JP2015232525A - Target substance capture device, production method of target substance capture device and detection device comprising target substance capture device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve dimensional accuracy of a reflection surface of a photonic crystal.SOLUTION: A target substance capture device 21 comprises: a target substance capture structure 25 having a surface on which, salient parts or recess parts are formed periodically; and a metal film 26 deposited on at least part of the surface, and forms a reflection surface 29. At least a part of the target substance capture structure 25 on the metal film 26 side is photocurable resin.

Description

  The present invention relates to a target substance capturing device, a method for manufacturing the target substance capturing device, and a detection apparatus including the target substance capturing device.

  Biosensors using photonic crystals are known as means for detecting target substances such as proteins and cells and measuring concentrations (for example, Non-Patent Document 1). The biosensor described in Non-Patent Document 1 irradiates light on a photonic crystal substrate on which a gold thin film is formed, and measures changes in the wavelength peak of reflected light reflected by the photonic crystal substrate. The target substance is detected and the concentration of the target substance is measured.

“Investigation of Plasmon resonances in metal films with nanohole arrays for biosensing applications”: Takumi Sannomiya, Olivier Scholder, Konstantins Jefimovs, Christian Hafner, and Andreas B. Dahlin, Received 10th December 2010, Revised 1th February 2011

  It is not easy to produce a photonic crystal substrate having a fine structure, and a slight defect may greatly affect the wavelength spectrum of reflected light. In order to form a highly accurate reflection surface of a photonic crystal, a nanoimprint technique for transferring a nanoscale uneven pattern to a molding material is useful.

  Nanoimprint technology includes thermal nanoimprint technology and optical nanoimprint technology. The thermal nanoimprint technology is a method of transferring a nanoscale uneven pattern by heating a material to be molded to a glass transition point or higher. In the thermal nanoimprint technique, since it is necessary to heat the glass transition point or higher, there is a possibility that the material to be molded may thermally shrink and the time for heating and cooling may hinder the realization of a high-speed process. Optical nanoimprint technology uses a photo-curing resin as the material to be molded, and is cured by irradiating the photo-curing resin with light (for example, ultraviolet rays) in a state where a nano mold (mold) having an uneven pattern is pressed against the photo-curing resin This is a method for transferring a nanoscale uneven pattern.

  Since the optical nanoimprint technology can transfer a fine pattern to a molding material at low temperature and low pressure, the heating and cooling time is not required and a high-speed process can be realized. According to the study by the present inventors, according to the optical nanoimprint technology, a sensor can be produced in about one-fourth the time of the thermal nanoimprint technology. Further, in the optical nanoimprint technique, since it is not necessary to heat the glass transition point or more, it is possible to reduce the possibility that the molding material is thermally contracted.

  Acrylic polymer or epoxy polymer known as photo-curing resin (after curing) or photo-curing resin (before curing) is uncured by ashing or strong alkaline solvent if uncured resin remains in the mold It is necessary to remove the resin, which causes damage to the mold. In order to avoid damage to the mold, it is necessary to produce a replica mold that is a duplicate of the original mold, which causes extra steps and costs. In the case of an acrylic polymer or an epoxy polymer, volume shrinkage of about 20% occurs during photocuring.

  An object of this invention is to improve the dimensional accuracy of the reflective surface of a photonic crystal.

  The present invention includes a target substance capturing structure having a surface in which concave portions or convex portions are periodically formed, and a metal film formed on at least a part of the surface to form a reflective surface, and the target The substance trapping structure is a target substance trapping device in which at least a part of the metal film side is a photocurable resin.

  In the present invention, since the target substance capturing structure is formed by photocuring, it is not necessary to heat it beyond the glass transition point. As a result, the time required for heating and cooling becomes unnecessary, and the process time can be shortened. Further, molding defects associated with heat shrinkage can be reduced.

  The photocurable resin is preferably an ultraviolet curable resin containing fluorine. The ultraviolet curable resin is preferably a ring-opening metathesis polymerization (FROMP) nanoimprint material containing fluorine.

  By using a ring-opening metathesis polymerization nanoimprint material containing fluorine, it is possible to improve the releasability after molding, and even when an uncured resin remains in the mold, it can be easily removed with an organic solvent. Therefore, the occurrence of mold damage can be reduced.

  The target substance is preferably a biological substance.

  The present invention is a detection apparatus including the target substance capturing apparatus described above.

  The detection apparatus includes a photonic crystal substrate including the target substance capturing structure and the metal film, and detects the presence and / or concentration of the target substance by changing the wavelength of reflected light from the photonic crystal substrate. It is preferable that it is a biosensor.

  The present invention presses a mold having a predetermined pattern against a sheet-like photocurable resin, irradiates the photocurable resin with light, releases the mold from the cured photocurable resin, In this method, a target substance capturing structure having a surface in which concave portions or convex portions are periodically formed is obtained, and a metal film is formed on at least a part of the surface.

  In the present invention, since the target substance capturing structure is formed by photocuring, it is not necessary to heat it beyond the glass transition point. As a result, the time required for heating and cooling becomes unnecessary, and the process time can be shortened. Further, molding defects associated with heat shrinkage can be reduced.

  The photocurable resin is an ultraviolet curable resin containing fluorine, and the light is preferably ultraviolet light. The ultraviolet curable resin is preferably a ring-opening metathesis polymerization (FROMP) nanoimprint material containing fluorine.

  By using a ring-opening metathesis polymerization nanoimprint material containing fluorine, it is possible to improve the releasability after molding, and even when an uncured resin remains in the mold, it can be easily removed with an organic solvent. Therefore, the occurrence of mold damage can be reduced.

  The present invention can improve the dimensional accuracy of the reflecting surface of the photonic crystal.

FIG. 1 is a diagram illustrating a target substance detection device. FIG. 2 is a perspective view of the target substance capturing device. FIG. 3 is a plan view of the target substance capturing device. FIG. 4 is a diagram showing a cross section taken along line AA in FIG. FIG. 5 is a partially enlarged view of the wall surface of the concave portion of the target substance capturing device. FIG. 6 is a partially enlarged view of the wall surface of another concave portion of the target substance capturing device. FIGS. 7-1 is a figure explaining an example of the manufacturing method of the target substance capture apparatus which concerns on this embodiment. 7-2 is a figure explaining an example of the manufacturing method of the target substance capture apparatus which concerns on this embodiment. FIGS. 7-3 is a figure explaining an example of the manufacturing method of the target substance capture apparatus which concerns on this embodiment. FIG. 8 is a diagram illustrating an example of a method for manufacturing the target substance capturing device according to the present embodiment. FIGS. 9-1 is a figure which shows the metal mold | die used by optical nanoimprint. FIG. 9-2 is a diagram illustrating a pattern transferred to a resin by a metal mold by optical nanoimprint. FIG. 10 is a schematic diagram for explaining each dimension of the unevenness transferred to the photo-curing resin. FIG. 11A is a diagram illustrating an AFM measurement result of a photonic crystal produced by optical nanoimprint. FIG. 11-2 is a diagram illustrating an AFM measurement result of a photonic crystal produced by thermal nanoimprint. 12-1 is a figure which shows the manufacturing method of the target substance capture apparatus which concerns on the modification of this embodiment. 12-2 is a figure which shows the manufacturing method of the target substance capture apparatus which concerns on the modification of this embodiment. 12-3 is a figure which shows the manufacturing method of the target substance capture apparatus which concerns on the modification of this embodiment. FIG. 13 is a diagram for explaining the principle of the photonic crystal biosensor. FIG. 14 is a diagram for explaining the principle of a photonic crystal biosensor. FIG. 15 is a diagram for explaining the principle of the photonic crystal biosensor. FIG. 16 is a diagram for explaining the principle of the photonic crystal biosensor. FIG. 17 is a diagram illustrating a photonic crystal biosensor. FIG. 18 is a diagram illustrating a photonic crystal biosensor. FIG. 19 is a diagram illustrating a photonic crystal biosensor. FIG. 20 is a diagram for explaining a photonic crystal biosensor fixing means. FIG. 21 is a diagram for explaining the photonic crystal biosensor fixing means. FIG. 22 is a diagram illustrating another embodiment of the photonic crystal biosensor. FIG. 23 is a diagram illustrating an example in which the light detection unit of the target substance detection device irradiates light to the photonic crystal biosensor. FIG. 24 is a diagram illustrating a structure of a measurement probe included in the light detection unit of the target substance detection device. FIG. 25 is a diagram illustrating the evaluation conditions of the light detection unit of the target substance detection device. FIG. 26 is a flowchart of the target substance detection method. FIG. 27 is a diagram for explaining the principle of a photonic crystal biosensor. FIG. 28 is a diagram for explaining the principle of a photonic crystal biosensor. FIG. 29 is a diagram illustrating the principle of a photonic crystal biosensor. FIG. 30 is a diagram for explaining the principle of the photonic crystal biosensor. FIG. 31 is a diagram for explaining the principle of a photonic crystal biosensor.

  Hereinafter, modes for carrying out the target substance detection device (hereinafter referred to as embodiments) will be described with reference to the drawings. In addition, this invention is not limited by the following embodiment. In addition, constituent elements in the following embodiments include those that can be easily assumed by those skilled in the art, those that are substantially the same, and those in a so-called equivalent range. Furthermore, the constituent elements disclosed in the following embodiments can be appropriately combined.

[Embodiment 1]
<Target substance detection device>
A target substance detection apparatus including the target substance capturing apparatus according to the first embodiment will be described. FIG. 1 is a diagram illustrating a target substance detection device. The target substance detection device 10 includes a photonic crystal biosensor 11 (target substance capture device 21) according to the present embodiment, a light detection unit 12, and a processing unit 13.

[Photonic crystal biosensor]
First, the photonic crystal biosensor 11 will be described. The photonic crystal biosensor 11 includes a target substance capturing device 21, an upper plate 22, and a lower plate 23. The upper plate 22 is provided with an opening 24. In the present embodiment, the photonic crystal biosensor 11 has a structure in which the target substance capturing device 21 is sandwiched between the upper plate 22 and the lower plate 23. In this embodiment, the photonic crystal biosensor 11 includes the upper plate 22 and the lower plate 23, but is not limited to this, and is formed only by the target substance capturing device 21. It may be.

(Target substance capture device)
FIG. 2 is a perspective view of the target substance capturing device 21. FIG. 3 is a plan view of the target substance capturing device 21. 4 is a diagram showing a cross section taken along the line AA in FIG. 3, and shows a cross section when the target substance capturing structure 25 is cut along a plane orthogonal to the surface 27 of the target substance capturing structure 25 as a main body of the photonic crystal. Show. The same applies to FIGS. 5 and 6 described later. 2 to 6 are diagrams schematically illustrating the thickness and size of the components constituting the target substance capturing device 21, which are different from actual ones. The same applies to this embodiment and other embodiments described later. As shown in FIGS. 2 to 4, the target substance capturing device 21 includes a target substance capturing structure 25 and a metal film 26. In the target substance capturing device 21, a cylindrical recess (hereinafter, appropriately referred to as a recess) 28 </ b> A is periodically formed on the surface 27 of the target substance capturing structure 25. The surface 27 is covered with a metal film 26 to form a reflection surface 29. The reflection surface 29 is the surface of the metal film 26. The reflection surface 29 reflects the light irradiated to the metal film 26 on the side opposite to the surface 27 side of the target substance capturing structure 25 of the metal film 26.

  First, the target substance capturing structure 25 will be described. A photonic crystal has a reflective surface in which concave portions with a predetermined depth or convex portions with a predetermined height are periodically formed on the surface, and when the reflective surface is irradiated with light of a specific wavelength (parallel light), the reflection It is a structure from which light can be obtained. A structure that obtains reflected light of a specific wavelength when light is irradiated onto a reflective surface having concave portions or convex portions formed periodically on the surface is generally called a photonic crystal.

  A photonic crystal is a structure having a lattice structure with sub-wavelength intervals. And when it irradiates the surface (reflection surface) of a photonic crystal with light of a wide wavelength range, it reflects or transmits light of a specific wavelength band depending on the surface state of the photonic crystal. The surface state of the photonic crystal depends on, for example, the shape and material of the photonic crystal. By reading the change in the reflected light or transmitted light, the change in the surface state of the photonic crystal can be quantified. Examples of changes in the surface state of the photonic crystal include adsorption of substances on the surface and structural changes. When a photonic crystal having a metal thin film formed on its surface is irradiated with light, an extreme value (maximum value or minimum value) appears in the light reflectance or light transmittance. This extreme value of reflectance or transmittance depends on the type of metal, the thickness of the metal, and the surface shape of the photonic crystal. The change in the surface state of the photonic crystal can be quantified by reading the light reflectance or light transmittance. The metal thin film will be described later. In order to quantify the change in the surface state of the photonic crystal from the change in reflected light or transmitted light, the following method can be used. For example, the amount of change in reflectance or transmittance at an extreme value (maximum value or minimum value) or the shift amount of a wavelength at which the reflectance or transmittance becomes an extreme value is obtained. Note that when there are a plurality of extreme values of reflectance or transmittance, attention is paid to arbitrary extreme values. Then, the change in the surface state of the photonic crystal can be quantified by obtaining the amount of change for the noticed extreme value or obtaining the shift amount of the wavelength that is the noticed extreme value.

  As shown in FIGS. 2 to 4, the target substance capturing structure 25 has a reflecting surface 29 in which concave portions 28 </ b> A are periodically formed on the surface 27. When the reflecting surface 29 is irradiated with light, light having a specific wavelength depending on the shape and material of the target substance capturing structure 25 is reflected.

  In the present embodiment, the recesses 28A are arranged in a triangular lattice shape. Further, the diameter D1 of the recess 28A is preferably 50 nm or more and 1000 nm or less, and more preferably 100 nm or more and 500 nm or less. The distance C1 between the centers of the recesses 28A is preferably 100 nm or more and 2000 nm or less, and more preferably 200 nm or more and 1000 nm or less. When the depth of the recess 28A is H1, the aspect ratio (H1 / D1) of the recess 28A is preferably 0.1 or more and 10 or less, more preferably 0.5 or more and 5.0 or less. is there. The dimensions of the recess 28A are not limited to those described above, and the recess 28A may be a quadrangular lattice, a hexagonal lattice, or the like. The recess 28A may be formed in a lattice shape by combining triangles, squares, and hexagons.

  The shape and dimensions of the target substance capturing structure 25 are not limited to the shapes shown in FIGS. For example, the target substance capturing structure 25 may have a rectangular or polygonal lattice pattern formed on the surface, or a parallel line pattern or a corrugated pattern formed on the surface (for details, see A pattern or the like formed periodically), or a combination of these patterns may be used.

  At least a part of the target substance capturing structure 25 on the metal film 26 side is a photocurable resin. All materials of the target substance capturing structure 25 may be a photo-curing resin. In producing the target substance capturing structure 25 using a resin, the temperature of the resin does not need to be higher than the glass transition point of the resin. Therefore, the volume change when the resin is cooled and solidified is reduced, so that the reduction in dimensional accuracy is suppressed.

  As the photocurable resin, an ultraviolet curable resin that is cured by irradiation with ultraviolet rays is preferable, and in particular, a ring-opening metathesis polymerization (FROMP) type ultraviolet curable resin containing fluorine in the structure is preferable. Specifically, a FROMP nanoimprint material (trade name) manufactured by Mitsui Chemicals, Inc. can be used. When the photo-curing resin contains fluorine, the releasability is improved, and the remaining uncured resin in the mold can be reduced. Moreover, since the photocurable resin containing fluorine has high releasability, it can be used without applying a release agent to the mold.

  The target substance capturing structure 25 is manufactured by performing fine processing on the surface of the material substrate. As a processing method, optical nanoimprint is preferable.

  Next, the metal film 26 will be described. In the present embodiment, as shown in FIG. 4, the target substance capturing structure 25 has a surface 27 covered with a metal film 26. The metal film 26 is preferably formed using one or more of gold (Au), silver (Ag), platinum (Pt), and aluminum (Al). In the present embodiment, the metal film 26 is made of Au. Au is preferable as the reflective surface 29 because it is excellent in stability. When one or more of silver (Ag) and aluminum (Al) is used for the metal film 26, the surface is preferably covered with gold. By doing in this way, the usage-amount of gold | metal | money can be reduced and the manufacturing cost of the target substance capture structure 25 can be suppressed.

  When the thickness of the metal film 26 is small, part of the incident light on the target substance capturing structure 25 may pass through the metal film 26. As a result, a large amount of unnecessary information may be included in the reflected light from the target substance capturing structure 25, such as a reduction in the amount of information obtained from the reflected light, diffracted light, or reflected light from the back surface of the target substance capturing structure 25. There is sex. By appropriately increasing the thickness of the metal film 26, unnecessary information contained in the reflected light from the target substance capturing structure 25 can be reduced, and the detection accuracy and concentration measurement precision of the target substance can be improved. it can. In addition, it is preferable that the thickness of the metal film 26 is moderately small because a detailed pattern shape can be easily formed on the surface 27 of the target substance capturing structure 25. For example, the corners of the pattern become sharp and it becomes easy to ensure the dimensions of the pattern. From this point of view, in the present embodiment, the thickness of the metal film 26 is preferably 30 nm or more and 1000 nm or less, more preferably 150 nm or more and 500 nm or less, and further preferably 200 nm or more and 400 nm or less. This is because the change of the reflectance with respect to the wavelength becomes almost the same when the thickness of the metal film 26 exceeds 200 nm.

The metal film 26 can be formed on the surface 27 of the target material capturing structure 25 by sputtering or vapor deposition. The outermost surface of the metal film 26 is preferably Au. When Ag, Pt, and Al are used for the metal film 26, the wavelength of reflected light at each extreme value is 1.5 times that when Au is used as the metal film 26. Thus, Ag, Pt, and Al have a sensitivity that is 1.5 times that of Au. Since Ag is easily oxidized, it is preferable to form an oxide thin film such as Au or SiO ₂ that is not easily oxidized after forming Ag on the surface 27 of the target substance capturing structure 25. In this case, an Au film having a thickness of 5 nm can be formed on the surface of the Ag film having a thickness of 200 nm. When an Au film having a thickness of 5 nm is formed on the surface of an Ag film having a thickness of 200 nm, the sensitivity is 1.5 times that of an Au film having a thickness of 200 nm. Further, no change in sensitivity was observed with or without the 5 nm Au film. Since Al is also easily oxidized like Ag, after forming an Al film on the surface 27 of the target substance capturing structure 25, it is preferable to form an oxide thin film such as Au or SiO ₂ that is not easily oxidized. In order to modify with an antibody or the like, it is preferable that Pt also forms an oxide thin film such as Au or SiO ₂ .

  The surface 27 of the target substance capturing structure 25 is preferably modified using 3-triethoxysilylpropylamine (APTES) or the like. When the Au or Ag metal film 26 is formed on the surface 27 of the target substance capturing structure 25, it is not APTES but has a thiol group at one end and a functional group such as an amino group or a carboxyl group at the other end. It is preferable to modify the surface 27 of the target substance capturing structure 25 by using a carbon chain having the following. When the metal film 26 other than Au or Ag is formed on the surface 27 of the target substance capturing structure 25, a silane coupling agent having a functional group at one end, such as APTES, is used to target the target substance capturing structure 25. It is preferable to modify the surface 27.

  Since the target substance capturing device 21 is formed by covering the surface 27 of the target substance capturing structure 25 with the metal film 26 to form the reflecting surface 29, the reflecting surface 29 corresponds to the recess 28 </ b> A of the target substance capturing structure 25. The recesses 28B of the target substance capturing device 21 are periodically formed. Similarly to the recess 28A, the recess 28B is arranged at a position where a plurality of adjacent triangular lattices are formed when viewed from above when adjacent recesses are connected by a line. Moreover, although the diameter D2 of the recessed part 28B is based also on the thickness of the metal film 26, it is preferable that they are 50 nm or more and 1000 nm or less, More preferably, they are 100 nm or more and 500 nm or less. Further, the distance C2 between the centers of the recesses 28B is preferably 100 nm or more and 2000 nm or less, more preferably 200 nm or more and 1000 nm or less, like the distance C1 between the centers of the recesses 28A. Further, when the depth of the recess 28B is H2, the aspect ratio (H2 / D2) of the recess 28B is preferably 0.1 or more and 10 or less, more preferably 0.5 or more and 5.0 or less. is there. In addition, the dimension of the recessed part 28B is not limited to what was mentioned above.

The recess 28B is formed such that the wall surface 28a of the recess 28B has a predetermined angle with the bottom surface 28b of the recess 28B. FIG. 5 is a partially enlarged view of the wall surface 28a of the recess 28B. FIG. 6 is a partially enlarged view of the wall surface of another concave portion of the target substance capturing device. In FIG. 5, the metal film 26 provided on the surface 27 of the target substance capturing structure 25 is omitted for convenience of explanation. As shown in FIG. 5, the wall surface 28 a of the recess 28 </ b> B has a predetermined angle with respect to the bottom surface 28 b where the recess 28 </ b> B is flat. In the cross section passing through the center of gravity of the bottom surface 28b of the recess 28B, the boundary between the wall surface 28a and the bottom surface 28b of the recess 28B is defined as a first boundary portion 31. A boundary between the surface 27 and the wall surface 28 a of the recess 28 </ b> B is defined as a second boundary portion 32. An intersection point between a straight line passing through the first boundary portion 31 in the direction perpendicular to the bottom surface 28b and a straight line passing through the second boundary portion 32 in the horizontal direction relative to the bottom surface 28b is defined as an intersection point A. A distance connecting the first boundary portion 31 and the second boundary portion 32 with a straight line is L1. A distance connecting the first boundary 31 and the intersection A with a straight line is L2. A distance connecting the second boundary portion 32 and the intersection A with a straight line is L3. Let θ be the angle formed by L1 and L2. At this time, in the recess 28B, an angle θ formed by L1 and L2 is formed so as to satisfy the following expressions (1) and (2).
tan θ = L3 / L2 (1)
0 ≦ tan θ ≦ 1.0 (2)

When light is irradiated onto the surface of a metal having a structure in which concave holes (holes) are periodically arranged, a peak is observed in the wavelength spectrum of the reflected light. The wavelength (peak wavelength) at which the reflectance with respect to the wavelength of the reflected light is maximized can be generally obtained by the following formula (i). In equation (i), λ _peak is the peak wavelength, a ₀ is the hole period, i, j are the diffraction orders, ε _m is the dielectric constant of the metal, and ε _d is The dielectric constant of the environment.

  According to the above formula (i), the peak wavelength can be obtained if the period in which the concave portion 28B is disposed is given. When observing the spectrum of the peak wavelength, the position of the peak wavelength can be easily specified when the width of the spectrum of the peak wavelength is smaller. Therefore, when the period in which the recesses 28B are arranged is clearly given, the spectrum width of the peak wavelength is reduced, and the position of the peak wavelength is easily specified.

  The target substance capturing device 21 has a periodic structure in which the recesses 28 </ b> B are periodically formed on the reflection surface 29. By forming the wall surface 28a of the recess 28B on the reflection surface 29 so as to satisfy the above formulas (1) and (2), the width of the shape of the wavelength spectrum of the reflected light becomes narrow, and the peak wavelength of the reflected light can be easily achieved. Can be specified. Thereby, the target substance can be detected with high accuracy. As a result, the sensor sensitivity of the photonic crystal biosensor 11 can be increased. The width of the shape of the wavelength spectrum of the reflected light is a half width.

Moreover, it is preferable that the recessed part 28B is formed so that following formula (2) 'may be satisfy | filled. By forming the wall surface 28a of the recess 28B so as to satisfy the above formula (1) and the following formula (2) ′, the shape of the wavelength spectrum of the reflected light is further narrowed, and the peak wavelength of the reflected light is further increased. Can be easily identified. As a result, the target substance can be detected with higher accuracy.
0 ≦ tan θ ≦ 0.7 (2) ′

  From Expressions (2) and (2) ′, θ is 0 degree or more. When θ = 0 degrees, the connection portion K between the surface 27 of the target substance capturing device 21 and the wall surface 28a of the recess 28B is approximately 90 degrees. When the connecting portion K is approximately 90 degrees, it becomes difficult to control the shape of the target substance capturing device 21, particularly the shape of the recess 28B. That is, it becomes difficult to obtain the desired shape of the recess 28B. It is preferable to make tan θ> 0, that is, θ larger than 0, since the desired shape of the recess 28B can be easily obtained. For example, when the target substance capturing structure 25 is made by an injection mold or imprint, a draft angle can be secured, which is preferable. Further, the target substance capturing device 21 is washed with water having a relatively high pressure. However, when the angle of the connection portion K is approximately 90 degrees, the angle is easily removed. As a result, the recess 28B may not have the desired shape. Since tan θ> 0, that is, by making θ larger than 0, the possibility that the corner of the connecting portion K can be taken can be reduced. Therefore, the concave portion 28B is preferable because it has an intended shape even after cleaning. Furthermore, by making tan θ> 0, that is, θ larger than 0, water easily enters the recess 28B, so that the target substance can be reliably captured in the recess 28B.

(Method for producing photonic crystal)
Next, an example of a process for producing the target substance capturing device 21 by optical nanoimprinting will be described. FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 8 are diagrams for explaining an example of the method for manufacturing the target substance capturing device according to the present embodiment. In step S <b> 101 shown in FIG. 7A, first, a sheet-like resin 100 in which the photocurable resin 102 is laminated on the resin sheet 101 is prepared. In optical nanoimprinting, a mold 103 having a nanometer-level microstructure or a nanometer-level periodic structure pattern is used. In step S102 shown in FIG. 7-2, the mold 103 is pressed against the photocurable resin 102 on the resin sheet 101, and is irradiated with ultraviolet rays UV for a predetermined time while being pressed with a predetermined pressure. Next, in step S103 shown in FIG. 7-3, after irradiating for a predetermined time, the mold 103 is released from the cured photocurable resin 102 ′ to obtain a photonic crystal 100 ′ to which a fine structure and a periodic structure are transferred. . The cured photo-curing resin 102 'corresponds to the target substance capturing structure 25 that is the main body of the photonic crystal in FIGS.

  After the target substance capturing structure 25 is produced, a metal film 26 is formed on the surface in contact with the mold 103 by sputtering or vapor deposition as shown in step S104 shown in FIG. Is completed.

  FIGS. 9-1 is a figure which shows the metal mold | die used by optical nanoimprint. FIG. 9-2 is a diagram illustrating a pattern transferred to a resin by a metal mold by optical nanoimprint. The mold 103 is a rectangular silicon (Si) substrate having an outer shape of 10 mm square and a thickness of about 0.6 nm, and has a dot pattern formation region 105 of 5 mm square near the center of the rectangle. In the dot pattern formation region 105, columnar protrusions 106 having a height of 200 nm are provided at predetermined intervals. The protrusion 106 is transferred to the photo-curing resin 102 ′ to form a recess 28 </ b> A.

  FIG. 10 is a schematic diagram for explaining each dimension of the unevenness transferred to the photo-curing resin. In FIG. 10, a is a cycle in which the unevenness is repeated, b is the diameter of the bottom of the recess, c is the diameter of the entrance of the recess, and d is the depth of the recess (or the height of the protrusion). The uneven | corrugated period of the used metal mold was 599 nm or more and 601 nm or less.

Table 1 shows the result of comparing the photonic crystal produced by optical nanoimprinting and the photonic crystal produced by thermal nanoimprinting for each dimension shown in FIG. Each dimension is an average value obtained by measuring a plurality of locations on the same test piece.

  As shown in Table 1, the photonic crystal produced by optical nanoimprint had a deeper recess depth d than the photonic crystal produced by thermal nanoimprint. The uneven period a indicating the lattice spacing was also closer to the mold.

  FIG. 11A is a measurement result of an atomic force microscope (AFM) with a photonic crystal produced by optical nanoimprint. FIG. 11-2 is an atomic force microscope measurement result of the photonic crystal produced by thermal nanoimprint. Also from this result, it can be seen that the uneven period a indicating the lattice interval is closer to the mold. Moreover, in the photonic crystal by optical nanoimprint, the variation of each dimension shown in FIG. 10 was hardly confirmed.

The peak ratio can be obtained from the length l in the measurement chart shown in FIGS. 11A and 11B and the distance m to the peak height by the following equation.
Peak ratio = (l−m) / l
It can be seen that the greater the peak ratio, the deeper the recess.

(Modification)
12A, 12B, and 12C are diagrams illustrating a method for manufacturing a target substance capturing device according to a modification of the present embodiment. In the method for manufacturing the target substance capturing device described above, the sheet-like resin 100 in which the photocurable resin 102 is laminated on the resin sheet 101 is prepared. The difference is that the photocurable resin 102 is directly applied to the mold 103 using 104 or the like. In step S <b> 201 shown in FIG. 12A, the photocurable resin 102 is directly applied onto the mold 103, and the roller 104 is made uniform. In step S202 shown in FIG. 12-2, the photocurable resin 102 is irradiated with ultraviolet light from a light source to be cured. In step S203 shown in FIG. 12C, by releasing from the mold 103, a photo-curing resin 102 ′ to which the pattern of the mold 103 is transferred is obtained. Here, a roller has been described as an example, but a squeegee may be used.

(Target substance capture substance)
Next, the target substance capturing substance that captures the target substance will be described. The target substance is an object to be detected by the target substance detection apparatus 10 and may be any of a polymer such as a protein, an oligomer, and a low molecule. The target substance is not limited to a single molecule, and may be a complex composed of a plurality of molecules. Examples of the target substance include pollutants in the atmosphere, harmful substances in water, biomarkers in the human body, and the like. Of these, cortisol and the like are preferable. Cortisol is a low molecular weight substance with a molecular weight of 362 g / mol. Cortisol is attracting attention as a substance that evaluates the degree of stress felt by humans because cortisol concentration in saliva increases when humans feel stress. If the concentration is measured using cortisol as a target substance, for example, the degree of stress can be evaluated by measuring the concentration of cortisol contained in human saliva. If the degree of stress is evaluated, it can be determined whether or not the subject is in a stress state at a level that leads to mental illness such as depression.

  The target substance capturing substance is a substance that binds to the target substance and captures the target substance. Here, the term “bonded” refers to a bond that is not chemically bonded, such as a bond by chemical adsorption or van der Waals force, in addition to the case of chemically bonding. Preferably, the target substance capturing substance is a substance that specifically reacts with the target substance to capture the target substance, and is preferably an antibody having the target substance as an antigen. Specific reaction means selectively forming a complex by reversibly or irreversibly binding to a target substance, and is not limited to a chemical reaction. Further, a substance that reacts specifically may exist in addition to the target substance. Even if there is a substance that reacts with the target substance capturing substance in addition to the target substance in the sample, the target substance can be quantified if the affinity is very small compared to the target substance. As the target substance capturing substance, an antibody using the target substance as an antigen, an artificially prepared antibody, a molecule composed of a substance constituting DNA such as adenine, thymine, guanine, or cytosine, a peptide, or the like can be used. When the target substance is cortisol, the target substance capturing substance is preferably a cortisol antibody.

  A known method can be employed to produce the target substance capturing substance. For example, the antibody can be produced by a serum method, a hybridoma method, or a phage display method. Molecules composed of substances constituting DNA can be prepared by, for example, the SELEX method (Systematic Evolution of Ligands by Exponential Enrichment). The peptide can be prepared by, for example, a phage display method. The target substance capturing substance does not need to be labeled with any enzyme / isotope. However, it may be labeled with an enzyme / isotope.

  In the present embodiment, the target substance capturing substance is fixed to the reflecting surface 29 of the target substance capturing apparatus 21 shown in FIG. Examples of means for fixing the target substance capturing substance to the reflection surface 29 of the target substance capturing apparatus 21 include chemical bonding such as covalent bonding, chemical adsorption, and physical adsorption, and a physical bonding method. These means can be appropriately selected according to the properties of the target substance-capturing substance. For example, when adsorption is selected as the fixing means, the adsorption operation is as follows. For example, a solution containing a target substance is dropped on the reflection surface 29 of the target substance capturing device 21, and the target substance capturing device 21 is cooled and heated for a predetermined time, at room temperature, or as necessary. The substance trapping substance is adsorbed on the reflecting surface 29.

  In the photonic crystal biosensor 11, an antibody (for example, cortisol antibody) that binds only to a specific antigen (for example, cortisol) is previously adsorbed (fixed) on the surface of the reflection surface 29 of the target substance capturing device 21. Thereby, the photonic crystal biosensor 11 can detect a specific antigen. This is because the optical characteristics of the target substance capturing structure 25 and various biological / chemical reactions that occur on or near the surface of the target substance capturing structure 25, for example, an antigen antibody in which a specific antigen reacts only with a specific antibody Reaction.

  The photonic crystal biosensor 11 may be one in which a blocking agent (protective substance) is immobilized on a reflective surface 29 on which an antibody that is a target substance capturing substance is immobilized. The blocking agent is immobilized before the target substance is brought into contact with the photonic crystal biosensor 11. The surface of the reflective surface 29 of the target substance capturing structure 25 is generally superhydrophobic. For this reason, impurities other than the antibody that is the target substance-capturing substance may be adsorbed on the reflecting surface 29 due to the hydrophobic interaction. Furthermore, since the optical characteristics of the target substance capturing structure 25 are greatly influenced by the surface state, it is preferable that no impurities are adsorbed on the reflection surface 29 of the target substance capturing structure 25. By fixing the blocking agent on the reflection surface 29 of the target substance capturing structure 25, the detection accuracy of the reflected light can be improved.

  Therefore, a so-called blocking agent is fixed in advance so that impurities and the like are not fixed to portions other than the portion where the antibody that is the target substance capturing substance is adsorbed (fixed) to the reflection surface 29 of the target substance capturing structure 25. It is preferable to keep it. In order to adsorb the blocking agent in advance, the blocking agent is brought into contact with the surface of the target substance capturing structure 25. As the blocking agent, skim milk, bovine serum albumin (BSA), or the like can be used.

  Next, the basic principle by which the photonic crystal biosensor 11 detects the target substance antigen and its concentration will be described. 13 to 16 are diagrams for explaining the principle of the photonic crystal biosensor 11. In general, the photonic crystal biosensor 11 uses the optical characteristics of the target substance capturing structure 25 and various biological / chemical reactions that occur on or near the surface of the target substance capturing structure 25, for example, a specific antigen is specified. A minute amount of protein or low molecular weight substance is detected by utilizing an antigen-antibody reaction that reacts only with the antibody. The photonic crystal biosensor 11 has the wavelength of the reflected light due to at least one of the surface plasmon resonance phenomenon and the localized surface plasmon resonance phenomenon when the reflecting surface 29 of the target substance capturing device 21 is irradiated with light of a specific wavelength. Use the phenomenon that the value shifts.

  As shown in FIG. 13, an antibody (target substance capturing substance) 34 is fixed to the reflecting surface 29 of the target substance capturing apparatus 21 by adsorption.

  Next, as shown in FIG. 14, a blocking agent (protective substance) 35 is preliminarily adsorbed on a portion of the reflective surface 29 other than the portion where the antibody 34 is adsorbed, that is, the reflective surface 29 other than the portion where the antibody 34 is adsorbed. . This prevents impurities or the like from being adsorbed on the reflective surface 29 other than the portion where the antibody 34 is adsorbed.

  Next, as shown in FIG. 15, an antigen (target substance) 36 is brought into contact with the photonic crystal biosensor 11 on which the antibody 34 and the blocking agent 35 are adsorbed, and an antigen-antibody reaction is performed. A complex 37 in which the antigen 36 is captured by the antibody 34 is fixed to the reflecting surface 29.

  Next, as illustrated in FIG. 16, the light detection unit 12 illustrated in FIG. 1 emits light (incident light) LI having a specific wavelength in a state where the antigen 36 is captured by the reflection surface 29 of the target substance capturing structure 25. The reflecting surface 29 of the target substance capturing device 21 is irradiated with parallel light. The light detection unit 12 illustrated in FIG. 1 detects the reflected light LR reflected by the reflecting surface 29 and obtains the wavelength of the extreme value of the reflected light LR. Then, the processing unit 13 shown in FIG. 1 obtains the wavelength at the extreme value of the intensity of the reflected light LR and the shift amount of the wavelength at the extreme value of the intensity, and acquires the antigen 36 captured on the reflection surface 29 of the target substance capturing device 21. The presence / absence of the antigen 36 is detected and the concentration of the antigen 36 is obtained.

  Based on the principle described above, the photonic crystal biosensor 11 can change the type of various biological substances such as proteins or low molecular weight substances, such as proteins, which are detection target substances, by changing the type of combination of the antibody 34 and the antigen 36. it can.

  In the photonic crystal biosensor 11, the antigen 36 is captured by the antibody 34 fixed to the reflecting surface 29, whereby the state of the reflecting surface 29 changes and the reflected light LR changes. The photonic crystal biosensor 11 outputs an optical physical quantity. This physical quantity correlates with a change in the surface state on the reflecting surface 29 of the target substance capturing device 21 and correlates with the amount of the complex 37 formed by capturing the antigen 36 on the antibody 34 fixed to the reflecting surface 29. The optical physical quantity is, for example, the shift amount of the wavelength at which the intensity of the reflected light LR is an extreme value, the change amount of the reflectance of the light, the shift amount of the wavelength at which the reflectance of the light is an extreme value, the intensity of the reflected light LR. And the amount of change in the extreme value of the intensity of the reflected light LR. In the present embodiment, the shift amount of the wavelength at which the intensity of the reflected light LR or the reflectance of the light becomes an extreme value is used.

  In order to output an optical physical quantity, for example, the following is performed. Light is incident perpendicularly to the reflecting surface 29 of the target substance capturing device 21, and the reflected light LR is detected. The reflected light LR can also be detected by making light incident at an angle with respect to the normal of the reflecting surface 29 of the target substance capturing device 21. By detecting the reflected light LR, the target substance detection device 10 shown in FIG. 1 can be made compact. In the case of detecting vertically incident and vertically reflected light, it is preferable to detect the reflected light LR by entering light using a bifurcated optical fiber. This structure will be described later.

(Production method of photonic crystal biosensor)
17, 18 and 19 are explanatory diagrams of the photonic crystal biosensor 11. FIG. An example of a method for producing the photonic crystal biosensor 11 shown in FIG. 1 will be described with reference to FIGS. 17, 18, and 19. As shown in FIG. 17, after the target substance capturing device 21 is installed on the lower plate 23, the upper plate 22 is installed on the lower plate 23 as shown in FIG. The photonic crystal biosensor 11 is manufactured by sandwiching the plate 23 and the upper plate 22. The end of the opening 24 on the lower plate 23 side is closed by the reflection surface 29 of the target substance capturing structure 25. With such a structure, the upper plate 22 has a droplet holding portion 38 having a constant volume, which is formed by being surrounded by the inner wall on the opening 24 side and the reflecting surface 29. The inner wall on the opening 24 side refers to the inner wall of the upper plate 22 that is a boundary surface between the upper plate 22 and the opening 24.

  FIG. 19 shows a state in which a predetermined solution is dropped on the droplet holder 38. In this case, since the droplet holding part 38 exhibits the droplet holding function, the solution is prevented from flowing out from the opening 24. Further, as long as the amount of the solution is such that it spreads to the droplet holding unit 38, the target substance can be detected and measured.

  The shape of the opening 24 is not limited to a cylindrical shape, and may be any other shape as long as a droplet can be held inside the opening 24. Further, when the opening 24 has a cylindrical shape, its diameter and the like may have various diameters according to the type of combination of the antibody 34 and the antigen 36, the required measurement accuracy, or the optical system of the reflected light detector. it can. The diameter of the opening 24 is preferably 0.5 mm or more and 10 mm or less, more preferably 2 mm or more and 6 mm or less in consideration of the operation when adsorbing the antigen 36 to the antibody 34 and the convenience of handling. It is.

  The material of the upper plate 22 and the lower plate 23 is not particularly limited. However, in consideration of the cleanliness of the surfaces of the upper plate 22 and the lower plate 23, it is preferably formed using stainless steel, polycycloolefin resin, silica, or the like.

  Next, another form of the photonic crystal biosensor 11 will be described. The upper plate 22 may be formed of a hydrophobic material. In particular, in detection and measurement of a so-called hydrophilic solution such as saliva, if the upper plate 22 is formed of a hydrophobic material, the solution can be accurately collected in the droplet holder 38. In the detection and measurement of so-called lipophilic solutions such as lipids, the solution can be accurately collected in the droplet holder 38 if the upper plate 22 is formed of a hydrophobic material.

  Furthermore, the upper plate 22 may be formed of a material having water repellency, oil repellency, or water / oil repellency. That is, a surface treatment or coating that exhibits hydrophobicity, lipophilicity, hydrophilicity, water repellency, and oil repellency may be applied to the upper plate 22 in accordance with the properties of the solution. By doing so, the solution can be accurately collected in the droplet holder 38.

  The photonic crystal biosensor 11 determines the position of the photonic crystal biosensor 11 relative to the light detection unit 12 illustrated in FIG. 1 and fixes the photonic crystal biosensor 11 below the photonic crystal biosensor 11. It is preferable to attach a fixing material (target substance capturing part fixing means, photonic crystal biosensor fixing means) for the purpose. As the fixing material, a magnet sheet, a double-sided tape, an adhesive, or the like can be used. Moreover, in order to fix the photonic crystal biosensor 11, a vacuum chuck or an electrostatic chuck may be used as a fixing mechanism instead of a fixing material. By fixing the photonic crystal biosensor 11, it is possible to reduce the displacement of the measurement position due to vibration during detection and measurement. As a result, more accurate detection and measurement can be realized.

  20 and 21 are diagrams for explaining the photonic crystal biosensor fixing means. 20 shows a state before the magnet sheet 39 is attached, and FIG. 21 shows a state after the magnet sheet 39 is attached. The photonic crystal biosensor 11 has a magnet sheet 39 attached to the lower side of the photonic crystal biosensor 11. The magnet sheet 39 functions as a photonic crystal biosensor fixing means.

  The photonic crystal biosensor 11 is uniformly produced by optical nanoimprint or the like. In order for the target substance detection device 10 to detect the reflected light more accurately, it is preferable that the incident site and the reflected site of the light irradiated on the photonic crystal biosensor 11 are accurately positioned.

  That is, the positional relationship at the time of measurement between the photonic crystal biosensor 11 and the measurement probe described later is preferably the same before and after the antigen-antibody reaction, and it is preferable to measure the same part. Therefore, the distance between the measurement probe and the reflecting surface 29 of the photonic crystal biosensor 11 is preferably the same before and after the antigen-antibody reaction, and is preferably fixed to 50 μm or more and 500 μm or less. Since the photonic crystal biosensor 11 includes the upper plate 22, the upper plate 22 functions as a spacer, and the distance between the measurement probe and the reflection surface 29 of the photonic crystal biosensor 11 can be made constant.

  The photonic crystal biosensor 11 may be marked with a positioning marker that displays a specific position on the reflecting surface 29. The marker can be attached by photolithography, sputtering, vapor deposition, a lift-off process using these, printing by ink or the like, or pattern formation by imprinting. The marker may be attached to either the front surface (the reflective surface 29 side) or the back surface (the opposite side of the reflective surface 29) of the photonic crystal biosensor 11 as long as the position can be read. Alternatively, the measurement part of the target substance capturing structure 25 may be removed and a marker may be attached to the target substance capturing structure 25 itself. Further, a marker may be attached to the upper plate 22 and the lower plate 23.

  Next, still another form of the photonic crystal biosensor 11 will be described. FIG. 22 is a diagram for explaining another form of the photonic crystal biosensor 11. As shown in FIG. 22, the photonic crystal biosensor 11 includes a member that closes the opening 24. The member that closes the opening 24 includes a cover 41 with a hole and a sheet 42. The cover 41 with a hole is a plate-like member having an opening 43, and the cover 41 with a hole is provided on the surface (the reflection surface 29 side) of the photonic crystal biosensor 11. The sheet 42 is provided on the opposite side (light incident side) of the cover 41 with holes from the photonic crystal biosensor 11. The sheet 42 functions as a covering member. In the photonic crystal biosensor 11, the openings 24 and 43 are closed by the cover 41 with a hole and the sheet 42.

  A space surrounded by the inner wall on the opening 43 side of the cover 41 with the hole, the inner wall on the opening 24 side, and the reflection surface 29 of the target substance capturing structure 25 is a droplet holding unit 44 having a constant volume. The inner wall on the opening 43 side refers to the inner wall of the holed cover 41 that is a boundary surface between the holed cover 41 and the opening 43. The opening 43 is covered with the sheet 42 after the target substance is disposed in the droplet holding unit 44. As a result, the droplet holder 44 is blocked by the sheet 42.

  The photonic crystal biosensor 11 includes the cover 41 with a hole and the sheet 42, thereby suppressing evaporation of the solution dropped on the opening 24 of the photonic crystal biosensor 11. For this reason, it can suppress that the density | concentration of the solution by evaporation at the time of an antigen antibody reaction changes. In addition, the photonic crystal biosensor 11 includes the cover 41 with a hole and the sheet 42, so that foreign matters can be prevented from entering the solution from the outside.

  Furthermore, by filling the droplet holder 44 with a solution, the reflected light can be measured more accurately in a state where the solution is filled. In this case, the sheet 42 is preferably a transparent material, and more preferably a sheet that absorbs light having a wavelength at the extreme value of the intensity of reflected light. For example, the material of the sheet 42 is preferably quartz (silica) or the like when measured with reflected light from the visible light region to the ultraviolet region.

[Photodetection section]
Next, the light detection unit 12 shown in FIG. 1 will be described. The light detection unit 12 illustrated in FIG. 1 includes a light source 51, a measurement probe 52, a light detection device 53, a first optical fiber 54, a second optical fiber 55, and a collimator lens 56. The light source 51 and the measurement probe 52 are optically connected by a first optical fiber 54. The measurement probe 52 and the light detection device 53 are optically connected by a second optical fiber 55. If necessary, a control device that is connected to the light source 51 and the light detection device 53 and that controls the light source 51 and processes a signal from the light detection device 53 may be provided.

  FIG. 23 is a diagram illustrating an example in which the light detection unit 12 irradiates the photonic crystal biosensor 11 with light. The first optical fiber 54 shown in FIG. 1 guides the light from the light source 51 shown in FIG. 1 to the measurement probe 52, and irradiates the reflection surface 29 of the target substance capturing device 21 of the photonic crystal biosensor 11 from the measurement probe 52. . The collimating lens 56 irradiates the reflecting surface 29 of the target substance capturing structure 25 as incident light LI after collimating the light emitted from the first optical fiber 54 and irradiated from the measurement probe 52. The second optical fiber 55 receives the light reflected by the reflecting surface 29 of the target substance capturing device 21 as reflected light LR and guides it to the light detecting device 53 shown in FIG. Although the kind of collimating lens 56 is not specifically limited, For example, the antireflection film which has a nanostructure can be used. The light detection device 53 is a device for detecting light, which includes a light receiving element such as a phototransistor or a CCD (Charge Coupled Device).

  FIG. 24 is a diagram showing the structure of the measurement probe 52 included in the light detection unit 12 shown in FIG. In the measurement probe 52, the first optical fiber 54 and the second optical fiber 55 are joined. In the measurement probe 52, the light exit surface 61 of the first optical fiber 54 and the incident surface 62 of the reflected light LR of the second optical fiber 55 are disposed on the same surface (incident / exit surface) 63. As described above, the measurement probe 52 includes the first optical fiber 54 and the second optical fiber 55 on the emission side (emission surface 61 side) of the first optical fiber 54 and the incident side (incident surface 62 side) of the second optical fiber 55. It is united. Then, the measurement probe 52 enters light using the first optical fiber 54 and the second optical fiber 55 and detects the reflected light LR.

  Since the measurement probe 52 has such a structure, the incident light LI that irradiates the reflective surface 29 of the target substance capturing structure 25 and the reflected light LR from the reflective surface 29 are emitted from substantially the same position and incident. Can be made. While the measurement probe 52 is configured as described above, and the collimator lens 56 is used to convert the light from the measurement probe 52 into parallel light, the light detection unit 12 converts the incident light LI of parallel light onto the reflection surface 29. It can be incident vertically. At the same time, the reflected light LR reflected perpendicularly from the reflecting surface 29 can be received. By doing in this way, the measurement probe 52 can suppress the fall of reflected light intensity to the minimum, and can mainly detect the 0th-order light component of the reflected light LR. Thereby, since the process part 13 can acquire the exact information of the reflective surface 29 of the target substance capture apparatus 21, the detection accuracy of a target substance and the measurement precision of a density | concentration improve. The method for detecting the reflected light LR is not limited to the measurement probe 52 as described above. For example, a half mirror may be disposed between the collimating lens 56 and the reflection surface 29, and the reflected light LR may be separated by the half mirror and guided from the second optical fiber 55 to the light detection device 53.

  Next, the evaluation conditions of the light detection unit 12 will be described. FIG. 25 is a diagram illustrating evaluation conditions of the light detection unit 12 of the target substance detection device 10 according to the present embodiment. As shown in FIG. 25, the light detection unit 12 arranges a collimator lens 56 between the incident / exit surface 63 of the measurement probe 52 and the reflection surface 29 of the target substance capturing device 21. The distance (measurement distance) between the collimating lens 56 and the reflecting surface 29 is h, the diameter of the parallel light emitted from the collimating lens 56 is d1, and the opening 24 through which the reflecting surface 29 of the target substance capturing structure 25 is exposed. Let d2 be the diameter of. In this evaluation, h was 15 mm or 40 mm, d1 was 3.5 mm, and d2 was 5 mm. The optical axis ZL of the light applied to the reflecting surface 29 and the optical axis ZL of the reflected light reflected by the reflecting surface 29 are both orthogonal to the reflecting surface 29. The diameter of the measurement probe 52 is 200 μm. White light was used as the irradiation light. The reflectance is the ratio of the standard material (aluminum plate) to the reflected light intensity.

[Processing part]
Next, the processing unit 13 shown in FIG. 1 will be described. The processing unit 13 obtains the extreme wavelength of the reflected light detected by the light detection unit 12. At the same time, the processing unit 13 detects at least the presence / absence of the target substance (for example, the antigen 36 shown in FIGS. 15, 16, and the like) based on the obtained extreme wavelength shift (amount of wavelength shift). The processing unit 13 is, for example, a microcomputer. There is a correlation between the wavelength shift amount and the concentration of the target substance captured by the reflection surface 29 of the target substance capturing device 21. For this reason, the processing unit 13 can obtain the concentration of the target substance captured by the reflection surface 29 from the wavelength shift amount.

(Method of detecting target substance)
Next, a method for detecting a target substance (target substance detection method) using the target substance detection apparatus 10 shown in FIG. 1 will be described. In this example, a case will be described in which a cortisol antibody is adsorbed on the reflection surface 29 of the target substance capturing device 21 to detect and measure cortisol in saliva as a target substance to be detected. As the target substance capturing structure 25, a sheet of a FROMP nanoimprint material (trade name, manufactured by Mitsui Chemicals) on which a predetermined fine structure is formed by optical nanoimprint is cut into a predetermined size.

  FIG. 26 is a flowchart showing an example of the target substance detection method according to the present embodiment. First, in step S <b> 11, a cortisol antibody solution (cortisol antibody concentration of 1 μg / ml to 50 μg / ml) is dropped onto the reflection surface 29 of the target substance capturing device 21. Then, the photonic crystal biosensor 11 is allowed to stand at a predetermined temperature for a predetermined time for a predetermined time or if necessary, and the cortisol antibody is adsorbed on the reflection surface 29 of the target substance capturing device 21.

  Next, in step S <b> 12, a phosphate buffer solution (PBS) is dropped onto the reflecting surface 29 of the target substance capturing device 21. Thereafter, a rinsing process is performed a plurality of times for removal by centrifugal force or the like.

  Next, in step S13, skim milk is dropped as the blocking agent 35 onto the reflecting surface 29 of the target substance capturing structure 25, and the photonic crystal biosensor 11 is allowed to stand for a predetermined time at a predetermined time or a predetermined temperature if necessary. The skim milk is adsorbed to the non-adsorbing portion of the cortisol antibody on the reflection surface 29 of the target substance capturing device 21.

  Thereafter, in step S14, the rinsing process is performed a plurality of times with a phosphate buffer solution, similarly to the rinsing process (step S12). By the above-described operation, a predetermined process is performed on the reflection surface 29 of the target substance capturing device 21, and the photonic crystal biosensor 11 is formed.

  Next, in step S15, the light detection unit 12 detects the reflected light LR from the reflection surface 29 when the reflection surface 29 of the target substance capturing structure 25 is irradiated with light, and the processing unit 13 reflects the reflected light LR. Measure. For example, the processing unit 13 measures the spectrum of the reflected light intensity of the reflected light LR. The wavelength of the light (incident light LI) applied to the reflecting surface 29 is, for example, not less than 300 nm and not more than 2000 nm.

  Next, in step S16, first, saliva is prepared as a solution containing cortisol. Pretreatment such as saliva sampling and impurity removal is performed using, for example, a commercially available saliva collection kit. The preparation of saliva may be performed at any time before the saliva is dripped onto the photonic crystal biosensor 11. For example, it may be performed before the photonic crystal biosensor 11 is formed, may be performed in parallel with the formation of the photonic crystal biosensor 11, or may be performed after the reflected light intensity is measured. 10 μL to 50 μL of saliva after sampling and pretreatment is dropped onto the photonic crystal biosensor 11.

  Next, in step S17, the photonic crystal biosensor 11 is allowed to stand for a predetermined time at a predetermined temperature for a predetermined time or, if necessary, for an antigen-antibody reaction.

  Thereafter, in step S18, the rinsing process is performed a plurality of times with a phosphate buffer solution, similarly to the rinsing process (step S14).

  Next, in step S <b> 19, the target material detection device 10 is used to irradiate the reflection surface 29 of the target material capturing device 21 with light. The light irradiated at this time is the same as the light irradiated on the reflecting surface 29 in step S15. Then, the target substance detection device 10 measures the reflected light LR from the reflecting surface 29, for example, the spectrum of the reflected light intensity.

  The wavelength at the extreme value of the reflected light intensity of the photonic crystal biosensor 11 is affected and changed by the antigen-antibody reaction or the like in the vicinity of the reflecting surface 29 or the reflecting surface 29. For this reason, cortisol in saliva can be detected from the difference in wavelength at the extreme value of reflected light intensity before and after the reaction, that is, the amount of wavelength shift. Further, the concentration of cortisol in saliva can be obtained from the wavelength shift amount.

  In step S20, the processing unit 13 obtains a wavelength shift (wavelength shift amount) at the extreme value (minimum value) of the reflected light intensity (or reflectance) measured in step S19. The wavelength shift amount is, for example, the extreme value (minimum value) of the wavelength λ2 after the target substance is captured on the reflecting surface 29 and the reflected light intensity (or reflectance) when the target substance is not captured on the reflecting surface 29. ) Is the difference λ2−λ1 with respect to the wavelength λ1.

  In step S <b> 21, the processing unit 13 determines that cortisol is present in saliva, for example, when there is a wavelength shift amount of a predetermined amount or more. The processing unit 13 determines the concentration of cortisol based on the amount of wavelength shift using, for example, a relational expression between the amount of wavelength shift and the concentration of cortisol. At this time, the relational expression is obtained in advance and stored in the storage unit of the processing unit 13.

  In the example described above, the wavelength shift amount is obtained using the extreme wavelength of the reflected light intensity on the reflection surface 29 in a state where the target substance is not captured, but the present invention is not limited to this. In Step S15 and Step S19, when there are a plurality of extreme values, the extreme value to be focused is appropriately selected. Then, the wavelength λ1 and the wavelength λ2 are obtained for the selected extreme value.

  As described above, the photonic crystal biosensor 11 includes the target substance capturing device 21 in which a plurality of concave portions 28B are periodically provided on the reflection surface 29. The recess 28B is formed on the reflection surface 29 of the target substance capturing device 21 so that the wall surface 28a of the recess 28B has a predetermined angle with respect to the bottom surface 28b of the recess 28B. Thereby, the period of the concave portion 28B becomes clear, and the width (for example, half-value width) of the spectrum shape of the wavelength of the reflected light LR becomes small, so that the peak wavelength can be easily specified. Therefore, the target substance detection device 10 can accurately detect the target substance (cortisol in this example) from at least the solution.

  Further, the target substance detection apparatus 10 irradiates light (incident light LI) perpendicularly to the reflection surface 29 of the photonic crystal biosensor 11 with parallel light, and receives the reflected light LR reflected vertically by the reflection surface 29. Then, the target substance (for example, cortisol) is detected, or the concentration of the target substance is obtained. Since the target substance capturing device 21 is formed on the reflection surface 29 so that the wall surface 28a of the recess 28B has a predetermined angle with respect to the bottom surface 28b of the recess 28B, the peak wavelength of the reflected light LR can be easily specified. Can be done. For this reason, the detection accuracy of the target substance and the measurement accuracy of the concentration can be further improved. Moreover, the usage-amounts, such as a cortisol antibody solution, saliva, and a rinse liquid, can be reduced significantly.

  In the present embodiment, the target substance capturing device 21 has the antibody 34 immobilized on the reflecting surface 29, but the present invention is not limited to this, and the target substance capturing device 21 has the antibody 34 on the reflecting surface 29. You may use without fixing.

[Embodiment 2]
Another method for detecting a target substance using the target substance detection apparatus 10 shown in FIG. 1 will be described. The target substance capturing device 21 according to this embodiment is the same as that of the first embodiment except that the antigen (target substance) 36 is fixed to the reflecting surface 29 and the antibody 34 is adsorbed to the antigen 36. Therefore, redundant description is omitted.

  27 to 31 are diagrams for explaining the principle of the photonic crystal biosensor. In this embodiment, the specific reaction between the antibody 34 and the antigen 36 will be described using cortisol as the antigen 36 and an anti-cortisol antibody as the antibody 34.

  First, as shown in FIG. 27, the photonic crystal biosensor 11 is the same as the means for immobilizing the antibody 34 on the reflection surface 29 as the means for immobilizing the antigen 36 on the reflection surface 29 of the target substance capturing device 21. Can do. Examples of means for fixing the antigen 36 to the reflecting surface 29 include chemical bonding and physical bonding methods such as covalent bonding, chemical adsorption, and physical adsorption. These means can be appropriately selected according to the properties of the antigen 36.

  The amount of the antigen 36 immobilized on the target substance capturing device 21 is a fixed amount. Thus, when the antibody 34 is adsorbed to the antigen 36 immobilized on the target substance capturing device 21 and a complex 65 (see FIGS. 29 and 30) is formed, it correlates with the amount of the complex 65 formed. The physical quantity can be output by the photonic crystal biosensor 11. The fixed amount of the antigen 36 to be fixed may be appropriately changed. For example, it can be set to an optimum amount according to the range of the amount of the antigen 36 contained in the sample S.

  Thereafter, as shown in FIG. 28, the blocking agent 35 is fixed to a portion of the reflecting surface 29 where the antigen 36 is not attached.

  Next, the reflective surface 29 of the target substance capturing structure 25 is irradiated with light (incident light) LI of, for example, 300 nm or more and 900 nm or less as parallel light so that the optical axis is orthogonal to the reflective surface 29. A wavelength at which the intensity or reflectance of the reflected light LR at this time becomes an extreme value (a minimum value in this example) is λ1.

  Next, as shown in FIG. 29, a mixture M including the complex 65 of the antigen 36 and the antibody 34 and the antibody 34 is prepared. The mixture M is obtained by mixing the sample S containing the antigen 36 and the solution G containing a known amount of the antibody 34. The complex 65 is obtained by reacting the antibody 34 and the antigen 36 by mixing the sample S containing the antigen 36 and the solution G containing the known amount of the antibody 34. The antibody 34 remains in the mixture M without reacting with the antigen 36 by increasing the known amount of the antibody 34 to the amount of the site to which the antigen 36 is contained in the sample S. The mixture M is brought into contact with the reflecting surface 29 of the target substance capturing device 21. Thereby, as shown in FIG. 30, a complex 65 is formed on the reflective surface 29 by the antigen 36 and the antibody 34 fixed on the reflective surface 29. Thereafter, as shown in FIG. 31, the light (incident light) LI of, for example, 300 nm or more and 2000 nm or less is irradiated to the reflecting surface 29 of the target substance capturing device 21 as parallel light and the optical axis is orthogonal to the reflecting surface 29. To do. At this time, the wavelength at which the reflected light intensity or reflectance of the reflected light LR becomes an extreme value (a minimum value in this example) is λ2.

  The wavelength shift amount of the wavelength at which the light reflectance is an extreme value is λ2−λ1. The amount of wavelength shift changes according to the change in the surface state of the reflection surface 29 of the target substance capturing device 21. Based on this wavelength shift amount, the antigen 36 is detected and quantified. The photonic crystal biosensor 11 outputs an optical physical quantity. This physical quantity correlates with a change in the surface state on the reflecting surface 29 and correlates with the amount of the complex 65 formed by the antigen 36 and the antibody 34 immobilized on the reflecting surface 29.

  In this embodiment, cortisol that is the antigen 36 is fixed to the target substance capturing device 21 and an anti-cortisol antibody that is the antibody 34 is reacted. Compared to the case where the antibody 34 is immobilized on the reflective surface 29 of the target substance capturing device 21 as in the first embodiment and then the antigen 36 is reacted with the antibody 34, the target substance is captured as in this embodiment. When cortisol is immobilized on the reflecting surface 29 of the device 21 and then the anti-cortisol antibody is reacted with cortisol, the change in the surface state of the target substance capturing device 21 becomes larger, and the sensitivity of the photonic crystal biosensor 11 is higher. improves. In the present embodiment, the concave portion 28B is formed on the reflecting surface 29 of the target substance capturing device 21 so that the wall surface 28a of the concave portion 28B has a predetermined angle with respect to the bottom surface 28b of the concave portion 28B. Can be easily identified. For this reason, according to this embodiment, the sensitivity of the photonic crystal biosensor 11 can be further increased.

  Next, a method for measuring the concentration of the antigen 36 will be described. Let X be the amount of the site to which antigen 36 is bound in sample S, and C be the known amount of antibody 34 in mixture M. At this time, the relationship between X and C is such that X is less than C (X <C). In the mixture M, the antigen 36 and the antibody 34 undergo an antigen-antibody reaction to form a complex 65. Since X is less than C (X <C), the amount of antibody 34 in mixture M is C-X. When the mixture M is brought into contact with the reflecting surface 29 on which a certain amount of antigen 36 is fixed, the antibody 34 in the mixture M reacts with the antigen 36 on the reflecting surface 29 to form a complex 65. . The amount of the antigen 36 fixed to the reflecting surface 29 is equal to or more than the amount CX of the antibody 34 in the mixture M.

  When all the antibodies 34 in the mixture M have undergone an antigen-antibody reaction with the antigen 36 on the reflecting surface 29, the amount of the complex 65 becomes C-X. The wavelength shift amount Δλ obtained from the wavelengths λ1 and λ2 measured before and after the mixture M is brought into contact with the reflecting surface 29 corresponds to the amount of the composite 65 fixed to the reflecting surface 29. Therefore, Δλ = k × (C−X). k is a constant for converting the wavelength shift amount Δλ into the amount of the complex 65. The relationship between the amount of the composite 65 fixed to the reflecting surface 29 and the wavelength shift amount Δλ is obtained in advance. From the above relational expression, the amount X of the antigen 36 can be determined by C−Δλ / k. The concentration of the antigen 36 can be determined based on the amount X of the antigen 36.

  In the present embodiment, the photonic crystal biosensor 11 is fixed to the reflection surface 29 of the target substance capturing device 21 using, for example, a secondary antibody that specifically reacts with the complex 65 as a complex binding substance. You may make it react with the composite_body | complex 65. FIG. The secondary antibody is brought into contact with the reflecting surface 29 of the target substance capturing device 21 in an amount more than the complex 65. Then, a secondary antibody is added to all the complexes 65 to form a second complex. By doing in this way, the change of the surface state of the target substance capture apparatus 21 becomes still larger. As a result, the sensitivity of the photonic crystal biosensor 11 further increases. The secondary antibody may be used as it is, or may be used after adding other substances. Since the change in the surface state of the target substance capturing device 21 increases as the secondary antibody increases, the sensitivity of the photonic crystal biosensor 11 is increased by reacting with the complex 65 after adding another substance to the secondary antibody. Becomes even larger.

  In the case of forming the second composite on the reflective surface 29, the reflective surface 29 after the formation of the second composite is irradiated with light. The wavelength at which the reflected light intensity or reflectance obtained as a result is an extreme value (minimum value in this example) is λ2. When there are a plurality of extreme values, the extreme value of interest is appropriately selected. The wavelength λ1 and the wavelength λ2 are obtained for the selected arbitrary extreme value. The photonic crystal biosensor 11 outputs an optical physical quantity. This physical quantity correlates with a change in the surface state on the reflecting surface 29 and correlates with the amount of the second complex fixed to the reflecting surface 29. Thereby, the second complex is detected and quantified. Since the amount of the second complex is the same as the amount of the complex 65, the complex 65 can be quantified.

  In the target substance capturing device described above, the target substance capturing structure 25 may be a photonic crystal including a reflective surface having convex portions formed periodically on the surface instead of the concave portions.

  As described above, according to the present invention, the dimensional accuracy of the reflecting surface of the photonic crystal can be improved by forming the photonic crystal by the optical nanoimprint technique using the photocurable resin. Therefore, the target substance can be detected with high accuracy in such a detection apparatus using the photonic crystal. In addition, the present invention reduces the residue of uncured resin in the mold by using a material having excellent releasability for the optical nanoimprint technology, and can be easily used with an organic solvent or the like even if it remains. Can be removed. Furthermore, according to the present invention, it is possible to reduce the volume shrinkage during photocuring.

DESCRIPTION OF SYMBOLS 10 Target substance detection apparatus 11 Photonic crystal biosensor 12 Optical detection part 13 Processing part 21 Target substance capture apparatus 22 Upper plate 23 Lower plate 24, 43 Opening part 25 Target substance capture structure 26 Metal film 27 Surface 28A, 28B Cylindrical shape Recess (recess)
28a Wall surface 28b Bottom surface 29 Reflecting surface 31 First boundary portion 32 Second boundary portion 34 Antibody (target substance capturing substance)
35 Blocking agent (protective substance)
36 Antigen (target substance)
37, 65 Complex 38, 44 Droplet holder 39 Magnet sheet 41 Cover with hole 42 Sheet 51 Light source 52 Measuring probe 53 Photodetector 54 First optical fiber 55 Second optical fiber 56 Collimating lens 61 Emission surface 62 Incident surface 63 Same Surface (input / output surface)
82, 93 Deepest part A Intersection M Mixture LI Incident light LR Reflected light

Claims (9)

A target substance capturing structure having a surface in which concave portions or convex portions are periodically formed;
A metal film formed on at least a part of the surface to form a reflective surface,
In the target substance capturing structure, at least a part of the metal film side is a photocurable resin,
Target substance capture device.   The target substance capturing device according to claim 1, wherein the photocurable resin is an ultraviolet curable resin containing fluorine.   The target substance capturing device according to claim 2, wherein the ultraviolet curable resin is a ring-opening metathesis polymerization nanoimprint material containing fluorine.   The target substance capturing device according to any one of claims 1 to 3, wherein the target substance is a biological substance.   The detection apparatus provided with the target substance capture apparatus of any one of Claims 1-4. A photonic crystal substrate comprising the target substance capturing structure and the metal film;
The detection apparatus according to claim 5, wherein the detection apparatus is a biosensor that detects at least one of the presence of a target substance and the concentration of the target substance by a change in wavelength of reflected light from the photonic crystal substrate. Press a mold having a predetermined pattern against a sheet-like photocurable resin,
Irradiating the photocurable resin with light,
The mold is released from the cured photo-curing resin to obtain a target substance capturing structure having one surface in which concave portions or convex portions are periodically formed,
Forming a metal film on at least a part of the one surface;
A method for producing a target substance capturing device.   The method for producing a target substance capturing device according to claim 7, wherein the photocurable resin is an ultraviolet curable resin containing fluorine, and the light is an ultraviolet ray.   The method for producing a target substance capturing device according to claim 8, wherein the ultraviolet curable resin is a ring-opening metathesis polymerization nanoimprint material containing fluorine.