Antigen measurement method, antigen measurement kit, and antigen measurement substrate

Abstract

This antigen measurement method comprises: a step for preparing a measurement substrate 10A which includes a substrate 110 that has a first main surface 110a and a second main surface 110b facing each other in the thickness direction, and a plurality of metal bodies 120 that is provided at intervals from each other along the first main surface 110a of the substrate 110; a step for preparing a measurement material 20 which contains metal particles 210; a step for bringing a measurement object containing an antigen 310 into contact with the measurement material 20, thereby preparing a measurement sample 30 which contains antigen-attached metal particles 410 in which the antigen 310 is bonded to the metal particles 210; a step for bringing the measurement sample 30 into contact with the measurement substrate 10A, thereby bonding the antigen-attached metal particles 410 to the measurement substrate 10A at positions that are different from the positions of the metal bodies 120 on the first main surface 110a of the substrate 110; and a step for measuring reflection absorption properties due to localized surface plasmon resonance from the first main surface 110a side of the substrate 110 of the measurement substrate 10A before and after the bonding of the antigen-attached metal particles 410 to the measurement substrate 10A, thereby measuring changes of the reflection absorption properties before and after the bonding of the antigen-attached metal particles 410 to the measurement substrate 10A.

Description

Antigen measurement method, antigen measurement kit, and antigen measurement substrate

[0001] The present invention relates to an antigen measurement method, an antigen measurement kit, and an antigen measurement substrate.

[0002] In Patent Document 1, a metal layer and an insulating layer are formed in this order on an insulating substrate, a single-layer metal colloid crystal composed of metal particles is formed on the insulating layer, the average particle diameter of the metal particles is 50 nm or more and 500 nm or less, and the interval between the metal particles is 50 nm or more and 1000 nm or less. A substrate for SPR measurement is disclosed.

[0003] Japanese Unexamined Patent Application Publication No. 2020-34543

[0004] In the substrate for SPR measurement described in Patent Document 1, a complicated patterning technique for controlling the arrangement of metal particles is not required, the manufacturing is easy, and a highly sensitive localized surface plasmon sensor can be constructed.

[0005] Surface plasmon is a疏密 wave based on the collective vibration of free electrons localized on the surface of a metal. When light is incident on the surface of a metal, when the condition is met that the surface plasmon localized on the surface of the metal and the incident light resonate, the energy of the incident light moves to the surface of the metal. This phenomenon is called surface plasmon resonance (SPR: Surface Plasmon Resonance). Surface plasmon resonance includes a propagating surface plasmon resonance using a metal thin film and a localized surface plasmon resonance using metal nanoparticles or metal nanostructures (hereinafter also referred to as localized surface plasmon resonance).

[0006] In the SPR measurement substrate described in Patent Document 1, for example, when an antigen is adsorbed onto a metal such as metal particles or a metal layer, the surface state of the metal changes, and it is thought that the refractive index (dielectric constant) near the surface of the metal changes, and the surface plasmon resonance wavelength changes in accordance with this change in refractive index (dielectric constant). In other words, it is thought that the SPR measurement substrate described in Patent Document 1 detects the change in refractive index (dielectric constant) near the surface of the metal due to the adsorption of an antigen onto the metal as a change in the surface plasmon resonance wavelength. Thus, it is thought that the SPR measurement substrate described in Patent Document 1 can perform sensing targeting antigen adsorption, etc., from the change in the surface plasmon resonance wavelength.

[0007] However, the SPR measurement substrate described in Patent Document 1 may have low antigen detection sensitivity due to reasons such as a small change in the resonance peak shift when detecting changes in the surface plasmon resonance wavelength. Therefore, there is room for improvement in the antigen detection sensitivity of the SPR measurement substrate described in Patent Document 1.

[0008] The present invention was made to solve the above problems and aims to provide an antigen measurement method with high antigen detection sensitivity. Furthermore, the present invention aims to provide an antigen measurement kit that can be effectively used with the above antigen measurement method. Moreover, the present invention aims to provide an antigen measurement substrate that can be effectively used with the above antigen measurement method.

[0009] In a first embodiment, the antigen measurement method of the present invention is characterized by comprising the steps of: preparing a measurement substrate having a substrate having a first main surface and a second main surface facing each other in the thickness direction, and a plurality of metal bodies provided at intervals from each other along the first main surface of the substrate; preparing a measurement material containing metal particles; producing a measurement sample containing antigen-attached metal particles in which the antigen is bound to the metal particles by bringing a measurement target object containing an antigen into contact with the measurement material; binding the antigen-attached metal particles to the measurement substrate at a position on the first main surface of the substrate different from the metal bodies by bringing the measurement sample and the measurement substrate into contact; and measuring the change in the reflection absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate by measuring the reflection absorption characteristics by localized surface plasmon resonance from the first main surface side of the substrate before and after binding the antigen-attached metal particles.

[0010] In a first embodiment, the antigen measurement kit of the present invention comprises a measurement substrate having a substrate having a first main surface and a second main surface opposite to each other in the thickness direction, a plurality of metal bodies provided at intervals from each other along the first main surface of the substrate, and a binding portion provided on the first main surface of the substrate at a position different from the metal bodies, and a measurement material containing metal particles, wherein the binding portion is a site for binding of antigen-attached metal particles in which an antigen is bound to the metal particles.

[0011] The antigen measurement substrate of the present invention, in the first embodiment of the first aspect, comprises a substrate having a first main surface and a second main surface opposite to each other in the thickness direction, a plurality of metal bodies provided at intervals from each other along the first main surface of the substrate, and a bonding portion provided on the first main surface of the substrate at a position different from the metal bodies, wherein the bonding portion is a site for binding antigen-attached metal particles to which an antigen has been bound.

[0012] The antigen measurement substrate of the present invention is characterized in that, in the second embodiment of the first embodiment, it comprises a substrate having a first main surface and a second main surface that are opposite to each other in the thickness direction, a plurality of metal bodies provided at intervals from each other along the first main surface of the substrate, a binding portion provided on the first main surface of the substrate at a position different from the metal bodies, and antigen-attached metal particles bound to the binding portion in a state in which the antigen is bound to the metal particles.

[0013] In a second embodiment, the antigen measurement method of the present invention is characterized by comprising the steps of: preparing a measurement substrate having a substrate having a first main surface and a second main surface facing each other in the thickness direction, wherein the substrate includes, in order from the second main surface side toward the first main surface side, an insulating substrate, a metal layer provided on the surface of the insulating substrate, and an insulating layer provided on the surface of the metal layer opposite to the insulating substrate; preparing a measurement material containing metal particles; producing a measurement sample containing antigen-attached metal particles in which the antigen is bound to the metal particles by bringing a measurement target object containing an antigen into contact with the measurement material; binding the antigen-attached metal particles to the measurement substrate on the first main surface of the substrate by bringing the measurement sample and the measurement substrate into contact; and measuring the change in the reflection absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate by measuring the reflection absorption characteristics by localized surface plasmon resonance from the first main surface side of the substrate before and after binding the antigen-attached metal particles.

[0014] In a second embodiment, the antigen measurement kit of the present invention comprises a substrate having a first main surface and a second main surface facing each other in the thickness direction, and a bonding portion provided on the first main surface of the substrate, wherein the substrate comprises a measuring substrate including, in order from the second main surface side toward the first main surface side, an insulating substrate, a metal layer provided on the surface of the insulating substrate, and an insulating layer provided on the surface of the metal layer opposite to the insulating substrate, and a measuring material containing metal particles, wherein the bonding portion is a portion for binding of antigen-attached metal particles in which the antigen is bound to the metal particles.

[0015] The antigen measurement substrate of the present invention, in the first embodiment of the second aspect, comprises a substrate having a first main surface and a second main surface facing each other in the thickness direction, and in order from the second main surface side toward the first main surface side, including an insulating substrate, a metal layer provided on the surface of the insulating substrate, an insulating layer provided on the surface of the metal layer opposite to the insulating substrate, and a bonding portion provided on the first main surface of the substrate, wherein the bonding portion is a portion for binding antigen-attached metal particles in which an antigen is bound to the metal particles.

[0016] The antigen measurement substrate of the present invention, in the second embodiment of the second aspect, is characterized in that it has a first main surface and a second main surface facing each other in the thickness direction, and comprises, in order from the second main surface side toward the first main surface side, an insulating substrate, a metal layer provided on the surface of the insulating substrate, an insulating layer provided on the surface of the metal layer opposite to the insulating substrate, a bonding portion provided on the first main surface of the substrate, and antigen-attached metal particles bound to the bonding portion in a state in which the antigen is bound to the metal particles.

[0017] The present invention provides an antigen measurement method with high antigen detection sensitivity. Furthermore, the present invention provides an antigen measurement kit that can be effectively used with the above antigen measurement method. Moreover, the present invention provides an antigen measurement substrate that can be effectively used with the above antigen measurement method.

[0018] Figure 1 is a schematic cross-sectional view illustrating an example of the process of preparing a measurement substrate for Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention. Figure 2 is a schematic cross-sectional view illustrating an example of the process of preparing measurement materials for Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention. Figure 3 is a schematic cross-sectional view illustrating an example of the process of preparing a measurement sample for Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention. Figure 4 is a schematic cross-sectional view illustrating an example of the process of binding antigen-attached metal particles to a measurement substrate for Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention. Figure 5 is a schematic cross-sectional view illustrating an example of the process of preparing a measurement substrate for Specific Example 1-1 relating to Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention. Figure 6 is a schematic cross-sectional view illustrating another example of the process of preparing a measurement substrate for Specific Example 1-1 relating to Specific Example 1-1 relating to Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention. Figure 7 is a schematic cross-sectional view illustrating an example of the process of preparing measurement materials for Specific Example 1-1 relating to Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention. Figure 8 is a schematic cross-sectional view showing an example of the process for preparing a measurement sample in Specific Example 1-1 of Embodiment 1-1 of the First Embodiment of the Antigen Measurement Method of the Present Invention. Figure 9 is a schematic cross-sectional view showing an example of the process for binding antigen-attached metal particles to a measurement substrate in Specific Example 1-1 of Embodiment 1-1 of the First Embodiment of the Antigen Measurement Method of the Present Invention. Figure 10 is a schematic cross-sectional view showing an example of the process for preparing a measurement substrate in Specific Example 1-2 of Embodiment 1-1 of the First Embodiment of the Antigen Measurement Method of the Present Invention. Figure 11 is a schematic cross-sectional view showing another example of the process for preparing a measurement substrate in Specific Example 1-2 of Embodiment 1-1 of the First Embodiment of the Antigen Measurement Method of the Present Invention. Figure 12 is a schematic cross-sectional view showing an example of the process for preparing a measurement material in Specific Example 1-2 of Embodiment 1-1 of the First Embodiment of the Antigen Measurement Method of the Present Invention. Figure 13 is a schematic cross-sectional view showing an example of the process for preparing a measurement sample in Specific Example 1-2 of Embodiment 1-1 of the First Embodiment of the Antigen Measurement Method of the Present Invention. Figure 14 is a schematic cross-sectional view showing an example of the step of binding antigen-attached metal particles to a measurement substrate, in a specific example 1-2 relating to Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention.Figure 15 is a schematic cross-sectional view showing an example of the process of preparing a measurement substrate in Embodiment 1-2 of the first embodiment of the antigen measurement method of the present invention. Figure 16 is a schematic cross-sectional view showing an example of the process of preparing a measurement material in Embodiment 1-2 of the first embodiment of the antigen measurement method of the present invention. Figure 17 is a schematic cross-sectional view showing an example of the process of preparing a measurement sample in Embodiment 1-2 of the first embodiment of the antigen measurement method of the present invention. Figure 18 is a schematic cross-sectional view showing an example of the process of binding antigen-attached metal particles to a measurement substrate in Embodiment 1-2 of the first embodiment of the antigen measurement method of the present invention. Figure 19 is a schematic cross-sectional view showing a modified measurement substrate in Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention. Figure 20 is a schematic cross-sectional view showing modified measurement substrate 1 in Embodiment 1-2 of the first embodiment of the antigen measurement method of the present invention. Figure 21 is a schematic cross-sectional view showing modified measurement substrate 2 in Embodiment 1-2 of the first embodiment of the antigen measurement method of the present invention. Figure 22 is a schematic cross-sectional view showing a modified example 3 of the measurement substrate for Embodiment 1-2 of the first embodiment of the antigen measurement method of the present invention. Figure 23 is a schematic plan view showing a preferred example of a state in which antigen-attached metal particles are bound to the measurement substrate. Figure 24 is a schematic cross-sectional view showing an example of the process of preparing the measurement substrate for Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention. Figure 25 is a schematic cross-sectional view showing an example of the process of preparing the measurement material for Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention. Figure 26 is a schematic cross-sectional view showing an example of the process of preparing the measurement sample for Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention. Figure 27 is a schematic cross-sectional view showing an example of the process of binding antigen-attached metal particles to the measurement substrate for Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention. Figure 28 is a schematic cross-sectional view showing an example of the process of preparing the measurement substrate for Specific Example 2-1 of Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention. Figure 29 is a schematic cross-sectional view showing another example of the step of preparing a measurement substrate in a specific example 2-1 of Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention.Figure 30 is a schematic cross-sectional view illustrating an example of the process of preparing measurement materials for Specific Example 2-1 of Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention. Figure 31 is a schematic cross-sectional view illustrating an example of the process of preparing a measurement sample for Specific Example 2-1 of Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention. Figure 32 is a schematic cross-sectional view illustrating an example of the process of binding antigen-attached metal particles to a measurement substrate for Specific Example 2-1 of Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention. Figure 33 is a schematic cross-sectional view illustrating an example of the process of preparing a measurement substrate for Specific Example 2-2 of Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention. Figure 34 is a schematic cross-sectional view illustrating another example of the process of preparing a measurement substrate for Specific Example 2-2 of Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention. Figure 35 is a schematic cross-sectional view illustrating an example of the process of preparing measurement materials for Specific Example 2-2 of Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention. Figure 36 is a schematic cross-sectional view showing an example of the process for preparing a sample for measurement in Specific Example 2-2 of Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention. Figure 37 is a schematic cross-sectional view showing an example of the process for binding antigen-attached metal particles to a measurement substrate in Specific Example 2-2 of Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention.

[0019] The following describes the antigen measurement method, antigen measurement kit, and antigen measurement substrate of the present invention. It should be noted that the present invention is not limited to the following configurations and may be modified as appropriate without departing from the spirit of the invention. Furthermore, combinations of several of the preferred configurations described below also constitute the present invention.

[0020] The drawings shown below are schematic representations, and their dimensions, aspect ratios, and scales may differ from those of the actual product.

[0021] In this specification, unless otherwise specified, terms describing relationships between elements (e.g., "parallel," "perpendicular," etc.) and terms describing the shape of elements mean not only their literal, exact form, but also a range that is substantially equivalent, for example, a range that includes differences of a few percent.

[0022] (1-1) First Embodiment of the Antigen Measurement Method of the Present Invention The antigen measurement method of the present invention, in a first embodiment, comprises the steps of: preparing a measurement substrate having a substrate having a first main surface and a second main surface opposite to each other in the thickness direction, and a plurality of metal bodies provided at intervals from each other along the first main surface of the substrate; preparing a measurement material containing metal particles; producing a measurement sample containing antigen-attached metal particles in which the antigen is bound to the metal particles by bringing a measurement target object containing an antigen into contact with the measurement material; binding the antigen-attached metal particles to the measurement substrate at a position on the first main surface of the substrate different from the metal bodies by bringing the measurement sample and the measurement substrate into contact; and measuring the change in the reflection absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate by measuring the reflection absorption characteristics by localized surface plasmon resonance from the first main surface side of the substrate before and after binding the antigen-attached metal particles.

[0023] In the first aspect of the antigen measurement method of the present invention, in the step of binding antigen-attached metal particles to a measurement substrate, the measurement sample containing the antigen-attached metal particles is brought into contact with the substrate and the measurement substrate having a plurality of metal bodies, thereby binding the antigen-attached metal particles to the measurement substrate at a position on the first main surface of the substrate that is different from the metal bodies. As a result, in the first aspect of the antigen measurement method of the present invention, the distance between metals changes before and after binding the antigen-attached metal particles to the measurement substrate, with respect to the plurality of metals including metal bodies that were previously provided on the first main surface of the substrate and antigen-attached metal particles (metal particles) newly bound to the first main surface of the substrate. Specifically, before binding the antigen-attached metal particles to the measurement substrate, the distance between metals corresponds to the distance between metal bodies, whereas after binding the antigen-attached metal particles to the measurement substrate, the distance between metals corresponds to the distance between the metal bodies and the antigen-attached metal particles (metal particles).

[0024] Furthermore, in the first aspect of the antigen measurement method of the present invention, in the step of measuring the change in reflection absorption characteristics, the change in reflection absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate is measured by measuring the reflection absorption characteristics due to localized surface plasmon resonance from the first main surface side of the measurement substrate before and after binding the antigen-attached metal particles to the measurement substrate. In the first aspect of the antigen measurement method of the present invention, as described above, the distance between the metals, including the metal body and the antigen-attached metal particles (metal particles), changes before and after binding the antigen-attached metal particles to the measurement substrate. Therefore, the reflection absorption characteristics due to localized surface plasmon resonance near the surface of the metal change significantly before and after binding the antigen-attached metal particles to the measurement substrate. Thus, in the first aspect of the antigen measurement method of the present invention, in order to significantly change the reflection absorption characteristics due to localized surface plasmon resonance near the surface of the metals, including the metal body and the antigen-attached metal particles (metal particles), the distance between the metals is changed by binding the antigen-attached metal particles to the measurement substrate, rather than changing the surface state of the metal body by binding the antigen to the metal body.

[0025] As described above, in the first embodiment of the antigen measurement method of the present invention, the change in reflection and absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate becomes large, making it easier to detect with high sensitivity that the antigen-attached metal particles have bound to the measurement substrate, that is, that the antigen on the antigen-attached metal particles has bound to the measurement substrate. Therefore, according to the first embodiment of the antigen measurement method of the present invention, an antigen measurement method with high antigen detection sensitivity can be realized.

[0026] In the first embodiment of the antigen measurement method of the present invention, the change in reflection absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate is large, so the reproducibility of antigen detection tends to be higher compared to the case where the change in reflection absorption characteristics is small.

[0027] In the first embodiment of the antigen measurement method of the present invention, the change in reflection and absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate is significant. Therefore, even if the performance (resolution) of the spectrometer used to measure the reflection and absorption characteristics is not high, for example, sensing that is sufficiently usable as a sensor such as a biosensor can be performed.

[0028] By utilizing the first aspect of the antigen measurement method of the present invention, it is possible to estimate the magnitude of, for example, the concentration of the antigen in the sample to be measured, based on the magnitude of the change in the reflection absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate.

[0029] A conventional method for measuring antigens is immunochromatography. In immunochromatography, a complex formed by the binding of the antigen in the sample to a metal colloid-labeled antibody binds to a capture antibody, and the antigen is detected by utilizing the resulting color reaction of the metal colloid. However, because immunochromatography relies on this color reaction, it is difficult to quantify the concentration of the antigen in the sample.

[0030] In contrast, the first embodiment of the antigen measurement method of the present invention does not utilize a color reaction like that of immunochromatography, but rather detects the antigen by utilizing the change in the reflectance absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate. Therefore, in the first embodiment of the antigen measurement method of the present invention, by measuring, for example, the change in resonance peak shift as the change in reflectance absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate, it is possible not only to quantify the change in reflectance absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate, but also to quantify the concentration of the antigen in the measurement sample.

[0031] The following describes an embodiment of the first aspect of the antigen measurement method of the present invention.

[0032] [Embodiment 1-1] In Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention, the substrate includes, in order from the second main surface side toward the first main surface side, an insulating substrate, a metal layer provided on the surface of the insulating substrate, and an insulating layer provided on the surface of the metal layer opposite to the insulating substrate.

[0033] Embodiment 1-1 of the first aspect of the antigen measurement method of the present invention comprises the following steps.

[0034] <Steps for preparing the measurement substrate> Figure 1 is a schematic cross-sectional view showing an example of the steps for preparing the measurement substrate in Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention.

[0035] In this process, the measurement substrate 10A shown in Figure 1 is prepared.

[0036] The measuring substrate 10A comprises a substrate 110 and a plurality of metal bodies 120.

[0037] The substrate 110 has a first main surface 110a and a second main surface 110b that are opposite to each other in the thickness direction (vertical direction in Figure 1).

[0038] The substrate 110 includes, in order from the second main surface 110b side toward the first main surface 110a side, an insulating substrate 111, a metal layer 112 provided on the surface of the insulating substrate 111, and an insulating layer 113 provided on the surface of the metal layer 112 opposite to the insulating substrate 111.

[0039] In the example shown in Figure 1, the surface of the insulating layer 113 opposite to the metal layer 112 constitutes the first main surface 110a of the substrate 110, and the surface of the insulating substrate 111 opposite to the metal layer 112 constitutes the second main surface 110b of the substrate 110.

[0040] The insulating substrate 111 is, for example, a glass substrate, a silicon substrate, a plastic substrate, etc.

[0041] Preferably, the metal layer 112 is provided on one main surface (the top surface in Figure 1) of the insulating substrate 111. In this case, the metal layer 112 may be provided on the entire main surface of the insulating substrate 111, or on a part of the main surface of the insulating substrate 111.

[0042] The metal layer 112 is preferably composed of a precious metal such as gold or silver that can exhibit strong localized surface plasmon resonance. In particular, the metal layer 112 is preferably composed of gold. Gold is easier to handle than silver and the like for reasons such as being less prone to oxidation.

[0043] The metal layer 112 is, for example, a metal film. The metal film is formed by methods such as vapor deposition, sputtering, or chemical plating.

[0044] The insulating layer 113 may be provided on the entire surface of the metal layer 112 on the side opposite to the insulating substrate 111, or may be provided on a part of the surface of the metal layer 112 on the side opposite to the insulating substrate 111. In the latter case, the insulating layer 113 may be provided at a position sandwiched between the metal layer 112 and the metal body 120.

[0045] The insulating layer 113 may be further provided on the other main surface of the insulating substrate 111 where the metal layer 112 is not provided.

[0046] The surface of the insulating layer 113 preferably has a positive charge or a negative charge.

[0047] The insulating layer 113 is preferably composed of a silane coupling agent. In this case, the metal body 120 is likely to be fixed to the surface of the insulating layer 113 on the side opposite to the metal layer 112 (for example, the metal body 120 is likely to be adsorbed).

[0048] When the insulating layer 113 is composed of a silane coupling agent, the insulating layer 113 may include, for example, a first silane coupling agent layer composed of 3 - mercaptopropyltrimethoxysilane (MPTMS) or the like and a second silane coupling agent layer composed of 3 - aminopropyltriethoxysilane (APTES) or the like in order from the side of the metal layer 112 toward the side opposite to the metal layer 112. In this case, the surface of the insulating layer 113 is likely to have a positive charge.

[0049] The thickness of the insulating layer 113 is not particularly limited and may be the same as the thickness of the metal layer 112 or different from the thickness of the metal layer 112. In the latter case, the thickness of the insulating layer 113 may be larger than the thickness of the metal layer 112 or smaller than the thickness of the metal layer 112.

[0050] The plurality of metal bodies 120 are provided at intervals along the first main surface 110a of the substrate 110. In the example shown in FIG. 1, the plurality of metal bodies 120 are provided at intervals along the surface of the insulating layer on the side opposite to the metal layer 112.

[0051] The multiple metal bodies 120 may be provided on the entire first main surface 110a of the substrate 110, or on a portion of the first main surface 110a of the substrate 110.

[0052] It is preferable that the multiple metal bodies 120 are arranged two-dimensionally on the first main surface 110a of the substrate 110. The multiple metal bodies 120 may be arranged two-dimensionally on the entire first main surface 110a of the substrate 110, or they may be arranged two-dimensionally on a part of the first main surface 110a of the substrate 110.

[0053] For example, when observing the measurement substrate 10A using a microscope such as an optical microscope or an electron microscope, if multiple metal bodies 120 are observed only when the height of the sample stage is set to a certain height, it can be determined that the multiple metal bodies 120 are arranged two-dimensionally on the first main surface 110a of the substrate 110.

[0054] It is more preferable that the multiple metal bodies 120 are arranged two-dimensionally on the first main surface 110a of the substrate 110, spaced apart from each other. In this case, the shortest distance between adjacent metal bodies 120 when viewed from the thickness direction of the substrate 110 may be the same, different from each other, or partially different. Furthermore, the average shortest distance between the metal bodies 120 is not particularly limited.

[0055] It is even more preferable that the multiple metal bodies 120 are arranged in a two-dimensional and regular manner with spacing between them on the first main surface 110a of the substrate 110. In this case, the multiple metal bodies 120 may be arranged to have a symmetry such as four-fold symmetry or six-fold symmetry.

[0056] It is preferable that the surface of the metal body 120 has a charge of a different sign than the surface of the insulating layer 113. For example, if the surface of the insulating layer 113 has a positive charge, it is preferable that the surface of the metal body 120 has a negative charge. In this case, the multiple metal bodies 120 are arranged with gaps between them along the surface of the insulating layer 113 opposite to the metal layer 112 due to the electrostatic repulsive force acting between the metal bodies 120. Furthermore, because the surface of the insulating layer 113 has a positive charge, the negatively charged metal bodies 120 are attracted to the surface of the insulating layer 113 opposite to the metal layer 112 by electrostatic attraction.

[0057] The metal body 120 is preferably composed of a precious metal such as gold or silver that can exhibit strong localized surface plasmon resonance. In particular, the metal body 120 is preferably composed of gold. Gold is easier to handle than silver and the like for reasons such as being less prone to oxidation.

[0058] As shown in Figure 1, the metal body 120 is preferably in particulate form. In other words, the metal body 120 is preferably in the form of metal particles.

[0059] When the metal body 120 is in particulate form, it is preferable that the metal body 120 consists of metal particles made of precious metals such as gold and silver that can exhibit strong localized surface plasmon resonance. In particular, it is preferable that the metal body 120 is made of gold particles. Gold is easier to handle than silver and the like for reasons such as being less prone to oxidation.

[0060] When the metal body 120 is in particulate form, the average particle size of the metal body 120 is not particularly limited, but is preferably 5 nm or more and 500 nm or less.

[0061] If the metal body 120 is in particulate form, the average particle size of the metal body 120 is measured using a scanning electron microscope (SEM) for 300 to 500 metal bodies 120, preferably all metal bodies 120, on the measuring substrate 10A.

[0062] When the metal bodies 120 are particulate, it is preferable that the multiple metal bodies 120 constitute a two-dimensional colloidal crystal regularly arranged on the first main surface 110a of the substrate 110. In this case, it is preferable that the multiple metal bodies 120 are spaced apart from each other and arranged two-dimensionally and regularly on the first main surface 110a of the substrate 110. In the two-dimensional colloidal crystal, the multiple metal bodies 120 may be arranged to have a symmetry such as four-fold symmetry or six-fold symmetry.

[0063] The metal body 120 may have a single-layer structure or a multi-layer structure. If the metal body 120 is particulate, it may have a single-layer structure such as a homogeneous structure or a multi-layer structure such as a core-shell structure.

[0064] In the example shown in Figure 1, the metal layer 112, the insulating layer 113, and the metal body 120 form a MIM (Metal-Insulator-Metal) structure. In other words, the measurement substrate 10A can be said to be an MIM structure substrate having an MIM structure.

[0065] <Steps for preparing measurement materials> Figure 2 is a schematic cross-sectional view showing an example of the steps for preparing measurement materials in Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention.

[0066] In this step, the measuring material 20 shown in Figure 2 is prepared.

[0067] The measuring material 20 contains metal particles 210.

[0068] The metal particles 210 are preferably composed of precious metals such as gold and silver that can exhibit strong localized surface plasmon resonance. In particular, the metal particles 210 are preferably gold particles. Gold is easier to handle than silver and the like for reasons such as being less prone to oxidation.

[0069] The average particle size of the metal particles 210 is not particularly limited, but is preferably between 5 nm and 500 nm.

[0070] The average particle size of the metal particles 210 is measured using a scanning electron microscope (SEM) after diluting the measurement material 20 with a solvent such as water, dropping it onto a substrate such as a glass substrate, drying it, and then measuring 300 to 500 metal particles 210, preferably all of the metal particles 210.

[0071] The average particle size of the metal particles 210 may be the same as the average particle size of the metal body 120 (when the metal body 120 is in particulate form), or it may be different from the average particle size of the metal body 120 (when the metal body 120 is in particulate form). In the latter case, the average particle size of the metal particles 210 may be larger than the average particle size of the metal body 120 (when the metal body 120 is in particulate form), or it may be smaller than the average particle size of the metal body 120 (when the metal body 120 is in particulate form).

[0072] The metal particles 210 may have a single-layer structure such as a homogeneous structure, or they may have a multi-layer structure such as a core-shell structure.

[0073] In this process, it is sufficient to prepare a measuring material 20 containing metal particles 210, and the measuring material 20 may contain substances other than metal particles 210. For example, the measuring material 20 may further contain a dispersion medium 220 for dispersing the metal particles 210. In this case, the measuring material 20 is produced, for example, by dispersing the metal particles 210 in the dispersion medium 220.

[0074] The dispersion medium 220 is not particularly limited and may be, for example, an inorganic solvent such as water, an organic solvent such as alcohol, etc.

[0075] <Steps for preparing a sample for measurement> Figure 3 is a schematic cross-sectional view showing an example of the steps for preparing a sample for measurement in Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention.

[0076] In this process, the measurement sample 30 shown in Figure 3 is prepared. Specifically, it is done as follows:

[0077] First, the object to be measured containing the antigen 310 is brought into contact with the measurement material 20 (see Figure 2).

[0078] Antigen 310 is not particularly limited and may be, for example, a GST antigen.

[0079] The object to be measured containing the antigen 310 is not particularly limited, and may be, for example, a sample of human body such as saliva, urine, or blood that is expected to be measured for the antigen 310. In this case, for example, the sample containing the antigen 310 can be dropped onto the measuring material 20 to bring the sample containing the antigen 310 into contact with the measuring material 20.

[0080] When bringing the object to be measured containing the antigen 310 into contact with the measurement material 20, one may be placed into the other (for example, one may be dropped onto the other as described above), or the two may be mixed together.

[0081] Then, by bringing the object to be measured containing the antigen 310 into contact with the measurement material 20, a measurement sample 30 is prepared, which includes antigen-attached metal particles 410 in which the antigen 310 is bound to the metal particles 210, as shown in Figure 3. In the example shown in Figure 3, a measurement sample 30 is prepared in which the antigen-attached metal particles 410 are present in the dispersion medium 220. At this time, antigen-attached metal particles 410 are prepared in proportion to the concentration of antigen 310 in the object to be measured. For example, if the concentration of antigen 310 in the object to be measured is high, a large amount of antigen-attached metal particles 410 are prepared in the measurement sample 30. On the other hand, if the concentration of antigen 310 in the object to be measured is low, a small amount of antigen-attached metal particles 410 are prepared in the measurement sample 30, and metal particles 210 without the antigen 310 bound remain in the measurement sample 30.

[0082] The method for obtaining the antigen-attached metal particles 410 is not particularly limited. For example, by pre-binding an antibody (not shown) to the metal particles 210 in the measurement material 20, and then utilizing the binding reaction between the antigen 310 and the antibody bound to the metal particles 210, antigen-attached metal particles 410 can be obtained in which the antigen 310 is bound to the metal particles 210 via the antibody.

[0083] In this process, as long as a measurement sample 30 containing antigen-attached metal particles 410 is prepared, a measurement sample 30 containing substances other than antigen-attached metal particles 410 may also be prepared. For example, in addition to antigen-attached metal particles 410, the measurement sample 30 may further contain at least one of metal particles 210 to which the antigen 310 is not bound, and antigen 310 to which the metal particles 210 are not bound. In other words, in addition to antigen-attached metal particles 410, the measurement sample 30 may further contain metal particles 210 to which the antigen 310 is not bound, or it may further contain antigen 310 to which the metal particles 210 are not bound, or it may further contain both metal particles 210 to which the antigen 310 is not bound, and antigen 310 to which the metal particles 210 are not bound.

[0084] If the sample 30 for measurement further contains substances other than the antigen-tagged metal particles 410, such as individual metal particles 210, the substances other than the antigen-tagged metal particles 410 may be removed by decantation or the like.

[0085] <Step of binding antigen-attached metal particles to a measurement substrate> Figure 4 is a schematic cross-sectional view showing an example of the step of binding antigen-attached metal particles to a measurement substrate in Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention.

[0086] In this process, antigen-coated metal particles 410 are bonded to the measurement substrate 10A. Specifically, this is done as follows.

[0087] First, the measurement sample 30 (see Figure 3) and the measurement substrate 10A (see Figure 1) are brought into contact.

[0088] The method for bringing the measurement sample 30 and the measurement substrate 10A into contact is not particularly limited. For example, the measurement sample 30 and the measurement substrate 10A can be brought into contact by dripping (dropping) the measurement sample 30 onto the measurement substrate 10A.

[0089] Then, by bringing the measurement sample 30 and the measurement substrate 10A into contact, the antigen-attached metal particles 410 are bonded to the measurement substrate 10A at a position on the first main surface 110a of the substrate 110 that is different from the metal body 120, as shown in Figure 4.

[0090] In this process, when bonding the antigen-attached metal particles 410 to the measurement substrate 10A, the shortest distance between adjacent metal particles 210, viewed from the thickness direction of the substrate 110, may be the same, may be different, or may be partially different. Furthermore, the average shortest distance between metal particles 210 is not particularly limited.

[0091] As shown in Figure 4, in this process, it is preferable to bind the antigen-attached metal particles 410 to the measurement substrate 10A such that the shortest distance D1 between the metal body 120 and the metal particles 210 is smaller than the shortest distance D2 between the metal bodies 120.

[0092] The method for binding the antigen-attached metal particles 410 to the measurement substrate 10A is not particularly limited. Specific examples of methods for binding the antigen-attached metal particles 410 to the measurement substrate 10A will be described later.

[0093] When binding the antigen-attached metal particles 410 to the measurement substrate 10A, it is preferable that the surface of the metal particles 210 of the antigen-attached metal particles 410 has the same charge sign as the surface of the metal body 120.

[0094] In this process, as long as the antigen-attached metal particles 410 are attached to the measurement substrate 10A, substances other than the antigen-attached metal particles 410 may be attached to the measurement substrate 10A.

[0095] In this process, as long as the antigen-attached metal particles 410 are bound to the measurement substrate 10A, it is not necessary to bind any substances other than the antigen-attached metal particles 410 to the measurement substrate 10A. For example, when the antigen-attached metal particles 410 are bound to the measurement substrate 10A, there may be substances such as metal particles 210 and antigens 310 that are not bound to the measurement substrate 10A. In this case, it is preferable to wash away the substances that are not bound to the measurement substrate 10A.

[0096] <Step to measure changes in reflection absorption characteristics> In this step, the reflection absorption characteristics due to localized surface plasmon resonance are measured from the first main surface 110a side of the substrate 110 of the measurement substrate 10A before and after the antigen-attached metal particles 410 are attached to the measurement substrate 10A, thereby measuring the change in reflection absorption characteristics before and after the antigen-attached metal particles 410 are attached to the measurement substrate 10A.

[0097] In this process, it is preferable to measure the change in resonance peak shift as a change in the reflection absorption characteristics before and after binding the antigen-attached metal particles 410 to the measurement substrate 10A. In this case, the change in reflection absorption characteristics before and after binding the antigen-attached metal particles 410 to the measurement substrate 10A can be easily quantified. The resonance peak shift may be a shift toward the longer wavelength side or a shift toward the shorter wavelength side.

[0098] In this process, the change in reflection absorption characteristics before and after binding the antigen-attached metal particles 410 to the measurement substrate 10A may be measured by measuring the change in resonance peak shift, measuring the change in resonance peak depth, or measuring the changes in both.

[0099] The method for measuring the reflection absorption characteristics due to localized surface plasmon resonance is not particularly limited. For example, first, incident light is shone from an optical fiber onto the first main surface 110a side of the substrate 110 of the measurement substrate 10A before and after binding the antigen-attached metal particles 410. Next, the reflected light reflected from the measurement substrate 10A is received by the optical fiber and guided to a spectrometer. The reflected light spectrally separated by the spectrometer is detected by a photodetector. Then, the reflection absorption characteristics are measured from the light detected by the photodetector.

[0100] In this process, the timing for measuring the reflection absorption characteristics by localized surface plasmon resonance on the measurement substrate 10A before the binding of the antigen-attached metal particles 410 is not particularly limited, as long as it is after the <step of preparing the measurement substrate> and before the <step of binding the antigen-attached metal particles to the measurement substrate>. Specifically, the timing for measuring the reflection absorption characteristics by localized surface plasmon resonance on the measurement substrate 10A before the binding of the antigen-attached metal particles 410 may be the same as the <step of preparing the measurement substrate>, or it may be after the <step of preparing the measurement substrate>, as long as it is before the <step of binding the antigen-attached metal particles to the measurement substrate>.

[0101] In this process, the timing for measuring the reflection absorption characteristics by localized surface plasmon resonance on the measurement substrate 10A after the binding of the antigen-attached metal particles 410 is not particularly limited as long as it is after the <step of binding the antigen-attached metal particles to the measurement substrate>. Specifically, the timing for measuring the reflection absorption characteristics by localized surface plasmon resonance on the measurement substrate 10A after the binding of the antigen-attached metal particles 410 may be the same as the <step of binding the antigen-attached metal particles to the measurement substrate>, or it may be after the <step of binding the antigen-attached metal particles to the measurement substrate>.

[0102] In Embodiment 1-1 of the first aspect of the antigen measurement method of the present invention, in the step of binding antigen-attached metal particles to a measurement substrate, the measurement sample 30 containing antigen-attached metal particles 410 is brought into contact with the measurement substrate 10A having a substrate 110 and a plurality of metal bodies 120, thereby binding the antigen-attached metal particles 410 to the measurement substrate 10A at a position on the first main surface 110a of the substrate 110 that is different from the metal bodies 120. As a result, in Embodiment 1-1 of the first aspect of the antigen measurement method of the present invention, the distance between the metals changes before and after binding the antigen-attached metal particles 410 to the measurement substrate 10A, with respect to the plurality of metals including the metal bodies 120 that were previously provided on the first main surface 110a of the substrate 110 and the antigen-attached metal particles 410 (metal particles 210) that are newly bound to the first main surface 110a of the substrate 110. Specifically, before the antigen-attached metal particles 410 are bound to the measurement substrate 10A, the distance between the metals corresponds to the distance between the metal bodies 120, whereas after the antigen-attached metal particles 410 are bound to the measurement substrate 10A, the distance between the metals corresponds to the distance between the metal body 120 and the antigen-attached metal particles 410 (metal particles 210).

[0103] Furthermore, in Embodiment 1-1 of the first aspect of the antigen measurement method of the present invention, in the step of measuring the change in reflection absorption characteristics, the change in reflection absorption characteristics before and after binding the antigen-attached metal particles 410 to the measurement substrate 10A is measured by measuring the reflection absorption characteristics due to localized surface plasmon resonance from the first main surface 110a side of the substrate 110 of the measurement substrate 10A before and after binding the antigen-attached metal particles 410 to the measurement substrate 10A. In Embodiment 1-1 of the first aspect of the antigen measurement method of the present invention, as described above, the distance between the metals, including the metal body 120 and the antigen-attached metal particles 410 (metal particles 210), changes before and after binding the antigen-attached metal particles 410 to the measurement substrate 10A. Therefore, the reflection absorption characteristics due to localized surface plasmon resonance near the surface of the metal change significantly before and after binding the antigen-attached metal particles 410 to the measurement substrate 10A. Thus, in Embodiment 1-1 of the first aspect of the antigen measurement method of the present invention, in order to significantly change the reflection absorption characteristics due to localized surface plasmon resonance near the surface of the metal body 120 and the antigen-attached metal particles 410 (metal particles 210), the distance between the metals is changed by attaching the antigen-attached metal particles 410 to the measurement substrate 10A, rather than changing the surface state of the metal body 120 by attaching the antigen 310 to the metal body 120.

[0104] As described above, in Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention, the change in reflection absorption characteristics before and after binding the antigen-attached metal particles 410 to the measurement substrate 10A is large, making it easier to detect with high sensitivity that the antigen-attached metal particles 410 have bound to the measurement substrate 10A, that is, that the antigen 310 of the antigen-attached metal particles 410 has bound to the measurement substrate 10A. Therefore, according to Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention, an antigen measurement method with high detection sensitivity for the antigen 310 can be realized.

[0105] The following describes a specific example of Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention described above, which includes a method for binding antigen-attached metal particles 410 to a measurement substrate 10A.

[0106] (Specific Example 1-1) Specific Example 1-1 relating to Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention has the following steps.

[0107] <Step to prepare the measurement substrate> In this step, a measurement substrate 10A is prepared, which further has one of biotin and avidin bonded to a position on the first main surface 110a of the substrate 110 that is different from the metal body 120.

[0108] Figure 5 is a schematic cross-sectional view showing an example of the step of preparing a measurement substrate in a specific example 1-1 relating to Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention.

[0109] In the example shown in Figure 5, a measurement substrate 10A is prepared which further has avidin 520 bonded to a position on the first main surface 110a of the substrate 110 that is different from the metal body 120.

[0110] Figure 6 is a schematic cross-sectional view showing another example of the step of preparing a measurement substrate in a specific example 1-1 relating to Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention.

[0111] As shown in Figure 6, in the example shown in Figure 5, avidin 520 may be bound to a position on the first main surface 110a of the substrate 110 that is different from the metal body 120, via biotin 510.

[0112] When both biotin 510 and avidin 520 are bound to a position on the first main surface 110a of the substrate 110 that is different from the metal body 120, the biotin 510 and avidin 520 may be located anywhere on the first main surface 110a of the substrate 110 that is different from the metal body 120, but it is preferable that one of them is located at the end of the substrate 110 opposite to the first main surface 110a (the upper end in Figure 6), rather than at the end of the substrate 110 on the first main surface 110a side (the lower end in Figure 6). In this case, it is preferable that one of the biotin 510 and avidin 520 located at the end of the substrate 110 opposite to the first main surface 110a in the above-mentioned area binds to the other biotin 510 and avidin 520 of the antigen-attached metal particle 410 in a later step.

[0113] The method for bonding biotin 510 to the first main surface 110a of the substrate 110 is not particularly limited and may be a known method. For example, if the first main surface 110a of the substrate 110 is a surface modified with 3-aminopropyltriethoxysilane (APTES) which constitutes the insulating layer 113, biotin 510 may be bonded to the first main surface 110a of the substrate 110 by performing a biotinylation reaction.

[0114] The method for bonding avidin 520 to the first main surface 110a of the substrate 110 is not particularly limited and may be a known method. For example, if the first main surface 110a of the substrate 110 is a surface modified with 3-aminopropyltriethoxysilane (APTES) which constitutes the insulating layer 113, biotin 510 may be bonded to the first main surface 110a of the substrate 110 by biotinization, and then avidin 520 (e.g., streptavidin) may be bonded to the biotin 510.

[0115] The following section describes the case using the measurement substrate 10A shown in Figure 5. The same applies to the case using the measurement substrate 10A shown in Figure 6.

[0116] <Steps for preparing measurement materials> In this step, measurement material 20 is prepared, which further contains biotin and the other avidin.

[0117] Figure 7 is a schematic cross-sectional view showing an example of the step of preparing measurement materials in a specific example 1-1 relating to Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention.

[0118] In the example shown in Figure 7, a measurement material 20 is prepared that includes antibody-coated metal particles 710, in which antibody 610 is bound to metal particles 210, and biotinylated antibody 810, in which biotin 510 is bound to antibody 620.

[0119] In the example shown in Figure 7, one type of material containing antibody-attached metal particles 710 and biotinylated antibody 810 integrally is prepared as the measurement material 20. However, two types of materials containing antibody-attached metal particles 710 and biotinylated antibody 810 separately may also be prepared.

[0120] The method for obtaining antibody-coated metal particles 710 is not particularly limited and may be a known method.

[0121] The method for obtaining biotinylated antibody 810 is not particularly limited and may be a known method.

[0122] Antibody 610 and antibody 620 are not particularly limited and may be anti-GST antibodies such as monoclonal antibodies or polyclonal antibodies.

[0123] <Process for preparing the sample for measurement> In this process, a sample for measurement 30 is prepared, which contains antigen-attached metal particles 410 to which biotin and avidin are bound.

[0124] Figure 8 is a schematic cross-sectional view showing an example of the process for preparing a sample for measurement in a specific example 1-1 of Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention.

[0125] In the example shown in Figure 8, a sample 30 for measurement is prepared, which includes antigen-attached metal particles 410 to which biotin 510 is bound. For example, by bringing a meter to be measured containing antigen 310 into contact with a measurement material 20 (see Figure 7), the antibody 610 of the antibody-attached metal particles 710 and the antibody 620 of the biotinylated antibody 810 are bound to the same antigen 310. In this case, antibodies 610 and 620 bind to different sites (epidopes) of antigen 310. This yields antigen-attached metal particles 410 to which biotin 510 is bound, preferably antigen-attached metal particles 410 to which biotin 510 is bound at the end.

[0126] If the sample 30 for measurement further contains substances other than the antigen-tagged metal particles 410, such as antibody-tagged metal particles 710 alone or biotinylated antibody 810 alone, the substances other than the antigen-tagged metal particles 410 may be removed by decantation or the like.

[0127] In the example shown in Figure 8, one type of material containing antibody-attached metal particles 710 and biotinylated antibody 810 integrally is prepared as the measurement material 20, and then a measurement sample 30 containing antigen-attached metal particles 410 with biotin 510 bound to them is prepared.

[0128] Unlike the example shown in Figure 8, if two types of materials containing antibody-attached metal particles 710 and biotinylated antibody 810 are prepared separately as the measurement material 20, a measurement sample 30 containing antigen-attached metal particles 410 with biotin 510 bound to them may be prepared, for example, as follows. First, the material containing the biotinylated antibody 810 is brought into contact with the object to be measured containing the antigen 310, thereby creating a material containing antigen-attached biotinylated antibody in which the antigen 310 is bound to the antibody 620 of the biotinylated antibody 810. Then, the material containing the remaining antibody-attached metal particles 710 is brought into contact with the material containing antigen-attached biotinylated antibody, thereby binding the antibody 610 of the antibody-attached metal particles 710 to the antigen 310 of the antigen-attached biotinylated antibody. This yields antigen-attached metal particles 410 with biotin 510 located at the end, similar to the example shown in Figure 8.

[0129] <Step of binding antigen-attached metal particles to the measurement substrate> In this step, the antigen-attached metal particles 410 are bound to the measurement substrate 10A by utilizing the binding reaction of biotin and avidin.

[0130] Figure 9 is a schematic cross-sectional view showing an example of the step of binding antigen-attached metal particles to a measurement substrate, in a specific example 1-1 of Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention.

[0131] In the example shown in Figure 9, the measurement sample 30 (see Figure 8) and the measurement substrate 10A (see Figure 5) are brought into contact, and the biotin 510 of the antigen-attached metal particle 410 is bound to the avidin 520 of the measurement substrate 10A by utilizing the binding reaction between biotin 510 and avidin 520. This causes the antigen-attached metal particle 410 to bind to the measurement substrate 10A at a position on the first main surface 110a of the substrate 110 that is different from the metal body 120. Although Figure 9 shows the antigen-attached metal particle 410 binding to one location on the measurement substrate 10A, the antigen-attached metal particle 410 may bind to multiple locations on the measurement substrate 10A.

[0132] In this process, when the antigen-attached metal particles 410 are bound to the measurement substrate 10A, substances such as antibody-attached metal particles 710 and biotinylated antibodies 810 that are not bound to the measurement substrate 10A may be present. In this case, it is preferable to wash away the substances that are not bound to the measurement substrate 10A.

[0133] <Step for measuring changes in reflection and absorption characteristics> This step is the same as described in Embodiment 1-1.

[0134] In the above steps, the relative positions of biotin 510 and avidin 520 may be reversed. That is, in the above steps, when the antigen-attached metal particles 410 are bound to the measurement substrate 10A by utilizing the binding reaction of biotin 510 and avidin 520, antigen-attached metal particles 410 with biotin 510 bound and measurement substrate 10A with avidin 520 bound are used, but antigen-attached metal particles 410 with avidin 520 bound and measurement substrate 10A with biotin 510 bound may also be used.

[0135] (Specific Example 1-2) Specific Example 1-2 relating to Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention has the following steps.

[0136] <Step to prepare the measurement substrate> In this step, a measurement substrate 10A is prepared, which further has antibodies bound to a position on the first main surface 110a of the substrate 110 that is different from the metal body 120.

[0137] Figure 10 is a schematic cross-sectional view showing an example of the step of preparing a measurement substrate in a specific example 1-2 relating to Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention.

[0138] In the example shown in Figure 10, a measurement substrate 10A is prepared which further has antibodies 620 bound to a position on the first main surface 110a of the substrate 110 that is different from the metal body 120.

[0139] Figure 11 is a schematic cross-sectional view showing another example of the step of preparing a measurement substrate in a specific example 1-2 relating to Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention.

[0140] As shown in Figure 11, in the example shown in Figure 10, the antibody 620 may be bound to a position on the first main surface 110a of the substrate 110 that is different from the metal body 120, via biotin 510 and avidin 520.

[0141] When the antibody 620 is bound to the first main surface 110a of the substrate 110 at a position different from the metal body 120, along with other substances, the antibody 620 may be located anywhere on the site where it is bound to the first main surface 110a of the substrate 110, but it is preferable that it is located at the end opposite to the first main surface 110a of the substrate 110, rather than at the end on the first main surface 110a side of the substrate 110.

[0142] The method for binding the antibody 620 to the first main surface 110a of the substrate 110 is not particularly limited and may be a known method.

[0143] The following section describes the case using the measurement substrate 10A shown in Figure 10. The same applies to the case using the measurement substrate 10A shown in Figure 11.

[0144] <Process for preparing measurement material> In this process, a measurement material containing metal particles 210 is prepared.

[0145] Figure 12 is a schematic cross-sectional view showing an example of the step of preparing measurement materials for a specific example 1-2 relating to Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention.

[0146] In the example shown in Figure 12, a measurement material 20 is prepared that includes antibody-coated metal particles 710, in which antibody 610 is bound to metal particles 210.

[0147] <Process for preparing a sample for measurement> In this process, a sample for measurement 30 containing antigen-attached metal particles 410 is prepared.

[0148] Figure 13 is a schematic cross-sectional view showing an example of the process for preparing a sample for measurement, in specific example 1-2 of Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention.

[0149] In the example shown in Figure 13, a measurement sample 30 is prepared, which includes antigen-attached metal particles 410 in which the antigen 310 is bound to the metal particles 210. For example, by bringing a measurement target containing the antigen 310 into contact with the measurement material 20 (see Figure 12), the antigen 310 is bound to the antibody 610 of the antibody-attached metal particles 710. This yields antigen-attached metal particles 410 in which the antigen 310 is bound to the metal particles 210 via the antibody 610.

[0150] If the sample 30 for measurement further contains substances other than the antigen-tagged metal particles 410, such as antibody-tagged metal particles 710 alone, the substances other than the antigen-tagged metal particles 410 may be removed by decantation or the like.

[0151] <Step of binding antigen-attached metal particles to the measurement substrate> In this step, antigen-attached metal particles 410 are bound to the measurement substrate 10A by utilizing the binding reaction between the antigen and the antibody.

[0152] Figure 14 is a schematic cross-sectional view showing an example of the step of binding antigen-attached metal particles to a measurement substrate, in a specific example 1-2 relating to Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention.

[0153] In the example shown in Figure 14, the measurement sample 30 (see Figure 13) and the measurement substrate 10A (see Figure 10) are brought into contact, and the binding reaction between the antigen 310 and the antibody 620 is utilized to bind the antigen 310 of the antigen-attached metal particle 410 to the antibody 620 of the measurement substrate 10A. This causes the antigen-attached metal particle 410 to bind to the measurement substrate 10A at a position on the first main surface 110a of the substrate 110 that is different from the metal body 120. Although Figure 14 shows the antigen-attached metal particle 410 binding to one location on the measurement substrate 10A, the antigen-attached metal particle 410 may bind to multiple locations on the measurement substrate 10A.

[0154] In this process, when the antigen-attached metal particles 410 are bound to the measurement substrate 10A, substances such as antibody-attached metal particles 710 that are not bound to the measurement substrate 10A may be present. In this case, it is preferable to wash away the substances that are not bound to the measurement substrate 10A.

[0155] <Step for measuring changes in reflection and absorption characteristics> This step is the same as described in Embodiment 1-1.

[0156] In the above, specific examples 1-1 and 1-2 of Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention have been described individually. However, in Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention, specific examples 1-1 and 1-2 may be combined. Specifically, in the <Step of preparing the measurement substrate>, a measurement substrate 10A may be prepared that further includes one of biotin 510 and avidin 520 bound to a position on the first main surface 110a of the substrate 110 that is different from the metal body 120 (Specific Example 1-1), and an antibody 620 bound to a position on the first main surface 110a of the substrate 110 that is different from the metal body 120 (Specific Example 1-2). In the <Step of preparing the measurement sample>, an antigen-coated metal particle 410 in which the other of biotin 510 and avidin 520 is bound (Specific Example 1-1), and biotin 510 and avidin A sample 30 for measurement may be prepared including antigen-attached metal particles 410 in a state where 520 is not bound (Specific Example 1-2), and in the step of binding antigen-attached metal particles to the measurement substrate, the antigen-attached metal particles 410 may be bound to the measurement substrate 10A by utilizing the binding reaction of biotin 510 and avidin 520 (Specific Example 1-1), and the antigen-attached metal particles 410 may be bound to the measurement substrate 10A by utilizing the binding reaction of antigen 310 and antibody 620 (Specific Example 1-2), both of which may be performed on the same measurement substrate 10A.

[0157] [Embodiment 1-2] In Embodiment 1-2 of the first embodiment of the antigen measurement method of the present invention, the substrate is an insulating substrate. Except for this point, Embodiment 1-2 of the first embodiment of the antigen measurement method of the present invention is the same as Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention.

[0158] Embodiment 1-2 of the first aspect of the antigen measurement method of the present invention comprises the following steps.

[0159] <Steps for preparing the measurement substrate> Figure 15 is a schematic cross-sectional view showing an example of the steps for preparing the measurement substrate in Embodiment 1-2 of the first embodiment of the antigen measurement method of the present invention.

[0160] In this step, the measurement substrate 10A' shown in Figure 15 is prepared.

[0161] The measuring substrate 10A' has a substrate 110 which is an insulating substrate 111, and a plurality of metal bodies 120.

[0162] <Steps for preparing measurement materials> Figure 16 is a schematic cross-sectional view showing an example of the steps for preparing measurement materials in Embodiment 1-2 of the first embodiment of the antigen measurement method of the present invention.

[0163] This process is the same as that described in Embodiment 1-1.

[0164] <Steps for preparing a sample for measurement> Figure 17 is a schematic cross-sectional view showing an example of the steps for preparing a sample for measurement in Embodiment 1-2 of the first embodiment of the antigen measurement method of the present invention.

[0165] This process is the same as that described in Embodiment 1-1.

[0166] <Step of binding antigen-attached metal particles to a measurement substrate> Figure 18 is a schematic cross-sectional view showing an example of the step of binding antigen-attached metal particles to a measurement substrate in Embodiment 1-2 of the first embodiment of the antigen measurement method of the present invention.

[0167] This process is the same as described in Embodiment 1-1, except that the substrate 110 is an insulating substrate 111.

[0168] In Embodiments 1-1 and 1-2, the metal body 120 is shown to be in particulate form, but the metal body 120 is not limited to being particulate.

[0169] Figure 19 is a schematic cross-sectional view showing a modified measurement substrate in Embodiment 1-1 of the first embodiment of the antigen measurement method of the present invention.

[0170] In the measuring substrate 10A shown in Figure 19, the metal body 120 is columnar (pole-shaped). In this case, the insulating layer 113 may be provided not on the entire surface of the metal layer 112 opposite to the insulating substrate 111, but on a part of the surface of the metal layer 112 opposite to the insulating substrate 111, in this case, in a position sandwiched between the metal layer 112 and the metal body 120.

[0171] Figure 20 is a schematic cross-sectional view showing a modified example 1 of the measurement substrate in Embodiment 1-2 of the first embodiment of the antigen measurement method of the present invention.

[0172] In the measuring substrate 10A' shown in Figure 20, the metal body 120 has a shape that covers the surface of the columnar (pole-shaped) insulating layer 113.

[0173] Figure 21 is a schematic cross-sectional view showing a modified example 2 of the measurement substrate in Embodiment 1-2 of the first embodiment of the antigen measurement method of the present invention.

[0174] In the measuring substrate 10A' shown in Figure 21, the metal layer 112 is provided on the first main surface 110a of the substrate 110 in a region between the metal bodies 120 so as to be separate from the metal bodies 120. Except for this point, the measuring substrate 10A' shown in Figure 21 is the same as the measuring substrate 10A' shown in Figure 20.

[0175] Figure 22 is a schematic cross-sectional view showing a modified example 3 of the measurement substrate in Embodiment 1-2 of the first embodiment of the antigen measurement method of the present invention.

[0176] In the measuring substrate 10A' shown in Figure 22, the metal layer 112 is provided in the region between the metal bodies 120 on the first main surface 110a of the substrate 110 so as to be in contact with the metal bodies 120. Except for this point, the measuring substrate 10A' shown in Figure 22 is the same as the measuring substrate 10A' shown in Figure 21.

[0177] The above shows a cross-sectional view (for example, Figure 4) of the state in which the antigen-attached metal particles 410 are bound to the measurement substrate 10A by the <step of binding antigen-attached metal particles to the measurement substrate>. Below, a preferred example of a plan view of the state in which the antigen-attached metal particles 410 are bound to the measurement substrate 10A by the <step of binding antigen-attached metal particles to the measurement substrate> is shown.

[0178] Figure 23 is a schematic plan view showing a preferred example of antigen-attached metal particles bound to a measurement substrate.

[0179] As shown in Figure 23, in the measurement substrate 10A, if a plurality of metal bodies 120 are arranged two-dimensionally and regularly with spacing between them on the first main surface 110a of the substrate 110, in the <step of bonding antigen-attached metal particles to the measurement substrate>, it is preferable to bond the antigen-attached metal particles 410 to the measurement substrate 10A such that when a virtual polygon is defined with three or more adjacent metal bodies 120 as vertices when viewed from the thickness direction of the substrate 110, the metal particles 210 are located inside the virtual polygon. In this case, in the <step of bonding antigen-attached metal particles to the measurement substrate>, it is particularly preferable to bond the antigen-attached metal particles 410 to the measurement substrate 10A such that the metal particles 210 are located at the geometric center of the virtual polygon.

[0180] In the example shown in Figure 23, the multiple metal bodies 120 are arranged to have a six-fold symmetry. In the step of binding antigen-attached metal particles to the measurement substrate, when a virtual triangle T is defined with three adjacent metal bodies 120 as vertices when viewed from the thickness direction of the substrate 110, the antigen-attached metal particles 410 are bound to the measurement substrate 10A such that the metal particles 210 are located inside the virtual triangle T, in this case at the geometric center of the virtual triangle T.

[0181] In the step of binding antigen-attached metal particles to the measurement substrate, when binding the antigen-attached metal particles 410 to the measurement substrate 10A such that the metal particles 210 are located inside the virtual polygon, it is sufficient to position at least one metal particle 210 inside the same virtual polygon, and it is preferable to position one metal particle 210 inside the same virtual polygon. In this case, the amount of charge on the surface of the metal body 120 and the amount of charge on the metal particles 210 may be the same or different.

[0182] The first embodiment of the antigen measurement method of the present invention is not limited to the above-described form, and various applications and modifications can be made within the scope of the present invention. Furthermore, the above-described forms are illustrative, and it goes without saying that partial substitution or combination of the configurations shown in different forms is possible.

[0183] (1-2) First Embodiment of the Antigen Measurement Kit of the Present Invention The antigen measurement kit of the present invention, in a first embodiment, comprises a measuring substrate having a substrate having a first main surface and a second main surface opposite to each other in the thickness direction, a plurality of metal bodies provided at intervals from each other along the first main surface of the substrate, and a binding portion provided on the first main surface of the substrate at a position different from the metal bodies, and a measuring material containing metal particles, wherein the binding portion is a site for binding of antigen-attached metal particles in which an antigen is bound to the metal particles.

[0184] In the first embodiment of the antigen measurement kit of the present invention, the measurement substrate has a binding portion provided on the first main surface of the substrate at a position different from that of the metal body. Furthermore, in the first embodiment of the antigen measurement kit of the present invention, the binding portion is a site for binding of antigen-attached metal particles, in which an antigen is bound to metal particles in the measurement material. Therefore, by using the first embodiment of the antigen measurement kit of the present invention, antigen-attached metal particles can be bound to the binding portion of the measurement substrate. Thus, for example, by using the first embodiment of the antigen measurement kit of the present invention in the first embodiment of the antigen measurement method of the present invention, it becomes easier to detect with high sensitivity that antigen-attached metal particles have bound to the binding portion of the measurement substrate, that is, that the antigen of the antigen-attached metal particles has bound to the binding portion of the measurement substrate. Therefore, according to the first embodiment of the antigen measurement kit of the present invention, an antigen measurement kit that can be effectively used in the first embodiment of the antigen measurement method of the present invention can be realized.

[0185] A first embodiment of the antigen measurement kit of the present invention may be any of the following embodiments, for example: (Embodiment A11) An embodiment having a measurement substrate 10A shown in Figure 5 and a measurement material 20 shown in Figure 7 (Embodiment A12) An embodiment having a measurement substrate 10A shown in Figure 6 and a measurement material 20 shown in Figure 7 (Embodiment A13) An embodiment having a measurement substrate 10A shown in Figure 10 and a measurement material 20 shown in Figure 12 (Embodiment A14) An embodiment having a measurement substrate 10A shown in Figure 11 and a measurement material 20 shown in Figure 12

[0186] In embodiments A11 and A12, the bonding portion corresponds to one of the biotin 510 and avidin 520 bonded to a position on the first main surface 110a of the substrate 110 that is different from the metal body 120.

[0187] In embodiments A13 and A14, the binding portion corresponds to the antibody 620 bound to a position on the first main surface 110a of the substrate 110 that is different from the metal body 120.

[0188] Embodiments A11, A12, A13, and A14 are not limited to a configuration in which the substrate 110 includes an insulating substrate 111, a metal layer 112, and an insulating layer 113 in order from the second main surface 110b side to the first main surface 110a side. For example, as shown in Figure 15, the substrate 110 may be an insulating substrate 111.

[0189] Other matters in the first embodiment of the antigen measurement kit of the present invention are the same as those described in the first embodiment of the antigen measurement method of the present invention.

[0190] The first embodiment of the antigen measurement kit of the present invention is not limited to the above-described form, and various applications and modifications can be made within the scope of the present invention. Furthermore, the above-described forms are illustrative, and it goes without saying that partial substitution or combination of the configurations shown in different forms is possible.

[0191] (1-3) First Embodiment of the Antigen Measurement Substrate of the Invention The antigen measurement substrate of the invention, in the first embodiment of the first embodiment, comprises a substrate having a first main surface and a second main surface opposite to each other in the thickness direction, a plurality of metal bodies provided at intervals from each other along the first main surface of the substrate, and a bonding portion provided on the first main surface of the substrate at a position different from the metal bodies, wherein the bonding portion is a portion for binding antigen-attached metal particles in which an antigen is bound to the metal particles.

[0192] The first embodiment of the first aspect of the antigen measurement substrate of the present invention has a binding portion provided on the first main surface of the substrate at a position different from that of the metal body. Furthermore, in the first embodiment of the first aspect of the antigen measurement substrate of the present invention, the binding portion is a site for binding of antigen-attached metal particles, in which an antigen is bound to the metal particles. Therefore, by using the first embodiment of the first aspect of the antigen measurement substrate of the present invention, antigen-attached metal particles can be bound to the binding portion of the antigen measurement substrate. Thus, for example, in the first aspect of the antigen measurement method of the present invention, if the first embodiment of the first aspect of the antigen measurement substrate of the present invention is used as a measurement substrate, it becomes easier to detect with high sensitivity that antigen-attached metal particles have bound to the binding portion of the antigen measurement substrate, that is, that the antigen of the antigen-attached metal particles has bound to the binding portion of the antigen measurement substrate. Therefore, according to the first embodiment of the first aspect of the antigen measurement substrate of the present invention, an antigen measurement substrate that can be effectively used in the first aspect of the antigen measurement method of the present invention can be realized.

[0193] The first embodiment of the first aspect of the antigen measurement substrate of the present invention may be, for example, any of the following embodiments: (Embodiment A21) Measurement substrate 10A shown in Figure 5 (Embodiment A22) Measurement substrate 10A shown in Figure 6 (Embodiment A23) Measurement substrate 10A shown in Figure 10 (Embodiment A24) Measurement substrate 10A shown in Figure 11

[0194] In embodiments A21 and A22, the bonding portion corresponds to one of the biotin 510 and avidin 520 bonded to a position on the first main surface 110a of the substrate 110 that is different from the metal body 120.

[0195] In embodiments A23 and A24, the binding portion corresponds to the antibody 620 bound to a position on the first main surface 110a of the substrate 110 that is different from the metal body 120.

[0196] Embodiments A21, A22, A23, and A24 are not limited to a configuration in which the substrate 110 includes an insulating substrate 111, a metal layer 112, and an insulating layer 113 in order from the second main surface 110b side to the first main surface 110a side. For example, as shown in Figure 15, the substrate 110 may be an insulating substrate 111.

[0197] Other matters in the first embodiment of the first aspect of the antigen measurement substrate of the present invention are the same as those described in the first aspect of the antigen measurement method of the present invention.

[0198] The first embodiment of the first aspect of the antigen measurement substrate of the present invention is not limited to the above-described embodiment, and various applications and modifications can be made within the scope of the present invention. Furthermore, the above embodiments are illustrative, and it goes without saying that partial substitution or combination of the configurations shown in different embodiments is possible.

[0199] (1-4) Second Embodiment of the First Embodiment of the Antigen Measurement Substrate of the Present Invention The antigen measurement substrate of the present invention, in the second embodiment of the first embodiment, is characterized by comprising: a substrate having a first main surface and a second main surface that are opposite to each other in the thickness direction; a plurality of metal bodies provided at intervals from each other along the first main surface of the substrate; binding portions provided on the first main surface of the substrate at positions different from the metal bodies; and antigen-attached metal particles bound to the binding portions in a state in which the antigen is bound to the metal particles.

[0200] The second embodiment of the first aspect of the antigen measurement substrate of the present invention has a binding portion provided at a position different from the metal body on the first main surface of the substrate, and an antigen-attached metal particle bound to the binding portion, with the antigen already bound to the metal particle. In the second embodiment of the first aspect of the antigen measurement substrate of the present invention, unlike the first embodiment of the first aspect of the antigen measurement substrate of the present invention, the antigen-attached metal particle is already bound to the binding portion. However, if there are binding portions to which the antigen-attached metal particle is not bound, in addition to the binding portion to which the antigen-attached metal particle is already bound, the antigen-attached metal particle can be bound to the binding portion to which the antigen-attached metal particle is not bound. Therefore, for example, if the second embodiment of the first aspect of the antigen measurement substrate of the present invention is used as a measurement substrate in the first aspect of the antigen measurement method of the present invention, it becomes easier to detect with high sensitivity that the antigen-attached metal particle has bound to a binding portion to which the antigen-attached metal particle is not bound, that is, that the antigen of the antigen-attached metal particle has bound to a binding portion to which the antigen-attached metal particle is not bound. Therefore, according to the second embodiment of the first aspect of the antigen measurement substrate of the present invention, an antigen measurement substrate that can be effectively used in the first aspect of the antigen measurement method of the present invention can be realized.

[0201] A second embodiment of the first embodiment of the antigen measurement substrate of the present invention may be any of the following embodiments, for example: (Embodiment A31) A substrate in which the antigen-attached metal particles 410 shown in Figure 8 are bound to the measurement substrate 10A shown in Figure 5 (the substrate shown in Figure 9) (Embodiment A32) A substrate in which the antigen-attached metal particles 410 shown in Figure 8 are bound to the measurement substrate 10A shown in Figure 6 (Embodiment A33) A substrate in which the antigen-attached metal particles 410 shown in Figure 13 are bound to the measurement substrate 10A shown in Figure 10 (the substrate shown in Figure 14) (Embodiment A34) A substrate in which the antigen-attached metal particles 410 shown in Figure 13 are bound to the measurement substrate 10A shown in Figure 11

[0202] In embodiments A31 and A32, the binding portion corresponds to one of biotin 510 and avidin 520 bound to a position on the first main surface 110a of the substrate 110 that is different from the metal body 120. In embodiments A31 and A32, the antigen-attached metal particle 410 is an antigen-attached metal particle in which the antibody 610 of the antibody-attached metal particle 710 and the antibody 620 of the biotinylated antibody 810 are bound to the same antigen 310.

[0203] In embodiments A33 and A34, the binding portion corresponds to the antibody 620 bound to a position on the first main surface 110a of the substrate 110 that is different from the metal body 120. In embodiments A33 and A34, the antigen-attached metal particle 410 is an antigen-attached metal particle in which the antigen 310 is bound to the metal particle 210 via the antibody 610.

[0204] Embodiments A31, A32, A33, and A34 are not limited to a configuration in which the substrate 110 includes an insulating substrate 111, a metal layer 112, and an insulating layer 113 in order from the second main surface 110b side to the first main surface 110a side. For example, as shown in Figure 15, the substrate 110 may be an insulating substrate 111.

[0205] Other matters in the second embodiment of the first embodiment of the antigen measurement substrate of the present invention are the same as those described in the first embodiment of the antigen measurement method of the present invention.

[0206] The second embodiment of the first aspect of the antigen measurement substrate of the present invention is not limited to the above-described embodiment, and various applications and modifications can be made within the scope of the present invention. Furthermore, the above embodiments are illustrative, and it goes without saying that partial substitution or combination of the configurations shown in different embodiments is possible.

[0207] (2-1) Second aspect of the antigen measurement method of the present invention The antigen measurement method of the present invention, in a second aspect, has a substrate having a first main surface and a second main surface facing each other in the thickness direction, and the substrate comprises the steps of preparing a measurement substrate which includes, in order from the second main surface side toward the first main surface side, an insulating substrate, a metal layer provided on the surface of the insulating substrate, and an insulating layer provided on the surface of the metal layer opposite to the insulating substrate, the steps of preparing a measurement material which includes metal particles, and bringing a measurement target object which includes an antigen into contact with the measurement material which includes the metal particles The method is characterized by comprising: a step of preparing a measurement sample containing antigen-bound metal particles; a step of bringing the measurement sample and the measurement substrate into contact so that the antigen-bound metal particles are bound to the first main surface of the substrate; and a step of measuring the change in the reflection absorption characteristics before and after the binding of the antigen-bound metal particles to the measurement substrate by measuring the reflection absorption characteristics by localized surface plasmon resonance from the first main surface side of the substrate before and after the binding of the antigen-bound metal particles.

[0208] In a second aspect of the antigen measurement method of the present invention, in the step of binding antigen-attached metal particles to a measurement substrate, the measurement sample containing the antigen-attached metal particles is brought into contact with the measurement substrate having the substrate, thereby binding the antigen-attached metal particles to the measurement substrate on the first main surface of the substrate. As a result, in the second aspect of the antigen measurement method of the present invention, the surface state of the measurement substrate changes before and after binding the antigen-attached metal particles to the measurement substrate.

[0209] Furthermore, in the second aspect of the antigen measurement method of the present invention, in the step of measuring the change in reflection absorption characteristics, the change in reflection absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate is measured by measuring the reflection absorption characteristics due to localized surface plasmon resonance from the first main surface side of the measurement substrate before and after binding the antigen-attached metal particles to the measurement substrate. In the second aspect of the antigen measurement method of the present invention, as described above, the surface state of the measurement substrate changes before and after binding the antigen-attached metal particles to the measurement substrate, so the reflection absorption characteristics due to localized surface plasmon resonance near the surface of the measurement substrate change significantly before and after binding the antigen-attached metal particles to the measurement substrate. Specifically, in the second aspect of the antigen measurement method of the present invention, the reflection absorption characteristics due to localized surface plasmon resonance near the surface of the measurement substrate are not detected before binding the antigen-attached metal particles to the measurement substrate, but are detected after binding the antigen-attached metal particles to the measurement substrate. Thus, in the second aspect of the antigen measurement method of the present invention, the surface state of the measurement substrate is changed by binding antigen-attached metal particles to the measurement substrate in order to significantly change the reflection absorption characteristics due to localized surface plasmon resonance near the surface of the measurement substrate.

[0210] As described above, in the second embodiment of the antigen measurement method of the present invention, the change in reflection and absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate becomes large, making it easier to detect with high sensitivity that the antigen-attached metal particles have bound to the measurement substrate, that is, that the antigen on the antigen-attached metal particles has bound to the measurement substrate. Therefore, according to the second embodiment of the antigen measurement method of the present invention, an antigen measurement method with high antigen detection sensitivity can be realized.

[0211] In the second aspect of the antigen measurement method of the present invention, the change in reflection absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate is large, so the reproducibility of antigen detection tends to be higher compared to the case where the change in reflection absorption characteristics is small.

[0212] In the second aspect of the antigen measurement method of the present invention, the change in reflection and absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate is significant. Therefore, even if the performance (resolution) of the spectrometer used to measure the reflection and absorption characteristics is not high, for example, sensing that is sufficiently usable as a sensor such as a biosensor can be performed.

[0213] By utilizing the second aspect of the antigen measurement method of the present invention, it is possible to estimate the magnitude of, for example, the concentration of the antigen in the sample to be measured, based on the magnitude of the change in the reflectance and absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate.

[0214] A conventional method for measuring antigens is immunochromatography. In immunochromatography, a complex formed by the binding of the antigen in the sample to a metal colloid-labeled antibody binds to a capture antibody, and the antigen is detected by utilizing the resulting color reaction of the metal colloid. However, because immunochromatography relies on this color reaction, it is difficult to quantify the concentration of the antigen in the sample.

[0215] In contrast, the second aspect of the antigen measurement method of the present invention does not utilize a color reaction like that of immunochromatography, but rather detects the antigen by utilizing the change in reflection absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate. Therefore, in the second aspect of the antigen measurement method of the present invention, by measuring, for example, the change in resonance peak shift as the change in reflection absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate, it is possible not only to quantify the change in reflection absorption characteristics before and after binding the antigen-attached metal particles to the measurement substrate, but also to quantify the concentration of the antigen in the measurement sample.

[0216] The following describes an embodiment of the second aspect of the antigen measurement method of the present invention.

[0217] [Embodiment 2-1] Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention has the following steps.

[0218] <Steps for preparing the measurement substrate> Figure 24 is a schematic cross-sectional view showing an example of the steps for preparing the measurement substrate in Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention.

[0219] In this step, the measurement substrate 10B shown in Figure 24 is prepared.

[0220] The measuring substrate 10B has a substrate 110.

[0221] The substrate 110 has a first main surface 110a and a second main surface 110b that are opposite to each other in the thickness direction (vertical direction in Figure 24).

[0222] The substrate 110 includes, in order from the second main surface 110b side toward the first main surface 110a side, an insulating substrate 111, a metal layer 112 provided on the surface of the insulating substrate 111, and an insulating layer 113 provided on the surface of the metal layer 112 opposite to the insulating substrate 111.

[0223] In the example shown in Figure 24, the surface of the insulating layer 113 opposite to the metal layer 112 constitutes the first main surface 110a of the substrate 110, and the surface of the insulating substrate 111 opposite to the metal layer 112 constitutes the second main surface 110b of the substrate 110.

[0224] <Steps for preparing measurement materials> Figure 25 is a schematic cross-sectional view showing an example of the steps for preparing measurement materials in Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention.

[0225] In this step, the measuring material 20 shown in Figure 25 is prepared.

[0226] The measuring material 20 contains metal particles 210.

[0227] The measuring material 20 may further include, for example, a dispersion medium 220 for dispersing metal particles 210.

[0228] <Steps for preparing a sample for measurement> Figure 26 is a schematic cross-sectional view showing an example of the steps for preparing a sample for measurement in Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention.

[0229] In this process, the measurement sample 30 shown in Figure 26 is prepared. Specifically, it is done as follows:

[0230] First, the object to be measured, which contains the antigen 310, is brought into contact with the measurement material 20 (see Figure 25).

[0231] Then, by bringing the object to be measured containing the antigen 310 into contact with the measurement material 20, a measurement sample 30 is prepared, which includes antigen-attached metal particles 410 in which the antigen 310 is bound to the metal particles 210, as shown in Figure 26. In the example shown in Figure 26, a measurement sample 30 is prepared in which the antigen-attached metal particles 410 are present in the dispersion medium 220.

[0232] <Step of binding antigen-attached metal particles to a measurement substrate> Figure 27 is a schematic cross-sectional view showing an example of the step of binding antigen-attached metal particles to a measurement substrate in Embodiment 2-1 of the second aspect of the antigen measurement method of the present invention.

[0233] In this process, the antigen-coated metal particles 410 are bound to the measurement substrate 10B. Specifically, this is done as follows.

[0234] First, the measurement sample 30 (see Figure 26) and the measurement substrate 10B (see Figure 24) are brought into contact.

[0235] Then, by bringing the measurement sample 30 and the measurement substrate 10B into contact, the antigen-attached metal particles 410 are bonded to the measurement substrate 10B on the first main surface 110a of the substrate 110, as shown in Figure 27.

[0236] <Step to measure changes in reflection absorption characteristics> In this step, the change in reflection absorption characteristics before and after bonding the antigen-attached metal particles 410 to the measurement substrate 10B is measured by measuring the reflection absorption characteristics due to localized surface plasmon resonance from the first main surface 110a side of the substrate 110 of the measurement substrate 10B before and after bonding the antigen-attached metal particles 410 to the measurement substrate 10B.

[0237] Other aspects of the above process are the same as those described in Embodiment 1-1.

[0238] In Embodiment 2-1 of the second aspect of the antigen measurement method of the present invention, in the step of binding antigen-attached metal particles to a measurement substrate, the measurement sample 30 containing antigen-attached metal particles 410 is brought into contact with the measurement substrate 10B having a substrate 110, thereby binding the antigen-attached metal particles 410 to the measurement substrate 10B on the first main surface 110a of the substrate 110. As a result, in Embodiment 2-1 of the second aspect of the antigen measurement method of the present invention, the surface state of the measurement substrate 10B changes before and after binding the antigen-attached metal particles 410 to the measurement substrate 10B.

[0239] Furthermore, in Embodiment 2-1 of the second aspect of the antigen measurement method of the present invention, in the step of measuring the change in reflection absorption characteristics, the change in reflection absorption characteristics before and after binding the antigen-attached metal particles 410 to the measurement substrate 10B is measured by measuring the reflection absorption characteristics due to localized surface plasmon resonance from the first main surface 110a side of the substrate 110 of the measurement substrate 10B before and after binding the antigen-attached metal particles 410 to the measurement substrate 10B. In Embodiment 2-1 of the second aspect of the antigen measurement method of the present invention, as described above, the surface state of the measurement substrate 10B changes before and after binding the antigen-attached metal particles 410 to the measurement substrate 10B, so the reflection absorption characteristics due to localized surface plasmon resonance near the surface of the measurement substrate 10B change significantly before and after binding the antigen-attached metal particles 410 to the measurement substrate 10B. Specifically, in Embodiment 2-1 of the second aspect of the antigen measurement method of the present invention, the reflection absorption characteristics due to localized surface plasmon resonance near the surface of the measurement substrate 10B are not detected before the antigen-attached metal particles 410 are bound to the measurement substrate 10B, but are detected after the antigen-attached metal particles 410 are bound to the measurement substrate 10B. Thus, in Embodiment 2-1 of the second aspect of the antigen measurement method of the present invention, the surface state of the measurement substrate 10B is changed by binding the antigen-attached metal particles 410 to the measurement substrate 10B in order to significantly change the reflection absorption characteristics due to localized surface plasmon resonance near the surface of the measurement substrate 10B.

[0240] As described above, in Embodiment 2-1 of the second aspect of the antigen measurement method of the present invention, the change in reflection absorption characteristics before and after binding the antigen-attached metal particles 410 to the measurement substrate 10B becomes large, making it easier to detect with high sensitivity that the antigen-attached metal particles 410 have bound to the measurement substrate 10B, that is, that the antigen 310 of the antigen-attached metal particles 410 has bound to the measurement substrate 10B. Therefore, according to Embodiment 2-1 of the second aspect of the antigen measurement method of the present invention, an antigen measurement method with high detection sensitivity for the antigen 310 can be realized.

[0241] The following describes a specific example of Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention described above, which includes a method for binding antigen-attached metal particles 410 to a measurement substrate 10B.

[0242] (Specific Example 2-1) Specific Example 2-1 relating to Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention has the following steps.

[0243] <Step to prepare the measurement substrate> In this step, a measurement substrate 10B is prepared, which further has one of biotin and avidin bonded to the first main surface 110a of the substrate 110.

[0244] Figure 28 is a schematic cross-sectional view showing an example of the step of preparing a measurement substrate in a specific example 2-1 of Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention.

[0245] In the example shown in Figure 28, a measurement substrate 10B is prepared, which further has avidin 520 bonded to the first main surface 110a of the substrate 110.

[0246] Figure 29 is a schematic cross-sectional view showing another example of the step of preparing a measurement substrate in a specific example 2-1 of Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention.

[0247] As shown in Figure 29, in the example shown in Figure 28, avidin 520 may be bound to the first main surface 110a of the substrate 110 via biotin 510.

[0248] The following section describes the case using the measurement substrate 10B shown in Figure 28. The same applies to the case using the measurement substrate 10B shown in Figure 29.

[0249] <Steps for preparing measurement materials> In this step, measurement material 20 is prepared, which further contains biotin and the other avidin.

[0250] Figure 30 is a schematic cross-sectional view showing an example of the step of preparing measurement materials in a specific example 2-1 of Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention.

[0251] In the example shown in Figure 30, a measurement material 20 is prepared that includes antibody-coated metal particles 710, in which antibody 610 is bound to metal particles 210, and biotinylated antibody 810, in which biotin 510 is bound to antibody 620.

[0252] <Process for preparing the sample for measurement> In this process, a sample for measurement 30 is prepared, which contains antigen-attached metal particles 410 to which biotin and avidin are bound.

[0253] Figure 31 is a schematic cross-sectional view showing an example of the process for preparing a sample for measurement, in specific example 2-1 of Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention.

[0254] In the example shown in Figure 31, a measurement sample 30 is prepared containing antigen-attached metal particles 410 to which biotin 510 is bound. For example, by bringing a meter containing antigen 310 into contact with a measurement material 20 (see Figure 30), the antibody 610 of the antibody-attached metal particles 710 and the antibody 620 of the biotinylated antibody 810 are bound to the same antigen 310. At this time, antibodies 610 and 620 bind to different sites (epidopes) of the antigen 310. This yields antigen-attached metal particles 410 to which biotin 510 is bound, preferably antigen-attached metal particles 410 to which biotin 510 is bound at the end.

[0255] <Step of binding antigen-attached metal particles to the measurement substrate> In this step, the antigen-attached metal particles 410 are bound to the measurement substrate 10B by utilizing the binding reaction of biotin and avidin.

[0256] Figure 32 is a schematic cross-sectional view showing an example of the step of binding antigen-attached metal particles to a measurement substrate, according to a specific example 2-1 of Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention.

[0257] In the example shown in Figure 32, the measurement sample 30 (see Figure 31) and the measurement substrate 10B (see Figure 28) are brought into contact, and the biotin 510 of the antigen-attached metal particle 410 is bound to the avidin 520 of the measurement substrate 10B by utilizing the binding reaction between biotin 510 and avidin 520. This causes the antigen-attached metal particle 410 to bind to the measurement substrate 10B on the first main surface 110a of the substrate 110. Although Figure 32 shows the antigen-attached metal particle 410 binding to one location on the measurement substrate 10B, the antigen-attached metal particle 410 may bind to multiple locations on the measurement substrate 10B.

[0258] <Step for measuring changes in reflection and absorption characteristics> This step is the same as the one described in Embodiment 2-1.

[0259] Other aspects of the above process are the same as those explained in Specific Example 1-1.

[0260] (Specific Example 2-2) Specific Example 2-2 relating to Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention has the following steps.

[0261] <Step to prepare the measurement substrate> In this step, a measurement substrate 10B is prepared, which further has antibodies bound to the first main surface 110a of the substrate 110.

[0262] Figure 33 is a schematic cross-sectional view showing an example of the step of preparing a measurement substrate in a specific example 2-2 relating to Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention.

[0263] In the example shown in Figure 33, a measurement substrate 10B is prepared, which further has an antibody 620 bound to the first main surface 110a of the substrate 110.

[0264] Figure 34 is a schematic cross-sectional view showing another example of the step of preparing a measurement substrate in a specific example 2-2 relating to Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention.

[0265] As shown in Figure 34, in the example shown in Figure 33, the antibody 620 may be bound to the first main surface 110a of the substrate 110 via biotin 510 and avidin 520.

[0266] The following section describes the case using the measurement substrate 10B shown in Figure 33. The same applies to the case using the measurement substrate 10B shown in Figure 34.

[0267] <Process for preparing measurement material> In this process, a measurement material containing metal particles 210 is prepared.

[0268] Figure 35 is a schematic cross-sectional view showing an example of the step of preparing measurement materials in a specific example 2-2 relating to Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention.

[0269] In the example shown in Figure 35, a measurement material 20 is prepared that includes antibody-coated metal particles 710, in which antibody 610 is bound to metal particles 210.

[0270] <Process for preparing a sample for measurement> In this process, a sample for measurement 30 containing antigen-attached metal particles 410 is prepared.

[0271] Figure 36 is a schematic cross-sectional view showing an example of the process for preparing a sample for measurement, in specific example 2-2 of Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention.

[0272] In the example shown in Figure 36, a measurement sample 30 is prepared, which includes antigen-attached metal particles 410 in which the antigen 310 is bound to the metal particles 210. For example, by bringing a measurement target containing the antigen 310 into contact with the measurement material 20 (see Figure 35), the antigen 310 is bound to the antibody 610 of the antibody-attached metal particles 710. This yields antigen-attached metal particles 410 in which the antigen 310 is bound to the metal particles 210 via the antibody 610.

[0273] <Step of binding antigen-attached metal particles to the measurement substrate> In this step, antigen-attached metal particles 410 are bound to the measurement substrate 10B by utilizing the binding reaction between the antigen and the antibody.

[0274] Figure 37 is a schematic cross-sectional view showing an example of the step of binding antigen-attached metal particles to a measurement substrate, in a specific example 2-2 relating to Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention.

[0275] In the example shown in Figure 37, the measurement sample 30 (see Figure 36) and the measurement substrate 10B (see Figure 33) are brought into contact, and the antigen 310 of the antigen-attached metal particle 410 is bound to the antibody 620 of the measurement substrate 10B by utilizing the binding reaction between the antigen 310 and the antibody 620. As a result, the antigen-attached metal particle 410 is bound to the measurement substrate 10B on the first main surface 110a of the substrate 110. Although Figure 37 shows the antigen-attached metal particle 410 binding to one location on the measurement substrate 10B, the antigen-attached metal particle 410 may bind to multiple locations on the measurement substrate 10B.

[0276] <Step for measuring changes in reflection and absorption characteristics> This step is the same as the one described in Embodiment 2-1.

[0277] Other aspects of the above process are the same as those explained in Specific Example 1-2.

[0278] In the above, specific examples 2-1 and 2-2 of Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention have been described individually. However, in Embodiment 2-1 of the second embodiment of the antigen measurement method of the present invention, specific examples 2-1 and 2-2 may be combined. Specifically, in the <step of preparing the measurement substrate>, a measurement substrate 10B may be prepared that further includes one of biotin 510 and avidin 520 bound to the first main surface 110a of the substrate 110 (Specific Example 2-1), and an antibody 620 bound to the first main surface 110a of the substrate 110 (Specific Example 2-2). In the <step of preparing the measurement sample>, an antigen-attached metal particle 410 in which the other of biotin 510 and avidin 520 is bound (Specific Example 2-1), and an antibody in which biotin 510 and avidin 520 are not bound. A sample 30 for measurement may be prepared containing antigen-attached metal particles 410 (Specific Example 2-2), and in the step of binding antigen-attached metal particles to the measurement substrate, the antigen-attached metal particles 410 may be bound to the measurement substrate 10B by utilizing the binding reaction of biotin 510 and avidin 520 (Specific Example 2-1), and the antigen-attached metal particles 410 may be bound to the measurement substrate 10B by utilizing the binding reaction of antigen 310 and antibody 620 (Specific Example 2-2), both of which may be performed on the same measurement substrate 10B.

[0279] The second embodiment of the antigen measurement method of the present invention is not limited to the above-described form, and various applications and modifications can be made within the scope of the present invention. Furthermore, the above-described forms are illustrative, and it goes without saying that partial substitution or combination of the configurations shown in different forms is possible.

[0280] (2-2) Second Embodiment of the Antigen Measurement Kit of the Present Invention The antigen measurement kit of the present invention, in a second embodiment, comprises a substrate having a first main surface and a second main surface opposite to each other in the thickness direction, and a bonding portion provided on the first main surface of the substrate, wherein the substrate comprises a measuring substrate including, in order from the second main surface side toward the first main surface side, an insulating substrate, a metal layer provided on the surface of the insulating substrate, and an insulating layer provided on the surface of the metal layer opposite to the insulating substrate, and a measuring material containing metal particles, wherein the bonding portion is a portion for binding of antigen-attached metal particles in which an antigen is bound to the metal particles.

[0281] In a second embodiment of the antigen measurement kit of the present invention, the measurement substrate has a binding portion provided on the first main surface of the substrate. Furthermore, in the second embodiment of the antigen measurement kit of the present invention, the binding portion is a site for binding of antigen-attached metal particles, in which an antigen is bound to metal particles in the measurement material. Therefore, by using the second embodiment of the antigen measurement kit of the present invention, antigen-attached metal particles can be bound to the binding portion of the measurement substrate. Thus, for example, by using the second embodiment of the antigen measurement kit of the present invention in a second embodiment of the antigen measurement method of the present invention, it becomes easier to detect with high sensitivity that antigen-attached metal particles have bound to the binding portion of the measurement substrate, that is, that the antigen of the antigen-attached metal particles has bound to the binding portion of the measurement substrate. Therefore, according to the second embodiment of the antigen measurement kit of the present invention, an antigen measurement kit that can be effectively used in the second embodiment of the antigen measurement method of the present invention can be realized.

[0282] A second embodiment of the antigen measurement kit of the present invention may be any of the following embodiments, for example: (Embodiment B11) An embodiment having a measurement substrate 10B shown in Figure 28 and a measurement material 20 shown in Figure 30 (Embodiment B12) An embodiment having a measurement substrate 10B shown in Figure 29 and a measurement material 20 shown in Figure 30 (Embodiment B13) An embodiment having a measurement substrate 10B shown in Figure 33 and a measurement material 20 shown in Figure 35 (Embodiment B14) An embodiment having a measurement substrate 10B shown in Figure 34 and a measurement material 20 shown in Figure 35

[0283] In embodiments B11 and B12, the bonding portion corresponds to one of the biotin 510 and avidin 520 bonded to the first main surface 110a of the substrate 110.

[0284] In embodiments B13 and B14, the binding portion corresponds to the antibody 620 bound to the first main surface 110a of the substrate 110.

[0285] Other matters in the second embodiment of the antigen measurement kit of the present invention are the same as those described in the second embodiment of the antigen measurement method of the present invention.

[0286] The second embodiment of the antigen measurement kit of the present invention is not limited to the above-described form, and various applications and modifications can be made within the scope of the present invention. Furthermore, the above-described forms are illustrative, and it goes without saying that partial substitution or combination of the configurations shown in different forms is possible.

[0287] (2-3) First Embodiment of the Second Aspect of the Antigen Measurement Substrate of the Present Invention The antigen measurement substrate of the present invention has a first main surface and a second main surface that are opposite to each other in the thickness direction, and comprises, in order from the second main surface side toward the first main surface side, an insulating substrate, a metal layer provided on the surface of the insulating substrate, an insulating layer provided on the surface of the metal layer opposite to the insulating substrate, and a bonding portion provided on the first main surface of the substrate, wherein the bonding portion is a portion for binding of antigen-attached metal particles in which an antigen is bound to a metal particle.

[0288] The first embodiment of the second embodiment of the antigen measurement substrate of the present invention has a binding portion provided on the first main surface of the substrate. Furthermore, in the first embodiment of the second embodiment of the antigen measurement substrate of the present invention, the binding portion is a site for binding of antigen-attached metal particles, in which an antigen is bound to a metal particle. Therefore, by using the first embodiment of the second embodiment of the antigen measurement substrate of the present invention, antigen-attached metal particles can be bound to the binding portion of the antigen measurement substrate. Thus, for example, in the second embodiment of the antigen measurement method of the present invention, if the first embodiment of the second embodiment of the antigen measurement substrate of the present invention is used as a measurement substrate, it becomes easier to detect with high sensitivity that antigen-attached metal particles have bound to the binding portion of the antigen measurement substrate, that is, that the antigen of the antigen-attached metal particles has bound to the binding portion of the antigen measurement substrate. Therefore, according to the first embodiment of the second embodiment of the antigen measurement substrate of the present invention, an antigen measurement substrate that can be effectively used in the second embodiment of the antigen measurement method of the present invention can be realized.

[0289] The first embodiment of the second aspect of the antigen measurement substrate of the present invention may be, for example, any of the following embodiments: (Embodiment B21) Measurement substrate 10B shown in Figure 28 (Embodiment B22) Measurement substrate 10B shown in Figure 29 (Embodiment B23) Measurement substrate 10B shown in Figure 33 (Embodiment B24) Measurement substrate 10B shown in Figure 34

[0290] In embodiments B21 and B22, the bonding portion corresponds to one of the biotin 510 and avidin 520 bonded to the first main surface 110a of the substrate 110.

[0291] In embodiments B23 and B24, the binding portion corresponds to the antibody 620 bound to the first main surface 110a of the substrate 110.

[0292] Other matters in the first embodiment of the second embodiment of the antigen measurement substrate of the present invention are the same as those described in the second embodiment of the antigen measurement method of the present invention.

[0293] The first embodiment of the second aspect of the antigen measurement substrate of the present invention is not limited to the above-described embodiment, and various applications and modifications can be made within the scope of the present invention. Furthermore, the above embodiments are illustrative, and it goes without saying that partial substitution or combination of the configurations shown in different embodiments is possible.

[0294] (2-4) Second Embodiment of the Antigen Measurement Substrate of the Invention The antigen measurement substrate of the invention, in the second embodiment of the second embodiment, has a first main surface and a second main surface that are opposite to each other in the thickness direction, and comprises, in order from the second main surface side toward the first main surface side, an insulating substrate, a metal layer provided on the surface of the insulating substrate, an insulating layer provided on the surface of the metal layer opposite to the insulating substrate, a bonding portion provided on the first main surface of the substrate, and antigen-attached metal particles bound to the bonding portion in a state in which the antigen is bound to the metal particles.

[0295] The second embodiment of the second aspect of the antigen measurement substrate of the present invention has a binding portion provided on a first main surface of the substrate, and an antigen-attached metal particle bound to the binding portion, with the antigen already bound to the metal particle. In the second embodiment of the second aspect of the antigen measurement substrate of the present invention, unlike the first embodiment, the antigen-attached metal particle is already bound to the binding portion. However, if there are binding portions to which the antigen-attached metal particle is not bound, in addition to the binding portion to which the antigen-attached metal particle is already bound, the antigen-attached metal particle can be bound to the binding portion to which the antigen-attached metal particle is not bound. Therefore, for example, if the second embodiment of the second aspect of the antigen measurement substrate of the present invention is used as a measurement substrate in the second aspect of the antigen measurement method of the present invention, it becomes easier to detect with high sensitivity that the antigen-attached metal particle has bound to a binding portion to which the antigen-attached metal particle is not bound, that is, that the antigen of the antigen-attached metal particle has bound to a binding portion to which the antigen-attached metal particle is not bound. Therefore, according to the second embodiment of the second aspect of the antigen measurement substrate of the present invention, an antigen measurement substrate that can be effectively used in the second aspect of the antigen measurement method of the present invention can be realized.

[0296] A second embodiment of the second aspect of the antigen measurement substrate of the present invention may be, for example, any of the following embodiments: (Embodiment B31) A substrate in which the antigen-attached metal particles 410 shown in Figure 31 are bound to the measurement substrate 10B shown in Figure 28 (the substrate shown in Figure 32) (Embodiment B32) A substrate in which the antigen-attached metal particles 410 shown in Figure 31 are bound to the measurement substrate 10B shown in Figure 29 (Embodiment B33) A substrate in which the antigen-attached metal particles 410 shown in Figure 36 are bound to the measurement substrate 10B shown in Figure 33 (the substrate shown in Figure 37) (Embodiment B34) A substrate in which the antigen-attached metal particles 410 shown in Figure 36 are bound to the measurement substrate 10B shown in Figure 34

[0297] In embodiments B31 and B32, the binding portion corresponds to one of biotin 510 and avidin 520 bound to the first main surface 110a of the substrate 110. In embodiments B31 and B32, the antigen-attached metal particle 410 is an antigen-attached metal particle in which the antibody 610 of the antibody-attached metal particle 710 and the antibody 620 of the biotinylated antibody 810 are bound to the same antigen 310.

[0298] In embodiments B33 and B34, the binding portion corresponds to the antibody 620 bound to the first main surface 110a of the substrate 110. In embodiments B33 and B34, the antigen-attached metal particle 410 is an antigen-attached metal particle in which the antigen 310 is bound to the metal particle 210 via the antibody 610.

[0299] Other matters in the second embodiment of the second aspect of the antigen measurement substrate of the present invention are the same as those described in the second aspect of the antigen measurement method of the present invention.

[0300] The second embodiment of the second aspect of the antigen measurement substrate of the present invention is not limited to the above-described embodiment, and various applications and modifications can be made within the scope of the present invention. Furthermore, the above embodiments are illustrative, and it goes without saying that partial substitution or combination of the configurations shown in different embodiments is possible.

[0301] This specification discloses the following:

[0302] <1> An antigen measurement method comprising the steps of: preparing a measuring substrate having a substrate having a first main surface and a second main surface opposite to each other in the thickness direction, and a plurality of metal bodies provided at intervals from each other along the first main surface of the substrate; preparing a measuring material containing metal particles; producing a measuring sample containing antigen-attached metal particles in which the antigen is bound to the metal particles by bringing a measuring object containing an antigen into contact with the measuring material; binding the antigen-attached metal particles to the measuring substrate at a position on the first main surface of the substrate different from the metal bodies by bringing the measuring sample and the measuring substrate into contact; and measuring the change in the reflection absorption characteristics before and after binding the antigen-attached metal particles to the measuring substrate by measuring the reflection absorption characteristics by localized surface plasmon resonance from the first main surface side of the substrate before and after binding the antigen-attached metal particles.

[0303] <2> The antigen measurement method according to <1>, wherein in the step of binding the antigen-attached metal particles to the measurement substrate, the antigen-attached metal particles are bound to the measurement substrate such that the shortest distance between the metal body and the metal particles is smaller than the shortest distance between the metal bodies.

[0304] <3> The antigen measurement method according to <1> or <2>, wherein in the step of preparing the above measurement substrate, the above measurement substrate is prepared having one of biotin and avidin bound to a position on the first main surface of the substrate different from the metal body, in the step of preparing the above measurement sample, the above measurement sample is prepared containing the above antigen-attached metal particles to which the other of biotin and avidin is bound, and in the step of binding the above antigen-attached metal particles to the above measurement substrate, the above antigen-attached metal particles are bound to the above measurement substrate by utilizing the binding reaction of biotin and avidin.

[0305] <4> The antigen measurement method according to any one of <1> to <3>, wherein in the step of preparing the measurement substrate, the measurement substrate is further having antibodies bound to a position on the first main surface of the substrate different from the metal body, and in the step of binding the antigen-attached metal particles to the measurement substrate, the antigen-attached metal particles are bound to the measurement substrate by utilizing the binding reaction between the antigen and the antibody.

[0306] <5> The antigen measurement method according to any one of <1> to <4>, wherein in the step of measuring the change in the reflection absorption characteristics, the amount of change in the resonance peak shift is measured as a change in the reflection absorption characteristics before and after bonding the antigen-attached metal particles to the measurement substrate.

[0307] <6> The antigen measurement method according to any one of <1> to <5>, wherein the substrate includes, in order from the second main surface side toward the first main surface side, an insulating substrate, a metal layer provided on the surface of the insulating substrate, and an insulating layer provided on the surface of the metal layer opposite to the insulating substrate.

[0308] <7> The antigen measurement method described in <6>, wherein the above metal layer is composed of gold.

[0309] <8> The antigen measurement method according to <6> or <7>, wherein the insulating layer is composed of a silane coupling agent.

[0310] <9> The antigen measurement method according to any one of <1> to <5>, wherein the substrate is an insulating substrate.

[0311] <10> The antigen measurement method according to any one of <1> to <9>, wherein the metal body is in particulate form.

[0312] <11> The antigen measurement method according to <10>, wherein the metal body is gold particles.

[0313] <12> The antigen measurement method according to any one of <1> to <11>, wherein the metal particles are gold particles.

[0314] <13> An antigen measurement kit comprising: a substrate having a first main surface and a second main surface opposite to each other in the thickness direction; a plurality of metal bodies provided at intervals from each other along the first main surface of the substrate; and a bonding portion provided on the first main surface of the substrate at a position different from the metal bodies; and a measurement material containing metal particles, wherein the bonding portion is a site for the binding of antigen-attached metal particles in which an antigen has been bound to the metal particles.

[0315] <14> An antigen measurement substrate comprising: a substrate having a first main surface and a second main surface opposite to each other in the thickness direction; a plurality of metal bodies provided at intervals from each other along the first main surface of the substrate; and a bonding portion provided on the first main surface of the substrate at a position different from the metal bodies, wherein the bonding portion is a site for binding of antigen-attached metal particles in which an antigen is bound to the metal particles.

[0316] <15> An antigen measurement substrate comprising: a substrate having a first main surface and a second main surface opposite to each other in the thickness direction; a plurality of metal bodies provided at intervals from each other along the first main surface of the substrate; a bonding portion provided on the first main surface of the substrate at a position different from the metal bodies; and antigen-attached metal particles bound to the bonding portion in a state in which the antigen is bound to the metal particles.

[0317] <16> An antigen measurement method characterized by comprising the steps of: preparing a measuring substrate having a substrate having a first main surface and a second main surface opposite to each other in the thickness direction, wherein the substrate includes, in order from the second main surface side toward the first main surface side, an insulating substrate, a metal layer provided on the surface of the insulating substrate, and an insulating layer provided on the surface of the metal layer opposite to the insulating substrate; preparing a measuring material containing metal particles; producing a measuring sample containing antigen-attached metal particles in which the antigen is bound to the metal particles by bringing a meter to be measured object containing an antigen into contact with the measuring material; binding the antigen-attached metal particles to the measuring substrate on the first main surface of the substrate by bringing the measuring sample and the measuring substrate into contact; and measuring the change in the reflection absorption characteristics before and after binding the antigen-attached metal particles to the measuring substrate by measuring the reflection absorption characteristics by localized surface plasmon resonance from the first main surface side of the substrate before and after binding the antigen-attached metal particles.

[0318] <17> An antigen measurement kit comprising: a substrate having a first main surface and a second main surface opposite to each other in the thickness direction; a bonding portion provided on the first main surface of the substrate, wherein the substrate comprises, in order from the second main surface side toward the first main surface side, an insulating substrate, a metal layer provided on the surface of the insulating substrate, and an insulating layer provided on the surface of the metal layer opposite to the insulating substrate; and a measurement material containing metal particles, wherein the bonding portion is a site for binding of antigen-attached metal particles in which an antigen is bound to the metal particles.

[0319] <18> An antigen measurement substrate having a first main surface and a second main surface opposite to each other in the thickness direction, and comprising, in order from the second main surface side toward the first main surface side, an insulating substrate, a metal layer provided on the surface of the insulating substrate, and an insulating layer provided on the surface of the metal layer opposite to the insulating substrate, and a bonding portion provided on the first main surface of the substrate, wherein the bonding portion is a portion for binding of antigen-attached metal particles in which an antigen is bound to a metal particle.

[0320] <19> An antigen measurement substrate having a first main surface and a second main surface opposite to each other in the thickness direction, and comprising, in order from the second main surface side toward the first main surface side, an insulating substrate, a metal layer provided on the surface of the insulating substrate, and an insulating layer provided on the surface of the metal layer opposite to the insulating substrate, a bonding portion provided on the first main surface of the substrate, and antigen-attached metal particles bonded to the bonding portion in a state where the antigen is bound to the metal particles.

[0321] 10A, 10A', 10B Measurement substrate 20 Measurement material 30 Measurement sample 110 Substrate 110a First main surface 110b Second main surface 111 Insulating substrate 112 Metal layer 113 Insulating layer 120 Metal body 210 Metal particles 220 Dispersion medium 310 Antigen 410 Antigen-coated metal particles 510 Biotin 520 Avidin 610, 620 Antibody 710 Antibody-coated metal particles 810 Biotinylated antibody D1 Shortest distance between metal bodies and metal particles D2 Shortest distance between metal bodies T Virtual triangle

Claims

1. An antigen measurement method comprising: preparing a measuring substrate having a substrate having a first main surface and a second main surface opposite to each other in the thickness direction, and a plurality of metal bodies provided at intervals from each other along the first main surface of the substrate; preparing a measuring material containing metal particles; producing a measuring sample containing antigen-attached metal particles in which the antigen is bound to the metal particles by bringing a meter to be measured object containing an antigen into contact with the measuring material; binding the antigen-attached metal particles to the measuring substrate at a position on the first main surface of the substrate different from the metal bodies by bringing the measuring sample and the measuring substrate into contact; and measuring the change in the reflection absorption characteristics before and after binding the antigen-attached metal particles to the measuring substrate by measuring the reflection absorption characteristics by localized surface plasmon resonance from the first main surface side of the substrate before and after binding the antigen-attached metal particles.

2. The antigen measurement method according to claim 1, wherein in the step of binding the antigen-attached metal particles to the measurement substrate, the antigen-attached metal particles are bound to the measurement substrate such that the shortest distance between the metal body and the metal particles is smaller than the shortest distance between the metal bodies.

3. The antigen measurement method according to claim 1 or 2, wherein in the step of preparing the measurement substrate, the measurement substrate is prepared having one of biotin and avidin bound to a position on the first main surface of the substrate different from the metal body; in the step of preparing the measurement sample, the measurement sample is prepared containing the antigen-attached metal particles to which the other of biotin and avidin is bound; and in the step of binding the antigen-attached metal particles to the measurement substrate, the antigen-attached metal particles are bound to the measurement substrate by utilizing the binding reaction of biotin and avidin.

4. The antigen measurement method according to any one of claims 1 to 3, wherein in the step of preparing the measurement substrate, the measurement substrate is prepared having an antibody bound to a position on the first main surface of the substrate different from the metal body, and in the step of binding the antigen-attached metal particles to the measurement substrate, the antigen-attached metal particles are bound to the measurement substrate by utilizing the binding reaction between the antigen and the antibody.

5. The antigen measurement method according to any one of claims 1 to 4, wherein in the step of measuring the change in the reflection absorption characteristics, the amount of change in the resonance peak shift is measured as a change in the reflection absorption characteristics before and after bonding the antigen-attached metal particles to the measurement substrate.

6. The antigen measurement method according to any one of claims 1 to 5, wherein the substrate includes, in order from the second main surface side toward the first main surface side, an insulating substrate, a metal layer provided on the surface of the insulating substrate, and an insulating layer provided on the surface of the metal layer opposite to the insulating substrate.

7. The antigen measurement method according to claim 6, wherein the metal layer is composed of gold.

8. The antigen measurement method according to claim 6 or 7, wherein the insulating layer is composed of a silane coupling agent.

9. The antigen measurement method according to any one of claims 1 to 5, wherein the substrate is an insulating substrate.

10. The antigen measurement method according to any one of claims 1 to 9, wherein the metal body is in the form of particulate matter.

11. The antigen measurement method according to claim 10, wherein the metal body is gold particles.

12. The antigen measurement method according to any one of claims 1 to 11, wherein the metal particles are gold particles.

13. An antigen measurement kit comprising: a substrate having a first main surface and a second main surface opposite to each other in the thickness direction; a plurality of metal bodies provided at intervals from each other along the first main surface of the substrate; and a bonding portion provided on the first main surface of the substrate at a position different from the metal bodies; and a measurement material containing metal particles, wherein the bonding portion is a site for the binding of antigen-laden metal particles in which an antigen has been bound to the metal particles.

14. An antigen measurement substrate comprising: a substrate having a first main surface and a second main surface opposite to each other in the thickness direction; a plurality of metal bodies provided at intervals from each other along the first main surface of the substrate; and a bonding portion provided on the first main surface of the substrate at a position different from the metal bodies, wherein the bonding portion is a site for binding antigen-attached metal particles in which an antigen is bound to the metal particles.

15. An antigen measurement substrate comprising: a substrate having a first main surface and a second main surface opposite to each other in the thickness direction; a plurality of metal bodies provided at intervals from each other along the first main surface of the substrate; a bonding portion provided on the first main surface of the substrate at a position different from the metal bodies; and antigen-attached metal particles bound to the bonding portion in a state where the antigen is bound to the metal particles.

16. An antigen measurement method characterized by comprising the steps of: preparing a measuring substrate having a substrate having a first main surface and a second main surface opposite to each other in the thickness direction, wherein the substrate includes, in order from the second main surface side toward the first main surface side, an insulating substrate, a metal layer provided on the surface of the insulating substrate, and an insulating layer provided on the surface of the metal layer opposite to the insulating substrate; preparing a measuring material containing metal particles; producing a measuring sample containing antigen-attached metal particles in which the antigen is bound to the metal particles by bringing a meter to be measured object containing an antigen into contact with the measuring material; binding the antigen-attached metal particles to the measuring substrate on the first main surface of the substrate by bringing the measuring sample and the measuring substrate into contact; and measuring the change in the reflection absorption characteristics before and after binding the antigen-attached metal particles to the measuring substrate by measuring the reflection absorption characteristics by localized surface plasmon resonance from the first main surface side of the substrate before and after binding the antigen-attached metal particles.

17. An antigen measurement kit comprising: a substrate having a first main surface and a second main surface opposite to each other in the thickness direction; a bonding portion provided on the first main surface of the substrate, wherein the substrate comprises, in order from the second main surface side toward the first main surface side, an insulating substrate, a metal layer provided on the surface of the insulating substrate, and an insulating layer provided on the surface of the metal layer opposite to the insulating substrate; and a measurement material containing metal particles, wherein the bonding portion is a site for binding of antigen-attached metal particles in which an antigen is bound to the metal particles.

18. An antigen measurement substrate having a first main surface and a second main surface opposite to each other in the thickness direction, and comprising, in order from the second main surface side toward the first main surface side, an insulating substrate, a metal layer provided on the surface of the insulating substrate, and an insulating layer provided on the surface of the metal layer opposite to the insulating substrate, and a bonding portion provided on the first main surface of the substrate, wherein the bonding portion is a portion for binding of antigen-attached metal particles in which an antigen is bound to a metal particle.

19. An antigen measurement substrate having a first main surface and a second main surface opposite to each other in the thickness direction, and comprising, in order from the second main surface side toward the first main surface side, an insulating substrate, a metal layer provided on the surface of the insulating substrate, and an insulating layer provided on the surface of the metal layer opposite to the insulating substrate, a bonding portion provided on the first main surface of the substrate, and antigen-attached metal particles bonded to the bonding portion in a state where the antigen is bound to the metal particles.

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