JP2007101308A - Target substance detecting element, device, and method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To further enhance the capacity of a chemical sensor for capturing a target substance. <P>SOLUTION: The target substance detecting element is used for detecting a target substance in a specimen using a plasmon resonance method and has a substrate, the metal thin film formed on the substrate, the dielectric layer formed on the metal thin film and the metal pattern layer formed on the dielectric layer. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a detection element, a detection apparatus, and a detection method for detecting a target substance in a specimen with high sensitivity using metal plasmon resonance.

  There are multiple markers in blood for specific diseases such as cancer, hepatitis, diabetes, and osteoporosis. When affected, the concentration of a specific protein increases during normal times. By monitoring these from normal times, it is possible to detect serious illnesses early. Therefore, monitoring of disease markers is expected as the next generation medical technology. One method for analyzing raw and unpurified proteins is based on sensors that use biological ligand-analyte interactions to identify specific compounds.

  There are various types of sensors using several detection methods such as fluorescence immunoassay, plasmon resonance, and optical interference. In any case, the ligand is immobilized on the sensor surface, and the analyte in the sample is selected with high sensitivity and selectivity to eliminate impurities, and only the target protein is immobilized on the substrate surface with high efficiency. Is a common step. In the fluorescence immunization method, a second ligand labeled with a fluorescent dye is further bound to the ligand-analyte complex, the fluorescent dye is excited, and the amount of fluorescence is measured to measure the concentration of the analyte. In the plasmon resonance method, the concentration of analyte bound to a ligand immobilized on a metal thin film or metal fine particle is measured by utilizing the fact that metal plasmon reacts sensitively to changes in the refractive index of the interface substance. ing.

Recently, a sensor using a metal microstructure has been developed for further performance improvement. Patent Document 1 discloses a sensor in which insulator microspheres are fixed on a metal thin film and the upper surface is coated with metal, and has an advantage of enabling high-sensitivity detection without requiring mechanical operation. . Patent Document 2 discloses an invention characterized in that, for the purpose of reducing the line width of a spectrum, metal fine particles are filled in micropores, and a metal thin film is formed on the surface to cause interaction.
Japanese Patent Laid-Open No. 11-326193 JP 2004-232027 A

  However, with the technology disclosed above, the peak width of the absorption spectrum used for sensing may be wide and the sensitivity may be low, and it is difficult for the sample liquid / cleaning liquid to reach the metal dots placed in the micropores, making it accurate. May not be detected. Therefore, further improvement in performance is desired as a chemical substance sensor for capturing a target substance.

  In view of the above problems, the present invention has been invented for the purpose of further improving the performance of a chemical sensor for capturing a target substance.

  A target substance detection element provided by the present invention is an element used for detecting a target substance in a specimen using a plasmon resonance method, and includes a substrate, a metal thin film formed on the substrate, A target substance detection element comprising a dielectric layer formed on the metal thin film and a metal pattern layer formed on the dielectric layer.

  A target substance detection apparatus provided by the present invention is an apparatus for detecting a target substance in a sample, and includes (1) a channel and (2) a liquid feeding means for supplying the sample to the channel. And (3) a target substance detection element having a capturing body for the target substance arranged in the flow path, and (4) a light irradiation means for irradiating the target substance detection element with light for detection by a plasmon resonance method And (5) a light detecting means for detecting a change in light emitted by the light irradiating means, wherein the target substance detecting element is that of the present invention. It is a detection device.

  The target substance detection method provided by the present invention is a method for detecting a target substance in a specimen, and the plasmon in the reaction field is fed while the specimen is fed to the reaction field in which the target substance detection element is arranged. A step of measuring a change in a resonance spectrum, and a step of detecting a target substance in the sample based on the change in the plasmon resonance spectrum, wherein the element of the present invention is used as the target substance detection element, This is a target substance detection method.

  By using the target substance detection element disclosed in the present invention, plasmon resonance can be used more effectively, and the target substance detection sensitivity can be improved.

  In the target substance detection element of the present invention, the metal thin film and the metal pattern are formed via the dielectric, so that the surface plasmon resonance generated in the metal thin film and the localized plasmon resonance generated in the metal pattern can be used. That is, in the present invention, sensitivity sufficient to detect a target substance in a specimen can be achieved by utilizing the interaction between surface plasmon resonance and localized plasmon resonance. Furthermore, since the accessibility between the target substance in the sample and the reaction region of the sensor is kept high, the time required for the reaction is reduced.

  Embodiments relating to the present invention will be described below with reference to the drawings. Here, in order to fully understand the present invention, specific embodiments will be described in detail, but the present invention is not limited to the contents described here.

<Target substance detection element>
An example of a preferable configuration of the target substance detection element according to the present invention will be described with reference to FIG. If the board | substrate 201 is a board | substrate which can detect the target substance by the target plasmon resonance method in this invention, there will be no restriction | limiting in particular. Here, it is preferable to use glass or plastic which is inexpensive and easily available. In the example shown here, a metal thin film 202 provided on a substrate 201 and a patterned metal layer 204 are stacked with a dielectric 203 interposed therebetween. Here, the dielectric 203 and the pattern-shaped metal layer 204 constitute a columnar body having the same pattern shape laminated in the thickness direction of the substrate.

  Thus, by arranging the pattern-shaped metal layer 204 on the metal thin film 202 via the dielectric 203, the surface plasmon resonance generated in the metal thin film 202 and the localized plasmon resonance generated in the pattern-shaped metal layer 204 are obtained. The detection sensitivity of the target substance can be improved by utilizing the interaction. Further, the patterned metal layer 204 can be provided at a position where interaction is easily obtained, and in this case, a further effect is desired.

  The metal thin films 202 and 204 are not particularly limited as long as they are made of a substance that generates plasmon at the interface with the specimen or buffer solution. In this configuration, it is preferable to use a single noble metal such as Au, Ag, Cu, or Pt, or an intermetallic compound or alloy composed of these elements. The thickness of the metal thin film 202 can be preferably in the range of 10 to 200 nm, and a metal material may be appropriately selected and used in consideration of the film thickness and the wavelength band of irradiation light. For example, in the case of Au, a film thickness of about 50 nm is preferable. The thickness of the metal thin film 204 can also be preferably in the range of 10 to 200 nm, and a metal material may be appropriately selected and used in consideration of the film thickness and the wavelength band of irradiation light.

The dielectric layer 203 is not particularly limited as long as it is a layer that enables detection by a desired plasmon resonance method. However, it is desirable to use a low dielectric constant material. Suitable materials include Al ₂ O ₃ , Sb ₂ O ₃ , BeO, Bi ₂ O ₃ , BiF ₃ , CaF ₂ , CeO ₂ , CeF ₃ , HfO ₂ , LaF ₃ , La ₂ O ₃ , PbCl ₂ , PbF. ₂ , LiF, MgF ₂ , MgO, NdF ₃ , Nd ₂ O ₃ , Pr ₆ O ₁₁ , Sc ₂ O ₃ , SiO, SiO ₂ , Si ₂ O ₃ , NaF, Ta ₂ O ₅ , TiO ₂ , TlCl, ThO ₂ , ThF ₄ , Y ₂ O ₃ , ZnSe, ZnS, ZrO ₂ , and various polymer materials. The layer thickness can be preferably in the range of 20 to 1000 nm, and a constituent material may be appropriately selected and used in consideration of the layer thickness and the wavelength band of irradiation light. For example, when SiO ₂ is used, it is preferable to use a columnar structure of about 100 nm. In addition, the thickness of the dielectric layer 203 and the patterned metal layer (204, 304, 310, etc.) provided thereon is 1/10 to 1/2 of the wavelength of light used to detect a change in optical characteristics. Range.

  The arrangement method of the columnar bodies may be a square arrangement, a triangular arrangement, or a hexagonal arrangement, and is not particularly limited as long as they are regularly arranged. The distance between the columnar bodies can be appropriately selected and used within the range of 50 to 1000 nm.

  The target substance capturing body 205 is not particularly limited as long as it forms a specific binding pair with the target substance. Here, it is preferable to use an antibody or a nucleic acid.

  As the target substance detection element, one having the structure shown in FIGS. 3A and 3B can be used separately from the element shown in FIG.

  In the target substance detection element 306 of FIG. 3A, a metal thin film is provided on a substrate 301, a continuous dielectric layer 303 is provided on the metal thin film 302, and an isolated (patterned) metal layer is formed thereon. 304 is provided. The capturing body 305 is fixed to the metal layer 304. As the materials for the substrate 301, the dielectric layer 303, and the metal layer 304, the same materials as those described above with reference to FIG. 2 can be used as appropriate. In the target substance detection element 312 shown in FIG. 3B, a metal thin film 308 is provided on a substrate 307, a continuous dielectric layer 309 is provided on the metal thin film 308, and holes are periodically opened in the upper layer. A metal layer 310 (a metal layer having openings (holes)) is provided. The capturing body 311 is fixed to the metal layer 310. In the target substance detection element having this configuration, the same material as described above can be used as appropriate.

  The patterned metal layer 304 and the metal layer 310 having holes are preferably aligned with each other using a lithography technique or an imprint technique.

  The columnar body having the metal pattern layer shown in FIG. 2, the pattern-like metal layer shown in FIG. 3A, the cross-sectional shape, size, arrangement density, and regular arrangement of the holes shown in FIG. Selection is made so that the target substance can be detected using the target plasmon resonance.

  The target substance is not particularly limited as long as it forms a specific binding pair with the target substance capturing body. Target substances contained in a specimen can be roughly classified into non-biological substances and biological substances.

  Examples of non-biological substances important as detection targets include PCBs having different chlorine substitution numbers / positions as environmental pollutants, dioxins having different chlorine substitution numbers / positions, and endocrine disrupting substances called so-called environmental hormones.

  The biological material is selected from nucleic acids, proteins, sugar chains, lipids, and complexes thereof. Specific examples include DNA, RNA, aptamer, gene, chromosome, cell membrane, virus, antigen, antibody, lectin, hapten, hormone, receptor, enzyme, peptide, sphingosaccharide, sphingolipid and the like.

  Furthermore, bacteria or cells themselves that contain or produce the “biological substance” and can be corrected by the target substance capturing element can also be detected.

<Target substance detection device>
An example of the target substance detection apparatus of the present invention will be described with reference to FIG. The light source 101 may be any light source that can be used for the intended measurement. For example, a white light source can be used. Among these, it is preferable to use a halogen lamp. The spectroscope 102 is not particularly limited as long as it can perform spectroscopy with a wavelength resolution of about 1 nm. In the present invention, it is preferable to use a multi-channel detector or a spectrophotometer. As the sample reservoir 103, a reservoir made of a material with less non-specific adsorption of the target substance, or a reservoir may be built in the chip. For example, it is preferable to use an Eppendorf tube coated with non-specific adsorption prevention. The structure of the cleaning liquid reservoir 104 and the constituent material thereof may be appropriately selected according to the form of the specimen and the target substance as the detection target. Here, it is preferable to use a glass or plastic tube for biochemistry. The flow path switching valve 105 is not particularly limited as long as the fluid tube can be switched. For example, it is preferable to use a three-way valve. As the liquid feeding means 106, a syringe pump, a tube pump, a diaphragm pump, or the like can be appropriately selected and used. When a syringe pump is used as the waste liquid tank 107, the syringe becomes a waste liquid tank as it is. When a tube pump or a diaphragm pump is used, a beaker or a bottle can be used as a waste tank. If there is only a small amount of sample, the sample may be circulated using a circulation channel or tube instead of using waste liquid. As the liquid feeding and waste liquid tubes 108 and 109, a tube made of a material having as little as possible adsorption of the target substance is preferable. As the target substance detection element 110, the plasmon optical element shown in FIG. 2 and the elements shown in FIGS. 3A and 3B are appropriately selected and used. The flow path 111 is preferably small so that the amount of the sample can be reduced as much as possible. In addition, it is preferable that the surface of the channel is coated so that the target substance is not adsorbed as much as possible. The cover 112 is preferably a transparent material and a material that can form a flow path with the substrate 113.

  Detection of a target substance using the apparatus having the configuration of FIG. 1 can be performed as follows. First, the valve 105 is adjusted to supply the cleaning liquid from the cleaning liquid reservoir 104 to the flow path 111 in which the target substance detection element 110 is disposed, thereby sufficiently cleaning the inside of the flow path. The cleaning waste liquid is collected in the waste liquid tank 107. After completion of the cleaning, the valve 105 is adjusted so that the specimen (liquid) 103 is supplied into the flow path 111 at a predetermined flow rate and is brought into contact with the target substance detection element 110. At this time, the specimen 103 is caused to flow through the flow path at a constant speed and is collected in the waste liquid tank. Considering the point in time when the specimen reaches the reaction field in contact with the capturing body on the target substance detection element, the measurement light is irradiated from the light source 101 and condensed at a predetermined position in the flow path via the collimator lens 115. Then, the reflected light is received by the spectroscope 102 to obtain the spectrum of the reflected light. When the target substance is captured by the capturing body of the target substance detection element 110, a spectrum change occurs due to plasmon resonance, and the presence or absence of the target substance in the sample can be detected based on the spectrum change. For example, it is possible to detect and record a spectrum at a predetermined time interval (interval), obtain a change in the spectrum over time, and use it to detect the target substance with high sensitivity.

  In addition, by providing a plurality of reaction fields in which a plurality of different capture bodies are divided and provided in the flow path, it is possible to detect a plurality of target substances corresponding to the plurality of capture bodies. A plurality of reaction fields may be provided in the same target substance capturing element, or, as shown in FIG. 6, target substance detection elements that can detect different target substances from the upstream to the downstream of the flow path in series. You may arrange in.

Examples of the present invention will be described below, but the present invention is not limited only to the contents described herein.
Example 1
<Target substance detection element>
The procedure for producing the target substance detection element used in this example will be described with reference to FIG. As the substrate 901, a silicon wafer having a thickness of 525 μm is used. A 50 nm Au thin film 902 is deposited on the substrate to form a film. Further, after forming a SiO ₂ layer 903 to a thickness of 50 nm by sputtering, positive type electron beam resist (ZEP520) 904 is coated with a spin coater. Then, using an electron beam drawing apparatus, an area of about 200 nm is exposed with an electron beam. Thereafter, development is performed to form a hole pattern, and then Au is further formed to a thickness of about 20 nm. Further, the resist and Au on the resist are removed by a lift-off process. Finally, the SiO ₂ layer is etched with SF 6 gas using Au as a mask to complete the device.

  After completion of the element, ultrasonic cleaning is performed using acetone, IPA, and ultrapure water. Further, an antibody is used as the capturing body 205, and is physically adsorbed and immersed by immersion for about 1 hour.

<Target substance detection device>
The apparatus used in this example will be described with reference to FIG. A halogen lamp is used as the light source 101, and a multi-channel detector (Hamamatsu Photonics) is used as the spectroscope 102. An Eppendorf tube is used as the specimen reservoir 103, and a biochemical glass bottle is used as the cleaning liquid reservoir 104. A three-way valve (GL Science) is used as the flow path switching valve 105, and a syringe pump (kd Scientific KDS200) is used as the liquid feeding means 106. A syringe is used as the waste liquid tank 107, and a Teflon tube is used as the liquid feeding and waste liquid tubes 108 and 109. As the target substance detection element 110, an element having the configuration shown in FIG. 1 is used. As the flow path 111, a 1 mm width, 100 μm width, and 40 mm length formed on the cover (PDMS substrate) 112 is used. Quartz glass is used as the substrate 113, and a plano-convex cylinder lens (Sigma light machine, 20 mm × 40 mm) is used as the collimating lens.

<Measurement of target substance>
In the apparatus of FIG. 1, the target substance detection element 206 on which the anti-PSA antibody is immobilized is set in the apparatus, and the following protocol is performed.
(1) The three-way valve is switched to the cleaning liquid side, and a buffer solution whose pH is adjusted to 7.4 is allowed to flow at a flow rate of 0.1 (ml / min.) For 10 minutes to thoroughly wash the target substance detection element.
(2) The valve is switched to the sample side, and the sample is allowed to flow at a flow rate of 0.1 (ml / min.) For 10 minutes to cause an antigen-antibody reaction.
(3) Measure the reflection spectrum at regular intervals while feeding the specimen.
(4) Switch the valve to the washing solution side again, and flow the buffer solution at a flow rate of 0.1 (ml / min.) For 10 minutes to wash away the non-specifically adsorbed antigen.

  According to the above protocol, a spectrum for each reaction time is obtained as shown in FIG. By fitting this spectrum with graph software and obtaining the peak value of the spectrum, the time course of the antigen-antibody reaction can be obtained as shown in FIG. Furthermore, it is possible to determine the concentration of the target substance in the specimen after washing.

  Since a disease marker is a protein whose concentration increases when a disease occurs, it can be diagnosed as to whether or not the disease marker is affected by comparison with a normal value. Further, by examining the reaction between the target protein and the antibody, it becomes possible to make a more accurate diagnosis.

(Example 2)
<Target substance detection element>
Two target substance detection elements shown in FIG. 2 are arranged in series, and different types of antibodies are immobilized on the target substance detection elements.
<Target substance detection device>
The apparatus used in this example will be described with reference to FIG. A halogen lamp is used as the light source 601 and a multi-channel detector capable of simultaneous measurement for 4 channels is used as the spectroscope 602. An Eppendorf tube is used as the specimen reservoir 603, and a glass bottle for biochemistry is used as the cleaning liquid reservoir 604. A three-way valve (GL Science) is used as the flow path switching valve 605, and a syringe pump (kd Scientific KDS200) is used as the pump 606. A syringe is used as the waste liquid tank 607, and a Teflon tube is used as the liquid feeding and waste liquid tubes 608 and 609. As the flow path 611, a 1 mm width, 100 μm width, and 40 mm length formed on the PDMS substrate 612 is used. Quartz glass is used as the substrate 613, and a plano-convex cylinder lens (Sigma light machine, 20 mm × 40 mm) is used as the collimating lens. The optical fiber 616 is not particularly limited as long as the amount of light from the light source is sufficient in each wavelength band. Here, it is preferable to use a fiber having a visible light wavelength band.

<Measurement of target substance>
In the apparatus of FIG. 6, the target substance detection element on which the anti-CEA antibody, anti-AFP antibody, anti-PSA antibody and anti-PAP antibody are immobilized is set in the apparatus and the following protocol is performed.
(1) The three-way valve is switched to the cleaning liquid side, and a buffer solution whose pH is adjusted to 7.4 is allowed to flow at a flow rate of 0.1 (ml / min.) For 10 minutes to thoroughly wash the target substance detection element.
(2) The valve is switched to the sample side, and the sample is allowed to flow at a flow rate of 0.1 (ml / min.) For 10 minutes to cause an antigen-antibody reaction.
(3) Measure the reflection spectrum at regular intervals while feeding the specimen.
(4) Switch the valve to the washing solution side again, and flow the buffer solution at a flow rate of 0.1 (ml / min.) For 10 minutes to wash away the non-specifically adsorbed antigen.

  According to the above protocol, a spectrum as shown in FIG. 6 is obtained for each reaction field. By fitting this spectrum with graph software and obtaining the peak value of the spectrum, the kinetics of the reaction can be measured for each marker protein as shown in FIG. Further, from the results of the kinetics measurement shown in FIG. 7, it is possible to derive the marker protein concentration as an index of the cause of the disease as shown in Table 1.

  As described above, by measuring the reaction profile of a plurality of disease marker proteins and their antibodies and examining the concentration of the target substance, it becomes possible to investigate the cause of the disease with higher accuracy than the measurement of one protein.

(Example 3)
<Target substance detection element>
Two target substance detection elements shown in FIG. 2 are arranged in series, and different types of antibodies are immobilized on the target substance detection elements.
<Target substance detection device>
The apparatus used in this embodiment will be described with reference to FIG. A halogen lamp is used as the light source 801, and a multi-channel detector capable of simultaneous measurement of 4 channels is used as the spectroscope 802. An Eppendorf tube is used as the specimen reservoir 803 and a biochemical glass bottle is used as the cleaning liquid reservoir 804. As the flow path switching valves 805 and 816, a three-way valve (GL Science) is used, and as the pump 806, a tube pump (kd Scientific KDS200) is used. A beaker is used as the waste liquid tank 807, and a Teflon tube is used as the liquid feeding and waste liquid tubes 808 and 809. As the flow path 811, a 1 mm width, 100 μm width, and 40 mm length formed on the PDMS substrate 212 is used. Quartz glass is used as the substrate 413, a plano-convex cylinder lens (Sigma light machine, 20 mm × 40 mm) is used as the collimating lens, and a Teflon tube is used as the circulation tube 817.

<Measurement of target substance>
In the apparatus of FIG. 8, the target substance detection element on which the anti-CEA antibody, anti-AFP antibody, anti-PSA antibody and anti-PAP antibody are immobilized is set in the apparatus and the following protocol is performed.
(1) Set the valve 1 on the washing liquid side and the valve 2 on the waste tank side, and flow the buffer solution adjusted to pH 7.4 at a flow rate of 0.1 (ml / min.) For 10 minutes to detect the target substance. Clean the element thoroughly.
(2) The valve 1 is switched to the sample side and the valve 2 is switched to the circulation tube side, and the sample is allowed to flow for 10 minutes at a flow rate of 0.1 (ml / min.) To cause an antigen-antibody reaction.
(3) Measure the reflection spectrum at regular intervals while feeding the specimen.
(4) Switch the valve to the washing solution side again, and flow the buffer solution at a flow rate of 0.1 (ml / min.) For 10 minutes to wash away the non-specifically adsorbed antigen.

  According to the above protocol, a spectrum as shown in FIG. 4 is obtained for each reaction field. By fitting this spectrum with graph software and obtaining the peak value of the spectrum, the kinetics of the reaction can be measured for each marker protein as shown in FIG. As described above, by measuring the reaction profile of a plurality of disease marker proteins and their antibodies, it becomes possible to investigate the cause of the disease with higher accuracy than the measurement of one protein.

It is a figure for demonstrating one Embodiment of this invention. It is a figure for demonstrating the example of the target substance detection element of this invention. It is a figure for demonstrating Examples 1-3 of this invention. It is a figure for demonstrating Example 1 of this invention. It is a figure for demonstrating Example 1 of this invention. It is a figure for demonstrating Example 2 of this invention. It is a figure for demonstrating Example 2 of this invention. It is a figure for demonstrating Example 3 of this invention. It is a figure for demonstrating Example 1 of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 101 Light source 102 Spectrometer 103 Specimen reservoir 104 Washing liquid reservoir 105 Flow path switching valve 106 Liquid feeding means 107 Waste liquid tank 108 Liquid feeding tube 109 Waste liquid tube 110 Target substance detection element 111 Flow path 112 Cover 113 Substrate 114 Target substance detection chip 115 Collimating lens DESCRIPTION OF SYMBOLS 201 Substrate 202 Metal thin film 203 Dielectric 204 Metal film 205 Capturer 206 Target substance detection element 301 Substrate 302 Metal thin film 303 Dielectric layer 304 Metal layer 305 Target substance capture body 306 Target substance detection element 307 Substrate 308 Metal layer 309 Dielectric layer 310 Metal layer (having holes)
311 Target substance capturing body 312 Target substance detection element 601 Light source 602 Spectroscope 603 Sample reservoir 604 Cleaning liquid reservoir 605 Flow path switching valve 606 Pump 607 Waste liquid tank 608 Liquid feed tube 609 Waste liquid tube 610 Target substance detection element 611 Flow path 612 Cover 613 Substrate 614 Target substance detection chip 615 Coupler 616 Optical fiber 801 Light source 802 Spectrometer 803 Specimen reservoir 804 Washing liquid reservoir 805 Channel switching valve 806 Pump 807 Waste liquid tank 808 Liquid feeding tube 809 Waste liquid tube 810 Target substance detection element 811 Flow path 812 Cover 814 Target substance detection chip 815 Coupler 817 Flow path switching valve 818 Sample circulation tube 901 Si substrate 902 Gold 03 SiO2 layer 904 positive resist 905 Gold

Claims (7)

An element used for detecting a target substance in a specimen using a plasmon resonance method,
A target substance detection comprising a substrate, a metal thin film formed on the substrate, a dielectric layer formed on the metal thin film, and a metal pattern layer formed on the dielectric layer element.   The target substance detection element according to claim 1, wherein a capture molecule for capturing the target substance is provided on the metal pattern layer.   The target substance detection element according to claim 1, wherein the dielectric layer has the same pattern shape as the metal pattern layer.   The target substance detection element according to claim 1, wherein optical characteristics of the target substance detection element change due to the presence of the target substance in the specimen.   5. The target substance detection element according to claim 4, wherein the thicknesses of the dielectric layer and the metal pattern layer are in the range of 1/10 to 1/2 of the wavelength of light used for detecting the change in the optical characteristics. An apparatus for detecting a target substance in a specimen,
(1) a flow path;
(2) liquid feeding means for supplying the specimen to the flow path;
(3) a target substance detection element having a capturing body for the target substance disposed in the flow path;
(4) a light irradiation means for irradiating the target substance detection element with light for detection by a plasmon resonance method;
(5) a light detection means for detecting a change in light emitted by the light irradiation means;
Have
The target substance detection device according to claim 1, wherein the target substance detection element is the one according to claim 1. A method for detecting a target substance in a specimen,
Measuring the change in the plasmon resonance spectrum in the reaction field while sending the sample to the reaction field in which the target substance detection element is arranged;
And a step of detecting a target substance in the specimen based on a change in the plasmon resonance spectrum, wherein the element according to claim 1 is used as the target substance detection element.

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