Substance detection device

Abstract

Provided is an easily-evaluated, immunochromatographic detection device 100 that detects a target substance 1 that may be included in a sample fluid 10. The detection device comprises: a flow path 101 for the sample fluid; and detection parts 106, 108 to which a capture substance 6, 8 is fixed. In one embodiment, when the target substance is included in the sample fluid, an MIM structure is formed at the detection parts, and a color is produced at the detection parts by resonance absorption. The MIM structure comprises a metal thin film 7 (an M layer) that is formed on a support 9, either a capture substance / target substance / standard substance composite layer or a capture substance / standard substance composite layer (an I layer), and metal fine particles 4 (an M layer) with which a standard substance 2 is labeled.

Description

[Technical Field]

[0001] The present disclosure relates to a substance detection device, and more particularly to a substance detection device that utilizes the resonance absorption phenomenon resulting from a light-absorbing nanostructure. [Background technology]

[0002] An antigen detection method known as "immunochromatography" has been developed that uses a standard antibody that specifically binds to a specific antigen and is labeled with gold colloid, enzymes, or fluorescent molecules, and a capture antibody that binds to the standard antibody (for example, Patent Document 1). Recently, a test kit for antibodies to the novel coronavirus (SARS-CoV-2), the virus that causes novel coronavirus disease (COVID-19), has also been developed (for example, Non-Patent Document 1).

[0003] On the other hand, the present inventor has developed a visible light absorption element (Patent Document 2), and has also disclosed a plasmon resonance measurement system (Patent Document 3).Furthermore, it has been disclosed that Si nanoparticles can independently become a color-developing body (Non-Patent Document 2). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2012 / 043746 [Patent Document 2] International Publication No. 2016 / 132979 [Patent Document 3] Japanese Patent Application Publication No. 2019-219272 [Non-patent literature]

[0005] [Non-Patent Document 1] "Fujifilm begins development of a highly sensitive and rapid COVID-19 antigen test kit using silver halide amplification technology from photographic development," Innervision Co., Ltd., October 5, 2020, https: / / www.innervision.co.jp / sp / products / release / 20201115, [online] Last searched: December 2, 2020 [Non-patent document 2] Yusuke Nagasaki, Masafumi Suzuki, and Junichi Takahara, "All-Dielectric Dual-Color Pixel with Subwavelength Resolution", Nano Letters 17, pp.7500-7506 (2017); DOI: 10.1021 / acs.nanolett.7b03421 Summary of the Invention [Problem to be solved by the invention]

[0006] In conventional immunochromatographic test kits, those labeled with fluorescent molecules require specialized light sources and imaging equipment to observe the fluorescence. In immunochromatographic kits labeled with colloidal gold, the pink coloring derived from the colloidal gold is observed visually. Even in this case, when the amount of antibody or other substance is small, a scanner is required to capture the faint coloration with high sensitivity, making it difficult for general users to easily use the test kit at home. In response to this, a method has been proposed to enhance the faint coloring of colloidal gold by reducing silver ions on the surface of gold nanoparticles, thereby amplifying the size of the gold particles by approximately 100 times, thereby improving visual detection ability (silver-amplified immunochromatography, Non-Patent Document 1). However, this silver ion sensitization process also requires specialized equipment, which makes it difficult for general users to easily use the test kit.

[0007] The present disclosure aims to solve any of the above problems, and by providing a detection device that can be easily used by general users, contributes to the spread of testing technology based on determination of the presence or absence of a substance to be detected. [Means for solving the problem]

[0008] The present inventors have conceived a new idea based on an antigen-antibody reaction, a type of physicochemical bond, that utilizes visible color development to enable detection of target substances. Specifically, the inventors combine the structure of a color-developing body using a light-absorbing nanostructure with immunochromatography. By utilizing the color development phenomenon resulting from the light-absorbing nanostructure, it is possible to display the presence or absence of a target substance (e.g., an antigen) in a sample as a color change that can be detected more sensitively by the naked eye. The inventors have discovered that a detection device based on this method can greatly enhance the practicality of tests that utilize antigen-antibody reactions, etc., and have completed the invention of this application.

[0009] That is, in one embodiment of the present disclosure, there is provided a detection device for detecting a target substance that may be contained in a sample fluid, the detection device comprising: a flow path for the sample fluid in which a standard substance capable of physicochemically binding to the target substance is dispersed; and at least one visible detection unit arranged in the flow path and having a capture substance fixed to at least a portion of the contact surface with the sample fluid, wherein the standard substance is labeled with microparticles, the capture substance is capable of physicochemically binding to at least either the target substance bound to the standard substance or the standard substance, and the microparticles form aggregates along the extent of the contact surface and develop color due to the target substance bound to the standard substance or the standard substance binding physicochemically to the capture substance.

[0010] In this application, the detectable substance is typically a molecule such as a virus protein or an antigen, and another typical example is an antibody. If the detectable substance is a molecule such as a virus protein or an antigen, the standard substance and capture substance that physicochemically bind to the detectable substance are typically antibodies. Furthermore, if the detectable substance is an antibody, the standard substance and capture substance that physicochemically bind to the detectable substance are typically antigens. Furthermore, in the description of this application, color change includes any optical change that can be detected visually by a person with normal color vision, such as a change in hue, brightness, or transmittance or reflectance, and also includes the visibly emergence of some pattern or mark. Therefore, color development and coloration also include the response of an object to visible light that can at least result in some kind of visual stimulus to the observer or modulation of the stimulus amount, and this response includes any response that changes at least one of brightness, hue, and saturation. The description of antigen-antibody reactions in this disclosure is primarily based on the case where a sample fluid is used as the specimen (analyte) and the antigen contained therein is the detection target. However, as will be apparent to those skilled in the art, the antigen-antibody reaction described in this disclosure can also be achieved by switching the antigen and antibody, and therefore the content of this disclosure also includes cases where an antibody is the detection target. Physicochemical binding is typically a selective binding reaction that has specificity for a combination of substances, such as an antigen-antibody reaction, which is an immunological reaction.

[0011] In this disclosure, microparticles refer to nanoparticles, primarily nanometer-sized particles, made of a material that can behave as a dielectric or metal in the electric or magnetic field of electromagnetic waves in the wavelength range included in visible light. A typical particle size is approximately 400 nm or less. When such microparticles form aggregates, they exhibit physico-optical effects. The physico-optical effects exhibited by these aggregates of microparticles differ somewhat from the physico-optical effects exhibited by the particles themselves when the microparticles are not aggregated. In this disclosure, an MIM structure has at least three metal-dielectric-metal (metal-insulator-metal) layers, with the dielectric layer being made of a material and thick enough to transmit electromagnetic waves in the visible range to at least some degree. It is not necessary for all layers in an MIM structure to be considered films or thin films in the usual sense. For example, this disclosure describes a structure in which one of the M layers in an MIM structure is composed of an aggregate of metal microparticles. In this disclosure, the optical changes detected, the device structure, and the function may be described using technical terms adapted or borrowed from any technical field that describes changes in hue or brightness in visible light. [Effects of the Invention]

[0012] In the detection device provided in any of the aspects of the present disclosure, the presence of antigens, including molecules such as viral proteins, can be indicated by a color change on the device. In any of the aspects of the present disclosure, simply by dropping a specimen sample onto the detection device, it becomes possible to quickly and easily determine or estimate the presence of the target antigen in the specimen by visually observing the color change on the device. [Brief explanation of the drawings]

[0013] [Figure 1A-B] FIG. 1A is a schematic cross-sectional view illustrating the principle of a conventional immunochromatography method using a gold colloid label, and FIG. 1B is a schematic plan view thereof. [Figure 2] FIG. 2 is an explanatory diagram illustrating the basic principle of an improved immunochromatography method employing a configuration utilizing an MIM structure in an embodiment of the present disclosure. [Figure 3] FIG. 3 is an explanatory diagram for explaining the resonance phenomenon in a MIM structure in a general case. [Figure 4] FIG. 4 is an explanatory diagram illustrating the basic principle of an improved immunochromatography method that employs color development by an aggregate of fine particles in an embodiment of the present disclosure. [Figure 5A-E] Figures 5A-E are a schematic diagram showing the overall structure of a detection device in an embodiment of the present disclosure (Figure 5A), an intermediate enlarged view of the test section located midway through the flow path (Figure 5B), a detailed enlarged view showing the layer structure in that island-shaped section (Figure 5C), an intermediate enlarged view of the control section located midway through the flow path (Figure 5D), and a detailed enlarged view showing the layer structure in that island-shaped section (Figure 5E). [Figure 6] FIG. 6 is a cross-sectional view illustrating the island configuration shown in FIGS. 5B and 4C in accordance with an embodiment of the present disclosure. [Figure 7] FIG. 7 is a cross-sectional view of a sensing device utilizing a transparent dielectric thin film in an embodiment of the present disclosure. [Figure 8] FIG. 8 is a cross-sectional view of another detection device according to an embodiment of the present disclosure. [Figure 9] FIG. 9 is a cross-sectional view of yet another detection device according to an embodiment of the present disclosure. [Figure 10A-D] Figure 10A is a schematic cross-sectional view illustrating the conditions for experimentally verifying the operating principle of a detection device according to an embodiment of the present disclosure, Figure 10B is an SEM photograph of the gold pattern that was actually formed, and Figures 10C and 10D are optical microscope images of the sample fluid before and after it was dripped. DETAILED DESCRIPTION OF THE INVENTION

[0014] The detection device according to the present disclosure will be described below. In the following description, in order to clearly explain the invention, the sample fluid is primarily referred to as a specimen (sample), and the antigen contained therein is primarily considered as the detection target. However, as will be apparent to those skilled in the art, the antigen-antibody reaction described in the present disclosure can also be achieved by swapping the antigen and antibody. It can also be applied to molecules that recognize and bind to each other. Furthermore, unless otherwise specified in the description, common parts or elements are designated by common reference symbols. Furthermore, in the drawings, the elements of each embodiment are not necessarily shown to scale.

[0015] 1. Concept The detection device provided in the present disclosure will be compared with a conventional immunochromatography method using a gold colloid label. Figure 1A is a schematic cross-sectional view illustrating the principle of a conventional immunochromatography method using a gold colloid label, and Figure 1B is a schematic plan view thereof. In this conventional method, a standard antibody 602 labeled with gold nanoparticles 604 is detachably positioned in advance at a position (not shown) where it will come into contact with a liquid specimen sample. This position may be, for example, a position where the specimen is dropped or an adjacent position accessible to the specimen from that position, and is called a conjugate portion. When gold nanoparticles 604 are dispersed alone in a medium such as water, they become colloidal, and even if the standard antibody 602 is labeled, they have the same properties. The specimen sample that comes into contact with the standard antibody 602 passes through a substrate 609 such as a cellulose membrane by diffusion, permeation, capillary action, or other means, while dispersing the gold nanoparticle-labeled standard antibody 602 in a colloidal state. The substrate forming the flow path for the sample serves as a chromatographic medium, on which capture antibodies A606 and B608 are arranged at intervals. The positions of capture antibodies A606 and B608, respectively, form the test section and control section. In many examples, the test section and control section are arranged in this order on the upstream and downstream sides, forming a visible line across the flow path through which the sample passes.

[0016] In conventional immunochromatography using gold nanoparticle labels, in a sample containing a target antigen 601, the antigen binds to a standard antibody 602 labeled with gold nanoparticles 604 as a result of an antigen-antibody reaction, and the antigen is dispersed throughout the sample. In contrast, in a sample lacking the target antigen 601, no antigen-antibody reaction occurs, and the standard antibody 602 labeled with gold nanoparticles 604 disperses without binding to the antigen 601. If the sample contains only a small amount of antigen 601, the excess standard antibody 602 also disperses without binding to the antigen 601. The sample passes through a flow path containing capture antibody A 606 in the test section and capture antibody B 608 in the control section, in that order, due to diffusion, permeation, capillary action, and other factors. The antigen portion of the antigen 601-standard antibody 602 complex in the sample binds to capture antibody A 606 in the upstream test section through an antigen-antibody reaction. In contrast, the standard antibody that has not bound to the antigen in the sample can bind to capture antibody B608 placed in the downstream control section through an antigen-antibody reaction, but cannot bind to capture antibody A606 located upstream. Because standard antibody 602 is labeled with gold nanoparticles 604, it turns pink, reflecting the absorption of green light associated with surface plasmon resonance.

[0017] In the conventional immunochromatography method using gold nanoparticle labels, if the downstream control section (capture antibody B608) turns pink, but the upstream test section (capture antibody A606) remains colorless, it is determined that the antigen 601 is not present in the sample. If two pink bands appear, one in the test section and one in the control section, it is determined that the antigen 601 is present (positive). If only the test section turns colored, or if both the control section and the test section are colorless, the test is a failure. In either case, the pink coloration is a phenomenon that occurs due to the surface plasmon resonance of the individual gold nanoparticles 604 themselves.

[0018] FIG. 2 is an explanatory diagram illustrating the basic principle of an improved immunochromatography method employing a configuration utilizing an MIM structure in this embodiment. This disclosure utilizes a color development phenomenon in which the color of particles changes at least from the color of the particles themselves due to their aggregated nature. Here, the basic principle is explained using a metal-(transparent) dielectric-metal (Metal-Insulator-Metal) structure, in which one metal layer is composed of an aggregate of metal particles. That is, in a typical detection device 100 of this embodiment, a metal thin film 7 is formed in advance on a substrate 9 disposed in a flow path 101 through which a sample passes. This flow path 101 can be any flow path through which a sample fluid 12 can develop in, for example, a chromatographic medium, but the formation of the metal thin film 7 may change its properties. The test section 106 and control section develop color; only the test section 106 is shown in FIG. 2. The surface of the metal thin film 7, or the surface of the capture antibody along that surface, serves as the contact surface with the sample fluid 12. In this disclosure, the test section 106 and the control section are sometimes collectively referred to as the detection section. In the test section 106, the antigen 1 already bound to the standard antibody 2 in the sample fluid 12 through an antigen-antibody reaction (antigen-antibody interaction) binds to the capture antibody 6 located there through a further antigen-antibody reaction. As a result, the expanding metal thin film 7 and an assembly of appropriate metal microparticles 4 labeling the standard antibody 2 form an MIM structure, sandwiching a composite layer of standard antibody 2, antigen 1, and capture antibody 6 between them. The assembly of metal microparticles 4 is formed along the expansion of the contact surface. Here, the layer corresponding to the dielectric located between the two metal layers is a single or multilayer layer with weak absorption and light transmission, referred to as a transparent dielectric layer. In the test section 106, a color corresponding to the absorption characteristics of the MIM structure can be visually observed. On the other hand, the standard antibody 2 that has not undergone an antigen-antibody reaction with the antigen 1 is not captured by the capture antibody 6 in the test area 106 because it does not bind to the antigen 1, and therefore does not form an MIM structure in the test area 106. In this case, the test area 106 remains the background color of the state in which the metal thin film 7 is formed on the substrate 9.In this embodiment, this may be expressed as not developing color or not showing color. In a typical example, once an MIM structure is formed, for example, the capture antibodies 6 are patterned to form island-like portions 107, and the wavelength characteristics of the resonance absorption, i.e., the color tone, are determined by the shape, arrangement, and size of the pattern. This is because the optical properties exhibited by the aggregate of metal microparticles 4 change through the patterning of the capture antibodies 6.

[0019] That is, the detection device of the present disclosure employing an MIM structure has the advantage that this absorption characteristic can be artificially changed by designing the shape, arrangement, and size of the pattern, as well as the thickness and dielectric properties of the dielectric layer. Figure 3 is an explanatory diagram for explaining the resonance phenomenon in an MIM structure in a general case. When the MIM structure is irradiated with indoor or other illumination light 200, free electron oscillations are induced in the upper metal structure 804 in response to the electromagnetic waves, and mirror-image free electron oscillations are also induced in the lower metal thin film 807. Each electron oscillation radiates electromagnetic waves, but by adjusting the thickness and properties of the transparent dielectric layer 805, the radiated waves can be made to cancel each other out in the reflection direction toward the top of the paper, i.e., to cause destructive interference in the electromagnetic waves. Meanwhile, illumination light 200 cannot pass through the lower metal thin film 807. The energy of light that cannot be reflected or transmitted is trapped between the two metal layers and ultimately absorbed by the metal of the upper metal structure 804 or the lower metal thin film 807, where it is converted into heat. This phenomenon is wavelength-dependent because it utilizes wave interference. In this way, the MIM structure can function as a light absorber, and its wavelength dependence can be adjusted by its geometric shape and material properties. The geometric shape that determines the wavelength dependence typically includes the shape and size of the overlapping portion between the upper metal structure 804 and the lower metal thin film 807. If the upper metal structure 804 has a repeating island pattern, such as the island-shaped portion 107 in FIG. 2, the geometric shape also includes the distance between the islands and the thickness of the transparent dielectric layer 805. Furthermore, the material properties that determine the wavelength dependence include the frequency response (dielectric function) of the dielectric properties of the upper metal structure 804, the lower metal thin film 807, and the transparent dielectric layer 805. The inventors have developed a general method for artificially designing color development by adjusting the geometric shape and material properties as described above (see, for example, Patent Document 2). In the detection device employing the MIM structure in this disclosure, the pattern corresponding to the upper metal structure 804 is realized by the aggregation of metal microparticles 4 in the island-shaped portion 107 (FIG. 2). Therefore, in order to color the detection device 100, the color of the metal fine particles 4 itself can be changed and artificially designed.

[0020] In addition to those employing MIM structures, the present disclosure also provides detection devices employing color development due to the aggregation of microparticles. Figure 4 is an explanatory diagram illustrating the basic principles of an improved immunochromatography method employing color development due to the aggregation of microparticles in this embodiment. The substrate 9 of the detection device 300 can be made of any material and can have any configuration. For example, the substrate 9 can be patterned with any desired patterning method to immobilize the capture antibodies 6 only in predetermined areas. Therefore, the surface of the substrate 9, or the surface with the capture antibodies 6 along that surface, serves as the contact surface with the sample fluid 12. If another layer is formed on the surface of the substrate 9, the surface of that layer serves as the contact surface. In the detection device 300, the flow channel 301 through which the sample passes does not necessarily require a metal thin film; this flow channel 301 can also be any flow channel through which the sample fluid 12 can flow. The test section 306 and control section develop color; Figure 4 shows only the test section 306. In the test area 306, the antigen 1 bound to the standard antibody 2 in the sample fluid 12 further binds to the capture antibody 6 located there through an antigen-antibody reaction. As a result, the aggregate of microparticles 34 that label the standard antibody 2 and form an aggregate along the spread of the contact surface develops a color corresponding to the size and shape of the aggregate. In the test area 106, the color corresponding to the absorption characteristics of the aggregate of microparticles 34 can be visually observed. The absorption characteristics of the aggregate of microparticles 34 vary depending on the shape, dielectric properties in the optical range, and size of the individual microparticles 34, as well as the shape and size of the island-like portion 307.

[0021] Here, one suitable material for the microparticles 34 is semiconductor microparticles. A preferred semiconductor material for the semiconductor microparticles of the microparticles 34 includes any one selected from the group consisting of silicon (Si), germanium (Ge), and gallium (Ga). Another suitable material for the microparticles 34 is compound microparticles. A preferred compound material for the microparticles 34 includes any one compound selected from the group consisting of titanium nitride (TiN), silicon carbide (SiC), gallium nitride (GaN), hafnium sulfide (HfS2), zinc sulfide (ZnS), barium titanate (BaTiO3), and vanadium dioxide (VO2). When the microparticles 34 are compound microparticles, the material of the microparticles 34 generally needs to have a large dielectric constant or a large imaginary part of the refractive index so that the aggregate of the microparticles 34 will appropriately exhibit color.

[0022] The color of the detection device 300 can also be adjusted based on the color of the aggregate of the particles 34. One suitable method is to form a thin metal film (not shown) on the substrate, thereby forming the substrate shown in FIG. 9 The object of the present invention is to adopt a structure similar to the laminate of the standard antibody 2, the antigen 1, and the metal thin film 7 as the base material of the detection device 300. In this structure, the formed metal thin film sandwiches a composite layer of standard antibody 2, antigen 1, and capture antibody 6 together with an assembly of microparticles 34. That is, in the MIM structure (FIG. 2), the lower M layer corresponds to the metal thin film, and the layer corresponding to the upper M layer is an assembly of microparticles 34 that is not necessarily metal. In this structure, the response of the assembly of microparticles 34 to visible light can be modulated by the metal thin film.

[0023] 2.Specific configuration A specific configuration employing an MIM structure in this embodiment will be described. FIG. 5A is a diagram showing the overall structure of a detection device 100 according to the present disclosure. In the detection device 100 according to the present disclosure, in the test section 106 or control section 108, metal microparticles 4, which may be microparticles that label the standard antibody 2, and a metal thin film 7 formed on a substrate 9 serving as a support are sandwiched between them to form an MIM structure, with either a composite layer of the standard antibody 2, antigen 1, and first capture antibody 6 (test section 106) or a composite layer of the standard antibody 2 and second capture antibody 8 (control section 108) sandwiched between them. The test section 106 and control section 108 are, for example, in the form of a line or a thin strip extending transversely to the flow direction of the flow channel 101. In this flow channel 101, the test section 106 is located upstream, and the control section 108 is located downstream. The substrate 9 is any material capable of supporting the metal thin film 7, and can be any solid or gel material, such as an insoluble membrane carrier, silicon substrate, glass substrate, or resin substrate.

[0024] A sample fluid 10 is dropped into the dropping section 102. When the sample fluid 10 is supplied to the dropping section 102, it also permeates the conjugate section 104. A standard antibody 2 is previously placed in the conjugate section 104 in a detachable manner. The standard antibody 2 is labeled with metal microparticles 4. In reality, the metal microparticles 4 may be larger than the standard antibody 2. If the sample fluid 10 contains an antigen 1, the standard antibody 2 labeled with the metal microparticles 4 will bind to the antigen 1 through an antigen-antibody reaction. The sample fluid 12, in which the standard antibody 2-antigen 1 complex is dispersed, moves through the flow path 101. The flow path 101 through which the sample fluid 12 moves is illuminated by some kind of light source or external light (not shown), such as natural light, and the reflected light is visible.

[0025] In the flow channel 101, a metal thin film 7 supported by a substrate 9 is formed. The sample fluid 12 flows while contacting the surface of the metal thin film 7. The metal thin film 7 may be subjected to additional treatments, such as surface treatments, to appropriately induce chromatographic phenomena, such as the flow and integration of the sample fluid 12, as long as they do not violate the detection principle described below. Figure 5B is an enlarged view of a test section 106 located midway through the flow channel 101. In the test section 106, island-shaped sections 107, each having a square planar shape, are appropriately arranged with a gap G1 between them. An example of this arrangement is a square lattice. The size of the island-shaped sections 107 is typically approximately 190 nm to 420 nm square, and the width of the gap G1 is approximately 75 nm to 180 nm. However, these sizes and shapes can be changed depending on the desired color and the refractive index of the sample fluid 12. When a sample fluid 12 containing antigen 1 passes through the island-shaped portion 107, an antigen-antibody reaction occurs between the antigen 1 and the first capture antibody 6. As a result, an MIM structure is formed, with the metal thin film 7 as the metal (M layer), the composite layer of the first capture antibody 6, antigen 1, and standard antibody 2 as the transparent dielectric layer (I layer), and the metal microparticles 4 as the metal (M layer). Figure 5C is a detailed enlarged view showing the layer structure of the island-shaped portion 107. In the island-shaped portion 107, the first capture antibody 6 is immobilized on the surface of the metal thin film 7. This first capture antibody 6 can bind to the antigen 1 through an antigen-antibody reaction, but cannot bind to the standard antibody 2 that is not bound to the antigen 1. To prevent the formation of an MIM structure in the island-shaped portion 107 and the gap position, the surface of the metal thin film 7 can be patterned so that the first capture antibody 6 binds only to the portions corresponding to the island-shaped portion 107. This patterning can be achieved by a method in which a substance to which the first capture antibody 6 is bound is attached in a pattern only to the range of the island-shaped portion 107 .

[0026] Before the sample fluid 12 reaches the island-shaped portion 107, it consists of, from bottom to top, the substrate 9, the metal thin film 7, and the first capture antibody 6; no MIM structure is formed. At this stage, the detection device 100 can be fabricated so that the test section 106 is visually indistinguishable from the rest of the flow path 101 excluding the test section 106 and the control section 108. The same applies even if the sample fluid 12 does not contain antigen 1 in the original sample fluid 10. On the other hand, if the original sample fluid 10 contains antigen 1, the standard antibody 2 in the sample fluid 12 binds to antigen 1, and antigen 1 binds to the first capture antibody 6, forming the MIM structure described above. Therefore, the MIM structure absorbs a portion of the visible wavelengths of the illumination light, causing the test section 106 to develop a color.

[0027] The control section 108 also has a structure similar to that of the test section 106, with island-shaped sections 109 formed. Figure 5D is an enlarged, intermediate view of the control section 108 located midway along the flow path 101. Similar to the test section 106 (Figure 5B), the control section 108 also has island-shaped sections 109, each with a square planar shape, arranged in an appropriate array, such as a square lattice, with a gap G2 between each. The typical size of the island-shaped sections 109 is similar to that of the island-shaped sections 107, and details of this will be described later. In the island-shaped sections 109, the standard antibody 2 contained in the sample fluid 12 forms an MIM structure, with the metal thin film 7 as the metal (M layer), the composite layer of the second capture antibody 8 and the standard antibody 2 as the transparent dielectric layer (I layer), and the metal microparticles 4 as the metal (M layer). Figure 5E is a detailed enlarged view showing the layer structure of the island-shaped sections 109. In the island-shaped sections 109, the second capture antibody 8 is also immobilized on the surface of the metal thin film 7. This second capture antibody 8 can bind to the standard antibody 2 that is not bound to the antigen 1 through an antigen-antibody reaction. As with the island-shaped portions 107, an MIM structure is formed in the island-shaped portions 109, and to prevent such an MIM structure from being formed at the position of the gap, the surface of the metal thin film 7 is patterned so that the second capture antibody 8 binds only to the portions that correspond to the island-shaped portions 109. This patterning method can be achieved by the same method as for the island-shaped portions 107.

[0028] The island-shaped portions 107 in the test section 106 and the island-shaped portions 109 in the control section 108 are arranged in a repeating island pattern as described above, allowing for adjustment of color development. Therefore, the arrangement and size of the island-shaped portions 109 can be made to match those of the island-shaped portions 107. Alternatively, the arrangement and size of the island-shaped portions 109 may be different from those of the island-shaped portions 107 depending on the desired color or reflecting differences in the MIM structure to be formed (e.g., differences in the composition of the composite layer that becomes the I layer). In other words, even if the island-shaped portions 107 and 109 have the same island pattern, arrangement, and gaps, they may not necessarily produce the same color due to differences in the presence or absence of antigen 1 and the first capture antibody 6 and the second capture antibody 8. In the detection device 100, the geometric shapes of the island-shaped portions 107 and 109 can be easily adjusted by controlling the shape during pattern formation. As a result, it is possible to color the test section 106 and the control section 108 the same color, to color the test section 106 and the control section 108 different colors, or to make the test section 106 and the control section 108 themselves stand out as a single band-like color, without forming them into a single color. Furthermore, by selecting the color itself in consideration of visibility or by using a combination of sharp colors for the coloring pattern, it is possible to lower the detection limit concentration of the antigen 1, increase the accuracy of visual determination, and improve visibility and practicality. These color adjustments and color combination changes can be made not only by selecting the material of the metal microparticles 4, but also by adjusting the geometric shapes of the test section 106 and the control section 108 and the island-like portions 107 and 109 in each of these sections, even if the metal microparticles 4 are fixed.

[0029] In other words, the detection device of this embodiment does not directly reflect the optical absorption of the reference antibody labeling substance, such as gold nanoparticles, as in conventional immunochromatographic detection devices. The detection device of the present disclosure utilizes the absorption reflecting the resonance interaction of the MIM structure formed between the metal nanoparticles labeling the reference substance and the metal thin film formed on the substrate, thereby enabling the design flexibility to be utilized to enhance practicality. Thus, in the test section 106 and control section 108, the difference in color of the device between when the MIM structure is formed and when it is not is visually distinguishable, forming the basis for judgment. The presence of antigen 1 in the sample fluid 10 is sensitively detected as a color change in the device due to the resonance absorption phenomenon of the designed metal structure. This does not require specialized equipment. The color change can be relatively freely adjusted during the design stage of the detection device and can be adjusted to be sensitively detectable by the naked eye.

[0030] As shown in Figures 5C and 5E, the metal microparticles 4 collectively function as the upper metal structure of the MIM structure. The material of the metal microparticles 4 can be appropriately selected to achieve the desired color. Preferred materials for the metal microparticles are metals selected from the group consisting of silver, aluminum, copper, chromium, and nickel. Silver and aluminum, in particular, have the advantage of having a high plasma frequency and no absorption band in the visible range, providing a wider color tuning range than gold. Gold can also be selected from the perspective of chemical stability. These metals can also be used for the metal thin film 7, which forms the lower metal structure of the MIM structure. Using these metals for the metal microparticles 4 or metal thin film 7 is advantageous for achieving the desired color. The metal microparticles 4 and metal thin film 7 can be made of the same metal or different metals.

[0031] The geometric shape of the MIM structure can be modified using several techniques to adjust the color development. Figure 6 is a cross-sectional view showing the configuration of the island portion 107 of the test section 106 and the island portion 109 of the control section 108 shown in Figures 5B and 4C in the detection device 100. As shown in Figure 6, the transparent dielectric layer (I layer) of the MIM structure is formed by a composite layer (test section 106) of the standard antibody 2, the antigen 1, and the first capture antibody 6. In the case of the control section 108, the composite layer of the standard antibody 2 and the second capture antibody 8 serves as the transparent dielectric layer.

[0032] There are no particular restrictions on the standard antibody 2, first capture antibody 6, and second capture antibody 8 for the optical operation described above. The standard antibody 2, first capture antibody 6, and second capture antibody 8 selected based on the immunological reaction (a type of physicochemical bond) associated with the target substance (antigen 1) do not necessarily result in a transparent dielectric layer (I layer) of a thickness suitable for color development. Therefore, the detection device of this embodiment can be improved to achieve a transparent dielectric layer of a thickness that exhibits favorable color development. Figure 7 is a cross-sectional view of a detection device 100A using a transparent dielectric thin film 3A in this embodiment. The transparent dielectric thin film 3A, together with the first capture antibody 6, antigen 1, and standard antibody 2, constitutes the transparent dielectric layer (I layer) of an MIM structure. The surface of the transparent dielectric thin film 3A is designed for patterning, forming an island-shaped region 107A for the test region 106A and an island-shaped region 109A for the control region 108A. The transparent dielectric thin film 3A is also formed around the island portions 107A and 109A. The transparent dielectric thin film 3A functions as a thickness adjustment layer that adjusts the thickness of the transparent dielectric layer (I layer) in the island portions 107A and 109A of the MIM structure. The transparent dielectric thin film 3A can be different between the test portion 106A and the control portion 108A. Various materials can be used for the transparent dielectric thin film 3A, including glass (silicon dioxide), resins, and inorganic materials such as magnesium fluoride, calcium fluoride, and silicon nitride. A suitable material for the transparent dielectric thin film 3A is one that exhibits some degree of transparency at any wavelength in the visible range, for example.

[0033] In the detection device of the present disclosure, it is also useful to pattern the metal thin film, which is the lower metal structure of the MIM structure, into a repeating island pattern. Figure 8 is a cross-sectional view of another detection device 100B of this embodiment. Even if it is difficult to finely pattern the capture antibody in the test or control section for some reason, the color development in the MIM structure can be adjusted by using, for example, a patterned lower metal thin film 7B in which the metal thin film is patterned. Any method employed in microfabrication technology can be used to pattern the patterned lower metal thin film 7B formed on the substrate 9. The patterned lower metal thin film 7B can be arranged as either an island-shaped portion 107B for the test section 106B or an island-shaped portion 109B for the control section 108B, as needed.

[0034] FIG. 9 is a cross-sectional view of yet another detection device 100C of this embodiment. The detection device of this embodiment can also be configured with an upper metal layer of an MIM structure that uses a patterned additional metal thin film layer in addition to metal microparticles. In the detection device 100C of FIG. 9, an additional metal thin film layer is disposed in a position that will become part of the upper metal layer of the MIM structure, and this is patterned to form a patterned upper metal thin film layer 5. Furthermore, the first capture antibody 6 or the second capture antibody 8 is disposed in a position where the patterned upper metal thin film layer 5 is not disposed. To pattern the patterned upper metal thin film layer 5, any method employed in fine processing technology can be used.

[0035] In the detection device 100C shown in Figure 9, the transparent dielectric thin film 3C has a step, with the area where the patterned upper metal thin film layer 5 is located being thicker and the other areas being thinner. This step can be determined based on the effective thickness of the composite layer formed by the standard antibody 2, antigen 1, first capture antibody 6, and second capture antibody 8. This step can also be formed using fine processing techniques. Furthermore, the transparent dielectric thin film 3C may be a single layer or a multilayer. In the case of a multilayer, the step can be easily adjusted by stacking layers with different etching rates. In addition, the transparent dielectric thin film 3C may be formed only in the area where the patterned upper metal thin film layer 5 is located. The metal microparticles 4 and the patterned upper metal thin film layer 5 can be arranged in both or either the test section 106C or the control section 108C, as needed.

[0036] In the detection devices 100, 100A, and 100B shown in Figures 5 to 8, the upper metal layer of the MIM structure is composed of metal microparticles 4 that can be positioned by the action of capture antibodies 6 and 8. In this case, the color change in the test and control sections depending on the presence or absence of metal microparticles 4 is utilized. For example, in the detection device 100 shown in Figure 5, the observed color of the test section 106 illuminated with white light is white for sample fluid 10 that does not contain antigen 1, and is non-white for sample fluid 10 that contains antigen 1 and forms an MIM structure due to the presence of metal microparticles 4. In contrast, in the test section 106C and control section 108C of the detection device 100C shown in Figure 9, the island section 107C forms an MIM structure with the patterned upper metal thin film layer 5 even when no metal microparticles 4 are present. When the sample fluid 10 contains antigen 1 and metal microparticles 4 are supplied, the metal microparticles 4 and the patterned upper metal thin film layer 5 together function as the upper metal layer of the MIM structure, changing the effective size of the upper metal layer and resulting in a color change. In this embodiment, the shape and size of the islands 107C, 109C including the patterned upper metal thin film layer 5 can be designed so that this color change is easily detected visually. That is, the observed color can be configured to be a non-white color (color A) for sample fluid 10 that does not contain antigen 1, and a different non-white color (color B) for sample fluid 10 that contains antigen 1 and forms an MIM structure due to the presence of metal microparticles 4. In detection device 100C, the presence or absence of metal microparticles 4 changes the size of the upper metal layer of the MIM structure, thereby causing a color change.

[0037] 3. Experimental Verification The expected color development phenomenon of the island-shaped portion 109 of the control section 108 in Figure 6 of this embodiment was experimentally confirmed. Figure 10A is a schematic cross-sectional view illustrating the conditions for this experimental confirmation, Figure 10B is an SEM photograph of the actual gold pattern formed, and Figures 10C and 10D are optical microscope images before and after the sample fluid was dispensed. As shown in Figure 10A, a silicon wafer was used as the substrate 9, and its surface was coated with a 100 nm thick aluminum layer. A gold circular patch array structure with a square lattice, 5 nm thick, 100 nm diameter, and 200 nm pitch, was then formed. The aluminum corresponds to the metal thin film 7, and the gold circular patch array structure was coated to selectively modify biotin molecules, as described below. Furthermore, while the island-shaped portion 109 in Figure 6 has a square pattern, experimental confirmation was performed based on a circular pattern. As shown in Figure 10B, each circular patch was approximately 100 nm in diameter. The gold circular patch array structure was formed only in an area of ​​800 μm square.

[0038] At this stage, the areas with the gold circular patch array structure cannot be distinguished visually from the surrounding areas where no gold patches have been formed. In other words, even if the gold patch structure is exposed to air or if a refractive index medium (such as water) is placed on top of it, the presence or absence of the gold patches is almost impossible to determine visually, as shown in Figure 10C, as they are achromatic like the surrounding area and only show a slight change in brightness. This is because the gold film thickness of the gold patches is only 5 nm, and the optical effects such as coloring that are characteristic of gold are hardly manifested.

[0039] Next, the substrate surface was modified with biotin molecules. The biotin molecules bound only to the gold circular patches, acting as the second capture antibody 8 (Figure 6) in the MIM structure. Even at this stage, the gold circular patch array structure was still visually indistinguishable from the surrounding areas where no gold patches had been formed.

[0040] A dispersion of 50 nm diameter gold nanoparticles whose surfaces were modified with avidin was prepared as a liquid corresponding to the sample fluid 10. In the MIM structure, avidin plays the role of the standard substance (standard antibody 2) of this embodiment, labeled with gold nanoparticles. This dispersion was dropped onto an area covering the portion of the substrate surface where the gold circular patches were formed, and allowed to react. Only the areas where the gold circular patches were formed turned a chromatic pink, while the areas without gold circular patches remained achromatic gray. This is shown in Figure 10D. Note that Figure 10D is a monochrome version of a color photograph, and the pink area in the color photograph (the square area in the center indicated by the range) is shown only as a gray that is lighter than the surrounding area.

[0041] The inventors of the present application believe that this coloration phenomenon is due to the fact that in the area where the gold circular patch is formed, biotin molecules and avidin molecules bind to each other, forming an MIM structure consisting of a gold nanoparticle layer (M layer) that labels the avidin molecules, a composite layer (I layer) of avidin molecules and biotin molecules, and a metal layer (M layer) of an aluminum thin film and the gold circular patch, and that this MIM structure is patterned.

[0042] Through this series of experiments, it was confirmed that it is possible to visually determine whether an MIM structure has actually been realized in the control section 108 in Figure 6. It is fully expected that an MIM structure can also be formed in the island-shaped section 107 of the test section 106 through an antigen-antibody reaction, and therefore the inventors believe that the operating principle of the detection device 100 of this embodiment is experimentally supported. In addition, the inventors believe that a general method developed separately by the inventors for artificially designing color development by adjusting the geometric shape and material (Patent Document 2) can also be applied to this embodiment.

[0043] 4. Variations In the above explanation, attention has been paid to the optical behavior of the test section and control section, which are the detection sections. In order to facilitate visual detection and increase sensitivity, a configuration can be adopted that makes it easier to determine the detection section at positions in the flow channel 101, including not only the detection section but also positions other than the detection section. For example, by forming the patterned upper metal thin film layer 5 shown in FIG. 9 at positions other than the detection section, the color tone that becomes the background of the flow channel 101 can be adjusted by the MIM structure. Metal particles 4 are arranged in the test section 106 and the control section 108. Ta The color tone of the image can be adjusted to a different designed color that contrasts well with the background color tone in a given situation. The detection device of this embodiment allows for such flexible color adjustment to be easily performed.

[0044] In the above description, the detection substance is an antigen, the standard substance is a standard antibody capable of physicochemically binding to the antigen, and the capture substance is a capture antibody capable of physicochemically binding to at least one of the antigen and the standard antibody. The detection device of this embodiment can also be used for immunological testing using a different combination. That is, the detection device can be implemented by using an antibody as the detection substance, a standard antigen capable of physicochemically binding to the antibody, and a capture substance capable of physicochemically binding to at least one of the antibody and the standard antigen. Similarly, the device can also be applied to molecules that recognize and bind to each other.

[0045] 5. Summary The embodiments of the present disclosure have been specifically described above. The above-described embodiments and configuration examples have been described for the purpose of explaining the invention, and the scope of the invention of this application should be determined based on the claims. Furthermore, modifications within the scope of the present disclosure, including other combinations of the embodiments, are also included in the claims. [Industrial Applicability]

[0046] The present disclosure is used to manufacture detection devices for testing techniques that determine the presence or absence of target substances through physicochemical binding, including immunological reactions. [Explanation of symbols]

[0047] 1. Antigen (substance to be detected) 2 Standard antibody (standard material) 3A , 3C transparent dielectric thin film (thickness adjustment layer) 34 Fine particles 4 Metal fine particles 5. Patterned top metal thin film layer 6. First capture antibody (capture substance) 7. Metallic thin film 7B Patterned lower metal thin film 8. Second capture antibody (capture substance) 9 Base material (support) 10 Sample fluid 12 Sample fluid (standard antibody dispersion) 100, 100A, 100B, 100C, 300 detection device 101, 301 flow channels 102 Dripping part 104 Conjugate part 106, 106A, 106B, 106C, 306 Test section (detection section) 107, 107A, 107B, 107C, 109A, 109B, 109C, 307 Island 108, 108A, 108B, 108C Control section (detection section) 200 illumination light

Claims

1. A detection device for detecting a target substance that may be contained in a sample fluid, comprising: a flow path for the sample fluid in which a standard substance capable of physicochemically binding to the target substance is dispersed; at least one visible detection unit disposed in the flow path and having a capture substance fixed to at least a portion of a contact surface with the sample fluid; It is equipped with the standard is labeled with a microparticle; the capture substance is capable of physicochemically binding to at least one of the analyte substance bound to the standard substance or the standard substance, the microparticles form aggregates along the extension of the contact surface and develop color when the target substance bound to the standard substance or the standard substance physicochemically binds to the capture substance, the fine particles are metal fine particles, the at least one detection unit includes a metal thin film formed on a support; When the sample fluid contains the target substance, the at least one detection unit the metal thin film; a composite layer consisting of the capture substance, the target substance, and the standard substance, or a composite layer consisting of the capture substance and the standard substance; The aggregate of metal fine particles; are configured to form an MIM structure. Detect devices.

2. the at least one detection unit includes a test unit provided upstream of the flow path and a control unit provided downstream of the flow path, the test part has a first capture substance, which is the capture substance for the test part, immobilized on a surface that contacts the sample fluid; the control part has a second capture substance, which is the capture substance for the control part, immobilized on a surface that contacts the sample fluid; the first capture substance is capable of physicochemically binding to the analyte substance bound to the standard substance; the second capture substance is capable of physicochemically binding to the standard substance; The detection device of claim 1 .

3. the MIM structure of the test section comprises the metal thin film, a composite layer of the first capture substance, the target substance, and the reference substance, and an aggregate of the metal fine particles; the MIM structure of the control section is composed of the metal thin film, a composite layer of the second capture substance and the standard substance, and an aggregate of the metal fine particles; The detection device of claim 2 .

4. The capture substance is patterned into a repeating island pattern, thereby adjusting the color development of the MIM structure in the detection section. The detection device according to claim 1 or claim 3.

5. The metal thin film is patterned into a repeating island pattern, thereby adjusting the color development of the MIM structure in the detection section. The detection device according to claim 1 or claim 3.

6. the repeating island pattern is an island pattern arranged with gaps between them, and the island pattern has at least one shape selected from the group consisting of a substantially square, a substantially circular, and a substantially rectangular shape; The detection device according to claim 4 or claim 5.

7. The metal fine particles are fine particles containing any one metal selected from the group consisting of silver, aluminum, copper, chromium, and nickel. A detection device according to any one of claims 1, 3 and 5.

8. The detecting section further includes a thickness adjustment layer made of a transparent dielectric material on the thin metal film, The capture substance is disposed on the thickness adjustment layer. A detection device according to any one of claims 1, 3, 5 and 7.

9. The detection unit, a transparent dielectric thin film disposed on the metal thin film; an additional patterned metal thin film layer disposed on the transparent dielectric thin film; It also has The capture substance is disposed at a position where the additional metal thin film layer is not disposed. A detection device according to any one of claims 1, 3, 5 and 7.

10. The particles are semiconductor particles. The detection device of claim 1 .

11. The semiconductor fine particles contain any one semiconductor material selected from the group consisting of silicon (Si), germanium (Ge), and gallium (Ga). The detection device of claim 10.

12. The fine particles are compound fine particles. The detection device of claim 1 .

13. The compound fine particles may be titanium nitride (TiN), silicon carbide (SiC), gallium nitride (GaN), hafnium sulfide (HfS ２ ), zinc sulfide (ZnS), barium titanate (BaTiO ３ ), vanadium dioxide (VO ２ ) and containing any one compound selected from the group consisting of The detection device of claim 12.

14. the substance to be detected is an antigen, the standard substance is a standard antibody capable of physicochemically binding to the antigen, The capture substance is a capture antibody that can be physicochemically bound to at least one of the antigen and the standard antibody. The detection device according to any one of claims 1 to 13.

15. the substance to be detected is an antibody, the standard substance is a standard antigen that can be physicochemically bound to the antibody, The capture substance is a capture antigen that can be physicochemically bound to at least one of the antibody and the standard antigen. The detection device according to any one of claims 1 to 13.

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