JP2009150708A - Target substance detection method and test kit - Google Patents

Abstract

To provide a method for detecting a target substance with improved detection sensitivity as compared with a conventional method using a sensor using a localized plasmon resonance phenomenon.
A method for detecting a target substance in a specimen, wherein fine metal structures capable of inducing localized plasmon resonance are separated from each other and arranged on a substrate surface, and separated from the fine metal structure on the substrate surface. Then, a test element in which the first probe is arranged, and a composite of a minute metal structure capable of inducing localized plasmon resonance and the second probe are prepared. When the first probe is immobilized on the surface of the test element by a reaction that forms a complex with the second probe via the target substance, the fine metal structure immobilized on each of the first probe and the second probe approaches each other. Since the plasmon resonance wavelength changes greatly, the target substance can be detected with high sensitivity.
[Selection] Figure 1

Description

  The present invention relates to a method for detecting a target substance and a detection kit.

  Nucleotide sequence analysis of genomes of various organisms including humans has been energetically promoted, and as a result, through detection of target substances such as genes and proteins, genetic diseases, cancer, infectious diseases, lifestyle-related diseases, etc. Diagnosis, decision of treatment policy, management after treatment, prognosis prediction, etc. have come to be performed.

  As a sensor technique used for detection of these target substances, a sensor using localized plasmon resonance is known. This sensor uses a device having a structure in which metal fine particles are fixed on the surface of a substrate as an inspection element, and means for irradiating the metal fine particles of the inspection element with light and spectrally divides the light reflected or transmitted by the metal fine particles. It is constituted by means for measuring the intensity of each.

  Local plasmon resonance is a phenomenon in which free electrons in metal fine particles resonate with an electric field of light of a specific wavelength and start to vibrate when light is irradiated onto metal fine particles smaller than the wavelength of light. When electrons start to vibrate and local plasmon resonance is induced, a strong electric field is generated around the metal fine particles, and scattering and absorption at a specific wavelength (resonance wavelength) are remarkably increased.

  The resonance wavelength depends on the refractive index of the substance around the metal fine particles. As the refractive index of the surrounding material increases, the resonance wavelength shifts to the longer wavelength side, and scattering and absorption of reflected light increase. Therefore, if the target substance is irradiated with light in a state where the target substance can be adsorbed or deposited on the metal fine particles, and the reflected or transmitted light intensity is measured, the target substance is observed with the change in the light intensity observed. Can be detected.

As a method for detecting a target substance dispersed in a solution using such a sensor, a method in which metal fine particles are also bound to the target substance side has been proposed (see Patent Document 1). In this method, when the target substance is adsorbed on the surface of the test element, the metal microparticles on the test element side and the metal microparticles on the target substance side are close to each other. Since the resonance wavelength of the localized plasmon also changes depending on the proximity state between the metal fine particles, the metal fine particles are also bonded to the target substance side, so that the change of the resonance wavelength can be easily observed.
JP 2005-195440 A

  When the concentration or molecular weight of the target substance is low and its detection is difficult, it is effective to use metal fine particles as a labeling agent as described in Patent Document 1, but this is applied to a localized plasmon resonance sensor. In this case, it is necessary to consider the electric field strength distribution of the fine metal structure fixed on the test element.

  In general, in the case of localized plasmon resonance that occurs when light is incident perpendicularly to the substrate surface, the electric field strength distribution of the fine metal structure disposed on the substrate surface is parallel to the substrate surface (XY axis direction). Strongly distributed. Therefore, as described in Patent Document 1, the metal fine particles are not labeled perpendicularly (Z-axis direction) to the fine metal structure on the substrate surface, but are labeled parallel to the fine metal structure on the substrate surface. Thus, the strong electric field possessed by the fine metal structure on the substrate can be used effectively, and the target substance can be detected with higher sensitivity.

  Accordingly, an object of the present invention is to overcome the above-mentioned problems and to provide a method and a test kit for improving the detection sensitivity of a localized plasmon resonance sensor.

The first detection method of the present invention comprises:
A method for detecting the presence / absence or amount of a target substance in a specimen, wherein a first micro metal structure capable of inducing localized plasmon resonance and a first probe are isolated from each other and arranged on a substrate surface A step of preparing an element;
Providing a labeling reagent in which a second fine metal structure capable of inducing localized plasmon resonance is fixed to the second probe;
Performing a reaction in which the first probe arranged in the test element forms a complex with the second probe of the labeling reagent through the target substance;
Detecting optical characteristics of the test element forming the composite; and
It is a detection method of the target substance characterized by including.

The second detection method of the present invention comprises:
A method for detecting the presence or amount of a target substance in a sample,
Providing a test element in which a first micro metal structure capable of inducing localized plasmon resonance and a first probe are arranged on the surface of the substrate so as to be isolated from each other; and a second inducing local plasmon resonance A step of preparing a labeling reagent in which a minute metal structure is fixed to the second probe, and a condition in which the target substance and the second probe of the labeling reagent are both present. Each performing a reaction to form a complex with the first probe;
Detecting optical characteristics of the test element,
The method of detecting a target substance, wherein the second probe is a substance that competes with the target substance and is captured by the first probe.

The test kit of the present invention comprises
A test element comprising a substrate on which a first micro metal structure capable of inducing localized plasmon resonance and a first probe that captures a target substance are arranged separated from each other;
A labeling reagent comprising a second fine metal structure capable of inducing localized plasmon resonance and a second probe;
It is a test kit for testing the presence or amount of a target substance characterized by having

  By using the method and test kit of the present invention, it is possible to detect a target substance having a low concentration or a low molecule, which is insufficient for the detection sensitivity of the localized plasmon sensor, with high sensitivity.

  Further, the electric field intensity distribution of the localized plasmon sensor element can be used effectively, and the target substance can be detected with high sensitivity.

  The present invention is a method for detecting the presence or absence or amount of a target substance in a specimen, wherein the first micro metal structure capable of inducing localized plasmon resonance and the first probe are isolated from each other on the surface of the substrate. A step of preparing an arranged test element, a step of preparing a labeling reagent in which a second micro metal structure capable of inducing localized plasmon resonance is fixed to the second probe, and a first arranged in the test element And a step of performing a reaction in which the probe forms a complex with the second probe of the labeling reagent through the target substance, and a step of detecting optical characteristics of the test element that has formed the complex. This is a method for detecting a target substance. The complex formed by the first probe and the second probe via the target substance is preferably formed by the first probe and the second probe spatially capturing different regions of the target substance. Such a configuration includes a case where the target substance is an antigen, the first probe is a primary antibody, and the second probe is a secondary antibody. At this time, the primary antibody and the secondary antibody each capture another region in the antigen. For example, if the antigen region captured by the primary antibody and the antigen region captured by the secondary antibody are different antigenic determinants, the present invention is applicable to the present invention. Further, even when the antigen regions to be captured are the same antigenic determinant, for example, when the antigen forms a multimer, it can be applied to the present invention. In other words, the effect of the present invention can be obtained if the first target substance capturing body and the second target substance capturing body spatially capture different regions of the target substance, not based on antigenic determinants. Become.

  Furthermore, the present invention relates to a method for detecting the presence or absence or amount of a target substance in a specimen, wherein the first micro metal structure capable of inducing localized plasmon resonance and the first probe are isolated from each other and Providing a test element disposed on the substrate, preparing a labeling reagent in which a second fine metal structure capable of inducing localized plasmon resonance is fixed to the second probe, the target substance and the label A step of performing a reaction in which each of the target substance and the second probe forms a complex with the first probe under a condition in which a second probe included in the reagent is present, and detecting optical characteristics of the test element And the second probe is a substance that is captured by the first probe in competition with the target substance. In this embodiment, a target substance can be used as the second probe.

  The micro metal structure provided in the test element is defined as a first micro metal structure, and the micro metal structure provided in the labeling reagent is defined as a second micro metal structure. However, this name is used to distinguish between the test element and the labeling reagent, and does not mean that different micro metal structures are used.

  The present invention is characterized in that, in the method using the above-described test element and labeling reagent, when the second fine metal structure is held on the base of the test element, the direction parallel to the base plane (XY axis) In addition, the first fine metal structure and the second fine metal structure can be close to each other. As a result, a large change occurs in the dielectric constant in the vicinity of the minute metal structure. By measuring this change in the dielectric constant using localized plasmon resonance, the presence or absence of the target substance can be detected with high sensitivity. And

  An outline of an example of the measuring method according to the present invention will be described with reference to the drawings. When using the test element shown in the figure, for example, the detection method of the present invention can be carried out according to the following procedure, but the present invention is not limited to this procedure.

  FIG. 1 illustrates an outline of a measurement method according to the present invention, taking a sandwich assay as an example.

Fig.1 (a) shows the test | inspection element 12 in this embodiment. The inspection element 12 is arranged or randomly arranged on the substrate 4 in a state where the first minute metal structures 1 capable of inducing localized plasmon resonance are separated from each other, and further, the surface of the substrate other than the minute metal structures. The first probes 3 are arranged or randomly arranged. Further, the surface of the substrate 4 and the surface of the first fine metal structure 1 are covered with the nonspecific adsorption inhibitor 2. (Fixing the first probe)
FIG. 1 (b) shows a conceptual diagram when the first probe 3 forms a complex with the second probe 5 via the target substance 6. A second minute metal structure 7 is fixed to the second probe 5.

  FIG. 1 (c) shows a change in the distance between the minute metal structures composed of the first minute metal structure 1 and the second minute metal structure 7 depending on the presence or absence of the target substance 6. When the target substance 6 is contained in the specimen, the first probe 3 and the second probe 5 form a complex via the target substance 6. At this time, as shown in FIG. 1 (c), the second micro metal structure forms a composite, thereby shortening the distance 8 between the micro metal structures. On the other hand, when the target substance 6 is not contained in the specimen, the complex 9 is not formed, and therefore the distance 9 between the minute metal structures does not change.

  That is, the minute metal structure 1 on the inspection element 12 side and the minute metal structure 7 on the target substance 5 side are close to each other, and the minute metal structure 7 is parallel to the plane of the base 4 (XY axis). Due to the proximity, a large change occurs in the dielectric constant in the vicinity of the minute metal structure, and this change in dielectric constant is measured using localized plasmon resonance to detect the presence or absence of the target substance with high sensitivity. Is possible.

  FIG. 2 illustrates an outline of the measurement method according to the present invention, taking a competitive assay as an example.

  FIG. 2A shows the test element 12 in the present embodiment. The inspection element 12 is arranged or randomly arranged on the substrate 4 in a state where the first minute metal structures 1 capable of inducing localized plasmon resonance are separated from each other, and further, the surface of the substrate other than the minute metal structures. The first probes 3 capable of capturing the target substance are arranged or randomly arranged. Further, the surface of the substrate 4 and the surface of the first minute metal structure 1 are covered with the nonspecific adsorption inhibitor 2.

  FIG. 2B shows a conceptual diagram of a reaction in which the target substance 6 and the second probe 5 form a complex with the first probe 3 in a competitive manner. A second minute metal structure 7 is fixed to the second probe. FIG. 2 (c) shows a conceptual diagram of a complex formed by the presence or absence of the target substance 6. When the target substance 6 is contained in the sample, the target substance 6 and the first probe 3 form a complex (11). On the other hand, when the target substance 6 is not contained in the specimen, the second probe 5 forms a complex with the first probe (10).

  That is, according to the present invention, the minute metal structure 1 on the test element 12 side and the minute metal structure 7 on the target substance 5 side are brought close to each other by various reactions such as a sandwich assay and a competitive assay. On the other hand, since the minute metal structure 7 approaches in the parallel (XY axis) direction, a large change occurs in the dielectric constant in the vicinity of the minute metal structure. Therefore, the presence or absence of the target substance can be detected with high sensitivity by measuring this change in dielectric constant using localized plasmon resonance.

  1 and 2 show the measurement method according to the present invention as an example of a sandwich assay and a competitive assay. However, the measurement method is not limited to these two measurement methods, and other hybridization reactions and ligations are also possible. Various reactions such as a reaction and a photocrosslinking reaction can be used.

  Next, the test element and kit of the present invention will be described in detail.

<First probe / second probe / specimen type>
In the present invention, a probe is a reagent used to detect a target substance, and may be a substance that directly captures the target substance (hereinafter referred to as a target substance capturing body) or competes with the target substance. It may be a substance that is captured by the target substance capturing body (also referred to as a capturing body binding substance). Competing means a case where only one substance is bound to one target substance capturing body under the condition that both the target substance and the capturing body binding substance exist. The target substance capturing body is preferably a substance that specifically binds to the target substance.

  A probe included in the test element is referred to as a first probe, and a probe included in the labeling reagent is referred to as a second probe. The first probe and the second probe may be different materials or the same material. As the first probe, a target substance capturing body is preferable. As the second probe, a target substance capturing body is preferable in the sandwich assay, and a capturing body binding substance is preferable in the competitive assay.

  Examples of the first and second probes include nucleic acids, proteins, peptides, amino acids, sugars, cells, antibodies, antigens, enzymes, receptors, environmental hormones, and the like. Moreover, as long as it is a nucleic acid, any of PNA, DNA, and RNA can be used, and also single-stranded and double-stranded nucleic acids can be used.

  In addition, the specimen to which the detection method of the present invention can be applied is not particularly limited as long as it may contain a target substance, and examples thereof include biological samples or environmental samples. it can. Biological samples include, for example, animal body fluids (eg, blood, serum, plasma, sputum, sweat, saliva, urine, etc.) or hair, excreta, organs, tissues, or animals and plants themselves or their A dry body etc. can be mentioned. Examples of the sample derived from the environment include river water, lake water, seawater, and soil.

<Micro metal structure>
The shape of the fine metal structure may be anything as long as it can perform measurement using localized plasmon resonance. For example, a spherical shape, a rod shape, a needle shape, a hollow element, a layered structure of different metals, a dielectric material, The layered structure, tube shape or the like can be used. Moreover, as long as localized plasmon resonance measurement can be performed, for example, it may have irregularities and protrusions.

  The material constituting the fine metal structure may be any metal element that can cause localized plasmon resonance, and is not limited thereto. Among them, gold, silver, copper, platinum, aluminum, or alloys thereof Etc. are preferable.

  In the present invention, the micro metal structure included in the test element is the first micro metal structure, and the micro metal structure included in the labeling reagent is the second micro metal structure.

  The first and second fine metal structures according to the present invention may be of any size as long as they can induce localized plasmon resonance, but may be in the range of 5 nm to 1450 nm. preferable.

<Inspection element>
As used in the present invention, the inspection element in which the first minute metal structures are separated from each other and arranged on the surface of the substrate means an optical device capable of inducing localized plasmon resonance. FIG. 4 shows a conceptual diagram of the test element 12 used in this embodiment. The test element 12 according to the present embodiment includes a first probe 3 that can form a complex with a target substance and a second probe, a first minute metal structure 1 that can induce localized plasmon resonance, and a substrate 4. Has been.

  The shape and material of the substrate 4 are not particularly limited as long as localized plasmon resonance measurement can be performed. For example, a shape selected from a planar structure, a porous structure, a fine particle aggregate, a columnar structure, a hollow column structure, a convex structure, a concave structure, a protruding structure, a fibrous structure, etc. Also good. Examples of the material constituting the base include glass and plastic.

  The fine metal structure according to the present invention is preferably arranged on the substrate so that the metal structures are separated from each other, more preferably 5 nm to 2000 nm.

  Moreover, when manufacturing the first fine metal structure, a fine processing technique or the like can be used. For example, a metal thin film is formed on the substrate by vacuum deposition or sputtering, and an electron beam resist is formed thereon by spin coating. Thereafter, the first fine metal structure can be formed on the substrate by exposure with an electron beam drawing apparatus, development, etching, and the like. Further, when forming a fine metal structure from a metal thin film, various fine processing techniques such as a focused ion beam processing apparatus, an X-ray exposure apparatus or an ultraviolet exposure apparatus can be used in addition to the electron beam drawing apparatus.

  For immobilization of the first probe on the substrate surface and immobilization of the second probe on the second micrometal structure, a known oligonucleotide, antigen, antibody, protein immobilization method or the like can be used. For example, when the first probe is immobilized on a substrate, various first probes are immobilized by an adsorption method, a coupling reaction, or the like by previously amination, carboxylation, or thiolation of the substrate surface with a silane agent or the like. be able to.

  When fixing the first probe to the base, it is necessary to prevent the first probe from being arranged on the fine metal structure provided on the base. Therefore, when the first probe is fixed before the minute metal structure is arranged, it is preferable that the first probe is not coupled to the position where the minute metal structure is arranged. In the case where the minute metal structure is provided before the first probe is fixed to the substrate, the first probe is not bonded to the minute metal structure, and the treatment may be performed only on the substrate surface. preferable.

  Moreover, when immobilizing a 2nd probe to a micro metal structure, the method using the organic substance which has a sulfur atom covalently couple | bonds with metal surfaces, such as gold | metal | money, via a sulfur atom is mentioned. In this case, the second probe can be fixed by introducing a thiol group into the second probe in advance and mixing it with the fine metal structure. Furthermore, a method in which streptavidin is previously adsorbed on a fine metal structure or substrate and a biotin-modified probe is selectively bound to streptavidin and immobilized can be used.

  For the dropping of the immobilization solution, an apparatus (for example, a DNA array, an ink jet apparatus, etc.) having a nozzle having a position control function capable of dropping a reagent only on a predetermined minute metal structure may be used. .

<Reagent for labeling>
The labeling reagent has a second probe and a second minute metal structure. The second probe is preferably fixed to the second fine metal structure.

  In the case of using a labeling reagent having a capturing body binding substance as the second probe, the capturing body binding substance competes with the target substance by mixing the labeling reagent with the sample liquid and bringing it into contact with the testing element. As the amount of the target substance in the specimen increases, the number of second fine metal structures held by the test element decreases. Further, the capturing substance binding substance may be the same as the target substance.

  On the other hand, a case where a complex of a probe (target substance capturing body) that can bind to a target substance and a second micro metal structure is used as a labeling reagent will be described. By using the labeling reagent having this configuration, the first probe performs a reaction to form a complex with the second probe via the target substance, whereby the second micro metal structure is held on the test element. Therefore, as the amount of the target substance in the specimen increases, the number of second fine metal structures held by the test element increases. Examples of the reaction step for forming the complex include the following three methods. In the first method, the sample solution and the test element are brought into contact with each other, thereby bringing the first probe on the test element into contact with the target substance, and the test element holding the target substance and the labeling reagent are brought into contact with each other. And a step of binding the second probe to the target substance bound to the first probe on the test element. In the second method, the first probe has a step of forming a complex with the second probe via the target substance by mixing the sample liquid and the labeling reagent and bringing them into contact with the test element. In the third method, a sample liquid and a labeling reagent are mixed to form a complex of the second minute metal structure and the target substance via the second probe of the labeling reagent; The step of separating the complex of the micro metal structure of 2 and the target substance, and contacting the analyte element with the complex of the second micro metal structure and the target substance, and the first probe causes the target substance to And forming a complex with the second probe.

  As described above, in any configuration of the labeling reagent, the probe included in the test element used in the present invention is fixed to the substrate surface excluding the first minute metal structure, so that the second minute The metal structure is close to the first minute metal structure in the direction parallel to the substrate plane (XY axis). Therefore, even if any of the above-described labeling reagents is used, the effect of the present invention is obtained in that the change in the dielectric constant in the vicinity of the fine metal structure is further changed. As a result, the presence or absence of the target substance can be detected with high sensitivity by measuring this change in dielectric constant using localized plasmon resonance.

<Inspection kit>
Next, the test kit of the present invention and this embodiment will be described.

The test kit of the present invention comprises
(1) A labeling reagent (2) is provided with at least a test element.

  The test element provided in the test kit of the present invention is the same as the test element disclosed in the detection method of the present invention. Here, the labeling reagent includes at least one second probe and at least one second micro metal structure. The reagent may be in a dry state or a solution state, and a reaction promoting reagent or the like may be added thereto.

  As one aspect of the test kit, a test element comprising a substrate in which a first micro metal structure capable of inducing localized plasmon resonance and a first probe for capturing a target substance are arranged separately from each other, It can be set as the structure which has a labeling reagent provided with the 2nd micro metal structure which can induce plasmon resonance, and a 2nd probe. As a combination of the first and second probes provided in this case, one in which the first probe and the second probe capture spatially different regions of the target substance may be used, or the second probe may be a target A substance that is captured by the first probe in competition with the substance can be used.

  In the test kit of the present invention, the first micro metal structure 1 and the second micro metal structure 7 are brought into close proximity by reaction with the target substance 6 and are further parallel to the plane of the substrate 4 (XY axis). Since the minute metal structure 7 is close to the direction, a large change occurs in the dielectric constant in the vicinity of the minute metal structure, and the change in the dielectric constant is measured using localized plasmon resonance. This is a test kit that detects the presence or absence with high sensitivity.

  Examples of the present invention will be described below.

Example 1
In this embodiment, a 96-well plate is used as a base, gold fine particles are fixed on the base as a first fine metal structure, and a first probe is fixed on the surface of the base to produce a test element. This is an example of a sandwich assay in which the target substance is reacted using, finally labeled with a second probe, and the presence or amount of the target substance is detected by local resonance plasmon measurement.

<Production of test element>
A 96-well aminated plate (manufactured by Sumitomo Bakelite) was introduced with a solution of gold fine particles having an average particle size of 100 nm (manufactured by Tanaka Kikinzoku Co., Ltd.) diluted to 30% with pure water and immersed in pure water for 24 hours. After cleaning in step 1, the substrate having the first fine metal structure on the surface is manufactured by drying with nitrogen gas. Thereafter, 100 μl each of 5 mg / ml PEG-SH reagent (average molecular weight 5000, manufactured by NOF Corporation) is added to each well of the plate and reacted at 30 ° C. for 2 hours. A non-specific adsorption prevention film is formed.

  Next, 100 μl of 10 μg / ml Anti-Rabbit IgG F (c) (manufactured by ROCKLAND) was added to each well of the plate and reacted overnight at 4 ° C. to immobilize on the substrate surface. A probe is used. The first probe is not disposed on the fine metal structure, but is disposed only on the surface of the substrate.

  Thereafter, 100 μl of 1% casein (manufactured by Technochemical) is added to each well of the plate and reacted at 30 ° C. for 2 hours to form a nonspecific adsorption preventing film on the substrate surface.

<Preparation of labeling reagent>
18 ml of a solution of gold fine particles having an average particle diameter of 100 nm (Tanaka Kikinzoku Co., Ltd.) is collected, 2 ml of a 100 mg / ml BSA solution is added, and then reacted at 30 ° C. for 2 hours to adsorb BSA to the gold fine particles.

  Next, the metal fine particle solution is centrifuged at 4000 g for 30 minutes, and the solution is replaced with 20 ml of 2 mM Borax solution.

Thereafter, centrifugation is again performed at 4000 g for 30 minutes, the solution is replaced with 10 ml of a phosphate buffer, and the BSA-adsorbed gold fine particles are used as a labeling reagent solution. <Detection reaction>
Anti-BSA (manufactured by ROCKLAND) was used as a target substance, a diluted solution of Anti-BSA (1 × 10 ⁻³ to 10 ⁻¹³ g / ml) was prepared, and each well of the test element (the 96-well plate) was adjusted. Add 100 μl each. Then, it is made to react at 37 degreeC for 2 hours, and is made to react with a 1st probe.

  Next, 100 μl of the labeling reagent solution is added to each well and reacted at 37 ° C. for 2 hours to form a complex with the first probe and the target substance.

<Localized plasmon resonance sensor not using the second fine metal structure (conventional method)>
A conventional test element is manufactured by the following procedure.

  First, a 96-hole amination plate (manufactured by Sumitomo Bakelite) was introduced with a solution of gold fine particles having an average particle size of 100 nm (Tanaka Kikinzoku Co., Ltd.) diluted to 30% with pure water, and immersed for 24 hours at room temperature. After washing with pure water, the substrate having the first fine metal structure on the surface is produced by drying with nitrogen gas.

  Next, 100 μl of 10 μg / ml Anti-Rabbit IgG F (c) (manufactured by ROCKLAND) was added to each well of the plate and reacted overnight at 4 ° C. to immobilize on the substrate surface. A probe is used. The first probe is disposed on the substrate surface and the fine metal structure.

  Thereafter, 100 μl of 1% casein (manufactured by Technochemical) is added to each well of the plate and reacted at 30 ° C. for 2 hours to form a nonspecific adsorption preventing film on the substrate surface.

Anti-BSA (manufactured by ROCKLAND) was used as a target substance, a diluted solution of Anti-BSA (1 × 10 ⁻³ to 10 ⁻¹³ g / ml) was prepared, and each well of the test element (the 96-well plate) was adjusted. Add 100 μl each. Then, it is made to react at 37 degreeC for 2 hours, and is made to react with the 1st probe arrange | positioned on the base-material surface and a micro metal structure.

<Detection of optical characteristics>
Next, an example of the detection device will be described. The present embodiment is an example in which detection is performed by light transmitted through a test element.

  FIG. 3A is a diagram schematically showing a detection method according to this embodiment. A detection device (not shown) includes an inspection chip holding mechanism, a light source, and a light receiving element.

  As schematically shown in FIG. 3A, the position of the light source 13 at the time of detection is at a position where the measurement light 14 can be applied to the inspection chip, and the position of the light receiving element 15 is the measurement light transmitted through the inspection element 12. It is in a position where 14 characteristics can be detected. In addition to this, a spectral detector (not shown) may be provided in the light receiving element. Furthermore, although not shown, it is preferable that an arithmetic device for calculating the detected characteristic change, a display means for displaying the detection result, and the like are provided.

  Next, an example of detecting Anti-BSA will be described.

  First, the detection reaction is performed using the inspection element, and the inspection element, the light source, and the light receiving element are arranged as described above, and a spectrum composed of the first and second fine metal structures is detected.

  The change in the spectrum due to the detection reaction originates from the change in the localized surface plasmon resonance state, which means that the second micro metal structure is indirectly bound on the test element. Therefore, it is possible to detect the presence and amount of Anti-BSA in the specimen by detecting the change in the spectrum.

  FIG. 5 shows the calibration curve 17 of Anti-BSA obtained in Example 1. As a comparison, an anti-BSA calibration curve 18 obtained by a localized plasmon resonance sensor (conventional method) that does not use the second fine metal structure is shown. It can be seen that the detection sensitivity of the present invention is improved as compared with the conventional method.

  Although described as a change in spectrum here, this change in spectrum may be a change in spectrum peak at a wavelength having the maximum value, or a change in peak shape such as a half-value width of a spectrum peak waveform. Furthermore, the light intensity at one or a plurality of wavelength points may be used.

(Example 2)
In this embodiment, a quartz substrate is used as a substrate, a gold dot pattern is formed on the substrate as a first fine metal structure, and a probe for capturing a target substance is immobilized on the substrate surface to produce a test element. This is an example of a competitive assay in which a competitive reaction with a target substance is carried out using this test element and a probe binding substance, and the presence or absence or amount of the target substance is detected by local plasmon resonance measurement.

<Production of test element>
On a quartz wafer (72 × 26 × 0.5 mm, manufactured by Shin-Etsu), a gold dot pattern (dot size: average major axis length 36.6 nm, average minor axis length 7.2 nm, An average aspect ratio of 5.0 and an average dot interval of 500 nm are formed, and this gold dot is used as a first fine metal structure.

  Next, the surface of the substrate is aminated using a silane coupling agent having an amino group. Further, 100 μl of 5 mg / ml PEG-SH reagent (molecular weight: 5000, manufactured by NOF Corporation) was added to the substrate and reacted at 30 ° C. for 2 hours to form a nonspecific adsorption preventing film on the surface of the first fine metal structure. Let it form.

  Next, 100 μl of 10 μg / ml Anti-Rabbit IgG F (c) (manufactured by ROCKLAND) is added to the substrate, reacted at 4 ° C. overnight, immobilized on the substrate surface, and the antibody is captured. This is the first probe. The first probe is not fixed to the surface of the fine metal structure, but is disposed only on the surface of the base.

  Thereafter, 100 μl of 1% casein (manufactured by Techno Chemical) is added to each well of the substrate and reacted at 30 ° C. for 2 hours to form a nonspecific adsorption preventing film on the substrate surface.

<Preparation of labeling reagent>
18 ml of a gold nanorod solution (rod shape: average major axis length 36.6 nm, average minor axis length 7.2 nm, average aspect ratio 5.0, manufactured by Mitsubishi Materials) was taken as a second probe, 100 μg / After adding 2 ml of ml of Anti-BSA solution (manufactured by ROCKLAND), the mixture is reacted at 30 ° C. for 2 hours. Next, the mixture is centrifuged at 4000 G for 30 minutes, and the solution is replaced with 20 ml of 2 mM Borax. Thereafter, centrifugation is again performed at 4000 g for 30 minutes, the solution is replaced with 10 ml of a phosphate buffer, and this Anti-BSA-immobilized gold nanorod is used as a labeling reagent. In this embodiment, the same substance as the target substance is used as the second probe.

<Detection reaction>
Prepare a diluted solution (1 × 10 ⁻³ to 10 ⁻¹³ g / ml) of Anti-BSA (manufactured by ROCKLAND), which is the target substance. Next, 50 μl of the diluted Anti-BSA solution is added to each well of the test element, and then 50 μl of the labeling reagent solution is added to each well of the test element. A competitive reaction is performed between the second probe provided in the labeling reagent and the target substance at 37 ° C. for 2 hours.

<Detection of optical characteristics>
Next, an example of the detection device will be described. The present embodiment is an example in which detection is performed by light reflected from the inspection element.

  FIG. 3B is a diagram schematically showing the detection method according to this embodiment. A detection device (not shown) includes an inspection chip holding mechanism, a light source, and a light receiving element.

  As shown schematically in FIG. 3B, the position of the light source 13 at the time of detection is at a position where the measurement light 16 can be irradiated to the inspection chip, and the position of the light receiving element 15 is the measurement light reflected from the inspection element 12. It is in a position where 16 characteristics can be detected. In addition to this, a spectral detector (not shown) may be provided in the light receiving element. Furthermore, although not shown, it is preferable that an arithmetic device for calculating the detected characteristic change, a display means for displaying the detection result, and the like are provided.

  Next, an example of detecting Anti-BSA will be described.

  First, the detection reaction is performed using the inspection element, and the inspection element, the light source, and the light receiving element are arranged as described above, and the spectra of the first and second fine metal structures are detected.

  The change in the spectrum due to the detection reaction originates from the change in the localized surface plasmon resonance state, which means that the second micro metal structure is indirectly bound on the test element. Therefore, it is possible to detect the presence and amount of Anti-BSA in the specimen by detecting the change in the spectrum.

Also in this embodiment, Anti-BSA can be detected with higher sensitivity than in the conventional method.
Although described as a change in spectrum here, this change in spectrum may be a change in spectrum peak at a wavelength having the maximum value, or a change in peak shape such as a half-value width of a spectrum peak waveform. Furthermore, the light intensity at one or a plurality of wavelength points may be used.

It is a conceptual diagram of the test | inspection method (sandwich assay) concerning one Embodiment of this invention. It is a conceptual diagram of the test | inspection method (competition assay) concerning one Embodiment of this invention. It is a conceptual diagram of the detection principle using the localized plasmon resonance in the test | inspection chip concerning this invention. (A) Transmitted light type (b) Reflected light type It is a conceptual diagram of the test | inspection element concerning this invention. It is an inspection example of Anti-BSA using the test | inspection element concerning this invention.

Explanation of symbols

1. First fine metal structure 2. 2. Non-specific adsorption inhibitor First probe 4. Substrate 5. Second probe 6. 6. Target substance Second fine metal structure 8. 8. Distance between minute metal structures when target substance is bonded 9. Distance between minute metal structures when target substance is not bound 10. a complex of the second probe and the first probe in which the second minute metal structure 7 is modified; 11. Complex of target substance and first probe Test element 13. Light source 14. Measuring light (after passing through the inspection element)
15. Light receiving element 16. Measuring light (after inspection element reflection)
17. Anti-BSA calibration curve according to the invention 18. Anti-BSA calibration curve by conventional method (unlabeled LSPR measurement)

Claims (7)

A method for detecting the presence or amount of a target substance in a sample,
Providing a test element in which a first micro metal structure capable of inducing localized plasmon resonance and a first probe are isolated from each other and disposed on a substrate surface;
Providing a labeling reagent in which a second fine metal structure capable of inducing localized plasmon resonance is fixed to the second probe;
Performing a reaction in which the first probe arranged in the test element forms a complex with the second probe of the labeling reagent through the target substance;
Detecting optical characteristics of the test element forming the composite; and
A method for detecting a target substance, comprising:   The detection method according to claim 1, wherein the complex is formed by spatially capturing different regions of the target substance by the first probe and the second probe. A method for detecting the presence or amount of a target substance in a sample,
Providing a test element in which a first micro metal structure capable of inducing localized plasmon resonance and a first probe are isolated from each other and disposed on a substrate surface;
Providing a labeling reagent in which a second fine metal structure capable of inducing localized plasmon resonance is fixed to the second probe;
Performing a reaction in which each of the target substance and the second probe forms a complex with the first probe under a condition where both the target substance and the second probe of the labeling reagent exist;
Detecting optical characteristics of the test element,
The method for detecting a target substance, wherein the second probe is a substance that competes with the target substance and is captured by the first probe.   The detection method according to claim 3, wherein the target substance is used as the second probe. A test element comprising a substrate on which a first micro metal structure capable of inducing localized plasmon resonance and a first probe that captures a target substance are arranged separated from each other;
A labeling reagent comprising a second fine metal structure capable of inducing localized plasmon resonance and a second probe;
An inspection kit for inspecting the presence or amount of a target substance characterized by comprising:   The test kit according to claim 5, wherein the first probe and the second probe spatially capture different regions of the target substance.   The test kit according to claim 5, wherein the second probe is a substance that competes with the target substance and is captured by the first probe.