Method for measuring test substance using electrochemical method

Abstract

In conventional techniques, it has been difficult to increase the reduction current of gold nanoparticles, i.e., to increase sensitivity. [Solution] An 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: a current measurement step of measuring a current value by controlling the potential of a working electrode to which an immune complex containing the test substance is immobilized; the immune complex comprising 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 a labeling substance that binds to the second binding substance; the labeling substance is a metal microparticle comprising a core and a metal layer covering the core; and the density of the material of the core is lower than the density of the material of the metal layer.

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, it was difficult to increase the reduction current of gold nanoparticles, i.e., to increase sensitivity, in conventional techniques including Patent Document 1. Therefore, we attempted to increase the reduction current by increasing the particle diameter of the gold nanoparticles.

[0007] However, it was found that when the particle diameter of gold nanoparticles is 100 nm or more, the background noise increases and the signal-to-noise ratio deteriorates. [Means for solving the problem]

[0008] As a result of extensive research, the inventors have found that by using a labeling substance composed of metal microparticles that include a non-conductive core and a metal layer covering the core, it is possible to increase the reduction current while suppressing background noise even when the particle diameter of the labeling substance is 100 nm or more, and have completed the present invention.

[0009] [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: a current measurement step of measuring a current value by controlling the potential of a working electrode to which the immune complex containing the test substance is immobilized; 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 a labeling substance that binds to the second binding substance; the labeling substance is a metal microparticle including a core and a metal layer covering the core, The density of the material of the core is lower than the density of the material of the metal layer. method The purpose is to provide

[0010] The method according to the present invention can obtain a higher reduction current than that obtained with conventional metal core particles while suppressing background noise.

[0011] [2] In the method described in [1], the core may be non-conductive.

[0012] [3] In the method described in [2], the core material may be a silica core.

[0013] [4] In the method according to [1], the thickness of the metal layer may be 10 nm to 20 nm.

[0014] [5] In the method according to [1], the particle size of the labeling substance is 100 nm or more.

[0015] In the method according to any one of [1] to [5], the metal layer may be a gold (Au) layer, a platinum (Pt) layer, a silver (Ag) layer, a copper (Cu) layer, a rhodium (Rh) layer, or a palladium (Pb) layer. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 shows a plan view of a printed electrode device. [Figure 2] FIG. 2 shows a bar graph of the average peak value (μA) of the reduction current for Sample 1 (particle diameter: 40 nm) and Sample 2 (particle diameter: 100 nm). [Figure 3] Figure 3(A) shows graphs illustrating the reduction current values ​​for gold nanoparticles (Graph 11: 0 ng / mL, Graph 12: 62.5 ng / mL, Graph 13: 125 ng / mL, and Graph 14: 250 ng / mL), while Figure 3(B) shows graphs illustrating the reduction current values ​​for gold nanoshell particles (Graph 21: 0 ng / mL, Graph 22: 62.5 ng / mL, Graph 23: 125 ng / mL, and Graph 24: 250 ng / mL). [Figure 4] FIG. 4 shows a dot plot graph plotting the peak value of the reduction current versus each antigen concentration when gold nanoparticles and gold nanoshell particles are used. DETAILED DESCRIPTION OF THE INVENTION

[0017] 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.

[0018] 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.

[0019] 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.

[0020] Measurement method of test substance The measurement method according to this embodiment is a method for measuring a test substance, characterized in that it examines the presence or concentration of the test substance. This measurement method includes a washing step of washing, with a washing buffer solution, a working electrode on which an immune complex containing the test substance is immobilized, and a current measurement step of controlling the potential of the working electrode in the washing buffer solution and measuring a current value.

[0021] 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.

[0022] 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 a 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.

[0023] The metal particle binding electrochemical immunoassay that forms the basis of the method according to this embodiment will be described in detail below.

[0024] 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 to form a labeled body (metallic fine particles). 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 label is then labeled with a labeling substance to prepare a labeled body.

[0025] 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.

[0026] 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.

[0027] The labeling substance is a metal microparticle comprising a core and a metal layer covering the core. The density of the material of the core is lower than the density of the material of the metal layer. In one embodiment, the core is non-conductive. The density of the material of the non-conductive core is lower than the density of the material of the metal layer. In one embodiment, the non-conductive core is a silica core. In one embodiment, the thickness of the metal layer is 10 nm to 20 nm. In one embodiment, the metal layer is a gold (Au) layer, a platinum (Pt) layer, a silver (Ag) layer, a copper (Cu) layer, a rhodium (Rh) layer, or a palladium (Pb) layer. In another embodiment, the metal layer is a gold (Au) layer. In one embodiment, the particle size of the labeling substance is 100 nm or more or more than 100 nm. In one embodiment, the particle size of the labeling substance is 100 to 200 nm (e.g., 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm, or a range between any two points selected from the group consisting of these). In one embodiment, the particle size of the labeling substance is 105 to 190 nm, 110 to 180 nm, 115 to 170 nm, or 120 to 160 nm.

[0028] After the antigen-antibody reaction is performed and the surface of the working electrode is washed with a washing buffer solution as necessary, the working electrode is brought into contact with, for example, an electrochemical measurement solution (a solution used for controlling the potential of the working electrode and for electrochemical measurements). 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.

[0029] The wash buffer may be PBS, acetate buffer, or MES buffer. In one embodiment, the wash buffer is a non-chelating buffer, and the pH of the wash buffer is 4 to 6. The wash buffer can be changed depending on the pH required for the electrochemical immunoassay to be performed. For example, if the required pH is around 5, acetate buffer may be selected, and if the required pH is around 5.5, MES buffer may be selected.

[0030] As the solution for electrochemical measurement, an acidic solution is preferably used because it can easily electrochemically oxidize metal microparticles. The acidic solution may be appropriately selected depending on the type of metal microparticles, and examples thereof 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 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.

[0031] On the other hand, in addition to acidic solutions, neutral solutions containing chlorine can also be used as electrochemical measurement solutions. 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 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 using KCl.

[0032] Current measurement process Next, the metal particles are electrochemically oxidized in the electrochemical measurement solution. For example, the potential of the working electrode relative to the reference electrode 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. At this time, the counter electrode and reference electrode are also in contact with the solution.

[0033] After electrochemically oxidizing metal particles, the presence or concentration of a test substance is measured based on the peak current value generated when the oxidized metal is reduced. Specifically, for example, the potential of the working electrode 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 aforementioned potential control, resulting in a reduction current, which is measured. The greater the amount of test substance in the test solution and the greater the number of metal particles collected near the working electrode, the greater the reduction current intensity, and thus the quantification or detection of the test substance can be achieved based on this. For example, the relationship between the reduction current value and a known concentration of the test substance can be determined in advance, and the concentration of the test substance can be determined by comparing the measured reduction current value. Furthermore, the presence or absence of the test substance in the test solution can be determined from the obtained reduction current value.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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 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.

[0038] By holding the potential of the working electrode 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.

[0039] 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.

[0040] 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.

[0041] 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]

[0042] 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).

[0043] 1. Materials and Methods antibody Anti-D-dimer antibody (Medix Biochemica) and anti-SARS-coronavirus (Cat: 40143-R040, Nippon Shinobiological Co., Ltd.) were used as the detection antibody and capture antibody. Electrode Device The electrode device used for measuring the analyte was a planar printed electrode device 1 (width 4 mm, length 12 mm, screen-printed carbon electrode (Yoshida Manufacturing Co., Ltd.)) 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.

[0044] After the capture antibody was dried and supported on the working electrode of the screen-printed electrode, the working electrode 2, counter electrode 3, and reference electrode 4 were blocked with a non-proteinaceous blocking solution. The resulting electrodes were placed in a sealed container containing a desiccant and stored at 4°C.

[0045] Preparation of antibody-labeled gold nanoparticles and gold nanoshell particles Gold nanoparticles with particle diameters of 40 nm and 100 nm (manufactured by BBI Solutions) and 150 nm gold nanoshell particles (core: Si, shell: Au, shell thickness: estimated 14 nm, manufactured by nanoComposix) were used. Gold nanoparticles are particles composed only of gold (Au).

[0046] To adjust the pH, 1 mL of 50 mM potassium dihydrogen phosphate solution, adjusted to pH 6.5 with sodium hydroxide, was added to 9 mL of gold nanoparticle dispersion (average particle diameter: 40 or 100 nm, optical density at 520 nm: 1.0) and gold nanoshell particle dispersion (average particle diameter: 150 nm, optical density at 590 nm: 20), and mixed. 1 mL of 0.1 mg / mL detection antibody diluted with pure water was added to 10 mL of each dispersion, and the mixture was left at room temperature for 1 hour to bind the detection antibody to the gold nanoparticles or gold nanoshell particles. Then, 1 mL of 10% bovine serum albumin (BSA, Sigma-Aldrich) was added to each dispersion, and the mixture was left at room temperature for 10 minutes to block the gold nanoparticle and gold nanoshell particle surfaces.

[0047] To remove unbound detection antibody and BSA, each dispersion was centrifuged at 8000 g at 4°C for 15 minutes, and the supernatant was removed. To each dispersion, 1 mL of 20 mM Tris-HCl buffer (pH 7.4) containing 1 wt% BSA was added to redisperse the gold nanoparticles and gold nanoshell particles. After centrifugation (8000 g at 4°C for 15 minutes), the supernatant was removed. This washing procedure was repeated once more, and the above buffer was added to each dispersion to redisperse the gold nanoparticles and gold nanoshell particles.

[0048] The optical density of the resulting antibody-labeled gold nanoparticle dispersion and antibody-labeled gold nanoshell particle dispersion was measured by ultraviolet-visible spectroscopy using a spectrophotometer at an irradiation wavelength of 520 nm (580 nm for particle diameters of 100 nm), and the dispersions were adjusted to an OD of 7.5. The antibody-labeled gold nanoparticle dispersion and antibody-labeled gold nanoshell particle dispersion were stored at 4°C.

[0049] 2. Experiment 1 Gold nanoparticles with a particle diameter of 40 nm or 100 nm were used. Anti-SARS-coronavirus antibody and its antigen protein (Recombinant SARS-CoV Nucleoprotein / NP Protein (Catalog #40143-V08B)) were used.

[0050] 15 μL of each dispersion of antibody-labeled gold nanoparticles (particle diameter 40 nm: Sample 1 or particle diameter 100 nm: Sample 2) bound to a detection antibody was mixed with 15 μL of conjugation buffer (20 mM Tris-HCl (pH 7.4), 1% BSA, 5% sucrose, 1% Tween 20), and the mixture was dried under reduced pressure for 15 minutes. 55 μL of an antigen solution (0.9% NaCl, 1% BSA) containing antigen protein (50 pg / mL) was added to each dried sample and allowed to react for 3 minutes. 4.5 μL of the mixture of each sample and antigen solution was contacted with working electrode 2, a screen-printed carbon electrode with immobilized capture antibody, for 15 minutes. After contact, working electrode 2 was washed with 250 μL of 2 M / L sodium chloride solution containing 0.05% Tween 20 and then dried by blowing air over it.

[0051] 25 μL of a 2M / L aqueous sodium chloride solution containing 0.05% tween was dropped onto the working electrode 2, counter electrode 3 and reference electrode 4 so that the surfaces of the electrodes were completely covered.

[0052] The gold atoms in the gold nanoparticles or gold nanoshell particles were oxidized using the potentiostat in DPV mode, followed by electrochemical measurements using differential pulse voltammetry. Electrochemical measurements were also performed on a control sample (0 pg / mL) that did not contain the detection antibody.

[0053] The oxidation of gold atoms was carried out by applying a voltage of 1200 mV to the working electrode 2 for 40 seconds relative to the reference electrode 4 made of silver / silver chloride. Differential pulse voltammetry was carried out by varying the base potential of the working electrode 2 from 700 mV to 0 mV relative to the reference electrode 4 at a pulse strength of 0.1 V, a potential step width of 8 mV, a pulse time of 0.4 seconds, a pulse width of 0.1 seconds, and a scan rate of 20 mV / second.

[0054] Table 1 summarizes the peak values ​​of the reduction current for gold nanoparticles and gold nanoshell particles measured twice. Figure 2 shows a bar graph of the average peak values ​​(μA) of the reduction current for Sample 1 (particle diameter: 40 nm) and Sample 2 (particle diameter: 100 nm).

[0055] [Table 1]

[0056] Although the peak value of the reduction current was larger in Sample 2 (particle diameter: 100 nm) than in Sample 1 (particle diameter: 40 nm), the S / N ratio (sample average peak value / control average peak value) was lower in Sample 2 (approximately 1.1) than in Sample 1 (approximately 2.1). For gold nanoparticles composed only of gold, the current value increased as the particle diameter of the gold particles increased, but the background noise also increased, and the detection limit could not be improved due to the worsening S / N ratio.

[0057] 3. Experiment 2 The D-dimer antigen was adjusted to 500, 250, 125, or 0 ng / mL in 20 mM Tris-HCl buffer (pH 7.4) containing 1 w / v% BSA. A dispersion of antibody-labeled gold nanoparticles (Comparative Example 1) or a dispersion of antibody-labeled gold nanoshell particles (Example 1) bound to a detection antibody was mixed with the D-dimer antigen solution at a 1:1 ratio and allowed to react for 3 minutes (final concentrations of D-dimer antigen were 250, 125, 62.5, or 0 ng / mL). After 3 minutes, 4.5 μL of the mixture of each dispersion and the D-dimer antigen solution was dropped onto the working electrode 2 and allowed to react for 3 minutes. After 3 minutes, each electrode was washed with 250 μL of 2 M NaCl solution.

[0058] A 2M / L aqueous sodium chloride solution was added dropwise to the surfaces of the working electrode 2, the counter electrode 3 and the reference electrode 4 so as to completely cover them.

[0059] Using the DPV mode of the potentiostat, oxidation of gold atoms in gold nanoparticles or gold nanoshell particles was performed, followed by electrochemical measurements using differential pulse voltammetry.

[0060] The oxidation of gold atoms was carried out by applying a voltage of 1250 mV to the working electrode 2 for 40 seconds relative to the reference electrode 4 made of silver / silver chloride. Differential pulse voltammetry was carried out by varying the base potential of the working electrode 2 from 700 mV to 0 mV relative to the reference electrode 4 at a pulse strength of 0.1 V, a potential step width of 8 mV, a pulse time of 0.4 seconds, a pulse width of 0.1 seconds, and a scan rate of 20 mV / second.

[0061] Figure 3(A) shows graphs (Graph 11: 0 ng / mL, Graph 12: 62.5 ng / mL, Graph 13: 125 ng / mL, and Graph 14: 250 ng / mL) illustrating the reduction current values ​​for Comparative Example 1 (gold nanoparticles). Figure 3(B) shows graphs (Graph 21: 0 ng / mL, Graph 22: 62.5 ng / mL, Graph 23: 125 ng / mL, and Graph 24: 250 ng / mL) illustrating the reduction current values ​​for Example 1 (gold nanoshell particles).

[0062] As shown in Figure 3(B), it was confirmed that the peak value of the reduction current of gold nanoshell particles, which appears around 300 to 500 mV, increases in a concentration-dependent manner, similar to gold nanoparticles.

[0063] Table 2 lists the peak value (μA) of the reduction current and the ratio (peak value of the example / peak value of the comparative example) for each concentration in Comparative Example 1 and Example 1. Figure 4 shows a dot plot graph in which the peak value of the reduction current for each concentration in Comparative Example 1 and Example 1 is plotted.

[0064] [Table 2]

[0065] It was revealed that Example 1 enhanced the signal by about 9 times compared to the conventional Comparative Example 1. Furthermore, unlike gold nanoparticles, the gold nanoshell particles showed a good rise in the calibration curve on the low concentration side. [Explanation of symbols]

[0066] 1. Printed electrode device 2...Working electrode 3. Opposite 4...Reference electrode 5. Insulating support 6. Insulation layer Reduction current value for 11··0 ng / mL gold nanoparticles The reduction current value for 12··62.5ng / mL gold nanoparticles 13··Reduction current value for 125ng / mL gold nanoparticles 14··Reduction current value for 250ng / mL gold nanoparticles The reduction current value for gold nanoshell particles at 21··0 ng / mL The reduction current value for gold nanoshell particles is 22··62.5ng / mL. The reduction current value for 23··125ng / mL gold nanoshell particles Reduction current values ​​for 24··250ng / mL gold nanoshell particles

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: a current measurement step of measuring a current value by controlling the potential of a working electrode to which an immune complex containing the test substance is immobilized; 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 a labeling substance that binds to the second binding substance; the labeling substance is a metal fine particle including a core and a metal layer covering the core, the density of the material of the core is lower than the density of the material of the metal layer; The core is non-conductive. method.

2. The method according to claim 1 , wherein the core material is a silica core.

3. The method of claim 1 , wherein the metal layer has a thickness of 10 nm to 20 nm.

4. The method according to claim 1 , wherein the particle size of the labeling substance is 100 nm or more.

5. 5. The method according to claim 1, wherein the metal layer is a gold (Au) layer, a platinum (Pt) layer, a silver (Ag) layer, a copper (Cu) layer, a rhodium (Rh) layer, or a palladium (Pb) layer.

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