JP2009168735A - Detection element and detection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection element and a detection method capable of detection spectrum changes steadily at high sensitivity, and which utilizes plasmon resonance. <P>SOLUTION: By using a detection element comprising a base substance, a flat metallic structure provided on the surface of the base substance, fine metallic particles secured to the upper surface of the metallic structure and a capturing object to selectively and specifically capture target substances of the liquid analyte, the target substances in the liquid analyte are captured by fine metallic particles, further, captured on the surface of the metal structure of the detection element, and thus the detection sensitivity by using plasmon resonance is improved. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a detection element and a detection method for detecting a target substance in a liquid 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 fine particles has been promoted as one method for meeting such requirements.

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. In Patent Document 2, as a configuration for detecting the adsorption of a substance to metal fine particles, the metal fine particles are arranged in the micropores of an alumina layer formed by anodizing a material mainly composed of aluminum. The configuration is disclosed. In Patent Document 2, the change in the plasmon resonance wavelength due to the proximity of the metal fine particles arranged in the micropores of the alumina layer and the metal fine particles in the sample solution through the detection target substance is measured.
Japanese Patent No. 03452837 JP 2005-195440 A

  When metal fine particles are fixed on a substrate as in Patent Document 1, metal fine particles cause plasmon interaction, and the peak width of the absorption spectrum is widened. Therefore, it is difficult to detect a minute change in absorption spectrum when the refractive index change of the medium around the metal fine particles is small or when the amount of adsorption of the substance is very small. If the fixing density of the metal fine particles is lowered so that the plasmon interaction between the metal fine particles on the substrate does not occur, the absorbance itself of the absorption spectrum becomes small, and in this case also, a minute change in the absorption spectrum is detected. It becomes difficult.

  On the other hand, in Patent Document 2, the metal fine particles in the sample solution are adsorbed on the uppermost side of the metal fine particles on the surface of the substrate or on the boundary with the substrate surface with respect to the metal fine particles exposed in a hemispherical shape on the surface of the substrate. Since the position is not determined, there is a problem that the change of the resonance condition is not stable. In the element structure in Patent Document 2, since the size of the fine metal particles on the substrate side is large in the depth direction of the micropores with respect to the size in the radial direction of the micropores, that is, the aspect ratio is large, the plasmon resonance condition is greatly changed. There is a problem that it cannot be done.

The detection element according to the present invention is an element that optically detects a target substance in a liquid specimen,
A substrate, a flat metal structure provided on the surface of the substrate, metal fine particles fixed on the upper surface of the metal structure, and a capturing body for selectively or specifically capturing a target substance of the liquid specimen;
A detection element comprising:

The detection method of the present invention is a method for optically detecting a target substance in a liquid specimen,
Preparing a detection element having the above-described configuration;
Dispersing the fine metal particles fixed with the target substance and a capturing body selectively or specifically captured in the liquid specimen;
Contacting the liquid specimen with the metal structure;
Irradiating the metal structure with detection light from a light source;
Measuring the intensity or spectrum of reflected or transmitted light from the metal structure;
And obtaining a characteristic of the liquid specimen from the intensity or spectrum of the reflected light or transmitted light.

  According to the present invention, the problem that the adsorption position of the metal fine particles is not fixed is determined by fixing a capture body that selectively or specifically captures the target substance only on the upper surface of the metal structure. The position can be limited to the top surface of the metal structure. Placing the metal structure in a flat shape on the substrate surface to increase its aspect ratio and preliminarily fixing metal fine particles on the upper surface of the metal structure can greatly change the plasmon resonance conditions. Can do. As a result, a larger plasmon resonance change can be obtained in accordance with the state change on the detection element surface, and the spectrum change in the detection light can be increased. As described above, it is possible to provide a detection element and a detection method capable of detecting a spectrum change stably and with high sensitivity.

  Hereinafter, the best mode 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 a detection element for optically detecting a target substance in a liquid specimen, and has a base and a flat metal structure provided on the surface of the base. At least the upper surface of the flat metal structure is exposed, and the metal fine particles and the capturing body for selectively or specifically capturing the target substance are fixed on the upper surface.

  Hereinafter, the substrate, the metal structure, the metal fine particles, the coupling layer for fixing the metal fine particles on the upper surface of the metal structure, the capturing body, and the target substance will be described.

(Substrate)
In the present invention, the substrate may be any substrate as long as it has a plane for providing a metal structure, but preferably has a high transmittance with respect to light irradiated from a light source. Examples of the material having a high transmittance with respect to the light emitted from the light source include silica, quartz, PMMA (polymethyl methacrylate), and polystyrene.

(Flat metal structure)
The flat metal structure in the present invention will be described. A flat metal structure means that the size 103 with respect to the thickness 104 of the metal structure 101 as shown in FIG. 1 is large. The thickness 104 is the maximum value of the distance between two points in the direction perpendicular to the plane of the base 102 on which the metal structure 101 is formed, and the size 103 is parallel to the plane on which the metal structure is formed. It is the maximum value of the distance between any two points in the metal structure in a plane.

  The size of the metal structure is preferably 20 nm or more and 1000 nm or less, and more preferably 40 nm or more and 450 nm or less. Furthermore, the thickness of the metal structure is preferably 10 nm or more and 100 nm or less. Moreover, it is preferable that the thickness with respect to the magnitude | size of a metal structure (henceforth an aspect ratio may be described) is 1/2 or less. More preferably, it is good that it is 1/4 or less. When metal fine particles are fixed on the upper surface of the metal structure, the resonance condition of localized surface plasmon resonance changes. At this time, when the thickness of the metal structure is very thin, the change in resonance condition changes greatly, and as a result, the peak change in the absorption spectrum also increases.

  If it mentions paying attention to a manufacturing method, what can be illustrated as a metal pattern in the metal structure in this invention is also mentioned. Specific examples include those shown in FIGS. 2A to 2I.

  The metal constituting the flat metal structure is preferably a metal that causes a localized surface plasmon resonance phenomenon. 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.

(Metal fine particles)
The metal fine particles in the present invention will be described. The following two types are used as the metal fine particles in the present invention.
(1) Those fixed to a metal structure in advance and used for detection.
(2) One having a capturing body that captures a target substance and being fixed to the upper surface of the metal structure via the target substance.

  The size of both the metal fine particles (1) and (2) is preferably 5 nm to 300 nm in diameter and smaller than the size of the metal structure. When the metal fine particle is not a true sphere, the particle diameter obtained by a commercially available particle size measuring apparatus is regarded as the diameter. That is, the diameter range does not strictly reflect an error in the measurement method, and fine particles having a particle size within this range may be selected for the purpose of measurement. The particle diameter of each metal fine particle fixed on the metal structure is preferably uniform.

  The metal constituting the metal fine particles is preferably a metal that causes a localized surface plasmon resonance phenomenon as in the metal structure. 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. It is more preferable to use metal fine particles made of the same metal as the metal structure.

(Coupling layer)
In fixing the metal fine particles to the upper surface of the metal structure, the metal fine particles can be fixed more firmly through the coupling layer. This coupling layer can be formed, for example, by using a coupling agent having a functional group that easily binds to a metal such as a thiol group, a disulfide group, or an amino group. Such a coupling layer may be formed on the upper surface of the metal structure, or may be formed on the surface of the metal fine particles.

(Capturer and target substance)
The capturing body and the target substance will be described. Any capture body can be used without particular limitation as long as it captures a target substance selectively or specifically. Examples of the capturing body include the following.
・ Recognizes the shape and size of the target substance using the three-dimensional structure of the polymer compound.
・ Recognize target substances using hydrogen bonds, coordination bonds, electrostatic interactions, hydrophobic fields, etc.
・ Recognize target substances by using some of the above structures, bonds, actions, etc. in combination.

  As described above, the term “capture” in the present invention and the present specification is a concept that broadly encompasses general substance recognition using various interactions. The target substance to be captured 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 of the target substance by 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), ligand-receptor interaction, DNA hybridization, DNA-protein (transcription factor, etc.) interaction, lectin-sugar chain interaction , Etc.

  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, the bacteria and cells themselves that produce the “biologically related substance” can also become target substances and capturing bodies as “biologically related substances”.

  Next, the detection element in the present invention in which only the upper surface of the metal structure is exposed will be described.

  In the present invention, the detection element in which only the upper surface of the metal structure is exposed is composed of the above-described substrate, metal structure, and metal fine particles. As a configuration example before the metal fine particles are fixed, a configuration of the metal structure 301 embedded in the base body 302 as shown in FIG. As another configuration example, the side surface of the metal structure 401 on the substrate 402 as shown in FIGS. 4A and 4B is covered with a layer 403 made of a substance to which metal particles and a capturing body do not bind. The structure currently made is also mentioned. Needless to say, the present invention is not limited to these examples, and various modifications exist within the scope of the present invention.

  In the present invention, the configuration in which only the upper surface of the metal structure is exposed on the substrate surface includes a configuration in which the upper surface of the metal structure projects from the substrate surface as shown in FIG. In this configuration, the end portion of the metal structure has a curved shape by chamfering so-called corner portions (edge portions) connected to the upper surface of the metal structure by drawing a curve from the substrate surface. Even in such a chamfered shape, the same effect as in FIG. 3A can be obtained.

  From the above configuration, the upper surface of the metal structure in the present invention is the maximum value of the distance between any two points in the metal structure in a plane parallel to the plane of the base on which the metal structure is formed. It refers to the surface of the metal structure exposed on the surface of the substrate on the surface passing through the point or on the upper surface.

  Next, FIG. 5 shows a schematic diagram of a measurement system for optically detecting the characteristics of a liquid specimen in the present invention. FIG. 5 is a schematic diagram of a measurement system as an example of a detection apparatus for the detection method of the present invention. This measurement system includes a light source 501, a detector 508, and a calculation device 509. Incident light 502 is irradiated from a light source 501 to a detection element 503 for optically determining the characteristics of a liquid specimen. The light 507 transmitted through the detection element 503 is measured by the detector 508. The arithmetic device 509 obtains the characteristics of the liquid specimen from the signal obtained by the detector 508. In this example, a measurement system that detects transmitted light is described. However, a measurement system that detects reflected light may be used.

  Hereinafter, a light source, a detector, and an arithmetic unit that constitute the detection device of the present invention will be described.

(light source)
The light source for irradiating the detection light to the detection element used in the present invention may be any wavelength band that causes plasmon resonance with the metal structure. Accordingly, various light sources such as lasers and LEDs are used as the light source. Preferred examples of the light source include a halogen lamp, a tungsten lamp, a xenon lamp, etc., which are broadband white light sources. Note that the detection apparatus of the present invention may have various optical systems between the light source 501 and the detection element 503. Examples of such an optical system include a lens optical system for adjusting the detection light irradiation area to the detection element 503, a collimating optical system (collimator), a polarizing plate for polarizing the detection light, A spectroscopic optical system such as a monochromator may be used.

(Detector)
The detector 508 used in the present invention may be any detector as long as it has a light receiving element capable of detecting the characteristics of the light transmitted through the detection element 503, but needs to be selected in accordance with a change in characteristics used for detection. is there. When detecting a change in the intensity of transmitted light or reflected light, a photodiode, a photomultiplier tube (PMT), or the like can be used. When detecting a spectral change, it is preferable to use a spectroscope using a spectroscopic optical system. When a spectroscopic optical system is used, spectroscopic measurement may be performed by passing light transmitted through or reflected by the detection element 503 through a spectroscope such as a monochromator or a polychromator.

(Arithmetic unit)
The calculation device of the detection apparatus according to the present invention is a device that calculates a change in the characteristics of transmitted or reflected light due to the liquid sample coming into contact with the detection element 503 from the signal obtained by the detector 508. More specifically, whether or not the specimen is in contact with the detection element 503 and the target substance is captured by the metal structure 505 by the capture body fixed on the metal structure 505, and any amount of the target substance when captured. It is a device that calculates whether or not is captured. It is preferable to provide a mechanism for calculating the intensity change when measuring the intensity of transmitted light or reflected light, and calculating the spectrum change when measuring the spectrum of transmitted light or reflected light.

  In addition, it is preferable that the detection device 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.

  Finally, a detection method in the present invention will be described. The detection method of the present invention is a method for optically detecting a target substance in a liquid specimen.

The detection method of the present invention can be carried out by using at least the following steps using, for example, the above-described detection apparatus.
(1) A step of preparing a metal structure in which metal fine particles and a capturing body are fixed on the upper surface (2) A step of dispersing metal fine particles having the capturing body fixed in a liquid sample (3) A liquid sample and a metal structure Contacting step (4) irradiating the metal structure with detection light from the light source (5) measuring intensity or spectrum of reflected or transmitted light from the metal structure (6) reflecting or transmitting light Steps for obtaining the characteristics of the liquid specimen from the intensity and spectrum will be described below.

(1) Step of preparing a metal structure in which metal fine particles and a capturing body are fixed on the upper surface This step selectively or specifically captures metal fine particles and a target substance on the upper surface, which is a detection element in the present invention. This is a step of preparing a flat metal structure to which the capturing body is fixed. Hereinafter, in order to prepare the detection element in the present invention, a specific example of a method for fixing the metal fine particles and the capturing body on the upper surface of the metal structure will be described, but the present invention is not limited thereto.

  First, a method for fixing metal fine particles on the upper surface of a flat metal structure will be described. The metal fine particles can be fixed by forming the coupling layer on the upper surface of the metal structure.

  Next, a method for fixing a capturing body that selectively or specifically captures a target substance on the upper surface of a metal structure having metal fine particles fixed on the upper surface will be described. Any capturing body that selectively or specifically captures the target substance may be used as long as it can be directly or indirectly immobilized on the upper surface of the metal structure. Further, any method may be used as a method for fixing the capturing body, as long as the function as the capturing body is not lost. When the capture body is directly fixed, the capture body needs to have not only a site for recognizing the target substance but also a site with good affinity for the material on the surface of the metal structure on the capture body. Since the surface of the metal structure contains a metal, it can be directly fixed if it has a functional group having a good affinity for the metal, such as a thiol group, a disulfide group, or an amino group, on the capturing body side.

  When the capturing body does not have a site for recognizing metal, the capturing body is indirectly fixed to the surface of the metal structure. In this case, the capturing body is fixed via the coupling layer. The coupling layer used in this step may be any layer as long as it has a site for recognizing the metal and the capturing body. For example, when the metal structure surface is gold and the capture body has a functional group having an affinity for an amino group, if aminoethanethiol having an amino group and a thiol group at both ends is used, the affinity between the thiol group and the gold Due to the strong nature, amino groups appear on the gold surface. For this reason, the amino group appearing on the surface of the metal structure and the capturing body are bonded to each other, and the capturing body can be fixed to the surface of the metal structure.

(2) Dispersing the metal fine particles on which the capturing body is fixed in the liquid sample This step is a process for dispersing the metal fine particles on which the capturing body is fixed in the liquid sample containing the target substance. First, metal fine particles to which a capturing body is fixed are prepared. The capturing method for capturing the target substance on the metal fine particles may be the same method as in step (1), in which the capturing material is immobilized on the metal structure. It may be used. A dispersion of metal fine particles prepared by this method and having a capturing body fixed thereto is dispersed in a liquid specimen containing a target substance. By doing so, the target substance in the liquid specimen is captured by the capturing body fixed to the metal fine particles.
(3) The step of bringing the liquid specimen into contact with the metal structure In this step, the contact between the metal structure and the liquid specimen means that the metal fine particles in which the target substance formed in the step (2) is captured are the metal structure. It is present near or in contact with the trap. When the target substance captured by the metal fine particles is captured by the capture body fixed to the metal structure, the metal fine particles are fixed to the upper surface of the metal structure. In the step (2), the step of dispersing the metal fine particles with the capturing body immobilized in the liquid sample is described. However, by bringing the liquid sample into contact with the metal structure first, the target substance in the liquid sample is converted into a metal. The metal fine particles are also fixed to the upper surface of the metal structure by performing a process of capturing with the capturing body fixed to the structure and bringing the dispersion liquid containing the metal fine particles to which the capturing body is fixed into contact with the detection element. It will be.

Steps (4) to (6) The above steps (4) to (6) can be performed by the above-described operation using the detection device shown in FIG.

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

  In this example, the production of the detection element, and the fixing and absorption spectrum of the metal fine particles in the production process will be described.

<Preparation of gold structure with exposed upper surface>
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 on the gold thin film, and 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, an ICP etcher is used to etch the gold thin film and the adhesive layer in the resist non-covered portion. After etching, the resist used for the mask is removed by an asher. Through this step, a 200 nm square square gold pattern 601 can be formed on the substrate 602 as shown in FIG. On the other hand, in the above process, before removing the resist used for the mask by the asher, SiO ₂ is deposited to a thickness of 20 nm, and then the resist on the gold pattern is removed. Thereby, it is possible to form a gold structure 601 in which only the upper surface as shown in FIG. 6B used in this embodiment is exposed and the side wall is covered with the SiO ₂ layer. Although an electron beam drawing apparatus is used here, a metal 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.

<Gold fine particle fixation>
Gold fine particles are fixed to the upper surface of the gold structure. First, an ethanol solution of aminoethanethiol having a thiol group having high binding property with gold is supplied to modify the upper surface of the gold structure. As a result, an amino group capable of binding to gold appears on the surface. A gold colloid solution (manufactured by Tanaka Kikinzoku Kogyo Co., Ltd.) having a diameter of 40 nm is supplied thereto, and gold fine particles are fixed on the upper surface of the gold structure, whereby the detection element in this example can be obtained.

<Gold fine particle fixation time and absorption spectrum>
Regarding the fixation of the gold fine particles, the number of gold fine particles fixed to the upper surface of the gold structure can be controlled by increasing the fixing time without changing the concentration of the gold fine particle solution. Hereinafter, the fixing time of the gold fine particles and the absorption spectrum of the detection element will be described.

  The relationship between the fixing time of the gold fine particle solution and the peak wavelength of the absorption spectrum of the detection element is as shown in FIG. By making it possible to freely select a fixed condition based on this calibration curve, the peak wavelength of the initial absorption spectrum of the detection element can be controlled. When the fixing time is short, there are few gold fine particles fixed on the upper surface of the gold structure, and the peak wavelength shifts to the long wavelength side. However, when the gold fine particles to be fixed increase, the shift to the short wavelength side is performed. This is because the gold fine particles to be fixed start to have a film property. As the number of fixed gold fine particles increases and the coverage of the upper surface of the gold structure increases, the degree of shift to the short wavelength side increases.

  When the fixing time becomes long, the reaction between the amino group on the gold structure and the gold fine particles reaches an equilibrium state, and therefore, a certain number or more of gold fine particles cannot be fixed. For this reason, when the same reaction system is used, the peak wavelength converges to a certain wavelength. When the gold fine particles are further fixed, it is possible to change the reaction system for fixing.

  In this embodiment, a sufficient amount of time is required to fix the gold fine particles on the upper surface of the gold structure until an equilibrium state is reached, and this is used as a detection element.

(Example 2)
Example 2 will be described with reference to FIG.
<Detection device>
The detection apparatus of the present embodiment will be described with reference to FIG. A light source unit 804 having a halogen lamp as a light source, a spectroscope 803 as a detector, an inlet 801, an outlet 802, and a liquid feed pump 805 for supplying and discharging a liquid specimen to and from a reaction region 806 in which elements are present; It comprises a waste liquid reservoir 809 for storing liquid specimens to be discharged. Further, a calculation device 808 for obtaining the characteristics of the liquid specimen from the signal from the spectroscope 803 for detection, and a display unit 807 for displaying the results obtained from the calculation device 808 are provided.
<Detection element>
In this example, the sensor manufactured in Example 1 is used as a detection element.
<Fixed body>
Thereafter, an aqueous glutaraldehyde solution is supplied to the device. Thereby, the aldehyde group is exposed on the surface of the metal structure. Subsequently, an amino group and an aldehyde group of the anti-human CRP antibody are bound by flowing in a Tris-HCl buffer solution (pH 8.0) to which an anti-human CRP antibody specific to the target substance is added as an antibody to be immobilized, Anti-human CRP antibody can be immobilized on the top surface of the gold structure.
<Contact with target substance metal structure>
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.
<Captured body fixed gold fine particles and contact with gold structure composite>
A solution of gold fine particles having an anti-human CRP antibody immobilized on the surface is supplied to the reaction region 806, and the gold fine particles are immobilized by an antigen-antibody reaction with human CPR on the upper surface of the gold structure. Immobilization of the anti-human CRP antibody on the gold fine particle surface is performed in advance. An aminoethanethiol ethanol solution and a glutaraldehyde aqueous solution are supplied to the surface of the gold fine particles, the aldehyde groups are represented on the surface, and an anti-human CRP antibody is supplied thereto to fix the gold fine particle surface.

<Measurement>
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 spectrum and the concentration of the target substance, the relationship between the change in spectrum and the concentration is obtained in advance using a plurality of detection elements prepared with the same amount of gold fine particles adsorbed and using a standard sample with a plurality of known concentrations. Keep it. 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. At this time, the measurement is preferably performed over time. This is because when using a detection element with a small amount of gold fine particles before adsorbing the target substance, the peak wavelengths of the absorption spectra may be equal at different concentrations of the target substance.

  Moreover, when measuring a low concentration target substance, it is preferable to adjust the fixed amount of the gold fine particles on the surface of the gold structure where the gradient of the spectrum change becomes maximum. This is because the spectrum change due to the adsorption of the gold fine particles to which the capturing body after the target substance adsorption is fixed becomes large.

  In addition, although it described as the change of a spectrum here, this spectrum change may be the change of the spectrum peak in the wavelength which has the maximum value, and the change of peak shape, such as the half width of the peak of the spectrum waveform, is used. Further, the light intensity at one or a plurality of wavelength points may be used.

It is a schematic diagram which shows an example of the detection element of this invention. It is a schematic diagram which shows the shape of the metal structure of this invention. It is a schematic diagram which shows the example of the detection element which only the surface of the metal structure of this invention exposed. It is a schematic diagram which shows the other example of the detection element which only the surface of the metal structure of this invention exposed. 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 graph showing the relationship between the fixed time when a metal microparticle is fixed to the metal structure surface in invention, and the peak wavelength of the absorption spectrum of a detection element. It is a block diagram regarding Example 2 of this invention.

Explanation of symbols

101 Metal Structure 102 Base 103 Size of Metal Structure 104 Metal Structure Thickness 301 Metal Structure 302 Base 401 Metal Structure 402 Base 501 Light Source 502 Incident Light 503 Detection Element 504 Base 505 Metal Structure 506 Metal Fine Particle 507 Transmitted light 508 Detector 509 Arithmetic device 601 Metal structure 602 Substrate 801 Inlet 802 Outlet 803 Detector 804 Light source unit 805 Liquid feed pump 806 Reaction area 807 Display unit 808 Central operator 809 Waste liquid reservoir

Claims (3)

An element for optically detecting a target substance in a liquid specimen,
A substrate, a flat metal structure provided on the surface of the substrate, metal fine particles fixed on the upper surface of the metal structure, and a capturing body for selectively or specifically capturing a target substance of the liquid specimen;
A detection element comprising:   The detection element according to claim 1, wherein an upper surface of the metal structure is exposed on a surface of the base. A method for optically detecting a target substance in a liquid specimen,
Preparing the detection element according to claim 1 or 2,
Dispersing the fine metal particles fixed with the target substance and a capturing body selectively or specifically captured in the liquid specimen;
Contacting the liquid specimen with the metal structure;
Irradiating the metal structure with detection light from a light source;
Measuring the intensity or spectrum of reflected or transmitted light from the metal structure;
And obtaining a characteristic of the liquid specimen from the intensity or spectrum of the reflected light or transmitted light.

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