JP2007327948A - Element, apparatus, and method for detection - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection element, a detection apparatus, and a detection method that can detect with high sensitivity and with stability. <P>SOLUTION: The detection element for detecting the characteristics of specimens through the use of detecting light comprises a plurality of metal structures separated from one another; a substrate having planes in which the plurality of metal structures separated from one another are present; and a specimen housing part, at least a part of which is constituted of a part of the planes. There are a plurality of the planes, and any one of the planes among the plurality of planes has a normal line which intersects with all the other planes. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a detection element, a detection apparatus, and a detection method for detecting a characteristic of a specimen.

  In recent years, along with the growing awareness of health and environmental issues, as well as food safety issues, substances related to these issues (so-called biological substances and other chemical substances (hereinafter referred to as target substances)) are detected. A way to do it has come to be desired.

  In order to detect a target substance, a highly sensitive detection technique is often required. This is because the amount of samples that contain target substances is often limited, and in addition, in the case of proteins in blood, only a very small amount of target substance is contained in a mixture of various substances. Because there are things. For these reasons, there is a need for a high-sensitivity detection technique that can detect a trace amount of a target substance contained in a trace amount of sample in a target substance detection method.

  Development of a measurement method using plasmon resonance of metal particles is underway as one method for meeting such demands.

  In Patent Document 1, a change in the refractive index of a medium is detected by measuring a change in the absorption spectrum of light transmitted through the medium using plasmon resonance of the metal fine particles immobilized on the substrate, and the adsorption of the substance to the metal fine particles is performed. And a technique for detecting deposition.

Patent Document 2 discloses a technique for measuring a change in the absorption spectrum of transmitted light with high sensitivity by disposing a plurality of metal fine particles in a micro-channel and entering measurement light into the channel. .
Japanese Patent No. 03452837 JP 2005-283556 A

  When the density of the metal fine particles on the substrate is reduced in Patent Document 1, the absorption spectrum of a single metal fine particle is measured and is less susceptible to the absorption spectrum due to the interaction between the metal fine particles. Is likely to be detected. However, in such a case, since the number of metal fine particles per unit area is reduced, it is considered that the absorbance of the spectrum is absolutely reduced. When the absorbance of the spectrum decreases, it becomes difficult to measure the characteristics of the absorption spectrum, and there is a problem that it is difficult to detect changes in the spectrum stably and with high sensitivity.

  On the other hand, in Patent Document 2, it is possible to increase the absorbance of the spectrum. However, in order to reduce the influence of the absorption spectrum due to the interaction between the metal fine particles, there is a problem that it is difficult to manufacture the device.

The present invention
A detection element that detects the characteristics of a specimen using detection light,
The detection element is
A plurality of metal structures spaced apart from each other;
A base having a plane on which a plurality of metal structures spaced apart from each other exist;
A specimen containing portion constituted at least in part by a part of the plane,
A plurality of said planes;
Any one of the plurality of planes has a normal line that intersects with all the other planes.

Another aspect of the present invention is:
An apparatus for detecting the characteristics of a specimen,
A sensing element;
A light source for irradiating the detection element with detection light;
A light receiving element that receives outgoing light emitted from the detection element;
An arithmetic unit for obtaining characteristics of the specimen from a signal obtained by the light receiving element;
Have
The detection element is
A plurality of metal structures spaced apart from each other;
A base having a plane on which a plurality of metal structures spaced apart from each other exist;
A specimen containing portion constituted at least in part by a part of the plane,
A plurality of said planes;
Any one of the plurality of planes has a normal line that intersects with all the other planes.

It is preferable that an arbitrary plane among the plurality of planes has a normal line that intersects with all the other planes.
It is preferable that a capture body for capturing the target substance in the specimen is provided on the surface of the metal structure included in the detection element.

  It is preferable that the plurality of planes exist in a traveling path of the detection light, and in each of the plurality of planes, an angle formed by a normal line of the plane and the detection light is within 20 °. .

According to another aspect of the present invention, there is provided a specimen storage portion that is at least partly constituted by a plurality of metal structures, a base having a plane on which the plurality of metal structures are separated from each other, and a part of the plane. A method for detecting the characteristics of a specimen using a detection element having
The detection element has a plurality of the planes;
Introducing the sample into the sample container;
Irradiating the detection light to the detection element so as to cross the plurality of planes;
Receiving light emitted from the detection element by a light receiving element;
And a step of obtaining a specimen characteristic from a signal output from the light receiving element.

Irradiating the detection element with the detection light so as to cross the plurality of planes,
A step of irradiating the detection element with the detection light such that the plurality of planes are present in a traveling path of the detection light, and an angle formed between a normal line in each of the plurality of planes and the detection light is within 20 °. It is preferable that

Another aspect of the present invention is a detection element for detecting characteristics of a liquid specimen using detection light,
A liquid specimen container, and a base having a plane on which a plurality of metal structures separated from each other are present,
The plane is a wall surface of the liquid specimen storage unit,
A plurality of the planes exist so as to cross the detection light.

Another aspect of the present invention is an apparatus for detecting characteristics of a liquid specimen,
A sensing element;
A light source for irradiating the detection element with detection light;
A light receiving element that receives light transmitted through the detection element;
An arithmetic device for obtaining the characteristics of the specimen from the signal obtained by the light receiving element,
The detection element includes a liquid specimen storage portion and a base having a plane on which a plurality of metal structures separated from each other are present,
The plane is a wall surface of the liquid specimen storage unit,
A plurality of the planes exist so as to cross the detection light.

Another aspect of the present invention is a method for detecting a characteristic of a liquid specimen,
A liquid specimen container, and a base having a plane on which a plurality of metal structures separated from each other exist,
A step of introducing the liquid sample into the liquid sample storage portion of the detection element in which the plane is a wall surface of the liquid sample storage portion and a plurality of the planes exist;
Irradiating the detection element with detection light so as to cross a plurality of the planes;
Receiving light transmitted through the detection element by a light receiving element;
And a step of obtaining characteristics of the liquid specimen from a signal output from the light receiving element.

  In the method for detecting the characteristics of the specimen or liquid specimen, the order of these steps is not specified.

  In the present invention described above, it is preferable that a capture body that selectively or specifically captures a target substance in a specimen is provided on the surface of the metal structure. Moreover, it is preferable that the said metal structure is a patterned flat structure.

  It is an object of the present invention to provide a detection element, a detection device, and a detection method that can perform detection with high sensitivity and stability.

  Hereinafter, modes for carrying out the present invention will be described. In addition, this invention is specified by a claim, Comprising: It is not limitedly interpreted to the following forms and Examples. For example, the present invention can be realized by freely changing materials, composition conditions, reaction conditions, arrangement of members and elements, and the like in the following forms and examples within a range that can be understood by those skilled in the art.

First, the detection element of the present invention will be described.
The detection element of the present invention is an element that detects the characteristics of a specimen using detection light. The detection of the characteristics of the specimen includes the detection of any characteristics such as physical characteristics and chemical characteristics of the specimen. As an easy-to-understand example of detecting the characteristics of a specimen, the chemical characteristics of the specimen (such as the presence or absence of interaction between the specimen and the constituent elements of the detection element) and the physical characteristics of the element (such as optical characteristics) are used. For example. As a more specific example, in order to measure the presence or concentration of a target substance in a specimen, after contacting the specimen and the element, the element is irradiated with detection light, and light emitted from the element is detected. In the case of measuring changes in detection light and outgoing light, and the like. Note that the specimen that is the detection target of the detection element of the present invention may be not only liquid but also non-liquid such as gaseous. However, from the viewpoint of easy detection, a liquid specimen is preferable.

  FIG. 1A shows an example of the detection element of the present invention.

  The detection element 100 includes a plurality of metal structures 101, a base 102, and a specimen storage unit 103, and the plurality of metal structures 101 are separated from each other on a plane 104 included in the base 102. That is, the base body 102 has a flat surface 104 on which a plurality of metal structures 101 spaced apart from each other exist. In addition, any one of the plurality of planes 104 on which the metal structure exists has a normal line that intersects with all the other planes. Hereinafter, “a plane on which a plurality of metal structures separated from each other” is simply referred to as “a plane on which a metal structure exists” may be described. Further, the specimen storage unit 103 is at least partially configured by a part of a plane on which the plurality of metal structures 101 are present.

  Specifically, for example, as shown in FIG. 2, a plurality of planes 104 on which the metal structures exist are present so as to cross light (detection light) 105 incident on the detection element 100. In the present invention, “the plane exists so as to cross the detection light” means that the plane and the detection light are non-parallel (more preferably vertical or substantially vertical), and the plane is the detection light. It exists in the traveling path. Here, “substantially vertical” means 90 ° ± 20 °. Therefore, it is more preferable that an angle formed between the normal line of the plane 104 on which the metal structure 101 exists and the detection light 105 is within 20 °.

  Here, when the detection light is not linear but exists in a three-dimensional shape such as a cylinder or a rectangular parallelepiped, the straight line existing at the center of the three-dimensional shape is considered as a reference for determining the angle.

  In addition, as a form of the element in which a plurality of planes on which the metal structures are present cross the detection light, as shown in FIGS. 1 (a) and 1 (d), opposing substrates are opposed to each other. The configuration is not limited to the element in which the metal structure 101 exists on each of the surfaces, and the specimen container 103 is sandwiched by the plane 104 on which the metal structure exists. For example, as shown in FIG. 1 (e), there are a plurality of bases 102 having a plane 104 where a metal structure exists, and planes 104 where a metal structure exists and planes 106 where no metal structure exists alternately exist. It may be an element. In addition, an element (such as the element shown in FIG. 1B or FIG. 1C) in which both surfaces of the substrate are the planes 104 on which the metal structures exist can be considered. Here, among the plurality of planes on which the metal structure exists, the metal structure existing on an arbitrary plane and the metal structure existing on a plane different from the plane are parallel to the plane on which the metal structure is formed. It may exist symmetrically with respect to the plane, or may not exist symmetrically. Specifically, the case where it exists symmetrically with respect to a plane parallel to the plane on which the metal structure is formed is as shown in (a), (b), (d), and (e) of FIG. The case where it does not exist symmetrically is a case as shown in FIG. 1 (c) or FIG. 1 (f).

  Moreover, it is preferable that the plane in which the plurality of metal structures exist has a normal line in which an arbitrary plane among the plurality of planes intersects with all the other planes. It suffices to have normals that intersect all the planes. Therefore, for example, as shown in FIG. 1G, the planes 104 on which the metal structures are present may be non-parallel.

  Needless to say, the present invention is not limited to these examples, and various modifications exist within the scope of the present invention. For example, as shown in FIG. 3, a sample flow path may be formed by combining a plurality of base bodies 202 having a plane on which the metal structure 201 exists.

  In any example, when the detection element of the present invention is used for measurement, the detection light incident on the element is emitted from the element after passing through a plurality of planes where the metal structure exists. By adopting such a configuration, the detection light is frequently absorbed by the metal structure, and a slight spectral change due to a minute refractive index change in the vicinity of the metal structure is detected stably and with high sensitivity. It becomes possible.

In the present invention, one of the great effects of allowing the metal structure to exist on the plane of the substrate is that the detection element can be easily manufactured. More specifically, when the surface is flat, it is possible to easily form a pattern in which the metal structures are separated from each other, or to perform a base treatment of a base for fixing the metal structures.
In addition, the detection element of the present invention preferably has a capturing body that selectively captures the target substance in the specimen on the surface of the detection element (more preferably, the surface of the metal structure). By having the capturing body, it is possible to detect the presence or absence or concentration of the target substance in the specimen.

  Hereinafter, each part which comprises the detection element 100 shown in FIG. 1 is demonstrated in detail.

(Substrate)
As long as the base | substrate 102 has the plane 104 for providing the some metal structure 101, what kind of thing may be sufficient as it. Among them, in particular, a material that does not hinder the transmission of detection light is preferable, and therefore, it is preferable to use a material having a high transmittance for detection light. Examples of the material having a high transmittance for detection light include silica, quartz, PMMA (polymethyl methacrylate), polystyrene, and the like. The substrate 102 may have a stacked structure of a plurality of layers, or may be formed by stacking the layers of the materials described above. In general, the substrate preferably has a polyhedral shape such as a rectangular parallelepiped shape, and more generally a flat plate shape (which is also a kind of rectangular parallelepiped). Further, the shape of the base is not limited to these, and as shown in FIG. 3, the base 202 having the metal structure 201 has a comb shape, and the bases 202 having the flat surface 204 having the metal structure are connected to each other. You may do it.

(Plane where the metal structure exists)
A plane 104 on which a plurality of metal structures are present is a part of the surface of the substrate 102.
For example, as shown in FIGS. 1A to 1D, the plane 104 on which a plurality of metal structures are present has two surfaces (hereinafter referred to as “base”) having the largest area among the planes of the flat base 102. In the case where two opposing surfaces of the surface of the surface have a larger area than the other surfaces, each of these two opposing surfaces can be referred to as a “main surface”). Here, in the present invention, the term “plane” refers to a flat surface (not a curved surface) having a surface roughness of 5 nm or less. As described above, since the shape of the base body 102 generally has a polyhedral shape such as a rectangular parallelepiped shape, one surface of the polyhedron can be a plane on which the metal structure exists. More generally, since the base body 102 has a flat plate shape, it can be formed as one surface of the flat plate base body.

  However, the plane on which the metal structure exists does not have to be one surface of the polyhedron. It suffices that at least the region of the substrate through which the detection light passes includes a plane on which the metal structure exists. Of course, it is not necessary that the entire surface of the substrate has a planar shape.

  Further, as will be described later, a substrate having a flat surface on which a metal structure exists is not necessarily a boundary region between the specimen and the outside. In other words, both sides of the base (both of the areas divided by the base) may be the specimen storage section. In this case, at the time of measurement, the specimen exists on both sides of the substrate.

  Further, as shown in FIG. 1 (d), when the plane 104 on which the metal structure exists is opposed to each other and is sandwiched between the planes 104 to form a sample container, and the sample is used by flowing into the sample container, The distance between the planes is preferably 300 nm to 1 mm. When it does, it is preferable that the space | interval of the said planes 104 is ... -... (please prescribe an upper limit and a lower limit). This is because if the distance between the planes 104 on which the metal structures are present is too narrow, the plasmons of the metal structures 101 interact with each other, affecting the spatial electric field distribution and intensity, resulting in sensor sensitivity. This is because it may decrease. Further, if the interval is too wide, the signal strength may be weakened due to the low density of the metal structure. For the same reason, when both surfaces of the substrate are flat surfaces on which the metal structures are present, the thickness of the substrate in the direction perpendicular to the metal structure forming surface is (specifies upper and lower limits). Preferably).

  Further, as shown in FIG. 1 (d), when the plane 104 on which the metal structure exists is opposed to each other and is sandwiched between the planes 104 to form a sample container, and the sample is used by flowing into the sample container, The distance between the planes is preferably 300 nm to 1 mm. This is because if the distance between the planes 104 on which the metal structures are present is too narrow, the plasmons of the metal structures 101 interact with each other, affecting the spatial electric field distribution and intensity, resulting in sensor sensitivity. This is because it may decrease. In addition, if the interval is too wide, the reaction efficiency between the target substance in the specimen and the capturing body existing on the surface of the metal structure decreases if the interval is too wide. In view of the distance between the planes in which the reaction efficiency does not decrease and the specimen can easily flow, it is more preferably 20 μm to 200 μm.

(Sample storage part)
The sample storage unit 103 has a role of storing a sample and, when the detection element has a capturing body, has a role as a place where the sample and the capturing body of the detection element come into contact. The specimen storage unit 103 is a void that is at least partially constituted by a part of the plane 104 on which the metal structure exists. More specifically, the specimen storage unit 103 is in contact with at least a part of a portion of the plane 104 where the metal structure 101 of the base body 102 is not present and the plane 104 of the metal structure 101. At least a part of the specimen storage unit 103 is constituted by a non-surface. More specifically, for example, in the example shown in FIGS. 1A, 1 </ b> D, and 1 </ b> F, the specimen storage unit 103 is a metal structure in the plane 104 where the metal structure 101 of the base body 102 exists. It is a gap sandwiched between portions constituted by a portion where 101 is not present and a surface of the metal structure 101 that is not in contact with the plane 104. Further, in the example shown in FIG. 1E, the structure is constituted by a portion of the plane 104 where the metal structure 101 of the base body 102 is present and the surface of the metal structure 101 not contacting the plane 104. And a gap sandwiched between the flat surfaces 106 where the metal structure of the base different from the base is not present. In the example shown in FIGS. 1B and 1C, the portion of the flat surface 104 where the metal structure 101 of the base 102 is present and the portion other than the base and the portion other than the base (for example, the detection element 100 flows). It is a gap sandwiched between the walls of the flow path when present inside the road.

  As described above, since the specimen storage unit 103 is a void, “A forms at least a part of the specimen storage unit 103” means that A (the surface of A when A is thick) is the specimen. It exists in the boundary of the accommodating part 103 and parts other than a specimen accommodating part, and A means that it is not contained in a specimen accommodating part.

  When a layer such as an adhesive layer exists between the base and the metal structure, the base is considered to have a laminated structure, and the surface of the layer is at least one of the planes on which the metal structure exists. Suppose that it constitutes a part. That is, a portion where the metal structure is present in the layer of the substrate is a portion where the metal structure is present on a plane where the metal structure of the substrate is present. Further, a portion where the metal structure does not exist in the layer of the base is a portion where the metal structure does not exist in a plane where the metal structure of the base exists.

  In addition, the detection element is configured by using a plurality of substrates and connecting surfaces perpendicular to the surface on which the metal structure exists with an adhesive or the like, or fixing the surface to the wall surface of the sample flow path. It can also be configured. In such a case, the specimen container includes at least a part of a plane where the metal structure of the base is present, a surface where the metal structure is not in contact with the plane, an adhesive layer, Thus, at least a part of the specimen storage unit is configured.

  In addition, the sample storage unit 103 may have a configuration (so-called batch configuration) that holds the sample in a state in which the sample is not moved, but preferably has a configuration in which the sample is held in a state in which the sample is moved. That is, the sample storage unit 103 is preferably a part of the flow path through which the sample passes. In this case, the detection light may be irradiated while the sample is circulated in the sample container, or the detection light may be irradiated after the sample is blocked.

  When the specimen storage part is a part of the flow path, the detection element 200 itself may be installed in the flow path 205 as shown in FIG. 4A, or as shown in FIG. In addition, at least a part of the flow path may be formed by the specimen storage unit 203 itself included in the detection element 200. Here, 206 indicates an inlet and 207 indicates an outlet.

  Note that it is preferable to form a flow path in the specimen storage unit 203 because introduction of the specimen into the detection element 200 and cleaning of the detection element 200 as necessary can be easily performed. For example, in the example of FIG. 4B, the sample can be introduced and washed using the inflow port 206 and the outflow port 207. The capturing body may be fixed before setting the detection element in the detection apparatus of the present invention, or may be fixed after setting the detection element.

(Metal structure)
The metal structure in the present invention may be any metal structure as long as it is disposed on the plane so as to be isolated from each other.

  The metal constituting the metal structure is preferably a metal exhibiting plasmon resonance in the context of the localized surface plasmon resonance method which is a preferred measurement method of the present invention. Specific examples include gold, silver, copper, aluminum, platinum, zinc, an alloy composed of two or more of these elements, and an alloy containing at least one of these elements. More preferably, gold | metal | money and silver which show a localized surface plasmon resonance notably are mentioned.

  In addition, the shape of the metal structure can be a spherical shape, a non-polyhedral shape such as a spherical shape, a spherical shape or a shape obtained by cutting a part of a substantially spherical shape, a cylindrical shape, a polygonal column, a cone, a pyramid, a ring shape with a thickness, Various polyhedron shapes such as well-shaped well-shaped and paddy-shaped shapes can be mentioned. Metal particles can also be used as the metal structure. The metal particles do not have to be true spheres, and may be polyhedrons.

  Here, when metal particles are used as the metal structure, core-shell type metal particles having a metal colloid or a dielectric as a core may be used. As an example of the core-shell type metal particle, as shown in FIG. 6A, a particle having a shell structure in which a cross section of a metal fine particle includes a metal core (metal colloid) 401, a dielectric shell layer 402, and a metal shell layer 403. Is given as an example. However, the number of shell layers, the material, the stacking order, the shape of the metal particles, and the like are not limited thereto.

  When the metal structure is a metal fine particle, the size is generally a size that may be called a metal fine particle, and the diameter is preferably 10 nm or more and 500 nm or less. When the metal particle is not a true sphere, the particle diameter obtained by a commercially available particle size measuring apparatus is regarded as the diameter.

  Further, when focusing attention on the manufacturing method, some examples of the metal structure of the invention can be called metal patterns. From the viewpoint of facilitating the production and improving the yield, it is preferable to form a metal pattern as a metal structure on the substrate as shown in FIGS. 5B and 5C. The example shown in FIG. 5A is an example of a detection element in which metal fine particles 301 are formed on two main surfaces of a flat substrate 302, and FIGS. 5B and 5C are flat plates. This is an example of a detection element in which a metal structure 303 made of a metal pattern is formed on two main surfaces of the substrate 302. The reason why it is better to use a metal pattern than a metal fine particle as the metal structure is as follows. As shown in FIG. 5A, when metal fine particles are used, partial aggregation may occur accidentally. In that case, since it exhibits a characteristic different from that of a single metal fine particle, variations in the detected spectrum occur, which may reduce the sensor sensitivity. On the other hand, when an element having a metal pattern is used, aggregation as described above can be prevented and the interval between the metal microstructures can be adjusted, so that it becomes easy to control the characteristics of the detected spectrum. Specifically, the accuracy of the spectrum is improved.

  Further, the example shown in FIG. 5C is an example similar to the example shown in FIG. 5B, but the metal structure 304 has a flat shape in FIG. 5B. Different from the example shown. It is preferable that the metal pattern has a flat shape. This is because in the metal structure, the smaller the aspect ratio, the greater the change in the plasmon resonance condition with respect to the surrounding refractive index change. The “aspect ratio of the metal structure” as used herein refers to a direction perpendicular to the metal structure formation surface of the substrate in a cross section obtained by cutting the metal structure along a plane perpendicular to the metal structure formation surface of the substrate. The length of the longest line segment among the line segments existing in the line is parallel to the metal structure forming surface and is perpendicular to the line segment existing in the direction perpendicular to the metal structure forming surface. Divide by the length of the longest line of minutes. In addition, the flat shape is a flat shape, and in the present invention, the aspect ratio is one half or less. More preferably, it is preferably 1/4 or less.

  Other examples of the shape of the metal pattern include those shown in FIGS. 6B to 6J. Here, FIG. 6 is a cross-sectional view of the metal structure cut along a plane parallel to the metal structure formation surface of the substrate.

  In addition, when producing a metal structure on a plane using various film-forming methods, it is difficult to measure the size with a particle size measuring apparatus. The preferable size of the metal structure in such a case is as follows. First, a preferable thickness (an average thickness in a direction perpendicular to a plane on which the metal structure is formed) is 10 nm or more and 100 nm or less. The size (maximum value of the distance between any two points in the metal structure in a plane parallel to the plane on which the metal structure is formed) is preferably 10 nm or more and 1450 nm or less, and 50 nm or more and 450 nm or less. It is more preferable that

  The distance between metal structures (the shortest distance between adjacent metal structures) is preferably 50 nm or more and 2 μm or less, and more preferably 150 nm or more and 1 μm. As described in the description of the distance between the planes where the metal structures exist, if the distance between the metal structures is too narrow, the plasmons of each metal structure interact with each other, and the spatial electric field distribution / It will affect the strength. As a result, the sensor sensitivity may decrease. On the other hand, if the distance is too wide, the signal intensity becomes weak because the density of the metal structure is low, and therefore a special optical system is required to increase the sensitivity.

  Further, if there is no plane having a certain size on the substrate (if the substrate does not have a plane having a certain size), it is difficult to form a metal pattern. Accordingly, a plane for allowing the metal structure to exist is also important from the viewpoint of facilitating the production of the detection element.

  Hereinafter, the capturing body and the target substance will be described.

(Capturer and target substance)
The capturing body is disposed on the surface of the metal structure or in the vicinity of the metal structure.
Any capture body can be used without particular limitation as long as it captures a target substance selectively or specifically. Note that “selectively capture target substances” means that a plurality of substances are captured, but the ability to capture target substances is particularly strong, and “specifically capture target substances” means target substances. Is only to capture.

  As the capturing body in the present invention, one that recognizes the shape and size of the target substance using the three-dimensional structure of the polymer compound, hydrogen bond, coordination bond, electrostatic interaction, hydrophobic field, etc. are used. And those that recognize the target substance. Further, some of these structures, bonds, actions, etc. may be used in combination to recognize the target substance. As described above, “capture” in the present invention and the present specification is a concept that broadly encompasses general substance recognition using various interactions. In addition, the target substance captured by the capturing body is not limited to a relatively small substance such as a molecule or an ion, but may be an assembly of molecules or a cell.

  Specific capture between the target substance and the target substance capturing body may be based on any kind of interaction as long as the physical / chemical change amount before and after the capture can be detected by the detection element of the present invention. Preferred interactions include antigen-antibody reaction, antigen-aptamer (RNA fragment having a specific structure) interaction, ligand-receptor interaction, DNA hybridization, DNA-protein (transcription factor, etc.) interaction, lectin-sugar chain Interaction, etc. Therefore, the combination of the target substance and the target substance capturing body can be a combination (for example, an antigen-antibody) that performs the interaction or the reaction.

  Any target substance can be detected as long as it is detected by the detection element of the present invention by being selectively or specifically captured by the capturing body by any interaction such as the above-described interaction. Become.

  A typical example of the target substance and the capturing body is a biological substance. Examples of the biological substance include biological molecules selected from nucleic acids, proteins, sugar chains, lipids, and complexes thereof. More specifically, a substance selected from any of DNA, RNA, aptamer, gene, chromosome, cell membrane, virus, antigen, antibody, lectin, hapten, hormone, receptor, enzyme, and peptide is the capturing body of the present invention. Or it is mentioned as a preferable example as a target substance. In addition, bacteria and cells themselves that produce the “biological substance” can also become a target substance and a capturing body as the “biological substance”.

Next, a detection apparatus using the detection element will be described.
7A and 7B are conceptual diagrams showing an example of the configuration of the detection apparatus according to the present invention. The detection apparatus of this example is an apparatus that optically detects the characteristics of a specimen, and includes the detection element 500, a light source 504 that irradiates the detection element 505 with detection light 505, and light transmitted through the detection element 500 (transmission). A light receiving element 506 that receives the light (light) 507, and an arithmetic unit (not shown) that obtains the characteristics of the specimen from the signal obtained by the light reception.

  The detection element 500 is the same as the element shown in FIG. 3, and is a combination of a plurality of bases 503 each having a flat surface 501 on which the metal structure 502 exists. In the drawing, reference numeral 508 denotes a specimen inlet, and 509 denotes a specimen outlet. A gap from the inlet 508 to the outlet 509 is a part of the specimen flow path (sample storage part 512). In addition, a capturing body (not shown) that captures the target substance in the specimen is fixed to the surface of the metal structure 502.

  By using the above-described configuration for the detection apparatus according to the present invention, an operation for detecting a target substance in a specimen can be continuously performed. In other words, a capturing body that captures a target substance is fixed to the detection element 500, a specimen containing the target substance is introduced into the detection element 500, and the detection light 505 is irradiated. Means for performing a series of steps until the target information is detected can be provided to one apparatus.

  Hereinafter, the light source 504, the light receiving element 506, and the arithmetic unit (not shown) which configure the detection apparatus 500 illustrated in FIG. 7 will be described in order.

(light source)
The detection light used in the present invention may be any wavelength as long as it causes a plasmon resonance with the metal structure. Accordingly, various light sources such as lasers and LEDs can be used as the light source. Preferred light sources include a broadband white light source such as a halogen lamp, a tungsten lamp, and a xenon lamp. The detection apparatus of the present invention may have various optical systems between the light source and the detection element. As an example of such an optical system, a lens optical system for adjusting an irradiation region of detection light to the detection element (in the example shown in FIG. 7A, also emitted light 505 of the light source), a collimating Examples include an optical system (collimator), a spectroscopic optical system such as a polarizing plate for polarizing detection light, and a monochromator. The detection apparatus illustrated in FIG. 7B is an example in which a spectroscopic optical system 510 including a monochromator is provided between a light source 504 and a detection element 501. Thereby, the emitted light 505 of the light source is made incident on the detection element as monochromatic light 511. The other points are the same as those of the apparatus shown in FIG.

  Various optical systems can be provided at arbitrary positions in the optical path of the detection light, not limited to between the light source 504 and the detection element 500.

(Light receiving element)
The light receiving element 506 may be any light receiving element that can detect the characteristic of the light 507 transmitted through the detection element 500, but it is necessary to select the light receiving element 506 in accordance with the characteristic change used for detection.

  When detecting a change in the intensity of transmitted light, a photodiode, a photomultiplier tube (PMT), or the like can be used as a light receiving element. When detecting a spectral change, it is preferable to use a detector (so-called spectroscope) using a spectroscopic optical system. When a spectroscopic optical system is used, spectroscopic measurement may be performed by passing light transmitted through the detection element through a spectroscope such as a monochromator or polychromator. Note that, as in the example shown in FIG. 7B described above, the light is split before irradiating the detection element using the spectroscopic optical system 510 such as a monochromator, the wavelength to be split is changed, and the intensity of transmitted light is changed. Spectrum measurement may be performed by measuring.

(Arithmetic unit)
The arithmetic device in the detection apparatus of this example is means for calculating a change in transmitted light characteristics due to the contact of the specimen with the metal structure 502 of the detection element 500 from the signal obtained by the light receiving element 506. More specifically, when the specimen comes into contact with the metal structure 502 of the detection element 500, whether or not the target substance in the specimen has been captured by the capturing body, and what amount of target substance has been captured. , Is calculated. It is a means for detecting the intensity change when the intensity of the transmitted light 507 is used as a signal, and detecting the spectrum change when the spectrum of the transmitted light 507 is used as a signal, and is preferably provided with a mechanism for signal processing the change in characteristics. . It is more preferable if a mechanism capable of quantitatively evaluating the characteristics by signal processing is provided. As such a mechanism, for example, there is a mechanism capable of calculating the amount of the target substance by comparing with the data indicating the correlation between the amount of the target substance acquired in advance and the characteristic change of the transmitted light. Can be mentioned.

  In addition, since the detection element of this invention has the largest frequency | count of detection light being absorbed in the metal structure vicinity, the structure which uses transmitted light as emitted light like FIG. 7 is preferable. However, it is also possible to adopt a configuration in which the detection light is reflected as the outgoing light after being transmitted through the plane where the metal structure is present a plurality of times and then reflected. For example, a substrate having a flat surface on which a metal structure farthest from the light source exists can be formed of a highly reflective material. In such a case, as shown in FIG. 7, the light receiving element is not present on the side opposite to the light source with reference to the detection element, but is present on the same side as the light source with reference to the detection element. When the detection light is incident perpendicularly or substantially perpendicularly to the detection element and the reflected light that has been transmitted through the plane on which the metal structure is present a plurality of times is used as the outgoing light, the detection light and the outgoing light are output by a beam splitter or the like. Can also be separated.

Moreover, it is preferable that the detection device of the present invention has a device (for example, a display device) that outputs a calculation result. The device that outputs the calculation result may exist separately from the calculation device, or may be integrated with the calculation device.
Next, the detection method in the present invention will be described.

The detection method of the present invention comprises:
Introducing the sample into the detection element described above;
Irradiating the detection element with detection light so as to cross a plane on which a plurality of metal structures separated from each other of the base of the detection element exists;
Receiving light emitted from the detection element by a light receiving element;
Obtaining the characteristics of the specimen from the signal output from the light receiving element;
Have

  For example, the detection method of the present invention can be realized by using the detection apparatus of the example shown in FIG.

Example 1
Example 1 will be described with reference to FIGS.

<Detection element fabrication>
A gold thin film with a thickness of 20 nm is formed by sputtering on a 0.725 mm thick quartz substrate having a Ti adhesive layer applied in advance to the surface. A negative resist is applied onto the gold thin film, and this is exposed to a square shape of 200 nm × 200 nm using an electron beam drawing apparatus to form a square resist pattern. Using this resist as a mask, the gold thin film and the adhesive layer in the resist non-covered portion are etched using an ICP etcher. After etching, the resist used for the mask is removed by an asher. By passing through this process, a base having a plurality of metal structures spaced apart from each other on one main surface is formed by forming a 200 nm square square gold pattern as shown in FIG. 6B. To manufacture. Although an electron beam drawing apparatus is used here, a pattern may be formed using a focused ion beam processing apparatus, an X-ray exposure apparatus, an ultraviolet exposure apparatus, or an excimer exposure apparatus. In addition, although the etching method is described here, it may be formed by a lift-off method.

  A detection element 600 as shown in FIG. 8 is manufactured by arranging three bases 602 each having a plurality of metal structures 601 made of gold separated from each other in parallel on one main surface.

<Detection device>
FIG. 9 is a configuration diagram of the detection apparatus of this embodiment.
It consists of a halogen lamp 701 as a light source, a detection element 700, and a spectroscope 708 as a light receiving element. The detection element 700 is the same as the detection element 600 of FIG. Reference numeral 709 denotes a plane on which the metal structure of the base body 703 exists, and reference numeral 710 denotes a specimen storage unit configured by the flat surface 709 on which the metal structure exists, the metal structure 704, and the base body 703. Note that a part of the flow path is formed in the detection element, and the specimen storage portion 710 is a part of the flow path. An inflow port 705 and an outflow port 706 for introducing and cleaning the specimen are provided. Reference numeral 704 denotes a metal structure made of gold, reference numeral 702 denotes detection light to the detection element, and reference numeral 707 denotes light transmitted through the detection element (transmitted light).

<Detection method>
The detection method in the present embodiment will be described in detail with reference to FIG.
As shown in FIG. 10, the inlet 801 and the outlet 802 of the detection element are connected to the other part of the flow path (the part connected to the liquid feed pump 805 and the part connected to the waste liquid reservoir 809). In addition, the detector element 803 having a spectroscope (light receiving element) and the light source unit 804 using a halogen lamp as a light source are positioned so that a reaction region (region where the metal structure exists) 806 is positioned on the optical axis. Arrange the position. In this state, the spectrum before the reaction is detected in advance using a spectroscope. Thereafter, the liquid feeding pump 805 is driven, and an ethanol solution of 11-mercaptodecanoic acid having a thiol group having a high affinity for gold is supplied into the element (inside the specimen storage portion), and the metal structure made of gold is surface-modified. Thereafter, 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 supplied into the device (in the specimen container). . Thereby, the succinimide group is exposed on the surface of the metal structure. Subsequently, an anti-human CRP antibody is used as a detection element by flowing in a Tris-HCl buffer solution (pH 8.0) to which an anti-human CRP antibody that specifically captures human CRP as a target substance is added as an immobilized antibody. Fix it. Next, human CRP is supplied to the reaction region 806 of the predetermined amount detection element, and human CRP as a target substance is captured by the anti-human CRP antibody by an antigen-antibody reaction between human CRP and the anti-human CRP antibody. After the reaction, the spectrum is measured with a spectroscope. The spectrum at this time and the spectrum before reaction are compared by a central operator 808 which is an arithmetic unit. This difference is a change in the local plasmon resonance state of the detection element due to the target substance being captured in the vicinity of the detection element. The change in the localized plasmon resonance state is displayed on the display unit 807 which is a display device.

  In this embodiment, the concentration of the target substance is obtained from the degree of change of the spectrum by the central operator 808 and displayed on the display unit 807. Regarding the relationship between the change in the spectrum and the concentration of the target substance, the relationship between the change in the spectrum and the concentration is obtained in advance using a standard sample having a plurality of known concentrations. Furthermore, a function of spectral change and concentration is obtained by creating a calibration curve based on this relationship. Using this function, the concentration of a target substance whose concentration is unknown can be determined based on the change in spectrum during actual measurement. In addition, although it described as the change of a spectrum here, the change of this spectrum may be a change of a spectrum peak (change of the wavelength with the maximum value of absorbance), or a change in peak shape such as a half width of the peak of the spectrum waveform. May be used. Furthermore, the light intensity at one or a plurality of wavelength points may be used.

(Example 2)
Example 2 will be described with reference to FIGS.

<Detection element fabrication>
A glass substrate 901 (manufactured by Matsunami Glass Co., Ltd.) whose entire surface has been modified with amino groups in advance is immersed in a solution of gold fine particles 902 having an average particle size of 40 nm (manufactured by Tanaka Kikinzoku Co., Ltd.) for 24 hours, washed with pure water, and then charged with nitrogen gas. Dry with.

  Thereafter, the capturing body for capturing the target substance is fixed to the surface of the gold fine particles by the following procedure. First, an ethanol solution of 11-Mercaptodecanoic acid having a thiol group having a high affinity for gold is added to modify the surface of the gold fine particles. Thereafter, the solution is brought into contact with 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). Thereby, the succinimide group is exposed on the surface of the gold fine particles. Subsequently, a Tris-HCl buffer solution (pH 8.0) to which an anti-human CRP antibody that specifically captures human CRP as a target substance is added as an antibody to be immobilized is dropped. The anti-human CRP antibody 903 is immobilized on the surface of the gold fine particle 902 by reacting the succinimide group disposed on the surface of the gold fine particle with the amino group of the anti-human CRP antibody. Unreacted succinimide groups on the surface of the gold fine particles are eliminated by adding Hydroxylamine Hydrochloride. As shown in FIG. 11, the substrate on which the gold fine particles on which the anti-human CRP antibody 903, which is a capturing body, is immobilized, is formed on two main surfaces facing each other as shown in FIG.

  Then, as shown in FIG. 12, both surfaces of such a substrate 1005 (the same as the substrate formed on the two main surfaces on which the gold fine particles on which the anti-human CRP antibody 903 in FIG. 11 is immobilized) are opposed to each other). A detection element 1003 is formed by disposing a polydimethylsiloxane (hereinafter referred to as PDMS) chip 1007 provided with a single groove on each. By disposing the groove on the substrate 1005 side, a specimen storage portion (a part of the flow path) 1006 can be formed. A part of the specimen storage unit 1006 becomes a reaction region 1004.

<Detection device configuration>
Next, the configuration of the detection apparatus used in this example will be described with reference to FIG.
The detection apparatus of this example includes a green semiconductor laser 1001 having a light emission center wavelength of 532 nm as a light source, a detection element 1003, and a photodiode 1008 as a light receiving element for transmitted light. These are arranged so that the detection light 1002 is irradiated onto the reaction region 1004 and light (transmitted light) 1009 transmitted through the detection element is incident on the photodiode 1008.

<Detection method>
In this example as well, the target substance can be detected through the same detection method as in Example 1. However, in this embodiment, a semiconductor laser is used for the light source unit, and a photodiode is used for the light receiving element.

  In the present example, since the anti-human CRP antibody is already immobilized on the gold microparticle as a target substance capturing body, the step of immobilizing the anti-human CRP antibody is omitted during detection.

It is typical sectional drawing which shows some examples of the base | substrate which has the plane where the several metal structure spaced apart from each other used suitably for this invention exists. It is a schematic diagram which shows the example of the relationship between detection light and a detection element. It is typical sectional drawing which shows the example which forms the flow path of a test substance by combining multiple bases which have the plane where a metal structure exists. It is a schematic diagram which shows the example of the relationship between a detection element and the flow path of a sample. It is typical sectional drawing which shows some examples of the base | substrate which has the plane in which the several metal structure mutually spaced apart used suitably for this invention exists. It is a schematic diagram which shows some examples of the shape of the metal structure used for this invention. It is a conceptual diagram which shows an example of a structure of the detection apparatus of this invention. It is a schematic diagram which shows the structure of the detection element regarding Example 1 of this invention. It is a schematic diagram which shows the structure of the detection apparatus regarding Example 1 of this invention. It is a block diagram regarding Example 1 of this invention. It is a schematic diagram which shows the structure of the detection element regarding Example 2 of this invention. It is a schematic diagram which shows the structure of the detection apparatus regarding Example 2 of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 Detection element 101 Metal structure 102 Base | substrate 103 Sample accommodating part 104 Plane in which a metal structure exists 105 Detection light 106 Plane in which a metal structure does not exist 200 Detection element 201 Metal structure 202 Base 203 Sample accommodating part 204 Metal structure is Presence plane 205 Channel 206 Inlet 207 Outlet 301 Metal fine particle 302 Base 303 Metal structure made of metal pattern 304 Metal structure made of metal pattern having flat shape 401 Metal core 402 Dielectric shell layer 403 Metal shell Layer 500 Detection element 501 Plane on which metal structure exists 502 Metal structure 503 Base 504 Light source 505 Detection light (light emitted from light source)
506 Light receiving element 507 Transmitted light (light transmitted through detection element)
508 Inflow port 509 Outflow port 510 Spectroscopic optical system 511 Monochromatic light 512 Specimen container 600 Detection element 601 Gold metal structure 602 Substrate (quartz substrate)
700 Detection Element 701 Halogen Lamp 702 Incident Light 703 Base 704 Metal Structure Made of Gold 705 Inlet 706 Outlet 707 Transmitted Light 708 Spectrometer 709 Plane Where Metal Structure Exists 710 Sample Storage Unit 801 Inlet 802 Outlet 803 Detection Device 804 Light source unit 805 Liquid feed pump 806 Reaction region 807 Display unit 808 Central operator 809 Waste liquid reservoir 901 Glass substrate modified with amino group 902 Gold fine particle 903 Anti-human CRP antibody 1001 Green semiconductor laser 1002 Incident light 1003 Detector element 1004 Reaction Region 1005 Substrate 1006 Liquid specimen container (part of flow path)
1007 PDMS chip 1008 Photodiode 1009 Transmitted light

Claims (10)

A detection element that detects the characteristics of a specimen using detection light,
The detection element is
A plurality of metal structures spaced apart from each other;
A base having a plane on which a plurality of metal structures spaced apart from each other exist;
A specimen containing portion constituted at least in part by a part of the plane,
A plurality of said planes;
Any one of the plurality of planes has a normal line intersecting with all the other planes.   The detection element according to claim 1, wherein an arbitrary plane among the plurality of planes has a normal line intersecting with all other planes.   The detection element according to claim 1, wherein the detection element has a capturing body that captures a target substance in the specimen on a surface of the metal structure.   The detection element according to claim 1, wherein the metal structure is a patterned flat structure. A detection device for detecting a characteristic of a specimen,
A sensing element;
A light source for irradiating the detection element with detection light;
A light receiving element that receives outgoing light emitted from the detection element;
An arithmetic unit for obtaining characteristics of the specimen from a signal obtained by the light receiving element;
Have
The detection element is
A plurality of metal structures spaced apart from each other;
A base having a plane on which a plurality of metal structures spaced apart from each other exist;
A specimen containing portion constituted at least in part by a part of the plane,
A plurality of said planes;
Any one of the plurality of planes has a normal line intersecting with all the other planes.   The detection apparatus according to claim 5, wherein an arbitrary plane among the plurality of planes has a normal line intersecting with all the other planes. The detection apparatus according to claim 5 or 6, further comprising a capturing body that captures a target substance in the specimen on a surface of the metal structure included in the detection element.   The plurality of planes exist in a traveling path of the detection light, and each of the plurality of planes exists at a position where an angle formed by a normal line of the plane and the detection light is within 20 °. The detection apparatus in any one of -7. Using a detection element having a plurality of metal structures, a base having a plane on which the plurality of metal structures are spaced apart from each other, and a specimen containing portion at least partially constituted by a part of the plane A method for detecting the characteristics of a specimen,
The detection element has a plurality of the planes;
Introducing the sample into the sample container;
Irradiating the detection light to the detection element so as to cross the plurality of planes;
Receiving light emitted from the detection element by a light receiving element;
And a step of obtaining a specimen characteristic from a signal output from the light receiving element. Irradiating the detection element with the detection light so as to cross the plurality of planes,
A step of irradiating the detection element with the detection light such that the plurality of planes are present in a traveling path of the detection light, and an angle formed between a normal line in each of the plurality of planes and the detection light is within 20 °. The detection method according to claim 9, wherein:

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