JP7699846B2 - Method for measuring a test substance using an electrochemical technique - Google Patents

Description

The present invention relates to a method for measuring a test substance using an electrochemical technique.

One method known for easily and sensitively measuring trace substances in a test solution is the immunoassay method that uses an antigen-antibody reaction. The ELISA method, which uses an enzyme-labeled antibody to detect and measure the concentration of a test substance by obtaining signals such as color development or luminescence resulting from an enzyme reaction, is used in a wide range of fields. However, the ELISA method requires an optical system to detect signals such as color development or 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.

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 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 electrochemical measurement, and (D) detecting the test substance in the sample based on the measurement result obtained in step (C), and is characterized in that step (B) is a step of washing the working electrode with a measurement solution having the same composition as the measurement solution used in step (C).

Patent Publication No. 2016-085168

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

Therefore, the inventors performed electrochemical immunoassays using the same buffer solution in the washing and current measurement steps to stabilize the pH of the solution in these steps. However, the use of a buffer solution did not necessarily result in successful electrochemical immunoassays.

After further investigation, the inventors discovered that electrochemical immunoassays are possible by using a buffer solution that does not have a chelating effect in the washing step and current measurement step, thus completing the present invention.

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 between 4 and 6.
The objective of the present invention is to provide a method.

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 it is also possible to stabilize the pH of the solution.

The washing buffer may be a buffer that does not have a chelating effect. The washing buffer may be an acetate buffer or an MES buffer.

FIG. 2 is a plan view of a printed electrode device used in the examples. 2(A) and (B) show the results of measuring the reduction peak current of gold colloid. FIG. 3 shows the results of measuring the reduction peak current of gold colloid in a 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). 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.

Definitions 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.

The numerical ranges and parameters set forth in the present invention are approximate, but 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 their respective testing measurements. 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.

The following describes an embodiment of the present invention. The following embodiment is merely an example, and the scope of the present invention is not limited to the embodiment described below. Note that explanations of similar content will be omitted to avoid repetition.

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.

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 the test substance that is different from the second binding substance.

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 to the surface of the working electrode, a labeling step of labeling a second binding substance with a labeling substance to create 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 the first binding substance is immobilized in advance, a labeling step of labeling a second binding substance with a labeling substance to create 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 includes a providing step of providing a working electrode to which a first binding substance has been immobilized in advance, 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 the test substance to the surface of the working electrode to which the first binding substance has been immobilized, thereby forming an immune complex on the surface of the working electrode.

The metal particle binding electrochemical immunoassay that is the basis of the method of this embodiment is described in detail below.

Metal particle binding electrochemical immunoassay Two types of specific binding substances for the test substance are prepared, one (first binding substance) is immobilized on the surface of a working electrode, and the other (second binding substance) is labeled with a labeling substance (e.g., metal particles) to form a label. Specifically, first, a primary antibody is immobilized on the surface of the working electrode used in the electrochemical measurement as the first binding substance for the test substance. The electrode surface is blocked to prevent non-specific adsorption. In addition, a secondary antibody is 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.

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, resulting in an amount of the label corresponding to the concentration of the test substance being collected near the working electrode 1.

Here, in the present invention, any substance, such as a biological substance or a synthetic substance, can be used as the test substance. An appropriate binding substance (first binding substance, second binding substance) that specifically binds to the test substance is selected according to the test substance. In order to collect an amount of metal microparticles corresponding to the test substance in the test solution, the present embodiment utilizes specific binding between an antigen and an antibody, 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.

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

After carrying out the antigen-antibody reaction, 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 method 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 has a pH of 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 depending on the pH required for the electrochemical immunoassay to be performed. 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.

Current measurement step 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.

After electrochemically oxidizing the 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 and the more metal particles are collected near the working electrode, the greater the reduction current strength becomes, 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 known concentration is determined in advance, and the concentration of the test substance can be determined by comparing the measured reduction current value. In addition, the presence or absence of the test substance in the test solution can be known from the obtained reduction current value.

When oxidizing the metal particles, the potential of the working electrode is set to a potential at which the metal particles can be oxidized. Specifically, the potential of the working electrode needs to be set to an appropriate optimal value depending on the type of metal particles used, but for example, it is preferable to set it to +1 V or more relative to a silver-silver chloride reference electrode. By setting the potential of the working electrode within the above range, the metal 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 will not appear during measurement, and conversely, if it exceeds the above range, diffusion due to migration of the oxidized metal particles occurs, the concentration of oxide near the working electrode decreases, and this may result in a smaller peak of the reduction current. A more preferable range is +1.2 V to +1.6 V.

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

When using gold particles with a particle size of 10 nm to 60 nm 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 for the working electrode relative to the silver-silver chloride reference electrode.

Here, to fully oxidize the metal microparticles, care must be taken to apply the optimal amount of charge depending on the amount of metal microparticles. Since the amount of charge is the integrated value of the current, if the potential applied to the working electrode is relatively low, it is necessary to apply this potential for a long period of time to fully 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 fully oxidize the metal microparticles.

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 set to 100 seconds or more, the obtained current value remains almost unchanged. Therefore, a time period of 1 second or more and 100 seconds or less is preferable. A more preferable range for the time period for holding the above potential is 40 seconds or more and 100 seconds or less.

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.

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 resulting 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.

In the above explanation, a method of collecting metal microparticles in an amount corresponding to the amount of the test substance in the test solution using a non-competitive reaction has been given as an example of a method of collecting metal microparticles in an amount corresponding to the amount of the test substance in the test solution, but a method of collecting metal microparticles in an amount corresponding to the amount of the test substance in the test solution using a competitive reaction may also be used.

Materials The following reagents were used in this example:

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] (Dojindo Chemical Industries, Ltd.)
Glycine (Fujifilm Wako Pure Chemical Industries, Ltd.)
Potassium hydrogen phthalate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

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 (mean particle size 40 nm, optical density at 520 nm: 1.0) and mixed. 1 mL of 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 antibody was bound to the gold nanoparticles by leaving it at room temperature for 10 minutes. Then, 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.

To remove unbound antibodies and BSA, the gold nanoparticle dispersion was centrifuged at 8000g at 4°C 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, and the gold nanoparticles were dispersed.

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

Preparation of antibody solid-phase electrode As an electrode device for measuring a 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 from carbon paste, a lead (not shown) formed from carbon paste, and a reference electrode 44 formed from silver/silver chloride on an insulating support 45, and a part of the surfaces of the working electrode 42, the counter electrode 43, and the reference electrode 44 are covered with an insulating layer 46 to define the effective electrode area.

After the capture 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 electrodes were placed in a sealed container containing a desiccant and stored at 4°C.

Gold-linked electrochemical immunoassay (GLEIA)
A dispersion of antibody-labeled gold nanoparticles bound to anti-D-dimer detection antibodies 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.

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

After leaving test solution 1 at room temperature for at least 10 minutes or test solution 2 and test solution 3 for 3 minutes, 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 left at room temperature for 3 minutes.

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.

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 resulting screen-printed electrodes so as to completely cover the working electrode, reference electrode, and counter electrode, and a potentiostat was used to oxidize the gold atoms in the gold nanoparticles, followed by electrochemical measurements using differential pulse voltammetry.

The gold atoms were oxidized by applying a voltage of +1.25 V to the working electrode for 40 seconds using a silver/silver chloride reference electrode as the standard, and differential pulse voltammetry was performed by changing the base potential of the working electrode from +0.7 V to 0 V using the same reference electrode as the standard, with 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 speed of 20 mV/second.

Reduction 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 3 to 8 shown in Table 2, that is, the results of measuring the blank three times each. The error bars in the figure show the standard deviation. At pH 4.0 or less, gold nanoparticles nonspecifically bound to the electrode, increasing the background noise.

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.

Peak reduction current in each buffer solution (pH 5.0 and pH 5.5) Next, the peak reduction current of the gold colloid 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)) was measured (the working electrode for Test 3 was used as the working electrode). 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 10 times. The current values shown in the figure are the peak reduction current values of the gold colloid calculated from the voltammogram after baseline subtraction.

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

Peak reduction current at pH 5.0 Next, the peak reduction current of gold colloid at pH 5.0 was measured. Figures 4 (A) to (C) show the results of measuring the peak reduction current of 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 used was the working electrode for Test 3). Each measurement was performed 10 times.

As shown in Figures 4 (A) to (C), the buffer solution adjusted to a pH of around 5.0 reduced the variability in the measured values more than a buffer-free solution with an unadjusted pH.

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

Claims (2)

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 gold particle ,
The pH of the wash buffer is between 4 and 6;
The washing buffer is a non-chelating buffer.
method. The method of claim 1, wherein the washing buffer is an acetate buffer or an MES buffer.