JP4915741B2 - Test substance measurement method - Google Patents

Description

  The present invention relates to a method for measuring a test substance using metal fine particles as a labeling substance.

  An immunoassay method using an antigen-antibody reaction is known as one method for measuring trace substances in a test solution easily and with high sensitivity. In an ELISA method, which is a typical immunoassay method, an antibody labeled with an enzyme is used to obtain a signal such as color development or luminescence derived from an enzyme reaction, thereby qualitatively or quantitatively determining a test substance. However, ELISA requires an optical system when detecting signals such as color development and light emission, and thus has a disadvantage of requiring a large measuring instrument. In addition, when performing accurate quantification, complicated processing such as conversion of measurement results such as color development into electrical signals may be required.

Thus, it has been proposed to use an electrochemical measurement method for detection in immunoassay methods using general-purpose labeling substances such as chromogenic labels and fluorescent labels. Since the apparatus used for electrochemical measurement can be reduced in size as compared with the apparatus used for ELISA or the like, it is expected that the measurement apparatus can be downsized and the detection sensitivity can be improved. For example, in Patent Document 1, after the metal fine particles are dissolved by chemical treatment, electrochemical measurement is performed, and the test substance is analyzed based on the peak current associated with the oxidation of the obtained metal fine particles.
Japanese translation of PCT publication No. 2004-512696

  However, in patent document 1, since the process which melt | dissolves metal microparticles | fine-particles completely by chemical treatment is required prior to electrochemical measurement, there exists a problem that measurement operation becomes complicated.

  On the other hand, as shown in FIG. 10, there is a method of measuring a test substance using a reduction current value as an index after electrochemically oxidizing metal fine particles. In this method, first, a test substance 103 is sandwiched between a primary antibody 102 immobilized on the surface of the working electrode 101 and a secondary antibody 104 labeled with metal fine particles 105 such as gold colloid particles, and the concentration of the test substance 103 is determined. The labeled metal fine particles 105 according to the above are localized at least on the surface of the working electrode 101 (FIG. 10A). Next, the metal fine particles 105 localized near the surface of the working electrode 1 are electrochemically oxidized and eluted in the measurement solution (FIG. 10B). Finally, the current value generated when the eluate eluted from the metal fine particles 105 as metal ions, complex ions, etc. is electrochemically reduced is measured (FIG. 10C).

  In this method, since electrochemical oxidation is performed in a state where the metal fine particles 105 are localized on the surface of the working electrode 101 using biological interaction, the metal fine particles involved in the reaction with the test substance All can be involved in the exchange of electrons with the working electrode surface, and as a result, highly sensitive measurement of the test substance is realized. Further, when the metal fine particles 105 localized on the surface of the working electrode 101 are directly oxidized and the oxidation current is measured, the metal (here, gold) is oxidized due to the distance between the metal fine particles 105 and the working electrode 101. Since the necessary conditions vary, no oxidation peak appears in the measured graph. On the other hand, the metal complex 105 is oxidized and eluted to generate gold complexes in the vicinity of the electrodes, and since the conditions necessary for the reduction of gold are almost constant, a reduction peak appears in the measured graph, Measurement is possible. Furthermore, since the labeled metal fine particles are eluted by controlling the potential of the working electrode, for example, complication of the measurement operation can be suppressed as compared with the case of oxidizing by chemical treatment.

  Incidentally, in the method shown in FIG. 10, it is necessary to sufficiently oxidize and elute the metal fine particles before measuring the reduction current or oxidation current. However, the present inventors have clarified that the eluate diffuses over time in this oxidation step, leading to a reduction in sensitivity.

  The present invention has been proposed in view of such a conventional situation, and provides a measurement method of a test substance capable of performing highly sensitive and accurate measurement without complicating the measurement operation. With the goal.

  In order to achieve the above-described object, a test substance measurement method according to the present invention uses a metal fine particle as a labeling substance, and uses a electrochemical measurement method to measure a test substance. Using the interaction to localize the metal fine particles at least on the surface of the working electrode, and electrochemically oxidizing and eluting the metal fine particles localized on the surface of the electrode. The diffusion of the eluate from the working electrode is suppressed at least in the metal fine particle oxidation step.

  In the above measurement method, metal ions and complex ions are generated in the vicinity of the electrode by oxidative elution of fine metal particles, and the conditions necessary for the reaction are almost constant compared to the state before oxidative elution. Therefore, by causing them to undergo a reduction precipitation reaction, a peak corresponding to the amount of metal fine particles appears in the graph in which the reduction reaction is measured, and accurate measurement is possible. Furthermore, since the metal fine particles are eluted by controlling the potential of the working electrode, for example, complication of the measurement operation can be suppressed as compared with the case of oxidizing by chemical treatment. Then, when electrochemically oxidizing at least the metal fine particles localized on the surface of the working electrode, the working electrode is prevented from diffusing from the surface of the working electrode etc. of the eluate such as ions generated from the metal fine particles. Since the eluate concentration on the surface is kept at a high concentration, a highly sensitive measurement is realized.

  According to the present invention, high-sensitivity and accurate measurement of a test substance in a test solution can be realized by a simple operation without requiring a large-sized measuring instrument used in, for example, an ELISA detection process. Can do. In addition, since the diffusion of the eluate eluted as metal ions or complex ions during the electrochemical oxidation of the metal fine particles is suppressed, further highly sensitive measurement is possible.

  Hereinafter, a method for measuring a test substance to which the present invention is applied will be described in detail with reference to the drawings.

<First Embodiment>
In the first embodiment, a case where an opposing member having a flat surface is used with reference to FIG. 1 will be described in order to suppress diffusion of eluents such as metal ions and complex ions.

  First, a planar electrochemical device in which a working electrode, a counter electrode, and a reference electrode are formed on the same surface is prepared. A primary antibody 2 that recognizes a test substance (antigen) 3 is fixed on the surface of the working electrode 1 having a substantially circular planar shape. The electrode surface is blocked to prevent nonspecific adsorption. Further, a secondary antibody 4 that recognizes different sites on the test substance 3 is prepared, and a labeled antibody is prepared by labeling, for example, gold colloidal particles as the metal fine particles 5.

  Next, a solution containing the labeled antibody and an unknown amount of the test substance 3 is supplied to the surface of the working electrode 1, brought into contact with the primary antibody 2, and an antigen-antibody reaction is performed on the working electrode 1. When the labeled antibody binds to the primary antibody 2 through the test substance 3, an amount of the metal fine particles 5 corresponding to the concentration of the test substance 3 is localized on the surface of the working electrode 1 (FIG. 1 (a)). Thereafter, the surface of the working electrode 1 is washed as necessary.

  Any substance such as a biological substance or a synthetic substance can be selected as the test substance 3. In the present embodiment, the antigen-antibody reaction is used to localize the metal fine particles 5 as the labeling substance on the surface of the working electrode 1. However, if the biological interaction is used, the antigen-antibody reaction can be performed. It is not limited. For example, specific binding of nucleic acid-nucleic acid, nucleic acid-nucleic acid binding protein, lectin-sugar chain, or receptor-ligand may be used.

  As the metal fine particles 5 as the labeling substance, metal fine particles used as the labeling substance can be arbitrarily used in addition to the colloidal gold particles. For example, fine particles such as gold, platinum, silver, copper, rhodium, and palladium, colloidal particles thereof, quantum dots, and the like can be used. Among them, it is preferable to use gold colloid particles having a particle diameter of 10 nm to 60 nm, particularly gold colloid particles having a particle diameter of about 40 nm.

Next, the working electrode 1, the counter electrode and the reference electrode are brought into contact with a measurement solution for electrochemical measurement, and the metal fine particles 5 localized on the surface of the working electrode 1 are electrochemically oxidized to form metal ions. And eluted as complex ions (FIG. 1B). When gold colloidal particles are used as the metal fine particles 5 and an aqueous hydrochloric acid solution is used as the measurement solution, the gold colloidal particles are eluted as AuCl ₄ ⁻ or the like by being electrochemically oxidized. At this time, a counter member 6 having a flat surface is prepared, and the flat surface of the counter member 6 is brought close to the surface of the working electrode 1 so as to cover the surface of the working electrode 1 with the counter member 6. Reduce the thickness of the measurement solution on the surface.

After the metal fine particles 5 are electrochemically oxidized and eluted, the eluate eluted as metal ions or complex ions such as AuCl ₄ ⁻ while keeping the facing member 6 close to the surface of the working electrode 1. A reduction current generated during electrochemical reduction is measured at the working electrode 1, and the presence or concentration of the test substance is measured based on the measurement result (FIG. 1 (c)). Specifically, for example, the potential of the working electrode 1 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 when an eluate such as a metal ion or a complex ion is reduced, and this is measured. Since the reduction current intensity increases as the number of fine metal particles 5 localized on the surface of the working electrode 1 increases, the determination or detection of the test substance 3 is realized based on this. For example, the concentration of the test substance 3 can be obtained by previously obtaining the relationship between the reduction current value and the test substance having a known concentration and comparing it with the measured reduction current value. Moreover, the presence or absence of the test substance 3 can be known from the obtained reduction current value.

  Examples of the method for measuring the current generated when the oxidized metal is electrochemically reduced include voltammetry such as differential pulse voltammetry and cyclic voltammetry, amperometry, chronometry and the like.

  As the counter member 6, any shape can be used as long as the region facing the working electrode 1 is a flat surface and a thin layer of the measurement solution can be formed on the surface of the working electrode 1. In order to further increase the sensitivity by preventing ion diffusion, the arithmetic average roughness Ra of the flat surface is preferably 20 μm or less. Examples of members that satisfy this condition include glass substrates and plastic substrates.

  The distance between the working electrode 1 and the facing member 6 is preferably 50 μm or less. By setting the thickness to 50 μm or less, the thickness of the measurement solution layer formed between the working electrode 1 and the counter member 6 is reduced, and the effluent from the metal fine particles 5 is present at a high concentration near the surface of the working electrode 1. Therefore, further higher sensitivity can be achieved.

  As the measurement solution, it is preferable to use an acidic solution because the metal fine particles 5 can be easily oxidized electrochemically. The acidic solution may be appropriately selected according to the type of the metal fine particles 5 and the like, and for example, an aqueous solution containing hydrochloric acid, nitric acid, acetic acid, phosphoric acid, citric acid, sulfuric acid and the like can be used. Considering the ease of electrochemical oxidation of the metal fine particles 5, it is preferable to use 0.05N to 2N hydrochloric acid aqueous solution, particularly 0.1N to 0.5N hydrochloric acid aqueous solution.

  In addition to the acidic solution, a neutral solution containing halogen ions such as chlorine ions can be used as the measurement solution. By using a neutral solution containing halogen ions such as chlorine ions, a larger amount of current change can be obtained in the measurement process described later than in the case of using an acidic solution, and as a result, a more sensitive measurement is achieved. . When an acidic solution is used, the peak shape may become asymmetrical, for example, the bottom of the reduction peak on the low potential side may increase, or noise may occur near 0.1 V, for example. On the other hand, by using a neutral solution containing halogen ions such as chlorine ions, the base of the reduction peak is flattened and the generation of the noise can be suppressed, so that the reduction peak intensity can be easily detected. Furthermore, it is possible to avoid the use of solutions that are difficult to handle, such as acidic solutions and alkaline solutions, and the measurement operation can be carried out safely and simply. As the neutral solution containing chlorine, for example, the above-mentioned effect can be obtained when KCl, NaCl, LiCl or the like is used, but the effect is particularly great when KCl is used.

  In order to oxidize the metal fine particles 5 electrochemically, the working electrode 1 is preferably set to +1 to +2 V with respect to, for example, a silver-silver chloride reference electrode. By setting the potential of the working electrode 1 within the above range, the metal fine particles 5 localized on the surface of the working electrode 1 can be completely oxidized and eluted, and the detection sensitivity of the test substance 3 is reliably improved. be able to. If the potential of the working electrode 1 is less than the above range, there may be no reduction current peak at the time of measurement. As a result of diffusion, the concentration of the eluate in the vicinity of the working electrode 1 is lowered, which may reduce the peak of the reduction current. A more preferable range is + 1.2V to + 1.6V.

  In order to oxidize and elute the metal fine particles 5 electrochemically, the potential of the working electrode 1 is maintained at a potential at which the metal fine particles 5 are oxidized and eluted for a predetermined time. The above operation is a preferable method because the metal fine particles 5 can be sufficiently oxidized. In addition to the above operation, the potential of the working electrode 1 may be changed over time. In this case, it is preferable to change the potential of the working electrode within the range of the potential at which the metal fine particles 5 are oxidized and eluted (for example, +1 V to +2 V with respect to the silver-silver chloride reference electrode). Furthermore, a potential at which the metal fine particles 5 are electrochemically oxidized and eluted may be applied to the working electrode 1 a plurality of times.

  For example, when gold colloidal particles having a particle diameter of 10 nm to 60 nm are used as the metal fine particles 5, when the gold colloidal particles are electrochemically oxidized, silver chloride is used in 0.1 N to 0.5 N hydrochloric acid solution. The potential of the working electrode with respect to the reference electrode is preferably set to + 1.2V to + 1.6V.

  When the metal fine particles 5 are sufficiently oxidized, care must be taken to give an optimum charge amount according to the amount of the metal fine particles 5. Since the amount of charge is a value obtained by integrating the current, if the potential applied to the working electrode 1 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 1 is a relatively high potential, the time required for sufficiently oxidizing the metal fine particles 5 may be short.

  By maintaining the potential of the working electrode 1 at the potential at which the metal fine particles 5 are electrochemically oxidized for 1 second or longer, the metal fine particles can be sufficiently oxidized, and the detection sensitivity can be improved reliably. it can. 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.

  In the present embodiment, when measuring the electrochemical oxidation and reduction current of the metal microparticles 5, the thickness of the measurement solution is reduced by disposing the opposing member 6 close to the surface of the working electrode 1. Loss due to diffusion of eluents such as metal ions and complex ions can be suppressed. As a result, it is possible to perform highly sensitive measurement compared to the case where the facing member 6 is not used. In addition, once the labeled metal fine particles are oxidized and eluted, the conditions necessary for the reduction of the metal ions become almost the same. Therefore, by reducing and precipitating them, the amount of fine metal particles is shown in the graph showing the reduction reaction. Corresponding peaks appear and accurate measurement is possible. Furthermore, by measuring the reduction current as in the present embodiment, it is possible to suppress the influence of noise derived from impurities and the like compared to the case of measuring the oxidation current.

<Second Embodiment>
In the second embodiment, the case where the counter electrode is brought close to the surface of the working electrode in order to prevent diffusion of eluents such as metal ions and complex ions and the reduction current is measured with the counter electrode will be described with reference to FIG. To do. In the following, the description overlapping with the previous description is omitted.

  First, a planar electrochemical device having a working electrode 1 on which a primary antibody 2 is immobilized is prepared, and a solution containing a secondary antibody 4 labeled with metal fine particles 5 and an unknown amount of a test substance 3 is applied to the surface of the working electrode 1. And the antigen-antibody reaction is carried out on the working electrode 1 (FIG. 2 (a)). The steps so far are the same as those in the first embodiment.

Next, the working electrode 1, the counter electrode, and the reference electrode are brought into contact with an electrochemical measurement solution, and the metal fine particles 5 localized near the surface of the working electrode 1 are electrochemically oxidized to form AuCl _4. ^- eluting metal ions or complex ions, and the like. In this embodiment, at this time, a counter electrode 7 different from the counter electrode and the reference electrode is prepared, and the counter electrode 7 is brought close to and opposed to the surface of the working electrode 1 so as to cover the surface of the working electrode 1 with this counter electrode 7. The eluate such as metal ions and complex ions eluted from the metal fine particles 5 is electrochemically reduced and deposited as metal 8 on the surface of the counter electrode 7 (FIG. 2B).

  The counter electrode 7 is an electrode for temporarily collecting eluents such as metal ions and complex ions eluted from the metal fine particles 5, and the surface facing the working electrode 1 is preferably flat. The planar shape is not particularly limited as long as a metal can be deposited on the surface, but is preferably substantially the same as the working electrode 1. The counter electrode 7 is preferably arranged so as to overlap the working electrode 1 in plan view.

  In order to deposit the metal 8 on the counter electrode 7, a potential capable of depositing the metal constituting the metal fine particles 5 may be applied to the counter electrode 7. For example, when gold colloidal particles are used as the metal fine particles 5, the counter electrode 7 is preferably set to 0 V to −1 V with respect to the silver / silver chloride reference electrode. In order to oxidize and elute the metal fine particles 5 electrochemically, it is preferable to apply +1 V to +2 V to the working electrode 1 with respect to the silver-silver chloride reference electrode as in the first embodiment.

  The timing for electrochemically eluting the metal fine particles 5 localized on the surface of the working electrode 1 and the timing for depositing the metal 8 on the surface of the counter electrode 7 may be different at the same time. It is preferable to set the potential of the counter electrode 7 to a potential at which the metal constituting the metal fine particles 5 is deposited, and then set the potential of the working electrode 1 to a potential at which the metal fine particles 5 are eluted. By setting it as such timing, precipitation on the surface of the counter electrode 7 can be made more reliable.

  The distance between the working electrode 1 and the counter electrode 7, that is, the thickness of the measurement solution layer, is preferably 50 μm or less. By setting the thickness to 50 μm or less, it is possible to more efficiently collect eluents such as metal ions and complex ions eluted from the metal fine particles 5 with the counter electrode.

  After elution of the metal fine particles 5 localized on the surface of the working electrode 1 and deposition of metal on the surface of the counter electrode 7, the state in which the counter electrode 7 was brought close to the surface of the working electrode 1 was maintained. The metal 8 deposited on the surface of the counter electrode 7 is electrochemically eluted (FIG. 2C). In order to elute the metal 8 on the surface of the counter electrode 7 electrochemically, a potential at which the metal 8 elutes may be applied to the counter electrode 7. When the metal 8 is eluted from the surface of the counter electrode 7, For example, when gold colloidal particles are used as the metal fine particles 5, the potential of the counter electrode 7 is preferably +1 V to +2 V with respect to the silver-silver chloride reference electrode. The time for setting the counter electrode 7 to a potential at which the metal 8 is eluted is preferably 0.1 seconds to 10 seconds. In addition, when the metal 8 deposited on the surface of the counter electrode 7 is eluted as metal ions or complex ions, there is a concern that the eluate such as metal ions or complex ions may be diffused. Since the time required for elution is shortened as compared with the case where the elution is performed, the loss due to diffusion during elution is small. Therefore, a decrease in detection sensitivity hardly causes a problem.

  After the metal 8 on the surface of the counter electrode 7 is eluted, a reduction current value generated when electrochemically reducing the eluted metal ions, complex ions, and the like is measured by the counter electrode 7, and based on this. The presence or absence or concentration of the test substance is measured (FIG. 2 (d)). The measurement of the reduction current at the counter electrode 7 is preferably performed immediately after the metal 8 on the surface of the counter electrode 7 is eluted.

  In the present embodiment, the eluate such as metal ions or metal complex ions eluted from the metal fine particles 5 is deposited on the counter electrode 7 and collected, so that the diffusion of the eluate is hindered, and the metal ions on the surface of the working electrode 1 The concentration of eluents such as metal complex ions is kept high. As a result, it is possible to perform highly sensitive measurement compared to the case where the counter electrode 7 is not used. Further, by measuring with the counter electrode 7 in a clean and activated state, measurement with higher sensitivity is possible as compared with measurement with the working electrode 1 in which an antibody or the like is immobilized or an antigen-antibody reaction is performed. . In addition, the conditions necessary for the reduction of metal ions and metal complex ions generated by once oxidizing and eluting the labeled metal fine particles are almost the same. Therefore, the reduction reaction is measured by reducing and precipitating them. The peak corresponding to the amount of fine metal particles appears in the graph, and accurate measurement is possible. Furthermore, by measuring the reduction current as in the present embodiment, it is possible to suppress the influence of noise derived from impurities and the like compared to the case of measuring the oxidation current.

<Third Embodiment>
In the first to second embodiments, the reduction current is measured in the last electrochemical measurement step, but the metal deposited on any electrode is oxidized and the oxidation current is measured. May be. In this embodiment, the case where the metal deposited on the counter electrode is oxidized electrochemically and the oxidation current is measured by the counter electrode will be described with reference to FIG.

  First, a planar electrochemical device having a working electrode 1 on which a primary antibody 2 is immobilized is prepared, and a solution containing a secondary antibody 4 labeled with metal fine particles 5 and an unknown amount of a test substance 3 is applied to the surface of the working electrode 1. And an antigen-antibody reaction is carried out on the working electrode 1 (FIG. 3A).

Next, the working electrode 1, the counter electrode, and the reference electrode are brought into contact with an electrochemical measurement solution, and the metal fine particles 5 localized near the surface of the working electrode 1 are electrochemically oxidized to form AuCl _4. ^- eluting metal ions or complex ions, and the like. In the present embodiment, at this time, the counter electrode 7 is brought close to and opposed to the surface of the working electrode 1 so that the surface of the working electrode 1 is covered with the counter electrode 7, and eluents such as metal ions and complex ions eluted from the metal fine particles 5 Is electrochemically reduced and deposited as metal 8 on the surface of the counter electrode 7 (FIG. 3B). The steps up to here are the same as those in the second embodiment.

  Next, the metal 8 deposited on the counter electrode 7 is electrochemically oxidized, its oxidation current is measured, and based on this, the presence or absence or concentration of the test substance is measured (FIG. 3C). The oxidation current is measured with the counter electrode 7. Examples of the method for measuring the oxidation current include voltammetry such as differential pulse voltammetry and cyclic voltammetry, amperometry, chronometry and the like.

  In the present embodiment, the eluate such as metal ions and complex ions eluted from the metal fine particles 5 is deposited on the counter electrode 7 and collected, so that the diffusion of the eluate is hindered. As a result, it is possible to perform highly sensitive measurement compared to the case where the counter electrode 7 is not used. Further, by measuring with the counter electrode 7 in a clean and activated state, measurement with higher sensitivity is possible as compared with measurement with the working electrode 1 in which an antibody or the like is immobilized or an antigen-antibody reaction is performed. . In addition, the conditions necessary for the reduction of metal ions and metal complex ions generated by once oxidizing and eluting the labeled metal fine particles are almost the same. Therefore, the reduction reaction is measured by reducing and precipitating them. The peak corresponding to the amount of fine metal particles appears in the graph, and accurate measurement is possible. Furthermore, by measuring the oxidation current as in the present embodiment, there is no diffusion due to oxidation elution compared to the first to second embodiments in which the reduction current is measured, and in principle, high sensitivity. It is thought that it becomes.

<Fourth Embodiment>
In the present embodiment, a method for suppressing the diffusion of eluents such as metal ions and complex ions using a pair of minute comb electrodes 1a and 1b as shown in FIG.

  First, a planar device in which a pair of comb electrodes 1a and 1b are formed on an insulating substrate 9 is prepared, and a primary antibody 2 that recognizes the test substance 3 is fixed to at least the surfaces of the comb electrodes 1a and 1b. . At this time, since it is difficult to fix the primary antibody 2 only to the surfaces of the minute comb-tooth electrodes 1a and 1b, the insulating substrate 9 on which the counter electrode (not shown) and the comb-tooth electrodes 1a and 1b are not formed. The primary antibody 2 may be immobilized on the surface.

  Next, a solution containing the secondary antibody 4 labeled with the metal fine particles 5 and an unknown amount of the test substance 3 is supplied to the surface on which the comb electrodes 1a and 1b of the device are formed, and an antigen-antibody reaction is performed. Thereby, the amount of the metal fine particles 5 corresponding to the concentration of the test substance 3 is localized on the surface of the working electrode 1 (FIG. 5A). The steps up to here are the same as those in the second embodiment except that the shape of the working electrode 1 is different.

  Next, the comb electrodes 1a and 1b, the counter electrode and the reference electrode are brought into contact with a measurement solution for electrochemical measurement. Thereafter, a potential at which the metal constituting the metal fine particles 5 is eluted is applied to one of the comb electrodes 1a and 1b to electrochemically oxidize the metal fine particles 5 localized on the surface. Eluted as metal ions or complex ions. At the same time, a potential at which the metal constituting the metal fine particles 5 precipitates is applied to the other comb-teeth electrode 1b, and the eluents such as metal ions and complex ions generated from the one comb-teeth electrode 1a side are reduced to form comb teeth. The metal 8 is deposited on the surface of the electrode 1b (FIG. 5B).

  Next, a potential opposite to the previous step is applied to each of the comb electrodes 1a and 1b. That is, a potential at which the metal constituting the metal fine particles 5 and the deposited metal 8 are eluted is applied to the other comb-tooth electrode 1b, and these are electrochemically oxidized and eluted as metal ions, complex ions, and the like. At the same time, a potential at which the metal constituting the metal fine particles 5 precipitates is applied to one of the comb electrodes 1a, and the eluate such as metal ions or complex ions is reduced and deposited on the surface of the comb electrodes 1a (FIG. 5 ( c)).

  Next, the metal 8 deposited on one comb electrode 1a is electrochemically oxidized, and the oxidation current is measured by the comb electrode 1a (FIG. 5D). Alternatively, the metal 8 on the surface of one comb electrode 1a is electrochemically eluted, and then the eluted metal ions and complex ions are electrochemically reduced, and the reduction current is reduced to the other comb tooth. You may measure with the electrode 1b.

  In the present embodiment, by performing the above-described operation using the comb electrodes 1a and 1b, eluents such as metal ions and complex ions are exchanged between minute regions between the comb electrodes 1a and 1b. Diffusion of eluents such as metal ions and complex ions is hindered, and highly sensitive measurement is possible. In addition, the conditions necessary for the reduction of metal ions and metal complex ions generated by once oxidizing and eluting the labeled metal fine particles are almost the same. Therefore, the reduction reaction is measured by reducing and precipitating them. The peak corresponding to the amount of fine metal particles appears in the graph, and accurate measurement is possible.

  In the first to fourth embodiments described above, the counter member, the counter electrode, or the micro comb-teeth electrode is exemplified as a means for suppressing the diffusion of eluents such as metal ions and complex ions from the electrode surface. The invention is not limited to these, and any means capable of suppressing diffusion of eluents such as metal ions and complex ions from the electrode surface can be arbitrarily adopted.

  Hereinafter, the measurement method of the present invention will be described with reference to experimental results.

Experiment 1
First, a planar type printed electrode device as shown in FIG. 6A was prepared. This printed electrode device is configured by forming a working electrode 12 made of carbon, a counter electrode made of carbon (not shown) and a reference electrode 13 made of Ag / AgCl on the surface of one end of a strip-shaped substrate 11. The An insulating film 15 made of a resist is formed on the substrate 11 and on the wiring 14 that connects the reference electrode 13 and a terminal (not shown), and the height A of the insulating film 15 from the substrate surface is 32 μm. It is said that. The distance B between the end of the insulating film 15 and the end of the substrate 11 on the side where the reference electrode 13 is formed is 3800 μm. Prior to the operations of Examples 1 to 3 and Comparative Example 1 below, the biological interaction is achieved by electrochemically depositing gold (Au) on the surface of the reference electrode 13 of the printed electrode device. By utilizing this, a state in which the gold fine particles are localized on the surface of the working electrode 12 was simulated.

<Example 1>
In Example 1, the electrochemical oxidation and reduction current of gold were measured in a state where the cover glass as the facing member 16 was brought close to the surface of the working electrode 12. That is, first, a 0.1 N hydrochloric acid aqueous solution was dropped as a measurement solution on the surface of the working electrode 12 of the printed electrode device, and droplets of the measurement solution were formed on the surface of the working electrode 12. Next, the thickness of the measurement solution was reduced by placing a cover glass as the facing member 16 so that a part of the film covered the insulating film 15 (FIG. 6B). Next, the potential of the working electrode 12 was kept at +1.2 V for 40 seconds, and gold on the surface of the working electrode 12 was electrochemically oxidized and eluted.

  Next, the current change with respect to the potential change was measured by cyclic voltammetry. In cyclic voltammetry, the potential scan range was -0.3 V to 1.2. Further, the potential of the working electrode 12 was changed from 0.8 V to −0.1 V by differential pulse voltammetry, and the current change with respect to the potential change was measured. The conditions for differential pulse voltammetry were an electric potential increase of 0.004 V, a pulse amplitude of 0.05 V, a pulse width of 0.05 S, and a pulse period of 0.2 S. The result of cyclic voltammetry is shown in FIG. 7, and the result of differential pulse voltammetry is shown in FIG.

<Example 2>
In Example 2, measurement of electrochemical oxidation and reduction current of gold was performed in a state where the distance between the cover glass as the facing member 16 and the working electrode 12 was wider than that in Example 1. That is, first, in order to widen the gap between the counter member 16 and the working electrode 12 in the vicinity of the end of the printed electrode device used in Comparative Example 1 and Example 1 on the side where the reference electrode 13 of the substrate 11 is formed. The convex part 17 was formed with the sealing material. The height C of the convex part 17 is 40 μm. A 0.1 N hydrochloric acid aqueous solution was dropped as a measurement solution on the surface of the working electrode 12 of the printed electrode device having the convex portions 17, and droplets of the measurement solution were formed on the surface of the working electrode 12. Next, the thickness of the measurement solution was reduced by placing the facing member 16 on the convex portion 17 and the insulating film 15 (FIG. 6C). Thereafter, electrochemical oxidation and reduction current were measured in the same manner as in Example 1 described above. The result of cyclic voltammetry is shown in FIG. 7, and the result of differential pulse voltammetry is shown in FIG.

<Example 3>
In Example 3, a glass epoxy substrate was used instead of the cover glass as the facing member 16, and the electrochemical oxidation and reduction current of gold were measured in the same manner as in Example 1. The result of differential pulse voltammetry is shown in FIG.

<Comparative Example 1>
In Comparative Example 1, the electrochemical oxidation of gold and the reduction current were measured without using a counter member. That is, first, a 0.1 N hydrochloric acid aqueous solution was dropped as a measurement solution on the surface of the working electrode 12 of the printed electrode device, and droplets of the measurement solution were formed on the surface of the working electrode 12. Next, the electrochemical oxidation and reduction currents of the measurement solution in the droplet state were measured in the same manner as in Example 1 described above. The result of cyclic voltammetry is shown in FIG. 7, and the result of differential pulse voltammetry is shown in FIG.

  As described above, in Example 1 and Example 2 where the cover glass as the facing member 16 was measured in the state of being close to the surface of the working electrode 12, the measurement solution was measured in the form of droplets based on the results of FIGS. As compared with Comparative Example 1, the peak current associated with the reduction of gold was obtained. In particular, in Example 1 in which the distance between the working electrode 12 and the facing member 16 was narrowed, a larger reduction peak current was obtained than in Example 2. From this, it can be seen that high-sensitivity detection is possible by bringing the opposing member close to the surface of the working electrode. Further, since the reduction peak current when the glass epoxy substrate was used as the counter member (Example 3) was smaller than that when the cover glass was used (Example 1), the working electrode was used as the counter member. It turned out that it is preferable to use the thing of the flatness of the surface of the part which opposes.

Experiment 2
In this experiment, colloidal gold particles that had been localized at the working electrode due to the antigen-antibody reaction were moved electrochemically to the counter electrode by elution and precipitation, and the amount of gold was measured by oxidation and reduction at each electrode. .

<Example 4>
HCG was selected as a model antigen for the sandwich immunoassay and its electrochemical detection was attempted. Colloidal gold particles were used as the labeling substance. A primary antibody (anti-α-subunit antibody) was immobilized on the surface of the working electrode of the same planar type printed electrode device as in Experiment 1. Next, a buffer solution containing the antigen was supplied onto the working electrode to capture the antigen, and a secondary antibody labeled with colloidal gold particles was bound to the antigen.

  Next, a saturated KCl solution was dropped on the surface of the printed electrode device including the working electrode, and then the counter electrode was installed so as to face the working electrode in proximity. In this state, +1.5 V was applied to the working electrode and -1 V was applied to the counter electrode for 120 seconds. The counter electrode is made of a carbon paste made of the same material as that of the working electrode, and can be operated with a potential independently of the working electrode and has the same electrode area as that of the working electrode. The distance between the working electrode and the counter electrode was about 20 μm. The surface roughness of each electrode is about 5 μm.

Next, the electrode was washed with pure water, dried with an N ₂ gun, and 0.1 N HCl was dropped onto the surface of the counter electrode. In order to elute the deposited gold, +1.2 V was applied to the counter electrode for 5 seconds. Thereafter, the current change with respect to the potential change was measured by differential pulse voltammetry (DPV) on the counter electrode side. The results are shown in FIG.

<Comparative example 2>
In the comparative example, first, an antigen-antibody reaction was performed in the same manner as in Example 4, and 0.1 N HCl was dropped onto the surface of the printed electrode device including the working electrode. Thereafter, 1.2 V was applied to the working electrode for 40 seconds as a pretreatment without installing a counter electrode. Thereafter, the current change with respect to the potential change was measured by differential pulse voltammetry (DPV) at the working electrode. The results are shown in FIG.

  From the result of FIG. 9, in Example 4 using the counter electrode, a reduction current value about three times larger than that in Comparative Example 2 without using the counter electrode is obtained, and therefore the method of the present invention is effective. I understand. In Example 4, compared with the case where metal fine particles are oxidized (Comparative Example 2), the oxidized and reduced gold is deposited on the electrode surface, so that the oxidation time may be as short as 5 seconds, which is due to diffusion. It is estimated that there was little loss. It is also presumed that the surface of the electrode (counter electrode) used for DPV measurement was in a highly active state without being contaminated by the antigen-antibody reaction. FIG. 9 also shows the results of applying 1.2 V to the counter electrode for 40 seconds as a pretreatment, and then measuring differential pulse voltammetry (DPV) on the working electrode side (reference example). Since no reduction current peak was observed in the measurement on the working electrode side, it can be seen that the metal was transferred to the counter electrode side by the pretreatment.

(A)-(c) is a schematic diagram for demonstrating 1st Embodiment of the measuring method to which this invention is applied. (A)-(d) is a schematic diagram for demonstrating 2nd Embodiment of the measuring method to which this invention is applied. (A)-(c) is a schematic diagram for demonstrating 3rd Embodiment of the measuring method to which this invention is applied. It is a principal part schematic plan view of the comb-tooth electrode used in 4th Embodiment. (A)-(d) is a schematic diagram for demonstrating 4th Embodiment of the measuring method to which this invention is applied. It is sectional drawing for demonstrating the positional relationship of the printed electrode device used in Experiment 1, and a counter member. FIG. 6 is a characteristic diagram showing the results of cyclic voltammetry in Experiment 1. FIG. 6 is a characteristic diagram showing the results of differential pulse voltammetry in Experiment 1. It is a characteristic view which shows the result of the differential pulse voltammetry of Experiment 2. (A)-(c) is a schematic diagram for demonstrating the conventional measuring method.

Explanation of symbols

  1 working electrode, 2 primary antibody, 3 test substance (antigen), 4 secondary antibody, 5 metal fine particle, 6 facing member, 7 facing electrode

Claims (11)

A method for measuring a test substance using an electrochemical measurement method while using metal fine particles as a labeling substance,
Utilizing the biological interaction to localize the metal microparticles at least on the surface of the working electrode;
And electrochemically oxidizing and eluting the metal fine particles localized on the surface of the working electrode,
A method for measuring a test substance, comprising suppressing diffusion of the eluate from the working electrode at least in the step of oxidizing the metal fine particles. A method for measuring a test substance using metal fine particles as a labeling substance,
Utilizing the biological interaction to localize the metal microparticles at least on the surface of the working electrode;
A step of electrochemically oxidizing and eluting the metal fine particles localized on the surface of the working electrode;
Measuring the current value at the time of electrochemically reducing the effluent by electrochemical oxidation at the working electrode,
Method of measuring the analyte you characterized thereby close the opposing member having a flat surface on the surface of the working electrode in the step of measuring the process and the reduction current for oxidizing the metal particles.   The method for measuring a test substance according to claim 2, wherein a distance between the working electrode and the facing member is 50 μm or less.   The method for measuring a test substance according to claim 2, wherein an arithmetic average roughness Ra of a surface of the facing member facing the working electrode is 20 μm or less. A method for measuring a test substance using metal fine particles as a labeling substance,
Utilizing the biological interaction to localize the metal microparticles at least on the surface of the working electrode;
In a state where a counter electrode different from the working electrode is brought close to the surface of the working electrode, the metal fine particles localized on the surface of the working electrode are electrochemically oxidized and eluted, and the counter electrode Electrochemically depositing the metal constituting the metal fine particles on the surface of
A step of electrochemically oxidizing and eluting the metal deposited on the counter electrode;
Method of measuring the analyte you, characterized in that the current value when electrochemically reduced the eluate precipitated metal by electrochemical oxidation after step of oxidizing a step of measuring with said counter electrode . A method for measuring a test substance using metal fine particles as a labeling substance,
Utilizing the biological interaction to localize the metal microparticles at least on the surface of the working electrode;
In a state where a counter electrode different from the working electrode is brought close to the surface of the working electrode, the metal fine particles localized on the surface of the working electrode are electrochemically oxidized and eluted, and the counter electrode Electrochemically depositing the metal constituting the metal fine particles on the surface of
Method of measuring the analyte you; and a step of measuring the current value at the time of electrochemically oxidizing the metal deposited on the counter electrode in the counter electrode.   The method for measuring a test substance according to claim 5 or 6, wherein an interval between the working electrode and the counter electrode is 50 µm or less.   The method for measuring a test substance according to claim 5 or 6, wherein a planar shape of the counter electrode is substantially the same as that of the working electrode. A method for measuring a test substance using an electrochemical measurement method using a metal fine particle as a labeling substance and a working electrode formed by a pair of comb electrodes,
Utilizing the biological interaction to localize the metal microparticles at least on the comb electrode surface;
The metal fine particles localized on the surface of one of the comb electrodes are electrochemically oxidized and eluted,
A first metal fine particle oxidation / reduction step for electrochemically depositing the metal constituting the metal fine particles on the surface of the other comb-shaped electrode;
Second metal fine particle oxidation / reduction that electrochemically oxidizes and elutes the metal fine particles localized on the surface of the other comb electrode and the deposited metal, and deposits metal on the surface of the one comb electrode method of measuring the analyte you; and a step.   10. The test substance according to claim 9, wherein a current value when electrochemically oxidizing the metal deposited on the one comb-shaped electrode after the second metal fine particle oxidation / reduction step is measured. Measuring method.   After the second metal fine particle oxidation / reduction step, a current value when the metal deposited on the one comb electrode is electrochemically oxidized and eluted, and the effluent is reduced by the other comb electrode. The method for measuring a test substance according to claim 9, wherein:

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