JP2008232720A - Detection element, detection element device, and detection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection element with its detection sensitivity on a target substance being high owing to its narrow band spectrum obtained, a detection element device, and a detection method. <P>SOLUTION: This detection element 8 consisting of a structure 7 formed on a support body 4, the structure 7 comprising a metal layer 2 with a capturing body 5 fixed thereon, is used for capturing the target substance in a specimen liquid by means of the capturing body 5. This detection element 8 is characterized in that a top dielectric layer 1 is formed on a surface of the metal layer 2 on the opposite side of its side facing the support body 4, and the capturing body 5 is fixed on a side surface 2a of the metal layer 2. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

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

<Target substance detection element>
There are multiple markers in blood for specific diseases such as cancer, hepatitis, diabetes, and osteoporosis. When affected, the concentration of a specific protein changes (increases or decreases) from normal. It is expected as a next-generation medical technology because it is possible to detect a serious illness early by monitoring these from normal times. In addition, it is considered that metastasis and recurrence can be detected at an early stage by monitoring the prognosis after tumor removal, and is expected to make a significant contribution to improving the quality of medical care.

  In recent years, the Food Sanitation Law has been amended to require businesses to display eggs, peanuts, buckwheat, wheat, and milk. However, few people know quantitatively how much they are allergic to what food. It is important to scientifically know these data as numerical values from the viewpoints of health promotion and preventive medicine for each person, and great expectations are placed on the technical field disclosed in the present invention.

  One method for analyzing raw and unpurified proteins is based on detection elements that use biological ligand-analyte interactions to identify specific compounds.

Typical detection element methods include fluorescence immunization, plasmon resonance, and optical interference. In either case, the ligand, which is one molecule, or the analyte, which is the other molecule, is immobilized on the surface of the detection element, and the ligand or the analyte in the specimen is selected and bonded with high selectivity. Thus, it is a common step to eliminate impurities and capture only the target substance of interest with high efficiency.
<Localized Surface Plasmon Resonance Sensor (LSPR)>
In the detection element using the plasmon resonance method, a ligand is immobilized on a metal thin film or a metal microparticle by utilizing the fact that metal plasmon reacts with high sensitivity to the refractive index change of the interface substance, and the ligand and the specific substance are specific. The concentration of the analyte bound to is measured. The ligand is immobilized on the surface of the detection element using chemical or physical means. By using this technique, it is possible to obtain information on the kinetics of the reaction between ligand analytes, and it is considered to be an advantage of LSPR together with the feature that labeling is unnecessary.

  In Patent Document 1, a localized surface plasmon detection element is produced by self-organizing and immobilizing metal fine particles on a glass substrate surface-modified with an amino group, and its basic characteristics are disclosed. . In addition, a technique for measuring the concentration and kinetics of a target substance by detecting an intensity change in an absorption spectrum is described.

  Patent Document 2 discloses a detection element for solving the problem that the change in the detection element output signal is weak and the problem that it takes a long time until the change is recognized. In other words, Patent Document 2 has a substrate in which a plurality of micropores are formed on one surface and metal fine particles are filled in the micropores, and at least a part of the metal microparticles is outside the substrate than one surface. Is disclosed.

  Non-Patent Document 1 discloses an LSPR detection element in which metal fine particles are immobilized on a chemically modified glass substrate as in Patent Document 1. Here, it is disclosed that streptavidin and biotin reaction can be observed from a change in the intensity of the absorption spectrum. Further, the refractive index response when the surrounding medium is changed is reported to be 76.4 nm / index.

Non-Patent Document 2 discloses a technique for producing an LSPR detection element by arranging Ag dots by using nanosphere lithography (lithography technique using polystyrene nanobeads). The refractive index response when the surrounding medium is changed is reported to be 200 nm / index.
Japanese Patent No. 3452837 JP 2004-245639 A Anal. Chem. 2002, 74, 504-509, A Colorimetric Gold Nanoparticle Sensor To Interrogate Biomolecular Interactions in Real Time on a Surface J. Phys. Chem. B 1999, 103, 9846-9853, Nanosphere Lithography: Effect of External Dielectric Medium on the Surface Plasmon Resonance Spectrum of a Periodic Array of Silver Nanoparticle

  However, the technique disclosed above has a problem that the line width of the plasmon resonance spectrum is very broad and the detection sensitivity of the target substance is low.

  Therefore, an object of the present invention is to provide a detection element, a detection element device, and a detection method in which a narrow band spectrum is obtained and the detection sensitivity of a target substance becomes high.

  In order to achieve the above object, the detection element of the present invention has a structure in which a structure having a metal layer on which a capture body is immobilized is formed on a support, and the capture body captures a target substance in a sample liquid. The element is characterized in that an upper surface dielectric layer is formed on a surface of the metal layer opposite to the side facing the support, and a capturing body is fixed on the side surface of the metal layer.

  In the detection element of the present invention, since the upper dielectric layer is formed on the metal layer, the capturing body is fixed only on the side surface of the metal layer. Thereby, the structure and the dielectric interface have substantially the same resonance condition. Therefore, when incident light is irradiated, the plasmon mode induced at the interface is reduced, the spectral line width (Q value) can be narrower than before, and the sensitivity of the detection element is further increased. Is possible.

The present invention will be described below with reference to the drawings.
<Target substance detection element>
1A and 1B are diagrams for explaining the outline of the detection element of the present invention, FIG. 1A is a side view, and FIG. 1B is a plan view.

  The detection element 8 includes a support 4 and a structure 7 that functions as a detection element disposed on the support 4. In the description in this specification, the detection element may be referred to as a target substance detection element. The structure 7 that is a metal nanostructure has an upper dielectric layer 1, a metal layer 2, and a lower dielectric layer 3, and the capturing body 5 is fixed only on the side surface 2 a of the metal layer 2. . The lower dielectric layer 3 is formed on the support 4, and the metal layer 2 is formed on the lower dielectric layer 3. In other words, the lower dielectric layer 3 is formed on the side of the metal layer 2 facing the support 4. The upper dielectric layer 1 is formed on the metal layer 2. In other words, the upper dielectric layer 1 is formed on the surface of the metal layer 2 opposite to the side facing the support 4.

  Thus, since the metal layer 2 is sandwiched between the upper dielectric layer 1 and the lower dielectric layer 3, the upper and lower surfaces of the metal layer 2 are not exposed, and only the side surface 2a is exposed. That is, since only the side surface 2a of the metal layer 2 is exposed, the capturing body 5 is fixed only to the exposed side surface 2a, and the capturing body 5 is fixed to the upper and lower surfaces of the unexposed metal layer 2. It has not been.

  The material of the metal layer 2 is not particularly limited as long as it is a metal in which plasmon resonance occurs. Here, it is preferable to select and use suitably from noble metals, such as Au, Ag, Cu, and Pt. The thickness of the metal layer 2 is not particularly limited as long as plasmon is generated at the interface between the sample liquid and the dielectric layer and the film operates as a detection element. Here, the film thickness is preferably in the range of 10 to 500 nm.

  The upper dielectric layer 1 and the lower dielectric layer 3 are made of an organic polymer material or an inorganic material.

  As the organic polymer material, a fluororesin can be used, and those having an amorphous structure are used from the viewpoint of optical properties such as transparency and refractive index. For example, it is composed of either Cytop (Asahi Glass Co., Ltd.), Teflon AF (DuPont), or a mixture thereof.

Examples of inorganic materials include SiO ₂ , SiOx, and fluorine compounds (CaF ₂ , MgF ₂ , LiF, etc.), and these materials are appropriately selected and used.

  Further, there is no particular problem whether the upper dielectric layer 1 and the lower dielectric layer 3 have the same thickness or different thicknesses.

Further, the wavelength dependency of the refractive index of the upper dielectric layer 1 and the lower dielectric layer 3 may satisfy the following relational expression over the entire wavelength range.

n Sample liquid: Refractive index of sample liquid n Upper surface derivative: Refractive index of upper surface derivative layer n Lower surface derivative: Refractive index of lower surface derivative layer n is a physical quantity of the substance and is a refractive index. n is wavelength-dependent and varies depending on the light used. In the present invention, the above relationship needs to be satisfied in the visible light to infrared region (about 350 to 1500 nm).

The refractive index is represented by a complex number as follows. In the case of a transparent substance such as a dielectric, κ may be set to almost 0, so that the refractive index is represented by the value of the real part only.
n = n + iκ
In the present invention, the dielectric constant is represented by ε = n ² .

  When the refractive indexes of the upper surface and the lower surface dielectric layer satisfy the above relationship, a spectrum having a high Q value can be obtained regardless of the refractive index of the sample solution.

  Moreover, the wavelength dependence of the dielectric constant of the upper surface dielectric layer 1 and the lower surface dielectric layer 3 may be substantially the same. Furthermore, the wavelength dependence of the dielectric constant of the upper dielectric layer 1 and the support 4 may be substantially the same.

  Since the refractive indexes of the upper surface dielectric layer 1 and the lower surface dielectric layer 3 are substantially the same, a spectrum having a higher Q value can be obtained, and the detection sensitivity of the target substance can be improved. It is also possible to reduce reflection on the substrate surface by making the wavelength dependence of the dielectric constant of the upper dielectric layer 1 and the support 4 substantially the same.

  The structure of the detection element of the present invention may be a two-layer structure 17 as shown in FIG. 2 in addition to the three-layer structure shown in FIG. In other words, the upper surface dielectric layer 1 may be formed on the surface of the metal layer 2 opposite to the side facing the support 4. In this case, the support 4 plays the role of the lower dielectric layer 3 of the structure 7 shown in FIG. The upper surface of the metal layer 2 is covered with the upper dielectric layer 1 and the lower surface is covered with the support 4 so that only the side surface 2a of the metal layer 2 is exposed.

  1 and 2 exemplify the detection element structure of the present invention having a square planar shape, but the present invention is not limited to this. The detection element 28 in FIG. 3A includes a structure 27 having a rectangular planar shape. The detection element 38 in FIG. 3B includes a structure 37 having a triangular planar shape. The detection element 48 in FIG. 3C includes a structure 47 having a circular planar shape. Note that each of the structures shown in FIGS. 3A to 3C has a three-layer structure, but may have a two-layer structure as shown in FIG.

  1 to 3, the trapping body 5 is illustrated as being uniformly fixed to the side surface 2a regardless of the deflection direction of the incident light 6, but the present invention is not limited to this. Absent. In other words, as shown in FIG. 4, it may be selectively fixed on a side surface perpendicular to the deflection direction (electric field vector). Preferably, the structure is selectively fixed to a region where the evanescent field is strong (in FIG. 4, a side surface orthogonal to the electric field vector). 4A shows the structure having the rectangular shape shown in FIG. 3A, and FIG. 4B shows the structure having the square shape shown in FIG. ing.

Further, the arrangement of the structures on the support is not particularly limited in any arrangement such as a tetragonal lattice arrangement, a triangular lattice arrangement, a hexagonal lattice arrangement, a rotation target arrangement, and a quasi-periodic arrangement. In addition, it is preferable that the pitch between structures exists in the range of 50-2000 nm.
<Process for manufacturing target substance detection element>
There are no particular problems with the target substance detection element manufacturing method as long as it is a method that can form a desired pattern. There is no problem even if it is a technique using electron beam (EB) lithography, a technique using nanoimprint technology, or a technique using photolithography or X-ray lithography. Furthermore, there is no problem even if the method is formed in a self-organized manner. In addition, the method of forming the metal-dielectric nanostructure by transferring the pattern may use etching, and there is no problem even if it is formed by lift-off.
<Capturer and target substance>
The capturing body 5 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.

  The target substance is not particularly limited as long as it forms a specific binding pair with the capturing body 5. Specifically, target substances contained in a specimen are roughly classified into non-biological substances and biological substances. Examples of non-biological substances that are of great industrial utility include PCBs with different chlorine substitution numbers / positions as environmental pollutants, dioxins with different chlorine substitution numbers / positions, and endocrine disrupting substances called so-called environmental hormones. It is done.

The biological material is selected from nucleic acids, proteins, sugar chains, lipids, and complexes thereof. Biological substances are included, and more specifically, biomolecules selected from nucleic acids, proteins, sugar chains, and lipids. Specifically, DNA, RNA, aptamer, gene, chromosome, cell membrane, virus, antigen, antibody, lectin, hapten, hormone, receptor, enzyme, peptide, sphingosaccharide, sphingolipid and the like. Further, the present invention can be applied to any substance as long as it includes a substance selected from any of these. Furthermore, bacteria and cells themselves that produce the above-mentioned “biological substances” can also be targeted substances as “biological substances” targeted by the present invention.
<Operating principle of detection element>
Next, the operation principle of the chemical detection element of the present invention will be described with reference to FIG.

In the figure, incident light 6 is incident from the upper direction to the lower direction (+ z direction) (FIG. 5A). Incident light 6 is free electrons and coupling in the structure 7, causing an evanescent field region E _F around the structure 7 (Figure 5 (b)). Evanescent field region E _F is sensitively respond to change in the refractive index of the surrounding medium.

  When the target substance in the specimen liquid is bound to the capturing body 5 fixed to the side surface 2a of the metal layer 2 of the structure 7, the refractive index around the structure 7 increases the refractive index of water. Then, the resonance frequency of plasmon resonance changes, and the peak of the absorption spectrum shifts to the long wavelength side (FIG. 5 (c)). This principle can be used to detect biochemical reactions such as antigen antibodies.

Detection device of the present invention, the capturing molecule 5 is immobilized only on the side surfaces 2a of the structure 7 has occurred an evanescent field region E _F. For this reason, the structure 7 and the dielectric interface have substantially the same resonance conditions. Therefore, when incident light 6 is irradiated, the plasmon mode induced at the interface is reduced, the spectral line width (Q value) can be narrower than that of the prior art, and the sensitivity of the detection element is further increased. It becomes possible.

Conventionally, an evanescent field region E _F spectral line width of the conventional detection device be immobilized capturing body 5 on the upper surface of the structure does not occur (Q value) was as broad. On the other hand, in the case of the present invention, a narrower band spectrum can be obtained as compared with the conventional configuration, so that sensing with higher sensitivity than the conventional detection element is possible.
<Target substance measuring device>
(Measurement device using reaction well)
A measurement apparatus of the present invention for measuring a target substance will be described with reference to FIG. FIG. 6A is a cross-sectional view of the measuring apparatus of the present invention, and FIG. 6B is a perspective view.

  The measurement apparatus of the present invention includes a light source 202, an objective lens 203, a deflection element 206, a substrate 200 on which a reaction well 201 is formed, a detection element of the present invention disposed in the reaction well 201, and a light receiving means 204. And have.

  The light source 202 is not particularly limited as long as it is a stable light source. In the present invention, it is preferable to use a halogen lamp. The objective lens 203 has no problem as long as the incident light from the light source 202 can be made substantially parallel. Here, it is more preferable to use one having no chromatic aberration. The polarizing means 206 is not particularly limited as long as it can generate polarized light. Here, it is preferable to use a polarizer that produces polarized light in the y direction. The light receiving means 204 is preferably a spectroscopic measurement apparatus having a wavelength resolution of about 1 nm. In the present invention, it is preferable to use a multi-channel detector or a spectrophotometer.

The reaction well 201 is preferably made of a material with little non-specific adsorption of the target substance (or a well member is coated with a non-specific adsorption prevention layer). In the reaction well 201, the detection element 8 of the present invention is arranged. The detection element in the reaction well 201 is not limited to the detection element 8, and may be the detection elements 18, 28, 38, and 48.
(Measurement device using microchannel)
A measuring apparatus using the microchannel of the present invention for measuring a target substance will be described with reference to FIG. Fig.7 (a) is a partial cross section figure of the measuring apparatus of this invention, FIG.7 (b) is a perspective view.

  The measuring device using the microchannel of the present invention includes an optical measurement system and a liquid channel system.

  The optical measurement system includes a light source 601, an objective lens 615, and a light receiving unit 602. The liquid flow path system includes a liquid supply system, a target substance detection chip 614, and a waste liquid recovery system.

  The light source 601 is not particularly limited as long as it is a stable light source. In the present invention, it is preferable to use a halogen lamp. The light receiving means 602 is preferably a spectroscopic measurement device having a wavelength resolution of about 1 nm. In the present invention, it is preferable to use a multi-channel detector or a spectrophotometer.

  The liquid supply system includes a specimen reservoir 603 and a cleaning liquid reservoir 604, which are connected to one end side of the flow path 611 of the target substance detection chip 614 via a flow path switching valve 605 and a liquid feeding tube 608. .

  The target substance detection chip 614 includes a substrate 613 and a cover 612 in which a flow path 611 and a reaction well 610 are formed.

  In the reaction well 610, the detection element 8 of the present invention is arranged. The detection element in the reaction well 610 is not limited to the detection element 8, and may be the detection elements 18, 28, 38, and 48.

  The sample reservoir 603 may be a reservoir made of a material with less non-specific adsorption of the target substance, or a reservoir built in the chip. In the present invention, it is preferable to use an Eppendorf tube coated with non-specific adsorption prevention.

  The cleaning liquid reservoir 604 is not particularly limited. Here, it is preferable to use a biochemical test grade glass or plastic tube.

  The flow path switching valve 605 is not particularly limited as long as the fluid tube can be switched. In the present invention, it is preferable to use a three-way valve.

  The waste liquid collection system includes a liquid feeding means 606 and a waste liquid tank 607, and is connected to the other end of the flow path 611 of the target substance detection chip 614 via a waste liquid tube 609.

  As the liquid feeding means 606, 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 a pump, the waste liquid tank 607 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.

  The liquid feeding and waste liquid tubes 608 and 609 are preferably tubes made of a material that can absorb the target substance as little as possible.

Hereinafter, the present invention will be described in more detail with reference to examples. In addition, each Example does not show an optimal value unless the present invention is limited.
(Example 1)
<1. Target substance detection element>
FIG. 8 is a process diagram for explaining the manufacturing method of the detection element of this embodiment.

As the support 4, a quartz wafer having a thickness of 525 μm is used. The material of the upper dielectric layer 1 and the lower dielectric layer 3 of the structure 7 is SiO ₂ and the material of the metal layer 2 is Au. Further, Ti is subtracted as an adhesion layer between the upper dielectric layer 1 and the metal layer 2 and between the metal layer 2 and the lower dielectric layer 3 (not shown). The shape of the structure 7 viewed from the xy plane is a square shape having a side of 150 nm. The thickness of the upper dielectric layer 1 and the lower dielectric layer 3 is 50 nm, respectively, and the thickness of the metal layer 2 is also 50 nm.

  A method for manufacturing the detection element 7 having the above configuration will be described below.

  First, a positive resist (ZEP520A) is spin-coated on the support 4 made of a quartz substrate to provide a resist layer 306. Next, as the conductive polymer layer 307, Espacer (manufactured by Showa Denko) is spin-coated (FIG. 8A).

  This is introduced into the chamber of the electron beam drawing apparatus, and a desired pattern is drawn (FIG. 8B).

  After the drawing is completed, the conductive polymer layer 307 is removed by washing with pure water (FIG. 8C).

  Then, it develops with a developing solution and the resist layer 306 is patterned (FIG.8 (d)).

  Using the patterned resist layer 306 as a mask, the lower dielectric layer 3, the Ti adhesion layer, the metal layer 2, the Ti adhesion layer, and the upper dielectric layer 1 are formed (FIG. 8E).

  Next, lift-off is performed to remove the lower dielectric layer 3, the Ti adhesion layer, the metal layer 2, the Ti adhesion layer, and the upper dielectric layer 1 other than the resist layer 306 and the structure 7 (FIG. 8F). .

Finally, the detection element 8 is completed by physically immobilizing the capturing body 5 on the side surface 2a of the metal layer 2 (FIG. 8G).
<2. Basic optical characteristics of target substance detection element>
This will be described with reference to FIG. FIG. 9 is a graph in which the transmittance with respect to wavelength of the detection element 8 having the structure 7 shown in FIG. 1 and the detection element having a structure without a dielectric layer is calculated by electromagnetic simulation software. In the figure, the bold line indicates the transmittance of the detection element 8 shown in FIG. 1, and the thin line indicates the transmittance of the detection element without the dielectric layer. Also, the transmission spectra in the atmosphere and pure water are shown for each structure.
The size of the laminated structure dots is x = y = 150 nm, and the thicknesses of the upper dielectric layer 1 and the lower dielectric layer 3 that are SiO ₂ layers and the metal layer 2 that is an Au layer are all 20 nm. The size of the Au dots is x = y = 150 nm, and the thickness of the Au layer is 20 nm.

From the graph, the spectral line width of the detection element 8 having the structure 7 of the present invention is narrower than the spectral line width of the detection element having a structure without a dielectric layer, both in the atmosphere and in pure water. Improved.
<3. Target substance detection kit / device>
In this example, the measuring apparatus shown in FIG. 6 was used.

A halogen lamp was used as the light source 202. As the objective lens 203, a plano-convex cylinder lens (Sigma light machine, 20 mm × 40 mm) was used. As the light receiving means 204, a multi-channel detector was used. The reaction well 201 is a processed PDMS substrate. The surface is subjected to treatment for preventing nonspecific adsorption. Further, the detection element 8 of the present invention which is a plasmon optical element is used as the target substance detection element.
<4. Measurement of target substance>
The detection element 8 on which the anti-IgE antibody is immobilized as the capturing body 5 is set in the reaction well 201, and the following protocol is performed.
(1) Wash the reaction well 201 and the detection element thoroughly with a buffer solution adjusted in pH.
(2) Inject 100 μL of the sample solution with a micropipette and bring it into contact with the detection element to cause an antigen-antibody reaction.
(3) After reacting for 2 hours, the sample solution is extracted with a micropipette and the nonspecifically adsorbed IgE antigen is washed with a buffer solution.
(4) The buffer solution is injected again, and the optical change of the detection element, which is a plasmon optical element, is measured, that is, the spectrum is measured.

When measurement is performed according to the above protocol, a plasmon resonance spectrum as shown in FIG. 5C is obtained. In the detection element having the configuration disclosed in the present invention, the spectral line width is narrower than that of a detection element in which gold colloid is immobilized or a detection element using Au dots. Furthermore, according to the detection element of the present invention, since the capturing body 5 is fixed only to the side surface 2a, the amount of the capturing body 5 is small and the detection element is more sensitive. By analyzing this spectrum, a calibration curve as shown in FIG. 10 is obtained. The practice is the calibration curve of the detection element of the present invention, and the broken line is the calibration curve of the conventional detection element in which the region where the capturing body is immobilized is not limited. By using the detection element of the present invention, the detection limit (LOD) can be reduced as compared with the conventional one.
(Example 2)
<1. Target substance detection element>
In this example, the sensitivity of the detection element 28 shown in FIG. 3A and the detection element 78 shown in FIG. The area of the upper dielectric layer 1 of the detection element 78 in the xy plane is smaller than the area of the metal layer 2 in the xy plane. Therefore, in the detection element 78, the capturing body 5 is fixed not only on the side surface 2 a of the metal layer 2 but also on the upper surface portion.
(First element)
In this embodiment, the detection element 28 shown in FIG. 3A in which the capturing body 5 is fixed only on the side surface 2a is used as the first element.

As the support 4 of the first element, a quartz wafer having a thickness of 525 μm was used. The material of the upper dielectric layer 1 and the lower dielectric layer 3 of the structure 7 was SiO ₂ and the material of the metal layer 2 was Au. Further, Ti is subtracted as an adhesion layer between the upper dielectric layer 1 and the metal layer 2 and between the metal layer 2 and the lower dielectric layer 3 (not shown).

The shape of the xy plane of the structure 7 is a rectangle having a major axis of 150 nm and a minor axis of 50 nm.
(Second element)
In this embodiment, the detection element 78 shown in FIG. 11A in which the capturing body 5 is fixed not only on the side surface 2a but also on the upper surface portion is a second element.

As the support 4 of the second element, a quartz wafer having a thickness of 525 μm was used. The material of the upper dielectric layer 1 and the lower dielectric layer 3 of the structure 7 was SiO ₂ and the material of the metal layer 2 was Au. Further, Ti is subtracted as an adhesion layer between the upper dielectric layer 1 and the metal layer 2 and between the metal layer 2 and the lower dielectric layer 3 (not shown).

The xy plane shape of the metal layer 2 and the lower dielectric layer 3 is a rectangle with a major axis of 150 nm and a minor axis of 50 nm. The shape of the xy plane of the upper dielectric layer 1 is a rectangle having a major axis of 100 nm and a minor axis of 50 nm.
<2. Target substance detection kit / device>
Measurement is performed using the same kit and apparatus as used in Example 1.
<3. Measurement of target substance>
In the apparatus of FIG. 4, the detection element 8 on which the anti-IgE antibody is immobilized is set in the apparatus, and the following protocol is performed.
(1) Wash the reaction well and the target substance detection element thoroughly with a buffer solution adjusted in pH.
(2) Inject 100 μL of the sample solution with a micropipette and bring it into contact with the detection element to cause an antigen-antibody reaction.
(3) After reacting for 2 hours, the sample solution is extracted with a micropipette and the non-specific CRP antigen is washed with a buffer solution.
(4) The buffer solution is injected again, and the optical change of the detection element, which is a plasmon optical element, is measured, that is, the spectrum is measured.

  When each element is measured according to the above protocol, a calibration curve as shown in FIG. 12 is obtained. In FIG. 12, the thick solid line represents the calibration curve of the detection element 8 provided with the first element, the thin solid line represents the calibration curve of the detection element 78 equipped with the second element, and the broken line represents the Au colloid without the dielectric layer. It is a calibration curve of the used detection element. As shown in the figure, the detection limit (LOD) of both elements can be made smaller than that of the conventionally disclosed detection element using Au colloid. Further, the plasmon element shown in FIG. 3A has a larger dielectric layer area and a narrower spectral line width than the plasmon element shown in FIG.

It should be noted that any one of the element in which the xy plane of the lower dielectric layer 3 shown in FIG. 11B is smaller than the metal layer 2 and the xy plane of the upper dielectric layer 1 and the lower dielectric layer 3 shown in FIG. The same result was obtained for an element that is smaller than the metal layer 2. That is, the plasmon element shown in FIG. 3 (a) has a larger dielectric layer area and a narrower spectral line width than the plasmon element shown in FIGS. 11 (b) and 11 (c). The value became smaller.
(Example 3)
<1. Target substance detection element>
The element configuration for detecting a target substance according to this example will be described with reference to FIG. As the support 304, a quartz wafer having a thickness of 525 μm is used. The nanostructures comprising a metal and a dielectric material of 301 and 303 using the SiO _2, the material of 302 using gold. Further, Ti is subtracted as an adhesion layer between SiO ₂ and Au (not shown).

The shape of the plasmon element on the xy plane is a square shape with a side of 150 nm. The thicknesses of the upper and lower dielectric layers are 50 nm and the gold thickness is 50 nm.
<2. Target substance detection kit / device>
The measuring apparatus shown in FIG. 7 was used.

  The light source 601 was a halogen lamp, and the light receiving means 602 was a multi-channel detector (Hamamatsu Photonics). The specimen reservoir 603 was an Eppendorf tube, and the cleaning liquid reservoir 604 was a biochemical glass bottle. The flow path switching valve 605 was a three-way valve (GL Science), the liquid feeding means 606 was a syringe pump (kd Scientific KDS200), and the waste liquid tank 607 was a syringe. Teflon tubes were used as the liquid feeding and waste liquid tubes 608 and 609. The channel 611 is formed in a cover (PDMS substrate) 612 with dimensions of 1 mm width, 100 μm width, and 40 mm length. The substrate 613 is made of quartz glass, and the objective lens 615 is a plano-convex cylinder lens (Sigma light machine, 20 mm × 40 mm).

The detection element 8 shown in FIG. 1 was used as the detection element.
<3. Measurement of target substance>
In the apparatus of FIG. 6, the target substance detection element 610 on which the anti-CRP antibody is immobilized is set in the apparatus, and the following protocol is performed.
(1) The three-way valve 605 is switched to the cleaning liquid reservoir 604 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 detection element 8.
(2) The three-way valve 605 is switched to the specimen reservoir 603 side, and the specimen 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) The three-way valve 605 is switched to the washing liquid reservoir 604 side again, and the buffer solution is allowed to flow for 10 minutes at a flow rate of 0.1 (ml / min.) To wash away the nonspecifically adsorbed antigen.

  With the above protocol, the spectrum shown in FIG. 5C is obtained at each reaction time.

A time course of the antigen-antibody reaction is obtained as shown in FIG. 13 by functionally fitting this spectrum with graph software to obtain the peak value of the spectrum.
Example 4
<1. Target substance detection element>
In this example, the time course of each antigen-antibody reaction of anti-CRP antibody, anti-IgG antibody, anti-IgM antibody, and anti-IgA antibody by the detection element 8 produced by the production process shown in FIG. 8 was obtained.

As the support 4, a quartz wafer having a thickness of 525 μm was used. The material of the upper dielectric layer 1 and the lower dielectric layer 3 of the structure 7 was SiO ₂ and the material of the metal layer 2 was Au. Further, Ti is subtracted as an adhesion layer between the upper dielectric layer 1 and the metal layer 2 and between the metal layer 2 and the lower dielectric layer 3 (not shown).

  The capturing body 5 uses an anti-CRP antibody, an anti-IgG antibody, an anti-IgM antibody, and an anti-IgA antibody.

The structure 7 has a square shape shown in FIG. 1 and has a side of 150 nm. The thicknesses of the upper dielectric layer 1, the metal layer 2, and the lower dielectric layer 3 are all 50 nm.
<2. Target substance measuring device>
In this example, the measuring apparatus shown in FIG. 14 was used.

  The measurement apparatus used in this example is composed of an optical measurement system and a liquid channel system.

  The optical measurement system of the measurement apparatus of the present embodiment is different from the measurement apparatus shown in FIG. 7 in that a multi-channel detector capable of 4ch simultaneous measurement is used, but other configurations are basically the same.

The optical measurement system includes a light source 601, a light receiving unit 602, and an optical fiber 716. The optical fiber 716 connected to the light source 601 and the optical fiber 716 connected to the spectroscope 602 are connected by a coupler 715. The optical fiber 716 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.
<3. Measurement of target substance>
A detection element 8 in which an anti-CRP antibody, an anti-IgG antibody, an anti-IgM antibody, and an anti-IgA antibody are immobilized as a capturing body 5 is prepared, and each detection element 8 is set in the measurement apparatus shown in FIG. Do.
(1) The three-way valve 605 is switched to the cleaning liquid reservoir 604 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 detection element 8.
(2) The three-way valve 605 is switched to the specimen reservoir 603 side, and the specimen is allowed to flow at a flow rate of 0.1 (ml / min.) For 10 minutes to cause an antigen-antibody reaction.
(3) The three-way valve 605 is switched to the washing liquid reservoir 604 side again, and the buffer solution is allowed to flow at a flow rate of 0.1 (ml / min.) For 10 minutes to wash away the nonspecifically adsorbed antigen.

  According to the above protocol, a spectrum as shown in FIG. 5C 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 the side view and top view for demonstrating the outline of an example of the detection element of this invention. It is the side view and top view for demonstrating the outline of the other example of the detection element of this invention. It is the side view and top view for demonstrating the outline of the other example of the detection element of this invention. It is the side view and top view for demonstrating the outline of the other example of the detection element of this invention. It is the figure and graph for demonstrating the principle of operation of the detection element of this invention. It is the schematic of an example of the measuring apparatus provided with the detection element of this invention. It is the schematic of an example of the measuring apparatus using the microchannel provided with the detection element of this invention. It is process drawing which shows an example of the manufacturing process of the detection element of this invention. It is the graph which calculated the transmittance | permeability with respect to the wavelength of the detection element which has a structure of this invention, and the detection element which has a structure without a dielectric layer with electromagnetic field simulation software. It is a graph which shows the calibration curve of the detection element which has a structure of the present invention, and the detection element which has a structure without a dielectric layer. It is the side view and top view which show the example of the detection element which has a structure provided with the dielectric material layer of the area smaller than the area of a metal layer. It is a graph which shows the calibration curve of the detection element which has a structure of the present invention, and the detection element which has a structure without a dielectric layer. It is a time course of the antigen antibody reaction in the detection element of this invention. It is the schematic of an example of the measuring apparatus using the multichannel detector provided with the detection element of this invention and capable of 4ch simultaneous measurement. It is a time course of the antigen antibody reaction in the detection element of this invention.

Explanation of symbols

2 Metal layer 2a Side surface 4 Support body 5 Capture body 7 Structure 8 Detection element

Claims (7)

In the detection element for capturing the target substance in the sample liquid by the capture body, the structure having the metal layer on which the capture body is immobilized is formed on the support.
The detection is characterized in that an upper surface dielectric layer is formed on a surface of the metal layer opposite to the side facing the support, and the capturing body is fixed on a side surface of the metal layer. element.   The detection element according to claim 1, wherein a lower dielectric layer is formed on a side of the metal layer facing the support. The refractive index of the upper dielectric layer and the lower dielectric layer is over the entire wavelength range,

The detection element according to claim 2, wherein: n specimen liquid: refractive index n of the specimen liquid n upper surface derivative: refractive index n of the upper surface derivative layer n lower surface derivative: refractive index of the lower surface derivative layer is satisfied.   The detection element according to claim 2 or 3, wherein the wavelength dependence of the dielectric constant of the upper surface dielectric layer and the lower surface dielectric layer is substantially the same.   The detection element according to claim 1, wherein the upper dielectric layer and the support have substantially the same wavelength dependency of dielectric constant. A detection element device for detecting a target substance in a sample liquid,
A light source;
The detection element according to any one of claims 1 to 5,
And a light receiving unit that receives light from the light source that has passed through the detection element. A detection method for detecting a target substance in a sample liquid,
Preparing a detection element device according to claim 6;
Contacting the target substance in the sample liquid with the detection element;
Detecting an optical change that occurs when the target substance comes into contact with the detection element.

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