JPH08247988A - Electrochemical detector and manufacture thereof - Google Patents

Abstract

PURPOSE: To improve the high current density and the S/N ratio by forming a plurality of infinitesimal patterns of conductor in which electrochemical reaction of substance to be measured easily occurs on a conductor in which the reaction scarcely occurs. CONSTITUTION: In the case of a conductor in which electro-chemical reaction scarcely occurs in substance to be measured such as hydrogen peroxide, as a conductor material in which the reaction easily occurs, a combination of metal having large electrochemical reaction velocity of hydrogen peroxide such as platinum, cobalt and other metal semiconductor such as carbon is included. As a method for forming a fine electrode pattern having 10μm or less on a conductor, since the conductor in which the reaction of the substance to be measured does not occur is substrate electrode, fine electrode pattern aggregate (fine array electrode) is formed on an insulating board by a plating method. Accordingly, since the substance to be measured is concentrically diffused on the fine electrode, a high current density is obtained. The substance to be measured is not reacted at the part except the patterns, and base line noise is extremely low. As a result, the S/N ratio is improved.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chemical sensor, a biosensor, an electrochemical detector applied to liquid phase chromatography and flow injection analysis, and a method for producing the same.

[0002]

2. Description of the Related Art Electrochemical detection is used to detect hydrogen ion concentration in a solution, ions present in a trace amount, and trace components in a living body such as hormones and sugars. For compounds existing in the living body, electrochemical measurement is widely used for measuring biogenic amines such as catecholamines and indoleamines, sugars such as glucose and fructose, acetylcholine and amino acids. Among these, biogenic amines directly undergo an oxidation-reduction reaction on the electrode, and thus are easily detected using a carbon electrode or the like. On the other hand, other substances do not react directly on the electrodes. Therefore, saccharides, acetylcholine, and choline are quantified by a method of generating hydrogen peroxide by an enzymatic reaction of an oxidase and detecting it by an electrode reaction, or by measuring a decrease amount of oxygen consumed by the enzymatic reaction. . On the other hand, amino acids are detected by electrochemical reaction after binding an electrochemically active substance to the substance to be measured and derivatization. Noble metals (gold, platinum) often used for electrochemical analysis in the measurement of hydrogen peroxide produced by enzymatic reaction and the measurement of oxygen consumed by enzymatic reaction.
Hydrogen peroxide and oxygen are widely used in carbon and carbon electrodes because they are most easily oxidized on platinum. However, the detection of saccharides, which is widely used in the diagnosis of diabetes, requires the quantification of a substance to be measured at a considerably high concentration, so the platinum electrode surface becomes contaminated during continuous measurement or measurement of many samples. Then, the reaction rate of hydrogen peroxide decreases significantly, and even if the concentration of the substance to be measured increases, the electrode reaction of hydrogen peroxide cannot follow and the signal is saturated. Further, when a sample having the same concentration is measured with the electrodes, the change of the signal with time is large.
In order to solve this, a method of plating platinum black on the electrode is known. Platinum black has an actual surface area that is 1000 times or more that of a normal platinum electrode when the apparent areas are the same, so that signal saturation is unlikely to occur even when a high concentration of hydrogen peroxide is measured. In the measurement of sugars, platinum black is generally plated on a platinum electrode to measure a sample with a high concentration of the substance to be measured, but conversely, a detector for detecting acetylcholine / choline, which requires measurement of a low concentration sample, is used. Improvement of the detection limit of is required. In acetylcholine and choline analysis, after separating both by column, acetylcholine is converted to choline by cholinesterase, then oxidized by choline oxidase, choline is directly oxidized by choline oxidase, and hydrogen peroxide generated is detected by platinum electrode. . Furthermore, as a biosensor electrode that can be used in a living body, a microelectrode plated with a platinum wire electrode in which platinum black is incorporated with an enzyme has been reported.

Since the platinum black electrode has a very large surface area,
The reaction rate of hydrogen peroxide per apparent electrode area is large and a very large current value can be obtained. However, the noise increases as the electrode surface area increases. As a result, it is suitable for saccharide detection that requires high concentration measurement, but it also increases noise when used for detection of neurotransmitters such as acetylcholine and choline, which require low detection limits.
It was not possible to obtain a low detection limit. On the other hand, if the platinum electrode is used as it is, the reactivity of the electrode with respect to hydrogen peroxide gradually decreases, and there is a drawback that the stability is poor. In recent years, there have been many methods for measuring trace substances with high sensitivity using microelectrodes. With a microelectrode, a high current density is obtained because the amount of the substance to be measured that diffuses per unit area is large, and S /
Since the N ratio is improved, the detection limit can be improved.
However, although a single microelectrode has a very small area, a high current density can be obtained, but the absolute current value is extremely small. By integrating a large number of microelectrodes into a microarray, it is possible to improve the current density and the detection limit while maintaining the absolute value of the current. 3 and 4
Is a schematic diagram of a microarray electrode that has been reported so far,
FIG. 3 is a schematic diagram of the structure of an electrode in which conductive fibers are enclosed in an insulator and the end face is polished, and FIG. 4 is a schematic diagram of the structure of an electrode produced by a lithography (fine processing) technique. Code 11
Is an insulating polymer, 12 is a platinum wire, 13 is a minute circular pattern electrode, 14 is an insulating film, 15 is a platinum thin film, and 16 is an insulating substrate. That is, the microarray electrode is obtained by bundling platinum wires as shown in FIG. 3 and enclosing it in a polymer and polishing the end face to obtain a microdisk electrode, or as shown in FIG. 4, insulating on a platinum thin film formed on a substrate. A method is used in which a film is formed, and thereafter, a large number of minute circular patterns or band-shaped patterns are formed by photolithography and dry etching to form a microarray. However, in the method of encapsulating platinum fiber, it is difficult to obtain a pattern of several microns or less because the electrode size is determined by the diameter of the fiber. Further, even in the case of using a lithography technique, it is necessary to use an electron beam lithography method to form an ultrafine array electrode of submicron or smaller, and it takes time to prepare a large number of electrode patterns. There was a drawback. Further, in the electrode production using the lithographic technique, there are drawbacks such as an increase in the electrode manufacturing cost due to the fact that the apparatus is expensive and the number of steps is increased.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to provide an electrochemical detector and a method of manufacturing the same which ameliorates the above-mentioned drawbacks of the prior art.

[0005]

The present invention will be summarized as follows. The first invention of the present invention is an invention relating to an electrochemical detector, in which an electrochemical reaction of a substance to be measured is extremely unlikely to occur. It is characterized in that a plurality of conductor patterns of 10 μm or less in which the substance to be measured easily causes an electrochemical reaction are formed. A second invention of the present invention relates to a method of manufacturing an electrochemical detector, wherein in the method of manufacturing the electrochemical detector of the first invention, an electrochemical reaction easily occurs with a substance to be measured. It is characterized in that the conductor pattern is produced by a plating method. A third invention of the present invention relates to another method for manufacturing an electrochemical detector, wherein in the method for manufacturing the electrochemical detector of the first invention, the substance to be measured and the electrochemical reaction are It is characterized in that a pattern of a conductor which is easily generated is formed by forming a thin film of an aromatic compound having metal molecules coordinated on an insulating substrate and thermally decomposing the thin film.

Among the electrochemically active substances, those having a low electron transfer reaction rate with the electrode have a great difference in the potential of the electrochemical reaction depending on the state of the electrode surface and the material of the electrode. For example, hydrogen peroxide has a sufficient oxidation reaction at 0.5 V with respect to a silver / silver chloride reference electrode at a platinum electrode, but a carbon electrode,
The gold electrode hardly reacts at the same potential. In the present invention, by utilizing this effect, a large number of microelectrode patterns are produced on an electric conductor on which a substance to be measured does not cause an electrochemical reaction by using an electrode material on which the substance to be measured easily causes an electrochemical reaction. As a result, the formed minute patterns act as ultra-fine electrodes for the substance to be measured, and the substance to be measured concentrates and diffuses only on the minute electrodes, so that a large current density is maintained and the same as with the minute electrodes. The inventors have found that a high current density can be achieved and high sensitivity can be realized, and the present invention has been completed.

Hereinafter, the present invention will be described in detail. In general, a fine island-shaped conductive pattern can be obtained on an insulating substrate by limiting the amount of a conductor to be formed by a thin film forming method such as an electroless plating method, a vapor deposition method and a sputtering method.
However, since the formed fine electrode patterns are not electrically connected to each other, they cannot be used as an assembly of microelectrodes without connecting each with a lead. In the present invention, since there is a conductor that does not cause an electrochemical reaction of the substance to be measured as the base electrode, an assembly of microelectrodes (plating method, vapor deposition, sputtering method, etc., without using a microfabrication technique such as a lithography method ( It has a feature that a micro array electrode) can be obtained.

In each of the following examples, when the substance to be measured is hydrogen peroxide, platinum or cobalt is used as a combination of a conductor in which the substance to be measured hardly causes an electrochemical reaction and a conductor material in which an electrochemical reaction easily occurs. Examples include a combination of a metal having a high electrochemical reaction rate of hydrogen peroxide and another metal, carbon, and a semiconductor such as tin oxide or indium oxide. When the substance to be measured is oxygen, a combination of a metal such as platinum and cobalt having a high electrochemical reaction rate of oxygen and a thin film of a conductor having a large overvoltage such as carbon can be mentioned. When the substance to be measured is carbon dioxide, a combination of a copper microelectrode and another metal, carbon, a semiconductor and a thin film thereof can be used. Next, as a method for forming a fine electrode pattern on a conductor, a plating method, an evaporation method or a sputtering method in which the amount is extremely limited, a thin film of an organic substance such as phthalocyanine or porphyrin containing metal, and then in a vacuum There is a method of forming fine metal particles having high reactivity with the substance to be measured in the carbon film by thermal decomposition.

[0009]

Embodiments of the present invention will be described below in detail with reference to the drawings. The present invention is not limited to the following examples.

Example 1 A silicon substrate (manufactured by Sumitomo Sitix Co., Ltd.) on the surface of which an oxide film having a thickness of 1 μm was formed was placed in a sputtering apparatus (manufactured by Nippon Seed Co.) with a metal mask, and chromium was deposited on the 10 nm substrate. After that, deposit 100 nm of gold and have a diameter of 3 mm
A gold thin film electrode was prepared. Then, the electrode substrate was connected to a working electrode terminal of a potentiostat (manufactured by Toho Giken) whose potential was controlled by a function generator (manufactured by Toho Giken), and silver / silver chloride was used as a reference electrode and a platinum wire was used as a counter electrode. Was immersed in the platinum plating solution. As the platinum plating solution, about 1 gram of chloroplatinic acid was dissolved in 30 ml of water, and then a solution containing 10 mg of lead acetate was diluted with water to a concentration of 1/10 for use. With respect to the gold thin film electrode connected to the working electrode terminal, with respect to the reference electrode, an electric potential of -1.5 V was applied for 1 second, an electric potential of 3.0 V was applied for 3 seconds, or an electrolysis voltage was -2.2 V for 1 second, 3 respectively. Platinum black was plated at 0.0 V for 3 seconds. 1 and 2 show schematic views of electrode structures produced under the respective electrolysis conditions. That is, FIGS. 1 and 2 are schematic views of the structure of platinum black formed on the gold thin film. Note that FIG. 1 shows a case where an ultra-fine platinum black array with a small plating amount is obtained, and FIG. 2 shows a case where the platinum black plating amount is large. Reference numeral 21 is a gold thin film, 22 is a platinum black pattern,
Reference numeral 23 denotes an insulating substrate. FIG. 1 shows an electrode in which platinum black is formed at a low voltage, and minute disk electrodes are formed at intervals on a gold thin film electrode substrate. On the other hand, FIG.
Shows that almost the entire surface of the gold thin film electrode is covered with the platinum black film, and the plating amount is large. This electrode was attached to a thin layer cell as an electrochemical detector of a chromatography device as shown in FIG. FIG. 5 is a schematic view of a chromatography device for acetylcholine choline analysis.
Is a pump for chromatography, 32 is a carrier solution, 33 is an injector for introducing a sample, 34 is a separation column for separating acetylcholine and choline, 35 is a reaction column in which acetylcholinesterase and choline oxidase are immobilized, and 36 is electrochemical detection. A thin layer cell equipped with a vessel, one block of which is made of stainless steel and also serves as a counter electrode. Further, 37 is a reference electrode, 38
Is an electrode of the present invention, 39 is a potentiostat, and 40 is a waste liquid container. In FIG. 5, acetylcholine and choline introduced from the injector are separated by a separation column so that acetylcholine is eluted first and then choline is eluted, and then introduced into the reaction column. In the reaction column, acetylcholine is converted to choline by cholinesterase, and then oxidized by betaine and hydrogen peroxide by choline oxidase, and the hydrogen peroxide is oxidized on the electrode to obtain a signal. On the other hand, choline is directly oxidized to betaine and hydrogen peroxide by choline oxidase, and the concentration is measured from the signal of hydrogen peroxide like acetylcholine. The electrode potential was set to 0.5 V against the silver / silver chloride electrode for the oxidation of hydrogen peroxide.

5 μl of an aqueous solution containing 1 pmol each of acetylcholine and choline was introduced, and the carrier solution was adjusted to pH 8 with a sodium hydroxide solution with an aqueous solution containing 50 mM sodium hydrogen phosphate and 0.5 mM EDTA. In addition, the flow rate was 120 μl / min and was −1.5 V
The measurement was performed using two types of electrodes plated with platinum black at 2.2V. FIG. 6 is a diagram showing a chromatogram when 1 pmol of acetylcholine and choline were injected. Two current peaks as shown in FIG. 6 were observed 7.6 minutes and 9.2 minutes after the introduction of the sample, the former corresponding to the peak of acetylcholine and the latter corresponding to the peak of choline. The absolute value of the peak is 6.3 nA for acetylcholine and 5.7 nA for choline for the electrodes prepared at -1.5 V.
Was obtained. On the other hand, in the electrodes manufactured at -2.2 V, peaks of 10.5 nA and 9.6 nA were obtained, respectively, and it was found that the peak current also increases as the platinum black increases. In contrast, when the gold thin film electrode was not plated with platinum black, almost no peak current was observed.
When the entire surface of the gold thin film was covered with platinum black, the peak value was almost saturated, and no increase in signal was observed even if the amount of platinum black deposited was increased thereafter. However, when the baseline noise was measured without injecting the sample, the noise increased as the platinum black plating amount increased,
21pA, -2.2V for electrodes plated at -1.5V
A noise of 70.5 pA was observed at the electrode plated with, and the electrode plated at -1.5 V had the highest S / N ratio, and a low detection limit of 10 fmol for acetylcholine and 10.9 fmol for choline was obtained. .
In addition, when the same measurement is performed using a commercially available platinum disk electrode having a diameter of 3 mm (manufactured by Bioanalytical Systems), the detection limit is 50 fmo.
It was l. In the electrode plated with −1.5V, hydrogen peroxide reacts only on the platinum black ultrafine electrode shown in FIG. 1, so that the diffusion of hydrogen peroxide on each platinum black disk electrode becomes hemispherical. A high current density can be obtained. on the other hand,
As the amount of platinum black increases, the number and size of microelectrodes increase, and the distance between the electrodes decreases, so that the diffusion layers overlap. As a result, the effect of the minute current is lost and the current density is reduced.
On the other hand, noise increases as the electrode surface area increases, so the detection limit cannot be improved even if the plating amount is increased.

Example 2 A gold thin film electrode was prepared in the same manner as in Example 1, and a platinum black disk array electrode was formed on the surface thereof. Then, the gold electrode on which the platinum black electrode was formed was put into a phosphate buffer solution (p
H7, 50 mM), and an aqueous glutamate oxidase solution (200 units / m 2) diluted with a phosphate buffer solution.
After dipping in l) for 30 minutes, the albumin solution (5%) and the glutaraldehyde solution (1%) were sequentially dipped to fix glutamate oxidase in the platinum black electrode. Also, an electrode in which the entire surface of the gold thin film electrode was covered with platinum black was prepared in the same manner as in Example 1, and glutamate oxidase was immobilized in the same process. As shown in FIG. 7, these electrodes were placed in a phosphate buffer solution (pH 7, 5) containing various concentrations of glutamate.
The sample was dipped in 0 mM) together with a reference electrode and a counter electrode, and the current value was measured. FIG. 7 is a schematic diagram of an electrochemical cell in which the relationship between glutamate concentration and current is measured. Reference numeral 51 is the electrode shown in Example 5 of the present invention, 52 is a counter electrode, 53 is a reference electrode, and 54 is glutamate. Means a solution containing.
As a result, the detection limit of 0.5 μM was obtained with the gold electrode detector formed with the platinum black disk array electrode, whereas the detection limit was 2 with the electrode entirely covered with the platinum black electrode.
The detection limit was greatly improved by forming a platinum black disk array structure with μM.

Example 3 A commercially available gold disk electrode (made by Bio-Analytical Systems) having a diameter of 3 mm for chromatography was connected to a working electrode terminal of potentiostat (made by Toho Giken Co., Ltd.),
Using a silver / silver chloride as a reference electrode and a platinum wire as a counter electrode, it was immersed in a platinum plating solution. Platinum plating solution is about 1
After dissolving 1 g of chloroplatinic acid in 30 ml of water, 1
The solution to which 0 mg of lead acetate was added was diluted with water to a concentration of 1/10 and used. To the gold thin film electrode connected to the working electrode terminal, a potential of -1.5 V is applied to the reference electrode by 1
Second, a potential of 3.0 V was applied for 3 seconds to prepare a number of submicron platinum black microdisk electrodes on the gold electrode. This electrode was attached to the same chromatography device as in Example 1, and acetylcholine / choline was detected under the same measurement conditions as in Example 1. When an aqueous solution (5 μl) containing 1 pmol of acetylcholine and choline was injected, peaks of 7.3 nA for acetylcholine and 6.6 nA for choline were obtained. Next, when a similar experiment was conducted using a platinum disk electrode having a diameter of 3 mm as a detector, peaks of 4.1 nA for acetylcholine and 3.7 nA for choline were obtained. The noise level was 0.03 nA for the gold electrode with the platinum black microelectrode pattern and 0.08 nA for the platinum disk electrode.
The following results were obtained, and the electrochemical detector of the present invention realized a lower detection limit.

Example 4 A 3-inch silicon wafer having an oxide film on its surface was placed in a quartz tube whose inside can be decompressed as shown in FIG. 8, and an electrode having a diameter of 3 mm and a metal mask having a lead portion were overlaid. It was That is, FIG. 8 is a schematic view of a carbon thin film electrode manufacturing method, in which reference numeral 61 is a quartz tube, and 62 is
4,9,10-perylene tetracarboxylic acid anhydride, 63
Is an electric furnace for sublimation (400 ° C.), 64 is an electric furnace for thermal decomposition (1000 ° C.), 65 is a 3-inch silicon wafer, 66
Means a metal mask. Then 3, 4, 9, 10-
Perylene tetracarboxylic acid anhydride was sublimated at 400 ° C., and a thermal decomposition product was deposited on the substrate on silicon under the conditions of 1000 ° C. and 0.001 Torr. 1 at 1000 ° C
After holding for 5 minutes, it was gradually cooled to obtain a conductive carbon thin film electrode having a surface resistance of about 300 ohms. A platinum black microdisk array was formed on the electrode by the same method as in Example 1. The platinum micro disk formed on the carbon thin film was used in Example 1.
In, a structure very similar to that formed on the gold thin film electrode was obtained. Also, an electrode in which the entire carbon thin film was covered with platinum black was prepared by increasing the amount of platinum black plating. Also,
A carbon thin film not plated with platinum black was also prepared. These electrodes were mounted on the same chromatography device as in Example 1, and acetylcholine / choline was detected under the same measurement conditions as in Example 1. When an aqueous solution (5 μl) containing 1 pmol of acetylcholine and choline was injected, peaks of 8.5 nA for acetylcholine and 7.0 nA for choline were obtained. On the other hand, in the case of electrodes whose entire surface is covered with platinum black, 12.
A peak of 3 nA and 11.2 nA was obtained, and it was found that the increase of platinum black also increased the peak current. Also,
A large peak current was hardly observed with the carbon thin film electrode alone. Next, without injecting the sample,
When the baseline noise was measured, the noise increased as the platinum black plating amount increased, and noise of 22.6 pA was observed at the electrode plated at -1.5 V and 115 pA at the electrode plated at -2.2 V. It was As a result, -1.
The electrode plated at 5V has the highest S / N ratio, 8 fmol for acetylcholine and 9.7 for choline.
It was found that a detection limit of fmol was obtained.

Embodiment 5 FIG. 9 shows a manufacturing process of an electrochemical detector according to Embodiment 5 of the present invention. That is, FIG. 9 is a process diagram of manufacturing a gold comb-shaped microelectrode partially modified with platinum black. Reference numeral 71 is a positive photoresist, 72 is a silicon oxide film, 73 is a silicon substrate,
74 is a chromium / gold laminated thin film, 75 is a resist insulating film, 7
6 means a platinum black microelectrode pattern. A photoresist (FH6400 manufactured by Fuji Hunt Co., Ltd.) was applied to a silicon wafer with a 1 μm oxide film (manufactured by Sumitomo Sitix Co., Ltd.) to a thickness of 1 μm (a). The resist-coated silicon wafer was put in an oven and baked at 90 ° C. for 90 seconds. After that, a mask aligner (Canon PL
F-501) was used for contact exposure for 15 seconds. The exposed silicon wafer was developed in a resist developer (Fuji Hunt Co., Ltd.) at 20 ° C. for 45 seconds, washed with water and dried to transfer the mask pattern to the resist (b). This substrate with resist was mounted in a predetermined position in a sputtering device (manufactured by Nippon Seed Co., Ltd.), and a chromium film was deposited to 10 nm, and then a gold or platinum film was deposited to 100 nm (c). Then, the substrate was immersed in methyl ethyl ketone and subjected to ultrasonic treatment, and the resist other than the electrode forming portion was peeled off to obtain a comb-shaped electrode pattern (d). Then, a silicon-based positive photoresist (SP3C, manufactured by Fuji Hunt Co., Ltd.) was spin-coated on the substrate to a thickness of 1 μm, baked on a hot plate at 90 ° C. for 90 seconds, and then exposed and developed using a mask. , Leaving the comb-shaped working electrode, reference electrode tip, and counter electrode part,
Covered with resist (e). Next, the resist was thermoset in an oven at 200 ° C. for 2 hours to form an insulating film. The prepared electrochemical detector had a comb-shaped working electrode having an electrode width of 2 μm, a gap of 2 μm, a comb length of 2 mm, and a number of combs of 250 pairs. After that, both terminals of the metal comb electrode were connected to the working electrode terminals of a potentiostat (manufactured by Toho Giken) whose potential was controlled by a function generator (manufactured by Toho Giken), and silver / silver chloride was used as a reference electrode. Using a platinum wire for the counter electrode, immersing in a platinum plating solution having the same composition as in Example 1,
When platinum black plating is performed at -1.5 V for 1 second, electrolysis concentrates at the edge of each comb part of the comb-shaped electrode, so that submicron-sized platinum micro band electrodes are formed at the comb edge. In the central part, a submicron or smaller platinum disk array electrode structure was obtained (f). This electrode was attached to a thin layer cell as an electrochemical detector of a chromatography device as shown in FIG. When 5 μl of Ringer's solution containing 1 pmol of acetylcholine and choline was injected under the same conditions as in Example 1, two current peaks corresponding to acetylcholine and choline were observed. The absolute values of the peaks were 3.1 nA for acetylcholine and 2.9 nA for choline. On the other hand, when a platinum comb-shaped electrode was used for the chromatographic detector instead of the platinum-plated gold comb-shaped electrode, the same measurement was performed, and peaks of 2.7 nA were obtained for acetylcholine and 2.5 nA for choline. In addition, comparing the noise of the baseline, it is 5 for the platinum black plated gold comb electrode.
A value of 13.5 pA was obtained for pA and platinum comb electrodes. Due to the relationship between the signal and noise, the signal has a noise level of 3
When the doubling value is set as the detection limit and calculation is performed for acetylcholine, it is 2.2 fm for the platinum black-plated gold comb electrode.
A value of 9.1 fmol was obtained for the ol and platinum comb electrodes, and it was found that the platinum black-plated gold comb electrode showed higher sensitivity and lower detection limit.

Example 6 By the same method as in Example 1, a gold thin film electrode having a diameter of 3 mm was formed on a silicon substrate with an oxide film. This electrode was placed in a sputtering device (manufactured by Nippon Seed Co., Ltd.), and platinum was formed by a sputtering method so that the film thickness was 5 nm or less. When the composition of the electrode surface was examined, 80% of gold and 20% of platinum were exposed on the surface, and it was confirmed that the platinum thin film was formed in an island shape on the gold thin film. When this electrode was attached as a detector of the same chromatographic apparatus as in Example 1 and 5 μl of Ringer's solution containing 1 pmol of acetylcholine and choline was injected, it was 2.1 for acetylcholine.
A peak of 1.9 nA was obtained for nA and choline. On the other hand, when a platinum comb-shaped electrode having a diameter of 3 mm was used for the detector, the current values for both acetylcholine and choline doubled. However, in the gold thin film electrode of the present invention in which platinum was sputtered in an island shape, the baseline noise was about 1/4 of the value obtained by using the platinum thin film electrode alone. As a result, in the electrode of the present invention, about 1 / compared to the platinum thin film electrode alone.
A detection limit of 2 was achieved.

Example 7 After purifying cobalt phthalocyanine (manufactured by Aldrich), 100 mg was placed on a boat of a vapor deposition apparatus, and a metal mask having a circular electrode pattern having a diameter of 3 mm, a lead and a pad portion was laid above it 3 inches. The quartz substrate was placed and the boat was heated to form a cobalt phthalocyanine film having a thickness of 0.3 μm on the substrate. Then, this substrate was put in a quartz tube capable of depressurizing the inside as shown in FIG. 8 and heat-treated for 30 minutes at 1000 ° C. and 0.001 Torr, and then gradually cooled to form a conductive carbon thin film disk electrode. Obtained. This carbon thin film is a scanning electron microscope JSM
Observation with -840 (manufactured by JEOL Ltd.) revealed that a large number of submicron cobalt particles were dispersed in the carbon film. This electrode substrate was mounted on the same chromatographic apparatus as in Example 1, and a Ringer's solution containing various concentrations of acetylcholine and choline was injected. As a result, good linearity was obtained from 2 fmol to 10 pmol for both acetylcholine and choline. Next, when a 3 mm platinum disk electrode was attached to the chromatography device and the same measurement was performed, the current value increased about 3 times, but the linearity between the acetylcholine and choline concentration and the current value was between 30 fmol and 10 pmol. It was found that the detection limit was greatly improved in the carbon thin film electrode in which a large number of cobalt fine particles were dispersed according to the present invention.

[0018]

As described above, in the electrochemical detector according to the present invention, the electrochemical reaction of the substance to be measured easily occurs on the conductor on which the electrochemical reaction of the substance to be measured hardly occurs. A characteristic is that a pattern of 10 μm or less is formed, and a substance to be measured is concentrated and diffused on a minute electrode, so that a high current density can be obtained. Further, the substance to be measured hardly reacts in the portion other than the fine pattern, and the baseline noise is extremely small. As a result, the S / N ratio is improved and the detection limit is improved. Such a fine structure can also be produced by finely processing a conductor thin film. However, in this method, a higher current density can be obtained because a finer electrode pattern can be formed by a plating method or thermal decomposition of an organic thin film in which a metal is coordinated, as compared with a normal photolithography method. Have. A method of measuring the concentration of a substance by measuring the increase or decrease of hydrogen peroxide or oxygen is widely used in the analysis and biosensor using the electrochemical reaction. This is because oxidase is often used for analysis using enzyme reaction,
This is because a lot of research has been conducted on biosensors that monitor the amount of hydrogen peroxide produced by enzymatic reactions and the amount of oxygen consumed by the reactions. The detector obtained in the present invention may be capable of measuring hydrogen peroxide and the like with high sensitivity as compared with conventional detectors, and thus is a detector for biosensors and chromatography devices used for measurement of physiologically active substances. It can be expected to have excellent effects such as great utility value.

[Brief description of drawings]

FIG. 1 is a schematic diagram of a structure in the case where a plating amount of platinum black formed on a gold thin film is small.

FIG. 2 is a schematic diagram of a structure in the case where a platinum black plating amount formed on a gold thin film is large.

FIG. 3 is a schematic diagram of a structure of a microarray electrode that has been reported in the past.

FIG. 4 is a schematic diagram of a structure of a microarray electrode that has been reported in the past.

FIG. 5 is a schematic diagram of a chromatography device for acetylcholine choline analysis.

FIG. 6 is a diagram showing a chromatogram when 1 pmol of acetylcholine and choline were injected.

FIG. 7 is a schematic diagram of an electrochemical cell in which the relationship between glutamate concentration and current is measured.

FIG. 8 is a schematic diagram of a carbon thin film electrode manufacturing method.

FIG. 9 is a process diagram of manufacturing a gold comb-shaped microelectrode partially modified with platinum black.

[Explanation of symbols]

11: insulating polymer, 12: platinum wire, 13: minute circular pattern electrode, 14: insulating film, 15: platinum thin film, 16:
Insulating substrate, 21: gold thin film, 22: platinum black pattern, 2
3: Insulating substrate, 31: Chromatography pump, 3
2: carrier solution holder, 33: injector, 34:
Acetylcholine / choline separation column, 35: reaction column, 36: thin layer cell, 37: reference electrode, 38: electrode shown in Example 3 of the present invention, 39: potentiostat, 4
0: waste liquid container, 51: electrode shown in Example 5 of the present invention,
52: counter electrode, 53: reference electrode, 54: solution containing glutamate, 61: quartz tube, 62: 3, 4, 9,
10-perylene tetracarboxylic acid anhydride, 63: electric furnace for sublimation (400 ° C.), 64: electric furnace for thermal decomposition (1000
C.), 65: 3 inch silicon wafer, 66: metal mask, 71: positive photoresist, 72: silicon oxide film, 73: silicon substrate, 74: chromium / gold laminated thin film,
75: Resist insulating film, 76: Platinum black microelectrode pattern

Claims (4)

[Claims] 1. A plurality of conductor patterns of 10 μm or less of an electric conductor that easily cause an electrochemical reaction of the substance to be measured are formed on the electric conductor on which the electrochemical reaction of the substance to be measured is extremely unlikely to occur. Electrochemical detector. 2. The electrochemical detector according to claim 1, wherein the substance to be measured is hydrogen peroxide. 3. The method for manufacturing an electrochemical detector according to claim 1, wherein a pattern of a conductor that easily causes an electrochemical reaction with a substance to be measured is produced by a plating method. Detector manufacturing method. 4. The method for manufacturing an electrochemical detector according to claim 1, wherein an aromatic substance in which a metal molecule is coordinated on an insulating substrate with a pattern of a conductor that easily causes an electrochemical reaction with a substance to be measured. A method for manufacturing an electrochemical detector, which comprises forming a thin film of a system-based compound, and producing the thin film by thermal decomposition thereof.