Substrate, analysis method, apparatus, and manufacturing method

Abstract

To improve the S / N ratio of a fluorescence signal.SOLUTION: A substrate comprises a plurality of projections including metal. On a first projection of the plurality of projections, a first metal part is provided which includes metal having at least one of gold, silver, platinum, copper, and palladium. On a second projection different from the first projection of the plurality of projections, a second metal part is provided which includes metal having at least one of gold, silver, platinum, copper, and palladium. Between the first projection and the first metal part and between the second projection and the second metal part, a dielectric part is provided. Between the first metal part and the second metal part, a gap is provided, and the distance between the first metal part and the second metal part is 50 nm or less. The first metal part and the second metal part have a functional group that can be adsorbed on or coupled to the first metal part and the second metal part.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a substrate. [Background technology]

[0002] When light hits a metal, plasma resonance occurs on the metal surface, and this resonance produces an electric field enhancement effect (localized plasmon resonance phenomenon). Development of electric field enhancement devices, such as sensor devices, Raman spectroscopy devices, and fluorescence devices, that utilize the electric field enhancement effect is progressing. Surface-enhanced fluorescence, which uses the optical electric field enhanced by localized plasmon resonance, is known to detect trace amounts of substances.

[0003] For example, Patent Document 1 discloses a configuration consisting of an optical substrate having a fine uneven structure and a metal film formed on the surface of the fine uneven structure. Patent Document 2 discloses a plasmon excitation sensor consisting of a substrate having a ligand and a plurality of metal protrusions covered with a self-assembled monolayer. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2007-240361 A [Patent Document 2] JP 2010-256161 A Summary of the Invention [Problem to be solved by the invention]

[0005] In the substrates disclosed in Patent Documents 1 and 2, when the amount of substance is small or when there are many impurities, the fluorescence enhancement effect is small and the background signal from the base material may be large, leaving room for improvement in the signal S / N ratio. Therefore, an object of the present invention is to improve the S / N ratio of a fluorescent signal in a fluorescence enhancement device. [Means for solving the problem]

[0006] A first means for solving the above problem is a substrate having a plurality of convex portions including a metal, wherein a first metal portion including a metal having at least one of gold, silver, platinum, copper, and palladium is provided on a first convex portion of the plurality of convex portions, and a second metal portion including a metal having at least one of gold, silver, platinum, copper, and palladium is provided on a second convex portion of the plurality of convex portions different from the first convex portion, and a dielectric portion is provided between the first convex portion and the first metal portion and between the second convex portion and the second metal portion, a gap is provided between the first metal portion and the second metal portion, the distance between the first metal portion and the second metal portion is 50 nm or less, and the first metal portion and the second metal portion have functional groups that can be adsorbed or bonded to the first metal portion and the second metal portion. Effect of the Invention

[0007] The present invention provides a technique that is advantageous in improving the S / N ratio of a fluorescent signal. [Brief description of the drawings]

[0008] [Figure 1] FIG. 2A is a schematic diagram of a substrate according to the present embodiment, FIG. 2B is an enlarged view of a main portion of FIG. 2A, and FIG. 2C is a schematic diagram showing a modified example of the substrate according to the present embodiment. [Diagram 2] FIG. 2 is a schematic diagram of a substrate according to the present embodiment. [Diagram 3] 5A to 5C are schematic diagrams illustrating a method for manufacturing a substrate according to the present embodiment. [Figure 4] FIG. 1 is a schematic diagram showing an example of an apparatus on which a substrate according to an embodiment of the present invention is mounted. [Diagram 5] FIG. 6 is a schematic view of a substrate according to a second embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Hereinafter, the embodiment for carrying out the present invention will be described with reference to the drawings. However, the embodiment described below is one embodiment of the invention, and the present invention is not limited to this. Then, common configurations will be described with mutual reference to multiple drawings, and the description of configurations with common symbols will be omitted as appropriate. Items with the same name but different can be distinguished by adding "No. 0", such as the first item and the second item.

[0010] First Embodiment The substrate 10 according to the present embodiment will be described with reference to FIG. 1. FIG. 1(a) is a schematic diagram of the substrate 10 according to the present embodiment, FIG. 1(b) is an enlarged view of the metal part 2 in FIG. 1(a), and FIG. 1(c) is a modified example of the substrate 10 according to the present embodiment. The substrate 10 includes a structure 1, a dielectric part 3 provided on the surface of the structure 1, a metal part 2 that forms an interface with the dielectric part 3, and a molecular layer 70 having a functional group 710 that can be adsorbed or bonded on the metal part 2. The structure 1 has an uneven structure, and the metal part 2 and the dielectric part 3 are provided on a plurality of protrusions 4 including protrusions 41 and 42 of the uneven structure. The dielectric part 3 is provided between the structure 1 and the metal part 2, and is preferably structured to form an interface with both the structure 1 and the metal part 2.

[0011] A gap 9 is provided between the metal portion 21 and the metal portion 22 adjacent to the metal portion 21, and the distance D between the metal portion 21 and the metal portion 22 is greater than 0 and 50 nm or less. The distance D between the metal portion 21 and the metal portion 22 is more preferably greater than 0 and 10 nm or less. The distance D is the shortest distance between the metal portion 21 and the metal portion 22. The distance D is more preferably the distance between the metal portion 21 and the metal portion 22, but it may be the distance between the metal portions 21 and 22 when a plurality of metal portions 21 are provided on the same convex portion 41. In addition, it may be the distance between the metal portion 2 provided on another convex portion 4, rather than the convex portion 42 adjacent to the convex portion 41. By providing the gap 9, it is possible to further enhance the optical electric field, and the intensity of the fluorescence can be improved on the substrate 10. The metal portion 21 and the metal portion 22 may be connected at a portion other than the gap 9 provided therebetween, but it is preferable that they are discontinuous.

[0012] The substrate 10 is provided with a molecular layer 70 having functional groups 710 capable of adsorbing or bonding onto the metal portion 21 and the metal portion 22. The molecular layer 70 is formed on a portion of the metal portion 2 where the metal portion 2 is not in contact with the dielectric portion 3. Alternatively, the molecular layer 70 is formed on the metal portion 2 and the dielectric portion 3 so as to cover a portion of the metal portion 2 and the dielectric portion 3 where the metal portion 2 and the dielectric portion 3 are not in contact with the protruding portion 4. Furthermore, as shown in FIG. 1(c), the molecular layer 70 may be formed on the side of the protruding portion 4 that is not in contact with the adhesive layer 6.

[0013] In Fig. 1(a), the surface of the metal portion 2 is covered with a molecular layer 70 having functional groups 710 capable of adsorbing or bonding as shown in Fig. 1(b). In Fig. 1(c), functional groups 710 are provided on the surfaces of the protrusion 4, the metal portion 2, and the dielectric portion 3. In other words, they are covered with the molecular layer 70. The coverage of the molecular layer 70 may be continuous or discontinuous.

[0014] The molecular layer 70 having the functional group 710 capable of adsorbing or binding to the metal part 2 is not particularly limited as long as it is composed of a molecule capable of stably adsorbing or binding to the metal part 2, but is preferably composed of an organic molecule or a protein, as described later. By providing the molecular layer 70 on the substrate 10, it becomes possible to appropriately arrange the fluorescent molecules to be detected, which are contained in the specimen on the base material, in the vicinity of the metal part 2. For example, it is possible to stably adsorb the fluorescent molecules contained in the specimen or the substance labeled with the fluorescent molecules. In this case, it is preferable that the molecular layer 70 contains a molecular recognition material capable of specifically binding to the fluorescent molecules contained in the specimen or the substance labeled with the fluorescent molecules. Here, the molecular recognition material is a molecule capable of specifically recognizing and binding to the substance to be measured in the specimen, and examples thereof include proteins, sugars, lipids, and nucleic acids. For example, an antibody can be used as the protein. By using the molecular layer 70 having an antibody, it becomes possible to fix an antigen for the antibody in the vicinity of the metal part 2. The size of the antibody molecule is preferably 5 nm or more and 15 nm or less, and the thickness of the molecular layer 70 having the antibody is preferably 5 nm or more and 15 nm or less (when the antibody is directly coated on the metal portion 2).

[0015] Another example of a protein is albumin. Albumin is a protein having a size of several nm, and can be used as a component of the molecular layer 70. By including albumin in the molecular layer 70, the molecular layer 70 becomes highly hydrophilic.

[0016] When the detection target is an antigen labeled with a fluorescent molecule (fluorescence-labeled antigen), the fluorescence-labeled antigen (generally several nm to several tens of nm in size) bound to the antibody in the molecular layer 70 is fixed via the antibody at a distance of 10 nm to 100 nm from the metal part 2. At a distance of 10 nm to 100 nm from the metal part 2, an enhanced electric field is localized, and the fluorescence-labeled antigen is fixed in this region. As a result, the S / N ratio of the fluorescence from the fluorescence-labeled antigen contained in the specimen is increased, making it possible to detect the fluorescence-labeled antigen with high sensitivity.

[0017] The molecular layer 70 having the functional group 710 capable of adsorbing or bonding to the metal part 2 can have organic molecular films or self-assembled monolayers as its components, as examples of organic molecules. Examples of organic molecular films include citric acid and amino acids. Examples of self-assembled monolayers include alkanethiols and silane coupling agents. The silane coupling agent is an organic silicon compound, and while various functional groups such as amino groups, carboxy groups, and hydroxyl groups can be selected, it exhibits strong bonding ability to the metal part 2, so that it is possible to form a stable molecular layer 70 with controlled physical properties. The alkanethiol is a molecule having a thiol group with about 4 to 20 carbon atoms, and in addition to having strong bonding affinity to metals, the molecule itself has strong intermolecular cohesive force, so that it is possible to form a stable monolayer on the metal part 2. When the metal part 2 is gold, it is particularly preferable as a component of the molecular layer. The alkanethiol has various functional groups such as amino groups, carboxy groups, and hydroxyl groups at its terminals.

[0018] An example of the molecular layer 70 of the present invention is characterized by having a functional group 710 that is not in contact with the metal portion 2. The functional group 710 is, for example, a group capable of interacting with the measurement target molecule, a functional group that binds an interacting molecular recognition material, or a functional group that does not substantially interact with impurities that are not the measurement target. The functional group 710 can be, for example, an amino group, a carboxy group, a hydroxyl group, a maleimide group, a thiol group, a methoxy group, a hydroxyl group, or the like, with the amino group, the carboxy group, and the hydroxyl group being particularly preferred.

[0019] One preferred embodiment of the molecular layer 70 having functional groups 710 that can be adsorbed or bonded onto the metal portion 2 is a molecular layer 70 made of a molecular recognition material and a self-assembled monolayer. FIG. 1(b) shows an example of this embodiment. FIG. 1(b) is an enlarged view of the metal portion 2 of the substrate 10. The metal portion 2 is coated with the molecular layer 70, which is made up of the functional groups 710, a self-assembled monolayer 711, and a self-assembled monolayer 712 having a molecular recognition material (the self-assembled monolayer to which the molecular recognition material is chemically bonded).

[0020] As an example of a preferred embodiment, there is a molecular layer 70 made of a molecular recognition material and albumin. In these examples of the embodiment, it is possible to bind the molecules to be measured while preventing non-specific binding of contaminants to the substrate 10. Non-specific binding of contaminants to the substrate 10 is undesirable because it causes noise in the detection of fluorescence (an increase in N) and inhibits binding of the molecular recognition material to the molecules to be measured (a decrease in S).

[0021] The thickness of the molecular layer 70 is not particularly limited as long as it is within the range of the localized enhanced electric field region, but is preferably 0.1 nm to 100 nm, more preferably 1 nm to 50 nm. If the thickness exceeds 100 nm, the distance between the fluorescent molecules and the metal part 2 increases, which may reduce the effect of improving the fluorescent signal intensity.

[0022] The substrate 10 of the present invention has a protruding structure consisting of a plurality of metal parts 2 provided with molecules having functional groups 710 capable of adsorption or binding, and thus a localized electric field enhancement region is generated in the vicinity of the protruding structure. This results in stronger fluorescence from fluorescent molecules present in the vicinity of the protruding structure. Furthermore, the characteristic structure and composition of the substrate of the present invention reduce background noise (e.g., scattered light noise, etc.) from the substrate 10, resulting in not only improved signal strength (improved S) due to the enhanced electric field, but also suppression of noise (reduced N), thereby improving the S / N ratio of the fluorescent signal.

[0023] In Fig. 1(a) and Fig. 1(c), the metal part 2 has a horsetail-like shape, but as shown in Fig. 2(a) and Fig. 2(b), the metal part 2 may be shaped to fit the dielectric part 3, and the shape of the metal part 2 is not limited. As shown in Fig. 1(c), the dielectric part 3 may be provided on the recess 43, and the dielectric part 3 may be connected to the dielectric part 3 on the protrusions 41 and 42. The metal of the protrusions 41 and 42 may be discontinuous. It is preferable that the metal part 2 does not cover the recess 43, and it is preferable that the dielectric part 3 is exposed in the space above the recess 43.

[0024] The uneven structure is preferably provided only on one surface of the structure 1, and the distance between the convex portion 41 and the concave portion 43, i.e., 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 height difference is preferably the average of the height differences of the uneven structure. The height difference may be the linear distance from the convex portion 41 to the concave portion 43, or may be the vertical distance from the convex portion 41 to the concave portion 43. The height difference can be obtained by observing the cross section of the substrate 10 with a scanning electron microscope or the like. The convex portion 41 and the convex portion 42 are preferably connected via the concave portion 43, but may be separated. In the structure 1, the convex portion 4 is metal, but the concave portion 43 does not have to be metal, and may be non-metallic, such as ceramics or resin.

[0025] The material of the structure 1 is preferably a highly conductive material, such as gold, silver, copper, aluminum, magnesium, tungsten, cobalt, zinc, nickel, and chromium, with nickel, zinc, and chromium being preferred, and nickel being particularly preferred.

[0026] The material of the metal part 2 includes any one of metals selected from gold, silver, platinum, copper, and palladium, and is particularly preferably gold or silver. The thickness of the metal part 2 is not particularly limited as long as it is thick enough to maintain a concave-convex structure capable of generating localized plasmons when irradiated with excitation light, but is preferably 5 nm or more and 50 nm or less.

[0027] The material of the dielectric portion 3 is preferably a metal oxide. The material of the metal oxide is not particularly limited to silica, alumina, zirconia, etc., but it is preferable that the main component is alumina, and more preferable that the plate crystals contain alumina as the main component. The plate crystals containing alumina as the main component are formed by plate crystals containing aluminum oxide or hydroxide or hydrates thereof as the main component, and a particularly preferable crystal is boehmite. Here, the plate crystals containing alumina as the main component may be plate crystals consisting of alumina alone, or may be plate crystals containing a trace amount of zirconium, silicon, titanium, zinc, etc. in the plate crystals of alumina. In the case of a plate structure of the plate crystals containing alumina as the main component, it is preferable that the plate crystals containing alumina as the main component are arranged in a direction perpendicular to the surface direction of the structure 1, and that the spatial occupancy rate of the plate crystals changes continuously. The metal oxide may also contain an amorphous gel of alumina. The dielectric portion 3 is preferably formed in a shape that follows the uneven structure or the protrusions 4 of the structure 1 as shown in FIG. 1(c). The thickness of the dielectric portion 3 is preferably not less than 30 nm and not more than 200 nm.

[0028] 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 calculated by the following formula. Sr=S / S0 formula (1)

[0029] In formula (1), S0 is the surface area when the measurement surface is assumed to be ideally flat, and S is the surface area of ​​the actual measurement surface. The specific surface area can be determined by observing a surface having an uneven structure using a scanning probe microscope or the like.

[0030] The metal elements of the structure 1 and the metal oxides in the dielectric portion 3 can be detected by energy dispersive X-ray analysis (EDX) when observing the surface or cross section with a scanning electron microscope (SEM) or a transmission electron microscope (TEM). They can also be detected by X-ray photoelectron spectroscopy (XPS). In the direction perpendicular to the surface direction of the structure 1, the proportion of metal oxides becomes relatively lower from the dielectric portion 3 toward the structure 1, and the proportion of metal elements constituting the structure 1 becomes higher, until finally only the metal elements are detected.

[0031] The substrate 10 has a base material 5 on the surface opposite to the side of the structure 1 on which the uneven structure is provided. The base material 5 is provided on the structure 1 via an adhesive layer 6, but the adhesive layer 6 can be omitted. The shape of the base material 5 may be any shape that can be formed according to the purpose of use, and includes, but is not limited to, a flat plate shape, a film shape, a sheet shape, and the like. The material of the base material 5 may be, but is not limited to, metal, glass, ceramics, wood, paper, resin, and the like. Examples of resins include, but are not limited to, polyester, triacetyl cellulose, cellulose acetate, polyethylene terephthalate, polypropylene, polystyrene, polycarbonate, polymethyl methacrylate, and ABS resin. In addition, films and molded products of thermoplastic resins such as polyphenylene oxide, polyurethane, polyethylene, and polyvinyl chloride; and thermosetting resins such as unsaturated polyester resin, phenolic resin, crosslinked polyurethane, crosslinked acrylic resin, and crosslinked saturated polyester resin may be used. The adhesive layer 6 may be any layer that can bond the base material 5 and the structure 1, and examples of the adhesive layer 6 include a layer made of a cured product of an adhesive resin (e.g., epoxy resin, etc.), and a double-sided tape.

[0032] Next, a method for manufacturing the substrate 10 will be described with reference to Fig. 3. The manufacturing method according to this embodiment includes a step of forming a structure 1, a step of forming a dielectric portion 3, a step of forming a metal portion 2, and a step of forming a molecular layer 70 having a functional group 710 capable of adsorbing or bonding on the metal portion.

[0033] The process of forming the dielectric portion 3 will be described with reference to Figures 3(a) and 3(b). Here, the dielectric portion 3 contains a metal oxide having alumina. An aluminum compound, and if necessary, other compounds, a stabilizer, and a water-soluble organic polymer compound are dissolved or suspended in an organic solvent to prepare a sol-gel coating liquid. This sol-gel coating liquid is applied to a base substrate 8 and dried to form an alumina gel film as an aluminum film 7 containing aluminum. Alternatively, an alumina gel film containing metallic aluminum as the aluminum film 7 is formed on the base substrate 8 by dry film formation such as vacuum deposition or sputtering.

[0034] Next, the aluminum film 7 is immersed in hot water to form an alumina uneven structure. By immersing the aluminum film 7 in hot water, the surface layer of the aluminum film 7 is peptized, and some components are dissolved. However, due to differences in the solubility of various hydroxides in hot water, plate-like crystals mainly composed of alumina are precipitated and grown on the surface layer of the aluminum film 7, forming the uneven structure of the dielectric part 3. In addition, when a film containing metallic aluminum is used instead of the aluminum film 7, the aluminum reacts with the hot water and is oxidized to alumina, and then the uneven structure of the dielectric part 3 is formed in the same manner as when the aluminum film 7 is used. Therefore, when the material of the base substrate 8 mainly contains aluminum or alumina, the formation of the aluminum film 7 on the base substrate 8 can be omitted. The temperature of the hot water is preferably 40° C. or higher and lower than 100° C. The immersion time is preferably about 5 minutes to about 24 hours. In the immersion treatment of the aluminum film 7 to which compounds other than the alumina component are added, the difference in solubility of each component in hot water is used to crystallize the plate-like crystals of alumina. Therefore, unlike the immersion treatment of the aluminum film 7 containing only alumina, the size of the plate-like crystals can be controlled over a wide range by changing the composition of the inorganic components. In addition, the height of the uneven shape of alumina can be adjusted by adjusting 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. As a result, it is possible to control the unevenness formed by the plate-like crystals over a wide range.

[0035] The material of the base substrate 8 is not particularly limited, and various materials such as glass, plastic, and metal can be used. When forming the aluminum film 7 using a sol-gel coating liquid that does not contain a stabilizer, it is preferable to use an inert gas atmosphere such as dry air or dry nitrogen for coating. The relative humidity of the dry atmosphere is preferably 30% or less. As a solution coating method for forming the aluminum film 7, a known coating method such as a dipping method, a spin coating method, a spray method, a printing method, a flow coating method, or a combination of these methods can be appropriately adopted. The film thickness can be controlled by changing the pulling speed in the dipping method or the substrate rotation speed in the spin coating method, and by changing the concentration of the sol-gel coating liquid. Drying can be performed at room temperature for about 30 minutes. In addition, drying or heat treatment can be performed at a higher temperature as necessary, and the higher the heat treatment temperature, the more stable the uneven structure of the dielectric part 3 can be formed by the immersion treatment described later. The suitable thickness of 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.

[0036] Next, the process of forming the structure 1 will be described with reference to FIG. 3(c). The structure 1 containing a metal is formed on the uneven structure of the dielectric portion 3 described in FIG. 3(b). As a method for forming the structure 1, a metal plating process is preferable, and an electroless plating process is more preferable. In the electroless plating process, an aqueous solution in which a palladium compound such as palladium chloride, a gold compound such as gold chloride, a silver compound such as silver chloride, a tin compound such as tin chloride, or the like is dissolved is applied to the uneven structure of the dielectric portion 3 to perform activation. The activation may be performed by immersing the uneven structure of the dielectric portion 3 together with the base material 8 in an aqueous solution in which a palladium compound is 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 of this embodiment, and an electroless plating solution containing nickel ions, chromium ions, and zinc ions is preferable, and a nickel plating solution containing nickel ions is particularly preferable. The nickel plating solution may contain a phosphorus component or a boron component in addition to the nickel component. The temperature of the plating solution in the electroless plating process is preferably 30°C or more and 98°C or less, more preferably 50°C or more and 90°C or less. The time for performing the electroless plating process can be adjusted according to the thickness of the structure 1 to be formed, and is usually 30 seconds to 1 hour. In this way, the structure 1 is formed so as to fill the gaps of the uneven structure, and the structure 1 having the uneven structure to which the uneven structure of the dielectric part 3 is transferred is formed. It is preferable to perform the electroless plating process so that the thickness of the structure 1 having the uneven structure is 200 nm or more and 15000 nm or less. In addition, the average height difference of the uneven structure corresponds to the average height difference of the uneven structure of the dielectric part 3, and is 100 nm or more and 1000 nm or less.

[0037] After the electroless plating process described above, in order to increase the thickness of the structure 1, an electroplating process may be performed on the opposite side of the structure 1 from the side on which the uneven structure is provided. A known electroplating solution may be used for the electroplating process, and for example, an electroplating solution containing nickel ions, iron ions, copper ions, etc. as metal ions may be used. When the same metal as that of the structure 1 is used for the electroplating process, the thickness of the structure 1 can be increased by the electroplating process. When a metal different from that of the structure 1 is used for the electroplating process, the metal provided by the electroplating process becomes the base material. In addition to inorganic salts that are raw materials for metal ions, conductive salts, salts for adjusting counter ions, carboxylic acid additives for increasing the homogeneity of the plating film, gloss agents, etc. may be added to the electroplating solution as necessary. In addition, in the electroplating process, the temperature, current density, and plating time of the electroplating solution may be adjusted to make the thickness of the structure 1 a desired thickness. If necessary, before the electroplating process, the opposite side of the structure 1 from the side on which the uneven structure is provided may be activated with an aqueous solution containing an acid, etc. Furthermore, in order to improve the quality of the film formed by the electroplating process, in addition to stirring the electroplating solution during the electroplating process, a step of removing foreign matter from the electroplating solution may also be provided.

[0038] Next, the process of adhering the substrate 5 to the structure 1 will be described with reference to FIG. 3(d). When the substrate 5 is made of a metal, the metal that will become the substrate 5 may be further laminated on the surface opposite to the surface of the structure 1 on which the concave-convex structure is provided. The metal may be laminated by the above-mentioned electroplating process or by physical vapor deposition such as sputtering. When the substrate 5 is made of a resin, the substrate 5 may be provided by depositing the resin that will become the substrate 5 on the surface opposite to the concave-convex structure of the structure 1 and then curing the resin. The substrate 5 may be adhered to the structure 1 by an adhesive layer 6. The adhesive used for the adhesive layer 6 is preferably a resin, but is not particularly limited as long as it is a material that can adhere the substrate 5 and the structure 1.

[0039] Next, the etching process of the base substrate 8, the aluminum film 7, and a part of the dielectric portion 3 will be described with reference to FIG. 3(e), FIG. 3(f), and FIG. 3(g). FIG. 3(e) is a view obtained by turning FIG. 3(d) upside down. First, the base substrate 8 is removed as shown in FIG. 3(f). When the aluminum film 7 is an alumina gel film, the aluminum film 7 can function as a part of the dielectric portion 3 of the substrate 10. The aluminum film 7 may be partially removed by etching. As an etching method, wet etching in which an aluminum-containing film is dissolved using an acid or an alkaline solution is preferable. Examples of acids include hydrochloric acid, nitric acid, and sulfuric acid. Examples of alkalis include sodium hydroxide and potassium hydroxide. From the viewpoint of work efficiency, an etching method using an alkaline solution is more preferable. The etching concentration is preferably in the range of several percent to several tens of percent, and the etching time is preferably in the range of several hours to several days. The remaining metal oxide such as alumina after etching can be detected by EDX during surface or cross-sectional observation by SEM or TEM, or by XPS measurement. In this step, the dielectric portion 3 is removed so that the distance H1 to the recesses of the uneven structure of the transferred structure 1 is smaller than the height difference H2 between the protrusions and the recesses of the structure 1 adjacent to the protrusions. The distance H1 may be 0, and the dielectric portion 3 in the recesses may be entirely etched. At this time, the protrusions of the uneven structure of the structure 1 are covered with the dielectric portion 3. The dielectric portion 3 is removed so that the surface of the dielectric portion 3 opposite the structure 1 has a shape that conforms to the uneven structure transferred to the structure 1. Before the bonding step of the substrate 5, the etching step of this step may be performed and then the substrate 5 may be bonded.

[0040] 3(h), a process for forming the metal part 2 is described. The metal part 2 containing any one selected from gold, silver, platinum, copper, and palladium is formed on the member obtained after etching by dry film formation such as vacuum deposition or sputtering.

[0041] Next, a process for forming a molecular layer 70 having functional groups 710 capable of adsorbing or bonding on a metal part will be described with reference to Fig. 3(i). In a method for forming a self-assembled monolayer using alkanethiol, a substrate (Fig. 3(h)) is immersed in an alkanethiol solution to form a self-assembled monolayer of alkanethiol on the surface of the metal part 2. In a method for forming a protein film, a substrate (Fig. 3(h)) is immersed in a protein solution to form an adsorption layer of protein on the surface of the metal part 2.

[0042] The process of forming the molecular layer 70 containing the molecular recognition material will be described. As a method utilizing physical adsorption, the substrate (FIG. 3(h)) is immersed in a solution containing the molecular recognition material, thereby forming an adsorption layer of the molecular recognition material on the surface of the metal part 2. For example, the substrate (FIG. 3(h)) is immersed in an aqueous solution containing an antibody, which is the molecular recognition material. After one hour at room temperature, the substrate (FIG. 3(h)) is removed and washed with water, thereby forming the molecular layer 70 consisting of an adsorption layer of the antibody. Furthermore, after this, the substrate on which the molecular layer 70 consisting of the adsorption layer of the antibody has been formed may be immersed in an aqueous solution of albumin, thereby covering the parts to which the antibody has not been adsorbed with albumin.

[0043] An example of a method using chemical bonds will be described. A substrate (FIG. 3(h)) is immersed in a solution of alkanethiol having a carboxylic acid at the end, and a self-assembled monolayer of alkanethiol having a carboxylic acid at the end is formed on the surface of the metal part 2. Next, this carboxylic acid is activated esterified using a water-soluble carbodiimide (WSC) (for example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)) and N-hydroxysuccinimide (NHS), and the activated esterified carboxyl group is chemically bonded (amide bonded) to an amino group of the molecular recognition material, thereby forming an adsorption layer of the molecular recognition material on the surface of the metal part 2. The molecular recognition material is stably fixed by adsorption through chemical bonds. After this, the substrate on which the molecular layer 70 consisting of the antibody adsorption layer is formed may be immersed in an aqueous solution of albumin to cover the part where the antibody is not adsorbed with albumin.

[0044] The thus obtained method for manufacturing the substrate 10 according to this embodiment provides high in-plane uniformity for the same sample, so that data with good reproducibility can be obtained, enabling highly reliable and effective measurements. In addition, because the manufacturing method is very simple, the production cost can be reduced compared to conventional devices.

[0045] The substrate 10 of this embodiment can be used as a fluorescence enhancing device in a fluorescence detection method. The substrate 10 of this embodiment can enhance the emission of fluorescence by an enhanced optical field associated with localized plasmon resonance.

[0046] Various adhesives can be used when providing the substrate 10 of this embodiment on the surface of a member or an article. Therefore, the substrate 10 of this embodiment can be provided on the surface of a member or an article depending on the purpose of use, and the surface of the member or article is not limited to being smooth, and may be a two-dimensional or three-dimensional curved surface.

[0047] Next, a fluorescence detection device will be described with reference to FIG. 4 as an example of an apparatus 100 on which the substrate 10 according to this embodiment can be mounted.

[0048] The device 100 includes a substrate 10, a light irradiating section 140 that irradiates the substrate 10 with light L1, and a light detecting section 150 that detects fluorescence L2 emitted from a specimen S.

[0049] The light irradiation section 140 has 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 irradiated onto the specimen S on the substrate 10. Light L2, which includes fluorescence emitted from the specimen S in response to irradiation with the light L1, is reflected by the dichroic mirror 143 toward the light detection section 150.

[0050] The light detection unit 150 includes an emission filter 151 and a detector 152. The emission filter 151 passes light having a wavelength in the range to be detected, among the light L2 reflected by the dichroic mirror 143. The light L2 containing fluorescence emitted from the specimen S passes through the emission filter 151 and can be detected by the detector 152.

[0051] More specifically, irradiation with light L1 induces localized plasmon resonance in the uneven structure of the substrate 10, generating an enhanced optical electric field on the surface of the metal part 2. Fluorescence L2 emitted from the specimen S and enhanced by this enhanced optical electric field is detected by the detector 152.

[0052] The wavelength of the light irradiated from the light source 141 to the specimen S can take any value depending on the fluorescent molecules to be detected, and can range from ultraviolet to visible to near infrared. It is preferable that the wavelength of the light irradiated to the specimen S be 300 nm or more and 850 nm or less.

[0053] Although a fluorescence detection device has been described here as an example of the device 100, the device is not limited to this device and can also be used in a fluorescence spectrometer, a fluorescence microscope, or the like.

[0054] <Second embodiment> Next, the substrate 10 according to this embodiment will be described with reference to FIG.

[0055] The substrate 10 according to this embodiment differs from the first embodiment in that the structure 1 has a hierarchical structure. The hierarchical structure is composed of at least two types of structures with different structural sizes, and means a structure having, for example, a first structure having a structural size on the order of microns and a second structure having a structural size on the order of submicrons. The height difference of the first structure having a structural size on the order of microns is, for example, 1 μm or more and 10 μm or less.

[0056] The structure 1 has a base 11 provided on an adhesive layer 6 and a concave-convex structure 12 provided on the base 11, and the concave-convex structure 12 is composed of a first concave-convex structure 121 and a second concave-convex structure 122 having a smaller scale than the first concave-convex structure 121. The second concave-convex structure 122 is provided on the first concave-convex structure 121, and the first concave-convex structure 121 and the second concave-convex structure 122 have a plurality of convex portions and concave portions provided between the plurality of convex portions.

[0057] Furthermore, similar to the first embodiment, a metal portion 2, a dielectric portion 3 between the structure 1 and the metal portion 2, and a molecular layer 70 having a functional group 710 capable of adsorbing or bonding onto the metal portion 2 are provided on the second uneven structure 122. By providing gaps between the metal portions 2 provided on the convex portions of the second uneven structure 122, it is possible to further enhance the optical electric field, thereby improving the intensity of fluorescence on the substrate 10. A molecular layer 70 may also be provided in the gaps between the first uneven structures 121.

[0058] It is preferable that the first uneven structure 121 and the second uneven structure 122 are each formed of the same material, and it is also preferable that the base 11 is made of the same material. The distance between the convex and concave portions of the second uneven structure 122, i.e., the height difference of the uneven structure, is preferably 100 nm or more and 1000 nm or less, and more preferably 100 nm or more and 500 nm or less.

[0059] When forming a concave-convex structure on a hierarchical structure as in this embodiment, the substrate 5 used may be any substrate having a micro-order concave-convex structure on the surface thereof, such as, but not limited to, ground glass roughened with an abrasive or an etching solution such as acid or alkali, or a substrate processed with an electron beam.

[0060] In FIG. 5, a horsetail-like hierarchical structure as shown in FIG. 1(a) is shown, but a form in which the metal portion 2 fits along the recess as shown in FIG. 2(a) may also be used.

[0061] <Example> Examples will be described below, but the present invention is not limited to the following examples.

[0062] Example 1 Aluminum sec-butoxide (hereinafter also referred to as "Al(O-sec-Bu)3") and ethyl acetoacetate (hereinafter also referred to as "EtOAcAc") were dissolved in 2-propanol (hereinafter also referred to as "IPA") and stirred at room temperature for about 3 hours to prepare an alumina sol solution. The molar ratio of each component in the alumina sol solution was Al(O-sec-Bu)3: EtOAcAc: IPA = 1: 1: 20. A 0.01M dilute hydrochloric acid solution was added to the alumina sol solution so that the amount of hydrochloric acid added was twice that of Al(O-sec-Bu)3 in terms of molar ratio, and the mixture was refluxed for about 6 hours to prepare a sol-gel coating solution. The sol-gel coating solution was applied to a mirror-polished quartz glass substrate, which is the base substrate, by spin coating to form a coating film. The coating film was then heat-treated at 100°C for 1 hour to obtain a transparent alumina gel film. Next, the alumina gel film was immersed in warm water at 80° C. for 30 minutes, and then dried at 100° C. for 10 minutes to form an alumina layer as the dielectric portion 3 having an uneven structure.

[0063] An aqueous palladium chloride solution was applied by spin coating onto the alumina layer having the concave-convex structure, and then dried at 100° C. Thereafter, the alumina layer was immersed in a nickel-phosphorus plating solution (phosphorus content: approximately 1 to 2 wt%) set at 80° C. for 40 minutes to form the concave-convex structure and a nickel layer as structure 1.

[0064] After the metal portion with the alumina layer was peeled off from the quartz glass substrate, an etching process was performed using a 3M aqueous sodium hydroxide solution at room temperature for 50 hours. SEM observation and XPS measurement showed that a nickel uneven structure was formed on the nickel layer, which was the metal layer, and alumina remained on the uneven structure as the dielectric portion 3. The average height difference of the uneven structure was 272 nm, the average surface roughness Ra' was 3.8 nm, and the specific surface area was 1.1.

[0065] Furthermore, a gold magnetron sputtering device (Quick Coater SC-701HMCII manufactured by Sanyu Electronics Co., Ltd.) was used to form a gold film on the surface of the obtained member. The gold film thickness was set to three levels: 5 nm, 10 nm, and 15 nm (Example 1-1, Example 1-2, and Example 1-3, respectively). Furthermore, an alkanethiol (12-mercaptododecanoic acid, manufactured by Sigma-Aldrich Co.) having a carboxylic acid at the end was used as a molecule having a functional group 710 that can be adsorbed or bonded to the metal part. The substrate was immersed in an ethanol solution of alkanethiol (alkanethiol concentration 1 mM) for 24 hours to form a self-assembled monolayer of alkanethiol. In this way, a substrate 10 provided with a molecular layer 70 having a carboxylic acid as the functional group 710 was obtained.

[0066] Example 2 The substrate in Example 2 is similar to Example 1 except that an alkanethiol having a carboxylic acid at its end (12-mercaptododecanoic acid, manufactured by Sigma-Aldrich) is used as a molecule having a functional group 710 capable of being adsorbed or bonded onto the metal portion 2, and then this carboxylic acid is activated and esterified using water-soluble carbodiimide (WSC) and N-hydroxysuccinimide (NHS), and the substrate is immersed in a solution (anti-mouse IgG goat antibody, manufactured by Sigma-Aldrich) containing an antibody as a molecular recognition material having an amino group (antibody concentration 1 mg / mL) to form a molecular layer 70. The substrate in Example 2 is capable of specifically recognizing and binding to mouse IgG as the molecular layer 70.

[0067] Comparative Example 1 The substrate used in Comparative Example 1 was prepared similarly to that in Example 1, except that the metal portion 2 and the molecular layer 70 were not formed.

[0068] Comparative Example 2 The substrate used in Comparative Example 2 was prepared in the same manner as in Example 1, except that the molecular layer 70 was not formed.

[0069] (Fluorescence measurement) A 100 μM aqueous solution of the dye rhodamine 6G (R6G) was dropped onto the surface of the above-mentioned substrate 10 as a specimen, and fluorescence measurement was performed. The measurement conditions were as follows. A fluorescent inverted microscope (Olympus, CKX) was used as the measurement device, and a fluorescent image was obtained with a G excitation filter for fluorescence observation, a mercury lamp for epifluorescence illumination (50 W), a 20x objective lens, and an exposure time of 1 s. The brightness value of the obtained fluorescent image was analyzed. The analysis software cellSens attached to the microscope was used to analyze the brightness value. Fluorescence of R6G was detected at all gold film thicknesses. For comparison, the fluorescence of a substrate without a gold film was measured in the same manner, and as a result, the fluorescence of R6G could not be observed. It was confirmed that the substrate 10 shown in the example has a fluorescence enhancement effect.

[0070] Furthermore, background fluorescence measurement was performed without dropping the R6G aqueous solution onto the substrate 10. As a result, the background value of the substrate 10 shown in the example was smaller than that of a substrate on which no gold film was formed.

[0071] From the above results, it was confirmed that the substrate 10 has a high S / N ratio in the fluorescence measurement of fluorescent molecules. Table 1 shows the fluorescence evaluation results. Those in which the fluorescent signal intensity was detected are marked with an ◯, and those in which it was not detected are marked with an X. In addition, those in which the fluorescence intensity ratio of R6G to the background was high (good S / N) are marked with an ◯, and those in which it was low (poor S / N) are marked with an X.

[0072] [Table 1]

[0073] (Evaluation Results) In Examples 1 and 2, by providing the functional group 710 on the metal part 2, the fluorescent signal was detected well and the fluorescent intensity ratio of R6G to the background was also high. In contrast, in Comparative Example 1, in which the metal part 2 and the functional group 710 were not provided, the fluorescent signal could not be detected well and the fluorescent intensity ratio of R6G to the background was also low. In Comparative Example 2, since the functional group 710 was not provided, fluorescence quenching was observed, the fluorescent signal could not be detected well, and the fluorescent intensity ratio of R6G to the background was also low.

[0074] The disclosure of the present invention is given below.

[0075] (Configuration 1) A substrate having a plurality of convex portions including a metal, the substrate comprising: a first metal portion including at least one of gold, silver, platinum, copper, and palladium on a first convex portion of the plurality of convex portions; a second metal portion including at least one of gold, silver, platinum, copper, and palladium on a second convex portion of the plurality of convex portions different from the first convex portion; a dielectric portion is provided between the first convex portion and the first metal portion, and between the second convex portion and the second metal portion; a gap is provided between the first metal portion and the second metal portion; a distance between the first metal portion and the second metal portion is 50 nm or less; and the first metal portion and the second metal portion have functional groups capable of adsorbing or bonding to the first metal portion and the second metal portion.

[0076] (Configuration 2) 2. The substrate according to claim 1, wherein the distance between the first metal portion and the second metal portion is 10 nm or less.

[0077] (Configuration 3) 3. The substrate according to claim 1, wherein the dielectric portion forms an interface with the first metal portion and the second metal portion.

[0078] (Configuration 4) 4. The substrate according to any one of claims 1 to 3, wherein the dielectric portion contains alumina.

[0079] (Configuration 5) 5. The substrate according to any one of configurations 1 to 4, wherein the surface of the dielectric portion opposite to the plurality of protrusions has a shape that conforms to the protrusions.

[0080] (Configuration 6) 6. The substrate according to any one of configurations 1 to 5, wherein the second convex portion is a convex portion adjacent to the first convex portion among the plurality of convex portions.

[0081] (Configuration 7) 7. The substrate according to any one of configurations 1 to 6, wherein the first metal portion and the second metal portion are discontinuous.

[0082] (Configuration 8) 8. The substrate according to any one of claims 1 to 7, wherein each of the plurality of protrusions includes a metal having at least one of nickel, chromium, and zinc.

[0083] (Configuration 9) The substrate according to any one of structures 1 to 8, characterized in that it has a concave-convex structure including the plurality of convex portions and concave portions between the plurality of convex portions, and the height difference of the concave-convex structure is 100 nm or more and 1000 nm or less.

[0084] (Configuration 10) 10. The substrate according to claim 9, wherein the dielectric portion is provided in the recess.

[0085] (Configuration 11) 11. The substrate according to structure 9 or 10, wherein the dielectric portion is exposed in the space above the recess.

[0086] (Configuration 12) 12. The substrate according to any one of configurations 1 to 11, wherein the dielectric portion forms an interface with the first protrusion.

[0087] (Configuration 13) 13. The substrate according to any one of configurations 1 to 12, wherein the first metal portion or the second metal portion has a thickness of 5 nm or more and 50 nm or less.

[0088] (Configuration 14) 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.

[0089] (Configuration 15) A substrate according to any one of structures 1 to 14, characterized in that the functional groups are provided on the surfaces of the first convex portion, the second convex portion, the first metal portion, the second metal portion, and the dielectric portion.

[0090] (Configuration 16) 16. The substrate according to any one of configurations 1 to 15, wherein the functional group is at least one of an amino group, a carboxy group, a hydroxyl group, a maleimide group, a thiol group, a methoxy group, and a hydroxyl group.

[0091] (Configuration 17) 17. The substrate according to any one of configurations 1 to 16, wherein the first metal portion and the second metal portion are provided with a molecular layer having the functional group.

[0092] (Configuration 18) 18. The substrate of claim 17, wherein the molecular layer comprises a molecular recognition material.

[0093] (Configuration 19) 19. A substrate according to any one of configurations 1 to 18, which enhances fluorescence.

[0094] (Analysis method 1) 19. An analytical method comprising the steps of placing a specimen on the substrate according to any one of configurations 1 to 18 and irradiating the specimen with light.

[0095] (Analysis method 2) The analytical method described in analytical method 1, wherein the wavelength of the light is 300 nm or more and 850 nm or less.

[0096] (device 1) 21. An apparatus comprising a light source for irradiating light and a substrate as described in configuration 1 or 20, wherein the light source is configured to irradiate light onto a specimen placed on the substrate.

[0097] (device 2) The apparatus described in Apparatus 1 is characterized in that it is equipped with a detector for detecting fluorescence emitted from the sample.

[0098] (Manufacturing method 1) forming a dielectric portion having a first uneven structure on a surface thereof; forming a metal-containing structure having a second uneven structure to which the first uneven structure is transferred on the first uneven structure; removing a part of the dielectric portion so that the dielectric portion covers the convex portions of the second uneven structure; removing a part of the dielectric portion so that a distance from a surface of the dielectric portion opposite the structure to a concave portion of the second uneven structure is smaller than a height difference between the convex portion and a concave portion of the structure adjacent to the convex portion; a step of forming a first metal portion containing at least one of gold, silver, platinum, copper and palladium on a second convex portion different from the first convex portion of the convex portions, and a step of providing functional groups capable of adsorption or bonding on the first and second metal portions, 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 50 nm or less.

[0099] (Manufacturing method 2) A method for manufacturing a substrate as described in manufacturing method 1, characterized in that the dielectric portion is removed so that the surface of the dielectric portion opposite the structure has a shape that conforms to the uneven structure transferred to the structure.

[0100] (Manufacturing method 3) The manufacturing method described in manufacturing method 1 or 2, characterized in that a gap of 10 nm or less is provided between the first metal portion and the second metal portion.

[0101] (Manufacturing method 4) 4. The manufacturing method described in any one of manufacturing methods 1 to 3, wherein the first metal portion and the second metal portion form an interface with the dielectric portion.

[0102] The above-described embodiments may be modified as appropriate without departing from the scope of the technical concept. For example, a plurality of embodiments may be combined. In addition, some of the features of at least one embodiment may be deleted or replaced.

[0103] In addition, new matters may be added to at least one embodiment. The disclosure of this specification includes not only what is explicitly described in this specification, but also all matters that can be understood from this specification and the drawings attached to this specification.

[0104] Furthermore, the disclosure of this specification includes the complement of each concept described in this specification. In other words, if this specification contains a statement that "A is greater than B," for example, even if the statement that "A is not greater than B" is omitted, this specification can be said to disclose that "A is not greater than B." This is because when a statement that "A is greater than B" is made, it is assumed that the case in which "A is not greater than B" is taken into consideration. [Explanation of symbols]

[0105] 3 Dielectric section 4 Convex 9 Gap 10 Substrate 11 First protrusion 12 Second protrusion 21 1st metal part 22 Second metal part D Distance

Claims

1. A substrate having a plurality of protrusions including a metal, a first metal portion including at least one of gold, silver, platinum, copper, and palladium is provided on a first convex portion of the plurality of convex portions; a second metal portion including at least one of gold, silver, platinum, copper, and palladium is provided on a second convex portion of the plurality of convex portions that is different from the first convex portion; a dielectric portion is 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 a distance between the first metal portion and the second metal portion is 50 nm or less; A substrate, characterized in that the first metal portion and the second metal portion have functional groups capable of being adsorbed to or bonded 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 10 nm or less.

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

4. The substrate according to claim 1 , wherein the dielectric portion comprises alumina.

5. The substrate according to claim 1 , wherein the surface of the dielectric portion opposite to the plurality of protrusions has a shape that conforms to the protrusions.

6. The substrate according to claim 1 , wherein the second convex portion is a convex portion adjacent to the first convex portion among the plurality of convex portions.

7. The substrate according to claim 1 , wherein the first metal portion and the second metal portion are discontinuous.

8. The substrate according to claim 1 , wherein each of the plurality of protrusions includes a metal having at least one of nickel, chromium, and zinc.

9. 2. The substrate according to claim 1, further comprising a concave-convex structure including the plurality of convex portions and concave portions between the plurality of convex portions, the concave-convex structure having a height difference of 100 nm or more and 1000 nm or less.

10. The substrate according to claim 9 , wherein the dielectric portion is provided in the recess.

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

12. The substrate according to claim 1 , wherein the dielectric portion forms an interface with the first protrusion.

13. The substrate according to claim 1 , wherein the first metal portion or the second metal portion has a thickness of 5 nm or more and 50 nm or less.

14. The substrate according to claim 1 , wherein the dielectric portion has a thickness of 30 nm or more and 200 nm or less.

15. The substrate according to claim 1 , wherein the functional group is provided on the surfaces of the first convex portion, the second convex portion, the first metal portion, the second metal portion, and the dielectric portion.

16. 2. The substrate according to claim 1, wherein the functional group is at least one of an amino group, a carboxy group, a hydroxyl group, a maleimide group, a thiol group, a methoxy group, and a hydroxyl group.

17. The substrate according to claim 1 , wherein the first metal portion and the second metal portion are provided with a molecular layer having the functional group.

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

19. 19. A substrate according to any one of claims 1 to 18, which enhances fluorescence.

20. 19. An analysis method comprising the steps of placing a specimen on the substrate according to claim 1 and irradiating the specimen with light.

21. 21. The analysis method according to claim 20, wherein the wavelength of the light is not less than 300 nm and not more than 850 nm.

22. An apparatus comprising a light source for irradiating light and a substrate according to any one of claims 1 to 18, The apparatus, wherein the light source is configured to irradiate light onto a specimen placed on the substrate.

23. 23. The apparatus of claim 22, further comprising a detector for detecting fluorescence from the analyte.

24. forming a dielectric portion having a first uneven structure on a surface thereof; forming a metal-containing structure having a second uneven structure on the first uneven structure, the second uneven structure being transferred to the first uneven structure; a step of removing a portion of the dielectric portion such that the dielectric portion covers a convex portion of the second uneven structure, and a step of removing a portion of the dielectric portion such that a distance from a surface of the dielectric portion opposite the structure to a concave portion of the second uneven structure is smaller than a height difference between the convex portion and a concave portion of the structure adjacent to the convex portion; forming a first metal part including a metal having at least one of gold, silver, platinum, copper, and palladium on a first convex part of the convex parts on the side of the dielectric part opposite to the structure, and a second metal part including a metal having at least one of gold, silver, platinum, copper, and palladium on a second convex part of the convex parts different from the first convex part; providing a functional group on the first metal portion and the second metal portion, the functional group being capable of adsorbing to or bonding to the first metal portion and the second metal portion; A method for manufacturing a substrate, characterized in that 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 50 nm or less.

25. The method for manufacturing a substrate according to claim 24, characterized in that the dielectric portion is removed so that the surface of the dielectric portion opposite the structure has a shape that conforms to the uneven structure transferred to the structure.

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

27. 26. The method according to claim 24, wherein the first metal portion and the second metal portion form an interface with the dielectric portion.

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