a single working electrode. (See Non-Patent Document 3). In this technique, the above-described carbon material is applied. However, in the comb-shaped electrode having a structure close to the order of μm, when the carbon material is applied as described above, a carbon material layer can be formed on the electrode portion. Is formed, and is electrochemically short-circuited. Thus, if the two comb electrodes are short-circuited, an electrochemical redox cycle cannot be generated on the electrodes, and high sensitivity cannot be realized.

  On the other hand, according to the present embodiment, a carbon material layer is selectively formed on the comb electrode portion, so that there is no short circuit between the two comb electrodes, and an electrochemical redox cycle is performed. It can be generated on the electrode. In addition, since the carbon material layer is formed on the electrode, high sensitivity can be achieved without reducing the size of the electrode pattern of the comb electrode by electrochemical measurement using the comb electrode.

  Hereinafter, the manufacturing method in this Embodiment is demonstrated in detail using FIG. 2A-FIG. 2E. 2A to 2D are cross-sectional views schematically showing the configuration of the electrochemical measurement electrode in each step for explaining the method for producing the electrochemical measurement electrode in Embodiment 1 of the present invention. FIG. 2E is a plan view showing a partial configuration of the electrode for electrochemical measurement.

  First, as shown in FIG. 2A, a resist pattern layer 202 is formed on the insulating layer 201. The resist pattern layer 202 has an opening at a location where a comb electrode to be described later is formed. Next, a metal layer 203 is formed on the insulating layer 201 and the resist pattern 202 as shown in FIG. 2B by depositing a metal on the resist pattern layer 202 by a deposition method such as sputtering.

  Next, by applying a dispersion solution in which small pieces of carbon material are dispersed, the carbon layer 203 is formed on the insulating layer 201 so as to cover the resist pattern layer 202 as shown in FIG. 2C. Layer 204 is formed. Thereafter, by removing the resist pattern layer 202, the metal layer 203 and the carbon layer 204 formed thereon are selectively removed. By this lift-off, as shown in FIGS. 2D and 2E, a state is obtained in which a carbon material layer 207 is formed on the comb-shaped electrodes 205 and 206 which are alternately arranged and opposed to each other.

  Next, it demonstrates in detail using an Example.

[Example 1]
First, a quartz substrate having a thickness of 0.5 mm is prepared. The quartz substrate is a substrate made of an insulating material. An electron beam resist is applied onto the quartz substrate to form a resist layer having a layer thickness of 0.5 mm. Next, the quartz substrate on which the resist layer is formed is pre-baked under heating conditions of 80 ° C. for 60 minutes. This heating may be performed using an oven, for example.

  Next, the pre-baked resist layer is patterned by an electron beam lithography technique. First, the quartz substrate is carried into an exposure chamber of an electron beam exposure apparatus and exposed to a pattern having a shape in which an opening is formed at a position where a comb electrode is formed. Further, after the exposure, development is performed to form a resist pattern layer. The dimension of the pattern of the formed resist pattern layer was set such that, for example, the length of the comb tooth portion of the comb-shaped electrode was 2 mm and the width was 10 μm. Further, the interval between the comb teeth (electrode interval) of two comb-shaped electrode patterns that are alternately arranged and opposed to each other is 0.5 μm.

  Next, a metal layer is formed on the quartz substrate and the formed pattern. For example, a titanium layer having a thickness of 10 nm is first formed by a vacuum vapor deposition apparatus, and a gold layer having a thickness of 100 nm is formed thereon. By forming the gold layer via the titanium layer, peeling of the gold layer is suppressed.

  Next, a dispersion solution in which small pieces of carbon material are dispersed is applied on the metal layer (gold layer) described above. For example, as the carbon material, graphene oxide formed by chemically oxidizing graphite is used. More specifically, first, natural graphite (1 g) pulverized by a ball mill and concentrated sulfuric acid (34.5 ml) are mixed, and sodium nitrate (0.75 g) is added to the mixture with a stirring bar. These mixtures are placed under ice cooling, and potassium permanganate (4.5 g) is gradually added, and stirring is continued for 2 hours. After this, the mixture is allowed to return to room temperature and stirring is continued for a further 5 days. This results in a dark gray product.

  Next, 5% dilute sulfuric acid (100 milliliters) and hydrogen peroxide (3 milliliters) were added to the resulting product and stirred to form a liquid, and this was further mixed with an excess amount of sulfuric acid (3%) / excess. Disperse it in a mixed solution of hydrogen oxide water (0.5%), and collect the precipitate by centrifugation. Subsequently, pure water is added to the precipitate for dispersion, and the precipitate is collected by centrifugation. These results in graphene oxide as a dark brown oily substance as the final product.

  The graphene oxide obtained as described above is added to pure water and stirred to obtain a dispersion solution (uniformly dispersed aqueous solution) in which the carbon material (graphene oxide) is dispersed. Here, the concentration may be about 0.01 to 0.1 wt%. The obtained dispersion aqueous solution becomes brown. From the atomic force microscope observation and the Raman spectroscopic analysis, it has been confirmed that the substance in this dispersion solution is a single-layer to three-layer graphene oxide piece (film piece) as a main component.

  Next, the dispersion solution (about 0.1 wt% aqueous solution) is applied to the resist pattern layer and the quartz substrate on which the metal layer is formed. For example, it may be applied by spin coating. By this application, the dispersed carbon material adheres and is fixed on the metal layer, and a carbon layer can be formed. As described above, since the carbon material is used as an oxidant, a state in which small pieces of the carbon material are uniformly dispersed is obtained in the dispersion solution, and since this dispersion solution is applied, the carbon layer is formed on the metal layer. A carbon layer composed of small pieces of material can be formed uniformly.

  Next, the quartz substrate on which the resist pattern layer, the metal layer, and the carbon layer are formed is sealed in a sealed container together with a solution in which a 35% hydrazine aqueous solution and a 28% ammonia aqueous solution are mixed at a volume ratio of 7:10. As a result, the carbon layer is exposed to hydrazine / ammonia vapor. By leaving this state at 95 ° C. for 1 hour, the carbon material of the carbon layer formed on the surface of the metal layer can be reduced. By this reduction, the carbon layer is composed of graphene (graphene oxide reductant) obtained by reducing single-layer to three-layer graphene oxide.

  After reducing the carbon layer as described above, the quartz substrate is immersed in methyl ethyl ketone, and subjected to ultrasonic treatment to peel off the resist pattern layer. By this peeling, the metal layer and the carbon layer on the resist pattern layer are selectively removed (lift-off), and a layer of carbon material is formed on the two comb-shaped electrodes that are alternately arranged and opposed to each other. State is obtained.

  Next, spin-on-glass is applied on the quartz substrate by spin coating, and heat-cured at 450 ° C. to form an insulating protective film. Next, an electron beam resist is applied again to form a resist layer having a layer thickness of 0.5 mm. Next, the quartz substrate on which the resist layer is formed is pre-baked under heating conditions of 80 ° C. for 60 minutes. Next, the pre-baked resist layer is patterned by an electron beam lithography technique.

  First, the quartz substrate is carried into an exposure chamber of an electron beam exposure apparatus and exposed. Further, after the exposure, development is performed to form a resist pattern layer. With this resist pattern layer, a part of the reference electrode and a part of the counter electrode formed on the comb electrode and the other region on the quartz substrate are covered.

Next, the insulating protective film is selectively removed by reactive ion etching with CF ₄ gas using the resist pattern layer as a mask, and the comb electrode, a part of the reference electrode, and a part of the counter electrode are exposed. The other regions except these are covered and protected with an insulating protective film.

  As described above, the electrochemical measurement electrode tip (electrode tip 304) shown in FIG. 3 was formed. The electrode chip 304 includes a working electrode 301, a counter electrode 302, and a reference electrode 303, which are two comb electrodes facing each other at a comb tooth portion. Each electrode is connected to a potentiostat 305 and a coulometer 306. The potentiostat 305 and the coulometer 306 are connected to the recorder 307. The potentiostat 305 controls the current flowing through the working electrode 301 and the counter electrode 302 so that the voltage between the working electrode 301 and the reference electrode 303 becomes a set value, and the coulometer 306 flows into the working electrode 301. Measure the current.

  For example, as shown in the perspective view of FIG. 4, the electrode chip 304 has a working electrode 301, a counter electrode 302, and a reference electrode 303 disposed on a substrate 300 having a plate thickness of 0.5 mm and a size of 12 × 20 (mm). Has been. For example, as shown in the perspective view of FIG. 4, a sample is dropped from a burette 401 onto two comb electrode portions facing each other at the comb tooth portion of the working electrode 301. Further, the electrode terminal 301 a and the electrode terminal 302 b are connected to the working electrode 301, the electrode terminal 302 a is connected to the counter electrode 302, and the electrode terminal 303 is connected to the reference electrode 303. By using the working electrode 301 composed of two comb-shaped electrodes facing each other at the above-described comb tooth portion, the amplification effect by the well-known redox cycle can be used.

  Electrochemical detection of the electrochemically active species obtained by the enzyme reaction was performed using the electrode tip described above. Alkaline phosphatase (ALP) was used as the enzyme, p-aminophenyl phosphate (pAPP) was reacted as a substrate, and the oxidation-reduction reaction of p-aminophenol (pAP) as a reaction product was detected on the electrode. Each electrode terminal is connected to a bipotentiostat (potentiostat 305) through a lead wire, and one pair of comb-shaped electrodes is scanned at a potential of 50 mV / sec from -0.3 V to 0.3 V, and the other The comb electrodes were fixed at a potential of −0.3 V, and the response current of each electrode was measured.

  One comb electrode exhibited a pAP oxidation reaction, and the other comb electrode exhibited a limiting current indicating a reduction reaction. As shown in FIG. 5, the magnitude of the peak current value of one comb-shaped electrode and the other comb-shaped electrode (solid line) is the case where the same-shaped comb-shaped electrode composed of gold without a carbon material layer is used (dotted line). Compared to, it increased about twice or more. Furthermore, from the magnitude of the current value of both, it can be seen that the ratio (trapping rate) of the molecules that have undergone the reduction reaction at the other comb electrode out of the pAP molecules oxidized at one comb electrode is 97%. Compared with the case where the same-shaped comb-shaped electrode in which no carbon material layer was formed was used, a higher capture rate of about 13% was obtained.

  The degree of improvement of the capture rate is almost comparable when the shape of the interdigitated electrodes is 2 mm in length, the electrode width is 2 μm, and the electrode interval is 2 μm. Thus, according to this Embodiment 1 (Example 1), a comb-shaped electrode is used, and a carbon material layer is formed on the surface of this electrode, whereby the reaction rate and diffusion rate can be increased, and the electrode width In addition, the capture rate is improved without miniaturizing the electrode spacing, and highly sensitive measurement is possible.

[Embodiment 2]
Next, a second embodiment of the present invention will be described. FIG. 6 is a flowchart for explaining a method for producing an electrode for electrochemical measurement according to Embodiment 2 of the present invention. In this manufacturing method, first, in step S601, two comb-shaped electrodes that alternately enter and face each other on the insulating layer are formed. Next, in step S602, a dispersion solution in which small pieces of carbon material are dispersed is applied on an insulating layer on which two comb-shaped electrodes are formed to form a carbon material layer. Here, it is assumed that small pieces of carbon material having a size smaller than the distance between the two comb electrodes are dispersed in the dispersion solution. In addition, the dimension of a small piece means the dimension of the longest location of a small piece.

  According to this manufacturing method, as in the first embodiment described above, the comb electrode used in the electrochemical measurement is formed with the carbon material layer thereon. Thus, by forming the carbon material layer on the comb-shaped electrode, high sensitivity can be achieved without changing the dimensions of the comb-shaped electrode.

  According to the second embodiment, the carbon material exists in a region other than the comb electrode, for example, between the two comb electrodes together with the comb electrode portion. However, since the size of the small pieces of the carbon material is smaller than the interval between the comb electrodes, the small pieces of the carbon material existing between the electrodes do not connect (short-circuit) between the electrodes. As described above, also in the second embodiment, as in the first embodiment described above, an electrochemical redox cycle can be generated on the electrodes without short-circuiting the two comb electrodes. . In addition, since the carbon material layer is formed on the electrode, high sensitivity can be achieved without reducing the size of the electrode pattern of the comb electrode by electrochemical measurement using the comb electrode.

  Hereinafter, the manufacturing method according to the second embodiment will be described in more detail with reference to FIGS. 7A to 7D. 7A to 7D are cross-sectional views schematically showing the configuration of the electrochemical measurement electrode in each step for explaining the method for manufacturing the electrochemical measurement electrode in Embodiment 2 of the present invention.

  First, as shown in FIG. 7A, a resist pattern layer 702 is formed on the insulating layer 701. The resist pattern layer 702 has an opening at a location where a comb electrode, which will be described later, is formed. Next, a metal layer 703 is formed on the insulating layer 701 and the resist pattern layer 702 as shown in FIG. 7B by depositing a metal on the resist pattern layer 702 by a deposition method such as sputtering. .

  Next, by removing the resist pattern layer 702, the metal layer 703 formed thereon is selectively removed. By this lift-off, as shown in FIG. 7C, two comb-shaped electrodes 705 and 706 are formed which are alternately placed and opposed to each other.

  Next, a dispersion solution in which a carbon material is dispersed is applied over the insulating layer 703 on which the comb electrodes 704 and 706 are formed. In the dispersion solution, small pieces of carbon material having a size smaller than the distance between the two comb electrodes are dispersed. By this application, as shown in FIG. 7D, a carbon material layer 708 made up of a plurality of pieces (film pieces) 707 of carbon material is formed. A plurality of small pieces 707 are formed over the entire area on the insulating layer 703 and the comb-shaped electrodes 704 and 706. However, since the size of the small piece 707 is smaller than the distance between the electrodes, the small piece 707 existing between the electrodes does not cause a short circuit.

  Next, it demonstrates in detail using an Example.

[Example 2]
Example 2 will be described below. First, a quartz substrate having a thickness of 0.5 mm is prepared. The quartz substrate is a substrate made of an insulating material. An electron beam resist is applied onto the quartz substrate to form a resist layer having a layer thickness of 0.5 mm. Next, the quartz substrate on which the resist layer is formed is pre-baked under heating conditions of 80 ° C. for 60 minutes. This heating may be performed using an oven, for example.

  Next, the pre-baked resist layer is patterned by an electron beam lithography technique. First, the quartz substrate is carried into an exposure chamber of an electron beam exposure apparatus and exposed to a pattern having a shape in which an opening is formed at a position where a comb electrode is formed. Further, after the exposure, development is performed to form a resist pattern layer. The dimension of the pattern of the formed resist pattern layer was such that the length of the comb tooth portion of the comb-shaped electrode was 2 mm and the width was 2 μm. Further, the distance between the opposing comb teeth (electrode distance) of the two comb-shaped electrode patterns that are alternately arranged and opposed to each other was 2 μm.

  Next, a metal layer is formed on the quartz substrate and the formed pattern. For example, a titanium layer having a thickness of 10 nm is first formed by a vacuum vapor deposition apparatus, and a gold layer having a thickness of 100 nm is formed thereon. By forming the gold layer via the titanium layer, peeling of the gold layer is suppressed. Next, the quartz substrate is immersed in methyl ethyl ketone, and ultrasonic treatment is performed to peel off the resist pattern layer. By this peeling, the metal layer on the resist pattern layer is selectively removed (lift-off), and two comb-shaped electrodes are formed which alternately enter and face each other. Thus, in Example 2, the comb electrode is made of gold.

  Next, a dispersion solution to be applied is prepared. First, natural graphite (1 g) pulverized by a ball mill and concentrated sulfuric acid (34.5 ml) are mixed, and sodium nitrate (0.75 g) is added to the mixture while stirring. These mixtures are placed under ice cooling, and potassium permanganate (4.5 g) is gradually added, and stirring is continued for 2 hours. After this, the mixture is allowed to return to room temperature and stirring is continued for a further 5 days. This results in a dark gray product.

  Next, 5% dilute sulfuric acid (100 milliliters) and hydrogen peroxide (3 milliliters) are added to the resulting product and stirred to form a liquid, and this is further mixed with an excess amount of sulfuric acid (3%) / excess. Disperse it in a mixed solution of hydrogen oxide water (0.5%), and collect the precipitate by centrifugation. Subsequently, pure water is added to the precipitate for dispersion, and the precipitate is collected by centrifugation. These results in graphene oxide as a dark brown oily substance as the final product.

  The graphene oxide obtained as described above is added to pure water and stirred to obtain a dispersion solution (uniformly dispersed aqueous solution) in which the carbon material (graphene oxide) is dispersed. Here, the concentration may be about 0.01 to 0.1 wt%. The obtained dispersion aqueous solution becomes brown. From the atomic force microscope observation and the Raman spectroscopic analysis, it has been confirmed that the substance in this dispersion solution is a single-layer to three-layer graphene oxide piece (film piece) as a main component.

  Next, 5 μl, 28% of 35% hydrazine aqueous solution is added to 10 ml of a dispersion solution (approximately 0.025 wt% aqueous solution) containing a small piece (film piece) of a carbon material mainly composed of the above-described single-layer to three-layer graphene oxide. Add 35 μl of aqueous ammonia and leave at 95 ° C. for 1 hour. By these things, the dispersion solution of the reduced body in which the graphene oxide was reduced is obtained.

  The dispersion was centrifuged at 5000 rpm to remove the precipitate, and then the supernatant obtained by further centrifuging the supernatant solution at 10,000 rpm was taken out. It was confirmed by atomic force microscope observation that this precipitate did not exceed 2 μm (the length of the longest part of the membrane piece). Using this precipitate, a dispersion solution (about 0.1 wt% aqueous solution) containing small pieces of graphene oxide reductant having a size not exceeding 2 μm is prepared.

  Next, the comb-shaped electrode formed on the quartz substrate is immersed in 1N dilute sulfuric acid, and in this state, the potential is swept to remove the surface oxide, and then the 2-naphthalenethiol ethanol solution adjusted to 10 mM is used. Immerse overnight to form a 2-naphthalenethiol layer (binder layer) on the surface of the comb-shaped electrode. Since 2-naphthalenethiol constituting the binder layer has a covalent bond between its own SH group and gold on the surface of the comb electrode, the binder layer is fixed (adsorbed) on the surface of the comb electrode. On the other hand, the binder layer is not formed on the surface (exposed surface) of the quartz substrate that does not form a covalent bond as described above.

  Next, on the quartz substrate provided with the comb-shaped electrode on which the binder layer is formed, the dispersion solution of the above-described reductant is applied. For example, it may be applied by spin coating. By this coating, small pieces of graphene (reduced form) in the dispersion solution are adsorbed by the interaction (hydrophobic interaction) of 2-naphthalenethiol constituting the binder layer with the naphthalene ring. As a result, a layer of carbon material made of graphene pieces is selectively formed on the surface of the comb-shaped electrode. In addition, since the graphene piece has a size smaller than the distance between the electrodes, even if it adheres to the quartz substrate between the electrodes, the electrodes are not short-circuited.

  In this way, the electrochemical measurement electrode tip (electrode tip 304) shown in FIG. 3 was formed. The electrode chip 304 includes a working electrode 301, a counter electrode 302, and a reference electrode 303, which are two comb electrodes facing each other at a comb tooth portion. Each electrode is connected to a potentiostat 305 and a coulometer 306. The potentiostat 305 and the coulometer 306 are connected to the recorder 307. The potentiostat 305 controls the current flowing through the working electrode 301 and the counter electrode 302 so that the voltage between the working electrode 301 and the reference electrode 303 becomes a set value, and the coulometer 306 flows into the working electrode 301. Measure the current.

  For example, as shown in the perspective view of FIG. 4, the electrode chip 304 has a working electrode 301, a counter electrode 302, and a reference electrode 303 disposed on a substrate 300 having a plate thickness of 0.5 mm and a size of 12 × 20 (mm). Has been. For example, as shown in the perspective view of FIG. 4, a sample is dropped from a burette 401 onto two comb electrode portions facing each other at the comb tooth portion of the working electrode 301. Further, the electrode terminal 301 a and the electrode terminal 302 b are connected to the working electrode 301, the electrode terminal 302 a is connected to the counter electrode 302, and the electrode terminal 303 is connected to the reference electrode 303. By using the working electrode 301 composed of two comb-shaped electrodes facing each other at the above-described comb tooth portion, the amplification effect by the well-known redox cycle can be used.

  Electrochemical detection of the electrochemically active species obtained by the enzyme reaction was performed using the electrode tip described above. Alkaline phosphatase (ALP) was used as the enzyme, p-aminophenyl phosphate (pAPP) was reacted as a substrate, and the oxidation-reduction reaction of p-aminophenol (pAP) as a reaction product was detected on the electrode. Each electrode terminal is connected to a bipotentiostat (potentiostat 305) through a lead wire, and one pair of comb-shaped electrodes is scanned at a potential of 50 mV / sec from -0.3 V to 0.3 V, and the other The comb electrodes were fixed at a potential of −0.3 V, and the response current of each electrode was measured.

  A limiting current indicating an oxidation reaction of pAP was observed in one comb-shaped electrode and a reduction reaction was observed in the other comb-shaped electrode. As shown in FIG. 8, the magnitude of the peak current value by one comb-shaped electrode and the other comb-shaped electrode (solid line) is compared with the case of using the same shaped comb-shaped electrode made of gold without a carbon raw material layer (dotted line). And increased more than twice.

  Furthermore, from the magnitude of the current value of one comb electrode and the other comb electrode, the ratio (trapping rate) of the molecules that have caused a reduction reaction in the other comb electrode out of the pAP molecules oxidized by the one comb electrode is It was found to be 99%, and a higher capture rate of about 2% was obtained compared to the case of using the same comb-shaped electrode in which no carbon material layer was formed.

  As described above, according to Example 2, using a comb-shaped electrode and forming a carbon material layer on the electrode surface, the reaction rate and the diffusion rate can be increased, and the electrode width and the electrode spacing can be reduced. The capture rate is improved without miniaturization, and highly sensitive measurement is possible.

[Example 3]
Next, Example 3 will be described. First, a quartz substrate having a thickness of 0.5 mm is prepared. The quartz substrate is a substrate made of an insulating material. An electron beam resist is applied onto the quartz substrate to form a resist layer having a layer thickness of 0.5 mm. Next, the quartz substrate on which the resist layer is formed is pre-baked under heating conditions of 80 ° C. for 60 minutes. This heating may be performed using an oven, for example.

  Next, the pre-baked resist layer is patterned by an electron beam lithography technique. First, the quartz substrate is carried into an exposure chamber of an electron beam exposure apparatus and exposed to a pattern having a shape in which an opening is formed at a position where a comb electrode is formed. Further, after the exposure, development is performed to form a resist pattern layer. The dimension of the pattern of the formed resist pattern layer was such that the length of the comb tooth portion of the comb-shaped electrode was 2 mm and the width was 10 μm. In addition, the distance between the opposing comb teeth (electrode distance) of the two comb-shaped electrode patterns that are alternately arranged and opposed to each other was set to 5 μm.

  Next, a metal layer is formed on the quartz substrate and the formed pattern. For example, a titanium layer having a thickness of 10 nm is first formed by a vacuum vapor deposition apparatus, and a gold layer having a thickness of 100 nm is formed thereon. Next, the quartz substrate is immersed in methyl ethyl ketone, and ultrasonic treatment is performed to peel off the resist pattern layer. By this peeling, the metal layer on the resist pattern layer is selectively removed (lift-off), and two comb-shaped electrodes are formed which alternately enter and face each other.

  Next, similarly to Example 2 described above, a dispersion solution including small pieces (film pieces) of a carbon material containing the above-described single-layer to three-layer graphene oxide as a main component is prepared. Further, similarly to Example 2 described above, reduction using a hydrazine aqueous solution and an ammonia aqueous solution produces a dispersion of a reduced product in which graphene oxide is reduced. Next, 10 ml of the prepared dispersion solution (about 0.025 wt% aqueous solution) is centrifuged at 5000 rpm to remove the precipitate, and then the supernatant solution is filtered using a filter having a pore size of 2 μm, and the filtrate is used to obtain 2 μm. A dispersion solution (about 0.1 wt% aqueous solution) containing small pieces (film pieces) of graphene (reduced form of graphene oxide) having a size not exceeding 1 mm is prepared. It was confirmed by atomic force microscope observation that the size of the precipitate (the size of the longest part of the film piece) did not exceed 2 μm.

  Next, the comb-shaped electrode formed on the quartz substrate is immersed in 1N dilute sulfuric acid, and in this state, the potential is swept to remove the surface oxide, and then the 2-naphthalenethiol ethanol solution adjusted to 10 mM is used. Immerse overnight to form a 2-naphthalenethiol layer (binder layer) on the surface of the comb-shaped electrode.

  Next, the above-mentioned dispersion solution is applied on a quartz substrate provided with a comb-shaped electrode on which a binder layer is formed. For example, it may be applied by spin coating. By this coating, the small pieces of graphene in the dispersion solution are adsorbed by the interaction (hydrophobic interaction) with the naphthalene ring of 2-naphthalenethiol constituting the binder layer. As a result, a layer of carbon material made of graphene pieces is selectively formed on the surface of the comb-shaped electrode. In addition, since the graphene piece has a size smaller than the distance between the electrodes, even if it adheres to the quartz substrate between the electrodes, the electrodes are not short-circuited.

In this way, the electrochemical measurement electrode tip (electrode tip 304) shown in FIG. 3 was formed. Using this electrode chip, the oxidation-reduction reaction of 1 mM hexaammineruthenium ion (Ru (NH ₃ ) ₆ ^{3 + / 2 +} ) KCl solution was detected. The terminal portion of the electrode for electrochemical measurement of the electrode tip is connected to the bipotentiostat via the lead wire, and the range from −0.4 V to 0.2 V is periodically changed to 50 mV / The potential was scanned in seconds, and the response current of the electrode was measured.

As shown by the solid line in FIG. 9, an oxidation reaction of Ru (NH ₃ ) ₆ ²⁺ was observed at −0.15 V, and a response indicating a reduction reaction of Ru (NH ₃ ) ₆ ³⁺ was observed at −0.22 V. Observed. The magnitude of the peak current value on the oxidation side increased about twice as compared with the case where the same comb-shaped electrode made of gold without a carbon raw material layer was used (dotted line in FIG. 9). Moreover, the magnitude of the peak current value on the reduction side increased 2.5 times or more as compared with the case where the same-shaped comb electrode made of gold without a carbon raw material layer was used.

This is because the detection target cation is attracted to the surface of the negatively charged carbon raw material layer (graphene) on the comb-shaped electrode, and the redox species is easily transported to the surface. Indicates an increase. In particular, in the reduction reaction of Ru (NH ₃ ) ₆ ³⁺ having a larger positive charge, the above-described effect is remarkable.

  As described above, according to Example 3, by using a comb-shaped electrode and forming a carbon material layer on the electrode surface, the reaction rate and the diffusion rate can be increased, and the electrode width and the electrode spacing can be reduced. The capture rate is improved without miniaturization, and highly sensitive measurement is possible.

  The present invention is not limited to the embodiment described above, and many modifications and combinations can be implemented by those having ordinary knowledge in the art within the technical idea of the present invention. It is obvious. For example, in the above description, the comb electrode has been described as an example, but the present invention is not limited to this. For example, even two ring disk electrodes that are used close to each other are the same as described above, and the present invention can be applied to at least two electrodes that are used close to, for example, about several μm.

  In addition, for example, in the above description, the carbon material layer is formed via the binder layer made of 2-naphthalenethiol. However, the present invention is not limited to this, and any organic compound having a thiol group can be used as the binder layer. Function. In such an organic compound, the thiol group is covalently bonded to gold constituting the electrode, and the carbon raw material is adsorbed to the organic group by interaction with the organic group (hydrophobic interaction). Alternatively, a carbon material layer may be formed directly on the electrode.

  10A, a recess 1001a is formed in the insulating layer 1001, an electrode 1002 is formed so as to be embedded in the recess 1001a, and a carbon material layer 1003 is formed over the electrode 1002. Good. Further, as shown in FIG. 10B, a carbon material layer 1003 may be formed on the electrode 1002 formed so as to be embedded in the recess 1001a with a binder layer 1004 interposed therebetween. By doing so, the surface of the insulating layer becomes flatter in a state where the electrode is formed, so that it is easier to form an electrode or the like by the lift-off method.

  The electrode material is not limited to gold, and copper, nickel, platinum, carbon, or the like may be used.

  In the above description, the case where the carbon material layer is made of graphene (reduced body) has been described. However, the present invention is not limited to this, and may be made of, for example, graphite. In this case, the graphite may be one in which about 2 to 10 layers of graphene are stacked.

  DESCRIPTION OF SYMBOLS 201 ... Insulating layer, 202 ... Resist pattern layer, 203 ... Metal layer, 204 ... Carbon layer, 205, 206 ... Comb-shaped electrode, 207 ... Layer of carbon material.

Claims (2)

A first step of forming a resist pattern layer on the insulating layer and then forming a metal layer on the resist pattern layer ;
A second step of applying a dispersion solution in which small pieces of carbon material made of graphene oxide are dispersed on the metal layer to form the carbon layer made of the carbon material on the metal layer;
After reducing the graphene oxide forming the carbon layer formed on the metal layer, the metal layer and the carbon layer are patterned by a lift-off method for removing the resist pattern layer, and two electrodes are formed. A method for producing an electrode for electrochemical measurement, comprising: a third step in which a layer of the carbon material is formed thereon. The manufacturing method of claim 1 Symbol placement of electrochemical measurement electrode,
The method of manufacturing an electrode for electrochemical measurement, wherein the two electrodes are two comb-shaped electrodes that alternately enter and face each other.