Method for producing composite particles, composite particles, base material particles, and base material for sensors

Abstract

Provided is a method for producing composite particles with which it is possible to enhance the light emission intensity of a light-emitting body by forming a plasmon structure in magnetic particles.　A method for producing composite particles comprising base material particles containing a magnetic body, and a metal-based particle assembly layer that is formed on the base material particles, a plurality of metal-based particles being arranged so as to be set away from each other, wherein: the method includes a step for immersing the base material particles in a metal-cation-containing liquid containing metal cations constituting the metal-based particles, and then adding a reducing agent to the metal-cation-containing liquid to form the metal-based particle assembly layer on the base material particles; and when a represents the valence of the metal cations, b represents the valence of anions when the reducing agent is ionized, m (mol) represents the abundance of the metal cations in a liquid obtained by adding the reducing agent to the metal-cation-containing liquid, and r (mol) represents the abundance of the ionized reducing agent in the liquid obtained by adding the reducing agent, relationship (1) is satisfied.

Description

Method for producing composite particles, composite particles, base material particles, and base material for sensors

[0001] The present invention relates to a method for producing composite particles, composite particles, base material particles, and a base material for sensors.

[0002] A technique for enhancing fluorescence utilizing localized plasmon resonance of metal nanoparticles is known (for example, Patent Document 1). Patent Document 1 describes that composite particles including an island layer (2) composed of a plurality of metal particles and a spacer layer (5) covering the layer are used in a photochemical fluorescence sensor.

[0003] Japanese Unexamined Patent Publication No. 08-271431

[0004] Meanwhile, in observation of an analyte using a microtube (for example, enzyme-linked immunosorbent assay (ELISA)), it has been studied to improve the observation efficiency and sensitivity of the analyte by using magnetic particles (particles containing a magnetic material) bound with a specific antibody. The present inventor studied combining plasmon resonance technology with analyte observation using magnetic particles, but it was difficult to form a plasmon structure capable of fluorescence enhancement on the surface of magnetic particles.

[0005] Accordingly, an object of one aspect of the present invention is to provide a method for producing composite particles having a plasmon structure on the surface of base material particles containing a magnetic material. Another object of the present invention is to provide composite particles obtained by the method for producing the composite particles, base material particles used in the method for producing the composite particles, and a sensor base material including the composite particles.

[0006] One aspect of the present invention includes, for example, the following: [1] A method for producing composite particles comprising: base particles containing a magnetic material; and a metal particle aggregate layer formed on the base particles, wherein a plurality of metal particles are spaced apart from each other, the method comprising the steps of: immersing the base particles in a metal cation-containing liquid containing metal cations constituting the metal particles; adding a reducing agent to the metal cation-containing liquid to form the metal particle aggregate layer on the base particles, wherein the manufacturing method satisfies the following formula (1) when the valence of the metal cation is a, the valence of the anion when the reducing agent is ionized is b, the amount of the metal cation in the liquid to which the reducing agent has been added is m (mol), and the amount of the ionized reducing agent in the liquid to which the reducing agent has been added is r (mol).

[0007] [2] The manufacturing method according to [1], wherein the base particles have an insulating layer, and the metal particle aggregate layer is formed on the insulating layer. [3] A composite particle comprising base particles and a metal particle aggregate layer formed on the base particles, wherein the base particles contain a magnetic material, and the metal particle aggregate layer is a layer in which a plurality of metal particles are arranged spaced apart from each other. [4] The composite particle according to [3], wherein the average particle size of the plurality of metal particles is 100 to 1600 nm, the average height is 20 to 500 nm, and the ratio of the average particle size to the average height is 1 to 8. [5] The composite particle according to [3] or [4], wherein the average distance between adjacent metal particles of the plurality of metal particles is 1 to 1000 nm, and the standard deviation of the average distance is 25 nm or less. [6] The composite particle according to any one of [3] to [5], wherein the magnetic material contains at least one selected from the group consisting of iron and iron oxide. [7] A composite particle according to any one of [3] to [6], wherein the base particle comprises an insulating layer, and the metal-based particle aggregate layer is formed on the insulating layer. [8] A composite particle according to [7], wherein the thickness of the insulating layer is 50 nm or more. [9] A composite particle according to any one of [3] to [8], wherein the elemental ratio of the component derived from the metal-based particle, measured by X-ray photoelectron spectroscopy on the surface of the composite particle, is 5 atomic percent or more.

[10] A composite particle according to any one of [3] to [9], wherein the elemental ratio of oxygen, measured by X-ray photoelectron spectroscopy on the surface of the composite particle, is 55 atomic percent or less.

[11] A composite particle according to any one of [3] to

[10] , wherein the elemental ratio of the component derived from the magnetic material, measured by X-ray photoelectron spectroscopy on the surface of the composite particle, is 1 atomic percent or less.

[12] A base particle containing a magnetic material, wherein the elemental ratio of the component derived from the magnetic material, measured by X-ray photoelectron spectroscopy on the surface of the base particle, is 1 atomic percent or less.

[13] A sensor substrate comprising composite particles described in any one of [3] to

[11] .

[0008] According to one aspect of the present invention, a method for producing composite particles having a plasmon structure on the surface of substrate particles containing a magnetic material can be provided. Another aspect of the present invention can provide composite particles obtained by the method for producing the composite particles, substrate particles used in the method for producing the composite particles, and a sensor substrate equipped with the composite particles.

[0009] Figure 1 is a schematic cross-sectional view of a composite particle according to one embodiment of the present invention. Figure 2 is a STEM cross-sectional image of the composite particle obtained in Example 3.

[0010] The present invention is not limited to the following examples.

[0011] <Composite Particles> A composite particle according to one embodiment of the present invention comprises a base particle and a metal-based particle aggregate layer formed on the base particle, wherein the base particle contains a magnetic material, and the metal-based particle aggregate layer is a layer in which a plurality of metal-based particles are arranged spaced apart from each other. According to the composite particle according to one embodiment, the luminescence intensity of a light-emitting body can be enhanced.

[0012] Figure 1 is a schematic cross-sectional view of a composite particle 100 according to one embodiment, the composite particle 100 comprising a base particle 10 and a metallic particle aggregate layer 20 formed on the base particle 10. The base particle 10 also comprises magnetic particles (particles containing magnetic material) 11 and an insulating layer 12 formed to cover the magnetic particles 11.

[0013] (Base Particles) The base particles contain magnetic material. A magnetic material is a substance that undergoes magnetization (orientation of magnetic moments) under the influence of an external magnetic field.

[0014] The shape of the base particles may be, for example, spherical, polyhedral, needle-shaped, etc. From the viewpoint of sample observation, the shape of the base particles is preferably spherical.

[0015] Examples of magnetic materials include ferromagnetic materials (iron, nickel, etc.), ferrimagnetic materials (magnetite (Fe3O4), maghemite (γ-Fe2O3), etc.), paramagnetic materials (aluminum, etc.), diamagnetic materials (gold, etc.), antiferromagnetic materials (α-Fe2O3, etc.), and superparamagnetic materials (iron oxide nanoparticles (Fe3O4, γ-Fe2O3), etc.). Among magnetic materials, at least one selected from the group consisting of ferromagnetic materials, ferrimagnetic materials, and superparamagnetic materials may be included, from the viewpoint of being attracted to a magnet even without an external magnetic field and having excellent operability when observing specimens. From the viewpoint of excellent cost, industrial applicability, and medical suitability, at least one magnetic material selected from the group consisting of iron and iron oxide may also be included.

[0016] The base particles may have an insulating layer. The insulating layer is formed on the outermost surface of the base particles and is formed to enclose particles containing magnetic material (magnetic particles). In this case, the base particles may be particles having a core-shell structure (core-shell particles) comprising a core portion made of magnetic particles and a shell portion made of an insulating layer.

[0017] The insulating layer may be formed on the entire surface of the substrate particles, or on only a portion of the surface of the substrate particles. The coverage rate of the insulating layer may be 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100%, from the viewpoint of facilitating the formation of a metallic particle aggregate layer. From the viewpoint of improving the fluorescence enhancement ratio, a coverage rate of 95% or more of the insulating layer is preferred. The coverage rate of the insulating layer may be calculated from the state of the substrate particles used to produce the composite particles, or from the state of the composite particles. When calculated from the state of the substrate particles, it can be evaluated, for example, by the following method. Using a Helios G4 UX (FIB-SEM) system manufactured by FEI, STEM cross-sectional images of substrate particles are acquired in BF-STEM mode. Ten substrate particles are randomly selected from each STEM cross-sectional image, and the cross-section of each substrate particle is observed. The following formula is calculated: (the outer circumference of the substrate particle minus the length of the portion where the magnetic particles or the magnetic material itself is exposed to the outside) / (the outer circumference of the substrate particle), and this is defined as the "insulating layer coverage rate". When calculating from the state of composite particles, the insulating layer coverage rate of the substrate particles used to produce the composite particles can be considered as the "insulating layer coverage rate". Specifically, it can be calculated by the method described in the examples.

[0018] The insulating layer may contain inorganic materials such as silicon dioxide (silica), silicon nitride, or silicon oxynitride, or organic materials such as epoxy resin, polyimide resin, or acrylic resin. The insulating layer may contain inorganic materials or silicon dioxide, from the viewpoint of easily forming a layer with high hydrophilicity and / or robustness. When the insulating layer contains silicon dioxide, substrate particles having an insulating layer containing silicon dioxide can be obtained by applying a silica sol-gel coating to magnetic particles.

[0019] The thickness of the insulating layer may be 40 nm or more, 50 nm or more, 60 nm or more, 80 nm or more, 100 nm or more, 150 nm or more, or 165 nm or more, from the viewpoint of easily forming a metallic particle aggregate layer and sufficiently covering the magnetic material of the substrate particles, thereby making it easier to further enhance the luminescence intensity of the light emitter. From the viewpoint of excellent operability when observing the sample, it may also be 1000 nm or less, 800 nm or less, 600 nm or less, or 500 nm or less.

[0020] The thickness of the insulating layer can be adjusted by changing the conditions for forming the insulating layer. For example, it can be adjusted by changing the content of the component that forms the insulating layer in the reaction solution, or by repeatedly performing the process of preparing the substrate particles with the insulating layer, as described below. It is preferable to repeatedly perform the process of preparing the substrate particles with the insulating layer so that the material forming the insulating layer adheres efficiently to the substrate particles and most of the material forming the insulating layer can be used for coating. When the substrate particles have an insulating layer made of silicon oxide, it is preferable to perform the process of preparing the substrate particles with the insulating layer two or more times, and more preferably four or more times, from the viewpoint of fluorescence enhancement ratio.

[0021] The thickness of the insulating layer can be measured by the method described in the examples below, but it may also be calculated from images acquired using a TEM (transmission electron microscope) instead of a SEM.

[0022] On the surface of the substrate particles, the elemental ratio of components derived from the magnetic material, as measured by X-ray photoelectron spectroscopy, may be 1 atomic% or less, 0.6 atomic% or less, 0.5 atomic% or less, or 0.1 atomic% or less, from the viewpoint of easily forming a metallic particle aggregate layer and easily enhancing the luminescence intensity of the light emitter. When the elemental ratio of components derived from the magnetic material is 1 atomic% or less, it can be said that the majority of the surface of the substrate particles is composed of an insulating layer, and the magnetic material is sufficiently covered by the insulating layer. By using such substrate particles in the manufacture of composite particles, it is easy to obtain composite particles that can enhance the luminescence intensity of the light emitter. That is, another embodiment of the present invention relates to substrate particles containing a magnetic material, wherein the elemental ratio of components derived from the magnetic material, as measured by X-ray photoelectron spectroscopy on the surface of the substrate particles, is 1 atomic% or less. The elemental ratio of components derived from the magnetic material may be 0 atomic% or more. In this specification, the elemental ratio of a specific component on the surface of the substrate particles is the elemental ratio (atomic %) calculated by narrow-scan analysis using an X-ray photoelectron spectrometer (hereinafter sometimes referred to as XPS). If elements of components derived from magnetic material are not detected, or are below the detection limit, the substrate particles can be said to be particles containing magnetic material.

[0023] The average particle size of the substrate particles may be 0.1 μm or larger, 0.5 μm or larger, or 1 μm or larger, from the viewpoint of easily forming a metallic particle aggregate layer with high plasmon function, or it may be 50 μm or smaller, 30 μm or smaller, or 10 μm or smaller, from the viewpoint of excellent handling when observing the sample. The average particle size of the substrate particles can be measured by observation using a microscope such as a scanning electron microscope (SEM).

[0024] (Metallic particle aggregate layer) The metallic particle aggregate layer is a layer in which multiple metallic particles are arranged spaced apart from each other. The multiple metallic particles may be arranged such that one metallic particle is stacked on top of another. From the viewpoint of not blocking light emission, it is preferable that the multiple metallic particles are not arranged such that one metallic particle is stacked on top of another.

[0025] Multiple metallic particles can constitute a plasmon structure. In this specification, "plasmon structure" means a structure consisting of a layer of metallic particle aggregates. In other words, it is a structure consisting of layers in which multiple metallic particles are spaced apart from each other. Metallic particles and plasmon structures can exhibit plasmon resonance. Plasmon resonance refers to compression and rarefaction waves of free electrons generated by the collective vibration of free electrons in a structure. In particular, in a plasmon structure, multiple metallic particles can constitute a plasmon structure, thereby exhibiting a specific plasmon resonance. For example, when a composite particle is applied to a sensor substrate, the intensity of luminescence (fluorescence, chemiluminescence, etc.) from a light-emitting element that labels the substance to be detected can be enhanced. Therefore, composite particles can be suitably used as a sensor substrate by introducing a capture substance that specifically binds to the substance to be detected onto the composite particle. By applying composite particles to a sensor substrate, the composite particle contains a layer of metallic particle aggregates that is a plasmon structure, and plasmon emission enhancement can be achieved, thereby improving detection sensitivity and detection accuracy.

[0026] To form a plasmon structure from multiple metallic particles, it is preferable that the metallic particles are 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 used as nanoparticles or aggregates of nanoparticles, exhibits a plasmon peak appearing in the ultraviolet to visible light region in absorption spectrum measurements by spectrophotometric method.

[0027] Examples of metallic materials capable of plasmon resonance in the ultraviolet to visible light region include precious metals such as gold, silver, copper, platinum, and palladium; non-precious metals such as aluminum and tantalum; alloys containing metals selected from precious metals and non-precious metals; and metallic compounds (metal oxides, metal salts, etc.) containing metals selected from precious metals and non-precious metals. Among these, precious metals such as gold, silver, copper, platinum, and palladium are preferred as metallic materials capable of plasmon resonance in the ultraviolet to visible light region from the viewpoint of easily enhancing the luminescence intensity of the light emitter, and silver is more preferred from the viewpoint of being inexpensive and having low absorption (small imaginary part of the dielectric function at visible light wavelengths).

[0028] From the viewpoint of fully exhibiting the effect of plasmon resonance and easily enhancing the luminescence intensity of the light emitter, it is preferable that the multiple metallic particles satisfy at least one of the following conditions (1) and (2), and more preferably both conditions. (1) The average particle size of the multiple metallic particles is 100 to 1600 nm, the average height of the multiple metallic particles is 20 to 500 nm, and the ratio of the average particle size to the average height (aspect ratio) is 1 to 8. (2) In the multiple metallic particles, the average distance between adjacent metallic particles is 1 to 1000 nm, and the standard deviation of the average distance is 25 nm or less.

[0029] The average particle size of the multiple metallic particles may be 100 nm or more, 150 nm or more, 200 nm or more, 220 nm or more, 230 nm or more, or 300 nm or more, from the viewpoint of easily enhancing the luminescence intensity of the light emitter. Alternatively, the average particle size of the multiple metallic particles may be 1200 nm or less, 900 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, or 350 nm or less. From the viewpoint of easily enhancing the emission wavelength used in the sensor, the average particle size of the multiple metallic particles is preferably 300 nm or less, more preferably 290 nm or less, even more preferably 270 nm or less, and even more preferably 240 nm or less. The average particle size of the multiple metallic particles may be, for example, 150-1600 nm, 150-1200 nm, 150-600 nm, 200-400 nm, 200-350 nm, 200-300 nm, 200-290 nm, 200-270 nm, or 200-240 nm. It is preferable that the average particle size of the metallic particles be appropriately selected depending on the type of metallic material constituting the metallic particles.

[0030] The average particle size of the multiple metallic particles described above is the average particle size of the selected metallic particles when, in the SEM observation image of the metallic particle aggregate layer of the composite particles, metallic particles are randomly selected, and five random tangent diameters are drawn within the image of each metallic particle (provided that all tangent diameter lines can pass only within the image of the metallic particle, and one of these lines must be the longest line that passes only within the metallic particle), and the average value of these values ​​(hereinafter, this average value will also be called the "average tangent diameter") is taken as the particle size of each metallic particle. The tangent diameter is defined as the distance when the contour (projected image) of a metallic particle is sandwiched between two parallel lines tangent to it (Nikkan Kogyo Shimbun, "Particle Measurement Technology", 1994, p. 5). The average particle size can be measured by the method described in the examples below, but it may also be calculated from images obtained using a TEM (transmission electron microscope) instead of an SEM.

[0031] The average height of the multiple metallic particles may be 20 nm or more, 30 nm or more, 40 nm or more, or 90 nm or more, from the viewpoint of making it easier to enhance the luminescence intensity of the light emitter. Alternatively, the average height of the multiple metallic particles may be 500 nm or less, 400 nm or less, 300 nm or less, or 200 nm or less. The average height of the multiple metallic particles may be 20 to 400 nm, 20 to 300 nm, 30 to 200 nm, or 90 to 200 nm. The average height can be measured by the method described in the examples below, but it may also be calculated from images acquired using a TEM (transmission electron microscope) instead of a SEM.

[0032] The aspect ratio of the multiple metallic particles may be 1 to 8, preferably 1.5 to 8, more preferably 2 to 8, and even more preferably 2.5 to 8, from the viewpoint of more easily enhancing the luminescence intensity of the light-emitting body. The aspect ratio of the metallic particles is defined as the ratio of the average particle size to the average height (average particle size / average height). The metallic particles may be perfectly spherical, but from the viewpoint of more easily enhancing the luminescence intensity of the light-emitting body, it is preferable that they have a flattened shape with an aspect ratio greater than 1.

[0033] In a plurality of metallic particles, the average distance between adjacent metallic particles (hereinafter also referred to as the "average interparticle distance") may be 1 to 1000 nm. In this specification, "adjacent metallic particles" means that when the metallic particle aggregate layer is viewed from the thickness direction of the composite particles, the plurality of metallic particles are adjacent in a two-dimensional direction. By arranging the plurality of metallic particles so that the average interparticle distance is within the above range, it is possible to easily obtain strong plasmon resonance and to further enhance the effect of extending the effective range of plasmon resonance. If the average interparticle distance is small, the electromagnetic field interaction between metallic particles becomes stronger and plasmon resonance is more likely to occur. Therefore, from the viewpoint of fluorescence enhancement ratio, it is preferably 1 to 250 nm, more preferably 1 to 150 nm, even more preferably 1 to 100 nm, even more preferably 1 to 50 nm, and particularly preferably 1 to 20 nm. If the average interparticle distance is 1 nm or more, electron transfer based on the Dexter mechanism is less likely to occur between particles, and the effect of plasmon resonance can be fully exerted. The average interparticle distance can be measured by the method described in the examples below, but it may also be calculated from images acquired using a TEM (transmission electron microscope) instead of a SEM.

[0034] The standard deviation of the average inter-particle distance may be 25 nm or less, 20 nm or less, or 15 nm or less. From the viewpoint of making it easier to further enhance the luminescence intensity of the light emitter, the standard deviation of the average inter-particle distance is preferably 0.1 nm or more, more preferably 0.2 nm or more, and even more preferably 0.3 nm or more. The standard deviation of the average inter-particle distance can be measured by the method described in the examples below, but it may also be calculated from images acquired using a TEM (transmission electron microscope) instead of a SEM.

[0035] From the viewpoint of easily exciting highly effective plasmons and further enhancing the luminescence intensity of the light emitter, it is preferable that the surface of the metallic particles consists of a smooth curved surface, and more preferably that they have a flattened shape consisting of a smooth curved surface. The surface of the metallic particles may contain minute irregularities (roughness), and in this sense, the metallic particles may have an amorphous shape.

[0036] The number of metallic particles forming the metallic particle aggregate layer is usually 10 or more, and preferably 30 or more from the viewpoint of more easily enhancing the luminescence intensity of the light emitter. A metallic particle aggregate layer formed by 10 or more metallic particles is prone to exhibiting strong plasmon resonance and extension of the plasmon resonance range due to interactions between localized plasmons of the metallic particles. The number of metallic particles forming the metallic particle aggregate layer may be, for example, 50 or more, 1000 or more, or 10000 or more. The number density of metallic particles in the metallic particle aggregate layer is preferably 7 particles / μm from the viewpoint of more easily enhancing the luminescence intensity of the light emitter. 2 More preferably, 15 particles / μm 2 That's all.

[0037] The metallic particle aggregate layer is preferably non-conductive as a layer, and more preferably, the metallic particles constituting the metallic particle aggregate layer are non-conductive between adjacent metallic particles. The non-conductivity between metallic particles in the metallic particle aggregate layer allows the plasmon resonance effect to be fully exhibited. Therefore, from the viewpoint of more easily enhancing the luminescence intensity of the light emitter, it is preferable that the metallic particles are reliably separated and that no conductive material is interposed between them. The metallic particles themselves may be conductive.

[0038] The fact that the metallic particle aggregate layer does not exhibit conductivity as a layer can be confirmed, for example, by bringing a pair of tester probes of a multimeter (e.g., Hewlett-Packard's "E2378A") into contact with the metallic particle aggregate layer at a distance of 10 mm to 15 mm, and when the range setting is "30 MΩ", the resistance value under those measurement conditions is 30 MΩ or higher, resulting in the display of "Overload".

[0039] (Protective layer) The composite particles may further include a protective layer formed so as to cover the surface of each metal-based particle in the metal-based particle aggregate layer. Including the protective layer in the composite particles is advantageous in the following aspects. [A] When metal-based particles are used as a luminescence enhancement element for enhancing the intensity of luminescence from a luminescent substance that labels a target substance to be detected, if the luminescent substance is in direct contact with the metal-based particles, quenching may occur due to electron tunneling from the luminescent substance to the metal-based particles, which may reduce the enhancement effect. Providing a protective layer on the metal-based particles can reliably separate the luminescent substance from the metal-based particles, thereby suppressing quenching and tending to result in more excellent luminescence enhancement characteristics. [B] The stability (such as oxidation resistance) and environmental resistance stability (for example, light resistance, humidity resistance, heat resistance, etc.) of the metal-based particles can be improved.

[0040] It is preferable that the protective layer is in direct contact with the surface of the metal-based particle. Usually, since a part of the surface of the metal-based particle is in contact with the base particle, the protective layer can cover at least a part of the portion of the surface of the metal-based particle that is not in contact with the base particle, and it is preferable that the protective layer covers the entire portion of the surface of the metal-based particle that is not in contact with the base particle. From the viewpoint of suppressing deterioration of the metal-based particles even when phosphate buffered saline (PBS) is used as a solvent, it is preferable that the metal-based particles do not have a surface that is not covered by either the base particle or the protective layer (that is, a surface exposed to the outside).

[0041] The protective layer may have a single-layer structure or a multi-layer structure. When the protective layer has a multi-layer structure, the protective layer may include a plurality of layers formed by different film formation methods. When the protective layer includes a first protective layer and a second protective layer, the exposed surface area of the metal-based particles can be reduced, deterioration of the metal-based particles can be suppressed even when PBS is used as a solvent, and a decrease in the luminescence intensity of the luminescent substance can be suppressed.

[0042] If the protective layer has a first protective layer and a second protective layer, the average thickness of the first protective layer may be, for example, 3 nm or more, 10 nm or more, 15 nm or more, 20 nm or more, or 25 nm or more, and may be 200 nm or less, 150 nm or less, 100 nm or less, or less than 50 nm. The average thickness of the second protective layer may be, for example, 1 nm or more, 3 nm or more, 5 nm or more, 10 nm or more, or 15 nm or more, and may be 200 nm or less, 150 nm or less, 100 nm or less, or less than 50 nm.

[0043] The surface shape of the protective layer may follow the surface shape of the metallic particles, from the viewpoint of more easily enhancing the luminescence intensity of the light-emitting material. For the surface shape of the protective layer to follow the surface shape of the metallic particles means that, in a cross-sectional view of the composite particles, the surface shape of the protective layer is approximately parallel to the surface shape of the metallic particles.

[0044] More specifically, in the surface shape of the protective layer described above, the thickness of the protective layer measured in a direction perpendicular to the surface of the substrate particles (i.e., the distance from the interface between the protective layer and the metal-based particles, or the interface between the protective layer and the substrate particles, to the surface of the protective layer opposite to the substrate particles) is preferably substantially uniform from the viewpoint of making it easier to enhance the luminescence intensity of the light-emitting element. A substantially uniform thickness of the protective layer means that the coefficient of variation (CV value) of the thickness of the protective layer, i.e., (standard deviation of the thickness of the protective layer / mean value of the thickness of the protective layer) is 20% or less.

[0045] The standard deviation and average value of the protective layer thickness can be determined as follows. Specifically, a cross-sectional image in a direction perpendicular to the surface of the substrate particles is acquired using a scanning microscope or the like. The cross-sectional image is an image obtained when observing a cross section at a magnification such that 10 to 250 metal-based particles are included in one screen. In the cross-sectional image, the direction along the surface of the substrate particle is divided into 51 equal parts, and surface points m (m is an integer from 1 to 50) are obtained in order from the left end of the cross-sectional image for the surface of the protective layer present on the equally dividing lines (the surface on the opposite side from the substrate particles). A straight line that minimizes the length from each surface point m to the surface of the metal-based particle or substrate particle is drawn. The length of the straight line drawn from each surface point is defined as protective layer thickness m (m is an integer from 1 to 50). The average value of the 50 protective layer thicknesses m is taken as the average thickness of the protective layer. Further, the standard deviation of the 50 protective layer thicknesses m is taken as the standard deviation of the protective layer thickness. The coefficient of variation (CV value) of the protective layer thickness is calculated from (standard deviation of protective layer thickness / average thickness of protective layer).

[0046] The coefficient of variation (CV value) of the protective layer thickness is preferably 18% or less, and more preferably 15% or less, from the viewpoint of obtaining more excellent emission enhancement characteristics due to increased uniformity of the protective layer thickness.

[0047] It is considered that a major factor for long-range plasmons is that the Whispering Gallery Mode propagating on the surface of individual metal-based particles resonates with that of adjacent particles. In the Whispering Gallery Mode propagating along the surface of a metal-based particle, the surface shape of the protective layer follows the surface shape of the metal-based particle. That is, it is presumed that when the thickness of the protective layer covering the metal-based particles is uniform, there is less disturbance in particle surface propagation and inter-particle propagation than when the metal-based particles are covered with a protective layer having non-uniform thickness, and inter-particle resonance between adjacent particles, which is a factor for long-range plasmons, is more likely to be expressed.

[0048] The average thickness of the protective layer may be, for example, 3 nm or more, 10 nm or more, 15 nm or more, 20 nm or more, 30 nm or more, or 40 nm or more. The average thickness of the protective layer may be, for example, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, 80 nm or less, or less than 50 nm.

[0049] The material forming the protective layer is preferably a non-conductive material, i.e., an insulating material. Examples of insulating materials include inorganic insulating materials such as SiO2, SiN, TiO2, Al2O3, and Si3N4; and organic insulating materials such as resin materials (e.g., polystyrene, acrylic resin, epoxy resin, etc.). The protective layer may be composed of two or more materials.

[0050] The protective layer may be a silicon atom-containing layer. If the protective layer is a silicon atom-containing layer, the elemental ratio of carbon measured by X-ray photoelectron spectroscopy on the surface of the protective layer (the surface opposite to the substrate particles) may be 20.0 atomic percent or less. The elemental ratio of carbon is the elemental ratio (atomic percent) calculated by narrow-scan analysis using XPS.

[0051] The protective layer is preferably amorphous. The amorphous nature of the protective layer can be confirmed by HAADF-STEM and electron diffraction patterns. Specifically, the amorphous nature of the protective layer can be confirmed by the absence of periodic contrast originating from crystals when an electron diffraction pattern is obtained in the area corresponding to the protective layer in a cross-sectional STEM image.

[0052] The composite particles may have yet another layer on top of the protective layer.

[0053] On the surface of the composite particles, the elemental ratio of components derived from metallic particles, as measured by X-ray photoelectron spectroscopy, may be 5 atomic% or more, 7 atomic% or more, 9 atomic% or more, or 10 atomic% or more, from the viewpoint of more easily enhancing the emission intensity of the emitter. An elemental ratio of 5 atomic% or more for components derived from metallic particles indicates that a sufficient number of metallic particles are arranged on the surface of the substrate particles. The elemental ratio of components derived from metallic particles may be 30 atomic% or less, 20 atomic% or less, 17 atomic% or less, or 15 atomic% or less. In this specification, the elemental ratio of a specific component on the surface of the composite particles is the elemental ratio (atomic%) calculated by XPS. Furthermore, if the composite particles are equipped with a protective layer, the elemental ratio (atomic%) is calculated from the values ​​obtained by the following measurement conditions after drilling the protective layer with a gas cluster ion beam (GCIB) until elements derived from the metallic particle aggregate layer (e.g., silver) are detected. (GCIB Measurement Conditions) Equipment: Thermo Fisher, K-Alpha X-ray source: Al-Kα (12kV / 6mA) X-ray size: 400μmφ Neutralizing gun: Yes (Beam / Emission / Focus / Extractor = 0.40V / 120μA / 25V / 40V) GCIB: Yes (Ion Energy, Cluster size, Raster size, Etch times = 6000eV / 300 / 1mm / 15s×200) Measurement mode: Snapshot mode

[0054] On the surface of the composite particles, the elemental ratio of oxygen measured by X-ray photoelectron spectroscopy may be 55 atomic% or less, 50 atomic% or less, or 45 atomic% or less, from the viewpoint of more easily enhancing the emission intensity of the light emitter. An elemental ratio of 55 atomic% or less of oxygen indicates that the surface of the substrate particles is sufficiently covered by the metallic particle aggregate layer. The elemental ratio of oxygen may also be 10 atomic% or more, 15 atomic% or more, 20 atomic% or more, or 25 atomic% or more.

[0055] On the surface of the composite particles, the elemental ratio of components derived from the magnetic material, as measured by X-ray photoelectron spectroscopy, may be 1 atomic percent or less, 0.5 atomic percent or less, 0.3 atomic percent or less, or 0.1 atomic percent or less, from the viewpoint of more easily enhancing the luminescence intensity of the light emitter. When the elemental ratio of components derived from the magnetic material is 1 atomic percent or less, it can be said that the majority of the surface of the composite particles is composed of an insulating layer or a metallic particle aggregate layer. The elemental ratio of components derived from the magnetic material may be 0 atomic percent or more.

[0056] (Effects) The composite particles according to this embodiment exhibit strong plasmon resonance in the metallic particle aggregate layer, and therefore, for example, a strong luminescence enhancement effect can be obtained. This effect is thought to be caused by the interaction between localized plasmons exhibited by multiple metallic particles.

[0057] The strength of the plasmon resonance exhibited by a composite particle is not simply the sum of the localized plasmon resonances exhibited by individual metallic particles at a specific wavelength, but rather a strength greater than that. In a composite particle, strong plasmon resonance is generated through the interaction of individual metallic particles. Such strong plasmon resonance is thought to arise from the interaction between localized plasmons of metallic particles.

[0058] Generally, when the absorption spectrum of a plasmon structure is measured by spectrophotometry, a plasmon resonance peak (hereinafter also referred to as the "plasmon peak") is observed as the peak at the longest wavelength in the ultraviolet to visible light region. The strength of the plasmon resonance of a 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. In particular, when the absorption spectrum of a metal-based particle aggregate layer having the above-described structure is measured by the spectrophotometry method described below, the absorbance at the maximum wavelength of the plasmon peak at the longest wavelength in the ultraviolet to visible light region may be 1 or more, 1.5 or more, or about 2.

[0059] The absorption spectrum of a plasmon structure can be measured by spectrophotometry. Specifically, the absorption spectrum is obtained by irradiating the back side (opposite side from the metallic particle aggregate layer) of a composite particle with a layer of metallic particle aggregates with incident light in the ultraviolet to visible light region from a direction perpendicular to the surface of the substrate particle, and measuring the intensity I of the transmitted light in all directions that has passed through to the metallic particle aggregate layer side, and by irradiating the same incident light from a direction perpendicular to the surface of a substrate particle of the same size and material as the substrate particle of the measurement sample, but without a layer of metallic particle aggregates, and measuring the intensity I0 of the transmitted light in all directions that has passed through from the opposite side of the incident surface, using an integrating sphere spectrophotometer. In this case, the absorbance, which is the vertical axis of the absorption spectrum, is given by the following formula: Absorbance = -log 10 It is expressed as (I / I0). The absorbance spectrum can be measured using a general spectrophotometer.

[0060] When measuring the maximum wavelength of the plasmon peak at the longest wavelength end in the ultraviolet-visible light region, and the absorbance at that maximum wavelength, an absorbance spectrum measurement may be performed using an objective lens and a spectrophotometer, with the measurement field narrowed.

[0061] <Method for Manufacturing Composite Particles> The composite particles described above can be obtained by the following method. That is, another embodiment of the present invention is a method for manufacturing composite particles comprising a base particle containing a magnetic material, and a metal particle aggregate layer formed on the base particle, wherein a plurality of metal particles are spaced apart from each other. The method for manufacturing composite particles comprises the steps of immersing the base particle in a metal cation-containing solution containing metal cations constituting the metal particles, and reducing the metal cations with a plating solution to which a reducing agent has been added to the metal cation-containing solution, thereby forming a metal particle aggregate layer on the base particle. When the valence of the metal cation is a, the valence of the anion when the reducing agent is ionized is b, the amount of metal cations in the plating solution is m (mol), and the amount of ionized reducing agent in the plating solution is r (mol), the following formula (1) is satisfied.

[0062]

[0063] The base particles may have an insulating layer. In this case, base particles having an insulating layer can be obtained, for example, by immersing magnetic particles (particles containing a magnetic material) in a reaction solution that forms the insulating layer. That is, the method for producing composite particles may further include a step of preparing base particles having an insulating layer, and the step of preparing base particles having an insulating layer may include immersing magnetic particles in a reaction solution that forms the insulating layer.

[0064] More specifically, when the base particles have an insulating layer made of silicon oxide, base particles with an insulating layer made of silicon oxide can be obtained by immersing magnetic particles in a reaction solution containing ammonia and tetraethyl orthosilicate, and heating and stirring while performing ultrasonic treatment. When forming an insulating layer containing elements other than silicon oxide, base particles with an insulating layer can be obtained, for example, by a sol-gel method using a metal-organic compound containing metal elements other than silicon as a raw material. In addition to the sol-gel method, base particles with an insulating layer can be obtained by dry methods, particularly powder sputtering and powder ALD methods. The sol-gel method is preferred from the viewpoint of high productivity because it can be applied in a liquid layer.

[0065] While commercially available magnetic particles contain magnetic material embedded in plastics, silica, etc., the magnetic material in such commercially available magnetic particles may be exposed. When the magnetic material is exposed, the reducing agent used to form the metallic particle aggregate layer is consumed to reduce the metallic components contained in the magnetic material, making it difficult to form the metallic particle aggregate layer on the magnetic particles. However, if the base particle has an insulating layer, the consumption of the reducing agent by the metallic components contained in the magnetic material is suppressed, making it easier to form the metallic particle aggregate layer.

[0066] Next, a layer of metallic particle aggregates is formed on the surface of the substrate particles. The metallic particle aggregate layer is formed by immersing the substrate particles in a metal cation-containing liquid and then adding a reducing agent. From the viewpoint of facilitating the uniform formation of the metallic particle aggregate layer, stirring may be performed after immersing the substrate particles in the metal cation-containing liquid.

[0067] Plating solutions contain metal cations that make up the metal particles. For example, if the metal particles contain silver, the plating solution contains silver cations, and more specifically, the plating solution contains silver nitrate, silver cyanide, etc.

[0068] The plating solution further contains a reducing agent. The reducing agent is capable of reducing the metal cations contained in the plating solution, which then ionize into anions. For example, if the metallic particles contain silver, examples of reducing agents include glucose, formaldehyde, ascorbic acid, sodium tartrate, and sodium sulfite. The type of reducing agent can be determined by considering the type of metal cations contained in the plating solution, the formation rate of the metallic particle aggregate layer, and other factors.

[0069] When the valence of a metal cation is a, the valence of the anion when the reducing agent is ionized is b, the amount of metal cation present in the plating solution is m (mol), and the amount of ionized reducing agent present in the plating solution is r (mol), then the value of [(m × a) / (r × b)] expressed by equation (1) is greater than 50. When the plating solution is prepared using an aqueous silver nitrate solution as the metal cation source, the amount of metal cation present in the plating solution m can be considered as the product of the concentration of the aqueous silver nitrate solution and the amount of aqueous silver nitrate solution added. When the plating solution is prepared using an aqueous glucose solution as the reducing agent, the amount of ionized reducing agent present in the plating solution r can be considered as the product of the concentration of the aqueous glucose solution and the amount of aqueous glucose solution added.

[0070] A value of [(m × a) / (r × b)] greater than 50 allows for a lower reaction rate of the reducing agent to the metal cation, making it easier to uniformly form a metal particle aggregate layer on the surface of the substrate particles. Furthermore, it becomes easier to form the metal particle aggregate layer even when there is a large variation in the particle size of the substrate particles.

[0071] The value of [(m × a) / (r × b)] may be 70 or more, 90 or more, or 150 or more, from the viewpoint of facilitating the uniform formation of a metallic particle aggregate layer on the surface of the base particles.

[0072] The plating solution may further contain additives other than reducing agents. For example, the plating solution may contain buffering agents, leveling agents, organic solvents, etc.

[0073] If the composite particles have a protective layer on a metal-based particle aggregate layer, the method for manufacturing the composite particles may further include a step of forming the protective layer on the metal-based particle aggregate layer. The method for forming the protective layer can be the same as the method for forming the insulating layer described above (e.g., the sol-gel method). From the viewpoint of forming a protective layer having a surface shape that follows the surface shape of the metal-based particles, dry film formation methods such as vapor deposition, sputtering, ion plating, CVD, and ALD; and wet film formation methods such as spray coating can be used. The sol-gel method is preferred from the viewpoint of high productivity because it can be applied in a liquid layer.

[0074] From the viewpoint of forming a surface shape that follows the surface shape of metallic particles, it is preferable to increase the energy of the composition ejected from the target (sputtered particles) by using a high power output in RF sputtering. For example, the discharge output (power) is 200 W or more, preferably 500 W or more, and more preferably 1000 W or more. By increasing the energy of the sputtered particles, the protective layer can be densified while improving its ability to follow the underlying structure, and high-speed film deposition becomes possible. RF sputtering is preferably carried out in an inert gas atmosphere such as argon gas, and it is preferable not to add oxygen to the inert gas atmosphere.

[0075] The protective layer may be a single-layer or multi-layer structure. If the protective layer is a multi-layer structure, it may have multiple layers formed by different deposition methods. For example, the protective layer may have a first protective layer formed on the surface of the metal particles by sputtering, and a second protective layer formed on the surface of the first protective layer by ALD, vacuum deposition, or ion plating. When the protective layer has a first protective layer and a second protective layer, the exposed surface of the metal particles can be reduced, thereby suppressing the degradation of the metal particles and preventing a decrease in the luminescence intensity of the light emitter.

[0076] <Sensor Substrates> Composite particles can be used as sensor substrates for sensors that detect substances to be detected. For example, a sensor substrate can be created by introducing a capture substance that specifically binds to the substance to be detected onto the composite particles.

[0077] The sensor can, for example, detect a substance to be detected. Detection may be qualitative or quantitative, meaning, for example, identification or quantification of the substance to be detected. The substance to be detected is identified or quantified by detecting the emission of a light-emitting element such as fluorescence or chemiluminescence, by labeling the substance to be detected with an antibody that recognizes the substance to be detected, or by labeling a secondary antibody of the antibody that recognizes the substance to be detected with an antibody that recognizes the substance to be detected, and then labeling the substance to be detected with an antibody that recognizes the substance to be detected. Preferably, the metallic particle aggregate layer of the sensor substrate, which is a plasmon structure, resonates with the light-emitting element, resulting in enhanced plasmon emission. By detecting this emission using a detector, the substance to be detected can be detected qualitatively or quantitatively. Therefore, the amount of the substance to be detected can be measured qualitatively or quantitatively by measuring the emission intensity.

[0078] According to the sensor comprising composite particles of the present invention, the composite particles include a layer of metallic particle aggregates that are plasmon structures, and since plasmon emission enhancement can be achieved, detection sensitivity and detection accuracy can be improved.

[0079] The substance to be detected is a substance that is the target of qualitative or quantitative detection and is a substance that specifically binds to the capture substance.

[0080] "Specific bonding" broadly refers to chemical bonds between substances, including non-covalent bonds, covalent bonds, and hydrogen bonds, such as interactions between protein molecules and electrostatic interactions between molecules.

[0081] The detection of the captured substance can be performed by pre-labeling the substance with a luminescent label and detecting the luminescence of the label itself or the luminescence of a luminescent material produced by a reaction of the label. The label may be a labeling material that specifically binds to a complex obtained by the specific binding of the captured substance and the substance to be detected. The luminescent material is a material that emits light upon injection of excitation energy by excitation light or through a chemical reaction. The principle of luminescence in the luminescent material is not limited and includes fluorescence, phosphorescence, chemiluminescence, etc. Conventionally known luminescent materials can be used.

[0082] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0083] (1) Preparation of base particles (Base particle 1) Magnetic particles (iron oxide particles) in a magnetic particle dispersion (manufactured by BIONEER CORPORATION, AccuBead, particle size: 1-5 μm) were precipitated by natural sedimentation, the precipitate was separated, the solvent remaining in the precipitate was removed on a hot plate at 100°C, and then the magnetic particle powder (iron oxide powder) was obtained by drying in a vacuum dryer for 24 hours.

[0084] An ammonia solution was obtained by dissolving 19 mL of a 25% by mass aqueous solution of ammonia (NH3) in 80 mL of ethanol. After raising the temperature of the ammonia solution to 50°C, 400 mg of magnetic particle powder and 64 μL of tetraethyl orthosilicate were added to the 50°C ammonia solution while stirring to prepare the reaction mixture. The reaction mixture was stirred at 50°C for 4 hours while being subjected to sonication. Next, the supernatant was removed using a centrifuge, washed with water, and then dried at 80°C to obtain base particle 1 in which an insulating layer of SiO2 was formed on the magnetic particles. The thickness of the insulating layer in base particle 1 was 69.5 nm.

[0085] (Base particle 2) Base particle 2 was obtained in the same manner as base particle 1, except that the amount of tetraethyl orthosilicate was changed to 108 μL. The thickness of the insulating layer in base particle 2 was 88.6 nm.

[0086] (Base particle 3) An ammonia solution was obtained by dissolving 19 mL of a 25% by mass aqueous solution of ammonia (NH3) in 80 mL of ethanol. After raising the temperature of the ammonia solution to 50°C, 400 mg of base particle 1 and 108 μL of tetraethyl orthosilicate were added to the ammonia solution at 50°C while stirring to prepare the reaction solution. The reaction solution was stirred at 50°C for 4 hours while being subjected to sonication. Next, the supernatant was removed using a centrifuge, washed with water, and then dried at 80°C. This operation was repeated a total of four times to obtain base particle 3. The thickness of the insulating layer in base particle 3 was 169 nm.

[0087] (2) Preparation of composite particles (Comparative example 1) 563 μL of 0.05 mol / L potassium hydroxide (KOH) aqueous solution was added dropwise to 112.5 mL of 0.12 mol / L silver nitrate (AgNO3) aqueous solution and then stirred. The solution changed from colorless and transparent to brown upon addition of potassium hydroxide aqueous solution. 1377 μL of 3.5 mol / L ethylenediamine (NH2CH2CH2NH2) aqueous solution was added dropwise to this solution while stirring, and the resulting solution was designated as silver ion solution A.

[0088] 37.35 mL of a 0.0075 mol / L glucose aqueous solution and 12.6 mL of methanol were mixed to obtain reducing agent solution B. Silver ion solution A was added to the tank, and then base material particles 3, which were to be used as base material particles, were added to the tank. Immediately after adding reducing agent solution B, the tank was stirred to immerse the base material particles 3 in the plating solution consisting of silver ion solution A and reducing agent solution B, and the growth of metallic particles made of silver on the surface of the base material particles 3 was initiated. At this time, the value of [(m × a) / (r × b)], expressed by the above formula (1), was 48.2. Here, a is the valence of the silver ion, and b is the valence of the glucose-derived anion (glucoside ion). m is the amount of silver ions present, which can be calculated by the product of the concentration of the silver nitrate aqueous solution and the amount of the silver nitrate aqueous solution, and r is the amount of glucoside ions, which can be calculated by the product of the concentration of the glucose aqueous solution and the amount of the glucose aqueous solution. The values ​​of a, b, m, and r are shown in Table 1. The plating solution was prepared and the substrate particles 3 were immersed in the plating solution under conditions of 25°C. Stirring was continued at 25°C. 28 minutes after the start of immersion, the substrate particles 3 (laminated material) having a layer of metallic particles was filtered off. This filtrate was then washed with a 1:1 volume mixture of acetone and ultrapure water. After removing the supernatant using a centrifuge, the material was dried at 80°C to obtain composite particles R1. The composite particles R1 consisted of linked metallic particles, and the metallic particles were not spaced apart.

[0089] (Example 1) 563 μL of 0.05 mol / L potassium hydroxide (KOH) aqueous solution was added dropwise to 112.5 mL of 0.12 mol / L silver nitrate (AgNO3) aqueous solution, and the mixture was stirred. The solution changed from colorless to brown upon addition of the potassium hydroxide aqueous solution. 1377 μL of 3.5 mol / L ethylenediamine (NH2CH2CH2NH2) aqueous solution was added dropwise to this solution while stirring to obtain silver ion solution A.

[0090] Reducing agent solution B was obtained by mixing 37.35 mL of a 0.001875 mol / L glucose aqueous solution with 12.6 mL of methanol. After adding base particle 1 to silver ion solution A, reducing agent solution B was added, and the mixture of silver ion solution A and reducing agent solution B (plating solution) was immediately stirred to initiate the growth of silver-based metallic particles on the surface of base particle 1 immersed in the plating solution. At this time, the value of [(m × a) / (r × b)], expressed by the above formula (1), was 192.8. Here, a is the valence of the silver ion, and b is the valence of the glucose-derived anion (glucoside ion). m is the amount of silver ions present, which can be calculated by the product of the concentration of the silver nitrate aqueous solution and the amount of the silver nitrate aqueous solution, and r is the amount of glucoside ions, which can be calculated by the product of the concentration of the glucose aqueous solution and the amount of the glucose aqueous solution. The values ​​of a, b, m, and r are shown in Table 1. The preparation of the plating solution and the immersion of base particle 1 into the plating solution were carried out in an environment of 25°C. The plating solution was stirred continuously at 25°C, and the particles were filtered off 28 minutes after the start of immersion. Next, the filtered material was washed with a solution of acetone and ultrapure water in a 1:1 volume ratio, the supernatant was removed using a centrifuge, and then the material was dried at 80°C to obtain composite particles E1. A layer of metallic particle aggregates, in which multiple metallic particles were arranged spaced apart from each other, was formed on the surface of composite particles E1.

[0091] (Example 2) Composite particle E2 was obtained in the same manner as in Example 1, except that base particle 1 was changed to base particle 2.

[0092] (Example 3) Composite particle E3 was obtained in the same manner as in Example 1, except that base particle 1 was changed to base particle 3.

[0093] (Example 4) Composite particle E4 was obtained in the same manner as in Example 1, except that base particle 1 was changed to base particle 3 and the concentration of the glucose aqueous solution was set to 0.00375 mol / L. At this time, the value of [(m × a) / (r × b)] represented by the above formula (1) was 96.4. The values ​​of a, b, m, and r are shown in Table 1. A layer of metallic particle aggregates, in which multiple metallic particles are arranged spaced apart from each other, was formed on the surface of composite particle E4. The average particle size was 292.6 nm and the average interparticle distance was 228.5 nm.

[0094] (Example 5) Composite particle E5 was obtained in the same manner as in Example 1, except that base particle 1 was changed to base particle 3 and the concentration of the glucose aqueous solution was set to 0.0009375 mol / L. At this time, the value of [(m × a) / (r × b)] represented by the above formula (1) was 385.5. The values ​​of a, b, m, and r are shown in Table 1. A layer of metal-based particles, in which multiple metal-based particles are arranged spaced apart from each other, was formed on the surface of composite particle E5.

[0095] [Evaluation of Fluorescence Intensity of Composite Particles Having Substrate Particles 3] A spin-on-glass (SOG) solution (manufactured by Tokyo Ohka Kogyo Co., Ltd., product name: OCD T-7 5500T) was spin-coated onto a glass substrate at 3000 rpm, and then substrate particles 3 were placed on top of the SOG coating. Next, the SOG coating was dried at 200°C to fix the substrate particles 3. Then, a rhodamine B solution was spin-coated onto the surface on which the substrate particles 3 were fixed at 2000 rpm for 100 seconds to support the fluorescent dye rhodamine B on the surface of the substrate particles 3. This was used as a comparative sample. As the rhodamine B solution, a 0.15 mM rhodamine B solution prepared by dissolving "Rhodamine B" manufactured by Exciton in ethanol was used.

[0096] Separately, the composite particle E1 was supported with the fluorescent dye rhodamine B on its surface using the same method as described above, except that the base particle 3 was replaced with composite particle E1. This was used as measurement sample 1.

[0097] Separately, the fluorescent dye rhodamine B was supported on the surface of composite particle E2 using the same method as described above, except that the base particle 3 was changed to composite particle E2. This was used as measurement sample 2.

[0098] Separately, the composite particle E3 was modified by replacing the base particle 3 with composite particle E3, and the same method as described above was used to support the surface of the composite particle E3 with the fluorescent dye rhodamine B. This was used as the measurement sample 3.

[0099] Separately, the composite particle E4 was modified by replacing the base particle 3 with composite particle E4, and the fluorescent dye rhodamine B was then supported on its surface using the same method as described above. This was used as measurement sample 4.

[0100] Separately, the composite particle E5 was modified by the same method as described above, except that the base particle 3 was replaced with composite particle E5. This was used as measurement sample 5.

[0101] Separately, the fluorescent dye rhodamine B was supported on the surface of composite particle R1 using the same method as described above, except that the base particle 3 was changed to composite particle R1. This was used as the reference sample.

[0102] For the comparison sample, measurement samples 1-5, and reference sample, fluorescence observation was performed using a fluorescence microscope (Nikon, product name: ECLIPSE Ni-L) through a fluorescence filter (long-pass filter ET570lp). The fluorescence intensity emitted from rhodamine B supported on substrate particle 3 (comparative sample), rhodamine B supported on composite particle E1 (measurement sample 1), rhodamine B supported on composite particle E2 (measurement sample 2), rhodamine B supported on composite particle E3 (measurement sample 3), rhodamine B supported on composite particle E4 (measurement sample 4), rhodamine B supported on composite particle E5 (measurement sample 5), and rhodamine B supported on composite particle R1 (reference sample) was measured.

[0103] In the fluorescence images obtained from the measurement of the comparison sample, 10 substrate particles 3 were randomly selected, and the average value of their signal intensities was calculated. Using this average value minus the background value, the comparative fluorescence intensity was calculated according to the following formula (A). Similarly, the average value of the signal intensities from the measurements of measurement samples 1 to 5 was calculated, and using this average value minus the background value, the fluorescence intensity was calculated according to the following formula (B), and these were defined as fluorescence intensities 1 to 5. Similarly, the average value of the signal intensities from the measurement of the reference sample was calculated, and using this average value minus the background value, the reference fluorescence intensity was calculated according to the following formula (C). Comparative fluorescence intensity = Average signal intensities of comparison samples - Background value of comparison samples (A) Fluorescence intensity = (Average signal intensities of measurement samples - Background value of measurement samples) / Comparative fluorescence intensity (B) Reference fluorescence intensity = (Average signal intensities of reference samples - Background value of reference samples) / Comparative fluorescence intensity (C)

[0104] The background values ​​were determined as follows: Fluorescence intensity was measured on untreated glass substrates (the same substrates used for the comparison sample, measurement samples 1-5, and the reference sample) under the same conditions as described above to obtain fluorescence images. Ten points were randomly selected from the obtained fluorescence images, and the average of their signal intensities was calculated. This average value was used as the background value.

[0105] Compared to the fluorescence intensity of the comparison sample, the fluorescence intensity of measurement sample 1 was 1.3 times, the fluorescence intensity of measurement sample 2 was 3.0 times, the fluorescence intensity of measurement sample 3 was 5.9 times, the fluorescence intensity of measurement sample 4 was 1.8 times, and the fluorescence intensity of measurement sample 5 was 2.5 times. The reference fluorescence intensity of the reference sample was 1.2 times.

[0106] For the obtained fluorescence intensities 1 to 5, the percentage of fluorescence intensity enhanced by the formation of the plasmon structure (hereinafter also referred to as the "fluorescence enhancement ratio") was calculated using the following formula (D). The obtained fluorescence enhancement ratios are shown in Table 1. Fluorescence enhancement ratio (%) = (fluorescence intensity / reference fluorescence intensity) × 100 ... (D)

[0107] [Measurement of Insulating Layer Thickness] Using a Helios G4 UX (FIB-SEM) instrument manufactured by FEI, STEM cross-sectional images of substrate particles were acquired in BF-STEM mode. In the STEM cross-sectional images, the insulating layer thickness was measured at 10 locations for randomly selected substrate particles, and the average value of the measurements was taken as the insulating layer thickness. The obtained insulating layer thicknesses are shown in Table 1.

[0108] [Insulating Layer Coverage] Using a Helios G4 UX (FIB-SEM) instrument manufactured by FEI, STEM cross-sectional images of composite particle R1 and composite particles E1 to E5 were acquired in BF-STEM mode. Ten composite particles were randomly selected from each STEM cross-sectional image, and the cross-section of each composite particle was observed. The "insulating layer coverage" was calculated as (the length obtained by subtracting the length of the area where the magnetic material is exposed to the outside, the length of the area where the metallic particles are in direct contact with the magnetic material, or the length of the area where the magnetic material is in direct contact with the protective layer from the outer circumference of the base particle in the composite particle) / (outer circumference of the base particle in the composite particle). The obtained insulating layer coverage is shown in Table 1.

[0109]

[0110] [Measurement of Average Particle Size of Metallic Particles] Using a scanning electron microscope "JSM-5500" manufactured by JEOL Ltd., SEM observation images of the metallic particle aggregate layer on the surface of the composite particle were acquired. In the observation images, 10 metallic particles were randomly selected, and five tangent lines were randomly drawn within each particle image (however, all lines representing the tangent diameters must pass only inside the particle image, and one of these lines must pass only inside the particle and be the longest line that can be drawn), and the average value (average tangent diameter) was calculated. The average tangent diameter was obtained for all 10 selected particles. The definition of tangent diameter is the same as described above. The length of 1 / 4 of the average tangent diameter of each particle is defined as the "edge region length" of each particle. In addition, the range from the contour line on the SEM observation image of each particle toward the inside of the particle, equal to the "edge region length", is defined as the "edge region" of each particle. Then, if any of the 10 particles overlapped even partially with the edge region of other particles, those particles were discarded, and the required number of newly selected particles were randomly chosen, and the average tangent diameter was obtained in the same manner as above. The above was repeated as needed to obtain the average tangent diameter for 10 particles that did not overlap with the edge region. The average of the obtained 10 average tangent diameters was taken as the average particle size. In the composite particle E3 of Example 3, the average particle size of the metallic particles was 237.3 nm. In the composite particle E2 of Example 2, the average particle size of the metallic particles was 241.0 nm. In the composite particle E4 of Example 4, the average particle size of the metallic particles was 292.6 nm.

[0111] [Measurement of Average Height of Metallic Particles] Using a Helios G4 UX (FIB-SEM) instrument manufactured by FEI, STEM cross-sectional images of the composite particles were acquired in BF-STEM mode. Six metallic particles were randomly selected from the STEM cross-sectional images, and the average height was obtained from the average height of the six selected metallic particles. In composite particle E1 of Example 1, the average height of the metallic particles was 103.9 nm. In composite particle E2 of Example 2, the average height of the metallic particles was 137.8 nm, and the ratio of average particle size to average height (aspect ratio) was 1.74. In composite particle E3 of Example 3, the average height of the metallic particles was 94.4 nm, and the ratio of average particle size to average height (aspect ratio) was 2.51. Figure 2 shows the STEM cross-sectional image of composite particle E3.

[0112] [Measurement of average interparticle distance and standard deviation of average interparticle distance of metallic particles] Using a scanning electron microscope "JSM-5500" manufactured by JEOL Ltd., SEM observation images of the metallic particle aggregate layer on the surface of composite particles were acquired. Next, the obtained observation images were read using the free image processing software "ImageJ" manufactured by the National Institutes of Health, USA, at a resolution of 1280 pixels wide x 960 pixels high. Then, using the random number generation function "RANDBETWEEN" in the spreadsheet software "Excel" manufactured by Microsoft, six random numbers (x1 to x6) were obtained from 1 to 1280, and six random numbers (y1 to y6) were obtained from 1 to 960. From the obtained six random numbers, six sets of random number combinations (x1, y1) were obtained to form (x6, y6). Random numbers generated from 1 to 1280 were used as the x-coordinate, and random numbers generated from 1 to 960 were used as the y-coordinate, obtaining six sets of coordinate points (x1, y1) to (x6, y6). Next, for each of the six metallic particle images containing these coordinate points, the interparticle distance between the metallic particle and the nearest adjacent metallic particle was obtained, and then the average interparticle distance was obtained as the average of the interparticle distances between these six adjacent metallic particles. In this process, if at least one of the six coordinate points from the six random number combinations was not included in the metallic particle image, or if two or more coordinate points were included in the same metallic particle, this random number combination was discarded, and random number generation was repeated until all six coordinate points were included in different metallic particle images. At the same time, the "edge region" described above was obtained from the same observation image. Then, if any of the six metallic particles have a metallic particle whose edge region partially overlaps with the edge region of another metallic particle in which each metallic particle is placed, that random number combination is discarded. Random number generation is repeated until all six coordinate points are contained within different metallic particle images and no metallic particles have a metallic particle whose edge region partially overlaps with the edge region of the substrate particle in which each metallic particle is placed, and the inter-particle distance is obtained in the same manner as above. The above is repeated as needed to obtain the inter-particle distance for six metallic particles that do not overlap with the edge region. In addition, the average inter-particle distance is obtained from the average inter-particle distance for the six metallic particles. Furthermore, the standard deviation of the average inter-particle distance is obtained from the inter-particle distance and the average inter-particle distance for the six metallic particles.In the composite particle E3 of Example 3, the average interparticle distance of the metallic particles was 36.3 nm, and the standard deviation of the average interparticle distance was 13.8 nm. In the composite particle E4 of Example 4, the average interparticle distance of the metallic particles was 228.5 nm.

[0113] [Measurement of elemental ratios by X-ray photoelectron spectroscopy] The elemental ratios of composite particles E1 to E3 and substrate particles 1 to 3 were measured using a K-Alpha (manufactured by Thermo Fisher) as the measuring device, according to the following measurement conditions. (Measurement conditions) X-ray source: Al-Kα (12kV / 6mA) X-ray size: 400μmφ Neutralizing gun: Yes (Beam / Emission / Focus / Extractor = 0.40V / 120μA / 25V / 40V) Measurement mode: Survey mode, Scan mode Survey step: 1.0eV Dwell Time: 25 ms Pass Energy: 200eV Number of scans: 4 times Scan (narrow scan) step: 0.1eV Dwell Time: 50 ms Pass Energy: 50eV Scan count: Si2p (10), C1s (10), Ag3d (10), O1s (10), Fe2p (20)

[0114] (Calculation of elemental ratios) The elemental ratio of oxygen (CO1) was measured based on the peak area (integral value) of the O1s spectrum. The elemental ratio of carbon (CC1) was measured based on the peak area of ​​the C1s spectrum. For M11, M12, and IL, appropriate spectra were selected according to their type. In addition, if peak overlap with other elements occurred in any of the selected spectra, peak separation was performed. Here, M11 is the main element of metallic particles, M12 is the main element of magnetic material, and IL is the main element of the insulating layer excluding oxygen.

[0115] In each example and comparative example, the main element of the metallic particles is silver, therefore Ag3d 3 / 2 Based on the peak area of ​​the spectrum, the elemental ratio of Ag (CM11) was measured, and since the main element of the magnetic material is iron, Fe2p 3 / 2Based on the peak area of ​​the spectrum, the elemental ratio of Fe (CM12) was measured, and since silicon is the main element of the insulating layer excluding oxygen, the elemental ratio of Si (CIL) was measured based on the peak area of ​​the Si2p spectrum.

[0116] Table 2 shows the elemental ratios CM11 (meaning the elemental ratio of silver in this example), the elemental ratio of oxygen CO1, and the elemental ratios CM12 (meaning the elemental ratio of iron in this example) derived from the metallic particles, measured by X-ray photoelectron spectroscopy on the surfaces of composite particles E1 to E3.

[0117]

[0118] Table 3 shows the elemental ratios derived from magnetic materials, measured by X-ray photoelectron spectroscopy on the surfaces of substrate particles 1 to 3. Table 3 also shows the fluorescence enhancement intensity values ​​of composite particles E1 to E3 using substrate particles 1 to 3 (Examples 1 to 3).

[0119]

[0120] Table 3 shows that, comparing Examples 1 to 3, there is a tendency for the fluorescence enhancement ratio to increase as the elemental ratio of Fe on the surface of the substrate particles decreases.

[0121] 10...Base particle, 11...Magnetic particle, 12...Insulating layer, 20...Metal particle aggregate layer, 100...Composite particle.

Claims

1. A method for producing composite particles comprising: base particles containing a magnetic material; and a metal particle aggregate layer formed on the base particles, wherein a plurality of metal particles are spaced apart from each other, the method comprising the steps of: immersing the base particles in a metal cation-containing liquid containing metal cations constituting the metal particles; adding a reducing agent to the metal cation-containing liquid to form the metal particle aggregate layer on the base particles, wherein the manufacturing method satisfies the following formula (1) when the valence of the metal cation is a, the valence of the anion when the reducing agent is ionized is b, the amount of the metal cation in the liquid to which the reducing agent has been added is m (mol), and the amount of the ionized reducing agent in the liquid to which the reducing agent has been added is r (mol).

2. The manufacturing method according to claim 1, wherein the base material particles comprise an insulating layer, and the metal-based particle aggregate layer is formed on the insulating layer.

3. A composite particle comprising a base particle and a metallic particle aggregate layer formed on the base particle, wherein the base particle contains a magnetic material and the metallic particle aggregate layer is a layer in which a plurality of metallic particles are arranged spaced apart from each other.

4. The composite particle according to claim 3, wherein the average particle size of the metal-based particles is 100 to 1600 nm, the average height is 20 to 500 nm, and the ratio of the average particle size to the average height is 1 to 8.

5. The composite particle according to claim 3 or 4, wherein, in the plurality of metallic particles, the average distance between adjacent metallic particles is 1 to 1000 nm, and the standard deviation of the average distance is 25 nm or less.

6. The composite particle according to claim 3 or 4, wherein the magnetic material comprises at least one selected from the group consisting of iron and iron oxide.

7. The composite particle according to claim 3 or 4, wherein the base particle comprises an insulating layer, and the metal-based particle aggregate layer is formed on the insulating layer.

8. The composite particle according to claim 7, wherein the thickness of the insulating layer is 50 nm or more.

9. The composite particle according to claim 3 or 4, wherein the elemental ratio of the component derived from the metal-based particle, as measured by X-ray photoelectron spectroscopy, on the surface of the composite particle is 5 atomic percent or more.

10. The composite particle according to claim 3 or 4, wherein the elemental ratio of oxygen measured by X-ray photoelectron spectroscopy on the surface of the composite particle is 55 atomic percent or less.

11. The composite particle according to claim 3 or 4, wherein the elemental ratio of the component derived from the magnetic material, as measured by X-ray photoelectron spectroscopy, on the surface of the composite particle is 1 atomic percent or less.

12. Substrate particles containing a magnetic material, wherein the elemental ratio of components derived from the magnetic material, as measured by X-ray photoelectron spectroscopy on the surface of the substrate particles, is 1 atomic percent or less.

13. A sensor substrate comprising the composite particles described in claim 3 or 4.

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