WO2019221298A1 - Electromagnetic wave enhancement element, producing method therefor, detection method using electromagnetic wave enhancement element, and amino acid sequence determination method - Google Patents

Electromagnetic wave enhancement element, producing method therefor, detection method using electromagnetic wave enhancement element, and amino acid sequence determination method Download PDF

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Publication number
WO2019221298A1
WO2019221298A1 PCT/JP2019/019810 JP2019019810W WO2019221298A1 WO 2019221298 A1 WO2019221298 A1 WO 2019221298A1 JP 2019019810 W JP2019019810 W JP 2019019810W WO 2019221298 A1 WO2019221298 A1 WO 2019221298A1
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electromagnetic wave
metal
dielectric
enhancing element
metal part
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PCT/JP2019/019810
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French (fr)
Japanese (ja)
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粟屋信義
西富雄
梶田浩志
田中覚
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Ｓｃｉｖａｘ株式会社
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Publication of WO2019221298A1 publication Critical patent/WO2019221298A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering

Definitions

  • the present invention relates to an electromagnetic wave enhancing element, a production method thereof, and an amino acid sequence determination method using the electromagnetic wave enhancing element.
  • Raman spectroscopy is a method for determining the molecular structure and crystal structure of a substance by examining the properties of Raman scattered light, which is light having a wavelength different from that of incident light.
  • the intensity of Raman scattered light is extremely weak, about 10 to the sixth power of the intensity of Rayleigh scattered light. Therefore, it is necessary to use a high-intensity light source such as a laser practically.
  • SERS surface-enhanced Raman scattering
  • SERS is a phenomenon in which Raman scattering of molecules adsorbed on a metal surface having a nano-order fine structure is remarkably enhanced.
  • the mechanism of this enhancement includes the enhancement of vibration (chemical enhancement) by charge transfer between metal and molecule, and the enhancement of the electric field formed when localized surface plasmons are excited by incident light (physical enhancement). ).
  • Patent Document 1 As a method for forming a nanogap structure in a self-organized manner by surface treatment, a method using a porous silicon structure (for example, Patent Document 1), a method for forming a nanostructure by embossing (for example, Patent Document 2) ), A method of forming a nanostructure by surface roughening by boehmite treatment (for example, Patent Document 3).
  • a method for forming a nanostructure in bulk a method for forming a nanocomposite (for example, Patent Document 4), a method for forming a metal nanosponge (for example, Patent Document 5), and a method for aggregating fullerenes (for example, Patent Documents) 6).
  • Patent Document 7 Also proposed is a technique in which metal particles are dispersed and fixed on the surface of a substrate to form nano-sized intervals in a self-organized manner (for example, Patent Document 7).
  • a metal film is deposited on the stepped pattern by a film forming method with insufficient step coverage such as vapor deposition or sputtering to form a nano-sized gap in a self-aligned manner in the stepped portion.
  • a film forming method with insufficient step coverage such as vapor deposition or sputtering to form a nano-sized gap in a self-aligned manner in the stepped portion.
  • Patent Document 8 There is a method of forming (for example, Patent Document 8).
  • Patent Document 9 As a structure using plasmon resonance in combination, a structure in which metal particles are periodically arranged via a metal mirror and an insulating film has been proposed (for example, Patent Document 9).
  • JP 2014-178327 A Special table 2009-501904 JP2014-202650 JP2015-68736 Special table 2011-533677 JP 2014-159364 A JP-A-2005-233637 JP2015-14547 Special table 2007-538264
  • the method of surface treatment, the method of forming nanostructures in bulk, and the method of spraying metal nanoparticles are not methods that can be processed uniformly and precisely, and thus the size of the nano-sized gap cannot be controlled. Therefore, there is a problem that the reproducibility of the SERS enhancement cannot be obtained.
  • the distance between the metal mirror and the metal particles can be controlled by controlling the thickness of the insulating film.
  • the shape of each uneven structure is not optimized, and the electric field enhancement effect is not sufficient.
  • an object of the present invention is to provide an electromagnetic wave enhancing element that brings about an electric field enhancement as compared with the conventional one and enables quantitative evaluation.
  • the electromagnetic wave enhancing element includes a dielectric concavo-convex structure formed by extending a dielectric portion in which a dielectric concavo-convex structure is formed and a concave portion whose width decreases toward the bottom side in one direction on the dielectric concavo-convex structure. And a metal part.
  • the concave portion when the angle formed by the tangents at the same height point of adjacent convex portions forming the concave portion is ⁇ , the concave portion preferably has a minimum value of ⁇ of 20 degrees or less.
  • the metal part is preferably A> B, where A is the vertical thickness of the uppermost point of the convex part of the metal concavo-convex structure, and B is the vertical thickness of the lowermost point. .9A> B is preferred.
  • the metal part is A> C, where A is the vertical thickness of the uppermost point of the convex part of the metal concave-convex structure and C is half the width of the lowest point of the concave part of the dielectric concave-convex structure. It is more preferable that A> 2C.
  • the metal part includes a metal part main body for defining the metal uneven structure, and a metal layer formed on a surface of the metal part main body and made of a metal having a higher electric field strength than the metal part main body. May be.
  • the pitch of the metal concavo-convex structure is preferably 200 nm or less.
  • the convex surface has a curved shape that swells toward the concave side.
  • the dielectric concavo-convex structure has a plurality of types of shapes.
  • the dielectric uneven structure may be composed of a plurality of regions, and the dielectric uneven structure may have a different shape for each region.
  • the metal concavo-convex structure is bonded with a fixing substance for fixing a specific substance.
  • the immobilizing substance can immobilize amino acids.
  • hydrophilic layer having hydrophilicity may be provided on the surface of the metal part.
  • an intermediate layer for improving the adhesion between the dielectric part and the metal part may be provided between the dielectric part and the metal part.
  • the method for manufacturing an electromagnetic wave enhancing element according to the present invention includes a dielectric part forming step of forming a dielectric uneven structure on the dielectric part, and a concave part that decreases in width toward the bottom side on the dielectric uneven structure.
  • the concave part is set so that the minimum value of ⁇ is 20 degrees or less. It is preferable to form the film.
  • the metal part is formed such that A> B, where A is the vertical thickness of the uppermost point of the convex part of the metal concavo-convex structure and B is the vertical thickness of the lowermost point. Is more preferable, and it is more preferable to form the metal portion so that 0.9A> B.
  • A> C where A is the thickness in the vertical direction of the top point of the convex portion of the metal concavo-convex structure, and C is the half width of the bottom portion of the concave portion of the dielectric concavo-convex structure.
  • a metal part main body for defining the metal concavo-convex structure is formed, and a metal layer made of a metal having a higher electric field strength than the metal part main body is formed on the surface of the metal part main body. It may be.
  • the dielectric part forming step is such that the pitch of the dielectric uneven structure is formed to be 200 nm or less.
  • the metal part forming step it is preferable to control the film thickness by using a film forming technique that increases the growth rate in the vertical direction of the metal part.
  • a film forming technique that increases the growth rate in the vertical direction of the metal part.
  • the metal part formed by the sputtering may be further subjected to electroless plating to adjust the shape of the metal part.
  • an intermediate layer forming step of forming an intermediate layer for improving the adhesion between the dielectric portion and the metal portion on the dielectric portion may be provided.
  • the metal part may have a fixed substance binding step of binding a fixed substance capable of fixing a specific substance.
  • It may have a hydrophilic layer forming step of forming a hydrophilic layer having hydrophilicity on the surface of the metal part.
  • the amino acid sequence determination method of the present invention includes a sequential decomposition step of sequentially decomposing amino acids from the N-terminus or C-terminus of a peptide or protein, a fractionation step of fractionating amino acids released by the sequential decomposition step, And an analysis step of analyzing the amino acid obtained by the fractionation step using the electromagnetic wave enhancing element of the present invention.
  • the sequential decomposition step may be performed using a protease.
  • the sequential decomposition step may be performed using a column on which the protease, the peptide, or the protein is immobilized.
  • the electromagnetic wave enhancing element of the present invention can bring about a large electric field enhancement by optimizing the shape of the metal concavo-convex structure.
  • 6 is a graph showing the maximum Raman intensity of Samples 1 to 3 and Comparative Samples 1 and 2. 6 is a graph showing the relationship between the size of the gap between samples 4 to 6 and the Raman intensity. It is sectional drawing which shows an electromagnetic wave enhancing element. It is sectional drawing which shows another electromagnetic wave enhancing element of this invention. It is sectional drawing which shows another electromagnetic wave enhancing element of this invention. It is sectional drawing which shows the electromagnetic wave enhancing element of the present invention. It is sectional drawing which shows another electromagnetic wave enhancing element of this invention.
  • the electromagnetic wave enhancing element of the present invention is a metal having a shape in which a dielectric portion 1 in which a dielectric concavo-convex structure 11 is formed and a concave portion 22 whose width decreases toward the bottom side is extended in one direction.
  • the recess 22 should have a minimum gap width of 25 nm or less, preferably 20 nm or less, between adjacent protrusions forming the recess 22. As shown in FIG.
  • the recess 22 is preferably in contact with adjacent projections 24A and 24B forming the recess 22, but as long as the electric field can be sufficiently enhanced, FIG. As shown in (b), there may be a gap. Further, in FIG. 1A, there is no gap between the dielectric part 1 and the metal part 2, but if the electric field can be sufficiently enhanced, the dielectric part 1 and the metal part 2 can be seen as shown in FIG. There may be a gap 29 between them.
  • the metal part 2 has a metal concavo-convex structure 21 constituting a concave part 22 for concentrating the electric field.
  • the metal concavo-convex structure 21 is formed in a shape obtained by extending the recess 22 in one direction, that is, linearly in a plan view.
  • an electromagnetic wave having a predetermined wavelength ⁇ is incident on the metal concavo-convex structure 21, a strong electric field is concentrated in a very narrow region of the concave portion 22 of the metal concavo-convex structure 21.
  • the SERS signal can be taken out using the portion showing the maximum value of the electric field enhancement intensity.
  • the angle formed between the tangents 25A and 25B at the same height point (the lowest point 23 of the concave portion 22 in the figure) of the adjacent convex portions 24A and 24B forming the concave portion 22 is defined.
  • the metal concavo-convex structure 21 having a large maximum value Ex of the electric field enhancement strength tends to be large when the minimum value of the angle ⁇ is 20 degrees or less.
  • the metal concavo-convex structure 21 having a large maximum value Ex of the electric field enhancement strength tends to increase when 0 ° ⁇ ⁇ 15 °, and tends to increase when 7 ° ⁇ ⁇ 13 °. .
  • the point of contact with the convex portion 24 of the tangent where ⁇ is the minimum value is half of the height of the convex portion 24 (the height of the highest point 26 of the convex portion 24 with respect to the lowest point 23 of the convex portion 24). It is better to be on the lower side, preferably lower than a quarter of the height of the convex portion 24, more preferably at the lowest point 23. Therefore, it is preferable that the shape of the convex portion 24 is a curved shape in which the surface of the convex portion 24 swells toward the concave portion 22 as shown in FIG.
  • the angle ⁇ may be measured by taking a cross-sectional photograph perpendicular to the extending direction of the metal concavo-convex structure 21 and analyzing the image.
  • the metal concavo-convex structure 21 having a large thickness tends to be large.
  • the metal concavo-convex structure 21 having a large maximum value Ex of the electric field enhancement strength tends to increase when 0.9A> B, and further tends to increase when 0.6A> B.
  • the electric field enhancement when the vertical thickness of the uppermost point 26 of the convex portion 24 of the metal concavo-convex structure 21 is A and the half width of the bottom of the concave portion 12 of the dielectric concavo-convex structure 11 is C, the electric field enhancement When the film thickness A is larger than C, the metal uneven structure 21 having a large maximum value Ex tends to increase. In particular, the metal concavo-convex structure 21 having a large maximum value Ex of the electric field enhancement strength tends to increase when A> 2C, and further tends to increase when A> 3.5C. In addition, as shown in FIG.
  • a gap is formed in the concave portion 22 of the metal concavo-convex structure 21, or a gap is formed between the dielectric portion 1 and the metal portion 2 as shown in FIG. Even in the case of 29, it is better that A> C, preferably A> 2C, and more preferably A> 3.5C.
  • the pitch P of the convex portions 24 of the metal concavo-convex structure 21 may be 1000 nm or less, preferably 200 nm or less, and more preferably 120 nm or less.
  • the material of the metal part 2 may be any material as long as it reflects electromagnetic waves.
  • metals such as gold, silver, copper, chromium, aluminum, platinum, and tungsten can be used. Moreover, these combinations may be sufficient.
  • the metal part 2 does not need to consist of a single metal, and a metal part body for defining a metal uneven structure and a metal layer made of different metals formed on the surface of the metal part body. It may be configured. For example, depending on the material of the metal part 2, there are some that have a large electric field enhancement but are difficult to form the metal relief structure 21. In this case, as shown in FIG. 22, the metal part 2 is formed on the surface of the metal part main body 2A for defining the metal uneven structure 21 and the metal part main body 2A, and has a higher electric field strength than the metal part main body 2A. And a metal layer 2B made of In this case, the metal layer is preferably 1 nm or more.
  • the dielectric portion 1 has a dielectric concavo-convex structure 11 for controlling the shape and pitch of the metal concavo-convex structure 21.
  • the material of the dielectric part 1 may be any dielectric material that can form the dielectric concavo-convex structure 11.
  • a resin such as an acrylic resin or a cyclic olefin resin, silicon (Si), An inorganic compound such as silicon dioxide (SiO 2 ) can be used.
  • a photocurable resin or a thermoplastic resin suitable for the imprint method may be used.
  • the shape of the dielectric concavo-convex structure 11 may be any shape as long as the shape of the metal concavo-convex structure 21 can be controlled so as to have a concave portion 22 whose width decreases toward at least the bottom side.
  • the shape of the dielectric concavo-convex structure 11 is preferably one that can control the shape of the metal concavo-convex structure 21 so that the minimum value of the angle ⁇ described above is 20 degrees or less.
  • the surface of the convex portion 14 of the dielectric concavo-convex structure 11 can be a curved shape swelled toward the concave portion 22 side.
  • the shape of the metal concavo-convex structure 21 can be controlled, a cross section having a trapezoidal shape, a rectangular shape such as a rectangle or a square, a triangular shape, or the like may be used.
  • the pitch of the dielectric concavo-convex structure 11 may be the same as the pitch P of the metal concavo-convex structure 21.
  • the other shapes of the dielectric concavo-convex structure 11 may be any shape as long as the metal concavo-convex structure 21 can be controlled so that the electromagnetic field can be enhanced, for example, the width and aspect ratio of the convex portion 14.
  • the width of the convex portion 14 of the dielectric concavo-convex structure 11 may be 30 to 60% of the pitch.
  • the aspect ratio of the convex portion 14 of the dielectric concavo-convex structure 11 is preferably 1 or more.
  • the enhancement of electromagnetic waves is proportional to the fourth power of the electric field, the electric field varies greatly depending on a slight difference in the shape of the metal concavo-convex structure 21.
  • the metal concavo-convex structure 21 has a sufficient number of recesses 22 so as to include a shape capable of enhancing electromagnetic waves.
  • the dielectric concavo-convex structure 11 formed on the electromagnetic wave enhancing element may have a plurality of types of shapes as shown in FIG.
  • the metal concavo-convex structure 21 to be formed also has a plurality of types of shapes, and the metal concavo-convex structure 21 that can enhance electromagnetic waves is reliably formed. can do.
  • the enhancement of electromagnetic waves is proportional to the fourth power of the electric field, the electromagnetic wave can be sufficiently enhanced if there is a metal uneven structure 21 having a suitable shape on the electromagnetic wave enhancing element.
  • the dielectric concavo-convex structure 11 formed on the electromagnetic wave enhancing element may have a plurality of regions, and the dielectric concavo-convex structure 11 may be formed so as to have a different shape for each region.
  • the dielectric portion 1 shown in FIG. 4 has a dielectric concavo-convex structure 11 in a line-and-space shape, and the pitch of the convex portion of the dielectric concavo-convex structure 11 is increased toward the right region in the figure, and the lower region is formed. Regions 91 to 99 are formed in which the line width of the convex portion decreases as the distance increases.
  • the metal concavo-convex structure 21 is preferably bonded with a fixing substance 5 for fixing the substance as shown in FIG.
  • the metal part 2 may be surface-treated with a coupling agent or the like having a functional group capable of forming a chemical bond with the surface of the metal part 2 and capable of chemical bonding or chemical adsorption with a substance to be fixed.
  • the film thickness is preferably 5 nm or less, preferably 1 nm or less.
  • a silane having a functional group capable of binding or adsorbing to an amino group or a carboxyl group of an amino acid As a specific example, when producing the electromagnetic wave enhancing element of the present invention for amino acid analysis, as shown in FIG. 23, a silane having a functional group capable of binding or adsorbing to an amino group or a carboxyl group of an amino acid. What is necessary is just to perform the surface treatment of the metal part 2 with a coupling agent.
  • the water-soluble reagent 6 such as rhodamine may be repelled on the silver surface and may not reach a hot spot where Raman enhancement is strong.
  • a hydrophilic layer made of a material that is at least more hydrophilic than the metal part 2 may be formed on the surface of the metal part 2.
  • a hydrophilic layer 7 made of a hydrophilic dielectric material such as silicon oxide (SiO 2 )
  • SiO 2 silicon oxide
  • the hydrophilic layer 7 should be coated with a film thickness of 5 nm or less, preferably 1 nm or less.
  • the intermediate layer 3 may be any material that improves the adhesion between the dielectric portion 1 and the metal portion 2, and for example, platinum can be used.
  • the method for manufacturing an electromagnetic wave enhancing element according to the present invention includes a dielectric part forming step for forming a dielectric concavo-convex structure 11 on a dielectric part 1 and a metal part 2 constituting a metal concavo-convex structure 21 on the dielectric concavo-convex structure 11. And a metal part forming step to be formed.
  • the dielectric part forming step is for forming the dielectric concavo-convex structure 11 for controlling the shape and pitch of the metal concavo-convex structure 21.
  • the dielectric part forming step may be performed by any method as long as the predetermined dielectric uneven structure 11 can be formed. For example, a method of forming the dielectric concavo-convex structure 11 composed of a curved line and space in which the surface of the convex portion 14 bulges toward the concave portion will be described.
  • a base material 10 made of a dielectric is prepared.
  • a dielectric concavo-convex structure 15 is formed on the base material 10 by imprinting.
  • FIG. 6A a base material 10 made of a dielectric is prepared.
  • a dielectric concavo-convex structure 15 is formed on the base material 10 by imprinting.
  • the dielectric uneven structure 11 may be formed by performing UV ozone irradiation.
  • a resin is applied to the dielectric concavo-convex structure 15 to form a reverse arched mask 16 in the concave portion 22 of the dielectric concavo-convex structure 15.
  • the dielectric uneven structure 11 may be formed by performing UV ozone irradiation.
  • the concave portion 22 is formed such that the minimum width of the gap between adjacent convex portions forming the concave portion 22 is 25 nm or less, preferably 20 nm or less. Is good. As shown in FIG.
  • the recess 22 is preferably in contact with adjacent projections 24A and 24B forming the recess 22, but as long as the electric field can be sufficiently enhanced, FIG. As shown in (b), there may be a gap. Further, in FIG. 1A, there is no gap between the dielectric part 1 and the metal part 2, but if the electric field can be sufficiently enhanced, the dielectric part 1 and the metal part 2 can be seen as shown in FIG. There may be a gap 29 between them.
  • the metal part forming step may be any process as long as the metal part 2 constituting the metal uneven structure 21 can be formed. For example, a film forming technique such as sputtering, vapor deposition, or plating may be used.
  • the minimum value of ⁇ is 20 degrees or less, preferably 0 ° ⁇ . It is better to form the recess 22 so that ⁇ 15 °, more preferably 7 ° ⁇ ⁇ 13 °.
  • the minimum value of the angle ⁇ is 20 degrees or less, for example, as shown in FIG. 6E, a film forming technique that increases the vertical growth rate of the metal part 2, for example, What is necessary is just to form the metal part 2 using anisotropic sputtering. Moreover, as shown in FIG.6 (f), on the metal part 2 formed by sputtering, electroless plating may be given further and the shape of the metal part 2 may be adjusted. Thereby, the minimum value of the angle ⁇ can be adjusted.
  • the metal part forming step when the vertical thickness of the uppermost point 26 of the convex part 24 of the metal concavo-convex structure 21 is A and the vertical thickness of the lowermost point 23 is B, the metal is formed so that A> B.
  • Part 2 may be formed. More preferably, the metal part 2 is formed so that 0.9A> B, and more preferably, the metal part 2 is formed so that 0.6A> B.
  • the metal part forming step when the vertical thickness of the uppermost point 26 of the convex part 24 of the metal concave-convex structure 21 is A and half of the bottom width of the concave part 12 of the dielectric concave-convex structure C is A> C, You may form the metal part 2 so that it may become. More preferably, the metal part 2 is formed so that A> 2C, and more preferably, the metal part 2 is formed so that A> 3.5C. In this case, the size of C may be determined by adjusting the width of the concave portion of the dielectric concavo-convex structure 11 formed in the dielectric portion forming step.
  • the metal part forming step as shown in FIG. 22, first, a metal part main body for defining the metal uneven structure 21 is formed using a metal that can easily form the metal uneven structure 21. Next, a metal layer made of a metal different from the metal part body, for example, a metal having a higher electric field strength than the metal part body may be formed on the surface of the metal part body.
  • the metal layer is preferably 1 nm or more.
  • an electromagnetic wave enhancing element having a metal concavo-convex structure in which the convex portion and the concave portion are extended in one direction can be manufactured (see FIG. 7).
  • an intermediate layer for improving the adhesion between the dielectric part 1 and the metal part 2 on the dielectric part 1 between the dielectric part forming step and the metal part forming step. 3 may be included.
  • the intermediate layer 3 formation step may be performed by any method, and examples thereof include film formation techniques such as sputtering, vapor deposition, and plating.
  • the metal part 2 is utilized by using a film forming technique that increases the vertical growth rate of the metal part 2, for example, anisotropic sputtering. May be formed.
  • FIG.8 (c) you may adjust the shape of the metal part 2 by giving an electroless plating further on the metal part 2 formed by sputtering.
  • the metal part 2 may have a fixed substance binding step of binding a fixed substance capable of fixing a specific substance.
  • the metal part 2 may be surface-treated with a coupling agent or the like having a functional group capable of forming a chemical bond with the surface of the metal part 2 and capable of chemical bonding or chemical adsorption with a specific substance to be fixed.
  • the film thickness is preferably 5 nm or less, preferably 1 nm or less.
  • a silane having a functional group capable of binding or adsorbing to an amino group or a carboxyl group of an amino acid As a specific example, when producing the electromagnetic wave enhancing element of the present invention for amino acid analysis, as shown in FIG. 23, a silane having a functional group capable of binding or adsorbing to an amino group or a carboxyl group of an amino acid. What is necessary is just to perform the surface treatment of the metal part 2 with a coupling agent.
  • a water-soluble reagent such as rhodamine may be repelled on the silver surface and may not reach a hot spot where Raman enhancement is strong.
  • a hydrophilic layer 7 made of a material having a higher hydrophilic property than the metal part 2 such as silicon oxide (SiO 2 )
  • the reagent can reach the hot spot, and high sensitivity can be obtained.
  • the hydrophilic layer 7 should be coated with a film thickness of 5 nm or less, preferably 1 nm or less.
  • the electromagnetic wave enhancing element of the present invention will be described using simulation.
  • the software DiffractMOD manufactured by Synopsys, Inc. was used.
  • the electromagnetic wave enhancing element includes a dielectric part 1 in which a dielectric concavo-convex structure 11 in which the convex part 14 is a semicircle having a radius r, and a semicircle and a concentric circle that constitute the convex part 14.
  • a metal part 2 having a metal concavo-convex structure 21 composed of a convex part 24 which is a semicircle or a semi-ellipse obtained by enlarging it in the vertical direction was used.
  • the horizontal dimension from the center of the semicircle or semi-ellipse constituting the convex portion 24 is set to r + 10 nm, and the width d of the concave portion 12 of the dielectric concavo-convex structure 11 is changed to overlap the side portions of the adjacent convex portions 24.
  • the thickness B in the vertical direction of the lowest point 23 of the metal part 2 was adjusted.
  • pitches of the metal uneven structure 21 were simulated for every 10 nm from 100 nm to 200 nm.
  • PMMA is assumed as the material of the dielectric part 1
  • gold is assumed as the material of the metal part 2.
  • the maximum value Ex of the electric field enhancement exceeds 80 times when the angle ⁇ formed by the metal part 2 in the concave part 22 of the metal concave-convex structure 21 is 20 degrees or less.
  • the maximum value Ex of the electric field enhancement strength tends to be large when 0 ° ⁇ ⁇ 15 ° and further large when 7 ° ⁇ ⁇ 13 °.
  • the pitch P is at least 200 nm or less, there is a case where the maximum value Ex of the electric field enhancement exceeds 80 times, and the maximum value Ex of the electric field enhancement tends to increase as the pitch P decreases. .
  • the maximum value Ex of the electric field enhancement intensity was large when the pitch P was 100 ⁇ P ⁇ 120 nm.
  • FIG. 10 indicates that the maximum value Ex of the electric field enhancement strength tends to increase as the film thickness A is greater than the film thickness B.
  • the maximum value Ex of the electric field enhancement strength exceeds 80 times, and when the thickness ratio B / A is smaller than 0.6, It was found that the maximum value Ex of the electric field enhancement intensity exceeds 100 times.
  • the pitch P is at least 200 nm or less, there is a case where the maximum value Ex of the electric field enhancement exceeds 80 times, and the maximum value Ex of the electric field enhancement tends to increase as the pitch P decreases. .
  • the maximum value Ex of the electric field enhancement intensity was large when the pitch P was 100 ⁇ P ⁇ 120 nm.
  • FIG. 11 shows that the maximum value Ex of the electric field enhancement tends to increase as A is greater than C.
  • the ratio A / C of A and C is larger than 2
  • the maximum value Ex of the electric field strength exceeds 100 times
  • the ratio A / C is larger than 3.5
  • the maximum value Ex of the electric field strength increases. It turned out that there was a thing exceeding 120 times.
  • the pitch P is at least 200 nm or less
  • the maximum value Ex of the electric field enhancement exceeds 80 times, and the maximum value Ex of the electric field enhancement tends to increase as the pitch P decreases.
  • the maximum value Ex of the electric field enhancement intensity was large when the pitch P was 100 ⁇ P ⁇ 120 nm.
  • Example 1 Next, the electromagnetic wave enhancing element of the present invention was actually created, and the intensity of Raman scattered light (hereinafter referred to as Raman intensity) was confirmed. Samples 1 to 3 described later were used as the electromagnetic wave enhancing elements. Moreover, in order to compare with the electromagnetic wave enhancing element of the present invention, comparative samples 1 and 2 described later were used.
  • Example 1 Silver was formed at 200 W for 245 seconds by sputtering on a line-and-space dielectric concavo-convex structure having a height of 120 nm, a line width of 50 nm, and a pitch of 100 nm, and a concave portion with a smaller width was stretched in one direction toward the bottom side.
  • An electromagnetic wave enhancing element having a metal uneven structure was formed.
  • the thickness in the vertical direction of the uppermost point of the convex portion of the metal concavo-convex structure was 130 nm.
  • FIG. 12 shows a plan photograph and a sectional photograph of the electromagnetic wave enhancing element.
  • Example 2 Silver was formed at 200 W for 245 seconds by sputtering on a line-and-space dielectric concavo-convex structure having a height of 140 nm, a line width of 70 nm, and a pitch of 140 nm, and a concave portion with a smaller width was stretched in one direction toward the bottom side.
  • An electromagnetic wave enhancing element having a metal uneven structure was formed.
  • the thickness in the vertical direction of the uppermost point of the convex portion of the metal concavo-convex structure was 125 nm.
  • FIG. 13 shows a plan photograph and a sectional photograph of the electromagnetic wave enhancing element.
  • Example 3 Silver was formed at 200 W for 245 seconds by sputtering on a line-and-space dielectric concavo-convex structure having a height of 200 nm, a line width of 100 nm, and a pitch of 200 nm, and a concave portion with a smaller width was stretched in one direction toward the bottom side.
  • An electromagnetic wave enhancing element having a metal uneven structure was formed. The thickness in the vertical direction of the uppermost point of the convex portion of the metal concavo-convex structure was 127 nm.
  • a plane photograph and a cross-sectional photograph of the electromagnetic wave enhancing element are shown in FIG.
  • Electrode 1 Silver was formed at 200 W for 245 seconds by sputtering on a dielectric concavo-convex structure in which a cylinder having a height of 500 nm and a bottom diameter of 230 nm was arranged in a triangle with a pitch of 460 nm, thereby forming an electromagnetic wave enhancing element.
  • the thickness in the vertical direction of the uppermost point of the convex portion of the metal concavo-convex structure was 120 nm.
  • FIG. 15 shows a plan photograph and a sectional photograph of the electromagnetic wave enhancing element.
  • each electromagnetic wave enhancing element was irradiated with a laser having a wavelength of 532 nm perpendicularly at 3 mW for 1 second, and the Raman intensity at a Raman shift of 1430 cm ⁇ 1 was measured.
  • a laser was incident on the electromagnetic wave enhancing element (samples 1 to 3) of the present invention, and the azimuth angle and the Raman intensity at a Raman shift of 1430 cm ⁇ 1 were measured by rotating a vertical line at the incident point as a rotation axis. The result is shown in FIG.
  • the azimuth angle represents the Raman intensity every 22.5 degrees with the position indicating the maximum value of the Raman intensity of each electromagnetic wave enhancing element as 0 degree.
  • the Raman scattered light detection method using the electromagnetic wave enhancing element of the present invention includes an irradiation step of making light incident on the electromagnetic wave enhancing element, and an electromagnetic wave having a vertical line at the point where the light is incident on the electromagnetic wave enhancing element as a rotation axis. It is preferable to have a rotation step of rotating the enhancement element by at least 90 degrees and a measurement step of measuring the maximum intensity of the Raman scattered light of the electromagnetic wave enhancement element during the rotation step. Thereby, the electromagnetic wave enhancing element of the present invention can detect the characteristics of the subject at the position with the highest sensitivity.
  • the electromagnetic wave enhancing element of the present invention may have an alignment mark 4 indicating the extending direction of the concave portion of the metal part 2 as shown in FIG.
  • the alignment mark 4 may be formed anywhere in the electromagnetic wave enhancing element as long as the extending direction of the recess of the metal part 2 is known.
  • the alignment mark 4 can be formed on the dielectric part 1 or on the metal part 2.
  • FIG. 19 shows the maximum Raman intensity of each electromagnetic wave enhancing element (samples 1 to 3 and comparative samples 1 and 2).
  • the electromagnetic wave enhancing elements (samples 1 to 3) of the present invention having a metal concavo-convex structure having a shape in which a concave portion whose width decreases toward the bottom side is extended in one direction, the metal concavo-convex structure is cylindrical. It can be seen that the present invention shows a very large Raman intensity compared to the electromagnetic wave enhancing element (Comparative Sample 1) and the conical electromagnetic wave enhancing element (Comparative Sample 2).
  • Example 2 Next, the relationship between the minimum width of the gap between adjacent convex portions forming the concave portion of the metal concavo-convex structure and the Raman intensity was examined.
  • the electromagnetic wave enhancement element samples 4 to 6 described later were used.
  • Example 4 Silver was deposited at 200 W for 170 seconds by sputtering on a line-and-space dielectric concavo-convex structure having a height of 140 nm, a line width of 70 nm, and a pitch of 140 nm, and a concave portion with a smaller width was stretched in one direction toward the bottom side. An electromagnetic wave enhancing element having a metal uneven structure was formed. At this time, the minimum width of the gap between adjacent convex portions forming the concave portion of the metal concave-convex structure was 25.9 nm.
  • Example 5 Silver was deposited at 200 W for 195 seconds by sputtering on a line-and-space dielectric concavo-convex structure having a height of 140 nm, a line width of 70 nm, and a pitch of 140 nm, and a concave portion with a smaller width was stretched in one direction toward the bottom side. An electromagnetic wave enhancing element having a metal uneven structure was formed. At this time, the minimum width of the gap between adjacent convex portions forming the concave portion of the metal concavo-convex structure was 23.7 nm.
  • Example 6 Silver was formed at 200 W for 245 seconds by sputtering on a line-and-space dielectric concavo-convex structure having a height of 140 nm, a line width of 70 nm, and a pitch of 140 nm, and a concave portion with a smaller width was stretched in one direction toward the bottom side.
  • An electromagnetic wave enhancing element having a metal uneven structure was formed.
  • the minimum width of the gap between adjacent convex portions forming the concave portion of the metal concavo-convex structure was 20 nm.
  • Each electromagnetic wave enhancing element was irradiated with a laser having a wavelength of 532 nm perpendicularly at 3 mW for 1 second, and the maximum value of the Raman intensity at a Raman shift of 1430 cm ⁇ 1 was measured. The result is shown in FIG.
  • the electromagnetic wave enhancing element of the present invention increases the Raman intensity when the minimum width of the gap between adjacent convex portions forming the concave portion of the metal concave-convex structure is reduced. Specifically, it can be seen that the Raman intensity exceeds 10,000 when the minimum width of the gap is 25 nm or less, and exceeds 35000 when it is 20 nm or less.
  • the amino acid sequence determination method of the present invention includes a sequential decomposition step for sequentially decomposing amino acids from the N-terminus or C-terminus of a peptide or protein, a fractionation step for fractionating amino acids released by the sequential degradation step, and a fractionation step And an analysis step of analyzing the amino acid obtained by the method using the electromagnetic wave enhancing element of the present invention.
  • the sequential decomposition step may be any method as long as it is a method for releasing an amino acid located at the N-terminus or C-terminus of a peptide or protein.
  • a method using a protease a phenylisothiocyanate (PITC) is reacted with a free amino group at the N-terminal part of a peptide or protein to form a phenylthiocarbamyl derivative (PTC amino acid), and then anilinothiazolinone with trifluoroacetic acid
  • a method of releasing as (ATZ) -amino acid (Edman degradation) can be used.
  • an exopeptidase that hydrolyzes a peptide bond at the N-terminal or C-terminal of a peptide or protein and sequentially releases amino acids from the terminal can be used.
  • the exopeptidase may be used by mixing two or more types having different characteristics.
  • carboxypeptidase Y that exhibits high catalytic activity when the second or terminal residue from the terminal is an aromatic or aliphatic amino acid, and a terminal residue that is a basic amino acid
  • carboxypeptidase B showing high catalytic action can be used in combination.
  • the fragment after performing an endopeptidase treatment first to obtain a fragment having an appropriate length, the fragment may be subjected to degradation by exopeptidase.
  • the sequential decomposition step of the present invention it is preferable that only one residue is released from the end of the peptide or protein.
  • the Raman scattering signal is analyzed even when two or more amino acids are bound, and the type and order of the amino acids are determined. Can be identified. Therefore, in this case, it is possible to identify an amino acid even in the case where 2 or more residues are released in a bound state from the end of the peptide or protein in the sequential decomposition step of the present invention. In some cases.
  • the sequential decomposition may be performed using a column on which a protease, peptide, or protein is immobilized. Immobilization of protease, peptide or protein to the carrier packed in the column may be performed by any method. By using the immobilized column, a sample that has been subjected to the sequential decomposition step, that is, a sample containing free amino acids can be easily obtained at a desired timing.
  • the fractionation step in the amino acid sequence determination method of the present invention may be any method as long as it is a method for fractionating amino acids released by the sequential decomposition step.
  • the eluate from the column may be collected over time using a fraction collector or the like.
  • the activity of the protease when controlling the activity of the protease, only the eluate that has undergone the degradation reaction by the protease may be collected in correspondence with the control. Thereby, a desired amino acid can be efficiently recovered.
  • the analysis step in the amino acid sequence determination method of the present invention is a step of analyzing the amino acid obtained by the fractionation step using the electromagnetic wave enhancing element of the present invention, and is performed, for example, as follows.
  • the free amino acid obtained by the sorting step is fixed to the end of the dielectric layer of the electromagnetic wave enhancing element of the present invention.
  • the amino acid can be fixed to the end portion of the dielectric layer by physical adsorption to the surface of the end portion or by bonding via polybrene or a silane coupling agent.
  • a solution containing the amino acid obtained by the fractionation step onto the electromagnetic wave enhancing element of the present invention in which a silane coupling agent is bonded to the end of the dielectric layer in advance this is appropriately washed, It can be dried.
  • the electromagnetic wave enhancing element to which the amino acid is fixed is irradiated with an electromagnetic wave having a predetermined wavelength, and the obtained Raman scattering signal is analyzed to identify the fixed amino acid.
  • various analyzes are performed as necessary to determine the amino acid sequence.
  • Example 3 Next, a method for producing the electromagnetic wave enhancing element of the present invention will be described.
  • FIG. 21 when an inexpensive sputter film formation is performed on a line-and-space dielectric concavo-convex structure, since the migration of atoms is small in gold, the film formation shape must correspond to the arrival amount of atoms on a one-to-one basis. become. Then, when adjacent gold contacts, a gentle contact angle is taken, and the signal cannot be amplified by the metal part 2. On the other hand, as shown in FIG.
  • Example 4 Next, another method for producing an electromagnetic wave enhancing element will be described.
  • the water-soluble reagent 6 such as rhodamine is sometimes repelled on the Ag surface and does not reach the hot spot. In this case, no Raman enhancement occurs. Therefore, as shown in FIG. 25, a hydrophilic layer 7 having a film thickness of 5 nm or less, preferably 1 nm or less, is formed of a hydrophilic dielectric material such as SiO 2 on the surface of Ag (metal part 2). As a result, the reagent reaches the hot spot and high sensitivity is obtained.

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Abstract

The purpose of the present invention is to provide an electromagnetic wave enhancement element which results in electric field enhancement compared to conventional elements and makes quantitative evaluation possible. The electromagnetic wave enhancement element mainly comprises: a dielectric part 1 in which a dielectric recess and protrusion structure 11 is formed; and a metal part 2 having, on the dielectric recess and protrusion structure 11, a metallic recess and protrusion structure 21, in which a recess portion 22 that decreases in width toward a bottom side thereof is elongated in one direction. Furthermore, when an angle between tangent lines 25A, 25B at a point 23 having the same height with neighboring protrusion portions 24A, 24B which form the recess portion 22 is θ, the recess portion 22 preferably has a 20 degrees or less minimum value of θ.

Description

電磁波増強素子およびその製造方法並びに電磁波増強素子を用いた検出方法およびアミノ酸配列決定方法Electromagnetic wave enhancing element, method for producing the same, detection method using electromagnetic wave enhancing element, and amino acid sequence determination method
　本発明は、電磁波増強素子およびその製造方法並びに電磁波増強素子を用いたアミノ酸配列決定方法に関するものである。 The present invention relates to an electromagnetic wave enhancing element, a production method thereof, and an amino acid sequence determination method using the electromagnetic wave enhancing element.
　光が物質に入射して分子と衝突すると、その一部は散乱される。この散乱光の波長を調べると、大部分の成分は入射光と同じ波長の光であるレイリー散乱光であるが、極わずかな成分として、入射光と異なった波長の光が含まれる。ラマン分光法とは、この入射光と異なった波長をもつ光であるラマン散乱光の性質を調べることにより、物質の分子構造や結晶構造などを知るための方法である。ここで、ラマン散乱光の強度は、レイリー散乱光の強度に対してわずか10のマイナス6乗程度と極めて微弱なため、実用的にはレーザーのような高強度光源を用いる必要がある。 When light enters a substance and collides with molecules, a part of it is scattered. When the wavelength of the scattered light is examined, most of the components are Rayleigh scattered light that is light having the same wavelength as the incident light, but light having a wavelength different from that of the incident light is included as an extremely small component. Raman spectroscopy is a method for determining the molecular structure and crystal structure of a substance by examining the properties of Raman scattered light, which is light having a wavelength different from that of incident light. Here, the intensity of Raman scattered light is extremely weak, about 10 to the sixth power of the intensity of Rayleigh scattered light. Therefore, it is necessary to use a high-intensity light source such as a laser practically.
　一方、近年、高感度な化学センサーやバイオセンサーに応用可能な分析手法として、表面増強ラマン散乱（Surface Enhanced Raman Scattering：以下、ＳＥＲＳと略する）を利用した分光法が注目されている。 On the other hand, in recent years, spectroscopy using surface-enhanced Raman scattering (hereinafter abbreviated as SERS) has been attracting attention as an analytical method applicable to highly sensitive chemical sensors and biosensors.
　ＳＥＲＳは、ナノオーダーの微細構造を持つ金属表面に吸着した分子のラマン散乱が、著しく増強される現象である。この増強のメカニズムとしては、金属－分子間の電荷移動による振動の増強（化学的な増強）と、局在表面プラズモンが入射光により励起されたときに形成される電場の増強（物理的な増強）であると言われている。 SERS is a phenomenon in which Raman scattering of molecules adsorbed on a metal surface having a nano-order fine structure is remarkably enhanced. The mechanism of this enhancement includes the enhancement of vibration (chemical enhancement) by charge transfer between metal and molecule, and the enhancement of the electric field formed when localized surface plasmons are excited by incident light (physical enhancement). ).
　従来、ＳＥＲＳの発生を目的として設計された種々の電磁波増強素子が知られている。 Conventionally, various electromagnetic wave enhancing elements designed for the purpose of generating SERS are known.
　例えば、表面処理により、自己組織的にナノギャップ構造をつくる方法として、多孔質シリコンの構造を利用するもの（例えば、特許文献１）、エンボス加工によってナノ構造を形成する方法（例えば、特許文献２）、ベーマイト処理による表面の粗化によりナノ構造を形成する方法（例えば、特許文献３）などがある。 For example, as a method for forming a nanogap structure in a self-organized manner by surface treatment, a method using a porous silicon structure (for example, Patent Document 1), a method for forming a nanostructure by embossing (for example, Patent Document 2) ), A method of forming a nanostructure by surface roughening by boehmite treatment (for example, Patent Document 3).
　また、バルクでナノ構造を作る方法として、ナノコンポジットを形成するもの（例えば、特許文献４）、金属ナノスポンジを形成するもの（例えば、特許文献５）、フラーレンを凝集させるもの（例えば、特許文献６）などがある。 In addition, as a method for forming a nanostructure in bulk, a method for forming a nanocomposite (for example, Patent Document 4), a method for forming a metal nanosponge (for example, Patent Document 5), and a method for aggregating fullerenes (for example, Patent Documents) 6).
　また、基板表面に金属粒子を散布して固定させ、自己組織的にナノサイズの間隔を作るものも提案されている（例えば、特許文献７）。 Also proposed is a technique in which metal particles are dispersed and fixed on the surface of a substrate to form nano-sized intervals in a self-organized manner (for example, Patent Document 7).
　一方、パターンを形成するものとしては、パターン形成した段差に蒸着やスパッタリング等の段差被覆が不十分な成膜方法で金属膜を堆積することで、段差部分に自己整合的にナノサイズのギャップを形成する方法がある（例えば、特許文献８）。 On the other hand, as a method for forming a pattern, a metal film is deposited on the stepped pattern by a film forming method with insufficient step coverage such as vapor deposition or sputtering to form a nano-sized gap in a self-aligned manner in the stepped portion. There is a method of forming (for example, Patent Document 8).
　更に、プラズモン共鳴を組み合わせて利用するものとして、金属ミラーと絶縁膜を介し金属粒子を周期的配置する構造も提案されている（例えば、特許文献９）。 Furthermore, as a structure using plasmon resonance in combination, a structure in which metal particles are periodically arranged via a metal mirror and an insulating film has been proposed (for example, Patent Document 9).
特開２０１４－１７８３２７JP 2014-178327 A 特表２００９－５０１９０４Special table 2009-501904 特開２０１４－２０２６５０JP2014-202650 特開２０１５－６８７３６JP2015-68736 特表２０１１－５３２３６７７Special table 2011-533677 特開２０１４－１５９３６４JP 2014-159364 A 特開２００５－２３３６３７JP-A-2005-233637 特開２０１５－１４５４７JP2015-14547 特表２００７－５３８２６４Special table 2007-538264
　しかしながら、表面処理による方法やバルクでナノ構造を作る方法、金属ナノ粒子の散布による方法は、精密に均一処理できる手法ではないため、ナノサイズのギャップの大きさを制御することができなかった。したがって、ＳＥＲＳの増強度の再現性が得られないという問題があった。 However, the method of surface treatment, the method of forming nanostructures in bulk, and the method of spraying metal nanoparticles are not methods that can be processed uniformly and precisely, and thus the size of the nano-sized gap cannot be controlled. Therefore, there is a problem that the reproducibility of the SERS enhancement cannot be obtained.
　また、金属ミラーと絶縁膜を介して金属粒子を周期的に配置する方法は、絶縁膜の厚さを制御することで金属ミラーと金属粒子の間の距離を制御することができる。しかしながら、周期性を持たせて共鳴させるだけでは、十分に強い電場増強効果を得ることができなかった。 Further, in the method of periodically arranging the metal particles via the metal mirror and the insulating film, the distance between the metal mirror and the metal particles can be controlled by controlling the thickness of the insulating film. However, it was not possible to obtain a sufficiently strong electric field enhancement effect only by resonating with periodicity.
　また、周期構造を利用するものであっても、各凹凸構造の形状を最適化したものではなく、電場増強効果は十分なものではなかった。 Moreover, even if the periodic structure is used, the shape of each uneven structure is not optimized, and the electric field enhancement effect is not sufficient.
　そこで本発明は、従来のものよりも電場増強をもたらし、定量的評価を可能にする電磁波増強素子を提供することを目的とする。 Therefore, an object of the present invention is to provide an electromagnetic wave enhancing element that brings about an electric field enhancement as compared with the conventional one and enables quantitative evaluation.
　本発明の電磁波増強素子は、誘電体凹凸構造が形成された誘電体部と、底部側に向かって幅の小さくなる凹部を一方向に延伸した形状の金属凹凸構造を前記誘電体凹凸構造上に有する金属部と、を具備することを特徴とする。 The electromagnetic wave enhancing element according to the present invention includes a dielectric concavo-convex structure formed by extending a dielectric portion in which a dielectric concavo-convex structure is formed and a concave portion whose width decreases toward the bottom side in one direction on the dielectric concavo-convex structure. And a metal part.
　この場合、前記凹部を形成する隣接する凸部の同じ高さの点における接線同士のなす角度をθとすると、当該凹部はθの最小値が２０度以下である方が好ましい。 In this case, when the angle formed by the tangents at the same height point of adjacent convex portions forming the concave portion is θ, the concave portion preferably has a minimum value of θ of 20 degrees or less.
　また、前記金属部は、前記金属凹凸構造の凸部の最上点の垂直方向の厚みをＡ、最下点の垂直方向の厚みをＢとすると、Ａ＞Ｂである方が好ましく、さらには０．９Ａ＞Ｂである方が好ましい。 Further, the metal part is preferably A> B, where A is the vertical thickness of the uppermost point of the convex part of the metal concavo-convex structure, and B is the vertical thickness of the lowermost point. .9A> B is preferred.
　また、前記金属部は、前記金属凹凸構造の凸部の最上点の垂直方向の厚みをＡ、誘電体凹凸構造の凹部の最下点の幅の半分をＣとすると、Ａ＞Ｃである方が好ましく、さらにはＡ＞２Ｃである方が好ましい。 The metal part is A> C, where A is the vertical thickness of the uppermost point of the convex part of the metal concave-convex structure and C is half the width of the lowest point of the concave part of the dielectric concave-convex structure. It is more preferable that A> 2C.
　また、前記金属部は、前記金属凹凸構造を定めるための金属部本体と、前記金属部本体の表面に形成され、当該金属部本体より電界増強度の大きい金属からなる金属層と、で構成してもよい。 The metal part includes a metal part main body for defining the metal uneven structure, and a metal layer formed on a surface of the metal part main body and made of a metal having a higher electric field strength than the metal part main body. May be.
　また、前記金属凹凸構造のピッチは、２００ｎｍ以下である方が好ましい。 The pitch of the metal concavo-convex structure is preferably 200 nm or less.
　また、前記凸部表面は前記凹部側に膨らんだ曲線状である方が好ましい。 Moreover, it is preferable that the convex surface has a curved shape that swells toward the concave side.
　また、前記誘電体凹凸構造は、複数種類の形状からなる方が好ましい。 Moreover, it is preferable that the dielectric concavo-convex structure has a plurality of types of shapes.
　また、前記誘電体凹凸構造は複数の領域からなり、前記誘電体凹凸構造は領域ごとに形状が異なってもよい。 The dielectric uneven structure may be composed of a plurality of regions, and the dielectric uneven structure may have a different shape for each region.
　また、前記金属部の凹部の延伸方向を示すアライメントマークを備えている方が好ましい。 Moreover, it is preferable to provide an alignment mark indicating the extending direction of the concave portion of the metal part.
　また、前記金属凹凸構造は、特定の物質を固定するための固定物質が結合されている方が好ましい。例えば、前記固定物質は、アミノ酸を固定するものとすることができる。 Further, it is preferable that the metal concavo-convex structure is bonded with a fixing substance for fixing a specific substance. For example, the immobilizing substance can immobilize amino acids.
　また、前記金属部の表面に、親水性を有する親水性層を具備してもよい。 Further, a hydrophilic layer having hydrophilicity may be provided on the surface of the metal part.
　また、前記誘電体部と前記金属部の間に前記誘電体部と前記金属部の密着性を向上するための中間層を有してもよい。 Further, an intermediate layer for improving the adhesion between the dielectric part and the metal part may be provided between the dielectric part and the metal part.
　また、本発明の電磁波増強素子の製造方法は、誘電体部に誘電体凹凸構造を形成する誘電体部形成工程と、前記誘電体凹凸構造上に、底部側に向かって幅の小さくなる凹部を一方向に延伸した形状の金属凹凸構造を有する金属部を形成する金属部形成工程と、を有することを特徴とする。 In addition, the method for manufacturing an electromagnetic wave enhancing element according to the present invention includes a dielectric part forming step of forming a dielectric uneven structure on the dielectric part, and a concave part that decreases in width toward the bottom side on the dielectric uneven structure. A metal part forming step of forming a metal part having a metal concavo-convex structure extending in one direction.
　この場合、前記金属部形成工程は、前記凹部を形成する隣接する凸部の同じ高さの点における接線同士のなす角度をθとすると、θの最小値が２０度以下となるように前記凹部を形成するものである方が好ましい。 In this case, in the metal part forming step, when the angle formed by tangents at the same height point of adjacent convex parts forming the concave part is θ, the concave part is set so that the minimum value of θ is 20 degrees or less. It is preferable to form the film.
　また、前記金属部形成工程は、前記金属凹凸構造の凸部の最上点の垂直方向の厚みをＡ、前記最下点の垂直方向の厚みをＢとすると、Ａ＞Ｂとなるように金属部を形成する方が好ましく、さらには０．９Ａ＞Ｂとなるように金属部を形成する方が好ましい。 In the metal part forming step, the metal part is formed such that A> B, where A is the vertical thickness of the uppermost point of the convex part of the metal concavo-convex structure and B is the vertical thickness of the lowermost point. Is more preferable, and it is more preferable to form the metal portion so that 0.9A> B.
　また、前記金属部形成工程は、前記金属凹凸構造の凸部の最上点の垂直方向の厚みをＡ、前記誘電体凹凸構造の凹部の底部の幅の半分をＣとすると、Ａ＞Ｃとなるように金属部を形成する方が好ましく、さらにはＡ＞２Ｃとなるように金属部を形成する方が好ましい。 In the metal portion forming step, A> C, where A is the thickness in the vertical direction of the top point of the convex portion of the metal concavo-convex structure, and C is the half width of the bottom portion of the concave portion of the dielectric concavo-convex structure. Thus, it is preferable to form the metal part, and it is more preferable to form the metal part so that A> 2C.
　また、前記金属部形成工程は、前記金属凹凸構造を定めるための金属部本体を形成し、前記金属部本体の表面に当該金属部本体より電界増強度の大きい金属からなる金属層を形成するものであってもよい。 In the metal part forming step, a metal part main body for defining the metal concavo-convex structure is formed, and a metal layer made of a metal having a higher electric field strength than the metal part main body is formed on the surface of the metal part main body. It may be.
　また、前記誘電体部形成工程は、前記誘電体凹凸構造のピッチを２００ｎｍ以下となるように形成するものである方が好ましい。 Further, it is preferable that the dielectric part forming step is such that the pitch of the dielectric uneven structure is formed to be 200 nm or less.
　また、前記金属部形成工程は、前記金属部の垂直方向の成長速度が大きくなる成膜技術を用いて膜厚をコントロールする方がよい。例えば、前記誘電体凹凸構造上にスパッタリングによって金属部を形成するものがある。また、前記金属部形成工程は、前記スパッタリングによって形成した金属部上に、更に無電界メッキを施して金属部の形状を調節するものであってもよい。 In the metal part forming step, it is preferable to control the film thickness by using a film forming technique that increases the growth rate in the vertical direction of the metal part. For example, there is one in which a metal portion is formed by sputtering on the dielectric uneven structure. In the metal part forming step, the metal part formed by the sputtering may be further subjected to electroless plating to adjust the shape of the metal part.
　また、前記誘電体部上に前記誘電体部と前記金属部の密着性を向上するための中間層を形成する中間層形成工程を有してもよい。 Further, an intermediate layer forming step of forming an intermediate layer for improving the adhesion between the dielectric portion and the metal portion on the dielectric portion may be provided.
　また、前記金属部に、特定の物質を固定可能な固定物質を結合させる固定物質結合工程を有してもよい。 Further, the metal part may have a fixed substance binding step of binding a fixed substance capable of fixing a specific substance.
　前記金属部の表面に、親水性を有する親水性層を形成する親水性層形成工程を有するものでもよい。 It may have a hydrophilic layer forming step of forming a hydrophilic layer having hydrophilicity on the surface of the metal part.
　また、本発明のアミノ酸配列決定方法は、ペプチド又はタンパク質のＮ末端又はＣ末端からアミノ酸を逐次的に分解する逐次分解工程と、前記逐次分解工程により遊離したアミノ酸を分取する分取工程と、前記分取工程により得られたアミノ酸を本発明の電磁波増強素子を用いて分析する分析工程と、を有することを特徴とする。 The amino acid sequence determination method of the present invention includes a sequential decomposition step of sequentially decomposing amino acids from the N-terminus or C-terminus of a peptide or protein, a fractionation step of fractionating amino acids released by the sequential decomposition step, And an analysis step of analyzing the amino acid obtained by the fractionation step using the electromagnetic wave enhancing element of the present invention.
　この場合、前記逐次分解工程は、プロテアーゼを用いて行えばよい。また、前記逐次分解工程は、前記プロテアーゼ又は前記ペプチド若しくは前記タンパク質を固定化したカラムを用いて行えばよい。 In this case, the sequential decomposition step may be performed using a protease. The sequential decomposition step may be performed using a column on which the protease, the peptide, or the protein is immobilized.
　本発明の電磁波増強素子は、金属凹凸構造の形状を最適化することにより、大きな電場増強をもたらすことができる。 The electromagnetic wave enhancing element of the present invention can bring about a large electric field enhancement by optimizing the shape of the metal concavo-convex structure.
本発明の電磁波増強素子を示す断面図である。It is sectional drawing which shows the electromagnetic wave enhancing element of the present invention. 本発明の電磁波増強素子を示す断面図である。It is sectional drawing which shows the electromagnetic wave enhancing element of the present invention. 本発明の別の電磁波増強素子を示す断面図である。It is sectional drawing which shows another electromagnetic wave enhancing element of this invention. 本発明の別の電磁波増強素子の誘電体部を示す平面図である。It is a top view which shows the dielectric material part of another electromagnetic wave enhancing element of this invention. 本発明の中間層を有する電磁波増強素子を示す断面図である。It is sectional drawing which shows the electromagnetic wave enhancement element which has an intermediate | middle layer of this invention. 本発明の電磁波増強素子の製造方法を示す断面図である。It is sectional drawing which shows the manufacturing method of the electromagnetic wave enhancement element of this invention. 本発明の電磁波増強素子を示す平面写真である。It is a plane photograph which shows the electromagnetic wave enhancing element of the present invention. 本発明の製造方法を説明するための電磁波増強素子の断面図および断面写真である。It is sectional drawing and the cross-sectional photograph of the electromagnetic wave enhancing element for demonstrating the manufacturing method of this invention. シミュレーション１の結果を示すグラフである。It is a graph which shows the result of simulation 1. シミュレーション２の結果を示すグラフである。It is a graph which shows the result of simulation 2. シミュレーション３の結果を示すグラフである。It is a graph which shows the result of simulation 3. 試料１の電磁波増強素子を示す（ａ）平面写真および（ｂ）断面写真である。It is (a) plane photograph and (b) cross-sectional photograph which show the electromagnetic wave enhancing element of Sample 1. 試料２の電磁波増強素子を示す（ａ）平面写真および（ｂ）断面写真である。It is (a) plane photograph and (b) cross-sectional photograph which show the electromagnetic wave enhancing element of Sample 2. 試料３の電磁波増強素子を示す（ａ）平面写真および（ｂ）断面写真である。It is (a) plane photograph and (b) cross-sectional photograph which show the electromagnetic wave enhancing element of Sample 3. 比較試料１の電磁波増強素子を示す（ａ）平面写真および（ｂ）断面写真である。It is (a) plane photograph and (b) cross-sectional photograph which show the electromagnetic wave enhancing element of Comparative Sample 1. 比較試料２の電磁波増強素子を示す（ａ）平面写真および（ｂ）断面写真である。It is (a) plane photograph and (b) cross-sectional photograph which show the electromagnetic wave enhancing element of Comparative Sample 2. 試料１～３の方位角とラマン強度の関係を示すグラフである。6 is a graph showing the relationship between the azimuth angle of samples 1 to 3 and the Raman intensity. 本発明の電磁波増強素子に係るアライメントマークを示す平面図である。It is a top view which shows the alignment mark which concerns on the electromagnetic wave enhancement element of this invention. 試料１～３および比較試料１，２のラマン強度の最大値を示すグラフである。6 is a graph showing the maximum Raman intensity of Samples 1 to 3 and Comparative Samples 1 and 2. 試料４～６の間隙の大きさとラマン強度の関係を示すグラフである。6 is a graph showing the relationship between the size of the gap between samples 4 to 6 and the Raman intensity. 電磁波増強素子を示す断面図である。It is sectional drawing which shows an electromagnetic wave enhancing element. 本発明の別の電磁波増強素子を示す断面図である。It is sectional drawing which shows another electromagnetic wave enhancing element of this invention. 本発明の別の電磁波増強素子を示す断面図である。It is sectional drawing which shows another electromagnetic wave enhancing element of this invention. 本発明の電磁波増強素子を示す断面図である。It is sectional drawing which shows the electromagnetic wave enhancing element of the present invention. 本発明の別の電磁波増強素子を示す断面図である。It is sectional drawing which shows another electromagnetic wave enhancing element of this invention.
　以下に、本発明の電磁波増強素子について説明する。本発明の電磁波増強素子は、図１に示すように、誘電体凹凸構造11が形成された誘電体部１と、底部側に向かって幅の小さくなる凹部22を一方向に延伸した形状の金属凹凸構造21を誘電体凹凸構造11上に有する金属部２と、で主に構成される。ここで、十分に強い電場増強効果を得るためには、凹部22は、当該凹部22を形成する隣接する凸部同士の間隙の最小幅が２５ｎｍ以下、好ましくは２０ｎｍ以下である方がよい。なお、凹部22は、図１（ａ）に示すように、凹部22を形成する隣接する凸部24Ａ，24Ｂが接している方が好ましいが、電界を十分に増強できる範囲であれば、図１（ｂ）に示すように隙間が空いていてもよい。また、図１（ａ）では、誘電体部１と金属部２の間に隙間はないが、電界を十分に増強できれば、図１（ｃ）に示すように誘電体部１と金属部２の間に空隙29があってもよい。 Hereinafter, the electromagnetic wave enhancing element of the present invention will be described. As shown in FIG. 1, the electromagnetic wave enhancing element of the present invention is a metal having a shape in which a dielectric portion 1 in which a dielectric concavo-convex structure 11 is formed and a concave portion 22 whose width decreases toward the bottom side is extended in one direction. And the metal part 2 having the concavo-convex structure 21 on the dielectric concavo-convex structure 11. Here, in order to obtain a sufficiently strong electric field enhancing effect, the recess 22 should have a minimum gap width of 25 nm or less, preferably 20 nm or less, between adjacent protrusions forming the recess 22. As shown in FIG. 1A, the recess 22 is preferably in contact with adjacent projections 24A and 24B forming the recess 22, but as long as the electric field can be sufficiently enhanced, FIG. As shown in (b), there may be a gap. Further, in FIG. 1A, there is no gap between the dielectric part 1 and the metal part 2, but if the electric field can be sufficiently enhanced, the dielectric part 1 and the metal part 2 can be seen as shown in FIG. There may be a gap 29 between them.
　金属部２は、電界を集中させる凹部22を構成する金属凹凸構造21を有する。金属凹凸構造21は、凹部22を一方向に延伸した形状、すなわち、平面図で直線状に形成される。この金属凹凸構造21に所定波長λの電磁波が入射すると、当該金属凹凸構造21の凹部22の極めて狭い領域に強い電界が集中する。この電界増強度の最大値を示す部分を利用してＳＥＲＳの信号を取り出すことができる。 The metal part 2 has a metal concavo-convex structure 21 constituting a concave part 22 for concentrating the electric field. The metal concavo-convex structure 21 is formed in a shape obtained by extending the recess 22 in one direction, that is, linearly in a plan view. When an electromagnetic wave having a predetermined wavelength λ is incident on the metal concavo-convex structure 21, a strong electric field is concentrated in a very narrow region of the concave portion 22 of the metal concavo-convex structure 21. The SERS signal can be taken out using the portion showing the maximum value of the electric field enhancement intensity.
　ここで、図２に示すように、凹部22を形成する隣接する凸部24Ａ，24Ｂの同じ高さの点（図中では凹部22の最下点23）における接線25Ａ，25Ｂ同士のなす角度をθとすると、電界増強度の最大値Ｅｘが大きい金属凹凸構造21は、角度θの最小値が２０度以下である場合に多い傾向があった。特に、電界増強度の最大値Ｅｘが大きい金属凹凸構造21は、０°＜θ＜１５°である場合に多い傾向があり、７°＜θ＜１３°である場合に更に多い傾向があった。なお、θが最小値をとる接線の凸部24との接点は、凸部24の高さ（凸部24の最下点23を基準とした凸部24の最上点26の高さ）の半分より下側にある方が良く、好ましくは凸部24の高さの４分の１より下側にある方が良く、更に好ましくは最下点23にあるのがよい。したがって、凸部24の形状は、図２に示すように、凸部24の表面が凹部22側に膨らんだ曲線状である方が好ましい。角度θの測定は、金属凹凸構造21の延伸方向に垂直な断面写真を撮影し、それを画像解析することによって求めればよい。 Here, as shown in FIG. 2, the angle formed between the tangents 25A and 25B at the same height point (the lowest point 23 of the concave portion 22 in the figure) of the adjacent convex portions 24A and 24B forming the concave portion 22 is defined. Assuming that θ, the metal concavo-convex structure 21 having a large maximum value Ex of the electric field enhancement strength tends to be large when the minimum value of the angle θ is 20 degrees or less. In particular, the metal concavo-convex structure 21 having a large maximum value Ex of the electric field enhancement strength tends to increase when 0 ° <θ <15 °, and tends to increase when 7 ° <θ <13 °. . Note that the point of contact with the convex portion 24 of the tangent where θ is the minimum value is half of the height of the convex portion 24 (the height of the highest point 26 of the convex portion 24 with respect to the lowest point 23 of the convex portion 24). It is better to be on the lower side, preferably lower than a quarter of the height of the convex portion 24, more preferably at the lowest point 23. Therefore, it is preferable that the shape of the convex portion 24 is a curved shape in which the surface of the convex portion 24 swells toward the concave portion 22 as shown in FIG. The angle θ may be measured by taking a cross-sectional photograph perpendicular to the extending direction of the metal concavo-convex structure 21 and analyzing the image.
　また、図２に示すように、金属凹凸構造21の凸部24の最上点26の垂直方向の厚みをＡ、最下点23の垂直方向の厚みをＢとすると、電界増強度の最大値Ｅｘが大きい金属凹凸構造21は、膜厚Ａが膜厚Ｂより大きい場合に多い傾向があった。特に、電界増強度の最大値Ｅｘが大きい金属凹凸構造21は、０．９Ａ＞Ｂである場合に多い傾向があり、０．６Ａ＞Ｂである場合に更に多い傾向があった。 As shown in FIG. 2, when the vertical thickness of the uppermost point 26 of the convex portion 24 of the metal concavo-convex structure 21 is A and the vertical thickness of the lowest point 23 is B, the maximum value Ex of the electric field enhancement intensity Ex When the film thickness A is larger than the film thickness B, the metal concavo-convex structure 21 having a large thickness tends to be large. In particular, the metal concavo-convex structure 21 having a large maximum value Ex of the electric field enhancement strength tends to increase when 0.9A> B, and further tends to increase when 0.6A> B.
　また、図２に示すように、金属凹凸構造21の凸部24の最上点26の垂直方向の厚みをＡ、誘電体凹凸構造11の凹部12の底部の幅の半分をＣとすると、電界増強度の最大値Ｅｘが大きい金属凹凸構造21は、膜厚ＡがＣより大きい場合に多い傾向があった。特に、電界増強度の最大値Ｅｘが大きい金属凹凸構造21は、Ａ＞２Ｃである場合に多い傾向があり、Ａ＞３．５Ｃである場合に更に多い傾向があった。なお、図１（ｂ）に示すように、金属凹凸構造21の凹部22に隙間が空いている場合や、図１（ｃ）に示すように、誘電体部１と金属部２の間に空隙29がある場合であっても同様に、Ａ＞Ｃである方が良く、好ましくはＡ＞２Ｃである方が良く、更に好ましくはＡ＞３．５Ｃである方がよい。 As shown in FIG. 2, when the vertical thickness of the uppermost point 26 of the convex portion 24 of the metal concavo-convex structure 21 is A and the half width of the bottom of the concave portion 12 of the dielectric concavo-convex structure 11 is C, the electric field enhancement When the film thickness A is larger than C, the metal uneven structure 21 having a large maximum value Ex tends to increase. In particular, the metal concavo-convex structure 21 having a large maximum value Ex of the electric field enhancement strength tends to increase when A> 2C, and further tends to increase when A> 3.5C. In addition, as shown in FIG. 1B, a gap is formed in the concave portion 22 of the metal concavo-convex structure 21, or a gap is formed between the dielectric portion 1 and the metal portion 2 as shown in FIG. Even in the case of 29, it is better that A> C, preferably A> 2C, and more preferably A> 3.5C.
　金属凹凸構造21のその他の形状、例えばピッチや凸部の幅、凸部のアスペクト比等は、電磁場増強をもたらすことができるものであればどのようなものでもよい。例えば、金属凹凸構造21の凸部24のピッチＰは１０００ｎｍ以下、好ましくは２００ｎｍ以下、更に好ましくは１２０ｎｍ以下とすればよい。 Other shapes of the metal concavo-convex structure 21, such as the pitch, the width of the convex portion, the aspect ratio of the convex portion, and the like may be any as long as the electromagnetic field can be enhanced. For example, the pitch P of the convex portions 24 of the metal concavo-convex structure 21 may be 1000 nm or less, preferably 200 nm or less, and more preferably 120 nm or less.
　金属部２の材質は、電磁波を反射するものであればどのようなものでもよいが、例えば、金、銀、銅、クロム、アルミニウム、プラチナ、タングステン等の金属を用いることができる。また、これらの組み合わせであってもよい。 The material of the metal part 2 may be any material as long as it reflects electromagnetic waves. For example, metals such as gold, silver, copper, chromium, aluminum, platinum, and tungsten can be used. Moreover, these combinations may be sufficient.
　また、金属部２は、単一の金属からなるものである必要はなく、金属凹凸構造を定めるための金属部本体と、金属部本体の表面に形成された異なる金属からなる金属層と、で構成してもよい。例えば、金属部２の材質によっては、電界増強度は大きいが金属凹凸構造21を形成するのが難しいものがある。この場合、金属部２は、図２２に示すように、金属凹凸構造21を定めるための金属部本体２Ａと、当該金属部本体２Ａの表面に形成され、金属部本体２Ａより電界強度の大きい金属からなる金属層２Ｂと、で構成することができる。この場合、金属層は１ｎｍ以上とするのが好ましい。 Moreover, the metal part 2 does not need to consist of a single metal, and a metal part body for defining a metal uneven structure and a metal layer made of different metals formed on the surface of the metal part body. It may be configured. For example, depending on the material of the metal part 2, there are some that have a large electric field enhancement but are difficult to form the metal relief structure 21. In this case, as shown in FIG. 22, the metal part 2 is formed on the surface of the metal part main body 2A for defining the metal uneven structure 21 and the metal part main body 2A, and has a higher electric field strength than the metal part main body 2A. And a metal layer 2B made of In this case, the metal layer is preferably 1 nm or more.
　誘電体部１は、図１に示すように、金属凹凸構造21の形状やピッチを制御するための誘電体凹凸構造11を有するものである。誘電体部１の材料としては、誘電体凹凸構造11を形成できる誘電体であればどのようなものでもよいが、例えば、アクリル系樹脂や環状オレフィン系樹脂等の樹脂や、ケイ素（Ｓｉ）や二酸化ケイ素（ＳｉＯ２）等の無機化合物を用いることができる。誘電体凹凸構造11をインプリント法によって成形するなら、当該インプリント方に適した光硬化性樹脂や熱可塑性樹脂などを用いればよい。 As shown in FIG. 1, the dielectric portion 1 has a dielectric concavo-convex structure 11 for controlling the shape and pitch of the metal concavo-convex structure 21. The material of the dielectric part 1 may be any dielectric material that can form the dielectric concavo-convex structure 11. For example, a resin such as an acrylic resin or a cyclic olefin resin, silicon (Si), An inorganic compound such as silicon dioxide (SiO 2 ) can be used. If the dielectric concavo-convex structure 11 is formed by the imprint method, a photocurable resin or a thermoplastic resin suitable for the imprint method may be used.
　誘電体凹凸構造11の形状は、少なくとも底部側に向かって幅の小さくなる凹部22を有するように金属凹凸構造21の形状を制御できるものであればよい。また、誘電体凹凸構造11の形状は、好ましくは、上述した角度θの最小値が２０度以下となるように金属凹凸構造21の形状を制御できるものがよい。例えば、金属凹凸構造21の延伸方向に垂直な断面において、誘電体凹凸構造11の凸部14表面が凹部22側に膨らんだ曲線状のものとすることができる。もちろん、金属凹凸構造21の形状を制御できるものであれば、断面が台形、長方形、正方形等の四角形状のもの、三角形状のもの等を用いてもよい。また、誘電体凹凸構造11のピッチは、金属凹凸構造21のピッチＰと同じピッチにすればよい。 The shape of the dielectric concavo-convex structure 11 may be any shape as long as the shape of the metal concavo-convex structure 21 can be controlled so as to have a concave portion 22 whose width decreases toward at least the bottom side. The shape of the dielectric concavo-convex structure 11 is preferably one that can control the shape of the metal concavo-convex structure 21 so that the minimum value of the angle θ described above is 20 degrees or less. For example, in the cross section perpendicular to the extending direction of the metal concavo-convex structure 21, the surface of the convex portion 14 of the dielectric concavo-convex structure 11 can be a curved shape swelled toward the concave portion 22 side. Of course, as long as the shape of the metal concavo-convex structure 21 can be controlled, a cross section having a trapezoidal shape, a rectangular shape such as a rectangle or a square, a triangular shape, or the like may be used. The pitch of the dielectric concavo-convex structure 11 may be the same as the pitch P of the metal concavo-convex structure 21.
　誘電体凹凸構造11のその他の形状、例えば凸部14の幅やアスペクト比等は、電磁場増強をもたらすことができるように金属凹凸構造21を制御できる形状であればどのようなものでもよい。例えば、誘電体凹凸構造11の凸部14の幅は、ピッチの３０～６０％とすればよい。また、誘電体凹凸構造11の凸部14のアスペクト比は１以上が好ましい。 The other shapes of the dielectric concavo-convex structure 11 may be any shape as long as the metal concavo-convex structure 21 can be controlled so that the electromagnetic field can be enhanced, for example, the width and aspect ratio of the convex portion 14. For example, the width of the convex portion 14 of the dielectric concavo-convex structure 11 may be 30 to 60% of the pitch. The aspect ratio of the convex portion 14 of the dielectric concavo-convex structure 11 is preferably 1 or more.
　ここで、電磁波の増強度は、電界の４乗に比例するため、金属凹凸構造21の形状のわずかな差で電界は大きく異なる。一方、金属凹凸構造21の形状を完全に制御して形成するのは難しいという問題がある。したがって、金属凹凸構造21は、電磁波を増強できる形状が含まれるように、十分な数の凹部22が形成されている方が好ましい。また、電磁波増強素子上に形成される誘電体凹凸構造11は、図３に示すように、複数種類の形状からなるものとしてもよい。これにより、誘電体凹凸構造11が複数種類の形状を有しているため、形成される金属凹凸構造21も複数種類の形状からなることになり、電磁波を増強できる金属凹凸構造21を確実に形成することができる。また、電磁波の増強度は、電界の４乗に比例するため、電磁波増強素子上にわずかでも適した形状の金属凹凸構造21があれば、十分に電磁波を増強することができる。なお、電磁波増強素子上に形成される誘電体凹凸構造11は、複数の領域を有し、誘電体凹凸構造11は当該領域ごとに形状が異なるように形成してもよい。例えば、図４に示す誘電体部１は、誘電体凹凸構造11をラインアンドスペース状とし、図中右の領域に行くほど誘電体凹凸構造11の凸部のピッチを大きくし、下の領域に行くほど凸部の線幅を小さくした領域91～99を形成したものである。 Here, since the enhancement of electromagnetic waves is proportional to the fourth power of the electric field, the electric field varies greatly depending on a slight difference in the shape of the metal concavo-convex structure 21. On the other hand, there is a problem that it is difficult to form the metal uneven structure 21 with complete control. Therefore, it is preferable that the metal concavo-convex structure 21 has a sufficient number of recesses 22 so as to include a shape capable of enhancing electromagnetic waves. Further, the dielectric concavo-convex structure 11 formed on the electromagnetic wave enhancing element may have a plurality of types of shapes as shown in FIG. As a result, since the dielectric concavo-convex structure 11 has a plurality of types of shapes, the metal concavo-convex structure 21 to be formed also has a plurality of types of shapes, and the metal concavo-convex structure 21 that can enhance electromagnetic waves is reliably formed. can do. In addition, since the enhancement of electromagnetic waves is proportional to the fourth power of the electric field, the electromagnetic wave can be sufficiently enhanced if there is a metal uneven structure 21 having a suitable shape on the electromagnetic wave enhancing element. The dielectric concavo-convex structure 11 formed on the electromagnetic wave enhancing element may have a plurality of regions, and the dielectric concavo-convex structure 11 may be formed so as to have a different shape for each region. For example, the dielectric portion 1 shown in FIG. 4 has a dielectric concavo-convex structure 11 in a line-and-space shape, and the pitch of the convex portion of the dielectric concavo-convex structure 11 is increased toward the right region in the figure, and the lower region is formed. Regions 91 to 99 are formed in which the line width of the convex portion decreases as the distance increases.
　また、金属凹凸構造21は、検出したい特定の物質を確実に検出するために、図２３に示すように、当該物質を固定するための固定物質５が結合されている方が好ましい。例えば、金属部２の表面と化学結合を形成でき、かつ、固定したい物質と化学結合又は化学吸着が可能な官能基を有するカップリング剤等で金属部２の表面処理をすればよい。当該表面処理は、膜厚を５ｎｍ以下、好ましくは１ｎｍ以下で被覆する方がよい。 Further, in order to reliably detect a specific substance to be detected, the metal concavo-convex structure 21 is preferably bonded with a fixing substance 5 for fixing the substance as shown in FIG. For example, the metal part 2 may be surface-treated with a coupling agent or the like having a functional group capable of forming a chemical bond with the surface of the metal part 2 and capable of chemical bonding or chemical adsorption with a substance to be fixed. In the surface treatment, the film thickness is preferably 5 nm or less, preferably 1 nm or less.
　具体的な例としては、アミノ酸の分析用に本発明の電磁波増強素子を製造する場合には、図２３に示すように、アミノ酸のアミノ基又はカルボキシル基と結合又は吸着可能な官能基を有するシランカップリング剤で金属部２の表面処理を行えばよい。 As a specific example, when producing the electromagnetic wave enhancing element of the present invention for amino acid analysis, as shown in FIG. 23, a silane having a functional group capable of binding or adsorbing to an amino group or a carboxyl group of an amino acid. What is necessary is just to perform the surface treatment of the metal part 2 with a coupling agent.
　また、金属部２を銀で作製した場合、図２４に示すように、ローダミンなど水溶性の試薬６は銀表面ではじかれて、ラマン増強が強く生じるホットスポットに到達しない場合がある。このような場合には、金属部２の表面に、少なくとも金属部２より親水性の高い材料からなる親水性層を形成してもよい。例えば、図２５に示すように、金属部２の表面に、酸化ケイ素（ＳｉＯ２）などの親水性の誘電体材料からなる親水性層７を被覆すれば、当該試薬をホットスポットに到着させることができ、高感度を得ることができる。当該親水性層７は、膜厚を５ｎｍ以下、好ましくは１ｎｍ以下で被覆する方がよい。 When the metal part 2 is made of silver, as shown in FIG. 24, the water-soluble reagent 6 such as rhodamine may be repelled on the silver surface and may not reach a hot spot where Raman enhancement is strong. In such a case, a hydrophilic layer made of a material that is at least more hydrophilic than the metal part 2 may be formed on the surface of the metal part 2. For example, as shown in FIG. 25, when the surface of the metal part 2 is covered with a hydrophilic layer 7 made of a hydrophilic dielectric material such as silicon oxide (SiO 2 ), the reagent reaches the hot spot. And high sensitivity can be obtained. The hydrophilic layer 7 should be coated with a film thickness of 5 nm or less, preferably 1 nm or less.
　また、誘電体部１と金属部２の間には、図５に示すように、誘電体部１と金属部２の密着性を向上するための中間層３を有してもよい。中間層３は誘電体部１と金属部２の密着性を向上させるものならどのようなものでもよいが、例えばプラチナを用いることができる。 Moreover, between the dielectric part 1 and the metal part 2, as shown in FIG. 5, you may have the intermediate | middle layer 3 for improving the adhesiveness of the dielectric part 1 and the metal part 2. As shown in FIG. The intermediate layer 3 may be any material that improves the adhesion between the dielectric portion 1 and the metal portion 2, and for example, platinum can be used.
　次に、本発明の電磁波増強素子の製造方法について図６を用いて説明する。本発明の電磁波増強素子の製造方法は、誘電体部１に誘電体凹凸構造11を形成する誘電体部形成工程と、当該誘電体凹凸構造11上に金属凹凸構造21を構成する金属部２を形成する金属部形成工程と、で主に構成される。 Next, a method for manufacturing the electromagnetic wave enhancing element of the present invention will be described with reference to FIG. The method for manufacturing an electromagnetic wave enhancing element according to the present invention includes a dielectric part forming step for forming a dielectric concavo-convex structure 11 on a dielectric part 1 and a metal part 2 constituting a metal concavo-convex structure 21 on the dielectric concavo-convex structure 11. And a metal part forming step to be formed.
　誘電体部形成工程は、金属凹凸構造21の形状やピッチを制御するための誘電体凹凸構造11を形成するためのものである。誘電体部形成工程は、所定の誘電体凹凸構造11を形成できるものであればどのような方法で行ってもよい。例えば、凸部14の表面が凹部側に膨らんだ曲線状のラインアンドスペースからなる誘電体凹凸構造11を形成する方法を説明する。まず、図６（ａ）に示すように、誘電体からなる基材10を用意する。当該基材10上に、図６（ｂ）に示すように、インプリント法によって誘電体凹凸構造15を形成する。次に、図６（ｃ）に示すように、誘電体凹凸構造15に樹脂を塗布して、誘電体凹凸構造15の凹部22に逆アーチ状のマスク16を形成する。次に、図６（ｄ）に示すように、ＵＶオゾン照射を行うことにより誘電体凹凸構造11を形成すればよい。なお、これらは一例であって、その他の技術、例えばフォトリソグラフィー技術やエッチング技術等の従来から知られる微細加工技術を組み合わせて用いてもよい。 The dielectric part forming step is for forming the dielectric concavo-convex structure 11 for controlling the shape and pitch of the metal concavo-convex structure 21. The dielectric part forming step may be performed by any method as long as the predetermined dielectric uneven structure 11 can be formed. For example, a method of forming the dielectric concavo-convex structure 11 composed of a curved line and space in which the surface of the convex portion 14 bulges toward the concave portion will be described. First, as shown in FIG. 6A, a base material 10 made of a dielectric is prepared. As shown in FIG. 6B, a dielectric concavo-convex structure 15 is formed on the base material 10 by imprinting. Next, as shown in FIG. 6C, a resin is applied to the dielectric concavo-convex structure 15 to form a reverse arched mask 16 in the concave portion 22 of the dielectric concavo-convex structure 15. Next, as shown in FIG. 6D, the dielectric uneven structure 11 may be formed by performing UV ozone irradiation. These are merely examples, and other techniques, for example, a conventionally known microfabrication technique such as a photolithography technique or an etching technique may be used in combination.
　金属部形成工程は、誘電体凹凸構造11上に、底部側に向かって幅の小さくなる凹部22を一方向に延伸した形状（平面図で直線状）の金属凹凸構造21を有する金属部２を形成するものである。ここで、十分に強い電場増強効果を得るためには、凹部22は、当該凹部22を形成する隣接する凸部同士の間隙の最小幅が２５ｎｍ以下、好ましくは２０ｎｍ以下となるように形成する方がよい。なお、凹部22は、図１（ａ）に示すように、凹部22を形成する隣接する凸部24Ａ，24Ｂが接している方が好ましいが、電界を十分に増強できる範囲であれば、図１（ｂ）に示すように隙間が空いていてもよい。また、図１（ａ）では、誘電体部１と金属部２の間に隙間はないが、電界を十分に増強できれば、図１（ｃ）に示すように誘電体部１と金属部２の間に空隙29があってもよい。金属部形成工程は、金属凹凸構造21を構成する金属部２を形成できればどのようなものでも良く、例えば、スパッタリングや蒸着、めっき等の成膜技術を用いればよい。 In the metal part forming step, the metal part 2 having the metal concave-convex structure 21 having a shape (linear in a plan view) in which the concave part 22 whose width decreases toward the bottom side is extended in one direction on the dielectric concave-convex structure 11 is formed. To form. Here, in order to obtain a sufficiently strong electric field enhancing effect, the concave portion 22 is formed such that the minimum width of the gap between adjacent convex portions forming the concave portion 22 is 25 nm or less, preferably 20 nm or less. Is good. As shown in FIG. 1A, the recess 22 is preferably in contact with adjacent projections 24A and 24B forming the recess 22, but as long as the electric field can be sufficiently enhanced, FIG. As shown in (b), there may be a gap. Further, in FIG. 1A, there is no gap between the dielectric part 1 and the metal part 2, but if the electric field can be sufficiently enhanced, the dielectric part 1 and the metal part 2 can be seen as shown in FIG. There may be a gap 29 between them. The metal part forming step may be any process as long as the metal part 2 constituting the metal uneven structure 21 can be formed. For example, a film forming technique such as sputtering, vapor deposition, or plating may be used.
　また、金属部形成工程は、凹部22を形成する隣接する凸部24の同じ高さの点における接線同士のなす角度をθとすると、θの最小値が２０度以下、好ましくは０°＜θ＜１５°、更に好ましくは７°＜θ＜１３°となるように凹部22を形成する方がよい。 Further, in the metal part forming step, when the angle formed by tangents at the same height point of the adjacent convex part 24 forming the concave part 22 is θ, the minimum value of θ is 20 degrees or less, preferably 0 ° <θ. It is better to form the recess 22 so that <15 °, more preferably 7 ° <θ <13 °.
　角度θの最小値が２０度以下になるように制御するためには、例えば、図６（ｅ）に示すように、金属部２の垂直方向の成長速度が大きくなるような成膜技術、例えば異方性のスパッタリングを利用して金属部２を形成すればよい。また、図６（ｆ）に示すように、スパッタリングによって形成した金属部２上に、更に無電界メッキを施して金属部２の形状を調節してもよい。これにより、角度θの最小値を調節することができる。 In order to control the minimum value of the angle θ to be 20 degrees or less, for example, as shown in FIG. 6E, a film forming technique that increases the vertical growth rate of the metal part 2, for example, What is necessary is just to form the metal part 2 using anisotropic sputtering. Moreover, as shown in FIG.6 (f), on the metal part 2 formed by sputtering, electroless plating may be given further and the shape of the metal part 2 may be adjusted. Thereby, the minimum value of the angle θ can be adjusted.
　また、金属凹凸構造21の角度θを制御するよりは、金属凹凸構造21の金属部２の膜厚を制御する方が容易である。したがって、金属部形成工程は、金属凹凸構造21の凸部24の最上点26の垂直方向の厚みをＡ、最下点23の垂直方向の厚みをＢとすると、Ａ＞Ｂとなるように金属部２を形成してもよい。より好ましくは０．９Ａ＞Ｂとなるように金属部２を形成する方が良く、更に好ましくは０．６Ａ＞Ｂとなるように金属部２を形成する方がよい。 Further, it is easier to control the film thickness of the metal portion 2 of the metal uneven structure 21 than to control the angle θ of the metal uneven structure 21. Therefore, in the metal part forming step, when the vertical thickness of the uppermost point 26 of the convex part 24 of the metal concavo-convex structure 21 is A and the vertical thickness of the lowermost point 23 is B, the metal is formed so that A> B. Part 2 may be formed. More preferably, the metal part 2 is formed so that 0.9A> B, and more preferably, the metal part 2 is formed so that 0.6A> B.
　また、金属部形成工程は、金属凹凸構造21の凸部24の最上点26の垂直方向の厚みをＡ、誘電体凹凸構造の凹部12の底部の幅の半分をＣとすると、Ａ＞Ｃとなるように金属部２を形成してもよい。より好ましくはＡ＞２Ｃとなるように金属部２を形成する方が良く、更に好ましくはＡ＞３．５Ｃとなるように金属部２を形成する方がよい。この場合、Ｃの大きさは、誘電体部形成工程で形成する誘電体凹凸構造11の凹部の幅を調節して行えばよい。 Further, in the metal part forming step, when the vertical thickness of the uppermost point 26 of the convex part 24 of the metal concave-convex structure 21 is A and half of the bottom width of the concave part 12 of the dielectric concave-convex structure C is A> C, You may form the metal part 2 so that it may become. More preferably, the metal part 2 is formed so that A> 2C, and more preferably, the metal part 2 is formed so that A> 3.5C. In this case, the size of C may be determined by adjusting the width of the concave portion of the dielectric concavo-convex structure 11 formed in the dielectric portion forming step.
　また、金属部２の材質によっては、電界増強度は大きいが金属凹凸構造21を形成するのが難しいものもある。例えば、金はマイグレーションが小さいため、スパッタ成膜を行うと成膜形状が原子の到達量に１対１で対応することになる。すると、隣接する金が接触するときに、図２１に示すように、緩やかな接触角度をとり易く、電界増強度が小さくなるという問題がある。この場合、金属部形成工程は、図２２に示すように、まず、金属凹凸構造21を形成し易い金属を用いて、金属凹凸構造21を定めるための金属部本体を形成する。次に、金属部本体の表面に当該金属部本体とは異なる金属、例えば、当該金属部本体より電界強度の大きい金属からなる金属層を形成すればよい。なお、金属層は１ｎｍ以上とするのが好ましい。 Depending on the material of the metal part 2, there are some which have a large electric field enhancement strength but it is difficult to form the metal uneven structure 21. For example, since migration of gold is small, when the sputter film formation is performed, the film formation shape corresponds to the arrival amount of atoms on a one-to-one basis. Then, when adjacent gold contacts, as shown in FIG. 21, there is a problem that it is easy to take a gentle contact angle and the electric field enhancement intensity becomes small. In this case, in the metal part forming step, as shown in FIG. 22, first, a metal part main body for defining the metal uneven structure 21 is formed using a metal that can easily form the metal uneven structure 21. Next, a metal layer made of a metal different from the metal part body, for example, a metal having a higher electric field strength than the metal part body may be formed on the surface of the metal part body. The metal layer is preferably 1 nm or more.
　このようにして凸部と凹部を一方向に延伸した金属凹凸構造を有する電磁波増強素子が製造できる（図７参照）。 Thus, an electromagnetic wave enhancing element having a metal concavo-convex structure in which the convex portion and the concave portion are extended in one direction can be manufactured (see FIG. 7).
　また、誘電体部形成工程と金属部形成工程の間に、図８（ａ）に示すように、誘電体部１上に誘電体部１と金属部２の密着性を向上するための中間層３を形成する中間層形成工程を有していてもよい。中間層３形成工程はどのような方法で行ってもよいが、スパッタリングや蒸着、めっき等の成膜技術が挙げられる。この場合、金属部形成工程では、図８（ｂ）に示すように、金属部２の垂直方向の成長速度が大きくなるような成膜技術、例えば異方性のスパッタリングを利用して金属部２を形成すればよい。また、図８（ｃ）に示すように、スパッタリングによって形成した金属部２上に、更に無電界メッキを施して金属部２の形状を調節してもよい。 Further, as shown in FIG. 8A, an intermediate layer for improving the adhesion between the dielectric part 1 and the metal part 2 on the dielectric part 1 between the dielectric part forming step and the metal part forming step. 3 may be included. The intermediate layer 3 formation step may be performed by any method, and examples thereof include film formation techniques such as sputtering, vapor deposition, and plating. In this case, in the metal part forming step, as shown in FIG. 8B, the metal part 2 is utilized by using a film forming technique that increases the vertical growth rate of the metal part 2, for example, anisotropic sputtering. May be formed. Moreover, as shown in FIG.8 (c), you may adjust the shape of the metal part 2 by giving an electroless plating further on the metal part 2 formed by sputtering.
　また、図２３に示すように、金属部２に、特定の物質を固定可能な固定物質を結合させる固定物質結合工程を有してもよい。例えば、金属部２の表面と化学結合を形成でき、かつ、固定したい特定物質と化学結合又は化学吸着が可能な官能基を有するカップリング剤等で金属部２の表面処理をすればよい。当該表面処理は、膜厚を５ｎｍ以下、好ましくは１ｎｍ以下で被覆する方がよい。 Further, as shown in FIG. 23, the metal part 2 may have a fixed substance binding step of binding a fixed substance capable of fixing a specific substance. For example, the metal part 2 may be surface-treated with a coupling agent or the like having a functional group capable of forming a chemical bond with the surface of the metal part 2 and capable of chemical bonding or chemical adsorption with a specific substance to be fixed. In the surface treatment, the film thickness is preferably 5 nm or less, preferably 1 nm or less.
　具体的な例としては、アミノ酸の分析用に本発明の電磁波増強素子を製造する場合には、図２３に示すように、アミノ酸のアミノ基又はカルボキシル基と結合又は吸着可能な官能基を有するシランカップリング剤で金属部２の表面処理を行えばよい。 As a specific example, when producing the electromagnetic wave enhancing element of the present invention for amino acid analysis, as shown in FIG. 23, a silane having a functional group capable of binding or adsorbing to an amino group or a carboxyl group of an amino acid. What is necessary is just to perform the surface treatment of the metal part 2 with a coupling agent.
　金属部の表面に、親水性を有する親水性層を形成する親水性層形成工程を有していてもよい。例えば、金属部２を銀で作製した場合、図２４に示すように、ローダミンなど水溶性の試薬は銀表面ではじかれて、ラマン増強が強く生じるホットスポットに到達しない場合がある。このような場合にも、図２５に示すように、金属部２の表面に、酸化ケイ素（ＳｉＯ２）などの少なくとも金属部２より親水性の高い材料からなる親水性層７を被覆すれば、当該試薬をホットスポットに到着させることができ、高感度を得ることができる。当該親水性層７は、膜厚を５ｎｍ以下、好ましくは１ｎｍ以下で被覆する方がよい。 You may have the hydrophilic layer formation process which forms the hydrophilic layer which has hydrophilic property on the surface of a metal part. For example, when the metal part 2 is made of silver, as shown in FIG. 24, a water-soluble reagent such as rhodamine may be repelled on the silver surface and may not reach a hot spot where Raman enhancement is strong. Even in such a case, as shown in FIG. 25, if the surface of the metal part 2 is covered with a hydrophilic layer 7 made of a material having a higher hydrophilic property than the metal part 2 such as silicon oxide (SiO 2 ), The reagent can reach the hot spot, and high sensitivity can be obtained. The hydrophilic layer 7 should be coated with a film thickness of 5 nm or less, preferably 1 nm or less.
◎シミュレーション
　次に、シミュレーションを用いて本発明の電磁波増強素子を説明する。シミュレーションには、シノプシス社（Synopsys, Inc）製のソフトDiffractMODを用いた。電磁波増強素子としては、図２に示すように、凸部14が半径ｒの半円である誘電体凹凸構造11が形成された誘電体部１と、凸部14を構成する半円と同心円の半円又はそれを垂直方向に拡大した半楕円である凸部24からなる金属凹凸構造21を有する金属部２と、を有するものを用いた。また、凸部24を構成する半円又は半楕円の中心から水平方向の大きさはｒ＋１０ｎｍとし、誘電体凹凸構造11の凹部12の幅ｄを変えて隣合う凸部24の側部を重ねることにより、金属部２の最下点23の垂直方向の厚みＢを調節した。 Simulation Next, the electromagnetic wave enhancing element of the present invention will be described using simulation. For the simulation, the software DiffractMOD manufactured by Synopsys, Inc. was used. As shown in FIG. 2, the electromagnetic wave enhancing element includes a dielectric part 1 in which a dielectric concavo-convex structure 11 in which the convex part 14 is a semicircle having a radius r, and a semicircle and a concentric circle that constitute the convex part 14. A metal part 2 having a metal concavo-convex structure 21 composed of a convex part 24 which is a semicircle or a semi-ellipse obtained by enlarging it in the vertical direction was used. Further, the horizontal dimension from the center of the semicircle or semi-ellipse constituting the convex portion 24 is set to r + 10 nm, and the width d of the concave portion 12 of the dielectric concavo-convex structure 11 is changed to overlap the side portions of the adjacent convex portions 24. Thus, the thickness B in the vertical direction of the lowest point 23 of the metal part 2 was adjusted.
　金属凹凸構造21のピッチは、１００ｎｍから２００ｎｍまで１０ｎｍごとに１１種類をシミュレーションした。また、誘電体部１の材料としては、ＰＭＭＡを想定し、金属部２の材料としては、金を想定した。 11 pitches of the metal uneven structure 21 were simulated for every 10 nm from 100 nm to 200 nm. In addition, PMMA is assumed as the material of the dielectric part 1, and gold is assumed as the material of the metal part 2.
［シミュレーション１］
　まず、金属凹凸構造21の凹部22における金属部２のなす角度θの最小値と当該凹部22における電界増強度の最大値Ｅｘ（倍）との関係をシミュレーションした。その結果を図９に示す。 [Simulation 1]
First, the relationship between the minimum value of the angle θ formed by the metal part 2 in the concave part 22 of the metal concave-convex structure 21 and the maximum value Ex (times) of the electric field enhancement intensity in the concave part 22 was simulated. The result is shown in FIG.
　図９によると、金属凹凸構造21の凹部22における金属部２のなす角度θが２０度以下である場合に電界増強度の最大値Ｅｘが８０倍を超えるものがあることがわかった。特に、電界増強度の最大値Ｅｘは、０°＜θ＜１５°で大きく、更に７°＜θ＜１３°で大きい傾向があった。また、ピッチＰは少なくとも２００ｎｍ以下であれば、電界増強度の最大値Ｅｘが８０倍を超えるものが存在し、ピッチＰが小さくなる程、電界増強度の最大値Ｅｘは大きくなる傾向があった。特にピッチＰが１００≦Ｐ≦１２０ｎｍの時に電界増強度の最大値Ｅｘは大きかった。 9 that the maximum value Ex of the electric field enhancement exceeds 80 times when the angle θ formed by the metal part 2 in the concave part 22 of the metal concave-convex structure 21 is 20 degrees or less. In particular, the maximum value Ex of the electric field enhancement strength tends to be large when 0 ° <θ <15 ° and further large when 7 ° <θ <13 °. Further, when the pitch P is at least 200 nm or less, there is a case where the maximum value Ex of the electric field enhancement exceeds 80 times, and the maximum value Ex of the electric field enhancement tends to increase as the pitch P decreases. . In particular, the maximum value Ex of the electric field enhancement intensity was large when the pitch P was 100 ≦ P ≦ 120 nm.
［シミュレーション２］
　次に、金属凹凸構造21の凸部24の最上点の垂直方向の厚みをＡ、最下点23の垂直方向の厚みをＢとしたときのＡとＢの膜厚比Ｂ／Ａと電界増強度の最大値Ｅｘ（倍）との関係をシミュレーションした。その結果を図１０に示す。 [Simulation 2]
Next, A / B film thickness ratio B / A and electric field enhancement, where A is the vertical thickness of the convex portion 24 of the metal concavo-convex structure 21 and B is the vertical thickness of the lowest point 23. The relationship with the maximum value Ex (times) of the degree was simulated. The result is shown in FIG.
　図１０によると、膜厚Ａが膜厚Ｂより大きいほど電界増強度の最大値Ｅｘが大きくなる傾向があることがわかった。また、厚みＡと厚みＢの膜厚比Ｂ／Ａが０．９より小さいと電界増強度の最大値Ｅｘが８０倍を超えるものがあり、膜厚比Ｂ／Ａが０．６より小さいと電界増強度の最大値Ｅｘが１００倍を超えるものがあることがわかった。また、ピッチＰは少なくとも２００ｎｍ以下であれば、電界増強度の最大値Ｅｘが８０倍を超えるものが存在し、ピッチＰが小さくなる程、電界増強度の最大値Ｅｘは大きくなる傾向があった。特にピッチＰが１００≦Ｐ≦１２０ｎｍの時に電界増強度の最大値Ｅｘは大きかった。 FIG. 10 indicates that the maximum value Ex of the electric field enhancement strength tends to increase as the film thickness A is greater than the film thickness B. In addition, when the thickness ratio B / A between the thickness A and the thickness B is smaller than 0.9, the maximum value Ex of the electric field enhancement strength exceeds 80 times, and when the thickness ratio B / A is smaller than 0.6, It was found that the maximum value Ex of the electric field enhancement intensity exceeds 100 times. Further, when the pitch P is at least 200 nm or less, there is a case where the maximum value Ex of the electric field enhancement exceeds 80 times, and the maximum value Ex of the electric field enhancement tends to increase as the pitch P decreases. . In particular, the maximum value Ex of the electric field enhancement intensity was large when the pitch P was 100 ≦ P ≦ 120 nm.
［シミュレーション３］
　次に、金属凹凸構造21の凸部24の最上点の垂直方向の厚みをＡ、誘電体凹凸構造の凹部の底部の幅の半分をＣとしたときのＡとＣの比Ａ／Ｃと電界増強度の最大値Ｅｘ（倍）との関係をシミュレーションした。その結果を図１１に示す。 [Simulation 3]
Next, A / C ratio A / C and electric field where A is the thickness in the vertical direction of the uppermost point of the convex portion 24 of the metal concavo-convex structure 21 and C is half the width of the bottom of the concave portion of the dielectric concavo-convex structure. The relationship with the maximum value Ex (times) of the enhancement was simulated. The result is shown in FIG.
　図１１によると、ＡがＣより大きいほど電界増強度の最大値Ｅｘが大きくなる傾向があることがわかった。また、ＡとＣの比Ａ／Ｃが２より大きいと電界増強度の最大値Ｅｘが１００倍を超えるものがあり、比Ａ／Ｃが３．５より大きいと電界増強度の最大値Ｅｘが１２０倍を超えるものがあることがわかった。また、ピッチＰは少なくとも２００ｎｍ以下であれば、電界増強度の最大値Ｅｘが８０倍を超えるものが存在し、ピッチＰが小さくなる程、電界増強度の最大値Ｅｘは大きくなる傾向があった。特にピッチＰが１００≦Ｐ≦１２０ｎｍの時に電界増強度の最大値Ｅｘは大きかった。 FIG. 11 shows that the maximum value Ex of the electric field enhancement tends to increase as A is greater than C. In addition, when the ratio A / C of A and C is larger than 2, there is a case where the maximum value Ex of the electric field strength exceeds 100 times, and when the ratio A / C is larger than 3.5, the maximum value Ex of the electric field strength increases. It turned out that there was a thing exceeding 120 times. Further, when the pitch P is at least 200 nm or less, there is a case where the maximum value Ex of the electric field enhancement exceeds 80 times, and the maximum value Ex of the electric field enhancement tends to increase as the pitch P decreases. . In particular, the maximum value Ex of the electric field enhancement intensity was large when the pitch P was 100 ≦ P ≦ 120 nm.
［実施例１］
　次に、実際に本発明の電磁波増強素子を作成し、ラマン散乱光の強度（以下、ラマン強度という）を確認した。電磁波増強素子としては、後述する試料１～３を用いた。また、本発明の電磁波増強素子と比較するために、後述する比較試料１，２を用いた。 [Example 1]
Next, the electromagnetic wave enhancing element of the present invention was actually created, and the intensity of Raman scattered light (hereinafter referred to as Raman intensity) was confirmed. Samples 1 to 3 described later were used as the electromagnetic wave enhancing elements. Moreover, in order to compare with the electromagnetic wave enhancing element of the present invention, comparative samples 1 and 2 described later were used.
［試料１］
　高さ１２０ｎｍ、線幅５０ｎｍ、ピッチ１００ｎｍであるラインアンドスペース状の誘電体凹凸構造にスパッタリングで銀を２００Wで２４５秒成膜し、底部側に向かって幅の小さくなる凹部を一方向に延伸した形状の金属凹凸構造を有する電磁波増強素子を形成した。金属凹凸構造の凸部の最上点の垂直方向の厚みは１３０ｎｍであった。当該電磁波増強素子の平面写真と断面写真を図１２に示す。 [Sample 1]
Silver was formed at 200 W for 245 seconds by sputtering on a line-and-space dielectric concavo-convex structure having a height of 120 nm, a line width of 50 nm, and a pitch of 100 nm, and a concave portion with a smaller width was stretched in one direction toward the bottom side. An electromagnetic wave enhancing element having a metal uneven structure was formed. The thickness in the vertical direction of the uppermost point of the convex portion of the metal concavo-convex structure was 130 nm. FIG. 12 shows a plan photograph and a sectional photograph of the electromagnetic wave enhancing element.
［試料２］
　高さ１４０ｎｍ、線幅７０ｎｍ、ピッチ１４０ｎｍであるラインアンドスペース状の誘電体凹凸構造にスパッタリングで銀を２００Wで２４５秒成膜し、底部側に向かって幅の小さくなる凹部を一方向に延伸した形状の金属凹凸構造を有する電磁波増強素子を形成した。金属凹凸構造の凸部の最上点の垂直方向の厚みは１２５ｎｍであった。当該電磁波増強素子の平面写真と断面写真を図１３に示す。 [Sample 2]
Silver was formed at 200 W for 245 seconds by sputtering on a line-and-space dielectric concavo-convex structure having a height of 140 nm, a line width of 70 nm, and a pitch of 140 nm, and a concave portion with a smaller width was stretched in one direction toward the bottom side. An electromagnetic wave enhancing element having a metal uneven structure was formed. The thickness in the vertical direction of the uppermost point of the convex portion of the metal concavo-convex structure was 125 nm. FIG. 13 shows a plan photograph and a sectional photograph of the electromagnetic wave enhancing element.
［試料３］
　高さ２００ｎｍ、線幅１００ｎｍ、ピッチ２００ｎｍであるラインアンドスペース状の誘電体凹凸構造にスパッタリングで銀を２００Wで２４５秒成膜し、底部側に向かって幅の小さくなる凹部を一方向に延伸した形状の金属凹凸構造を有する電磁波増強素子を形成した。金属凹凸構造の凸部の最上点の垂直方向の厚みは１２７ｎｍであった。当該電磁波増強素子の平面写真と断面写真を図１４に示す。
［比較試料１］
　高さ５００ｎｍ、底面の直径２３０ｎｍである円柱をピッチ４６０ｎｍで三角配置とした誘電体凹凸構造にスパッタリングで銀を２００Wで２４５秒成膜し、電磁波増強素子を形成した。金属凹凸構造の凸部の最上点の垂直方向の厚みは１２０ｎｍであった。当該電磁波増強素子の平面写真と断面写真を図１５に示す。 [Sample 3]
Silver was formed at 200 W for 245 seconds by sputtering on a line-and-space dielectric concavo-convex structure having a height of 200 nm, a line width of 100 nm, and a pitch of 200 nm, and a concave portion with a smaller width was stretched in one direction toward the bottom side. An electromagnetic wave enhancing element having a metal uneven structure was formed. The thickness in the vertical direction of the uppermost point of the convex portion of the metal concavo-convex structure was 127 nm. A plane photograph and a cross-sectional photograph of the electromagnetic wave enhancing element are shown in FIG.
[Comparative sample 1]
Silver was formed at 200 W for 245 seconds by sputtering on a dielectric concavo-convex structure in which a cylinder having a height of 500 nm and a bottom diameter of 230 nm was arranged in a triangle with a pitch of 460 nm, thereby forming an electromagnetic wave enhancing element. The thickness in the vertical direction of the uppermost point of the convex portion of the metal concavo-convex structure was 120 nm. FIG. 15 shows a plan photograph and a sectional photograph of the electromagnetic wave enhancing element.
［比較試料２］
　高さ２５０ｎｍである円錐をピッチ２５０ｎｍで三角配置とした誘電体凹凸構造にスパッタリングで銀を２００Wで２４５秒成膜し、電磁波増強素子を形成した。金属凹凸構造の凸部の最上点の垂直方向の厚みは１２０ｎｍであった。当該電磁波増強素子の平面写真と断面写真を図１６に示す。 [Comparative sample 2]
Silver was formed at 200 W for 245 seconds by sputtering on a dielectric concavo-convex structure in which a cone having a height of 250 nm was triangularly arranged at a pitch of 250 nm to form an electromagnetic wave enhancing element. The thickness in the vertical direction of the uppermost point of the convex portion of the metal concavo-convex structure was 120 nm. FIG. 16 shows a plan photograph and a sectional photograph of the electromagnetic wave enhancing element.
　まず、測定する対象物として、上記各電磁波増強素子の金属凹凸構造の表面に４－アミノベンゼンチオールを成膜した。そして、各電磁波増強素子に対して垂直に波長が５３２ｎｍのレーザーを３ｍＷで１秒照射して、ラマンシフト１４３０ｃｍ－１のラマン強度を測定した。 First, as an object to be measured, 4-aminobenzenethiol was formed on the surface of the metal concavo-convex structure of each electromagnetic wave enhancing element. Each electromagnetic wave enhancing element was irradiated with a laser having a wavelength of 532 nm perpendicularly at 3 mW for 1 second, and the Raman intensity at a Raman shift of 1430 cm −1 was measured.
　本発明の電磁波増強素子（試料１～３）にレーザーを入射させ、入射した点における垂直方向の線を回転軸として回転させて方位角とラマンシフト１４３０ｃｍ－１のラマン強度を測定した。その結果を図１７に示す。なお、方位角は、各電磁波増強素子のラマン強度の最大値を示す位置を０度とし、２２．５度ごとのラマン強度を表したものである。 A laser was incident on the electromagnetic wave enhancing element (samples 1 to 3) of the present invention, and the azimuth angle and the Raman intensity at a Raman shift of 1430 cm −1 were measured by rotating a vertical line at the incident point as a rotation axis. The result is shown in FIG. The azimuth angle represents the Raman intensity every 22.5 degrees with the position indicating the maximum value of the Raman intensity of each electromagnetic wave enhancing element as 0 degree.
　図１７に示すように、本発明の電磁波増強素子は、方位角を変えるとラマン強度が変化することがわかる。したがって、本発明の電磁波増強素子を用いるラマン散乱光検出方法としては、当該電磁波増強素子に光を入射させる照射工程と、光が電磁波増強素子に入射した点における垂直方向の線を回転軸として電磁波増強素子を少なくとも９０度以上回転させる回転工程と、回転工程中に、電磁波増強素子のラマン散乱光の最大強度を測定する測定工程と、を有する方が好ましい。これにより、本発明の電磁波増強素子は、最も感度の高い位置で被検体の特性を検出することができる。 As shown in FIG. 17, it can be seen that the Raman intensity of the electromagnetic wave enhancing element of the present invention changes when the azimuth angle is changed. Accordingly, the Raman scattered light detection method using the electromagnetic wave enhancing element of the present invention includes an irradiation step of making light incident on the electromagnetic wave enhancing element, and an electromagnetic wave having a vertical line at the point where the light is incident on the electromagnetic wave enhancing element as a rotation axis. It is preferable to have a rotation step of rotating the enhancement element by at least 90 degrees and a measurement step of measuring the maximum intensity of the Raman scattered light of the electromagnetic wave enhancement element during the rotation step. Thereby, the electromagnetic wave enhancing element of the present invention can detect the characteristics of the subject at the position with the highest sensitivity.
　また、本発明の電磁波増強素子は、図１８に示すように、金属部２の凹部の延伸方向を示すアライメントマーク４を有していてもよい。これにより、電磁波増強素子の最も感度の高い位置を把握し易くすることができる。なお、当該アライメントマーク４は、金属部２の凹部の延伸方向がわかれば電磁波増強素子のどこに形成してもよい。例えば、アライメントマーク４を誘電体部１の上に形成したり、金属部２の上に形成したりすることができる。 Moreover, the electromagnetic wave enhancing element of the present invention may have an alignment mark 4 indicating the extending direction of the concave portion of the metal part 2 as shown in FIG. Thereby, it is possible to easily grasp the position with the highest sensitivity of the electromagnetic wave enhancing element. The alignment mark 4 may be formed anywhere in the electromagnetic wave enhancing element as long as the extending direction of the recess of the metal part 2 is known. For example, the alignment mark 4 can be formed on the dielectric part 1 or on the metal part 2.
　また、各電磁波増強素子（試料１～３および比較試料１，２）のラマン強度の最大値を図１９に示す。図１９に示すように、底部側に向かって幅の小さくなる凹部を一方向に延伸した形状の金属凹凸構造を有する本発明の電磁波増強素子（試料１～３）は、金属凹凸構造が円柱状の電磁波増強素子（比較試料１）や円錐状である電磁波増強素子（比較試料２）と比べて非常に大きなラマン強度を示すことがわかる。 Further, FIG. 19 shows the maximum Raman intensity of each electromagnetic wave enhancing element (samples 1 to 3 and comparative samples 1 and 2). As shown in FIG. 19, the electromagnetic wave enhancing elements (samples 1 to 3) of the present invention having a metal concavo-convex structure having a shape in which a concave portion whose width decreases toward the bottom side is extended in one direction, the metal concavo-convex structure is cylindrical. It can be seen that the present invention shows a very large Raman intensity compared to the electromagnetic wave enhancing element (Comparative Sample 1) and the conical electromagnetic wave enhancing element (Comparative Sample 2).
［実施例２］
　次に、金属凹凸構造の凹部を形成する隣接する凸部同士の間隙の最小幅の大きさとラマン強度との関係を調べた。電磁波増強素子としては、後述する試料４～６のものを用いた。 [Example 2]
Next, the relationship between the minimum width of the gap between adjacent convex portions forming the concave portion of the metal concavo-convex structure and the Raman intensity was examined. As the electromagnetic wave enhancement element, samples 4 to 6 described later were used.
［試料４］
　高さ１４０ｎｍ、線幅７０ｎｍ、ピッチ１４０ｎｍであるラインアンドスペース状の誘電体凹凸構造にスパッタリングで銀を２００Wで１７０秒成膜し、底部側に向かって幅の小さくなる凹部を一方向に延伸した形状の金属凹凸構造を有する電磁波増強素子を形成した。この際、金属凹凸構造の凹部を形成する隣接する凸部同士の間隙の最小幅の大きさは、２５．９ｎｍであった。 [Sample 4]
Silver was deposited at 200 W for 170 seconds by sputtering on a line-and-space dielectric concavo-convex structure having a height of 140 nm, a line width of 70 nm, and a pitch of 140 nm, and a concave portion with a smaller width was stretched in one direction toward the bottom side. An electromagnetic wave enhancing element having a metal uneven structure was formed. At this time, the minimum width of the gap between adjacent convex portions forming the concave portion of the metal concave-convex structure was 25.9 nm.
［試料５］
　高さ１４０ｎｍ、線幅７０ｎｍ、ピッチ１４０ｎｍであるラインアンドスペース状の誘電体凹凸構造にスパッタリングで銀を２００Wで１９５秒成膜し、底部側に向かって幅の小さくなる凹部を一方向に延伸した形状の金属凹凸構造を有する電磁波増強素子を形成した。この際、金属凹凸構造の凹部を形成する隣接する凸部同士の間隙の最小幅の大きさは、２３．７ｎｍであった。 [Sample 5]
Silver was deposited at 200 W for 195 seconds by sputtering on a line-and-space dielectric concavo-convex structure having a height of 140 nm, a line width of 70 nm, and a pitch of 140 nm, and a concave portion with a smaller width was stretched in one direction toward the bottom side. An electromagnetic wave enhancing element having a metal uneven structure was formed. At this time, the minimum width of the gap between adjacent convex portions forming the concave portion of the metal concavo-convex structure was 23.7 nm.
［試料６］
　高さ１４０ｎｍ、線幅７０ｎｍ、ピッチ１４０ｎｍであるラインアンドスペース状の誘電体凹凸構造にスパッタリングで銀を２００Wで２４５秒成膜し、底部側に向かって幅の小さくなる凹部を一方向に延伸した形状の金属凹凸構造を有する電磁波増強素子を形成した。この際、金属凹凸構造の凹部を形成する隣接する凸部同士の間隙の最小幅の大きさは、２０ｎｍであった。 [Sample 6]
Silver was formed at 200 W for 245 seconds by sputtering on a line-and-space dielectric concavo-convex structure having a height of 140 nm, a line width of 70 nm, and a pitch of 140 nm, and a concave portion with a smaller width was stretched in one direction toward the bottom side. An electromagnetic wave enhancing element having a metal uneven structure was formed. At this time, the minimum width of the gap between adjacent convex portions forming the concave portion of the metal concavo-convex structure was 20 nm.
　測定する対象物として、上記各電磁波増強素子の金属凹凸構造の表面に４－アミノベンゼンチオールを成膜した。そして、各電磁波増強素子に対して垂直に波長が５３２ｎｍのレーザーを３ｍＷで１秒照射して、ラマンシフト１４３０ｃｍ－１のラマン強度の最大値を測定した。その結果を図２０に示す。 As an object to be measured, 4-aminobenzenethiol was formed on the surface of the metal relief structure of each of the electromagnetic wave enhancing elements. Each electromagnetic wave enhancing element was irradiated with a laser having a wavelength of 532 nm perpendicularly at 3 mW for 1 second, and the maximum value of the Raman intensity at a Raman shift of 1430 cm −1 was measured. The result is shown in FIG.
　図２０に示すように、本発明の電磁波増強素子は、金属凹凸構造の凹部を形成する隣接する凸部同士の間隙の最小幅の大きさが小さくなるとラマン強度が大きくなることがわかる。具体的には、間隙の最小幅の大きさが２５ｎｍ以下となるとラマン強度が１００００を超え、２０ｎｍ以下となると３５０００を超えることがわかる。 As shown in FIG. 20, it can be seen that the electromagnetic wave enhancing element of the present invention increases the Raman intensity when the minimum width of the gap between adjacent convex portions forming the concave portion of the metal concave-convex structure is reduced. Specifically, it can be seen that the Raman intensity exceeds 10,000 when the minimum width of the gap is 25 nm or less, and exceeds 35000 when it is 20 nm or less.
　次に、本発明の電磁波増強素子をペプチド又はタンパク質のアミノ酸配列の決定に用いる場合について説明する。 Next, the case where the electromagnetic wave enhancing element of the present invention is used for determining the amino acid sequence of a peptide or protein will be described.
　本発明のアミノ酸配列決定方法は、ペプチド又はタンパク質のＮ末端又はＣ末端からアミノ酸を逐次的に分解する逐次分解工程と、逐次分解工程により遊離したアミノ酸を分取する分取工程と、分取工程により得られたアミノ酸を本発明の電磁波増強素子を用いて分析する分析工程と、で主に構成される。 The amino acid sequence determination method of the present invention includes a sequential decomposition step for sequentially decomposing amino acids from the N-terminus or C-terminus of a peptide or protein, a fractionation step for fractionating amino acids released by the sequential degradation step, and a fractionation step And an analysis step of analyzing the amino acid obtained by the method using the electromagnetic wave enhancing element of the present invention.
　逐次分解工程は、ペプチド又はタンパク質のＮ末端又はＣ末端に位置するアミノ酸を遊離させる方法であればどのような方法であってもよい。例えば、プロテアーゼを用いる方法や、ペプチド又はタンパク質のＮ末端部の遊離アミノ基にフェニルイソチオシアネート（ＰＩＴＣ）を反応させ、フェニルチオカルバミル誘導体（ＰＴＣアミノ酸）とし、次いでトリフルオロ酢酸によってアニリノチアゾリノン（ＡＴＺ）-アミノ酸として遊離させる方法（エドマン分解）を用いることができる。 The sequential decomposition step may be any method as long as it is a method for releasing an amino acid located at the N-terminus or C-terminus of a peptide or protein. For example, a method using a protease, a phenylisothiocyanate (PITC) is reacted with a free amino group at the N-terminal part of a peptide or protein to form a phenylthiocarbamyl derivative (PTC amino acid), and then anilinothiazolinone with trifluoroacetic acid A method of releasing as (ATZ) -amino acid (Edman degradation) can be used.
　プロテアーゼを用いる方法の場合、例えば、ペプチド又はタンパク質のＮ末端又はＣ末端のペプチド結合を加水分解し、当該末端からアミノ酸を順次遊離させるエキソペプチダーゼを用いることができる。当該エキソペプチダーゼは、特性の異なる２種以上のものを混合して用いてもよい。例えば、Ｃ末端に作用するカルボキシペプチダーゼを用いる場合、末端から２番目又は末端の残基が芳香族か脂肪族アミノ酸の場合に高い触媒作用を示すカルボキシペプチダーゼＹと、末端の残基が塩基性アミノ酸の場合に高い触媒作用を示すカルボキシペプチダーゼＢとを組み合わせて用いることができる。なお、最初にエンドペプチダーゼ処理を行い、適当な長さのフラグメントを取得した後、当該フラグメントをエキソペプチダーゼによる分解に処してもよい。 In the case of a method using a protease, for example, an exopeptidase that hydrolyzes a peptide bond at the N-terminal or C-terminal of a peptide or protein and sequentially releases amino acids from the terminal can be used. The exopeptidase may be used by mixing two or more types having different characteristics. For example, when using a carboxypeptidase that acts on the C-terminal, carboxypeptidase Y that exhibits high catalytic activity when the second or terminal residue from the terminal is an aromatic or aliphatic amino acid, and a terminal residue that is a basic amino acid In this case, carboxypeptidase B showing high catalytic action can be used in combination. In addition, after performing an endopeptidase treatment first to obtain a fragment having an appropriate length, the fragment may be subjected to degradation by exopeptidase.
　プロテアーゼを用いた逐次分解においては、当該プロテアーゼの活性の保持と喪失を制御することにより、ペプチド又はタンパク質の末端から１残基のみを遊離させ、これを分取することが容易となる。当該制御は、プロテアーゼ活性の至適範囲に影響を与える温度、ｐＨ、時間、イオン存在の有無等の操作により可能である。例えば、Ｃｏ２＋存在下かつ９０℃で高い活性を示し、Ｃｏ２＋非存在下かつ３７℃でほぼ活性を示さない耐熱性のプロテアーゼを用いれば、温度とＣｏ２＋存在の有無により制御することができる。また、１分で１残基を遊離させるプロテアーゼを用いれば、時間により容易に制御することができる。 In sequential decomposition using a protease, by controlling the retention and loss of the activity of the protease, only one residue is released from the end of the peptide or protein, and it becomes easy to fractionate it. The control can be performed by operations such as temperature, pH, time, presence / absence of ions, which affect the optimum range of protease activity. For example, highly active in the Co 2+ presence and 90 ° C., the use of the heat-resistant proteases do not show nearly activity Co 2+ absence and 37 ° C., can be controlled by the presence or absence of temperature and Co 2+ exists . In addition, if a protease that liberates one residue in 1 minute is used, it can be easily controlled by time.
　なお、本発明の逐次分解工程においては、ペプチド又はタンパク質の末端から１残基のみが遊離されることが好ましい。しかしながら、本発明の電磁波増強素子において強い電界の集中が生じる範囲の大きさによっては、アミノ酸が２残基以上結合した状態であってもラマン散乱シグナルを解析して、当該アミノ酸の種類や順番を同定することができる。したがって、この場合には、本発明の逐次分解工程において、ペプチド又はタンパク質の末端から２残基又はそれ以上の残基が結合した状態で遊離された場合であっても、アミノ酸の同定が可能な場合もある。 In the sequential decomposition step of the present invention, it is preferable that only one residue is released from the end of the peptide or protein. However, depending on the size of the range where strong electric field concentration occurs in the electromagnetic wave enhancing element of the present invention, the Raman scattering signal is analyzed even when two or more amino acids are bound, and the type and order of the amino acids are determined. Can be identified. Therefore, in this case, it is possible to identify an amino acid even in the case where 2 or more residues are released in a bound state from the end of the peptide or protein in the sequential decomposition step of the present invention. In some cases.
　プロテアーゼを用いた逐次分解を行う場合、当該逐次分解は、プロテアーゼ又はペプチド若しくはタンパク質を固定化したカラムを用いても行ってもよい。カラムに充填する担体へのプロテアーゼ又はペプチド若しくはタンパク質の固定は、任意の手法により行えばよい。当該固定化したカラムを用いることにより、逐次分解工程を経たサンプル、すなわち遊離アミノ酸を含むサンプルを、所望のタイミングで容易に取得することができる。 When performing sequential decomposition using a protease, the sequential decomposition may be performed using a column on which a protease, peptide, or protein is immobilized. Immobilization of protease, peptide or protein to the carrier packed in the column may be performed by any method. By using the immobilized column, a sample that has been subjected to the sequential decomposition step, that is, a sample containing free amino acids can be easily obtained at a desired timing.
　本発明のアミノ酸配列決定方法における分取工程は、前記逐次分解工程により遊離したアミノ酸を分取する方法であればどのような方法であってもよい。例えば、前述のように、プロテアーゼ又はペプチド若しくはタンパク質を固定化したカラムを用いる場合、カラムからの溶出液を、フラクションコレクター等を用いて経時的に分取すればよい。また、前述したように、プロテアーゼの活性を制御する場合には、当該制御に対応させて、プロテアーゼによる分解反応がなされた溶出液のみを分取すればよい。これにより、所望のアミノ酸を効率良く回収することができる。 The fractionation step in the amino acid sequence determination method of the present invention may be any method as long as it is a method for fractionating amino acids released by the sequential decomposition step. For example, as described above, when using a column on which a protease, peptide, or protein is immobilized, the eluate from the column may be collected over time using a fraction collector or the like. Further, as described above, when controlling the activity of the protease, only the eluate that has undergone the degradation reaction by the protease may be collected in correspondence with the control. Thereby, a desired amino acid can be efficiently recovered.
　本発明のアミノ酸配列決定方法における分析工程は、分取工程により得られたアミノ酸を本発明の電磁波増強素子を用いて分析する工程であり、例えば、以下のように行う。 The analysis step in the amino acid sequence determination method of the present invention is a step of analyzing the amino acid obtained by the fractionation step using the electromagnetic wave enhancing element of the present invention, and is performed, for example, as follows.
　まず、前記分取工程により得られた遊離アミノ酸を本発明の電磁波増強素子の誘電体層の端部に固定する。当該誘電体層の端部へのアミノ酸の固定は、当該端部の表面への物理的吸着や、ポリブレンやシランカップリング剤を介した結合により行うことができる。例えば、誘電体層の端部に予めシランカップリング剤を結合させておいた本発明の電磁波増強素子に、分取工程により得られたアミノ酸を含む溶液を滴下した後、これを適宜、洗浄、乾燥すればよい。次に、当該アミノ酸が固定した電磁波増強素子に所定波長の電磁波を照射し、得られたラマン散乱シグナルを解析して、当該固定されたアミノ酸の同定を行う。当該得られたアミノ酸の情報を用い、必要であれば適宜各種の解析を行い、アミノ酸配列を決定する。 First, the free amino acid obtained by the sorting step is fixed to the end of the dielectric layer of the electromagnetic wave enhancing element of the present invention. The amino acid can be fixed to the end portion of the dielectric layer by physical adsorption to the surface of the end portion or by bonding via polybrene or a silane coupling agent. For example, after dropping a solution containing the amino acid obtained by the fractionation step onto the electromagnetic wave enhancing element of the present invention in which a silane coupling agent is bonded to the end of the dielectric layer in advance, this is appropriately washed, It can be dried. Next, the electromagnetic wave enhancing element to which the amino acid is fixed is irradiated with an electromagnetic wave having a predetermined wavelength, and the obtained Raman scattering signal is analyzed to identify the fixed amino acid. Using the obtained amino acid information, various analyzes are performed as necessary to determine the amino acid sequence.
［実施例３］
　次に、実際に本発明の電磁波増強素子の作成方法について述べる。図２１に示すように、ラインアンドスペース状の誘電体凹凸構造に安価なスパッタ成膜を行うと金は原子のマイグレーションが小さいため、成膜形状は原子の到達量に１対１に対応することになる。すると、隣接する金が接触する時には緩やかな接触角度をとり、金属部２で信号が増幅することができない。一方、図２２のように、スパッタ時、原子がマイグレーションを行うＡｇは、マイグレーションにより金属部２（金属部本体２Ａ）はドロップ状の形に成長し、隣接するＡｇ同士は急峻な角度で接する。この表面に１ｎｍ以上のＡｕの金属層２Ｂを形成すればＡｕのラマン増幅特性が得られる。 [Example 3]
Next, a method for producing the electromagnetic wave enhancing element of the present invention will be described. As shown in FIG. 21, when an inexpensive sputter film formation is performed on a line-and-space dielectric concavo-convex structure, since the migration of atoms is small in gold, the film formation shape must correspond to the arrival amount of atoms on a one-to-one basis. become. Then, when adjacent gold contacts, a gentle contact angle is taken, and the signal cannot be amplified by the metal part 2. On the other hand, as shown in FIG. 22, during the sputtering, Ag that undergoes migration of atoms causes the metal part 2 (metal part body 2A) to grow into a drop shape by migration, and adjacent Ags contact each other at a steep angle. If an Au metal layer 2B of 1 nm or more is formed on this surface, Au Raman amplification characteristics can be obtained.
［実施例４］
　次に、別の電磁波増強素子の作成方法について述べる。図２４に示すように、ローダミンなど水溶性の試薬６はＡｇ表面ではじかれてホットスポットに到達しない場合がある。この場合はラマン増強がおきない。そこで、図２５に示すように、Ａｇ（金属部２）の表面にＳｉＯ２など親水性の誘電体材料で、膜厚が５ｎｍ以下、好ましくは１ｎｍ以下の親水性層７を形成する。これにより試薬はホットスポットに到着し、高感度が得られる。 [Example 4]
Next, another method for producing an electromagnetic wave enhancing element will be described. As shown in FIG. 24, the water-soluble reagent 6 such as rhodamine is sometimes repelled on the Ag surface and does not reach the hot spot. In this case, no Raman enhancement occurs. Therefore, as shown in FIG. 25, a hydrophilic layer 7 having a film thickness of 5 nm or less, preferably 1 nm or less, is formed of a hydrophilic dielectric material such as SiO 2 on the surface of Ag (metal part 2). As a result, the reagent reaches the hot spot and high sensitivity is obtained.
１　誘電体部
２　金属部
２Ａ　金属部本体
２Ｂ　金属層
３　中間層
４　アライメントマーク
５　固定物質
６　試薬
７　親水性層
10　基材
11　誘電体凹凸構造
12　凹部
14　凸部
15　誘電体凹凸構造
16　マスク
21　金属凹凸構造
22　凹部
23　最下点
24　凸部
24Ａ　凸部
24Ｂ　凸部
25Ａ　接線
25Ｂ　接線
26　最上点
91～99　領域 DESCRIPTION OF SYMBOLS 1 Dielectric part 2 Metal part 2A Metal part main body 2B Metal layer 3 Intermediate layer 4 Alignment mark 5 Fixed substance 6 Reagent 7 Hydrophilic layer
10 Substrate
11 Dielectric relief structure
12 Recess
14 Convex
15 Dielectric relief structure
16 mask
21 Metal relief structure
22 recess
23 Bottom point
24 Convex
24A Convex
24B Convex
25A Tangent
25B Tangent
26 Top point
91-99 area

Claims (35)

  1. 　誘電体凹凸構造が形成された誘電体部と、
    　底部側に向かって幅の小さくなる凹部を一方向に延伸した形状の金属凹凸構造を前記誘電体凹凸構造上に有する金属部と、
    を具備することを特徴とする電磁波増強素子。     A dielectric portion formed with a dielectric concavo-convex structure;
    A metal portion having a metal concavo-convex structure on the dielectric concavo-convex structure in a shape extending in one direction with a concave portion having a width that decreases toward the bottom side;
    An electromagnetic wave enhancing element comprising:
  2. 　前記凹部は、前記凹部を形成する隣接する凸部同士の間隙の最小幅が２５ｎｍ以下であることを特徴とする請求項１記載の電磁波増強素子。 The electromagnetic wave enhancing element according to claim 1, wherein the concave portion has a minimum width of 25 nm or less between adjacent convex portions forming the concave portion.
  3. 　前記凹部を形成する隣接する凸部の同じ高さの点における接線同士のなす角度をθとすると、当該凹部はθの最小値が２０度以下であることを特徴とする請求項１又は２記載の電磁波増強素子。 The minimum value of the said recessed part is 20 degrees or less, when the angle which the tangents in the point of the same height of the adjacent convex part which forms the said recessed part make is (theta), The said recessed part is 2 or less. Electromagnetic wave enhancing element.
  4. 　前記金属部は、前記金属凹凸構造の凸部の最上点の垂直方向の厚みをＡ、最下点の垂直方向の厚みをＢとすると、Ａ＞Ｂであることを特徴とする請求項１ないし３のいずれかに記載の電磁波増強素子。 2. The metal part according to claim 1, wherein A> B, where A is a vertical thickness of the uppermost point of the convex part of the metal concavo-convex structure and B is a vertical thickness of the lowest point. 4. The electromagnetic wave enhancing element according to any one of 3 above.
  5. 　前記金属部は、前記金属凹凸構造の凸部の最上点の垂直方向の厚みをＡ、最下点の垂直方向の厚みをＢとすると、０．９Ａ＞Ｂであることを特徴とする請求項４記載の電磁波増強素子。 The metal part has a relation of 0.9A> B, where A is the vertical thickness of the uppermost point of the convex part of the metal concavo-convex structure and B is the vertical thickness of the lowest point. 4. The electromagnetic wave enhancing element according to 4.
  6. 　前記金属部は、前記金属凹凸構造の凸部の最上点の垂直方向の厚みをＡ、誘電体凹凸構造の凹部の最下点の幅の半分をＣとすると、Ａ＞Ｃであることを特徴とする請求項１ないし５のいずれかに記載の電磁波増強素子。 The metal part is characterized by A> C, where A is the thickness in the vertical direction of the uppermost point of the convex part of the metal concave-convex structure, and C is half the width of the lowest point of the concave part of the dielectric concave-convex structure. The electromagnetic wave enhancing element according to any one of claims 1 to 5.
  7. 　前記金属部は、前記金属凹凸構造の凸部の最上点の垂直方向の厚みをＡ、誘電体凹凸構造の凹部の最下点の幅の半分をＣとすると、Ａ＞２Ｃであることを特徴とする請求項６記載の電磁波増強素子。 The metal part has A> 2C, where A is the thickness in the vertical direction of the uppermost point of the convex part of the metal concave-convex structure, and C is half the width of the lowest point of the concave part of the dielectric concave-convex structure. The electromagnetic wave enhancing element according to claim 6.
  8. 　前記金属部は、前記金属凹凸構造を定めるための金属部本体と、前記金属部本体の表面に形成され当該金属部本体より電界増強度の大きい金属からなる金属層と、からなることを特徴とする請求項１ないし７のいずれかに記載の電磁波増強素子。 The metal part is composed of a metal part main body for defining the metal concavo-convex structure, and a metal layer formed on a surface of the metal part main body and made of a metal having a larger electric field strength than the metal part main body. The electromagnetic wave enhancing element according to any one of claims 1 to 7.
  9. 　前記金属凹凸構造のピッチは、２００ｎｍ以下であることを特徴とする請求項１ないし８のいずれかに記載の電磁波増強素子。 The electromagnetic wave enhancing element according to any one of claims 1 to 8, wherein a pitch of the metal concavo-convex structure is 200 nm or less.
  10. 　前記凸部表面は前記凹部側に膨らんだ曲線状であることを特徴とする請求項１ないし９のいずれかに記載の電磁波増強素子。 The electromagnetic wave enhancing element according to any one of claims 1 to 9, wherein the convex surface has a curved shape bulging toward the concave side.
  11. 　前記誘電体凹凸構造は、複数種類の形状からなることを特徴とする請求項１ないし１０のいずれかに記載の電磁波増強素子。 11. The electromagnetic wave enhancing element according to claim 1, wherein the dielectric uneven structure has a plurality of types of shapes.
  12. 　前記誘電体凹凸構造は複数の領域からなり、前記誘電体凹凸構造は領域ごとに形状が異なることを特徴とする請求項１ないし１１のいずれかに記載の電磁波増強素子。 12. The electromagnetic wave enhancement element according to claim 1, wherein the dielectric concavo-convex structure includes a plurality of regions, and the shape of the dielectric concavo-convex structure is different for each region.
  13. 　前記金属部の凹部の延伸方向を示すアライメントマークを具備することを特徴とする請求項１ないし１２のいずれかに記載の電磁波増強素子。 The electromagnetic wave enhancing element according to claim 1, further comprising an alignment mark indicating an extending direction of the concave portion of the metal part.
  14. 　前記金属凹凸構造は、特定の物質を固定するための固定物質が結合されていることを特徴とする請求項１ないし１３のいずれかに記載の電磁波増強素子。 14. The electromagnetic wave enhancing element according to claim 1, wherein a fixed substance for fixing a specific substance is bonded to the metal concavo-convex structure.
  15. 　前記固定物質は、アミノ酸を固定するものであることを特徴とする請求項１４記載の電磁波増強素子。 15. The electromagnetic wave enhancing element according to claim 14, wherein the immobilizing substance is for immobilizing amino acids.
  16. 　前記金属部の表面に、親水性を有する親水性層を具備することを特徴とする請求項１ないし１３のいずれかに記載の電磁波増強素子。 14. The electromagnetic wave enhancing element according to claim 1, further comprising a hydrophilic layer having hydrophilicity on a surface of the metal part.
  17. 　前記誘電体部と前記金属部の間に前記誘電体部と前記金属部の密着性を向上するための中間層を有することを特徴とする請求項１ないし１６のいずれかに記載の電磁波増強素子。 The electromagnetic wave enhancing element according to claim 1, further comprising an intermediate layer for improving adhesion between the dielectric part and the metal part between the dielectric part and the metal part. .
  18. 　誘電体部に誘電体凹凸構造を形成する誘電体部形成工程と、
    　前記誘電体凹凸構造上に、底部側に向かって幅の小さくなる凹部を一方向に延伸した形状の金属凹凸構造を有する金属部を形成する金属部形成工程と、
    を有することを特徴とする電磁波増強素子の製造方法。     A dielectric part forming step of forming a dielectric uneven structure in the dielectric part;
    A metal part forming step of forming a metal part having a metal concavo-convex structure in a shape extending in one direction on the dielectric concavo-convex structure, a concave part having a width that decreases toward the bottom side;
    A method for producing an electromagnetic wave enhancing element, comprising:
  19. 　前記金属部形成工程は、前記凹部を形成する隣接する凸部の同じ高さの点における接線同士のなす角度をθとすると、θの最小値が２０度以下となるように前記凹部を形成するものであることを特徴とする請求項１８記載の電磁波増強素子の製造方法。 The metal part forming step forms the concave part so that a minimum value of θ is 20 degrees or less, where θ is an angle formed by tangents at the same height point of adjacent convex parts forming the concave part. The method of manufacturing an electromagnetic wave enhancing element according to claim 18, wherein
  20. 　前記金属部形成工程は、前記金属凹凸構造の凸部の最上点の垂直方向の厚みをＡ、前記最下点の垂直方向の厚みをＢとすると、Ａ＞Ｂとなるように金属部を形成するものであることを特徴とする請求項１８又は１９記載の電磁波増強素子の製造方法。 In the metal part forming step, the metal part is formed so that A> B, where A is the vertical thickness of the uppermost point of the convex part of the metal concavo-convex structure and B is the vertical thickness of the lowermost point. 20. The method of manufacturing an electromagnetic wave enhancing element according to claim 18 or 19, wherein
  21. 　前記金属部形成工程は、前記金属凹凸構造の凸部の最上点の垂直方向の厚みをＡ、前記最下点の垂直方向の厚みをＢとすると、０．９Ａ＞Ｂとなるように金属部を形成するものであることを特徴とする請求項２０記載の電磁波増強素子の製造方法。 In the metal part forming step, when the vertical thickness of the uppermost point of the convex part of the metal concavo-convex structure is A and the vertical thickness of the lowest point is B, the metal part is 0.9A> B. The method for manufacturing an electromagnetic wave enhancing element according to claim 20, wherein:
  22. 　前記金属部形成工程は、前記金属凹凸構造の凸部の最上点の垂直方向の厚みをＡ、前記誘電体凹凸構造の凹部の底部の幅の半分をＣとすると、Ａ＞Ｃとなるように金属部を形成するものであることを特徴とする請求項１８ないし２１記載の電磁波増強素子の製造方法。 In the metal part forming step, A> C, where A is the thickness in the vertical direction of the top point of the convex part of the metal concave-convex structure, and C is half the width of the bottom part of the concave part of the dielectric concave-convex structure. The method for manufacturing an electromagnetic wave enhancing element according to claim 18, wherein the metal part is formed.
  23. 　前記金属部形成工程は、前記金属凹凸構造の凸部の最上点の垂直方向の厚みをＡ、前記誘電体凹凸構造の凹部の底部の幅の半分をＣとすると、Ａ＞２Ｃとなるように金属部を形成するものであることを特徴とする請求項２２記載の電磁波増強素子の製造方法。 The metal part forming step is such that A> 2C, where A is the thickness in the vertical direction of the highest point of the convex part of the metal concavo-convex structure and C is the half width of the bottom part of the concave part of the dielectric concavo-convex structure. The method of manufacturing an electromagnetic wave enhancing element according to claim 22, wherein the metal part is formed.
  24. 　前記金属部形成工程は、前記金属凹凸構造を定めるための金属部本体を形成し、前記金属部本体の表面に当該金属部本体より電界増強度の大きい金属からなる金属層を形成するものであることを特徴とする請求項１８ないし２３のいずれかに記載の電磁波増強素子の製造方法。 The metal part forming step forms a metal part main body for defining the metal uneven structure, and forms a metal layer made of a metal having a larger electric field strength than the metal part main body on the surface of the metal part main body. The method for manufacturing an electromagnetic wave enhancing element according to any one of claims 18 to 23, wherein:
  25. 　前記誘電体部形成工程は、前記誘電体凹凸構造のピッチを２００ｎｍ以下となるように形成するものであることを特徴とする請求項１８ないし２４のいずれかに記載の電磁波増強素子の製造方法。 The method for manufacturing an electromagnetic wave enhancing element according to any one of claims 18 to 24, wherein the dielectric part forming step is performed such that a pitch of the dielectric uneven structure is 200 nm or less.
  26. 　前記金属部形成工程は、前記金属部の垂直方向の成長速度が大きくなる成膜技術を用いることを特徴とする請求項１８ないし２５のいずれかに記載の電磁波増強素子の製造方法。 The method for manufacturing an electromagnetic wave enhancing element according to any one of claims 18 to 25, wherein the metal part forming step uses a film forming technique that increases a growth rate in a vertical direction of the metal part.
  27. 　前記金属部形成工程は、前記誘電体凹凸構造上にスパッタリングによって金属部を形成するものであることを特徴とする請求項２６記載の電磁波増強素子の製造方法。 27. The method of manufacturing an electromagnetic wave enhancing element according to claim 26, wherein the metal part forming step forms a metal part by sputtering on the dielectric concavo-convex structure.
  28. 　前記金属部形成工程は、前記スパッタリングによって形成した金属部上に、更に無電界メッキを施して金属部の形状を調節するものであることを特徴とする請求項２７記載の電磁波増強素子の製造方法。 28. The method of manufacturing an electromagnetic wave enhancing element according to claim 27, wherein the metal part forming step further includes electroless plating on the metal part formed by the sputtering to adjust the shape of the metal part. .
  29. 　前記誘電体部上に前記誘電体部と前記金属部の密着性を向上するための中間層を形成する中間層形成工程を有することを特徴とする請求項１８ないし２８のいずれかに記載の電磁波増強素子の製造方法。 The electromagnetic wave according to any one of claims 18 to 28, further comprising an intermediate layer forming step of forming an intermediate layer for improving adhesion between the dielectric portion and the metal portion on the dielectric portion. A method for manufacturing an enhancement element.
  30. 　前記金属部に、特定の物質を固定可能な固定物質を結合させる固定物質結合工程を有することを特徴とする請求項１８ないし２９のいずれかに記載の電磁波増強素子の製造方法。 30. The method of manufacturing an electromagnetic wave enhancing element according to claim 18, further comprising a fixed substance binding step of binding a fixed substance capable of fixing a specific substance to the metal part.
  31. 　前記金属部の表面に、親水性を有する親水性層を形成する親水性層形成工程を有することを特徴とする請求項１８ないし２９のいずれかに記載の電磁波増強素子。 30. The electromagnetic wave enhancing element according to claim 18, further comprising a hydrophilic layer forming step of forming a hydrophilic layer having hydrophilicity on the surface of the metal part.
  32. 　請求項１ないし１７のいずれかに記載の電磁波増強素子に光を入射させる照射工程と、
    　前記光が前記電磁波増強素子に入射した点における垂直方向の線を回転軸として前記電磁波増強素子を少なくとも９０度以上回転させる回転工程と、
    　前記回転工程中に、前記電磁波増強素子のラマン散乱光の最大強度を測定する測定工程と、
    を具備することを特徴とするラマン散乱光検出方法。     An irradiation step of causing light to enter the electromagnetic wave enhancing element according to any one of claims 1 to 17,
    A rotation step of rotating the electromagnetic wave enhancement element by at least 90 degrees about a vertical line at a point where the light is incident on the electromagnetic wave enhancement element;
    A measuring step of measuring the maximum intensity of Raman scattered light of the electromagnetic wave enhancing element during the rotating step;
    A Raman scattered light detection method comprising:
  33. 　ペプチド又はタンパク質のＮ末端又はＣ末端からアミノ酸を逐次的に分解する逐次分解工程と、
    　前記逐次分解工程により遊離したアミノ酸を分取する分取工程と、
    　前記分取工程により得られたアミノ酸を請求項１４記載の電磁波増強素子を用いて分析する分析工程と、
    を有することを特徴とするアミノ酸配列決定方法。     A sequential degradation step of sequentially degrading amino acids from the N-terminus or C-terminus of the peptide or protein;
    A fractionation step of fractionating amino acids released by the sequential decomposition step;
    An analysis step of analyzing the amino acid obtained by the fractionation step using the electromagnetic wave enhancing element according to claim 14;
    A method for determining an amino acid sequence, comprising:
  34. 　前記逐次分解工程は、プロテアーゼを用いて行うことを特徴とする請求項３３記載のアミノ酸配列決定方法。 The amino acid sequence determination method according to claim 33, wherein the sequential decomposition step is performed using a protease.
  35. 　前記逐次分解工程は、前記プロテアーゼ又は前記ペプチド若しくは前記タンパク質を固定化したカラムを用いて行うことを特徴とする請求３４記載のアミノ酸配列決定方法。 The amino acid sequence determination method according to claim 34, wherein the sequential decomposition step is performed using a column on which the protease, the peptide, or the protein is immobilized.
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