Method for measuring a test substance using an electrochemical technique

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 Art

[0002] As one of the methods for simply and highly sensitively measuring trace substances in a test solution, an immunoassay method using an antigen-antibody reaction is known. As an immunoassay method, an ELISA method that uses an antibody labeled with an enzyme and detects or measures the concentration of a test substance by obtaining a signal such as color development or luminescence derived from an enzyme reaction is adopted in a wide range of fields. However, in the ELISA method, since an optical system is required for signal detection such as color development or luminescence, a large measuring instrument is required. In addition, when accurate quantification is performed, complicated processing such as converting the measurement result of color development or the like into an electrical signal is required.

[0003] Patent Document 1 discloses an electrochemical immunoassay method that uses a working electrode and a counter electrode to detect a test substance contained in a sample. The electrochemical immunoassay method disclosed in Patent Document 1 includes: (A) a step of forming a complex containing the test substance contained in the sample and metal fine particles on the working electrode; (B) a step of washing the working electrode; and (C) adding a measurement solution to the working electrode and measuring, by an electrochemical measurement method, a current, voltage, or charge caused by the metal fine particles contained in the complex on the working electrode in the measurement solution; (D) a step of detecting the test substance in the sample based on the measurement result obtained in the step (C), and the step (B) is a step of washing the working electrode using 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

Summary of the Invention

Problems to be Solved by the Invention

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

[0006] Therefore, in order to stabilize the pH of the solution in the washing step and the current measurement step, the present inventors performed electrochemical immunoassay using the same buffer solution in the washing step and the current measurement step. However, even when using a buffer solution, the electrochemical immunoassay did not always succeed.

Means for Solving the Problems

[0007] As a result of further investigation, the present inventors found that electrochemical immunoassay can be achieved by using a buffer solution having no chelating action in the washing step and the current measurement step, and thus completed the present invention.

[0008] An object of the present invention is A method for measuring a test substance, characterized by examining the presence or concentration of the test substance, the method comprising: A washing step of washing a working electrode on which an immune complex containing the test substance is immobilized with a washing buffer; A current measurement step of controlling the potential of the working electrode in the washing buffer and measuring a current value; including The immune complex includes the test substance, a first binding substance specifically binding to the test substance, a second binding substance specifically binding to the test substance, and a labeling substance bound to the second binding substance; The labeling substance is metal fine particles; The pH of the washing buffer is from 4 to 6; method is to provide.

[0009] By using this method, in the washing step and the current measurement step in the electrochemical immunoassay method, the same solution can be used, and the pH of the solution can be stabilized.

[0010] The above washing buffer may be a buffer having no chelating action. The above washing buffer may be an acetate buffer or a MES buffer.

Brief Description of Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0012] Definition For convenience, the specific terms used in this application are collected here. Unless otherwise specified, all technical terms and scientific terms used in this application have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains. Unless otherwise clearly stated in the context, the singular forms "a", "an" and "the" include plural references.

[0013] The numerical ranges and parameters shown in the present invention are approximate values. Although the numerical values shown in specific examples are described as accurately as possible, any numerical value inherently contains certain errors that inevitably result from the standard deviation found in each test measurement value. Further, 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 when considered by those skilled in the art.

[0014] Hereinafter, embodiments of the present invention will be described. The following embodiments are illustrative, and the scope of the present invention is not limited to those shown in the following embodiments. Note that the description of the same content is omitted to avoid repetition.

[0015] Method for measuring a test substance The measurement method according to this embodiment is a measurement method for a test substance, characterized by examining the presence or concentration of the test substance. This measurement method includes a washing step of washing a working electrode on which an immune complex containing the test substance is immobilized with a washing buffer, and a current measurement step of controlling the potential of the working electrode in the washing buffer and measuring the current value.

[0016] The immune complex includes 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 labeling substance that binds to the second binding substance. In one embodiment, the first binding substance recognizes a site on a different test substance than the second binding substance.

[0017] In one embodiment, the measurement method according to this embodiment uses a working electrode on which an immune complex is fixed in advance. In another embodiment, the measurement method according to this embodiment further includes a fixing step of fixing a first binding substance on the surface of the working electrode, a labeling step of preparing a labeled body by labeling a second binding substance with a labeling substance, and a forming step of supplying the labeled body and a test substance onto the surface of the working electrode on which the first binding substance is fixed to form an immune complex on the surface of the working electrode. In still another embodiment, the measurement method according to this embodiment further includes a providing step of providing a working electrode on which a first binding substance is fixed in advance, a labeling step of preparing a labeled body by labeling a second binding substance with a labeling substance, and a forming step of supplying the labeled body and a test substance onto the surface of the working electrode on which the first binding substance is fixed to form an immune complex on the surface of the working electrode. In still another embodiment, the measurement method according to this embodiment further includes a providing step of providing a working electrode on which a first binding substance is fixed in advance and a labeled body in which a second binding substance is labeled with a labeling substance in advance, and a forming step of supplying the labeled body and a test substance onto the surface of the working electrode on which the first binding substance is fixed to form an immune complex on the surface of the working electrode.

[0018] Hereinafter, the metal particle-bound electrochemical immunoassay that is the basis of the method according to this embodiment will be described in detail.

[0019] Metal Particle-Bound Electrochemical Immunoassay Prepare two types of specific binding substances for the test substance. Fix one (the first binding substance) on the surface of the working electrode, and label the other (the second binding substance) with a labeling substance (e.g., metal particles) to obtain a labeled body. Specifically, first, fix a primary antibody as the first binding substance for the test substance on the surface of the working electrode used in the electrochemical measurement. The electrode surface is blocked to prevent non-specific adsorption. Also, prepare a secondary antibody as the second binding substance that recognizes a site on the test substance different from the first binding substance, and prepare a labeled body by labeling this with a labeling substance.

[0020] Next, a test solution containing the above-described labeling substance and an analyte of an unknown amount is supplied to the surface of the working electrode and brought into contact with the primary antibody, and an antigen-antibody reaction is carried out on the working electrode. By binding the labeling substance to the primary antibody via the analyte, an amount of the labeling substance corresponding to the concentration of the analyte is collected in the vicinity of 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 analyte. For the binding substances (first binding substance, second binding substance) that specifically bind to the analyte, an appropriate one is selected according to the test substance. In order to collect an amount of metal fine particles corresponding to the analyte in the test solution, specific binding between an antigen and an antibody is utilized in the present embodiment. However, the combination is not limited to this as long as substances specifically bind to each other. For example, specific binding between nucleic acid-nucleic acid, nucleic acid-nucleic acid binding protein, lectin-sugar chain, or receptor-ligand may be utilized. The order of the analyte-specific binding substance relationship may be reversed from the above.

[0022] Examples of the labeling substance include metal fine particles. The metal fine particles are not particularly limited, and for example, fine particles such as gold, platinum, silver, copper, rhodium, palladium, and their colloidal particles, 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. After washing, the washing buffer is brought into contact with the working electrode. To bring the solution into contact with the working electrode, any means such as dropping the solution onto the surface of the working electrode or immersing the working electrode in the solution can be taken. The washing buffer is a buffer having no chelating action, and the pH of the washing buffer is from 4 to 6. The washing buffer may be an acetate buffer or a MES buffer. The washing buffer can be changed according to the pH required for the electrochemiluminescence immunoassay to be performed. For example, if the required pH is around 5, an acetate buffer may be selected, and if the required pH is around 5.5, a MES buffer may be selected.

[0024] Current measurement step Next, the metal fine particles are electrochemically oxidized in the washing buffer solution. For example, the potential of the working electrode with respect to the reference electrode is held at the potential at which the metal fine particles are electrochemically oxidized for a predetermined time. By doing this, the metal fine particles collected near the surface of the working electrode are completely oxidized. At this time, the counter electrode and the reference electrode are also brought into contact with the solution.

[0025] After electrochemically oxidizing the metal fine particles, the presence or concentration of the analyte is measured based on the peak current value generated when reducing the oxidized metal. Specifically, for example, the potential of the working electrode is changed in the negative direction, and the current change accompanying the potential change is measured. When the electrode potential is changed in the negative direction, a reduction current flows due to the reduction of the metal oxidized by the above-described potential control, and this is measured. Since the more analyte is present in the test solution and the more metal fine particles are collected near the working electrode, the greater the reduction current intensity, quantification or detection of the analyte is realized based on this. For example, the relationship between the reduction current value and the analyte with a known concentration is obtained in advance and compared with the measured reduction current value, whereby the analyte concentration can be determined. Also, the presence or absence of the analyte 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, although it is necessary to appropriately set the potential of the working electrode to an optimal value according to the type of metal fine particles used, for example, it is preferably +1 V or more with respect to the silver / silver chloride reference electrode. 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 analyte can be surely improved. If the potential of the working electrode is less than the above range, there is a possibility that no peak of the reduction current appears during measurement. Conversely, if it exceeds the above range, diffusion due to the migration of the oxidized metal fine particles occurs, and the concentration of the oxide near the working electrode decreases, which may result in a smaller peak of the reduction current. A more preferable range is +1.2 V to +1.6 V.

[0027] As a specific means of electrochemically oxidizing metal fine particles, there is an example of holding the potential of the working electrode at the 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 preferable method because the metal fine particles can be sufficiently oxidized. When applying the potential at which the metal fine particles are electrochemically oxidized to the working electrode, in addition to the method of holding the potential of the working electrode at a predetermined potential as described above, for example, by cyclic voltammetry or the like, the potential of the working electrode may be changed over time. When changing the potential of the working electrode over time, it is preferable to change the potential of the working electrode within the range of the potential at which the metal fine particles are oxidized (for example, +1 to +2 V with respect to the silver-silver chloride reference electrode). Further, when oxidizing the metal fine particles, the potential at which the metal fine particles are electrochemically oxidized may be applied to the working electrode a plurality of times.

[0028] When using gold fine particles with a particle size of 10 nm to 60 nm as the metal fine particles, when electrochemically oxidizing the gold fine particles, it is preferable to set the potential of the working electrode with respect to the silver-silver chloride reference electrode to +1.2 V to +1.6 V in a 0.1 N to 0.5 N hydrochloric acid solution.

[0029] Here, when sufficiently oxidizing the metal fine particles, it is necessary to pay attention to applying an optimal charge amount according to the amount of the metal fine particles. Since the charge amount is the value obtained by integrating the current, if the potential applied to the working electrode is a relatively low potential, it is necessary to apply the potential for a long time in order to sufficiently oxidize the metal fine particles. On the other hand, if the potential applied to the working electrode is a relatively high potential, the time required to sufficiently oxidize the metal fine particles may be short.

[0030] By setting the holding time of the potential of the working electrode at the potential at which the metal fine particles are electrochemically oxidized to 1 second or more, the metal fine particles can be sufficiently oxidized and the detection sensitivity can be reliably improved. On the other hand, even if the application time is set to 100 seconds or more, the obtained current value hardly changes. Therefore, 1 second or more and 100 seconds or less is preferable. A more preferable range of the holding time of the potential is 40 seconds or more and 100 seconds or less.

[0031] As a method for measuring the current generated when an oxidized metal is electrochemically reduced, for example, voltammetry such as differential pulse voltammetry and cyclic voltammetry, amperometry, chronometry, etc. can be mentioned.

[0032] Since an antigen-antibody reaction or the like is performed on the working electrode to collect metal fine particles near the surface of the working electrode, and the reduction peak current derived from the metal fine particles contained in the labeling substance is measured, the analyte in the test solution can be measured simply and with high sensitivity.

[0033] In the above description, as a method for collecting an amount of metal fine particles corresponding to the amount of the analyte, a method for collecting an amount of metal fine particles corresponding to the amount of the analyte in the test solution using a non-competitive reaction was given as an example, but a method for collecting an amount of metal fine particles corresponding to the amount of the analyte in the test solution using a competitive reaction may also be adopted.

Example

[0034] Materials In this example, the following reagents were used. Anti-D dimer antibody for detection (manufactured by St John's Laboratory) Anti-D dimer antibody for capture (manufactured by TRINA Bioreactives) Gold nanoparticles, particle diameter 40 nm (manufactured by BBI Solutions) Bovine serum albumin (BSA) (manufactured by Sigma-Aldrich) Screen-printed carbon electrode (manufactured by BioDevice Technology) Non-protein-based blocking solution PBS D dimer calibrator (manufactured by Sekisui Medical) Tween20 (polyoxyethylene (20) sorbitan monolaurate) (manufactured by Fujifilm Wako Pure Chemical Corporation) MES [2-(N-morpholino)ethanesulfonic acid monohydrate] (manufactured by Dojindo Laboratories) Glycine (manufactured by Fujifilm Wako Pure Chemical Corporation) Potassium hydrogen phthalate (manufactured by FUJIFILM Wako Pure Chemical Corporation)

[0035] Preparation of antibody-labeled gold nanoparticles For pH adjustment, 1 mL of a 50 mM potassium dihydrogen phosphate solution adjusted to pH 6.5 with sodium hydroxide was added to 9 mL of a gold nanoparticle dispersion (average particle diameter 40 nm, optical density at 520 nm: 1.0) and mixed. 1 mL of a 0.1 mg / mL anti-D dimer antibody for detection diluted with pure water was added to 10 mL of this gold nanoparticle dispersion, and the mixture was allowed to stand at room temperature for 10 minutes to bind the antibody to the gold nanoparticles. Then, 1 mL of 10% BSA was added to the gold nanoparticle dispersion, and the mixture was allowed to stand at room temperature for 10 minutes to perform a blocking treatment on the surface of the gold nanoparticles.

[0036] To remove unbound antibody and BSA, the gold nanoparticle dispersion was centrifuged at 8000 g for 15 minutes at 4°C, and the supernatant was removed. 1 mL of a 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 to redisperse the gold nanoparticles. After centrifugation (at 8000 g for 15 minutes at 4°C), the supernatant of the gold nanoparticle dispersion was removed. After repeating this washing operation once more, the above buffer was added to the gold nanoparticle dispersion from which the supernatant had been removed to disperse the gold nanoparticles.

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

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

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

[0040] Gold-linked electrochemical immunoassay (GLEIA) The dispersion of antibody-labeled gold nanoparticles conjugated with the detection anti-D dimer antibody 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 solutions shown in Table 1 below at a ratio of 1:1.

Table 1

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

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

[0043] The electrodes were washed with 250 μL of various cleaning solutions (Test 1 (Test Solution 1): 6 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 solutions in Table 4). After washing, the electrodes were blocked with a blocking solution, and the blocking solution on the electrodes was blown off and dried with an air blow (air spray).

[0044]

Table 2

[0045] To the obtained screen-printed electrodes, 25 μL of various measurement solutions (Test 1: 0.2 M HCl, Test 2: 6 solutions in Table 3, Test 3: 7 solutions in Table 4) were dropped so as to completely cover the working electrode, reference electrode, and counter electrode, and electrochemical measurements were performed using a potentiostat for the oxidation of gold atoms in gold nanoparticles and subsequent differential pulse voltammetry.

[0046]

Table 3

[0047]

Table 4

[0048] Note that the oxidation of gold atoms was performed by applying a voltage of +1.25 V to the working electrode for 40 seconds with reference to a reference electrode composed of silver / silver chloride, and differential pulse voltammetry was performed by changing the base potential of the working electrode from +0.7 V to 0 V with reference to the same reference electrode at a pulse intensity 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.

[0049] Reduction peak current at each pH Figure 2(A) shows the results of measuring the reduction peak current of gold colloids using the working electrode for Test 1 and the solutions with pH values from 3 to 8 shown in Table 2, that is, the results of measuring the blank three times each. The error bars in the figure represent the standard deviation. At pH 4.0 or lower, gold nanoparticles non-specifically bound to the electrode, increasing the background noise.

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

[0051] Reduction peak current in each buffer (pH 5.0 and pH 5.5) Next, the reduction peak current of gold colloids in the buffers 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)) was measured (using the working electrode for Test 3 as the working electrode). Figure 4 shows the results of measuring the sample with a D-dimer calibrator concentration of 0.5 μg / mL three times each. The measurement was performed 10 times. The current values shown in the figure are the reduction peak current values of gold colloids 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) had large variations in the measured values, and phthalic acid B (pH 5.0) with chelating action had poor redox efficiency. On the other hand, acetic acid B (pH 5.0) and MES (pH 5.5), which are buffers without chelating action, had small variations in the measured values at the pH values where they exerted buffering action (5.0 and 5.5 respectively) and good redox efficiency.

[0053] Reduction 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 the buffer-free solution (pH unadjusted), acetic acid B (pH 5.0), and MES (pH 5.0) shown in Table 4, respectively (the working electrode regarding Test 3 was used as the working electrode). The measurements were each performed 10 times.

[0054] As shown in Figures 4(A) to (C), the buffer adjusted to around pH 5.0 was able to reduce the variation in the measured values compared to the buffer-free solution without pH adjustment.

Explanation of Signs

[0055] 1 Printed electrode device 2 Working electrode 3 Counter electrode 4 Reference electrode 5 Insulating support 6 Insulating layer

Claims

1. A method for measuring a test substance, characterized by examining the presence or concentration of the test substance, the method comprising: a washing step of washing a working electrode on which an immune complex containing the test substance is immobilized with a washing buffer; a current measurement step of controlling the potential of the working electrode in the washing buffer and measuring a current value; including the immune complex includes the test substance, a first binding substance specifically binding to the test substance, a second binding substance specifically binding to the test substance, and a labeling substance binding to the second binding substance; the labeling substance is gold fine particles; the pH of the washing buffer is from 4 to 6; the washing buffer is a buffer having no chelating action; method.

2. The method according to claim 1, wherein the washing buffer is an acetate buffer or a MES buffer.

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