Laminated substrate

Abstract

A laminated substrate providing excellent fluorescence enhancement properties. The laminated substrate comprises: a plate-shaped substrate having a pair of opposing main surfaces; a plurality of metal-based particles disposed on one main surface of the substrate at intervals from each other along a direction parallel to one main surface; and a protective layer covering the surface of each metal-based particle, wherein the plurality of metal-based particles satisfy at least one of the following (1) and (2), and the surface shape of the protective layer follows the surface shape of the metal-based particles. (1) The average particle size of the plurality of metal-based particles is 200 to 1600 nm, the average height of the plurality of metal-based particles is 55 to 500 nm, and the aspect ratio defined as the ratio of the average particle size to the average height is 1 to 8. (2) The plurality of metal-based particles are disposed on one main surface of the substrate such that the average distance between them and adjacent metal-based particles is 1 to 1000 nm, and the standard deviation of the average distance is 30 nm or less.

Description

Technical Field

[0001] This invention relates to laminated substrates. Background Technology

[0002] It is known that techniques for enhancing fluorescence by utilizing localized plasmon resonance of metal nanoparticles exist (e.g., Patent Document 1). Patent Document 1 describes the use of a laminate comprising an island layer (2) composed of multiple metal particles and a spacer layer (5) covering the island layer for a photochemical fluorescence sensor.

[0003] Existing technical documents

[0004] Patent documents

[0005] Patent Document 1: Japanese Patent Application Publication No. 08-271431 Summary of the Invention

[0006] The problem the invention aims to solve

[0007] However, in the aforementioned laminates, there is still room for improvement in the degree of fluorescence enhancement. The object of this invention is to provide a laminated substrate with excellent fluorescence enhancement properties.

[0008] Solution for solving the problem

[0009] [1] A laminated substrate comprising:

[0010] A plate-shaped substrate having a pair of main surfaces facing each other;

[0011] A plurality of metallic particles are arranged spaced apart from each other on a main surface of the substrate along a direction parallel to the main surface; and

[0012] A protective layer covering the surface of each of the aforementioned metal particles.

[0013] The plurality of metallic particles satisfy at least one of the following (1) and (2),

[0014] The surface shape of the protective layer follows the surface shape of the metallic particles.

[0015] (1) The average particle size of the plurality of metal particles is 200~1600nm, the average height of the plurality of metal particles is 55~500nm, and the aspect ratio defined by the ratio of the average particle size to the average height is 1~8.

[0016] (2) The plurality of metal particles are respectively disposed on one main surface of the substrate with an average distance of 1 to 1000 nm between them and the adjacent metal particles, and the standard deviation of the average distance is less than 30 nm.

[0017] [2] According to the metal-based particle aggregate laminate substrate described in [1], the coefficient of variation (CV value) of the thickness of the protective layer is less than 30%.

[0018] [3] The metal-based particle aggregate laminated substrate according to [1] or [2], wherein the average thickness of the protective layer is less than 300 nm.

[0019] [4] According to the metal-based particle aggregate laminate substrate described in [3], the average thickness of the protective layer is less than 50 nm.

[0020] [5] A metal-based particle aggregate laminated substrate according to any one of [1] to [4], wherein the protective layer is amorphous.

[0021] The effects of the invention

[0022] A multilayer substrate that provides excellent fluorescence enhancement properties. Attached Figure Description

[0023] Figure 1 This is a cross-sectional view schematically illustrating an example of a laminated substrate of the present invention. Detailed Implementation

[0024] <Laminated substrate>

[0025] (1) Composition of layers

[0026] The laminated substrate disclosed herein comprises:

[0027] A plate-shaped substrate having a pair of opposing main surfaces;

[0028] A plurality of metallic particles arranged spaced apart from each other along a direction parallel to the main surface on one surface of the substrate; and

[0029] A protective layer covering the surface of each of the aforementioned metal particles.

[0030] Reference Figure 1 A laminated substrate according to one embodiment will be described.

[0031] Figure 1 This is a cross-sectional view schematically representing an example of a laminated substrate. Figure 1 The stacked substrate 100 shown includes: a substrate 10 having a pair of opposing main surfaces 10a and 10b; a plurality of metal particles 20 disposed on the main surface 10a of the substrate 10 at intervals from each other in a direction parallel to the main surface 10a; and a protective layer 30 covering the surface of each metal particle 20.

[0032] (2) Substrate

[0033] The substrate 10 is plate-shaped. Plate-shaped means having a pair of opposing main surfaces 10a and 10b, and side surfaces connecting the main surfaces. The distance between the main surfaces, i.e., the thickness, is less than 1 / 5 of the maximum diameter of the main surface (e.g., the diagonal length if it is a rectangle). The thickness can be less than 1 / 10, less than 1 / 20, or less than 1 / 30 of the maximum diameter of the main surface. The area of ​​the main surface is larger than the area of ​​the other surfaces constituting the side surfaces.

[0034] There is no specific limit to the lower limit of the area of ​​the main surface; for example, it can be 0.25 μm. 2 That's all. The laminated substrate can be a long roll, therefore there is no particular limit to the upper limit of the area of ​​the main surface; for example, it can be 1m². 2 the following.

[0035] The thickness of the substrate 10 is not particularly limited, for example, it is 10μm to 10mm, preferably 20μm to 5mm, and more preferably 30μm to 1mm.

[0036] The substrate 10 is preferably non-conductive. This is because when the substrate 10 is conductive, electron transfer can occur between the metallic particles formed thereon via the substrate, thus tending to reduce the plasmon resonance effect. Examples of non-conductive materials constituting the substrate 10 include inorganic insulating materials such as mica, SiO2, ZrO2, and glass, as well as thermoplastic resins.

[0037] The substrate 10 can be a light-transmitting or optically transparent substrate, or it can be a non-light-transmitting (light-absorbing) substrate.

[0038] The substrate 10 can be a single-layer structure or a multi-layer structure.

[0039] (2) Metallic particles

[0040] Multiple metallic particles 20 are arranged on the main surface 10a of the substrate 10, spaced apart from each other in a direction parallel to the main surface 10a. It should be noted that the multiple metallic particles 20 are preferably not arranged apart from each other in a direction perpendicular to the main surface 10a. In other words, the metallic particles 20 are preferably arranged two-dimensionally along the main surface 10a.

[0041] like Figure 1 As shown, the metal particles 20 are preferably in contact with the main surface 10a of the substrate 10. The metal particles 20 are preferably not disposed on the other main surface 10b of the substrate 10.

[0042] Multiple metallic particles 20 constitute a metallic particle aggregate layer 21. These metallic particles 20 can form a plasmonic structure. A "platinonic structure" refers to a structure capable of exhibiting plasmonic resonance. Plasmons are the compression-sparse waves of free electrons generated by the collective vibration of free electrons within a structure. By making the multiple metallic particles 20 plasmonic structures, for example, when a laminated substrate is applied to a sensor element, the intensity of luminescence (fluorescence, etc.) from a luminescent body labeled with the detected substance can be enhanced. Therefore, the laminated substrate is suitable as a luminescence enhancement element for various sensor elements. By applying the laminated substrate to a sensor element, the sensitivity, quantitative accuracy, and / or reproducibility (stability) of the sensor element can be improved.

[0043] In order to make the metal-based particle aggregate layer 21 a plasmonic structure, the metal-based particles 20 are preferably formed of a material capable of plasmonic resonance in the ultraviolet to visible light region. A material capable of plasmonic resonance in the ultraviolet to visible light region refers to a material that, when formed into nanoparticles or their aggregates, shows a plasmonic peak in the ultraviolet to visible light region in an absorption spectroscopy measurement based on spectrophotometry.

[0044] Examples of metallic materials capable of plasmon resonance in the ultraviolet to visible light region include noble metals such as gold, silver, copper, platinum, and palladium; metals other than noble metals such as aluminum and tantalum; alloys containing metals selected from the noble metals and other metals; and metal compounds (metal oxides, metal salts, etc.) containing metals selected from the noble metals and other metals. Among these, noble metals such as gold, silver, copper, platinum, and palladium are preferred as metallic materials capable of plasmon resonance in the ultraviolet to visible light region, and silver is more preferred from the viewpoint of being inexpensive and having low absorption (small imaginary part of the dielectric function at visible light wavelengths).

[0045] The plurality of metal particles 20 satisfy at least one of (1) and (2) below, preferably both.

[0046] (1) The average particle size D of the multiple metal particles is 200~1600nm, the average height H of the multiple metal particles is 55~500nm, and the aspect ratio AR, defined by the ratio of the average particle size to the average height, is 1~8.

[0047] (2) Multiple metal particles are disposed on a main surface of a substrate such that the average distance L between them and the adjacent metal particles is in the range of 1 to 1000 nm, and the standard deviation LCV of the average distance is less than 30 nm.

[0048] The average particle size D of the plurality of metal particles 20 constituting the metal particle aggregate layer 21 is preferably in the range of 200-1200 nm, more preferably 250-500 nm, and even more preferably 300-500 nm. The average particle size of the metal particles 20 is preferably appropriately selected according to the type of metal material constituting the metal particles 20.

[0049] The average particle size D of the aforementioned multiple metallic particles 20 refers to the average particle size of the selected 10 metallic particles when, in an SEM image viewed directly above the metallic particle aggregate layer 21 composed of multiple metallic particles 20, 10 metallic particles are randomly selected, and 5 tangent diameters are randomly drawn within each metallic particle image (wherein, the straight lines that become tangent diameters can only pass through the interior of the metallic particle image, and one of them is the longest straight line that can be drawn, passing only through the interior of the metallic particle). The average value of these tangent diameters (hereinafter also referred to as the "average tangent diameter") is used as the particle size of each metallic particle. The tangent diameter is defined as the perpendicular line connecting the contour (projected image) of the metallic particle by the interval between two parallel lines tangent to the contour of the metallic particle (Nikkan Kogyo Shimbun Co., Ltd., "Particle Measurement Technology", 1994, p. 5).

[0050] The method for determining the average particle size will be explained in more detail below. First, the SEM images were measured using a JSM-5500 scanning electron microscope manufactured by Nippon Electron Ltd., or an equivalent device. Next, the obtained images were read into the free image processing software "ImageJ" manufactured by the National Institutes of Health (NIH) at a resolution of 1280 pixels horizontally × 960 pixels vertically. Then, the random number generation function "RANDBETWEEN" in the spreadsheet software "Excel" manufactured by Microsoft was used to obtain 10 random numbers from 1 to 1280 (x1, x2, x3, x4, x5, x6, x7, x8, x9, x...). 10 ), obtain 10 random numbers from 1 to 960 (y1, y2, y3, y4, y5, y6, y7, y8, y9, y 10 From the 10 random numbers obtained, 10 combinations of random numbers are generated: (x1, y1), (x2, y2), (x3, y3), (x4, y4), (x5, y5), (x6, y6), (x7, y7), (x8, y8), (x9, y9), and (x... 10 y 10The values ​​of random numbers generated from 1 to 1280 are set as the x-coordinates, and the values ​​of random numbers generated from 1 to 960 are set as the y-coordinates, resulting in 10 sets of coordinate points: (x1, y1), (x2, y2), (x3, y3), (x4, y4), (x5, y5), (x6, y6), (x7, y7), (x8, y8), (x9, y9), and (x... 10 y 10 Then, the average tangent diameter is obtained for each of the 10 metal-based particle images containing the coordinate point. This average tangent diameter is then used as the average of the 10 average tangent diameters to obtain the average particle size. If at least one of the 10 coordinate points in the 10 sets of random number combinations is not included in the metal-based particle image, or if more than two coordinate points are contained within the same metal-based particle, the random number combination is discarded, and random numbers are repeatedly generated until all 10 coordinate points are included in different metal-based particle images.

[0051] The average height H of the plurality of metal-based particles 20 is preferably 55-300 nm, more preferably in the range of 70-150 nm. The average height H of the metal-based particles 20 refers to the average of 10 measured values ​​when 10 metal-based particles are randomly selected from the AFM observation image of the metal-based particle aggregate layer 21 and the height of these 10 metal-based particles is measured.

[0052] The aspect ratio AR of the plurality of metallic particles 20 is preferably between 2 and 8, and more preferably between 2.5 and 8. The aspect ratio AR of the metallic particles 20 is defined by the ratio of the average particle size D to the average height H (average particle size D / average height H). The metallic particles 20 may be perfectly spherical, but are preferably flattened with an aspect ratio AR greater than 1.

[0053] In the metallic particle aggregate layer 21, the average distance L (hereinafter also referred to as "average interparticle distance") between each of the plurality of metallic particles 20 and its adjacent metallic particles is 1 to 1000 nm, but preferably in the range of 1 to 150 nm. In this specification, adjacent metallic particles mean adjacent in a direction parallel to the main surface 10a. By arranging the plurality of metallic particles 20 with such an average interparticle distance, strong plasmon resonance can be easily obtained, and the effect of extending the range of plasmon resonance can be further improved. The average interparticle distance is more preferably 1 to 100 nm, more preferably 1 to 50 nm, and even more preferably 1 to 20 nm. If the average interparticle distance is less than 1 nm, electron transfer based on the Dexter mechanism occurs between the particles, which is detrimental to the quenching of local plasmons.

[0054] The average interparticle distance refers to the average of the interparticle distances of 10 randomly selected metal particles in a SEM image taken directly above the metal particle aggregate layer 21 composed of multiple metal particles 20, where the interparticle distances between each selected metal particle and its adjacent metal particles are calculated. The interparticle distance with adjacent metal particles is the average of the distances between each of the selected metal particles (the minimum distance between the surfaces of adjacent metal particles).

[0055] The method for determining the average interparticle distance will be explained in more detail below. First, the SEM images were measured using a JSM-5500 scanning electron microscope manufactured by Nippon Electron Ltd., or an equivalent device. Next, the obtained images were read in at a width of 1280 pixels × height of 960 pixels using the free image processing software "ImageJ" manufactured by the National Institutes of Health, USA. Then, the random number generation function "RANDBETWEEN" of the spreadsheet software "Excel" manufactured by Microsoft was used to obtain 10 random numbers (x1~x2) from 1 to 1280. 10 ), obtain 10 random numbers (y1~y60) from 1 to 960. 10 From the 10 random numbers obtained, obtain 10 sets of random number combinations (x1, y1) ~ (x 10 y 10 The values ​​of random numbers generated from 1 to 1280 are set as the x-coordinates, and the values ​​of random numbers generated from 1 to 960 are set as the y-coordinates, resulting in 10 sets of coordinate points (x1, y1) ~ (x... 10 y 10 Then, for a total of 10 images of metallic particles containing the coordinate point, the inter-particle distance between the metallic particle and its adjacent metallic particles is obtained. This distance is then used as the average of the 10 inter-particle distances with adjacent metallic particles to obtain the average inter-particle distance. If at least any one of the 10 coordinate points generated as 10 random number combinations is not included in the metallic particle image, or if more than 2 coordinate points are contained within the same metallic particle, the random number combination is discarded, and random numbers are generated repeatedly until all 10 coordinate points are contained in different metallic particle images.

[0056] The standard deviation of the average interparticle distance is preferably 0.1 nm or more, more preferably 0.2 nm or more, and even more preferably 0.3 nm or more.

[0057] The standard deviation of the average interparticle distance is defined as follows. In a SEM image viewed directly above a layer 21 of metallic particles 20, one metallic particle is randomly selected. For this particle, the interparticle distance between it and its adjacent metallic particles is calculated. The interparticle distance between it and its adjacent metallic particles is the average of the distances (minimum distances between surfaces) between each of the particles. In the SEM image, nine metallic particles, different from the one mentioned above, are randomly selected. For these nine particles, the interparticle distances between them and their adjacent metallic particles are calculated in the same manner. The standard deviation of the total interparticle distances between these ten metallic particles and their adjacent metallic particles is defined as the standard deviation of the average interparticle distance.

[0058] The method for determining the standard deviation of the average interparticle distance is explained in more detail below. First, the SEM images were measured using a JSM-5500 scanning electron microscope manufactured by Nippon Electron Ltd., or an equivalent device. Next, the obtained images were read in at a width of 1280 pixels × height of 960 pixels using the free image processing software "ImageJ" manufactured by the National Institutes of Health, USA. Then, the random number generation function "RANDBETWEEN" of the spreadsheet software "Excel" manufactured by Microsoft was used to obtain 10 random numbers (x1~x2) from 1 to 1280. 10 ), obtain 10 random numbers (y1~y60) from 1 to 960. 10 From the 10 random numbers obtained, obtain 10 sets of random number combinations (x1, y1) ~ (x 10 y 10 The values ​​of random numbers generated from 1 to 1280 are set as the x-coordinates, and the values ​​of random numbers generated from 1 to 960 are set as the y-coordinates, resulting in 10 sets of coordinate points (x1, y1) ~ (x... 10 y 10 Then, for a total of 10 images of metallic particles containing the coordinate point, the inter-particle distance between the metallic particle and its adjacent metallic particles is obtained. This distance is then used as the standard deviation of the 10 inter-particle distances to their adjacent metallic particles to obtain the standard deviation of the average inter-particle distance. If at least one of the 10 coordinate points generated as 10 random number combinations is not included in the metallic particle image, or if more than two coordinate points are contained within the same metallic particle, the random number combination is discarded. Random numbers are generated repeatedly until all 10 coordinate points are included in different metallic particle images.

[0059] From the viewpoint of highly effective plasmon excitation, the metallic particles 20 preferably have a surface composed of smooth curved surfaces, and more preferably have a flat shape composed of smooth curved surfaces. However, the surface may also contain some minor bumps (roughness). In this sense, the metallic particles may also be irregular in shape.

[0060] The number of metal-based particles 20 contained in the metal-based particle aggregate layer 21 is typically 10 or more, preferably 30 or more. By forming a metal-based particle aggregate layer containing 10 or more metal-based particles, strong plasmon resonance and an extended range of plasmon resonance can be easily achieved through the interaction between local plasmons of the metal-based particles. The number of metal-based particles 20 contained in the metal-based particle aggregate layer 21 can be, for example, 50 or more, more preferably 1000 or more, and even more preferably 10000 or more. The number density of metal-based particles 20 in the metal-based particle aggregate layer 21 is preferably 7 particles / μm. 2 The above, more preferably 15 per μm 2 above.

[0061] Preferably, the metal-based particle aggregate layer 21 does not exhibit conductivity, and more preferably, the metal-based particles 20 constituting the metal-based particle aggregate layer 21 are non-conductive with their adjacent metal-based particles. In the metal-based particle aggregate layer 21, if there are sites in the metal-based particle aggregate layer 21 where electrons can be donated and accepted between the metal-based particles 20, there is a tendency for the plasmon resonance effect to decrease. Therefore, it is preferable that the metal-based particles 20 are reliably separated, and that there is no conductive material between the metal-based particles 20. The metal-based particles 20 themselves may also be conductive.

[0062] The metallic particle aggregate layer 21 does not exhibit conductivity as a layer, for example, this can be confirmed by the following method: when a pair of tester probes of a multimeter (tester (Hewlett-Packard "E2378A")) are in contact with the metallic particle aggregate layer 21 at a distance of 10 mm to 15 mm, and the resistance value is above 30 MΩ under the test conditions when the range is set to "30 MΩ", the result will show "overload", thus confirming this.

[0063] (3) Protective layer

[0064] A protective layer 30 covers the surface of each metal-based particle 20. The protective layer 30 preferably has direct contact with the surface of the metal-based particles 20. The protective layer 30 can be a single-layer structure or a multi-layer structure. Typically, a portion of the surface of each metal-based particle 20 ( Figure 1The lower surface side of the metal particles 20 is covered by the substrate 10, so the protective layer 30 can cover at least a portion of the exposed portion of the surface of the metal particles 20 that is not covered by the substrate 10, and preferably covers all of the exposed portion of the surface of the metal particles 20 that is not covered by the substrate 10. The metal particles 20 preferably do not have a surface exposed to the outside that is not covered by either the substrate 10 or the protective layer 30.

[0065] The surface shape of the protective layer 30 follows the surface shape of the metallic particles 20. This means that the surface shape of the protective layer 30 follows the surface shape of the metallic particles 20. Figure 1 In the cross-sectional view shown, the surface shape of the protective layer 30 is approximately parallel to the surface shape of the metallic particles 20. Figure 1 This is a schematic diagram of a cross-section perpendicular to the main surface 10a of the substrate 10.

[0066] More specifically, in the surface shape of the protective layer 30 described above, it is preferable that the thickness T of the protective layer 30, measured in a direction perpendicular to the main surface 10a, is substantially uniform. That is, the distance between the interface 20a of the protective layer 30 and the metal particles and the surface 30a of the protective layer 30 opposite to the substrate 10, perpendicular to the main surface 10a, is substantially uniform. The thickness T of the protective layer being substantially uniform means that the coefficient of variation (CV value) of the protective layer thickness, i.e., (standard deviation of the protective layer thickness T / average value of the protective layer thickness T), is 30% or less.

[0067] The standard deviation and average value of the thickness T can be calculated as follows. Specifically, a microscope image of the cross-section of the laminated substrate perpendicular to the main surface 10a is prepared using a scanning microscope or similar instrument. The microscope image is set to a magnification containing 10 to 250 metallic particles. The length 51 of the straight line on the main surface 10a is divided into equal parts in the microscope image, and coordinates 1, 2, ..., 50 are obtained from the left end of the cross-sectional image. Starting from these points, a group of straight lines perpendicular to the main surface 10a is drawn, and the intersection points of these lines with the interfaces of the metallic particles 20 and the protective layer 30 (from intersection point 1-1 to intersection point 50-1) are obtained. Additionally, the intersection points of these lines with the surface 30a of the protective layer 30 opposite to the substrate 10 (from intersection point 1-2 to intersection point 50-2) are also obtained. The distance between intersection point 1-1 and intersection point 1-2 is defined as the protective layer thickness T1, and the distance between intersection point 2-1 and intersection point 2-2 is defined as the protective layer thickness T2, thus obtaining protective layer thicknesses T1 to T50. The average of these 50 values ​​is taken as the average thickness of the protective layer. Additionally, the standard deviation of these 50 values ​​is taken as the standard deviation of the protective layer thickness, and (standard deviation of protective layer thickness / average thickness of protective layer) is taken as the coefficient of variation (CV) of the protective layer thickness.

[0068] It should be noted that in microscope images, the thickness of a straight line is not measured if it does not intersect with metallic particles.

[0069] From the viewpoint of improving the uniformity of the protective layer thickness and increasing the luminescence enhancement ratio, the coefficient of variation (CV value) of the protective layer thickness can be less than 20%, preferably less than 18%, and more preferably less than 15%.

[0070] There are no particular restrictions on the average thickness of the protective layer; for example, it can be above 3nm, above 10nm, above 15nm, above 20nm, above 30nm, or above 40nm. The thickness of the protective layer can be below 300nm, below 250nm, below 200nm, below 150nm, below 100nm, below 80nm, or less than 50nm.

[0071] There is no particular limitation on the surface roughness of the protective layer. The arithmetic mean roughness Ra of the protective layer surface can be above 10 nm, above 15 nm, or above 20 nm. The arithmetic mean roughness Ra can be below 500 nm, below 300 nm, below 200 nm, or below 100 nm.

[0072] like Figure 1 As shown, the protective layer 30 may also have a portion that contacts and covers the portion of the main surface 10a of the substrate 10 that is not covered by the metal particles 20.

[0073] The protective layer is preferably made of a non-conductive material, i.e., an insulating material. Examples of insulating materials include inorganic insulating materials such as SiO2, SiN, TiO2, Al2O3, and Si3N4; and organic insulating materials such as resin materials (e.g., polystyrene, acrylic resins, epoxy resins). The protective layer can be composed of two or more materials. The protective layer can be a single-layer structure or a multi-layer structure.

[0074] The protective layer is preferably amorphous. The amorphous nature of the protective layer 30 can be confirmed by HAADF-STEM and electron diffraction patterns. Regarding the protective layer 30, when the electron diffraction pattern of the region corresponding to the protective layer in the cross-sectional STEM image is obtained, the protective layer 30 can be determined to be an amorphous layer based on the absence of periodic contrast originating from a crystal.

[0075] The water contact angle of the protective layer surface is not particularly limited, and can be 40° or less, preferably 30° or less, and more preferably 20° or less. By reducing the water contact angle of the protective layer surface, it is easier for the aqueous solution to contact the protective layer. The aqueous solution can be, for example, a treatment solution containing the trapping substance or its precursor used when immobilizing the trapping substance on the surface of the protective layer, or optionally a sample containing the substance to be detected supplied to the surface of the protective layer to which the trapping substance is immobilized.

[0076] Forming a protective layer is advantageous in the following ways.

[0077] [A] When using metallic particles as a luminescence enhancement element to increase the luminescence intensity of a luminescent body from a labeled substance being tested, if the luminescent body is in direct contact with the metallic particles, extinction may occur due to electron tunneling from the luminescent body to the metallic particles, potentially reducing the enhancement effect. By providing a protective layer on the metallic particles, the luminescent body can be reliably separated from the metallic particles, thus suppressing extinction.

[0078] [B] It can improve the stability (oxidation resistance, etc.) and environmental stability (such as light resistance, humidity resistance, heat resistance, etc.) of metal particles.

[0079] The laminated substrate may have further layers on the protective layer.

[0080] (Effects)

[0081] The laminated substrate of this embodiment can exhibit the features described in [a] and [b] below. These features can be considered to be manifested through the interaction between localized plasmons exhibited by the plurality of metallic particles 20.

[0082] [a] The plasmon resonance exhibited by the metallic particle aggregate layer 21 has a wide range of influence. As a result, for example, the range of the plasmon-based luminescence enhancement effect can be expanded, and thus the luminescence of luminescent bodies located in the range of hundreds of nm (e.g., 200 nm) from the surface of the metallic particle aggregate layer 21 can also be enhanced.

[0083] [b] The metallic particle aggregate layer 21 exhibits strong plasmon resonance. As a result, for example, a strong luminescence enhancement effect can be obtained.

[0084] Regarding [a] above, the laminated substrate according to the present invention can enhance the luminescence of light-emitting bodies disposed at a distance of, for example, 10 nm or more from the metal particle aggregate layer 21, then tens of nm (e.g., 20 nm, 30 nm or 40 nm or more), then 100 nm or more or 200 nm or more.

[0085] Regarding [b] above, the intensity of the plasmon resonance exhibited by the laminated substrate is not simply the sum of the localized plasmon resonances exhibited by the individual metallic particles at a specific wavelength, but rather an intensity greater than their sum. In the laminated substrate, the individual metallic particles interact to exhibit strong plasmon resonances. This strong plasmon resonance can be considered to be manifested through the interaction between the localized plasmons of the metallic particles.

[0086] Typically, when the absorption spectrum of a plasmonic structure is measured using spectrophotometry, the plasmonic resonance peak (hereinafter also referred to as the "plasmonic peak") is observed as the peak on the longest wavelength side in the ultraviolet to visible region. The intensity of the plasmonic resonance of the plasmonic structure can be evaluated based on the absorbance at the maximum wavelength of the plasmonic peak. There is a tendency that the greater the absorbance value, the greater the intensity of the plasmonic resonance. When the absorption spectrum of the metallic particle aggregate layer 21 with the above-described structure is measured by the spectrophotometric method described below, the absorbance at the maximum wavelength of the plasmonic peak on the longest wavelength side in the ultraviolet to visible region can be 1 or more, further 1.5 or more, and even more approximately 2.

[0087] The absorption spectrum of plasmonic structures can be determined by spectrophotometry. Specifically, the absorption spectrum can be obtained by measuring the following intensities using an integrating sphere spectrophotometer: the intensity I of transmitted light in all directions transmitted toward the metal particle aggregate layer side when incident light in the ultraviolet to visible light region is irradiated from a direction perpendicular to the substrate surface on the back side (opposite to the metal particle aggregate layer) of a substrate with a stacked metal particle aggregate layer; and the intensity I0 of transmitted light in all directions transmitted from the opposite side of the incident surface when the same incident light is irradiated from a direction perpendicular to the surface of a substrate with the same thickness and material as the substrate of the test sample and without a stacked metal particle aggregate layer.

[0088] At this point, the absorbance, which forms the vertical axis of the absorption spectrum, is expressed by the following formula:

[0089] Absorbance = -log 10 (I / I0).

[0090] Absorption spectra can be measured using a general spectrophotometer.

[0091] In addition, when measuring the maximum wavelength and absorbance of the plasmon peak located on the longest wavelength side in the ultraviolet to visible light region, an objective lens and spectrophotometer can be used to narrow the field of view for absorption spectroscopy measurement.

[0092] The laminated substrate according to this embodiment can improve the luminous enhancement ratio. The reason is not yet clear, but the following reasons can be considered.

[0093] It can be argued that the main cause of long-range plasmon resonance lies in the resonance between the whispering gallery modes propagating on the surfaces of individual metallic particles and the whispering gallery modes of adjacent particles. This can be speculated to be because, regarding the whispering gallery modes propagating on the surfaces of metallic particles, if the surface shape of the protective layer follows the surface shape of the metallic particles—in other words, if the thickness of the protective layer covering the metallic particles is uniform—there is less disturbance during propagation on the particle surface and between particles compared to when covered by a protective layer of uneven thickness. This makes it easier to exhibit the inter-particle resonance that is the main cause of long-range plasmon resonance.

[0094] (Manufacturing method of laminated substrate)

[0095] The aforementioned laminated substrate can be fabricated, for example, by the following method.

[0096] First, prepare a plate-shaped substrate 10. The surface of the substrate can be pre-cleaned by degreasing. The degreasing cleaning of the substrate can be performed using appropriate methods depending on the substrate material; for example, cleaning using a liquid selected from organic solvents and water can be used. Examples of cleaning methods include immersion in the liquid, ultrasonic cleaning while immersing in the liquid, and wiping with a cloth (woven fabric, non-woven fabric, etc.) impregnated with the liquid. Degreasing cleaning can also be a combination of cleaning steps using two or more liquids. For example, in the case of a glass substrate, degreasing cleaning can sequentially include: ultrasonic cleaning using alcohols (methanol, ethanol, isopropanol, etc.), ultrasonic cleaning using ketones (acetone, methyl ethyl ketone, or methyl isobutyl ketone, etc.), and ultrasonic cleaning using ultrapure water.

[0097] Next, a metal-based microparticle layer is formed on the main surface of the substrate. Examples of this method are shown in [A] to [C] below.

[0098] [A] A bottom-up method in which multiple metal-based particles 20 are gradually grown from tiny seeds on a substrate 10;

[0099] [B] A method of forming a film on a substrate 10 by covering multiple metal particles 20 with a protective film of a specified thickness made of an amphiphilic material;

[0100] [C] Others: Methods for post-processing thin films produced by vapor deposition or sputtering, resist processing, etching processing, casting using a dispersion of metal particles, etc.

[0101] In the above method [A], it is preferable to include a process of growing metal-based particles at an extremely low speed on a substrate 10 adjusted to a specified temperature (hereinafter also referred to as the "particle growth process"). According to the manufacturing method including this particle growth process, a metal-based particle aggregate layer 21 having the above-mentioned preferred average particle size, average height, aspect ratio, average interparticle distance, and standard deviation of the average interparticle distance can be obtained with good control.

[0102] In the particle growth process, the growth rate of metal particles on the substrate 10 is preferably less than 1 nm / min, more preferably less than 0.5 nm / min, in terms of average height growth rate.

[0103] The average height growth rate mentioned here can also be called the average deposition rate or the average thickness growth rate of metallic particles, and is defined by the following formula: average height of metallic particles / growth time of metallic particles. The definition of "average height of metallic particles" is as described above.

[0104] The growth time of metallic particles refers to the time from the start to the end of the growth process; specifically, it refers to the supply time of the metallic material. When the metallic particle aggregate layer 21 is considered as a film, the growth time can also be called the film formation time. When the method for growing metallic particles is sputtering, the growth time is called the sputtering time.

[0105] The temperature of the substrate 10 in the particle growth process is preferably 100°C or higher and 450°C or lower, more preferably 200°C or higher and 450°C or lower, even more preferably 250°C or higher and 350°C or lower, and even more preferably 300°C or around (approximately 300°C ± 10°C).

[0106] By adjusting the average height growth rate, substrate temperature, and / or metal particle growth time, the average interparticle distance, standard deviation, average particle size, average height, and aspect ratio of the multiple metal particles 20 grown on the substrate 10 can be controlled.

[0107] The pressure (pressure within the apparatus chamber) during the growth of metallic particles is not particularly limited as long as it is sufficient for particle growth, and is generally less than atmospheric pressure. There is no particular limitation on the lower limit of the pressure, but from the perspective of easily adjusting the average height growth rate to the aforementioned range, 0.5 Pa or more is preferred, 6 Pa or more is more preferred, and 10 Pa or more is even more preferred.

[0108] There are no particular limitations on the specific method for growing metallic particles on the substrate 10, as long as the particle growth rate is less than 1 nm / min. Examples include sputtering, vacuum evaporation, and other vapor deposition methods. From the perspective of being able to grow a metallic particle aggregate layer relatively easily and maintaining an average height growth rate of less than 1 nm / min, DC sputtering is preferred in sputtering methods.

[0109] There are no particular restrictions on the sputtering method; DC argon ion sputtering, which uses an ion gun or plasma discharge to accelerate argon ions in an electric field and irradiate them onto the target, can be used. By appropriately adjusting various conditions in the sputtering method, such as the current value, voltage value, and substrate-target distance, particle growth can be achieved at an average height growth rate of less than 1 nm / min.

[0110] It should be noted that, in order to obtain a metallic particle aggregate layer 21 with the above-mentioned preferred average particle size, average height, aspect ratio, average interparticle distance, and standard deviation of the average interparticle distance, it is preferable to keep the average height growth rate less than 1 nm / min and the average particle size growth rate less than 5 nm during the particle growth process. However, when the average height growth rate is less than 1 nm / min, the average particle size growth rate is typically less than 5 nm. More preferably, the average particle size growth rate is 1 nm / min or less.

[0111] The average particle size growth rate is defined by the following formula: average particle size of the metal-based particles / growth time of the metal-based particles. The definitions of "average particle size of the metal-based particles" and "growth time of the metal-based particles" are as described above.

[0112] In order to obtain a metal-based particle aggregate layer 21 with the above-mentioned preferred average particle size, average height, aspect ratio, average interparticle distance, and standard deviation of the average interparticle distance, it is preferable to appropriately adjust the metal-based particle growth time in the particle growth process while taking into account the above-mentioned preferred manufacturing conditions.

[0113] (Formation of the protective layer)

[0114] Next, a protective layer is formed on the surface of the metallic particles.

[0115] There are no particular limitations on the method of forming the protective layer. From the viewpoint of forming a surface shape that follows the surface shape of the metal particles, dry film formation methods such as vapor deposition, sputtering, ion plating, CVD, and ALD are preferred; wet film formation methods such as spraying are also preferred.

[0116] There are no particular restrictions on the sputtering method, but high-frequency (RF) sputtering is preferred. Argon or similar gases can be used as the sputtering gas. It should be noted that, from the viewpoint of reducing the contact angle of water on the protective layer surface, it is preferable to form a SiO2 film at least on the outermost surface of the protective layer using sputtering.

[0117] From the viewpoint of forming a surface shape that follows the surface shape of the metal-based particles, it is preferable to increase the energy of the composition (sputtered particles) ejected from the target by setting a high output power in RF sputtering, for example, the discharge output (power) is 200W or more, preferably 500W or more, and more preferably 1000W or more. By increasing the energy of the sputtered particles, it is possible to improve the following of the underlying structure while densifying the film, and film formation can be carried out at high speed.

[0118] In RF sputtering, it is preferable to carry out the process in an atmosphere of inert gas such as argon, and preferably without adding oxygen to the inert gas atmosphere.

[0119] <Sensor Components>

[0120] The laminated substrate can be used as a sensor element in a sensor device for detecting a substance. For example, after cleaning the laminated substrate with a piranha solution or the like, a trapping substance that specifically binds to the substance being detected is introduced at a predetermined position on the surface of the protective layer 30 (the surface opposite to the side of the substrate 10) using a photolithography method or the like, thereby fabricating a sensor element.

[0121] The aforementioned sensor element can, for example, detect a substance as follows. Detection can be qualitative or quantitative, such as the identification or quantification of the substance. When the substance being detected, which has been labeled with a substance-specific binding capability of the sensor element, is irradiated with excitation light, the label, acting as a luminescent body, is excited. Furthermore, the metallic particle aggregate layer of the sensor element, preferably a plasmonic structure, resonates with the excited luminescent body, exhibiting enhanced plasmonic luminescence. By detecting the luminescence from the excited luminescent body using a detector, the substance being detected can be qualitatively or quantitatively determined. For example, by measuring the luminescence intensity, the amount of the substance being detected can be qualitatively or quantitatively determined.

[0122] According to the sensor element having the stacked substrate of the present invention, the stacked substrate preferably includes a layer of metallic particle aggregates as a plasmonic structure, thereby exhibiting plasmonic luminescence enhancement, and thus improving detection sensitivity and detection accuracy.

[0123] The substance being detected is the target substance for qualitative or quantitative detection; it is the substance that specifically binds to the target substance. There are no particular limitations on the substances that can be detected; examples include nucleosides, nucleotides, nucleic acids, proteins, carbohydrates, glycoproteins, lectins, viruses, cells, antibodies, and exosomes. Sensor elements whose detected substances are biologically derived or biologically related are also called biosensor elements.

[0124] Nucleic acids are polymers (nucleotide chains) of phosphate esters of nucleosides formed by glycosidic bonds between purine or pyrimidine bases and sugars. These include oligonucleotides containing probe DNA, polynucleotides, DNA (full-length or fragments thereof) polymerized from purine and pyrimidine nucleotides, RNA, and polyamide nucleotide derivatives (PNA). Nucleosides are compounds formed by glycosidic bonds between bases and sugars, while nucleotides are compounds with phosphate groups bonded to nucleosides. Both nucleosides and nucleotides are compounds containing bases.

[0125] "Specific binding" in a broad sense refers to chemical bonding, including non-covalent bonding, covalent bonding, and hydrogen bonding between substances. Examples include interactions between protein molecules and electrostatic interactions between molecules.

[0126] The detection of the captured analyte can be performed by pre-labeling the analyte with a marker acting as a luminescent agent and detecting the luminescence emanating from that marker. The marker can be a labeling substance that specifically binds to a complex formed by the specific binding of the captured substance and the analyte. The luminescent agent is a substance that emits light through the injection of excitation energy based on excitation light. The luminescence principle of the luminescent agent is not limited; examples include fluorescence, phosphorescence, and chemiluminescence. Conventionally known luminescent agents can be used as the luminescent agent.

[0127] A capturing substance is a substance that functions to specifically bind to and capture a analyte. The capturing substance may be, for example, fixed to the surface of the protective layer 30. The capturing substance may be, for example, a substance having a binding active group capable of specifically binding to the analyte. Examples of binding active groups include carboxyl groups, hydroxyl groups, etc., which can interact electrostatically with the analyte. There are no particular limitations on the capturing substance; examples include nucleosides, nucleotides, nucleic acids, proteins, sugars, glycoproteins, etc.

[0128] Examples of sensor devices include DNA sequencers, DNA microarrays, virus sensors, ion sensors, enzyme-linked immunosorbent assay (ELISA) readers (protein chips, glycan chips, lectin chips, etc.), microspectroscopic spectrometers, glucose sensors, and other biosensor devices.

[0129] Example

[0130] The present invention will be described in more detail below with reference to specific embodiments, but the present invention is not limited to these embodiments.

[0131] <Example 1>

[0132] (1) Degreasing and cleaning of glass substrate

[0133] Prepare a square sodium glass substrate with one side measuring 50 mm and a thickness of 0.7 mm. As a degreasing and cleaning process, the sodium glass substrate is cleaned with isopropanol and acetone, followed by ultrapure water cleaning.

[0134] (2) Formation of metallic particle aggregate layers

[0135] Using a DC magnetron sputtering apparatus, silver particles were grown extremely slowly on a sodium glass substrate that had undergone degreasing and cleaning treatment, thereby forming a metal-based particle aggregate layer on the entire surface of the substrate.

[0136] Gas used: Argon

[0137] Chamber pressure (sputtering gas pressure): 10 Pa

[0138] Distance between substrate and target: 100mm

[0139] Sputtering power: 4W

[0140] Average particle size growth rate (average particle size / growth time of metal-based particles): 0.9 nm / min

[0141] Average height growth rate (= average deposition rate = average height / metal-based particle growth time): 0.25 nm / min

[0142] Substrate temperature: 300℃

[0143] Metal-based particle growth time: 360 minutes

[0144] Based on the above definitions, the average particle size of the silver particles constituting the metallic particle aggregate layer was determined from SEM images to be 335 nm, the average interparticle distance was 16.7 nm, and the standard deviation of the average interparticle distance was 27.8 nm. The SEM images also show that the metallic particle aggregate layer has a particle size of approximately 6.25 × 10⁻⁶. 10 (approximately 25 per μm) 2 Silver particles. Furthermore, based on AFM images captured using a KEYENCE VN-8010 camera, the average height of the silver particles was determined to be 96.2 nm. Therefore, the aspect ratio (average particle size / average height) of the silver particles was calculated to be 3.48.

[0145] The conductivity was confirmed by connecting a tester (a multimeter (Hewlett-Packard "E2378A")) to the surface of the metal particle aggregate layer formed on the substrate, and the result confirmed that it was not conductive.

[0146] (3) Formation of the protective layer

[0147] Under conditions of argon atmosphere without oxygen introduction, discharge output (power) of 1000W, and protective layer growth rate of 1.8nm / min, a SiO2 layer as a protective layer is formed on the surface of a substrate with multiple metal particles by using RF sputtering with a SiO2 target.

[0148] It should be noted that a substrate much larger than the target is rotated on the target to form a protective layer over a wide area on the substrate. The growth rate of the protective layer is the average rate of the total area of ​​the precipitated protective layer.

[0149] (4) Evaluation of the protective layer

[0150] (4-1) Coverage state of the protective layer over the metallic particle aggregate layer and the average thickness of the protective layer

[0151] The laminated substrate was cut along a plane parallel to the stacking direction of the layers, and the cross-sectional image of the cut portion was observed using a scanning electron microscope "JSM-5500" manufactured by Nippon Egis Corporation. This confirmed that the protective layer covers the entire surface of the metal particles opposite to the substrate. Furthermore, using the apparatus and measurement conditions described later, X-ray photoelectron spectroscopy was performed on the surface of the laminated substrate opposite to the glass substrate. No signal of silver atoms was detected, thus confirming that the protective layer covers the entire surface of the metal particles opposite to the glass substrate. Additionally, it was confirmed that the protective layer is formed not only on the surface of the metal particles but also on the surface of the substrate exposed between the metal particles.

[0152] (4-2) Standard deviation and average value of the thickness T of the protective layer

[0153] Furthermore, based on the above measurement method using a scanning electron microscope "JSM-5500" manufactured by Nippon Electron Ltd., the standard deviation and mean value of the protective layer thickness T were 4.0 nm and 28.0 nm, respectively. The coefficient of variation (CV) of the protective layer thickness, i.e. (standard deviation of protective layer thickness / mean value of protective layer thickness), was 14.3%.

[0154] (4-3) Arithmetic mean roughness Ra of the protective layer surface

[0155] The arithmetic mean roughness Ra of the protective layer surface of the laminated substrate was determined using AFM. Specifically, an AFM (KEYENCE VN-8000) was used to obtain a 5μm × 5μm uneven shape on the protective layer side surface of the laminated substrate in contact mode. The entire area was designated as the effective area, and the arithmetic mean roughness Ra value of the protective layer surface was analyzed and output by "3D Measurement". The arithmetic mean roughness Ra of the protective layer surface in Example 1 was 20.1 nm.

[0156] (4-4) Water contact angle of the protective layer surface

[0157] The contact angle of ultrapure water on the surface of the protective layer of the laminated substrate was obtained using a fully automatic contact angle meter DM-701 (manufactured by Kyowa Interface Science Co., Ltd.). The water contact angle on the surface of the protective layer in Example 1 was 15.6°.

[0158] (4-5) Crystallization state of the protective layer

[0159] The crystallinity of the protective layer's cross-section was confirmed by analyzing the electron diffraction pattern of a STEM image obtained using the following measurement conditions and apparatus. Since no diffraction contrast originating from a crystal structure was observed, the protective layer was confirmed to be amorphous.

[0160] (Apparatus and Measurement Conditions)

[0161] • Device: ARM200F manufactured by NEC Corporation

[0162] • Measurement method: Observation of electron diffraction patterns

[0163] Accelerating voltage: 200kV

[0164] (4-6) Evaluation of the luminous enhancement ratio of the multilayer substrate

[0165] <Example A>

[0166] By growing silver particles under the same conditions as in Example 1, a metal-based particle aggregate layer, identical to that in Example 1, was formed on a 0.5 mm thick sodium glass substrate. This metal-based particle aggregate layer had the same particle shape and average interparticle distance as in Example 1.

[0167] Then, a SiO2 protective layer with an average thickness of 28.0 nm was immediately formed under the same conditions as in Example 1. Rhodamine B solution was spin-coated onto the surface of this protective layer at 2000 rpm, and then allowed to dry, thus fixing the Rhodamine B pigment (as the luminescent agent) onto the surface of the protective layer, resulting in photoexcited luminescent element A. The Rhodamine B solution was prepared by dissolving Rhodamine B pigment (Exciton Rhodamine 110) in ethanol at a concentration of 0.15 mM. The standard deviation and mean of the protective layer thickness T were 4.0 nm and 28.0 nm, respectively. The surface shape of the SiO2 protective layer followed the surface shape of the metal-based particles. The coefficient of variation (CV) of the protective layer thickness, i.e., (standard deviation of the protective layer thickness / mean of the protective layer thickness), was 14.3%.

[0168] <Reference Example A'>

[0169] Except for the absence of a metal-based particle aggregate layer, a reference-based photoluminescent element A' is obtained by the same method as in Example A.

[0170] <Comparative Example B>

[0171] In addition to fabricating the protective layer using a spin-coating method with SOG solution, a comparative photoluminescent element B with fixed rhodamine pigment was fabricated using the same method as in Example A. Specifically, an SOG solution was spin-coated onto a metal-based particle aggregate layer, and a SiO2 protective layer with an average thickness of 35.3 nm was stacked. The SOG solution used was a solution obtained by diluting "OCD T-7 5500T" manufactured by Tokyo Ohka Kogyo Co., Ltd., an organic SOG material, with ethanol. The surface shape of the SiO2 protective layer did not follow the surface shape of the metal-based particles and was generally smooth. The standard deviation, mean, and coefficient of variation of the protective layer thickness T were 16.1 nm, 35.3 nm, and 45.7%, respectively. The arithmetic mean roughness Ra of the protective layer surface was 5.2 nm, and the water contact angle of the protective layer surface (before rhodamine pigment fixation) was 64.1°.

[0172] <Comparison with Example B>

[0173] Except for lacking a layer of metallic particle aggregates, a reference frame comparative photoluminescent element B' was obtained using the same method as Comparative Example B.

[0174] Next, construct Figure 1 The optical sensing device shown is used. Detector D is a fluorescence spectrophotometer (trade name: PMA-12, manufactured by Hamamatsu Photonics).

[0175] exist Figure 1In the optical sensing device shown, an excitation light L1 with a wavelength of 532 nm is irradiated onto the laminated substrate 100 using a light source. For the emitted light L2, a wavelength cutoff filter F, which filters out the wavelength of the excitation light L1, is used by a detector D for measurement.

[0176] For the emission spectrum obtained from detector D, calculate the integral value of the emission spectrum from wavelength 550 nm to 950 nm.

[0177] (Evaluation of luminescence enhancement effect)

[0178] The integral value of the emission spectrum in Example A was calculated when the integral value of the emission spectrum in Reference Example A' was set to 1, thus obtaining the luminescence enhancement factor of Example A. Furthermore, the integral value of the emission spectrum in Comparative Example B was calculated when the integral value of the emission spectrum in Comparative Reference Example B' was set to 1, thus obtaining the luminescence enhancement factor of Comparative Example B. The luminescence enhancement factors were 51.1 times and 17.1 times, respectively.

[0179] Explanation of reference numerals in the attached figures

[0180] 10…substrate, 20…metal particles, 21…metal particle aggregate layer, 30…protective layer, 100…stacked substrate.

Claims

1. A laminated substrate comprising: A plate-shaped substrate having a pair of main surfaces facing each other; A plurality of metallic particles arranged spaced apart from each other along a direction parallel to the main surface of the substrate; and A protective layer covering the surface of each of the aforementioned metal particles. The plurality of metallic particles satisfy at least one of the following (1) and (2), The surface shape of the protective layer follows the surface shape of the metallic particles. (1) The average particle size of the plurality of metal particles is 200~1600nm, the average height of the plurality of metal particles is 55~500nm, and the aspect ratio defined by the ratio of the average particle size to the average height is 1~8; (2) The plurality of metal particles are respectively disposed on one main surface of the substrate with an average distance of 1 to 1000 nm between them and the adjacent metal particles, and the standard deviation of the average distance is less than 30 nm.

2. The laminated substrate according to claim 1, wherein The coefficient of variation (CV) of the thickness of the protective layer is less than 30%.

3. The laminated substrate according to claim 1 or 2, wherein, The average thickness of the protective layer is less than 300 nm.

4. The laminated substrate according to claim 3, wherein, The average thickness of the protective layer is less than 50 nm.

5. The laminated substrate according to claim 1 or 2, wherein, The protective layer is amorphous.

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