Method for measuring test substance using electrochemical method

Abstract

In the prior art, the reduction current of the gold nanoparticles was observed by sweeping the potential in the negative direction immediately after applying the oxidation potential, but this resulted in differences in the current value between the electrodes. [Solution] The object of the present invention is to provide a method for measuring a test substance, characterized by examining the presence or concentration of the test substance, the method comprising: an oxidation step of applying a first potential to a working electrode to which an immune complex containing the test substance and metal microparticles is immobilized; a low potential step of changing the first potential to a second potential lower than the first potential; and a current measurement step of measuring the reduction current generated when the oxidized metal microparticles are reduced, wherein the immune complex comprises the test substance, a first binding substance specifically bound to the test substance, a second binding substance specifically bound to the test substance, and the metal microparticles bound to the second binding substance.

Description

[Technical Field]

[0001] The present invention relates to a method for measuring a test substance using an electrochemical technique. [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 measuring a test substance, which comprises collecting metal microparticles in a test solution in an amount corresponding to the test substance near the surface of a working electrode, electrochemically oxidizing the metal microparticles, measuring the current value generated when the oxidized metal is electrochemically reduced, and determining the presence or concentration of the test substance based on the current value. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2007 / 116811 Summary of the Invention [Problem to be solved by the invention]

[0006] However, in the prior art including Patent Document 1, the reduction current of the gold nanoparticles was observed by sweeping the potential in the negative direction immediately after applying the oxidation potential, which resulted in a difference in the current value between the electrodes. [Means for solving the problem]

[0007] As a result of extensive research, the inventors of the present invention have succeeded in suppressing the inter-electrode difference in voltammograms and the associated current values ​​by applying an oxidation potential, followed by temporarily applying a potential lower than the oxidation potential, and then sweeping the potential in the negative direction. Furthermore, they have succeeded in suppressing or reducing interference peaks caused by the application of an oxidation potential.

[0008] [1] The object of the present invention is to A method for measuring a test substance, characterized by examining the presence or concentration of the test substance, the method comprising: an oxidation step of applying a first potential to the working electrode on which the immune complex containing the test substance and metal fine particles is immobilized; a low potential step of changing the first potential to a second potential lower than the first potential; a current measuring step of measuring a reduction current generated when the oxidized metal fine particles are reduced; Including, The immune complex comprises the test substance, a first binding substance that specifically binds to the test substance, a second binding substance that specifically binds to the test substance, and the metal fine particles that bind to the second binding substance. method The purpose is to provide

[0009] According to this method, the difference between electrodes in the voltammogram can be suppressed, enabling more accurate measurements.

[0010] [2] In the method according to [1], the second potential may be maintained for a certain period of time.

[0011] [3] In the method described in [2], the predetermined time may be 1 to 60 seconds.

[0012] [4] In the method according to any one of [1] to [3], the second potential may be 20% to 60% lower than the first potential.

[0013] [5] The method according to any one of [1] to [4] may further include a potential changing step of changing the second potential to a third potential that is lower or higher than the second potential. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows a plan view of a printed electrode device. [Figure 2] Figure 2 shows the voltammograms of differential pulse voltammetry. The dashed line in Figure 2 shows the voltammogram obtained by applying 1250 mV for 40 seconds and sweeping from 1100 mV to 0 V relative to a silver / silver chloride reference electrode. The solid line in Figure 2 shows the voltammogram obtained by applying an oxidation potential of 1250 mV for 40 seconds, followed by 700 mV for 10 seconds, and sweeping from 1100 mV to 0 V. [Figure 3] FIG. 3 shows a voltammogram of differential pulse voltammetry performed by applying 1250 mV for 40 seconds, followed by 700 mV for 10 seconds, and sweeping from 700 mV to 0 V relative to a silver / silver chloride reference electrode. [Figure 4] FIG. 4 shows a voltammogram of differential pulse voltammetry performed by applying 1250 mV for 40 seconds to a silver / silver chloride reference electrode, followed by a sweep from 700 mV to 0 V. [Figure 5] Figure 5 shows the voltammograms obtained by differential pulse voltammetry, in which a potential of 1250 mV was applied for 40 seconds relative to a silver / silver chloride reference electrode, followed by various potentials for 10 seconds and sweeping from 1100 mV to 0 V. [Figure 6] Figure 6 shows the voltammograms obtained by differential pulse voltammetry, in which a potential of 1250 mV was applied for 40 seconds relative to a silver / silver chloride reference electrode, followed by various potentials for 10 seconds and sweeping from 1100 mV to 0 V. DETAILED DESCRIPTION OF THE INVENTION

[0015] 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.

[0016] 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.

[0017] 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 those shown in the following embodiments. Note that, to avoid repetition, explanations of similar content will be omitted as appropriate.

[0018] Measurement method of test substance The measurement method according to this embodiment is a method for measuring a test substance, characterized in that the presence or absence or concentration of the test substance is examined. an oxidation step of applying a first potential to the working electrode on which the immune complex containing the test substance and metal fine particles is immobilized; a low potential step of changing the first potential to a second potential lower than the first potential; a current measuring step of measuring a reduction current generated when the oxidized metal fine particles are reduced; Including, The immune complex comprises the test substance, a first binding substance that specifically binds to the test substance, a second binding substance that specifically binds to the test substance, and the metal microparticles that bind to the second binding substance. Includes:

[0019] The immune complex comprises a test substance, a first binding substance that specifically binds to the test substance, a second binding substance that specifically binds to the test substance, and a labeled substance that binds to the second binding substance. In one embodiment, the first binding substance recognizes a site on the test substance that is different from that of the second binding substance.

[0020] In one embodiment, the measurement method according to this embodiment uses a working electrode on which an immune complex has been immobilized in advance. In another embodiment, the measurement method according to this embodiment further comprises an immobilization step of immobilizing a first binding substance on the surface of the working electrode, a labeling step of labeling a second binding substance with a labeling substance to form a label, and a formation step of supplying a label and a test substance to the surface of the working electrode on which the first binding substance has been immobilized, thereby forming an immune complex on the surface of the working electrode. In yet another embodiment, the measurement method according to this embodiment further comprises a providing step of providing a working electrode on which a first binding substance has been immobilized in advance, a labeling step of labeling a second binding substance with a labeling substance to form a label, and a formation step of supplying a label and a test substance to the surface of the working electrode on which the first binding substance has been immobilized, thereby forming an immune complex on the surface of the working electrode. In yet another embodiment, the measurement method according to this embodiment further includes a providing step of providing a working electrode to which a first binding substance has been immobilized in advance, and a labeled entity in which a second binding substance has been labeled in advance with a labeling substance, and a forming step of supplying the labeled entity and the test substance to the surface of the working electrode to which the first binding substance has been immobilized, thereby forming an immune complex on the surface of the working electrode.

[0021] The metal particle binding electrochemical immunoassay that forms the basis of the method according to this embodiment will be described in detail below.

[0022] Metal particle-binding electrochemical immunoassay Two types of specific binding substances for the test substance are prepared, one (first binding substance) is immobilized on the surface of a working electrode, and the other (second binding substance) is labeled with a labeling substance (e.g., metal fine particles) to form a labeled substance. Specifically, a primary antibody is first immobilized on the surface of the working electrode used in electrochemical measurement as the first binding substance for the test substance. The electrode surface is blocked to prevent nonspecific adsorption. A secondary antibody is also prepared as a second binding substance that recognizes a site on the test substance different from the first binding substance, and a labeled substance is then labeled to this to prepare a labeled substance.

[0023] Next, a test solution containing the label and an unknown amount of the test substance is supplied to the surface of the working electrode and brought into contact with the primary antibody, causing an antigen-antibody reaction on the working electrode. The label binds to the primary antibody via the test substance, causing an amount of the label corresponding to the concentration of the test substance to be collected near the working electrode 1.

[0024] 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 in the test solution. However, this combination is not limited to this, and any combination that specifically binds between substances may be used, such as specific binding between nucleic acids, nucleic acids, nucleic acid-nucleic acid binding proteins, lectins, and sugar chains, or receptors and ligands. The order of the relationship between the test substance and the specific binding substance may be reversed.

[0025] Examples of the labeling substance include metal fine particles. There are no particular limitations on the metal fine particles, but for example, fine particles of gold, platinum, silver, copper, rhodium, palladium, etc., or colloidal particles or quantum dots thereof can be used. Among these, it is preferable to use gold fine particles with a particle size of 10 nm to 60 nm, and particularly gold fine particles with a particle size of about 40 nm.

[0026] After the antigen-antibody reaction is carried out and the surface of the working electrode is washed with a washing solution as necessary, the working electrode is brought into contact with, for example, an electrochemical measurement solution. To bring the electrochemical measurement solution into contact with the working electrode, any method can be used, such as dropping the electrochemical measurement solution onto the surface of the working electrode or immersing the working electrode in the electrochemical measurement solution.

[0027] The solution used for controlling the potential of the working electrode and for electrochemical measurement (electrochemical measurement solution) is preferably an acidic solution, since it can easily electrochemically oxidize 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 metal particles, 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.

[0028] On the other hand, as the solution used for controlling the potential of the working electrode and performing electrochemical measurements (electrochemical measurement solution), in addition to acidic solutions, neutral solutions containing chlorine can also be used. The use of a neutral solution containing chlorine results in a larger current change than the use of an acidic solution, resulting in more sensitive measurements. Furthermore, when an acidic solution is used, the peak shape may become asymmetric, e.g., the base of the reduction peak may rise at low potentials, or noise may occur, for example, around 0.1 V. In contrast, the use of a neutral solution containing chlorine flattens the base of the reduction peak and suppresses the generation of the above-mentioned 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 procedures. The above-mentioned effects can be obtained when using neutral solutions containing chlorine, such as KCl, NaCl, and LiCl, but the effect is particularly pronounced when KCl is used.

[0029] oxidation process Next, to electrochemically oxidize the metal microparticles, a first potential (oxidation potential) is applied to the working electrode on which the immune complex containing the test substance is immobilized. The first potential is a potential that electrochemically induces oxidation of the metal microparticles. Oxidation of the metal microparticles can be induced by maintaining the potential of the working electrode relative to the reference electrode at a potential at which the metal microparticles are electrochemically oxidized for a predetermined period of time. By applying the first potential to the working electrode on which the immune complex is immobilized, the metal microparticles collected near the surface of the working electrode are completely oxidized. At this time, the counter electrode and reference electrode are also in contact with the electrochemical measurement solution.

[0030] When oxidizing metal microparticles, the potential of the working electrode is set to a potential at which the metal microparticles can be oxidized. Specifically, the potential of the working electrode must be set to an optimal value depending on the type of metal microparticles used. For example, it is preferable to set the potential of the working electrode to +1 V or higher relative to a silver-silver chloride reference electrode. By setting the potential of the working electrode within the above range, the metal microparticles collected near the surface of the working electrode can be completely oxidized and eluted, thereby reliably improving the detection sensitivity of the analyte. If the potential of the working electrode 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 migrate and diffuse, reducing the oxide concentration near the working electrode and resulting in a smaller reduction current peak. A more preferable range is +1.2 V to +1.6 V.

[0031] A specific method for electrochemically oxidizing metal microparticles is to maintain the potential of the working electrode 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, in addition to maintaining the potential of the working electrode at a predetermined potential as described above, the potential of the working electrode may be varied over time, for example, by cyclic voltammetry. When varying the potential of the working electrode over time, it is preferable to vary the potential of the working electrode within a potential range at which the metal microparticles are oxidized (for example, +1 to +2 V relative to a silver-silver chloride reference electrode). Furthermore, when oxidizing the metal microparticles, a potential at which the metal microparticles are electrochemically oxidized may be applied to the working electrode multiple times.

[0032] 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 0.1N to 0.5N hydrochloric acid solution at a potential of +1.2 V to +1.6 V of the working electrode relative to a silver-silver chloride reference electrode.

[0033] 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 with respect to time, if the potential applied to the working electrode 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 is relatively high, only a short time is required to sufficiently oxidize the metal microparticles.

[0034] By setting the time (first time) for which the potential of the working electrode is maintained at a potential (first potential) at which the metal microparticles are electrochemically oxidized to 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 first time is set to 100 seconds or more, the obtained current value remains almost unchanged. Therefore, the first time is preferably 1 second or more and 100 seconds or less, and more preferably 40 seconds or more and 100 seconds or less.

[0035] Low potential process Next, the first potential is changed to a second potential that is lower than the first potential. In one embodiment, the second potential is 20% to 60% lower than the first potential (e.g., 20, 25, 30, 35, 40, 45, 50, 55, or 60%, or a range between any two points selected from the group consisting of these). In one embodiment, the first potential is changed to a second potential that is lower than the first potential, and the second potential is maintained for a certain period of time. The certain period of time is the time during which the potential of the working electrode is maintained at the second potential, and is preferably 1 second or more, more preferably 1 to 60 seconds (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 seconds, or a range between any two points selected from the group consisting of these). In one embodiment, the time required to change from the first potential to the second potential (first change time) is preferably as short as technically possible, for example, within 1 second.

[0036] Current measurement process Next, the reduction current generated when the oxidized metal fine particles are reduced is measured. In one embodiment, the method according to this embodiment further includes a potential changing step of changing the second potential to a third potential lower or higher than the second potential. The potential changing step is performed after the low potential step and before the current measuring step. In one embodiment, the third potential is a potential lower or higher than the second potential by 20% to 60% (e.g., 20, 25, 30, 35, 40, 45, 50, 55, or 60%, or a range between any two points selected from the group consisting of these).

[0037] In one embodiment, the potential (second or third potential) of the working electrode is changed in the negative direction while measuring the reduction current generated when the oxidized metal particles are reduced. The presence or concentration of the analyte can be determined based on the peak current value generated when the oxidized metal particles are reduced. Specifically, for example, the potential (second or third potential) of the working electrode is changed in the negative direction while measuring the current change associated with the potential change. As the electrode potential is changed in the negative direction, the oxidized and eluted metal particles are reduced by the aforementioned potential control, 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, 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.

[0038] 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.

[0039] An antigen-antibody reaction or the like is carried out on the working electrode to collect metal particles near the surface of the working electrode, and the reduction peak current derived from the metal particles contained in the label is measured, allowing for simple and highly sensitive measurement of the test substance in the test solution.

[0040] In the above explanation, a method of collecting an amount of metal microparticles corresponding to the amount of test substance in a test solution 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 in a test solution, but a method of collecting an amount of metal microparticles corresponding to the amount of test substance in a test solution using a competitive reaction may also be used. [Example]

[0041] Examples of the present invention will be described below with reference to experimental results (for details of the experimental apparatus, method, conditions and reagents, see JP 2025-006568 A).

[0042] (Experiment 1) material In this example, the following reagents were used: Anti-NT-proBNP antibody for detection (Medix Biochemica) Anti-NT-proBNP antibody for capture (manufactured by Medix Biochemica) Gold nanoparticles, particle diameter 40 nm (BBI Solutions) Bovine serum albumin (BSA) (Sigma-Aldrich) Screen-printed carbon electrode (Yoshida Manufacturing Co., Ltd.) Non-protein blocking solution

[0043] Preparation of antibody-labeled gold nanoparticles To adjust the pH, 9 mL of gold nanoparticle dispersion (mean particle size 40 nm, optical density at 520 nm: 1.0) was mixed with 1 mL of 50 mM potassium dihydrogen phosphate solution adjusted to pH 6.5 with sodium hydroxide. 1 mL of 0.1 mg / mL anti-NT-proBNP antibody for detection, diluted with pure water, was added to 10 mL of this gold nanoparticle dispersion and allowed to stand at room temperature for 10 minutes to bind the antibody to the gold nanoparticles. Subsequently, 1 mL of 10% BSA was added to the gold nanoparticle dispersion and allowed to stand at room temperature for 10 minutes to block the gold nanoparticle surface.

[0044] To remove unbound antibody and BSA, the gold nanoparticle dispersion was centrifuged at 8000 g at 4°C for 15 minutes, and the supernatant was removed. 1 mL of 20 mM Tris-HCl buffer (pH 7.4) containing 1 wt% BSA was added to the gold nanoparticle dispersion from which the supernatant had been removed, and the gold nanoparticles were re-dispersed. After centrifugation (8000 g at 4°C for 15 minutes), the supernatant of the gold nanoparticle dispersion was removed. This washing procedure was repeated once more, and the above buffer was added to the gold nanoparticle dispersion from which the supernatant had been removed to disperse the gold nanoparticles.

[0045] The optical density of the antibody-labeled gold nanoparticle dispersion was measured at an irradiation wavelength of 520 nm using a spectrophotometer. The antibody-labeled gold nanoparticle dispersion was stored at 4°C.

[0046] Preparation of antibody solid-phase electrodes The electrode device used for measuring the analyte was a planar printed electrode device 1 (width 4 mm, length 12 mm) as shown in Figure 1. The printed electrode device 1 has a working electrode 2 and a counter electrode 3 formed from carbon paste, a lead (not shown) also formed from carbon paste, and a reference electrode 4 formed from silver / silver chloride, all mounted on an insulating support 5. Parts of the surfaces of the working electrode 2, counter electrode 3, and reference electrode 4 are covered with an insulating layer 6, thereby defining the effective electrode area.

[0047] After the capture anti-NT-proBNP antibody was dried and loaded onto the working electrode of the screen-printed electrode, the working electrode, reference electrode, and counter electrode were blocked with a non-proteinaceous blocking solution. The resulting electrode was placed in a sealed container containing a desiccant and stored at 4°C.

[0048] Gold-linked electrochemical immunoassay (GLEIA) A dispersion of antibody-labeled gold nanoparticles bound to anti-NT-proBNP antibodies for detection was diluted in 20 mM Tris-HCl buffer (pH 7.4) containing 1 w / v% BSA to a final concentration of 500 pg / mL of NT-proBNP recombinant protein, and the diluted solution was brought into contact with the working electrode of a screen-printed carbon electrode for 7 minutes.

[0049] After washing with a 2 mol / L sodium chloride solution, gold-binding electrochemical immunoassay was performed. Specifically, differential pulse voltammetry was performed by applying 1250 mV for 40 seconds relative to a silver / silver chloride reference electrode, followed by a sweep from 1100 mV to 0 V (Comparative Example 1). In the potential application test of the present invention, differential pulse voltammetry was performed by applying an oxidation potential of 1250 mV for 40 seconds, followed by a 700 mV application for 10 seconds, followed by a sweep from 1100 mV to 0 V (Example 1). The results are shown in Figure 2.

[0050] result When 1250 mV was applied for 40 seconds and then the potential was swept from 1100 mV to 0 V, a broad peak was observed in the region from 700 mV to 1000 mV (dashed line in Figure 2). This broad peak is an interference peak, the wavelength range and magnitude of which vary depending on the screen-printed carbon electrode. Applying 700 mV for 10 seconds after applying the oxidizing potential suppressed or reduced this broad interference peak (solid line in Figure 2).

[0051] (Experiment 2) Experiment 2 was the same as Experiment 1 except for the conditions of the gold-linked electrochemical immunoassay.

[0052] Differential pulse voltammetry was performed by applying 1250 mV for 40 seconds, followed by 700 mV for 10 seconds, and then sweeping from 700 mV to 0 V, relative to a silver / silver chloride reference electrode (Example 2). As a control, differential pulse voltammetry was performed by applying 1250 mV for 40 seconds, followed by sweeping from 700 mV to 0 V, relative to a silver / silver chloride reference electrode (Comparative Example 2). The results are shown in Figures 3 and 4 (N=3).

[0053] result Table 1 shows the average values, standard deviations, and coefficients of variation of the measured values ​​obtained using the measurement methods and conditions of Example 2 and Comparative Example 2.

[0054] [Table 1]

[0055] The change in the measured value was slight even when the measurement method and conditions of Example 2 were applied. By applying the measurement method and conditions of Example 2, the coefficient of variation could be reduced.

[0056] Figure 3 shows voltammograms obtained by measuring samples containing 0, 30, 100, and 500 pg / mL of NT-proBNP using the measurement method and conditions of Example 2. Figure 4 shows voltammograms obtained by measuring the same sample as in Example 2 using the measurement method and conditions of Comparative Example 2. Figures 3 and 4 show voltammograms obtained by measuring samples containing 0 pg / mL, 30 pg / mL, 100 pg / mL, and 500 pg / mL of NT-proBNP.

[0057] It can be seen that the variation in the voltammogram in the region from 500 mV to 700 mV could be significantly suppressed or reduced by the measurement method and conditions of Example 2. By applying 700 mV for 10 seconds after application of the oxidation potential, the interference peak could be suppressed or reduced.

[0058] (Experiment 3) Experiment 3 was the same as Experiment 1 except for the conditions of the gold-linked electrochemical immunoassay.

[0059] Differential pulse voltammetry was performed by applying 1250 mV for 40 seconds to a silver / silver chloride reference electrode, followed by applying various potentials (450, 500, 700, 800, and 900 mV) for 10 seconds, and then sweeping from 1100 mV to 0 V. The results are shown in Figure 5.

[0060] result Applying a potential of 500 to 800 mV for 10 seconds after applying the oxidation potential suppressed or reduced the interference peak. Applying a potential of 900 mV for 10 seconds after applying the oxidation potential had limited effect on suppressing or reducing the interference peak. Applying a potential of 450 mV for 10 seconds after applying the oxidation potential, which is included in the reduction peak of gold nanoparticles, reduced the reduction current of the gold nanoparticles.

[0061] (Experiment 4) Experiment 4 was the same as experiment 1 except for the conditions of the gold-linked electrochemical immunoassay.

[0062] Differential pulse voltammetry was performed by applying 1250 mV for 40 seconds to a silver / silver chloride reference electrode, followed by applying various potentials (700, 1250, and 1400 mV) for 10 seconds, and then sweeping from 1100 mV to 0 V. The results are shown in Figure 6.

[0063] result In Figure 6, the solid line shows a voltammogram measured by applying 1250 mV for 40 seconds followed by 1400 mV for 10 seconds, the dashed line shows a voltammogram measured by applying 1250 mV for 40 seconds followed by 700 mV for 10 seconds, and the dotted line shows a voltammogram measured after applying 1250 mV for 40 seconds.

[0064] When an oxidizing potential of 1250 mV was applied for 40 seconds, followed by an oxidizing potential of 1400 mV for 10 seconds, the interference peak increased (solid line in Figure 6). In other words, applying an oxidizing potential multiple times did not contribute to suppressing or reducing the interference peak. [Explanation of symbols]

[0065] 1. Printed electrode device 2...Working electrode 3. Opposite 4...Reference electrode 5. Insulating support 6. Insulation layer

Claims

1. A method for measuring a test substance, characterized in that the presence or concentration of the test substance is examined, the method comprising: an oxidation step of applying a first potential to a working electrode on which an immune complex containing the test substance and metal fine particles is immobilized; a low potential step of changing the first potential to a second potential lower than the first potential; a current measuring step of measuring, at the working electrode, a reduction current generated when the oxidized metal fine particles are reduced; Including, the immune complex comprises the test substance, a first binding substance that specifically binds to the test substance, a second binding substance that specifically binds to the test substance, and the metal fine particle that binds to the second binding substance; The second potential is maintained for a period of time; The predetermined time is from 1 second to 60 seconds. method.

2. The method of claim 1 , wherein the second potential is 20% to 60% lower than the first potential.

3. The method of claim 1 , further comprising a potential-changing step of changing the second potential to a third potential that is lower or higher than the second potential.

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