JP2014206463A - Target substance capture device - Google Patents

Abstract

A target substance capturing device capable of increasing the probability of obtaining an appropriate output as a sensor and a target substance detecting device including the target substance capturing device are provided. A plurality of non-flat portions are arranged and include a reflecting surface that captures a target substance and reflects irradiated light. When a natural number of 2 or more is set to M and a natural number of 2 or more different from M is set to N, an array in which a plurality of non-flat portions are arranged is arranged at a vertex position in a figure in which the non-flat portions are M times symmetric. A plurality of unit arrays arranged so that the centers of the non-flat portions are overlapped one by one, and the center of gravity of the figure that is M times symmetrical is one at the intersection of the lattice pattern in which the plurality of unit arrays are N times symmetrical They are arranged so as to overlap each other. The reflected light reflected by the reflecting surface exhibits an extreme value at a specific wavelength, and there are a plurality of extreme values. [Selection] Figure 3

Description

  The present invention relates to a target substance capturing apparatus that detects a target substance and a target substance detection apparatus including the target substance capturing apparatus.

  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 the reflected light reflected by the photonic crystal substrate, thereby detecting target substances and targets. Measuring the concentration of substances.

“Development of a mass-producible on-chip plasmonic nanohole array biosensor”: Kohei Nakamoto, Ryoji Kurita, Osamu Niwa, Toshiyuki Fujiicd and Munehiro Nishida, Received 20th July 2011, Accepted 27th September 2011

  The biosensor described in Non-Patent Document 1 performs sensing using one extreme value among the extreme values of reflected light of irradiated light. However, it is not easy to fabricate a photonic crystal substrate having a fine structure, and a few defects may greatly affect the wavelength spectrum of reflected light, and an appropriate output as a sensor may not be obtained. For this reason, a biosensor capable of obtaining an appropriate output as a sensor even if there is a slight defect in the photonic crystal substrate is desired.

  An object of the present invention is to provide a target substance capturing device that can increase the probability of obtaining an appropriate output as a sensor, and a target substance detection device including the target substance capturing device.

  The present invention is a target substance capturing device in which a plurality of non-flat portions are arranged, includes a reflecting surface that captures a target substance and reflects irradiated light, and a natural number of 2 or more is M, which is different from M When the natural number of 2 or more is N, the array in which the plurality of non-flat portions are arranged is such that the centers of the non-flat portions are one by one at the positions of the vertices in the figure in which the non-flat portions are M-fold symmetric. Including a plurality of unit arrays arranged so as to overlap each other, the plurality of unit arrays are arranged to overlap one by one the center of gravity of the figure that is M times symmetrical at the position of the intersection of the lattice pattern that is N times symmetrical, The reflected light reflected by the reflecting surface exhibits an extreme value at a specific wavelength, and there are a plurality of the extreme values.

  The reflected light of the light irradiated to the target substance detection device according to the present invention exhibits an extreme value at a specific wavelength, and there are a plurality of such extreme values. Accordingly, the target substance detection device according to the present invention can obtain an appropriate output as a sensor if measurement can be performed using any one of a plurality of extreme values. Therefore, the target substance detection device according to the present invention has a defect in the shape of the non-flat portion, and even if it is not possible to measure one extreme value among a plurality of extreme values, measurement of other extreme values is possible. Therefore, there is a high possibility that an appropriate output as a sensor can be obtained. Therefore, the target substance detection device according to the present invention can increase the probability that it can function as a sensor.

  In the present invention, it is preferable that the M is 3 and the N is 6.

  If the target substance capturing device of the present invention is used, the probability of obtaining an appropriate output as a sensor can be increased. The target substance detection apparatus provided with the target substance capturing apparatus of the present invention can increase the probability of obtaining an appropriate output as a sensor.

FIG. 1 is a diagram illustrating a target substance detection device. FIG. 2 is a perspective view of a metal film-coated photonic crystal. FIG. 3 is a plan view of the metal film-coated photonic crystal. FIG. 4 is a diagram showing a cross section taken along line AA in FIG. FIG. 5 is a diagram illustrating the reflectance with respect to the wavelength of the reflected light in the example. FIG. 6 is a diagram illustrating a cross section taken along the line AA in FIG. 3 when the non-flat portion is a convex portion. FIG. 7 is a diagram illustrating a method for manufacturing a photonic crystal. FIG. 8 is a diagram illustrating a method for manufacturing a photonic crystal. FIG. 9 is a diagram illustrating a method for manufacturing a photonic crystal. FIG. 10 is a diagram for explaining the principle of the photonic crystal biosensor. FIG. 11 is a diagram for explaining the principle of a photonic crystal biosensor. FIG. 12 is a diagram for explaining the principle of the photonic crystal biosensor. FIG. 13 is a diagram for explaining the principle of the photonic crystal biosensor. FIG. 14 is a diagram illustrating a photonic crystal biosensor. FIG. 15 is a diagram illustrating a photonic crystal biosensor. FIG. 16 is a diagram illustrating a photonic crystal biosensor. FIG. 17 is a diagram for explaining a photonic crystal biosensor fixing means. FIG. 18 is a diagram for explaining the photonic crystal biosensor fixing means. FIG. 19 is a diagram illustrating another embodiment of the photonic crystal biosensor. FIG. 20 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. 21 is a diagram illustrating a structure of a measurement probe included in the light detection unit of the target substance detection device. FIG. 22 is a diagram illustrating the evaluation conditions of the light detection unit of the target substance detection device. FIG. 23 is a flowchart of the target substance detection method. FIG. 24 is a diagram for explaining the principle of the photonic crystal biosensor. FIG. 25 is a diagram for explaining the principle of the photonic crystal biosensor. FIG. 26 is a diagram illustrating the principle of the photonic crystal biosensor. 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.

  DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments for carrying out a target substance detection apparatus according to the present invention (hereinafter referred to as embodiments) will be described in detail based on 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.

[First Embodiment]
<Target substance detection device>
A target substance detection apparatus provided with a 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 (target substance capture device) 11, a light detection unit 12, and a processing unit 13 according to the first embodiment.

(Photonic crystal biosensor)
First, the photonic crystal biosensor 11 will be described. The photonic crystal biosensor 11 includes a metal film-coated photonic crystal 21, an upper plate 22, and a lower plate 23. The upper plate 22 is provided with an opening 24. In the first embodiment, the photonic crystal biosensor 11 has a structure in which a metal film-covered photonic crystal 21 is sandwiched between an upper plate 22 and a lower plate 23. In the first embodiment, the photonic crystal biosensor 11 includes the upper plate 22 and the lower plate 23. However, the present invention is not limited to this, and the metal film-covered photonic crystal 21 is not limited thereto. It may be formed only by.

(Metal film coated photonic crystal)
FIG. 2 is a perspective view of the metal film-coated photonic crystal 21. FIG. 3 is a plan view of the metal film-covered photonic crystal 21. FIG. 4 is a diagram showing a cross section taken along the line AA in FIG. 3, and shows a cross section when the photonic crystal 25 is cut along a plane orthogonal to the surface 27 of the photonic crystal 25. 2 to 4 are diagrams schematically illustrating the thickness, size, and the like of components constituting the metal film-coated photonic crystal 21. The same applies to the first embodiment and other embodiments described later. As shown in FIGS. 2 to 4, the metal film-covered photonic crystal 21 includes a photonic crystal 25 and a metal film 26. The metal film-covered photonic crystal 21 covers a reflective surface 29 in which a plurality of non-flat portions 28 </ b> A are arranged on the surface 27 of the photonic crystal 25 with a metal film 26. The non-flat portion 28 </ b> A is a cylindrical recess that is recessed with respect to the surface 27. The metal film 26 may not be provided.

  First, the photonic crystal 25 will be described. Generally, a photonic crystal has a reflective surface in which concave portions having a predetermined depth or convex portions having a predetermined height are periodically formed on the surface, and light of a specific wavelength (parallel light) is irradiated onto the reflective surface. Then, it is a structure from which the reflected 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 of a structure (henceforth a reflective surface) with the light of a wide region wavelength, it reflects or permeate | transmits the light of the specific wavelength band depending on the surface state of a 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. Even in a photonic crystal having a metal thin film formed on its surface, when it is irradiated with light, an extreme value (maximum value or minimum value) appears in 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 the extreme value (maximum value or minimum value) or the shift amount of the 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 with respect to the extreme value of interest or by obtaining the amount of shift of the wavelength that is the extreme value of interest. In the following description, the term “extreme value” is used to mean an extreme value suitable for use in quantifying a change in the surface state of a photonic crystal.

  As shown in FIGS. 2 to 4, the photonic crystal 25 has a reflection surface 29 in which a plurality of non-flat portions 28 </ b> A are arranged on the surface 27. When the reflection surface 29 is irradiated with light, light having a specific wavelength depending on the shape and material of the photonic crystal 25 is reflected.

  In the first embodiment, the arrangement in which the plurality of non-flat portions 28A are arranged is such that the center G1 of the non-flat portion 28A is located at the vertex position in the equilateral triangle Po in which the three non-flat portions 28A are three-fold symmetrical figures. A plurality of unit arrays U arranged so as to be stacked one by one are included. In the arrangement in which the plurality of non-flat portions 28A are arranged, the plurality of unit arrays U are arranged so that the centroids G2 of the regular triangles Po overlap one by one at the position of the intersection of the lattice pattern La that is six-fold symmetric. Thereby, the unit arrangement | sequence U is arrange | positioned so that the lattice pattern La may be formed. Here, the wavelength at which the reflected light of the light incident on the reflecting surface 29 of the photonic crystal 25 exhibits an extreme value depends on the period of the lattice pattern La on the reflecting surface 29. On the reflecting surface 29, the period of the lattice pattern La is equal to the distance between the centroids G2 of the adjacent regular triangles Po. Therefore, in the first embodiment, the wavelength at which the reflected light of the light incident on the reflecting surface 29 exhibits an extreme value depends on the distance between the centroids G2 of the adjacent regular triangles Po.

  Further, the number of non-flat portions 28A included in the unit array U may not be three. For example, the number of non-flat portions 28A included in the unit array U may be two, or four or more. Further, the position where the center G1 of the non-flat portion 28A overlaps in the unit array U may not be the vertex of the regular triangle Po. For example, the position where the center G1 of the non-flat portion 28A overlaps in the unit array U may be the vertex of a figure that is rotationally symmetric other than an equilateral triangle. Further, the position where the center of gravity G2 of the equilateral triangle Po overlaps may not be the intersection of the lattice pattern La that is 6-fold symmetric. For example, the position where the center of gravity G2 of the regular triangle Po overlaps may be an intersection of lattice patterns that are rotationally symmetric other than 6-fold symmetry. However, the natural numbers of 2 or more are M and N, the position where the center G1 of the non-flat portion 28A overlaps in the unit array U is the vertex of the M-fold symmetrical figure, and the position where the center of gravity G2 of the regular triangle Po overlaps is N-fold symmetrical. When the intersection of a certain lattice pattern is used, M and N need to be different.

  Further, the diameter D1 of the non-flat portion 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 non-flat portions 28A is preferably 100 nm or more and 2000 nm or less, and more preferably 200 nm or more and 1000 nm or less. In addition, when the depth of the non-flat portion 28A is H1, the aspect ratio (H1 / D1) of the non-flat portion 28A is preferably 0.1 or more and 10 or less, more preferably 0.5 or more and 5 or less. 0.0 or less. The dimension of the non-flat portion 28A is not limited to the above.

  As a material of the photonic crystal 25, an organic material such as a synthetic resin or an inorganic material such as a metal / ceramic can be used.

  Synthetic resins include polyethylene, polypropylene, polymethylpentene, polycycloolefin, polyamide, polyimide, acrylic, polymethacrylic acid ester, polycarbonate, polyacetal, polytetrafluoroethylene, polybutylene terephthalate, polyethylene terephthalate, polyvinyl chloride, polyvinyl chloride Thermosetting resins such as vinylidene, polystyrene, polyphenylene sulfide, polyether sulfone, and polyether ether ketone, and phenol resins, urea resins, and epoxy resins can be used.

  As the ceramic, ceramics such as silica, alumina, zirconia, titania and yttria can be suitably used.

  As the metal, various alloys including steel materials can be used. Specifically, stainless steel, titanium, a titanium alloy, or the like can be preferably used.

  Considering optical properties, processability, resistance to solutions containing target substances (target substances), adsorptivity of target substance capture substances (specific binding substances), resistance to cleaning agents, etc. Polycycloolefin synthetic resin or silica ceramic is more preferable. Among these, the polycycloolefin synthetic resin is most suitable because of its excellent processability.

  The photonic crystal 25 is manufactured by performing fine processing on the surface of the material substrate. As a processing method, laser processing, thermal nanoimprint, optical nanoimprint, a combination of a photomask and etching, or the like can be used. In particular, when a thermoplastic resin such as a polycycloolefin-based synthetic resin is used as a material, a method using thermal nanoimprinting is preferable.

  Next, the metal film 26 will be described. For example, in the first embodiment, as shown in FIG. 4, the reflective surface 29 of the photonic crystal 25 is covered with a metal film 26. The metal film 26 is preferably formed using at least one of gold (Au), silver (Ag), platinum (Pt), and aluminum (Al). In the first 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 photonic crystal 25 can be suppressed.

  When the thickness of the metal film 26 is small, part of the incident light on the photonic crystal 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 photonic crystal 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 photonic crystal 25. By appropriately increasing the thickness of the metal film 26, unnecessary information contained in the reflected light from the photonic crystal 25 can be reduced, and the detection accuracy and concentration measurement accuracy of the target substance can be improved. Further, 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 photonic crystal 25. For example, the corners of the pattern become sharp and it becomes easy to ensure the dimensions of the pattern. From such a viewpoint, in the first 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.

Further, the metal film 26 can be formed on the reflection surface 29 of the photonic crystal 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 reflective surface 29 of the photonic crystal 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 photonic crystal 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 reflective surface 29 of the photonic crystal 25 is preferably modified using 3-triethoxysilylpropylamine (APTES) or the like. When the Au or Ag metal film 26 is formed on the reflective surface 29 of the photonic crystal 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 reflecting surface 29 of the photonic crystal 25 using the carbon chain that it has. When the metal film 26 other than Au or Ag is formed on the reflective surface 29 of the photonic crystal 25, a silane coupling agent having a functional group at one end, for example, APTES is used to reflect the reflective surface of the photonic crystal 25. It is preferable to modify 29.

  Since the metal film-coated photonic crystal 21 is obtained by coating the reflective surface 29 of the photonic crystal 25 with the metal film 26, the metal film-coated photo is applied to the reflective surface 29 corresponding to the non-flat portion 28A of the photonic crystal 25. A non-flat portion 28B of the nick crystal 21 is formed. In addition, although the diameter D2 of the non-flat portion 28B depends on the thickness of the metal film 26, it is preferably 50 nm or more and 1000 nm or less, and more preferably 100 nm or more and 500 nm or less. Further, the distance C2 between the centers of the non-flat portions 28B is preferably 100 nm or more and 2000 nm or less, more preferably 200 nm or more and 1000 nm or less, similarly to the distance C1 between the centers of the non-flat portions 28A. Further, when the depth of the non-flat portion 28B is H2, the aspect ratio (H2 / D2) of the non-flat portion 28B is preferably 0.1 or more and 10 or less, and more preferably 0.5 or more and 5 or less. 0.0 or less. The dimension of the non-flat portion 28B is not limited to the above.

  Next, the characteristics of the reflected light of the light irradiated on the metal film-coated photonic crystal 21 of the first embodiment shown in FIGS. FIG. 5 is a diagram showing the reflectance with respect to the wavelength of the reflected light of the light irradiated on the metal film-coated photonic crystal 21 of the first embodiment. In FIG. 5, the extreme values are the minimum values at two locations where the shape of the spectrum of the reflected light changes near the wavelength of 700 nm. The change in the shape of the spectrum of the reflected light that appears near the wavelength of 500 nm is caused by the metal film 26 formed of Au, and appears regardless of the presence or absence of the non-flat portions 28A and 28B. For this reason, the change in the shape of the spectrum of reflected light that appears in the vicinity of a wavelength of 500 nm is not counted as the extreme value of the first embodiment. The reflected light of the light irradiated on the metal film-coated photonic crystal 21 of the first embodiment shows an extreme value at a specific wavelength, and the number of the extreme values is two. Thus, if the sensor using the metal film-coated photonic crystal 21 of the first embodiment can measure using at least one extreme value out of two extreme values, the sensor can output an appropriate output. can get. Therefore, the sensor using the metal film-covered photonic crystal 21 according to the first embodiment has a defect in the shape of the non-flat portions 28A and 28B, and measures one of the two extreme values. Even if it is not possible to measure other extreme values, it is highly possible that an appropriate output as a sensor can be obtained. Therefore, the sensor using the metal film-coated photonic crystal 21 of the first embodiment can increase the probability that it can function as a sensor. In addition, the reflected light of the light irradiated to the metal film covering photonic crystal 21 of the first embodiment shows an extreme value at a specific wavelength, and the number of the extreme values may be three or more.

  In the above description, the non-flat portion according to the first embodiment is a concave portion as shown in FIG. 4, but may be a convex portion as shown in FIG. At this time, the non-flat portions 28 </ b> A and 8 </ b> B are cylindrical convex portions that protrude from the surface 27. FIG. 9 is a diagram illustrating an AA cross section in FIG. 3 when the non-flat portion is a convex portion.

(Method for producing photonic crystal)
Next, an example of a process for producing the metal film-coated photonic crystal 21 by thermal nanoimprint will be described. 7, 8 and 9 are diagrams for explaining a method of manufacturing a photonic crystal. As shown in FIG. 7, in thermal nanoimprint, a mold DI having a nanometer-level microstructure or a nanometer-level periodic structure pattern is used. Then, as shown in FIG. 8, the heated mold DI is pressed against the sheet-shaped resin P, pressed at a predetermined pressure for a predetermined time, and released when the surface temperature of the mold DI reaches a predetermined temperature. The structure and the periodic structure are transferred to the sheet-like resin P. Thereby, the photonic crystal 25 is obtained.

  When the resin P is a cycloolefin polymer, the mold DI is heated to about 160 ° C., pressed at a pressure of about 12 MPa for a predetermined time, and released when the surface temperature of the mold DI reaches about 60 ° C. It is preferable.

  After producing the photonic crystal 25, as shown in FIG. 9, a metal film 26 is formed on the surface in contact with the mold DI by a sputtering or vapor deposition apparatus or the like, and the metal film-covered photonic crystal 21 is completed. .

(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. By measuring the concentration of 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, and cytosine, a peptide, and 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 first embodiment, the target substance trapping substance is fixed to the reflection surface 29 of the metal film-coated photonic crystal 21 shown in FIG. Examples of means for fixing the target substance capturing substance to the reflecting surface 29 of the metal film-coated photonic crystal 21 include chemical bonds such as covalent bonding, chemical adsorption, and physical adsorption, and physical bonding methods. 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-capturing substance is dropped on the reflective surface 29 of the metal film-coated photonic crystal 21, and the metal film-coated photonic crystal 21 is cooled at a predetermined time, at room temperature, or as necessary. Heating causes the target substance capturing substance to be 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) to the surface of the reflective surface 29 of the metal film-coated photonic crystal 21. Thereby, the photonic crystal biosensor 11 can detect a specific antigen. This utilizes the optical characteristics of the photonic crystal 25 and various biological / chemical reactions that occur on or near the surface of the photonic crystal 25, such as an antigen-antibody reaction in which a specific antigen reacts only with a specific antibody. To do.

  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 reflecting surface 29 of the photonic crystal 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 photonic crystal 25 are greatly influenced by the surface state, it is preferable that no impurities are adsorbed on the reflection surface 29 of the photonic crystal 25. Since the blocking agent is fixed to the reflection surface 29 of the photonic crystal 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 a portion other than the portion where the antibody that is the target substance capturing substance is adsorbed (fixed) to the reflection surface 29 of the photonic crystal 25. Is preferred. In order to adsorb the blocking agent in advance, the blocking agent is brought into contact with the surface of the photonic crystal 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. 10 to 13 are diagrams for explaining the principle of the photonic crystal biosensor 11. In general, the photonic crystal biosensor 11 has optical characteristics of the photonic crystal 25 and various biological / chemical reactions that occur on or near the surface of the photonic crystal 25, for example, a specific antigen is only a specific antibody. By utilizing the antigen-antibody reaction of reacting, a minute amount of protein or low molecular weight substance is detected. The photonic crystal biosensor 11 reflects the wavelength of the reflected light due to the surface plasmon resonance phenomenon and / or the localized surface plasmon resonance phenomenon when the reflection surface 29 of the metal film-coated photonic crystal 21 is irradiated with light of a specific wavelength. Use the phenomenon of extreme values shifting.

  As shown in FIG. 10, an antibody (target substance-capturing substance) 34 is fixed to the surface of the reflection surface 29 of the metal film-coated photonic crystal 21 by adsorption.

  Next, as shown in FIG. 11, a blocking agent (protective substance) 35 is adsorbed in advance on a portion of the reflective surface 29 other than the portion where the antibody 34 is adsorbed, that is, on 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. 12, 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. 13, the light detection unit 12 illustrated in FIG. 1 converts light (incident light) LI having a specific wavelength into parallel light in a state where the antigen 36 is captured by the reflection surface 29 of the photonic crystal 25. Then, the reflective surface 29 of the metal film-coated photonic crystal 21 is irradiated. 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 illustrated 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 is captured by the reflective surface 29 of the metal film-covered photonic crystal 21. The presence or absence of the antigen 36 is detected, or the concentration of the antigen 36 is obtained.

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

  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 of the reflective surface 29 of the metal film-coated photonic crystal 21, and correlates with the amount of the complex 37 formed by capturing the antigen 36 on the antibody 34 immobilized on the reflective surface 29. To do. 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 first 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, it is performed as follows. Light is incident perpendicularly to the reflective surface 29 of the metal film-covered photonic crystal 21, and the reflected light LR is detected. It is also possible to detect the reflected light LR by entering light at an angle with respect to the normal of the reflecting surface 29 of the metal film-covered photonic crystal 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)
Next, an example of production of the photonic crystal biosensor 11 shown in FIG. 1 will be described. FIGS. 14, 15, and 16 are explanatory diagrams of the photonic crystal biosensor 11. As shown in FIG. 14, after the metal film-coated photonic crystal 21 is placed on the lower plate 23, the upper plate 22 is placed on the lower plate 23 as shown in FIG. The photonic crystal biosensor 11 is produced by sandwiching 21 between the lower 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 photonic crystal 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. 16 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. In addition, as long as the amount of the solution spreads to the droplet holding unit 38, the target substance can be sufficiently 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. In addition, when the opening 24 is cylindrical, its diameter may be varied depending on 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 to 10 mm, more preferably 2 mm to 6 mm in consideration of the operation when adsorbing the antigen 36 to the antibody 34 and the convenience of handling. .

  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 preferable to use 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, when a so-called hydrophilic solution such as saliva is detected and measured, if the upper plate 22 is made of a hydrophobic material, the solution can be accurately collected in the droplet holder 38. In addition, when detecting and measuring a so-called lipophilic solution such as lipid, the solution can be accurately collected in the droplet holding unit 38 if the upper plate 22 is formed of a hydrophobic material.

  Further, the upper plate 22 may be formed of a material having water repellency, oil repellency or water / oil repellency. Further, a surface treatment or coating that exhibits hydrophobicity, hydrophilicity, water repellency, and oil repellency may be applied to the upper plate 22. 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. Further, 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 performed.

  17 and 18 are diagrams for explaining the photonic crystal biosensor fixing means. FIG. 17 shows a state before the magnet sheet 39 is attached, and FIG. 18 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 thermal nanoimprint or the like. In order for the target substance detection apparatus 10 to detect reflected light more accurately, it is preferable to accurately position the incident site and the reflected site of the light irradiated to the photonic crystal biosensor 11.

  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 reflection 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 to 500 μm. 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.

  Further, 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 with 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 photonic crystal 25 may be removed and a marker may be attached to the photonic crystal 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. 19 is a diagram for explaining another form of the photonic crystal biosensor 11. As shown in FIG. 19, 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 holes, the inner wall on the opening 24 side, and the reflection surface 29 of the photonic crystal 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 holed cover 41 and the sheet 42, thereby preventing foreign matters from being mixed into the solution from the outside.

  Further, by filling the droplet holder 44 with the solution, it is possible to measure the reflected light more accurately with the solution 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 unit 12)
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. 20 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 from the measurement probe 52 to the reflection surface 29 of the metal film-coated photonic crystal 21 included in the photonic crystal biosensor 11. Irradiate. The collimating lens 56 irradiates the reflecting surface 29 of the photonic crystal 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 reflection surface 29 of the metal film-covered photonic crystal 21 as reflected light LR, and guides it to the light detection 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. 21 is a diagram illustrating a structure of the measurement probe 52 included in the light detection unit 12 illustrated 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 irradiated to the reflection surface 29 of the photonic crystal 25 and the reflection light LR from the reflection surface 29 are emitted from substantially the same position and are incident. Can do. 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 metal film covering photonic crystal 21, the detection accuracy of a target substance and the measurement accuracy 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. 22 is a diagram illustrating the evaluation conditions of the light detection unit 12 of the target substance detection device 10 according to the first embodiment. As shown in FIG. 22, 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 metal film-covered photonic crystal 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 diameter of the opening 24 through which the reflecting surface 29 of the photonic crystal 25 is exposed. Is d2. 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 unit 13)
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 the presence / absence of at least the target substance (for example, the antigen 36 shown in FIGS. 12, 13, and the like) based on the obtained extreme wavelength shift (wavelength shift amount). The processing unit 13 is, for example, a microcomputer. There is a correlation between the amount of wavelength shift and the concentration of the target substance trapped on the reflection surface 29 of the metal film-coated photonic crystal 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 cortisol antibody is adsorbed on the reflective surface 29 of the metal film-coated photonic crystal 21 and cortisol in saliva is detected and measured as a target substance to be detected. As the photonic crystal 25, a cycloolefin polymer sheet having a predetermined fine structure formed on the surface by thermal nanoimprint is cut into a predetermined size.

  FIG. 23 is a flowchart illustrating an example of the target substance detection method according to the first embodiment. First, in step S11, the light detection unit 12 detects the reflected light LR from the reflection surface 29 when the reflection surface 29 of the photonic crystal 25 is irradiated with light, and the processing unit 13 measures the reflected light LR. (Step S11). 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 S <b> 12, a cortisol antibody solution (cortisol antibody concentration of 1 μg / ml to 50 μg / ml) is dropped onto the reflective surface 29 of the metal film-coated photonic crystal 21. The photonic crystal biosensor 11 is allowed to stand at a predetermined temperature for a predetermined time or if necessary, and the cortisol antibody is adsorbed on the reflection surface 29 of the metal film-coated photonic crystal 21.

  Next, in step S <b> 13, a phosphate buffer solution (PBS: Phosphate buffered saline) is dropped onto the reflective surface 29 of the metal film-coated photonic crystal 21. Thereafter, a rinsing process is performed a plurality of times for removal by centrifugal force.

  Next, in step S14, skim milk is dropped as the blocking agent 35 onto the reflecting surface 29 of the photonic crystal 25, and the photonic crystal biosensor 11 is left 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 metal film-coated photonic crystal 21.

  Thereafter, in step S15, the rinsing process is performed a plurality of times with a phosphate buffer solution, similarly to the rinsing process (step S13). By the above-described operation, the reflective surface 29 of the metal film-coated photonic crystal 21 is subjected to a predetermined treatment, and the photonic crystal biosensor 11 is formed.

  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 S15).

  Next, in step S <b> 19, the target substance detection device 10 is used to irradiate light onto the reflection surface 29 of the metal film-covered photonic crystal 21. The light irradiated at this time is the same as the light irradiated on the reflecting surface 29 in step S11. 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 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 material is captured on the reflective surface 29 and the reflected light intensity (or reflectance) when the target material is not captured on the reflective 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. For example, the wavelength shift amount may be obtained using the extreme wavelength of the intensity of the reflected light from the reflecting surface 29 after the rinsing process (step S13 or step S15) is finished. Further, when there are a plurality of extreme values in step S11 and step S19, the extreme value to be noted is appropriately selected. Then, the wavelength λ1 and the wavelength λ2 are obtained for the selected extreme value.

  In the first embodiment, the metal film-covered photonic crystal 21 has the antibody 34 immobilized on the reflection surface 29. However, the present invention is not limited to this. The antibody 34 may be used without being fixed to the surface 29.

[Second Embodiment]
A target substance detection apparatus provided with a target substance capturing apparatus according to a second embodiment will be described. In the target substance capturing apparatus according to the second embodiment, the substance (target substance) 36 that is fixed to the reflecting surface 29 of the metal film-coated photonic crystal 21 is used as an antigen (target substance) 36, and the antibody 34 is adsorbed to the antigen 36. Except for this, the second embodiment is the same as the first embodiment, and a duplicate description is omitted.

  24 to 28 are diagrams illustrating the principle of the photonic crystal biosensor. In the second 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. 24, the photonic crystal biosensor 11 is similar to 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 metal film-coated photonic crystal 21. It can be carried out. 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 metal film-coated photonic crystal 21 is a fixed amount. Thus, when the antibody 34 is adsorbed to the antigen 36 fixed to the metal film-coated photonic crystal 21 to form a complex 65 (see FIGS. 26 and 27), the amount of the complex 65 formed and The photonic crystal biosensor 11 can output the correlated physical quantity. 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. 25, 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 photonic crystal 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 (minimum value in this example) is λ1.

  Next, as shown in FIG. 26, a mixture M including a 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 a solution 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 a solution containing a 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 reflection surface 29 of the metal film-coated photonic crystal 21. As a result, as shown in FIG. 27, 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. 28, light (incident light) LI of, for example, 300 nm or more and 2000 nm or less is parallel light and the optical axis is orthogonal to the reflection surface 29 on the reflection surface 29 of the metal film-coated photonic crystal 21. Irradiate. 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 metal film-coated photonic crystal 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 the second embodiment, cortisol that is the antigen 36 is immobilized on the metal film-coated photonic crystal 21, and an anti-cortisol antibody that is the antibody 34 is reacted. Compared to the case where the antibody 34 is reacted with the antibody 34 after the antibody 34 is fixed to the reflective surface 29 of the metal film-coated photonic crystal 21 as in the first embodiment, the second embodiment is compared with the case of the second embodiment. As described above, when cortisol is immobilized on the reflective surface 29 of the metal film-coated photonic crystal 21, the change in the surface state of the metal film-coated photonic crystal 21 becomes larger when the anti-cortisol antibody is reacted with cortisol. The sensitivity of the photonic crystal biosensor 11 is improved.

  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 second embodiment, the photonic crystal biosensor 11 uses, for example, a secondary antibody that specifically reacts with the complex 65 as a complex binding substance, and the reflective surface 29 of the metal film-coated photonic crystal 21. You may make it react with the composite_body | complex 65 fixed to. The secondary antibody is brought into contact with the reflecting surface 29 of the metal film-covered photonic crystal 21 in an excessive amount than the first complex 65. Then, a secondary antibody is added to all the complexes 65 to form a second complex. By doing so, the change in the surface state of the metal film-coated photonic crystal 21 is further increased. 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 metal film-coated photonic crystal 21 increases as the secondary antibody increases, the photonic crystal biosensor 11 can be reacted with the complex 65 after adding another substance to the secondary antibody. The sensitivity is further increased.

  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.

10 Target Substance Detection Device 11 Photonic Crystal Biosensor (Target Substance Capture Device)
DESCRIPTION OF SYMBOLS 12 Light detection part 13 Processing part 21 Metal film covering photonic crystal 22 Upper plate 23 Lower plate 24, 43 Opening part 25 Photonic crystal 26 Metal film 27 Surface 28A, 28B Non-flat part 29 Reflecting surface 34 Antibody (Target substance capture substance) )
35 Blocking agent (protective substance)
36 Antigen (target substance)
37, 65 Composite 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 Outgoing surface 62 Incident surface 63 Same Surface (input / output surface)
M mixture U unit array LI incident light LR reflected light

Claims (2)

A target substance capturing device including a reflective surface in which a plurality of non-flat portions are arranged, captures a target substance, and reflects irradiated light,
When a natural number of 2 or more is M and a natural number of 2 or more different from M is N,
The arrangement in which the plurality of non-flat portions are arranged includes a plurality of unit arrays arranged so that the centers of the non-flat portions are overlapped one by one at the positions of vertices in a figure in which the non-flat portions are M-fold symmetric. A plurality of unit arrays are arranged so as to overlap the center of gravity of the figure having M times symmetry one by one at the position of the intersection of the lattice pattern having N times symmetry,
The target substance capturing device, wherein the reflected light reflected by the reflecting surface exhibits an extreme value at a specific wavelength, and there are a plurality of extreme values.   The target substance capturing apparatus according to claim 1, wherein the M is 3 and the N is 6.

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