Substrate, analysis method, apparatus, and manufacturing method

Abstract

The present application provides a substrate including a plurality of protrusions including a metal, in which a first metal portion including a metal including at least one of gold, silver, platinum, copper, and palladium is disposed on a first protrusion of the plurality of protrusions, and a second metal portion including a metal including at least one of gold, silver, platinum, copper, and palladium is disposed on a second protrusion of the plurality of protrusions. Wherein a second metal portion including a metal including at least one of gold, silver, platinum, copper, and palladium is provided on a second protrusion different from the first protrusion among the plurality of protrusions, wherein dielectric portions are disposed between the first protrusion and the first metal portion and between the second protrusion and the second metal portion, and wherein a gap is disposed between the first metal portion and the second metal portion, and a distance between the first metal moiety and the second metal moiety is 50 nm or less, and wherein the first metal moiety and the second metal moiety comprise functional groups that are adsorbable or bondable to the first metal moiety and the second metal moiety.

Description

Technical Field

[0001] This invention relates to a substrate. Background Technology

[0002] When light shines on a metal, a plasmonic resonance is generated on the metal's surface, and an electric field enhancement effect is applied through this resonance (localized plasmonic resonance phenomenon). Progress is being made in the development of electric field enhancement devices (such as sensor devices, Raman spectrometers, and fluorescence devices) that utilize this electric field enhancement effect. Surface-enhanced fluorescence methods are known, which use an optical electric field enhanced by localized plasmonic resonance to detect small amounts of matter.

[0003] For example, Patent Document 1 describes a form comprising an optical substrate constituting a finely textured structure and a metal film formed on the surface of the finely textured structure. Patent Document 2 describes a plasma excitation sensor comprising a substrate having a plurality of metal protrusions coated with ligands and self-assembled monomolecular films.

[0004] List of cited references

[0005] Patent documents

[0006] PTL1: Japanese Patent Publication No. 2007-240361

[0007] PTL2: Japanese Patent Publication No. 2010-256161 Summary of the Invention

[0008] Technical issues

[0009] In the substrates described in Patent Document 1 or 2, when the amount of material is small or the amount of foreign matter is large, the fluorescence enhancement effect may decrease and the background signal from the substrate may increase, leaving room for improving the signal-to-noise ratio (S / N ratio). Therefore, the present invention aims to improve the S / N ratio of the fluorescence signal in a fluorescence enhancement device.

[0010] Solution to the problem

[0011] According to an aspect for solving the above-mentioned problems, a substrate is provided, comprising a plurality of protrusions containing a metal, wherein a first protrusion of the plurality of protrusions is provided with a first metal portion comprising at least one of gold, silver, platinum, copper and palladium, wherein a second metal portion comprising at least one of gold, silver, platinum, copper and palladium is provided on a second protrusion of the plurality of protrusions different from the first protrusion, wherein a dielectric portion is provided between the first protrusion and the first metal portion and between the second protrusion and the second metal portion, wherein a gap is provided between the first metal portion and the second metal portion, and the distance between the first metal portion and the second metal portion is less than 50 nm, and wherein the first metal portion and the second metal portion include functional groups adsorbed or bound to the first metal portion and the second metal portion.

[0012] Beneficial effects of the invention

[0013] It provides a technique that is beneficial for improving the S / N ratio of fluorescence signals. Attached Figure Description

[0014] Figure 1A This is a schematic diagram of the substrate according to this embodiment.

[0015] Figure 1B yes Figure 1A An enlarged view of the main parts shown.

[0016] Figure 1C This is a schematic diagram showing a variation of the substrate according to this embodiment.

[0017] Figure 2A This is a schematic diagram showing the substrate according to this embodiment.

[0018] Figure 2B This is a schematic diagram showing the substrate according to this embodiment.

[0019] Figure 3A This is a schematic diagram illustrating a method for manufacturing a substrate according to this embodiment.

[0020] Figure 3B This is a schematic diagram illustrating a method for manufacturing a substrate according to this embodiment.

[0021] Figure 3C This is a schematic diagram illustrating a method for manufacturing a substrate according to this embodiment.

[0022] Figure 3D This is a schematic diagram illustrating a method for manufacturing a substrate according to this embodiment.

[0023] Figure 3E This is a schematic diagram illustrating a method for manufacturing a substrate according to this embodiment.

[0024] Figure 3F This is a schematic diagram illustrating a method for manufacturing a substrate according to this embodiment.

[0025] Figure 3G This is a schematic diagram illustrating a method for manufacturing a substrate according to this embodiment.

[0026] Figure 3H This is a schematic diagram illustrating a method for manufacturing a substrate according to this embodiment.

[0027] Figure 3I This is a schematic diagram illustrating a method for manufacturing a substrate according to this embodiment.

[0028] Figure 4 This is a schematic diagram illustrating an example of a device mounted on a substrate according to this embodiment.

[0029] Figure 5 This is a schematic diagram of the substrate according to the second embodiment. Detailed Implementation

[0030] Some embodiments of the invention are described below with reference to the accompanying drawings. However, the embodiments described below are embodiments of the invention, and the invention is not limited thereto. Common components are described by cross-referencing multiple drawings, and descriptions of components indicated by common reference numerals are appropriately omitted. Different items with the same name can be distinguished from each other by assigning ordinal numbers (such as first item and second item).

[0031] <First Embodiment>

[0032] refer to Figures 1A to 1C The substrate 10 according to the first embodiment is described. Figure 1A This is a schematic diagram of the substrate 10 according to this embodiment. Figure 1B yes Figure 1A An enlarged view of the metal part 2 shown, and Figure 1C A variation of the substrate 10 according to this embodiment is shown. The substrate 10 includes a structure 1, a dielectric portion 3 disposed on the surface of the structure 1, a metal portion 2 forming an interface with the corresponding dielectric portion 3, and a molecular layer 70 having functional groups 710 that can be adsorbed or bonded to the metal portion 2. The structure 1 has an uneven structure. The metal portion 2 and the dielectric portion 3 are disposed on a plurality of protrusions 4 (including protrusions 41 and protrusions 42) of the uneven structure. The dielectric portion 3 is disposed between the structure 1 and the metal portion 2, and preferably has a structure that forms an interface with both the structure 1 and the metal portion 2.

[0033] A gap 9 is provided between metal portion 21 and metal portion 22 adjacent to metal portion 21. The distance D between metal portion 21 and metal portion 22 is greater than 0 and less than or equal to 50 nm. More preferably, the distance D between metal portion 21 and metal portion 22 is greater than 0 and less than or equal to 10 nm. Distance D is the shortest distance between metal portion 21 and metal portion 22. Distance D is more preferably the distance between metal portion 21 and metal portion 22; however, multiple metal portions 21 can be provided on the same protrusion 41, and distance D can be the distance between two adjacent metal portions among the multiple metal portions 21. In addition, distance D can be the distance between metal portion 21 and another metal portion 2, which is not provided on the protrusion 42 adjacent to protrusion 41 but on another protrusion 4. The gap 9 is provided to further enhance the optical electric field and improve the fluorescence intensity on the substrate 10. Metal portions 21 and metal portions 22 can be connected to each other except for the portion provided between them by the gap 9, but preferably they are not continuous.

[0034] The substrate 10 includes a molecular layer 70 having functional groups 710 that can be adsorbed or bonded to the metal portions 21 and 22. The molecular layer 70 is formed on the portion of the metal portion 2 that is not in contact with the dielectric portion 3. Alternatively, the molecular layer 70 is formed on both the metal portion 2 and the dielectric portion 3 to coat the portions of the metal portion 2 and the dielectric portion 3 that are not in contact with the protrusion 4. Furthermore, as... Figure 1C As shown, the molecular layer 70 can be formed on the side of the protrusion 4 that does not contact the adhesive layer 6.

[0035] exist Figure 1A In the process, a molecular layer 70 having adsorbable or bindable functional groups 710 is coated on the surface of the metal portion 2, such as... Figure 1B As shown. In Figure 1C In this process, functional groups 710 are disposed on the surfaces of the protrusion 4, the metal portion 2, and the dielectric portion 3. In other words, the surfaces of the protrusion 4, the metal portion 2, and the dielectric portion 3 are coated with a molecular layer 70. The coating with the molecular layer 70 can be continuous or discontinuous.

[0036] While the molecular layer 70, including the functional groups 710 that can adsorb or bind to the metal portion 2, is not particularly limited, as long as the molecular layer 70 is made of molecules capable of stably adsorbing or binding to the metal portion 2, it is preferred, as described below, that the molecular layer 70 is made of organic molecules and proteins. When the substrate 10 includes the molecular layer 70, fluorescent molecules contained in the sample as detection targets can be suitably arranged on the substrate near the metal portion 2. For example, fluorescent molecules contained in the sample or substances labeled with fluorescent molecules can be stably adsorbed. In this case, the molecular layer 70 preferably contains a molecular recognition material that specifically binds to the fluorescent molecules contained in the sample or substances labeled with fluorescent molecules. Molecular recognition materials are molecules that specifically recognize and are capable of binding to the measurement target substance in the sample, and examples of molecular recognition materials include proteins, carbohydrates, lipids, and nucleic acids. As a protein, for example, an antibody can be used. When using a molecular layer 70 including an antibody, an antigen against the antibody can be immobilized near the metal portion 2. The size of the antibody molecule is preferably 5 nm or more and 15 nm or less, and the thickness of each molecular layer 70 including the antibody is preferably 5 nm or more and 15 nm or less (in the case where the metal part 2 is directly coated with the antibody).

[0037] As another example of a protein, albumin can be used. Albumin is a protein with a size of a few nanometers and can be used as a component of molecular layer 70. When molecular layer 70 contains albumin, molecular layer 70 becomes highly hydrophilic.

[0038] When the target for detection is an antigen labeled with a fluorescent molecule (fluorescently labeled antigen), the fluorescently labeled antigen (typically several nanometers to tens of nanometers each) bound to the antibody in the molecular layer 70 is immobilized at a distance of more than 10 nm and less than 100 nm from the metal part 2. At this distance of more than 10 nm and less than 100 nm from the metal part 2, the electric field is enhanced and localized. Because the fluorescently labeled antigen is immobilized in this region, the signal-to-noise ratio (S / N) of the fluorescence from the fluorescently labeled antigen contained in the sample is improved, and the fluorescently labeled antigen can be detected with high sensitivity.

[0039] The molecular layer 70, having functional groups 710 that can adsorb onto or bind to the metal moiety 2, can comprise organic molecular films and self-assembled monolayers. Examples of organic molecules include citric acid and amino acids. Examples of self-assembled monolayers include alkyl thiols and silane coupling agents. Silane coupling agents are organosilicon compounds that can selectively use various functional groups (e.g., amino, carboxyl, and hydroxyl) while exhibiting a high binding affinity to the metal moiety 2. Thus, a stable molecular layer 70 with controlled physical properties can be formed. Alkyl thiols are molecules with a thiol group having about 4 to 20 carbon atoms, exhibiting high affinity for metals and high intermolecular cohesion, thus enabling the formation of stable monolayers on the metal moiety 2. When the metal moiety 2 is made of gold, alkyl thiols are particularly preferred as components of the molecular layer. Alkyl thiols have various functional groups at their ends, such as amino, carboxyl, and hydroxyl groups.

[0040] As an example, the molecular layer 70 according to the invention has functional groups 710 that do not contact the metal portion 2. Examples of functional groups 710 include functional groups that interact with the target molecule, functional groups that bind to the interacting molecule recognition material, and functional groups that substantially do not interact with foreign matter that is not the target molecule. For example, amino, carboxyl, hydroxyl, maleimide, thiol, methoxy, or hydroxyl groups can be used as functional groups 710, with amino, carboxyl, or hydroxyl groups being particularly preferred.

[0041] As an example of a preferred embodiment of a molecular layer 70 having functional groups 710 that can be adsorbed or bound to the metal portion 2, a molecular layer 70 made of molecular recognition material and a self-assembled monolayer can be used. Figure 1B An example of it is shown. Figure 1B This is an enlarged view of a metal portion 2 of the substrate 10. The metal portion 2 is coated with a molecular layer 70, and the molecular layer 70 includes functional groups 710, a self-assembled monomolecular film 711, and a self-assembled monomolecular film 712 including a molecular recognition material (obtained by chemically bonding the molecular recognition material to the self-assembled monomolecular film).

[0042] Furthermore, as an example of a preferred embodiment, a molecular layer 70 made of molecular recognition material and albumin can be used. In these examples of embodiments, the binding of the target molecule can be measured while preventing non-specific binding of foreign matter to the substrate 10. In fluorescence detection, non-specific binding of foreign matter to the substrate 10 can cause noise (increased N) and inhibit the binding of the molecular recognition material to the target molecule (decreased S), which is undesirable.

[0043] While the thickness of each molecular layer 70 is not particularly limited, it is acceptable as long as it is within the range that can achieve the effect of improving fluorescence intensity; in other words, it is acceptable within the range of the locally enhanced electric field region. For example, the thickness is preferably 0.1 nm or more and 100 nm or less, more preferably 1 nm or more and 50 nm or less. If the thickness exceeds 100 nm, the distance between the fluorescent molecules and the metal part 2 also increases, and the effect of improving the fluorescence signal intensity may deteriorate.

[0044] The substrate 10 according to the invention includes a protrusion structure comprising a plurality of metal portions 2, each metal portion comprising molecules having adsorbable or bindable functional groups 710. Therefore, a localized electric field enhancement region is generated near the protrusion structure. Consequently, fluorescence from fluorescent molecules present near the protrusion structure is increased. Furthermore, the characteristic structure and composition of the substrate according to the invention reduce background noise (e.g., scattered light noise) from the substrate 10. As a result, not only is signal intensity increased (increased S) through electric field enhancement, but noise is also suppressed (reduced N), leading to an improved S / N ratio of the fluorescence signal.

[0045] exist Figure 1A and Figure 1C In this configuration, each metal part 2 has a protruding portion with a circular head; however, each metal part 2 may have, for example, a protruding portion with a circular head. Figure 2A and Figure 2B The shape of the dielectric portion 3 is shown, and the shape of each metal portion 2 is not limited. Furthermore, as... Figure 1C As shown, the dielectric portion 3 can be disposed on the recess 43, and the dielectric portion 3 can be connected to the dielectric portions 3 on the protrusions 41 and 42. The metal portions of the protrusions 41 and 42 can be discontinuous. Preferably, the metal portion 2 does not cover the recess 43, and the dielectric portion 3 is exposed in the space above the recess 43.

[0046] Preferably, the uneven structure is provided only on one of the surfaces of the structure 1. The distance between the protrusion 41 and the recess 43, that is, the height difference of the uneven structure, is preferably 100 nm or more and 1000 nm or less, more preferably 100 nm or more and 500 nm or less. The above-mentioned height difference is preferably the average value of the height difference of the uneven structure. The height difference can be a linear distance from the protrusion 41 to the recess 43, or it can be a vertical distance from the protrusion 41 to the recess 43. The height difference can be determined by observing the cross-section of the substrate 10 using a scanning electron microscope or the like. The protrusion 41 and the protrusion 42 are preferably connected by the recess 43, but they can also be separated from each other. In the structure 1, the protrusion 4 is made of metal, but the recess 43 may not be made of metal, but may be made of non-metal such as ceramic or resin.

[0047] The material of structure 1 is preferably a highly conductive material, such as gold, silver, copper, aluminum, magnesium, tungsten, cobalt, zinc, nickel, or chromium. Nickel, zinc, and chromium are preferred, and nickel is particularly preferred.

[0048] The material of the metal portion 2 is a metal selected from at least one of gold, silver, platinum, copper and palladium, with gold or silver being particularly preferred. Although there is no particular limitation on the thickness of each metal portion 2 as long as the uneven structure that can receive excitation light to generate local plasma is maintained, the thickness of each metal portion 2 is preferably 5 nm or more and 50 nm or less.

[0049] The dielectric portion 3 is preferably made of a metal oxide. While the metal oxide can be silicon dioxide, alumina, zirconium oxide, etc., and is not particularly limited, it is preferable to include alumina as a main component, and more preferably, it includes plate-like crystals containing alumina as a main component. The plate-like crystals containing alumina as a main component are made of plate-like crystals containing aluminum oxide, hydroxide, or their hydrates as main components, and boehmite is a particularly preferred crystal. The plate-like crystals containing alumina as a main component can be plate-like crystals made of alumina alone, or plate-like crystals containing small amounts of zirconium, silicon, titanium, zinc, etc. In the case of a plate-like structure containing alumina as a main component, it is preferable to arrange the plate-like crystals containing alumina as a main component in a direction perpendicular to the surface direction of structure 1, and their space occupancy is preferably continuously varied. Furthermore, the metal oxide can include an amorphous gel of alumina. Figure 1C As shown, the dielectric portion 3 is preferably formed as a concave-convex structure or protrusion 4 following the structure 1. The thickness of each dielectric portion 3 is preferably 30 nm or more and 200 nm or less.

[0050] The substrate 10 according to this embodiment preferably has a specific surface area Sr of 1.0 or more and 3.0 or less. The specific surface area Sr is determined by the following formula.

[0051] Formula (1) for Sr=S / S0

[0052] In formula (1), S0 is the surface area assuming the measured surface is ideally flat, and S is the actual surface area of ​​the measured surface. The specific surface area can be determined by observing a surface with an uneven structure using a scanning probe microscope or similar device.

[0053] When observing the surface or cross-section using scanning electron microscopy (SEM) or transmission electron microscopy (TEM), the metallic elements in structure 1 and the metal oxides in dielectric portion 3 can be detected by energy-dispersive X-ray analysis (EDX). Furthermore, the metallic elements in structure 1 and the metal oxides in dielectric portion 3 can be detected by X-ray photoelectron spectroscopy (XPS). In the direction perpendicular to the surface direction of structure 1, from dielectric portion 3 towards structure 1, the proportion of metal oxides relatively decreases, the proportion of metallic elements constituting structure 1 increases, and ultimately, only metallic elements are detected.

[0054] The substrate 10 includes a base material 5 on the surface of the structure 1 opposite to the side where the uneven structure is provided. The base material 5 is disposed above the structure 1 and has an adhesive layer 6 between them, but the adhesive layer 6 may be omitted. The shape of the base material 5 may be a shape corresponding to the application, and may be plate-like, film-like, sheet-like, etc., but is not limited thereto. Examples of materials for the base material 5 include metal, glass, ceramic, wood, paper, and resin, but the materials for the base material 5 are not limited thereto. Examples of resins include polyester, triacetyl cellulose, cellulose acetate, polyethylene terephthalate, polypropylene, polystyrene, polycarbonate, polymethyl methacrylate, and acrylonitrile-butadiene-styrene (ABS) resin. In addition, the resin may be a film or molding made of thermoplastic resin such as polyphenylene ether, polyurethane, polyethylene, or polyvinyl chloride, or a film or molding made of thermosetting resin such as unsaturated polyester resin, phenolic resin, crosslinked polyurethane, crosslinked acrylic resin, or crosslinked saturated polyester resin. The adhesive layer 6 can be any layer as long as it can bond the substrate 5 and the structure 1 together. Examples of adhesive layers include layers made of cured adhesive resin (e.g., epoxy resin) and double-sided tape.

[0055] Next, refer to Figures 3A to 3I A method for manufacturing substrate 10 is described. The manufacturing method according to this embodiment includes forming a structure 1, forming a dielectric portion 3, forming a metal portion 2, and forming a molecular layer 70 having functional groups 710 that can be adsorbed or bound to the metal portion.

[0056] refer to Figure 3A and Figure 3B Describes the formation of dielectric portion 3. Dielectric portion 3 comprises a metal oxide including aluminum oxide. A sol-gel coating solution is prepared by dissolving or suspending an aluminum compound, along with other compounds, stabilizers, and water-soluble organic polymer compounds as needed, in an organic solvent. The sol-gel coating solution is applied to a substrate 8 and dried to form an alumina gel film serving as an aluminum film 7. Alternatively, an alumina gel film containing metallic aluminum, serving as an aluminum film 7, is formed on the substrate 8 by dry film formation, such as vacuum deposition or sputtering.

[0057] Subsequently, the aluminum film 7 is immersed in hot water to form an uneven structure of alumina. When the aluminum film 7 is immersed in hot water, its surface is subjected to a gelling and similar treatment, and some components are washed away. However, due to the differences in solubility of various hydroxides in hot water, plate-like crystals containing alumina as the main component precipitate and grow on the surface of the aluminum film 7, forming the uneven structure of the dielectric portion 3. When a film containing metallic aluminum is used instead of the aluminum film 7, the aluminum reacts with hot water and oxidizes to alumina, then forms the uneven structure of the dielectric portion 3 in a manner similar to that used with the aluminum film 7. Therefore, when the substrate 8 mainly contains aluminum or alumina, the formation of the aluminum film 7 on the substrate 8 can be omitted. The temperature of the hot water is preferably 40°C or higher and less than 100°C. The immersion treatment time is preferably about 5 minutes to about 24 hours. In the immersion treatment where compounds other than the alumina component are added to the aluminum film 7, the differences in solubility of each component in hot water are utilized to crystallize the plate-like crystals of alumina. Therefore, unlike the immersion treatment of aluminum film 7 containing a single alumina component, the size of the plate-like crystals can be controlled within a wide range by changing the composition of the inorganic components. Furthermore, the height of the uneven shape of the alumina can be adjusted by regulating the film thickness of the aluminum film 7. The average height of the uneven structure of the dielectric portion 3 is preferably 100 nm or more and 1000 nm or less, more preferably 100 nm or more and 500 nm or less. The thickness of the dielectric portion 3 is preferably 30 nm or more and 200 nm or less. This allows for control over the unevenness formed by the plate-like crystals within a wide range.

[0058] There are no particular limitations on the material of the substrate 8, and various materials such as glass, plastic, and metal can be used. When forming the aluminum film 7 using a sol-gel coating solution without stabilizers, the coating atmosphere is preferably an inert gas atmosphere such as dry air or dry nitrogen. The relative humidity of the drying atmosphere is preferably 30% or less. As a solution coating method for forming the aluminum film 7, known coating methods such as dip coating, spin coating, spray coating, printing, and flow coating, or combinations thereof, can be appropriately employed. The film thickness can be controlled by changing the pull-out speed in dip coating, the substrate rotation speed in spin coating, and the concentration of the sol-gel coating solution. Drying at room temperature for about 30 minutes is sufficient. In addition, drying or heat treatment at higher temperatures can be performed as needed. With higher heat treatment temperatures, a more stable uneven structure of the dielectric portion 3 can be formed by the dip treatment described below. A suitable film thickness for the aluminum film 7 is 100 nm or more and 600 nm or less, preferably 100 nm or more and 300 nm or less, and more preferably 100 nm or more and 200 nm or less.

[0059] Next, refer to Figure 3C Describe the formation of structure 1. (Refer to...) Figure 3BA metal-containing structure 1 is formed on the uneven structure of the described dielectric portion 3. As a method for forming the structure 1, metal plating is preferred, and electroless plating is more preferred. In electroless plating, activation is performed by applying a solution obtained by dissolving palladium compounds such as palladium chloride, gold compounds such as gold chloride, silver compounds such as silver chloride, and tin compounds such as tin chloride to the uneven structure of the dielectric portion 3. Activation can be performed by immersing the uneven structure of the dielectric portion 3 together with the substrate 8 in a solution in which palladium compounds are dissolved. Thereafter, the structure 1 is deposited on the uneven structure of the dielectric portion 3 using an electroless plating solution. The metal ions in the electroless plating solution correspond to the structure 1 of the substrate 10 according to this embodiment. Electroless plating solutions containing nickel ions, chromium ions, and zinc ions are preferred, and nickel plating solutions containing nickel ions are particularly preferred. In addition to the nickel component, the nickel plating solution may also contain phosphorus and boron components. The temperature of the plating solution in electroless plating is preferably 30°C or higher and 98°C or lower, more preferably 50°C or higher and 90°C or lower. The processing time for electroless plating can be adjusted according to the thickness of the structure 1 to be formed, and is typically from 30 seconds to one hour. The structure 1 is formed to fill the gaps in the uneven structure as described above, and a structure 1 having an uneven structure transferred from the uneven structure of the dielectric portion 3 is formed. Electroless plating is preferably performed such that the thickness of the structure 1 including the uneven structure is 200 nm or higher and 15000 nm or lower. Furthermore, the average value of the height difference of the uneven structure corresponds to the average value of the height difference of the uneven structure of the dielectric portion 3, and is 100 nm or higher and 1000 nm or lower.

[0060] After performing the above-described electroless plating, electroplating can be performed on the surface of structure 1 opposite to the surface with the uneven structure to increase the thickness of structure 1. In electroplating, a known electroplating solution can be used, and for example, an electroplating solution containing nickel ions, iron ions, copper ions, etc., as metal ions can be used. When electroplating is performed using the same metal as structure 1, the thickness of structure 1 can be increased by electroplating. When electroplating is performed using a metal different from the metal of structure 1, the metal provided by electroplating serves as the substrate. In addition to inorganic salts used as raw materials for metal ions, conductive salts, salts for adjusting counterions, carboxylic acid additives for improving coating uniformity, brighteners, etc., can be added to the electroplating solution as needed. During electroplating, the thickness of structure 1 can be adjusted to the desired thickness by adjusting the solution temperature, current density, and plating time of the electroplating solution. If necessary, before electroplating, an activation treatment can be performed on the surface of structure 1 opposite to the surface with the uneven structure using an aqueous solution containing acid, etc. In addition, to improve the quality of the film formed by electroplating, besides stirring the electroplating solution during electroplating, it is also possible to remove foreign matter from the electroplating solution.

[0061] Next, refer to Figure 3D The description describes the bonding of the substrate 5 to the structure 1. When the substrate 5 is made of metal, the metal to be used as the substrate 5 can be further stacked on the surface of the structure 1 opposite to the surface where the uneven structure is provided. As a method of stacking the metal, it can be done by electroplating as described above, or by physical vapor deposition, such as sputtering. When the substrate 5 is made of resin, the substrate 5 can be formed by depositing the resin to be used as the substrate 5 on the surface of the structure 1 opposite to the surface where the uneven structure is provided, and then curing the resin. The substrate 5 can be bonded to the structure 1 by an adhesive layer 6. The adhesive used for the adhesive layer 6 is preferably resin, but is not particularly limited, as long as the material bonds the substrate 5 and the structure 1.

[0062] Next, refer to Figure 3E , Figure 3F and Figure 3G Describes a portion of the etched substrate 8, aluminum film 7, and dielectric portion 3. Figure 3E It is achieved by reversing vertically. Figure 3D The obtained image. First, as... Figure 3F As shown, substrate 8 is removed. In the case where aluminum film 7 is an alumina gel film, aluminum film 7 can be used as part of the dielectric portion 3 of substrate 10. Aluminum film 7 can be partially removed by etching. As an etching method, wet etching is preferred, which uses an acid or alkali solution to dissolve the aluminum-containing film. Examples of acids include hydrochloric acid, nitric acid, and sulfuric acid. Examples of alkalis include sodium hydroxide and potassium hydroxide. From the viewpoint of efficiency, etching methods using alkaline solutions are more preferred. The etching concentration is preferably in the range of a few percent to several tens of percent, and the etching time is preferably in the range of several hours to several days. When the surface or cross-section is observed by SEM or TEM, residual metal oxides such as alumina after etching can be detected, for example, by measurements using EDX or XPS. In etching, the dielectric portion 3 is removed such that the distance H1 from the recess of the uneven structure of the transferred structure 1 is less than the height difference H2 between the protrusion of structure 1 and the recess adjacent to the protrusion. The distance H1 can be zero, and the dielectric portion 3 in the recess can be completely etched. At this time, the protrusion of the uneven structure of structure 1 is covered by the dielectric portion 3. The dielectric portion 3 is removed, so that the surface of the dielectric portion 3 on the side opposite to the structure 1 has a shape that follows the uneven structure transferred to the structure 1. Etching can be performed before bonding the substrate 5.

[0063] Next, refer to Figure 3H Describes the formation of metallic portion 2. A metallic portion 2, comprising any one of gold, silver, platinum, copper, and palladium, is formed on the component obtained after etching by dry film formation such as vacuum deposition or sputtering.

[0064] Next, refer to Figure 3IDescribes the formation of a molecular layer 70 having functional groups 710 that can be adsorbed or bound to a metal portion. As a method for forming a self-assembled monolayer using alkyl thiols, a substrate ( Figure 3H The substrate ( ) is immersed in an alkyl thiol solution to form a self-assembled monolayer of alkyl thiols on the surface of the metal portion 2. As a method for forming a protein membrane, the substrate ( ) is immersed in an alkyl thiol solution to form a self-assembled monolayer of alkyl thiols on the surface of the metal portion 2. Figure 3H Immerse it in a protein solution to form a protein adsorption layer on the surface of the metal part 2.

[0065] Describes the formation of a molecular layer 70 containing a molecular recognition material. As a method using physical adsorption, the substrate ( Figure 3H The substrate is immersed in a solution containing molecular recognition material to form an adsorption layer of molecular recognition material on the surface of the metal part 2. For example, the substrate ( Figure 3H The substrate was immersed in a solution containing antibodies as molecular recognition materials. After being left at room temperature for one hour, the substrate was... Figure 3H The substrate is removed and washed with water to form a molecular layer 70 containing an antibody adsorption layer. Thereafter, the substrate on which the molecular layer 70 containing the antibody adsorption layer is formed can be immersed in an albumin solution to cover the portions of the antibody that were not adsorbed with albumin.

[0066] An example of using a chemical bonding method is described. When the substrate ( Figure 3H When immersed in a solution of alkyl thiols terminally containing carboxylic acids, a self-assembled monolayer of alkyl thiols terminally containing carboxylic acids is formed on the surface of metal part 2. Subsequently, the carboxylic acid is converted into an active ester using water-soluble carbodiimide (WSC) (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)) and N-hydroxysuccinimide (NHS), and the carboxyl groups thus converted into active esters are chemically bonded (amide bonding) to the amino groups included in the molecular recognition material. As a result, an adsorption layer of molecular recognition material is formed on the surface of metal part 2. The molecular recognition material is stably immobilized by adsorption through chemical bonds. Furthermore, the substrate on which the molecular layer 70, including an adsorption layer of antibodies, is subsequently formed can be immersed in an albumin solution to cover the portions of the antibody that are not adsorbed with albumin.

[0067] By using the substrate 10 manufacturing method according to this embodiment obtained in the manner described above, a sample with high planar uniformity can be obtained. Therefore, data with excellent measurement reproducibility can be obtained, and efficient measurements with high reliability can be performed. Furthermore, this method is an extremely simple manufacturing method, which allows for cost reduction compared to existing equipment.

[0068] The substrate 10 according to this embodiment can be used as a fluorescence enhancement device in a fluorescence detection method. The substrate 10 according to this embodiment can enhance fluorescence emission by using an optical electric field enhanced by localized plasmon resonance.

[0069] To mount the substrate 10 according to this embodiment onto the surface of a component or article, any of a variety of adhesives can be used. Therefore, the substrate 10 according to this embodiment can be mounted onto the surface of a component or article depending on the application. The surface of the component or article is not limited to a smooth surface and can have a two-dimensional or three-dimensional curved surface.

[0070] Next, as an example of a device 100 on which the substrate 10 according to this embodiment can be mounted, refer to Figure 4 Describe the fluorescence detection equipment.

[0071] The device 100 includes a substrate 10, a light irradiation unit 140 that irradiates the substrate 10 with light L1, and a light detection unit 150 that detects fluorescence L2 emitted from the sample S.

[0072] The light irradiation unit 140 includes a light source 141 that emits light L1, and an excitation filter 142 that adjusts the excitation wavelength of the light L1 emitted from the light source 141. The light L1 passes through a dichroic mirror 143 and is applied to a sample S on the substrate 10. Light L2, which contains fluorescence emitted from the sample S by irradiating it with light L1, is reflected by the dichroic mirror 143 toward the light detection unit 150.

[0073] The light detection unit 150 includes a light emission filter 151 and a detector 152. The light emission filter 151 allows light with wavelengths within a certain range in the light L2 reflected by the dichroic mirror 143 to be detected. Light L2 containing fluorescence emitted from the sample S passes through the light emission filter 151 and can be detected by the detector 152.

[0074] More specifically, in the uneven structure of the substrate 10, local plasmon resonance is induced by irradiation with light L1, and an enhanced optical electric field is generated on the surface of the metal portion 2. Detector 152 detects the fluorescence L2 emitted from the sample S and enhanced by the enhanced optical electric field.

[0075] The wavelength of the light applied from the light source 141 to the sample S can have any value depending on the fluorescent molecule to be detected. Ultraviolet light to visible light and further to near-infrared light are available, and the wavelength of the light applied to the sample S is preferably above 300 nm and below 850 nm.

[0076] The fluorescence detection device is described as an example of device 100; however, device 100 is not limited to fluorescence detection devices, and device 100 can be a fluorescence spectrometer, fluorescence microscope, etc.

[0077] <Second Embodiment>

[0078] Next, refer to Figure 5 The substrate 10 according to the second embodiment is described.

[0079] The substrate 10 according to this embodiment differs from the substrate 10 according to the first embodiment in that the structure 1 has a hierarchical structure. The hierarchical structure includes at least two types of structures with different structural dimensions, and for example, refers to a structure including a first structure with micrometer-level structural dimensions and a second structure with sub-micrometer-level structural dimensions. The height difference of the first structure with micrometer-level structural dimensions is, for example, 1 μm or more and 10 μm or less.

[0080] The structure 1 includes a base 11 disposed on an adhesive layer 6, and a convex-concave structure 12 disposed on the base 11. The convex-concave structure 12 includes a first convex-concave structure 121 and a second convex-concave structure 122 that is smaller in size than the first convex-concave structure 121. The second convex-concave structure 122 is disposed on the first convex-concave structure 121, and each of the first convex-concave structure 121 and the second convex-concave structure 122 includes a plurality of protrusions and a recess disposed between the plurality of protrusions.

[0081] Furthermore, as in the first embodiment, the metal portion 2, the dielectric portion 3 between the structure 1 and the metal portion 2, and the molecular layer 70 having functional groups 710 that can be adsorbed or bonded to the metal portion 2 are disposed on the second uneven structure 122. When gaps are provided between the metal portions 2 disposed on the protrusions of the second uneven structure 122, the optical electric field can be further enhanced, and the intensity of fluorescence in the substrate 10 can be increased. The molecular layer 70 can be disposed in the gaps of the first uneven structure 121.

[0082] The first and second concave-convex structures 121 and 122 are preferably made of the same material, and the base 11 is also preferably made of the same material. The distance between the protrusion and the concave portion of the second concave-convex structure 122, that is, the height difference between the concave and convex structures, is preferably 100 nm or more and 1000 nm or less, more preferably 100 nm or more and 500 nm or less.

[0083] In the case where a textured structure is formed in the hierarchical structure as in this embodiment, the substrate 5 to be used has a micron-level textured structure on the surface of the substrate. For example, the substrate may be frosted glass roughened by an abrasive or acid or alkali etchant, or a substrate treated with an electron beam, etc., but is not limited to these.

[0084] exist Figure 5 In this context, hierarchical structures have the following characteristics: Figure 1A The protrusion shown has a rounded head, but the metal part 2 can be as follows: Figure 2AThe recessed area is shown in the diagram.

[0085] <Example>

[0086] Examples are described below. However, the invention is not limited to the examples described below.

[0087] (Example 1)

[0088] An alumina sol solution was prepared by dissolving aluminum sec-butoxyl (hereinafter, also referred to as "Al(O-sec-Bu)3") and ethyl acetoacetate (hereinafter, also referred to as "EtOAcAc") in 2-propanol (hereinafter, also referred to as "IPA") and stirring the mixture at room temperature for approximately three hours. The molar ratio of the components in the alumina sol solution was Al(O-sec-Bu)3:EtOAcAc:IPA = 1:1:20. A sol-gel coating solution was prepared by adding 0.01M hydrochloric acid aqueous solution to the alumina sol solution such that the amount of hydrochloric acid added was twice the molar ratio of Al(O-sec-Bu)3, and then refluxing the resulting solution for approximately six hours. The sol-gel coating solution was applied to a mirror-polished quartz glass substrate used as the substrate by spin coating to form a coating film. Subsequently, the coating film was heat-treated at 100°C for one hour to obtain a transparent alumina gel film. Subsequently, the alumina gel film was immersed in hot water at 80°C for 30 minutes and then dried at 100°C for 10 minutes to form an alumina layer that serves as the dielectric portion 3 with an uneven structure.

[0089] After applying an aqueous palladium chloride solution to an alumina layer with an uneven structure by spin coating, the substrate is dried at 100°C. Then, the substrate is immersed in a nickel-phosphorus plating solution (phosphorus content of about 1 wt% to about 2 wt%) set at 80°C for 40 minutes to form a nickel layer that serves as the uneven structure and structure 1.

[0090] After the metal portion, including the alumina layer, was peeled from the quartz glass substrate, etching was performed for 50 hours at room temperature using a 3M sodium hydroxide aqueous solution as an etching step. SEM observation and XPS measurements revealed a nickel-coated roughness structure formed at the nickel layer used as the metal layer, and alumina, serving as dielectric portion 3, remained on the roughness structure. The average height difference of the roughness structure was 272 nm, the average surface roughness Ra' was 3.8 nm, and the specific surface area was 1.1.

[0091] Furthermore, a gold film was formed on the surface of the obtained component using a gold magnetron sputtering system (Quick Coater SC-701HMCII manufactured by Sanyu Electron Co., Ltd.). The thickness of the gold film was set to three levels: 5 nm, 10 nm, and 15 nm (corresponding to Examples 1-1, 1-2, and 1-3, respectively). Additionally, an alkyl thiol (12-mercaptododecanoic acid, manufactured by Sigma-Aldrich Co. LLC) with a carboxylic acid terminal was used as the molecule having a functional group 710 that can be adsorbed or bound to the metal portion. The substrate was immersed in an alkyl thiol ethanol solution (alkyl thiol concentration 1 mM) for 24 hours to form a self-assembled monolayer of alkyl thiol. In this way, a substrate 10 comprising a molecular layer 70 having a carboxylic acid as a functional group 710 was obtained.

[0092] (Example 2)

[0093] After using an alkyl thiol (12-mercaptododecanoic acid, manufactured by Sigma-Aldrich Co. LLC) with a carboxylic acid terminal as a molecule with a functional group 710 that can be adsorbed or bound to the metal moiety 2, the carboxylic acid is converted into an active ester using water-soluble carbodiimide (WSC) and N-hydroxysuccinimide (NHS), and molecular layer 70 is formed by immersing the substrate in a solution (antibody concentration 1 mg / mL) containing an antibody (anti-mouse IgG goat antibody, manufactured by Sigma-Aldrich Co. LLC) as a molecular recognition material with an amino group. Other processes are similar to those in Example 1. The substrate in Example 2 is able to specifically recognize and bind to mouse IgG as molecular layer 70.

[0094] (Comparative Example 1)

[0095] As a substrate used in Comparative Example 1, a substrate similar to the substrate in Example 1 was prepared, except that the metal portion 2 and the molecular layer 70 were not formed.

[0096] (Comparative Example 2)

[0097] As a substrate used in Comparative Example 2, a substrate similar to the substrate in Example 1 was prepared, except that no molecular layer 70 was formed.

[0098] (Fluorescence measurement)

[0099] A 100 μM solution of Rhodamine dye 6G (R6G) was dropped onto the surface of the substrate 10, and fluorescence measurements were performed. The measurement conditions were as follows: A fluorescence inverted microscope (CKX, manufactured by Olympus Corporation) was used as the measurement device, and fluorescence images were acquired using a fluorescence observation G excitation filter, an epifluorescence illumination mercury lamp (50W), and a 20x objective lens with an exposure time of 1 second. The brightness values ​​of the obtained fluorescence images were analyzed. CellSens analysis software, provided with the microscope, was used to analyze the brightness values. R6G fluorescence was detected at any gold film thickness. For comparison, similar fluorescence measurements were performed on substrates without a gold film. No R6G fluorescence was observed. The fluorescence enhancement effect of substrate 10 in each example was confirmed.

[0100] Furthermore, background fluorescence measurements were performed without dropping the R6G solution onto substrate 10. As a result, the background values ​​of substrate 10 in each example were lower compared to substrates without a gold film.

[0101] The above results confirm that substrate 10 achieved a high S / N ratio in the fluorescence measurement of fluorescent molecules. Table 1 shows the fluorescence evaluation results. Results with detected fluorescence signal intensity are indicated by ○, while results with no detected fluorescence signal intensity are indicated by ×. Furthermore, results with a high R6G fluorescence intensity to background ratio (high S / N ratio) are indicated by ○, while results with a low R6G fluorescence intensity to background ratio (low S / N ratio) are indicated by ×.

[0102] [Table 1]

[0103] (Assessment Results)

[0104] In Examples 1 and 2, the functional group 710 was disposed on the metal portion 2, thus the fluorescence signal was sufficiently detected, and the ratio of R6G fluorescence intensity to background was also high. Conversely, in Comparative Example 1, where neither the metal portion 2 nor the functional group 710 was disposed, the fluorescence signal was not sufficiently detected, and the ratio of R6G fluorescence intensity to background was also low. Furthermore, in Comparative Example 2, since the functional group 710 was not disposed, fluorescence quenching was observed, the fluorescence signal was not sufficiently detected, and the ratio of R6G fluorescence intensity to background was also low.

[0105] The above embodiments can be appropriately modified without departing from the technical concept. For example, multiple embodiments can be combined. Furthermore, a portion of the items described in at least one embodiment can be deleted or replaced.

[0106] Furthermore, new items may be added to at least one embodiment. The disclosure of this specification includes not only the items explicitly described herein, but also all items that can be understood from this specification and the accompanying drawings.

[0107] The disclosure in this specification includes the complement of the various concepts described herein. More specifically, for example, when the description "A is greater than B" exists in this specification, even if the description "A is not greater than B" is omitted, this specification still discloses "A is not greater than B". This is because the existence of the description "A is greater than B" presupposes the case of "A is not greater than B".

[0108] This invention is not limited to the embodiments described above, and various changes and modifications can be made within the spirit and scope of this invention. Therefore, in order to inform the public of the scope of this invention, the following claims are made.

[0109] This application claims priority to Japanese Patent Application No. 2023-181745, filed on October 23, 2023, the entire contents of which are incorporated herein by reference.

Claims

1. A substrate comprising a plurality of protrusions containing metal, The first protrusion among the plurality of protrusions is provided with a first metal portion comprising at least one of gold, silver, platinum, copper, and palladium. The second protrusion, which is different from the first protrusion among the plurality of protrusions, is provided with a second metal portion comprising at least one of gold, silver, platinum, copper, and palladium. Dielectric portions are provided between the first protrusion and the first metal portion, and between the second protrusion and the second metal portion. A gap is provided between the first metal portion and the second metal portion, and the distance between the first metal portion and the second metal portion is less than 50 nm. The first metal portion and the second metal portion include functional groups adsorbed or bound to the first metal portion and the second metal portion.

2. The substrate according to claim 1, wherein the distance between the first metal portion and the second metal portion is less than 10 nm.

3. The substrate according to claim 1 or 2, wherein the dielectric portion forms an interface with each of the first metal portion and the second metal portion.

4. The substrate according to any one of claims 1 to 3, wherein the dielectric portion comprises aluminum oxide.

5. The substrate according to any one of claims 1 to 4, wherein the dielectric portion has a shape following the protrusions on a surface on the side opposite to the plurality of protrusions.

6. The substrate according to any one of claims 1 to 5, wherein the second protrusion is one of the plurality of protrusions adjacent to the first protrusion.

7. The substrate according to any one of claims 1 to 6, wherein the first metal portion and the second metal portion are discontinuous with each other.

8. The substrate according to any one of claims 1 to 7, wherein each of the plurality of protrusions comprises a metal including at least one of nickel, chromium and zinc.

9. The substrate according to any one of claims 1 to 8, wherein a concave-convex structure is provided, comprising the plurality of protrusions and the recesses between the plurality of protrusions, and the height difference of the concave-convex structure is 100 nm or more and 1000 nm or less.

10. The substrate according to claim 9, wherein the dielectric portion is disposed on the recess.

11. The substrate according to claim 9 or 10, wherein the dielectric portion is exposed in the space above the recess.

12. The substrate according to any one of claims 1 to 11, wherein the dielectric portion forms an interface with the first protrusion.

13. The substrate according to any one of claims 1 to 12, wherein the thickness of the first metal portion or the second metal portion is 5 nm or more and 50 nm or less.

14. The substrate according to any one of claims 1 to 13, wherein the thickness of the dielectric portion is 30 nm or more and 200 nm or less.

15. The substrate according to any one of claims 1 to 14, wherein the functional groups are disposed on the surfaces of the first protrusion, the second protrusion, the first metal portion, the second metal portion, and the dielectric portion.

16. The substrate according to any one of claims 1 to 15, wherein the functional group is at least one selected from amino, carboxyl, hydroxyl, maleimide, thiol, methoxy, and hydroxyl groups.

17. The substrate according to any one of claims 1 to 16, wherein a molecular layer having the functional groups is disposed on the first metal portion and the second metal portion.

18. The substrate of claim 17, wherein the molecular layer comprises a molecular recognition material.

19. The substrate according to any one of claims 1 to 18, wherein the substrate is a fluorescence-enhancing substrate.

20. An analytical method comprising: Place the sample on the substrate according to any one of claims 1 to 19; as well as The sample was irradiated with light.

21. The analytical method according to claim 20, wherein the wavelength of the light is above 300 nm and below 850 nm.

22. An apparatus comprising: A light source, configured to emit light; as well as The substrate according to any one of claims 1 to 19, The light source is configured to illuminate a sample placed on the substrate with the light.

23. The apparatus of claim 22, further comprising a detector configured to detect fluorescence from the sample.

24. A method for manufacturing a substrate, the method comprising: A dielectric portion with a first uneven structure is formed on the surface; A structure is formed on a first convex-concave structure, the structure comprising metal and having a second convex-concave structure formed by transferring the first convex-concave structure; Remove a portion of the dielectric portion so that the dielectric portion covers the protrusion of the second concave-convex structure, and remove a portion of the dielectric portion so that the distance from the surface of the dielectric portion on the side opposite to the structure to the recess of the second concave-convex structure is less than the height difference between the protrusion and the recess of the structure adjacent to the protrusion; A first metal portion comprising at least one of gold, silver, platinum, copper, and palladium is formed on a first protrusion on the side of the dielectric portion opposite to the structure, and a second metal portion comprising at least one of gold, silver, platinum, copper, and palladium is formed on a second protrusion in the protrusion that is different from the first protrusion; as well as Functional groups that can adsorb or bind to the first metal portion and the second metal portion are provided on the first metal portion and the second metal portion. A gap is provided between the first metal part and the second metal part, and the distance between the first metal part and the second metal part is less than 50 nm.

25. The method of manufacturing a substrate according to claim 24, wherein the dielectric portion is removed so that the surface of the dielectric portion on the side opposite to the structure has a shape that follows the uneven structure transferred to the structure.

26. The method of manufacturing a substrate according to claim 24 or 25, wherein a gap of less than 10 nm is provided between the first metal portion and the second metal portion.

27. The method of manufacturing a substrate according to any one of claims 24 to 26, wherein the first metal portion and the second metal portion form an interface with the dielectric portion.

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