Electrochemical immunosensor

Abstract

In the prior art, a conjugate pad carrying a gold colloid-labeled antibody is essential, the number of man-hours is large in the manufacturing process and assembly process, and the conjugate pad dominates the cost. The purpose of the present invention is to provide an electrochemical immunosensor comprising a case that is provided with a flow path through which a sample solution including a test substance can move, an electrode that is disposed in the flow path so as to be capable of contacting the sample solution, and a labeling part that includes a labeling body having metal microparticles to which a first binding substance that specifically binds to the test substance is bonded, the electrode being provided with an electrode part, and the electrode part being provided with a second binding substance that is different from the first binding substance and that specifically binds to a site on the test substance, and the labeling part.

Description

Electrochemical immunosensor

[0001] The present invention relates to an electrochemical immunosensor for measuring a test substance in an electrochemical manner.

[0002] Immunoassays utilizing antigen-antibody reactions are known as one of the methods for easily and sensitively measuring trace substances in test solutions. ELISA, which uses enzyme-labeled antibodies to detect and measure the concentration of test substances by obtaining signals such as color development or luminescence resulting from an enzyme reaction, is widely used in various fields. However, ELISA requires an optical system for detecting signals such as color development or luminescence, necessitating a large measuring instrument. Furthermore, accurate quantification requires complex processing, such as converting measurement results such as color development into electrical signals.

[0003] Therefore, methods have been proposed that utilize electrochemical measurement for detection in immunoassays and the like that use general-purpose labeling substances such as colorimetric labels or fluorescent labels. Since the devices used in electrochemical measurements can be made smaller than those used in ELISA and the like, it is expected that both miniaturization of the measuring device and improvement of detection sensitivity can be achieved.

[0004] Patent Document 1 discloses a method for electrochemical detection in which an electrode portion, a conductive portion for transmitting current from the electrode portion, and a connection portion for connecting to an electrical measuring device for measuring the current value are arranged on a resin sheet support, and a plurality of pads are partially stacked on the support, and a sample solution is made to flow across the plurality of pads, and the flow is controlled at the position of the electrode portion 5.

[0005] Patent No. 6714256

[0006] However, the measurement method disclosed in Patent Document 1 had variations in measurement results when pads were used. In particular, the technology disclosed in Patent Document 1 required a conjugation pad carrying a gold colloid-labeled antibody, which required many steps in the manufacturing and assembly processes and was a major factor in the cost.

[0007] The present inventors performed electrochemical immunoassay by immobilizing gold colloid-labeled antibodies in the flow channel without using a conjugation pad.

[0008] However, it was difficult to measure simply by removing the conjugation pad.

[0009] As a result of extensive research, the inventors have discovered that by using an electrode part that includes both a label part containing a label having metal microparticles bound to a first binding substance that specifically binds to a test substance, and a second binding substance that specifically binds to a test substance, it is possible to immobilize a gold colloid-labeled antibody in a flow path without using a conjugation pad, and have completed the present invention.

[0010] [1] An object of the present invention is to provide an electrochemical immunosensor comprising: a case having a flow path through which a sample solution containing a test substance can move; an electrode arranged in the flow path so as to be in contact with the sample solution; and a labeling unit including a label having metal microparticles bound to a first binding substance that specifically binds to the test substance, wherein the electrode comprises an electrode unit, and the electrode unit comprises: a second binding substance that specifically binds to a site on the test substance different from the first binding substance; and the labeling unit.

[0011] By using the electrochemical immunosensor according to the present invention, it is possible to carry out electrochemical immunoassays by immobilizing gold colloid-labeled antibodies in the flow channel without using a conjugation pad.

[0012] [2] In the electrochemical immunosensor described in [1], the electrode may comprise a working electrode, a counter electrode, and a reference electrode, and the second binding substance and the labeling portion may be provided at least on the surface of the working electrode.

[0013] [3] In the electrochemical immunosensor described in [1], the flow path may include an upstream flow path and a downstream flow path located downstream of the upstream flow path, the upstream flow path may include an electrode compartment, and the electrode section may be arranged in the electrode compartment.

[0014] [4] In the electrochemical immunosensor according to [3], the downstream flow channel may include an absorber that absorbs the sample solution.

[0015] FIG. 1A shows a planar perspective view of an electrochemical immunosensor 1 according to this embodiment. FIG. 1B shows a planar perspective view of an electrochemical immunosensor 1 equipped with a lid 50. FIG. 2A(a) shows a planar view of the electrochemical immunosensor 1 according to this embodiment, and FIG. 2A(b) shows a cross-sectional view of the electrochemical immunosensor 1 taken along line A-A in FIG. 2A(a). FIG. 2B(a) shows a planar view of the electrochemical immunosensor 1 equipped with a lid 50, and FIG. 2B(b) shows a cross-sectional view of the electrochemical immunosensor 1 taken along line A-A in FIG. 2B(a). FIG. 3 shows a developed view of the electrochemical immunosensor 1 according to this embodiment. FIG. 4A shows details of the electrodes 20, their arrangement, and the position of the labeled portion 41. FIG. 4B shows a labeled portion 41' positioned differently from the labeled portion 41 shown in FIG. 4A. FIG. 5 shows graphs of the average (ave) peak values ​​of Example 1 and Comparative Examples 1 and 2.

[0016] DEFINITIONS For convenience, certain terms used in this application are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0017] Although the numerical ranges and parameters set forth in the present invention are approximate, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in each test measurement. Also, as used herein, the term "about" generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error as considered by one of ordinary skill in the art.

[0018] Hereinafter, embodiments of the present invention will be described. The following embodiments are merely examples, and the scope of the present invention is not limited to the following embodiments. Note that similar content will not be described again to avoid repetition.

[0019] 1A shows a planar perspective view of an electrochemical immunosensor 1 according to this embodiment. FIG. 1A shows a planar perspective view of an electrochemical immunosensor 1 including a lid 50. FIG. 2A(a) shows a plan view of the electrochemical immunosensor 1 according to this embodiment, and FIG. 2A(b) shows a cross-sectional view of the electrochemical immunosensor 1 taken along line A-A in FIG. 2A(a). FIG. 2B(a) shows a plan view of the electrochemical immunosensor 1 including a lid 50, and FIG. 2B(b) shows a cross-sectional view of the electrochemical immunosensor 1 taken along line A-A in FIG. 2B(a). FIG. 3 shows an exploded view of the electrochemical immunosensor 1 according to this embodiment. FIG. 4A shows details of the electrodes 20, their arrangement, and the position of the labeled portion 41. FIG. 4B shows a labeled portion 41' positioned differently from the labeled portion 41 shown in FIG. 4A. The dashed lines in FIGS. 4A and 4B conveniently indicate the electrode portion 21 hidden by the marking portions (41 and 41').

[0020] Electrochemical immunosensor 1 The electrochemical immunosensor 1 of this embodiment comprises a case 10 having a flow path 100 through which a sample solution (first solution) containing a test substance can move, an electrode 20 arranged within the flow path 100 so as to be able to come into contact with the sample solution, and a labeling section 41 containing a label having metal microparticles bound to a first binding substance that specifically binds to the test substance.

[0021] Case 10 In one embodiment, the flow channel 100 is a recess formed in the case 10. In one embodiment, the case 10 includes a mold plate 11 that defines the shape of the flow channel 100 (such as the length, width, and depth of the flow channel) and a container portion 12 that houses the mold plate 11. The mold plate 11 may be detachable from the container portion 12, or may be bonded to the container portion 12 via any bonding means (such as adhesive or welding). The electrochemical immunosensor 1 may include a cover 50 that has a shape that covers at least the flow channel 100.

[0022] Electrode 20 The electrode 20 comprises an electrode portion 21 that comprises both a second binding substance that specifically binds to a site on the test substance that is different from the first binding substance, and a label portion 41 .

[0023] In one embodiment, the electrode 20 further includes a connection portion 22 that connects to an electrical measuring instrument that measures the current value of the current from the electrode portion 21, and a conductive portion 23 that is located between the electrode portion 21 and the connection portion 22 and transmits the current from the electrode portion 21 to the connection portion 22.

[0024] In one embodiment, the electrode unit 21 includes a working electrode 21 a, a counter electrode 21 b, and a reference electrode 21 c. The working electrode 21 a is connected to the working electrode conductive unit 23 a, the counter electrode 21 b is connected to the counter electrode conductive unit 23 b, and the reference electrode 21 c is connected to the reference electrode conductive unit 23 c. In one embodiment, the first binding substance is provided on the surface of the working electrode 21 a.

[0025] The connection portion 22 of the electrode 20 is configured to protrude on the downstream D side of the case 10, but such a configuration is not essential, and the connection portion 22 may be arranged inside the case 10.

[0026] Flow Channel 100 The flow channel 100 includes an upstream flow channel 110 and a downstream flow channel 120 located downstream D from the upstream flow channel 110. In an embodiment, the downstream flow channel 120 includes a first downstream flow channel 130 and a second downstream flow channel 140 located downstream D from the first downstream flow channel 130. In an embodiment, the flow channel 100 is water-repellent. In another embodiment, the first downstream flow channel 130 includes a water-repellent region. In an embodiment, the flow channel 100 is hydrophilic. In another embodiment, the first downstream flow channel 130 includes a hydrophilic region. The flow channel 100 according to this embodiment has a constant flow channel width, but the first downstream flow channel 130 may have a shape (tapered shape) in which its width increases from the upstream U to the downstream D. In an embodiment, the width of the second downstream flow channel 140 is wider than the width of the upstream flow channel 110. In an embodiment, the width of the second downstream flow channel 140 may be constant. In one embodiment, the upstream width of first downstream flow channel 130 is the same as the downstream width of upstream flow channel 110 , and the downstream width of first downstream flow channel 130 is the same as the width of second downstream flow channel 140 .

[0027] The upstream flow path 110 includes an upstream section 111, an electrode section 112, and a downstream section 113. The electrode section 112 is located downstream D from the upstream section 111, and has an electrode unit 21 disposed therein. The downstream section 113 is located downstream D from the electrode section 112, and is connected to the downstream flow path 120.

[0028] In one embodiment, the upstream compartment 111 includes an introduction compartment 114 into which a sample solution is introduced, and a transfer compartment 116 located downstream D from the introduction compartment 114 and upstream U from the electrode compartment 112 .

[0029] The downstream section 113 is a section that separates the sample solution that has flowed from the electrode section 21 to the downstream flow path 120, and by separating the sample solution, the sample solution remaining in the electrode section 21 can be sufficiently replaced with a second solution (such as a cleaning solution or a solution for electrochemical measurement).

[0030] Labeling portion 41 The labeling portion 41 is provided on (is in contact with) the electrode portion 21. In one embodiment, the labeling portion 41 is provided on at least the working electrode 21a. In one embodiment, the labeling portion 41 is provided only on the working electrode 21a in the electrode portion 21. In other words, the labeling portion 41 is provided on the working electrode 21a, but not on the counter electrode 21b or the reference electrode 21c. The labeling portion 41 can fix the label to the electrode portion 21 by drying a solution containing the label on the electrode portion 21.

[0031] Downstream Flow Channel 120 In one embodiment, the downstream flow channel 120 includes an absorbent 30 that absorbs the sample solution. In another embodiment, the second downstream flow channel 140 includes an absorbent 30 that absorbs the sample solution. The absorbent 30 may be made of, for example, nitrocellulose. It is preferable that the absorbent 30 does not contact the inner wall of the downstream flow channel 120.

[0032] Cover 50 The cover 50 can cover or partially cover the case 10. The cover 50 has a shape that at least covers the flow channel 100. In one embodiment, the cover 50 has a shape that covers or partially covers the mold plate 11. In another embodiment, the cover 50 has a shape that covers or partially covers the container 12. When the cover 50 covers the flow channel 100, the flow channel 100 has a tunnel structure, and the flow channel 100 is protected from the external environment. The cover 50 has a sample hole 51 that allows fluid communication between the external environment and the upstream compartment 113, and a sample solution can be introduced into the upstream compartment 113 through the sample hole 51. The surface 52 of the cover 50 may be flat, and the back surface (not shown) of the cover 50 may also be flat.

[0033] Electrochemical Treatment First, a second binding substance (e.g., a primary antibody) for the test substance is immobilized on the surface of an untreated working electrode 21a of the electrode 20 used in electrochemical measurement. The untreated working electrode 21a is a working electrode 21a to which a second binding substance has not been immobilized, and may include an element that facilitates immobilization of the second binding substance to the working electrode 21a. Next, the surface of the working electrode 21a is blocked to prevent nonspecific adsorption.

[0034] Alternatively, a label is prepared by labeling a first binding substance (e.g., a secondary antibody) that recognizes a site on the test substance different from that of the second binding substance (e.g., a primary antibody) with metal fine particles. Next, a solution containing the label is dried on the electrode section 21, thereby applying (fixing) the label to the electrode section 21.

[0035] The electrodes 20 are set in the case 10, and the assembled electrochemical immunosensor 1 is set in an electrical measuring device.

[0036] A sample solution is introduced into the introduction section 114. The sample solution introduced into the introduction section 114 flows downstream D due to capillary action and gravity, and reaches the labeled section 41 of the electrode section 112.

[0037] If the sample solution contains an analyte that binds to the first binding substance, an analyte-first binding substance-metal fine particle complex is formed. The complex in the sample solution then comes into contact with the working electrode 21a of the electrode 20.

[0038] When the analyte in the complex comes into contact with the second binding substance on the working electrode 21a, an antigen-antibody reaction occurs on the working electrode 21a. As the label binds to the second binding substance via the analyte, an amount of metal microparticles corresponding to the concentration of the analyte is collected near the working electrode 21a. To shorten the operation time, it is preferable that the antigen-antibody reaction be carried out while the sample solution is passing over the working electrode 21a, but this does not exclude the possibility of carrying out the antigen-antibody reaction while the flow of the sample solution is stopped.

[0039] The sample solution passes through the downstream section 113 and flows into the downstream flow path 120, where it is absorbed by the absorber 30 disposed in the downstream flow path 120. The sample solution is quickly discharged from the upstream flow path 110 to the downstream flow path 120 by the first downstream flow path 130, allowing a second solution such as a cleaning solution or a solution for electrochemical measurement to be quickly introduced. Note that, because the working electrode 21a is washed with the second solution, the sample solution may remain on the working electrode 21a to the extent that it covers the working electrode 21a.

[0040] In the present invention, any substance, such as a biological substance or a synthetic substance, can be used as the test substance. The binding substances (first binding substance, second binding substance) that specifically bind to the test substance are selected appropriately depending on the test substance. In this embodiment, the specific binding between an antigen and an antibody is used to collect an amount of metal microparticles that corresponds to the test substance, but this combination is not limiting as long as it results in specific binding between substances. For example, specific binding between nucleic acid and nucleic acid, nucleic acid and nucleic acid binding protein, lectin and sugar chain, or receptor and ligand may also be used. The order of the relationship between the test substance and the specific binding substance may be reversed from that described above.

[0041] The metal fine particles used as the labeling substance are not particularly limited, and examples thereof include fine particles of gold, platinum, silver, copper, rhodium, palladium, etc., colloidal particles thereof, quantum dots, etc. Among these, it is preferable to use gold fine particles having a particle size of 10 nm to 100 nm, and particularly gold fine particles having a particle size of about 40 nm.

[0042] After the sample solution is discharged into the downstream flow path 120, a second solution is introduced into the introduction compartment 114 to wash the surface of the working electrode 21a. Further, a second solution, or a different type of second solution, may be introduced into the introduction compartment 114. For electrochemical measurement, the second solution is left on the working electrode 21a, the counter electrode 21b, and the reference electrode 21c to such an extent that it covers the working electrode 21a, the counter electrode 21b, and the reference electrode 21c.

[0043] The metal particles are electrochemically oxidized. For example, the potential of the working electrode 21a relative to the reference electrode 21c is maintained for a predetermined time at a potential at which the metal particles are electrochemically oxidized. This completely oxidizes the metal particles collected near the surface of the working electrode 21a.

[0044] After electrochemically oxidizing metal particles, the presence or concentration of the analyte is measured based on the peak current value generated when the oxidized metal is reduced. Specifically, for example, the potential of the working electrode 21a is shifted negatively, and the change in current accompanying the potential shift is measured. As the electrode potential is shifted negatively, the oxidized and eluted metal is reduced by the potential control described above, resulting in a reduction current, which is measured. The greater the amount of analyte in the test solution and the greater the number of metal particles collected near the working electrode 21a, the greater the reduction current intensity, and thus the quantification or detection of the analyte can be achieved based on this. For example, the relationship between the reduction current value and a known concentration of the analyte can be determined in advance, and the concentration of the analyte can be determined by comparing the measured reduction current value. Furthermore, the presence or absence of the analyte in the test solution can be determined from the obtained reduction current value.

[0045] The solution used for potential control of the working electrode 21a and electrochemical measurement is preferably an acidic solution, since it can easily electrochemically oxidize the metal microparticles. The acidic solution may be selected appropriately depending on the type of metal microparticles, and examples of the acidic solution include aqueous solutions containing hydrochloric acid, nitric acid, acetic acid, phosphoric acid, citric acid, sulfuric acid, etc. Considering the ease of electrochemical oxidation of the metal microparticles, it is preferable to use a 0.05N to 2N hydrochloric acid aqueous solution, and more preferably a 0.1N to 0.5N hydrochloric acid aqueous solution.

[0046] On the other hand, in addition to acidic solutions, neutral solutions containing chlorine can also be used as the solutions used for potential control of the working electrode 21a and electrochemical measurement. Using a neutral solution containing chlorine results in a larger current change than using an acidic solution, resulting in more sensitive measurements. Furthermore, when using an acidic solution, the peak shape may become asymmetric, e.g., the base of the reduction peak may rise on the low potential side, and noise may occur, for example, around 0.1 V. In contrast, using a neutral solution containing chlorine flattens the base of the reduction peak and suppresses the generation of the noise, simplifying the detection of the reduction peak intensity. Furthermore, the use of solutions that are difficult to handle, such as acidic or alkaline solutions, can be avoided, allowing for safe and simple measurement operations. The above-mentioned effects can be obtained when using a neutral solution containing chlorine, such as KCl, NaCl, or LiCl, but the effect is particularly pronounced when using KCl.

[0047] When oxidizing the metal microparticles, the potential of the working electrode 21a is set to a potential at which the metal microparticles can be oxidized. Specifically, the potential of the working electrode 21a must be set to an optimal value depending on the type of metal microparticles used; for example, it is preferably set to +1 to +2 V relative to the silver-silver chloride reference electrode 21c. By setting the potential of the working electrode 21a within the above range, the metal microparticles collected near the surface of the working electrode 21a can be completely oxidized and eluted, thereby reliably improving the detection sensitivity of the analyte. If the potential of the working electrode 21a is set below the above range, the reduction current peak may not appear during measurement. Conversely, if the potential exceeds the above range, the oxidized metal microparticles may diffuse due to migration, reducing the oxide concentration near the working electrode 21a and thereby reducing the reduction current peak. A more preferred range is +1.2 V to +1.6 V.

[0048] A specific method for electrochemically oxidizing the metal microparticles includes maintaining the potential of the working electrode 21a at a potential at which the metal microparticles oxidize for a predetermined period of time. Maintaining the potential for a predetermined period of time is a preferred method because it allows the metal microparticles to be sufficiently oxidized. Furthermore, when applying a potential at which the metal microparticles electrochemically oxidize to the working electrode 21a, in addition to the method of maintaining the potential of the working electrode 21a at a predetermined potential as described above, the potential of the working electrode 21a may be varied over time, for example, by cyclic voltammetry. When varying the potential of the working electrode 21a over time, it is preferable to vary the potential of the working electrode 21a within a potential range at which the metal microparticles oxidize (e.g., +1 to +2 V relative to the silver-silver chloride reference electrode 21c). Furthermore, when oxidizing the metal microparticles, a potential at which the metal microparticles electrochemically oxidize may be applied to the working electrode 21a multiple times.

[0049] When using gold microparticles with a particle size of 10 nm to 60 nm as the metal microparticles, it is preferable to electrochemically oxidize the gold microparticles in a 0.1 to 0.5 normal hydrochloric acid solution by setting the potential of the working electrode 21a relative to the silver-silver chloride reference electrode 21c to +1.2 V to +1.6 V.

[0050] Here, when sufficiently oxidizing the metal microparticles, it is necessary to take care to apply an optimal amount of charge depending on the amount of metal microparticles. Since the amount of charge is a value obtained by integrating the current, if the potential applied to the working electrode 21a is relatively low, the potential must be applied for a long time to sufficiently oxidize the metal microparticles. On the other hand, if the potential applied to the working electrode 21a is relatively high, only a short time is required to sufficiently oxidize the metal microparticles.

[0051] By holding the potential of the working electrode 21a at a potential at which the metal microparticles are electrochemically oxidized for 1 second or more, the metal microparticles can be sufficiently oxidized, and the detection sensitivity can be reliably improved. On the other hand, even if the application time is 100 seconds or more, the obtained current value remains almost unchanged. Therefore, 1 second or more and 100 seconds or less is preferable. A more preferable range for the holding time of the potential is 40 seconds or more and 100 seconds or less.

[0052] Examples of methods for measuring the current generated when an oxidized metal is electrochemically reduced include voltammetry such as differential pulse voltammetry and cyclic voltammetry, amperometry, and chronometry.

[0053] In the above-described embodiment, an antigen-antibody reaction or the like is carried out on the working electrode 21a to collect metal microparticles near the surface of the working electrode 21a, and the reduction peak current derived from the metal microparticles contained in the label is measured, thereby enabling simple and highly sensitive measurement of the analyte in the test solution.

[0054] In another embodiment, in order to collect an amount of metal microparticles corresponding to the test substance near the surface of the working electrode 21a, two types of binding substances for the test substance are prepared, one (first binding substance) is immobilized on the surface of magnetic microparticles, and the other (second binding substance) is labeled with metal microparticles to form a labeled substance, and the magnetic microparticles after reacting with the labeled substance are collected on the surface of the working electrode 21a.

[0055] In the above explanation, a method of collecting an amount of metal microparticles corresponding to the amount of test substance using a non-competitive reaction has been given as an example of a method of collecting an amount of metal microparticles corresponding to the amount of test substance, but a method of collecting an amount of metal microparticles corresponding to the amount of test substance using a competitive reaction may also be used.

[0056] Examples of the present invention will be described below with reference to experimental results (for details of the experimental apparatus, methods, conditions and reagents, see WO2007 / 116811).

[0057] (Experiment 1) 1. Immobilization of antibody on working electrode In this experiment, we attempted to measure human gonadotropin (hCG) diluted with PBS (phosphate buffer solution) using a printed electrode with a primary antibody (anti-hCG antibody) immobilized on the surface of the working electrode. hCG is a type of pregnancy diagnostic marker. Gold colloid-labeled anti-hαS antibody was used as the gold colloid-labeled secondary antibody (label).

[0058] The electrode devices used for measuring the analyte were an electrochemical immunosensor 1 shown in Figures 1A and 1B and an electrode 20 (printed electrode) shown in Figure 4A. The electrode part of the electrode had a working electrode and a counter electrode made of carbon paste, a lead made of carbon paste, and a reference electrode made of silver / silver chloride, all mounted on an insulating support. Parts of the surfaces of the working electrode, counter electrode, and reference electrode were covered with an insulating layer, thereby defining the effective electrode area.

[0059] 2 μL of anti-hCG antibody (primary antibody) solution prepared at a concentration of 100 μg / mL was dropped onto the working electrode and allowed to stand in a cool, dark place at 4° C. for 12 hours or more to immobilize the anti-hCG antibody on the surface of the working electrode. After washing the printed electrode device with PBS, blocking was performed with 0.1% bovine serum albumin.

[0060] 2. Drying and Supporting of Gold Colloid-Labeled Secondary Antibody on Electrode As a gold colloid-labeled secondary antibody, a gold colloid-labeled anti-hαS antibody was dried and supported on the electrode at the following positions: Example 1 (directly above the working electrode): Position on the working electrode Comparative Example 1 (upstream of the working electrode): Position upstream of the working electrode Comparative Example 2 (downstream of the working electrode): Position downstream of the working electrode

[0061] 3. Measurement of Test Substances A solution containing hCG as a test substance was prepared by diluting it with PBS (phosphate buffer solution) to a concentration of 0 ng / mL or 100 ng / mL. 4.5 μL of the solution was dropped onto the printed electrode device through the sample hole. After the solution had passed through the electrode section, the electrode section was washed with PBS.

[0062] After washing, 30 μL of a 0.1 M aqueous hydrochloric acid solution was introduced into the printed electrode device treated as described above so that the entire surfaces of the working electrode, reference electrode, and counter electrode were completely covered, and the potential of the working electrode relative to the silver-silver chloride reference electrode was maintained at +1.2 V. The holding time was 40 seconds.

[0063] Next, the potential of the working electrode was changed from 0.8 V to −0.1 V by differential pulse voltammetry, and the change in current relative to the potential change was measured. The voltammetry conditions were a potential increase of 0.004 V, a pulse amplitude of 0.05 V, a pulse width of 0.05 S, and a pulse period of 0.2 S. The results are shown in Table 1.

[0064]

[0065] Figure 5 shows a graph of the average (ave) peak values ​​for Example 1 and Comparative Examples 1 and 2. As shown in Figure 5, the reduction peak current could be measured even when the gold colloid label was dried and supported directly on the working electrode.

[0066] DESCRIPTION OF SYMBOLS 1 Electrochemical immunosensor 10 Case 11 Mold plate 12 Container portion 20 Electrode 21 Electrode portion 21a Working electrode 21b Counter electrode 21c Reference electrode 22 Connection portion 23 Conductive portion 23a Conductive portion for working electrode 23b Conductive portion for counter electrode 23c Conductive portion for reference electrode 30 Absorbent 41, 41' Label portion 50 Cover 51 Sample hole 52 Surface 100 Flow path 110 Upstream flow path 111 Upstream compartment 112 Electrode compartment 113 Downstream compartment 114 Introduction compartment 116 Transfer compartment 120 Downstream flow path 130 First downstream flow path 140 Second downstream flow path U Upstream D Downstream

Claims

1. An electrochemical immunosensor comprising: a case having a flow path through which a sample solution containing a test substance can move; an electrode arranged within the flow path so as to be in contact with the sample solution; and a labeling unit including a label having metal microparticles bound to a first binding substance that specifically binds to the test substance, wherein the electrode comprises an electrode unit, and the electrode unit comprises: a second binding substance that specifically binds to a site on the test substance different from the first binding substance; and the labeling unit.

2. The electrochemical immunosensor according to claim 1, wherein the electrodes comprise a working electrode, a counter electrode, and a reference electrode, and the second binding substance and label are provided at least on the surface of the working electrode.

3. The electrochemical immunosensor described in claim 1, wherein the flow path comprises an upstream flow path and a downstream flow path located downstream of the upstream flow path, the upstream flow path comprises an electrode compartment, and the electrode section is arranged in the electrode compartment.

4. The electrochemical immunosensor according to claim 3, wherein the downstream flow path is provided with an absorbent that absorbs the sample solution.

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