Electrochemical immunosensor

Abstract

To solve the problem in which electrochemical immunosensor, excluding pads, suffered from poor drainage of a first liquid, leading to turbidity with a second liquid and making measurement difficult.SOLUTION: The electrochemical immunosensor includes: a case equipped with a flow channel through which a sample solution containing a test substance can move; an electrode arranged within the flow channel such that it can come into contact with the sample solution; and a reaction section including a marker having metal microparticles to which a first binding substance, which specifically binds to the test substance, is bound. The electrodes includes an electrode section equipped with a second binding substance that specifically binds to the test substance, which is different from the first binding substance. The flow channel includes an upstream flow channel and a downstream flow channel located further downstream than the upstream flow channel. The downstream flow channel includes a drainage region to facilitate complete drainage of the sample solution. The upstream flow channel includes a reaction compartment containing the reaction section and an electrode compartment located downstream of the reaction compartment and housing the electrode section.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

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

[0002] Immunoassays using antigen-antibody reactions are known as a simple and highly sensitive method for measuring trace substances in test solutions. ELISA, an immunoassay method that uses enzyme-labeled antibodies to detect and measure the concentration of test substances by obtaining signals such as color development or luminescence resulting from the enzyme reaction, is widely used in various fields. However, ELISA requires an optical system for detecting signals such as color development and luminescence, necessitating large-scale measuring equipment. 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 that use general-purpose labeling substances such as colorimetric or fluorescent labels. Because the equipment used for electrochemical measurement can be made smaller than that used for ELISA, it is expected that both miniaturization of the measuring equipment 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 multiple pads are partially stacked on the support, and a sample solution is made to flow across the multiple pads, and the flow is controlled at the position of the electrode portion 5. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6714256 Summary of the Invention [Problem to be solved by the invention]

[0006] However, the measurement method disclosed in Patent Document 1 results in variations in measurement results when pads are used.

[0007] The present inventors prepared an electrochemical immunosensor without pads and performed measurements using two or more types of solutions.

[0008] However, in the electrochemical immunosensor without the pads, the first liquid did not drain well, and the second liquid became cloudy, making measurement difficult. [Means for solving the problem]

[0009] As a result of extensive research, the inventors have discovered that by providing an electrochemical immunosensor with an upstream flow path, a downstream flow path located downstream of the upstream flow path, and an intermediate flow path located between the upstream and downstream flow paths and which widens from upstream to downstream, it is possible to improve the runout of the first liquid and prevent it from becoming cloudy with the second liquid, and have completed the present invention.

[0010] [1] The object of the present invention is to a case having a flow path through which a sample solution containing a test substance can move; an electrode disposed in the flow channel so as to be in contact with the sample solution; a reaction section containing a label having metal fine particles bound to a first binding substance that specifically binds to the test substance; Equipped with the electrode includes an electrode portion having a second binding substance that specifically binds to the test substance and is different from the first binding substance; The flow path is an upstream flow path; a downstream flow path located downstream of the upstream flow path; Equipped with the downstream flow path includes a drainage area that promotes complete drainage of the sample solution; The upstream flow path is a reaction compartment comprising the reaction section; an electrode compartment located downstream of the reaction compartment and in which the electrode unit is disposed; Equipped with Electrochemical immunosensor The purpose is to provide

[0011] By using the electrochemical immunosensor according to the present invention, it is possible to improve the draining of the first liquid and prevent it from becoming cloudy with the second liquid.

[0012] [2] [1] The electrochemical immunosensor according to The downstream flow path is a first downstream flow path in which the liquid cut-off region is located; a second downstream flow path located downstream of the first downstream flow path; The device may also include:

[0013] [3] [2] The electrochemical immunosensor according to The first downstream flow path may have a shape in which the width increases from upstream to downstream.

[0014] [4] [1] The electrochemical immunosensor according to The device may further include a cover having a shape that covers at least the flow path.

[0015] [5] [4] The electrochemical immunosensor according to [4], The cover may have a recess on its rear surface, The recess may be provided on the rear surface so as to be positioned opposite the downstream flow path or a part of the downstream flow path when the cover is placed on the case.

[0016] [6] [2] The electrochemical immunosensor according to The first downstream flow path may include a water-repellent region.

[0017] [7] [1] The electrochemical immunosensor according to The electrodes may include a working electrode, a counter electrode, and a reference electrode; The second binding substance may be provided on the surface of the working electrode.

[0018] [8] [2] In the electrochemical immunosensor according to The second downstream flow path may include an absorbent that absorbs the sample solution.

[0019] [9] [2] In the electrochemical immunosensor according to The first downstream flow path may not be provided with an absorbent that absorbs the sample solution. [Brief explanation of the drawings]

[0020] [Figure 1A] FIG. 1A shows a plan perspective view of an electrochemical immunosensor 1 according to this embodiment. [Figure 1B] FIG. 1B shows a top perspective view of the electrochemical immunosensor 1 with the lid 50. FIG. [Figure 2A] 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 AA in FIG. 2A(a). [Figure 2B] Figure 2B(a) shows a plan view of the electrochemical immunosensor 1 with the lid 50, and Figure 2B(b) shows a cross-sectional view of the electrochemical immunosensor 1 with the lid 50 taken along line AA in Figure 2B(a). [Figure 3] FIG. 3 shows a development view of the electrochemical immunosensor 1 according to this embodiment. [Figure 4] FIG. 4(a) shows the structure of the forming plate 11 in detail, and FIG. 4(b) shows the details of the electrodes 20 and their arrangement. [Figure 5]FIG. 5(a) shows a perspective view of the front surface 52A side of the cover 50A, and FIG. 5(b) shows a perspective view of the back surface 53A side of the cover 50A. [Figure 6] FIG. 6(a) shows a plan view of the electrochemical immunosensor 1 with the cover 50A installed, and FIG. 6(b) shows a cross-sectional view of the electrochemical immunosensor 1 taken along line AA in FIG. 6(a). [Figure 7] FIG. 7(a) shows in detail the structure of a forming plate 11B according to another embodiment, and FIG. 7(b) shows in detail the structure of a forming plate 11C according to another embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0021] definition For convenience, certain terms used in this application are collected here. Unless otherwise defined, all technical and scientific terms used in this application 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.

[0022] 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.

[0023] 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 repeatedly explained to avoid cumbersome repetition.

[0024] Embodiment FIG. 1A shows a plan perspective view of an electrochemical immunosensor 1 according to this embodiment. FIG. 1A shows a plan 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 AA in FIG. 2A(a). FIG. 2B(a) shows a plan view of the electrochemical immunosensor 1 including the lid 50, and FIG. 2B(b) shows a cross-sectional view of the electrochemical immunosensor 1 taken along line AA in FIG. 2B(a). FIG. 3 shows an exploded view of the electrochemical immunosensor 1 according to this embodiment. FIG. 4(a) shows the structure of the plate 11 in detail, and FIG. 4(b) shows the details of the electrodes 20 and their arrangement.

[0025] 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 in contact with the sample solution, and a reaction section 40 containing a label having metal microparticles bound to a first binding substance that specifically binds to the test substance and a substrate (e.g., nitrocellulose).

[0026] 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.

[0027] electrode 20 The electrode 20 includes an electrode portion 21 that includes a second binding substance that specifically binds to a test substance different from the first binding substance.

[0028] In one embodiment, the electrode 20 further comprises 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.

[0029] In one embodiment, the electrode unit 21 includes a working electrode 21a, a counter electrode 21b, and a reference electrode 21c. The working electrode 21a is connected to the working electrode conductive unit 23a, the counter electrode 21b is connected to the counter electrode conductive unit 23b, and the reference electrode 21c is connected to the reference electrode conductive unit 23c. In one embodiment, the first binding substance is provided on the surface of the working electrode 21a.

[0030] 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 disposed inside the case 10.

[0031] Channel 100 The flow path 100 includes an upstream flow path 110 and a downstream flow path 120 located downstream D from the upstream flow path 110. The downstream flow path 120 includes a liquid-cutting region that promotes complete drainage of the sample solution. Providing the liquid-cutting region in the downstream flow path 120 promotes complete drainage of the sample solution (first liquid) flowing through the flow path 100, and even if a second liquid (a solution different from the sample solution) is introduced into the flow path 100 early, it is possible to avoid the first liquid and the second liquid becoming cloudy.

[0032] The term "complete liquid cutoff" refers to a state in which the continuity of the sample solution is lost after the sample solution has flowed into the liquid cutoff region, and there is a region in the upstream flow channel 110 (e.g., the downstream section 113 described below) and / or the downstream flow channel 120 (e.g., the first downstream flow channel 130 described below) where the continuity of the sample solution is lost. The state in which the continuity of the sample solution is lost allows the sample solution to remain in the upstream flow channel 110, for example, allowing the sample solution to remain on the electrode section 21 (or the electrode section 112 described below). The state in which the continuity of the sample solution is lost can be confirmed visually or by the presence or absence of the sample solution being absorbed by paper such as filter paper. The state in which the continuity of the sample solution is lost occurs within a typical time required by a skilled artisan in the art (e.g., 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, or within a range between any two points selected from these values) from the time the sample solution reaches the liquid cutoff region until the second liquid is introduced into the flow channel 100.

[0033] In one embodiment, the downstream flow path 120 includes a first downstream flow path 130 in which a liquid-cutting region is located and a second downstream flow path 140 located downstream D from the first downstream flow path 130. In one embodiment, the flow path 100 is water-repellent. In another embodiment, the first downstream flow path 130 includes a water-repellent region. In one embodiment, the flow path 100 is hydrophilic. In another embodiment, the first downstream flow path 130 includes a hydrophilic region.

[0034] In one embodiment, the first downstream flow channel 130 has a shape (tapered shape) in which its width increases from the upstream U to the downstream D. The increase in the width of the first downstream flow channel 130 from the upstream U to the downstream D promotes complete drainage of the sample solution (first liquid) flowing through the flow channel 100, and prevents the first and second liquids from becoming turbid even if the second liquid is introduced into the flow channel 100 early. In one embodiment, the width of the second downstream flow channel 140 is wider than the width of the upstream flow channel 110. In one embodiment, the width of the second downstream flow channel 140 may be constant. In one embodiment, the upstream width of the first downstream flow channel 130 is the same as the downstream width of the upstream flow channel 110, and the downstream width of the first downstream flow channel 130 is the same as the width of the second downstream flow channel 140.

[0035] In one embodiment, the first downstream flow channel 130 has a shape (tapered shape) in which the width increases from the upstream U to the downstream D, and has a water-repellent region or a hydrophilic region.

[0036] Upstream channel 110 The upstream flow path 110 includes an upstream compartment 111, an electrode compartment 112, and a downstream compartment 113. The upstream compartment 111 includes a reaction section 40. The electrode compartment 112 is located downstream D from the upstream compartment 111, and has an electrode section 21 disposed therein. The downstream compartment 113 is located downstream D from the electrode compartment 112, and is connected to the downstream flow path 120.

[0037] In one embodiment, the upstream section 111 includes an introduction section 114 into which a sample solution is introduced, and a reaction section 115 located downstream D from the introduction section 114 and in which the reaction section 40 is disposed.

[0038] In one embodiment, the electrode unit 21 is disposed adjacent to the reaction unit 40 or disposed on the downstream D side of the reaction unit 40 via an additional compartment. The length of the additional space is the distance between the upstream U end of the electrode unit 21 and the downstream D end of the reaction unit 40, and may be, for example, 0.5 mm to 10 mm, or may be 0.5 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm, or may be within a range between any two points selected from these lengths.

[0039] The downstream section 113 is a section that divides the sample solution that has flowed from the electrode section 21 to the downstream flow path 120, and by dividing 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).

[0040] In one embodiment, the upstream flow path 110 does not include a water-absorbent structure (e.g., a nitrocellulose pad) capable of absorbing a solution, such as a sample solution. In another embodiment, the electrode compartment 112 and / or the downstream compartment 113 does not include a water-absorbent structure.

[0041] Downstream flow path 120 In one embodiment, the downstream flow path 120 (particularly the second downstream flow path 140) includes an absorber 30 that absorbs the sample solution. In another embodiment, the first downstream flow path 130 does not include an absorber 30. In yet another embodiment, the first downstream flow path 130 does not include an absorber 30, and the second downstream flow path 140 includes an absorber 30. The absorber 30 may be made of, for example, nitrocellulose. It is preferable that the absorber 30 does not contact the inner wall of the downstream flow path 120.

[0042] An example of the first downstream flow channel 130 will be described in detail using the mold plate 11 as an example. The mold plate 11 includes a first connecting wall 123 and a second connecting wall 124. The upstream flow channel 110 includes a first flow channel end 116 and a second flow channel end 117 located on the downstream D side of the upstream flow channel 110. The downstream flow channel 120 includes a first flow channel end 126 and a second flow channel end 127C located on the upstream U side of the downstream flow channel 120. The first flow channel end 116 of the upstream flow channel 110C is connected to the first flow channel end 126 of the downstream flow channel 120 via the first connecting wall 123. The second flow channel end 117 of the upstream flow channel 110 is connected to the second flow channel end 127 of the downstream flow channel 120 via the second connecting wall 124.

[0043] The first downstream flow path 130 has a shape whose width increases from the upstream U to the downstream D, with the first flow path end 126 of the downstream flow path 120 located outside the upstream flow path 110 and located downstream D from the first flow path end 116 of the upstream flow path 110, and the second flow path end 127 of the downstream flow path 120 located outside the upstream flow path 110 and located downstream D from the second flow path end 127 of the downstream flow path 120.

[0044] 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.

[0045] 5(a) and 5(b) show a cover 50A according to another embodiment and its cross-sectional view, respectively. The cover 50A differs from the cover 50 in that a recess 54A is provided on the rear surface 53A. The recess 54A is provided on the rear surface 53A so as to face (cover) the downstream flow channel 120 or a portion of the downstream flow channel 120 (e.g., the first downstream flow channel 130) when the cover 50A is placed in the case 10. In one embodiment, the planar shape of the recess 54A is substantially the same as the planar shape of the downstream flow channel 120. By covering the flow channel 100 with the cover 50A, the volume of the downstream flow channel 120 is increased by the volume of the recess 54A, further promoting the separation of the sample solution in the downstream compartment 113. In one embodiment, the planar shape of the recess 54A may be substantially the same as the first downstream flow channel 130.

[0046] Flow paths 100B and 100C according to another embodiment Fig. 7(a) shows a mold plate 11B having a flow path 100B according to another embodiment, and Fig. 7(b) shows a mold plate 11C having a flow path 100C according to another embodiment. The flow paths 100B and 100C shown in Figs. 7(a) and 7(b) are merely illustrative shapes of the mold plate and can also be applied to the case.

[0047] Flow path 100B The flow path 100B of the mold plate 11B includes an upstream flow path 110B and a downstream flow path 120B located downstream D from the upstream flow path 110B. The width of the downstream flow path 120B is wider than the width of the upstream flow path 110B. The downstream flow path 120B includes a first sub-section 121B and a second sub-section 122B that protrude upstream U on both sides of the downstream D side of the upstream flow path 110B.

[0048] The mold plate 11B has a first connecting wall 123B and a second connecting wall 124B, the upstream flow path 110B has a first flow path end 116B and a second flow path end 117B located on the downstream D side of the upstream flow path 110B, the downstream flow path 120B has a first flow path end 126B and a second flow path end 127B located on the upstream U side of the downstream flow path 120B, and the first flow path end 116B of the upstream flow path 110B is connected to the first flow path end 126B of the downstream flow path 120B via the first connecting wall 123B, and the second flow path end 117B of the upstream flow path 110B is connected to the second flow path end 127B of the downstream flow path 120B via the second connecting wall 124B.

[0049] The first sub-section 121B is defined by the first flow path end 126B of the downstream flow path 120B being located outside the upstream flow path 110B and on the upstream U side of the first flow path end 116B of the upstream flow path 110B, and the second sub-section 122B is defined by the second flow path end 127B of the downstream flow path 120B being located outside the upstream flow path 110B and on the upstream U side of the second flow path end 127B of the downstream flow path 120B.

[0050] In one embodiment, the flow channel 100B includes a water-repellent region on the downstream D side of the upstream flow channel 110B, on the upstream U side of the downstream flow channel 120B, and / or between the upstream flow channel 110B and the downstream flow channel 120B.

[0051] Channel 100C The flow path 100C of the mold plate 11C includes an upstream flow path 110C and a downstream flow path 120C located downstream D from the upstream flow path 110C. The width of the downstream flow path 120C is wider than the width of the upstream flow path 110C.

[0052] The mold plate 11C has a first connecting wall 123C and a second connecting wall 124C, the upstream flow path 110C has a first flow path end 116C and a second flow path end 117C located on the downstream D side of the upstream flow path 110C, and the downstream flow path 120C has a first flow path end 126C and a second flow path end 127C located on the upstream U side of the downstream flow path 120C, and the first flow path end 116C of the upstream flow path 110C is connected to the first flow path end 126C of the downstream flow path 120C via the first connecting wall 123C, and the second flow path end 117C of the upstream flow path 110C is connected to the second flow path end 127C of the downstream flow path 120C via the second connecting wall 124C.

[0053] The first connecting wall 123C and the second connecting wall 124C are arranged on the same line in the width direction of the flow channel.

[0054] In one embodiment, the flow channel 100C includes a water-repellent region on the downstream D side of the upstream flow channel 110C, on the upstream U side of the downstream flow channel 120C, and / or between the upstream flow channel 110C and the downstream flow channel 120C.

[0055] Electrochemical Treatment

[0056] 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.

[0057] Furthermore, 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, the label is detachably bound to reaction section 40.

[0058] The electrode 20 and the reaction section 40 are set in the case 10, and the assembled electrochemical immunosensor 1 is set in an electric measuring device.

[0059] The 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 reaction section 40.

[0060] 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. This complex in the sample solution leaves the reaction section 40 and comes into contact with the working electrode 21a of the electrode 20 disposed in the electrode compartment 112.

[0061] 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.

[0062] 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 downstream section 113 allows the sample solution to be quickly discharged from the upstream flow path 110 to the downstream flow path 120, 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.

[0063] 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 corresponding 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.

[0064] 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.

[0065] After the sample solution is discharged into the downstream flow path 120, a second solution is introduced into the introduction section 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 section 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.

[0066] 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.

[0067] 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 gradually shifted in the negative direction, and the change in current accompanying the potential shift is measured. As the electrode potential is shifted in the negative direction, the oxidized and eluted metal is reduced by the potential control described above, resulting in a reduction current, which is then 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. Therefore, 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.

[0068] The solution used for controlling the potential of the working electrode 21a and for electrochemical measurement is preferably an acidic solution, since it can easily electrochemically oxidize the metal particles. The acidic solution may be selected appropriately depending on the type of metal particles, 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 particles, it is preferable to use a 0.05-2N hydrochloric acid aqueous solution, and more preferably a 0.1-0.5N hydrochloric acid aqueous solution.

[0069] On the other hand, in addition to acidic solutions, neutral solutions containing chlorine can also be used as solutions 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 at low potentials, 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, this avoids the use of solutions that are difficult to handle, such as acidic or alkaline solutions, 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.

[0070] 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, a potential of +1 to +2 V relative to the silver-silver chloride reference electrode 21c is preferable. 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 test substance. 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 concentration of oxides near the working electrode 21a and thereby reducing the reduction current peak. A more preferable range is +1.2 to +1.6 V.

[0071] A specific method for electrochemically oxidizing the metal microparticles is to maintain the potential of the working electrode 21a at a potential at which the metal microparticles are oxidized 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. When applying a potential at which the metal microparticles are electrochemically oxidized to the working electrode 21a, in addition to 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 are oxidized (for example, +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 are electrochemically oxidized may be applied to the working electrode 21a multiple times.

[0072] When gold microparticles having a particle size of 10 nm to 60 nm are used as the metal microparticles, it is preferable to electrochemically oxidize the gold microparticles in a 0.1-0.5 normal hydrochloric acid solution at a potential of +1.2 V to +1.6 V of the working electrode 21a relative to the silver-silver chloride reference electrode 21c.

[0073] Here, to fully oxidize the metal microparticles, it is necessary to carefully 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 fully 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 fully oxidize the metal microparticles.

[0074] 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, a holding time of the potential is preferably 1 second or more and 100 seconds or less. A more preferable range for the holding time of the potential is 40 seconds or more and 100 seconds or less.

[0075] 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.

[0076] In the above-described embodiment, an antigen-antibody reaction or the like is carried out on the working electrode 21a to collect metal particles near the surface of the working electrode 21a, and the reduction peak current derived from the metal particles contained in the label is measured, thereby enabling simple and highly sensitive measurement of the analyte in the test solution.

[0077] 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.

[0078] 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. [Explanation of symbols]

[0079] 1. Electrochemical immunosensor 10 cases 11, 11B, 11C type plates 12 Container section 20 electrodes 21 Electrode section 21a Working electrode 21b Opposite 21c reference electrode 22 Connection 23 Conductive part 23a Conductive part for working electrode 23b Counter electrode conductive part 23c Conductive part for reference electrode 30 absorber 40 Reaction section 50, 50A cover 51 Sample hole 52 Surface 52A surface 53A back 54A Recess 100, 100B, 100C flow path 110, 110B, 110C upstream flow path 111 Upstream Section 112 Electrode compartment 113 Downstream Section 114 Introduction area 115 Reaction Compartment 116B First flow path end of upstream flow path 117B Second flow end of upstream flow channel 120, 120B, 120C downstream flow path 121B First Subdivision 122B Second Subdivision 123B First connecting wall 124B Second connecting wall 126B First end of downstream flow channel 127B Second flow end of downstream flow channel 130 First downstream channel 140 Second downstream channel U Upstream D downstream

Claims

1. a case having a flow path through which a sample solution containing a test substance can move; an electrode disposed in the flow channel so as to be able to come into contact with the sample solution; a reaction section containing a label having metal fine particles bound to a first binding substance that specifically binds to the test substance; Equipped with the electrode includes an electrode portion having a second binding substance that specifically binds to the test substance and is different from the first binding substance; The flow path is an upstream flow path; a downstream flow path located downstream of the upstream flow path; Equipped with the downstream flow path includes a drainage area that promotes complete drainage of the sample solution; The upstream flow path is a reaction compartment comprising the reaction section; an electrode compartment located downstream of the reaction compartment and in which the electrode unit is disposed; Equipped with Electrochemical immunosensor.

2. The downstream flow path is a first downstream flow path in which the liquid-cutting region is located; a second downstream flow path located downstream of the first downstream flow path; The electrochemical immunosensor of claim 1 , comprising:

3. The electrochemical immunosensor according to claim 2 , wherein the first downstream channel has a shape in which its width increases from upstream to downstream.

4. The electrochemical immunosensor according to claim 1 , further comprising a cover having a shape that covers at least the flow channel.

5. The cover has a recess on its rear surface, The electrochemical immunosensor according to claim 4 , wherein the recess is provided on the rear surface so as to face the downstream flow channel or a part of the downstream flow channel when the cover is placed on the case.

6. The electrochemical immunosensor according to claim 2 , wherein the first downstream flow channel comprises a water-repellent region.

7. The electrodes include a working electrode, a counter electrode, and a reference electrode; the second binding substance is provided on the surface of the working electrode; The electrochemical immunosensor according to claim 1 .

8. The electrochemical immunosensor according to claim 2 , wherein the second downstream flow path includes an absorbent that absorbs the sample solution.

9. The electrochemical immunosensor according to claim 2 , wherein the first downstream channel does not include an absorbent that absorbs the sample solution.

Images