Method for measuring measurement-target substance using electrochemical method

Abstract

To solve the problem that in the conventional technique, measurement-target solution for use in an electrochemical immunity measurement method is not buffer solution, therefore the pH of the measurement-target solution is not stabilized.SOLUTION: A method for measuring a measurement-target is provided, for inspecting presence / absence or density of the measurement-target, the method comprises: a washing step in which a working electrode to which an immune composite including the measurement-target is fixed, is washed by washing buffer solution; and a current measuring step in which a current value is measured by controlling a potential of the working electrode in the washing buffer solution. The immune composite includes: the measurement-target; a first coupled object specifically coupled to the measurement-target; a second coupled object specifically coupled to the measurement-target; and a labeled substance coupled to the second coupled object, the labeled substance is metal fine particles, and pH of the washing buffer solution is 4 to 6.SELECTED DRAWING: Figure 3

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 and luminescence derived from enzyme reactions, is used in a wide range of fields. However, ELISA requires an optical system to detect signals such as color development and luminescence, which necessitates a large measuring machine. In addition, to perform accurate quantification, complex processing is required, such as converting the measurement results such as color development into electrical signals.

[0003] Patent Document 1 discloses an electrochemical immunoassay method for detecting a test substance contained in a sample using a working electrode and a counter electrode. The electrochemical immunoassay method disclosed in Patent Document 1 includes the steps of (A) forming a complex containing the test substance contained in the sample and metal particles on a working electrode, (B) washing the working electrode, and (C) adding a measurement solution to the working electrode and measuring the current, voltage, or charge caused by the metal particles contained in the complex on the working electrode in the measurement solution by an electrochemical measurement method, and (D) detecting the test substance in the sample based on the measurement result obtained in the step (C), and is characterized in that the step (B) is a step of washing the working electrode with a measurement solution having the same composition as the measurement solution used in the step (C). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 2016-085168 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the measurement solution in Patent Document 1 has a problem in that the pH is not stable because it is not a buffer solution.

[0006] Therefore, the present inventors performed electrochemical immunoassays using the same buffer solution in the washing step and the current measurement step in order to stabilize the pH of the solution in the washing step and the current measurement step. However, the use of a buffer solution did not necessarily result in a successful electrochemical immunoassay. [Means for solving the problem]

[0007] As a result of further investigation, the inventors discovered that electrochemical immunoassay becomes possible by using a buffer solution that does not have a chelating effect in the washing step and the current measurement step, and thus completed the present invention.

[0008] The object of the present invention is to A method for measuring a test substance, comprising the steps of: determining the presence or absence or concentration of the test substance; a washing step of washing the working electrode on which the immune complex containing the test substance is immobilized with a washing buffer; a current measuring step of measuring a current value by controlling the potential of the working electrode in the washing buffer solution; Including, 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 a labeling substance bound to the second binding substance; the labeling substance is a metal fine particle, The pH of the washing buffer is 4 to 6. method The purpose of this project is to provide

[0009] By using this method, it is possible to use the same solution in the washing step and the current measurement step in an electrochemical immunoassay, and to stabilize the pH of the solution.

[0010] The wash buffer may be a non-chelating buffer. The wash buffer may be an acetate buffer or an MES buffer. [Brief description of the drawings]

[0011] [Figure 1] FIG. 2 is a plan view of a printed electrode device used in the examples. [Diagram 2] 2(A) and (B) show the results of measuring the reduction peak current of gold colloid. [Diagram 3] FIG. 3 shows the results of measuring the reduction peak current of gold colloid in a buffer-free solution, citrate B (pH 5.0 and pH 5.5), phthalate B (pH 5.0), acetate B (pH 5.0) and MES (pH 5.5). [Figure 4] 4(A) to (C) show the results of measuring the reduction peak current of gold colloids using a buffer-free solution (pH unadjusted), acetic acid B (pH 5.0), and MES (pH 5.0), respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] 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.

[0013] The numerical ranges and parameters given in the present invention are approximate values, but the numerical values ​​given in the specific examples are described as precisely as possible. However, any numerical value inherently contains a certain error that necessarily results from the standard deviation found in each test measurement. Also, the term "about" as used herein 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 those skilled in the art.

[0014] Hereinafter, an embodiment of the present invention will be described. The following embodiment is merely an example, and the scope of the present invention is not limited to the following embodiment. Note that, in order to avoid repetition and complexity, explanations of similar contents will be omitted.

[0015] Measurement method of test substance The measurement method according to the present embodiment is a method for measuring a test substance, characterized in that it examines the presence or absence 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 to measure a current value.

[0016] The immune complex comprises a test substance, a first binding substance specifically bound to the test substance, a second binding substance specifically bound to the test substance, and a labeling substance bound to the second binding substance. In one embodiment, the first binding substance recognizes a site on the test substance that is different from the site on the test substance from that of the second binding substance.

[0017] In one embodiment, the measurement method according to the present embodiment uses a working electrode on which an immune complex is immobilized in advance. In another embodiment, the measurement method according to the present embodiment further includes a fixing step of fixing 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 prepare a label, and a forming step of supplying a label and a test substance to the surface of the working electrode on which the first binding substance is immobilized to form an immune complex on the surface of the working electrode. In yet another embodiment, the measurement method according to the present embodiment further includes a providing step of providing a working electrode on which a first binding substance is immobilized in advance, a labeling step of labeling a second binding substance with a labeling substance to prepare a label, and a forming step of supplying a label and a test substance to the surface of the working electrode on which the first binding substance is immobilized to form 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 to which a first binding substance has been immobilized in advance, and a labeled body in which a second binding substance has been labeled in advance with a labeling substance, and a forming step of supplying the labeled body 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.

[0018] The metal particle binding electrochemical immunoassay that is the basis of the method according to this embodiment will be described in detail below.

[0019] Metal particle-binding electrochemical immunoassay Two types of specific binding substances for the test substance are prepared, one of which (the first binding substance) is immobilized on the surface of a working electrode, while the other (the second binding substance) is labeled with a labeling substance (e.g., metal fine particles) to prepare a label. 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 non-specific 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 prepared by labeling this with a labeling substance.

[0020] Next, a test solution containing the above-mentioned 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, and an antigen-antibody reaction occurs on the working electrode. The label binds to the primary antibody via the test substance, and an amount of the label corresponding to the concentration of the test substance is collected near the working electrode 1.

[0021] Here, 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 according to the test substance. In order to collect metal particles in an amount corresponding to the test substance in the test solution, the specific binding between an antigen and an antibody is used in this embodiment, but this combination is not limited to this, and any combination that specifically binds between substances may be used, for example, specific binding between nucleic acid-nucleic acid, nucleic acid-nucleic acid binding protein, lectin-sugar chain, or receptor-ligand. The order of the relationship between the test substance and the specific binding substance may be reversed from that described above.

[0022] The labeling substance may be metal fine particles. The metal fine particles are not particularly limited, but for example, fine particles of gold, platinum, silver, copper, rhodium, palladium, etc., colloidal particles thereof, quantum dots, etc., can be used. Among them, it is preferable to use gold fine particles having a particle size of 10 nm to 60 nm, particularly gold fine particles having a particle size of about 40 nm.

[0023] An antigen-antibody reaction is carried out, and the surface of the working electrode is washed with a washing buffer solution. After washing, the washing buffer solution is brought into contact with the working electrode. Any means can be used to bring the solution into contact with the working electrode, such as dropping the solution onto the surface of the working electrode or immersing the working electrode in the solution. The washing buffer solution is a buffer solution that does not have a chelating effect, and the pH of the washing buffer solution is 4 to 6. The washing buffer solution may be an acetate buffer solution or an MES buffer solution. The washing buffer solution can be changed according to the pH required for the electrochemical immunoassay to be carried out. For example, if the required pH is around 5, an acetate buffer solution may be selected, and if the required pH is around 5.5, an MES buffer solution may be selected.

[0024] Current measurement process Next, the metal particles are electrochemically oxidized in the washing buffer 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 the reference electrode are also in contact with the solution.

[0025] After electrochemically oxidizing metal particles, the presence or absence or concentration of the test substance is measured based on the peak current value that occurs when the oxidized metal is reduced. Specifically, for example, the potential of the working electrode is changed in the negative direction, and the change in current accompanying the potential change is measured. When the electrode potential is changed in the negative direction, the metal that has been oxidized and dissolved is reduced by the above-mentioned potential control, and a reduction current flows, which is measured. The more the test substance in the test solution is, and the more the metal particles that are collected near the working electrode, the greater the reduction current intensity, and the quantification or detection of the test substance is realized based on this. For example, the relationship between the reduction current value and the test substance of a known concentration is determined in advance, and the concentration of the test substance can be determined by comparing the reduction current value measured. In addition, the presence or absence of the test substance in the test solution can be known from the obtained reduction current value.

[0026] When oxidizing the metal fine particles, the potential of the working electrode is set to a potential at which the metal fine particles can be oxidized. Specifically, the potential of the working electrode needs to be set to an optimal value depending on the type of metal fine particles used, and is preferably set to +1V or more relative to a silver-silver chloride reference electrode, for example. By setting the potential of the working electrode within the above range, the metal fine particles collected near the surface of the working electrode can be completely oxidized and eluted, and the detection sensitivity of the test substance can be reliably improved. If the potential of the working electrode is set below the above range, there is a risk that the peak of the reduction current does not appear during measurement, and conversely, if it exceeds the above range, diffusion due to migration of the oxidized metal fine particles occurs, the concentration of the oxide near the working electrode decreases, and this may result in a smaller peak of the reduction current. A more preferable range is +1.2V to +1.6V.

[0027] A specific method for electrochemically oxidizing metal fine particles includes holding the potential of the working electrode at a potential at which the metal fine particles are oxidized for a predetermined time. The operation of holding the potential for a predetermined time is a preferred method because the metal fine particles can be sufficiently oxidized. When applying a potential at which the metal fine particles are electrochemically oxidized to the working electrode, the potential of the working electrode may be changed over time, for example, by cyclic voltammetry, in addition to the method of holding the potential of the working electrode at a predetermined potential as described above. When changing the potential of the working electrode over time, it is preferable to change the potential of the working electrode within a potential range at which the metal fine particles are oxidized (for example, +1 to +2 V relative to a silver-silver chloride reference electrode). Furthermore, when oxidizing the metal fine particles, the potential at which the metal fine particles are electrochemically oxidized may be applied to the working electrode multiple times.

[0028] When gold particles having a particle size of 10 nm to 60 nm are used as the metal particles, it is preferable to electrochemically oxidize the gold particles 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.

[0029] Here, when oxidizing the metal fine particles sufficiently, it is necessary to pay attention to provide an optimal charge amount according to the amount of the metal fine particles. Since the charge amount 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 oxidize the metal fine particles sufficiently. On the other hand, if the potential applied to the working electrode is relatively high, the time required to oxidize the metal fine particles sufficiently is short.

[0030] By holding the potential of the working electrode at a potential at which the metal particles are electrochemically oxidized for 1 second or more, the metal particles 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 is almost unchanged. Therefore, a holding time of the potential is preferably 1 second or more and 100 seconds or less. A more preferable range of the holding time of the potential is 40 seconds or more and 100 seconds or less.

[0031] Methods for measuring the current generated when an oxidized metal is electrochemically reduced include, for example, voltammetry such as differential pulse voltammetry and cyclic voltammetry, amperometry, and chronometry.

[0032] 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, thereby enabling the measurement of the test substance in the test solution simply and with high sensitivity.

[0033] In the above explanation, a method of collecting an amount of metal microparticles corresponding to the amount of analyte 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 analyte in a test solution, but a method of collecting an amount of metal microparticles corresponding to the amount of analyte in a test solution using a competitive reaction may also be used. EXAMPLES

[0034] material In this example, the following reagents were used: Anti-D-dimer antibody for detection (St John's Laboratory) Anti-D-dimer antibody for capture (TRINA Bioreactives) Gold nanoparticles, particle size 40 nm (BBI Solutions) Bovine serum albumin (BSA) (Sigma-Aldrich) Screen-printed carbon electrodes (BioDevice Technology) Non-protein based blocking solution PBS D-dimer calibrator (Sekisui Medical Co., Ltd.) Tween 20 (Polyoxyethylene (20) Sorbitan Monolaurate) (Fujifilm Wako Pure Chemical Industries, Ltd.) MES [2-(N-morpholino)ethanesulfonic acid monohydrate] (manufactured by Dojindo Chemical Industries, Ltd.) Glycine (Fujifilm Wako Pure Chemical Industries, Ltd.) Potassium hydrogen phthalate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

[0035] Preparation of antibody-labeled gold nanoparticles 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 size 40 nm, optical density at 520 nm: 1.0) and mixed. 1 mL of anti-D-dimer antibody for detection diluted with 0.1 mg / mL pure water was added to 10 mL of this gold nanoparticle dispersion, and the antibody was bound to the gold nanoparticles by leaving it at room temperature for 10 minutes. After that, 1 mL of 10% BSA was added to the gold nanoparticle dispersion, and the gold nanoparticle surface was blocked by leaving it at room temperature for 10 minutes.

[0036] To remove unbound antibodies and BSA, the gold nanoparticle dispersion was centrifuged at 4°C and 8000g for 15 minutes, and the supernatant was removed. 1 mL of 20 mM Tris-HCl buffer (pH 7.4) containing 1 w / v % BSA was added to the gold nanoparticle dispersion from which the supernatant had been removed, and the gold nanoparticles were dispersed again. After centrifugation (4°C, 8000g 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.

[0037] The optical density of the antibody-labeled gold nanoparticle dispersion was measured at an irradiation wavelength of 520 nm using a spectrophotometer by ultraviolet-visible spectroscopy. The antibody-labeled gold nanoparticle dispersion was stored at 4°C.

[0038] Preparation of antibody solid-phase electrodes A planar type printed electrode device 41 (width 4 mm, length 12 mm) as shown in Fig. 1 was used as the electrode device for measuring the test substance. The printed electrode device 41 has a working electrode 42 and a counter electrode 43 formed from carbon paste, a lead (not shown) also formed from carbon paste, and a reference electrode 44 formed from silver / silver chloride, all on an insulating support 45. Parts of the surfaces of the working electrode 42, counter electrode 43, and reference electrode 44 are covered with an insulating layer 46 to define the effective electrode area.

[0039] After the anti-D-dimer antibody was dried and supported on the working electrode of the screen-printed electrode, the working electrode, reference electrode, and counter electrode were blocked with a non-protein blocking solution. The resulting electrode was placed in a sealed container containing a desiccant and stored at 4°C.

[0040] Gold-linked electrochemical immunoassay (GLEIA) A dispersion of antibody-labeled gold nanoparticles bound to anti-D-dimer antibody for detection was diluted with 20 mM Tris-HCl buffer (pH 7.4) containing 1 w / v % BSA so that the optical density at an irradiation wavelength of 520 nm was 4.0 or 2.0. Each diluted dispersion was mixed with the solution shown in Table 1 below in a 1:1 ratio. [Table 1]

[0041] The concentration of D-dimer calibrator in test solutions 2 and 3 is 0.5 μg / mL.

[0042] Test solution 1 was allowed to stand at room temperature for 10 minutes or more, or test solutions 2 and 3 for 3 minutes, after which a 4.5 μL aliquot of each solution was dropped onto the working electrode of a screen-printed electrode with immobilized anti-D-dimer capture antibody and allowed to stand at room temperature for 3 minutes.

[0043] The electrodes were washed with 250 μL of various washing solutions (Test 1 (Test Solution 1): 6 types of solutions in Table 2, Test 2 (Test Solution 2): 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl and 0.05 v / v% Tween 20, Test 3 (Test Solution 3): 7 types of solutions in Table 4). After washing, the electrodes were blocked with a blocking solution, and the blocking solution on the electrodes was blown off with an air blower (air spray) and dried.

[0044] [Table 2]

[0045] 25 μL of each of the measurement solutions (Test 1: 0.2 M HCl, Test 2: 6 types of solutions in Table 3, Test 3: 7 types of solutions in Table 4) was dropped onto the obtained screen-printed electrodes so as to completely cover the working electrode, reference electrode, and counter electrode. A potentiostat was used to oxidize the gold atoms in the gold nanoparticles, followed by electrochemical measurement by differential pulse voltammetry.

[0046] [Table 3]

[0047] [Table 4]

[0048] The oxidation of gold atoms was carried out by applying a voltage of +1.25 V to the working electrode for 40 seconds relative to a silver / silver chloride reference electrode, and differential pulse voltammetry was carried out by changing the base potential of the working electrode from +0.7 V to 0 V relative to the same reference electrode at a pulse strength of 0.1 V, potential step width of 8 mV, pulse time of 0.4 seconds, pulse width of 0.1 seconds, and scan rate of 20 mV / second.

[0049] Redox peak current at each pH Figure 2(A) shows the results of measuring the reduction peak current of gold colloid using the working electrode for Test 1 and solutions with pH values ​​from 3 to 8 shown in Table 2, i.e., the blank was measured three times each. The error bars in the figure show the standard deviation. At pH 4.0 or lower, gold nanoparticles bound nonspecifically to the electrode, increasing the background noise.

[0050] Figure 2(B) shows the results of measuring the reduction peak current of gold colloid three times each using the working electrode for Test 2 and solutions of pH 3 to 8 shown in Table 3. The error bars in the figure represent the standard deviation. When a sample with a D-dimer calibrator concentration of 0.5 μg / mL was measured, the measured values ​​at pH 7.0 and 8.0 were significantly smaller than the other pH values, and at pH 6.0 or lower, the measured values ​​increased as the pH decreased.

[0051] Redox peak current in each buffer solution (pH 5.0 and pH 5.5) Next, the reduction peak current of the gold colloid was measured in the buffer solutions shown in Table 4 (buffer-free solution, citric acid B (pH 5.0 and pH 5.5), phthalic acid B (pH 5.0), acetic acid B (pH 5.0), and MES (pH 5.5)) (the working electrode used was the working electrode for Test 3). Figure 4 shows the results of measuring each sample with a D-dimer calibrator concentration of 0.5 μg / mL three times. The measurement was performed ten times. The current values ​​shown in the figure are the reduction peak current values ​​of the gold colloid calculated from the voltammogram after baseline subtraction.

[0052] As shown in Figure 3, the buffer-free solution and citric acid B (pH 5.0 and pH 5.5) showed large variations in the measured values, and phthalic acid B (pH 5.0), which has a chelating effect, showed poor redox efficiency. On the other hand, acetate B (pH 5.0) and MES (pH 5.5), which are buffers without a chelating effect, showed small variations in the measured values ​​at the pH values ​​at which they exert a buffering effect (5.0 and 5.5, respectively), and also showed good redox efficiency.

[0053] Redox peak current at pH 5.0 Next, the reduction peak current of the gold colloid at pH 5.0 was measured. Figures 4(A) to (C) show the results of measuring the reduction peak current of the gold colloid using a buffer-free solution (pH unadjusted), acetic acid B (pH 5.0), and MES (pH 5.0) shown in Table 4, respectively (the working electrode for Test 3 was used as the working electrode). Each measurement was performed 10 times.

[0054] As shown in Figures 4(A) to (C), the buffer solution adjusted to around pH 5.0 was able to reduce the variability in the measurement values ​​more than a buffer-free solution with an unadjusted pH. [Explanation of symbols]

[0055] 1. Printed electrode device 2 working electrode 3. Opposite 4 Reference electrode 5 Insulating support 6 Insulating layer

Claims

1. A method for measuring a test substance, comprising the steps of: determining the presence or absence or concentration of the test substance; a washing step of washing the working electrode on which the immune complex containing the test substance is immobilized with a washing buffer; a current measuring step of measuring a current value by controlling the potential of the working electrode in the washing buffer solution; Including, 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 a labeling substance bound to the second binding substance; the labeling substance is a metal fine particle, The pH of the wash buffer is between 4 and 6. method.

2. The method of claim 1 , wherein the wash buffer is a non-chelating buffer.

3. The method of claim 2 , wherein the washing buffer is an acetate buffer or an MES buffer.

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