Kit for detecting detection target substance, and detection method

Abstract

Disclosed is a kit for detecting a detection target substance, said kit comprising: a plasmon resonance chip; a first protein that specifically binds to the detection target substance; and a second protein that specifically binds to the detection target substance, wherein the plasmon resonance chip is provided with a substrate, a metallic particle aggregate layer that is formed on the substrate, and a protective layer that covers the metallic particle aggregate layer. Also disclosed is a detection method for a detection target substance which uses said kit.

Description

Kit and detection method for detecting target substance

[0001] The present disclosure relates to a kit and a detection method for detecting a target substance.

[0002] A technology for enhancing fluorescence by utilizing localized plasmon resonance of metal nanoparticles is known. Patent Document 1 describes a metal-based particle assembly that is a plasmonic material useful for improving the luminous efficiency of light-emitting elements (organic electroluminescence (EL) elements, inorganic EL elements, inorganic light-emitting diode (LED) elements, etc.) and the conversion efficiency of photoelectric conversion elements (solar cell elements). Patent Document 2 describes a metal-based particle assembly that is a new structure that can be used in optical sensing devices and is useful as an enhancing element for enhancing the luminescence of a light-emitting body.

[0003] Enzyme-Linked Immunosorbent Assay (ELISA), particularly a technique typified by sandwich ELISA, is one of the general-purpose measurement techniques for detecting biomolecules such as proteins. In sandwich ELISA, a biomolecule is brought into contact with a molecule immobilized on a substrate that specifically binds to the biomolecule to be detected, and the biomolecule is then captured. The biomolecule is then further brought into contact with a molecule that specifically binds to the biomolecule and is labeled so that its optical signal can be detected, thereby labeling the biopolymer, and the biopolymer is quantified and evaluated using the intensity of the optical signal as an index.

[0004] JP 2013-177665 A JP 2021-156591 A

[0005] As described in Patent Document 1, signal enhancement using a plasmonic material capable of generating localized plasmon resonance has been mainly used to enhance luminescence signals in optical organic and inorganic materials, such as those used in displays, or to improve the energy conversion efficiency on a solid phase in devices that use light as input and / or output, such as improving the power generation efficiency in photoelectric conversion elements, such as those used in solar cells.

[0006] An object of the present disclosure is to provide a kit and a detection method for detecting a target substance, which have high detection sensitivity for the target substance.

[0007] The present inventors have found that a plasmonic material capable of generating localized plasmon resonance can be applied to sandwich ELISA, and that the application of a plasmonic material to sandwich ELISA significantly improves the detection sensitivity of a target substance.

[0008] The present disclosure relates to, for example, the following: [1] A kit for detecting a substance to be detected, comprising: a plasmon resonance chip; a first protein that specifically binds to the substance to be detected; and a second protein that specifically binds to the substance to be detected, and further satisfying the following (i) or (ii): (i) the second protein is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; (ii) the kit further comprises a third protein that binds to the second protein and is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; the plasmon resonance chip comprises: a substrate; a metal-based particle assembly layer formed on the substrate; and a protective layer that covers the metal-based particle assembly layer, the metal-based particle assembly layer comprising a particle assembly comprising 30 or more metal-based particles that are spaced apart from one another and arranged two-dimensionally, The metal-based particles have an average particle size in the range of 200 to 1600 nm, an average height in the range of 55 to 500 nm, and an aspect ratio, defined as the ratio of the average particle size to the average height, in the range of 1 to 8. [2] A kit for detecting a substance to be detected, comprising: a plasmon resonance chip; a first protein that specifically binds to the substance to be detected; and a second protein that specifically binds to the substance to be detected, and further satisfying the following (i) or (ii): (i) the second protein is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; (ii) the kit further comprises a third protein that binds to the second protein and is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; wherein the plasmon resonance chip comprises: a substrate; a metal-based particle assembly layer formed on the substrate; and a protective layer that covers the metal-based particle assembly layer, wherein the metal-based particle assembly layer is a metal-based particle assembly comprising a plurality of metal-based particles that are arranged so as to be spaced apart from one another, and the plurality of metal-based particles are arranged such that the average distance between adjacent metal-based particles is 1 nm or more and 1000 nm or less, and the standard deviation of the average distance is 25 nm or less. Kit.[3] A kit for detecting a substance to be detected, comprising: a plasmon resonance chip; a first protein that specifically binds to the substance to be detected; and a second protein that specifically binds to the substance to be detected, and further satisfying the following (i) or (ii): (i) the second protein is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; (ii) the kit further comprises a third protein that binds to the second protein and is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; wherein the plasmon resonance chip comprises: a substrate; a metal-based particle assembly layer formed on the substrate; and a protective layer that covers the metal-based particle assembly layer, wherein the metal-based particle assembly layer is a metal-based particle assembly formed of a plurality of metal-based particles arranged spaced apart from one another, and the protective layer has a first surface, and the carbon atom concentration of the first surface measured by X-ray photoelectron spectroscopy is 20.0 atomic % or less. [4] The kit according to any one of [1] to [3], wherein the protective layer has a first surface, and the first protein is immobilized on the first surface. [5] The kit according to any one of [1] to [4], wherein, in detecting the analyte, the number of molecules contained in the analyte-protein complex formed on the plasmon resonance chip, the analyte, the first protein, and the second protein, is eight or less per molecule of the analyte. [6] The kit according to any one of [1] to [5], wherein the protective layer has an average thickness of 10 nm or more and 300 nm or less. [7] The kit according to any one of [1] to [6], wherein the plurality of metal-based particles have an average particle size in the range of 200 nm or more and 1600 nm or less, an average height in the range of 55 nm or more and 500 nm or less, an aspect ratio defined as the ratio of the average particle size to the average height in the range of 1 or more and 8 or less, and in the metal-based particle assembly layer, the plurality of metal-based particles are arranged such that an average distance between adjacent metal-based particles is in the range of 1 nm or more and 150 nm or less.[8] A method for detecting a substance to be detected, comprising the steps of: contacting the substance to be detected with a plasmon resonance chip having a first surface immobilized with a first protein that specifically binds to the substance to be detected; and contacting the plasmon resonance chip after the contacting of the substance to be detected with a second protein that specifically binds to the substance to be detected, and further satisfying the following (I) or (II): (I) the second protein is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; (II) contacting the plasmon resonance chip after the contacting of the second protein with a third protein that binds to the second protein and is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; wherein the plasmon resonance chip comprises: a substrate; a metal-based particle assembly layer formed on the substrate; and a protective layer that covers the metal-based particle assembly layer, the metal-based particle assembly layer comprises a particle assembly including 30 or more metal-based particles arranged two-dimensionally and spaced apart from one another, the metal-based particles having an average particle size in the range of 200 to 1600 nm, an average height in the range of 55 to 500 nm, and an aspect ratio defined as the ratio of the average particle size to the average height in the range of 1 to 8, and the protective layer has the first surface.[9] A method for detecting a substance to be detected, comprising the steps of: contacting the substance to be detected with a plasmon resonance chip having a first surface immobilized with a first protein that specifically binds to the substance to be detected; and contacting the plasmon resonance chip after the contacting of the substance to be detected with a second protein that specifically binds to the substance to be detected, and further satisfying the following (I) or (II): (I) the second protein is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; (II) contacting the plasmon resonance chip after the contacting of the second protein with a third protein that binds to the second protein and is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; wherein the plasmon resonance chip comprises: a substrate; a metal-based particle assembly layer formed on the substrate; and a protective layer that covers the metal-based particle assembly layer, the metal-based particle assembly layer is a metal-based particle assembly made of a plurality of metal-based particles arranged to be spaced apart from one another, the plurality of metal-based particles are arranged such that an average distance between adjacent metal-based particles is equal to or greater than 1 nm and equal to or less than 1000 nm, a standard deviation of the average distance is equal to or less than 25 nm, and the protective layer has the first surface.

[10] A method for detecting a substance to be detected, comprising the steps of: contacting the substance to be detected with a plasmon resonance chip having a first surface immobilized with a first protein that specifically binds to the substance to be detected; and contacting the plasmon resonance chip after the contacting of the substance to be detected with a second protein that specifically binds to the substance to be detected, and further satisfying the following (I) or (II): (I) the second protein is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; (II) contacting the plasmon resonance chip after the contacting of the second protein with a third protein that binds to the second protein and is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; wherein the plasmon resonance chip comprises: a substrate; a metal-based particle assembly layer formed on the substrate; and a protective layer that covers the metal-based particle assembly layer, a detection method in which the metal-based particle assembly layer is a metal-based particle assembly including a plurality of metal-based particles arranged at a distance from one another, and the protective layer has the first surface, and a carbon atom concentration of the first surface measured by X-ray photoelectron spectroscopy is 20.0 atomic % or less.

[11] The detection method according to any one of [8] to

[10] , further comprising the step of immobilizing the first protein on the first surface of the plasmon resonance chip before the step of contacting the substance to be detected with the plasmon resonance chip.

[12] The detection method according to any one of [8] to

[11] , wherein the protective layer has an average thickness of 10 nm or more and 300 nm or less.

[13] The detection method according to any one of [8] to

[12] , wherein the plurality of metal-based particles have an average particle size in the range of 200 nm or more and 1600 nm or less, and an average height in the range of 55 nm or more and 500 nm or less, an aspect ratio defined as the ratio of the average particle size to the average height in the range of 1 or more and 8 or less, and in the metal-based particle assembly layer, the plurality of metal-based particles are arranged such that an average distance between adjacent metal-based particles is in the range of 1 nm or more and 150 nm or less.

[14] The detection method according to any one of [8] to

[13] , wherein the step of contacting the plasmon resonance chip with the second protein or the third protein, which is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance, comprises adding a solution containing the second protein or the third protein onto the first surface of the plasmon resonance chip so that the height from the first surface to the liquid surface of the solution is less than 500 μm.

[15] Use or application of a plasmon resonance chip in detecting a substance to be detected; a first protein that specifically binds to the substance to be detected; and a second protein that specifically binds to the substance to be detected, wherein the plasmon resonance chip comprises: a substrate; a metal-based particle assembly layer formed on the substrate; and a protective layer that covers the metal-based particle assembly layer, wherein the metal-based particle assembly layer comprises a particle assembly of 30 or more metal-based particles that are spaced apart from one another and arranged two-dimensionally, and the metal-based particles have an average particle size in the range of 200 to 1600 nm, an average height in the range of 55 to 500 nm, and an aspect ratio, defined as the ratio of the average particle size to the average height, in the range of 1 to 8.

[16] Use or application of a plasmon resonance chip in detecting a substance to be detected in combination with: a first protein that specifically binds to the substance to be detected; and a second protein that specifically binds to the substance to be detected, wherein the plasmon resonance chip comprises: a substrate; a metal-based particle assembly layer formed on the substrate; and a protective layer that covers the metal-based particle assembly layer, wherein the metal-based particle assembly layer comprises a particle assembly in which 30 or more metal-based particles are arranged two-dimensionally and spaced apart from one another, and the metal-based particles have an average particle size in the range of 200 to 1600 nm, an average height in the range of 55 to 500 nm, and an aspect ratio, defined as the ratio of the average particle size to the average height, in the range of 1 to 8.

[17] Use or application of a plasmon resonance chip in detecting a substance to be detected; a first protein that specifically binds to the substance to be detected; and a second protein that specifically binds to the substance to be detected, wherein the plasmon resonance chip comprises: a substrate; a metal-based particle assembly layer formed on the substrate; and a protective layer that covers the metal-based particle assembly layer, wherein the metal-based particle assembly layer is a metal-based particle assembly formed of a plurality of metal-based particles arranged to be spaced apart from one another, the plurality of metal-based particles are arranged such that an average distance between adjacent metal-based particles is 1 nm or more and 1000 nm or less, and the standard deviation of the average distance is 25 nm or less.

[18] Use or application of a plasmon resonance chip in detecting a substance to be detected in combination with: a first protein that specifically binds to the substance to be detected; and a second protein that specifically binds to the substance to be detected, wherein the plasmon resonance chip comprises: a substrate; a metal-based particle assembly layer formed on the substrate; and a protective layer covering the metal-based particle assembly layer, wherein the metal-based particle assembly layer is a metal-based particle assembly formed of a plurality of metal-based particles arranged to be spaced apart from one another, the plurality of metal-based particles are arranged such that an average distance between adjacent metal-based particles is 1 nm or more and 1000 nm or less, and the standard deviation of the average distance is 25 nm or less.

[19] Use or application of a plasmon resonance chip in detecting a substance to be detected; a first protein that specifically binds to the substance to be detected; and a second protein that specifically binds to the substance to be detected, wherein the plasmon resonance chip comprises: a substrate; a metal-based particle assembly layer formed on the substrate; and a protective layer that covers the metal-based particle assembly layer, wherein the metal-based particle assembly layer is a metal-based particle assembly formed of a plurality of metal-based particles arranged at a distance from one another, and the protective layer has a first surface, and a carbon atom concentration of the first surface measured by X-ray photoelectron spectroscopy is 20.0 atomic % or less.

[20] Use or application of a plasmon resonance chip in detecting an analyte in combination with: a first protein that specifically binds to the analyte; and a second protein that specifically binds to the analyte, wherein the plasmon resonance chip comprises: a substrate; a metal-based particle assembly layer formed on the substrate; and a protective layer covering the metal-based particle assembly layer, wherein the metal-based particle assembly layer is a metal-based particle assembly formed of a plurality of metal-based particles arranged at a distance from one another, and the protective layer has a first surface, and a carbon atom concentration of the first surface measured by X-ray photoelectron spectroscopy is 20.0 atomic % or less.

[21] Use or application of any one of

[15] to

[20] , wherein the protective layer has a first surface, and the first protein is immobilized on the first surface.

[22] Use or application of any one of

[15] to

[21] , wherein the protective layer has an average thickness of 10 nm or more and 300 nm or less.

[23] The use or application of any one of

[15] to

[22] , wherein the plurality of metal-based particles have an average particle size in the range of 200 nm or more and 1600 nm or less, an average height in the range of 55 nm or more and 500 nm or less, an aspect ratio defined as the ratio of the average particle size to the average height in the range of 1 or more and 8 or less, and the plurality of metal-based particles in the metal-based particle assembly layer are arranged such that an average distance between adjacent metal-based particles is in the range of 1 nm or more and 150 nm or less.

[24] The use or application of any one of

[15] to

[23] , comprising, in this order, a step of contacting the analyte with a plasmon resonance chip having a first surface on which a first protein that specifically binds to the analyte is immobilized, and a step of contacting the plasmon resonance chip with a second protein that specifically binds to the analyte.

[25] The use or application of any one of

[15] to

[24] , further comprising a step of immobilizing the first protein on the first surface of the plasmon resonance chip before the step of contacting the substance to be detected with the plasmon resonance chip.

[26] The use or application of any one of

[15] to

[25] , wherein the second protein is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance.

[27] The use or application of any one of

[15] to

[25] , wherein the second protein is used or applied in combination with a third protein that binds to the second protein and is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance.

[28] The use or application of

[27] , further comprising a step of contacting a third protein that binds to the second protein and is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance, with the plasmon resonance chip after contacting the second protein.

[29] The use or application of

[26] or

[28] , wherein the step of contacting the plasmon resonance chip with the second protein or the third protein, which is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance, comprises adding a solution containing the second protein or the third protein onto the first surface of the plasmon resonance chip so that the height from the first surface to the liquid level of the solution is less than 500 μm.

[0009] According to the present disclosure, there are provided a kit and a detection method for detecting a target substance, which have high detection sensitivity for the target substance. According to the present disclosure, there are provided a kit and a detection method for detecting a target substance, which have high detection sensitivity for the target substance compared to a kit including a general substrate that does not have a metal particle assembly layer.

[0010] 1 is a cross-sectional view schematically showing one example of a plasmon resonance chip according to an embodiment of a first aspect of the present invention. 2 is SEM images (10,000x and 50,000x scales) of a metal-based particle assembly film in a metal-based particle assembly film-layered substrate obtained in Preparation Example 1, viewed from directly above. 3 is an AFM image of a metal-based particle assembly film in a metal-based particle assembly film-layered substrate obtained in Preparation Example 1. 4 is a graph showing the relationship between antigen concentration and luminescence intensity in Example 1 (Control) and Comparative Example 1 (LRLP). 5 is a graph showing luminescence intensity at an antigen concentration of 100 ng / mL in Example 1 (Control) and Comparative Example 1 (LRLP).

[0011] Hereinafter, embodiments for carrying out the present disclosure will be described, but the present disclosure is not limited to the following embodiments.

[0012] A first aspect of the present disclosure relates to a kit for detecting an analyte, comprising a plasmon resonance chip, a first protein, and a second protein. The kit detects the analyte by bringing the analyte into contact with the plasmon resonance chip on which the first protein is immobilized (solid-phased) to capture the analyte, and then bringing the analyte into contact with the plasmon resonance chip to bind the analyte, and detecting the presence of the second protein as a fluorescent signal or a luminescent signal.

[0013] <Plasmon Resonance Chip> In the present disclosure, a plasmon resonance chip refers to a chip (substrate, sensor chip, functional scaffold) that can enhance the fluorescence intensity of fluorescent substances and the luminescence intensity of luminescent substances present near the chip through plasmon resonance. Such a plasmon resonance chip enhances the fluorescent signal or luminescence signal generated by the presence of a target substance. This makes it possible to detect, for example, extremely low concentrations of target substances that were difficult to detect using conventional technology. This can be expected to expand the range of target substances and significantly reduce the time required to detect the target substance.

[0014] A plasmon resonance chip according to a first aspect of the present disclosure includes a substrate, a metal-based particle assembly layer formed on the substrate, and a protective layer covering the metal-based particle assembly layer.

[0015] Fig. 1 is a cross-sectional view that schematically shows an example of a plasmon resonance chip according to an embodiment of the first aspect. The plasmon resonance chip shown in Fig. 1 includes a substrate 10, a metal-based particle assembly layer 20 that is disposed on a surface of the substrate 10, and a protective layer 30 that is disposed on the metal-based particle assembly layer 20. The metal-based particle assembly layer 20 is an assembly of a plurality of metal-based particles 21, and is a layer that is constituted by the plurality of metal-based particles 21 that are disposed spaced apart from one another. The protective layer 30 has a first surface 31.

[0016] <Substrate> The material of the substrate 10 is not particularly limited and may be, for example, glass, silicon wafer, or plastic. The light transmittance of the substrate 10 may be, for example, transparent, colored, or black, and in a preferred embodiment, the substrate 10 may be transparent.

[0017] <Metal-Based Particle Assembly Layer> Metal-based particle assembly layer 20 is a layer made up of a plurality of metal-based particles 21 arranged spaced apart from one another, and is a plasmon structure. A "plasmon structure" refers to a structure that can exhibit plasmon resonance. Plasmons are compressional waves of free electrons that are generated by the collective vibration of free electrons in a structure. When metal-based particle assembly layer 20 is a plasmon structure, the fluorescence intensity of a fluorescent substance or the luminescence intensity of a light-emitting substance can be enhanced.

[0018] In order to form metal-based particle assembly layer 20 into a plasmonic structure, metal-based particles 21 are preferably made of a material capable of plasmon resonance in the ultraviolet to visible light region. A material capable of plasmon resonance in the ultraviolet to visible light region means a material that, when made into nanoparticles or an assembly thereof, exhibits a plasmon peak that appears in the ultraviolet to visible light region in absorption spectrum measurement by absorptiometry.

[0019] Examples of metal-based materials capable of plasmon resonance in the ultraviolet to visible light region include precious metals such as gold, silver, copper, platinum, and palladium; metals other than precious metals such as aluminum and tantalum; alloys containing a metal selected from the precious metals and metals other than precious metals; and metal compounds (metal oxides, metal salts, etc.) containing a metal selected from the precious metals and metals other than precious metals. Among these, precious metals such as gold, silver, copper, platinum, and palladium are preferred as metal-based materials capable of plasmon resonance in the ultraviolet to visible light region, and silver is more preferred from the viewpoints of being inexpensive and having small absorption (the imaginary part of the dielectric function at visible light wavelengths is small).

[0020] Metal-based particle assembly layer 20 in a plasmon resonance chip according to the first aspect of the present disclosure satisfies at least one of the following (a) to (c): (a): the metal-based particle assembly layer comprises a particle assembly including 30 or more metal-based particles arranged two-dimensionally and spaced apart from one another, the metal-based particles having an average particle size in the range of 200 to 1600 nm, an average height in the range of 55 to 500 nm, and an aspect ratio defined as the ratio of the average particle size to the average height in the range of 1 to 8; (b): the metal-based particle assembly layer is a metal-based particle assembly including a plurality of metal-based particles arranged apart from one another, the plurality of metal-based particles being arranged such that the average distance between adjacent metal-based particles is at least 1 nm and not more than 1000 nm, and the standard deviation of the average distance is not more than 25 nm; (c): the metal-based particle assembly layer is a metal-based particle assembly including a plurality of metal-based particles arranged apart from one another.

[0021] In a preferred embodiment, metal-based particle assembly layer 20 in the plasmon resonance chip according to the first aspect of the present disclosure further satisfies the following (d); (d): the plurality of metal-based particles have an average particle size in the range of 200 nm or more and 1600 nm or less, an average height in the range of 55 nm or more and 500 nm or less, and an aspect ratio defined as the ratio of the average particle size to the average height in the range of 1 or more and 8 or less, and in the metal-based particle assembly layer, the plurality of metal-based particles are arranged such that the average distance between adjacent metal-based particles is in the range of 1 nm or more and 150 nm or less.

[0022] When metal-based particle assembly layer 20 satisfies at least one of the above conditions (a) to (d), the range of action of plasmon resonance exhibited by metal-based particle assembly layer 20 is widened in the plasmon resonance chip. This, for example, makes it possible to widen the range over which the plasmon emission enhancement effect extends, and therefore it is possible to enhance the emission of fluorescent substances and luminescent substances located within, for example, a range of several hundred nanometers (e.g., 200 nm) from the surface of metal-based particle assembly layer 20. This makes it possible for the plasmon resonance chip according to the first aspect of the present disclosure to enhance the emission of fluorescent substances and luminescent substances located at a distance of, for example, 10 nm or more, or even several tens of nanometers (e.g., 20 nm, 30 nm, or 40 nm) or more, or even 100 nm or more or 200 nm or more. Such an expansion of the range of action of plasmon resonance occurs preferably when metal-based particle assembly layer 20 satisfies the above (a), when (b), or when (c) and (d), and occurs more preferably when metal-based particle assembly layer 20 satisfies the above (a) and (d), when (b) and (d), or when all of the above (a) to (d) are satisfied.

[0023] When the metal-based particle assembly layer 20 satisfies at least one of the above conditions (a) to (d), the metal-based particle assembly layer 20 exhibits strong plasmon resonance. This allows, for example, a strong luminescence enhancement effect to be obtained. The strength of the plasmon resonance exhibited by a plasmon resonance chip is not simply the sum of the localized plasmon resonances exhibited by the individual metal-based particles at a specific wavelength, but is even stronger. In a plasmon resonance chip, strong plasmon resonance occurs due to interactions between the individual metal-based particles. It is believed that such strong plasmon resonance occurs due to interactions between the localized plasmons of the metal-based particles. Such plasmon resonance enhancement is preferably achieved when the metal-based particle assembly layer 20 satisfies the above condition (a), (b), or (c) and (d), and more preferably when the metal-based particle assembly layer 20 satisfies the above conditions (a) and (d), (b) and (d), or all of the above conditions (a) to (d).

[0024] Generally, when the absorption spectrum of a plasmon structure is measured by absorptiometry, a plasmon resonance peak (hereinafter also referred to as "plasmon peak") is observed as the peak located at the longest wavelength in the ultraviolet to visible light region. The strength of the plasmon resonance of the plasmon structure can be evaluated from the magnitude of the absorbance at the maximum wavelength of the plasmon peak. The greater the absorbance value, the greater the strength of the plasmon resonance tends to be. When the absorption spectrum of metal-based particle assembly layer 20 that satisfies at least one of (a) to (d) above is measured by the following absorptiometry, the absorbance at the maximum wavelength of the plasmon peak located at the longest wavelength in the ultraviolet to visible light region can be 1 or more, or even 1.5 or more, or even about 2.

[0025] The absorption spectrum of the plasmon structure can be measured by absorptiometry. Specifically, the absorption spectrum can be measured by irradiating the back side (the side opposite the metal-based particle assembly layer) of a glass substrate on which a metal-based particle assembly layer has been laminated with incident light in the ultraviolet to visible light region from a direction perpendicular to the substrate surface, and measuring the intensity I of transmitted light in all directions that has passed through the metal-based particle assembly layer side, and measuring the intensity I of transmitted light in all directions that has passed through the opposite side of the incident surface, when a substrate having the same thickness and material as the substrate of the measurement sample is irradiated with the same incident light from a direction perpendicular to the surface of the substrate on which the metal-based particle assembly layer has not been laminated. ０ and are measured using an integrating sphere spectrophotometer. In this case, the absorbance, which is the vertical axis of the absorption spectrum, is calculated using the following formula: absorbance = -log １０ (I / I ０ The absorption spectrum can be measured using a general spectrophotometer.

[0026] Furthermore, when measuring the maximum wavelength of the plasmon peak at the longest wavelength side in the ultraviolet to visible light region and its absorbance, an objective lens and a spectrophotometer may be used to narrow the measurement field and perform absorption spectrum measurement.

[0027] The number of metal-based particles 21 contained in metal-based particle assembly layer 20 is typically 10 or more, and preferably 30 or more. By forming a metal-based particle assembly layer containing 10 or more metal-based particles, strong plasmon resonance and an extension of the range of action of plasmon resonance are likely to occur due to interactions between localized plasmons of the metal-based particles. The number of metal-based particles 21 contained in metal-based particle assembly layer 20 may be, for example, 50 or more, or even 1,000 or more, or even 10,000 or more. The number density of metal-based particles 21 in metal-based particle assembly layer 20 is preferably 7 particles / μm ２ More preferably, 15 particles / μm ２ The term "plurality of metal-based particles" in the present disclosure encompasses "30 or more metal-based particles" in (a) above.

[0028] The plurality of metal-based particles 21 constituting metal-based particle assembly layer 20 preferably have an average particle size in the range of 200 nm or more and 1600 nm or less, more preferably 200 nm or more and 1200 nm or less, even more preferably 250 nm or more and 500 nm or less, and even more preferably 300 nm or more and 500 nm or less. The average particle size of metal-based particles 21 is preferably selected appropriately depending on the type of metal-based material constituting metal-based particles 21. When the average particle size of the plurality of metal-based particles 21 is within the above range, the range of action of plasmon resonance in the plasmon resonance chip is expanded, and the plasmon resonance is enhanced.

[0029] The average particle size of the plurality of metal-based particles 21 is the average particle size of the 10 selected metal-based particles when 10 metal-based particles are randomly selected in an SEM image observed from directly above metal-based particle assembly layer 20 composed of the plurality of metal-based particles 21, five tangential diameters are randomly drawn within each metal-based particle image (wherein all of the tangential diameter lines can only pass through the interior of the metal-based particle image, and one of these lines must be the longest line that can be drawn and passes only through the interior of the metal-based particle), and the average value (hereinafter, this average value will also be referred to as the "average tangential diameter") is taken as the particle size of each metal-based particle. The tangential diameter is defined as the perpendicular line connecting the distance between two parallel lines tangent to the outline (projected image) of a metal-based particle ("Particle Measurement Technology," Nikkan Kogyo Shimbun, 1994, p. 5).

[0030] To explain the method for measuring the average particle size in more detail, first, an SEM observation image is measured using a scanning electron microscope "JSM-5500" manufactured by JEOL Ltd. or an equivalent device. Next, the obtained observation image is read in 1280 horizontal pixels x 960 vertical pixels using free image processing software "ImageJ" manufactured by the National Institutes of Health. Next, 10 random numbers (x) are generated from 1 to 1280 using the random number generation function "RANDBETWEEN" in the spreadsheet software "Excel" manufactured by Microsoft Corporation. １ , x ２ , x ３ , x ４ , x ５ , x ６ , x ７ , x ８ , x ９ , x １０ ), 10 random numbers from 1 to 960 (y １ , y ２ , y ３ , y ４ , y ５ , y ６ , y ７ , y ８ , y ９ , y １０ ) are obtained. From each of the 10 random numbers obtained, 10 sets of random number combinations (x １ , y １ ), (x ２ , y ２ ), (x ３ , y ３ ), (x ４ , y ４ ), (x ５ , y ５ ), (x ６ , y ６ ), (x ７ , y ７ ), (x ８ , y ８ ), (x ９ , y ９ ) and (x １０ , y １０ The random numbers generated from 1 to 1280 are used as the x coordinates, and the random numbers generated from 1 to 960 are used as the y coordinates. １ , y１ ), (x ２ , y ２ ), (x ３ , y ３ ), (x ４ , y ４ ), (x ５ , y ５ ), (x ６ , y ６ ), (x ７ , y ７ ), (x ８ , y ８ ), (x ９ , y ９ ) and (x １０ , y １０ ) is obtained. The above-mentioned average tangent diameter is then obtained for each of a total of 10 metal-based particle images that include the coordinate point, and the average particle diameter is then obtained as the average of the 10 average tangent diameters. If at least one of the 10 coordinate points that make up the 10 random number combinations is not included in a metal-based particle image, or if two or more coordinate points are included in the same metal-based particle, the random number combination is discarded, and random number generation is repeated until all 10 coordinate points are included in different metal-based particle images.

[0031] The plurality of metal-based particles 21 preferably have an average height in the range of 55 nm or more and 500 nm or less, more preferably 55 nm or more and 300 nm or less, and even more preferably 70 nm or more and 150 nm or less. The average height of metal-based particles 21 is the average value of 10 measurements obtained by randomly selecting 10 metal-based particles in an AFM observation image of metal-based particle assembly layer 20 and measuring the heights of these 10 metal-based particles. When the average height of the plurality of metal-based particles 21 is within the above range, the range of action of plasmon resonance in the plasmon resonance chip is expanded, and the plasmon resonance is enhanced.

[0032] The aspect ratio of the plurality of metal-based particles 21 is preferably in the range of 1 or more and 8 or less, more preferably 2 or more and 8 or less, and even more preferably 2.5 or more and 8 or less. The aspect ratio of the metal-based particles 21 is defined as the ratio of the average particle diameter to the average height (average particle diameter / average height). When the aspect ratio of the plurality of metal-based particles 21 is within the above range, the range of action of plasmon resonance in the plasmon resonance chip is expanded and the plasmon resonance is enhanced. The metal-based particles 21 may be spherical, but from the viewpoint of expanding the range of action of plasmon resonance in the plasmon resonance chip and enhancing the plasmon resonance, it is preferable that the metal-based particles 21 have a flat shape with an aspect ratio of greater than 1.

[0033] In the metal-based particle assembly layer 20, the plurality of metal-based particles 21 are arranged such that the average distance between adjacent metal-based particles (hereinafter also referred to as the "average inter-particle distance") is in the range of 1 nm to 1,000 nm, and is preferably arranged in the range of 1 nm to 150 nm, more preferably 1 nm to 100 nm, even more preferably 1 nm to 50 nm, and still more preferably 1 nm to 20 nm. Arranging the plurality of metal-based particles 21 at such an average inter-particle distance makes it easier to obtain strong plasmon resonance and further enhances the effect of extending the range of action of plasmon resonance. Conversely, if the average inter-particle distance is less than 1 nm, electron transfer based on the Dexter mechanism occurs between the particles, which is disadvantageous in terms of deactivation of localized plasmons.

[0034] The average interparticle distance is the average value of the interparticle distances of 10 metal-based particles obtained by randomly selecting 10 metal-based particles in an SEM image taken directly above metal-based particle assembly layer 20 made up of a plurality of metal-based particles 21 and determining the interparticle distance between each of the selected metal-based particles and adjacent metal-based particles. The interparticle distance between adjacent metal-based particles is the average value obtained by measuring the distances between all adjacent metal-based particles (the smallest distance between the surfaces of adjacent metal-based particles).

[0035] To explain the method for measuring the average interparticle distance in more detail, first, an SEM observation image is measured using a scanning electron microscope "JSM-5500" manufactured by JEOL Ltd. or an equivalent device. Next, the obtained observation image is read in 1280 horizontal pixels x 960 vertical pixels using free image processing software "ImageJ" manufactured by the National Institutes of Health. Next, 10 random numbers (x) are generated from 1 to 1280 using the random number generation function "RANDBETWEEN" in the spreadsheet software "Excel" manufactured by Microsoft Corporation. １ ~x １０ ), 10 random numbers from 1 to 960 (y １ ~y １０ ) are obtained. From each of the 10 random numbers obtained, 10 sets of random number combinations (x １ , y １ ) to (x １０ , y １０ The random numbers generated from 1 to 1280 are used as the x coordinates, and the random numbers generated from 1 to 960 are used as the y coordinates. １ , y １ ) ~ (x １０ , y １０ ) is obtained. Then, for each of a total of 10 metal-based particle images that include the coordinate point, the inter-particle distance between the metal-based particle and the adjacent metal-based particle is obtained, and then the average inter-particle distance is obtained as the average value of the inter-particle distances between the 10 adjacent metal-based particles. If at least one of the 10 coordinate points that make up the 10 random number combinations is not included in the metal-based particle image, or if two or more coordinate points are included in the same metal-based particle, the random number combination is discarded, and random number generation is repeated until all 10 coordinate points are included in different metal-based particle images.

[0036] In metal-based particle assembly layer 20, the standard deviation of the average inter-particle distance among a plurality of metal-based particles 21 may be preferably 35 nm or less, more preferably 30 nm or less, and even more preferably 25 nm or less, and is preferably 0.1 nm or more, more preferably 0.2 nm or more, and even more preferably 0.3 nm or more. When the standard deviation of the average inter-particle distance among a plurality of metal-based particles 21 is equal to or less than the above upper limit and / or equal to or more than the above lower limit, the range of action of plasmon resonance in the plasmon resonance chip is expanded, and plasmon resonance is enhanced.

[0037] The standard deviation of the average interparticle distance is defined as follows. In an SEM image taken directly above metal-based particle assembly layer 20 made up of a plurality of metal-based particles 21, one metal-based particle is first selected at random, and the interparticle distance between that metal-based particle and adjacent metal-based particles is determined. The interparticle distance between adjacent metal-based particles is the average value obtained by measuring the distances between all adjacent metal-based particles (the smallest distances between surfaces). In the SEM image, nine metal-based particles different from the one selected at random are selected, and the interparticle distance between adjacent metal-based particles for these nine metal-based particles is determined in the same manner as above. The standard deviation of the interparticle distances between adjacent metal-based particles for a total of 10 metal-based particles obtained in this manner is defined as the standard deviation of the average interparticle distance.

[0038] To explain more specifically the method for measuring the standard deviation of the average interparticle distance, first, an SEM observation image is measured using a scanning electron microscope "JSM-5500" manufactured by JEOL Ltd. or an equivalent device. Next, the obtained observation image is read in 1280 horizontal pixels x 960 vertical pixels using free image processing software "ImageJ" manufactured by the National Institutes of Health. Next, 10 random numbers (x) are generated from 1 to 1280 using the random number generation function "RANDBETWEEN" in the spreadsheet software "Excel" manufactured by Microsoft Corporation. １ ~x １０ ), 10 random numbers from 1 to 960 (y １ ~y １０ ) are obtained. From each of the 10 random numbers obtained, 10 sets of random number combinations (x１ , y １ ) to (x １０ , y １０ The random numbers generated from 1 to 1280 are used as the x coordinates, and the random numbers generated from 1 to 960 are used as the y coordinates. １ , y １ ) ~ (x １０ , y １０ ) is obtained. Then, for each of a total of 10 metal-based particle images that include the coordinate point, the interparticle distance between the metal-based particle and the adjacent metal-based particle is obtained, and the standard deviation of the average interparticle distance is then obtained as the standard deviation of the interparticle distances between the 10 adjacent metal-based particles. If at least one of the 10 coordinate points that make up the 10 sets of random number combinations is not included in a metal-based particle image, or if two or more coordinate points are included in the same metal-based particle, the random number combination is discarded, and random number generation is repeated until all 10 coordinate points are included in different metal-based particle images.

[0039] From the viewpoint of exciting plasmons with high efficiency, it is preferable that the metal-based particles 21 have a smoothly curved surface, and it is particularly preferable that the surface has a flat shape with a smoothly curved surface, but the surface may include some minute irregularities (roughness), and in this sense the metal-based particles may be amorphous.

[0040] It is preferable that metal-based particle assembly layer 20 does not exhibit electrical conductivity as a layer, and it is more preferable that metal-based particles 21 constituting metal-based particle assembly layer 20 are each electrically non-conductive with adjacent metal-based particles. If there are locations in metal-based particle assembly layer 20 where electrons can be exchanged between metal-based particles 21, the plasmon resonance effect tends to be reduced. Therefore, it is preferable that metal-based particles 21 are reliably spaced apart and that no electrically conductive material is present between metal-based particles 21. Metal-based particles 21 themselves may be electrically conductive.

[0041] That metal-based particle assembly layer 20 does not exhibit conductivity as a layer can be confirmed, for example, by the fact that when a pair of tester probes of a multimeter [tester (Hewlett-Packard Company's "E2378A")] is brought into contact with metal-based particle assembly layer 20 at a distance of 10 mm to 15 mm, and the range is set to "30 MΩ," the resistance value under the measurement conditions is 30 MΩ or greater, resulting in a display of "overload."

[0042] In the metal-based particle assembly layer 20 according to an embodiment of the first aspect of the present disclosure, the maximum wavelength of the plasmon peak in the visible light region can exhibit a unique shift in the absorption spectrum depending on the average particle size and the average inter-particle distance of the metal-based particles 21, which may enable particularly enhanced emission in a specific (desired) wavelength region. Specifically, as the average inter-particle distance is kept constant and the average particle size of the metal-based particles 21 is increased, the maximum wavelength of the plasmon peak located on the longest wavelength side in the visible light region shifts to the short wavelength side (blue shift). Similarly, as the average particle size of large metal-based particles is kept constant and the average inter-particle distance is reduced (i.e., the metal-based particles 21 are arranged more densely), the maximum wavelength of the plasmon peak located on the longest wavelength side in the visible light region shifts to the short wavelength side. This unique phenomenon contradicts the Mie scattering theory, which is generally accepted for plasmonic materials (according to this theory, as the particle size increases, the maximum wavelength of the plasmon peak shifts to the long wavelength side (red shift)).

[0043] The above-described peculiar blue shift is also thought to occur when metal-based particle assembly layer 20 has a structure in which large metal-based particles 21 are densely arranged at specific intervals, resulting in interactions between localized plasmons of metal-based particles 21. Metal-based particle assembly layer 20 (in a state where it is laminated on a glass substrate) according to the present embodiment can have a plasmon peak on the longest wavelength side in an absorption spectrum in the visible light region measured by absorptiometry, which plasmon peak can exhibit a maximum wavelength, for example, in a wavelength region of 350 to 550 nm, depending on the shape of metal-based particles 21 and the distance between particles. Furthermore, metal-based particle assembly layer 20 according to the present embodiment can exhibit a blue shift of typically about 30 to 500 nm (for example, 30 to 250 nm) compared to when metal-based particles 21 are arranged at sufficiently long inter-particle distances (for example, 1 μm).

[0044] A sensor chip including metal-based particle assembly layer 20 in which the maximum wavelength of the plasmon peak has been blue-shifted is extremely advantageous, for example, in the following respects: Even when a blue-emitting substance with relatively low luminous efficiency is used as the labeling substance, the luminous efficiency can be increased to a sufficient level.

[0045] <Method of Manufacturing Metal-Based Particle Assembly Layer> Metal-based particle assembly layer 20 can be produced by, for example, the following methods: [A] a bottom-up method in which a plurality of metal-based particles 21 are grown from minute seeds on substrate 10; [B] a method in which a plurality of metal-based particles 21 are coated with a protective film made of an amphiphilic material having a predetermined thickness, and then this is formed into a film on substrate 10 by the Langmuir Blodgett (LB) film method; [C] other methods such as post-treatment of a thin film produced by vapor deposition or sputtering, resist processing, etching, and a casting method using a dispersion liquid in which metal-based particles are dispersed.

[0046] The above method [A] preferably includes a step of growing metal-based particles at an extremely slow rate on substrate 10 adjusted to a predetermined temperature (hereinafter also referred to as a "particle growth step"). According to a production method including such a particle growth step, metal-based particle assembly layer 20 having the above-mentioned preferred average particle size, average height, aspect ratio, average interparticle distance, and standard deviation of the average interparticle distance can be obtained with good control.

[0047] In the particle growth step, the rate at which the metal-based particles are grown on substrate 10 is preferably less than 1 nm / min, and more preferably 0.5 nm / min or less, in terms of average height growth rate. The average height growth rate here can also be referred to as the average deposition rate or the average thickness growth rate of the metal-based particles, and is defined by the following formula: average height of metal-based particles / metal-based particle growth time. The definition of "average height of metal-based particles" is as described above. The metal-based particle growth time refers to the time from the start to the end of growth of the metal-based particles, and specifically refers to the supply time of the metal-based material. When metal-based particle assembly layer 20 is considered as a film, the metal-based particle growth time can also be referred to as the film formation time. When the method for growing the metal-based particles is a sputtering method, the metal-based particle growth time is the sputtering time.

[0048] The temperature of the substrate 10 in the particle growth process is preferably 100°C or higher and 450°C or lower, more preferably 200°C or higher and 450°C or lower, even more preferably 250°C or higher and 350°C or lower, and even more preferably 300°C or thereabouts (approximately 300°C ± 10°C).

[0049] By adjusting the average height growth rate, the substrate temperature and / or the metal-based particle growth time, it is possible to control the average interparticle distance and its standard deviation, average particle size, average height, and aspect ratio of the plurality of metal-based particles 21 grown on the substrate 10.

[0050] The pressure (pressure in the chamber of the apparatus) when growing the metal-based particles is not particularly limited as long as it is a pressure that allows particle growth, but is usually less than atmospheric pressure. The lower limit of the pressure is not particularly limited, but is preferably 0.5 Pa or more, more preferably 6 Pa or more, and even more preferably 10 Pa or more, so that the average height growth rate can be easily adjusted within the above range.

[0051] The specific method for growing metal-based particles on substrate 10 is not particularly limited as long as it allows particles to grow at an average height growth rate of less than 1 nm / min, and examples of such a method include sputtering and vapor deposition methods such as vacuum deposition. Among sputtering methods, direct current (DC) sputtering is preferably used because it allows a metal-based particle assembly layer to grow relatively easily and makes it easy to maintain an average height growth rate of less than 1 nm / min.

[0052] The sputtering method is not particularly limited, and examples thereof include direct current argon ion sputtering, in which argon ions generated by an ion gun or plasma discharge are accelerated by an electric field and irradiated onto a target. Other conditions in the sputtering method, such as the current value, voltage value, and substrate-target distance, are appropriately adjusted so that particles grow at an average height growth rate of less than 1 nm / min.

[0053] In order to obtain, with good control, metal-based particle assembly layer 20 having the above-mentioned preferred average particle size, average height, aspect ratio, average inter-particle distance, and standard deviation of the average inter-particle distance, it is preferable to set the average height growth rate to less than 1 nm / min in the particle growth step, as well as the average particle size growth rate to less than 5 nm. However, when the average height growth rate is less than 1 nm / min, the average particle size growth rate will usually be less than 5 nm. The average particle size growth rate is more preferably 1 nm / min or less. The average particle size growth rate is defined by the following formula: average particle size of metal-based particles / growth time of metal-based particles. The definitions of "average particle size of metal-based particles" and "growth time of metal-based particles" are as described above.

[0054] In order to obtain metal-based particle assembly layer 20 having the above-mentioned preferred average particle size, average height, aspect ratio, average interparticle distance, and standard deviation of the average interparticle distance, it is preferable to appropriately adjust the metal-based particle growth time in the particle growth step while taking into consideration the above-mentioned preferred manufacturing conditions.

[0055] <Protective Layer> As shown in FIG. 1 , the plasmon resonance chip according to the first aspect of the present disclosure includes a protective layer 30 that covers the surfaces of each metal-based particle 21 constituting the metal-based particle assembly layer 20 and protects the metal-based particles 21. The protective layer 30 is preferably insulating. Being insulating ensures the non-conductivity of the metal-based particle assembly layer 20 (non-conductivity between the metal-based particles) and also provides electrical insulation between the metal-based particle assembly layer 20 and other adjacent layers. Since no current flows through the metal-based particle assembly layer 20, the luminescence enhancement effect due to plasmon resonance can be fully achieved. Furthermore, the protective layer 30 that covers the metal-based particles can prevent the metal-based particles 21 from coming into direct contact with layers other than the protective layer 30 or with the external environment, thereby preventing deterioration of the metal-based particles 21.

[0056] In the example of the plasmon resonance chip shown in FIG. 1 , protective layer 30 is disposed on the side opposite substrate 10 with respect to metal-based particle assembly layer 20, and so as to cover the entire surface of metal-based particle assembly layer 20 opposite substrate 10. Thus, from the standpoint of protecting metal-based particle assembly layer 20, protective layer 30 is preferably disposed so as to cover the entire surface of metal-based particle assembly layer 20 opposite substrate 10, i.e., it is preferable that a portion of the surface of the plasmon resonance chip opposite substrate 10 is not constituted by the surface of metal-based particle assembly layer 20. In the plasmon resonance chip shown in FIG. 1 , the surface of protective layer 30 opposite substrate 10 is first surface 31, and the entire surface of the plasmon resonance chip opposite substrate 10 is constituted by the surface of protective layer 30 (first surface). Furthermore, protective layer 30 is preferably formed so as to fill gaps between metal-based particles 21. Protective layer 30 is a layer having a first surface. The first surface here refers to at least a part of the surface that defines the outer edge of the protective layer 30 and is at least a part of the surface opposite to the substrate 10 side.

[0057] The material constituting the protective layer 30 is preferably one having good insulating properties, such as spin-on glass (SOG; for example, containing an organic siloxane material) or SiO ２ and Si ３ N ４ There are no particular restrictions on the thickness of protective layer 30 as long as it can prevent metal-based particles 21 from coming into direct contact with layers other than protective layer 30 or with the external environment, but since it is preferable that the distance between metal-based particle assembly layer 20 and a labeling substance present in the vicinity of the specific bond between the capture substance and the analyte, as will be described later, is within a predetermined range, the thinner the thickness, the better as long as the desired protection is ensured.

[0058] The first surface 31 of the protective layer 30 has a carbon atom signal intensity (carbon atom concentration) of 20.0 atomic % or less as measured by X-ray photoelectron spectroscopy. This allows the plasmon resonance chip to have excellent resistance to cleaning with piranha solution. From the viewpoint of enhancing this resistance, the carbon atom signal intensity is preferably 18 atomic % or less, more preferably 15 atomic % or less, even more preferably 12 atomic % or less, and even more preferably 10 atomic % or less.

[0059] The signal intensity of the carbon atoms is preferably 0.2 atomic % or more, more preferably 0.5 atomic % or more, even more preferably 1 atomic % or more, and may be 5 atomic % or more. By forming protective layer 30 using such a composition, the coatability of the composition and, therefore, the mass productivity of plasmon resonance chips can be improved, and the smoothness of the surface of protective layer 30 (the surface opposite to substrate 10, first surface 31) can be increased.

[0060] The signal intensity of the carbon atoms on the first surface 31 can be measured by X-ray photoelectron spectroscopy. The signal intensity of the carbon atoms is defined as the ratio (%) of the area of ​​the signal of the carbon atoms to the total area of ​​all signals in the spectrum obtained by X-ray photoelectron spectroscopy.

[0061] The surface of the plasmon resonance chip opposite to the substrate 10 has a signal intensity of the metal atoms constituting the metal-based particles 21 measured by X-ray photoelectron spectroscopy of preferably 7.0 atomic % or less, more preferably 5.0 atomic % or less, even more preferably 3.0 atomic % or less, even more preferably 2.0 atomic % or less, particularly preferably 1.5 atomic % or less, even more particularly preferably 1.0 atomic % or less, even more particularly preferably 0.7 atomic % or less, and most preferably below the detection limit. This can more reliably protect the metal-based particle assembly layer 20 and can provide the plasmon resonance chip with excellent resistance to cleaning with piranha solution. The signal intensity of the metal atoms can be measured as the intensity (atomic %) of the metal atomic signal observed from the surface and is defined as the ratio (%) of the area of ​​the metal atom signal to the total area of ​​all signals in the spectrum obtained by X-ray photoelectron spectroscopy.

[0062] A plasmon resonance chip according to an embodiment of the first aspect of the present disclosure may have excellent resistance to piranha solution cleaning. A test for evaluating the resistance to piranha solution cleaning may be a piranha solution cleaning test in which the plasmon resonance chip is immersed in a piranha solution at a temperature of 25°C for 30 minutes. The piranha solution used in the piranha solution cleaning test is a mixed solution obtained by mixing concentrated sulfuric acid with a concentration of 98% by mass and 30% by mass of hydrogen peroxide solution in a volume ratio of 4:1. A plasmon resonance chip having excellent resistance has no locations in the metal-based particle assembly layer 20 where hole-like (pinhole-like) dissolution has occurred after the piranha solution cleaning test, or, even if such locations exist, the number of holes is small or the diameter of the holes is small. Furthermore, a plasmon resonance chip having excellent resistance tends to have a small change in surface roughness of the surface of the protective layer 30 (the surface opposite to the substrate 10, i.e., the first surface 31) before and after the piranha solution cleaning test. When the above-described piranha solution cleaning test is performed on a plasmon resonance chip that has extremely low resistance to cleaning with piranha solution, metal-based particle assembly layer 20 below protective layer 30 may be eroded to the extent that it penetrates through.

[0063] The protective layer 30 may contain silicon (Si) atoms and carbon (C) atoms as atoms identifiable by X-ray photoelectron spectroscopy, and may also contain oxygen (O) atoms, nitrogen (N) atoms, etc. in addition to silicon (Si) and carbon (C) atoms. Specific constituent materials of the protective layer 30 include, for example, Si ａ O ｂ C ｃ , Si ｄ N ｅ C ｆ , Si ｇ O ｈ N ｉ C ｊ , Si ｋ O ｌ , Si ｍ N ｎ , Si ｏ O ｐ N ｑ and the like, and preferably, Si ａ O ｂ C ｃ , Si ｄ N ｅ C ｆ , Si ｇ O ｈ N ｉ C ｊ where a to q represent the composition ratio of each atom in each compound. The protective layer 30 may be made of two or more materials. The protective layer 30 may have a single layer structure or a multi-layer structure made of different materials.

[0064] The first surface 31 of the protective layer 30 has an oxygen atomic signal intensity measured by X-ray photoelectron spectroscopy of, for example, 30 atomic % to 90 atomic %. From the viewpoint of improving the resistance of the laminate to cleaning with a piranha solution, the oxygen atomic signal intensity is preferably 40 atomic % to 80 atomic %, more preferably 50 atomic % to 70 atomic %, even more preferably 55 atomic % to 65 atomic %, and particularly preferably 60 atomic % to 65 atomic %.

[0065] The first surface 31 of the protective layer 30 has a signal intensity of silicon atoms measured by X-ray photoelectron spectroscopy of, for example, 5 atomic % to 50 atomic %. From the viewpoint of increasing the resistance of the laminate to cleaning with a piranha solution, the signal intensity of silicon atoms is preferably 10 atomic % to 45 atomic %, more preferably 20 atomic % to 40 atomic %, and even more preferably 25 atomic % to 35 atomic %.

[0066] The protective layer 30 is preferably an amorphous layer. According to the manufacturing method of the plasmon resonance chip described below, it is possible to form the protective layer 30 into an amorphous layer, even though the method includes a heat treatment step at a relatively high temperature. The fact that the protective layer 30 is an amorphous layer is advantageous in that a material that can be applied to a coating process can be selected as the raw material for the protective layer 30, and industrially high-throughput production can be performed.

[0067] The fact that the protective layer 30 is an amorphous layer can be confirmed by X-ray diffraction. That is, when the protective layer 30 is measured by X-ray diffraction under the following conditions, the protective layer 30 can be determined to be an amorphous layer if there is no peak with a full width at half maximum (FWHM) of 5° or less due to crystals in the range of 2θ = 5° to 85° (preferably, the range of 0° to 90°): Apparatus: Rigaku SmartLab; Measurement method: θ / 2θ measurement method; Measurement range: 2θ = 5° to 90°; Tube voltage: 45 kV; Tube current: 200 mA.

[0068] The average thickness of protective layer 30 is, for example, 10 nm or more and 300 nm or less, preferably 15 nm or more and 250 nm or less, and more preferably 20 nm or more and 200 nm or less. The average thickness of protective layer 30 may be 30 nm or more, 40 nm or more, 50 nm or more, 55 nm or more, or 60 nm or more. When the average thickness of protective layer 30 is within the above range, it is possible to provide a function of protecting metal-based particle assembly layer 20 and sufficient durability of the plasmon resonance chip, and it is also possible to flatten the surface irregularities of metal-based particle assembly layer 20.

[0069] Protective layer 30 preferably has an average thickness within the above range and a thickness sufficient to cover the entire surface of metal-based particle assembly layer 20 opposite substrate 10. Coverage of the entire surface of metal-based particle assembly layer 20 opposite substrate 10 by protective layer 30 can be confirmed by obtaining a surface image of the plasmon resonance chip using a scanning electron microscope "JSM-5500" manufactured by JEOL Ltd. or an equivalent device. Alternatively, similar to the measurement of the signal intensity of carbon atoms on the first surface, this can also be confirmed by measuring the surface of the plasmon resonance chip opposite substrate 10 by X-ray photoelectron spectroscopy and finding that the signal intensity of metal atoms constituting metal-based particles 21 is 1 atomic % or less (preferably below the detection limit).

[0070] The average thickness of the protective layer 30 is the average value of thicknesses measured at 10 arbitrary points on the protective layer. The thickness of the protective layer can be measured by measuring the step height using an AFM or by observing the cross section using an SEM. If the surface on which the protective layer is formed is not flat, the thickness can be measured at 10 arbitrary points on the protective layer by observing an SEM cross section image, and the average value can be used as the average thickness of the protective layer.

[0071] The protective layer 30 is preferably a wet coating layer. A wet coating layer refers to a layer formed through a process of applying a coating liquid (a protective layer-forming composition, described below). Having the protective layer 30 as a wet coating layer is advantageous in smoothing the surface of the protective layer 30 (the surface opposite the substrate 10, the first surface). Having the protective layer 30 as a wet coating layer makes it easier to obtain a plasmon resonance chip having a protective layer 30 with low surface roughness (the surface opposite the substrate 10, the first surface). A low surface roughness of the protective layer 30 makes it easier to improve the uniformity of subsequent processing, such as coating, when subsequent processing is performed on the surface, and also makes it easier to reduce defects due to the occurrence of defects associated with subsequent processing.

[0072] Protective layer 30 may be formed by vapor deposition, sputtering, CVD, or the like. In this case, the resulting protective layer 30 is likely to be a layer having surface irregularities (large surface roughness) that follow the surface irregularities of metal-based particle assembly layer 20, and is also likely to be a crystalline protective layer 30.

[0073] The arithmetic mean roughness Ra (hereinafter also referred to as "Ra (before testing)") of the surface of the protective layer 30 (the surface opposite the substrate 10, the first surface) measured in accordance with JIS B 0601:2001 is preferably 10 nm or less, more preferably 8 nm or less, even more preferably 5 nm or less, still more preferably 3 nm or less, and particularly preferably 2 nm or less. Ra (before testing) may be 0.1 nm or more.

[0074] A plasmon resonance chip according to one embodiment of the first aspect of the present disclosure may have excellent resistance to piranha solution cleaning. The plasmon resonance chip has an arithmetic mean roughness Ra (hereinafter also referred to as "Ra (after test)") measured in accordance with JIS B 0601:2001 after the piranha solution cleaning test. The arithmetic mean roughness Ra (after test) is preferably 10 nm or less, more preferably 8 nm or less, even more preferably 5 nm or less, even more preferably 3 nm or less, and particularly preferably 2 nm or less. Ra (after test) may be 0.1 nm or more.

[0075] The plasmon resonance chip according to the present invention may have an Ra (before the test) of 10 nm or less, 8 nm or less, 5 nm or less, 3 nm or less, or 2 nm or less, and an Ra (after the test) of 10 nm or less, 8 nm or less, 5 nm or less, 3 nm or less, or 2 nm or less. Furthermore, the plasmon resonance chip according to the present invention tends to have a small change in the surface roughness of the surface of the protective layer 30 (the surface opposite to the substrate 10, the first surface) before and after the test.

[0076] <Method of Forming Protective Layer> Such protective layer 30 can be formed by applying a protective layer-forming composition onto metal-based particle assembly layer 20 formed on substrate 10 to form a coating layer, followed by heat treatment. The protective layer-forming composition can be applied by, for example, spin coating, slit coating, or slit and spin coating, and the use of such a coating method makes it easier to form a protective layer having a desired average thickness and a smooth surface.

[0077] Examples of materials contained in the protective layer-forming composition include silicon atom-containing materials such as spin-on glass (SOG), polysilazane, tetraethyl orthosilicate (TEOS), methyltrimethoxysilane (MTMS), etc. Spin-on glass (SOG) has a siloxane structure, and examples thereof include silica glass, alkylsiloxane polymers, alkylsilsesquioxane polymers, hydrogenated silsesquioxane polymers, and hydrogenated alkylsilsesquioxane polymers.

[0078] In particular, from the viewpoint of forming a protective layer having a carbon atom signal intensity equal to or greater than the above-mentioned preferred lower limit, the composition for forming a protective layer preferably contains a protective layer-forming component having a carbon atom, such as an organic SOG having an organic group or an organic structure. Examples of the organic SOG include SOG having a methyl group or an ethyl group. Use of the organic SOG can also improve the coatability of the composition for forming a protective layer.

[0079] The protective layer-forming composition may further contain a solvent, a reaction catalyst, water, a surfactant, and the like in addition to the protective layer-forming components.

[0080] After forming the coating layer, a step of heat-treating the coating layer at a temperature of 300°C or higher and 800°C or lower is carried out. The heat treatment temperature is preferably 350°C or higher and 700°C or lower, more preferably 400°C or higher and 650°C or lower, even more preferably 450°C or higher and 600°C or lower, and particularly preferably 500°C or higher and 600°C or lower. If the heat treatment temperature is too low, when a protective layer-forming component having carbon atoms is used, the signal intensity of the carbon atoms in protective layer 30 is likely to be higher than the preferred range, and in this case, the resistance of the plasmon resonance chip to cleaning with piranha solution is low. If the heat treatment temperature is too high, oxidation of metal-based particle assembly layer 20 occurs, and the temperature may exceed the decomposition temperature of the oxidized metal-based particles or the melting temperature of the metal-based particle assembly layer. In this case, there is a risk of damaging metal-based particle assembly layer 20 during the heat treatment step.

[0081] The heat treatment time is, for example, 1 minute to 720 minutes, preferably 2 minutes to 480 minutes, more preferably 2 minutes to 240 minutes. The heat treatment can be performed, for example, in air or an inert gas (e.g., nitrogen, argon) atmosphere. The heat treatment pressure may be atmospheric pressure.

[0082] It should be noted that the plasmon resonance chip according to an embodiment of the first aspect of the present disclosure may have a layer other than substrate 10, metal-based particle assembly layer 20, and protective layer 30. Examples of such layers include a layer interposed between metal-based particle assembly layer 20 and protective layer 30.

[0083] <Detectable Substance> The term "detectable substance" refers to a substance that is detected by the kit or detection method according to the present disclosure. The term "detection" as used herein includes both qualitative detection, i.e., detection aimed at assessing the presence or absence of the detectable substance, and quantitative detection, i.e., detection aimed at measuring the amount (e.g., concentration) of the detectable substance. The detectable substance is not particularly limited as long as it is an organic or inorganic substance that can be specifically captured by a protein. Examples of the detectable substance include proteins, peptides, glycoproteins, lipids, sugars, small molecules, nucleic acids, nucleotides, nucleosides, and other biologically derived substances, viruses, and cells. In a preferred embodiment, the detectable substance may be a protein, peptide, lipid, sugar, virus, or cell. In a more preferred embodiment, the detectable substance may be a protein, peptide, virus, or cell. In an even more preferred embodiment, the detectable substance may be a protein or peptide. Examples of such detectable substances include phosphorylated tau protein and peptide fragments thereof; T-cell co-inhibitory molecule programmed cell death 1 (PD-1) protein and peptide fragments thereof; troponin, its subunits, and their peptide fragments; and prostate-specific antigen (PSA) and peptide fragments thereof.

[0084] The target substance according to an embodiment of the present disclosure may be contained in a biological sample. Examples of such biological samples include plasma, serum, cerebrospinal fluid, urine, lymph, and saliva. Such biological samples are not necessarily limited to those containing the target substance, but may be any biological sample that may potentially contain the target substance. The origin of such biological samples is not particularly limited, and may be, for example, human or non-human animals, such as mice, rats, hamsters, rabbits, dogs, cats, and monkeys.

[0085] <First Protein> The first protein is a protein that specifically binds to the analyte. The first protein is not particularly limited as long as it specifically binds to the analyte, and may be, for example, an antibody against the analyte or an antigen-binding fragment thereof, a protein that forms a complex with the analyte under physiological conditions, or a receptor for the analyte, such as a protein, peptide, glycoprotein, lipid, sugar, or small molecule. However, from the viewpoint of high specificity and enabling highly sensitive and accurate detection of the analyte, the first protein is preferably an antibody against the analyte or an antigen-binding fragment thereof.

[0086] When the first protein is an antibody against the analyte, the antibody may be a monoclonal or polyclonal antibody, preferably a monoclonal antibody from the viewpoint of specifically and accurately quantifying the analyte. These antibodies can be prepared by methods commonly used by those skilled in the art using materials commonly used by those skilled in the art, such as animals administered (immunized) with an antigen and myeloma. Alternatively, commercially available antibodies can be used.

[0087] When the first protein is an antigen-binding fragment of an antibody against the substance to be detected, the type of the antigen-binding fragment is not particularly limited, and may be, for example, Fab, Fab', F(ab') ２ These antigen-binding fragments can be prepared by methods commonly used by those skilled in the art using Escherichia coli or the like into which a nucleic acid (e.g., a plasmid) encoding the antigen-binding fragment has been introduced.

[0088] The first protein may be modified to facilitate immobilization on the first surface 31 of the plasmon resonance chip. Examples of such modifications include modification with a molecule (e.g., biotin) that binds with high affinity to a modification (e.g., streptavidin modification) applied to the surface of the first surface 31.

[0089] In a kit according to an embodiment of the first aspect of the present disclosure, the first protein may be immobilized on the first surface 31 of the protective layer 30 of the plasmon resonance chip. The immobilization (solid-phase) of the first protein on the first surface 31 may be by physical adsorption via hydrophobic interaction, by binding or interaction of a high-affinity molecule as described above, or by binding to a primary antibody (first protein) by modifying the first surface 31 with a secondary antibody. However, from the viewpoints of simplicity and economy, physical adsorption via hydrophobic interaction may also be used. Such physical adsorption can be achieved, for example, by contacting the first surface 31 with a solution containing the first protein for a predetermined time (e.g., 1 hour).

[0090] <Second Protein / Third Protein> The second protein is a protein that specifically binds to the analyte. The second protein is not particularly limited as long as it specifically binds to the analyte, and may be, for example, an antibody against the analyte or an antigen-binding fragment thereof, a protein that forms a complex with the analyte under physiological conditions, or a receptor for the analyte, such as a protein, peptide, glycoprotein, lipid, sugar, or small molecule. However, from the viewpoint of high specificity and enabling highly sensitive and accurate detection of the analyte, the second protein is preferably an antibody against the analyte or an antigen-binding fragment thereof.

[0091] When the second protein is an antibody against the analyte, the antibody may be a monoclonal or polyclonal antibody, preferably a monoclonal antibody from the viewpoint of specifically and accurately quantifying the analyte. These antibodies can be prepared by methods commonly used by those skilled in the art using materials commonly used by those skilled in the art, such as animals administered (immunized) with an antigen and myeloma. Commercially available antibodies can also be used.

[0092] When the second protein is an antigen-binding fragment of an antibody against the substance to be detected, the type of the antigen-binding fragment is not particularly limited, and may be, for example, Fab, Fab', F(ab') ２These antigen-binding fragments can be prepared by methods commonly used by those skilled in the art using Escherichia coli or the like into which a nucleic acid (e.g., a plasmid) encoding the antigen-binding fragment has been introduced.

[0093] In detecting an analyte substance by sandwich ELISA, the first protein and the second protein must simultaneously bind to the analyte substance. Therefore, the first protein and the second protein are proteins that can simultaneously bind to the analyte substance. In other words, the second protein is a protein that can bind to the analyte substance in a state where it is bound to the first protein. In other words, the second protein is a protein that binds to a site on the analyte substance that is different from the binding site of the first protein.

[0094] In one embodiment of the present disclosure, the second protein may be modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance. When the second protein is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance, the analyte can be detected based on the fluorescent signal or the luminescent signal without further labeling.

[0095] On the other hand, in another embodiment of the present disclosure, when the second protein is not modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance, the kit according to the first aspect of the present disclosure may further include a third protein. Here, the third protein is a protein that binds to the second protein and is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance. In one embodiment, the third protein is a protein that binds to the second protein, does not bind to the analyte substance or the first protein, and is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance. Examples of such third proteins include proteins that are modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance and bind to a site on the second protein other than the binding site for the analyte substance. For example, when the second protein is an antibody, an example of the third protein is a secondary antibody (e.g., an anti-IgG antibody) that is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance. Furthermore, for example, when the second protein is a biotinylated antibody, the third protein can be streptavidin modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance.

[0096] In the above cases, the second protein or third protein modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance is preferably modified with an enzyme that produces a fluorescent substance or a luminescent substance, or with a luminescent substance or an enzyme that produces it, from the viewpoint of increasing the detection sensitivity of the analyte in detection using the kit, and is particularly preferably modified with an enzyme that produces a luminescent substance. In particular, when the second protein or third protein is modified with an enzyme that produces a luminescent substance, the detection sensitivity of the analyte is significantly increased compared to when the second protein or third protein is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance, thereby enabling, for example, the detection of extremely low concentrations of the analyte that were difficult to detect using conventional techniques. This can be expected to, for example, expand the range of analyte substances and significantly reduce the time required for detection of the analyte.

[0097] The fluorescent substance is not particularly limited as long as it can detect (evaluate the presence or absence of or quantify) the analyte substance based on the emitted fluorescence, and may be, for example, a fluorescent dye such as a fluorescein, rhodamine, cyanine, or coumarin. When the second protein or the third protein is modified with a fluorescent substance, the analyte substance can be detected, for example, after contact with the second protein or the third protein, by irradiating the second protein or the third protein with excitation light corresponding to the excitation wavelength of the fluorescent substance that modifies the second protein or the third protein, and measuring the intensity of the emitted fluorescence.

[0098] The luminescent substance is not particularly limited as long as it can detect (evaluate the presence or absence of or quantify) the analyte substance based on the chemiluminescence it generates, and examples thereof include luminol, pyrogallol, etc. When the second protein or the third protein is modified with a luminescent substance, the analyte substance can be detected, for example, by contacting the second protein or the third protein with the luminescent substance, adding a chemical substance (e.g., hydrogen peroxide) that excites the luminescent substance modifying the second protein or the third protein, and measuring the intensity of the chemiluminescence generated.

[0099] The enzyme that produces a fluorescent substance is not particularly limited as long as it can detect (evaluate the presence or absence of or quantify) the analyte based on the fluorescence from the fluorescent substance produced by the enzyme upon addition of a substrate for the enzyme, and may be, for example, horseradish peroxidase (HRP). In this case, an example of an HRP substrate is 10-acetyl-3,7-dihydroxyphenoxazine, which, when metabolized by HRP, produces fluorescent 7-hydroxy-3H-phenoxazin-3-one. When the second protein or third protein is modified with an enzyme that produces a fluorescent substance, the analyte can be detected, for example, by contacting the second protein or third protein with the enzyme, adding a substrate for the enzyme that produces the fluorescent substance and a substance necessary for the enzymatic reaction (e.g., hydrogen peroxide), irradiating the second protein or third protein with excitation light corresponding to the excitation wavelength of the fluorescent substance produced when the substrate is metabolized by the enzyme that modifies the second protein or third protein, and measuring the intensity of the emitted fluorescence.

[0100] The enzyme that produces a luminescent substance is not particularly limited as long as it can detect (evaluate the presence or absence of or quantify) the analyte based on the luminescence (bioluminescence) from the luminescent substance produced by the enzyme upon addition of a substrate for the enzyme, and may be, for example, luciferase or horseradish peroxidase (HRP). In this case, an example of a luciferase substrate is luciferin, and an example of an HRP substrate is luminol. When the second protein or third protein is modified with an enzyme that produces a luminescent substance, the analyte can be detected, for example, by contacting the second protein or third protein with the analyte, adding a substrate for the enzyme that produces the luminescent substance and a substance necessary for the enzymatic reaction (e.g., hydrogen peroxide), and then measuring the intensity of luminescence (bioluminescence) produced by the luminescent substance produced when the substrate is metabolized by the enzyme that modifies the second protein or third protein.

[0101] A kit according to an embodiment of the first aspect of the present disclosure may be configured such that, in detecting a substance to be detected, the number of molecules contained in a substance-protein complex formed on a plasmon resonance chip and including the substance to be detected, a first protein, and a second protein, is a predetermined number or less per molecule of the substance to be detected. In other words, a kit according to an embodiment of the first aspect of the present disclosure may be configured such that, in detecting a substance to be detected, the number of elements in a substance-protein complex formed on a plasmon resonance chip and including the substance to be detected, a first protein, and a second protein, is a predetermined number or less per molecule of the substance to be detected. The predetermined number may be, for example, 8, preferably 6, and more preferably 5. The molecules constituting the substance-protein complex formed on the plasmon resonance chip may include the substance to be detected, a first protein, a second protein, and a third protein, but do not include an enzyme substrate. For example, if the second protein is a monoclonal antibody and the kit contains a third protein, the number of molecules (number of elements) per molecule of the analyte will be at least four (first protein, analyte, second protein, third protein). Alternatively, if the second protein is a polyclonal antibody, there are two binding sites for the second protein on one analyte molecule, and the kit contains a third protein, the number of molecules (number of elements) per molecule of the analyte will be at least six (first protein, analyte, two molecules of the second protein, two molecules of the third protein). In one embodiment, the number of molecules per molecule of the analyte contained in the analyte-protein complex formed on the plasmon resonance chip may be the total number of molecules of the analyte, first protein, second protein, and third protein per molecule of the analyte.

[0102] The kit according to one embodiment of the first aspect of the present disclosure may further include a substrate for the enzyme that produces a fluorescent or luminescent substance. The substrate may be one that is commonly used by those skilled in the art in accordance with the enzyme that produces the fluorescent or luminescent substance.

[0103] The kit according to one embodiment of the first aspect of the present disclosure may further include substances necessary for the enzymatic reaction of the enzyme that produces the fluorescent or luminescent substance. The substances may be those commonly used by those skilled in the art in accordance with the enzyme that produces the fluorescent or luminescent substance. Examples of such substances include coenzymes and peroxides such as hydrogen peroxide.

[0104] The kit according to an embodiment of the first aspect of the present disclosure may further include instructions, which may be electronic, and which may include content corresponding to the detection method according to an embodiment of the second aspect of the present disclosure, which will be described later.

[0105] The kit described above allows for highly sensitive detection of a target substance. For example, a kit according to an embodiment of the first aspect of the present disclosure can detect a target substance with at least two, three, five, eight, or ten times the sensitivity of a kit including a general plate (e.g., a microwell plate) instead of a plasmon resonance chip. That is, for example, a kit according to an embodiment of the first aspect of the present disclosure can detect a target substance at a concentration that is at most half, at most one-third, at most one-fifth, at most one-eighth, or at most one-tenth the lower limit of the concentration detectable by a kit including a general plate (e.g., a microwell plate) instead of a plasmon resonance chip.

[0106] <Detection method> Next, a method for detecting a target substance using the kit according to an embodiment of the first aspect of the present disclosure described above will be described. A second aspect of the present disclosure is a method for detecting a target substance using the kit according to an embodiment of the first aspect of the present disclosure. The plasmon resonance chip, first protein, second protein, third protein, fluorescent substance, luminescent substance, enzyme that produces a fluorescent substance or luminescent substance, and its substrate in the detection method according to the second aspect of the present disclosure can be the same as those described in the kit according to the first aspect of the present disclosure.

[0107] A detection method according to a second aspect of the present disclosure includes, in this order, a step of contacting the substance to be detected with a plasmon resonance chip having a first surface immobilized with a first protein that specifically binds to the substance to be detected (a capture step), and a step of contacting the plasmon resonance chip after the contact with the substance to be detected with a second protein that specifically binds to the substance to be detected (a labeling step). In one embodiment, the detection method according to the second aspect of the present disclosure further includes a step of immobilizing the first protein on the first surface of the plasmon resonance chip (a solid-phase step) before the capture step.

[0108] In the solid-phasing step, the first protein is immobilized (solid-phased) on the first surface of the plasmon resonance chip. The solid-phasing step can be performed by a method commonly used by those skilled in the art. For example, the solid-phasing step may be performed by physical adsorption via hydrophobic interaction as described in the first aspect of the present disclosure, by binding or interaction of high-affinity molecules such as biotin-avidin interaction, or by binding a primary antibody (first protein) to a first surface modified with a secondary antibody. However, from the viewpoint of simplicity and economy, physical adsorption via hydrophobic interaction is also preferred. Such physical adsorption may be performed, for example, by contacting a solution containing the first protein with the first surface for a predetermined time (e.g., 1 hour). The concentration of the first protein in the solution contacted with the first surface in the solid-phasing step may be, for example, 0.1 μg / mL to 1000 μg / mL or 0.1 μg / mL to 100 μg / mL. The solid-phasing step may be performed by a method commonly used by those skilled in the art. In addition, in a preferred embodiment, the molar concentration of the first protein contacted with the plasmon resonance chip may be higher than the molar concentration of the analyte contacted in the capture step, from the viewpoint of preventing saturation of the first protein on the plasmon resonance chip in the subsequent capture step, which would make it impossible to accurately quantify the analyte. After the immobilization step, the first protein that has not been immobilized on the plasmon resonance chip is removed from the system. Furthermore, washing may be performed, as necessary, to remove the unimmobilized first protein.

[0109] In the capture step, the substance to be detected is brought into contact with a plasmon resonance chip having a first protein immobilized on its first surface. As a result, the substance to be detected is captured (bound) by the first protein immobilized on the surface of the plasmon resonance chip. Such a plasmon resonance chip having a first protein immobilized on its first surface can be obtained, for example, by a solid-phase step. The capture step is usually performed by adding a solution containing the substance to be detected onto the first surface of the plasmon resonance chip having the first protein immobilized. The concentration of the substance to be detected in the solution containing the substance to be detected added in the capture step is, for example, 1.0 x 10 －７ ng / mL ~ 1.0×10 －２ ng / mL, 1.0×10 －７ ng / mL ~ 1.0×10 －３ ng / mL, 1.0×10 －７ ng / mL ~ 1.0×10 －４ ng / mL, 3.0×10 －７ ng / mL ~ 1.0×10 －５ ng / mL or 7.0 x 10 －７ ng / mL ~ 1.0×10 －６ The concentration may be as low as ng / mL. The detection method using a plasmon resonance chip according to the present disclosure has high detection sensitivity, making it possible to detect low concentrations of the analyte, such as in the above-mentioned range. The capture step may be performed by a method commonly used by those skilled in the art. In the capture step, the time for contacting the analyte with the plasmon resonance chip having the first protein immobilized on its first surface may be, for example, 1 minute or more, 1 hour or more, or 3 hours or more, or 168 hours or less, 48 ​​hours or less, or 24 hours or less. After the capture step, the analyte that was not captured by the first protein is removed from the system. Furthermore, washing may be performed, if necessary, to remove the uncaptured analyte.

[0110] In the labeling step, a second protein is contacted with the plasmon resonance chip after the contact with the analyte in the capture step. If the analyte captured by the first protein is present on the plasmon resonance chip as a result of the labeling step, a complex of the first protein, the analyte, and the second protein is formed on the plasmon resonance chip. The labeling step is typically performed by adding a solution containing the second protein to the first surface of the plasmon resonance chip after the contact with the analyte in the capture step. The labeling step may be performed by a method typically performed by those skilled in the art. In a preferred embodiment, the molar concentration of the second protein contacted with the plasmon resonance chip in the labeling step may be higher than the molar concentration of the analyte contacted in the capture step, in order to prevent the generation of a large amount of analyte that is captured by the first protein but not labeled with the second protein, thereby preventing accurate detection. Furthermore, the contact time of the second protein in the labeling step may be, for example, 1 minute to 168 hours, 5 minutes to 24 hours, or 10 minutes to 6 hours, and may be, for example, 1 hour.

[0111] In one embodiment, the second protein contacted in the labeling step may be modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance. In this case, the analyte can be detected based on a fluorescent signal or a luminescent signal without further labeling.

[0112] In another embodiment, if the second protein contacted in the labeling step is not modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance, the detection method according to the second aspect of the present disclosure may further include, after the labeling step, a step (dual labeling step) of contacting the plasmon resonance chip after the contact of the second protein with a third protein that binds to the second protein and is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance. If a substance to be detected captured by the first protein is present on the plasmon resonance chip through the dual labeling step, a complex of the first protein, the substance to be detected, the second protein, and the third protein is formed on the plasmon resonance chip. This makes it possible to detect the substance to be detected as a fluorescent or luminescent signal using the substance or enzyme labeled on the third protein. The dual labeling step is typically performed by adding a solution containing the modified third protein to the first surface of the plasmon resonance chip after the contact of the second protein in the labeling step. The double labeling step can be carried out according to a method commonly used by those skilled in the art. For example, the contact time may be 1 minute to 168 hours, 3 minutes to 24 hours, 5 minutes to 6 hours, or 10 minutes to 2 hours.

[0113] In one embodiment, the labeling step may include adding a solution containing a second protein or a third protein modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance onto the first surface of the plasmon resonance chip so that the height from the first surface to the liquid level of the solution is less than 500 μm. This allows for more sensitive detection of the target substance.

[0114] In one embodiment, the detection method according to the second aspect of the present disclosure may include a step of detecting the analyte based on a fluorescent signal or a luminescence signal (detection step) after the labeling step if the method does not include a dual-labeling step, or after the dual-labeling step if the method includes a dual-labeling step. In the detection step, for example, if fluorescence or luminescence is observed in the detection step, the presence of the analyte can be evaluated, and the analyte can be quantified based on the fluorescence intensity or luminescence intensity observed in the detection step. The detection step can be performed, for example, by measuring the fluorescence intensity or luminescence intensity of the solution on the plasmon resonance chip after the labeling step or dual-labeling step. The amount of solution may be reduced by diluting the solution or removing a portion of the solution as needed before the detection step or before measuring the fluorescence intensity or luminescence intensity.

[0115] When the second protein or the third protein is labeled with a fluorescent substance, the detection of the analyte in the detection step is performed based on a fluorescent signal. In this case, the detection step includes, in this order, irradiating the plasmon resonance chip with excitation light corresponding to the excitation wavelength of the fluorescent substance and observing the fluorescence emitted by the fluorescent substance.

[0116] When the second protein or the third protein is labeled with a luminescent substance, the detection of the analyte in the detection step is performed based on the luminescence signal. In this case, the detection step includes, in this order, contacting the plasmon resonance chip with a substance that excites the luminescent substance (e.g., hydrogen peroxide) and observing the luminescence emitted by the luminescent substance. The substance that excites the luminescent substance can be selected by those skilled in the art depending on the type of luminescent substance. Contacting the substance that excites the luminescent substance can be performed, for example, by adding a solution containing the substance to the solution on the plasmon resonance chip after the labeling step or double-labeling step. Conditions for contacting the substance that excites the luminescent substance can be conditions that are commonly used by those skilled in the art.

[0117] When the second protein or the third protein is labeled with an enzyme that produces a fluorescent substance, the detection of the analyte in the detection step is performed based on the fluorescent signal. In this case, the detection step includes, in this order, contacting the plasmon resonance chip with a substrate of the enzyme, irradiating it with excitation light corresponding to the excitation wavelength of the produced fluorescent substance, and observing the fluorescence emitted by the fluorescent substance. In one embodiment, in addition to the enzyme substrate, a substance necessary for the enzymatic reaction, such as a coenzyme of the enzyme, may also be contacted. Contacting the enzyme substrate and the substance necessary for the enzymatic reaction with the plasmon resonance chip can be performed, for example, by adding a solution containing them to the solution on the plasmon resonance chip after the labeling step or double-labeling step. Conditions for contacting the enzyme substrate and the substance necessary for the enzymatic reaction with the plasmon resonance chip can be conditions commonly used by those skilled in the art.

[0118] When the second protein or the third protein is labeled with an enzyme that produces a luminescent substance, the detection of the analyte in the detection step is performed based on the luminescence signal. In this case, the detection step includes, in this order, contacting the plasmon resonance chip with a substrate of the enzyme and observing the luminescence generated by the luminescent substance. In one embodiment, in addition to the enzyme substrate, a substance necessary for the enzymatic reaction, such as a coenzyme of the enzyme, may also be contacted. Contacting the plasmon resonance chip with the enzyme substrate and the substance necessary for the enzymatic reaction is performed, for example, by adding a solution containing them to the solution on the plasmon resonance chip after the labeling step or double-labeling step. Conditions for contacting the plasmon resonance chip with the enzyme substrate and the substance necessary for the enzymatic reaction can be conditions commonly used by those skilled in the art.

[0119] In one embodiment, the measurement of fluorescence or luminescence in the detection step may be performed under conditions such that the height of the solution on the plasmon resonance chip from the first surface to the solution surface is less than 500 μm. Under such conditions, signals derived from fluorescent or luminescent substances present in the solution are more likely to be amplified, allowing the detection of the target substance with higher sensitivity.

[0120] In one embodiment, the detection method according to the second aspect of the present disclosure may be a non-diagnostic method. Furthermore, in one embodiment, the detection method according to the second aspect of the present disclosure may be a diagnostic aid method or a method for obtaining diagnostic data, in which, in a case where a biological specimen that may contain a detectable substance is used as a sample, if the detectable substance is detected or if a large amount of the detectable substance is detected, the subject from whom the sample was collected is likely to suffer from a disease that causes an increase in the detectable substance. In a preferred embodiment, such diseases include cancer and Alzheimer's disease.

[0121] The present invention will be described in detail below based on examples, but these are given as examples only and the present invention is not limited to these examples in any way.

[0122] Preparation Example 1: Preparation of peptide immunizing antigen Tau150-170. Synthesis of peptides corresponding to amino acid residues 150-170 of human Tau protein was outsourced to Sigma-Aldrich Japan. These were mixed with carrier proteins, bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), at a weight ratio of 1:1 in 50 mM phosphate buffer, and a peptide-carrier crosslinking reaction was carried out using 3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS). Completion of crosslinking between the peptide and carrier protein was confirmed by SDS-PAGE.

[0123] Preparation Example 2: Preparation 2 of peptide immunizing antigens Tau145-190 and Tau145-190(T181ph) Using peptides corresponding to amino acid residues 145 to 190 of human Tau protein (Sigma-Aldrich Japan), two peptides, Tau145-190 and Tau145-190(T181ph), which differ in the phosphorylation state of threonine, the 181st amino acid of Tau protein, were obtained by outsourcing their synthesis to Sigma-Aldrich Japan. These peptides were conjugated to BSA or KLH using a crosslinking reaction with MBS, as in Preparation Example 1.

[0124] Preparation Example 3: Production of rat anti-Tau150-170 monoclonal antibody The human Tau150-170-KLH protein solution (2.5 mg / mL) prepared in Preparation Example 1 was mixed and emulsified with Freund's complete adjuvant FCA at a ratio of 1:2 to obtain an antigen protein solution. 200 μL of this antigen protein solution was administered subcutaneously to both footpads of WKY / Izm rats. Two weeks after the initial administration, an additional 100 μL of the antigen protein solution was administered to the base of the tail of the immunized animals, and three days later, the animals were euthanized and their iliac lymph nodes were isolated. Lymphocytes were isolated from the isolated iliac lymph nodes, and the isolated lymphocytes were fused with mouse myeloma cells Sp2 / 0 according to standard methods to obtain hybridoma cells. These hybridomas were selected by ELISA using the human Tau150-170-BSA protein solution prepared in Preparation Example 1 as an antigen, and finally clones with high affinity and high specificity were obtained.

[0125] Preparation Example 4: Production of rabbit anti-Tau145-190 (T181ph) monoclonal antibody The human Tau145-190 (T181ph)-KLH protein solution (2.5 mg / mL) prepared in Preparation Example 2 was mixed and emulsified with TiterMax Gold (Funakoshi) at a 1:1 ratio to obtain an antigen protein solution. 400 μL of this antigen protein solution was administered subcutaneously to both footpads of JW / CSK rabbits for immunization. Two weeks after the initial immunization, an additional 400 μL of the antigen protein solution was administered to the same sites of the immunized animals, and one week later, the animals were euthanized and their popliteal lymph nodes were isolated. Lymphocytes were isolated from the isolated popliteal lymph nodes, and lymphocytes producing antibodies that strongly reacted with human Tau145-190 (T181ph)-BSA prepared in Preparation Example 2 were isolated from the lymphocytes using a single cell picking system (AS-PS, AS ONE). Antibody genes were isolated and identified from these isolated lymphocytes using single-cell PCR, and then cloned into mammalian expression vectors to enable the production of recombinant antibodies. These recombinant antibodies were produced in cultured cells (HEK293 and CHO), and high-affinity, high-selectivity clones were selected using ELISA.

[0126] Production Example 1 Production of Metal-Based Particle Assembly and Metal-Based Particle Assembly Film-Layered Substrate Using a DC magnetron sputtering device under the conditions below, silver particles were grown very slowly on a soda glass substrate, and a thin film of metal-based particle assembly was formed over the entire surface of the substrate, thereby obtaining a metal-based particle assembly layer-layered substrate.

[0127] Gas used: argon, Chamber pressure (sputtering gas pressure): 10 Pa, Distance between substrate and target: 100 mm, Sputtering power: 4 W, Average grain size growth rate (average grain size / sputtering time): 0.9 nm / min, Average height growth rate (= average deposition rate = average height / sputtering time): 0.25 nm / min, Substrate temperature: 300°C, Substrate size and shape: square with sides of 5 cm.

[0128] Figure 2 is an SEM image of the metal-based particle assembly film in the obtained metal-based particle assembly film-layered substrate, viewed from directly above. Figure 2(a) is an enlarged image at a scale of 10,000 times, and Figure 2(b) is an enlarged image at a scale of 50,000 times. Figure 3 is an AFM image showing the metal-based particle assembly film in the obtained metal-based particle assembly film-layered substrate. A Keyence Corporation's "VN-8010" was used to capture the AFM image (same below). The size of the image shown in Figure 3 is 5 μm × 5 μm.

[0129] From the SEM image shown in Figure 2, the average particle size, based on the above definition, of the silver particles constituting the metal-based particle assembly of this example was determined to be 335 nm, and the average interparticle distance was determined to be 16.7 nm. Furthermore, from the AFM image shown in Figure 3, the average height was determined to be 96.2 nm. From these, the aspect ratio (average particle size / average height) of the silver particles was calculated to be 3.48, and it can also be seen from the acquired image that the silver particles have a flat shape. Furthermore, from the SEM image, the metal-based particle assembly of this example had a particle size of approximately 6.25 x 10 １０ pieces (approximately 25 pieces / μm ２ ) silver particles.

[0130] Furthermore, when the conductivity was checked by connecting a tester (multimeter (E2378A manufactured by Hewlett-Packard Company) to the surface of the metal-based particle assembly film in the obtained metal-based particle assembly film-layered substrate, it was confirmed that the film had no conductivity.

[0131] Example 1 Detection of Tau145-190 (T181ph) by Sandwich ELISA Using a Microplate with a Long-Range Plasmon Substrate Affixing of a Long-Range Plasmon Substrate to a Microplate A long-range plasmon substrate (thickness: 1 mm, size: 25 mm × 25 mm, outermost surface protective coating: SiO ) molded from the metal-based particle assembly film laminated substrate obtained in Preparation Example 1 was attached to the bottom surface of a bottomless 96-well microplate (CS Crea Inc.) to which an acrylic adhesive for bonding had been attached. ２ Since a luminescent substrate is used for ELISA detection using this plate, a seal-type aluminum tape was attached to the plasmon substrate in order to block light transmission.

[0132] [Antibody] The rat anti-Tau150-170 monoclonal antibody clone prepared and selected in Preparation Example 3 was used as the capture antibody (first protein), and the rabbit anti-Tau145-190 (T181ph) monoclonal antibody clone prepared and selected in Preparation Example 4 was used as the primary antibody (second protein) in the following tests. The secondary antibody (third protein) used to detect the primary antibody was a goat anti-rabbit IgG antibody-HRP-labeled (111-035-144, Jackson ImmunoResearch).

[0133] [Immobilization of antibodies onto plates and blocking] The capture antibody was adjusted to 5 μg / mL with 0.2 mM phosphate buffer, and 100 μL of this solution was added per well to the long-range plasmon substrate-attached plate and allowed to stand for 2 hours at 37° C. to achieve immobilization via hydrophobic interactions. The 96-well plate on which immobilization was completed was then washed three times with PBS, and 100 μL of 10% BSA / TBS solution was added per well. Blocking was performed by allowing the plate to stand overnight at 4° C.

[0134] [Detection of Tau145-190 (T181ph)] The blocking solution in a 96-well microplate was removed, and 100 μL of a solution of the Tau145-190 (T181ph) peptide (carrier-free) prepared in Preparation Example 2 diluted 10-fold with 10% BSA / PBS solution at concentrations ranging from 100 ng / mL to 0.01 ng / mL, and a negative control solution containing no antigen, were added to each well and allowed to stand for 1 hour at 25 ° C. After washing three times with PBS, 100 μL of a solution of the primary antibody, rabbit anti-Tau145-190 (T181ph) monoclonal antibody, adjusted to 1 μg / mL with 10% BSA / PBS solution, was added to each well and allowed to stand overnight at 4 ° C. After washing three times with PBS, 100 μL of a solution containing goat anti-rabbit IgG antibody-HRP (secondary antibody) adjusted to 50 ng / mL in 10% BSA / TBS solution was added to each well and allowed to stand at room temperature for 30 minutes. After washing three times with PBS, 25 μL of a luminescence reagent for detecting HRP activity (ELISA-star, Fujifilm Wako Pure Chemical Industries) was added to each well, and activity was measured using a luminescence measurement plate reader, Synergy LX (Agilent) (integration time: 1 sec). The luminescence intensity at each antigen concentration was obtained by subtracting the luminescence intensity value for the negative control from the luminescence intensity value observed at each antigen concentration.

[0135] Comparative Example 1: Detection of Tau145-190 (T181ph) by sandwich ELISA using a glass substrate without a long-range plasmon structure 2 All steps from antibody immobilization to detection were carried out under the same conditions as in Example 1, except that the long-range surface plasmon resonance substrate was changed to a glass substrate provided with only a protective layer.

[0136] The relationship between antigen concentration and luminescence intensity in Example 1 (Control) and Comparative Example 1 (LRLP) is shown in Figure 4. Furthermore, the luminescence intensity at an antigen concentration of 100 ng / mL in Example 1 (Control) and Comparative Example 1 (LRLP) is shown in Figure 5 (n = 3). Comparing the luminescence intensity at an antigen concentration of 100 ng / mL, Example 1, which includes a long-range plasmon substrate, exhibited a 2.6-fold increase in luminescence intensity compared to Comparative Example 1, which lacked a long-range plasmon structure. Furthermore, in Comparative Example 1, when the antigen concentration was reduced to 10 ng / mL, the luminescence intensity decreased to the negative control level, but significant luminescence was observed even at 1 ng / mL in Example 1. In other words, Example 1 demonstrated higher detection sensitivity than Comparative Example 1.

[0137] DESCRIPTION OF SYMBOLS 10... Substrate, 20... Metal-based particle aggregate layer, 21... Metal-based particles, 30... Protective layer, 31... First surface.

Claims

1. A kit for detecting a substance to be detected, comprising: a plasmon resonance chip; a first protein that specifically binds to the substance to be detected; and a second protein that specifically binds to the substance to be detected, and further satisfying the following (i) or (ii): (i) the second protein is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; (ii) the kit further comprises a third protein that binds to the second protein and is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; wherein the plasmon resonance chip comprises: a substrate; a metal-based particle assembly layer formed on the substrate; and a protective layer that covers the metal-based particle assembly layer, wherein the metal-based particle assembly layer is composed of a particle assembly composed of 30 or more metal-based particles that are spaced apart from one another and arranged two-dimensionally, the metal-based particles have an average particle size in the range of 200 to 1600 nm, an average height in the range of 55 to 500 nm, and an aspect ratio, defined as the ratio of the average particle size to the average height, in the range of 1 to 8.

2. A kit for detecting a substance to be detected, comprising: a plasmon resonance chip; a first protein that specifically binds to the substance to be detected; and a second protein that specifically binds to the substance to be detected, and further satisfying the following (i) or (ii): (i) the second protein is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; (ii) the kit further comprises a third protein that binds to the second protein and is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; wherein the plasmon resonance chip comprises: a substrate; a metal-based particle assembly layer formed on the substrate; and a protective layer that covers the metal-based particle assembly layer; wherein the metal-based particle assembly layer is a metal-based particle assembly comprising a plurality of metal-based particles arranged to be spaced apart from one another, the plurality of metal-based particles are arranged such that the average distance between adjacent metal-based particles is 1 nm or more and 1000 nm or less, and the standard deviation of the average distance is 25 nm or less. Kit.

3. A kit for detecting a substance to be detected, comprising: a plasmon resonance chip; a first protein that specifically binds to the substance to be detected; and a second protein that specifically binds to the substance to be detected, and further satisfying the following (i) or (ii): (i) the second protein is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; (ii) the kit further comprises a third protein that binds to the second protein and is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; wherein the plasmon resonance chip comprises: a substrate; a metal-based particle assembly layer formed on the substrate; and a protective layer that covers the metal-based particle assembly layer, wherein the metal-based particle assembly layer is a metal-based particle assembly comprising a plurality of metal-based particles arranged at a distance from one another, and the protective layer has a first surface, and the carbon atom concentration of the first surface measured by X-ray photoelectron spectroscopy is 20.0 atomic % or less.

4. The kit according to any one of claims 1 to 3, wherein the protective layer has a first surface, and the first protein is immobilized on the first surface.

5. A kit according to any one of claims 1 to 3, configured so that in detecting the target substance, the number of molecules contained in a target substance-protein complex formed on the plasmon resonance chip, which complex comprises the target substance, the first protein, and the second protein, is 8 molecules or less per molecule of the target substance.

6. The kit according to any one of claims 1 to 3, wherein the protective layer has an average thickness of 10 nm or more and 300 nm or less.

7. The kit according to any one of claims 1 to 3, wherein the plurality of metal-based particles have an average particle size in the range of 200 nm or more and 1600 nm or less, an average height in the range of 55 nm or more and 500 nm or less, an aspect ratio defined as the ratio of the average particle size to the average height in the range of 1 or more and 8 or less, and in the metal-based particle assembly layer, the plurality of metal-based particles are arranged such that the average distance between adjacent metal-based particles is in the range of 1 nm or more and 150 nm or less.

8. A method for detecting an analyte, comprising the steps of: contacting the analyte with a plasmon resonance chip having a first surface immobilized with a first protein that specifically binds to the analyte; and contacting the plasmon resonance chip after the contacting with the analyte with a second protein that specifically binds to the analyte, and further comprising the step of: (I) the second protein is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; (II) contacting the plasmon resonance chip after the contacting with the second protein with a third protein that binds to the second protein and is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; wherein the plasmon resonance chip comprises: a substrate; a metal-based particle assembly layer formed on the substrate; and a protective layer that covers the metal-based particle assembly layer, the metal-based particle assembly layer comprises a particle assembly including 30 or more metal-based particles arranged two-dimensionally and spaced apart from one another, the metal-based particles having an average particle size in the range of 200 to 1600 nm, an average height in the range of 55 to 500 nm, and an aspect ratio defined as the ratio of the average particle size to the average height in the range of 1 to 8, and the protective layer has the first surface.

9. A method for detecting an analyte, comprising the steps of: contacting the analyte with a plasmon resonance chip having a first surface immobilized with a first protein that specifically binds to the analyte; and contacting the plasmon resonance chip after the contacting with the analyte with a second protein that specifically binds to the analyte, and further comprising the step of: (I) the second protein is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; (II) contacting the plasmon resonance chip after the contacting with the second protein with a third protein that binds to the second protein and is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; wherein the plasmon resonance chip comprises: a substrate; a metal-based particle assembly layer formed on the substrate; and a protective layer that covers the metal-based particle assembly layer, the metal-based particle assembly layer is a metal-based particle assembly made of a plurality of metal-based particles arranged to be spaced apart from one another, the plurality of metal-based particles are arranged such that an average distance between adjacent metal-based particles is equal to or greater than 1 nm and equal to or less than 1000 nm, a standard deviation of the average distance is equal to or less than 25 nm, and the protective layer has the first surface.

10. A method for detecting an analyte, comprising the steps of: contacting the analyte with a plasmon resonance chip having a first surface immobilized with a first protein that specifically binds to the analyte; and contacting the plasmon resonance chip after the contacting with the analyte with a second protein that specifically binds to the analyte, and further comprising the step of: (I) the second protein is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; (II) contacting the plasmon resonance chip after the contacting with the second protein with a third protein that binds to the second protein and is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance; wherein the plasmon resonance chip comprises: a substrate; a metal-based particle assembly layer formed on the substrate; and a protective layer that covers the metal-based particle assembly layer, the metal-based particle assembly layer is a metal-based particle assembly made of a plurality of metal-based particles arranged spaced apart from one another, and the protective layer has the first surface, and a carbon atom concentration of the first surface measured by X-ray photoelectron spectroscopy is 20.0 atomic % or less.

11. The detection method according to any one of claims 8 to 10, further comprising the step of immobilizing the first protein on the first surface of the plasmon resonance chip before the step of contacting the substance to be detected with the plasmon resonance chip.

12. The detection method according to any one of claims 8 to 10, wherein the protective layer has an average thickness of 10 nm or more and 300 nm or less.

13. The detection method according to any one of claims 8 to 10, wherein the plurality of metal-based particles have an average particle size in the range of 200 nm or more and 1600 nm or less, an average height in the range of 55 nm or more and 500 nm or less, an aspect ratio defined as the ratio of the average particle size to the average height in the range of 1 or more and 8 or less, and in the metal-based particle assembly layer, the plurality of metal-based particles are arranged such that an average distance between adjacent metal-based particles is in the range of 1 nm or more and 150 nm or less.

14. The detection method according to any one of claims 8 to 10, wherein the step of contacting the second protein or the third protein, which is modified with a fluorescent substance, a luminescent substance, or an enzyme that produces a fluorescent substance or a luminescent substance, with the plasmon resonance chip comprises adding a solution containing the second protein or the third protein onto the first surface of the plasmon resonance chip so that the height from the first surface to the liquid surface of the solution is less than 500 μm.

Images