JP2007192806A - Target substance detection element substrate, target substance detection element, target substance detection apparatus and method using the same, and kit for the same - Google Patents

Abstract

To further improve the performance of a chemical sensor for capturing a target substance.
An element used for detecting a target substance in a specimen using a plasmon resonance method, wherein a metal pattern layer is provided on an underlayer having a refractive index equivalent to that of water, and the metal pattern layer A target substance detection element is prepared by fixing a target substance capturing body to the substrate.
[Selection] Figure 1

Description

  The present invention relates to an element for detecting a target substance useful for detecting the presence or absence of a target substance in a sample solution, a substrate for producing the element, a target substance detection apparatus, a detection method, and a detection kit.

  One form of sensor is a biosensor. A biosensor is a measuring device that uses the excellent biomolecule recognition ability of a living body or a biomolecule, and has recently been expected to be widely applied not only to the medical field but also to the environment, food products, and the like. In general, a biosensor detects a capture object that recognizes and captures a substance to be measured (hereinafter referred to as a target substance) and physical and chemical changes that occur at that time, and detects electrical signals, optical signals, and the like. And a detection element that converts the signal into a possible signal. In the living body, there are, for example, enzyme-substrate, antigen-antibody, DNA-DNA and the like as combinations of substances having affinity for each other. Biosensors use the principle that one of these combinations can be immobilized or supported on a substrate and used as a trap component to selectively measure the other substance. In addition, various types of detection elements such as an oxygen electrode, a hydrogen peroxide electrode, an ion electrode, an ISFET, and a thermistor have been proposed. Recently, a crystal resonator or a SAW element capable of detecting a mass change of about nanograms, A plasmon resonance element may be used.

Among various sensors, a sensor using a detection system using plasmon resonance has the following features.
(1) Since a labeling molecule such as a fluorescent dye is not required, the assay configuration is simple.
(2) The process of the adsorption reaction on the surface of the metal fine particles can be directly monitored in real time.

  Therefore, this measurement method is expected to be applied to various assays.

  This measurement method utilizes the fact that metal particles such as gold and silver are immobilized on the substrate surface, and when a light is incident on the metal particles, a characteristic resonance spectrum appears due to localized plasmon resonance induced there. Thus, the substance in the vicinity of the metal fine particles is detected. It is known that the resonance wavelength depends on the dielectric constant of the medium near the metal fine particles. As the dielectric constant increases, the absorbance at the resonance peak increases and shifts to the longer wavelength side. For example, Japanese Patent No. 342837 proposes a system using a gold colloid having a diameter of about 20 nm. This is to detect the refractive index of the medium up to the distance of the diameter of the metal fine particles fixed to the substrate, and as a result, it is possible to detect the adsorption and deposition of substances on the surface of the metal fine particles. .

  On the other hand, as described in Felicia Tam et al. (J. Phys. Chem. B., 2004, Vol. 108, No. 45, p. 17290-17294), core-shell type fine particles in which a gold thin film is coated on a dielectric core are formed. There is also an example of an element structure that is manufactured and improved in detection capability.

Japanese Patent Laid-Open No. 2003-270132 includes a metal film provided with a plurality of openings to which a sensor material is fixed, a light source, and a photodetector that detects light transmitted through or reflected from the openings. A sensor device is disclosed that is periodically arranged in a first direction in the plane.
Japanese Patent No. 3452837 JP 2003-270132 A J. Phys. Chem. B., 2004, 108, 45, p. 17290-17294

  Sufficient detection sensitivity can be obtained when an affinity assay such as an immunoassay using the specificity of an antigen-antibody reaction is performed using an element for detecting a target substance using conventional plasmon resonance. There was no case. This problem relating to detection sensitivity is one of the problems to be solved when a detection method using plasmon resonance is applied to an affinity assay in place of the existing fluorescence immunization method or chemiluminescence immunization method. That is, the problem concerning the above-mentioned detection sensitivity is a big subject to the expansion of the use of the measuring method using localized plasmon resonance.

  The present invention has been made in view of the above-described problems, and an object of the present invention is to produce a target substance detection element capable of improving detection sensitivity in detection of a target substance using localized plasmon resonance, and the same. It is to provide a substrate. Another object of the present invention is to provide a target substance detection device and a target substance detection method using the target substance detection element. A further object of the present invention is to provide a kit for this target substance detection method.

A substrate for a target substance detection element according to the present invention is:
A target substance having a metal pattern layer provided on the surface of the support, and a target substance capturing body capable of capturing a target substance in a sample solution containing at least water as a liquid medium is bonded to the metal pattern layer; A substrate used to form a target substance detection element that detects a target substance using plasmon resonance,
The metal pattern layer is formed on an underlayer provided on the support surface, and the underlayer is represented by the following formula:
0.90na ≦ nb ≦ 1.05na
na: a refractive index of water nb: a substrate for a target substance detection element having a refractive index (nb) represented by a refractive index of an underlayer.

  The target substance detection element of the present invention is a target substance detection element characterized in that a target substance capturing body is bound to the metal pattern layer of the substrate.

The target substance detection apparatus of the present invention is an apparatus for detecting a target substance in a sample solution,
The target substance detection element, sample solution supply means for supplying the sample solution to the target substance detection element, and detection for detecting that the target substance capturing body of the target substance detection element has captured the target substance And a detecting device.

The method for detecting a target substance of the present invention is a detection method for detecting the presence or absence of a target substance or the amount of the target substance in a sample liquid containing at least water as a liquid medium,
Contacting the target substance detection element with the sample solution;
Detecting the capture of the target substance on the target substance detection element when the target substance is contained in the sample solution;
A method for detecting a target substance characterized by comprising:

  A kit for detecting a target substance of the present invention is a kit for detecting the presence or absence of a target substance in a sample solution or the amount of the target substance, the target substance detection element having the above-described configuration, and the target of the target substance A kit for detecting a target substance, comprising a reagent necessary for capture by a substance detection element.

  Effective use of incident light to obtain plasmon resonance by providing a metal pattern layer that binds the target substance trapping body via an underlayer having a refractive index equivalent to that of water on the support Thus, it is possible to improve the detection sensitivity.

  The element for detecting a target substance and the detection apparatus using the target substance of the present invention can be suitably used for detecting biological substances (proteins, nucleic acids, sugar chains, lipids, etc.), allergens, bacteria, viruses and the like. Furthermore, the detection device of the present invention can be suitably applied as a so-called biosensor using a biologically derived substance or a similar substance as a capturing body component regardless of medical use, industrial use, and home use.

In the target substance detection element of the present invention, the metal pattern layer provided on the support is light transmissive (transparent) having a refractive index equivalent to that of water in an environment (temperature, pressure, etc.) in which measurement is performed. Provided on the underlayer. Here, as a refractive index equivalent to water, the following formula:
0.90na ≦ nb ≦ 1.05na
na: Refractive index of water nb: Preferably selected from the refractive index (nb) defined by the refractive index of the underlayer. The refractive index of water (na: refractive index at the interface with the air phase) is 1.34 at 589 nm. Then, the effect in this invention is acquired by selecting the refractive index (refractive index in the interface with an air phase) on the same conditions of the base layer concerning this invention from the range of 1.20-1.40. Can do. Further, this refractive index is preferably selected from the range of 1.30 to 1.36. Hereinafter, the target substance detection element of the present invention, a detection apparatus and method using the same, and a kit for the same will be further described.

(Substrate for target substance detection element)
A substrate for preparing a target substance detection element includes a support, and a metal pattern layer provided on the support via an underlayer having a refractive index equivalent to that of water. The shape (planar shape) and structure of the metal pattern layer can be appropriately determined according to the type of detection system. When applied to a detection system using the plasmon resonance method, the metal pattern layer is provided on the support as an isolated (independent) layer, or the metal layer on the support is provided with an opening (hole). Is preferred. Each of these preferred embodiments will be described below.
(1) Isolated Metal Pattern FIG. 1 shows an example of a substrate for a target substance detection element provided with a metal pattern layer 1 whose planar shape is a rectangular parallelepiped. This target substance detection element substrate has a structure in which an isolated metal pattern layer (metal structure) 1 is disposed on an underlayer 2 provided on the surface of a support 3. The underlayer 2 has a refractive index equivalent to that of water. FIG. 1A is a perspective view of the substrate surface, and FIG. 1B is a cross-sectional view in a direction perpendicular to the substrate. The underlying layer 2 may be patterned in the same manner as the metal pattern layer 1 as shown in FIG. The structure represented by FIGS. 1A and 1B is the simplest form. In addition, the two-dimensional (planar) shape of the metal pattern layer can be a shape as typified by FIGS. In the manufacturing method, when the outer shape and the inner shape are not the same shape (FIG. 2 (K)), the metal pattern layer (FIG. 2) that is rounded and blunted without the intersection of the linear metal pattern layers being formed at an acute angle. (L)). These metal pattern layers are made of any of gold, silver, copper, aluminum, and platinum, preferably noble metals, or alloys thereof, and these materials are selected from the viewpoint of adhesion to the underlayer. It is preferable. When the underlayer is formed of a material containing fluorine, a thin film such as chromium or titanium may be formed between the underlayer and the metal layer.

  The size of the metal pattern layer in the planar shape, that is, the distance from an arbitrary point on the outer peripheral portion to another point is preferably in the range of 10 nm to 1450 nm. Furthermore, it is preferable that it exists in the range of 50 nm-450 nm. In this case, the maximum distance between any two points only needs to be within this range. For example, in the case of the cross-shaped pattern shown in FIG. 13, the distance between the points XY is the maximum. Within the range. Also in the case of the rectangular ring pattern shown in FIG. 13, the distance of the diagonal line L between the outer peripheral shapes XY is similarly the maximum, so L is within the above range. In the circular ring pattern shown in FIG. 2 (G), the diameter of the outer circumference circle is in the above range. When the size of the planar shape of the metal pattern layer 1 is within this range, localized plasmon resonance capable of achieving the target detection sensitivity can be obtained more effectively.

  On the other hand, when the metal pattern layer 1 is configured to have a band-shaped portion, for example, in the shapes of FIGS. 2E to 2L, the width (band width) of the band-shaped portion is the target localization of the present invention. There is no particular limitation as long as plasmon resonance can be obtained. The band width is preferably in the range of 10 nm to 100 nm. For example, in the case of the circular ring pattern of FIG. 2G, this band width is the difference between the radii of the outer circumference circle and the inner circumference circle, and in the case of the pattern shown in FIG. In addition, even if the width | variety of a strip | belt-shaped pattern is the same in the same metal pattern layer, there may exist a different part in strip | belt width.

  One or more metal pattern layers are provided on the substrate as necessary. When a plurality of metal pattern layers are provided, the interval (d) between the metal pattern layers is (λmax−50 nm) ≦ d ≦ (λmax + 50 nm) with reference to the maximum absorption wavelength (λmax) when one metal pattern layer is provided on the support. ). Specifically, the distance is preferably selected from a range of 50 nm to 2000 nm, more preferably 400 nm to 1500 nm. This is because the spatial electric field distribution / intensity is affected by the plasmon interaction between the metal pattern layers. Moreover, since the density of a metal pattern layer will fall and signal intensity will become weak when a space | interval becomes large, it will be necessary to apply a special optical system, Therefore It is preferable to exist in the said range.

  When a plurality of metal pattern layers are provided, a plurality of types of metal pattern layers that differ in at least one of the planar shape and the size thereof can be provided on the substrate. When considering the manufacturing efficiency of the metal pattern layer and the point of simplifying the configuration of the detection system, as shown in FIG. 3, the metal pattern layer 1 having the same shape and size is regularly arranged in a region of about several mm square. It is preferable to arrange them on the support 3 in an array. With such an arrangement, measurement of transmitted light, scattered light, and reflected light can be facilitated.

Since the thickness, outer shape, band width, and distance between adjacent metal pattern layers affect the peak position of the localized plasmon resonance spectrum, they should be formed in a size suitable for measurement. Among the shapes shown in FIG. 2, a shape having a loop portion and / or a branch portion is preferable for further improving detection sensitivity. The reason is considered to be synergistically affected by the following points.
(A) The contour length in one metal pattern layer is increased.
(B) The number of corners further increases when a branching part is used.
(C) The distance between edges in the planar shape of the metal pattern layer can be reduced.

An isolated metal pattern (metal structure) as shown in FIGS. 1 and 2 can be formed to a thickness of about 10 nm to 200 nm.
(2) Opening type metal pattern layer FIG. 15 shows an example of a metal pattern layer in which an opening is provided in the metal layer provided on the support surface. As shown in the plan view of FIG. 15 and the cross-sectional view in the direction perpendicular to the substrate, the metal pattern layer 201 in this substrate is provided on the support 203 via the base layer 202. A number of identical circular openings are regularly arranged in the metal pattern layer 201. The opening can be formed by removing a part of the metal pattern layer using an etching process or the like so that the base layer 202 is exposed at the bottom. FIG. 16 shows a configuration in which the refractive index of the support 302 is equivalent to that of water.

  The planar shape of the opening (hole) of the metal pattern layer can be, for example, a band shape as shown in FIGS. 17 (a: micro slit aperture array) and (c: compound periodic micro slit aperture array). Furthermore, various shapes such as a strip shape as shown in FIG. 17 (b: two-dimensional array of minute openings) or a triangle shape as shown in FIG. 17 (d: two-dimensional array of minute openings) can be used. When providing a plurality of openings in the metal layer, openings having different shapes may be provided in the same metal layer. However, considering the advantages of the manufacturing method, it is preferable to regularly arrange openings having the same shape.

  In this case, the metal pattern layer is also composed of any metal of gold, silver, copper, aluminum and platinum, preferably a noble metal, or an alloy thereof, and these materials are used from the viewpoint of adhesion to the underlayer. Is preferably selected. When the underlayer is formed of a material containing fluorine, a thin film such as chromium or titanium may be formed between the underlayer and the metal layer.

  The thickness of the metal pattern layer in this case can also be formed with a film thickness of about 10 nm to 200 nm, and is appropriately selected depending on the wavelength band of the irradiation light. For example, in the case of Au, about 50 nm is preferable. The shape of the pattern is preferably a pattern shape in which light interacting with metal free electrons is a main component, and a shape having a plurality of openings in the film is preferable. The cross-sectional shape of the opening can take various forms such as a circle, an ellipse, a triangle, a rectangle, and a polygon.

  The distance between two adjacent openings when providing a plurality of openings is preferably selected according to a desired detection system as a distance equal to or less than the wavelength of the irradiation light. The distance between the openings can be set in the same manner as the distance between the isolated metal patterns described above.

(Underlayer)
The underlayer is made of a material having a refractive index equivalent to that of water. As this material, it is desirable to use a transparent low dielectric constant material. Such materials include inorganic materials and organic materials. Examples of the inorganic material include magnesium fluoride (refractive index: 1.37) or lithium fluoride (refractive index: 1.39).

  Examples of the organic material include various polymers.

  As a material for forming the underlayer, a material containing fluorine is preferable. As the organic material containing fluorine, a fluororesin is preferable. Furthermore, a fluororesin having an amorphous structure (for example, PTFE (polytetrafluoroethylene, refractive index: 1.35)) is more preferable from the viewpoint of optical properties such as transparency and refractive index. Specific examples of such a fluororesin include Cytop (manufactured by Asahi Glass Co., Ltd.), Teflon AF (manufactured by DuPont), or a mixture thereof.

  In the present invention, the metal pattern layer is surrounded by a material having the same refractive index by providing the base layer having the same refractive index as water (refractive index: 1.34) containing the target substance. It becomes. Thereby, reflection from different refractive index layers (dielectric layers) is suppressed, turbulence of plasmon resonance is suppressed, and the plasmon resonance peak becomes sharp.

  The thickness of the underlayer can be appropriately selected depending on the form of the base layer. When a base layer is provided on a separately provided support (for example, in the case of the form (II) of the support described later), it is preferably 10 nm to 50 μm, more preferably 100 nm to 30 μm. .

  The refractive index of the underlayer is generally defined by a method for obtaining a refractive index from spectral reflectance and transmittance using a microspectrophotometer and a method by ellipsometry.

(Support)
The support on which the underlayer and the metal pattern layer are provided may be any support that supports these layers and enables detection of a target substance using these layers. Further, the support itself may also serve as an underlayer, or a plurality of layers may be formed on the support, and the uppermost layer may be used as the underlayer. Examples of such a configuration include the following aspects.
(I) The support is composed of a single layer and functions as an underlayer.
(II) An underlayer is provided on the support.

  Examples of the support that can be used in the form (II) include an optically transparent glass plate, a quartz plate, and a resin plate such as polycarbonate and polystyrene. In addition, as the support that also serves as the base layer in the form (I), a plate made of a fluororesin having a refractive index equivalent to that of water is preferably used.

(Manufacturing method of substrate)
(I) Isolated metal pattern layer An example of a method for forming an isolated metal pattern layer will be described with reference to the drawings. As shown in FIG. 5, first, a base layer is formed on the support using a material for forming the base layer (FIG. 5B). When using a resin, a spin coat method or the like is used. In the case of an inorganic material, a vapor deposition method or the like can be used. Subsequently, a metal thin film is formed by sputtering or vapor deposition (FIG. 5C). An electron beam resist is formed thereon by spin coating (FIG. 5D), and is exposed by an electron beam drawing apparatus to obtain a resist pattern after development (FIG. 5E). Thereafter, unnecessary metal thin film is etched (FIG. 5F), the resist is removed, and an array-shaped metal pattern layer having a fluorine-containing material as a base is formed (FIG. 5G). . In addition to the electron beam lithography apparatus described above, the metal pattern layer can be formed by a patterning method using a focused ion beam processing apparatus, an X-ray exposure apparatus, or an EUV exposure apparatus.

  Further, as shown in FIG. 6, a manufacturing method using a support body (FIG. 6A) having fine unevenness manufactured by a molding method using a fluororesin material is also possible. As shown in FIG. 6, a metal thin film is formed on the concave / convex formation surface of the support by sputtering or vapor deposition (FIG. 6B). Next, the metal thin film on the surface is polished to form a metal structure having a desired layer structure on the substrate (FIG. 6C). FIG. 7 shows a manufacturing method when the thickness of the metal pattern layer is thinner than the height of the unevenness. In this case, the convex portion of the support may be located above the metal pattern layer, or a metal thin film may be formed on the wall surface of the concave and convex portion. Here, instead of polishing, the metal film can be removed by using etch back by dry etching.

(Ii) Opened metal pattern layer Basically, it can be produced in the same manner as the above-described isolated metal pattern layer. That is, as shown in FIGS. 5A to 5D, an underlayer, a metal layer, and a resist layer are provided in this order on a support, and a portion of the resist layer at a position corresponding to the opening is removed. An opening type metal pattern layer can be obtained by forming a resist pattern and etching the metal layer through the resist pattern.

(Target substance detection element and target substance)
The target substance detection element can be produced by binding the target substance capturing body to the metal pattern layer of the substrate that can take the above-described various configurations. 4 and 15 show a state where the target capturing body is fixed to the metal pattern layer.

  The target substance is not particularly limited as long as it forms a specific binding pair with the target substance capturing body. Specifically, the target substance contained in the sample solution is roughly classified into a non-biological substance and a biological substance. Industrially valuable non-biological substances include PCBs with different chlorine substitution numbers / positions as environmental pollutants, dioxins with different chlorine substitution numbers / positions, endocrine disrupting substances called so-called environmental hormones, etc. Can be mentioned. The biological material is selected from nucleic acids, proteins, sugar chains, lipids, and complexes thereof. More specifically, there are those comprising a biomolecule selected from nucleic acids, proteins, sugar chains, and lipids. 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 and cells themselves that produce the above-mentioned “biological substances” can also be target substances as “biological substances” targeted by the present invention.

  As the target substance capturing body, a substance capable of specifically binding to the target substance is used. For example, when the relationship between the target substance and the target substance capturing body is enzyme-substrate, antigen-antibody, nucleic acid-nucleic acid, compound-its receptor, compound-substance having specific affinity for it, etc. One can be a target substance capturing body and the other can be a target substance. These capturing bodies can be immobilized on the surface of the metal pattern layer by a physical or chemical method.

  4 and 15 show a case where an antibody is used as the target substance capturing bodies 5 and 204. FIG. When the antibody is immobilized on the metal pattern layers 1, 201, a complex is specifically formed when the target substance (4) approaches the metal pattern layers 1, 201. As a result, the dielectric constant (refractive index) on the detection element surface changes. The antibody is a member of any immunoglobulin class, and in the present invention, an IgG class derivative is more preferable. Further, the antibody as the capturing body may be an antibody fragment (antibody fragment) fragmented by any technique. The term “antibody fragment” or “antibody fragment” refers to any molecule or complex of an antibody that is less than the full length of the antibody or immunoglobulin. Preferably, antibody fragments retain at least a significant portion of the specific binding ability of the full length antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab ′, F (ab ′) 2, scFv, Fv, multispecific multivalent antibodies (diabodies, triabodies, etc.) and Fd fragments. is not. In the case of using an antibody fragment, the detection sensitivity can be improved because it can be captured in the vicinity of the detection element. Further, when a multispecific multivalent antibody is used, it has specific recognition ability for each of the detection element and the target substance, so that the capturing body can be immobilized on the detection element simply and efficiently. Examples other than the antigen-antibody reaction in such complex formation include enzyme-substrate complex, complementary base pairing by DNA hybridization, and one of these complexes is captured by the other. Can be used as

  These capturing bodies are immobilized on the surface of the detection element by a physical or chemical method. In addition, the surface of the detection element is coated with skim milk, casein, bovine serum albumin, phospholipid, polyethylene glycol, and derivatives thereof to prevent unnecessary signals due to adsorption of contaminants due to so-called non-specific adsorption. It is still preferred to do so.

  In the target substance detection element according to the present invention, the refractive index of the underlayer has the same refractive index as that of water contained in the sample solution. It is possible to extract only the result of interaction with light, and in the case of an isolated metal pattern layer, by adopting a configuration in which the interval between adjacent metal pattern layers is close to the resonance wavelength, Coherent localized plasmon interaction between adjacent metal pattern layers can be obtained. As a result, a sharper peak waveform can be obtained and the spectral shift can be detected more sensitively. Thereby, the detection element which can detect the trace amount target substance in a sample solution, the detection method of the target substance using the same, and the detection apparatus and kit for it can be provided.

  In addition, when an aperture-type metal pattern layer is used, it is possible to further amplify changes in the intensity of transmitted light that passes through the aperture, and sufficient sensitivity and wide dynamics to detect the target substance in the sample solution A range can be achieved.

  The present invention can exhibit the various effects described above while maintaining the merit of detecting change over time during the reaction, which plasmon resonance originally has.

(Detection apparatus and detection method)
Next, a target substance detection apparatus using the element configured as described above will be described. The detection apparatus according to the present invention includes at least holding means for holding the element having the above-described configuration and detection means for detecting a signal from the element. An example of a detection device according to the present invention is shown in FIG. The detection means includes an optical detection system including a light source, a spectroscope, and lenses. In addition, this apparatus further includes a liquid feeding system including a reaction well, a channel, a liquid feeding mechanism, and the like for moving the sample solution to the detection element and causing a reaction with the element.

  As a light source, what can cover the wavelength range from a visible region to a near infrared region can be used. The optical measurement can use an absorption spectrum, a transmission spectrum, a scattering spectrum, and a reflection spectrum. Most preferably, the peak wavelength of the absorption spectrum or the peak absorption intensity is used.

  In the detection element having an isolated metal pattern layer, when the target substance is specifically bound, the localized plasmon resonance changes with respect to the unbound state, and the peak wavelength of the absorption spectrum shifts to the longer wavelength side and absorbs at the same time. Strength increases. Depending on the degree of the shift amount, the amount of the target substance can be determined from a calibration curve for the target substance prepared in advance. Further, detection and quantification can be performed by reflected light and scattered light as well as the transmitted light. Since the illustrated detection apparatus utilizes localized plasmon resonance, local electric field enhancement occurs in the vicinity of the isolated metal pattern layer. This phenomenon can be applied to measurement methods such as surface-enhanced Raman spectroscopy (SERS) and surface plasmon fluorescence spectroscopy (SPFS), and the target substance can be quantified by these methods.

  The reaction well and the flow path can be easily produced by using a polydimethylsiloxane (PDMS) substrate used in a so-called μTAS (Micro Total Analysis Systems) type apparatus. This PDMS substrate is bonded to the substrate on which the detection element is manufactured and used in the shape as shown in FIG. As a liquid feeding mechanism, a micro piston pump, a syringe pump, or the like can be used.

  Next, a typical usage pattern is shown in FIG. First, a sample solution containing a target substance is introduced from an inlet 11 through a flow path 8 into a reaction well portion 9 having a detection element 10 having a capture ability. Incubation is performed for a fixed time, and the transmission spectrum at that time is measured by the spectrophotometer 15. The central processing unit 16 displays the measurement results such as concentration and reaction rate on the display unit 17 by comparison with a calibration curve prepared in advance. If necessary, the reaction well 9 may be washed by introducing a phosphate buffer solution or the like as a washing solution from the inlet 1 before measurement. Here, in addition to statically measuring the spectral change after a certain time, it is also possible to dynamically measure the change in real time. In that case, the time change rate and the like can be acquired as new information.

  FIG. 14 shows an apparatus for measuring transmitted light with respect to an element using an aperture type metal pattern. The light source 101 is not particularly limited as long as it is a stable light source. For example, it is preferable to use a halogen lamp. As the light receiving means 102, a spectroscopic measurement apparatus having a wavelength resolution of about 1 nm is desirable. For example, it is preferable to use a multi-channel detector or a spectrophotometer. The sample solution reservoir 103 may be a reservoir made of a material with less non-specific adsorption of a target substance, or a structure in which a reservoir is built in a chip. For example, it is preferable to use an Eppendorf tube coated with non-specific adsorption prevention. The cleaning liquid reservoir 104 is not particularly limited. 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. Moreover, when there is only a small amount of the sample solution, the sample solution may be circulated using a circulation channel or a tube without using the waste solution. As the liquid feeding and waste liquid tubes 108 and 109, tubes made of a material having as little as possible the adsorption of the target substance are preferable. As the target substance detection element 110, elements shown in FIGS. 15 to 17 are appropriately selected and used. The channel 111 is preferably minute so that the amount of the sample solution 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.

  Next, the principle of operation of the detection device using the element using the aperture type metal pattern will be described with reference to FIGS. In FIG. 15, incident light 205 is incident on the array of minute openings 201 from the upper direction to the lower direction (+ z direction). At this time, since the size and interval of the openings are both equal to or less than the wavelength of the light used for detection, the component polarized in the x direction in the figure of the electric field of the incident light 205 interacts with the electrons in the metal thin film 201. Then, surface plasmon is generated on the inner surface of the minute slit opening. The generated surface plasmon propagates down the inner surface of the minute slit (+ z direction in the figure), is converted again to light at the lower end of the minute opening 201, and is transmitted downward (+ z direction) as transmitted light 206.

  When light is transmitted through the plurality of micro openings 201 through the surface plasmon state, the spectrum with respect to the wavelength change of the incident light is absorbed by resonance with the electric multipole generated in the micro openings (local plasmon resonance). Has a resonant peak. The shape of this localized plasmon resonance peak varies depending on the width and interval of the minute apertures.

  In the element having the opening-type metal pattern, a base layer having a refractive index comparable to that of water, which is a liquid medium of a sample solution containing a target substance, is disposed as a base layer of a metal thin film having a minute opening. Incident light is efficiently converted into transmitted light via plasmons. With this configuration, when the target substance is bound to the capturing body, the change in the integrated intensity of the transmitted light is increased.

  FIG. 22 shows an example of a transmitted light intensity spectrum obtained by a detection device in which an element having this aperture type metal pattern is arranged. (A) is a transmitted light spectrum when the refractive index of a foundation layer and water is not equivalent, (b) is a transmitted light spectrum when the refractive index of a foundation layer and water is equivalent. From the figure, it can be seen that when the refractive indices of water and the underlayer are made equal, the change in the integrated intensity of the transmitted light spectrum increases. FIG. 23 is a diagram showing a change in signal intensity after reacting specimens of various concentrations for a certain period of time and then washing the element surface with a buffer solution, and (b) shows a lower detection limit than (a). And it can be confirmed that the dynamic range is large. The dotted line represents the spectrum before the target substance reacts with the complementary molecule. The element shown in FIG. 15 is brought into contact with a specimen containing a target substance, and the element and the target substance are reacted. Depending on the reaction time, the integrated intensity of the transmitted light spectrum increases. A graph showing the change in the integrated intensity with time is shown in FIG. 20, and the reaction dynamics between the target substance and the antibody can be observed by using the sensor having the configuration shown in the present invention.

  A sample solution for detecting the presence or absence of the target substance or its amount is supplied as an aqueous solution. The aqueous solution is composed of water as a solvent (liquid medium). The dissolved solute mainly includes proteins and salts (phosphate, acetate, borate, tris hydrochloride, chloride salt, calcium salt, magnesium salt, etc.). In addition, saccharides (such as trehalose), cryoprotectants such as glycerol and ethylene glycol, preservatives such as sodium azide and thimerosal, protease inhibitors such as PMSF (phenylmethanesulfonyl fluoride) and leupeptin, EDTA (ethylenediaminetetraacetic acid) A metal chelating agent such as disodium or disodium ethylenediaminetetraacetate) or a reducing agent such as DTT (dithiothreitol) may be included.

(Detection kit)
The target substance detection kit of the present invention can be provided in the following various forms.
(I) A target substance detection kit having a substrate for a target substance detection element and a target substance capturing body.
(II) A target substance detection kit comprising a target substance detection element, a detection device, and a reagent necessary for capturing the target substance by the target substance detection element.

  The target substance according to the present invention can be detected by using the kit having the constitution (II). According to the kit having the above configuration (I), a new target substance detection element can be provided to a user who already has a detection device and a reagent.

  Hereinafter, the present invention will be described more specifically with reference to examples. In addition, this invention is not limited only to a following example.

Example 1
FIG. 10 shows a schematic structure used in this example. Here, first, a 5% Cytop solution (CTL-809M (Asahi Glass Co., Ltd.)) (solvent: CTL-180 (Asahi Glass Co., Ltd.)) is spin-coated on a 625 μm thick quartz substrate at 2500 rpm. Holding for 45 seconds gives a film thickness of 500 nm, followed by baking at 70 ° C. for 40 minutes, followed by baking at 190 ° C. for 1 hour, to form a fluororesin layer. A film is formed on the fluororesin layer on the quartz substrate.

  A negative electron beam resist (SAL601 (made by Rohm & Haas)) on a gold thin film is spin-coated and patterned by an electron beam drawing apparatus. Then, the gold thin film is patterned using the resist as a mask to form an isolated metal pattern layer. Each metal pattern layer is a 200 nm × 200 nm square pattern (planar shape). An interval between adjacent metal pattern layers is 800 nm, and a large number of metal pattern layers are arranged in a 3 mm × 3 mm region in a regular array.

  Next, an anti-AFP (α-fetoprotein) antibody, which is a target substance capturing body, is immobilized on the surface of the metal pattern layer made of gold in order to impart a capture capability to the surface of the metal pattern layer. First, an ethanol solution of 11-Mercaptodecanoic acid having a thiol group having a high affinity for gold is dropped with a spotter or the like to modify the surface of the metal pattern layer. Thereby, the carboxyl group is exposed on the surface of the metal pattern layer. In this state, an aqueous solution of N-Hydroxysulfuccinimide (manufactured by Dojindo Laboratories) and an aqueous solution of 1-Ethyl-3- [3-dimethylamino] propyl] carbohydrate hydride (manufactured by Dojindo Laboratories) are similarly applied to the reaction region using a spotter. Dripping. Thereby, the succinimide group is exposed on the surface of the metal pattern layer. Further, by binding streptavidin, the surface of the metal pattern layer is modified with streptavidin. A biotinylated anti-AFP antibody is immobilized on the metal pattern layer.

  A plurality of regions having a large number of metal pattern layers are prepared on a substrate, different antibodies are immobilized on each of them, and different target substances in a sample solution are detected on the same substrate (multi-sensor configuration). It is also possible. In this case, such a configuration can be obtained by performing different immobilization operations for each region using different antibodies.

  Using the element obtained as described above, the AFP concentration in the sample solution can be specifically measured by the following operation.

  First, a sample solution containing AFP, which is a target substance, is introduced from the inlet 11 into the manufactured element 10 to capture AFP on the structure. The sample solution is discharged from the outlet 12, and then a phosphate buffer is introduced from the inlet 11 to wash the inside of the reaction well 9. Finally, a phosphate buffer solution is filled and the absorption spectrum of the gold metal pattern layer is measured. As shown in FIG. 11, the absorption spectrum is shifted when the target substance binds to the surface of the detection element due to a specific antigen-antibody reaction when comparing before and after the reaction. Here, the correlation between the peak intensity of the absorption spectrum or the shift amount of the peak wavelength and the AFP concentration is obtained in advance using a known AFP control solution as shown in the calibration curve of FIG. A trace AFP concentration can be determined.

Example 2
In this embodiment, a substrate with fine unevenness is produced by a molding method using a fluororesin substrate to form a detection element. The fine uneven pattern can be produced in the same way as manufacturing a general optical disc. Cytop (manufactured by Asahi Glass Co., Ltd.) is used as the fluororesin. The outer shape of the metal pattern layer is a rectangular pattern of 300 nm × 100 nm. The thickness of the gold thin film is 20 nm, the distance between each metal pattern layer is 1400 nm, and a large number of metal pattern layers are arranged in a regular array of 3 mm × 3 mm. The gold thin film is formed by a vapor deposition method. Next, the unnecessary gold thin film on the surface is polished, and a metal pattern layer having a desired layer structure is formed on a support having a function as an underlayer (FIG. 6C). In order to give the capture capability to the surface of the metal pattern layer, the same treatment as in Example 1 is performed. In this example, an anti-PSA (prostate specific antigen) antibody, which is a target substance capturing body, is immobilized on the surface of a gold structure.

  As in Example 1, the absorption spectrum is shifted by the binding of the target substance to the surface of the detection element due to the specific antigen-antibody reaction when comparing before and after the reaction. The correlation between the peak intensity of the absorption spectrum or the shift amount of the peak wavelength and the PSA concentration is obtained in advance by a known PSA control solution, and thereby, the trace PSA concentration of the sample solution having an unknown concentration can be obtained.

Example 3 and Comparative Example 1
In the present embodiment, the shape of the metal pattern layer is a ring-shaped circumferential structure (FIG. 2E). This metal pattern layer is characterized by having a localized plasmon enhancement effect due to the ring shape. The device is manufactured in the same manner as in Example 1 except that a magnesium fluoride thin film formed by vapor deposition is used instead of the fluororesin. The film thicknesses of the magnesium fluoride thin film and the gold thin film are 500 nm and 20 nm, respectively. When confirmed with a scanning electron microscope (SEM) image, the outer shape (planar shape) of the metal pattern layer is a square of 200 nm × 200 nm, and the ring-shaped line width is 50 nm. Due to the high and low resolution of the drawing process, the shape of the intersecting portion is not necessarily produced at a right angle, but it does not cause a problem in function. The interval between adjacent metal pattern layers is 1350 nm, and a large number of metal pattern layers are arranged in a 3 mm × 3 mm region in a regular array. In the present example in which the metal structure is arranged on the magnesium fluoride thin film, the half width of the spectrum of localized plasmon absorption is 20 to 40% as compared with the case where the magnesium fluoride thin film is not provided (Comparative Example 1). Decrease degree.

Example 4
In this embodiment, the shape of the metal pattern layer is formed as a structure having an intersection (FIG. 2I). This structure is characterized in that it has a local plasmon enhancement effect due to having an intersection. A metal pattern layer is formed on a 625 μm thick quartz substrate. First, a lithium fluoride thin film (50 nm) is formed on a quartz substrate by vapor deposition. After that, the metal pattern layer is obtained by sequentially performing the gold thin film formation (film thickness 20 nm) by the sputtering method, the patterning of the structure using the electron beam drawing apparatus, and the etching process as in Example 1. When confirmed with a scanning electron microscope (SEM) image, the outer shape (planar shape) of 500 nm is a square of 200 nm × 200 nm, and the line width of the belt-like portion is 50 nm. Due to the level of resolution of the drawing process, the shape of the intersection may not necessarily be formed at a right angle, but there is no problem as a function. An interval between adjacent metal pattern layers is 1250 nm, and a large number of metal pattern layers are arranged in a 3 mm × 3 mm region in a regular array. The structure of the present embodiment can also be used as a biosensor element as in the above-described embodiments.

Example 5
<Target substance detection element>
This will be described with reference to FIG. A glass plate is used as the support 203. MgF ₂ is deposited to 200 nm on the support 203 to form the underlayer 202. A metal thin film 201 is vapor-deposited thereon by 200 nm. Further, a positive electron beam resist (ZEP520) is spin-coated on the metal film, and patterning is performed by electron beam drawing. The metal layer is etched using the resist pattern as a mask, and 300 nm × 50 nm rectangles are arranged in an array with a period of 1000 nm. An antibody is used as the capturing body 205.

<Target substance measuring device>
This will be described with reference to FIG. A halogen lamp is used as the light source 101, a multi-channel detector (Hamamatsu Photonics) is used as the spectroscope 102, and an Eppendorf tube is used as the sample solution reservoir 103. A glass bottle for biochemistry is used as the cleaning liquid reservoir 104, and a three-way valve (GL Science) is used as the flow path switching valve 105. 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 as shown in FIG. 15 described above is used, and 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. 14, 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. Immobilization of the antibody on the surface of the gold thin film can be performed in the same manner as in Example 1.
(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 solution side, and the sample solution is allowed to flow for 10 minutes at a flow rate of 0.1 (ml / min.) To cause the antigen-antibody reaction.
(3) Measure the reflection spectrum at regular intervals while feeding the sample solution.
(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 similar to that shown in FIG. 22 is obtained. By integrating this spectrum with graph software and obtaining the integral value at each time, the time course of the antigen-antibody reaction can be obtained as shown in FIG.

Example 6
<Target substance detection element>
Four target substance detection elements shown in FIG. 15 are arranged in series, and different types of antibodies are immobilized on each of the target substance detection elements. Each element is manufactured in the same manner as in Example 5 except that a predetermined antibody is used.
<Target substance measuring device>
This will be described with reference to FIG. A halogen lamp is used as the light source 601, and a multi-channel detector capable of 4ch simultaneous measurement is used as the spectroscope 602. An Eppendorf tube is used as the sample solution reservoir 603, a glass bottle for biochemistry is used as the cleaning solution reservoir 604, and a three-way valve (GL Science) is used as the flow path switching valve 605. A syringe pump (kd Scientific KDS200) is used as the pump 606, a syringe is used as the waste liquid tank 607, and Teflon tubes are used as the liquid feeding and waste liquid tubes 608 and 609. As the target substance detection element 610, a chip in which the four elements shown in FIG. 15 are arranged in series is used. As the flow path 611, a 1 mm width, 100 μm width, and 40 mm length formed on the PDMS substrate 612 is used. Use. 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. 19, 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 solution side, and the sample solution is allowed to flow for 10 minutes at a flow rate of 0.1 (ml / min.) To cause the antigen-antibody reaction.
(3) Measure the reflection spectrum at regular intervals while feeding the sample solution.
(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. 22 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.

Example 7
<Target substance detection element>
Four target substance detection elements shown in FIG. 15 are arranged in series, and different types of antibodies are immobilized on each of the target substance detection elements. Each element is manufactured in the same manner as in Example 4 except that a predetermined antibody is used.
<Target substance measuring device>
This will be described with reference to FIG. A halogen lamp is used as the light source 801, a multi-channel detector capable of simultaneous measurement of 4ch is used as the spectroscope 802, and an Eppendorf tube is used as the sample solution reservoir 803. A glass bottle for biochemistry is used as the cleaning liquid reservoir 804, a three-way valve (GL Science) is used as the flow path switching valves 805 and 816, and a tube pump (kd Scientific KDS200) is used as the pump 806. 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 target substance detection element 810, a chip in which the four elements shown in FIG. 15 described above are arranged in series is used, and the flow path 811 is a 1 mm width, 100 μm width, 40 mm length formed on the PDMS substrate 212. Use. 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. 21, 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 solution side and the valve 2 is switched to the circulation tube side, and the sample solution 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 sample solution.
(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. 22 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.

(Example 8)
The apparatus shown in FIG. 10 is configured in the same manner as in Example 1 except that the bispecific antibody (diabody) described in JP-A-2005-31446 is used as the target substance capturing body. This diabody is used for imparting capturing ability to the surface of the metal structure of the detection element, and is composed of gold, which is a constituent material of the metal structure of the detection element, and HEL (chicken egg white lysozyme), which is a target substance. Specific affinity for one substance. The prepared diabody is added to the detection element portion together with a phosphate buffer solution, incubated for about 30 minutes, and then washed with a buffer solution before use. According to the method using the diabody of the present embodiment, it is possible to fix without impairing the affinity as compared with the chemical immobilization method, and as a result, the amount of capture bodies necessary for immobilization on the detection element is reduced. it can.

Next, using this apparatus, the HEL concentration in the specimen can be specifically measured by the following operation.
(1) A specimen containing HEL, which is a target substance, is introduced from the inlet 8 into the manufactured element, and HEL is captured on the structure.
(2) The specimen is discharged, a phosphate buffer is introduced from the inlet 8, and the inside of the reaction well 7 is washed.
(3) Finally, a phosphate buffer solution is filled and the absorption spectrum of the gold structure is measured.
Comparing the absorption spectrum before and after the reaction, as shown in FIG. 11, the absorption spectrum shifts due to the target substance binding to the detection element surface by a specific antigen reaction. Here, the correlation between the peak intensity of the absorption spectrum or the shift amount of the peak wavelength and the HEL concentration is obtained in advance by a known HEL control solution, and the trace HEL concentration of a sample whose concentration is unknown can be obtained.

It is a schematic diagram which shows the metal structure in embodiment of this invention. It is a schematic diagram which shows the example of the planar shape of the metal structure in embodiment of this invention. It is a schematic diagram which shows an example of the detection element in embodiment of this invention. It is a schematic diagram which shows the example of the capturing body on the detection element in embodiment of this invention. It is a figure explaining the manufacturing method of the detection element in embodiment of this invention. It is a figure explaining the manufacturing method of the detection element in embodiment of this invention. It is a figure explaining the manufacturing method of the detection element in embodiment of this invention. It is a schematic diagram showing a part of detection device in an embodiment of the present invention. It is a block diagram of the detection apparatus in embodiment of this invention. 1 is a schematic diagram of a detection device of Example 1. FIG. It is an example of the change of the detection spectrum (absorption spectrum) of Example 1. FIG. 2 is an example of a calibration curve for a sample solution of Example 1. FIG. It is a figure for demonstrating the magnitude | size of a metal pattern. 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 the example of the target substance detection element (separate structure) of this invention. It is a figure for demonstrating embodiment 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 embodiment of this invention. It is a figure for demonstrating embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Metal pattern layer 2 Underlayer 3 Support body 4 Target substance 5 Target substance capture body 6 Metal thin film 7 Electron beam resist 9 Reaction well 10 Detection element 11 Inlet 12 Outlet 13 Liquid feed pump 14 Light source unit 15 Spectrophotometer 16 Central processing unit 17 Display unit 18 Liquid feed pump 19 Flow path 20 Waste liquid reservoir 21 Tungsten lamp 22 Collimating lens 23 Substrate 101 Light source 102 Spectrometer 103 Sample reservoir 104 Cleaning liquid reservoir 105 Flow path switching valve 106 Liquid feed means 107 Waste liquid tank 108 Liquid feed tube 109 Waste liquid Tube 110 Target substance detection element 111 Flow path 112 Cover 113 Substrate 114 Target substance detection chip 115 Collimator lens 201 Metal pattern 202 Underlayer 203 Support body 204 Capture body 205 Incident light 206 Transmitted light 30 DESCRIPTION OF SYMBOLS 1 Metal pattern 302 Support body 303 Capture body 304 Incident light 305 Transmitted light 401 Metal thin film 402 having a micro slit aperture array Metal thin film 403 having a micro aperture two-dimensional array Metal thin film 404 having a complex periodic aperture array Circular micro aperture two-dimensional Metal thin film 601 having array Light source 602 Spectrometer 603 Sample reservoir 604 Cleaning liquid reservoir 605 Flow path switching valve 606 Pump 607 Waste liquid tank 608 Liquid feeding tube 609 Waste liquid tube 610 Target substance detection element 611 Channel 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 h Tube 810 Target substance detection element 811 Channel 812 Cover 813 Substrate 814 Target substance detection chip 815 Coupler 817 Channel switching valve 818 Sample circulation tube

Claims (17)

A target substance having a metal pattern layer provided on the surface of the support, and a target substance capturing body capable of capturing a target substance in a sample solution containing at least water as a liquid medium is bonded to the metal pattern layer; A substrate used to form a target substance detection element that detects a target substance using plasmon resonance,
The metal pattern layer is formed on an underlayer provided on the support surface, and the underlayer is represented by the following formula:
0.90na ≦ nb ≦ 1.05na
na: Refractive index of water nb: Substrate for target substance detection element having a refractive index (nb) represented by the refractive index of the underlayer.   The substrate according to claim 1, wherein the refractive index nb is in a range of 1.20 or more and 1.40 or less.   The substrate according to claim 1, wherein the underlayer is made of a material containing fluorine.   The substrate according to claim 3, wherein the fluorine-containing material is a fluororesin.   The substrate according to claim 3, wherein the fluorine-containing material is magnesium fluoride or lithium fluoride.   The substrate according to claim 1, wherein the metal pattern layer includes a plurality of isolated metal patterns formed on the base layer.   The substrate according to claim 6, wherein a distance between the plurality of isolated metal patterns is in a range of 400 nm to 1500 nm.   A target substance detection element, wherein a target substance capturing body is bonded to the metal pattern layer of the substrate according to claim 1.   The target substance detection element according to claim 8, wherein the target substance capturing body is an antibody.   The target substance detection element according to claim 9, wherein the antibody is an antibody fragment.   The device for detecting a target substance according to claim 10, wherein the antibody fragment is a multispecific multivalent antibody. An apparatus for detecting a target substance in a sample solution,
9. The target substance detection element according to claim 8, a sample solution supply means for supplying the sample solution to the target substance detection element, and a target substance capturing body possessed by the target substance detection element captures the target substance. And a detection means for detecting the detection device.   The detection device according to claim 12, wherein the detection unit is a unit that optically detects capture of a target substance by the target substance capture body.   The detection apparatus according to claim 13, wherein the optical detection unit measures transmitted light, scattered light, or reflected light from the target substance detection element.   The detection apparatus according to claim 12, wherein the target substance detection element is arranged in a flow path of the sample solution. A detection method for detecting the presence or absence of a target substance in a sample liquid containing at least water as a liquid medium or the amount of the target substance,
A step of bringing the target substance detection element according to claim 8 into contact with the sample solution;
Detecting the capture of the target substance on the target substance detection element when the target substance is contained in the sample solution;
A method for detecting a target substance, comprising:   A kit for detecting the presence or absence of a target substance in a sample solution or the amount of the target substance, which is necessary for capturing the target substance detection element according to claim 8 and the target substance with the target substance detection element. And a reagent for detecting a target substance.

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