JP2566173B2 - Electrochemical detector - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an electrochemical detector applied to a flow cell, liquid phase chromatography or the like.

[Conventional technology]

Usually, in blood glucose level measurement and the like, a device called a flow cell is used in which a sample sample is injected into a carrier solvent flowing at a constant flow rate and the sample is measured by a detector placed in a flow path. Also, in liquid phase chromatography, a column for chromatography is inserted between the sample inlet and the detector, where the injected sample is separated and each component is detected. .

Known methods for detecting this sample include ultraviolet and visible spectroscopic methods, refractive index measurements, electrical conductivity measurements, and electrochemical methods. In the electrochemical method, an electrode is placed in the flow path, a constant potential is applied to it, and when the sample flowing through the carrier reaches the electrode, the redox current that occurs with the electrode is monitored. By doing so, detection is performed.
As described above, the electrochemical detection is simple and relatively sensitive to the device, and does not respond to an electrochemically inactive substance or a substance having a redox potential lower than the applied potential. It has a feature that it can be selectively detected. Furthermore, even an electrochemically inactive substance such as glucose can be detected by modifying the electrode with an enzyme such as glucose oxidase.

[Problems to be Solved by the Invention]

In a flow cell or the like, the longer the length of the electrode in the flow direction, the longer the contact time with the detection substance, which causes a problem that the response becomes slow. Further, if the distance between the reference electrode and the working electrode is long, a voltage drop corresponding to the electric resistance of the liquid between them occurs, and the signal becomes dull. In order to suppress these, miniaturize the electrode and shorten the contact time,
Although it suffices to shorten the distance between the working electrode and the reference electrode, this has the drawback that the current value becomes small and detection becomes difficult due to the small electrode area. Furthermore, the current response depends on the electrode shape, so different shapes will show different responses. Since many microelectrodes are manufactured by encapsulating metal wires such as platinum and gold, carbon fibers, metal chlorides, etc. in a glass thin tube, it is not possible to obtain electrodes with exactly the same electrode shape, which is troublesome to manufacture. Since it is difficult to obtain a large amount and the variation among the prepared electrodes is large, it is necessary to calibrate the electrodes in advance when quantitative data is required, which requires a great amount of measurement time. For this reason, it has been very difficult to obtain quantitative data when the measurement cannot be performed due to contamination or corrosion of the electrodes.

To solve such a problem, it has been proposed to use a microelectrode manufactured by applying a lithographic technique (for example, Analytical Chemistry, Volume 60, 2770).
Page 1988). According to this method, a large number of microelectrodes having an arbitrary shape and a constant distance between electrodes can be produced on the substrate with good reproducibility. Using this method, a microelectrode in which two comb-shaped electrodes are interlocked with each other is prepared, and one of the electrodes is set to the oxidation potential of the target substance and the other is set to the reduction potential, whereby the oxidation and reduction are repeated between the two electrodes, and the current Have been proposed (Journal of Electroanalytical Che
mistry, 256, p. 269, 1988). But with this method,
A dual potentiostat that can control two working electrodes at different potentials at the same time is required, and since it has to be connected to two working electrodes, it has drawbacks such as a complicated structure of the detection unit. The device could not be used as it was.

[Means for solving the problem]

In order to solve such a problem, the electrochemical detector according to the present invention has a plurality of working electrodes composed of patterned thin film electrodes formed on an insulating substrate, and an oxidation-reduction potential is applied to a specimen, A first working electrode that oxidizes or reduces the analyte, and a first portion that reduces or oxidizes the analyte that has been oxidized or reduced by the first working electrode,
And, when the first portion is electrically connected to the first portion and the first portion reduces or oxidizes the specimen, the second portion oxidizes or reduces the specimen in the specimen sample solution in the vicinity and has a potential of A second working electrode that is not applied,
The working electrode and the first portion of the second working electrode are separated by a minute planar interval and / or a minute interval due to a three-dimensional step through the insulating layer.

[Action]

In the present invention, two working electrodes have been connected to a dual potentiostat and a different potential has been applied to each of them, whereas one working electrode (first working electrode) is a normal potentiostat. Of the two working electrodes by connecting the two working electrodes (second working electrode) to the first working electrode close to the first working electrode and the second part separated from the first working electrode. I found that the same effect as connecting to the tatto can be obtained.

In other words, when the target substance is oxidized or reduced by the first working electrode to which an electric potential is applied, the target substance diffuses to the first portion of the second working electrode separated by a minute interval, while the second substance separated from the first part of the second working electrode is separated. Since the oxidized or reduced target substance has not diffused to the second portion of the working electrode, the concentration difference is generated between the first portion and the second portion. Due to this concentration difference, an electric potential is generated in the second working electrode, and the target substance oxidized or reduced in the first working electrode to which the electric potential is applied is reduced or oxidized in the first portion of the second working electrode. We found that so-called redox cycling, which returns to the original substance, occurred and the sensitivity could be increased.

At this time, the electrons generated or consumed in the electrochemical reaction that occurs in the first portion of the second working electrode are consumed or generated in the reverse electrochemical reaction that occurs in the second portion.
Note that the smaller the minute gap between the first working electrode and the first portion of the second working electrode, the more effective it is, but it is preferably at least 50 μm or less.

The second portion of the second working electrode is the electrode itself or the liquid passage provided at an appropriate position of the liquid passage of the flow cell or the chromatograph if it is a conductive material such as a stainless pipe, and further, the second portion. It is also possible to extend the wiring from the first portion, connect a metal such as platinum as the second portion to this wiring, and immerse the wiring in a carrier liquid reservoir such as a flow cell or its waste liquid receiver. Also, the first part and the second part are 10
It is preferable that they are separated by at least μm.

Further, if an ammeter is provided in the middle of the wiring, the same measurement as in the case of using the dual potentiostat can be performed. For example, in a sample containing ascorbic acid in addition to the target substance, ascorbic acid and the target substance are simultaneously allowed to participate in the first working electrode, and the reduction current of the target substance in the first portion of the second working electrode is set to the above-mentioned value. It is possible to know the concentration of only the target substance by measuring with the ammeter.

As the first and second working electrodes, the first working electrode is a microelectrode, the second working electrode is a normal size electrode, and these electrodes are arranged in parallel or opposite to each other at a minute interval. Good.

In addition, in a minute disk array electrode in which a large number of minute disk electrodes are arranged in a matrix,
May be used as the second working electrode, in which the insulating film between the disk electrodes is electrically isolated from the disk electrodes and replaced with a conductor, and only the electrode part of the conventional measuring device is used. Should be changed.

〔Example〕

Hereinafter, the present invention will be described in detail with reference to the drawings. The present invention is not limited to the following examples.

(Example 1) FIGS. 1 (a) to 1 (f) are cross-sectional views of steps for explaining the structure of an example of the electrochemical detector according to the present invention based on its manufacturing method. In the figure, first, as shown in (a) of the figure, a titanium film having a thickness of 5 nm is sandwiched between a silicon substrate (made by Osaka Titanium Co., Ltd.) 2 having an oxide film 1 having a thickness of 1 μm and a thickness of 100 nm. The platinum thin film 3 having a thickness was formed by using an electron beam heating vapor deposition apparatus (VI-451 manufactured by Anelva). Next, as shown in FIG.
Photoresist (MP1400-27, manufactured by Shipley Co., Ltd.) was applied on the top to a thickness of 1 μm. The resist-coated silicon substrate 2 was baked on a hot plate at 80 ° C. for 2 minutes. After that, the electrode pattern of the comb-shaped structure that meshes with a chrome mask is applied to the mask aligner (Canon: P
Contact exposure was performed with LA-501) for 15 seconds. The exposed silicon substrate 2 is a resist developer (made by Shipley Co., Ltd .: MF319).
It was developed at 20 ° C. for 60 seconds, washed with water and dried to transfer the mask pattern to a resist to form a resist pattern 4. Next, as shown in FIG. 7C, this silicon substrate 2 is attached to a predetermined position in an ion milling device (Millatron manufactured by Commonwealth Scientific), and milling is performed with argon gas 2 × 10 ⁻⁴ Torr and extraction voltage 550V. A comb-shaped working electrode in which the platinum and the underlying titanium film not covered by the resist pattern 4 are removed, and then the resist pattern 4 is removed by an assuring device (Plasma Ashia made by Tokyo Ohka) to mesh with each other. Formed 5,6. Next, as shown in FIG. 3D, the silicon substrate 2 is placed on a plasma CVD apparatus (AMP-made by Applied Materials).
3300), silane gas 23SCCM, ammonia gas 48S
Flow each gas at CCM flow rate, gas pressure 0.2Torr, input power 500
W, silicon substrate temperature 300 ℃, 10 minutes deposition, thickness 4
It was covered with a silicon nitride film 7 of 00 nm. Next, as shown in FIG. 3E, a resist was spin-coated on the silicon nitride film 7 again, exposed by a mask aligner, and developed to obtain a resist pattern 8. Next, as shown in FIG. 6 (f), the silicon substrate 2 is placed in a reactive ion etching apparatus (DEM-451 manufactured by Anelva) and CF ₄ gas is supplied at a flow rate of 25 SCCM and pressure.
Using the resist pattern as a mask under the conditions of 0.25Pa and 150W 1
The silicon nitride film 7 was etched for 5 minutes to expose the comb-shaped working electrodes 5, 6 and their connecting pads. The shape of the manufactured comb-shaped working electrodes 5 and 6 is 1.5 for each comb.
μm, comb spacing 1.5 μm, comb length 2 mm and number of combs
It was 100 each.

The electrochemical detector thus formed was attached to a flow cell system (manufactured by BAS: LC-4B / 17AT), and one of the two comb-shaped working electrodes 5 and 6 was used as a potentiostat for the flow cell system. The other side was connected to a platinum wire immersed in a waste reservoir containing ferrocene. When the potential of the comb-shaped working electrode was set to 0.7 V with respect to the reference electrode and 100 μ of 1 μmol / ferocene was injected at a flow rate of 0.8 ml / min, the comb-shaped working electrode responded immediately and peaked at 30 msec. Current value
It showed 55nA and returned to its original value in 50msec. Also, using a dial potentiostat, similar results were obtained by performing similar experiments with one potential of the comb-shaped working electrode set to 0.7 V and the other potential of -0.1 V with respect to the reference electrode. . On the other hand, when a similar experiment was conducted using circular electrodes of the same area,
The peak current reached 0.29nA in msec, and returned to the original value in 500msec.

(Embodiment 2) FIGS. 2 (a) to 2 (f) are sectional views of steps for explaining another embodiment of the method for manufacturing an electrochemical detector according to the present invention. The same reference numerals are attached. In the figure, first, as shown in FIG.
A silicon substrate 2 having an m-thick oxide film 1 was attached to a predetermined position in a stepper device (Anerva: SPF-332H) together with a metal mask, pressure 1.3Pa, in argon, power.
Sputtering chromium at 50 W for 10 seconds, then spattering platinum at power 70 W for 1 minute without breaking the vacuum, and as shown in FIG. 7B, the lower electrode 9 of chromium-platinum film having a thickness of 100 nm is formed. Formed. Next, the metal mask was removed, and the silicon dioxide film 10 was deposited by the sputtering method as shown in FIG. Next, as shown in FIG.
W, spatter for 10 minutes, silicon dioxide film 10 300n
After setting the film thickness to m, another metal mask was mounted again, and a chromium-platinum surface electrode 11 was deposited to 100 nm. Then, a photoresist (MP1400-27 manufactured by Shipley Co., Ltd.) was applied on the silicon substrate 2 to a thickness of 1 μm. And
Put this resist-coated silicon substrate 2 in the oven 80
Baking was carried out under the conditions of ℃ and 30 minutes. Then, reduction projection exposure was performed for 0.3 seconds by a stepper (manufactured by Nikon: NSR1010G) using a reticle. The exposed silicon substrate 2 is developed in a resist developer (MF-319 manufactured by Shipley Co., Ltd.) at 20 ° C. for 60 seconds, washed with water and dried to transfer the reticle pattern to the resist as shown in FIG. A resist pattern 12 was formed. Next, after development, the silicon substrate 2 is attached at a predetermined position in the ion milling apparatus, and the argon gas pressure 2
Milling was performed at × 10 ^-4 Torr and extraction voltage of 550 V, and the upper working electrode 13 was formed as shown in (f) of the figure, and then it was put into the reactive ion etching device, CF ₄ gas, flow rate 25 SCCM, pressure. The silicon dioxide film 10 is etched for 10 minutes under the conditions of 0.25 Pa and 150 W, and a large number of minute disk array electrodes having fine holes as shown in FIG.
14 formed. In this case, the diameter of each disk array electrode 14 is 1 μm, the distance between the disk array electrodes 14 is 10 μm, and the number is
The number was set to 10,000. Then, this silicon substrate 2 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 an electrode pattern.
An outline of the electrochemical measurement cell thus manufactured is shown in a perspective view in FIG. In the figure, 9A and 13A are connection pads.

The electrochemical detector configured as described above was attached to the flow cell system, and the lower microdisk array electrode 14 was connected to the potentiostat of the flow cell system.
When the electric potential of the lower minute disk array electrode 14 serving as the working electrode with respect to the reference electrode was set to 0.7 V and 100 μ of 1 μmol /% ferrocene was injected at a flow rate of 0.8 ml / min,
This minute disk array electrode 14 reacts immediately, 30msec
Showed a peak current value of 55 nA and returned to the original value at 50 msec. On the other hand, by using a dual potentiostat, the potential of the minute disk array electrode 14 is set to 0.7 V with respect to the reference electrode. In addition to this, in this case, the upper working electrode 13 around it is also − When a similar experiment was conducted by setting a potential of 0.1 V, similar results were obtained.

(Embodiment 3) FIGS. 4 (a) to 4 (g) are cross-sectional views of steps for explaining still another embodiment of the method for manufacturing an electrochemical detector according to the present invention. Are given the same reference numerals. In the figure, first, as shown in (a) of the figure, a silicon substrate 2 having an oxide film 1 having a thickness of 1 μm on its surface is attached at a predetermined position in a sputtering apparatus, and chromium and platinum are sequentially deposited by sputtering. . Then, sputtering was performed under a pressure of 10 ^-2 Torr and an argon atmosphere for 50 W of chromium for 10 seconds and 70 W of platinum for 1 minute to obtain a platinum / chromium thin film having a film thickness of 100 nm. After that, a photoresist is formed on the silicon substrate 2.
It was applied to a thickness of 1.0 μm. The resist-coated silicon substrate 2 was baked on a hot plate at 90 ° C. for 2 minutes. After that, contact exposure was carried out for 15 seconds by a mask aligner. The exposed silicon substrate 2 is stored in a resist developer for 20
After development at 60 ° C for 60 seconds, the mask pattern was transferred to the resist by washing with water and drying. Next, this resist-transferred silicon substrate 2 is attached to a predetermined position in the ion milling machine, a platinum / chromium thin film is milled for 2 minutes at an argon gas pressure of 2 × 10 ⁻⁴ Torr and a withdrawal voltage of 550 V, and the resist is removed by an assing machine. After removal, a lower comb-shaped working electrode 15 as shown in FIG. Next, this silicon substrate 2 was placed again in the sputtering apparatus, and the entire surface of the substrate was covered with a 100 nm silicon dioxide film 10 as shown in FIG.
After that, the silicon substrate 2 is attached to the sputtering device again, and chromium and platinum are sequentially deposited by sputtering to obtain a film thickness.
A 100 nm platinum / chrome thin film was formed. After that, a photoresist was applied to the silicon substrate 2 to a thickness of 1 μm, alignment was performed, and a comb-shaped electrode pattern was exposed by contact. After development, the platinum / chromium thin film was milled again, and the resist was stripped by assing to form the upper comb-shaped working electrode 16 as shown in FIG. Next, this silicon substrate 2 is put into the sputter device again, and the same figure (e)
As shown in, the entire surface of the substrate was covered with a 100 nm silicon dioxide film 10. Next, a photoresist is applied to this silicon substrate 2 to a thickness of 1 μm, and only a comb-shaped electrode portion (1 mm × 0.25 mm) and a pad portion that are vertically engaged with each other are exposed and developed using a chrome mask. The part was exposed as shown in FIG. Next, this silicon substrate 2 is put into a reactive ion etching apparatus, CF ₄ gas, flow rate 25 SCCM, pressure 0.
Using the resist pattern 8 as a mask under the conditions of 25 Pa and 150 W, the silicon dioxide film 10 is etched for 5 minutes to expose the upper comb-shaped working electrode 16 and the lower comb-shaped working electrode 15 as shown in FIG. Let As a result, an interdigitated comb-shaped electrode chemical measurement cell having a very small space between two upper and lower working electrodes was obtained. The shapes of the comb-shaped working electrodes 15 and 16 thus produced were: electrode width of each comb: 1.5 μm, step difference between comb electrodes: 0.3 μm, comb length: 2 mm, 200 combs each. FIG. 5 is a perspective view showing a schematic view of the electrode portion of the produced comb-shaped electrochemical measurement cell.

Attach the electrochemical detector thus prepared to a high performance liquid chromatography device (manufactured by JASCO Corporation: 800 series with a catechol pack attached as a column),
The upper comb-shaped working electrode 16 was connected to platinum immersed in the waste liquid reservoir containing the target sample, and the potential of the lower comb-shaped working electrode 15 was set to 0.7 V with respect to the saturated calomel electrode. Noradrenaline, epinephrine, and dopamine were dissolved in 1 ml of PH3.1 phosphate buffer (400 pg each), and 100 μl of the solution was supplied at a flow rate of 1 ml / min.
Each sample was separated by a column when injected under the condition of 5 nA and showed a peak current of 5 nA. When normal glassy carbon is used as the electrode, the peak current is 0.
It was 5 nA.

(Example 4) The electrochemical detector prepared in Example 3 was attached to liquid phase chromatography, the potential of the upper comb-shaped working electrode 16 was set to 0.7 V with respect to the reference electrode, and the lower comb-shaped. Working electrode 15
An ammeter was attached between the electrode and the platinum in the waste liquid reservoir containing dopamine so that the current of the lower comb-shaped working electrode 15 could be measured. 100 μmol / ascorbic acid and 100 μmol / dopamine mixed solution 100 μ were injected at a flow rate of 0.8 ml / min. As a result, 3 minutes after the injection, an ascorbic acid oxidation current was observed only on the upper comb-shaped working electrode 16 side, and almost no current was observed on the lower comb-shaped working electrode 15. 8 minutes after the injection, the upper comb-shaped working electrode 16 side had a dopamine oxidation current (85 nA), and the lower comb-shaped working electrode 15
On the side, a reduction current of oxidized dopamine was observed.
Upper comb-shaped working electrode without connecting lower comb-shaped working electrode 15
When the potential of 16 was set to 0.7 V and the same thing was done, the oxidation current of dopamine was 1/20 (4.2 nA) of that when the lower comb-shaped working electrode 15 was connected. It was

(Example 5) In Example 4, the electrochemical detector was installed in a flow cell instead of liquid phase chromatography, and the potential of the upper comb-shaped working electrode 16 was set to 0.7 V with respect to the reference electrode, and the lower part was set. An ammeter was attached between the comb-shaped working electrode 15 and the platinum in the waste liquid reservoir containing dopamine so that the current of the lower comb-shaped working electrode 15 could be measured. 100 μmol / ascorbic acid, 100
100 μl of a mixed solution of μmol / dopamine at a flow rate of 0.8 ml / m
Injected under in. As a result, 3 minutes after the injection, the oxidation currents of ascorbic acid and dopamine were observed on the side of the upper comb-shaped working electrode 16 and the current of only dopamine was observed on the lower comb-shaped working electrode 15. When the same operation was performed by setting the potential of the upper comb-shaped working electrode 16 to 0.7 V without connecting the lower comb-shaped working electrode 15, the oxidation current was measured by connecting the lower comb-shaped working electrode 15. 20 minutes for Natsuta 1 (4.2nA)
It was below, and moreover, it could not be separated because it overlapped with the signal of ascorbic acid.

In the above-mentioned embodiment, the silicon substrate with an oxide film is used as the substrate whose surface or the whole is insulative, but other than this, a quartz plate, an aluminum oxide substrate, a glass substrate, a plastic substrate and the like can be mentioned. . Further, examples of the metal for the electrode include gold, platinum, silver, chromium, titanium, stainless steel, and the like. Further, as semiconductors for electrodes, p and n type silicon, p and n
Type germanium, cadmium sulfide, titanium dioxide, zinc oxide, gallium phosphide, gallium arsenide, indium phosphide,
Cadmium serine, cadmium tellurium, molybdenum dioxide, tungsten selenide, copper dioxide, tin oxide, indium oxide, indium tin oxide and the like can be mentioned. Conductive carbon can be mentioned as a semimetal. Examples of the insulating film include silicon oxide, silicon dioxide, silicon nitride, silicone resin, polyimide and its derivatives, epoxy resin, and polymer thermosetting material. As the reference substance on the reference electrode, silver,
Examples thereof include silver chloride and polyvinyl ferrocene.

Further, the microelectrodes are manufactured by combining lithographic techniques such as a thin film forming method, a resist pattern forming method and an etching method. The thin film forming method may be a vapor deposition method, a sputtering method, a CVD method or a coating method. As the resist pattern forming method, photolithography, electron beam lithography, X-ray lithography and the like can be used. As the pattern forming method, first, a thin film is formed on the entire surface of the substrate, a resist pattern is formed on the thin film, and an etching method or a resist pattern for etching a lower thin film with this as a mask is formed, and then the thin film is formed thereon. A lift-off method or the like can be used in which a thin film pattern is formed only on the cormorant that is not covered with the resist by depositing and peeling the resist. After the electrode is manufactured, it is also possible to form a metal as a supporting material or an organic redox polymer on one of the manufactured electrodes by plating or an electrolytic polymerization method to form a reference electrode.

〔The invention's effect〕

As described above, the electrochemical detector according to the present invention has two working electrodes, does not require a dual potentiostat, and can be used by replacing a conventional detection electrode with a redox cycle. As a result, the current increased, and the sensitivity could be improved 10 to 100 times more than the conventional detector. In the case of samples for which ascorbic acid was an interfering substance and was difficult to measure, the ascorbic acid was oxidized at one electrode to make it electrochemically inactive, and then the target substance at the other electrode. By detecting, without being disturbed by ascorbic acid,
I was able to detect it. Furthermore, because it is manufactured using the lithographic technique, it is possible to obtain a large number of measuring cells with working electrodes, reference electrodes, and counter electrodes of any size, shape, and interelectrode distance at low cost, and to measure with a simple device. Since such advantages can be obtained, an extremely excellent effect such as extremely high utility value as an electrochemical detection for a flow cell or liquid phase chromatography can be obtained.

[Brief description of drawings]

1 (a) to 1 (f) are cross-sectional views of steps for explaining an embodiment of the method for manufacturing an electrochemical detector according to the present invention, and FIGS. 2 (a) to 2 (f) are other sectional views of the present invention. FIG. 3 is a perspective view showing a structure of an electrochemical detector according to an embodiment of the present invention, and FIGS. 4 (a) to 4 (g) show still another embodiment of the present invention. FIG. 5 is a sectional view of a process for explaining an embodiment, and FIG. 5 is a schematic view of a main part showing a structure according to still another embodiment of the present invention. 1 ... Silicon oxide film, 2 ... Silicon substrate, 3 ... Platinum thin film, 4 ... Resist pattern, 5,6 ... Comb-shaped working electrode, 7 ... Silicon nitride film, 8 ... Resist pattern, 9 ... … Lower electrode, 9A …… Connecting pad, 10 …… Silicon dioxide film, 11 …… Surface electrode, 12 …… Resist pattern, 13 …… Upper working electrode, 13A …… Connecting pad, 14 ……
Micro disk array electrode, 15 ... Lower comb-shaped working electrode,
16 ... Upper comb-shaped working electrode.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hisao Tabei 1-1-6 Uchisaiwaicho, Chiyoda-ku, Tokyo Inside Nippon Telegraph and Telephone Corporation (56) Reference JP-A-1-301159 (JP, A) JP-A 2-19757 (JP, A) JP-A-2-268265 (JP, A) JP-A-2-179248 (JP, A) Actual Development Sho 61-50263 (JP, U)

Claims (1)

(57) [Claims] 1. An electrochemical having a working electrode composed of a plurality of patterned thin-film electrodes formed on an insulating substrate, for inflowing a sample solution to electrically detect a sample dissolved therein. In the detector, a first working electrode for applying an oxidation-reduction potential to the analyte to oxidize or reduce the analyte, and a first portion for reducing or oxidizing the analyte oxidized or reduced by the first working electrode , And the first
Is electrically connected to the portion of, and when the first portion reduces or oxidizes the specimen, the second portion oxidizes or reduces the specimen in the specimen sample solution in the vicinity,
A second working electrode to which an electric potential is not applied, and the first working electrode and the first portion of the second working electrode are formed by a minute planar space and / or a three-dimensional step through an insulating layer. An electrochemical detector characterized by being separated by minute intervals.